Method for predicting efficacy of immune checkpoint inhibitor treatment for lung cancer patients

Abstract

This method for predicting the efficacy of treatment of a lung cancer patient using an immune checkpoint inhibitor comprises: isolating exosomes from a biological sample derived from the lung cancer patient; and determining the amount of expression of a protein present in the exosomes by mass spectrometry. The protein is one or more proteins selected from the group of proteins shown in tables 1-1 to 1-6.

Description

[Technical field]

[0001] The present invention relates to a method for predicting the efficacy of immune checkpoint inhibitor treatment for a lung cancer patient. This application claims priority based on Japanese Patent Application No. 2020-056752, filed on March 26, 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] As companion diagnostics for immune checkpoint inhibitors (ICIs) such as anti-PD-1 / PD-L1 antibodies, diagnosis by PD-L1 expression in tumor tissues and diagnosis by microsatellite instability (MSI) testing have been put to practical use. However, in diagnosis by PD-L1 expression, the results vary depending on the antibody, and there are cases where administration of ICIs is effective even in cases where the result is negative. In diagnosis by MSI testing, the percentage of patients with microsatellite instability (MSI-High) is less than 10% in most types of cancer, and there are cases where MSI-High patients show drug resistance. Therefore, it is difficult to say that the sensitivity and specificity of these diagnostic methods are sufficient, and the search for biomarkers to predict treatment efficacy is a major challenge.

[0003] On the other hand, cells are known to release exosomes, which are small lipid bilayer vesicles that contain various cellular contents such as proteins, nucleic acids, lipids, and sugars. Exosomes are important clues for understanding the pathology of the body, and there is active research into how exosomes are involved in various diseases.

[0004] Patent Document 1 discloses the use of perforin in blood samples as a biomarker for evaluating the effect of cancer treatment with ICI.

[0005] Non-Patent Document 1 discloses PD-L1-positive exosomes as a biomarker for predicting the therapeutic effect of PD-1 inhibitors in metastatic gastric cancer.

[0006] Although there are companion diagnostic biomarkers for ICI in lung cancer, there is a need to develop a method for predicting the efficacy of ICI in lung cancer with higher sensitivity and specificity. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent No. 6630026 [Non-patent literature]

[0008] [Non-Patent Document 1] Kim ST et al., “Comprehensive molecular characterization of clinical responses to PD-1 inhibition in metastatic gastric cancer.”, Nat med., Vol. 24, pp. 1449-1458, 2018. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention has been made in consideration of the above circumstances, and provides a novel method for predicting the effectiveness of treatment with an immune checkpoint inhibitor for a lung cancer patient. [Means for solving the problem]

[0010] That is, the present invention includes the following aspects. (1) A method for predicting the efficacy of immune checkpoint inhibitor treatment for a lung cancer patient, comprising: isolating exosomes from a biological sample derived from the lung cancer patient, and determining the expression level of proteins present in the exosomes by mass spectrometry; The method, wherein the protein is one or more proteins selected from the group of proteins shown in the following Tables 1-1 to 1-6.

[0011] [Table 1-1]

[0012] [Table 1-2]

[0013] [Table 1-3]

[0014] [Table 1-4]

[0015] [Table 1-5]

[0016] [Table 1-6]

[0017] The method according to (1), wherein the protein is one or more membrane proteins selected from the group of proteins shown in Table 2 below.

[0018] [Table 2]

[0019] (3) The method according to (1) above, wherein the protein is one or more proteins selected from the group of proteins shown in Table 3 below.

[0020] [Table 3]

[0021] (4) The method according to any one of (1) to (3), wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody. (5) The method according to any one of (1) to (4), wherein the immune checkpoint inhibitor is one or more selected from the group consisting of nivolumab, pembrolizumab, and atezolizumab. (6) The method according to any one of (1) to (5), wherein the method for isolating exosomes from a biological sample derived from a lung cancer patient uses a mixture of magnetic particles bound to an anti-CD9 antibody, an anti-CD81 antibody, or an anti-CD63 antibody. Effect of the Invention

[0022] According to the above aspect, a novel method for predicting the effectiveness of treatment with an immune checkpoint inhibitor for a lung cancer patient can be provided. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] <Immune checkpoint> As used herein, the term "immune checkpoint (system)" refers to a mechanism that suppresses immune responses by suppressing the activity of immune-related cells (including T cells). This system suppresses inappropriate immune responses and excessive inflammatory responses to self-cells and tissues. An immune checkpoint molecule is a molecule that is expressed on immune-related cells and transmits a signal to the immune-related cells to suppress immune responses by binding to a ligand. Examples of immune checkpoint molecules and their ligands include, but are not limited to, PD-1, CTLA-4, TIM-3 (T-cell immunoglobulin and mucin containing protein-3), LAG-3 (lymphocyte activation gene-3), PD-L1, PD-L2, BTNL2, B7-H3, B7-H4, B7-H5 (VISTA), CD48, CD80, 2B4, BTLA, CD160, CD60, CD86, and TIGIT (T cell immunoreceptor with Ig and ITIM domain). As used herein, the term "immune checkpoint inhibitor" refers to a drug that inhibits the binding of an immune checkpoint molecule to its ligand, thereby inhibiting signal transduction by the immune checkpoint molecule. Many cancer cells express immune checkpoint molecule ligands PD-L1, PD-L2, and CD80 on the cell surface, which helps them avoid attacks by immune-related cells such as T cells. It is therefore believed that, under normal conditions, the immune function of the body alone is unable to eliminate cancer tissues. Immune checkpoint inhibitors enable the body's immune function to efficiently eliminate cancer by inhibiting ligand-receptor interactions, which are responsible for the transmission of inhibitory signals from such cancer tissues to the immune function.

[0024] <Immune checkpoint inhibitors> Immune checkpoint inhibitors include PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, and the like. Examples of PD-1 inhibitors include, but are not limited to, anti-PD-1 antibodies such as Nivolumab (sold as Obdivo (registered trademark)) and Pembrolizumab (sold as Keytruda (registered trademark)).

[0025] Examples of PD-L1 inhibitors include, but are not limited to, anti-PD-L1 antibodies such as avelumab, durvalumab, and atezolizumab (sold as Tecentriq (registered trademark)).

[0026] CTLA-4 inhibitors include, but are not limited to, anti-CTLA-4 antibodies such as ipilimumab and tremelimumab.

[0027] Other examples of immune checkpoint inhibitors include drugs that target immune checkpoint proteins such as TIM-3, LAG-3, B7-H3, B7-H4, B7-H5 (VISTA), and TIGIT.

[0028] Among them, the immune checkpoint inhibitor applied to the method of the present embodiment is preferably a PD-1 inhibitor or a PD-L1 inhibitor, more preferably an anti-PD-1 antibody or an anti-PD-L1 antibody. As for the anti-PD-1 antibody or the anti-PD-L1 antibody, it is also preferable to apply it to one or more types selected from the group consisting of nivolumab, pembrolizumab, and atezolizumab described in this embodiment.

[0029] <Exosome-derived proteins for immune checkpoint inhibitor sensitivity assessment> As shown in the Examples below, the inventors analyzed exosomes in the blood of 10 responders and 10 non-responders of lung cancer patients whose sensitivity to immune checkpoint inhibitors was known. As a result, 2406 types of proteins present in exosomes were identified, and among them, exosome-derived proteins with an AUC (Area Under the Curve) value of 0.8 or more of the ROC curve, which is an index of discriminatory ability in determining drug sensitivity, were found. Furthermore, an algorithm was constructed in Python based on the AUC value of the ROC curve and the best cutoff value, and a multimarker with sensitivity and specificity that can be used in clinical practice was found by combining the above proteins.

[0030] <Method for predicting efficacy of immune checkpoint inhibitor treatment for lung cancer patients> A method for predicting the efficacy of treatment with an immune checkpoint inhibitor for a lung cancer patient according to one embodiment of the present invention (hereinafter, sometimes simply referred to as the "method of this embodiment") comprises isolating exosomes from a biological sample derived from a lung cancer patient and determining the expression level of a protein present in the exosome by mass spectrometry (hereinafter, sometimes referred to as "this step"). In this specification and claims, "companion diagnostic agent" refers to a diagnostic agent used in an examination performed before actually starting medication in order to predict the effect of a pharmaceutical product on an individual lung cancer patient, the risk of side effects, and the appropriate dosage.

[0031] The protein is one or more proteins selected from the protein group shown in Tables 4-1 to 4-19 below. As shown in the Examples described later, these proteins have an AUC value of 0.8 or more of the ROC curve, which is an index of discriminatory ability in drug sensitivity assessment, and can be said to be biomarkers with excellent sensitivity and specificity for predicting the effectiveness of treatment with an immune checkpoint inhibitor for lung cancer patients. Therefore, according to the method of this embodiment, the effectiveness of treatment with an immune checkpoint inhibitor for lung cancer patients can be predicted with high accuracy.

[0032] The protein groups shown in the following Tables 4-1 to 4-19 are the same as the protein groups shown in Tables 1-1 to 1-6 above, and for the protein groups shown in Tables 1-1 to 1-6 above, the following information is shown: the Genbank accession number of the mRNA base sequence encoding each human protein, the Genbank accession number of the amino acid sequence of each human protein, the name of the gene encoding each human protein, and the UniProt accession number of the amino acid sequence of each human protein.

[0033] [Table 4-1]

[0034] [Table 4-2]

[0035] [Table 4-3]

[0036] [Table 4-4]

[0037] [Table 4-5]

[0038] [Table 4-6]

[0039] [Table 4-7]

[0040] [Table 4-8]

[0041]

Table 4-9

[0042]

Table 4-10

[0043]

Table 4-11

[0044]

Table 4-12

[0045]

Table 4-13

[0046]

Table 4-14

[0047]

Table 4-15

[0048]

Table 4-16

[0049]

Table 4-17

[0050]

Table 4-18

[0051] [Table 4-19]

[0052] Among the protein groups shown in Tables 4-1 to 4-19, for example, one or more membrane proteins selected from the protein group shown in Table 2 can be preferably used in the method of this embodiment. The protein group shown in Table 2 is a group consisting of 36 membrane proteins having an ROC curve AUC value of 0.8 or more, which is an index of discriminatory ability in drug susceptibility determination, as shown in the Examples described later.

[0053] Among the protein groups shown in Tables 4-1 to 4-19, for example, one or more proteins selected from the protein group shown in Table 3 can be preferably used in the method of this embodiment. The protein group shown in Table 3 is a group consisting of 11 proteins that have an AUC value of 0.8 or more of the ROC curve, which is an index of discriminatory ability in drug susceptibility determination, and do not produce false negatives based on the threshold value calculated by Youden idex for patients with drug susceptibility (responders), as shown in the Examples described later.

[0054] The method of this embodiment will be described in detail below.

[0055] Examples of biological samples used in the method of this embodiment include various liquids such as body fluids, cell culture supernatants, and tissue cell lysates. Of these, body fluids or cell culture supernatants are preferred. Examples of body fluids include whole blood, serum, plasma, various blood cells, blood clots, platelets, and the like, as well as urine, semen, breast milk, sweat, interstitial fluid, interstitial lymphatic fluid, bone marrow fluid, tissue fluid, saliva, gastric juice, synovial fluid, pleural effusion, bile, ascites, and amniotic fluid. Of these, plasma is preferred. The plasma may be treated with an anticoagulant such as citric acid, heparin, or EDTA.

[0056] The biological sample may be pretreated by adding it to a known buffer, or may be taken from a living body and used as it is.

[0057] The amount of the sample can be adjusted appropriately, and may be, for example, 1 μL or more and 1000 μL or less, for example, 1 μL or more and 500 μL or less.

[0058] Methods for isolating exosomes from biological samples include, but are not limited to, PEG precipitation, isolation methods using an ultracentrifuge, immunoprecipitation using exosome-capturing molecules, etc. Among these, it is preferable to use the immunoprecipitation method using exosome-capturing molecules, since it allows selective isolation of exosomes easily without pretreatment.

[0059] The exosome-capturing molecule consists of a substance that specifically binds to an antigen expressed on the surface of an exosome.

[0060] Examples of antigens expressed on the surface of exosomes include, but are not limited to, CD9, CD63, and CD81.

[0061] Exosome-capturing molecules consist of substances that specifically bind to these antigens. Examples of specific binding substances include substances that specifically bind to antigens, such as antibodies and aptamers.

[0062] The antibody may be any antibody that has the ability to bind to an antigen sequence (epitope) on the surface of exosome, and specifically includes, for example, anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody. These antibodies may be monoclonal or polyclonal, but are preferably monoclonal. A mixture of multiple antibodies may also be used. Furthermore, these antibodies may be bispecific antibodies. Examples of antibody classes include IgG and IgM, but are preferably IgG. The antibody may also be a chimeric antibody such as a humanized antibody. The antibody may be derived from an animal species such as a mammal, such as a human, mouse, rabbit, cow, pig, horse, sheep, or goat, or a bird, such as a chicken, and is not particularly limited. The antibody may be prepared, for example, from serum derived from the animal species by a conventionally known method, or an antibody synthesized from known antibody gene sequence information may be used, or a commercially available antibody may be used.

[0063] Moreover, antibody fragments can be used instead of antibodies, including, for example, Fv, Fab, Fab', F(ab')2, rIgG, and scFv.

[0064] An aptamer is a substance that has a specific binding ability to a target substance. Examples of aptamers include nucleic acid aptamers and peptide aptamers. Nucleic acid aptamers that have a specific binding ability to an antigen can be selected, for example, by the systematic evolution of ligand by exponential enrichment (SELEX) method. Furthermore, peptide aptamers that have a specific binding ability to an antigen can be selected, for example, by the two-hybrid method using yeast.

[0065] It is preferable that the exosome-capturing molecule is immobilized on a solid support.

[0066] Examples of materials for the solid phase carrier include polymeric compounds such as polystyrenes, polyethylenes, polypropylenes, polyesters, poly(meth)acrylonitriles, styrene-butadiene copolymers, poly(meth)acrylic esters, fluororesins, cross-linked dextran, and polysaccharides; glass; metals; magnetic materials; resin compositions containing magnetic materials; and combinations of these.

[0067] The shape of the solid phase carrier is not particularly limited, and examples include tray-like, spherical, particulate, fibrous, rod-like, disc-like, container-like, cell, microplate, microfluidic device, and test tube. In this process, magnetic particles are preferred.

[0068] The magnetic particles include metals such as iron oxide (Fe3O4), iron sesquioxide (γ-Fe2O3), various ferrites, iron, manganese, nickel, cobalt, and chromium; magnetic fine particles made of alloys of cobalt, nickel, manganese, and the like; and magnetic particles containing these magnetic materials in a resin. Examples of the resin include hydrophobic polymers and hydrophilic polymers. Among them, magnetic particles containing a magnetic material in a resin are preferred, and those having a polymer layer formed on the surface of mother particles containing superparamagnetic fine particles are more preferred. For example, there are magnetic particles described in JP 2008-32411 A (Reference 1), in which a hydrophobic first polymer layer is formed on the surface of mother particles containing superparamagnetic fine particles, a second polymer layer having at least a glycidyl group on the surface is formed on the first polymer layer, and polar groups are introduced by chemically modifying the glycidyl group.

[0069] Here, the superparamagnetic fine particles are typically iron oxide fine particles having a particle diameter of 20 nm or less (preferably 5 nm to 20 nm), and are represented by XFe2O4 (X=Mn, Co, Ni, Mg, Cu, Li 0.5 Fe 0.5 Examples of the magnetite include ferrite represented by Fe3O4, magnetite represented by Fe3O4, and γ-Fe2O3. In terms of high saturation magnetization and low residual magnetization, it is preferable to include either γ-Fe2O3 or Fe3O4.

[0070] Moreover, the monomers for forming the hydrophobic first polymer layer are roughly classified into monofunctional monomers and crosslinkable monomers. Examples of the monofunctional monomer include aromatic vinyl monomers such as styrene, α-methylstyrene, and halogenated styrene; and ethylenically unsaturated carboxylic acid alkyl ester monomers such as methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, stearyl acrylate, stearyl methacrylate, cyclohexyl acrylate, cyclohexyl methacrylate, isobornyl acrylate, and isobornyl methacrylate. Examples of the crosslinkable monomer include polyfunctional (meth)acrylates such as ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, pentaerythritol triacrylate, pentaerythritol trimethacrylate, dipentaerythritol hexaacrylate, and dipentaerythritol hexamethacrylate; conjugated diolefins such as butadiene and yprene; and divinylbenzene, diallyl phthalate, allyl acrylate, and allyl methacrylate.

[0071] The monomer for forming the second polymer layer is intended mainly for the purpose of introducing functional groups to the particle surface, and includes a glycidyl group-containing monomer. The content of the glycidyl group-containing monomer in the monomer for forming the second polymer layer is preferably 20% by mass or more. Here, examples of the copolymerizable monomer containing a glycidyl group include glycidyl acrylate, glycidyl methacrylate, and allyl glycidyl ether.

[0072] The polar group introduced by chemically modifying the glycidyl group of the second polymer layer is preferably a functional group capable of reacting with a substance that specifically binds to an antigen expressed on the surface of cancer cells, and more preferably contains at least one atom selected from the group consisting of oxygen, nitrogen, and sulfur atoms. Among these, an amino group, an aldehyde group, a carboxyl group, or an active ester group is more preferable. In particular, when the second polymer layer of the magnetic particle has the polar group and a 2,3-dihydroxypropyl group, the binding property with a substance that specifically binds to an antigen expressed on the surface of exosomes (especially an antibody to an antigen expressed on the surface of exosomes) is improved.

[0073] The method of binding a specific binding substance for an antigen expressed on the surface of exosomes to a solid-phase carrier includes a physical adsorption method, a covalent bond method, an ionic bond method, and other chemical binding methods. Examples of physical adsorption methods include a method of directly immobilizing a specific binding substance for an antigen expressed on the surface of exosomes on a solid-phase carrier, and a method of chemically binding the substance to other proteins such as albumin and then adsorbing and immobilizing the substance. Examples of chemical binding methods include a method of directly binding the substance to the solid-phase carrier using a functional group that is introduced onto the surface of the solid-phase carrier and is capable of reacting with a specific binding substance for an antigen expressed on the surface of exosomes, a method of chemically introducing a spacer molecule (such as a carbodiimide compound) between the solid-phase carrier and the specific binding substance for the antigen expressed on the surface of exosomes and then binding the substance, and a method of binding a specific binding substance for an antigen expressed on the surface of exosomes to other proteins such as albumin and then chemically binding the protein to the solid-phase carrier.

[0074] The immunoprecipitation method using an exosome-capturing molecule may be carried out using salts, proteins such as albumin, surfactants, and the like, in addition to the above-mentioned components, as necessary.

[0075] In the immunoprecipitation method using an exosome-capturing molecule, the reaction temperature is usually within the range of 2° C. to 42° C., and the reaction time is usually 5 minutes to 24 hours. When the reaction is performed at 2° C. to 8° C., the reaction time is preferably 8 hours to 30 hours, and when the reaction is performed at 8° C. to 42° C., the reaction time is preferably 5 minutes to 24 hours.

[0076] In the immunoprecipitation method using an exosome-capturing molecule, the pH in the system is not particularly limited, but is preferably in the range of pH 5 to 10, more preferably pH 6 to 8. In order to maintain the desired pH, a buffer solution is usually used, such as a phosphate buffer, a tris(hydroxymethyl)aminomethane buffer, a HEPES buffer, or an MES buffer. Furthermore, when one or more types of solid phase carriers to which exosome-capturing molecules are immobilized are used, the amount used and the mixing ratio are not particularly limited, but are preferably 0.00001 to 10 w / v%, more preferably 0.00001 to 5 w / v%, and even more preferably 0.001 to 0.1 w / v%, relative to the total amount of the liquid phase in the system.

[0077] Mass spectrometry is then used to determine the expression levels of proteins present in the isolated exosomes. The protein for which the expression level is to be determined is one or more proteins selected from the group consisting of the proteins shown in Tables 4-1 to 4-19 above.

[0078] When using mass spectrometry, a pretreatment may be performed to separate substances present in exosomes prior to mass spectrometry. As a separation method, various chromatographic separation techniques can be used, more specifically, for example, liquid chromatography (LC), high performance liquid chromatography (HPLC), gas chromatography (GC), thin layer chromatography, and size exclusion chromatography are included, but are not limited to these. Among them, liquid chromatography or high performance liquid chromatography is preferred.

[0079] Mass spectrometry methods include tandem quadrupole mass spectrometry (MS / MS), direct injection mass spectrometry, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), capillary electrophoresis mass spectrometry (CE-MS), and inductively coupled plasma mass spectrometry. spectrometry (ICP-MS), pyrolysis mass spectroscopy (Py-MS), ion mobility mass spectrometry, time-of-flight mass spectrometry (TOF MS), and the like. Among these, the mass spectrometry method is preferably a mass spectrometry method combined with the above-mentioned separation method, and examples thereof include, but are not limited to, GC-MS, LC-MS, HPLC-MS, LC-MS / MS, and HPLC-MS / MS.

[0080] The expression level of a protein can be determined, for example, by analysis using the DIA (data-independent acquisition) method in mass spectrometry. The DIA method is a data-independent analysis method that comprehensively analyzes all ions within a certain mass range by MS / MS. Among them, the SWATH method, which is one of the DIA methods, is a method in which, for example, the task of acquiring MS / MS 10 times while shifting a 100 Da wide window is repeated, and the MS / MS spectrum of all ions within a total width of 1,000 Da is recorded (the window width and number of acquisitions are variable).

[0081] In the method of this embodiment, the effectiveness of treatment with an immune checkpoint inhibitor in a lung cancer patient from which the biological sample to be evaluated is derived is predicted based on the determined expression level of the protein. Specifically, when the expression level of the protein is equal to or lower than a preset reference value, it can be evaluated or predicted that the lung cancer patient from which the biological sample to be evaluated is derived may have sensitivity to an immune checkpoint inhibitor, i.e., that treatment with an immune checkpoint inhibitor may be effective. On the other hand, when the expression level of the protein is higher than a preset reference value, it can be evaluated or predicted that the lung cancer patient from which the biological sample to be evaluated is derived may not have sensitivity to an immune checkpoint inhibitor, i.e., that treatment with an immune checkpoint inhibitor may not be effective. The reference value is a reference value for distinguishing between a lung cancer patient group that is sensitive to an immune checkpoint inhibitor (responder group) and a lung cancer patient group that is not sensitive to an immune checkpoint inhibitor (non-responder group).

[0082] The reference value can be experimentally determined as a threshold value that can distinguish between the responder group and the non-responder group by measuring the expression level of a specific protein present in the exosomes of the responder group and the non-responder group. The method for determining the reference value of the expression level of a specific protein in the method of this embodiment is not particularly limited, and can be determined, for example, using a general statistical method. The specific protein referred to here means one or more proteins selected from the protein group shown in Tables 4-1 to 4-19 above.

[0083] Specifically, as a method for determining the reference value, for example, for a patient diagnosed with lung cancer by a commonly used method such as imaging diagnosis, the expression level of a specific protein present in exosomes in a biological sample collected in advance before administration of an immune checkpoint inhibitor is measured. After measurement for multiple patients, the expression level of a specific protein present in exosomes in these biological samples is calculated from the average or median, and the value including this can be used as the reference value. In addition, it is preferable that the reference value is described in the instructions of the kit related to the evaluation or prediction method.

[0084] In addition, the expression levels of specific proteins present in exosomes in biological samples collected in advance before administration of an immune checkpoint inhibitor can be measured for multiple responder groups and multiple non-responder groups, and the expression levels and variance of specific proteins present in exosomes in the biological samples of the responder and non-responder groups can be calculated from the average value, median value, etc., and then a threshold value that distinguishes between the two values ​​while taking the variance into account can be determined and used as the reference value.

[0085] <Other embodiments> In one embodiment, the present invention provides a biomarker for predicting or diagnosing the effectiveness of treatment with an immune checkpoint inhibitor for a lung cancer patient, the biomarker consisting of one or more proteins selected from the group of proteins shown in Tables 1-1 to 1-6 above. In one embodiment, the present invention provides a biomarker for predicting or diagnosing the effectiveness of treatment with an immune checkpoint inhibitor for a lung cancer patient, the biomarker consisting of one or more membrane proteins selected from the group of proteins shown in Table 2 above. In one embodiment, the present invention provides a biomarker for predicting or diagnosing the effectiveness of treatment with an immune checkpoint inhibitor for a lung cancer patient, the biomarker consisting of one or more proteins selected from the group of proteins shown in Table 3 above.

[0086] In one embodiment, the present invention provides a method for diagnosing the effectiveness of a treatment with an immune checkpoint inhibitor for a lung cancer patient, i.e., a companion diagnostic method for an immune checkpoint inhibitor for a lung cancer patient, comprising: and isolating exosomes from a biological sample derived from the lung cancer patient and determining the expression level of proteins present in the exosomes by mass spectrometry. The method further comprises the step of: providing said protein comprising one or more proteins selected from the group of proteins shown in Tables 1-1 to 1-6 above.

[0087] In one embodiment, the present invention provides a method for diagnosing the effectiveness of a treatment with an immune checkpoint inhibitor for a lung cancer patient, i.e., a companion diagnostic method for an immune checkpoint inhibitor for a lung cancer patient, comprising: and isolating exosomes from a biological sample derived from the lung cancer patient and determining the expression level of proteins present in the exosomes by mass spectrometry. The protein is one or more proteins selected from the group of proteins shown in Table 2 above.

[0088] In one embodiment, the present invention provides a method for diagnosing the effectiveness of a treatment with an immune checkpoint inhibitor for a lung cancer patient, i.e., a companion diagnostic method for an immune checkpoint inhibitor for a lung cancer patient, comprising: isolating exosomes from a biological sample derived from the lung cancer patient, and determining the expression level of proteins present in the exosomes by mass spectrometry; The protein is one or more proteins selected from the group of proteins shown in Table 3 above. EXAMPLES

[0089] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0090] [Example 1] 1. LC-MS analysis of exosomes (1) Preparation of biological samples EDTA plasma from 20 Japanese lung cancer patients (before administration of nivolumab, pembrolizumab, or atezolizumab) was used in the following study. For the lung cancer patient group, 10 patients who were judged to have a PR (partial response) after administration of nivolumab (12 cases), pembrolizumab (6 cases), or atezolizumab (2 cases) according to the RECIST guidelines (5 patients administered nivolumab, 4 patients administered pembrolizumab, and 1 patient administered atezolizumab) were classified as the responder group, and 3 patients who were judged to have SD (stable disease) (3 patients administered nivolumab) and 7 patients who were judged to have PD (progression) (4 patients administered nivolumab, 2 patients administered pembrolizumab, and 1 patient administered atezolizumab) were classified as the non-responder group.

[0091] (2) Antibody-bound particles for capturing exosomes Magnetic particles (Magnosphere, manufactured by JSR Life Sciences Corporation) TM Exosome capture antibodies (Anti-CD9mAb (MBL, MEX001-3), Anti-CD63 (LAMP-3)mAb (MBL, MEX002-3), or Anti-CD81 (TAPA1)mAb (MBL, MEX003-3)) were bound to the antibody-binding protein MS300 / Carboxyl to prepare three types of antibody-binding particles. Equal amounts of the three types of antibody-binding particles were then mixed, and the solution with a final particle concentration of 0.06% by mass was used as the antibody-binding particle solution in the following tests.

[0092] (3) Exosome isolation The frozen and thawed EDTA plasma was filtered through a membrane filter with a pore size of 0.22 μm (Merck Millipore, SLGV033RS), and 0.5 mL was added to a 15 mL low-adsorption tube (Sumitomo Bakelite, MS-52150). Next, 4.5 mL of TBS was added to dilute the solution 10-fold, and 5 mL of the antibody-bound particle solution was added, followed by stirring at room temperature overnight. After the reaction, the antibody-bound particles were transferred to a 2 mL low-adsorption tube (Sumitomo Bakelite Co., Ltd., MS-4220M) and washed with a washing solution (ExoCap TM The particles were washed four times with Ultracentrifugation / Storage Booster (MBL, MEX-USB). The antibody-bound particles were transferred to a new 2 mL low-adsorption tube and washed twice with TBS. Then, 50 μL of 2% by mass sodium dodecyl sulfate (SDS) solution was added and shaken at 25 °C for 10 minutes. The supernatant after the reaction was added to a 100K ultrafiltration spin column (PALL, OD100C34) and centrifuged at 10,000 × g for 5 minutes, after which the filtrate was collected in a 2 mL low-adsorption tube and used as the exosome extract.

[0093] (4) LC-MS / MS analysis of exosome proteins 25 μL of the exosome extract was transferred to a 1.5 mL tube (Eppendorf, 0030123328), 8 times the amount of cold acetone was added, and the mixture was left to stand at -20 ° C for 2 hours. Then, after centrifugation at 4 ° C and 15,000 × g for 15 minutes, 20 μL of 100 mM Tris (pH 8.5) solution containing 0.5% by mass sodium dodecanoate was added to the precipitate, and the protein was dissolved by a closed ultrasonic crusher. In order to cleave the SS bond of the protein, dithiothreitol (DTT) was added to a final concentration of 10 mM and reacted at 50 ° C for 30 minutes. In order to alkylate the cysteine ​​residue, iodoacetamide was added to a final concentration of 30 mM and reacted at room temperature for 30 minutes under light-shielded conditions. To stop the reaction, cysteine ​​was added to a final concentration of 60 mM and left to stand at room temperature for 10 minutes. After that, 150 μL of 50 mM ammonium bicarbonate was added, followed by the addition of 250 ng of Lys-C (Fujifilm Wako Pure Chemical Industries, Ltd., 125-05061) and 250 ng of trypsin (Promega, V5113), and the reaction was allowed to proceed overnight at 37 ° C. to degrade the protein into peptides. To remove sodium dodecanoate, 5% by mass of trifluoroacetic acid (TFA) was added to make the solution acidic, and the solution was centrifuged at 15,000 × g for 10 minutes at room temperature to collect the supernatant. After desalting using a C18 column, the solution was dried in a centrifugal evaporator, a solution containing 3% by mass acetonitrile-0.1% by mass formic acid was added, and the peptides were dissolved in a sample sealed ultrasonic disintegrator, followed by LC-MS analysis. LC-MS analysis was performed using Thermo Fisher Scientific's Q Exactive TM Protein identification and relative quantification were performed using HF-X and Scaffold DIA software.

[0094] 2. Calculation of the area under the receiver operating characteristic (ROC) curve (AUC) Proteins analyzed using the Scaffold DIA software in "1.(4)" were extracted with ProteinFDR of 1% or less, and the immunoglobulin and keratin groups, which were considered to be contaminations, were excluded, and the relative quantitative values ​​were normalized. 2,406 proteins were identified from lung cancer patients. For these proteins, the AUC, which is an index of discriminatory ability, was calculated using Scikit-learn, an open source library of the programming language Python, and the threshold for discriminating between the two groups was calculated using a method using the Youden index. 825 proteins with AUC values ​​of 0.7 or more and their AUC values ​​are shown in Tables 5-1 to 5-54. The amino acid sequence of "Putative nucleoside diphosphate kinase" in Table 5-19 is shown in SEQ ID NO:1.

[0095] [Table 5-1]

[0096] [Table 5-2]

[0097] [Table 5-3]

[0098] [Table 5-4]

[0099] [Table 5-5]

[0100] [Table 5-6]

[0101]

Table 5-7

[0102]

Table 5-8

[0103]

Table 5-9

[0104]

Table 5-10

[0105]

Table 5-11

[0106]

Table 5-12

[0107]

Table 5-13

[0108]

Table 5-14

[0109]

Table 5-15

[0110]

Table 5-16

[0111]

Table 5-17

[0112]

Table 5-18

[0113]

Table 5-19

[0114]

Table 5-20

[0115]

Table 5-21

[0116]

Table 5-22

[0117]

Table 5-23

[0118]

Table 5-24

[0119]

Table 5-25

[0120]

Table 5-26

[0121]

Table 5-27

[0122]

Table 5-28

[0123]

Table 5-29

[0124]

Table 5-30

[0125]

Table 5-31

[0126]

Table 5-32

[0127]

Table 5-33

[0128]

Table 5-34

[0129]

Table 5-35

[0130]

Table 5-36

[0131]

Table 5-37

[0132]

Table 5-38

[0133]

Table 5-39

[0134]

Table 5-40

[0135]

Table 5-41

[0136]

Table 5-42

[0137]

Table 5-43

[0138]

Table 5-44

[0139]

Table 5-45

[0140]

Table 5-46

[0141]

Table 5-47

[0142] [Table 5-48]

[0143] [Table 5-49]

[0144] [Table 5-50]

[0145] [Table 5-51]

[0146] [Table 5-52]

[0147] [Table 5-53]

[0148] [Table 5-54]

[0149] As shown in Tables 5-1 to 5-54, a group of proteins that can be used to determine drug sensitivity was found among the proteins present in exosomes in the blood of lung cancer patients.

[0150] 3. Extraction of membrane proteins Based on the predicted membrane protein information from The Human Protein Atlas (https: / / www.proteinatlas.org / ), membrane protein groups were extracted from the protein groups in Tables 5-1 to 5-54. The obtained membrane protein lists are shown in Tables 6-1 to 6-15.

[0151] [Table 6-1]

[0152] [Table 6-2]

[0153] [Table 6-3]

[0154] [Table 6-4]

[0155] [Table 6-5]

[0156] [Table 6-6]

[0157] [Table 6-7]

[0158] [Table 6-8]

[0159] [Table 6-9]

[0160] [Table 6-10]

[0161] [Table 6-11]

[0162] [Table 6-12]

[0163] [Table 6-13]

[0164] [Table 6-14]

[0165] [Table 6-15]

[0166] As shown in Tables 6-1 to 6-15, 36 types of membrane proteins were detected with an AUC of 0.8 or more, and 168 types were detected with an AUC of 0.7 or more.

[0167] 4. Extraction of protein groups that do not produce false negatives for drug-sensitive patients (responders) Protein groups that did not produce false negatives were extracted based on the threshold calculated using the Youden index for the 10 responders, and the results were sorted in order of highest AUC. Tables 7-1 to 7-4 show the results.

[0168] [Table 7-1]

[0169] [Table 7-2]

[0170] [Table 7-3]

[0171] [Table 7-4]

[0172] As shown in Tables 7-1 to 7-4, there were 11 proteins with an AUC of 0.8 or more, and 47 proteins with an AUC of 0.7 or more. It was thought that by combining these markers, it would be possible to construct a multi-marker that is resistant to errors. [Industrial Applicability]

[0173] According to the method of this embodiment, the effectiveness of treatment with immune checkpoint inhibitors for lung cancer patients can be predicted with high accuracy.

Claims

1. 1. A method for predicting the efficacy of immune checkpoint inhibitor treatment for a lung cancer patient, comprising: and isolating exosomes from a biological sample derived from the lung cancer patient and determining the expression level of proteins present in the exosomes by mass spectrometry. The method, wherein the protein is one or more proteins selected from the group of proteins shown in Tables 1-1 to 1-6 below. 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 1-5】 【Table 1-6】

2. The method of claim 1, wherein the protein is one or more membrane proteins selected from the group of proteins shown in Table 2 below. 【Table 2】

3. The method of claim 1, wherein the protein is one or more proteins selected from the group of proteins shown in Table 3 below. 【Table 3】

4. The method according to any one of claims 1 to 3, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

5. The method according to any one of claims 1 to 4, wherein the immune checkpoint inhibitor is one or more selected from the group consisting of nivolumab, pembrolizumab, and atezolizumab.

6. The method according to any one of claims 1 to 5, wherein the method for isolating exosomes from a biological sample derived from a lung cancer patient uses a mixture of magnetic particles bound to an anti-CD9 antibody, an anti-CD81 antibody, or an anti-CD63 antibody.

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