Use of BMMF1 REP protein as a diagnostic marker for pancreatic cancer and type 2 diabetes

Abstract

The present invention relates to the use of BMMF Rep protein as a diagnostic marker for pancreatic cancer and diabetes.

Description

Technical Field

[0001] The present invention relates to the use of DNA replication-related (REP) proteins as diagnostic markers for pancreatic cancer and type 2 diabetes.

Background Art

[0002] The pancreas functions as an exocrine gland that secretes pancreatic juice transported to the digestive tract through the pancreatic duct, and at the same time functions as an endocrine gland that secretes hormones such as insulin and glucagon into the blood. Therefore, pancreatic diseases are often the cause of diabetes or are closely related to diabetes.

[0003] Since the pancreas is surrounded by many organs such as the stomach, duodenum, small intestine, liver, and gallbladder, pancreatic cancer is not only difficult to detect early, but also has properties such as lack of symptoms, very rapid progression, and metastasis to other organs, and has a very poor prognosis compared to other cancers. Despite some new cancer treatment options, such as immunotherapy approaches using checkpoint inhibitors or bispecific antibodies, the 5-year relative survival rate by cancer type remains the lowest among pancreatic cancers, between 6% and 10%.

[0004] The basic treatment for pancreatic cancer is performed by surgery, systemic chemotherapy, radiotherapy, or a combination of these, depending on the degree of progression. 15 - 20% of pancreatic cancer patients undergo surgery for potential complete cure, but the majority of patients do not benefit from surgery due to local progression or metastasis.

[0005] The UICC (Union Internationalis Contra Cancrum) stage of pancreatic cancer progression is classified into stages 0, IA, IB, IIA, IIB, III, IVa, and IVb. Stages I - III account for more than half of the 5-year survival numbers, and stages IVa and IVb account for more than 70% of the progression stage at diagnosis. Therefore, early detection and treatment of pancreatic cancer are necessary.

[0006] Abdominal ultrasound examination is a very useful, convenient, and minimally invasive examination method for patient care or health diagnosis. However, it is often difficult to visualize pancreatic cancer with a small tumor size or lesions in the tail of the pancreas. In health examinations, the abnormal detection rate in pancreatic images by abdominal ultrasound examination is about 1%, and the detection rate of pancreatic cancer is about 0.05% or less.

[0007] On the other hand, cystic diseases occurring in the pancreas are known to progress to invasive cancer through malignant transformation and can be regarded as pancreatic cancer precursor lesions. The malignancy of cystic diseases is evaluated based on cyst diameter, wall thickening, main pancreatic duct diameter, intramural nodules, main pancreatic duct stenosis, lymph node enlargement, cystic lesions, etc. Patients with mucinous cystic neoplasm (also called mucinous cystadenoma), which is a type of benign cystic disease, have a high possibility of developing malignant cancer. Therefore, even if no malignant tumor of the neoplasm is present when they are detected, it is recommended that they undergo regular follow-up or tumor resection.

Summary of the Invention

Problems to be Solved by the Invention

[0008] In order to improve early detection, biomarkers that promote the early detection of pancreatic cancer and further insight into its etiology are needed.

Means for Solving the Problems

[0009] The inventors have found that the intake of BMMF (bovine meat and milk factor) agents within the first few months of life, either by replacing breastfeeding during weaning with dairy products or by the intake of dairy or beef products, generally leads to early infection by neonatal BMMF antigens. It is hypothesized that initial exposure induces an immunological state in which BMMF ingested through nutrition in later life is not removed or neutralized as efficiently as in individuals who benefit from longer breastfeeding periods. Within the next few or several decades, depending on the host immune system, additional BMMF antigens accumulate within the stroma of pancreatic tissue. This accumulation can also be induced by the uptake of specific molecules that represent receptors for BMMF. These molecules are also taken up by the consumption of bovine products and incorporated into lectin receptors on the surface of host cells. The combination of continuous intake of BMMF and local spread of infection leads to a stable increase in diffusible reactive oxygen species (ROS) and cyclooxygenase-2 (Cox-2) when a certain level of antigen is reached, which induces chronic and local inflammatory conditions. This dramatically increases the probability of uncontrolled cell proliferation, accompanied by the fixation of random mutations in surrounding cells induced by ROS (de Villiers and zur Hausen, 2021). In particular, cells with inherently high replication activity can represent targets rich in random DNA mutations that allow for the stochastic occurrence of mutations as a fundamental requirement for tumor formation and the development of pancreatic cancer. Thus, BMMF represents a specific and local trigger for the induction of chronic inflammation within the tissue stroma, which leads to an increase in ROS, induces proliferation and mutations in surrounding replicating cells, and ultimately results in the formation of hyperplasia as a precursor to cancer.

[0010] Specifically, the structural properties of polymorphic vesicles that were regularly identified by staining prostate cancer patient tissues for the expression of Rep proteins in a subgroup of BMMF1 were observed. The latter represents a protein involved in the replication of the small single-stranded circular plasmid of BMMF and is most likely to contribute to the polymorphic vesicle structure found at the periphery of pancreatic cancer. After staining with a gold-labeled monoclonal antibody against BMMF1 Rep, gold-labeled structurally dense regions are shown in preselected regions of pancreatic cancer. Similar structures were observed in human embryonic cells (HEK293TT) overexpressing Rep. These data suggest that Rep or Rep isoforms contribute to vesicle morphogenesis. Furthermore, colocalization of the anti-Rep staining signal with CD68-positive macrophages was observed. The regions where the Rep-specific antibody is most detected correlate with the regions where the detection level of CD68-positive cells, which indicate the localization of the Rep-specific antigen in the inflammatory tissue region, is the highest, i.e., the regions where the levels of inflammatory monocytes, circulating macrophages, or resident tissue macrophages are particularly high.

[0011] Bovine serum was first subjected to density gradient centrifugation. Individual fractions of these gradients were analyzed by electron microscopy for structural particles as well as for DNA extraction, amplification and sequencing. Comparison with all data banks revealed DNA sequence homology to the transmissible spongiform encephalopathy (TSE)-associated isolates Sphinx 1.76 (1,758 bp; accession number HQ444404) and Sphinx 2.36 (2,360 bp; accession number HQ444405) (Manuelidis L. 2011). The full-length plasmid-like episomal DNA genomes of the bovine and milk factor (BMMF) were obtained using PCR amplification with adjacent primers and partial DNA fragments. The major open reading frames of all isolates were identified in silico as putative replication genes (Rep), revealing a significant degree of similarity to specific plasmids of Acinetobacter baumannii and Psychrobacter species. Subsequently, more than 130 BMMF DNA genomes were isolated from milk and other dairy products, and three isolates were also isolated from brain biopsies and sera from multiple sclerosis patients. These isolates were classified as BMMF1, BMMF2 and BMMF4 based on DNA sequence homology (Funk et al. 2014, Gunst et al. 2014, Whitley et al. 2014, Falida et al., 2017, de Villiers et al., 2019). Detailed analysis of their genomes revealed features of both viral and bacterial plasmids required for replication, transcription and translation, with many all present on a single genome, thereby constituting a novel class of pathogens (de Villiers et al. 2019).

[0012] Two BMMF1 isolates from brain biopsies of MS patients were MSBI1.176 (MSBI, multiple sclerosis brain isolate) (1766 bp) and MSBI2.176 (1766 bp), which were named "MSBI1 genome" and "MSBI2 genome", respectively. MSBI1.176 shares 98% nucleotide similarity with the sequence of Sphinx1.76. The sequences of isolates MSBI1.176 and MSBI2.176 have been deposited in the EMBL data bank under accession numbers LK931491 (MSBI1.176) and LK931492 (MSBI2.176) (Whitley C. et al. 2014) and are sequenced and described in International Publication No. WO 2016 / 005054.

[0013] Additional isolates were obtained from milk, dairy products and bovine serum. These milk isolates (CMI) were CMI1.252, CMI2.214, CMI3.168, CMI4.158, HCBI3.108, HCBI4.296, HCBI6.252, CMI.M3.1 and CMI.9M.1, designated "CMI1 genome", "CMI2 genome", "CMI3 genome", "CMI4 genome", "HCBI3 genome", "HCBI4 genome", "HCBI6 genome", "C1MI.3M.1 genome", "C1MI.9M.1 genome", respectively. The sequences of the isolates have been deposited in the EMBL data bank under accession numbers LK931487 (CMI1.252), LK931488 (CMI2.214), LK931489 (CMI3.168), LK931490 (CMI4.158), LK931495 (HCBI3.108), LK931496 (HCBI4.296), LK931493 (HCBI6.252), LR215499 (C1M1.3M.1), LR215496 (C1M1.9M.1) and are partially sequenced and described in International Publication No. WO 2016 / 005054. The CMI5 genome is described in Falida et al., 2017.

[0014] The inventors have found that both the CMI genome and the MSBI genome replicate in vitro and show significant production of transcribed RNA. Antibodies against MSBI1.176 Rep show expression in the peripheral tissues surrounding pancreatic cancer tissues. The inventors have found that the Rep proteins encoded by a subgroup of BMMF1 infectious agents (MSBI1 Rep, MSBI2 Rep, CMI1 Rep, CMI2 Rep, CMI3 Rep, CMI4 Rep, HCBI3 Rep, HCBI4 Rep, HCBI6 Rep, C1MI.3M.1 Rep, C1MI.9M.1 Rep) are diagnostic markers for pancreatic cancer.

[0015] The inventors produced monoclonal antibodies against the Rep protein using a consensus peptide derived from the full-length MSBI1.176 Rep or two conserved regions of the MSBI1.176 Rep sequence. In certain embodiments, the anti-Rep antibody binds to an epitope of the Rep protein illustrated in FIG. 5. Particularly preferred antibodies bind to an epitope within an amino acid sequence selected from the group consisting of amino acids 1-136, 137-229, and 230-324 of SEQ ID NO: 1. For example, the antibody binds to an epitope contained in SEQ ID NO: 2 or SEQ ID NO: 3.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0017] The present invention teaches that the Rep protein can be a diagnostic marker for indicating an increased risk of developing pancreatic cancer, or for diagnosing or confirming the diagnosis of pancreatic cancer.

[0018] The term "pancreatic cancer" means cancer that develops as a result of uncontrolled cell growth in the pancreas gland. These malignant tumors can develop as a result of existing benign cysts and hyperplasia, where genetic mutations promote the transition from normal to cancerous growth. The term "pancreatic cancer" refers to the pre-disease stage, early or late stage of the disease, and metastases arising therefrom. "Pancreatic cancer" encompasses all malignant neoplasms of the head, body, tail of the pancreas, or pancreatic duct. This can relate to exocrine or endocrine cells and encompasses all types of exocrine or endocrine neoplasms. The most common exocrine tumors are pancreatic ductal adenocarcinoma (PDAC), ampullary cancer, intraductal papillary mucinous neoplasm (IPMT), or acinar cell carcinoma. The most common endocrine tumors are insulinoma, gastrinoma, somatostatinoma, or glucagonoma.

[0019] In an alternative embodiment, the invention may also include a systematic examination of healthy pancreatic tissue (tissue from an individual without a cancer diagnosis or specific signs of disease) to assess future disease risk. This means that the invention is also suitable for determining the predisposition to develop pancreatic cancer by using BMMF Rep as an early diagnostic marker.

[0020] "Predisposition" means a tendency to a medical condition, in accordance with the accepted meaning in medicine. It is a state of pharmaceutical susceptibility or special susceptibility to a particular disease, usually based on the combined effects of genetic and environmental factors.

[0021] As used herein, the "Rep protein" refers to a DNA replication-related protein. The Rep protein includes DNA binding activity and may be essential for the initiation of replication of episomal / viral DNA molecules. Generally, the Rep protein refers to Rep proteins from the group of BMMF1 proteins (de Villiers et al., 2019). In particular, the Rep protein is the MSBI1 genome-encoded Rep protein (MSBI1 Rep), the MSBI2 genome-encoded Rep protein (MSBI2 Rep), the CMI1 genome-encoded Rep protein (CMI1 Rep), the CMI2 genome-encoded Rep protein (CMI2 Rep), the CMI3 genome-encoded Rep protein (CMI3 Rep), the CMI4 genome-encoded Rep protein (CMI4 Rep), the HCBI4 genome-encoded Rep protein (HCBI4 Rep), the HCBI6-encoded Rep protein (HCBI6 Rep), the C1MI.3M.1 genome-encoded Rep protein (C1MI.3M.1 Rep), the C1MI.9M.1 genome-encoded protein (C1MI.9M.1 Rep). Preferably, the MSBI1 Rep protein is encoded by MSBI1.176 deposited with the EMBL Data Bank under accession number LK931491 and has the amino acid sequence shown in SEQ ID NO: 1, or the Rep protein is encoded by MSBI2.176 deposited with the EMBL Data Bank under accession number LK931492 and has the amino acid sequence shown in SEQ ID NO: 8 (Whitley, Gunst et al. 2014). In another preferred embodiment, the CMI1 Rep protein is encoded by CMI1.252 deposited with the EMBL Data Bank under accession number LK931487 and has the amino acid sequence shown in SEQ ID NO: 10. In another preferred embodiment, the CMI2 Rep protein is encoded by CMI2.214 deposited with the EMBL Data Bank under accession number LK931488 and has the amino acid sequence shown in SEQ ID NO: 11. In another preferred embodiment, the CMI3 Rep protein is encoded by CMI3.168 deposited with the EMBL Data Bank under accession number LK931489 and has the amino acid sequence shown in SEQ ID NO: 12.In another preferred embodiment, the CMI4 Rep protein is encoded by CMI4.158 deposited with the EMBL database under accession number LK931490 and has the amino acid sequence shown in SEQ ID NO: 16. In another preferred embodiment, HCBI3 is encoded by HCBI3.108 deposited with the EMBL database under accession number LK931495 and has the amino acid sequence shown in SEQ ID NO: 17. In another preferred embodiment, HCBI4 is encoded by HCBI4.296 deposited with the EMBL database under accession number LK931496 and has the amino acid sequence shown in SEQ ID NO: 18. In another preferred embodiment, C1M1.3M.1 is deposited with the EMBL database under accession number LR215499 and has the amino acid sequence shown in SEQ ID NO: 19. In another preferred embodiment, C1M1.9M.1 is deposited with the EMBL database under accession number LR215496 and has the amino acid sequence shown in SEQ ID NO: 20.

[0022] In a particularly preferred embodiment, the Rep protein comprises an N-terminal region conserved among BMMF1 genomes consisting essentially of amino acids 1 to 229 of SEQ ID NO: 1 and a C-terminal variable region specific to MSBI1.176 consisting essentially of amino acids 230 to 324 of SEQ ID NO: 1. The N-terminal conserved region comprises a putative first DNA-binding domain consisting essentially of amino acids 1 to 136 of SEQ ID NO: 1 and a second putative DNA-binding domain consisting essentially of amino acids 137 to 229 of SEQ ID NO: 1. The C-terminal domain shows little sequence homology with any known protein and consists of amino acids 230 to 324.

[0023] The "Rep protein" also encompasses fragments and variants of proteins having SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and / or SEQ ID NO: 20 that can bind to anti-Rep antibodies specific for the Rep protein having the amino acid sequence of SEQ ID NO: 1. Preferably, such fragments are immunogenic fragments of proteins having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and / or SEQ ID NO: 20, contain at least one epitope of the anti-Rep protein antibody against the Rep protein of SEQ ID NO: 1, and preferably contain at least 7, 8, 9, 10, 15, 20, 25 or 50 consecutive amino acids. In certain embodiments, the fragment comprises or consists essentially of a domain of the Rep protein, such as an N-terminal conserved region, a C-terminal variable region, a first or second DNA binding domain. Variants of proteins having SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and / or SEQ ID NO: 20 contain one or more deletions, substitutions or additions of amino acids compared to their respective sequences, and have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and / or SEQ ID NO: 20, and the variants can bind to anti-Rep antibodies specific for the Rep protein having the amino acid sequence of SEQ ID NO: 1. The definition of variants includes, for example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids, peptide nucleic acids (PNA), etc.), polypeptides having substituted linkages, and polypeptides having other modifications known in the art both for naturally occurring and non-naturally occurring ones. The term "Rep protein" includes fusion proteins with heterologous amino acid sequences, leader sequences or Tag sequences, etc.In certain embodiments of the present invention, the protein tag is genetically grafted onto a Rep protein selected from the group consisting of the above-mentioned Rep proteins, such as MSBI1, MSBI2, CMI1, CMI2, CMI3, CMI4, HCBI4, HCBI6, C1MI.3M.1 or C1MI.9M.1. In particular, at least one protein tag is bound to a polypeptide consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1-3, 8-12, 14 and 16-20. Such protein tags can be removable by chemical agents or enzymatic means. Examples of protein tags are affinity tags or chromatography tags for purification. For example, the Rep protein can be fused to a Tag sequence selected from the group consisting of, for example, His6-Tag (SEQ ID NO: 4), T7-Tag (SEQ ID NO: 5), FLAG-Tag (SEQ ID NO: 6) and Strep-II-Tag (SEQ ID NO: 7), His-Tag (SEQ ID NO: 4), T7-Tag (SEQ ID NO: 5), FLAG-Tag (SEQ ID NO: 6) or StrepII-Tag (SEQ ID NO: 7). Furthermore, a fluorescent tag such as green fluorescent protein (GFP) or a variant thereof can be attached to the Rep protein according to the present invention.

[0024] In a particularly preferred embodiment, the MSBI1 genomic code Rep protein (MSBI1 Rep) is codon-optimized for production in human cell lines (such as HEK-293, HEK293TT, HEK293T, HEK293FT, HaCaT, HeLa, SiHa, CaSki, HDMEC, L1236, L428, BJAB, MCF7, Colo678) as well as bovine (such as MAC-T) or mouse cell lines (such as GT1-7) or any other cell line. This is described in detail in the specification of International Patent Application No. PCT / EP2017 / 075774.

[0025] The Rep proteins of the present invention containing the above-mentioned Rep fragments and Rep variants can be prepared by classical chemical synthesis. The synthesis can be carried out in a homogeneous solution or in the solid phase. The polypeptides according to the present invention can also be prepared by recombinant DNA technology. As used herein, "subject" refers to an individual or patient of a mammal including mice, cows, e.g., different cow species, monkeys, and humans. Preferably, the subject is a human patient.

[0026] As used herein, "anti-Rep antibody" refers to an antibody that binds to the Rep protein of the present invention with a stronger affinity than to non-Rep proteins at a detectable level. Preferably, the antigenic affinity for the Rep protein is at least 2-fold, more preferably 5-fold or 10-fold greater than the background binding. In particular, the anti-Rep antibody detects MSBI1 Rep having the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the antibody detects MSBI1 Rep, MSBI2 Rep, CMI1 Rep, CMI2 Rep, CMI3 Rep, CMI4 Rep, HCBI4 Rep, HCBI6 Rep, C1MI.3M.1 Rep, and / or C1MI.9M.1 Rep. In certain specific embodiments, the anti-Rep antibody detects at least two, preferably all, of MSBI1 Rep, MSBI2 Rep, CMI1 Rep, CMI2 Rep, CMI3 Rep, CMI4 Rep HCBI4 Rep, HCBI6 Rep, C1MI.3M.1 Rep, and / or C1MI.9M.1 Rep.

[0027] The inventors also tested the antibody levels of pancreatic cancer patients by contacting the Rep protein with a sample suspected of containing an anti-Rep protein antibody under conditions that allow the Rep protein to bind to any such antibody present in the sample. Such conditions are typically physiological temperature, pH, and ionic strength using an excess of the Rep protein. Following incubation of the Rep protein with the sample, immune complexes composed of the antigen are detected. In certain embodiments, the Rep protein is conjugated to a signal generating compound, such as a detectable label, or an additional binding agent conjugated to the signal generating compound, such as a secondary anti-human antibody, is used to detect the immune complex.

[0028] Anti-Rep antibodies can be detected and quantified in an assay based on the Rep protein as a protein antigen, which serves as a target for antibodies of a mammal, such as a human, suspected in a sample. Preferably, the Rep protein is purified and the sample can be, for example, serum or plasma. The method includes immobilization of the Rep protein on a matrix and subsequent incubation of the immobilized Rep protein with the sample. Finally, the Rep-binding antibody of the immunological complex formed between the Rep protein and the antibody of the sample is quantified by a secondary HRP-conjugated detection antibody that enables quantification based on an HRP (horseradish peroxidase) substrate conjugated to a signal generating compound. This signal generating compound or label is either detectable itself or can react with an additional compound to generate a detectable product.

[0029] The design of immunoassays is diverse, and many formats are known in the art. The protocol can use, for example, a solid support or immunoprecipitation. Most assays involve the use of a binding agent, such as a labeled antibody or labeled Rep protein, bound to a signal generating compound; the label can be, for example, an enzyme molecule, a fluorescent molecule, a chemiluminescent molecule, a radioactive molecule, or a dye molecule. Assays that amplify the signal from the immune complex are also known; examples thereof are assays that utilize biotin and avidin or streptavidin, as well as enzyme labels and mediated immunoassays such as ELISA assays.

[0030] Immunoassays can be in a heterologous or homologous format and can be in a standard or competitive format. Both standard and competitive formats are known in the art.

[0031] In an immunoprecipitation or agglutination assay format, the reaction between the Rep protein and the anti-Rep antibody forms a network that precipitates out of solution or suspension, forming a visible layer or film of precipitate. If the anti-Rep antibody is not present in the sample, no visible precipitate is formed.

[0032] In a further embodiment, the inventors used a method in which an increased amount of Rep protein in a sample correlates with the diagnosis or predisposition to pancreatic cancer. In such an embodiment, the Rep protein in the sample is detected by an anti-Rep antibody.

[0033] As used herein, "sample" refers to a biological sample including cancerous pancreatic tissue, peripheral tissue surrounding the cancerous tissue, and (benign) hyperplasia. Samples include tissue samples such as tissue cultures or biopsy specimens.

[0034] Such a method (ex vivo or in vitro) includes the step of detecting the Rep protein in a sample from a subject by an anti-Rep antibody. In such a method, the Rep protein is detected in the tissue sample by immunohistochemical methods or immunofluorescence microscopy.

[0035] In certain embodiments, the anti-Rep antibody is used for the detection or capture of Rep protein in a sample.

[0036] The term "antibody" preferably relates to antibodies consisting essentially of pooled polyclonal antibodies having different epitope specificities, as well as distinct monoclonal antibody preparations. As used herein, the terms "antibody" (Ab) or "monoclonal antibody" (Mab) mean intact immunoglobulin molecules as well as antibody fragments (e.g., Fab and F(ab')2 fragments, etc.) that can specifically bind to the Rep protein. Fab and F(ab')2 fragments lack the Fc fragment of the intact antibody, are removed more rapidly from the circulation, and may have less non-specific tissue binding than intact antibodies. Accordingly, these fragments, as well as products of FAB or other immunoglobulin expression libraries, are preferred. Furthermore, antibodies useful for the purposes of the present invention include chimeric, single-chain, multifunctional (e.g., bispecific) and humanized or human antibodies.

[0037] In certain embodiments, the antibody or antigen-binding fragment thereof is conjugated to a signal generating compound, e.g., carrying a detectable label. The antibody or antigen-binding fragment thereof can be detectably labeled directly or indirectly, e.g., with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelating agent or an enzyme. Those skilled in the art will know or be able to ascertain other suitable labels for binding to the antibody using routine experimentation.

[0038] The anti-Rep antibody is preferably produced (generated) against the Rep protein having the amino acid sequence of SEQ ID NO: 1 or a fragment thereof by methods well known to those skilled in the art.

[0039] In certain embodiments, anti-Rep antibodies that can bind to some or all types of Rep proteins from the group of BMMF1 proteins are used in the methods of the present invention. Such antibodies bind to epitopes within the conserved N-terminal region of the Rep protein from amino acids 1 to 229 of SEQ ID NO: 1. In certain embodiments, anti-BMMF1 Rep-type anti-Rep antibodies that bind to epitopes within SEQ ID NO: 2 (amino acids 32 to 49 of SEQ ID NO: 1) or SEQ ID NO: 3 (amino acids 197 to 216 of SEQ ID NO: 1) are used. The peptide fragments of SEQ ID NO: 2 and SEQ ID NO: 3 are highly conserved among Rep proteins from BMMF1 and appear to be exposed due to their hydrophilicity. This anti-BMMF1 type of anti-Rep antibody can be produced by a peptide consisting essentially of the amino acid sequence shown in SEQ ID NO: 2 or 3, or by another immunogenic fragment containing preferably at least 8 to 15 amino acids derived from the conserved N-terminal Rep protein region from amino acids 1 to 229 of SEQ ID NO: 1, for example, by immunization of mice or guinea pigs.

[0040] In further embodiments, anti-Rep antibodies specific for the MSBI1 Rep protein are used. Such antibodies can be prepared, for example, by immunizing a mammal such as a mouse or a guinea pig with the full-length Rep protein having the amino acid sequence of SEQ ID NO: 1.

[0041] Preferably, the methods of the present invention use anti-Rep antibodies that can detect Rep proteins in the range from picograms to femtograms.

[0042] An example of such a group of anti-Rep antibodies is shown in Table 1: [Table 1]

[0043] The localization of antibody epitopes within MSBI1.176 Rep is shown in FIG. 8.

[0044] The anti-Rep antibodies of Group A have an epitope within the amino acid sequence shown in SEQ ID NO: 3 (aa198 - 217 of SEQ ID NO: 1) and can detect MSBI1 Rep and Rep proteins containing this conserved epitope of the BMMF1 group (e.g., MSBI2, CMI1, CMI2, CMI3, CMI4, HCBI4, HCBI6, C1MI.3M.1, C1MI.9M.1). In an immunofluorescence assay, such anti-Rep antibodies detect a specific Rep localization pattern, with the main localization being homogeneously distributed across the cytoplasm and nuclear membrane; additional weakly homogeneous localization is seen in the nucleus. An example of such a Group A antibody is the antibody AB01523 - 1 - 1 (also called antibody 1 - 5; DSM ACC3327) used as a Group A antibody in the examples.

[0045] The anti-Rep antibodies of Group B have an epitope within the amino acid sequence shown in SEQ ID NO: 2 (aa33 - 50 of SEQ ID NO: 1) and can detect MSBI1 Rep and Rep proteins containing this conserved epitope of the BMMF1 group (e.g., MSBI2, CMI1, CMI2, CMI3, CMI4, HCBI4, HCBI6, C1MI.3M.1, C1MI.9M.1). In an immunofluorescence assay, such anti-Rep antibodies specifically detect speckles (cytoplasmic aggregates) of the Rep protein (often around the nuclear membrane). An example of such a Group B antibody is the antibody designated AB02 304 - 4 - 1 (also called antibody 5 - 2; DSM ACC3328) used as a Group B antibody in the examples.

[0046] The anti-Rep antibodies of Group C specifically detect the structural epitope of MSBI1 (SEQ ID NO: 1). In an immunofluorescence assay, such anti-Rep antibodies detect a specific Rep localization pattern, where the main localization is homogeneously distributed across the cytoplasm and nuclear membrane; a further weakly homogeneous distribution is seen in the nucleus. An example of such a Group C antibody is the antibody MSBI1381-6-2 (also called antibody 3-6 or antibody 3) used in the examples as a Group C antibody having an epitope in the sequence of aa230 - 324; DSM ACC3329. Another example of an antibody of Group C antibodies is the antibody MBSI1572-13-19 (also called antibody 10-3 or antibody 10) that detects an epitope in the C-terminal domain of MSB11 Rep (aa230-324). Another example of an antibody of Group C antibodies is the antibody MBSI1617-1-3 (also called antibody 11-5) that detects an epitope in the N-terminal domain of MSB11 Rep (aa1-136).

[0047] The anti-Rep antibodies of Group D specifically detect the structural epitope of MSBI1 (SEQ ID NO: 1), where the antibody MSBI1961-2-2 (also called antibody 9-2; DSM ACC3331) designated as "D1" detects the epitope shown in SEQ ID NO: 9 (aa281 - 287) in the C-terminal domain of MSBI1. The antibody MSBI1761-5-1 (also called antibody 13; DSM ACC3328) designated as "D2" detects a 3D structural epitope of MSBI1 that is exclusively accessible under in vivo conditions and not accessible by Western blot. In an immunofluorescence assay, such anti-Rep antibodies specifically detect speckles (cytoplasmic aggregates) of the Rep protein (often present around the nuclear membrane).

[0048] The availability of BMMF-specific Rep monoclonal antibodies and the demonstration of the DNA of these infectious agents in the tissue surrounding the tumor have prompted the inventors to investigate the presence of specific structural components in patient tissues by immunogold electron microscopy using the primary Rep monoclonal antibody as a tool. The identification of such gold-labeled structures in the tissue surrounding the pancreatic cancer of a patient is described in detail in the following examples.

[0049] The ultrastructural analysis of the present invention by immunoelectron microscopy (IEM) demonstrates BMMF Rep expression in macrophages in the tissue surrounding pancreatic cancer. The gold-decorated targets are of different sizes and shapes, showing a polymorphic appearance distinct from the surrounding area. Polymorphic structures with a diameter of about 50 - 200 nm were found in the tissue surrounding the pancreatic cancer tumor. Both cryo-IEM and conventional resin TEM allow for more delicate preservation of the structure compared to pre-embedding IEM of FFPE (formalin-fixed paraffin-embedded tissue) samples, showing MSBI1.176 Rep overexpression-dependent isolates in the cytoplasm of HEK293TT cells that may associate with membranes. In all modes of sample preparation, detailed observation of the aggregates reveals ultrastructures such as voids and filaments showing higher-order organization.

[0050] Using semi - automated quantification of Rep, CD68, and CD163 immunofluorescence microscopy staining of individual FFPE tissues, the levels of Rep+, Rep+CD68+, Rep+CD68+CD163+ and CD68+ cells were compared in pancreatic cancer tumors and tissues from tumor - adjacent regions verified by pathologists, as well as in tissues from age - matched chronic pancreatitis patients and age - matched non - tumor individuals. A significant increase in the number of Rep+CD68+ cells (average of about 7.4% of total cells) was observed in the tumors of pancreatic cancer patients when compared to non - cancerous healthy controls (average < 1%) and when compared to tumor - adjacent tissues (average < 1%). When identifying Rep - positive cells as the fraction of M2 - like CD163 - positive macrophages within the entire macrophage population, an increase in the number of Rep+CD68+CD163+ cells (average of about 19.5% of all macrophages) was observed in the tumors of pancreatic cancer patients compared to healthy controls (average of 5.5% of all macrophages). A large number of Rep+ Mfs are represented by M2 - like macrophages triple - positive stained with Rep, CD68, and CD163 antibodies (Figure 9). Cell - based quantification of the same parameters for chronic pancreatitis patients showed that the amounts of Rep+CD68+ cells (average of about 4.0%) and Rep+CD68+CD163+ cells (average of about 9.5%) within all macrophages were localized between the values detected for pancreatic cancer tumor tissues (maximum values were 7.4% and 19.5% respectively) and the values detected for healthy controls (minimum values were < 1% and 5.5% respectively). Quantification of Rep+CD68+ cells and Rep+CD68+CD163+ cells within all macrophages in tumor - adjacent tissues (average < 1% and about 5.5%) was comparable to the cell numbers in healthy individuals. The numbers of Rep+, CD68+ and Rep+CD68+ cells were significantly increased around the tumors when compared to the respective quantification of stained cells in paired tumors (median of 6.3% vs 0.5% Rep+, 11.0% vs 2.5% CD68+, 2.2% vs < 0.1% Rep+CD68+ cells) (Figure 10).

[0051] Comparison of Rep staining area and staining intensity in the islets of Langerhans based on semi - automatic quantification of tissues from healthy donors, PDAC patients, and patients with chronic pancreatitis (a representative DAB IHC staining example is shown in Fig. 11) revealed a significant increase (by approximately 38% on average) in the Rep staining area in the islets of Langerhans of PDAC patients compared to healthy controls (by approximately 18% on average) (Fig. 12). The staining area in chronic pancreatitis (by approximately 25% on average) was significantly smaller when compared to PDAC, but still increased when compared to the group of healthy donors. The same trend was observed for Rep staining intensity, which was generally at a higher level. The Rep staining intensity increased significantly in the islets of Langerhans of PDAC patients (by approximately 160 a.u. on average) compared to healthy controls (by approximately 135 a.u. on average). The staining intensity in chronic pancreatitis (by approximately 140 a.u. on average) was significantly smaller when compared to PDAC, but still increased when compared to the healthy group.

[0052] Detection of Rep positivity in triple - positive macrophages (Rep+CD68+CD163) was used to characterize the differentiation of Rep - positive macrophages, generally distinguishing between more M1 - like (CD68+CD163 -) macrophage polarization and M2 - like (CD68+CD163+) macrophage polarization. Quantification of triple - positive macrophages may clarify future diagnostic differentiation and help identify the (pathogenic) involvement of the BMMF - positive macrophage population in cancer. M1 - like macrophages are expected to act in an inflammation - inducing (e.g., mobilizing tumor - cell - reactive lymphocytes) and anti - tumor - forming manner, while M2 - like macrophages are considered to be tumor - promoting, releasing cytokines and chemokines involved in the mobilization of tumor - promoting regulatory T cells.

[0053] The above analysis can be regarded as evidence that the Rep protein is a structural protein that can potentially be targeted by antibodies for both preventive and therapeutic purposes.

[0054] Furthermore, the inventors have found an association between BMMF infection and type 2 diabetes, because it has been shown by the inventors that BMMF infection spreads in insulin-producing pancreatic β-cells in the endocrine islets of Langerhans. As a result, large aggregates of the BMMF Rep protein are formed, which potentially interfere with insulin production. At a later stage, β-cell depletion occurs, leading to dependence on insulin injections. The association between type 2 diabetes and pancreatic cancer is illustrated in FIG. 7.

[0055] The present invention is further illustrated by the following examples, but is not limited thereto.

Example

[0056] Example 1: Detection of BMMF Protein Targets in Pancreatic Cancer Surrounding Tissues Tissue Staining Tissue surrounding pancreatic cancer was obtained as FFPE sections by EPZ-Pancobank / BioMaterial Bank Heidelberg according to the regulations of the tissue bank (Ethic votes S-708 / 2019 and S-083 / 2021) approved by the Ethics Committee of Heidelberg University, after obtaining the consent of the patients.

Table 2

[0057] FFPE IHC DAB staining was performed based on the Zytomed ChemPlus (HRP) staining kit (Zytomed Systems) including epitope retrieval (30 minutes, 95 °C) using EDTA (pH 8.5, Sigma) as previously described (Bund et al., 2021). Following Rep staining with AB3 (1 hour, room temperature), the slides were stained with a DAB high contrast kit (Zytomed Systems) and hematoxylin counterstain. The slides were scanned using a Hamamatsu Nanozoomer slide scanner (Hamamatsu) and analyzed with NDP.view2 Plus software (Hamamatsu).

[0058] Staining of tissue PDAC tissue samples with anti-Rep antibodies (e.g., mAb 10; Table 1) shows the detection of protein targets in the tissue area around the tumor in pancreatic cancer patient samples. Generally, anti-Rep detection resulted in strong staining of smaller-sized aggregated structures mainly within the cytoplasmic region of insulin-positive beta cells in the endocrine islets of Langerhans (Figure 1A). Additionally, a second staining pattern was observed in the exocrine tissue area. Here, co-localization of the anti-Rep staining signal with CD68-positive macrophages (distributed throughout the exocrine tissue area and the stroma) was observed (Figure 1B). The area with the highest Rep-specific antibody detection correlates with the area having the highest detection level for CD68-positive cells indicating the localization of the Rep-specific antigen in the inflammatory tissue area, i.e., the area having particularly high levels of inflammatory monocytes, circulating macrophages, or resident tissue macrophages. No signal detection was observed in the control staining with antibody isotype controls.

[0059] Example 2: Pre-embedding Immunoelectron Microscopy (IEM) Using tissues from pancreatic cancer patients that were positively stained for BMMF Rep in IHC, the localization and physical expression of the Rep target were analyzed at ultrastructural resolution. The inventors first performed IHC DAB staining using monoclonal anti-BMMF1 Rep AB10 on one of a series of FFPE sections to identify the positive tissue area (Figure 2A). Based on Example 1, serial sections including both exocrine and endocrine tissue areas around the tumor were then gold-labeled by pre-embedding IEM (pre-IEM) (Figure 2B).

[0060] Therefore, the standard procedure for immunolabeling of FFPE materials and subsequent resin embedding for ultrathin sectioning was as follows: FFPE sections (3 μm thick) placed on glass slides were deparaffinized with xylene and rehydrated in ethanol with gradually changing concentrations. The immunoreaction was performed by incubation in polyclonal rabbit anti-mouse (1:150, Z0259, Dako) as a linker for 40 mM glycine (aldehyde block), 1% BSA (protein block), primary antibody AB3 and reporter protein A-gold (CMC, nominal gold particle size 5 nm or 10 nm). Experiments without the primary antibody were included as reporter-only controls. Sections on glass slides were post-fixed in 2.5% glutaraldehyde, subsequently treated with 1% OsO4, dehydrated by a stepwise process of ethanol, and subjected to flat embedding in epoxy (Serva). Ultrathin sections taken at a nominal thickness of 60 nm were stained with uranyl and lead for direct visualization by electron microscopy.

[0061] Pre-embedding IEM staining revealed distinct dense particle structures with high-density exclusive gold labeling of polymorphic nature in the range of approximately 50 - 200 nm in size in both exocrine and endocrine tissue regions. The identified polymorphic structures differed in size and shape and appeared to include a filamentous substructure highlighted by arrows (Figure 3). Apart from these structures, gold particles did not accumulate in other regions of the tissue. The cell localization context was not identified because the structure preservation inherent to the FFPE protocol was compromised.

[0062] Example 3: Cryo-IEM (Tokuyasu method) after Overexpression of MSBI1.176Rep in HEK293TT for Verification of Pre-IEM Cell culture and transfection HEK293TT cells were cultured as previously reported ((Buck et al. 2005, de Villiers et al. 2011), transfected with polyethyleneimine (PEI, Sigma) at 70 - 80% confluence in 6-well plates with 1 μg of plasmid DNA per well, and overexpressed untagged H1MSB.1 Rep or 3xFlag-H1MSB.1 Rep protein (control: mock transfection with pcDNA(-)). The overexpression plasmids were designed based on PCR cloning of the H1MSB.1 Rep gene (cloning via BamHI / KpnI, PCR primers: pcDNA3.1(-)H1MSB.1 Rep fwd: GC GGA TCC GCC ATG AGC GAC CTG ATC GTG AAA G (SEQ ID NO: 21), rev: GC GGT ACC TCA AAA CAC GAC TCC AAA CTC TTC C (SEQ ID NO: 22); pcDNA3.1(-)3xFlag H1MSB.1-Rep fwd: GT GGA TCC GCC ATG GAC TAC AAA GAC CAT GAC GGT GAT TAT AAA GAT CAT GAC ATC GAT TAC AAG GAT GAC GAT GAC AAG GGA GCA AGC GAC CTG ATC GTG AAA GAC AAT GC (SEQ ID NO: 23), Rep rev: GA GGT ACC TCA AAA CAC GAC TCA AAC TCT TCC AGT TTA G (SEQ ID NO: 24)). Cells were harvested 48 hours after transfection. One part was analyzed by SDS-PAGE and immunoblotting as previously described (Bund et al. 2021), and the other part was fixed for cryoIEM (see below).

[0063] Western blot analysis Western blot analysis was performed as previously described (Bund et al. 2021), except that cells were harvested 48 h later and subsequently lysed in Lammli buffer. Lysates were separated by SDS-PAGE (Sigma True-PAGE 4–20%) on a precast gel and then electrotransferred onto nitrocellulose. Immunodetection by Western blot was performed using two primary mouse monoclonal antibodies against Rep AB3 and AB7 (DKFZ Heidelberg (Bund et al. 2021)), or anti-Flag M2 (F3165, Sigma Aldrich), respectively, with an HRP-conjugated goat anti-mouse IgG secondary antibody (115-035-205, Dianova).

[0064] Rep protein expression was verified by WB analysis using anti-Flag as well as anti-Rep AB3 or AB7 antibodies. Full-length Rep protein bands with molecular weights of 40 kDa and 37 kDa were seen for 3xFlag Rep and untagged Rep, respectively. Furthermore, smaller molecular weight bands described in a previous study (Bund et al. 2021) were positively stained (Figure 4).

[0065] Cryo IEM (Tokuyasu method) The protocol of Slot & Geuze (Slot and Geuze 2007) was followed. HEK293TT cells transfected with each plasmid were fixed in buffered aldehyde (2% formaldehyde, 0.2% glutaraldehyde, 100 mM phosphate buffer, pH 7.2) and embedded in 10% gelatin. Vibratome sections (200 μm thick) of patient biopsies or gelatin-embedded cell blocks were perfused with 2.3 M sucrose in 50 mM phosphate buffer and frozen in liquid nitrogen. Cryosections with a nominal thickness of 60 nm were cut at 160 K using a UCT7 / FC7 cryo-ultramicrotome (Leica). After buffer washing and incubation in 40 mM glycine and 1% BSA, immunoreactions were performed on ice. A polyclonal rabbit anti-mouse antibody (1:150, Z0259, DAKO) was used to bridge the primary antibody of mouse origin to enable the binding of protein A-gold reporter (1:50, CMC, nominal size of gold particles: 5 nm or 10 nm). Control samples were stained with reporter only (without primary antibody), or with either primary antibody isotype IgG1 (MG1-45, Biolegend) or anti-Flag.

[0066] Overexpression of both constructs led to the formation of large separated complexes in the cytoplasm of transfected cells (Figure 5). These complexes were stained positively (anti-Rep AB3, AB7 and anti-Flag respectively), while the remaining cytoplasm remained free of gold labeling. Tagless H1MSB.1 Rep was further separated to the nucleoplasmic position (Figure 6A) and showed association with the membrane (Figure 6B). Isotype antibodies did not react with the target structures (Figure 5). Transfection with the pcDNA3.1(-) vector (without Rep insert) did not result in separated structures.

[0067] Resin TEM (without immunolabeling) Transfected HEK293TT cells expressing H1MSB.1 Rep and cultured on punched Aklar-fluoropolymer films (EMS) were embedded in resin for ultrathin sectioning according to standard procedures. This included primary fixation in buffered aldehyde (4% formaldehyde, 2% glutaraldehyde, 1 mM CaCl2, 1 mM MgCl2, pH 7.2 in 100 mM sodium cacodylate), post-fixation in buffered 1% osmium tetroxide and en bloc staining in 1% uranyl acetate, dehydration in stepwise concentrations of ethanol, and embedding in epoxy (glycidyl ether, NMA, DDSA; Serva). Ultrathin sections with a nominal thickness of 60 nm and counterstained with lead citrate and uranyl acetate were analyzed by EM910 at 80 kV (Carl Zeiss), and micrographs were taken using a slow-scan CCD camera (TRS).

[0068] Overexpression of untagged MSBI1.176 Rep led to the formation of large segregation complexes in the cytoplasm of transfected cells, which was not observed after mock transfection with pcDNA3.1(-). The segregation structures showed association with the membrane (asterisk, see Fig. 6C).

[0069] Example 4: Cell-based Quantification by Immunofluorescence Microscopy of Rep-, CD68- and CD163-positive Cells in Tumors and Tumor Surrounding Tissues of Pancreatic Ductal Adenocarcinoma Patients, Chronic Pancreatitis Patients and Non-cancer Controls Tissue Staining and Tissue Analysis Tissues from pancreatic cancer patients, chronic pancreatitis patients, and healthy transplant donors were obtained as FFPE sections at the PancoBank of the European Pancreatic Center (EPZ) in Heidelberg, Germany, in accordance with the regulations of the tissue bank, including patient consent and approval by the Ethics Committee of Heidelberg University (ethics vote S-708 / 2019), and in full compliance with the criteria defined in the Declaration of Helsinki. The FFPE sections were stained overnight at room temperature with primary anti-BMMF1 Rep (monoclonal AB3 and AB10, DKFZ Heidelberg, 1:1 mixture), anti-CD68 (Cell signaling #76437), and anti-CD163 (Novusbio, NB110-40686) antibodies at dilutions of 1:250 (Rep), 1:500 (CD68), and 1:200 (CD163) as described by Bund et al., 2021. Secondary goat anti-mouse Alexa Fluor 594 (Invitrogen #A11032), goat anti-rabbit Alexa Fluor 488 (Invitrogen #A11034), and donkey anti-mouse Alexa Fluor 647 (Invitrogen #A31571) were incubated at room temperature for 60 minutes. For negative control staining, a PBT dilution containing PBS, BSA, Tween20, and sodium azide was used. The slides were scanned with a Hamamatsu Nanozoomer slide scanner (Hamamatsu) and analyzed with NDP.view2 Plus software (Hamamatsu).

[0070] Based on the signal intensities of DAPI nuclei and antibody staining of cells in the digitized scans after Rep / CD68 / CD163 co-immunodetection, semi-automatic quantification of Rep, CD68, and CD163 staining of individual FFPE tissues was performed using an in-house developed script for ImageJ / Fiji (v1.52). Cell density was measured in 8 - 12 randomly selected microscopic fields (a total of approximately 15,000 cells per sample). The data were expressed as the mean percentage (average) of Rep, CD68, CD163 positive cells and / or their combinations normalized to either the total number of nuclei (or the total number of CD68+ macrophages), as described by Bund et al., 2021 and Nikitina et al, 2023 for similar quantification in the context of colorectal cancer.

[0071] The results are shown in Figures 9 and 10.

Table 3

[0072] Example 5: Semi-automatic Quantification by Immunofluorescence Microscopy of Rep+ Insulin+ Cells in Tumors and Tumor Surrounding Tissues of Pancreatic Ductal Adenocarcinoma Patients, Chronic Pancreatitis Patients and Non-cancer Controls Tissue Staining and Tissue Analysis The tissue (the same as described in Example 4) was stained with primary anti-BMMF1 Rep (monoclonal AB10, DKFZ Heidelberg, anti-BMMF1 Rep (monoclonal AB10, DKFZ Heidelberg, diluted 1:500, incubated for 30 minutes at room temperature) and anti-insulin antibody (in serial tissue sections, to assign pancreatic islets, Santa Cruz Biotechnology, Dallas, Texas, sc-8033, mouse, 1:200, incubated for 180 minutes at room temperature). The secondary rabbit anti-mouse (Abcam, Cambridge, UK, #125904) was incubated for 20 minutes at room temperature. Detection was performed using the Bond Polymer Refine Detection Kit (Leica Biosystems, #DS9800) including DAB chromogen and hematoxylin counterstain. Both DAB brightfield slides and immunofluorescence slides were scanned with a Hamamatsu Nanozoomer slide scanner (Hamamatsu, Shizuoka, Japan) and analyzed with ndp.view2 plus software (Hamamatsu).

[0073] For the analysis of the Rep antibody staining positive area and staining intensity of pancreatic islets, semi-automatic quantitative Rep staining was performed using an in-house developed script of ImageJ / Fiji (v1.52) based on the DAB signal area and average intensity of each selected islet of the tissue. 20 - 40 islets (maximum number) per tissue were included in the analysis. The data were expressed as the % (average) of the Rep staining area in the islets of Langerhans and the Rep staining intensity in the islets of Langerhans as described by Nikitina et al., 2022.

[0074] The results are shown in Figure 11 (representative DAB IHC tissue staining) and Figure 12.

[0075] Summary of the sequence TIFF2025518482000005.tif232170TIFF2025518482000006.tif215170TIFF2025518482000007.tif220170TIFF2025518482000008.tif195170References

[0076] Eilebrecht, S., et al. (2018),”Expression and replication of virus-like DNA in human cells”, Scientific Reports 8:2851 Funk, M., et al. (2014). “Isolation of protein-associated circular DNA from healthy cattle serum”. Genome Announc 2(4) Giraldo, R., et al. (2011). “RepA-WH1 prionoid: a synthetic amyloid proteinopathy in a minimalist host.” Prion 5(2):60-64 Gunst, K., et al. (2014). “Isolation of bacterial plasmid-related replication-associated cirular DNA from a serum sample of a multiple sclerosis patient.” Genome Announc 2(4). Lamberto, I., et al. (2014). “Mycovirus-like DNA virus sequences from cattle serum and human brain and serum samples from multiple sclerosis patients.” Genome Announc 2(4). Manuelidis L., 2011. “Nuclease resistant circular DNAs co-purify with infectivity in scrapie and CJD”. J. Neurovirol. 17:131-145. Torreira, E., et al. (2015). “Amyloidogenesis of bacterial prionoid RepA-WH1 recaptiulates dimer to monomer transitions of RepA in DNA replication initiation.” Structure 23(1):183-189 Whitley, C., et al. (2014). “Novel replication-competent cirulara DNA molecules from healthy cattle serum and milk and multiple sclerosis-affected human brain tissue.” Genome Announc 2(4). zur Hausen, H., Bund, T., de Villiers, E.-M. (2017). “Infectious agents in bovine red meat and milk and their potential role in cancer and other chronic diseases.” Curr. Top.Microbiol. Immunol., Volume 407, 83-116. Bund, T., E. Nikitina, D. Chakraborty, C. Ernst, K. Gunst, B. Boneva, C. Tessmer, N. Volk, A. Brobeil, A. Weber, M. Heikenwalder, H. zur Hausen and E. M. de Villiers (2021). Analysis of chronic inflammatory lesions of the colon for BMMF Rep antigen expression and CD68 macrophage interactions. Proc Natl Acad Sci U S A 118(12):e2025830118. doi: 10.1073 / pnas.2025830118. Slot, J. W. and H. J. Geuze (2007). Cryosectioning and immunolabeling. Nat Protoc 2(10): 2480-2491. Falida, K., S. Eilebrecht, K. Gunst, H. zur Hausen and E. M. de Villiers (2017). Isolation of Two Virus-Like Circular DNAs from Commercially Available Milk Samples. Genome Announc 5(17):e00266-17. doi: 10.1128 / genomeA.00266-17. Koenig, M. T., R. Fux, E. Link, G. Sutter, E. Maertlbauer and A. Didier (2021a). Circular Rep-Encoding Single-Stranded DNA Sequences in Milk from Water Buffaloes (Bubalus arnee f. bubalis). Viruses 13(6):1088. doi: 10.3390 / v13061088. Koenig, M. T., R. Fux, E. Link, G. Sutter, E. Maertlbauer and A. Didier (2021b). Identification and Characterization of Circular Single-Stranded DNA Genomes in Sheep and Goat Milk. Viruses 13(11):2176. doi: 10.3390 / v13112176. De Villiers and zur Hausen (2021), Bovine Meat and Milk Factors (BMMFs): Their proposed role in common human cancers and type 2 diabetes mellitus, Cancers (Basel), 13(21): 5407 De Villiers, E.M., Gunst, K., Chakraborty D., Ernst, C., Bund, T., zur Hausen, H., (2019), A specific class of infectious agents isolated from bovine serum an diary products and peritumoral colon cancer tissue, Emerging Microbes and Infections 8(1), 1205-1218 Nikitina, E., Burk-Koerner, A., Wiesenfarth, M., Alwers, E., Heide, D., Tessmer, C., Ernst, C., Krunic, D., Schrotz-King, P., Chang-Claude, J., von Winterfeld, M., Herpel, E., Brobeil, A., Brenner, H., Heikenwalder, M., Hoffmeister, M., Kopp-Schneider, A., Bund, T., Bovine meat and milk factor protein expression in tumor-free mucosa of colorectal cancer patients coincides with macrophages and might interfere with patient survival. Molecular Oncology, 2023. DOI: 10.1002 / 1878-0261.13390 Nikitina, E., Alikhanyan, K., Nessling, M., Richter, K., Kaden, S., Ernst, C., Seitz, S., Chuprikova, L., Haefele, L., Gunst, K., Rahbari, N., Birgin, E., Rasbach, E., Rahbari, M., Brobeil, A., Schenk, M., Buechler, M., de Villiers, E.-M., Bund, T.*, zur Hausen, H. Structural Expression of BMMF in tissues of colorectal, lung and pancreatic cancer patients. International Journal of Cancer, 2022. DOI: 10.1002 / ijc.34374

Claims

1. Use of beef and milk factor group 1 (BMMF1) Rep protein as a diagnostic marker for pancreatic cancer.

2. The use according to claim 1, characterized in that the Rep protein is MSBI1 genomic code Rep protein (MSBI1 Rep), MSBI2 genomic code Rep protein (MSBI2 Rep), CMI1 genomic code Rep protein (CMI1 Rep), CMI2 genomic code Rep protein (CMI2 Rep), CMI3 genomic code Rep protein (CMI3 Rep) or CMI4 genomic code Rep protein (CMI4 Rep).

3. A method for diagnosing or providing a predisposition to pancreatic cancer in a subject, comprising the step of detecting the Rep protein in a sample obtained from the subject with an anti-Rep antibody that binds to an epitope contained in SEQ ID NO: 2 or SEQ ID NO:

3. A method characterized by this.

4. The method according to claim 3, characterized in that an antibody specific for the Rep protein binds to an epitope within an amino acid sequence selected from the group consisting of amino acids 1 to 136, 137 to 229 and 230 to 324 of SEQ ID NO:

1.

5. The method according to claim 3 or 4, characterized in that the sample derived from the subject is selected from the group consisting of cancerous pancreatic tissue, peripheral tissue surrounding the cancerous tissue, and (benign) cysts.

6. The method according to any one of claims 3 to 5, characterized in that CD68-positive cells are further detected in the sample with an anti-CD68 antibody.

7. The method according to any one of claims 3 to 6, characterized in that CD163-positive cells are further detected in the sample with an anti-CD163 antibody.

8. The method according to any one of claims 3 to 7, wherein insulin-positive cells are further detected in the sample by an anti-insulin antibody. **Claim 9** Use of beef and milk factor group 1 (BMMF1) Rep protein as a diagnostic marker for diabetes.

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