JPH05304987A - Human type monoclonal antibody and gene coding the same, hybridoma and antitumor agent - Google Patents

Abstract

PURPOSE:To provide the novel monoclonal antibody useful for producing an antitumor agent by bonding the antibody to a liposome in which a carcinostatic agent is emcapsuled. CONSTITUTION:The human type monoclonal antibody, e.g. GAH antibody (IgG), is produced by the cell fusion of a cancer patient-originated lymphocyte with a mouse myeloma cell, and specifically binds to the membrane surface antigen of a cancer cell. An example of antigen is produced by culturing a hybridoma GAH prepared by fusing the mouse myeloma cell with the lymphocyte originated from a lymph node belonging to the cancer site of the cancer patient.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel monoclonal antibody which can be used for diagnosis and treatment of cancer, and D which encodes it.
The present invention relates to a hybridoma that produces NA and its antibody, and an antitumor agent obtained by combining the antibody with a liposome encapsulating a cancer drug and the like.

[0002]

2. Description of the Related Art At present, there is no anti-cancer agent showing a sufficient effect as a therapeutic agent for solid cancer. On the other hand, there has been a concept of targeting a drug for a long time by concentrating the drug on the tissue or organ to which it is applied, and it has been expected that solid cancer can be treated if the drug can be specifically concentrated on the cancer by this method. .. Established production method of mouse monoclonal antibody that can be mass-produced (Milstein & Koehler, Nature
e, 1975), many attempts have been made to accumulate anticancer agents, toxins and the like in cancer tissues by utilizing their specificity, and their effects have been recognized.

Up to now, the binding of an antibody and a drug has been attempted by a method of chemically modifying the drug, that is, a method of directly binding the antibody and the drug, a method of binding via a water-soluble polymer such as dextran, and the like. Has been. However, it has been pointed out that these methods have a problem that the amount of a drug that can be bound per antibody molecule is small and that the activity is reduced due to the modification of the drug.

On the other hand, as a means for delivering a large amount of a drug without modifying it, a method of encapsulating a drug in a liposome and binding an antibody to the surface thereof, that is, an antibody-bound liposome has been proposed, which has many excellent antitumor effects. It has been reported [Konno et al., Cancer Research 47 , 44.
71 (1987), Hashimoto et al., JP-A-58-134032.
Gazette].

[0005]

However, regarding the antibody, in the actual treatment, it is difficult to continuously administer the mouse monoclonal antibody due to side effects such as anaphylaxis caused by the immune response [A. Lo Bu
gli et al. Proc. Natl. Acad. Sci.
U. S. A. , 86, 4220, (1989)], the use of a humanized monoclonal antibody is more preferable. However, compared with the method for producing a mouse monoclonal antibody, the method for producing a human monoclonal antibody is difficult to carry out an active immunization method for efficiently obtaining human B cells that produce the desired antibody. It has been difficult to obtain a humanized monoclonal antibody that reacts sufficiently with tumor cells, because an efficient method for infinite proliferation of erythrocytes has not been established.

[0006]

[Means for Solving the Problems] The inventors of the present invention are eager to provide a humanized monoclonal antibody which enables the targeting treatment of an anticancer drug, toxin or the like to cancer tissue without the above problems. As a result of the examination, in particular, by producing a hybridoma that produces a novel human monoclonal antibody having the antigen on the membrane surface of cancer cells, and further binding the antibody to a ribosome encapsulating an antitumor agent, etc., it is effective in targeting therapy. It was found that an antitumor agent that can be used was obtained, and the present invention was completed.

[0007] That is, the gist of the present invention is a human-type monoclonal antibody produced by a fused cell of a lymphocyte derived from a cancer patient and a mouse myeloma cell, which specifically binds to a membrane surface antigen of the cancer cell, and a gene encoding the same. , A hybridoma producing the monoclonal antibody, and an antitumor agent containing the monoclonal antibody. Examples of the human monoclonal antibody of the present invention include those in which the heavy chain variable region contains the amino acid sequences of SEQ ID NOs: 13, 14 and 15 in the sequence listing, and more specifically, the heavy chain variable region is the sequence in the sequence listing. SEQ ID NO: 19 of the sequence listing, which contains the amino acid sequences of SEQ ID NOs: 16, 17 and 18, and the light chain variable region is
Those containing the amino acid sequences of 20 and 21, the heavy chain variable region containing the amino acid sequences of SEQ ID NOs: 22, 23 and 24 of the sequence listing, and the light chain variable region of the sequence listing SEQ ID NO: 2
Examples thereof include those containing the amino acid sequences of 5, 26 and 27, but are not limited thereto. Of course, in the present invention, the monoclonal antibody of the present invention also includes those in which alterations such as substitution, insertion, deletion or addition of a part of amino acids have been carried out within the range not impairing the reactivity with the above-mentioned antigen.

The present invention will be described in detail below. The hybridoma according to the present invention comprises A. The method of Imam et al. [Can
cer Research 45 , 263 (1985)]
First, lymphocytes are isolated from cancer-affected lymph nodes extracted from a cancer patient and fused with mouse myeloma cells using polyethylene glycol. Using the obtained hybridoma supernatant, a hybridoma that produces an antibody that is positive by an enzyme immunoassay against various cancer cell lines fixed with paraformaldehyde is selected and cloned.

Further, from the supernatant of the hybridoma, a conventional method [R. C. Duhamel et al. Immunol. M
Methods 31 , 211 (1979)], the monoclonal antibody was purified, labeled with a fluorescent substance, and the reactivity to live cancer cell lines, various red blood cells, white blood cells, etc. was detected by flow cytometry to detect anticancer cell lines. For reactive red blood cells and white blood cells,
Antibodies showing no reactivity are selected. Further, by comparing the reactivity with respect to the cancer cells isolated from the cancer tissue extracted from the cancer patient and the normal cells isolated from the non-cancerous part of the same tissue of the same patient, the cancer cells have a larger amount of antibody. Combine,
An antibody that does not react with normal cells or is as reactive as an antibody derived from a healthy person is selected.

The nucleotide sequence of the DNA encoding the antibody produced by the hybridoma thus selected is, for example,
It is obtained by the following method. From antibody-producing hybridomas, the guanidine thiocyanate-lithium chloride method [Ca
sara et al., DNA, 2 , 329 (1983)] mR.
NA is prepared and its cDNA library is made using oligo (dT) primers. Then the cDNA
(DG) tailing is performed, and an antibody is prepared by PCR using poly C that hybridizes to this dG tail and a sequence part common to the human antibody heavy chain gene and light chain gene whose genes have already been obtained as a probe. Code c
Amplify the DNA. After that, the ends of the DNA are blunted, and the DNA cut out from the gel by electrophoresis is cut into p
Insert into cloning vector such as UC119
Ger et al.'s dideoxy method [Proc. Natl. Aca
d. Sci. U. S. A. , 74 , 5463, (197
7)] determines the base sequence.

As a result, the antibody of the present invention includes, for example, those in which the heavy chain variable region contains the amino acid sequences of SEQ ID NOs: 13, 14 and 15 in the sequence listing, and specifically, the heavy chain variable region is in the sequence listing. SEQ ID NO: 1 of the sequence listing including the amino acid sequences of SEQ ID NOs: 16, 17 and 18
Those containing the amino acid sequences of 9, 20 and 21, the heavy chain variable regions containing the amino acid sequences of SEQ ID NOs: 22, 23 and 24 of the sequence listing, and the light chain variable regions of SEQ ID NOs: 25, 26 and 27 of the sequence listing. However, the present invention is not limited to these.

The amino acid sequences of SEQ ID NOs: 13, 14 and 15 in the Sequence Listing are the amino acid sequences of 16, 17 and 18 and the amino acid sequences of 22, 23 and 24 are called "hypervariable regions" among the heavy chain variable regions. Similarly, the amino acid sequences of SEQ ID NOs: 19, 20 and 21 in the sequence listing, 25,
The amino acid sequences of 26 and 27 are called "hypervariable regions" among the light chain variable regions. Such a region determines the specificity of the immunoglobulin as an antibody and the binding affinity between the antigenic determinant and the antibody. Therefore, in the present invention, the heavy chain variable region other than the hypervariable region may be derived from another antibody.

Particularly preferred monoclonal antibodies in the present invention are those in which the heavy chain variable region and the light chain variable region are represented by the amino acid sequences of SEQ ID NOs: 5 and 6, respectively, and the amino acid sequences of SEQ ID NOs: 11 and 12. It is represented by. The nucleotide sequences of the constant regions of the heavy and light chains are Nucleic Acids Research
14 , 1779 (1986), The Journal.
of Biological Chemistry
257 , 1516 (1982) and Cell 22 ,
197 (1980).

The present antibody is obtained by culturing a hybridoma producing the present antibody using eRDF containing fetal bovine serum, RPMI 1640 culture medium, or the like, or SEQ ID NO: 3 in the sequence listing.
And 4, or 9 and 10 or the like, the DNA encoding the variable region represented by the nucleotide sequence and the DNA encoding the constant regions of the heavy chain and the light chain are further ligated, respectively, to chemically synthesize the gene, and the expression of the gene is expressed. Various known publicly available expression vectors, for example, pKCRH2 [Sanshin et al., Nature,
307, 605, pKCR which can be constructed by the procedure shown from (1984)] in FIG. 1 or FIG. 2 (Delta] E) /
Inserted in H and pKCRD, CHO cells (Chinese
It can be obtained by expressing in a host such as Hyster ovary cell). For example, H at both ends of the heavy chain gene
in d III pKCR those obtained by adding the site (ΔE) / H
At the Hind III site of S.
A selectable marker gene such as the DHFR gene is inserted into the al I site. On the other hand, an Eco RI site added to both ends of the light chain gene was inserted into the Eco RI site of pKCRD, and DHFR was also added to the Sal I site of this plasmid.
Insert the gene. Both plasmids are CHOdhfr
^- [Urlaub G. & Chasin L. et al. A. P
roc. Natl. Acad. Sci. U. S. A. ，
77 , 4216 (1980)] and the like by the calcium phosphate method, and by further selecting antibody-producing cells from cells that grow in αMEM culture solution containing no nucleotides. The antibody is purified by adsorbing Protein A to a column or the like in which Protein A is bound to a support such as cellulofine or agarose and eluting it from the culture solution in which these cells are cultured.

In the antitumor agent of the present invention, the liposome carrying the above-mentioned antibody comprises a double lipid layer, and the lipid layer may be a multi-layer or a single layer. As the constituent components, phosphatidylcholine, cholesterol, phosphatidylethanolamine, and phosphatidic acid as a substance giving an electric charge are used. For example, as the usage ratio of the constituents, cholesterol is 0.3 to 1 mol, preferably 0.4 to 1 mol of phosphatidylcholine.
-0.6 mol, phosphatidylethanolamine is 0.01-0.2 mol, preferably 0.02-0.1 m
For ol and phosphatidic acid, a composition ratio of 0 to 0.4 mol, preferably 0 to 0.15 mol can be used.
A known method can be used as a method for producing liposomes. For example, the lipid mixture obtained by removing the solvent is emulsified with a homogenizer or the like and freeze-thawed to obtain multilamellar liposomes. Further, in order to adjust to an appropriate particle size, a method of ultrasonic treatment, high speed homogenization, or pressure filtration with a membrane having uniform pores [Hope M. J. La Bi
ochimica et Biophysica Ac
ta 812 , 55 (1985)] and the like. At this time, liposomes having a size of 30 nm to 200 nm are selected as a preferable size.

Examples of the drug to be encapsulated in liposomes are anticancer agents such as adriamycin, daunomycin, mitomycin, cisplatin, pinklistin, evirubicin, methotrexate, 5Fu and maculacinomycin, toxins such as ricin A and diphtheria toxin, and antisense R.
NA or the like can be used. The drug can be encapsulated in the liposome by hydrating the lipid with an aqueous drug solution. For adriamycin, daunomycin, and epirubicin, a remote loading method utilizing a pH gradient (Lawrence DM et al., Cancer).
r Research 49 , 5922 (1989)).

As a method for binding the monoclonal antibody to the surface of the liposome, there are a method of inserting a purified substance with a hydrophobic substance to insert it into the liposome, a method of crosslinking phosphatidylethanolamine and the antibody with glutar, and the like. Preferably, after thiolation of the antibody, a liposome containing a maleimide group-introduced lipid is prepared, and a toxin for a cancer drug or a tumor cell is bound to the surface of the liposome by reacting with the liposome having the maleimide group. You can It is also possible to modify the surface of the liposome by reacting the remaining maleimide group with a compound containing a thiolated polyalkylene glycol moiety.

To give a thiol group to an antibody, N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), iminothiolane, mercaptoalkylimidate, etc., which is usually used for thiolation of protein with respect to solid amino group, is used. The method using the compound of 1) or the method of reducing the endogenous dithiol group of the antibody to a thiol group is used, and the method of using the endogenous thiol group is more preferable from the viewpoint of maintaining the activity. An antibody is converted into F (ab) ' _{2 by} an enzyme such as pepsin, further reduced by dithiothreitol (DTT), etc., and converted into Fab' to newly generate 1 '.
~ 3 thiol groups can be subjected to a binding reaction with liposomes. The binding between the maleimide group-containing liposome and the thiolated antibody is a neutral buffer solution (pH 6.5-7.5).
It is achieved by reacting for 2 to 16 hours.

For the formulation of the antitumor agent of the present invention, a known method, that is, a dehydration method (JP-A-2-502348),
A method of adding a stabilizer and using it as a liquid preparation (Japanese Patent Laid-Open No. 64-9-9).
No. 331) and a freeze-drying method (Japanese Patent Laid-Open No. 64-9931) can be used. The antitumor agent of the present invention can be used by an intravascular administration method or a local administration method such as intraperitoneal administration, and the dose thereof can be optimized depending on the drug contained in the liposome. Taking an adriamycin inclusion body as an example, the dose can be 50 mg / kg or less, preferably 10 mg / kg or less, more preferably 5 mg / kg or less as the amount of adriamycin.

[0020]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the invention is not limited thereto unless it exceeds the gist.

Example 1 (Human Monoclonal Antibody GAH by Cell Fusion of Lymphocytes Derived from Cancer Patients' Lymph Nodes with Mouse Myeloma)
(1) Preparation of lymphocytes (1) Preparation of lymphocytes Lymph nodes belonging to the cancer excised from a colorectal cancer patient are finely disentangled with scissors and a scalpel, and then the culture medium A (eRDF) is used.
The cells were dispersed using a stainless net in [Far East Pharmaceutical Co., Ltd.] +50 μg / ml gentamicin sulfate. The cell suspension was centrifuged at 1000 rpm for 10 minutes to remove the supernatant, further suspended in fresh culture medium A and washed by centrifugation to finally obtain 2.6 × 10 ⁷ cells. ..

(2) Cell fusion Lymphocytes are polyethylene glycol (Boehringer /
Mouse myeloma cells (manufactured by Mannheim) (1 x 1
0 ⁷ cells) according to a conventional method. The fused cells contained 5 μM hypoxanthine, 0.04 μM aminopterin, 1.6 μM thymidine, and 10% fetal calf serum (FCS) in culture medium A (culture medium containing HAT) and the number of lymphocytes was 5. Suspend to a density of 4 × 10 ⁵ / ml, inoculate 100 μl each into a 96-well plate,
Cultured in a CO ₂ incubator at 7 ° C. The culture solution was replaced with the above-mentioned HAT-added culture solution in an amount of half each time, and the culture was continued until hybridoma colonies appeared. The appearance of hybridoma colonies was confirmed in all wells. Regarding the culture supernatant of each well, according to the method of Test Example 1 below, a fixed cancer cell line, that is, a colon cancer line C
-1 [Sato et al., History of Medicine, 96 , 876, (197
6)] [obtained from Institute for Immunological Biology], gastric cancer strain MKN45
[Naito et al., Cancer and Chemotherapy, 5 , 89, (1978)]
The reactivity to [obtained from Institute for Immunological Biology] was measured. The appearance rate of positive wells was 7.
3% (35 wells), 4.6 for MKN45 strain
% (22 wells), and 6 wells showed reactivity with both cell lines. Hybridomas were cloned from the wells showing reactivity with both cell lines. Cloning was carried out by the limiting dilution method.
This was repeated and the hybridoma clone GAH was established.

Example 2 (Purification and labeling of monoclonal antibody GAH) (1) Culture of hybridoma GAH and monoclonal G
Purification of AH antibody Fetal bovine serum was applied to a protein A-agarose [Repligen] column, and the column was passed through to prepare a serum from which substances adsorbed to the protein A-agarose column were removed. For culturing the hybridoma GAH, an eRDF (Kyoto Pharmaceutical Co.) culture solution containing 3% of this serum was used. The GAH antibody was purified by subjecting the culture solution in which the hybridoma GAH was cultured to a protein A-agarose column to adsorb the antibody, followed by elution. By using the above-mentioned serum for culturing, a serum-derived antibody and a purified GAH antibody free from a substance adsorbed to protein A-agarose were obtained. The GAH antibody was confirmed to be pure IgG by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

(2) Fluorescent labeling of GAH antibody The purified GAH antibody was added to Coons A. et al. H. FITC (fluorescein isothiocyanate), which is a fluorescent substance, was labeled by the method described in 1. Carbonate buffer (pH 9.5)
The antibody was dialyzed and reacted with the FITC solution, and then the fluorescently labeled antibody and free FITC were separated by gel filtration. The absorbance, OD _{280 nm} and OD _{495 nm} of the fraction from which the fluorescently labeled antibody had been collected were measured to calculate the labeling rate. The binding molar ratio of the labeled antibody to the FITC (F / P
The ratio) was 0.93.

Test Example 1 (Study on Reactivity of Human Monoclonal Antibody to Cancer Cell Line) (1) Cancer Cell Line and Maintenance As human cancer cell lines, colon cancer line C-1 and gastric cancer line MKN4
5 cell lines were used. These cell lines are culture medium B
(10% FCS-containing eRDF) was maintained and grown under the conditions of 37 ° C. and 5% CO ₂ .

(2) Examination of reactivity to cancer cell line a. Measurement of Reactivity Using Fixed Cancer Cell Lines Cancer cell lines were 96-well plates for 3-4 days,
After culturing to a single layer and removing the supernatant, 10 mM phosphate buffer pH 7.4, 0.15 M NaCl solution (P
After washing twice with BS), 2% -paraformaldehyde was fixed (room temperature, 20 minutes). After washing 5 times with PBS, PBS containing 5% -BSA (bovine serum albumin)
The solution was added at 200 μl / well and left at 37 ° C. for 2 hours for blocking. This plate is 5 with PBS
After washing twice, 50 μl of hybridoma culture supernatant was added thereto. After reacting at 37 ° C. for 2 hours, the plate was washed 5 times with PBS, and 50 μl of goat antibody against human antibody conjugated with alkaline phosphatase (1000-fold dilution, manufactured by Capel) was added, and the reaction was carried out at 37 ° C. for 1 hour. It was After the reaction
After washing 5 times with PBS, 25 mM p-nitrophenyl phosphate-containing 0.05 M carbonate buffer-1 mM
After adding 50 μl / well of MgCl (pH 9.5) and reacting at room temperature for 1 hour to overnight, the absorbance at 405 nm was measured by a microplate photometer [Corona]. As in (2) of Example 1 above, the cultured cancer cell lines, C-1 and MKN4, whose reactivity was examined
Cloning is performed from the well where positive is confirmed in 5,
A hybridoma strain GAH was obtained. Similar reactivity was obtained with the antibody purified from the culture supernatant of GAH.

B. Reactivity to Live Cancer Cells The supernatant of cancer cells cultured in a flask or a petri dish was removed, 0.02% -EDTA-containing PBS solution was added, and the mixture was left at room temperature for 30 minutes to suspend the cells. The cells were centrifugally washed with culture medium B, so that the cell density of about 1 × 10 ⁶ / 200μl, of Example 2 Fluorescence-labeled GAH antibody obtained in (2) (final concentration 50 [mu] g / ml) It was suspended in the contained serum (derived from a healthy person) and reacted at 0 ° C. for 60 minutes. Then, the mixture was centrifuged at 2000 rpm for 2 minutes to remove the supernatant, and the cells were suspended in 1 ml of PBS and washed by centrifugation. The cells were suspended in 300 μl of PBS containing 10 μg / ml propidium iodide (PI). did. This cell suspension is flow cytometer (FC
M) and FACS440 (manufactured by Becton Dickinson), and the amount of fluorescence (FITC and PI) bound to each cell was measured. The dead cells contained in the system were PI
Are taken into the nucleic acid and emit fluorescence of PI, so that dead cell groups having fluorescence of PI can be removed in data processing. A marker (quantitative kit) of fluorescent amount (five kinds) [manufactured by Ortho Diagnostic Systems] was applied to FCM under the same conditions, and the average bound FITC amount per cell was calculated using it as an index, and The average number of bound antibodies per living cancer cell was calculated from the value and the F / P ratio of the labeled antibody calculated in (2) of Example 2, and the results are shown in Table 1.

[0028]

[Table 1] Table 1 ───────────────────────────────── Antibodies Cancer cell lines ─────── ──────────────────── GAH Control IgG ─────────────────────────── ────── MKN45 3.5 × 10 ⁴ 0.15 × 10 ⁴ C-1 0.6 × 10 ⁴ <0.1 × 10 ⁴ ──────────────── ────────────────── When a GAH antibody is used, IgG derived from healthy human serum used as a control for gastric cancer cell lines and colon cancer cell lines
About 6 to 23 times as much antibody as the antibody (fluorescently labeled by the same method as GAH) bound.

Test Example 2 (Reactivity of Human Monoclonal Antibody GAH to Blood Cells) From peripheral blood of 7 healthy subjects and 3 cancer patients,
Kinoshita's method [separation of erythroid cells, the new edition of Japan Hematology
13 , 800 (1979)]. For white blood cells, peripheral blood of healthy individuals was collected by adding heparin, 2 ml of 6% dextran physiological saline solution was added to 10 ml, mixed, and allowed to stand at room temperature for 50 minutes to obtain a plasma layer, 1500 rpm, 5 minutes White blood cells were obtained by centrifugation. The reactivity to these cells was measured by FCM as in the method of measuring the reactivity to live cancer cell lines. However, PI is not added. Regarding white blood cells, the forward scatter and side scatter of the cells in the FCM revealed that the main cell type, lymphocyte (limphocyt).
e), granulocyte, monocyte (m)
onion) and platelets [Bio / Tec
hnology 3 , 337 (1985)], and the reactivity to each of them was examined. The results are shown in Table 2.

[0030]

[Table 2] Table 2 ────────────────────────────────── Antibodies ──────── ─────────────────── GAH Control IgG ───────────────────────────── ───── White blood cells Small lymphocytes Negative Negative Condylar spheres 0.49 × 10 ^{4 *} 0.48 × 10 ^{4 *} Monocytes 0.41 × 10 ^{4 *} 0.43 × 10 ^{4 *} Blood platelets Negative negative erythrocytes (10 types) Negative Negative ───────────────────────────────── *: Average number of antibody bound per cell GAH antibody was not found to react with red blood cells and small lymphocytes. The reactivity of granulocytes and monocytes was equivalent to that of the control antibody (same as in Test Example 1).

Test Example 3 (Reactivity of human monoclonal antibody GAH to cells derived from fresh cancer tissue and non-cancerous tissue) To examine the cancer cell binding specificity of GAH antibody, it was removed at the same time as the cancer tissue of a cancer patient. Cells were isolated from fresh tissues of the same patient and the same organ, and the reactivity of GAH antibody to each cell was measured. Isolation of cells from the tissue was performed according to the method of Tokita et al. [Clinics of Cancer, 32 , 1803 (1986)]. Place the tissue on a Teflon sheet laid on a rubber plate, beat it with a razor to shred it, transfer it to a 1 mm stainless steel mesh, sieve it in a petri dish containing the culture solution, and remove the microcell clumps that have passed through the mesh. The liquid was centrifuged at 1000 rpm, the floating fat and the necrotic portion suspended were discarded, and the centrifugal washing was repeated. Cell clumps were pumped and dispersed with a syringe attached to a 23 gauge cathelan needle. The reactivity to the cells thus obtained was measured by FCM as in the case of examining the reactivity to the above-mentioned live cancer cell line. The results are shown in Table 3.

[0032]

[Table 3] Table 3 ─────────────────────────────────── Escherichia coli gastric cancer (Borr.IV) antibody ─ ─────────────────────────── Cancer Non-cancer Cancer Non-cancer ────────────────── ────────────────── GAH 1.1 × 10 ⁴ 0.03 × 10 ⁴ 180 × 10 ⁴ 4.6 × 10 ⁴ Control 0.15 × 10 ⁴ 0.04 × 10 ⁴ 3.5 × 10 ⁴ 0.9 × 10 ⁴ IgG ─────────────────────────────────── *: Average antibody binding per cell Indicates a number. The average number of bound antibodies to cancer tissue-derived cells of GAH antibody was remarkably higher than the number of bound to non-cancerous part-derived cells, and it was 51 times for gastric cancer and 7 times for colon cancer even when compared with control antibody. It was suggested that the GAH antibody recognizes an antigen that appears more frequently on the membrane of cancer cells.

Example 3 (Determination of subclass of human monoclonal antibody GAH light chain) The GAH antibody obtained in (1) of Example 2 was subjected to SDS-PAGE in a reducing state to separate it into a heavy chain and a light chain. The electrophoresed protein is transferred to a transfer membrane (Poly
vinylide-ne difluoride film)
After blotting on [Millipore] and blocking this membrane with a 5% BSA solution, a goat antibody against human κ chain or human λ chain to which peroxidase was bound [Cappel] was reacted. After washing, as an enzyme substrate, 0.
A 0.05% (w / v) 4-chloronaphthol solution containing 015% H ₂ O ₂ was reacted. As a result, it was shown that the light chain of the GAH antibody reacted with the anti-human κ chain antibody, a band appeared due to color development, and was a κ chain.

(Acquisition of human monoclonal antibody GAH gene and determination of nucleotide sequence) a. Polymerase chain reaction (hereinafter abbreviated as PCR)
Preparation of cDNA encoding the antibody GAH by the method The guanidine thiocyanate-lithium chloride method (D) from the GAH antibody-producing hybridoma obtained in Example 1 (2)
RNA having poly (A) was prepared according to NA 2 , 329 (1983)) as follows. Hybridoma 1x
10 ⁷ cells were treated with 5 M guanidine thiocyanate, 10 mM
EDTA, 50 mM Tris-HCl (pH 7) and 8
% (V / v) β-mercaptoethanol was solubilized in 7.5 ml of the solution, and cesium chloride was further added so as to be 1 g / 2.5 ml and mixed. 8.0 ml of this solubilized product was gently placed on 3.5 ml of 5.7 M cesium chloride solution contained in a centrifuge tube, and Hitachi RP
After centrifugation at 30,000 rpm for 23.5 hours with an S40T rotor, RNA was recovered as a precipitate. The RNA precipitate was treated with 0.1% sodium lauryl sulfate, 1 mM EDT.
A, dissolved in 400 μl of a solution containing 10 mM Tris-hydrochloric acid (pH 7.5), extracted with phenol-chloroform, and recovered by ethanol precipitation. About 64 μg of the obtained RNA was dissolved in 40 μl of a solution containing 10 mM Tris-hydrochloric acid (pH 8.0) and 1 mM EDTA. From 21 μl of this, about 2.64 μg of mRNA having poly (A) was obtained by using an mRNA purification kit (manufactured by Pharmacia).

1.1 μg of this poly (A) mRNA was added to 1
Dissolved in 0 μl of water, oligo d (T) 12-18 primer (Pharmacia) 1.5 μg, 10 mM 4d
NTP [manufactured by Takara Shuzo] 3 μl, reverse transcriptase [manufactured by Life Science] 40u, R
NA-degrading enzyme inhibitor [manufactured by Takara Shuzo] 30u, 5 times concentration of reverse transcriptase buffer [250 mM Tris-hydrochloric acid (pH 8.
3), 40 mM magnesium chloride, 250 mM potassium chloride] 6 μl, and water to make a total of 30 μl system,
The reaction was carried out at 41 ° C for 1 hour. Ethanol precipitation was performed from the above reaction solution to obtain cDNA.

The obtained cDNA was dissolved in 15.3 μl of water. To this solution, a 5-fold concentrated terminal deoxynucleotide transferase buffer solution [250 mM Tris-hydrochloric acid (pH
7.5), 50 mM magnesium chloride] 4.8 μl, terminal deoxynucleotide transferase (manufactured by Pharmacia)
12 u, 10 mM dGTP [Takara Shuzo] 2.4 μl
Was added to make a total of 24 μl, and the mixture was reacted at 37 ° C. for 1.5 hours to add poly d (G) to the 3 ′ end of cDNA. Then, the enzyme was inactivated by heating at 70 ° C for 15 minutes.

PCR was carried out using the thus obtained cDNA as a substrate. PCR was performed using Perkin Elmer Cetus DNA Cycler (Perkin Elmer Cetus DNA T
The thermal cycler was used according to the instructions for use. That is, poly C (15 nucleotides) 4 that hybridizes to the dG tail added to the 3'end of the cDNA as a primer for amplifying the cDNA encoding the variable region of the heavy chain in 2 μl of the above reaction termination solution
0 pmoles and human IgG antibody are all common. Part of the variable region (1 of the amino acid sequence of SEQ ID NO: 5
13-119) to a single-stranded DNA primer (SEQ ID NO: 1,37 nucleotides) corresponding to the region spanning the constant region [Nucleic Acids Rear]
ch 14 , 1779 (1986)] 25 pmole
s, and poly C 40 pmole as a primer for amplifying the cDNA encoding the variable region of the light chain.
s and a single-stranded DNA primer (SEQ ID NO: 2, corresponding to a region spanning a part of the J region of human κ chain (113th to 114th of the amino acid sequence of SEQ ID NO: 6) to the constant region).
21 nucleotides) (The Journal of
Biological Chemistry 257 ,
1516 (1982), Cell 22 , 197 (19).
80)) 40 pmoles, 10 times concentration of PCR buffer (100 mM Tris-HCl (pH 8.3), 500 mM)
Potassium chloride, 15 mM magnesium chloride, 0.1%
(W / v) gelatin) 10 μl, 10 mM 4dNTP
Water was added to 2 μl of [Takara Shuzo] and 2.5 u of Taq DNA polymerase (Takara Shuzo) to make a 100 μl system. Reaction is at 94 ° C for 1 minute (denaturation step), 55
After 30 cycles of incubation at 2 ° C. for 2 minutes (annealing step), 72 ° C. for 3 minutes (extension step), incubation was further performed at 72 ° C. for 7 minutes. The obtained reaction solution was ethanol-precipitated, the precipitate was dissolved in 30 μl of water, and Klenow fragment [Takara Shuzo] 2u, 1 mM 4dNTP 4 μl, a 10-fold concentrated end blunting buffer (500 mM Tris-HCl (pH 7.
6), 4 μl of 100 mM magnesium chloride) was added to make a total reaction system of 40 μl, and the reaction was performed at 37 ° C. for 30 minutes.
A cDNA having a blunt-ended single strand was obtained.

B. Determination of nucleotide sequence of cDNA As a result of analysis of the obtained cDNA solution by 2% agarose electrophoresis, a band of about 500 bp was recognized. This approximately 500 bp band was cut out from an agarose gel according to a conventional method, and cloned into pUC119 of the cloning vector.
It was inserted into the Sma I site and its nucleotide sequence was determined by the dideoxy method. As a result, in the base sequence of the PCR fragment, the base sequences encoding the variable regions of the heavy chain and the light chain were as shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The amino acid sequences of the variable regions of the heavy chain and the light chain of the GAH antibody produced by the hybridoma predicted from the determined nucleotide sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. Also, from the results of the base sequence,
The subclass of GAH antibody was found to be IgGl. This amino acid sequence and base sequence were clarified D
The NA fragment can be used for DN without repeating the above method.
It can be obtained with good reproducibility using the A synthesizer.

Example 4 (Human Monoclonal Antibody 1-3 by Cell Fusion of Lymphocytes Derived from Cancer Patients' Lymph Nodes with Mouse Myeloma)
-1 Establishment of -1 Producing Strain) (1) Preparation of Lymphocytes Example 1 was obtained from lymph nodes belonging to the cancer excised from a lung cancer patient.
Lymphocytes were prepared in the same manner as in (1) above, and 3 × 10 ⁷ cells were finally obtained.

(2) Cell fusion Lymphocytes are polyethylene glycol (Boehringer /
Mouse myeloma cells (8 x 1) manufactured by Mannheim
0 ⁶ cells) and fused according to a conventional method. The fused cells were suspended in a HAT-added culture medium at a density of 5.2 × 10 ⁵ / ml in the same manner as in (2) of Example 1, and
Each 96 μl-plate was inoculated with 00 μl of each well, and when appropriate, half the amount was replaced with the above HAT-added culture medium, and the culture was continued until hybridoma colonies appeared. The appearance of hybridoma colonies was confirmed in all wells. With respect to the culture supernatant of each well, a reaction against a fixed cancer cell line, that is, a colon cancer cell line C-1 and a gastric cancer cell line MKN45 was carried out by the method described in (2) of Test Example 1 as in (2) of Example 1. The sex was measured. The appearance rate of positive wells was 16.3% (94 wells) for the C-1 strain and 6.3% (36 wells) for the MKN45 strain, showing reactivity to both cell lines. The wells shown were 4 wells. Hybridomas were cloned from the wells showing reactivity with both cell lines. Cloning was performed 3 times by the limiting dilution method, and the hybridoma clone 1-
3-1 was established.

Example 5 (Purification and Labeling of Monoclonal Antibody 1-3-1) (1) Culture of Hybridoma 1-3-1 and Purification of Monoclonal Antibody 1-3-1 For Culture of Hybridoma 1-3-1 The eRDF (manufactured by Kyokuto Pharmaceutical Co., Ltd.) culture solution containing 3% of the serum shown in (1) of Example 2 was used as the culture medium. The 1-3-1 antibody was purified by subjecting the culture solution in which the hybridoma 1-3-1 was cultured to a protein A-agarose column to adsorb the antibody, and then eluting it. The 1-3-1 antibody is SDS-
It was confirmed to be pure IgM by PAGE.

(2) Fluorescent labeling of 1-3-1 antibody Purified 1-3-1 antibody was labeled with FITC in the same manner as in (2) of Example 2 to recover the fluorescently labeled antibody. The absorbance, OD _{280 nm} and OD _{495 nm} of the _collected fractions were measured and the labeling rate was calculated. The F / P ratio was 6.7.

Test Example 4 (Study of Reactivity of Human Monoclonal Antibody to Cancer Cell Line) (1) Cancer Cell Line and Maintenance Human colon cancer line C-1 and gastric cancer line MKN45 are the same as in Test Example 1 (1). Similarly to the above, the culture medium B was maintained and grown under the conditions of 37 ° C. and 5% CO ₂ .

(2) Examination of Reactivity to Cancer Cell Line Reactivity to Live Cancer Cells The supernatant of cancer cells cultured in a flask or a petri dish was removed, and a 0.02% -EDTA-containing PBS solution was added to the reaction solution at room temperature for 30 minutes. It was left to stand and the cells were suspended. The cells were centrifugally washed with culture medium B, so that the cell density of about 1 × 10 ⁶ / 200μl, were suspended into PBS, final concentration, 50 [mu] g
1- obtained in (1) of Example 5 above so that
The antibody 3-1 was added and reacted at 0 ° C. for 60 minutes. Then, centrifuge at 2000 rpm for 2 minutes to remove the supernatant,
200 μl of a 500-fold diluted FITC-labeled anti-human antibody (manufactured by Kapel) was added to PBS containing% BSA to suspend the cells, and the cells were further reacted at 0 ° C. for 60 minutes. After the reaction, the mixture was centrifuged at 2000 rpm for 2 minutes to remove the supernatant, the cells were suspended in 1 ml of PBS, and the cells were washed by centrifugation. Then, the cells were PBS containing 10 μg / ml of PI.
Suspended in 300 μl. This cell suspension was subjected to FCM and the amount of fluorescence bound to individual cells (FITC and PI)
Was measured. The reactivity of the 1-3-1 antibody against each of the colon cancer cell line Cl and the gastric cancer cell line MKN45 is shown in FIGS. 3 and 4. The horizontal axis shows the fluorescence intensity per cancer cell, and the vertical axis shows the number of cancer cells. As a control, a commercially available human IgM antibody (manufactured by Kapel) was used, and the reactivity to the cancer cell line was similarly examined. In the figure, the reactivity of the 1-3-1 antibody is shown by a dotted line, and the control is shown by a solid line. From this, it is found that the 1-3-1 antibody has a strong binding property to cancer cells.

Test Example 5 (Fresh cancer tissue of human monoclonal antibody 1-3-1,
Reactivity to cells derived from non-cancerous tissue) To examine the cancer cell binding specificity of the 1-3-1 antibody, cells were isolated from fresh tissue of the same patient and the same organ that was extracted at the same time as the cancerous tissue of the cancer patient. The cells were separated and the reactivity of the 1-3-1 antibody to each cell was measured. Isolation of cells from the tissue was performed according to the method of Tokita et al. As in Test Example 3 above. The reactivity to the cells thus obtained was measured by FCM as in the case of examining the reactivity to the above-mentioned live cancer cell line. However, these cells were washed with the culture medium B by centrifugation,
About 1 × 10 ⁶ / 200μl of to be a cell density, serum containing the Example 5 (2) obtained in fluorescently labeled 1-3-1 antibody (final concentration 50 [mu] g / ml) (healthy subjects from) Suspended in water and reacted at 0 ° C for 60 minutes, then 2000
The supernatant was removed by centrifugation at rpm for 2 minutes, the cells were suspended in 1 ml of PBS, and the cells were washed by centrifugation to give 10 μg / cell.
Suspended in 300 μl PBS containing ml PI. Then, this cell suspension was subjected to FCM, and the amount of fluorescence bound to each cell (FITC and PI) was measured. Fluorescent (5 types) markers (quantitative kit: described above)
Cells to FCM under the same conditions, and cell 1
The average amount of bound FITC per cell was calculated, and the average number of bound antibody per living cancer cell was calculated from the value and the F / P ratio of the labeled antibody calculated in (2) of Example 5 and the result was obtained. Are shown in Table 4 below.

[0046]

[Table 4] Table 4 ─────────────────────────────────── Escherichia coli gastric cancer antibody ─────── ─────────────────────── Cancer Non-cancer Cancer Non-cancer ─────────────────────── ───────────── 1-3-1 1.5 × 10 ⁴ 0.04 × 10 ⁴ 1.8 × 10 ³ 0.05 × 10 ³ Control 0.15 × 10 ⁴ 0.04 × 10 ⁴ 0.2 × 10 ³ 0.3 × 10 ³ ─────────────────────────────────── 1-3-1 Antibody reaction to cells derived from non-cancerous part As for the sex, an antibody (1-3-
In the same manner as in (1), the average number of bound antibodies to cancer tissue-derived cells was significantly higher than that to non-cancer-derived cells, and When compared with the control antibody, both gastric cancer and colon cancer were 10 times higher, suggesting that the 1-3-1 antibody recognizes an antigen that appears more frequently on the membrane of cancer cells.

Example 6 (Determination of subclass of human type monoclonal antibody 1-3-1 light chain) The 1-3-1 antibody obtained in (1) of Example 5 was used as the antibody instead of the GAH antibody. The subclass of 1-3-1 antibody light chain was determined in the same manner as in Example 3 except for the above. As a result, it was shown that the light chain of the 1-3-1 antibody reacted with the anti-human λ chain antibody, a band appeared due to color development, and was a λ chain.

(Human antibody 1-3-1 gene acquisition and nucleotide sequence determination) a. Polymerase chain reaction (hereinafter abbreviated as PCR)
Preparation of cDNA encoding antibody 1-3-1 by the method from the 1-3-1 antibody-producing hybridoma obtained in (2) of Example 4 guanidine thiocyanate-lithium chloride method (DNA 2,329 (1983)) According to poly (A)
Was prepared. The RNA was prepared in the same manner as in Example 3 except that the number of hybridoma cells used was 2 × 10 ⁸ . RNA obtained
About 1.8 mg was dissolved in 1 ml of a solution consisting of 10 mM Tris-hydrochloric acid (pH 8.0) and 1 mM EDTA.
From this, from 230 μl, mRN containing poly (A) was prepared using the mRNA Purification Kit (described above).
A about 20 μg was obtained.

1 μg of 4.3 μg of this poly (A) mRNA
It was dissolved in 0 μl of water and oligo d (T) was prepared in the same manner as in Example 3.
12-18 primer 0.6 μg, 10 mM 4dNT
P2 μl, reverse transcriptase 40 u, RNA degrading enzyme inhibitor 3
6 μl of a reverse transcriptase buffer solution having a concentration of 0 u and a 5-fold concentration was added, and water was added to make a system of 30 μl in total, and the mixture was reacted at 42 ° C. for 1 hour. Ethanol precipitation was performed from the above reaction solution to obtain cDNA
Got

The obtained cDNA was dissolved in 20 μl of water. To 10 μl of this, 5 μl of 5 times concentration of terminal deoxynucleotide transferase buffer, terminal deoxynucleotide transferase 11 u, 10 mM dGTP 2.5 μ
l, and 6.5 μl of water was added to make a total system of 25 μl.
After reacting at 7 ° C for 1 hour, poly d was added to the 3'end of the cDNA.
(G) was added. Then, the enzyme was inactivated by heating at 70 ° C for 10 minutes.

PCR was carried out using the thus obtained cDNA as a substrate. For PCR, 2.5μ of the above reaction completion solution
d added to the 3'end of the cDNA as a primer for amplifying the cDNA encoding the variable region of the heavy chain
25 pmoles of poly C (14 nucleotides) that hybridizes to the G tail, and a single-stranded DNA primer (SEQ ID NO: 7, corresponding to the base sequence part of the constant region of IgM)
17 nucleotides) [Nucleic Acids R
essearch 18,4278 (1990)] 25p
moles, and cDN encoding the variable region of the light chain
Poly C 25p as a primer for amplifying A
moles and a single-stranded primer corresponding to the base sequence part of the constant region of the λ chain (SEQ ID NO: 8, 19 nucleotides)
[Nature 294 , 536 (1981)] 25p
The same procedure as in Example 3 was performed except that moles was used to obtain a cDNA having a blunt-ended single strand.

B. Determination of nucleotide sequence of cDNA As a result of analysis of the obtained cDNA solution by 2% agarose electrophoresis, a band of about 500 bp was recognized. This approximately 500 bp band was cut out from an agarose gel according to a conventional method, and cloned into pUC119 of the cloning vector.
It was inserted into the Sma I site and its nucleotide sequence was determined by the dideoxy method. As a result, in the base sequence of the PCR fragment, the base sequences encoding the variable regions of the heavy chain and the light chain were as shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. The amino acid sequences of the variable regions of the heavy chain and the light chain of the 1-3-1 antibody produced by the hybridoma, which are predicted from the determined nucleotide sequences, are as shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. The DNA fragment whose amino acid sequence and base sequence have been clarified can be obtained with good reproducibility by a DNA synthesizer without repeating the above-mentioned method.

Example 7 (Preparation of GAH antibody-bound liposomes containing adriamycin) a. Preparation of thiolated antibody Anti-tumor cell-reactive GAH antibody (IgG) was added with 0.1M-acetate buffer (pH 4.0) in an amount of 1/40 mol of pepsin [Cooper Biomedical],
The reaction was carried out overnight at 37 ° C. to form F (ab ′) ₂ , and F (ab ′) ₂ was isolated by chromatographic separation using a cation exchange resin (mono S) [manufactured by Pharmacia]. Separation conditions were performed by a linear gradient of the same buffer containing 0 to 0.5 M NaCl in 0.1 M acetate buffer (pH 4.0). The isolated F (ab ') ₂ was added to 0.15M
12 μl of 10% DTT was added per mg of antibody in 0.1 M acetate buffer (pH 4.5) containing NaCl,
It was left at room temperature for 80 minutes. After the reaction was completed, desalting was performed using a gel filtration column (PD-10) equilibrated with PBS to prepare a thiolated Fab 'antibody.

B. Preparation of thiolated polyethylene glycol L-cystine 48 mg was added to 0.4 M borate buffer 10 ml.
Dissolved in 2,4-bis (polyethylene glycol)-
200 mg of 6-chloro-s-triazine (activated PEG2) [Seikagaku Corporation] was added, and the mixture was reacted overnight at room temperature. 62 mg of DTT was added to the obtained cysteine-bonded PEG and reacted at 37 ° C. for 6 hours to give cysteine-bonded PEG.
A solution containing was obtained. Furthermore, the reaction solution is subjected to gel filtration (GH-2
5 Seikagaku Co., Ltd., etc., and desalted, 10 mM phosphate buffer (p
H7.4), 0.15 M NaCl (PBS) and then equilibrated with PBS thiopropyl sepharose 6B
(Pharmacia) was added to 7 ml, and unbound substances were washed away with PBS. Cysteine-conjugated PEG bound to gel
Is eluted with PBS containing 50 mM-DTT, and then excess DTT is desalted and removed by gel filtration or the like to prepare a standard product (thiolated PE).
G) was obtained.

C. Preparation of Maleimidated-Dipalmitoylphosphatidylethanolamine Dipalmitoylphosphatidylethanolamine 12
7 mg, N- (ε-maleimidocaproyloxy) succinimide (EMCS) 80 mg, triethylamine 4
4 ul was added to a solution of chloroform / methanol: 5/1 and reacted under a nitrogen stream for 3 hours. Then 2 more
0 mg of EMCS was added and further reacted at room temperature for 3 hours. After confirming that the ninhydrin reaction of the reaction solution became negative, it was dried under reduced pressure and redissolved in a small amount of chloroform. The maleimidated dipalmitoylphosphatidylethanolamine was purified by chromatography using UNISIL [manufactured by Gascro Industrial Co., Ltd.]. Add to the same column equilibrated with chloroform and add chloroform / methanol:
The target substance was obtained by developing with an eluent of 10/1.

D. Preparation of Maleimide Group-Containing Adriamycin Encapsulated Liposomes DPPC (Dipalmitoylphosphatidylcholine) /
chol (cholesterol) / maleimidated DPPE
(Dipalmitoylphosphatidylethanolamine)
However, 1 ml of 0.4M citrate buffer pH 4 was added to 100 mg of a solid lipid mixture (manufactured by Nippon Seika Co., Ltd.) consisting of 18/10/3 (mol ratio), and the mixture was stirred and freeze-thaw was repeated 5 times for hydration. The multilamellar liposomes were prepared by
Pressurizing device (e) equipped with a polycarbonate membrane with a pore size of
xtrude; Lipex Biomembrane
Granulated liposomes were obtained by repeating pressure filtration 10 times while heating to 60 ° C. in s). This liposome solution was neutralized with a 1 M NaOH solution and heated to 60 ° C., and adriamycin (manufactured by Kyowa Hakko Co., Ltd.) was added at 1/10 weight of the lipid weight. 97 according to the pH gradient inside and outside the liposome
% Or more of adriamycin was actively encapsulated in the liposome to prepare a maleimide group-containing adriamycin-encapsulated liposome.

E. Binding of thiolated antibody to maleimide group-containing adriamycin-encapsulated liposome and PEG modified thiolated Fab ′ antibody, 5 m to the maleimide-containing adriamycin-encapsulated liposome consisting of 100 mg of lipid.
g, and allowed to react at 37 ° C. for 8 hours, and further 5 μmol
The PEG-modified antibody-conjugated adriamycin-encapsulated liposome was prepared by adding the thiolated PEG of Example 1 and reacting in PBS at room temperature for 6 hours. Further Sepharose CL6
Gel filtration was performed using B (Pharmacia) to separate unreacted cysteine-bonded PEG.

Test Example 6 (Confirmation of drug efficacy of adriamycin-encapsulated GAH antibody-bound PEG-modified liposome) The antitumor effect was confirmed by using the human gastric cancer cell line MKN45 in which reactivity of GAH antibody was observed and accumulation was observed in the nude mouse transplantation system. investigated. The treatment experiment was started 10 days after the transplantation of 1 × 10 ⁶ cultured MKN45 cells subcutaneously in nude mice and when the tumor weight reached about 100 mg. The results are shown in Fig. 5. On day 3, 3 and 7 days after the start of treatment, the adriamycin-encapsulated GAH antibody-bound PEG-modified liposome was administered at 5 mg / kg in terms of adriamycin by tail vein (indicated by ⋄ in the figure). As a control group, phosphate buffered saline (indicated by ◆ in the figure) and adriamycin alone (in the figure,
□) and adriamycin-encapsulated PEG-modified liposome (indicated by × in the figure) administration groups (6 to 7 mice in each group) were provided. In order to measure the change over time in the tumor growth, the estimated tumor weight was calculated according to the Batter-Columbus method according to the formula of minor axis x minor axis x major axis / 2, and the transition was based on the tumor weight at the start of treatment. Indicated. The horizontal axis shows the number of days elapsed after the start of the treatment experiment. In the figure (↓) shows the drug administration date.
From this, a strong antitumor effect of the adriamycin-encapsulated monoclonal antibody-bonded PEG-modified liposome was shown.

[0059]

EFFECT OF THE INVENTION By using the humanized monoclonal antibody obtained in the present invention, it is possible to carry out the targeting treatment of cancer tissues with anticancer agents, toxins and the like. Further, since the antitumor agent of the present invention contains a human type monoclonal antibody,
It is specific to cancer tissue and can be administered continuously.

[0060]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 37 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Human IgG antibody Sequence G GCC CTT GGT GGA GGC TGA AGA GAC GGT GAC CAT TCT 37

SEQ ID NO: 2 Sequence length: 21 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Human IgG antibody Sequence TGG TGC AGC CAC AGT TCG TTT 21

SEQ ID NO: 3 Sequence length: 357 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human antibody GAH producing hybridoma cell sequence CAG GTG CAG CTG CAG GAG TCG GGC CCA GGA CTG GTG AAG CCT TCA 45 CAG ACC CTG TCC CTC ACC TGC ACT GTC TCT GGT GGC TCC ATC AGC 90 AGT TGT GGT TTC TAC TGG AAC TGG ATC CGC CAG CAC CCA GGG AAG 135 GGC CTG GAG TGG ATT GGG TAC ATC TAT TAC AGT GGG AGC ACC TAC 180 TAC AAC CCG TCC CTC AAG AGT CGA GTT ACC ATA TCG CTA GAC ACG 225 TCT AAG AGC CAG TTC TCC CTG AAG CTG AGC TCT CTG ACT GCC GCG 270 GAC ACG GCC GTG TAT TAC TGT GCG AGG TCT ACC CGA CTA CTA CTA GGG 315 GCT GAC TAC TGG GGC CAG GGA ACA ATG GTC ACC GTC TCT TCA 357

SEQ ID NO: 4 Sequence length: 342 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human antibody GAH producing hybridoma cell Sequence GAC ATC GTG ATG ACC CAG TCT CCA GAC TCC CTG GCT GTG TCT CTG 45 GGC GAG AGG GCC ACC ATC AAC TGC AAG TCC AGC CAG AGT GTT TTA 90 TAC AAC TCC AAC AAT AAG AAA TAC TTA GCT TGG TAC CAG CAG AAA 135 CCA GGA CAG CCT CCT AAG CTG CTC ATT TAC TGG GCA TCT ACC CGG 180 GAA TCC GGG GTC CCT GAC CGA TTC AGT GGC AGC GGG TCT GGG ACA 225 GAT TTC ACT CTC ACC ATC AGC AGC CTG CAG GCT GAA GAT GTG GCA 270 GTT TAT TAC TGT CAG CAG TAT TAT AGT ACT CCG TGG ACG GGC 315 CAA GGG ACC AAG GTG GAA ATC AAA CGA 342

SEQ ID NO: 5 Sequence length: 119 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human antibody GAH producing hybridoma cell sequence Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5 10 15 Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser 20 25 30 Ser Cys Gly Phe Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys 35 40 45 Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr 50 55 60 Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Leu Asp Thr 65 70 75 Ser Lys Ser Gln Phe Ser Leu Lys Leu Ser Ser Leu Thr Ala Ala 80 85 90 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Thr Arg Leu Arg Gly 95 100 105 Ala Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 110 115 119

SEQ ID NO: 6 Sequence length: 114 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human antibody GAH producing hybridoma cell sequence Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu 1 5 10 15 Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu 20 25 30 Tyr Asn Ser Asn Asn Lys Lys Tyr Leu Ala Trp Tyr Gln Gln Lys 35 40 45 Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 50 55 60 Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 65 70 75 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala 80 85 90 Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Trp Thr Phe Gly 95 100 105 Gln Gly Thr Lys Val Glu Ile Lys Arg 110 114

SEQ ID NO: 7 Sequence length: 17 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin human IgM antibody Sequence C GAG GGG GAA AAG GGT T 17

SEQ ID NO: 8 Sequence length: 19 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin human IgM antibody Sequence G AAG CTC CTC AGA GGA GGG 19

SEQ ID NO: 9 Sequence length: 366 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human antibody 1-3-1 producing hybridoma cell Sequence CAG CTG CAG CTG CAG GAG TCG GGC CCA GGA CTG GTG AAG CCT TCG 45 GAG ACC CTG TCC CTC ACC TGC ACT GTC TCT GGT GGC TCC ATC AGC 90 AGT AGT AGT TAC TAC TGG GGC TGG ATC CGC CAG CCC CCA GGG AAG 135 GGG CTG GAG TGG ATT GGG AGT ATC TAT TAT AGT GGG AGC ACC TAC 180 TAC AAC CCG TCC CTC AAG AGT CGA GTC ACC ATA TCC GTA GAC ACG 225 TCC AAG AAC CAG TTC TCC CTG AAG CTG AGC TCT GTG ACC GCC GCA 270 GAC ACG GCT GTG TAT TAC TGT GCG AGG AGC TAC GGG GGC TAC 315 TAC TAC GGT ATG GAC GTC TGG GGC CAA GGG ACC ACG GTC ACC GTC 360 TCC TCA 366

SEQ ID NO: 10 Sequence length: 324 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human antibody 1-3-1 producing hybridoma cell sequence TAT GAG CTG ACA CAG CCA CCC TCG GTG TCA GTG TCC CCA GGA CAG 45 ACG GCC AGG ATC ACC TGC TCT GGA GAT GCA TTG CCA AAG CAA TAT 90 GCT TAT TGG TAC CAG CAG AAG CCA GGC CAG GCC CCT GTG CTG GTG 135 ATA TAT AAA GAC AGT GAG AGG CCC TCA GGG ATC CCT GAG CGA TTC 180 TCT GGC TCC AGC TCA GGG ACA ACA GTC ACG TTG ACC ATC AGT GGA 225 GTC CAG GCA GAA GAC GAG GCT GAC TAT TAC TGT CAA TCA GCA GAC 270 AGC AGT GGT ACT TAT GAG GTA TTC GGC GGG ACC AAG CTG ACC 315 GTC CTA GGT 324

SEQ ID NO: 11 Sequence length: 122 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human antibody 1-3-1 producing hybridoma cell Sequence Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5 10 15 Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser 20 25 30 Ser Ser Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys 35 40 45 Gly Leu Glu Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr 50 55 60 Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr 65 70 75 Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala 80 85 90 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ser Tyr Gly Gly Tyr 95 100 105 Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val 110 115 120 Ser Ser 122

SEQ ID NO: 12 Sequence length: 108 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human antibody 1-3-1 producing hybridoma cell sequence Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Gln Tyr 20 25 30 Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val 35 40 45 Ile Tyr Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe 50 55 60 Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly 65 70 75 Val Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp 80 85 90 Ser Ser Gly Thr Tyr Glu Val Phe Gly Gly Gly Thr Lys Leu Thr 95 100 105 Val Leu Gly 108

SEQ ID NO: 13 Sequence length: 8 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human type monoclonal antibody producing hybridoma cell having its antigen on the membrane surface of cancer cell Sequence Ile Ser Ser Xaa Xab Xac Tyr Trp 1 5 Xaa: Cys or Ser, Xab: Gly or Ser, Xac: Phe or Tyr

SEQ ID NO: 14 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human type monoclonal antibody producing hybridoma cell having its antigen on the membrane surface of cancer cell

SEQ ID NO: 15 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human type monoclonal antibody producing hybridoma cell having its antigen on the membrane surface of cancer cell

SEQ ID NO: 16 Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human antibody GAH producing hybridoma cell

SEQ ID NO: 17 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human antibody GAH producing hybridoma cell

SEQ ID NO: 18 Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human antibody GAH producing hybridoma cell

SEQ ID NO: 19 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human antibody GAH producing hybridoma cell sequence Lys Ser Ser Gln Ser Val Leu Tyr Asn Ser Asn Asn Lys Lys Tyr Leu Ala 1 5 10 15

SEQ ID NO: 20 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human antibody GAH producing hybridoma cell

SEQ ID NO: 21 Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human antibody GAH producing hybridoma cell

SEQ ID NO: 22 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human antibody 1-3-1 producing hybridoma cell

SEQ ID NO: 23 Sequence length: 14 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human antibody 1-3-1 producing hybridoma cell sequence Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro 1 5 10

SEQ ID NO: 24 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human antibody 1-3-1 producing hybridoma cell

SEQ ID NO: 25 Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human antibody 1-3-1 producing hybridoma cell

SEQ ID NO: 26 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin of cell type: Human antibody 1-3-1 producing hybridoma cell

SEQ ID NO: 27 Sequence length: 11 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Cell type: Human antibody 1-3-1 producing hybridoma cell

SEQ ID NO: 28 Sequence length: 24 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human type monoclonal antibody producing hybridoma cell having its antigen on the membrane surface of cancer cell

SEQ ID NO: 29 Sequence length: 36 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human type monoclonal antibody producing hybridoma cell having its antigen on the membrane surface of cancer cell Array ATT GGG WRY ATC TAT TAY AGT GGG AGC ACC TAC TAC 36 W: T or A, R: A or G, Y: C or T

SEQ ID NO: 30 Sequence length: 12 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human type monoclonal antibody producing hybridoma cell having its antigen on the membrane surface of cancer cell Array GGK RYK GAC KWC 12 K: G or T, R: G or A, Y: C or TW: A or T

SEQ ID NO: 31 Sequence length: 27 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human antibody GAH producing hybridoma cell Sequence ATC AGC AGT TGT GGT TTC TAC TGG 27

SEQ ID NO: 32 Sequence length: 36 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human antibody GAH producing hybridoma cell sequence ATT GGG TAC ATC TAT TAC AGT GGG AGC ACC TAC TAC 36

SEQ ID NO: 33 Sequence length: 27 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human antibody GAH producing hybridoma cell Sequence TCT ACC CGA CTA CGG GGG GCT GAC TAC 27

SEQ ID NO: 34 Sequence length: 51 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human antibody GAH producing hybridoma cell Sequence AAG TCC AGC CAG AGT GTT TTA TAC AAC TCC AAC AAT AAG AAA TAC TTA GCT 51

SEQ ID NO: 35 Sequence length: 21 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human antibody GAH producing hybridoma cell Sequence TGG GCA TCT ACC CGG GAA TCC 21

SEQ ID NO: 36 Sequence length: 27 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human antibody GAH producing hybridoma cell Sequence CAG CAG TAT TAT AGT ACT CCG TGG ACG 27

SEQ ID NO: 37 Sequence length: 30 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human antibody 1-3-1 producing hybridoma cell sequence ATC AGC AGT AGT AGT TAC TAC TGG GGC TGG 30

SEQ ID NO: 38 Sequence length: 42 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human antibody 1-3-1 producing hybridoma cell Sequence ATT GGG AGT ATC TAT TAT AGT GGG AGC ACC TAC TAC AAC CCG 42

SEQ ID NO: 39 Sequence length: 36 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human antibody 1-3-1 producing hybridoma cell Sequence GGG AGC TAC GGG GGC TAC TAC TAC GGT ATG GAC GTC 36

SEQ ID NO: 40 Sequence length: 27 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human antibody 1-3-1 producing hybridoma cell sequence GAT GCA TTG CCA AAG CAA TAT GCT TAT 27

SEQ ID NO: 41 Sequence length: 12 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human antibody 1-3-1 producing hybridoma cell Sequence AAA GAC AGT GAG 12

SEQ ID NO: 42 Sequence length: 33 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA Origin Cell type: Human antibody 1-3-1 producing hybridoma cell Sequence CAA TCA GCA GAC AGC AGT GGT ACT TAT GAG GTA 33

[Brief description of drawings]

1 shows a schematic diagram of the construction of the vector pKCRD.

FIG. 2 shows a schematic diagram of the construction of the vector pKCR (ΔE) / H.

FIG. 3 shows the reactivity of 1-3-1 antibody against colorectal cancer cell line C1.

FIG. 4 shows the reactivity of 1-3-1 antibody against gastric cancer cell line MKN45.

FIG. 5 shows the antitumor effect of GAH antibody-bonded PEG-modified liposomes encapsulating adriamycin against cancer transplanted in nude mice.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI Technical indication C12N 15/13 ZNA // A61B 10/00 T C12N 15/08 G01N 33/577 B 9015-2J ( C12P 21/08 C12R 1:91) 8931-4B C12N 15/00 C (72) Inventor Norihiko Ito 1000 Kamoshida-cho, Midori-ku, Yokohama-shi, Kanagawa Sanryo Kasei Co., Ltd. General Research Institute (72) Inventor Kazuhiro Nagaike Sanryo Kasei Co., Ltd. Research Institute, 1000 Kamoshida-cho, Midori-ku, Yokohama, Kanagawa

Claims (29)

[Claims] 1. A humanized monoclonal antibody produced by cell fusion of lymphocytes derived from a cancer patient and mouse myeloma cells and specifically binding to a membrane surface antigen of the cancer cells. 2. The heavy chain variable region is represented by SEQ ID NO: 1 in the sequence listing.
The monoclonal antibody according to claim 1, comprising the amino acid sequences of 3, 14, and 15. 3. The heavy chain variable region is SEQ ID NO: 1 in the sequence listing.
2. The monoclonal antibody according to claim 1, which comprises the amino acid sequences of 6, 17 and 18 and the light chain variable region comprises the amino acid sequences of SEQ ID NOs: 19, 20 and 21 of the sequence listing. 4. The monoclonal antibody according to claim 1, wherein the heavy chain variable region and the light chain variable region are represented by the amino acid sequences of SEQ ID NOs: 5 and 6 in the sequence listing, respectively. 5. The heavy chain variable region is represented by SEQ ID NO: 2 in the sequence listing.
2. The monoclonal antibody according to claim 1, which comprises the amino acid sequences of 2, 23 and 24 and the light chain variable region comprises the amino acid sequences of SEQ ID NOs: 25, 26 and 27 of the sequence listing. 6. The monoclonal antibody according to claim 1, wherein the heavy chain variable region and the light chain variable region are represented by the amino acid sequences of SEQ ID NOs: 11 and 12 in the sequence listing, respectively. 7. A DNA encoding the monoclonal antibody according to claim 1. 8. A DNA encoding the monoclonal antibody according to claim 2. 9. The heavy chain variable region is SEQ ID NO: 2 in the sequence listing.
The DNA according to claim 8, which comprises the nucleotide sequences of 8, 29 and 30. 10. A DNA encoding the monoclonal antibody according to claim 3. 11. The heavy chain variable region is represented by SEQ ID NO: 3 in the sequence listing.
11. The monoclonal antibody according to claim 10, comprising the nucleotide sequences of 1, 32 and 33, and the light chain variable region comprises the nucleotide sequences of SEQ ID NOs: 34, 35 and 36 of the sequence listing. 12. A DNA encoding the monoclonal antibody according to claim 4. 13. The heavy chain variable region and the light chain variable region,
13. The DNA according to claim 12, which is represented by the nucleotide sequences of SEQ ID NOS: 3 and 4 in the sequence listing, respectively. 14. A DNA encoding the monoclonal antibody according to claim 5. 15. The heavy chain variable region is SEQ ID NO: 3 in the sequence listing.
15. The monoclonal antibody according to claim 14, comprising the nucleotide sequences of 7, 38 and 39, and the light chain variable region comprises the nucleotide sequences of SEQ ID NOs: 40, 41 and 42 in the sequence listing. 16. A DNA encoding the monoclonal antibody according to claim 6. 17. The heavy chain variable region and the light chain variable region are
The DNA according to claim 16, which is represented by the nucleotide sequences of SEQ ID NOs: 9 and 10 of the Sequence Listing, respectively. 18. A hybridoma which produces the monoclonal antibody according to claim 1. 19. A hybridoma which produces the monoclonal antibody according to claim 2. 20. A hybridoma producing the monoclonal antibody according to claim 3. 21. A hybridoma that produces the monoclonal antibody according to claim 4. 22. A hybridoma which produces the monoclonal antibody according to claim 5. 23. A hybridoma which produces the monoclonal antibody according to claim 6. 24. An antitumor agent having the monoclonal antibody according to claim 1 carried on the surface of a liposome encapsulating a toxin for a tumor cell or a carcinostatic agent. 25. An antitumor agent having the monoclonal antibody according to claim 2 carried on the surface of a liposome encapsulating a toxin or an antitumor agent against tumor cells. 26. An antitumor agent comprising the monoclonal antibody according to claim 3 carried on the surface of a liposome encapsulating a toxin against a tumor cell or a carcinostatic agent. 27. An antitumor agent comprising the monoclonal antibody according to claim 4 carried on the surface of a liposome encapsulating a toxin for a tumor cell or a carcinostatic agent. 28. An antitumor agent in which the monoclonal antibody according to claim 5 is carried on the surface of a liposome encapsulating a toxin for a tumor cell or an antitumor agent. 29. An antitumor agent in which the monoclonal antibody according to claim 6 is carried on the surface of a liposome encapsulating a toxin for tumor cells or an anticancer agent.