Method for screening olfactory receptor protein or combination thereof

Abstract

To provide a method for screening an olfactory receptor protein or its combination useful in cancer examination.SOLUTION: A method for screening an olfactory receptor protein or a combination thereof, comprising testing the reactivity of a test olfactory receptor protein to cancer, and selecting at least one selected from the group consisting of (1) a combination of the olfactory receptor protein A and the olfactory receptor protein C, (2) a combination of the olfactory receptor protein A ' and the olfactory receptor protein B, and (3) the olfactory receptor protein B '.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for screening olfactory receptor proteins or combinations thereof. [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 screening method for olfactory receptor proteins or combinations thereof that are useful in cancer testing. [Means for solving the problem]

[0007] The present inventors have discovered that there are various olfactory receptors, each with different response intensities to samples derived from a subject, cancer patients, and healthy individuals. Based on this finding, the inventors conducted further research and discovered that some 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). Further research based on this finding revealed that some receptors are reactive to one cancer but not to another, or that their reactivity is reversed, and that some receptors are reactive to multiple types of cancer. Based on these findings, the inventors conducted extensive research and discovered that the use of olfactory receptors characterized by cancer reactivity, or a combination thereof, can improve the accuracy of cancer testing and / or enable more accurate determination of whether or not a cancer is present when multiple types of cancer are suspected. The present invention encompasses the following aspects.

[0008] Item 1. Testing the reactivity of a test olfactory receptor protein against cancer, and (1) a combination of an olfactory receptor protein A that is reactive to cancer type a and an olfactory receptor protein C that is not reactive to cancers including cancer type a; (2) Reactive to cancer type a and at least one type of cancer other than cancer type a A combination of an olfactory receptor protein A' that has no reactivity or has an opposite reactivity to at least one type of cancer other than cancer type a, and an olfactory receptor protein B that has reactivity to multiple types of cancer including cancer type a and the direction of the reactivity is the same; and (3) Olfactory receptors that are reactive to multiple types of cancer and have the same direction of reactivity Protein B', Selecting at least one selected from the group consisting of: A method for screening for olfactory receptor proteins or combinations thereof.

[0009] Item 2. The method for screening olfactory receptor proteins according to Item 1, wherein the selected olfactory receptor proteins or a combination thereof are receptors for use in cancer testing.

[0010] Item 3. The olfactory receptor protein A reacts with at least one type of cancer other than cancer type a. Item 3. The screening method according to Item 1 or 2, wherein the antibody has no responsiveness to the antibody.

[0011] Item 4. The screening method according to any one of Items 1 to 3, wherein the olfactory receptor protein C has no reactivity to multiple types of cancer including cancer type a.

[0012] Item 5. The screening method according to any one of Items 1 to 4, wherein the olfactory receptor is an insect olfactory receptor.

[0013] Item 6. Contacting the test olfactory receptor protein with a sample derived from a subject with cancer and measuring the response intensity P; and contacting the test olfactory receptor protein with a sample derived from a healthy subject without cancer and measuring the response intensity Q; 6. The screening method according to any one of Items 1 to 5, comprising:

[0014] Item 7. Determining that the test olfactory receptor protein has reactivity to cancer when the response intensity P and the response intensity Q are different; and / or If the response intensity P and the response intensity Q are comparable, it is determined that the test olfactory receptor protein does not have reactivity to cancer. Item 7. The screening method according to Item 6, comprising: [Effects of the Invention]

[0015] According to the present disclosure, a screening method for finding olfactory receptor proteins and / or combinations thereof useful in cancer testing can be provided. DETAILED DESCRIPTION OF THE INVENTION

[0016] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0017] As used herein, amino acid mutations are, for example, amino acid substitutions, insertions, additions, or deletions, preferably substitutions, and particularly preferably conservative substitutions.

[0018] As used herein, "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 include 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; and amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan. Conservative substitutions also include substitutions of amino acid residues with one another; amino acid residues with beta-branched side chains, such as threonine, valine, and isoleucine; and amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine.

[0019] 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 called BLASTP and BLASTX based on the BLAST algorithm 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 / ).

[0020] 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.

[0021] In one aspect, the present disclosure includes testing the reactivity of a test olfactory receptor protein to cancer; and (1) A combination of olfactory receptor protein A that is reactive to cancer type a and olfactory receptor protein C that is not reactive to cancers including cancer type a, (2) A combination of an 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 that has an opposite reactivity to at least one type of cancer other than cancer type a, and an olfactory receptor protein B that is reactive to multiple types of cancer including cancer type a and has the same direction of reactivity; and (3) Olfactory receptors that are reactive to multiple types of cancer and have the same direction of reactivity Protein B', Selecting at least one selected from the group consisting of: The present invention relates to a method for screening for olfactory receptor proteins or combinations thereof (sometimes referred to herein as the "screening method of the present disclosure"). This will be described below.

[0022] The cancer is not particularly limited. Examples of cancer include colon cancer, 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 / 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 includes cancers of all grades (e.g., mild, moderate, severe) and stages.

[0023] Cancer type A is a cancer type that is distinguished from other cancer types in terms of tissue origin, cell origin, etc. Insofar as this is the case, there is no particular limitation.

[0024] Multiple types of cancer are cancers that are distinguished from each other in terms of tissue origin, cell origin, etc. do.

[0025] Olfactory receptor proteins are proteins that can detect the presence of chemicals, such as ionotropic receptors and / or G protein-coupled receptors. is preferably an olfactory receptor protein of a vertebrate such as a mammal, bird, reptile, amphibian, or fish, or an insect or nematode, and is more preferably an insect olfactory receptor protein from the viewpoint that it is easier to obtain the receptor targeted by the screening method of the present disclosure.

[0026] 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).

[0027] Insects from which insect olfactory receptor proteins are derived preferably include dipteran insects such as Culicidae and Drosophilidae; lepidopteran insects such as Bombycidae; hymenopteran insects such as Apidae; orthopteran insects such as Acrididae; and hemipteran insects such as Cimex. More preferably, dipteran insects such as Culicidae and Drosophilidae; orthopteran insects such as Acrididae; and hemipteran insects such as Cimex. Examples of Culicidae insects 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).

[0028] Various wild-type insect olfactory receptor proteins are known, or can be easily identified by a sequence identity search based on known sequences.

[0029] The test olfactory receptor is an olfactory receptor protein subjected to the screening method of the present disclosure. The test olfactory receptor may comprise a wild-type olfactory receptor amino acid sequence, or may comprise an amino acid sequence into which a mutation has been introduced into the wild-type olfactory receptor amino acid sequence, for example, an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more identity thereto.

[0030] In the screening method of the present disclosure, the number of types of olfactory receptors to be tested is not particularly limited, and may be, for example, 1 or more, 2 or more, 10 or more, 20 or more, 40 or more, 60 or more, 100 or more, 200 or more, or 300 or more. and can be, for example, 5000 or less, 3000 or less, 2000 or less, 1000 or less, or 700 or less. It can be.

[0031] Insect olfactory receptor protein A has reactivity to cancer type a (i.e., cancer type a The response intensity differs between patient-derived samples and healthy subject-derived samples.

[0032] 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.

[0033] Insect olfactory receptor protein C has no reactivity to cancers, including cancer type a (i.e., the response intensity is equivalent between samples derived from cancer patients, including cancer type a, and samples derived from healthy individuals).

[0034] 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.

[0035] Insect olfactory receptor protein C corrects the response strength derived from the receptor for individual differences between samples. This makes it possible to improve the accuracy of cancer type a determination using insect olfactory receptor protein A. In addition, insect olfactory receptor protein C can be used to detect cancer types derived from the receptor. By using the response intensity as an index (for example, the response intensity being within a certain range (for example, not being an abnormal value)), it is possible to detect the presence of an insect olfactory receptor protein A. It is possible to improve the accuracy of cancer type a determination when using the accuracy of cancer type a determination.

[0036] Therefore, the combination of insect olfactory receptor protein A and insect olfactory receptor protein C is useful in cancer testing.

[0037] Insect olfactory receptor protein A' has reactivity to cancer type a (i.e., cancer type a The response intensity differs between patient-derived samples and healthy subject-derived samples, and there are few cases of cancers other than cancer type a. have no reactivity to at least one (preferably at least two) of the cancer types (i.e., The response intensity is equivalent between samples from patients with at least one type of cancer other than cancer and samples from healthy individuals. or the reactivity to at least one type of cancer other than cancer type a is opposite (i.e., the response strength of the sample derived from a patient with cancer type a is higher than that of the sample derived from a patient with at least one type of cancer other than cancer type a). The response intensity of the samples from healthy subjects is higher than that of the samples from healthy subjects.

[0038] 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).

[0039] Insect olfactory receptor protein B has reactivity to multiple types of cancer (two or more, preferably three or more) including cancer type a, and the reactivity is in the same direction (i.e., cancer type a patients). The response intensity of the sample derived from a subject and the response intensity of the sample derived from a patient with at least one type of cancer other than cancer type a are equal to the response intensity of the sample derived from a healthy subject. Insect olfactory receptor protein B, e.g. The response strength to samples from multiple cancer patients, including cancer type a, is higher than that to samples from healthy subjects. receptors (receptor BX) that tend to have a higher response intensity than the receptors The response strength to samples from multiple cancer patients, including cancer type a, is higher than that to samples from healthy subjects. receptors with a tendency to have a lower response strength than the ... is.

[0040] The response strength of insect olfactory receptor protein A' is used as an index (for example, the response strength is The type of cancer is determined by the index (whether the level is above or below the cut-off value). More specifically, for example, determining that the subject has cancer type a when the response intensity of receptor A'X1 and / or receptor A'X2 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 A'Y1 and / or receptor A'Y2 to a sample derived from the subject is equal to or lower than a cutoff value; can be done.

[0041] Furthermore, it is possible to combine the response strength of insect olfactory receptor protein B as an index (for example, For example, the response intensity may be determined to be equal to or greater than a cutoff value (using the response intensity as an index), thereby enabling the cancer type a to be determined with higher accuracy. In addition to the response intensity of protein A' as an index, determining that the subject has cancer type a when the response intensity of receptor BX 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 BY to a sample derived from the subject is equal to or lower than a cutoff value; can be done.

[0042] Therefore, the combination of insect olfactory receptor protein A' and insect olfactory receptor protein B is useful in cancer testing.

[0043] Insect olfactory receptor protein B' is effective against multiple types of cancer (two or more, preferably three or more). The reactivity is the same as that of a certain cancer (cancer type a) and the direction of the reactivity is the same. The response intensity of the patient-derived sample and the response intensity of the sample derived from a patient with at least one type of cancer other than cancer type a are the same as the response intensity of the sample derived from a healthy subject.

[0044] Insect olfactory receptor protein B', e.g. A receptor (receptor B'X) whose response intensity to samples derived from multiple types of cancer patients tends to be higher than its response intensity to samples derived from healthy individuals, or A receptor (receptor B'Y) whose response intensity to samples derived from multiple cancer patients tends to be lower than that to samples derived from healthy individuals. is.

[0045] The response strength of insect olfactory receptor protein B' is used as an index (for example, the response strength is By using the level above or below the cut-off value as an index, it is possible to make a more thorough determination of whether or not a cancer is present among multiple possible types of cancer. determining that the subject has cancer when the response intensity of receptor B'X to a sample derived from the subject is equal to or greater than a cutoff value; and / or determining that the subject has cancer when the response intensity of receptor B'Y to a sample derived from the subject is equal to or lower than a cutoff value; can be done.

[0046] Therefore, insect olfactory receptor protein B' is useful in cancer testing.

[0047] Specifically, the reactivity of the test insect olfactory receptor protein to cancer can be evaluated by, for example, The test olfactory receptor protein is contacted with a sample derived from a subject having cancer, and the response intensity P is measured. Measuring, and contacting the test olfactory receptor protein with a sample derived from a healthy subject without cancer and measuring the response intensity Q; The test can be performed by a method including:

[0048] 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.

[0049] 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.

[0050] The above determination is performed by using a subject having a certain cancer (cancer type a) and a subject having any cancer other than cancer type a as subjects from which the sample for measuring the response intensity P is derived. Insect olfactory receptor proteins A, C, A', B, and B' can be screened.

[0051] 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.

[0052] In the screening method of the present disclosure, the test olfactory receptor protein is not particularly limited as long as it is in a form that can exhibit odorant response activity. Odorant response activity refers to the property of an olfactory receptor protein recognizing an odorant and transmitting a signal (e.g., influx of cations, increase in cyclic AMP, etc.). For this reason, it is usually preferable that the test olfactory receptor protein be in a form that is retained in a membrane.

[0053] 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.

[0054] The test olfactory receptor protein is preferably used in the form of a cell that expresses the test olfactory receptor protein.

[0055] The cells are not particularly limited, but animal cells such as insect cells and mammalian cells are preferred from the viewpoint of suitability for detecting chemical substances.

[0056] The cell preferably contains an exogenous polynucleotide comprising a coding sequence for the test olfactory receptor protein. The exogenous polynucleotide is a polynucleotide derived from the genomic DNA (particularly, chromosomal genomic DNA) of the cell. It is a polynucleotide that contains a base sequence that is not derived from human DNA, and to that extent, it is not subject to any particular restrictions. Not limited to.

[0057] When the olfactory receptor protein is an insect olfactory receptor protein, the exogenous polynucleotide preferably comprises 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, and 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. When activated, it transports sodium ions (Na + ), calcium ions (Ca 2+ ) This allows cations such as ATP to flow into the cell.

[0058] The exogenous polynucleotide preferably contains a coding sequence for a protein that emits fluorescence or luminescence in response to ions (e.g., calcium ions) that flow into the cell when the olfactory receptor protein responds, or to second messengers such as cyclic AMP that increase within the cell. Examples of such proteins include aequorin, yellow camelon, and GCaMP. Alternatively, ion-dependent fluorescent dyes such as calcium ion-dependent fluorescent dyes (e.g., Fura-2, Fluo-3, Fluo-4, etc.) are also preferred.

[0059] In the screening method of the present disclosure, the olfactory receptor protein / the cell is preferably contained in a compartment.

[0060] A compartment is an area where 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 desiccation resistance of the insect olfactory receptor proteins / cells, retention of the insect olfactory receptor proteins / cells, production efficiency, or odorant detectability, the compartment is preferably in a form separated by walls (e.g., well-shaped and / or separated from the surrounding area by an outer wall). Furthermore, by making the surface outside the compartment a surface on which cells cannot be arranged (e.g., adhere) (or on which adhesion is significantly reduced), it is possible to distinguish the compartment from a compartment on which cells can be arranged (e.g., adhere).

[0061] 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).

[0062] The subject-derived sample is not particularly limited, and may be a body fluid of the subject or a sample derived therefrom (body fluid-derived sample), or exhaled breath.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.).

[0067] After contacting the sample derived from the subject with the test olfactory receptor protein, the response intensity of the test olfactory receptor protein can be measured to obtain the response intensity to the sample derived from the subject.

[0068] 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 olfactory receptor protein. For example, the sample derived from the subject can be brought into contact with the olfactory receptor protein by adding the sample derived from the subject to a compartment containing a membrane that retains the olfactory receptor protein to be tested or cells that express the olfactory receptor protein.

[0069] 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 test olfactory receptor protein or the amount of cyclic AMP production. One example is a method in which the amount of a factor responsible for signal transduction (e.g., cations flowing into the membrane due to ion channel activity) is converted into a signal (e.g., luminescence, light emission, etc.) and the signal amount is measured.

[0070] The type of response intensity used as an index in the screening method of the present disclosure is not particularly limited, and may 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 may 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.

[0071] The selected olfactory receptor protein or a combination thereof can be used in cancer testing. For example, it can be used in cancer testing using the above-mentioned cutoff values.

[0072] The cutoff value can be appropriately set by a person skilled in the art from the viewpoint of various indices of accuracy of cancer detection (e.g., Accuracy, F1-score, Matthews, Precision, ROC-AUC, Recall, Specificity, etc.). The cutoff value may be either a value set on a case-by-case 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 samples collected from subjects determined not to have cancer or subjects determined to have cancer.

[0073] In one embodiment, the cutoff value refers to a value that provides a sufficiently high accuracy in determining whether or not a cancer is present when the presence or absence of a cancer is determined based on that value. For example, a cutoff value that shows a high positive rate in individuals with cancer is a value that provides a sufficiently high accuracy in determining whether or not a cancer is present. Furthermore, a value that shows a high negative rate in individuals without cancer can be set as the cutoff value.

[0074] Techniques for setting cutoff values ​​are well known in the art. More specifically, cutoff values ​​can be set, for example, by measuring the amount or concentration of a target biomarker in a biological sample collected from a subject determined not to have cancer or a subject determined to have cancer, and using the measured value to perform statistical analysis based on, for example, receiver operating characteristic (ROC) curve analysis (more specifically, a method using the Youden index is exemplified).

[0075] Such a cutoff value does not have a specific value, but varies depending on the subject population used when setting the cutoff value.

[0076] 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. [Example]

[0077] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0078] 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 Patent No. 6875815. The resulting 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). 25 urine samples from patients with HIV infection and 25 urine samples from healthy individuals were used.

[0079] 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, there was one that was specifically targeted to cancer type X patients. The results included receptors with higher luminescence intensity in the patients with glaucoma and receptors with higher luminescence intensity in the healthy controls.

[0080] 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.

[0081] 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.

[0082] 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 that of healthy individuals. The results are shown in Table 1.

[0083] [Table 1]

[0084] 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.

[0085] 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

[0086] 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.

[0087] Test Example 3. Determination of Cancer Type X 1 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:

[0088] 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.

[0089] 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: .

[0090] 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.

[0091] Next, for each correction receptor (correction receptor i1, i2, i3, i4, i5, i6,), The geometric mean of the luminescence intensity of urine from patients with cancer type X and the geometric mean of the luminescence intensity of urine from healthy subjects measured in Test Example 1 in the above was calculated, and the geometric mean of both geometric means was further geometrically averaged to obtain the time change in luminescence intensity as a reference. For each correction receptor, the AUC of the time change in luminescence intensity of the test sample and the standard The AUC of the time change of the 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 α i3 , ratio α i4 , ratio α i5 , ratio α i6 The geometric mean of ( , ) was defined as the correction coefficient α.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] [Table 2]

[0096] From the above, it was shown that the accuracy of determining cancer type X can be improved by combining receptors. was done.

Claims

1. testing the reactivity of the test olfactory receptor protein to cancer; and (1) A combination of olfactory receptor protein A that is reactive to cancer type a and olfactory receptor protein C that is not reactive to cancers including cancer type a, (2) A combination of an 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 that has an opposite reactivity to at least one type of cancer other than cancer type a, and an olfactory receptor protein B that is reactive to multiple types of cancer including cancer type a and has the same direction of reactivity; (3) Olfactory receptors that are reactive to multiple types of cancer and whose reactivity is consistent Protein B', Selecting at least one selected from the group consisting of: A method for screening for olfactory receptor proteins or combinations thereof.

2. The method for screening olfactory receptor proteins according to claim 1, wherein the selected olfactory receptor protein or a combination thereof is a receptor for use in cancer testing.

3. The olfactory receptor protein A has reactivity to at least one type of cancer other than cancer type a. The screening method according to claim 1, wherein the method does not include the step of:

4. The screening method according to claim 1, wherein the olfactory receptor protein C is not reactive to multiple types of cancer, including cancer type a.

5. The screening method according to claim 1, wherein the olfactory receptor is an insect olfactory receptor.

6. contacting the test olfactory receptor protein with a sample derived from a subject with cancer and measuring the response intensity P; and contacting the test olfactory receptor protein with a sample derived from a healthy subject without cancer and measuring the response intensity Q; The screening method according to claim 1, comprising:

7. determining that the test olfactory receptor protein has reactivity to cancer when the response intensity P and the response intensity Q are different; and / or If the response intensity P and the response intensity Q are comparable, it is determined that the test olfactory receptor protein does not have reactivity to cancer. The screening method according to claim 6, comprising: