WO2021045224A1 - がん免疫療法等における免疫関連有害事象の予測 - Google Patents

がん免疫療法等における免疫関連有害事象の予測 Download PDF

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WO2021045224A1
WO2021045224A1 PCT/JP2020/033698 JP2020033698W WO2021045224A1 WO 2021045224 A1 WO2021045224 A1 WO 2021045224A1 JP 2020033698 W JP2020033698 W JP 2020033698W WO 2021045224 A1 WO2021045224 A1 WO 2021045224A1
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cancer
lung
risk
gprc5a
treatment
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French (fr)
Japanese (ja)
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博嘉 西川
ビタリー コチン
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Tokai National Higher Education and Research System NUC
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Tokai National Higher Education and Research System NUC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a biomarker useful for prediction and diagnosis of lung disorders (particularly interstitial pneumonia).
  • the present invention relates to biomarkers that can be used for predicting lung disorders as immune-related adverse events in cancer immunotherapy and the like, and their uses.
  • Interstitial pneumonia is a disease that causes inflammation mainly in the stroma of the lung, and is known to develop due to various causes (for example, autoimmune diseases such as collagen disease, drugs, and infectious diseases). Unspecified interstitial pneumonia is called "idiopathic interstitial pneumonia". Idiopathic interstitial pneumonia is classified into subtypes such as idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia (NSIP), and specific organizing pneumonia (COP). Idiopathic interstitial pneumonia is designated as an intractable disease, and careful treatment is required for its diagnosis / differentiation and treatment. In addition, antifibrotic drugs (pi veneidone, nintedanib), steroids, immunosuppressive drugs, etc.
  • Idiopathic interstitial pneumonia is classified into subtypes such as idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia (NSIP), and specific organizing pneumonia (COP). Idiopathic interstitial pneumonia is designated as an intractable disease, and careful treatment is required for its diagnosis
  • cancer immunotherapy generally attacks cancer cells and obtains a therapeutic effect by activating the antitumor immune response in the body.
  • the immune response to the self is also unintentionally activated, which has been a problem of inducing the autoimmune response.
  • interstitial pneumonia which is an autoimmune reaction to lung tissue
  • ICB immune checkpoint inhibitors
  • ICB immune checkpoint blockade
  • irAE immune-related adverse events
  • serious lung damage is recognized, and countermeasures are urgently needed. If the risk (risk) of lung injury represented by interstitial pneumonia is known, it will be possible to avoid it in advance (change of treatment method, etc.) and take early measures, and it will be possible to provide more appropriate and highly effective treatment. ..
  • a determination method using a polymorphism of the STAT3 gene has been proposed as a means for predicting the risk of developing interstitial pneumonia due to a molecular target drug or an immune checkpoint inhibitor (Patent Document 1).
  • the present invention poses a problem as an adverse event such as the onset of lung disorders represented by interstitial pneumonia (particularly, idiopathic interstitial pneumonia) (particularly in cancer immunotherapy and the like).
  • the main task is to find a means for predicting the risk of developing or exacerbating lung damage) and to utilize and apply it.
  • an immune checkpoint inhibitor activates only the antitumor immune response, attacking cancer cells but not normal cells, but sometimes in the thymus. It may activate an immune response (autoimmune response) to normal cells that have bypassed education, which causes immune-related adverse events (irAEs). Therefore, it is important to identify antigens recognized by immune cells attacking normal cells and regulate the immune response to them in order to control irAE.
  • autoimmune response immune response to normal cells that have bypassed education, which causes immune-related adverse events (irAEs). Therefore, it is important to identify antigens recognized by immune cells attacking normal cells and regulate the immune response to them in order to control irAE.
  • HLA-2402 which is a specific HLA (human leukocyte antigen) characteristic of Japanese, has a causative antigen. Therefore, the antigen presented by HLA-2402 was comprehensively analyzed while limiting it to lung-specific peptide antigens.
  • GPRC5A G protein-coupled receptor class C group 5 member A
  • lung damage can also occur in cancer immunotherapy other than immune checkpoint inhibitors and cancer treatment using anticancer drugs.
  • complications are also observed in autoimmune diseases such as rheumatoid arthritis, and there are many cases in which the disease worsens with treatment.
  • GPRC5A is expressed specifically in lung tissue
  • the presumed mechanism that lung tissue is damaged by an immune response involving HLA-A2402 and GPRC5A identified as its antigen peptide, as well as anti-cancer agents If we consider that the same immune response may occur in lung disorders caused by treatment and in autoimmune diseases, the onset and exacerbation of lung disorders in various cancer treatments and autoimmune diseases It can be reasonably understood that anti-GPRC5A antibody and HLA-A2402 binding GPRC5A peptide-specific cytotoxic T cells are useful as biomarkers for predicting.
  • anti-GPRC5A antibody and HLA-A2402 restrictive GPRC5A peptide are also biomarkers for predicting the risk of developing idiopathic interstitial pneumonia, which requires careful diagnosis / differentiation and is often difficult to treat. It is suggested that specific cytotoxic T cells and the like are extremely useful. Thus, patients undergoing various cancer treatments, patients with autoimmune diseases, etc., who have lung disorders or who may have exacerbated lung disorders, and patients with idiopathic interstitial pneumonia / potential It is possible to provide a patient with a means for predicting the risk of onset or exacerbation by an objective criterion, that is, the level of a biomarker consisting of a specific substance in the living body.
  • the means include, for example, selection / identification of treatment targets (exclusion of patients who should not be treated) and determination of treatment policy before treatment, and early treatment of lung disorders during treatment (for example, early treatment by monitoring the pathological condition). It is useful for (intervention), review or change of treatment policy, estimation of prognosis after treatment, etc., and can maximize the treatment effect and improve the QOL of patients.
  • a biomarker for lung disease consisting of anti-GPRC5A antibody or HLA-A2402 restrictive GPRC5A peptide-specific cytotoxic T cells.
  • the biomarker according to [1] which is used for predicting the risk of lung injury.
  • [6] A method for predicting the risk of lung injury using the level of a biomarker consisting of anti-GPRC5A antibody or HLA-A2402 binding GPRC5A peptide-specific cytotoxic T cells in a blood sample derived from a subject as an index.
  • the risk prediction method according to [6] wherein the lung disorder is a lung disorder associated with cancer treatment or treatment of an autoimmune disease.
  • the risk prediction method according to [7] wherein the cancer treatment is cancer immunotherapy.
  • cancer immunotherapy is treatment with an immune checkpoint inhibitor.
  • the cancers to be treated are non-small cell lung cancer, small cell lung cancer, breast cancer, cervical cancer, uterine body cancer, endometrial cancer, ovarian cancer, vaginal cancer, gastric cancer, duodenal cancer.
  • the cancers to be treated are non-small cell lung cancer, small cell lung cancer, gastric cancer, colon cancer, esophageal cancer, malignant melanoma, or hepatocellular carcinoma, [7] to [9].
  • Autoimmune diseases include rheumatoid arthritis, systemic erythematosus, rheumatic fever, scleroderma, dermatomyositis, polymyositis, nodular polyarteritis, Sjogren's syndrome, mixed connective tissue disease (MCTD), and multiple occurrences.
  • Vascular granulomatosis (Wegener's granulomatosis), eosinophilic polyangiitis granulomatosis (Charg-Strauss syndrome), microscopic polyangiitis, Takayasu's arteritis (aortitis syndrome), giant cell arteritis Flame (temporal arteritis), rheumatic polymyositis, eosinophilic granulomatitis, adult Still's disease, tonic spondylitis, psoriatic arteritis, recurrent polychondritis, Bechet's disease or sarcoidosis, [ 7] The risk prediction method described in.
  • the risk prediction method according to any one of [6] to [12], wherein the lung disorder is interstitial pneumonia.
  • the risk prediction method according to [6], wherein the lung disorder is idiopathic interstitial pneumonia.
  • a kit for predicting, diagnosing or differentiating the risk of lung injury which comprises a reagent for measuring an anti-GPRC5A antibody.
  • the kit according to [21] which is a kit based on the principle of enzyme immunoassay or fluorescence immunoassay.
  • a kit for predicting, diagnosing or differentiating the risk of lung injury which comprises a probe labeled with a multimerized HLA-A2402-peptide complex or a reagent for preparing the probe.
  • HLA-A24 antigenic peptides.
  • Peptides presented on HLA-A24 by various cancer cell lines were recovered using immunoprecipitation (center) and analyzed by mass spectrometry (right). Expression of the three identified candidate peptides in each tissue. It was detected and compared by the RT-PCR method.
  • BRAIN brain
  • COLON colon
  • HEART heart
  • INTEST small intestine
  • KIDONEY kidney
  • LEUK white blood cells
  • LIVER liver
  • LUNG lungs
  • OVARY ovary
  • PNCR. PLCNT
  • PROST. Progester
  • the vertical axis shows the average value of the difference in fluorescence intensity between the case with and without the primary antibody, and represents the detected amount of GPRC5A peptide.
  • the horizontal axis shows the peptide concentration at the time of pulse. Detection of GPRC5A antibody in patients with lung injury. It was detected and compared by flow cytometry.
  • the vertical axis shows the number of cells.
  • the horizontal axis indicates the fluorescence intensity and represents the amount of GPRC5A antibody detected. Arrows show a sample of patients who have experienced interstitial pneumonia. Detection of GPRC5A antibody in patients with lung injury. Detected and compared by ELISA.
  • the horizontal line in each column indicates the median value, the cross indicates the average value, the upper end of each column indicates the 75th percentile value, the lower end of each column indicates the 25th percentile value, and the upper end of the line extending from each column is the maximum. The value is shown, and the lower end of the line extending from each column shows the minimum value.
  • Biomarkers of lung injury As described above, according to the study by the present inventors, an immune response targeting GPRC5A is involved in lung injury (particularly interstitial pneumonia) in cancer treatment using immunity checkpoint inhibitors. It was revealed that "the presence of an immune response to GPRC5A” reflects the risk (risk / possibility) of lung damage (particularly interstitial pneumonia). Based on this result, the first aspect of the present invention is a "biomarker of lung injury” (hereinafter, abbreviated as “biomarker of the present invention”, which is a marker substance useful for detecting or grasping the presence of the immunity response. (Sometimes called).
  • the biomarker is particularly useful for predicting the risk of interstitial pneumonia among lung disorders, and can be used, for example, for diagnosing / assessing idiopathic interstitial pneumonia and predicting the risk of developing it.
  • it has high utility value as a means for predicting the risk of lung injury associated with cancer treatment or treatment of autoimmune disease, that is, the biomarker of the present invention is "lung associated with cancer treatment or treatment of autoimmune disease”. It is also extremely useful as a "biomarker that predicts the risk of disability.”
  • Biomarkers that predict the risk of lung damage associated with cancer treatment or treatment of autoimmune diseases are the risks of lung damage or exacerbation of lung damage associated with treatment in cancer treatment or treatment of autoimmune diseases.
  • the biomarkers of the present invention provide an objective judgment material for distinguishing patients at high risk of developing lung injury as an adverse event from other patients.
  • the biomarker of the present invention has high utility value in identifying or selecting a patient who is at high risk of developing lung injury as an adverse event.
  • biomolecule means a molecule (compound) found in a living body.
  • a specific biomolecule is used as a biomarker, but when using it (typically, application to a risk prediction method), a biomolecule in a blood sample / sample separated from the living body is used. ..
  • the present invention utilizes "anti-GPRC5A antibody” or "HLA-A2402 restrictive GPRC5A peptide-specific cytotoxic T cells” that are useful for detecting the presence of an immune response to GPRC5A. That is, the biomarker of the present invention is composed of "anti-GPRC5A antibody” or "HLA-A2402 restrictive GPRC5A peptide-specific cytotoxic T cell".
  • GPRC5A has been reported to be associated with esophageal cancer, pancreatic cancer, etc. (see, for example, Non-Patent Documents 1 and 2).
  • the amino acid sequence of GPRC5A NCBI, Gene ID: 9052 registered in the public database and the gene sequence encoding it are shown in the attached sequence listing.
  • SEQ ID NO: 1 Amino acid sequence of GPRC5A (NCBI Reference Sequence: NP_003970.1 retinoic acid-induced protein 3 [Homo sapiens])
  • SEQ ID NO: 2 cDNA sequence of GPRC5A (NCBI Reference Sequence: NM_003979.3, Homo sapiens G protein-coupled receptor class C group 5 member A (GPRC5A), mRNA)
  • HLA-A2402 binding GPRC5A peptide-specific cytotoxic T cells (hereinafter, may be referred to as "GPRC5A-specific CTL" for convenience of explanation) specifically recognize the complex of HLA-A2402 and GPRC5A peptide. It is a T cell (CTL) that exhibits cytotoxic activity. It is unique and characteristic that a particular CTL becomes a biomarker.
  • CTL T cell
  • the "lung disorder” in the present invention typically occurs with the treatment of cancer or autoimmune diseases, that is, is positioned as an adverse event in these treatments.
  • the term "pulmonary disorder” should be interpreted in a broad sense, and its type, degree, degree of progression, etc. are not particularly limited.
  • interstitial pneumonia is the target of risk prediction.
  • the present invention can also be used for risk prediction, diagnosis / differentiation, etc. of idiopathic interstitial pneumonia.
  • Cancer treatment is not particularly limited as long as lung damage as an adverse event may occur or the lung damage may be exacerbated when it is applied (that is, when it is used for treating a patient). Therefore, cancer immunotherapy, drug therapy targeting cancer (chemotherapy), etc. correspond to cancer treatment. From the mechanism (estimated) that GPRC5A is released and diffused from cancer cells crushed by cancer treatment and an immune response using GPRC5A peptide as an antigen is triggered to cause lung damage, cancer is treated. As far as the subject is concerned, not only immunotherapy and drug therapy (chemotherapy), but also radiotherapy and surgical therapy (surgical resection) can be classified as "cancer treatment” in the present invention.
  • Immunotherapy in cancer treatment includes treatment with immune checkpoint inhibitors (typically antibodies), antibody therapy, and tumor-specific T cells (CAR-).
  • immune checkpoint inhibitors typically antibodies
  • CAR- tumor-specific T cells
  • adoptive immunotherapy using non-specific effector cells adoptive immunotherapy using non-specific effector cells, agonist antibody against costimulatory molecule
  • cytokine therapy cytokine gene therapy, etc.
  • the biomarker of the present invention is particularly useful for predicting the risk of lung injury (particularly interstitial pneumonia) associated with treatment with an immune checkpoint inhibitor or antibody therapy.
  • antibody drugs used for these treatments include anti-PD-1 antibody drugs such as nivolumab (Opdivo®) and pembrolizumab (Keytruda®), avelumab (Bavencio®), and atezolizumab ( Anti-PD-L1 antibody drugs such as Tecentriq (registered trademark) and Durvalumab (Imfinzi (registered trademark)), anti-CTLA-4 antibody drugs such as ipilimumab (Yervoy (registered trademark)) and tremelimumab, rituximab (Rituxan (registered trademark)) ), Trastuzumab (Herceptin®), Alemtuzumab (Campath®), Cetuximab (Erbitux®), Panitumumab (Vectibi
  • cancers are non-small cell lung cancer, small cell lung cancer, breast cancer, cervical cancer, uterine body cancer, endometrial cancer, ovarian cancer, vaginal cancer, gastric cancer, duodenal cancer, small intestinal cancer , Colorectal cancer, rectal cancer, colon cancer, anal cancer, esophageal cancer, oral cancer, tongue cancer, pharyngeal cancer, head and neck cancer, gastrointestinal stromal tumor (GIST), malignant melanoma (Melanoma), hepatocellular carcinoma, bile duct cancer (extrahepatic bile duct cancer, intrahepatic bile duct cancer), bile sac cancer, biliary tract cancer, pancreatic cancer, renal cell cancer, bladder cancer, urinary tract , Prostate cancer, thyroid cancer, adrenal cancer, pituitary tumor, pineapple tumor, neuroblastoma, glioma, gliogry cell tumor, myeloid blast species, hodgkin lymphoma, B-cell non-ho
  • the biomarker of the present invention can also be used to predict the risk of lung damage associated with the treatment of autoimmune diseases.
  • autoimmune diseases include rheumatoid arthritis, systemic erythematosus, rheumatic fever, scleroderma, dermatomyositis, polymyositis, nodular polyarteritis, Sjogren's syndrome, mixed connective tissue disease (MCTD), and polyangiitis.
  • Germocytosis (Wegener's granulomatosis), eosinophilia polyangiitis granulomatosis (Charg-Strauss syndrome), microscopic polyangiitis, Takayasu's arteritis (aortitis syndrome), giant cell arteritis ( Temporal arteritis), rheumatic polymyositis, eosinophilic myocarditis, adult Still's disease, tonic spondylitis, psoriatic arteritis, recurrent polychondritis, Bechet's disease and sarcoidosis.
  • a second aspect of the present invention relates to a method for predicting the risk of lung damage with respect to the use of the biomarker of the present invention (hereinafter, may be referred to as "risk prediction method of the present invention").
  • risk prediction method of the present invention the level of the biomarker (anti-GPRC5A antibody or HLA-A2402 binding GPRC5A peptide-specific cytotoxic T cell) of the present invention in a blood sample derived from a subject is used as an index for lung injury.
  • Predict risk Typically, the following steps (1) to (3) will be carried out.
  • a step of preparing a blood sample collected from a subject (2) A step of measuring the biomarker in the blood sample (3) A step of determining the risk of lung injury based on the measured value. A step in which high levels of the biomarker represent a high risk of lung injury
  • Step (1) a blood sample derived from the subject is prepared.
  • cancer treatment or treatment of autoimmune disease is planned or considered, and lung injury as an adverse event occurs or lung injury.
  • patients who are concerned about the possibility of exacerbation are the subjects.
  • the present invention is particularly useful for predicting the risk of interstitial pneumonia among lung disorders, and when the present invention is used for predicting the risk of interstitial pneumonia, for example, interstitial pneumonia (particularly idiopathic) Those who are suspected of developing (interstitial pneumonia) or who may develop it in the future are the subjects.
  • the allele in the antigen and its combination in HLA possessed by the subject are not particularly limited.
  • Examples of alleles in the antigen include A * 02: 01, A * 02: 06, A * 02: 07, A * 02: 10, A * 02: 18, A * 02: 03, A * 24: 02, A.
  • the subject's blood for example, peripheral blood
  • plasma or serum can be used as the blood sample.
  • Blood samples may be collected and prepared by a conventional method. Blood samples are prepared prior to the practice of the present invention. That is, the risk prediction method of the present invention does not include treatment (blood sampling) for a patient to prepare a sample.
  • Step (2) the biomarker in the blood sample is measured.
  • An appropriate measurement method is used depending on the biomarker to be adopted.
  • anti-GPRC5A antibody is adopted as a biomarker, measurement is usually performed using an immunological method. According to the immunological method, rapid and sensitive measurement is possible. Also, the operation is simple. Immunological measurements use substances that have specific binding to the biomarker anti-GPRC5A antibody. As the substance, GPRC5A or an antibody-binding fragment thereof is typically used.
  • the "antibody-binding fragment” is an "GPRC5A fragment” containing an epitope that binds to an anti-GPRC5A antibody, and has a binding property to an anti-GPRC5A antibody as well as a full-length GPRC5A.
  • the measurement method examples include fluorescence immunoassay (FIA method), enzyme immunoassay (EIA method), radioimmunoassay (RIA method), Western blotting, and latex agglutination method.
  • Preferred measurement methods include the FIA method and the EIA method. According to these methods, it is possible to measure with high sensitivity, quickly and easily.
  • FIA method a fluorescent label is used to detect an antigen-antibody complex (immune complex) using fluorescence as a signal.
  • an enzyme label is used to detect an immune complex using color development or luminescence based on an enzyme reaction as a signal.
  • FIA method or the EIA method include an ELISA method (Enzyme Linked Immunosolvent Assay), an indirect fluorescent antibody method (IIF), a fluorescent enzyme immunoassay (FEIA), and a chemiluminescent enzyme immunoassay (CLEIA). ..
  • the ELISA method has many advantages such as high detection sensitivity, high specificity, excellent quantification, easy operation, and suitable for simultaneous processing of multiple samples.
  • An example of a specific operation method when using the ELISA method is shown below.
  • an antigen GPRC5A (eg, human recombinant GPRC5A) or an antibody-binding fragment thereof) is immobilized on an insoluble support.
  • the surface of a microplate is sensitized (coated) with an antigen (GPRC5A or an antibody-binding fragment thereof).
  • the sample is brought into contact with the immobilized antigen.
  • the anti-GPRC5A antibody As a result of this operation, if the anti-GPRC5A antibody is present in the sample, the immobilized antigen and the anti-GPRC5A antibody bind to each other to form an immune complex. After removing the non-specific binding component by a washing operation, the immune complex is detected using a labeled secondary antibody. Instead of such an indirect method, the detection may be performed by a direct method, a sandwich method, a competitive method, or the like. The details of the ELISA method are described in many books and treatises, which can be referred to when setting the experimental procedure and experimental conditions of each method.
  • HLA-A2402 binding GPRC5A peptide-specific cytotoxic T cells are adopted as biomarkers, for example, the binding between biotin and avidin (preferably streptavidin) is used.
  • GPRC5A-specific CTL in a sample is measured using a reagent obtained by tetramerizing the complex of HLA and antigen peptide (HLA tetramer) as a probe.
  • a multimerized probe in which a plurality of MHC-peptide complexes are bound to a fluorescently labeled polymer (for example, a dextran polymer) has also been developed, and measurement using the probe is also possible.
  • the amount of GPRC5A-specific CTL is very small even if it is present in the sample, it is usually amplified prior to detection in order to improve measurement accuracy and detection sensitivity.
  • contact between the complex of the HLA-A2402 molecule and the GPRC5A peptide and the GPRC5A-specific CTL that may be present in the sample in the presence of the HLA / antigen peptide is required. Incubate the specimen under the conditions that arise. The presence of GPRC5A-specific CTLs in the sample results in specific amplification due to the addition of stimulation by the GPRC5A peptide presented on HLA-A2402.
  • Step (3) the risk of lung injury is determined based on the measurement result of step (2), that is, the level of the biomarker.
  • the “level” here typically means “amount” or “concentration”. However, the term “level” is also used to indicate whether or not the biomarker of the present invention can be detected (that is, whether or not it is apparently present) in accordance with conventional practice and common general knowledge.
  • the "risk of lung disorder” in the present invention means a specific treatment (cancer treatment or self) received by a subject when predicting the risk of lung disorder associated with cancer treatment or treatment of an autoimmune disease. It is a risk that lung damage will occur or worsen with the treatment of immune diseases).
  • the criterion that "a high level of biomarker represents a high risk of lung injury" is adopted.
  • low levels of biomarkers determine a low risk of lung injury.
  • the determination is made by comparing the measured value obtained in step (2) with the measured value of the control (control sample) or by comparing with the reference value set based on the measured value of the control or the like.
  • the control for example, a biomarker level (positive control) of a patient with pulmonary disorder, a biomarker level (negative control) of a patient without pulmonary disorder, and a bimarker level (negative control) of a healthy person can be used. ..
  • the reference value is set based on the measured values obtained from a plurality of control subjects (preferably 50 or more, more preferably 100 or more).
  • the risk of lung injury may be determined either qualitatively or quantitatively. An example of determination is shown below.
  • the judgment in the present invention is automatic / mechanical without the judgment of a person having specialized knowledge such as a doctor or a laboratory technician. Can be done.
  • Biomarker measurement ⁇ a risk of lung injury 10% or less
  • a ⁇ biomarker measurement ⁇ b risk of lung injury 20% to 30%
  • b ⁇ biomarker measurement ⁇ c 40% to 50% risk of lung injury
  • c ⁇ biomarker measurement ⁇ d risk of lung injury 60% to 70%
  • d ⁇ biomarker measurement 80% or more risk of lung injury
  • Biomarker measurement ⁇ a risk of exacerbation of lung injury 10% or less
  • a ⁇ biomarker measurement ⁇ b risk of exacerbation of lung injury 20% to 30%
  • b ⁇ biomarker measurement ⁇ c 40% to 50% risk of exacerbation of lung injury
  • c ⁇ biomarker measurement ⁇ d risk of exacerbation of lung injury 60% to 70%
  • d ⁇ biomarker measurement 80% or more risk of exacerbation of lung damage
  • the current measured value is compared with the measured value at a certain point in the past, and the measured value fluctuates, that is, whether or not the biomarker level is increased or decreased and / or increased or decreased.
  • the resulting data on changes in biomarker levels can be useful information for monitoring the risk of lung injury or for prognosis estimation. Specifically, for example, it can be determined that the risk of lung injury has increased, decreased, or has not changed between the previous examination and the current examination based on the fluctuation of the biomarker level. .. If such an evaluation is performed in parallel with the treatment, the signs of lung damage can be grasped at an early stage, and a more appropriate treatment policy can be determined. As a result, the therapeutic effect can be maximized while avoiding adverse events, which can contribute to the improvement of the patient's QOL.
  • the determination result is that when cancer or autoimmune disease is treated, lung injury as an adverse event occurs or the lung. It provides useful information in identifying patients with exacerbated disabilities. Therefore, in one aspect of the present invention, a subject at risk of or at high risk of adverse events is excluded from the treatment target based on the determination result (step (a)). Excluding subjects at risk of or at high risk of adverse events from treatment means identifying subjects with no or low risk of adverse events as treatment targets and selecting them as treatment targets. Is synonymous with. Therefore, this step makes it possible to narrow down the patients who are suitable for treatment.
  • the judgment result can also be used to determine the treatment policy of the patient. Therefore, in one aspect of the present invention, the treatment policy of the subject is determined based on the determination result (step (b)).
  • the treatment policy is designed or selected according to the judgment result.
  • the subject (patient) is not recommended for scheduled treatment and should be excluded from treatment if it is determined that there is a risk or high risk of adverse events.
  • Other treatments are recommended.
  • the planned treatment is treatment with an immune checkpoint inhibitor
  • other treatments include, for example, antimetabolites, alkylating agents, treatment with anticancer agents such as microtubule agents, and surgical resection. Can be mentioned.
  • the subject can recommend the application of the planned treatment. If the subject is treated at risk or at high risk of adverse events, the course should be carefully monitored and early action should be taken.
  • the determination result can be used for reviewing or changing the treatment policy of the patient. Therefore, in one aspect of the present invention, the treatment policy of the subject is reviewed or changed based on the determination result (step (c)).
  • the determination result may be used for prognosis estimation.
  • a more appropriate treatment policy can be established for each subject (patient).
  • therapeutic intervention can be performed earlier, and the therapeutic effect (for example, prevention of progression or exacerbation of pathological condition / pathological condition) can be increased. ..
  • kits useful for predicting, diagnosing or distinguishing risk of lung disorder.
  • the risk prediction method of the present invention can be easily implemented.
  • the kit of the present invention can also be used for simple diagnosis or differentiation of interstitial pneumonia (particularly, idiopathic interstitial pneumonia).
  • One aspect of the kit of the present invention targets an anti-GPRC5A antibody and includes a reagent for measuring an anti-GPRC5A antibody.
  • a reagent for measuring an anti-GPRC5A antibody is constructed by using a substance exhibiting specific binding property to an anti-GPRC5A antibody, that is, GPRC5A or an antibody-binding fragment thereof (hereinafter, these are collectively referred to as a “binding molecule”).
  • the binding molecule can be prepared, for example, as a recombinant protein.
  • it can be prepared by transforming a suitable host cell with DNA encoding the target binding molecule (GPRC5A or an antibody binding fragment thereof) and recovering the protein expressed in the transformant.
  • the recovered protein is appropriately purified according to the purpose. If a binding molecule is obtained as a recombinant protein in this way, various modifications are possible.
  • a DNA encoding a binding molecule and another suitable DNA are inserted into the same vector and a recombinant protein is produced using the vector, a bond consisting of the recombinant protein to which an arbitrary peptide is linked is produced.
  • Sex molecules can be obtained.
  • modifications may be made so as to add sugar chains and / or lipids, or to process the N-terminal or C-terminal. With the above modifications, it is possible to extract the recombinant protein, simplify the purification, add a biological function, and the like.
  • the binding molecule may be prepared using a cell-free synthesis system.
  • Cell-free synthesis system (cell-free transcription system, cell-free transcription / translation system) does not use living cells, but ribosomes derived from living cells (or obtained by genetic engineering methods), transcription / translation factors, etc. Is used to synthesize the mRNA or protein encoded by the template nucleic acid (DNA or mRNA) in vitro.
  • the method for preparing the binding molecule is not limited to the genetic engineering method. For example, if it is naturally occurring, the binding molecule constituting the reagent of the present invention can be prepared from a natural material by a standard method (crushing, extraction, purification, etc.).
  • the kit of the present invention for measuring the anti-GPRC5A antibody is constructed as, for example, an ELISA kit.
  • a reagent for measuring an anti-GPRC5A antibody (including a binding molecule) may be immobilized in advance.
  • the insoluble support used for immobilization is not particularly limited.
  • a resin such as polystyrene resin, polycarbonate resin, silicon resin, nylon resin, or an insoluble support made of a water-insoluble substance such as glass or magnetic material can be used.
  • the shape of the insoluble support is also not particularly limited, and examples thereof include a dish (culture dish), a multi-well plate, and particles (for example, magnetic beads). Support on an insoluble support can be performed by physical adsorption or chemisorption.
  • the enzyme-labeled secondary antibody may be included in the kit.
  • Horseradish peroxidase, microperoxidase, alkaline phosphatase, ⁇ -D-galactosidase and the like can be used for the enzyme labeling.
  • a reagent for measuring anti-GPRC5A antibody may be constructed using the labeled binding molecule, and a kit capable of directly measuring the amount of anti-GPRC5A antibody using the labeled amount as an index may be used.
  • labeling substances in this case are fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), europium, 7-AAD, Alexa Fluor® 488, Alexa Fluor® 350, Alexa.
  • the kit of the present invention is configured with HLA-A2402 restrictive GPRC5A peptide-specific cytotoxic T cells (GPRC5A-specific CTL) as measurement targets.
  • a probe labeled with a multimerized HLA-peptide complex or a reagent for preparing the probe (for example, HLA heavy chain ( ⁇ chain), ⁇ 2-microglobulin light chain, antigen peptide (GPRC5A peptide), etc.) is included in the kit.
  • HLA-A2402 is used as the HLA molecule so that the detection and measurement characteristic of the present invention can be performed.
  • HLA-A2402 may be a mutant or a variant as long as it retains the binding property to the peptide antigen.
  • the probe include a probe in which the HLA-peptide complex is tetramerized using fluorescently labeled avidin (preferably streptavidin), and a plurality of MHC-peptide complexes on a fluorescently labeled polymer (for example, dextran polymer).
  • fluorescently labeled avidin preferably streptavidin
  • MHC-peptide complexes on a fluorescently labeled polymer for example, dextran polymer
  • kits In order to enhance the convenience of the kit, other reagents (buffer solution, reaction reagent, purification reagent, etc.), containers, devices, etc. used when using the kit (for example, when implementing the risk prediction method of the present invention) are used. It may be included in the kit of the present invention. It is also preferable to include the standard sample in the kit. An instruction manual is usually attached to the kit of the present invention.
  • HLA-A24 1.
  • Various cancer cell lines (lung cancer; Sq-1, LHK2, colon cancer; SW480, HCT15, Colo320, gastric cancer; Kato-3, MKN45, hepatocellular carcinoma; HepG2, malignant melanoma; SK-MEL-128)
  • the peptide presented in HLA-A24 was recovered by immunoprecipitation, and identification was attempted by mass spectrometry.
  • HLA-A24 non-expressing colorectal cancer cell line HCT116 was used as a negative control.
  • GPRC5A is highly expressed in a lung-specific manner is an indicator that the presence of antibodies and CTLs against GPRC5A causes lung damage, that is, a general-purpose biomarker for lung damage (particularly interstitial pneumonia). Suggest that
  • Peptide bond assay (1) Method Pulse T2-A24 cells with a synthetic peptide (control peptide: HPGPRPAL (SEQ ID NO: 18), LAMB3 peptide: KYAELKDRL (SEQ ID NO: 4), GPRC5A peptide: LYAPYSTHF (SEQ ID NO: 5)). It was incubated at 27 ° C for 3 hours and then incubated at 37 ° C for 2.5 hours. Cells were incubated with anti-HLA-A24 mAb (C7709A2), followed by PE-conjugated anti-mouse antibody and then analyzed by flow cytometry.
  • control peptide control peptide: HPGPRPAL (SEQ ID NO: 18)
  • LAMB3 peptide KYAELKDRL (SEQ ID NO: 4)
  • GPRC5A peptide LYAPYSTHF (SEQ ID NO: 5)
  • GPRC5A antibody in patients with lung injury 1
  • Method Eight patients who received T2 cells that stably express GPRC5A and immune checkpoint inhibitors (nivolumab (Niv), pembrolizumab (Pem), ipilimumab (Ipi), or durvalumab (Dur)).
  • the name was subsequently experienced with lung injury (interstitial pneumonia)) serum PBS 1/10 dilution was incubated on ice, washed with PBS, and then secondary BV510-conjugated anti-human antibody (BD Horizon, BD Horizon,). Incubated with # 563247). After washing several times, it was analyzed by flow cytometry.
  • the present invention provides a means for predicting the risk of lung injury.
  • the present invention is useful for determining and changing the treatment policy, and can contribute to maximizing the therapeutic effect and improving the QOL of patients.
  • diagnosis / differentiation of idiopathic interstitial pneumonia, etc. is expected.

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CN114839377A (zh) * 2022-05-31 2022-08-02 山东大学齐鲁医院 用于评估胆管癌进展的标志物及其应用
WO2023214086A1 (fr) * 2022-05-06 2023-11-09 Centre National De La Recherche Scientifique (Cnrs) Nouvel actif de cicatrisation et son utilisation
CN119534843A (zh) * 2024-10-15 2025-02-28 中国医学科学院肿瘤医院 预测非小细胞肺癌与免疫相关不良事件的标志物以及应用

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WO2005068632A1 (ja) * 2004-01-20 2005-07-28 Aichi Prefecture ＨＬＡ−Ａ２４０２拘束性Ｅｐ−ＣＡＭ特異的ＣＴＬが認識するエピトープ・ペプチド及びその用途
JP2006204220A (ja) * 2005-01-28 2006-08-10 Mie Tlo Co Ltd Ｇｐｒｃ５ａ遺伝子発現を高める方法
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WO2023214086A1 (fr) * 2022-05-06 2023-11-09 Centre National De La Recherche Scientifique (Cnrs) Nouvel actif de cicatrisation et son utilisation
FR3135267A1 (fr) * 2022-05-06 2023-11-10 Centre National De La Recherche Scientifique (Cnrs) Nouvel actif de cicatrisation et son utilisation
CN114839377A (zh) * 2022-05-31 2022-08-02 山东大学齐鲁医院 用于评估胆管癌进展的标志物及其应用
CN114839377B (zh) * 2022-05-31 2023-04-21 山东大学齐鲁医院 用于评估胆管癌进展的标志物及其应用
CN119534843A (zh) * 2024-10-15 2025-02-28 中国医学科学院肿瘤医院 预测非小细胞肺癌与免疫相关不良事件的标志物以及应用

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