Method for predicting risk of tumor growth, kit used in said method, and model animal exhibiting tumor growth

Abstract

Provided is a data acquisition method for predicting the risk of tumor growth due to SITH-1, the method including testing for SITH-1 of human herpesvirus 6 (HHV-6) in a sample collected from a subject, wherein the risk of tumor growth due to SITH-1 is predicted to be high when it is determined that the expression of SITH-1 is high.

Description

Method for predicting risk of cancer growth, kit for use in said method, and model animal exhibiting cancer growth

[0001] The present invention relates to a method for predicting the risk of onset, proliferation, metastasis and recurrence of cancer for which SITH-1 (Small protein encoded by the Intermediate stage Transcript of HHV-6-1) of human herpesvirus 6 (HHV-6) serves as a risk factor (hereinafter referred to as cancer growth induced by SITH-1), a test kit for the risk of cancer growth induced by SITH-1, a model animal for cancer growth induced by SITH-1, and prevention of cancer growth induced by SITH-1 based on the risk prediction described above.

[0002] It is well known that cancer and depression are associated. While it is generally believed that cancer causes the onset of depression, there is also a theory that depression patients are more likely to develop cancer. No conclusion has been reached on whether depression promotes the onset, proliferation, metastasis and recurrence of cancer.

[0003] The inventors discovered SITH-1, a viral protein expressed during latent infection of human herpesvirus 6 (HHV-6) or when reactivation is induced, as a risk factor for depression. The expression of SITH-1 can be detected by anti-SITH-1 antibodies in peripheral blood, and the anti-SITH-1 antibody positive rate in depression patients was about 80% (Patent Document 1, Non-Patent Documents 1 and 2).

[0004] In a mouse model in which SITH-1 was expressed, symptoms characteristic of the pathological condition of depression such as depression-like behavior and hyperactivation of the hypothalamic-pituitary-adrenal axis (HPA axis) were observed, indicating that SITH-1 is one of the causes of depression.

[0005] Japanese Patent Application No. 2024-150783 "Method for Predicting Depression and Test Kit for Depression"

[0006] iScience 2020 Jun 26;23(6):101187iScience 2024 Mar 15; 27(3): 109203

[0007] The present invention aims to provide a method for predicting the risk of cancer growth, a cancer growth testing kit, a model animal for cancer growth, and a method for preventing cancer growth.

[0008] The inventors diligently investigated the above problems and found that anti-SITH-1 antibodies, an indicator of SITH-1 expression, are frequently elevated in cancer patients. Furthermore, they observed that cancer development, proliferation, and metastasis were promoted in model mice expressing SITH-1, and found that the host's immune mechanism against cancer was suppressed. The inventors completed the present invention by discovering for the first time that the SITH-1 protein, a risk factor for depression, has the function of promoting cancer growth and metastasis by suppressing the host's immune mechanism against cancer, and that these phenomena can be suppressed by improving the decrease in acetylcholine production in the brain.

[0009] In other words, the present invention is as follows: [1] A method for acquiring data to predict the risk of cancer growth, comprising the step of testing the amount of human herpesvirus 6 (HHV-6) SITH-1 or the amount of antibodies against SITH-1 in a sample taken from a subject, wherein a high amount of SITH-1 or antibodies against SITH-1 is determined to indicate a high risk of cancer growth. [2] The method according to [1], wherein the testing step comprises measuring the amount of antibodies against HHV-6 SITH-1. [3] The method according to [2], wherein the amount of antibodies against SITH-1 is measured using a polypeptide containing an amino acid sequence of 15 or more amino acids that shows 90% or more homology to the amino acid sequence shown in SEQ ID NO: 1. [4] The method according to [1], wherein the testing step comprises detecting a repeat sequence polymorphism in the repeat element region of HHV-6. [5] The method according to any one of [1] to [4], wherein the cancer growth due to SITH-1 is selected from the group consisting of cancer onset, proliferation, metastasis, and recurrence. [6] A kit for testing the risk of cancer growth, comprising a reagent for detecting an antibody against the SITH-1 protein of human herpesvirus 6 (HHV-6) or a reagent for detecting a repeat sequence polymorphism in the repeat element region of HHV-6. [7] The kit according to [6], wherein the reagent comprises a polypeptide having an amino acid sequence of 15 or more amino acids that exhibits 90% or more homology to the amino acid sequence shown in SEQ ID NO: 1. [8] The kit according to [6], wherein the reagent comprises an oligonucleotide primer set for amplifying the repeat element region or an oligonucleotide primer for sequencing the region. [9] The kit according to any one of [6] to [8] for testing a sample including blood.

[10] The kit according to any one of [6] to [8], wherein the cancer growth due to SITH-1 is selected from the group consisting of cancer development, proliferation, metastasis, and recurrence.

[11] A non-human model animal capable of evaluating the effect of SITH-1 on cancer growth, the non-human model animal being artificially modified to express the SITH-1 protein in the olfactory bulb.

[12] A non-human animal model according to

[11] that highly expresses factors that promote cancer growth, inflammatory cytokines, sympathetic nerve molecules, immune checkpoint molecules, and / or immunosuppressive factors.

[13] A method for obtaining data for determining a cancer treatment strategy, comprising detecting the risk of cancer growth due to SITH-1 by the method according to any one of [1] to [5], wherein, when such risk is detected, it is determined that treatment or prevention is necessary to suppress cancer growth with a drug that suppresses the effect of SITH-1.

[14] The method according to

[13] , wherein the drug that suppresses the effect of SITH-1 is a drug that stimulates acetylcholine receptors, a drug that inhibits the degradation of acetylcholine, or a drug that promotes the synthesis of acetylcholine.

[0010] While it has been previously hypothesized that stress and depression are related to cancer growth (development, proliferation, metastasis, and recurrence), there has been no objective method to evaluate the causes of stress and depression and to suppress cancer growth. This invention allows for the assessment of SITH-1 expression in patients, thereby evaluating the risk of cancer growth due to SITH-1, and enabling diagnosis, prevention, and treatment of the risk of cancer growth by suppressing the effects of SITH-1.

[0011] Figure 1 shows the anti-SITH-1 antibody titers of cancer patients. Healthy individuals are not cancer patients, but subjects who are not suffering from depression, post-COVID-19 symptoms, inflammatory bowel disease, or chronic fatigue syndrome, and who are known to have elevated anti-SITH-1 antibody titers. For cancer patients, gastric cancer patients, esophageal cancer patients, and lung cancer patients are shown as representatives. All patients are shown before treatment. Figure 2 shows the increase in tumor weight due to SITH-1. Human gastric cancer cells (85As2) were subcutaneously inoculated into nude mice, and the tumor weight was measured after 2 weeks (A) and 8 weeks (B). Comparisons are shown between uninoculated (Control) and inoculated (85As2) mice, and between administration of an empty adenovirus vector (emp) and a SITH-1 expressing adenovirus vector (SITH) into the nasal cavity of mice. Figure 3 shows the promotion of metastasis by SITH-1. C57BL / 6 mice were inoculated with mouse melanoma cells B16F10 via the tail vein, and the number of melanomas engrafted in the lungs was measured. (A) A comparison of an empty adenovirus vector (emp) and (B) a SITH-1 expressing adenovirus vector (SITH) is shown. The bar in (C) represents the median. Figure 4 shows the promotion of inflammation by SITH-1. Human gastric cancer cells (85As2) were inoculated into the peritoneal cavity of nude mice, and mRNA expression of IL-1β (A), IL-6 (B), and TNFα (C) in mouse cells obtained from cancer tissue was measured 2 weeks later. A comparison of administration of PBS into the peritoneal cavity, administration of 85As2 cells, and administration of an empty adenovirus vector (emp) and a SITH-1 expressing adenovirus vector (SITH) into the nasal cavity of mice is shown. A, B, C: Mann-Whitney U test (bars represent the median). Figure 5 shows the enhancement of the sympathetic nervous system by SITH-1. Figure 6 shows the expression levels of Tyrosine hydroxylase (A) and Tropomyosin receptor kinase B (B) mRNA. A, B: Mann-Whitney U test (bars indicate median), *: p < 0.05. Figure 6 shows the effect of SITH-1 on cancer immune checkpoints. Human gastric cancer cells (85As2) were inoculated into the peritoneal cavity of nude mice, and the expression levels of CD274 (also known as PD-L1) (A) or PD-1 (B) mRNA were measured in mouse cells obtained from cancer tissue two weeks later.Figure 7 shows a comparison of the effects of administering an empty adenovirus vector (emp) to mice and an adenovirus vector expressing SITH-1 (SITH) to mice. A, B: Mann-Whitney U test (bars indicate median), *: p < 0.05. Figure 7 shows the increase in factors that suppress cancer immunity by SITH-1. Human gastric cancer cells (85As2) were inoculated into the peritoneal cavity of nude mice, and the expression levels of CD80 mRNA (A) and Galectin-9 mRNA (B) were measured in mouse cells obtained from cancer tissue two weeks later. Figure 8 shows a comparison of the effects of administering an empty adenovirus vector (emp) to mice and an adenovirus vector expressing SITH-1 (SITH) to mice. A, B: Mann-Whitney U test (bars indicate median), *: p < 0.05. Figure 8 shows the increase in factors that promote cancer growth by SITH-1. Human gastric cancer cells (85As2) were inoculated into the peritoneal cavity of nude mice, and the expression levels of MMP9 mRNA (A), VEGFA mRNA (B), and TGF-β1 mRNA (C) were measured in mouse cells obtained from cancer tissue two weeks later. Furthermore, in a lung metastasis model in which mouse melanoma cells B16F10 were inoculated via the tail vein into C57BL / 6 mice, the expression levels of MMP8 mRNA (D) and HAVCR2 mRNA (E) were measured one week after inoculation. Additionally, human gastric cancer cells (85As2) were subcutaneously inoculated into the peritoneal cavity of nude mice, and TGF-β1 mRNA (F) was measured in mouse cells obtained from cancer tissue two weeks later. A, D, E: T-test (bars indicate mean, error bars indicate standard error), B, C, F: Mann-Whitney U test (bars indicate median).

[0012] <Classification of HHV-6> In this invention, HHV-6 is preferably HHV-6B, but HHV-6A is also acceptable.

[0013] <SITH-1> In this invention, SITH-1 protein means Small PROTEIN encoded by the Intermediate Transcript of HHV-6 protein, and includes its variants and analogues.

[0014] The SITH-1 protein binds to the host protein CAML (calcium-modulating cyclophyllin ligand), increasing intracellular calcium concentration in glial cells. CAML is a protein that is abundant in the brain and lymphocytes in the host body and is known to increase intracellular calcium concentration. Furthermore, the increase in intracellular calcium concentration due to SITH-1 protein expression is thought to lead to the activation of overall signal transduction within latent infected cells, contributing to the efficient reactivation of HHV-6.

[0015] The SITH-1 protein is preferably selected from the following groups: (a) a protein having the amino acid sequence of SEQ ID NO: 1; (b) a protein having an amino acid sequence in which one or more (e.g., 1 to 15, preferably 1 to 10, more preferably 1 to 5) amino acid deletions, substitutions, insertions and / or additions are made in the amino acid sequence of SEQ ID NO: 1, and which has the activity to increase intracellular calcium concentration; where the substitution is preferably a conservative substitution. A conservative substitution is the replacement of a specific amino acid residue with a residue having similar physicochemical characteristics, but any substitution is acceptable as long as it does not substantially alter the structural characteristics of the original sequence. Non-limiting examples of conservative substitutions include substitutions between aliphatic group-containing amino acid residues, such as substitutions between Ile, Val, Leu, or Ala; and substitutions between polar residues, such as substitutions between Lys and Arg, Glu and Asp, and Glun and Asn. (c) A protein having an amino acid sequence that has 90% or more, preferably 95% or more, identity with the amino acid sequence of Sequence ID No. 1, and that has the activity to increase intracellular calcium concentration; where, "identity" of the sequence means the percentage of identical amino acid residues to all overlapping amino acid residues in the optimal alignment (preferably, the algorithm may consider introducing gaps into one or both of the sequences for the optimal alignment) when the two amino acid sequences are aligned using a mathematical algorithm known in the art.

[0016] <Subjects> The subjects are mammals, preferably primates, and more preferably humans.

[0017] <Sample> The sample is not particularly limited as long as it is derived from the subject and may contain the SITH-1 protein antibody, SITH-1 mRNA, or latent gene coding region in HHV-6 (preferably HHV-6B) that is the target of detection. Specifically, examples include samples containing cells, tissues or their fragments, and body fluids (blood, serum, saliva, sweat, urine, etc.). When measuring the amount of SITH-1 antibody, body fluid is preferred, and serum is more preferred. When measuring gene polymorphisms in HHV-6, body fluid is preferred, and saliva is more preferred.

[0018] <Cancer> There are no particular restrictions on the types of cancer, but examples include lung cancer, stomach cancer, colon cancer, prostate cancer, esophageal cancer, breast cancer, pancreatic cancer, biliary tract cancer, ovarian cancer, adrenal cancer, uterine cancer, skin cancer, melanoma, neuroblastoma, glioma, mesothelioma, myeloma, lymphoma, and leukemia.

[0019] <Method for predicting cancer growth caused by SITH-1> One embodiment of the present invention is a data acquisition method for predicting the risk of cancer growth caused by SITH-1, which includes detecting antibodies against the SITH-1 protein, which are indicators of the amount of SITH-1 protein produced by human herpesvirus 6 (HHV-6), in a sample taken from a subject, and detecting gene polymorphisms that affect the expression level of SITH-1 protein or mRNA, or the expression level of SITH-1 in HHV-6, wherein a high amount of SITH-1 or antibodies against SITH-1 is determined to indicate a high risk of cancer growth.

[0020] This can also be described as a method for obtaining data to predict the risk of cancer growth, which includes a step of examining the amount of HHV-6 SITH-1 (mRNA or protein), gene polymorphisms affecting SITH-1 expression levels, or the amount of antibodies against SITH-1 in a sample taken from a subject.

[0021] Here, cancer growth includes the onset of cancer, proliferation (increase in cancer tissue), metastasis, and recurrence. In other words, data can be obtained to predict the risk of cancer onset, proliferation, metastasis, and recurrence. The data obtained by this method can be used to assist physicians and other medical professionals in predicting the risk of cancer growth.

[0022] First, we will describe a method for measuring antibodies against the SITH-1 protein. Antibodies against the SITH-1 protein can be measured, for example, by an immunoassay using an antigen containing a partial peptide of the SITH-1 protein.

[0023] Immunological assays using the SITH-1 protein as an antigen include Western blotting, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassays, fluorescence immunoassays, immunoprecipitation, immunoturbidimetry, flow cytometry, fluorescence staining, immunostaining, and lateral flow immunoassays.

[0024] In the case of Western blotting, proteins can be separated based on the molecular weight and isoelectric point of the antigen, allowing for easy determination of the presence or absence of anti-SITH-1 antibodies from the band size. In the case of immunoassay, the antigen immobilized on a support or cultured cells containing the antigen is fixed with an appropriate solvent, and the antibody can be quantified by contacting it with a sample derived from the test subject and binding the anti-SITH-1 antibody. In flow cytometry, the anti-SITH-1 antibody can be quantified by contacting cells overexpressing the antigen with a sample derived from the test subject to bind the anti-SITH-1 antibody, followed by a reaction with a fluorescently labeled anti-human IgG antibody. To measure the amount of antibody, a secondary antibody against the anti-SITH-1 antibody specifically bound to the antigen on the transfer membrane or solid phase can be used to enhance the reaction, and it is preferable that the secondary antibody is labeled.

[0025] The antigens used for detection of the SITH-1 protein in this invention may include the full-length SITH-1 protein, a fusion protein (SITH-CAML) of the full-length SITH-1 protein and the host protein calcium-modulating cyclophilin ligand (CAML), a part of SITH-1, or a part of SITH-CAML. However, in this invention, any antigen recognized by an anti-SITH-1 antibody may be used. When using a partial peptide of the SITH-1 protein, a polypeptide containing an amino acid sequence of 15 or more amino acids showing 90% or more homology to the amino acid sequence shown in SEQ ID NO: 1 is preferred.

[0026] A high level of antibodies against SITH-1 is detected, which is associated with a high risk of cancer growth. Here, cancer growth includes the onset of cancer, proliferation (increase in cancer tissue), metastasis, and recurrence. Therefore, the method of the present invention includes testing for the presence or absence of cancer onset, testing for the risk of cancer progression or metastasis in cancer patients, and testing for the risk of postoperative recurrence in cancer patients (the same applies when prediction is based on mRNA levels, protein levels, and genetic polymorphisms).

[0027] Prediction can be made by comparing the measured amount of SITH-1 with a reference value. When obtaining data for predicting the occurrence (onset) of cancer, if the amount of SITH-1 antibody in the subject is equal to or greater than the amount of SITH-1 antibody measured in cancer patients in advance, a high risk of cancer occurrence (onset) can be predicted. Similarly, if the amount of SITH-1 antibody in the subject is significantly higher than the amount of SITH-1 antibody measured in healthy individuals in advance, or the cutoff value for the amount of SITH-1 antibody that distinguishes healthy individuals from cancer patients, a high risk of cancer occurrence (onset) can be predicted. The cutoff value can be set, for example, by the following methods: (i) setting the cutoff value as the mean value of anti-SITH-1 antibody titers in the healthy group plus twice the standard deviation (mean + 2SD). (ii) creating an ROC curve from the measured values ​​of the healthy group and the cancer patient group, and setting the value at the point where the Youden index is maximum as the cutoff value. The above method for setting cutoff values ​​can be applied not only to anti-SITH-1 antibody titers, but also to measurements of SITH-1 protein levels and SITH-1 mRNA levels, as well as to the assessment of the risk of proliferation, metastasis, or recurrence in cancer patients.

[0028] When obtaining data to predict cancer growth, metastasis, and recurrence in cancer patients, a high risk of cancer growth, metastasis, and / or recurrence can be predicted if the amount of SITH-1 antibody in the subject is equal to or greater than the amount of SITH-1 antibody in cancer patients whose cancer has grown, metastasized, and / or recurred, as measured in advance. A high risk of cancer growth, metastasis, and / or recurrence can also be predicted if the amount of SITH-1 antibody in the subject is significantly higher than the amount of SITH-1 antibody in cancer patients whose cancer has not grown, metastasized, and / or recurred, as measured in advance, or compared to the cutoff value of the amount of SITH-1 antibody that distinguishes patients with cancer growth, metastasis, and / or recurrence from those without. If a prediction of a high risk of cancer growth, metastasis, and / or recurrence is obtained, preventive measures using antibodies such as immune checkpoint inhibitors, or therapeutic measures such as surgical treatment, chemotherapy, and radiotherapy can be taken.

[0029] Next, we will describe a method of testing based on the amount of SITH-1. The amount of SITH-1 may be the amount of SITH-1 protein or the expression level of the SITH-1 gene, i.e., the amount of SITH-1 mRNA. For example, the amount of SITH-1 protein can be measured using an anti-SITH-1 antibody, and the amount of SITH-1 mRNA can be measured using a set of oligonucleotide primers or oligonucleotide probes that hybridize to the SITH-1 gene. When the amount of SITH-1 mRNA or protein is determined to be high, a high risk of cancer growth is predicted. Comparison of the measured amount of SITH-1 mRNA or protein with reference values ​​can be performed in the same manner as described above for the amount of anti-SITH-1 antibody. That is, in the above description, "amount of SITH-1 antibody" can be read as "amount of SITH-1 mRNA or protein".

[0030] Alternatively, instead of measuring the expression levels of the SITH-1 protein or the SITH-1 gene, the method may be performed by detecting gene polymorphisms that increase the expression level of the SITH-1 gene. That is, one embodiment of the present invention is a data acquisition method for predicting the risk of cancer growth, which includes detecting repeat sequence polymorphisms in the repeat element region of the latent gene coding region related to the expression of the SITH-1 gene of HHV-6 in a sample taken from a subject, wherein a high risk of cancer growth is predicted when a gene polymorphism that increases the expression level of the SITH-1 gene is detected.

[0031] Here, a repeating element region refers to a region in the latent gene coding region of HHV-6B that contains multiple repetitions of a specific set of sequences. A repeating sequence polymorphism refers to a genetic polymorphism resulting from a difference in the number of repeats of a base sequence. Containing multiple repetitions means that the same sequence may be repeated consecutively multiple times, or other sequences may be included between consecutive identical sequences. It may also consist of a random distribution of specific set of sequences. Typically, the length per unit of a repeating sequence is 12 bases, but this may be increased or decreased by one or more bases.

[0032] The R1, R2, and R3 regions are examples of repeating element regions in the latent gene coding region of HHV-6B. The detected gene polymorphism may be a gene polymorphism contained in any of the R1, R2, or R3 regions, but it is preferable that it be a gene polymorphism contained in the R1 region, which is located upstream of exon 1 of the SITH-1 protein mRNA and partially overlaps with exon 1 (iScience. 2024 Feb 10;27(3):109203.). The sequence of the R1 region is shown in Sequence ID No. 8.

[0033] The repeating sequence polymorphism in the R1 region is not particularly limited, but examples include polymorphisms that repeat any one or more sequences selected from the R1A to R1U sequences represented by sequence numbers 9 to 29, respectively. It is preferable that the polymorphism repeats the R1A sequence represented by sequence number 9. For example, a high risk of cancer growth can be predicted by using as an indicator that the number of repeats of the R1A sequence is below a certain number, for example, 20 or less, or 17 or less. In this case, the lower limit of the number of repeats of the R1A sequence is not particularly limited, but may be any of 2 or more to 16 or more. For example, a high risk of cancer growth can be predicted when the number of repeats of the R1A sequence is 2 or more and 20 or less, 5 or more and 20 or less, 10 or more and 20 or less, 13 or more and 20 or less, or 16 or more and 20 or less. Alternatively, for example, a high risk of cancer growth can be predicted when the number of repeats of the R1A sequence is 2 or more and 17 or less, 5 or more and 17 or less, 10 or more and 17 or less, 13 or more and 17 or less, 16 or more and 17 or less, or 17. Alternatively, the risk of cancer growth can be predicted by combining the number of repeats in the R1A sequence with the number of repeats in the R1B to R1U sequences, etc.

[0034] For example, when detecting repeat sequence polymorphisms in the R1 region of the SITH-1 gene expression region (repeatable element region) in HHV-6B, it is preferable that the detection reagent includes a primer for amplifying the R1 region and a primer for sequencing the amplified R1 region. Although not particularly limited, when performing nested PCR, the primers for amplifying the R1 region can be those of SEQ ID NOs. 2-3 in the primary PCR and those of SEQ ID NOs. 4-5 in the secondary PCR, and using these primers allows for more efficient amplification of the target DNA. Furthermore, the primers for sequencing the amplified R1 region DNA can be those of SEQ ID NOs. Note that repeat sequence polymorphisms in the R1 region of the SITH-1 gene expression region (repeatable element region) in HHV-6B can also be detected by hybridization methods using DNA chips immobilized with oligonucleotide probes containing complementary sequences.

[0035] Based on the sequencing results of the R1 region of DNA, for example, if the number of repeats in the R1A sequence is below a certain number, such as 20 or less, or 17 or less, a high risk of cancer growth can be predicted. In this case, the lower limit of the number of repeats in the R1A sequence is not particularly limited, but may be any of 2 or more to 16 or more. For example, when the number of repeats in the R1A sequence is 2 or more and 20 or less, 5 or more and 20 or less, 10 or more and 20 or less, 13 or more and 20 or less, or 16 or more and 20 or less, a high risk of cancer growth can be predicted. Alternatively, for example, when the number of repeats in the R1A sequence is 2 or more and 17 or less, 5 or more and 17 or less, 10 or more and 17 or less, 13 or more and 17 or less, 16 or more and 17 or less, or 17, a high risk of cancer growth can be predicted. Furthermore, the risk of cancer growth can be predicted by combining the number of repeats in the R1A sequence with the number of repeats in the R1B to R1U sequences, etc.

[0036] <Kit> This disclosure also provides a kit for testing the risk of cancer growth. This can also be described as a kit for use in risk assessment. Specifically, the kit includes a reagent for detecting an antibody against the SITH-1 protein of HHV-6 or a reagent for detecting repeat sequence polymorphisms in the repeat element region of HHV-6.

[0037] Reagents for detecting antibodies against the HHV-6 SITH-1 protein (anti-SITH-1 antibodies) include antigen polypeptides derived from SITH-1. These may be the full-length SITH-1 protein, a fusion protein (SITH-CAML) of the full-length SITH-1 protein and the full-length host protein calcium-modulating cyclophilin ligand (CAML), or a polypeptide containing a partial sequence of SITH-1. Examples of such partial peptides include polypeptides containing 15 or more amino acids that show 90% or more homology to the amino acid sequence shown in SEQ ID NO: 1.

[0038] In addition to the antigen polypeptide derived from SITH-1, the kit may include other substances necessary for the reaction to detect antibody levels that can be stored in coexistence without adversely affecting the reaction, such as chromogenic reagents, labeled secondary antibodies, and various blocking agents. The kit may also further include anti-SITH-1 antibody-positive serum as a positive control, negative serum as a negative control, buffer, washing solution, instructions for use, etc.

[0039] The antigen polypeptide derived from the antigen protein SITH-1 is preferably provided immobilized on a suitable solid support. Examples of solid supports include various carriers made of insoluble polysaccharides (e.g., agarose, dextran, cellulose, etc.) commonly used in antigen-antibody reactions, synthetic resins, glass, metals, etc., such as microplates, tubes, membranes, columns, beads, sensor chips, etc. Immobilization may be performed by physical adsorption or by chemical bonding, which is commonly used to insolubilize and immobilize proteins.

[0040] Reagents for detecting repeat sequence polymorphisms in the repeat element region of HHV-6 include oligonucleotide primer sets for amplifying the repeat element region, such as the R1 region, oligonucleotide primers for sequencing the amplified R1 region, and DNA chips immobilized with oligonucleotide probes containing complementary sequences of the R1 region. In this case, the kit may also include reagents for gene amplification and instructions for use. This disclosure also relates to the use of SITH-1 derived antigen polypeptides, reagents for detecting anti-SITH-1 antibodies, reagents for detecting SITH-1 mRNA, or reagents for detecting repeat sequence polymorphisms in the repeat element region of HHV-6 in applications for obtaining information to assess the risk of cancer development, growth, metastasis, or recurrence.

[0041] <Method for determining cancer growth induced by SITH-1 using animal models> Animal models capable of evaluating the effect of SITH-1 expression on cancer growth include, for example, non-human mammalian models created by artificially modifying the SITH-1 protein to express in the olfactory bulb.

[0042] As model animals, non-human mammals are preferred, and rodents such as mice and rats are preferred.

[0043] To evaluate the effects of SITH-1 on cancer, specifically its effect on promoting cancer growth and metastasis, it is preferable to express the SITH-1 protein in the olfactory bulb of animal models that have mutations that cause cancer or animal models in which cancer has been transplanted.

[0044] Examples include introducing (administering) the SITH-1 gene into a non-human mammal in a form that allows the gene to be expressed in the olfactory bulb. For example, this includes introducing a gene construct comprising the coding region of the SITH-1 gene operably linked downstream of a promoter that functions in astrocytes or the like. Examples of the promoter include the GFAP promoter described later. The gene transfer can be performed using a known gene transfer technique, and is preferably performed using a vector, more preferably using a viral vector. Examples of the viral vector include retroviral vectors, adenoviral vectors, adeno-associated viral vectors and the like.

[0045] The effect of SITH-1 on cancer growth using a model animal can be determined based on the weight of the cancer and the number of metastatic foci. It can also be determined by the expression of factors that promote cancer growth.

[0046] The model animal can also be used for efficacy evaluation of therapeutic agents for cancer and prophylactic agents for preventing the onset, recurrence and metastasis of cancer. For example, a therapeutic agent for cancer or a candidate substance thereof is administered to the model animal, subsequent cancer proliferation and metastasis are evaluated, and it can be determined that the agent has efficacy when the cancer proliferation and metastasis are suppressed as compared with the case of no administration.

[0047] The model animal highly expresses inflammatory cytokines such as IL-1β, IL-6 and TNFα, sympathetic nerve molecules such as Tyrosine hydroxylase, immune checkpoint molecules such as PD-L1 and PD-1, immunosuppressive factors such as CD80, and cancer growth-promoting factors such as Galectin-9, MMP8, MMP9, VEGFA, TGF-β1 and HAVCR2, and thus the cancer growth by SITH-1 and the effect of a drug may be evaluated based on the amounts of these molecules.

[0048] <Method for preventing cancer growth caused by SITH-1 based on prediction of cancer growth caused by SITH-1> One embodiment of the present invention is the prevention of cancer growth by administering drugs, etc., to subjects who have been determined to be at high risk of cancer growth caused by SITH-1 based on the method for predicting cancer growth caused by SITH-1. For example, after cancer resection surgery, if the amount of SITH-1 or SITH-1 antibody is examined and the amount of SITH-1 or SITH-1 antibody is high (higher than the reference value) and it is determined that there is a high risk of cancer growth (especially recurrence) caused by SITH-1, a policy can be established that it is necessary to prevent or treat cancer growth by administering drugs, etc.

[0049] The agents used in this invention to prevent cancer growth caused by SITH-1 may be any of the following: chemical drugs, herbal medicines and their components, crude drugs and their components, or food components.

[0050] The agents used in this invention to prevent SITH-1-induced cancer growth are not particularly limited as long as they can prevent SITH-1-induced cancer growth. Preferably, the agents are those that can suppress cancer growth when administered to animals expressing SITH-1.

[0051] In the present invention, preferred agents for preventing cancer growth caused by SITH-1 are those that enhance the action of acetylcholine. Examples include agents that inhibit the breakdown of acetylcholine and agents that promote the synthesis of acetylcholine. Agents that promote the cholinergic anti-inflammatory pathway or have parasympathetic nerve stimulating effects, which are the actions of acetylcholine, may also be used. Specifically, examples include cholinesterase inhibitors (such as donepezil, galantamine, and rivastigmine), α7 nicotinic receptor agonists, and herbal medicine components such as Angelica sinensis and Polygala tenuifolia.

[0052] The examples are provided for disclosure purposes only and are not intended to limit the scope of the invention.

[0053] <Subjects> 16 healthy individuals, 16 gastric cancer patients, 16 esophageal cancer patients, 16 lung cancer patients

[0054] <Recombinant Adenovirus> Recombinant adenoviruses were constructed to infect animal models and express SITH-1. The construction of recombinant adenoviruses followed the standard protocol of the Adenovirus Expression Vector Kit (Takara Bio). The GFAP promoter obtained from the pGfa2Lac plasmid containing the human glial fibrillary acidic protein (GFAP) promoter (M. Brenner et al., J Neurosci. 1994, 14(3):1030-1037.) and the SITH-1 gene amplified by PCR were cloned into an adenovirus cosmid vector using a standard method (Ad-GFAP-SITH1). This Ad-GFAP-SITH1 cosmid vector, and a control cosmid vector without the target gene insertion (pAxcwit), were transfected into HEK293 cells. Recombinant adenoviruses were prepared in 293 cells and purified using Adeno-X Virus Purification Kits (Clontech). Furthermore, the titer of the purified virus was determined using Adeno-X rapid titer kits (Clontech).

[0055] <Model Animals> 8-9 week old C57BL / 6 mice or BALB / cAJcl-nu / nu nude mice were anesthetized with isoflurane and 1 x 10⁻¹⁶ mice were used. 9 25 μL of SITH-1 / Adv solution at ifu / mL was administered bilaterally into the nostrils and allowed to aspirate naturally. The mice were then returned to their home cages and reared for one week. As a control, mice administered an empty adenovirus vector (empty / Adv) that did not express any adenovirus were used via nasal administration.

[0056] <Measurement of Anti-SITH-1 Antibody Titer> HEK293T cells were transfected with an expression plasmid encoding a SITH-1 and CAML fusion protein. After 30 hours, the cells were dried and fixed with 100% methanol to be used as the antigen. Antibody measurement was performed using immunofluorescence. Human serum was stained green using an anti-human secondary antibody labeled with Alexa Fluor 488 (Thermo Fisher Scientific), and the CAML portion was reacted with rabbit anti-CAML antibody as the primary antibody, followed by staining red using an anti-rabbit secondary antibody labeled with Alexa Fluor 594 (Thermo Fisher Scientific). Antibody titers were analyzed using BZ-H4C software after fluorescence microscopy imaging with a Keyence BZ-H4XD. The average antibody titer of healthy individuals was set to 1, and the titer was expressed as a ratio of fluorescence intensity.

[0057] <mRNA Expression Measurement> RNA was purified using the RNeasy Mini Kit (QIAGEN) from organs collected from model animals. cDNA was synthesized using the PrimeScript RT reagent Kit (Takara Bio) with the purified RNA as a template. Various gene expressions were analyzed using RT-qPCR with the synthesized cDNA. The 18S rRNA measurement results were used for standardization.

[0058] <Example 1> Analysis of anti-SITH-1 antibody titers in cancer patients Anti-SITH-1 antibody titers were measured using serum obtained from the peripheral blood of 16 healthy individuals, 16 gastric cancer patients, 16 esophageal cancer patients, and 16 lung cancer patients. Blood samples were collected from all cancer patients before treatment. The results showed that the anti-SITH-1 antibody titers in gastric cancer patients, esophageal cancer patients, and lung cancer patients were all significantly higher than those in healthy individuals. The statistical significance levels, as measured by the Mann-Whitney U test, were p=0.0000328 for gastric cancer patients, p=0.0000020 for esophageal cancer patients, and p=0.00027 for lung cancer patients (Figure 1). The above results indicate that SITH-1 expression is closely related to cancer.

[0059] <Example 2> Increase in tumor weight due to SITH-1 Human gastric cancer cells (85As2) were subcutaneously inoculated into nude mice, and the tumor weight was measured at 2 weeks and 8 weeks. One week before tumor harvesting, a comparison was made between administering an empty adenovirus vector into the nasal cavity of mice and administering a SITH-1 expressing adenovirus vector. The results showed that the tumor weight tended to be heavier when the SITH-1 expressing adenovirus vector was administered (Figure 2). The above results indicate that SITH-1 expression tends to promote tumor growth.

[0060] <Example 3> Promotion of metastasis by SITH-1 As a model of cancer metastasis, mouse melanoma cells B16F10 were inoculated into the tail vein of C57BL / 6 mice, and the number of melanomas that engrafted in the lungs after one week was measured. The number of engrafted cells was compared between mice that were inoculated into the nasal cavity with an empty adenovirus vector at the same time as the tail vein inoculation of B16F10, and mice that were inoculated into the nasal cavity with a SITH-1 expressing adenovirus vector. The measurement results showed that mice inoculated into the nasal cavity with a SITH-1 expressing adenovirus vector tended to have a higher number of cancers that engrafted in the lungs (Figure 3). The above results indicate that SITH-1 expression tends to promote tumor metastasis.

[0061] <Example 4> Promotion of inflammation by SITH-1 Local inflammation in the presence of a tumor is known to reduce immunity against the tumor and promote tumor growth. Human gastric cancer cells (85As2) were inoculated into the peritoneal cavity of nude mice, and the expression levels of the inflammatory cytokines IL-1β, IL-6, and TNFα were measured by the expression of their respective mRNAs in mouse cells obtained from the cancer tissue two weeks later. The cases in which an empty adenovirus vector and a SITH-1 expressing adenovirus vector were administered into the nasal cavity of mice simultaneously with the intraperitoneal administration of 85As2 were compared. The measurement results showed that administration of the SITH-1 expressing adenovirus vector resulted in high expression of inflammatory cytokines (Figure 4). The above results indicate that SITH-1 expression promotes inflammation caused by tumors.

[0062] <Example 5> Sympathetic nervous system enhancement by SITH-1 It is known that enhanced sympathetic nervous system response reduces immunity against tumors and promotes tumor growth. SITH-1-expressing adenovirus vectors or empty adenovirus vectors were administered intranasally to C57BL / 6 mice, and the expression levels of Tyrosine hydroxylase and Tropomyosin receptor kinase B, markers of sympathetic nervous system enhancement, were measured by their respective mRNA expression levels in the adrenal cells one week later. The results of administering empty adenovirus vectors and SITH-1-expressing adenovirus vectors into the nasal cavity of mice were compared. The measurement results showed high expression of Tyrosine hydroxylase when administered with SITH-1-expressing adenovirus vectors (Figure 5). The above results indicate that SITH-1 expression promotes the sympathetic nervous system.

[0063] <Example 6> Effect of SITH-1 on immune checkpoints in cancer It is known that the expression of immune checkpoint molecules reduces the host's immune function against cancer and promotes cancer growth. Human gastric cancer cells (85As2) were inoculated into the peritoneal cavity of nude mice, and the expression levels of the immune checkpoint molecules CD274 (also known as PD-L1) and PD-1 in mouse cells obtained from cancer tissue two weeks later were measured by the expression of their respective mRNAs. The results were compared when an empty adenovirus vector and a SITH-1 expressing adenovirus vector were administered into the nasal cavity of mice. The measurement results showed that CD274 (also known as PD-L1) was highly expressed when the SITH-1 expressing adenovirus vector was administered (Figure 6). The above results indicate that SITH-1 expression promotes the expression of the immune checkpoint molecule CD274 (also known as PD-L1).

[0064] <Example 7> Suppression of cancer immunity by SITH-1 It is known that the expression of factors that suppress cancer immunity is enhanced in relation to the factors of cancer growth. Human gastric cancer cells (85As2) were inoculated into the peritoneal cavity of nude mice, and the expression levels of the immunosuppressive factors CD80 and Galectin-9, which are expressed in mouse cells obtained from cancer tissue two weeks later, were measured by the expression of their respective mRNAs. The cases in which an empty adenovirus vector and a SITH-1 expressing adenovirus vector were administered into the nasal cavity of mice were compared. The measurement results showed that CD80 expression was high when the SITH-1 expressing adenovirus vector was administered (Figure 7). The above results indicate that the expression of SITH-1 promotes the expression of the immunosuppressive factor CD80.

[0065] <Example 8> Increase in factors promoting cancer growth by SITH-1 Regarding factors in cancer growth, it is known that the expression of factors promoting cancer growth is enhanced. Human gastric cancer cells (85As2) were inoculated into the peritoneal cavity of nude mice, and the expression levels of cancer growth promoting factors, MMP9, VEGFA, and TGF-β1, in mouse cells obtained from cancer tissue two weeks later were measured by the expression of their respective mRNAs. In addition, in a lung metastasis model in which mouse melanoma cells B16F10 were inoculated into the tail vein of C57BL / 6 mice, the expression levels of MMP8 and HAVCR2 one week after inoculation were measured by the expression of their respective mRNAs. Furthermore, human gastric cancer cells (85As2) were subcutaneously inoculated into the peritoneal cavity of nude mice, and TGF-β1 in mouse cells obtained from cancer tissue two weeks later was measured by the expression of their mRNAs. The case in which an empty adenovirus vector and a SITH-1 expressing adenovirus vector were administered into the nasal cavity of mice were compared. The measurements revealed that administration of a SITH-1-expressing adenovirus vector resulted in high expression of MMP9, VEGFA, MMP8, and HAVCR2. Furthermore, administration of a SITH-1-expressing adenovirus vector also showed changes in TGF-β1 expression levels that promoted cancer growth (Figure 8). These results indicate that SITH-1 expression promotes the expression of molecules that promote tumor growth.

Claims

1. A method for acquiring data to predict the risk of cancer growth, comprising the step of examining the amount of human herpesvirus 6 (HHV-6) SITH-1 or the amount of antibodies against SITH-1 in a sample taken from a subject, wherein a high amount of SITH-1 or antibodies against SITH-1 is determined to indicate a high risk of cancer growth.

2. The method according to claim 1, wherein the testing step includes measuring the amount of HHV-6 antibody against SITH-1.

3. The method according to claim 2, wherein the amount of antibody against SITH-1 is measured using a polypeptide containing an amino acid sequence of 15 or more amino acids that shows 90% or more homology to the amino acid sequence shown in SEQ ID NO:

1.

4. The method according to claim 1, wherein the inspection step includes detecting repeat sequence polymorphisms in the repeat element region of HHV-6.

5. The method according to any one of claims 1 to 4, wherein the growth of cancer by SITH-1 is selected from the group consisting of cancer development, proliferation, metastasis, and recurrence.

6. A kit for testing the risk of cancer growth, comprising a reagent for detecting an antibody against the SITH-1 protein of human herpesvirus 6 (HHV-6) or a reagent for detecting repeat sequence polymorphisms in the repeat element region of HHV-6.

7. The kit according to claim 6, wherein the reagent comprises a polypeptide having an amino acid sequence of 15 or more amino acids that exhibits 90% or more homology to the amino acid sequence shown in SEQ ID NO:

1.

8. The kit according to claim 6, wherein the reagent comprises an oligonucleotide primer set for amplifying the repeating element region, or an oligonucleotide primer for sequencing the region.

9. A kit according to any one of claims 6 to 8 for testing a sample containing blood.

10. The kit according to any one of claims 6 to 8, wherein the growth of cancer by SITH-1 is selected from the group consisting of cancer onset, proliferation, metastasis, and recurrence.

11. A non-human animal model capable of evaluating the effects of SITH-1 on cancer growth, wherein the SITH-1 protein has been artificially modified to express in the olfactory bulb.

12. A non-human model animal according to claim 11, which highly expresses factors that promote cancer growth, inflammatory cytokines, sympathetic nerve molecules, immune checkpoint molecules, and / or immunosuppressive factors.

13. A method for obtaining data for determining a cancer treatment strategy, comprising detecting the risk of cancer growth due to SITH-1 by the method of claim 1, wherein, when such risk is detected, it is determined that treatment or prevention is necessary to suppress cancer growth with a drug that suppresses the effects of SITH-1.

14. The method according to claim 13, wherein the agent that suppresses the effect of SITH-1 is an agent that stimulates acetylcholine receptors, an agent that inhibits the degradation of acetylcholine, or an agent that promotes the synthesis of acetylcholine.

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