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 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 receptor protein B', which has reactivity to multiple types of cancer and the reactivity is in the same direction; 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 screening method according to Item 1 or 2, wherein the olfactory receptor protein A has no reactivity to at least one type of cancer other than cancer type a.

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

[0019] As used herein, the "identity" of an amino acid sequence refers to the degree of correspondence between 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 S F. "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 S F. "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 / ).

[0020] 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.

[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 receptor protein B', which has reactivity to multiple types of cancer and the reactivity is in the same direction; 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., and is not particularly limited in that respect.

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

[0025] The olfactory receptor protein is a protein capable of detecting the presence of a chemical substance, such as an ionotropic receptor and / or a G protein-coupled receptor. The olfactory receptor protein is preferably an olfactory receptor protein of vertebrates such as mammals, birds, reptiles, amphibians, and fish, as well as insects and nematodes. From the viewpoint of easier acquisition of receptors targeted by the screening method of the present disclosure, an insect olfactory receptor protein is more preferred.

[0026] Insect olfactory receptor proteins are membrane proteins with seven transmembrane domains that function as insect odor sensors. From the amino terminus (hereinafter sometimes referred to as the "N-terminus") to the carboxyl terminus (hereinafter 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).

[0027] Insects from which insect olfactory receptor proteins are derived preferably include Diptera insects such as Culicidae and Drosophilidae, Lepidoptera insects such as Bombycidae, Hymenoptera insects such as Apidae, Orthoptera insects such as Acrididae, and Hemiptera insects such as Cimex, and more preferably Diptera insects such as Culicidae and Drosophilidae, Orthoptera insects such as Acrididae, and Hemiptera insects such as Cimex. 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).

[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 to be 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 in 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 can 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 also be, for example, 5000 or less, 3000 or less, 2000 or less, 1000 or less, or 700 or less.

[0031] Insect olfactory receptor protein A has reactivity to cancer type a (i.e., the response intensity differs between samples derived from cancer type a patients and samples derived from healthy individuals).

[0032] Insect olfactory receptor protein A, e.g. A receptor (receptor AX) whose response intensity to samples derived from cancer type a patients tends to be higher than that to samples derived from healthy individuals, or Receptors whose response intensity to samples derived from cancer type a patients tends to be lower than that to samples derived from healthy individuals (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 intensity of the insect olfactory receptor protein A can be used as an index (for example, the response intensity being equal to or greater than a cutoff value or less as an index), thereby determining the type of cancer a. 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] The response strength derived from the insect olfactory receptor protein C can be used to correct for individual differences between specimens. This makes it possible to improve the accuracy of cancer type a determination using the insect olfactory receptor protein A. Furthermore, by using the response strength derived from the insect olfactory receptor protein C as an index (for example, by using the response strength within a certain range (for example, not being an abnormal value) as an index), it is possible to improve the accuracy of cancer type a determination when using the insect olfactory receptor protein A for 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' 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 individuals), but is unreactive 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 individuals), or has 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 that of samples derived from healthy individuals).

[0038] Insect olfactory receptor protein A', e.g. A receptor (receptor A'X1) whose response intensity to samples derived from patients with cancer type a tends to be higher than its response intensity to samples derived from healthy subjects, and whose response intensity to samples derived from patients with at least one type of cancer (preferably at least two types) other than cancer type a tends to be equivalent to its response intensity 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 type of cancer (preferably at least two types) 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 its response intensity to samples derived from healthy subjects, and whose response intensity to samples derived from patients with at least one type of cancer (preferably at least two types) other than cancer type a tends to be equivalent to its response intensity 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 its response intensity to samples derived from healthy subjects, and whose response intensity to samples derived from patients with at least one type of cancer (preferably at least two types) other than cancer type a tends to be higher than its response intensity to samples derived from healthy subjects (i.e., whose 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 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). Insect olfactory receptor protein B, e.g. 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 that to samples derived from healthy subjects. is.

[0040] The response intensity of the insect olfactory receptor protein A' can be used as an index (for example, the response intensity being equal to or greater than a cutoff value or less). 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, by combining the response intensity of insect olfactory receptor protein B as an index (for example, by 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' as an index, for example, 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' has reactivity to multiple types of cancer (two or more, preferably three or more) and the direction of the reactivity is the same (i.e., the response intensity of a sample derived from a patient with a certain cancer (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).

[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] By using the response intensity of the insect olfactory receptor protein B' as an index (for example, by using the response intensity as an index that is equal to or greater than a cutoff value), it is possible to make a more thorough determination of whether or not a cancer is present among multiple possible types of cancer. More specifically, for example, 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, contacting a test olfactory receptor protein with a sample derived from a subject having cancer and measuring the response intensity P; 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 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.

[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] By performing the above-mentioned determination using a subject with a certain cancer (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, insect olfactory receptor proteins A, C, A', B, and B' can be screened.

[0051] 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.

[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 not particularly limited as long as it is a polynucleotide comprising a base sequence that is not derived from the genomic DNA (particularly chromosomal genomic DNA) of the cell.

[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. Insect olfactory receptor co-receptors are membrane proteins with a seven-transmembrane structure, similar to olfactory receptors, and function 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+ ) 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 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 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 cancer is determined based on that value. For example, a value that shows a high positive rate in individuals with cancer and 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 Japanese Patent No. 6875815. The resulting expression plasmid was introduced into cells, which were then seeded onto a 384-well plate for luminescence intensity measurement. Luminescence intensity was measured 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 a certain type of cancer (cancer type X) and 25 urine samples from healthy individuals.

[0079] 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 for which the maximum luminescence intensity measured for 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 the difference between the maximum luminescence intensity for cancer type X patients and the maximum luminescence intensity for healthy subjects was greater than 1000 were selected. In this way, insect olfactory receptors reactive to cancer were identified. Among these insect olfactory receptors, there were receptors with higher luminescence intensity in cancer type X patients and receptors with higher luminescence intensity in healthy subjects.

[0080] 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.

[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 the urine of healthy subjects. 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 are not reactive to other cancer types (a and b), or the direction of the reaction is opposite (d). Therefore, by using the strength of the response derived from the receptor as an index (for example, by using the strength of the response as an index above or below a cutoff value), it is possible to determine the specific cancer with higher accuracy, even when there are multiple possible types of cancer.

[0085] 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 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 higher 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.

[0086] Furthermore, since receptors in group c in Table 1 are reactive to multiple types of cancer, by using the strength of the response derived from the receptor as an indicator (using the strength of the response as an indicator that is equal to or greater than the cutoff value), it is possible to more thoroughly determine whether or not a cancer is present 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 (the geometric mean of the time change in luminescence intensity of urine from patients with cancer type X and urine from healthy subjects measured in Test Example 1) was used to determine whether or not a specimen was cancer type X. The specific method is as follows.

[0088] First, for the test sample to be determined, the time change in luminescence intensity for each receptor selected in Test Example 1 was measured. Next, the absolute values ​​of the differences between the geometric mean of the luminescence intensity of the urine of cancer type X patients and the geometric mean of the luminescence intensity of the urine of healthy subjects measured at each time in Test Example 1 and the luminescence intensity of the test sample were calculated, and the cumulative sums for each were calculated. The smaller cumulative sum for the urine of cancer type X patients and the urine of healthy subjects was used as the determination result. As a result, there were three receptors with an accuracy rate of 0.80 or higher.

[0089] Next, individual differences were corrected using the luminescence intensity (measured in Test Example 1) of the insect olfactory receptor (correction receptor) that was not reactive to cancer found in Test Example 1, and the luminescence intensity (measured in Test Example 1) of the insect olfactory receptor (determination receptor) that was reactive to cancer found in Test Example 1 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 receptors i1, i2, i3, i4, i5, i6), the geometric mean of the luminescence intensity of urine from patients with cancer type X measured in Test Example 1 at each time point and the geometric mean of the luminescence intensity of urine from healthy subjects were calculated, and the two geometric means were further geometrically averaged to obtain the time change in the reference luminescence intensity. For each correction receptor, the AUC of the time change in the luminescence intensity of the test sample and the AUC of the time change in the reference luminescence intensity were 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 determination receptor, the luminescence intensity of the test sample at each time was multiplied by a correction coefficient α to obtain a corrected luminescence intensity. For each determination receptor, the absolute value of the difference between the geometric mean of the luminescence intensity of the urine of cancer type X patients and the geometric mean of the luminescence intensity of the urine of healthy subjects measured at each time and 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 cancer type X patients and the urine of healthy subjects was used as the determination result. As a result, six receptors had an accuracy rate of 0.80 or higher.

[0093] From the above, it was demonstrated that the accuracy of determining cancer type X can be improved by using the insect olfactory receptors that are not reactive to cancer, as discovered in Test Example 1, to correct for individual differences.

[0094] Next, whether or not the specimen was cancer type X was determined using the top 1 to 5 receptor combinations, top 1 to 8 receptor combinations, top 1 to 9 receptor combinations, and top 1 to 10 receptor combinations, which had high accuracy rates after correction for individual differences. Specifically, each determination receptor was used to determine whether or not the specimen was cancer type X after correction for individual differences in the same manner as above, and whether or not the specimen was cancer type X was determined by majority vote of the determination results for each of the above combinations of determination receptors. The results are 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.

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 receptor protein B', which has reactivity to multiple types of cancer and the reactivity is in the same direction; 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 screening method according to claim 1, wherein the olfactory receptor protein A has no reactivity to at least one type of cancer other than cancer type a.

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:

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