JP-2026514347-A - PRO-C17 assay

Abstract

Disclosed is an immunoassay method ("PRO-C17 assay") for measuring the level of type XVII collagen ectodomains present in patient samples, which can be used to detect and/or monitor cancer in patients and/or to assess the severity of cancer. Also disclosed are monoclonal antibodies and assay kits suitable for use in carrying out the method. [Selection Diagram] Figure 7

Inventors

• ウィルムセン，ニコラス
• ブラヴォ，マリナ，クレスポ
• ペーターセン，ラスムス，スンド
• 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 (12)

1. An immunoassay method for detecting and/or monitoring cancer in a patient and/or assessing the severity of cancer in a patient, wherein the immunoassay method is: i) Contacting the patient's sample with a monoclonal antibody that specifically binds to the amino acid sequence QGMAPAAGA (SEQ ID NO: 1); ii) Detecting the binding between the monoclonal antibody and the peptide in the sample and determining the amount of binding; and, iii) The method includes 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.
2. The immunoassay method according to claim 1, wherein the monoclonal antibody specifically binds to the amino acid sequence LQGMAPAAGA (SEQ ID NO: 2).
3. The immunoassay method according to any of the above claims, wherein the monoclonal antibody does not specifically bind to the amino acid sequence GMAPAAGADL (SEQ ID NO: 3).
4. The immunoassay method according to any of the above claims, wherein the monoclonal antibody does not specifically bind to the amino acid sequence LQGLAPLGSE (SEQ ID NO: 4).
5. The immunoassay method according to any of the above claims, wherein the monoclonal antibody does not specifically bind to the amino acid sequence LQGMAYTVQG (SEQ ID NO: 5).
6. The immunoassay method according to any of the above claims, wherein the monoclonal antibody is produced by counteracting a synthetic peptide having the amino acid sequence LQGMAPAAGA (SEQ ID NO: 2).
7. The immunoassay method according to any one of the above claims, wherein the cancer is pancreatic cancer, colorectal cancer, kidney cancer, ovarian cancer, bladder cancer, lung cancer, melanoma, breast cancer, head and neck cancer, prostate cancer, or gastric cancer.
8. The immunoassay method according to any of the above claims, wherein the cancer is colorectal cancer.
9. The immunoassay method according to any one of the above claims, wherein the immunoassay method is a method for evaluating the severity of cancer in a patient.
10. The immunoassay method according to any of the above claims, wherein the sample from the patient is selected from blood, serum, or plasma.
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.

Description

This invention relates to an immunoassay method suitable for use in the detection and/or monitoring of cancer and/or assessment of its severity, and to antibodies and assay kits suitable for use in carrying out said method. Cancer is the leading cause of death worldwide, accounting for nearly 10 million deaths in 2020. The use of biomarkers is important for early diagnosis and the selection of appropriate treatments to reduce the burden of cancer [1] . In recent years, attention has been drawn to the tumor microenvironment, more specifically the extracellular matrix (ECM), which has been reported to promote tumor progression and reduce the response to cancer treatment [2] . Collagen is a major component of the extracellular matrix (ECM), and 28 different types of collagen have been identified to date [3] . In healthy tissues, collagen production and degradation are highly regulated to maintain tissue homeostasis. However, in cancer, excessive metabolic turnover and remodeling occur, resulting in a loss of systematic cellular behavior in the tissue and leading to unbalanced angiogenesis that can promote disease progression and tumorigenesis [4] . Protein fragments resulting from this excessive ECM remodeling are released into the circulation and can be non-invasively measured by liquid biopsy and used as biomarkers. Extensive research and biomarker discovery have been directed towards the most dominant collagens of the ECM, such as types I, III, and IV, but little is known about the less abundant, highly specialized groups of collagen. Interrupted triple-helical membrane-associated collagens (MACITs), including type XIII, XVII, XXIII, and XXV collagens, are type II proteins containing large ectodomains that are not present in other collagens and can be shed and released into the pericellular matrix [5,6] . Overall, MACITs are expressed in small amounts in adult tissues but are upregulated both during embryogenesis and tumorigenesis. While MACITs may contribute to maintaining ECM stability, there is growing evidence that they play an essential role in the process of determining cell fate by binding to ECM components such as cell surface receptors or growth factors in both healthy and cancerous tissues [7-9] . MACITs consist of an N-terminal cytoplasmic domain comprising distinct collagenous (COL) and non-collagenous (NC) domains, a transmembrane domain, and a C-terminal ectodomain [10] . Type XIII, XXIII, and XXV collagens are structurally similar, and their ectodomains are cleaved by furin proteases, whereas type XVII collagen has a different structure and is cleaved by ADAM9, 10, and 17 [11-13] . Type XVII collagen, also known as BP180 or BPAG2, consists of three identical 180 kDa α-chains and is part of the hemidesmosome, providing stable adhesion between basal keratinocytes and the basement membrane, thus playing an important role in the skin [14] . Several vesicular skin diseases are associated with both hereditary and acquired dysfunction of type XVII collagen. For example, mutations in the COL17A1 gene can cause junctional epidermolysis bullosa (JEB), while autoimmunity against the NC16A domain of shed type XVII collagen can induce bullous pemphigoid (BP) [15-17] . One study showed that a polyclonal antibody produced in opposition to the peptide sequence LQGMAPAAG (corresponding to amino acids 524-532 of the NC16A domain of type XVII collagen) preferentially reacts with the detached ectodomain of type XVII collagen, and that serum from bullous pemphigoid reacts with this same peptide, suggesting that the neoepitope of the detached ectodomain of type XVII collagen is located in this amino acid range and could be a target for blister-inducing antibodies [24] . In cancer, type XVII collagen and its detached ectodomain appear to play a major role in the development and metastasis of epithelial carcinomas [18] . Abnormal expression of the COL17A1 gene has been detected in many epithelial tumors, such as colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, or squamous cell carcinoma, and is associated with poor prognosis [19-22] . Furthermore, recent studies on the role of type XVII collagen in cancer stem cell dormancy have revealed that COL17A1-KO colorectal cancer organoids tend to form larger colonies than wild-type organoids due to increased proliferation rates. These studies also found that COL17A1-KO colorectal cancer organoids eliminate dormant LGR + p27 + cells and are more sensitive to chemotherapy, suggesting that COL17A1 may play a role in maintaining cell dormancy [23] . The inventors developed and validated an ELISA (also referred to herein as the "PRO-C17 assay") targeting the ectodomain of type XVII collagen, and studied the potential of PRO-C17 as a biomarker in cancer patients by measuring the levels of circulating PRO-C17 in serum samples (i.e., the level of type XVII collagen ectodomain detected by the PRO-C17 assay). The assay was found to be technically robust, and the d