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EP-3748001-B1 - PEPTIDE LIBRARY AND USE THEREOF

EP3748001B1EP 3748001 B1EP3748001 B1EP 3748001B1EP-3748001-B1

Inventors

  • NISHIMIYA, DAISUKE
  • HASHIMOTO, RYUJI

Dates

Publication Date
20260513
Application Date
20130807

Claims (14)

  1. A peptide library comprising a plurality of different peptides: wherein the peptides each comprise an amino acid sequence encoded by a nucleotide sequence consisting of the sequence represented by SEQ ID NO: 14 in the Sequence Listing; wherein the nucleotide sequence has been modified by replacing a nucleotide sequence consisting of the 43rd base thymine to the 93rd base thymine with a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing; wherein the peptide library has a diversity of at least 1 × 10 5 , and wherein: i) each of the 1st Xaa to the 5th Xaa, the 7th Xaa, the 9th Xaa, and the 10th Xaa counting from the amino terminus of SEQ ID NO: 1 in the Sequence Listing in each peptide of said plurality of peptides is independently any amino acid other than cysteine and proline; ii) each of the 6th Xaa and the 8th Xaa counting from the amino terminus of SEQ ID NO: 1 in the Sequence Listing in each peptide of said plurality of peptides is independently any amino acid other than cysteine; iii) the 11th Xaa counting from the amino terminus of SEQ ID NO: 1 in the Sequence Listing in each peptide of said plurality of peptides is independently an amino acid selected from the group consisting of tyrosine, serine, phenylalanine, leucine, and threonine; and iv) the 12th Xaa counting from the amino terminus of SEQ ID NO: 1 in the Sequence Listing in each peptide of said plurality of peptides is independently an amino acid selected from the group consisting of asparagine, aspartic acid, leucine, lysine, glutamine, alanine, and glutamic acid.
  2. The peptide library according to claim 1, wherein the library has a diversity of at least 1 × 10 8 , preferably at least 1 × 10 10 .
  3. The peptide library according to claim 1 or 2, wherein the library is a phage display library, a ribosome display library, or a nucleic acid display library.
  4. A method for identifying a peptide that binds to a target molecule, comprising the following steps (i) and (ii): (i) contacting peptides contained in the peptide library according to any one of claims 1 to 3 with the target molecule; and (ii) recovering a peptide that binds to the target molecule.
  5. The method according to claim 4, wherein the target molecule is derived from a human.
  6. The method according to claim 4 or 5, wherein the target molecule is not trypsin and/or acrosin.
  7. The method according to claim 4 or 5, wherein the target molecule is a serine protease other than trypsin and/or acrosin, wherein the peptide inhibits the proteolytic activity of the serine protease, and wherein the method comprises the further step (iii): (iii) determining that the peptide is positive for inhibition, when the peptide inhibits the proteolytic activity of the serine protease.
  8. A nucleic acid library comprising a plurality of different nucleic acids: wherein the nucleic acids each comprise a nucleotide sequence encoding an amino acid sequence encoded by a nucleotide sequence consisting of the sequence represented by SEQ ID NO: 14 in the Sequence Listing; wherein each nucleotide sequence has been modified by replacing a nucleotide sequence consisting of the 43rd base thymine to the 93rd base thymine with a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing, wherein the nucleic acid library has a diversity of at least 1 × 10 5 , and wherein: i) each of the 1st Xaa to the 5th Xaa, the 7th Xaa, the 9th Xaa, and the 10th Xaa counting from the amino terminus of SEQ ID NO: 1 in the Sequence Listing in each peptide of said plurality of peptides is independently any amino acid other than cysteine and proline; ii) each of the 6th Xaa and the 8th Xaa counting from the amino terminus of SEQ ID NO: 1 in the Sequence Listing in each peptide of said plurality of peptides is independently any amino acid other than cysteine; iii) the 11th Xaa counting from the amino terminus of SEQ ID NO: 1 in the Sequence Listing in each peptide of said plurality of peptides is independently an amino acid selected from the group consisting of tyrosine, serine, phenylalanine, leucine, and threonine; and iv) the 12th Xaa counting from the amino terminus of SEQ ID NO: 1 in the Sequence Listing in each peptide of said plurality of peptides is independently an amino acid selected from the group consisting of asparagine, aspartic acid, leucine, lysine, glutamine, alanine, and glutamic acid.
  9. A nucleic acid library according to claim 8, wherein the nucleic acids each comprise a nucleotide sequence consisting of the sequence represented by SEQ ID NO: 14 in the Sequence Listing, and wherein each nucleotide sequence has been modified by replacing a nucleotide sequence consisting of the 43rd base thymine to the 93rd base thymine with a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing.
  10. The nucleic acid library according to claim 8 or 9, wherein the nucleic acid library contains a plurality of nucleic acids which encode a single amino acid sequence.
  11. The nucleic acid library according to any one of claims 8 to 10, wherein the nucleic acids are comprised in a plurality of microorganisms.
  12. The nucleic acid library according to claim 11, wherein the plurality of microorganisms are selected from (a) cells, preferably Escherichia coli; (b) virus, phages or phage-like molecules, or particles thereof.
  13. The nucleic acid library according to claim 12, wherein the nucleic acids comprise codons which encode the amino acid sequence, and wherein the codons are selected according to codon usage of the cells or host cells.
  14. The nucleic acid library according to any one of claims 8 to 10, wherein the nucleic acids are comprised in a plurality of vectors, preferably wherein the vectors are phagemids, cosmids, or plasmids.

Description

Technical Field The present invention relates to a peptide, a derivative of the peptide, a nucleic acid corresponding to the peptide or the derivative of the peptide, a vector comprising the nucleic acid, a cell in which the vector and/or the nucleic acid is introduced, a method for producing the peptide and/or the derivative thereof comprising culturing the cell, a peptide library comprising the peptide and/or the derivative thereof, a method for identifying a peptide and/or a derivative thereof binding to a target molecule, a method for producing a peptide and/or a derivative thereof that binds to a target molecule, a method for determining whether or not a test peptide and/or derivative thereof binds to a target molecule, a nucleic acid library comprising the nucleic acid, a composition comprising the peptide or the derivative thereof, the nucleic acid, the vector, or the cell, a reagent comprising the peptide or the derivative thereof, the nucleic acid, the vector, or the cell, etc. Background Art SPINK2 (serine protease inhibitor Kazal-type 2) is a 7 kDa protein composed of Kazal-type domains having three disulfide bonds. In the human body, this protein is expressed in the testis or the seminal vesicle and functions as a trypsin/acrosin inhibitor (Non Patent Literature 1). In 1991, Winter et al., reported antibody screening using phage display, and this phage display had a great impact on the development of antibody drugs by providing a method for developing fully human antibodies (Non Patent Literature 2). With recent advances in protein engineering, non-antibody scaffolds have been increasingly actively developed using display techniques such as phage display or ribosome display. These non-antibody scaffolds, also called engineering binding (affinity) proteins, etc., refer to artificial proteins provided with antibody variable region (CDR)-like binding regions. The non-antibody scaffolds have binding activity against protein X to be targeted and permit protein-protein interaction. In addition, the non-antibody scaffolds have beneficial characteristics from the viewpoint of productivity, immunogenicity, tissue infiltration, etc. As typified by Kunitz domain library (Dyax Corp.) (Patent Literature 1), anticalin (Pieris AG), or the like, only limited types of non-antibody scaffolds have been developed, though their scientific or industrial rise has been desired. Thus, the development of a novel non-antibody scaffold against a disease-related molecule has been desired (Non Patent Literature 3). Patent Literature 2 relates to the human SPINK2 protein. Non Patent Literature 4 relates to the inhibition mechanism of the MRPINK protein, that was found to inhibit chymotrypsin specifically but not trypsin or thrombin. Citation List Patent Literature Patent Literature 1: U.S. Patent Application Publication No. US2008/0020394 A1 (or Japanese National Publication of International Patent Application Publication No. 2007-524348)Patent Literature 2: PCT Patent Application Publication No. WO2006/125195 A2. Non Patent Literature Non Patent Literature 1: Chen T, Lee TR, Liang WG, Chang WS, Lyu PC. (2009) Identification of trypsin-inhibitory site and structure determination of human SPINK2 serine proteinase inhibitor. Proteins. 77 (1):209-19.Non Patent Literature 2: James D. Marks, Hennie R. Hoogenboom, Timothy P. Bonnert, John McCafferty, Andrew D. Griffiths, Greg Winter (1991) By-passing immunization: Human antibodies from V-gene libraries displayed on phage. J Mol Biol. 222(3):581-97.Non Patent Literature 3: Skerra A. (2007) Alternative non-antibody scaffolds for molecular recognition. Curr Opin Biotechnol. 18:295-304.Non Patent Literature 4: Ye Li et al. "Inhibition Mechanism and the Effects of Structure on Activity of Male Reproduction-Related Peptidase Inhibitor Kazal-Type (MRPINK) of Macrobrachium rosenbergii", Marine Biotechnology, Springer-Verlag, NE, vol. 11, no. 2, 16 September 2008, pages 252-259. Summary of Invention Technical Problem The present inventors have conducted diligent studies on SPINK2 and a variant thereof, and consequently completed the present invention, for example, by preparing a library comprising a peptide that exhibits high binding activity against a molecule other than an endogenous target of SPINK2 and isolating, from the library, a peptide that exhibits high binding activity against a target molecule other than the endogenous target. Solution to Problem The present invention relates to: (1) a peptide library comprising a plurality of different peptides: wherein the peptides each comprise an amino acid sequence encoded by a nucleotide sequence derived from SEQ ID NO: 14 in the Sequence Listing;wherein the nucleotide sequence has been modified by replacing a nucleotide sequence consisting of the 43rd base thymine to the 93rd base thymine with a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing; and wherein the peptide library has a d