Use of BMMF1 REP protein as a diagnostic marker for lung cancer

Abstract

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

Description

Technical Field

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

Background Art

[0002] Lung cancer is the second most commonly diagnosed cancer and a major cause of cancer-related death worldwide. Known risk factors include tobacco smoke and other chemical carcinogens, as well as red meat consumption. Adenocarcinoma is the most common histological subtype. According to the classification system of the International Association for the Study of Lung Cancer (IASLC), lung adenocarcinoma is classified as adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), and invasive adenocarcinoma (IA). The IASLC classification system has been demonstrated to have great value in predicting the prognosis of patients with resected lung adenocarcinoma. Patients with AIS and MIA and those who have undergone complete surgical resection have a favorable prognosis, with a 5-year disease-free survival (DFS) rate higher than 90%. In contrast, patients with the IA subtype have a worse prognosis with an average 5-year DFS of less than 45%. Successfully classifying lung cancer patients into subtypes with differences related to the prognosis of disease-free survival is useful for stratifying patients for adjuvant therapy before or after surgical resection.

Summary of the Invention

Problems to be Solved by the Invention

[0003] Once a lung tumor is identified, the chances of cure under current treatment modalities are limited. Therefore, methods to improve early diagnosis are extremely important, and there is a need for diagnostic markers that facilitate the early detection of lung cancer and further insight into its etiology.

Means for Solving the Problems

[0004] 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 results in 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 lung tissue stroma. This accumulation can also be induced by the uptake of specific molecules that represent BMMF receptors. 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 BMMF intake 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 intrinsically high replication activity can represent targets rich in random DNA mutations that allow for the stochastic occurrence of mutations, which are fundamental requirements for tumor formation and the development of lung 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.

[0005] Specifically, polymorphic vesicle structural characteristics that were regularly identified by staining lung cancer patient tissues for the expression of the Rep protein of the BMMF1 subgroup 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 lung cancer. After staining with a gold-labeled monoclonal antibody against BMMF1 Rep, gold-labeled structurally dense regions are shown in preselected areas of lung 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 highest, i.e., the regions where the levels of inflammatory monocytes, circulating macrophages, or resident tissue macrophages are particularly high.

[0006] 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 genome of the bovine and milk factor (BMMF) was 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).

[0007] 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 were deposited in the EMBL data bank under accession numbers LK931491 (MSBI1.176) and LK931492 (MSBI2.176) (Whitley C. et al. 2014) and were sequenced and described in International Publication No. WO 2016 / 005054.

[0008] 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, C1MI.M3.1 and C1MI.9M.1, which were 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 were 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 were partially sequenced and described in International Publication No. WO 2016 / 005054. The CMI5 genome is described in Falida et al., 2017.

[0009] The inventors have shown that both the CMI genome and the MSBI genome replicate in vitro and exhibit significant production of transcribed RNA. Antibodies against MSBI1.176 Rep show Rep protein expression in peripheral tissues surrounding lung cancer tissues. The inventors have found that the Rep proteins encoded by the BMMF1 infectious agent (MSBI1 Rep, MSBI2 Rep, CMI1 Rep, CMI2 Rep, CMI3 Rep, CMI4 Rep, CMI5 Rep, HCBI4 Rep, HCBI6 Rep, C1MI.3M.1 Rep, C1MI.9M.1 Rep) are diagnostic markers for lung cancer.

[0010] The inventors produced monoclonal antibodies against the BMMF1 Rep protein using a consensus peptide derived from the full-length MSBI1.176 Rep or two conserved regions of the MSBI1.176 Rep amino acid sequence. In certain embodiments, the anti-Rep antibody binds to an epitope of the MSBI1.176 Rep protein exemplified in FIG. 5. Particularly preferred antibodies bind to epitopes within amino acid sequences 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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BEST MODE FOR CARRYING OUT THE INVENTION

[0012] The present invention teaches that the Rep protein can serve as a diagnostic marker indicating an increased risk of developing lung cancer, diagnosing lung cancer, or confirming the diagnosis of lung cancer.

[0013] The term "lung cancer" means cancer that develops as a result of uncontrolled cell growth in the lungs and bronchial system. These malignancies can develop as a result of existing benign hyperplasias (e.g., chondroma, lipoma, fibroma) in which genetic mutations promote the transition from normal to cancerous growth. The term "lung cancer" means the pre-disease stage, early or late stage of the disease, and metastases therefrom. "Lung cancer" encompasses all malignant neoplasms of the lungs and bronchial system. Most lung tumors are "carcinomas," i.e., malignant tumors arising from epithelial cells.

[0014] The present invention encompasses a systematic examination of healthy lung tissue (tissue from individuals without a cancer diagnosis or specific signs of disease) for assessing future disease risk. This means that the present invention is suitable for determining the predisposition to develop lung cancer by using BMMF Rep as an early diagnostic marker.

[0015] "Predisposition" means a tendency to a medical condition, according to 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.

[0016] As used herein, "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), or 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 in the EMBL database 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 in the EMBL database 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 in the EMBL database 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 in the EMBL database 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 Data Bank 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 Data Bank 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 Data Bank 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 Data Bank 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 Data Bank under accession number LR215496 and has the amino acid sequence shown in SEQ ID NO: 20.

[0017] 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.

[0018] The "Rep protein" also encompasses fragments and variants of a protein having SEQ ID NO: 1 or 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 an anti-Rep antibody specific for the Rep protein having the amino acid sequence of SEQ ID NO: 1. Preferably, such a fragment is an immunogenic fragment of a protein having the amino acid sequence 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, includes at least one epitope of an anti-Rep protein antibody against the Rep protein of SEQ ID NO: 1, and preferably contains 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 the N-terminal conserved region, the C-terminal variable region, the first or second DNA binding domain. Variants of a protein 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 as compared to their respective sequences, 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 an anti-Rep antibody specific for the Rep protein having the amino acid sequence of SEQ ID NO: 1. The definition of a variant includes, for example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids, peptide nucleic acids (PNAs), etc.), polypeptides having substituted linkages, and polypeptides having other modifications known in the art both of 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 may 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.

[0019] 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 International Patent Application No. PCT / EP2017 / 075774.

[0020] The Rep proteins of the present invention, including the above-mentioned Rep fragments and Rep variants, can be prepared by classical chemical synthesis. The synthesis can be carried out in homogeneous solution or in the solid phase. The polypeptides according to the present invention can also be prepared by recombinant DNA technology.

[0021] As used herein, "subject" refers to an individual or patient of a mammal, including a mouse, a cow, such as different cow species, a monkey, and a human. Preferably, the subject is a human patient.

[0022] As used herein, "anti-Rep antibody" refers to an antibody that binds to the Rep protein of the present invention at a detectable level with a stronger affinity for the Rep protein than for non-Rep proteins. Preferably, the antigen affinity for the Rep protein is at least 2-fold, 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, HCBI6, C1MI.3M.1, and / or C1MI.9M.1. In certain embodiments, the anti-Rep antibody cross-detects at least two, preferably all, of MSBI1 Rep, MSBI2 Rep, CMI1 Rep, CMI2 Rep, CMI3 Rep, CMI4 Rep, HCBI4, HCBI6, C1MI.3M.1, and / or C1MI.9M.1.

[0023] The inventors also tested the antibody levels in lung 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.

[0024] Anti-Rep antibodies can be detected and quantified in assays based on the Rep protein as a protein antigen, which serves as a target for antibodies of mammals suspected in a sample, such as humans. 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-binding detection antibody that enables quantification based on an HRP (horseradish peroxidase) substrate of a detection binder conjugated to a signal-generating compound. This signal-generating compound or label is either detectable itself or can react with a further compound to generate a detectable product.

[0025] The design of immunoassays is diverse and many formats are known in the art. The protocol can use, for example, a solid-phase support or immunoprecipitation. Most assays involve the use of a binder conjugated to a signal-generating compound, such as a labeled antibody or a labeled Rep protein; 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-labeled and mediated immunoassays such as ELISA assays.

[0026] Immunoassays can be of a heterologous or homologous format and can be of a standard or competitive type. Both standard and competitive formats are known in the art.

[0027] 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.

[0028] In a further embodiment, the inventors used a method in which the increased amount of Rep protein in a sample correlates with the diagnosis or predisposition of lung cancer. In such an embodiment, the Rep protein in the sample is detected by an anti-Rep antibody.

[0029] As used herein, "sample" refers to a biological sample including cancerous lung tissue, peripheral tissue surrounding the cancerous tissue, and (benign) hyperplasia. Samples include tissue samples such as tissue cultures or biopsy specimens. Further, the term "sample" includes sputum, breath condensate, and bronchoalveolar lavage (BAL).

[0030] 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.

[0031] In certain embodiments, the anti-Rep antibody is used for the detection or capture of the Rep protein in a sample.

[0032] The term "antibody" preferably relates to antibodies consisting essentially of pooled polyclonal antibodies having different epitope specificities, as well as separate 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. Thus, these fragments, as well as products of FAB or other immunoglobulin expression libraries, are preferred. Further, antibodies useful for the purposes of the present invention include chimeric, single-chain, multifunctional (e.g., bispecific) and humanized antibodies or human antibodies.

[0033] 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 chelator, or an enzyme. One of ordinary skill in the art will know or be able to ascertain other suitable labels for binding to the antibody using routine experimentation.

[0034] In a preferred embodiment, BMMF protein detection is possible in sputum and exhaled condensate and bronchoalveolar fluid (BAL) using an anti-Rep antibody and a reader compatible with immunoassay methods such as immunoblotting and ELISA, or an antibody-independent detection method such as mass spectrometry or amplified protein detection methods in proteomics (such as NGS-linked O-Link technology).

[0035] In certain embodiments, detection of BMMF protein based on an anti-Rep antibody-conjugated contrast agent (reacting with Rep-positive cells) in low-dose CT is possible.

[0036] 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 of ordinary skill in the art.

[0037] 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 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.

[0038] In further embodiments, anti-Rep antibodies specific for the MSBI1 Rep protein are used. Such antibodies can be produced, 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.

[0039] Preferably, the methods of the invention use anti-Rep antibodies that can detect Rep proteins in the range from picograms to femtograms.

[0040] An example of such a group of anti-Rep antibodies is shown in Table 1:

Table 1

[0041] The localization of antibody epitopes within MSBI1.176 Rep is shown in FIG. 8.

[0042] 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.

[0043] 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.

[0044] 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 localization 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; DSM ACC3329) used in the examples as a Group C antibody having an epitope in the sequence of aa230 - 324. 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).

[0045] 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 the speckles (cytoplasmic aggregates) of the Rep protein (often present around the nuclear membrane).

[0046] The availability of BMMF-specific Rep monoclonal antibodies and the demonstration of the DNA of these infectious agents in the tissues surrounding tumors have prompted the inventors to investigate the presence of specific structural components in patient tissues by immunogold electron microscopy using primary Rep monoclonal antibodies as tools. The identification of such gold-labeled structures in the tissues surrounding lung tumors from lung cancer patients is described in detail in the following examples.

[0047] The ultrastructural analysis of the present invention by immunoelectron microscopy (IEM) demonstrates BMMF Rep expression in macrophages in the tissues surrounding lung cancer. The gold-decorated targets are of different sizes and shapes, showing a polymorphic appearance distinct from the surrounding regions. Polymorphic structures with diameters of approximately 50 - 200 nm were found in the tissues surrounding lung tumors. Both cryo-IEM and conventional resin TEM allow for more delicate preservation of structure compared to pre-embedding IEM of formalin-fixed paraffin-embedded (FFPE) 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 that show higher-order organization.

[0048] 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 in lung cancer tumors and paired peritumoral tissues as well as tissues from non - tumor individuals were compared (Figure 9). A significant increase in the number of Rep+CD68+ cells (mean of about 2.2% of total cells) was observed peritumorally in lung cancer patients when compared to healthy controls (mean <0.1%). Similarly, an increase in the number of Rep+CD68+CD163+ cells (median of about 0.75%) was observed peritumorally in lung cancer patients compared to healthy controls (mean <0.1%), indicating that a large number of Rep+ Mfs are represented by M2 - like macrophages that were positively stained with both CD68 and CD163 antibodies. Approximately 40% of Rep+CD68+ macrophages in peritumoral lung tissues are also CD163+. On average, 22% of all peritumoral CD68+ macrophages were Rep+. The number of CD68+ cells did not show a significant change between peritumoral tissues of lung cancer patients (median 11.0%) and non - tumor controls (median 4.5%). In conclusion, the data support the detection of lung cancer individuals based on increased levels of peritumoral Rep+ macrophages. The numbers of Rep+, CD68+, and Rep+CD68+ cells were significantly increased peritumorally 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, respectively) (Figure 9). In total, 19 out of 23 peritumoral lung tissues were tested IHC - positive with anti - Rep antibody 3 - 6.

[0049] Using the detection of Rep positivity in triple - positive macrophages (Rep+CD68+CD163), the differentiation of Rep - positive macrophages was characterized, generally distinguishing more M1 - like (CD68+CD163 -) macrophage polarization from 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 tumor - promoting, releasing cytokines and chemokines involved in the mobilization of tumor - promoting regulatory T cells.

[0050] The ultrastructural analysis of the present invention can be regarded as evidence that the Rep protein is a structural protein that can potentially be targeted by antibodies, both for prophylactic and therapeutic purposes.

[0051] The present invention is further illustrated by, but not limited to, the following examples.

Example

[0052] Example 1: Detection of the BMMF protein target in the tissue surrounding lung cancer by immunohistochemistry Tissue staining and tissue analysis Lung cancer tissues were obtained as FFPE sections from the University Pathology Institute Heidelberg according to the regulations of the tissue bank, including patient consent and approval by the Ethics Committee of the University of Heidelberg (Ethic approval S - 206 / 2005).

Table 2

[0053] FFPE IHC DAB staining was performed based on the Zytomed ChemPlus (HRP) staining kit (Zytomed Systems) including epitope retrieval (30 min, 95 °C) with EDTA (pH 8.5, Sigma) as previously described (Bund et al., 2021). Following Rep staining with AB3 (1 h, room temperature), slides were stained with the DAB high contrast kit (Zytomed Systems) and hematoxylin counterstain. Slides were scanned using a Hamamatsu Nanozoomer slide scanner (Hamamatsu) and analyzed with NDP.view2 Plus software (Hamamatsu). Staining with anti-Rep antibodies (e.g., mAb 3-6; Table 2) showed detection of the protein target in the peritumoral and bronchial tissue areas within lung cancer patient samples. In general, anti-Rep detection resulted in strong staining of smaller-sized aggregated structures mainly within the cytoplasmic region of the stained cells (Figure 1, center). Staining with anti-Rep antibodies was consistent with CD68 staining in CD68-positive macrophages (Figure 1, left). The regions with the highest Rep-specific antibody detection correlate with the regions having the highest detection levels for CD68-positive cells indicating the localization of the Rep-specific antigen in the inflammatory tissue area, i.e., areas with 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 (Figure 1, right). Staining with anti-Rep antibodies also resulted in co-staining with CD68-positive macrophages in tissue areas with still intact alveolar morphology in the peritumoral region (Figure 2).

[0054] Example 2: Pre-embedding immunoelectron microscopy (IEM) Using lung cancer patient-derived tissues that were positively stained for BMMF Rep in IHC, the localization and physical expression of the Rep target were analyzed with ultrastructural resolution. The inventors first performed IHC DAB staining on one of a series of consecutive FFPE sections using monoclonal anti-BMMF1 Rep AB3 to identify positive tissue regions (Figure 3, left). Based on Example 1, consecutive sections containing the identified positively stained regions were then gold-labeled by pre-embedding IEM (pre-IEM) (right IEM magnification series in Figure 3).

[0055] Thus, the standard procedures for immunolabeling FFPE materials and subsequent resin embedding for ultrathin sectioning were as follows: FFPE sections (3 μm thick) mounted on glass slides were de-waxed with xylene and rehydrated in ethanol with gradually changing concentrations. The immunoreaction was performed by incubation in 40 mM glycine (aldehyde block), 1% BSA (protein block), primary antibody AB3, and polyclonal rabbit anti-mouse (1:150, Z0259, Dako) as a linker for 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.

[0056] Pre-embedding IEM staining revealed a high density of exclusive gold labeling on distinct dense particle structures that were polymorphic in the size range of approximately 50 - 200 nm. The identified structures differed in size and shape and appeared to contain filamentous substructures highlighted by arrows (Figure 4). Apart from these structures, gold particles did not accumulate in other regions of the tissue. Due to the impairment of structure preservation inherent to the FFPE protocol, the cellular localization context was not identified.

[0057] Example 3: Cryo-IEM (Tokuyasu method) after overexpression of MSBI1.176Rep in HEK293TT for validation of pre-IEM Cell culture and transfection HEK293TT cells were cultured as previously reported ((Buck et al. 2005, de Villiers et al. 2011), and transfected with polyethyleneimine (PEI, Sigma) at 70 - 80% confluence in 6-well plates with 1 μg of plasmid DNA per well to overexpress 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).

[0058] 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, followed by an HRP-conjugated goat anti-mouse IgG secondary antibody (115-035-205, Dianova).

[0059] 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 of 40 kDa and 37 kDa were seen for 3xFlag Rep and untagged Rep, respectively. Additionally, smaller molecular weight bands, as described in a previous study (Bund et al. 2021), were positively stained (Figure 5).

[0060] 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. Frozen sections 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.

[0061] Overexpression of both constructs led to the formation of large separated complexes in the cytoplasm of transfected cells (Figure 6). 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 7A) and showed association with the membrane (Figure 7B). Isotype antibodies did not react with the target structure (Figure 6). Transfection with the pcDNA3.1(-) vector (without Rep insert) did not result in separated structures.

[0062] 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).

[0063] 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 membranes (asterisks, see Fig. 7C).

[0064] Example 4: Cell-based quantification by immunofluorescence microscopy of Rep-, CD68- and CD163-positive cells in tumors and tumor surrounding tissues of lung cancer patients and non-cancer controls Tissue staining and tissue analysis Lung cancer patient tissues were obtained as FFPE sections from the University Pathology Institute Heidelberg in accordance with the regulations of the tissue bank, including patient consent and approval by the Ethics Committee of the University of Heidelberg (ethics approval S-206 / 2005), and in full compliance with the standards 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).

[0065] Based on the signal intensities of DAPI nuclei and antibody staining of cells in the digitized scans after simultaneous immunodetection of Rep / CD68 / CD163, semi - automated quantification of Rep, CD68, and CD163 staining in individual FFPE tissues was performed using an in - house developed script of ImageJ / Fiji (v1.52). Cell density was measured in 30 randomly selected microscopic fields. The data were represented as the median percentage 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.

Table 3

[0066] Summary of sequences TIFF2025518483000005.tif232170TIFF2025518483000006.tif215170TIFF2025518483000007.tif220170TIFF2025518483000008.tif195170References

[0067] 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) 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). Manuelidis L., 2011. “Nuclease resistant circular DNAs co - purify with infectivity in scrapie and CJD”. J. Neurovirol. 17:131 - 145. 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. de Villiers, E. M. and H. 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. doi: 10.3390 / cancers13215407. de Villiers, E.-M., K. Gunst, D. Chakraborty, C. Ernst, T. Bund and H. zur Hausen (2019). "A specific class of infectious agents isolated from bovine serum and dairy products and peritumoral colon cancer tissue." Emerging microbes & 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

Claims

1. Use of bovine meat and milk factor group 1 (BMMF1) Rep protein as a diagnostic marker for lung cancer.

2. The use according to claim 1, wherein 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), CMI4 genomic code Rep protein (CMI4 Rep), HCBI6 genomic code Rep protein (HCBI6 Rep), HCBI4 genomic code Rep protein (HCBI4 Rep), C1MI3M.1 genomic code Rep protein, C1MI.9M.1 genomic code protein (C1MI.9M.1 Rep).

3. A method for diagnosing or providing a predisposition to lung cancer in a subject, comprising the step of detecting a 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 in that.

4. The method according to claim 3, wherein 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, wherein the sample derived from the subject is selected from the group consisting of cancerous lung tissue, peripheral tissue surrounding the cancerous tissue, and (benign) cysts.

6. The method according to any one of claims 3 to 5, wherein 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, wherein CD163-positive cells are further detected in the sample by an anti-CD163 antibody.

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