Cancer diagnostic agent

Abstract

The present invention addresses the problem of providing a cancer diagnosis technology using an olfactory receptor. Said problem is solved by a diagnostic agent for a cancer type a, the diagnostic agent comprising: an insect olfactory receptor protein A' that exhibits a reaction to the cancer type a and does not exhibit a reaction to at least one type of cancer other than the cancer type a, or that exhibits a reverse reaction to at least one type of cancer other than the cancer type a; and an insect olfactory receptor protein B that exhibits reactions to a plurality of types of cancer including the cancer type a and in which the directions of said reactions are the same.

Description

Cancer testing agents

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

[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.

[0005] International Publication No. 2022 / 024902

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

[0007] The present inventors conducted research focusing on insect olfactory receptors as olfactory receptors and discovered that various insect olfactory receptors exist, with different response intensities to samples derived from test subjects, cancer patients, and healthy individuals. Based on this finding, further research led to the discovery that some insect olfactory receptors are cancer-reactive (i.e., their response intensities differ between samples derived from cancer patients and healthy individuals), while others are not cancer-reactive (i.e., their response intensities are equivalent between samples derived from cancer patients and healthy individuals). Based on this finding, further research led to the discovery of receptors that are reactive to a certain cancer but not to another cancer, or whose reactivity is reversed, as well as receptors that are reactive to multiple types of cancer, including a certain cancer. The present disclosure encompasses the following aspects:

[0008] Item 1. A test agent for cancer type a, comprising an insect olfactory receptor protein A' that is reactive to cancer type a and not reactive to at least one type of cancer other than cancer type a, or whose reactivity to at least one type of cancer other than cancer type a is opposite to that of cancer type a, and an insect olfactory receptor protein B that is reactive to multiple types of cancer including cancer type a and whose reactivity is in the same direction.

[0009] Item 2. The test agent according to Item 1, wherein the insect olfactory receptor protein A' has no reactivity to at least two types of cancer other than cancer type a.

[0010] Item 3. The test agent according to Item 1 or 2, comprising a cell A' expressing the insect olfactory receptor protein A' and a cell B expressing the insect olfactory receptor protein B.

[0011] Item 4. The test agent according to Item 3, wherein the cell A' and the cell B are contained in compartments.

[0012] Item 5. The test agent according to Item 3 or 4, wherein the cell A' and the cell B are separated into separate compartments.

[0013] Item 6. The testing agent according to any one of Items 1 to 5, which is used to test for cancer type a based on the reactivity of the insect olfactory receptor protein A' and the insect olfactory receptor protein B to a sample derived from a subject.

[0014] Item 7. The test agent according to any one of Items 3 to 5, comprising a cell chip including a compartment containing the cells.

[0015] Item 8. 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 B, the composition comprising an insect olfactory receptor protein A' that is reactive to cancer type a and not reactive to at least one type of cancer other than cancer type a, or whose reactivity to at least one type of cancer other than cancer type a is opposite to that of the cancer type a, and a composition comprising an insect olfactory receptor protein B that is reactive to multiple types of cancer including cancer type a and whose reactivity is in the same direction.

[0016] Item 9. A cell chip 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 B, which have reactivity to cancer type a and no reactivity to at least one type of cancer other than cancer type a, or have an opposite reactivity to at least one type of cancer other than cancer type a, and a compartment containing insect olfactory receptor protein B that have reactivity to multiple types of cancer including cancer type a and the reactivity is in the same direction.

[0017] Item 10. A method for testing for cancer type a, comprising contacting a sample derived from a subject with insect olfactory receptor protein A' that is reactive to cancer type a and not reactive to at least one type of cancer other than cancer type a, or whose reactivity to at least one type of cancer other than cancer type a is opposite to that of the cancer type a, and insect olfactory receptor protein B that is reactive to multiple types of cancer including cancer type a and whose reactivity is in the same direction.

[0018] According to the present disclosure, a cancer testing technology that utilizes insect olfactory receptors can be provided.

[0019] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0020] As used herein, amino acid mutations are, for example, amino acid substitutions, insertions, additions, or deletions, preferably substitutions, and particularly preferably conservative substitutions.

[0021] As used herein, the term "conservative substitution" refers to the substitution of an amino acid residue 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 constitutes a conservative substitution. Other examples of conservative substitutions include 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.

[0022] As used herein, "identity" of amino acid sequences refers to the degree of correspondence between the amino acid sequences of two or more comparable amino acid sequences. Therefore, the greater the identity between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity between amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using 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)). Based on this BLAST algorithm, programs called BLASTP and BLASTX have been developed. Specific techniques for these analysis methods are known, and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ).

[0023] In this specification, OR indicates an odorant receptor, Aa indicates that it is derived from 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.

[0024] In one aspect, the present disclosure relates to a test agent for cancer type a (sometimes referred to herein as the "test agent of the present disclosure"), which comprises an insect olfactory receptor protein A' that is reactive to cancer type a and not reactive to at least one type of cancer other than cancer type a, or whose reactivity to at least one type of cancer other than cancer type a is opposite to that of cancer type a, and an insect olfactory receptor protein B that is reactive to multiple types of cancer including cancer type a and whose reactivity is in the same direction. This will be explained below.

[0025] The cancer type A to be tested is a cancer type that is distinguishable from other cancer types in terms of tissue origin, cell origin, etc., and is not particularly limited insofar as such. Examples of cancer type A include 32 types, such as colorectal cancer, gastric 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 / 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 encompasses cancers of all grades (e.g., mild, moderate, severe) and stages. In one embodiment, cancer type A is a solid cancer, and in another embodiment, it is a blood cancer. In one embodiment, cancer type a is more preferably a digestive cancer, more preferably gastric cancer, colon cancer, esophageal cancer, liver cancer, pancreatic cancer, or biliary tract cancer, and even more preferably gastric cancer, colon cancer, or esophageal cancer. In one embodiment, cancer type a is more preferably colon cancer, prostate cancer, bladder cancer, lung cancer, stomach cancer, breast cancer, liver cancer, pancreatic cancer, biliary tract cancer, kidney cancer (renal cell carcinoma), cervical cancer, endometrial cancer, uterine sarcoma, esophageal cancer, skin cancer, thyroid cancer, ovarian cancer, or head and neck cancer.

[0026] The cancer type a may be one type alone or a combination of two or more types. The cancer type a may be at least one type selected from the group consisting of, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 21 or more, or 32 types of cancer among the 32 specific cancer types listed above. The number of species may be at most one, and may be, for example, at least one species selected from the group consisting of 32 or less, 31 or less, 30 or less, 29 or less, 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 species. The above lower and upper limits can be combined arbitrarily.

[0027] Insect olfactory receptor proteins are membrane proteins with seven transmembrane domains that function as insect odor sensors. From the amino terminus (hereafter sometimes referred to as the "N-terminus") to the carboxyl terminus (hereafter sometimes referred to as the "C-terminus") of an olfactory receptor protein, they are composed of the N-terminal region (NT), the first transmembrane domain (TM1), the first extracellular loop (EC1), the second transmembrane domain (TM2), the first intracellular loop (IC1), the third transmembrane domain (TM3), the second extracellular loop (EC2), the fourth transmembrane domain (TM4), the second intracellular loop (IC2), the fifth transmembrane domain (TM5), the third extracellular loop (EC3), the sixth transmembrane domain (TM6), the third intracellular loop (IC3), the seventh transmembrane domain (TM7), and the C-terminal region (CT). In the present disclosure, each region is 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 preferably include Diptera insects such as Culicidae and Drosophilidae; Lepidoptera insects such as Bombycidae, Pyralidae, Noctuidae, and Tortricidae; Hymenoptera insects such as Apidae; Orthoptera insects such as Acrididae; Hemiptera insects such as Bedbug and Delphacidae, and more preferably Diptera insects such as Culicidae and Drosophilidae; Orthoptera insects such as Acrididae; Hemiptera insects such as Bedbug. Examples of Culicidae insects include Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus. Examples of insects in the Drosophilidae family include Drosophila melanogaster, Drosophila pseudoobscura, and Drosophila virillis. Examples of insects in the Bombycidae family include Bombyx mori, Bombyx mandarina, and Trilocha varians. Examples of insects in the Apidae family include Apis mellifera, Apis florea, Apis dorsata, and Bombus terrestris. Examples of insects belonging to the Acrididae family include the migratory locust (Locusta migratoria), and examples of insects belonging to 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' can contain amino acid mutations in the wild-type amino acid sequence, so long as it is reactive to cancer type a (i.e., the response intensity differs between samples derived from patients with cancer type a and samples derived from healthy subjects) and not reactive to at least one (preferably at least two) types of cancer other than cancer type a (i.e., the response intensity is equivalent between samples derived from patients with at least one type of cancer other than cancer type a and samples derived from healthy subjects), or has the opposite reactivity to at least one type of cancer other than cancer type a (i.e., the response intensity of samples derived from patients with cancer type a is higher or lower than the response intensity of samples derived from patients with at least one type of cancer other than cancer type a compared to the response intensity of samples derived from healthy subjects). The reactivity can be determined, for example, according to or in accordance with the method of Test Example 1 described below.

[0031] Insect olfactory receptor protein A' can include a wild-type amino acid sequence, or an amino acid sequence that has, for example, 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more identity to the wild-type amino acid sequence.

[0032] The insect olfactory receptor protein A' may be a single type or a combination of two or more types. From the viewpoint of accuracy in determining cancer type a, the combination may preferably be two or more types, three or more types, four or more types, five or more types, six or more types, seven or more types, eight or more types, nine or more types, or ten or more types, or may be 30 or less types, 25 or less types, 20 or less types, 15 or less types, 12 or less types, or 11 or less types.

[0033] Insect olfactory receptor protein A' is, for example, a receptor (receptor A'X1) whose response intensity to samples derived from patients with cancer type a tends to be higher than that to samples derived from healthy subjects, and whose response intensity to samples derived from patients with at least one (preferably at least two) types of cancer other than cancer type a tends to be equivalent to that to samples derived from healthy subjects; a receptor (receptor A'X2) whose response intensity to samples derived from patients with cancer type a tends to be higher than that to samples derived from healthy subjects, and whose response intensity to samples derived from patients with at least one (preferably at least two) types of cancer other than cancer type a tends to be lower than that to samples derived from healthy subjects (i.e., whose reactivity is opposite to that of cancer type a); A receptor (receptor A'Y1) whose response intensity to samples derived from patients with cancer type a tends to be lower than that to samples derived from healthy subjects, and whose response intensity to samples derived from patients with at least one (preferably at least two) types of cancer other than cancer type a tends to be equivalent to that to samples derived from healthy subjects, or a receptor (receptor A'Y2) whose response intensity to samples derived from patients with cancer type a tends to be lower than that to samples derived from healthy subjects, and whose response intensity to samples derived from patients with at least one (preferably at least two) types of cancer other than cancer type a tends to be higher than that to samples derived from healthy subjects (i.e., whose reactivity is opposite to that of cancer type a).

[0034] Insect olfactory receptor protein B can contain amino acid mutations in the wild-type amino acid sequence, so long as it is reactive to multiple (two or more, preferably three or more) types of cancer, including cancer type a, and the direction of the reactivity is the same (i.e., the response intensity of a sample derived from a patient with cancer type a is the same as the response intensity of a sample derived from a patient with at least one type of cancer other than cancer type a, relative to the response intensity of a sample derived from a healthy subject). The reactivity can be determined, for example, according to or in accordance with the method of Test Example 1 described below.

[0035] Insect olfactory receptor protein B can include a wild-type amino acid sequence, or an amino acid sequence that has, for example, 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more identity to the wild-type amino acid sequence.

[0036] The insect olfactory receptor protein B may be a single protein or a combination of two or more proteins. From the viewpoint of accuracy in determining cancer type a, the combination may preferably be two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more proteins, or may be 30 or less, 25 or less, 20 or less, 15 or less, 12 or less, or 11 or less proteins.

[0037] Insect olfactory receptor protein B is, for example, a receptor (receptor BX) whose response intensity to samples derived from multiple types of cancer patients, including cancer type a, tends to be higher than the response intensity to samples derived from healthy individuals, or a receptor (receptor BY) whose response intensity to samples derived from multiple types of cancer patients, including cancer type a, tends to be lower than the response intensity to samples derived from healthy individuals.

[0038] As long as the insect olfactory receptor proteins A' and B have the above-mentioned cancer reactivity, other amino acid sequences, for example, proteins or peptides such as protein tags, fluorescent proteins, luminescent proteins, signal sequences, etc. Examples of protein tags include biotin, His tags, FLAG tags, Halo tags, MBP tags, HA tags, Myc tags, V5 tags, and PA tags.

[0039] The insect olfactory receptor proteins A' and B may be chemically modified as long as they have the above-mentioned cancer reactivity.

[0040] Insect olfactory receptor proteins A' and B have a C-terminus containing a carboxyl group (-COOH) or a carboxylate group (-COO － ), amide (-CONH2) or ester (-COOR).

[0041] 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-naphthyl-C such as α-naphthylmethyl 1－2 C such as alkyl group 7－14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0042] The insect olfactory receptor proteins A' and B may have amidated or esterified carboxyl groups (or carboxylates) other than those at the C-terminus. In this case, the esters used may be, for example, the C-terminal esters described above.

[0043] Furthermore, in the insect olfactory receptor proteins A' and B, the amino group of the N-terminal amino acid residue is protected by a protecting group (e.g., a C group such as a formyl group or an acetyl group). 1－6 C such as alkanoyl 1－6 those in which the N-terminal glutamine residue that can be generated by cleavage in vivo is pyroglutamated; those in which the 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 an appropriate protecting group (e.g., C group 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 bound.

[0044] The insect olfactory receptor proteins A' and B 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.

[0045] The insect olfactory receptor proteins A' and B may be in the form of a solvate. The solvent is not particularly limited, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0046] 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. Therefore, in the test agent of the present disclosure, it is usually preferable that the insect olfactory receptor protein of the present disclosure be in a form retained in a membrane.

[0047] The membrane is preferably a lipid membrane. The 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.

[0048] 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 B expressing insect olfactory receptor protein B.

[0049] 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.

[0050] Examples of insect cells include Sf cells, MG1 cells, and High Five cells. TM Examples of Sf cells that can be used include Sf9 cells (ATCC CRL1711) and Sf21 cells. Among insect cells, cells derived from insects of the family Arctiidae are particularly preferred.

[0051] 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.

[0052] Examples of the Arctiidae family include the Arctiinae subfamily, Lithosiinae subfamily, and Syntominae subfamily, with the Arctiinae being preferred among these. Examples of the Arctiinae subfamily include the genera Spilosoma, Spilarctia, and Rhagonis, with the Rhagonis genus being particularly preferred. The Rhagonis genus is not particularly limited, but the Rhagonis genus is particularly preferred.

[0053] Cells derived from an insect of the family Arctiidae can be obtained from a known biobank, or can be obtained by collecting and culturing from a living insect of the family Arctiidae according to or in accordance with a known method, or by establishing a line if necessary.

[0054] 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.

[0055] Examples of mammalian cells include COS7 cells, CHO cells, HEK293 cells, HEK293FT cells, Hela cells, PC12 cells, N1E-115 cells, and SH-SY5Y cells.

[0056] The cell of the present disclosure preferably contains an exogenous polynucleotide comprising a coding sequence for the insect olfactory receptor protein of the present disclosure. The exogenous polynucleotide is a polynucleotide comprising a base sequence not derived from the genomic DNA (particularly chromosomal genomic DNA) of the cell, and is not particularly limited insofar as it does so.

[0057] 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 with a seven-transmembrane structure, similar to the olfactory receptor, and functions by forming a heterocomplex with the olfactory receptor. The olfactory receptor complex, which is a heterocomplex composed 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 + ), calcium ions (Ca 2+ As the insect olfactory receptor co-receptor, the olfactory receptor co-receptor described in Japanese Patent No. 6875815 may be used.

[0058] 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 in response to an insect olfactory receptor protein. Examples of such proteins include aequorin, yellow camelon, GCaMP, etc. Alternatively, the cells of the present disclosure preferably contain an ion-dependent fluorescent dye such as a calcium ion-dependent fluorescent dye (e.g., Fura-2, Fluo-3, Fluo-4, etc.).

[0059] Coding sequences such as insect olfactory receptor protein coding sequences, insect olfactory receptor co-receptor coding sequences, and coding sequences for fluorescent or luminescent proteins are preferably contained in the exogenous polynucleotide in the form of an expression cassette (a promoter and a coding sequence placed under the control of the promoter). The promoter of the expression cassette can be shared among multiple coding sequences.

[0060] 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.

[0061] A compartment is an area in which insect olfactory receptor proteins / cells are arranged. The form of the compartment is not particularly limited as long as it allows the insect olfactory receptor proteins / cells to be arranged. From the viewpoints of the desiccation resistance of the insect olfactory receptor proteins / cells, the retention of the insect olfactory receptor proteins / cells, production efficiency, or the detectability of odorants, it is preferable that the compartment 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, it is also possible to distinguish the surface outside the compartment from a compartment in which cells can be arranged (for example, adhere) by making the surface on which cells cannot be arranged (for example, adhere) (or the adhesion is significantly reduced).

[0062] 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).

[0063] The material of the compartment is not particularly limited as long as it can hold cells, and can be, for example, resin, metal, etc.

[0064] 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 , 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 2The following is the result.

[0065] From the viewpoint of detection sensitivity or production efficiency, the number of compartments is preferably 1 to 2000, more preferably 4 to 1600, and even more preferably 8 to 400. In a preferred embodiment of the present disclosure, the number is 10 or more, 20 or more, or 50 or more.

[0066] From the viewpoint of detection sensitivity, a compartment usually contains multiple cells. 2 The number of cells per 1000 mm is, for example, 50 to 20,000 cells / mm 2 From the viewpoint of detection sensitivity, cell viability, etc., the cell density 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 , particularly preferably 1000 to 5000 cells / mm 2 is.

[0067] The testing agent of the present disclosure preferably contains two or more types of cells (preferably 3 to 25 types, more preferably 3 to 12 types, and even more preferably 3 to 10 types) that express different insect olfactory receptor proteins of the present disclosure. In this case, it is preferable that the two or more types of cells are separated into separate compartments (for example, cell A' is placed in one compartment (compartment A') and cell B is placed in another compartment (compartment B)).

[0068] Furthermore, in the test agent of the present disclosure, it is preferable that two or more different insect olfactory receptor proteins of the present disclosure are separated into separate compartments (for example, insect olfactory receptor protein A is placed in one compartment (compartment A') and insect olfactory receptor protein B is placed in another compartment (compartment B)).

[0069] 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.

[0070] 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).

[0071] 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.

[0072] 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, a subject for which information regarding cancer type a is unknown, etc. When the subject is a human, any person can be the subject, regardless of their past medical history, including those considered to be healthy, and examples of subjects include those who undergo health checkups or comprehensive medical examinations, and those who have undergone cancer testing using interview methods, questionnaire tests, score methods, pathological diagnostic methods, imaging diagnostic methods, blood biochemistry tests, etc.

[0073] 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.

[0074] 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.

[0075] 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.).

[0076] 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.

[0077] The manner of contact is not particularly limited, as long as the components in the sample derived from the specimen can come into contact with the insect olfactory receptor protein of the present disclosure. For example, the sample derived from the specimen can be contacted with the insect olfactory receptor protein of the present disclosure by adding the sample to a compartment (compartment A' containing insect olfactory receptor protein A', and compartment B containing insect olfactory receptor protein B) (each of which further contains a liquid, if necessary) containing a membrane that holds the insect olfactory receptor protein of the present disclosure (membrane A' that holds insect olfactory receptor protein A', or membrane B that holds insect olfactory receptor protein B) or a cell that expresses the insect olfactory receptor protein of the present disclosure (cell A' that expresses insect olfactory receptor protein A', or cell B that expresses insect olfactory receptor protein B).

[0078] 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.

[0079] The type of response intensity used as an indicator for cancer type a testing is not particularly limited, and can be, for example, the maximum signal amount, the integrated value of the signal amount, the rate of increase in the signal amount, etc. More specifically, it can be, for example, the maximum signal amount, the integrated value, and / or the rate of increase in the signal amount within a predetermined time or after a predetermined time has elapsed after contact with the sample derived from the subject.

[0080] By using the response intensity of the insect olfactory receptor protein A' as an index (e.g., by using the response intensity being equal to or greater than a cutoff value as an index), cancer type a can be determined. More specifically, for example, if the response intensity of receptor A'X1 and / or receptor A'X2 to a sample derived from a subject is equal to or greater than a cutoff value, the subject can be determined to have cancer type a, and / or if the response intensity of receptor A'Y1 and / or receptor A'Y2 to a sample derived from a subject is equal to or less than the cutoff value, the subject can be determined to have cancer type a.

[0081] Furthermore, by combining the response intensity of insect olfactory receptor protein B as an index (for example, using the response intensity being equal to or greater than a cutoff value as an index), cancer type a can be determined with higher accuracy. More specifically, in addition to the response intensity of the insect olfactory receptor protein A' described above as an index, it is possible to determine that the subject has cancer type a if the response intensity of receptor BX to a sample derived from the subject is equal to or greater than a cutoff value, and / or to determine that the subject has cancer type a if the response intensity of receptor BY to a sample derived from the subject is equal to or less than the cutoff value.

[0082] The cutoff value can be appropriately set by a person skilled in the art from the viewpoint of various indices of accuracy of cancer type a determination (e.g., Accuracy, F1-score, Matthews, Precision, ROC-AUC, Recall, Specificity, etc.). The cutoff value may be either a value set on an individual basis depending on race, age, etc., or a preset value. The cutoff value may be, for example, a value based on the maximum, average, percentile, or minimum value of the response intensity to a sample collected from a subject determined not to be affected by cancer type a or a subject determined to be affected by cancer type a.

[0083] In one embodiment, the cutoff value refers to a value that provides a sufficiently high accuracy in determining the presence or absence of cancer type a when that value is used as a standard. For example, 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 can be set as the cutoff value.

[0084] Such a cutoff value does not have a specific value, but varies depending on the subject population used when setting the cutoff value.

[0085] 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.

[0086] Use of the test agent of the present disclosure can provide an indicator for cancer type A testing, thereby assisting in cancer type A testing. Furthermore, use of a cutoff value can determine the presence or absence of cancer type A.

[0087] 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.

[0088] The test agent of the present disclosure can be in the form of a kit. The kit may include instruments, reagents, instructions, and the like that can be used to perform a cancer type a test.

[0089] 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.

[0090] Specifically, screening can be performed by a method including, for example, contacting a test insect olfactory receptor protein with a sample derived from a subject with cancer and measuring the response intensity P, and contacting a test insect olfactory receptor protein with a sample derived from a healthy subject without cancer and measuring the response intensity Q.

[0091] The response intensity P can be a value (e.g., an average value such as a geometric mean) obtained by, for example, obtaining multiple response intensities (response intensity P1, response intensity P2, response intensity P3, ...) using samples derived from multiple subjects (subject P1, subject P2, subject P3, ...) and performing arithmetic processing using these multiple response intensities. The same applies to the response intensity Q.

[0092] The above-mentioned "plurality" means, for example, 3 or more, preferably 5 or more, more preferably 10 or more, even more preferably 15 or more, more preferably 20 or more, and for example, 500 or less, 300 or less, 200 or less, 100 or less, or 50 or less. By using multiple specimens, variations between specimens (e.g., variations in specimen concentration, condition (e.g., stage of cancer), etc.) can be averaged out, allowing the insect olfactory receptor protein of the present disclosure to be screened with higher accuracy.

[0093] More specifically, in this method, if the response intensity P and the response intensity Q are different, it can be determined that the test insect olfactory receptor protein has reactivity to cancer, and / or if the response intensity P and the response intensity Q are comparable, it can be determined that the test insect olfactory receptor protein does not have reactivity to cancer.

[0094] Insect olfactory receptor proteins A' and B can be screened by performing the above-mentioned determination using a subject with cancer type a and a subject with any cancer other than cancer type a as the subject from which the sample used to measure the response intensity P is derived.

[0095] The description of the test agent of the present disclosure is incorporated herein for the embodiment of the screening method.

[0096] Whether or not the response intensity P and the response intensity Q differ can be determined by setting a certain reference value, as in Test Example 1 described below. That is, if the difference between the response intensity P and the response intensity Q exceeds (or is 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 (or less than) the reference value, it can be determined that there is no reactivity to cancer.

[0097] In one aspect, the present disclosure relates to: 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 B, the combination comprising a composition containing insect olfactory receptor protein A' and a composition containing insect olfactory receptor protein B; a cell chip 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 B, the cell chip comprising a compartment containing insect olfactory receptor protein A' and a compartment containing insect olfactory receptor protein B; a method for testing for cancer type a, the method comprising contacting a sample derived from a subject with the insect olfactory receptor protein A' and the insect olfactory receptor protein B; etc. The explanation of the testing agent in the present disclosure is incorporated herein by reference for these aspects.

[0098] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0099] Test Example 1. Screening for Cancer-Reactive Insect Olfactory Receptors 1. Cancer-reactive and non-reactive insect olfactory receptors were selected by screening from Aedes aegypti-derived olfactory receptors, Anopheles gambiae-derived olfactory receptors, Drosophila melanogaster-derived olfactory receptors, Bombyx mori-derived olfactory receptors, Locusta migratoria-derived olfactory receptors, and Cimex lectularius-derived olfactory receptors. Expression plasmids containing the GFP-Aequorin coding sequence, the insect olfactory receptor co-receptor Orco coding sequence, and the insect olfactory receptor OR coding sequence were prepared according to the method described in Japanese Patent No. 6875815. The resulting expression plasmids were transfected into cells, which were then seeded on a 384-well plate and subjected to luminescence intensity measurements. Luminescence intensity measurements were performed using an FDSS / μCELL (Hamamatsu Photonics) by adding the test sample and measuring the luminescence intensity over time. The test samples used were 25 urine samples from patients with solid cancer of a certain tissue (cancer type X) and 25 urine samples from healthy individuals.

[0100] First, the geometric mean of the luminescence intensity of each test sample in each OR was calculated for urine from cancer type X patients and healthy subjects. Receptors whose maximum luminescence intensity in cancer type X patients and / or healthy subjects was 2000 or greater within 100 seconds of the start of measurement (the test sample was added 10 seconds after the start of measurement) and whose maximum luminescence intensity in cancer type X patients and / or healthy subjects differed by more than 1000 were selected. In this way, insect olfactory receptors with cancer reactivity were identified. Among these insect olfactory receptors, some exhibited higher luminescence intensity in cancer type X patients and others in healthy subjects.

[0101] Next, receptors were selected that had a maximum luminescence intensity of 2000 or more in patients with cancer type X and / or in healthy subjects, and that were determined to have no difference between the maximum luminescence intensity in patients with cancer type X and the maximum luminescence intensity in healthy subjects. In this way, insect olfactory receptors that are not reactive to cancer were discovered.

[0102] 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.

[0103] Test Example 2. Screening for Cancer-Reactive Insect Olfactory Receptors 2 For solid cancers (cancer types Y and Z) derived from tissues different from cancer type X, 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 that of healthy subjects. The results are shown in Table 1. In Table 1, the first two letters of the receptor name indicate the biological species from which the insect olfactory receptor originated: Aa indicates Aedes aegypti, Ag indicates Anopheles gambiae, Dm indicates Drosophila melanogaster, Lm indicates Locusta migratoria, and Cl indicates Cimex lectularius.

[0104]

[0105] Receptors in groups a, b, and d in Table 1 are reactive to a specific cancer (cancer type X), but are not reactive to other cancer types (a and b), or the direction of the reaction is opposite (d). Therefore, by using the response strength derived from the receptor as an index (for example, by using the response strength being above or below a cutoff value as an index), it is possible to determine the specific cancer with higher accuracy, even when there are multiple possible types of cancer.

[0106] Receptors in Group c in Table 1 are reactive to multiple types of cancer, including a specific cancer (cancer type X). Therefore, by combining the response intensity derived from this receptor with the response intensities derived from receptors in Groups a, b, and d (for example, using as an indicator that the response intensity derived from receptors in Group c is equal to or greater than a cutoff and that the response intensities derived from receptors in Groups a, b, and d are equal to or greater than a cutoff), the specific cancer can be diagnosed with greater accuracy. Furthermore, by further testing subjects diagnosed as having cancer by testing with receptors in Group c with receptors in Groups a, b, and d, it is possible to reduce unnecessary tests for identifying cancer type.

[0107] Furthermore, since receptors in Group c in Table 1 have reactivity to multiple types of cancer, by using the response strength derived from the receptor as an index (using the response strength being equal to or greater than a cutoff value as an index), it is possible to more thoroughly determine whether or not a cancer is present when there are multiple possible types of cancer.

Claims

1. A testing agent for cancer type a, comprising an insect olfactory receptor protein A' that is reactive to cancer type a and not reactive to at least one type of cancer other than cancer type a, or whose reactivity to at least one type of cancer other than cancer type a is opposite, and an insect olfactory receptor protein B that is reactive to multiple types of cancer including cancer type a and whose reactivity is in the same direction.

2. The test agent according to claim 1, wherein the insect olfactory receptor protein A' has no reactivity to at least two types of cancer other than cancer type a.

3. The test agent according to claim 1, comprising cells A' expressing the insect olfactory receptor protein A' and cells B expressing the insect olfactory receptor protein B.

4. The test agent according to claim 3, wherein said cell A' and said cell B are contained in compartments.

5. The test agent according to claim 3, wherein said cells A' and said cells B are separated into separate compartments.

6. The test agent according to claim 1, which is used to test for cancer type a based on the reactivity of the insect olfactory receptor protein A' and the insect olfactory receptor protein B to a sample derived from a subject.

7. The test agent according to claim 3, comprising a cell chip containing compartments containing the cells.

8. A combination of compositions for use in contacting a sample derived from a subject suspected of having cancer type a with insect olfactory receptor protein A' and insect olfactory receptor protein B, the combination comprising a composition containing insect olfactory receptor protein A' that is reactive to cancer type a and not reactive to at least one type of cancer other than cancer type a, or whose reactivity to at least one type of cancer other than cancer type a is opposite to that of the cancer type a, and a composition containing insect olfactory receptor protein B that is reactive to multiple types of cancer including cancer type a and whose reactivity is in the same direction.

9. A cell chip for use in contacting a sample derived from a subject suspected of having cancer type a with insect olfactory receptor protein A' and insect olfactory receptor protein B, the cell chip comprising a compartment containing insect olfactory receptor protein A' that is reactive to cancer type a and not reactive to at least one type of cancer other than cancer type a, or whose reactivity to at least one type of cancer other than cancer type a is opposite to that of the cancer type a, and a compartment containing insect olfactory receptor protein B that is reactive to multiple types of cancer including cancer type a and whose reactivity is in the same direction.

10. A method for testing for cancer type a, comprising contacting a sample derived from a subject with insect olfactory receptor protein A', which is reactive to cancer type a and not reactive to at least one type of cancer other than cancer type a, or whose reactivity to at least one type of cancer other than cancer type a is opposite to that of the cancer type a, and insect olfactory receptor protein B, which is reactive to multiple types of cancer including cancer type a and whose reactivity is in the same direction.

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