HK1181781A - Anti-cd27 antibody - Google Patents

Description

anti-CD 27 antibodies

Technical Field

The present invention relates to a humanized antibody or a fragment thereof that specifically recognizes a polypeptide encoded by the CD27 gene comprising an O-linked sugar chain to which galactose is not bound and binds to the extracellular domain of the polypeptide; and a hybridoma producing the humanized antibody, a DNA encoding the humanized antibody, a vector containing the DNA, a transformant obtained by transforming the vector, a method for producing a humanized antibody or the antibody fragment using the hybridoma or the transformant, and a diagnostic agent comprising the humanized antibody or the antibody fragment or a therapeutic agent comprising the humanized antibody or the antibody fragment as an active ingredient.

Background

In recent years, the following examples have been reported: with the onset of various diseases and the progress of the disease, the sugar chain structure attached to a protein expressed by cells associated with the diseases and the disease is changed. In this example, the expression of Tn antigen, which is one of O-linked (serine threonine type) sugar chain antigens, and sialylated Tn antigen in which sialic acid is added to the Tn antigen, which are expressed in more than 80% of human cancer species, is typical (non-patent document 2).

These sugar chain antigens are known to be hardly expressed in normal cells, and studies are being conducted to apply them to medical treatment as target molecules for cancer-specific vaccine therapy (non-patent document 1). The expression of these cancer-specific sugar chain antigens is regulated by enzyme activities constituting complex sugar chain biosynthetic pathways and sugar chain metabolic pathways existing in the living body. As an example, the following are known: in cancer cells, the expression pattern of genes encoding proteins involved in the sugar chain biosynthetic pathway is changed, and as a result, the sugar chain biosynthetic pathway is interrupted halfway; and the like.

The Tn antigen is known as an intermediate product of the biosynthetic pathway of O-linked sugar chains in normal cells, and has: the structure of N-acetylgalactosamine (GalNAc) having an alpha linkage to a hydroxyl group present in the side chain of a specific serine (Ser) residue or threonine (Thr) residue in the amino acid sequence of the protein (GalNAc. alpha. -Ser/Thr).

Biosynthesis of a normal type O-linked sugar chain (TF antigen or the like) is carried out by transferring one molecule of galactose to the non-reducing terminal side of Tn antigen by the activity of core 1. beta.3 galactosyltransferase (core 1. beta.3 Gal-T, T-synthetase). It is considered that in a variety of cancer cell lines, the activity of core1 β 3 galactosyltransferase in cells is decreased, and as a result, the biosynthetic pathway of sugar chains is blocked, thereby expressing Tn antigen or sialyl Tn antigen.

The mechanism of the decrease in the activity of core 1 β 3 galactosyltransferase in cancer cells has been complicated and not completely understood until now. However, one of the mechanisms is presumed to be as follows: a gene encoding a specific chaperone protein (Cosmc) required for the expression of the core 1 β 3 galactosyltransferase activity is mutated, and as a result, the intracellular core 1 β 3 galactosyltransferase activity is greatly reduced (non-patent document 6).

Since the expression of Tn antigen is commonly observed in a plurality of cancer species, it is considered that an abnormality in the intracellular sugar chain biosynthetic pathway or sugar chain metabolic pathway is a major cause of common changes in the sugar chain structures attached to a large number of different glycoproteins expressed in the cell.

A typical disease in which a change in the sugar chain structure is closely related to the progress of a disease state is known as cancer. In addition to cancer, IgA nephropathy is known as a disease in which a change in sugar chain structure is known to be closely related to the progress of disease. IgA nephropathy is chronic glomerulonephritis characterized by pathological observation that immunoglobulin a (IgA) as one of immunoglobulins is deposited in the glomerular mesangial region in granular form, and was first reported by Berger in 1968 (non-patent document 2).

IgA nephropathy is a representative nephritis of about half of chronic glomerulonephritis patients in japan. It is said that about four patients diagnosed with IgA nephropathy develop terminal renal failure within the following 20 years, and thus artificial dialysis and kidney transplantation have to be performed. As can be seen, although IgA nephropathy is generally regarded as a disease with a poor prognosis, a clinically effective treatment method has not yet been established.

In IgA nephropathy patients, it is known that mainly IgA1 of the two IgA isotypes (IgA1 and IgA2) deposits in the kidney. Further, as one of the causes of the deposition, there are reported: the structure of the O-linked sugar chain attached to the hinge region, which is present in IgA1 molecules but not in IgA2 molecules, is changed from a normal type to a Tn antigen or a sialylated Tn antigen (non-patent document 3, non-patent document 4).

It has been demonstrated that when an O-linked sugar chain attached to the IgA1 hinge region lacks galactose and becomes a Tn antigen or sialylated Tn antigen, the self-aggregation ability of the IgA1 molecule is enhanced and the deposition of the IgA1 molecule in the renal membrane region is enhanced (non-patent document 5).

Further, it is reported that: in IgA-producing cells isolated from IgA nephropathy patients, the reduction of expression level of Cosmc leads to a reduction in core 1 β 3 galactosyltransferase activity (non-patent document 6).

That is, in IgA nephropathy patients, IgA production cells are disrupted in the sugar chain biosynthesis pathway halfway, and as a result, IgA1 having a normal sugar chain cannot be produced, and instead, sugar chain-deficient IgA1 is produced. As one of the pathogenesis of IgA nephropathy, the following mechanism is proposed: this sugar chain-deficient IgA1 deposits on the glomerulus, and as a result, inflammation is induced.

Generally, IgA is produced by B cells in blood or plasma cells into which B cells differentiate. It is known that plasma cells, which are the final stage of B cell differentiation, are distributed in secondary lymphoid tissues, systemic mucosal tissues and bone marrow, etc. and produce a large amount of antibodies, while IgA-producing plasma cells are mainly distributed in mucosal tissues.

On the other hand, it is also known that, in the germinal center of the secondary lymphoid tissue, memory B cells or plasma cells are differentiated from B cell clones that have acquired the ability to produce high-affinity IgA antibodies, and distributed in target organs throughout the body to produce antibodies continuously over a long period of time.

However, it has not been clarified in which stage of the differentiation process of B cells a cell producing sugar chain-deficient IgA involved in the onset of IgA nephropathy is produced and in which tissue in the body a B cell or plasma cell producing sugar chain-deficient IgA is distributed.

Among proteins known as cell membrane surface molecules expressed on B cells or plasma cells, CD27 is known as one of molecules to which O-linked sugar chains are bound (non-patent document 7). The CD27 molecule is a type I membrane protein having a molecular weight of about 55kDa belonging to the Tumor Necrosis Factor Receptor (TNFR) superfamily, and exists as a dimer in which two monomers are linked by a disulfide bond (non-patent document 8).

CD27 is known to be expressed on a part of T lymphocytes in addition to plasma cells and B cells, and is known to be elevated particularly in the differentiation process of B cells, when differentiated into memory B cells and plasma cells. It is known that CD27 to which O-linked sugar chains are bonded is expressed on cells in these differentiation processes, but the amino acid residues to which sugar chains are bonded have not been clarified (non-patent document 9).

As a ligand molecule of CD27, CD70, which is one of TNF families, is known. It is known that CD70 binds to part of CD27 expressed on B cells or T cells to introduce a cell proliferation signal or promote antibody production by B cells (non-patent document 10).

It is also known that CD27 is expressed not only in normal cells but also in some cancer cells with increased expression. As the type of CD 27-expressing cancer, various non-hodgkin lymphomas such as mantle cell lymphoma, chronic lymphocytic leukemia, small lymphocytic leukemia, burkitt (バ — キツト) lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, diffuse large cell lymphoma, and plasmacytoma have been reported (non-patent document 11).

It is known that, in most cancer cells, sugar chain-deficient proteins containing sugar chains, such as Tn antigen or sialylated Tn antigen, are expressed as described above.

As an antibody specifically recognizing CD27, there are reported: an S152 antibody obtained by immunizing leukemia cells isolated from a patient with Sezary syndrome (non-patent document 8). However, the S152 antibody showed affinity also for normal B cells and T cells. To date, no antibody has been known that specifically recognizes a CD27 molecule containing an O-linked sugar chain to which galactose is not bound.

It is generally known that when a non-Human Antibody such as a Mouse Antibody or the like is administered to a Human, it is recognized as a foreign substance, thereby inducing an Anti-Mouse Antibody Human Antibody (Human Anti-Mouse Antibody; HAMA) in the Human.

It is known that HAMA reacts with a mouse antibody to be administered to cause side effects (non-patent documents 12 to 15), or accelerates the disappearance of the mouse antibody in vivo (non-patent documents 16 to 18), and reduces the therapeutic effect of the mouse antibody (non-patent documents 19 and 20).

In order to solve the above problems, attempts have been made to produce human chimeric antibodies and humanized antibodies from non-human antibodies by gene recombination techniques.

Humanized antibodies have many advantages for administration to humans over non-human antibodies such as mouse antibodies. For example, it has been reported that in experiments using monkeys, the immunogenicity is reduced and the blood half-life is extended as compared with mouse antibodies (non-patent documents 21 and 22). That is, the humanized antibody has fewer side effects on humans than non-human antibodies, and the therapeutic effect thereof can be expected to be sustained for a long period of time.

In addition, since a humanized antibody is produced by gene recombination technology, it can be produced as a molecule in various forms. For example, when the γ 1 subclass is used as a heavy chain (hereinafter referred to as H chain) constant region (hereinafter referred to as C region) of a human antibody (the H chain C region is referred to as CH), a humanized antibody having a high equivalent function to antibody-dependent cellular cytotoxicity (hereinafter referred to as ADCC activity) can be produced (non-patent document 23), and the half-life in blood can be expected to be longer than that of a mouse antibody (non-patent document 24).

In particular, in the treatment of removing Tn antigen type CD27 positive cells from the body, cytotoxic activities such as complement-dependent cytotoxic activity (hereinafter referred to as CDC activity) and ADCC activity mediated by the Fc region of the antibody (region after the hinge region of the antibody heavy chain) are important in order to accumulate effector cells in the vicinity of target cells by the antibody and specifically kill the target cells. In human therapy, it is preferable to use a human chimeric antibody, a humanized antibody or a humanized antibody in order to exert cytotoxic activity (non-patent documents 25 and 26).

Furthermore, with the recent progress in protein engineering and genetic engineering, humanized antibodies can also be prepared as Fab, Fab ', F (ab') ₂Small-molecular-weight antibody fragments such as single-chain antibodies (hereinafter referred to as scFv) (non-patent document 27), dimerized V-region fragments (hereinafter referred to as Diabody) (non-patent document 28), disulfide-stabilized V-region fragments (hereinafter referred to as dsFv) (non-patent document 29), and peptides containing CDRs (non-patent document 30). These antibody fragments have excellent migration properties to target tissues as compared with intact antibody molecules (non-patent document 31).

Documents of the prior art

Non-patent document

Non-patent document 1: crit Rev oncog.,6,57(1995)

Non-patent document 2: j Urol Nephrol, 74,694(1968)

Non-patent document 3: clin Exp Immunol, 100,470(1995)

Non-patent document 4: j Am Soc Neph, 7,955(1996)

Non-patent document 5: nephrol Dial transfer, 17,50(2002)

Non-patent document 6: j Intern Med.,258,467(2005)

Non-patent document 7: current Opinion in Immunology 17,275(2005)

Non-patent document 8: j Immunol, 141,21(1988)

Non-patent document 9: eur J Immunol, 22,447(1992)

Non-patent document 10: proc.Natl.Acad.Sci.,94.6346(1997)

Non-patent document 11: leukemia and Lymphoma, 43,1855(2002)

Non-patent document 12: hum. Pathol, 38,564(2007)

Non-patent document 13: hem. Pathol, 36,886(2005)

Non-patent document 14: FEBS Lett.,579,6179(2005)

Non-patent document 15: cancer Res.,65,7378(2005)

Non-patent document 16: hem. Pathol, 36,886(2005)

Non-patent document 17: oncogene,13,2328(2006)

Non-patent document 18: virchows arch, 448,52(2006)

Non-patent document 19: j. Immunol.,135,1530(1985)

Non-patent document 20: cancer Res, 46,6489(1986)

Non-patent document 21: cancer Res, 56,1118(1996)

Non-patent document 22: immunol.,85,668(1995)

Non-patent document 23: cancer Res, 56,1118(1996)

Non-patent document 24: immunol.,85,668(1995)

Non-patent document 25: J.Immunol.,144,1382(1990)

Non-patent document 26: nature,322,323(1988)

Non-patent document 27: science,242,423(1988)

Non-patent document 28: nature Biotechnol.,15,629(1997)

Non-patent document 29: molecular immunol.,32,249(1995)

Non-patent document 30: j.biol.chem.,271,2966(1996)

Non-patent document 31: cancer Res.,52,3402(1992)

Disclosure of Invention

Problems to be solved by the invention

As described above, no antibody has been known so far which specifically recognizes CD27 molecule containing an O-linked sugar chain to which galactose is not bound. The antibody specifically recognizing the CD27 molecule containing an O-linked sugar chain to which galactose is not bound is very effective for diagnosing and treating diseases and the like associated with CD27 containing an O-linked sugar chain to which galactose is not bound.

Accordingly, an object of the present invention is to provide a monoclonal antibody that specifically recognizes CD27 comprising an O-linked sugar chain to which galactose is not bound and binds to the extracellular domain of CD27, or a method for using the same.

Means for solving the problems

The present invention relates to the following (1) to (23).

(1) A humanized antibody or an antibody fragment thereof, wherein the VH of the antibody is a VH comprising the amino acid sequence of CDR 1-3 represented by SEQ ID Nos. 58-60 and the VL of the antibody is a VL comprising the amino acid sequence of CDR 1-3 represented by SEQ ID Nos. 61-63, which antibody or antibody fragment thereof recognizes and binds to the extracellular domain of a polypeptide encoded by the CD27 gene comprising an O-linked sugar chain to which galactose is not bound.

(2) The humanized antibody or an antibody fragment thereof according to (1), wherein the VH of the humanized antibody comprises an amino acid sequence into which at least one modification selected from the following modifications is introduced: substitution of Ser at position 30 into Asn, substitution of Val at position 48 into Ile, substitution of Ser at position 49 into Ala, substitution of Asn at position 77 into Gly, substitution of Val at position 93 into Thr, substitution of Ala at position 97 into Thr, and substitution of Thr at position 117 into Val in the amino acid sequence represented by SEQ ID NO. 96; and the VL of the humanized antibody comprises an amino acid sequence into which at least one modification selected from the following modifications is introduced: substitution of Ile at position 21 to Leu, substitution of Pro at position 40 to Leu, substitution of Val at position 58 to Ile, substitution of Thr at position 85 to Ala, and substitution of Tyr at position 87 to Phe in the amino acid sequence shown in SEQ ID NO. 97.

(3) The humanized antibody or an antibody fragment thereof according to (1), wherein VH of the humanized antibody comprises an amino acid sequence represented by any one of sequence Nos. 96, 105 and 107, and VL of the humanized antibody comprises an amino acid sequence represented by sequence No. 97.

(4) A DNA encoding the humanized antibody or the antibody fragment according to any one of (1) to (3).

(5) A recombinant vector comprising the DNA according to (4).

(6) A transformant obtained by introducing the recombinant vector of (5) into a host cell.

(7) A method for producing the humanized antibody or the antibody fragment thereof according to any one of (1) to (3), which comprises culturing the transformant according to (6) in a culture medium, producing and accumulating the humanized antibody or the antibody fragment according to any one of (1) to (3) in the culture, and collecting the antibody or the antibody fragment from the culture.

(8) An immunological detection or measurement method for CD27 comprising an O-linked sugar chain to which a galactose is not bonded, which uses the humanized antibody or the antibody fragment according to any one of (1) to (3).

(9) A detection reagent for CD27 comprising an O-linked sugar chain to which galactose is not bound, which comprises the humanized antibody or the antibody fragment according to any one of (1) to (3).

(10) A diagnostic agent for a disease associated with CD27 comprising an O-linked sugar chain to which a galactose is not bonded, comprising the humanized antibody or the antibody fragment according to any one of (1) to (3).

(11) The diagnostic agent according to (10), wherein the disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound is IgA nephropathy.

(12) The diagnostic agent according to (10), wherein the disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound is cancer.

(13) A therapeutic agent for a disease associated with CD27 comprising an O-linked sugar chain to which a galactose is not bonded, comprising the humanized antibody or antibody fragment according to any one of (1) to (3) as an active ingredient.

(14) The therapeutic agent according to (13), wherein the disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound is IgA nephropathy.

(15) The therapeutic agent according to (13), wherein the disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound is cancer.

(16) A method for diagnosing a disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound, comprising: a cell expressing CD27 comprising an O-linked sugar chain to which galactose is not bound, which is detected or measured using the humanized antibody or the antibody fragment according to any one of (1) to (3).

(17) A method for diagnosing a disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound, comprising: CD27 comprising an O-linked sugar chain to which galactose is not bound is detected or measured using the humanized antibody or the antibody fragment according to any one of (1) to (3).

(18) The diagnostic method according to (16) or (17), wherein the disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound is IgA nephropathy.

(19) The diagnostic method as described in (16) or (17), wherein the disease associated with CD27 containing an O-linked sugar chain to which galactose is not bound is cancer.

(20) Use of the humanized antibody or the antibody fragment of any one of (1) to (3) for the production of a diagnostic agent for a disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound.

(21) Use of the humanized antibody or the antibody fragment of any one of (1) to (3) for producing a therapeutic agent for a disease associated with CD27, which contains an O-linked sugar chain to which galactose is not bound.

(22) The use of the humanized antibody or the antibody fragment according to (20) or (21), wherein the disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound is IgA nephropathy.

(23) The use of the humanized antibody or the antibody fragment of (20) or (21), wherein the disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound is cancer.

Effects of the invention

The humanized antibody or the antibody fragment of the present invention is a monoclonal antibody that specifically recognizes the extracellular domain of a polypeptide encoded by the CD27 gene (hereinafter referred to as CD27) containing an O-linked sugar chain to which galactose is not bound, and binds to the extracellular domain.

By detecting or quantifying the sugar chain-deficient CD27 or cells expressing the polypeptide using the humanized antibody or the antibody fragment of the present invention, a disease associated with the sugar chain-deficient CD27 can be diagnosed.

In addition, since the humanized antibody or the antibody fragment of the present invention can bind to the extracellular domain of the sugar chain-deficient CD27 polypeptide, it can be suitably used for detecting cells expressing the polypeptide. In particular, since the antibody or the antibody fragment of the present invention can bind to the extracellular domain of the sugar chain-deficient CD27, it can be preferably used for the analysis by flow cytometry for detecting CD27 expressed on the cell membrane while maintaining the natural type steric structure.

In addition, the antibody or the antibody fragment having an effector activity as the humanized antibody or the antibody fragment of the present invention can kill cells expressing the sugar chain-deficient CD27 polypeptide both in vivo and in vitro. In addition, the above-mentioned humanized antibody or the antibody fragment of the present invention can kill cells expressing a sugar chain-deficient CD27 polypeptide in vivo to reduce them, and therefore, can be used particularly effectively as a therapeutic agent.

Drawings

FIG. 1 shows a method for producing a plasmid vector pCR2.1CD27 containing a DNA sequence encoding human CD27 protein.

FIG. 2 shows a method for preparing a plasmid vector pCR CD27axb comprising a DNA sequence encoding the extracellular domain of human CD27 protein.

FIG. 3 shows a method for preparing plasmid vector pBShC γ 4SP having a mutant human IgG4Fc fragment in which the constant region of human IgG4 has been amino acid-substituted.

FIG. 4 shows a method for preparing a plasmid vector pCR IgG4Fc BamHI comprising a DNA sequence having a restriction enzyme recognition sequence inserted into the DNA sequence of mutant human IgG4Fc fragment.

FIG. 5 shows a method for preparing pKANTEX XhoI/SalI into which a DNA sequence of CD27-Fc was inserted.

FIG. 6 shows a method for preparing a CD27-Fc protein expression vector pKANTEX CD27-hIgG4 Fc.

FIGS. 7(a) and (b) show the results of SDS-PAGE analysis of CD27-Fc protein expressed in CHO/DG44 cells and Lec8 cells as host cells. FIG. 7(a) shows a non-reduced state in which no β -mercaptoethanol is added, and FIG. 7(b) shows a reduced state after β -mercaptoethanol is added. In FIGS. 7(a) and (b), the fractions of the marker, Lec8 cells and CHO/DG44 cells are shown from the left.

FIG. 8(a) shows the results of SDS-PAGE analysis of CD27-Fc protein expressed in CHO/DG44 cells and Lec8 cells as host cells. FIG. 8(b) shows the results of Western blotting of CD27-Fc protein expressed by CHO/DG44 cells and Lec8 cells as host cells. In FIGS. 8(a) and (b), samples of the marker, CHO/DG44 cells and Lec8 cells are shown from the left lane. In Western immunoblotting, staining was performed using anti-RCAS 1 antibody 22-1-1 antibody.

FIG. 9 shows a method for producing a plasmid vector pCR CD27axc containing DNA encoding CD27 protein for expression in animal cells.

FIG. 10 shows a method for preparing animal cell expression vector pKANTEX CD 27.

FIGS. 11(a) and (b) show the results of flow cytometry of anti-CD 27 monoclonal antibodies against CHO/DG44 cells expressing CD27 and Lec8 cells expressing CD 27. FIG. 11(a) shows the results of the histogram for CD27/Lec8-4, and FIG. 11(b) shows the results of the histogram for CD27/DG 44-8. In all histograms, the vertical axis represents the number of cells, and the horizontal axis represents the fluorescence intensity.

FIGS. 12(a) and (b) are graphs showing the results of measuring the binding activity of the anti-sugar chain-deficient CD27 monoclonal antibodies KM4030 and KM4031 to Lec8 cells, CD27/Lec8-4 cells and CD27/DG44-8 cells by fluorescent cell staining. Fig. 12(a) shows the measurement results using the ABI cell detection system, with the vertical axis indicating the fluorescence intensity and the horizontal axis indicating the reacted antibody. Fig. 12(b) shows the measurement results using a Flow Cytometer (FCM), with the average fluorescence intensity on the vertical axis and the reacted antibody on the horizontal axis.

Fig. 13(a) and (b) show the results of competition ELISA for anti-sugar chain-deficient CD27 monoclonal antibodies KM4030 and KM 4031. FIG. 13(a) shows the reactivity of anti-sugar chain-deficient CD27 monoclonal antibody KM4030, and FIG. 13(b) shows the reactivity of anti-sugar chain-deficient CD27 monoclonal antibody KM 4031. The vertical axis represents cell proliferation, and the horizontal axis represents antibody concentration.

FIGS. 14(a) and (b) show the gene cloning method for the anti-sugar chain-deficient CD27 monoclonal antibody.

FIG. 15 shows a method for producing an anti-sugar chain-deficient CD27 chimeric antibody expression vector.

FIG. 16 shows a method for producing an anti-sugar chain-deficient CD27 chimeric antibody expression vector.

FIG. 17 shows a method for producing an anti-sugar chain-deficient CD27 chimeric antibody expression vector.

FIG. 18 shows a method for producing an anti-sugar chain-deficient CD27 chimeric antibody expression vector.

FIGS. 19(A) (a) and (b) are graphs showing the results of measuring the binding activity of various anti-sugar chain-deficient CD27 chimeric KM4026 (. diamond.), chimeric KM4028 (. DELTA.), chimeric KM4030 (. smallcircle.), chimeric KM4031(□) to CD27/Lec8-4 cells using a Flow Cytometer (FCM). FIG. 19(A) and (b) shows the results of measuring the binding activity of a commercially available anti-CD 27 antibody O323(●) to CD27/Lec8-4 cells using a Flow Cytometer (FCM). The vertical axis represents the mean fluorescence intensity, and the horizontal axis represents the final concentration of the reacted antibody.

FIG. 19(B) (a) is a diagram showing the results of measuring the binding activity of various anti-sugar chain-deficient CD27 chimeric KM4026 (. DELTA.), chimeric KM4028 (. DELTA.), chimeric KM4030 (. smallcircle.), chimeric KM4031(□) to CD27/DG44-4 cells using Flow Cytometry (FCM). FIG. 19(B) and (B) show the results of measuring the binding activity of a commercially available anti-CD 27 antibody O323(●) to CD27/DG44-4 cells using a Flow Cytometer (FCM). The vertical axis represents the mean fluorescence intensity, and the horizontal axis represents the final concentration of the reacted antibody.

FIG. 19(C) (a) shows the results of measuring the binding activity of various anti-sugar chain-deficient CD27 chimeric KM4026 (. diamond.), chimeric KM4028 (. DELTA.), chimeric KM4030 (. smallcircle.), chimeric KM4031(□) to Lec8 cells using Flow Cytometry (FCM). FIG. 19(C) and (b) show the results of measuring the binding activity of a commercially available anti-Tn antibody 22-1-1(■) to Lec8 cells using a Flow Cytometer (FCM). The vertical axis represents the mean fluorescence intensity, and the horizontal axis represents the final concentration of the reacted antibody.

FIG. 20 shows antibody-dependent cellular cytotoxicity (ADCC activity) against CD27/Lec8-4 cells of various chimeric antibodies against sugar chain-deficient CD27 KM4026 (. DELTA.), chimeric KM4028 (. DELTA.), chimeric KM4030 (. smallcircle.), and chimeric KM4031(□). The vertical axis represents the cytotoxic activity (%), and the horizontal axis represents the final concentration of each antibody.

FIG. 21 shows complement-dependent cytotoxic activity (CDC activity) of various anti-sugar chain-deficient CD27 chimeric antibodies, chimeric KM4026, chimeric KM4028, chimeric KM4030 and chimeric KM4031 against CD27/Lec8-4 cells. The vertical axis represents cytotoxic activity (%), and the horizontal axis represents the reacted antibody.

FIG. 22 shows a method for preparing a plasmid vector pCRmfCD27 containing a DNA encoding cynomolgus monkey CD27 protein.

FIG. 23 shows a method for preparing a plasmid vector mfCD27His comprising a DNA encoding cynomolgus monkey CD27 protein.

FIG. 24 shows a method for preparing cynomolgus monkey CD27 expression vector pKANTEX mfCD27 His.

FIG. 25 shows the results of measurement of the binding activity of each of the chimeric KM4026, chimeric KM402, chimeric KM4030, chimeric KM4031 and commercially available anti-CD 27 antibody O323 against the chimeric CD27/Lec8 cells or the chimeric CD27/DG44 cells of anti-sugar chain-deficient CD27 chimeric antibodies using a Flow Cytometer (FCM). The vertical axis represents the mean fluorescence intensity, and the horizontal axis represents the reacted antibody.

FIG. 26 shows antibody-dependent cellular cytotoxicity (ADCC activity) against cynomolgus monkey CD27/Lec8 cells of various anti-sugar chain-deficient CD27 chimeric antibodies chimeric KM4026 (. diamond-solid.), chimeric KM4028 (. tangle-solidup.), chimeric KM4030(●) and chimeric KM4031(■). The vertical axis represents the cytotoxic activity (%), and the horizontal axis represents the final concentration of each antibody.

FIG. 27 shows antibody-dependent cellular cytotoxicity (ADCC activity) of various anti-sugar chain-deficient CD27 chimeric antibody chimeric KM4030 against cynomolgus monkey CD27/Lec8 cells (●) or human CD27/Lec8 cells (. smallcircle.). The vertical axis represents the cytotoxic activity (%), and the horizontal axis represents the final concentration of each antibody.

FIG. 28(A) (a) is a BIACORE sensorgram showing the binding of the anti-sugar chain-deficient CD27 antibody KM4026 to the sugar chain-deficient CD27-Fc (Tn antigen type CD 27-Fc). FIG. 28(A) (b) is a BIACORE sensor chart showing the binding between the anti-sugar chain-deficient CD27 antibody KM4027 and the sugar chain-deficient CD27-Fc (Tn antigen type CD 27-Fc). FIG. 28(A) (c) is a BIACORE sensor chart showing the binding between the anti-sugar chain-deficient CD27 antibody KM4028 and the sugar chain-deficient CD27-Fc (Tn antigen type CD 27-Fc). FIG. 28(A) (d) is a BIACORE sensorgram showing the binding of the anti-sugar chain-deficient CD27 antibody KKM4030 to the sugar chain-deficient CD27-Fc (Tn-antigen type CD 27-Fc). FIG. 28(A) (e) is a BIACORE sensor chart showing the binding between the anti-sugar chain-deficient CD27 antibody KM4031 and the sugar chain-deficient CD27-Fc (Tn antigen type CD 27-Fc). The vertical axis represents RU (resonance unit), and the horizontal axis represents reaction time (seconds).

FIG. 28(B) (a) is a BIACORE sensorgram showing the binding of anti-sugar chain-deficient CD27 antibody KM4026 to sialylated Tn antigen type CD 27-Fc. FIG. 28(B) (B) is a BIACORE sensorgram showing the binding of various anti-sugar chain-deficient CD27 antibodies KM4027 to sialylated Tn antigen type CD 27-Fc. FIG. 28(B) (c) is a BIACORE sensorgram showing the binding of anti-sugar chain-deficient CD27 antibody KM4028 to sialylated Tn antigen type CD 27-Fc. FIG. 28(B) (d) is a BIACORE sensorgram showing the binding of the anti-sugar chain-deficient CD27 antibody KM4030 to the sialylated Tn antigen type CD 27-Fc. FIG. 28(B) (e) is a BIACORE sensorgram showing the binding of the anti-sugar chain-deficient CD27 antibody KM4031 to the sialylated Tn antigen type CD 27-Fc. The vertical axis represents RU (resonance unit), and the horizontal axis represents reaction time (seconds).

FIG. 29(a) is a BIACORE sensor diagram showing the binding between the anti-sugar chain-deficient CD27 chimeric antibody KM4030 and the sugar chain-deficient CD27-Fc (Tn antigen type CD 27-Fc). FIG. 29(b) is a BIACORE sensorgram showing the binding of the humanized antibody HV0LV0 to sugar chain-deficient CD27-Fc (Tn antigen type CD 27-Fc). FIG. 29(c) is a BIACORE sensorgram showing the binding of the humanized antibody HV5LV0 to sugar chain-deficient CD27-Fc (Tn antigen type CD 27-Fc). FIG. 29(d) is a BIACORE sensorgram showing the binding of the humanized antibody HV7LV0 to sugar chain-deficient CD27-Fc (Tn antigen type CD 27-Fc). The vertical axis represents RU (resonance unit), and the horizontal axis represents reaction time (seconds).

FIG. 30(A) is a graph showing the results of measuring the binding activity of the anti-sugar chain-deficient CD27 chimeric antibody KM4030 (. smallcircle.) and the anti-sugar chain-deficient CD27 humanized antibodies HV0LV0(□), HV5LV0 (. DELTA.), HV7LV0 (. diamond.) to CD27/Lec8-M19 cells using a Flow Cytometer (FCM). The vertical axis represents the mean fluorescence intensity, and the horizontal axis represents the final concentration of the reacted antibody.

FIG. 30(B) is a graph showing the results of measuring the binding activity of the anti-sugar chain-deficient CD27 chimeric antibody KM4030 (. smallcircle.) and the anti-sugar chain-deficient CD27 humanized antibodies HV0LV0(□), HV5LV0 (. DELTA.), HV7LV0 (. diamond.) to CD27/DG44-4 cells using a Flow Cytometer (FCM). The vertical axis represents the mean fluorescence intensity, and the horizontal axis represents the final concentration of the reacted antibody.

FIG. 31 shows antibody-dependent cellular cytotoxicity (ADCC activity) against CD27/Lec8-M19 cells of the anti-sugar chain-deficient CD27 chimeric antibody KM4030 (. smallcircle.) and the anti-sugar chain-deficient CD27 humanized antibody HV0LV0(□), HV5LV0 (. DELTA.), HV7LV0 (. diamond.). The vertical axis represents the cytotoxic activity (%), and the horizontal axis represents the final concentration of each antibody.

FIG. 32 shows complement-dependent cytotoxic activity (CDC activity) against CD27/Lec8-M19 cells of the anti-sugar chain-deficient CD27 chimeric antibody KM4030 (. smallcircle.) and the anti-sugar chain-deficient CD27 humanized antibody HV0LV0(□), HV5LV0 (. DELTA.), HV7LV0 (. diamond.). The vertical axis represents cytotoxic activity (%), and the horizontal axis represents the reacted antibody.

FIG. 33(A) is a graph showing the results of measuring the binding activity of the anti-sugar chain-deficient CD27 chimeric antibody KM4030 (. smallcircle.) and the anti-sugar chain-deficient CD27 humanized antibodies HV0LV0(□), HV5LV0 (. DELTA.), HV7LV0 (. diamond.) to cynomolgus monkey CD27/Lec8 cells using a Flow Cytometer (FCM). The vertical axis represents the mean fluorescence intensity, and the horizontal axis represents the final concentration of the reacted antibody. The vertical axis represents the mean fluorescence intensity, and the horizontal axis represents the reacted antibody.

FIG. 33(B) is a graph showing the results of measuring the binding activity of the anti-sugar chain-deficient CD27 chimeric antibody KM4030 (. smallcircle.) and the anti-sugar chain-deficient CD27 humanized antibodies HV0LV0(□), HV5LV0 (. DELTA.), HV7LV0 (. diamond.) to cynomolgus monkey CD27/DG44 cells using a Flow Cytometer (FCM). The vertical axis represents the mean fluorescence intensity, and the horizontal axis represents the final concentration of the reacted antibody. The vertical axis represents the mean fluorescence intensity, and the horizontal axis represents the reacted antibody.

Detailed Description

The present invention relates to a monoclonal antibody (hereinafter, also referred to as a humanized antibody of the present invention or an antibody of the present invention) that specifically recognizes an extracellular domain of a polypeptide encoded by the CD27 gene (hereinafter, also referred to as CD27) containing an O-linked sugar chain to which galactose is not bound and binds to the extracellular domain.

The CD27 gene may be any gene encoding CD27, and includes a gene having the nucleotide sequence represented by SEQ ID NO. 1. In addition, a gene that hybridizes to DNA comprising the base sequence represented by seq id No. 1 under stringent conditions and encodes a polypeptide having the function of CD27, and the like are also included in the CD27 gene of the present invention.

The DNA that hybridizes under stringent conditions is DNA that can hybridize using DNA having the base sequence represented by SEQ ID NO. 1 as a probe and obtained by colony hybridization, plaque hybridization, southern blot hybridization, DNA microarray, or the like.

Specifically, there may be mentioned DNAs that can be identified by the following methods: the DNA derived from the colony or plaque by hybridization, or the PCR product or the oligo DNA having the sequence is hybridized with a filter or a slide glass in the presence of 0.7 to 1.0 mol/L sodium chloride at 65 ℃, and then the filter or the slide glass is washed with 0.1 to 2-fold concentration of SSC solution (the composition of the 1-fold concentration of SSC solution includes 150 mmol/L sodium chloride and 15 mmol/L sodium citrate) at 65 ℃ to identify the DNA.

Hybridization can be according to [ Molecular Cloning, A Laboratory Manual, second edition (Cold spring harbor Laboratory Press, 1989); current Protocols in molecular Biology (latest molecular Biology laboratory methods) (John Wilmington publishing Co., 1987-1997); DNA Cloning 1: Core Techniques, A practical approach (DNA Cloning 1: Core technology and practical methods), second edition (Oxford university, 1995), and the like.

The DNA that can hybridize is preferably a DNA having at least 60% homology with the base sequence represented by SEQ ID NO. 1, more preferably a DNA having at least 80% homology with the base sequence represented by SEQ ID NO. 1, and still more preferably a DNA having at least 95% homology with the base sequence represented by SEQ ID NO. 1.

Polymorphisms of genes are often observed in the base sequences of genes encoding proteins of eukaryotes. The CD27 gene used in the present invention also includes a gene whose nucleotide sequence is slightly mutated due to gene polymorphism.

As CD27, there may be mentioned: a polypeptide having an amino acid sequence represented by SEQ ID No. 2; a polypeptide having a function of CD27, which comprises an amino acid sequence in which one or more amino acids are deleted, substituted or added from the amino acid sequence represented by SEQ ID NO. 2; and a polypeptide having a function of CD27, which comprises an amino acid sequence having preferably 60% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more homology with the amino acid sequence represented by seq id No. 2.

The polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO. 2 can be obtained by the following method: use [ Molecular Cloning, A Laboratory Manual, second edition (Cold spring harbor Laboratory Press, 1989); current Protocols in Molecular Biology (John Willtd. father publishing Co., 1987-1997); nucleic Acids Research,10,6487 (1982); proc.natl.acad.sci.usa,79,6409 (1982); gene,34,315 (1985); nucleic Acids Research,13,4431 (1985); site-directed mutagenesis described in Proc.Natl.Acad.Sci.USA,82,488(1985) et al, for example, introduces site-directed mutagenesis into DNA encoding a polypeptide having an amino acid sequence represented by SEQ ID NO. 2.

The number of amino acids to be deleted, substituted or added is not particularly limited, but is preferably one to several tens, for example, 1 to 20 amino acids, and more preferably one to several, for example, 1 to 5 amino acids.

Unless otherwise specified, the homology numerical value described in the present invention may be a numerical value calculated using a homology search program known to those skilled in the art, and the nucleotide sequence may include: numerical values calculated using default parameters in BLAST [ j.mol.biol.,215,403(1990) ], and the like, for amino acid sequences, there are: using BLAST2[ Nucleic acids sres, 25,3389 (1997); genome res, 7,649 (1997); http:// www.ncbi.nlm.nih.gov/duration/BLASTInfo/information 3.html ] as default parameters.

As default parameters, G (Cost to open gap) is 5 in the case of a base sequence and 11 in the case of an amino acid sequence; -E (last tofxtended gap) is 2 in case of base sequence and 1 in case of amino acid sequence; -q (nucleotide mismatch Penalty, Penalty for nucleotide mismatch) is-3; -r (nucleotide match score, rewarded for nucleotide match) is 1; e (expected value) is 10; -W (word size) is 11 residues in the case of base sequence and 3 residues in the case of amino acid sequence; y (threshold for non-vacancy extension fall, Dropoff (X) for blast extensions in bits) is 20 in blastn and 7 in programs other than blastn; -X (drop threshold for gap alignment, X dropoff value for gapped alignment in bits) is 15; z (final X drop value for gap alignment in bits) is 50 in blastn and 25 in programs other than blastn (http:// www.ncbi.nlm.nih.gov/blast/html/blastgihelp. html).

The polypeptide comprising a partial sequence of the amino acid sequence represented by SEQ ID NO. 2 can be produced by a method known to those skilled in the art, for example, by deleting a part of the DNA encoding the amino acid sequence represented by SEQ ID NO. 2 and culturing a transformant into which an expression vector comprising the DNA is introduced.

Further, based on the thus-prepared polypeptide or DNA, a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted or added from the partial sequence of the amino acid sequence represented by SEQ ID NO. 2 can be obtained by the same method as described above.

As the extracellular domain of CD27, there can be mentioned: and a region corresponding to The 1 st to 171 st positions of The ectodomain predicted in The literature [ The Journal of immunology,147,3165(1991) ].

In the present invention, the extracellular domain of the polypeptide encoded by the CD27 gene, which contains an O-linked sugar chain to which galactose is not bound, may be any CD27 containing an O-linked sugar chain to which galactose is not bound. Specifically, examples thereof include: an extracellular domain of CD27 encoded by the base sequence represented by SEQ ID NO. 1, comprising an O-linked sugar chain to which galactose is not bound.

The O-linked sugar chain refers to a structure in which a sugar chain is bonded to an amino acid residue of serine (Ser) or threonine (Thr) in a protein via an — OH group contained in each amino acid side chain.

Among O-linked sugar chains, an O-linked sugar chain in which N-acetylgalactosamine (GalNAc) is bonded to the-OH group of the Ser or Thr amino acid side chain of a polypeptide is referred to as a mucin-type sugar chain. Specific examples of the O-linked sugar chain include: t antigen, sialylated T antigen, Tn antigen, sialylated Tn antigen, and the like (table 1).

[ Table 1]

(NeuNAc: N-acetylneuraminic acid)

In the present invention, the O-linked sugar chain to which galactose is not bonded refers to an O-linked sugar chain to which galactose (Gal) is not bonded to N-acetylgalactosamine (GalNAc) bonded through the-OH group of the amino acid residue of Ser or Thr of the protein. Specific examples thereof include the above-mentioned Tn antigen and sialylated Tn antigen.

The O-linked sugar chain to which galactose is not bonded is an intermediate in the synthetic pathway of a normal O-linked sugar chain, and is usually scarcely present in a glycoprotein in normal cells, while expression is confirmed in some specific diseases such as cancer and renal diseases.

Hereinafter, in the present invention, an O-linked sugar chain to which galactose is not bonded may be referred to as an abnormal sugar chain, a protein to which an abnormal sugar chain is bonded may be referred to as a sugar chain-deficient protein, and CD27 to which an abnormal sugar chain is bonded may be referred to as sugar chain-deficient CD 27.

Examples of the amino acid residue in the polypeptide to which an O-linked sugar chain is bonded include: the amino acid residues of serine (Ser) and threonine (Thr) in the amino acid sequence of the extracellular domain of the CD27 protein.

Further, the consensus sequence for O-linked sugar chain binding can be confirmed by using sequence search software such as NetOGlyc3.1Server (http:// www.cbs.dtu.dk/services/NetOGlyc /) for amino acid residues in the polypeptide to which the O-linked sugar chain binds. Alternatively, specific sugar chain binding sites can be determined by Mass Spectrometry (MS) analysis of glycoproteins having O-linked sugar chains.

In the present invention, as the amino acid residue in the polypeptide to which an O-linked sugar chain in the CD27 protein binds, an O-linked sugar chain can be bound to any one of Ser or Thr residues in the amino acid sequence of the CD27 protein, and preferred examples thereof include sugar chain binding sites comprising at least one amino acid residue selected from the group consisting of Thr at position 118, Ser at position 127, Thr at position 129, Ser at position 132, Ser at position 133, Ser at position 137, Thr at position 143, Ser at position 149, Thr at position 156, Thr at position 162, Thr at position 173, Ser at position 175, and Thr at position 176 in the CD27 protein represented by seq id No. 2.

The number of O-linked sugar chains bonded to the extracellular domain of the CD27 protein is not limited as long as at least one Ser residue or Thr residue is bonded to an O-linked sugar chain.

As a method for obtaining a cell expressing CD27 (hereinafter referred to as sugar chain-deficient CD27) comprising an O-linked sugar chain to which galactose is not bound of the present invention, it can be produced, for example, by the following method: a cell expressing a sugar chain-deficient CD27 was prepared by introducing a DNA encoding CD27 into a cell strain in which the activity of an enzyme that adds Gal to N-acetylgalactosamine (GalNAc) bonded to Ser/Thr in the polypeptide in the O-linked sugar chain synthesis process, a protein involved in the activity of the enzyme, a protein involved in the transport of uridine 5' -diphosphate galactose (UDP-galactose), or the like was reduced or deleted.

Alternatively, cells expressing CD27 having an O-linked sugar chain to which galactose is not bound can also be prepared by allowing a sugar chain cleaving enzyme such as sialidase or galactosidase to act on cells expressing CD27 containing normal O-type sugar chains.

Specific examples of enzymes that add Gal to GalNAc bound to Ser or Thr on a polypeptide include β 1, 3-galactosyltransferase [ The Journal of biological chemistry,277,178-186(2002) ], and The like.

In addition, as proteins involved in the activity of an enzyme that adds Gal to GalNAc binding to Ser or Thr on a polypeptide, mention may be made of Cosmc [ proceedings of the National Academy of Sciences of the United states of America,99,16613-16618(2002) ] which is a partner involved in protein folding of the enzyme.

CD 27-expressing cells derived from IgA nephropathy patients can be used as CD 27-expressing cells because of the reduction or deletion of the activity of an enzyme that adds Gal to GalNAc bound to Ser/Thr on the polypeptide, a protein involved in the activity of the enzyme, a protein involved in the transport of UDP-galactose, or the like, by addition, deletion, or substitution to DNA encoding the enzyme.

Examples of the protein involved in the transport of UDP-galactose include UDP-galactose transporter and the like. Examples of cell lines in which the activity of the UDP-galactose transporter is reduced or deleted include Lec8 cells [ Glycobiology, 1,307-14(1991) ].

In the present invention, examples of the cells expressing the sugar chain-deficient CD27 include: cells that exist endogenously in the human body, cell lines established from cells that exist endogenously in the human body, or cells obtained by gene recombination techniques, and the like.

Preference is given to the following: such cell lines as described above, which have reduced or deleted activities of an enzyme that adds Gal to GalNAc bound to Ser/Thr on the polypeptide in the O-linked sugar chain synthesis process, a protein involved in the activity of the enzyme, a protein involved in the transport of UDP-galactose, and the like, and cells that have the same properties and exist endogenously in humans, and the like.

Preferred examples of the cells endogenously present in the human body include cells having reduced or deleted activities such as an enzyme that adds Gal to GalNAc bound to Ser/Thr in the polypeptide during O-linked sugar chain synthesis, a protein involved in the activity of the enzyme, and a protein involved in the transport of UDP-galactose.

Specifically, for example, a cell expressing the CD27 protein in IgA nephropathy patients or cancer patients can be mentioned. Examples of such cells include cells expressing the CD27 protein in immune-related cells or tumor cells obtained by biopsy or the like.

Examples of the cells obtained by the gene recombination technique include the following: a host cell is prepared in which the activity of an enzyme that adds Gal to GalNAc bonded to Ser/Thr on a polypeptide in the O-linked sugar chain synthesis process, a protein involved in the activity of the enzyme, a protein involved in the transport of UDP-galactose, or the like is reduced or deleted, and an expression vector containing cDNA encoding a target polypeptide is introduced into the host cell, thereby expressing the sugar chain-deficient CD 27.

Specific examples of the host cell include: and Lec8 cells in which the activity of the UDP-galactose transporter is reduced, or IgA antibody-expressing cells derived from IgA nephropathy patients in which the activity of the enzyme is reduced or deleted by abnormality of β 1, 3-galactosyltransferase or Comsc chaperone protein associated with the activity of the enzyme.

Further, examples of the method for producing the sugar chain-deficient CD27 protein include: a method of expressing the sugar chain-deficient CD27 protein using the above-mentioned CD 27-expressing cells and purifying the protein, and the like.

As a method for obtaining the sugar chain-deficient CD27 protein, it can be obtained, for example, by expression and purification in the form of a fusion protein of the CD27 protein with other substances.

Examples of the substance fused with the CD27 protein include: and polypeptides such as an antibody constant region, an antibody Fc fragment, a GST tag, a histidine tag (also referred to as His tag), and a Myc tag. The fusion protein can be isolated and purified by using an affinity chromatography column such as protein A, a nickel column, or a specific antibody column.

The monoclonal antibody or the antibody fragment of the present invention has a binding activity to the sugar chain-deficient CD27 cell or the sugar chain-deficient CD27 obtained as described above.

The binding of the antibody or the antibody fragment of the present invention to the sugar chain-deficient CD27 protein can be confirmed, for example, by the following method: a method of confirming the binding property between a cell expressing a specific antigen and an antibody against the specific antigen by using a known immunological detection method, preferably a fluorescent cell staining method.

Alternatively, a known immunological detection method [ monoclonal antibodies-Principles and practice of monoclonal antibodies, third edition, academic Press (1996); Antibodies-A Laboratory Manual (antibody Laboratory Manual), Cold spring harbor Laboratory (1988); クロ experiments on ン antibodies were performed in combination of experiments マニユアル (guidance for monoclonal antibody experiments), , サイエンテイフイツク (1987), and the like.

The monoclonal antibody of the present invention includes: antibodies produced by hybridomas, and recombinant antibodies produced by transformants transformed with expression vectors containing antibody genes.

Hybridomas can be prepared, for example, by the following method: cells expressing the above-described sugar chain-deficient CD27 or the like are prepared as an antigen, an antibody-producing cell having antigen specificity is induced from an animal immunized with the antigen, and the antibody-producing cell is fused with a myeloma cell. The anti-sugar chain-deficient CD27 antibody can be obtained by culturing the hybridoma or by administering the hybridoma to an animal to produce cancerous ascites in the animal, and isolating and purifying the culture solution or ascites.

As the animal to be immunized with the antigen, any animal can be used as long as it can produce a hybridoma, and mice, rats, hamsters, rabbits, and the like are preferably used. In addition, antibodies and the like produced by hybridomas prepared as follows are also included in the antibodies of the present invention: cells having antibody-producing ability are obtained from the above-mentioned animals, immunized in vitro, and then fused with myeloma cells to prepare hybridomas.

A monoclonal antibody refers to an antibody secreted by a single clone of an antibody-producing cell, which recognizes only one epitope (also referred to as an antigenic determinant), and the amino acid sequences (primary structures) constituting the monoclonal antibody are identical.

As epitopes, for example: a single amino acid sequence recognized and bound by the monoclonal antibody, a three-dimensional structure composed of amino acid sequences, an amino acid sequence to which a sugar chain is bound, a three-dimensional structure composed of an amino acid sequence to which a sugar chain is bound, and the like. The epitope of the monoclonal antibody of the present invention is exemplified by the steric structure of the sugar chain-deficient CD27 protein.

The monoclonal antibody of the present invention may be any antibody as long as it recognizes and binds to the extracellular domain of the sugar chain-deficient CD27, and specifically, examples thereof include: monoclonal antibodies KM4026, KM4027, KM4028, KM4030, KM4031 and the like.

More specifically, for example: the monoclonal antibody KM4026 produced by hybridoma KM4026, a monoclonal antibody that binds to the extracellular domain of sugar chain-deficient CD27 in competition with monoclonal antibody KM4026, and a monoclonal antibody that binds to an epitope present in the extracellular domain of sugar chain-deficient CD27 to which monoclonal antibody KM4026 binds.

The monoclonal antibody of the present invention may include, for example: the monoclonal antibody KM4027 produced by hybridoma KM4027, a monoclonal antibody that binds to the extracellular domain of sugar chain-deficient CD27 in competition with monoclonal antibody KM4027, and a monoclonal antibody that binds to an epitope present in the extracellular domain of sugar chain-deficient CD27 to which monoclonal antibody KM4027 binds.

The monoclonal antibody of the present invention may include, for example: the monoclonal antibody KM4028 produced by hybridoma KM4028, a monoclonal antibody that binds to the extracellular domain of sugar chain-deficient CD27 in competition with monoclonal antibody KM4028, and a monoclonal antibody that binds to an epitope present in the extracellular domain of sugar chain-deficient CD27 to which monoclonal antibody KM4028 binds.

In addition, as the monoclonal antibody of the present invention, there can be mentioned, for example: monoclonal antibody KM4030 produced by hybridoma KM4030, a monoclonal antibody that binds to the extracellular domain of sugar chain-deficient CD27 in competition with monoclonal antibody KM4030, and a monoclonal antibody that binds to an epitope present in the extracellular domain of sugar chain-deficient CD27 to which monoclonal antibody KM4030 binds.

In addition, as the monoclonal antibody of the present invention, there may be mentioned: monoclonal antibody KM4031 produced by hybridoma KM4031, a monoclonal antibody that binds to the extracellular domain of sugar chain-deficient CD27 in competition with monoclonal antibody KM4031, and a monoclonal antibody that binds to an epitope present in the extracellular domain of sugar chain-deficient CD27 to which monoclonal antibody KM4031 binds, and the like.

Specific examples of the monoclonal antibody that competes with the monoclonal antibody of the present invention include: the monoclonal antibodies having a competitive reaction with the various monoclonal antibodies and the epitope present in the extracellular domain of the sugar chain-deficient CD27 were as described above.

In addition, specific examples of the monoclonal antibody that binds to the epitope bound by the monoclonal antibody of the present invention include: the monoclonal antibodies described above bound to the epitopes present in the extracellular domain of sugar chain-deficient CD27 recognized by the respective monoclonal antibodies.

Hybridoma KM4030 was deposited under the Budapest treaty at 5.6.2008 at the national institute of Integrated services and technology patent Collection, Japan (305. sup. 856, 6. sup. st. Central No. 1 of 1 atm, 1T, 1N, 1, Tokyo, Kyowa, Japan) under the accession number FERM BP-10976.

Examples of the recombinant antibody include antibodies produced by gene recombination techniques, such as human chimeric antibodies, humanized antibodies, human antibodies, and fragments of the antibodies. Among the recombinant antibodies, those having antigen-binding activity, low antigenicity and prolonged half-life in blood are preferred as therapeutic agents.

The human chimeric antibody is an antibody comprising a heavy chain variable region (hereinafter referred to as "VH") and a light chain variable region (hereinafter referred to as "VL") of a non-human antibody, and a heavy chain constant region (hereinafter referred to as "CH") and a light chain constant region (hereinafter referred to as "CL") of a human antibody.

The human chimeric antibody of the present invention can be produced as follows. That is, the human chimeric antibody is prepared by obtaining cdnas encoding VH and VL from the monoclonal antibody of the present invention that specifically recognizes the sugar chain-deficient CD27 and binds to the extracellular domain or a hybridoma that produces a monoclonal antibody that specifically recognizes the sugar chain-deficient CD27 and binds to the extracellular domain, inserting these into an expression vector for animal cells having genes encoding CH and CL of a human antibody, constructing a human chimeric antibody expression vector, and introducing the human chimeric antibody expression vector into animal cells to express the antibody.

The CH of the human chimeric antibody may be any of human immunoglobulin (hereinafter referred to as "hIg"), preferably a CH of the hIgG class, and may be any of subclasses such as hIgG1, hIgG2, hIgG3, and hIgG4 belonging to the hIgG class.

The CL of the human chimeric antibody may be any CL belonging to the hIg family, and either a kappa-class or a lambda-class CL may be used.

The human chimeric antibody of the present invention includes the following human chimeric antibodies (1) to (5).

(1) Human chimeric antibody having VH of antibody as amino acid sequence represented by SEQ ID No. 25 and VL of antibody as amino acid sequence represented by SEQ ID No. 35

(2) Human chimeric antibody in which VH of antibody is amino acid sequence represented by sequence No. 26 and VL of antibody is amino acid sequence represented by sequence No. 36

(3) Human chimeric antibody in which VH of antibody is amino acid sequence represented by sequence No. 27 and VL of antibody is amino acid sequence represented by sequence No. 37

(4) Human chimeric antibody in which VH of antibody is amino acid sequence represented by sequence No. 28 and VL of antibody is amino acid sequence represented by sequence No. 38

(5) Human chimeric antibody in which VH of antibody is amino acid sequence represented by sequence No. 29 and VL of antibody is amino acid sequence represented by sequence No. 39

The human chimeric antibody of the present invention includes the following human chimeric antibodies (1) to (5).

(1) The antibody VH is a human chimeric antibody comprising the amino acid sequences of CDR 1-3 represented by sequence numbers 40-42, and the antibody VL is a human chimeric antibody comprising the amino acid sequences of CDR 1-3 represented by sequence numbers 43-45

(2) The antibody VH is a human chimeric antibody comprising amino acid sequences of CDRs 1-3 represented by sequence numbers 46-48, and the antibody VL is a human chimeric antibody comprising amino acid sequences of CDRs 1-3 represented by sequence numbers 49-51

(3) The antibody VH is a human chimeric antibody comprising the amino acid sequences of CDR 1-3 represented by sequence numbers 52-54, and the antibody VL is a human chimeric antibody comprising the amino acid sequences of CDR 1-3 represented by sequence numbers 55-57

(4) The antibody VH is a human chimeric antibody comprising the amino acid sequences of CDR 1-3 represented by sequence numbers 58-60, and the antibody VL is a human chimeric antibody comprising the amino acid sequences of CDR 1-3 represented by sequence numbers 61-63

(5) The antibody VH is a human chimeric antibody comprising amino acid sequences of CDRs 1-3 represented by sequence numbers 64-66, and the antibody VL is a human chimeric antibody comprising amino acid sequences of CDRs 1-3 represented by sequence numbers 67-69

The humanized antibody is an antibody obtained by grafting amino acid sequences of CDRs of VH and VL of a non-human antibody to appropriate positions of VH and VL of a human antibody, and is also called a human CDR-grafted antibody or a reshaped antibody (reshaped antibody).

The humanized antibody of the present invention can be produced as follows: a cDNA encoding an antibody variable region (hereinafter, referred to as a V region) produced by grafting the amino acid sequences of the CDRs of VH and VL of a non-human antibody produced by a hybridoma producing a monoclonal antibody that specifically recognizes a sugar chain-deficient CD27 protein of the present invention and binds to the extracellular domain into the framework (hereinafter, referred to as FR) of VH and VL of an arbitrary human antibody is constructed, and a humanized antibody expression vector is constructed by inserting the cDNA into an expression vector for animal cells having genes encoding CH and CL of a human antibody, and then introducing the expression vector into animal cells to express the antibody, thereby producing a humanized antibody.

The amino acid sequences of the FRs of VH and VL of the human antibody of the present invention may be those of the FRs of VH and VL derived from a human antibody. For example, it is possible to use: amino acid Sequences of FRs of VH and VL of human antibodies registered in databases such as Protein Data Bank, or consensus amino acid Sequences of Proteins of immunological interest, respective subclasses of FRs of VH and VL of human antibodies described in the United states department of health and public service (1991), etc.

The CH of the humanized antibody of the present invention may be any CH as long as it belongs to the hIg, and preferably is a CH of the hIgG class. Any one of subclasses such as hIgG1, hIgG2, hIgG3, and hIgG4 belonging to the hIgG class can be used.

The CL of the humanized antibody of the present invention may be any CL as long as it belongs to the hIg, and either a kappa-based CL or a lambda-based CL may be used.

The humanized antibody of the present invention includes the following humanized antibodies (1) to (5).

(1) Humanized antibody wherein CDRs 1-3 of VH of the antibody are amino acid sequences represented by sequence numbers 40-42, and CDRs 1-3 of VL of the antibody are amino acid sequences represented by sequence numbers 43-45

(2) Humanized antibody wherein CDRs 1-3 of VH of the antibody are amino acid sequences represented by sequence numbers 46-48 and CDRs 1-3 of VL of the antibody are amino acid sequences represented by sequence numbers 49-51

(3) Humanized antibody wherein CDRs 1-3 of VH of the antibody are amino acid sequences represented by sequence numbers 52-54 and CDRs 1-3 of VL of the antibody are amino acid sequences represented by sequence numbers 55-57

(4) Humanized antibody wherein CDRs 1-3 of VH of the antibody are amino acid sequences represented by sequence numbers 58-60 and CDRs 1-3 of VL of the antibody are amino acid sequences represented by sequence numbers 61-63

(5) Humanized antibody wherein CDRs 1-3 of VH of the antibody are amino acid sequences represented by sequence numbers 64-66 and CDRs 1-3 of VL of the antibody are amino acid sequences represented by sequence numbers 67-69

Specifically, the humanized antibody of the present invention is preferably a humanized antibody comprising at least one of the following (a) VH and (b) VL. In the following (a) and (b), the number of modifications to be introduced is not limited.

(a) VH comprising an amino acid sequence in which Ser at position 30, Val at position 48, Ser at position 49, Asn at position 77, Val at position 93, Ala at position 97 and Thr at position 117 in the amino acid sequence represented by SEQ ID NO. 96 or the amino acid sequence represented by SEQ ID NO. 96 are substituted with another amino acid residue

(b) VL comprising an amino acid sequence represented by SEQ ID NO. 97 or an amino acid sequence in which Ile at position 21, Pro at position 40, Val at position 58, Thr at position 85 and Tyr at position 87 in the amino acid sequence represented by SEQ ID NO. 97 are substituted with other amino acid residues

Examples of the VH contained in the humanized antibody of the present invention include a VH comprising an amino acid sequence in which Ser at position 30, Val at position 48, Ser at position 49, Asn at position 77 and Ala at position 97 are substituted with another amino acid residue in the amino acid sequence represented by SEQ ID NO. 96.

Among them, preferred as the VH contained in the humanized antibody of the present invention are the following VH (1) and VH (2).

(1) VH comprising an amino acid sequence in which Val at position 48, Ser at position 49 and Ala at position 97 in the amino acid sequence represented by SEQ ID NO. 96 are substituted with other amino acid residues

(2) VH comprising an amino acid sequence in which Ser at position 30 and Ala at position 97 in the amino acid sequence represented by SEQ ID NO. 96 are substituted with other amino acid residues

Examples of the amino acid sequence of the VH include an amino acid sequence into which at least one modification selected from the following modifications is introduced: substitution of Ser at position 30 into Asn, substitution of Val at position 48 into Ile, substitution of Ser at position 49 into Ala, substitution of Asn at position 77 into Gly, substitution of Val at position 93 into Thr, substitution of Ala at position 97 into Thr, and substitution of Thr at position 117 into Val in the amino acid sequence represented by SEQ ID NO. 96.

Specific examples of the amino acid sequence of the VH include amino acid sequences to which 1 to 7 modifications are introduced, as described below.

Specific examples of the amino acid sequence into which 7 modified VH have been introduced include: an amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Ser at position 49 is substituted with Ala, Asn at position 77 is substituted with Gly, Val at position 93 is substituted with Thr, Ala at position 97 is substituted with Thr, and Thr at position 117 is substituted with Val.

Specific examples of the amino acid sequence of VH into which 6 modifications have been introduced include the following amino acid sequences (1) to (7).

(1) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Ser at position 49 is substituted with Ala, Asn at position 77 is substituted with Gly, Val at position 93 is substituted with Thr, and Ala at position 97 is substituted with Thr

(2) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Ser at position 49 is substituted with Ala, Asn at position 77 is substituted with Gly, Val at position 93 is substituted with Thr, and Thr at position 117 is substituted with Val

(3) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Ser at position 49 is substituted with Ala, Asn at position 77 is substituted with Gly, Ala at position 97 is substituted with Thr, and Thr at position 117 is substituted with Val

(4) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Ser at position 49 is substituted with Ala, Val at position 93 is substituted with Thr, Ala at position 97 is substituted with Thr, and Thr at position 117 is substituted with Val

(5) An amino acid sequence comprising substitution of Ser at position 30 with Asn, substitution of Val at position 48 with Ile, substitution of Asn at position 77 with Gly, substitution of Val at position 93 with Thr, substitution of Ala at position 97 with Thr, and substitution of Thr at position 117 with Val in the amino acid sequence represented by SEQ ID NO. 96

(6) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Ser at position 49 is substituted with Ala, Asn at position 77 is substituted with Gly, Val at position 93 is substituted with Thr, Ala at position 97 is substituted with Thr, and Thr at position 117 is substituted with Val

(7) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Ile, Ser at position 49 is substituted by Ala, Asn at position 77 is substituted by Gly, Val at position 93 is substituted by Thr, Ala at position 97 is substituted by Thr, and Thr at position 117 is substituted by Val

Specific examples of the amino acid sequence of VH into which 5 modifications are introduced include the following amino acid sequences (1) to (21).

(1) An amino acid sequence in which Ser at position 49 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ala, Asn at position 77 is substituted with Gly, Val at position 93 is substituted with Thr, Ala at position 97 is substituted with Thr, and Thr at position 117 is substituted with Val

(2) An amino acid sequence in which Val at position 48, Asn at position 77, Val at position 93, Thr at position 97, Ala at position 117 and Val at position 117 are substituted in the amino acid sequence represented by SEQ ID NO. 96 by Ile, Gly, and Thr, respectively

(3) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Ile, Ser at position 49 is substituted by Ala, Val at position 93 is substituted by Thr, Ala at position 97 is substituted by Thr, and Thr at position 117 is substituted by Val

(4) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Ile, Ser at position 49 is substituted by Ala, Asn at position 77 is substituted by Gly, Ala at position 97 is substituted by Thr, and Thr at position 117 is substituted by Val

(5) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Ile, Ser at position 49 is substituted by Ala, Asn at position 77 is substituted by Gly, Val at position 93 is substituted by Thr, and Thr at position 117 is substituted by Val

(6) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Ile, Ser at position 49 is substituted by Ala, Asn at position 77 is substituted by Gly, Val at position 93 is substituted by Thr, and Ala at position 97 is substituted by Thr

(7) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Asn at position 77 is substituted with Gly, Val at position 93 is substituted with Thr, Ala at position 97 is substituted with Thr, and Thr at position 117 is substituted with Val

(8) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Asn, Ser at position 49 is substituted by Ala, Val at position 93 is substituted by Thr, Ala at position 97 is substituted by Thr, and Thr at position 117 is substituted by Val

(9) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Asn, Ser at position 49 is substituted by Ala, Asn at position 77 is substituted by Gly, Ala at position 97 is substituted by Thr, and Thr at position 117 is substituted by Val

(10) An amino acid sequence comprising an amino acid sequence in which the 30 th Ser in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Asn, the 49 th Ser by Ala, the 77 th Asn by Gly, the 93 th Val by Thr, and the 117 th Thr by Val

(11) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Ser at position 49 is substituted with Ala, Asn at position 77 is substituted with Gly, Val at position 93 is substituted with Thr, and Ala at position 97 is substituted with Thr

(12) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Val at position 93 is substituted with Thr, Ala at position 97 is substituted with Thr, and Thr at position 117 is substituted with Val

(13) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Asn at position 77 is substituted with Gly, Ala at position 97 is substituted with Thr, and Thr at position 117 is substituted with Val

(14) An amino acid sequence comprising substitution of Ser at position 30 with Asn, substitution of Val at position 48 with Ile, substitution of Asn at position 77 with Gly, substitution of Val at position 93 with Thr, and substitution of Thr at position 117 with Val in the amino acid sequence represented by SEQ ID NO. 96

(15) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Asn at position 77 is substituted with Gly, Val at position 93 is substituted with Thr, and Ala at position 97 is substituted with Thr

(16) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Ser at position 49 is substituted with Ala, Ala at position 97 is substituted with Thr, and Thr at position 117 is substituted with Val

(17) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Ser at position 49 is substituted with Ala, Val at position 93 is substituted with Thr, and Thr at position 117 is substituted with Val

(18) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Ser at position 49 is substituted with Ala, Val at position 93 is substituted with Thr, and Ala at position 97 is substituted with Thr

(19) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Ser at position 49 is substituted with Ala, Asn at position 77 is substituted with Gly, and Thr at position 117 is substituted with Val

(20) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Ser at position 49 is substituted with Ala, Asn at position 77 is substituted with Gly, and Ala at position 97 is substituted with Thr

(21) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Ser at position 49 is substituted with Ala, Asn at position 77 is substituted with Gly, and Val at position 93 is substituted with Thr

Specific examples of the amino acid sequence of VH into which 4 modifications are introduced include the following amino acid sequences (1) to (35).

(1) An amino acid sequence in which Asn at position 77 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Gly, Val at position 93 is substituted with Thr, Ala at position 97 is substituted with Thr, and Thr at position 117 is substituted with Val

(2) An amino acid sequence in which Ser at position 49 in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Ala, Val at position 93 is substituted by Thr, Ala at position 97 is substituted by Thr, and Thr at position 117 is substituted by Val

(3) An amino acid sequence in which Ser at position 49 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ala, Asn at position 77 is substituted with Gly, Ala at position 97 is substituted with Thr, and Thr at position 117 is substituted with Val

(4) An amino acid sequence in which Ser at position 49 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ala, Asn at position 77 is substituted with Gly, Val at position 93 is substituted with Thr, and Thr at position 117 is substituted with Val

(5) An amino acid sequence in which Ser at position 49 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ala, Asn at position 77 is substituted with Gly, Val at position 93 is substituted with Thr, and Ala at position 97 is substituted with Thr

(6) An amino acid sequence in which Val at position 48, Val at position 93, Thr at position 97, Ala at position 117 and Val at position 117 are substituted in the amino acid sequence represented by SEQ ID NO. 96

(7) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Ile, Asn at position 77 is substituted by Gly, Ala at position 97 is substituted by Thr, and Thr at position 117 is substituted by Val

(8) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Ile, Asn at position 77 is substituted by Gly, Val at position 93 is substituted by Thr, and Thr at position 117 is substituted by Val

(9) An amino acid sequence in which the 48 th Val, the 77 th Asn, the 93 th Val and the 97 th Ala are substituted by Ile, Gly, Thr, etc. in the amino acid sequence represented by SEQ ID NO. 96

(10) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Ile, Ser at position 49 is substituted by Ala, Ala at position 97 is substituted by Thr, and Thr at position 117 is substituted by Val

(11) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Ile, Ser at position 49 is substituted by Ala, Val at position 93 is substituted by Thr, and Thr at position 117 is substituted by Val

(12) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Ile, Ser at position 49 is substituted by Ala, Val at position 93 is substituted by Thr, and Ala at position 97 is substituted by Thr

(13) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ile, Ser at position 49 is substituted with Ala, Asn at position 77 is substituted with Gly, and Thr at position 117 is substituted with Val

(14) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Ile, Ser at position 49 is substituted by Ala, Asn at position 77 is substituted by Gly, and Ala at position 97 is substituted by Thr

(15) An amino acid sequence in which the 48 th Val, the 49 th Ser, the 77 th Asn, the 93 th Val and the 49 th Thr in the amino acid sequence represented by SEQ ID NO. 96 are substituted with Ile, Ala, Gly and Thr, respectively

(16) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 93 is substituted with Thr, Ala at position 97 is substituted with Thr, and Thr at position 117 is substituted with Val

(17) An amino acid sequence in which the amino acid sequence represented by SEQ ID NO. 96 has Asn substituted for Ser at position 30, Gly substituted for Asn at position 77, Thr substituted for Ala at position 97, and Val substituted for Thr at position 117

(18) An amino acid sequence in which the 30 th Asn, the 77 th Asn, the 93 th Thr and the 117 th Thr are substituted by the Gly, the Asn, the 77 th Asn, the 93 th Val and the 117 th Thr in the amino acid sequence represented by SEQ ID NO. 96, respectively

(19) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Asn at position 77 is substituted with Gly, Val at position 93 is substituted with Thr, and Ala at position 97 is substituted with Thr

(20) An amino acid sequence in which the 30 th Ser in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Asn, the 49 th Ser by Ala, the 97 th Ala by Thr, and the 117 th Thr by Val

(21) An amino acid sequence in which the 30 th Ser in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Asn, the 49 th Ser by Ala, the 93 th Val by Thr, and the 117 th Thr by Val

(22) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Ser at position 49 is substituted with Ala, Val at position 93 is substituted with Thr, and Ala at position 97 is substituted with Thr

(23) An amino acid sequence in which the 30 th Ser in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Asn, the 49 th Ser by Ala, the 77 th Asn by Gly, and the 117 th Thr by Val

(24) An amino acid sequence in which the amino acid sequence represented by SEQ ID NO. 96 has the substitution of Ser at position 30 with Asn, the substitution of Ser at position 49 with Ala, the substitution of Asn at position 77 with Gly, and the substitution of Ala at position 97 with Thr

(25) An amino acid sequence in which the 30 th Ser in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Asn, the 49 th Ser by Ala, the 77 th Asn by Gly, and the 93 th Val by Thr

(26) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Ala at position 97 is substituted with Thr, and Thr at position 117 is substituted with Val

(27) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Val at position 93 is substituted with Thr, and Thr at position 117 is substituted with Val

(28) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Val at position 93 is substituted with Thr, and Ala at position 97 is substituted with Thr

(29) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Asn at position 77 is substituted with Gly, and Thr at position 117 is substituted with Val

(30) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Asn at position 77 is substituted with Gly, and Ala at position 97 is substituted with Thr

(31) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Asn at position 77 is substituted with Gly, and Val at position 93 is substituted with Thr

(32) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Ser at position 49 is substituted with Ala, and Thr at position 117 is substituted with Val

(33) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Ser at position 49 is substituted with Ala at position 97 is substituted with Thr

(34) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Ser at position 49 is substituted with Ala, and Val at position 93 is substituted with Thr

(35) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Ser at position 49 is substituted with Ala, and Asn at position 77 is substituted with Gly

Specific examples of the amino acid sequence of VH into which 3 modifications have been introduced include the following amino acid sequences (1) to (35).

(1) An amino acid sequence in which the 30 th Ser in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Asn, the 48 th Val is substituted by Ile, and the 49 th Ser is substituted by Ala

(2) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, and Asn at position 77 is substituted with Gly

(3) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, and Val at position 93 is substituted with Thr

(4) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, and Ala at position 97 is substituted with Thr

(5) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, and Thr at position 117 is substituted with Val

(6) An amino acid sequence in which the 30 th Asn, 49 th Ala and 77 th Gly Asn in the amino acid sequence represented by SEQ ID NO. 96 are substituted with Ser

(7) An amino acid sequence in which the 30 th Ser in the amino acid sequence shown in SEQ ID NO. 96 is substituted by Asn, the 49 th Ser by Ala and the 93 th Val by Thr

(8) An amino acid sequence in which the 30 th Ser in the amino acid sequence shown in SEQ ID NO. 96 is substituted by Asn, the 49 th Ser by Ala and the 97 th Ala by Thr

(9) An amino acid sequence in which the 30 th Ser in the amino acid sequence shown in SEQ ID NO. 96 is substituted by Asn, the 49 th Ser is substituted by Ala, and the 117 th Thr is substituted by Val

(10) An amino acid sequence in which the 30 th Asn, the 77 th Asn and the 93 rd Val in the amino acid sequence represented by SEQ ID NO. 96 are substituted with one another by one or more amino acids selected from the group consisting of Ser, Asn, Gly, Val and Thr

(11) An amino acid sequence in which the 30 th Asn, the 77 th Asn and the 97 th Ala in the amino acid sequence shown in SEQ ID NO. 96 are substituted by Asn, Gly and Thr, respectively

(12) An amino acid sequence in which the 30 th Asn, the 77 th Asn and the 117 th Thr are substituted by the Gly, respectively, in the amino acid sequence represented by SEQ ID NO. 96

(13) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 93 is substituted with Thr, and Ala at position 97 is substituted with Thr

(14) An amino acid sequence in which the 30 th Ser is substituted by Asn, the 93 th Val is substituted by Thr, and the 117 th Thr is substituted by Val in the amino acid sequence represented by SEQ ID NO. 96

(15) An amino acid sequence in which the 30 th Ser is substituted by Asn, the 97 th Ala is substituted by Thr, and the 117 th Thr is substituted by Val in the amino acid sequence represented by SEQ ID NO. 96

(16) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ile, Ser at position 49 is substituted with Ala, and Asn at position 77 is substituted with Gly

(17) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ile, Ser at position 49 is substituted with Ala, and Val at position 93 is substituted with Thr

(18) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted by Ile, Ser at position 49 is substituted by Ala, and Ala at position 97 is substituted by Thr

(19) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ile, Ser at position 49 is substituted with Ala, and Thr at position 117 is substituted with Val

(20) An amino acid sequence in which the 48 th Val, the 77 th Asn, and the 93 rd Val are substituted by Ile, Gly, and Thr, respectively, in the amino acid sequence represented by SEQ ID NO. 96

(21) An amino acid sequence in which Val at position 48, Asn at position 77, and Ala at position 97 are substituted by Thr in the amino acid sequence represented by SEQ ID NO. 96

(22) An amino acid sequence in which Val at position 48, Asn at position 77, and Thr at position 117 are substituted by Val in the amino acid sequence represented by SEQ ID NO. 96

(23) An amino acid sequence in which Val at position 48, Val at position 93 and Ala at position 97 are substituted by Ile, Thr and the like in the amino acid sequence represented by SEQ ID NO. 96

(24) An amino acid sequence in which Val at position 48, Val at position 93 and Thr at position 117 are substituted by Val in the amino acid sequence represented by SEQ ID NO. 96

(25) An amino acid sequence in which Val at position 48, Ala at position 97 and Thr at position 117 are substituted by Val in the amino acid sequence represented by SEQ ID NO. 96

(26) An amino acid sequence in which Ser at position 49 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ala, Asn at position 77 is substituted with Gly, and Val at position 93 is substituted with Thr

(27) An amino acid sequence in which Ser at position 49 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ala, Asn at position 77 is substituted with Gly, and Ala at position 97 is substituted with Thr

(28) An amino acid sequence in which Ser at position 49 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ala, Asn at position 77 is substituted with Gly, and Thr at position 117 is substituted with Val

(29) An amino acid sequence in which Ser at position 49 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ala, Val at position 93 is substituted with Thr, and Ala at position 97 is substituted with Thr

(30) An amino acid sequence in which Ser at position 49 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ala, Val at position 93 is substituted with Thr, and Thr at position 117 is substituted with Val

(31) An amino acid sequence in which Ser at position 49 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ala, Ala at position 97 is substituted with Thr, and Thr at position 117 is substituted with Val

(32) An amino acid sequence in which Asn at position 77 of the amino acid sequence represented by SEQ ID NO. 96 is substituted with Gly, Val at position 93 is substituted with Thr, and Ala at position 97 is substituted with Thr

(33) An amino acid sequence in which Asn at position 77 of the amino acid sequence represented by SEQ ID NO. 96 is substituted with Gly, Val at position 93 is substituted with Thr, and Thr at position 117 is substituted with Val

(34) An amino acid sequence in which Asn at position 77 of the amino acid sequence represented by SEQ ID NO. 96 is substituted with Gly, Ala at position 97 is substituted with Thr, and Thr at position 117 is substituted with Val

(35) An amino acid sequence in which Val at position 93 is substituted with Thr, Ala at position 97 is substituted with Thr, and Thr at position 117 is substituted with Val in the amino acid sequence represented by SEQ ID NO. 96

Specific examples of the amino acid sequence of VH into which 2 modifications have been introduced include the following amino acid sequences (1) to (21).

(1) An amino acid sequence obtained by substituting Ser at position 30 into Asn and substituting Val at position 48 into Ile in the amino acid sequence represented by SEQ ID NO. 96

(2) An amino acid sequence obtained by substituting Ser at position 30 into Asn and substituting Ser at position 49 into Ala in the amino acid sequence represented by SEQ ID NO. 96

(3) An amino acid sequence obtained by substituting Asn for Ser at position 30 and Asn for Gly for Asn at position 77 in the amino acid sequence represented by SEQ ID NO. 96

(4) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn and Val at position 93 is substituted with Thr

(5) An amino acid sequence in which Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn and Ala at position 97 is substituted with Thr

(6) An amino acid sequence in which the 30 th Ser in the amino acid sequence shown in SEQ ID NO. 96 is substituted by Asn and the 117 th Thr is substituted by Val

(7) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ile and Ser at position 49 is substituted with Ala

(8) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ile and Asn at position 77 is substituted with Gly

(9) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ile and Val at position 93 is substituted with Thr

(10) An amino acid sequence in which Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ile and Ala at position 97 is substituted with Thr

(11) An amino acid sequence in which Val at position 48 is substituted with Ile and Thr at position 117 is substituted with Val in the amino acid sequence represented by SEQ ID NO. 96

(12) An amino acid sequence in which Ser at position 49 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ala, and Asn at position 77 is substituted with Gly

(13) An amino acid sequence in which Ser at position 49 in the amino acid sequence shown in SEQ ID NO. 96 is substituted with Ala and Val at position 93 is substituted with Thr

(14) An amino acid sequence in which Ser at position 49 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Ala and Ala at position 97 is substituted with Thr

(15) An amino acid sequence in which Ser at position 49 in the amino acid sequence shown in SEQ ID NO. 96 is substituted with Ala and Thr at position 117 is substituted with Val

(16) An amino acid sequence obtained by substituting Asn at position 77 into Gly and substituting Val at position 93 into Thr in the amino acid sequence represented by SEQ ID NO. 96

(17) An amino acid sequence obtained by substituting Gly for Asn at position 77 and Thr for Ala at position 97 in the amino acid sequence represented by SEQ ID NO. 96

(18) An amino acid sequence obtained by substituting Asn at position 77 into Gly and substituting Thr at position 117 into Val in the amino acid sequence represented by SEQ ID NO. 96

(19) An amino acid sequence in which Val at position 93 is substituted with Thr and Ala at position 97 is substituted with Thr in the amino acid sequence represented by SEQ ID NO. 96

(20) An amino acid sequence in which Val at position 93 is substituted with Thr and Thr at position 117 is substituted with Val in the amino acid sequence represented by SEQ ID NO. 96

(21) An amino acid sequence in which Ala at position 97 in the amino acid sequence shown in SEQ ID NO. 96 is substituted by Thr and Thr at position 117 is substituted by Val

Specific examples of the amino acid sequence of VH into which 1 modification is introduced include the following amino acid sequences (1) to (7).

(1) An amino acid sequence obtained by substituting Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 with Asn

(2) Substitution of Val at position 48 in the amino acid sequence represented by SEQ ID NO. 96 with an amino acid sequence of Ile

(3) An amino acid sequence obtained by substituting Ser at position 49 in the amino acid sequence represented by SEQ ID NO. 96 with Ala

(4) An amino acid sequence obtained by substituting Asn at position 77 in the amino acid sequence represented by SEQ ID NO. 96 with Gly

(5) An amino acid sequence in which Val at position 93 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Thr

(6) An amino acid sequence obtained by substituting Ala at position 97 in the amino acid sequence represented by SEQ ID NO. 96 with Thr

(7) An amino acid sequence obtained by substituting Thr at position 117 in the amino acid sequence represented by SEQ ID NO. 96 with Val

More specifically, the amino acid sequences of VH of the humanized antibody of the present invention include the amino acid sequences represented by SEQ ID Nos. 96, 105 and 107.

Examples of the VL contained in the humanized antibody of the present invention include a VL comprising an amino acid sequence in which Ile at position 21, Pro at position 40, Val at position 58, Thr at position 85 and Tyr at position 87 in the amino acid sequence represented by SEQ ID NO. 97 are substituted with another amino acid residue.

Among them, the VL contained in the humanized antibody of the present invention is preferably an amino acid sequence in which Pro at position 40, Val at position 58 and Tyr at position 87 in the amino acid sequence represented by SEQ ID NO. 97 are substituted with other amino acid residues.

Examples of the amino acid sequence of VL include an amino acid sequence into which at least one modification selected from the following modifications is introduced: substitution of Ile at position 21 to Leu, substitution of Pro at position 40 to Leu, substitution of Val at position 58 to Ile, substitution of Thr at position 85 to Ala, and substitution of Tyr at position 87 to Phe in the amino acid sequence shown in SEQ ID NO. 97.

Specific examples of the amino acid sequence of VL include the following amino acid sequences into which 1 to 5 modifications have been introduced.

Specific examples of the amino acid sequence into which 5 modified VL is introduced include an amino acid sequence in which Ile at position 21 is substituted with Leu, Pro at position 40 is substituted with Leu, Val at position 58 is substituted with Ile, Thr at position 85 is substituted with Ala, and Tyr at position 87 is substituted with Phe in the amino acid sequence represented by SEQ ID NO. 97.

Specific examples of the amino acid sequence into which 4 modified VLs are introduced include the following amino acid sequences (1) to (5).

(1) An amino acid sequence in which Pro at position 40 is substituted with Leu, Val at position 58 is substituted with Ile, Thr at position 85 is substituted with Ala, and Tyr at position 87 is substituted with Phe in the amino acid sequence represented by SEQ ID NO. 97

(2) An amino acid sequence in which Ile at position 21, Val at position 58, Thr at position 85, and Tyr at position 87 are substituted with Leu, Ile at position 58, Ala at position 85, and Phe at position 87 in the amino acid sequence represented by SEQ ID NO. 97

(3) An amino acid sequence in which Ile at position 21 in the amino acid sequence shown in SEQ ID NO. 97 is substituted with Leu, Pro at position 40 is substituted with Leu, Thr at position 85 is substituted with Ala, and Tyr at position 87 is substituted with Phe

(4) An amino acid sequence in which Ile at position 21 in the amino acid sequence shown in SEQ ID NO. 97 is substituted with Leu, Pro at position 40 is substituted with Leu, Val at position 58 is substituted with Ile, and Tyr at position 87 is substituted with Phe

(5) An amino acid sequence in which Ile at position 21 in the amino acid sequence represented by SEQ ID NO. 97 is substituted with Leu, Pro at position 40 is substituted with Leu, Val at position 58 is substituted with Ile, and Thr at position 85 is substituted with Ala

Specific examples of the amino acid sequence into which 3 modified VLs are introduced include the following amino acid sequences (1) to (9).

(1) An amino acid sequence in which Ile at position 21 in the amino acid sequence shown in SEQ ID NO. 97 is substituted with Leu, Pro at position 40 is substituted with Leu, and Val at position 58 is substituted with Ile

(2) An amino acid sequence in which Ile at position 21 in the amino acid sequence shown in SEQ ID NO. 97 is substituted with Leu, Pro at position 40 is substituted with Leu, and Thr at position 85 is substituted with Ala

(3) An amino acid sequence in which Ile at position 21 in the amino acid sequence shown in SEQ ID NO. 97 is substituted with Leu, Pro at position 40 is substituted with Leu, and Tyr at position 87 is substituted with Phe

(4) An amino acid sequence in which Ile at position 21, Val at position 58, and Thr at position 85 are substituted by Leu, Ile at position 58, and Ala at position 85 in the amino acid sequence represented by SEQ ID NO. 97

(5) An amino acid sequence in which Ile at position 21, Val at position 58, and Tyr at position 87 are substituted with Leu, Ile at position 21, and Phe at position 87 in the amino acid sequence represented by SEQ ID NO. 97

(6) An amino acid sequence in which Ile at position 21, Thr at position 85, and Tyr at position 87 are substituted with Leu, Ala, and Tyr at position 85, in the amino acid sequence represented by SEQ ID NO. 97

(7) An amino acid sequence in which Pro at position 40 is substituted with Leu, Val at position 58 is substituted with Ile, and Thr at position 85 is substituted with Ala in the amino acid sequence represented by SEQ ID NO. 97

(8) An amino acid sequence in which Pro at position 40 is substituted with Leu, Val at position 58 is substituted with Ile, and Tyr at position 87 is substituted with Phe in the amino acid sequence represented by SEQ ID NO. 97

(9) An amino acid sequence in which Val at position 58, Thr at position 85, and Tyr at position 87 are substituted with Ile, Ala, and Tyr at position 85 in the amino acid sequence represented by SEQ ID NO. 97

Specific examples of the amino acid sequence into which 2 modified VLs are introduced include the following amino acid sequences (1) to (10).

(1) An amino acid sequence in which Ile at position 21 in the amino acid sequence shown in SEQ ID NO. 97 is substituted with Leu, and Pro at position 40 is substituted with Leu

(2) An amino acid sequence in which Ile at position 21 in the amino acid sequence represented by SEQ ID NO. 97 is substituted with Leu and Val at position 58 is substituted with Ile

(3) An amino acid sequence in which Ile at position 21 in the amino acid sequence represented by SEQ ID NO. 97 is substituted with Leu and Thr at position 85 is substituted with Ala

(4) An amino acid sequence in which Ile at position 21 in the amino acid sequence represented by SEQ ID NO. 97 is substituted with Leu and Tyr at position 87 is substituted with Phe

(5) An amino acid sequence in which Pro at position 40 is substituted with Leu and Val at position 58 is substituted with Ile in the amino acid sequence represented by SEQ ID NO. 97

(6) An amino acid sequence in which Pro at position 40 is substituted with Leu and Thr at position 85 is substituted with Ala in the amino acid sequence represented by SEQ ID NO. 97

(7) An amino acid sequence in which Pro at position 40 is substituted with Leu and Tyr at position 87 is substituted with Phe in the amino acid sequence represented by SEQ ID NO. 97

(8) An amino acid sequence in which Val at position 58 is substituted with Ile and Thr at position 85 is substituted with Ala in the amino acid sequence represented by SEQ ID NO. 97

(9) An amino acid sequence in which Val at position 58 is substituted with Ile and Tyr at position 87 is substituted with Phe in the amino acid sequence represented by SEQ ID NO. 97

(10) An amino acid sequence in which Thr at position 85 in the amino acid sequence represented by SEQ ID NO. 97 is substituted with Ala and Tyr at position 87 is substituted with Phe

Specific examples of the amino acid sequence into which 1 modified VL is introduced include: an amino acid sequence in which Ile at position 21 is substituted with Leu, an amino acid sequence in which Pro at position 40 is substituted with Leu, an amino acid sequence in which Val at position 58 is substituted with Ile, an amino acid sequence in which Thr at position 85 is substituted with Ala, and an amino acid sequence in which Tyr at position 87 is substituted with Phe, in the amino acid sequence represented by SEQ ID NO. 97.

More specifically, VL of the humanized antibody of the present invention includes an amino acid sequence represented by SEQ ID NO. 97.

Specific examples of the humanized antibody of the present invention include the antibodies (1) to (5) below.

(1) Humanized antibody comprising at least one of H chain comprising variable region of amino acid sequence represented by SEQ ID NO. 96 and L chain comprising variable region of amino acid sequence represented by SEQ ID NO. 97

(2) Humanized antibody comprising at least one of H chain comprising variable region of amino acid sequence represented by SEQ ID NO. 101 and L chain comprising variable region of amino acid sequence represented by SEQ ID NO. 97

(3) Humanized antibody comprising at least one of H chain comprising variable region of amino acid sequence represented by SEQ ID NO. 103 and L chain comprising variable region of amino acid sequence represented by SEQ ID NO. 97

(4) Humanized antibody comprising at least one of H chain comprising variable region of amino acid sequence represented by SEQ ID NO. 105 and L chain comprising variable region of amino acid sequence represented by SEQ ID NO. 97

(5) Humanized antibody comprising at least one of H chain comprising variable region of amino acid sequence represented by SEQ ID NO. 107 and L chain comprising variable region of amino acid sequence represented by SEQ ID NO. 97

The human antibody originally refers to an antibody existing endogenously in the human body, and includes antibodies obtained from a human antibody phage library and a human antibody-producing transgenic animal, which have been prepared by recent technological advances in genetic engineering, cell engineering and developmental engineering.

The antibody endogenously present in the human body can be obtained, for example, as follows: the antibody can be purified from the culture supernatant by isolating human peripheral blood lymphocytes, infecting them with EB virus or the like to immortalize them, and cloning them.

A human antibody phage library is a library obtained by inserting an antibody gene prepared from a human B cell into a phage gene and expressing an antibody fragment such as Fab or scFv on the phage surface. Phage expressing an antibody fragment having a desired antigen-binding activity on the surface can be recovered from the library using the binding activity of the antibody fragment to the antigen-immobilized substrate as an index. The antibody fragment can be further converted into a human antibody molecule comprising two complete H chains and L chains by genetic engineering methods.

The human antibody-producing transgenic animal refers to an animal having a human antibody gene recombined in a cell. Specifically, for example, a human antibody gene is introduced into a mouse ES cell, and the ES cell is transplanted into an early embryo of a mouse and then developed, whereby a human antibody-producing transgenic mouse can be prepared.

In the method for producing a human antibody from a human antibody-producing transgenic animal, a human antibody-producing hybridoma is obtained by a normal hybridoma production method performed in an animal other than a human and cultured, whereby the human antibody can be produced and accumulated in a culture supernatant.

An antibody or an antibody fragment thereof, which has one or more amino acids deleted, added, substituted, or inserted in the amino acid sequence constituting the above antibody or antibody fragment and has the same activity as the above antibody or antibody fragment thereof, is also included in the antibody or antibody fragment thereof of the present invention.

The number of amino Acids to be deleted, substituted, inserted and/or added is not particularly limited, and is a number that can be deleted, substituted or added to a certain extent by using known techniques such as site-directed mutagenesis described in molecular cloning, second edition, latest molecular biology laboratory methods, Nucleic Acids Research,10,6487(1982), proc.Natl.Acad.Sci., USA,79,6409(1982), Gene,34,315(1985), Nucleic Acids Research,13,4431(1985), proc.Natl.Acad.Sci USA,82,488(1985), and the like. For example, one to several tens, preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5.

The amino acid sequence of the above antibody in which one or more amino acid residues are deleted, substituted, inserted or added is defined as follows. That is, it means that there is deletion, substitution, insertion and/or addition of one or more amino acid residues in any and one or more amino acid sequences in the same sequence. In addition, there are cases where deletion, substitution, insertion, or addition occurs simultaneously, and there are cases where the amino acid residue to be substituted, inserted, or added is either a natural type or an unnatural type.

Examples of the natural amino acid residue include: l-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine, and the like.

Preferred examples of mutually substitutable amino acid residues are shown below. Amino acid residues contained in the same group may be substituted for each other.

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutyric acid, methionine, O-methylserine, tert-butylglycine, tert-butylalanine, cyclohexylalanine

Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid

Group C: asparagine and glutamine

Group D: lysine, arginine, ornithine, 2, 4-diaminobutyric acid, 2, 3-diaminopropionic acid

Group E: proline, 3-hydroxyproline, 4-hydroxyproline

And F group: serine, threonine, homoserine

Group G: phenylalanine, tyrosine

Examples of the effector activity of the present antibody include: ADCC activity, CDC activity, antibody dependent phagocytosis (ADCP) activity, opsonization, and the like can be controlled by various methods.

As a method for regulating the effector activity, for example, there can be mentioned: a method of controlling a sugar chain bound to the Fc region of an antibody, a method of modifying an amino acid residue in the Fc region of an antibody, and the like.

Examples of the method for controlling the sugar chain bound to the Fc fragment of the antibody include: a method of reducing ADCC and CDC activity by removing the sugar chain at the 297 position of an IgG antibody [ Molecular Immunology,32,1311, (1995), International publication No. 2008/030564 ], and a method of reducing CDC activity by reducing the binding of galactose to the Fc region of an antibody.

In addition, as a method for controlling a sugar chain bound to the Fc fragment of an antibody, for example, the following methods can be mentioned: a method for producing an antibody comprising a sugar chain to which fucose is not bound on N-acetylglucosamine (GlcNAc) of a base to which a sugar chain is bound, among N-linked sugar chains bound on asparagine at position 297 of an Fc fragment of an IgG antibody (US7,214,775, US6,946,292); a method of producing an antibody comprising a sugar chain to which bisecting (bisecting) GlcNAc is bound [ Nature Biotechnology,17,176, (1999) ]; a method for producing an antibody having a sugar chain to which galactose (Gal) bonded to a non-reducing end is bound [ hum.antibody.Hybridomas, 5,143-151, (1994) ]; and the like.

Examples of the method for amino acid modification of an amino acid residue in the Fc region of an antibody include the following methods: a method of modulating effector activity by performing amino acid modification of an Fc fragment of an antibody (J.B.C.,277,26733-26740,2002, U.S. Pat. No. 6,737,056, U.S. Pat. No. 7,297,775, U.S. Pat. No. 2007/0020260, International publication No. 2005/070963); and a method of regulating effector activity by performing domain exchange between the respective subclasses of the Fc fragment of an antibody (International publication No. 2007/011041).

The antibody fragment of the present invention includes: fab, Fab ', F (ab')₂scFv, diabody, dsFv and the like.

The antibody fragment of the present invention includes: fab, F (ab')₂Fab', scFv, diabodies, dsFvs and CDR-containing peptides, etc.

The Fab is an antibody fragment having an antigen binding activity and a molecular weight of about 5 ten thousand, which is obtained by treating IgG with papain, a protease, and in which about half of the H chain on the N-terminal side is bonded to the entire L chain by disulfide bonds.

The Fab of the present invention can be obtained by treating the monoclonal antibody of the present invention that specifically recognizes the sugar chain-deficient CD27 protein and binds to the extracellular domain with papain, a protease. Alternatively, the antibody can be produced by inserting a DNA encoding the Fab of the antibody into an expression vector for prokaryote or an expression vector for eukaryote, introducing the vector into a prokaryote or eukaryote, and expressing the vector.

F(ab’)₂The fragment is a fragment having an antigen-binding activity and a molecular weight of about 10 ten thousand, which is composed of two Fab regions bound to the hinge portion, and which is obtained by digesting the lower part of two disulfide bonds in the hinge portion of IgG with pepsin.

F (ab') of the present invention₂The monoclonal antibody of the present invention, which specifically recognizes the sugar chain-deficient CD27 protein and binds to the extracellular domain, can be obtained by treating with pepsin, a proteolytic enzyme. Alternatively, the Fab' described below may be produced by thioether bonding or disulfide bonding.

Fab 'is the same as the above F (ab')₂The disulfide bond at the hinge region of (2) is cleaved to obtain an antibody fragment having an antigen-binding activity and a molecular weight of about 5 ten thousand.

The Fab 'of the present invention can be produced by binding the F (ab') of the present invention, which specifically recognizes the sugar chain-deficient CD27 protein and binds to the extracellular domain₂Treating with reducing agent dithiothreitol. Alternatively, the antibody can be produced by inserting DNA encoding Fab' fragments of the antibody into prokaryotesThe expression vector is used in an expression vector for a eukaryote or the like, and the vector is introduced into a prokaryote or a eukaryote and expressed.

An scFv is a VH-P-VL or VL-P-VH polypeptide formed by linking one VH to one VL using an appropriate peptide linker (hereinafter referred to as P), and is an antibody fragment having antigen binding activity.

The scFv of the present invention can be produced by the following method: the expression can be carried out by obtaining cdnas encoding VH and VL of the monoclonal antibody of the present invention that specifically recognizes the sugar chain-deficient CD27 protein and binds to the extracellular domain, constructing DNA encoding scFv, inserting the DNA into an expression vector for prokaryote or an expression vector for eukaryote, and introducing the expression vector into prokaryote or eukaryote.

The diabody is an antibody fragment obtained by dimerization of scFv, and is an antibody fragment having a bivalent antigen binding activity. The divalent antigen-binding activities may be the same, or one of them may have different antigen-binding activities.

The bivalent antibody of the present invention can be produced by the following method: the monoclonal antibody of the present invention, which specifically recognizes the sugar chain-deficient CD27 protein and binds to the extracellular domain, is obtained by constructing DNA encoding scFv such that the amino acid sequence of P is 8 residues or less in length, inserting the DNA into an expression vector for prokaryote or an expression vector for eukaryote, and introducing the expression vector into prokaryote or eukaryote to express the antibody.

The dsFv refers to an antibody fragment in which a polypeptide having one amino acid residue in each of VH and VL substituted with a cysteine residue is bound via a disulfide bond between the cysteine residues. The amino acid residue substituted for a cysteine residue can be selected based on the prediction of the steric structure of the antibody according to the method disclosed by Reiter et al [ Protein Engineering,7,697(1994) ].

The dsFv of the present invention can be produced by the following method: the expression of the dsFv is carried out by obtaining cdnas encoding VH and VL of the monoclonal antibody of the present invention which specifically recognizes the sugar chain-deficient CD27 protein and binds to the extracellular domain, constructing a DNA encoding dsFv, inserting the DNA into an expression vector for prokaryote or an expression vector for eukaryote, and introducing the expression vector into prokaryote or eukaryote.

The peptide comprising a CDR is composed of at least one region in the CDR of VH or VL. Peptides comprising multiple CDRs can be conjugated directly or via a peptide linker.

The CDR-containing peptide of the present invention can be produced by the following method: the expression can be carried out by constructing DNAs encoding CDRs of VH and VL of the monoclonal antibody which specifically recognizes the sugar chain-deficient CD27 protein of the present invention and binds to the extracellular domain, inserting the DNAs into an expression vector for prokaryote or an expression vector for eukaryote, and introducing the expression vector into prokaryote or eukaryote.

Furthermore, the peptide containing CDR can also be produced by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method).

The invention comprises the following steps: the antibody-bound product of the present invention is obtained by binding a drug, a protein, a radioisotope, or the like to the monoclonal antibody or antibody fragment that specifically recognizes the sugar chain-deficient CD27 protein and binds to the extracellular domain, by a chemical method or a genetic engineering method.

The conjugate of the present invention is produced by binding a drug, a protein, a radioisotope or the like to the N-terminal side or the C-terminal side of the H chain or the L chain of the monoclonal antibody or the antibody fragment thereof of the present invention which specifically recognizes the sugar chain-deficient CD27 protein and binds to the extracellular domain, an appropriate substituent or side chain in the antibody or the antibody fragment thereof, and a sugar chain in the antibody or the antibody fragment thereof by a chemical method [ antibody engineering, gold photofinishing, shinkanji (1994) ].

Alternatively, the protein may be produced by a genetic engineering method in which a DNA encoding the monoclonal antibody or antibody fragment thereof that specifically recognizes the sugar chain-deficient CD27 protein of the present invention and binds to the extracellular domain is ligated to a DNA encoding a protein to be bound, and inserted into an expression vector, and the expression vector is introduced into a host cell of a prokaryote or eukaryote.

Examples of the drug include: chemotherapeutic agents, antibody drugs, immunostimulants, polymeric agents, and the like.

Examples of the protein include: cytokines, proliferation factors, and toxic proteins, among others.

Furthermore, the agent bound to the antibody or the antibody fragment may be in the form of a prodrug. The prodrug in the present invention is a drug that is chemically modified by an enzyme or the like present in a tumor environment to be converted into a substance having an action of killing cancer cells.

Examples of the chemotherapeutic agent include any of alkylating agents, nitrosourea agents, metabolic antagonists, anticancer antibiotics, plant-derived alkaloids, topoisomerase inhibitors, hormone therapy preparations, hormone antagonists, aromatase inhibitors, P-glycoprotein inhibitors, platinum complex derivatives, M-phase inhibitors, and kinase inhibitors.

Examples of the chemotherapeutic agent include: amifostine (amifostine), cisplatin, Dacarbazine (DTIC), dactinomycin, dichloromethyldiethylamine (mechlorethamine), streptozotocin, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), doxorubicin liposome (Doxil), epirubicin, gemcitabine (gemcitabine), daunorubicin liposome (Daunoxyme), procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, daunomycin, pelomycin, estramustine, paclitaxel (taxol), docetaxel (docetaxel), aldesleukin, asparaginase, busulfan, carboplatin, oxaliplatin, nedaplatin, cladribine, camptothecin, CPT-11, 10-hydroxy-SN 7-ethyl-camptothecin (38), 5-fluorodeoxyuridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna, irinotecan, topotecan (ノギテカン), mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, hydroxyurea, plicamycin, mirtutan, pemphidase, pentostatin, pipobroman, streptozotocin, tamoxifen, goserelin, leuprorelin, flutamide, teniposide, testolactone, thioguanine, thiotepa, uramustine, vinorelbine, chlorambucil, hydrocortisone, prednisolone, methylprednisolone, vindesine, nimustine, semustine, capecitabine, raltitrexed, azacitidine, Flt T, oxaliplatin, gefitinib (Iressa), imatinib (3), an inhibitor of Vascular Endothelial Growth Factor (VEGFR) receptor (VEGFR) inhibitors, Fibroblast Growth Factor Receptor (FGFR) inhibitors, Epidermal Growth Factor Receptor (EGFR) inhibitors (e.g., Iressa, Tarceva, etc.), radicicol, 17-allylamino-17-demethoxygeldanamycin, rapamycin, amsacrine, all-trans retinoic acid, thalidomide, anastrozole, fadrozole, letrozole, exemestane, gold thiomalate, D-penicillamine, buciramine, azathioprine, mizoribine, cyclosporine, rapamycin, hydrocortisone, bexarotene (e.g., tamidetin), tamoxifen, alcalasone, kininones, estrogens, anastrozole (e.g., Reiningde), Lipran, aspirin, indomethacin, celecoxib, azathioprine, penicillamine, gold thiomalate, chlorpheniramine maleate, chlorpheniramine, clemastine, tretinoin, bexarotene, arsenic, bortezomib, allopurinol, gemtuzumab ozogamicin, ibritumomab tiuxetan, 131 tositumomab, tamerastine, oxaziclovir, clarithromycin, oncovin, ifosfamide, ketoconazole, aminoglutethimide, suramin, methotrexate and Maytansinoid (Maytansinoid) and derivatives thereof, and the like.

Examples of the method for binding a chemotherapeutic agent to an antibody include: a method of binding a chemotherapeutic agent to an amino group of an antibody by glutaraldehyde; and a method of binding an amino group of a chemotherapeutic agent to a carboxyl group of an antibody using a water-soluble carbodiimide.

Examples of antibody pharmaceuticals include: an antibody having resistance to an antigen that induces apoptosis by binding of the antibody, an antigen associated with the pathological formation of a tumor or an antigen that regulates an immune function, an antigen associated with angiogenesis at a diseased site.

Examples of the antigen that induces apoptosis by binding of an antibody include: differentiation antigen (hereinafter referred to as CD)19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79B, CD80(B7.1), CD81, CD82, CD83, CD 84, CD85, CD86(B7.2), Human Leukocyte Antigen (HLA) class II, EGFR and the like.

Examples of antigens involved in the pathological formation of tumors or antigens that modulate immune function include: CD4, CD40, CD40 ligand, B7 family molecules (e.g., CD80, CD86, CD274, B7-DC, B7-H2, B7-H3, B7-H4, etc.), ligands for B7 family molecules (e.g., CD28, CTLA-4, ICOS, PD-1, BTLA, etc.), OX-40 ligand, CD137, Tumor Necrosis Factor (TNF) receptor family molecules (e.g., DR4, DR5, TNFR1, TNFR2, etc.), TNF-related apoptosis-inducing ligand receptor (TRAIL) family molecules, receptor family of TRAIL family molecules (TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4), nuclear factor kappa B receptor activating factor ligand (RANK), RANK ligand, CD25, folate receptor 4, cytokine [ e.g., interleukin-1 alpha (hereinafter, IL-1. beta. IL 4, IL-5, IL-4-5, IL-5, etc.), IL-6, IL-10, IL-13, Transforming Growth Factor (TGF) beta, TNF alpha, etc. ], receptors for the above cytokines, chemokines (e.g., SLC, ELC, I-309, TARC, MDC, CTACK, etc.), and receptors for the above chemokines.

Examples of antigens of antibodies that inhibit angiogenesis in a lesion include: vascular Endothelial Growth Factor (VEGF), angiogenin, Fibroblast Growth Factor (FGF), EGF, Platelet Derived Growth Factor (PDGF), insulin-like growth factor (IGF), Erythropoietin (EPO), TGF beta, IL-8, Ephilin and SDF-1, and the like, and receptors thereof.

The immunostimulant may be a natural product known as an immunoadjuvant, and specific examples thereof include: beta-1, 3-glucan (e.g. lentinan and cilostase), alpha-galactosyl ceramide (KRN7000), thallus powder (e.g. streptolysin and BCG), thallus extract (e.g. Coriolus versicolor polysaccharide).

Examples of the polymer drug include: polyethylene glycol (hereinafter referred to as PEG), albumin, dextran, polyethylene oxide, styrene maleic acid copolymer, polyvinylpyrrolidone, pyran copolymer, hydroxypropyl methacrylamide, and the like.

By binding these polymeric drugs to antibodies or antibody fragments, the following effects can be expected: (1) improving stability to various chemical, physical or biological factors; (2) the half-life period in blood is obviously prolonged; (3) inhibiting the disappearance of immunogenicity and inhibiting antibody production; etc. [ バイオユンジユゲ - ト medicine, Guang Chuan bookshop (1993) ].

Examples of a method for binding PEG to an antibody include a method of reacting with a PEG-modified reagent [ バイオユンジユゲ - ト medicinal herbs, Guangchuan bookshop (1993) ]. Examples of the pegylation modification reagent include: a modifier for the epsilon-amino group of lysine (Japanese patent application laid-open No. 61-178926), a modifier for the carboxyl groups of aspartic acid and glutamic acid (Japanese patent application laid-open No. 56-23587), and a modifier for the guanidine group of arginine (Japanese patent application laid-open No. 2-117920).

The cytokine or growth factor may be any cytokine or growth factor that enhances cells such as NK cells, macrophages, and neutrophils, and examples thereof include: interferons (hereinafter referred to as IFN) - α, INF- β, INF- γ, IL-2, IL-12, IL-15, IL-18, IL-21, IL-23, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and the like.

Examples of toxic proteins include: ricin, diphtheria toxin, ONTAK, etc., and also includes protein toxins obtained by introducing mutations into proteins to regulate toxicity.

Examples of the radioactive isotope include: 131I, 125I, 90Y, 64Cu, 199Tc, 77Lu, 211At, Re186, Re188, and In111, etc. The radioisotope can be directly bound to the antibody by the chloramine-T method or the like. In addition, a substance for chelating a radioisotope may be bound to the antibody. Examples of the chelating agent include methylphenyldiethylenetriaminepentaacetic acid (MX-DTPA).

In the present invention, the antibody used in the present invention may be administered in combination with one or more other agents, or may be combined with irradiation. Examples of the other agents include the above-mentioned chemotherapeutic agents, antibody drugs, immunostimulants, cytokines, growth factors, and the like.

As the radiation irradiation, there are included: photon (electromagnetic wave) irradiation such as X-ray and γ -ray; and particle beam irradiation such as electron beam, proton beam, and heavy particle beam.

As a method of combined administration, administration may be simultaneous with the antibody used in the present invention, or may be performed before or after the antibody used in the present invention is administered.

In the detection method, the quantitative method, the detection reagent, the quantitative reagent or the diagnostic reagent of the present invention, a method in which a specific labeling substance is labeled on the antibody of the present invention can be exemplified. The label may be a label used in a general immunological detection or measurement method, and includes: enzymes such as alkaline phosphatase, peroxidase, and luciferase; acridineLuminescent materials such as esters and loxapine; fluorescent substances such as Fluorescein Isothiocyanate (FITC) and trimethylrhodamine (RITC); and the like.

The method for producing the antibody of the present invention will be specifically described below.

1. Method for producing monoclonal antibody

(1) Preparation of antigens

By introducing an expression vector containing a cDNA encoding full-length or partial-length CD27 into a yeast, insect cell, animal cell, or the like, in which the activity of an enzyme that adds Gal to GalNAc bound to Ser/Thr on a polypeptide in the O-linked sugar chain synthesis process, a protein involved in the activity of the enzyme, a protein involved in the transport of UDP-galactose, or the like is reduced or deleted, a sugar chain-deficient CD27 or a cell expressing a sugar chain-deficient CD27 can be obtained as an antigen, by the method described below.

In addition, a method of producing an antigen by purifying sugar chain-deficient CD27 from cultured cells of various human sources, human tissues, or the like that express sugar chain-deficient CD27 in large amounts on cell membranes or in a culture solution, or a synthetic peptide having a partial sequence of sugar chain-deficient CD27 may be used as an antigen. Furthermore, the sugar chain-deficient CD27 can be obtained by adding a sugar chain to CD27 which has been expressed and purified in a test tube using a prokaryote such as Escherichia coli which does not have the ability to add sugar chains.

In addition, by introducing an expression vector containing a cDNA encoding full-length or partial-length CD27 into a host cell such as yeast, insect cell, animal cell, etc. having a normal O-linked sugar chain synthesis process in the same manner, a cell expressing CD27 having a normal O-linked sugar chain can be obtained, and CD27 protein having a normal O-linked sugar chain can be purified from the cell.

The sugar chain-deficient CD27, CD27 protein having normal O-linked sugar chains, or the expression cells obtained as described above can be used for screening of target antibodies, confirming the reactivity of the obtained antibodies to antigens.

The polypeptide used IN the present invention can be produced by expressing DNA encoding the polypeptide IN a host cell by using the method described IN Molecular Cloning, laboratory Manual, second edition, Cold spring harbor laboratory Press (1989), Currentprotocols IN Molecular Biology, John Willd parent-son publishing Co., 1987-1997, and the like.

For example, it can be produced by the following method. First, a cDNA encoding the full-length polypeptide is inserted downstream of a promoter of an appropriate expression vector, thereby creating a recombinant vector. In this case, if necessary, a DNA fragment of an appropriate length including a portion encoding the polypeptide may be prepared based on the full-length cDNA, and the DNA fragment may be used in place of the full-length cDNA. Subsequently, the recombinant vector is introduced into a host cell suitable for the expression vector, whereby a transformant producing the polypeptide can be obtained.

Any host cell can be used as long as it has the ability to add an O-linked sugar chain and can express a desired gene, such as Escherichia coli, yeast, insect cells, and animal cells.

As the expression vector, an expression vector capable of autonomous replication in the host cell to be used or capable of integrating into the chromosome and containing an appropriate promoter at a position where DNA encoding the polypeptide can be transcribed is used.

When a prokaryote such as escherichia coli is used as a host cell, the recombinant vector containing a DNA encoding the polypeptide used in the present invention is preferably a vector which is autonomously replicable in the prokaryote and contains a promoter, a ribosome binding sequence, the DNA used in the present invention, and a transcription termination sequence. The recombinant vector may further contain a gene for regulating a promoter.

Examples of expression vectors include: pBTrp2, pBTac1, pBTac2 (all manufactured by Roche diagnostics Co.), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN), pKK 10 (Japanese patent application laid-open No. 58-110600), pKK 200[ Agricutural biological Chemistry,48,669(1984) ], pLSA1[ Agric.biol. chem.,53,277(1989) ], pGEL1[ Proc.Natl.Acad.Sci.USA,82,4306(1985) ], pBluescript II SK (-) (manufactured by Stratagene), pTrs30[ prepared by Escherichia coli JM 109/pTS 30(FERM BP-545407) ], pGTARM 7336 (prepared by Escherichia coli JM 6711-36 32, pGRE-369634 (JP-7) and pGHA-369685 (JP-7,9626,5411,5436,9626,9685), US4939094, US5160735), pSupex, pUB 110, pTP5, pC194, pEG400[ j.bacteriol.,172,2392(1990) ], pGEX (manufactured by Pharmacia), pET system (manufactured by Novagen), pME18SFL3, and the like.

Any promoter can be used as long as it is capable of functioning in the host cell to be used. Examples thereof include: promoters derived from Escherichia coli, phage, and the like, such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, and T7 promoter. Further, a tandem promoter in which two Ptrp are connected in series, a tac promoter, a lac T7 promoter, a let I promoter, or the like may be used, and a modified promoter may be used.

In addition, as the recombinant vector, a plasmid in which the distance between the Shine-Dalgarno (Shine-Dalgarno) sequence as a ribosome binding sequence and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 nucleotides) is preferably used.

In the nucleotide sequence of the DNA encoding the polypeptide used in the present invention, nucleotides may be substituted to form codons optimal for expression in a host, thereby improving the productivity of the target polypeptide. Further, the transcription termination sequence is not necessarily required for the expression of the gene in the above recombinant vector, but it is preferable to arrange the transcription termination sequence immediately downstream of the structural gene.

Examples of the host cell include microorganisms belonging to the genus Escherichia and the like, for example, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, and Escherichia coli DH5 α.

As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing a DNA into the above-mentioned host cell, and examples thereof include: methods using calcium ion [ Proc. Natl. Acad. Sci. USA,69,2110(1972) ], Gene,17,107(1982), and Molecular & general genetics,168,111(1979), and the like.

In the case of using an animal cell as a host, examples of the expression vector include: pcDNAI, pcDM8 (sold by フナユシ Co.), pAGE107[ Japanese patent application laid-open No. Hei 3-22979; cytotechnology,3,133, (1990), pAS3-3 (Japanese patent laid-open No. H2-227075), pCDM8[ Nature,329,840, (1987) ], pcDNAI/Amp (manufactured by Invitrogen), pcDNA3.1 (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103[ J.biochemistry,101,1307(1987) ], pAGE210, pME18SFL3, pKANTEX93[ International publication No. 97/10354 ], and the like.

As the promoter, any promoter can be used as long as it can function in animal cells, and examples thereof include: a promoter of an IE (immediate early) gene of Cytomegalovirus (CMV), an early promoter of SV40, a promoter of retrovirus, a metallothionein promoter, a heat shock promoter, an SR α promoter, a promoter and an enhancer of moloney murine leukemia virus (moloney murineukemia virus), and the like. In addition, an enhancer of the IE gene of human CMV may be used together with a promoter.

Any host cell can be used as long as it has a reduced or absent activity, such as an enzyme that adds Gal to N-acetylgalactosamine (GalNAc) bound to Ser/Thr in the polypeptide in the sugar chain synthesis pathway, a protein involved in the activity of the enzyme, or a protein involved in the transport of uridine 5' -diphosphate galactose (UDP-galactose). Specifically, there may be mentioned: lec8 mutant as UDP-galactose transporter deleted Chinese hamster cell (CHO cell) [ ACSSymp. Ser.128,214(1980) ].

In addition, a cell line in which the function of a UDP-galactose transporter (also known as UDP-galactose transporter, UGALT) or an enzyme such as glycoprotein-N-acetylgalactosamine 3-. beta. -galactosyltransferase (C1GALT1, also known as core 1-. beta. -3-gal-t, t-synthase) or C1GALT 1-specific chaperonin 1(C1GALT1C1, also known as core 1. beta. -3-galactosyltransferase-specific chaperone (COSMC), C1GALT2) is reduced or deleted in a cell in which the activity of an enzyme or transporter involved in the sugar chain synthesis pathway is not deleted can be used.

Examples of cells in which the activity of an enzyme or a transporter involved in the sugar chain synthesis pathway is not lost include: namalwa (Namalwa) cells, COS cells which are monkey cells, CHO cells which are Chinese hamster cells, HBT5637 (Japanese patent application laid-open No. Sho 63-299), and the like.

Examples of the enzyme group to be used include the above-mentioned ones.

Examples of the method for reducing the gene function include: antisense, ribozyme [ proc.Natl.Acad.Sci.U.S.A.,96,1886(1999) ], homologous recombination [ Manipulating the mouse Embryo manipulation Laboratory Manual, second edition, Cold spring harbor Laboratory Press (1994); gene Targeting, A Practical Approach (Gene Targeting: Practical discussion), IRL Press (1993) under the heading of Oxford university Press, RNA-DNA oligonucleotide (RDO) method, RNA interference (RNAi) method [ Nature,391,806 (1998); proc.natl.acad.sci.usa 95,15502, (1998); nature,395,854, (1998); proc.natl.acad.sci.usa,96,5049, (1999); cell,95,1017, (1998); proc.natl.acad.sci.usa,96,1451, (1999); roc. natl. acad. sci. usa,95,13959, (1998); nature Cell biol.,2,70, (2000) ], a method using a retrovirus, and a method using a transposon [ Nature Genetics,25,35, (2000) ], and the like.

As a method for introducing a vector, any method can be used as long as it is a method for introducing DNA into animal cells, and examples thereof include: electroporation [ Cytotechnology,3,133(1990) ], calcium phosphate method (Japanese patent laid-open No. 2-227075), and lipofection method [ Proc.Natl.Acad.Sci.USA,84,7413(1987) ].

As a method for expressing a gene, in addition to direct expression, secretory production, expression of a fusion protein, and the like can be performed according to the method described in molecular cloning, A Laboratory Manual, second edition, Cold spring harbor Laboratory Press (1989), and the like. When the expression is carried out in a cell derived from a eukaryote, a polypeptide having a sugar or a sugar chain attached thereto can be obtained.

The transformant obtained as described above is cultured in a medium, the polypeptide is produced and accumulated in the culture, and the polypeptide is collected from the culture, whereby the polypeptide can be produced. The method of culturing the transformant in a medium can be performed according to a conventional method used for culturing a host.

When a microorganism transformed with a recombinant vector using an inducible promoter is cultured, an inducer may be added to the culture medium as needed. For example, when a microorganism transformed with a recombinant vector using a lac promoter is cultured, isopropyl-. beta. -D-thiogalactopyranoside or the like can be added to the medium, and when a microorganism transformed with a recombinant vector using a trp promoter is cultured, indoleacrylic acid or the like can be added to the medium.

As a medium for culturing a transformant obtained by using an animal cell as a host, a commonly used RPMI1640 medium [ The Journal of The American medical Association,199,519(1967) ], Eagle's MEM medium [ Science,122,501(1952) ], Dulbecco's modified MEM medium [ Virology,8,396(1959) ], 199 medium [ Proc. Soc. exp. biol. Med.,73,1(1950) ], a medium obtained by adding FCS or The like to these media, and The like can be used.

The culture is usually carried out at a pH of 6-8, a temperature of 30-40 ℃ and 5% CO₂In the presence of the catalyst for 1 to 7 days. In addition, during the culture, if necessary, antibiotics such as kanamycin and penicillin may be added to the medium.

As described above, the polypeptide used in the present invention can be produced by culturing a transformant derived from a microorganism, an animal cell, or the like, which comprises a recombinant vector in which a DNA encoding the polypeptide used in the present invention is recombined, according to a usual culture method, producing and accumulating the polypeptide, and collecting the polypeptide from the culture.

As a method for expressing a gene, in addition to direct expression, secretory production, expression of a fusion protein, and the like can be performed according to a method described in molecular cloning, Alaberration Manual, second edition, Cold spring harbor laboratory Press (1989), and the like.

As a method for producing a polypeptide, the following methods are mentioned: the method of production in a host cell, the method of secretion out of a host cell, and the method of production on the outer membrane of a host cell can be selected as appropriate by changing the structures of the host cell used and the polypeptide produced.

In the case of producing a polypeptide in a host cell or on the outer membrane of a host cell, the polypeptide can be produced by using the method of pallson et al [ j.biol.chem.,264,17619(1989) ], the method of roc et al [ proc.natl.acad.sci., USA,86,8227 (1989); genes Develop, 4,1288(1990), or the gene product can be secreted out of the host cell by the methods described in Japanese patent application laid-open No. 05-336963 and International publication No. 94/23021. Further, according to the method described in Japanese patent application laid-open No. 2-227075, the amount of production can be increased by using a gene amplification system using a dihydrofolate reductase gene or the like.

The polypeptide can be isolated and purified, for example, as follows.

When the polypeptide is expressed in a solubilized state in the cells, the cells are recovered by centrifugation after the completion of the culture, suspended in an aqueous buffer solution, and then disrupted by an ultrasonic disrupter, French press, MANTON-GULIN homogenizer, horizontal sand mill, or the like, to thereby obtain a cell-free extract.

The cell-free extract is centrifuged, and the purified product is obtained from the supernatant obtained by a separation and purification method using a usual enzyme, that is, a method using a solvent extraction method, a salting-out method using ammonium sulfate or the like, a desalting method, a precipitation method using an organic solvent, an anion exchange chromatography method using a resin such as Diethylaminoethyl (DEAE) -agarose or DIAION HPA-75 (manufactured by mitsubishi chemical corporation), a cation exchange chromatography method using a resin such as S-Sepharose FF (manufactured by Pharmacia), a hydrophobic chromatography method using a resin such as butyl Sepharose or phenyl Sepharose, a gel filtration method using a molecular sieve, an affinity chromatography method, a focusing chromatography method, an electrophoresis method such as isoelectric point electrophoresis, or the like, alone or in combination.

When the polypeptide is expressed as inclusion bodies in the cells, the cells are similarly collected, disrupted, and centrifuged, whereby the inclusion bodies of the polypeptide are collected as a precipitate fraction. The inclusion bodies of the polypeptide thus recovered are solubilized with a protein denaturing agent. The solubilized solution is diluted or dialyzed to restore the normal spatial structure of the polypeptide, and then a purified preparation of the polypeptide is obtained by the same separation and purification method as described above.

The polypeptide used in the present invention can also be produced by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method). Further, chemical synthesis can be carried out using a peptide synthesizer such as Advanced ChemTech, パ - キン. エルマ, Pharmacia, Protein Technology Instrument, synthetic cell-Vega, PerSeptive, Shimadzu corporation, or the like.

(2) Immunization of animals and production of antibody-producing cells

Mice, rats or hamsters of 3 to 20 weeks old were immunized with the antigen prepared as described above, and antibody-producing cells in the spleen, lymph nodes, and peripheral blood of the animals were collected. In addition, in the case where the immunogenicity is low and thus a sufficient increase in antibody titer is not observed in the above-mentioned animals, there is also a method of using a CD27 knock-out animal as an immunized animal.

Immunization is carried out by administering the antigen subcutaneously, intravenously or intraperitoneally in animals together with an appropriate Adjuvant [ e.g., complete Freund's Adjuvant, aluminum hydroxide gel, pertussis vaccine, etc. ].

In the case where the antigen is a partial peptide, it is reacted with BSA (bovine serum albumin) or KLH (keyhole limpet) Hemocyanin, Keyhole Limpet Hemocyanin) and the like as a conjugate, and they were used as an immunogen.

For administration of the antigen, 5 to 10 times are administered every 1 to 2 weeks after the first administration. Blood was collected from the fundus venous plexus on days 3 to 7 after each administration, and the reaction of the serum with the antigen was examined by an enzyme immunoassay [ Antibodies-A Laboratory Manual, Cold spring harbor Laboratory, 1988] or the like. A mouse, rat or hamster whose serum exhibits a sufficient antibody titer against an antigen used for immunization is used as a source of spleen cells.

When the fusion of spleen cells and myeloma cells is carried out, spleens are removed from immunized mice, rats or hamsters 3 to 7 days after the last administration of the antigen substance, and the spleen cells are collected. The spleen was finely chopped in MEM medium (manufactured by japan water pharmaceutical company), stirred with forceps and centrifuged (1200rpm, 5 minutes), and then the supernatant was discarded, treated with Tris-ammonium chloride buffer (ph7.65) for 1 to 2 minutes to remove erythrocytes, and washed 3 times with MEM medium, thereby providing splenocytes for fusion.

(3) Preparation of myeloma cells

As myeloma cells, established cell lines obtained from mice were used. For example, it is possible to use: 8-azaguanine-resistant mice (BALB/c derived) myeloma cell line P3-X63Ag8-U1(P3-U1) [ Current Topics in Microbiology and Immunology,18,1(1978) ], P3-NS1/1-Ag41(NS-1) [ European J.immunology,6,511(1976) ], -SP2/O-Ag 14(SP-2) [ Nature,276,269(1978) ], P3-X63-Ag8653(653) [ J.154immunology, 123, 8(1979) ], P3-X63-Ag8(X63) [ Nature,256,495 (5) ], and the like.

As described aboveThe cell line used 8-azaguanine medium [ glutamine (1.5mM) and 2-mercaptoethanol (5X 10) were added to RPMI-1640 medium^-5M), gentamicin (10. mu.g/mL) and Fetal Calf Serum (FCS) (hereinafter referred to as "Normal Medium"), and 8-azaguanine (15. mu.g/mL) was further added thereto]Passaging is carried out, and the cells are passaged into a normal culture medium 3-4 days before cell fusion so as to ensure that the cells reach 2 multiplied by 10 on the day of fusion⁷More than one cell.

(4) Cell fusion

The antibody-producing cells and myeloma cells are thoroughly washed with MEM medium or PBS (disodium hydrogenphosphate 1.83g, potassium dihydrogenphosphate 0.21g, salt 7.65g, distilled water 1 liter, pH7.2) to mix the cells so that the number of cells reaches the number of antibody-producing cells, myeloma cells =5 to 10:1, centrifuged (1200rpm, 5 minutes), the supernatant is discarded, the precipitated cell population is thoroughly stirred and dispersed, and then 0.2 to 1mL/10 is added at 37 ℃ while stirring⁸To an antibody-producing cell, a mixed solution of 2g of polyethylene glycol-1000 (PEG-1000), 2mL of MEM and 0.7mL of dimethyl sulfoxide was added, and 1 to 2mL of MEM medium was added every 1 to 2 minutes, and after repeating the above steps several times, the MEM medium was added to a total amount of 50 mL.

After centrifugation (900rpm, 5 minutes), the supernatant was discarded, the cells were gently stirred, sucked and blown out with a graduated pipette, and the cells were gently suspended in 100mL of HAT medium [ to which hypoxanthine (10) (added to normal medium)^-4Mole/liter), thymidine (1.5X 10)^-5Mole/liter) and aminopterin (4X 10)^-7Mole/liter) of culture medium]In (1). The suspension was dispensed into 96 well plates at 100. mu.L/well in 5% CO₂Culturing for 7-14 days at 37 ℃ in an incubator.

After the culture, a part of the culture supernatant is taken, and cells that react with an antigen containing the polypeptide used in the present invention but do not react with an antigen containing no polypeptide are selected by a binding assay described later or the like.

Then, cloning was repeated twice by the limiting dilution method [ first using HT medium (medium obtained by removing aminopterin from HAT medium) and second using normal medium ], to select cells in which strong antibody titer was stably observed as a hybridoma strain producing a monoclonal antibody.

(5) Preparation of monoclonal antibodies

Applying 0.5mL 2,6,10, 14-tetramethylpentadecane (pristane) into pristane chamber, and feeding for 2 weeks]The mice or nude mice of later 8-10 weeks old are 2 × 10 ⁶~5×10⁷The hybridoma cells producing the anti-CD 27 monoclonal antibody obtained in (4) were intraperitoneally injected in a single amount. The hybridoma produces cancerous ascites within 10-21 days.

Ascites is collected from the above mouse, centrifuged (3000rpm, 5 minutes) to remove solid content, then salted out with 40 to 50% ammonium sulfate, and then purified by caprylic acid precipitation, DEAE-agarose column, protein a column or gel filtration column to collect IgG or IgM fraction as purified monoclonal antibody.

Determination of antibody subclasses was performed by enzyme immunoassay using a subclass typing kit. The quantification of the amount of protein was calculated by the Lory method and absorbance at 280 nm.

(6) Binding assays

As antigens, use was made of: a gene-introduced cell or recombinant protein obtained by introducing an expression vector comprising cDNA encoding the CD27 polypeptide used in the present invention into Escherichia coli, yeast, insect cells, animal cells, or the like, or a purified polypeptide or partial peptide obtained from human tissue, by the method described in item (1).

In the case of partial peptides as antigens, BSA (bovine serum albumin) and KLH (keyhole limpet)Hemocyanin, Keyhole Limpet Hemocyanin), and the like, and the conjugates are used.

The antigen is dispensed into a 96-well plate and made into a solid phase, and then serum of an immunized animal, a culture supernatant of a hybridoma producing a monoclonal antibody, or a purified antibody is dispensed as a first antibody and reacted. After washing with PBS or PBS-0.05% tween, an anti-immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent substance, a radioactive compound, or the like is dispensed as a second antibody, and reaction is performed. After washing well with PBS-Tween, a reaction corresponding to the labeling substance of the second antibody was carried out.

An antibody that competes with a monoclonal antibody that recognizes CD27 containing an O-linked sugar chain to which galactose is not bound and binds to the extracellular domain can be obtained by adding a test antibody to the above-described binding assay system and reacting the same. That is, an antibody in which the binding of the monoclonal antibody is inhibited when the test antibody is added is selected, whereby a monoclonal antibody that competes with the obtained monoclonal antibody in binding to the extracellular domain of the sugar chain-deficient CD27 can be obtained.

Further, an antibody that binds to an epitope recognized by a monoclonal antibody that recognizes the sugar chain-deficient CD27 and binds to the extracellular domain can be obtained by the following method: the epitope of the antibody obtained in the binding assay system is identified, and a part of the identified epitope, a sugar chain-binding peptide that mimics the steric structure of the epitope, or the like is prepared and immunized.

2. Production of recombinant antibody

As an example of producing a recombinant antibody, methods for producing a human chimeric antibody and a humanized antibody are shown below.

(1) Construction of vector for expression of recombinant antibody

The vector for expression of a recombinant antibody is an expression vector for animal cells into which DNAs encoding CH and CL of a human antibody are recombined, and can be constructed by cloning DNAs encoding CH and CL of a human antibody into expression vectors for animal cells, respectively.

The C region of a human antibody can be any of CH and CL of a human antibody. Examples thereof include: and the human antibody has a gamma 1 subclass of CH and a kappa class of CL. As the DNA encoding CH and CL of a human antibody, chromosomal DNA including exons and introns may be used, cDNA may also be used, and cDNA is preferably used.

Any expression vector for animal cells may be used as long as it can express a gene encoding the C region of a human antibody by recombination. Examples thereof include: pAGE107[ Cytotechnol, 3,133(1990) ], pAGE103[ J.biochem, 101,1307(1987) ], pHSG274[ Gene,27,223(1984) ], pKCR [ Proc.Natl.Acad.Sci.USA,78,1527(1981) ], pSG1bd2-4[ Cytotechnol, 4,173(1990) ], and pSE1UK1Sed1-3[ Cytotechnol, 13,79(1993) ], and the like.

Examples of promoters used in expression vectors for animal cells include: the early promoter of SV40 [ J.Biochem.,101,1307(1987) ], LTR of Moloney murine leukemia virus [ biochem.Biophys.Res.Commun.,149,960(1987) ], and the promoter of immunoglobulin H chain [ Cell,41,479(1985) ]. Examples of enhancers to be used in expression vectors for animal cells include enhancers [ Cell,33,717(1983) ].

The vector for expression of a recombinant antibody can be either a type in which an antibody H chain and an antibody L chain are present on different vectors or a type in which they are present on the same vector (tandem type), and is preferably a tandem type vector for expression of a recombinant antibody from the viewpoints of ease of construction of a recombinant antibody expression vector, ease of introduction into animal cells, and balance between the expression levels of an antibody H chain and an antibody L chain in animal cells [ J.Immunol.methods,167,271(1994) ].

Examples of the vector for expressing a tandem type recombinant antibody include: pKANTEX93[ International publication No. 97/10354 ] and pEE18[ Hybridoma,17,559(1998) ], and the like.

(2) Acquisition of cDNA encoding V region of antibody derived from non-human animal and analysis of amino acid sequence

cDNAs encoding VH and VL of a non-human antibody can be obtained as follows.

mRNA is extracted from hybridoma cells producing a non-human antibody, and cDNA is synthesized. The synthesized cDNA is cloned into a vector such as phage or plasmid to prepare a cDNA library. Recombinant phages or recombinant plasmids having cdnas encoding VH or VL, respectively, were isolated from the library using DNA encoding the C region part or V region part of a mouse antibody as a probe.

The full-length nucleotide sequence of VH or VL of the target mouse antibody on the recombinant phage or recombinant plasmid is determined, and the full-length amino acid sequence of VH or VL is deduced from the nucleotide sequence.

As the non-human animal, any animal can be used as long as it can produce hybridoma cells of mouse, rat, hamster, rabbit, and the like.

Examples of a method for producing total RNA from hybridoma cells include guanidine isothiocyanate-cesium trifluoroacetate method [ Methods in enzymol, 154,3(1987) ]. Examples of the kit include RNAeasy kit (manufactured by QIAGEN corporation).

Examples of the method for preparing mRNA from total RNA include a cellulose column method [ Molecular Cloning, A Laboratory Manual, second edition (Cold spring harbor Laboratory Press, 1989) ] in which oligo (dT) is immobilized. Further, as the kit, for example, OligoTM-dT30< Super > mRNA purification kit (manufactured by Takara) and the like can be cited.

Examples of a kit for preparing mRNA from hybridoma cells include: a FastTrackmRNA isolation kit (Invitrogen) and a QuickPrep mRNA purification kit (Pharmacia), etc.

Examples of the method for synthesizing cDNA and preparing a cDNA library include conventional methods [ Molecular Cloning, A Laboratory Manual, second edition (Cold spring harbor Laboratory Press, 1989); current Protocols in Molecular Biology, appendix 1-34]. In addition, SuperScript for cDNA synthesis and plasmid cloning using a commercially available kit can be exemplified^TMPlasmid system (Invitrogen) and ZAP-cDNA Synthesis kit (Stratagene).

In the preparation of a cDNA library, any vector can be used as long as it can recombine cDNA synthesized using mRNA extracted from hybridoma cells as a template.

For example, it is possible to use: ZAP Express [ Strategies,5,58(1992) ], pBluescript IISK (+) [ Nucleic Acids Research,17,9494(1989) ], lambda ZAPII (Stratagene Co., Ltd.), lambda gt10, lambda gt11(1985), lambda BlueMid (Clontech Co., Ltd.), lambda ExCell, pT7T318U (Pharmacia Co., Ltd.), pcD2[ mol.cell.biol.,3,280(1983) ], and pUC18[ Gene,33,103(1985) ], and the like.

Coli into which a cDNA library constructed from a phage or a plasmid vector is introduced may be any E.coli into which the cDNA library can be introduced, expressed and retained.

For example, it is possible to use: XL1-Blue MRF' [ Strategies,5,81(1992) ], C600[ Genetics,39,440(1954) ], Y1088, Y1090 [ Science,222,778(1983) ], NM522[ J.mol.biol.,166,1(1983) ], K802[ J.mol.biol.,16,118(1966) ], and JM105[ Gene,38,275(1985) ], and the like.

As a method for screening cDNA clones encoding VH or VL of a non-human antibody from a cDNA library, screening can be performed by a colony hybridization method or a plaque hybridization method using an isotope-or fluorescence-labeled probe [ Molecular Cloning, A Laboratory Manual, second edition (Cold spring harbor Laboratory Press, 1989) ].

Alternatively, a primer may be prepared and used as a template to prepare a cDNA or cDNA library synthesized from mRNA by polymerase chain reaction [ hereinafter referred to as PCR method; molecular Cloning, laboratory Manual, second edition, Cold spring harbor laboratory Press (1989); CurrentProtocolysin Molecular Biology, appendix 1-34] to prepare cDNAs encoding VH or VL.

The cDNA selected by the above method is cleaved with an appropriate restriction enzyme or the like, and then cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), and the base sequence of the cDNA can be determined by performing a reaction by a commonly used base sequence analysis method such as dideoxy method [ proc.natl.acad.sci.usa,74,5463(1977) ] of Sanger (Sanger, F.) or the like, and analyzing the reaction by using an automatic base sequence analyzer such as a.l.f. dna sequencer (manufactured by Pharmacia) or the like.

From the determined nucleotide Sequences, the full-length amino acid Sequences of VH and VL, respectively, were deduced and compared with the full-length amino acid Sequences of VH and VL of known antibodies [ Sequences of Proteins of immunological Interest, American Ministry of health and public service (1991) ], whereby it was confirmed whether the obtained cDNA encoded the complete amino acid Sequences of VH and VL of an antibody containing a secretion signal sequence, respectively.

With respect to the complete amino acid Sequences of VH and VL of an antibody comprising a secretion signal sequence, the length and N-terminal amino acid sequence of the secretion signal sequence can be estimated by comparing with the full-length amino acid Sequences of VH and VL of known antibodies [ Sequences of proteins of Immunological Interest, U.S. department of health and public service (1991) ], and the subclass to which they belong can be known.

The amino acid sequence of each CDR of VH and VL can also be found by comparison with the amino acid Sequences of VH and VL of known antibodies [ Sequences of Proteins of immunologicalcatel interest, U.S. department of health and public service (1991) ].

Furthermore, it is possible to confirm whether or not the amino acid sequence used is a new amino acid sequence by performing a sequence homology search such as the BLAST method [ J.mol.biol.,215,403(1990) ] on an arbitrary database such as SWISS-PROT and PIR-Protein using the complete amino acid sequences of VH and VL.

(3) Construction of human chimeric antibody expression vector

A human chimeric antibody expression vector can be constructed by cloning cDNA encoding VH or VL of a non-human antibody into the upstream of each gene encoding CH or CL of a human antibody of the vector for expression of a recombinant antibody described in (1) of item 2.

For example, in order to link the 3 '-terminal side of cDNA encoding VH or VL of a non-human antibody to the 5' -terminal side of CH or CL of a human antibody, cDNA of VH and VL designed as follows is prepared: the base sequence of the linker encodes the appropriate amino acids and becomes the appropriate restriction enzyme recognition sequence.

The human chimeric antibody expression vector can be constructed by cloning each of the prepared cDNAs for VH and VL into the upstream of each gene encoding CH or CL of the human antibody of the vector for expression of humanized antibody described in (1) of item 2, and expressing them in an appropriate form.

Alternatively, cDNAs encoding VH or VL of a non-human antibody can be amplified by PCR using synthetic DNAs having recognition sequences for suitable restriction enzymes at both ends, and cloned into the vector for expression of recombinant antibody according to item 2 (1).

(4) Construction of cDNA encoding V region of humanized antibody

A cDNA encoding VH or VL of a humanized antibody can be constructed as follows. First, the amino acid sequence of the framework region (hereinafter referred to as "FR") of VH or VL of a human antibody to which the amino acid sequence of CDR of VH or VL of a non-human antibody is grafted is selected. The amino acid sequence of the selected FR may be any amino acid sequence derived from a human antibody.

Examples thereof include: amino acid Sequences of FRs of human antibodies registered in databases such as Protein Data Bank (Sequences of Proteins of Immunological Interest, U.S. department of health and public services (1991)), and the like.

In order to suppress the decrease in the binding activity of an antibody, an amino acid sequence of an FR having as high homology as possible (at least 60% or more) with the amino acid sequence of the FR of VH or VL of the original antibody is selected. Then, the amino acid sequences of the CDRs of the original antibody are grafted to the amino acid sequences of the FRs of the VH or VL of the selected human antibody, respectively, thereby designing the amino acid sequences of the VH or VL of the humanized antibody, respectively.

DNA Sequences encoding the amino acid Sequences of VH and VL of humanized antibodies were designed by converting the designed amino acid Sequences into DNA Sequences in consideration of the frequency of codon usage appearing in the base sequence of the antibody gene [ Sequences of Proteins of Immunological Interest, U.S. department of health and public service (1991) ].

Based on the designed DNA sequence, a plurality of synthetic DNAs each having a length of about 100 bases were synthesized, and PCR was performed using these synthetic DNAs. In this case, it is preferable to design 6 synthetic DNAs for both H chain and L chain in view of the reaction efficiency in PCR and the length of DNA that can be synthesized.

Furthermore, by introducing recognition sequences for appropriate restriction enzymes into the 5' -ends of the synthetic DNAs located at both ends, cDNA encoding VH or VL of a humanized antibody can be easily cloned into the vector for expression of a humanized antibody constructed in (1) of item 2.

After the PCR reaction, the amplified products were cloned into plasmids such as pBluescript SK (-) (manufactured by Stratagene Co., Ltd.), and the nucleotide sequences were determined by the method described in (2) of item 2, whereby plasmids having DNA sequences encoding the amino acid sequences of VH or VL of the desired humanized antibody were obtained.

(5) Modification of amino acid sequence of V region of humanized antibody

It is known that: when only CDRs of VH and VL of a non-human antibody are grafted into FRs of VH and VL of a human antibody, the antigen binding activity of the humanized antibody is reduced as compared with that of the original non-human antibody [ BIO/TECHNOLOGY,9,266(1991) ].

This is considered to be because, in VH and VL of an original nonhuman antibody, not only the CDR but also the amino acid residues in the FR are directly or indirectly related to the antigen binding activity, and therefore, when the amino acid residues of the FR of the nonhuman antibody are substituted with the amino acid residues of the FR of a human antibody by humanization, the antigen binding activity is decreased.

In order to solve this problem, the following method is implemented: in the humanized antibody, amino acid residues directly involved in the binding to the antigen, amino acid residues interacting with the amino acid residues of the CDR, and amino acid residues involved in the binding indirectly to the antigen while maintaining the steric structure of the antibody are identified in the amino acid sequences of the FRs of VH and VL of a human antibody, and these amino acid residues are substituted with the amino acid residues of the original non-human antibody, whereby the reduced antigen-binding activity is increased [ BIO/TECHNOLOGY,9,266(1991) ].

In order to solve the problem of how to efficiently identify the amino acid residues of these FRs involved in the antigen-binding activity in the production of humanized antibodies, the construction and analysis of the three-dimensional structure of the antibody were carried out by X-ray crystallography [ j.mol.biol.,112,535(1977) ], or computer modeling [ Protein Engineering,7,1501(1994) ], or the like.

The information on the three-dimensional structure of the above-mentioned antibody provides a lot of useful information for the production of a humanized antibody, but a method for producing a humanized antibody that can be applied to all antibodies has not yet been established, and various attempts have been made to produce a plurality of modified antibodies for each antibody and to study the correlation between the modified antibodies and the antigen-binding activity.

The amino acid residues of the FRs of VH and VL of a human antibody can be modified by the PCR method described in (4) of item 2 using a synthetic DNA for modification. The amplified product after PCR was confirmed to have been modified as desired by determining the nucleotide sequence by the method described in (2) of item 2.

(6) Construction of humanized antibody expression vector

A humanized antibody expression vector can be constructed by cloning each of the constructed cDNAs encoding VH or VL of the recombinant antibody into the upstream of each of the genes encoding CH or CL of the human antibody of the vector for expression of a recombinant antibody described in (1) of item 2.

For example, by introducing suitable recognition sequences for restriction enzymes into the 5' -ends of synthetic DNAs located at both ends of synthetic DNAs used for constructing VH or VL of a humanized antibody in (4) and (5) of item 2, cDNA of the constructed VH or VL can be cloned upstream of each gene encoding CH or CL of a human antibody in the vector for expression of a humanized antibody described in (1) of item 2, and expressed in a suitable form.

(7) Transient expression of recombinant antibodies

In order to efficiently evaluate the antigen binding activity of the various humanized antibodies produced, transient expression of the recombinant antibody can be carried out using the recombinant antibody expression vectors described in (3) and (6) of item 2 or modified expression vectors. As the host cell for introducing the expression vector, any cell can be used as long as it can express the recombinant antibody.

Among them, COS-7 cells (ATCCRL 1651) [ Methods in Nucleic Acids Res., CRC press,283(1991) ] are generally preferred from the viewpoint of high expression level.

Examples of the method for introducing the expression vector into COS-7 cells include: DEAE-dextran method [ Methods in Nucleic Acids Res., CRC Press,283(1991) ], Lipofectin method [ Proc. Natl. Acad. Sci. USA,84,7413(1987) ], and the like.

After the introduction of the expression vector, the expression amount and the antigen-binding activity of the recombinant antibody in the culture supernatant can be measured by an enzyme-linked immunosorbent assay [ hereinafter referred to as ELISA; MonoclonaLantibodies-Principles and practice, third edition, academic Press USA (1996); Antibodies-A Laboratory Manual, Cold spring harbor Laboratory (1988); クロ experiments performed in experiments on ン antibody were performed in マニユアル, Co., Ltd, サイエンテイフイツク (1987), and the like.

(8) Stable expression of recombinant antibodies

A transformant stably expressing the recombinant antibody can be obtained by introducing the recombinant antibody expression vector described in (3) or (6) of item 2 into an appropriate host cell.

Examples of methods for introducing an expression vector into a host cell include electroporation methods [ Japanese patent application laid-open No. 2-257891; cytotechnology,3,133(1990), etc.

As the host cell for introducing the expression vector of the recombinant antibody, any cell can be used as long as it can express the recombinant antibody.

Examples thereof include: mouse SP2/0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), two Chinese hamster ovary cells CHO/dhFr-cells (ATCC CRL9096) and CHO/DG44 cells [ colloidal Cell and Molecular Genetics,12,555(1986) ], lectin-resistant Lec13[ colloidal and Cell Molecular Genetics,12,55(1986) ], alpha 1, 6-fucosyltransferase gene-deficient CHO cells (International publication No. 05/35586, International publication No. 02/31140), and rat YB2/3HL.P2.G 11.16Ag.20 cells (ATCC CRL 1662), and the like.

In addition to the above-mentioned host cells, host cells in which the activity of a protein such as an enzyme involved in the synthesis of intracellular ribonucleotide GDP-fucose, a protein involved in the modification of a sugar chain in which the 1-position of fucose is α -linked to the 6-position of N-acetylglucosamine at the reducing end of an N-glycosidically linked complex sugar chain, a protein involved in the transport of intracellular ribonucleotide GDP-fucose to the golgi, or the like is reduced or deleted may be used, and CHO cells in which the α 1, 6-fucosyltransferase gene described in international publication No. 05/35586, international publication No. 02/31140, or the like is deleted, or the like may be preferably used.

After introduction of the expression vector, a transformant which stably expresses the recombinant antibody can be selected by culturing the vector in a medium for animal cell culture containing a drug such as G418 sulfate (hereinafter referred to as G418, manufactured by SIGMA) according to the method disclosed in Japanese patent application laid-open No. 2-257891.

As the culture medium for animal cell culture, there can be used: RPMI1640 medium (manufactured by Invitrogen corporation), GIT medium (manufactured by Nippon pharmaceutical Co., Ltd.), EX-CELL301 medium (manufactured by JRH Co., Ltd.), IMDM medium (manufactured by Invitrogen corporation), hybridoma SFM medium (manufactured by Invitrogen corporation), and a medium containing various additives such as fetal calf serum (hereinafter, FCS) added to the above medium.

By culturing the obtained transformant in a medium, the recombinant antibody can be expressed and accumulated in the culture supernatant. The expression level and antigen-binding activity of the recombinant antibody in the culture supernatant can be measured by ELISA method or the like. Furthermore, the expression level of the recombinant antibody of the transformant can be increased by the method disclosed in Japanese patent application laid-open No. 2-257891 using a DHFR amplification system or the like.

The recombinant antibody can be purified from the culture supernatant of the transformant using a protein A column [ Monoclonal Antibodies-Principles and practice, third edition, American academic Press (1996); antibodies-organic Manual, Cold spring harbor laboratory (1988) ].

In addition, a purification method generally used for protein purification may be used. For example, purification can be performed by combining gel filtration, ion exchange chromatography, ultrafiltration, and the like.

The molecular weight of the H chain, L chain or whole antibody molecule of the purified recombinant antibody can be determined by polyacrylamide gel electrophoresis [ hereinafter referred to as SDS-PAGE; nature,227,680(1970) and Western immunoblotting [ Monoclonal Antibodies-Principles and dpracts, third edition, academic Press USA (1996); Antibodies-A laboratory Manual, Cold spring harbor laboratory (1988), and the like.

3. Evaluation of Activity of antibody or antibody fragment of the present invention

The reaction specificity of the purified antibody or antibody fragment of the present invention can be evaluated as follows.

A CD 27-expressing cell expressing a nucleotide sequence (SEQ ID NO: 1) encoding CD27 can be prepared by using, as a host, a cell line in which activities of an enzyme that adds Gal to GalNAc bound to Ser/Thr on a polypeptide in the O-linked sugar chain synthesis process, a protein involved in the activity of the enzyme, a protein involved in the transport of uridine 5' -diphosphate galactose (UDP-galactose), and the like are reduced or deleted.

Thus, cells expressing CD27 having normal O-linked sugar chains and cells expressing CD27 deficient in sugar chains can be prepared, and the reactivity of the cell line expressing each CD27 with purified antibodies can be measured by ELISA method and fluorescent antibody method [ cancer immunol.

In addition, soluble CD27 proteins with a three-dimensional structure can be prepared by expressing the extracellular domain of CD27 in the form of a soluble substance such as a fusion protein in the above host cells and purifying the expressed extracellular domain under appropriate conditions.

As the fusion protein, there can be mentioned: a CD27 protein fused to another polypeptide such as an antibody constant region (also referred to as Fc), GST tag, histidine tag (also referred to as His tag), or Myc tag. The fusion protein can be isolated and purified by using an affinity chromatography column such as protein a, a nickel column, or a specific antibody column.

It is also possible to use BIAcore by using Surface Plasmon Resonance (SPR)^TMELISA, immunoprecipitation, etc. to determine the reactivity of these purified soluble CD27 proteins with purified Antibodies [ Monoclonal Antibodies-Principles and practice, third edition, U.S. academic Press (1996); Antibodies-A Laboratory Manual, Cold spring harbor Laboratory (1988) ]。

The cytotoxic activity against a cultured cell line expressing a sugar chain-deficient CD27 can be evaluated by measuring CDC activity, ADCC activity, and the like using a known method [ Cancer immunol.

4. Method for diagnosing disease using monoclonal antibody or antibody fragment thereof of the present invention that specifically recognizes sugar chain-deficient CD27 and binds to the extracellular domain

By detecting or quantifying the sugar chain-deficient CD27 or cells expressing the polypeptide using the antibody or the antibody fragment of the present invention, a disease associated with the sugar chain-deficient CD27 can be diagnosed.

Any disease associated with the sugar chain-deficient CD27 is included as long as cells expressing the sugar chain-deficient CD27 polypeptide are found in vivo. Specifically, IgA nephropathy or cancer is exemplified. Examples of the cancer include cancers derived from the differentiation process of B cells or T cells.

Specifically, examples thereof include: and various non-hodgkin lymphomas such as mantle cell lymphoma, chronic lymphocytic leukemia, small lymphocytic leukemia, burkitt (バ - キツト) lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, diffuse large cell lymphoma, and plasmacytoma.

In the present invention, the biological sample to be detected or measured for the sugar chain-deficient CD27 polypeptide is not particularly limited as long as it is a sample that may contain the polypeptide, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, and culture fluid.

Among diseases related to the sugar chain-deficient CD27, for example, the diagnosis of IgA nephropathy can be performed as follows.

The antibody, the antibody fragment or the derivative thereof of the present invention is used for detecting or measuring a sugar chain-deficient CD27 polypeptide in a biological sample collected from the body of a plurality of healthy persons by the following immunological method, thereby confirming the expression level of the polypeptide in the biological sample of a healthy person.

The expression level of the polypeptide is also examined in a biological sample of a subject, and the expression level is compared with that of a healthy person. When the expression level of the polypeptide in a subject is increased as compared with that in a healthy person, the possibility of diagnosing IgA nephropathy or future IgA nephropathy is high.

In the case of a disease associated with sugar chain-deficient CD27, for example, diagnosis of cancer can be performed as follows.

The expression level of the polypeptide in a biological sample of a healthy person is confirmed by detecting or measuring a sugar chain-deficient CD27 using the antibody of the present invention, the antibody fragment thereof, or a derivative thereof according to the following immunological method.

The expression level of the polypeptide is also examined in a biological sample of a subject, and the expression level is compared with that of a healthy person. In the case where the expression level of the polypeptide in a subject is increased as compared with that in a healthy person, the cancer can be diagnosed as positive.

The diagnostic agent comprising the antibody of the present invention, the antibody fragment or a derivative thereof may contain a reagent for carrying out an antigen-antibody reaction or a reagent for detecting the reaction, depending on the target diagnostic method. Examples of the reagent for carrying out an antigen-antibody reaction include: buffers and salts, and the like.

Examples of the detection reagent include: a labeled secondary antibody that recognizes the antibody or the antibody fragment or a derivative of the derivative, a substrate for a labeled substance, and the like.

In the present invention, any known method can be used to detect or measure the amount of sugar chain-deficient CD 27. Examples thereof include immunological detection and measurement methods.

The immunological detection or measurement method is a method of detecting or measuring the amount of an antibody or the amount of an antigen using an antigen or an antibody subjected to labeling. Examples of immunological detection or measurement methods include: radioimmunoassay (RIA), enzyme immunoassay (EIA or ELISA), Fluorescence Immunoassay (FIA), luminescence immunoassay (luminescence immunoassay), western immunoblotting, and physicochemical methods (e.g., TIA, LAPIA, PCIA, etc.), etc.

Examples of Radioimmunoassay (RIA) include the following methods: the antibody or the antibody fragment of the present invention is reacted with an antigen or a cell expressing an antigen, or the like, and further, an anti-immunoglobulin antibody or a binding fragment labeled with an irradiation beam is reacted, and then, the reaction is measured by a scintillation counter or the like.

Examples of the enzyme immunoassay method (EIA or ELISA) include the following methods: reacting the antibody or the antibody fragment of the present invention with an antigen or a cell expressing an antigen, further reacting the labeled anti-immunoglobulin antibody or the binding fragment, and then measuring a luminescent dye by an absorptiometer; for example, sandwich ELISA method or the like can be used.

As the label used in the enzyme immunoassay, any of the known enzyme labels (Shichuanrong et al, enzyme immunoassay, medical Booth) as described above can be used. For example, it is possible to use: alkaline phosphatase label, peroxidase label, luciferase label, biotin label, and the like.

The sandwich ELISA method is as follows: after the antibody is bound to the solid phase, it is allowed to capture the antigen to be detected or measured, and the second antibody is allowed to react with the captured antigen. In this ELISA method, two antibodies or antibody fragments that recognize antigens to be detected or measured are prepared, and either one of the antibodies or antibody fragments is adsorbed to a plate (for example, a 96-well plate) in advance, and the second antibody or antibody fragment is labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, biotin, or the like in advance.

On the plate on which the above-mentioned antibody is adsorbed, cells or a disrupted solution thereof, tissues or a disrupted solution thereof, cell culture supernatant, serum, pleural effusion, ascites, ocular fluid, and the like separated from the living body are reacted, and then the labeled monoclonal antibody or antibody fragment is reacted to carry out a detection reaction corresponding to the labeled substance.

When the antigen concentration in the test sample is measured by the above-described method, the antigen concentration in the test sample can be calculated from a calibration curve prepared by gradient-diluting an antigen whose concentration is known.

As the antibody used in the sandwich ELISA method, for example, any of polyclonal antibodies and monoclonal antibodies, Fab ', and F (ab')₂And the like. The combination of two antibodies used in the sandwich ELISA method may be a combination of monoclonal antibodies or antibody fragments recognizing different epitopes, or a combination of polyclonal antibodies and monoclonal antibodies or antibody fragments.

Examples of the fluorescence immunoassay method (FIA) include the literature [ MonoclonaAntibodies-Principles and practice, third edition, American academy of academic Press (1996); クロ implementation of the protocol for experiments on ン antibody was performed in experiments マニユアル, Co., Ltd, サイエンテイフイツク (1987), etc.

As the label used in the fluorescence immunoassay method, any known fluorescent label (the fluorescent antibody method, ソフトサイエンス, inc.) as described above can be used. For example, FITC labeling, RITC labeling, and the like can be used.

As the luminescence immunoassay method (luminescence immunoassay), for example, [ Monoclonal Antibodies-Principles and practice, third edition, American academic Press (1996); クロ implementation of the protocol for experiments on ン antibody was performed in experiments マニユアル, Co., Ltd, サイエンテイフイツク (1987), etc.

Examples of the label used in the luminescence immunoassay method include any of those known as described above [ Jinjing-Yangtze code, bioluminescence and chemiluminescence, Guangchuan bookshop; clinical examination 42(1998)]Is labeled with a light emitting substance of (1). For example, acridine can be usedEster labeling and loxphenol base labelingAnd recording and the like.

The western blotting method is as follows: the antigen or the cells expressing the antigen is confirmed by separating the fractions by SDS-polyacrylamide gel electrophoresis [ Antibodies-A Laboratory Manual (Cold spring harbor Laboratory, 1988) ], blotting the gel onto a PVDF membrane or a nitrocellulose membrane, reacting the antibody or antibody fragment recognizing the antigen with the membrane, further reacting the anti-mouse IgG antibody or binding fragment labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, a biotin, or the like, and visualizing the label. An example of western blotting is shown below.

Cells or tissues expressing a polypeptide having an amino acid sequence represented by SEQ ID No. 2 were lysed, and the amount of protein per lane was adjusted to 0.1 to 30. mu.g under reducing conditions, followed by electrophoresis by SDS-PAGE. The electrophoresed proteins were transferred to a PVDF membrane, and reacted in PBS containing 1% BSA (hereinafter referred to as BSA-PBS) at room temperature for 30 minutes to carry out blocking operation.

Here, the monoclonal antibody of the present invention was reacted, washed with PBS containing 0.05% tween-20 (hereinafter, referred to as tween-PBS), and peroxidase-labeled goat anti-mouse IgG was reacted at room temperature for 2 hours. The band to which the monoclonal antibody has been bound is washed with tween-PBS, and detected using an ECLTM western blot detection reagent (manufactured by Amersham) or the like, whereby a polypeptide having the amino acid sequence represented by sequence No. 2 can be detected.

As an antibody used for detection by western blotting, an antibody capable of binding to a polypeptide that does not retain a native steric structure can be used.

The physicochemical method may be specifically carried out as follows: the antibody or the antibody fragment of the present invention is used to bind CD27 binding to a galactose-deficient O-type sugar chain as an antigen to the antibody or the antibody fragment of the present invention, thereby forming an aggregate, and the aggregate is detected.

In addition, examples of the physicochemical method include: capillary method, one-dimensional immunodiffusion method, immunoturbidimetry, and latex immunoturbidimetry [ outline of clinical examination, gold-source publication, 499(1998) ].

For example, in the latex immunoturbidimetry, a carrier such as polystyrene latex having a particle size of about 0.1 μm to about 1 μm sensitized with an antibody or an antigen is used, and when an antigen-antibody reaction is caused by the corresponding antigen or antibody, scattered light in the reaction solution increases and transmitted light decreases. The change is detected as absorbance or integrating sphere turbidity, whereby the antigen concentration or the like in the test sample can be measured.

Since the antibody or the antibody fragment of the present invention can bind to the extracellular domain of the sugar chain-deficient CD27 polypeptide, it can be suitably used for detecting cells expressing the polypeptide. For detection of cells expressing the polypeptide, a known immunological detection method may be used, and immunoprecipitation, immunocytostaining, immunohistostaining, and the like are preferably used. In addition, a fluorescent antibody staining method using FMAT8100HTS system (manufactured by applied biosystems, usa) and the like may be applied.

The immunoprecipitation method refers to the following method: cells expressing the polypeptide or the like are reacted with the monoclonal antibody or antibody fragment of the present invention, and then a carrier having a specific binding ability to immunoglobulin, such as protein G-agarose, is added to precipitate an antigen-antibody complex. Alternatively, the method may be performed as follows.

The antibody or the antibody fragment of the present invention is immobilized on a 96-well plate for ELISA, and then blocked with BSA-PBS. When the antibody is in an unpurified state, for example, an unpurified state such as a hybridoma culture supernatant, an anti-mouse immunoglobulin, a rat immunoglobulin, protein a or G, or the like is immobilized in advance on a 96-well plate for ELISA, and after blocking with BSA-PBS, the hybridoma culture supernatant is dispensed and bound.

BSA-PBS was discarded, and after washing with PBS sufficiently, a cell or tissue lysate expressing a polypeptide having an amino acid sequence represented by SEQ ID NO. 2 was reacted. Immunoprecipitates were extracted from the well-washed plates with sample buffer for SDS-PAGE and detected by Western blotting as described above.

The immunocytostaining method and the immunohistological staining method refer to a fluorescent antibody staining method as follows: cells or tissues expressing an antigen are treated with a surfactant, methanol, or the like as necessary to increase the permeability of the antibody, the antibody of the present invention is reacted, an anti-immunoglobulin antibody or a binding fragment, which is labeled with a fluorescent label such as FITC, an enzyme label such as peroxidase, a biotin label, or the like, is reacted, and the label is visualized and observed microscopically, or the cells are reacted with a fluorescent-labeled antibody and analyzed by a flow cytometer (flow cytometry).

For example, the literature [ monoconal Antibodies-Principles and practice, third edition, academic Press of America (1996); クロ implementation of the protocol for experiments on ン antibody was performed in experiments マニユアル, Co., Ltd, サイエンライフイツク (1987), etc.

In particular, since the antibody or the antibody fragment of the present invention can bind to the extracellular domain of the sugar chain-deficient CD27, it can be preferably used for the analysis by flow cytometry for detecting CD27 expressed on the cell membrane while maintaining the natural type steric structure.

In addition, by using the FMAT8100HTS system (manufactured by applied biosystems, usa) using the principle of fluorescent antibody staining, the amount of antigen or the amount of antibody can be measured without separating the formed antibody-antigen complex from free antibody or antigen that does not participate in the formation of the antibody-antigen complex.

5. Method for treating diseases using the monoclonal antibody or the antibody fragment of the present invention that specifically recognizes and binds to a sugar chain-deficient CD27 polypeptide

The monoclonal antibody or antibody fragment thereof of the present invention, which specifically recognizes the sugar chain-deficient CD27 polypeptide and binds to the extracellular domain, can be used for the treatment of diseases associated with the sugar chain-deficient CD27 polypeptide.

The disease related to the sugar chain-deficient CD27 polypeptide may be any disease as long as a cell expressing the polypeptide is found in vivo, and examples thereof include IgA nephropathy, cancer, and the like.

In addition, as the disease, a disease which has IgA nephropathy and shows nephrotic syndrome and a disease which shows renal failure can be cited.

Examples of the cancer include a tumor of hematopoietic origin (also referred to as hematological cancer) and a solid cancer of epithelial origin.

Specific examples of the hematological cancer include: leukemia and lymphoma (hodgkin's lymphoma, non-hodgkin's lymphoma), multiple myeloma, and the like.

Specific examples of non-hodgkin's lymphoma include: mantle cell lymphoma, chronic lymphocytic leukemia, small lymphocytic leukemia, burkitt's lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, diffuse large cell lymphoma, and plasmacytoma.

Specific examples of the solid cancer include: breast cancer, uterine cancer, colorectal cancer, gastric cancer, ovarian cancer, lung cancer, renal cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer, pancreatic cancer, and the like.

The therapeutic agent of the present invention includes a cancer therapeutic agent containing the antibody or the antibody fragment of the present invention as an active ingredient. The therapeutic agents of the present invention include cancer therapeutic agents having an effect activity such as ADCC activity and CDC activity, cancer therapeutic agents utilizing apoptosis induction, and the like.

The antibody or the antibody fragment of the present invention is capable of recognizing a sugar chain-deficient CD27 polypeptide expressed on the cell membrane, and therefore, is capable of recognizing cells expressing a sugar chain-deficient CD27 polypeptide present in the body.

Therefore, the antibody or the antibody fragment having an effector activity as the antibody or the antibody fragment of the present invention can kill cells expressing the sugar chain-deficient CD27 polypeptide in vivo and in vitro.

In addition, the above-described antibody or the antibody fragment of the present invention can kill cells expressing the sugar chain-deficient CD27 polypeptide in vivo to reduce them, and therefore, can be used particularly effectively as a therapeutic agent.

The therapeutic agent containing the antibody or the antibody fragment or the derivative thereof of the present invention may be provided in the form of a pharmaceutical preparation containing the antibody or the antibody fragment or the derivative thereof as an active ingredient alone, and usually, preferably, in the form of a pharmaceutical preparation prepared by mixing the antibody or the antibody fragment or the derivative thereof with one or more pharmacologically acceptable carriers and producing the mixture by any method known in the art of pharmaceutical technology.

The route of administration is preferably the most effective route when using therapy, and can be listed as: oral administration or non-oral administration such as oral, intratracheal, intrarectal, subcutaneous, intramuscular, and intravenous. In the case of antibody or peptide preparations, intravenous administration may be preferably cited.

As administration forms, mention may be made, for example, of: spray, capsule, tablet, granule, syrup, emulsion, suppository, injection, ointment, and adhesive tape.

Examples of formulations suitable for oral administration include: emulsion, syrup, capsule, tablet, powder, granule, etc. Liquid preparations such as emulsions and syrups can be manufactured using as additives: saccharides such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and polypropylene glycol, oils such as sesame oil, olive oil and soybean oil, preservatives such as parabens, flavors such as strawberry flavor and mint.

Capsules, tablets, powders, granules and the like can be produced using the following ingredients as additives: excipients such as lactose, glucose, sucrose and mannitol, disintegrating agents such as starch and sodium alginate, lubricants such as magnesium stearate and talc, binders such as polyvinyl alcohol, hydroxypropyl cellulose and gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerol.

Examples of formulations suitable for parenteral administration include: injection, suppository, spray, etc. Injections are prepared using carriers comprising sodium chloride solution, glucose solution, and a mixture of both, and the like. Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids. The spray is prepared using the antibody or antibody fragment itself, a carrier that does not irritate the oral and tracheal mucosa of the recipient and that disperses the antibody or antibody fragment into fine particles for easy absorption, and the like.

Specific examples of the carrier include lactose and glycerin. Depending on the nature of the antibody or antibody fragment and the carrier used, formulations such as aerosols and dry powders may be used. Further, to these non-oral preparations, components exemplified as additives for oral preparations may be added.

The amount or frequency of administration varies depending on the target therapeutic effect, administration method, treatment time, age, body weight, etc., and is usually 10. mu.g/kg to 8mg/kg per day for an adult.

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the following examples.

Examples

Example 1 preparation of soluble CD27 ectodomain containing O-linked galactose-free sugar chains (hereinafter sometimes referred to as sugar chain-deficient CD27)

(1) Cloning of the human CD27 Gene

The gene encoding CD27 was isolated from a cDNA library derived from human peripheral blood purchased from クロ ン テ ツ ク according to the following procedure. In a reaction solution containing 1-fold concentration of BD Advantage PCR buffer (クロ ン テ ツ ク Co.) and 1-fold concentration of attached dNTP, 25ng of human peripheral blood mononuclear cell-derived single-stranded cDNA, 0.2. mu.M cd27fw (SEQ ID NO: 3), 0.2. mu.M cd27809B (SEQ ID NO: 4) and 1-fold concentration of Advantage 2PCR polymerase mixture (クロ ン テ ツ ク Co.) were used, and the total volume was adjusted to 50. mu.L to perform PCR.

The reaction conditions are as follows: the cycle was 1 at 98 ℃ for 15 seconds and 68 ℃ for 30 seconds, and the cycle was performed for 30 times. The reaction mixture was separated by 2% agarose gel electrophoresis, and then the PCR product of about 1kbp was inserted into the pCR-2.1 vector using TOPO TA cloning kit (manufactured by Invitrogen corporation) according to the instructions attached thereto. Coli was transformed with a plasmid containing the PCR-amplified fragment, and plasmids obtained from the respective clones were prepared and the DNA sequence was confirmed, whereby PCR 2.1CD27 (fig. 1) having the DNA sequence shown in sequence No. 1 was obtained.

(2) Construction of plasmid cloned with Gene having human CD27 ectodomain

Subsequently, by performing PCR as described below, cDNA encoding CD27 was isolated after removal of the transmembrane region present at the C-terminal side. In a reaction solution containing 0.2 mmol/L dNTPs and 1 mmol/L magnesium chloride, 1ng of pCR 2.1CD27, 1. mu.mol/L CD27-A (SEQ ID NO: 5), 1. mu.mol/L CD27-B (SEQ ID NO: 6) and 2.5 units of KOD polymerase (manufactured by Toyobo Co.) were used, and the total volume was adjusted to 50. mu.L, and PCR was carried out under the following conditions: the cycle was set to 1 cycle at 98 ℃ for 15 seconds and 68 ℃ for 30 seconds, and the cycle was set to 25 cycles.

The reaction solution was separated by 2% agarose gel electrophoresis, and then a PCR product of about 600bp was introduced into the pCR-Blunt vector using a ZeroBlunt PCR cloning kit (Invitrogen corporation) according to the instructions attached thereto. The plasmid in which the gene having the extracellular domain of human CD27 was cloned was designated pCRCD27axb (FIG. 2).

(3) Construction of vector pBShC gamma 4SP having mutant human IgG4Fc fragment

A plasmid pBShC γ 4SP having a C region of mutant human IgG4 subclass in which Ser at position 108 of the C region (hinge region) of wild-type human IgG4 subclass is substituted with Pro was constructed using the plasmid pBShC γ 4 having cDNA encoding the C region of wild-type human IgG4 subclass described in international publication No. 97/10354. It is known that this modification stabilizes dimer formation by the IgG hinge region (molecular immunology, 30,105,1993).

A50. mu.L reaction solution containing 1ng of plasmid pBShC.gamma.4 as a template [10mM Tris-HCl (pH8.3), 50mM potassium chloride, 1.5mM magnesium chloride, 0.001% gelatin, 200. mu.M dNTPs, 0.5. mu.M primer 1 (SEQ ID NO: 7), 0.5. mu.M primer 2 (SEQ ID NO: 8), and 2 units of TaKaRa Ex Taq DNA polymerase ] was prepared, and 30 cycles were carried out using GeneAmp PCR System 9700 (manufactured by パ - キン エルマ Co.) at 1 cycle of 2 minutes at 94 ℃, 2 minutes at 55 ℃ and 2 minutes at 72 ℃.

The reaction solution was purified using a QIAquick PCR purification kit (manufactured by キアゲン) according to the instructions attached thereto, treated with the restriction enzyme EcoT14I (manufactured by タカラバイオ), separated by 0.8% agarose gel electrophoresis, and then the amplified fragment was recovered using a QIAquick gel extraction kit (manufactured by キアゲン) according to the instructions attached thereto.

The plasmid pBShC.gamma.4 was cleaved with the restriction enzyme EcoT14I, treated with alkaline phosphatase (タカラバイオ Co.) to remove the 5' -terminal phosphate, separated by 0.8% agarose gel electrophoresis in the same manner, and then the plasmid fragment was recovered using QIAquick gel extraction kit according to the instructions attached thereto. The recovered amplified fragment was ligated with a plasmid fragment derived from plasmid pBShC γ 4 to construct plasmid pBShC γ 4SP containing a cDNA of interest (FIG. 3).

(4) Cloning of cDNA comprising partial sequence of human IgG4Fc

A DNA fragment encoding human IgG4Fc, which had a restriction enzyme BamHI site at the 5 'end and a SalI restriction enzyme at the 3' end, was amplified by performing the PCR reaction described below. PCR was carried out using 25ng of pBShC γ 4SP constructed in (3) above, 1. mu. mol/L g4A (SEQ ID NO: 9), 1. mu. mol/L g4B (SEQ ID NO: 10), and 2.5 units of KOD polymerase (manufactured by Toyo Co.) in a reaction solution containing 0.2 mmol/L dNTPs and 1 mmol/L magnesium chloride, to adjust the total volume to 50. mu.L.

The reaction conditions are as follows: the cycle was set to 1 cycle at 98 ℃ for 15 seconds and 68 ℃ for 30 seconds, and the cycle was set to 25 cycles. The reaction solution was separated by 2% agarose gel electrophoresis, and then a PCR product of about 700bp was introduced into a pCR-Blunt vector using a Zero Blunt PCR cloning kit (manufactured by Invitrogen corporation) according to the attached instructions. This plasmid was designated pCRIgG4FcBamHISalI (FIG. 4).

(5) Construction of expression vector pKANTEX XhoI/SalI for animal cells

The humanized antibody expression vector pKANTEX93 described in International publication No. 97/10354 was digested with restriction enzyme XhoI (タカラバイオ) and restriction enzyme SalI (タカラバイオ), separated by 0.8% agarose gel electrophoresis, and a plasmid fragment of about 9.8kbp was recovered using a gel extraction kit (キアゲン). The 5 '-end and the 3' -end of the recovered DNA fragment were ligated using a DNA ligation kit (manufactured by タカラバイオ Co.), and E.coli DH 5. alpha. strain (manufactured by Toyo Boseki Co.) was transformed using the resulting recombinant plasmid DNA.

Recombinant plasmid DNA was isolated from the obtained plurality of ampicillin-resistant colonies using QIAprep Spin miniprep kit (manufactured by キアゲン Co.), and digestion was performed with restriction enzymes NotI (manufactured by タカラバイオ Co.) and KpnI (manufactured by タカラバイオ Co.) to confirm that the expression unit of the antibody L chain was removed. This plasmid was designated pKANTEX XhoI/SalI (FIG. 5).

(6) Preparation of soluble CD27 ectodomain expression plasmid pKANTEX CD27IgG4Fc

A plasmid pKANTEXCD27IgG4Fc (FIG. 6) for expressing CD27-Fc was obtained by ligating a fragment of about 600bp obtained by digesting pCR 2.1CD27axb prepared in (2) above with NotI and BamHI, a DNA fragment of about 700bp obtained by digesting pCRIgG4FcBamHI SalI prepared in (3) above with BamHI and SalI, and a DNA fragment of about 8.8kbp obtained by digesting pKANTEX XhoI/SalI prepared in (5) above with NotI and SalI.

The gene sequence of the soluble CD27-Fc fusion protein (hereinafter sometimes referred to as CD27-Fc) encoded by the above plasmid is referred to as SEQ ID No. 11, and the amino acid sequence of the soluble CD27-Fc fusion protein is referred to as SEQ ID No. 12. Coli transformed with the expression vector was inoculated into 100mL of LB medium, cultured overnight, and then recovered, and the Plasmid was purified according to the protocol attached thereto using a Qiafilter Plasmid median purification kit (Qiafilter Plasmid midi kit) (manufactured by キアゲン).

After purification, 30. mu.g of the plasmid vector was digested with the restriction enzyme AatII, thereby linearizing it. After linearization, phenol/chloroform extraction and ethanol precipitation were performed, and the DNA was dissolved in 1/10 concentration of TE buffer (1mM Tris HCl,0.1mM EDTA), and the DNA concentration was measured and used for gene transfer.

(7) Expression of CD27-Fc

The introduction of the CD27-Fc expression plasmid pKANTEX CD27IgG4Fc into CHO/DG44 cells (physical Cell and Molecular Genetics,12,555 (1986); hereinafter referred to as DG44) or Lec8 cells can be carried out according to the following procedure, following electroporation [ サイトテクノロジ I, 3,133(1990) ].

First, Iscove's Modified Dulbecco's Medium (Invitro corporation) supplemented with 10% fetal bovine dialysis serum (Invitro corporation), 50. mu.g/mL gentamicin (ナカライテスク corporation) and 1 XHT additive (Invitro corporation) was added to a basal medium]DG44 cells from passage No. [137 mmol/L KCl, 2.7 mmol/L NaCl, 8.1 mmol/L Na ] were suspended in K-PBS buffer₂HPO₄1.5 mmol/l KH₂PO₄4.0 mmole/l MgCl₂]In order to reach 8 × 10⁶Cell suspension was prepared per mL.

200. mu.L (1.8X 10) ⁶One) the prepared cell suspension was mixed with 10. mu.g of the linearized plasmid pKANTEXCD27IgG4Fc prepared in (6) above. However, the passage of Lec8 cells was performed in basal medium (hereinafter referred to as HT-medium) without 1 XHT additive.

The cell DNA mixture was transferred to a Gene Pulser cuvette (distance between electrodes: 2mm) (BIO-RAD Co.), and then Gene transfer was carried out using a Gene transfer apparatus Genepulser (BIO-RAD Co.) under conditions of a pulse voltage of 0.35KV and a capacitance of 250. mu.F.

The cell suspension was mixed with 10mL of HT-medium [ Elcisco modified Dulbecco's medium (Invitrogen Co., Ltd.) supplemented with 10% fetal bovine dialysis serum (Invitrogen Co., Ltd.) and 50. mu.g/mL gentamicin (ナカライテスク Co., Ltd.) ]]Inoculating to 75cm²In a flask for adherent cells (manufactured by Gelina), 5% CO was added at 37 ℃₂The incubator of (2) for cultivation. After 3 days of culture, G418 (manufactured by Sigma) was added to the culture to a final concentration of 0.5mg/mL, and the culture was continued for 10 days.

After 10 days, passage was carried out to 182cm²The adherent cells of (1) were cultured in a flask for adherent cells (manufactured by Gerania) until they were confluent. When the medium reached confluency, the medium was replaced with a serum-free medium EXCELL301 (manufactured by JRH Bioscience Co., Ltd.), and after one week of culture, the culture supernatant was collected and purified by the following method.

(8) Purification of CD27-Fc

The culture supernatant obtained by the culture in (7) was centrifuged at 3000rpm and 4 ℃ for 10 minutes, and the supernatant was collected and then filtered using a PES membrane (manufactured by Asahi テクノグラス) having a pore size of 0.22. mu.m. 0.5mL of Mab Select (Amersham Pharmacia Biotech) was packed into a column having an inner diameter of 0.8cm, and 3.0mL of purified water and 3.0mL of 0.2M boric acid-0.15M NaCl buffer (pH7.5) (hereinafter referred to as boric acid buffer) were sequentially passed through the column.

Further, the carrier was equilibrated by washing with 2.0mL of 0.1M citric acid buffer (pH3.5) and 1.5mL of boric acid buffer in this order. Subsequently, the culture supernatant was passed through a column, and then washed with 3.0mL of a boric acid buffer. After washing, the antibody adsorbed on the carrier was eluted using 1.25mL of 0.1M citric acid buffer (pH 3.5).

Upon elution, 5 fractions of 250. mu.L each were obtained. Subsequently, the resulting purified fractions were subjected to SDS-PAGE analysis, and fractions in which the target protein was confirmed to elute were pooled and dialyzed against PBS buffer at 4 ℃ for one day and night.

After dialysis, the CD27-Fc solution was recovered, sterile-filtered using Millex GV (Millipop) having a pore size of 0.22 μ M, and then absorbance at 280nm (OD280nm) was measured using an absorptiometer (SHIMADZU UV-1700), and the concentration of CD27-Fc was calculated (OD280nm =1.0, converted to 0.68 mg/mL; calculated from ε M =134655, MW = 92840).

3.6mg of CD27-Fc derived from Lec8 and 2.2mg of CD27-Fc derived from CHO/DG44 were obtained from approximately 300mL of serum-free culture supernatant of CD27-Fc expressing cells derived from each host cell. The results of SDS-PAGE analysis of the eluted fractions of each protein are shown in FIG. 7.

As a result, under reducing conditions, CD27-Fc was observed at a position having a molecular weight of about 65kDa in the case of DG44 as a host, and CD27-Fc was observed at a position having a molecular weight of about 48kDa in the case of Lec8 as a host. On the other hand, under non-reducing conditions, the respective bands were observed at positions having a molecular weight about 2 times that of the band observed under reducing conditions, and thus it could be confirmed that CD27-Fc exists as a dimer.

(9) Confirmation of sugar chain Structure

To 16. mu.L of the solution obtained by diluting CD27-Fc purified in (8) above to 10 times with PBS, 4. mu.L of SDS sample buffer at 5 times was added, and the mixture was subjected to SDS-PAGE at 90 ℃ for 5 minutes. For electrophoresis, 5 to 20% SDS-polyacrylamide gel E-PAGEL (manufactured by ATTO Co., Ltd., catalog No. E-T520L) was used, and electrophoresis was carried out for 90 minutes at a current of 20 mA/piece in an ATTO ラピダス. ミニスラブ electrophoresis tank.

The transfer onto the PVDF membrane (manufactured by Millipore Immobilon, catalog No. IPVH304F0) was carried out under 180mA for 90 minutes using ATTO ホライズブロツト. The transferred membrane was immersed in PBS containing 10% BSA (hereinafter referred to as 10% BSA-PBS), and left overnight at 4 ℃.

Then, anti-RCAS 1 antibody clone 22-1-1 (manufactured by MBL Co., Ltd., catalog No. D060-3) prepared to 5. mu.g/mL using 1% BSA-PBS was added thereto, and the reaction was carried out at room temperature for 2 hours. After washing with 0.05% Tween-20-PBS (Wako pure chemical Co., Ltd., catalog No. 167-11515) at room temperature for 30 minutes, the rabbit anti-mouse immunoglobulin (DAKO Co., Ltd., catalog No. P0161) labeled with secondary antibody peroxidase after diluted 2000-fold with 1% BSA-PBS was reacted at room temperature for 1 hour.

After washing with 0.05% Tween-20-PBS at room temperature for 30 minutes, detection was carried out using ECL western blotting detection reagent (Amersham Pharmacia Biotech, Cat. No. RPN 2106). The results are shown in fig. 8.

As is clear from the results of SDS-PAGE analysis, the molecular weight of CD27-Fc derived from DG44 was higher than that of CD27-Fc derived from Lec8, and the structures of sugar chains binding to CD27-Fc were made different by using DG44 cells and Lec8 cells as host cells.

In addition, it was clarified that the anti-RCAS 1 antibody 22-1-1 recognizes a Tn antigen as an O-linked sugar chain, which is known as an anti-Tn antibody [ J.B.C.,278,22998-23007, (2003) ].

The anti-RCAS-1 antibody clone 22-1-1 as an anti-Tn antibody did not bind to CD27-Fc derived from DG44 at all, but specifically bound to CD27-Fc derived from Lec 8. It was confirmed that Tn antigen as an O-type sugar chain to which galactose was not bound was bound to CD27-Fc produced by Lec 8.

[ example 2]

Preparation of CHO cells expressing CD27 on cell Membrane

(1) Construction of CD27 expression plasmid pKANTEX CD27

A cDNA fragment obtained by removing a cDNA portion unnecessary for gene expression was prepared from pCR 2.1CD27 prepared in example 1 by the PCR reaction described below.

PCR was carried out using 1ng of pCR 2.1CD27, 1. mu.M of CD27-A (SEQ ID NO: 5), 1. mu.M of CD27-C (SEQ ID NO: 13) and 2.5 units of KOD polymerase (manufactured by Toyo Co.) in a reaction solution containing 0.2 mmol/L of dNTPs and 1 mmol/L of magnesium chloride, to adjust the total volume to 50. mu.L.

The reaction conditions are as follows: the cycle was set to 1 cycle at 98 ℃ for 15 seconds and 68 ℃ for 30 seconds, and the cycle was set to 25 cycles. This reaction solution was separated by 2% agarose gel electrophoresis, and then a PCR product of about 800bp was introduced into a pCR-Blunt vector using a ZeroBlunt PCR cloning kit (manufactured by Invitrogen corporation) according to the instructions attached thereto, thereby obtaining pCR27axc having the DNA sequence shown in SEQ ID NO. 1 (FIG. 9).

Subsequently, a DNA fragment of about 780bp obtained by digesting pCR27axc with restriction enzymes NotI and SalI was ligated to a DNA fragment of about 8.9kbp obtained by digesting pKANTEX XhoI/SalI prepared in example 1 with restriction enzymes NotI and SalI, thereby constructing a plasmid pKANTEX CD27 for expressing CD27 (FIG. 10).

The sequence of the CD27 gene encoded by this plasmid is shown as sequence No. 1, and the amino acid sequence translated from this gene is shown as sequence No. 2. Coli transformed with the expression vector was inoculated into 100mL of LB medium and cultured overnight. After the culture, the cells were collected, and the plasmid was purified according to the protocol attached thereto using Qiafilter plasmid quantitative purification kit (キアゲン).

After purification, 30. mu.g of the plasmid vector was digested with the restriction enzyme AatII, thereby linearizing it. After linearization, phenol/chloroform extraction and ethanol precipitation were performed, and the resulting solution was dissolved in 1/10 concentration of TE buffer (1mM Tris HCl,0.1mM EDTA), and the concentration was measured to conduct gene transfer.

(2) Introduction of CD27 expression plasmid pKANTEX CD27

The CD 27-expressing plasmid pKANTEX CD27 gene prepared in (1) above was introduced into Lec8 cells and CHO/DG44 cells, thereby establishing Lec8 cells and DG44 cells for expressing CD 27. The procedure of example 1 was repeated, except that pKANTEXCD27 was used as the plasmid for introduction.

However, the cells after gene transfer were suspended in 30mL of HT-medium and plated in 3 96-well plates at 100. mu.L/well. After 2 days of inoculation, the medium was replaced with a passaging medium containing 500. mu.g/mL G418, and culture was carried out for 10 days. After 10 days, the medium was changed to HT-medium containing 50 nMX (manufactured by Sigma aldrich Co.), thereby obtaining MTX-resistant strains. CD 27-expressing strain derived from Lec8 strain was named CD27/Lec8-4, while CD 27-expressing cell derived from DG44 cell was named CD27/DG 44-8.

(3) Confirmation of CD 27-expressing cells

In order to confirm the expression of CD27 in the CD 27-expressing cells prepared in (2), analysis was performed using a Flow Cytometer (FCM) as follows.

Divide and fetch 1~ 5X 10⁶The CD 27-expressing cells were transferred into a 15mL tube (manufactured by BD Co., Ltd.), centrifuged (1500rpm, 5 minutes), the supernatant was removed, and the cells were suspended in 50. mu.L of 1% bovine serum albumin-containing PBS buffer [ hereinafter referred to as 1% BSA-PBS (manufactured by ユ - ジンバイオ Co., Ltd.) ]]In (1).

To this was added 10. mu.L of an anti-CD 27 mouse monoclonal antibody labeled with PC5 (manufactured by Beckmann Coulter Co., Ltd., catalog No. 6607107) or a PC 5-labeled mouse IgG1 isotype control (Beckmann Coulter, catalog No. 6607012) as a primary antibody, and the mixture was reacted at ice temperature for 60 minutes. After the reaction, the cells were washed twice with 1mL of 1% BSA-PBS, suspended in 500. mu.L of 1% BSA-PBS, and the fluorescence intensity was measured by Flow Cytometry (FCM) (manufactured by BD Co.).

The results are shown in fig. 11. As shown in fig. 11, it was confirmed that: CD27/Lec8-4 and CD27/DG44-8 express CD27 to approximately the same extent on the cell membrane.

[ example 3]

Preparation of monoclonal antibody against CD27 having galactose-unbound O-linked sugar chain (hereinafter referred to as anti-sugar chain-deficient CD27 monoclonal antibody)

(1) Preparation of immunogens

Mu.g of CD27-Fc produced by Lec8 strain obtained in example 1 was mixed with 2mg of aluminum hydroxide adjuvant (Antibodies-A Laboratory Manual, Cold spring harbor Laboratory, page 99, 1988) and 1X 10⁹Individual cells of pertussis vaccine (manufactured by qianye county serum study) were administered together to 3 female SD rats of 4 weeks of age.

Starting 2 weeks after the administration, 50. mu.g of CD27-Fc produced by Lec8 strain was administered once a week for a total of 3 times. Blood was partially collected from the tail vein, and the binding activity of the obtained antiserum was measured by fluorescent cell staining using an ABI8200 cell detection system (manufactured by applied biosystems usa) or a flow cytometer (Cytomics FC500MPL, manufactured by beckmann coulter corporation), and 3 days after the last immunization, the spleen was removed from the mice showing sufficient antibody titer.

The spleen was finely chopped in MEM (minimum essential medium) medium (manufactured by Nippon pharmaceutical Co., Ltd.), scattered with forceps, and centrifuged (1200rpm, 5 minutes). To the obtained pellet fraction, Tris-ammonium chloride buffer (pH7.6) was added and treated for 1 to 2 minutes, thereby removing erythrocytes. The resulting pellet fraction (cell fraction) was washed 3 times with MEM medium for cell fusion.

(2) Binding ELISA

The antigen for binding ELISA used was CD27-Fc produced by the Lec8 strain obtained in example 1 and CD27-Fc produced by DG44 cells, respectively. The CD27-Fc protein (5. mu.g/mL) was dispensed at 50. mu.L/well into a 96-well ELISA plate (Kurana) and allowed to stand overnight at 4 ℃ for adsorption.

After washing the plate, 1% BSA-PBS was added at 100. mu.L/well and left at room temperature for 1 hour to block the residual active groups. After the standing, 1% BSA-PBS was discarded, and 50. mu.L/well of the plate was dispensed with antiserum for immunized animals or hybridoma culture supernatant as a primary antibody and allowed to stand for 2 hours.

The plate was washed with 0.05% polyoxyethylene (20) sorbitan monolaurate [ (a product corresponding to the trademark "tween 20" of ICI corporation, manufactured by wako pure chemical industries, Ltd.) -PBS (hereinafter, referred to as tween-PBS) ], and then peroxidase-labeled rabbit-anti-rat immunoglobulin (Zymed corporation) was added thereto at 50 μ L/well as a secondary antibody, and the plate was left at room temperature for 1 hour.

The plate was washed with Tween-PBS, and ABTS [2, 2-azinebis (3-ethylbenzothiazole-6-sulfonic acid) ammonium was added]Substrate solution [1 mmol/L ABTS-0.1 mol/L citric acid buffer solution (pH4.2), 0.1% H₂O₂]The resultant was developed, and the absorbance of OD415nm was measured using a microplate reader (Emax; molecular instruments Co.).

(3) Fluorescent cell staining method (ABI8200 cell detection System analysis)

CD27/Lec8-4 and CD27/DG44-8 prepared in example 2 were used as cells for analysis. CD27/Lec8-4 and CD27/DG44-8 passaged using a passaging medium supplemented with 50nM MTX and 500ng/mL G418 were peeled off with a 0.05% trypsin solution (manufactured by Invitrogen) at 1X 10 per well⁴An amount of each 100. mu.L of the medium was inoculated into black 96-well plates for ABI8200 and cultured overnight.

To the plate, 10. mu.L/well of the immunized rat antiserum or hybridoma culture supernatant was dispensed as a primary antibody, 100. mu.L/well of ALEXA 647-labeled anti-rat immunoglobulin G (H + L) (manufactured by Invitrogen corporation) was added as a secondary antibody, and the plate was left for 4 hours in the dark. Fluorescence at 650 to 685nm excited by laser 633He/Ne was measured using an ABI8200 cell detection system (manufactured by applied biosciences).

(4) Fluorescent cell staining method (flow cytometry analysis)

CD27/Lec8-4 and CD27/DG44-8 prepared in example 2 were used as cells for analysis. CD27/Lec8-4 and CD27/DG44-8 passaged using HT-medium supplemented with 50nM MTX and 500. mu.g/mL G418 were peeled off with 0.02% EDTA solution (ナカライテスク Co., Ltd.), and then, each cell was washed with PBS.

After washing, 1 to 5X 10⁵The cells were suspended in 50. mu.L of 1% BSA-PBS, and then 50. mu.L/well of the immune rat antiserum or hybridoma culture supernatant was dispensed as a primary antibody, and reacted at ice temperature for 30 minutes.

Bovine serum albumin, a mouse IgG1 isotype control (ユスモバイオ), and a rat IgG2a isotype control (ユスモバイオ) were used as negative controls, and an anti-RCAS 1 antibody 22-1-1 (MBL) and an anti-CD 27 mouse monoclonal antibody (beckmarkett) were used as positive controls, and were allowed to react at the same time.

After the reaction, the cells were washed by centrifugation twice using PBS, and ALEXA 488-labeled anti-rat immunoglobulin G (H + L) (Invitrogen) as a secondary antibody was added thereto at 50. mu.L/well, and the reaction was carried out for 30 minutes at ice temperature in the absence of light. The cells were again washed by centrifugation twice using PBS, and then suspended in 500. mu.L of 1% BSA-PBS, and fluorescence at 510 to 530nm excited by 488nm argon laser was measured using a flow cytometer (Cytomics FC500MPL, manufactured by Beckmann Coulter).

(5) Preparation of mouse myeloma cells

The 8-azaguanine-resistant mouse myeloma cell line P3X63Ag8U.1 (P3-U1; purchased from ATCC) was cultured in normal medium (10% FCS RPMI medium) to ensure 2X 10 cells fused together ⁷More than one cell, and used as parent strain for cell fusion.

(6) Preparation of hybridomas

The mouse spleen cells obtained in (1) above and the myeloma cells obtained in (5) above were mixed at a ratio of 10:1, and centrifuged (250 Xg, 5 minutes). The cell population of the obtained pellet fraction was sufficiently stirred and then, while stirring, at 37 ℃ at every 10 ℃⁸To a mouse spleen cell volume of 0.5mL, a mixed solution of 1g polyethylene glycol-1000 (PEG-1000), 1mL MEM medium, and 0.35mL dimethylsulfoxide was added, and 1mL MEM medium was added to the suspension every 1 to 2 minutes, and after repeating the above steps several times, MEM medium was further added until the total volume reached 50 mL.

The suspension was centrifuged (900rpm, 5 minutes), the cells of the obtained pellet fraction were gently scattered, and then the cells were gently suspended in 100mL of HAT medium [ a medium obtained by adding HAT medium additives (manufactured by invitrogen) to RPMI medium supplemented with 10% fetal bovine serum ] by sucking and blowing the mixture with a graduated pipette.

The suspension was dispensed into a 96-well plate at 200. mu.L/well and incubated in 5% CO₂Culturing for 8-10 days at 37 ℃ in an incubator. After the culture, wells in which the culture supernatants reacted with CD27/Lec8-4 but not with CD27/DG44-8 and Lec8 cells were selected by the fluorescent cell staining method described in the above (3) and (4), and the cells contained in the wells were cloned twice by the limiting dilution method to obtain single cell clones.

As a result, hybridomas KM4030 and KM4031 producing monoclonal antibody KM4030 or KM4031 specifically binding to sugar chain-deficient CD27 were established (fig. 12). By the same method, hybridomas KM4026, KM4027 and KM4028 which produce monoclonal antibodies KM4026, KM4027 or KM4028 specifically reactive with sugar chain-deficient CD27 were established.

The above hybridoma producing a monoclonal antibody specifically binding to sugar chain-deficient CD27 is obtained by the following method: recombinant cells expressing normal sugar chains CD27 and sugar chain-deficient CD27 were prepared by using enzymes involved in the sugar chain synthesis pathway, the DG44 strain in which the activity of the transporter is not deleted, and the Lec8 strain in which the activity of the UDP-galactose transporter is reduced or deleted, and a system capable of screening monoclonal antibodies that specifically bind only cells expressing sugar chain-deficient CD27 without binding to cells expressing normal sugar chains CD27 and Lec8 strain was designed, and screening analysis was performed on an approximately 6000-well scale.

(7) Purification of monoclonal antibodies

The hybridoma obtained in the above (6) is cultured in a medium of 5 to 20X 10⁶Individual cells/quantity were injected intraperitoneally into 7 week old female mice (ICRs) after pristane treatment. After 10 to 21 days, the hybridomas produce cancerous ascites, and ascites (1 to 8 mL/mouse) is collected from the mice in which the ascites had accumulated. The ascites was filtered with a syringe filter (pore size: 5 μm) to remove solid components.

Purified IgG monoclonal Antibodies were obtained by purification using the caprylic acid precipitation method [ Antibodies-laboratory Manual, Cold spring harbor laboratory (1988) ]. The determination of the subclass of the monoclonal antibody was performed by a binding ELISA using a subclass typing kit (rat monoclonal antibody isotype typing kit, manufactured by DS ファ - マバイオメデイカル Co.).

The results showed that, with respect to the subclasses of the respective antibodies, anti-sugar chain-deficient CD27 monoclonal antibody KM4026 was of rat IgG2a class, anti-sugar chain-deficient CD27 monoclonal antibody KM4027 was of rat IgG2b class, anti-sugar chain-deficient CD27 monoclonal antibody KM4028 was of rat IgG1 class, anti-sugar chain-deficient CD27 monoclonal antibody KM4030 was of rat IgG2a class, and anti-sugar chain-deficient CD27 monoclonal antibody KM4031 was of rat IgG1 class.

[ example 4]

Examination of reactivity of monoclonal antibody specific to anti-sugar chain-deficient CD27

The reaction specificity of the anti-sugar chain-deficient CD27 monoclonal antibodies KM4030 and KM4031 was investigated using a competition ELISA system shown below. CD27-Fc produced by Lec8 strain obtained in example 1 was dispensed at a concentration of 5. mu.g/mL at 50. mu.L/well into a 96-well ELISA plate (manufactured by Glaina), and allowed to stand overnight at 4 ℃ for adsorption.

After washing the plate, 1% BSA-PBS was added at 200. mu.L/well and left at room temperature for 1 hour to block the residual active groups. After the standing, 1% BSA-PBS was discarded, and the diluted anti-sugar chain-deficient CD27 monoclonal antibody KM4030 purified antibody or KM4031 purified antibody was dispensed to the plate at 50. mu.L/well as a primary antibody.

Meanwhile, CD27-Fc protein produced by Lec8, CD27-Fc protein produced by DG44 or human immunoglobulin was added as a binding competitive substance at a concentration of 20. mu.g/mL, 2. mu.g/mL, 0.2. mu.g/mL or 0.02. mu.g/mL, and the antibody and the binding competitive substance were allowed to coexist.

After the plate was left at room temperature for 2 hours, it was washed with 0.05% polyoxyethylene (20) sorbitan monolaurate [ (a product corresponding to the trademark "tween 20" of ICI corporation, manufactured by wako pure chemical industries, Ltd.) -PBS (hereinafter, referred to as tween-PBS), and then peroxidase-labeled rabbit anti-rat immunoglobulin (manufactured by Zymed) was added at 50 μ L/well as a secondary antibody, and left at room temperature for 1 hour.

The plate was washed with Tween-PBS, and ABTS [2, 2-azinebis (3-ethylbenzothiazole-6-sulfonic acid) ammonium was added]Substrate solution [1 mmol/L ABTS-0.1 mol/L citric acid buffer solution (pH4.2), 0.1% H ₂O₂]The resultant was developed, and then the absorbance of OD415nm was measured using a microplate reader (Emax; molecular instruments Co.).

The results of competition ELISA for the anti-sugar chain-deficient CD27 monoclonal antibodies KM4030 and KM4031 are shown in fig. 13. From this result, it was revealed that CD27-Fc and human immunoglobulin produced by DG44 did not inhibit the binding of the anti-sugar chain-deficient CD27 monoclonal antibodies KM4030 and KM4031 to CD27-Fc binding to Tn antigen, but CD27-Fc binding to Tn antigen inhibited the binding.

In addition, the same results were obtained for the anti-sugar chain-deficient CD27 monoclonal antibodies KM4026, KM4027 and KM 4028. From the above results, it was revealed that the anti-sugar chain-deficient CD27 monoclonal antibodies KM4026, KM4027, KM4028, KM4030 and KM4031 of the present invention specifically recognize the sugar chain-deficient CD 27.

The results obtained by evaluating the binding activity of anti-sugar chain-deficient CD27 monoclonal antibodies KM4026, KM4027, KM4028, KM4030 and KM4031 to the sugar chain-deficient CD27-Fc of the anti-sugar chain-deficient CD27 chimeric antibody using BIACORE are shown in FIGS. 28(A) and (B). The binding activity was measured by using Biacore T100 (manufactured by GE healthcare group Life sciences) and using a surface plasmon resonance method (SPR method).

An anti-human IgG4 antibody (Pharmingen) was immobilized on a CM5 sensor chip (GE healthcare group life science department) by amine coupling using an amine coupling kit (BIACORE corporation) according to the protocol attached thereto.

Sialidase A or beta (1-4) galactosidase obtained by using a Prozyme Glyco enzyme deglycosylation kit (manufactured by Prozyme) and a ProO-Link Extender deglycosylation additive (manufactured by Prozyme) and Tn antigen type CD27-Fc or sialylated Tn antigen type CD27-Fc obtained by subjecting CD27-Fc generated from DG44 obtained in example 1(7) to a sugar chain digesting enzyme treatment in accordance with the protocol attached were added, and the mixture was captured on a chip on which an anti-human IgG4 antibody was immobilized to achieve 200-250RU (resonance unit).

Then, measurement samples (anti-sugar chain defect CD27 monoclonal antibodies KM4026, KM4027, KM4028, KM4030, and KM4031) diluted in five gradients from 20000ng/mL were passed over the chip at a flow rate of 30 μ L/min to obtain sensorgrams at respective concentrations, and analyzed using analysis software Biacore t100 evaluation software (manufactured by Biacore corporation) attached to the apparatus.

The results showed that the anti-sugar chain-deficient CD27 monoclonal antibody showed binding activity to Tn antigen-type CD27-Fc and sialylated Tn antigen-type CD 27-Fc. From the above results, it was revealed that the anti-sugar chain-deficient CD27 monoclonal antibody of the present invention binds to both Tn antigen and sialylated Tn antigen.

[ example 5]

Isolation and analysis of cDNA encoding the variable region of anti-sugar chain-deficient CD27 monoclonal antibody

(1) Preparation of mRNA from hybridoma producing anti-sugar chain-deficient CD27 monoclonal antibody

Using RNAeasy mini-extraction kit (manufactured by QIAGEN Co.) and OligotexTM-dT30<Super>mRNA purification kits (manufactured by TaKaRa Co., Ltd.) of 5X 10 obtained in example 3 according to the respective instructions attached thereto⁷~1×10⁸Each of hybridomas KM4026, KM4027, KM4028, KM4030 and KM4031 of individual cells produced mRNA of each anti-sugar chain-deficient CD27 monoclonal antibody.

(2) Gene cloning of variable regions of H chain and L chain of anti-sugar chain-deficient CD27 monoclonal antibody

The cDNA was obtained from the mRNA of the monoclonal antibody obtained in example 5(1) using a BD SMART RACE cDNA amplification kit (BD Biosciences), according to the attached instructions.

Using the obtained cDNA as a template, a cDNA fragment of a heavy chain variable region (hereinafter referred to as VH) of each antibody was amplified by performing PCR reaction using a rat IgG 1-specific primer (seq id No. 14), a rat IgG2 a-specific primer (seq id No. 15), a rat IgG2 b-specific primer (seq id No. 16), or a rat CH 1-specific primer (seq id No. 17).

In addition, a rat Ig (. kappa.) specific primer (SEQ ID NO: 18) and a rat Ig (kappa.) specific primer (SEQ ID NO: 19) were used in place of each subclass-specific primer of the antibody to perform PCR, thereby amplifying a cDNA fragment of the light chain variable region (hereinafter referred to as VL) of each antibody.

The PCR reactions for amplifying VL and VH of KM4026, KM4030, KM4031 were performed using the advantaged 2PCR kit (Clontech) according to the instructions attached thereto, and the PCR reactions for amplifying VL and VH of KM4027, KM4028 were performed using KOD Plus polymerase (dongyo) according to the instructions attached thereto.

Subsequently, in order to clone and determine the base sequence, the obtained PCR products were separated by agarose gel electrophoresis, and then PCR products derived from KM4026, KM4030 and KM4031 were inserted into PCR vectors using TOPO TA cloning kit (manufactured by invitrogen) according to the attached instructions, and PCR products derived from KM4027 and KM4028 were inserted into PCR vectors using Zero Blunt TOPO PCR cloning kit (manufactured by invitrogen) for sequencing according to the attached instructions.

Coli was transformed with a plasmid into which a PCR amplified fragment was inserted, a plasmid obtained from each clone was prepared, and the DNA sequence was confirmed. As a result, a plasmid containing cDNA of full length VH in which ATG sequence presumed to be initiation codon exists at 5 'end of cDNA and a plasmid containing cDNA of full length VL in which ATG sequence presumed to be initiation codon exists at 5' end of cDNA were obtained. The brief process of cloning is shown in FIG. 14.

(3) Analysis of Gene sequence of variable region of anti-CD 27 monoclonal antibody

The full-length nucleotide sequences of VH of the anti-sugar chain defective CD27 monoclonal antibodies KM4026, KM4027, KM4028, KM4030 and KM4031 contained in the plasmid obtained in example 5(2) are represented by seq id nos. 20 to 24, the full-length amino acid sequences of VH including signal sequences estimated from the sequences are represented by seq id nos. 25 to 29, the full-length nucleotide sequences of VL of the anti-sugar chain defective CD27 monoclonal antibodies KM4026, KM4027, KM4028, KM4030 and KM4031 contained in the plasmid obtained in example 5(2) are represented by seq id nos. 30 to 34, and the full-length amino acid sequences of VL including signal sequences estimated from the sequences are represented by seq id nos. 35 to 39.

In addition, identification was performed by comparing the CDRs of VH and VL of each monoclonal antibody with the amino acid sequences of known antibodies. The amino acid sequences of CDR1, CDR2 and CDR3 of VH of anti-sugar chain-deficient CD27 monoclonal antibody KM4026 are represented by sequence No. 40, sequence No. 41 and sequence No. 42, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL of anti-sugar chain-deficient CD27 monoclonal antibody KM4026 are represented by sequence No. 43, sequence No. 44 and sequence No. 45, respectively.

The amino acid sequences of CDR1, CDR2 and CDR3 of VH of the anti-sugar chain-deficient CD27 monoclonal antibody KM4027 are represented by the sequence numbers 46, 47 and 48, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL of the anti-sugar chain-deficient CD27 monoclonal antibody KM4027 are represented by the sequence numbers 49, 50 and 51, respectively.

The amino acid sequences of CDR1, CDR2 and CDR3 of VH of anti-sugar chain defect CD27 monoclonal antibody KM4028 are represented by sequence No. 52, sequence No. 53 and sequence No. 54, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL of anti-sugar chain defect CD27 monoclonal antibody KM4028 are represented by sequence No. 55, sequence No. 56 and sequence No. 57, respectively.

The amino acid sequences of CDR1, CDR2 and CDR3 of VH of anti-sugar chain defect CD27 monoclonal antibody KM4030 are represented by sequence No. 58, sequence No. 59 and sequence No. 60, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL of anti-sugar chain defect CD27 monoclonal antibody KM4030 are represented by sequence No. 61, sequence No. 62 and sequence No. 63, respectively.

The amino acid sequences of CDR1, CDR2 and CDR3 of VH of anti-sugar chain defect CD27 monoclonal antibody KM4031 are represented by sequence No. 64, sequence No. 65 and sequence No. 66, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL of anti-sugar chain defect CD27 monoclonal antibody KM4031 are represented by sequence No. 67, sequence No. 68 and sequence No. 69, respectively.

[ example 6]

Production of anti-sugar chain-deficient CD27 chimeric antibody

(1) Construction of anti-sugar chain-deficient CD27 chimeric antibody expression vector

The chimeric antibody produced in the present invention was obtained by ligating the heavy chain constant region of human IgG1 and the light chain constant region of human kappa disclosed in US97/10354 to the variable regions of the heavy chain and light chain of the anti-CD 27 rat monoclonal antibody obtained in example 5 (3).

As a method, an anti-sugar chain-deficient CD27 chimeric antibody expression vector was constructed in the following manner using the pCR vector containing VL or VH of each monoclonal antibody obtained in example 5(3) and the antibody expression vector pKANTEX93 described in US97/10354, which contains a heavy chain constant region of human IgG1 and a light chain constant region of human κ (fig. 15, 16, 17, 18).

The following solutions were prepared: 10ng of pCR vector containing VL or VH of KM4026, KM4027, KM4028, KM4030, KM4031 was used as a template, and contained 2. mu.L of 10 XKODPlus buffer, 2. mu.L of 2 mM dNTP, 1. mu.L of 25 mM magnesium sulfate, 1. mu.L of LKOD Plus polymerase (manufactured by Toyo Co., Ltd.), and 1. mu.L each of 10. mu.M VL and VH specific primers for each anti-CD 27 monoclonal antibody, and the total amount was 20. mu.L. The following PCR reaction was performed using this preparation solution: after heating at 94 ℃ for 5 minutes, 30 cycles of 1 cycle of 94 ℃ for 1 minute and 68 ℃ for 2 minutes were carried out.

Primers for VL of KM4026 are represented by sequence numbers 70 and 71, and primers for VH are represented by sequence numbers 72 and 73; primers for VL of KM4027 are represented by sequence numbers 74 and 75, and primers for VH are represented by sequence numbers 76 and 77; primers for VL of KM4028 are represented by sequence numbers 78 and 79, and primers for VH are represented by sequence numbers 80 and 81; primers for VL of KM4030 are represented by sequence numbers 82 and 83, and primers for VH are represented by sequence numbers 84 and 85; primers for VL of KM4031 are represented by sequence numbers 86 and 87, and primers for VH are represented by sequence numbers 88 and 89.

After separating each PCR reaction product by 1% agarose gel electrophoresis, a specific PCR amplification band was collected and inserted into a pCR Blunt-TOPO vector using a Zero Blunt TOPO PCR cloning kit for sequencing (Invitrogen) according to the attached instructions.

The VL of each antibody thus obtained was digested with restriction enzymes EcoRI (New England Biolabs) and BsiWI (New England Biolabs) to obtain an EcoRI-BsiWI fragment of VL. In addition, VH of each antibody was digested with restriction enzymes NotI (New England Biolabs) and ApaI (New England Biolabs) to obtain a NotI-ApaI fragment.

Each EcoRI-BsiWI fragment of VL of the anti-CD 27 monoclonal antibodies KM4026, KM4027, KM4028, KM4030 or KM4031 was ligated with a DNA fragment obtained by digesting pKANTEX93 with restriction enzymes EcoRI and BsiWI using Ligation High (manufactured by toyobo co., ltd.) according to the attached instructions.

Coli DH5 α (manufactured by toyobo) was transformed with the ligated DNA fragment, plasmids obtained from each clone were prepared, reaction was performed using BigDye terminator cycle sequencing FSReady reaction kit (manufactured by PE バイオシステムズ) according to the attached instructions, and then the nucleotide sequence was analyzed using a sequencer ABI PRISM3700 of the company.

As a result, pKANTEX93 into which a cDNA encoding VL of the anti-CD 27 monoclonal antibodies KM4026, KM4027, KM4028, KM4030, or KM4031 was inserted was obtained.

Subsequently, using Ligation High (manufactured by toyobo corporation), each NotI-ApaI fragment of VH of the anti-CD 27 monoclonal antibodies KM4026, KM4027, KM4028, KM4030, or KM4031 and a DNA fragment obtained by digesting pKANTEX93 of each VL into which the anti-CD 27 monoclonal antibody has been inserted with restriction enzymes NotI and ApaI were ligated according to the attached instructions.

Coli DH5 α (manufactured by toyobo) was transformed with the ligated DNA fragment, plasmids obtained from each clone were prepared, reaction was performed using BigDye terminator cycle sequencing FSReady reaction kit (manufactured by PE バイオシステムズ) according to the attached instructions, and then the nucleotide sequence was analyzed using a sequencer ABI PRISM3700 of the company.

As a result, an anti-CD 27 chimeric antibody expression vector into which cdnas encoding VL and VH of the anti-CD 27 monoclonal antibodies KM4026, KM4027, KM4028, KM4030, or KM4031, respectively, were inserted was obtained.

(2) Expression of anti-sugar chain-deficient CD27 chimeric antibody Using animal cells

Using the anti-sugar chain-deficient CD27 chimeric Antibody expression vector obtained in the above (1), expression of an anti-sugar chain-deficient CD27 chimeric Antibody in animal cells was carried out by a conventional method [ Antibody Engineering, A Practical Guide (Antibody Engineering practice Guide), Williams H & Frahmann (1992) ], to obtain transformants which produce anti-sugar chain-deficient CD27 chimeric antibodies (chimeric KM4026, chimeric KM4028, chimeric KM4030 and chimeric KM 4031).

As an animal cell line for expression, a CHO/DG44 cell line in which the α 1, 6-fucosyltransferase (FUT8) gene was double-knocked out (hereinafter referred to as FUT8 knock-out CHO cell) was used. It is known that fucose is not added to a core part of an N-linked complex sugar chain of an antibody expressed in the host cell strain (International publication No. 2002/31140).

(3) Obtaining purified chimeric antibody

After culturing the transformants obtained in (2) above, chimeric KM4026, chimeric KM4028, chimeric KM4030 and chimeric KM4031, by a usual culture method, the cell suspension was recovered, centrifuged at 3000rpm and 4 ℃ for 20 minutes, the culture supernatant was recovered, and then filtered and sterilized through a Millex GV filter having a pore size of 0.22. mu.m.

Chimeric KM4026, chimeric KM4028, chimeric KM4030 and chimeric KM4031, which are anti-sugar chain-deficient CD27 chimeric antibodies, were purified from the resulting culture supernatants by the same method as in example 1(7) using MabSelect.

(4) Determination of fucose content of anti-sugar chain-deficient CD27 chimeric antibody

The method described in International publication No. 2002/31140 was used to examine the ratio of sugar chains not alpha-linked to the 1-position of fucose at the 6-position of N-acetylglucosamine present at the reducing end of the N-linked complex sugar chain in the Fc fragment of each anti-sugar chain-deficient CD27 chimeric antibody. The results are shown in table 2.

[ Table 2] fucose content of anti-sugar chain-deficient CD27 chimeric antibody

As shown in table 2, it was found that fucose was not added to the chimeric antibody produced in example 6 (3).

[ example 7]

Evaluation of Activity of anti-sugar chain-deficient CD27 chimeric antibody

In the following (1) to (3), the activity of the anti-sugar chain defect CD27 chimeric antibody chimeric KM4026, chimeric KM4028, chimeric KM4030 and chimeric KM4031 obtained in example 6 was evaluated.

(1) BIACORE was used to evaluate the binding activity of the anti-sugar chain-deficient CD27 chimeric antibody to human sugar chain-deficient CD27-Fc

In order to analyze the binding activity of the chimeric KM4026, chimeric KM4028 and chimeric KM4030, which are anti-sugar chain-deficient CD27 chimeric antibodies, to human sugar chain-deficient CD27-Fc in terms of reaction velocity, the binding activity was measured by using the surface plasmon resonance method (SPR method). All of the following operations were performed using biacore t100 (manufactured by GE medical group life science).

The Lec 8-derived CD27-Fc obtained in example 1(7) was immobilized on a CM5 sensor chip (manufactured by GE healthcare group life science). Based on an automated procedure of single reaction kinetics, measurement was performed by successively adding five concentrations of measurement samples (chimeric KM4026, chimeric KM4028, chimeric KM4030, and chimeric KM4031) prepared by 3-fold dilution from 9 μ g/mL to a chip on which CD27-Fc was immobilized, in order from a low concentration.

The binding rate constant ka and dissociation rate constant kd of each antibody to human sugar chain-deficient CD27-Fc were calculated by analysis using Biacore T100 evaluation software (manufactured by Biacore corporation) attached to the apparatus and using a Bivalent Analyte (Bivalent analysis) model.

As a result, the binding rate constant ka1, dissociation rate constant KD1 and dissociation constant KD (KD1/ka1) of each of the obtained antibodies are shown in Table 3.

[ Table 3]

As shown in Table 3, each of the chimeric KM4026, chimeric KM4028, chimeric KM4030 and chimeric KM4031 showed 1X 10 to the human sugar chain defect CD27-Fc^-81×10^-9High affinity in mol/l.

(2) Evaluation of reaction specificity of anti-sugar chain-deficient CD27 chimeric antibody by fluorescent cell staining method (flow cytometry analysis)

CD27/DG44-4 cells, CD27/Lec8-4 cells and Lec8 cells prepared in the same manner as in example 2 were used as cells for analysis. CD27/DG44-4 passaged using HT-medium supplemented with 500. mu.g/mL G418 and CD27/Lec8-4 passaged using HT-medium supplemented with 50 nmol/L MTX and 500. mu.g/mL G418 were stripped with 0.02% EDTA solution, and then, each cell was washed with PBS.

After washing, 5X 10⁵After suspending the cells in 50. mu.L of 1% BSA-PBS, 50. mu.L/well of the chimeric antibodies against sugar chain-deficient CD27 (chimeric KM4026, chimeric KM4028, chimeric KM4030 and chimeric KM4031) was dispensed, and antibody solutions prepared at 0.02. mu.g/mL, 0.2. mu.g/mL, 2. mu.g/mL and 10. mu.g/mL were used as primary antibodies and reacted at ice temperature for 1 hour.

As positive controls, an anti-RCAS 1 mouse antibody 22-1-1 (manufactured by MBL Co.) and an anti-CD 27 mouse antibody O323 (manufactured by Santa Cruzbiotechnology Co.) were used as anti-Tn antibodies.

After the reaction, the cells were washed by centrifugation twice using PBS, and ALEXA Fluoro 488-labeled anti-human immunoglobulin G (H + L), ALEXA Fluoro 488-labeled anti-mouse immunoglobulin G (H + L) or ALEXA Fluoro 488-labeled anti-human immunoglobulin M (μ) was added as a secondary antibody at 50 μ L/well, and the reaction was carried out for 30 minutes at ice temperature in the absence of light.

The cells were again washed by centrifugation twice using PBS, and then suspended in 500. mu.L of 1% BSA-PBS, and fluorescence at 510 to 530nm, which was excited by 488nm argon laser, was measured using a flow cytometer (CytomicsFC 500MPL, manufactured by Beckmann Coulter). The results are shown in FIGS. 19(A) to (C).

As a result, neither the anti-sugar chain-deficient CD27 chimeric antibody bound to Lec8 cells nor CD27/DG44-4 cells, but only binding to CD27/Lec8-4 cells expressing sugar chain-deficient CD27 was observed.

From the above results, it was revealed that the anti-sugar chain-deficient CD27 chimeric antibodies of the present invention chimeric KM4026, chimeric KM4028, chimeric KM4030 and chimeric KM4031 specifically recognize the sugar chain-deficient CD27 expressed on the cell surface.

(3) Evaluation of antibody-dependent cellular cytotoxicity (ADCC Activity) of anti-sugar chain-deficient CD27 chimeric antibody

ADCC activity of anti-sugar chain-deficient CD27 chimeric antibody chimeric KM4026, chimeric KM4028, chimeric KM4030 and chimeric KM4031 against CD27/Lec8-4 cells prepared in example 2 was measured by the following method.

(3) -1 preparation of a suspension of target cells

CD27/Lec8-4 cells passaged using HT-medium supplemented with 50 nmol/l MTX and 500 μ G/mL G418 were peeled off with a 0.02% EDTA solution, washed with PBS, washed with RPMI1640 medium (manufactured by invitrogen) containing 5% dialyzed fetal bovine serum (dfs) (manufactured by invitrogen) and containing no phenol red (hereinafter referred to as ADCC medium), and prepared to an optimum concentration using the medium to prepare a target cell suspension.

(3) -2 preparation of Effector cell suspension

Peripheral Blood Mononuclear Cells (PBMC) were isolated from Peripheral blood of healthy persons by the following method. Peripheral blood of 50mL of healthy persons was collected from healthy persons using a syringe to which 0.5mL of heparin sodium injection "シミズ" (manufactured by Wako pure chemical industries, Ltd.) recorded in Japanese pharmacopoeia was added.

3.5mL of the collected peripheral blood was gently layered on 3mL of each mononuclear-polymorphonuclear separation solution (DS ファ - マバイオメデイカル) dispensed into a 15mL tube, and centrifuged at 400 Xg and under a brake-off condition at room temperature for 20 minutes to separate a mononuclear cell layer. The monocyte fraction thus obtained was washed twice with a medium for ADCC, and the optimal number of cells was prepared using the medium, thereby preparing an effector cell suspension.

(3) -3 determination of ADCC Activity

The measurement was carried out by using LDH-cytoxic Test Wako (manufactured by Wako Co., Ltd.) according to the following procedure in accordance with the attached instructions.

50. mu.L of an antibody solution obtained by diluting each antibody from 30. mu.g/mL to a concentration of 0.003. mu.g/mL in a 10-fold dilution manner was dispensed into each well of a 96-well U-shaped substrate (manufactured by FALCON Co., Ltd.), followed by 1X 10 ⁴Each cell/50. mu.L/well was dispensed with the target cell suspension prepared in (3) -1, and finally, at 2.5X 10⁵Each cell/50. mu.L/well was dispensed with the effector cell suspension prepared in (3) -2, adjusted to a total amount of 150. mu.L, and reacted at 37 ℃ for 4 hours. Thus, the experiment was performed under conditions where the ratio of effector cells (E) to target cells (T) was 25: 1. The ADCC activity was determined by the following formula. The results are shown in fig. 20.

(formula (II))

ADCC activity (%) = { ([ absorbance of sample ] - [ absorbance of target cell free in nature ] - [ absorbance of effector cell free in nature ])/([ absorbance of target cell free in total ] - [ absorbance of target cell free in nature ]) } × 100

As a result, the anti-sugar chain-deficient CD27 chimeric antibodies chimeric KM4026, chimeric KM4028, chimeric KM4030 and chimeric KM4031 in which core fucose was not bound to the N-linked complex sugar chains of the Fc fragment of the antibody all exhibited high ADCC activity on CD27/Lec8-4 cells.

From the above results, it was revealed that the anti-sugar chain-deficient CD27 chimeric antibodies of the present invention, chimeric KM4026, chimeric KM4028, chimeric KM4030 and chimeric KM4031 all had high ADCC activity against cells expressing sugar chain-deficient CD 27.

(4) Evaluation of complement-dependent cytotoxic Activity (CDC Activity) of anti-sugar chain-deficient CD27 chimeric antibody

CDC activity of anti-sugar chain-deficient CD27 chimeric antibody chimeric KM4026, chimeric KM4028, chimeric KM4030 and chimeric KM4031 against CD27/Lec8-4 cells prepared in example 2 was measured by the following methods.

CD27/Lec8-4 cells were washed with PBS, then washed with RPMI1640 medium containing 10% dFBS, and prepared to an optimum concentration with the medium, thereby preparing a target cell suspension. The target cell suspension was dispensed at 50. mu.L/well into a 96-well flat bottom plate (manufactured by Gerania) so that each well became 5X 10⁴The cells were then supplemented with anti-sugar chain-deficient CD27 chimeric antibody solution and human complement (manufactured by SIGMA corporation) prepared at appropriate concentrations, and the total amount was 150. mu.L/well.

In addition, reaction wells containing no antibody (0% cytotoxic activity wells) were prepared as negative controls, and reaction wells containing no cells (100% cytotoxic activity wells) were prepared as positive controls. At 37 deg.C, 5% CO²The reaction was carried out in the incubator of (1) for 2 hours.

After completion of the reaction, 15. mu.L of WST-1 reagent (manufactured by Roche) was added to each reaction well, and reacted at 37 ℃ for about 4 hours, and the absorbance (OD450nm-OD690nm) in each well was measured using a microplate reader (Emax). CDC activity (cytotoxic activity [% ]) was calculated from the absorbance of each well using the following formula. The results are shown in fig. 21.

(formula (II))

CDC activity (cytotoxic activity [% ]) = {1- (absorbance of reaction well-100% cell lysis well)/(absorbance of 0% cell lysis well-100% cell lysis well) } × 100

As a result, chimeric KM4026, chimeric KM4028, chimeric KM4030 and chimeric KM4031, which are chimeric antibodies against sugar chain-deficient CD27 obtained in the present invention, all showed CDC activity.

[ example 8]

Preparation of CHO cell expressing cynomolgus monkey CD27 on cell Membrane

(1) Cloning of cynomolgus monkey CD27 Gene

The gene encoding CD27 was isolated from cynomolgus monkey peripheral blood-derived RNA according to the following procedure. The following solutions were prepared: mu.g of RNA isolated from peripheral blood of cynomolgus monkeys by Trizol (manufactured by Invitrogen corporation) was used as a template, and 1. mu.L of a mixture containing oligo dT 1. mu. L, dNTP attached to SuperScriptIII first strand synthesis kit (manufactured by Invitrogen corporation) was contained, and the total amount was 5. mu.L.

The prepared solution was reacted at 65 ℃ for 5 minutes, then quenched on ice for 1 minute, and then added with 2. mu. L, DTT 2. mu.L of 10 XT buffer and 2. mu.L of RNase OUT 1. mu. L, RT 1. mu.L of the SuperScriptIII first strand synthesis kit, followed by reverse transcription at 50 ℃ for 50 minutes.

After the reaction, the reaction mixture was heated at 85 ℃ for 5 minutes to inactivate the reverse transcriptase, and then 1. mu.L of RNase H attached to the first strand synthesis kit of SuperScriptIII was added thereto, and the reaction mixture was reacted at 37 ℃ for 20 minutes to completely decompose the RNA. Before use, the single-stranded cDNA derived from peripheral blood of the cynomolgus monkey was stored at-20 ℃.

The following solutions were prepared: using 1.25. mu.L of the cynomolgus monkey peripheral blood-derived single-stranded cDNA prepared as described above as a template, 2.5. mu.L of 10 XKOD Plus buffer, 2.5. mu.L of 2 mM dNTP, 1. mu.L of 25 mM magnesium sulfate, 0.5. mu.L of KOD Plus polymerase (manufactured by Toyobo Co., Ltd.), 20 picomoles of each of mfCD27_5UTR (SEQ ID NO: 90) designed from the 5 '-side untranslated region sequence of macaque CD27 and mfCD27_3UTR (SEQ ID NO: 91) designed from the 3' -side untranslated region sequence of macaque CD27, the total amount was 25. mu.L.

The following PCR reaction was performed using this preparation solution: after heating at 94 ℃ for 5 minutes, 30 cycles of 1 cycle of 94 ℃ for 30 seconds and 68 ℃ for 2 minutes were carried out. The reaction solution was separated by 1% agarose gel electrophoresis, and then, a PCR product of about 800bp was inserted into the pCR Blunt-TOPO vector using a Zero BluntTOPO PCR cloning kit for sequencing (manufactured by Invitrogen) according to the attached instructions.

Coli DH5 α (manufactured by toyobo) was transformed with a vector into which a PCR amplification fragment was inserted, plasmids obtained from each clone were prepared, reaction was performed using BigDye terminator cycle sequencing FS Ready reaction kit (manufactured by PE バイオシステムズ) according to the attached instructions, and then the base sequence was analyzed using a sequencer ABI PRISM3700 of the company. As a result, plasmid pCR mfCD27 (FIG. 22) into which cDNA encoding cynomolgus monkey CD27 (SEQ ID NO: 92) was cloned was obtained.

(2) Construction of monkey CD27 expression plasmid pKANTEX mfCD27His

The cynomolgus monkey CD27 expression vector pKANTEX mfCD27His with His tag attached to the C-terminus was prepared by the following method.

The following solutions were prepared: 10ng of pCR mfCD27 was used as a template, and contained 5. mu.L of 10 XKOD Plus buffer, 5. mu.L of 2 mM dNTP, 2. mu.L of 25 mM magnesium sulfate, 1. mu.L of KOD Plus polymerase (manufactured by Toyo Boseki Co., Ltd.), 0.2. mu.L each of 100. mu.M mfCD27 toKAN-5 (SEQ ID NO: 93) and mfCD27 HisKAN-3 (SEQ ID NO: 94), and the total amount was 50. mu.L.

The following PCR reaction was performed using this preparation solution: after heating at 94 ℃ for 5 minutes, 30 cycles of 1 cycle of 94 ℃ for 30 seconds and 68 ℃ for 2 minutes were carried out. The reaction solution was separated by 1% agarose gel electrophoresis, and then, a PCR product of about 800bp was inserted into the pCR Blunt-TOPO vector using a Zero BluntTOPO PCR cloning kit for sequencing (manufactured by Invitrogen) according to the attached instructions.

Coli DH5 α (manufactured by toyobo) was transformed with a vector into which a PCR amplification fragment was inserted, plasmids obtained from each clone were prepared, reaction was performed using BigDye terminator cycle sequencing FS Ready reaction kit (manufactured by PE バイオシステムズ) according to the attached instructions, and then the base sequence was analyzed using a sequencer ABI PRISM3700 of the company.

As a result, plasmid pCR mfCD27His (FIG. 23) into which cDNA (SEQ ID NO: 95) encoding cynomolgus monkey CD27 having a His tag attached to the C-terminus was cloned was obtained.

Using Ligation High (manufactured by Toyo Boseki Co., Ltd.), an approximately 800bp DNA fragment obtained by digesting pCR mfCD27His with restriction enzymes NotI and SalI (manufactured by タカラバイオ Co., Ltd.) was ligated with an approximately 10kbp DNA fragment obtained by digesting pKANTEX CD27 prepared in example 2 with restriction enzymes NotI and SalI (manufactured by タカラバイオ Co., Ltd.) according to the attached instructions.

Coli DH5 α (manufactured by toyobo) was transformed with the ligated DNA fragment, plasmids obtained from each clone were prepared, reaction was performed using BigDye terminator cycle sequencing FSReady reaction kit (manufactured by PE バイオシステムズ) according to the attached instructions, and then the nucleotide sequence was analyzed using a sequencer ABI PRISM3700 of the company.

As a result, a plasmid pKANTEX mfCD27His for expressing cynomolgus monkey CD27 having a His tag attached to the C-terminus was obtained (fig. 24).

DH 5. alpha. obtained by transformation with pKANTEX mfCD27His (manufactured by Toyo Boseki Co.) was inoculated into 200mL of LB medium and cultured overnight. After the culture, the cells were collected, and the plasmid was purified using a QIAfilter plasmid middle-size extraction kit (キアゲン Co.) according to the instructions attached thereto. 50. mu.g of the purified plasmid was digested with the restriction enzyme AatII (New England Biolabs) to linearize the plasmid.

(3) Introduction of monkey CD27 expression plasmid pKANTEX mfCD27His

The cynomolgus monkey CD27 expression plasmid pKANTEX mfCD27His gene prepared in (2) above was introduced into Lec8 cells and CHO/DG44 cells, thereby establishing Lec8 cells and DG44 cells expressing cynomolgus monkey CD 27.

The procedure of example 1(6) was followed except that pKANTEX mfCD27His was used as the plasmid for introduction. However, the cells after gene transfer were suspended in 30mL of HT-medium and plated in 3 96-well plates at 100. mu.L/well.

After 1 day of inoculation, the medium for passaging containing 500. mu.g/mL of G418 was replaced, and culture was carried out for 10 days to obtain G418-resistant clones. The cynomolgus monkey CD 27-expressing strain derived from the Lec8 strain was designated as cynomolgus monkey CD27/Lec8 cells, while the cynomolgus monkey CD 27-expressing cell derived from the DG44 cell was designated as cynomolgus monkey CD27/DG44 cells.

[ example 9]

Evaluation of Cross-reactivity of anti-sugar chain-deficient CD27 chimeric antibody with monkey CD27 protein

The cynomolgus monkey CD27/DG44 cells and cynomolgus monkey CD27/Lec8 cells prepared in example 8 were used as cells for analysis.

(1) Evaluation of reactivity of anti-sugar chain-deficient CD27 chimeric antibody with monkey CD 27-expressing cells by fluorescent cell staining method (flow cytometry analysis)

The binding activity of the chimeric KM4026, chimeric KM4028, chimeric KM4030 and chimeric KM4031, which are anti-sugar chain-deficient CD27 chimeric antibodies, to cynomolgus monkey CD 27-expressing cells was measured by the following method.

Cynomolgus monkey CD27/DG44 cells and cynomolgus monkey CD27/Lec8 cells passaged using HT-medium supplemented with 500. mu.g/mL G418 were detached with 0.02% EDTA solution, and then each cell was washed with PBS.

After washing, 5X 10⁵The cells were suspended in 50. mu.L of 1% BSA-PBS, and 50. mu.L/well of the chimeric antibody against sugar chain-deficient CD27 (chimeric KM4026, chimeric K)M4028, chimeric KM4030 and chimeric KM4031) was prepared in 10. mu.g/mL and reacted as a primary antibody at ice temperature for 1 hour.

As a positive control, an anti-CD 27 mouse antibody O323 was used. After the reaction, the cells were washed by centrifugation twice using PBS, and ALEXA fluor 488-labeled anti-human immunoglobulin G (H + L) or ALEXA fluor 488-labeled anti-mouse immunoglobulin G (H + L) (both manufactured by BioLegend corporation) was added as a secondary antibody at 50 μ L/well, and the reaction was performed for 30 minutes at ice temperature in the dark.

The cells were again washed by centrifugation twice using PBS, and then suspended in 500. mu.L of 1% BSA-PBS, and fluorescence at 510 to 530nm, which was excited by 488nm argon laser, was measured using a flow cytometer (CytomicsFC 500MPL, manufactured by Beckmann Coulter). The results are shown in fig. 25.

The results showed that chimeric KM4030 and chimeric KM4031, which are chimeric antibodies against sugar chain-deficient CD27, reacted with cynomolgus monkey CD27/Lec8 cells expressing sugar chain-deficient CD 27.

On the other hand, the chimeric KM4026 and chimeric KM4028, which are anti-sugar chain-deficient CD27 chimeric antibodies, showed less reactivity with cynomolgus monkey CD27/Lec8 cells. In addition, it was confirmed that none of the anti-sugar chain-deficient CD27 chimeric antibodies bound to cynomolgus monkey CD27/DG44 cells.

(2) Evaluation of ADCC Activity of anti-sugar chain-deficient CD27 chimeric antibody on monkey CD 27-expressing cells

Cynomolgus monkey CD27/Lec8 cells passaged using HT-medium supplemented with 500. mu.g/mL G418 were detached with 0.02% EDTA solution, washed with PBS, then washed with ADCC medium, and prepared to an optimum concentration using the medium to prepare a target cell suspension. The preparation of effector cell suspensions and the measurement of ADCC activity were performed by the same methods as in example 7. The results are shown in fig. 26.

As a result, the chimeric KM4026, chimeric KM4028, chimeric KM4030 and chimeric KM4031, which are anti-sugar chain-deficient CD27 chimeric antibodies, all exhibited ADCC activity against cynomolgus monkey CD27/Lec8 cells (sugar chain-deficient cynomolgus monkey CD27 cells) containing an O-linked sugar chain to which galactose was not bound.

From the above results, it was revealed that the chimeric KM4026, chimeric KM4028, chimeric KM4030 and chimeric KM4031, which are chimeric antibodies against the sugar chain-deficient CD27, all showed cross-reactivity to the sugar chain-deficient cynomolgus monkey CD27 cells. The strong or weak reactivity of each anti-sugar chain-deficient CD27 chimeric antibody to sugar chain-deficient cynomolgus monkey CD27 was observed, suggesting that there was a difference in the recognized epitope.

In addition, ADCC activity of the anti-sugar chain-deficient CD27 chimeric antibody chimeric KM4030 was measured using cells in which the expression level of CD27 on the cell surface was approximately the same in the human CD27/Lec8 cells prepared in example 2 and the cynomolgus monkey CD27/Lec8 cells prepared in example 8, which were confirmed by flow cytometry analysis. Effector cell suspensions were prepared from peripheral blood of the same healthy human. The results are shown in fig. 27.

The results suggest that chimeric KM4030, which is a chimeric antibody of CD27 having a sugar chain defect, exhibited the same ADCC activity on cynomolgus monkey CD27/Lec8 cells and human CD27/Lec8 cells.

[ example 10]

Production of humanized antibody

(1) Design of amino acid sequences of VH and VL of humanized antibody against sugar chain-deficient CD27

The amino acid sequence of VH of the humanized antibody against sugar chain defect CD27 was designed as follows.

First, the amino acid sequence of FR of VH of a human antibody for grafting the amino acid sequences of CDRs 1-3 of the anti-sugar chain-deficient CD27 rat monoclonal antibody KM4030VH represented by SEQ ID Nos. 58, 59 and 60, respectively, was selected.

Human antibodies having high homology to the anti-sugar chain-deficient CD27 rat monoclonal antibody KM4030 were searched in the amino Acid database of existing proteins by the BLASTP method [ Nucleic Acid Res, 25,3389(1997) ] using the GCG software package (manufactured by Genetics Computer Group Co., Ltd.) as a sequence analysis system.

Comparison of the homology scores with the actual amino acid sequence homology results in SWISSPROT database accession numbers: the antibody spectrum of a neutralizing monoclonal antibody against H3N2 human influenza virus of BAH04525 (hereinafter referred to as BAH04525) showed 83.9% homology and was the most homologous human antibody, and therefore, the amino acid sequence of the FR of this antibody was selected.

The amino acid sequences of the CDRs of the VH of the anti-sugar chain-deficient CD27 rat monoclonal antibody KM4030 represented by SEQ ID Nos. 58 to 60 were grafted to the appropriate positions of the amino acid sequences of the FRs of the human antibodies thus determined. Thus, the amino acid sequence HV0 of VH of the humanized antibody against sugar chain defect CD27, which is represented by SEQ ID NO. 96, was designed.

Next, the amino acid sequence of VL of the humanized antibody against sugar chain defect CD27 was designed as follows.

Selecting an amino acid sequence of FR of VL of a human antibody for grafting an amino acid sequence of CDRs 1-3 of an anti-sugar chain-deficient CD27 rat monoclonal antibody KM4030VL represented by SEQ ID Nos. 61-63, respectively.

Kabat et al, classified VL of various known human antibodies into four subgroups (HSG I-IV) based on homology of amino acid Sequences thereof, and reported consensus Sequences of the above subgroups [ Sequences of Proteins of Immunological Interest, United states department of health and public service (1991) ]. Therefore, the amino acid sequences of the FRs of the consensus sequences of subgroups I to IV of VL of the human antibody and the amino acid sequence of the FR of VL of the anti-sugar chain-deficient CD27 rat antibody KM4030 were subjected to homology search.

As a result of homology search, the homologies of HSGI, HSGII, HSGIII and HSGIV were 86.3%, 60.0%, 73.8% and 73.8%, respectively. Therefore, the amino acid sequence of FR of VL of KM4030 has the highest homology with subgroup I.

From the above results, the amino acid sequences of the CDRs of VL of the anti-sugar chain-deficient CD27 rat monoclonal antibody KM4030 were grafted to the appropriate positions of the amino acid sequences of FRs of the consensus sequence of subgroup I of VL of human antibodies.

However, Leu at position 124 in the amino acid sequence VL of the anti-sugar chain-deficient CD27 rat monoclonal antibody KM4030 shown in sequence No. 38 is not the most frequently used amino acid residue at the corresponding position in the amino acid sequence of the human antibody FR exemplified by kabat et al, but is also a relatively frequently used amino acid residue, and therefore, it is decided to use the amino acid residue described above appearing in the amino acid sequence of the anti-sugar chain-deficient CD27 rat monoclonal antibody KM 4030.

Thus, the amino acid sequence LV0 of VL of the anti-sugar chain defect CD27 humanized antibody represented by SEQ ID NO. 97 was designed.

The amino acid sequences HV0 of VH and LV0 of VL of the designed humanized antibody against sugar chain defect CD27 were sequences obtained by grafting only the amino acid sequences of CDRs of the anti-sugar chain defect CD27 rat monoclonal antibody KM4030 to the amino acid sequences of FRs of a selected human antibody.

However, in general, when a humanized antibody is prepared, the binding activity is often reduced only by simply grafting the amino acid sequence of the CDR of the rat antibody to the FR of the human antibody. Therefore, in order to avoid the decrease in binding activity, the amino acid sequences of the CDRs are grafted and amino acid residues which are thought to affect the binding activity among amino acid residues of FRs of human antibodies and rat antibodies are modified.

Therefore, in this example, the amino acid residues of the FRs considered to have an influence on the binding activity were also identified as follows.

First, using a computer modeling method, a three-dimensional structure of an antibody V region (HV0LV0) comprising the amino acid sequence HV0 of VH and the amino acid sequence LV0 of VL of the above-designed anti-sugar chain-defect CD27 humanized antibody was constructed. Using software Discovery Studio (manufactured by Accelrys corporation), three-dimensional structure coordinates were created in accordance with the attached instructions and the three-dimensional structure was displayed.

In addition, a computer model of the three-dimensional structure of the V region of the anti-sugar chain-deficient CD27 rat monoclonal antibody KM4030 was constructed in the same manner. Furthermore, amino acid residues different from those of the anti-sugar chain-deficient CD27 rat antibody KM4030 were selected from the amino acid sequences of the FRs of VH and VL of HV0LV0, an amino acid sequence modified to the amino acid residues of the anti-sugar chain-deficient CD27 rat monoclonal antibody KM4030 was prepared, and a three-dimensional structure model was similarly constructed.

The three-dimensional structures of the V regions of the anti-sugar chain-deficient CD27 rat monoclonal antibodies KM4030 and HV0LV0 and the modified antibodies prepared as described above were compared, and amino acid residues predicted to have an influence on the binding activity of the antibodies were identified.

As a result, of the amino acid residues in the FR of HV0LV0, those which were thought to affect the binding activity of the antibody by changing the three-dimensional structure of the antigen-binding site were selected as: ser at position 30, Val at position 48, Ser at position 49, Asn at position 77, Val at position 93, Ala at position 97 and Thr at position 117 in HV0, Ile at position 21, Pro at position 40, Val at position 58, Thr at position 85 and Tyr at position 87 in LV 0.

At least one or more of the amino acid sequences of these selected amino acid residues were modified to the amino acid residues present at the same site of the anti-sugar chain-deficient CD27 rat monoclonal antibody KM4030, thereby designing VH and VL of humanized antibodies having various modifications.

Specifically, at least one of the following amino acid modifications was introduced into the antibody VH: substitution of Ser at position 30 with Asn, substitution of Val at position 48 with Ile, substitution of Ser at position 49 with Ala, substitution of Asn at position 77 with Gly, substitution of Val at position 93 with Thr, substitution of Ala at position 97 with Thr, and substitution of Thr at position 117 with Val in the amino acid sequence represented by SEQ ID NO. 96.

In addition, for VL, at least one of the following amino acid modifications was introduced: substitution of Ile at position 21 to Leu, substitution of Pro at position 40 to Leu, substitution of Val at position 58 to Ile, substitution of Thr at position 85 to Ala, and substitution of Tyr at position 87 to Phe in the amino acid sequence shown in SEQ ID NO. 97.

Amino acid sequences of variable regions of HV2LV0, HV3LV0, HV5LV0 and HV7LV0, which were obtained by modifying at least one amino acid residue present in FR of HV0LV0, were designed, and the amino acid sequences of H chain variable regions HV2, HV3, HV5 and HV7 are represented by sequence numbers 101, 103, 105 and 107, respectively.

(2) Production and evaluation of humanized antibody against sugar chain-deficient CD27

In the case of amino acid modification of the DNA encoding the amino acid sequence of the variable region of the anti-sugar chain-deficient CD27 humanized antibody using codons used in the DNA encoding the amino acid sequence of VH or VL of anti-sugar chain-deficient CD27 rat monoclonal antibody KM4030, codons that are frequently used in mammalian cells were used for the preparation.

DNA sequences encoding the amino acid sequences of HV0 and LV0 of the humanized antibody against sugar chain defect CD27 are represented by SEQ ID Nos. 98 and 99, respectively, and DNA sequences encoding the amino acid sequences of variable regions HV2, HV3, HV5 and HV7 obtained by amino acid modification are represented by SEQ ID Nos. 100, 102, 104 and 106, respectively.

(3) Construction of cDNA encoding VH of humanized antibody against sugar chain-deficient CD27

cDNAs encoding the amino acid sequences HV0, HV5 and HV7 of the VH of the humanized antibody against sugar chain defect CD27 represented by SEQ ID Nos. 98, 104 and 106 designed in this example (1) were prepared by full-length synthesis.

(4) Construction of cDNA encoding VL of humanized antibody against sugar chain-deficient CD27

A cDNA encoding the amino acid sequence LV0 of the VL of the humanized antibody against sugar chain defect CD27 designated by SEQ ID NO. 99 designed in example (1) was prepared by full-length synthesis.

(5) Construction of anti-sugar chain-deficient CD27 humanized antibody expression vector

Various anti-sugar chain-deficient CD27 humanized antibody expression vectors were constructed by inserting a cDNA encoding any one of HV0, HV5, and HV7 obtained in examples (2) and (3) and a cDNA encoding LV0 into appropriate positions of the humanized antibody expression vector pKANTEX93 described in international publication No. 97/10354.

(6) Stable expression of anti-sugar chain-deficient CD27 humanized antibody and obtaining of purified antibody using animal cells

The stable expression of the humanized antibody against sugar chain-deficient CD27 using animal cells and the purification of the antibody from the culture supernatant were carried out by the same methods as those described in examples 6(2) and (3).

As a result, an anti-sugar chain-deficient CD27 humanized antibody HV0LV0 having VH HV0 and VL LV0 was produced; three antibodies, i.e., HV5 for VH, LV0 for VL 0, HV7 for VH, and HV7LV0 for VL 0, were used.

[ example 11]

Evaluation of Activity of anti-sugar chain-deficient CD27 humanized antibody

The activities of the anti-sugar chain-deficient CD27 chimeric antibody KM4030 obtained in example 6 and the anti-sugar chain-deficient CD27 humanized antibodies HV0LV0, HV5LV0 and HV7LV0 obtained in example 10 were evaluated in the following (1) to (5).

(1) BIACORE was used to evaluate the binding activity of the anti-sugar chain-deficient CD27 chimeric antibody and humanized antibody to human sugar chain-deficient CD27-Fc

In order to analyze the binding activity of the humanized antibody against sugar chain defect CD27 to human sugar chain defect CD27 in terms of reaction rate, the measurement was carried out in the same manner as in example 7(1) using BIACORE T100 (manufactured by BIACORE Co.).

As a result, the binding rate constant Ka1, dissociation rate constant Kd1 and dissociation constant KD (Kd1/Ka1) of each of the obtained antibodies are shown in Table 4. In addition, the sensing diagram is shown in fig. 29.

Such as a watch4, humanized antibodies HV0LV0, HV5LV0 and HV7LV0 all showed 2X 10^-9The affinity was high at mol/liter, and the antigen-binding activity was maintained at a level equivalent to that of the anti-sugar chain-deficient CD27 chimeric antibody KM 4030.

[ Table 4]

Binding Activity of anti-sugar chain-deficient CD27 chimeric antibody and humanized antibody to human sugar chain-deficient CD27-Fc

(2) Evaluation of reaction specificity of humanized antibody against sugar chain deficiency CD27 by fluorescent cell staining method (flow cytometry analysis)

The reactivity specificity of the humanized antibody against sugar chain-deficient CD27 was measured in the same manner as in example 7 (2). The cell lines used CD27/DG44-4 cells and CD27/Lec8-M19 cells, which were prepared in the same manner as in example 2 and had approximately the same antigen expression level, as the cells for analysis.

The primary antibody was prepared using humanized antibodies HV0LV0, HV5LV0 or HV7LV0 against the sugar chain-deficient CD27 to final concentrations of 0.0001. mu.g/mL, 0.001. mu.g/mL, 0.003. mu.g/mL, 0.01. mu.g/mL, 0.03. mu.g/mL, 0.1. mu.g/mL, 1. mu.g/mL and 10. mu.g/mL. The results are shown in fig. 30(a) and 30 (B).

As a result, none of the humanized antibodies HV0LV0, HV5LV0 and HV7LV0 against sugar chain-deficient CD27 bound to CD27/DG44-4 cells, while binding was observed only to CD27/Lec8-M19 cells expressing sugar chain-deficient CD 27. In addition, the reactivity of the humanized antibody against sugar chain-deficient CD27 to CD27/Lec8-M19 cells expressing sugar chain-deficient CD27 was substantially the same as that of the chimeric KM4030 antibody against sugar chain-deficient CD 27.

(3) Evaluation of antibody-dependent cellular cytotoxicity (ADCC Activity) of humanized antibody against sugar chain-deficient CD27

ADCC activities of the humanized antibodies HV0LV0, HV5LV0 and HV7LV0 against sugar chain-deficient CD27 were measured in the same manner as in example 7 (3). The target cells used were CD27/DG44-4 cells and CD27/Lec8-M19 cells prepared in the same manner as in example 2. In addition, experiments were performed under conditions where the ratio of effector cells (E) to target cells (T) was 12.5: 1. The results are shown in fig. 31.

As a result, the ADCC activity of the humanized antibody HV0LV0 was slightly decreased as compared with the anti-sugar chain-deficient CD27 chimeric KM4030 antibody, but the anti-sugar chain-deficient CD27 humanized antibodies HV5LV0 and HV7LV0 showed substantially the same or higher ADCC activity as the anti-sugar chain-deficient CD27 chimeric KM4030 antibody than the anti-sugar chain-deficient CD27 chimeric KM4030 antibody.

(4) Evaluation of complement-dependent cytotoxic Activity (CDC Activity) of anti-sugar chain-deficient CD27 humanized antibody

CDC activity of the humanized antibodies HV0LV0, HV5LV0 and HV7LV0 against sugar chain-deficient CD27 was determined by the same method as in example 7 (4). The antibody was prepared to a final concentration of 100. mu.g/mL. The results are shown in fig. 32.

As a result, the anti-sugar chain-deficient CD27 humanized antibodies HV0LV0, HV5LV0 and HV7LV0 obtained in the present invention showed stronger CDC activity than the anti-sugar chain-deficient CD27 chimeric KM4030 antibody.

(5) Evaluation of Cross-reactivity of humanized antibody against sugar chain-deficient CD27 with monkey CD27 protein

The binding activity of the anti-sugar chain-deficient CD27 humanized antibodies HV0LV0, HV5LV0 and HV7LV0 to cynomolgus monkey CD 27-expressing cells was measured by fluorescent cell staining (flow cytometry analysis) in the same manner as in example 9 (1). Cynomolgus monkey CD 27-expressing cells the cynomolgus monkey CD27/DG44 cells and cynomolgus monkey CD27/Lec8 cells prepared in example 8 were used. The results are shown in fig. 33(a) and 33 (B).

As a result, none of the humanized antibodies HV0LV0, HV5LV0 and HV7LV0 against sugar chain-deficient CD27 bound to cynomolgus monkey CD27/DG44 cells. On the other hand, humanized antibodies HV0LV0, HV5LV0 and HV7LV0 against sugar chain-deficient CD27 reacted with cynomolgus monkey CD27/Lec8 cells expressing sugar chain-deficient CD27 in the same manner as the anti-sugar chain-deficient CD27 chimeric KM4030 antibody. From the above results, it was revealed that the humanized antibodies HV0LV0, HV5LV0 and HV7LV0 against the sugar chain-deficient CD27 all showed cross-reactivity to the sugar chain-deficient cynomolgus monkey CD27 cells.

This application is based on us provisional patent application 61/290542, filed on 12/29/2009, the content of which is incorporated in the present specification by reference.

Industrial applicability

The present invention can provide a monoclonal antibody or antibody fragment that specifically recognizes CD27 containing an O-linked sugar chain to which galactose is not bound and binds to the extracellular domain, a hybridoma that produces the antibody, a DNA encoding the antibody, a vector containing the DNA, a transformant obtained by transforming the vector, a method for producing an antibody or antibody fragment using the hybridoma or transformant, and a diagnostic agent or therapeutic agent for a CD 27-related disease containing the antibody or antibody fragment as an active ingredient.

Deposit number

IPOD FREM BP-10976

Sequence Listing free text

SEQ ID NO. 1-human CD27 base sequence

SEQ ID NO. 2-human CD27 amino acid sequence

Sequence No. 3-description of artificial sequence: CD27 Forward primer

Sequence No. 4-description of artificial sequence: CD27809B

Sequence No. 5-manual sequence: CD27-A primer

Sequence number 6-description of artificial sequence: CD27-B primer

Sequence No. 7-manual sequence: primer 1

Sequence number 8-record of artificial sequence: primer 2

Sequence number 9-description of artificial sequence: g4A primer

Sequence number 10-description of artificial sequence: g4B primer

Sequence number 11-description of artificial sequence: CD27-Fc protein base sequence

Sequence number 12-description of artificial sequence: CD27-Fc protein amino acid sequence

Sequence No. 13-manual sequence: CD27-C primer

Sequence number 14-description of artificial sequence: rat IgG1 specific primer

Sequence number 15-description of artificial sequence: rat IgG2a specific primer

Sequence number 16-description of artificial sequence: rat IgG2b specific primer

Sequence number 17-description of artificial sequence: rat CH1 specific primer

Sequence number 18-description of artificial sequence: rat Ig (kappa) -specific primer 1

Sequence number 19-record of artificial sequence: rat Ig (kappa) -specific primer 2

The nucleotide sequence of SEQ ID NO. 20-KM4026VH

The base sequence of SEQ ID NO. 21-KM4027VH

The nucleotide sequence of SEQ ID NO. 22-KM4028VH

The base sequence of SEQ ID NO. 23-KM4030VH

Sequence number 24-KM4031VH

Amino acid sequence of sequence No. 25-KM4026VH

Amino acid sequence of sequence No. 26-KM4027VH

Amino acid sequence of sequence No. 27-KM4028VH

Sequence number 28-KM4030VH amino acid sequence

Amino acid sequence of sequence No. 29-KM4031VH

The nucleotide sequence of SEQ ID NO. 30-KM4026VL

The nucleotide sequence of SEQ ID NO. 31-KM4027VL

The nucleotide sequence of SEQ ID NO. 32-KM4028VL

The base sequence of SEQ ID NO. 33-KM4030VL

The base sequence of SEQ ID NO. 34-KM4031VL

Amino acid sequence of sequence No. 35-KM4026VL

Amino acid sequence of sequence number 36-KM4027VL

Amino acid sequence of sequence No. 37-KM4028VL

Amino acid sequence of sequence No. 38-KM4030VL

Sequence number 39-KM4031VL amino acid sequence

SEQ ID NO. 40-KM4026VH CDR1

SEQ ID NO. 41-KM4026VH CDR2

Sequence number 42-KM4026VH CDR3

Sequence number 43-KM4026VL CDR1

SEQ ID NO. 44-KM4026VL CDR2

SEQ ID NO. 45-KM4026VL CDR3

SEQ ID NO. 46-KM4027VH CDR1

SEQ ID NO. 47-KM4027VH CDR2

SEQ ID NO. 48-KM4027VH CDR3

SEQ ID NO. 49-KM4027VL CDR1

SEQ ID NO. 50-KM4027VL CDR2

SEQ ID NO. 51-KM4027VL CDR3

SEQ ID NO. 52-KM4028VH CDR1

SEQ ID NO. 53-KM4028VH CDR2

SEQ ID NO. 54-KM4028VH CDR3

SEQ ID NO. 55-KM4028VL CDR1

SEQ ID NO. 56-KM4028VL CDR2

SEQ ID NO. 57-KM4028VL CDR3

SEQ ID NO. 58-KM4030VH CDR1

SEQ ID NO. 59-KM4030VH CDR2

SEQ ID NO. 60-KM4030VH CDR3

SEQ ID NO. 61-KM4030VL CDR1

Sequence number 62-KM4030VL CDR2

SEQ ID NO. 63-KM4030VL CDR3

Sequence number 64-KM4031VH CDR1

SEQ ID NO. 65-KM4031VH CDR2

Sequence number 66-KM4030VH CDR3

Sequence number 67-KM4031VL CDR1

Sequence number 68-KM4031VL CDR2

SEQ ID NO. 69-KM4031VL CDR3

Sequence number 70-record of artificial sequence: KM4026VL chimeric primer 1

Sequence number 71-description of artificial sequence: KM4026VL chimeric primer 2

Sequence number 72-description of artificial sequence: KM4026VH chimeric primer 1

Sequence number 73-description of artificial sequence: KM4026VH chimeric primer 2

Sequence number 74-description of artificial sequence: KM4027VL chimeric primer 1

Sequence No. 75-manual sequence: KM4027VL chimeric primer 2

Sequence number 76-description of artificial sequence: KM4027VH chimeric primer 1

Sequence No. 77-description of artificial sequence: KM4027VH chimeric primer 2

Sequence number 78-record of artificial sequence: KM4028VL chimeric primer 1

Sequence number 79-description of artificial sequence: KM4028VL chimeric primer 2

Sequence No. 80-manual sequence: KM4028VH chimeric primer 1

Sequence No. 81-manual sequence: KM4028VH chimeric primer 2

Sequence number 82-description of artificial sequence: KM4030VL chimeric primer 1

Sequence number 83-manual sequence description: KM4030VL chimeric primer 2

Sequence number 84-description of artificial sequence: KM4030VH chimeric primer 1

Sequence number 85-manual sequence: KM4030VH chimeric primer 2

Sequence number 86-description of artificial sequence: KM4031VL chimeric primer 1

Sequence No. 87-description of artificial sequence: KM4031VL chimeric primer 2

Sequence number 88-description of artificial sequence: KM4031VH chimeric primer 1

Sequence No. 89-manual sequence: KM4031VH chimeric primer 2

Sequence number 90-manual sequence: primer mfCD2_75UTR

Sequence number 91-manual sequence entry: primer mfCD2_73UTR

SEQ ID NO. 92-Macaca fascicularis CD27cDNA sequence

Sequence number 93-manual sequence entry: primer mfCD27toKAN _5

Sequence number 94-record of artificial sequence: primer mfCD27HisKAN _3

Sequence number 95-description of artificial sequence: his-tagged cynomolgus monkey CD27cDNA sequence

Sequence number 96-description of artificial sequence: KM4030HV0 amino acid sequence

Sequence number 97-description of artificial sequence: KM4030LV0 amino acid sequence

Sequence number 98-record of artificial sequence: KM4030HV0 base sequence

Sequence No. 99-manual sequence: KM4030LV0 base sequence

Sequence number 100-description of artificial sequence: KM4030HV2 base sequence

Sequence number 101-description of artificial sequence: KM4030HV2 amino acid sequence

Sequence number 102-description of artificial sequence: KM4030HV3 base sequence

Sequence number 103-description of artificial sequence: KM4030HV3 amino acid sequence

Sequence number 104-record of artificial sequence: KM4030HV5 base sequence

Sequence number 105-description of artificial sequence: KM4030HV5 amino acid sequence

Sequence number 106-description of artificial sequence: KM4030HV7 base sequence

Sequence number 107-description of artificial sequence: KM4030HV7 amino acid sequence

PCT/RO/134 Table

Claims (23)

1. A humanized antibody or an antibody fragment thereof, wherein,

the VH of the antibody is a VH comprising an amino acid sequence of CDR 1-3 represented by SEQ ID Nos. 58-60 and the VL of the antibody is a VL comprising an amino acid sequence of CDR 1-3 represented by SEQ ID Nos. 61-63,

the antibody or antibody fragment thereof recognizes and binds to an extracellular domain of a polypeptide encoded by the CD27 gene comprising an O-linked sugar chain to which galactose is not bound.

2. The humanized antibody or antibody fragment thereof of claim 1,

the VH of the humanized antibody comprises an amino acid sequence into which at least one modification selected from the following modifications is introduced:

wherein Ser at position 30 in the amino acid sequence represented by SEQ ID NO. 96 is substituted with Asn, Val at position 48 is substituted with Ile, Ser at position 49 is substituted with Ala, Asn at position 77 is substituted with Gly, Val at position 93 is substituted with Thr, Ala at position 97 is substituted with Thr, and Thr at position 117 is substituted with Val, and,

the VL of the humanized antibody comprises an amino acid sequence into which at least one modification selected from the following modifications is introduced:

substitution of Ile at position 21 to Leu, substitution of Pro at position 40 to Leu, substitution of Val at position 58 to Ile, substitution of Thr at position 85 to Ala, and substitution of Tyr at position 87 to Phe in the amino acid sequence shown in SEQ ID NO. 97.

3. The humanized antibody or an antibody fragment thereof according to claim 1, wherein VH of the humanized antibody comprises an amino acid sequence represented by any one of sequence nos. 96, 105 and 107, and VL of the humanized antibody comprises an amino acid sequence represented by sequence No. 97.

4. A DNA encoding the humanized antibody or the antibody fragment according to any one of claims 1 to 3.

5. A recombinant vector comprising the DNA according to claim 4.

6. A transformant obtained by introducing the recombinant vector according to claim 5 into a host cell.

7. A method for producing an antibody or an antibody fragment according to any one of claims 1 to 3, comprising culturing the transformant according to claim 6 in a medium, producing and accumulating the humanized antibody or the antibody fragment according to any one of claims 1 to 3 in the culture, and collecting the antibody or the antibody fragment from the culture.

8. An immunological detection or assay method for CD27 comprising an O-linked sugar chain to which galactose is not bound, which uses the humanized antibody or the antibody fragment according to any one of claims 1 to 3.

9. A detection reagent for CD27 comprising an O-linked sugar chain to which galactose is not bound, which comprises the humanized antibody or the antibody fragment according to any one of claims 1 to 3.

10. A diagnostic agent for a disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound, comprising the humanized antibody or the antibody fragment according to any one of claims 1 to 3.

11. The diagnostic agent according to claim 10, wherein the disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound is IgA nephropathy.

12. The diagnostic agent according to claim 10, wherein the disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound is cancer.

13. A therapeutic agent for a disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound, comprising the humanized antibody or antibody fragment according to any one of claims 1 to 3 as an active ingredient.

14. The therapeutic agent according to claim 13, wherein the disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound is IgA nephropathy.

15. The therapeutic agent according to claim 13, wherein the disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound is cancer.

16. A method for diagnosing a disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound, comprising: a cell expressing CD27 comprising an O-linked sugar chain to which galactose is not bound, which is detected or measured using the humanized antibody or the antibody fragment according to any one of claims 1 to 3.

17. A method for diagnosing a disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound, comprising: the humanized antibody or the antibody fragment according to any one of claims 1 to 3, wherein CD27 comprising an O-linked sugar chain to which galactose is not bound is detected or measured.

18. The diagnostic method according to claim 16 or 17, wherein the disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound is IgA nephropathy.

19. The diagnostic method according to claim 16 or 17, wherein the disease related to CD27 comprising an O-linked sugar chain to which galactose is not bound is cancer.

20. Use of the humanized antibody or the antibody fragment according to any one of claims 1 to 3 for the production of a diagnostic agent for a disease associated with CD27 containing an O-linked sugar chain to which galactose is not bound.

21. Use of the humanized antibody or the antibody fragment according to any one of claims 1 to 3 for producing a therapeutic agent for a disease associated with CD27 containing an O-linked sugar chain to which galactose is not bound.

22. The use of the humanized antibody or the antibody fragment according to claim 20 or 21, wherein the disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound is IgA nephropathy.

23. The use of the humanized antibody or the antibody fragment according to claim 20 or 21, wherein the disease associated with CD27 comprising an O-linked sugar chain to which galactose is not bound is cancer.