JPH05192181A - Human monoclonal antibody - Google Patents

Abstract

PURPOSE:To provide a human monoclonal antibody useful as a diagnostic or treating agent for melanoma. CONSTITUTION:The objective human monoclonal antibody has a class IgM immunoglobulin H chain and a type kappa L-chain, specifically recognizes the common antigen of ganglioside GM3 (N-acetylneuraminic acid), GT1b, GD3, GD1a and GQ1b existing on the surface of melanoma cell MeWo, M12, M14 or G-361 and exhibits CDC activity on melanoma cell having the common antigen. There are also provided its production method, a human cell strain transformed with EBV producing the antibody, a composition for the diagnosis or treatment of melanoma containing the antibody, a gene containing the antibody gene, a plasmid containing the gene, a recombinant cell containing the plasmid, a process for producing an antibody with the recombinant cell, an antibody containing H chain and L chain each having V region containing a specific amino acid sequence and the use of the antibody.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention is used as a diagnostic or therapeutic drug for melanoma, and as a test drug for studying the relationship between sugar chains and canceration mechanism, the role of sugar chains in vivo, etc. It relates to a useful human monoclonal antibody, a method for producing the same, a manufacturing material, and a pharmaceutical composition.

[0002]

2. Description of the Related Art A method for establishing a cell line producing a uniform antibody having specificity for a specific antigenic determinant was reported by Milstein et al. [Nature, Vol. 25
6, P. 495-497 (1975)], qualitative and quantitative determination of trace substances became possible. On the other hand, in order to search for cancer antigens, many monoclonal antibodies specific to cancer cells were produced using this technique, and it was revealed that some of them are antibodies that recognize sugar chains of lipids or glycoproteins. I was killed. For example, as a monoclonal antibody reactive with human melanoma, an antibody reactive with ganglioside GD3 or a glycolipid of ganglioside GM3 has been obtained [J. Exp. Med. (Journal of Experimental Medicine), Vol. 155, P.I. 53
92-5396 (1983) and Gann, V
ol. 75, P.I. 418-426 (1984)]. These antibodies are useful for diagnosing cancer and have been attempted to be used for therapy. However, since these antibodies are heterologous (mouse) proteins for humans, there is a problem that they may cause serious side effects such as anaphylaxis and other allergic reactions when transferred into the human body.

As a means for solving such a problem, human-derived immunoglobulins are being developed, not from xenogeneic animals, and several human monoclonal antibodies against melanoma have already been reported [P.
roc. Natl. Acad. Sci. , USA (Proceedings of the National Academy)
Of Sciences of the USA), Vo
l. 84, P.P. 2416-2420 (1987), the same V
ol. 79, P. 7626-7633 (1982) and the same Vol. 80, P.I. 5392-5396 (198
3)].

[0004]

However, at present, these antibodies do not always give satisfactory results for all melanoma patients. The reason for this is that even if a single antibody cannot respond to changes in the sugar chain structure on the cancer cell surface during treatment and the diversity of sugar chain structure on the cancer cell surface, even if the antibody reacts with cancer cells. It does not exhibit effective cell killing activity.

[0005]

In order to solve such problems, it is considered as a useful means to provide an antibody that recognizes a common part of various sugar chain structures that can be expressed by melanoma. From the above, the present inventors recognized a sugar chain structure on melanoma different from known human monoclonal antibodies, and complement-dependent cell killing (Complement D) to cells having the recognized antigen on the surface thereof.
dependent Cytotoxicity, hereinafter C
Efforts have been made to produce and search for human monoclonal antibodies having activity (abbreviated as DC).

The present inventors collected lymphocytes from the peripheral blood of a melanoma patient and used the lymphocytes for Epstein-
Epstein-Barr virus (hereinafter EB
Abbreviated as V) to obtain an EBV-transformed human cell line, from which CD for melanoma is obtained.
When a human cell line producing an antibody having C activity was established and the reaction specificity of this antibody for various cells was examined, it was found that this antibody was MeWo, M12, M14, G-361.
, While showing CDC activity against melanoma, it shows almost no CDC activity against human normal skin fibroblasts, human adenocarcinoma, human neuroblastoma, African green monkey kidney cells, etc. Obtained the finding that it is useful as a therapeutic drug,
The sugar chain recognized by this antibody is ganglioside GM3.
(N-acetylneuraminic acid), GT1b, GD3, G
Since it is D1a and GQ1b, the knowledge that it is a novel antibody was also obtained, and the present invention was completed. Furthermore, the present inventors
Obtained mRNA from BV-transformed human cell line to obtain cDNA
Through this, the amino acid sequence of each V region of H chain and L chain which is antigen-specific for this human monoclonal antibody and the nucleotide sequence encoding the same were elucidated, and the present invention was also completed by a gene manipulation technique.

[0007]

DISCLOSURE OF THE INVENTION The present invention provides that immunoglobulin class is IgM and L chain type is κ, and MeWo, M
A melanoma that recognizes the common antigen of ganglioside GM3 (N-acetylneuraminic acid), GT1b, GD3, GD1a and GQ1b existing on the surface of melanoma cells of 12, M14 or G-361, and further has this common antigen on its surface. Human monoclonal antibody showing CDC activity against cells, but hardly showing against human normal skin fibroblasts, human adenocarcinoma, human neuroblastoma or African green monkey kidney cells, a process for producing the same, A diagnostic or therapeutic composition for melanoma containing the produced EBV-transformed human cell line and this antibody.

The present invention also provides the human monoclonal antibody gene, a plasmid containing the same, a recombinant cell containing the same, a method for producing an antibody by this recombinant cell, and the V region of the H chain is represented by SEQ ID NO: 1 in the sequence listing. First Gl
The 1st As represented by SEQ ID NO: 2 in which the V region of the L chain contains an amino acid sequence from u to 117th Val
An antibody containing the amino acid sequence from p to the 114th Arg, a diagnostic composition or a therapeutic composition containing this antibody.
Further, the present invention provides the first Gl represented by SEQ ID NO: 1.
A gene encoding the amino acid sequence from u to the 117th Val, 123 to 12 represented by SEQ ID NO: 1
Nucleotide sequence from the 5th GAG to the 471 to 473 GTC, the 1st Asp represented by SEQ ID NO: 2
To a gene encoding the amino acid sequence from Arg to 114th Arg or 66 to 6 represented by SEQ ID NO: 2
It is also a gene containing the nucleotide sequence from the 8th GAC to the 405th to 407th CGA.

The antibodies of the present invention belong to the IgM class.
The basic structure of IgM is composed of a pentamer of units consisting of two heavy chains (H chains) and two light chains (L chains) and one J chain. The L chain is a polypeptide having a molecular weight of about 25,000, and the H chain is a polypeptide having a molecular weight of about 51,000. The J chain is a polypeptide having a molecular weight of about 15,000 and has a sugar chain. Approximately 1 from the N-terminus of the H and L chains
The 00 amino acid sequence is called the variable region (V region),
It is antigen-specific. Approximately 400 each following V area
And about 150 amino acid sequences are called constant region (C region) and are constant in class and subclass.

Here, the production and establishment of an EBV-transformed human cell line producing the antibody of the present invention and the acquisition of the antibody will be described.

First, lymphocytes including B lymphocytes are collected from the peripheral blood of melanoma patients by a normal gravity centrifuge method, etc., and antibodies are stably produced from these lymphocytes and continue to proliferate indefinitely. Transformation with EBV is performed to obtain the cell line. EBV is known as a virus that transforms human normal B lymphocytes into a proliferative cell line [Nature, Vol. 269, P.I. 410 (1
973)]. Examples of the EBV-containing material include marmoset cell line B95-8 [Proc. Natl. Acad.
Sci. , USA, Vol. 87, P.I. 3333-33
37 (1990)]. When infected with EBV, a T cell proliferation inhibitor such as cyclosporin A is added to a lymphocyte suspension containing the target B lymphocytes, and the cells are further cultured. Proliferation-inducing IL-6 is added and cultured to obtain an EBV-transformed human cell line [Science, Vol. 239, P.I.
502-504 (1988)].

Next, with respect to the culture solution of the cell line obtained by the above operation, melanoma cells (MeWo, M12, M14)
Alternatively, G-361) is used to screen for the presence or absence of CDC activity to establish the target EBV-transformed human cell line. These melanoma cells are identified as follows. MeWo cell Delivered from Tokyo Metropolitan Institute of Clinical Medicine M12 cell Delivered from Kyushu University Institute of Biological Defense Medicine M14 cell Same as above G361 cell ATCC No. CRL1424 [Am
erican Type Culture Colle
deposit (ATCC) deposit No. ]

Further, the EBV-transformed human cell line obtained by the above procedure is cultured to produce the desired antibody. Purification of the antibody in the culture broth can be carried out by combining known biochemical and immunochemical techniques, such as ammonium sulfate precipitation, gel filtration, ion exchange chromatography and affinity chromatography.

The purified antibody obtained as described above is homogeneous and has high titer, and is suitable for use as a biochemical preparation. For example, after filtration and sterilization operation using a membrane filter or the like, it is mixed with itself or a suitable pharmacologically acceptable carrier, excipient, diluent, etc. with an amount of antibody sufficient for diagnosis or treatment. It is possible to

The therapeutic composition can be prepared, for example, as an injection, but the effective dose depends on the degree of symptoms of the patient, age and weight, administration method, etc. Usually determined by
The range is from about 10 ng to about 100 mg / kg body weight. Since the antibody of the present invention shows specificity to various melanoma cells, the antibody can be labeled with, for example, an enzyme or a radioisotope and used as an immunodiagnostic agent.

[0016] Next, the embodiment of the antibody gene of the present invention will be described. Regarding the gene manipulation technique, the methods and means described in many documents are applied. The documents are listed for reference. T. Maniatis et al: Molecular
r Cloning, Cold Spring Har
bor Laboratory (1982), R.B. Wu
et al: Methods in Enzymol
Ogy, 100 and 101 (1983), R.A. Wue
tal: Methods in Enzymolog
y, 153, 154 and 155 (1987), F.I. M.
Ausubel et al: Current Pro
tocols in Molecular Biolo
gy, John Wiley & Sons, Inc.

For the antibody gene of the present invention, a cDNA library is prepared using mRNA derived from EBV-transformed human cells producing the above-mentioned human monoclonal antibody, and cD encoding the desired human monoclonal antibody is prepared from the cDNA library.
A plasmid containing NA is isolated and its nucleotide sequence is determined. The method and operation are carried out according to a conventional method as described later in Examples 2 and 3. mRNA is the EBV
It is obtained by culturing transformed human cells, lysing the cells with a predetermined guanidinium isothiocyanate solution, and then extracting. CDNA using this mRNA as a template
Is prepared and incorporated into an appropriate vector to prepare a cDNA library, and plasmid DNA containing this cDNA is immobilized on a nylon filter. Regarding the C region of the H chain of human IgM, its nucleotide sequence and amino acid sequence are [T. H. Rabbitts, A.M. Foster, C.I.
P. Milstein: Nucleic Acids
Res. , Vol. 9, P.I. 4509-4524 (19
81)] for the C region of the L chain [P. A. Hie
ter, E.I. E. Max, J .; G. Seidman,
J. V. Maizel Jr. , P .; Leder: Ce
ll, Vol. 22, P.I. 197-207 (198
0)] and for the J chain [J. E. Mole,
A. S. Bown, J. et al. C. Bennett: Bio
chemistry, Vol. 16, P.I. 3507-3
513 (1977)],
An oligonucleotide corresponding to a part of those domains is chemically synthesized. These are used as probes, and a strain containing a plasmid DNA that reacts with the probe is isolated by screening. A plasmid DNA fragment containing the H chain and L chain cDNAs of the antibody is obtained from these strains, and the nucleotide sequence of the cDNA portion is determined.

In the gene of the antibody of the present invention, the V region of the H chain is represented by the first Glu to 1 in which the V region of the H chain is represented by SEQ ID NO: 1.
It is a nucleotide sequence encoding an amino acid sequence up to the 17th Val and a V region of the L chain is a nucleotide sequence encoding an amino acid sequence from the 1st Asp to the 114th Arg represented by SEQ ID NO: 2. In addition, the V region of the H chain is represented by SEQ ID NO: 1 to the 123rd to 125th GAs.
66 to the base sequence from G to the 471st to 473rd GTC, and the V region of the L chain is represented by SEQ ID NO: 2
It is also the base sequence from the 68th GAC to the 405th to 407th CGA.

On the other hand, since the nucleotide sequence and amino acid sequence of the antibody gene of the present invention have been elucidated, the antibody gene of the present invention can be easily produced by the methods and operations commonly used in chemical synthesis of genes. For example, a gene containing an appropriate base sequence (upper chain) that encodes a predetermined amino acid sequence represented by SEQ ID NO: 1 or 2 and a base sequence (lower chain) complementary to it can be obtained by converting those genes into several oligonucleotides. It can be produced by a method of separately chemically synthesizing and sequentially sequentially connecting each block. As a method for synthesizing the oligonucleotide, the diester method [H. G. Khorana: Some Recent
Developments in Chemistry
of Phosphate Esters of B
iological Interest, John W
iley and Sons, Inc. , New Yo
rk (1961)], triester method [R. L. Let
Singer et al. Am. Chem. So
c. , Vol. 89, P.I. 4801 (1967)] and the phosphite method (MD Matteucci et al.
al: Tetrahedron Lett. , Vol.
21, P.I. 719 (1980)], but the phosphite method using a fully automatic DNA synthesizer is preferably used from the viewpoint of operability and the like.

The synthesized oligonucleotide is purified by a conventional purification method such as high performance liquid chromatography using a reverse phase chromatography column and electrophoresis using a polyacrylamide gel. The oligonucleotides are then phosphorylated, eg using T4 polynucleotide kinase, annealed and then ligated using T4 DNA ligase. Here, the oligonucleotide is divided into several blocks, sequentially ligated to obtain a desired antibody gene sequence, cleaved with a restriction enzyme or blunted with T4 DNA polymerase, and then purified by electrophoresis or the like. Regarding the obtained DNA fragments, for example, pUC8, ibid 9, ibid 1
8 and 19 [J. Messing et al: Gen
e, Vol. 19, P.I. 259 (1982)], and competent cells are transformed by a conventional method and cloned. Plasmid DNA is extracted and purified from the obtained clone according to a known method, and it is checked whether or not the nucleotide sequence of the DNA fragment inserted into the vector has achieved the desired gene sequence. Each part of the achieved antibody gene is excised from the plasmid vector containing each by using a restriction enzyme, ligated again into the vector, and then incorporated to obtain a plasmid vector having the desired antibody gene. The plasmid vector thus obtained is cleaved with a restriction enzyme and then separated and purified by gel electrophoresis to obtain a desired antibody gene.

Then, the antibody gene of the present invention is appropriately inserted into an expression vector to construct an antibody expression vector. The expression vector has a promoter region and an enhancer sequence upstream of the translation initiation codon (ATG), and has a poly A signal region downstream of the translation termination codon (TAA, TGA or TAG). When inserting the antibody gene into the expression vector, the predetermined H chain and L chain, which are antibody genes, and if necessary, the J chain genes can be integrated into one plasmid, or into separate plasmids. You can also Plasmids are pRc / CMV, pRc / RS
V etc. can be used.

An antibody can be expressed by introducing the expression vector into a host cell by a known method and culturing the transformed host cell. E. coli, yeast or human or other mammalian cells are used as the host cell, and the plasmid is also selected to be applied to this host cell. When an antibody non-secreting human or other mammalian cell having a J chain gene is used as the host cell, the expression vector containing only the H chain and L chain genes according to the present invention is introduced into the host cell. Thus, the antibody of the present invention can be expressed.

By culturing the transformed host cell, the desired antibody is produced and accumulated in the culture to be separated. When animal cells are used as the host cells, they can be generally cultured for several days at 37 ° C. in an atmosphere of 5% CO ₂ according to a conventional method.

After culturing, the antibody of the present invention is usually obtained by centrifuging to collect the culture solution and remove the cells. Further, the thus obtained product can be purified by a combination of filtration with a filter, affinity chromatography, cation or anion exchange chromatography and other conventional methods for separating and purifying proteins. The antibody thus obtained can be formulated as in the case of the aforementioned antibody produced by culturing an EBV-transformed human cell line and used as a diagnostic or therapeutic composition. Since the antibody of the present invention is of human origin and has low antigenicity to the human body, it is useful as a therapeutic composition for melanoma.

[0025]

EXAMPLES The present invention will be described in more detail with reference to examples below, but these do not limit the present invention.

Example 1 (1) Preparation and establishment of EBV-transformed human cell line Target B lymphocytes from peripheral blood of melanoma patients by density centrifugation using Ficoll-Hypaque as a density gradient solution Were collected. About 0.5 ~ lymphocytes obtained here
An appropriate amount of the culture supernatant of the marmoset cell line B95-8 was added to 5 × 10 ⁷ cells, and the cells were infected at 37 ° C. for 3 to 4 hours with gentle shaking. Then, the lymphocytes were suspended in RPMI1640 culture solution containing 20% fetal bovine serum (hereinafter abbreviated as FCS-RPMI medium), and cyclosporin A was further added to 2 μm.
IL-6, which is added at a concentration of g / ml and simultaneously induces the growth of EBV-transformed cells, is 1.9 × 10 ³ U.
To a concentration of 1 ml / ml, and culture in a 96-well cell culture plate for 2-3 weeks (incubator, 3
7 ° C., 5% CO ₂ ) EBV-transformed human cell line was obtained.

The supernatant of the culture fluid of the cell line obtained by the above operation was screened for the presence or absence of CDC activity against melanoma, and cloned by the limiting dilution method.
The BV transformed cell line AKH11 was established.

The CDC activity was measured by the following method.

1.5 × 10 ⁶ melanoma cells (Me
Wo, M12, M14, G-361) 5% F each
Addition to CS-RPMI medium (0.3 ml), ⁵¹ Cr
0.5mCi / m of sodium chromate labeled with
It was added to give a concentration of 1 and incubated at 37 ° C. for 1 hour. As each melanoma cell, the one specified as described above was used. Cells containing ⁵¹ Cr were washed 4 times with Hank's solution, and the cells were washed with 5% FCS-RPMI.
Resuspended in medium (2 ml). This cell suspension 0.
To 1 ml, 0.1 ml of the supernatant obtained by culturing the above-mentioned transformed cell line was diluted to an antibody dilution of 1/300, 1/100, 1/30, 1/10 and 1/3 and added. After reacting at 37 ° C for 30 minutes, centrifuge (5000r
0.15 ml of the supernatant was removed. An equal amount of rabbit complement serum was mixed with the remaining 0.05 ml fraction containing cells, reacted at 37 ° C. for 45 minutes, and then centrifuged (5000 rpm, 5 minutes). This supernatant 0.05
The radioactivity (cpm) of ml (A) and the remaining fraction (B) containing cells was measured by a gamma counter, and the concentration of the antibody at each concentration in the supernatant obtained by culturing the EBV-transformed cell line for each melanoma cell was measured. CDC activity (% cell lysis) was calculated as follows and these results are shown in FIG.

Cell lysis% = [(A-Ac) × 2 / (A +
B) -Ac × 2] × 100 (where Ac represents the background radioactivity of the control which reacted only with complement)

Further, instead of melanoma cells, human normal skin fibroblasts, human adenocarcinoma (SW-1
3), human neuroblastoma (IMR-32) and African green monkey kidney cells (Vero cell) were used to measure radioactivity in the same manner as described above to calculate CDC activity (% cell lysis). The results are shown in FIG. These cells were used as specified below. Normal human skin fibroblasts: From the Cell Engineering Center of Osaka University SW-13 cells: ATCC No. CCL105 (AT
CC Deposit No. ) IMR-32 cells: ATCC No. CCL127 (A
TCC deposit no. ) Vero cell: ATCC No. CCL81 (A
TCC deposit no. )

In FIG. 1, MeWo results (a), M12 results (b), M14 results (c), G-361 results (d), human normal skin fibroblast results. (E), the result of SW-13 is (f), I
The results of MR-32 are shown as (g) and the results of Verocell are shown as (h), respectively.

From these results, it can be seen that the antibody of the present invention specifically recognizes melanoma cells and thus can be used as a diagnostic agent for melanoma. In addition, since it binds to melanoma cells and exhibits CDC activity, it can be used as a therapeutic drug for melanoma.

(2) Acquisition and identification of monoclonal antibody produced by EBV-transformed cell line AKH11 EBV-transformed cell line AKH11 established in (1) above
Was cultured for 1 week in a 20% FCS-RPMI medium using a 10 cm Petri dish (in an incubator, 37 ° C.,
5% _CO 2). This culture solution is centrifuged (1000 rp
for 5 minutes) to obtain a culture supernatant containing the desired antibody.

The immunoglobulin H chain class and L chain type of the antibody of the present invention will be specified below.

Goat anti-human IgA antibody (Kappel; N
o. 0201-0021), goat anti-human IgG antibody (the same; No. 0201-0121), goat anti-human IgM
Antibody (No. 0201-0201), goat anti-human I
g-κ antibody (same; No. 0201-0241) or goat anti-human Ig-λ antibody (same; No. 0201-0281)
To 0.15 ml of CNBr-activated Sepharose 4B (trade name: manufactured by Pharmacia) in which each of the above was coupled to each dilution of the above culture supernatant (1 to 100-fold dilution)
An absorption experiment was conducted by adding 0.5 ml and reacting at 4 ° C. for 16 to 18 hours. Each sample was centrifuged (1000 rpm, 5 minutes), and the CDC activity of each supernatant was measured using MeWo as target cells. From these results, this antibody was specifically absorbed only by goat anti-human IgM antibody and goat anti-human Ig-κ antibody. Therefore, it was revealed that the antibody of the present invention has an immunoglobulin H chain class of IgM and an L chain type of κ.

The specification of the sugar chain antigen recognized by the antibody of the present invention will be described below.

The sugar chain antigen recognized by the antibody of the present invention was identified by enzyme immunoassay as described below using various glycolipids as antigens. First, after adsorbing 0.5 μg / well of various glycolipids on a 96-well microplate, 50 mM Tris-buffered saline (pH: 0.1% bovine serum albumin) was added.
7.2) blocked non-specific binding (4 ° C,
18 hours). Next, 50 μl / well of a 4-fold diluted solution of the supernatant obtained by culturing AKH11 cells was added and reacted at 37 ° C. for 1 hour. Furthermore, a 200-fold diluted peroxidase-labeled goat anti-human IgM antibody (manufactured by Kappel: No. 3601-020) was used.
1) 50 μl / well was added and reacted at 37 ° C. for 1 hour. Ortho-phenylenediamine 0.4 as enzyme substrate
100 μl / well of a substrate buffer solution (0.1 M citric acid, 0.2 M disodium phosphate, 0.005% hydrogen peroxide solution) containing mg / ml was reacted, and the absorbance at 492 nm was measured to obtain various The reaction of the antibody against the glycolipid antigen was examined.

As a result, the antibody of the present invention was found to be ganglioside GM3 (N-acetylneuraminic acid)>GT1b> G.
D3>GD1a> GQ1b were reacted in the order of strength, but lactosylceramide, GM3 (N-glycosylneuraminic acid), GM2, GM1, GD2, GD1b, 9-o-A.
It was found not to react with c-GD3, SSEA-1 (Le ^x ), and sialylparagloboside. Below, AK
The antibody of the present invention produced by H11 cells is designated as AKH11 antibody.

The above measurement was carried out according to the procedure described in Protein Nucleic Acid Enzyme, No. 31, Supplement, P. 183-192 (1987) as a reference.

The following examples were carried out according to the following experimental documents unless otherwise specified. (1) T. Maniatis, E .; F. Fritsc
h, J. Sambrook: Molecular Cl
oning, Cold Spring Harbor
Laboratory (USA) (2) F.I. M. Ausabel, et al. : Cur
rent Protocols in Molecule
ar Biology, John Wiley & Son
s, Inc. (USA)

Example 2 Preparation of cDNA Library Using mRNA Derived from AKH11 Antibody-Producing Cells EBV-transformed cell line A obtained in Example 1 (1) above
KH11 in 20% FCS-RPMI medium at 37 ° C for 9
Cultured for 6 hours. Thereafter, 1 × 10 ⁸ AKH11 cells were treated with 20 mM sodium acetate 0.1 mM DTT, 0.
It was homogeneously dissolved using a 20-G needle in 3.5 ml of a 4M guanidinium isothiocyanate solution containing 5% N-lauryl sarcosine. 5.7M of the mixture
Cesium chloride solution (5.7M cesium chloride, 0.1m
M EDTA) 1.5 ml and layer at 35 ° C. at 35 ° C.
Centrifugation was performed at 000 rpm for 21 hours to obtain a precipitate containing mRNA. This precipitate was added to 5 mM EDTA, 1% SDS.
It was dissolved in a 10 mM Tris-HCl (pH 7.4) solution containing EDTA and precipitated with ethanol to obtain mRNA.

This mRNA was adsorbed on an oligo (dT) cellulose column and 0.5 M NaCl, 1 mM ED was added.
TA, containing 0.1% sodium lauryl sarcosine 2
The mRNA containing poly (A) was fractionated by elution with a 0 mM Tris-HCl (pH 7.6) solution. This mRNA was further precipitated with ethanol and 1 mM
10 mM Tris-HCl (pH 8.
0) dissolved in solution. Using this poly (A) mRNA as a template, cDNA is synthesized with a cDNA synthesis kit (Amer S
manufactured by ham: RPN1256Y)
After adding EcoRI linker to DNA, pUC1
It was incorporated into 9 vector to prepare a cDNA library. The plasmid vector containing the circularized cDNA is E. coli JM109 (Takara Shuzo: E. coli JM109
Competent cells) to obtain a cDNA library using E. coli JM109 as a host.

Example 3 Isolation of plasmid containing cDNA of AKH11 antibody and determination of its nucleotide sequence The AKH11 cDNA library using Escherichia coli JM109 was nylon filter (manufactured by Amersham:
Hybond-N), and a replica filter was prepared in which two filters each using this filter as a master filter were set as one set. E. coli on this replica filter was treated with a denaturing solution (0.5 M NaOH,
Filter paper (Whatman) moistened with 1.5M NaCl)
3MM paper) for 7 minutes, then neutralizing solution [1.5M NaCl, 0.5M Tr
is-HCl (pH 7.4)] on filter paper (same as above) for 3 minutes, then fresh neutralizing solution on filter paper (same as above) for 3 minutes, then double SSC Solution (0.06M NaCl, 6mM
After washing with sodium citrate, UV irradiation (UV
Stratalinker type 1800, manufactured by SCS) was used to immobilize the plasmid DNA on the filter.

On the other hand, the nucleotide sequence of the human IgM antibody μ chain gene that has been reported [T. H. Rabbitts,
A. Forster, C.I. P. Milstein: Nu
cleic Acids Res. , Vol. 9, P.I.
4509 (1981)], part of the CH1 domain (3
Oligonucleotide 5'-AATTTCC of SEQ ID NO: 3 in the sequence listing, which is complementary to the nucleotide sequence corresponding to the 3rd to 52nd positions)
ACAGGAGACGAGG-3 ′ and a part of the CH4 domain (a nucleotide sequence corresponding to the 338th to 357th positions) which is complementary to the oligonucleotide 5′- of SEQ ID NO: 4 in the sequence listing.
AGGCCAGCAGGGTCAGTAGC-3 ′ was synthesized and both were used as probes for screening μ chain cDNA. In addition, in the same manner as described above, human IgM
Nucleotide sequence of antibody κ chain gene [P. A. Hieter,
E. E. Max, J .; G. Seidman, J .; V. M
aizel Jr. , P .; Leder: Cell, Vo
l. 22, P.I. 197 (1980)] (345-
Oligonucleotide 5'-AAGATGAA of SEQ ID NO: 5 in the sequence listing, which is complementary to the nucleotide sequence corresponding to position 364)
GACAGATGGTGC-3 'and (647-666
5'-ACTTCTCCCTCC of SEQ ID NO: 6 in the sequence listing, which is complementary to the nucleotide sequence corresponding to
TAACACTCT-3 'was synthesized and both were combined with κ chain cDNA
It was used as a probe for screening A. The 5'ends of these oligonucleotide probes were labeled with T4 polynucleotide kinase and [γ-32p] ATP to ^{32 '.}
Labeled with P. The labeled probes were separately associated with a replica filter on which plasmid DNA was immobilized.

The association reaction was performed with 5 × Denhardt's 0.1% SDS containing 10 μCi of labeled probe.
5-fold SS supplemented with 0 μg / ml denatured salmon sperm DNA
It was carried out in 10 ml of C (0.15 M NaCl, 0.015 M sodium citrate) solution at 40 ° C. for 16 hours. After the reaction, 6 times SSC containing 0.1% SDS (0.18MN
aCl, 0.018M sodium citrate) solution at room temperature for 30 minutes three times, and at 43 ° C. for 60 minutes twice twice [Experimental Manual (1) Molecu
lar Cloning p. 309]. A radioautogram was taken from the washed filter, and a strain that reacts with two kinds of probes was selected by superimposing a set of radioautograms of two replica filters. According to this method, E. E. coli that reacts with the JM109 strain and a probe of the κ chain gene portion. coli JM1
09 strains were obtained.

Plasmid DNA was prepared from these strains by the alkaline method [H. C. Birnboim, J .; Dolly: N
ucleic Acids Res. , Vol. 7,
P. 1513 (1979)]. The plasmid DNA was cleaved with the restriction enzyme EcoRI (Takara Shuzo) and fractionated by agarose gel electrophoresis, and then the DNA fragments were separated from the agarose gel by a nylon filter (Amers).
manufactured by ham: Hybond-N). When this filter was allowed to associate with the oligonucleotide probe described above, the DNA fragment reacted with the probe. Therefore, of these, the largest EcoRI DNA fragment (cDNA part)
Harboring the E. coli JM109
2-14 (which reacted with the probe of the μ chain gene portion) and E. coli JM109K5-1-1 (κ
Those that reacted with the probe of the chain gene portion) were selected. These were designated as JMp2-14 and JMpK5-1-1 at the Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology, respectively.
As a deposit (Ministry of Industrial Science and Technology No. 3638 and No. 36)
39). In addition, these contain p2-14 and pK5-1-1 as recombinant plasmids, respectively.

E. E. coli JM109 2-14 and E. coli. The nucleotide sequence of the cDNA portion of the plasmid DNA (p2-14 and pK5-1-1) of E. coli JM109K5-1-1 was determined by the dideoxynucleotide synthetic chain termination method [J. Messing et al: Nucleic A
cids Res. , Vol. 9: P.I. 309 (198
1)]. As a result, the document [T.
H. Rabbitts, A.M. Foster, C.I. P. M
ilstein: Nucleic Acids Re
s. , Vol. 9, P.I. 4509-4524 (198
1) and P.I. A. Hieter, E.I. E. Max,
J. G. Seidman, J .; V. MaizelJ
r. , P .; Leder: Cell, Vol. 22, P.I.
197-207 (1980)], the base sequence and amino acid sequence of the μ chain V region of the AKH11 antibody were determined to be SEQ ID NO: 1 in the sequence listing, and the base sequence and amino acid sequence of the κ chain V region were determined. And described in SEQ ID NO: 2 in the sequence listing.

Example 4 Expression Vector 2-14Rc / C
Construction of MV Plasmid p2-14 prepared in Example 3 was digested with restriction enzyme N
Cut with coI (Takara Shuzo) and SalI (Takara Shuzo),
A DNA fragment containing the μ chain gene of the AKH11 antibody was isolated. This DNA fragment was reacted with Klenow fragment (Takara Shuzo) to blunt the ends, and then a synthetic linker was ligated with T4 DNA ligase. The synthetic linker has an oligonucleotide of 8 bases having a sequence represented by 5'-CTCTAAAG-3 'and a sequence complementary to the oligonucleotide, and is represented by 5'-CACA-3' on its 3'side. Sequence added 12
It was obtained by synthesizing base oligonucleotides, phosphorylating their 5'ends with polynucleotide kinases respectively, and then annealing both. T4DN
A ligase was inactivated by heat treatment at 65 ° C. for 10 minutes, and then AKH to which a linker was bound by agarose electrophoresis.
A DNA fragment containing the μ chain gene of 11 antibody was isolated. On the other hand, plasmid pRc / CMV (Invitrogen
Digested with the restriction enzyme BstXI and mixed with this open-loop plasmid and a DNA fragment containing the μ chain gene of the AKH11 antibody to which the above synthetic linker is bound, and T4 DNA ligase is allowed to act on the AKH11 downstream of the cytomegalovirus promoter. The expression vector 2-14Rc / CMV in which the μ chain gene of the antibody was inserted was constructed.

Example 5 Expression vector K5-1-1Rc
/ CMV Construction The plasmid pK5-1-1 prepared in Example 3 was digested with the restriction enzyme EcoRI to separate a DNA fragment containing the AK chain gene of the AKH11 antibody. This DNA fragment was reacted with Klenow fragment to blunt the ends, and then a synthetic linker (the same as in Example 4 above) was added to T4.
Ligated by DNA ligase. After inactivating T4 DNA ligase by heat treatment at 65 ° C. for 10 minutes, a DNA fragment containing a κ chain gene of the AKH11 antibody to which a linker was bound was separated by agarose electrophoresis. On the other hand, the plasmid pRc / CMV was cut with the restriction enzyme BstXI,
AK to which this open-circle plasmid and the above synthetic linker are bound
A DNA fragment containing the κ chain gene of the H11 antibody was mixed, and T
4DNA ligase was allowed to act to construct an expression vector K5-1-1Rc / CMV in which the AKH11 antibody κ chain gene was inserted downstream of the cytomegalovirus promoter.

Example 6 Transfection of Expression Vector into Mouse Myeloma Cell Line XA653 Mouse myeloma cell XA653 (ATCC No. C
RL-1580) was cultured in DMEM medium containing 10% fetal bovine serum, the cells were replaced with serum-free DMEM medium, and 5 ml of a cell suspension having a cell density of 5 × 10 ⁵ cells / ml was added to a 6 cm dish. 5 μl of the expression vector 2-14Rc / CMV (2
8.5 μg) DNA, 5 μl of expression vector K5-1-
1 Rc / CMV (18.5 μg) DNA, 20 μl D
EAE-dextran and 70 μl TE [10 mM
Tris-HCl (pH 8.0), 1 mM EDTA]
Was added. Finally, add chloroquine to a final concentration of 100
After adding so that the concentration became μM, the cells were cultured for 4 hours, then the culture solution was replaced with a 10% FCS-RPMI medium, and further cultured for 4 days. The culture solution was collected and collected, and it was confirmed by transfecting the expression vector whether or not the recombinant AKH11 antibody was produced by measuring the biological activity.

Example 7 Confirmation of biological activity of recombinant AKH11 antibody Recombinant AKH contained in the culture solution obtained in Example 6
The biological activity of the 11 antibody was measured by measuring the CDC activity, and the IgM amount of the recombinant AKH11 antibody produced was further measured by the ELISA method (enzyme linked immunosorbent assay).

The CDC activity was measured by the method described in Example 1 (1) above. As a result, melanoma cells M
Cell lysis rate of eWo is 41.2%, melanoma cell M1
The% cytolysis of 2 was 43.8% and that of melanoma human neuroplastoma (IMR-32) was 0%.
%Met.

Next, the amount of IgM was measured by the ELISA method. First, IgG fraction goat anti-human IgG (Fab fragment specific, manufactured by Kappel: No. 0201-0101) 1 on a 96-well microplate.
A 0 μg / ml solution was adsorbed at 100 μl / well for 18 hours at 4 ° C., and then nonspecific binding was blocked with a borate buffer solution (pH 8.0) containing 1 mg / ml ovalbumin (4 ° C. for 18 hours). Next, 50 μl / well of a culture solution containing recombinant AKH11 antibody was added, and the mixture was reacted at 37 ° C. for 1 hour, and further 10 μg / ml peroxidase-labeled goat anti-human IgM (μ chain specific, manufactured by Kappel: No. 3).
601-0201) 50 μl / well was added and reacted at 37 ° C. for 1 hour. Substrate buffer containing 0.1 mg / ml of ortho-phenylenediamine as an enzyme substrate (0.1 M
Citric acid, 0.2M disodium phosphate, 0.00
5% hydrogen peroxide solution) 100 μl / hole react, 450
The absorbance at nm was measured to calculate the amount of IgM. As a control for calculating the amount of IgM, human IgM (manufactured by Kappel:
No. 6001-1590) was used. Recombinant AK
The amount of IgM in the culture medium containing the H11 antibody was 100 ng / m
It was l.

From these results, the expression vector 2 was added to the mouse myeloma cell line XA653, which is originally a non-secretory antibody type.
-14Rc / CMV and expression vector K5-1-1Rc /
By transfecting CMV, AKH11
It can be demonstrated that an antibody can be produced that has similar action characteristics to the antibody.

[0056]

The antibody of the present invention is ganglioside GM3.
(N-acetylneuraminic acid), GT1b, GD3, G
It is a novel one that recognizes a common antigen of D1a and GQ1b, and exhibits CDC activity against melanomas such as MeWo, M12, M14, and G-361, while human normal skin fibroblasts, human adenocarcinoma, human Since it exhibits almost no CDC activity against neuroplastoma, African green monkey kidney cells, etc., it recognizes melanoma specifically and has CDC activity. Further, since it is of human origin, it has low antigenicity to the human body and has almost no side effects. Further, by using the gene of the antibody of the present invention, the encoded antibody can be produced by using a gene manipulation technique. Therefore, the antibody of the present invention is useful as a diagnostic or therapeutic composition for melanoma, and also as a test drug for studying the relationship between sugar chains and canceration mechanism, the role of sugar chains in vivo, etc. It is useful.

[Sequence list]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[Brief description of drawings]

FIG. 1 is a diagram showing the CDC activity (% of cell lysis) against various cells by the antibody of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12N 15/00 8931-4B 15/13 15/70 G01N 33/53 N 8310-2J D 8310-2J 33/574 B 9015-2J 33/577 B 9015-2J // (C12P 21/08 C12R 1:91)

Claims (21)

[Claims] 1. The immunoglobulin class is IgM, the L chain type is κ, and MeWo, M12, M1.
4 or ganglioside GM3 (N-acetylneuraminic acid) present on the surface of melanoma cells of G-361, G
A human monoclonal antibody, which recognizes a common antigen of T1b, GD3, GD1a and GQ1b. 2. MeWo, M1 having said common antigen
2, M-14 or G-361 showing complement-dependent cytocidal activity against melanoma cells, but against human normal skin fibroblasts, human adenocarcinoma, human neuroblastoma or African green monkey kidney cells Is a human monoclonal antibody according to claim 1, which shows almost no complement-dependent cell-killing activity. 3. The human monoclonal antibody according to claim 1, wherein the antibody is an AKH11 antibody. 4. B lymphocytes are collected from the peripheral blood of a melanoma patient and transformed with Epstein-Barr virus to establish a human cell line producing the human monoclonal antibody according to claim 1, which is used as a medium. A method for producing a human monoclonal antibody, which comprises culturing at 1 to recover the antibody. 5. A diagnostic or therapeutic composition for melanoma, comprising the human monoclonal antibody according to claim 4 in an amount sufficient for diagnosing or treating melanoma. 6. A human cell line transformed with Epstein-Barr virus, which produces the human monoclonal antibody according to claim 1. 7. An antibody gene comprising the gene encoding the human monoclonal antibody according to claim 1 or 2. 8. A gene, which comprises a gene encoding an amino acid sequence from Glu at position 1 to Val at position 117 at Val represented by SEQ ID NO: 1 in the sequence listing. 9. 123 represented by SEQ ID NO: 1 in Sequence Listing
~ 125th GAG to 471st to 473th GTC
A gene characterized by including the nucleotide sequences up to. 10. A gene comprising a gene encoding an amino acid sequence from the 1st Asp to the 114th Arg represented by SEQ ID NO: 2 in the sequence listing. 11. 66 represented by SEQ ID NO: 2 in the Sequence Listing
A gene comprising a base sequence from the 68th GAC to the 405th to 407th CGA. 12. The method according to claim 7, 8, 9, 10 or 11.
A recombinant plasmid comprising the gene of 13. The recombinant plasmid according to claim 12, wherein the recombinant plasmid is p2-14 or pK5-1-1. 14. The expression vector is 2-14Rc / CMV.
Alternatively, the recombinant plasmid according to claim 12, which is K5-1-1Rc / CMV. 15. A recombinant cell comprising the recombinant plasmid according to claim 12, 13 or 14. 16. The recombinant cell according to claim 15, wherein the host cell is Escherichia coli or a mammalian cell. 17. The recombinant cell is JMp2-14 or JM.
pK5-1-1 (Ministry of Mechanical Engineers Article No. 3638 or No. 36)
39), the recombinant cell according to claim 15. 18. The recombinant cell is an expression vector 2-14R.
The recombinant cell according to claim 15, which comprises c / CMV or K5-1-1Rc / CMV. 19. A method for producing a human monoclonal antibody, which comprises culturing the recombinant cell according to claim 15, 16 or 18 in a medium to recover the antibody. 20. The V region of the H chain contains the amino acid sequence from Glu at the 1st position to Val at the 117th position represented by SEQ ID NO: 1 in the sequence listing, and the V region of the L chain is at the 1st position represented by SEQ ID NO: 2. A human monoclonal antibody comprising an amino acid sequence from Asp to Arg at position 114. 21. A diagnostic or therapeutic composition for melanoma, comprising the human monoclonal antibody according to claim 20 in an amount sufficient for diagnosing or treating melanoma.