WO2015079709A1 - Anti-fluorescent-pigment monoclonal antibody - Google Patents
Anti-fluorescent-pigment monoclonal antibody Download PDFInfo
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- WO2015079709A1 WO2015079709A1 PCT/JP2014/005969 JP2014005969W WO2015079709A1 WO 2015079709 A1 WO2015079709 A1 WO 2015079709A1 JP 2014005969 W JP2014005969 W JP 2014005969W WO 2015079709 A1 WO2015079709 A1 WO 2015079709A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
Definitions
- the present invention encodes an anti-fluorescent dye monoclonal antibody capable of binding to the epitope of Alexa Fluor (registered trademark, manufactured by Invitrogen) 647, a polypeptide constituting the monoclonal antibody, and the polypeptide It relates to DNA.
- Alexa Fluor registered trademark, manufactured by Invitrogen
- Enormous types of antibodies have been developed and sold as research reagents, diagnostic reagents, various substance monitoring reagents, antibody drugs, and the like.
- Antibodies are generally prepared from the sera of animals immunized with antigens, but antigens often have multiple epitopes (antigenic determinants), so if prepared simply from serum, antibodies against each epitope Is mixed, and this is called a polyclonal antibody.
- a monoclonal antibody has a uniform immunoglobulin molecular species itself and becomes a single molecular species for one epitope, and therefore has exactly the same antigen specificity.
- monoclonal antibodies are produced by fusing antibody-producing lymphocyte B cells taken from the spleen and lymph nodes of animals immunized with antigen and plasma cell myeloma cells that proliferate indefinitely to produce immunoglobulins.
- a method has been employed in which a hybridoma cell is used to separate a cell population grown from a single hybridoma cell producing the target antibody by a limiting dilution method and purify an antibody secreted by the cell population.
- a labeled antigen formed by labeling an antigen recognized by a target antibody is brought into contact with a cell population containing a target cell producing the antibody, and the labeled antigen is contacted with the target cell.
- Antibody production method in which the labeled target cells obtained are separated, the antibody genes possessed by the separated labeled target cells are prepared, and the prepared antibody genes are expressed using an expression vector [Patent Document 1] and the like are known.
- the present inventors have detected and identified B cells that produce already established antigen-specific human antibodies at the single cell level, and obtained a direct antibody gene by PCR cloning [Patent Document 2]. It has been shown that it is possible to detect B cells that produce specific human antibodies against infectious diseases and cancer-associated antigens.
- Alexa Fluor 647 is a fluorescent material having the following structural formula and having a maximum absorption wavelength of 650 nm. Further, as an application of a monoclonal antibody that recognizes a fluorescent substance, use of an alkaline phosphatase-labeled anti-FITC (fluorescein isothiocyanate) antibody as a secondary antibody in immunohistochemistry [Patent Document 3] or anti-Cy (registered trademark) 5 Known is an indirect magnetic labeling separation method [Non-patent Document 1] for cells stained with a primary antibody labeled with Cy5, PE-Cy5 or Alexa Fluor 647 using a microbead to which a monoclonal antibody is bound.
- FITC fluorescein isothiocyanate
- JP 2006-180708 A Japanese Patent No. 5205597 Japanese Patent No. 4004385
- An object of the present invention is to provide a novel monoclonal antibody that recognizes Alexa Fluor 647.
- the present inventors have measured autoantibody titers against cancer-associated antigens in blood against several types of cancer-associated antigen proteins (EGFR, VEGFR2, VEGFR3, CDH11, etc.) in 19 cases of malignant glioma patients.
- EGFR cancer-associated antigen proteins
- VEGFR2 peripheral blood B cells of the case (patient) with the highest antibody titer against VEGFR2
- staining with Alexa Fluor 647-labeled VEGFR2 protein (extracellular domain) and PE-labeled mouse anti-human IgG antibody gave VEGFR2 specific B cells with antibodies were selected, and mRNA for each cell was collected using Single® cell® sorter® (FACSAria, BD Bioscience), and cDNA cloning was performed.
- the cloned antibody gene was incorporated into an expression vector, and a full-length antibody protein was expressed and secreted in a baculovirus expression system, and the antibody was purified.
- a human monoclonal antibody showing binding activity specifically to Alexa Fluor 647, a fluorescent dye, was identified accidentally. It was.
- the present invention has been completed based on these findings.
- the present invention is as follows. (1) A monoclonal antibody that recognizes Alexa Fluor 647. (2) A polypeptide constituting the heavy chain variable region of an antibody that recognizes Alexa Fluor 647, wherein the heavy chain variable region includes a hypervariable region comprising the amino acid sequence shown in SEQ ID NO: 2. Polypeptide. (3) A polypeptide constituting the heavy chain variable region of an antibody that recognizes Alexa Fluor 647, wherein the heavy chain variable region is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 4 A polypeptide comprising an amino acid sequence.
- a polypeptide that constitutes the heavy chain of an antibody that recognizes Alexa Fluor 647 the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 6
- a polypeptide characterized by the above A polypeptide constituting a light chain variable region of an antibody that recognizes Alexa Fluor 647, wherein the light chain variable region includes a hypervariable region consisting of the amino acid sequence shown in SEQ ID NO: 8 Polypeptide.
- a polypeptide comprising an amino acid sequence (7)
- An anti-alexafluor 647 monoclonal comprising the polypeptide according to any one of (2) to (4) above and the polypeptide according to any one of (5) to (7) above. antibody.
- the anti-alexafluor 647 monoclonal according to (9) above comprising the polypeptide according to (2) or (3) above and the polypeptide according to (5) or (6) above antibody.
- a substance labeled with Alexa Fluor 647 can be monitored or a substance labeled with Alexa Fluor 647 can be quantified. Is possible.
- FIG. 3 is a diagram showing RT-PCR of mRNA collected for each cell using a single cell sorter (FACSAria, BD Bioscience).
- the cloned antibody gene is incorporated into an expression vector, and a full-length antibody protein is expressed and secreted in a baculovirus expression system.
- Purified 15 clone antibodies have binding activity against VEGFR2-Alexafluor 647 and VEGFR2 protein by ELISA. It is the figure evaluated by the law. It is a figure which shows the Coomassie brilliant blue dyeing
- SPR Surface plasmid * resonance
- the polypeptide of the present invention is a polypeptide constituting the heavy chain variable region or heavy chain, and the light chain variable region or light chain of an antibody that recognizes Alexa Fluor 647, and 1) the amino acid shown in SEQ ID NO: 2 Polypeptide [P1] constituting a heavy chain variable region comprising a hypervariable region consisting of a sequence (complementarity determining region: CDR3), 2) the amino acid sequence shown in SEQ ID NO: 4, or at least 97% identical to SEQ ID NO: 4, preferably Is a polypeptide [P2] comprising a heavy chain variable region consisting of an amino acid sequence that is 98% or more identical, more preferably 99% or more identical, 3) the amino acid sequence shown in SEQ ID NO: 6, or at least 95% with SEQ ID NO: 6
- DNA of the present invention examples include polypeptides [P1] and [P2] constituting the heavy chain variable region of the antibody recognizing the above-described Alexa Fluor 647 of the present invention, polypeptides [P3] constituting the heavy chain, light chains
- the DNA is not particularly limited as long as it encodes the polypeptides [P4] and [P5] constituting the variable region and the polypeptide [P6] constituting the light chain, and more specifically, 1) heavy chain DNA [D1] encoding a heavy chain variable region including the base sequence shown in SEQ ID NO: 1 encoding the hypervariable region, 2) a base sequence shown in SEQ ID NO: 3 encoding the heavy chain variable region, or at least SEQ ID NO: 3 and DNA [D2] comprising a nucleotide sequence that is 95% identical, preferably 98% or more identical, more preferably 99% or more identical, 3) the nucleotide sequence shown in SEQ ID NO: 5 encoding the heavy chain, Or DNA [D3]
- DNA [D4] encoding a light chain variable region containing the nucleotide sequence shown [5], 5) the nucleotide sequence shown in SEQ ID NO: 9 encoding the light chain variable region, or at least 95% identical, preferably 98% or more identical More preferably, DNA [D5] comprising a nucleotide sequence that is 99% or more identical, 6) the nucleotide sequence shown in SEQ ID NO: 11 encoding the light chain, or at least 90% identical, preferably 95% or more identical to SEQ ID NO: 11. More preferably, DNA [D6] consisting of a base sequence that is 98% or more identical can be mentioned.
- amino acid sequence or base sequence for example, at least 95% identity is to be compared when the sequence in question is counted as only one difference in each amino acid or nucleotide insertion, deletion, substitution, etc. Means containing less than 5% different amino acids and nucleotides from the sequence.
- the monoclonal antibody of the present invention is not particularly limited as long as it is an anti-alexafluor 647 monoclonal antibody that specifically binds to alexafluor 647, and a polypeptide constituting the heavy chain variable region of the anti-alexafluor 647 monoclonal antibody [P1] and [P2], one of the polypeptides [P3] constituting the heavy chain, the polypeptides [P4] and [P5] constituting the light chain variable region, and the polypeptide [P6] constituting the light chain Monoclonal antibodies containing any of them, for example, [P1] and [P4], [P2] and [P5], [P3] and [P6], [P1] and [P5], [P2] and [P4], etc. Specific examples thereof include monoclonal antibodies.
- the anti-alexafluor 647 monoclonal antibody of the present invention includes monoclonal antibodies having any isotype such as IgG, IgA, IgM, IgD, and IgE.
- the anti-alexafluor 647 monoclonal antibody of the present invention includes In addition to full-body monoclonal antibodies, active fragments of monoclonal antibodies such as F (ab ′) 2 , Fab ′, Fab, Fv (variable fragment of antibody), sFv, dsFv (disulphide stabilized Fv), dAb ( single domain antibody) and the like.
- DNA [D3] encoding the heavy chain of anti-alexafluor 647 monoclonal antibody and DNA [D6] encoding the light chain were incorporated. It can be obtained by transforming a host cell with an expression vector and culturing the transformed cell in an appropriate medium for expression.
- the production of the Fab antibody includes a conventional method in which the purified full-length antibody is digested with papain and only the Fab fragment is purified again.
- scFv single-chain Fv fragment
- the anti-alexafluor 647 monoclonal antibody of the present invention includes chimeric antibodies and humanized antibodies that combine antibody variable regions (Fv) and antibody constant regions (Fc) derived from various animal species.
- a chimeric antibody or humanized antibody can be prepared by binding and expressing a gene encoding an Fv region and a gene encoding an Fc region.
- animal species include mice, rats, hamsters, guinea pigs, rabbits, goats, sheep, donkeys, pigs, cows, horses, dogs, cats, chickens, monkeys, humans, etc., which are general antibody animal species. be able to.
- the constant region may be the same as the monoclonal antibody from which the variable region is derived, or may be derived from a different monoclonal antibody.
- a known human IgG1 or mouse IgG1 Fc region can be used as the Fc region.
- the capture antibody has a constant region derived from an animal species different from the constant region of the detection antibody. And the cross-reaction between the detection antibody and the detection antibody can be further prevented.
- an anti-human antibody such as an enzyme-labeled anti-human IgG antibody
- a human-mouse chimeric antibody may be used as the capture antibody.
- the human-mouse chimeric antibody is, for example, SEQ ID NO: 13 in which the Fc region sequence of a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6 derived from human is replaced with an artificially synthesized mouse-derived Fc region sequence.
- An expression vector that expresses a gene encoding a polypeptide consisting of the amino acid sequence shown in FIG. 5 is prepared, and introduced into a mammalian cell such as exp293 cells together with a human-derived gene encoding the amino acid sequence shown in SEQ ID NO: 12. It can be obtained by producing in.
- a DNA encoding the polypeptide of the present invention is expressed in host cells such as Escherichia coli, hay, yeast, insect cells, plant cells, and animal cells.
- host cells such as Escherichia coli, hay, yeast, insect cells, plant cells, and animal cells.
- preferred are baculovirus Autographaifcalifornica nuclear polyhedrosis virus (AcNPV) vector and baculovirus shuttle vector (pFastBac Dual ⁇ ⁇ vector).
- BmN4 of a Bombyxmori host cell is preferably a baculovirus nuclear polyhedrosis virus Bombyxmori polynucledrosis virus (BmNPV) vector.
- the expression vector may contain a drug resistance gene as a promoter, terminator, and marker gene.
- Examples of the method for introducing a vector into a host cell include In-Fusion cloning system (Clontech), liposome method, lipofection method, microinjection method, DEAE dextran method, calcium phosphate method, electroporation method and the like.
- Lipofectin Reagent registered trademark
- Lipofectamine registered trademark
- Lipofectamine registered trademark
- 2000 Reagent manufactured by Invitrogen
- SuperFect registered trademark
- Transfection Reagent manufactured by Qiagen
- FuGENE registered trademark
- HD Transfection Reagent Examples of methods widely used in the art using commercially available transfection reagents such as Roche Diagnostics
- FuGENE® 6 Transfection Reagent Roche Diagnostics
- the anti-Alexafluor 647 monoclonal antibody of the present invention can be advantageously used for monitoring a substance labeled with Alexa Fluor 647 administered to a test non-human animal subject. More specifically, for the purpose of confirming sensitivity to pharmaceuticals and adjusting dosage intervals and dosages, for example, when used in experimental animals for the development of biological products (medicines) for specific diseases It can be advantageously used for monitoring the efficacy by biological formulation concentration and the localization of biological products in vivo / in situ. Specifically, after the development candidate drug labeled with Alexa Fluor 647 is administered to an experimental animal, an organ to be observed is appropriately taken out, and an immunohistochemistry method, an immunoprecipitation method, an ELISA method using the antibody of the present invention. A method for identifying, visualizing, and quantifying the drug localized in the target organ by Western blotting or the like can be exemplified.
- the anti-alexafluor 647 monoclonal antibody of the present invention can be used for the purpose of cell separation / concentration based on the magnetic cell separation method, such as MACS (registered trademark) cell separation system (Miltenyi Biotec), Dynabeads (Registered trademark) cell separation system (Invitrogen), BD IMag (trademark) cell separation system, etc. can be mentioned suitably.
- MACS registered trademark
- Dynabeads Registered trademark
- BD IMag trademark
- the present invention will be described more specifically by way of examples.
- the technical scope of the present invention is not limited to these examples.
- the patient specimens (cells and plasma) used in the examples are those of studies approved by the institutional clinical ethics review committee with the consent of the patient being treated at the Shizuoka Cancer Center. Used within range.
- VEGFR2-Alexafluor 647-specific antibody-producing B cells using a single cell sorter
- Whole blood from GB-SCC008 cases was subjected to density gradient centrifugation using Ficoll-Paque TM plus (manufactured by GE Healthcare).
- CD19 positive cells were selected with an AutoMACS (Miltenyi) apparatus using CD19 microbeads (Miltenyi).
- VEGFR2 extracellular domain
- PE-labeled mouse anti-human IgG antibody manufactured in this laboratory
- PerCP-labeled mouse anti-human CD14 monoclonal antibody clone: MfP9 (BD Bioscience)
- PI propidium iodide
- the detection sensitivity of the cell sorter was examined using patient peripheral blood mononuclear cells containing anti-CEACAM5 mouse hybridoma cells.
- the contained anti-CEACAM5 mouse hybridoma cells were detected using Alexa Fluor 647-labeled CEACAM5 antigen and anti-mouse CD138 antibody.
- the detection limit of the cell sorter was 10 cells / 10 6 peripheral blood mononuclear cells, 0.001% (FIG. 5A).
- the antibody gene of 22 clones was incorporated into an expression vector, and the full-length antibody protein was expressed and secreted by the baculovirus expression system and purified.
- the antibody gene (H chain and L chain) was incorporated into a baculovirus shuttle vector (pFastBac Dual vector), introduced into Escherichia coli DH10Bac, and a recombinant bacmid was prepared by homologous recombination.
- the bacmid DNA was introduced into insect-derived Sf9 cells to produce a recombinant baculovirus.
- the amplified virus was infected with insect-derived High Five cells, which are high-protein-producing strains, and cultured in a serum-free medium at 27 ° C. for 64 hours.
- the antibody protein produced in the culture supernatant was purified using a Protein A prepack column (manufactured by GE Healthcare). About 15 clones that could be obtained, the binding activity to Alexa Fluor 647-labeled VEGFR2 and VEGFR2 protein was evaluated by ELISA (FIG. 7). Five clones that strongly bound to Alexa Fluor 647-labeled VEGFR2 protein were observed (# 48, 51, 54, 55, 56). Among them, clones # 48 and # 51 bound to VEGFR2.
- Affinity measurement of antibody clone using SPR method The affinity between # 48, # 51, and # 55 purified antibody of antibody clone and VEGFR2 protein and Alexa Fluor 647-labeled VEGFR2 protein was measured using BIAcore X100 (manufactured by GE Healthcare). ) Using the surface plasmon resonance method (SPR analysis). The amount of ligand (each antibody clone) immobilized on the chip was between 1000 and 10,000 response units (RU). The antibody was dissolved in HBS buffer (0.15 M NaCl, 3 mM EDTA, 0.05% Tween 20) and measured at a flow rate of 10 ⁇ l / min or 30 ⁇ l / min at 25 ° C. and a maximum concentration of 150 ⁇ M. When regenerating the chip, 3 molar magnesium chloride was used. Kinetic constants were calculated from the measured data using BIAcore X100 evaluation software.
- the association rate constant (Ka), dissociation rate constant (Kd) and dissociation constant (KD) were determined in a 1: 1 binding model, and for antibody clone # 55, 1: 1 binding was performed.
- the dissociation constant was obtained by the two-state reaction model using the difference in the dissociation rate of the reaction. The results are shown in Table 1. From the results of SPR analysis, the clone # 48 and # 51 antibodies showed relatively strong binding activity to VEGFR2, whereas the clone # 55 antibody showed no affinity for the VEGFR2 protein and the Alexa Fluor 647-labeled VEGFR2 protein. It became clear that it only combined. Thus, clone # 55 antibody was found to be a human monoclonal antibody that specifically recognizes Alexa Fluor 647, which is a fluorescent dye (FIG. 9).
- Alexa Fluor 647 labeled trastuzumab was prepared by the following method. First, the genes encoding the polypeptide (SEQ ID NO: 14) constituting the heavy chain of trastuzumab (anti-Her2 antibody) and the polypeptide (SEQ ID NO: 15) constituting the light chain were respectively pcDNA3.3 vectors (Thermo Fisher Scientific) And introduced into Expi293 cells, cultured for 5-7 days, and then recovered and purified from the culture supernatant using protein A to obtain trastuzumab.
- Alexa Fluor 647 Protein Labeling Kit manufactured by Thermo Fisher Scientific
- Alexa Fluor 647 labeling operation is performed on the obtained trastuzumab according to the manual attached to the product, and Alexa Fluor 647 Labeled Trastuzumab is labeled. Obtained.
- Alexa Fluor 647-labeled trastuzumab was measured by the following method.
- a 96-well immobilizer amino plate manufactured by Thermo Fisher Scientific
- 10 ⁇ g / ml anti-Alexafluor 647 antibody (antibody clone # 55) diluted in PBS as a capture antibody at room temperature.
- the plate was washed 3 times with a washing solution (PBS containing 0.05% Tween-20), and blocked by reacting overnight at 4 ° C. with PBS containing 3% BSA.
- 0.2 ⁇ g / ml horseradish peroxidase-labeled streptavidin (PN21130, manufactured by Thermo Fisher Scientific) was added at 50 ⁇ l / well, reacted for 30 minutes, washed 7 times with a washing solution, and then a substrate solution (TMB (substrate).
- TMB substrate solution
- reagent set manufactured by BD Bioscience was added at 100 ⁇ l / well, incubated at room temperature for 30 minutes, and 1 M sulfuric acid aqueous solution was added at 50 ⁇ l / well to stop the color reaction.
- the absorbance (450 nm) of each well was measured using a plate reader (ImmunoMini NJ-2300, manufactured by Biotech).
- a graph was prepared by plotting the absorbance (450 nm) on the X-axis and the concentration (ng / ml) of Alexa Fluor 647-labeled trastuzumab on the Y-axis (FIG. 10).
- a calibration curve having a high correlation coefficient can be created from the absorbance and the concentration of Alexa Fluor 647-labeled trastuzumab. Therefore, a calibration curve of Alexa Fluor 647-labeled antibody is prepared by sandwich ELISA using antibody clone # 55, the test substance is processed in the same manner as described above, and the absorbance (450 nm) is measured. It was revealed that the concentration of Alexa Fluor 647-labeled antibody in the substance can be quantified.
- Quantifying the concentration of Lexafluor 647-labeled antibody by sandwich ELISA can be applied to the development of antibody drugs.
- an antibody that specifically recognizes the tumor cell labeled with Alexa Fluor 647 (hereinafter also referred to as “Alexa label-tumor cell recognition antibody”) labeled with Alexa Fluor 647 on a normal animal and an animal transplanted with tumor cells.
- blood is collected from each animal after a predetermined period.
- the concentration of the Alexa label-tumor cell recognition antibody contained in each blood is quantitatively compared by sandwich ELISA using the antibody of the present invention, thereby comparing the Alexa label that binds to tumor cells in the animal body.
- the amount of tumor cell recognition antibody can be measured. By measuring the amount of the Alexa-labeled tumor-cell-recognizing antibody that binds to such tumor cells, it is possible to determine the dose and timing of administration of the antibody when used as an antibody drug.
- the present invention enables selection of target cells as a secondary antibody against Alexa Fluor 647-labeled antibody and construction of an ELISA system that can quantify Alexa Fluor 647-labeled protein.
- it can be suitably used for monitoring the blood concentration of Alexa Fluor 647-labeled antibody in vivo, and is expected to be useful for analysis of metabolism and pharmacokinetics of antibody drugs.
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Abstract
The present invention addresses the problem of providing a novel monoclonal antibody that recognizes Alexa Fluor (registered trademark) 647. A single cell sorter is used to recover mRNA from each individual B cell, cDNA cloning is performed, and DNA that codes an H-chain hypervariable region and an L-chain hypervariable region of an anti-Alexa-Fluor-647 monoclonal antibody, DNA that codes the H-chain variable region and the L-chain variable region, and DNA that codes the H-chain and the L-chain, are identified. The related antibody gene is inserted into an expression vector, a full length antibody protein expressed by baculovirus is expressed/secreted, and the antibody is purified.
Description
本発明は、アレクサフルオール（Alexa Fluor；登録商標、インビトロジェン社製）６４７のエピトープに結合することができる抗蛍光色素モノクローナル抗体や、該モノクローナル抗体を構成するポリペプチドや、該ポリペプチドをコードするＤＮＡに関する。
The present invention encodes an anti-fluorescent dye monoclonal antibody capable of binding to the epitope of Alexa Fluor (registered trademark, manufactured by Invitrogen) 647, a polypeptide constituting the monoclonal antibody, and the polypeptide It relates to DNA.
抗体は研究用試薬、診断用試薬、各種物質モニター用試薬、抗体医薬等として、膨大な種類が開発・販売されている。抗体は、一般的に抗原で免疫した動物の血清から調製されるが、抗原は複数のエピトープ（抗原決定基）をもつことが多いため、単に血清から調製されたままでは、各々のエピトープに対する抗体が混ざっており、これをポリクローナル抗体という。一方、モノクローナル抗体は、免疫グロブリン分子種自体が均一であり、一つのエピトープに対する単一の分子種となるため、抗原特異性が全く同一の抗体となる。
Enormous types of antibodies have been developed and sold as research reagents, diagnostic reagents, various substance monitoring reagents, antibody drugs, and the like. Antibodies are generally prepared from the sera of animals immunized with antigens, but antigens often have multiple epitopes (antigenic determinants), so if prepared simply from serum, antibodies against each epitope Is mixed, and this is called a polyclonal antibody. On the other hand, a monoclonal antibody has a uniform immunoglobulin molecular species itself and becomes a single molecular species for one epitope, and therefore has exactly the same antigen specificity.
従来、モノクローナル抗体の作製には、抗原で免疫した動物の脾臓やリンパ節から取り出した抗体産生リンパ球のＢ細胞と、無限に増殖して免疫グロブリンを産生する形質細胞腫瘍の骨髄腫細胞を融合した、ハイブリドーマ細胞を用い、目的の抗体を産生する１個のハイブリドーマ細胞から増殖した細胞集団を、限界希釈法により分離し、該細胞集団が分泌する抗体を精製する方法がとられてきた。
Conventionally, monoclonal antibodies are produced by fusing antibody-producing lymphocyte B cells taken from the spleen and lymph nodes of animals immunized with antigen and plasma cell myeloma cells that proliferate indefinitely to produce immunoglobulins. Thus, a method has been employed in which a hybridoma cell is used to separate a cell population grown from a single hybridoma cell producing the target antibody by a limiting dilution method and purify an antibody secreted by the cell population.
その他のモノクローナル抗体の作製法としては、目的の抗体が認識する抗原を標識化してなる標識化抗原を、前記抗体を産生するターゲット細胞を含む細胞集団に接触させ、前記標識化抗原を前記ターゲット細胞に結合させ、得られる標識化ターゲット細胞を分離し、分離した標識化ターゲット細胞を用いて、それが保有する抗体遺伝子を調製し、調製した抗体遺伝子を、発現ベクターを用いて発現させる抗体作製方法［特許文献１］等が知られる。
As another method for producing a monoclonal antibody, a labeled antigen formed by labeling an antigen recognized by a target antibody is brought into contact with a cell population containing a target cell producing the antibody, and the labeled antigen is contacted with the target cell. Antibody production method in which the labeled target cells obtained are separated, the antibody genes possessed by the separated labeled target cells are prepared, and the prepared antibody genes are expressed using an expression vector [Patent Document 1] and the like are known.
また、本発明者らは、既に確立した抗原特異的なヒト抗体を産生するＢ細胞を１細胞レベルで検出・同定し、ＰＣＲクローニング法により、直接抗体遺伝子を取得する技術［特許文献２］により、感染症やがん関連抗原に対する特異的なヒト抗体を産生するＢ細胞の検出が可能であることを示してきた。
In addition, the present inventors have detected and identified B cells that produce already established antigen-specific human antibodies at the single cell level, and obtained a direct antibody gene by PCR cloning [Patent Document 2]. It has been shown that it is possible to detect B cells that produce specific human antibodies against infectious diseases and cancer-associated antigens.
アレクサフルオール６４７は、以下の構造式を有し、その極大吸収波長が６５０ｎｍの蛍光物質である。また、蛍光物質を認識するモノクローナル抗体の応用としては、免疫組織化学におけるアルカリフォスファターゼ標識抗ＦＩＴＣ（フルオレセインイソチオシアネート）抗体の二次抗体としての使用［特許文献３］や、抗Ｃｙ（登録商標）５モノクローナル抗体を結合させたマイクロビーズによる、Ｃｙ５、ＰＥ－Ｃｙ５又はアレクサフルオール６４７標識の一次抗体で染色した細胞の間接磁気標識分離法［非特許文献１］等が知られている。
Alexa Fluor 647 is a fluorescent material having the following structural formula and having a maximum absorption wavelength of 650 nm. Further, as an application of a monoclonal antibody that recognizes a fluorescent substance, use of an alkaline phosphatase-labeled anti-FITC (fluorescein isothiocyanate) antibody as a secondary antibody in immunohistochemistry [Patent Document 3] or anti-Cy (registered trademark) 5 Known is an indirect magnetic labeling separation method [Non-patent Document 1] for cells stained with a primary antibody labeled with Cy5, PE-Cy5 or Alexa Fluor 647 using a microbead to which a monoclonal antibody is bound.
本発明の課題は、アレクサフルオール６４７を認識する新規なモノクローナル抗体を提供することにある。
An object of the present invention is to provide a novel monoclonal antibody that recognizes Alexa Fluor 647.
本発明者らは、悪性グリオーマ患者１９症例において数種類のがん関連抗原タンパク（ＥＧＦＲ，ＶＥＧＦＲ２，ＶＥＧＦＲ３，ＣＤＨ１１等）に対する血液中のがん関連抗原に対する自家抗体価を測定していた。ＶＥＧＦＲ２に対する抗体価が最も高かった症例（患者）の末梢血Ｂ細胞を用いて、アレクサフルオール６４７標識ＶＥＧＦＲ２タンパク（細胞外ドメイン）とＰＥ標識マウス抗ヒトＩｇＧ抗体による染色にて、ＶＥＧＦＲ２特異的な抗体をもつＢ細胞を選別し、更に、Single cell sorter (FACSAria, BD Bioscience)を用いて１細胞ごとのｍＲＮＡを回収し、ｃＤＮＡクローニングを行った。クローニングされた抗体遺伝子を発現ベクターに組み込んで、バキュロウイルス発現系にて完全長抗体タンパクを発現・分泌させ、抗体を精製した。ＥＬＩＳＡ法にてＶＥＧＦＲ２－アレクサフルオール６４７及びＶＥＧＦＲ２タンパクに対する、精製抗体の結合活性を評価したところ、蛍光色素であるアレクサフルオール６４７特異的に結合活性を示すヒトモノクローナル抗体が、偶発的に同定された。本発明はこれらの知見に基づき完成に至ったものである。
The present inventors have measured autoantibody titers against cancer-associated antigens in blood against several types of cancer-associated antigen proteins (EGFR, VEGFR2, VEGFR3, CDH11, etc.) in 19 cases of malignant glioma patients. Using the peripheral blood B cells of the case (patient) with the highest antibody titer against VEGFR2, staining with Alexa Fluor 647-labeled VEGFR2 protein (extracellular domain) and PE-labeled mouse anti-human IgG antibody gave VEGFR2 specific B cells with antibodies were selected, and mRNA for each cell was collected using Single® cell® sorter® (FACSAria, BD Bioscience), and cDNA cloning was performed. The cloned antibody gene was incorporated into an expression vector, and a full-length antibody protein was expressed and secreted in a baculovirus expression system, and the antibody was purified. As a result of evaluating the binding activity of the purified antibody to VEGFR2-Alexafluor 647 and VEGFR2 protein by ELISA, a human monoclonal antibody showing binding activity specifically to Alexa Fluor 647, a fluorescent dye, was identified accidentally. It was. The present invention has been completed based on these findings.
すなわち、本発明は以下のとおりである。
（１）アレクサフルオール６４７を認識するモノクローナル抗体。
（２）アレクサフルオール６４７を認識する抗体の重鎖可変領域を構成するポリペプチドであって、該重鎖可変領域が配列番号２に示すアミノ酸配列からなる超可変領域を含むことを特徴とするポリペプチド。
（３）アレクサフルオール６４７を認識する抗体の重鎖可変領域を構成するポリペプチドであって、該重鎖可変領域が配列番号４に示すアミノ酸配列、又は配列番号４と少なくとも９７％同一であるアミノ酸配列からなることを特徴とするポリペプチド。
（４）アレクサフルオール６４７を認識する抗体の重鎖を構成するポリペプチドであって、該重鎖が配列番号６に示すアミノ酸配列、又は配列番号６と少なくとも９５％同一であるアミノ酸配列からなることを特徴とするポリペプチド。
（５）アレクサフルオール６４７を認識する抗体の軽鎖可変領域を構成するポリペプチドであって、該軽鎖可変領域が、配列番号８に示すアミノ酸配列からなる超可変領域を含むことを特徴とするポリペプチド。
（６）アレクサフルオール６４７を認識する抗体の軽鎖可変領域を構成するポリペプチドであって、該軽鎖可変領域が、配列番号１０に示すアミノ酸配列、又は配列番号１０と少なくとも９７％同一であるアミノ酸配列からなることを特徴とするポリペプチド。
（７）アレクサフルオール６４７を認識する抗体の軽鎖を構成するポリペプチドであって、該軽鎖が、配列番号１２に示すアミノ酸配列、又は配列番号１２と少なくとも９５％同一であるアミノ酸配列からなることを特徴とするポリペプチド。
（８）上記（２）～（７）のいずれかに記載のポリペプチドをコードすることを特徴とするＤＮＡ。
（９）上記（２）～（４）のいずれかに記載のポリペプチド、及び上記（５）～（７）のいずれかに記載のポリペプチドを含むことを特徴とする抗アレクサフルオール６４７モノクローナル抗体。
（１０）上記（２）又は（３）に記載のポリペプチド、及び上記（５）又は（６）に記載のポリペプチドを含むことを特徴とする上記（９）記載の抗アレクサフルオール６４７モノクローナル抗体。
（１１）上記（４）に記載のポリペプチド、及び上記（７）に記載のポリペプチドを含むことを特徴とする上記（９）記載の抗アレクサフルオール６４７モノクローナル抗体。 That is, the present invention is as follows.
(1) A monoclonal antibody that recognizes Alexa Fluor 647.
(2) A polypeptide constituting the heavy chain variable region of an antibody that recognizes Alexa Fluor 647, wherein the heavy chain variable region includes a hypervariable region comprising the amino acid sequence shown in SEQ ID NO: 2. Polypeptide.
(3) A polypeptide constituting the heavy chain variable region of an antibody that recognizes Alexa Fluor 647, wherein the heavy chain variable region is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 4 A polypeptide comprising an amino acid sequence.
(4) A polypeptide that constitutes the heavy chain of an antibody that recognizes Alexa Fluor 647, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 6 A polypeptide characterized by the above.
(5) A polypeptide constituting a light chain variable region of an antibody that recognizes Alexa Fluor 647, wherein the light chain variable region includes a hypervariable region consisting of the amino acid sequence shown in SEQ ID NO: 8 Polypeptide.
(6) A polypeptide constituting the light chain variable region of an antibody that recognizes Alexa Fluor 647, wherein the light chain variable region is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 10 A polypeptide comprising an amino acid sequence.
(7) A polypeptide that constitutes the light chain of an antibody that recognizes Alexa Fluor 647, wherein the light chain is represented by SEQ ID NO: 12 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 12 A polypeptide characterized by comprising:
(8) A DNA encoding the polypeptide according to any one of (2) to (7) above.
(9) An anti-alexafluor 647 monoclonal comprising the polypeptide according to any one of (2) to (4) above and the polypeptide according to any one of (5) to (7) above. antibody.
(10) The anti-alexafluor 647 monoclonal according to (9) above, comprising the polypeptide according to (2) or (3) above and the polypeptide according to (5) or (6) above antibody.
(11) The anti-alexafluor 647 monoclonal antibody according to (9) above, comprising the polypeptide according to (4) above and the polypeptide according to (7) above.
（１）アレクサフルオール６４７を認識するモノクローナル抗体。
（２）アレクサフルオール６４７を認識する抗体の重鎖可変領域を構成するポリペプチドであって、該重鎖可変領域が配列番号２に示すアミノ酸配列からなる超可変領域を含むことを特徴とするポリペプチド。
（３）アレクサフルオール６４７を認識する抗体の重鎖可変領域を構成するポリペプチドであって、該重鎖可変領域が配列番号４に示すアミノ酸配列、又は配列番号４と少なくとも９７％同一であるアミノ酸配列からなることを特徴とするポリペプチド。
（４）アレクサフルオール６４７を認識する抗体の重鎖を構成するポリペプチドであって、該重鎖が配列番号６に示すアミノ酸配列、又は配列番号６と少なくとも９５％同一であるアミノ酸配列からなることを特徴とするポリペプチド。
（５）アレクサフルオール６４７を認識する抗体の軽鎖可変領域を構成するポリペプチドであって、該軽鎖可変領域が、配列番号８に示すアミノ酸配列からなる超可変領域を含むことを特徴とするポリペプチド。
（６）アレクサフルオール６４７を認識する抗体の軽鎖可変領域を構成するポリペプチドであって、該軽鎖可変領域が、配列番号１０に示すアミノ酸配列、又は配列番号１０と少なくとも９７％同一であるアミノ酸配列からなることを特徴とするポリペプチド。
（７）アレクサフルオール６４７を認識する抗体の軽鎖を構成するポリペプチドであって、該軽鎖が、配列番号１２に示すアミノ酸配列、又は配列番号１２と少なくとも９５％同一であるアミノ酸配列からなることを特徴とするポリペプチド。
（８）上記（２）～（７）のいずれかに記載のポリペプチドをコードすることを特徴とするＤＮＡ。
（９）上記（２）～（４）のいずれかに記載のポリペプチド、及び上記（５）～（７）のいずれかに記載のポリペプチドを含むことを特徴とする抗アレクサフルオール６４７モノクローナル抗体。
（１０）上記（２）又は（３）に記載のポリペプチド、及び上記（５）又は（６）に記載のポリペプチドを含むことを特徴とする上記（９）記載の抗アレクサフルオール６４７モノクローナル抗体。
（１１）上記（４）に記載のポリペプチド、及び上記（７）に記載のポリペプチドを含むことを特徴とする上記（９）記載の抗アレクサフルオール６４７モノクローナル抗体。 That is, the present invention is as follows.
(1) A monoclonal antibody that recognizes Alexa Fluor 647.
(2) A polypeptide constituting the heavy chain variable region of an antibody that recognizes Alexa Fluor 647, wherein the heavy chain variable region includes a hypervariable region comprising the amino acid sequence shown in SEQ ID NO: 2. Polypeptide.
(3) A polypeptide constituting the heavy chain variable region of an antibody that recognizes Alexa Fluor 647, wherein the heavy chain variable region is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 4 A polypeptide comprising an amino acid sequence.
(4) A polypeptide that constitutes the heavy chain of an antibody that recognizes Alexa Fluor 647, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 6 A polypeptide characterized by the above.
(5) A polypeptide constituting a light chain variable region of an antibody that recognizes Alexa Fluor 647, wherein the light chain variable region includes a hypervariable region consisting of the amino acid sequence shown in SEQ ID NO: 8 Polypeptide.
(6) A polypeptide constituting the light chain variable region of an antibody that recognizes Alexa Fluor 647, wherein the light chain variable region is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 10 A polypeptide comprising an amino acid sequence.
(7) A polypeptide that constitutes the light chain of an antibody that recognizes Alexa Fluor 647, wherein the light chain is represented by SEQ ID NO: 12 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 12 A polypeptide characterized by comprising:
(8) A DNA encoding the polypeptide according to any one of (2) to (7) above.
(9) An anti-alexafluor 647 monoclonal comprising the polypeptide according to any one of (2) to (4) above and the polypeptide according to any one of (5) to (7) above. antibody.
(10) The anti-alexafluor 647 monoclonal according to (9) above, comprising the polypeptide according to (2) or (3) above and the polypeptide according to (5) or (6) above antibody.
(11) The anti-alexafluor 647 monoclonal antibody according to (9) above, comprising the polypeptide according to (4) above and the polypeptide according to (7) above.
本発明によると、アレクサフルオール６４７と特異的に結合するモノクローナル抗体を用いることから、アレクサフルオール６４７で標識された物質をモニタリングすることや、アレクサフルオール６４７で標識された物質を定量することが可能となる。
According to the present invention, since a monoclonal antibody that specifically binds to Alexa Fluor 647 is used, a substance labeled with Alexa Fluor 647 can be monitored or a substance labeled with Alexa Fluor 647 can be quantified. Is possible.
本発明のポリペプチドとしては、アレクサフルオール６４７を認識する抗体の重鎖可変領域や重鎖、及び軽鎖可変領域や軽鎖を構成するポリペプチドであって、１）配列番号２に示すアミノ酸配列からなる超可変領域（相補性決定領域：ＣＤＲ３）を含む重鎖可変領域を構成するポリペプチド［Ｐ１］、２）配列番号４に示すアミノ酸配列、若しくは配列番号４と少なくとも９７％同一、好ましくは９８％以上同一、より好ましくは９９％以上同一であるアミノ酸配列からなる重鎖可変領域を構成するポリペプチド［Ｐ２］、３）配列番号６に示すアミノ酸配列、又は配列番号６と少なくとも９５％同一、好ましくは９８％以上同一、より好ましくは９９％以上同一であるアミノ酸配列からなる重鎖を構成するポリペプチド［Ｐ３］、又は、４）配列番号８に示すアミノ酸配列からなる超可変領域（相補性決定領域：ＣＤＲ３）を含む軽鎖可変領域を構成するポリペプチド［Ｐ４］、５）配列番号１０に示すアミノ酸配列、若しくは配列番号１０と少なくとも９７％同一、好ましくは９８％以上同一、より好ましくは９９％以上同一であるアミノ酸配列からなる軽鎖可変領域を構成するポリペプチド［Ｐ５］、６）配列番号１２に示すアミノ酸配列、若しくは配列番号１２と少なくとも９５％同一、好ましくは９８％以上同一、より好ましくは９９％以上同一であるアミノ酸配列からなる軽鎖を構成するポリペプチド［Ｐ６］を挙げることができる。
The polypeptide of the present invention is a polypeptide constituting the heavy chain variable region or heavy chain, and the light chain variable region or light chain of an antibody that recognizes Alexa Fluor 647, and 1) the amino acid shown in SEQ ID NO: 2 Polypeptide [P1] constituting a heavy chain variable region comprising a hypervariable region consisting of a sequence (complementarity determining region: CDR3), 2) the amino acid sequence shown in SEQ ID NO: 4, or at least 97% identical to SEQ ID NO: 4, preferably Is a polypeptide [P2] comprising a heavy chain variable region consisting of an amino acid sequence that is 98% or more identical, more preferably 99% or more identical, 3) the amino acid sequence shown in SEQ ID NO: 6, or at least 95% with SEQ ID NO: 6 A polypeptide [P3] constituting a heavy chain consisting of amino acid sequences that are identical, preferably 98% or more identical, more preferably 99% or more identical, or 4) Polypeptide constituting the light chain variable region comprising the hypervariable region (complementarity determining region: CDR3) consisting of the amino acid sequence shown in SEQ ID NO: 8 [5] 5) Amino acid sequence shown in SEQ ID NO: 10, or SEQ ID NO: A polypeptide comprising a light chain variable region consisting of an amino acid sequence that is at least 97% identical, preferably 98% or more identical, more preferably 99% or more identical to 10 [6], 6) the amino acid sequence shown in SEQ ID NO: 12, Another example is a polypeptide [P6] constituting a light chain consisting of an amino acid sequence that is at least 95% identical, preferably 98% or more identical, more preferably 99% or more identical to SEQ ID NO: 12.
本発明のＤＮＡとしては、上記本発明のアレクサフルオール６４７を認識する抗体の重鎖可変領域を構成するポリペプチド［Ｐ１］及び［Ｐ２］、重鎖を構成するポリペプチド［Ｐ３］、軽鎖可変領域を構成するポリペプチド［Ｐ４］及び［Ｐ５］、並びに、軽鎖を構成するポリペプチド［Ｐ６］をそれぞれコードするＤＮＡであれば特に制限されず、より具体的には、１）重鎖超可変領域をコードする配列番号１に示す塩基配列を含む重鎖可変領域をコードするＤＮＡ［Ｄ１］、２）重鎖可変領域をコードする配列番号３に示す塩基配列、若しくは配列番号３と少なくとも９５％同一、好ましくは９８％以上同一、より好ましくは９９％以上同一である塩基配列を含むＤＮＡ［Ｄ２］、３）重鎖をコードする配列番号５に示す塩基配列、若しくは配列番号５と少なくとも９０％同一、好ましくは９５％以上同一、より好ましくは９８％以上同一である塩基配列からなるＤＮＡ［Ｄ３］、４）軽鎖超可変領域をコードする配列番号７に示す塩基配列を含む軽鎖可変領域をコードするＤＮＡ［Ｄ４］、５）軽鎖可変領域をコードする配列番号９に示す塩基配列、若しくは配列番号９と少なくとも９５％同一、好ましくは９８％以上同一、より好ましくは９９％以上同一である塩基配列を含むＤＮＡ［Ｄ５］、６）軽鎖をコードする配列番号１１に示す塩基配列、若しくは配列番号１１と少なくとも９０％同一、好ましくは９５％以上同一、より好ましくは９８％以上同一である塩基配列からなるＤＮＡ［Ｄ６］を挙げることができる。
Examples of the DNA of the present invention include polypeptides [P1] and [P2] constituting the heavy chain variable region of the antibody recognizing the above-described Alexa Fluor 647 of the present invention, polypeptides [P3] constituting the heavy chain, light chains The DNA is not particularly limited as long as it encodes the polypeptides [P4] and [P5] constituting the variable region and the polypeptide [P6] constituting the light chain, and more specifically, 1) heavy chain DNA [D1] encoding a heavy chain variable region including the base sequence shown in SEQ ID NO: 1 encoding the hypervariable region, 2) a base sequence shown in SEQ ID NO: 3 encoding the heavy chain variable region, or at least SEQ ID NO: 3 and DNA [D2] comprising a nucleotide sequence that is 95% identical, preferably 98% or more identical, more preferably 99% or more identical, 3) the nucleotide sequence shown in SEQ ID NO: 5 encoding the heavy chain, Or DNA [D3] consisting of a base sequence that is at least 90% identical to SEQ ID NO: 5, preferably 95% or more identical, more preferably 98% or more identical, and 4) SEQ ID NO: 7 encoding the light chain hypervariable region. DNA [D4] encoding a light chain variable region containing the nucleotide sequence shown [5], 5) the nucleotide sequence shown in SEQ ID NO: 9 encoding the light chain variable region, or at least 95% identical, preferably 98% or more identical More preferably, DNA [D5] comprising a nucleotide sequence that is 99% or more identical, 6) the nucleotide sequence shown in SEQ ID NO: 11 encoding the light chain, or at least 90% identical, preferably 95% or more identical to SEQ ID NO: 11. More preferably, DNA [D6] consisting of a base sequence that is 98% or more identical can be mentioned.
また、上記アミノ酸配列や塩基配列において、例えば、少なくとも９５％同一とは、問題の配列が、アミノ酸やヌクレオチドの各挿入、欠失、置換等をただ１つの違いとして数えたとき、比較しようとしている配列と５％未満の異なるアミノ酸やヌクレオチドを含むことを意味する。
In addition, in the above amino acid sequence or base sequence, for example, at least 95% identity is to be compared when the sequence in question is counted as only one difference in each amino acid or nucleotide insertion, deletion, substitution, etc. Means containing less than 5% different amino acids and nucleotides from the sequence.
本発明のモノクローナル抗体としては、アレクサフルオール６４７に特異的に結合する抗アレクサフルオール６４７モノクローナル抗体であれば特に制限されず、抗アレクサフルオール６４７モノクローナル抗体の重鎖可変領域を構成するポリペプチド［Ｐ１］及び［Ｐ２］、重鎖を構成するポリペプチド［Ｐ３］のいずれかと、軽鎖可変領域を構成するポリペプチド［Ｐ４］及び［Ｐ５］、軽鎖を構成するポリペプチド［Ｐ６］のいずれかを含むモノクローナル抗体、例えば、［Ｐ１］と［Ｐ４］、［Ｐ２］と［Ｐ５］、［Ｐ３］と［Ｐ６］、［Ｐ１］と［Ｐ５］、［Ｐ２］と［Ｐ４］等をそれぞれ含むモノクローナル抗体を具体的に挙げることができる。
The monoclonal antibody of the present invention is not particularly limited as long as it is an anti-alexafluor 647 monoclonal antibody that specifically binds to alexafluor 647, and a polypeptide constituting the heavy chain variable region of the anti-alexafluor 647 monoclonal antibody [P1] and [P2], one of the polypeptides [P3] constituting the heavy chain, the polypeptides [P4] and [P5] constituting the light chain variable region, and the polypeptide [P6] constituting the light chain Monoclonal antibodies containing any of them, for example, [P1] and [P4], [P2] and [P5], [P3] and [P6], [P1] and [P5], [P2] and [P4], etc. Specific examples thereof include monoclonal antibodies.
本発明の抗アレクサフルオール６４７モノクローナル抗体には、ＩｇＧ、ＩｇＡ、ＩｇＭ、ＩｇＤ、ＩｇＥ等のいずれのアイソタイプを有するモノクローナル抗体が含まれ、また、本発明の抗アレクサフルオール６４７モノクローナル抗体には、完全長（Full body）モノクローナル抗体の他、モノクローナル抗体の活性フラグメント、例えば、Ｆ（ａｂ′）２、Ｆａｂ′、Ｆａｂ、Ｆｖ（variable fragment of antibody）、ｓＦｖ、ｄｓＦｖ（disulphide stabilised Fv）、ｄＡｂ（single domain antibody）等も含まれる。
The anti-alexafluor 647 monoclonal antibody of the present invention includes monoclonal antibodies having any isotype such as IgG, IgA, IgM, IgD, and IgE. The anti-alexafluor 647 monoclonal antibody of the present invention includes In addition to full-body monoclonal antibodies, active fragments of monoclonal antibodies such as F (ab ′) 2 , Fab ′, Fab, Fv (variable fragment of antibody), sFv, dsFv (disulphide stabilized Fv), dAb ( single domain antibody) and the like.
本発明の抗アレクサフルオール６４７モノクローナル抗体の完全長抗体の作製方法としては、抗アレクサフルオール６４７モノクローナル抗体の重鎖をコードするＤＮＡ［Ｄ３］と軽鎖をコードするＤＮＡ［Ｄ６］を組み込んだ発現ベクターにより宿主細胞を形質転換し、形質転換細胞を適切な培地で培養して発現させることにより得ることができる。Ｆａｂ抗体の作製には、精製された上記完全長抗体をパパイン消化して、Ｆａｂ断片のみを再度精製する従来法が挙げられる。また、一本鎖Ｆｖフラグメント（ｓｃＦｖ）抗体の作製には、抗アレクサフルオール６４７モノクローナル抗体の重鎖可変領域をコードするＤＮＡ［Ｄ１］又は［Ｄ２］と軽鎖可変領域をコードするＤＮＡ［Ｄ４］又は［Ｄ５］とをリンカーで連結して宿主細胞内で発現させる方法等を挙げることができる。
As a method for producing the full-length antibody of the anti-alexafluor 647 monoclonal antibody of the present invention, DNA [D3] encoding the heavy chain of anti-alexafluor 647 monoclonal antibody and DNA [D6] encoding the light chain were incorporated. It can be obtained by transforming a host cell with an expression vector and culturing the transformed cell in an appropriate medium for expression. The production of the Fab antibody includes a conventional method in which the purified full-length antibody is digested with papain and only the Fab fragment is purified again. In addition, for the production of a single-chain Fv fragment (scFv) antibody, DNA [D1] or [D2] encoding the heavy chain variable region of anti-alexafluor 647 monoclonal antibody and DNA [D4] encoding the light chain variable region. ] Or [D5] linked by a linker and expressed in a host cell.
本発明の抗アレクサフルオール６４７モノクローナル抗体には、抗体可変領域（Ｆｖ）と、様々な動物種由来の抗体定常領域（Ｆｃ）を組み合わせたキメラ抗体やヒト化抗体も含まれる。かかるキメラ抗体やヒト化抗体は、Ｆｖ領域をコードする遺伝子とＦｃ領域をコードする遺伝子と結合させて発現させることにより、調製することができる。かかる動物種としては、一般的な抗体の動物種であるマウス、ラット、ハムスター、モルモット、ウサギ、ヤギ、ヒツジ、ロバ、ブタ、ウシ、ウマ、イヌ、ネコ、ニワトリ、サル、ヒト等を例示することができる。定常領域は、可変領域の由来するモノクローナル抗体と同一のものであっても、あるいは、異なるモノクローナル抗体に由来するものであってもよい。例えば、Ｆｃ領域として、公知のヒトのＩｇＧ１、マウスのＩｇＧ１のＦｃ領域が使用可能である。
The anti-alexafluor 647 monoclonal antibody of the present invention includes chimeric antibodies and humanized antibodies that combine antibody variable regions (Fv) and antibody constant regions (Fc) derived from various animal species. Such a chimeric antibody or humanized antibody can be prepared by binding and expressing a gene encoding an Fv region and a gene encoding an Fc region. Examples of such animal species include mice, rats, hamsters, guinea pigs, rabbits, goats, sheep, donkeys, pigs, cows, horses, dogs, cats, chickens, monkeys, humans, etc., which are general antibody animal species. be able to. The constant region may be the same as the monoclonal antibody from which the variable region is derived, or may be derived from a different monoclonal antibody. For example, a known human IgG1 or mouse IgG1 Fc region can be used as the Fc region.
なお、サンドイッチＥＬＩＳＡ法において本発明のヒト抗アレクサフルオール６４７モノクローナル抗体を捕獲抗体として用いる場合には、かかる捕獲抗体の定常領域が検出抗体の定常領域と異なる動物種由来とすることで、捕獲抗体と検出抗体との交差反応をより防ぐことが可能となる。例えば、図１に示すように、検出抗体として酵素標識抗ヒトＩｇＧ抗体等の抗ヒト抗体を用いる場合には、捕獲抗体としてヒト－マウスキメラ抗体（マウス化抗体）を用いればよい。前記ヒト－マウスキメラ抗体は、例えば、ヒト由来の配列番号６に示すアミノ酸配列からなるポリペプチドコードする遺伝子のうちＦｃ領域配列を人工合成されたマウス由来のＦｃ領域配列と入れ替えた、配列番号１３に示すアミノ酸配列からなるポリペプチドをコードする遺伝子を発現する発現ベクターを作製し、ヒト由来の配列番号１２に示すアミノ酸配列をコードする遺伝子と共にｅｘｐｉ２９３細胞のような哺乳類細胞に導入して培養上清中に産生させることにより得ることができる。
In the sandwich ELISA method, when the human anti-alexafluor 647 monoclonal antibody of the present invention is used as a capture antibody, the capture antibody has a constant region derived from an animal species different from the constant region of the detection antibody. And the cross-reaction between the detection antibody and the detection antibody can be further prevented. For example, as shown in FIG. 1, when an anti-human antibody such as an enzyme-labeled anti-human IgG antibody is used as the detection antibody, a human-mouse chimeric antibody (mouse antibody) may be used as the capture antibody. The human-mouse chimeric antibody is, for example, SEQ ID NO: 13 in which the Fc region sequence of a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6 derived from human is replaced with an artificially synthesized mouse-derived Fc region sequence. An expression vector that expresses a gene encoding a polypeptide consisting of the amino acid sequence shown in FIG. 5 is prepared, and introduced into a mammalian cell such as exp293 cells together with a human-derived gene encoding the amino acid sequence shown in SEQ ID NO: 12. It can be obtained by producing in.
本発明のモノクローナル抗体の製造に用いることができる発現ベクターとしては、本発明のポリペプチドをコードするＤＮＡを、大腸菌、枯草金、酵母、昆虫細胞、植物細胞、動物細胞等の宿主細胞において発現しうるものが好ましく、バキュロウイルスのAutographa californica nuclear polyhedrosis virus（AcNPV）ベクター及びバキュロウイルス用のシャトルベクター（pFastBac Dual vector）を好適に挙げることができる。また、宿主細胞がカイコ（Bombix mori）細胞のＢｍＮ４では、バキュロウイルスの核多角体病ウイルスBombix mori nuclear polyhedrosis virus（BmNPV）ベクターを好適に挙げることができる。また、上記発現ベクターには、プロモーター、ターミネーター、マーカー遺伝子としての薬剤耐性遺伝子を含んでいてもよい。
As an expression vector that can be used for production of the monoclonal antibody of the present invention, a DNA encoding the polypeptide of the present invention is expressed in host cells such as Escherichia coli, hay, yeast, insect cells, plant cells, and animal cells. Among them, preferred are baculovirus Autographaifcalifornica nuclear polyhedrosis virus (AcNPV) vector and baculovirus shuttle vector (pFastBac Dual 好 適 vector). In addition, BmN4 of a Bombyxmori host cell is preferably a baculovirus nuclear polyhedrosis virus Bombyxmori polynucledrosis virus (BmNPV) vector. The expression vector may contain a drug resistance gene as a promoter, terminator, and marker gene.
また、宿主細胞へのベクターの導入法としては、In-Fusionクローニングシステム（Clontech社）、リポソーム法、リポフェクション法、マイクロインジェクション法、ＤＥＡＥデキストラン法、リン酸カルシウム法、エレクトロポレーション法等を挙げることができ、Lipofectin Reagent（登録商標）、Lipofectamine（登録商標）、Lipofectamine（登録商標）2000 Reagent（インビトロジェン社製）や、SuperFect（登録商標）Transfection Reagent（キアゲン社製）、FuGENE（登録商標）HD Transfection Reagent（ロシュ・ダイアグノスティックス社製）、FuGENE（登録商標）6 Transfection Reagent（ロシュ・ダイアグノスティックス社製）等の市販のトランスフェクション試薬を用いる当技術分野で広く用いられている手法を挙げることができる。
Examples of the method for introducing a vector into a host cell include In-Fusion cloning system (Clontech), liposome method, lipofection method, microinjection method, DEAE dextran method, calcium phosphate method, electroporation method and the like. , Lipofectin Reagent (registered trademark), Lipofectamine (registered trademark), Lipofectamine (registered trademark) 2000 Reagent (manufactured by Invitrogen), SuperFect (registered trademark) Transfection Reagent (manufactured by Qiagen), FuGENE (registered trademark) HD Transfection Reagent ( Examples of methods widely used in the art using commercially available transfection reagents such as Roche Diagnostics) and FuGENE® 6 Transfection Reagent (Roche Diagnostics). Can do.
本発明の抗アレクサフルオール６４７モノクローナル抗体は、被検非ヒト動物対象に投与された、アレクサフルオール６４７で標識された物質のモニタリングに有利に使用することができる。より詳細には、特定疾患の生物学的製剤（医薬品）の開発に向け実験動物に使用する場合を例として、医薬品への感受性の確認や投与間隔及び投与量の調整等を目的とした、生物学的製剤濃度による有効性、及びin vivo／in situにおける生物学的製剤の局在に対するモニタリングに有利に使用することができる。具体的には、アレクサフルオール６４７で標識された開発候補医薬品を実験動物に投与したのち、適宜観察対象の臓器を取り出し、本発明の抗体を用いた免疫組織化学法、免疫沈降法、ＥＬＩＳＡ法、ウェスタンブロッティング法等により、対象の臓器に局在する該医薬品を同定、可視化、定量する手法を例示することができる。
The anti-Alexafluor 647 monoclonal antibody of the present invention can be advantageously used for monitoring a substance labeled with Alexa Fluor 647 administered to a test non-human animal subject. More specifically, for the purpose of confirming sensitivity to pharmaceuticals and adjusting dosage intervals and dosages, for example, when used in experimental animals for the development of biological products (medicines) for specific diseases It can be advantageously used for monitoring the efficacy by biological formulation concentration and the localization of biological products in vivo / in situ. Specifically, after the development candidate drug labeled with Alexa Fluor 647 is administered to an experimental animal, an organ to be observed is appropriately taken out, and an immunohistochemistry method, an immunoprecipitation method, an ELISA method using the antibody of the present invention. A method for identifying, visualizing, and quantifying the drug localized in the target organ by Western blotting or the like can be exemplified.
本発明の抗アレクサフルオール６４７モノクローナル抗体は、磁気細胞分離法に基づく細胞分離・濃縮を目的とした利用を例示することができ、ＭＡＣＳ（登録商標）細胞分離システム（Miltenyi Biotec社）や、Dynabeads（登録商標）細胞分離システム（インビトロジェン社）、ＢＤ ＩＭａｇ（商標）細胞分離システム等好適に挙げることができる。（ａ）アレクサフルオール６４７で標識された任意の抗体と特異的に結合している濃縮対象細胞を含む、細胞懸濁液試料を調製し；（ｂ）前記試料に、本発明の抗アレクサフルオール６４７モノクローナル抗体が結合した磁気ビーズを添加し、濃縮対象細胞をビーズに結合させ；（ｃ）前記試料を磁場の存在下で強磁性マトリックスに通過させ、強磁性マトリックスに結合しない濃縮非対象細胞を回収し；（ｄ）濃縮対象細胞を提供するために、実質的に磁場の非存在下で該マトリックスから結合細胞を溶出し、濃縮対象細胞を回収する方法を挙げることができる。磁気細胞分離法に基づく細胞分離・濃縮方法の概略を図２に示す。
The anti-alexafluor 647 monoclonal antibody of the present invention can be used for the purpose of cell separation / concentration based on the magnetic cell separation method, such as MACS (registered trademark) cell separation system (Miltenyi Biotec), Dynabeads (Registered trademark) cell separation system (Invitrogen), BD IMag (trademark) cell separation system, etc. can be mentioned suitably. (A) preparing a cell suspension sample containing cells to be enriched specifically bound to any antibody labeled with Alexa Fluor 647; (b) adding the anti-alexaflu of the present invention to the sample; Add magnetic beads bound with all 647 monoclonal antibody to bind the cells to be enriched to the beads; (c) pass the sample through the ferromagnetic matrix in the presence of a magnetic field and do not bind to the ferromagnetic matrix And (d) a method of eluting the bound cells from the matrix in the absence of a magnetic field and recovering the enrichment target cells in order to provide the enrichment target cells. An outline of the cell separation / concentration method based on the magnetic cell separation method is shown in FIG.
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。また、実施例において使用した患者検体（細胞および血漿）は、静岡県立静岡がんセンターにて治療中の患者の同意のもと、施設内の臨床研究倫理審査委員会の承認を受けた研究の範囲内で使用された。
Hereinafter, the present invention will be described more specifically by way of examples. However, the technical scope of the present invention is not limited to these examples. In addition, the patient specimens (cells and plasma) used in the examples are those of studies approved by the institutional clinical ethics review committee with the consent of the patient being treated at the Shizuoka Cancer Center. Used within range.
１．悪性グリオーマ患者血液中の自家抗体測定
悪性グリオーマ１９症例においてがん関連抗原タンパクの上皮成長因子受容体（ＥＧＦＲ）、血管内皮細胞増殖因子受容体（ＶＥＧＦＲ２及びＶＥＧＦＲ３）、カドヘリン（ＣＤＨ１１）、血小板凝集因子（Ｐｏｄｏｐｌａｎｉｎ）及び陰性コントロールのグルタチオンＳ－トランスフェラーゼ（ＧＳＴ）に対する、患者血液中の自家抗体の測定を、特開２０１０－２３７１２６号公報に開示される、本発明者が開発した「ＥＬＩＳＡ法を用いたヒト血清中の抗原特異的なＩｇＧ抗体力価測定の標準化法」にて実施した（図３）。
各組換えタンパク２０ｎｇを９６ウェルプレートに固相化し、４℃で一晩インキュベートした後、３％ＢＳＡ溶液にてブロッキングを行った。次に希釈した患者由来の血漿１００ｕｌを添加し、２時間室温にてインキュベートした。洗浄後、１０００倍希釈のＨＲＰ標識ヒツジ抗ヒトＩｇＧ抗体（GEヘルスケア社製）、その後発色基質を添加し、イムノリーダー（Immuno Mini NJ-2300、ナルジェヌンク社製）にて吸光度の測定を行った。
その結果、ＧＢ－ＳＣＣ００８症例の血液中には、ＥＧＦＲ、ＶＥＧＦＲ２及びＶＥＧＦＲ３について自家抗体が顕著に存在することが示された。また、ＧＢ－ＳＣＣ００８症例の血液中の各種がん関連抗原タンパクに対する自家抗体は、ウェスタンブロッティングによっても確認できた（図４）。 1. Autoantibody measurement in blood of malignant glioma patients In 19 cases of malignant glioma, cancer-associated antigen protein epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptors (VEGFR2 and VEGFR3), cadherin (CDH11), platelet aggregation factor Measurement of autoantibodies in patient blood against (Podoplanin) and negative control glutathione S-transferase (GST) was performed by the inventors using the “ELISA method” disclosed in Japanese Patent Application Laid-Open No. 2010-237126. This was carried out by “standardization method of antigen-specific IgG antibody titer measurement in human serum” (FIG. 3).
20 ng of each recombinant protein was immobilized on a 96-well plate and incubated overnight at 4 ° C., followed by blocking with a 3% BSA solution. Next, 100 ul of diluted patient plasma was added and incubated for 2 hours at room temperature. After washing, 1000-fold diluted HRP-labeled sheep anti-human IgG antibody (GE Healthcare) was added, and then a chromogenic substrate was added, and the absorbance was measured with an immunoleader (Immuno Mini NJ-2300, manufactured by Nargenunk). .
As a result, it was shown that autologous antibodies for EGFR, VEGFR2 and VEGFR3 were significantly present in the blood of GB-SCC008 cases. In addition, autoantibodies against various cancer-related antigen proteins in the blood of GB-SCC008 cases were confirmed by Western blotting (FIG. 4).
悪性グリオーマ１９症例においてがん関連抗原タンパクの上皮成長因子受容体（ＥＧＦＲ）、血管内皮細胞増殖因子受容体（ＶＥＧＦＲ２及びＶＥＧＦＲ３）、カドヘリン（ＣＤＨ１１）、血小板凝集因子（Ｐｏｄｏｐｌａｎｉｎ）及び陰性コントロールのグルタチオンＳ－トランスフェラーゼ（ＧＳＴ）に対する、患者血液中の自家抗体の測定を、特開２０１０－２３７１２６号公報に開示される、本発明者が開発した「ＥＬＩＳＡ法を用いたヒト血清中の抗原特異的なＩｇＧ抗体力価測定の標準化法」にて実施した（図３）。
各組換えタンパク２０ｎｇを９６ウェルプレートに固相化し、４℃で一晩インキュベートした後、３％ＢＳＡ溶液にてブロッキングを行った。次に希釈した患者由来の血漿１００ｕｌを添加し、２時間室温にてインキュベートした。洗浄後、１０００倍希釈のＨＲＰ標識ヒツジ抗ヒトＩｇＧ抗体（GEヘルスケア社製）、その後発色基質を添加し、イムノリーダー（Immuno Mini NJ-2300、ナルジェヌンク社製）にて吸光度の測定を行った。
その結果、ＧＢ－ＳＣＣ００８症例の血液中には、ＥＧＦＲ、ＶＥＧＦＲ２及びＶＥＧＦＲ３について自家抗体が顕著に存在することが示された。また、ＧＢ－ＳＣＣ００８症例の血液中の各種がん関連抗原タンパクに対する自家抗体は、ウェスタンブロッティングによっても確認できた（図４）。 1. Autoantibody measurement in blood of malignant glioma patients In 19 cases of malignant glioma, cancer-associated antigen protein epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptors (VEGFR2 and VEGFR3), cadherin (CDH11), platelet aggregation factor Measurement of autoantibodies in patient blood against (Podoplanin) and negative control glutathione S-transferase (GST) was performed by the inventors using the “ELISA method” disclosed in Japanese Patent Application Laid-Open No. 2010-237126. This was carried out by “standardization method of antigen-specific IgG antibody titer measurement in human serum” (FIG. 3).
20 ng of each recombinant protein was immobilized on a 96-well plate and incubated overnight at 4 ° C., followed by blocking with a 3% BSA solution. Next, 100 ul of diluted patient plasma was added and incubated for 2 hours at room temperature. After washing, 1000-fold diluted HRP-labeled sheep anti-human IgG antibody (GE Healthcare) was added, and then a chromogenic substrate was added, and the absorbance was measured with an immunoleader (Immuno Mini NJ-2300, manufactured by Nargenunk). .
As a result, it was shown that autologous antibodies for EGFR, VEGFR2 and VEGFR3 were significantly present in the blood of GB-SCC008 cases. In addition, autoantibodies against various cancer-related antigen proteins in the blood of GB-SCC008 cases were confirmed by Western blotting (FIG. 4).
２．シングルセルソーターによるＶＥＧＦＲ２－アレクサフルオール６４７特異的抗体産生Ｂ細胞の分取
ＧＢ－ＳＣＣ００８症例の全血からFicoll-PaqueTM plus（GEヘルスケア社製）処理による密度勾配遠心分離を行い、末梢血単核球を分離した後、CD19マイクロビーズ（Miltenyi社製）を用いてAutoMACS (Miltenyi社製）装置にてCD19陽性細胞を選別した。その後、アレクサフルオール６４７標識ＶＥＧＦＲ２（細胞外ドメイン）タンパク(当研究室にて作製)、ＰＥ標識マウス抗ヒトＩｇＧ抗体（BD Bioscience社製）、ＰｅｒＣＰ標識マウス抗ヒトＣＤ１４モノクローナル抗体（クローン：MfP9）（BD Bioscience社製）及びＰＩ（propidium iodide）を用いてＶＥＧＦＲ２抗体を産生する細胞分取用の染色を行った。また抗CEACAM5マウスハイブリドーマ細胞（クローン：T84.66A3.1A.1F2, ATCC, cat. HB-8747）を使用した実験では、アレクサフルオール６４７標識ヒトCEA-related cell adhesion molecules（ＣＥＡＣＡＭ５）タンパク(当研究室にて作製)、ＰＥ標識ラット抗マウスＣＤ１３８モノクローナル抗体（クローン：２８１－２）（BD Bioscience社製）を使用して細胞の染色を行った。
シングルセルソーター(FACSAria，BD Bioscience社製)を用いて、ＰＩ陰性生細胞のＣＤ１４陰性かつＣＤ１９陽性Ｂ細胞のうち、ＶＥＧＦＲ２（細胞外ドメイン）タンパクに結合する免疫グロブリンＧ（ＩｇＧ）が細胞膜に結合しているＢ細胞にゲートを設定したところ、ＣＤ１９陽性細胞の０．０６％がＶＥＧＦＲ２特異的抗体産生Ｂ細胞であった（図５Ｂ）。セルソーターの検出感度は、抗ＣＥＡＣＡＭ５マウスハイブリドーマ細胞を含む患者末梢血単核球を用いて検討した。含まれる抗ＣＥＡＣＡＭ５マウスハイブリドーマ細胞は、アレクサフルオール６４７標識ＣＥＡＣＡＭ５抗原と抗マウスＣＤ１３８抗体を用いて検出した。セルソーターの検出限界は、１０細胞／１０６末梢血単核球であり、０．００１％であった（図５Ａ）。 2. Separation of VEGFR2-Alexafluor 647-specific antibody-producing B cells using a single cell sorter Whole blood from GB-SCC008 cases was subjected to density gradient centrifugation using Ficoll-Paque TM plus (manufactured by GE Healthcare). After separating the nuclei, CD19 positive cells were selected with an AutoMACS (Miltenyi) apparatus using CD19 microbeads (Miltenyi). Then, Alexa Fluor 647-labeled VEGFR2 (extracellular domain) protein (produced in this laboratory), PE-labeled mouse anti-human IgG antibody (manufactured by BD Bioscience), PerCP-labeled mouse anti-human CD14 monoclonal antibody (clone: MfP9) (BD Bioscience) and PI (propidium iodide) were used for staining for cell sorting to produce VEGFR2 antibody. In experiments using anti-CEACAM5 mouse hybridoma cells (clone: T84.66A3.1A.1F2, ATCC, cat. HB-8747), Alexa Fluor 647-labeled human CEA-related cell adhesion molecules (CEACAM5) protein (this study) The cells were stained using PE labeled rat anti-mouse CD138 monoclonal antibody (clone: 281-2) (BD Bioscience).
Using a single cell sorter (FACSAria, manufactured by BD Bioscience), immunoglobulin G (IgG) that binds to VEGFR2 (extracellular domain) protein among CD14 negative and CD19 positive B cells of PI negative live cells binds to the cell membrane. When the B cells were gated, 0.06% of CD19 positive cells were VEGFR2-specific antibody-producing B cells (FIG. 5B). The detection sensitivity of the cell sorter was examined using patient peripheral blood mononuclear cells containing anti-CEACAM5 mouse hybridoma cells. The contained anti-CEACAM5 mouse hybridoma cells were detected using Alexa Fluor 647-labeled CEACAM5 antigen and anti-mouse CD138 antibody. The detection limit of the cell sorter was 10 cells / 10 6 peripheral blood mononuclear cells, 0.001% (FIG. 5A).
ＧＢ－ＳＣＣ００８症例の全血からFicoll-PaqueTM plus（GEヘルスケア社製）処理による密度勾配遠心分離を行い、末梢血単核球を分離した後、CD19マイクロビーズ（Miltenyi社製）を用いてAutoMACS (Miltenyi社製）装置にてCD19陽性細胞を選別した。その後、アレクサフルオール６４７標識ＶＥＧＦＲ２（細胞外ドメイン）タンパク(当研究室にて作製)、ＰＥ標識マウス抗ヒトＩｇＧ抗体（BD Bioscience社製）、ＰｅｒＣＰ標識マウス抗ヒトＣＤ１４モノクローナル抗体（クローン：MfP9）（BD Bioscience社製）及びＰＩ（propidium iodide）を用いてＶＥＧＦＲ２抗体を産生する細胞分取用の染色を行った。また抗CEACAM5マウスハイブリドーマ細胞（クローン：T84.66A3.1A.1F2, ATCC, cat. HB-8747）を使用した実験では、アレクサフルオール６４７標識ヒトCEA-related cell adhesion molecules（ＣＥＡＣＡＭ５）タンパク(当研究室にて作製)、ＰＥ標識ラット抗マウスＣＤ１３８モノクローナル抗体（クローン：２８１－２）（BD Bioscience社製）を使用して細胞の染色を行った。
シングルセルソーター(FACSAria，BD Bioscience社製)を用いて、ＰＩ陰性生細胞のＣＤ１４陰性かつＣＤ１９陽性Ｂ細胞のうち、ＶＥＧＦＲ２（細胞外ドメイン）タンパクに結合する免疫グロブリンＧ（ＩｇＧ）が細胞膜に結合しているＢ細胞にゲートを設定したところ、ＣＤ１９陽性細胞の０．０６％がＶＥＧＦＲ２特異的抗体産生Ｂ細胞であった（図５Ｂ）。セルソーターの検出感度は、抗ＣＥＡＣＡＭ５マウスハイブリドーマ細胞を含む患者末梢血単核球を用いて検討した。含まれる抗ＣＥＡＣＡＭ５マウスハイブリドーマ細胞は、アレクサフルオール６４７標識ＣＥＡＣＡＭ５抗原と抗マウスＣＤ１３８抗体を用いて検出した。セルソーターの検出限界は、１０細胞／１０６末梢血単核球であり、０．００１％であった（図５Ａ）。 2. Separation of VEGFR2-Alexafluor 647-specific antibody-producing B cells using a single cell sorter Whole blood from GB-SCC008 cases was subjected to density gradient centrifugation using Ficoll-Paque TM plus (manufactured by GE Healthcare). After separating the nuclei, CD19 positive cells were selected with an AutoMACS (Miltenyi) apparatus using CD19 microbeads (Miltenyi). Then, Alexa Fluor 647-labeled VEGFR2 (extracellular domain) protein (produced in this laboratory), PE-labeled mouse anti-human IgG antibody (manufactured by BD Bioscience), PerCP-labeled mouse anti-human CD14 monoclonal antibody (clone: MfP9) (BD Bioscience) and PI (propidium iodide) were used for staining for cell sorting to produce VEGFR2 antibody. In experiments using anti-CEACAM5 mouse hybridoma cells (clone: T84.66A3.1A.1F2, ATCC, cat. HB-8747), Alexa Fluor 647-labeled human CEA-related cell adhesion molecules (CEACAM5) protein (this study) The cells were stained using PE labeled rat anti-mouse CD138 monoclonal antibody (clone: 281-2) (BD Bioscience).
Using a single cell sorter (FACSAria, manufactured by BD Bioscience), immunoglobulin G (IgG) that binds to VEGFR2 (extracellular domain) protein among CD14 negative and CD19 positive B cells of PI negative live cells binds to the cell membrane. When the B cells were gated, 0.06% of CD19 positive cells were VEGFR2-specific antibody-producing B cells (FIG. 5B). The detection sensitivity of the cell sorter was examined using patient peripheral blood mononuclear cells containing anti-CEACAM5 mouse hybridoma cells. The contained anti-CEACAM5 mouse hybridoma cells were detected using Alexa Fluor 647-labeled CEACAM5 antigen and anti-mouse CD138 antibody. The detection limit of the cell sorter was 10 cells / 10 6 peripheral blood mononuclear cells, 0.001% (FIG. 5A).
３．１細胞レベルでの抗体遺伝子の解析及び同定
上記により、セルソーターで１細胞ごとに分取したＶＥＧＦＲ２－アレクサフルオール６４７特異的抗体産生Ｂ細胞の、１細胞レベルでの抗体遺伝子の解析を、特許番号第５２０５５９７号公報に開示される、本発明者が開発した「１細胞レベルでの抗体遺伝子の解析・同定方法」に準拠して実施した。まずセルソーターにて１細胞ごとに９６ウェルプレートに分取した後、細胞を破砕させ、直接１細胞ごとにｍＲＮＡを回収した。その後ＲＴ－ＰＣＲを行ったところ、採取した６０個のＢ細胞のうち４０個でＩｇＨ遺伝子の増幅に成功した（図６ 最上段）。また、分取したウェルごとの細胞の有無を確認するために実施した、βアクチンのＲＴ－ＰＣＲでは、３８個で成功した（図６ 最下段）。最終的に同一細胞でＩｇＨ及びＩｇＬ遺伝子がともに増幅された細胞は、２２個であった。これらのすべての細胞からＨ鎖及びＬ（kappa）鎖の抗体遺伝子のｃＤＮＡがクローニングされた。 3. Analysis and identification of antibody genes at the level of 1 cell According to the above, analysis of antibody genes at the level of 1 cell of BEGFR2-Alexafluor 647-specific antibody-producing B cells sorted for each cell with a cell sorter, This was carried out in accordance with “A method for analyzing and identifying antibody genes at the level of one cell” developed by the present inventors and disclosed in Japanese Patent No. 5205597. First, after sorting each cell into a 96-well plate using a cell sorter, the cells were disrupted, and mRNA was directly collected for each cell. Subsequently, RT-PCR was performed, and the IgH gene was successfully amplified in 40 of the collected 60 B cells (the top row in FIG. 6). In addition, the β-actin RT-PCR carried out to confirm the presence of cells in each sorted well was successful with 38 cells (the bottom in FIG. 6). Finally, 22 cells in which both IgH and IgL genes were amplified in the same cell. From all these cells, cDNAs of antibody genes of H chain and L (kappa) chain were cloned.
上記により、セルソーターで１細胞ごとに分取したＶＥＧＦＲ２－アレクサフルオール６４７特異的抗体産生Ｂ細胞の、１細胞レベルでの抗体遺伝子の解析を、特許番号第５２０５５９７号公報に開示される、本発明者が開発した「１細胞レベルでの抗体遺伝子の解析・同定方法」に準拠して実施した。まずセルソーターにて１細胞ごとに９６ウェルプレートに分取した後、細胞を破砕させ、直接１細胞ごとにｍＲＮＡを回収した。その後ＲＴ－ＰＣＲを行ったところ、採取した６０個のＢ細胞のうち４０個でＩｇＨ遺伝子の増幅に成功した（図６ 最上段）。また、分取したウェルごとの細胞の有無を確認するために実施した、βアクチンのＲＴ－ＰＣＲでは、３８個で成功した（図６ 最下段）。最終的に同一細胞でＩｇＨ及びＩｇＬ遺伝子がともに増幅された細胞は、２２個であった。これらのすべての細胞からＨ鎖及びＬ（kappa）鎖の抗体遺伝子のｃＤＮＡがクローニングされた。 3. Analysis and identification of antibody genes at the level of 1 cell According to the above, analysis of antibody genes at the level of 1 cell of BEGFR2-Alexafluor 647-specific antibody-producing B cells sorted for each cell with a cell sorter, This was carried out in accordance with “A method for analyzing and identifying antibody genes at the level of one cell” developed by the present inventors and disclosed in Japanese Patent No. 5205597. First, after sorting each cell into a 96-well plate using a cell sorter, the cells were disrupted, and mRNA was directly collected for each cell. Subsequently, RT-PCR was performed, and the IgH gene was successfully amplified in 40 of the collected 60 B cells (the top row in FIG. 6). In addition, the β-actin RT-PCR carried out to confirm the presence of cells in each sorted well was successful with 38 cells (the bottom in FIG. 6). Finally, 22 cells in which both IgH and IgL genes were amplified in the same cell. From all these cells, cDNAs of antibody genes of H chain and L (kappa) chain were cloned.
４．バキュロウイルス発現系による完全長抗体タンパクの作製
２２クローンの抗体遺伝子を発現ベクターに組み込んで、バキュロウイルス発現系にて完全長抗体タンパクを発現・分泌させ、精製を行った。
抗体遺伝子（Ｈ鎖及びＬ鎖）をバキュロウイルス用のシャトルベクター（pFastBac Dual vector）に組み込んで、大腸菌DH10Bacに導入し、相同組換えにより組換えバクミドの作製を行った。バクミドＤＮＡは、昆虫由来のＳｆ９細胞に導入され、組換えバキュロウイルスの作製を行った。増幅されたウイルスは、タンパクの高産生株である昆虫由来のHigh Five細胞に感染させ、２７℃で６４時間無血清培地にて培養した。培養上清中に産生された抗体タンパクは、プロテインＡプレパックカラム（GEヘルスケア社製）を用いて精製を行った。取得できた１５クローンについて、アレクサフルオール６４７標識ＶＥＧＦＲ２及びＶＥＧＦＲ２タンパクに対する結合活性をＥＬＩＳＡ法にて評価した（図７）。アレクサフルオール６４７標識ＶＥＧＦＲ２タンパクに対して強く結合したクローンが５種類認められた（＃４８，５１，５４，５５，５６）。このうちＶＥＧＦＲ２にも結合したものが、クローン＃４８，＃５１であった。従ってこの結果から、＃５４，＃５５，＃５６のクローンは、ＶＥＧＦＲ２タンパクではなく、アレクサフルオール６４７標識特異的に結合活性のある抗体である可能性が示された。ＶＥＧＦＲ２タンパクに対する抗体クローンの＃４８，＃５１，＃５５精製抗体を、ＳＤＳ－ＰＡＧＥ法にて電気泳動し、泳動後のゲルをクマシーブリリアントブルー染色したところ、Ｈ鎖、Ｌ鎖に分かれた明瞭なバンドを確認できた（図８）。 4). Production of full-length antibody protein by baculovirus expression system The antibody gene of 22 clones was incorporated into an expression vector, and the full-length antibody protein was expressed and secreted by the baculovirus expression system and purified.
The antibody gene (H chain and L chain) was incorporated into a baculovirus shuttle vector (pFastBac Dual vector), introduced into Escherichia coli DH10Bac, and a recombinant bacmid was prepared by homologous recombination. The bacmid DNA was introduced into insect-derived Sf9 cells to produce a recombinant baculovirus. The amplified virus was infected with insect-derived High Five cells, which are high-protein-producing strains, and cultured in a serum-free medium at 27 ° C. for 64 hours. The antibody protein produced in the culture supernatant was purified using a Protein A prepack column (manufactured by GE Healthcare). About 15 clones that could be obtained, the binding activity to Alexa Fluor 647-labeled VEGFR2 and VEGFR2 protein was evaluated by ELISA (FIG. 7). Five clones that strongly bound to Alexa Fluor 647-labeled VEGFR2 protein were observed (# 48, 51, 54, 55, 56). Among them,clones # 48 and # 51 bound to VEGFR2. Therefore, from this result, it was shown that the clones of # 54, # 55, and # 56 were not VEGFR2 protein but an antibody having specific binding activity to Alexa Fluor 647 label. The # 48, # 51, and # 55 purified antibodies of antibody clones against VEGFR2 protein were electrophoresed by SDS-PAGE, and the gel after electrophoresis was stained with Coomassie brilliant blue. A band was confirmed (FIG. 8).
２２クローンの抗体遺伝子を発現ベクターに組み込んで、バキュロウイルス発現系にて完全長抗体タンパクを発現・分泌させ、精製を行った。
抗体遺伝子（Ｈ鎖及びＬ鎖）をバキュロウイルス用のシャトルベクター（pFastBac Dual vector）に組み込んで、大腸菌DH10Bacに導入し、相同組換えにより組換えバクミドの作製を行った。バクミドＤＮＡは、昆虫由来のＳｆ９細胞に導入され、組換えバキュロウイルスの作製を行った。増幅されたウイルスは、タンパクの高産生株である昆虫由来のHigh Five細胞に感染させ、２７℃で６４時間無血清培地にて培養した。培養上清中に産生された抗体タンパクは、プロテインＡプレパックカラム（GEヘルスケア社製）を用いて精製を行った。取得できた１５クローンについて、アレクサフルオール６４７標識ＶＥＧＦＲ２及びＶＥＧＦＲ２タンパクに対する結合活性をＥＬＩＳＡ法にて評価した（図７）。アレクサフルオール６４７標識ＶＥＧＦＲ２タンパクに対して強く結合したクローンが５種類認められた（＃４８，５１，５４，５５，５６）。このうちＶＥＧＦＲ２にも結合したものが、クローン＃４８，＃５１であった。従ってこの結果から、＃５４，＃５５，＃５６のクローンは、ＶＥＧＦＲ２タンパクではなく、アレクサフルオール６４７標識特異的に結合活性のある抗体である可能性が示された。ＶＥＧＦＲ２タンパクに対する抗体クローンの＃４８，＃５１，＃５５精製抗体を、ＳＤＳ－ＰＡＧＥ法にて電気泳動し、泳動後のゲルをクマシーブリリアントブルー染色したところ、Ｈ鎖、Ｌ鎖に分かれた明瞭なバンドを確認できた（図８）。 4). Production of full-length antibody protein by baculovirus expression system The antibody gene of 22 clones was incorporated into an expression vector, and the full-length antibody protein was expressed and secreted by the baculovirus expression system and purified.
The antibody gene (H chain and L chain) was incorporated into a baculovirus shuttle vector (pFastBac Dual vector), introduced into Escherichia coli DH10Bac, and a recombinant bacmid was prepared by homologous recombination. The bacmid DNA was introduced into insect-derived Sf9 cells to produce a recombinant baculovirus. The amplified virus was infected with insect-derived High Five cells, which are high-protein-producing strains, and cultured in a serum-free medium at 27 ° C. for 64 hours. The antibody protein produced in the culture supernatant was purified using a Protein A prepack column (manufactured by GE Healthcare). About 15 clones that could be obtained, the binding activity to Alexa Fluor 647-labeled VEGFR2 and VEGFR2 protein was evaluated by ELISA (FIG. 7). Five clones that strongly bound to Alexa Fluor 647-labeled VEGFR2 protein were observed (# 48, 51, 54, 55, 56). Among them,
５．抗体クローンのＳＰＲ法を用いた親和性測定
抗体クローンの＃４８，＃５１，＃５５精製抗体と、ＶＥＧＦＲ２タンパク及びアレクサフルオール６４７標識ＶＥＧＦＲ２タンパクとの親和性を、BIAcore X100（GEヘルスケア社製）の機器を用いた表面プラズモン共鳴法（ＳＰＲ解析）にて測定した。チップへのリガンド（各抗体クローン）固定化量は１０００～１００００レスポンスユニット（ＲＵ）の間で行われた。抗体はＨＢＳ緩衝液（０．１５Ｍ ＮａＣｌ、３ｍＭ ＥＤＴＡ、０．０５％ Ｔｗｅｅｎ２０）に溶解し、流速１０μｌ／ｍｉｎ又は３０μｌ／ｍｉｎ、２５℃、最大１５０μＭの濃度で測定した。チップの再生を行う際には３molar塩化マグネシウムを用いた。BIAcore X100エバリュエーションソフトウェアを用いて測定データから動態定数を算出した。 5. Affinity measurement of antibody clone using SPR method The affinity between # 48, # 51, and # 55 purified antibody of antibody clone and VEGFR2 protein and Alexa Fluor 647-labeled VEGFR2 protein was measured using BIAcore X100 (manufactured by GE Healthcare). ) Using the surface plasmon resonance method (SPR analysis). The amount of ligand (each antibody clone) immobilized on the chip was between 1000 and 10,000 response units (RU). The antibody was dissolved in HBS buffer (0.15 M NaCl, 3 mM EDTA, 0.05% Tween 20) and measured at a flow rate of 10 μl / min or 30 μl / min at 25 ° C. and a maximum concentration of 150 μM. When regenerating the chip, 3 molar magnesium chloride was used. Kinetic constants were calculated from the measured data using BIAcore X100 evaluation software.
抗体クローンの＃４８，＃５１，＃５５精製抗体と、ＶＥＧＦＲ２タンパク及びアレクサフルオール６４７標識ＶＥＧＦＲ２タンパクとの親和性を、BIAcore X100（GEヘルスケア社製）の機器を用いた表面プラズモン共鳴法（ＳＰＲ解析）にて測定した。チップへのリガンド（各抗体クローン）固定化量は１０００～１００００レスポンスユニット（ＲＵ）の間で行われた。抗体はＨＢＳ緩衝液（０．１５Ｍ ＮａＣｌ、３ｍＭ ＥＤＴＡ、０．０５％ Ｔｗｅｅｎ２０）に溶解し、流速１０μｌ／ｍｉｎ又は３０μｌ／ｍｉｎ、２５℃、最大１５０μＭの濃度で測定した。チップの再生を行う際には３molar塩化マグネシウムを用いた。BIAcore X100エバリュエーションソフトウェアを用いて測定データから動態定数を算出した。 5. Affinity measurement of antibody clone using SPR method The affinity between # 48, # 51, and # 55 purified antibody of antibody clone and VEGFR2 protein and Alexa Fluor 647-labeled VEGFR2 protein was measured using BIAcore X100 (manufactured by GE Healthcare). ) Using the surface plasmon resonance method (SPR analysis). The amount of ligand (each antibody clone) immobilized on the chip was between 1000 and 10,000 response units (RU). The antibody was dissolved in HBS buffer (0.15 M NaCl, 3 mM EDTA, 0.05% Tween 20) and measured at a flow rate of 10 μl / min or 30 μl / min at 25 ° C. and a maximum concentration of 150 μM. When regenerating the chip, 3 molar magnesium chloride was used. Kinetic constants were calculated from the measured data using BIAcore X100 evaluation software.
抗体クローン＃４８及び＃５１については、１：１結合モデルにて結合速度定数（Ｋａ）、解離速度定数（Ｋｄ）及び解離定数（ＫＤ）を求め、抗体クローン＃５５については、１：１結合反応の解離速度の差を利用したTwo-state reactionモデルにて解離定数を求めた。結果を表１に示す。ＳＰＲ解析の結果から、クローン＃４８及び＃５１抗体は、ＶＥＧＦＲ２に比較的強く結合活性を示すが、一方クローン＃５５抗体は、ＶＥＧＦＲ２タンパクには親和性を示さず、アレクサフルオール６４７標識ＶＥＧＦＲ２タンパクのみに結合することが明らかとなった。これよりクローン＃５５抗体は、蛍光色素であるアレクサフルオール６４７を特異的に認識するヒトモノクローナル抗体であることがわかった（図９）。
For antibody clones # 48 and # 51, the association rate constant (Ka), dissociation rate constant (Kd) and dissociation constant (KD) were determined in a 1: 1 binding model, and for antibody clone # 55, 1: 1 binding was performed. The dissociation constant was obtained by the two-state reaction model using the difference in the dissociation rate of the reaction. The results are shown in Table 1. From the results of SPR analysis, the clone # 48 and # 51 antibodies showed relatively strong binding activity to VEGFR2, whereas the clone # 55 antibody showed no affinity for the VEGFR2 protein and the Alexa Fluor 647-labeled VEGFR2 protein. It became clear that it only combined. Thus, clone # 55 antibody was found to be a human monoclonal antibody that specifically recognizes Alexa Fluor 647, which is a fluorescent dye (FIG. 9).
６．サンドイッチＥＬＩＳＡ法を用いたアレクサフルオール６４７標識抗体濃度の測定
アレクサフルオール６４７標識抗体として、アレクサフルオール６４７標識トラスツズマブを次の方法で作製した。まずトラスツズマブ（抗Ｈｅｒ２抗体）の重鎖を構成するポリペプチド（配列番号１４）と軽鎖を構成するポリペプチド（配列番号１５）をコードする遺伝子をそれぞれpcDNA3.3ベクター（サーモフィッシャーサイエンティフィック社製）に組み込み、Expi293細胞に遺伝子導入し、５～７日間培養した後に培養上清からプロテインＡを用いて回収精製することでトラスツズマブを得た。次に、得られたトラスツズマブに対して、Alexa Fluor 647 Protein Labeling Kit(サーモフィッシャーサイエンティフィック社製）を用い、製品付属のマニュアルに従ってアレクサフルオール６４７標識操作を行い、アレクサフルオール６４７標識トラスツズマブを得た。 6). Measurement of Alexa Fluor 647 Labeled Antibody Concentration Using Sandwich ELISA Method As Alexa Fluor 647 labeled antibody, Alexa Fluor 647 labeled trastuzumab was prepared by the following method. First, the genes encoding the polypeptide (SEQ ID NO: 14) constituting the heavy chain of trastuzumab (anti-Her2 antibody) and the polypeptide (SEQ ID NO: 15) constituting the light chain were respectively pcDNA3.3 vectors (Thermo Fisher Scientific) And introduced into Expi293 cells, cultured for 5-7 days, and then recovered and purified from the culture supernatant using protein A to obtain trastuzumab. Next, using the Alexa Fluor 647 Protein Labeling Kit (manufactured by Thermo Fisher Scientific), Alexa Fluor 647 labeling operation is performed on the obtained trastuzumab according to the manual attached to the product, and Alexa Fluor 647 Labeled Trastuzumab is labeled. Obtained.
アレクサフルオール６４７標識抗体として、アレクサフルオール６４７標識トラスツズマブを次の方法で作製した。まずトラスツズマブ（抗Ｈｅｒ２抗体）の重鎖を構成するポリペプチド（配列番号１４）と軽鎖を構成するポリペプチド（配列番号１５）をコードする遺伝子をそれぞれpcDNA3.3ベクター（サーモフィッシャーサイエンティフィック社製）に組み込み、Expi293細胞に遺伝子導入し、５～７日間培養した後に培養上清からプロテインＡを用いて回収精製することでトラスツズマブを得た。次に、得られたトラスツズマブに対して、Alexa Fluor 647 Protein Labeling Kit(サーモフィッシャーサイエンティフィック社製）を用い、製品付属のマニュアルに従ってアレクサフルオール６４７標識操作を行い、アレクサフルオール６４７標識トラスツズマブを得た。 6). Measurement of Alexa Fluor 647 Labeled Antibody Concentration Using Sandwich ELISA Method As Alexa Fluor 647 labeled antibody, Alexa Fluor 647 labeled trastuzumab was prepared by the following method. First, the genes encoding the polypeptide (SEQ ID NO: 14) constituting the heavy chain of trastuzumab (anti-Her2 antibody) and the polypeptide (SEQ ID NO: 15) constituting the light chain were respectively pcDNA3.3 vectors (Thermo Fisher Scientific) And introduced into Expi293 cells, cultured for 5-7 days, and then recovered and purified from the culture supernatant using protein A to obtain trastuzumab. Next, using the Alexa Fluor 647 Protein Labeling Kit (manufactured by Thermo Fisher Scientific), Alexa Fluor 647 labeling operation is performed on the obtained trastuzumab according to the manual attached to the product, and Alexa Fluor 647 Labeled Trastuzumab is labeled. Obtained.
サンドイッチＥＬＩＳＡ法を用いて、アレクサフルオール６４７標識トラスツズマブを次の方法で測定した。９６穴イモビライザーアミノプレート（サーモフィッシャーサイエンティフィック社製）に、ＰＢＳ中に希釈した１０μｇ／ｍｌの抗アレクサフルオール６４７抗体（抗体クローン＃５５）を捕獲抗体として５０μｌ／ウェルずつ分注し室温で２時間反応固定化した後、洗浄液（０．０５％Ｔｗｅｅｎ－２０含有ＰＢＳ）を用いて３回洗浄し、３％ＢＳＡを含むＰＢＳを用いて４℃で一晩反応させブロッキングを行った。上記プレートを洗浄液で３回洗浄した後、０、１５．６２５、３１．２５、６２．５、１２５、２５０、５００及び１０００ｎｇ／ｍｌのアレクサフルオール６４７標識トラスツズマブを各ウェルに加え、室温で２時間反応させた。次に上記プレートを、洗浄液で３回洗浄後、検出抗体として５μｇ／ｍｌのビオチン標識した抗アレクサフルオール６４７抗体（抗体クローン＃５５）を５０μｌ／ウェルずつ加えて３０分間反応させ、洗浄液で３回洗浄した。さらに、０．２μｇ／ｍｌのホースラディッシュペルオキシダーゼ標識ストレプトアビジン（PN21130、サーモフィッシャーサイエンティフィック社製）を５０μｌ／ウェルずつ加えて３０分間反応させ、洗浄液で７回洗浄した後、基質液（TMB substrate reagent set, BDバイオサイエンス社製）を１００μｌ／ウェルずつ加え、室温で３０分間インキュベートし、１Ｍの硫酸水溶液を５０μｌ／ウェルずつ添加し発色反応を停止させた。各ウェルの吸光度（４５０ｎｍ）をプレートリーダー（ImmunoMini NJ-2300、バイオテック社製)を用いて測定した。
Using a sandwich ELISA method, Alexa Fluor 647-labeled trastuzumab was measured by the following method. A 96-well immobilizer amino plate (manufactured by Thermo Fisher Scientific) was dispensed with 50 μl / well of 10 μg / ml anti-Alexafluor 647 antibody (antibody clone # 55) diluted in PBS as a capture antibody at room temperature. After immobilizing the reaction for 2 hours, the plate was washed 3 times with a washing solution (PBS containing 0.05% Tween-20), and blocked by reacting overnight at 4 ° C. with PBS containing 3% BSA. After washing the plate three times with wash solution, 0, 15.625, 31.25, 62.5, 125, 250, 500 and 1000 ng / ml Alexa Fluor 647-labeled trastuzumab was added to each well and 2 at room temperature. Reacted for hours. Next, after washing the plate three times with a washing solution, 5 μg / ml of biotin-labeled anti-Alexafluor 647 antibody (antibody clone # 55) as a detection antibody was added in an amount of 50 μl / well and reacted for 30 minutes. Washed twice. Further, 0.2 μg / ml horseradish peroxidase-labeled streptavidin (PN21130, manufactured by Thermo Fisher Scientific) was added at 50 μl / well, reacted for 30 minutes, washed 7 times with a washing solution, and then a substrate solution (TMB （substrate). reagent set (manufactured by BD Bioscience) was added at 100 μl / well, incubated at room temperature for 30 minutes, and 1 M sulfuric acid aqueous solution was added at 50 μl / well to stop the color reaction. The absorbance (450 nm) of each well was measured using a plate reader (ImmunoMini NJ-2300, manufactured by Biotech).
吸光度（４５０ｎｍ）をＸ軸に、アレクサフルオール６４７標識トラスツズマブの濃度（ｎｇ／ｍｌ）をＹ軸にしてプロットしたグラフを作成した（図１０）。その結果、吸光度とアレクサフルオール６４７標識トラスツズマブの濃度から相関係数が高い検量線を作成できることが確認できた。したがって、抗体クローン＃５５を用いたサンドイッチＥＬＩＳＡ法により、アレクサフルオール６４７標識抗体の検量線を作成し、被検物質を上記と同様に処理して吸光度（４５０ｎｍ）を測定することで、被検物質中のアレクサフルオール６４７標識抗体の濃度を定量することが可能であることが明らかとなった。
A graph was prepared by plotting the absorbance (450 nm) on the X-axis and the concentration (ng / ml) of Alexa Fluor 647-labeled trastuzumab on the Y-axis (FIG. 10). As a result, it was confirmed that a calibration curve having a high correlation coefficient can be created from the absorbance and the concentration of Alexa Fluor 647-labeled trastuzumab. Therefore, a calibration curve of Alexa Fluor 647-labeled antibody is prepared by sandwich ELISA using antibody clone # 55, the test substance is processed in the same manner as described above, and the absorbance (450 nm) is measured. It was revealed that the concentration of Alexa Fluor 647-labeled antibody in the substance can be quantified.
サンドイッチＥＬＩＳＡ法によりレクサフルオール６４７標識抗体の濃度を定量することは、抗体医薬の開発に応用できる。例えば、正常な動物と腫瘍細胞を移植した動物に対して、アレクサフルオール６４７で標識した、前記腫瘍細胞を特異的に認識する抗体（以下、「アレクサ標識－腫瘍細胞認識抗体」ともいう）を投与した後、所定期間後にそれぞれの動物から血液を採取する。次に、本発明の抗体を用いたサンドイッチＥＬＩＳＡ法により、それぞれの血液中に含まれるアレクサ標識－腫瘍細胞認識抗体の濃度を定量して比較することで、動物体内の腫瘍細胞に結合するアレクサ標識－腫瘍細胞認識抗体量を測定することができる。かかる腫瘍細胞に結合するアレクサ標識－腫瘍細胞認識抗体量の測定により、抗体医薬として用いた場合の抗体の投与量、投与時期等を判断することが可能となる。
Quantifying the concentration of Lexafluor 647-labeled antibody by sandwich ELISA can be applied to the development of antibody drugs. For example, an antibody that specifically recognizes the tumor cell labeled with Alexa Fluor 647 (hereinafter also referred to as “Alexa label-tumor cell recognition antibody”) labeled with Alexa Fluor 647 on a normal animal and an animal transplanted with tumor cells. After administration, blood is collected from each animal after a predetermined period. Next, the concentration of the Alexa label-tumor cell recognition antibody contained in each blood is quantitatively compared by sandwich ELISA using the antibody of the present invention, thereby comparing the Alexa label that binds to tumor cells in the animal body. -The amount of tumor cell recognition antibody can be measured. By measuring the amount of the Alexa-labeled tumor-cell-recognizing antibody that binds to such tumor cells, it is possible to determine the dose and timing of administration of the antibody when used as an antibody drug.
本発明は、アレクサフルオール６４７標識抗体に対する２次抗体として標的細胞の選別や、アレクサフルオール６４７標識タンパクを定量しうるＥＬＩＳＡ系の構築が可能である。特にアレクサフルオール６４７標識抗体のin vivoでの血中濃度のモニタリング等に対し好適に利用することができ、抗体医薬の代謝や薬物動態の解析に有用性を発揮しうると期待される。
The present invention enables selection of target cells as a secondary antibody against Alexa Fluor 647-labeled antibody and construction of an ELISA system that can quantify Alexa Fluor 647-labeled protein. In particular, it can be suitably used for monitoring the blood concentration of Alexa Fluor 647-labeled antibody in vivo, and is expected to be useful for analysis of metabolism and pharmacokinetics of antibody drugs.
Claims (11)
- アレクサフルオール６４７を認識するモノクローナル抗体。 A monoclonal antibody that recognizes Alexa Fluor 647.
- アレクサフルオール６４７を認識する抗体の重鎖可変領域を構成するポリペプチドであって、該重鎖可変領域が配列番号２に示すアミノ酸配列からなる超可変領域を含むことを特徴とするポリペプチド。 A polypeptide constituting the heavy chain variable region of an antibody that recognizes Alexa Fluor 647, wherein the heavy chain variable region includes a hypervariable region consisting of the amino acid sequence shown in SEQ ID NO: 2.
- アレクサフルオール６４７を認識する抗体の重鎖可変領域を構成するポリペプチドであって、該重鎖可変領域が配列番号４に示すアミノ酸配列、又は配列番号４と少なくとも９７％同一であるアミノ酸配列からなることを特徴とするポリペプチド。 A polypeptide that constitutes the heavy chain variable region of an antibody that recognizes Alexa Fluor 647, wherein the heavy chain variable region is represented by SEQ ID NO: 4 or an amino acid sequence that is at least 97% identical to SEQ ID NO: 4 A polypeptide characterized by comprising:
- アレクサフルオール６４７を認識する抗体の重鎖を構成するポリペプチドであって、該重鎖が配列番号６に示すアミノ酸配列、又は配列番号６と少なくとも９５％同一であるアミノ酸配列からなることを特徴とするポリペプチド。 A polypeptide that constitutes the heavy chain of an antibody that recognizes Alexa Fluor 647, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence that is at least 95% identical to SEQ ID NO: 6 A polypeptide.
- アレクサフルオール６４７を認識する抗体の軽鎖可変領域を構成するポリペプチドであって、該軽鎖可変領域が、配列番号８に示すアミノ酸配列からなる超可変領域を含むことを特徴とするポリペプチド。 A polypeptide constituting the light chain variable region of an antibody that recognizes Alexa Fluor 647, wherein the light chain variable region comprises a hypervariable region consisting of the amino acid sequence shown in SEQ ID NO: 8 .
- アレクサフルオール６４７を認識する抗体の軽鎖可変領域を構成するポリペプチドであって、該軽鎖可変領域が、配列番号１０に示すアミノ酸配列、又は配列番号１０と少なくとも９７％同一であるアミノ酸配列からなることを特徴とするポリペプチド。 A polypeptide that constitutes the light chain variable region of an antibody that recognizes Alexa Fluor 647, wherein the light chain variable region is the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence that is at least 97% identical to SEQ ID NO: 10 A polypeptide characterized by comprising:
- アレクサフルオール６４７を認識する抗体の軽鎖を構成するポリペプチドであって、該軽鎖が、配列番号１２に示すアミノ酸配列、又は配列番号１２と少なくとも９５％同一であるアミノ酸配列からなることを特徴とするポリペプチド。 A polypeptide constituting the light chain of an antibody that recognizes Alexa Fluor 647, wherein the light chain consists of the amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence that is at least 95% identical to SEQ ID NO: 12. Characteristic polypeptide.
- 請求項２～７のいずれかに記載のポリペプチドをコードすることを特徴とするＤＮＡ。 A DNA encoding the polypeptide according to any one of claims 2 to 7.
- 請求項２～４のいずれかに記載のポリペプチド、及び請求項５～７のいずれかに記載のポリペプチドを含むことを特徴とする抗アレクサフルオール６４７モノクローナル抗体。 An anti-alexafluor 647 monoclonal antibody comprising the polypeptide according to any one of claims 2 to 4 and the polypeptide according to any one of claims 5 to 7.
- 請求項２又は３に記載のポリペプチド、及び請求項５又は６に記載のポリペプチドを含むことを特徴とする請求項９記載の抗アレクサフルオール６４７モノクローナル抗体。 The anti-alexafluor 647 monoclonal antibody according to claim 9, comprising the polypeptide according to claim 2 or 3, and the polypeptide according to claim 5 or 6.
- 請求項４に記載のポリペプチド、及び請求項７に記載のポリペプチドを含むことを特徴とする請求項９記載の抗アレクサフルオール６４７モノクローナル抗体。 The anti-alexafluor 647 monoclonal antibody according to claim 9, comprising the polypeptide according to claim 4 and the polypeptide according to claim 7.
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