Pro-c12 immunoassay for determining the c-terminus of type xii collagen

Abstract

Disclosed herein are methods of immunoassay for detecting in a patient sample (such as for example a blood, serum or plasma sample) a biomarker (PRO-C12) originating from Type XII Collagen. Said methods may be used for detecting and / or monitoring cancer or a level of severity thereof. Also disclosed are monoclonal antibodies and assay kits for use in said methods of immunoassay.

Description

[0001] PRO-C12 AssayField of the invention The present invention relates to methods of immunoassay fordetecting in a patient sample (such as for example a blood,serum or plasma sample) a biomarker originating from Type XIICollagen, and to the use of said methods of immunoassay fordetecting and / or monitoring cancer or a level of severitythereof. The present invention also relates to monoclonalantibodies and assay kits for use in said methods ofimmunoassay. Background Cancer remains a global health challenge, with its incidence steadily rising worldwide[1]. The complexity of cancer, together with the lack of efficient diagnostic and prognostic tools, underscores the urgent need for innovative approaches to enhance early detection and prognosis. Traditional diagnostic methods often pose challenges, including invasiveness and limitations in providing comprehensiveinformation. In this context, the development of non-invasivebiomarkers emerges as a promising avenue, offering the potential to revolutionize cancer diagnosis and monitoring. Non-invasive biomarkers not only contribute to the early detection of cancer but also facilitate personalized treatment strategies. In recent years, the tumor microenvironment (TME) consisting of tumor cells, stromal cells, immune cells and extracellular matrix (ECM) has garnered significant attention due to its critical role in tumor progression and impact in treatment outcomes. Numerous studies have revealed variations in the ECM composition between the different cancer types which highlights the pivotal role of the TME in cancer development and underscores the need for targeted therapeutic strategies that consider the intricacies of this microenvironment[2]–[6]. Collagens stand as the predominant components of the ECM, shaping the structural framework of tissues and organs. 28 different types of collagens have been identified to date, providing essential structural support, tensile strength, and elasticity to tissues, influencing cell behavior and multiple physiological processes. Beyond their structural role, collagens actively participate in cell signaling, migration and differentiation[7], [8]. Moreover, collagen fragments originated from excessive remodeling of the ECM during tumor progression have been previously proposed as cancer biomarkers. For instance, high levels of the type III collagen pro-peptides in circulation reflect tumor fibrosis activity and predicted poor prognosis in patients with different solid cancer types[9]–

[0014] . Although less abundant than fibrillar collagens, FACIT collagens (fibril associated collagens with interrupted triple-helices) including type IX, XII, XIV, XIX, XX, XXI and XXII are associated with fibrillar collagens and act as molecular bridges, playing a crucial role in organizing and maintaining the stability of ECM. FACITs have multiple triple-helical domain separated by non- triple-helical domains, and unlike fibrillar collagens, they do not undergo proteolytic processing from a larger precursor form

[0015] ,

[0016] . Type XII collagen was first discovered by cDNA cloning with partial homology to the α1 chain of type IX collagen associated with type I collagen fibrils

[0017] –

[0019] . Type XII collagen is composed of two small collagenous domains (COL1 and COL2) separated by non-collagenous domains (NC1 and NC2), and a large N-terminus domain (NC3)

[0020] ,

[0021] . Splicing in the NC3 domain results in two isoforms, XIIA (large isoform) and XIIB (small isoform), and therefore type XII collagen molecules can assemble as homotrimers or as a combination of XIIA and XIIB heterotrimers and their expression depends on tissue localization and developmental stage

[0018] ,

[0022] ,

[0023] . Increased expression of type XII collagen has been reported upon mechanical stress but further research is warranted to explore the molecular mechanisms in this process

[0024] –

[0026] . Due to its molecular structure and tissue distribution, type XII collagen seems to play a role in type I collagen fibrillogenesis, fibril organization and interactions with other ECM molecules

[0015] ,

[0027] –

[0029] . Additionally, type XII collagen is expressed in bone, muscle and tendons and regulates tissue regeneration and communication between cells during development

[0030] –

[0033] . Moreover, mutations in the COL12A1 are associated with myopathic type Ehlers-Danlos syndrome (mEDS) characterized by muscle weakness, distal joint hypermobility and delayed tendon reflexes

[0034] . In recent years, multiple studies have reported that type XII collagen is involved in cell migration and invasion and tumor growth

[0035] -

[0037] . COL12A1 overexpression has been detected and was predictive of poor prognosis in multiple cancer types including colorectal (CRC), gastric, breast cancer, renal, ovarian and pancreatic cancer

[0035] ,

[0037] –

[0048] . Further, in patients with breast cancer high expression of COL12A1 predicted poor response to immunotherapy treatment

[0044] ,

[0049] . Interestingly, a recent study demonstrated that type XII collagen secreted by cancer associated fibroblasts (CAFs) modifies type I collagen organization surrounding the tumor and stablishing a pro-metastatic environment for cancer cells dissemination and proposed type XII as a tool to identify patients that are more prone to breast cancer relapse

[0043] . Summary of the Invention The present inventors have developed and validated an enzyme- linked immunosorbent assay (ELISA) targeting a biomarker consisting of a C-terminus sequence of type XII collagen (“PRO-C12”), and have demonstrated that said biomarker ispresent and can be detected by said assay in patient serumsamples. The present inventors have moreover demonstrated that said biomarker is present in elevated levels in serumsamples from patients with different cancer types and thatthe assay can be used to distinguish between cancer patients and healthy controls, thereby demonstrating the biological relevance and utility of the biomarker and assay.Accordingly, in a first aspect the present invention providesa method of immunoassay comprising;i) contacting a patient sample with a monoclonalantibody that specifically binds to the C-terminusamino acid sequence YNGQGYPGSG (SEQ ID NO: 1) (alsoreferred to herein as “PRO-C12”, the “PRO-C12 target sequence”, or simply the “target sequence”); and ii) detecting and determining the amount of bindingbetween said monoclonal antibody and peptides in the sample. In a preferred embodiment, the method is a method ofimmunoassay for detecting and / or monitoring a cancer or alevel of severity thereof in a patient, the method furthercomprising; iii) correlating said amount of binding with valuesassociated with normal healthy subjects and / or values associated with known disease severity and / or values obtained from said patient at a previous time point and / or with a predetermined cut-off value.As used herein the term “C-terminus sequence” refers to a C-terminal peptide sequence at the extremity of a polypeptide, i.e. at the C-terminal end of the polypeptide, and is not tobe construed as meaning in the general direction thereof. Asused herein the term “N-terminus sequence” refers to an N-terminal peptide sequence at the extremity of a polypeptide, i.e. at the N-terminal end of the polypeptide, and is not to be construed as meaning in the general direction thereof. As used herein, the terms “peptide” and “polypeptide” are used synonymously. As used herein the term “monoclonal antibody” refers to both whole antibodies and to fragments thereof that retain the binding specificity of the whole antibody, such as for example a Fab fragment, F(ab’)2 fragment, single chain Fv fragment, or other such fragments known to those skilled in the art. As is well known, whole antibodies typically have a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair made up of one "light" and one "heavy" chain. The N-terminal regions of each light chain and heavy chain contain the variable region, while the C-terminal portions of each of the heavy and light chains make up the constant region. The variable region comprises three complementarity determining regions (CDRs), which are primarily responsible for antigen recognition. The constant region allows the antibody to recruit cells and molecules of the immune system. Antibody fragments retaining binding specificity comprise at least the CDRs and sufficient parts of the rest of the variable region to retain said binding specificity. In the present invention, a monoclonal antibody comprising any constant region known in the art can be used. In the case of mouse antibodies and human antibodies, the constant light chains are classified as either kappa or lambda light chains. Heavy constant chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG isotype has several subclasses, including, but not limited to IgGl, IgG2,IgG3, and IgG4 in the case of humans and IgGl, IgG2a, IgG2b,IgG2c and IgG3 in the case of mice. The monoclonal antibodymay preferably be of the IgG isotype, including any one of the IgG subclasses. The CDR of an antibody can be determined using methods known in the art such as that described by Kabat et al. Antibodies can be generated from B cell clones. The isotype of the antibody can be determined by ELISA specific for IgM, IgG or IgA isotype, or subclass. The amino acid sequence of the antibodies generated can be determined using standard techniques. For example, RNA can be isolated from the cells, and used to generate cDNA by reverse transcription. The cDNA is then subjected to PCR using primers which amplify the heavy and light chains of the antibody. For example primers specific for the leader sequence for all VH (variable heavy chain) sequences can be used together with primers that bind to a sequence located in the constant region of the isotype which has been previously determined. The light chain can be amplified using primers which bind to the 3’ end of the Kappa or Lamda chain together with primers which anneal to the V kappa or V lambda leader sequence. The full length heavy and light chains can be generated and sequenced. As used herein the term “amount of binding” refers to the quantification of binding between the antibody and peptides in the patient sample. Said quantification may for example be determined by comparing the measured values of binding in the patient sample against a calibration curve produced using measured values of binding in standard samples containing known concentrations of a peptide to which the antibodyspecifically binds, in order to determine the quantity ofpeptide to which the antibody specifically binds in the patient sample. Any suitable analytical method can be usedfor measuring the amount of binding. For example, an ELISAmethod can be used in which spectrophotometric analysis is used to measure the amount of binding both in the patient samples and when producing the calibration curve.As used herein the term “predetermined cut-off value” meansan amount of binding that is determined statistically to beindicative of a high likelihood of a disease (i.e. a cancer)or a particular severity thereof in a patient, in that a measured value of the target peptide in a patient sample that is (as appropriate) above or below the statistical cut-off value corresponds to at least a 70% probability, preferably at least an 75% probability, more preferably at least an 80% probability, more preferably at least an 85% probability, more preferably at least a 90% probability, and most preferably at least a 95% probability of the presence of saiddisease or particular severity thereof.As used herein, the term “values associated with normal healthy subjects” means standardised quantities of binding determined by the method described supra for samples fromsubjects considered to be healthy, i.e. without disease (i.e.a without cancer); and the term “values associated with known disease severity” means standardised quantities of binding determined by the method described supra for samples frompatients known to have disease (i.e. a cancer) of a knownseverity.In certain embodiments, the method may be a method fordetecting and / or monitoring a cancer or a level of severitythereof in a patient wherein the cancer is prostate cancer,breast cancer, melanoma, lung cancer, gastric cancer, pancreatic cancer, head and neck cancer or colorectal cancer.In preferred embodiments, the patient sample is a humanbiofluid sample. Preferably the sample is a blood-basedsample, such as blood (whole blood), plasma or serum. The monoclonal antibody preferably does not specifically bind to a peptide having the C-terminus amino acid sequenceYNGQGYPGSGA (SEQ ID NO: 2) (i.e. an elongated version of thetarget sequence extended at its C-terminus by the addition ofan alanine residue). Preferably, the ratio of the affinity ofsaid antibody for the target sequence to the affinity of said antibody for the elongated version of the target sequence isat least 10 to 1, and more preferably is at least 20 to 1, atleast 30 to 1, at least 40 to 1, at least 50 to 1 or at least100 to 1. The monoclonal antibody preferably does not specifically bind to a peptide having the C-terminus amino acid sequenceYNGQGYPGS (SEQ ID NO: 3) (i.e. a truncated version of thetarget sequence truncated by removal of the last glycine residue). Preferably, the ratio of the affinity of said antibody for the target sequence to the affinity of said antibody for the truncated version of the target sequence isat least 10 to 1, and more preferably is at least 20 to 1, atleast 30 to 1, at least 40 to 1, at least 50 to 1 or at least100 to 1.The monoclonal antibody may for example be raised against a synthetic peptide having the C-terminus amino acid sequenceYNGQGYPGSG (SEQ ID NO: 1). For example, the monoclonalantibodies may be raised by: (a) immunizing a rodent (or other suitable mammal) with a synthetic peptide comprisingthe C-terminus sequence YNGQGYPGSG (SEQ ID NO: 1), whichpeptide may optionally be linked at its N-terminus to an immunogenic carrier protein (such as keyhole limpet hemocyanin); (b) isolating and cloning a single antibody producing cell; and (c) assaying the resulting monoclonal antibodies to ensure that they have the desired specificity. In preferred embodiments the immunoassay is a competition assay or a sandwich assay. The immunoassay may, for example, be a radio-immunoassay or an enzyme-linked immunosorbent assay (ELISA). Such assays are techniques known to the person skilled in the art. In a second aspect, the present invention provides a method of treating a cancer in a patient in need thereof, the method comprising:(a) carrying out a method of immunoassay for detectinga cancer or a level of severity thereof in accordance with the first aspect of the present invention on a sample from a patient; and(b) administering to the patient a therapy for thetreatment of said cancer if it is determined in step (a) that the patient has said cancer or aparticular level of severity thereof. Preferred embodiments of the method in accordance with the second aspect will be apparent from the foregoing discussion of preferred embodiments of the methods according to the first aspect. For example, step (a) may in particular comprise carrying out the method of immunoassay to detect a prostate cancer, breast cancer, melanoma, lung cancer, gastric cancer, pancreatic cancer, head and neck cancer, colorectal cancer or a particular level of severity thereof. The therapy may be any therapy suitable for treating the cancer in question. The therapy may for example comprise or consist of one or more surgeries, one or more radiation therapies, one or more medicaments (such as for example one or more chemotherapies, one or more immunotherapies and / or one or more hormonal therapies), or combinations thereof. Medicaments may be formulated for topical or systemic administration. Topical medicaments may for example be formulated as creams, foams, gels, lotions, or ointments for administration. Systemic medicaments may for example be formulated for enteral or parenteral administration. Surgeries may be curative surgeries, preventative surgeries, debulking surgeries, palliative surgeries and / or restorative surgeries. For example, where the cancer is prostate cancer, suitable therapies may for example comprise one or more of:radiotherapy; chemotherapy using chemotherapeutic agents,such as for example docetaxel, cabazitaxel, docetaxel, thalidomide, and combinations thereof; immunotherapy, such asfor example the monoclonal antibody bevacizumab; hormonaltherapies such as for example abiraterone and enzalutamide;external beam radiation therapy; particle therapy; high-intensity focused ultrasound; cryotherapy; and surgicalprocedures such as for example a radical prostatectomy.Where the cancer is breast cancer, suitable therapies may forexample comprise one or more of: a mastectomy, quadrantectomyor lumpectomy; estrogen receptor blockers (such astamoxifen); aromatase inhibitors (such as anastrozole or letrozole) that block production of estrogen; CDK inhibitors; one or more chemotherapeutic agents such as a combination ofcyclophosphamide, doxorubicin and optionally a taxane (suchas docetaxel), or a combination of cyclophosphamide, methotrexate, and fluorouracil; one or more monoclonalantibodies such as trastuzumab and / or pertuzumab; andradiotherapy.Where the cancer is melanoma, suitable therapies may forexample comprise one or more of: surgical excision of thetumour, and optionally lymph nodes in the area of the tumour;interferon treatment; chemotherapy using agents such as forexample dacarbazine; small-molecule targeted therapies usingfor example BRAF inhibitors (such as vemurafenib anddabrafenib), MEK inhibitors (trametinib), C-Kit inhibitorsand / or NRAS inhibitors; immunotherapy using cytokines (e.g.IL-2 and / or IFN-α), immune check point inhibitors (such asfor example anti-CTLA-4 monoclonal antibodies such as ipilimumab or tremelimumab, toll-like receptor (TLR)agonists, CD40 agonists, anti-PD-1 antibodies such aspembrolizumab, pidilizumab or nivolumab, LAG-3 inhibitorssuch as relatlimab, and / or PD-L1 antibodies), and / or adoptivecell transfer (using for example pre-stimulated, modified Tcells or dendritic cells); and radiotherapy.Where the cancer is lung cancer, suitable therapies may forexample comprise one or more of: surgery, such as performinga lobectomy, a sublobar excision (wedge resection) or removalof a whole lung (pneumonectomy); radiotherapy, examples ofwhich include radiotherapy given together with chemotherapy, post-operative radiotherapy, brachytherapy (localized radiotherapy) given directly inside the airway, prophylactic cranial irradiation, stereotactic radiation and palliativeradiotherapy; chemotherapy using for example one more agentssuch as cisplatin / carboplatin, etoposide, gemcitabine,paclitaxel, docetaxel, vinorelbine, topotecan, irinotecan andpemetrexed; epidermal growth factor receptor (EGFR) inhibitors drugs such as erlotinib, gefitinib, afatinib, dacomitinib or osimertinib; and immunotherapy, using forexample anti PD-L1 monoclonal antibodies such asatezolizumab, nivolumab or pembrolizumab, monoclonalantibodies targeting cytotoxic T-lymphocyte-associatedprotein 4 (CTLA-4) such as ipilimumab, and / or monoclonalantibodies that targets vascular endothelial growth factorsuch as bevacizumab. Where the cancer is gastric cancer, suitable therapies may for example comprise one or more of: surgical procedures such as for example an endoscopic mucosal resection. endoscopic submucosal dissection or gastrectomy; chemotherapy usingagents such as fluorouracil, capecitabine, BCNU, methyl-CCNU, and doxorubicin, mitomycin C, cisplatin, Taxotere orcombinations thereof; targeted therapy using epidermal growthfactor receptor 2 inhibitors, such as trastuzumab; and radiotherapy. For example where the cancer is a pancreatic cancer, suitable therapies may for example comprise one or more of: surgical resection, such as for example tumor resection, the Whipple procedure, total pancreatectomy or distal pancreatectomy; radiotherapy, examples of which include but are not limited to radiotherapy given together with chemotherapy, post-operative radiotherapy, brachytherapy (localizedradiotherapy), stereotactic radiation and palliativeradiotherapy; chemotherapy using for example one more agentssuch as gemcitabine, 5-FU , erlotinib, FOLFIRINOX, nab-paclitaxel or combinations thereof; the somatostatin analogclass of medications; lanreotide; targeted therapy using everolimus or sunitinib; nuclear medicine therapy with radiolabeled peptides or hormones such as iobenguane; and techniques such as radiofrequency ablation (RFA), cryoablation, or hepatic artery embolization. Where the cancer is head and neck cancer, suitable therapiesmay for example comprise one or more of: surgery, includingbut not limited to laser surgery; radiation therapy, including but not limited to 3D conformal radiation therapy, intensity-modulated radiation therapy, particle beam therapy and brachytherapy; one or more chemotherapy agents such asfor example, paclitaxel, carboplatin, cetuximab, docetaxel,cisplatin and fluorouracil; photodynamic therapy utilizing amphinex; monoclonal antibodies such as cetuximab,bevacizumab, erlotinib, pembrolizumab or nivolumab; gene therapies such as gendicine; and immune checkpoint inhibitors. Where the cancer is colorectal cancer, suitable therapies may for example comprise one or more of: endoscopic mucosal resection or endoscopic submucosal dissection; a partial colectomy (or proctocolectomy for rectal lesions); chemotherapy agents such as for example capecitabine, fluorouracil, irinotecan, oxaliplatin or UFT; antiangiogenic drugs such as for example bevacizumab; epidermal growth factor receptor inhibitors, such as for example aflibercept, cetuximab and panitumumab; radiation therapy; immunecheckpoint inhibitors; and monoclonal antibodies such aspembrolizumab or dostarlimab.In a third aspect, the present invention provides amonoclonal antibody that specifically binds to the C-terminusamino acid sequence YNGQGYPGSG (SEQ ID NO: 1).The antibody according to the third aspect of the invention is, in particular, suitable for use in carrying out the methods of immunoassay according to the first aspect of the invention. Preferred embodiments and features of the antibody according to the third aspect will therefore be apparent from the above discussion of the preferred embodiments of the methods according to the first aspect. In a fourth aspect, the present invention provides an immunoassay kit comprising a monoclonal antibody in accordance with the third aspect of the present invention, and at least one of: -a streptavidin coated well plate- a biotinylated peptide Biotin-L-YNGQGYPGSG (SEQ ID NO:4), wherein L is an optional linker -a secondary antibody for use in a sandwich immunoassay- a calibrator protein comprising the C-terminus aminoacid sequence YNGQGYPGSG (SEQ ID NO: 1)- an antibody biotinylation kit- an antibody HRP labelling kit- an antibody radiolabelling kitThe immunoassay kit according to the fourth aspect of the invention is, in particular, suitable for use in carrying out the method of immunoassay according to the first aspect of the invention. Further preferred embodiments and features of the immunoassay kit according to the fourth aspect will therefore be apparent from the above discussion of the preferred embodiments of the methods according to the first aspect. Figures Figure 1: Specificity of the PRO-C12 assay. Inhibitioncurve for the standard peptide(IRGPPGPPGYCDSSQCASIPYNGQGYPGSG (SEQ ID NO: 5)), deselectionpeptide (PGLPGYPGSP (SEQ ID NO: 6)), elongated peptide(YNGQGYPGSGA (SEQ ID NO: 2)) and truncated peptide (YNGQGYPGS(SEQ ID NO: 3)). Peptides were diluted twofold. Figure 2: Quantification of PRO-C12 in serum frompatients with bladder (19), breast (19), colorectal (19), head and neck (20), kidney (16), lung (17), ovarian (20), pancreatic (17), prostate (19), stomach cancer (17) ormelanoma (20) and age-matched healthy controls (33). PRO-C12levels are shown as Tukey-style boxplots. For samples with measurements falling below the LLOQ were given the LLOQ value determined during assay validation. Differences in the PRO- C12 levels among groups were analyzed using Kruskal-Wallis test corrected for multiple comparisons using Dunn’s test. **** indicates a p-value below 0.0001. *** indicates a p- value below 0.001. ** indicates a p-value below 0.01. * indicates a p-value below 0.05. Figure 3: Western blot results of type XII collagen insupernatant from pancreatic CAFs (pCAF), pancreatic NFs (pNF), breast CAFs (bCAF), lung CAFs (lCAF) and lung NFs (lNF) treated and untreated with tgf-β1. A 333kDa fragment corresponding to the full size of type XII collagen was detected. Figure 4: COL12A1 gene expression obtained from the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. The normal groups included normal samples from GTEx and tumor-adjacent normal samples TCGA. Figure 5: Levels of C12 in cancer patients compared to healthy donors. Differences in the C12 levels among groups were analyzed using Kruskal-Wallis test corrected for multiple comparisons using Dunn’s test. **** indicates a p- value below 0.0001. *** indicates a p-value below 0.001. ** indicates a p-value below 0.01. * indicates a p-value below 0.05. ns indicates not significant. Figure 6: Correlation between PRO-C12 and C12M levels.Pearson’s correlation coefficient (r). Examples The presently disclosed embodiments are described in the following Examples, which are set forth to aid in the understanding of the disclosure, and should not be construed to limit in any way the scope of the disclosure as defined in the claims which follow thereafter. The following examples are put forth 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 scope of the present disclosure nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts,temperature, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. Materials and methods Monoclonal antibody development A monoclonal antibody (also referred to hereinafter as the “PRO-C12 antibody” or “PRO-C12 mAb”) targeting the PRO-C12 target sequence was generated in the following manner.A 10 amino acid peptide 3053YNGQGYPGSG3063 (SEQ ID NO: 1)consisting of the amino acid sequence found at the C-terminus of the alpha-1 chain of type XII collagen (UniProtKB: Q99715) was purchased from Genscript (Piscataway, NJ, USA) and used for immunization. This peptide was linked at its N-terminus to Keyhole Limpet Hemocyanin (KLH) carrier protein via cross- linking using sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, Thermo Scientific, Waltham,MA, USA, cat. No. 22322) to form an immunogenic peptide(“KLH”-CGG-YNGQGYPGSG (SEQ ID NO: 7)). Glycine and cysteineresidues were added at the N-terminal end of the sequence for proper binding of the peptide to the carrier protein. Monoclonal antibodies were produced by immunizing six-week- old Balb / C mice with subcutaneous injection of 200 uL emulsified antigen containing 100 ug of immunogenic peptide mixed with Sigma Adjuvant System (Sigma cat. No. S6322). Immunizations were administered at 2-week intervals until stable titers of sera were achieved. The mouse exhibiting the highest titer was given a four-week rest period before receiving a booster dose of 100 ug immunogenic peptide in 100 uL 0.9% NaCl solution intravenously. Hybridoma cells were generated by fusing spleen cells with SP2 / 0 myeloma cells and the hybridoma cells were then cultured in 96-well microtiter plates. Limited dilution was employed to ensure the monoclonal growth. The selection of the antibody clone targeting the epitope of interest was chosen based on a preliminary competitive ELISA assessing reactivity towardsthe selection peptide (YNGQGYPGSG (SEQ ID NO: 1)), atruncated peptide (YNGQGYPGS (SEQ ID NO: 3)), an elongatedpeptide (YNGQGYPGSGA (SEQ ID NO: 2)), and a deselectionpeptide (PGLPGYPGSP (SEQ ID NO: 6)) corresponding to asequence from the type IV collagen alpha 4 chain. Subsequently, the monoclonal antibodies were purified using protein-G-columns following the manufacturer’s guidelines (GE Healthcare Life Sciences, Little Chalfont, UK, cat. No. 17- 0404-01). To complete the process, the purified antibodies were labelled with horseradish peroxidase (HRP) using a peroxidase labelling kit (Roche Diagnostics GmbH, Mannheim, Germany, cat. no. 11829696001). PRO-C12 ELISA protocol A competitive ELISA targeting PRO-C12 (also referred to hereinafter as the “PRO-C12 assay” or “PRO-C12 ELISA”) was developed, using the PRO-C12 antibody described above. The ELISA underwent multiple optimizations including the selection of assay buffer, incubation duration and temperature and antibody and peptide concentration. The final PRO-C12 ELISA protocol consisted of the following steps.A 96-well streptavidin-coated ELISA plate was coated with 100uL / well of 5 ng / mL biotinylated peptide (biotin-YNGQGYPGSG(SEQ ID NO: 8)) in assay buffer (50 mM PBS, 1% BSA (w / v),0.018% bronidox (v / v), 0.1% Tween-20 (w / v), 4 g / L NaCl, pH 7.4) and incubated for 30 minutes at 20 ◦C with shaking at 300 RPM. After 5 washes with washing buffer (25 mM Tris, 50mM NaCl, pH 7.2), 20 uL / well of the sample diluted 1:2 inassay buffer was added in duplicates, followed by 100 uL / well of 25 ng / mL of the HRP-labelled monoclonal antibody (PRO-C12 mAb) in assay buffer. The mixture was incubated for 20 h at 4 ◦C with shaking at 300 RPM. Following a second washing cycle, 100 uL / well of 3,30,5,50-tetramethylbenzidine (TMB) was added and incubated for 15 minutes in darkness at 20 ◦C with shaking at 300 RPM. The reaction was quenched by adding 100 uL / well of 1% H2SO4(v / v). Absorbance was measured at 450 nm with 650 nm as the reference. For the generation of the standard curve, 20 uL / well of 100 ng / mL of 30aa standardpeptide (IRGPPGPPGYCDSSQCASIPYNGQGYPGSG (SEQ ID NO: 5)),serially diluted twofold, was added to the appropriate wells, and a four-parametric logistic regression model (4PL) was used for curve fitting. Each plate included five quality control samples, comprising two human serum, one sheep serum, one pig serum and one human serum spiked with standard peptide. Technical validation of the PRO-C12 ELISA Antibody specificity evaluation involved testing of the 30aastandard peptide (IRGPPGPPGYCDSSQCASIPYNGQGYPGSG (SEQ ID NO:5)) assessed in twofold dilution series. Additionally,peptides consisting of an elongated version (YNGQGYPGSGA (SEQID NO: 2)) and a truncated version(YNGQGYPGS (SEQ ID NO: 3))of the PRO-C12 target sequence were tested as well as adeselection peptide (PGLPGYPGSP (SEQ ID NO: 6)). To assesslinearity of dilution, twofold dilution of human serum samples were measured, and the percentage recovery of the measured concentration relative to the predicted concentration was calculated. Accuracy was evaluated by spiking a known quantity of the standard peptide into human serum samples or by spiking one human serum sample into another at different ratios (100:0, 75:25, 50:50, 25:75, or 0:100), followed by calculating the percentage recovery of the spiked sample relative to the non-spiked sample. The impact of interfering substances commonly found in serum including hemoglobin, lipids and biotin was assessed by spiking human serum samples with known quantities of these substances (hemoglobin low = 2.5 mg / mL, high = 5 mg / mL; lipids low = 1.5 mg / mL, high = 5 mg / mL; and biotin low = 5 ng / mL, high = 100 ng / mL), and the recovery relative to the non-spiked sample was calculated. Assay variation was tested through ten independent runs using ten quality control samples in double determinations. Intra-assay variation was calculated as the mean coefficient of variance (CV%) between double determinations in each run (CV% <10), while inter- assay variation was calculated as the mean CV% across all ten runs (CV% <15). The lower (LLMR) and upper (ULMR) limits of the measurement range were determined across the ten independent runs, representing the boundaries of the linear range observed in the standard curve. The lower limit of detection (LLOB) was calculated as the mean interpolated concentration of 60 blank samples in single determinations containing only assay buffer plus three standard deviations while the upper limit of detection (ULOQ) was calculated as the mean interpolated concentration of the standard peptide corresponding to the highest concentration of the standard curve minus three standard deviations. The lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) were determined based on the minimum concentrations at which the average coefficient of variation (CV%) for serum samples remained below 20%. Analyte stability was evaluated for three human serum samples incubated at either 4, 18 and 36 C after 4 hours, 24 hours and 48 hours of storage and the percentage recovery of the incubated samples relative to the corresponding control sample kept at -20 C was calculated. Freeze-thaw stability was assessed by repeatedly freezing and thawing human serum samples for up to five rounds, and the percentage recovery of the samples relative to the corresponding control samples subjected to a single freeze- thaw round was calculated.Fibroblasts cell culture – Scar-In-A-JarThe Scar-In-A-Jar (SiaJ) methodology used in this study was described previously

[0050] ,

[0051] . Native human quiescent normal fibroblasts (NFs) and cancer associated fibroblasts (CAFs) from pancreas, breast and lung were purchased from Vitro biopharma (cat# SC00A5, cat# CAF06, cat# CAF07-AD, cat# CAF08, cat# CAF05 Golden, CO, USA), and Lonza (cat# CC-2512, Morrisville, NC, USA). Fibroblasts were cultured in flasks coated with 5 µg / cm2type I collagen purified from rat tail tendon (cat# P8188, Innoprot, Derio, Biscay, Spain). When the confluency reached 80%, 30,000 fibroblatsts per well were seeded in 48-well plates, and the medium was replaced with Gibco DMEM + GlutaMAX (cat# 31966047, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (cat# F7524, Sigma Aldrich, St. Louis, MO, USA) and 1% penicillin / streptomycin (P / S) (cat# P4333, Sigma Aldrich, USA). After 24 hours, the culture medium was substituted with ficoll medium containing 50% Gibco DMEM + GlutaMAX supplemented with 0.4% FBS, 1% P / S, 50% 70 and 400 kDa Ficoll (cat#17031050 and cat#17030050, Cytvia, Marlborough, MA, USA) and 0.05 mg / mL of L-ascorbic acid (cat#013-12061, Fujifilm, Tokyo, Japan). Fibroblasts were either treated with 1 ng / ml tgf-β1 (cat# 7754-BH / CF, Bio- techne, Minneapolis, MN, USA) or not treated. Cell supernatant was collected and preserved at -20 C for analysis on days 3,6,9 and 12. After removal of cell supernatant, new ficoll medium with or without tgf-β1 was added to the respective wells. Western blot of SiaJ supernatant SiaJ Supernatant on day 9 was electro-phoresed on a NuPAGE 4– 12% Bis–Tris gel (Invitrogen, Carlsbad, CA, US) under reducing conditions usingNuPAGE® MES SDS running buffer (Invitrogen, Carlsbad, CA, US). The proteins from the polyacrylamide gel were then transferred onto an iBlot® nitrocellulose membrane (Life Technologies, Bengaluru, India) using the iBlot® Dry blotting system (Life Technologies, Carlsbad, CA, US). Following this, the membrane was blocked for 1 hour with 5% skim milk (Sigma–Aldrich, St. Louis, MO, USA) in TBST (Tris-Buffered Saline (TBS) with 0.1% Tween-20). The membrane was incubated overnight at 4 °C with type XIIcollagen monoclonal antibody (PRO-C12 mAb but not labelledwith horseradish peroxidase) and GAPDH loading Control monoclonal antibody (GA1R) (Cat # MA5-15738, Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, the membrane was washed in TBST three times for 10 minutes and then incubated with the secondary peroxidase-conjugated antibody (1:5000, Jackson Immunoresearch, West Grove, PA, US) for 1 hour. After another wash in TBST, the membrane was incubated for 3 minutes with Clarity Max Western ECL substrate (Cat # 1705062, Bio-Rad Laboratories Inc, Waltham, MA, USA). Thebands were visualized using the C-DiGit™ Blot Scanner (LI-CORBiosciences, Lincoln, NE, USA). ImageLab software version 6.1 (Bio-Rad) was employed for image acquisition. Patient samples The cohort consisted of serum samples obtained from healthy controls and patients diagnosed with cancer. This cohort wascategorized into 12 groups, each group consisting of patientswith a specific cancer type or healthy controls, said groupsbeing as follows: bladder cancer patients (19 individuals),breast cancer patients (19 individuals), colorectal cancerpatients (19 individuals), head and neck cancer patients (20individuals), kidney cancer patients (16 individuals), lungcancer patients (17 individuals), ovarian cancer patients (20individuals), pancreatic cancer patients (17 individuals),prostate cancer patients (19 individuals), stomach cancerpatients (17 individuals), melanoma patients (20individuals), and age-matched healthy controls (33 individuals). The serum samples from cancer patients and healthy controls were sourced from Proteogenex (Los Angeles, CA, USA) and BioIVT (Westbury, NY, USA), respectively, and were stored at -80 ◦C before analysis. According to the vendors, the sample collection process received approval from an Institutional Review Board or Independent Ethical Committee, and patients provided informed consent at the Russian Oncological Research Centre n.a. Blokhin RAMS (PG-ONC 2003 / 1) and Western Institutional Review Board, Inc. (WIRB®Protocol #20161665). All investigations adhered to theprinciples outlined in the Helsinki Declaration. Analysis of Publicly Available Genomics Databases The TCGA (https: / / www.cancer.gov / tcga) and GTEx (https: / / www.gtexportal.org / ) datasets were accessed through the UCSC Xena browser (http: / / xena.ucsc.edu / ). Within the Xena browser, the inventors focused on the TCGA TARGET GTEx combined cohort, specifically filtering down to TCGA and GTEx patients. For COL12A1 gene expression analysis, the inventors used RSEM expected count (DESeq2 normalized) UCSC Toil RNA- seq Recomputed data. The processing and normalization details of this data have been previously described

[0052] –

[0055] . Two comparison groups were created by combining the GTEx normal data with TCGA normal data, facilitating comparison with the TCGA primary tumor data. Statistics Differences in the PRO-C12 levels among groups were analyzed using Kruskal-Wallis test corrected for multiple comparisons using Dunn’s test. Differences in COL12A1 gene expressionbetween healthy and cancer tissue were analyzed using unpaired t-test. Diagnostic accuracy was tested by the AUROC curve. Sensitivity and specificity were determined at the estimated optimal cut-off value according to the Youden index. A p value below 0.05 was considered significant. Asterisks indicate the following significance levels: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. When doing multiple comparisons tests, multiplicity adjusted p-values are reported. Statistical analysis and graphs were done in GraphPad Prism (version 10.1.1 for Windows, GraphPad Software, San Diego, CA, USA, www.graphpad.com), MedCalc (MedCalc Statistical Software version 22.003 (MedCalc Software Ltd, Ostend, Belgium; https: / / www.medcalc.org; 2023)) and R version 4.3.1 (R Core Team (2023), R Foundation for Statistical Computing, Vienna, Austria, https: / / www.R- project.org.Comparison of PRO-C12 to another type XII collagen biomarkertarget To gain insight into the significance of the PRO-C12biomarker target, the inventors also compared the biologicalrelevance and correlation between measuring the serums levels of PRO-C12 using the PRO-C12 assay (that as noted abovetargets the sequence aa3053'YNGQGYPGSG'aa3063 (SEQ ID NO: 1) atthe C-terminus of the alpha-1 chain of type XII collagen) versus measuring the serum levels of another type XIIcollagen fragment, namely aa2887'GLKGEKGDRGDIASQNM'aa2903 (SEQ IDNO: 9), using another assay (referred to herein as the “C12”) targeting said other fragment. Accordingly, the levels of C12 were measured in the same serum cancer samples in which PRO-C12 had been measured previously (as described above). Results PRO-C12 ELISA development and validation The process conditions for the finalised PRO-C12 ELISA protocol were selected based on their ability to achieve optimal sensitivity in human serum samples while adhering to specified technical criteria and included determining the optimal incubation time and temperature, selecting the most suitable assay buffer, and adjusting concentrations of kit components. Technical validation testing of the finalised PRO-C12 ELISA produced the following results. The specificity of the PRO-C12 ELISA was evaluated based on the ability of peptides to compete for binding to the monoclonal antibody. The tested peptides included the 30aastandard peptide (IRGPPGPPGYCDSSQCASIPYNGQGYPGSG (SEQ ID NO:5)), an elongated peptide (YNGQGYPGSGA (SEQ ID NO: 2)), atruncated peptide (YNGQGYPGS (SEQ ID NO: 3)) and adeselection peptide (PGLPGYPGSP (SEQ ID NO: 6)). Notably,only the standard peptide exhibited a dose-dependentinhibition of the signal (Figure 1). Overall, these resultsdemonstrate the assay’s specificity for the PRO-C12 targetsequence (YNGQGYPGSG (SEQ ID NO: 1)) at the C-terminus oftype XII collagen. Linearity of dilution and parallelism to the standard curve was confirmed when serum samples were diluted at a ratio of 1:2, with an average recovery of 91 %. Accuracy testing through spiking recovery tests demonstrated a good recovery of the standard peptide in human serum, achieving an average recovery of 90 %. Similarly, matrix-in-matrix spiking which involves the addition of one human serum sample into another human serum sample, yielded an average recovery rate of 97 %. Even at high concentrations of commonly interfering substances in the serum such as hemoglobin, lipids and biotin, recovery rates remained within the 15 % range. Assayvariation, both inter- and intra-assay was 12 and 5 %respectively. Analyte stability, conducted over a span of up to 48 h at either 4, 20 or 36 °C showed recoveries within the 15 % range. Furthermore, stability after five freeze-thaw cycles demonstrated an average recovery rate of 98 %. Themeasurement range was 1.9 ng / mL- 100 ng / mL, with a lowerlimit of detection of 0.55 ng / mL. PRO-C12 in serum of patients with solid cancers To investigate the use of the PRO-C12 assay in a cancer context, PRO-C12 levels (i.e. the levels of PRO-C12 detected by the PRO-C12 assay) were measured in serum samples from a cohort of patients with various cancer types, namely bladder (19), breast (19), colorectal (19), head and neck (20), kidney (16), lung (17), ovarian (20), pancreatic (17), prostate (19), stomach cancer (17) or melanoma (20), alongside healthy controls (33). PRO-C12 levels were significantly elevated in patients with prostate (p < 0.0001), breast (p < 0.0001), melanoma (p < 0.0001), lung (p = 0.0001), gastric (p = 0.0004), pancreatic (p = 0.0019), head and neck (p = 0.0036) and colorectal cancer (p = 0.0048) compared to healthy controls (Figure 2), whereas patients with ovarian, kidney and bladder showed similar levels of PRO-C12 compared to healthy controls. Regarding diagnostic accuracy, PRO-C12 was especially good at discriminating healthy controls from cancer patients, in particular patients with breast (AUROC = 0.95 ), prostate (AUROC = 0.92), lung (AUROC = 0.92), gastric (AUROC = 0.88), melanoma (AUROC = 0.83), pancreatic (AUROC = 0.80) and head and neck cancer (AUROC = 0.76) (table X). In summary, these findings indicate elevated levels of PRO- C12 and type XII collagen in serum from patients with different cancer types. Type XII collagen is expressed by CAFs and NFs Western blot analysis on the supernatant obtained from different types of CAFs and NFs treated and untreated with tgf-β1 to induce fibrosis revealed the presence of type XII collagen. The observed band, appearing at approximately333kDa, corresponds to the full-size protein. Althoughdiscerning differences can be challenging, our analysis suggests a potentially elevated expression of type XII collagen when fibroblasts are stimulated with tgf-β1, with a notable emphasis on lung derived CAFs and NFs (Figure 3). COL12A1 gene expression in TCGA and GTEx databases To validate the relevance of type XII collagen in cancer, we examined the expression levels of COL12A1 normal and tumortissue, in the specific cancer types were we previouslyobserved increased PRO-C12 levels, using publicly accessibledata from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) initiatives

[0056] ,

[0057] . The normal data set included 2833 normal samples and 4121 tumor samples from the GTEx dataset and tumor-adjacent normal tissue from the TCGA dataset (table 1). COL12A1 expression was significantly increased in patients with breast (p < 0.0001), colorectal (p = <0.0001), head and heck (p = <0.0001), lung (p = <0.0001), pancreas (p = <0.0001), prostate (p = <0.0001) and gastric (p = <0.0001) cancer (Figure 4). While the difference did not reach statistical significance, the expression of COL12A1 was also higher in patients with melanoma compared to healthy tissue (p > 0.05). Taking together, these findings are in agreement with our PRO-C12 measurements in circulation.Table 1. Summary of the samples obtained from the CancerGenome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Comparison of PRO-C12 to another type XII collagen biomarkertarget To gain insight into the significance of the PRO-C12biomarker target, the inventors also compared the biologicalrelevance and correlation between measuring the serums levels of PRO-C12 using the PRO-C12 assay (that as noted abovetargets the sequence aa3053'YNGQGYPGSG'aa3063 (SEQ ID NO: 1) atthe C-terminus of the alpha-1 chain of type XII collagen) versus measuring the serum levels of another type XIIcollagen fragment, namely aa2887'GLKGEKGDRGDIASQNM'aa2903 (SEQ IDNO: 9), using another assay (referred to herein as the “C12”) targeting said other fragement. Accordingly, the levels of C12 were measured in the same serum cancer samples in which PRO-C12 had been measured previously (as described above), and the results are shown in figure 5. Whereas, as described above, increased levels of PRO-C12 were found to be present in several cancer types including breast, CRC, head and neck, lung, melanoma, pancreatic, prostate and gastric cancer(Figure 2), no difference was observed compared to healthyindividuals when measuring C12 in any of the cancerindications (Figure 5). Furthermore, no correlation was foundbetween PRO-C12 and C12 levels (Figure 6). Discussion In this study, the inventors developed and validated an ELISAassay (“PRO-C12 assay”) targeting the C-terminus of type XIIcollagen that proved to be sensitive, specific and accurate in serum samples. PRO-C12 was elevated in serum from patients diagnosed with different cancer types including prostate, breast, melanoma, lung, gastric, pancreatic, head and neck and CRC. To corroborate our results obtained measuring PRO-C12, the inventors employed COL12A1 gene expression data from the TCGA and GTEx databases and confirmed the increased levels of type XII collagen expression in those specific groups of patients. Further, the PRO-C12 assay demonstrated diagnostic accuracy by discriminating between healthy donors and patients with several cancer types, particularly prostate cancer. In humans, COL12A1 is expressed during the developmental stage in mesenchymal tissues whereas in adults its presence is limited to basement membrane (BS) and fascia of muscle, kidney and dermis

[0058] . In a COL12A1 knockout mouse model osteoblasts displayed abnormal polarization and poor mineralization emphasizing the role of type XII collagen in cell-cell interaction during osteogenesis

[0030] ,

[0031] . Interestingly, prostate cancer frequently metastasizes to bone and, although the mechanisms driving metastasis to theosteoblastic niche remain unclear, the ECM possibly plays asignificant role in facilitating this process

[0059] . Tenascin-C, an ECM protein expressed in the nervous system during bone development and essential for wound healing in adults has been proposed as key hallmark of reactive stroma response in prostate cancer

[0060] –

[0065] . Studies have shown that overexpression of tenascin-C by myofibroblasts and CAFs could play an essential role prostate cancer dissemination to the bone by promoting EMT induction as well as colony formation mediated by integrin α9β1

[0066] –

[0068] . Moreover, in a 3D model using osteogenic organoids, prostate cancer cells increased proliferation and colony formation in tenascin-C rich regions mimicking the bone microenvironment. Further, α9 integrin knockout cells resulted in less adhesion and lower type XII collagen expression, implying an association an association between cell binding and type XII collagen production. One could speculate the stimulation of osteoblast differentiation and the deposition of osteoid at metastatic sites might be prompted by the tenascin-C-induced production of type XII collagen in metastatic cells and high PRO-C12 levels could be result from this stimulation. In this context, PRO-C12 could be used as a tool to monitor the efficacy of anticancer treatments that disrupt the α9 integrin-tenascin-C interaction and improve the prognosis of patients with metastatic prostate cancer

[0068] . Type XII collagen has been proposed as potential prognosis biomarker for predicting clinical outcomes and as anti-cancer treatment target in other tumor types. For instance, a study reported that in patients with gastric cancer type XII collagen expression was increased both at the mRNA and protein levels and that in was mainly expressed by the CAFs in surrounding the tumor

[0069] . Furthermore, overexpression of type XII collagen correlated with advanced TNM stage, metastasis and worse clinical outcomes

[0069] . Even though the molecular mechanisms underlying type XII in gastric cancer have not been fully elucidated, some studies suggest that IDO1 and type XII collagen together could induce gastric metastasis

[0037] . IDO1 is an enzyme essential for catabolism of tryptophan along the kynurenine pathway

[0070] . Elevated levels of IDO1 may lead to a depletion of tryptophan levels withing the TME

[0071] –

[0073] . This, in turn, directly hampers the proliferation and function of immune cells. Consequently, an increased IDO1 level may be implicated as a factor contributing to immune evasion in cancer

[0074] . By knocking down both IDO1 and COL12A1 in SGC- 7901 cells higher ERK phosphorylation inhibition than by knocking them individually was observed, leading decreased cell migration. MAPK pathway inhibition was reversed by integrin β1 overexpression, implying that both IDO1 and type XII collagen-integrin β1 activate the MAPK pathway. Overall, they propose a mechanism where type XII collagen could facilitate interactions between collagen fibrils and integrin β1, leading to ERK phosphorylation in gastric cancer cells and tumor dissemination. In this context, IDO1 and type XII collagen might be promising targets to treat gastric cancer, and PRO-C12 could help to assess which patients respond to treatment

[0037] . Remarkably, the present inventors’ study revealed elevated levels of PRO-C12 in patients diagnosed with PDAC. This observation aligns with previous research and could be indicative of CAF activation. CAFs are known contributors to the tumor microenvironment and have been specifically identified as expressing type XII collagen. In the present study, the inventors detected the presence of type XII collagen in the supernatant of both CAFs and NFs. Notably, upon treatment of fibroblasts with TGF-b1 which induces tumor fibrosis and collagen synthesis, the inventors observed a discernible trend towards and increased production of type XII collagen. This suggests a potential regulatory role of TGF-b1 in the synthesis of type XII collagen by fibroblasts providing insight into the dynamic nature of extracellular matrix remodeling in response to TGF-β1 stimulation. In earlier investigations involving patients with PDAC, higher expression of type XII collagen was detected stages III and IV compared to stages I and II and correlated with poor prognosis. When analyzing genetic alterations in PDAC tissues they found that samples containing P53 and KRAS mutations had higher type XII collagen expression than the wild-type tissue, and qPCR proved that type XII collagen mainly derived from CAFs and not by tumor cells. Moreover, type XII collagen expression exhibited a positive correlation with genes linked to fibroblast activation such as FAP, vimentin, α-SMA and ACTA2. Additionally, pathway correlation analyses revealed a significant association between type XII collagen expression and processes such as collagen formation, ECM-related genes, the TGF-β pathway, and the inflammation signature and interestingly, knockdown of COL12A1 resulted in inhibition of CAF invasion and reduction of CAF associated biomarker expression. This interconnected relationship underscores the potential role of type XII collagen in influencing various molecular pathways associated with the intricate dynamics of the ECM and inflammatory responses

[0075] . Moreover, type XII collagen is expressed not only by CAFs but also by CRC cells in the desmoplastic front, and one could speculate that beyond its potential role as CAF biomarker, may also an indicator for cancer cells in the invasive front

[0035] . In studies in breast cancer, it was confirmed that CAFs deposit type XII collagen in the TME. Additionally, they observed that the heightened stiffness attributed to increases type XII collagen deposition, induced a change of phenotype of normal fibroblasts into myofibroblasts, thus facilitating the formation of a pro-invasive TME that facilitate tumor invasion

[0043] . As type XII collagen deposition increases as the tumor develops PRO-C12 may serve as a biomarker of early dissemination, and it could help to identify those patients with breast cancer that are at high risk of tumor metastasis. Type XII collagen belongs to the FACIT family and plays a role the fibril assembly of fibrillar collagens like type I and III. Type XII collagen contains a domain that binds to fibrillar collagens and contributes to the interaction and organization of fibril structures, providing additional stabilization. Recent studies have highlighted how desmoplastic lesions are characterized by chaotic collagenous deposition surrounding the tumor and that this altered ECM organization differs from normal structure in healthy tissue. One could speculate that CAFs recruited by cancer cells would attempt the reorganization of the ECM surrounding the tumor through various mechanisms, potentially involving production of type XII collagen that would be reflected by high levels of PRO-C12 in circulation. The PRO-C12 ELISA was designed to target the C-terminus of type XII collagen, but the cleavage process releasing the protein fragment into the bloodstream remains unclear. The inventors hypothesize that the complete type XII collagen protein would remain in the ECM organizing fibrillar collagens and that the fragments found in circulation might have lost their anchoring properties and are result from excessive ECM remodeling occurring in the TME. Increased levels of PRO-C12 in patients with cancer could reflect the excessive production of type XII collagen by CAFs and tumor cells in the invasive front as discussed previously. Nevertheless, the intricacies of this process remain poorly described. In this specification, unless expressly otherwise indicated, the word ‘or’ is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator ‘exclusive or’ which requires that only one of the conditions is met. The word ‘comprising’ is used to mean ‘including or consisting of’. All prior teachings acknowledged above are hereby incorporated by reference. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof. References[1] World Health Organization, “Cancer,”https: / / www.who.int / en / news-room / fact-sheets / detail / cancer.[2] J. J. F. Sleeboom, G. S. van Tienderen, K. Schenke-Layland, L. J. W. van der Laan, A. A. Khalil, and M. M. A. Verstegen, “The extracellular matrix as hallmark of cancer and metastasis: From biomechanics to therapeutic targets,” Sci. Transl. Med., vol. 16, no. 728, p. 3840, 2024, doi: 10.1126 / scitranslmed.adg3840.[3] R. G. Rowe and S. J. Weiss, “Navigating ECM barriers atthe invasive front: The cancer cell-stroma interface,” Annu. Rev. Cell Dev. Biol., vol. 25, pp. 567–595, 2009, doi: 10.1146 / annurev.cellbio.24.110707.175315.[4] T. Zhang, Y. Jia, Y. Yu, B. Zhang, F. Xu, and H. Guo,“Targeting the tumor biophysical microenvironment to reduce resistance to immunotherapy,” Adv. Drug Deliv. Rev., vol. 186, p. 114319, Jul. 2022, doi: 10.1016 / j.addr.2022.114319.[5] D. Hanahan and R. A. Weinberg, “The Hallmarks ofCancer,” Cell, vol. 100, no. 1, pp. 57–70, Jan. 2000, doi: 10.1016 / S0092-8674(00)81683-9.[6] D. Hanahan and R. A. Weinberg, “Hallmarks of cancer: Thenext generation,” Cell, vol. 144, no. 5, pp. 646–674, 2011, doi: 10.1016 / j.cell.2011.02.013.[7] Biochemistry of Collagens, Laminins and Elastin, 3rdEditio. 2023. [Online]. Available: https: / / shop.elsevier.com / books / biochemistry-of-collagens- laminins-and-elastin / karsdal / 978-0-443-15617-5[8] C. Frantz, K. M. Stewart, and V. M. Weaver, “Theextracellular matrix at a glance,” J. Cell Sci., vol. 123, no. 24, pp. 4195–4200, Dec. 2010, doi: 10.1242 / jcs.023820.[9] N. Willumsen et al., “Fibrotic activity quantified inserum by measurements of type III collagen pro-peptides can be used for prognosis across different solid tumor types,” Cell. Mol. Life Sci., vol. 79, no. 4, p. 204, Apr. 2022, doi: 10.1007 / s00018-022-04226-0.

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Claims

Claims1. A method of immunoassay, said method comprising;i) contacting a patient sample with a monoclonalantibody that specifically binds to the C-terminusamino acid sequence YNGQGYPGSG (SEQ ID NO: 1); andii) detecting and determining the amount of bindingbetween said monoclonal antibody and peptides in the sample.

2. A method as claimed in claim 1, wherein the method is amethod of immunoassay for detecting and / or monitoring acancer or a level of severity thereof in a patient, the method further comprising;iii) correlating said amount of binding with valuesassociated with normal healthy subjects and / or values associated with known disease severity and / or values obtained from said patient at a previous time point and / or with a predetermined cut-off value.

3. A method as claimed in claim 2, wherein the cancer isprostate cancer, breast cancer, melanoma, lung cancer, gastric cancer, pancreatic cancer, head and neck cancer or colorectal cancer.

4. A method as claimed in any preceding claim, wherein thepatient sample is selected from blood, plasma or serum.

5. A method as claimed in any preceding claim, wherein themonoclonal antibody does not specifically bind to a peptide having the C-terminus amino acid sequence YNGQGYPGSGA (SEQ ID NO: 2).

6. A method as claimed in any preceding claim, wherein themonoclonal antibody does not specifically bind to a peptide having the C-terminus amino acid sequence YNGQGYPGS (SEQ ID NO: 3).

7. A method as claimed in any preceding claim, wherein themonoclonal antibody is raised against a synthetic peptide having the C-terminus amino acid sequence YNGQGYPGSG (SEQ ID NO: 1).

8. A method as claimed in any preceding claim, wherein theimmunoassay is a competition assay or a sandwich assay.

9. A method as claimed in any preceding claim, wherein theimmunoassay is a radio-immunoassay or an enzyme-linked immunosorbent assay.

10. A monoclonal antibody that specifically binds to theC-terminus amino acid sequence YNGQGYPGSG (SEQ ID NO:1).

11. A monoclonal antibody as claimed in claim 10, whereinthe monoclonal antibody does not specifically bind to a peptide having the C-terminus amino acid sequence YNGQGYPGSGA (SEQ ID NO: 2).

12. A monoclonal antibody as claimed in claim 10 or 11,wherein the monoclonal antibody does not specifically bind to a peptide having the C-terminus amino acid sequence YNGQGYPGS (SEQ ID NO: 3).

13. A monoclonal antibody as claimed in any one of claims 10to 12, wherein the monoclonal antibody is raised againsta synthetic peptide having the C-terminus amino acid sequence YNGQGYPGSG (SEQ ID NO: 1).

14. An immunoassay kit comprising a monoclonal antibody asclaimed in any one of claims 10 to 13, and at least oneof: -a streptavidin coated well plate- a biotinylated peptide Biotin-L-YNGQGYPGSG (SEQ IDNO: 4), wherein L is an optional linker -a secondary antibody for use in a sandwichimmunoassay -a calibrator protein comprising the C-terminusamino acid sequence YNGQGYPGSG (SEQ ID NO: 1)- an antibody biotinylation kit- an antibody HRP labelling kit- an antibody radiolabelling kit

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