Top > Search of International Patents > POLYPEPTIDE EXHIBITING AFFINITY TO ANTIBODIES FORMING NON-NATURAL THREE-DIMENSIONAL STRUCTURE

POLYPEPTIDE EXHIBITING AFFINITY TO ANTIBODIES FORMING NON-NATURAL THREE-DIMENSIONAL STRUCTURE meetings

Foreign code F180009477
File No. 2017002014
Posted date Sep 18, 2018
Country WIPO
International application number 2017JP031315
International publication number WO 2018043629
Date of international filing Aug 31, 2017
Date of international publication Mar 8, 2018
Priority data
  • P2016-170867 (Sep 1, 2016) JP
Title POLYPEPTIDE EXHIBITING AFFINITY TO ANTIBODIES FORMING NON-NATURAL THREE-DIMENSIONAL STRUCTURE meetings
Abstract 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.
Outline of related art and contending technology 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.
Scope of claims (In Japanese)請求の範囲
[請求項1]
 下記式1:
 P-Q-x-I-x-L-x-[IL]-[NT]-[YW] (配列番号1) (1)
(式中、xは任意のアミノ酸残基を表す。[ ]は[ ]内のアミノ酸残基のいずれか一つを表す)で示されるアミノ酸配列からなるポリペプチドであるか、あるいは該アミノ酸配列において1個若しくは数個のアミノ酸残基が付加されたアミノ酸配列からなるポリペプチドであって、免疫グロブリンGのCH1-CLドメインに親和性を示すポリペプチド。
[請求項2]
 配列番号2~3:
 PQEIRLILNW (配列番号2)
 PQWITLTITY (配列番号3)
のいずれかで表されるアミノ酸配列からなるポリペプチド、または 配列番号2~3のいずれかで表されるアミノ酸配列において、1個のアミノ酸残基が、付加、削除、置換又は挿入されたアミノ酸配列からなるポリペプチドであって、免疫グロブリンG のCH1-CLドメインに親和性を示すポリペプチドである、請求項1に記載のポリペプチド。
[請求項3]
 下記式2:
 Y-D-P-E-T-G-T-W-P-Q-x-I-x-L-x-[IL]-[NT]-[YW] (配列番号4) (2)
(式中、xは任意のアミノ酸残基を表す。[ ]は[ ]内のアミノ酸残基のいずれか一つを表す)で示されるアミノ酸配列からなるポリペプチドであるか、あるいは該アミノ酸配列において1個若しくは数個のアミノ酸残基が付加されたアミノ酸配列からなるポリペプチドであって、免疫グロブリンG のCH1-CLドメインに親和性を示すポリペプチド。
[請求項4]
 配列番号5~6及び36~41:
 YDPETGTWPQEIRLILNW (配列番号5)
 YDPETGTWPQWITLTITY (配列番号6)
 HNFTLPLWMYYDPETGTWPQEIRLILNW (配列番号36)
 RFPLMFGPSWYDPETGTWPQEIRLILNW (配列番号37)
 RFYVLLDSSWYDPETGTWPQEIRLILNW (配列番号38)
 VSKFYPLWTRYDPETGTWPQEIRLILNW (配列番号39)
 VFLVLMGPEFYDPETGTWPQEIRLILNW (配列番号40)
 FLLFCPRSLCYDPETGTWPQEIRLILNW (配列番号41)
のいずれかで表されるアミノ酸配列からなるポリペプチド、または
 配列番号5~6及び36-41のいずれかで表されるアミノ酸配列において、1個若しくは2個のアミノ酸残基が、付加、削除、置換又は挿入されたアミノ酸配列からなるポリペプチドであって、免疫グロブリンG のCH1-CLドメインに親和性を示すポリペプチドである、請求項3に記載のポリペプチド。
[請求項5]
 下記式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] (配列番号7) (3)(式中、xは任意のアミノ酸残基を表す。[ ]は[ ]内のアミノ酸残基のいずれか一つを表す)で示されるアミノ酸配列からなるポリペプチドであるか、あるいは該アミノ酸配列において1個若しくは数個のアミノ酸残基が付加されたアミノ酸配列からなるポリペプチドであって、免疫グロブリンGのCH1-CLドメインに親和性を示すポリペプチド。
[請求項6]
 配列番号8~12:
 PNSGGGGSYDPETGTWPQEIRLILNW (配列番号8)
 PNSGGGGSYDPETGTWPQWITLTITY (配列番号9)
 PNSGGGGSYDPETGTWAQEIRLILNW (配列番号10)
 PNSGGGGSYDPETGTWPAEIRLILNW (配列番号11)
 PNSGGGGSYDPETGTWPQEIRLIANW (配列番号12)
のいずれかで表されるアミノ酸配列からなるポリペプチド、または
 配列番号8~12のいずれかで表されるアミノ酸配列において、1~3個のアミノ酸残基が、付加、削除、置換又は挿入されたアミノ酸配列からなるポリペプチドであって、免疫グロブリンGのCH1-CLドメインに親和性を示すポリペプチドである、請求項5に記載のポリペプチド。
[請求項7]
 請求項1~6のいずれかに記載のポリペプチドのアミノ末端あるいはカルボキシル末端あるいは両末端に第2のポリペプチドを伸張した連結型ポリペプチドであって、免疫グロブリンGのCH1-CLドメインに親和性を示すポリペプチド。
[請求項8]
 請求項1~6のいずれかに記載のポリペプチドのアミノ末端あるいはカルボキシル末端あるいは両末端にタンパク質を結合した融合タンパク質であって、免疫グロブリンGのCH1-CLドメインに親和性を示すタンパク質。
[請求項9]
 配列番号13~15及び42~46のいずれかで表されるアミノ酸配列からなる、請求項8に記載のタンパク質。
[請求項10]
 請求項1~7のいずれかに記載のポリペプチドあるいは請求項8又は9に記載のタンパク質をコードする核酸。
[請求項11]
 配列番号16~18及び47~51のいずれかで表される塩基配列からなる、請求項10に記載の核酸。
[請求項12]
 請求項10又は11に記載の核酸を含有する組換えベクター。
[請求項13]
 請求項12に記載の組換えベクターが導入された形質転換体。
[請求項14]
 請求項10又は11に記載の核酸を含有する組換えファージ又は組換えウイルス。
[請求項15]
 請求項1~7に記載のポリペプチド、及び請求項8に記載のタンパク質のいずれかに有機化合物あるいは無機化合物あるいは有機化合物と無機化合物の両方が結合した修飾ポリペプチド又は修飾タンパク質であって、免疫グロブリンG のCH1-CLドメインに親和性を示すポリペプチド又はタンパク質。
[請求項16]
 請求項1~7のいずれかに記載のポリペプチド、請求項8又は9に記載のタンパク質、又は請求項15に記載のポリペプチドもしくはタンパク質が、水不溶性の固相支持体に固定化されていることを特徴とする、固定化ポリペプチド又は固定化タンパク質。
[請求項17]
 請求項1~7のいずれかに記載のポリペプチド、請求項8又は9に記載のタンパク質、請求項10又は11に記載の核酸、請求項12に記載の組換えベクター、請求項13に記載の形質転換体、請求項14に記載の組換えファージ又は組換えウイルス、請求項15に記載のポリペプチド又はタンパク質、請求項16に記載の固定化ポリペプチド又は固定化タンパク質からなる群より選択される少なくとも1つを含む、免疫グロブリンG のCH1-CLドメインを部分として含み当該CH1-CLドメインが非天然型立体構造を形成しているタンパク質を検出、精製、固定化又は除去するためのキット。
[請求項18]
 免疫グロブリンG のCH1-CLドメインを部分として含み当該CH1-CLドメインが非天然型立体構造を形成しているタンパク質を検出するための方法であって、
 (1) 該CH1-CLドメイン含有タンパク質の混入が疑われる被検試料を、請求項1~7のいずれかに記載のポリペプチド、請求項8~9のいずれかに記載のタンパク質、請求項13に記載の形質転換体、請求項14に記載の組換えファージ若しくは組換えウイルス、請求項15に記載のポリペプチドもしくはタンパク質、又は請求項16に記載の固定化ポリペプチド若しくは固定化タンパク質と接触させる工程、及び
 (2) 該CH1-CLドメイン含有タンパク質と、ポリペプチド、タンパク質、形質転換体、組換えファージ若しくは組換えウイルス、又は固定化ポリペプチド若しくは固定化タンパク質との間で結合が生じたか否かを判定する工程、
を含む方法。
[請求項19]
 免疫グロブリンG のCH1-CLドメインを部分として含み当該CH1-CLドメインが非天然型立体構造を形成しているタンパク質を精製するための方法であって、
 (1) 該CH1-CLドメイン含有タンパク質を含む試料を請求項1~7のいずれかに記載のポリペプチド、請求項8又は9に記載のタンパク質、請求項13に記載の形質転換体、請求項14に記載の組換えファージ若しくは組換えウイルス、請求項15に記載のポリペプチドもしくはタンパク質、又は請求項16に記載の固定化ポリペプチド若しくは固定化タンパク質と接触させて、該CH1-CLドメイン含有タンパク質を、ポリペプチド、タンパク質、形質転換体、組換えファージ若しくは組換えウイルス、又は固定化ポリペプチド若しくは固定化タンパク質と結合させる工程、及び
 (2) ポリペプチド、タンパク質、形質転換体、組換えファージ若しくは組換えウイルス、又は固定化ポリペプチド若しくは固定化タンパク質と結合した該CH1-CLドメイン含有タンパク質を試料から回収する工程、
を含む方法。
[請求項20]
 免疫グロブリンG のCH1-CLドメインを部分として含み当該CH1-CLドメインが非天然型立体構造を形成しているタンパク質を除去するための方法であって、
 (1) 該CH1-CLドメイン含有タンパク質を含む試料を請求項1~6のいずれかに記載のポリペプチド、請求項8又は9に記載のタンパク質、請求項13に記載の形質転換体、請求項14に記載の組換えファージ若しくは組換えウイルス、請求項15に記載のポリペプチドもしくはタンパク質、又は請求項16に記載の固定化ポリペプチド若しくは固定化タンパク質と接触させて、該CH1-CLドメイン含有タンパク質を、ポリペプチド、タンパク質、形質転換体、組換えファージ若しくは組換えウイルス、又は固定化ポリペプチド若しくは固定化タンパク質と結合させる工程、及び
 (2) ポリペプチド、タンパク質、形質転換体、組換えファージ若しくは組換えウイルス、又は固定化ポリペプチド若しくは固定化タンパク質と結合した該CH1-CLドメイン含有タンパク質を試料から除去する工程、
を含む方法。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY
  • Inventor
  • WATANABE HIDEKI
  • Honda Shinya
IPC(International Patent Classification)
Specified countries 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
Reference ( R and D project ) Biomedical Research Institue,Molecular and Cellular Breeding Research Group

PAGE TOP

close
close
close
close
close
close