JP5939855B2 - Transferrin receptor antibody - Google Patents

Description

  The present invention relates to an antibody that specifically recognizes a transferrin receptor and use thereof.

  The success of Herceptin (Non-patent Document 1) for breast cancer and Rituxan (Non-patent document 2) for malignant B lymphoma has shown that antibodies are effective as therapeutic agents for cancer. Certain antibodies exert an ADCC action (Non-patent Document 3) and / or a CDC action (Non-Patent Document 4) by forming a complex with an antigen molecule present on the cell membrane, and by this action, target cells ( Cells that express the antigen). ADCC action and CDC action may trigger apoptosis. The action of such antibodies is antigen specific. In other words, as long as the antigen that it recognizes is expressed, the antibody acts without distinction between cancer cells and normal cells. Therefore, the success or failure of the development of antibody therapeutics against cancer depends on how to find an antigen that is specifically expressed in cancer and that causes the ADCC action or CDC action when the antibody is recognized. Antibodies against such antigens are promising candidates for therapeutic agents that can reliably kill target cancer cells while minimizing the effects (side effects) on normal cells.

  By the way, in antibody drug discovery, it is indispensable to obtain an antibody that recognizes an “intact” target cancer antigen existing on the cell membrane surface. However, since the target cancer antigen is a membrane protein, It was difficult to obtain even antibodies against cancer antigens. In order to solve such problems, the present inventors have created a huge human antibody library consisting of 100 billion independent clones so far, and present it on the cell membrane surface of cancer cells and tissues. We established a comprehensive method for obtaining antibodies against proteins (cell surface antigens). (Patent Documents 1 to 3).

International Publication No. 01/062907 Pamphlet International Publication No. 2001/096401 Pamphlet JP 2005-185281 A

Mass R, et al .: The Concordance Between the Clinical Trials Assay (CTA) and Fluorescence in Situ Hybridization (FISH) in the Herceptin Pivotal Trials .: Proc Am Soci Clin Oncol 19, 75a, 2000 Berinstein NL, Grillo-Lopez AJ, White CA, Bence-Bruckler I, Maloney D, Czuczman M, et al. Association of serum Rituximab (IDEC-C2B8) concentration and anti-tumor response in the treatment of recurrent low-grade or follicular non -Hodgkin's lymphoma. Annals of Oncology 1998, 9: 995-1001. Bruggemann M., Williams GT, Bindon CI, Clark MR, Walker MR, Jefferis R., Waldmann H., Neuberger MS (1987). Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies.J. Exp. Med., 166, 1351-1361. Loos M. (1982) .The classical complement pathway: mechanism of activation of the first component by antigen-antibody complexes.Prog.Allergy, 30, 135-192. Mol Immunol. 1982 May; 19 (5): 651-7.

  Adult T cell leukemia (ATL) is an intractable blood cancer caused mainly by the virus HTLV1 (human T cell leukemia virus Type 1) that is transmitted via breast milk. There are more than 1 million domestically infected people, the largest in developed countries, and the incubation period is as long as 55 years on average. The lifetime incidence of infected persons is about 5%, and the annual number of patients is about 1000. Traditionally, it was a common disease in the Kyushu area, but due to the mobility of people, the number of infected people is increasing in Tokyo and Osaka. With existing multi-drug chemotherapy, 90% died within a year, and bone marrow transplantation has a high age of onset, so the cure rate is low and a new treatment is urgently needed. In addition, recent reports indicate that HTLV-1 carriers are increasing in the Kanto Kansai region, and that the number of ATL patients and deaths is increasing. The government has also created an ATL mission team and is taking measures. On the other hand, the only therapeutic drugs for ATL currently on the market are anticancer drugs (cyclophosphamide, adriamycin, vincristine, etc.) that are commonly used in combination with multiple drugs, and the 5-year survival rate is 10% or less. It has strong side effects such as bone marrow suppression, infection, and bleeding, and is denied as a useful product for treatment. When it develops, ATL cells proliferate in the blood, resulting in decreased immune function and lymph node tumors. This is the most difficult incurable disease among leukemias. There is no standard treatment, the average survival time after onset is as short as one year, and about 1,000 people die every year. Infected people continue to be intimidated by the onset, and the development of treatments and new drugs is an urgent issue. It is. An object of the present invention is to provide an antibody capable of specifically recognizing a transferrin receptor, which is a protein expressed in ATL cells.

  In order to solve the above-mentioned problems, the inventors proceeded with antibody analysis by the following approach. First, a cell line expected to express a cell surface antigen for the obtained antibody was prepared, and the antibody was isolated against the antigen on the cell membrane. As a result, they succeeded in finding a novel antibody that can specifically recognize the transferrin receptor.

That is, according to the present invention, the heavy chain first complementarity determining region (VH CDR1), the heavy chain second complementarity determining region (VH CDR2), and the heavy chain third complementarity determining region (VH CDR3), respectively, 1-3, 7-9, 13-15, 19-21, 25-27, 31-33, 37-39, 43-45, 49-51, 55-57, 61-63, 67-69, 73- A heavy chain variable region having the amino acid sequence of any of 75, 79-81, or 85-87, or an amino acid sequence substantially identical thereto;
SEQ ID NOs: 4-6, 10-12 as a light chain first complementarity determining region (VL CDR1), a light chain second complementarity determining region (VL CDR2), and a light chain third complementarity determining region (VL CDR3), respectively 16-18, 22-24, 28-30, 34-36, 40-42, 46-48, 52-54, 58-60, 64-66, 70-72, 76-78, 82-84, or 88 A light chain variable region having any amino acid sequence of -90 or an amino acid sequence substantially identical thereto;
Isolated antibodies having specific binding to TfR specifically expressed on cancer cell membranes are provided.

Preferably, the antibody of the present invention is an isolated antibody having a specific binding property to TfR specifically expressed on a cancer cell membrane, selected from the following (1) to (15).
(1) Heavy chain first complementarity determining region (VH CDR1) of SEQ ID NO: 1, heavy chain second complementarity determining region (VH CDR2) of SEQ ID NO: 2, heavy chain third complementarity determining region of SEQ ID NO: 3 ( VH CDR3) or a heavy chain variable region having substantially the same CDR, a light chain first complementarity determining region (VL CDR1) of SEQ ID NO: 4, and a light chain second complement of SEQ ID NO: 5 An antibody having a determining region (VL CDR2), a CDR comprising the light chain third complementarity determining region (VL CDR3) of SEQ ID NO: 6, or a light chain variable region having a CDR substantially the same as the CDR;
(2) heavy chain first complementarity determining region (VH CDR1) of SEQ ID NO: 7, heavy chain second complementarity determining region (VH CDR2) of SEQ ID NO: 8, heavy chain third complementarity determining region of SEQ ID NO: 9 ( VH CDR3) or a heavy chain variable region having substantially the same CDR, a light chain first complementarity determining region (VL CDR1) of SEQ ID NO: 10, and a light chain second complement of SEQ ID NO: 11 An antibody having a determining region (VL CDR2), a CDR comprising the light chain third complementarity determining region (VL CDR3) of SEQ ID NO: 12, or a light chain variable region having a CDR substantially the same as the CDR;
(3) heavy chain first complementarity determining region (VH CDR1) of SEQ ID NO: 13, heavy chain second complementarity determining region (VH CDR2) of SEQ ID NO: 14, heavy chain third complementarity determining region of SEQ ID NO: 15 ( VH CDR3) or a heavy chain variable region having substantially the same CDR, a light chain first complementarity determining region (VL CDR1) of SEQ ID NO: 16, and a light chain second complement of SEQ ID NO: 17 An antibody having a determining region (VL CDR2), a CDR comprising the light chain third complementarity determining region (VL CDR3) of SEQ ID NO: 18, or a light chain variable region having a CDR substantially the same as the CDR;
(4) SEQ ID NO: 19 heavy chain first complementarity determining region (VH CDR1), SEQ ID NO: 20 heavy chain second complementarity determining region (VH CDR2), SEQ ID NO: 21 heavy chain third complementarity determining region ( VH CDR3) or a heavy chain variable region having substantially the same CDR, a light chain first complementarity determining region (VL CDR1) of SEQ ID NO: 22, and a light chain second complement of SEQ ID NO: 23 An antibody having a determining region (VL CDR2), a CDR comprising the light chain third complementarity determining region (VL CDR3) of SEQ ID NO: 24, or a light chain variable region having a CDR substantially the same as the CDR;
(5) heavy chain first complementarity determining region (VH CDR1) of SEQ ID NO: 25, heavy chain second complementarity determining region (VH CDR2) of SEQ ID NO: 26, heavy chain third complementarity determining region of SEQ ID NO: 27 ( VH CDR3) or a heavy chain variable region having substantially the same CDR, a light chain first complementarity determining region (VL CDR1) of SEQ ID NO: 28, and a light chain second complement of SEQ ID NO: 29 An antibody having a determining region (VL CDR2), a CDR comprising the light chain third complementarity determining region (VL CDR3) of SEQ ID NO: 30, or a light chain variable region having a CDR substantially the same as the CDR;
(6) SEQ ID NO: 31 heavy chain first complementarity determining region (VH CDR1), SEQ ID NO: 32 heavy chain second complementarity determining region (VH CDR2), SEQ ID NO: 33 heavy chain third complementarity determining region ( VH CDR3) or a heavy chain variable region having substantially the same CDR, a light chain first complementarity determining region of SEQ ID NO: 34 (VL CDR1), and a light chain second complement of SEQ ID NO: 35 An antibody having a determining region (VL CDR2), a CDR comprising the light chain third complementarity determining region (VL CDR3) of SEQ ID NO: 36, or a light chain variable region having a CDR substantially the same as the CDR;
(7) heavy chain first complementarity determining region (VH CDR1) of SEQ ID NO: 37, heavy chain second complementarity determining region (VH CDR2) of SEQ ID NO: 38, heavy chain third complementarity determining region of SEQ ID NO: 39 ( VH CDR3) or a heavy chain variable region having substantially the same CDR, a light chain first complementarity determining region of SEQ ID NO: 40 (VL CDR1), and a light chain second complement of SEQ ID NO: 41 An antibody having a determining region (VL CDR2), a CDR comprising the light chain third complementarity determining region (VL CDR3) of SEQ ID NO: 42, or a light chain variable region having a CDR substantially the same as the CDR;
(8) SEQ ID NO: 43 heavy chain first complementarity determining region (VH CDR1), SEQ ID NO: 44 heavy chain second complementarity determining region (VH CDR2), SEQ ID NO: 45 heavy chain third complementarity determining region ( VH CDR3) or a heavy chain variable region having substantially the same CDR, a light chain first complementarity determining region of SEQ ID NO: 46 (VL CDR1), and a light chain second complement of SEQ ID NO: 47 An antibody having a determining region (VL CDR2), a CDR comprising the light chain third complementarity determining region (VL CDR3) of SEQ ID NO: 48, or a light chain variable region having a CDR substantially the same as the CDR;
(9) Heavy chain first complementarity determining region (VH CDR1) of SEQ ID NO: 49, heavy chain second complementarity determining region (VH CDR2) of SEQ ID NO: 50, heavy chain third complementarity determining region of SEQ ID NO: 51 ( VH CDR3) or a heavy chain variable region having substantially the same CDR, a light chain first complementarity determining region of SEQ ID NO: 52 (VL CDR1), and a light chain second complement of SEQ ID NO: 53 An antibody having a determining region (VL CDR2), a CDR comprising the light chain third complementarity determining region (VL CDR3) of SEQ ID NO: 54, or a light chain variable region having a CDR substantially the same as the CDR;
(10) heavy chain first complementarity determining region (VH CDR1) of SEQ ID NO: 55, heavy chain second complementarity determining region (VH CDR2) of SEQ ID NO: 56, heavy chain third complementarity determining region of SEQ ID NO: 57 ( VH CDR3) or a heavy chain variable region having substantially the same CDR, a light chain first complementarity determining region (VL CDR1) of SEQ ID NO: 58, and a light chain second complement of SEQ ID NO: 59 An antibody having a determining region (VL CDR2), a CDR comprising the light chain third complementarity determining region (VL CDR3) of SEQ ID NO: 60, or a light chain variable region having a CDR substantially the same as the CDR;
(11) Heavy chain first complementarity determining region (VH CDR1) of SEQ ID NO: 61, heavy chain second complementarity determining region (VH CDR2) of SEQ ID NO: 62, heavy chain third complementarity determining region of SEQ ID NO: 63 ( VH CDR3) or a heavy chain variable region having substantially the same CDR, a light chain first complementarity determining region of SEQ ID NO: 64 (VL CDR1), and a light chain second complement of SEQ ID NO: 65 An antibody having a determining region (VL CDR2), a CDR consisting of the light chain third complementarity determining region (VL CDR3) of SEQ ID NO: 66, or a light chain variable region having a CDR substantially identical thereto;
(12) heavy chain first complementarity determining region (VH CDR1) of SEQ ID NO: 67, heavy chain second complementarity determining region (VH CDR2) of SEQ ID NO: 68, heavy chain third complementarity determining region of SEQ ID NO: 69 ( VH CDR3) or a heavy chain variable region having substantially the same CDR, a light chain first complementarity determining region (VL CDR1) of SEQ ID NO: 70, and a light chain second complement of SEQ ID NO: 71 An antibody having a determining region (VL CDR2), a CDR comprising the light chain third complementarity determining region (VL CDR3) of SEQ ID NO: 72, or a light chain variable region having a CDR substantially the same as the CDR;
(13) heavy chain first complementarity determining region (VH CDR1) of SEQ ID NO: 73, heavy chain second complementarity determining region (VH CDR2) of SEQ ID NO: 74, heavy chain third complementarity determining region of SEQ ID NO: 75 ( VH CDR3) or a heavy chain variable region having substantially the same CDR, a light chain first complementarity determining region (VL CDR1) of SEQ ID NO: 76, and a light chain second complement of SEQ ID NO: 77 An antibody having a determining region (VL CDR2), a CDR consisting of the light chain third complementarity determining region (VL CDR3) of SEQ ID NO: 78, or a light chain variable region having a CDR substantially identical to the CDR;
(14) heavy chain first complementarity determining region (VH CDR1) of SEQ ID NO: 79, heavy chain second complementarity determining region (VH CDR2) of SEQ ID NO: 80, heavy chain third complementarity determining region of SEQ ID NO: 81 ( VH CDR3) or a heavy chain variable region having substantially the same CDR, a light chain first complementarity determining region of SEQ ID NO: 82 (VL CDR1), and a light chain second complement of SEQ ID NO: 83 An antibody having a determining region (VL CDR2), a CDR comprising the light chain third complementarity determining region (VL CDR3) of SEQ ID NO: 84, or a light chain variable region having a CDR substantially the same as the CDR;
(15) heavy chain first complementarity determining region (VH CDR1) of SEQ ID NO: 85, heavy chain second complementarity determining region (VH CDR2) of SEQ ID NO: 86, heavy chain third complementarity determining region of SEQ ID NO: 87 ( VH CDR3) or a heavy chain variable region having substantially the same CDR, a light chain first complementarity determining region of SEQ ID NO: 88 (VL CDR1), and a light chain second complement of SEQ ID NO: 89 An antibody having a determining region (VL CDR2), a CDR comprising the light chain third complementarity determining region (VL CDR3) of SEQ ID NO: 90, or a light chain variable region having a CDR substantially the same as the CDR;

  Preferably, the antibody of the present invention is a human antibody or a humanized antibody.

The present invention further provides a pharmaceutical composition comprising the antibody of the present invention described above.
Preferably, a cytotoxic substance is bound to the antibody.
Preferably, the cytotoxic substance is a drug, a toxin, or a radioactive substance.
Preferably, the pharmaceutical composition of the present invention is used as an anticancer agent.

Preferably, the cancer is blood cancer or solid cancer.
Preferably, the blood cancer is adult T cell leukemia (ATL).
Preferably, the solid cancer is lung cancer, colon cancer, stomach cancer, bladder cancer, pancreatic cancer, prostate cancer, liver cancer, cervical cancer, uterine cancer, ovarian cancer, breast cancer, head and neck cancer. , Skin cancer.

  The antibody capable of specifically recognizing the transferrin receptor of the present invention is useful as a molecular target drug targeting a protein expressed in ATL cells, and can significantly improve the QOL and treatment of ATL patients.

FIG. 1 shows the results of confirming the TfR binding property of an anti-TfR phage antibody by immunoprecipitation and Western blotting. FIG. 2 shows the results of immunostaining using clinical specimens surgically excised from cancer patients. FIG. 3 shows the tumor growth inhibitory effect of anti-TfR antibody in the ATL model. FIG. 4 shows the tumor growth inhibitory effect of an anti-TfR antibody in a pancreatic cancer model.

Hereinafter, the present invention will be described in more detail.
The antibody of the present invention is an antibody that specifically binds to TfR.

  Human transferrin receptor (TfR) is a single transmembrane protein encoded by human chromosome 3 and composed of 760 amino acids. This protein, also known as TFR antigen, is involved in cellular iron uptake and is thought to be involved in cell proliferation. In addition, it is considered that activated B cells and T cells in blood cells present TfR on the surface upon activation.

  TfR has been studied as a cancer target molecule since the 1980s using naked antibodies, drug-added antibodies, and transferrin-drug additions. In fact, Phase I clinical trials using mouse IgA antibody 42/6 have been conducted. It has been broken. TfR expression correlates with cell proliferation, and this has been suggested to explain the high proportion of tumors positively stained with TfR antibodies and the limited staining of normal tissues. TfR antibodies are generally accepted to inhibit cell proliferation by reducing iron uptake into cells. This can be achieved by inhibiting the interaction between iron-added transferrin and transferrin receptor, or by altering the dynamic state of the TfR cycle and cell surface presentation. Inhibition of iron uptake in tumor cells has been observed to manifest primarily as a cell cycle arrest in S phase followed by accumulation of cells in G1 phase, and finally up to induction of cell death .

  In vivo, a rat antibody that recognizes mouse TfR has been tested in a syngeneic mouse leukemia model, which improves survival compared to controls and is visible to normal tissue recognized by the antibody over a 4-week administration period. There were no signs of injury or damage. Furthermore, there was no change in the number of red blood cells or white blood cells. However, analysis of bone marrow progenitor cells showed a reduction in CFU-e (erythroid colony forming unit). Knowledge of inhibiting TfR can be obtained by evaluating the results of a phase I clinical trial conducted using mouse antibody 42/6. In this study, signs of tumor response were observed despite a shorter course of mouse antibody administration and weak pharmacokinetics. As a side effect, a decrease in bone marrow CFU-e after antibody administration was observed, but the result was not statistically significant.

  In Examples described later in this specification, experiments such as ELISA, FACS, immunoprecipitation test, Western blot, mass spectrometry, and tissue staining were performed. Hereinafter, these experimental methods will be described.

<Immunoassay such as ELISA and FACS>
The contact operation between each sample antibody and cells is performed in an appropriate solution. At that time, it is preferable to set conditions that do not affect the characteristics of the sample antibody and do not damage the cells. For example, in a culture solution suitable for the survival / proliferation of the cell, in a phosphate or citrate buffer, physiological saline, or the like, under a temperature condition of 20 ° C to 40 ° C (preferably 30 ° C to 37 ° C), Cells and sample antibody are allowed to coexist for 30 minutes to 1 hour. During this time, the solution may be stirred. In order to obtain highly reliable data, the conditions for bringing each sample antibody into contact with the cells are in principle unified.

After the above contact operation, labeling is performed as necessary (that is, except when a labeled sample antibody is used). Here, “labeling” refers to labeling the sample antibody bound to the cell surface. For example, the labeling can be carried out by reacting (contacting) a cell with a labeling substance (detection antibody) bound to an antibody having a specific binding ability to the sample antibody with the cell after the contact operation. Instead of directly binding the detection antibody to the sample antibody, another antibody or the like may be interposed between them. Thus, various labeling methods can be employed, and those skilled in the art can select an appropriate method. In flow cytometric analysis, a fluorescent dye is usually used as a labeling substance. For example, fluorescent dyes such as Alexa488, AMCA, Cascade Blue (registered trademark), FITC, PerCP ^™ , Cy ^™ 3, Texas Red (registered trademark), Cy ^™ 5, APC, TRITC, and the like can be used.

  Basic operating methods, analysis conditions, etc. may be in accordance with the instruction manual attached to the device. There are also many papers and books on flow cytometry analysis, such as Cao TM, et al. Cancer. 2001 Jun 15; 91 (12): 2205-13., Storek KJ, et al. Blood 97: 3380- 3389, WEIR'S HANDBOOK OF EXPERIMENTAL IMMUNOLOGY Vol.II <Blackwell Science>, Little MT and R. Storb Nture Reviews Cancer 2002 2: 231-238, etc.

  A typical procedure for flow cytometry analysis is shown below. After reacting the sample antibody and the cells, the detection antibody labeled with a fluorescent dye is reacted to label the cells fluorescently. Depending on the amount of antigen present on the cell surface, the amount of sample antibody to be bound varies, resulting in a difference in the amount of fluorescent labeling on the cells. Therefore, by measuring the fluorescence intensity, the affinity between the antigen present on the surface of the cell and the sample antibody and the amount of the antigen can be estimated. Usually, prior to detection of fluorescence intensity, the forward scattered light (Forward Scatter: FSC) and side scattered light (Side Scatter: SSC) are measured and gated, and only the fluorescence intensity of the target cell population is measured. . Specifically, for example, taking forward scattered light and side scattered light on the X-axis and Y-axis, respectively, a cell population that seems to be a living cell in the data obtained from the dot plot development (when using cell lines or cultured cells) A highly homogenous cell population), and the fluorescence intensity in the gate is measured. The measurement result is displayed in a format such as a histogram.

<Antigen identification>
Antigen identification of selected antibodies (hereinafter referred to as “selected antibodies”) is performed by immunoprecipitation test, Western blot, affinity chromatography, and proteomic methods (electrophoresis, mass spectrometry, genome database search, bioinformatics analysis). , And expression analysis of the corresponding gene. Among these, the method based on the proteomic technique based on mass spectrometry is suitable for identification of unknown antigens, and is preferable as the identification method employed in the method of the present invention. Note that these methods are not mutually exclusive and can be used in combination of two or more as necessary.

<Mass spectrometry>
Mass spectrometry is a method for determining the mass of a sample by separating ions generated from a sample such as protein or peptide according to mass / charge (m / z) and then measuring the intensity. Analysis of biological samples such as proteins and peptides by the development of soft ionization methods such as ESI (Electro Spray Ionization) and MALDI (Matrix Assisted Laser Deporption Ionization) Mass spectrometry has become widely used.

  A mass spectrometer is generally composed of a combination of an ion source, a mass spectrometer, and a detector, and various mass spectrometers are commercially available depending on the type of sample and the purpose of analysis. For identification of proteins and peptides, MS / MS (Mass spectrometry / mass spectrometry) such as ESI Q-TOF MS, MALDI-TOF MS, etc. are used. Measurement methods combining liquid chromatography and mass spectrometry (LC-MAS (liquid chromatography / electrospray ionization mass spectrometer), LC-MS / MS, etc.) can also be used.

  In the tandem mass spectrometer, two mass spectrometers are connected in series, and the ions generated by the ion source are separated by the first mass spectrometer (MS1) and only a single ion peak is selectively passed. After that, an inert gas particle or the like collides with the product ion to be decomposed into product ions, and the product ions are analyzed by a second mass spectrometer (MS2). Tandem mass spectrometers such as Q-TOF, TOF-TOF, QQ, and Q-IT (Iontrap) exist by combining the first mass spectrometer (MS1) and the second mass spectrometer (MS2) To do. Q-TOF (quadrupole mass spectrometer (Q-MS) and TOF mass spectrometer (TOD-MS) tandem mass spectrometer connected in series) As described above, a hybrid tandem mass spectrometer composed of two different mass spectrometers has excellent MS / MS measurement ability and is suitable for identification of amino acid sequences of proteins and peptides.

  In order to identify amino acid sequences from the results of the mass spectrometer, PMF method (peptide mass fingerprinting method) or MS / MS ion search method for searching genome data using experimental results is used. Further, a de novo sequencing method in which an amino acid sequence is determined by mathematical operation from an MS / MS spectrum without genome data search may be used.

<Immunoprecipitation, affinity chromatography>
On the other hand, immunoprecipitation tests, Western blotting, affinity chromatography and the like are effective methods when the selected antibody is expected to recognize a known antigen. In these methods, the reactivity between the selected antibody and the known antigen is examined. That is, in the immunoprecipitation test, it is examined whether the selected antibody and a specific known antigen form an immunoprecipitate. If an immunoprecipitate is formed, it is determined that the known antigen is the antigen of the selected antibody. The On the other hand, in Western blotting, it is examined whether the selected antibody recognizes the antigen protein transferred to the PVDF membrane or the like. In affinity chromatography, the adsorptivity of a selected antibody to a column carrying a specific known antigen is examined, and a determination is made based on the presence or absence of the adsorptivity. In addition, procedures such as immunoprecipitation tests, Western blotting, affinity chromatography and the like may follow conventional methods.

If disease-related molecules (disease gene products, etc.) are obtained in some form, such as purified protein or recombinant protein, intermolecular interactions with antibodies can be performed in vitro (fluorescence spectroscopy, gel filtration, ultracentrifugation). Classic method used, method using surface plasmon resonance phenomenon, method using crystal oscillator microbalance, etc., or in vivo (single molecule tracking method, fluorescence resonance energy transfer (FRET) observation method , Etc.).
When specific reactivity is recognized between the identified antigen and each antibody by the above confirmation test, it is determined that the above estimation is correct.

<Tissue staining>
Reactivity with patient-derived cells or tissues can be detected and evaluated using immunohistochemical staining, immunoprecipitation, flow cytometry analysis, Western blot, ELISA, and the like. These methods are not mutually exclusive and can be used in combination of two or more as required. Among these, it is preferable to employ an immunohistochemical staining method. According to the immunohistochemical staining method, rapid and sensitive detection is possible. Also, the operation is relatively simple.

In immunohistochemical staining, a tissue collected from a patient is brought into contact with an antibody, and then a specifically bound antibody is detected. Specifically, the method of the present invention can be carried out according to the immunohistochemical staining method shown below.
In general, immunohistochemical staining of a living tissue is performed by the following procedures (a) to (j). Various documents and books can be referred to for immunohistochemical staining of biological tissues (for example, “Enzyme Antibody Method, Revised 3rd Edition”, edited by Keiichi Watanabe and Kazuho Nakane, interdisciplinary project).

(A) Fixing / embedding with paraffin A tissue collected surgically from a living body is fixed with formalin, paraformaldehyde, anhydrous ethyl alcohol, or the like. Then embedded in paraffin. Generally dehydrated with alcohol, treated with xylene, and finally embedded in paraffin. A specimen embedded in paraffin is sliced into a desired thickness (for example, 3 to 5 μm thick) and spread on a glass slide. In place of the paraffin-embedded specimen, an alcohol-fixed specimen, a dry-sealed specimen, a frozen specimen, or the like may be used.
(B) Deparaffinization Generally, treatment is performed sequentially with xylene, alcohol, and purified water.
(C) Pretreatment (antigen activation)
If necessary, enzyme treatment, heat treatment and / or pressure treatment are performed for antigen activation.
(D) Endogenous peroxidase removal When peroxidase is used as a labeling substance for staining, endogenous peroxidase activity is removed by treatment with hydrogen peroxide.
(E) Nonspecific reaction inhibition The section is treated with bovine serum albumin solution (for example, 1% solution) for several minutes to several tens of minutes to inhibit nonspecific reaction. Note that this step may be omitted by carrying out the next primary antibody reaction using an antibody solution containing bovine serum albumin.
(F) Primary antibody reaction An antibody diluted to an appropriate concentration is dropped onto a section on a slide glass, and then reacted for several tens of minutes to several hours. After completion of the reaction, wash with an appropriate buffer such as phosphate buffer.
(G) Addition of labeling reagent Peroxidase is frequently used as a labeling substance. A secondary antibody to which peroxidase is bound is dropped onto a section on a slide glass, and then allowed to react for several tens of minutes to several hours. After completion of the reaction, wash with an appropriate buffer such as phosphate buffer.
(H) Color reaction DAB (3,3'-diaminobenzidine) is dissolved in Tris buffer. Subsequently, hydrogen peroxide is added. The coloring solution thus prepared is allowed to infiltrate into the section for several minutes (for example, 5 minutes) to develop a color. After color development, the section is thoroughly washed with tap water to remove DAB.
(I) Nuclear staining Nuclear staining is performed by reacting Meyer's hematoxylin for several seconds to several tens of seconds. Wash with running water and color (usually for a few minutes).
(J) Dehydration, clearing, sealing After dehydrating with alcohol, clearing with xylene, and finally sealing with synthetic resin, glycerin, rubber syrup or the like.

<Epitope>
Here, even for cancers of the same organ, the pathological condition (grade of malignancy) may vary greatly depending on the patient. This pathological difference is expected to involve a specific antigen expression mode. On the other hand, the antibody set obtained by the method of this embodiment typically comprises a set of antibodies that differ from each other at the epitope level, although there is no difference in the level of the antigen to be recognized. That is, an antibody set including a plurality of antibodies that recognize different epitopes. Such an antibody set makes it possible to detect or evaluate the expression mode of an antigen from various aspects, and is useful for, for example, detection of a specific disease state in cancer or the like and determination of malignancy. It should be noted that specificity (cross-reactivity) or the like may be compared among the antibodies in the antibody group, and the antibody having the most excellent characteristics may be finally selected.

Next, the antibody of the present invention will be described in more detail.
In the present specification, the following abbreviations (in parentheses) are used in accordance with the convention as required.
Heavy chain (H chain), light chain (L chain), heavy chain variable region (VH), light chain variable region (VL), complementarity determining region (CDR), first complementarity determining region (CDR1), second Complementarity determining region (CDR2), third complementarity determining region (CDR3) heavy chain first complementarity determining region (VH CDR1), heavy chain second complementarity determining region (VH CDR2), heavy chain third Complementarity determining region (VH CDR3) light chain first complementarity determining region (VL CDR1), light chain second complementarity determining region (VL CDR2), light chain third complementarity determining region (VL CDR3)

  The sequence of the framework region (FR region) in the variable region of the antibody of the present invention is not particularly limited as long as it does not substantially affect the specific binding to the corresponding antigen. For example, when the antibody of the present invention is constructed as a humanized antibody, the FR region of a known human antibody can be used. In addition, when constructing an antibody used as a reagent for detection or an antibody used for application to animal species other than humans, the expected effect can be achieved without using the human antibody FR region or It may be rather inappropriate to use the human antibody FR region. In such a case, the FR region of a non-human animal species (for example, mouse or rat) can be used.

  In one embodiment, the antibody of the present invention includes a constant region in addition to a variable region (for example, in the case of an IgG type antibody). The sequence of the constant region in this embodiment is not particularly limited. For example, as described below, when the antibody of the present invention is constructed as a humanized antibody, a known human antibody constant region can be used. Similarly to the FR region, a constant region of an animal species other than human (eg, mouse or rat) can also be used.

  On the other hand, a human CDR-grafted antibody can be prepared, for example, by the following method. First, the amino acid sequences of the H chain variable region and the L chain variable region of an antibody against a specific antigen and the base sequence encoding the same are determined by the method described in the column of the method for producing a chimeric antibody. In addition, the amino acid sequence and base sequence of each CDR region are determined.

  Next, an FR (framework region) that exists across the CDR region is selected. There are approximately three methods for selecting FR. The first method is a method using a human antibody frame, such as NEWM and REI, whose three-dimensional structure has already been clarified (Riechmann L. et al., Nature 332, 323-3Z7 (1988); Tempst, PR. Et. al., Protein Engineering 7, 1501-1507 (1994); Ellis JH. etal., J. Immunol 155, 925-937 (1995)). In the second method, a human antibody variable region having the highest homology with the mouse antibody variable region of interest is selected from a database, and its FR is used (Queen C. et al., Proc Natl Acad SciUSA 86, 10029). -10033 (1989); Rozak MJ. Et al., J Biol Chem 271, 22611-22618 (1996); Shearman CW. Et al., J. Immunol 147, 4366-4373 (1991)). The third method is to select the most commonly used amino acid in human antibody FRs (Sato K. et al., Mol Immunol 31, 371-381 (1994); Kobinger F. et al., Protein Engineering 6, 971-980 (1993); Kettleborough CA. et al., Protein Engineering 4, 773-783 (1991)). Any of these methods can be used in the present invention.

  Even if the amino acid sequence of the selected human FR is modified, it can be used as the FR amino acid sequence as long as the finally obtained human CDR-grafted antibody has a specific binding property to TfR. it can. In particular, when a part of the amino acid of the selected human FR is changed to the FR amino acid of the antibody derived from CDR, it is highly possible that the characteristics of the antibody are maintained. The number of amino acids to be modified is preferably 30% or less of the entire FR, more preferably 20% or less of the entire FR, and still more preferably 10% or less of the entire FR.

  Next, a DNA encoding the H chain variable region and the L chain variable region is designed by combining the FR selected by any of these methods and the CDR. Based on this design, DNA encoding the H chain variable region and DNA encoding the L chain variable region are respectively prepared by chemical synthesis, biochemical cleavage / recombination, and the like. Then, the DNA encoding the H chain variable region is incorporated into an expression vector together with the DNA encoding the human immunoglobulin H chain constant region to construct an H chain expression vector. Similarly, an L chain expression vector is constructed by incorporating DNA encoding the L chain variable region into an expression vector together with DNA encoding the human immunoglobulin L chain constant region. Examples of expression vectors include, but are not limited to, SV40 virus based vectors, EB virus based vectors, BPV (papilloma virus) based vectors, and the like.

  A host cell is cotransformed with the H chain expression vector and the L chain expression vector prepared by the above method. CHO cells (Chinese hamster ovary) (A. Wright & SL Morrison, J. Immunol. 160, 3393-3402 (1998)), SP2 / 0 cells (mouse myeloma) (K. Motmans et al., Eur. J. Cancer Prev. 5, 512-519 (1996), RP Junghans et al., Cancer Res. 50, 1495-1502 (1990)) are preferably used. For transformation, the lipofectin method (RWMalone et al., Proc. Natl. Acad. Sci. USA 86, 6077 (1989), PLFelgner et al., Proc. Natl. Acad. Sci. USA 84, 7413 ( 1987), electroporation method, calcium phosphate method (FL Graham & AJvan der Eb, Virology 52, 456-467 (1973)), DEAE-Dextran method and the like are preferably used.

  After culturing the transformant, the human CDR-grafted antibody is isolated from the transformant cells or from the culture solution. For separation and purification of antibodies, methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, affinity chromatography, ion exchange chromatography, gel filtration chromatography and the like can be used in appropriate combination.

Antibody fragments can be prepared based on the antibody of the present invention or based on the sequence information of the gene encoding the antibody of the present invention. Antibody fragments include Fab, Fab ′, F (ab ′) ₂ , scFv, and dsFv antibodies.

Fab has a molecular weight of about 50,000 composed of L chain and H chain variable region obtained by digesting IgG with papain in the presence of cysteine, and H chain fragment consisting of C _H 1 domain and part of hinge part. It is a fragment. In the present invention, the antibody can be obtained by digesting with papain. Alternatively, Fab can be prepared from a transformant obtained by incorporating a DNA encoding part of the H chain and L chain of the above antibody into an appropriate vector and transforming using the vector.

Fab ′ is a fragment having a molecular weight of about 50,000 obtained by cleaving a disulfide bond between the H chains of F (ab ′) ₂ described later. In the present invention, the antibody is obtained by digesting with pepsin and cleaving disulfide bonds using a reducing agent. Similarly to Fab, it can also be prepared by genetic engineering using DNA encoding Fab ′.

F (ab ′) ₂ is a fragment (Fab) composed of an L chain and an H chain variable region obtained by digesting IgG with pepsin, and an H chain fragment consisting of a C _H 1 domain and a part of the hinge region. ') Is a fragment having a molecular weight of about 100,000 bonded by a disulfide bond. In the present invention, the antibody is obtained by pepsin digestion. Similarly to Fab, it can be prepared by genetic engineering using DNA encoding F (ab ′) ₂ .

scFv is an antibody fragment in which an Fv comprising an H chain variable region and an L chain variable region is made into a single chain by linking the C terminus of one chain and the other N terminus with an appropriate peptide linker. As the peptide linker, for example, (GGGGS) ₃ having high flexibility can be used. For example, a DNA encoding an scFv antibody is constructed using DNA encoding the H chain variable region and L chain variable region of the above antibody and DNA encoding a peptide linker, and this is incorporated into an appropriate vector, and the vector is used. ScFv can be prepared from the transformant transformed as described above.

  dsFv is an Fv fragment in which a Cys residue is introduced at an appropriate position in the H chain variable region and the L chain variable region, and the H chain variable region and the L chain variable region are stabilized by disulfide bonds. The position of Cys residue introduction in each chain can be determined based on the three-dimensional structure predicted by molecular modeling. In the present invention, for example, a three-dimensional structure is predicted from the amino acid sequences of the H chain variable region and the L chain variable region of the above-described antibody, and DNAs encoding the H chain variable region and L chain variable region into which mutations are introduced are constructed based on the prediction. Then, this is incorporated into an appropriate vector, and dsFv can be prepared from a transformant transformed with the vector.

  It is also possible to multimerize antibody fragments by linking scFv antibody, dcFv antibody, etc. using an appropriate linker, or by fusing streptavidin.

A fusion antibody or a labeled antibody can be constructed by fusing or binding a low molecular compound, protein, labeling substance or the like to the antibody (including antibody fragment) of the present invention. As the labeling substance, radioactive substances such as ¹²⁵ I, peroxidase, β-D-galactosidase, microperoxidase, horseradish peroxidase (HRP), fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC), alkaline phosphatase, biotin, etc. are used. be able to.

Furthermore, the antibody of the present invention can also be used as a medium (carrier) for specifically delivering a drug or the like to a specific cancer cell. That is, as a use of the antibody of the present invention, a DDS (Drug Delivery System) targeting a specific cancer cell is assumed.
In addition, each use of the antibody of this invention is explained in full detail below.
According to the present invention, cancer can be examined using the antibody of the present invention.
Step (1): A step of preparing a test cell separated from a living body.
Step (2): A step of detecting a TFR antigen for a test cell.

  The information obtained by the test method of the present invention is useful for diagnosis of biliary liver cancer or pancreatic cancer. For example, information obtained by carrying out the above method for patients with biliary liver cancer can be used for evaluation or grasping of the patient's disease state, evaluation of therapeutic effect, and the like. For example, if the method of the present invention is performed in parallel with the treatment of biliary liver cancer, the therapeutic effect can be evaluated based on the information obtained as a result. Specifically, by implementing the method of the present invention after drug administration, the change in the expression level of TFR antigen in lung cancer can be examined, and the therapeutic effect can be determined from the increase or decrease in the expression level. Thus, you may utilize the method of this invention for the monitoring of a therapeutic effect.

  On the other hand, information obtained when targeting a person other than a patient, that is, a person who is not certified for biliary liver cancer, can be used for determination of the presence or absence of morbidity in the biliary liver cancer, evaluation of morbidity risk, and the like. According to the method of the present invention, since the diagnosis of liver cancer can be performed based on an index of gene expression that is excellent in objectivity, it can be said that its value is very high.

Hereafter, each step to comprise is demonstrated in detail.
1. In step (1), cells separated from the subject (subject, living body) are prepared. Not only patients but also healthy individuals can be targeted here. For example, a part of a target tissue collected by biopsy (biopsy) can be used as a test cell for the method of the present invention.

  In the present invention, the “test cell” is a cell that serves as a sample (target) for detection in the method of the present invention. The test cell is separated from the living body. That is, the present invention is applied to a test cell that is separated from a living body. “Separated from the living body” means a state in which the test cell is completely isolated from the living body from which the test cell is extracted by removing a part of the living tissue in which the test cell exists. When an immunological detection method is employed in step (2), the test cells are usually prepared in a state where they exist in the living body, that is, in a state of being bound to surrounding cells (as a tissue piece). Used in the method. The test cells may be used in the method of the present invention after being separated (isolated) from surrounding cells.

2. In step (2), the TFR antigen is detected for the prepared test cell. “Detecting a TFR antigen” means examining whether or not the TFR antigen is expressed (presence or absence of expression), or grasping the expression level of the TFR antigen as an absolute amount or a relative amount. The reference of the relative amount here can be, for example, the TFR antigen amount of a standard sample prepared according to the malignancy. Usually, the presence or absence of the expression of the TFR antigen, and the amount thereof when it is expressed will be examined. It is not essential to strictly quantify the amount of TFR antigen when detecting the TFR antigen.

  In one embodiment of the present invention, a detection method targeting mRNA that is a transcription product of a TFR antigen is performed. For detection (measurement) of mRNA, conventional methods such as RT-PCR and various hybridization methods using specific probes (eg, Northern hybridization, in situ hybridization) can be employed. In another embodiment of the present invention, a detection method targeting a TFR antigen expression product (protein) is performed.

  It is preferable to detect the TFR antigen by immunological techniques (for example, immunohistochemical staining). In the immunological technique, an anti-TFR antigen antibody is used, and CD46 antigen protein is detected using the binding property (binding amount) of the antibody as an index. The immunological detection method enables rapid and sensitive detection. Also, the operation is simple. Examples of the detection method include ELISA, radioimmunoassay, FCM, immunoprecipitation, immunoblotting, and the like.

  According to the immunohistochemical staining method, the TFR antigen can be detected quickly and with high sensitivity. Also, the operation is simple. Therefore, the burden on the subject (patient) accompanying the detection of the TFR antigen is also reduced.

  If a labeled antibody is used as the anti-TFR antibody of the present invention, it is possible to directly detect the amount of bound antibody using the labeled amount as an index. Therefore, it becomes a simpler method. On the other hand, in addition to the need to prepare an anti-TFR antibody to which a labeling substance is bound, there is a problem that detection sensitivity is generally lowered. Therefore, it is preferable to use an indirect detection method such as a method using a secondary antibody to which a labeling substance is bound or a method using a polymer to which a secondary antibody and a labeling substance are bound. The secondary antibody here is an antibody having a specific binding property to an anti-TFR antibody. For example, when an anti-TFR antibody is prepared as a rabbit antibody, an anti-rabbit IgG antibody can be used. Labeled secondary antibodies that can be used for various types of antibodies such as rabbits, goats, and mice are commercially available (for example, Funakoshi Co., Ltd., Cosmo Bio Co., Ltd., etc.), depending on the anti-TFR antibody used in the present invention. Can be appropriately selected and used.

  Examples of labeling substances include peroxidase, β-D-galactosidase, microperoxidase, horseradish peroxidase (HRP), fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC), alkaline phosphatase, biotin, and radioactive substances. What is selected is preferably used. In particular, highly sensitive detection is possible by the method of reacting avidin peroxidase using biotin as a labeling substance.

(Therapeutic use)
ADVANTAGE OF THE INVENTION According to this invention, by using TfR as a target, the chemical | medical agent (cancer therapeutic agent) which can act and damage specifically for cancer cells, and the treatment method using the same are provided. In one embodiment of the agent of the present invention, an anti-TfR antibody is contained as an active ingredient. In a preferred embodiment of the drug of the present invention, an anti-TFR antibody having ADCC activity is contained as an active ingredient. With the drug of this aspect, a therapeutic effect can be obtained by cell damage utilizing ADCC activity. The anti-TFR antibody having ADCC activity is an antibody shown in the examples described later (may be partially modified as long as it maintains specific binding properties and ADCC activity to TFR), or is constructed based on this. Different types of antibodies (eg, IgG type) can be used. This antibody has both specific binding to TFR and ADCC activity. Therefore, it is possible to specifically bind to a cancer cell expressing TFR, and then exhibit ADCC activity, thereby damaging the cancer cell. Although the target cancer cells of the drug of this embodiment are not particularly limited, for example, lung cancer, colon cancer, stomach cancer, bladder cancer, pancreatic cancer, prostate cancer, liver cancer, cervical cancer, uterine cancer, ovary Can target ATL cells that are cancer, breast cancer, head and neck cancer, skin cancer, and blood cancer.

  The antibody of the present invention can be used as a carrier for DDS. That is, this embodiment provides an immune complex obtained by binding a drug (cytotoxic etc.) or a radioisotope (collectively referred to as “active ingredient”) to an anti-HER1 antibody or the like. An immune complex containing a drug (cytotoxin) having a cell-killing or cytotoxic activity is generally called an immunotoxin.

Examples of drugs used in the present invention include, for example, duocarmycin, duocarmycin analogs and inducers, CC-1065, duocarmycin analogs based on CBI, duocarmycin analogs based on MCBI, Duocarmycin analogs based on CCBI, doxorubicin, doxorubicin conjugate, morpholino-doxorubicin, cyanomorpholino-doxorubicin, dolastatin, dressatin-10, combretastatin, calicheamicin, maytansine, maytansine analog, DM1, DM2, DM3 , DM4, DMI, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), 5-benzoylvalerin AE ester (AEVB), tubulin, disorazole, epothilone, paclitaxel, docetaxel, SN-38, topotecan, lysoxine, echinomycin, colchicine, vinblastine, vindesine, estramustine, semadotine, eluterobin, methotrexate, methotrexate, methotrexate , 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leulosin, lurocidine, actinomycin, daunorubicin, daunorubicin conjugate, mitomycin C, mitomycin A, carminomycin, aminopterin, thalisomycin, podophyllo Toxin, podophyllotoxin derivative, etoposide, etoposide phosphate, vincristine, taxol, Kiso tail retinoic acid, butyric acid, N ⁸ - can be exemplified acetyl spermidine and camptothecin like, but is not limited thereto.

  In the present invention, the aforementioned drug and antibody can be bound by a known method. The antibody and the drug may be directly bonded via a linking group or the like that they have, or may be indirectly bonded via another substance such as a linker.

  Examples of the linking group when the drug is directly bonded include a disulfide bond using an SH group and a bond via maleimide. For example, the intramolecular disulfide bond in the Fc region of the antibody and the disulfide bond of the drug are reduced, and both are bonded by a disulfide bond. There is also a method via maleimide. Another method is to introduce cysteine into the antibody by genetic engineering.

  It is also possible to bind the antibody and the drug indirectly via another substance (linker). The linker preferably has one or more functional groups that react with the antibody or drug or both. Examples of functional groups include amino groups, carboxyl groups, mercapto groups, maleimide groups, pyridinyl groups, and the like. The linker may be a peptide linker. Techniques for conjugating active moieties to antibodies are known, e.g. Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (Eds.), Pp. 243-56 (Alan R. Liss, Inc. 1985), Controlled Drug Delivery (2nd edition.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987), Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 ( Academic Press 1985), Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62: 119-58 (1982).

  As another embodiment of the present invention, a so-called immunotoxin in which a toxin (toxin) is chemically or genetically bound to an antibody can be mentioned.

  Examples of the toxin used in the present invention include the following. Diphtheria toxin A chain, Pseudomonas endotoxin, lysine chain; sugar-free ricin A chain Gelonin (Saporin) and the like can be mentioned.

  Another embodiment of the antibody of the present invention is a so-called RI-labeled antibody in which the antibody of the present invention and a radioactive substance are bound.

  The radioactive substance is preferably a cytotoxic radioactive metal when used as a cancer therapeutic agent, and preferably a non-cytotoxic radioactive metal when used as a cancer diagnostic agent. There is also a method using iodine 123 (123I) and iodine 131 (I131).

  Examples of such cytotoxic radioactive metals include yttrium 90 (90Y), rhenium 186 (186Re), rhenium 188 (188Re), copper 67 (67Cu), iron 59 (59Fe), strontium 89 (89Sr), and gold 198. (198Au), mercury 203 (203Hg), lead 212 (212Pb), dysprosium 165 (165Dy), ruthenium 103 (103Ru), bismuth 212 (212Bi), bismuth 213 (213Bi), holmium 166 (166Ho), samarium 153 (153Sm) ), Lutetium 177 (177Lu) and the like.

  Among these radioactive metals, 90Y, 153Sm, and 177Lu are preferable from the viewpoints of half-life, radiation energy, easy labeling reaction, labeling rate, and complex stability.

  On the other hand, non-cytotoxic radioactive metals used for diagnostic agents include technesium 99m (99mTc), indium 111 (111In), indium 113m (113mIn), gallium 67 (67Ga), gallium 68 (68Ga), and thallium 201 (201Tl). Chromium 51 (51Cr), cobalt 57 (57Co), cobalt 58 (58Co), cobalt 60 (60Co), strontium 85 (85Sr), mercury 197 (197Hg), and copper 64 (64Cu) are preferably used. It is not limited to.

  In order to bind these radioactive metal elements to the antibody of the present invention, it is preferable to react the metal chelate reagent with the antibody and react with the radioactive metal element to form a complex. In the modified antibody thus obtained, a radioactive metal element is bound to the antibody of the present invention via a metal chelating reagent.

  Examples of metal chelating reagents used for such complex formation include (1) 8-hydroxyquinoline, 8-acetoxyquinoline, 8-hydroxyquinaldine, oxyquinoline sulfate, O-acetyloxin, O-benzoyloxin, Op-nitrobenzoyloxins, quinoline derivatives having a quinoline skeleton such as norfloxacin, ofloxacin, enoxacin, ciprofloxacin, lomefloxacin, tosufloxacin, fleroxacin, sparfloxacin and the like; (2) chloranilic acid, aluminone , Thiourea, pyrogallol, cuperone, bismuthiol (II), galloyl gallic acid, thiolide, 2-mercaptobenzothiazole, tetraphenylarsonium chloride, etc .; (3) ethylenediamine Acetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) and dihydroxyethylglycine, diaminopropanoltetraacetic acid, ethylenediaminediacetic acid, ethylenediaminedipropionate hydrochloride, hydroxyethylethylenediaminetriacetic acid, ethylenediaminetetrakis (methylenephosphone) Acid), glycol etherdiaminetetraacetic acid, hexamethylenediaminetetraacetic acid, hydroxyethyliminodiacetic acid, iminodiacetic acid, diaminopropanetetraacetic acid, nitrilotriacetic acid, nitrilotripropionic acid, nitrilotris (methylenesulfonic acid) trisodium salt, Triethylenetetramine hexaacetic acid, methyl DTPA, cyclohexyl DTPA, aminobenzyl EDTA, isothiocyanobenzyl EDTA, isothiocyanobe Gilt DTPA, methylisothiocyanobenzyl DTPA, cyclohexylisothiocyanobenzyl DTPA, maleimide propylamide benzyl EDTA, maleimide pentylamide benzyl EDTA, maleimide decylamide benzyl EDTA, maleimide pentylamide benzyl DTPA, maleimide decylamide benzyl EDTA, maleimide decylamide (4) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7- Triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane (Cycle), 1 , 4,8,11-teto Examples include laazacyclotetradecane (Cyclam), isothiocyanobenzyl DOTA, and isothiocyanobenzyl NOTA.

  Among these metal chelate reagents, isothiocyanobenzyl DOTA, methylisothiocyanobenzyl DTPA, and cyclohexylisothiocyanobenzyl DTPA are easy to introduce metal chelates into antibodies, labeling rate, complex stability, etc. preferable.

  Those skilled in the art can bind the radioactive metal element to the antibody of the present invention according to a conventional method. For example, the reaction can be performed by reacting the antibody of the present invention with a metal chelate reagent, preparing a labeling precursor in advance, and then reacting with a radioactive metal element.

  As an active ingredient to be contained in the pharmaceutical composition of the present invention, a protein or peptide having a desired biological activity may be used. Proteins that can be used for such purposes include abrin, ricin A, Pseudomonas exotoxin, diphtheria toxin, tumor necrosis factor, interferon-γ, interleukin-1 (IL-1), interleukin-2 ( IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF) lymphokine.

  The pharmaceutical composition of the present invention can be formulated according to a conventional method. In the case of formulating, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological Saline solution and the like). As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, diethylin sulfite, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

  The dosage form for formulation is not particularly limited, and can be prepared as, for example, tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, and suppositories.

  In the treatment using the drug of the present invention, the drug of the present invention is administered to a subject (patient) having cancer cells or adult T cell leukemia cells. The agent of the present invention can be applied to a subject (patient) by oral administration or parenteral administration (intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal injection, direct introduction into a target cell, etc.) depending on the form.

  The dose of the drug varies depending on symptoms, patient age, sex, weight, and the like, but those skilled in the art can appropriately set an appropriate dose. For example, the dose can be set so that the amount of active ingredient per day is about 0.001 mg to about 100 mg for adults (body weight about 60 kg). As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In setting the administration schedule, it is possible to take into account the patient's medical condition and the duration of the drug effect.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Example 1: Phage antibody screening using cancer cell lines (1) Screening of phage antibodies that bind to cancer cells (hepatoma cell line HepG2)
First, HepG2 cells were cultured in a 15 cm dish, and then dissociated from the dish with 2 mg / ml collagenase I (Gibco BRL) / cell dissociation buffer (Gibco BRL). It was washed with cold PBS and 4 × 10 ⁷ was used. This was mixed with a 1 × 10 ¹³ cfu human antibody phage library (see Japanese Patent Application Laid-Open No. 2005-185281, WO2008 / 007648, and WO2006 / 090750), and the final concentration of the reaction solution was 1% BSA-0.1% NaN3. / MEM, with a volume of 1.6 ml, and reacted by slowly rotating at 4 ° C. for 4 hours. After completion of the reaction, the reaction solution is divided into two parts, each layer is layered on 0.6 ml organic solution (dibutyl phtalate cycloheximide 9: 1), and the cells are allowed to act on the microcentrifuge at 3000 rpm for 2 minutes. Sedimented to the bottom of the tube. For each tube, the solution was discarded and the cells were suspended in 0.7 ml of 1% BSA / MEM, layered on 0.7 ml of organic solvent and centrifuged. After repeating this operation once more, the solution was discarded, and the cells were suspended in 0.3 ml of PBS, frozen in liquid nitrogen, and thawed at 37 ° C.

This was infected with 20 ml of OD0.5 Escherichia coli DH12S for 1 hour, and a portion thereof was placed on an ampicillin plate to calculate the titer of the recovered phage. The phage-infected Escherichia coli was cultured overnight at 30 ° C. in 600 ml of 2 × YTGA medium (2 × YT, 200 μg / ml ampicillin sulfate, 1% glucose). 10 ml of this overnight culture was mixed with 200 ml of 2 × YTA medium (2 × YT, 200 μg / ml ampicillin sulfate), cultured at 37 ° C. for 1.5 hours, added with 1 × 10 ¹¹ helper phage KO7, cultured at 37 ° C. for 1 hour, and then 800 ml of 2 × YTGAK 2xYT, 200 μg / ml ampicillin sulfate, 0.05% glucose, 50 μg / ml kanamycin) was added and cultured overnight at 30 ° C. This was centrifuged at 8000 rpm for 10 minutes to prepare 1 l of supernatant, and 200 ml of PEG solution (20% polyetyleneglycol 6000, 2.5M NaCl) was mixed with it and stirred well, followed by centrifugation at 8000 rpm for 10 minutes to precipitate phages. . This was suspended in 10 ml of PBS, and a part thereof was used to examine the number of E. coli infections. This is the 1st screening phage.

For 2ed screening, cultured cells 2 × 10 ⁷ and 1st phage 1 × ^{10 10} were used, and the volume of the reaction solution was 0.8 ml. The reaction solution was 1% BSA-0.1% NaN3 / MEM, and the whole scale was half of the 1st screening.

The 3rd screening was performed under the same conditions as the 2nd screening except that the 2nd phage 1 × 10 ⁹ was used.

(2) Analysis of antibody expression ability of phage antibody Dilute Escherichia coli obtained by screening, spread it on a normal agar medium containing 100 μg / ml ampicillin, pick up the obtained colonies, and overnight at 30 ° C. in 2 × YTGA medium Culture, extraction of DNA with Kurabo Industries PI-50, and determination of nucleotide sequence by dideoxy method. In addition, 0.05 ml of this overnight culture was planted in 1.2 ml of 2xYTAI (2xYT, 200 μg / ml ampicillin sulfate, 0.5 mM IPTG), cultured overnight at 30 ° C, and centrifuged at 15000 rpm for 5 minutes in a microcentrifuge to obtain the supernatant. .

  Since the antibody is expressed as a cp3 fusion protein, expression studies using it were performed. That is, first, the obtained supernatant was reacted with Maxisorp (NUNC) at 37 ° C. for 2 hours, and then the solution was discarded, and blocking was performed by reacting 5% BSA at 37 ° C. for 2 hours. The solution was discarded, and a rabbit anti-cp3 antibody (Medical and Biological Laboratories Inc.) diluted 2000 times with 0.05% Tween / PBS was reacted at room temperature for 1 hour, washed with PBS, and 2000 times with 0.05% Tween / PBS. The diluted HRP-labeled goat anti-rabbit IgG antibody (Medical and Biological Laboratories Inc.) was reacted at room temperature for 1 hour, washed with PBS, and reacted with 100 μl of OPD solution at room temperature for 15 minutes, with 2M ammonium sulfate. The reaction was stopped and the absorbance at 492 nm was measured with SPECTRAmax 340PC (Molecular Devices). As a result of positive absorbance of 0.5 or more, 1012 clones were positive.

(3) DNA sequence analysis With respect to 1012 types of phages whose antibody expression was confirmed by (2) above, DNA sequences were analyzed by existing methods, and incomplete antibodies retaining defective regions and antibodies with overlapping sequences were eliminated. Then, 410 phage antibodies having independent antibody sequences were selected.

  By the same method, regarding the 21 types of cancer cells shown in Table 1 below, the following 1863 phage antibodies having independent sequences were established by the screenings (1) to (3) above.

Example 2: Antibody binding evaluation test by ELISA using secreted antigen of TfR (1) Preparation of TfR-secreting cells From the cancer cells # MIAPaCa2, SKOv3 (TfR high-expressing strain), TfR extracellular domain by a conventional method After the cDNA was prepared, it was inserted into pCMV-Script (Clontech) to create a TfR expression vector. This expression vector was introduced into cell line # 293T to prepare expression cells that secrete TfR.

(2) Preparation of ELISA system Using the TfR antigen obtained from TfR-secreting cells, the binding activity was evaluated by ELISA in the following manner. The antigen was sensitized to the Maxisorp immuno module. Specifically, the reaction was performed at 50 μl / well at 37 ° C. for 2 hours at a concentration of 10 μg / ml. Thereafter, the supernatant was discarded and the blocking operation was started. Specifically, Blocking solution (5% skim milk / 0.05% NaN ₃ / PBS) was reacted at 200 μl / well at 37 ° C. for 2 hours. Thereafter, the blocking solution was removed and washed once with a phosphate buffer. Thereafter, the sample antibody expression culture supernatant as a primary antibody was reacted at 100 μl / well at 37 ° C. for 1 hour. Thereafter, the supernatant was removed and washed 5 times with a phosphate buffer. Next, Rabbit anti-cp3 polyclonal as a secondary antibody diluted 5000 times with PBS / 0.05% Tween20 was reacted at 100 μl / well at 37 ° C. for 1 hour, the supernatant was removed, and washed 5 times with phosphate buffer. Next, anti-rabbit IgG (H + L) -HRP as a secondary antibody diluted 2000 times with PBS / 0.05% Tween20 was reacted at 100 μl / well at 37 ° C. for 1 hour. Thereafter, the supernatant was removed and washed 5 times with a phosphate buffer. Thereafter, OPD in 0.1 M citrate phosphate buffer pH 5.1 0.01% H ₂ O ₂ 100 μl / well was added as a color developing reagent and allowed to react at room temperature for 5 minutes. Thereafter, 2N H ₂ SO ₄ was added at 100 μl / well to stop the color reaction. Thereafter, the absorbance at 492 nm was measured with SPECTRAmax 340PC (Molecular Devices).

(3) Selection of anti-TfR antibody by TfR solid-phase ELISA As a result of evaluating 1863 phage antibodies obtained in Example 2 by ELISA, antibodies having the following 15 independent sequences are specific for human TfR. It was confirmed that it showed mechanical reactivity. The amino acid sequence of each antibody clone is as follows.
(1) Tfr001 antibody
VH CDR1: SEQ ID NO: 1, VH CDR2: SEQ ID NO: 2, VH CDR3: SEQ ID NO: 3
VL CDR1: SEQ ID NO: 4, VL CDR2: SEQ ID NO: 5, VL CDR3: SEQ ID NO: 6
(2) Tfr002 antibody
VH CDR1: SEQ ID NO: 7, VH CDR2: SEQ ID NO: 8, VH CDR3: SEQ ID NO: 9
VL CDR1: SEQ ID NO: 10, VL CDR2: SEQ ID NO: 11, VL CDR3: SEQ ID NO: 12
(3) Tfr003 antibody
VH CDR1: SEQ ID NO: 13, VH CDR2: SEQ ID NO: 14, VH CDR3: SEQ ID NO: 15
VL CDR1: SEQ ID NO: 16, VL CDR2: SEQ ID NO: 17, VL CDR3: SEQ ID NO: 18
(4) Tfr004 antibody
VH CDR1: SEQ ID NO: 19, VH CDR2: SEQ ID NO: 20, VH CDR3: SEQ ID NO: 21
VL CDR1: SEQ ID NO: 22, VL CDR2: SEQ ID NO: 23, VL CDR3: SEQ ID NO: 24
(5) Tfr005 antibody
VH CDR1: SEQ ID NO: 25, VH CDR2: SEQ ID NO: 26, VH CDR3: SEQ ID NO: 27
VL CDR1: SEQ ID NO: 28, VL CDR2: SEQ ID NO: 29, VL CDR3: SEQ ID NO: 30

(6) Tfr006 antibody
VH CDR1: SEQ ID NO: 31, VH CDR2: SEQ ID NO: 32, VH CDR3: SEQ ID NO: 33
VL CDR1: SEQ ID NO: 34, VL CDR2: SEQ ID NO: 35, VL CDR3: SEQ ID NO: 36
(7) Tfr007 antibody
VH CDR1: SEQ ID NO: 37, VH CDR2: SEQ ID NO: 38, VH CDR3: SEQ ID NO: 39
VL CDR1: SEQ ID NO: 40, VL CDR2: SEQ ID NO: 41, VL CDR3: SEQ ID NO: 42
(8) Tfr008 antibody
VH CDR1: SEQ ID NO: 43, VH CDR2: SEQ ID NO: 44, VH CDR3: SEQ ID NO: 45
VL CDR1: SEQ ID NO: 46, VL CDR2: SEQ ID NO: 47, VL CDR3: SEQ ID NO: 48
(9) Tfr009 antibody
VH CDR1: SEQ ID NO: 49, VH CDR2: SEQ ID NO: 50, VH CDR3: SEQ ID NO: 51
VL CDR1: SEQ ID NO: 52, VL CDR2: SEQ ID NO: 53, VL CDR3: SEQ ID NO: 54
(10) Tfr010 antibody
VH CDR1: SEQ ID NO: 55, VH CDR2: SEQ ID NO: 56, VH CDR3: SEQ ID NO: 57
VL CDR1: SEQ ID NO: 58, VL CDR2: SEQ ID NO: 59, VL CDR3: SEQ ID NO: 60

(11) Tfr011 antibody
VH CDR1: SEQ ID NO: 61, VH CDR2: SEQ ID NO: 62, VH CDR3: SEQ ID NO: 63
VL CDR1: SEQ ID NO: 64, VL CDR2: SEQ ID NO: 65, VL CDR3: SEQ ID NO: 66
(12) Tfr012 antibody
VH CDR1: SEQ ID NO: 67, VH CDR2: SEQ ID NO: 68, VH CDR3: SEQ ID NO: 69
VL CDR1: SEQ ID NO: 70, VL CDR2: SEQ ID NO: 71, VL CDR3: SEQ ID NO: 72
(13) Tfr013 antibody
VH CDR1: SEQ ID NO: 73, VH CDR2: SEQ ID NO: 74, VH CDR3: SEQ ID NO: 75
VL CDR1: SEQ ID NO: 76, VL CDR2: SEQ ID NO: 77, VL CDR3: SEQ ID NO: 78
(9) Tfr014 antibody
VH CDR1: SEQ ID NO: 79, VH CDR2: SEQ ID NO: 80, VH CDR3: SEQ ID NO: 81
VL CDR1: SEQ ID NO: 82, VL CDR2: SEQ ID NO: 83, VL CDR3: SEQ ID NO: 84
(15) Tfr015 antibody
VH CDR1: SEQ ID NO: 85, VH CDR2: SEQ ID NO: 86, VH CDR3: SEQ ID NO: 87
VL CDR1: SEQ ID NO: 88, VL CDR2: SEQ ID NO: 89, VL CDR3: SEQ ID NO: 90

Example 3: Confirmation of TfR binding property of anti-TfR phage antibody (1) Immunoprecipitation and Western blotting 15 types of phages obtained in Example 2 were infected with E. coli to express antibody scFV, followed by column purification Thus, 15 types of antibodies were obtained. The antibody was solid-phased on the glass filter at a ratio of 1 ml of the gel after swelling with respect to 5 mg of the antibody amount of CNBr-activated Sepharose 4B. About 60 μl of the immobilized antibody (hereinafter referred to as antibody beads) (about 150 μl as a solution) was placed in a 2 ml tube, and 1/10 volume (about 15 μl) of 4 mM biotin was added thereto. To this, a mixture of 0.5 plate lysate (600 μl) and 60 μl of biotin solution was added and reacted for several hours with stirring at 4 ° C., and then the tube was centrifuged (5500 g, 1 min, 4 ° C. And the supernatant was removed. Add 800 μl of biotin / lysis-T buffer for washing (0.5 mM biotin, 0.1% Tween20 / PBS), mix by inversion a few times, and centrifuge the tube (5500 g, 1 min, 4 ° C.) The supernatant was removed. After performing this washing operation again, add 30 μl of elution citric acid solution (50 mM citric acid pH 2.5) to the antibody beads, and after stirring, centrifuge the tube (5500 g, 1 min, 4 ° C.) and collect the supernatant. did. 30 μl of elution citric acid solution was again added to the remaining antibody beads, the mixture was stirred, the tube was centrifuged (5500 g, 1 min, 4 ° C.), and the supernatant was collected. This elution operation was repeated three more times to collect a sample solution, which was neutralized by adding 3M Tris. This sample was run on SDS-PAGE, and the band was confirmed by silver staining. This sample was simultaneously subjected to Western blotting using streptavidin-HRP (Anti-Streptavidin, IgG Fraction, Conjugated to Peroxidase CORTEX biochem) to detect biotinylated membrane protein bands.

  The results of immunoprecipitation and Western blot are shown in FIG. FIG. 1 is a band pattern obtained by separating a sample after immunoprecipitation by SDSPAGE. The band surrounded by the square frame was digested in the gel and measured with a mass spectrometer.

(2) Identification of antigenic proteins obtained by immunoprecipitation by mass spectrometry (in-gel trypsin digestion)
The portion corresponding to the detected membrane protein was digested with trypsin in the gel, and the peptide was recovered. SDS polyacrylamide gel electrophoresis was performed according to a conventional method, and a band obtained by staining with Coomassie brilliant blue was cut out. This was immersed in a 200 mM ammonium bicarbonate-50% acetonitrile solution, shaken at 37 ° C. for 45 minutes, discarded, and the same operation was repeated twice to remove Coomassie brilliant blue. The gel was dried under reduced pressure, and 4 μl of trypsin (20 μg / ml) dissolved in 40 mM ammonium bicarbonate (pH 8.1) -10% acetonitrile was added per unit area (mm 2) of the gel slice and left at room temperature for 1 hour. Fully infiltrated. To this, 2.5 times the amount of trypsin solution added earlier was added and allowed to stand at 37 ° C. for 18 hours. This was filtered through a tube with a filter having a pore size of 0.22 μm, and the peptide produced by cleaving the antigen with trypsin was recovered.

(Identification of antigen by mass spectrometry)
Samples obtained by in-gel trypsin digestion were subjected to HPLC connected to an electrospray ionization ion trap quadrupole mass spectrometer. From the HPLC reversed-phase chromatography column, each peptide sequentially eluted by the difference in hydrophobicity is ionized by electrospray method by changing the linear concentration gradient of acetonitrile from 0% to 80% containing 0.1% TFA. Mass and internal amino acid sequence were determined. The set of amino acid internal sequences obtained was searched using the published TfR amino acid sequence database. As a result, it was confirmed that 13 phages obtained by Examples bind to TfR.

Example 4: Evaluation of TfR high-expressing cell line binding (FACS)
The reactivity of each isolated antibody clone against various cell lines was confirmed by FCM as follows. The experimental procedure was as follows. First, for adherent cell lines, in 6-well plates (Falcon 3516), for floating cell lines such as ATL-derived cell lines, in suspension culture flasks (70 ml (slant neck)), in medium (RPMI-1640: Sigma-Aldrich) , 10% fetal bovine serum, 1% penicillin-streptomycin solution) and cultured at 37 ° C. in a CO ₂ incubator.

The adherent cell line was dissociated from the culture dish with 2 mg / ml collagenase I (Gibco BRL) / cell dissociation buffer (Gibco BRL), and then recovered with 10% FBS / D MEM. On the other hand, in the case of suspension cells, the medium was once centrifuged to remove the medium (400 × g, 4 ° C., 2 minutes). After such operations, each cell was washed with 2.5% BSA, 0.05% NaN ₃ / PBS (BSA solution), suspended in 100 μl of 2.5% normal goat serum / BSA solution and allowed to stand on ice for 30 minutes. The solution was dispensed at 10 ⁶ cells / well and centrifuged (400 × g, 4 ° C., 2 minutes) to remove the supernatant.

  The antibody was added to 5 μg / ml and left on ice for 1 hour. This was washed once with BSA solution, suspended in 100 μl of 5 μg / ml BSA solution of anti-cp3 mouse monoclonal antibody (Medical and Biological Laboratories), and left on ice for 1 hour. This was washed once with BSA solution, suspended in Alexa488-conjugated anti-mouse IgG goat antibody (Molecularprobe) 5 μg / ml BSA solution 100 μl, and allowed to stand on ice for 1 hour. This was washed twice with BSA solution, suspended in 500 μl of BSA solution, added with 50 μl of fixative (formaldehyde), and allowed to stand for 10 minutes. Thereafter, 150 μl of PBS was added, treated with a cell strainer (Becton Dickinson), and then the fluorescence intensity of the cell population was analyzed with a Becton Dickinson FACScaliver (FCM). As a result, the 15 types of antibodies showed significant peak shifts in all the cancer cell lines (A431, PANC1, KATOIII, MIAPaCa2) that expressed high TfR for which the binding was evaluated.

Example 5: Immunostaining using a clinical specimen surgically excised from a cancer patient (1) Section preparation The excised tissue is about 5 mm x 5 mm x 10 mm, 4% PFA / 0.01% at 4 ° C Put in glutaraldehyde / 0.1M cacodylate buffer (PFA is Wako Pure Chemical, glutaraldehyde is Kanto Chemical, sodium cacodylate is SIGMA), microwave fix using microwave oven (SHARP), And fixed again at 4 ° C. for 1 hour. Then, transfer to 10% sucrose / PBS, soak at 4 ° C for 4 hours, replace with 15% sucrose / PBS, soak at 4 ° C for 4 hours, and then replace with 20% / sucrose / PBS at 4 ° C. It was immersed overnight, embedded in OTC compound, and rapidly frozen in dry ice / hexane. This was sliced to a thickness of 4 μm with a cryostat (Reichert-Jung 2800 FRIGCUT E), attached to a silane-coated slide glass (MATSUNAMI), and air-dried with a cold air dryer for 30 minutes.

(2) Staining The slide glass on which the section was affixed was hydrophilized by being immersed in PBS three times for 5 minutes each. Next, 50 μl of 0.3% H ₂ O ₂ /0.1% NaN ₃ was added dropwise and reacted at room temperature for 10 minutes to block endogenous peroxidase, and then washed 3 times for 5 minutes each with PBS. Then, the mixture was reacted in 2% BSA / PBS at room temperature for 10 minutes to block nonspecific reaction. Thereafter, 50 μl of a phage antibody sample was dropped into the place where excess liquid was dropped, and the mixture was reacted at room temperature for 1 hour, and then washed 3 times for 5 minutes each with PBS. Next, 50 μl of anti-CP3 rabbit antibody 5 μg / ml was dropped, followed by a secondary antibody reaction at room temperature for 45 minutes, followed by washing with PBS three times for 5 minutes each. Then, 50 μl of peroxidase-labeled dextran-conjugated anti-rabbit immunoglobulin / goat polyclonal antibody (DAKO) was added dropwise, and a tertiary antibody reaction was performed at room temperature for 30 minutes. This was washed with PBS three times for 5 minutes, and then 50 μl of DAB / H ₂ O ₂ coloring solution was added dropwise. After the color was changed to brown, the reaction was stopped by transferring to a vat filled with distilled water. After washing with water for 10 minutes, after nuclear staining with hematoxylin, it was dehydrated and transparent, sealed with marinol, and microscopically examined. An example of the result of immunostaining is shown in FIG.

Example 6: Conversion to IgG type antibody (construction of IgG type antibody gene)
In order to investigate the effectiveness as an antibody drug, some antibodies were converted to IgG type.
First, using the VH and VL genes of scFVcp3-type antibody, it was confirmed that there were no restriction enzyme sites necessary for cloning into the Fc region of IgG1 and the nucleotide sequence of the gene. PCR was performed using the antibody gene as a template and primers for amplifying H and L chains. The amplified product was ligated downstream of the CM1 promoter of the IgG1 construction vector to obtain a plasmid DNA containing an IgG type antibody gene.

(Expression of IgG type antibody)
GenePORTER Reagent (Gene Therapy Systems: T201007) was used for transfection of plasmid DNA into CHO-K1 cells. First, prepare CHO-K1 cells at 2 × 10 ⁴ cells / ml in a 60 mm culture dish from the day before transfection (the medium is α-MEM (Invitrogen: 12561-056) in 10% FCS). (Exitec Inc .: 268-1) used).
8 μg of plasmid DNA was dissolved in 250 μL of a serum-free medium (Serum Free Medium, hereinafter abbreviated as SFM— (Invtrogen: 12052-098 CHO-S-SFMII)) and applied to a 0.22 μm filter. GenePORTER Reagent 40 μL was added to 250 μL SFM.

Plasmid DNA dissolved in SFM and GenePORTER Reagent were quickly mixed and allowed to stand at room temperature for 30 min.
The cells were washed twice with 2 ml of SFM, and plasmid DNA-GenePORTER mixture (Transfection Medium) was slowly added to the plate containing the cells, and cultured in an incubator at 37 ° C. for 5 hours.
The transfection medium was aspirated, washed twice with αMEM 10% FCS, 5 ml of αMEM 10% FCS was added, and the cells were cultured in an incubator at 37 ° C. for 48 hours.

  The medium was replaced with 10 mL of αMEM 10% FCS + 700 μg / ml G418 (Sigma: G7034), and selection was started (the following medium used αMEM 10% FCS + 700 μg / mL G418). After culturing at 37 ° C. for 48 hours, the cells were washed with 10 mL of PBS, treated with 750 μL of 0.25% Trypsin-EDTA (Sigma T4049), added with 5 mL of αMEM, detached and collected from the plate, and the number of cells was measured. Based on the results, limiting dilution was performed under the conditions of 10 cells / 200 μL / well 96 well 2 plates. After culturing for 14 days, ELISA was performed using the culture supernatant of each well to confirm the expression of IgG type antibody.

(Purification of expressed protein (IgG) from culture supernatant)
1 mL of Protein G Sepharose 4 Fast Flow (amersham pharmacia biotech: 17-0618-01) was packed in a column and equilibrated with 5 mL of PBS. The culture supernatant was applied and fed at a flow rate of 1 drop / 2 seconds to bind the expressed protein (IgG) to the column. Deliver 10 mL of PBS at a flow rate of 1 drop / 2 seconds, wash away non-adsorbed components, and then feed 6 mL of elution buffer (0.2 M glycine-HCl, pH 3) at a flow rate of 1 drop / second, and 1 mL of the eluate. Collected in 1.5 ml tubes. 400 μL of neutralization buffer (3M Tris-HCl) was added to the collection tube in advance, and neutralization was performed simultaneously with the collection. The eluates were combined and concentrated to 750 μL, solution replacement (PBS, complete, 0.01% NaN ₃ ) was performed, and the antibody protein concentration was calculated by SDS-PAGE.

Example 7: In vivo drug efficacy evaluation (1) Anti-tumor effect of anti-TfR antibody in ATL model ATL cell line MT-2 cells were cultured in RPMI1640 culture medium supplemented with 10% FBS. Cells were collected by centrifugation at the time of transplantation, and the cells were suspended in RPMI 1640 at 5 × 10 ⁷ / ml. 100 μl of the above cell suspension was transplanted subcutaneously into the right flank of NOG / Jic mice (female, 7 weeks old, Central Laboratory for Animal Experiments). After transplantation, the tumor diameter was measured with a caliper twice a week, and when the average tumor volume reached about 120 mm 3, the mice were divided into two groups (n = 5) by randomization to the tumor volume. The TfR006 antibody diluted with PBS was administered to the antibody administration group from the tail vein so that the mouse was 15 mg / kg. To the negative control group, 0.2 mL PBS was administered from the tail vein. Administration was performed twice a week (every 3 days or 4 days) for a total of 6 times. Similarly, after administration, the tumor diameter was measured twice a week with a caliper to determine the tumor volume of each group. The antitumor effect was determined by a parametric Dunnet multiple comparison test using the PBS group as a control for the tumor volume on the last measurement day.
Tumor volume was calculated by the following formula.
Tumor volume = (minor axis) ² x major axis x 0.5
The time course of the mean value of the tumor volume is shown in FIG. As shown in the figure, the TfR006 antibody showed a strong tumor reduction effect in the ATL model.

(2) Anti-tumor effect of anti-TfR antibody in pancreatic cancer model Pancreatic cancer-derived cell line PK-45 was cultured in PRPMI1640 + 10% FBS. At the time of transplantation, the cells were detached from the plate with 0.25% trypsin, washed once with PBS, and then suspended in RPMI1640. SCID mice (female, 7 weeks old, CLEA Japan) were transplanted subcutaneously into the right ventral region at 5 × 10 ⁶ cells / mouse at a cell suspension of 200 μl / mouse. After transplantation, the tumor diameter was measured every day with calipers, and the volume was determined by the following formula. When the average tumor volume reached 150 mm 3 or more, the mice were divided into two groups (n = 5) using a grouping software (EXSAS version 7.6 CB). The TfR006 antibody diluted with PBS was administered to the antibody administration group from the tail vein so that the mouse was 15 mg / kg. To the negative control group, 0.2 mL PBS was administered from the tail vein. Administration was performed twice a week (every 3 days or 4 days) for a total of 6 times. Tumor diameter was measured with a caliper twice a week after administration, and the tumor volume of each group was determined. The antitumor effect was determined by a parametric Dunnet multiple comparison test using the PBS group as a control for the tumor volume on the last measurement day. Tumor volume was calculated by the following formula.
Tumor volume = (minor axis) ² x major axis x 0.5
The change over time of the average value of the tumor volume is shown in FIG. As shown in the figure, in the pancreatic cancer xenograft model, suppression of tumor growth was observed in the antibody administration group. From these results, the TfR006 antibody suggested a strong inhibitory effect on the proliferation of TfR positive expressing cancer cells.

Claims (9)

SEQ ID NO: 31 heavy chain first complementarity determining region (VH CDR1), SEQ ID NO: 32 heavy chain second complementarity determining region (VH CDR2), SEQ ID NO: 33 heavy chain third complementarity determining region (VH CDR3) A light chain first complementarity determining region (VL CDR1) of SEQ ID NO: 34, a light chain second complementarity determining region (VL CDR2) of SEQ ID NO: 35, and a light chain of SEQ ID NO: 36. An antibody having a light chain variable region having a third chain complementarity determining region (VL CDR3) , binding to cancer cell NCI-H441, and specifically binding to TfR. The antibody according to claim 1, which is a human antibody or a humanized antibody. A pharmaceutical composition comprising the antibody according to claim 1 or 2 . The pharmaceutical composition according to claim 3 , wherein a cytotoxic substance is bound to the antibody. The pharmaceutical composition according to claim 4 , wherein the cytotoxic substance is a drug, a toxin, or a radioactive substance. The pharmaceutical composition according to any one of claims 3 to 5 , which is used as an anticancer agent. The pharmaceutical composition according to claim 6 , wherein the cancer is blood cancer or solid cancer. The pharmaceutical composition according to claim 7, wherein the blood cancer is adult T-cell leukemia (ATL). Solid cancer is lung cancer, colon cancer, stomach cancer, bladder cancer, pancreatic cancer, prostate cancer, liver cancer, cervical cancer, uterine cancer, ovarian cancer, breast cancer, head and neck cancer, skin The pharmaceutical composition according to claim 7, wherein