WO2006057339A1 - Method of producing antibody - Google Patents

Method of producing antibody Download PDF

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Publication number
WO2006057339A1
WO2006057339A1 PCT/JP2005/021684 JP2005021684W WO2006057339A1 WO 2006057339 A1 WO2006057339 A1 WO 2006057339A1 JP 2005021684 W JP2005021684 W JP 2005021684W WO 2006057339 A1 WO2006057339 A1 WO 2006057339A1
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Prior art keywords
peptide
antibody
site
solution
loop structure
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PCT/JP2005/021684
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French (fr)
Japanese (ja)
Inventor
Seima Itami
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Mitsubishi Pharma Corporation
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Priority to JP2006547853A priority Critical patent/JPWO2006057339A1/en
Publication of WO2006057339A1 publication Critical patent/WO2006057339A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

  • the present invention relates to a method for efficiently producing an antibody against a site that forms a loop structure under physiological conditions of a protein.
  • Antibodies against proteins are usually produced by immunizing non-human mammals with whole proteins, which are linear peptides having a partial sequence thereof. If an antibody is obtained against a particular domain of the protein, it is usually immunized with a peptide of sequence within that domain.
  • proteins form complex three-dimensional structures in vivo, there are many cases in which antibodies that recognize a protein in a three-dimensional manner cannot be obtained by immunization with linear peptides.
  • a protein itself is immunized, it is difficult to obtain an antibody that recognizes only a certain specific domain.
  • the homology of the region other than the complementarity determining region (CDR) is very high. It was difficult to obtain an antibody that recognizes only the antibody.
  • An object of the present invention is to provide a method for efficiently obtaining an antibody capable of specifically recognizing a site forming a loop structure under physiological conditions of a protein.
  • the present inventor has intensively studied to solve the above problems. As a result, a complex loop structure is formed in vivo by immunizing a non-human mammal with a peptide having a cyclic structure and an amino acid sequence that forms a loop structure under the physiological conditions of the protein. The inventors have found that a specific antibody against a protein having a site can be efficiently obtained, and have completed the present invention.
  • the present invention is as follows.
  • An antibody against a protein having a site that forms a loop structure under physiological conditions A method comprising the steps of immunizing a non-human mammal with a peptide having a cyclic structure, including the amino acid sequence of the site forming the loop structure, and recovering the non-human mammal antibody. .
  • a method for producing an antibody against a protein having a site that forms a loop structure under physiological conditions, the peptide comprising the amino acid sequence of the site that forms the loop structure, and having a cyclic structure and non-human comprising: immunizing a mammal; recovering B cells from the non-human mammal and fusing them with myeloma cells to obtain a hyperidoma; and recovering the antibody from the culture supernatant of the hybridoma.
  • a method for producing an antibody against a protein having a site that forms a loop structure under physiological conditions comprising a peptide having a cyclic structure and a non-human peptide comprising the amino acid sequence of the site that forms the loop structure
  • a method comprising the steps of immunizing a mammal, recovering B cells from the non-human mammal, infecting with EBV and immortalizing, and recovering an antibody from the culture supernatant.
  • FIG. 1 is a graph showing the results of titer measurement of mouse antiserum.
  • FIG. 2 is a graph showing the results of competition assay of whole Ab 3-3 and E2tag with mouse antiserum. The reactivity when each serum was allowed to compete was shown by absorbance.
  • FIG. 3 is a diagram (photograph) showing the results of subclass test of No. 61 strain.
  • FIG. 4 is a graph showing the results of reactivity of monoclonal antibody 61-11F to whole Ab3-3.
  • FIG. 5 is a schematic diagram of the three-dimensional structure of an antibody.
  • the method for producing an antibody of the present invention is a method for producing an antibody against a protein having a site that forms a loop structure under physiological conditions, comprising the amino acid sequence of the site. And immunizing a non-human mammal with a peptide having a cyclic structure, and recovering the antibody from the non-human mammal.
  • the protein having a site having a loop structure under physiological conditions is not particularly limited, and examples thereof include an antibody, a growth factor, an enzyme, and a receptor. Among these, an antibody is particularly preferred.
  • the loop structure may be a stem-and-loop structure or the like that does not necessarily need to be circular.
  • the length of the site having the loop structure is not particularly limited, but 1 to 50 amino acids are preferred, 1 to 36 amino acids are more preferred, and 1 to 18 amino acids are more preferred.
  • physiological conditions means physiological conditions such as in cells or body fluids, and includes solutions such as physiological saline.
  • the antibody has a variable region and constant region force, and the CDR complementation determining region of the hypervariable region in the variable region has a loop structure (Fig. 5).
  • CDRs are examples of sites that take loop structures under physiological conditions in antibodies.
  • an antibody against CDR is prepared, an antibody that specifically recognizes CDR is hardly obtained even if a peptide having the amino acid sequence of CDR is used as an antigen as it is.
  • a peptide having a cyclic structure mimics the loop structure of CDR, an antibody that specifically recognizes CDR can be obtained efficiently.
  • the crystal structure analysis of protein three-dimensional structure is not powerful, it is possible to immunize the peptide sequence by estimating the loop structure based on the three-dimensional structure prediction data. Even if the three-dimensional structure is not clear, the three-dimensional structure can be determined by X-ray crystal structure analysis, etc., and based on this, the part having the loop structure can be selected.
  • a peptide having an amino acid sequence at a site forming the loop structure and having a cyclic structure is used.
  • the amino acid sequence of the site forming the loop structure may be the amino acid sequence of the entire loop, but the amino acid sequence of a part of the loop or preferably a part of the loop including the amino acid at the tip (folding point) of the loop. It may be a mino acid sequence.
  • the peptide used in the present invention may have an amino acid sequence ability at the above site, or may be an amino acid sequence added with an arbitrary number of amino acids.
  • the length of the peptide used in the present invention is not particularly limited, but 1 to 50 amino acids are preferable, 1 to 36 amino acids are more preferable, and 1 to 17 amino acids are more preferable.
  • the cyclic structure may be included in a part of the peptide used in the present invention, or the whole peptide may form a cyclic structure.
  • the peptide having the amino acid sequence and having a cyclic structure can be obtained, for example, by first synthesizing a linear peptide containing the amino acid sequence and circularizing it by the following method. it can. However, peptides containing a cyclic structure may be synthesized from the beginning.
  • Cyclization is achieved by forming a disulfide bond between the thiol groups of two cysteine residues present in the peptide.
  • side chain amino group such as ⁇ -amino group of lysine existing in the peptide
  • side chain carboxyl group such as 13 carboxyl group of aspartic acid or ⁇ -carboxyl group of glutamic acid
  • Cyclic formation occurs by crosslinking between an amino group and a thiol group.
  • amino acid sequence of a site having a loop structure under physiological conditions and a cyclic structure-containing peptide containing the amino acid sequence.
  • peptides that can be used in the present invention are not limited to this example.
  • This sequence is the sequence of CDR3 of anti-HCV (hepatitis C virus) antibody.
  • cyclic peptide containing this sequence examples include peptides as shown below which are made cyclic by disulfide bonding of the 6th and 11th cysteines.
  • a non-human mammal is immunized with a peptide having a cyclic structure as described above.
  • the type of non-human mammal is not particularly limited, but rabbits, mice, rats, goats, hidges, horses, rabbits, monkeys and the like are preferable.
  • the antibody produced by the method of the present invention may be an antiserum, a polyclonal antibody, or a monoclonal antibody.
  • a general method for example, a method of immunizing by injecting a cyclic structure-containing peptide combined with an adjuvant into a non-human mammal preferably 2 to 3 times. It is done.
  • the polyclonal antibody can be recovered from the serum of the immunized non-human mammal using, for example, a protein A column. Polyclonal antibodies are not necessarily purified. It may be collected as an antiserum containing polyclonal antibodies that need not be manufactured. Whether the recovered polyclonal antibody or the serum containing it specifically recognizes the target protein can be evaluated by, for example, Western blotting or ELISA.
  • Monoclonal antibodies are produced from, for example, antibodies produced by hybridomas produced by fusing lymphocytes isolated from non-human mammals immunized with a cyclic structure-containing peptide with myeloma cells. It can be obtained by selecting an antibody that specifically recognizes the target protein. The evaluation of antibody specificity can be confirmed by Western blotting or ELISA.
  • monoclonal antibodies were obtained by, for example, producing immortalized cells by infecting lymphocytes isolated from non-human mammals immunized with a cyclic structure-containing peptide with EBV (Epstein Barr virus). It can also be obtained by selecting an antibody that specifically recognizes the target protein from the antibodies produced by the cells. The specificity of the antibody can be confirmed by Western blotting or ELISA.
  • monoclonal antibodies obtained from antisera, polyclonal antibodies, hyperpridoma cells or EBV immortalized cells are used as antibody fragments, for example, F (ab ′)-modified antibodies, F (ab ') Antibody, short chain antibody (scFv), diabodies and mi
  • the monoclonal antibody obtained by the hyperidoma cell may be chimerized or humanized.
  • a monoclonal antibody is chimerized or engineered using a recombinant antibody-producing technique for producing a human constant antibody constant region or a humanized antibody producing technique for a human other than the hypervariable region.
  • the antibody obtained by the production method of the present invention can be used as an antibody drug, a diagnostic agent, a research reagent and the like.
  • an anti-CDR antibody can be obtained.
  • the anti-CDR antibody is suitable for pharmacokinetic measurement of an antibody drug or for identifying an antibody derived from the same animal species. Can be used.
  • VLR17S-S cyclic peptide a cyclic peptide (hereinafter referred to as VLR17S-S cyclic peptide) in which the peptide was cross-linked intramolecularly with two cysteines.
  • 40 mg of VLR17SH free peptide obtained by the above operation was dissolved in 400 mL of degassed water and stirred slowly. Three days later, 10 mL of the solution was collected and lyophilized. Since the SH group was measured using Ellman's reagent and a value of 0.005 was obtained, it was considered that the free SH group in the VLR17SH free peptide was sufficiently reduced by cyclization. The next day, the solvent was concentrated to about 80 mL, fractionated by HPLC, and the resulting solution was lyophilized. VLR17S-S cyclic peptide was obtained by this process
  • the whole Ab 3-3 solution was replaced with 0.1 M NaCl, 0.1 M Acetate buffer (pH 3.2) using Centricon 30. The final concentration was 12.6 mg / mL and stored at 4 ° C.
  • reaction composition was prepared and incubated at 37 ° C for 3 hours.
  • F (a) of human IgGl / kappa used as an ELISA negative control was prepared.
  • VLR17S-S cyclic peptide lmg powder was dissolved in 500 ⁇ l of EDC conditioning buffer (0.1 M MES buffer ( ⁇ 4.7) 0.9 ⁇ NaCl 0.02% NaN3).
  • KLH (powder) purchased from Pierce
  • 100 l (lmg) was added to the peptide solution.
  • 5 mg of EDC reagent purchased from Pierce
  • VLR17SH free—KLH Maleimide cross-linked antigen (VLR17SH free—KLH) solution 100 1 (corresponding to 100 g peptide) + PBS 400 ⁇ l + FCA (purchased from Difco) 500 ⁇ 1 500 ⁇ l of 8 week old abdominal cavity was administered.
  • mice were administered 500 1 at a time into the abdominal cavity of 8 weeks old.
  • VLR17SH free—KLH Maleimide cross-linked antigen (VLR17SH free—KLH) solution 100 1 (corresponding to 100 g of peptide) + PBS 800 ⁇ 1 + Libi-adjuvant (purchased from RIBI / Corixa) 100 ⁇ 1 were mixed, and 2 immunized BALBZc mice 500 500 1 doses were administered to the abdominal cavity at 10 weeks of age.
  • EDC cross-linked product antigen (VLR 17S—S cyclic—KLH) solution 100 1 (equivalent to 125 g of peptide) + PBS 800 ⁇ 1 + Libi adjuvant 100 ⁇ 1 were mixed, and 2 immunized BALBZc mice were placed in the abdominal cavity of 10 weeks old. 500 ⁇ l was administered.
  • Ribi adjuvant (RIBI: 67060-121801004) 100 ⁇ 1 was mixed, and 250 ⁇ l was administered to the peritoneal cavity of 4 immunized BALB Zc mice 10 weeks old.
  • Tertiary immunization was performed at intervals of 2 weeks after secondary immunization.
  • each mouse is subjected to Nanno ring (ear punching), and blood is collected by performing 3 times 40 to 50 1 blood collection from the orbital vein using a capillary blood collection tube. It was. After standing at room temperature for 1 hour, serum was separated using a centrifuge (10000 rpm, 5 minutes).
  • ELISA plates were prepared, each immobilized with cyclic peptide, linear peptide, whole Ab 3-3, and human Ig Gl / kappa F (ab ′) for negative subjects.
  • VLR17S-S cyclic peptide 600 ⁇ g powder was weighed and autoclaved sterilized water (600 ⁇ 1) was added to prepare a lmgZml peptide solution.
  • Block Ace TM with preservative purchased from Dainippon Pharmaceutical was added at 200 uel and stored in a low-temperature room at 4 ° C until use.
  • Aminobrate (purchased from Sumitomo Beichi Clyde) was prepared and diluted with PBS to prepare 2 % Dartalaldehyde (purchased from Nacalai Tester) was charged with 50 ⁇ 1Z wells, reacted at room temperature for 1 hour, and aldehyded.
  • Block Ace TM with preservative purchased from Dainippon Pharmaceutical was added in 200 1Z well and stored at 4 ° C until use.
  • Block ace TM containing preservatives (purchased from Dainippon Pharmaceutical Co., Ltd.) in aminoblate was added at 200 ⁇ ⁇ / well and stored at 4 ° C until use.
  • the blocking solution of the 5 kinds of antigen-immobilized plate prepared was discarded, and the solution was thoroughly cut off with Kim towel.
  • Each of the collected mouse partial sera was diluted 10-fold with Block Ace TM stock solution. 40 1 diluted serum was added to each well and allowed to react for 1 hour at room temperature (20 ° C). Each well was washed 3 times with PBS. Drain the solution thoroughly with Kim Towel.
  • Anti-mouse IgG—POD-labeled antibody purchased from Zymed
  • Chromogenic substrate ABTS was added at 50 / z lZ well and allowed to react for 10 minutes at room temperature. Add 150 mM oxalic acid solution with 50 1Z well to stop the reaction, molecular Absorbance at A405nm was measured using M-Tmax (device number UVT06000) from Device Corporation.
  • mice immunized with VLR17S-S cyclic peptide sera reactive only with whole Ab 3-3 could be obtained efficiently.
  • E2tag solution (described in WO 03/014728) is diluted to 0.05 ⁇ g / mL with 0.05M bicarbonate buffer (pH 9.6), added to a 96-well plate at 50 ⁇ L / well, and 1 ° C at 37 ° C. Incubated for hours. The solution in each well was discarded with a decantation, and PBS containing 250 ⁇ L of 5% BSA was placed in each well and incubated at 4 ° C for 16 hours or more. After removing the solution, each well was washed 5 times with PBS containing 0.05% Tween20.
  • Phosphate Citrate Buffer taenoic acid (anhydrous)
  • 20 mg of 0-phenylenediamine purchased from SIGMA
  • the reaction was allowed to proceed for about 5 minutes at room temperature, protected from light. After stopping the reaction by adding an equal volume of 10% sulfuric acid, the absorbance A1 at wavelength 1 (490 nm) and the absorbance A2 at wavelength 2 (650 nm) of each well were measured using an immuno reader. (A1-A2) Asked.
  • whole Ab 3-3 immunized mouse serum inhibited the interaction between whole Ab 3-3 and E2tag in both sera reactive with whole Ab 3-3. Of these, one case was weaker with an antibody that specifically recognizes only whole Ab 3-3.
  • an idiotype antiserum against the target antibody was efficiently obtained in a mouse immunized with a peptide obtained by cyclizing the CDR3 of the target antibody. Furthermore, the obtained antiserum efficiently inhibited the binding between the target antibody and the antigen.
  • mice immunized with a peptide cyclized with CDR3 also removed the spleen and attempted to produce a hyperidoma and a monoclonal antibody.
  • a fourth immunization was performed at an interval of one month from the third immunization.
  • the spleen was removed from the mouse 3 days later, and blood was collected from the heart at the same time.
  • the serum was collected in a small cooling centrifuge (10000 rpm, 5 minutes).
  • the excised spleen was crushed with a glass rod sterilized with dry heat while adding 10 ml of serum-free medium on a metal mesh sterilized with dry heat. Placed in a 50 ml centrifuge tube and washed 3 times with serum-free medium.
  • mouse myeloma cells P3U1 were cultured in six 9cm dishes as a fusion partner, collected, washed three times in the same serum-free medium, and mixed together to prepare 50% PEG (Nacalai Tester).
  • the last eye of the eye had no cell suspension applied for antiserum as a positive control.
  • HAT medium ICN: 16-808-49 and Sanko Junyaku: CM-B
  • CM-B CM-B
  • the antigen plate was prepared by adding 13 ⁇ m of Imunoplate (Narugenunk: code.468667) at 50 ⁇ 1 uel.
  • Block Ace TM (Dainippon Pharmaceutical: code.UK-B25) stock solution was added in 200 ⁇ l Zwell and allowed to stand overnight at 4 ° C for blocking operation.
  • anti-mouse IgG—POD labeled antibody (Zymed: 61-6520) was diluted 500 times with Block Ace TM , put in 50 1Z well and left at room temperature for 1 hour. Then, the secondary reaction was performed.
  • the positive strain (No. 61) was cloned by the limiting dilution method. Colonies were picked up under a microscope to separate from competing cells. The activity of the cell group separated by pick-up was confirmed again, and the group force with positive reaction also started crawling. Cloning was performed by using a 96-well culture plate containing 100 1 of cloning medium and diluted cell solution 100 1 diluted to 2 cells, 1 cell, and 0.5 cells per well. I went in the usual way.
  • VLR17S-S cyclic peptide plate was prepared and subjected to cloning on the diluted wells.
  • the culture supernatant obtained by culturing the 61-11F strain was purified using a Protein A column.
  • the culture supernatant was applied to a Protein A column (column height 1 cm, column diameter 1 cm) at a linear speed of 0.5 cmZmin. Thereafter, 50 ml of PBS was flowed at a linear speed of 0.5 cmZmin to wash the column. Next, about 30 ml of Elution solution and 0.1 M Acetate buffer (pH 3.0) were flowed at a linear speed of 0.5 cmZmin. Dispense the Elution solution into 14 flasks of 2 ml each. Abs 280 nm of the dispensed Elution solution was measured using a spectrophotometer (Hitachi, double beam spectrophotometer U-2001). Combine the Elution solution of the Fracolle tube that had absorbance in the peak range into one antibody samp le.
  • the obtained antibody sample was replaced with PBS containing 0.1% azide by a conventional method using Centricon 50.
  • the solution 9 was added at 100 L / well and allowed to react for about 5 minutes while protected from light at room temperature. After stopping the reaction by adding an equal volume of 10% sulfuric acid, immunowell reader (Inter Med, Immuno Reader NJ-2000) was used for each well at wavelength 1 (490 °) and wavelength 2 (650 °). Absorbances A1 and A2 were measured to determine (Al-A2). As a result, it was shown that the 61-11F-derived monoclonal antibody specifically binds only to whole Ab 3_3 (Fig. 4).
  • composition of each solution is as follows.
  • Solution 1 PBS containing 0.1% Sodium Azide
  • Solution 2 PBS containing 1% BSA and 0.1% Sodium Azide
  • Solution 3 PBS containing 0.05% Tween20
  • Solution 5 HRP-labeled anti-mouse immunoglobulin goat antibody (purchased from Zymed) 2 ⁇ L of stock solution dissolved in 10 ml of solution 4 (prepared at time of use)
  • Solution 6 Take 14.2 g of sodium monohydrogen phosphate and add water to make 500 ml.
  • Solution 7 Take 10.5g of citrate monohydrate and add water to make 500ml
  • Solution 8 Add 243 ml of solution 6 to 257 ml of solution 5 and add water to make 1000 ml.
  • Solution 9 0 -Furendiamine (purchased from Wako Pure Chemicals) 4.0mg, take solution 7 10ml and 30
  • a specific antibody against a protein having a site that forms a complex loop structure in a living body can be efficiently produced.

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Abstract

An antibody, which specifically recognizes a site in a protein capable of forming a loop structure under physiological conditions, is efficiently acquired by immunizing a nonhuman mammal with a peptide containing the amino acid sequence of the site in the protein capable of forming a loop structure under physiological conditions and having a cyclic structure.

Description

明 細 書  Specification
抗体の製造方法  Antibody production method
技術分野  Technical field
[0001] 本発明は、タンパク質の生理的条件下でループ構造を形成する部位に対する抗体 を効率よく製造する方法に関する。  The present invention relates to a method for efficiently producing an antibody against a site that forms a loop structure under physiological conditions of a protein.
背景技術  Background art
[0002] タンパク質に対する抗体は、通常、その部分配列を有する直鎖状ペプチドある 、は wholeのタンパク質で非ヒト哺乳動物を免疫することによって作製される。タンパク質の ある特定のドメインに対する抗体を取得した 、場合は、通常はそのドメイン内の配列 のペプチドを免疫する。しかし、タンパク質は生体内で複雑な 3次元構造を形成して いるため、直鎖状ペプチドの免疫ではタンパク質を立体的特異的に認識する抗体が 得られない場合が多カゝつた。さらに、タンパク質そのものを免疫した場合にはそのタ ンパク質を立体的に認識する抗体が得られることはある力 ある特定のドメインのみを 認識する抗体を得ることは困難であった。特に、抗体に対する抗体を取得する場合 は相補性決定領域 (CDR)以外の部分のホモロジ一が非常に高いため、同動物種の 他の抗体にはまったく反応性を示さず、特定の 1種類の抗体のみを認識する抗体を 取得することは困難であった。  [0002] Antibodies against proteins are usually produced by immunizing non-human mammals with whole proteins, which are linear peptides having a partial sequence thereof. If an antibody is obtained against a particular domain of the protein, it is usually immunized with a peptide of sequence within that domain. However, since proteins form complex three-dimensional structures in vivo, there are many cases in which antibodies that recognize a protein in a three-dimensional manner cannot be obtained by immunization with linear peptides. Furthermore, when a protein itself is immunized, it is difficult to obtain an antibody that recognizes only a certain specific domain. In particular, when obtaining an antibody against an antibody, the homology of the region other than the complementarity determining region (CDR) is very high. It was difficult to obtain an antibody that recognizes only the antibody.
発明の開示  Disclosure of the invention
[0003] 本発明はタンパク質の生理的条件下でループ構造を形成する部位を特異的に認 識できる抗体を効率よく取得できる方法を提供することを課題とする。  [0003] An object of the present invention is to provide a method for efficiently obtaining an antibody capable of specifically recognizing a site forming a loop structure under physiological conditions of a protein.
[0004] 本発明者は上記課題を解決すベぐ鋭意検討を行った。その結果、タンパク質の生 理的条件下でループ構造を形成する部位のアミノ酸配列を含み、かつ環状構造を 有するペプチドで非ヒト哺乳動物を免疫することにより、生体内で複雑なループ構造 を形成する部位を有するタンパク質に対する特異的抗体を効率よく取得できることを 見出し、本発明を完成するに至った。  [0004] The present inventor has intensively studied to solve the above problems. As a result, a complex loop structure is formed in vivo by immunizing a non-human mammal with a peptide having a cyclic structure and an amino acid sequence that forms a loop structure under the physiological conditions of the protein. The inventors have found that a specific antibody against a protein having a site can be efficiently obtained, and have completed the present invention.
[0005] すなわち、本発明は以下のとおりである。  That is, the present invention is as follows.
(1)生理的条件下でループ構造を形成する部位を有するタンパク質に対する抗体を 製造する方法であって、前記ループ構造を形成する部位のアミノ酸配列を含み、環 状構造を有するペプチドで非ヒト哺乳動物を免疫するステップと、該非ヒト哺乳動物 力 抗体を回収するステップを含む方法。 (1) An antibody against a protein having a site that forms a loop structure under physiological conditions A method comprising the steps of immunizing a non-human mammal with a peptide having a cyclic structure, including the amino acid sequence of the site forming the loop structure, and recovering the non-human mammal antibody. .
(2)生理的条件下でループ構造を形成する部位を有するタンパク質に対する抗体を 製造する方法であって、前記ループ構造を形成する部位のアミノ酸配列を含み、か つ環状構造を有するペプチドで非ヒト哺乳動物を免疫するステップと、該非ヒト哺乳 動物から B細胞を回収しミエローマ細胞と融合させてハイプリドーマを得るステップと、 ノ、イブリドーマの培養上清力も抗体を回収するステップを含む方法。  (2) A method for producing an antibody against a protein having a site that forms a loop structure under physiological conditions, the peptide comprising the amino acid sequence of the site that forms the loop structure, and having a cyclic structure and non-human A method comprising: immunizing a mammal; recovering B cells from the non-human mammal and fusing them with myeloma cells to obtain a hyperidoma; and recovering the antibody from the culture supernatant of the hybridoma.
(3)生理的条件下でループ構造を形成する部位を有するタンパク質に対する抗体を 製造する方法であって、前記ループ構造を形成する部位のアミノ酸配列を含み、か つ環状構造を有するペプチドで非ヒト哺乳動物を免疫するステップと、該非ヒト哺乳 動物から B細胞を回収し EBVを感染させて不死化するステップと、その培養上清から 抗体を回収するステップを含む方法。  (3) A method for producing an antibody against a protein having a site that forms a loop structure under physiological conditions, comprising a peptide having a cyclic structure and a non-human peptide comprising the amino acid sequence of the site that forms the loop structure A method comprising the steps of immunizing a mammal, recovering B cells from the non-human mammal, infecting with EBV and immortalizing, and recovering an antibody from the culture supernatant.
(4)前記ペプチドが、前記アミノ酸配列に含まれる 2個のシスティン残基のチオール 基間のジスルフイド結合を介する環状構造を有するペプチドである、 (1)〜（3)の 、 ずれかの方法。  (4) The method according to any one of (1) to (3), wherein the peptide is a peptide having a cyclic structure via a disulfide bond between thiol groups of two cysteine residues contained in the amino acid sequence.
(5)タンパク質が抗体であり、ループ構造をとる部位力 CDRである、（1)〜（4)のいず れかの方法。  (5) The method according to any one of (1) to (4), wherein the protein is an antibody and is a site force CDR having a loop structure.
図面の簡単な説明  Brief Description of Drawings
[0006] [図 1]マウス抗血清の力価測定の結果を示すグラフ図。 [0006] FIG. 1 is a graph showing the results of titer measurement of mouse antiserum.
[図 2]マウス抗血清による whole Ab 3-3と E2tagの competition assayの結果を示すグラ フ図。各血清を競合させたときの反応性を吸光度で示した。  FIG. 2 is a graph showing the results of competition assay of whole Ab 3-3 and E2tag with mouse antiserum. The reactivity when each serum was allowed to compete was shown by absorbance.
[図 3]No.61株のサブクラス検定結果を示す図（写真)。  FIG. 3 is a diagram (photograph) showing the results of subclass test of No. 61 strain.
[図 4]モノクローナル抗体 61-11Fの whole Ab3-3への反応性の結果を示すグラフ図。  FIG. 4 is a graph showing the results of reactivity of monoclonal antibody 61-11F to whole Ab3-3.
[図 5]抗体の立体構造の模式図。  FIG. 5 is a schematic diagram of the three-dimensional structure of an antibody.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0007] 本発明の抗体の製造方法は、生理的条件下でループ構造を形成する部位を有す るタンパク質に対する抗体を製造する方法であって、前記部位のアミノ酸配列を含み 、かつ環状構造を有するペプチドで非ヒト哺乳動物を免疫するステップと、該非ヒト哺 乳動物から抗体を回収するステップを含む方法である。 [0007] The method for producing an antibody of the present invention is a method for producing an antibody against a protein having a site that forms a loop structure under physiological conditions, comprising the amino acid sequence of the site. And immunizing a non-human mammal with a peptide having a cyclic structure, and recovering the antibody from the non-human mammal.
[0008] 生理的条件下でループ構造をとる部位を有するタンパク質としては特に制限されな いが、抗体、増殖因子、酵素、受容体などが挙げられ、その中では抗体が特に好まし い。ループ構造は必ずしも環状になっている必要はなぐステムアンドループ構造な どであってもよい。ループ構造をとる部位の長さは特に制限されないが、 1〜50アミノ 酸が好ましぐ 1〜36アミノ酸がより好ましぐ 1〜18アミノ酸がさらに好ましい。なお、 「生理的条件下」とは、細胞内や体液中などの生理的条件下を意味し、生理食塩水 などの溶液中も含む。  [0008] The protein having a site having a loop structure under physiological conditions is not particularly limited, and examples thereof include an antibody, a growth factor, an enzyme, and a receptor. Among these, an antibody is particularly preferred. The loop structure may be a stem-and-loop structure or the like that does not necessarily need to be circular. The length of the site having the loop structure is not particularly limited, but 1 to 50 amino acids are preferred, 1 to 36 amino acids are more preferred, and 1 to 18 amino acids are more preferred. The term “physiological conditions” means physiological conditions such as in cells or body fluids, and includes solutions such as physiological saline.
抗体は可変領域と定常領域力 なり、可変領域の中の超過変領域の CDR湘補性 決定領域）がループ構造をとっている（図 5)。したがって、抗体における生理的条件 下でループ構造をとる部位としては CDRが挙げられる。 CDRに対する抗体を作製す る場合、 CDRのアミノ酸配列を有するペプチドをそのまま抗原として用いても CDRを 特異的に認識する抗体がほとんど得られない。本発明の製造法によれば、環状構造 を有するペプチドが CDRのループ構造を模倣するため、 CDRを特異的に認識する抗 体が効率よく得られる。  The antibody has a variable region and constant region force, and the CDR complementation determining region of the hypervariable region in the variable region has a loop structure (Fig. 5). Thus, CDRs are examples of sites that take loop structures under physiological conditions in antibodies. When an antibody against CDR is prepared, an antibody that specifically recognizes CDR is hardly obtained even if a peptide having the amino acid sequence of CDR is used as an antigen as it is. According to the production method of the present invention, since a peptide having a cyclic structure mimics the loop structure of CDR, an antibody that specifically recognizes CDR can be obtained efficiently.
[0009] その他のタンパク質についても、現在、 X線結晶構造解析により、数多くのタンパク 質について 3次元構造が明らかになっており、それを参照することによりループ構造 をとる部位を選択することができる。例えば、 sbi.imim.es/cgi- bin/archdb/loops.plから アクセスすることができる ArcDB (Nucleic Acids Res. 2004 Jan 1;32 Database issue:D 185-8.)においては X線結晶構造解析のされている蛋白質のループ構造を取る部分 を検索することが可能である。上記データベースでは蛋白質のループ構造を取って V、る部分のペプチドの配列、すなわち本発明における免疫すべきペプチドを特定す ることが可能である。上記データベースをもとにループ状構造を検索すると、ミオシン 2重鎖の 264〜295アミノ酸の部分、ペプシンの 48〜74アミノ酸部分、ヘモグロビン の 21〜70アミノ酸部分などが検索される。  [0009] With regard to other proteins as well, three-dimensional structures of many proteins have been clarified by X-ray crystal structure analysis, and a site having a loop structure can be selected by referring to them. . For example, in ArcDB (Nucleic Acids Res. 2004 Jan 1; 32 Database issue: D 185-8.) Accessible from sbi.imim.es/cgi-bin/archdb/loops.pl, X-ray crystal structure analysis It is possible to search for a portion of a protein that has a loop structure. In the above database, it is possible to take the loop structure of the protein and specify the peptide sequence of V, that is, the peptide to be immunized in the present invention. When the loop structure is searched based on the above database, the 264-295 amino acid portion of myosin duplex, the 48-74 amino acid portion of pepsin, the 21-70 amino acid portion of hemoglobin, and the like are searched.
さらに、 X線結晶構造解析のデータベースは種々存在するので（Protein Data Bank Japanなど）、それらを任意に用いて立体構造の推定およびループ構造の決定をす ることち可會である。 Furthermore, there are various databases for X-ray crystal structure analysis (Protein Data Bank Japan, etc.), so they can be used arbitrarily to estimate steric structures and determine loop structures. It's pretty cute.
また、蛋白質の立体構造の結晶解析が分力 ていない場合でも立体構造予測デ ータをもとにループ構造を推定しそのペプチド配列を免疫することも可能である。 また、 3次元構造が明らかになっていないものでも、 X線結晶構造解析などによって 3次元構造を決定し、それに基づいて、ループ構造をとる部位を選択することができ る。  In addition, even when the crystal structure analysis of protein three-dimensional structure is not powerful, it is possible to immunize the peptide sequence by estimating the loop structure based on the three-dimensional structure prediction data. Even if the three-dimensional structure is not clear, the three-dimensional structure can be determined by X-ray crystal structure analysis, etc., and based on this, the part having the loop structure can be selected.
[0010] 本発明の抗体の製造方法においては、上記ループ構造を形成する部位のアミノ酸 配列を含み、かつ環状構造を有するペプチドを用いる。ループ構造を形成する部位 のアミノ酸配列としては、ループ全体のアミノ酸配列でもよいが、ループの一部のアミ ノ酸配列や好ましくはループの先端 (折り返し点）のアミノ酸を含むループの一部のァ ミノ酸配列であってもよい。  [0010] In the method for producing an antibody of the present invention, a peptide having an amino acid sequence at a site forming the loop structure and having a cyclic structure is used. The amino acid sequence of the site forming the loop structure may be the amino acid sequence of the entire loop, but the amino acid sequence of a part of the loop or preferably a part of the loop including the amino acid at the tip (folding point) of the loop. It may be a mino acid sequence.
本発明で用いるペプチドは上記部位のアミノ酸配列力もなるものであってもよいが、 上記部位のアミノ酸配列にさらに任意の数のアミノ酸が付加されたものであってもよ い。本発明で用いるペプチドの長さは特に制限されないが、 1〜50アミノ酸が好まし く、 1〜36アミノ酸がより好ましぐさらに好ましくは 1〜17アミノ酸がよい。なお、環状 構造は本発明で用いるペプチドの一部に含まれて 、てもよ 、し、ペプチド全体が環 状構造を形成していてもよい。  The peptide used in the present invention may have an amino acid sequence ability at the above site, or may be an amino acid sequence added with an arbitrary number of amino acids. The length of the peptide used in the present invention is not particularly limited, but 1 to 50 amino acids are preferable, 1 to 36 amino acids are more preferable, and 1 to 17 amino acids are more preferable. The cyclic structure may be included in a part of the peptide used in the present invention, or the whole peptide may form a cyclic structure.
[0011] 上記アミノ酸配列を含み、かつ環状構造を有するペプチドは、例えば、まず、上記 アミノ酸配列を含む直鎖上のペプチドを合成し、以下のような方法で環状ィ匕すること によって得ることができる。ただし、最初から環状構造を含むペプチドを合成してもよ い。  [0011] The peptide having the amino acid sequence and having a cyclic structure can be obtained, for example, by first synthesizing a linear peptide containing the amino acid sequence and circularizing it by the following method. it can. However, peptides containing a cyclic structure may be synthesized from the beginning.
1.ペプチド内に存在する 2つのシスティン残基のチオール基間にジスルフイド結合 を形成させることにより環状化する。  1. Cyclization is achieved by forming a disulfide bond between the thiol groups of two cysteine residues present in the peptide.
2.ペプチド内に存在するリジンの εーァミノ基などの側鎖アミノ基と、ァスパラギン酸 の 13 カルボキシル基、グルタミン酸の γ—カルボキシル基などの側鎖カルボキシ ル基とをペプチド結合させて環状化する。  2. Peptide bond the side chain amino group such as ε-amino group of lysine existing in the peptide and side chain carboxyl group such as 13 carboxyl group of aspartic acid or γ-carboxyl group of glutamic acid to cyclize.
3.ホモ二価架橋試薬、ヘテロ二価架橋試薬などの化学的架橋剤を用い、ペプチド 内の側鎖間、例えば、アミノ基間、カルボキシル基間、アミノ基ーカルボキシル基間、 アミノ基—チオール基間等で架橋することにより環状ィ匕する。 3.Using chemical cross-linking agents such as homo-bivalent cross-linking reagents and hetero-bivalent cross-linking reagents, between side chains in peptides, for example, between amino groups, between carboxyl groups, between amino groups and carboxyl groups, Cyclic formation occurs by crosslinking between an amino group and a thiol group.
4.ループ構造をとる部位のアミノ酸配列にシスティンなどの架橋可能なアミノ酸を付 カロした配列を有するペプチドを合成し、付加されたアミノ酸を介して環状ィ匕することも 可能である。例えばシスティンのな 、ペプチドの両末端にシスティンを入れて架橋す る。  4. It is also possible to synthesize a peptide having a sequence in which a crosslinkable amino acid such as cysteine is attached to the amino acid sequence of a site having a loop structure and circulate through the added amino acid. For example, without cysteine, cysteine is inserted at both ends of the peptide to crosslink.
[0012] 以下に、生理的条件下でループ構造をとる部位のアミノ酸配列とそれを含む環状 構造含有ペプチドの一例を挙げる。ただし、本発明に用いることのできるペプチドは この例に限定されない。  [0012] The following is an example of an amino acid sequence of a site having a loop structure under physiological conditions and a cyclic structure-containing peptide containing the amino acid sequence. However, peptides that can be used in the present invention are not limited to this example.
Val Leu Arg uly Tyr Cys Arg Arg uly Serし ys Tyr Asp Trp Leu Asp Pro (目歹 U¾=号 1)  Val Leu Arg uly Tyr Cys Arg Arg uly Ser ys Tyr Asp Trp Leu Asp Pro (target U¾ = number 1)
この配列は抗 HCV (C型肝炎ウィルス）抗体の CDR3の配列である。  This sequence is the sequence of CDR3 of anti-HCV (hepatitis C virus) antibody.
この配列を含む環状ペプチドとしては 6番目と 11番目のシスティンをジスルフイド結 合させることによって環状にした下記に示すようなペプチドが挙げられる。  Examples of the cyclic peptide containing this sequence include peptides as shown below which are made cyclic by disulfide bonding of the 6th and 11th cysteines.
[0013] [化 1] [0013] [Chemical 1]
Arg-Gly Arg-Gly
Arg Ser  Arg Ser
ValLeuArgGlyTyrCys CysTyrAspTrpLeuAspPro  ValLeuArgGlyTyrCys CysTyrAspTrpLeuAspPro
[0014] 本発明の抗体の製造方法では、上述したような環状構造を有するペプチドで非ヒト 哺乳動物を免疫する。非ヒト哺乳動物の種類は特に制限されないが、ゥサギ、マウス 、ラット、ャギ、ヒッジ、ゥマ、ゥシ、サルなどが好ましい。本発明の方法によって製造さ れる抗体は抗血清、ポリクローナル抗体であってもよいし、モノクローナル抗体であつ てもよい。 [0014] In the method for producing an antibody of the present invention, a non-human mammal is immunized with a peptide having a cyclic structure as described above. The type of non-human mammal is not particularly limited, but rabbits, mice, rats, goats, hidges, horses, rabbits, monkeys and the like are preferable. The antibody produced by the method of the present invention may be an antiserum, a polyclonal antibody, or a monoclonal antibody.
免疫する方法は一般的な方法を採用することができ、たとえば、アジュバントを配合 した環状構造含有ペプチドを、好ましくは 2〜3回、非ヒト哺乳動物に注射すること〖こ より免疫する方法が挙げられる。  As a method for immunization, a general method can be adopted, for example, a method of immunizing by injecting a cyclic structure-containing peptide combined with an adjuvant into a non-human mammal preferably 2 to 3 times. It is done.
ポリクローナル抗体は、免疫された非ヒト哺乳動物の血清から、例えば、プロテイン Aカラムなどを用いて回収することができる。なお、ポリクローナル抗体は必ずしも精 製されたものである必要はなぐポリクローナル抗体を含む抗血清として回収されても よい。回収されたポリクローナル抗体またはそれを含む血清が目的タンパク質を特異 的に認識するかどうかの評価は、例えば、ウェスタンブロットや ELISAによって行うこと ができる。 The polyclonal antibody can be recovered from the serum of the immunized non-human mammal using, for example, a protein A column. Polyclonal antibodies are not necessarily purified. It may be collected as an antiserum containing polyclonal antibodies that need not be manufactured. Whether the recovered polyclonal antibody or the serum containing it specifically recognizes the target protein can be evaluated by, for example, Western blotting or ELISA.
モノクローナル抗体は、例えば、環状構造含有ペプチドで免疫された非ヒト哺乳動 物から単離されたリンパ球をミエローマ細胞と融合させてハイプリドーマを作製し、得 られたハイブリドーマが産生する抗体の中から、目的タンパク質を特異的に認識する 抗体を選択することによって得ることができる。抗体の特異性の評価は、ウェスタンブ ロットや ELISAなどによって確認することができる。  Monoclonal antibodies are produced from, for example, antibodies produced by hybridomas produced by fusing lymphocytes isolated from non-human mammals immunized with a cyclic structure-containing peptide with myeloma cells. It can be obtained by selecting an antibody that specifically recognizes the target protein. The evaluation of antibody specificity can be confirmed by Western blotting or ELISA.
さらに、モノクローナル抗体は、例えば、環状構造含有ペプチドで免疫された非ヒト 哺乳動物から単離されたリンパ球を EBV (ェプスタインバーウィルス）で感染させて不 死化細胞を作製し、得られた細胞が産生する抗体の中から、目的タンパク質を特異 的に認識する抗体を選択することによって得ることもできる。抗体の特異性の評価は 、ウェスタンブロットや ELISAなどによって確認することができる。  Furthermore, monoclonal antibodies were obtained by, for example, producing immortalized cells by infecting lymphocytes isolated from non-human mammals immunized with a cyclic structure-containing peptide with EBV (Epstein Barr virus). It can also be obtained by selecting an antibody that specifically recognizes the target protein from the antibodies produced by the cells. The specificity of the antibody can be confirmed by Western blotting or ELISA.
[0015] さらに、本発明においては、抗血清、ポリクローナル抗体やハイプリドーマ細胞や E BV不死化細胞から得られたモノクローナル抗体を、抗体のフラグメント、例えば、 F (a b ' )化抗体、 F (ab' )化抗体、短鎖抗体（scFv)、ダイアポディ (Diabodies)およびミFurthermore, in the present invention, monoclonal antibodies obtained from antisera, polyclonal antibodies, hyperpridoma cells or EBV immortalized cells are used as antibody fragments, for example, F (ab ′)-modified antibodies, F (ab ') Antibody, short chain antibody (scFv), diabodies and mi
2 2
二ボディ（Minibodies)などにしてもよい。また、ハイプリドーマ細胞によって得られた モノクローナル抗体を、キメラ化又はヒト化してもよい。具体的には、遺伝子組み換え によって抗体の定常領域をヒト由来のものとするキメラ抗体作成技術や、超可変領域 以外をヒト由来とするヒト化抗体作成技術などを用いて、モノクローナル抗体をキメラ 化又はヒト化することができる。  Two bodies (Minibodies) may be used. In addition, the monoclonal antibody obtained by the hyperidoma cell may be chimerized or humanized. Specifically, a monoclonal antibody is chimerized or engineered using a recombinant antibody-producing technique for producing a human constant antibody constant region or a humanized antibody producing technique for a human other than the hypervariable region. Can be humanized.
[0016] 本発明の製造法によって得られる抗体は、抗体医薬、診断薬、研究用試薬などとし て使用することができる。また、環状構造を有するペプチドが抗体の CDRに基づくぺ プチドである場合、抗 CDR抗体が得られるが、抗 CDR抗体は抗体医薬の薬物動態測 定ゃ同動物種由来抗体の識別などに好適に使用することができる。 [0016] The antibody obtained by the production method of the present invention can be used as an antibody drug, a diagnostic agent, a research reagent and the like. In addition, when the peptide having a cyclic structure is a peptide based on the CDR of an antibody, an anti-CDR antibody can be obtained. The anti-CDR antibody is suitable for pharmacokinetic measurement of an antibody drug or for identifying an antibody derived from the same animal species. Can be used.
実施例  Example
[0017] 以下、実施例を挙げて本発明を具体的に説明する。ただし、本発明は以下の実施 例に限定されるものではない。 Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is as follows. It is not limited to examples.
[0018] [実施例 1]ポリクローナル抗体 (抗血清)の作製と評価  [0018] [Example 1] Preparation and evaluation of polyclonal antibody (antiserum)
<免疫用ペプチドの調製 >  <Preparation of peptide for immunization>
抗ペプチド抗体作製用として、 Pioneer (Applied Biosystems社）を用いて、 F-moc 固相合成法により whole Ab 3- 3 (完全抗 HCV抗体; WO 03/014728に記載）の CDR3 のアミノ酸配列である" VLRGYCRRGS CYDWLDP"のペプチド（配列番号 1)を 、 80%の純度で合成し、精製を実施した。  The amino acid sequence of CDR3 of whole Ab 3-3 (complete anti-HCV antibody; described in WO 03/014728) by F-moc solid-phase synthesis using Pioneer (Applied Biosystems) for the production of anti-peptide antibodies. A peptide of “VLRGYCRRGS CYDWLDP” (SEQ ID NO: 1) was synthesized with a purity of 80% and purified.
ペプチド合成時に脱保護は TFA(4.5mL)、チオア-ノール (0.25mL)、エタンジチォ ール（EDT) (0.15mL)、ァ-ソール (O.lmL)の混液を用い 3.5時間行った。榭脂をろ過 で除き、エーテル (無水）を用いて遠沈により 2回洗浄後、乾燥をした。水 75mLとァセ トニトリル 5mLの混液に本品を溶解し分取用 HPLC(Waters 600E; column u- Bondasp here 150 X 19mm)を用いて相当部分を分取した。分取部分を凍結乾燥し、精製直鎖 状ペプチド（以下、 VLR17SHフリーペプチドと呼ぶ）約 48mgを得た。本品のうち 8mg を分取しこれ以降の操作に使用した。  During peptide synthesis, deprotection was performed for 3.5 hours using a mixture of TFA (4.5 mL), thioanol (0.25 mL), ethanediol (EDT) (0.15 mL), and arsol (O.lmL). The resin was removed by filtration, washed twice by centrifugation with ether (anhydrous) and then dried. This product was dissolved in a mixture of 75 mL of water and 5 mL of acetonitrile, and a corresponding portion was separated using preparative HPLC (Waters 600E; column u-Bondasp here 150 X 19 mm). The fractionated portion was lyophilized to obtain about 48 mg of a purified linear peptide (hereinafter referred to as VLR17SH free peptide). 8 mg of this product was collected and used for subsequent operations.
さらにペプチドを 2つのシスティンで分子内架橋した環状ペプチド（以下 VLR17S —S環状ペプチドと呼ぶ）の合成を実施した。上記操作で得られた VLR17SHフリー ペプチド 40mgを脱気した水 400mLに溶解しゆっくり撹拌を行った。 3日後に溶液のう ち 10mLを分取し、凍結乾燥を行った。 Ellman試薬を用いて SH基の測定を行い、 0.00 5という値を得たので、環状化により VLR17SHフリーペプチド中のフリーの SH基は 十分少なくなつたと考えた。翌日、溶媒を 80mL程度に濃縮し HPLCにより分取を行い 、得られた溶液を凍結乾燥した。本工程により VLR17S— S環状ペプチドを取得した  Furthermore, we synthesized a cyclic peptide (hereinafter referred to as VLR17S-S cyclic peptide) in which the peptide was cross-linked intramolecularly with two cysteines. 40 mg of VLR17SH free peptide obtained by the above operation was dissolved in 400 mL of degassed water and stirred slowly. Three days later, 10 mL of the solution was collected and lyophilized. Since the SH group was measured using Ellman's reagent and a value of 0.005 was obtained, it was considered that the free SH group in the VLR17SH free peptide was sufficiently reduced by cyclization. The next day, the solvent was concentrated to about 80 mL, fractionated by HPLC, and the resulting solution was lyophilized. VLR17S-S cyclic peptide was obtained by this process
[0019] <免疫用 F(a ）の作製 > [0019] <Production of F (a) for immunization>
2  2
whole Ab 3- 3溶液をセントリコン 30を用いて、 0.1M NaCl, 0.1M Acetate buffer(pH 3 .2)に置換した。最終濃度を 12.6mg/mLとし、 4°Cで保存した。  The whole Ab 3-3 solution was replaced with 0.1 M NaCl, 0.1 M Acetate buffer (pH 3.2) using Centricon 30. The final concentration was 12.6 mg / mL and stored at 4 ° C.
以下の反応組成液を作製し、 37°Cで 3時間インキュベートした。  The following reaction composition was prepared and incubated at 37 ° C for 3 hours.
(組成）  (Composition)
whole Ab 3—3 10.4mg ペプシン 15 μ g/1.2mL 0.1M NaCl, O.IM Acetate buffer(pH 3.2) 反応後、直ちに 200 μ Lづつに小分けし、 1M Tris baseを 25 μ L加えた後- 80°Cに保 存した。 whole Ab 3—3 10.4mg Pepsin 15 μg / 1.2 mL 0.1 M NaCl, O.IM Acetate buffer (pH 3.2) Immediately after reaction, aliquot 200 μL, add 25 μL of 1 M Tris base, and store at -80 ° C. .
Gel濾過精製前に 1本ずつ融解し、 TOSOH TSKgel G3000SWXLにて精製および P BSへの置換を実施した。 Gelろ過により約 120(kDa)の whole Ab 3-3の F(a ）のみに  Prior to gel filtration purification, each was melted and purified with TOSOH TSKgel G3000SWXL and replaced with PBS. Only about 120 (kDa) whole Ab 3-3 F (a) by Gel filtration
2 精製し免疫用抗原とした。  2 Purified and used as an antigen for immunization.
さらに、 ELISAの陰性対照として用いるヒト IgGl/kappaの F(a ）を作製した。  In addition, F (a) of human IgGl / kappa used as an ELISA negative control was prepared.
2  2
ヒト IgGl/kappa(THE BINDING SITE社より購入） 4mgをセントリコン 30を用いて、 0.1 M NaCl, O.IM Acetate buffer(pH 3.2)に置換する操作を 4回行った。最終濃度を 9.9m g/mLとし、 4°Cで保存した。  The operation of replacing 4 mg of human IgGl / kappa (purchased from THE BINDING SITE) with 0.1 M NaCl, O.IM Acetate buffer (pH 3.2) using Centricon 30 was performed 4 times. The final concentration was 9.9 mg / mL and stored at 4 ° C.
以下の反応組成液を 2本作製し、 37°Cで 3時間インキュベートした。  Two reaction composition solutions below were prepared and incubated at 37 ° C for 3 hours.
(組成） (Composition)
ヒ卜 Igtjl/kappa 2mg  卜 Igtjl / kappa 2mg
ペプシン 2.9 μ g/231 L O.IM NaCl, O.IM Acetate buffer(pH 3.2) 反応後、直ちに 1M Tris baseを 29 Lカ卩えた後- 80°Cに保存した。  Pepsin 2.9 μg / 231 L O.IM NaCl, O.IM Acetate buffer (pH 3.2) Immediately after the reaction, 29 L of 1M Tris base was immediately stored and stored at −80 ° C.
Gel濾過精製前に 1本づっ融解し、 TOSOH TSKgel G3000SWXLにて精製および P BSへの置換を実施した。 Gelろ過により 120(kDa)のヒト IgGl/kappaの F(a ）のみに精  Before gel filtration purification, one was melted and purified with TOSOH TSKgel G3000SWXL and replaced with PBS. By gel filtration, only 120 (kDa) human IgGl / kappa F (a) was purified.
2 製した。  2 made.
<ペプチドの KLHへの架橋 > <Crosslinking of peptides to KLH>
(方法） (Method)
(1)マレイミド法による SHフリーペプチドと KLH(keyhole limpet hemocyanin)の架橋 VLR17SHフリーペプチド lmg粉末を 500 μ 1のマレイミドコンジユゲーシヨンバー ファー（80mMリン酸緩衝液（pH7) 0.1M EDTA 0.15M NaCl)で溶解した。マレイミド K LH (ピアス社より購入） lmg粉末を 500 μ 1の純水で溶解し、ペプチド溶液と直ちに 混合した。室温（25°C)で 3時間反応させた。反応後、 100 1ずつ 6本に分注して、 残りはひとまとめのまま 20°Cで保存した。 [0021] (2) EDC (1— ethy— 3— (3— dimethylaminopropyl) carbodiimide hydrochloride)法による 環状ペプチドと KLHの架橋 (1) Crosslinking of SH-free peptide and KLH (keyhole limpet hemocyanin) by maleimide method VLR17SH-free peptide lmg powder was added to 500 μ1 of maleimide conduit buffer (80 mM phosphate buffer (pH 7) 0.1 M EDTA 0.15 M NaCl ). Maleimide K LH (Purchased from Pierce) lmg powder was dissolved in 500 μl of pure water and immediately mixed with the peptide solution. The reaction was carried out at room temperature (25 ° C) for 3 hours. After the reaction, 100 1 was dispensed into 6 bottles, and the rest were stored together at 20 ° C. [0021] (2) Crosslinking of cyclic peptide and KLH by EDC (1— ethy— 3— (3— dimethylaminopropyl) carbodiimide hydrochloride) method
VLR17S— S環状ペプチド lmg粉末を 500 μ 1の EDCコンジユゲーシヨンバーフ ァー（0.1M MES緩衝液 (ρΗ4.7) 0.9Μ NaCl 0.02%NaN3)で溶解した。 KLH (粉末）（ ピアス社より購入）は、純水で lOmgZmlとして、そのうち 100 l (lmg分）をぺプチ ド溶液に添加した。 EDC試薬 (ピアス社より購入） 5mgを、 500 μ 1の純水で溶解し 、そのうち 50 1を、ペプチド 'KLH混合溶液に添カ卩し、室温（25°C)で 3時間反応さ せた。反応後、透析チューブ (分子量 10000カット）に移し、 2Lの PBSに対してー晚 透析した。翌日、透析チューブよりマイクロチューブ内に取り出し、さらに 100 1ずつ 6本に分注して、残りはひとまとめのまま 20°Cで保存した。  VLR17S-S cyclic peptide lmg powder was dissolved in 500 μl of EDC conditioning buffer (0.1 M MES buffer (ρΗ4.7) 0.9Μ NaCl 0.02% NaN3). KLH (powder) (purchased from Pierce) was added as pure lOmgZml, and 100 l (lmg) was added to the peptide solution. 5 mg of EDC reagent (purchased from Pierce) was dissolved in 500 μl of pure water, 50 1 of which was added to peptide KLH mixed solution and reacted at room temperature (25 ° C) for 3 hours. . After the reaction, it was transferred to a dialysis tube (molecular weight: 10000 cut) and dialyzed against 2 L of PBS. The next day, it was taken out from the dialysis tube into a microtube, and further dispensed into 6 bottles of 100 1 each, and the rest were stored together at 20 ° C.
[0022] <抗原のマウスへの免疫 >  <0022> <Immunization of mice with antigen>
KLH架橋 VLR17SHフリーペプチド、 KLH架橋 VLR17S— S環状ペプチド、 whol e Ab 3-3 F(ab')の 3種類の抗原を用いて免疫作業を実施した。  Immunization was performed using three types of antigens: KLH-crosslinked VLR17SH free peptide, KLH-crosslinked VLR17S-S cyclic peptide, whol Ab 3-3 F (ab ').
2  2
[0023] 一次免疫  [0023] Primary immunity
マレイミド架橋物抗原 (VLR17SHフリ一— KLH)溶液 100 1 (ペプチド 100 g相 当） +PBS400 μ l+FCA(Difco社より購入） 500 μ 1を混合し、ェマルジヨン化後、 2 匹の BALBZcマウス孚 8週令の腹腔〖こ 500 μ 1ずつ投与した。  Maleimide cross-linked antigen (VLR17SH free—KLH) solution 100 1 (corresponding to 100 g peptide) + PBS 400 μl + FCA (purchased from Difco) 500 μ 1 500 μl of 8 week old abdominal cavity was administered.
EDC架橋物抗原 (VLR 17S— S環状— KLH)溶液 100 1 (ペプチド 125 g相当 ) +PBS400 μ 1+FCA 500 μ 1を混合し、ェマルジヨン化後、 2匹の BALBZcマウ ス孚 8週令の腹腔に 500 μ 1ずつ投与した。  EDC cross-linked antigen (VLR 17S—S cyclic—KLH) solution 100 1 (equivalent to 125 g of peptide) + PBS 400 μ 1 + FCA 500 μ 1 mixed, after emulsification, 2 BALBZc mice 8 weeks old 500 μl was administered to the peritoneal cavity.
whole Ab 3-3 F(ab，）溶液（0. 869mg/ml) 250 /z 1 (86. 9 目当） +PBS750  whole Ab 3-3 F (ab,) solution (0.869 mg / ml) 250 / z 1 (86. 9 eyes) + PBS750
2  2
/z l+FCAl. 0mlを混合し、ェマルジヨン化後、 4匹の BALBZcマウス孚 8週令の腹 腔に 500 1ずつ投与した。  / z l + FCAl. 0 ml was mixed, and after emulsification, 4 BALBZc mice were administered 500 1 at a time into the abdominal cavity of 8 weeks old.
[0024] 二次免疫から三次免疫 [0024] Secondary to tertiary immunity
一次免疫から 2週間後、二次免疫を行った。  Two weeks after the primary immunization, secondary immunization was performed.
マレイミド架橋物抗原 (VLR17SHフリ一— KLH)溶液 100 1 (ペプチド 100 g相 当） +PBS800 μ 1+リビアジュバント（RIBI/Corixa社より購入） 100 μ 1を混合し、既 免疫 2匹の BALBZcマウス孚 10週令の腹腔に 500 1ずつ投与した。 EDC架橋物抗原 (VLR 17S— S環状— KLH)溶液 100 1 (ペプチド 125 g相当 ) +PBS800 μ 1+リビアジュバント 100 μ 1を混合し、既免疫 2匹の BALBZcマウス 孚 10週令の腹腔に 500 μ 1ずつ投与した。 Maleimide cross-linked antigen (VLR17SH free—KLH) solution 100 1 (corresponding to 100 g of peptide) + PBS 800 μ 1 + Libi-adjuvant (purchased from RIBI / Corixa) 100 μ 1 were mixed, and 2 immunized BALBZc mice 500 500 1 doses were administered to the abdominal cavity at 10 weeks of age. EDC cross-linked product antigen (VLR 17S—S cyclic—KLH) solution 100 1 (equivalent to 125 g of peptide) + PBS 800 μ1 + Libi adjuvant 100 μ 1 were mixed, and 2 immunized BALBZc mice were placed in the abdominal cavity of 10 weeks old. 500 μl was administered.
whole Ab 3—3 F(ab')溶液（0.869 mg/ml) 250 1 (86. 9 μ g相当） +PBS650 μ 1  whole Ab 3-3 F (ab ') solution (0.869 mg / ml) 250 1 (equivalent to 86.9 μg) + PBS650 μ 1
2  2
+リビアジュバント（RIBI:67060- 121801004) 100 μ 1を混合し、既免疫 4匹の BALB Zcマウス孚 10週令の腹腔に 250 μ 1ずつ投与した。  + Ribi adjuvant (RIBI: 67060-121801004) 100 μ1 was mixed, and 250 μl was administered to the peritoneal cavity of 4 immunized BALB Zc mice 10 weeks old.
二次免疫より 2週間の間隔をあけて三次免疫を行った。  Tertiary immunization was performed at intervals of 2 weeks after secondary immunization.
[0025] <血清の採取 > <0025> <Serum collection>
3次免疫の一週間後に、各マウスにナンノ《リング（耳パンチを施す)を実施し、キヤ ピラリー採血管を用いて、眼窩静脈より 40〜50 1採血する操作を 3回実施し血液を 集めた。室温 1時間放置後、遠心機（10000rpm、 5分）により血清を分離した。  One week after the third immunization, each mouse is subjected to Nanno ring (ear punching), and blood is collected by performing 3 times 40 to 50 1 blood collection from the orbital vein using a capillary blood collection tube. It was. After standing at room temperature for 1 hour, serum was separated using a centrifuge (10000 rpm, 5 minutes).
[0026] <力価測定 > [0026] <Titration>
力価測定のため、環状ペプチド、直鎖ペプチド、 whole Ab 3-3、陰性対象用ヒト Ig Gl/kappa F(ab')をそれぞれ固定化した ELISAプレートを調製した。  For titer measurement, ELISA plates were prepared, each immobilized with cyclic peptide, linear peptide, whole Ab 3-3, and human Ig Gl / kappa F (ab ′) for negative subjects.
2  2
[0027] (1)VLR17S— S環状ペプチド固定化プレート  [0027] (1) VLR17S—S cyclic peptide-immobilized plate
(i) VLR17S- S環状ペプチド、 600 μ g粉末を秤量してオートクレーブ滅菌水 600 μ 1を 加えて lmgZmlのペプチド溶解液とした。  (i) VLR17S-S cyclic peptide, 600 μg powder was weighed and autoclaved sterilized water (600 μ1) was added to prepare a lmgZml peptide solution.
(ii)ァミノプレート（住友べ一クライド社より購入）を用意し、 0. 1MMES緩衝液 (pH4 . 7)により、ペプチド濃度が 5 /z g/mlとなるように希釈し、 50 1/ゥエルで投入した  (ii) Prepare an amino plate (purchased from Sumitomo Beichi Clyde), dilute with 0.1MMES buffer (pH 4.7) to a peptide concentration of 5 / zg / ml, and inject at 50 1 / well. did
(iii)純水で lmgZmlの濃度に調製した EDC試薬 (ピアス社より購入)を 10 μ lZゥェ ルで (ii)で調製したアミノブレートに添加し、 4°C一晩架橋反応した。 (iii) EDC reagent (purchased from Pierce) adjusted to a concentration of lmgZml with pure water was added to the aminoblate prepared in (ii) at 10 μlZwell and allowed to crosslink overnight at 4 ° C.
(iv)翌日、 PBSにて各ゥエルを 3回洗浄後、防腐剤入りブロックエース™ (大日本製薬 より購入)を、 200 ゥエルで投入し、使用まで、低温室 4°C保管した。  (iv) The next day, each well was washed 3 times with PBS, then Block Ace ™ with preservative (purchased from Dainippon Pharmaceutical) was added at 200 uel and stored in a low-temperature room at 4 ° C until use.
[0028] (2)VLR17SHフリーペプチド固定化プレート  [0028] (2) VLR17SH free peptide immobilization plate
(0VLR17 SHフリーペプチド、 600 μ g粉末を秤量してオートクレーブ滅菌水 600 μ 1 をカ卩えて lmg/mlのペプチド溶解液とした。  (0 VLR17 SH free peptide, 600 μg powder was weighed and 600 μ 1 of autoclaved sterilized water was added to prepare a lmg / ml peptide solution.
(ii)アミノブレート (住友べ一クライド社より購入)を用意し、 PBSで希釈して調製した 2 %ダルタルアルデヒド (ナカライテスタより購入)を 50 μ 1Zゥエル投入し、室温で 1時 間反応させ、アルデヒドィ匕した。 (ii) Aminobrate (purchased from Sumitomo Beichi Clyde) was prepared and diluted with PBS to prepare 2 % Dartalaldehyde (purchased from Nacalai Tester) was charged with 50 μ1Z wells, reacted at room temperature for 1 hour, and aldehyded.
(iii) PBSにて各ゥエルを 3回洗浄後、 0. 2M炭酸緩衝液 (pH9. 5)で 5 g/mlに希 釈した SHフリーペプチドを 50 1Zゥエルで投入し、 4°C一晩架橋反応した。  (iii) After washing each well three times with PBS, add SH free peptide diluted to 5 g / ml with 0.2 M carbonate buffer (pH 9.5) at 50 1Z well, and overnight at 4 ° C. Cross-linking reaction occurred.
(iv)翌日、 PBSにて各ゥエルを 3回洗浄後、防腐剤入りブロックエース™ (大日本製薬 より購入)を、 200 1Zゥエルで投入し、使用まで 4°C保管した。  (iv) The next day, each well was washed 3 times with PBS, then Block Ace ™ with preservative (purchased from Dainippon Pharmaceutical) was added in 200 1Z well and stored at 4 ° C until use.
[0029] (3) whole Ab 3-3 F(ab')自然吸着プレート  [0029] (3) Whole Ab 3-3 F (ab ') natural adsorption plate
2  2
(i) whole Ab 3—3 F(ab') 869 μ gZmlを PBSにて、 5 μ gZmlに希釈し、 50 μ lZゥ  (i) Whole Ab 3-3 F (ab ') 869 μgZml is diluted with PBS to 5 μgZml, and 50 μlZ
2  2
エルでィムノプレート（ナルジェヌンクより購入）に投入し、 4°Cー晚放置した。  It was put into the Imunoplate (purchased from Nargenunk) at L and left at 4 ° C- 晚.
(ii)翌日、 PBSにて各ゥエルを 3回洗浄後、防腐剤入りブロックエース™ (大日本製薬 より購入)を、 200 1Zゥエルで投入し、使用まで 4°C保管した。  (ii) The next day, each well was washed 3 times with PBS, then Block Ace ™ with preservative (purchased from Dainippon Pharmaceutical) was added in 200 1Z well and stored at 4 ° C until use.
[0030] (4)ヒト IgGl/kappaZF(a ） 2 自然吸着プレート  [0030] (4) Human IgGl / kappaZF (a) 2 natural adsorption plate
(i)ヒト IgGl/kappa F(ab') 728 μ gZmlを PBSにて、 5 μ gZmlに希釈し、 50 μ \/  (i) Dilute human IgGl / kappa F (ab ') 728 μgZml with PBS to 5 μgZml, 50 μ \ /
2  2
ゥエルでィムノプレート（ナルジェヌンクより購入）に投入し、 4°Cー晚放置した。  We put it on the Imunoplate (purchased from Nargenunk) at the well and left it at 4 ° C.
(ii)翌日、 PBSにて各ゥエルを 3回洗浄後、防腐剤入りブロックエース™ (大日本製薬 より購入)を、 200 1Zゥエルで投入し、使用まで 4°C保管した。  (ii) The next day, each well was washed 3 times with PBS, then Block Ace ™ with preservative (purchased from Dainippon Pharmaceutical) was added in 200 1Z well and stored at 4 ° C until use.
[0031] (5)ブランクプレート  [0031] (5) Blank plate
(i)アミノブレートに防腐剤入りブロックエース™ (大日本製薬より購入)を、 200 μ \/ ゥエルで投入し、使用まで 4°C保管した。  (i) Block ace ™ containing preservatives (purchased from Dainippon Pharmaceutical Co., Ltd.) in aminoblate was added at 200 μ \ / well and stored at 4 ° C until use.
[0032] 力価測定は以下の方法で実施した。  [0032] The titer measurement was performed by the following method.
作製した 5種の抗原固定ィ匕プレートのブロッキング液を捨て、キムタオルで液をよく 切った。採取したマウス部分血清をそれぞれ、ブロックエース™原液で 10倍希釈した 。各ゥエルに 40 1ずつ、希釈した血清を投入し、室温（20°C) 1時間反応させた。 PB Sで各ゥエル 3回洗浄した。キムタオルで液をよく切った。抗マウス IgG— POD標識抗 体 (Zymed社より購入）をブロックエース™で 500倍希釈して 50 μ lZゥエルで投入し 、室温（20°C) 1時間反応させた。 PBSで各ゥエル 3回洗浄した。キムタオルで液をよ く切った。発色基質: ABTSを 50 /z lZゥエルで投入し、室温にて 10分発色反応させ た。 150mMシユウ酸溶液を 50 1Zゥエルで添カ卩し、反応を停止させ、モレキュラー デバイス社の M- Tmax (機体番号 UVT06000)を用いて A405nmの吸光度を測定した The blocking solution of the 5 kinds of antigen-immobilized plate prepared was discarded, and the solution was thoroughly cut off with Kim towel. Each of the collected mouse partial sera was diluted 10-fold with Block Ace ™ stock solution. 40 1 diluted serum was added to each well and allowed to react for 1 hour at room temperature (20 ° C). Each well was washed 3 times with PBS. Drain the solution thoroughly with Kim Towel. Anti-mouse IgG—POD-labeled antibody (purchased from Zymed) was diluted 500-fold with Block Ace ™, added in 50 μl Zwell, and reacted at room temperature (20 ° C.) for 1 hour. Each well was washed 3 times with PBS. The liquid was thoroughly cut with a Kim towel. Chromogenic substrate: ABTS was added at 50 / z lZ well and allowed to react for 10 minutes at room temperature. Add 150 mM oxalic acid solution with 50 1Z well to stop the reaction, molecular Absorbance at A405nm was measured using M-Tmax (device number UVT06000) from Device Corporation.
[0033] 吸光度測定結果を図 1に示す。 [0033] The absorbance measurement results are shown in FIG.
VLR17S-S環状ペプチド免疫マウス血清 2例はともに陰性対象であるヒト IgGl/kapp aには反応性を示さず whole Ab 3-3にのみ反応性を示した。  Both VLR17S-S cyclic peptide immunized mouse sera showed no reactivity to human IgGl / kappa, which was a negative target, but only to whole Ab 3-3.
VLR17 SHフリーペプチド免疫マウス血清 2例はともに whole Ab 3- 3にもヒト IgGl/ka ppaにも反応性を示さなかった。  Neither of the two VLR17 SH-free peptide-immunized mouse sera showed reactivity to whole Ab3-3 or human IgGl / ka ppa.
whole Ab 3-3免疫マウス血清 4例は 2例が whole Ab 3-3に反応性を示したがそのう ちの 1例はヒト IgGl/kappaにも反応性を示した。  Four cases of whole Ab 3-3 immunized mouse serum were reactive to whole Ab 3-3, one of which was also reactive to human IgGl / kappa.
ゆえに、 VLR17S-S環状ペプチドを免疫したマウスでは whole Ab 3-3のみに反応性 を示す血清が効率的に取得できた。  Therefore, in mice immunized with VLR17S-S cyclic peptide, sera reactive only with whole Ab 3-3 could be obtained efficiently.
[0034] <マウス抗血清による whole Ab 3—3と E2tagの competition assay > [0034] <Competition assay of whole Ab 3-3 and E2tag with mouse antiserum>
E2tag溶液（WO 03/014728に作製法を記載）を 0.05M Bicarbonate buffer(pH9.6)で 3.3 μ g/mLに希釈し、 50 μ L/wellにて 96wellプレートに加え、 37°Cで 1時間インキュべ ートした。各 wellの溶液をデカンテーシヨンで廃棄し、各 wellに 250 μ Lの 5% BSAを含 む PBSをカ卩え、 4°Cで 16時間以上保温した。溶液除去後、 0.05% Tween20を含む PBS で各 wellを 5回づっ洗浄した。同時に各免疫マウス血清を 5% BSAを含む PBSを用いて 50倍、 500倍、 5000倍希釈した溶液 50 μ Lと 50ng/mLの whole Ab 3- 3溶液 50 μ Lを混 合し 37°Cで 1時間インキュベートした。本溶液 50 Lを各 wellにて加え、 37°Cで 1時間 インキュベートした。溶液除去、 0.05% Tween20を含む PBSで 5回洗浄後、 5% BSAを含 む PBSを用いて HRP標識抗ヒト IgG(Fc)を 2000倍希釈した溶液を各 wellに 50 Lカロえ 、室温で 1時間保温した。液除去、 PBSで 5回洗浄後、 0-フエ-レンジァミン（SIGMAよ り購入） 20mgと 30%%H 0 20 Lをカ卩えた Phosphate Citrate Buffer (タエン酸（無水  E2tag solution (described in WO 03/014728) is diluted to 0.05 μg / mL with 0.05M bicarbonate buffer (pH 9.6), added to a 96-well plate at 50 μL / well, and 1 ° C at 37 ° C. Incubated for hours. The solution in each well was discarded with a decantation, and PBS containing 250 μL of 5% BSA was placed in each well and incubated at 4 ° C for 16 hours or more. After removing the solution, each well was washed 5 times with PBS containing 0.05% Tween20. At the same time, mix 50 μL of each immunized mouse serum diluted 50-fold, 500-fold, and 5000-fold with PBS containing 5% BSA and 50 μL of 50 ng / mL whole Ab3-3 solution at 37 ° C. Incubated for 1 hour. 50 L of this solution was added to each well and incubated at 37 ° C for 1 hour. Remove the solution, wash 5 times with PBS containing 0.05% Tween20, and then add 50 L of HRP-labeled anti-human IgG (Fc) diluted 2000 times with PBS containing 5% BSA at room temperature. Incubated for 1 hour. After removing the solution and washing with PBS 5 times, Phosphate Citrate Buffer (taenoic acid (anhydrous)) containing 20 mg of 0-phenylenediamine (purchased from SIGMA) and 30 %% 0-20 L
2 2  twenty two
) 4.7g、Na HPO 7.3gを MilliQ水 1Lに溶解した buffer (pH5.0) ) 50 Lを各 wellにカロえ  ) 4.7 g, Na HPO 7.3 g dissolved in 1 L MilliQ water buffer (pH 5.0)) 50 L in each well
2 4  twenty four
室温で遮光して約 5分間反応させた。等量の 10%硫酸を加えて反応を停止させた後、 ィムノリーダーを用いて各 wellの波長 1 (490nm)における吸光度 A1及び波長 2 (650nm )における吸光度 A2を測定し、（A1-A2)を求めた。  The reaction was allowed to proceed for about 5 minutes at room temperature, protected from light. After stopping the reaction by adding an equal volume of 10% sulfuric acid, the absorbance A1 at wavelength 1 (490 nm) and the absorbance A2 at wavelength 2 (650 nm) of each well were measured using an immuno reader. (A1-A2) Asked.
[0035] 結果を図 2に示す。 VLR17環状ペプチド免疫マウス血清は 2例ともに whole Ab 3-3と E2tagの interaction を阻害した。 The results are shown in FIG. VLR17 cyclic peptide immunized mouse sera inhibited the interaction between whole Ab 3-3 and E2tag in both cases.
また、 whole Ab 3-3免疫マウス血清は whole Ab 3-3に反応性を示した血清 2例とも に whole Ab 3-3と E2tagの interactionを阻害した。し力し、そのうち 1例は whole Ab 3-3 のみを特異的に認識する抗体ではな力つた。  In addition, whole Ab 3-3 immunized mouse serum inhibited the interaction between whole Ab 3-3 and E2tag in both sera reactive with whole Ab 3-3. Of these, one case was weaker with an antibody that specifically recognizes only whole Ab 3-3.
以上の結果より、目的抗体の CDR3を環状ィ匕したペプチドを免疫したマウスにおい て効率よく目的抗体に対するイデォタイプ抗血清が取得された。さら〖こ、得られた抗 血清は目的抗体と抗原の結合を効率よく阻害した。  From the above results, an idiotype antiserum against the target antibody was efficiently obtained in a mouse immunized with a peptide obtained by cyclizing the CDR3 of the target antibody. Furthermore, the obtained antiserum efficiently inhibited the binding between the target antibody and the antigen.
[0036] [実施例 2]モノクローナル抗体の作製と評価 [0036] [Example 2] Preparation and evaluation of monoclonal antibody
さらに CDR3を環状ィ匕したペプチドを免疫したマウス力も脾臓を摘出しハイプリドー マおよびモノクローナル抗体の作製を試みた。  In addition, the mouse immunized with a peptide cyclized with CDR3 also removed the spleen and attempted to produce a hyperidoma and a monoclonal antibody.
[0037] <抗原のマウスへの免疫 II > <0037> <Immune Immunization of Mice II>
四次免疫から五次免疫  Fourth to fifth immunity
三次免疫から 1ヶ月の間隔をあけて四次免疫を実施した。  A fourth immunization was performed at an interval of one month from the third immunization.
さらに、脾臓摘出前の追加免疫として四次免疫の二週間後に EDC架橋物抗原 (V LR17環状— KLH)溶液 100 1 (ペプチド 125 μ g相当） + PBS 800 μ 1+リビアジュ バント（RIBI:67060-121801004) 100 μ 1を混合し、 EDC架橋物抗原既免疫 2匹の ΒΑ LBZcマウス孚 10週令の腹腔に 500 1ずつ投与し五次免疫を実施した。  Furthermore, as a booster before splenectomy, two weeks after the fourth immunization, EDC cross-linked antigen (V LR17 cyclic—KLH) solution 100 1 (corresponding to 125 μg of peptide) + PBS 800 μ1 + Libijjuvant (RIBI: 67060- 121801004) 100 μ1 was mixed, and EDC cross-linked product antigen pre-immunization Two LBZc mice were administered to the abdominal cavity of 10 weeks old 500 1 at a time to carry out the fifth immunization.
[0038] <ハイブリドーマ作製と抗体の機能解析 > [0038] <Hybridoma production and antibody functional analysis>
( 1)細胞融合  (1) Cell fusion
五次免疫を施し、 3日後のマウスより脾臓を摘出し、同時に心臓より採血を実施し、 のちほど小型冷却遠心機にて（10000rpm、 5分)血清を採取した。摘出した脾臓は 、乾熱滅菌した金属メッシュ上で、無血清培地 10mlを加えながら、乾熱滅菌したガラ ス棒により脾臓をつぶした。 50ml遠心チューブに入れ、無血清培地でのベ 3回の洗 浄 '遠心を行った。一方、融合パートナーとしてマウス骨髄腫細胞 P3U1を 9cmシャ ーレ 6枚分培養したものを回収し、同じく無血清培地でのベ 3回の洗浄を行い、両者 を混合して 50%PEG (ナカライテスタ： code.28219-55、オートクレーブにて滅菌） /R PMI1640 (無血清)存在下で、細胞融合を実施した。 融合細胞は、血清培地 100mlに懸濁し、 100 1Zゥエルずつ 96穴培養プレート（ Falcon： code.3072)、 10枚分にまきこみ、 37°C、 5%COで培養を開始した。 10枚 After the fifth immunization, the spleen was removed from the mouse 3 days later, and blood was collected from the heart at the same time. The serum was collected in a small cooling centrifuge (10000 rpm, 5 minutes). The excised spleen was crushed with a glass rod sterilized with dry heat while adding 10 ml of serum-free medium on a metal mesh sterilized with dry heat. Placed in a 50 ml centrifuge tube and washed 3 times with serum-free medium. On the other hand, mouse myeloma cells P3U1 were cultured in six 9cm dishes as a fusion partner, collected, washed three times in the same serum-free medium, and mixed together to prepare 50% PEG (Nacalai Tester). : Code.28219-55, sterilized by autoclave) / R PMI1640 (serum-free) was present in the presence of cell fusion. The fused cells were suspended in 100 ml of serum medium, and 100 1Z wells were seeded in 96-well culture plates (Falcon: code.3072), 10 plates, and culture was started at 37 ° C, 5% CO. 10 sheets
2  2
目の最終ゥエル 1つは、陽性コントロールとしての抗血清用に細胞懸濁液は未投入と しておいた。  The last eye of the eye had no cell suspension applied for antiserum as a positive control.
[0039] (2) HAT選択  [0039] (2) HAT selection
細胞融合の翌日より、 HAT培地（ICN： 16- 808- 49と三光純薬： CM- B)を 100 μ \/ ゥエルで添加し、融合した細胞のみの選択培養を開始した。翌日から、培地交換を 実施した。  On the next day after cell fusion, HAT medium (ICN: 16-808-49 and Sanko Junyaku: CM-B) was added at 100 μ \ / well, and selective culture of only the fused cells was started. The medium was changed from the next day.
途中、顕微鏡観察によりコロニーの増殖を観察し、細胞融合の成功を確認した。  On the way, the growth of colonies was observed by microscopic observation to confirm the success of cell fusion.
[0040] (3)ハイブリドーマスクリーニング [0040] (3) Hybridoma screening
(i)抗原プレートの作製  (i) Preparation of antigen plate
whole Ab 3-3 F(ab')抗体とブランク抗体をそれぞれ PBS溶液により 10 g/ml  Whole Ab 3-3 F (ab ') antibody and blank antibody each in PBS solution at 10 g / ml
2  2
、 50 μ 1Ζゥエルで、ィムノプレート（ナルジェヌンク： code.468667)各 13枚に投入し、 抗原プレートを作製した。  The antigen plate was prepared by adding 13 μm of Imunoplate (Narugenunk: code.468667) at 50 μ1 uel.
(ii)翌日、ブロックエース TM (大日本製薬： code.UK- B25)原液を、 200 μ lZゥエルで 投入し、 4°C一晩放置しブロッキング操作を実施した。 (ii) On the next day, Block Ace ™ (Dainippon Pharmaceutical: code.UK-B25) stock solution was added in 200 μl Zwell and allowed to stand overnight at 4 ° C for blocking operation.
(iii)翌日、融合細胞 959株の培養上清を無菌下で、 110 1ずつ全ゥエルにつ ヽて 回収した。回収した上清を、 whole Ab 3-3 F(ab')抗体とブランク抗体プレートのそれ  (iii) On the next day, the culture supernatant of 959 strains of fused cells was collected and collected in 1101 aliquots under aseptic conditions. Collect the collected supernatant from whole Ab 3-3 F (ab ') antibody and blank antibody plate.
2  2
ぞれに 50 1ずつ投入し、室温で 1時間放置して、一次反応を実施した。  50 1 was added to each and left at room temperature for 1 hour to carry out the primary reaction.
(iv) PBSで各ゥエルを 3回洗浄後、抗マウス IgG— POD標識抗体（Zymed : 61-6520 )をブロックエース TMにて 500倍希釈し、 50 1Zゥエルで投入し、室温で 1時間放置 して、二次反応を実施した。 (iv) After washing each well three times with PBS, anti-mouse IgG—POD labeled antibody (Zymed: 61-6520) was diluted 500 times with Block Ace ™ , put in 50 1Z well and left at room temperature for 1 hour. Then, the secondary reaction was performed.
(V) PBSで各ゥエルを 4回洗浄後、 ABTSけ力ライテスタ試薬を使用した自家調製 品）を 100 /z lZゥエルで投入し、室温で 5〜 10分程度放置して、発色反応を実施し た。  (V) After washing each well 4 times with PBS, add ABTS strength lyesta reagent (self-prepared product) using 100 / z lZ well and leave at room temperature for 5-10 minutes to perform color reaction did.
[0041] 吸光度測定を行い発色が強力つた 63株について二次スクリーニングを実施した。  [0041] Absorbance measurement was performed, and secondary screening was performed on 63 strains with strong color development.
一次スクリーニングで陽性と思われた 63株について、再度、両抗原とあらたに環状 ペプチド抗原に対しての反応を調べた。二次スクリーニングでは二次抗体のバックグ ラウンドを回避するために環状ペプチド抗原を抗原としたスクリーニングを実施した。 環状ペプチドと whole Ab 3-3 F(ab')抗体に反応し、ブランク抗体に反応しない No. For 63 strains that appeared to be positive in the primary screening, the reaction against both antigens and a new cyclic peptide antigen was examined again. In secondary screening, secondary antibody background In order to avoid rounds, screening was performed using cyclic peptide antigens as antigens. Reacts with cyclic peptide and whole Ab 3-3 F (ab ') antibody, but does not react with blank antibody
2  2
61を選抜した。  61 were selected.
[0042] (4)陽性株のクローユング [0042] (4) Cloning of positive strains
限界希釈法により、陽性株 (No. 61)のクローユングを実施した。競合する細胞群と 分離するために、顕微鏡下でコロニーをピックアップした。ピックアップにより分離した 細胞群の活性を再度確認し、反応陽性の群力もクローユングを開始した。クローニン グは、あら力じめクロー-ングメディウム 100 1を入れた 96穴培養プレートに、 1穴あ たり 2細胞、 1細胞、 0. 5細胞となるように、希釈した細胞液 100 1をカ卩えて定法どお り行った。  The positive strain (No. 61) was cloned by the limiting dilution method. Colonies were picked up under a microscope to separate from competing cells. The activity of the cell group separated by pick-up was confirmed again, and the group force with positive reaction also started crawling. Cloning was performed by using a 96-well culture plate containing 100 1 of cloning medium and diluted cell solution 100 1 diluted to 2 cells, 1 cell, and 0.5 cells per well. I went in the usual way.
[0043] (5)サブクラス検定 [0043] (5) Subclass test
陽性株（クローユング前の No. 61)の培養上清を用いて、 IDZSP KIT(Zymed 9 3-6550)の説明書にしたがって、検定したところ IgG2aと判断された。（図 3)  Using a culture supernatant of a positive strain (No. 61 before cloning), according to the instructions of IDZSP KIT (Zymed 9 3-6550), it was judged as IgG2a. (Figure 3)
[0044] (6)クローユングアツセィとリクローニング [0044] (6) Cloning time and recloning
希釈培養したゥエルに対して、 VLR17S— S環状ペプチドプレートを作製してクロ 一二ングアツセィを実施した。  A VLR17S-S cyclic peptide plate was prepared and subjected to cloning on the diluted wells.
完全なシングルセルになるまで、限界希釈クローユングとアツセィを繰り返した。  Limit dilution clotting and assembly were repeated until a complete single cell was achieved.
[0045] (7)シングルクローン榭立 [0045] (7) Single clone establishment
シングルクローンを 3種独立して取得し、増殖してきた順に 本株 1種（61— 11F)、 亜株 2種 (61— 12H、 61— 9G)と名づけ、拡大培養を実施し凍結保存した。  Three single clones were obtained independently and named as one strain (61-11F) and two substrains (61-12H, 61-9G) in the order in which they were grown, expanded and stored frozen.
[0046] (8)培養上清の精製 [8] Purification of culture supernatant
61 - 11F株を培養し得られた培養上清を ProteinAカラムを用いて精製した。  The culture supernatant obtained by culturing the 61-11F strain was purified using a Protein A column.
ProteinAカラム（カラム高 lcm、カラム径 1cm)に線速 0.5cmZminで培養上清を appl yした。その後、 PBSを線速 0.5cmZminで 50ml流しカラムを洗浄した。次に Elution溶 液、 0.1M Acetate buffer(pH3.0)を線速 0.5cmZminで約 30ml流した。 Elution溶液を 2 mlづっフラコレチューブ 14本に分注した。分注した Elution溶液の Abs280nmを分光光 度計（日立製作所製、ダブルビーム分光光度計 U-2001)を用いてを測定した。ピー クの範囲で吸光度があったフラコレチューブの Elution溶液を 1つにまとめて抗体 samp leとした。 The culture supernatant was applied to a Protein A column (column height 1 cm, column diameter 1 cm) at a linear speed of 0.5 cmZmin. Thereafter, 50 ml of PBS was flowed at a linear speed of 0.5 cmZmin to wash the column. Next, about 30 ml of Elution solution and 0.1 M Acetate buffer (pH 3.0) were flowed at a linear speed of 0.5 cmZmin. Dispense the Elution solution into 14 flasks of 2 ml each. Abs 280 nm of the dispensed Elution solution was measured using a spectrophotometer (Hitachi, double beam spectrophotometer U-2001). Combine the Elution solution of the Fracolle tube that had absorbance in the peak range into one antibody samp le.
得られた抗体 sampleはセントリコン 50を用いて定法により 0.1%のァザイドを含む PBS に置換した。  The obtained antibody sample was replaced with PBS containing 0.1% azide by a conventional method using Centricon 50.
[0047] (9)モノクローナル抗体の活性測定 [0047] (9) Measurement of monoclonal antibody activity
whole Ab 3-3 F(ab')溶液およびヒト IgGl/kappa F(ab')溶液 (溶液 1で 10 g/mlに  Whole Ab 3-3 F (ab ') solution and human IgGl / kappa F (ab') solution (to 10 g / ml with solution 1
2 2  twenty two
希釈）を L/wellで 96wellプレートに加え、 37°Cで 2時間処理した。その後、 wellの 溶液をすて、各 wellに 250 1の溶液 2を加え、 4°Cで 16時間以上保温した。溶液除去 後、溶液 3で各 wellを 5回ずつ洗浄し、溶液 4で 100, 50, 25, 12.5, 6.25, 3.125(ng/mL )に希釈した 61-1 IF上清由来のモノクローナル抗体溶液を 50 L/wellでカ卩えて、 37 °Cで 1時間保温した。溶液除去、溶液 3で 5回洗浄後、溶液 5を L/wellでカロえ、 室温で 1時間保温した。液除去、 PBSで 5回洗浄後、溶液 9を 100 L/wellでカ卩えて室 温で遮光して約 5分間反応させた。等量の 10%硫酸を加えて反応を停止させた後、ィ ムノリーダー（Inter Med社製、 Immuno Reader NJ-2000)を用いて各 wellの波長 1 (490 應)及び波長 2 (650應）における吸光度 A1及び A2を測定し、（Al- A2)を求めた。そ の結果、 61-11F由来のモノクローナル抗体は whole Ab 3_3のみに特異的に結合す ることが示された（図 4)。  Dilution) was added to a 96-well plate at L / well and treated at 37 ° C for 2 hours. Thereafter, the well solution was rinsed, 250 1 solution 2 was added to each well, and the mixture was incubated at 4 ° C for 16 hours or longer. After removing the solution, wash each well 5 times with solution 3 and dilute the monoclonal antibody solution from 61-1 IF supernatant diluted to 100, 50, 25, 12.5, 6.25, 3.125 (ng / mL) with solution 4. Covered at 50 L / well and incubated at 37 ° C for 1 hour. After removing the solution and washing with Solution 3 five times, Solution 5 was prepared with L / well and incubated at room temperature for 1 hour. After removing the solution and washing 5 times with PBS, the solution 9 was added at 100 L / well and allowed to react for about 5 minutes while protected from light at room temperature. After stopping the reaction by adding an equal volume of 10% sulfuric acid, immunowell reader (Inter Med, Immuno Reader NJ-2000) was used for each well at wavelength 1 (490 °) and wavelength 2 (650 °). Absorbances A1 and A2 were measured to determine (Al-A2). As a result, it was shown that the 61-11F-derived monoclonal antibody specifically binds only to whole Ab 3_3 (Fig. 4).
[0048] なお、各溶液の組成は以下の通りである。 [0048] The composition of each solution is as follows.
溶液 1 : 0.1% Sodium Azideを含む PBS  Solution 1: PBS containing 0.1% Sodium Azide
溶液 2 : 1% BSA、 0.1% Sodium Azideを含む PBS  Solution 2: PBS containing 1% BSA and 0.1% Sodium Azide
溶液 3： 0.05%の Tween20を含む PBS  Solution 3: PBS containing 0.05% Tween20
溶液 4 : 1% BSAを含む PBS  Solution 4: PBS containing 1% BSA
溶液 5： HRP標識抗マウス免疫グロブリンャギ抗体 (Zymed社より購入)原液 2 μ Lを 10 mlの溶液 4に溶力したもの（用時調製）  Solution 5: HRP-labeled anti-mouse immunoglobulin goat antibody (purchased from Zymed) 2 μL of stock solution dissolved in 10 ml of solution 4 (prepared at time of use)
溶液 6：リン酸一水素ナトリウム 14.2gを取り、水をカ卩えて 500mlとする  Solution 6: Take 14.2 g of sodium monohydrogen phosphate and add water to make 500 ml.
溶液 7：クェン酸 1水和物 10.5gを取り水を加えて 500mlとする  Solution 7: Take 10.5g of citrate monohydrate and add water to make 500ml
溶液 8 :溶液 5 257mlに溶液 6を 243ml加え、水を加えて 1000mlとする  Solution 8: Add 243 ml of solution 6 to 257 ml of solution 5 and add water to make 1000 ml.
溶液 9： 0 -フ -レンジァミン（和光純薬より購入) 4.0mgを取り、溶液 7を 10ml及び 30 Solution 9: 0 -Furendiamine (purchased from Wako Pure Chemicals) 4.0mg, take solution 7 10ml and 30
%(v/v)過酸ィ匕水素溶液 5マイクロリットルを加えて溶かす (調製後 10分以内に用いた 産業上の利用の可能性 Add 5 microliters of% (v / v) peracid-hydrogen peroxide solution to dissolve (use within 10 minutes after preparation) Industrial applicability
本発明の方法によれば、生体内で複雑なループ構造を形成する部位を有するタン パク質に対する特異的抗体を効率よく製造することができる。  According to the method of the present invention, a specific antibody against a protein having a site that forms a complex loop structure in a living body can be efficiently produced.

Claims

請求の範囲 The scope of the claims
[1] 生理的条件下でループ構造を形成する部位を有するタンパク質に対する抗体を製 造する方法であって、前記ループ構造を形成する部位のアミノ酸配列を含み、かつ 環状構造を有するペプチドで非ヒト哺乳動物を免疫するステップと、該非ヒト哺乳動 物から抗体を回収するステップを含む方法。  [1] A method for producing an antibody against a protein having a site that forms a loop structure under physiological conditions, comprising an amino acid sequence of the site that forms the loop structure, and a peptide having a cyclic structure and non-human A method comprising the steps of immunizing a mammal and recovering an antibody from the non-human mammal.
[2] 生理的条件下でループ構造を形成する部位を有するタンパク質に対する抗体を製 造する方法であって、前記ループ構造を形成する部位のアミノ酸配列を含み、かつ 環状構造を有するペプチドで非ヒト哺乳動物を免疫するステップと、該非ヒト哺乳動 物から B細胞を回収しミエローマ細胞と融合させてハイプリドーマを得るステップと、ハ イブリドーマの培養上清力も抗体を回収するステップを含む方法。  [2] A method for producing an antibody against a protein having a site that forms a loop structure under physiological conditions, comprising an amino acid sequence of the site that forms the loop structure, and a peptide having a cyclic structure and non-human A method comprising the steps of immunizing a mammal, recovering B cells from the non-human mammal and fusing them with myeloma cells to obtain a hyperidoma, and recovering antibodies from the culture supernatant of the hybridoma.
[3] 生理的条件下でループ構造を形成する部位を有するタンパク質に対する抗体を製 造する方法であって、前記ループ構造を形成する部位のアミノ酸配列を含み、かつ 環状構造を有するペプチドで非ヒト哺乳動物を免疫するステップと、該非ヒト哺乳動 物から B細胞を回収しェプスタインバーウィルスを感染させて不死化するステップと、 その培養上清力 抗体を回収するステップを含む方法。  [3] A method for producing an antibody against a protein having a site that forms a loop structure under physiological conditions, comprising an amino acid sequence of the site that forms the loop structure, and a peptide having a cyclic structure and non-human A method comprising immunizing a mammal, recovering B cells from the non-human mammal, infecting with Epstein-Barr virus, and immortalizing, and recovering the culture supernatant antibody.
[4] 前記ペプチドが、前記アミノ酸配列に含まれる 2個のシスティン残基のチオール基間 のジスルフイド結合を介する環状構造を有するペプチドである、請求項 1〜3の 、ず れか一項に記載の方法。  [4] The peptide according to any one of claims 1 to 3, wherein the peptide is a peptide having a cyclic structure via a disulfide bond between thiol groups of two cysteine residues contained in the amino acid sequence. the method of.
[5] タンパク質が抗体であり、ループ構造をとる部位力 CDRである、請求項 1〜4のいず れか一項に記載の方法。  [5] The method according to any one of claims 1 to 4, wherein the protein is an antibody and is a site force CDR having a loop structure.
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JP2016026211A (en) * 2009-10-27 2016-02-12 ユセベ ファルマ ソシエテ アノニム FUNCTION MODIFYING Nav1.7 ANTIBODIES

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JP2000229997A (en) * 1999-02-10 2000-08-22 Nissui Pharm Co Ltd Cyclic peptide and aids vaccine
WO2003014728A1 (en) * 2001-08-10 2003-02-20 Mitsubishi Pharma Corporation Remedies for hepatitis c

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JP2000229997A (en) * 1999-02-10 2000-08-22 Nissui Pharm Co Ltd Cyclic peptide and aids vaccine
WO2003014728A1 (en) * 2001-08-10 2003-02-20 Mitsubishi Pharma Corporation Remedies for hepatitis c

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016026211A (en) * 2009-10-27 2016-02-12 ユセベ ファルマ ソシエテ アノニム FUNCTION MODIFYING Nav1.7 ANTIBODIES

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