JP2017109965A - Anti-pancreatic polypeptide monoclonal antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop monoclonal antibody that does not cross-react with peptide YY and specifically reacts with pancreatic polypeptide.SOLUTION: An anti-pancreatic polypeptide monoclonal antibody or a fragment containing the antigen binding site that binds to a pancreatic polypeptide but does not bind to peptide YY is obtained from a hybridoma using antibody-producing cells obtained by immunizing a pancreatic polypeptide gene-deficient mouse with a pancreatic polypeptide.SELECTED DRAWING: None

Description

  The present invention relates to monoclonal antibodies specific for pancreatic polypeptides.

  The pancreatic islets (pancreatic islets) that play a central role in adult blood glucose control are mainly composed of four types of endocrine cells, that is, α, β, δ, and PP cells. Although there are various reports (Non-patent Documents 1 and 2) regarding the plasticity of these islet cells, the exact cell lineage in adults from the developmental stage and the physiological role of PP cells have not been clarified at all. Pancreatic polypeptide (PP) is one of the islet hormones belonging to the NPY family composed of pancreatic polypeptide, peptide YY (PYY), and neuropeptide Y.

  In order to elucidate the physiological roles of such PP cells and pancreatic polypeptides, antibodies specific for pancreatic polypeptides are required. Antibodies against pancreatic polypeptides have been reported (Non-Patent Documents 3 to 15).

Genes Dev. 26, 1680-1685, 2011 Cell 138, 449-462, 2009 Development 125, 2213-2221, 1998 PLoS Biology 5 (7), e163, 2007 Nature 463, 775-780, 2010 Development Cell 4,383-393, 2003 Genes & Development 17, 2591-2603, 2003 J. et al. Clin. Invest. 117,961-970,007 Nature 23, 71-75, 1999 Nature 466, 627-633, 2008 PNAS 109 (10), 3915-3920, 2012 Diabetes 63,224-236,2014 Nature 455 (7213), 627-632, 2008 Endocrinology 154 (2), 4493-4502, 2103 Cell 153, 747-758, 2013

However, since most of the known anti-pancreatic polypeptide antibodies are polyclonal antibodies, it is difficult to obtain antibodies with the same specificity again. In addition, as a result of the immunohistochemistry of the existing anti-pancreatic polypeptide antibody by using the pancreatic tissue section of the pancreatic polypeptide gene (Ppy) -deficient mouse, the present inventor found that clear positive cells were observed. Was found to cross-react with peptide YY as well as pancreatic polypeptide.
Therefore, there is no antibody specific to mouse pancreatic polypeptide that does not cross-react with peptide YY, and the development of such a monoclonal antibody has been desired.

  Accordingly, as a result of various studies to solve the above problems, the present inventor obtained a hybridoma using antibody-producing cells obtained by immunizing a Ppy-deficient non-human animal with a pancreatic polypeptide, and obtained a monoclonal antibody obtained from the hybridoma. The present inventors have found that the antibody does not react with peptide YY in liquid phase or in immunohistochemistry, is specific for pancreatic polypeptide, and is useful for detection of pancreatic polypeptide, thereby completing the present invention.

  That is, the present invention provides the following [1] to [10].

[1] An anti-pancreatic polypeptide monoclonal antibody which binds to pancreatic polypeptide but does not bind to peptide YY or a fragment containing an antigen-binding site thereof.
[2] (1) binds to pancreatic polypeptide by ELISA but does not bind to peptide YY, (2) detects pancreatic polypeptide by Western blot, but does not detect peptide YY, (3) immunohistochemistry [2] The anti-pancreatic polypeptide monoclonal antibody according to [1] or a fragment containing an antigen-binding site thereof, wherein pancreatic polypeptide-containing tissue is detected but pancreatic polypeptide-free tissue is not detected
[3] The anti-pancreatic polypeptide monoclonal antibody according to [1] or [2], which recognizes the amino acid sequence represented by SEQ ID NO: 1, or a fragment containing the antigen-binding site thereof.
[4] The anti-pancreatic polypeptide monoclonal antibody according to [1] or [2] produced by the hybridoma deposited under accession number NITE P-02175 or a fragment containing the antigen-binding site thereof.
[5] Production of an anti-pancreatic polypeptide monoclonal antibody according to any one of [1] to [4], comprising a step of immunizing a Ppy-deficient non-human animal with a polypeptide having the sequence represented by SEQ ID NO: 1 as an immunizing antigen Method.
[6] A reagent for detecting pancreatic polypeptide or pancreatic polypeptide-containing tissue, comprising the anti-pancreatic polypeptide monoclonal antibody according to any one of [1] to [4] or a fragment containing an antigen-binding site thereof.
[7] A method for detecting pancreatic polypeptide or pancreatic polypeptide-containing tissue, comprising using the anti-pancreatic polypeptide monoclonal antibody according to any one of [1] to [4] or a fragment containing an antigen-binding site thereof.
[8] The method for detecting pancreatic polypeptide or pancreatic polypeptide-containing tissue according to [7], wherein the anti-pancreatic polypeptide monoclonal antibody or a fragment containing the antigen-binding site thereof is labeled with a labeling substance [9] [1 ] Or a hybridoma producing the monoclonal antibody according to [2].
[10] The hybridoma according to [9], deposited with a deposit number of NITE P-02175.

  The monoclonal antibody of the present invention is specific to pancreatic polypeptide, does not react with the peptide YY cross-reacted with conventional antibodies, and can detect pancreatic polypeptide in liquid phase or immunohistochemistry. Therefore, the use of the monoclonal antibody of the present invention makes it possible to study the physiological functions of pancreatic polypeptides and the role of PP cells.

The confirmation result by SDS-PAGE after the peptide conjugation of the synthetic peptide and carrier protein which were used for immunization of a Ppy gene deficient mouse is shown. The result of the Western blot of PP peptide and PYY peptide is shown. The detection result of the positive cell by the monoclonal antibody concerning this invention in the pancreatic islet of a Ppy gene-deficient mouse | mouth (Ppycre ^{/ cre} ) and a wild type mouse | mouth (Ppy ^{+ / +} ) is shown.

  The anti-pancreatic polypeptide monoclonal antibody of the present invention binds to pancreatic polypeptide but does not bind to peptide YY. Specifically, (1) binds to pancreatic polypeptide by ELISA but does not bind to peptide YY; (2) detects pancreatic polypeptide by Western blot but does not detect peptide YY; (3) immunization Anti-pancreatic polypeptide monoclonal antibodies that detect pancreatic polypeptide-containing tissue in histochemistry but do not detect pancreatic polypeptide-free tissue are preferred.

  ELISA is an abbreviation for Enzyme-Linked Immunosorbent Assay, and pancreatic polypeptide can be quantified by a competitive method or a sandwich method. In the case of the competitive method, the sample and enzyme-labeled antigen are added to the solid-phased anti-pancreatic polypeptide monoclonal antibody to cause an antigen-antibody reaction, and after washing, react with the enzyme substrate, develop color, and the absorbance is measured for the sample. The amount of antigen in it is measured. In the case of the sandwich method, a specimen is added to an immobilized anti-pancreatic polypeptide antibody to cause an antigen-antibody reaction. Further, an enzyme-labeled second anti-pancreatic polypeptide antibody is added to cause an antigen-antibody reaction. After washing, it reacts with the enzyme substrate, develops color, measures the absorbance, and measures the amount of antigen in the sample. Since the anti-pancreatic polypeptide monoclonal antibody of the present invention binds to the pancreatic polypeptide by these ELISAs and does not bind to the peptide YY, only the pancreatic polypeptide can be accurately quantified.

  Western blotting is a method of detecting proteins separated by electrophoresis with an antibody. The anti-pancreatic polypeptide monoclonal antibody of the present invention detects pancreatic polypeptide by Western blot but does not detect peptide YY.

  Moreover, if the anti-pancreatic polypeptide monoclonal antibody of the present invention is used, pancreatic polypeptide-containing pancreatic tissue can be detected by immunohistochemistry, and pancreatic tissue not containing pancreatic polypeptide is not detected. More specifically, when the anti-pancreatic polypeptide monoclonal antibody of the present invention is an antibody against mouse pancreatic polypeptide, positive cells are observed in pancreatic islets in pancreatic tissue sections of wild-type mice, but in pancreatic tissue sections of Ppy-deficient mice. There is no positive reaction. Moreover, it is preferable to detect a pancreatic polypeptide in the immunohistochemistry of a human pancreatic tissue section even if it is an antibody against mouse pancreatic polypeptide.

  The anti-pancreatic polypeptide monoclonal antibody of the present invention is obtained using the polypeptide represented by SEQ ID NO: 1, that is, the polypeptide having amino acids 30 to 65 of the mouse pancreatic polypeptide (mPP) (SEQ ID NO: 1) as an antigen. Which recognize these polypeptides.

The anti-pancreatic polypeptide monoclonal antibody of the present invention is produced by a hybridoma prepared using the polypeptide represented by SEQ ID NO: 1 as a sensitizing antigen and using antibody-producing cells derived from Ppy-deficient non-human animals as antibody-producing cells. Is preferred.
That is, the anti-pancreatic polypeptide monoclonal antibody of the present invention can be obtained, for example, by fusing antibody-producing cells obtained by immunizing a Ppy-deficient non-human animal with the polypeptide represented by SEQ ID NO: 1 and myeloma cells. Can be obtained by culturing the hybridoma.

  The non-human animal immunized with the sensitizing antigen may be any Ppy-deficient non-human animal. For example, a Ppy-deficient mammal such as a Ppy-deficient mouse is used. A Ppy-deficient mouse can be produced, for example, by replacing the coding region of Ppy with a cre sequence by a genome editing technique in which significant technological progress has recently been made.

  In order to immunize an animal with a sensitizing antigen, a known method is performed. For example, as a general method, a sensitizing antigen is injected into a mammal intraperitoneally or subcutaneously. Specifically, an appropriate amount of a normal adjuvant, for example, Freund's complete adjuvant, is mixed with a sensitizing antigen diluted to an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline, etc. Administer several times every 4-21 days. In addition, an appropriate carrier can be used during immunization with the sensitizing antigen.

  Thus, after immunizing a mammal and confirming that a desired antibody level rises in serum, immune cells are collected from the mammal and cell fusion is performed. Preferable immune cells include spleen cells.

  Mammalian myeloma cells are used as parent cells to be fused with the immune cells. This myeloma cell is known from various known cell lines such as P3 (P3x63Ag8.653) (Kearney et al., J Immunol 1979; 123: 1548-1550), NS-1 (Kohler. G. and Milstein, C Eur. J Immunol 1976; 6: 511-519), SP2 / 0 (Shulman, M. et al., Nature 1978; 276: 269-270) and the like are preferably used.

  The cell fusion between the immune cells and myeloma cells is basically performed by a known method, for example, the method of Kohler and Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol 1981; 73: 3-46). It can carry out according to etc.

  More specifically, the cell fusion is performed in a normal culture solution in the presence of a cell fusion promoter, for example. For example, polyethylene glycol (PEG), Sendai virus (HVJ), or the like is used as a fusion promoter, and an auxiliary agent such as dimethyl sulfoxide can be used to increase the fusion efficiency as desired.

  The use ratio of immune cells and myeloma cells can be arbitrarily set. For example, the number of immune cells is preferably 1 to 10 times that of myeloma cells. As the culture solution used for the cell fusion, for example, RPMI1640 culture solution suitable for growth of the myeloma cell line, MEM culture solution, and other normal culture solutions used for this kind of cell culture can be used. Serum such as fetal bovine serum (FBS) may be added.

  In cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture solution, and a polyethylene glycol solution (for example, an average molecular weight of about 1000 to 6000) preheated to about 37 ° C. is usually 30 to 60%. A target fused cell (hybridoma) is prepared by adding and mixing at a concentration of (w / v). Subsequently, cell fusion agents and the like that are undesirable for the growth of the hybridoma are removed by sequentially adding an appropriate culture medium and centrifuging to remove the supernatant.

  The hybridoma thus obtained is selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). The culture in the HAT culture solution is continued for a sufficient time (usually several days to several weeks) for cells other than the target hybridoma (non-fused cells) to die. Subsequently, the usual limiting dilution method is performed, and screening and single cloning of the hybridoma producing the target antibody are performed.

  For the screening of hybridomas, for example, monoclonal antibodies that react with pancreatic polypeptides can be selected by ELISA using pancreatic polypeptides used as sensitizing antigens.

  The hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution, and can be stored for a long time in liquid nitrogen.

  In order to obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a usual method and obtained as a culture supernatant, or the hybridoma is administered to a mammal compatible therewith to proliferate, and as ascites The method of obtaining is adopted. The former method is suitable for obtaining highly pure antibodies, while the latter method is suitable for mass production of antibodies.

As described above, the anti-pancreatic polypeptide monoclonal antibody of the present invention or a labeled body thereof reacts specifically with the pancreatic polypeptide and does not cross-react with peptide YY or the like, so that detection of pancreatic polypeptide in the liquid phase and tissue is possible. Useful for.
The label of the antibody of the present invention may be either an enzyme or a radioisotope. Detection of pancreatic polypeptide in the liquid phase includes ELISA and Western blot, and detection of pancreatic polypeptide in tissue includes immunohistochemistry.
As immunohistochemical means, for example, a fluorescent antibody method, an enzyme antibody method and the like are used.

  EXAMPLES Next, an Example is given and this invention is demonstrated still in detail.

Example 1
(1) Production of Ppy gene-deficient mouse The mouse Ppy gene encoding the mouse PP peptide is present on chromosome 11. First, a Ppy-cre knock-in mouse (Ppy ^{cre / +} ) in which the coding sequence of the Ppy gene was replaced with a cre sequence was prepared. This mouse expresses cre instead of expressing the endogenous Ppy gene. Therefore, the knock-in allele is functionally a Ppy null allele. As a method of knocking in the cre sequence at the Ppy locus, genome editing using ZFN (Zinc finger nuclease) (Geurts et al. Science. 2009; 325: 433.) Was used. A targeting construct was prepared in which the first methionine of the PP peptide was replaced with the first methionine of the cre gene in the coding region of the endogenous Ppy gene.
The designed ZFN and targeting construct were injected into a 1-cell stage fertilized egg, and the fertilized egg was returned to the uterus of a pseudopregnant mouse to obtain a litter. By analyzing the Ppy locus of the genomic gene from several tens of pups, one line of mice (Ppy ^{cre / +} ) in which the cre sequence was correctly knocked into the coding sequence of the target Ppy gene was identified. Furthermore, Ppy ^{cre / cre} mice were obtained by crossing these mice. It was confirmed by in situ hybridization that the Ppy gene mRNA was detected that this mouse was deficient in the Ppy gene.

(2) Sensitization A KLH-mPP peptide conjugate (KLH was bound to a peptide having Cys at the N-terminus of the mPP peptide) solution and Freund's complete adjuvant were mixed at a volume ratio of about 1: 2 to prepare an emulsion. The above-mentioned 5 PPY knockout mice (PPY ^{cre / cre} ) were immunized with KLH-peptide solution (2.5 mg / mL) in the tail at a rate of 0.05 mL / animal.
Further, the base of the tail was boosted using 0.05 mL / animal (concentration: 2.5 mg / mL) of the KLH-mPP peptide conjugate solution.

(3) Cell fusion 1) Recovery of SP2 cells (mouse myeloma) SP2 cells were recovered using a scraper, centrifuged (1000 rpm, 5 minutes), the supernatant was aspirated and Dulbecco's modified Eagle medium (D-MEM) 20 mL (serum-free) was added and the number of cells was counted.
2) The immunized animals were euthanized by inhaling isoflurane in excess. Disinfecting alcohol was applied to the whole and fixed to the operating table.
3) The mouse was laparotomized, and the retroperitoneum and the surrounding adipose tissue were cut open with tweezers, and a pair of left and right iliac lymph nodes were collected.
4) Lymph nodes were lined using a net. Lymphocytes were collected in a tube while washing with D-MEM (serum-free) so that the final volume was 30 mL.
5) Cell fusion SP2 cells were added to the lymphocyte tube of 4) so that the lymphocyte: SP2 cell = 5: 1 cell ratio was obtained. After centrifugation (1280 rpm, 10 minutes), the supernatant was aspirated and incubated at 37 ° C. for 2 minutes. Thereafter, 1 mL of polyethylene glycol was added dropwise over 1 minute. The reaction was carried out at 37 ° C. for 2 minutes. D-MEM4.5mL was dripped over 3 minutes. D-MEM4.5mL was dripped over 2 minutes. After centrifugation (1000 rpm, 5 minutes), the supernatant was aspirated.
6) The fused cells were suspended in the HAT culture solution, seeded in a 96-well plate, cultured, and hybridomas were selected.

(4) Hybridoma Screening i) Primary Screening by ELISA As solid-phased antigens, BSA-mouse PP peptide for positive plates and BSA-mouse PYY peptide for negative plates were used. Here, the amino acid sequence of the mouse PYY peptide is shown in SEQ ID NO: 2.
1) Antigen (3 μg / mL) was immobilized on a plate for assay using 10 mM phosphate buffer (pH 7).
2) Incubated at 37 ° C for 60 minutes (or overnight at 4 ° C).
3) The antigen solution was removed, and a blocking solution (1% BSA / PBS) was added at 100 μL / well.
4) Incubated overnight at 4 ° C. (or 30 minutes at room temperature).
5) A blocking solution was added to the primary antibody (culture supernatant) and added at 100 μL / well.
6) Incubated at room temperature for 1 hour.
7) The secondary antibody ^*) was diluted 20,000 times with a blocking solution and added at 50 μL / well.
8) Incubated at room temperature for 30 minutes.
9) A color former (color former using TMBZ as a substrate) was added at 50 μL / well.
10) The absorbance was measured (450 nm), and three types of candidate wells were selected.

  * Anti-mouse IgG (HRP label) and anti-mouse IgG L chain (HRP label) were used as secondary antibodies.

  Since many ELISA positive antibodies that reacted with mPP but did not react with mPYY were obtained, the top 20 positive wells were selected from each mouse for a secondary screening.

ii) Secondary screening for narrowing down target antibodies Using the candidate antibodies obtained in the primary screening, the following secondary screening was performed to obtain three types of monoclonal antibodies that satisfy these conditions. That is, (1) PP is detected by Western blot, but PYY is not detected; (2) PP in wild-type mouse islets is detected in immunohistochemistry, but does not cross-react with the islets of Ppy-deficient mice. One of the obtained hybridomas was deposited as NITE P-02175 at the Patent Microorganism Deposit Center.

Example 2
Characteristics of the obtained monoclonal antibody (1) ELISA
The confirmation result by SDS-PAGE of the peptide conjugation of the synthetic peptide and carrier protein used for immunization of a Ppy gene-deficient mouse is shown in FIG. (Method: MBS method, carrier protein: KLH (for immunization) / BSA (for confirmation of consumption))
The obtained monoclonal antibody reacted with BSA-mPP in ELISA but did not react with BSA-mPYY.
(2) Western blot By Western blot using BSA-mPP peptide conjugate used for ELISA screening and BSA-mPYY peptide conjugate as negative control, the obtained monoclonal antibody detects mPP but not mPYY. It was an antibody (Figure 2).
(3) Immunohistochemistry When the obtained monoclonal antibody is used, positive cells are observed in the pancreatic islets in the pancreatic tissue section of the wild type mouse (Ppy ^{+ / +} ), but in the Ppy deficient mouse (Ppy ^{cre / cre} ). No positive cells were observed in the pancreatic tissue section (FIG. 3).

Claims (10)

  An anti-pancreatic polypeptide monoclonal antibody or a fragment containing an antigen-binding site thereof that binds to pancreatic polypeptide but does not bind to peptide YY.   (1) binds to pancreatic polypeptide by ELISA, but does not bind to peptide YY, (2) detects pancreatic polypeptide by Western blot, but does not detect peptide YY, (3) pancreatic polyp The anti-pancreatic polypeptide monoclonal antibody according to claim 1 or a fragment containing an antigen-binding site thereof, which detects a peptide-containing tissue but does not detect a pancreatic polypeptide-free tissue.   The anti-pancreatic polypeptide monoclonal antibody according to claim 1 or 2, which recognizes the amino acid sequence represented by SEQ ID NO: 1, or a fragment containing the antigen-binding site thereof.   3. The anti-pancreatic polypeptide monoclonal antibody according to claim 1 or 2, which is produced by a hybridoma deposited under accession number NITE P-02175, or a fragment containing the antigen-binding site thereof.   The method for producing an anti-pancreatic polypeptide monoclonal antibody according to any one of claims 1 to 4, comprising a step of immunizing a Ppy-deficient non-human animal with a polypeptide having the sequence represented by SEQ ID NO: 1 as an immunizing antigen.   A reagent for detecting pancreatic polypeptide or pancreatic polypeptide-containing tissue, comprising the anti-pancreatic polypeptide monoclonal antibody according to any one of claims 1 to 4 or a fragment containing the antigen-binding site thereof.   A method for detecting pancreatic polypeptide or pancreatic polypeptide-containing tissue, comprising using the anti-pancreatic polypeptide monoclonal antibody according to any one of claims 1 to 4 or a fragment containing an antigen-binding site thereof.   The method for detecting pancreatic polypeptide or pancreatic polypeptide-containing tissue according to claim 7, wherein the anti-pancreatic polypeptide monoclonal antibody or a fragment containing the antigen-binding site thereof is labeled with a labeling substance.   A hybridoma producing the monoclonal antibody according to claim 1 or 2.   The hybridoma according to claim 9, which is deposited under the deposit number NITE P-02175.