JP2007252372A - Monoclonal antibody, gene encoding the antibody, hybridoma, pharmaceutical composition and diagnostic reagent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a monoclonal antibody which can be used as a cancer therapeutic agent which acts selectively on a cancer tissue of non-small cell lung cancer, pancreatic cancer, gastric cancer or the like, with a reduced side effect. <P>SOLUTION: The invention relates to the monoclonal antibody having a heavy chain variable region and a light chain variable region each contains a specific amino acid sequence, and to the DNA encoding the antibody, the recombinant vector containing the DNA, the transformant containing the vector, the hybridoma producing the antibody and the pharmaceutical composition containing the antibody as the therapeutic agent and diagnostic reagent. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a novel monoclonal antibody that can be used for treatment and diagnosis of cancer, a DNA encoding the antibody, a hybridoma producing the antibody, a pharmaceutical composition containing the antibody, and a diagnostic reagent.

  In the field of cancer treatment, research on targeting therapy targeting specific cancer cells has been conducted for solid cancers that do not have sufficient therapeutic agents. Monoclonal antibodies that specifically recognize cancer cells are effective for such targeting. However, when mouse monoclonal antibodies are used, there are problems such as difficulty in continuous administration due to side effects such as anaphylaxis caused by immune responses ( For example, refer nonpatent literature 1.).

  And in order to solve such a problem, acquisition of the monoclonal antibody with few side effects has been tried. There are technologies for producing chimeric antibodies in which the constant region of an antibody is derived from a human by genetic recombination, and technologies for creating a humanized antibody in which a non-hypervariable region is derived from a human. It has been more desirable. As a method for obtaining such a fully human monoclonal antibody, there is a hybridoma method using human-derived lymphocytes (see, for example, Non-Patent Document 2), but the production of a fully human monoclonal antibody is compared with that of a mouse monoclonal antibody. Since active immunization for efficiently obtaining human B cells producing the target antibody is difficult and an efficient method for infinite proliferation of antibody-producing cells has not been established, tumors are sufficiently produced. The acquisition of human monoclonal antibodies that react with cells has been reported very little (see, for example, Patent Document 1), and it still remains difficult.

Even under such circumstances, monoclonal antibodies having a killing effect or a growth-inhibiting effect against specific cancer cells alone or in combination with an anticancer agent using a humanized antibody production technique or the like gradually. Recently, application of anti-Her2 humanized antibody to breast cancer (for example, see Non-patent Document 3), anti-EGF receptor antibody (for example, see Non-Patent Document 4) and anti-VEGF ( Clinical trials using vascular endothelial growth factor) antibodies (see, for example, Non-Patent Document 5) are underway. However, there is still no antibody that can be used for specific targeting therapy for non-small cell lung cancer, which has a large number of affected individuals, and pancreatic cancer, which is a particularly refractory cancer type. In addition, acquisition of a monoclonal antibody with few side effects and high specificity for cancer tissues has been desired.
As such an antibody, Patent Document 2 discloses a monoclonal antibody having high specificity to cancer tissue, but it has been desired to obtain a further antibody.

Japanese Patent No. 3236667 JP-A-2005-040126 Proc.Natl.Acad.Sci.U.S.A.vol.86, p4220, 1989 Cancer Res.vol.45, p263, 1985 Oncology vol. 63 Suppl 1, pp25-32, 2002 Semin Oncol.vol.29, No.5 Suppl 14, pp18-30, 2002 Semin Oncol.vol.29, No.6 Suppl 16, pp10-14, 2002 Mol Cell Biol. 1987, vol. 7, No. 11, p3908-15

  An object of the present invention is to provide a monoclonal antibody that is useful for diagnosis and treatment of cancer, particularly non-small cell lung cancer, pancreatic cancer or gastric cancer, and has few side effects.

  As a result of intensive studies to provide a monoclonal antibody for use in targeting therapy for cancer tissue, the present inventors have discovered a novel human that specifically recognizes cancer cells such as HLC-1, PANC-1, HT29 and MKN45. A hybridoma producing a monoclonal antibody was prepared, and by using this antibody, it was found that a therapeutic drug for cancer useful for targeting therapy was obtained, and the present invention was completed.

That is, the present invention is as follows.
(1) A monoclonal antibody comprising the heavy chain variable region comprising the amino acid sequences of SEQ ID NOs: 74, 76 and 78.
(2) The monoclonal antibody according to (1), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 72.
(3) The monoclonal antibody according to (1), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 104.
(4) A monoclonal antibody comprising the amino acid sequences of SEQ ID NOs: 80, 82 and 84 in the light chain variable region.
(5) The monoclonal antibody according to (4), wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 70.
(6) The monoclonal antibody according to (4), wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 106.
(7) A monoclonal antibody, wherein the heavy chain variable region includes the amino acid sequences of SEQ ID NOs: 74, 76, and 78, and the light chain variable region includes the sequences of SEQ ID NOs: 80, 82, and 84.
(8) The monoclonal antibody according to (7), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 72, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 70.
(9) The monoclonal antibody according to (7), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 104, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 106.
(10) The monoclonal antibody according to any one of (1) to (9), which is a human antibody.
(11) A DNA encoding the monoclonal antibody according to any one of (1) to (10).
(12) The region encoding the heavy chain variable region includes the nucleotide sequences of SEQ ID NOs: 73, 75 and 77, and the region encoding the light chain variable region includes the nucleotide sequences of SEQ ID NOs: 79, 81 and 83 (11) DNA characterized by these.
(13) The region encoding the heavy chain variable region includes the base sequence of SEQ ID NO: 71, and the region encoding the light chain variable region includes the base sequence of SEQ ID NO: 69, (11) Or DNA of (12).
(14) The region encoding the heavy chain variable region includes the base sequence of SEQ ID NO: 103, and the region encoding the light chain variable region includes the base sequence of SEQ ID NO: 105, (11) Or DNA of (12).
(15) A recombinant vector comprising the DNA of any one of (11) to (14).
(16) A transformant comprising the recombinant vector of (15).
(17) A hybridoma that produces the monoclonal antibody according to any one of (1) to (10).
(18) A pharmaceutical composition comprising the monoclonal antibody according to any one of (1) to (10).
(19) The pharmaceutical composition according to (18), which is a cancer therapeutic agent.
(20) The pharmaceutical composition according to (19), wherein the cancer therapeutic agent is a cancer therapeutic agent for one or more cancers selected from the group consisting of non-small cell lung cancer, pancreatic cancer, and gastric cancer.
(21) The pharmaceutical composition according to any one of (18) to (20), wherein the monoclonal antibody is carried on the surface of a liposome encapsulating a toxin or an anticancer agent.
(22) A diagnostic reagent comprising the monoclonal antibody according to any one of (1) to (10).

  By using the monoclonal antibody obtained in the present invention, it is possible to provide a cancer therapeutic agent that selectively attacks non-small cell lung cancer, pancreatic cancer, gastric cancer or the like. Furthermore, when the human monoclonal antibody of the present invention is used, it is possible to provide a cancer therapeutic drug that can be continuously administered with few side effects.

Hereinafter, the present invention will be described in detail.
<1> Monoclonal antibody of the present invention The monoclonal antibody of the present invention is a monoclonal antibody that specifically recognizes a cancer cell-specific antigen. Specific examples of the monoclonal antibody of the present invention include those containing the amino acid sequences of SEQ ID NOs: 74, 76 and 78 in the heavy chain variable region and the amino acid sequences of SEQ ID NOs: 80, 82 and 84 in the light chain variable region. . Here, in the amino acid sequence of SEQ ID NO: 76, the second amino acid may be Ile or Thr. Similarly, the 16th amino acid in the amino acid sequence of SEQ ID NO: 76 may be Lys or Asn. That is, the heavy chain variable region includes (2Ile, 16Lys), (2Ile, 16Asn), (2Thr, 16Lys), (2Thr, 16Asn) in total of four types of sequences.
The above six types of sequences are sequences of regions called “hypervariable regions” in the variable regions of the heavy chain and the light chain. An antibody consists of a heavy chain and a light chain, and each chain consists of a constant region and a variable region. The variable region further includes a hypervariable region, which determines the specificity of the immunoglobulin as an antibody and the binding affinity between the antigenic determinant and the antibody. Therefore, the antibody of the present invention contains each of the above sequences in the hypervariable region, but regions other than the hypervariable region may be derived from other antibodies. Here, other antibodies include antibodies derived from organisms other than humans, but those derived from humans are preferable from the viewpoint of reducing side effects.
Particularly preferred monoclonal antibodies in the present invention include antibodies containing the amino acid sequence of SEQ ID NO: 72 in the heavy chain variable region and the amino acid sequence of SEQ ID NO: 70 in the light chain variable region. Here, in the sequence of SEQ ID NO: 72, the 51st amino acid may be either Ile or Thr, and the 65th amino acid may be either Lys or Asn.
As long as the specificity of the antibody that recognizes a cancer cell-specific antigen is not impaired, the sequence of the variable region of the antibody of the present invention is the sequence of SEQ ID NOs: 72 (heavy chain) and 70 (light chain). It may be a sequence into which one or several amino acid substitutions, deletions or additions have been introduced. Here, several means preferably 2 to 5, more preferably 2 or 3, particularly preferably 2. Such substitutions, deletions and additions may be introduced into the hypervariable region, but are preferably introduced into regions other than the hypervariable region of the variable region.

In the present invention, a monoclonal antibody refers to a monoclonal antibody, a fragment of a monoclonal antibody, an F (ab ′) ₂ antibody, an F (ab ′) antibody, a short chain antibody (scFv), a diabody (Diabodies), and a minibody ( Minibodies). In the case of including the constant region, the amino acid sequences of the heavy and light chain constant regions are Nucleic Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982 and Cell vol.22, P197, 1980 is preferred. As an antibody of this invention containing a constant region and a variable region, the antibody which has an amino acid sequence of sequence number 104 (heavy chain) or sequence number 106 (light chain) can be mentioned, for example.

  The monoclonal antibody of the present invention can be obtained by, for example, culturing a hybridoma that produces the antibody using a fetal bovine serum-containing RPMI1640 culture solution or the like, or a DNA encoding a variable region having the nucleotide sequences of SEQ ID NOs: 71 and 69. In addition, a gene (for example, the gene of SEQ ID NO: 103 (heavy chain) or 105 (light chain)) in which DNAs encoding heavy chain and light chain constant regions are linked is synthesized by PCR or chemical synthesis, The antibody is produced by incorporating into a known expression vector (such as pcDNA3.1 (invitrogen)) that enables the expression of the gene and expressing it in a host such as CHO cells (Chinese highster ovary cells) or E. coli. From these cultures, antibodies can be purified using a Protein A column or the like. It can be obtained by.

<2> DNA of the present invention
The DNA of the present invention is a DNA encoding the monoclonal antibody of the present invention. As an example, the region encoding the heavy chain variable region includes sequences encoding the amino acid sequences of SEQ ID NOs: 74, 76, and 78, respectively. In addition, DNAs in which the region encoding the light chain variable region includes sequences encoding the amino acid sequences of SEQ ID NOs: 80, 82, and 84, respectively. Among them, the region encoding the heavy chain variable region includes the nucleotide sequences of SEQ ID NOs: 73, 75 and 77, respectively, and the region encoding the light chain variable region includes the nucleotide sequences of SEQ ID NOs: 79, 81 and 83, respectively. DNA is preferred. Here, in the sequence of SEQ ID NO: 75, the fifth base may be t or c. Similarly, in the sequence of SEQ ID NO: 75, the 48th base may be g or t.
Since the hypervariable region encoded by the DNA of these sequences is a region that determines the specificity of the antibody, the sequence encoding the other region may be a sequence derived from another antibody. Here, other antibodies include antibodies derived from organisms other than humans, but those derived from humans are preferable from the viewpoint of reducing side effects.
Particularly preferred DNA in the present invention includes a region encoding the heavy chain variable region comprising a sequence encoding the amino acid sequence of SEQ ID NO: 72, and a region encoding the light chain variable region encoding the amino acid sequence of SEQ ID NO: 70. DNA containing the sequence to be processed. Among them, particularly preferred DNA is DNA in which the sequence encoding the heavy chain variable region includes the base sequence of SEQ ID NO: 71, and the region encoding the light chain variable region includes the base sequence of SEQ ID NO: 69. Here, in the array of SEQ ID NO: 71, the 152nd position may be t or c. Similarly, in the sequence of SEQ ID NO: 71, the 195th may be g or t.
The DNA of the present invention hybridizes under stringent conditions with the DNA comprising the nucleotide sequence of SEQ ID NO: 71 and the nucleotide sequence of SEQ ID NO: 69 as long as it encodes a monoclonal antibody that recognizes a cancer cell-specific antigen. It may be a thing. Here, stringent conditions correspond to, for example, 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, which is a washing condition for Southern hybridization. The conditions for hybridizing at a salt concentration are included.
The DNA of the present invention may encode all of the heavy chain and light chain constant regions and variable regions, or may encode only the heavy chain and light chain variable regions. Nucleic Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982 and Cell, in the case of encoding all of the constant region and the variable region. Those described in vol.22, p197, 1980 are preferable. Examples of the DNA of the present invention encoding the constant region and the variable region include DNAs having the base sequences of SEQ ID NO: 103 (heavy chain) and SEQ ID NO: 105 (light chain).
The DNA of the present invention can be obtained, for example, by the following method. First, total RNA is prepared from cells such as the hybridoma of the present invention using a commercially available RNA extraction kit, and cDNA is synthesized with reverse transcriptase using a random primer or the like. Next, in the variable regions of known human antibody heavy chain genes and light chain genes, cDNAs encoding the antibodies are amplified by PCR using oligonucleotides having conserved sequences as primers. The sequence encoding the constant region can be obtained by amplifying a known sequence by the PCR method. The base sequence of DNA can be determined by a conventional method by incorporating it into a sequencing plasmid. The DNA encoding the monoclonal antibody of the present invention can also be obtained by chemically synthesizing a variable region or a partial sequence thereof and binding it to a sequence containing a constant region.
The present invention also provides a recombinant vector containing the DNA of the present invention and a transformant containing the recombinant vector. The recombinant vector may be a vector that can be expressed in a prokaryotic cell such as Echerichia coli (for example, pBR322, pUC119, or a derivative thereof), but a vector that can be expressed in a eukaryotic cell is preferable. More preferred are vectors that can be expressed in mammalian cells. Examples of vectors that can be expressed in mammalian cells include plasmid vectors such as pcDNA3.1 (Invitrogen) and virus vectors such as pDON-AI DNA (Takara Bio). The transformant into which the recombinant vector of the present invention is introduced may be a prokaryotic cell such as E. coli, but is preferably a eukaryotic cell, more preferably a mammal-derived cell. Examples of mammalian cells include Chinese hamster ovary cells (CHO cells).

<3> Hybridoma of the Present Invention The hybridoma of the present invention is a hybridoma that produces a monoclonal antibody as described above. Examples of the hybridoma of the present invention include the hybridomas 020630-18-1 and 020630-18-1-1F8-1 shown in the examples described later. The hybridoma of the present invention can be obtained by the following method. First, A. According to the method of Imam et al. (Cancer Research vol. 45, 263 1985), lymphocytes are isolated from lymph nodes belonging to cancer removed from gastric cancer patients, and cells containing lymphocytes are fused with mouse myeloma cells using polyethylene glycol. To obtain a hybridoma. Next, an enzyme immunoassay is performed using the obtained hybridoma supernatant to select a hybridoma producing an antibody that is positive for various cancer cell lines immobilized with paraformaldehyde, and the hybridoma is further cloned by limiting dilution. .

<4> Pharmaceutical composition of the present invention The pharmaceutical composition of the present invention comprises the monoclonal antibody of the present invention together with a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include soluble carriers such as known physiologically acceptable buffers (eg, phosphate buffer) or solid state carriers such as latex beads.

  The pharmaceutical composition of the present invention is suitably used as a therapeutic agent for cancer, particularly non-small cell lung cancer, pancreatic cancer or gastric cancer. In addition, the pharmaceutical composition of the present invention may utilize the cell killing effect or growth inhibitory effect of the monoclonal antibody itself, or the anti-cancer agent may be bound to a cancer tissue by binding the monoclonal antibody of the present invention to an anti-cancer agent such as adriamycin. It may be targeted.

  A particularly suitable pharmaceutical composition in the present invention is one in which the antibody of the present invention is bound to a liposome containing a toxin, an anticancer agent or the like. The liposome carrying the antibody is composed of a double lipid layer, and any one in which the lipid layer is a multilayer or a single layer can be used. As the components of the liposome, phosphatidylcholine, cholesterol, phosphatidylethanolamine, phosphatidic acid as a substance that imparts electric charge, and the like are used. As the use ratio of the constituent components, for example, cholesterol is 0.3 to 1 mol, preferably 0.4 to 0.6 mol, and phosphatidylethanolamine is 0.01 to 0.2 mol, preferably 0 with respect to 1 mol of phosphatidylcholine. 0.02-0.1 mol and phosphatidic acid can be used in a composition ratio of 0-0.4 mol, preferably 0-0.15 mol.

  A well-known method can be used for the manufacturing method of a liposome. For example, the lipid mixture from which the solvent has been removed is emulsified with a homogenizer, etc., and after freezing and thawing, multilamellar liposomes are obtained. It can be produced by using a pressure filtration method (Biochimica et Biophysica Acta vol. 812, p55, 1985). The size of the liposome is preferably 30 nm to 200 nm.

  Examples of drugs to be encapsulated in liposomes include adriamycin, daunomycin, mitomycin, cisplatin, vincristine, epirubicin, methotrexate, 5Fu (5-fluorouracil), anticancer agents such as acracinomycin, toxins such as ricin A and diphtheria toxin, antisense RNA, etc. Can be used. Drugs can be encapsulated in liposomes by hydrating lipids with aqueous drug solutions. Adriamycin, daunomycin, and epirubicin can be encapsulated using a remote loading method using a pH gradient (Cancer Res. Vol. 49, p5922, 1989).

  Examples of the method for binding the monoclonal antibody on the liposome surface include a method of inserting a hydrophobic substance into the purified antibody and inserting it into the liposome, a method of cross-linking phosphatidylethanolamine and the antibody with glutar, etc. After thiolation of the antibody, a liposome containing a lipid having a maleimide group introduced therein is prepared, an anticancer agent or a toxin is encapsulated, and both are reacted to bind the antibody to the surface of the liposome. In addition, a water-soluble polymer derivative having a reactive site with an amino group and a thiol group or a latent thiol group portion can also be suitably used (Japanese Patent Laid-Open No. 11-152234). On the other hand, it is also possible to modify the surface of the liposome by reacting a residual maleimide group with a compound containing a thiolated polyalkylene glycol moiety.

Giving a thiol group to an antibody includes N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), iminothiolane, mercaptoalkylimidate, etc., which are commonly used for protein thiolation with respect to the amino group of the antibody. A method using a compound or a method in which an endogenous dithiol group of an antibody is reduced to a thiol group is used, and a method using an endogenous thiol group is more preferable from the viewpoint of maintaining activity. The antibody is converted to F (ab ′) ₂ with an enzyme such as pepsin, further reduced with dithiothreitol (DTT) or the like, and converted into F (ab ′) to form 1 to 3 newly produced thiol groups as liposomes. It can also be used for the coupling reaction. Binding of the maleimide group-containing liposome and the thiolated antibody can be achieved by reacting in a neutral buffer (pH 6.5-7.5) for 2 to 16 hours.

  For the formulation of the cancer therapeutic agent of the present invention, a known method, that is, a dehydration method (Japanese Patent Publication No. 2-502348), a method in which a stabilizer is added and used as a liquid, a freeze-drying method (Japanese Patent Laid-Open No. 64-9931) And the like can be used. The therapeutic agent for cancer of the present invention can be used in an intravascular administration method, a local administration method such as intraperitoneal injection, and the dosage 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.

<5> Diagnostic reagent of the present invention The diagnostic reagent of the present invention includes a diagnostic reagent that utilizes the cancer cell specificity of the antibody of the present invention. Specifically, the antibody, secondary antibody and detection of the present invention Examples include cancer diagnostic reagents comprising a substrate and the like.

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

(1) Preparation of hybridoma by cell fusion of lymphocytes derived from cancer-affiliated lymph nodes and mouse myeloma (1) -1. Preparation of lymphocytes Cancer removed from a gastric cancer patient on a metal mesh in a petri dish filled with culture solution B in which 10% fetal calf serum (FCS) was added to culture solution A (RPMI1640 or e-RDF + 50 μg / ml gentamicin sulfate) Lymphocytes were dispersed from regional lymph nodes. This cell suspension was centrifuged at 3000 rpm for 5 minutes to obtain 4.8 × 10 ⁷ cancer-affected lymph node-derived lymphocytes.

(1) -2. Cell fusion Lymphocytes derived from cancer-affiliated lymph nodes are fused with mouse myeloma cells (approximately the same number as lymphocytes) using polyethylene glycol 1500 (Roche Diagnostics) according to the standard method (Cancer Research vol. 45, 263, 1985). I let you. The fused cells were suspended in the culture medium B so that the number of cells was 5 × 10 ⁵ / ml, 100 μl each was seeded on a 96-well plate, and culture was started in a CO ₂ incubator at 37 ° C. On the second day, 100 μl of culture medium B (HAT-added culture medium) supplemented with 10 μM hypoxanthine, 0.04 μM aminopterin, and 1.6 μM thymidine was added to each well, and the cells were cultured until hybridoma colonies appeared. . As a result, hybridoma colonies were confirmed in 23 wells.

(2) Examination of reactivity of human monoclonal antibody to cancer cell line (2) -1. Cancer cell line and maintenance Using the culture supernatant of the emerging hybridoma, the reactivity to the fixed gastric cancer cell line MKN45 (Cancer and Chemotherapy, vol.5, p89, 1978, obtained from the Institute for Immunobiology) was measured. Hybridomas were selected. The cancer cell line was maintained and grown under the conditions of 37 ° C. and 5% CO ₂ in the culture solution D in which 5% FCS was added to the culture solution C (D-MEM / F12 + 50 μg / ml gentamicin sulfate).

(2) -2. Measurement of reactivity to cancer cell lines The above cancer cell lines were cultured in 96-well plates for 3 to 4 days until they became one layer, and after removing the supernatant, 10 mM phosphate buffer (pH 7.4, 0). After washing once with 15M NaCl) (PBS), fixation with 2% paraformaldehyde (room temperature, 20 minutes) was performed. After washing 5 times with PBS, blocking was performed by adding 150 μl / well of PBS solution containing 5% BSA (bovine serum albumin). This plate was washed 5 times with PBS, 50 μl of hybridoma culture supernatant was added, and reacted at 37 ° C. for 1 hour and a half. Next, it was washed 5 times with PBS, and a goat antibody (1000 culture diluted, Capel) against human antibody bound with 50 μl of horseradish peroxidase (HRP) was added, followed by reaction at 37 ° C. for 1 hour. Next, the plate was washed with PBS containing 0.05% Tween 20 (PBS-T) and contained o-phenylenediamine dihydrochloride (5.2%) and H ₂ O ₂ (0.015%). Phosphate citrate buffer was added at 50 μl / well and reacted at room temperature until color development could be confirmed, and then the absorbance at 490 nm was measured with a microphotometer (Nihon Intermed). Cloning was carried out by limiting dilution from wells that were confirmed to be reactive, and hybridoma strain 020630-18-1 was obtained. Hereinafter, the monoclonal antibody obtained from this strain is referred to as 020630-18-1. Further cloning was repeated to obtain a hybridoma strain 020630-18-1-1F8-1. Hereinafter, the monoclonal antibody obtained from this strain is referred to as 1F8 antibody.

(3) Purification and labeling of monoclonal antibodies 020630-18-1 and 1F8 (3) -1. Culture of hybridomas 020630-18-1 and 020630-18-1-1F8-1 and purification of monoclonal antibodies 020630-18-1 and 1F8 First, fetal bovine serum was purified from protein A-glass bead column (procep A) (bio PROCESSING) And a hybridoma 020630-18-1 or 020630-18-1-1F8-1 was prepared using the culture solution A supplemented with 4 to 10% of the serum. Cultured. Next, the culture solution in which the hybridoma 020630-18-1 or the hybridoma 020630-18-1-1F8-1 is cultured is applied to the process A to adsorb the process A adsorbed polypeptide, and then the elution is performed. The adsorbed polypeptide was purified. This process A adsorbed polypeptide was used as 020630-18-1 antibody or 1F8 antibody. It was considered that purified 020630-18-1 antibody or 1F8 antibody free from contamination of substances adsorbed to process A, such as serum-derived antibody, was obtained by using the above serum for culture. Although not shown in the figure, 020630-18-1 antibody and 1F8 antibody were confirmed to be pure IgG by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

(3) -2.020630-18-1 antibody or 1F8 antibody biotin labeling 020630-18-1 antibody or 1F8 antibody purified by Prosep A using biotinylation reagent (Amersham Pharmacia Biotech) according to the instructions After labeling, the labeled antibody and free biotin were separated by gel filtration.

(4) Examination of reactivity to various tissue sections (4) -1. Preparation of various tissue sections Various cancer cell lines (lung cancer cell line: HLC-1 (cancer vol.67, No.4, pp483-492, 1976, obtained from Keio University School of Medicine, Mr. Suzuki), A549, PC-3, PC-9 (all for immunobiological laboratories), pancreatic cancer cell lines: SUIT2 (obtained from National Hospital Kyushu Cancer Center, Mr. Iguchi), HPAC (ATCC No. CRL-2119), PANC-1 (ATCC No. CRL-1469) ) And PK8 (obtained from Medical Cell Resource Center, Institute of Aging Medicine, Tohoku University), gastric cancer cell lines: MKN45, MKN74, HSC-3 (all of which are Institute for Immunobiology), colon cancer cell line: HT29 (ATCC No. ), DLD-1 (ATCC No. CCL221), LoVo (ATCC No. CCL229), and CoLo205 (ATCC No. CCL222), respectively. In this culture medium D, the cells were grown under conditions of 37 ° C. and 5% CO ₂ , and about 1 × 10 ⁶ to 1 × 10 ⁷ cells were transplanted subcutaneously into nude mice (Claire Japan) to form tumors. The formed tumor was excised, fixed with a formalin solution according to a conventional method, and embedded in paraffin to prepare sections.

(4) -2. Detection of Reactivity to Various Tissue Sections The prepared various tissue sections were deparaffinized according to a conventional method and subjected to a blocking operation, and then Example (3) -2. The biotin-labeled 020630-18-1 antibody described in 1) was reacted. For detection, a DAKO Catalyzed Signal Amplification (CSA) System (DAKO) was used, and the reactivity was detected as a reddish brown staining of diaminobenzidine. Further, the tissue sections subjected to the immunostaining were stained with nuclei of cells in the tissue with hematoxylin in order to confirm the tissue image.
Stained images of various cancer tissue sections of the 020630-18-1 antibody are shown in FIG. The 020630-18-1 antibody showed clear reactivity to cancer cells in any tumor tissue section. In particular, the nuclei of cancer cells were strongly stained. On the other hand, no reactivity was observed against non-cancer cells derived from nude mice in tissue sections.
Although not shown in FIG. 1, as a control, staining using a human antibody purified from human serum using a process A column in the same manner as 020630-18-1 antibody was also performed in the same procedure as described above. . This control antibody did not show any specific reactivity with each tissue section.

(5) Binding activity of 1F8 antibody to the surface of live cancer cells The small lumps of fresh lung cancer tissue and non-lung cancer tissue removed by surgery from lung cancer patients are loosened with a razor blade and suspended in the culture medium D. Tissue-derived viable cells were prepared by turbidity and passing through a mesh. The live cells were suspended in human serum (containing 0.05% sodium azide), and the biotin-labeled 1F8 antibody described in Example (3) -2 was added to the cell solution to a concentration of 50 μg / ml. The total amount was adjusted to ˜100 μl (cell number: 10 ⁵ to 10 ⁶ ). The reaction was allowed to proceed for 60 minutes under ice cooling. After removing the antibody-containing solution from the supernatant by centrifugation (2000 rpm, 5 minutes), 20 nM-QdotTM565 Streptavidin-labeled (manufactured by Quantm Dot Corporation) solution (containing 0.05% sodium azide) was added at room temperature, Reaction was performed for 30 minutes, this solution was removed, PBS (containing 0.05% sodium azide) was added, and observation with a confocal fluorescence microscope (CSU10, manufactured by Yokogawa Electric Corporation) was performed. The binding activity of the 1F8 antibody to the surface of each living cell is shown in FIG. The 1F8 antibody showed binding activity on the surface of lung cancer tissue-derived living cells (FIG. 2-A), but did not show binding activity on non-cancerous tissue-derived living cells (FIG. 2-B). Although not shown in FIG. 2, the human antibody used as a control did not show binding activity to any cells.

(6) Examination of action on cancer cell line (6) -1. Cancer Cell Lines and Vascular Endothelial Cells and Maintenance Lung cancer line HLC-1, gastric cancer line MKN45 and pancreatic cancer line PANC-1 were used as human cancer cell lines. These cancer cell lines were maintained and grown in culture medium D at 37 ° C. and 5% CO ₂ .

(6) -2. Examination of growth inhibitory effect on cancer cell line Lung cancer cell line HLC-1, gastric cancer cell line MKN45 and pancreatic cancer cell line PANC-1 were each diluted with culture medium C containing 10% human serum, and the number of cells in the well was 1.5 × 10 ^3/100 [mu] l / well, the concentration of 1F8 antibody was adjusted to be equal magnification diluted from 100 [mu] g / ml. The cells were cultured in an environment of 37 ° C. and 5% CO ₂ , and the culture supernatant was exchanged three times every other day so that the conditions were the same as described above, and the MTT assay (J. Immunol. Methods vol. , P257, 1984), and compared the number of live cancer cells.
The results are shown in FIG. The inhibitory effect was shown with the number of cells under control conditions where no antibody was added as 100%. It was confirmed that by adding the 1F8 antibody, cell proliferation was suppressed in a concentration-dependent manner for any cancer cell line. In particular, a strong growth inhibitory activity was confirmed in the lung cancer cell line HLC-1. Although not shown in the figure, the human antibody used as a comparison did not show the effect of inhibiting the growth of the cancer cell line due to the addition of the same concentration.

(7) Acquisition of human monoclonal antibody 1F8 gene and determination of base sequence Total RNA was prepared from 1F8 antibody-producing hybridoma using RNeasy Protect Mini kit (QIAGEN). Reverse transcription reaction was performed using ThermoScript RT-PCR System (Invitrogen) using random 6mer as a primer to synthesize cDNA.
Primers for PCR amplification are based on known antibody sequences (J Mol Biol. Vol. 222, pp581-597, 1991). For amplification of the heavy chain variable region, the frame 1 of the human antibody heavy chain variable region is used. PCR primers VH1 (SEQ ID NO: 1), VH2 (SEQ ID NO: 3), VH3 (SEQ ID NO: 5) corresponding to the conserved N-terminal amino acid sequences (SEQ ID NOs: 2, 4, 6, 8, 10 and 12), respectively. , VH4 (SEQ ID NO: 7), VH5 (SEQ ID NO: 9), VH6 (SEQ ID NO: 11) equivalence mixture on the 5 ′ side, C-terminal amino acid sequence conserved in frame 4 of the human antibody heavy chain variable region 3 equivalent mixtures of PCR primers JH1 (SEQ ID NO: 13), JH2 (SEQ ID NO: 15), JH3 (SEQ ID NO: 17), JH4 (SEQ ID NO: 19) respectively corresponding to (SEQ ID NOs: 14, 16, 18 and 20) 'Use for side .
PCR primers corresponding to the N-terminal amino acid sequences (SEQ ID NOs: 22, 24, 26, 28, 30 and 32) conserved in frame 1 of the human antibody κ chain variable region are used as primers for amplification of the κ chain variable region. Equivalent mixture of primers VK1 (SEQ ID NO: 21), VK2 (SEQ ID NO: 23), VK3 (SEQ ID NO: 25), VK4 (SEQ ID NO: 27), VK5 (SEQ ID NO: 29), VK6 (SEQ ID NO: 31) on the 5 ′ side PCR primers JK1 (SEQ ID NO: 33) and JK2 (SEQ ID NO: 33) corresponding to the C-terminal amino acid sequences (SEQ ID NOs: 34, 36, 38, 40 and 42) conserved in frame 4 of the human antibody κ chain variable region, respectively. No. 35), an equivalent mixture of JK3 (SEQ ID NO: 37), JK4 (SEQ ID NO: 39), JK5 (SEQ ID NO: 41) was used on the 3 ′ side.
The primers for amplification of the λ chain variable region correspond to the N-terminal amino acid sequences (SEQ ID NOs: 44, 46, 48, 50, 52, 54 and 56) conserved in frame 1 of the human antibody λ chain variable region, respectively. PCR primers VL1 (SEQ ID NO: 43), VL2 (SEQ ID NO: 45), VL3 (SEQ ID NO: 47), VL4 (SEQ ID NO: 49), VL5 (SEQ ID NO: 51), VL6 (SEQ ID NO: 53), VL7 (SEQ ID NO: 55) ) At the 5 ′ end, and PCR primer JL1 (SEQ ID NO: 57) corresponding to the C-terminal amino acid sequence (SEQ ID NO: 58, 60 and 62) conserved in frame 4 of the human antibody λ chain variable region. ), An equal mixture of JL2 (SEQ ID NO: 59), JL3 (SEQ ID NO: 61) was used at the 3 ′ end.
The PCR reaction was performed using the Perkin Elmer Gene Amp PCR System.
Using 2400, ThermoScript RT-PCR System (Invitrogen) was used according to the instructions. As a result, the light chain was amplified by PCR using a primer for λ chain and not amplified by a primer for κ chain. Therefore, it was found that the light chain of the antibody produced from the hybridoma was a λ chain.
The amplified variable region DNA fragments of each heavy chain and light chain were purified using MiniElute PCR Purification kit (QIAGEN). Next, TOPO
Using TA cloning kit (Invitrogen), pCR2.1-TOPO and the purified PCR product were ligated to transform E. coli. The resulting colony was picked up, the plasmid was purified using QIAGEN Plasmid mini kit (QIAGEN), digested with EcoRI at 37 ° C. for 30 minutes, and confirmed that the DNA fragment of interest was incorporated by 2% agarose electrophoresis did.
The base sequence was analyzed by CEQ 2000 DNA Analysis System (Beckman) using a M13 primer for a plurality of colonies in which the target DNA fragment was incorporated. As a result, from analysis of the heavy chain, three types of sequences, 1F8-HA (SEQ ID NO: 63), 1F8-H-B (SEQ ID NO: 65), and 1F8-HC (SEQ ID NO: 67) are obtained. It was. From the analysis of λ chain, the sequence of 1F8-L (SEQ ID NO: 69) was obtained. By comparing these base sequences and the corresponding amino acid sequences (heavy chain: SEQ ID NO: 64, 66, 68, λ chain: SEQ ID NO: 70), the base sequence of the variable region (heavy chain and heavy chain respectively) The chain: SEQ ID NO: 71, light chain: SEQ ID NO: 69) and amino acid sequence (heavy chain: SEQ ID NO: 72, light chain: SEQ ID NO: 70) were determined.
The boundary between the hypervariable region (CDR) and the framework was determined with reference to Kabat et al. (Sequences of Proteins of Immunological Interest, fifth edition National Institutes of Health, Bethesda, MD 1991). As a result, the heavy chain CDRs were HCDR1 (base sequence: SEQ ID NO: 73, amino acid sequence: SEQ ID NO: 74), HCDR2 (base sequence: SEQ ID NO: 75, amino acid sequence: SEQ ID NO: 76) and HCDR3 (base sequence: SEQ ID NO: 77). , Amino acid sequence: SEQ ID NO: 78), light chain CDRs are LCDR1 (base sequence: SEQ ID NO: 79, amino acid sequence: SEQ ID NO: 80), LCDR2 (base sequence: SEQ ID NO: 81, amino acid sequence: SEQ ID NO: 82) and LCDR3 ( Base sequence: SEQ ID NO: 83, amino acid sequence: SEQ ID NO: 84).

(8) γ-1F8. Preparation of (Recombinant 1F8 Antibody) (8-1) Preparation of cDNA and Acquisition of 1F8 Variable Region Fragment Preparation of cDNA from total RNA (described in Example (7)) and PCR reaction were performed using the Perkin Elmer Gene Amp PCR System. 2400 was used according to the instructions using Thermoscript RT-PCR System, High Fidelity (Invitrogen). Random 6mer attached to the kit was used as a primer for preparing heavy chain cDNA.
For amplification of the variable region of heavy chain and λ chain, a primer into which a restriction enzyme site for ligation with an expression plasmid was introduced was used. For amplification of the heavy chain variable region, P1 (SEQ ID NO: 89) containing an AflII site on the 5 ′ end side and P2 (SEQ ID NO: 90) containing an NheI site on the 3 ′ end side were used. For amplification of the λ chain variable region, P3 containing a BsrGI site on the 5 ′ end side (equal mixture of SEQ ID NOs: 91, 92, 93, 94, 95, 96, 97) and an AvrII site on the 3 ′ end side P4 (equal mixture of SEQ ID NO: 98, 99, 100) was used.
The amplified heavy chain and λ chain variable region DNA fragments were purified using QIAquick PCR Purification Kit (QIAGEN). Next, the DNA fragment was digested with a restriction enzyme for ligation with an expression vector. The heavy chain DNA fragment was digested with AflII and NheI at 37 ° C. for 2 hours. The λ chain DNA fragment was digested with BsrGI and AvrII at 37 ° C. for 2 hours. The digested DNA fragment was subjected to 1.5% agarose electrophoresis, and the target DNA fragment was purified using QIAquick Gel Extraction Kit (QIAGEN).

(8-2) Cleavage of Expression Plasmid and Acquisition of Plasmid Fragment In order to express a 1F8 human IgG1-type recombinant antibody, pEX-G1- containing a heavy chain constant region sequence and a dhfr gene sequence as a heavy chain expression plasmid As the sig-dhfr ″, λ chain expression plasmid, pEX-lamda-sig containing the λ chain constant region sequence was used. pEX-G1-sig-dhfr ″ was digested with AflII and SpeI at 37 ° C. for 2 hours. pEX-lamda-sig was digested with BsrGI and AvrII for 2 hours. The digested fragment was subjected to 0.8% agarose electrophoresis, and the target fragment was purified using QIAquick Gel Extraction Kit (QIAGEN).

(8-3) Ligation of 1F8 variable region to expression plasmid The DNA fragment containing 1F8 heavy chain variable region obtained by the above method and the cleaved expression plasmid pEX-G1-sig-dhfr ″ are Ligation Kit Ver . 2.1 (TAKARA) was used for ligation according to the instructions, and E. coli DH5α-T1 (Invitrogen) was transformed according to the instructions. The resulting colonies were picked up and the plasmid was purified using QIAprep Spin Miniprep Kit (QIAGEN). The plasmid containing the heavy chain base sequence was digested with HindIII and NheI at 37 ° C. for 1.5 hours and subjected to 1% agarose electrophoresis to obtain the desired plasmid pEX-1F8-H. The nucleotide sequence of the obtained plasmid was confirmed using P5 (SEQ ID NO: 101) on the 5 ′ end side and P6 (SEQ ID NO: 102) on the 3 ′ end side. The base sequence of the heavy chain structural gene of the 1F8 recombinant antibody is shown in SEQ ID NO: 103.
Meanwhile, an expression plasmid pKS-lamda-sig cleaved with a DNA fragment containing the 1F8 λ chain variable region was obtained from Ligation Kit Ver. Using 2.1 (TAKARA), ligation was performed according to the instructions, and E. coli DH5α-T1 (Invitrogen) was transformed. The resulting colonies were picked up and the plasmid was purified using QIAprep Spin Miniprep Kit (QIAGEN). The plasmid containing the λ chain base sequence was digested with BsrGI and AvrII at 37 ° C. for 2 hours and subjected to 1% agarose electrophoresis to obtain the desired plasmid pEX-1F8-L. The obtained plasmid was confirmed for the base sequence using P5 on the 5 ′ end side. The base sequence of the λ chain structural gene of the 1F8 recombinant antibody is shown in SEQ ID NO: 105.

(8-3) Ligation of plasmid containing 1F8 heavy chain and 1F8 λ chain In order to link 1F8 heavy chain and λ chain to one plasmid, the plasmid obtained above was digested with restriction enzymes. pEX-1F8-H was digested with NheI at 37 ° C. for 1.5 hours, and pEX-1F8-L was digested with NheI and SpeI at 37 ° C. for 1.5 hours. The obtained fragment was purified using QIAquick PCR Purification Kit (QIAGEN). Each fragment was transferred to Ligation Kit Ver. 2.1 (TAKARA) was used for ligation according to the instructions, and E. coli DH5α-T1 (Invitrogen) was transformed. The resulting colonies were picked up and the plasmid was purified using QIAprep Spin Miniprep Kit (QIAGEN). The obtained plasmid was digested with NheI at 37 ° C. for 1 hour and subjected to 1.5% agarose electrophoresis to select a plasmid of the desired size. Further, they were digested with HindIII at 37 ° C. for 1 hour and subjected to 1.5% agarose electrophoresis to check the plasmids, thereby obtaining the desired plasmid pEX-1F8-HL.

(8-4) Preparation of γ-1F8 Production Recombinant A CHO (DG325) cell line that can be cultured in a serum-free medium was used as a cell line for preparing a γ-1F8 production recombinant. CHO (DG325) was adjusted to 1 × 10 ⁶ cells / ml with CHO-S-SFMII (GIBCO). 2 μg of plasmid pEX-1F8-HL DNA and 6 μl of FuGENE 6 (Roche) were mixed and transfected into CHO (DG325) according to the instructions. EX-CELL325-PF (Nichirei) was added 5.5 hours after transfection. After culturing for 2 days, G418 (Promega) 400 μg / ml and methotrexate (Sigma) 0 or 25 nM were added to the medium as a selection reagent and cultured. After cloning, a cell line producing γ-1F8 was obtained.

(8-5) Screening of antibody producing strain using ELISA Assay Screening of antibody producing cells was performed by sandwich ELISA. First, goat anti-human immunoglobulin antibody (CAPPEL) was diluted to 50 μg / ml with PBS, added to a 96-well plate (FALCON) at 50 μl / well, and treated at 37 ° C. for 2 hours. After washing 5 times with PBS, blocking was performed by adding 150 μl / well of PBS solution containing 5% BSA (bovine serum albumin). The plate was washed 5 times with PBS, 50 μl of recombinant culture supernatant was added, and reacted at 37 ° C. for 2 hours. Next, it was washed 5 times with PBS, and a goat antibody (1000 culture diluted, Capel) against human antibody bound with 50 μl of horseradish peroxidase (HRP) was added, followed by reaction at 37 ° C. for 1 hour. Next, the plate was washed with PBS containing 0.05% Tween 20 (PBS-T) and contained o-phenylenediamine dihydrochloride (5.2%) and H ₂ O ₂ (0.015%). Phosphate citrate buffer was added at 50 μl / well and reacted at room temperature until color development could be confirmed. Then, 1N—H ₂ SO ₄ was added at 50 μl / well, the reaction was stopped, and the absorbance at 490 nm was measured using a microphotograph. A cell line with high production was selected by measuring with a meter (Nihon Intermed).

(8-6) Purification of γ-1F8 Using the culture solution A containing 4% of Prosep A-treated serum to which G418 (Promega) was added, the γ-1F8-producing cell line obtained above was cultured, and γ-1F8 was A culture broth containing was obtained. Next, the culture solution was subjected to process A to adsorb γ-1F8, and then eluted to purify γ-1F8. Although not shown in the figure, γ-1F8 was confirmed to be pure IgG by SDS-PAGE.

(8-7) Confirmation of binding activity of γ-1F8 to cancer cells Binding of purified γ-1F8 described in (8-6) to HLC-1 cells fixed by the same method as described in (2-2) The activity was confirmed. Each concentration of antibody was reacted with fixed cancer cells at 37 ° C. for 1 hour. The plate was washed with PBS, a goat antibody against human antibody bound with HRP (1000-fold diluted, capel) was added, and the reaction was further carried out at 37 ° C. for 1 hour. As a result of detecting the reactivity of γ-1F8, it was confirmed that γ-1F8 showed binding activity to HLC-1 cells in a concentration-dependent manner (FIG. 4).

The figure (photograph) which shows the reactivity with respect to various cancer tissue sections of 020630-18-1 antibody. The figure (photograph) which shows the binding activity of the 1F8 antibody to the lung cancer live cell surface (A) and the lung normal live cell surface (B). The graph which shows the growth inhibitory effect with respect to the cultured cancer cell lines HLC-1, PANC-1, and MKN45 of 1F8 antibody. The graph which shows the binding activity with respect to the fixed cancer cell surface of (gamma) -1F8 antibody.

Claims (22)

A monoclonal antibody comprising an amino acid sequence of SEQ ID NOs: 74, 76 and 78 in a heavy chain variable region. The monoclonal antibody according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 72. The monoclonal antibody according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 104. A monoclonal antibody comprising the amino acid sequences of SEQ ID NOs: 80, 82 and 84 in the light chain variable region. The monoclonal antibody according to claim 4, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 70. The monoclonal antibody according to claim 4, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 106. A monoclonal antibody, wherein the heavy chain variable region comprises the amino acid sequences of SEQ ID NOs: 74, 76, and 78, and the light chain variable region comprises the sequences of SEQ ID NOs: 80, 82, and 84. The monoclonal antibody according to claim 7, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 72, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 70. The monoclonal antibody according to claim 7, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 104, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 106. The monoclonal antibody according to any one of claims 1 to 9, which is a human antibody. DNA encoding the monoclonal antibody according to any one of claims 1 to 10. The region encoding the heavy chain variable region includes the nucleotide sequences of SEQ ID NOs: 73, 75 and 77, and the region encoding the light chain variable region includes the nucleotide sequences of SEQ ID NOs: 79, 81 and 83, The DNA according to claim 11. The region encoding the heavy chain variable region includes the base sequence of SEQ ID NO: 71, and the region encoding the light chain variable region includes the base sequence of SEQ ID NO: 69. 12. DNA according to 12. The region encoding the heavy chain variable region includes the base sequence of SEQ ID NO: 103, and the region encoding the light chain variable region includes the base sequence of SEQ ID NO: 105. 12. DNA according to 12. A recombinant vector comprising the DNA according to any one of claims 11 to 14. A transformant comprising the recombinant vector according to claim 15. The hybridoma which produces the monoclonal antibody as described in any one of Claims 1-10. A pharmaceutical composition comprising the monoclonal antibody according to any one of claims 1 to 10. The pharmaceutical composition according to claim 18, which is a cancer therapeutic agent. 20. The pharmaceutical composition according to claim 19, wherein the cancer therapeutic agent is a cancer therapeutic agent for one or more cancers selected from the group consisting of non-small cell lung cancer, pancreatic cancer, and gastric cancer. 21. The pharmaceutical composition according to any one of claims 18 to 20, wherein the monoclonal antibody is carried on the surface of a liposome encapsulating a toxin or an anticancer agent. The diagnostic reagent containing the monoclonal antibody as described in any one of Claims 1-10.

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