Biomarker for diagnosing progressive pulmonary fibrosis, progressive pulmonary fibrosis testing method using said biomarker, and use of same

Abstract

The purpose of the present invention is to provide: a biomarker useful for rapid testing and / or early testing of progressive pulmonary fibrosis (PPF); a PPF testing method using the biomarker as an index; a PPF diagnostic agent containing a detection agent for the biomarker; a PPF diagnostic kit including the detection agent for the biomarker; and the like. This progressive pulmonary fibrosis testing method comprises a step for measuring the expression level of a gene product of at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, APOC1, LGMN, SLC40A1, FOLR2, MS4A6A, MARCKS, TMEM176B, DAB2, STAB1, GPNMB, CSTB, SDS, LIPA, and SGK1.

Description

Biomarker for diagnosing progressive pulmonary fibrosis, method for examining progressive pulmonary fibrosis using the biomarker, and use thereof

[0001] It relates to a biomarker for diagnosing progressive pulmonary fibrosis, a method for examining progressive pulmonary fibrosis using the biomarker, and use thereof.

[0002] Pulmonary fibrosis is a disease in which fibrosis of the lungs progresses, the tissue hardens, the alveolar structure collapses, and the lung function, including respiratory function, deteriorates. In the process of progression to pulmonary fibrosis, it passes through a group of diseases called interstitial lung diseases (ILDs) and reaches a single disease called pulmonary fibrosis. The number of patients with pulmonary fibrosis is increasing annually worldwide, the median survival is extremely low, and the 5-year survival rate is said to be around 40%, making it a refractory disease equivalent to lung cancer. Today, the number of patients with pulmonary fibrosis is increasing, and further increases in the number of patients are expected in the future. Also, among ILDs, there are patients with extremely rapid progression of fibrosis. In the "International Clinical Practice Guidelines for Idiopathic Pulmonary Fibrosis and Progressive Pulmonary Fibrosis 2022" published in 2022, patients with extremely rapid progression of fibrosis among ILDs were defined as progressive pulmonary fibrosis (PPF).

[0003] On the other hand, currently, there is only one anti-fibrotic drug clinically available for PPF, and it is a symptomatic treatment rather than a radical treatment. There is no way to suppress the decline in lung function other than early treatment intervention. However, in the diagnosis according to the current PPF diagnostic guidelines, it is essential to follow up symptoms, etc. over a long period of one year or less, and the diagnosis is made based on indicators such as deterioration of respiratory function and progression of fibrosis level (Non-Patent Documents 1 and 2). There is a major clinical problem in that, although early treatment intervention must be carried out, the current PPF diagnosis allows the disease state to worsen. Also, since it is impossible to diagnose PPF with a single test, the burden associated with the number of hospital visits and the number of test items, etc. significantly reduces the patient's QOL. Also, today, there is no marker that can diagnose PPF.

[0004] Boehringer Ingelheim website, "Idiopathic Pulmonary Fibrosis and Progressive Pulmonary Fibrosis: International Clinical Practice Guideline 2022 (Still Images)," Boehringer Plus, January 30, 2023, https: / / pro.boehringer-ingelheim.com / jp / product / ofev / ild-international-clinical-practice-guideline-2022. Raghu G, et al., Am J Respir Crit Care Med. 2022; 205(9):e18-e47.

[0005] The objective is to provide a biomarker useful for rapid and / or early detection of progressive pulmonary fibrosis (PPF), a PPF testing method using the biomarker as an indicator, a PPF diagnostic reagent containing the biomarker, and a PPF diagnostic kit containing the biomarker.

[0006] As a result of diligent research, the inventors have found that the gene product of at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, APOC1, LGMN, SLC40A1, FOLR2, MS4A6A, MARCKS, TMEM176B, DAB2, STAB1, GPNMB, CSTB, SDS, LIPA, and SGK1 is useful as a biomarker for rapid and / or early detection of PPF. The present invention was completed by further research based on this finding, and this disclosure includes, for example, the inventions represented below. Item 1. A method for testing for progressive pulmonary fibrosis, comprising the step of measuring the expression level of the gene product of at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, APOC1, LGMN, SLC40A1, FOLR2, MS4A6A, MARCKS, TMEM176B, DAB2, STAB1, GPNMB, CSTB, SDS, LIPA, and SGK1 in a sample taken from a subject. Item 2. The testing method according to item 1, wherein the gene is at least one selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, and APOC1.Item 3. The testing method according to Item 1 or 2, wherein the gene product is a protein, and the protein is a protein expressed from at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, and APOC1. Item 4. The testing method according to claim 3, wherein the protein is at least one selected from the group consisting of hepcidin, CXCL8 / IL8, SPP1, RNASE1, CCL13, SELENO, PLTP, CCL18, APOE, cathepsin B, cathepsin Z, heme oxygenase 1, coagulation factor XIII A chain, CD163, chitinase 3 like 1, phospholipase A2 group VII, chitinase 1, FABP5, cathepsin L, matrix metallopeptidase 9, apolipoprotein C2, TREM2, TREM-2V, CCL2, and apolipoprotein C1. Claim 5. The testing method according to claim 1 or 2, wherein the gene product is RNA. Claim 6. The testing method according to any one of claims 1 to 5, wherein the sample is at least one selected from the group consisting of bronchoalveolar lavage fluid and serum. Claim 7. The testing method according to any one of claims 1 to 6, further comprising the step of determining whether the subject has progressive pulmonary fibrosis or is at high risk of progressive pulmonary fibrosis based on the expression level of the gene product measured in the above step. Claim 8. The testing method according to any one of claims 1 to 7, further comprising the step of determining whether the subject has progressive pulmonary fibrosis or is at high risk of progressive pulmonary fibrosis if the expression level of the gene product measured in the above step (test value) is higher than the expression level of the gene product in a non-PPF-derived sample (reference value) or higher than a preset cutoff value.Item 9. A biomarker for the diagnosis of progressive pulmonary fibrosis comprising the gene product of at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, APOC1, LGMN, SLC40A1, FOLR2, MS4A6A, MARCKS, TMEM176B, DAB2, STAB1, GPNMB, CSTB, SDS, LIPA, and SGK1. Item 10. The biomarker according to item 9, wherein the gene is at least one selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, and APOC1. Item 11. The biomarker according to claim 9 or 10, wherein the gene product is a protein, and the protein is a protein expressed from at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, and APOC1. Claim 12. The biomarker according to claim 9 or 10, wherein the gene product is RNA. Claim 13. A diagnostic agent for progressive pulmonary fibrosis, comprising a detection agent for the gene product of at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, APOC1, LGMN, SLC40A1, FOLR2, MS4A6A, MARCKS, TMEM176B, DAB2, STAB1, GPNMB, CSTB, SDS, LIPA, and SGK1.Item 14. A diagnostic kit for progressive pulmonary fibrosis containing a detection agent for the gene product of at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, APOC1, LGMN, SLC40A1, FOLR2, MS4A6A, MARCKS, TMEM176B, DAB2, STAB1, GPNMB, CSTB, SDS, LIPA, and SGK1. Item 15. (A) A method for diagnosing progressive pulmonary fibrosis, comprising the step of measuring the expression level of the gene product of at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, APOC1, LGMN, SLC40A1, FOLR2, MS4A6A, MARCKS, TMEM176B, DAB2, STAB1, GPNMB, CSTB, SDS, LIPA, and SGK1 in a sample taken from a subject requiring diagnosis of progressive pulmonary fibrosis. Item 16. The diagnostic method according to claim 15, further comprising (B) determining that the subject has progressive pulmonary fibrosis or is at high risk of progressive pulmonary fibrosis if the expression level of the gene product measured in step (A) (test value) is higher than the expression level of the gene product in a non-PPF-derived sample (reference value) or higher than a preset cutoff value. Claim 17. The diagnostic method according to claim 15 or 16, further comprising (C) providing treatment for progressive pulmonary fibrosis to the subject determined to have progressive pulmonary fibrosis in step (B).

[0007] This disclosure provides a biomarker useful for rapid and / or early detection of PPF, a PPF testing method using the biomarker as an indicator, a PPF diagnostic reagent containing the biomarker, and a PPF diagnostic kit containing the biomarker.

[0008] Figure 1 shows the expression level of Hepcidin (a protein encoded by HAMP) (Test Example 1). Figure 2 shows the expression level of HAMP mRNA (Test Example 2). Figure 3 shows the expression level of CXCL8 protein (Test Example 3). Figure 4 shows the expression levels of genes and SPP1 protein that were highly expressed in samples derived from PPF patients (Test Example 4).

[0009] The embodiments included in this disclosure are described in further detail below. In this disclosure, “contains” also means “substantially consists of” or “consists of.”

[0010] This disclosure provides a method for testing progressive pulmonary fibrosis (PPF), which includes measuring the expression level of the gene product of at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, APOC1, LGMN, SLC40A1, FOLR2, MS4A6A, MARCKS, TMEM176B, DAB2, STAB1, GPNMB, CSTB, SDS, LIPA, and SGK1 in a sample taken from a subject. This method may be referred to as "the testing method of this disclosure" below.

[0011] The sample collected from the subject is not particularly limited, but bronchoalveolar lavage fluid, serum, etc., are preferred examples. The sample may be collected from the subject according to a known collection procedure. The sample may be used alone or in combination of two or more types. The subject is not limited as long as it is a subject that requires PPF testing, but preferably, subjects suspected of having interstitial lung disease (ILD) or diagnosed with ILD are examples. Mammals such as humans are examples of subjects, and humans are preferred.

[0012] In this disclosure, "gene" means double-stranded DNA, single-stranded DNA (sense strand or antisense strand), and fragments thereof.

[0013] HAMP (hepcidin antimicrobial peptide) is a known gene that codes for the protein hepcidin. The nucleotide sequence of HAMP can be found in publicly available databases. For example, the nucleotide sequence of HAMP is registered as HGNC ID: 15598 in the HUGO Gene Nomenclature Committee (HGNC).

[0014] CXCL8 (CXC motif chemokine ligand 8) is a known gene that encodes the protein CXCL8 / IL8. The nucleotide sequence of CXCL8 can be found in publicly available databases. For example, the nucleotide sequence of CXCL8 is registered as HGNC ID: 6025 in the HUGO Gene Nomenclature Committee (HGNC).

[0015] SPP1 (secreted phosphoprotein 1) is a known gene that codes for the protein SPP1. The nucleotide sequence of SPP1 can be found in publicly available databases. For example, the nucleotide sequence of SPP1 is registered with the HUGO Gene Nomenclature Committee (HGNC) as HGNC ID: 11255.

[0016] RNASE1 (ribonuclease A family member 1, pancreatic) is a known gene that codes for the protein RNASE1. The nucleotide sequence of RNASE1 can be found in publicly available databases. For example, the nucleotide sequence of RNASE1 is registered as HGNC ID: 10044 in the HUGO Gene Nomenclature Committee (HGNC).

[0017] CCL13 (CC motif chemokine ligand 13) is a known gene that codes for the protein CCL13. The nucleotide sequence of CCL13 can be found in known databases. For example, the nucleotide sequence of CCL13 is registered as HGNC ID: 10611 in the HUGO Gene Nomenclature Committee (HGNC).

[0018] SELENOP (selenoprotein P) is a known gene that codes for the protein SELENOP. The nucleotide sequence of SELENOP can be found in publicly available databases. For example, the nucleotide sequence of SELENOP is registered with the HUGO Gene Nomenclature Committee (HGNC) as HGNC ID: 10751.

[0019] PLTP (phospholipid transfer protein) is a known gene that codes for the protein PLTP. The nucleotide sequence of PLTP can be found in publicly available databases. For example, the nucleotide sequence of PLTP is registered as HGNC ID: 9093 in the HUGO Gene Nomenclature Committee (HGNC).

[0020] CCL18 (CC motif chemokine ligand 18) is a known gene that codes for the protein CCL18. The nucleotide sequence of CCL18 can be found in known databases. For example, the nucleotide sequence of CCL18 is registered as HGNC ID: 10616 in the HUGO Gene Nomenclature Committee (HGNC).

[0021] APOE (apolipoprotein E) is a known gene that codes for the protein APOE. The nucleotide sequence of APOE can be found in publicly available databases. For example, the nucleotide sequence of APOE is registered as HGNC ID: 613 in the HUGO Gene Nomenclature Committee (HGNC).

[0022] CTSB (cathepsin B) is a known gene that codes for the protein cathepsin B. The nucleotide sequence of CTSB can be found in publicly available databases. For example, the nucleotide sequence of CTSB is registered as HGNC ID:2527 in the HUGO Gene Nomenclature Committee (HGNC).

[0023] CTSZ (cathepsin Z) is a known gene that codes for the protein cathepsin Z. The nucleotide sequence of CTSZ can be found in publicly available databases. For example, the nucleotide sequence of CTSZ is registered as HGNC ID: 2547 in the HUGO Gene Nomenclature Committee (HGNC).

[0024] HMOX1 (heme oxygenase 1) is a known gene that codes for the protein heme oxygenase 1. The nucleotide sequence of HMOX1 can be found in publicly available databases. For example, the nucleotide sequence of HMOX1 is registered as HGNC ID: 5013 in the HUGO Gene Nomenclature Committee (HGNC).

[0025] F13A1 (coagulation factor XIII A chain) is a known gene that codes for the protein coagulation factor XIII A chain. The nucleotide sequence of F13A1 can be found in publicly available databases. For example, the nucleotide sequence of F13A1 is registered as HGNC ID: 3531 in the HUGO Gene Nomenclature Committee (HGNC).

[0026] CD163 (the CD163 molecule) is a known gene that codes for the protein CD163. The nucleotide sequence of CD163 can be found in publicly available databases. For example, the nucleotide sequence of CD163 is registered as HGNC ID: 1631 in the HUGO Gene Nomenclature Committee (HGNC).

[0027] CHI3L1 (chitinase 3 like 1) is a known gene that codes for the protein chitinase 3 like 1. The nucleotide sequence of CHI3L1 can be found in publicly available databases. For example, the nucleotide sequence of CHI3L1 is registered as HGNC ID: 1932 in the HUGO Gene Nomenclature Committee (HGNC).

[0028] PLA2G7 (phospholipase A2 group VII) is a known gene that encodes the protein phospholipase A2 group VII. The nucleotide sequence of PLA2G7 can be found in known databases. For example, the nucleotide sequence of PLA2G7 is registered as HGNC ID: 9040 in the HUGO Gene Nomenclature Committee (HGNC).

[0029] CHIT1 (chitinase 1) is a known gene that codes for the protein chitinase 1. The nucleotide sequence of CHIT1 can be found in publicly available databases. For example, the nucleotide sequence of CHIT1 is registered as HGNC ID: 1936 in the HUGO Gene Nomenclature Committee (HGNC).

[0030] FABP5 (fatty acid binding protein 5) is a known gene that codes for the protein FABP5. The nucleotide sequence of FABP5 can be found in publicly available databases. For example, the nucleotide sequence of FABP5 is registered as HGNC ID: 3560 in the HUGO Gene Nomenclature Committee (HGNC).

[0031] CTSL (cathepsin L) is a known gene that codes for the protein cathepsin L. The nucleotide sequence of CTSL can be found in publicly available databases. For example, the nucleotide sequence of CTSL is registered as HGNC ID: 2537 in the HUGO Gene Nomenclature Committee (HGNC).

[0032] MMP9 (matrix metallopeptidase 9) is a known gene that codes for the protein matrix metallopeptidase 9. The nucleotide sequence of MMP9 can be found in publicly available databases. For example, the nucleotide sequence of MMP9 is registered as HGNC ID: 7176 in the HUGO Gene Nomenclature Committee (HGNC).

[0033] APOC2 (apolipoprotein C2) is a known gene that codes for the protein apolipoprotein C2. The nucleotide sequence of APOC2 can be found in publicly available databases. For example, the nucleotide sequence of APOC2 is registered as HGNC ID: 609 in the HUGO Gene Nomenclature Committee (HGNC).

[0034] TREM2 (triggering receptor expressed on myeloid cells 2) is a known gene that encodes the protein TREM2. The nucleotide sequence of TREM2 can be found in publicly available databases. For example, the nucleotide sequence of TREM2 is registered with the HUGO Gene Nomenclature Committee (HGNC) as HGNC ID: 17761. The TREM2 gene is also known to express TREM-2V, an isoform of the TREM2 protein.

[0035] CCL2 (CC motif chemokine ligand 2) is a known gene that codes for the protein CCL2. The nucleotide sequence of CCL2 can be found in publicly available databases. For example, the nucleotide sequence of CCL2 is registered as HGNC ID: 10618 in the HUGO Gene Nomenclature Committee (HGNC).

[0036] APOC1 (apolipoprotein C1) is a known gene that codes for the protein apolipoprotein C1. The nucleotide sequence of APOC1 can be found in publicly available databases. For example, the nucleotide sequence of APOC1 is registered as HGNC ID: 607 in the HUGO Gene Nomenclature Committee (HGNC).

[0037] LGMN (legumain) is a known gene that codes for the protein legumain. The nucleotide sequence of LGMN can be found in publicly available databases. For example, the nucleotide sequence of LGMN is registered as HGNC ID: 9472 in the HUGO Gene Nomenclature Committee (HGNC).

[0038] SLC40A1 (solute carrier family 40 member 1) is a known gene that encodes the protein ferroportin. The nucleotide sequence of SLC40A1 and the like can be known from known databases and the like. For example, the nucleotide sequence of SLC40A1 is registered as HGNC ID: 10909 of the HUGO Gene Nomenclature Committee (HGNC).

[0039] FOLR2 (folate receptor beta) is a known gene that encodes the protein FOLR2. The nucleotide sequence of FOLR2 and the like can be known from known databases and the like. For example, the nucleotide sequence of FOLR2 is registered as HGNC ID: 3793 of the HUGO Gene Nomenclature Committee (HGNC).

[0040] MS4A6A (membrane spanning 4-domains A6A) is a known gene that encodes the protein MS4A6A. The nucleotide sequence of MS4A6A and the like can be known from known databases and the like. For example, the nucleotide sequence of MS4A6A is registered as HGNC ID: 13375 of the HUGO Gene Nomenclature Committee (HGNC).

[0041] MARCKS (myristoylated alanine rich protein kinase C substrate) is a known gene that encodes the protein MARCKS. The nucleotide sequence of MARCKS and the like can be known from known databases and the like. For example, the nucleotide sequence of MARCKS is registered as HGNC ID: 6759 of the HUGO Gene Nomenclature Committee (HGNC).

[0042] TMEM176B (transmembrane protein 176B) is a known gene that encodes the protein TMEM176B. The nucleotide sequence of TMEM176B can be known from known databases and the like. For example, the nucleotide sequence of TMEM176B is registered as HGNC ID: 1916348 of the HUGO Gene Nomenclature Committee (HGNC).

[0043] DAB2 (DAB adaptor protein 2) is a known gene that encodes the protein DAB2. The nucleotide sequence of DAB2 can be known from known databases and the like. For example, the nucleotide sequence of DAB2 is registered as HGNC ID: 2662 of the HUGO Gene Nomenclature Committee (HGNC).

[0044] STAB1 (stabilin 1) is a known gene that encodes the protein stabilin 1. The nucleotide sequence of STAB1 can be known from known databases and the like. For example, the nucleotide sequence of STAB1 is registered as HGNC ID: 18628 of the HUGO Gene Nomenclature Committee (HGNC).

[0045] GPNMB (glycoprotein nmb) is a known gene that encodes the protein GPNMB. The nucleotide sequence of GPNMB can be known from known databases and the like. For example, the nucleotide sequence of GPNMB is registered as HGNC ID: 4462 of the HUGO Gene Nomenclature Committee (HGNC).

[0046] CSTB (cystatin B) is a known gene that encodes the protein cystatin B. The nucleotide sequence of CSTB can be known from known databases and the like. For example, the nucleotide sequence of CSTB is registered as HGNC ID: 2482 of the HUGO Gene Nomenclature Committee (HGNC).

[0047] SDS (serine dehydratase) is a known gene that codes for the protein serine dehydratase. The nucleotide sequence of SDS can be found in publicly available databases. For example, the nucleotide sequence of SDS is registered as HGNC ID: 67376 in the HUGO Gene Nomenclature Committee (HGNC).

[0048] LIPA (lipase A, lysosomal acid type) is a known gene that encodes the protein lipase A, lysosomal acid type. The nucleotide sequence of LIPA can be found in publicly available databases. For example, the nucleotide sequence of LIPA is registered as HGNC ID: 6617 in the HUGO Gene Nomenclature Committee (HGNC).

[0049] SGK1 (serum / glucocorticoid regulated kinase 1) is a known gene that codes for the protein SGK1. The nucleotide sequence of SGK1 can be found in publicly available databases. For example, the nucleotide sequence of SGK1 is registered with the HUGO Gene Nomenclature Committee (HGNC) as HGNC ID: 10810.

[0050] Examples of gene products include RNA (mRNA, mRNA precursors, non-coding RNAs such as miRNA and lncRNA), proteins (including isoforms), etc., with proteins and mRNA being preferred examples. The procedure for measuring the expression level of gene products in a sample is not limited as long as the measurement is possible, and it may be measured according to conventionally known procedures. The expression level of gene products may be measured directly or indirectly, as long as it reflects the expression level of the gene product. The expression level may be absolute or relative. The gene product to be measured may be only one type, or a combination of two or more types.

[0051] The measurement of gene product expression levels is not limited to any method that can specifically measure the expression level of the target gene product, and can be performed according to conventionally known procedures. For example, for protein measurement, all known measurement methods based on antigen-antibody reactions such as ELISA (Enzyme-linked immunosorbent assay), immunostaining, Western blotting, flow cytometry, dot blotting, and immunoprecipitation, as well as mass spectrometry, can be used. For mRNA measurement, known detection methods such as RT (Reverse Transcription)-PCR, quantitative PCR, in situ hybridization, microarrays (nucleic acid chips, etc.), RNA-Seq, and Northern blotting can be used. In addition, substances or activities that reflect the expression level of gene products may be measured, and in this case, the measurement method should be appropriately determined. Samples may be used directly for measurement, or pre-treatment may be performed as necessary. Extraction of mRNA or proteins from samples can also be performed according to conventionally known procedures in this field. The measurement may be performed using commercially available kits, as exemplified by the test examples described later.

[0052] Without limiting this disclosure, for example, ELISA is a protein detection (measurement) method that utilizes the antigen-antibody reaction principle to detect (measure) the activity derived from the target of measurement using a labeled antigen or labeled antibody. For example, quantitative PCR is an mRNA detection (measurement) method that prepares cDNA from mRNA derived from a sample according to a conventional method, and performs PCR using techniques such as adding a fluorescently labeled primer (probe) that can amplify the target region using the cDNA as a template, or a fluorescent protein that is inserted between base pairs of double-stranded DNA amplified as a PCR product in combination with a primer as an indicator, and quantitatively detects (measures) the PCR amplification product.

[0053] From the viewpoint of genes encoding secreted proteins, preferred examples of genes include HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, and APOC1.

[0054] When measuring the expression level of a protein as a gene product, preferred examples of proteins include those expressed from HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, or APOC1.

[0055] Therefore, when measuring the expression level of a protein as a gene product, examples of secreted proteins expressed from the aforementioned gene include hepcidin, CXCL8 / IL8, SPP1, RNASE1, CCL13, SELENO, PLTP, CCL18, APOE, cathepsin B, cathepsin Z, heme oxygenase 1, coagulation factor XIII A chain, CD163, chitinase 3 like 1, phospholipase A2 group VII, chitinase 1, FABP5, cathepsin L, matrix metallopeptidase 9, apolipoprotein C2, TREM2, TREM-2V, CCL2, and apolipoprotein C1. Hepcidin is a more preferred example of such a protein. As mentioned above, for example, hepcidin is a protein expressed from HAMP. TREM-2V is an isoform encoded by the aforementioned TREM2 gene. The amino acid sequences of TREM2 and TREM-2V can be found in known databases, etc. For example, the amino acid sequence of the TREM2 protein is registered as ID:Q9NZC2-1 in Uniprot, and the amino acid sequence of the TREM-2V protein is registered as ID:Q9NZC2-2 in UniProt.

[0056] The testing method of this disclosure, including step (1) described above, can provide the expression level of a gene product useful as a testing indicator for PPF. The testing method of this disclosure, including step (1) described above, is useful in considering the determination of PPF in a subject, the necessity of treatment for the subject, and the selection of a treatment method.

[0057] The testing method of this disclosure may further include a step (sometimes referred to as step (2)) of determining whether the subject is PPF or at high risk of PPF based on the expression level of the gene product measured in step (1). The testing method of this disclosure, further including this step, can determine whether the subject is PPF or at high risk of PPF. As one embodiment of step (2), it is preferably exemplified that the determination of whether the subject is PPF or at high risk of PPF is based on whether the expression level of the gene product measured in step (1) (test value) is higher than the expression level of the gene product in a non-PPF-derived sample (reference value) or higher than a preset cutoff value.

[0058] If the test value is higher than the reference value or a predetermined cutoff value, the subject is determined to have PPF or be at high risk of PPF. Without limiting this disclosure, for example, if the test value is higher than the reference value or a predetermined cutoff value, but the subject has not been diagnosed with PPF based on conventional PPF diagnostic guidelines at the time of the test, the subject may also be determined to be at high risk of PPF (highly likely to develop PPF in the future or likely to have PPF). Thus, the testing method of this disclosure can be said to be a method for determining whether a subject has PPF or is at high risk of PPF at least one month, at least two months, at least three months, at least four months, at least five months, or at least six months earlier than a diagnosis based on conventional PPF diagnostic guidelines.

[0059] In this disclosure, non-PPF refers to patients who have ILD but were not diagnosed with PPF or were not diagnosed as being at high risk of PPF. Diagnosis according to conventional PPF diagnostic guidelines involves long-term follow-up, within one year of diagnosis of ILD, to determine whether or not a patient has PPF. Therefore, conventionally, a diagnosis of PPF is made at least several months after the date of diagnosis (diagnosis date) when ILD is suspected or diagnosed. Accordingly, non-PPF refers to patients who have been diagnosed with ILD but were not diagnosed as having PPF or were not diagnosed as being at high risk of PPF within at least several months from the date of diagnosis. This period (at least several months) is typically exemplified as between one month and twelve months, with the date of diagnosis (day 0) being considered day 0. Preferably, the lower limit is 1, 2, 3, 4, or 5 months, and preferably the upper limit is 11, 10, 9, 8, 7, or 6 months, more preferably 1 month or more and 6 months or less. The timing of sample collection from non-PPF is not limited as long as it is after a diagnosis of suspected or diagnosed ILD in non-PPF. Preferably, the collection period is within several months from the date of suspected or diagnosed ILD, and this period is described in the same manner as described above. The gene product in a non-PPF-derived sample means the gene product of the gene in a sample collected from non-PPF. The sample is described in the same manner as the sample collected from the subject described above, as long as it is from non-PPF, and bronchoalveolar lavage fluid, serum, etc. are preferably exemplified. The gene product is also described in the same manner as described above, as long as it is from non-PPF, and preferably proteins, mRNA, etc.Furthermore, while there are no restrictions as long as the test value and the reference value can be compared, it is generally preferred that the same type of sample be used for both the test value and the reference value (for example, if the sample for the test value is bronchoalveolar lavage fluid, then the sample for the reference value is also bronchoalveolar lavage fluid), and it is also preferred that the same type of gene product be used (for example, if the test value is a protein of a certain gene, then the reference value is also a protein of the same gene), and it is also preferred that the expression level measurement be performed using the same measurement procedure.

[0060] In this disclosure, ILD and PPF are based on the reliable definitions of ILD and PPF in this field as presented in the "International Clinical Practice Guidelines for Idiopathic Pulmonary Fibrosis and Progressive Pulmonary Fibrosis 2022," etc. The causes of ILD are diverse and, for example, classified according to the cause into idiopathic interstitial pneumonia (cause unknown), autoimmune interstitial lung disease (caused by collagen diseases such as rheumatoid arthritis), occupational interstitial lung disease (caused by dust inhalation, hypersensitivity pneumonitis, etc.), iatrogenic interstitial lung disease (caused by drugs, radiation therapy, etc.), and other interstitial lung diseases (caused by sarcoidosis, etc.), and these are collectively referred to as ILD. Whether or not a patient is non-PPF can be determined by a medical institution based on the reliable definitions presented in the aforementioned guidelines, etc.

[0061] In this disclosure, "higher than the reference value" is not limited to the extent that the test value exceeds the reference value. "Higher than the reference value" preferably means that the test value is 1.1 times or more the reference value, and more preferably means that it is 1.2 times or more, or 1.3 times or more, etc. This value may be appropriately changed depending on the type of gene product, the measurement method, and whether the test value and reference value are absolute or relative values. In this disclosure, "less than or equal to the reference value" as described later is not limited to the extent that the test value is equal to or lower than the reference value.

[0062] In this disclosure, the cutoff value can be set according to a conventionally known cutoff value setting procedure, preferably based on an ROC curve (Receiver Operating Characteristic curve) created from two elements (specificity and sensitivity). While not limiting this disclosure, examples of cutoff values ​​include those where the specificity is 100% and the sensitivity is 60% or higher based on the ROC curve; more preferably, a specificity of 100% and sensitivity of 70% or higher; even more preferably, a specificity of 100% and sensitivity of 75% or higher; particularly preferably, a specificity of 100% and sensitivity of 80% or higher; a specificity of 100% and sensitivity of 85% or higher; or a specificity of 100% and sensitivity of 90% or higher. Setting cutoff values ​​based on an ROC curve created from specificity and sensitivity is widely practiced in this art, the procedure is easily understood by those skilled in the art, and statistical software is available on the market. Examples of statistical software include Graphpad prism (manufactured by GraphPad Software).

[0063] While not limiting this disclosure, in Test Example 1 described below, using the gene product of HAMP as an indicator, the cutoff value at 100% specificity and 91.6% sensitivity was 18.98 pg / ml. In Test Example 3 described below, using the gene product of CXCL8 as an indicator, the cutoff value at 100% specificity and 75% sensitivity was 51.97 pg / ml. In Test Example 4 described below, using the gene product of SPP1 as an indicator, the cutoff value at 100% specificity and 66.67% sensitivity was 174.9 pg / ml. In this disclosure, the cutoff value may be appropriately determined depending on the type of biomarker. Furthermore, as a biomarker to be used in PPF testing, additional desired biomarkers may be appropriately selected based on the aforementioned specificity and sensitivity.

[0064] Based on these considerations, step (2) described above can also be described as a step in which, if the test value is higher than the reference value or higher than a predetermined cutoff value, the sample is determined to be from a subject with PPF or from a subject at high risk of PPF.

[0065] Without limiting this disclosure, the testing method of this disclosure may further include a step of determining that the subject may not have PPF or is not at high risk of PPF if the test value is below a reference value or below a predetermined cutoff value. Alternatively, this step may be defined as determining that the sample may not be from a subject with PPF or is from a subject not at high risk of PPF if the test value is below the reference value or below a predetermined cutoff value. Thus, the testing method of this disclosure can determine whether a subject has PPF at the time the method is performed.

[0066] Furthermore, the testing method of this disclosure can determine whether a subject has PPF or the risk thereof based on the expression level, that is, using the expression level as an indicator. Thus, this disclosure can also be said to provide a PPF testing method that uses the expression level of the gene product in a sample taken from a subject as an indicator. In addition, the testing method of this disclosure may be performed in combination with or as an aid to conventional imaging diagnostics such as CT scans used for the diagnosis of PPF. Thus, the testing method of this disclosure can also be said to be a method that assists PPF testing.

[0067] Furthermore, this indicates that the gene product can be considered a biomarker for testing PPF. Accordingly, this disclosure can also be said to provide a method for measuring a biomarker, which includes the step of measuring the expression level of the gene product in a sample collected from a subject. Furthermore, this disclosure can also be said to provide a method for using the gene product in a sample collected from a subject as a biomarker for testing PPF. In these methods, the subject, sample, gene product, expression level, measurement, etc., are all described in the same manner as described above. Therefore, this indicates that the gene product can also be useful as a diagnostic biomarker for PPF.

[0068] Biomarkers for the Diagnosis of Progressive Pulmonary Fibrosis Therefore, this disclosure also provides biomarkers for the diagnosis of PPF, comprising the gene product of at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, APOC1, LGMN, SLC40A1, FOLR2, MS4A6A, MARCKS, TMEM176B, DAB2, STAB1, GPNMB, LGMN, CSTB, SDS, LIPA, and SGK1.

[0069] As described above, PPF in a subject can be determined using the expression level of the gene product as an indicator, and furthermore, PPF can be diagnosed in the subject based on this determination.

[0070] Therefore, this disclosure can also be said to provide a method for using the gene product as a biomarker for the diagnosis of PPF. This disclosure can also be said to provide a method for using the expression level of the gene product as an indicator for the diagnosis of PPF.

[0071] Furthermore, this means that the present disclosure also provides a method for diagnosing PPF, which includes a step (sometimes referred to as step (A)) of measuring the expression level of the gene product in a sample taken from a subject requiring diagnosis of PPF.

[0072] The diagnostic method of this disclosure may further include a step of determining that the subject is PPF or at high risk of PPF based on the expression level of the gene product measured in step (A), and more preferably a step (sometimes referred to as step (B)) of determining that the subject is PPF or at high risk of PPF if the expression level of the gene product measured in step (A) (test value) is higher than the expression level of the gene product in a non-PPF sample (reference value) or higher than a preset cutoff value. In this diagnostic method, the subject, sample, gene product, expression level, test value, reference value, non-PPF, cutoff value, etc., are all described in the same way as in the testing method described above. The diagnostic method may further include a step of determining that the subject may not be PPF or is not at high risk of PPF if the test value is below the reference value or below a preset cutoff value.

[0073] The diagnostic method of this disclosure may further include a step (sometimes referred to as step (C)) of providing treatment for PPF to a subject determined to have PPF in step (B). Examples of treatment include drug therapy using steroids or antifibrotic drugs, oxygen administration, respiratory rehabilitation, and lung transplantation. Treatment may be performed individually or in combination of two or more treatments. Treatment may include, for example, administering one or more therapeutic agents commonly used for PPF to the subject. Therapeutic agents that can be used for PPF include, for example, antifibrotic agents and steroids, and are preferably antifibrotic agents. The antifibrotic agent is not limited to these, but for example, nintedanib ethanesulfonate, marketed by Boehringer Ingelheim under the trade name Ofev®, can be used.

[0074] The diagnostic method of this disclosure may further include a step of recording the judgment results and / or matters relating to treatment (for example, at least one piece of information selected from examination records, diagnostic records, treatment records, surgical records, nursing records, etc., including disease status, treatment plan, treatment progress, treatment effect, and final treatment results) on a medium. Examples of mediums include medical records (paper medical records, electronic medical records (including medical records obtained by scanning paper medical records, etc.)) recorded by medical professionals including physicians, and materials presented to patients (paper, etc.), and can be easily recorded using electronic devices such as writing instruments, personal computers, tablet terminals, and mobile phones. The recorded information may be printed out, copied, etc., as needed.

[0075] Furthermore, the inspection method of this disclosure may also include a step of providing treatment for PPF to a subject determined to be PPF in the above step, a step of recording the determination result and / or matters (information) related to the treatment in the above step on a medium, and / or a step of printing out and / or copying the information.

[0076] Diagnostic Agents and Kits for Progressive Pulmonary Fibrosis This disclosure further encompasses diagnostic agents for progressive pulmonary fibrosis that include a detection agent for the gene product of at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, APOC1, LGMN, SLC40A1, FOLR2, MS4A6A, MARCKS, TMEM176B, DAB2, STAB1, GPNMB, LGMN, CSTB, SDS, LIPA, and SGK1. The gene, gene product, and progressive pulmonary fibrosis are described in the same manner as above.

[0077] The detection agent for gene products is not limited as long as it can specifically detect the gene product, and examples include antibodies, probes, aptamers (peptide aptamers, nucleic acid aptamers, etc.), primers, etc. that specifically detect gene products. The antibody may be a polyclonal antibody, a monoclonal antibody, or a fragment thereof (e.g., Fab, Fab', F(ab')2, etc.). As explained above in the measurement of gene product expression levels, the detection agent may directly or indirectly detect the gene product (preferably protein, mRNA). The detection agent is not limited as long as it can determine the presence or amount of the gene product.

[0078] The detection reagent may be modified as needed. Examples of modifications include the addition of labels (e.g., fluorescent dyes, enzymes, proteins, radioisotopes, chemiluminescent substances, biotin, etc.). The detection reagent may also be immobilized on any solid phase (e.g., microplates, magnetic beads, etc.). Therefore, the diagnostic reagent of this disclosure can also be provided in the form of a substrate on which the detection reagent is immobilized (e.g., a microplate on which antibodies are immobilized). The detection reagent may be used alone or in combination of two or more types.

[0079] The diagnostic agent of this disclosure may contain any additional components as long as they do not interfere with the effects of this disclosure. Examples of such components include carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, colorants, fragrances, chelating agents, pH adjusters, and preservatives. These components may be used individually or in combination of two or more, and their amounts may be appropriately determined as long as they do not interfere with the effects of this disclosure.

[0080] In the diagnostic reagents of this disclosure, the content of the detection agent is not limited as long as it can detect the gene product, and may be appropriately limited depending on the type of antibody, probe, primer, etc. Furthermore, the form of the diagnostic reagent may be solid (powder, granules, tablets, etc.), semi-solid, or liquid.

[0081] Detection using the diagnostic reagents of this disclosure may be performed by referring to conventionally known detection procedures depending on the antibody, probe, primer, etc., as long as the gene product can be detected, and for example, it may be performed in the same manner as the measurement procedure in the aforementioned testing method. In this disclosure, detection also includes the meaning of measurement.

[0082] The diagnostic agent of this disclosure may also be in kit form, and therefore, this disclosure can also be said to provide a PPF diagnostic kit containing a detection agent for the gene product. The kit may, along with the diagnostic agent, optionally further include a buffer usable for detecting the gene product, a nucleic acid amplification reagent, a reverse transcriptase, a substrate, a primary antibody, a secondary antibody, an aptamer (peptide aptamer, nucleic acid aptamer, etc.), a positive control, a negative control, instructions for use, etc. For example, the instructions for use may contain a URL or a reading code for a webpage, and the instructions for use may be obtainable via the URL or reading code. The diagnostic kit of this disclosure can also be used in the aforementioned testing method, and therefore, the diagnostic kit of this disclosure can also be said to be a PPF testing kit. The diagnostic agent or diagnostic kit of this disclosure is useful in that it allows the aforementioned test or diagnosis to be carried out easily.

[0083] As described above, this disclosure provides PPF biomarkers useful for rapid and / or early testing and / or diagnosis of PPF, as well as methods for testing and diagnosing PPF using these biomarkers.

[0084] As mentioned above, PPF is a disease in which fibrosis progresses extremely rapidly among ILDs, and currently there is only one antifibrotic drug available for use in clinical practice for PPF. Furthermore, it is a symptomatic treatment rather than a curative treatment, and there is no way to suppress the decline in lung function other than early therapeutic intervention. In addition, currently, a diagnosis of ILD alone does not allow for the determination of whether or not a person has PPF, or whether or not they are at high risk of developing PPF. Diagnosing PPF requires long-term follow-up of less than one year, using indicators such as deterioration of respiratory function and progression of fibrosis levels, which means that despite the need for early therapeutic intervention, the condition is forced to worsen. In addition, current PPF diagnosis requires imaging equipment such as CT scans, so examinations can only be performed at limited institutions such as large hospitals, and multiple examinations are required, which places a heavy burden on patients and presents challenges from a healthcare financial perspective. It is important to screen for PPF rapidly and / or early, and by extension, to diagnose PPF rapidly and / or early.

[0085] The biomarkers and testing methods utilizing these biomarkers are extremely useful in that they enable rapid and early PPF testing and, consequently, PPF diagnosis compared to current PPF diagnostic methods. In particular, they eliminate the need for long-term follow-up after diagnosis of ILD, as is currently required, thus enabling early therapeutic intervention for patients determined to have PPF using the biomarkers disclosed. Furthermore, as mentioned above, the methods disclosed are based on the expression level of gene products, allowing test results to be obtained within a few hours to a few days. In this respect as well, the biomarkers disclosed are useful for early intervention in treatment using antifibrotic drugs, etc. Moreover, the biomarkers disclosed enable highly accurate determination of PPF with a single test. In addition, for example, testing and / or diagnosis may be performed by combining image interpretation (CT images), which is left to the physician's judgment, with interpretation based on the expression level of the biomarkers disclosed. In this case, testing and / or diagnosis becomes possible using a clear indicator of expression level in addition to image interpretation.

[0086] The embodiments of this disclosure will be described in more detail below with examples, but the embodiments of this disclosure are not limited to the examples below.

[0087] Test Example 1 (Hepcidin ELISA protocol) 1-1. Test Procedure As the test sample, bronchoalveolar lavage fluid (BALF) (-80°C frozen, unconcentrated) derived from human interstitial lung disease was used, collected from subjects suffering from ILD. Specifically, the subjects' lungs were washed with 150 mL to 200 mL of physiological saline to obtain bronchoalveolar lavage fluid (hereinafter referred to as bronchoalveolar lavage fluid). The recovery rate was less than 30%, and bronchoalveolar lavage fluid containing a large amount of blood was excluded. The subjects were Japanese (males and females aged 20 years or older). At the time of sample collection, each subject had been diagnosed with ILD, but whether or not they had PPF had not been diagnosed. Subjects diagnosed with PPF by a physician based on current PPF diagnostic guidelines six months or more after sample collection were classified as "PPF" (n=12), and subjects diagnosed as not having PPF were classified as "non-PPF" (n=12).

[0088] The expression level of hepcidin protein in the samples was measured using the commercially available hepcidin detection kit, Human Hepcidin DuoSet® ELISA (R&D Systems: DY8307-05). Specifically, the following was performed: Reagents used in the kit: • PBS: D-PBS (Nacalai Tesque: 14249-24) • Tween20: Polyoxyethylene (20) sorbitan monolaurate (FUJIFILM: 160-21211) • BSA: 30(w / v)% bovine serum albumin solution, fatty acid-free (Nacalai Tesque: 19361-84) • Substrate solution: ELISA POD substrate TMB solution (Easy) (Nacalai Tesque: 05299-54) • 2N H2SO4: 1 mol / L sulfuric acid (2N) (Nacalai Tesque: 95626-06) Preparation reagents: • Wash buffer: 0.05% Tween (registered trademark) 20 in PBS • Reagent Diluent: 1% BSA in PBS Equipment used: • Measuring instrument: Varioskan LUX (Thermo Fisher Scientific) ・Concentration calculation: SkanIt Microplate Reader 6.1 Software (Thermo Fisher Scientific) ・Statistical analysis: Graph pad prism10.0.3 (Graphpad)

[0089] The expression level of Hepcidin protein in the sample was measured according to the following procedure in accordance with the kit's instructions: 1) Capture antibody was diluted to the indicated concentration in PBS and immobilized on 100 μL (room temperature (25°C), overnight). 2) After aspiration, the sample was washed three times with 400 μL of wash buffer. 3) Blocking was performed with 300 μL of Regent Diluent (room temperature, 1 hour). 4) Washing procedure (same as in 2) above) was performed. 5) 100 μL of standard or sample serially diluted with Regent Diluent was added (room temperature, 2 hours). 6) Washing procedure (same as in 2) above) was performed. 7) 100 μL of detection antibody diluted with Regent Diluent was added (room temperature, 2 hours). 8) Washing procedure (same as in 2) above) was performed. 9) 100 μL of Streptavidin-HRP diluted with Regent Diluent was added (room temperature, 20 minutes). 10) Washing procedure (same as in 2 above) was performed. 11) 100 μL of Substrate solution was added (room temperature, 10 minutes). 12) 50 μL of H2SO4 was added, and absorbance was immediately measured at 450 nm. 13) A calibration curve was created using 4PL, and the concentration was calculated. 14) Statistical analysis was performed using the Unpaired t-test. The error bars in the quantitative data of this test example or the test examples described later indicate the mean ± standard error of the mean, and the difference between groups was calculated using the two-sided unpaired Student's t-test or one-way analysis of variance with Tukey's correction, with statistical significance set at p<0.05 (GraphPad Prism 10.0.3 software (GraphPad Software) was used).

[0090] Setting the Cutoff Value (Threshold): The cutoff value was determined according to a conventionally known method of setting cutoff values ​​using ROC curves. Specifically, an ROC curve was created based on the specificity and sensitivity, two elements that constitute test accuracy, from the non-PPF and PPF data (expression levels). The ROC curve was created using Graphpad prism 10.0.3 (GraphPad Software). In this study, the threshold (cutoff value) was set to the point with the highest sensitivity among the concentrations that result in 100% (zero false positives). Graphpad prism sets the threshold mechanically using a predetermined calculation formula, so there is no human intervention, and the cutoff value can be set objectively.

[0091] 1-2. Results The results are shown in Figure 1. In Figure 1, the graph on the left shows the measurement results using all diluted samples, and the graph on the right shows the measurement results using diluted samples in the low concentration range (Hepcidin concentration < 200 pg / ml). As shown in Figure 1, it was found that the expression level of Hepcidin protein was significantly increased in PPF compared to non-PPF. Furthermore, as shown in the graph on the right of Figure 1, the expression level of Hepcidin was significantly increased in PPF even in the low concentration range. In the graph on the right, the maximum value of Hepcidin expression was 10.01 pg / ml in non-PPF, and the minimum value above the dotted line in the graph was 24.88 pg / ml in PPF, which is 2.48 times higher than in non-PPF. Furthermore, as mentioned above, by referring to the ROC curve and setting a threshold, a hepcidin concentration of 18.98 pg / ml was used as the threshold, resulting in a sensitivity of 91.6% and a specificity of 100%. From these results, it can be said that hepcidin is an excellent biomarker for PPF. Rapid influenza tests already used in clinical practice are known to have a specificity of over 90% and a sensitivity of around 50-70%. Considering that even a sensitivity of around 50% is currently used as a cutoff value, it can be understood that hepcidin, which allows for the setting of a highly accurate cutoff value (combining high specificity and high sensitivity), is extremely useful as a biomarker for PPF. The ability to set a cutoff value that can accurately distinguish between non-PPF and PPF is clinically very useful because it allows for judgment based on individual patient measurements rather than comparison with other patients.

[0092] These results demonstrate that hepcidin is highly useful as a biomarker for PPF testing or diagnosis. Furthermore, this study compared subjects with non-PPF individuals (those with ILD but who were not diagnosed with PPF according to conventional PPF diagnostic guidelines for at least several months after sample collection), rather than comparing them to healthy individuals. In other words, hepcidin was used to further identify PPF among patients already diagnosed with ILD. The ability to determine PPF in comparison to non-PPF individuals, rather than just healthy individuals, is highly significant in clinical practice. Moreover, as mentioned above, the subjects in this study were those who had ILD but could not be determined to have PPF at the time of sample collection, and were only diagnosed with PPF by a physician several months later based on current PPF diagnostic guidelines. This indicates that hepcidin is highly useful as a biomarker because it can determine PPF in its early stages (i.e., before fibrosis has progressed significantly). The association between hepcidin and the progression of ILD was previously unknown and has now been revealed for the first time through this study.

[0093] Test Example 2 (Hepcidin qPCR protocol) 2-1. Test Procedure As the sample, cells containing alveolar lavage fluid derived from human interstitial lung disease (ILD) were used (liquid nitrogen frozen). Specifically, the lungs of the subjects were washed with 150 mL to 200 mL of physiological saline to obtain alveolar lavage fluid. The recovery rate was less than 30%, and alveolar lavage fluid containing a large amount of blood was excluded. After centrifugation of the alveolar lavage fluid, the precipitated cell clumps were resuspended in cell cryopreservation solution (CELLBANKER1: ZENOGEN PHARMA) and frozen in liquid nitrogen for storage. Cells collected from the alveolar lavage fluid in this way are referred to as alveolar lavage fluid-containing cells in this test example. The subjects were Japanese (males and females aged 20 years or older). As described above, subjects diagnosed with PPF by a physician based on current PPF diagnostic guidelines six months or more after sample collection were classified as "PPF" (n=8), and subjects diagnosed as not having progressive pulmonary fibrosis were classified as "non-PPF" (n=10).

[0094] The expression level of hepcidin-encoding mRNA (HAMP mRNA) was measured using the following kits. The gene name for hepcidin is HAMP. Kits used: SuperPrep Cell Lysis & RT Kit for qPCR (TOYOBO: SCQ-101), THUNDERBIRD Probe qPCR Mix (TOYOBO: QPS-101), TaqMan Gene Expression Assays HAMP (Thermo Fisher Scientific: Hs00221783_m1). Equipment used: Measurement device: Quantstudio6 (Thermo Fisher Scientific), Analysis tool: Design & Analysis Software (Thermo Fisher Scientific), Statistical analysis: Graphpad prism 10.0.3 (Graphpad).

[0095] The HAMP mRNA expression level in the sample was measured according to the following procedure, in accordance with the kit's instructions: 1) The sample (cells) stock was thawed from liquid nitrogen. 2) 5 mL of PBS was added to the thawed cells and centrifuged (1500 rpm, 3 minutes). 3) 10 μL was taken from the suspended cells in 1 mL of PBS. 4) 50 μL of Lysis Solution (containing gDNA Remover) was added, shaken for 30 seconds, and then allowed to stand (room temperature, 5 minutes). 5) 10 μL of Stop Solution (containing RNase Inhibitor) was added, shaken for 30 seconds, and then allowed to stand (room temperature, 2 minutes). 6) RT Master Mix was prepared (5x RT Mix 160 μL + Nuclease-free water 4800 μL). 7) 8 μL of the lysate from step 5) was added to 40 μL of the Master Mix and mixed. 8) The reaction was carried out in a Thermal Cycler at 37°C for 15 minutes → 50°C for 5 minutes → 98°C for 5 minutes → 4°C, Hold. 3 μL of the reaction solution from 9) + 10 μL of Thunderbird probe qPCR Mix + 1 μL of Taqman probe + 0.4 μL of ROX reference dye + 6 μL of sterile water were added and mixed. 10) PCR was performed for 50 cycles of initial denaturation at 95°C for 20 seconds → (95°C for 1 second + 60°C for 20 seconds). 11) ΔCt was calculated from the obtained Ct values ​​(ΔCt = HAMP Ct - 18S Ct). 12) ΔΔCt was calculated from the obtained ΔCt using one non-PPF sample as a reference. (ΔΔCt = non-PPF reference sample ΔCt - each sample ΔCt) 13) Two samples were used for expression comparison. －△ΔCt The following was calculated. 14) Statistical analysis was performed using the Unpaired t-test.

[0096] 2-2. Results The results are shown in Figure 2. In Figure 2, the graph on the left shows the measurement results using all diluted samples, and the graph on the right shows the measurement results using diluted samples in the low expression range (HAMP expression level < 30). As shown in Figure 2, it was found that HAMP expression levels were significantly increased in PPF compared to non-PPF. From these results, it was found that HAMP is useful as a biomarker for PPF testing or diagnosis. In particular, similar to Test Example 1, Test Example 2 was compared with non-PPF, not with healthy individuals, meaning that HAMP is very useful as an indicator for PPF diagnosis, etc., in that it can distinguish PPF among patients diagnosed with ILD. Also, similar to Test Example 1, the subjects in Test Example 2 were diagnosed by a physician several months after sample collection based on PPF diagnostic guidelines to determine whether or not they had PPF. From this, it was found that HAMP is also very useful as a biomarker that can determine PPF in the early stages (i.e., when the progression of fibrosis has not progressed much).

[0097] Test Example 33-1. Test Procedure

[0098] <Cytokine array protocol> As a sample, human interstitial lung disease-derived bronchoalveolar lavage fluid (BALF) (-80°C frozen, unconcentrated) collected from subjects suffering from ILD was used, in the same manner as in Test Example 1. The subjects were Japanese (non-PPF (n=1), PPF (n=1)). Kit used: Pierce TM BCA Protein Assay Kits (ThermoFisher Scientific: 23225) • Human Cytokine Array C1000 (RayBiotech: AAH-CYT-1000-2) Kit Reagents • PBS: D-PBS (Nacalai Tesque: 14249-24) • UltraPure TM DNase / RNase-Free Distilled Water (Invitrogen: 10977015) Measurement Equipment: Varioskan LUX (Thermo Fisher Scientific), ImageQuant TM LAS4000 (Cytiva)

[0099] The mRNA expression levels of each gene were measured using the following procedure, in accordance with the kit's instructions: 1) After thawing the sample, 25 μL was used to perform a Pierce metering. TM 1) Concentration was measured using BCA Protein Assay Kits. 2) Concentrations were calculated from a calibration curve and non-PPF and PPF concentrations were equalized using PBS. 3) Membranes were removed from the Human Cytokine Array C1000 and incubated in the provided wells with Blocking Buffer at 25°C for 30 minutes. 4) 1 ml of sample was added to each well and incubated at 4°C for one day. 5) Washing was repeated three times with 2 ml of Wash Buffer I at 25°C for 5 minutes each. 6) Washing was repeated twice with 2 ml of Wash Buffer II at 25°C for 5 minutes each. 7) 1 ml of Biotinylated Antibody Cocktail was added to each well and incubated at 4°C for one day. 8) Washing procedures 5) and 6) were performed. 9) 2 ml of 1X HRP-Streptavidin was added to each well and incubated at 4°C for one day. 10) Washing procedures 5) and 6) were performed. 11) Detection Buffers C and D were mixed in a 1:1 ratio, 500 μL was added to each well, and allowed to stand for 2 minutes. 12) ImageQuant TM The photos were taken using the LAS4000.

[0100] <CXCL8 ELISA protocol> As a sample, human interstitial lung disease-derived bronchoalveolar lavage fluid (BALF) (-80°C frozen, unconcentrated) collected from subjects suffering from ILD was used, in the same manner as in Test Example 1. The subjects were Japanese (males and females aged 20 years or older). As described above, after sample collection, subjects diagnosed with PPF by a physician based on the current PPF diagnostic guidelines were classified as "PPF" (n=12), and subjects diagnosed as not having PPF were classified as "non-PPF" (n=12). The expression level of CXCL8 in the samples was measured using the commercially available kit Human IL-8 / CXCL8 DuoSet® ELISA (R&D SYSTEMS: DY208-05). Specifically, the following was done. Reagents used in the kit: • PBS: D-PBS (Nacalai Tesque: 14249-24) • Tween 20: Polyoxyethylene (20) sorbitan monolaurate (FUJIFILM: 160-21211) • BSA: 30(w / v)% bovine serum albumin solution, fatty acid-free (Nacalai Tesque: 19361-84) • Substrate solution: ELISA POD substrate TMB solution (Easy) (Nacalai Tesque: 05299-54) • 2N H2SO4: 1 mol / l sulfuric acid (2N) (Nacalai Tesque: 95626-06) Preparation reagents: • Wash buffer: 0.05% Tween (registered trademark) 20 in PBS • Reagent Diluent: 0.1% BSA, 0.05% Tween 20 in PBS • Block buffer: 1% BSA in PBS Equipment / Measurement equipment: Varioskan LUX (Thermo Fisher Scientific) - Concentration calculation: SkanIt Microplate Reader Software (Thermo Fisher Scientific) - Statistical analysis: Graph pad prism10.0.3 (Graphpad)

[0101] The expression level of CXCL8 protein in the sample was measured according to the following procedure in accordance with the kit's instructions: 1) Capture antibody was diluted to the indicated concentration in PBS and immobilized on 100 μL (room temperature, overnight). 2) After aspiration, the sample was washed three times with 400 μL of wash buffer. 3) Blocking was performed with 300 μL of Regent Diluent (room temperature, 1 hour). 4) Washing procedure (same as in 2) above) was performed. 5) 100 μL of standard or sample serially diluted with Regent Diluent was added (room temperature, 2 hours). 6) Washing procedure (same as in 2) above) was performed. 7) 100 μL of detection antibody diluted with Regent Diluent was added (room temperature, 2 hours). 8) Washing procedure (same as in 2) above) was performed. 9) 100 μL of Streptavidin-HRP diluted with Regent Diluent was added (room temperature, 20 minutes). 10) Washing procedure (same as in 2) above) was performed. 11) 100 μL of Substrate solution was added (room temperature, 10 minutes). 12) 50 μL of H2SO4 was added, and absorbance was immediately measured at 450 nm. 13) A calibration curve was created using 4PL, and the concentration was calculated. 14) Statistical analysis was performed using the Unpaired t test.

[0102] 3-2. Results The results are shown in Figure 3. As shown in Figure 3, it was found that the expression level of CXCL8 was significantly increased in PPF compared to non-PPF. Furthermore, it was found that threshold setting based on non-PPF was possible even when using CXCL8 as an indicator. Specifically, when the maximum value of 50.83 pg / ml in non-PPF was set as the threshold, the lowest value in PPF above the threshold was 53.1 pg / ml, which was 1.06 times higher than that of non-PPF. As described above, when threshold setting was performed by referring to the ROC curve, a CXCL8 concentration of 51.97 pg / ml was set as the threshold, with a sensitivity of 75% and a specificity of 100%, indicating that CXCL8 is an excellent biomarker for PPF. The ability to set a threshold that can distinguish between non-PPF and PPF with high accuracy is clinically very useful because it allows for judgment based on individual patient measurements rather than comparison with other patients. From this, it was found that CXCL8 is also very useful as an indicator for PPF diagnosis, etc.

[0103] Test Example 44-1. Test Procedure The following test was conducted focusing on macrophages. In the same manner as described above, bronchoalveolar lavage fluid was collected from subjects suffering from ILD (20 Japanese subjects (non-PPF (n=11) and PPF (n=9))) and scRNA-seq, mass cytometry analysis, viSNE analysis, etc. were performed to classify and select cell populations in the bronchoalveolar lavage fluid independently, and macrophages common to PPF were identified. Furthermore, focusing on two specific types of macrophages (Fibrotic macrophage 1 and Fibrotic macrophage 2), mRNA expression in these macrophages was examined.

[0104] Furthermore, the expression level of the SPP1 protein was measured according to the following procedure. As a sample, bronchoalveolar lavage fluid (BALF) (-80°C frozen, unconcentrated) collected from subjects suffering from ILD was used, as described above. The subjects were Japanese (non-PPF (n=12), PPF (n=12)).

[0105] Kit used: Human Osteopontin ELISA Kit (abcam: ab269374) Reagents used in the kit: UltraPure TM DNase / RNase-Free Distilled Water (Invitrogen: 10977015) Measuring equipment / Measuring equipment: Varioskan LUX (Thermo Fisher Scientific) - Concentration calculation: SkanIt Microplate Reader Software (Thermo Fisher Scientific) - Statistical analysis: Graph pad prism10.0.3 (Graphpad)

[0106] The expression level of the SPP1 protein was measured using the following procedure, in accordance with the kit's instructions: 1) 50 μL of calibration standard and sample were placed in each well. 2) 50 μL of Antibody Cocktail was added to each well and incubated at 25°C for 1 hour. 3) Three 10-second washes were performed using 350 μL of wash buffer. 4) 100 μL of TMB Development Solution was added and incubated at 25°C for 10 minutes. 5) 100 μL of Stop Solution was added and mixed for 1 minute, after which the absorbance was measured at 450 nm. 6) A calibration curve was created using 4PL and the concentration was calculated. 7) Statistical analysis was performed using an unpaired t-test.

[0107] 4-2. Results The results of the above test confirmed that the genes shown in Figure 4 were highly expressed in the identified macrophages. Among the genes shown in Figure 4, those enclosed in a box indicate genes that encode secreted proteins. Furthermore, when protein measurement was performed using SPP1 as a representative example of the highly expressed gene, it was found that the expression level of SPP1 protein was significantly increased in PPF compared to non-PPF, as shown in Figure 4. As described above, threshold setting was performed by referring to the ROC curve, and with an SPP1 concentration of 174.9 pg / ml as the threshold, the sensitivity was 66.67% and the specificity was 100%, indicating that SPP1 is an excellent biomarker for PPF. In addition, regarding HAMP (the gene name for Hepcidin (protein)), the results consistently showed that the number of macrophages expressing Hepcidin was significantly increased in PPF compared to non-PPF, consistent with the results demonstrated in Test Example 1 above. From these results, it was found that the gene products of each gene shown in Figure 4 are useful as biomarkers for testing or diagnosing PPF. In particular, the gene products (mainly mRNA and proteins) of HAMP, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, and APOC1, which encode secreted proteins, were found to be even more useful as biomarkers for testing or diagnosing PPF.

Claims

1. A method for testing progressive pulmonary fibrosis, comprising the step of measuring the expression level of the gene product of at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, APOC1, LGMN, SLC40A1, FOLR2, MS4A6A, MARCKS, TMEM176B, DAB2, STAB1, GPNMB, CSTB, SDS, LIPA, and SGK1 in a sample taken from a subject.

2. The testing method according to claim 1, wherein the gene is at least one selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, and APOC1.

3. The testing method according to claim 1, wherein the gene product is a protein, and the protein is a protein expressed from at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, and APOC1.

4. The testing method according to claim 1, wherein the sample is at least one selected from the group consisting of bronchoalveolar lavage fluid and serum.

5. The testing method according to claim 1, further comprising the step of determining whether the subject has progressive pulmonary fibrosis or is at high risk of progressive pulmonary fibrosis based on the expression level of the gene product measured in the above step.

6. The testing method according to claim 1, further comprising the step of determining that the subject has progressive pulmonary fibrosis or is at high risk of progressive pulmonary fibrosis if the expression level of the gene product measured in the above step (test value) is higher than the expression level of the gene product in a non-PPF-derived sample (reference value) or higher than a preset cutoff value.

7. A biomarker for the diagnosis of progressive pulmonary fibrosis comprising the gene product of at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, APOC1, LGMN, SLC40A1, FOLR2, MS4A6A, MARCKS, TMEM176B, DAB2, STAB1, GPNMB, CSTB, SDS, LIPA, and SGK1.

8. The biomarker according to claim 7, wherein the gene is at least one selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, and APOC1.

9. The biomarker according to claim 7, wherein the gene product is a protein, and the protein is a protein expressed from at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, and APOC1.

10. A diagnostic agent for progressive pulmonary fibrosis containing a detection agent for the gene product of at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, APOC1, LGMN, SLC40A1, FOLR2, MS4A6A, MARCKS, TMEM176B, DAB2, STAB1, GPNMB, CSTB, SDS, LIPA, and SGK1.

11. A diagnostic kit for progressive pulmonary fibrosis containing a detection agent for the gene product of at least one gene selected from the group consisting of HAMP, CXCL8, SPP1, RNASE1, CCL13, SELENOP, PLTP, CCL18, APOE, CTSB, CTSZ, HMOX1, F13A1, CD163, CHI3L1, PLA2G7, CHIT1, FABP5, CTSL, MMP9, APOC2, TREM2, CCL2, APOC1, LGMN, SLC40A1, FOLR2, MS4A6A, MARCKS, TMEM176B, DAB2, STAB1, GPNMB, CSTB, SDS, LIPA, and SGK1.

Images