WO2023042875A1 - ゲル形成キット及びその利用 - Google Patents

ゲル形成キット及びその利用 Download PDF

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Publication number
WO2023042875A1
WO2023042875A1 PCT/JP2022/034520 JP2022034520W WO2023042875A1 WO 2023042875 A1 WO2023042875 A1 WO 2023042875A1 JP 2022034520 W JP2022034520 W JP 2022034520W WO 2023042875 A1 WO2023042875 A1 WO 2023042875A1
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Prior art keywords
gel
cancer
group
cancer cells
peptide
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English (en)
French (fr)
Japanese (ja)
Inventor
香織 武田
武彦 横堀
遼 村主
高行 浅尾
憲 調
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Fujifilm Corp
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Fujifilm Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to a gel-forming kit, a gel-forming solution, and a gel composition for use in preparing non-human model animals with cancer.
  • the present invention further relates to a method for producing a gel composition for use in producing non-human model animals with cancer.
  • the present invention further relates to a non-human model animal having cancer and a method for producing a non-human model animal having cancer.
  • the present invention further relates to a method for evaluating a test substance using the non-human model animal having cancer.
  • Non-Patent Document 1 A common method for verifying the effects of cancer treatment is to form a tumor in a non-human animal by the above-mentioned transplantation, and to evaluate suppression of tumor volume when treatment such as drug administration is performed.
  • Non-Patent Document 2 the survival rate of tumors and cells in non-human animals is generally low (Non-Patent Document 2), and the proliferation rate after engraftment often varies between tests, so the time required for tumor growth is not constant. (Non-Patent Document 3). These factors make the generation of tumor-bearing non-model animals a cost and time hurdle in conducting preclinical studies.
  • Patent Document 1 describes a non-human model animal implanted with a cell structure consisting of cells and polymer blocks.
  • a block (mass) made of a biocompatible polymer is used.
  • the problem to be solved by the present invention is that in the production of non-human model animals having cancer, a gel-forming kit for use in producing non-human animals that can form tumors in a short period of time and with high efficiency, To provide a gel-forming solution and a gel composition. Furthermore, the problem to be solved by the present invention is to provide a method for producing a gel composition for use in producing a non-human model animal having cancer. Furthermore, the problem to be solved by the present invention is to provide a non-human model animal having cancer and a method for producing a non-human model animal having cancer. Furthermore, the problem to be solved by the present invention is to provide a method for evaluating a test substance using the non-human model animal having cancer.
  • a non-human model animal having cancer comprising a peptide consisting of only one chain and a cross-linking agent having at least two chains containing a functional group capable of covalently bonding with an amino group and a hydrophilic linking group.
  • Gel forming kit for use in making (2) The gel-forming kit according to (1), wherein the chain containing a functional group capable of covalently bonding with the amino group and a hydrophilic linking group is represented by Formula 1 below.
  • A represents an arbitrary amino acid or amino acid sequence
  • B represents an arbitrary amino acid or amino acid sequence
  • n X's each independently represent any amino acid
  • n Y's each independently represent an amino acid.
  • n represents an integer of 3 to 100
  • m represents an integer of 2 to 10.
  • the n Gly-XY may be the same or different.
  • the gel-forming kit according to (8) wherein the cancer cells are selected from the group consisting of a patient-derived tumor tissue, a suspension of established cancer cells, or a suspension of patient-derived cancer cells. .
  • the cancer cells are selected from the group consisting of liver cancer cells, biliary tract cancer cells, pancreatic cancer cells, colon cancer cells, osteosarcoma cells, chondrosarcoma cells, or angiosarcoma cells, (8 ) or the gel-forming kit according to (9).
  • (11) A non-human model animal having cancer, comprising a peptide consisting of only a single chain and a cross-linking agent having at least two chains containing a functional group capable of covalently bonding to an amino group and a hydrophilic linking group.
  • a method for producing a non-human model animal having cancer comprising implanting a product or gel-forming solution into a non-human animal.
  • a method for producing a non-human model animal having cancer comprising transplanting a gel-forming solution containing and into the non-human animal.
  • (20) A method for producing a non-human model animal having cancer according to (18) or (19), wherein the gel composition or gel-forming solution is subcutaneously, intraperitoneally, or implanted into an organ or tissue of a non-human animal.
  • (21) A method for evaluating a test substance, comprising administering the test substance to the non-human model animal having cancer according to (17).
  • the tumor formation rate in a short period of time was improved by using a peptide consisting of only a single chain.
  • the gel composition is rapidly degraded by enzymes secreted by cancer cells. Therefore, it is speculated that tumor formation is promoted by securing an appropriate space for cancer cells to proliferate.
  • the gel-forming kit, gel-forming solution, and gel composition of the present invention provide a scaffold that allows tumors to engraft with high efficiency and allows tumor growth to be obtained in a short period of time in the production of non-human model animals having cancer. It can be used as material.
  • the non-human model animal having cancer and the method for evaluating a test substance using the non-human model animal having cancer of the present invention are useful in verifying the effects of cancer treatment.
  • a gel-forming kit for use in preparing a non-human model animal having cancer according to the present invention comprises: (a) a peptide consisting of only one chain (hereinafter also referred to as a single chain peptide); (b) a cross-linking agent having at least two chains containing a functional group capable of covalently bonding with an amino group and a hydrophilic linking group; including.
  • Matrigel® a commonly used scaffolding material, is mainly composed of type IV collagen (which contains a three-stranded structure unique to collagen), and contains MMP (matrix metalloprotease), a process necessary for tumor growth. Scaffold degradation by scaffolds requires two steps: degradation of collagen (collagenase) and degradation of gelatin (gelatinase) (Laronha, H., & Caldeira, J. (2020). Structure and Function of Human Matrix Metalloproteinases. Cells, 9, 1076.p3).
  • the gel composition produced by the gel-forming kit of the present invention consists of a single-chain peptide, it is considered to be rapidly decomposed by MMP in one step, like gelatin.
  • the gel-forming kit By using the gel-forming kit according to the present invention, it is possible to produce a non-human model animal that has a high tumor engraftment rate and allows tumors to grow in a short period of time.
  • the gel composition produced by the gel-forming kit of the present invention forms a homogeneous network structure of single-chain peptides dispersed in the gel composition, and is easily accessible by MMPs, so that it is degraded at an appropriate timing. considered to be easy.
  • the combination of cross-linking agent and single-chain peptide may be a combination that exhibits time-dependent gelling ability.
  • the time-dependent gelling ability means gelation in 1 to 60 minutes after mixing the single-chain peptide and the cross-linking agent at 15 to 40°C, and 3 to 30 minutes. is more preferable.
  • the cross-linking agent and the single-chain peptide preferably gel at 10°C to 50°C, more preferably at 15°C to 40°C, and from the viewpoint of gelation at body temperature of the animal, 30°C. C. to 40.degree. C. is most preferred.
  • the cross-linking agent used in the present invention has at least two chains containing functional groups capable of covalent bonding with amino groups and hydrophilic linking groups. Although the chain may be single-ended with no branching, it is preferred that the chain is branched.
  • the number of chains is not particularly limited as long as it is two or more. 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, preferably 2 to 8, more preferably 2 to 6 book, more preferably four. Since a uniform three-dimensional network structure can be formed, it is most preferable to use a tetra-branched cross-linking agent in which the above four chains are branched at one point.
  • the crosslinker has two or more functional groups capable of covalently bonding with amino groups.
  • the weight average molecular weight of the cross-linking agent is not particularly limited, but is preferably from 5,000 to 40,000, more preferably from 5,000 to 30,000, still more preferably from 10,000 to 30,000, and particularly preferably from the viewpoint of forming a uniform network structure. It is 15000 to 25000, and 20000 can be mentioned as an example.
  • a chain comprising a functional group capable of covalently bonding with an amino group and a hydrophilic linking group is preferably represented by Formula 1 below.
  • Z-(A 1 ) w -(B 1 ) x -(C 1 ) y - Formula 1 wherein Z is a functional group that can covalently bond with an amino group, A 1 is a hydrophobic linking group, B 1 is a hydrophilic linking group, C 1 is a hydrophobic linking group, and w is an integer of 1 or more, x is an integer of 1 or more, and y is an integer of 0 or more.
  • the cross-linking agent is preferably represented by Formula 2 below.
  • [Z-(A 1 ) w -(B 1 ) x -(C 1 ) y -] v -CH n Formula 2 wherein Z is a functional group that can covalently bond with an amino group, A 1 is a hydrophobic linking group, B 1 is a hydrophilic linking group, C 1 is a hydrophobic linking group, and w is an integer of 1 or more, x is an integer of 1 or more, y is an integer of 0 or more, v is an integer of 2 to 4, and n is an integer of 0 to 2. However, v+n is 4.
  • Z, A 1 , B 1 and C 1 may be the same or different in each branch or between branches, and w, x and y may be the same or different between branches.
  • w is preferably an integer of 1-10, more preferably an integer of 1-5.
  • x is preferably an integer of 10-300, more preferably an integer of 20-200.
  • y is preferably an integer of 0-5, more preferably an integer of 0-3.
  • v is preferably 4 and n is preferably 0.
  • Functional groups that can covalently bond with amino groups are functional groups that react with amino groups, such as succinimidyl groups, isocyanates, isothiocyanates, sulfonyl chlorides, aldehydes, acyl azides, acid anhydrides, imidoesters, epoxides, active esters, and the like.
  • a succinimidyl group is preferred, and more preferred from the viewpoint that the reaction proceeds easily at the pH of the body.
  • the cross-linking agent has two or more functional groups capable of covalently bonding with amino groups, and the functional groups may be the same or different.
  • Examples of the hydrophilic linking group represented by B 1 include an ethylene oxide group (--CH 2 CH 2 O--) and a group containing an ethylene oxide unit.
  • the hydrophilic linking group is an ethylene oxide group (--CH 2 CH 2 O--) or a group containing ethylene oxide units
  • the cross-linking agent is also called a polyethylene glycol (PEG) cross-linking agent.
  • the cross-linking agent is preferably a PEG cross-linking agent, more preferably a four-branched PEG cross-linking agent having a succinimidyl group at the terminal.
  • Hydrophobic linking groups represented by A 1 include hydrocarbon groups having 1 to 10 carbon atoms, preferably 1 to 5 carbon atoms.
  • the hydrophobic linking group represented by A 1 may have a linking group such as —O—, —CO— or —COO— at its terminal.
  • a 1 may have a connecting group selected from -O-, -CO- and -COO- connecting groups at both ends.
  • the hydrophobic linking group represented by C 1 includes a hydrocarbon group having 1 to 3 carbon atoms, preferably a methylene group or an ethylene group.
  • the hydrophobic linking group represented by C 1 may have a linking group such as —O—, —CO— or —COO— at its terminal.
  • C 1 may have a connecting group selected from -O-, -CO- and -COO- connecting groups at both ends.
  • the cross-linking agent used in the present invention preferably has tissue adhesiveness.
  • Tissue adhesiveness means that the cross-linking agent can chemically bond with the tissue at the site of installation. Preferred is chemical bonding between the cross-linking agent and the amino groups of the tissue surface protein.
  • a peptide consisting of only a single chain used in the present invention means that it is formed of only a single chain and does not contain a three-stranded structure unique to collagen. It is also preferred that the single-chain peptides are non-multimeric.
  • the monomer content of the single-chain peptide is preferably 50-100%, more preferably 80-100%.
  • the single-chain peptide used in the present invention preferably has biocompatibility. Biocompatibility means that it does not cause significant adverse reactions such as long-term and chronic inflammatory reactions when in contact with living organisms.
  • Preferred single-chain peptides are recombinant peptides.
  • the single-chain peptide may be crosslinked between peptide molecules or non-crosslinked, but non-crosslinked peptides are preferable because they are easily degraded in vivo at appropriate timing.
  • the single-chain peptide in an uncrosslinked state preferably contains 40% to 100%, more preferably 60% to 100%, random structure in an aqueous solution.
  • the random structure is a structure (irregular structure) when the polymer chain exists in a solution without forming a specific higher-order structure, and its content is measured by circular dichroism (CD) spectrum measurement, etc. measured by
  • a peptide containing lysine is preferable as the single-chain peptide, and a peptide containing 5% or more of lysine is more preferable as the single-chain peptide from the viewpoint of reaction of functional groups (such as succinimidyl groups) capable of covalently bonding with amino groups.
  • single-chain peptide is not particularly limited, gelatin, elastin, fibronectin, pronectin, tenascin, fibrin, fibroin, entactin, thrombospondin, and retronectin are preferred, and gelatin is more preferred.
  • Gelatin may be a recombinant peptide. Recombinant peptides are described later in this specification.
  • gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen can be used.
  • those described in EP1014176, US Pat. No. 6,992,172, International Publication WO2004/85473, International Publication WO2008/103041, etc. can be used, but are not limited thereto.
  • Preferable recombinant peptides for use in the present invention are peptides of the following aspects.
  • the recombinant peptide is not naturally derived, there is no concern about bovine spongiform encephalopathy (BSE), and it is highly non-infectious.
  • BSE bovine spongiform encephalopathy
  • the recombinant peptide is more uniform than natural gelatin and has a determined sequence, it is possible to precisely design the strength and degradability of the peptide with little variation due to cross-linking or the like.
  • the molecular weight of the recombinant peptide is not particularly limited, but is preferably 2000 or more and 100000 or less (2 kDa or more and 100 kDa or less), more preferably 2500 or more and 95000 or less (2.5 kDa or more and 95 kDa or less), and still more preferably 5000 or more and 90000 or less. (5 kDa or more and 90 kDa or less), most preferably 10000 or more and 90000 or less (10 kDa or more and 90 kDa or less).
  • the molecular weight distribution of the recombinant peptide is not particularly limited, it preferably contains a recombinant peptide in which the area of the maximum molecular weight peak in molecular weight distribution measurement is 70% or more of the total area of all molecular weight peaks, and 90% or more is more. Preferably, 95% or more is most preferred.
  • the molecular weight distribution of recombinant peptides can be measured by the method described in PCT/JP2017/012284.
  • the recombinant peptide preferably has repeats of the Gly-XY sequence characteristic of collagen.
  • a plurality of Gly-XY may be the same or different.
  • Gly-XY Gly represents glycine
  • X and Y represent any amino acid (preferably any amino acid other than glycine).
  • the Gly-XY sequence characteristic of collagen is a very specific partial structure in the amino acid composition and sequence of gelatin-collagen compared to other proteins. Glycine occupies about one-third of the whole in this portion, and in the amino acid sequence, it is repeated one in three.
  • Glycine is the simplest amino acid, is less constrained to the configuration of the molecular chain, and greatly contributes to the regeneration of the helical structure during gelation.
  • the proportion of uncharged amino acids in the polar amino acids is preferably 5% or more and less than 20%, preferably 5% or more and less than 10%.
  • Sequences of IKVAV, LRE, DGEA and HAV sequences are preferred. More preferred are the RGD sequence, YIGSR sequence, PDSGR sequence, LGTIPG sequence, IKVAV sequence and HAV sequence, and particularly preferred is the RGD sequence. Among the RGD sequences, the ERGD sequence is preferred. By using a peptide having a cell adhesion signal, the amount of substrate produced by cells can be improved.
  • the ratio of the RGD motif to the total number of amino acids is preferably at least 0.4%.
  • each stretch of 350 amino acids comprises at least one RGD motif.
  • the ratio of RGD motifs to total amino acids is more preferably at least 0.6%, more preferably at least 0.8%, more preferably at least 1.0%, more preferably at least 1.2%. and most preferably at least 1.5%.
  • the number of RGD motifs in the peptide is preferably at least 4, more preferably at least 6, more preferably at least 8, more preferably 12 to 16 per 250 amino acids.
  • a proportion of 0.4% of RGD motifs corresponds to at least one RGD sequence per 250 amino acids.
  • a recombinant peptide of 251 amino acids must contain at least two RGD sequences to satisfy at least 0.4% of the characteristics.
  • the recombinant peptide comprises at least 2 RGD sequences per 250 amino acids, more preferably at least 3 RGD sequences per 250 amino acids, even more preferably at least 4 RGD sequences per 250 amino acids.
  • a further aspect of the recombinant peptide of the present invention comprises at least 4 RGD motifs, preferably at least 6, more preferably at least 8, even more preferably 12 to 16 RGD motifs.
  • the recombinant peptide may be partially hydrolyzed.
  • the recombinant peptide used in the present invention is represented by A-[(Gly-XY) n ] m -B.
  • Each of n Xs independently represents any amino acid, and each of n Ys independently represents any amino acid.
  • m preferably represents an integer of 2-10, more preferably an integer of 3-5.
  • n is preferably an integer of 3-100, more preferably an integer of 15-70, and most preferably an integer of 50-65.
  • A represents any amino acid or amino acid sequence and B represents any amino acid or amino acid sequence.
  • the n Gly-XY may be the same or different.
  • the recombinant peptide used in the present invention has the formula: Gly-Ala-Pro-[(Gly-XY) 63 ] 3 -Gly (wherein each of the 63 Xs independently represents any amino acid). Each of the 63 Y's independently represents an amino acid, and the 63 Gly-XY's may be the same or different.).
  • naturally occurring collagen may be any naturally occurring collagen, but is preferably type I, II, III, IV, or V collagen. More preferred are type I, type II, or type III collagen. According to another aspect, the origin of said collagen is preferably human, bovine, porcine, mouse or rat, more preferably human.
  • the recombinant peptide is not deaminated.
  • the recombinant peptide does not have a telopeptide.
  • the recombinant peptide is a substantially pure polypeptide prepared by a nucleic acid encoding amino acid sequence.
  • Particularly preferred recombinant peptides used in the present invention are: (1) a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; (2) a peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 1 and having biocompatibility; or (3) a peptide set forth in SEQ ID NO: 1
  • a peptide consisting of an amino acid sequence having a sequence identity of 80% or more (more preferably 90% or more, particularly preferably 95% or more, most preferably 98% or more) with the amino acid sequence, and having biocompatibility; is either
  • amino acid sequence in which one or several amino acids are deleted, substituted or added is preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5 number, particularly preferably 1 to 3.
  • Recombinant peptides used in the present invention can be produced by genetic recombination techniques known to those skilled in the art. For example, see EP1014176A2, US Pat. It can be produced according to the described method. Specifically, a gene encoding an amino acid sequence of a given recombinant peptide is obtained, incorporated into an expression vector to prepare a recombinant expression vector, and introduced into an appropriate host to prepare a transformant. . By culturing the resulting transformant in an appropriate medium, the recombinant peptide is produced, and the recombinant peptide used in the present invention can be prepared by recovering the recombinant peptide produced from the culture. .
  • the peptide may be in solution or powder.
  • a solution it can be included in the kit at a concentration of preferably 1-100 mg/mL, more preferably 1-80 mg/mL, even more preferably 5-80 mg/mL.
  • the gel-forming kit of the present invention may further contain cancer cells.
  • cancer cells may include cancer cells.
  • human-derived cancer cells are preferred, and human-derived solid cancer cells are more preferred.
  • Cancer cell states include, but are not limited to, cell suspensions, spheroids, organoids, or tumor tissues.
  • the cancer cells may be patient-derived tumor tissue, established cancer cell lines, or patient-derived cancer cells. Turbidity is preferred.
  • the gel-forming kit of the present invention further includes a cross-linking agent having at least two chains containing a functional group capable of covalently bonding with an amino group and a hydrophilic linking group, and instructions for administering a single-chain peptide to a subject. may be included.
  • the gel-forming kit of the present invention may further contain a solvent for dissolving the powdery peptide or the powdery cross-linking agent.
  • the pH of the solvent is preferably 4.0 to 9.0, particularly preferably 6.0 to 8.0 from the viewpoint of biocompatibility.
  • the gel-forming kit of the present invention may further include instruments such as syringes, filters, containers, gel-shaping jigs, films, and tweezers used from gel formation to implantation.
  • a non-human having cancer comprising a peptide consisting of only a single chain and a cross-linking agent having at least two chains containing a functional group capable of covalently bonding with an amino group and a hydrophilic linking group.
  • Gel-forming solutions for use in making model animals are provided.
  • a gel-forming solution means a solution obtained by mixing a single-chain peptide and a cross-linking agent until gelation occurs. Gelation means that a solution changes from a fluid state to a non-fluid state. Details and preferred embodiments for peptides consisting of only one chain and crosslinkers are as described herein above.
  • the total concentration of the single-chain peptide and the cross-linking agent in the gel-forming solution is preferably 1 mg/mL to 200 mg/mL, more preferably 1.5 mg/mL to 150 mg/mL, still more preferably 20 mg/mL to 100 mg/mL. It is particularly preferred that the total concentration of the single-stranded peptide and the cross-linking agent in the gel-forming solution at the time of implantation is 20 mg/mL to 100 mg/mL.
  • the active terminal molar concentration ratio [amino group (—NH 2 ) of single-chain peptide]:[functional group of crosslinker capable of covalent bonding with amino group] is in the range of 1:2 to 4:1. is preferably 4:1 from the viewpoint of
  • the concentration of the cross-linking agent in the gel-forming solution is preferably in the range of 1 mg/mL to 150 mg/mL, more preferably 3 mg/mL to 100 mg/mL, even more preferably 3 mg/mL to 50 mg/mL, Especially preferred is 9 mg/mL to 50 mg/mL. It is particularly preferred that the concentration of the cross-linking agent in the gel-forming solution at the time of implantation is between 9 mg/mL and 50 mg/mL.
  • the concentration range of the single-chain peptide in the gel-forming solution is 1 mg/mL to 100 mg/mL, preferably 5 mg/mL to 70 mg/mL, and particularly preferably 10 mg/mL to 50 mg/mL. It is particularly preferred that the single-chain peptide concentration of the gel-forming solution at the time of implantation is between 10 mg/mL and 50 mg/mL.
  • the gel-forming solution of the present invention may further contain cells other than cancer cells.
  • cancer is formed from a peptide consisting of only one chain and a cross-linking agent having at least two or more chains containing a functional group capable of covalently bonding with an amino group and a hydrophilic linking group.
  • a gel composition is provided for use in generating a non-human model animal.
  • the gel composition contains a single-chain peptide and a cross-linking agent, and the gel-forming solution undergoes gelation as described above. Details and preferred embodiments for peptides consisting of only one chain and crosslinkers are as described herein above.
  • the gel composition of the present invention can be used for the production of non-human model animals with cancer.
  • the concentration of the cross-linking agent in the gel composition at the time of implantation of the present invention is preferably in the range of 1 mg/mL to 150 mg/mL, more preferably 3 mg/mL to 100 mg/mL, still more preferably 9 mg/mL. ⁇ 50 mg/mL.
  • the gel composition at the time of transplantation of the present invention may contain cells other than cancer cells.
  • the volume of tumor tissue used is preferably 10-1000 mm 3 , more preferably 50-500 mm 3 .
  • the timing of embedding the tumor tissue in the gel-forming solution is preferably immediately to 60 minutes after mixing the single-chain peptide and the cross-linking agent, and more preferably 1 to 60 minutes from the viewpoint of cytotoxicity. preferable.
  • Tumor tissue may be embedded in a gel-forming solution to form a gel, or may be embedded in a gel composition that does not contain cancer cells.
  • a tumor tissue is embedded in a gel-forming solution to gel, it is preferable to allow the tissue to stand at 10 to 40° C. after embedding to gel before implantation.
  • the gel composition of the present invention contains growth factors (e.g., epidermal growth factor (EGF)), basic fibroblast growth factor (bFGF), nerve growth factor (nerve growth factor) , Platelet-Derived Growth Factor (PDGF), Insulin-like Growth Factor 1, Transforming Growth Factor ⁇ (TGF- ⁇ ), etc.), Enzymes (MMPs, etc.), Cells Other components such as culture medium, serum, blood, ascites, and pleural effusion may be included.
  • growth factors e.g., epidermal growth factor (EGF)
  • bFGF basic fibroblast growth factor
  • nerve growth factor nerve growth factor
  • PDGF Platelet-Derived Growth Factor
  • TGF- ⁇ Transforming Growth Factor ⁇
  • Enzymes MMPs, etc.
  • Cells Other components such as culture medium, serum, blood, ascites, and pleural effusion may be included.
  • the non-human model animal having cancer of the present invention comprises a peptide consisting of only one chain, and at least two chains containing a functional group capable of covalently bonding with an amino group and a hydrophilic linking group. It can be produced by implanting a gel composition or gel-forming solution formed from the cross-linking agent having the above and containing cancer cells into a non-human animal.
  • Animals used for non-human model animal production are not particularly limited as long as they are non-human animals, but mammals are preferred.
  • mammals are preferred.
  • rodents such as mice, rats, rabbits and hamsters are preferred from the viewpoint of ease of handling, and mice are particularly preferred.
  • test substance is not particularly limited and can be appropriately selected according to the purpose. cleansing agents, plant extracts, microbial products, and the like. Libraries for compounds, peptides, proteins, and antibodies can also be used. For example, compound libraries prepared using combinatorial chemistry techniques, random peptide libraries or antibody libraries prepared by solid-phase synthesis or phage display methods can be used.
  • the route of administration of the test substance may be oral or parenteral.
  • Parenteral administration includes, for example, systemic administration such as intravenous, intraarterial or intramuscular administration, or local administration.
  • the dose, dosing interval, start time of administration, and dosing period of the test substance are not particularly limited, and can be appropriately selected according to the purpose. It can be selected as appropriate.
  • a test substance that can reduce cancer (tumor) or suppress cancer (tumor) growth is selected as a candidate substance for cancer treatment. can be done. Reduction of cancer (tumor) or inhibition of cancer (tumor) growth can be evaluated by measuring the size (volume, etc.) of cancer (tumor).
  • symptoms caused by cancer or numerical values of cancer-related markers may be used as indicators.
  • evaluation of cancer pathology in a non-human model animal can be performed using a non-human animal to which no test substance is administered as a negative control.
  • a test substance selected as described above can be a candidate for a therapeutic drug for cancer.
  • CBE3 Recombinant Peptide
  • CBE3 Molecular weight: 51.6 kD Structure: GAP[(GXY) 63 ] 3G Number of amino acids: 571 RGD sequence: 12 Imino acid content: 33% Nearly 100% of the amino acids are GXY repeats.
  • the amino acid sequence of CBE3 does not contain serine, threonine, asparagine, tyrosine and cysteine.
  • CBE3 has an ERGD sequence.
  • Examples 1 to 3 Preparation of non-human model animals using human liver cancer-derived cell lines Water for injection (Otsuka Pharmaceutical Co., Ltd.) was mixed to prepare 200 mmol/L phosphate buffer (pH 6.8). PBS for gel preparation (phosphate buffered saline) was prepared. The lyophilized CBE3 was dissolved in PBS at room temperature for 5 hours to the concentration shown in Table 1, and then heated at 37°C for 30 minutes to dissolve completely. Then, it was filtered through a 0.2 ⁇ m filter to prepare a CBE3 solution.
  • Water for injection Otsuka Pharmaceutical Co., Ltd.
  • PBS for gel preparation phosphate buffered saline
  • the tetra-branched PEG cross-linking agent (SUNBRIGHT PTE-200HS, Yuka Sangyo Co., Ltd.) shown in FIG.
  • a PEG solution was prepared by dissolving in PBS and filtering through a 0.2 ⁇ m filter.
  • a PEG solution was added to the resulting CBE3 solution at a volume ratio of 1:1 and vortexed for 30 seconds to obtain a gel-forming solution used as a scaffold as shown in Table 1.
  • the solid content concentration in Table 1 indicates the total concentration of CBE3 and the cross-linking agent.
  • Human liver cancer-derived cell line HuH-7 (1.5 ⁇ 10 7 cells/mL) suspended in PBS (Thermo Fisher Scientific, pH 7.4) was added to each gel-forming solution at a volume ratio of 2 (gel-forming solution): 1 (cell suspension) and mixed well by pipetting to obtain a gel-forming solution for transplantation.
  • 200 ⁇ L/site 1.0 ⁇ 10 6 cells/site
  • NOD SCID mice female, 7-week-old
  • the mean tumor volumes of the groups using each scaffold were 1020 mm 3 , 483 mm 3 and 185 mm 3 respectively.
  • a graph of the tumor volume of each specimen and the average value of each group is shown in FIG.
  • necropsy was performed to collect the tissue, fixation was performed in 10% neutral buffered formalin (Fujifilm Wako Pure Chemical Industries, Ltd.) at room temperature for 48 hours, and then the cell-implanted site was examined on the surface including the long side of the tumor. It was cut out and immersed in 80% ethanol for 24 hours for degreasing.
  • Dispensing console (Sakura Seiki Co., Ltd.) 100% ethanol (Fuji Film Wako Pure Chemical Industries, Ltd.), xylene (Fuji Film Wako Pure Chemical Industries, Ltd.), paraffin (Sakura Fine Tech Japan Co., Ltd.) in this order after solvent substitution, A paraffin section was obtained by embedding in paraffin using a closed automatic fixing and embedding apparatus and slicing with a microtome to a thickness of 3 ⁇ m.
  • a tumor with a tumor area of 10 mm 2 or more was determined to be tumor formation, and the ratio of the number of tumor-formed sites to the number of transplanted sites was calculated as the tumor formation rate. Tumor formation rates were 83%, 83% and 67% in each group. Representative histopathological images taken with an optical microscope are shown in FIGS.
  • Comparative Examples 1-3 Production of non-human model animals using human liver cancer-derived cell lines (using Matrigerl (registered trademark) and PBS) High concentration Matrigel® (Corning, protein concentration 18-22 mg/mL), Matrigel® (Corning, protein concentration 8-12 mg/mL), PBS (Thermo Fisher Scientific, pH 7.4) Using it as a scaffold, non-human model animals were prepared in the same manner as in Examples 1 to 3, and the tumor volume, tumor area, and tumor formation rate were evaluated. The mean tumor volumes of the groups using each scaffold were 134 mm 3 , 35 mm 3 and 111 mm 3 respectively. A graph of the tumor volume of each specimen and the average value of each group is shown in FIG. The tumor formation rate was 0% in all groups.
  • Example 3 A summary of the results of Examples 1-3 and Comparative Examples 1-3 is shown in Table 2.
  • the tumor formation rate was 50% or more, the tumor formation rate was judged as “good”, and when the tumor formation rate was less than 50%, the judgment was made as "poor”. It can be seen that the use of a scaffold made of a peptide consisting of a single chain improves the tumor formation rate in a short period of time (21 days) after transplantation, making it possible to produce a non-human model animal with high efficiency.
  • accumulation of phagocytic cells in the scaffold was confirmed in Example 3.
  • the solid content concentration in the gel is low, it is easy for cells to enter the gel, and the gel is phagocytosed by phagocytic cells.
  • Examples 4-7 Preparation of non-human model animals from human colorectal cancer liver metastases
  • PEG solution was added to CBE3 solution at a volume ratio of 1:1 and vortexed for 30 seconds.
  • a gel-forming solution to be used as a scaffold was prepared by doing so.
  • the concentration of each solution was as shown in Table 3.
  • the solid content concentration in Table 3 indicates the total concentration of CBE3 and the cross-linking agent.
  • Establishment of Human Colorectal Cancer Liver Metastasis Model A tumor tissue collected from a mouse was cut into 5 mm squares. Tumor piece embedding in gel-forming solution and transplantation into mice were performed by two methods (Imbed method or Syringe method).
  • the Imbed method first, a parafilm was laid on a hot plate at 37° C., 200 ⁇ L of a gel-forming solution (before gelation) was dropped, and a tumor piece was embedded and allowed to stand for 10 minutes. After confirming the gelation of the scaffold material, the back of the mouse was incised, and the embedded tumor pieces were subcutaneously implanted with forceps and then sutured.
  • 200 ⁇ L of gel-forming solution was first placed in a truncated 1 mL syringe, and tumor pieces were embedded in the syringe. After standing at room temperature (20° C.) for 60 minutes to confirm gelation of the scaffold material, the back of the mouse was incised, transplanted while being pushed out from the syringe, and sutured.
  • a histopathological specimen was prepared in the same manner as in Examples 1-3. Images of the obtained sections were taken with an optical microscope, the tumor area was measured, and the tumor area and tumor formation rate were evaluated in the same manner as in Examples 1-3. The tumor formation rates were 100%, 100%, 100% and 75% in each group. Representative histopathological images taken with an optical microscope are shown in FIGS.
  • Example 8 Evaluation of Scaffold Decomposition in Collagenase Solution
  • a gel-forming solution having the same composition as in Example 4 (CBE3 concentration: 18.6 mg/mL) was prepared. 150 ⁇ L of the gel-forming solution was allowed to stand at 37° C. for 1 hour on a parafilm for gelation, and then allowed to stand at 37° C. for 1 hour in PBS (Thermo Fisher Scientific, pH 7.4). It was removed from the PBS and allowed to stand at 37° C. for a predetermined time in a 50 U/L Clostridium histolyticum-derived collagenase solution. Degradation in the collagenase solution was evaluated by measuring the weight of the gel and calculating the "weight ratio to the gel left standing in PBS to which no collagenase was added" as % of residual gel. The evaluation results are shown in FIG.
  • Examples 9 to 11 Measurement of storage elastic modulus
  • Gel-forming solutions as shown in Table 4 were obtained with the same formulations as in Examples 5, 6 and 7.
  • the solid content concentration in Table 4 indicates the total concentration of CBE3 and the cross-linking agent.
  • the storage modulus was measured at 37° C. using a rheometer HAAKE MARS40 manufactured by Thermo Scientific.
  • the storage elastic modulus after 60 minutes is shown in Table 4, and the change in storage elastic modulus over time is shown in FIG.
  • Example 12 Structural Analysis of CBE3 by Circular Dichroism (CD) Spectroscopy
  • a lyophilized CBE3 was dissolved in water for injection at 37° C. to prepare a 0.2 mg/mL measurement solution.
  • a measurement solution was placed in a cell with a layer length of 1 mm, and a CD spectrum measurement in the far ultraviolet region (250 to 200 nm) was performed at 25 ° C. using a circular dichroism spectrometer (J-820) manufactured by JASCO Corporation. .
  • Water for injection was used for blank measurement.
  • Table 5 shows the results of secondary structure analysis of the measurement results using JASCO Corporation's software (JWSSE-480).

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JP2013526297A (ja) * 2010-05-07 2013-06-24 ユニバーシティー オブ ノース カロライナ アット チャペル ヒル 実質組織からの細胞の移植方法
WO2015172073A1 (en) * 2014-05-08 2015-11-12 Cornell University Bio-adhesive gels and methods of use
WO2017022613A1 (ja) * 2015-08-03 2017-02-09 富士フイルム株式会社 細胞構造体、非ヒトモデル動物、非ヒトモデル動物の製造方法、及び被験物質の評価方法
WO2020050205A1 (ja) * 2018-09-03 2020-03-12 富士フイルム株式会社 ゲル形成キット、ゲルおよびゲルの製造方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013526297A (ja) * 2010-05-07 2013-06-24 ユニバーシティー オブ ノース カロライナ アット チャペル ヒル 実質組織からの細胞の移植方法
WO2015172073A1 (en) * 2014-05-08 2015-11-12 Cornell University Bio-adhesive gels and methods of use
WO2017022613A1 (ja) * 2015-08-03 2017-02-09 富士フイルム株式会社 細胞構造体、非ヒトモデル動物、非ヒトモデル動物の製造方法、及び被験物質の評価方法
WO2020050205A1 (ja) * 2018-09-03 2020-03-12 富士フイルム株式会社 ゲル形成キット、ゲルおよびゲルの製造方法

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