Novel epitope-specific assays to measure protease-mediated degradation of collagen type IV

Abstract

Described herein is an assay for measuring protease-mediated degradation of type IV collagen and its biomarker potential for identifying cancer patients with a T-cell permissive tumor microenvironment.

Description

Technical Field

[0001] This invention relates to a method for measuring protease-mediated type IV collagen degradation and its biomarker potential, for the identification of cancer patients with a T-cell permissive tumor microenvironment. Background Technology

[0002] Immunotherapy with immune checkpoint inhibitors has revolutionized cancer treatment by providing the opportunity for a durable response (1). Immune checkpoint inhibitor therapy involves antibodies, such as anti-CTLA-4, anti-PD1, and anti-PD-L1 antibodies, which can reactivate cytotoxic T lymphocytes, thereby enabling them to eliminate tumor cells. However, despite the clinical success of these immune checkpoint inhibitors, only a subset of cancer patients experience a long-term survival benefit. Therefore, it is important to identify non-invasive biomarkers that can identify cancer patients who will respond to immune checkpoint inhibitor therapy in order to avoid mistreatment and adverse events. To identify predictive biomarkers, understanding some factors that influence response and resistance is crucial.

[0003] Three distinct immune profiles of patients prior to treatment have been identified as being associated with responses to immune checkpoint therapy (2). Clinical responses are most frequently observed in patients with an immune-inflammatory tumor type characterized by the presence of CD4 and CD8-expressing T cells in the tumor microenvironment near the tumor cells. The immune-exempt phenotype is characterized by the presence of immune cells, but these are retained in the surrounding matrix, thus preventing T cell infiltration. In the immune-desert phenotype, there are no T cells in either the tumor parenchyma or the matrix. Patients with either the immune-exempt or immune-desert phenotypes rarely respond to immune checkpoint inhibitor therapy, making biomarkers for identifying immune-inflammatory tumor types potentially very useful predictive tools. For effective cancer immunotherapy, it is important that T cells are both activated and recruited to the tumor microenvironment that defines the immune-inflammatory phenotype (“hot tumor”) (3).

[0004] The composition of the extracellular matrix (ECM) has been shown to influence T cell localization and migration, and its key role in immunotherapy resistance has been identified (4–7). We have previously shown that the serum biomarker PRO-C3, reflecting excessive type III collagen formation (connective tissue hyperplasia), and the biomarker C4M, reflecting type IV collagen degradation by matrix metalloproteinase (MMP)-9, are associated with poor responses to immune checkpoint blockade (8). In addition, C4M is elevated in various cancer patients (9,10).

[0005] Interestingly, T cells also express proteases that induce invasive behavior in T cells (11,12). It has been shown that migrating T cells secrete MMPs and serine proteases (granzyme B) to cross the basement membrane and enter underlying tissues (11,13-15). Summary of the Invention

[0006] Type IV collagen is a major component of the basement membrane; therefore, the inventors hypothesized that type IV collagen fragments produced by specific proteases are released into the circulatory system of cancer patients as part of T cell migration from the circulatory system to the tumor microenvironment. Thus, these type IV collagen fragments could potentially possess the potential to be biomarkers for identifying cancer patients with a T-cell permissive tumor microenvironment responding to immune checkpoint inhibitor therapy. The inventors have now developed a competitive electrochemiluminescence immunoassay (ECLIA) that targets novel epitopes of protease-mediated type IV collagen degradation and demonstrated elevated levels in the serum of patients with metastatic melanoma who were successfully treated with the immune checkpoint inhibitor ipilimumab. To further evaluate its potential, this biomarker was also evaluated in the serum of patients with different types of cancer. This biomarker could also be used to provide survival prognosis for patients with cancer, particularly pancreatic ductal adenocarcinoma.

[0007] Therefore, in a first aspect, the present invention relates to a peptide that specifically recognizes and binds to a peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) (also referred to herein as the target peptide or C4aa). 1355 The peptide sequence represents the result of using a protease such as a serine protease (e.g., granzyme B) or a matrix metalloproteinase (e.g., MMP-9) (leading to amino acid F). 1354 and M 1355 (The cleavage between them) produces new epitopes by digesting the α2 chain of type IV collagen.

[0008] Preferably, the monoclonal antibody does not recognize or specifically binds to the extended form of the N-terminal amino acid sequence, which is XMGNTGPTGAV (SEQ ID No. 2), where X is any amino acid. Preferably, X is F. Preferably, the monoclonal antibody does not recognize or specifically binds to the truncated form of the N-terminal amino acid sequence, particularly the peptide of GNTGPTGAV (SEQ ID No. 3). Preferably, the monoclonal antibody does not recognize or specifically bind to the mutant form of the N-terminal amino acid sequence, which is MGQTGPTGAV (SEQ ID No. 4), MGNSGPTGAV (SEQ ID No. 5), and / or QGNTGPTGAV (SEQ ID No. 6).

[0009] Preferably, the ratio of the antibody's affinity for the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) to the antibody's affinity for the extended N-terminal amino acid sequence FMGNTGPTGAV (SEQ ID No. 7) is at least 10:1, more preferably at least 50:1, at least 100:1, at least 500:1, at least 1,000:1, at least 10,000:1, at least 100,000:1, or at least 1,000,000:1.

[0010] Preferably, the ratio of the antibody's affinity for the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) to the antibody's affinity for the truncated N-terminal amino acid sequence GNTGPTGAV (SEQ ID No. 3) is at least 10:1, more preferably at least 50:1, at least 100:1, at least 500:1, at least 1,000:1, at least 10,000:1, at least 100,000:1, or at least 1,000,000:1.

[0011] Preferably, the ratio of the antibody's affinity for the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) to the antibody's affinity for the mutated N-terminal amino acid sequence MGQTGPTGAV (SEQ ID No. 4) is at least 10:1, more preferably at least 50:1, at least 100:1, at least 500:1, at least 1,000:1, at least 10,000:1, at least 100,000:1, or at least 1,000,000:1.

[0012] Preferably, the ratio of the antibody's affinity for the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) to the antibody's affinity for the mutated N-terminal amino acid sequence MGNSGPTGAV (SEQ ID No. 5) is at least 10:1, more preferably at least 50:1, at least 100:1, at least 500:1, at least 1,000:1, at least 10,000:1, at least 100,000:1, or at least 1,000,000:1.

[0013] Preferably, the ratio of the antibody's affinity for the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) to the antibody's affinity for the mutated N-terminal amino acid sequence QGNTGPTGAV (SEQ ID No. 6) is at least 10:1, more preferably at least 50:1, at least 100:1, at least 500:1, at least 1,000:1, at least 10,000:1, at least 100,000:1, or at least 1,000,000:1.

[0014] Monoclonal antibodies that specifically bind to the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) can be generated using any suitable technique known in the art. For example, monoclonal antibodies can be generated against a synthetic peptide having the amino acid sequence MGNTGPTGAV (SEQ ID No. 1), for example, by: immunizing rodents (or other suitable mammals) with a synthetic peptide comprising the sequence MGNTGPTGAV (SEQ ID No. 1), which may optionally be linked to an immunogenic carrier protein (e.g., keyhole hemocyanin); isolating and cloning individual antibody-generating cells; and determining the resulting monoclonal antibodies to ensure they have the desired specificity. Exemplary protocols for generating monoclonal antibodies that specifically bind to the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) are described below.

[0015] Preferably, the monoclonal antibody or a fragment thereof may preferably contain one or more complementarity-determining regions (CDRs) selected from:

[0016] CDR-L1:KSSQSLLYSDGKTYLN(SEQ ID No.8)

[0017] CDR-L2: LVSKLDS (SEQ ID No. 9)

[0018] CDR-L3:WQGTHFVT(SEQ ID No.10)

[0019] CDR-H1:TYNIGVG(SEQ ID No.11)

[0020] CDR-H2:HIWYNDIKYYNTALKS(SEQ ID No.12)

[0021] CDR-H3:LRPSFDY(SEQ ID No.13)

[0022] Preferably, the antibody or fragment thereof contains at least 2, 3, 4, 5 or 6 of the listed CDR sequences.

[0023] Preferably, the monoclonal antibody or a fragment thereof has a light chain variable region comprising the following CDR sequence:

[0024] CDR-L1:KSSQSLLYSDGKTYLN(SEQ ID No.8)

[0025] CDR-L2: LVSKLDS (SEQ ID No.9) and

[0026] CDR-L3:WQGTHFVT(SEQ ID No.10)

[0027] Preferably, the monoclonal antibody or fragment thereof has a light chain comprising a framework sequence between CDRs, wherein the framework sequence is substantially identical or substantially similar to the framework sequence between CDRs in the following light chain sequence (wherein CDRs are shown in bold and underline, and the framework sequence is shown in italics).

[0028]

[0029] Preferably, the monoclonal antibody or a fragment thereof has a heavy chain variable region comprising the following CDR sequence:

[0030] CDR-H1:TYNIGVG(SEQ ID No.11)

[0031] CDR-H2: HIWYNDIKYYNTALKS (SEQ ID No. 12) and

[0032] CDR-H3:LRPDSFDY(SEQ ID No.13)

[0033] Preferably, the monoclonal antibody or fragment thereof has a heavy chain comprising a framework sequence between CDRs, wherein the framework sequence is substantially identical or substantially similar to the framework sequence between CDRs in the following light chain sequence (wherein CDRs are shown in bold and underline, and the framework sequence is shown in italics).

[0034]

[0035] As used herein, if the framework amino acid sequence between the CDRs of one antibody has at least 70%, 80%, 90%, or at least 95% similarity or identity with the framework amino acid sequence between the CDRs of another antibody, then the framework amino acid sequences between the CDRs of the two antibodies are substantially identical or substantially similar. The similar or identical amino acids may be continuous or discontinuous.

[0036] A framework sequence may contain one or more amino acid substitutions, insertions, and / or deletions. Amino acid substitutions can be conserved, meaning that the substituted amino acid has similar chemical properties to the original amino acid. Those skilled in the art will understand which amino acids have similar chemical properties. For example, the following groups of amino acids have similar chemical properties, such as size, charge, and polarity: Group 1: Ala, Ser, Thr, Pro, Gly; Group 2: Asp, Asn, Glu, Gln; Group 3: His, Arg, Lys; Group 4: Met, Leu, Ile, Val, Cys; Group 5: Phe, Thy, Trp.

[0037] Programs such as CLUSTAL can be used to compare amino acid sequences. This program compares amino acid sequences and finds the best alignment by appropriately inserting spaces in either sequence. Amino acid identity or similarity (identity plus conservation of amino acid types) can be calculated to obtain the best alignment. Programs like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. Therefore, comparisons can be made to obtain a comparison of several regions where similarity is found, each region having a different score. Both types of analysis are considered in this invention. It is preferable to calculate identity or similarity over the entire length of the frame sequence.

[0038] In some preferred embodiments, the monoclonal antibody or a fragment thereof may contain a light chain variable region sequence:

[0039]

[0040] (CDR displays in bold and underline; frame sequences are displayed in italics)

[0041] and / or heavy chain variable region sequences:

[0042]

[0043] (CDR displays in bold and underline; frame sequences are displayed in italics)

[0044] In a second aspect, the present invention relates to a method for identifying whether a subject with cancer responds to immunotherapy, the method comprising detecting the presence of a peptide having the N-terminal amino acid MGNTGPTGAV (SEQ ID No. 1) in a sample obtained from the subject. The sample is preferably a biofluid sample, particularly a human biofluid sample.

[0045] Preferably, the immunotherapy comprises at least one immune checkpoint inhibitor. Immune checkpoint inhibitors target molecules on immune cells that need to be activated (or inactivated) to initiate an immune response. These checkpoint proteins include PD-1, PD-L1, and CTLA-4. Immune checkpoint inhibitors may target any one or more of these molecules. Immune checkpoint inhibitors include ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cimiprimab, or combinations thereof. Preferably, the immune checkpoint inhibitor may target CTLA-4, such as ipilimumab. Alternatively, the immune checkpoint inhibitor may target PD-1, such as pembrolizumab, nivolumab, and / or cimiprimab. Alternatively, immune checkpoint inhibitors can target PD-L1, such as atezolizumab, avelumab, or durvalumab. Preferably, immunotherapy with at least one immune checkpoint inhibitor comprises administration of ipilimumab.

[0046] Preferably, the method is an immunoassay. More preferably, the method includes contacting a biofluid sample obtained from a subject with a monoclonal antibody according to the first aspect of the invention, and detecting the binding between the monoclonal antibody and a peptide in the sample.

[0047] The method of the second aspect utilizes the monoclonal antibody of the first aspect, and therefore the preferred embodiment of the second aspect will be apparent from the above discussion of the preferred embodiment of the first aspect.

[0048] Preferably, the detection is quantitative. Therefore, the method can include detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample.

[0049] Preferably, the immunoassay is a competitive immunoassay.

[0050] Preferably, the immunoassay is an enzyme-linked immunosorbent assay (ELISA) or an electrochemiluminescence immunoassay (ECLIA). Preferably, the ELISA is a competitive ELISA. Preferably, the ECLIA is a competitive ECLIA.

[0051] The biofluid sample may be, but is not limited to, blood, serum, plasma, urine, or supernatant from cell or tissue cultures. Preferably, the biofluid is serum or plasma, and most preferably serum.

[0052] In the second aspect of the method, the sample is obtained from a subject who has been diagnosed with cancer. The cancer may be metastatic. The cancer is preferably selected from melanoma, breast cancer, colorectal cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, prostate cancer, or pancreatic cancer (including pancreatic ductal adenocarcinoma). Preferably, the subject has melanoma, particularly metastatic melanoma.

[0053] The method may further include correlating the amount of detected peptides with values ​​associated with normal healthy subjects and / or values ​​obtained from cancer patients who have clinically responded to immunotherapy, such as those who have experienced prolonged survival, tumor shrinkage, and / or symptom improvement after treatment. Elevated peptide levels indicate that the subject has an immune-inflammatory tumor type and will therefore respond to immunotherapy.

[0054] As used herein, the term “values ​​associated with normal healthy subjects and / or values ​​obtained from oneself in response to immunotherapy in cancer patients” refers to standardized values ​​determined by the above methods for subjects considered healthy, i.e., without cancer; and / or standardized values ​​determined by the above methods for subjects known to have cancer and who have clinically responded to immunotherapy, preferably immune checkpoint inhibitor therapy, such as tumor size reduction, symptom improvement, and / or extended overall survival.

[0055] In some embodiments of the method according to the second aspect, the amount of binding of a monoclonal antibody specific to the epitope of type IV collagen peptide to the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) is correlated with one or more predetermined cutoff values.

[0056] As used herein, a “cutoff value” refers to a statistically determined amount of binding that indicates a high probability of response to immunotherapy using an immune checkpoint inhibitor. A measurement of biomarker binding in a patient sample equal to or higher than the following statistical cutoff values, corresponding to a probability of at least 70%, preferably at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%, of the presence or probability of response to immunotherapy, preferably indicated by tumor size reduction, symptom improvement, and / or increased overall survival, using an immune checkpoint inhibitor. A “cutoff value” can be calculated by comparing results obtained from patients diagnosed with cancer and responding to immunotherapy with results obtained from patients diagnosed with the same cancer but not responding to immunotherapy.

[0057] If the amount of binding of a monoclonal antibody specific to the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) is in the top three quartiles (Q2+Q3+Q4) of the level measured in cancer patients, especially those with the same type of cancer, this suggests that the patient may be responding to treatment with an immune checkpoint inhibitor.

[0058] The predetermined cutoff value for the binding amount of a monoclonal antibody specific to the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) can be in the range of 10.0-20.0 ng / mL. Preferably, the predetermined cutoff value for the binding amount of a monoclonal antibody specific to the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) is at least 14.5 ng / mL. In this regard, statistical analysis has shown that a measured binding amount of at least 14.5 ng / mL or greater of a monoclonal antibody specific to the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) can be a determinant of a patient's likelihood of responding to immunotherapy, preferably immunotherapy using an immune checkpoint inhibitor. With a statistical cutoff value of at least 14.5 ng / mL, the method of the present invention can be used to predict the response to immunotherapy with high confidence. In particular, at least 14.5 ng / mL or greater can be a determinant of a melanoma patient's likelihood of responding to immunotherapy. Applying such a statistical cutoff is particularly advantageous because it produces independent diagnostic analyses; that is, it eliminates the need for any direct comparisons with healthy individuals and / or patients known to have responded to immunotherapy, preferably those who have responded to immune checkpoint inhibitor therapy, to draw conclusions. Rapid, conclusive diagnosis can lead to a response in patients who are more likely to be treated early, which in turn improves overall survival and / or reduces the risk of hospitalization.

[0059] The method may further include administering immunotherapy to subjects identified as having elevated levels of the peptide.

[0060] In a third aspect, the present invention relates to an assay kit comprising a monoclonal antibody that specifically recognizes and binds to a peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1), and at least one of the following:

[0061] -Streptavidin-coated well plates;

[0062] - A C-terminal biotinylated peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1);

[0063] - A calibration peptide with the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1),

[0064] - Antibody biotinylation kit;

[0065] - Antibody HRP labeling kit;

[0066] - Antibody radiolabeling kit; and

[0067] - Visualization kit for assays.

[0068] This kit can be used to identify cancer patients who will respond to immunotherapy, preferably with immune checkpoint inhibitors.

[0069] The immunoassay kit is suitable for implementing the method according to the second aspect and contains the monoclonal antibody according to the first aspect; therefore, the preferred embodiments of the third aspect will become apparent from the above discussion of the preferred embodiments of the first and second aspects.

[0070] In a fourth aspect, the invention also relates to the use of immunotherapy to treat a patient diagnosed with cancer who is known to have elevated levels of peptide C4-aa. 1355 Subjects. Preferably, the immunotherapy comprises at least one immune checkpoint inhibitor. As used herein, "elevated levels of peptide C4-aa..." 1355 "This refers to a level of peptide that is significantly higher than that detected in normal healthy controls and / or subjects who have been diagnosed with cancer but have not yet responded to immunotherapy, particularly immune checkpoint inhibitors."

[0071] The cancer can be metastatic. The cancer is preferably selected from melanoma, breast cancer, colorectal cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, prostate cancer, or pancreatic cancer, including pancreatic ductal adenocarcinoma. Preferably, the subject has melanoma, particularly metastatic melanoma.

[0072] In a fifth aspect, the invention also relates to a method for predicting the survival outcome of a subject with cancer, the method comprising detecting the presence of a peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1).

[0073] Preferably, the method is an immunoassay. More preferably, the method includes contacting a biofluid sample obtained from a subject with a monoclonal antibody according to the first aspect of the invention, and detecting the binding between the monoclonal antibody and a peptide in the sample.

[0074] The method of the fifth aspect utilizes the monoclonal antibody of the first aspect, and therefore the preferred embodiment of the fifth aspect will be apparent from the above discussion of the preferred embodiment of the first aspect.

[0075] Preferably, the detection is quantitative. Therefore, the method can include detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample.

[0076] Preferably, the immunoassay is a competitive immunoassay.

[0077] Preferably, the immunoassay is an enzyme-linked immunosorbent assay (ELISA) or an electrochemiluminescence immunoassay (ECLIA). Preferably, the ELISA is a competitive ELISA. Preferably, the ECLIA is a competitive ECLIA.

[0078] The biofluid sample may be, but is not limited to, blood, serum, plasma, urine, or supernatant from cell or tissue cultures. Preferably, the biofluid is serum or plasma, and most preferably, serum.

[0079] In the second aspect of the method, the sample is obtained from a subject who has been diagnosed with cancer. The cancer is preferably selected from melanoma, breast cancer, colorectal cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, prostate cancer, or pancreatic cancer, including pancreatic ductal adenocarcinoma. Preferably, the subject has pancreatic cancer, more preferably pancreatic ductal adenocarcinoma.

[0080] This method may further include correlating the amount of the detected peptide with values ​​associated with normal healthy subjects and / or values ​​obtained from cancer patients, such as those already diagnosed with the same type of cancer. If the amount of binding of the monoclonal antibody specific to the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) is at the top or bottom quartile (Q1 or Q4) of the level measured in cancer patients, particularly those with the same type of cancer, this indicates a likely poor prognosis and an increased risk of death. If the amount of binding of the monoclonal antibody specific to the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) is at the middle quartile (Q2+Q3) of the level measured in cancer patients, particularly those with the same type of cancer, this indicates a reduced risk of death.

[0081] As used herein, the terms “values ​​associated with normal healthy subjects and / or values ​​obtained from cancer patients” refer to standardized quantities determined by the above methods for subjects considered healthy (i.e., without cancer) and / or standardized quantities determined by the above methods for subjects known to have cancer.

[0082] In some embodiments of the method according to the fifth aspect, the amount of binding of a monoclonal antibody specific to the epitope of type IV collagen peptide to the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) is associated with one or more related predetermined cutoff values.

[0083] As used herein, a “cutoff value” refers to a statistically determined amount of binding to indicate a reduction in the risk of death. Measurements of biomarker binding in patient samples within the statistical cutoff value can correspond to a probability of at least 70%, preferably at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of a reduction in the risk of death. The “cutoff value” can be calculated by comparing results obtained from patients diagnosed with cancer and with known survival times.

[0084] definition

[0085] As used in this article, the terms “peptide” and “polypeptide” are used synonymously.

[0086] As used herein, the term "monoclonal antibody" refers to an intact antibody and a fragment thereof that retains the binding specificity of the intact antibody, such as a Fab fragment, an F(ab')2 fragment, a single-chain Fv fragment, or other such fragments known to those skilled in the art. It is well known that intact antibodies typically have a "Y-shaped" structure of two pairs of identical polypeptide chains, each pair consisting of a "light" chain and a "heavy" chain. The N-terminal regions of each light and heavy chain contain variable regions, while the C-terminal portions of each heavy and light chain constitute constant regions. The variable regions contain three complementarity-determining regions (CDRs) primarily responsible for antigen recognition. The constant regions allow the antibody to recruit cells and molecules of the immune system. An antibody fragment that retains binding specificity contains at least a portion of the CDRs and the remaining variable regions sufficient to retain said binding specificity.

[0087] In this invention, the monoclonal antibody may contain any constant region known in the art. The human constant light chain is divided into κ and λ light chains. The constant heavy chain is divided into μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG isotypes have several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. The monoclonal antibody is preferably an IgG isotype, including any one of IgG1, IgG2, IgG3, or IgG4.

[0088] Methods known in the art, such as those by Kabat et al., can be used. 19The described method determines the CDR of an antibody. As described in the examples, antibodies can be generated from B cell clones. The isotype of the antibody can be determined by an ELISA specific to human IgM, IgG, or IgA isotypes or human IgG1, IgG2, IgG3, or IgG4 subclasses. The amino acid sequence of the generated antibody can be determined using standard techniques. For example, RNA can be isolated from cells and used to generate cDNA by reverse transcription. The cDNA is then PCR-paired using primers that amplify the antibody heavy and light chains. For example, primers specific to the leader sequence of all VH (variable heavy chain) sequences can be used in conjunction with primers that bind to sequences located in previously determined isotype constant regions. The light chain can be amplified using primers that bind to the 3' end of the κ or λ chain and primers that anneal to the Vκ or Vλ leader sequence. Full-length heavy and light chains can be generated and sequenced.

[0089] As used herein, the term "C-terminus" refers to the end of a polypeptide, specifically at the C-terminus, and should not be interpreted in terms of its general orientation. Similarly, the term "N-terminus" refers to the end of a polypeptide, specifically at the N-terminus, and should not be interpreted in terms of its general orientation.

[0090] As used herein, the term “competitive immunoassay” refers to an immunoassay in which a target peptide present in a sample (if present) competes with a known amount of a peptide target (e.g., bound to or labeled with a fixed substrate) for binding to an antibody, a technique known to those skilled in the art.

[0091] As used herein, the term "ELISA" (enzyme-linked immunosorbent assay) refers to an immunoassay in which an antibody linked to an enzyme such as horseradish peroxidase or alkaline phosphatase is used to detect the presence of a target peptide (if present) in a sample. The enzyme activity is then assessed by incubation with a substrate that produces a measurable product. Thus, the presence and / or amount of the target peptide in the sample can be detected and / or quantified. ELISA is a technique known to those skilled in the art.

[0092] As used herein, the term "ECLIA" (electrochemically linked immunosorbent assay) refers to an immunoassay in which an antibody linked to an electrochemiluminescent label, such as the SULFO-Tag system, is used to detect the presence of a target peptide (if any) in a sample. An electric current is applied to the sample, causing the electrochemiluminescent label to emit light. The light intensity is then measured to quantify the target peptide in the sample. Thus, the presence and / or amount of the target peptide in the sample can be detected and / or quantified. ECLIA is a technique known to those skilled in the art.

[0093] As used herein, the term "binding amount" refers to the quantification of the binding between a monoclonal antibody and a target peptide, wherein such quantification is determined by comparing a measured value of the target peptide in a biofluid sample with a calibration curve generated using a standard sample of the target peptide at a known concentration. In the specific assay disclosed herein for measuring a target peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) in a biofluid, a calibration curve is generated using a standard sample of a calibration peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) at a known concentration (and which may specifically consist of the amino acid sequence MGNTGPTGAV (SEQ ID No. 1)). The measured value in the biofluid sample is compared with the calibration curve to determine the actual amount of the target peptide in the sample.

[0094] As used herein, “immunotherapy” refers to a method of artificially stimulating the immune system in cancer treatment. Many different types of immunotherapy exist, including but not limited to T-cell connectives, CAR T-cell therapy, cytokines, and immune checkpoint inhibitors. Preferably, immunotherapy involves administering at least one immune checkpoint inhibitor, such as ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cimiprimab, or combinations thereof. Preferably, the immune checkpoint inhibitor is ipilimumab.

[0095] As used in this article, the term "C4-aa" 1355 "or "C4G" refers to the amino acid F that has the effect of being caused by the amino acid F 1354 and M 1355 The protease that cleaves between the proteins produces a novel epitope peptide of the type VI collagen α2 chain with the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1). Preferably, the protease is a serine protease, such as granzyme B, or a matrix metalloproteinase, such as MMP-9. Attached Figure Description

[0096] The invention will now be described in the following embodiments with reference to the following figures:

[0097] Figure 1 C4-aa was displayed 1355 The specificity of monoclonal antibodies.

[0098] In competitive C4-aa 1355In ECLIA, the reactivity of monoclonal antibodies to the following: A) selected peptides (MGNTGPTGAV (SEQ ID No. 1)), extended peptides (FMGNTGPTGAV (SEQ ID No. 7)), truncated peptides (GNTGPTGAV (SEQ ID No. 3)), nonsense-selective peptides (LLARDFEKNY (SEQ ID No. 18)), and nonsense-coating peptides (LLARDFEKNY-K-biotin); and (B) selected peptides (MGNTGPTGAV (SEQ ID No. 1)) and deselective peptides 1 (MGQTGPTGAV (SEQ ID No. 4)), 2 (MGNSGPTGAV (SEQ ID No. 5)), and 3 (QGNTGPTGAV (SEQ ID No. 6)). %B / B0: B equals the intensity of the sample well (x ng / ml peptide OD), and B0 equals the maximum intensity (OD, 0 ng / ml peptide OD).

[0099] Figure 2 The proteolytic degradation of type IV collagen α2 chains containing MMP-9 or granzyme B was shown.

[0100] Type IV collagen α2 chain was incubated with MMP-9(A) or granzyme B(GzB)(B) for 72 hours, and then C4-aa was measured. 1355 ECLIA level.

[0101] Figure 3 Serum C4-aa was shown in patients with metastatic melanoma. 1355 level.

[0102] C4-aa levels in serum at baseline and 3 weeks after ipilimumab treatment 1355 Levels (n=41). Serum levels were compared using the Wilcoxon paired-rank test.

[0103] Figure 4 The Kaplan-Meier analysis of overall survival is shown.

[0104] Overall survival in patients with metastatic melanoma treated with ipilimumab, for C4-aa 1355 For Q1, the pre-treatment level is the upper quartile (Q2+Q3+Q4) compared to the lower quartile (Q1) (cutoff: 14.5 ng / ml) (A). For C4M, it is the upper quartile (Q4) compared to the lower quartile (Q1+Q2+Q3) (cutoff: 35.0 ng / ml) (B). A log-rank test was used to determine the differences between survival curves, where a p-value < 0.05 was considered statistically significant.

[0105] Figure 5 It showed C4-aa in patients with metastatic melanoma. 1355 Correlation between C4M levels and C4M levels.

[0106] Pearson correlation analysis was performed to characterize pretreatment serum C4-aa levels in metastatic melanoma patients treated with ipilimumab (n=54). 1355 The relationship between and C4M levels.

[0107] Figure 6 Serum C4-aa1355 levels were shown in cancer patients and healthy controls.

[0108] A) Serum C4-aa levels in healthy controls (n=40), breast cancer (n=13), colorectal cancer (CRC) (n=7), gastric cancer (n=9), non-small cell lung cancer (NSCLC) (n=12), small cell lung cancer (SCLC) (n=7), melanoma (n=7), ovarian cancer (n=10), pancreatic cancer (n=2), and prostate cancer (n=13). 1355 Levels. Groups were compared using the Kruskal-Wallis test adjusted for multiple comparisons against Dunn. B) C4-aa levels in serum from healthy controls (n=40) were compared using the unpaired Mann-Whitney test. 1355 The levels were compared with those in the combined group of cancer patients (n=80). The black horizontal line represents the median of the duplicate measurements. C) Pearson correlation analysis was performed to characterize serum C4-aa levels from the combined group of cancer patients (n=80). 1355 The relationship between and C4M levels.

[0109] Figure 7 The serum C4G (C4-aa) levels were shown based on early and late pancreatic ductal adenocarcinoma (PDAC). 1355 (p<0.05)

[0110] Figure 8 The results show the baseline assessment versus C4G (C4-aa) by grouping at the 25th and 75th percentiles (Q1+Q4 vs. Q2+Q3) (dichotomy). 1355 Kaplan-Meier curves for overall survival (OS) related to this. Detailed Implementation

[0111] Various embodiments are described and disclosed in the following examples. These embodiments are intended to aid in understanding this disclosure and should not be construed as limiting the scope of the invention in any way as defined in the following claims. The following examples are presented to provide those skilled in the art with a complete disclosure and a description of how the described embodiments can be made and used, and are not intended to limit the scope of this disclosure, nor are they intended to represent that the following experiments are all or only the experiments performed. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise stated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is degrees Celsius, and pressure is atmospheric pressure or close to atmospheric pressure.

[0112] Materials and methods

[0113] Unless otherwise stated, all reagents used in the experiments were standard chemicals from Merck (Whitehouse station, NJ, USA) and Sigma-Aldrich (St. Louis, MO, USA).

[0114] Identification of peptides by mass spectrometry

[0115] Type IV collagen from human placenta (Sigma Aldrich, catalog number C5533) was proteolytically digested at 37°C for 24 hours and 72 hours at a ratio of 10:1, and then stored at -80°C until mass spectrometry analysis.

[0116] 1 μg of sample (corresponding to 100 μl of digested or undigested collagen in 50 mM Tris, 150 mM NaCl, pH 7.5 buffer) was reduced with 10 mM dithiothreitol for 30 min at 56 °C, followed by alkylation with 40 mM iodoacetamide in the dark at room temperature for 60 min. Any remaining iodoacetamide was quenched with 10 mM dithiothreitol for 5 min at room temperature. The sample was then digested with Lys-C at 37 °C on a shaker at a 1:20 enzyme:substrate ratio (Wako Chemicals, catalog number 125-05061) for 16 h. After adding 100 μl of 1M NaCl containing 1% formic acid to the digest, the sample was passed through a 30 kDa filter (PALL LifeSciences, catalog OD030C34) to remove GAGs, and desalted using a reverse-phase Vydac UltraMicroSpin C18 column (Harvard Apparatus, catalog 74-7206) according to the manufacturer's instructions. Non-targeted mass spectrometry analysis was performed on a quadrupole Orbitrap benchtop mass spectrometer QExactive (ThermoScientific) equipped with an Easy nano-LC 1000 system (ThermoFisher Scientific). Analysis was performed on a 75 μm × 25 cm Acclaim Pepmap filled with 2 μm particles. TM Separation was performed on an RSLC C18 capillary column (ThermoFisher Scientific). Desolvation was performed using a spray voltage of +2000V and a heated ion transfer setting of 275°C. In-line reversed-phase separation was performed using a linear binary gradient at a flow rate of 300 nL / min for 85 min. The gradient started at 3% solvent B, proceeded for 4 min, then increased to 35% solvent B over 64 min, and then to 45% solvent B over 5 min. Finally, the organic solvent concentration was increased to 90% over 5 min and held at 90% for 7 min. MS scans (400–1200 m / z) were recorded in an Orbitrap mass analyzer set to 200 m / z with a resolution of 70,000, 1 × 10⁻⁶. 6 Automatic gain control (AGC) target and 100 ms maximum ion implantation time. After MS, at 2 × 10 4Data-related collision-induced dissociation MS / MS scans of the 15 most potent multicharged ions were performed at a resolution of 17,500 with an intensity threshold, a 2 m / z isolation width, and 30-second dynamic exclusion enabled. Identification of the Homo sapiens proteome (UniProt proteome ID UP000005640) was performed using Proteome Discoverer 2.1 software (ThermoFisher Scientific). The processing workflow consists of the following nodes: a Spectrum Selector for spectral preprocessing (precursor mass range: 300–30000 Da; S / N threshold: 1.5), a Sequest-HT search engine (protein database: see above; enzyme: Lys-C (half); maximum deletion cleavage site: 2; peptide length range: 6–144 amino acids; precursor mass tolerance: 10 ppm; fragment mass tolerance: 0.02 Da; static modification: cysteine ​​carbamidomethylation), and a permeabilizer for peptide validation (FDR < 1% based on peptide q value). The results are filtered to retain only major proteins with at least one unique peptide, and protein grouping is allowed according to the principle of parsimony. For label-free quantification (LFQ), the sum of the top 3 peptides for each protein is taken to reflect protein strength. Peptide strength is quantified using a proprietary algorithm developed in Proteome Discoverer 2.1 (ThermoFisher Scientific).

[0117] Peptide selection

[0118] The first six amino acids from the N-terminus and C-terminus of each peptide from type IV collagen, identified by mass spectrometry, were considered novel epitopes produced by the protease. Homology of the protease-generated sequence with other proteins and species was analyzed using NPS@: Network protein sequence analysis (16) of the Uniprot / Swiss-Prot database. A cleavage site F was found. 1354 ↓M 1355 ( 1355 MGNTGPTGAV 1364 The C-terminal amino acid sequence of C4-aa is unique to the human type IV collagen α2 chain and has been selected as a target for antibody production. This is used for monoclonal antibody production and measurement of protease-mediated type IV collagen (C4-aa) chains. 1355 The synthetic peptides used for technical evaluation of the degraded ECLIA were purchased from Genscript, as shown in Table 1.

[0119] Table 1: For C4-aa 1355 Test, develop and validate synthetic peptides

[0120]

[0121]

[0122] KLH, keyhole hemocyanin

[0123] The target sequence was used as the select peptide (MGNTGPTGAV (SEQ ID No. 1)). The immunogenic peptide (MGNTGPTGAV-GGC-KLH) was generated by covalently linking the select peptide to the keyhole hemocyanin (KLH) carrier protein, with glycine and cysteine ​​residues added in between to ensure proper linkage. A biotinylated peptide (MGNTGPTAV-K-Biotin) was used as the coating peptide. Antibody specificity was tested using peptides including the extended peptide (FMGNTGPTGAV (SEQ ID No. 7)), the truncated peptide (GNTGPTGAV (SEQ ID No. 3)), the nonsense select peptide (LLARDFEKNY (SEQ ID No. 18)), and the nonsense coating peptide (LLARDFEKNY-K-Biotin). To test for potential cross-reactivity with other ECM proteins with similar sequences, three peptides with an amino acid mismatch at one of the N-terminal positions 1 (QGNTGPTGAV (SEQ ID No. 6)), 3 (MGQTGPTGAV (SEQ ID No. 4)), or 4 (MGNSGPTGAV (SEQ ID No. 5)) were included in the specificity test. Antibody specificity was calculated as the percentage of signal inhibition of a two-fold diluted peptide.

[0124] Monoclonal antibody production and clonal characterization

[0125] Subcutaneous immunization of 6-7 week old female Balb / C mice was repeated every two weeks with 200 μl of emulsified antigen containing 100 μg of immunogenic peptide (MGNTGPTGAV-GGC-KLH) and Stimune adjuvant (Thermo Fisher, catalog number 7925000) until stable titer levels were achieved. Mice with the highest antibody titers rested for four weeks and were then boosted intraperitoneally with immunogenic peptide. Three days later, spleen cells were isolated and fused with mouse SP2 / 0 myeloma cells to generate hybridoma cells as described above (17). Hybridoma cells were cultured in 96-well microtiter plates with limiting dilutions to ensure monoclonal growth. The reactivity of supernatants from monoclonal antibody-producing hybridoma cells to select peptides and human serum samples was screened in a preliminary competitive ELISA using biotinylated coated peptides on streptavidin-coated microtiter plates (Roche, catalog number 11940279). Purify clones that exhibit the best reactivity to the selected peptide using the manufacturer’s instructions (GE Healthcare Life Sciences, catalog number 17-0404-01).

[0126] C4-aa 1355 ECLIA Solution

[0127] During the assay development process, the optimal incubation buffer, time, temperature, and concentrations of antibody and coating peptide were determined, ultimately leading to the competitive C4-aa assay. 1355The ECLIA protocol is as follows: Incubate an MSD GOLD 96-well streptavidin pre-coated plate (Meso Scale Discovery, catalog number L15SA-1) at 20°C in the dark with 150 μl / well blocking buffer (containing bovine serum albumin (BSA) (5% w / v) and 5-bromo-5-nitro-1,3-dioxane (bronidox) (0.36% v / v)), 10 mM phosphate-buffered saline (PBS) with 8 g / L NaCl, pH 7.4) and shake (300 rpm) for 60 minutes. The plates were coated with 25 μl / well of biotinylated coating peptide dissolved in assay buffer (50 mM PBS containing BSA (1% w / v), Tween-20 (0.1% w / v), and 5-bromo-5-nitro-1,3-dioxane (0.36% v / v), 8 g / L NaCl, pH 7.4) to a final concentration of 2 ng / ml, and incubated at 20°C in the dark with shaking (750 rpm) for 60 min. Next, 25 μl / well of select peptide, assay control, or pre-diluted serum / plasma sample (1:2) was added, followed immediately by 25 μl / well of SULFO-TAG (MSD GOLD SULFO-TAGNHS-ester conjugated, Meso Scale Discovery, catalog number R31AA-1)-labeled monoclonal antibody diluted in assay buffer to a final concentration of 25 ng / ml, and the plates were incubated at 4°C in the dark with shaking (300 rpm) for 20 h. After all incubation steps, the plate was washed three times in wash buffer (20 mM Tris, 50 mM NaCl, pH 7.2). Finally, 150 μl / well of MSD GOLD read buffer (Meso Scale Discovery, catalog number R92TG-2) was added, and the plate was read immediately in a Sector Imager 6000 (Meso Scale Discovery) within 2 minutes. SULFO-TAG was activated for luminescence upon power-up, and the luminescence data were analyzed using MSD DiscoveryWorkbench 4.0 software. Analyte concentrations were calculated using a 4-parameter curve fitting model.

[0128] C4-aa 1355 Technical evaluation of the measurement

[0129] The lower limit of detection was determined from ten independent runs using the background mean plus 2.5 times the standard deviation. The upper limit of detection was determined from the same ten runs using the reverse calibration concentration of the highest concentration of the selected peptide minus 2.5 times the standard deviation. Intra-assay and inter-assay variability were determined from ten independent runs of seven samples, in duplicate, covering the entire linear range of the standard curve. The samples consisted of four samples with different amounts of the selected peptide in the assay buffer and three different healthy human serum samples. Intra-assay variability was calculated as the mean coefficient of variation (CV%) within the plate, and inter-assay variability was calculated as the mean CV% across ten plates. To determine the linearity of the assay, human serum (n=3) or EDTA plasma samples (n=3) were 2-fold diluted, and linearity was calculated as the percentage of recovery of the undiluted sample. Analyte stability was tested by four repeated freeze / thaw cycles of human serum (n=3 per cycle), and analyte recovery was calculated using the first cycle as a reference. In addition, analyte stability was tested by incubating human serum samples at 4°C or 20°C for 24 or 48 hours (n=3 at each time point), and recoveries were calculated using samples stored at -20°C as a reference. Interference was tested by adding low / high concentrations of biotin (3.0 / 9.0 ng / ml), lipemia (1.5 / 5.0 mg / ml), and hemoglobin (2.5 / 5.0 mg / ml) to serum samples, and recoveries were calculated using serum samples as a reference.

[0130] In vitro lysis of type IV collagen

[0131] Recombinant type IV collagen α2 chain (MyBioSource) and MMP-9 (Giotto, catalog number G04MP09C) or granzyme B (Abcam, catalog number ab168093) were incubated at 37°C at a 10:1 ratio (10 μg type IV collagen and 1 μg protease) in MMP buffer (50 mM Tris-HCl, 150 nM NaCl, 10 mM CaCl2, 10 μM ZnCl, 0.05% Brij 35, pH 7.5) or GzB buffer (50 mM Tris, 150 mM NaCl, pH 7.5) for 72 hours, and then stored at -80°C until analysis. Carboxymethylated transferrin digestion with MMP-9 or GzB served as positive controls, while MMP buffer with MMP-9 alone and GzB buffer with GzB alone served as negative controls. The activity of MMP-9 and GzB was confirmed by Coomassie brilliant blue staining (data not shown).

[0132] C4-aa 1355 Clinical validation of the test

[0133] Following informed consent, serum samples were collected from 54 patients with stage IV melanoma treated with ipilimumab (3 mg / kg body weight) as the standard of care at Herlev Hospital and Aarhus University Hospital, Denmark. The study was approved by the Ethics Committee for the Capital Region of Denmark (H-2-2012-058) and complies with the 1975 Helsinki Declaration. Serum samples were collected at baseline and 3 weeks after the first treatment (before the second dose).

[0134] Serum samples from patients with other cancers were obtained from the commercial supplier Asterand Bioscience (Detroit, MI, USA) and included breast cancer (n=13), colorectal cancer (CRC) (n=7), gastric cancer (n=9), non-small cell lung cancer (NSCLC) (n=12), small cell lung cancer (SCLC) (n=7), melanoma (n=7), ovarian cancer (n=10), pancreatic cancer (n=2), and prostate cancer (n=13). These samples were collected with informed consent and approval from the appropriate institutional review committee in accordance with the Declaration of Helsinki.

[0135] C4M was evaluated in serum samples from cancer patients to correlate with the newly developed biomarker C4-aa. 1355 Comparison. Based on a novel monoclonal antibody specific to MMP-9-mediated type IV collagen degradation manufactured by Nordic Bioscience (Herlev, Denmark), the C4M competitive ELISA is a well-characterized assay and is measured according to the manufacturer’s specifications (18).

[0136] Statistical analysis

[0137] The Wilcoxon matched pairs signed rank test was used to compare biomarker levels in melanoma patients at baseline and at week 3. Kaplan-Meier survival curves were used to analyze overall survival (OS) in melanoma patients, for C4-aa... 1355 For Q1, the pre-treatment level is the upper quartile (Q2+Q3+Q4) compared to the lower quartile (Q1). For C4M, it is the upper quartile (Q4) compared to the lower quartile (Q1+Q2+Q3).

[0138] The Kruskal-Wallis test, adjusted for Dunn multiple comparisons, was used to analyze C4-aa in serum samples from different cancer patients. 1355 Levels were compared with healthy controls. An unpaired Mann-Whitney test was used to compare the combined group of healthy controls and cancer patients. Pearson correlation analysis was performed to characterize serum C4-aa levels from both the combined group of metastatic melanoma and cancer patients, separately. 1355 The relationship between C4M levels and p-values. A p-value < 0.05 was considered statistically significant. Graphs and statistical analyses were performed using GraphPad Prism version 7 (GraphPad Software, CA, USA).

[0139] result

[0140] New C4-aa 1355 Specificity of the assay

[0141] The novel competitive C4-aa was tested 1355 The specificity of monoclonal antibodies in ECLIA. Selected peptides inhibit signaling in a dose-dependent manner, while extended peptides, truncated peptides, and nonsense selected peptides do not inhibit signaling. Figure 1 A). No signal was observed when using nonsense biotinylated peptides. Figure 1 A). When reactivity was tested against peptides with only one amino acid mismatch compared to the selected peptide, no reactivity was detected at peptide concentrations of 0–30 ng / ml, while deselected peptide 2 suppressed the signal to 65% at the highest concentration. Figure 1 B). In summary, these data demonstrate that monoclonal antibodies exhibit high specificity for novel epitopes on selected peptides.

[0142] MMP-9-mediated proteolytic degradation of type IV collagen

[0143] To confirm C4-aa 1355 Antibody / Assay recognizes a new type IV collagen epitope, C4-aa, produced by the protease. 1355 Measurements were taken in undigested type IV collagen, MMP-9-digested type IV collagen, and GzB-digested type IV collagen. Figure 2 As shown, C4-aa 1355 The antibody was detected only in MMP-9-digested and GzB-digested samples, but not in undigested (protease-free) samples, indicating that the antibody is specific for novel epitopes produced by the protease.

[0144] C4-aa 1355 Technical evaluation of the measurement

[0145] The different technical verification steps summarized in Table 2 were used to further evaluate C4-aa. 1355 The technical performance of the ECLIA assay was as follows: The detection range was 0.6–832 ng / ml. Intra-assay and inter-assay variability were 6% and 8%, respectively, below the acceptance criteria of 10% and 15%. Linearity was detected at dilutions from undiluted to 1:4, with recoveries of 94% and 106% in serum and EDTA plasma, respectively. The analyte recovery in serum was 96% after four freeze / thaw cycles. After prolonged storage at 4°C or 20°C for 48 hours, the analyte recoveries in human serum were 122% and 109%, respectively. No interference was detected in low or high concentrations of lipemia or hemoglobin in serum, with recoveries ranging from 92–111%. Low levels of biotin did not interfere with the analyte, while high levels did, with recoveries of 94% and 71%, respectively. The acceptable recovery rate was within 100 ± 20%. In summary, these results indicate that C4-aa 1355 ECLIA is a technically robust measurement.

[0146] Table 2: C4-aa 1355 Technical verification of the measurement

[0147]

[0148] Percentage reports are averages

[0149] C4-aa in patients with metastatic melanoma treated with ipilimumab 1355 Clinical assessment of the test

[0150] To evaluate C4-aa 1355 The biomarker potential of C4-aa was measured in the serum of patients with metastatic melanoma at baseline and 3 weeks after ipilimumab treatment. 1355 When biomarker levels were paired, C4-aa was observed 3 weeks post-treatment. 1355 The level increased slightly (p = 0.090). Figure 3 ).

[0151] Next, C4-aa was evaluated using the Kaplan-Meier curve. 1355 Associations between biomarkers and survival outcomes. Compared to low levels (Q1), C4-aa 1355 Higher baseline levels (Q2+Q3+Q4) were significantly associated with longer overall survival (OS) (p = 0.040). Figure 4 A) The median overall survival (OS) was 646 days for patients with high biomarkers, while it was 290 days for patients with low biomarkers.

[0152] Due to C4-aa 1355The study also assessed the association between C4M and OS by measuring novel epitopes produced by two different proteases on type IV collagen and C4M biomarkers. These findings on C4M have been previously published. (8) The results of C4M measured in 54 patients were as follows: Figure 4 As shown in B. For C4M biomarkers, compared with low levels (Q1+Q2+Q3), high baseline levels (Q4) were significantly associated with shorter OS (p=0.005). Figure 4 B). Interestingly, these two biomarkers showed opposite associations with the results.

[0153] In addition, the baseline C4-aa levels of patients with metastatic melanoma were studied. 1355 The correlation between C4-aa and C4M levels. 1355 Uncorrelated with C4M (r = 0.021, p = 0.883) Figure 5 ).

[0154] C4-aa 1355 Clinical assessment in other cancer patients

[0155] To further evaluate C4-aa 1355 The potential of C4-aa as a biomarker was investigated in the serum of patients with various cancers, including breast cancer, CRC, gastric cancer, NSCLC, SCLC, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer, and in the serum of healthy controls. 1355 Measurements were taken. C4-aa was observed in each group of patients. 1355 Inter-patient changes in biomarker levels Figure 6 A). The median C4-aa in healthy controls was... 1355 No significant differences were observed when comparing levels with each group of cancer patients. Figure 6 A). However, when comparing C4-aa in the combined group of healthy controls and cancer patients... 1355 At that time, compared with healthy controls (12.0 ng / ml), C4-aa in cancer patients was significantly lower. 1355 The level was significantly elevated (14.8 ng / ml) (p = 0.006). Figure 6 B).

[0156] As shown in the first cohort, C4-aa in these cancer patients 1355 The C4M level was again uncorrelated (r = 0.197, p = 0.080). Figure 6 C).

[0157] Serum C4-aa measured at baseline 1355 (C4G) predicts outcomes for patients with pancreatic ductal adenocarcinoma (PDAC) treated with chemotherapy.

[0158] C4-aa was measured in pretreatment serum samples from 40 patients with pancreatic ductal adenocarcinoma (PDAC). 1355 (C4G). All patients were from the Danish BIOPAC study “Biomarkers in Patients with Pancreatic Cancer” (NCT03311776). Patients were recruited from six Danish hospitals between December 2008 and September 2017. Patients with pancreatic cancer (PDAC) had histologically confirmed tumors. PDAC patients were treated with various types of chemotherapy according to national guidelines (www.gicancer.dk). The study was conducted based on the recommendations of the Danish Regional Committee on Health Research Ethics. The BIOPAC protocol was approved by the Danish Regional Committee on Health Research Ethics (VEK ref. KA-20060113) and the Data Protection Authority (j.nr. 2006-41-6848). All subjects signed written informed consent in accordance with the Declaration of Helsinki, 8th edition. Blood samples were obtained at diagnosis or before surgery. Samples were processed according to nationally approved blood standard operating procedures (www.herlevhospital.dk / biopac.dk). Serum samples and clinical data from patients were collected prospectively. Blind testing was performed on serum samples.

[0159] according to Figure 7 The results showed that the disease stage was different in patients with advanced PDAC compared to those with early-stage PDAC. 1355 Significantly reduced (Mann-Whitney test, p = 0.0132). The effects of PDAC on baseline and overall survival (OS) compared to C4-aa were evaluated. 1355 Possible associations. The 25th and 75th percentile tangents are used to define "extreme" C4-aa. 1355 In the groups analyzed by Kaplan and Meier, C4-aa was found to be at levels (<25th percentile +>75th percentile, i.e., quartile 1 and quartile 4, Q1 + Q4) and "non-extreme" levels (>25th percentile to <75th percentile, Q2 + Q3). 1355 Patients improved overall survival ( Figure 8 Univariate Cox regression analysis showed that it had a "non-extreme" C4-aa value. 1355 The risk of death was reduced in the subgroup of patients with elevated C4-aa levels (Table 3). Furthermore, multivariate Cox regression analysis showed that C4-aa... 1355 The predicted values ​​are independent of the stage.

[0160]

[0161] Discussion and Conclusion

[0162] A robust and specific competitive ECLIA has been developed and validated, which enables protease-mediated type IV collagen (C4-aa) to... 1355 The newly generated epitopes from degradation were measured non-invasively. C4-aa was found in these melanoma patients. 1355 High baseline levels of C4M were associated with a clinical response to immune checkpoint inhibitor therapy (longer overall survival). Conversely, high baseline levels of C4M were associated with shorter overall survival. C4-aa in these samples... 1355 It is not correlated with C4M levels. C4-aa 1355 Both C4-aa and C4M measure novel epitopes on type IV collagen, but at two different sites. Interestingly, these data indicate that these distinct cleavage products are released during two different pathological events, one associated with a favorable outcome and the other with a poor outcome when measured at baseline. Furthermore, C4-aa was detected in the serum of metastatic melanoma patients 3 weeks after immune checkpoint inhibitor treatment, compared to baseline. 1355 The level has risen slightly.

[0163] C4-aa 1355 Findings associated with the clinical response of these melanoma patients to immune checkpoint inhibitor therapy suggest that C4-aa 1355 To determine the potential of C4-aa as a biomarker in an immuno-oncology setting to identify cancer patients with a T-cell-permissive tumor microenvironment who respond to therapy. 1355 This can reflect the protease-mediated migration of T cells from the circulatory system to the basal matrix. Conversely, supporting previous findings that C4M is associated with adverse responses suggests that C4M is involved in tumor activity and reactive matrix. (8-10) .

[0164] Interestingly, this study showed that one novel type IV collagen epitope fragment is associated with tumorigenesis (C4M), and another novel type IV collagen epitope fragment is associated with T cell infiltration (C4-aa). 1355 This supports the measurement of pathology-specific novel epitopes rather than just the value of total protein.

[0165] C4-aa 1355 Elevated levels were also observed in other cancer types besides melanoma, suggesting the potential of this biomarker for other indications. Furthermore, C4-aa was also found in these cancer patients. 1355 The lack of correlation between these biomarkers and C4M levels again confirms that these biomarkers reflect different pathological aspects of the tumor microenvironment.

[0166] C4-aa was found 1355Elevated levels were observed in patients with early-stage pancreatic ductal adenocarcinoma (PDAC). Furthermore, the marker C4-aa was found in those with extreme levels (i.e., in the 1st or 4th quartile). 1355 The overall survival rate of patients with this condition is reduced.

[0167] To the best of our knowledge, this indicates that this specific protease-mediated type IV collagen (C4-aa) 1355 The degradation of ) has biomarker potential in cancer and is the first study related to response to immune checkpoint inhibitor therapy.

[0168] References

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[0173] 5. Nissen NI, Karsdal M, Willumsen N, Collagens and Cancer-associated fibroblasts in the reactive stroma and its relation to cancer biology, Journal of Experimental and Clinical Cancer Research. 2019.

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[0176] 8. Jensen C, Madsen DH, Hansen M, Schmidt H, Svane IM, Karsdal MA, et al. Non-invasive biomarkers derived from the extracellular matrix associated with response to immune checkpoint blockade (anti-CTLA-4) in metastatic melanoma patients. Journal for ImmunoTherapy of Cancer. 2018.

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[0178] 10. Bager CL, Willumsen N, Leeming DJ, Smith V, Karsdal MA, Dornan D, et al., Collagen degradation products measured in serum can separate ovarian and breast cancer patients from healthy controls: A preliminary study, Cancer Biomarkers. 2015; 15(6):783–8.

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Claims

1. A monoclonal antibody or its antigen-binding fragment thereof, which specifically binds to a peptide consisting of the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1), wherein The monoclonal antibody or its antigen-binding fragment does not specifically bind to a peptide having the N-terminal amino acid sequence XMGNTGPTGAV (SEQ ID No. 2), where X represents any amino acid, and / or the monoclonal antibody or its antigen-binding fragment does not specifically bind to a peptide having the N-terminal amino acid sequence MGQTGPTGAV (SEQ ID No. 4), wherein the monoclonal antibody or its antigen-binding fragment contains the following complementarity-determining region, i.e., CDR: CDR-L1: KSSQSLLYSDGKTYLN (SEQ ID No. 8) CDR-L2: LVSKLDS (SEQ ID No. 9) CDR-L3: WQGTHFVT (SEQ ID No. 10) CDR-H1: TYNIGVG (SEQ ID No. 11) CDR-H2: HIWYNDIKYYNTALKS (SEQ ID No. 12) CDR-H3: LRPDSFDY (SEQ ID No. 13).

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, wherein, The monoclonal antibody or its antigen-binding fragment is a monoclonal antibody generated against a synthetic peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1).

3. The monoclonal antibody or its antigen-binding fragment according to claim 1, wherein, The monoclonal antibody or its antigen-binding fragment does not specifically bind to a peptide having the N-terminal amino acid sequence GNTGPTGAV (SEQ ID No. 3).

4. The monoclonal antibody or its antigen-binding fragment according to claim 1, wherein, The monoclonal antibody or its antigen-binding fragment does not specifically bind to a peptide having the N-terminal amino acid sequence QGNTGPTGAV (SEQ ID No. 6).

5. The monoclonal antibody or its antigen-binding fragment according to claim 1, wherein, The monoclonal antibody or its antigen-binding fragment does not specifically bind to a peptide having the N-terminal amino acid sequence MGNSGPTGAV (SEQ ID No. 5).

6. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 in the preparation of a kit for detecting the presence or absence of a peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) using an immunoassay method, wherein, The immunoassay method is used to determine whether a subject with cancer responds to immunotherapy.

7. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 in the preparation of a kit for detecting the presence or absence of a peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1) using an immunoassay method, wherein, The immunoassay method is used to predict the survival outcome of subjects with cancer.

8. The use according to claim 6 or claim 7, wherein, The immunoassay method includes contacting a biofluid sample obtained from a subject with a monoclonal antibody that specifically binds to a peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1), and detecting the binding between the monoclonal antibody and the peptide in the sample.

9. The use according to claim 6 or claim 7, wherein, The detection is quantitative.

10. The use according to claim 6 or claim 7, wherein, The immunoassay method is a competitive immunoassay method.

11. The use according to claim 6 or claim 7, wherein, The subjects had been diagnosed with cancer selected from melanoma, breast cancer, colorectal cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, prostate cancer, or pancreatic cancer.

12. The use according to claim 11, wherein, The subject had been diagnosed with melanoma.

13. The use according to claim 11, wherein, The subject had been diagnosed with metastatic melanoma.

14. The use according to claim 6 or claim 7, wherein, The method further includes: correlating the amount of the peptide with values ​​associated with normal healthy subjects and / or values ​​obtained from cancer patients who have responded to immunotherapy.

15. The use according to claim 14, wherein, The method also includes administering immunotherapy to subjects with elevated levels of the present peptides.

16. The use according to claim 6, wherein, The immunotherapy includes at least one immune checkpoint inhibitor.

17. The use according to claim 16, wherein, The immune checkpoint inhibitor is ipilimumab.

18. An assay kit comprising a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5, and at least one of the following: -Streptavidin-coated well plates; - A C-terminal biotinylated peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1); - A calibration peptide with the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID No. 1), - Antibody biotinylation kit; - Antibody HRP labeling kit; - Antibody radiolabeling kit; and - Visualization kit for assays.

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