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WO2014136443A1 - Anticorps monoclonal anti-aβ42 - Google Patents

Anticorps monoclonal anti-aβ42 Download PDF

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Publication number
WO2014136443A1
WO2014136443A1 PCT/JP2014/001187 JP2014001187W WO2014136443A1 WO 2014136443 A1 WO2014136443 A1 WO 2014136443A1 JP 2014001187 W JP2014001187 W JP 2014001187W WO 2014136443 A1 WO2014136443 A1 WO 2014136443A1
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peptide
monoclonal antibody
antibody
amino acid
seq
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Japanese (ja)
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崇臣 福原
誠司 曽我部
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PHC Corp
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Panasonic Healthcare Co Ltd
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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

Definitions

  • the present invention relates to a monoclonal antibody that specifically reacts with A ⁇ 42 peptide, a hybridoma that can produce the monoclonal antibody, and a kit containing the monoclonal antibody.
  • AD Alzheimer's disease
  • a ⁇ peptide In order to diagnose Alzheimer's disease at an early stage, a method for detecting ⁇ amyloid (hereinafter also referred to as A ⁇ ) peptide present in body fluids such as blood and spinal fluid is known.
  • a ⁇ peptide is a peptide composed of 40 to 43 amino acids, and it is known that in Alzheimer's disease, the A ⁇ peptide aggregates to form amyloid and deposits in the brain.
  • the A ⁇ 42 peptide which is a peptide consisting of 42 amino acids, is said to be a factor marker for Alzheimer's disease.
  • a ⁇ 43 peptide which is a peptide consisting of 43 amino acids, is localized at the center of cerebral senile plaques. It has been clarified through research (see Non-Patent Document 1).
  • Monoclonal antibodies that react with A ⁇ peptides are already known (see, for example, Patent Document 1), and A ⁇ peptide detection kits containing such monoclonal antibodies are already commercially available.
  • Non-Patent Document 2 immunohistochemical staining using autopsy tissue from AD patients has shown that A ⁇ is also present in tissues other than the brain, such as the nasal mucosa (see Non-Patent Document 2).
  • a ⁇ 42 peptide and A ⁇ 43 peptide are considered to have different functions among A ⁇ peptides
  • a ⁇ 42 peptide and A ⁇ 43 peptide are used in detecting A ⁇ peptide present in body fluids such as blood and cerebrospinal fluid. It would be preferable to be able to detect them separately.
  • a ⁇ 42 which controls the total amount of the elderly group, is considered to be more highly related to AD than A ⁇ 43, which is localized at the center of the elderly group. It is desirable that it can be detected automatically.
  • conventionally known monoclonal antibodies that react with the antigen A ⁇ peptide react with both the A ⁇ 42 peptide and the A ⁇ 43 peptide.
  • a monoclonal antibody that does not react with A ⁇ 43 peptide but specifically reacts with A ⁇ 42 peptide, which is a factor marker of Alzheimer's disease, has not been known. Therefore, the problem to be solved by the present invention is to provide an antibody for distinguishing and detecting A ⁇ 42 peptide from A ⁇ 43 peptide.
  • the present inventors have conducted extensive research and succeeded in establishing a specific hybridoma that produces a monoclonal antibody that does not substantially react with the A ⁇ 43 peptide but reacts specifically with the A ⁇ 42 peptide, thereby completing the present invention. It came to do.
  • the present invention provides the following: (1) a monoclonal antibody that reacts with A ⁇ 42 peptide and does not react with A ⁇ 43 peptide; (2) From the heavy chain complementarity determining region (CDR) 1 consisting of the amino acid sequence represented by SEQ ID NO: 8, the heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 9, and the amino acid sequence represented by SEQ ID NO: 10 A heavy chain variable region composed of a heavy chain CDR3 and a heavy chain framework sequence derived from an immunoglobulin heavy chain; and a light chain CDR1 composed of an amino acid sequence represented by SEQ ID NO: 13, represented by SEQ ID NO: 14 A light chain CDR2 comprising an amino acid sequence, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 15, and a light chain variable region comprising a light chain framework sequence derived from an immunoglobulin light chain, (1) A monoclonal antibody according to claim 1; (3) The monoclonal antibody according to (1), comprising a heavy chain complement
  • the monoclonal antibody provided by the present invention does not substantially react with the A ⁇ 43 peptide, but reacts specifically with the A ⁇ 42 peptide, so that the A ⁇ 42 peptide that is a factor marker of Alzheimer's disease can be specifically detected.
  • the use of the monoclonal antibody of the present invention or the A ⁇ detection kit containing the antibody makes it possible to specifically detect or measure the A ⁇ 42 peptide, which was impossible with conventional antibodies or detection kits, and thus is extremely useful.
  • the present invention relates to a monoclonal antibody that reacts with A ⁇ 42 peptide and does not react with A ⁇ 43 peptide.
  • the present invention provides: Heavy chain complementarity determining region (CDR) 1 consisting of the amino acid sequence represented by SEQ ID NO: 8, heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 9, and heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 10
  • the present invention relates to a monoclonal antibody comprising a light chain CDR2 comprising: a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15, and a light chain variable region comprising a light chain framework sequence derived from an immunoglobulin light chain.
  • the monoclonal antibody of the present invention is represented by the heavy chain CDR sequence consisting of the amino acid sequence represented by SEQ ID NO: 8 and SEQ ID NO: 9 as long as it is a monoclonal antibody that reacts with the A ⁇ 42 peptide but does not react with the A ⁇ 43 peptide. Also included are antibodies comprising at least one heavy chain CDR sequence selected from the group consisting of a heavy chain CDR sequence consisting of an amino acid sequence and a heavy chain CDR sequence consisting of the amino acid sequence represented by SEQ ID NO: 10.
  • the monoclonal antibody of the present invention is represented by the heavy chain CDR sequence consisting of the amino acid sequence represented by SEQ ID NO: 8, represented by SEQ ID NO: 9, as long as it is a monoclonal antibody that reacts with the A ⁇ 42 peptide and does not react with the A ⁇ 43 peptide.
  • antibodies comprising one light chain CDR sequence.
  • the monoclonal antibody of the present invention is a monoclonal antibody that reacts with the A ⁇ 42 peptide and does not react with the A ⁇ 43 peptide, the amino acid sequence of the CDR (heavy chain CDR1 to 3 and light chain CDR1 to 3) of the monoclonal antibody of the present invention described above. For example, at least 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, or 93% sequence identity. Also included are antibodies comprising a CDR sequence having an amino acid sequence.
  • the monoclonal antibody of the present invention includes the CDRs of the above-described monoclonal antibodies of the present invention (heavy chain CDR1 to 3 and light chain CDR1 to 3) as long as the monoclonal antibody reacts with the A ⁇ 42 peptide and does not react with the A ⁇ 43 peptide. Also included are antibodies comprising a CDR sequence having an amino acid sequence in which one or several, eg, 2 or 3, amino acids are substituted, deleted or added for each amino acid sequence.
  • “does not react with a peptide” or “does not substantially react with a peptide” means that in a test for confirming the reactivity of an antibody and / or antibody fragment with respect to a peptide, no result showing the reactivity is obtained. Or the result is below the detection limit.
  • a test is, for example, a test as described in Example 2 below.
  • sequence identity refers to a case where two or more sequences (base sequence or amino acid sequence) are aligned in consideration of gaps and insertions so that the degree of sequence match is maximized. Of the same base or amino acid at corresponding positions in two or more sequences.
  • the method of determining identity is designed to give the greatest degree of match between aligned sequences.
  • a method for determining identity between two sequences is not limited, and includes BLASTP, BLASTN, FASTA and the like. It can also be determined using DNASIS (manufactured by Hitachi Software Engineering Co., Ltd.) or GENETYX (manufactured by Genetics Co., Ltd.). Alternatively, in the case of a short chain peptide, it can also be determined by simply comparing the sequences.
  • One skilled in the art can determine identity between sequences by the methods described above.
  • the present invention also relates to a polynucleotide encoding the amino acid sequence of the CDR (heavy chain CDR1 to 3 and light chain CDR1 to 3) of the monoclonal antibody of the present invention described above.
  • One skilled in the art can determine and generate sequences for such polynucleotides using techniques known in the art.
  • the monoclonal antibody of the present invention is a monoclonal antibody produced by a hybridoma whose accession number is NITE P-1463.
  • the invention relates to a hybridoma that produces the monoclonal antibody of the invention.
  • the hybridoma was devisated on November 8, 2012 at the depositary organization (NITE), the National Institute for Product Evaluation and Technology (NITE), which has an address in 2-5-8 Kazusa Kamashika, Kisarazu City, Chiba Prefecture, Japan. (NPMD)) and the deposit number is NITE P-1463.
  • the monoclonal antibody produced by the hybridoma does not react with A ⁇ 40 peptide and A ⁇ 43 peptide, but reacts only with A ⁇ 42 peptide. Therefore, A ⁇ 42 peptide can be specifically detected among A ⁇ , and is extremely useful.
  • the monoclonal antibody produced by the hybridoma exhibits excellent characteristics such as high reactivity with the A ⁇ 42 peptide as compared with a commercially available anti-A ⁇ antibody. Furthermore, it shows the characteristic that the reactivity with respect to the A ⁇ 42 peptide in the nasal sample is higher than that of the A ⁇ 42 peptide in the brain sample.
  • the production method (immunization method and selective collection method) of the hybridoma of the present invention is not particularly limited. Using an A ⁇ 42 peptide or a part thereof as an antigen, it can be produced by a usual method for producing a hybridoma. That is, the hybridoma of the present invention is injected by injecting an antigen into an immunized animal such as a rat, taking out lymphocytes related to the production of antibodies thereto, mixing it with myeloma, and culturing it on a HAT medium. Can be manufactured.
  • a ⁇ 42 peptide When using a part of the A ⁇ 42 peptide as an antigen, it is particularly preferable to use an amino acid sequence containing the C-terminus of the A ⁇ 42 peptide. Since A ⁇ 40 peptide (SEQ ID NO: 16), A ⁇ 42 peptide (SEQ ID NO: 17), and A ⁇ 43 peptide (SEQ ID NO: 18) have different C-terminal amino acid sequences, respectively, by using an amino acid sequence including the C-terminal of A ⁇ 42 peptide, It becomes possible to obtain a hybridoma that produces a monoclonal antibody that specifically reacts with the A ⁇ 42 peptide. In the examples described later, the amino acid sequence containing the amino acid sequence from the 33rd to the 42nd position on the C-terminal side of the amino acid sequence of the A ⁇ 42 peptide was used as the antigen peptide.
  • the monoclonal antibody produced by the hybridoma is labeled with, for example, a general labeled protein, and the reactivity with respect to each of A ⁇ 42 peptide and A ⁇ 43 peptide is measured. .
  • a monoclonal antibody that specifically reacts with the A ⁇ 42 peptide can be confirmed.
  • the inventors determined the amino acid sequence of the monoclonal antibody produced by the hybridoma of the present invention, and succeeded in identifying the complementarity determining region (CDR), which is a hypervariable region.
  • CDR complementarity determining region
  • Three CDRs contained in the variable region of the heavy chain of the monoclonal antibody have an amino acid sequence represented by SEQ ID NOs: 8 to 10.
  • the three CDRs contained in the variable region of the light chain of the monoclonal antibody have an amino acid sequence represented by the amino acid sequence represented by SEQ ID NOs: 13-15.
  • a heavy chain CDR consisting of the amino acid sequence represented by SEQ ID NOs: 8 to 10 is incorporated into a heavy chain framework sequence derived from an immunoglobulin heavy chain to constitute a heavy chain variable region, and is represented by SEQ ID NOs: 13 to 15
  • An antibody can be prepared by constructing a light chain variable region by incorporating a light chain CDR comprising an amino acid sequence represented by the amino acid sequence described above into a light chain framework sequence derived from an immunoglobulin light chain.
  • a conventionally known method can be adopted as a method for producing such an antibody.
  • the present invention relates to a chimeric monoclonal antibody or a humanized monoclonal antibody having the CDR sequence of the monoclonal antibody of the present invention described above.
  • a chimeric antibody typically comprises a variable region of an antibody derived from a certain species and a constant region of an antibody derived from a different species, and can be easily constructed by a gene recombination technique. Methods for making chimeric antibodies are known in the art. For example, it can be produced by the method described in “Roguska et al., Proc Natl Acad Sci USA A.
  • a humanized antibody is typically an antibody having one or more CDRs from a non-human species, a framework region (FR) from a human immunoglobulin, and a constant region from a human immunoglobulin.
  • Methods for making humanized antibodies are known in the art.
  • a humanized antibody can be produced by applying a human heavy chain framework sequence and a human light chain framework sequence as the heavy chain framework sequence and the light chain framework sequence.
  • Almagro et al. FRONT Biosci. 2008 Jan 1; 13: 1619-1633. See etc.
  • CDR As a method of determining the definition of CDR and its position, for example, the definition of Kabat (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institute of Health 91, or National Institute of Health.
  • the definition Chothia et al., J. Mol. Biol., 1987; 196: 901-917) may be adopted. In some cases, it may be determined in consideration of both the Kabat definition and the Chothia definition. For example, an overlapping part of CDRs according to each definition or a part including both CDRs according to each definition may be determined. It can also be a CDR.
  • Martin et al.'S method Proc. Natl. Acad. Sci.
  • the present invention relates to an antibody fragment derived from the antibody of the present invention described above.
  • the “antibody fragment derived from the antibody of the present invention” is a part (partial fragment) of the antibody of the present invention or a peptide containing a part of the antibody of the present invention, and retains the action of the antibody of the present invention on the antigen.
  • Such antibody fragments include Fab, Fab ′, F (ab ′) 2 , single chain antibody (scFv), dimerized V region (diabody), disulfide stabilized V region (dsFv), and the present invention.
  • Fab is an antibody fragment having antigen-binding activity in which about half of the N-terminal side of the H chain and the entire L chain are linked by a disulfide bond among fragments obtained by treating immunoglobulin protein with papain. Therefore, the Fab of the present invention can be obtained by treating the monoclonal antibody of the present invention with papain.
  • Fab encoding is produced by inserting DNA encoding Fab of the monoclonal antibody of the present invention into a prokaryotic expression vector or eukaryotic expression vector, and introducing the vector into prokaryotic or eukaryotic organisms. You can also
  • F (ab ′) 2 is a fragment having antigen-binding activity, which is obtained by treating the lower part of the disulfide bond of the hinge region of an immunoglobulin protein with pepsin, and is composed of two Fab regions joined by a hinge portion. is there. Therefore, F (ab ′) 2 of the present invention can be obtained by treating the monoclonal antibody of the present invention with pepsin. Alternatively, Fab ′ described later can be produced by thioether bond or disulfide bond.
  • Fab ′ is an antibody fragment having antigen-binding activity obtained by cleaving the disulfide bond in the hinge region of F (ab ′) 2 described above. Therefore, Fab ′ of the present invention can be obtained by treating F (ab ′) 2 of the present invention with a reducing agent such as dithiothreitol.
  • DNA encoding the Fab ′ fragment of the monoclonal antibody of the present invention is inserted into a prokaryotic expression vector or eukaryotic expression vector, and the vector is introduced into prokaryotic or eukaryotic organisms to be expressed. 'Can also be manufactured.
  • a single chain antibody is a polypeptide in which one heavy chain variable region (VH) and one light chain variable region (VL) are linked using an appropriate peptide linker, and has an antigen binding activity. It is an antibody fragment having The scFv of the present invention obtains cDNA encoding the VH and VL of the monoclonal antibody of the present invention, constructs a DNA encoding scFv, and inserts the DNA into a prokaryotic expression vector or eukaryotic expression vector.
  • the expression vector can be expressed and produced by introducing it into a prokaryotic or eukaryotic organism.
  • the dimerization V region (diabody) is an antibody fragment obtained by dimerizing the above-mentioned scFv, and is an antibody fragment having a bivalent antigen-binding activity.
  • the bivalent antigen binding activity may be the same or different.
  • the dimerized V region of the present invention is obtained by obtaining cDNA encoding the VH and VL of the monoclonal antibody of the present invention, constructing DNA encoding scFv so as to contain an appropriate short peptide linker, It can be expressed and produced by inserting it into a prokaryotic expression vector or eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism.
  • the disulfide-stabilized V region refers to a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue and 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 a known method (for example, Protein Engineering, 7, 697 (1994)).
  • the dsFv of the present invention obtains cDNA encoding the VH and VL of the monoclonal antibody of the present invention, constructs a DNA encoding dsFv, and inserts the DNA into a prokaryotic expression vector or a eukaryotic expression vector.
  • the expression vector can be expressed and produced by introducing it into a prokaryotic or eukaryotic organism.
  • the peptide containing CDR is composed of at least one region of CDR sequence of VH or VL. Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker.
  • the peptide containing the CDR of the present invention constructs the DNA encoding the VH and VL CDRs of the monoclonal antibody of the present invention, inserts the DNA into a prokaryotic expression vector or eukaryotic expression vector, and the expression vector Can be expressed and produced by introducing it into a prokaryotic or eukaryotic organism.
  • the peptide containing CDR can also be manufactured by chemical synthesis methods, such as Fmoc method or tBoc method.
  • the monoclonal antibody of the present invention includes a derivative of an antibody in which a radioisotope, a low molecular drug, a high molecular drug, a protein or the like is chemically or genetically bound to the monoclonal antibody of the present invention or an antibody fragment thereof.
  • a normal immunological detection or measurement method as a drug that binds to the monoclonal antibody of the present invention or an antibody fragment thereof.
  • alkaline phosphatase, peroxidase or luciferase is used.
  • the present invention relates to a method for producing an antibody or antibody fragment of the present invention comprising culturing a hybridoma having a deposit number of NITE P-1463.
  • a conventionally well-known method can be employ
  • the present invention relates to an A ⁇ 42 detection or measurement kit comprising the antibody or antibody fragment of the present invention described above.
  • the A ⁇ 42 detection kit of the present invention includes a buffer solution, a diluent, a washing solution, a secondary antibody solution, a calibration curve reagent necessary for concentration regression of the detected measurement value, a plate, and instructions for use Documents, specimen collection devices, specimen collection tubes, and the like.
  • the kit of the present invention is a system that detects A ⁇ 42 peptide by a color development reaction
  • the kit of the present invention can further contain a color developing solution, a color developing stop solution and the like.
  • the kit of the present invention is a system that detects A ⁇ 42 peptide by fluorescence or color development, it is not necessary to include a color development solution and a color development stop solution.
  • the kit of the present invention is an ELISA kit.
  • the present invention relates to a method for detecting or measuring A ⁇ 42 in a specimen, including the use of the antibody, antibody fragment or kit of the present invention described above.
  • the “specimen” is an animal, preferably a mammal, more preferably a primate, even more preferably a human, particularly preferably a human who may be suffering from AD, or a mild cognitive impairment of AD type. Collected from human or AD patients.
  • the site from which the specimen is collected is not particularly limited as long as it is a site where A ⁇ 42 is known to exist in AD patients. Accordingly, the specimen is, for example, a sample collected from the nose (eg, nasal mucosa) such as cerebrospinal fluid, blood, or nasal wiping solution, or a sample collected from the oral cavity (eg, oral mucosa) such as the oral wiping solution.
  • these specimens may be prepared in a state suitable for use in the kit or method of the present invention using a lysate, a buffer or a diluent.
  • the specimen used in the kit or method of the present invention is collected from the nose (eg, nasal mucosa).
  • the specimen used in the kit or method of the present invention is a nasal wipe.
  • Example 1 Acquisition of hybridoma A hybridoma producing the monoclonal antibody of the present invention was obtained by the following procedure.
  • a peptide having the amino acid sequence represented by SEQ ID NO: 1 was synthesized as an antigen peptide. This peptide is obtained by adding Cys to the N-terminus of the amino acid sequence of the A ⁇ 42 peptide from the 33rd to the 42nd amino acid sequence on the C-terminal side. The addition of Cys is for conjugation with the carrier protein.
  • Hybridomas were cloned from the 12 positive wells, and the following tests were performed. ⁇ Cell seeding by limiting dilution ⁇ Reactivity check by ELISA (confirms reactivity with A ⁇ 42 peptide and no reactivity with A ⁇ 40 peptide) -Checking for single colony formation by microscopic observation of ELISA positive wells By the above test, one hybridoma of an ELISA positive single clone was obtained. The name of the hybridoma was 6C10H3.
  • hybridoma was cryopreserved and deposited with the National Institute of Technology and Evaluation (NITE) and the Patent Microorganism Deposit Center (NPMD). The deposit number is NITE P-1463, and the deposit date is November 8, 2012.
  • NITE National Institute of Technology and Evaluation
  • NPMD Patent Microorganism Deposit Center
  • Example 2 Specificity evaluation of monoclonal antibody (1) Preparation of labeled antibody Monoclonal antibody 6C10H3 was obtained from hybridoma 6C10H3. Subsequently, the monoclonal antibody 6C10H3 was labeled by the following procedure to obtain a labeled antibody.
  • Processing was performed according to the protocol of Peroxidase Labeling Kit-SH manufactured by Dojindo Laboratories. That is, a sample solution containing 200 ⁇ g of antibody and 100 ⁇ L of Solution A attached to the kit were added to Filtration Tube attached to the kit. After light mixing by pipetting, centrifugation was performed at 8000 ⁇ g for 10 minutes. 100 ⁇ L of Solution A was added to the reducing agent included in the kit and dissolved by pipetting. 100 ⁇ L of this solution was added onto a Filtration Tube membrane in which the antibody was concentrated, and mixed well with the antibody by pipetting. After reacting at 37 ° C.
  • the labeled antibody was diluted 1000 times, and the reactivity to each of A ⁇ 40 peptide, A ⁇ 42 peptide, and A ⁇ 43 peptide was measured by the following procedure.
  • the reactivity with respect to each peptide was similarly measured using the monoclonal antibody contained in a commercially available A ⁇ detection kit (manufactured by Wako Pure Chemical Industries, Ltd.).
  • HRP-labeled antibody (BC05) solution (provided with a kit manufactured by Wako Pure Chemical Industries, Ltd.) was added to each well in one set.
  • HRP-labeled antibody (6C10H3) solution was added to each well. After the addition, the reaction was allowed to proceed for 5 minutes at room temperature with stirring. After washing was performed 5 times, 100 ⁇ L of TMB solution was added and allowed to react for 30 minutes in the dark at room temperature.
  • FIG. 1 shows that neither the monoclonal antibody 6C10H3 of the present invention nor the commercially available monoclonal antibody shows reactivity to the A ⁇ 40 peptide, and from FIG. 2, both antibodies show reactivity to the A ⁇ 42 peptide.
  • FIG. 3 shows that the monoclonal antibody 6C10H3 of the present invention does not show reactivity with the A ⁇ 43 peptide, whereas the commercially available monoclonal antibody shows reactivity with the A ⁇ 43 peptide.
  • the monoclonal antibody 6C10H3 of the present invention shows reactivity to A ⁇ 42 peptide, but A ⁇ 43 peptide Since it does not show any reactivity to the A ⁇ 42 peptide, it can be seen that the A ⁇ 42 peptide can be distinguished from the A ⁇ 43 peptide.
  • Example 3 Evaluation of recognition ability of monoclonal antibody Comparison of recognition ability of A ⁇ 42 peptide using monoclonal antibody 6C10H3 and 1F115 antibody (manufactured by IBL) which is generally an anti-A ⁇ antibody frequently used by researchers did.
  • a 4G8 antibody was biotinylated using a biotin labeling kit (SH label, manufactured by Dojindo).
  • HRP labeling of 6C10H3 antibody and 1F115 antibody was performed using an HRP labeling kit (SH labeling, manufactured by Dojindo).
  • a ⁇ 42 peptide Human, 1-42, manufactured by Peptide Laboratories was diluted to obtain an A ⁇ 42 peptide solution having a concentration of 0, 5, 10, 20, 50, 100, 200, or 500 pM.
  • each of the prepared A ⁇ 42 peptide solution (0, 5, 10, 20, 50, 100, 200, 500 pM) was added to the plate and reacted at room temperature for 2 hours, and then the A ⁇ 42 peptide solution was removed from the plate. did. The plate was washed 5 times using the washing solution.
  • HRP-labeled antibodies (6C10H3 antibody and 1F115 antibody) at the same concentration was added to each plate and reacted. Thereafter, the plate was washed 5 times using the washing solution (5 times each).
  • HRP enzyme substrate manufactured by SIGMA
  • a stop solution manufactured by KPL
  • color development was measured at a wavelength of 450 nm to 620 nm.
  • FIG. 4 shows the color values of HRP-labeled 6C10H3 antibody (black square) and HRP-labeled 1F115 antibody (white square) when an A ⁇ 42 peptide solution diluted 2500 times is used. From this figure, it can be seen that the 6C10H3 antibody of the present invention is more reactive with the A ⁇ 42 peptide than the 1F115 antibody, which is a commercially available anti-A ⁇ antibody.
  • Example 4 CDR sequencing of monoclonal antibody The sequence of the complementarity determining region (CDR) of the 6C10H3 antibody of the present invention was determined by the following procedure.
  • RNA Extraction Procedure Using hybridoma 6C10H3 (about 1 ⁇ 10 7 cells), the following procedure was performed in this order to collect total RNA.
  • -Homogenization step 1 mL of TRIzol Reagent (manufactured by life technologies (Invitrogen), No. 15596-026) was added by pipetting and treated at room temperature for 5 minutes.
  • RNA extraction step 0.2 mL of chloroform was added, mixed by inversion, and treated at room temperature for 3 minutes. Thereafter, centrifugation (12000 g, 4 ° C., 15 minutes) was performed, and the aqueous layer was recovered.
  • First washing step 0.5 mL of isopropanol is added, mixed by inversion, and treated at room temperature for 10 minutes. Thereafter, centrifugation (12000 g, 4 ° C., 10 minutes) is performed, and the precipitate is collected (the supernatant is removed by suction).
  • -Second washing step 1 mL of 75% ethanol was added and mixed by inversion. Thereafter, centrifugation (12000 g, 4 ° C., 5 minutes) is performed, and the precipitate is collected (the supernatant is removed by suction) and air-dried.
  • -Adjustment process Rnase free water 0.02mL is added and it processes at 55 degreeC for 10 minute (s).
  • cDNA preparation procedure Using the total RNA recovered in the above (1), cDNA was prepared by the following procedure. Denaturation step: Dilute 5 ⁇ g of total RNA to 20 ⁇ L. Then, after processing at 65 degreeC for 10 minutes, it cools rapidly. Then, 11 ⁇ L of Bulk first-strand reaction mix, 1 ⁇ L of DTT solution, and 1 ⁇ L of primer (Not Id (T) 18) were added from First-Strand cDNA synthesis kit manufactured by GE Healthcare. Incubation step: held at 37 ° C. for 1 hour.
  • RT-PCR RT-PCR was performed using the cDNA obtained in (2) above.
  • primers having sequences represented by SEQ ID NOs: 2 and 3 were used as heavy chain primers
  • primers having sequences represented by SEQ ID NOs: 4 and 5 were used as light chain primers.
  • Sequencing Sequencing was performed from BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit manufactured by Life technologies (Invitrogen) using primers specific to pGEM-T Easy Vector.
  • the heavy chain of the 6C10H3 antibody consists of the amino acid sequence represented by SEQ ID NO: 7, and of which the heavy chain CDR consists of the amino acid sequence represented by SEQ ID NOs: 8 to 10.
  • the light chain of the 6C10H3 antibody consists of the amino acid sequence represented by SEQ ID NO: 12, of which the light chain CDR consists of the amino acid sequence represented by SEQ ID NOs: 13-15.
  • SEQ ID NO: 6 is the base sequence encoding the heavy chain of the 6C10H3 antibody
  • SEQ ID NO: 11 is the base sequence encoding the light chain of the 6C10H3 antibody.
  • the CDR region was amplified by PCR using the pGEM-T vector incorporating the 6C10H3 antibody gene (heavy chain and light chain) as a template and the following primers.
  • primers having sequences represented by SEQ ID NOs: 19 and 20 were used as heavy chain primers
  • primers having sequences represented by SEQ ID NOs: 21 and 22 were used as light chain primers.
  • each PCR product treated with a restriction enzyme was incorporated into the following vector treated with the same restriction enzyme.
  • the human IgG1 gene Fc region has already been incorporated.
  • Heavy chain pCAGGS vector
  • Light chain pCAGGS vector
  • the obtained vector containing 6C10H3 antibody-derived heavy chain CDR comprises a rat-derived heavy chain variable region (VH) and a human-derived heavy chain constant region (CH1, CH2 and CH3).
  • the obtained vector containing the 6C10H3 antibody-derived light chain CDR contains a rat-derived light chain variable region (VL) and a human-derived light chain constant region (CL).
  • HEK293T Human embryonic kidney cell HEK293T was used as a host and cultured on a 5 mL scale. All the cultures were performed under conditions of 37 ° C. and 5% CO 2.
  • HEK293T cells were cultured in a DMEM containing 10% FBS per 6 cm dish. When 80-90% confluent, transfection was performed. Before transfection, 293 ⁇ L of DMEM (0% FBS) mixed with 7.0 ⁇ g of plasmid (heavy chain: 3.5 ⁇ g, light chain: 3.5 ⁇ g) and DMEM (0% FBS) in 293 ⁇ l A mixture of 10.6 ⁇ L of a 1 mg / mL polyethyleneimine solution was prepared.
  • Example 6 Evaluation of recognition ability for A ⁇ 42 derived from different tissues Using a specimen collected from tissues around the nose and a specimen collected from the brain, the recognition ability of the monoclonal antibody 6C10H3 to A ⁇ 42 derived from different tissues was evaluated. For the measurement, the labeled monoclonal antibody 6C10H3 prepared in Example 2 was used. As a comparative example, a monoclonal antibody contained in a commercially available A ⁇ detection kit (manufactured by Wako Pure Chemical Industries, Ltd.) was used.
  • the labeled antibody was diluted 1000 times, and the reactivity with respect to each of the specimen collected from the tissue around the nose and the specimen collected from the brain was measured by the following procedure.
  • a ⁇ detection kit human ⁇ amyloid (1-42) ELISA kit Wako, high-sensitivity product, manufactured by Wako Pure Chemical Industries, Ltd.
  • Sex was measured.
  • HRP-labeled antibody (BC05) solution (provided with a kit manufactured by Wako Pure Chemical Industries, Ltd.) was added to each well in one set.
  • HRP-labeled antibody (6C10H3) solution was added to each well. After the addition, the reaction was allowed to proceed for 5 minutes at room temperature with stirring. After washing was performed 5 times, 100 ⁇ L of TMB solution was added and allowed to react for 30 minutes in the dark at room temperature.
  • the monoclonal antibody 6C10H3 of the present invention has a higher reactivity with respect to the sample collected from the tissue around the nose than the sample collected from the brain.
  • the commercially available monoclonal antibody there was no difference between the reactivity to the specimen collected from the brain and the reactivity to the specimen collected from the tissues around the nose. From this, it was shown that the monoclonal antibody of the present invention reacts more specifically with A ⁇ 42 derived from nasal tissue.
  • the monoclonal antibody provided by the present invention enables detection of A ⁇ 42 peptide contained in a sample when performing AD diagnosis outside the body using a sample collected from an AD patient or a healthy person who has the possibility of AD. Further, the monoclonal antibody provided by the present invention may be used as an antibody drug by making it a humanized antibody. Therefore, the present invention can be used in fields such as pharmaceuticals, diagnostic agents, and test agents.

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Abstract

L'invention fournit un anticorps monoclonal réagissant à un peptide Aβ42, mais ne réagissant pas à un peptide Aβ43. En outre, l'invention fournit notamment un hybridome produisant ledit anticorps, un fragment d'anticorps dérivé dudit anticorps, un procédé de fabrication dudit anticorps ou du fragment d'anticorps, un kit contenant ledit anticorps ou ledit fragment d'anticorps, et un procédé destiné à mesurer un Aβ42 à l'aide dudit anticorps, dudit fragment d'anticorps ou dudit kit.
PCT/JP2014/001187 2013-03-08 2014-03-04 Anticorps monoclonal anti-aβ42 Ceased WO2014136443A1 (fr)

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JP2013-046216 2013-03-08
JP2013046216A JP2016103980A (ja) 2013-03-08 2013-03-08 ハイブリドーマ及びモノクローナル抗体

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10509736A (ja) * 1995-02-14 1998-09-22 バイエル コーポレイション βＡ４ペプチドに特異的なモノクローナル抗体
WO2005080435A1 (fr) * 2004-02-20 2005-09-01 Immuno-Biological Laboratories Co., Ltd. Anticorps monoclonal et utilisation de celui-ci
JP2005531509A (ja) * 2002-02-28 2005-10-20 マインドセット・バイオファーマスーティカルズ・ユーエスエイ・インコーポレイテッド アミロイドβペプチドに対する特異性抗体、医薬組成物、及びその使用方法
WO2006046644A1 (fr) * 2004-10-28 2006-05-04 Sanko Junyaku Co., Ltd. Procédé d’examen de la maladie d’alzheimer et réactif de diagnostic
WO2009016734A1 (fr) * 2007-07-31 2009-02-05 University Of Tsukuba Procédé pour la détection d'un trouble cognitif léger

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10509736A (ja) * 1995-02-14 1998-09-22 バイエル コーポレイション βＡ４ペプチドに特異的なモノクローナル抗体
JP2005531509A (ja) * 2002-02-28 2005-10-20 マインドセット・バイオファーマスーティカルズ・ユーエスエイ・インコーポレイテッド アミロイドβペプチドに対する特異性抗体、医薬組成物、及びその使用方法
WO2005080435A1 (fr) * 2004-02-20 2005-09-01 Immuno-Biological Laboratories Co., Ltd. Anticorps monoclonal et utilisation de celui-ci
WO2006046644A1 (fr) * 2004-10-28 2006-05-04 Sanko Junyaku Co., Ltd. Procédé d’examen de la maladie d’alzheimer et réactif de diagnostic
WO2009016734A1 (fr) * 2007-07-31 2009-02-05 University Of Tsukuba Procédé pour la détection d'un trouble cognitif léger

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