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JP-2026514348-A - C3F assay

JP2026514348AJP 2026514348 AJP2026514348 AJP 2026514348AJP-2026514348-A

Abstract

This document discloses an immunoassay method for detecting fibroblast-activating protein (FAP)-producing fragments of type III collagen in patient samples, and its use for detecting and/or monitoring cancer or arthritis or a specific level of their severity in patients. It also describes monoclonal antibodies and assay kits for use with the immunoassay method. [Selection Diagram] Figure 6

Inventors

  • ペーターセン，ラスムス，スンド
  • ウィルムセン，ニコラス
  • カルスダル，モルテン，アッセール

Assignees

  • ノルディック・ビオサイエンス・エー／エス

Dates

Publication Date
20260511
Application Date
20240322
Priority Date
20230322

Claims (17)

  1. An immunoassay method including the following steps; i) Contacting the patient's sample with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence AGPAGAPGPA (SEQ ID NO: 1); and, ii) Detect the binding between the monoclonal antibody and the peptide in the sample, and determine the amount of binding.
  2. The immunoassay method is an immunoassay method for detecting and/or monitoring cancer or a specific level of severity of said cancer in a patient, the method further comprising the following steps, according to claim 1; iii) Correlating the binding amount with a value associated with a normal healthy person, and/or a value associated with a known disease severity, and/or a value obtained from the patient at a past point in time, and/or a predetermined cutoff value.
  3. The immunoassay method according to claim 2, wherein the cancer is lung cancer.
  4. The immunoassay method according to claim 3, wherein the lung cancer is non-small cell lung cancer.
  5. The immunoassay method described above is a method for detecting and/or monitoring arthritis or a specific level of severity of said arthritis in a patient, the method further comprising the following steps, according to claim 1; iii) Correlating the binding amount with a value associated with a normal healthy person, and/or a value associated with a known disease severity, and/or a value obtained from the patient at a past point in time, and/or a predetermined cutoff value.
  6. The immunoassay method according to claim 5, wherein the arthritis is spondyloarthritis.
  7. The immunoassay method according to any of the above claims, wherein the sample from the patient is selected from blood, plasma, or serum.
  8. The immunoassay method according to any one of the claims, wherein the monoclonal antibody does not specifically bind to the peptide having the N-terminal amino acid sequence PAGPAGAPGPA (SEQ ID NO: 2).
  9. The immunoassay method according to any one of the claims, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence GPAGAPGPA (SEQ ID NO: 3).
  10. The immunoassay method according to any of the above claims, wherein the monoclonal antibody is produced in opposition to a synthetic peptide having the N-terminal amino acid sequence AGPAGAPGPA (SEQ ID NO: 1).
  11. The immunoassay method according to any one of the above claims, wherein the immunoassay method is a competitive assay or a sandwich assay.
  12. The immunoassay method according to any one of the above claims, wherein the immunoassay method is a radioimmunoassay or an enzyme-linked immunosorbent assay.
  13. A monoclonal antibody that specifically binds to the N-terminal amino acid sequence AGPAGAPGPA (SEQ ID NO: 1).
  14. The monoclonal antibody according to claim 13, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence PAGPAGAPGPA (SEQ ID NO: 2).
  15. The monoclonal antibody according to claim 13 or claim 14, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminal amino acid sequence GPAGAPGPA (SEQ ID NO: 3).
  16. The monoclonal antibody described above is produced by counteracting a synthetic peptide having the N-terminal amino acid sequence AGPAGAPGPA (SEQ ID NO: 1), as described in any one of claims 13 to 15.
  17. An immunoassay kit comprising a monoclonal antibody according to any one of claims 13 to 16, and at least one of the following: - Well plate coated with streptavidin - Biotinylated peptide AGPAGAPGPA-L-biotin (SEQ ID NO: 14), where L is an optional linking group - Second antibody used in sandwich immunoassay - Calibration protein containing the N-terminal amino acid sequence AGPAGAPGPA (SEQ ID NO: 1) - Antibody biotinylation kit - Antibody HRP labeling kit - Antibody radiolabeling kit.

Description

This invention relates to an immunoassay method for detecting fibroblast-activating protein (FAP)-producing fragments of type III collagen in patient samples, and to its use for detecting and/or monitoring cancer or arthritis or a specific level of their severity in patients. The invention also relates to monoclonal antibodies and assay kits for use in said immunoassay method. In the United States alone, it is estimated that there will be more than 230,000 new cases of lung cancer and more than 130,000 deaths from lung cancer in 2022, making lung cancer still one of the leading causes of cancer-related deaths. [1] The tumor microenvironment (TME) is well established to be important for tumor progression and patient survival, and therefore there is growing interest in the investigation and treatment of various cancers [2-5] . The TME includes the extracellular matrix (ECM), tumor cells, immune cells, and stromal cells [6] . Cancer-associated fibroblasts (CAFs) are a major component of the tumor stroma [6,7] . CAFs are involved in cancer progression, immunosuppression, and ECM remodeling [8-10] . CAFs affect ECM remodeling by over-synthesizing collagen and other ECM components, and by secreting metalloproteinases (MMPs) [10,11] . Fibroblast-activating protein (FAP) is a type II transmembrane serine protease that is expressed almost exclusively in the interstitium under pathological conditions including arthritis, fibrosis, and cancer, and is one of the major biological markers of CAF [3, 12, 13] . Several studies have investigated the diagnostic and prognostic value of FAP [2, 14–17] . While the results of these studies were somewhat inconclusive regarding the general prognostic value of FAP, FAP has shown potential as a prognostic marker for outcomes in lung, gastric, and colon cancer [16–18] . FAP has also been shown to be involved in ECM reconstruction and fibrosis [19] . For example, excessive ECM degradation due to MMP-mediated cleavage during tumor progression generates protein fragments that are released into circulation. These fragments can be non-invasively evaluated as fluid biopsies and quantified as disease biomarkers [20, 21] . Since FAP has been shown to cleave ECM components including type I and type III collagen, fragments derived from these cleavages can be expected to appear in circulation [12, 22] . FAP belongs to the dipeptidyl peptidase 4 (DPP4) protein family, and like other enzymes in the DPP4 family, FAP has dipeptidyl peptidase activity. Furthermore, FAP also has endopeptidase activity, which distinguishes FAP from other DPP4 proteins and leads to potential unique cleavage targets [3, 13] . Zhang et al. used terminal amine isotope labeling (TAILS) on mouse embryonic fibroblast substrates to identify the native substrates of human FAP. Using this degradomics approach, Zhang et al. identified multiple potential FAP cleavage sites in various ECM-related proteins, including 1069 AGPSGAPGPA 1078 (SEQ ID NO: 20) on mouse type III collagen [22] . The inventors have now developed an enzyme-linked immunosorbent assay (ELISA) targeting a fragment of the alpha-1 chain of human type III collagen, which is generated by cleavage of the alpha-1 chain between amino acid residues P 1069 and A 1070 by fibroblast-activating protein (FAP). This fragment may serve as a biomarker for FAP activity in the extracellular matrix (ECM). The inventors further measured the levels of this biomarker in a lung cancer cohort and compared the levels in patients with non-small cell lung cancer (including adenocarcinoma and squamous cell carcinoma) with those in healthy individuals. The inventors also measured the levels of this biomarker in patients with spondyloarthritis. The inventors demonstrated that the levels of this biomarker were elevated in patients with cancer and elevated in patients with arthritis. Therefore, in a first embodiment, the present invention provides an immunoassay method comprising contacting a sample of a patient with a monoclonal antibody that specifically binds to a fibroblast-activating protein (FAP)-producing neoepitope of the alpha-1 chain FAP-producing fragment of human type III collagen, and detecting the binding between the monoclonal antibody and a peptide in the sample and determining the amount of binding, wherein the FAP-producing neoepitope consists of an N-terminal sequence or a C-terminal sequence of the FAP-producing fragment that is present at the end of the FAP-producing fragment cleaved by FAP. In a preferred embodiment, the immunoassay method includes the following steps: i) Contacting the patient's sample with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence AGPAGAPGPA (SEQ ID NO: 1) (the sequence is also referred to herein as the “target sequence” and/or “C3F”); and ii) Detecting the binding between the monoclonal antibody and the peptide in the sample and determining the amount of binding. In a preferred embodiment, the immunoassay metho