Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

• the present invention relates to antibodies that specifically bind to mammalian angiotensin II type 1 receptor (AT1R).
• AT1R mammalian angiotensin II type 1 receptor
• angiotensin II receptors There are two types of angiotensin II receptors: type 1 and type 2, and they are used to regulate physiological responses such as salt and water balance, blood pressure, and vascular tone, and to form pathological conditions such as diabetes, hypertension, myocardial infarction, congestive heart failure, and stroke. Many of the previously recognized effects of angiotensin II are mediated by the angiotensin II type 1 receptor (AT1R). Accurate detection of AT1R protein is very important for elucidating the molecular mechanism of AT1R in physiological reactions and pathomorphisms.
• AT1R angiotensin II type 1 receptor
• AT1R angiotensin II type 1 receptor
• AT1aR angiotensin II type 1a receptor
• AT1bR angiotensin II type 1b receptor
• the present invention is applicable to the detection of angiotensin II type 1 receptor in mammals and does not exhibit non-specific binding like currently commercially available anti-angiotensin II type 1 receptor antibodies.
• the purpose of the present invention is to provide antibodies that specifically bind to type 1 receptors.
• the present inventors focused on the amino acid sequence at positions 317-335, which is the intracellular domain of the mammalian angiotensin II type 1 receptor, and discovered that antibodies that bind to peptides having this specific amino acid sequence are capable of detecting angiotensin II type 1 receptors. They discovered that it specifically binds to the human body, and completed the present invention. Angiotensin II type 1 receptor can be specifically detected using the antibody that binds to a peptide having a specific amino acid sequence of the present invention.
• the present invention is as follows.
• An antibody against mammalian angiotensin II type 1 receptor (a) a peptide consisting of the amino acid sequence of SEQ ID NO: 1; or (b) a peptide consisting of the amino acid sequence of SEQ ID NO: 1 in which one or several amino acids have been substituted, deleted, added and/or inserted; An antibody obtained by using as an antigen.
• An antibody against the mammalian angiotensin II type 1 receptor of the present invention comprising a predetermined amino acid sequence of the present invention (amino acid residues 317-335 of the intracellular domain of the mammalian angiotensin II type 1 receptor, "AT1aR (317-335)) is applicable to the detection of angiotensin II type 1 receptor in mammals, and is currently commercially available.
• Antibodies that specifically bind to angiotensin II type 1 receptors that do not exhibit non-specific binding like available anti-angiotensin II type 1 receptor antibodies can be provided.
• 1 is a fluorescence micrograph showing the results of immune cell staining for anti-rAT1aR (317-335) polyclonal antibody in Test 1.
• 1 is a fluorescence micrograph showing the results of immune cell staining for anti-rAT1aR (317-335) monoclonal antibody in Test 1.
• 1 is a fluorescence micrograph showing the results of immunocytochemical staining using a commercially available anti-angiotensin II type 1 receptor antibody in Test 1.
• 2 is a fluorescence micrograph showing the results of immunohistological staining of mouse liver tissue for anti-rAT1aR (317-335) polyclonal antibody in Test 2.
• the antibody of the present invention is an antibody against a mammalian angiotensin II type 1 receptor, and is preferably an antibody that specifically recognizes a mammalian angiotensin II type 1 receptor.
• the mammalian angiotensin II type 1 receptor is preferably a human, rat or mouse angiotensin II type 1 receptor, more preferably a rat or mouse angiotensin II type 1 receptor, even more preferably a rat or mouse angiotensin II type 1 receptor. It is an angiotensin II type 1a receptor.
• the antibody against the mammalian angiotensin II type 1 receptor obtained in the present invention may be a polyclonal antibody or a monoclonal antibody.
• the antibody against the mammalian angiotensin II type 1 receptor of the present invention has as an antigen: (a) A peptide consisting of the amino acid sequence of SEQ ID NO: 1, (b) a peptide consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, added and/or inserted in the amino acid sequence of SEQ ID NO: 1; or (c) at least 80% of the amino acid sequence of SEQ ID NO: 1. , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 Peptides having amino acid sequences that are %, 98% or 99% identical can be used.
• the antibody against the mammalian angiotensin II type 1 receptor of the present invention recognizes amino acid residues 317-335 of the intracellular domain of the mammalian angiotensin II type 1 receptor.
• amino acid sequence described by SEQ ID NO: 1 is the intracellular domain amino acid residue position 317-335 (or “rAT1aR (317-335)”). This sequence is identical to amino acid residues 317-335 of the intracellular domain of the mouse angiotensin II type 1a receptor shown in SEQ ID NO:3. Furthermore, this sequence is the one in which asparagine at position 329 is replaced with serine among the amino acid residues 317-335 of the intracellular domain of the human angiotensin II type 1 receptor shown by SEQ ID NO: 4, and the sequence is shown by SEQ ID NO: 5.
• amino acid residues 317-335 of the intracellular domain of rat angiotensin II type 1b receptor threonine at position 325 is replaced with lysine, alanine at position 328 is replaced with serine, and glycine at position 329 is replaced with serine
• SEQ ID NO: Among the amino acid residues 317-335 of the intracellular domain of the mouse angiotensin II type 1b receptor shown in 6, arginine at position 325 was replaced with lysine, alanine at position 328 was replaced with serine, and glycine at position 329 was replaced with serine. It is the same as the thing.
• a peptide having the amino acid sequence consisting of SEQ ID NO: 1 can be chemically synthesized by conventionally known methods, or can be synthesized by recombinant technology using microorganisms.
• the antigen-antibody reaction can be carried out using the above-mentioned antigen by administering a substance serving as an antigen to an immunized animal, as is conventionally known.
• Antibodies against the mammalian angiotensin II type 1 receptor of the present invention can be produced by administering a substance that serves as an antigen and eliciting an immune response in the immunized animal.
• immunized animals include, but are not limited to, guinea pigs, rats, mice, rabbits, sheep, etc. used as experimental animals.
• the method of administering the antigen is not particularly limited, but includes, for example, subcutaneous, intraperitoneal, intravenous, intramuscular, and intradermal routes.
• the administration schedule of the antigen is not particularly limited, but for example, by administering the antigen about 2 to 10 times at an interval of 2 weeks, antibodies against the angiotensin II type 1 receptor of mammals can be produced. can be done.
• a serum sample can be collected from the immunized animal and the amount of antibody produced can be confirmed to confirm whether a sufficient immune response has been induced.
• serum is collected from the immunized animal and purified by a conventionally known method to obtain an antibody against the mammalian angiotensin II type 1 receptor.
• the dose of the antigen for the initial immunization and booster immunization is not particularly limited, but the dose of the antigen can be, for example, 10 to 200 ⁇ g per mouse.
• an adjuvant may be co-administered.
• a conventionally known adjuvant capable of eliciting an immune reaction at a high titer can be used. Examples include Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide, and the like.
• a polyclonal antibody can be obtained by the method for producing an antibody of the present invention, and subsequently, the antibody can be purified by a conventionally known method to obtain a highly pure antibody.
• hybridomas can be produced by collecting antibody-producing cells from the spleen or lymph nodes of immunized animals that have produced antibodies, and monoclonal antibodies can also be obtained.
• an antigen in which a large number of peptides are covalently bonded to a carrier protein is used.
• the bond between the peptide and the carrier protein can be formed by a conventionally known method.
• carrier proteins include keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), mouse serum albumin (MSA), rabbit serum albumin (RSA), and the like.
• KLH keyhole limpet hemocyanin
• BSA bovine serum albumin
• OVA ovalbumin
• MSA mouse serum albumin
• RSA rabbit serum albumin
• Such a linker is not particularly limited as long as it is a linker that can covalently bond the carrier protein and the cysteine-added peptide, but examples include N-hydroxysuccinimide activated ester (NHS ester), maleimide, and carbodiimide. Examples include linkers having functional groups such as.
• the method of administering the antigen is not particularly limited, but the antibody titer may be measured and the antigen may be administered until the desired antibody titer is obtained.
• Antibody titer can be measured by absorbance.
• the antibody titer can be measured using a blood sample appropriately collected from the immunized animal, and it is preferable to use a serum sample from which impurities have been removed by centrifugation or the like.
• the antibody titer can be measured by a conventionally known method such as ELISA (enzyme linked immunosorbent assay), although it is not particularly limited.
• ELISA enzyme linked immunosorbent assay
• chromatographic purification such as ion exchange chromatography, gel filtration chromatography, and affinity chromatography, and salting-out methods can be used.
• This is a polyclonal antibody that recognizes amino acid residues 317-335 of the intracellular domain of animal angiotensin II type 1 receptor.
• the antigen specificity of polyclonal antibodies can be confirmed by methods such as ELISA.
• an antibody that recognizes amino acid residues 317-335 of the intracellular domain of the mammalian angiotensin II type 1 receptor can be obtained, it can be obtained from the spleen or lymph nodes of the immunized animal using conventional methods.
• Antibody-producing cells can be collected.
• Hybridomas (fused cells) can be obtained by performing cell fusion using the obtained antibody-producing cells.
• Hybridomas can be obtained by fusing antibody-producing cells with myeloma cells. As myeloma cells to be fused with antibody-producing cells, commonly available cell lines of animals such as mice can be used.
• myeloma cells examples include mouse-derived myeloma P3/X63-AG8, P3/NSI/1-Ag4-1, P3/X63-Ag8.U1, SP2/0-Ag14, F0 or BW5147, and rat-derived myeloma 210RCY3-Ag1. .2.3. , human-derived myeloma U-266AR1, GML500-6TG-A1-2, UC729-6, CEM-AGR, D1R11, or CEM-T15.
• hybridomas By screening after cell fusion, only hybridomas can be selectively grown.
• a screening method it is preferable to culture in HAT medium or the like.
• the hybridoma may be cultured together with feeder cells such as thymocytes in order to support the proliferation of the hybridoma.
• the antigen specificity of the antibodies produced by the obtained hybridomas can be confirmed by measuring the culture supernatant by a known method such as ELISA, and only hybridomas that have been confirmed to produce the desired antibodies can be selected.
• a hybridoma consisting of a single clone can be obtained by removing mixed cells other than the intended one.
• hybridomas can be cryopreserved by a conventionally known method.
• Monoclonal antibodies can be produced from the hybridomas after cloning by conventionally known methods. For example, hybridomas can be grown intraperitoneally in mice and ascites containing monoclonal antibodies can be purified and produced. Alternatively, it can be produced by culturing hybridomas in a serum-free medium and purifying the culture supernatant.
• the antibody obtained in the present invention is a mouse antibody when a mouse is used as the immunized animal, but it can be made into a chimeric antibody, humanized antibody, or human antibody by conventionally known methods.
• Example 1 Preparation of anti-rAT1aR (317-335) polyclonal antibody
• a peptide (rAT1aR(317-335), SEQ ID NO: 1) having an amino acid sequence from positions 317 to 335 was chemically synthesized.
• (2) Preparation of polyclonal antibody The cysteine moiety of rAT1aR (317-335) and maleimide group-introduced keyhole limpet hemocyanin (KLH) were crosslinked in an aqueous solution to obtain a complex.
• KLH keyhole limpet hemocyanin
• the resulting peptide-KLH complex solution was mixed with an equivalent amount of Freund's complete adjuvant and administered subcutaneously to the back of rabbits four times at two-week intervals. Two weeks later, whole blood was collected, and polyclonal antibody "rAT1aR (317-335)/p" was obtained. Titer measurements were performed by ELISA.
• Example 2 Preparation of anti-rAT1aR (317-335) monoclonal antibody
• Immunization A peptide (rAT1aR (317-335)) bound to keyhole limpet hemocyanin (KLH) was mixed with an equal volume of Freund's complete adjuvant. , was administered intramuscularly to mice (Balb/c, male, 4 weeks old, CLEA Japan) twice at 2-week intervals. After more than two weeks, KLH-peptide alone was administered intravenously.
• KLH-peptide alone was administered intravenously.
• Cell fusion Four days after intravenous administration, the mice were euthanized by excessive administration of pentobarbital, and the spleens were removed.
• Splenocytes were isolated, counted, and mixed with twice the number of SP2/O (mouse myeloma-derived cell line, cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS)). did.
• the cells were collected by centrifugation, and 1 ml of 50% polyethylene glycol (Roche) pre-warmed at 37°C was added dropwise. After stirring at 37° C. for 1 minute, 20 ml of pre-warmed DMEM was added dropwise. After centrifugation at room temperature, the mixture was left at 37°C for 5 minutes.
• SP2/O mouse myeloma-derived cell line, cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS)
• HAT medium DMEM supplemented with 15% FCS and 1x HAT (Roche)
• the mixture was dispensed into a 96-well microplate at 100 ⁇ l/well and cultured at 37°C. After 4 days, 50 ⁇ l of HAT medium was added.
• Screening When the diameter of the cell colony reached approximately 200 to 300 ⁇ m, a portion of the supernatant was collected and subjected to ELISA using peptide (rAT1aR (317-335)) as an antigen.
• Test 1 Evaluation of specificity of antibody against angiotensin II type 1a receptor in immunocytostaining HEK293 cells in which rat angiotensin II type 1a receptor was forcibly expressed and a plasmid containing no angiotensin II AT1a receptor gene (empty vector)
• the anti-rAT1aR (317-335) polyclonal antibody and anti-rAT1aR (317-335) monoclonal antibody obtained in Example 1 and Example 2 were used to label the introduced angiotensin II type 1a receptor gene in HEK293 cells introduced with Immune cell staining was performed for HA (Hemagglutinin). Nuclear staining with DAPI was also performed.
• the results for the anti-rAT1aR (317-335) polyclonal antibody are shown in FIG. 1, and the results for the anti-rAT1aR (317-335) monoclonal antibody are shown in FIG.
• three commercially available antibodies against angiotensin II type 1 receptor c-572 (manufactured by Santa Cruz), AB15552 (manufactured by Merck & Co., Ltd.) and G- 3: Immunocyte staining was performed on sc-515884 (manufactured by Santa Cruz).
• the fluorescence of the fluorescent protein DsRed contained in the vector for introducing the rat angiotensin II type 1 receptor gene was measured. The results are shown in Figure 3.
• anti-rAT1aR (317-335) polyclonal antibody and anti-rAT1aR (317-335) monoclonal antibody can specifically stain cells expressing rat angiotensin II type 1a receptor.
• all commercially available antibodies against angiotensin II type 1 receptor stain cells that have not been introduced with the vector indicating that the staining of cells with commercially available antibodies is nonspecific. Shown.
• Test 2 Evaluation of specificity of anti-rAT1aR (317-335) polyclonal antibody and anti-rAT1aR (317-335) monoclonal antibody in immunohistological staining
• Anti-rAT1aR (317-335) polyclonal antibody obtained in Example 1 and Example 2 Immunohistochemical staining was performed on mouse liver tissue using the antibody and anti-rAT1aR (317-335) monoclonal antibody.
• Angiotensin II type 1a receptor gene-deficient mice (referred to as “AT1aR KO mice"), angiotensin II type 1a receptor gene-deficient mice and angiotensin II type 1b receptor gene-deficient mice (referred to as “AT1abR KO mice”) each have These are so-called knockout mice in which both the angiotensin II type 1a receptor gene, the angiotensin II type 1a receptor gene, and the angiotensin II type 1b receptor gene are deleted or rendered dysfunctional.
• AT1abR KO mice were produced using the method described in the literature (Proc. Natl. Acad. Sci. USA (1998) 95:15496-15501).
• FIG. 4 is a photograph showing the results of immunohistological staining of mouse liver tissue using an anti-rAT1aR (317-335) polyclonal antibody
• FIG. 5 is a photograph showing the results of immunohistological staining of mouse liver tissue using an anti-rAT1aR (317-335) monoclonal antibody.
• binding to type 1b receptors is not thought to be a problem in normal mouse tissues, but in AT1aR KO mice, expression of angiotensin II type 1b receptors increases, so tissue staining of AT1aR KO mice does not show stained images. It is considered that this has been approved.
• the anti-rAT1aR (317-335) polyclonal antibody can specifically stain angiotensin II type 1 receptor in immunohistological staining of mouse liver tissue. Immunohistochemical staining using anti-rAT1aR (317-335) monoclonal antibody gave similar results, but the stained image was clearer compared to that using anti-rAT1aR (317-335) polyclonal antibody.
• Test 3 Evaluation of specificity of anti-rAT1aR (317-335) polyclonal antibody and anti-rAT1aR (317-335) monoclonal antibody in Western blot Angiotensin II type 1a receptor in HEK293 cells forced to express rat angiotensin II type 1a receptor
• the expression levels of body proteins were determined by labeling the anti-rAT1aR (317-335) polyclonal antibody and anti-rAT1aR (317-335) monoclonal antibody obtained in Example 1 and Example 2, and the introduced rat angiotensin II type 1a receptor gene. It was measured by Western blotting using HA (Hemagglutinin).
• HA Hemagglutinin
• FIG. 6 is a photograph showing the results of Western blotting using an anti-rAT1aR (317-335) polyclonal antibody
• FIG. 7 is a photograph showing the results of Western blotting using an anti-rAT1aR (317-335) monoclonal antibody.
• Western blotting using anti-rAT1aR (317-335) polyclonal antibody and anti-rAT1aR (317-335) monoclonal antibody revealed that rat angiotensin II type 1a receptor was expressed in HEK293 cells.

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