WO2013031619A1 - Agent de diagnostic de tumeur à base d'image contenant un anticorps du récepteur d'anti-transferrine humaine - Google Patents

Agent de diagnostic de tumeur à base d'image contenant un anticorps du récepteur d'anti-transferrine humaine Download PDF

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WO2013031619A1
WO2013031619A1 PCT/JP2012/071260 JP2012071260W WO2013031619A1 WO 2013031619 A1 WO2013031619 A1 WO 2013031619A1 JP 2012071260 W JP2012071260 W JP 2012071260W WO 2013031619 A1 WO2013031619 A1 WO 2013031619A1
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antibody
seq
diagnostic agent
cdr2
cdr1
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PCT/JP2012/071260
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Japanese (ja)
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良和 黒澤
恒夫 佐賀
厚至 辻
須藤　幸夫
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学校法人藤田学園
独立行政法人放射線医学総合研究所
株式会社ペルセウスプロテオミクス
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Priority to JP2013531245A priority Critical patent/JP5990523B2/ja
Publication of WO2013031619A1 publication Critical patent/WO2013031619A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the present invention relates to a diagnostic agent for tumors for imaging, comprising an antibody that recognizes human transferrin receptor (TfR).
  • TfR human transferrin receptor
  • 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.
  • 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.
  • 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.
  • 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.
  • CT and MRI are mainly used in tumor image diagnosis.
  • 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.
  • PET Positron Emission Tomography
  • 18F-FDG 18F-2-fluoro-2-deoxyglucose
  • 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.
  • Patent Document 1 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 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
  • 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.
  • TfR transferrin receptor
  • 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.
  • an imaging tumor diagnostic agent comprising an antibody obtained by labeling an antibody recognizing human transferrin receptor with a radionuclide.
  • a diagnostic imaging agent kit for imaging comprising an antibody that recognizes a human transferrin receptor and a radionuclide.
  • 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.
  • 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.
  • 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.
  • the antibody is an antibody comprising the constant region of a human antibody.
  • the constant region of the human antibody consists of a constant region of the human antibody IgG1 class.
  • 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.
  • 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.
  • the antibody used in the present invention is an antibody that specifically recognizes a transferrin receptor (TfR) on the cell surface.
  • TfR transferrin receptor
  • 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.
  • CD71 antigen also known as the CD71 antigen
  • human antibodies human chimeric antibodies, human CDR-grafted antibodies, non-human animal antibodies, and the like can be used.
  • 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.
  • 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.
  • 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.
  • 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.
  • the target protein can be immobilized on beads coated with streptavidin.
  • the target protein 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.
  • 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.
  • ATCC American Tissue Culture Collection
  • 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.
  • 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
  • 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.
  • the antibody used in the present invention may be an antibody fragment.
  • 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.
  • 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.
  • 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).
  • P an appropriate peptide linker
  • 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)).
  • 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.
  • 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.
  • 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 ⁇ g / 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.
  • 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).
  • a linker metal chelating reagent
  • 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.
  • the metal chelate 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.
  • 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
  • isothiocyanobenzyl DOTA isothiocyanobenzyl DOTA, methylisothiocyanobenzyl DTPA, cyclohexylisothiocyanobenzyl DTPA, and isothiocyanobenzyl deferoxamine
  • isothiocyanobenzyl DOTA isothiocyanobenzyl DOTA, methylisothiocyanobenzyl DTPA, cyclohexylisothiocyanobenzyl DTPA, and isothiocyanobenzyl deferoxamine.
  • 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.
  • 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.
  • a pre-labeled preparation an imaging tumor diagnostic agent containing a labeled anti-TfR antibody can be used for administration as it is.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the phage-infected Escherichia coli was cultured overnight at 30 ° C. in 600 ml of 2 ⁇ YTGA medium (2 ⁇ YT, 200 ⁇ g / ml ampicillin sulfate, 1% glucose). 10 ml of this overnight culture was mixed with 200 ml of 2 ⁇ YTA medium (2 ⁇ YT, 200 ⁇ g / ml ampicillin sulfate), cultured at 37 ° C.
  • 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.
  • the antibody 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.
  • NUNC Maxisorp
  • 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 ⁇ l 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.
  • 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.
  • sample antibody expression culture supernatant as a primary antibody was reacted at 100 ⁇ l / well at 37 ° C. for 1 hour. Thereafter, the supernatant was removed and washed 5 times with a phosphate buffer.
  • Rabbit anti-cp3 polyclonal as a secondary antibody diluted 5000 times with PBS / 0.05% Tween20 was reacted at 100 ⁇ l / well at 37 ° C. for 1 hour, the supernatant was removed, and washed 5 times with phosphate buffer.
  • anti-rabbit IgG (H + L) -HRP as a secondary antibody diluted 2000 times with PBS / 0.05% Tween20 was reacted at 100 ⁇ l / 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 ⁇ l / 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 ⁇ l / well to stop the color reaction. Thereafter, the absorbance at 492 nm was measured with SPECTRAmax 340PC (Molecular Devices).
  • 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)
  • VH CDR1: TSGVGVG (SEQ ID NO: 7)
  • VL CDR1: GGNNIGSKSVH (SEQ ID NO: 10)
  • 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.
  • FACS high expression cell line binding
  • 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.
  • 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 ⁇ l 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 ⁇ g / ml and left on ice for 1 hour. This was washed once with BSA solution, then suspended in 100 ⁇ l of 5 ⁇ g / 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 ⁇ l of 5 ⁇ g / ml BSA solution of Alexa488-conjugated anti-mouse IgG goat antibody (Molecularprobe) and allowed to stand on ice for 1 hour.
  • Example 4 Conversion to IgG type antibody (construction of IgG type antibody gene)
  • IgG type antibody construction of IgG type antibody gene
  • 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.
  • GenePORTER Reagent (Gene Therapy Systems: T201007) was used for transfection of plasmid DNA into CHO-K1 cells.
  • the medium is ⁇ -MEM (Invitrogen: 12561-056) in 10% FCS). (Exitec Inc .: 268-1) used).
  • SFM- Human Free Medium
  • GenePORTER Reagent 40 ⁇ L was applied to 250 ⁇ L 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 ⁇ MEM 10% FCS, 5 ml of ⁇ MEM 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 ⁇ MEM 10% FCS + 700 ⁇ g / ml G418 (Sigma: G7034), and selection was started (hereinafter, ⁇ MEM 10% FCS + 700 ⁇ g / mL G418 was used).
  • ⁇ MEM 10% FCS + 700 ⁇ g / mL G418 was used.
  • the cells were washed with 10 mL of PBS and treated with 750 ⁇ L of 0.25% Trypsin-EDTA (Sigma T4049), then 5 mL of ⁇ MEM 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 ⁇ L / 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne un agent de diagnostic de tumeur à base d'image à haute précision, qui contient un anticorps pouvant reconnaître spécifiquement un récepteur de la transferrine (TfR) sur une membrane cellulaire. L'invention porte en outre sur un agent de diagnostic de tumeur à base d'image qui contient ledit anticorps, un anticorps reconnaissant un récepteur de la transferrine humaine étant marqué par un radionucléide.
PCT/JP2012/071260 2011-08-26 2012-08-23 Agent de diagnostic de tumeur à base d'image contenant un anticorps du récepteur d'anti-transferrine humaine WO2013031619A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014073641A1 (fr) * 2012-11-08 2014-05-15 国立大学法人 宮崎大学 Anticorps capable de reconnaître spécifiquement un récepteur de transferrine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005121179A2 (fr) * 2004-06-07 2005-12-22 Raven Biotechnologies, Inc. Anticorps vis-a-vis du recepteur de transferine

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AU2006304790B2 (en) * 2005-10-20 2013-06-27 Georgetown University Tumor-targeted nanodelivery systems to improve early MRI detection of cancer

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005121179A2 (fr) * 2004-06-07 2005-12-22 Raven Biotechnologies, Inc. Anticorps vis-a-vis du recepteur de transferine

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014073641A1 (fr) * 2012-11-08 2014-05-15 国立大学法人 宮崎大学 Anticorps capable de reconnaître spécifiquement un récepteur de transferrine
JPWO2014073641A1 (ja) * 2012-11-08 2016-09-08 国立大学法人 宮崎大学 トランスフェリン受容体を特異的に認識できる抗体
US9593165B2 (en) 2012-11-08 2017-03-14 University Of Miyazaki Antibody capable of specifically recognizing transferrin receptor

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