Neoepitope-specific assay to measure protease-mediated degradation of type IV collagen

Abstract

An assay measuring protease-mediated degradation of type IV collagen and its potential as a biomarker for identifying cancer patients with a T cell-permissive tumor microenvironment are described.

Description

[Technical Field]

[0001] The present invention relates to assays and biomarkers that measure protease-mediated degradation of type IV collagen, which can identify cancer patients with a T-cell permissive tumor microenvironment.

[0002] Introduction Immunotherapy using immune checkpoint inhibitors has revolutionized cancer treatment by providing the opportunity for durable responses (Reference 1). Immune checkpoint inhibitor treatment involves antibodies, such as anti-CTLA-4, anti-PD1, and anti-PD-L1, that can reactivate cytotoxic T lymphocytes to eliminate tumor cells. However, despite the clinical success of these immune checkpoint inhibitors, only a small subset of cancer patients benefit from long-term survival. Therefore, to avoid mistreatment and adverse events, it is important to identify noninvasive biomarkers that can identify cancer patients who will respond to immune checkpoint inhibitor therapy. To identify predictive biomarkers, it is essential to understand several factors that influence response and resistance.

[0003] Three distinct immune profiles have been identified in patients prior to treatment that correlate with their response to immune checkpoint therapy (Reference 2). Clinical responses occur most frequently in patients with immune-inflamed tumor types, which are characterized by the presence of CD4 and CD8-expressing T cells in the tumor microenvironment near tumor cells. The immune-excluded phenotype is characterized by the presence of immune cells, but they are retained in the surrounding stroma, preventing T cell infiltration. In the immune-desert phenotype, T cells are absent from either the tumor parenchyma or stroma. Because patients with immune-excluded or immune-desert phenotypes rarely respond to immune checkpoint inhibitor therapy, biomarkers identifying immune-inflamed tumor types can be very useful predictive tools. For effective cancer immunotherapy, it is important that T cells are activated and recruited to the tumor microenvironment, a hallmark of the immune-inflamed phenotype ("hot tumor") (Reference 3).

[0004] The composition of the extracellular matrix (ECM) has been shown to influence the location and migration of T cells and is recognized to play an important role in resistance to immunotherapy (References 4-7). We previously demonstrated that the serological biomarker PRO-C3, which reflects excessive type III collagen formation (fibrosis), and the biomarker C4M, which reflects matrix metalloproteinase (MMP)-9-degraded type IV collagen, are associated with poor response to immune checkpoint blockade (Reference 8). In addition, C4M is elevated in patients with various cancers (References 9, 10).

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

[0006] Because type IV collagen is a major component of basement membranes, we hypothesized that T cell migration from the circulation to the tumor microenvironment might result in the release of specific protease-generated type IV collagen fragments into the circulation of cancer patients. Therefore, these type IV collagen fragments may have the potential to identify cancer patients with a T cell-permissive tumor microenvironment that responds to immune checkpoint inhibitor treatment. We therefore developed a competitive electrochemiluminescence immunoassay (ECLIA) targeting a neoepitope of protease-mediated degradation of type IV collagen and demonstrated that its levels were elevated in serum from metastatic melanoma patients successfully treated with the immune checkpoint inhibitor ipilimumab. To further evaluate this potential, we also evaluated this biomarker in serum from patients with different types of cancer. This biomarker can also be used to provide a prognosis for survival in cancer patients, particularly pancreatic ductal adenocarcinoma.

[0007] Thus, in a first aspect, the present invention provides a peptide (herein referred to as the target peptide or C4aa) having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1). 1355 The present invention relates to a monoclonal antibody that specifically recognizes and binds to a serine protease (e.g., granzyme B) or a matrix metalloproteinase (e.g., MMP-9), and the peptide sequence is selected from the group consisting of amino acid F, ... 1354 and M 1355 represents a neoepitope generated by digestion of type IV collagen α2 chain by a protease causing cleavage between

[0008] Preferably, the monoclonal antibody does not recognize or specifically bind to an extension 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 bind to truncations of the N-terminal amino acid sequence, in particular the peptide GNTGPTGAV (SEQ ID NO: 3). Preferably, the monoclonal antibody does not recognize or specifically bind to variants of the N-terminal amino acid sequence which are MGQTGPTGAV (SEQ ID NO: 4), MGNSGPTGAV (SEQ ID NO: 5) and / or QGNTGPTGAV (SEQ ID NO: 6).

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

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

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

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

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

[0014] Monoclonal antibodies that specifically bind to the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1) can be produced by any suitable technique known in the art. For example, monoclonal antibodies can be produced by immunizing rodents (or other suitable mammals) against a synthetic peptide having the amino acid sequence MGNTGPTGAV (SEQ ID NO: 1), e.g., a synthetic peptide consisting of the sequence MGNTGPTGAV (SEQ ID NO: 1), optionally linked to an immunogenic carrier protein (such as keyhole limpet hemocyanin), isolating and cloning single antibody-producing cells, and assaying the resulting monoclonal antibodies to determine that they have the desired specificity. An exemplary protocol for producing monoclonal antibodies that specifically bind to the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1) is described below.

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

[0016] Preferably, the antibody or fragment thereof comprises at least two, three, four, five, or six of the above CDR sequences.

[0017] Preferably, the monoclonal antibody or fragment thereof has a light chain variable region comprising the following CDR sequences: CDR-L1: KSSQSLLYSDGKTYLN (SEQ ID NO: 8) CDR-L2: LVSKLDS (SEQ ID NO: 9) and CDR-L3: WQGTHFVT (SEQ ID NO: 10)

[0018] Preferably, the monoclonal antibody or fragment thereof has a light chain comprising inter-CDR framework sequences that are substantially identical to or substantially similar to the inter-CDR framework sequences of the light chain sequence below (CDRs are shown in bold and underlined, framework sequences are shown in italics):

[0019] [ka]

[0020] Preferably, the monoclonal antibody or fragment thereof has a heavy chain variable region comprising the following CDR sequences: CDR-H1: TYNIGVG (SEQ ID NO: 11) CDR-H2: HIWYNDIKYYNTALKS (SEQ ID NO: 12) and CDR-H3: LRPDSFDY (SEQ ID NO: 13)

[0021] Preferably, the monoclonal antibody or fragment thereof has a heavy chain comprising framework sequences between the CDRs, said framework sequences being heavy chain The sequences are substantially identical to or substantially similar to the framework sequences between the CDRs of the sequences (CDRs are shown in bold and underlined, framework sequences are shown in italics).

[0022] [ka]

[0023] As used herein, the amino acid sequence of a framework present between the CDRs of an antibody is considered to be substantially identical or substantially similar to the amino acid sequence of a framework present between the CDRs of another antibody if it has at least 70%, 80%, 90%, or at least 95% similarity or identity to the amino acid sequence of a framework present between the CDRs of the other antibody. The similar or identical amino acids may be contiguous or non-contiguous.

[0024] The framework sequence may contain one or more amino acid substitutions, insertions, and / or deletions. The amino acid substitutions may be conservative, 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 share similar chemical properties. For example, the following groups of amino acids share similar chemical properties in terms of size, charge, polarity, etc.: 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.

[0025] Programs such as the CLUSTAL program can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in any appropriate sequence. For optimal alignment, it is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type). Programs such as BLASTx align the longest stretch of similar sequences and assign values ​​to matching positions. In this way, a comparison can be made, and several regions of similarity, each with a different score, are found. It is contemplated that these two types of analysis can be used in the present invention. Identity or similarity is preferably calculated over the entire length of the framework sequence.

[0026] In certain preferred embodiments, the monoclonal antibody or fragment thereof comprises the light chain variable region sequence:

[0027] [ka]

[0028] (CDRs are in bold and underlined, framework sequences are in italics) and / or heavy chain variable region sequence:

[0029] [ka]

[0030] (CDRs are in bold and underlined, framework sequences are in italics) may include:

[0031] In a second aspect, the present invention relates to a method for identifying whether a cancer patient will respond to immunotherapy, said method comprising detecting the presence of a peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1) in a sample obtained from the patient, the sample being preferably a biological fluid sample, in particular a human biological fluid sample.

[0032] Preferably, the immunotherapy includes at least one immune checkpoint inhibitor. Immune checkpoint inhibitors target molecules present on certain 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 can target any one or more of these molecules. Immune checkpoint inhibitors include ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cemiplimab, or a combination thereof. Preferably, the immune checkpoint inhibitor can target CTLA-4, such as ipilimumab. Alternatively, the immune checkpoint inhibitor can target PD-1, such as pembrolizumab, nivolumab, and / or cemiplimab. Alternatively, the immune checkpoint inhibitor can target PD-L1, such as atezolizumab, avelumab, or durvalumab. Preferably, the immunotherapy with at least one immune checkpoint inhibitor comprises administering ipilimumab.

[0033] Preferably, the method is an immunoassay. More preferably, the method comprises contacting a sample of biological fluid obtained from a subject with a monoclonal antibody according to the first aspect of the invention and detecting binding between the monoclonal antibody and a peptide in the sample.

[0034] The method of the second aspect utilises the monoclonal antibody of the first aspect and therefore preferred embodiments of the second aspect will be apparent from the above discussion of preferred embodiments of the first aspect.

[0035] Preferably, the detection is quantitative, and therefore the method may comprise detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample. Preferably, the immunoassay is a competitive immunoassay. 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.

[0036] The biological fluid sample may be, but is not limited to, blood, serum, plasma, urine, or supernatant obtained from cell or tissue culture. Preferably, the biological fluid is serum or plasma, most preferably serum.

[0037] In the method of the second embodiment, the sample is obtained from a patient diagnosed with cancer.Cancer can be metastatic.Cancer is preferably selected from melanoma, breast cancer, colon 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 patient has melanoma, particularly metastatic melanoma.

[0038] The method may further include correlating the amount of the detected peptide with values ​​associated with normal healthy individuals and / or values ​​obtained from cancer patients who have clinically responded to immunotherapy, e.g., patients who have experienced extended survival, tumor shrinkage, and / or symptom improvement after treatment. Elevated levels of the peptide may indicate that the subject has an immunoinflammatory tumor type and is therefore responsive to immunotherapy.

[0039] As used herein, the term "values ​​relevant to normal healthy individuals and / or values ​​obtained from cancer patients who have clinically responded to immunotherapy" refers to standardized amounts determined by the above methods in subjects who are considered healthy, i.e., cancer-free, and / or in subjects known to have cancer who have clinically responded to immunotherapy, preferably immune checkpoint inhibitor treatment, e.g., experiencing a reduction in tumor size, improvement in symptoms, and / or longer overall survival.

[0040] In some embodiments of the method of the second aspect, the amount of binding of a monoclonal antibody specific for an epitope of a type IV collagen peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1) correlates with one or more predetermined cutoff values.

[0041] As used herein, the term "cutoff value" refers to a statistically determined amount of binding that indicates a high likelihood of responding to immunotherapy using an immune checkpoint inhibitor. A measured value of biomarker binding in a patient sample that is equal to or exceeds the statistical cutoff value may correspond to at least a 70% probability, preferably at least an 80% probability, preferably at least an 85% probability, more preferably at least a 90% probability, and most preferably at least a 95% probability of a response to immunotherapy, preferably an immune checkpoint inhibitor, as indicated by a reduction in tumor size, improvement in symptoms, and / or longer overall survival. The "cutoff value" can be calculated by comparing results obtained from patients diagnosed with cancer who responded to immunotherapy with results obtained from patients diagnosed with the same cancer who did not respond to immunotherapy.

[0042] If the measured amount of binding of a monoclonal antibody specific for the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1) is in the top three-quarters of levels (Q2+Q3+Q4) measured in cancer patients, particularly in patients with the same type of cancer, this indicates that the patient is likely to respond to treatment with an immune checkpoint inhibitor.

[0043] The predetermined cutoff value for the amount of bound monoclonal antibody specific to the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1) can be within the range of 10.0 to 20.0 ng / mL. Preferably, the predetermined cutoff value for the amount of bound 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, it has been found through the use of statistical analysis that a measured amount of bound monoclonal antibody specific to the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1) of at least 14.5 ng / mL or greater may be identified as a patient likely to respond to immunotherapy, preferably immunotherapy using an immune checkpoint inhibitor. By using a statistical cutoff value of at least 14.5 ng / mL, the method of the present invention can be used to predict response to immunotherapy with a high level of confidence. In particular, a value of at least 14.5 ng / mL or greater may be identified as a patient with melanoma likely to respond to immunotherapy. The application of such a statistical cutoff value is particularly advantageous because it provides a stand-alone diagnostic assay. That is, it eliminates the need for direct comparison with healthy individuals and / or patients known to have responded to immunotherapy, preferably with immune checkpoint inhibitor treatment, in order to reach a diagnostic conclusion. Facilitating conclusive predictions allows patients to potentially respond to treatment earlier, which in turn may improve their overall chances of survival and / or reduce the risk of hospitalization.

[0044] The method may further include administering immunotherapy to a subject determined to have elevated levels of the peptide present.

[0045] In a third aspect, the present invention provides 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: - streptavidin-coated well plates; - a C-terminally biotinylated peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1); - a calibration peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1); - Antibody biotinylation kit; - Antibody HRP labeling kit; - antibody radiolabeling kit; and - Assay Visualization Kit The present invention relates to an assay kit comprising:

[0046] The kit may be used to identify cancer patients who will respond to immunotherapy, preferably treatment with immune checkpoint inhibitors. The immunoassay kit is suitable for carrying out the method of the second aspect and comprises the monoclonal antibody of the first aspect; therefore, preferred embodiments of the third aspect will be apparent from the above discussion of preferred embodiments of the first and second aspects.

[0047] In a fourth aspect, the present invention also provides a method for treating cancer diagnosed with peptide C4-aa 1355 The present invention relates to a method of immunotherapy for treating a patient known to have elevated levels of C4-aa. Preferably, the immunotherapy comprises at least one immune checkpoint inhibitor. 1355 "Elevated levels" refers to amounts of peptides that are significantly higher than those detected in normal healthy controls and / or patients diagnosed with cancer who have not responded to immunotherapy, particularly immune checkpoint inhibitors.

[0048] The cancer may be metastatic. The cancer is preferably selected from melanoma, breast cancer, colon 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 patient has melanoma, particularly metastatic melanoma.

[0049] In a fifth aspect, the present invention also relates to a method of predicting survival outcome in a cancer patient, said method comprising detecting the presence of a peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1).

[0050] Preferably, the method is an immunoassay. More preferably, the method comprises contacting a sample of biological fluid obtained from a subject with a monoclonal antibody according to the first aspect of the invention, and detecting binding between the monoclonal antibody and a peptide in the sample.

[0051] The method according to the fifth aspect utilises the monoclonal antibody of the first aspect and therefore preferred embodiments of the fifth aspect will be apparent from the above discussion of preferred embodiments of the first aspect.

[0052] Preferably, the detection is quantitative, and thus the method may involve detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample. Preferably, the immunoassay is a competitive immunoassay. 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.

[0053] The biological fluid sample may be, but is not limited to, blood, serum, plasma, urine, or supernatant obtained from cell or tissue culture. Preferably, the biological fluid is serum or plasma, most preferably serum.

[0054] In the method of the second embodiment, the sample is obtained from a patient diagnosed with cancer.The cancer is preferably selected from melanoma, breast cancer, colon 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 patient has pancreatic cancer, more preferably pancreatic ductal adenocarcinoma.

[0055] The method may further include correlating the amount of the detected peptide with values ​​associated with normal healthy individuals and / or values ​​obtained from cancer patients, e.g., patients diagnosed with the same cancer. If the measured amount of binding of a monoclonal antibody specific for the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1) is in the upper or lower quartile (Q1 or Q4) of levels measured in cancer patients, particularly patients with the same type of cancer, this indicates that the patient is likely to have a poor prognosis and an increased risk of death. If the measured amount of binding of a monoclonal antibody specific for the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1) is in the middle quartile (Q2 + Q3) of levels measured in cancer patients, particularly patients with the same type of cancer, this indicates that the patient has a low risk of death.

[0056] As used herein, the term "values ​​associated with normal healthy individuals and / or values ​​obtained from cancer patients" refers to standardized amounts determined by the above methods for subjects considered to be healthy, i.e., not having cancer, and / or standardized amounts determined by the above methods for subjects known to have cancer.

[0057] In some embodiments of the method of the fifth aspect, the amount of binding of a monoclonal antibody specific for an epitope of a type IV collagen peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1) is correlated with one or more predetermined cutoff values.

[0058] As used herein, "cutoff value" refers to the amount of binding that is statistically determined to indicate a reduced risk of death. Measurements that fall within the statistical cutoff value for biomarker binding in patient samples are: The reduced risk of death may correspond to at least a 70% probability, preferably at least an 80% probability, preferably at least an 85% probability, more preferably at least a 90% probability, and most preferably at least a 95% probability. A "cut-off value" may be calculated by comparing results obtained from patients diagnosed with cancer and with known survival times.

[0059] definition As used herein, the terms "peptide" and "polypeptide" are used interchangeably.

[0060] As used herein, the term "monoclonal antibody" refers to both whole antibodies and fragments thereof that retain the binding specificity of the whole antibody, such as Fab fragments, F(ab')2 fragments, single-chain Fv fragments, or other such fragments known to those skilled in the art. As is well known, whole antibodies typically have a "Y-shaped" structure consisting of two identical paired polypeptide chains, each of which is composed of one "light" chain and one "heavy" chain. The N-terminal regions of each of the light and heavy chains comprise the variable region, while the C-terminal portions of each of the heavy and light chains constitute the constant region. The variable regions contain three complementarity-determining regions (CDRs), which are primarily responsible for antigen recognition. The constant region enables the antibody to recruit cells and molecules of the immune system. Antibody fragments that retain binding specificity contain at least the CDRs and a sufficient portion of the remainder of the variable region to retain binding specificity.

[0061] In the present invention, the monoclonal antibody may contain any constant region known in the art. Human constant light chains are classified as kappa and lambda light chains. Constant heavy chains are classified as mu, delta, gamma, alpha, or epsilon, which define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. The IgG isotype has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. The monoclonal antibody may preferably belong to the IgG isotype, including any one of IgG1, IgG2, IgG3, or IgG4.

[0062] The CDRs of an antibody can be determined using methods known in the art, such as those described by Kabat et al. (reference 19). Antibodies can be generated from B cell clones as described in those examples. The isotype of the antibody can be determined by ELISA specific for 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 can then be subjected to PCR using primers that amplify the heavy and light chains of the antibody. For example, primers specific to the leader sequences of all VH (variable heavy) sequences can be used together with primers that bind to sequences located in the constant region of a predetermined isotype. Light chains can be amplified using a primer that binds to the 3' end of the kappa or lambda chain together with a primer that anneals to the leader sequence of Vkappa or Vlambda. Full-length heavy and light chains can be generated and sequenced.

[0063] As used herein, the term "C-terminus" refers to, and should not be construed as meaning, at the terminus of a polypeptide, i.e., at the C-end of a polypeptide, in its general direction. Similarly, the term "N-terminus" refers to, and should not be construed as meaning, at the terminus of a polypeptide, i.e., at the N-end of a polypeptide, in its general direction.

[0064] As used herein, the term "competitive immunoassay" refers to an immunoassay in which a target peptide (if any) present in a sample competes with a known amount of peptide target (e.g., bound to an immobilized substrate or labeled) for antibody binding, a technique known to those skilled in the art.

[0065] As used herein, the term "ELISA" (enzyme-linked immunosorbent assay) refers to an immunoassay that uses an antibody linked to an enzyme, such as horseradish peroxidase or alkaline phosphatase, to detect a target peptide (if any) present in a sample. The activity of the enzyme is then assessed by incubation with a substrate that produces a measurable product. This allows the presence and / or amount of the target peptide in the sample to be detected and / or quantified. ELISA is a technique known to those skilled in the art.

[0066] As used herein, the term "ECLIA" (electrochemically linked immunosorbent assay) refers to an immunoassay that detects a target peptide (if any) present in a sample using an antibody conjugated to an electrochemiluminescent label, such as the SULFO-Tag system. When electricity is applied to the sample, the electrochemiluminescent label emits light. The intensity of the light is then measured to quantify the target peptide in the sample. This allows the presence and / or amount of the target peptide in the sample to be detected and / or quantified. ECLIA is a technique known to those skilled in the art.

[0067] The term "bound amount" as used herein refers to the quantification of binding between a monoclonal antibody and a target peptide, and the quantification is determined by comparing the measured value of the target peptide in a biological fluid sample with a calibration curve, which is prepared using a standard sample of the target peptide with 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 biological fluid, a calibration curve is prepared using a standard sample of a known concentration of a calibration peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1) (which may specifically be a peptide consisting of the amino acid sequence MGNTGPTGAV (SEQ ID NO: 1)). The value measured in the biological fluid sample is compared with the calibration curve to determine the actual amount of the target peptide in the sample.

[0068] As used herein, the term "immunotherapy" refers to a method of artificially stimulating the immune system in the treatment of cancer. There are several different types of immunotherapy, including, but not limited to, T cell engagers, CAR T cell therapy, cytokines, and immune checkpoint inhibitors. Preferably, the immunotherapy involves administering at least one immune checkpoint inhibitor, such as ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cemiplimab, or a combination thereof. Preferably, the immune checkpoint inhibitor is ipilimumab.

[0069] As used herein, the term "C4-aa 1355 " or "C4G" is the amino acid F 1354 and M 1355 This refers to a type VI collagen α2 chain neoepitope peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1) produced by a protease that causes cleavage between α2 and β2. Preferably, the protease is a serine protease, such as granzyme B, or a matrix metalloproteinase such as MMP-9.

[0070] figure The invention will now be illustrated in the following examples with reference to the following figures: [Brief explanation of the drawings]

[0071] [Figure 1] Figure 1 shows the specificity of the C4-aa1355 monoclonal antibody. The reactivity of the monoclonal antibody in a competitive C4-aa1355 ECLIA was tested against (A) the selected peptide (MGNTGPTGAV (SEQ ID NO: 1)), the extended peptide (FMGNTGPTGAV (SEQ ID NO: 7)), the truncated peptide (GNTGPTGAV (SEQ ID NO: 3)), the nonsense selected peptide (LLARDFEKNY (SEQ ID NO: 18)), and the nonsense coating peptide (LLARDFEKNY-K-biotin), and (B) the selected peptide (MGNTGPTGAV (SEQ ID NO: 1)) and nonselected 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 (OD at x ng / ml peptide), and B0 equals the maximum intensity (OD at 0 ng / ml peptide). [Figure 2] Figure 2 shows the proteolysis of type IV collagen α2 chain using MMP-9 or granzyme B. Type IV collagen α2 chain was incubated with MMP-9 (A) or granzyme B (GzB) (B) for 72 hours, and then C4-aa1355 ECLIA levels were measured. [Figure 3] Figure 3 shows serum C4-aa1355 levels in patients with metastatic melanoma. Serum C4-aa1355 levels at baseline and 3 weeks after ipilimumab treatment (n=41). Serum levels were compared using the Wilcoxon matched-pairs rank test. [Figure 4]Figure 4 shows Kaplan-Meier analysis of overall survival in patients with metastatic melanoma treated with ipilimumab. For C4-aa1355, the overall survival was compared between those with pretreatment levels in the upper quartile (Q2 + Q3 + Q4) and those with pretreatment levels in the lower quartile (Q1) (cutoff value: 14.5 ng / ml) (A). For C4M, the overall survival was compared between those with pretreatment levels in the upper quartile (Q4) and those with pretreatment levels in the lower quartile (Q1 + Q2 + Q3) (cutoff value: 35.0 ng / ml) (B). Differences between survival curves were determined using the log-rank test, and a p-value of p<0.05 was considered statistically significant. [Figure 5] Figure 5 shows the correlation between C4-aa1355 and C4M levels in metastatic melanoma patients. Pearson's correlation analysis was performed to describe the relationship between C4-aa1355 and C4M levels in pretreatment serum from metastatic melanoma treated with ipilimumab (n=54). [Figure 6] Figure 6 shows serum C4-aa1355 levels in cancer patients and healthy controls. A) Serum C4-aa1355 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). Groups were compared using the Kruskal-Wallis test adjusted for Dunn's multiple comparisons. B) Serum C4-aa1355 levels from healthy controls (n = 40) were compared with the combined group of cancer patients (n = 80) using an unpaired Mann-Whitney test. The horizontal black line represents the median value for patients measured twice. C) Pearson's correlation analysis was performed to describe the relationship between C4-aa1355 and C4M levels in serum from a combined group of cancer patients (n=80). [Figure 7] Figure 7 shows serum C4G (C4-aa1355) levels in early and late stage pancreatic ductal adenocarcinoma (PDAC). *p<0.05 [Figure 8]Figure 8 shows Kaplan-Meier plots for assessing overall survival (OS) associated with C4G (C4-aa1355) at baseline by dichotomization at the 25th and 75th percentiles (Q1+Q4 vs. Q2+Q3). [Example]

[0072] Various embodiments are described and disclosed in the following examples. These examples are presented to aid in understanding the disclosure and should not be construed in any way to limit the scope of the invention as defined in the claims that follow. The examples set forth below are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the disclosure or to imply that the experiments described below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0073] Materials and Methods All reagents used in the experiments were standard chemicals from Merck (Whitehouse Station, NJ, USA) and Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.

[0074] Peptide identification by mass spectrometry Type IV collagen from human placenta (Sigma-Aldrich, Cat. No. C5533) was proteolytically digested at a 10:1 ratio for 24 and 72 hours at 37°C and then stored at -80°C until mass spectrometry analysis.

[0075] One microgram of sample (corresponding to digested or undigested collagen in 100 μl of 50 mM Tris, 150 mM NaCl, pH 7.5 buffer) was reduced with 10 mM dithiothreitol for 30 minutes at 56°C and alkylated with 40 mM iodoacetamide for 60 minutes in the dark at room temperature. Any residual iodoacetamide was quenched with 10 mM dithiothreitol for 5 minutes at room temperature. The sample was digested with Lys-C (Wako Chemicals, catalog no. 125-05061) at an enzyme:substrate ratio of 1:20 for 16 hours on a shaker at 37°C. After adding 100 μl of 1 M NaCl containing 1% formic acid to the digest, the mixture was passed through a 30 kDa filter (Pall Life Sciences, catalog number OD030C34) to remove GAGs and desalted using a reversed-phase Vydac UltraMicro Spin C18 column (Harvard Apparatus, catalog number 74-7206) according to the manufacturer's instructions. Untargeted mass analysis was performed on a quadrupole-orbitrap benchtop mass spectrometer, QExactive (Thermo Scientific), equipped with an Easy nano-LC 1000 system (Thermo Fisher Scientific). Separation was performed on a 75 μm x 25 cm Acclaim Pepmap™ RSLC C18 capillary column packed with 2 μm particles (Thermo Fisher Scientific). A spray voltage of +2000 V was used with a heated ion transfer setting of 275 °C for desolvation. Online reversed-phase separation was performed using a flow rate of 300 nl / min with a linear binary gradient of 85 min. The gradient started at 3% solvent B for 4 min, then increased to 35% solvent B at 64 min, and then increased to 45% solvent B at 5 min. Finally, the organic solvent concentration was increased to 90% in 5 min and maintained at 90% for 7 min. MS scans (400–1200 m / z) were performed at 200 m / z, 1 × 10 6 The Orbitrap mass spectrometer was recorded at a resolution of 70,000, with an automatic gain control (AGC) target and a maximum ion injection time of 100 ms. MS was followed by 2 × 10 4Data-dependent collision-induced dissociation MS / MS scans were performed at a resolution of 17,500 on the 15 most intense multiply charged ions, with an intensity threshold of 1 m / z, an isolation width of 2 m / z, and dynamic exclusion allowed for 30 seconds. Identification from the discovery data was performed using the Homo sapiens proteome (UniProt proteome ID UP000005640) with Proteome Discoverer 2.1 software (Thermo Fisher Scientific). The processing workflow consisted of the following nodes: Spectrum Selector for spectrum preprocessing (precursor mass range: 300-30,000 Da; S / N threshold: 1.5), Sequest-HT search engine (protein database: see above; enzyme: Lys-C(hemimeric); max; missed cleavage sites: 2; peptide length range: 6-144 amino acids; precursor mass tolerance: 10 ppm; fragment mass tolerance: 0.02 Da; static modification: cysteine ​​carbamidomethylation); and Percolator for peptide validation (FDR < 1% based on peptide q-values). Results were filtered to retain only master proteins with at least one unique peptide, allowing protein grouping according to the parsimony principle. For label-free quantification (LFQ), the sum of the top three peptides for each protein was taken to reflect the protein's intensity. Peptide intensities were quantified using a proprietary algorithm developed in Proteome Discoverer 2.1 (ThermoFisher Scientific).

[0076] Peptide selection The first six amino acids from the N- and C-termini of each peptide derived from type IV collagen, identified by mass spectrometry, were considered as protease-generated neoepitopes. The protease-generated sequences were analyzed for homology with other human proteins and species using NPS@:Network Protein Sequence Analysis in conjunction with the Uniprot / Swiss-Prot database (reference 16). Cleavage site F 1354 ↓M 1355 ( 1355 MGNTGPTGAV 1364The amino acid sequence C-terminal from α2 chain of human type IV collagen was found to be unique to the α2 chain of human type IV collagen and was selected as a target for antibody production. The synthetic peptides used for monoclonal antibody production and the protease-mediated degradation of type IV collagen (C4-aa 1355 The technical evaluation of ECLIA for measuring ) was purchased from GenScript and is shown in Table 1.

[0077] [Table 1]

[0078] The target sequence was used as the selection peptide (MGNTGPTGAV (SEQ ID NO: 1)). The immunogenic peptide (MGNTGPTGAV-GGC-KLH) was generated by covalently linking the selection peptide to the keyhole limpet hemocyanin (KLH) carrier protein, with the addition of glycine and cysteine ​​residues between them to ensure correct linkage. A biotinylated peptide (MGNTGPTAV-K-biotin) was used as the coating peptide. Antibody specificity was tested by including an extended peptide (FMGNTGPTGAV (SEQ ID NO: 7)), a truncated peptide (GNTGPTGAV (SEQ ID NO: 3)), a nonsense selection peptide (LLARDFEKNY (SEQ ID NO: 18)), and a nonsense coating peptide (LLARDFEKNY-K-biotin). To test for potential cross-reactivity to other ECM proteins with similar sequences, three peptides with one amino acid mismatch at either the first (QGNTGPTGAV (SEQ ID NO: 6)), third (MGQTGPTGAV (SEQ ID NO: 4)), or fourth (MGNSGPTGAV (SEQ ID NO: 5)) position from the N-terminus were included in the specificity test. Antibody specificity was calculated as the percentage of signal inhibition of two-fold diluted peptides.

[0079] Monoclonal antibody production and clone characterization Six- to seven-week-old female Balb / C mice were subcutaneously immunized every other week with 200 μl of emulsified antigen containing 100 μg of the immunogenic peptide (MGNTGPTGAV-GGC-KLH) in Stimune Immunogenic Adjuvant (ThermoFisher, catalog no. 7925000) until a stable titer level was achieved. Mice with the highest antibody titers were rested for 4 weeks and then boosted intraperitoneally with the immunogenic peptide. Three days later, splenocytes were isolated and fused with mouse SP2 / 0 myeloma cells to generate hybridoma cells as previously described (reference 17). Hybridoma cells were cultured in 96-well microtiter plates, and limiting dilution was used to ensure monoclonal growth. Supernatants from monoclonal antibody-producing hybridoma cells were screened for reactivity to selected peptides and human serum samples in a preliminary competitive ELISA using biotinylated coated peptides in streptavidin-coated microtiter plates (Roche, catalog no. 11940279). Clones with the best reactivity to the selected peptides were purified using a Protein G column (GE Healthcare Life Sciences, catalog no. 17-0404-01) according to the manufacturer's instructions.

[0080] C4-aa 1355 ECLIA Protocol During assay development, the optimal incubation buffer, time, temperature, and concentration of antibody and coating peptide were determined to determine the final competitive C4-aa 1355The ECLIA protocol was as follows: MSD GOLD 96-well streptavidin-precoated plates (Meso Scale Discovery, catalog number L15SA-1) were incubated with 150 μl / well of blocking buffer (10 mM phosphate-buffered saline (PBS) containing bovine serum albumin (BSA) (5% w / v) and bronidox (0.36% v / v), 8 g / L NaCl, pH 7.4) for 60 minutes at 20°C with shaking (300 rpm) in the dark. Plates were coated with 25 μl / well of biotinylated coating peptide at a concentration of 2 ng / ml in assay buffer (50 mM PBS, 8 g / L NaCl, pH 7.4 containing BSA (1% w / v), Tween-20 (0.1% w / v), and bronidox (0.36% v / v)) and incubated for 60 min at 20°C with shaking (750 rpm) in the dark. Next, 25 μl / well of selected peptide, assay control, or prediluted serum / plasma sample (1:2) was added, followed by 25 μl / well of SULFO-TAG (MSD GOLD SULFO-TAG NHS-Ester Conjugation, Meso Scale Discovery, Catalog No. R31AA-1)-labeled monoclonal antibody diluted in assay buffer to a final concentration of 25 ng / ml. The plate was incubated for 20 hours at 4°C with shaking (300 rpm) in the dark. After all incubation steps, the plate was washed three times with wash buffer (20 mM Tris, 50 mM NaCl, pH 7.2). Finally, 150 μl / well of MSD GOLD Read Buffer (Meso Scale Discovery, Catalog No. R92TG-2) was added, and the plate was immediately read within 2 minutes on a Sector Imager 6000 (Meso Scale Discovery). SULFO-TAG was capable of emitting light when electricity was applied, and the luminescence data was analyzed using MSD Discovery Workbench 4.0 software. Analyte concentrations were calculated using a four-parametric curve-fitting model.

[0081] C4-aa1355 Technical evaluation of the assay The lower limit of detection was determined from 10 independent assay runs using the background mean plus 2.5 times the standard deviation. The upper limit of detection was determined from 10 identical assay runs using the inverse calibration concentration of the selected peptide at the highest concentration minus 2.5 times the standard deviation. Intra- and inter-assay variability was determined from 10 independent assay runs of seven identical samples at concentrations covering the entire linear range of the standard curve. Samples consisted of four samples containing different amounts of the selected peptide in assay buffer and three different serum samples from healthy humans. Intra-assay variability was calculated as the average coefficient of variation (CV%) within a plate, and inter-assay variability was calculated as the average CV% across 10 plates. To determine assay linearity, two-fold dilutions of human serum samples (n = 3) or EDTA plasma samples (n = 3) were performed, and linearity was calculated as the percent recovery of the undiluted samples. Analyte stability was tested by four repeated freeze / thaw cycles of human serum (n=3 for each cycle), and analyte recovery was calculated based on the first cycle. Furthermore, specimen stability was tested by human serum samples (n=3 for each time point) incubated at 4°C or 20°C for 24 or 48 hours, and recovery was calculated based on the sample stored at -20°C. Interferences were tested by adding low / high amounts of biotin (3.0 / 9.0 ng / ml), hyperlipidemia (1.5 / 5.0 mg / ml), and hemoglobin (2.5 / 5.0 mg / ml) to the serum samples, and recovery was calculated based on the serum samples.

[0082] In vitro cleavage of type IV collagen Recombinant type IV collagen α2 chain (Mybiosource) and MMP-9 (Giotto, catalog no. G04MP09C) or granzyme B (GzB) (Abcam, catalog no. ab168093) were incubated at 37°C for 72 h at a 10:1 ratio (10 μg of type IV collagen and 1 μg of protease) in MMP buffer (50 mM Tris-HCl, 150 nM NaCl, 10 mM CaCl2, 10 μM ZnCl2, 0.05% Brij35, pH 7.5) or GzB buffer (50 mM Tris, 150 mM NaCl, pH 7.5), respectively, and then stored at -80°C until analysis. Digestion of carboxymethylated transferrin with MMP-9 or GzB was included as a positive control, and MMP-9 alone in MMP buffer and GzB alone in GzB buffer were included as negative controls. The activity of MMP-9 and GzB was confirmed by Coomassie blue staining (data not shown).

[0083] C4-aa 1355 Clinical validation of the assay After obtaining informed consent, serum samples were collected from stage IV melanoma patients (n=54) treated with ipilimumab (3 mg / kg body weight) as standard care at Herlev Hospital and Aarhus University Hospital, Denmark. This study was approved by the Danish Capital Region Ethics Committee (H-2-2012-058) in accordance with the 1975 Declaration of Helsinki. Serum samples were collected at baseline and 3 weeks after the first treatment (before the second dose of treatment).

[0084] Serum samples from other cancer patients were obtained from a commercial vendor, Asterand Biosciences (Detroit, Michigan, USA). These 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). Serum samples from healthy controls (n = 40) were obtained from a commercial vendor, Valley Biomedical (Winchester, Virginia, USA). These samples were collected after informed consent and approval by the appropriate institutional review boards in accordance with the Declaration of Helsinki.

[0085] Newly developed biomarker C4-aa 1355 C4M was assessed in serum samples from cancer patients for comparison. The C4M competitive ELISA is a well-characterized assay based on a monoclonal antibody specific for a neoepitope in MMP-9-mediated degradation of type IV collagen and manufactured by Nordic Biosciences (Hallev, Denmark), and measurements were performed according to the manufacturer's specifications (reference 18).

[0086] statistical analysis Wilcoxon matched-pairs signed-rank test was used to compare biomarker levels in melanoma patients at baseline and week 3. Kaplan-Meier survival curves were used to analyze overall survival (OS) of melanoma patients and C4-aa 1355 For Q4M, we contrasted those whose pre-treatment levels were in the upper quartile (Q2+Q3+Q4) with those whose pre-treatment levels were in the lower quartile (Q1), and for C4M, we contrasted those whose pre-treatment levels were in the upper quartile (Q4) with those whose pre-treatment levels were in the lower quartile (Q1+Q2+Q3).

[0087] C4-aa in serum samples from patients with different cancers 1355Levels of C4-aa in serum from each of the combined metastatic melanoma and cancer patients were compared using a Kruskal-Wallis test adjusted for Dunn's multiple comparisons. Healthy controls were compared with the combined cancer patients using an unpaired Mann-Whitney test. Pearson's correlation analysis was performed to compare the C4-aa in serum from each of the combined metastatic melanoma and cancer patients. 1355 The relationship between IL-1 and C4M levels was described. A p-value of p<0.05 was considered statistically significant. Graphing and statistical analysis were performed using GraphPad Prism version 7 (GraphPad Software, CA, USA).

[0088] result Novel C4-aa 1355 Assay specificity The specificity of the monoclonal antibody was confirmed by the novel competitive C4-aa 1355 The selected peptides inhibited the signal in a dose-dependent manner, whereas extended, truncated, and nonsense selected peptides did not (Figure 1A). No signal was observed when a nonsense biotinylated peptide was used (Figure 1A). When reactivity was tested against peptides with only a single amino acid mismatch with the selected peptides, no reactivity was detected at peptide concentrations between 0 and 30 ng / ml, whereas nonselected peptide 2 inhibited the signal by 65% ​​at the highest concentration (Figure 1B). Collectively, these data suggest that the monoclonal antibody is highly specific for the neoepitope on the selected peptide.

[0089] Proteolysis of type IV collagen by MMP-9 C4-aa 1355 To confirm that the antibody / assay recognized the protease-generated type IV collagen neoepitope, C4-aa 1355 was measured for undigested type IV collagen, MMP-9-digested type IV collagen, and GzB-digested type IV collagen. 1355was detectable only in the MMP-9-digested and GzB-digested samples, whereas neither level was detected in the undigested (no protease) samples, indicating that the antibody was specific for a protease-generated neoepitope.

[0090] C4-aa 1355 Technical evaluation of the assay C4-aa 1355 The technical performance of the ECLIA assay was further evaluated through different technical validation steps, summarized in Table 2. The detection range of the assay was 0.6 to 832 ng / mL. The intra- and inter-assay variations were 6% and 8%, respectively, below the acceptance criteria of the 10% and 15% range, respectively. Linearity was detected from undiluted to a 1:4 dilution, with dilution recoveries of 94% and 106% for serum and EDTA plasma, respectively. After four freeze / thaw cycles, the analyte recovery in serum was 96%. After long-term storage of human serum at 4°C or 20°C for 48 hours, the analyte recoveries were 122% and 109%, respectively. No interference was detected from serum with high or low lipid or hemoglobin contents, with recoveries ranging from 92 to 111%. Low biotin content did not interfere with the analyte, but high biotin content did, with recoveries of 94% and 71%, respectively. The acceptance criteria for recovery was within 100±20%. 1355 The ECLIA has been shown to be a technically robust assay.

[0091] [Table 2]

[0092] C4-aa in patients with metastatic melanoma treated with ipilimumab 1355 Clinical evaluation of the assay C4-aa 1355 To assess the biomarker potential of C4-aa in serum from patients with metastatic melanoma at baseline and 3 weeks after ipilimumab treatment, 1355 The biomarker levels were compared.1355 The levels of were slightly elevated after 3 weeks of treatment (p=0.090) (Figure 3).

[0093] Next, C4-aa 1355 The association between biomarkers and survival outcomes was assessed by Kaplan-Meier curves. 1355 Higher baseline levels of Q2 + Q3 + Q4 were significantly associated with longer overall survival (OS) compared with lower levels (Q1) (p = 0.040) (Figure 4A). Median OS was 646 days in patients with high biomarkers compared with 290 days in patients with low biomarkers.

[0094] C4-aa 1355 Because the C4M and C4M biomarkers measure two distinct protease-generated neoepitopes on type IV collagen, we also evaluated the association between C4M and OS. While these findings regarding C4M have been published previously (reference 8), the results of C4M measured in 54 patients are shown in Figure 4B. For the C4M biomarker, high baseline levels (Q4) were significantly associated with shorter OS compared with low levels (Q1 + Q2 + Q3) (p = 0.005) (Figure 4B). Interestingly, the two biomarkers showed opposite associations with outcome.

[0095] Furthermore, C4-aa in patients with metastatic melanoma at baseline 1355 The correlation between C4-aa levels and C4M levels was examined. 1355 and C4M did not correlate (r = 0.021, p = 0.883) (Figure 5).

[0096] C4-aa in patients with other cancers 1355 Clinical evaluation of the assay C4-aa 1355 To further evaluate the biomarker potential of C4-aa, it was measured in serum from patients with different cancers, either breast cancer, CRC, gastric cancer, NSCLC, SCLC, melanoma, ovarian cancer, pancreatic cancer or prostate cancer, as well as in serum from healthy controls.1355 Interpatient variability in biomarker levels was observed (Figure 6A). 1355 When comparing the median levels of C4-aa in each group of cancer patients, no significant differences were observed (Figure 6A). 1355 When compared with the combination group of cancer patients, C4-aa 1355 Levels were significantly elevated in cancer patients (14.8 ng / ml) compared to healthy controls (12.0 ng / ml) (p=0.006) (Fig. 6B). As shown in the first cohort, C4-aa in these cancer patients 1355 and C4M levels were not correlated (r = 0.197, p = 0.080) (Figure 6C).

[0097] Serum C4-aa measured at baseline 1355 (C4G) predicts outcome in patients with pancreatic ductal adenocarcinoma (PDAC) treated with chemotherapy. C4-aa 1355C4G was measured in pretreatment serum samples from 40 patients with pancreatic ductal adenocarcinoma (PDAC). All patients were provided by the Danish BIOPAC study "BIOmarkers in Patients with Pancreatic Cancer" (NCT03311776). Patients were recruited from six Danish hospitals between December 2008 and September 2017. PDAC patients had histologically confirmed tumors. PDAC patients were treated with various types of chemotherapy according to national guidelines (www.gicancer.dk). The study was conducted in accordance with the recommendations of the Danish Regional Committee for Health Research Ethics. The BIOPAC protocol was approved by the Danish Regional Committee for Health Research Ethics (VEK ref. KA-20060113) and the Data Protection Agency (j.nr.2006-41-6848). All subjects provided written informed consent in accordance with the 8th edition of the Declaration of Helsinki. Blood samples were obtained at the time of diagnosis or before surgery. Samples were processed according to nationally approved standard operating procedures for blood (www.herlevhospital.dk / biopac.dk). Serum samples and clinical data from patients were collected prospectively. Serum samples were measured blinded.

[0098] The results are shown in Figure 7 according to disease stage. 1355 was significantly lower in serum from patients with late-stage PDAC compared with early-stage PDAC (Mann-Whitney test, p-value = 0.0132). 1355 The possible association with "extreme" C4-aa 1355 Using the 25th and 75th percentile cutpoints to define groups of levels (<25th percentile + >75th percentile, i.e., quartile 1 and quartile 4, Q1 + Q4), Kaplan-Meier analysis identified "non-extreme" C4-aa 1355 Patients with "non-extreme" C4-aa levels (>25th percentile to <75th percentile, Q2+Q3) were found to have improved overall survival (Figure 8). Univariate Cox regression analysis supported this, demonstrating that "non-extreme" C4-aa levels were associated with improved overall survival (Figure 8). 1355The subgroup of patients with C4-aa levels showed a decreased risk of death (Table 3). Furthermore, multivariate Cox regression analysis showed that C4-aa 1355 showed that the predictive value of was independent of disease stage.

[0099] [Table 3]

[0100] Discussion and Conclusion Neoepitopes (C4-aa) generated by protease-mediated degradation of type IV collagen 1355 A robust and specific competitive ECLIA has been developed and validated that allows for the noninvasive measurement of C4-aa in these melanoma patients. 1355 High baseline levels of C4-aa in these samples were associated with clinical response (longer overall survival) to immune checkpoint inhibitor treatment. In contrast, high baseline levels of C4M were associated with shorter overall survival. 1355 C4-aa levels were not correlated with C4M levels. 1355 Both C4-aa and C4M measure neoepitopes of type IV collagen, but at two different sites. Interestingly, these data suggest that these different cleavage products are released during two different pathological events, one associated with a good outcome and one associated with a poor outcome when measured at baseline. Furthermore, after 3 weeks of immune checkpoint inhibitor treatment, slightly elevated levels of C4-aa were detected in serum from metastatic melanoma patients compared to baseline. 1355 was detected.

[0101] C4-aa 1355 The finding that C4-aa is associated with clinical response to immune checkpoint inhibitor treatment in these melanoma patients is supportive of the findings. 1355 These results demonstrate that the assay has biomarker potential in the immuno-oncology setting to identify cancer patients with a T cell-permissive tumor microenvironment that will respond to treatment.1355 This may reflect protease-mediated T cell migration from the circulation to the underlying stroma. Conversely, C4M was associated with poor reactivity, supporting previous findings (references 8-10) suggesting that C4M is associated with tumor activity and reactive stroma.

[0102] Interestingly, this study demonstrated that one type of type IV collagen neoepitope fragment (C4M) is associated with tumorigenesis, while another type of type IV collagen neoepitope fragment (C4-aa 1355 ) has been shown to be associated with T cell infiltration, supporting the value of measuring pathology-specific neoepitopes and not simply total protein.

[0103] C4-aa 1355 is also elevated in other cancer types besides melanoma, suggesting that the biomarker has potential in other indications. Furthermore, in patients with these cancers, C4-aa 1355 Again, there was no correlation between IL-1 and C4M levels, validating that these biomarkers reflect pathologically distinct aspects of the tumor microenvironment.

[0104] C4-aa 1355 was found to be elevated in patients with early stage pancreatic ductal adenocarcinoma (PDAC). 1355 Patients at the extreme levels (ie, first or fourth quartile) were found to have reduced overall survival.

[0105] To the best of our knowledge, this is the first study to investigate such specific protease-mediated degradation of type IV collagen (C4-aa 1355 This is the first study to demonstrate that IL-16 has the potential to be a biomarker in cancer and is associated with response to immune checkpoint inhibitor therapy.

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[0108] 13. El-Shabrawi Y, Walch A, Hellman J, Egger G, Foster CS. Selective metalloproteinase 2 and 9 inhibitors inhibit MMP-dependent chemotaxis and ameliorate experimental autoimmune uveitis Journal of Neuroimmunology. 2004; 14. Leppert D, Hauser SL, Kishiyama JL, An S, Zeng L, Gotzula EJ. Stimulation of matrix metalloproteinase-dependent migration of T cells by eicosanoids. FASEB Journal. 1995; 15. Edspur K, Basse PH, Goldfarb RH, Albertson P. Cytotoxic lymphocyte matrix metalloproteinases influence tumor invasion and immune regulation Cancer Microenvironment. 2011. 16. Combet C, Blanchet C, Geojon C, Deléage G. NPS@: Network Protein Sequence Analysis Trends in biochemical sciences. 2000 Mar;25(3):147-50. 17. Gefter ML, Margulies DH, Scharf MD. A simple method for polyethylene glycol-facilitated hybridization of mouse myeloma cells. Somatic Cell Genetics. 1977;3(2):231-6. 18. Sand JM, Larsen L, Hogaboam C, Martinez F, Han M, Larsen MR et al. MMP-mediated degradation of type IV collagen alpha 1 and alpha 3 chains reflects basement membrane remodeling in experimental and clinical fibrosis - Validation of two novel biomarker assays PLoS ONE. 2013;8(12). 19. Kabat, E. A., T. T. Wu, H. M. Perry, K. S. Gottesman, and C. Forer (1987). Sequence of the protein of immunological interest U.S. Department of Health and Human Services, Bethesda, Maryland, p. 1.

Claims

1. A monoclonal antibody which specifically recognizes and binds to the N-terminal amino acid sequence of a peptide which is produced by degrading type IV collagen α2 chain with a protease and has the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1), The following complementarity determining regions (CDRs): 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); A monoclonal antibody comprising:

2. 2. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1).

3. The monoclonal antibody comprises the following peptides (i) to (v): (i) a peptide having the N-terminal amino acid sequence XMGNTGPTGAV (SEQ ID NO: 2), where X represents an amino acid; (ii) a peptide having the N-terminal amino acid sequence GNTGPTGAV (SEQ ID NO: 3); (iii) a peptide having the N-terminal amino acid sequence MGQTGPTGAV (SEQ ID NO: 4); (iv) a peptide having the N-terminal amino acid sequence QGNTGPTGAV (SEQ ID NO: 6); (v) a peptide having the N-terminal amino acid sequence MGNSGPTGAV (SEQ ID NO: 5); 3. The monoclonal antibody according to claim 1 or 2, which does not specifically recognize or bind to any one or more peptides among the above.

4. A method for identifying whether a cancer patient will respond to immunotherapy, the method being an immunoassay comprising contacting a biological fluid sample obtained from the patient with the monoclonal antibody of claim 1, and detecting the presence of a peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1) by detecting binding between the monoclonal antibody and the peptide in the sample.

5. A method for predicting survival outcome in a cancer patient, the method being an immunoassay comprising contacting a biological fluid sample obtained from the patient with the monoclonal antibody described in claim 1, and detecting the presence of a peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1) by detecting binding between the monoclonal antibody and the peptide in the sample.

6. The method of claim 4 or claim 5, wherein the detection is quantitative.

7. The method of any one of claims 4 to 6, wherein the immunoassay is a competitive immunoassay.

8. 8. The method of any one of claims 4 to 7, wherein the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1).

9. 9. The method of claim 4, wherein the patient is diagnosed with a cancer selected from melanoma, breast cancer, colon cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, prostate cancer, and pancreatic cancer.

10. 10. The method of claim 9, wherein the patient is a patient diagnosed with melanoma.

11. The method of claim 10, wherein the patient is diagnosed with metastatic melanoma.

12. 12. The method of any one of claims 4, 6 to 11, wherein the method further comprises correlating the amount of the peptide with values ​​associated with normal healthy individuals and / or values ​​obtained from cancer patients who have responded to immunotherapy.

13. 10. The monoclonal antibody of claim 1 and at least one of the following: - streptavidin-coated well plates; a C-terminally biotinylated peptide with 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, - Assay Visualization Kit An assay kit comprising:

14. 14. The assay kit of claim 13, wherein the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having the N-terminal amino acid sequence MGNTGPTGAV (SEQ ID NO: 1).

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