JP2003012544A - Medicine for preventing and treating cancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a human cancer-preventing and treating agent using a cancer cell-specific HLA-F antigen, and to provide an anti-HLA-F antibody. SOLUTION: This cancer-preventing and treating agent contains a part or all of a DNA sequence described in the specific sequence of the sequence table as an active ingredient. The cancer-preventing and treating agent can derive cytotoxic T lymphocytes against human cancer cells in vivo or in vitro. Further, an anti-HLA-F monoclonal antibody can be utilized for an HLA-F antigen assay and for a target chemotherapeutic using the antibody.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cytotoxic T-lymphocyte for human cancer cells.
s, hereinafter referred to as "CTL". ) In vivo or in vitro
For preventing and / or treating human cancer using a cancer cell-specific HLA-F antigen that can be induced in
DNA vaccine containing DNA encoding F antigen protein, cell vaccine expressing HLA-F antigen, HLA-
The present invention relates to cytotoxic (damaged) T cells induced using cells expressing F antigen, and anti-HLA-F antibody.

[0002]

2. Description of the Related Art Up to now, the treatment of cancer has been mainly "surgery",
The three major therapies, "radiation" and "chemotherapy" have been given. Cancers have been classified according to the organ in which they develop, and further treated by histologically subdividing them according to the type of cells that became the mother of the development, and treatment methods have been selected. However, in these treatment methods, even if the subjects have the same cancer histologically, it is often the case that a certain effect cannot be obtained for each individual patient, and since these treatment methods cause residual disorders and strong side effects, The problem arises that patients who have failed these treatments themselves suffer great disadvantages.

In recent years, methods such as "gene therapy" and "immunotherapeutic" have advanced as new therapeutic methods for cancer, and have achieved therapeutic results. The main subject of the latter "immunotherapy" is to identify the cancer antigens expressed in individual patients and induce the immune response of the living body against them. Cancer antigen
An abnormal protein produced by the canceration of cells appears on the cell surface along with the flow of the antigen presentation mechanism. In order to cure cancer by inducing an immune response, it is essential to identify this "cancer antigen" that CTL recognizes with MHC (major histocompatibility complex) molecules on the cell surface.

The proteins and peptides identified as cancer antigens capable of inducing CTL so far include MAGE family (J. Exp. Med. 178: 489-495,199).
3), BAGE (Immunity 2: 167-175, 1995), GAGE
(J.Exp.Med.182: 689-698, 1995), tyrosinase (J.Exp.M.
ed.178: 489-495,1993), MART-1 (Proc. Natl. Acad.
Sci. USA 91: 3515, 1994), gp100 (J.Exp.Med. 17)
9: 1005-1009, 1994), gp75 (J. Exp. Med. 181: 799)
-804, 1995) etc. are known. However, these cancer antigens are not common to various cancers, and their immune response is also restricted to a specific MHC {HLA (human leukocyte antigen)} type. In addition, as a mechanism for escape from the immunity of cancer cells, it is known that the expression of MHC molecules on the surface of cancer cells is reduced. In such a case, CTL cannot be induced even by using the above MHC-restricted peptide. .

[0005]

SUMMARY OF THE INVENTION Therefore, the object of the present invention is to produce CTLs in a living body in a cancer-specific and non-organ-specific manner, and to suppress MHC non-restriction of cancer cells and antigen-presenting cells.
The purpose of the present invention is to provide a drug that can be used for cancer treatment and cancer prevention regardless of the difference in organs or the cause of carcinogenesis, by using a "cancer common antigen" capable of inducing cancer.

[0006]

That is, the present invention provides the following. (1) A prophylactic / therapeutic agent for cancer, which comprises, as an active ingredient, a part or all of the DNA sequence shown in SEQ ID NO: 1 of the Sequence Listing. (2) A prophylactic / therapeutic agent for cancer, which comprises, as an active ingredient, a plasmid containing a part or all of the DNA sequence shown in SEQ ID NO: 1 of the Sequence Listing. (3) A prophylactic / therapeutic agent for cancer, which comprises, as an active ingredient, a viral vector having a part or all of the DNA sequence set forth in SEQ ID NO: 1 of the Sequence Listing. (4) The DN according to (1), (2) or (3) above
A, a cell presenting a cancer antigen transformed by a plasmid or a viral vector. (5) Induction using the cell that presents the cancer antigen according to (4) above, or the cell that presents the cancer antigen pulsed with a polypeptide containing at least a part of the amino acid sequence of SEQ ID NO: 4 in the sequence listing Cytotoxic (damaged) T cells (hereinafter referred to as CTL). (6) A CTL inducer that is non-organ-specific and can induce cancer cell-specific CTL. (7) A CTL inducer capable of inducing cancer cell-specific CTL without MHC restriction. (8) A CTL-inducing agent which is organ-specific and non-MHC-constrained and can induce cancer cell-specific CTL.

(9) An anti-HLA-F antibody which recognizes any of the sequences of SEQ ID NOs: 4, 5 and 6 in the sequence listing. (10) The antibody is a monoclonal antibody.
The anti-HLA-F antibody described in 1. (11) A 3A5 antibody that recognizes the amino acid sequence of Arg-Asn-Leu-Leu-Arg-Arg-Tyr-Asn-Gln-Ser. (12) A 3H5 antibody that recognizes the amino acid sequence of Glu-Gly-Pro-Gln-Tyr-Trp-Glu-Trp-Thr-Thr. (13) A hybridoma or cell line that produces the antibody according to any one of (9) to (12). (14) The hybridoma has an accession number FERM P
The hybridoma according to the above (13), which is -18795 or FERM P-18796. (15) A composition for treating or preventing cancer, tumor and / or leukemia, which comprises an anti-HLA-F antibody. (16) A detection agent for detecting a mutant cell group of cancer, a tumor focus site, and / or leukemia, which comprises a labeled anti-HLA-F antibody. (17) A method for measuring an HLA-F antigen, which comprises measuring an HLA-F antigen using an anti-HLA-F antibody. (18) A gene therapy vector containing an anti-HLA-F antibody. (19) The desired substance was HL-treated using an anti-HLA-F antibody.
A method of delivering to the site where the AF antigen is present, particularly a method using an anti-HLA-F antibody that recognizes any one of SEQ ID NOs: 4, 5, and 6 in the sequence listing.

The present inventor has identified a human cancer-specific antigen,
Its amino acid sequence and DNA sequence were clarified (Japanese Patent Application No. 11-279566). This cancer cell-specific HLA
The -F antigen is one of the non-classical MHC class I antigens, but its expression pattern in the living body is a molecule whose expression is induced by carcinogenesis in contrast to the classical MHC class I antigen.

Cancer cell-specific HLA-F antigen is originally human M
Since it is an HC class I antigen, it is considered possible to induce CTL if it can be expressed alone on the cell surface. That is, it is considered that cells in which the expression of classical MHC class I antigen is reduced due to canceration can also be the target of CTL, and any cell line can be used as a method for inducing CTL regardless of MHC positive or negative.

Therefore, the present inventor has found that HLA-specific for cancer cells is used.
The F antigen gene inserted into an expression vector was introduced into and expressed in a human cell line to establish cancer cell-specific HLA-F antigen-positive cells. On the other hand, as a result of attempting to induce CTLs from peripherally hemolyzed lymphocytes of subjects with different MHC types, CTLs showing cytotoxic activity only to cells expressing cancer cell-specific HLA-F antigen were obtained in all cases. I was able to. From this fact, it was confirmed that the cancer cell-specific HLA-F antigen-expressing cells could be used as a cancer cell-specific and non-MHC-restricted CT.
The inventors have found that L can be induced and completed the present invention.

Therefore, the present invention provides a cancer preventive / therapeutic agent capable of inducing an immune response in a cancer cell-specific manner. The cancer cell-specific HLA-F antigen or the DNA encoding the HLA-F antigen has a specific amino acid sequence or base sequence and is commonly expressed in cancer. Those having such a specific sequence are Even if the name is different from that of the present invention or is derived from a source different from that disclosed in the present invention, a cancer preventive / therapeutic agent using the same is included in the present invention.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION A cancer cell-specific HLA-F antigen is
Specifically, it is a part of the amino acid sequence of SEQ ID NO: 4 in the sequence listing as disclosed in Japanese Patent Application No. 11-279566. More specifically, at least SEQ ID NO: 5 in the sequence listing
The amino acid sequence having the amino acid sequence of SEQ ID NO: 6 is more preferable. Amino acid sequences may have a few amino acids or fewer deleted or replaced, inserted, or added with other amino acids or amino acid sequences without altering their properties due to species homology or mutation. , Such sequences are also within the scope of the polypeptides used in the present invention as long as they do not materially affect the properties of the invention. The present inventors have found that cancer cell-specific H
It is the amino acid sequence of SEQ ID NO: 6 that is specified as the extracellular domain of the HLA-F antigen α chain, α1, α2, and α3, which is involved in the antigenicity of the LA-F antigen. Have specified that.

The DNA encoding the cancer cell-specific HLA-F antigen is the DNA of SEQ ID NO: 1 in the sequence listing, specifically, the DNA containing at least the DNA of SEQ ID NO: 3, and the DNA of at least SEQ ID NO: 2. DNA containing SEQ ID NO: 2, even if it is a part of the DNA of SEQ ID NO: 3.
May be a part of DNA. In addition, cancer cell-specific H
As long as it is a DNA encoding the LA-F antigen, it may be a DNA within the degeneracy range of the DNAs of SEQ ID NOs: 1, 2, and 3. The amino acid sequence of the cancer cell-specific HLA-F antigen does not change its characteristics due to interspecies differences or mutations, a few amino acids or less are deleted, or other amino acids or amino acid sequences are used. D that may be substituted, inserted or added and encodes such an amino acid sequence
NA is also the range of DNA used in the present invention as long as it does not essentially affect the properties of the present invention. For DNA, c
DNA, genomic DNA and synthetic DNA are included.
In addition, the DNA may be single-stranded or double-stranded, and when single-stranded, may include both sense strand and antisense strand.

(A) A preparation HLA-F antigen containing a part or all of DNA encoding a cancer cell-specific HLA-F antigen as an active ingredient, or a preparation containing a plasmid containing these DNAs as an active ingredient. Encoding DNA is the genomic HLA
It may be the -F gene region or cDNA obtained by synthesis from mRNA. The method for synthesizing cDNA from mRNA of cultured cells will be described below. (1) Synthesis of cDNA Human cancer cells [eg human myeloid leukemia cells HL-60
(Details are described in Cancer, Vol. 28, pp. 1300-1310 (1968)), U937 (Details are J. Exp. Med., Vol. 143,
pp1528-1533 (1976)). The present invention cites these documents and constitutes the contents of the present specification. ], Etc. are cultured, mRNA is extracted, and cDNA for this is synthesized using reverse transcriptase.

(2) Preparation of HLA-F cDNA The obtained cDNA was used as a template DNA and HL was used as a primer.
Amplification of HLA-F cDNA by PCR (polymerase chain reaction) method using deoxyoligonucleotide specific to AF gene (for details, see J. Immunolog
y, Vol. 149, pp.668-676 (1992). The present invention cites these documents and constitutes the contents of the present specification. ).
The present inventor has identified that it is part of the extracellular domain of the HLA-F antigen α chain that is involved in the antigenicity of the cancer cell-specific HLA-F antigen. Therefore, HLA-Fc
As the DNA, it is preferable to amplify the site encoding the extracellular domain of HLA-F antigen α chain. Specifically, at least HLA-F gene No. 64 of SEQ ID NO: 1
~ HLA containing No. 885 (ie, the sequence of SEQ ID NO: 2)
Amplification is carried out using a primer designed so as to obtain FcDNA, more preferably an HLA-FcDNA fragment containing HLA-F gene containing No. 130 to No. 774 of SEQ ID NO: 1 (that is, the sequence of SEQ ID NO: 3). The obtained cDNA
Is analyzed and compared with the base sequence of the HLA-F gene to confirm whether HLA-F cDNA was obtained.

(3) Preparation of expression plasmid HLA-FcDNA is replicated in a host cell to prepare an expression plasmid for producing a protein. the above
A translation initiation codon is added upstream of the HLA-F cDNA obtained by the method (2), and a translation termination codon is added downstream thereof to control transcription (for example, trp, lac, T7,
SV40 early promoter and other regulatory genes)
Incorporate into vector. As a vector, specifically,
pBR322, pUC19, pSV / SPORT1, p
Rc-CMV, pZeo-SV2 and the like are mentioned, and particularly p
Rc-CMV and pZeo-SV2 are preferred. Such a plasmid is used in Molecular Cloning A LABOLATORY MAN
It can be prepared by a general method described in UAL SECOND EDITION (1989) and the like. The obtained expression plasmid is introduced into an appropriate host cell to prepare a transformant cell. Further, in order to more reliably express the sequence, the sequence encoding the intracellular region of HLA-F was replaced with another HLA class I.
It can also be inserted into the vector by substituting the sequence of the intracellular region of the a molecule.

(B) Preparation containing a viral vector containing a part or all of DNA encoding a cancer cell-specific HLA-F antigen as an active ingredient The cDNA obtained by the method of (a) (2) above is a viral vector. To insert. As the viral vector, for example, retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus,
RNA viruses or DNA viruses such as poliovirus and Sindbis virus are mentioned, and retrovirus, adenovirus, adeno-associated virus, and vaccinia virus are particularly preferable. For the method of inserting the cDNA into a viral vector, see Molecular Cloning A LABOLATORY MAN.
It can be performed by a general method described in UAL SECOND EDITION (1989) and the like.

When a formulation containing the virus vector of the present invention as an active ingredient is administered by an in vivo method,
For example, it can be administered into veins, arteries, subcutaneously, intramuscularly and the like. Generally, it is administered in the form of an injection or the like containing the virus vector of the present invention as an active ingredient, and a conventional carrier may be added if necessary.

[0019] In addition, as a method for administering a preparation containing a part or all of DNA encoding a cancer cell-specific HLA-F antigen as an active ingredient, a liposome method can also be mentioned. A liposome, a membrane-fusion liposome (Sendai virus (HVJ) -liposome, etc.) containing a part or all of the DNA encoding the cancer cell-specific HLA-F antigen is suspended, frozen, or concentrated by centrifugation. And the like.

(C) Cells presenting cancer antigens transformed with the plasmid or viral vector of the present invention (1) Transformant with the plasmid An expression plasmid having HLA-FcDNA inserted is introduced into an appropriate host cell to transform it. Create a transformant. Examples of host cells include cells of prokaryotes such as Escherichia coli, single cell eukaryotes such as yeast, and multicellular eukaryotes such as insects and animals. Examples of gene transfer methods into host cells include the calcium phosphate method, the DEAE-dextran method, the electric pulse method, the lipofection method and the like. The transformant produces the target protein by culturing in a suitable medium. The produced protein can be isolated and purified by a general biochemical method, and the purified protein can be used to purify antibodies and pulse antigen-presenting cells.

(2) Transformant with virus vector The virus vector in which the HLA-F cDNA is inserted is introduced into an appropriate host cell to prepare HLA-F antigen-expressing cells. From the viewpoint of using this transformant as a cancer preventive / therapeutic agent and a cell for inducing CTL, an antigen-presenting cell using an autologous cell of a cancer disease patient or a healthy person who desires cancer prevention is preferable as a host cell, Also, any HLA-negative autologous or other human cell line can be used. Dendritic cells are particularly preferable as the antigen-presenting cells, and HLA-F antigen-expressing dendritic cells obtained by introducing a virus vector into which the HLA-F gene has been inserted can also be administered to a living body as a vaccine.

Dendritic cells can be induced to differentiate from progenitor cells such as peripheral blood-derived mononuclear cells, bone marrow cells, or cord blood-derived undifferentiated cells, and can be easily collected. It is preferable to use. Although these cells are preferably autologous, non-autologous cells may be used as long as they have the same HLA type. As a method for separating and purifying nucleated cells from the collected peripheral blood, Ficoll-
Ficoll-Paque density gradient centrifugation or elution, a solution that selectively lyses red blood cells, such as ammonium chloride-pot.
assium), ammonium oxalate (ammonium oxalat
Examples include a method of suspending in e) and separating. In addition, separation can be performed using antibody-immobilized beads using an antibody against an appropriate surface antigen, for example, CD34 antigen, magnetic beads, a column packed with these, and a flow cytometer. As a medium for inducing differentiation of dendritic cells, AIM-V, RPMI-1640, DMEM, IM
DM etc. are mentioned. Appropriately 5 to 20% of bovine serum, fetal bovine serum, human serum, bovine albumin (BSA), human albumin (HSA) or the like is added. In addition, appropriate antibiotics, antibodies, pyruvic acid (0.1-5 mM), glutamine (0.5-5 mM), 2-mercaptoethanol (10
˜100 μM) may be added. A differentiation inducing agent is added to this in an amount of 1 to 1000 ng / ml. As the differentiation inducer, GM-CSF (granulocyte macrophage colony stimulating factor), IL-4, SCF (stem cell factor),
IL-13, TNF-α, Flt3-Ligand, I
Cytokines such as L-1, IL-2 and IL-3 are preferable, and in particular, a combination of GM-CSF and IL-4, GM-.
Combination of CSF, IL-4 and TNF-α, GM
A combination of -CSF, SCF and TNF-α is preferred. Chemokines and the like may be added to prepare cells that effectively present antigens. Precursor cells in such a medium at about 34 to 38 ° C. under about 5% carbon dioxide gas atmosphere for 5 to 5
It is cultured for 21 days, preferably 7 to 14 days. The target dendritic cells were obtained (1) Morphological features were protrusions extending and dendritic cells, (2) HLA-
DR (high), CD1a (low), CD14
(-), CD25 (+), CD83 (+), CD86
It can be confirmed by showing the expression pattern of cell surface markers such as (high) and CD80 (+).

The obtained dendritic cells are brought into contact with a complex with a virus vector into which HLA-F cDNA has been inserted, ex vivo, and as a result, HLA-F cDNA is introduced into the dendritic cells to obtain cancer cell-specific HLA-F. The antigen is expressed on dendritic cells.

Cord blood-derived undifferentiated cells or peripheral blood-derived
Mononuclear cells have extremely low proliferative potential by inducing differentiation.
Induction of differentiation as described above
The obtained dendritic cells may be used as they are.
When administered as a vaccine in humans,
In some cases it may be preferable to
The following method may be performed. The heating method is
Processing, radiation treatment, or mitomycin C treatment.
You can When using X-ray irradiation, the X-ray irradiation tube
Place a flask containing dendritic cells under the
Irradiate at 00 to 3300 Rad. Mitomycin C treatment
The method is, for example, 1 to 3 × 10 dendritic cells. ⁷Individual cells / m
Suspend at a density of 1 ml per 1 ml of cell suspension
Syn C added at a ratio of 25 to 50 μg, at 37 ° C., 30 to
Incubate for 60 minutes. For example, the method of treating cells with heat is
If the viable cell concentration is 1 x 10⁷Cells / ml adjusted to
Heat treatment of centrifuge tube containing suspension for 20 minutes at 50-65 ℃
It is prepared by performing.

Cells presenting antigen pulsed with cancer cell-specific HLA-F antigen peptide In addition to the above, cells presenting the cancer antigen of the present invention are dendritic cells in vitro with HLA-F antigen peptide. It is also possible to produce cells that present an antigen by pulsing under the conditions described below.

(D) C induced using cancer cell-specific HLA-F antigen expression-positive cells or cells presenting cancer antigens pulsed with a cancer cell-specific HLA-F antigen peptide
TL (1) CTL induced using cancer cell-specific HLA-F antigen expression-positive cells The cancer cell-specific HLA-F antigen expression-positive cells obtained in (a) or (b) above are separated from peripheral blood. By culturing with lymphocytes, CTLs that recognize the cancer cell-specific HLA-F antigen are induced. If necessary, it is preferable to introduce a plasmid for expressing an auxiliary molecule such as B7 into the host cell in advance, or to use a cell expressing such an auxiliary molecule. The CTL induction can be performed in vitro or in vivo in an animal or human body.

(2) CTL Induced Using Cells Presenting Cancer Antigen Pulsed with Cancer Cell-Specific HLA-F Antigen Peptide The cancer cell-specific HLA-F antigen peptide is one of the sequences of SEQ ID NO: 4. Part or all consist of at least 6 to 11 amino acids. The peptide may be added at the time of culturing to induce dendritic cell differentiation, or may be added after differentiation.
Dendritic cells are incubated at room temperature or 37 ° C. for at least about 1-6 hours for peptide antigens and at least about 3-12 hours for intact protein antigens.
Using the cells presenting the antigen thus obtained,
CTL can be induced in the same manner as (1).

(3) The CTL obtained by induction is organ-nonspecific, and the CTL obtained by the method by peptide stimulation is MHC-restricted, but it is stimulated by expressing HLA-F antigen on the cell surface. When used, it is a non-MHC-restricted and cancer cell-specific CTL that has not been previously obtained. When such a CTL is used as a cancer preventive / therapeutic agent, a cancer-specific cellular immune response effectively suppresses the growth and metastasis of cancer cells, and thus has therapeutic / preventive effects.

(E) Antibody that specifically reacts with cancer cell-specific HLA-F antigen (1) The antibody may be a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody, The invention provides the following antibodies. An anti-HLA-F antibody, which is a polyclonal or monoclonal antibody that specifically binds to an amino acid sequence of at least a part of any one of SEQ ID NOs: 4, 5, and 6.

Anti-HLA-F antibodies which recognize and bind the following sequences: 3A5 antibody and 3H5 antibody. Sequence recognized by 3A5 antibody: 1-letter code: RNLLRRYNQS 3-letter code: Arg-Asn-Leu-Leu-Arg-Arg-Tyr-Asn-Gln-Ser Sequence recognized by 3H5 antibody: 1-letter code: EGPQYWEWTT 3-letter code : Glu-Gly-Pro-Gln-Tyr-Trp-Glu-Trp-Thr-Thr The above sequence is the amino acid sequence at the corresponding position in the sequence number in the sequence listing. Sequence number 4: No. 100 to 109 (3A5) No. 76 to 85 (3H5) Sequence number 5: No. 79 to 88 (3A5) No. 55 to 64 (3H5) Sequence number 6: No. 57 to 66 ( 3A5) No.33 to 42 (3H5) The sequences recognized above were each converted into SWISS PL of GenomeNet.
An OT search revealed that it was not present in other reported human proteins.

Neutralizing antibody against HLA-F antigen From the results described in Example 5 below, the 3A5 antibody and 3H
5 antibody was confirmed to be an antibody that neutralizes the cytotoxic activity of CTL. The hybridomas that produce the above-mentioned antibodies have the accession numbers FERM P-18795, respectively.
(Identification display F3A5), and FERM P-1879
6 (identification F3H5) has been deposited on March 25, 2002, at the Patent Biological Depositary Center, National Institute of Advanced Industrial Science and Technology, Central 1st, 1-1 1-1 Higashi, Tsukuba, Ibaraki, Japan.

(2) The antibody of the present invention can be used in a method for treating or preventing cancer, tumor and / or leukemia.
Since the anti-HLA-F antibodies show strong reactivity to the cancer-specific antigen HLA-F, these antibodies are useful for designing targeted therapeutic agents used in the treatment of human cancer. An antibody against a cancer cell-specific HLA-F antigen can be coupled to a substance having cytotoxicity to be applied to missile therapy.

These include a drug delivery system using an anti-HLA-F antibody, the anti-HLA-F antibody of the present invention, cytotoxic substances exemplified below, and, if necessary, sterile water and physiological saline. It may contain water, excipients and the like. Since the anti-HLA-F antibody of the present invention shows strong reactivity with the HLA-F antigen,
A cytotoxic substance can be effectively delivered to an HLA-F antigen that is expressed regardless of the type of cancer, and a therapeutic or prophylactic effect can be obtained in a smaller amount than when a cytotoxic substance is used alone. . The drug to be delivered is D for gene therapy.
It may be NA. Examples of gene therapy DNAs include plasmid DNAs that express cytotoxic proteins. A composition for treating or preventing cancer and / or tumor, comprising an anti-HLA-F antibody. The anti-HLA-F antibody of the present invention, a cytotoxic substance exemplified below, and, if necessary, sterile water, physiological saline, an excipient and the like may be contained. Examples of the substance having cytotoxicity include an endogenous substance (various cytokines, Fas ligand, perforin, etc.), a chemotherapeutic agent, a radionuclide and the like. Examples of the chemotherapeutic agent include anti-cancer agents such as adriamycin, cyclophosphamide, 5-fluorouracil and cisplatin.

(3) The antibody of the present invention can be used in a method for detecting HLA-F antigen. Since the anti-HLA-F antibody shows strong reactivity to the cancer-specific antigen HLA-F, these antibodies can be used as a detecting agent used in the diagnosis of human cancer. In particular, the antibody of the present invention can be used for detecting various cancers. The amount of cancer cell-specific HLA-F antigen can be measured in vitro or in vivo by using a sandwich method or a competition method with a known amount of an antibody against the cancer cell-specific HLA-F antigen. It can be used for diagnosis. A detection agent for detecting a mutant cell group of cancer, a lesion site of a tumor, and / or leukemia, which comprises a labeled anti-HLA-F antibody. By adding a label to the antibody of the present invention, a complex that can be detected during image diagnosis can be obtained. The label is a radioactive label,
A labeling substance such as an enzyme label or a fluorescent label can be used to add it to the antibody by a known method used in immunoassay. A method for measuring an HLA-F antigen, which comprises measuring an HLA-F antigen using an anti-HLA-F antibody. Anti-HLA-F antibody was used to target the desired substance to HLA-F.
A method of delivering to the site where the antigen is present. This method is not limited to diagnostic / therapeutic methods, but as a screening method, various substances can be tested in vitro or in vivo on HL.
Use as a screening method for developing an effective new drug by simulating or examining whether or not it can be delivered to the site or place where the AF antigen is present, or whether it can be delivered and exert its activity can do.

[0035]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. (Example 1) (1) Preparation of HLA-F expression plasmid FpHE donated by Dr. Daniel Geraghty of Canada
B® (J. Immunol. 142: 3320-3328 (1989), a 5.4 kbp HindIII fragment containing the HLA-F gene region was added to pHEBo.
Cloned to pUC118 and EcoR I
3.7 kbp HLA-F obtained by digestion with HindIII
The fragment was ligated with the 3.2 kbp pUC118 fragment by a ligation reaction. E. coli JM109
Strain, the same applies hereinafter) and subcloned.
9 kbp of pUC118 / F (E-Hw) was obtained. This pUC118 / F (E-
Hw) was digested with KpnI to remove an intron sequence of about 150 bp, self-ligated, and introduced into Escherichia coli for subcloning. Furthermore, this is Sma
Digested with I to remove about 70 bp, Sal I linker-ligated product was introduced into E. coli, and cloned to obtain 6.7 kbp of pUC118 / F (E-Hr1).

The obtained pUC118 / F (E-Hr1) was treated with EcoR I and Sal.
About 730 bp was removed by digestion with I, and a 6.6 kbp linear fragment was recovered. On the other hand, using pUC118 / F (E-Hr1) as a template, an F primer (5 ') containing 4 bp upstream of the initiation codon was used.
-tatgaattcggtcatggcgccccgaag-3 ') and R primer containing Sal I linker site (5'-attgtcgacggtccgtggcggat
PCR reaction was performed using gg-3 ′), and the obtained PCR product was digested with EcoR I and Sal I to prepare a fragment of about 510 bp. This fragment and a 6.6 kbp linear fragment were ligated by ligation reaction, introduced into E. coli, and subcloned to obtain 6.7 kbp pUC118 / F (E-Hr2). Next, pUC118 / F (E-Hr2) was digested with EcoR I, Hind III and Bgl.
After digestion with I, a 3.5 kbp fragment containing the HLA-F gene as a blunt end was recovered. With this 3.5 kbp fragment
The pRC-CMV vector was digested with HindIII, ligated with a 5.5 kbp fragment which was blunt-ended, and introduced into Escherichia coli, subcloned, confirmed the sequence, and confirmed to have a 9.0 kbp HLA-F expression plasmid. The vector pRC-CMV / F was obtained. Separately, pZeo-SV2
It was digested with Nhe I and Pst I, treated with T4 DNA polymerase to make blunt ends, ligated by self ligation, subcloned once, and then digested with Eco RV. This and HLA-F obtained from pUC118 / F (E-Hr2)
The gene fragment of 3.5 kbp is ligated by a ligation reaction,
Subcloning, restriction enzyme pattern and DNA
The sequence was confirmed and pZeo-SV2 / F was obtained.

<Preparation of novel expression plasmid with enhanced extracellular expression efficiency> mR from peripheral blood mononuclear cells of healthy humans known to express HLA-B7 in advance
NA was prepared, this was used as a template, and c
DNA was synthesized. Using this cDNA as a template, HLA-
B7-specific primer (5'-TAAGAGCTCAGGAGCCGTCTTCCCA
ATCCA-3 'and 5'-TATGATATCTTTTCAAGCTGTGAGAGACAC-3')
And a 0.2 kbp cDNA fragment (HL
A-B7 intracellular region) was amplified and then digested with SacI and EcoRV. pUC118 / F (E-Hr2) was digested with SacI and EcoRV to remove a sequence encoding the intracellular region of HLA-F to prepare a 5.7 kbp fragment, and the HLA-B7 intracellular region (0.2 kbp fragment) was prepared. After ligation by ligation reaction and introduction into E. coli, subcloning is performed once and pUC1
18 / F-B7 was obtained. This (pUC118 / F-B7) is EcoRI and Hi
It was digested with ndIII and blunt-ended with T4 DNA polymerase to prepare a 2.3 kbp F-B7 fragment. Also pZ
eo-SV2 was digested with NheI and PstI to make blunt ends, and then ligated by self-ligation to obtain pZeo-SV2 (-NP). This was digested with EcoRV and dephosphorylated, and the 2.3 kbp F-B7 fragment was ligated, introduced into E. coli and subcloned to obtain pZeo-SV2 / F-B7.
Sequence the insert sequence of the resulting plasmid,
It was confirmed that the fusion protein has HLA-F in the extracellular region and HLA-B7 in the intracellular region.

(2) Recombinant adenovirus vector expressing HLA-F A 3.5 kbp fragment obtained by digesting the above-mentioned pUC118 / F (E-Hr2) with EcoRI, HindIII and BglI was treated with T4 DNA polymerase to terminate the ends. Blunted fragment and cosmid vector pAxC
A ligation reaction of a 45 kbp fragment obtained by digesting Awt (Takara Shuzo) with Swa I was introduced into Escherichia coli, subcloned, and the sequence was confirmed to obtain 48.5 kbp pAxCAwt / F. . According to the Takara Shuzo manual, pAxCAwt / F introduced into 293 cells was cultured, and an HLA-F antigen-expressing recombinant adenovirus vector was obtained from the culture supernatant.

(3) Preparation of HLA-F antigen-expressing positive cells HLA-F expression plasmid vector pRC-CMV / was added to human cell line VA-13 confirmed to have no HLA-F expression by RT-PCR. Cells into which F 4 has been introduced, which has been cultured in the presence of G418 and has survived to form colonies,
That is, HLA-F stable expression positive cells VA-13 / F were obtained by selecting cells (transformants) into which the HLA-F gene had been introduced. The expression of VA-13 / F HLA-F antigen was confirmed by Western blotting using anti-HLA-F monoclonal antibodies (3A5 and 3H5).

(4) Preparation of CTL-inducing HLA-F antigen expression-positive cells HLA antigen-negative human cell line RPMI8226 was added to HL
A-F expression plasmid vector pZeo-SV2 / F was introduced by the lipofection method, and 2
HLA-F stable expression cell line RPMI822 after weekly culture
6 / F-B7 was established. RPM by Western blotting method using anti-HLA-F monoclonal antibody (3A5 and 3H5) and flow cytometer
The expression of the extracellular region of the HLA-F antigen of I8226 / F-B7 was confirmed. In addition, as a result of the screening of the natural HLA-F antigen expression-positive cells, C of the B cell leukemia cell line was
It was revealed that 1R cells express a large amount of HLA-F antigen on the cell surface. These cells were used as HLA-F antigen expression positive cells for inducing CTL.

(5) Three healthy persons (A, B,
Lymphocytes were separated from 15 cc of peripheral blood collected from C) using a Vacutainer (manufactured by Becton Dickinson) and suspended in RPMI1640 medium supplemented with 10% serum. RPMI8226 / F-B7 cells were mixed with lymphocytes of each sample at a ratio of 1:10, and cultured in RPMI1640 medium supplemented with 10% serum for 3 weeks at 37 ° C. and 5% CO ₂ for 3 weeks. Got

(6) Measurement of cytotoxic activity RPMI8226 / F-B7 cells (H
LA-F expression positive cells) and RPMI8226 (control cells) were labeled with 400 nmole of europium (Eu ³⁺ ) immediately before the measurement of cytotoxic activity. This was rinsed 3 times with PBS and then 5% with RPMI1640 medium supplemented with 10% serum.
The concentration was adjusted to × 10 ⁴ / ml, and 100 µl / well was added to each 96-well round bottom plate (Sumitomo). C obtained above
Let TL be an Effector cell, and the ratio (E / T ratio) of Effector cell to Target cell shown in the table below is 20: 1, 10: 1,
Dilute to 5: 1 with RPMI1640 medium supplemented with 10% serum, and add 200 μl / into the 96-well round bottom plate (Sumitomo).
well was added.

[0043] After the plate was cultured for 18 hours, the release activity of europium was measured using the DELFIA system (Wallac). The cytotoxic activity was according to the following formula. Cytotoxic activity (%) = [(release activity of test group−spontaneous release activity) / (100% release activity−spontaneous release activity)] × 10
0 results are shown in FIGS. From Figures 1-3, which CT
L also showed no toxicity to RPMI8226 (control cells), but RPMI8226 / F-B7
It was highly toxic to cells. Furthermore, in the range examined, the cells were toxic to cells expressing HLA-F in all cases, but cells not expressing HLA-F were not toxic.

(Example 2) (1) Preparation of dendritic cells 20 cc of heparinized peripheral blood from 3 healthy volunteers was TI
Diluted 2-fold with L-Media (manufactured by IBL) and dispensed into two centrifuge tubes filled with 15 ml each of Ficoll.
It was centrifuged at 00 rpm for 20 minutes. The separated mononuclear cell layer was collected and transferred to a new centrifuge tube. TIL-M here
After adding dia, the mixture was centrifuged at 1800 rpm for 10 minutes, the supernatant was removed, and the precipitated cells were collected and suspended in AIM-V medium (manufactured by GIBCO). After centrifuging this at 1200 rpm for 5 minutes, the supernatant was removed, the precipitated cells were collected, suspended in 20 ml of AIM-V medium in the flask, and left standing at 37 ° C. for 2 hours.

The culture solution containing non-adherent cells in the flask was removed, and the flask was washed with AIM-V medium. In a flask with adherent cells still attached, IL-4 (IL004
CHEMICON) 500U / ml and GM-CSF (GF004
CHIMICON made) 800 U / ml added AIM-V
A medium was added, and the cells were cultured in a CO ₂ incubator at 37 ° C. for 7 days, and the cells detached from the flask wall surface were collected to obtain dendritic cells. The surface antigen of the obtained dendritic cells was detected by using a flow cytometer.
83, HLA-DR positive, and confirmed to be mature dendritic cells.

(2) Preparation of Sensitive Antigen (i) Preparation of HLA-F cDNA from Cultured Cancer Cells Method devised by JM Houlihan et al. (J. Immunology, Vo
l.149, pp.668-676 (1992)), human leukemia-derived cells U937 (J. Exp. Med., Vol.143, pp 1528-1533).
(1976)), the mRNA was extracted from the cultured cancer cells and the polydT column (Oligotex-dT30, JSR Corporation) was used to
An mRNA fraction having a dA structure was obtained. With this as a mold,
CDNA was synthesized by reverse transcriptase. This cDNA
HLA-F α chain specific primer (5'-ACA
TCGCCGTGGAGTACGTAGACG-3 'and 3'-GAACACCTCTGGTCCGG
ACGTCCC-5 ') was used for amplification. The nucleotide sequence of the obtained cDNA was determined and compared with the nucleotide sequence of the HLA-F gene. As a result, HLA-F of 647 bp from exon2 to exon4
It was confirmed to be a cDNA fragment. It should be noted that this sequence is D with ac added to the 5'end of the DNA sequence of SEQ ID NO: 3.
It was an NA sequence.

(Ii) Purification of cancer cell-specific HLA-F antigen The HLA-F cDNA fragment obtained in (i) was ligated to the downstream of the histidine tag gene (His x6) of the expression vector pQE31 and cloned into a recombinant plasmid. Obtained. Escherichia coli (E. coli JM109 strain) was transformed with this recombinant plasmid to obtain IPTG (isopropyl β-D-thiogalactopy).
expression is induced by ranoside), His x6 and HLA-F
A fusion protein with the fragment was produced. The fusion protein-producing Escherichia coli was suspended in PBS, disrupted by ultrasonic waves, and the insoluble fraction was collected by centrifugation. This insoluble fraction was solubilized with urea and the fusion protein was purified by Ni-chelate affinity chromatography. SD this
When analyzed by S-PAGE, the molecular weight was 31 KD and the degree of purification was 95%. In addition, the N-terminal amino acid sequence was determined, and it was confirmed that the amino acid sequence was translated as designed.

(3) Sensitization 100 μg of HLA-F antigen sensitizing this dendritic cell /
It was added to the medium at a concentration of ml and reacted at room temperature for 3 hours.

(4) Introduction of HLA-F antigen-expressing recombinant adenovirus vector According to the Takara Shuzo manual, the titer of the HLA-F-expressing adenovirus vector prepared in the 293 cell supernatant was measured. Based on the results, MOI (multiplicities of in
(infection) = 1000 and added to the medium of dendritic cells and cultured for 48 hours. The expression of HLA-F was confirmed by the presence of mRNA by RT-PCR and the Western blotting method using anti-HLA-F monoclonal antibodies (3A5 and 3H5).

(5) Two healthy persons (D, E) having different types of CTL-induced MHC class I antigen
Lymphocytes were separated from 15 cc of peripheral blood collected from the above using a Vacutainer and suspended in RPMI1640 medium supplemented with 10% serum. The dendritic cells obtained in (3) above and the lymphocytes of each sample were mixed at a ratio of 1:10, and the mixture was added to RPMI1640 medium supplemented with 10% serum at 37 ° C. under 5% CO ₂ conditions for 3 weeks. The cells were cultured to obtain CTL.

(6) Measurement of cytotoxic activity The CTL obtained in (5) above was measured for cytotoxic activity in the same manner as in (6) of Example 1. The results are shown in FIG. 4 and FIG.
Shown in.

(Example 3) (1) Preparation of HLA-F cDNA from cultured cancer cells Method devised by JM Houlihan et al. (J. Immunology, Vo
l. 149, pp. 668-676 (1992)), human leukemia-derived cells U937 (J. Exp. Med., Vol. 143, pp 1528-15).
33 (1976)), the mRNA was extracted from the cultured cancer cells and polydT
Using a column (Oligotex-dT30, JSR Corporation), poly
An mRNA fraction having an A structure was obtained. With this as a mold,
CDNA was synthesized by reverse transcriptase. Using this cDNA as a template, an HLA-Fα chain-specific primer (5'-ACATC
GCCGTGGAGTACGTAGACG-3 'and 3'-GAACACCTCTGGTCCGGAC
It was amplified using GTCCC-5 '). The nucleotide sequence of the obtained cDNA was determined and compared with the nucleotide sequence of the HLA-F gene. As a result, 647 bp HLA-F from exon2 to exon4 was obtained.
It was confirmed to be a cDNA fragment.

(2) Purification of cancer cell-specific HLA-F antigen The HLA-F cDNA fragment obtained in (1) was ligated downstream of the histidine tag gene (His x6) of the expression vector pQE31, and cloned into a recombinant plasmid. Obtained.
E. coli JM109 strain with this recombinant plasmid
Was transformed with IPTG (Isopropyl thiogalactopyran
expression was induced to produce a fusion protein of His x6 and HLA-F fragment.

The fusion protein-producing Escherichia coli was suspended in PBS, disrupted by sonication, and the insoluble fraction was collected by centrifugation. This insoluble fraction was solubilized with urea and the fusion protein was purified by Ni chelate affinity chromatography. When analyzed by SDS PAGE, the molecular weight was 30 KD and the degree of purification was 95%.
Further, the N-terminal amino acid sequence was determined, and it was confirmed that the translation was performed as designed.

(3) Preparation of anti-HLA-F mouse monoclonal antibody 1) Immunization, cell fusion (a) Preparation of antigen solution for immunization Recombinant HLA-F antigen (prepared by the method described in Japanese Patent Laid-Open No. 2001-95584) was physiologic. A water-in-oil emulsion was prepared using a glass syringe equipped with a luer lock in which 1 mg / ml prepared with saline and an equivalent amount of an adjuvant (Titer Max Gold, manufactured by Sigma) were connected. (b) Immunization of mouse BALB / c mouse (6 weeks old, female, purchased from CLEA Japan, Inc.)
The above antigen solution (0.1 ml) was subcutaneously or intraperitoneally administered to the mice two to three times every two weeks to sufficiently sensitize the mice. Three days before cell fusion, 30 μl of the antigen solution was intraperitoneally administered as a final immunization to prepare for cell fusion. (c) Culture of myeloma cells Frozen mouse myeloma cells (P3U1) are thawed in water at 37 ° C to start culture. RPMI1640 for culture
A conditioned medium (containing pyruvic acid and glutamine) to which FCS was added so as to be 15% was used, and a cell at a time corresponding to the logarithmic growth phase of the sufficiently grown cells was prepared for fusion. (d) The cell fusion-sensitized mouse was killed by collecting blood from the heart and the spleen or lymph node was aseptically removed. Tissue is thawed using scissors and stainless mesh, suspended in RPMI1640 medium, and 1200 rp
Sensitized lymphocytes were prepared by centrifuging at m 10 min and performing a washing step of suspending the pellet in RPMI1640 again twice. Myeloma cells were prepared at a ratio of 2: 5 with the obtained sensitized lymphocytes, and washed once with RPMI1640 at 1000 rpm for 5 min. Mixed sensitized lymphocytes and myeloma cells 1200
A cell pellet was obtained by centrifugation at rpm 10 min. Cell fusion was performed according to a conventional method. 1 ml of polyethylene glycol (Boehringer Mannheim) heated to 37 ° C
Was added to the cell pellet, gently stirred, and cell fusion was performed for 1 minute. Then 1 ml of RPMI1640 heated to 37 ℃
Was added little by little over 1 minute, the same operation was repeated twice, and then 10 ml was added over 3 minutes for dilution.
After the cells were placed at 37 ° C. for 5 minutes, the cells were collected by centrifugation at 1000 rpm for 5 minutes. The fused cells were seeded on five 96-well plates and cultured under the conditions of 37 ° C., 5% CO ₂ and saturated steam. RPMI 1640 with 15% FCS added to the medium
HAT reagent and 5% Blai clone (manufactured by Dainippon Pharmaceutical Co., Ltd.) were used. Hybridoma colonies were confirmed from 5 to 7 days after cell fusion, and on the 11th day, the cells grew to a state where antibody assay was possible.

2) Antibody assay (a) Preparation of antigen plate for ELISA To detect monoclonal antibody-producing cells, an ELISA method in which an antigen was immobilized was used. 50 μl of each recombinant HLA-F antigen solution adjusted to 5 μg / ml with PBS was added to the ELISA plate and reacted at room temperature for 2 hours for immobilization. Then discard the antigen solution and use as blocking
100 μl of Block Ace (manufactured by Dainippon Pharmaceutical Co., Ltd.) diluted 4-fold with PBS was added, and the mixture was reacted at room temperature for 1 hour (or 4 ° C. overnight) or more. (b) Assay Discard the blocking solution from the plate, add 50 μl of supernatant from the cell fusion culture plate to each well, and incubate at 37 ℃
Reacted for hours. A 100-fold diluted antiserum obtained at the time of blood collection was used as a positive control, and a culture solution was used as a negative control. After the reaction, the culture supernatant was discarded and washed once with physiological saline. Second antibody diluted 1000 times (alkaline phosphatase labeled anti-mouse IgG)
50 μl was added and the reaction was performed for 1 hour in the same manner. After washing 3 times with physiological saline, 50 μl of ALP rose (manufactured by Shinotest) substrate solution was added and reacted at room temperature for 20 minutes, and then color solution 5
Color was developed by adding 0 μl, and the absorbance at 510 nm was measured. (c) After confirming that the value of the determination negative control was 0.05 or less, the well showing the same reaction as the positive control was selected as the well containing the antibody positive clone.

(4) Protocol for Epitope Analysis Analysis of Epitopes Recognized by 3A5 Monoclonal Antibody and 3H5 Monoclonal Antibody In order to cover the entire region of the purified antigen used for immunization of mice, a peptide of 10 amino acids was added every 3 amino acids. A total of 77 ways were synthesized by shifting. Table 1 shows a list of sequences of the synthesized peptides. (Synthesis of the peptide was requested to MIMOTOPES by "PepSet Kit" handled by Kurabo Industries.) The 3A5 monoclonal antibody and the peptide to which the 3H5 monoclonal antibody binds were confirmed by the ELISA method. 27) RNLLRR
The 3H5 monoclonal antibody bound to the YNQS peptide (Table 1 # 19) and to the EGPQYWEWTT peptide. From this result, the epitope recognized by each monoclonal antibody was determined. GenomeNe for each epitope
A SWISS PLOT search of t revealed that it was not present in other reported human proteins.

[0058]

Example 4 Primary autologous mixed lymphocytes using HLA-F antigen-expressing dendritic cells obtained by introducing a recombinant adenovirus vector expressing HLA-F antigen into dendritic cells as stimulator cells. Culture method,
By repeatedly stimulating the proliferating cell fraction with HLA-F antigen-expressing dendritic cells, HLA-F antigen-specific CD8
Positive cytotoxic T cells were established and their cytotoxic activity was examined by the following method. Measurement of cytotoxic activity 1) Target cells: C1R cell line (HLA-F antigen positive)
And RPMI8226 cell line (HLA-F antigen negative: negative control cells) Effector cells: HLA-F antigen-specific CD8-positive cytotoxic T cells and antigen non-specific CD8-positive T cells (control cells) Effector / Target ratio = 10 1/1 and 20/1, ⁵¹ Cr release asa
It was measured by the ay method. That is, the Target Cells ⁵¹
After labeling with Cr, it was washed 3 times with RPMI1640 medium (Gibco), and 5x10 with RPMI1640 medium supplemented with 10% human AB serum.
Adjust to ⁴ / ml and add 100 to 96-hole round bottom plate (Sumitomo)
μl / well was added (cells per well, 5x10 ³ ce
lls). Further, the above Effector cells were diluted with RPMI1640 medium supplemented with 10% human AB serum so as to have the above-mentioned ratio, and 100 μl / well was added thereto. After culturing this plate for 6 hours, the supernatant was collected and
The release of ⁵¹ Cr was measured. The results are shown in FIGS. 6 and 7. 2) The ratio of cytotoxic activity was in accordance with the above formula.

Results of cytotoxic activity From the results of FIG. 6, C1R cell line (HLA-F antigen positive)
Alternatively, the ratio (%) of the cytotoxic activity of HLA-F antigen-specific CD8-positive cytotoxic T cells when the RPMI8226 cell line (HLA-F antigen negative: negative control cells) was used as Target cells was shown, and HLA- It can be seen that F antigen-specific CD8-positive cytotoxic T cells injure the HLA-F antigen-positive C1R cell line but not the HLA-F antigen-negative RPMI8226 cell line. From the results of FIG. 7, cells of HLA-F antigen-specific CD8-positive cytotoxic T cells and antigen non-specific CD8-positive T cells (control) when C1R cell line (HLA-F antigen-positive) was used as Target cells The percentage (%) of the damaging activity is shown, and HLA-F antigen-specific C
It can be seen that only D8 positive cytotoxic T cells injure the C1R cell line.

(Example 5) <Inhibition test of cytotoxic activity> HLA-used in Example 4
Inhibition using anti-HLA-F monoclonal antibody (3H5) to confirm whether CD8-positive T cells established by repeated stimulation with F antigen-expressing dendritic cells are specific to HLA-F antigen After doing the test,
By treating the established CD8-positive T cells with the anti-HLA-F monoclonal antibody, inhibition of the cytotoxic activity against C1R was observed as compared with that before the treatment.
The anti-HLA-F monoclonal antibody of the present invention is HLA-
It was confirmed that the antibody neutralizes the damage to F antigen-positive cells.

Example 6 <Antibody Dependent Cytotoxicity (ADCC) Test> Using C1R expressing HLA-F on the cell surface as a target cell, ⁵¹ C
After r-labeling, the plate was washed 3 times with RPMI1640 medium (Gibco), adjusted to 5x10 ⁴ / ml with RPMI1640 medium containing 10% FBS, and 100 μl / well on a 96-well round bottom plate (Sumitomo).
Each (cell number per well, 5 × 10 ³ cells).
To this, 3H5, 3A5 monoclonal antibody or non-specific mouse IgG was added at a concentration of 1 μg / ml, and monocytes obtained from peripheral blood of healthy subjects were added at an E / T ratio of 5, 10, 20. After culturing for 6 hours, the supernatant was collected and the release of ⁵¹ Cr was measured by a γ-counter. The results are shown in Figure 8.
Shown in. From the results of FIG. 8, when antibodies 3H5 and 3A5 that specifically recognize HLA-F bind to HLA-F of C1R cells, monocytes having an Fc receptor that recognizes the Fc portion binds to the Fc portion, Since the HLA-F specific antibodies 3H5 and 3A5 show the activity of damaging C1R cells, the damaging activity to the C1R cells was measured, and when the non-specific mouse IgG antibody was used, the damage to the C1R cells was measured. It can be seen that no activity is observed.

[0063]

INDUSTRIAL APPLICABILITY The present invention is a cancer cell-specific HLA-F capable of inducing cytotoxic T cells (Cytotoxic T Lymphocytes, hereinafter referred to as "CTL") to human cancer cells in vivo or in vitro. Provided is a human cancer preventive / therapeutic agent using an antigen. It also provides an anti-HLA-F monoclonal antibody specific for the HLA-F antigen. The DNA vaccine containing the DNA encoding the cancer cell-specific HLA-F antigen protein provided by the present invention, the cell vaccine expressing the HLA-F antigen, and the cell damage induced by the cells expressing the HLA-F antigen (damage) ) Sex T cells are not organ-specific and are effective in the prevention and treatment of cancer. Anti-HLA-
The F monoclonal antibody is useful for the measurement of HLA-F antigen, the diagnosis of cancer using the same, and the targeted therapeutic agent.

[0064]

[Sequence list]                             SEQUENCE LISTING <110> K. EGAWA et. Al. <120> Cancer prophylactic / treatment agent <130> MEDI007 <160> 6 <210> SEQ ID No: 1 <211> 1089 <212> DNA <213> human <400> 1 atggcgcccc gaagcctcct cctgctgctc tcaggggccc tggccctgac cgatacttgg 60 gcgggctccc actccttgag gtatttcagc accgctgtgt cgcggcccgg ccgcggggag 120 ccccgctaca tcgccgtgga gtacgtagac gacacgcaat tcctgcggtt cgacagcgac 180 gccgcgattc cgaggatgga gccgcgggag ccgtgggtgg agcaagaggg gccgcagtat 240 tgggagtgga ccacagggta cgccaaggcc aacgcacaga ctgaccgagt ggccctgagg 300 aacctgctcc gccgctacaa ccagagcgag gctgggtctc acaccctcca gggaatgaat 360 ggctgcgaca tggggcccga cggacgcctc ctccgcgggt atcaccagca cgcgtacgac 420 ggcaaggatt acatctccct gaacgaggac ctgcgctcct ggaccgcggc ggacaccgtg 480 gctcagatca cccagcgctt ctatgaggca gaggaatatg cagaggagtt caggacctac 540 ctggagggcg agtgcctgga gttgctccgc agatacttgg agaatgggaa ggagacgcta 600 cagcgcgcag atcctccaaa ggcacacgtt gcccaccacc ccatctctga ccatgaggcc 660 accctgaggt gctgggccct gggcttctac cctgcggaga tcacgctgac ctggcagcgg 720 gatggggagg aacagaccca ggacacagag cttgtggaga ccaggcctgc aggggatgga 780 accttccaga agtgggccgc tgtggtggtg ccttctggag aggaacagag atacacatgc 840 catgtgcagc acgaggggct gccccagccc ctcatcctga gatgggagca gtctccccag 900 cccaccatcc ccatcgtggg catcgttgct ggccttgttg tccttggagc tgtggtcact 960 ggagctgtgg tcgctgctgt gatgtggagg aagaagagct cagatagaaa cagagggagc 1020 tactctcagg ctgcagtcac tgacagtgcc cagggctctg gggtgtctct cacagctaat 1080 aaagtgtga 1089

[0065] <210> SEQ ID No: 2 <211> 822 <212> DNA <213> human <400> 2 ggctcccact ccttgaggta tttcagcacc gctgtgtcgc ggcccggccg cggggagccc 60 cgctacatcg ccgtggagta cgtagacgac acgcaattcc tgcggttcga cagcgacgcc 120 gcgattccga ggatggagcc gcgggagccg tgggtggagc aagaggggcc gcagtattgg 180 gagtggacca cagggtacgc caaggccaac gcacagactg accgagtggc cctgaggaac 240 ctgctccgcc gctacaacca gagcgaggct gggtctcaca ccctccaggg aatgaatggc 300 tgcgacatgg ggcccgacgg acgcctcctc cgcgggtatc accagcacgc gtacgacggc 360 aaggattaca tctccctgaa cgaggacctg cgctcctgga ccgcggcgga caccgtggct 420 cagatcaccc agcgcttcta tgaggcagag gaatatgcag aggagttcag gacctacctg 480 gagggcgagt gcctggagtt gctccgcaga tacttggaga atgggaagga gacgctacag 540 cgcgcagatc ctccaaaggc acacgttgcc caccacccca tctctgacca tgaggccacc 600 ctgaggtgct gggccctggg cttctaccct gcggagatca cgctgacctg gcagcgggat 660 ggggaggaac agacccagga cacagagctt gtggagacca ggcctgcagg ggatggaacc 720 ttccagaagt gggccgctgt ggtggtgcct tctggagagg aacagagata cacatgccat 780 gtgcagcacg aggggctgcc ccagcccctc atcctgagat gg 822

[0066] <210> SEQ ID No: 3 <211> 645 <212> DNA <213> human <400> 3 atcgccgtgg agtacgtaga cgacacgcaa ttcctgcggt tcgacagcga cgccgcgatt 60 ccgaggatgg agccgcggga gccgtgggtg gagcaagagg ggccgcagta ttgggagtgg 120 accacagggt acgccaaggc caacgcacag actgaccgag tggccctgag gaacctgctc 180 cgccgctaca accagagcga ggctgggtct cacaccctcc agggaatgaa tggctgcgac 240 atggggcccg acggacgcct cctccgcggg tatcaccagc acgcgtacga cggcaaggat 300 tacatctccc tgaacgagga cctgcgctcc tggaccgcgg cggacaccgt ggctcagatc 360 acccagcgct tctatgaggc agaggaatat gcagaggagt tcaggaccta cctggagggc 420 gagtgcctgg agttgctccg cagatacttg gagaatggga aggagacgct acagcgcgca 480 gatcctccaa aggcacacgt tgcccaccac cccatctctg accatgaggc caccctgagg 540 tgctgggccc tgggcttcta ccctgcggag atcacgctga cctggcagcg ggatggggag 600 gaacagaccc aggacacaga gcttgtggag accaggcctg caggg 645

[0067] <210> SEQ ID No: 4 <211> 362 <212> PRT <213> human <400> 4 Met Ala Pro Arg Ser Leu Leu Leu Leu Leu Ser Gly Ala Leu Ala Leu  1 5 10 15 Thr Asp Thr Trp Ala Gly Ser His Ser Leu Arg Tyr Phe Ser Thr Ala              20 25 30 Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Tyr Ile Ala Val Glu Tyr          35 40 45 Val Asp Asp Thr Gln Phe Leu Arg Phe Asp Ser Asp Ala Ala Ile Pro      50 55 60 Arg Met Glu Pro Arg Glu Pro Trp Val Glu Gln Glu Gly Pro Gln Tyr  65 70 75 80 Trp Glu Trp Thr Thr Gly Tyr Ala Lys Ala Asn Ala Gln Thr Asp Arg                  85 90 95 Val Ala Leu Arg Asn Leu Leu Arg Arg Tyr Asn Gln Ser Glu Ala Gly             100 105 110 Ser His Thr Leu Gln Gly Met Asn Gly Cys Asp Met Gly Pro Asp Gly         115 120 125 Arg Leu Leu Arg Gly Tyr His Gln His Ala Tyr Asp Gly Lys Asp Tyr     130 135 140 Ile Ser Leu Asn Glu Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Val 145 150 155 160 Ala Gln Ile Thr Gln Arg Phe Tyr Glu Ala Glu Glu Tyr Ala Glu Glu                 165 170 175 Phe Arg Thr Tyr Leu Glu Gly Glu Cys Leu Glu Leu Leu Arg Arg Tyr             180 185 190 Leu Glu Asn Gly Lys Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Ala         195 200 205 His Val Ala His His Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys     210 215 220 Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg 225 230 235 240 Asp Gly Glu Glu Gln Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro                 245 250 255 Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser             260 265 270 Gly Glu Glu Gln Arg Tyr Thr Cys His Val Gln His Glu Gly Leu Pro         275 280 285 Gln Pro Leu Ile Leu Arg Trp Glu Gln Ser Pro Gln Pro Thr Ile Pro     290 295 300 Ile Val Gly Ile Val Ala Gly Leu Val Val Leu Gly Ala Val Val Thr 305 310 315 320 Gly Ala Val Val Ala Ala Val Met Trp Arg Lys Lys Ser Ser Asp Arg                 325 330 335 Asn Arg Gly Ser Tyr Ser Gln Ala Ala Val Thr Asp Ser Ala Gln Gly             340 345 350 Ser Gly Val Ser Leu Thr Ala Asn Lys Val         355 360

[0068] <210> SEQ ID No: 5 <211> 274 <212> PRT <213> human <400> 5 Gly Ser His Ser Leu Arg Tyr Phe Ser Thr Ala Val Ser Arg Pro Gly  1 5 10 15 Arg Gly Glu Pro Arg Tyr Ile Ala Val Glu Tyr Val Asp Asp Thr Gln              20 25 30 Phe Leu Arg Phe Asp Ser Asp Ala Ala Ile Pro Arg Met Glu Pro Arg          35 40 45 Glu Pro Trp Val Glu Gln Glu Gly Pro Gln Tyr Trp Glu Trp Thr Thr      50 55 60 Gly Tyr Ala Lys Ala Asn Ala Gln Thr Asp Arg Val Ala Leu Arg Asn  65 70 75 80 Leu Leu Arg Arg Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln                  85 90 95 Gly Met Asn Gly Cys Asp Met Gly Pro Asp Gly Arg Leu Leu Arg Gly             100 105 110 Tyr His Gln His Ala Tyr Asp Gly Lys Asp Tyr Ile Ser Leu Asn Glu         115 120 125 Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Val Ala Gln Ile Thr Gln     130 135 140 Arg Phe Tyr Glu Ala Glu Glu Tyr Ala Glu Glu Phe Arg Thr Tyr Leu 145 150 155 160 Glu Gly Glu Cys Leu Glu Leu Leu Arg Arg Tyr Leu Glu Asn Gly Lys                 165 170 175 Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Ala His Val Ala His His             180 185 190 Pro Ile Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe         195 200 205 Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Glu Gln     210 215 220 Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr 225 230 235 240 Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg                 245 250 255 Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Gln Pro Leu Ile Leu             260 265 270 Arg Trp

[0069] <210> SEQ ID No: 6 <211> 215 <212> PRT <213> human <400> 6 Ile Ala Val Glu Tyr Val Asp Asp Thr Gln Phe Leu Arg Phe Asp Ser  1 5 10 15 Asp Ala Ala Ile Pro Arg Met Glu Pro Arg Glu Pro Trp Val Glu Gln              20 25 30 Glu Gly Pro Gln Tyr Trp Glu Trp Thr Thr Gly Tyr Ala Lys Ala Asn          35 40 45 Ala Gln Thr Asp Arg Val Ala Leu Arg Asn Leu Leu Arg Arg Tyr Asn      50 55 60 Gln Ser Glu Ala Gly Ser His Thr Leu Gln Gly Met Asn Gly Cys Asp  65 70 75 80 Met Gly Pro Asp Gly Arg Leu Leu Arg Gly Tyr His Gln His Ala Tyr                  85 90 95 Asp Gly Lys Asp Tyr Ile Ser Leu Asn Glu Asp Leu Arg Ser Trp Thr             100 105 110 Ala Ala Asp Thr Val Ala Gln Ile Thr Gln Arg Phe Tyr Glu Ala Glu         115 120 125 Glu Tyr Ala Glu Glu Phe Arg Thr Tyr Leu Glu Gly Glu Cys Leu Glu     130 135 140 Leu Leu Arg Arg Tyr Leu Glu Asn Gly Lys Glu Thr Leu Gln Arg Ala 145 150 155 160 Asp Pro Pro Lys Ala His Val Ala His His Pro Ile Ser Asp His Glu                 165 170 175 Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Thr             180 185 190 Leu Thr Trp Gln Arg Asp Gly Glu Glu Gln Thr Gln Asp Thr Glu Leu         195 200 205 Val Glu Thr Arg Pro Ala Gly     210 215

[Brief description of drawings]

FIG. 1 is a graph showing the cytotoxic activity of CTL derived from peripheral blood of healthy person A.

FIG. 2 is a graph showing the cytotoxic activity of CTL derived from the peripheral blood of healthy person B.

FIG. 3 is a graph showing the cytotoxic activity of CTL derived from peripheral blood of healthy person C.

FIG. 4 is a graph showing the cytotoxic activity of CTL derived from peripheral blood of healthy person D.

FIG. 5 is a graph showing the cytotoxic activity of CTL derived from peripheral blood of healthy human E.

FIG. 6 C1R cell line (HLA-F antigen positive) or
It is a graph which shows the ratio (%) of the cytotoxic activity of the HLA-F antigen specific CD8 positive cytotoxic T cell when RPMI8226 cell line (HLA-F antigen negative: negative control cell) is made into Target cell.

FIG. 7: C1R cell line (HLA-F antigen positive) was Targ
It is a graph which shows the ratio (%) of the cytotoxic activity of HLA-F antigen-specific CD8-positive cytotoxic T cells and antigen non-specific CD8-positive T cells (control) when the cells are et cells.

FIG. 8 is a graph showing the results of an antibody-dependent cellular cytotoxicity (ADCC) test.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] April 5, 2002 (2002.4.5)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0064

[Correction method] Change

[Correction content]

[0064]

[Sequence Listing] SEQUENCE LISTING <110> K. EGAWA et. Al. <120> Cancer prophylactic / treatment agent <130> MEDI011 <160> 6 <210> SEQ ID No: 1 <211> 1089 <212> DNA <213 > human <400> 1 atggcgcccc gaagcctcct cctgctgctc tcaggggccc tggccctgac cgatacttgg 60 gcgggctccc actccttgag gtatttcagc accgctgtgt cgcggcccgg ccgcggggag 120 ccccgctaca tcgccgtgga gtacgtagac gacacgcaat tcctgcggtt cgacagcgac 180 gccgcgattc cgaggatgga gccgcgggag ccgtgggtgg agcaagaggg gccgcagtat 240 tgggagtgga ccacagggta cgccaaggcc aacgcacaga ctgaccgagt ggccctgagg 300 aacctgctcc gccgctacaa ccagagcgag gctgggtctc acaccctcca gggaatgaat 360 ggctgcgaca tggggcccga cggacgcctc ctccgcgggt atcaccagca cgcgtacgac 420 ggcaaggatt acatctccct gaacgaggac ctgcgctcct ggaccgcggc ggacaccgtg 480 gctcagatca cccagcgctt ctatgaggca gaggaatatg cagaggagtt caggacctac 540 ctggagggcg agtgcctgga gttgctccgc agatacttgg agaatgggaa ggagacgcta 600 cagcgcgcag atcctccaaa ggcacacgtt gcccaccacc ccatctctga ccatgaggcc 660 accctgaggt gctgggccct gggcttctac cctgcggaga tcacgctg ac ctggcagcgg 720 gatggggagg aacagaccca ggacacagag cttgtggaga ccaggcctgc aggggatgga 780 accttccaga agtgggccgc tgtggtggtg ccttctggag aggaacagag atacacatgc 840 catgtgcagc acgaggggct gccccagccc ctcatcctga gatgggagca gtctccccag 900 cccaccatcc ccatcgtggg catcgttgct ggccttgttg tccttggagc tgtggtcact 960 ggagctgtgg tcgctgctgt gatgtggagg aagaagagct cagatagaaa cagagggagc 1020 tactctcagg ctgcagtcac tgacagtgcc cagggctctg gggtgtctct cacagctaat 1080 aaagtgtga 1089

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 35/02 A61P 35/02 4C087 43/00 105 43/00 105 4H045 C07K 16/32 C07K 16/32 C12N 5/10 G01N 33/574 A 15/02 D 15/09 ZNA C12P 21/08 G01N 33/574 G01N 33/577 B C12N 15/00 ZNAA // C12P 21/08 C G01N 33/577 5/00 B ( 72) Inventor Koji Egawa 3-1-11 F-term, Setagaya-ku, Tokyo 4B024 AA01 AA12 BA36 BA44 CA04 DA03 EA02 EA04 GA11 GA13 HA15 4B064 AG27 CA20 CC24 DA01 DA14 4B065 AA26X AA93X AA93Y AB01 BA02 BA12 BA12 BA12 BA12 BA12 BA12 BA05 CA44 CA46 4C084 AA13 NA14 ZB212 ZB261 ZB271 4C085 AA14 BB01 CC02 CC12 DD23 DD62 EE01 4C087 AA01 AA02 BB64 BC83 CA12 NA14 ZB21 ZB26 ZB27 4H045 AA 10 AA11 AA20 AA30 BA10 CA41 DA76 DA86 EA20 EA51 FA74

Claims (15)

[Claims] 1. A preventive / therapeutic agent for cancer, which comprises, as an active ingredient, part or all of the DNA sequence set forth in SEQ ID NO: 1 of the Sequence Listing. 2. A preventive / therapeutic agent for cancer, which comprises, as an active ingredient, a plasmid having a part or all of the DNA sequence set forth in SEQ ID NO: 1 in the Sequence Listing. 3. A preventive / therapeutic agent for cancer, which comprises, as an active ingredient, a viral vector having a part or all of the DNA sequence set forth in SEQ ID NO: 1 in the Sequence Listing. 4. The DNA according to claim 1,
A cell presenting a cancer antigen transformed with a plasmid or a viral vector. 5. A cell that presents the cancer antigen according to claim 4,
Alternatively, a cytotoxic (damaged) T cell (hereinafter referred to as CTL) induced by using a cell presenting a cancer antigen pulsed with a polypeptide containing at least a part of the amino acid sequence of SEQ ID NO: 4 in the sequence listing. 6. A CTL inducer capable of inducing cancer cell-specific CTL, which is organ-specific and MHC-free. 7. An anti-HLA-F antibody which recognizes any of the sequences of SEQ ID NOs: 4, 5 and 6 in the sequence listing. 8. The anti-HLA-F antibody according to claim 7, wherein the antibody is a monoclonal antibody. 9. Arg-Asn-Leu-Leu-Arg-Arg-Tyr-Asn-Gln-Se
A 3A5 antibody that recognizes the amino acid sequence of r. 10. Glu-Gly-Pro-Gln-Tyr-Trp-Glu-Trp-Thr-
A 3H5 antibody that recognizes the amino acid sequence of Thr. 11. A hybridoma or cell line which produces the antibody according to any one of claims 7 to 10. 12. The hybridoma has an accession number FER.
The hybridoma according to claim 11, which is MP-18795 or FERMP-18796. 13. A composition for treating or preventing cancer, tumor and / or leukemia, which comprises an anti-HLA-F antibody. 14. A labeled anti-HLA-F antibody is included.
A detecting agent for detecting a mutant cell group of cancer, a lesion site of a tumor, and / or leukemia. 15. A method for measuring HLA-F antigen, which comprises measuring HLA-F antigen using an anti-HLA-F antibody.