WO2013031619A1 - Image-based tumor diagnosis agent containing anti-human transferrin receptor antibody - Google Patents

Abstract

The present invention relates to a high-precision image-based tumor diagnosis agent which contains an antibody capable of specifically recognizing a transferrin receptor (TfR) on a cell membrane. Provided is the image-based tumor diagnosis agent which contains the antibody in which an antibody that recognizes a human transferring receptor is marked by a radionuclide.

Description

Imaging diagnostic agent containing anti-human transferrin receptor antibody

The present invention relates to a diagnostic agent for tumors for imaging, comprising an antibody that recognizes human transferrin receptor (TfR).

Cancer is an important disease that accounts for the top causes of death. Surgical excision is the most effective treatment for cancer, and in particular, if the primary lesion is excised before infiltrating surrounding organs and tissues, the possibility of healing is high. It is important to detect it early, but most cancers are difficult to treat because early symptoms are extremely difficult because they do not show symptoms early, and they have already infiltrated surrounding organs and tissues at the time of diagnosis. There are not many cases. For example, pancreatic cancer, which is known to have a poor prognosis, has one of the most probable causes of early prognosis, and in many cases, it has already infiltrated outside the pancreas and metastasized to the liver when diagnosed. That is. In a nationwide survey in Japan, CT is the most common method of finding pancreatic cancer at 44%, and ultrasound is 41%. Therefore, these two examination methods are currently important for the diagnosis of pancreatic cancer. However, in the case of CT generally used at present, it is difficult to distinguish between cancerous tissue and non-cancerous tissue, and diagnosis by a skilled image diagnostician is required. Tumor markers for pancreatic cancer include CA19-9, DUPAN-2, Span-1, and CEA. Although these markers are all positive in advanced cancer, the early diagnosis rate is low, and there is no marker that can be said to be useful for early diagnosis. Further, since CA19-9 increases in blood concentration even in non-malignant tumors such as hepatitis, cirrhosis, and pancreatitis, it is known that CA19-9 is not suitable for use in the diagnosis of pancreatic cancer. Although early diagnosis of pancreatic cancer is very important, there is currently no method for diagnosing pancreatic cancer before it invades surrounding organs.

Currently, CT and MRI are mainly used in tumor image diagnosis. However, benign / malignant differentiation, diagnosis of recurrence after surgery, differentiation from other lesions, etc. may be difficult even with a contrast agent using CT or MRI. Recently, as diagnostic imaging for assisting CT and MRI, Positron Emission Tomography (PET) diagnosis using 18F-2-fluoro-2-deoxyglucose (18F-FDG) has been used. However, 18F-FDG often accumulates in normal tissues (such as the brain) with active glucose metabolism, active inflammatory tissues, and granulation tissues because of its diagnostic mechanism, and therefore often suffers from diagnosis. Therefore, development of a highly specific and highly accurate diagnostic method that specifically recognizes only cancer tissue and does not recognize normal tissue is desired.

In recent years, cancer diagnosis methods and treatment methods targeting proteins specifically expressed in cancer cells have been actively developed. That is, by using a cell surface protein that is highly expressed in cancer cells but less or not expressed in normal tissues, and using an antibody that specifically recognizes this cell surface protein, This is a method of performing treatment (Patent Document 1).

Transferrin receptor (TfR) was first identified on reticulocytes as a cell membrane structure for taking up iron bound to transferrin (Tf) into cells (Non-patent Document 1). Since then, it has been expressed in various tumor cells (Non-Patent Documents 2 to 5), placental trophoblast cells (Non-Patent Document 6 and Non-Patent Document 7), activated lymphocytes (Non-Patent Document 8), and the like. It has been reported that

JP 2008-290996 A

An object of the present invention is to provide a high-accuracy tumor diagnostic agent for imaging, which includes an antibody capable of specifically recognizing transferrin receptor (TfR) expressed on a cell membrane.

The present inventors obtained scFv that reacts with TfR using phage® Display, and analyzed the amino acid sequence. These CDRs were found to have a novel amino acid sequence and converted to IgG. Furthermore, IgG antibody was labeled with a radionuclide, and specific accumulation in TfR-positive tumors was confirmed in tumor-bearing mice. As described above, the usefulness of the imaging tumor diagnostic agent using the antibody in use in a human living body is shown, and the present invention has been completed.

That is, according to the present invention, there is provided an imaging tumor diagnostic agent comprising an antibody obtained by labeling an antibody recognizing human transferrin receptor with a radionuclide.
Furthermore, according to the present invention, there is provided a diagnostic imaging agent kit for imaging, comprising an antibody that recognizes a human transferrin receptor and a radionuclide.

Preferably, CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of the antibody include the amino acid sequences represented by SEQ ID NOs: 1, 2, and 3, respectively, and CDR1, CDR2, and CDR1, CDR2 of the light chain variable region (VL) of the antibody, CDR3 includes the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively.
Preferably, CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of the antibody include the amino acid sequences represented by SEQ ID NOs: 7, 8, and 9, respectively, and CDR1, CDR2, and CDR2 of the antibody light chain variable region (VL), CDR3 comprises the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, respectively.
Preferably, CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of the antibody include the amino acid sequences represented by SEQ ID NOs: 13, 14, and 15, respectively, and CDR1, CDR2, and CDR2 of the light chain variable region (VL) of the antibody, CDR3 comprises the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, respectively.

Preferably, the antibody is an antibody comprising the constant region of a human antibody.
Preferably, the constant region of the human antibody consists of a constant region of the human antibody IgG1 class.
Preferably, the antibody is from a peptide comprising Fab, Fab ′, F (ab ′) ₂ , single chain antibody (scFv), dimerized V region (Diabody), disulfide stabilized V region (dsFv) and CDR. An antibody fragment selected from the group consisting of
Preferably, the radionuclide is ⁸⁹ Zr, ^99m Tc, ¹¹¹ In, ^{113 m} In, ⁶⁷ Ga, ⁶⁸ Ga, ²⁰¹ Tl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁶⁰ Co, ⁸⁵ Sr, ¹⁹⁷ Hg, ⁶⁴ Cu, ¹²³ Selected from the group consisting of I, ¹²⁵ I, and ¹³¹ I.

According to the present invention, an imaging tumor diagnostic agent using an antibody that recognizes human TfR is provided. The diagnostic agent for imaging tumors of the present invention specifically binds to human TfR and accumulates well in tumors where TfR is highly expressed. A tumor can be imaged from outside the body by using the imaging diagnostic agent for images of the present invention, and a lesion, infiltration and metastasis can be examined. That is, the tumor diagnostic agent for images of the present invention is useful for early detection and diagnosis of diseases.

FIG. 1 shows that ⁸⁹ Zr-DF anti-TfR antibody was administered to nude mice transplanted subcutaneously with tumors that highly expressed human TfR and tumors that did not express human TfR. The results of PET imaging after 6 days are shown.

Hereinafter, the present invention will be described in more detail.
The antibody used in the present invention is an antibody that specifically recognizes a transferrin receptor (TfR) on the cell surface. 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 the CD71 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.

As the antibody used in the present invention, human antibodies, human chimeric antibodies, human CDR-grafted antibodies, non-human animal antibodies, and the like can be used. Preferably, it is a human antibody.

Human chimeric antibodies are composed of non-human animal antibody H chain V regions (hereinafter also referred to as VH) and antibody L chain V regions (hereinafter also referred to as VL) and human antibody H chain C regions (hereinafter referred to as CH). And an L chain C region of a human antibody (hereinafter also referred to as CL). Any animal other than humans can be used as long as it can produce hybridomas such as mice, rats, hamsters, rabbits and the like.

A human CDR-grafted antibody is a cDNA encoding a V region in which the VH and VL CDR amino acid sequences of non-human animal antibodies that specifically react with human TfR are grafted onto the VH and VL FRs of any human antibody. Are constructed, inserted into animal cell expression vectors having DNA encoding human antibody CH and CL, respectively, to construct human CDR-grafted antibody expression vectors, which are introduced into animal cells to be expressed and produced. Can do.

Human antibody means an antibody acquired by various acquisition methods. For example, human lymphocytes are sensitized in vitro with a desired antigen or cells expressing the desired antigen, and the sensitized lymphocytes are fused with human myeloma cells, such as U266, to form a desired human antibody having binding activity to the antigen. (See Japanese Patent Publication No. 1-59878). Further, a desired human antibody can be obtained by immunizing a transgenic animal having all repertoires of human antibody genes with a desired antigen (WO93 / 12227, WO92 / 03918, WO94 / 02602, WO94 / 25585). WO96 / 34096, WO96 / 33735). Preferably, it is a human antibody obtained by the phage display method.

Acquisition of antibodies by phage display will be described in detail.
1. Construction of phage display library Human B cell cDNA can be obtained from patients or healthy individuals using bone marrow, lymph nodes or peripheral blood. Antibody gene fragments are amplified by PCR (Polymerase Chain Reaction) using primers of the variable regions of the antibody heavy chain and light chain antibody genes, respectively. These gene fragments are artificially linked and expressed as a fusion protein with the outer shell protein g3p of filamentous bacterial phage M13.

A human single chain antibody gene library is prepared by integrating a heavy chain fragment-linker sequence-light chain fragment or light chain fragment-linker sequence-heavy chain fragment, which is a human scFv fragment, into a phagemid vector or a phage vector. can do.

Introduction of this phagemid vector incorporating the human single-chain antibody fragment into E. coli was performed by Davis et al. (BASIC METHODS IN MOLECULAR BIOLOGY, 1986) and Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed. Press, Cold Spring Harbor, NY, 1989) and other methods described in many standard laboratory manuals such as electroporation, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micro It can be performed by injection, cationic lipid-mediated transfection, transduction, scrape loading, ballistic introduction, infection, and the like.

2. Biopanning Biopanning is an operation for selecting a phage for a target protein of interest from an antibody phage library. Various panning methods are known. The target protein is immobilized, reacted with an antibody phage library, and unbound phages are removed by washing. Thereafter, the phage specific to the target protein may be concentrated by performing several operations of eluting the bound phage, infecting E. coli and allowing it to proliferate. The target protein can be immobilized by directly adsorbing the target protein on a plastic surface such as an immunotube, or by biotinylating the target protein and immobilizing it via immobilized streptavidin. Alternatively, the target protein can be immobilized on beads coated with streptavidin. Other than that, when panning a molecule on the cell surface, there is a cell panning in which the antibody phage library is directly reacted with the cell. Biopanning is performed in this way, and phages that react with the target protein are selected.

3. Analysis of phage antibodies By analyzing the genes of the phages that react with the selected target protein, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. If the DNA sequence of scFv that binds to the antigen is clarified, an appropriate expression vector can be prepared from the sequence to obtain a human antibody. These methods are already well known, and WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, and WO95 / 15388 can be referred to.

The antibody used in the present invention can be obtained by introducing an antibody gene into an appropriate host cell system and expressing the protein. Many host cell systems used for protein expression are of mammalian origin in antibody production systems. The producer can preferentially determine the particular host cell system that is most suitable for the gene product he wishes to express. Common host cell lines include CHO-derived cell lines (Chinese hamster ovary cell line), CV1 (monkey kidney line), COS (derivative of CV1 that carries SV40T antigen), SP2 / 0 (mouse myeloma), P3x63-Ag3 .653 (mouse myeloma), and 293 (human kidney), 293T (a derivative of 293 that carries the SV40T antigen), but is not limited thereto. Host cell lines can be obtained from commercial facilities, the American Tissue Culture Collection (ATCC), or from published publication agencies.

Preferably, the host cell line is either a CHO-derived cell line that is dgfr gene expression deficient or SP2 / 0. Orlando, G.M. , Et al., Effect of gamma rays at the dihydroformat reduce locus: deletions and inversions, Soma. Cell. Mol. Genet. Vol. 12, 1986, p5555-566, and Schulman, M .; Et al., A letter cell line for making hybridomas secreting specific antigens, Nature Vol. 276, 1978, p269-270. Most preferably, the host cell line is DHFR deficient CHO. Transfection of the plasmid into the host cell can be accomplished using any technique. Specific methods include transfection (including calcium phosphate method, DEAE method, lipofection, and electroporation), a method of introducing DNA using an envelope such as Sendai virus, microinjection, retrovirus virus, adeno Examples include, but are not limited to, infection using virus vectors such as viruses. Current Protocols in Molecular Biology, Chapter 9 Introduction of DNA into Mammalian Cells, John Wiley and Sons, Inc. checking ... Most preferred is the introduction of the plasmid into the host by electroporation.

(Antibody fragment)
The antibody used in the present invention may be an antibody fragment. Examples of antibody fragments include Fab, Fab ′, F (ab ′) ₂ , scFv, dsFv, and peptides containing CDRs.
Fab is a fragment obtained by treating IgG with the proteolytic enzyme papain (cleaved at the 224th amino acid residue of the H chain), and about half of the N chain side of the H chain and the entire L chain are disulfide bonds. It is an antibody fragment having an antigen binding activity with a molecular weight of about 50,000.

The Fab of the present invention can be obtained by treating an antibody that specifically reacts with TfR with the proteolytic enzyme papain. Alternatively, the Fab can be produced by inserting a DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the antibody. it can.

F (ab ′) ₂ is a fragment obtained by treating IgG with proteolytic enzyme pepsin (cleaved at the 234th amino acid residue of the H chain), and Fab is bound via a disulfide bond in the hinge region. It is an antibody fragment having an antigen-binding activity having a molecular weight of about 100,000, which is slightly larger than those obtained.

Fab ′ is an antibody fragment having an antigen-binding activity having a molecular weight of about 50,000, which is obtained by cleaving the disulfide bond in the hinge region of F (ab ′) ₂ .
The Fab ′ of the present invention can be obtained by treating F (ab ′) ₂ that specifically reacts with TfR with a reducing agent dithiothreitol. Alternatively, the DNA encoding the Fab ′ fragment of the antibody is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and Fab ′ is expressed by introducing the vector into a prokaryotic or eukaryotic organism. can do.

ScFv represents a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (hereinafter referred to as P). As VH and VL contained in the scFv of the present invention, an antibody that specifically reacts with the TfR of the present invention, for example, a humanized antibody or a human antibody can be used.

The scFv of the present invention obtains cDNAs encoding VH and VL of an antibody that specifically reacts with TfR, constructs DNA encoding scFv, and uses the DNA as a prokaryotic expression vector or eukaryotic expression vector. ScFv can be produced by inserting the expression vector into a prokaryotic organism or a eukaryotic organism and expressing the vector.

dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue, which are bonded via a disulfide bond between the cysteine residues. The amino acid residue substituted for the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to the method shown by Reiter et al. (Protein Engineering, 7 , 697 (1994)). As VH and VL contained in the dsFv of the present invention, antibodies that specifically react with the TfR of the present invention, for example, humanized antibodies or human antibodies can be used.

The dsFv of the present invention obtains cDNAs encoding VH and VL of an antibody that specifically reacts with TfR, constructs a DNA encoding dsFv, and uses the DNA as a prokaryotic expression vector or eukaryotic expression vector. DsFv can be produced by inserting the expression vector into a prokaryote or a eukaryote and expressing the vector.

The peptide containing CDR is configured to include at least one region of H chain or L chain CDR. Multiple CDRs can be linked directly or via a suitable peptide linker.

The CDR-containing peptide of the present invention is obtained by obtaining cDNAs encoding VH and VL of an antibody that specifically reacts with CDH3, and then constructing a DNA encoding CDR, and using the DNA as a prokaryotic expression vector or eukaryotic A peptide containing CDR can be produced by inserting the expression vector into a biological expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism.

Further, a peptide containing CDR can also be produced by a chemical synthesis method such as Fmoc method (fluorenylmethyloxycarbonyl method), tBoc method (t-butyloxycarbonyl method), or the like.

(Reactivity to TfR expressing cells)
In order to examine the reactivity to TfR-expressing cells, TfR expressed on the cell surface can be assayed using a flow cytometer.
The antibody used in the flow cytometer may be a fluorescent substance such as FITC, an antibody labeled with biotin, or an unlabeled antibody. A fluorescently labeled avidin, a fluorescently labeled anti-human immunoglobulin antibody, or the like is used depending on whether or not the antibody used is labeled. Reactivity is evaluated by adding a sufficient amount of anti-TfR antibody (usually a final concentration of 0.1 to 10 μg / ml) to the sample and comparing the reactivity with the negative control antibody and the positive control antibody. Can do.

The tumor diagnostic agent for imaging of the present invention is composed of an antibody that specifically recognizes TfR on the cell surface and a radionuclide.

Radionuclides include zirconium 89 ( ⁸⁹ Zr), technetium 99m ( ^99m Tc), indium 111 ( ¹¹¹ In), indium 113m ( ^113m In), gallium 67 ( ⁶⁷ Ga), gallium 68 ( ⁶⁸ Ga), thallium 201 ( ²⁰¹ Tl), chromium 51 ( ⁵¹ Cr), cobalt 57 ( ⁵⁷ Co), cobalt 58 ( ⁵⁸ Co), cobalt 60 ( ⁶⁰ Co), strontium 85 ( ⁸⁵ Sr), mercury 197 ( ¹⁹⁷ Hg), copper 64 ( ⁶⁴ Cu), iodine 123 ( ¹²³ I), iodine 125 ( ¹²⁵ I), and iodine 131 ( ¹³¹ I) are preferably used, but are not limited thereto. Among these, ⁸⁹ Zr and ¹¹¹ In are preferable.

In one form of the present invention, the diagnostic agent for imaging tumor of the present invention comprises an antibody that specifically recognizes TfR, a radionuclide, and a linker (metal chelating reagent). As another form of the present invention, ¹²³ I, ¹²⁵ I, ¹³¹ I and the like may be bound to an antibody that specifically recognizes TfR without using a metal chelating reagent.

In order to bind the radionuclide described above to the anti-TfR antibody, it is preferable to react the metal chelate reagent with the antibody and react with the radiometal element to form a complex. In the modified antibody thus obtained, the radionuclide is bound to the anti-TfR antibody 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 Tetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) and dihydroxyethylglycine, diaminopropanoltetraacetic acid, ethylenediaminediacetic acid, ethylenediaminedipropionate hydrochloride, hydroxyethylethylenediaminetriacetic acid, ethylenediaminetetrakis (methylene) Phosphonic 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, isothiocyano NDPA, 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-te Examples include traazacyclotetradecane (Cyclam), isothiocyanobenzyl DOTA, isothiocyanobenzyl NOTA, and dethioxamine-based compounds such as isothiocyanobenzyl deferoxamine.

Among these metal chelating reagents, isothiocyanobenzyl DOTA, methylisothiocyanobenzyl DTPA, cyclohexylisothiocyanobenzyl DTPA, and isothiocyanobenzyl deferoxamine are metal chelate-introducing reactions, labeling rates, It is preferable in terms of stability and the like. (The appropriate chelate varies depending on the nuclide to be labeled)

The binding of the radiometal nuclide to the anti-TfR antibody can be performed according to a conventional method. For example, it can be carried out by reacting an anti-TfR antibody with a metal chelate reagent, preparing a labeling precursor in advance, and then reacting with a radioactive metal nuclide.

In the tumor diagnostic agent for imaging of the present invention, the molar ratio of the anti-TfR antibody to the metal chelating reagent is important for cancer cell accumulation, and the molar ratio (antibody: chelating reagent) is 1: 0.1 to 1: 4.5 is preferable, and 1: 0.5 to 1: 3 is more preferable. In order to achieve such a molar ratio, it is preferable to react the antibody and the chelating reagent in a molar ratio of 1: 0.1 to less than 1: 5, particularly 1: 1 to 1: 3. The number of chelate modifications of an antibody can be calculated by measuring the molecular weight using MALDI-TOF mass spectrometry or the like, and comparing the molecular weight of an unmodified antibody with a modified antibody (US Pat. No. 7514078, Lu et al. J Pharm Sci. 94 (4), 2005, p788-797, Tedesco et al., J Clin Onco. 23 (16S), 2005, 4765). The number of chelate modifications of the antibody can also be measured by chelate titration method. A method using an alkaline earth metal colorimetric reagent (Arsenazo III) is known (Bradyr et al., Nucl Med Biol. 31, 795-802, 2004, Dadachova et al., Nucl Med Biol. 26, 977-982, 1999. ).

The imaging diagnostic agent for an image of the present invention includes a method of providing as a pre-labeled preparation and a method of providing as a kit preparation containing a labeling precursor, and any method may be used in the present invention. When provided as a pre-labeled preparation, an imaging tumor diagnostic agent containing a labeled anti-TfR antibody can be used for administration as it is. When provided as a kit preparation, it can be used for administration after labeling with a desired radionuclide.

The antibody of the present invention labeled with a radionuclide may be used as a tumor diagnostic agent by mixing, dissolving, emulsifying or the like together with a pharmaceutically acceptable carrier. For example, an antibody labeled with a radionuclide is formulated into a dosage form such as an injectable solution, suspension, emulsion, etc. together with a pharmaceutically acceptable solvent, excipient, binder, stabilizer, dispersant and the like. Can be In an injectable formulation, the monoclonal antibody of the invention labeled with a radionuclide is in an aqueous solution, preferably a physiologically compatible buffer such as Hanks's solution, Ringer's solution, or physiological saline buffer. Can be dissolved. In addition, the composition can take the form of a suspension, solution, emulsion or the like in an oily or aqueous vehicle.

The route of administration of the imaging diagnostic agent for imaging is not particularly limited, but is usually parenteral, for example, administered by injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.), transdermal, transmucosal, etc. be able to.

The dose and frequency of administration vary depending on the patient's age, weight, purpose of diagnosis, etc., but in general, the monoclonal antibody of the present invention is in the range of about 0.1 mg to 1000 mg, preferably about 0, per kg of body weight per dose. It can be administered in the range of 1 mg to 100 mg.

The tumor is a malignant tumor with high TfR expression. Examples include colon cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, ovarian cancer, cervical cancer, bladder cancer, prostate cancer, and adult leukemia.

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these 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). The dissociated cells were washed with chilled PBS and collected by centrifugation. Harvested cells 4 × 10 ⁷ were used. This was mixed with a human antibody phage library of 1 × 10 ¹³ cfu (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, then 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 were performed using it. 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, the following 1863 phage antibodies having independent sequences were established by screening (1) to (3) above for the 21 types of cancer cells shown in Table 1 below.

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 1 by ELISA, antibodies having the following three independent sequences are specific for human TfR. It was confirmed that it showed mechanical reactivity. The gene sequence of the phage antibody was read, and the CDR amino acid sequences of antibody PPAT-061-01, antibody PPAT-061-02, and antibody PPAT-061-03 were obtained from the gene sequence.

Antibody PPAT-061-01:
VH:
CDR1: SYGMH (SEQ ID NO: 1)
CDR2: VISFDGSSKYYADSVKG (SEQ ID NO: 2)
CDR3: DSNFWSGYYSPVDV (SEQ ID NO: 3)
VL:
CDR1: TRSSGSIASNSVQ (SEQ ID NO: 4)
CDR2: YEDTQRPS (SEQ ID NO: 5)
CDR3: QSYDSAYHWV (SEQ ID NO: 6)

Antibody PPAT-061-02
VH:
CDR1: TSGVGVG (SEQ ID NO: 7)
CDR2: LIYWDDDKHYSPSLKS (SEQ ID NO: 8)
CDR3: NGDYGIEFDY (SEQ ID NO: 9)
VL:
CDR1: GGNNIGSKSVH (SEQ ID NO: 10)
CDR2: YDSDRPS (SEQ ID NO: 11)
CDR3: QVWDSSSDHVV (SEQ ID NO: 12)

Antibody PPAT-061-03
VH:
CDR1: NYGMS (SEQ ID NO: 13)
CDR2: WISAYNGNTNYGEKLQG (SEQ ID NO: 14)
CDR3: DDYYGSGVDAFDI (SEQ ID NO: 15)
VL:
CDR1: GGNKIGSKSVH (SEQ ID NO: 16)
CDR2: YDRDRPS (SEQ ID NO: 17)
CDR3: QVWDSSSDVV (SEQ ID NO: 18)

Example 3: Evaluation of TfR high expression 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.

Antibody was added to 5 μg / ml and left on ice for 1 hour. This was washed once with BSA solution, then 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, then suspended in 100 μl of 5 μg / ml BSA solution of Alexa488-conjugated anti-mouse IgG goat antibody (Molecularprobe) and allowed to stand on ice for 1 hour. This was washed twice with BSA solution, suspended in 500 μl of BSA solution, 50 μl of fixative (formaldehyde) was added, and the mixture was 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 three types of antibodies showed significant peak shifts in all the cancer cell lines (A431, PANC1, KATOIII, MIAPaCa2) expressing TfR that were evaluated for binding.

Example 4: 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 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 applied to 250 μL of SFM. added.

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 (hereinafter, αMEM 10% FCS + 700 μg / mL G418 was used). After culturing at 37 ° C. for 48 hours, the cells were washed with 10 mL of PBS and treated with 750 μL of 0.25% Trypsin-EDTA (Sigma T4049), then 5 mL of αMEM was added, 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 5: Preparation of labeled antibody (1) Binding of Desferrioxamine (DF) to antibody The antibody was dissolved in a buffer solution (0.1 M Na2CO3) to adjust the antibody concentration to 5 mg / mL. On the other hand, p-scn-DF (B-705 manufactured by Macrocyclics) was dissolved in DMSO to a concentration of 0.753 mg / mL. The mixture was stirred and mixed so that the molar ratio of antibody to DF was 1: 3, and allowed to stand at 37 ° C. for 30 minutes. After completion of the reaction, purification was performed using PBS on a Sephadex G50 (GE Healthcare 17-0041-01) column. The antibody used is antibody PPAT-061-01.

(2) Confirmation of chelate introduction rate 1 μL Ferric chloride (Fe-59, Perkin Elmer NEZ037) was added to the chelate-antibody reaction solution (5 μL) before purification in (1) and allowed to stand at room temperature for 30 minutes. 1 μL was applied to a desalting column (PD-10, GE Healthcare 17-0435-01), and 30 fractions were obtained with 0.5 mL of PBS. 30 fractions were measured with a gamma counter (Aroka). The chelate introduction rate was calculated from the ratio of the antibody fraction and the chelate fraction.

(3) Preparation of ⁸⁹ Zr-labeled antibody (i) ⁸⁹ Zr-labeled Purified antibody PPAT-061-01 was adjusted to 4 mg / mL PBS, ⁸⁹ Zr-oxalate was added, and the mixture was allowed to stand at room temperature for 1 hour.
(Ii) Confirmation of the labeling rate The labeling rate of the labeling reaction solution (0.5 μL) was confirmed using thin layer chromatography (Merck). The developing solvent was DTPA water (pH 7), the plate was exposed to an imaging plate (Fuji Film) for 5 seconds, and an image was acquired with an image scanner (Fuji Film). The labeling rate was calculated by measuring the intensity of the antibody and DPTA with the attached software.

Example 6: Tumor Imaging The ⁸⁹ Zr-DF anti-TfR antibody prepared in Example 5 is a tumor that highly expresses human TfR (MIA Paca-2, yellow arrowhead) and does not express human TfR The tumor (A4, light blue arrowhead) was administered to nude mice transplanted subcutaneously, and PET imaging was performed 1 day, 2 days, 4 days, and 6 days after administration. From 1 day after administration, ⁸⁹ Zr-DF anti-TfR antibody (antibody PPAT-061-01) was clearly accumulated in the MIA Paca-2 transplanted tumor, and the accumulation increased until 6 days after administration. On the other hand, accumulation in A4 transplanted tumors was low and gradually decreased with time. ⁸⁹ Zr-DF anti-TfR antibody (antibody PPAT-061-01) was shown to be suitable for imaging of tumors expressing TfR.

Claims (16)

1. A diagnostic agent for tumors for imaging, comprising an antibody obtained by labeling an antibody recognizing human transferrin receptor with a radionuclide.
2. CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of the antibody each include the amino acid sequences represented by SEQ ID NOs: 1, 2, and 3, and CDR1, CDR2, and CDR3 of the light chain variable region (VL) of the antibody are: The diagnostic agent for tumors for imaging according to claim 1, comprising the amino acid sequences represented by SEQ ID NOs: 4, 5, and 6, respectively.
3. CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of the antibody include the amino acid sequences represented by SEQ ID NOs: 7, 8, and 9, respectively, and CDR1, CDR2, and CDR3 of the light chain variable region (VL) of the antibody are: The diagnostic agent for tumors for imaging according to claim 1, comprising the amino acid sequences represented by SEQ ID NOs: 10, 11, and 12, respectively.
4. CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of the antibody include the amino acid sequences shown by SEQ ID NOs: 13, 14, and 15, respectively, and CDR1, CDR2, and CDR3 of the light chain variable region (VL) of the antibody are: The tumor diagnostic agent for imaging according to claim 1, comprising the amino acid sequences represented by SEQ ID NOs: 16, 17, and 18, respectively.
5. The diagnostic agent for tumor according to any one of claims 1 to 4, wherein the antibody is an antibody containing a constant region of a human antibody.
6. The diagnostic agent for tumor according to claim 5, wherein the constant region of the human antibody is composed of a constant region of the human antibody IgG1 class.
7. The antibody consists of a peptide comprising Fab, Fab ′, F (ab ′) ₂ , single chain antibody (scFv), dimerized V region (Diabody), disulfide stabilized V region (dsFv) and CDR. The diagnostic agent for imaging tumors according to any one of claims 1 to 4, which is an antibody fragment selected.
8. The radionuclide is ⁸⁹ Zr, ^99m Tc, ¹¹¹ In, ^{113 m} In, ⁶⁷ Ga, ⁶⁸ Ga, ²⁰¹ Tl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁶⁰ Co, ⁸⁵ Sr, ¹⁹⁷ Hg, ⁶⁴ Cu, ¹²³ I, ¹²⁵ The diagnostic agent for tumors for imaging according to any one of claims 1 to 7, which is selected from the group consisting of I and ^131I .
9. A tumor diagnostic agent kit for imaging, comprising an antibody recognizing a human transferrin receptor and a radionuclide.
10. CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of the antibody each include the amino acid sequences represented by SEQ ID NOs: 1, 2, and 3, and CDR1, CDR2, and CDR3 of the light chain variable region (VL) of the antibody are: The tumor diagnostic agent kit for images according to claim 9, comprising the amino acid sequences represented by SEQ ID NOs: 4, 5, and 6, respectively.
11. CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of the antibody include the amino acid sequences represented by SEQ ID NOs: 7, 8, and 9, respectively, and CDR1, CDR2, and CDR3 of the light chain variable region (VL) of the antibody are: The tumor diagnostic agent kit for imaging according to claim 9, comprising the amino acid sequences represented by SEQ ID NOs: 10, 11, and 12, respectively.
12. CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) of the antibody include the amino acid sequences shown by SEQ ID NOs: 13, 14, and 15, respectively, and CDR1, CDR2, and CDR3 of the light chain variable region (VL) of the antibody are: The tumor diagnostic agent kit for imaging according to claim 9, comprising the amino acid sequences represented by SEQ ID NOs: 16, 17, and 18, respectively.
13. The diagnostic imaging agent for an image according to any one of claims 9 to 12, wherein the antibody is an antibody containing a constant region of a human antibody.
14. The diagnostic agent kit for imaging according to claim 13, wherein the constant region of the human antibody is composed of a constant region of the human antibody IgG1 class.
15. The antibody consists of a peptide comprising Fab, Fab ′, F (ab ′) ₂ , single chain antibody (scFv), dimerized V region (Diabody), disulfide stabilized V region (dsFv) and CDR. The tumor diagnostic agent kit for imaging according to any one of claims 9 to 12, which is a selected antibody fragment.
16. The radionuclide is ⁸⁹ Zr, ^99m Tc, ¹¹¹ In, ^{113 m} In, ⁶⁷ Ga, ⁶⁸ Ga, ²⁰¹ Tl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁶⁰ Co, ⁸⁵ Sr, ¹⁹⁷ Hg, ⁶⁴ Cu, ¹²³ I, ¹²⁵ The tumor diagnostic agent kit for imaging according to any one of claims 9 to 15, which is selected from the group consisting of I and ^131I .

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