WO2005040799A1 - 相補性ペプチド人工抗体 - Google Patents
相補性ペプチド人工抗体 Download PDFInfo
- Publication number
- WO2005040799A1 WO2005040799A1 PCT/JP2004/015140 JP2004015140W WO2005040799A1 WO 2005040799 A1 WO2005040799 A1 WO 2005040799A1 JP 2004015140 W JP2004015140 W JP 2004015140W WO 2005040799 A1 WO2005040799 A1 WO 2005040799A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- peptide
- complementary
- peptides
- antibody
- artificial antibody
- Prior art date
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2318/00—Antibody mimetics or scaffolds
- C07K2318/20—Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
Definitions
- the present invention relates to a method for detecting and analyzing a target protein by using a technique for designing a complementary peptide having a binding property to a protein, and using the complementary peptide as an artificial antibody instead of an antibody.
- One of the evaluation indices for utilizing a complementary peptide in a Genetic Algorithm is a hydrophobicity value.
- For the amino acid at each position of the peptide add the amino acids before and after it (5 or several amino acids before and after it, and 0 in the case of the terminal amino acid), calculate the average of the hydrophobicity values, and calculate the hydrophobicity.
- the sex value is the opposite value! If it is +3.0, -3.0 is the full score of the hydrophobicity evaluation at that amino acid position.
- the second evaluation index is the correspondence of the bulkiness of the amino acid side chain at the corresponding position.
- Ex carbons of the corresponding amino acids do not prevent them from approaching each other near 5 angstroms, resulting in a side chain volume. If no volume inhibition occurs, a full score shall be given, and a penalty score shall be assessed according to the degree of inhibition.
- As the third evaluation index an evaluation point is given using the degree of coincidence of the backbone alignment of the peptide skeleton as an evaluation index.
- a predetermined number e.g., 300
- randomly designed peptides consisting of the same number of amino acids as the target peptide is generated as a candidate peptide library, and their complementarity to the target peptide is evaluated. Record in memory.
- the top two peptides with the highest evaluation scores were selected from among them, and the amino acid sequences of those peptides were scrambled into another sequence.
- a next-generation peptide library is created.
- the complementarity to the target peptide is evaluated, the evaluation value is recorded in the combi- rator memory, the top two peptides are selected, and the next-generation peptide library is prepared in the same manner as described above. create. Repeat this continuously, ideally Is repeated continuously until a peptide with a full score is obtained.
- This computer program software is called MIMETIC! At or near the full score, a peptide is synthesized, the binding activity to the protein having the target peptide is evaluated, and a peptide with high binding affinity is adopted as an artificial antibody peptide.
- a mouse or a heron can be immunized with an antigen, and sera from animals that produce antibodies can be used as antisera to purify the antibodies with serum ability.
- Lymphocytes such as spleen cells of an animal such as a mouse immunized with an antigen can be fused with a myeloma cell line to prepare a hybridoma, and a hybridoma that produces the desired antibody can be cloned to produce a monoclonal antibody.
- the phage display method is used to prepare an antibody having reactivity with an epitope having no antigenicity to an immunized animal, but it is an extremely complicated method.
- a complementary peptide that is expected to have reactivity with a target peptide portion of an antigen protein is designed using a computer program such as MIMETIC. By synthesizing the designed peptide and confirming that it has specific binding to the target protein, it can be used as an artificial antibody peptide. Means for solving the problem
- a peptide having complementarity to the amino acid sequence of a target peptide portion of an antigen protein is designed using a computer program such as MIMETIC.
- the peptide prepared above is allowed to react with the target protein, and the specific binding to the target protein is examined.
- the peptide having specifically high binding is defined as an artificial antibody peptide.
- the complementarity binding to the amino acid sequence of the target site of any protein molecule is A peptide is prepared and used as an artificial antibody peptide for detecting an antigen protein.
- MIMETIC a design program software, can be used to design complementary peptides. Therefore, it is possible to freely design an artificial antibody peptide that reacts to an arbitrary site of the protein selected as a target, and it is not necessary to use a complicated antibody preparation technique such as immunizing an animal! / ⁇ . Since antibodies and the like are prepared, artificial antibody peptides for new proteins can be quickly created, so that it can be used to construct detection methods for new proteins and the like. When producing antibodies by immunizing animals, it is difficult to expect antibody production at sites common to animal species.
- the artificial antibody peptide of the present invention can create a peptide having reactivity also to a site common to animal species, and thus can easily construct a method for detecting an antigen protein.
- the present invention when constructing a detection system such as an ELISA method or a protein array method using two or more antibodies, the present invention, which can design and create an artificial antibody peptide for a distant site of a target molecule, is extremely useful. .
- Example 1 A peptide complementary to a peptide at each site of reverse transcriptase (hereinafter abbreviated as RT) of Human immunodeficiency virus-1 (HIV-1), which is a pathogenic virus of AIDS, was analyzed by the analysis program of the present invention ( As a result of automatic design by MIMETIC), each of the peptides shown in Table 1 could be designed.
- the synthesis of the designed peptide was carried out using a peptide synthesizer (AMS 422 Multiple Poptide Synthesizer) manufactured by ABiMED by an ordinary solid-phase method in which 9-Fluorenylmethoxycarbonyl (Fmoc) amino acids were sequentially bonded.
- a peptide synthesizer AMS 422 Multiple Poptide Synthesizer
- the synthesized peptide was cut out like a mold, and the residue block was removed.
- the resulting peptide was recovered by precipitation with ether, the ether was removed by drying and purified by reverse phase HPLC.
- Virus RNA was extracted by the guanidine isothiophene method (homezynski, P. & 3 ⁇ 4acchi, N.
- the amount of full-length viral genomic RNA contained in the total RNA sample is determined by the ribonuclease protection method (Kaye, JF, et al. Cis-acting sequences involved in human immunodeficiency virus type 1 RNA packaging.J. Virol. 69: 6588 6592, 1995 and Huang, Y., et al. The role of nucleocapsid and U5 stem / A rich loop sequences in tRNA (3Lys) genomic placement and initiation of reverse trans criptation in human immunodeficiency virus type 1.J. Virol. 72:
- the viral genomic RNA binds tRNA Lys3 in the cell and can be used for measuring RT activity. About 50 million viral genomic RNAs were added to 20 ul RT buffer (50 mM Tris—HC1, pH 7.5, 60 mM KC1, 3 mM MgCl, 10 mM
- tRNA Lys3 0.2 mM dCTP, 0.2 mM dTTP, 5 uCi 32 P-dGTP and 0.05 mM ddATP were reacted by extending one base with 32 p-dCTP in 5 microliters and extending it by 6 bases.
- the extended primer was recovered by precipitation with ethanol, and electrophoresed on a 6% polyacrylamide gel containing 7 Murea, and the extended primer was detected and analyzed by autoradiography. A test peptide for examining the effect on RT activity was added to this RT reaction system, and the presence or absence and the amount of the base sequence added by RT were examined. As a result, TLMA2993,
- Table 1 shows the amino acid sequences of various sites considered to be involved in the activity of the reverse transcriptase (RT) of HIV-1, and the amino acid sequences of peptides automatically designed by MIMETIC for these sites. Out of the ten peptides, three peptides suppressed the RT activity.
- the 50X inhibitory concentration for RT is 17 ⁇ ⁇ for ESLA2340. 15 ⁇ for TLMAZ993. 15 ⁇ for PSTW1594.
- HIV reverse transcriptase HIV reverse transcriptase
- a 96-well plate was coated with ESLA2340, and a diluted solution of HIV-RT was added thereto. The plate was incubated at 4 ° C overnight. After washing the plate, the plate was washed with biotin-labeled TLMA2993. And left at room temperature for 1 hour. After the plate was washed again, avidin labeled with peroxidase was added and reacted for 1 hour at room temperature.
- Pro-carboxypeptidase R is an active form of carboxypeptidase R, which is excised from the amino terminal to the 92nd arginine with a trypsin-like enzyme such as thrombin or plasmin.
- Peptidase R is an active form of carboxypeptidase R, which is excised from the amino terminal to the 92nd arginine with a trypsin-like enzyme such as thrombin or plasmin.
- the peptide complementary to the amino acid sequence consisting of 5 or 11 amino acids was automatically designed by the analysis program of the present invention (MIMETIC), and the peptides shown in Table 2 could be designed respectively.
- MIMETIC analysis program of the present invention
- peptides consisting of 20 and 15 amino acids peptides (Note 1) and (Note 2) in Table 2) were activated by T / TM of ProCPR. The effect on was analyzed.
- the synthesized peptides were synthesized using ABiMED's peptide synthesizer (AMS 422 Multiple Poptide Synthesizer).
- ProCPR was also activated by elastase-trypsin and the like, but these peptides suppressed only the activation of T / TM and did not inhibit the activation of elastase-trypsin.
- Table 2 summarizes the designed peptides complementary to ProCPR. Table 2 shows mimetic peptides automatically designed by MIMETIC for 11, 15, 20, 24, and 30 amino acid sequences after the 87th amino acid of ProCPR. Since it is considered that a sugar chain has been added to the amino acid at position 86, the peptide after amino acid 87 is removed.
- VGGRRTRARRVLLLVLTETH (hereinafter abbreviated as VGG) showed a binding reaction to ProCPR using a surface plasmon resonance analyzer (Biacore).
- a surface plasmon resonance analyzer (Biacore)
- human plasma containing ProCPR was diluted and added, and reacted at room temperature for 1 hour.
- 10G1 which was a monoclonal antibody against ProCPR labeled with biotin was added to the plate and reacted for 1 hour at room temperature.
- avidin labeled with peroxidase was added to react.
- a peroxidase coloring reagent was added to form a color according to the strength of the enzyme reaction of peroxidase as in the form, and the measurement was carried out with a plate reader. Color development was observed according to the concentration of plasma including ProPCR, confirming that VGG coated on the plate can be used as an artificial peptide antibody.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
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Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2005514931A JPWO2005040799A1 (ja) | 2003-10-29 | 2004-10-14 | 相補性ペプチド人工抗体 |
EP04792372A EP1681568A1 (en) | 2003-10-29 | 2004-10-14 | Artificial antibody comprising complementary peptide |
US11/414,713 US20060275826A1 (en) | 2003-10-29 | 2006-04-28 | Artificial antibody comprising complementary peptide |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2003-368875 | 2003-10-29 | ||
JP2003368875 | 2003-10-29 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/414,713 Continuation-In-Part US20060275826A1 (en) | 2003-10-29 | 2006-04-28 | Artificial antibody comprising complementary peptide |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2005040799A1 true WO2005040799A1 (ja) | 2005-05-06 |
Family
ID=34510360
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2004/015140 WO2005040799A1 (ja) | 2003-10-29 | 2004-10-14 | 相補性ペプチド人工抗体 |
Country Status (5)
Country | Link |
---|---|
US (1) | US20060275826A1 (ja) |
EP (1) | EP1681568A1 (ja) |
JP (1) | JPWO2005040799A1 (ja) |
CN (1) | CN1871515A (ja) |
WO (1) | WO2005040799A1 (ja) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008018355A1 (fr) * | 2006-08-08 | 2008-02-14 | Sharp Kabushiki Kaisha | biodétecteur, son procédé de fabrication et procédé de détection utilisant le biodétecteur |
WO2009050486A2 (en) * | 2007-10-19 | 2009-04-23 | The University Of Surrey | Peptide manipulators of regulatory t cell activity |
EP2174948A1 (en) * | 2008-09-26 | 2010-04-14 | Centre National De La Recherche Scientifique | Antiviral polypeptides |
US11918624B2 (en) | 2020-06-10 | 2024-03-05 | Kelsius Laboratories LLC | Therapeutic composition for use in the treatment of COVID-19 and other cytokine storm associated disorders |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH08506320A (ja) * | 1992-11-11 | 1996-07-09 | モスバッハ、クラウス | 人工抗体、その製造方法および使用 |
JPH09127116A (ja) * | 1995-11-01 | 1997-05-16 | Agency Of Ind Science & Technol | タンパク質分子識別機能を有する物質 |
JP2000074900A (ja) * | 1998-08-28 | 2000-03-14 | Toshibumi Takeuchi | 人工レセプターの評価方法 |
JP2003313200A (ja) * | 2002-02-21 | 2003-11-06 | Hidechika Okada | 活性ペプチドの評価システムと新規活性ペプチド |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
SE8704185L (sv) * | 1987-10-28 | 1989-04-29 | Ferring Ab | Nya peptider, artificiella antigener och immunoanalystestsatser |
US5639626A (en) * | 1994-11-15 | 1997-06-17 | Chiron Diagnostics Corporation | Reagents for specific binding assays |
-
2004
- 2004-10-14 EP EP04792372A patent/EP1681568A1/en not_active Ceased
- 2004-10-14 JP JP2005514931A patent/JPWO2005040799A1/ja not_active Withdrawn
- 2004-10-14 WO PCT/JP2004/015140 patent/WO2005040799A1/ja not_active Application Discontinuation
- 2004-10-14 CN CNA2004800306789A patent/CN1871515A/zh active Pending
-
2006
- 2006-04-28 US US11/414,713 patent/US20060275826A1/en not_active Abandoned
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH08506320A (ja) * | 1992-11-11 | 1996-07-09 | モスバッハ、クラウス | 人工抗体、その製造方法および使用 |
JPH09127116A (ja) * | 1995-11-01 | 1997-05-16 | Agency Of Ind Science & Technol | タンパク質分子識別機能を有する物質 |
JP2000074900A (ja) * | 1998-08-28 | 2000-03-14 | Toshibumi Takeuchi | 人工レセプターの評価方法 |
JP2003313200A (ja) * | 2002-02-21 | 2003-11-06 | Hidechika Okada | 活性ペプチドの評価システムと新規活性ペプチド |
Non-Patent Citations (1)
Title |
---|
CAMPBELL ET AL.: "A novel genetic algorithm for designing mimetic peptides that interfere with the function of a target molecule", MICROBIOLOGY AND IMMUNOLOGY, vol. 46, no. 3, 2002, pages 211 - 215, XP002969372 * |
Also Published As
Publication number | Publication date |
---|---|
JPWO2005040799A1 (ja) | 2007-04-19 |
CN1871515A (zh) | 2006-11-29 |
EP1681568A1 (en) | 2006-07-19 |
US20060275826A1 (en) | 2006-12-07 |
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