Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
  • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0018—Culture media for cell or tissue culture

Definitions

• the present invention relates to a cyclic peptide or a salt thereof that has excellent binding properties to FGFR (Fibroblast Growth Factor Receptor) protein and excellent stability.
• the present invention further relates to a cyclic peptide complex or a salt thereof in which two or more molecules of the above cyclic peptide or a salt thereof are linked by a linker.
• the present invention further relates to the use of the above cyclic peptide or a salt thereof and the cyclic peptide complex or a salt thereof.
• Cell therapy products which administer cells with healing functions into the body, are being developed as a treatment for diseases that could not be treated with conventional small molecule drugs and antibody drugs. Furthermore, in recent years, research and development has been conducted toward the practical application of cell products such as cultured meat (cellular agriculture) and cells for drug discovery support, which are expected to be measures against food shortages and greenhouse gases. In the research and production of these cell therapy products and cell products, it is important to efficiently grow cells while maintaining their quality, such as their undifferentiated ability. Among the components of the medium used for cell growth, a group of proteins called growth factors (also called growth factors or cytokines) are important components that greatly affect the quality and cell growth rate.
• growth factors also called growth factors or cytokines
• growth factors are generally very expensive and have low stability, which is the reason for the high manufacturing cost of cell therapy products and cell products.
• the production of cultured meat is costly and difficult to produce commercially, and a very important cost factor is the need for large amounts of specialized cell culture medium, especially suitable growth factors, which tend to be the most expensive components of cell culture medium.
• the majority of production costs (up to 96% or more) are determined by the cost of growth factors, making production economically unfeasible.
• FGFs fibroblast growth factors
• bFGF basic fibroblast growth factor
• iPSCs induced pluripotent stem cells
• bFGF's ability to maintain undifferentiated potential and promote proliferation is expressed when it binds to the extracellular domain of the FGFR (Fibroblast Growth Factor Receptor) protein. Therefore, with the aim of reducing the price and improving the stability of bFGF, substances that bind to the extracellular domain of the FGFR protein have long been researched.
• FGFR Fibroblast Growth Factor Receptor
• Patent Document 1 describes a disulfide-type cyclic peptide that binds to FGFR protein.
• Patent Document 2 describes a thioether-type cyclic peptide as a substance that combines binding ability and stability with proteins other than FGFR.
• Patent Document 1 The peptide described in Patent Document 1 has extremely low binding affinity to FGFR protein and is therefore not of practical use.
• disulfide bonds contained in disulfide-type cyclic peptides are known to be unstable and easily decomposed in cell culture environments, etc.
• the amino acid sequence that binds to FGFR in Patent Document 1 is combined with the stable thioether bond in Patent Document 2, the peptide has extremely low binding affinity to FGFR protein and is therefore not of practical use.
• the present invention aims to provide a cyclic peptide or a salt thereof, and a cyclic peptide complex or a salt thereof, which have excellent binding properties to FGFR proteins.
• a further aim of the present invention is to provide a medium composition, a medium additive, a material for purification, a material for labeling, a material for cell control, and a material for accumulation, which utilize the above-mentioned cyclic peptide or a salt thereof, and the cyclic peptide complex or a salt thereof.
• a peptide comprising an amino acid sequence represented by X1-Xm-X2-Xn-X3, comprising a cyclized portion cyclized by a covalent bond, the cyclized portion being represented by formula (2):
• a cyclic peptide or a salt thereof comprising the structure represented by: X1 represents a glutamine residue or a histidine residue; Xm represents a peptide residue containing any 2 to 4 amino acid residues;
• X2 represents an alanine residue, a leucine residue, an isoleucine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, a glutamine residue, a serine residue, a threonine residue, an asparagine residue, a glutamic acid residue, an arginine residue, or a histidine residue;
• Xn represents an amino acid residue or a peptide residue, containing any one to three amino acid residues;
• X3
• Z1 and Z2 each independently represent a linking group.
• m represents an integer of 1 to 10.
• the cyclic peptide according to ⁇ 1> which is represented by the following formula (1-1) or formula (1-2), or a salt thereof.
• W represents -Xm-X1-Y1- or Xk, where Xm is bonded to X2 and Y1 is bonded to CO;
• W is -Xm-X1-Y1-, V is -NH-Y3 and L0 is L, or V is R and L0 is L1;
• W represents Xk V represents --NH--X1-Y5, and L0 represents L.
• V1 represents NH-Y3 or R; When V1 represents NH-Y3, L0 represents L, and when V1 represents R, L0 represents L1.
• X1 represents a glutamine residue or a histidine residue;
• Xm represents a peptide residue containing any 2 to 4 amino acid residues;
• Xk represents a peptide residue containing any one to three amino acid residues;
• X2 represents an alanine residue, a leucine residue, an isoleucine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, a glutamine residue, a serine residue, a threonine residue, an asparagine residue, a glutamic acid residue, an arginine residue, or a histidine residue;
• Xn represents an amino acid residue or a peptide residue, containing any one to three amino acid residues;
• Xi represents a single bond or an amino acid or peptid
• X1 represents a glutamine residue or a histidine residue
• Xm represents a peptide residue containing any 2 to 4 amino acid residues
• Xk represents a peptide residue containing any one to three amino acid residues
• X2 represents an alanine residue, a leucine residue, an isoleucine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, a glutamine residue, a serine residue, a threonine residue, an asparagine residue, a glutamic acid residue, an arginine residue, or a histidine residue
• Xn represents an amino acid residue or a peptide residue, containing any one to three amino acid residues
• Xi represents a single bond or an amino acid or peptide residue
• X1 represents a glutamine residue or a histidine residue
• Xm represents a peptide residue containing any 2 to 4 amino acid residues
• Xk represents a peptide residue containing any one to three amino acid residues
• X2 represents an alanine residue, a leucine residue, an isoleucine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, a glutamine residue, a serine residue, a threonine residue, an asparagine residue, a glutamic acid residue, an arginine residue, or a histidine residue
• Xn represents an amino acid residue or a peptide residue, containing any one to three amino acid residues
• Xi represents a single amino acid residue or a peptide residue, containing any one to three amino acid residues
• Xi represents a single amino acid residue or a peptide residue, containing any one to three amino acid residues
• Xm represents a group represented by -Xo-X4-, Xo represents an amino acid residue or a peptide residue, including any one to three amino acid residues; X4 represents an alanine residue, an isoleucine residue, a phenylalanine residue, a tyrosine residue, an asparagine residue, a histidine residue, or a methionine residue; The cyclic peptide or a salt thereof according to ⁇ 1>.
• Xm represents a group represented by -Xo-X4-, Xo represents an amino acid residue or a peptide residue, including any one to three amino acid residues;
• X4 represents an isoleucine residue, a phenylalanine residue, a tyrosine residue, an asparagine residue, a histidine residue, or a methionine residue;
• Xk represents a group represented by -Xj-X4-;
• Xj represents a single bond or an amino acid residue or a peptide residue containing 1 to 2 arbitrary amino acid residues;
• the cyclic peptide or a salt thereof according to ⁇ 1>.
• Xm represents a group represented by -X5-X6-X4-, X5 represents an alanine residue, a proline residue, a leucine residue, an isoleucine residue, a valine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, an asparagine residue, a serine residue, a glutamine residue, a threonine residue, an aspartic acid residue, a glutamic acid residue, an arginine residue, a histidine residue, a methionine residue, a thiol group-containing amino acid residue forming a cyclized portion, or a halocarboxyl group-containing amino acid residue forming a cyclized portion; X6 represents any amino acid residue; X4 represents an alanine residue, an isoleucine residue, a phenylalanine residue, a tyrosine residue, an asparagine residue,
• Xm represents a group represented by -X5-X6-X4-
• X5 represents an alanine residue, a proline residue, a leucine residue, an isoleucine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, an asparagine residue, a glutamine residue, a threonine residue, an aspartic acid residue, a glutamic acid residue, an arginine residue, a histidine residue, or a methionine residue;
• X6 represents any amino acid residue;
• X4 represents an isoleucine residue, a phenylalanine residue, a tyrosine residue, an asparagine residue, a histidine residue, or a methionine residue;
• Xk represents a group represented by -X6-X4-; The cyclic peptide or a salt thereof according to ⁇ 1>.
• L is, or Z11 and Z12 each independently represent a linking group; m represents an integer of 1 to 10; * represents the position of the ⁇ carbon atom in the amino acid main chain; L1 is, Z12 represents a linking group; m represents an integer of 1 to 10; * represents the position of the ⁇ -carbon atom of the amino acid main chain;
• ⁇ 11> The cyclic peptide or a salt thereof according to ⁇ 1>, which contains a homocysteine residue at the cyclized portion.
• a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 37 to 450 and 452 to 454 in Tables 4 to 6, or any one of the amino acid sequences of SEQ ID NOs: 37 to 450 and 452 to 454 in Tables 4 to 6 in which 1 to 4 amino acids have been substituted, deleted or inserted, and having binding ability to FGFR protein.
• ⁇ 15> The cyclic peptide or a salt thereof according to ⁇ 1>, which is modified with another substance.
• ⁇ 16> A cyclic peptide complex or a salt thereof, in which two or more molecules of the cyclic peptide or a salt thereof according to any one of ⁇ 1> to ⁇ 15> are linked via a linker.
• ⁇ 17> A cyclic peptide complex or a salt thereof, in which two molecules of the cyclic peptide or a salt thereof according to any one of ⁇ 1> to ⁇ 15> are linked via a linker.
• ⁇ 18> The cyclic peptide complex or a salt thereof according to ⁇ 16>, which is modified with another substance.
• a medium composition or a medium additive comprising the cyclic peptide or a salt thereof according to any one of ⁇ 1> to ⁇ 15>.
• a purification material comprising the cyclic peptide or a salt thereof according to any one of ⁇ 1> to ⁇ 15>.
• a labeling material comprising the cyclic peptide or a salt thereof according to any one of ⁇ 1> to ⁇ 15>.
• ⁇ 22> A material for cell regulation, comprising the cyclic peptide or a salt thereof according to any one of ⁇ 1> to ⁇ 15>.
• ⁇ 23> A material for accumulation, comprising the cyclic peptide or a salt thereof according to any one of ⁇ 1> to ⁇ 15>.
• ⁇ 24> A medium composition or a medium additive comprising the cyclic peptide complex or a salt thereof according to ⁇ 16>.
• ⁇ 25> A purification material comprising the cyclic peptide complex or a salt thereof according to ⁇ 16>.
• ⁇ 26> A labeling material comprising the cyclic peptide complex or a salt thereof according to ⁇ 16>.
• ⁇ 27> A material for cell control, comprising the cyclic peptide complex or a salt thereof according to ⁇ 16>.
• ⁇ 28> A material for accumulation, comprising the cyclic peptide complex or a salt thereof according to ⁇ 16>.
• the cyclic peptide or salt thereof of the present invention has excellent binding properties to FGFR proteins.
• FIG. 1 shows the structures of the compounds used in the examples.
• FIG. 2 shows the cell proliferation promoting effect of the cyclic peptide complex (SEQ ID NO: 458).
• FIG. 3 shows the bovine cell proliferation effect of the cyclic peptide conjugate (SEQ ID NO: 458).
• a "step” or a word expressing a step includes not only a step that is independent of other steps, but also a step that cannot be clearly distinguished from other steps as long as the purpose of the step is achieved.
• a numerical range indicated using "to” indicates a range that includes the numerical values before and after "to" as the minimum and maximum values, respectively.
• the upper or lower limit value described in one numerical range may be replaced with the upper or lower limit value of another numerical range described in stages.
• the upper or lower limit value of the numerical range may be replaced with a value shown in the examples.
• amino acids are represented by the names, abbreviations, etc. adopted by the International Union of Pure and Applied Chemistry and International Union of Biochemistry and Molecular Biology IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN).
• Amino acid residues are represented by the abbreviations of the amino acids from which they are derived.
• the amino acid residues may be N-terminal amino acids (N-terminal residues) or C-terminal amino acids (C-terminal residues).
• amino acid sequence also called the "primary structure" of a peptide or protein is represented by a line of amino acid residues arranged from left to right from N-terminus to C-terminus.
• Amino acids include, but are not limited to, ⁇ -amino acids, ⁇ -amino acids, ⁇ -amino acids, and amino acids in which the amino or carboxyl group is substituted with a similarly reactive group (e.g., a primary amine with a secondary or tertiary amine, or a carboxyl group with an ester).
• ⁇ -amino acids refer to molecules containing an amino group and a carboxyl group bonded to a carbon designated as the ⁇ -carbon.
• ⁇ -amino acids refer to molecules containing both an amino group and a carboxyl group in the ⁇ configuration.
• ⁇ -amino acids refer to molecules containing both an amino group and a carboxyl group in the ⁇ configuration.
• ⁇ -amino acids are preferred.
• Amino acids may be either natural or unnatural amino acids, and may be either D- or L-isomers.
• an amino acid is represented by its name, and there exist enantiomeric isomers, i.e., L- and D-isomers, the L-isomer will generally be represented unless the distinction between L and D isomers is explicitly stated.
• "isoleucine” will represent “L-isoleucine”
• the enantiomer of "isoleucine” will be represented as "D-isoleucine”.
• amino acid residues may be either natural or unnatural amino acids, and may be either D- or L-isomers.
• Table 1 shows the names and abbreviations (one-letter and three-letter abbreviations) of amino acids for which one-letter and three-letter abbreviations are officially recognized.
• the amino acids are not limited to those listed in Table 1, and amino acids called unnatural amino acids can also be used. Examples of unnatural amino acids are listed in Table 2 below, but are not limited thereto.
• the amino acid may also be an N-alkyl amino acid in which the hydrogen atom on the N-terminal amino group is replaced with an alkyl group (e.g., a methyl group or a prokyl group).
• An example of an N-alkyl amino acid is N-methylleucine (hereinafter referred to as meL ).
• Amino acids containing aromatic residues include phenylalanine, tryptophan, tyrosine, histidine, and unnatural amino acids containing a benzene ring, an imidazole ring, or a pyridine ring in the side chain structure, and preferably phenylalanine, tryptophan, and tyrosine.
• methionine, cysteine, and lysine may be used, but it is preferable not to use methionine and cysteine from the viewpoint of oxidation resistance, and it is preferable not to include cysteine and lysine from the viewpoint of the cost of chemically synthesizing peptides.
• a peptide generally refers to a structure consisting of 3 to 100 amino acids, but may contain structures other than amino acids at the termini and/or inside. Any substance containing 3 to 100 amino acids is a peptide.
• a cyclic peptide refers to a peptide containing a closed ring structure consisting of three or more amino acid residues.
• the cyclic peptide of the present invention is a peptide comprising an amino acid sequence represented by X1-Xm-X2-Xn-X3, and comprising a cyclized portion cyclized by a covalent bond, the cyclized portion being represented by formula (2): It is a cyclic peptide comprising the structure shown below.
• X1 represents a glutamine residue or a histidine residue
• Xm represents a peptide residue containing any 2 to 4 amino acid residues
• X2 represents an alanine residue, a leucine residue, an isoleucine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, a glutamine residue, a serine residue, a threonine residue, an asparagine residue, a glutamic acid residue, an arginine residue, or a histidine residue
• Xn represents an amino acid residue or a peptide residue, containing any one to three amino acid residues
• X3 represents an amino acid residue containing an aromatic residue in the side chain.
• Z1 and Z2 each independently represent a linking group.
• m represents an integer of 1 to 10.
• the cyclic peptide of the present invention is represented by the following formula (1-1) or formula (1-2).
• W represents -Xm-X1-Y1- or Xk, where Xm is bonded to X2 and Y1 is bonded to CO;
• W is -Xm-X1-Y1-, V is -NH-Y3 and L0 is L, or V is R and L0 is L1;
• W represents Xk
• V represents --NH--X1-Y5, and L0 represents L.
• V1 represents NH-Y3 or R;
• V1 represents NH-Y3, L0 represents L, and when V1 represents R, L0 represents L1.
• X1 represents a glutamine residue or a histidine residue
• Xm represents a peptide residue containing any 2 to 4 amino acid residues
• Xk represents a peptide residue containing any one to three amino acid residues
• X2 represents an alanine residue, a leucine residue, an isoleucine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, a glutamine residue, a serine residue, a threonine residue, an asparagine residue, a glutamic acid residue, an arginine residue, or a histidine residue
• Xn represents an amino acid residue or a peptide residue, containing any one to three amino acid residues
• Xi represents a single bond or an amino acid or peptide residue, including 1 to 2 any amino acid residues
• X3 represents an amino acid residue containing an aromatic residue in the side chain
• Y1 represents a single bond or an amino
• the cyclic peptide of the present invention is represented by the following formula (1A), formula (1B), or formula (1C).
• X1 represents a glutamine residue or a histidine residue
• Xm represents a peptide residue containing any 2 to 4 amino acid residues
• Xk represents a peptide residue containing any one to three amino acid residues
• X2 represents an alanine residue, a leucine residue, an isoleucine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, a glutamine residue, a serine residue, a threonine residue, an asparagine residue, a glutamic acid residue, an arginine residue, or a histidine residue
• Xn represents an amino acid residue or a peptide residue, containing any one to three amino acid residues
• Xi represents a single bond or an amino acid or peptide residue, including 1 to 2 any amino acid residues
• the cyclic peptide of the present invention is represented by the following formula (1A1), formula (1A2), formula (1B), formula (1C1) or formula (1C2).
• X1 represents a glutamine residue or a histidine residue
• Xm represents a peptide residue containing any 2 to 4 amino acid residues
• Xk represents a peptide residue containing any one to three amino acid residues
• X2 represents an alanine residue, a leucine residue, an isoleucine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, a glutamine residue, a serine residue, a threonine residue, an asparagine residue, a glutamic acid residue, an arginine residue, or a histidine residue
• Xn represents an amino acid residue or a peptide residue, containing any one to three amino acid residues
• Xi represents a single bond or an amino acid or peptide residue
• Xm represents a group represented by -Xo-X4-;
• Xo represents an amino acid residue or a peptide residue, including any one to three amino acid residues;
• X4 represents an alanine residue, an isoleucine residue, a phenylalanine residue, a tyrosine residue, an asparagine residue, a histidine residue, or a methionine residue;
• Xk represents a group represented by -Xj-X4-; Xj represents a single bond, or an amino acid residue or peptide residue containing any one or two amino acid residues.
• Xm represents a group represented by -Xo-X4-;
• Xo represents an amino acid residue or a peptide residue, including any one to three amino acid residues;
• X4 represents an isoleucine residue, a phenylalanine residue, a tyrosine residue, an asparagine residue, a histidine residue, or a methionine residue;
• Xk represents a group represented by -Xj-X4-;
• Xj represents a single bond, or an amino acid residue or peptide residue containing any one or two amino acid residues.
• Xm represents a group represented by -X5-X6-X4-;
• X5 represents an alanine residue, a proline residue, a leucine residue, an isoleucine residue, a valine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, an asparagine residue, a serine residue, a glutamine residue, a threonine residue, an aspartic acid residue, a glutamic acid residue, an arginine residue, a histidine residue, a methionine residue, a thiol group-containing amino acid residue forming a cyclized portion, or a halocarboxyl group-containing amino acid residue forming a cyclized portion;
• X6 represents any amino acid residue;
• X4 represents an alanine residue, an isoleucine residue, a phenylalanine residue, a tyrosine residue, an asparag
• Xm represents a group represented by -X5-X6-X4-;
• X5 represents an alanine residue, a proline residue, a leucine residue, an isoleucine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, an asparagine residue, a glutamine residue, a threonine residue, an aspartic acid residue, a glutamic acid residue, an arginine residue, a histidine residue, or a methionine residue;
• X6 represents any amino acid residue;
• X4 represents an isoleucine residue, a phenylalanine residue, a tyrosine residue, an asparagine residue, a histidine residue, or a methionine residue;
• Xk represents a group represented by -X6-X4-.
• L is, or Z11 and Z12 each independently represent a linking group; m represents an integer of 1 to 10; * represents the position of the ⁇ carbon atom in the amino acid main chain; L1 is, Z12 represents a linking group; m represents an integer of 1 to 10; and * represents the position of the ⁇ carbon atom of the amino acid main chain.
• X1 preferably represents a glutamine residue.
• X5 is preferably a phenylalanine residue, a tyrosine residue, a tryptophan residue, a glutamine residue, a histidine residue, a thiol group-containing amino acid residue that forms a cyclized portion, or a halocarboxyl group-containing amino acid residue that forms a cyclized portion.
• X5 is more preferably a phenylalanine residue, a tyrosine residue, a tryptophan residue, a histidine residue, a thiol group-containing amino acid residue that forms a cyclized portion, or a halocarboxyl group-containing amino acid residue that forms a cyclized portion.
• X5 is particularly preferably a phenylalanine residue, a tyrosine residue, a tryptophan residue, or a histidine residue.
• X6 preferably represents an alanine residue, a glycine residue, a leucine residue, a isoleucine residue, a valine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, an asparagine residue, a glutamine residue, a serine residue, a threonine residue, an aspartic acid residue, a glutamic acid residue, an arginine residue, a histidine residue, a lysine residue, or a methionine residue.
• X6 more preferably represents an alanine residue, a glycine residue, a leucine residue, a isoleucine residue, a valine residue, a tyrosine residue, a tryptophan residue, an asparagine residue, a glutamine residue, a serine residue, a threonine residue, an aspartic acid residue, a glutamic acid residue, an arginine residue, a histidine residue, a lysine residue, or a methionine residue.
• X4 preferably represents an alanine residue, a phenylalanine residue, or a tyrosine residue. X4 more preferably represents a phenylalanine residue or a tyrosine residue.
• X2 preferably represents a leucine residue, an isoleucine residue, a phenylalanine residue, or a tyrosine residue. X2 more preferably represents a leucine residue, a phenylalanine residue, or a tyrosine residue. X2 particularly preferably represents a leucine residue or a phenylalanine residue.
• Xn preferably represents a peptide residue consisting of two amino acid residues represented by Xn1-Xn2.
• Xi preferably represents an amino acid residue represented by Xn1.
• Xn1 preferably represents an alanine residue, a glycine residue, a leucine residue, a isoleucine residue, a valine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, an asparagine residue, a glutamine residue, a serine residue, a threonine residue, an aspartic acid residue, a glutamic acid residue, an arginine residue, a histidine residue, a lysine residue, or a methionine residue.
• Xn1 more preferably represents an alanine residue, a leucine residue, a isoleucine residue, a valine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, an asparagine residue, a glutamine residue, a serine residue, a threonine residue, an aspartic acid residue, a glutamic acid residue, an arginine residue, a histidine residue, a lysine residue, or a methionine residue.
• Xn2 is preferably an alanine residue, a glycine residue, a proline residue, a leucine residue, a isoleucine residue, a valine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, an asparagine residue, a glutamine residue, a serine residue, a threonine residue, an aspartic acid residue, a glutamic acid residue, an arginine residue, a histidine residue, a lysine residue, a methionine residue, a thiol group-containing amino acid residue forming a cyclized portion, or a halocarboxyl group-containing amino acid residue forming a cyclized portion.
• Xn2 is more preferably an alanine residue, a glycine residue, a leucine residue, a isoleucine residue, a valine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, an asparagine residue, a glutamine residue, a serine residue, a threonine residue, an aspartic acid residue, a glutamic acid residue, an arginine residue, a histidine residue, a lysine residue, or a methionine residue.
• Xn2 particularly preferably represents an alanine residue, a leucine residue, a valine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, an asparagine residue, a glutamine residue, a serine residue, a threonine residue, an aspartic acid residue, a glutamic acid residue, an arginine residue, a histidine residue, a lysine residue, or a methionine residue.
• X3 preferably represents a phenylalanine residue, a tyrosine residue, or a tryptophan residue.
• Y1 preferably represents a group represented by Ym-Y11-Yn, Ym represents an amino acid residue or a peptide residue containing any one to three amino acid residues; Y11 represents an alanine residue, a glycine residue, a tyrosine residue, an asparagine residue, a serine residue, a threonine residue, a glutamic acid residue, or a histidine residue; Yn represents a peptide residue consisting of any five amino acid residues.
• Y11 preferably represents an asparagine residue.
• Ym preferably represents an amino acid residue represented by Ym3, a peptide residue consisting of two amino acid residues represented by Ym2-Ym3, or a peptide residue consisting of three amino acid residues represented by Ym1-Ym2-Ym3.
• Ym more preferably represents a peptide residue consisting of two amino acid residues represented by Ym2-Ym3, or a peptide residue consisting of three amino acid residues represented by Ym1-Ym2-Ym3.
• Ym1 preferably represents an alanine residue.
• Ym2 preferably represents an alanine residue or a proline residue.
• Ym3 preferably represents an alanine residue or a glutamine residue.
• Yn preferably represents a peptide residue consisting of five amino acid residues represented by Yn1-Yn2-Yn3-Yn4-Yn5.
• Yn1 preferably represents an alanine residue or a phenylalanine residue. Yn1 more preferably represents a phenylalanine residue.
• Yn2 preferably represents an alanine residue or an isoleucine residue. Yn2 more preferably represents an isoleucine residue.
• Yn3 preferably represents an alanine residue or an asparagine residue. Yn3 more preferably represents an asparagine residue.
• Yn4 preferably represents an alanine residue or a proline residue.
• Yn5 preferably represents an alanine residue or a valine residue.
• Y2 preferably represents a single bond.
• Y3 preferably represents any one amino acid residue.
• Y4 preferably represents any one amino acid residue.
• Z1 and Z2 each independently represent a linking group, preferably an alkylene group having 1 to 10 carbon atoms, more preferably an alkylene group having 1 to 5 carbon atoms, and particularly preferably an alkylene group having 2 to 4 carbon atoms.
• m is preferably an integer of 1 to 5, more preferably an integer of 1 to 3, and particularly preferably an integer of 1.
• Z11 and Z12 each independently represent a linking group, preferably an alkylene group having 1 to 10 carbon atoms, more preferably an alkylene group having 1 to 5 carbon atoms, and particularly preferably an alkylene group having 2 to 4 carbon atoms.
• Cyclic peptides are formed by binding multiple amino acid residues contained in a polypeptide chain to each other through covalent bonds other than main chain peptide bonds to form a closed ring structure.
• a cyclic peptide a partial structure consisting of the above-mentioned multiple amino acid residues bound to each other is called a cyclized portion.
• the cyclized portion of the cyclic peptide must not contain a disulfide bond.
• An example of a cyclized portion that does not contain a disulfide bond is a structure that contains a thioether bond.
• a thiol bond can be formed by reacting a side chain thiol group of an amino acid residue derived from an amino acid having a thiol group in its side chain with a side chain chloroacetyl group of an amino acid residue derived from an amino acid having a chloroacetyl group in its side chain.
• a linear polypeptide can be cyclized by forming a thiol bond between the side chain thiol group of an L-homocysteine residue and the side chain chloroacetyl group of an N- ⁇ -chloroacetyl-L-lysine residue.
• an amino acid having a halocarbonyl group such as a chloropropionyl group
• an amino acid having a haloacetyl group such as a chloroacetyl group
• an amino acid having a halocarbonyl group, such as a chloropropionyl group, in the side chain, which has more methylene units than a haloacetyl group is preferred because the fewer the number of methylene units, the higher the cyclization efficiency.
• a thioether it is preferable to use an amino acid having a halocarbonyl group in the side chain rather than an amino acid having a halocarbonyl group in the main chain from the viewpoint of FGFR binding.
• the thiol donor amino acid is preferably homocysteine rather than cysteine from the standpoints of both stability and suppression of the formation of racemized compounds during the peptide chemical synthesis process.
• the cyclization portion contains a homocysteine residue.
• the number of amino acid residues constituting the ring of the cyclic peptide is preferably 10 to 22, more preferably 14 to 22, and particularly preferably 15 to 20.
• the amino acids used in the cyclic peptide may have similar structures (generally considered to be similar) in terms of FGFR protein binding. Similar structures include unnatural amino acids. Unnatural amino acids may have an extended carbon chain (e.g., homoserine), a methyl group (e.g., N-methylalanine), or a single or multiple modifications (e.g., phosphorylated tyrosine). In the cyclic peptide, the amino acids at positions not specifically defined may be natural amino acids, non-natural amino acids, or amino acid analogs (peptoids).
• cyclic peptides include:
• the present invention relates to a method for producing a FGFR protein comprising the steps of: (a) administering to a subject an antibody or antibody composition comprising: a) administering to a subject a antibody or antibody composition comprising an antibody having an affinity for an FGFR protein; Mention may be made of cyclic peptides.
• cyclic peptides include: SEQ ID NOs: 37 to 51, 53 to 55, 57 to 61, 63 to 68, 70 to 89, 92 to 93, 103 to 104, 106 to 109, 114 to 115, 117, 119, 121 to 123, 125 to 133, 136 to 181, 187 to 189, 194 to 199, 201 to 203, 205 to 207, 211 to 218, 220 to 235, 237 to 250, 254 to 270, 272,
• the present invention relates to a method for preparing a nucleic acid sequence comprising the amino acid sequences of SEQ ID NOs: 275-276, 278-291, 293-324, 328-337, 339, 341-354, 356, 358-360, 385-386, 391-392, 394-398, 400, 402-403, 406-407, 409-411, 430, 433-435, 437-444, 446-450, and 452,
• cyclic peptide include: SEQ ID NO:37-42, SEQ ID NO:45, SEQ ID NO:47-51, SEQ ID NO:53-55, SEQ ID NO:60-61, SEQ ID NO:63, SEQ ID NO:65-68, SEQ ID NO:71-88, SEQ ID NO:92-93, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:114-115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125-133, SEQ ID NO:136-170, SEQ ID NO:172-180, SEQ ID NO:187-188, SEQ ID NO:194-199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205-207, SEQ ID NO:211-212, SEQ ID NO:214-218, SEQ ID NO:220-235, SEQ ID NO:237-2 50, SEQ ID NO:254-260
• the number of amino acids substituted, deleted or inserted may be 1 to 4, but is preferably 1, 2 or 3, and more preferably 1 or 2.
• the binding affinity to FGFR protein is indicated by the amount of cyclic peptide bound to FGFR protein when a certain concentration of the cyclic peptide is applied to a certain amount of FGFR protein.
• the measurement is performed using ELISA and SPR, but other methods (ITC, alphascreen, etc.) can also be used for the measurement.
• the FGFR protein may be a full-length protein, a partial domain, or a mutation may be introduced.
• the molecular stability of cyclic peptides is measured using reduction resistance and alkali resistance as indicators, but the same applies to resistance to other stimuli (e.g., X-ray resistance, gamma ray resistance, ultraviolet resistance, heat resistance, and chemical resistance). This is because molecular stability basically indicates that the molecule is more stable in terms of free energy.
• the cyclic peptide may be modified with other substances, such as those exemplified below.
• the cyclic peptide may have 1 to 20 amino acids and/or modified structures. From the viewpoint of the cost of chemically synthesizing peptides, it is preferable that no amino acids are added. From the viewpoint of stability, it is preferable that a modified structure is added. Examples of modified structures include acetylation and its related structures at the N-terminus, and amidation and its related structures at the C-terminus.
• the cyclic peptide may be modified, for example, by phosphorylation, methylation, adenylylation, ADP (adenosine diphosphate) ribosylation, or glycosylation depending on the application.
• the cyclic peptide may be provided with a functional structure.
• the functional structure include water-soluble polymers such as polyethylene glycol (PEG), IgG and other proteins, various affinity tags (e.g., histidine (His) tag, FLAG tag), solid-phase carriers (e.g., Sepharose beads, magnetic beads, various gels), fluorescent dyes, and radioisotopes.
• Amino acids and/or modified structures may be missing from the cyclic peptide. From the viewpoint of FGFR protein binding, the number of missing amino acids must be four or less, two or less is preferable, and no missing amino acids is particularly preferable.
• the cyclic peptide of the present invention may be a salt.
• the salt is preferably a salt with physiologically acceptable inorganic and organic acids and bases.
• suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecyl sulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, pamoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate, trifluoroacetate, and undecanoate.
• Salts derived from appropriate bases include alkali metal (e.g
• the method for producing the cyclic peptide is not particularly limited, and the cyclic peptide can be produced by chemical synthesis, genetic engineering synthesis, or enzymatic synthesis using a cell-free translation system. From the viewpoint of purity, chemical synthesis is more preferable.
• Chemical peptide synthesis may be by solid-phase synthesis or liquid-phase synthesis.
• solid-phase synthesis is preferred, and solid-phase synthesis using an automated peptide synthesizer is simple and preferred.
• liquid-phase synthesis is preferred from the standpoint of workability.
• a good guide for large-scale synthesis is 1 g or more, but this is not limited to this.
• Solid-phase synthesis of peptides involves, for example, an esterification reaction between the hydroxyl group of a resin having a hydroxyl group and the carboxyl group of a first amino acid (usually the C-terminal amino acid of the target peptide) whose ⁇ -amino group is protected with a protecting group.
• esterification catalyst known dehydration condensation agents such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), etc., can be used.
• the protecting group of the ⁇ -amino group of the first amino acid is removed, and a second amino acid in which all functional groups except the carboxyl group of the main chain are protected is added, the carboxyl group is activated, and the first and second amino acids are bonded. Furthermore, the ⁇ -amino group of the second amino acid is deprotected, and a third amino acid in which all functional groups except the carboxyl group of the main chain are protected is added, the carboxyl group is activated, and the second and third amino acids are bonded. This process is repeated until a peptide of the desired length is synthesized, after which all functional groups are deprotected.
• Resins for solid-phase synthesis include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin (Merck), HMPA-PEGA resin (Merck), etc. These resins may be washed with a solvent (dimethylformamide (DMF), 2-propanol, methylene chloride, etc.) before use.
• a solvent dimethylformamide (DMF), 2-propanol, methylene chloride, etc.
• Examples of protecting groups for ⁇ -amino groups include benzyloxycarbonyl (Cbz or Z) groups, tert-butoxycarbonyl (Boc) groups, fluorenylmethoxycarbonyl (Fmoc) groups, benzyl groups, allyl groups, and allyloxycarbonyl (Alloc) groups.
• the Cbz group can be deprotected by hydrofluoric acid, hydrogenation, etc.
• the Boc group can be deprotected by trifluoroacetic acid (TFA)
• the Fmoc group can be deprotected by treatment with piperidine.
• the ⁇ -carboxyl group can be protected using methyl esters, ethyl esters, benzyl esters, tert-butyl esters, cyclohexyl esters, etc.
• the hydroxyl groups of serine and threonine can be protected with benzyl groups or tert-butyl groups, and the hydroxyl groups of tyrosine are protected with 2-bromobenzyloxycarbonyl groups or tert-butyl groups.
• the amino group of the lysine side chain and the carboxyl group of glutamic acid or aspartic acid can be protected in the same manner as the ⁇ -amino group and ⁇ -carboxyl group.
• the activation of the carboxyl group can be carried out using a condensation agent.
• the condensation agent include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU).
• the peptide chain can be cleaved from the resin by treatment with an acid such as TFA or hydrogen fluoride (HF).
• Genetic engineering synthesis is a method of synthesizing peptides by introducing genes into cells, such as bacteria, yeast, nematode cells, insect cells, and animal cells (e.g., mammalian cells). For example, synthesis can be performed by introducing an unnatural amino acid using the four-base codon method. Alternatively, synthesis can be performed by synthesizing a linear peptide and cyclizing it by reacting a crosslinkable functional group in the side chain of the amino acid residue introduced into the cyclic portion.
• Cell-free translation systems also known as cell-free protein synthesis systems, are translation systems that do not use cells such as E. coli as they are, but utilize components present within the cells of E. coli and other bacteria.
• the reconstituted cell-free translation system can be constructed using purified ribosomal proteins, aminoacyl-tRNA synthetases (ARS), ribosomal RNA, amino acids, GTP, ATP, translation initiation factors (IFs), elongation factors (EFs), release factors (RFs), ribosome recycling factors, and other factors necessary for translation.
• the translation system may be continuously energized using dialysis.
• RNA polymerase may be added to effect transcription from DNA.
• cell-free translation systems include RTS-100 (registered trademark) from Roche Diagnostics, a system derived from E. coli, products from Zoigene and Cell Free Sciences, a system based on wheat germ extract, and PURESYSTEM (registered trademark) from PGI, Purefrex (registered trademark) from GeneFrontier, and PUREExpress (registered trademark) In Vitro Protein Synthesis Kit from New England BioLabs, among others, as reconstituted translation systems.
• RTS-100 registered trademark
• E. coli products from Zoigene and Cell Free Sciences
• PURESYSTEM registered trademark
• Purefrex registered trademark
• GeneFrontier GeneFrontier
• PUREExpress registered trademark
• a cyclic peptide complex or a salt thereof in which two or more molecules of the cyclic peptide or a salt thereof of the present invention are linked by a linker.
• the number of molecules of the cyclic peptide or a salt thereof linked by the linker is not particularly limited as long as it is two or more molecules, but is, for example, 2 to 10 molecules, preferably 2 to 5 molecules, more preferably 2 to 4 molecules, even more preferably 2 or 3 molecules, and particularly preferably 2 molecules.
• FGFR is a single-pass transmembrane receptor tyrosine kinase, and FGFR dimerizes upon binding to FGF, bringing the intracellular kinase domains of FGFR into close proximity, which causes mutual phosphorylation and promotes cell proliferation and/or differentiation state control. Therefore, it is believed that by polymerizing a cyclic peptide that has FGFR protein binding ability via a linker structure (the polymerized cyclic peptide is hereinafter referred to as a cyclic peptide complex) and allowing it to act on cells expressing FGFR, FGFR multimerization can occur via the cyclic peptide complex, and FGFR can be phosphorylated. In this case, considering that phosphorylation is mediated by the proximity of the intracellular kinase domains of FGFR, it is believed preferable to multimerize FGFR at an appropriate distance and orientation.
• FGFR phosphorylation intended to maintain the proliferation or differentiation state of cells expressing FGFR may require the inclusion of multiple cyclic peptides.
• the multiple cyclic peptides contained in the cyclic peptide complex may be conjugated by covalent bonds or may be bonded by non-covalent bonds (e.g., complex formation, affinity binding, nucleic acid hybridization). From the viewpoint of synthesis costs, the above bonds may be non-covalent bonds, but from the viewpoint of complex stability, covalent bonds (e.g., amide bonds, various click chemistry methods) are preferred.
• the multiple cyclic peptides may be complexed by direct bonding between the cyclic peptides, or may be complexed via other molecules.
• the multiple cyclic peptides contained in the cyclic peptide complex may have the same structure or different structures. From the viewpoint of promoting cell proliferation and/or controlling the cell differentiation state, they may have different structures, but from the viewpoint of synthesis costs, it is preferable that they have the same structure.
• the length of the linker is not particularly limited, but from the viewpoint of FGFR phosphorylation, it is preferably 10 to 200 ⁇ , more preferably 20 to 100 ⁇ .
• the bending of the molecular chain was taken into account and a unit distance of 1.33 ⁇ per single bond (4 ⁇ per amino acid residue) was calculated. By using this unit distance, the length of the linker can be calculated based on the molecular structure.
• the linker can be attached to either the N-terminus or the C-terminus of the cyclic peptide, but it is more preferable that it is attached to the C-terminus.
• the linker structure may be PEG (polyethylene glycol), an alkyl chain (polyethylene), a polypeptide, a polyester, a polyacrylamide, a polycarbonate, a polypropylene, a polystyrene, and/or a polyurethane.
• the linker structure may also form a salt.
• the salt is preferably the above-mentioned salt.
• PEG polyethylene glycol
• an alkyl chain (polyethylene) polypeptide
• a polypeptide a polyester
• a polyacrylamide a polycarbonate
• a polypropylene a polystyrene
• a polystyrene and/or a polyurethane.
• the linker structure may also form a salt.
• the salt is preferably the above-mentioned salt.
• PEG polyethylene glycol
• an alkyl chain polyethylene
• a polypeptide a polyester
• a polyacrylamide is more preferable.
• the cyclic peptide complex can be produced, for example, by linking two cyclic peptides with one amino group using a linker molecule with two NHS-activated carboxyl groups (hereinafter referred to as Bis-NHS linker).
• Bis-NHS linker Various companies sell Bis-NHS linkers with various linker lengths, examples of which include BS(PEG)5 (PEGylated bis(sulfosuccinimidyl) suberate) (Thermo, 21581) and Bis(NHS)PEG9 (Tokyo Chemical Industry, B4688).
• Linking cyclic peptides using a Bis-NHS linker can be carried out by mixing the cyclic peptide and linker in water at a pH of around 7.
• a cyclic peptide complex by using a branched amino acid with multiple amino groups in solid-phase and liquid-phase peptide synthesis.
• branched amino acids include lysine and 2,4-diaminobutanoic acid.
• the multiple amino groups are deprotected and the peptide chain is extended on both, allowing the cyclic peptide complex to be synthesized on the solid-phase resin or on the tag for liquid-phase synthesis.
• the peptide chain can be extended on all amino groups simultaneously, or one at a time.
• the cyclic peptide complex of the present invention may be modified with other substances.
• the other substances are the same as those described above for the modification of the cyclic peptide.
• the cyclic peptide or salt thereof of the present invention and the cyclic peptide complex or salt thereof of the present invention can be used for functional analysis, functional control, labeling, growth promotion, differentiation state control, or purification of biological materials (proteins, cells, tissues, etc.) including FGFR proteins.
• the growth promotion effect of the cyclic peptide complex or salt thereof of the present invention contributes to reducing the production costs of various cell therapy products and cultured meat, and is therefore of high industrial value.
• commercial production can be achieved by replacing the growth factors, which account for the majority of the production costs (up to 96% or more), with the cyclic peptide complex or salt thereof of the present invention.
• the subject on which the cyclic peptide or its salt, or the cyclic peptide complex or its salt is applied may be FGFR protein or other proteins, compounds and/or cells from the viewpoints of binding ability, function controllability, labeling efficiency, purification, proliferation promotion and/or differentiation state control.
• cells expressing FGFR protein and/or FGFR, cell secretions and/or cell lysates, and cells known to have proliferation and undifferentiation maintenance effects due to bFGF are preferred.
• Cells expressing FGFR2 or FGFR4 protein and/or FGFR2 or FGFR4, cell secretions and/or cell lysates are more preferred.
• Cells expressing FGFR2 protein and/or FGFR2 cell secretions and/or cell lysates are particularly preferred.
• the subject on which the cyclic peptide or its salt, or the cyclic peptide complex or its salt is to act may be a substance derived from a human, a mouse, a cow, or another animal. From the viewpoints of binding ability, function controllability, labeling efficiency, purification, proliferation promotion, and/or differentiation state control, a substance derived from an animal having a high homology between the amino acid sequence constituting FGFR and that of humans is preferred. Examples of animals having a high homology between the amino acids constituting FGFR and that of humans include cows, pigs, chickens, and tuna.
• the cyclic peptide or its salt, or the cyclic peptide complex or its salt may be used by dissolving it in an aqueous solution or organic solvent, or may be used by binding it to a solid-phase carrier (e.g., a plate, beads).
• a solid-phase carrier e.g., a plate, beads.
• a reactive group for binding e.g., an amino group, a thiol group, biotin
• the cyclic peptide or a salt thereof of the present invention and the cyclic peptide complex or a salt thereof of the present invention can be used as a medium composition, a medium additive, a purification material, a labeling material, a cell control material, or an accumulation material.
• the cyclic peptide or its salt, or the cyclic peptide complex or its salt is used as a medium composition, it can be prepared by mixing the components by a conventional method, and the form is not particularly limited as long as the desired effects such as cell proliferation promoting action and/or cell control can be obtained, and for example, it can be prepared in the form of a liquid medium, a semi-liquid medium, or a solid medium.
• the medium composition of the present invention may also be prepared in a powder form. By preparing it in a powder form, transportation and storage can be made extremely easy. Furthermore, by adding sterilized water and/or agar, etc. at the time of use, a liquid, semi-liquid, or solid medium can be easily prepared.
• the preparation concentration of the cyclic peptide or its salt, or the cyclic peptide complex or its salt is preferably 0.001 to 100 nmol/L, more preferably 0.01 to 10 nmol/L, and particularly preferably 0.1 to 10 nmol/L, from the viewpoint of FGFR phosphorylation.
• the medium composition of the present invention can be used in any culture method, such as adhesion culture, suspension culture, embedded culture, and tissue culture.
• cyclic peptide or its salt, or the cyclic peptide complex or its salt is used as a medium composition
• components generally used in cell culture such as amino acids, vitamins, buffers, inorganic salts, carbon sources, serum and serum substitutes, may be appropriately used.
• the cyclic peptide or a salt thereof, or the cyclic peptide complex or a salt thereof is used as a medium composition, it can be dissolved in a medium before use.
• the dissolved concentration is preferably 0.001 to 100 nmol/L, more preferably 0.01 to 10 nmol/L, and particularly preferably 0.1 to 10 nmol/L, from the viewpoint of FGFR phosphorylation.
• the medium may be one that is generally used for cell culture, but from the viewpoint of cell growth promotion and/or cell control, a medium whose components are prepared to be suitable for culturing the cells to be used is more preferable.
• the medium may contain serum, but when used in the production of cell products such as cell therapy products and cultured meat, a serum-free medium is preferable from the viewpoint of safety.
• the serum-free medium does not contain serum, but may contain purified components derived from serum, or may contain recombinant proteins of serum-derived components.
• the serum-free medium may contain a serum substitute, for example, a medium that appropriately contains serum albumin, transferrin, fatty acid, collagen precursor, trace elements, 2-mercaptoethanol or 3' thiolglycerol, or equivalents thereof.
• a serum substitute can be prepared, for example, by the method described in WO 98/30679.
• the cyclic peptide or a salt thereof or the cyclic peptide complex or a salt thereof of the present invention can also be used as a serum substitute.
• Commercially available products may be used as serum substitutes. Examples of such commercially available serum replacements include KnockoutTM Serum Replace (Life Technologies), Chemically Defined Lipid Concentrated (Life Technologies), and GlutamaxTM (Life Technologies).
• suitable ion exchange resins include, but are not limited to, B27 (Life Technologies), B27 (Life Technologies), and N2 (Life Technologies).
• the cyclic peptide or its salt, or the cyclic peptide complex or its salt When used as a medium additive, it can be used when adding the cyclic peptide or its salt, or the cyclic peptide complex or its salt to a medium.
• the medium additive of the present invention may further contain an appropriate amount of any additive, such as a stabilizer, an isotonicity agent, a pH adjuster, etc., in addition to the cyclic peptide or its salt, or the cyclic peptide complex or its salt of the present invention, so long as it does not impair the desired effects such as cell proliferation promotion and/or cell control.
• the medium additive of the present invention may be in any dosage form, such as a solution, solid, or powder, as long as the desired effect can be obtained. From the viewpoint of reducing transportation costs, a solid or powder form is preferable.
• the medium additive When the medium additive is in a solid or powder form, it may be dissolved to the desired concentration using an appropriate buffer solution, solvent, or the like, and then used, or it may be used in its solid or powder form as is.
• the medium additive is in a solution, it is preferable to sterilize the solution, such as by filtration sterilization using a membrane filter, or the like.
• the number of times and timing of adding the medium additive of the present invention to the medium are not particularly limited, and may be added before, during, or after cell culture. From the viewpoint of promoting proliferation and/or controlling the differentiation state, however, it is preferable to add the medium additive before and/or during cell culture.
• the medium composition or medium additive can be used for producing a cell therapy product or cultured meat.
• a method for producing a cell therapy product or cultured meat can include, for example, a step of culturing cells using a medium containing the cyclic peptide or a salt thereof, or the cyclic peptide complex or a salt thereof.
• Cultured meat refers to meat produced via cell culture. It is an innovative technology that uses cell engineering to produce animal meat from cultured tissues instead of the traditional method of raising livestock. The production process involves culturing animal cells that are engineered to grow into muscle tissue or fat. This technology results in a protein-rich food that is materially and nutritionally equivalent to conventional meat and fish-based foods. Raising livestock is very resource-intensive, with the obvious animal welfare concerns, as well as the ever-increasing use of water and land for the cultivation of feed crops. Also, due to greenhouse gas emissions, deforestation, pollution and antibiotic resistance, growing awareness of the ecological impact of meat production and growing interest in animal welfare, "cultured meat", “clean meat” or “cultured meat” have been proposed to replace the consumption of conventional meat with "lab-grown meat”.
• meat refers to an aggregate of muscle fibers, connective tissue, and fat. It is preferable for cultured meat to mimic the structure of meat, but it does not have to contain all of the so-called components of meat, as long as it contains cultured cells selected from the group consisting of fibroblasts, cells derived from adipose tissue, and cells derived from muscle tissue. It is more preferable for cultured meat to contain cultures of multiple types of cells. Cultured meat may contain extracellular matrix in addition to cultured cells selected from the group consisting of fibroblasts, adipose tissue-derived cells, and muscle tissue-derived cells.
• a method for producing cultured meat includes, for example, the following steps of culturing cultured cells selected from the group consisting of fibroblasts, adipose tissue-derived cells, and muscle tissue-derived cells, and recovering and collecting the cultured cells.
• a method for producing cultured meat may further include a differentiation induction step and a culture step after collection.
• the cells can be cultured by seeding the cells in a medium containing the cyclic peptide or its salt, or the cyclic peptide complex or its salt.
• the culture is performed under conditions well known in the art, for example, in a 37°C CO2 incubator.
• the culture may be plate culture or suspension culture.
• the grown cells can be collected as a culture by trypsin treatment or the like, and may be subcultured after collection.
• the cells can also be cultured by seeding the cells on a detachable structure.
• the structure to which the grown cells are attached can be collected as a culture.
• Such a structure can be constructed of an extracellular matrix such as collagen, elastin, fibronectin, laminin, entactin, etc., and the cultured meat may be formed by accumulating the structure to which the cells are attached.
• the accumulation step includes shaping the culture of one or more types of cells collected.
• the culture formed in the accumulation step may be a piece of meat such as a steak, a carcass, or minced meat.
• the accumulation step includes accumulating the cell culture together with at least one substance selected from the group consisting of other cells, blood, and tissues.
• the other cells may be cultured cells or cells collected from animals. More specifically, the cell culture may be shaped together with other cells cultured in a medium containing the cyclic peptide or a salt thereof, or the cyclic peptide complex or a salt thereof of the present invention.
• muscle tissue-derived cells cultured in a medium containing the cyclic peptide or a salt thereof, or the cyclic peptide complex or a salt thereof of the present invention may be accumulated with adipose tissue-derived cells and/or fibroblasts cultured in a medium containing the cyclic peptide or a salt thereof, or the cyclic peptide complex or a salt thereof of the present invention.
• Co-culture may also be performed after accumulation.
• the culture of one or more types of cells collected may be mixed and seeded on an extracellular matrix for co-culture. Examples of the extracellular matrix that can be used include collagen, elastin, fibronectin, laminin, and entactin.
• the medium used in this case can also be a medium containing the cyclic peptide or a salt thereof, or a cyclic peptide complex or a salt thereof, of the present invention.
• the culture of one or more types of cells that have been collected may be accumulated with blood and/or tissue.
• the tissue may be obtained from an animal or may be cultured.
• blood, fat tissue, or muscle tissue separated during meat processing may be accumulated with the culture to produce cultured meat.
• the differentiation induction step may be performed after cell culture, or may be performed before, during, or after the accumulation step.
• the differentiation induction step allows mononuclear muscle satellite cells and myoblasts to differentiate into multinuclear myotube cells, which can then be matured into muscle fibers.
• Differentiation induction may be performed by a method known in the art, and one example is a method of culturing cells under a high carbon dioxide concentration. For example, differentiation into myotube cells can be promoted by culturing cells under a 5 to 10% (v/v) CO2 atmosphere.
• the medium containing the cyclic peptide or its salt, or the cyclic peptide complex or its salt can be used to culture any animal cell.
• cells derived from livestock such as cows, pigs, goats, sheep, rabbits, chickens, ostriches, and ducks can be used.
• bovine cells any of the following types of cells may be used: Holstein, Jersey, Japanese Black, Japanese Brown, Shorthorn, Japanese Polled, and their crossbreeds.
• cells of meat breeds such as Japanese Black, Japanese Brown, Shorthorn, and Japanese Polled are preferred.
• a medium containing a cyclic peptide or a salt thereof, or a cyclic peptide complex or a salt thereof can also be used to culture tissues in which cells are assembled.
• the animal cells may be primary cells obtained from an animal, and passaged cells passaged from primary cells, or may be established cells or genetically modified cells in which genes have been deleted or inserted.
• Primary cells can be obtained by culturing animal tissue in a medium. They may also be cells differentiated from stem cells such as stem cells. From the viewpoint of producing cell therapy products, it is preferable to culture somatic stem cells or induced pluripotent stem cells, including mesenchymal stem cells. From the viewpoint of producing cultured meat, it is preferable to culture cells selected from the group consisting of fibroblasts, cells derived from adipose tissue, and cells derived from muscle tissue.
• Fibroblasts are cells that make up connective tissue and produce extracellular matrix such as collagen and elastin. Fibroblasts present in muscles are specifically called myofibroblasts. Myofibroblasts form the connective tissue that surrounds bundles of muscle fibers in skeletal muscles. Myofibroblasts express ⁇ -SMA, produce extracellular matrix, and can accumulate fat, contributing to texture and flavor.
• Adipose tissue-derived cells are cells that make up adipose tissue and are isolated from adipose tissue and cultured.
• the adipose tissue-derived cells are at least one cell selected from the group consisting of adipose stem cells, multilocular adipocytes, and unilocular adipocytes.
• Adipose stem cells are mesenchymal stem cells that have the ability to differentiate into various cells, and can differentiate into muscle cells, fat cells, and connective tissue cells.
• Multilocular adipocytes also known as brown adipocytes, contribute to fat burning in the body.
• Unilocular adipocytes also known as white adipocytes, can store lipid droplets within the cells.
• Adipose tissue-derived cells contain fat, which contributes to the taste of meat.
• Muscle tissue-derived cells are cells that make up muscle tissue and are separated from muscle tissue and cultured. Examples of muscle tissue-derived cells include myoblasts, satellite cells, and myotubes. However, myotubes do not have the ability to proliferate, so from the perspective of proliferation, myoblasts and/or satellite cells are preferred. Satellite cells are somatic stem cells contained in muscle, and can proliferate and differentiate into myoblasts. Myoblasts are cells from which muscle fibers are derived, and are mononuclear cells with the ability to proliferate. When myoblasts differentiate, they fuse with each other to form multinucleated myotubes, which then mature into muscle fibers.
• Muscle fibers are made up of myofibrils, which are made up of actin and myosin fibers, which are proteins that make up muscle. They are classified into red muscle fibers (type I, type IIA) and white muscle fibers (IIB) depending on the isoform of myosin, and contribute to the difference in the taste of meat.
• the cyclic peptide may be bound to a solid phase support or may be used without being bound.
• Methods for using the cyclic peptide without being bound include a method of imparting a structure for separation (e.g., a biotin tag, an antibody epitope, a polar residue for electrostatic separation) to the cyclic peptide, or a method of imparting a toxin to the cyclic peptide to kill non-target cells.
• a fluorescent dye e.g., a radioisotope, a nuclear polarized molecule, an affinity tag (e.g., biotin, FLAG tag), or an enzyme (e.g., luciferase) may be used as the labeling group.
• an affinity tag e.g., biotin, FLAG tag
• an enzyme e.g., luciferase
• the cyclic peptide When the cyclic peptide or its salt, or the cyclic peptide complex or its salt is used as a cell control material, the cyclic peptide may be used by binding to a solid support (e.g., flask, plate) or may not be bound. Examples of cell control include promotion of FGFR phosphorylation, inhibition of FGFR phosphorylation, promotion of cell proliferation, inhibition of cell proliferation, promotion of cell differentiation, and inhibition of cell differentiation. In addition, by linking the cyclic peptide to another cell stimulant, it is possible to express the effect of the cell stimulant selectively linked to cells having FGFR.
• a solid support e.g., flask, plate
• a cyclic peptide or a salt thereof, or a cyclic peptide complex or a salt thereof as a material for accumulating a specific molecule in the vicinity of FGFR, it is necessary to link the cyclic peptide to the molecule to be accumulated.
• the linkage may be one molecule to one molecule or one molecule to multiple molecules, and the linkage may be a reversible bond or an irreversible bond.
• Example 1 Obtaining FGFR-binding candidate peptides using mRNA display method
• Candidate sequences of cyclic peptides that bind to FGFR were obtained using the mRNA display method.
• the sequence of SEQ ID NO: 1 was used as a library for the mRNA display method.
• translation begins from the initiation codon (ATG) at positions 86 to 88 from the 5' end, the group of bases from the 107th position onwards (NNNTGT(NNN) 6-18 TAGNNN (NNN is a trimer oligonucleotide, where N each independently represents A, T, G or C)) is a random sequence, and the group of bases located on the 3' end side of the random sequence is a binding site for a puromycin linker and a stop codon.
• the group of bases before the 85th position from the 5' end is a sequence necessary for transcription and translation initiation, such as a T7 promoter sequence or a Shine-Dalgarno sequence.
• the trimer oligonucleotide represented by NNN is an equal mixture of trimer oligonucleotides corresponding to the 18 types of codons shown in Table 1, in which one type of codon is assigned to one type of amino acid. (NNN) For parts 6 to 18 , 13 types of libraries with different repeat numbers ranging from 6 to 18 were individually prepared, and mRNA display was performed for each.
• NNN is a trimer oligonucleotide, where each N independently represents A, T, G, or C.
• the library of sequence number 1 was prepared by overlap extension PCR. Specifically, three types of DNA, DNA of sequence number 2, DNA of sequence number 3, and DNA of any one of sequence numbers 4 to 16, were mixed at 3 ⁇ mol/L, 1 ⁇ mol/L, and 1 ⁇ mol/L, respectively, and in the presence of Platinum SuperFi II DNA Polymerase (Thermo, 12361010), three steps of 98°C/30 seconds, 98°C/10 seconds, 60°C/10 seconds, and 72°C/10 seconds were repeated for seven cycles, and finally, 72°C/5 minutes were treated, thereby linking the three DNAs and preparing the desired library.
• the prepared library was purified and diluted to 10 ng/ ⁇ L.
• the trimer oligonucleotide represented by NNN is an equal mixture of trimer oligonucleotides corresponding to the 18 types of codons shown in Table 1, with one type of codon assigned to one type of amino acid.
• SEQ ID NO: 2 GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGGTTAAAAAAACAAAC
• SEQ ID NO: 3 ATACTCAAGCTTATTATTTATTTATTACCCCCCGCCGCCCCCGTCCTGCTACCGCCAGAACCACC
• SEQ ID NO: 4 AAGAAGGAGATATACATATGGTTAAAAAAACAAAACANNNTGT (NNN) 6 TAGNNNGGCGGTTCTGGCGGTAGC SEQ ID NO: 5: AAGAAGGAGATATACATATGGTTAAAAAAACAAAACANNNTGT (NNN) 7 TAGNNNGGCGGTTCTGGCGGTAGC SEQ ID NO: 6: AAGAAGGAGATATACATATGGTTAAAAAAACAAAACANNNTGT (NNN) 8 TAGNNNGGCGGTTCTGGCGGTAGC SEQ ID NO: 7: AAGAAGGAGATATACATATGGTTAAAAAAACAAAACANN
• FGFR recombinant human FGFR2 alpha (IIIc) Fc Chimera Protein, CF (R&D systems, 712-FR-050) was used and immobilized (FGFR concentration at immobilization: 1 ⁇ g/ ⁇ L) on magnetic beads (NHS Mag Sepharose, Cytiva, 28951380) using the protocol specified by the manufacturer (Cytiva).
• IgG1 Fc, Human recombinant (Fujifilm Wako Pure Chemical, 098-07141) was immobilized on the magnetic beads in the same manner as above.
• the prepared library (SEQ ID NO: 1) was reacted in the presence of T7 RNA Polymerase (TaKaRa, 2540A) at 37° C. for 30 minutes to prepare a library transcript. This DNA fragment was purified and diluted to 10 ⁇ mol/L.
• the library transcripts final concentration 5 ⁇ mol/L
• the puromycin linker of SEQ ID NO:17 final concentration 10 ⁇ mol/L
• TBS buffer 1.25 mmol/L Tris, 25 mmol/L NaCl, pH 7.5
• UV 365 nm
• SEQ ID NO: 17 (PsoralenC6)- UACCCCCCGCCGCCCCCCCGUCCU- (Sp18)-(Sp18)-(Sp18)-CC-(Puro) (See FIG. 1 for the structures of PsoralenC6, Sp18, and Puro.
• the underlined nucleotides are those in which the 2'OH of RNA has been converted to 2'OMe, and the non-underlined nucleotides represent unmodified DNA.
• a tRNA with an anticodon of CUA that pairs with the UAG codon of mRNA was prepared by transcribing the DNA of SEQ ID NO: 18.
• This tRNA was aminoacylated with an N-chloroacetylated lysine pdCpA (phospho 2'deoxyribocytidylylriboadenosine) ester.
• This aminoacyl-tRNA is called aminoacyl-tRNA(1).
• SEQ ID NO: 18 GTTGTAAAAACGACGGCCAGTGCCAAGCTTGGGCTAATACGACTCACTATAGGGAGAGTAGTTCAATGGTAGAACGTCGGTCTCTAAAAACCGAGCGTTGAGGGTTCGATTCCTTTCTCTCCAC
• the complex of the library transcript and puromycin linker was translated in a translation solution containing PUREfrex2.0 (Gene Frontier, PF201-0.25-5) and aminoacyl-tRNA. 5.25 ⁇ L of the complex, 7.5 ⁇ L of PUREfrex2.0 Solution I, 0.75 ⁇ L of Solution II, 1.5 ⁇ L of Solution III, and dried aminoacyl-tRNA (1) (final concentration 0.5 ⁇ g/ ⁇ L) were mixed and reacted at 37°C for 60 minutes to produce an mRNA-cyclic peptide conjugate.
• reaction volume 100 ⁇ L 22.5 ⁇ L was mixed with 10 ⁇ L of magnetic bead-immobilized IgG1 Fc in TBS buffer (20 mmol/L Tris, 150 mmol/L NaCl, 0.1% BSA, 0.05% Tween 20, pH 7.4) (reaction volume 100 ⁇ L), and the reaction was carried out at room temperature for 45 minutes. The supernatant was then collected to remove the cDNA-mRNA-cyclic peptide conjugates bound to the magnetic beads.
• the supernatant was mixed with 10 ⁇ L of magnetic bead-immobilized FGFR (reaction volume 50 ⁇ L) and reacted at room temperature for 45 minutes. After that, the magnetic beads were washed three times with 100 ⁇ L of TBS buffer to extract the cDNA-mRNA-cyclic peptide conjugate that binds to FGFR.
• the extracted cDNA-mRNA-cyclic peptide conjugate was amplified by the following two-step PCR.
• the first PCR amplification product (final concentration 10 nmol/L) was mixed with DNA of sequence number 2 (final concentration 0.5 ⁇ mol/L) and DNA of sequence number 3 (final concentration 0.5 ⁇ mol/L), and in the presence of Platinum SuperFi II DNA Polymerase (Thermo, 12361010), 3 steps of 98°C/30 seconds, 98°C/10 seconds, 60°C/10 seconds, and 72°C/10 seconds were repeated for six cycles, and finally 72°C/5 minutes were performed to obtain DNA with the same sequence as the original library, except for the random sequence.
• the library was purified, diluted to 2 ng/ ⁇ L, and used in the next round.
• the base sequence of the 8th round first-stage PCR product was identified using MiSeq (Illumina) and Miseq Reagent kit v2 (300 cycles) (Illumina, MS-102-2022) according to the standard protocol of Illumina. Analysis of the identified cyclic peptides revealed many cyclic peptides containing the following amino acid sequences: X1-Xm-X2-Xn-X3 X1: glutamine residue or histidine residue Xm: peptide residue containing any 2 to 4 amino acid residues X2: alanine residue, leucine residue, isoleucine residue, phenylalanine residue, tyrosine residue, tryptophan residue, glutamine residue, serine residue, threonine residue, asparagine residue, glutamic acid residue, arginine residue, or histidine residue Xn: amino acid residue or peptide residue containing any 1 to 3 amino acid residues X3: phenylalanine residue, tryptophan residue
• Example 2 Evaluation of binding of peptides (enzymatically synthesized products)
• the FGFR binding properties of the peptides were evaluated using ELISA.
• the peptides used for the evaluation were enzymatically synthesized by the following method.
• a fusion peptide (SEQ ID NO: 25) was designed in which a translation-promoting sequence (SEQ ID NO: 22), a peptide, a Myc tag (SEQ ID NO: 23), and a HiBiT tag (SEQ ID NO: 24) were linked in this order.
• a template DNA sequence (SEQ ID NO: 26) was designed in which a sequence necessary for transcription and translation initiation, such as a T7 promoter sequence, a Shine-Dalgarno sequence, and an initiation codon (ATG) were added to the 5'-end side of the DNA sequence encoding the fusion peptide, and a group of stop codons were added to the 3'-end side.
• a sequence necessary for transcription and translation initiation such as a T7 promoter sequence, a Shine-Dalgarno sequence, and an initiation codon (ATG) were added to the 5'-end side of the DNA sequence encoding the fusion peptide, and a group of stop codons were added to the 3'-end side.
• the codon is assigned to a chloroacetylated lysine codon, and therefore, in the peptide encoded by the DNA sequence, a thiol group of cysteine and a chloroacetyl group of chloroacetylated lysine spontaneously form a thioether bond, forming a cyclic peptide.
• SEQ ID NO: 22 VKKTKT SEQ ID NO:23: EQKLISEEDL SEQ ID NO: 24: VSGWRLFKKIS SEQ ID NO: 25: MVKKTKT [HCSYERLQFHGHEAPFRVXV] GSGSGSEQKLISEEDLGGSVSGWRLFKKIS (X is chloroacetylated lysine, and the part in [ ] is an example of the amino acid sequence of the peptide to be evaluated, and the part in [ ] is different for each peptide.)
• SEQ ID NO: 26 GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAAATAATTTTGTTTAACTTTAAGAAGGGAGATATACCAATGGTTAAAAAACAAAAACA [CATTGCTCT TACGAACGTCTGCAGTTCCATGGTCATGAAGCTCCGTTCCGTGTTTAGGTT] GGTTCTGGCAGTGGTTCCGAACAGAAACTGATCAGCGAAGAAGATCTGGGTGGCTCT
• Template DNA was generated by two-step overlap extension PCR.
• four kinds of DNA, DNA of SEQ ID NO:27, DNA of SEQ ID NO:28, DNA of SEQ ID NO:29, and DNA of SEQ ID NO:30 were mixed at 0.3 ⁇ mol/L, 0.3 ⁇ mol/L, 0.05 ⁇ mol/L, and 0.05 ⁇ mol/L, respectively, and in the presence of PrimeSTAR Max (TaKaRa, R045B), three steps of 98°C/10 seconds, 39°C/5 seconds, 72°C/5 seconds, followed by 98°C/10 seconds, 58°C/5 seconds, and 72°C/5 seconds were repeated for 27 cycles to ligate DNA of SEQ ID NO:29 and DNA of SEQ ID NO:30.
• the ligated DNA was purified and diluted to 50 ng/ ⁇ L.
• SEQ ID NO: 27 GAAATTAATACGACTCACTATAGG
• SEQ ID NO: 28 GAACCACTGCCAGAACC
• SEQ ID NO: 29 GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATAACCAATGGTTAAAAAACAAAAC
• DNA of SEQ ID NO: 27 DNA of SEQ ID NO: 31, DNA of SEQ ID NO: 32, and the purified product of the first step were mixed at 0.3 ⁇ mol/L, 0.3 ⁇ mol/L, 0.0025 ⁇ mol/L, and 0.4 ng/ ⁇ L, respectively, and in the presence of PrimeSTAR Max (TaKaRa, R045B), three steps of 98°C/10 seconds, 58°C/5 seconds, and 72°C/5 seconds were repeated for 30 cycles to ligate DNA of SEQ ID NO: 32 and the purified product of the first step to obtain a template DNA (SEQ ID NO: 25).
• the template DNA was purified and diluted to 50 ng/ ⁇ L.
• SEQ ID NO: 31 GGATTAGTTATTCATTAGCTAATC
• SEQ ID NO: 32 GGTTCTGGCAGTGGTTCCGAACAGAAACTGATCAGCGAAGAAGATCTGGGTGGCTCTGTAAGTGGATGGCGATTATTCAAGAAGATTAGCTAATGAATAACTAATCC
• the concentration of the enzymatically synthesized peptide was measured by diluting the peptide with Can Get Signal Immunoreaction Enhancer Solution I (TOYOBO, NKB-101) using a Nano Glo HiBiT Lytic Detection System (Promega, N3040) and a chemically synthesized peptide (SEQ ID NO: 33) for a calibration curve of known concentration, according to the standard protocol of the Nano Glo HiBiT Lytic Detection System.
• SEQ ID NO: 33 EQKLISEEDLGGSVSGWRLFKKIS
• the FGFR binding ability of the enzymatically synthesized peptides was measured by ELISA to evaluate the amount of peptide binding to FGFR.
• 5 ng of Recombinant Human FGFR2 alpha (IIIc) Fc Chimera Protein, CF (R&D systems, 712-FR-050) was immobilized in each well of a 96-well plate, and the wells were blocked with Pierce Protein-Free (PBS) Blocking Buffer (Thermo, 37572). Then, the peptide diluted with Can Get Signal Immunoreaction Enhancer Solution I (TOYOBO, NKB-101) was added and reacted at room temperature for 3 hours.
• PBS Pierce Protein-Free
• the dose-response relationship was calculated from the difference between the amount of luminescence in the wells where FGFR was immobilized and the amount of luminescence in the wells where FGFR was not immobilized, and the peptide concentration at the time of reaction, and the FGFR binding was evaluated according to the following evaluation criteria. Evaluation criteria A, B or C are preferred.
• the luminescence signal value emitted by the cyclic peptide of SEQ ID NO: 47 at a reaction concentration of 10 nmol/L is normalized to 1, and the luminescence signal value emitted by the cyclic peptide at a reaction concentration of 10 nmol/L is A: 0.7 or more.
• the evaluation results of the cyclic peptides are shown in Table 4.
• the amino acid residues in parentheses indicate amino acid residues that form a cyclized structure by a thioether bond or a disulfide bond between side chains.
• K(acetyl) indicates a structure in which the side chain of a lysine residue is modified with a haloacetyl group
• the cyclic peptide has a cyclized structure in which the halogen atom of the haloacetyl group is replaced by a thiol group of another residue.
• SEQ ID NO:34 is a cyclic peptide reported in International Publication WO2000/003245 (Patent Document 1), and SEQ ID NO:36 is a cyclic peptide obtained by converting SEQ ID NO:34 into a thioether-type cyclized structure.
• SEQ ID NO: 35 is a linear peptide reported in Dev. Neurobiol. (2009) 69 (13): 837-854 (Agonists of fibroblast growth factor receptor induce neurite outgrowth and survival of cerebellar granule neurons).
• SEQ ID NO:37 onwards are the cyclic peptides covered by the present invention.
• Example 3 Evaluation of binding of cyclic peptides (chemically synthesized products)
• the FGFR binding properties of the cyclic peptides were evaluated using the surface plasmon resonance (SPR) method.
• the cyclic peptides used for the evaluation were custom synthesized by Toray Research Center, Inc.
• CM5 sensor chip (Cytiva, 29149603) was set on a surface plasmon resonance device Biacore-T200 (manufactured by Cytiva), and HBS-EP buffer (Cytiva, BR100826) was added as a running buffer at a flow rate of 10 ⁇ L/min to equilibrate the flow path.
• the cyclic peptide diluted to 10 nmol/L using HBS-EP was added to each flow channel of the FGFR solid-state sensor chip prepared above at 25°C for 10 minutes, and then HBS-EP was run as a running buffer for 30 minutes to measure the binding of the cyclic peptide to FGFR. After that, a regeneration solution in which sodium chloride powder was dissolved in Glysine 1.5 (Cytiva, BR100354) to a final concentration of 0.15 mol/L was run twice through each flow channel for 1 minute to perform a regeneration process to remove the bound cyclic peptide.
• the amount of binding of the cyclic peptide was evaluated from the difference between the measurement value measured by Biacore-T200 in the flow channel with FGFR immobilized when 10 nmol/L of the cyclic peptide was run and the measurement value measured by Biacore-T200 in the flow channel without FGFR immobilized.
• the amount of the cyclic peptide bound evaluated above was normalized to the amount of cyclic peptide molecules bound per FGFR molecule using the molecular weights of the cyclic peptide and FGFR, and the FGFR binding was evaluated according to the following evaluation criteria.
• Evaluation criteria A, B, or C are preferable.
• (Criteria for evaluation of FGFR binding) A: The binding amount is 0.7 cyclic peptide molecules/1 FGFR molecule or more.
• B The binding amount is 0.5 cyclic peptide molecules/FGFR molecule or more and less than 0.7 molecules/FGFR molecule.
• C The binding amount is 0.1 cyclic peptide molecule/FGFR molecule or more and less than 0.5 molecule/FGFR molecule.
• D The binding amount is less than 0.1 cyclic peptide molecule/1 FGFR molecule.
• Example 4 Evaluation of FGFR versatility of cyclic peptides (chemically synthesized products) The binding of the cyclic peptides to various types of FGFR was evaluated using the surface plasmon resonance (SPR) method. The cyclic peptides used for the evaluation were custom synthesized by Toray Research Center, Inc.
• CM5 sensor chip (Cytiva, 29149603) was set on a surface plasmon resonance device Biacore-T200 (manufactured by Cytiva), and HBS-EP buffer (Cytiva, BR100826) was added as a running buffer at a flow rate of 10 ⁇ L/min to equilibrate the flow path.
• GFR3 Protein, Human, Recombinant (ECD, His Tag) (Sino Biological, 16044-H08H) and FGFR4 Protein, Human, Recombinant (His Tag) (Sino Biological, 10538-H08H) were added for 10 minutes, and then HBS-EP was run as a running buffer for 30 minutes to measure the binding of various FGFRs to the cyclic peptides.
• the amount of FGFR binding evaluated above was normalized to the amount of FGFR molecule binding per molecule of cyclic peptide using the molecular weights of the cyclic peptide and FGFR, and the FGFR binding was evaluated according to the following evaluation criteria.
• Evaluation criteria A, B, or C are preferable.
• B The binding amount is 0.01 FGFR molecule/1 cyclic peptide molecule or more and less than 0.05 FGFR molecule/1 cyclic peptide molecule.
• the binding amount is 0.005 FGFR molecule/1 cyclic peptide molecule or more and less than 0.01 FGFR molecule/1 cyclic peptide molecule.
• D The binding amount is less than 0.005 FGFR molecules/1 molecule of cyclic peptide.
• Example 5 Evaluation of stability of cyclic peptides The molecular stability of the cyclic peptide was evaluated by analyzing an aqueous solution of the cyclic peptide treated with a reducing agent by LC (liquid chromatography analysis). The reducing agent treatment was carried out under the following conditions based on the glutathione concentration contained in the cells. 25 ⁇ L of a 0.2 mg/mL cyclic peptide aqueous solution was prepared, and 58.8 ⁇ L of a 12.6 mmol/L DTT aqueous solution was added to this aqueous solution, and the solution was incubated at room temperature for 1 hour to obtain a reducing agent-treated cyclic peptide aqueous solution.
• LC liquid chromatography analysis
• the DTT aqueous solution was prepared by diluting a 1 mol/L (+-)-dithiothreitol (DTT) solution (FUJIFILM Wako Pure Chemical Industries, 044-33871) with water.
• DTT dithiothreitol
• the total area of all peaks in the LC/MS of the cyclic peptide before the reducing agent treatment was taken as 100%, and the ratio of the total area of all peaks in the LC/MS of the reducing agent-treated cyclic peptide aqueous solution was calculated to calculate the cyclic peptide residual rate, and the molecular stability was evaluated according to the following evaluation criteria. Evaluation criteria A, B, or C are preferable.
• LC device Prominence series (pump, column oven, autosampler, detector) (Shimadzu Corporation) Detector: Photodiode array detector (SPD-M20A), measurement wavelength 280 nm
• LC device Prominence series (pump, column oven, autosampler, detector) (Shimadzu Corporation)
• Detector Photodiode array detector (SPD-M20A), measurement wavelength 280 nm
• Eluent A 0.1% trifluoroacetic acid as a solute, 100% water as a solvent
• Eluent B 0.1% trifluoroacetic acid as a solute, 100% acetonitrile as a solvent
• Flow rate 1.0 mL/min
• Injection volume 25 ⁇ L
• Gradient 20-50%: eluent B (0-15 min
• the evaluation results are shown in Table 7.
• the amino acid residues in parentheses indicate amino acid residues that form a cyclized structure with a thioether bond or disulfide bond between the side chains.
• K acetyl indicates a structure in which the side chain of a lysine residue is modified with a haloacetyl group, and the cyclic peptide has a cyclized structure in which the halogen atom of the haloacetyl group is replaced by the thiol group of another residue.
• a cyclic peptide containing a cysteine residue at the cyclization site (SEQ ID NO: 450) and a cyclic peptide containing a homocysteine residue at the cyclization site (SEQ ID NO: 442) was compared under harsher conditions, such as alkaline treatment as described below.
• the alkali treatment was carried out in the following manner.
• the residual rate of SEQ ID NO: 450 was 36%, and the residual rate of SEQ ID NO: 442 was 61%, indicating that the cyclic peptide containing a homocysteine residue in the cyclization portion is more stable than the cyclic peptide containing a cysteine residue in the cyclization portion.
• the LC conditions used for evaluating the molecular stability were the same as those used for measuring the reducing agent-treated cyclic peptide described above.
• Example 6 Evaluation of cell proliferation promoting effect of cyclic peptide complex
• the FGFR phosphorylation ability of the cyclic peptide complex was evaluated using the proliferation-promoting effect on BaF3 cells stably expressing FGFR.
• BaF3 cells stably expressing FGFR were generated by inserting the FGFR gene into vectors (human: SinoBiologicals, HG10824-UT, bovine: the sequence of Accession No. F1MNW2 was inserted into pcDNA3.1/Hygro(+) Mammalian Expression Vector (Thermo, V87020), porcine: the sequence of Accession No.
• A0A4X1SNN3 was inserted into pcDNA3.1/Hygro(+) Mammalian Expression Vector (Thermo, V87020), chicken: the sequence of Accession No. A0A8V0XB17 was inserted into pcDNA3.1/Hygro(+) Mammalian Expression Vector (Thermo, V87020)).
• the expression vector (Thermo, V87020) was introduced into BaF3 cells using Nucleofector (Lonza), and cells into which the FGFR gene had been introduced were established by drug selection using hygromycin.
• the cyclic peptide complex used for evaluation was custom synthesized by Toray Research Center, Inc.
• RPMI-1640 with L-Glutamine and Phenol Red (FUJIFILM Wako Pure Chemical Industries, 189-02025) and fetal bovine serum (Thermo, 10270-106) were mixed at a volume ratio of 9:1, and then heparin (STEM CELL, 07980) was added to a final concentration of 8 ⁇ g/mL to prepare a medium for evaluation (hereinafter referred to as RPMI (10% FBS) medium).
• BaF3 cells stably expressing FGFR were washed twice with RPMI (10% FBS) medium, and then suspended in RPMI (10% FBS) medium in which the cyclic peptide complex was dissolved to a final concentration of 0.001 to 10 nmol/L, and seeded on a 384-well plate. At this time, a condition in which the cyclic peptide complex was not added was prepared as a negative control.
• A The number of viable cells is 1,000 cells/well or more.
• B The number of viable cells is 400 cells/well or more and less than 1000 cells/well.
• C The number of viable cells is 200 cells/well or more and less than 400 cells/well.
• D The number of viable cells is less than 200 cells/well.
• the amino acid residues in parentheses indicate amino acid residues that form a cyclized structure with a thioether bond or disulfide bond between the side chains.
• K acetyl indicates a structure in which the side chain of a lysine residue is modified with a haloacetyl group, and the cyclic peptide has a cyclized structure in which the halogen atom of the haloacetyl group is replaced by the thiol group of another residue.
• Example 7 Evaluation of the activity of cyclic peptide complexes to promote bovine cell proliferation
• the cyclic peptide complex was evaluated for its ability to promote bovine cell (myoblast) proliferation.
• Bovine myoblasts were isolated from bovine muscle tissue by the following method. First, bovine muscle tissue from Japanese black cattle was chopped in HBSS (-) (phenol red-free) (Fujifilm Wako Pure Chemical Industries, 085-09355), and then pronase from Streptomyces Griseus (Sigma-Aldrich, 10165921001) was added to a final concentration of 1 mg/mL, and the mixture was left to stand at 37°C for 1 hour. After standing, HBSS containing 10% FBS was added, and cells were obtained by centrifugation.
• HBSS phenol red-free
• the obtained cells were suspended in D-MEM (high glucose) (containing L-glutamine and phenol red) (Fujifilm Wako Pure Chemical Industries, 044-29765) containing a final concentration of 10% bovine serum and 30 ng/mL bFGF.
• the suspended cells were cultured in a culture flask coated with iMatrix-511 solution (Fujifilm Wako Pure Chemical Industries, 381-07363) at 0.55 ⁇ g/cm 2.
• Bovine myoblasts that had been passaged at least twice after the start of culture were used for evaluation.
• the proliferation-promoting effect on bovine myoblasts was evaluated by the following method. Subcultured bovine myoblasts were detached using Trypsin-EDTA (0.05%) and phenol red (Thermo, 25300-054), suspended in 10% FBS-containing D-MEM (high glucose) (containing L-glutamine and phenol red) diluted with bFGF to a final concentration of 30 ng/mL or with the cyclic peptide complex to a final concentration of 0.03 to 10 nmol/L, and seeded on a 96-well plate coated with iMatrix-511 to a concentration of 0.55 ⁇ g/cm 2 .
• a condition in which neither bFGF nor the cyclic peptide complex was added was prepared as a negative control.
• the medium supernatant was removed, and 10% FBS-containing D-MEM (high glucose) (containing L-glutamine and phenol red) containing Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water (Thermo, H3570), and -Cellstain (registered trademark)- Calcein-AM solution (1 mg/mL DMSO solution) (DOJINDO, C396) at final concentrations of 1/10,000 and 1/4,000 by volume, respectively, was added, and the cells were left to stand at 37°C (5% CO2 ) for 30 minutes to stain all cells and live cells, and images were taken using a confocal microscope (Yokogawa Electric, CQ1).
• the number of viable cells was calculated from the image data, and the number of viable cells was normalized by setting the number of viable cells in the well to which 30 ng/mL bFGF was added as 100 and the number of viable cells in the negative control well as 0.
• the evaluation results are shown in FIG.
• the cyclic peptide complex (SEQ ID NO: 458) exhibited a bovine myoblast proliferation effect of approximately 40% of that of 30 ng/mL bFGF, indicating that the cyclic peptide complex of the present invention can also be used to culture bovine myoblasts.

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