WO2023042875A1

WO2023042875A1 - Gel formation kit and use thereof

Info

Publication number: WO2023042875A1
Application number: PCT/JP2022/034520
Authority: WO
Inventors: 香織武田; 武彦横堀; 遼村主; 高行浅尾; 憲調
Original assignee: 富士フイルム株式会社
Priority date: 2021-09-16
Filing date: 2022-09-15
Publication date: 2023-03-23
Also published as: JPWO2023042875A1

Abstract

The present invention addresses the problem of providing: a gel formation kit which can be used for the production of a non-human animal and enables the formation of a tumor with high efficiency within a short period of time in the production of a non-human model animal with cancer; a gel formation solution; a gel composition; and a method for producing the gel composition. The present invention also addresses the problem of providing: a non-human model animal with cancer; a method for producing the non-human model animal; and a method for evaluating a test substance. According to the present invention, a gel formation kit which can be used for the production of a non-human model animal with cancer is provided, which comprises a peptide composed only of a single strand and a crosslinking agent having at least two chains each having a functional group capable of covalently bonding to an amino group and a hydrophilic linking group.

Description

Gel forming kit and its use

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.

In preclinical studies of novel drugs targeting cancer diseases, non-human animal models are used in which patient-derived tumor tissue, patient-derived cancer cells, and established cancer cell lines are transplanted into immunodeficient mice. (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.

In tumorigenesis, engraftment of transplanted tumor tissue and cells, and growth of tumor tissue are necessary. However, 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.

Engelbreth-Holm-Swarm (EHS) mouse sarcoma-derived scaffolds such as Matrigel (registered trademark) are commercially available and widely used as scaffolds that assist tumor formation in the production of non-model animals. EHS-derived scaffolds contain laminin and type IV collagen, which are the main components, as well as various growth factors (Non-Patent Document 4). It is expected that scaffold injection into the site of tumor tissue transplantation will improve the tumor formation rate. However, even when EHS-derived scaffolds are used, the tumor formation rate may remain as low as 10 to 20% depending on the test system, and it takes several months from transplantation to tumor formation (non-patent literature 2).

On the other hand, Patent Document 1 describes a non-human model animal implanted with a cell structure consisting of cells and polymer blocks. In Patent Literature 1, a block (mass) made of a biocompatible polymer is used.

International publication WO2017/022613

　There is a demand for the development of new scaffold materials that can improve the survival rate of tumors in the production of non-human model animals and allow tumors to grow in a short period of time after transplantation.

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.

As a result of intensive studies by the present inventors in order to solve the above problems, 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, and a single chain We have found that gels formed by cross-linking peptides improve the rate of tumorigenesis in the short term. The present invention has been completed based on these findings.

That is, according to the present invention, the following inventions are provided.
(1) 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.
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.
(3) The gel-forming kit according to (1) or (2), wherein the hydrophilic linking group contains an ethylene oxide unit.
(4) the functional group capable of covalent bonding with the amino group is selected from the group consisting of succinimidyl group, isocyanate, isothiocyanate, sulfonyl chloride, aldehyde, acylazide, acid anhydride, imidoester, epoxide and active ester; The gel-forming kit according to any one of (3).
(5) The gel-forming kit according to any one of (1) to (4), wherein the peptide consisting of only one chain is a recombinant peptide.
(6) The gel-forming kit according to (5), wherein the recombinant peptide is represented by the following formula.
A-[(Gly-X-Y) _n ] _m -B
In the formula, 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, and n Y's each independently represent an amino acid. n represents an integer of 3 to 100, and m represents an integer of 2 to 10. The n Gly-XY may be the same or different.
(7) the recombinant peptide is
A peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1;
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 of SEQ ID NO: 1 and having biocompatibility; or 80% or more of the amino acid sequence of SEQ ID NO: 1 A peptide consisting of an amino acid sequence having the sequence identity of and having biocompatibility;
The gel-forming kit according to (5) or (6), which is either
(8) The gel-forming kit according to any one of (1) to (7), further comprising cancer cells.
(9) 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. .
(10) 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. Gel-forming solution for use in making.
(12) The gel-forming solution according to (11), wherein the total concentration of the single-stranded peptide and the cross-linking agent in the gel-forming solution is 1 to 200 mg/mL.
(13) The gel-forming solution according to (11) or (12), further comprising cancer cells.
(14) A non-human cancer model formed from 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 gel composition for use in making animals.
(15) The gel composition according to (14), further comprising cancer cells.
(16) Mixing a peptide consisting of only one chain with 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 to produce a gel-forming solution. ,
embedding cancer cells with the gel-forming solution;
A method for producing a gel composition for use in producing a non-human model animal having cancer.
(17) A gel composition formed from 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, and containing cancer cells. A non-human model animal having cancer, which has a substance as an implant.
(18) A gel composition formed from 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, and containing cancer cells. A method for producing a non-human model animal having cancer, comprising implanting a product or gel-forming solution into a non-human animal.
(19) Transplantation of cancer cells into a non-human animal, and a cross-linking agent having at least two or more chains containing a single-chain peptide, 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 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).

According to the present invention, the tumor formation rate in a short period of time was improved by using a peptide consisting of only a single chain. Although the reason why such an effect is exhibited is not clear in detail, the present inventors found that 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. In addition, 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. In addition, 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.

FIG. 1 shows the chemical structure of a tetra-branched polyethylene glycol cross-linking agent used in Examples. FIG. 2 shows the tumor volumes of Examples 1-3 and Comparative Examples 1-3. 3 shows representative histopathological images of Example 1. FIG. 4 shows representative histopathological images of Example 2. FIG. 5 shows representative histopathological images of Example 3. FIG. 6 shows a typical histopathological image of Comparative Example 1. FIG. 7 shows a typical histopathological image of Comparative Example 2. FIG. 8 shows a typical histopathological image of Comparative Example 3. FIG. 9 shows representative histopathological images of Example 4. FIG. 10 shows representative histopathological images of Example 5. FIG. 11 shows a representative histopathological image of Example 6. FIG. 12 shows representative histopathological images of Example 7. FIG. FIG. 13 shows scaffold degradation in collagenase solution. FIG. 14 shows the change in storage modulus of the gel over time.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail.
[Gel formation kit]
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). On the other hand, since 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. It is presumed that this contributes to ensuring the space required for cell growth and promotes cell growth. 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.

In the gel-forming kit of the present invention, each of the cross-linking agent and the single-chain peptide can be included in the kit as an injectable form. Injectables are substances that can be passed through a syringe needle, preferably fluid solutions or suspensions, particularly preferably homogeneous aqueous solutions. Injectable forms include, but are not limited to, solutions, suspensions, powders, and the like. In the case of powder, it can be used after dissolving or suspending in a liquid at the time of use. As an example, a kit can include a cross-linking agent in powder form and a single peptide chain in solution.

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.

(crosslinking agent)
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.
By having two or more of the above chains, 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. When 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. In addition, when using a PEG cross-linking agent, you may use other cross-linking agents other than a PEG cross-linking agent together.

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. In addition, 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. In addition, 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.

(single-chain peptide)
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.

Although the type of 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.

(recombinant peptide)
Preferred single-chain peptides are recombinant peptides.
A recombinant peptide means a polypeptide or protein-like substance having an amino acid sequence similar to that of gelatin produced by gene recombination technology. Recombinant peptides that can be used in the present invention preferably have repeats of a collagen-characteristic Gly-XY sequence (X and Y each independently represent an amino acid). Here, a plurality of Gly-XY may be the same or different. Preferably, one molecule contains two or more sequences of cell adhesion signals. As the recombinant peptide used in the present invention, gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen can be used. For example, 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.

Because the recombinant peptide is not naturally derived, there is no concern about bovine spongiform encephalopathy (BSE), and it is highly non-infectious. In addition, since 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).
Although 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. Here, a plurality of Gly-XY may be the same or different. In Gly-XY, Gly represents glycine, and 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. Amino acids represented by X and Y contain a large amount of imino acids (proline, oxyproline), and preferably account for 10% to 45% of the total. Preferably, 80% or more, more preferably 95% or more, and most preferably 99% or more of the amino acids in the sequence of the recombinant peptide are Gly-XY repeat structures.

In general gelatin, charged and uncharged polar amino acids are present at a ratio of 1:1. Here, the polar amino acids specifically refer to cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, serine, threonine, tyrosine and arginine, among which the polar uncharged amino acids are cysteine, asparagine, glutamine and serine. , threonine and tyrosine. In the recombinant peptide used in the present invention, the proportion of polar amino acids in all constituent amino acids is 10-40%, preferably 20-30%. In addition, 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%. Furthermore, it is preferred that any one of serine, threonine, asparagine, tyrosine and cysteine amino acids, preferably two or more amino acids, is not included in the sequence.

Generally, in polypeptides, the minimum amino acid sequence that functions as a cell adhesion signal is known (for example, published by Nagai Publishing Co., Ltd. "Pathophysiology" Vol. 9, No. 7 (1990) p. 527). The peptide used in the present invention preferably has two or more of these cell adhesion signals in one molecule. Cell adhesion signals may be the same or different. Specific sequences include the RGD sequence, LDV sequence, REDV sequence, YIGSR sequence, PDSGR sequence, RYVVLPR sequence, LGTIPG sequence, RNIAEIIKDI sequence, which are represented by amino acid single letter notation, in that there are many types of cells to which it adheres. 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.

As for the arrangement of the RGD sequences in the recombinant peptides used in the present invention, the number of amino acids between RGDs is preferably between 0 and 100, preferably between 25 and 60, and is not uniform.
The content of this minimum amino acid sequence is preferably 3 to 50, more preferably 4 to 30, particularly preferably 5 to 20 per protein molecule from the viewpoint of cell adhesion and proliferation. Twelve are most preferred.

In the recombinant peptide used in the present invention, the ratio of the RGD motif to the total number of amino acids is preferably at least 0.4%. Where the recombinant peptide comprises 350 or more amino acids, it is preferred that 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. Since the number of RGD motifs is an integer, a recombinant peptide of 251 amino acids must contain at least two RGD sequences to satisfy at least 0.4% of the characteristics. Preferably, 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. include. 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.

Preferably, 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.

More preferably, 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.).

It is preferable to bind multiple naturally occurring collagen sequence units to the repeating unit. The term "naturally occurring collagen" as used herein 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 isoelectric point of the recombinant peptide used in the present invention is preferably 5-10, more preferably 6-10, still more preferably 7-9.5. The isoelectric point of the recombinant peptide is measured according to isoelectric focusing (Maxey, C. R. (1976; see Phitogr. Gelatin 2, Editor Cox, P. J. Academic, London, Engl.)). % recombinant peptide solution through a mixed crystal column of cation and anion exchange resins and then measuring the pH.

Preferably, the recombinant peptide is not deaminated.
Preferably, the recombinant peptide does not have a telopeptide.
Preferably, 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

"One or several" in the "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. .

In the gel-forming kit of the present invention, the peptide may be in solution or powder.
In the case of 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.
Although the type of cancer cells does not matter, human-derived cancer cells are preferred, and human-derived solid cancer cells are more preferred. For example, malignant melanoma, malignant lymphoma, gastrointestinal cancer, lung cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, colon cancer, ureteral tumor, gallbladder cancer, bile duct cancer, biliary tract cancer, Breast cancer, liver cancer, pancreatic cancer, testicular cancer, maxillary cancer, tongue cancer, lip cancer, oral cavity cancer, pharyngeal cancer, laryngeal cancer, ovarian cancer, uterine cancer, prostate cancer, thyroid cancer Cancer, brain tumor, Kaposi's sarcoma, hemangioma, leukemia, polycythemia vera, neuroblastoma, retinoblastoma, myeloma, cystoma, sarcoma, osteosarcoma, sarcoma, skin cancer, basal cell carcinoma, skin Examples include, but are not limited to, adnexal cancer, skin metastasis cancer, and the like. Among them, liver cancer cells, biliary tract cancer cells, pancreatic cancer cells, colon cancer cells, osteosarcoma cells, chondrosarcoma cells, and angiosarcoma cells are 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.

[Gel-forming solution]
According to the present invention, 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 cancer cells. Details and preferred embodiments of cancer cells are as described herein above.

The gel-forming solution of the present invention may further contain cells other than cancer cells.

The gel-forming solution 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 β (TGF-β), etc.), Enzymes (MMPs, etc.), Cells Other components such as culture medium, serum, blood, ascites, and pleural effusion may be included.

A gel-forming solution can be produced by mixing 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. .
The gel-forming solution contains a peptide consisting of only one chain, 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 a cell suspension containing cancer cells. It can also be produced by mixing

[Gel composition and method for producing gel composition]
According to the present invention, 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 may further contain cancer cells. Details and preferred embodiments of cancer cells 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 total concentration of the single-chain peptide and the cross-linking agent in the gel composition at the time of implantation of the present invention is preferably 1 mg/mL to 200 mg/mL, more preferably 1.5 mg/mL to 150 mg/mL, More preferably 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, preferably 4:1.

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 concentration of the single-chain peptide in the gel composition at the time of implantation of the present invention is 1 mg/mL to 100 mg/mL, preferably 5 mg/mL to 70 mg/mL, particularly 10 mg/mL to 50 mg/mL. preferable.

The gel composition at the time of transplantation of the present invention may contain cells other than cancer cells.

The gel composition of the present invention is prepared by mixing a peptide consisting of only one chain and a cross-linking agent having at least two chains each containing a functional group capable of covalently bonding with an amino group and a hydrophilic linking group, and causing a cross-linking reaction. It can be manufactured by doing.

The gel composition of the present invention can also be prepared by mixing 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 to form a gel. It can be produced by preparing a solution and embedding cancer cells in the gel-forming solution or a gel composition that does not contain cancer cells. Embedding is the mixing of a cell suspension with a gel-forming solution or the encapsulation of spheroids, organoids, tumor tissue with a gel-forming solution or a gel composition that does not contain cancer cells.
The gel composition may contain a solvent. The solvent is preferably an aqueous one, preferably an aqueous buffer or a cell culture medium, most preferably a phosphate buffer. The pH of the solvent is preferably 4.0 to 9.0, particularly preferably 6.0 to 8.0 from the viewpoint of biocompatibility.

When a gel composition containing a suspension of cancer cells is produced, the cell concentration in the gel composition is preferably 5.0×10 ² to 5.0×10 ⁸ cells/mL, and 5.0. More preferably 10 ⁴ to 2.5 x 10 ⁸ cells/mL, most preferably 5.0 x 10 ⁵ to 5.0 x 10 ⁷ cells/mL.
The number of cells to be transplanted may be any number as long as the cells are contained, but the number of cells per gel composition to be transplanted to one site is preferably 1.0×10 ² to 1.0×10 ⁸ cells, and 1.0×10 ⁴ to 5.0×10 8 cells. 0×10 ⁷ cells are more preferred, and 1.0×10 ⁵ to 1.0×10 ⁷ cells are most preferred.

When the gel-forming solution is mixed with the cell suspension, the mixing timing is preferably immediately to 60 minutes, more preferably immediately to 30 minutes, after mixing the single-chain peptide and the cross-linking agent. , from the viewpoint of cytotoxicity and uniform cell suspension, 1 to 10 minutes is most preferred.

When mixing the gel-forming solution with the cell suspension, it is preferable to operate on ice or at room temperature (10 to 30°C), and from the viewpoint of cell viability, operation on ice is more preferable.

When a gel composition containing tumor tissue is produced, 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.
When embedding the tumor in the gel-forming solution, it is preferable to operate on ice or at room temperature (10-30° C.). When 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 β (TGF-β), etc.), Enzymes (MMPs, etc.), Cells Other components such as culture medium, serum, blood, ascites, and pleural effusion may be included.

[Non-human model animal having cancer and method for producing the same]
The gel composition or gel-forming solution of the present invention can be implanted in non-human animals for the production of non-human model animals having cancer.

According to the present invention, a peptide comprising 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, and containing cancer cells. A non-human model animal having cancer is provided that has the gel composition as an implant.
The cancer is preferably human cancer.

As for cancer diseases, specific examples of cancers derived from cancer cells include those mentioned above in this specification, but are not limited to these.

As a first example, 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.

As a second example, the non-human model animal having cancer of the present invention is prepared by transplanting cancer cells into a non-human animal, and a cross-linking agent having at least two chains containing a hydrophilic linking group, and a gel-forming solution can be produced by implanting the gel-forming solution into a non-human animal.

The site of implantation of the gel composition or gel-forming solution in the non-human model animal is not particularly limited. and preferably subcutaneous.

Animals used for non-human model animal production are not particularly limited as long as they are non-human animals, but mammals are preferred. Among mammals, rodents such as mice, rats, rabbits and hamsters are preferred from the viewpoint of ease of handling, and mice are particularly preferred.

As non-human model animals, animals with weakened immunity or immunodeficient animals are preferred. The non-human model animal with weakened immunity may be an animal whose immunity is weakened to the extent that the cell structure can engraft. For example, nude mice, nude rats, SCID (severe combined immunodeficiency) mice, NOD (non-obese diabetic)-SCID mice, immune deficiencies such as ALY (hereditary lymph node deficiency) mice Animals can be mentioned, and commercial products can also be used. Non-human animals may be irradiated with radiation such as X-rays to reduce immunity. Non-human model animals to be prepared can be 4- to 12-week-old mice or rats, for example. Non-human animals can be kept in an infection-protected environment, depending on the degree of immunocompromise. Antibiotics may be administered to non-human animals for the purpose of protection against infection.

As a method of implanting the gel composition or gel-forming solution, a method of injecting the composition before gelation with a syringe or a method of implanting the composition after gelation into the incision site can be considered. When a cell suspension is to be implanted, it is preferable to embed cells by suspending them in a gel-forming solution prior to gelation and injecting the cells into the implantation site with a syringe. When spheroids, organoids, or tumor tissues are transplanted, the gel-forming solution before gelation may be injected into the same site with a syringe after transplanting the implant into the incision site, or the cancer cells may be embedded in the gel. The composition may be prepared and then transplanted to the incision site. When transplanting to the incision site, the composition may be directly transplanted to the transplant site using tweezers or the like, or may be injected using an instrument such as a trocar.

The time from embedding the cells in the gel-forming solution to transplantation is 12 hours or less, preferably 3 hours or less.

The non-human animal of the present invention is useful for the development of cancer therapeutic drugs, verification of cancer therapeutic effects, verification of side effects of cancer therapy, development of reagents for detecting and imaging cancer, and the like.

[Test substance evaluation method]
According to the present invention, a method for evaluating a test substance is provided, which comprises administering the test substance to the non-human model animal having cancer of the present invention. When candidate substances for cancer therapeutic agents such as anticancer agents are used as test substances, cancer therapeutic agents can be screened.

The method for evaluating a test substance according to the present invention preferably includes the steps of administering the test substance to the non-human model animal of the present invention, and evaluating the pathology of cancer in the non-human model animal.

The 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.

In the process of evaluating cancer pathology in non-human model animals, 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). Alternatively, in the step of evaluating the pathological condition of cancer in a non-human model animal, symptoms caused by cancer or numerical values of cancer-related markers may be used as indicators.
In addition, 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.

The present invention will be described more specifically with the following examples, but the present invention is not limited by the examples.

Reference Example 1: Recombinant Peptide The following CBE3 was prepared as a recombinant peptide (described in International Publication WO2008/103041).
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.
Isoelectric point: 9.34
GRAVY value: -0.682
1/IOB value: 0.323
Amino acid sequence (SEQ ID NO: 1 in the sequence listing) (same as SEQ ID NO: 3 in International Publication WO2008/103041, except that X at the end is changed to "P")
GAP(GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADG
APGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAA
GLPGPKGERGDAGPKGADGAPGKDGVRGLAGPP)3G

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 μm filter to prepare a CBE3 solution.

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 μm 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. Within 5 minutes thereafter, 200 μL/site (1.0×10 ⁶ cells/site) were transplanted into NOD SCID mice (female, 7-week-old) on both sides of the back (total of 2 sites), thereby forming a non-human model. animals were produced. Twenty-one days after transplantation, the minor axis, major axis, and height of the tumor were measured with vernier calipers, and the tumor volume was calculated as (long axis)×(short axis)×(height)×π/6. 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.

After measurement of the tumor volume, 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 μm. After sufficiently drying the section at 50°C, it was immersed in hematoxylin (Sakura Fine Tech Japan Co., Ltd.) for 1 minute, washed with water, and then immersed in 0.1% eosin (Fujifilm Wako Pure Chemical Industries, Ltd.) for 1 minute. Hematoxylin and eosin (H&E) staining was performed by washing with water again. After dehydration with 100% ethanol, clearing with xylene, and encapsulation with marinol, a histopathological specimen was obtained. After taking an image of the obtained specimen with an optical microscope, the major axis of the largest tumor mass in the tissue and the minor axis perpendicular to the major axis were measured. Area was calculated. 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. However, in the evaluation of histopathological specimens, there were some specimens in which aggregates of cells with an area of about 0.1 to 1.0 mm ² were observed. Therefore, if kept for a long period of time, such as several months, tumors may form, but it is considered unsuitable for short-term tumor formation. Representative histopathological images taken by light microscopy are shown in Figures 6-8.

A summary of the results of Examples 1-3 and Comparative Examples 1-3 is shown in Table 2. When 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. In the evaluation of the histopathological specimen, accumulation of phagocytic cells in the scaffold was confirmed in Example 3. When 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. This is thought to hinder the performance of the gel as a scaffold. Therefore, in order to particularly increase the tumor formation rate, it is effective to suppress the infiltration of phagocytic cells by setting the total concentration of the single-chain peptide and the cross-linking agent contained in the scaffold at the time of transplantation to 20 mg/mL or more. .

Examples 4-7: Preparation of non-human model animals from human colorectal cancer liver metastases As in Examples 1-3, 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). In the Imbed method, first, a parafilm was laid on a hot plate at 37° C., 200 μL 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. In the Syringe method, 200 μL 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.

NOD SCID mice (male, 7 weeks old) were used as mice, and transplanted to one site on each side of the back (2 sites in total). On the 43rd day after transplantation, the minor axis and major axis of the tumor were measured with vernier calipers, and the tumor volume was calculated by (long axis)×(short axis) ² ×1/2. The mean tumor volumes in each implantation condition were 1339 mm ³ , 1133 mm ³ , 824 mm ³ and 167 mm ³ respectively.

After measuring the tumor volume, 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.

The results of Examples 4 to 7 are summarized in Table 3. When 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 a non-human model animal can be produced with high efficiency by transplanting tumor tissue with a high engraftment rate using a peptide scaffold consisting of a single chain. There is no difference between the Imbed method and the Syringe method for embedding the tumor piece in the gel-forming solution and transplanting it to the mouse. From the fact that the tumor formation rate was particularly high in Examples 4, 5, and 6, in order to particularly promote tumor formation, the total concentration of the single-chain peptide and the cross-linking agent contained in the scaffold at the time of transplantation is 20 mg/mL or more.

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 μL 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.

Comparative Example 4 Evaluation of Scaffold Degradation in Collagenase Solution was used to evaluate degradation in a collagenase solution in the same manner as in Example 8. The evaluation results are shown in FIG.

From Example 8 and Comparative Example 4, it can be seen that scaffolding materials made of peptides consisting of only a single chain are susceptible to degradation by degrading enzymes.

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. Immediately after mixing the CBE3 solution and the PEG solution, 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.

It was confirmed that the higher the solid content concentration in the gel-forming solution, the higher the storage elastic modulus after gelation. As described above, infiltration of phagocytic cells is suppressed by setting the solid content concentration of the gel-forming solution to 20 mg/mL or more when transplanted into mice. This is because the gel has a high storage elastic modulus. Conceivable. The solid content concentration in Table 4 indicates the total concentration of CBE3 and the cross-linking agent.

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).

Claims

Used for preparing 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 with an amino group and a hydrophilic linking group. Gel Forming Kit for.
2. The gel-forming kit according to claim 1, wherein the chain comprising a functional group capable of covalently bonding with the amino group and a hydrophilic linking group is 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.
3. A gel-forming kit according to claim 1 or 2, wherein said hydrophilic linking group comprises ethylene oxide units.
3. The method according to claim 1 or 2, wherein said functional group capable of covalently bonding with an amino group is selected from the group consisting of succinimidyl group, isocyanate, isothiocyanate, sulfonyl chloride, aldehyde, acylazide, acid anhydride, imidoester, epoxide and active ester. Gel forming kit as described.
3. The gel-forming kit according to claim 1 or 2, wherein the peptide consisting of only one chain is a recombinant peptide.
6. The gel-forming kit according to claim 5, wherein the recombinant peptide is represented by the following formula.
A-[(Gly-X-Y) n ] m -B
In the formula, 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, and n Y's each independently represent an amino acid. n represents an integer of 3 to 100, and m represents an integer of 2 to 10. The n Gly-XY may be the same or different.
The recombinant peptide is
A peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1;
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 of SEQ ID NO: 1 and having biocompatibility; or 80% or more of the amino acid sequence of SEQ ID NO: 1 A peptide consisting of an amino acid sequence having the sequence identity of and having biocompatibility;
The gel-forming kit according to claim 5, which is any one of
3. The gel-forming kit according to claim 1, further comprising cancer cells.
9. The gel-forming kit according to claim 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.
9. The cancer cells of claim 8, wherein 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. gel forming kit.
Used for preparing 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 with an amino group and a hydrophilic linking group. gel-forming solution for
The gel-forming solution according to claim 11, wherein the total concentration of said single chain peptide and said cross-linking agent in the gel-forming solution is 1-200 mg/mL.
13. The gel-forming solution according to claim 11 or 12, further comprising cancer cells.
Preparation of a non-human model animal having cancer, which is formed from 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. A gel composition for use in
15. The gel composition of claim 14, further comprising cancer cells.
preparing a gel-forming solution by mixing 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;
embedding cancer cells with the gel-forming solution;
A method for producing a gel composition for use in producing a non-human model animal having cancer.
Implanting a gel composition formed from 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, and containing cancer cells. A non-human model animal having cancer as an object.
A gel composition or gel formed from 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, and containing cancer cells. A method for producing a non-human model animal having cancer, comprising transplanting a forming solution into a non-human animal.
Transplanting cancer cells into a non-human animal, and comprising 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 to an amino group and a hydrophilic linking group. A method for producing a non-human model animal having cancer, comprising transplanting a gel-forming solution into the non-human animal.
20. The method for producing a non-human model animal having cancer according to claim 18 or 19, wherein the gel composition or gel-forming solution is subcutaneously, intraperitoneally, or implanted into an organ or tissue of the non-human animal.
A method for evaluating a test substance, comprising administering the test substance to the non-human model animal having cancer according to claim 17.