Test agent for cancer

Abstract

To provide a cancer inspection technique using an olfactory receptor.SOLUTION: A test agent for a cancer type a, comprising an insect olfactory receptor protein A having reactivity with the cancer type a and an insect olfactory receptor protein C having no reactivity with a cancer including the cancer type a.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a cancer diagnostic agent and the like. [Background technology]

[0002] Cancer is the leading cause of death in Japan, with around one million people contracting the disease each year. With the aging population, these numbers are expected to continue to increase. Early diagnosis leads to early treatment, which is expected to improve prognosis, so efforts are underway to develop simpler cancer risk testing technologies.

[0003] Groups of odorants that characterize specific human diseases and mental states have been identified, and because of their high utility as test markers, the development of various odor sensors targeting these has become active. Because biological olfactory receptors have superior properties in terms of diversity, sensitivity, selectivity, etc. that are not found in conventional odor sensor elements such as semiconductors, there are high expectations for the development of new odor sensors that use olfactory receptors as sensor elements.

[0004] Patent Document 1 discloses the use of cells expressing modified olfactory receptors or lipid bilayer membranes comprising modified olfactory receptors as odor sensors. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2022 / 024902 Summary of the Invention [Problem to be solved by the invention]

[0006] An objective of the present disclosure is to provide a cancer testing technology that utilizes olfactory receptors. [Means for solving the problem]

[0007] The present inventors have conducted research focusing on insect olfactory receptors as olfactory receptors, and have found that there are various insect olfactory receptors that differ in response strength to samples derived from test subjects, samples derived from cancer patients, samples derived from healthy individuals, etc. Based on this finding, the inventors have conducted further research and found that among insect olfactory receptors, there are receptors that are reactive to cancer (i.e., the response strength differs between samples derived from cancer patients and samples derived from healthy individuals), and receptors that are not reactive to cancer (i.e., the response strength is equivalent between samples derived from cancer patients and samples derived from healthy individuals), and have found that the accuracy of cancer testing can be improved by using these receptors in combination. The present disclosure encompasses the following aspects.

[0008] Section 1. Insect olfactory receptor protein A reactive to cancer type a, and cancer type a A test agent for cancer type a, which contains insect olfactory receptor protein C that has no reactivity to cancer.

[0009] Item 2. The test agent according to Item 1, comprising a cell A expressing the insect olfactory receptor protein A and a cell C expressing the insect olfactory receptor protein C.

[0010] Item 3. The test agent according to Item 2, wherein the cell A and the cell C are contained in compartments.

[0011] Item 4. The test agent according to Item 3, wherein the cells A and the cells C are separated into separate compartments. .

[0012] Item 5. The insect olfactory receptor protein A is effective against at least one type of cancer other than cancer type a. Item 5. The testing agent according to any one of Items 1 to 4, which has no reactivity to at least one type of cancer other than cancer type a, or has an opposite reactivity to at least one type of cancer other than cancer type a.

[0013] Item 6. The test agent according to any one of Items 1 to 5, wherein the insect olfactory receptor protein C has no reactivity to multiple types of cancer including cancer type a.

[0014] Item 7. Any of Items 1 to 6, which is used in testing for cancer type a based on the response intensity of the insect olfactory receptor protein A and the insect olfactory receptor protein C to a sample derived from a subject. The test agent described in

[0015] Item 8. The test agent according to any one of Items 2 to 4, comprising a cell chip including a compartment containing the cells.

[0016] Item 9. A combination of compositions for use in contacting a sample derived from a subject suspected of having cancer type a with the insect olfactory receptor protein A and the insect olfactory receptor protein C, the combination comprising a composition comprising an insect olfactory receptor protein A that is reactive to cancer type a and a composition comprising an insect olfactory receptor protein C that is not reactive to cancers including cancer type a.

[0017] Item 10. A cell chip comprising a compartment containing an insect olfactory receptor protein A reactive to cancer type a, and a compartment containing an insect olfactory receptor protein C unreactive to cancers including cancer type a, for use in contacting a sample derived from a subject suspected of having cancer type a with said insect olfactory receptor protein A and said insect olfactory receptor protein C.

[0018] Item 11. Insect olfactory receptor protein A reactive to cancer type a, and cancer type a a sample derived from a subject, and an insect olfactory receptor protein C that is not reactive to cancer and contains the A method for testing for cancer type a, comprising contacting a [Effects of the Invention]

[0019] According to the present disclosure, a cancer testing technology that utilizes insect olfactory receptors can be provided. DETAILED DESCRIPTION OF THE INVENTION

[0020] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0021] As used herein, amino acid mutations are, for example, amino acid substitutions, insertions, additions, or deletions, preferably substitutions, and particularly preferably conservative substitutions.

[0022] In this specification, "conservative substitution" means that an amino acid residue is substituted with an amino acid residue having a similar side chain.For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is considered to be conservative substitution.Other than this, substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine are also considered to be conservative substitutions.

[0023] As used herein, "identity" of amino acid sequences refers to the degree of identity between two or more comparable amino acid sequences. The degree of identity of the amino acid sequences of two amino acid sequences is called the degree of identity of the two amino acid sequences of two amino acid sequences. The higher the match, the higher the identity or similarity of the sequences. The level of identity of amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87:2264 -2268(1990), Karlin S, Altschul SF. “Applications and statistics for multiple "High-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90:5873-7 (1993)). Programs such as BLASTX and BLASTX have been developed. Specific techniques for these analysis methods are publicly known, and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ).

[0024] In this specification, OR indicates odorant receptor, and Aa indicates Aedes aegypti. Ag indicates that it is derived from Anopheles gambiae, Dm indicates that it is derived from Drosophila melanogaster, Bm indicates that it is derived from Bombyx mori, Lm indicates that it is derived from Locusta migratoria, and CI indicates that it is derived from Cimex lectularius.

[0025] In one aspect, the present disclosure provides an insect olfactory receptor protein having reactivity to cancer type a. Insect olfactory receptor protein C lacks reactivity to protein A and cancers, including cancer type a A diagnostic agent for cancer type a (sometimes referred to herein as the "diagnostic agent of the present disclosure") comprising ), which will be explained below.

[0026] The cancer type a being tested is distinguished from other cancer types in terms of tissue and cell origin, etc. Cancer type a is not particularly limited in that respect. Cancer type a is, for example, colon cancer. These include stomach cancer, lung cancer, small cell lung cancer, breast cancer, prostate cancer, malignant lymphoma, pancreatic cancer, liver cancer, biliary tract cancer, esophageal cancer, bladder cancer, renal pelvis and ureter cancer, kidney cancer (renal cell carcinoma), skin cancer, thyroid cancer, ovarian cancer, mesothelioma, leukemia, chronic lymphocytic leukemia, multiple myeloma, melanoma, sarcoma, cervical cancer, endometrial cancer, uterine sarcoma, head and neck cancer, GIST (gastrointestinal stromal tumor), salivary gland cancer, small intestine cancer, brain tumor, and cancer of unknown primary origin. Cancer type a includes cancers of all grades (e.g., mild, moderate, severe) and stages.

[0027] Insect olfactory receptor proteins are membrane proteins with seven transmembrane structures, and they are involved in the detection of insect odors. The amino terminus (hereinafter referred to as the "N terminus") of the olfactory receptor protein acts as a powerful sensor. ) to the carboxyl terminus (hereinafter sometimes referred to as the "C terminus"). N-terminal region (NT), first transmembrane domain (TM1), first extracellular loop (EC1), second transmembrane domain (TM2), first intracellular loop (IC1), third transmembrane domain (TM3), second extracellular loop (EC2), fourth transmembrane domain (TM4), second intracellular loop (IC2), fifth transmembrane domain (TM5), third extracellular loop (EC3), sixth transmembrane domain (TM6), third intracellular loop (IC3) , the seventh transmembrane domain (TM7), and the C-terminal region (CT). In this study, each region was determined by structure prediction (default conditions) using TMpred (K. Hofmann, W. Stoffel, TMbase - a database of membrane spanning protein segments, Biol. Chem. Hoppe-Seyler, 374 (1993), p. 166, https: / / embnet.vital-it.ch / software / TMPRED_form.html).

[0028] Insects from which insect olfactory receptor proteins are derived are preferably Diptera insects such as Culicidae and Drosophilidae; Lepidoptera insects such as Bombycidae; Hymenoptera insects such as Apidae; Orthoptera insects such as Acrididae; Hemiptera insects such as Cimex; and the like, and more preferably Culicidae and Siberian dinosaurs. Examples of insects that belong to the family Culicidae include Diptera such as Melanogonia; Orthoptera such as Acrididae; and Hemiptera such as Cimex. Examples of insects that belong to the family Culicidae include Anopheles gambiae. , Aedes aegypti, Culex quinquefasciatus, etc. Examples of insects of the Drosophilidae family include Drosophila melanogaster, Drosophila pseudoobscura, Examples of insects in the Bombyx family include the black fruit fly (Drosophila virillis). Examples include the silkworm moth (Bombyx mori), the mulberry silkworm (Bombyx mandarina), and the fig umbrella moth. Examples of insects of the Apidae family include the European honeybee (Apis mellifera), the oriental honeybee (Apis florea), the giant honeybee (Apis dorsata), and the European bumblebee (Bombus terrestris). Examples of insects of the Acrididae family include the migratory locust (Locusta migratoria), and examples of insects of the Cimex family include the bedbug (Cimex lectularius).

[0029] Various wild-type insect olfactory receptor proteins are known, or can be easily identified by a sequence identity search based on known sequences.

[0030] Insect olfactory receptor protein A has reactivity to cancer type a (i.e., cancer type a The reactivity may include an amino acid mutation relative to the wild-type amino acid sequence, as long as the reactivity differs between samples derived from patients and samples derived from healthy individuals. The determination may be made in accordance with or pursuant to the law.

[0031] Insect olfactory receptor protein A has a wild-type amino acid sequence, For example, it may comprise an amino acid sequence having an identity of 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 98% or more, and particularly preferably 99% or more.

[0032] The insect olfactory receptor protein A may be one type alone or a combination of two or more types. From the viewpoint of accuracy in determining the type of cancer, it is preferable to use a combination of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more types. Alternatively, the number of combinations may be 30 or less, 25 or less, 20 or less, 15 or less, 12 or less, or 11 or less.

[0033] Insect olfactory receptor protein A, e.g. The response intensity to samples derived from cancer type a patients is greater than the response intensity to samples derived from healthy subjects. Receptors that tend to be high (receptor AX), or The response intensity to samples derived from cancer type a patients is greater than the response intensity to samples derived from healthy subjects. receptors that tend to be low (receptor AY), It can be.

[0034] The insect olfactory receptor protein A is preferably used for at least one type of cancer other than cancer type a. or at least one type of cancer other than cancer type a (preferably at least two The receptor (insect olfactory receptor protein A') has the opposite reactivity to the olfactory receptor (insect species).

[0035] Insect olfactory receptor protein A', e.g. The response intensity to samples derived from cancer type a patients is greater than the response intensity to samples derived from healthy subjects. The incidence of cancer tends to be high, and at least one type (preferably at least two types) of cancer other than cancer type a A receptor (receptor A'X1) whose response strength to patient-derived samples tends to be equivalent to that to healthy subject-derived samples. The response intensity to samples derived from cancer type a patients is greater than the response intensity to samples derived from healthy subjects. The incidence of cancer tends to be high, and at least one type (preferably at least two types) of cancer other than cancer type a A receptor (receptor A'X2) whose response intensity to patient-derived samples tends to be lower than that to healthy subject-derived samples (i.e., whose reactivity is opposite to that of cancer type a), The response intensity to samples derived from cancer type a patients is greater than the response intensity to samples derived from healthy subjects. The incidence of cancer tends to be low, and at least one type (preferably at least two types) of cancer other than cancer type a A receptor (receptor A'Y1) whose response intensity to a patient-derived sample tends to be equivalent to that to a healthy subject-derived sample, or The response intensity to samples derived from cancer type a patients is greater than the response intensity to samples derived from healthy subjects. The incidence of cancer tends to be low, and at least one type (preferably at least two types) of cancer other than cancer type a The receptor (receptor A'Y2) tends to have a higher response intensity to samples derived from patients than to samples derived from healthy individuals (i.e., the reactivity is opposite to that of cancer type a).

[0036] Insect olfactory receptor protein C has no reactivity to cancers, including cancer type a (i.e., the response intensity is equivalent between samples from cancer patients and healthy subjects, including cancer type a). The reactivity can be determined, for example, according to or in accordance with the method of Test Example 1 described below.

[0037] Insect olfactory receptor protein C has a wild-type amino acid sequence, For example, it may comprise an amino acid sequence having an identity of 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 98% or more, and particularly preferably 99% or more.

[0038] The insect olfactory receptor protein C may be one type alone or a combination of two or more types. From the viewpoint of accuracy in determining the type of cancer, the combination may preferably be 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more types, or may alternatively be 30 or less, 25 or less, 20 or less, 15 or less, 12 or less, or 10 or less types.

[0039] Insect olfactory receptor protein C preferably has no reactivity to multiple types of cancer (preferably at least two types, more preferably at least three types) including cancer type a. As long as insect olfactory receptor protein A has reactivity to cancer type a, and as long as insect olfactory receptor protein C has reactivity to cancer type a, other amino acid sequences, for example, proteins or peptides such as protein tags, fluorescent proteins, luminescent proteins, and signal sequences, may be added. Examples of protein tags include biotin, His tags, FLAG tags, Halo tags, MBP tags, HA tags, Myc tags, V5 tags, and PA tags. Examples include:

[0040] The insect olfactory receptor protein A may be chemically modified as long as it has reactivity to cancer type a, and the insect olfactory receptor protein C may be chemically modified as long as it has no reactivity to cancer type a.

[0041] Insect olfactory receptor proteins A and C have a carboxyl group (-COOH) at the C-terminus, Rate (-COO - ), amide (-CONH2) or ester (-COOR).

[0042] Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1-6 Alkyl groups; for example, C groups such as cyclopentyl and cyclohexyl 3-8 Cycloalkyl groups such as phenyl and α-naphthyl 6-12 aryl groups; For example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl group; α-naphthylmethyl α-naphthyl-C 1-2 C such as alkyl group 7-14 Aralkyl group; Pivaloyloxymethyl A methyl group is used.

[0043] Insect olfactory receptor proteins A and C have carboxyl groups (or carboxyl groups) other than the C-terminus. The C-terminal ester may be amidated or esterified. In this case, the ester may be, for example, the above-mentioned C-terminal ester.

[0044] Furthermore, insect olfactory receptor proteins A and C retain the amino group of the N-terminal amino acid residue. Protective groups (e.g., formyl, acetyl, etc.) 1-6 C such as alkanoyl 1-6 acyl group, etc.), and the N-terminal glutamine residue that can be generated by cleavage in vivo is Substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by suitable protecting groups (e.g., C groups such as formyl group, acetyl group, etc.). 1-6 C such as alkanoyl group 1-6 Also included are those protected by an acyl group or other suitable glycan, or conjugated proteins such as glycoproteins to which sugar chains are attached.

[0045] The insect olfactory receptor proteins A and C may be in the form of a salt with an acid or a base. The salt is not particularly limited, and either an acid salt or a basic salt can be used. Examples of acid salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0046] The insect olfactory receptor proteins A and C may be in the form of a solvate. The solvent is not particularly limited, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0047] In the test agent of the present disclosure, the insect olfactory receptor protein (the insect olfactory receptor protein of the present disclosure) is not particularly limited as long as it is contained in a form capable of exhibiting odorant response activity. Odorant response activity refers to the property in which an insect olfactory receptor protein recognizes an odorant, and the olfactory receptor complex formed by the olfactory receptor and the olfactory receptor co-receptor is activated, thereby exhibiting ion channel activity. For this reason, in the test agent of the present disclosure, it is generally preferable that the insect olfactory receptor protein of the present disclosure be in a form retained in a membrane.

[0048] The membrane is preferably a lipid membrane. A lipid membrane refers to a membranous body composed of lipids. The lipid membrane may form a planar membrane or a sac-shaped membrane (small vesicle) such as a vesicle (liposome) or a micelle. The lipid membrane may be composed of a single lipid membrane layer or two or more lipid membrane layers, and is preferably a lipid bilayer membrane. When the lipid membrane structure is a cell, the lipid membrane can also be referred to as, for example, a cell membrane.

[0049] The test agent of the present disclosure preferably contains cells expressing the insect olfactory receptor protein of the present disclosure (cells of the present disclosure). The test agent of the present disclosure more preferably contains cells A expressing insect olfactory receptor protein A and cells C expressing insect olfactory receptor protein C.

[0050] The cells are not particularly limited. From the viewpoint of suitability for detecting odorants, animal cells such as insect cells and mammalian cells are preferred. In one embodiment, insect cells are preferred.

[0051] Examples of insect cells include Sf cells, MG1 cells, and High Five cells. TM Cells, BmN cells, etc. Examples of Sf cells include Sf9 cells (ATCC CRL1711) and Sf21 cells. Among insect cells, cells derived from insects of the family Arctiidae are particularly preferred.

[0052] The cells derived from an insect of the family Arctiidae are primary cultured cells or established cell lines of cells derived from an insect of the family Arctiidae that constitute the living body, and are not particularly limited as long as they are so.

[0053] Examples of the Arctiidae family include the Arctiinae subfamily, the Lithosiinae subfamily, and the Syntominae subfamily, and among these, the Arctiinae subfamily is preferred. As the Arctiinae subfamily, the Spilosoma (the genus of the fall moth) is preferred. The genus Spilactia is not particularly limited, but Spilactia imparilis is particularly preferred.

[0054] Cells derived from insects of the family Arctiidae can be obtained from known biobanks, or can be obtained by collecting and culturing live insects of the family Arctiidae according to or in accordance with known methods, and can also be established as lines if necessary.

[0055] Examples of cells derived from Mulberry butterfly include FFPRI-SpIm-2AM-SF cells (MAFF number: 275052) and FFPRI-SpIm-2AM-IPL411 cells (MAFF number: 275053) from the National Agriculture and Food Research Organization Genebank.

[0056] Mammalian cells include COS7 cells, CHO cells, HEK293 cells, HEK293FT cells, and Hela cells. , PC12 cells, N1E-115 cells, SH-SY5Y cells, etc.

[0057] The cells of the present disclosure preferably contain an exogenous polynucleotide comprising a coding sequence for an insect olfactory receptor protein of the present disclosure. The exogenous polynucleotide is defined as a polynucleotide that is not part of the genomic DNA of the cell (especially the genomic DNA of the cell). A polynucleotide containing a base sequence that is not derived from a specific DNA (e.g., chromosomal genomic DNA), There are no particular restrictions on the type.

[0058] The exogenous polynucleotide preferably contains a coding sequence for an insect olfactory receptor co-receptor. The insect olfactory receptor co-receptor is a membrane protein having a seven-transmembrane structure, similar to the olfactory receptor. The olfactory receptor complex, which is a heterocomplex consisting of an olfactory receptor and an olfactory receptor co-receptor, has ion channel activity that is activated by odorants, and when activated, it transports sodium ions (Na + ),mosquito Calcium ions (Ca 2+ The olfactory receptor co-receptor of insects may be the olfactory receptor co-receptor described in Japanese Patent No. 6875815.

[0059] The exogenous polynucleotide preferably contains a coding sequence for a protein that emits fluorescence or luminescence in response to ions (such as calcium ions) that flow into the cell when the insect olfactory receptor protein responds. Examples of such proteins include aequorin, Yellow Cameleon Alternatively, the cells of the present disclosure may be modified by expressing calcium ion-dependent fluorescent dyes ( It is preferred to include an ion-dependent fluorescent dye such as Fura-2, Fluo-3, Fluo-4, etc.

[0060] Coding sequences such as insect olfactory receptor protein coding sequences, insect olfactory receptor co-receptor coding sequences, and fluorescent or luminescent protein coding sequences are preferably contained in exogenous polynucleotides in the form of expression cassettes (promoters and coding sequences placed under the control of the promoters). The promoters of the expression cassettes can be shared among multiple coding sequences.

[0061] In the test agent of the present disclosure, the insect olfactory receptor protein of the present disclosure / the cell of the present disclosure are preferably contained in a compartment.

[0062] The compartments are the areas where insect olfactory receptor proteins / cells are located. There are no particular limitations on the form as long as it allows the placement of insect olfactory receptor proteins / cells. From the viewpoints of desiccation resistance of insect olfactory receptor proteins / cells, retention of insect olfactory receptor proteins / cells, production efficiency, or odorant detectability, it is preferable that the compartments be in a form separated by walls (for example, in the form of a well and / or separated from the surrounding area by an outer wall). Furthermore, by making the surface outside the compartments a surface on which cells cannot be placed (for example, adhere) (or on which adhesion is significantly reduced), it is also possible to distinguish the compartments from those on which cells can be placed (for example, adhere).

[0063] The compartment can be, for example, a compartment within a cell chip that comprises cells and a device for holding the cells (such as a dish or well plate).

[0064] The material of the compartment is not particularly limited as long as it can hold cells, and can be, for example, resin, metal, etc.

[0065] The area of ​​the compartment is preferably 0.5 to 100 mm from the viewpoint of detection sensitivity or production efficiency. 2 , More preferably, 1 to 50 mm 2 , and more preferably 1.5 to 40 mm 2 , and even more preferably 2 to 40 mm 2 In a preferred embodiment of the present disclosure, the area is 50 mm 2 Below, 35mm 2 or less, or 15 mm 2 The following is the result.

[0066] The number of compartments is preferably 1 to 2000, more preferably 1 to 2000, from the viewpoint of detection sensitivity or production efficiency. The number is preferably 4 to 1600, and more preferably 8 to 400. In this case, the number is 10 or more, 20 or more, or 50 or more.

[0067] From the viewpoint of detection sensitivity, a compartment usually contains multiple cells. 2 ) The number of cells per unit area is, for example, 50 to 20,000 cells / mm 2 From the viewpoint of detection sensitivity, cell viability, etc., it is preferably 100 to 15,000 cells / mm 2 , more preferably 100 to 10,000 cells / mm 2 , and more preferably 200 to 10,000 cells / mm 2 , and even more preferably 500 to 7000 cells / mm 2 , and particularly preferably 1000 to 5000 cells / mm 2 is.

[0068] The test agent of the present disclosure is a test agent for detecting two or more insect olfactory receptor proteins of the present disclosure that are different from each other. The above (preferably 3 to 25 types, more preferably 3 to 12 types, and even more preferably 3 to 10 types) In this case, the two or more types of cells are preferably separated into separate compartments. (e.g., cell A is placed in one compartment (compartment A) and cell C is placed in another compartment (compartment B). It is preferable that the ion exchange layer is arranged as shown in FIG. C).

[0069] In addition, the test agent of the present disclosure may contain two or more different insect olfactory receptor proteins of the present disclosure. Proteins are separated into separate compartments (e.g., insect olfactory receptor protein A) The insect olfactory receptor protein C is placed in another compartment (compartment C). It is preferable to

[0070] The test agent of the present disclosure can be used to test for cancer type a based on the response intensity of the insect olfactory receptor protein of the present disclosure to a sample derived from a subject.

[0071] The subject-derived sample is not particularly limited as long as it is a body fluid of the subject or a sample derived therefrom (body fluid-derived sample).

[0072] The subject is not particularly limited and may be, for example, a variety of mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, horses, cows, and pigs, with humans being preferred.

[0073] The condition of the subject is not particularly limited, and examples include a subject suspected of having cancer type a, a subject unknown as to whether or not the subject has cancer type a, a subject determined to have cancer type a, a subject determined not to have cancer type a, and a subject for which information regarding cancer type a is unknown. When the specimen is a human, any individual can be the subject, regardless of their past medical history, including individuals considered to be healthy. For example, subjects can include those undergoing health checkups or comprehensive medical examinations, and those undergoing cancer testing using interview methods, questionnaire tests, score methods, pathological diagnostic methods, imaging diagnostic methods, blood biochemistry tests, etc.

[0074] Examples of body fluids include urine, blood, saliva, sweat, tears, tissue fluid, synovial fluid, follicular fluid, cerebrospinal fluid, semen, milk, vaginal fluid, etc. Among these body fluids, urine is preferred from the viewpoint of ease of collection.

[0075] Body fluids can be collected from living organisms according to or in accordance with known methods. The collected body fluids can be used immediately for preparing samples of the present disclosure, or can be stored (e.g., refrigerated or frozen) before being used for preparing samples of the present disclosure.

[0076] A body fluid-derived sample is not a body fluid itself, but a sample obtained by subjecting a body fluid to some treatment that affects the component composition within the body fluid, and is not particularly limited insofar as such treatment can include various treatments such as dilution with a solvent or solution, purification, etc. Purification methods include, for example, treatments to remove salts, proteins, etc. (e.g., enzyme treatment, chromatography column purification, centrifugation, etc.).

[0077] After contacting a sample derived from a subject with the insect olfactory receptor protein of the present disclosure, the response strength of the insect olfactory receptor protein of the present disclosure can be measured to obtain the response strength to the sample derived from a subject.

[0078] The manner of contact is not particularly limited as long as the components in the sample derived from the subject can come into contact with the insect olfactory receptor protein of the present disclosure. For example, a membrane that retains the insect olfactory receptor protein of the present disclosure (membrane A that retains insect olfactory receptor protein A, or membrane C that retains insect olfactory receptor protein C) or a cell that expresses the insect olfactory receptor protein of the present disclosure (insect olfactory receptor protein A) or a cell C expressing an insect olfactory receptor protein C) (if necessary). The specimen-derived sample can be brought into contact with the insect olfactory receptor protein of the present disclosure by adding the sample to each of the compartments (compartment A containing insect olfactory receptor protein A and compartment C containing insect olfactory receptor protein C, which may further contain a liquid if necessary).

[0079] The method for measuring the response intensity is not particularly limited as long as it is a method that can detect the ion channel activity of the insect olfactory receptor protein of the present disclosure. One example is a method in which the amount of cations that flow into the membrane due to ion channel activity is converted into a signal (e.g., luminescence, fluorescence, etc.) amount and the signal amount is measured.

[0080] The type of response intensity used as an index for cancer type a testing is not particularly limited, and may be, for example, the maximum signal amount. The signal amount may be, for example, the maximum value, the integrated value of the signal amount, or the rate of increase of the signal amount, and more specifically, the signal amount may be, for example, the maximum value, the integrated value, and / or the rate of increase of the signal amount within a predetermined time or after a predetermined time has elapsed after contact with the sample derived from the subject.

[0081] The response strength of insect olfactory receptor protein A is used as an index (for example, the response strength is The type of cancer can be determined by the above-mentioned (the index is whether the value is above or below the threshold value). More specifically, for example, determining that the subject has cancer type a when the response intensity of receptor AX to a sample derived from the subject is equal to or greater than a cutoff value; and / or determining that the subject has cancer type a when the response intensity of receptor AY to a sample derived from the subject is equal to or lower than a cutoff value; can be done.

[0082] Insect olfactory receptor protein C corrects the response strength derived from the receptor for individual differences between samples. This can be used to improve the accuracy of cancer type a determination using insect olfactory receptor protein A.

[0083] The response strength of insect olfactory receptor protein A' is used as an index (for example, the response strength is By using the level above or below the cut-off value as an indicator, cancer type a can be determined with higher accuracy even when there are multiple possible types of cancer.

[0084] The cutoff value is determined by various indices of accuracy for cancer type a determination (e.g., Accuracy, F1-score, Matthews , Precision, ROC-AUC, Recall, Specificity, etc.) should be appropriately set by a person skilled in the art. The cutoff value may be a value set each time depending on race, age, etc., or a value set in advance. The cutoff value may be, for example, a value at which it is determined that a person is not affected by cancer type a. For samples collected from subjects who have been diagnosed with cancer type a or subjects who have been diagnosed with cancer type a The value may be based on the maximum, average, percentile, or minimum of the response strengths.

[0085] In one embodiment, the cutoff value is a value used as a standard to determine the presence or absence of cancer type a. For example, the cutoff value is a value that shows a high positive rate in individuals with cancer type a and a high negative rate in individuals without cancer type a. It can be set as follows.

[0086] Such a cutoff value does not have a specific value, but varies depending on the subject population used when setting the cutoff value.

[0087] Even when the same subject is used, the measured values ​​may differ depending on the analytical method used, so the cutoff value is set according to the analytical method used.

[0088] By using the test agent of the present disclosure, an index for cancer type a testing can be provided, This can assist in cancer type a testing. Also, by using a cutoff value, It is possible to determine whether or not cancer type A is present.

[0089] The testing agent of the present disclosure may be in the form of a composition. The composition may contain other components as needed. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, etc.

[0090] The test agent of the present disclosure can be in the form of a kit. It may also include instruments, reagents, instructions, etc. that can be used in the practice.

[0091] The insect olfactory receptor proteins of the present disclosure can be screened by testing the reactivity of the test insect olfactory receptor protein against cancer.

[0092] Specifically, for example, contacting a test insect olfactory receptor protein with a sample derived from a subject having cancer and measuring the response intensity P; contacting the test insect olfactory receptor protein with a sample derived from a healthy subject without cancer and measuring the response intensity Q; The screening can be carried out by a method comprising:

[0093] The response intensity P may be, for example, a signal from a plurality of subjects (subject P1, subject P2, subject P3, . . . ). Using each sample, multiple response intensities (response intensity P1, response intensity P2, response intensity P3, ...) can be obtained, and a value (e.g., an average value such as a geometric mean) can be obtained by arithmetic processing using these multiple response intensities. The same applies to the response intensity Q.

[0094] More specifically, in this method, determining that the test insect olfactory receptor protein has reactivity to cancer when the response intensity P and the response intensity Q are different; and / or determining that the test insect olfactory receptor protein does not have reactivity to cancer when the response intensity P and the response intensity Q are comparable; can be done.

[0095] The above judgment is performed by using a subject having a certain cancer (cancer type a) as the subject from which the sample for measuring the response intensity P is derived, and, if necessary, a subject having any cancer other than cancer type a. By doing so, insect olfactory receptor proteins A and C can be screened.

[0096] The description of the test agent of the present disclosure is incorporated herein for the embodiment of the screening method.

[0097] The determination of whether the response intensity P and the response intensity Q are different is based on a certain criterion, as in Test Example 1 described later. In other words, if the difference between the response intensity P and the response intensity Q is greater than (or equal to or greater than) the reference value, it can be determined that there is reactivity to cancer, and if it is equal to (or less than) the reference value, it can be determined that there is no reactivity to cancer.

[0098] The present disclosure, in one aspect, comprises: A combination of compositions comprising an insect olfactory receptor protein A and an insect olfactory receptor protein C, the combination being used to contact a sample derived from a subject suspected of having cancer type a with the insect olfactory receptor protein A and the insect olfactory receptor protein C; a cell chip comprising a compartment containing an insect olfactory receptor protein A and a compartment containing an insect olfactory receptor protein C, the cell chip being used for contacting a sample derived from a subject suspected of having cancer type a with the insect olfactory receptor protein A and the insect olfactory receptor protein C; A method for testing for cancer type a, which comprises contacting insect olfactory receptor protein A and insect olfactory receptor protein C with a sample derived from a subject; For these aspects, the description of the test agent of the present disclosure is incorporated herein by reference. [Example]

[0099] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0100] Test Example 1. Screening of insect olfactory receptors with cancer reactivity 1 An expression plasmid containing the GFP-Aequorin coding sequence, the insect olfactory receptor co-receptor Orco coding sequence, and the insect olfactory receptor OR coding sequence was prepared according to the method described in Japanese Patent No. 6875815. The obtained expression plasmid was introduced into cells, seeded on a 384-well plate, and the luminescence intensity was measured. The luminescence intensity was measured by adding the test sample to FDSS / μCELL (Hamamatsu Photonics). The test sample was a cancer of a certain tissue (cancer type X). Twenty-five urine samples from patients and 25 urine samples from healthy individuals were used.

[0101] First, the luminescence intensity of each test sample in each OR was compared between the urine of cancer type X patients and healthy subjects. The geometric mean was calculated for 100 seconds from the start of measurement (the test sample was added 10 seconds after the start of measurement). In this case, the maximum luminescence intensity in the case of a patient with cancer type X and / or the maximum luminescence intensity in the case of a healthy subject is The measured value is 2000 or more, and the maximum luminescence intensity in patients with cancer type X is different from the maximum in healthy individuals. Receptors with a difference of more than 1000 in luminescence intensity were selected. In this way, insect olfactory receptors with reactivity to cancer were discovered. Among these insect olfactory receptors, those with reactivity to cancer type X were selected. The results included receptors with higher luminescence intensity in the patients with glaucoma and receptors with higher luminescence intensity in the healthy controls.

[0102] Next, the maximum luminescence intensity in the case of a patient with cancer type X and / or the maximum luminescence intensity in the case of a healthy subject The measured value is 2000 or more, and the maximum luminescence intensity in patients with cancer type X is different from the maximum in healthy individuals. Receptors that were judged to have no difference in luminescence intensity were selected. In this way, insect olfactory receptors that are not reactive to cancer were discovered.

[0103] The response strength of insect olfactory receptors that are not reactive to cancer can be used to correct for individual differences between specimens, thereby improving the accuracy of cancer detection using insect olfactory receptors that are reactive to cancer.

[0104] Test Example 2: Screening for insect olfactory receptors with cancer reactivity 2 For cancers (cancer types Y and Z) derived from tissues different from cancer type X, the luminescence intensity was measured in the same manner as in Test Example 1, and it was determined whether the luminescence intensity was higher or equal between the urine of cancer patients and the urine of healthy subjects. The results are shown in Table 1.

[0105] [Table 1]

[0106] Receptors in groups a, b, and d in Table 1 are reactive to a specific cancer (cancer type X), but not to other cancer types (a and b), or the direction of the reaction is opposite (d). Therefore, the strength of the response derived from the receptor should be used as an index (for example, if the strength of the response is By using the level above or below the threshold as an indicator, it is possible to determine the specific cancer with higher accuracy even when there are multiple possible types of cancer.

[0107] The receptors in group c in Table 1 have reactivity to multiple types of cancer, including a specific cancer (cancer type X). Therefore, the response strength derived from this receptor and the responses derived from receptors in groups a, b, and d are (e.g., if the response intensity from group c receptors is below the cutoff, and the response strength from receptors of groups a, b, and d is above or below the cutoff. By using the receptors of group c as an index, it is possible to diagnose the specific cancer with higher accuracy. By examining receptors of groups a, b, and d in detail, unnecessary tests for identifying cancer types can be avoided. It is also possible to reduce

[0108] In addition, receptors in group c in Table 1 have reactivity to multiple types of cancer. By using the receptor-derived response strength as an indicator (using the response strength being equal to or greater than a cutoff value as an indicator), it is possible to more thoroughly determine whether or not a patient has cancer, even when there are multiple possible types of cancer.

[0109] Test Example 3: Determining Cancer Type X The luminescence intensity of the receptor found in Test Example 1 (measured in Test Example 1) was The geometric mean of the time change in the luminescence intensity of the subject's urine is used to determine whether the specimen is cancer type X. The specific method is as follows:

[0110] First, for the test sample to be evaluated, the luminescence intensity of each receptor selected in Test Example 1 was measured. The time change was measured. Next, the absolute value of the difference between the geometric mean of the luminescence intensity of the urine of the cancer type X patient and the geometric mean of the luminescence intensity of the urine of the healthy subject measured in Test Example 1 at each time and the luminescence intensity of the test sample was calculated, and the cumulative sum for each was calculated. For urine, the smaller cumulative sum was used as the judgment result. As a result, there were three receptors with a correct answer rate of 0.80 or higher.

[0111] Next, we investigated the insect olfactory receptors (correction receptors) that are not reactive to cancer, which were found in Test Example 1. The luminescence intensity (measured in Test Example 1) of the insect olfactory receptor (receptor for evaluation) having reactivity to cancer found in Test Example 1 was used to correct for individual differences. ) was used to determine whether the specimen was cancer type X. The specific method is as follows: .

[0112] First, the time-dependent changes in luminescence intensity in the test sample to be evaluated were measured in the evaluation receptor and the correction receptor.

[0113] Next, for each correction receptor (correction receptors i1, i2, i3, i4, i5, i6), the geometric mean of the luminescence intensity of urine from cancer type X patients and the geometric mean of the luminescence intensity of urine from healthy subjects measured in Test Example 1 at each time point were calculated, and the two geometric means were further geometrically averaged to obtain a reference time change in luminescence intensity. For each correction receptor, the AUC of the time change in luminescence intensity of the test sample and the reference time change were calculated. The AUC of the time change in luminescence intensity was calculated, and the ratio of the two AUCs (ratio α i1 , ratio α i2 , ratio α i3 , ratio α i4 , ratio α i5 , ratio α i6 ,) were calculated. All ratios (ratio α i1 , ratio α i2 , ratio αi3 , ratio α i4 , ratio α i5 , ratio α i6 The geometric mean of ( , ) was defined as the correction coefficient α.

[0114] Next, for each receptor for determination, the luminescence intensity of the test sample at each time was multiplied by the correction coefficient α to obtain the corrected luminescence intensity. The geometric mean of the measured luminescence intensity of urine from patients with cancer type X and that of healthy individuals. The absolute value of the difference from the corrected luminescence intensity of the test sample was calculated, and the cumulative sum for each was calculated. The smaller cumulative sum for the urine of the cancer type X patient and the urine of the healthy subject was used as the judgment result. As a result, there were six receptors with a correct answer rate of 0.80 or higher.

[0115] From the above, it was found that the insect olfactory receptors that are not reactive to cancer, which were found in Test Example 1, can be used in individuals. It was shown that using this for difference correction improves the accuracy of determining cancer type X.

[0116] Next, we selected the top 1 to 5 receptor combinations, the top 1 to 8 receptor combinations, the top 1 to 9 receptor combinations, and the top 1 to 10 receptor combinations, which have high accuracy rates after correcting for individual differences. In this way, it was determined whether the specimen was cancer type X. Specifically, using each determination receptor, After correcting for individual differences in the same manner as above, it is determined whether the specimen is cancer type X, and the above combination The majority vote of the results of each receptor was used to determine whether the cancer was type X or not. is shown in Table 2.

[0117] [Table 2]

[0118] From the above, it was shown that the accuracy of determining cancer type X can be improved by combining receptors. was done.

Claims

1. Insect olfactory receptor protein A with reactivity to cancer type a, and cancers including cancer type a A test agent for cancer type a containing insect olfactory receptor protein C that has no reactivity to

2. Cell A expressing the insect olfactory receptor protein A, and cell C expressing the insect olfactory receptor protein C. The test agent according to claim 1, comprising a cell C expressing the gene.

3. The test agent according to claim 2 , wherein the cell A and the cell C are contained in compartments.

4. The test agent according to claim 3 , wherein the cells A and the cells C are separated into separate compartments.

5. The insect olfactory receptor protein A is reactive to at least one type of cancer other than cancer type a. The test agent according to claim 1, wherein the test agent has no reactivity to at least one type of cancer other than cancer type a, or has an opposite reactivity to at least one type of cancer other than cancer type a.

6. The test agent according to claim 1, wherein the insect olfactory receptor protein C has no reactivity to multiple types of cancer including cancer type a.

7. The test agent according to claim 1, which is used to test for cancer type a based on the response intensity of the insect olfactory receptor protein A and the insect olfactory receptor protein C to a sample derived from a subject.

8. The test agent according to claim 2 , comprising a cell chip containing a compartment containing the cells.

9. A composition comprising an insect olfactory receptor protein A having reactivity to cancer type a, and a The composition includes an insect olfactory receptor protein C that has no reactivity to cancer, A combination of compositions for use in contacting a sample derived from a subject suspected of having cancer type a with the insect olfactory receptor protein A and the insect olfactory receptor protein C.

10. A compartment containing insect olfactory receptor protein A reactive to cancer type a, and cancer type a a compartment containing insect olfactory receptor protein C that has no reactivity to the cancer, A cell chip for use in contacting a sample derived from a subject suspected of being species a with the insect olfactory receptor protein A and the insect olfactory receptor protein C.

11. Insect olfactory receptor protein A with reactivity to cancer type a, and cancers including cancer type a The sample derived from the test subject is contacted with an insect olfactory receptor protein C that has no reactivity to A method for testing for cancer type A, including:

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