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POLYPEPTIDE EXHIBITING AFFINITY TO ANTIBODIES FORMING NON-NATURAL THREE-DIMENSIONAL STRUCTURE コモンズ 新技術説明会

外国特許コード F180009477
整理番号 2017002014
掲載日 2018年9月18日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2017JP031315
国際公開番号 WO 2018043629
国際出願日 平成29年8月31日(2017.8.31)
国際公開日 平成30年3月8日(2018.3.8)
優先権データ
  • 特願2016-170867 (2016.9.1) JP
発明の名称 (英語) POLYPEPTIDE EXHIBITING AFFINITY TO ANTIBODIES FORMING NON-NATURAL THREE-DIMENSIONAL STRUCTURE コモンズ 新技術説明会
発明の概要(英語) The present invention relates to a novel polypeptide and to the use thereof, the polypeptide exhibiting an affinity to proteins that partially contain CH1-CL domains forming a non-natural three-dimensional structure, and being capable of being suitably used to detect, immobilize, or remove such proteins. Specifically, disclosed herein are a polypeptide having an amino acid sequence represented by the following formulas 1–3: (1) P-Q-x-I-x-L-x-[IL]-[NT]-[YW] (SEQ ID NO.1); (2) Y-D-P-E-T-G-T-W-P-Q-x-I-x-L-x-[IL]-[NT]-[YW] (SEQ ID NO.4); (3) P-N-S-G-G-G-G-S-Y-D-P-E-T-G-T-W-P-Q-x-I-x-L-x-[IL]-[NT]-[YW] (SEQ ID NO.7) (wherein x represents any amino acid residue, and brackets represent any one of the amino acid residues within the brackets); and a method of using said polypeptide to detect, purify, and remove proteins partially containing CH1-CL domains forming a non-natural three-dimensional structure.
従来技術、競合技術の概要(英語) BACKGROUND ART
Monoclonal antibodies used in therapeutic applications, the medicament is a so-called antibody, sales year exceeds $300 billion is, the largest magnitude among the bio-pharmaceuticals, pharmaceutical industry or in the entire hard disk drives have the most rapid growth in the segment.Until now, a monoclonal antibody of the 23 type which uses a full, some of sales in the year 10 already and the blockbuster than billions of dollars. 1995 Years 2007 years from the start of the trial drug candidate monoclonal antibody may be increased to greater than 3 times and, continues to extend further the number of (non-patent document 1).
In addition, the progress of the clinical application of the antibody, the antibody molecules of the tissue than the high transition, the manufacturing cost is reduced than in the form of an antibody molecule, antibody Fab regions of antibodies such as a low molecular weight has been the development of next generation type (non-patent document 2) as, the year 2015, as a therapeutic antibody Fab region 3 in one of the FDA approved.IgG antibody molecule in such a low-molecular weight is in the form of complement the disadvantages of the antibody type is being developed in the future is expected.
Use of such antibodies in accordance with the increase of market growth, antibody molecules having affinity for the creation of, and improved research and development are actively performed.This is because, such a molecule, antibody studies, manufacturing, and from that is useful for analysis, particularly when the affinity purification of the antibody pharmaceutical manufacturing, and quality control in a large demand is expected in this specification.
Current, various approaches in relation to the polypeptide antibody affinity and the research and development, to name only a few of such.
Suzuki et al., 7 residues or 12 residues of the linear peptide library M13 presented using a filamentous bacteriophage, human IgG Fc region of the identified plurality of polypeptide with affinity, the affinity of the Fc region of an enzyme linked immunosorbent assay (ELISA) whether or not measured by (Patent Document 1).They are identified from a plurality of the sequences were made and the common sequence is extracted, in addition to binding to human IgG, horses, sheep, rabbits, guinea pigs, goats, cats, dogs, cows, pigs, IgG Fc region of the mouse from the affinity was confirmed by ELISA.
DeLano et al., by disulfide bonds in the equation i j k is circularized r.Xaai Cys Xaaj Cys Xaak (i+j+k=18) cyclic peptide library M13 presented using a filamentous bacteriophage, for binding to human IgG protein from Staphylococcus aureus A competing reaction of the cyclic peptide of 20 residues and acquiring a plurality.They further, the common sequence of these peptides extracted cyclic peptide residues of Fc-III 13 produce, in the competitive reaction of protein A and the ability to find competitive inhibition of Ki=100 nm (non-patent document 3) and, in the Fab fragment of IgG antigen binding site capable of fuzing Fc-III, Fab in in vivo experiments using a rabbit in the half-life can be improved (patent document 2) disclosed.Dias et al. is also, in this cyclic peptide D to L and a Fc-III of the remaining groups can be used to further circulari Pro introduced to prepare FcBP-2, affinity IgG (Fc-III bond dissociation constant KD=185 nm) to raise the KD=2 nm (Non-Patent Document 4) has succeeded.
Fassina et al., Lys residue having a branched structure represented by the synthesis and screening (Arg Thr Xaa) 4 Lys2 Lys Gly tetra polypeptide library, Protein A (PAM) to compete with the protein A mimetic peptide was made (non-patent document 5).Rabbit IgG KD=300 nm to one of the PAM TG19318 is a peptide that has an affinity for and, further it is immobilized by affinity chromatography, human, bovine, equine, porcine, mouse, rat, goat, sheep serum IgG purification can be included in the (non-patent document 6) shown.
Ehrlich et al., similar to the method of Suzuki et al., 7 residues or 12 residues of the linear peptide library M13 presented using a filamentous bacteriophage, humanized IgG obtained by pepsin digestion pFc ' fragment affinity peptides were isolated (non-patent document 7).
Krook et al., 10 residues in length of the linear peptide library M13 presented using a filamentous bacteriophage, human IgG Fc region according to the affinity of the peptide was made.They are human and the peptide by ELISA from porcine strong affinity IgG was confirmed to be shown (non-patent document 8).
Verdoliva et al., Lys residue Cys residue and branch structure is introduced into the annular (Cys Xaa3) can be represented by the 2 Lys Gly mouse monoclonal IgG screened against synthetic peptide, affinity peptides FcRM near the hinge region was prepared.They are further immobilized FcRM constructed affinity chromatography, purification of the IgG from mice and humans (Non-Patent Document 9) reported.
Watanabe et al., 10 residues comprising the amino acid sequence and protein micro shinyorin randomly (Patent Document 3) an artificial protein library of human IgG Fc region with a low affinity for the high affinity of the linear peptide can be reduced, without having a cyclic structure to improve its affinity to 40,600 times, KD=1.6 nm high affinity for a polypeptide of 54 (Patent Document 4) to prepare AF.p17 (non-patent document 10).
Μg et al., cyclic peptides Cys Xaa7-10 Cys T7 bacteriophage library presented used, human IgG Fc region according to the affinity of the peptide was made (non-patent document 11).To produce their peptides, the native Fc region not having a three-dimensional structure, caused by the acid treatment to form a non-naturally occurring three-dimensional structure in the Fc region recognizes, made up of the different IgG affinity peptides.Itoh et al., this peptide pharmaceutical human antibody, immunoglobulin preparation, IgG reagent included in the non-naturally occurring three-dimensional structure produced by the acid treatment of the content can be examined (Patent Document 5) disclosed.
Watanabe et al., an artificial protein including the 10 residue protein shinyorin small (Patent Document 3) a library of human IgG Fc region with affinity for an artificial protein residues 25 to prepare a AF.2A1 (non-patent document 12).AF.2A1 Is, the acid treatment, heat treatment, the reducing agent treatment occurs in the non-naturally occurring three-dimensional structure is formed in the high affinity Fc indicates a specific area, the native conformation of the Fc region of the non-naturally occurring three-dimensional structure (Patent Document 4) and identify exactly.
As described above, a plurality of IgG affinity peptides are developed, their molecular diversity is not sufficient.IgG is, the light chain and Heavy chain (Light chain) configured as a hetero tetramer, heavy chain is VH, CH1, CH2, CH3 of type 4, the light VL is, the type of CL 2, total 6 types of domains has a complex structure composed of a certain reason.An antibody detection, purification, immobilized, analysis, or the removal or the like in the embodiment, having characteristics suitable for the usage of each of the antibody affinity molecule is required.Specifically, any portion of a protein comprising the antibody and antibody to bind to, or to what extent the specificity of molecular recognition, as well as the difference in the amino acid sequence of the difference of the conformational change can be identified, how much the intensity of the affinity, dissociation of the binding to the change of the condition/solution can be controlled, if the stability and solubility, or can be mass-produced, and having suitable properties in the viewpoint of the antibody affinity molecule is required.
Is an enlarged view of the pharmaceutical market with an antibody, the antibody molecules can be subject to further separation and analysis techniques of the techniques have been strongly desired to be enhanced.
With respect to the analysis technique, a technique is strongly desired in the future as the sophistication of the particular (1) glycosylation post-translational modification of a molecule with a variety of non-uniformity analysis techniques, (2) the antibody molecule with the conformational changes of the non-uniformity analysis techniques, (3) aggregates, agglomerates to form a molecule with a non-uniformity analysis techniques, of the expected development of the region 3 (non-patent document 13).Antibody molecule by various physical or chemical stress, which is different from the native conformation of the normal called alternatively folded state (AFS) non-naturally occurring three-dimensional structure is formed have been reported (Non-Patent Document 14) (Non-Patent Document 15).Such non-naturally occurring three-dimensional structure is not only causes the loss of the activity of the antibody, the protein can also be a cause.As a result, not only reduction of efficacy, side effects and the cause of the cause of the immunogenic suggested risk (non-patent document 16), non-naturally occurring antibody-dimensional structure analysis technique, on the pharmaceutical quality control of the antibody in the evolution as a technique indispensable is demanded (non-patent document 17).
Protein molecular shape or a three-dimensionally to demonstrate the structure of the analytical techniques include, X-ray crystal structure analysis, nuclear magnetic resonance, electron microscope, an ultra-centrifugal analysis, isoelectric focusing, a dynamic light scattering, circular dichroism spectrum, liquid chromatography and the like (Non-Patent Document 17).Each of the analysis techniques is the quickness of the measurement throughput and accuracy of the analysis, each of the detection sensitivity is an advantage such as, in general, analysis accuracy and throughput have a trade-off relationship and, in both the spectroscopic and chromatographic methods is satisfied is not a method.For example, atomic-level accuracy is capable of giving a three-dimensional structure information of the X-ray crystal structure analysis or nuclear magnetic resonance analysis time on the order of several months is required.In addition, dynamic light scattering measurement is completed in a few minutes can be in liquid chromatography and the molecular structure of a small amount of change or fine mixing cannot be detected.Therefore, analysis accuracy and throughput of the analysis technique according to both a solution of the problem is demanded.
On the other hand, as a technique for separation and purification of the antibody, antibody specific affinity to bind molecules that have an affinity ligand used in the art.This application is as a ligand binding, protein A or protein from bacteria such as g a natural protein, or antibody produced artificially are used as the affinity molecule.These affinity binding of the antibody molecule, becomes possible to collect, often an antibody Fc region of the non-naturally occurring or native conformation of the three-dimensional structure and the affinity (JP-1-5) (Non-Patent Document 3-12), Fab antibody constant region of a portion of the native conformation CH1-CL domains non-naturally occurring three-dimensional structure can not be specifically identified.
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY
  • 発明者(英語)
  • Watanabe Hideki
国際特許分類(IPC)
指定国 National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
参考情報 (研究プロジェクト等) Biomedical Research Institue,Molecular and Cellular Breeding Research Group

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