USE OF BMMF1 REP PROTEIN AS A BIOMARKER FOR BREAST CANCER

Abstract

The present invention relates to the use of BMMF Rep-protein as biomarker for breast cancer.

Claims

1) A biomarker for breast cancer comprising a Bovine Meat and Milk Factor Group 1 (BMMF1) Rep Protein.

2) The biomarker of claim 1 wherein the Rep protein is a MSBI1 genome-encoded Rep protein (MSBI1 Rep), a MSBI2 genome-encoded Rep protein (MSBI2 Rep), a CMI1 genome-encoded Rep protein (CMI1 Rep), a CMI2 genome-encoded Rep protein (CMI2 Rep) or CMI3 genome-encoded Rep protein (CMI3 Rep).

3) A method for providing a diagnosis or predisposition for breast cancer in a subject, comprising detecting Rep protein in a sample from a subject by anti-Rep antibodies that bind to an epitope comprising SEQ ID NO:2 or SEQ ID NO:3.

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

5) The method of claim 3, wherein the sample from a subject is selected from the group consisting of a cancerous breast tissue, peripheral tissue surrounding the cancerous tissue, (benign) hyperplasias.

6) The method of claim 4, wherein the sample from a subject is selected from the group consisting of a cancerous breast tissue, peripheral tissue surrounding the cancerous tissue, (benign) hyperplasias.

7) The method of claim 3, wherein additionally CD68 positive cells are detected in the sample by an anti-CD68 antibody.

8) The method of claim 4, wherein additionally CD68 positive cells are detected in the sample by an anti-CD68 antibody.

9) The method of claim 5, wherein additionally CD68 positive cells are detected in the sample by an anti-CD68 antibody.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0022] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0023] FIG. 1 shows a proposed model for breast cancer development.

[0024] FIG. 2 shows IHC detection of BMMF1 Rep on breast cancer patient tissue 7G6MJX (scale bar=100 μm) in consecutive tissue sections.

[0025] FIG. 3 shows IHC detection of BMMF1 Rep on breast cancer patient tissue 1HHY7K (scale bar=100 μm) in consecutive tissue sections.

[0026] FIG. 4 shows characteristics of the raised antibodies and the localization of epitopes within Rep.

[0027] FIG. 5 depicts a bar diagram showing the Immunoreactive Score based on BMMF1 Rep staining (X-axis: Immunoreactive Score; Y-axis: number of patients).

DETAILED DESCRIPTION OF THE INVENTION

[0028] The invention provides the teaching that Rep proteins may represent biomarkers for an enhanced risk to develop breast cancer and are useful as a marker for determining the overall survival prognosis of breast cancer patients.

[0029] The terms “breast cancer” means a cancer that evolved as a consequence of uncontrolled cell growth in the female or male breast tissue. These malignancies may develop as a consequence of pre-existing benign adenomas and hyperplasias where genetic alterations promote the transition from normal to cancerous growth. The term “breast cancer” means pre-stages, early stages or late stages of the disease and metastases derived therefrom.

[0030] In an alternative embodiment the present invention may also encompass the systematic testing of healthy breast tissue (tissue from individuals without cancer diagnosis or a specific hint for the disease) to assess the disease risk in the future. This means that the present invention is also suitable to determine the predisposition for developing breast cancer.

[0031] “Rep protein” as used herein refers to a DNA-replication-associated protein (RepB). The Rep protein may comprise DNA binding activity and could be essential for initiation of replication of episomal/viral DNA molecules. In general Rep protein refers to a Rep protein from the group of the Small Sphinx Genome (Whitley et al., 2014). In particular, the Rep protein is a MSBI1 genome-encoded Rep protein (MSBI1 Rep), a MSBI2 genome-encoded Rep protein (MSBI2 Rep), a CMI1 genome-encoded Rep protein (CMI1 Rep), a CMI2 genome-encoded Rep protein (CMI2 Rep) or CMI3 genome-encoded Rep protein (CMI3 Rep). Preferably, the MSBI1 Rep protein is encoded by MSBI1.176 deposited in the EMBL databank under the acc. no. LK931491 and has the amino acid sequence as depicted in SEQ ID NO:1 or the Rep protein is MSBI2 encoded by MSBI2.176 deposited in the EMBL databank under the acc. no. LK931492 and has the amino acid sequence as depicted in SEQ ID NO:8 (Whitley, Gunst et al. 2014). In another preferred embodiment the CMI1 Rep protein is encoded by CMI1.252 deposited in the EMBL databank under the acc. no. LK931487 and has the amino acid sequence as depicted in SEQ ID NO:10. In another preferred embodiment the CMI2 Rep protein is encoded by CMI2.214 deposited in the EMBL databank under the acc. no. LK931488 and has the amino acid sequence as depicted in SEQ ID NO:11. In another preferred embodiment the CMI3 Rep protein is encoded by CMI3.168 deposited in the EMBL databank under the acc. no. LK931489 and has the amino acid sequence as depicted in SEQ ID NO:12. In a particular preferred embodiment the Rep protein may comprise a N-terminal region conserved among BMMF1 genomes consisting essentially of amino acids from 1 to 229 of SEQ ID NO:1 and a C-terminal variable region specific for MSBI1.176 consisting essentially from amino acids 230 to 324 of SEQ ID NO:1. The N-terminal conserved region may comprise a putative, first DNA binding domain consisting essentially of amino acids from 1 to 136 of SEQ ID NO: 1 and a second putative DNA binding domain consisting essentially of amino acids from 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.

[0032] “Rep protein” also encompasses fragments and variants of the protein with SEQ ID NO:1 or SEQ ID NO:8 which are capable of binding an anti-Rep antibody specific for Rep protein having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:8. Preferably, such a fragment is an immunogenic fragment of the protein having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:8 which encompasses at least one epitope for an anti-Rep protein antibody against the Rep protein of SEQ ID NO:1 or SEQ ID NO:8 and, preferably, may comprise at least 7, 8, 9, 10, 15, 20, 25 or 50 contiguous amino acids. In particular embodiments the fragment may comprise or consists essentially of a domain of the Rep protein, for example, the N-terminal conserved region, the C-terminal variable region, the first or second DNA binding domain. A variant of the protein with SEQ ID NO:1 or SEQ ID NO:8 may comprise one or more amino acid deletions, substitutions or additions compared to SEQ ID NO:1 and has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or

[0033] 99% to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:8, wherein the variant is capable of binding an anti-Rep antibody specific for a Rep protein having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:8. Included within the definition of variant are, for example, polypeptides containing one or more analogues of an amino acid (including, for example, unnatural amino acids, peptide nucleic acid (PNA), etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. The term Rep protein includes fusion proteins with a heterologous amino acid sequence, with a leader sequence or with a Tag-sequence and the like. In certain embodiments of the invention protein tags are genetically grafted onto the Rep protein described above, for example the Rep protein selected from the group consisting of MSBI1, MSBI2, CMI1, CMI2 or CMI3. In particular at least one protein tag is attached to a polypeptide consisting of an amino acid sequence as depicted in any one of SEQ ID NOs:1-3,8-12,14. Such protein tags may be removable by chemical agents or by enzymatic means. Examples of protein tags are affinity or chromatography tags for purification. For example the Rep protein may be fused to a Tag-sequence, for example, selected from the group consisting of His.sub.6-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). a His-Tag (SEQ ID No:4), a T7-Tag (SEQ ID NO:5), FLAG-Tag (SEQ ID NO:6) or StrepII-Tag (SEQ ID NO:7). Further, fluorescence tags such as green fluorescence protein (GFP) or its variants may be attached to a Rep-protein according to the invention.

[0034] In a particular preferred embodiment the MSBI1 genome-encoded Rep protein (MSBI1 Rep) is codon-optimized for the production in human cell lines (e.g. HEK-293, HEK293TT, HEK293T, HEK293FT, HaCaT, HeLa, SiHa, CaSki, HDMEC, L1236, L428, BJAB, MCF7, Colo678, any primary cell lines) as well as bovine (e.g. MAC-T) or murine cell lines (e.g. GT1-7). This is described in detail in PCT/EP2017/075774.

[0035] The Rep protein of the invention, including the Rep fragments and Rep variants as defined above, can be prepared by classical chemical synthesis. The synthesis can be carried out in homogeneous solution or in solid phase. The polypeptides according to this invention can also be prepared by means of recombinant DNA techniques.

[0036] “Subject” as used herein refers to a mammalian individual or patient, including murines, cattle, for example bovines, simians and humans. Preferably, the subject is a human patient.

[0037] “Anti-Rep antibody” as used herein refers to an antibody binding at a detectable level to Rep protein which affinity is more strongly to the Rep protein of the invention than to a non-Rep protein. Preferably, the antigen affinity for Rep protein is at least 2 fold larger than background binding. In particular the anti-Rep antibody is specific for the MSBI1 Rep having the amino acid sequence of SEQ ID NO:1 or MSBI2 Rep. In particular embodiments the antibody is cross-specific for MSBI1 Rep, MSBI2 Rep, CMI1 Rep, CMI2 Rep and/or CMI3 Rep. In certain embodiments the anti-Rep antibody is cross-specific for at least two, preferably all, off MSBI1 Rep, MSBI2 Rep, CMI1 Rep, CMI2 Rep and/or CMI3 Rep.

[0038] The inventors also tested the antibody level of breast cancer patients by contacting the Rep protein with a specimen suspected of containing anti-Rep protein antibodies under conditions that permit the Rep protein to bind to any such antibody present in the specimen. Such conditions will typically be physiologic temperature, pH and ionic strength using an excess of Rep protein. The incubation of the Rep protein with the specimen is followed by detection of immune complexes which may comprise the antigen. In certain embodiments either the Rep protein is coupled to a signal generating compound, e.g. detectable label, or an additional binding agent, e.g. secondary anti-human antibody, coupled to a signal generating compound is used for detecting the immune complex.

[0039] Anti-Rep antibodies can be detected and quantified in assays based on Rep protein as protein antigen, which serves as target for the mammalian, e.g. human, antibodies suspected in the specimen. Preferably, the Rep protein is purified and the specimen can be, for example, serum or plasma. The methods include immobilization of Rep protein on a matrix followed by incubation of the immobilized Rep protein with the specimen. Finally, the Rep-bound antibodies of the formed immunological complex between Rep protein and antibodies of the specimen are quantified by a detection binding agent coupled to a signal generating compound, e.g. secondary HRP-(horseradish-peroxidase)-coupled detection antibody allowing for HRP-substrate based quantification. This signal generating compound or label is in itself detectable or may be reacted with an additional compound to generate a detectable product.

[0040] Design of the immunoassay is subject to a great deal of variation, and many formats are known in the art. Protocols may, for example, use solid supports, or immunoprecipitation. Most assays involve the use of binding agents coupled to signal generating compounds, for example labelled antibody or labelled Rep protein; the labels may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the immune complex are also known; examples of which are assays which utilize biotin and avidin or streptavidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.

[0041] The immunoassay may be in a heterogeneous or in a homogeneous format, and of a standard or competitive type. Both standard and competitive formats are known in the art.

[0042] In an immunoprecipitation or agglutination assay format the reaction between the Rep protein and the anti-Rep antibody forms a network that precipitates from the solution or suspension and forms a visible layer or film of precipitate. If no anti-Rep antibody is present in the specimen, no visible precipitate is formed.

[0043] In further embodiments the inventors used methods wherein an increased amount of Rep protein in a sample correlates with a diagnosis or predisposition of breast cancer. In such embodiments the Rep protein in the sample is detected by anti-Rep antibodies.

[0044] “Sample” as used herein refers to a biological sample encompassing cancerous breast tissue, peripheral tissue surrounding the cancerous tissue and (benign) hyperplasias. The samples encompass tissue samples such as tissue cultures or biopsy specimen.

[0045] Such methods (ex-vivo or in-vitro) may comprise the steps of detecting Rep protein in a sample from a subject by anti-Rep antibodies. In such methods Rep protein is detected in tissue samples by immunohistochemical methods or immunofluoresence microscopy.

[0046] In certain embodiments anti-Rep antibodies are used for the detection or capturing of the Rep protein in the sample.

[0047] The term “antibody”, preferably, relates to antibodies which consist essentially of pooled polyclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations. As used herein, the term “antibody”(Ab) or “monoclonal antibody” (Mab) is meant to include intact immunoglobulin molecules as well as antibody fragments (such as, for example, Fab and F(ab′)2 fragments) which are capable of specifically binding to Rep protein. Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody. Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies useful for the purposes of the present invention include chimeric, single chain, multifunctional (e.g. bispecific) and humanized antibodies or human antibodies.

[0048] In certain embodiments the antibody or antigen binding fragment thereof is coupled to a signal generating compound, e.g., carries a detectable label. The antibody or antigen binding fragment thereof can be directly or indirectly detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme. Those of ordinary skill in the art will know of other suitable labels for binding to the antibody, or will be able to ascertain such, using routine experimentation.

[0049] Anti-Rep antibodies are, preferably, raised (generated) against a Rep protein having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:8 or a fragment thereof by methods well known to those skilled in the art.

[0050] In certain embodiments anti-Rep antibodies are used in the methods of the invention which are capable of binding to several or all kinds of Rep proteins from the group of the Small Sphinx Genome (anti-Small-Sphinx-like Rep antibody or anti-SSLRep antibody). Such anti-SSLRep antibody binds to an epitope within the conserved N-terminal region of the Rep protein from amino acids 1 to 229 of SEQ ID NO:1. In particular embodiments anti-Rep antibodies of the anti-SSLRep type are used which bind to an epitope within SEQ ID NO:2 (amino acids 32-49 of SEQ ID NO:1) or SEQ ID NO:3 (amino acids 197-216 of SEQ ID NO:1). The peptide fragments of SEQ ID NO:2 and SEQ ID NO:3 are highly conserved among the Rep proteins from the Small Sphinx Genome group and appear to be exposed due to their hydrophilic character. Anti-Rep antibodies of the anti-SSLRep type may be produced by immunization, for example of mice or guinea pig, by peptides consisting essentially of the amino acid sequences as depicted in SEQ ID NOs:2 or 3; or by other immunogenic fragments, preferably which may comprise at least 8-15 amino acids, derived from the conserved N-terminal Rep protein region from amino acids 1 to 229 of SEQ ID NO:1.

[0051] In further embodiments anti-Rep antibodies specific for MSBI1 Rep protein are used. Such antibodies may be produced, for example, by immunization of a mammal such as mice or guinea pig with a full-length Rep protein having the amino acid sequence of SEQ ID NO:1.

[0052] Preferably, the methods of the invention use anti-Rep antibodies which are capable of detecting Rep protein up to ranges from picogramm to femtogramm.

[0053] Examples of such groups of anti-Rep antibodies are shown in Table 1:

TABLE-US-00001 Antibody Rep-Protein DSMZ Group Localisation Specificity Antibody deposit Group A cytoplasm + MSBI1 + AB01 523-1-1 DSM nuclear small-sphinx- (Ab 1-5) ACC3327 membrane like (+nucleus) All BMMF1 Reps Group B speckles in MSBI1 AB02 304-4-1 DSM cytoplasm small-sphinx- (Ab 5-2) ACC3328 like Group C cytoplasm + MSBI1 MSBI1 381-6-2 DSM nuclear specific (Ab 3-6) ACC3329 membrane (+ MSBI1 572-13-19 nucleus) (Ab 10-3) MSBI1 617-1-3 (Ab 11-5) Group D speckles in MSBI1 D1: MSBI1 961- DSM cytoplasm specific 2-2 (Ab 9-2) ACC3331 D2: MSBI1 761- DSM 5-1 (Ab 13) ACC3330

[0054] Anti-Rep antibodies of group A have an epitope within the amino acid sequence depicted in SEQ ID NO:3 (aa 198-217 of SEQ ID NO:1) and are capable of detecting MSBI1 Rep and Rep proteins which may comprise this conserved epitope of the Small Sphinx Genome group (e.g. MSBI2, CMI1, CMI4). In immunofluoresence assays such anti-Rep antibodies detect a specific Rep localisation pattern, wherein the main localisation is homogeneously distributed over the cytoplasm and nuclear membrane; and additional weak and homogeneously distributed localisation is seen in the nucleus. An example of such a group A antibody is antibody AB01 523-1-1 (also called antibody 1-5; DSM ACC3327) which was employed in the examples as group A antibody.

[0055] Anti-Rep antibodies of group B have an epitope within the amino acid sequence depicted in SEQ ID NO:2 (aa 33-50 of SEQ ID NO:1) and are capable of detecting MSBI1 Rep and Rep proteins which may comprise this conserved epitope of the Small Sphinx Genome group (e.g. MSBI2, CMI1, CMI4). In immunofluoresence assays such anti-Rep antibodies detect specifically speckles (cytoplasmatic aggregations) of the Rep protein (often in the periphery of the nuclear membrane). An example of such a group B antibody is the antibody designated as ABO2 304-4-1 (also called antibody 5-2; DSM ACC3328) which was employed in the examples as group B antibody.

[0056] Anti-Rep antibodies of group C detect specifically a structural epitope of MSBI1 (SEQ ID NO:1). In immunofluoresence assays such anti-Rep antibodies detect a specific Rep localisation pattern, wherein the main localisation is homogeneously distributed over the cytoplasm and nuclear membrane; and additional weak and homogeneously distributed localisation is seen in the nucleus. An example of such a group C antibody is antibody MSBI1 381-6-2 (also called antibody 3-6; DSM ACC3329) which was employed in the Example as group C antibody with an epitope in the sequence of aa 230-324. Another example of an antibody of a group C antibody is antibody MBSI1 572-13-19 (also called antibody 10-3) detecting an epitope in the C-terminal domain of MSBI 1 Rep (aa 230-324). Another example of an antibody of a group C antibody is antibody MBSI1 617-1-3 (also called antibody 11-5) detecting an epitope in the N-terminal domain of MSBI 1 Rep (aa 1-136).

[0057] Anti-Rep antibodies of group D detect specifically a structural epitope of MSBI1 (SEQ ID NO:1), where antibody MSBI1 961-2-2 designated as “D1” (also called antibody 9-2; DSM ACC3331) detects an epitope depicted in SEQ ID NO:9 (aa 281-287) in the C-terminal domain of MSBI1. Antibody MSBI1 761-5-1 (also called antibody 13; DSM ACC3328) designated as “D2” detects a 3D structural epitope of MSBI1 which is exclusively accessible under in vivo conditions and is not accessible in Western Blots. In immunofluoresence assays such anti-Rep antibodies detect specifically speckles (cytoplasmatic aggregations) of the Rep protein (often in the periphery of the nuclear membrane.

[0058] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

[0059] The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.

Example 1: Detection of BMMF Protein Targets in Breast Tissue

[0060] All tissue samples were provided by the tissue bank of the National Center for Tumor Diseases (NCT, Heidelberg, Germany and Institute of Pathology, Heidelberg University Hospital, Germany) in accordance with the regulations of the tissue bank and the approval of the ethics committee of Heidelberg University.

[0061] Tissue Staining

[0062] The paraffin-embedded tissue sections (˜4 μm thickness) were stained with the Zytomed Chem-Plus HRP Polymer-Kit (Zytomed, POLHRP-100) and the DAB Substrate Kit High Contrast (Zytomed, DAB500plus) after EDTA epitope retrieval (Sigma E1161) with the given antibody incubations (c.f. Table 1) and hemytoxylin counterstain. Slides were scanned with a digital slide scanner (Hamamatsu) and analyzed based on with NDP.view2 Plus software (Hamamatsu).

TABLE-US-00002 TABLE 1 Final concentration Incubation Antibody Source Host Dilution in μg/ml time Primary Rep mAb #3-6 T. Bund, mouse 1:500 3.9 30 min at DKFZ room Rep mAb T. Bund, mouse 1:500 3.9 temperature #10-3 DKFZ CD68 Cell rabbit 1:1000 signaling #76437 Secondary rabbit anti- Abcam rabbit 1:500 20 min at mouse #125904 room temperature

[0063] Staining with anti-Rep antibodies (e.g. mAb 10-3, mAb 3-6) shows specific detection of protein targets in stromal tumor tissue regions within breast cancer patient samples 7G6MJX and 1HHY7K (FIGS. 2 and 3). In general, the anti-Rep detection resulted in intense staining of smaller sized aggregates mainly within the cytoplasmic regions of cells within the stroma. Additionally, a colocalization of the anti-Rep stained signals with CD68-positive macrophages was observed. The regions with highest Rep-specific antibody detection correlate with regions with highest detection levels for CD68 positive cells pointing towards a localization of the Rep-specific antigens in inflammatory tissue areas, i.e. regions with especially high levels of inflammatory monocytes, circulating macrophages, or resident tissue macrophages. No signal detection was observed in control stainings with an antibody isotype control.

Example 2: Tissue Staining and Tissue Analysis

[0064] Tissue microarray ZTMA26 was generated and provided by courtesy of Universitätsspital Zürich. In this data set 1 peritumoral tissue spot per patient was contained.

[0065] ZTMA26 was stained fully automatically on a BOND MAX machine (Leica Biosystems) with EDTA epitope retrieval buffer (Abcam, #ab93680). Primary antibody anti-BMMF1 Rep (#3-6, monoclonal, DKFZ Heidelberg) and isotype control antibody (Biolegend IgG1, MG1-45) were incubated for 30 min at room temperature (4 μg/ml). Secondary rabbit anti-mouse (Abcam #125904) was incubated for 20 min at room temperature. Detection was performed by using Bond Polymer Refine Detection Kit (Leica #DS9800) including DAB chromogen and hematoxylin counterstain. Slides were scanned using a Hamamatsu Nanozoomer slide scanner (Hamamatsu) and analyzed with NDP.view2 Plus software (Hamamatsu).

[0066] Tissue Analysis

[0067] For analysis of BMMF1 Rep staining on the TMAs, the antibody staining was characterized based on two parameters: the percentage of stained cells (positivity) and intensity (I) of the signal within interstitial/stromal parts of the tissue spots. Epithelial parts and tumor cells were not included into analysis as they are not the target of BMMF positivity, in general. The positivity (POS) of BMMF1 Rep staining was assessed using a three-level scale in which 0 indicated no positive tissue parts at all, 1 indicated 1-10% positive, 2 indicated 11-30%, 3 indicated more than 30% positive cells distributed in several regions of the tissue spot. Intensity (I) was graded as follows: 0=no detection, 1=moderate, 2=intense staining. For statistical analysis, the immunoreactive score (IRS) was calculated as follows: IRS=I×POS; minimum value=0, maximum value=6 (Tab. 2).

TABLE-US-00003 TABLE 2 Scoring parameters for quantification of BMMF1 Rep staining on TMAs. Positivity Intensity (proportion POS (Staining 1 of positive Target intensity, 1) cells, POS) BMMF1 Rep 0 no detection 0 0 1 moderate 1 1-10% 2 strong 2 11-30% 3 >31%

IRS=I×POS

[0068] IRS=immunoreactive score Using these scoring criteria the samples from peritumoral tissue (16 patients) based on BMMF1 Rep staining are:

[0069] 12% negative (IRS 0)

[0070] 88% positive (at least IRS 1) [with 44% significantly positive=at least IRS 2]

[0071] These results are shown as bar diagrams in FIG. 5.

TABLE-US-00004 SEQUENCE SUMMARY SEQ ID NO SEQUENCE 1 Amino acid sequence of Rep protein encoded by MSBI1.176 MSDLIVKDNALMNASYNLALVEQRLILLAIIEARETGKGINANDPLTVHASS YINQFNVERHTAYQALKDACKDLFARQFSYQEKRERGRINITSRWVSQIGYM DDTATVEIIFAPAVVPLITRLEEQFTQYDIEQISGLSSAYAVRMYELLICWRST GKTPIIELDEFRKRIGVLDTEYTRTDNLKMRVIELALKQINEHTDITASYEQHK KGRVITGESEKEKHKKQNSDKTPKNSDSSPRIVKHSQIPTNIVKQPENAKMSD LEHRASRVTGEIIVIRNRLSDRFKQGDESAIDMMKRIQSEIITDAIADQWESKLE EFGVVF 2 Amino acid sequence of Rep peptide fragment EARETGKGINANDPLTVH 3 Amino acid sequence of Rep peptide fragment KQINEHTDITASYEOHKKGRT 4 His-Tag (with two neutral stuffer amino acids) GAHHHHHH 5 T7-Tag MASMTGGQQMG 6 FLAG-Tag DYKDDDDK 7 Strep-II-Tag WSHPQTEK 8 Amino acid sequence of Rep protein encoded by MSBI2.176 MSKINVKDNALMNASYNLDLVEQRLILLAIIEARESGKGINANDPLTVHA ESYINQFGVHRVTAYQALKDACDNLFARQFSYQSKSEKGNIQNHRSRWVS EIIYIDTEATVKIIFAPAIVPLITRLEEQFKYDIEQISDLSSAYAIRLY ELLICWRSTGKTPIIGLGEFRNRVGVLDSEYHRIAHLKERVIEHSIKQIN EHTDITATYEQHKKGRTITGFSFKFKQKKPKQAEIATETPKTATNDPDTT KPLTEPQIAKYSMILCKLGSISDLSNFPDYPAFANWIGNILRNIPEKADEQ IAKRIFTALKTETDYSKKN 9 MSBI.1 specific epitope NRLSDRF 10 Amino acid sequence of Rep protein encoded by CMI1.252 MSDLIVKDNALMNASYNLALVEQRLILLAILEARETGKGINANDPLTVHASS YINQFNVERHTAYQALKDACKDLFARQFSYQEKRERGRINITSRWVSQIGYM DDTATVEIIFAPAVVPLITRLEEQFTQYDIEQISELSSAYAVRLYELLICWRSTG KTPIIDLTEFRKRLGVLDTEYTRTDNLKMRVIELGLKQINEHTDITASYEQHK KGRTITGESFKFKQKKKTGAEMPKNSDSSPHIEKPSQIPANIAKQPENAKKDD LGHRASKITGLIMSNGLADRFKRGDESVIDMMKRIKEEITTDTTADQWENKL EEFGVIFQS 11 Amino acid sequence of Rep protein encoded by CMI2.214 MSDLIVKDNALMNASYNLDLVEQRLILLAILEARETGKGINANDPLTVHAES YINQFGVARQTAYQALKDACKDLFARQFSYQEKRERGRANITSRWVSQIAYI DETATVEVIFAPAVVPLITRLEEQFTQYDIEQISGLSSAYAVRLYELLICWRST GKTPVIELAEFRKRLGVLNDEYTRSDNFKKWIIENPIKQINEHTDITASYEQH KKGRTITGFSFKFKQKKKTEPETPKNSDSSQRIEKPSQIPANIVKQPENANLSD LQHRASKITGLIMSNRLSDRFKQGDESIMQMMARIQSETTTDSIADQWQSKLE EFGVVF 12 Amino acid sequence of Rep protein encoded by CMI3.168 MSDLIVKDNALMNASYNLALVEQRLILLAILEARETGKGINANDPLTVHASS YINQFNVERHTAYQALKDACKDLFARQFSYQEKRERGRANITSRWVSQIAYI DETATVEVIFAPAVVPLITRLEEQFTQYDIEQISGLSSAYAVRLYELLICWRTT GKTPVLDLTEFRKRLGVLDTEYTRTDNLKMRVIEQSLKQINKHTDITASYEQ HKKGRTITGFSFKFKQKKKTEPETPKNNDSGVSKPKTVEIPAEVVKQPKNTN LSDLEKRVRMITGAIAKNNLASRFQHGNESPLDMMKRIQSEITSDETADLWQ NKLESMGVVF 13 DNA sequence MSBI1 Rep codon-optimized ATGAGCGACCTGATCGTGAAAGACAATGCCCTGATGAACGCCTCCTACA ACCTGGCACTGGTCGAACAGAGACTGATTCTGCTGGCTATCATCGAGGCA AGGGAGACCGGCAAGGGCATCAACGCCAATGACCCCCTGACAGTGCACG CCAGCTCCTACATCAACCAGTTTAATGTGGAGCGCCACACCGCCTATCAG GCCCTGAAGGACGCCTGCAAGGATCTGTTTGCCCGGCAGTTCAGCTACCA GGAGAAGCGGGAGAGAGGCAGGATCAACATCACAAGCAGATGGGTGTC CCAGATCGGCTATATGGACGATACCGCCACAGTGGAGATCATCTTTGCAC CAGCAGTGGTGCCTCTGATCACCAGGCTGGAGGAGCAGTTCACACAGTA CGACATCGAGCAGATCTCCGGACTGTCTAGCGCCTACGCCGTGCGCATGT ATGAGCTGCTGATCTGTTGGCGGTCTACCGGCAAGACACCTATCATCGAG CTGGATGAGTTCCGCAAGCGGATCGGCGTGCTGGACACCGAGTACACCA GAACAGATAACCTGAAGATGAGAGTGATCGAGCTGGCCCTGAAGCAGAT CAATGAGCACACCGATATCACAGCCTCTTATGAGCAGCACAAGAAGGGC CGCGTGATCACCGGCTTCAGCTTTAAGTTCAAGCACAAGAAGCAGAACTC TGACAAGACACCAAAGAATAGCGATTCCTCTCCCCGGATCGTGAAGCAC AGCCAGATCCCTACCAACATCGTGAAGCAGCCAGAGAATGCCAAGATGT CCGACCTGGAGCACAGGGCATCTAGGGTGACAGGCGAGATCATGAGAAA TAGGCTGAGCGATCGGTTCAAGCAGGGCGACGAGTCCGCCATCGATATG ATGAAGAGAATCCAGTCCGAGATCATCACCGACGCCATCGCCGATCAGT GGGAATCTAAACTGGAAGAGTTTGGAGTCGTGTTTGGAGCACATCACCAT CATCATCACTGA 14 Protein sequence MSBI1 Rep codon-optimized MSDLIVKDNALMNASYNLALVEQRLILLAIIEARETGKGINANDPLTVHASS YINQFNVERHTAYQALKDACKDLFARQFSYQEKRERGRINITSRWVSQIGYM DDTATVEIIFAPAVVPLITRLEEQFTQYDIEQISGLSSAYAVRMYELLICWRST GKTPIIELDEFRKRIGVLDTEYTRTDNLKMRVIELALKQINEHTDITASYEQHK KGRVITGFSEKEKHKKQNSDKTPKNSDSSPRIVKHSQIPTNIVKQPENAKMSD LEHRASRVTGEIMRNRLSDRFKQGDESAIDMMKRIQSEIITDAIADQWESKLE EFGVVFGA 15 DNA sequence MSBI1 Rep wild-type ATGAGCGATTTAATAGTAAAAGATAACGCCCTAATGAATGCTAGTTATAA CTTAGCTTTGGTTGAACAGAGGTTAATTCTATTAGCAATCATAGAAGCGA GAGAAACAGGCAAAGGGATTAATGCCAATGATCCTTTAACAGTTCATGC AAGTAGCTATATCAATCAATTTAACGTAGAAAGGCATACGGCATATCAA GCCCTCAAAGATGCTTGTAAAGACTTGTTTGCCCGTCAATTCAGTTACCA AGAAAAGCGAGAACGAGGACGAATTAATATTACAAGTCGATGGGTTTCG CAAATTGGCTATATGGACGATACAGCAACCGTTGAGATTATTTTTGCCCC TGCGGTTGTTCCTCTGATTACACGGCTAGAGGAACAGTTCACCCAGTACG ATATTGAGCAAATTAGCGGTTTATCGAGTGCATATGCTGTTCGTATGTAC GAACTGCTGATTTGTTGGCGTAGCACAGGCAAAACACCAATTATTGAGCT AGACGAGTTTAGAAAGCGAATAGGTGTTTTAGATACTGAATACACTAGA ACAGATAATTTAAAGATGCGAGTTATTGAATTAGCCCTAAAACAAATCA ACGAACATACAGACATCACAGCAAGCTATGAACAACACAAAAAAGGGC GAGTGATTACAGGATTCTCATTCAAGTTTAAGCACAAGAAACAAAACAG CGATAAAACGCCAAAAAATAGCGATTCTAGCCCACGTATCGTAAAACAT AGTCAAATCCCTCCAACATTGTAAAACAGCCTGAAAACGCCAAAATGAG CGATTTAGAACATAGAGCGAGCCGTGTTACAGGGGAAATAATGCGAAAT CGTCTGTCAGATCGGTTTAAACAAGGCGATGAATCAGCAATCGACATGAT GAAACGTATTCAAAGTGAAATAATAACCGATGCAATAGCAGACCAGTGG GAAAGCAAACTGGAGGAGTTTGGCGTGGTTTTTTAG

REFERENCES

[0072] Eilebrecht, S., et al. (2018),“Expression and replication of virus-like DNA in human cells”, Scientific Reports 8:2851.

[0073] Funk, M., et al. (2014). “Isolation of protein-associated circular DNA from healthy cattle serum”. Genome Announc 2 (4).

[0074] Giraldo, R., et al. (2011). “RepA-WH1 prionoid: a synthetic amyloid proteinopathy in a minimalist host.” Prion 5 (2):60-64.

[0075] 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).

[0076] 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).

[0077] Manuelidis L., 2011. “Nuclease resistant circular DNAs co-purify with infectivity in scrapie and CJD”. J. Neurovirol. 17:131-145.

[0078] 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.

[0079] The invention is further described by the following numbered paragraphs:

[0080] 1) Use of Bovine Meat and Milk Factor Group 1 (BMMF1) Rep Protein as a biomarker for breast cancer.

[0081] 2) The use of paragraph 1 wherein the Rep protein is a MSBI1 genome-encoded Rep protein (MSBI1 Rep), a MSBI2 genome-encoded Rep protein (MSBI2 Rep), a CMI1 genome-encoded Rep protein (CMI1 Rep), a CMI2 genome-encoded Rep protein (CMI2 Rep) or CMI3 genome-encoded Rep protein (CMI3 Rep).

[0082] 3) A method for providing a diagnosis or predisposition for breast cancer in a subject, comprising the step of detecting Rep protein in a sample from a subject by anti-Rep antibodies that bind to an epitope comprised by SEQ ID NO:2 or SEQ ID NO:3.

[0083] 4) The method of paragraph 3, wherein the antibody specific for Rep protein binds to an epitope that is within an amino acid sequence selected from the group consisting of amino acids from 1 to 136, from 137 to 229 and from 230 to 324 of SEQ ID NO:1.

[0084] 5) The method of paragraph 3 or 4, wherein the sample from a subject is selected from the group consisting of a cancerous breast tissue, peripheral tissue surrounding the cancerous tissue, (benign) hyperplasias.

[0085] 6) The method of any of paragraphs 3 to 5, wherein additionally CD68 positive cells are detected in the sample by an anti-CD68 antibody.

[0086] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.