Reagents against human endogenous retroviruses to target cancer cells

Abstract

The present invention relates to affinity reagents that are capable of specifically binding to a surface antigen of a virus belonging to the family of human endogenous retroviruses (HERV), in particular to a HERV envelope protein, for use in the prophylactic and / or therapeutic treatment of a cancer in a subject in need thereof. The invention relates also to the use of such affinity reagents in the diagnosis or prognosis of cancer, in particular lymphoma.

Description

[0001] REAGENTS AGAINST HUMAN ENDOGENOUS RETROVIRUSES TO TARGET

[0002] CANCER CELLS

[0003] DESCRIPTION

[0004] Technical field of the invention

[0005] The present invention relates to affinity reagents that are capable of specifically binding to a surface antigen of a virus belonging to the family of human endogenous retroviruses (HERV), in particular to the HERV envelope protein, for use in the prophylactic and / or therapeutic treatment of a cancer in a subject in need thereof. The invention relates also to the use of such affinity reagents in the diagnosis or prognosis of cancer, in particular lymphoma.

[0006] State of the art

[0007] No vaccine or other universally successful method for the prevention or treatment of cancer is currently available. Management of the disease currently relies on a combination of early diagnosis (through routine screening procedures) and aggressive treatment, which may include one or more of a variety of treatments such as surgery, radiotherapy, chemotherapy, and hormone therapy. The course of treatment for a particular cancer is often selected based on a variety of prognostic parameters, including an analysis of specific-tumor markers.

[0008] In particular, lymphomas are among the ten most frequent causes of cancer-related deaths. Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma, accounting for approximately 30% of all of cases. DLBCL is an heterogenous group of diseases that has been molecularly, phenotypically, and clinically defined in multiple molecular subtypes. Despite more than half of DLBCL can be cured, approximately 40% of DLBCL patients show refractory disease of relapse after an initial response. For many of these cases the outcome remains poor, even after the introduction of the most recent antibody-based or cellular therapies and there is a need of new therapeutic targets for this type of cancer.

[0009] Tumor-associated antigens recognized by the immune system are a very attractive target for human cancer diagnostics and therapy. However, few immunotherapy approaches have been used for the treatment and prevention of cancers. One problem limiting the success of cancer vaccines is that the immune system generally does not recognize cancer cells as being foreign, which is a requirement for initiating an immune response. Cancer immunotherapy, however, is limited due in part to the limited number of tumor-associated antigens identified to date. Accordingly, there remains an urgent need for potent, broad-spectrum therapeutics for use in therapy, prophylaxis, and diagnosis of cancers, particularly of lymphomas.

[0010] Summary of the Invention

[0011] The present invention is based on the discovery that surface antigens of human endogenous retroviruses (HERVs) and particularly those belonging to the K-family of HERVs, are differently expressed in normal and neoplastic samples from cancer patients, in particular those suffering from lymphoma, with a significantly higher expression in neoplastic than in normal samples, thus making them of potential use in cancer diagnosis, prognosis, and treatment.

[0012] HERVs are the result of germline infections from exogenous retroviruses that became incorporated into germ-line DNA millions of years ago. HERVs have been detected in humans and they encompass up to 8% of our genome. Due to technical issues, human endogenous retroviruses are discarded by default pipelines used for transcriptome analysis. Many HERV families have been described and HERV-K family is a human specific HERV family with intact open reading frames (ORF). HERV-K family comprises subgroups called HML-1 to HML-10 (human endogenous Mouse Mammary Tumor Virus-like, MMTV-like), representing separate germ-line infections. Members of the HML-2 subgroup have the highest coding competence and are the most studied in relation to diseases. Two HERV-K elements, part of the HML-2 subgroup, HERV-K113 and HERVK115, are of particular interest since they are full-length proviruses. HERV-K113 has complete open reading frames for all viral proteins. HERV-K 113 and HERV-K 115 were found in the genome in one copy located on chromosome 19p 13.11 and 8p23.1, respectively. Their frequencies depend on the geographical region, with the highest prevalence in Africans around 22% for HERV-K113 and 27% for HERV-K115, while HERV-K113 and HERV-K115 in the EU group was 8.26% and 5.71%, respectively.

[0013] As will be further detailed in the experimental section of the present specification, the inventors have surprisingly found that the envelope proteins of endogenous retroviruses of the K family, and particularly of HERV-K113, were among the top expressed with a significantly higher expression in neoplastic than in normal samples in subjects suffering from lymphoma. Using a phage display screening against four possible peptides of the envelope protein of HERV-K113 virus, the inventors have particularly identified and obtained a humanized nanobody with high affinity to the envelope protein of endogenous retroviruses, herein named as FF-01.

[0014] In certain aspects, the invention thus provides an affinity reagent capable of specifically binding to a surface antigen of a virus belonging to human endogenous retroviruses (HERV), preferably of at least one virus belonging to the HML-2 subgroup of the HERV-K family, more preferably of HERV-K113, for use in a prophylactic and / or therapeutic treatment of a cancer in a subject in need thereof. According to a preferred embodiment, said cancer is a lymphoma, more preferably is a diffuse large B-cell lymphoma (DLBCL).

[0015] In certain aspects, the invention provides an interfering agent capable of preventing and / or inhibiting the expression or activity of a target gene of a HERV, preferably of a target gene encoding for a surface antigen of at least one virus belonging to the HML-2 subgroup of the HERV-K family, more preferably of HERV-K 113, for use in a prophylactic and / or therapeutic treatment of a cancer in a subject in need thereof, preferably wherein said cancer is a lymphoma, more preferably wherein said cancer is DLBCL.

[0016] In certain aspects, the invention provides a method of preventing or treating a cancer, in particular lymphoma and more specifically DLBCL, or conditions or disorders resulting from such disease, comprising administering an affinity reagent and / or an interfering agent according to any embodiments herein disclosed, to a subject in need thereof.

[0017] The invention further provides an affinity reagent according to any embodiments herein disclosed for use in the diagnosis, prophylaxis and / or treatment of a subject having, or at risk of developing, a cancer, in particular lymphoma, more in particular DLBCL. Furthermore, the invention pertains to the use of the affinity reagents and / or the nucleic acid molecules of the invention in the diagnosis / detection of such cancers.

[0018] In certain aspects, the invention provides a pharmaceutical composition comprising at least one or more affinity reagents, and / or one or more interfering agents according to any one of the embodiments herein disclosed and a pharmaceutically acceptable carrier and its use in the prevention and / or treatment of a cancer in a subject in need thereof, in particular a lymphoma such as DLBCL.

[0019] In certain aspects, the invention provides an isolated cell line that produces the affinity reagent and / or an interfering agent according to any one of the embodiments herein disclosed. In certain aspects, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes the affinity reagent according to any one of the embodiments herein disclosed, as well as its use in a prophylactic and / or therapeutic treatment of a cancer in a subject in need thereof, in particular lymphoma such as DLBCL.

[0020] In certain aspects, the invention provides a vector comprising the nucleic acid molecule encoding the affinity reagent according to any one of the embodiments herein disclosed and / or comprising an interfering agent according to any one of the embodiments herein disclosed, wherein the vector optionally comprises an expression control sequence operably linked to the nucleic acid molecule.

[0021] The invention further provides the use of a vector according to any of the embodiments disclosed herein in a prophylactic and / or therapeutic treatment of a cancer in a subject in need thereof, in particular a lymphoma such as DLBCL.

[0022] In certain aspects, the invention provides a non-human transgenic animal or transgenic plant comprising the nucleic acid and / or interfering agent according to any one of the preceding embodiments, wherein the non-human transgenic animal or transgenic plant expresses said nucleic acid and / or interfering agent. In certain embodiments, said non-human transgenic animal is a mammal.

[0023] In certain aspects, the invention provides the in vitro use of a surface antigen of a HERV, preferably a surface antigen of at least one virus belonging to the HML-2 subgroup of the HERV-K family, more preferably of HERV-K113, as a biomarker for the diagnosis and / or prognosis and / or to monitor the development of a cancer in a subject, in particular wherein said cancer is a lymphoma such as DLBCL.

[0024] In certain aspects, the invention provides an in vitro method for revealing the presence of a HERV in a sample comprising the following steps: i) Contacting an affinity reagent according to any one of the embodiments herein disclosed; ii) Detecting the binding of said affinity reagent with a surface antigen of said HERV, in particular with the HERV envelope protein.

[0025] In certain aspects, the invention provides an in vitro method for the diagnosis and / or prognosis and / or to monitor the development of a cancer in a subject comprising the following steps: i) determining and / or quantifying the expression levels of a surface antigen of a HERV, preferably a surface antigen of at least one virus belonging to the HML-2 subgroup of the HERV-K family, more preferably of HERV-K113, in a sample isolated from said subject; preferably wherein said cancer is a lymphoma, more preferably wherein said cancer is a DLBCL. In certain aspects, the invention further provides a diagnostic or prognostic kit comprising as a specific reagent an affinity reagent according to any of the embodiments disclosed herein. Preferably, said kit is for use in a method for determining and / or quantifying, in a sample isolated from a subject, the expression levels of a surface antigen of a HERV, preferably a surface antigen of at least one virus belonging to the HML-2 subgroup of the HERV-K family, more preferably of HERV-K113.

[0026] The invention contemplates combinations of any of the foregoing aspects and embodiments of the invention.

[0027] Brief description of the drawings

[0028] Fig. 1. (A) Multidimensional scaling (MDS) plot showing normal and tumoral samples clustering differently based on HERV expression. (B) Multidimensional scaling (MDS) plot on tumoral samples based on HERV expression. Patients are coloured differently based on subtype: ABC, GCB or unclassified DLBCL. (C) Multidimensional scaling (MDS) plot on tumoral samples clustering them in 4 different groups based on HERV expression. (D) Alluvional plot showing the distribution of ABC, GCB, and unclassified patients in the four clusters. (E) Survival curve of the patients divided in the 4 different clusters

[0029] Fig. 2. (A) HERV-K (HML-2) RNA expression in tumoral versus normal samples. (B) HERV-K113 RNA expression in tumoral versus normal samples. * = p value <0.05. Icpm = log counts per million

[0030] Fig- 3. Multidimensional scaling (MDS) plot on cell lines divided by subtype.

[0031] Fig. 4. (A) Real time PCR for HERV-K env expression. Expression of env RNA in DLBCL cell lines. GAPDH was used as positive control. Expression normalized on non- retrotranscribed samples. (B) Immunoblot for HERV-K env expression in DLBCL cell lines with the respective quantification (column graph on the bottom). GAPDH was used as housekeeping gene

[0032] Fig- 5. Surface expression of HERVK envelope in lymphoma cell lines, with the respective quantification (MFI=median fluorescence intensity). Anti-HERVK envelope antibody showed envelope surface expression in some lymphoma cell lines. Rabbit polyclonal IgG was used as negative control Fig- 6. Schematic approach for the identification of HERV-K113 insertion in genomic DNA. Fig. 7. Pattern of KI 13 insertion in the genome. Agarose gel electrophoresis after PCR showing a full-length heterozygous insertion of HERV-K113 in RCK8 and no insertion in the U2932.

[0033] Fig. 8. Pattern of KI 13 insertion in the genome. Agarose gel electrophoresis after PCR showing a full-length heterozygous insertion of HERV-K113 in RCK8.

[0034] Fig- 9. SiRNA sequences used for HERV-K downregulation.

[0035] Fig. 10. (A) Nucleofection efficiency control showing around 100% of nucleofection efficiency. (B) Downregulation control after siRNAs compared to Ctr treated cells (SCR = scramble siRNA sequence). GAPDH was used as housekeeping gene. (C) Proliferation curve of cell lines treated with CTR siRNA (scramble) or siRNA2 or siRNA3 (D) Percentage of living cells after siRNA2 or siRNA3 treatment.

[0036] Fig. 11. (A) HERV-K113 protein sequence. In Bold highlight the sequences corresponding to the identified peptides (B) 3D protein structure modelling of HERV-K113. In the circle highlighted the part of the protein representing peptide2 and peptide3.

[0037] Fig- 12. ELISA performed on serum samples from lymphoma patients against the four identified peptides. Y-axes, 450nm absorbance.

[0038] Fig. 13. ELISA validations performed with serial serum dilutions.

[0039] Fig. 14. (A) FF-01 binding data obtained by Surface plasmon resonance on immobilized peptide of HERV-K envelope. (B) FACS data of FF-01 binding extracellular envelope in RCK-8 cell line.

[0040] Fig. 15. Sequence of peptide 3 is conserved among several HER-K family members.

[0041] Fig. 16. Antibody-Dependent Cell-Mediated Cytotoxicity experiment. ADCC performed coculturing lymphoma cell line RCK8 or U2932 with effector cells in the presence of negative IgG control (CTR-), non-binder Ab (FF-00) and envelope binder antibody (FF-01) for 6 and 24 hours.

[0042] Fig. 17. (A) FF-01 internalization in RCK-8 cell lines. (B) FF-01 internalization in RCK-8, U2932 and Toledo cell lines, normalized on FF-00 internalization. Cell lines incubated with FF-01 or neg CTR and FabFluor were followed for 40 hours with live cell imaging Incucyte. The increase in red fluorescent area was monitored over time and normalized on confluency. FF-01 internalization was presented as ratio compared to negative control. Detailed description of the invention

[0043] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, second ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), incorporated herein by reference.

[0044] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0045] As will be further illustrated in the detailed description of the invention, the term “affinity reagent” as used herein encompasses peptides, proteins, polypeptides, oligonucleotides, polynucleotides, aptamers, or any other molecular compound that is capable of specifically binding to a surface antigen of a human endogenous retrovirus (HERV) according to any of the variants disclosed in the present specification and in the claims, particularly to the envelope protein of at least one virus belonging to the HML-2 subgroup of the HERV-K family, more preferably of HERV-K113.

[0046] Affinity reagents in the form of proteins or polypeptides according to the present invention also encompass affinity reagents analogues comprising a conservative amino acid substitution (or insertion or deletion) with respect to the native sequence of the affinity reagent. Some embodiments of the invention include affinity reagent analogues with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 substitutions from the germline amino acid sequence. In certain embodiments, amino acid substitutions to an affinity reagent or antigenbinding portion thereof according to the invention are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity to form protein complexes, and (4) confer or modify other physicochemical or functional properties of such analogues, but still retain specific binding to a HERV surface antigen according to the invention, and particularly to a HERV envelope protein. Analogues can include various muteins of a sequence other than the normally occurring peptide sequence. For example, single or multiple amino acid substitutions, preferably conservative amino acid substitutions, may be made in the normally occurring sequence, preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence; e.g., a replacement amino acid should not alter the anti-parallel [beta]-sheet that makes up the immunoglobulin binding domain that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence. In general, glycine and proline would not be used in an anti-parallel [beta]-sheet. Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et ai, Nature 354: 105 (1991), incorporated herein by reference.

[0047] The term "polypeptide" encompasses native or artificial proteins, protein fragments and polypeptide analogues of a protein sequence. A polypeptide may be monomeric or polymeric. The term "isolated protein", "isolated polypeptide" or "isolated antibody" is a protein, polypeptide or antibody that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. A protein or polypeptide is "substantially pure", "substantially homogeneous", or "substantially purified" when at least about 60 to 75% of a sample exhibits a single polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% W / W of a protein sample, more usually about 95%, and preferably will be over 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

[0048] The term "antigen-binding portion" as used with reference to an affinity reagent according to any of the embodiments disclosed herein encompasses any portion of the affinity reagent that retains its capability of specifically binding to a surface antigen of a HERV according to the present invention under suitable binding conditions, and particularly to the envelope protein of said HERV, more preferably to the envelope protein of at least one virus belonging to the HML-2 subgroup of the HERV-K family, even more preferably of HERV-K113. In some embodiments, antigen-binding portions are peptides of at least 5, 6, 8 or 10 amino acids long. In other embodiments, the antigen-binding portions are at least 14, at least 20, at least 50, or at least 70, 80, 90, 100, 150 or 200 amino acids long. In other embodiments the antigen-binding portions are at least 20 or 25 amino acids long, preferably at least 50, 60, 70, 80, 90, 100, 150 or 200 amino acids long or longer, and can often be as long as a full- length polypeptide.

[0049] Where an "antibody" is referred to herein with respect to the invention, it is normally understood that an antigen-binding portion thereof may also be used. An antigen-binding portion of an antibody competes with the intact antibody for specific binding. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., second ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes).

[0050] Merely by way of example, antigen-binding portions according to the present invention may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some embodiments, antigen-binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, nanobodies and any polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the HERV surface antigen.

[0051] From N-terminus to C-terminus, both the mature light and heavy chain variable domains of an antibody according to the invention comprise the regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain herein is in accordance with the definitions of IMGT convention described in Lefranc et al. (2003), Developmental & Comparative Immunology 27.1 (2003): 55-77.

[0052] As used herein, a “Fd fragment” means an antibody fragment that consists of the VH and CH 1 domains; an “Fv fragment” consists of the VL and VH domains of a single arm of an antibody; and a “dAb fragment” (Ward et al, Nature 341 :544-546 (1989)) consists of a VH domain.

[0053] As used herein, the term "human antibody" means any antibody in which the variable and constant domain sequences are human sequences or any of the CDRs of the variable domain sequences are human sequences. The term encompasses antibodies with sequences derived from human genes, but which have been changed, e.g., to decrease possible immunogenicity, increase affinity, eliminate cysteines that might cause undesirable folding, etc. The term encompasses such antibodies produced recombinantly in non-human cells, which might impart glycosylation not typical of human cells.

[0054] The term "chimeric antibody" as used herein means an antibody that comprises regions from two or more different antibodies.

[0055] The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Epitopes or antigenic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three- dimensional structural characteristics, as well as specific charge characteristics. An epitope may be "linear" or "conformational." In a linear epitope, all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another.

[0056] The term "polynucleotide" as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms.

[0057] The term "isolated polynucleotide" as used herein means a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated polynucleotide" (1) is not associated with all or a portion of a polynucleotides with which the "isolated polynucleotide" is found in nature, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

[0058] The term "naturally occurring nucleotides" as used herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" as used herein includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkages" referred to herein includes oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See e.g., LaPlanche et al., Nucl. Acids Res. 14:9081 (1986); Stec et al, J. Am. Chem. Soc. 106:6077 (1984); Stein et al., Nucl. Acids Res. 16:3209 (1988); Zon et al., Anti-Cancer Drug Design 6:539 (1991); Zon et al.. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); U.S. Patent No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990), the disclosures of which are hereby incorporated by reference. An oligonucleotide can include a label for detection, if desired.

[0059] "Operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0060] The term "expression control sequence" as used herein means polynucleotide sequences that are necessary to affect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence.

[0061] The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression and processing and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0062] The term "vector", as used herein, means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, the vector is a plasmid, i.e., a circular double stranded piece of DNA into which additional DNA segments may be ligated. In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").

[0063] The term "recombinant host cell" (or simply "host cell"), as used herein, means a cell into which a recombinant expression vector has been introduced. It should be understood that "recombinant host cell" and "host cell" mean not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0064] The term "percent sequence identity" in the context of nucleotide or aminoacidic sequences means the residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs available, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132: 185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996); Pearson, J Mol. Biol 276:71-84 (1998); incorporated herein by reference).

[0065] The term "substantial similarity" or "substantial sequence similarity," when referring to a nucleic acid or fragment thereof, or aminoacidic means that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above. As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights as supplied with the programs, share at least 70%, 75% or 80% sequence identity, preferably at least 90% or 95% sequence identity, and more preferably at least 97%, 98% or 99% sequence identity. In certain embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

[0066] A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31 (1994). Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulphur-containing side chains: cysteine and methionine. Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al, Science 256: 1443-45 (1992), incorporated herein by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 loglikelihood matrix. Sequence identity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap" and "Bestfit" which can be used with default parameters as specified by the programs to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof.

[0067] As used herein, the terms "label" or "labelled" refers to incorporation of another molecule in the antibody. In one embodiment, the label is a detectable marker, e.g., incorporation of a radiolabelled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In another embodiment, the label or marker can be therapeutic, e.g., a drug conjugate or toxin. Various methods of labelling polypeptides and glycoproteins are known in the art and may be used.

[0068] As used herein, the term “interfering agent” is intended to encompass any agent which interferes with, prevents, inhibits or silence expression or activity of a target gene or genomic sequence of a target gene of a HERV, for example resulting in a reduction or suppression of cell proliferation.

[0069] As used herein, "inhibiting expression of target gene" includes any decrease in expression or protein activity or level of the target gene or protein encoded by the target gene. The decrease may be of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the expression of a target gene or the activity or level of the protein encoded by a target gene which has not been targeted by an interfering agent.

[0070] "Short interfering RNA" (siRNA), also referred to herein as "small interfering RNA" is defined as an agent which functions to inhibit expression of a target gene, e. g, by RNA interference. An siRNA may be chemically synthesized, may be produced by in vitro transcription, or may be produced within a host cell. In one embodiment, siRNA is a double stranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides in length, and more preferably about 19,20, 21, or 22 nucleotides in length, and may contain a 3'and / or 5'overhang on each strand having a length of about 0,1, 2, 3,4, or 5 nucleotides. The length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand. Preferably the siRNA is capable of promoting RNA interference through degradation or specific post- transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA).

[0071] "RNA interference" is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target gene results in the sequence specific degradation or specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene (see Cobum, G. and Cullen, B. (2002) J. of Virology 76 (18): 9225), thereby inhibiting expression of the target gene.

[0072] As used herein, the term "subject" refers to animal, preferably a mammal. The mammalian subject may be a non-human mammal, but is generally a human, such as a human adult. The subject will in general be a living subject. However, whilst the uses, methods and compositions of the present invention are especially suited for diagnosis and prognosis of a living subject, they may also be used for post-mortem diagnosis in a subject, for example, to identify family members at risk of developing the same disease.

[0073] According to an additional aspect of the invention, said subject can be an asymptomatic subject, or a subject who has mild, moderate, or severe symptoms. Said subject, according to an additional aspect of the invention, can include a subject who has been subjected to anticancer therapy, for example radiotherapy or chemotherapy.

[0074] The term “human endogenous retrovirus” herein abbreviated as “HERV” encompasses complex and heterogeneous multicopy families of genetic elements which are remnants of ancestral retroviral infections having entered the genome of certain species through insertions within germline cells. Altogether HERV elements represent about 8% of the human genome and, when HERV elements have retained transcriptional activity, they rarely cause complete retroviral genome expression, and this is barely expected to arise from completely functional proviral copies. HERVs contain over 200 distinct groups and subgroups. The accumulation of mutations has led to a loss of infectivity of HERVs, and in general they are largely noninfectious retroviral remnants. However, open reading frames (ORFs) have been observed for ERV3, HERV-E 4-1, and HERV-K, but their significance is unknown. As used herein the abbreviation “HERV-K” refers to the HERV K-type family, which is characterized by the presence of primer binding sites for lysine tRNA. Only HERV-K appears to have the full complement of open reading frames typical of replication competent mammalian retroviruses. The K family contains a central open reading frame (cORF) and is comparable to HIV-I Rev protein. HERV-K was originally identified by its homology to the mouse mammary tumor virus (MMTV).

[0075] ***

[0076] Affinity reagents capable of binding to a surface antigen of a human endogenous retrovirus (HERV)

[0077] In one embodiment, the invention provides an affinity reagent or an antigen-binding portion thereof capable of specifically binding to a surface antigen of a virus belonging to human endogenous retroviruses (HERV), for use in a prophylactic and / or therapeutic treatment of a cancer in a subject in need thereof.

[0078] Preferably, in one embodiment the invention provides an affinity reagent or an antigenbinding portion thereof capable of specifically binding to a surface antigen of a virus belonging to the type-K human endogenous retrovirus family (HERV-K).

[0079] In one preferred embodiment, said virus belongs to one of the following subgroups of the HERV-K family: HML-1, HML-2, HML-3, HML-4, HML-5, HML-6, HML-7, HML-8, HML-9 and HML-10.

[0080] In some aspects, the affinity reagent or antigen-binding portion thereof according to any of the embodiments disclosed in the present specification and in the claims specifically binds to a surface antigen of at least one virus, at least two viruses, at least three viruses, at least four viruses or at least five viruses belonging to the HERV-K family according to any of the variants disclosed in the present specification and in the claims.

[0081] According to a more preferred embodiment, the invention provides an affinity reagent or antigen-binding portion thereof that specifically binds to a surface antigen of at least one virus selected among the following viruses belonging to the HML-2 subgroup of the HERV- K family: HERV-K113, HERV-K 115, HERV- K102, HERV-K50A, HERV-K107, HERV- K108, HERV-K 109, HERV-K36, HERV-K41, HERV-K101, HERV-C19, HERV-K33, HERV-K 18, HERV-K42, and HERV-K 103, more preferably of HERV-K 113. In particular, said HERV-K113 is located on chromosome 19pl3.11, said HERV-K102 is located on chromosome lq22, said HERV-K50A is located on chromosome lq22, said HERV-K107 is located on chromosome 5q33.3, said HERV-K108 is located on chromosome 7p22.1, said HERV-K109 is located on chromosome 6ql4.1, said HERV-K115 is located on chromosome 8p23.1, said HERV-K36 is located on chromosome 1 lq22.1, said HERV-K41 is located on chromosome 12ql4, said HERV-K101 is located on chromosome 22ql l.21, said HERV-KC19 is located on chromosome 19ql 1, said HERV- K33 is located on chromosome 10pl4, said HERV-K18 is located on chromosome lq23.3, said HERV-K42 is located on chromosome 12q24.33, and said HERV-K103 is located on chromosome 10pl2.2.

[0082] In one preferred aspect, the surface antigen as defined in the present specification and in the claims is the envelope protein of a HERV virus according to any of the embodiments herein described, more preferably is the envelope protein of at least one virus belonging to the HML-2 subgroup of the HERV-K family, even more preferably of HERV-K113, even more preferably is the envelope protein having the aminoacidic sequence set forth in SEQ ID NO. 6.

[0083] According to one preferred aspect, the affinity reagent or antigen-binding portion thereof according to any of the embodiments disclosed in the present specification and in the claims specifically binds to the envelope protein or fragment thereof of at least one HERV according to any of the variants described in the present specification and in the claims.

[0084] More preferably, the affinity reagent or antigen-binding portion thereof specifically binds to the envelope protein or fragment thereof of HERV -K 113. Even more preferably, said affinity reagent or antigen-binding portion thereof specifically binds to the HERV envelope protein having the aminoacidic sequence set forth in SEQ ID NO. 6.

[0085] In another preferred aspect, the affinity reagent or antigen-binding portion thereof according to any of the embodiments disclosed in the present specification and in the claims specifically binds to an epitope comprising or consisting of a peptide selected from peptide 2 having the sequence set forth in SEQ ID No. 1, peptide 3 having the sequence set forth in SEQ ID No. 2, peptide 4 having the sequence set forth in SEQ ID No. 3, peptide 5 having the sequence set forth in SEQ ID No. 4, peptide 3 having the sequence set forth in SEQ ID No. 7, peptide 3 having the sequence set forth in SEQ ID No. 8, peptide 3 having the sequence set forth in SEQ ID No. 9, and peptide 3 having the sequence set forth in SEQ ID No. 10. More preferably, the affinity reagent or antigen-binding portion thereof according to any of the embodiments disclosed in the present specification and in the claims specifically binds to an epitope comprising or consisting of peptide 2 having the sequence set forth in SEQ ID No. 1.

[0086] In one preferred aspect, the affinity reagent according to the present invention is an antibody, hereinafter also named as an anti-HERV antibody.

[0087] The affinity reagent according to the present invention may, for example, be selected from a monoclonal antibody, a multi-specific antibody, a bi-specific antibody, a three-specific antibody, a scFV, a diabody, a triabody, a tetrabody, a minibody, a linear antibody, a chelating recombinant antibody, a tribody, a bibody, an intrabody, a nanobody, an antibodydrug conjugate, a binding-domain immunoglobulin fusion protein, a fusion antibody, a chimeric antibody, an immunoadhesin, or an antigen binding portion thereof.

[0088] In one particular embodiment, said antibody or antigen binding portion is a Fab, a F(ab') 2, a Fv or a single-chain antibody fragment.

[0089] In certain embodiments the antibodies or antigen binding fragments thereof of any of the embodiments disclosed herein are bispecific, three-specific or multi-specific. Bi-, three- or multi-specific antibodies of the invention may be formed using methods well known in the art, e.g., chemical conjugation of one or more antibodies or antigen binding variable domains disclosed herein to each other and / or to differing epitope binding polypeptides, wherein the binding domains of the bi-, three- or multi-specific molecule exhibit affinity for at least two different antigens. For example, an antibody according to the present invention may comprise a first and a second VL domain, or a first and second VH domain, wherein said first and second domain have differing binding specificities (i.e., bind to different antigens). According to one preferred embodiment, an antibody or antigen binding fragment thereof according to the present invention comprises a first and a second VL domain, or a first and second VH domain according to any of the embodiments disclosed in the present specification and in the claims, wherein said first and second domain have differing binding specificities for HERV (i.e., bind to different antigens).

[0090] In some embodiments, the affinity reagent is a single-chain antibody (scFv) in which a VL and VH domains are paired to form a monovalent molecule via a synthetic linker that enables them to be made as a single protein chain. (Bird et al, Science 242:423-426 (1988) and Huston et al, Proc. Natl Acad. ScL USA 85:5879-5883 (1988)). In certain embodiments of the invention, at least one antigen binding domain of the bi-, three- or multispecific antibody or antigen binding portion of the invention immunospecifically binds to a HERV surface antigen, in particular to the envelope protein of said HERV, preferably of at least one virus belonging to the HML-2 subgroup of the HERV-K family according to any of the variants disclosed herein, even more preferably of HERV-K113.

[0091] In another embodiment, the invention provides a fusion antibody or immunoadhesin made that comprises all or a portion of at least an anti-HERV antibody of the invention according to any of the embodiments disclosed herein, linked to another polypeptide. In one embodiment, only the variable domains of at least one, at least two or at least three anti- HERV antibodies according to any of the embodiments disclosed herein are linked to the polypeptide. In still another embodiment, the VH domain of an anti-HERV antibody is linked to a first polypeptide, while the VL domain of an anti-HERV antibody is linked to a second polypeptide that associates with the first polypeptide in a manner such that the VH and VL domains can interact with one another to form an antigen binding site. In still another embodiment, the VH domain is separated from the VL domain by a linker such that the VH and VL domains can interact with one another (see below under Single Chain Antibodies). The VH-linker-VL antibody is then linked to the polypeptide of interest. The fusion antibody is useful for directing a polypeptide to a HERV surface antigen-expressing cell or tissue. The polypeptide may be a therapeutic agent, such as a toxin, chemokine, or other regulatory protein, or may be a diagnostic agent, such as an enzyme that may be easily visualized, such as horseradish peroxidase. In addition, fusion antibodies can be created in which two (or more) single-chain antibodies are linked to one another.

[0092] This is useful if one wants to create a divalent or polyvalent antibody on a single polypeptide chain, or if one wants to create a bispecific antibody or nanobody.

[0093] In some embodiments, the affinity reagent is a diabody, i.e., are bivalent antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites. (See e.g., Holliger P. et al, Proc. Natl. Acad. ScL USA 90:6444-6448 (1993), and Poljak R. J. et al, Structure 2: 1121-1123 (1994)). In such embodiments, the CDR(s) may be incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be incorporated noncovalently. In embodiments having one or more binding sites, the binding sites may be identical to one another or may be different.

[0094] In one preferred aspect, the affinity reagent or antigen binding portion thereof according to the present invention is a nanobody or single-domain antibody (sdAb), namely an antibody fragment consisting of a single monomeric variable antibody domain, more particularly is a camelid or humanized nanobody.

[0095] More preferably, the affinity reagent or antigen binding portion thereof according to the present invention is a nanobody having the aminoacidic sequence set forth in SEQ ID No. 5 or else having an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98% or 99% identical in amino acid sequence to SEQ ID No. 5.

[0096] An antibody or nanobody that is specific for human HERV-K envelope proteins may recognize a wild-type or mutant form of the HERV-K protein. In particular embodiments, antibodies or nanobodies are produced to, but not limited to, HERV-K envelope proteins and variants thereof, preferably to HERV-K113 and / or HERV-K115 envelope protein.

[0097] Various procedures known in the art may be used for the production of polyclonal antibodies to polypeptides, derivatives, or analogs. For the production of antibody, various host animals, including but not limited to rabbits, mice, rats, sheep, goats, etc, can be immunized by injection with the polypeptide or a derivative (e.g., fragment or fusion protein). The polypeptide or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

[0098] Monoclonal antibodies directed toward a HERV-K envelope polypeptide, fragment, analog, or derivative thereof, may be prepared by any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used.

[0099] One type of amino acid substitution that may be made is to change one or more cysteines in an affinity reagent in the form of an antibody or nanobody according to the present invention, which may be chemically reactive, to another residue, such as, without limitation, alanine, or serine. In one embodiment, there is a substitution of a non-canonical cysteine. The substitution can be made in a CDR or framework region of a variable domain or in the constant domain of an antibody or nanobody according to the invention.

[0100] Accordingly, as will be further reported below, the present disclosure is also directed to hybridoma cell lines that produce an antibody or particularly a monoclonal antibody that specifically binds to an antigen (e.g., HERV-K envelope protein, and preferably the envelope protein of at least one virus belonging to the HML-2 subgroup of the HERV-K family, even more preferably of HERV-K 113) of a HERV-K positive cancer.

[0101] Another type of amino acid substitution that may be made is to change any potential proteolytic sites in the affinity reagent such as the nanobody according to the present invention. Such sites may occur in a CDR or framework region of a variable domain or in the constant domain of an antibody. Substitution of cysteine residues and removal of proteolytic sites may decrease the risk of any heterogeneity in the antibody product and thus increase its homogeneity. Another type of amino acid substitution is to eliminate asparagineglycine pairs, which form potential deamidation sites, by altering one or both of the residues. In one aspect, an antibody or nanobody according to any of the variants disclosed in the present specification and in the claims is a human or humanized antibody or nanobody.

[0102] In cases when the affinity reagent of the invention is an antibody, the class and subclass of such antibody may be determined by any method known in the art. In general, the class and subclass of an antibody may be determined using antibodies that are specific for a particular class and subclass of antibody. Such antibodies are commercially available. The class and subclass can be determined by ELISA, or Western blot (immunoblot) as well as other techniques. Alternatively, the class and subclass may be determined by sequencing all or a portion of the constant domains of the heavy and / or light chains of the antibodies, comparing their amino acid sequences to the known amino acid sequences of various class and subclasses of immunoglobulins, and determining the class and subclass of the antibodies.

[0103] In some embodiments, the affinity reagent of the invention is an IgG, an IgM, an IgE, an IgA, or an IgD molecule. In one embodiment, the affinity reagent is an IgG and is an IgGl, IgG2, IgG3, or IgG4 subclass. In still another embodiment, the human antibody subclass is IgGl.

[0104] In the production and use of antibodies, screening for or testing with the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.

[0105] One aspect of the invention is an affinity reagent capable of specific binding to a surface antigen of the invention or fragments thereof, which contains or is conjugated to a detectable label or is conjugated to a therapeutic drug or moiety such as a cytotoxic moiety.

[0106] In a specific embodiment, affinity reagents of the present disclosure are conjugated to a secondary component, such as, for example, a small molecule, polypeptide, or polynucleotide. The conjugation may be produced through a chemical modification of the affinity reagent, which conjugates the affinity reagent to the secondary component. The conjugated affinity reagent may allow for targeting of the secondary component, such as, for example, a cytotoxic agent or an anti -tumor agent or an imaging agent, to the site of interest. The secondary component may be of any size or length. Examples of secondary components include, but are not limited to, chemotherapeutic agents, toxins, photo-activated toxins (e.g., dihydropyridine- and omega-conotoxin), radioactive isotopes, mitotic inhibitors, cell-cycle regulators, and anti -microtubule disassembly compounds (e.g., taxol).

[0107] Those skilled in the art will realize that there are numerous radioisotopes and chemocytotoxic agents that can be coupled to tumor specific affinity reagents by well-known techniques and delivered to specifically destroy tumor tissue or cells.

[0108] Other covalent and non-covalent modifications of the affinity reagents or portions thereof of the present disclosure are embraced herein, including agents which are co-administered or administered after the affinity reagent or fragments, to induce growth inhibition or killing of the cells to which the affinity reagent or portion thereof has previously bound.

[0109] According to another aspect of the invention, the affinity reagent is a hybridizing agent capable of hybridizing to nucleic acid encoding the HERV surface antigen according to any of the variants herein disclosed, for example, a hybridizing agent which contains or is conjugated to a detectable label. One example of a hybridizing agent is an inhibitory RNA (RNAi) or siRNA. Other examples include anti-sense oligonucleotides and ribozymes. In some embodiments of the invention, the affinity reagent of the invention according to any of the variants disclosed herein binds to a surface antigen of a HERV according to any of the embodiments disclosed herein with high affinity. In some embodiments, the affinity reagents of the invention bind with high affinity to an envelope protein or peptide of at least one virus belonging to the HERV-K family according to any of the variants disclosed herein, particularly to the envelope protein of at least one virus belonging to the HML-2 subgroup of the HERV-K family, even more preferably of HERV-K113.

[0110] An affinity reagent according to the present invention is said to specifically bind a HERV surface antigen when the dissociation constant is for example < 1 mM, preferably < 100 nM and most preferably < 10 nM. The binding affinity and dissociation rate of an affinity reagent to a surface antigen according to any of the embodiments disclosed herein can be determined by methods known in the art. The binding affinity can be measured by ELISAs, RIAs, flow cytometry, surface plasmon resonance, such as BIACORE(TM). The dissociate rate can be measured by surface plasmon resonance. Preferably, the binding affinity and dissociation rate is measured by surface plasmon resonance. More preferably, the binding affinity and dissociation rate are measured using BIACORE(TM). One can determine whether an antibody has substantially the same KD as an affinity reagent according to the present invention by using methods known in the art.

[0111] The invention provides a human anti-HERV monoclonal antibody that binds to a surface antigen of a HERV according to any of the embodiments disclosed herein, in particular a surface antigen of at least one virus belonging to the HERV-K family according to any of the variants disclosed herein, such as the HERV-K113 and HERV-K115, and competes or cross-competes with and / or binds the same epitope as an affinity reagents according to any of the embodiments of the invention, in particular the nanobody herein disclosed as FF-01. If two antibodies reciprocally compete with each other for binding to HERV surface antigen, they are said to cross-compete.

[0112] One can determine whether an antibody binds to the same epitope or cross competes for binding with an anti-HERV antibody by using methods known in the art. In one embodiment, one allows the anti-HERV antibody of the invention to bind to HERV surface antigens under saturating conditions and then measures the ability of the test antibody to bind to HERV surface antigens. If the test antibody is able to bind to HERV surface antigens at the same time as the anti-HERV antibody, then the test antibody binds to a different epitope as the anti-HERV antibody. However, if the test antibody is not able to bind to the HERV surface antigen at the same time, then the test antibody binds to the same epitope, an overlapping epitope, or an epitope that is in close proximity to the epitope bound by the anti-HERV antibody, or the binding of the human anti-HERV antibody may induce a conformational change in the surface antigen that prevents or reduces binding of the test antibody. This experiment can be performed using ELISA, RIA, BIACORE(TM), flow cytometry or other methods known in the art.

[0113] To test whether an anti-HERV antibody cross-competes with another anti-HERV antibody, one may use the competition method described above in two directions i.e. determining if the reference antibody blocks the test antibody and vice versa. In one embodiment, the experiment is performed using ELISA. Methods of determining KD are discussed further below.

[0114] In another embodiment, the invention provides an anti-HERV affinity reagent, in particular an anti-HERV antibody or nanobody, that destroys, inhibits, blocks, neutralizes or decreases the action or function of cancer cells expressing HERV surface antigens, in particular the envelope protein of said HERV, according to any of the variants disclosed in the present specification and in the claims, in particular kills cancer cells expressing at least one HERV- K surface antigen, more particularly kills cancer cells expressing the envelope protein of said HERV-K such as HERV-K113 and / or HERV-K115.

[0115] In certain aspects, the affinity reagent or antigen-binding portion thereof that specifically binds to a surface antigen of HERV according to any of the embodiments disclosed herein, exhibits cytotoxicity on cancer cells expressing a surface antigen of a HERV according to any of the variants disclosed in the present specification and in the claims, particularly the envelope protein of said HERV, such as the envelope protein of HERV-K113 and / or HERV-K115.

[0116] As disclosed in more details in the Examples, the inventors found that the most potent affinity reagent FF-01, surprisingly exhibits the ability to induce antibody-dependent cellular cytotoxicity in the lymphoma cancer cell line RCK8.

[0117] In certain embodiments, the invention provides an affinity reagent that specifically binds to a surface antigen of HERV according to any of the variants disclosed herein, particularly to the envelope protein of HERV-K113 and / or HERV-K115, that inhibits, blocks, or decreases in severity for a day, a week, a month, 6 months, a year, or for the remainder of the subjects’ life symptoms or conditions resulting from a cancer, particularly a lymphoma such as DLBCL, by 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%. In certain embodiments, the invention provides an affinity reagent or antigen-binding portion thereof that may perform any combination of the preceding embodiments.

[0118] Interfering agents

[0119] In one aspect, the invention further provides an interfering agent capable of preventing and / or inhibiting the expression or activity of a target gene or sequence of a human endogenous retrovirus (HERV) according to any of the variants disclosed in the present specification and in the claims, for use in a prophylactic and / or therapeutic treatment of a cancer in a subject in need thereof, preferably wherein said cancer is a lymphoma selected among any of the variants described in the present specification, more preferably wherein said cancer is a diffuse large B-cell lymphoma (DLBCL).

[0120] According to one preferred aspect, the target gene or sequence is a target gene or sequence encoding for at least one surface antigen of at least one virus belonging to the K-family of HERV, more preferably is a target gene or sequence encoding for at least one surface antigen of at least one virus belonging to one of the following subgroups of the HERV-K family: HML-1, HML-2, HML-3, HML-4, HML-5, HML-6, HML-7, HML-8, HML-9 and HML- 10.

[0121] In some aspects, the target gene or sequence is a target gene or sequence encoding for at least one surface antigen of HERV-K113 and / or HERV-K115, preferably encodes for the envelope protein of HERV-K113 and / or HERV-K115, more preferably encodes for the HERV envelope protein having the aminoacidic sequence set forth in SEQ ID No. 6.

[0122] According to one preferred aspect, the interfering agent is selected from an antisense oligonucleotide, a small interfering RNA (siRNA), a short hairpin RNA (shRNA), an aptamer, or a ribozyme, preferably is a siRNA.

[0123] In particular, the present invention relates to siRNA molecules of about 15 to about 40 or about 15 to about 28 nucleotides in length, which are homologous to said target gene or sequence and mediate RNA interference of the target gene or sequence. Preferably, the siRNA molecules have a length of about 19 to about 25 nucleotides. The siRNA molecules of the present invention can also comprise a 3' hydroxyl group. The siRNA molecules can be single-stranded or double stranded; such molecules can be blunt ended or comprise overhanging ends (e.g., 5', 3'). In specific embodiments, the RNA molecule is double stranded and either blunt ended or comprises overhanging ends.

[0124] According to a more preferred aspect, the interfering agent is a siRNA comprising or consisting of a sense sequence set forth in SEQ ID No. 11 and an antisense sequence set forth in SEQ ID No. 13 or is a siRNA comprising or consisting of a sense sequence set forth in SEQ ID No. 12 and an antisense sequence set forth in SEQ ID No. 14.

[0125] Column 3: sense siRNA (5p --> 3p) Column 4: antisense siRNA (5p --> 3p)

[0126] GCAGCUAACUAUACCUACUTT AGUAGGUAUAGUUAGCUGCTT siRNA 2 (SEQ ID No. 11) (SEQ ID No. 13)

[0127] CGAGGUCAAUUCUACCACATT UGUGGUAGAAUUGACCUCGTT siRNA 3 (SEQ ID No. 12) (SEQ ID No. 14)

[0128] TT dovrebbero essere UU?

[0129] Synthetic siRNA molecules of the present invention can be obtained using a number of techniques known to those of skill in the art. For example, the siRNA molecule can be chemically synthesized or recombinantly produced using methods known in the art, such as using appropriately protected ribonucleoside phosphoramidites and a conventional DNA / RNA synthesizer.

[0130] Therapeutic methods of Use of the affinity reagents and / or interfering agents

[0131] As already mentioned, the affinity reagents or antigen-binding portion thereof according to any of the embodiments disclosed in the present specification and in the claims, as well as the interfering agents according to any of the embodiments disclosed in the present specification and in the claims, can be used in a prophylactic and / or therapeutic treatment of a cancer, or conditions or disorders resulting from such disease, in a subject in need thereof. Preferably, said cancer is a HERV-positive cancer, namely a cancer that is characterized, associated, or caused by expression of at least one surface antigen of a HERV according to any of the variants disclosed herein, preferably a virus belonging to the K family of HERVs. In particular, a HERV positive cancer refers to cancers that are characterized by expression of a HERV gene, in particular a HERV-K gene, more preferably a gene of at least one virus belonging to the HML-2 subgroup of the HERV-K family, even more preferably a HERV- K113 gene , or a polymorphism or sequence variant thereof, which results in an antigen derived from the HERV or HERV-K gene, or a polymorphism or sequence variant thereof. More preferably, a HERV positive cancer refers to cancers that are characterized by expression of a HERV-K gene encoding for the envelope protein of said HERV-K, for example a gene encoding for the envelope protein having the aminoacidic sequence set forth in SEQ ID No. 6. Examples of a HERV-K positive cancers include, but are not limited to, breast cancers, ovarian cancers, teratocarcinomas, and melanomas.

[0132] As shown in the experimental section of the present specification, the inventors have particularly demonstrated the presence of HERV-K envelope antigens on the surface of lymphoma cells, and that tumoral and normal specimens have distinct HERV-K expression profiles. In particular, they have demonstrated that human endogenous retroviruses envelope proteins can not only be expressed but are also exposed on the cell surface of lymphoma cancer cells.

[0133] One preferred embodiment thus refers in particular to an affinity reagent or antigen-binding portion thereof according to any of the variants disclosed in the present specification and in the claims, as well as to an interfering agent according to any of the variants disclosed in the present specification and in the claims, for use in a prophylactic and / or therapeutic treatment of a lymphoma in a subject in need thereof.

[0134] Preferably, the lymphoma is selected from diffuse large B-cell lymphoma (DLBCL), such as ABC-DLBCL, GCB-DLBCL, mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), chronic lymphocytic leukemia (CLL), Burkitt lymphoma (BL) and primary mediastinal B-cell lymphoma (PMBCL), more preferably the lymphoma is DLBCL.

[0135] Any type of the affinity reagents or antigen-binding portion thereof or interfering reagents disclosed herein can be used therapeutically.

[0136] In one particular embodiment, the invention provides a method for neutralizing or inhibiting or killing, or reducing or suppressing proliferation of cancer cells by administering one or more affinity reagents or antigen binding portions thereof, and / or one or more interfering agents according to any of the embodiments disclosed herein to a subject in need thereof.

[0137] In various embodiments, the affinity reagent is an antibody or nanobody according to any of the variants disclosed herein. In some embodiments, the antibody or nanobody, or antigenbinding portion thereof, binds to the envelope protein of at least one virus belonging to the HERV-K family according to any of the variants disclosed in the present specification, in particular HERV-K113, more preferably is the nanobody having the aminoacidic sequence set forth in SEQ ID No. 5.

[0138] As mentioned above, HERV-K envelope proteins or fragments thereof may serve as a tumor- associated antigen which can be used to elicit T cell and B cell responses. In therapeutic applications, this may be used to reduce immune tolerance in, for example, a cancer patient. For example, the HERV-K envelope protein is expressed on both the cell surface and cytoplasm of lymphoma cells, therefore providing a target for both B cells and T cells, and potentially greatly increasing the effectiveness of HERV-K envelope proteins, preferably HERV-K113 envelope proteins, as tumour-associated antigens.

[0139] In one embodiment, the invention provides methods of treating, aiding in the treatment, preventing or aiding in the prevention of, a cancer according to any of the variants disclosed herein, and conditions or disorders resulting from such disease, in a subject by administering to the subject a therapeutically effective or prophylactically effective amount of one or more affinity reagents or interfering agents of the invention. Antibodies and antigen-binding portions thereof which are antagonists of a HERV or a surface antigen of a HERV, preferably HERV-K113, can be used as therapeutics for cancer treatment according to the invention.

[0140] The affinity reagents or interfering agents of the invention may be administered locally or systemically.

[0141] In some aspects, the method according to any of the embodiments disclosed herein comprises administering the nanobody FF-01 having the aminoacidic sequence set forth in SEQ ID No. 5.

[0142] In particular embodiments, affinity reagents specific for the HERV-K envelope protein or fragments thereof according to the present invention may be used in conjunction with other expressed HERV antigens. This may be particularly useful for immunotherapy and antibody treatments of diseases in which several different HERVs are expressed. The affinity reagents or antigen binding portions thereof according to any of the embodiments disclosed in the present specification and in the claims can also be used in an in vitro method for the design of a vaccine against at least one HERV according to any of the variants disclosed in the present specification and in the claims, in particular a virus belonging to the K-family of HERVs such as HERV-K113. Structural characterization of the affinity reagent-antigen complex may be used to instruct antigen design.

[0143] Nucleic Acids, Vectors, Host Cells, and Recombinant Methods of Making Nucleic Acids The present invention also encompasses nucleic acid molecules encoding an affinity reagent or antigen-binding portions thereof according to any of the variants disclosed herein.

[0144] In some embodiments, the affinity reagent is an immunoglobulin and different nucleic acid molecules encode a heavy chain and a light chain of said immunoglobulin. In other embodiments, the same nucleic acid molecule encodes a heavy chain and a light chain of an anti-HERV immunoglobulin. In one embodiment, the nucleic acid encodes an anti-HERV antibody or nanobody, or antigen-binding portion thereof, of the invention. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a VL amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions and / or 1, 2, or 3 non- conservative substitutions compared to germline.

[0145] Substitutions may be in the CDR regions, the framework regions, or in the constant domain. In some embodiments, the nucleic acid molecule encodes a nanobody having the aminoacidic sequence as set forth in SEQ ID No. 5 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID No. 5.

[0146] A nucleic acid molecule encoding the heavy or light chain of an anti-HERV antibody or nanobody or portions thereof can be isolated from any source that produces such antibody or nanobody. In various embodiments, the nucleic acid molecules are isolated from a B cell isolated from an animal immunized with surface antigens or from an immortalized cell derived from such a B cell that expresses or encodes an anti-HERV antibody or nanobody. Methods of isolating mRNA encoding an antibody are well known in the art. See, e.g., Sambrook et al. The mRNA may be used to produce cDNA for use in the polymerase chain reaction (PCR) or cDNA cloning of antibody genes. In one embodiment, the nucleic acid molecule is isolated from a hybridoma that has as one of its fusion partners a human immunoglobulin-producing cell from a non-human transgenic animal. In an even more preferred embodiment, the human immunoglobulin producing cell is isolated from a XENOMOUSE animal. In another embodiment, the human immunoglobulin-producing cell is from a non-human, non-mouse transgenic animal, as described above. In another embodiment, the nucleic acid is isolated from a non-human, non-transgenic animal.

[0147] The nucleic acid molecules isolated from a non-human, non-transgenic animal may be used, e.g., for humanized antibodies or nanobodies.

[0148] In some embodiments, a nucleic acid encoding a heavy chain of an anti-HERV antibody of the invention can comprise a nucleotide sequence encoding a VH domain of the invention joined in-frame to a nucleotide sequence encoding a heavy chain constant domain from any source. Similarly, a nucleic acid molecule encoding a light chain of an anti-HERV antibody of the invention can comprise a nucleotide sequence encoding a VL domain of the invention joined in-frame to a nucleotide sequence encoding a light chain constant domain from any source. In a further aspect of the invention, nucleic acid molecules encoding the variable domain of the heavy (VH) and / or light (VL) chains are "converted" to full-length antibody genes.

[0149] In one embodiment, nucleic acid molecules encoding the VH or VL domains are converted to full-length antibody genes by insertion into an expression vector already encoding heavy chain constant (CH) or light chain constant (CL) domains, respectively, such that the VH segment is operatively linked to the CH segment(s) within the vector, and / or the VL segment is operatively linked to the CL segment within the vector. In another embodiment, nucleic acid molecules encoding the VH and / or VL domains are converted into full-length antibody genes by linking, e.g., ligating, a nucleic acid molecule encoding a VH and / or VL domains to a nucleic acid molecule encoding a CH and / or CL domain using standard molecular biological techniques. Nucleotide sequences of human heavy and light chain immunoglobulin constant domain genes are known in the art. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publ. No. 91-3242, 1991. Nucleic acid molecules encoding the full-length heavy and / or light chains may then be expressed from a cell into which they have been introduced and the anti-HERV antibody or nanobody isolated.

[0150] The nucleic acid molecules may be used to recombinantly express large quantities of anti- HERV affinity reagents, in particular anti-HERV antibodies. The nucleic acid molecules also may be used to produce chimeric antibodies, bispecific antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies and antibody derivatives, as described further below. If the nucleic acid molecules are derived from a non-human, non- transgenic animal, the nucleic acid molecules may be used for antibody humanization, also as described below.

[0151] In another embodiment, a nucleic acid molecule of the invention is used as a probe or PCR primer for a specific antibody sequence. For instance, the nucleic acid can be used as a probe in diagnostic methods or as a PCR primer to amplify regions of DNA that could be used, inter alia, to isolate additional nucleic acid molecules encoding variable domains of anti- HERV antibodies. In some embodiments, the nucleic acid molecules are oligonucleotides. In some embodiments, the oligonucleotides are from highly variable domains of the heavy and light chains of the antibody of interest. The nucleic acid molecules herein disclosed may be DNA or RNA molecules.

[0152] Vectors

[0153] The invention provides vectors comprising nucleic acid molecules that encode an affinity reagent of the invention or an antigen-binding portion thereof.

[0154] The invention also provides vectors comprising nucleic acid molecules that encode the heavy chain and / or the light chain of an affinity reagent in the form of an antibody or antigenbinding portion thereof according to any of the variants disclosed herein.

[0155] The invention further provides vectors comprising nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments, and probes thereof. In some embodiments, the affinity reagents or antigen-binding portions of the invention are expressed by inserting DNAs encoding partial or full-length light and heavy chains of the same, obtained as described above, into expression vectors such that the genes are operatively linked to necessary expression control sequences such as transcriptional and translational control sequences.

[0156] The invention further provides vectors comprising at least one interfering agent according to any of the embodiments disclosed in the present specification and in the claims, particularly an interfering agent in the form of siRNA according to any of the variants disclosed in the present specification and in the claims.

[0157] The siRNA molecules of the invention can be inserted into vectors as described herein and known in the art. The pharmaceutical preparation of the siRNA vector can include the siRNA vector in an acceptable diluent or can comprise a slow-release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e. g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0158] Expression vectors as defined herein include plasmids, retroviruses, adenoviruses, adeno- associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV derived episomes, and the like. The gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors. In one embodiment, both genes are inserted into the same expression vector.

[0159] The genes are inserted into the selected expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).

[0160] For antibodies, a convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can easily be inserted and expressed, as described above. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C domain, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The recombinant expression vector also can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e. a signal peptide from a non-immunoglobulin protein). In addition to the genes encoding for an affinity reagent or else in addition to the sequence of the interfering agent of the invention, the recombinant expression vectors of the invention can carry regulatory sequences that control the expression of the genes of interest in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV promoter / enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus, (e.g. the adenovirus major late promoter (AdMLP)), polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters. For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. Patent No. 5,168,062, U.S. Patent No. 4,510,245 and U.S. Patent No. 4,968,615. Methods for expressing antibodies in plants, including a description of promoters and vectors, as well as transformation of plants is known in the art. See, e.g., United States Patent 6,517,529, incorporated herein by reference. Methods of expressing polypeptides in bacterial cells or fungal cells, e.g., yeast cells, are also well known in the art. In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017, incorporated herein by reference). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfir- host cells with methotrexate selection / amplification), the neo gene (for G418 selection), and the glutamate synthetase gene.

[0161] In some embodiments said vector is a selected from RNA virus vectors, DNA virus vectors, plasmid viral vectors, adenovirus vectors, adenovirus associated virus vectors, herpes virus vectors and retrovirus vectors.

[0162] In some aspects, the invention provides compositions (e.g., pharmaceutical compositions), methods, kits and reagents, comprising one or more isolated nucleic acid molecules or vectors according to any one of the embodiments herein disclosed for use in the prevention and / or treatment of cancer, in particular in humans and other mammals, in particular a lymphoma such as DLBCL. In some embodiments such nucleic acid molecules and vectors are formulated in a nanoparticle, for example in lipid nanoparticle, cationic lipid nanoparticle, examples of such formulations can be found in US2020197510 herein incorporated by reference.

[0163] Non-Hybridoma Host Cells and Methods of Recombinantly Producing Protein

[0164] Nucleic acid molecules encoding an affinity reagent of the invention or an antigen-binding portion thereof, an interfering agent according to any of the variants disclosed in the present specification and in the claims as wells as vectors comprising nucleic acid molecules and / or interfering agents according to any of the variants disclosed herein can be used for transfection or transformation of a suitable mammalian, plant, bacterial or yeast host cell. Transfection or transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art (see, e.g., U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, incorporated herein by reference). Methods for transforming plant cells are well known in the art, including, e.g., Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art. Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, N50 cells, SP2 cells, HEK-293T cells, NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 or Sf21 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods. Plant host cells include, e.g., Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris. Further, expression of antibodies of the invention from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0 216 846, 0 256 055, 0 323 997 and 0 338 841. It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from each other. However, all affinity reagents encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein are part of the instant invention, regardless of the glycosylation of the antibodies.

[0165] Transgenic Animals and Plants

[0166] Affinity reagents or antigen-binding portions thereof of the invention also can be produced transgenically through the generation of a mammal or plant that is for example transgenic for the immunoglobulin heavy and light chain sequences of interest and production of an affinity reagent in a recoverable form therefrom.

[0167] Merely by way of example, in connection with the transgenic production in mammals, anti- HERV affinity reagents according to the present invention can be produced in, and recovered from, the milk of goats, cows, or other mammals. See, e.g., U.S. Patent Nos. 5,827,690, 5,756,687, 5,750,172, and 5,741,957, incorporated herein by reference. In some embodiments, non- human transgenic animals that comprise human immunoglobulin loci are immunized with HERV whole cells or an immunogenic portion thereof, such as one or more surface antigens as described above. Methods for making antibodies in plants are described, e.g., in U.S. patents 6,046,037 and 5,959,177, incorporated herein by reference. In some embodiments, non-human transgenic animals or plants are produced by introducing one or more nucleic acid molecules encoding an anti-HERV antibody of the invention into the animal or plant by standard transgenic techniques. See Hogan and United States Patent 6,417,429, supra. The transgenic cells used for making the transgenic animal can be embryonic stem cells or somatic cells or a fertilized egg. The transgenic non-human organisms can be chimeric, nonchimeric heterozygotes, and nonchimeric homozygotes. See, e.g., Hofian et al. Manipulating the Mouse Embryo: A Laboratory Manual second ed., Cold Spring Harbor Press (1999); Jackson et al, Mouse Genetics and Transgenics: A Practical Approach, Oxford University Press (2000); and Pinkert, Transgenic Animal Technology: A Laboratory Handbook, Academic Press (1999), all incorporated herein by reference. In some embodiments, the transgenic non-human animals have a targeted disruption and replacement by a targeting construct that encodes a heavy chain and / or a light chain of interest.

[0168] In one embodiment, the transgenic animals comprise and express nucleic acid molecules encoding heavy and light chains that specifically bind to HERV, and preferably to an envelope peptide or protein of a virus belonging to HERV-K according to any of the embodiments disclosed herein.

[0169] In one embodiment, the transgenic animals comprise and express nucleic acid molecules encoding heavy and light chains that specifically bind to a surface antigen of at least one HERV-K virus according to any of the variants disclosed herein, preferably to at least two, at least three, at least four or at least five viruses belonging to the HERV-K family. In some embodiments, the transgenic animals comprise nucleic acid molecules encoding a modified antibody such as a single-chain antibody, a chimeric antibody or a humanized antibody or nanobody. The anti-HERV antibodies may be made in any transgenic animal. In one embodiment, the non-human animals are mice, rats, sheep, pigs, goats, cattle, camel or horses. The non-human transgenic animal expresses said encoded polypeptides in blood, milk, urine, saliva, tears, mucus and other bodily fluids.

[0170] Pharmaceutical compositions

[0171] Another aspect of the invention provides a pharmaceutical composition comprising one or more affinity reagents or an antigen-binding portion thereof, and / or one or more interfering agents according to any of the embodiments disclosed in the present specification and in the claims, and a pharmaceutically acceptable carrier.

[0172] In another embodiment, the invention provides a pharmaceutical composition comprising one or more nucleic acid molecules or vectors according to any of the variants disclosed in the present specification and in the claims, and a pharmaceutically acceptable carrier.

[0173] In a preferred embodiment the pharmaceutical composition comprises at least one affinity reagent in the form of an antibody or nanobody according to any of the variants disclosed in the present specification and in the claims, in particular a nanobody having the aminoacidic sequence set forth in SEQ ID No. 5.

[0174] In another embodiment, the pharmaceutical composition comprises at least one interfering agent in the form of a siRNA according to any of the variants disclosed in the present specification and in the claims. In particular, The RNA interfering agents, e.g., the siRNAs of the invention, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise one or more RNA interfering agents, e.g., siRNAs, such as one or more siRNA inhibiting or silencing the expression or activity of a gene encoding for the envelope protein of at least one virus belonging to the HML-2 subgroup of the HERV-K family, even more preferably of HERV-K 113, and a pharmaceutically acceptable carrier.

[0175] The compositions according to any of the embodiments disclosed herein are particularly for use in the prevention and / or treatment of a cancer, more preferably a HERV positive cancer such as a HERV-K113 positive cancer, in particular a lymphoma, more particularly a lymphoma selected from diffuse large B-cell lymphoma (DLBCL), such as ABC-DLBCL, GCB-DLBCL, mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), chronic lymphocytic leukemia (CLL), Burkitt lymphoma (BL) and primary mediastinal B-cell lymphoma (PMBCL), preferably DLBCL.

[0176] In certain embodiments the one or more affinity reagents and / or the one or more interfering agents of the invention are in a composition comprising a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the affinity reagents.

[0177] In another embodiment, one or more of the affinity reagents and / or interfering agents of the invention are administered in combination with one or more additional antagonistic antibodies that bind different surface antigens of HERV or different epitopes on the surface antigen of HERV and / or that bind the surface antigen from different isolates of HERV.

[0178] The compositions according to any of the embodiments of the invention may be in a variety of forms, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).

[0179] In one embodiment, the affinity reagent or antigen-binding portion thereof, and / or the interfering agent, or pharmaceutical compositions thereof is administered by intravenous infusion or injection. In still another embodiment, the affinity reagent or antigen-binding portion thereof, and / or the interfering agent, or pharmaceutical composition thereof is administered by intramuscular or subcutaneous injection.

[0180] In another embodiment the pharmaceutic compositions comprising the affinity reagent or antigen-binding portion thereof and / or the interfering agent of the invention may be administered to the subject, orally, nasally, vaginally, buccally, rectally, via the eye, or via the pulmonary route, in a variety of pharmaceutically acceptable dosing forms, which will be familiar to those skilled in the art.

[0181] Before administration, a formulation containing the affinity reagent and / or the interfering agent, may be conveniently contained in a two-compartment unit dose container, one compartment containing a freeze-dried affinity reagent preparation and the other compartment containing normal saline.

[0182] Pharmaceutical compositions are typically sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the affinity reagent and / or the interfering agent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

[0183] Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0184] In certain embodiments, the affinity reagent or antigen-binding portion thereof and / or the interfering agent or the pharmaceutical compositions thereof may be prepared with a carrier that will protect the affinity reagent and / or the interfering agent against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems (J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978).

[0185] The invention also provides compositions suitable for administration by inhalation, which comprise one or more affinity reagents or antigen-binding portion thereof and / or one or more interfering agents as described herein. The affinity reagents and / or the interfering agents may be conveniently delivered to a subject in the form of an aerosol spray presentation from pressurized packs or from a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. Dellamary et al. (2004) J Control Release. ;95(3): 489-500 describes formulations for the pulmonary delivery of antibodies.

[0186] The invention also provides pharmaceutical compositions comprising one or more affinity reagents or antigen-binding portion thereof and / or the interfering agent described herein suitable for administration through the oral mucosa. Oral transmucosal delivery refers to the delivery of a delivery vehicle across a mucous membrane in the oral cavity, pharyngeal cavity, or esophagus, and may be contrasted, for example, with traditional oral delivery, in which absorption of a drug occurs in the intestine. Accordingly, routes of administration in which the affinity reagents are absorbed through the buccal, sublingual, gingival, pharyngeal, and / or esophageal mucosa are all encompassed within "oral transmucosal delivery," as that term is used herein. For administration through the transmucosal mucosa, the affinity reagents or antigen-binding portion thereof of the invention may be formulated, for example, into chewing gums (see U.S. Pat No. 5,711,961) or buccal patches (see e.g. U.S. Patent No. 5,298,256).

[0187] The invention also provides compositions suitable for administration through the vaginal mucosa, which comprise one or more affinity reagents or antigen-binding portion thereof and / or one or more interfering agents as described herein. For example, the affinity reagents of the invention may be formulated into a vaginal suppository, foam, cream, tablet, capsule, ointment, or gel. In certain embodiments, the compositions comprising the affinity reagents of the invention are formulated with permeants appropriate to the transmucosal barrier to be permeated. Such penetrants are generally known in the art, and include, for example, for trans mucosal administration bile salts and fusidic acid derivatives.

[0188] In certain embodiments, an affinity reagent and / or interfering agent of the invention can be orally administered, for example, with an inert diluent or an assailable edible carrier. The compound (and other ingredients, if desired) can also be enclosed in a hard- or soft-shell gelatine capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the one or more affinity reagents and / or interfering agents can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.

[0189] In another embodiment, the affinity reagents and / or interfering agents of the present invention or pharmaceutical compositions according to any of the variants disclosed herein are administered to the subject in combination with other therapeutic agents.

[0190] Additional active compounds or therapeutic agents also can be incorporated into the pharmaceutical compositions according to any of the variants disclosed herein. The compositions herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise a cytotoxic agent, cytokine, growth inhibitory agent and / or cardioprotectant. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. These therapeutic agents include, without limitation, antibodies that bind other targets, anticancer agents, photosensitizers, androgen, oestrogen, nonsteroidal anti-inflammatory agents, antihypertensive agents, analgesic agents, antidepressants, antibiotics, anaesthetics, antiemetics, anti-infectants, contraceptives, antidiabetic agents, steroids, anti-allergy agents, chemotherapeutic agents, anti-migraine agents, agents for smoking cessation, anti-viral agents, immunosuppressants, thrombolytic agent, cholesterol-lowering agents and antiobesity agents.

[0191] In one embodiment, the additional therapeutic agents may be treating the symptoms of the selected cancer on their own and may optionally synergize with the effects of the affinity reagent. The additional agent that is administered may be selected by one skilled in the art for treating the selected cancer, in particular lymphoma.

[0192] Merely by way of example, co-administration of the affinity reagent and / or the interfering agent of the invention with an additional therapeutic agent (combination therapy) encompasses administering a composition comprising an anti-HERV antibody or nanobody of the invention and the additional therapeutic agent as well as administering two or more separate compositions, one comprising the anti-HERV antibody or nanobody and the other(s) comprising the additional therapeutic agent(s). Further, although co-administration or combination therapy generally means that the affinity reagent and / or the interfering agent and additional therapeutic agents are administered at the same time as one another, it also encompasses instances in which the affinity reagent and / or the interfering agent and additional therapeutic agents are administered at different times.

[0193] For instance, an affinity reagent and / or the interfering agent according to the present invention may be administered once every three days, while the additional therapeutic agent is administered once daily. Alternatively, the affinity reagent may be administered prior to or after treatment with the additional therapeutic agent, for example after a patient has failed therapy with the additional agent. Similarly, administration of the affinity reagent may be administered prior to or after other therapy. The affinity reagent and / or the interfering agent or a pharmaceutical composition thereof and one or more additional therapeutic agents (the combination therapy) may be administered once, twice, or at least the period of time until the disease is treated, palliated or cured. Preferably, the combination therapy is administered multiple times. The combination therapy may be administered from three times daily to once every six months. The administering may be on a schedule such as three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once every month, once every two months, once every three months and once every six months, or may be administered continuously via a minipump. The combination therapy may be administered via an oral, mucosal, buccal, intranasal, inhalable, intravenous, subcutaneous, intramuscular, or parenteral.

[0194] Such combination therapy may require lower dosages of the affinity reagent and / or the interfering agent of the invention as well as the co-administered agents, thus avoiding possible toxicities or complications associated with the various monotherapies.

[0195] The compositions according to any of the embodiments of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of one or more affinity reagent or antigen-binding portion thereof and / or of one or more interfering agents of the invention. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the affinity reagent or portion thereof and / or of the interfering agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the affinity reagent or portion to elicit a desired response in the selected subject. A therapeutically effective amount is also one in which any toxic or detrimental effects of the affinity reagent or portion thereof and / or of the interfering agent are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.

[0196] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the one or more affinity reagent or portion thereof and / or the interfering agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an affinity reagent and / or interfering agent for the treatment of sensitivity in individuals.

[0197] In one preferred embodiment, the pharmaceutical composition comprising equal or less than 400 mg for dosage unit of one or more affinity reagents or antigen-binding portion thereof and / or one or more interfering agents according to any of the variants described herein, more preferably less than 400, 350, 300, 250, 200, 150, 100, 50, 25, 10 mg for dosage unit.

[0198] The pharmaceutical composition comprising such dosage unit is preferably for parental administration, for example for intravenous, subcutaneous, intraperitoneal, or intramuscular administration. In one preferred embodiment, the pharmaceutical composition is in the liquid form in a concentration between 20 and 200 mg / ml, more preferably between 40 and 80 mg / ml. An exemplary, non-limiting range for a therapeutically or prophylactically- effective amount of an affinity reagent or portion thereof of the invention is 0.025 to 50 mg / kg, more preferably 0.1 to 5 mg / kg, more preferably 0.1-5, 0.1 to 4 or 0.25 to 3 mg / kg.

[0199] In one embodiment, the invention provides one or more affinity reagent or an antigenbinding portion and / or one or more interfering agents according to any one of the embodiments herein disclosed, for use in a method for prophylactic or therapeutic treatment of a cancer or conditions or disorders resulting from such disease, in particular a lymphoma according to any of the variants disclosed in the present specification, wherein said method comprising the step of administering to a subject a therapeutically effective amount of said affinity reagent or antigen-binding portion thereof and / or said interfering agent, preferably between 0.025 to 50 mg / kg, more preferably 0.1 to 5 mg / kg, more preferably 0.1-5, 0.1 to 4 or 0.25 to 3 mg / kg once a day, for example for at least one, two, three, four, five, six, seven, eight, nine, ten, eleven days. Such subject is a mammal, preferably a human. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0200] In another embodiment of the present disclosure, compositions are provided that comprise the affinity reagent, or antigen binding portions described herein, and / or the interfering agent bound to a solid support. A solid support for use in the present disclosure will be inert to the reaction conditions for binding. A solid phase support for use in the present disclosure must have reactive groups or activated groups in order to attach the affinity reagent or its binding partner thereto. In another embodiment, the solid phase support may be a useful chromatographic support, such as the carbohydrate polymers SEPHAROSE(R), SEPHADEX(R), or agarose. As used herein, a solid phase support is not limited to a specific type of support. Rather, a large number of supports are available and are known to one of ordinary skill in the art. Solid phase supports include, for example, silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, magnetic beads, membranes (including, but not limited to, nitrocellulose, cellulose, nylon, and glass wool), plastic and glass dishes or wells, and the like.

[0201] Another aspect of the present invention provides a therapeutic kit comprising one or more affinity reagent, or antigen-binding portion thereof, and / or one or more interfering agent according to any of the embodiments of the invention or a composition comprising such one or more affinity reagents or antigen-binding portion and / or one or more interfering agent thereof according to the invention, as well as its use in a prophylactic and / or therapeutic treatment of a cancer in a subject in need thereof. The cancer can be any of the cancers disclosed in the present specification and in the claims, in particular is a lymphoma, more preferably DLBCL.

[0202] The kit may include, in addition to the affinity reagent and / or interfering agent or compositions thereof, diagnostic or therapeutic agents. A kit can also include instructions for use in a diagnostic or therapeutic method, as well as packaging material such as, but not limited to, ice, dry ice, styrofoam, foam, plastic, cellophane, shrink wrap, bubble wrap, cardboard, and starch peanuts. In still another embodiment, the kit includes one or more affinity reagents or a composition comprising it and one or more therapeutic agents that can be used in a method as previously disclosed.

[0203] Diagnostic and Prognostic Methods of Use

[0204] The affinity reagents or antigen-binding portion thereof according to the invention may be used also as diagnostic and / or prognostic tools for rapid detection or monitoring of the development of a cancer according to any of the variants disclosed herein, in particular a lymphoma, more particularly a lymphoma selected from diffuse large B-cell lymphoma (DLBCL), such as ABC-DLBCL, GCB-DLBCL, mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), chronic lymphocytic leukemia (CLL), Burkitt lymphoma (BL) and primary mediastinal B-cell lymphoma (PMBCL), more preferably DLBCL.

[0205] In one aspect, the invention provides diagnostic methods. The affinity reagent or antigenbinding portion thereof can be used to detect a surface antigen of a HERV according to any of the variants disclosed herein in a biological sample in vitro or in vivo. In one embodiment, the invention provides a method for diagnosing the presence or location of a surface antigen of HERV, preferably the envelope protein of said HERV, according to any of the variants disclosed herein in a subject in need thereof. The affinity reagents can be used in a conventional immunoassay, including, without limitation, an ELISA, an RIA, flow cytometry, tissue immunohistochemistry, Western blot (immunoblot) or immunoprecipitation.

[0206] In one particular embodiment, the invention provides the in vitro or in vivo use of a surface antigen of a HERV according to any of the variants disclosed herein, preferably the use of the envelope protein of said HERV, as a biomarker for the diagnosis and / or prognosis and / or to monitor the development of a cancer in a subject, in particular wherein said cancer is a lymphoma, preferably DLBCL. Preferably, said surface antigen of HERV is a surface antigen of at least one virus belonging to the HML-2 subgroup of the HERV-K family, even more preferably of HERV-K113, more preferably is the envelope protein of a virus belonging to the K-family of HERVs, even more preferably the envelope protein of at least one virus belonging to the HML-2 subgroup of the HERV-K family, even more preferably the envelope protein of HERV-K113. In a particular embodiment, said surface antigen is the protein having the aminoacidic sequence set forth in SEQ ID NO. 6. In another aspect, the invention thus provides an in vitro or ex vivo method for revealing the presence and / or quantifying the expression levels of a HERV, preferably of at least one virus belonging to the HML-2 subgroup of the HERV-K family, even more preferably of HERV- K113, in a sample comprising the following steps: i) Contacting an affinity reagent or an antigen-binding portion thereof according to any of the variants disclosed in the present specification and in the claims with said sample; ii) Detecting the binding of said affinity reagent or an antigen-binding portion thereof with a surface antigen of said HERV.

[0207] In another embodiment, the invention provides an in vitro or ex vivo method for the diagnosis and / or prognosis and / or to monitor the development of a cancer in a subject, in particular a lymphoma and more preferably a DCBLC, wherein the method comprises the following step: i) determining and / or quantifying the expression levels of a surface antigen of a human endogenous retrovirus (HERV), preferably a surface antigen of at least one virus belonging to the HML-2 subgroup of the HERV-K family, even more preferably of HERV-K113, in a sample isolated from said subject.

[0208] According to an aspect of the invention, the herein described methods can be used for the diagnosis and / or to monitor the development and / or to provide a prognosis of any type of HERV positive cancer of a subject such as those exemplified in the present specification. Preferably said cancer is a lymphoma, more particularly a lymphoma selected from diffuse large B-cell lymphoma (DLBCL), such as ABC-DLBCL, GCB-DLBCL, mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), chronic lymphocytic leukemia (CLL), Burkitt lymphoma (BL) and primary mediastinal B-cell lymphoma (PMBCL), more preferably DLBCL.

[0209] Preferred examples of samples to use in the diagnostic or prognostic methods herein disclosed are urine, stool, whole blood, plasma, serum, saliva, biopsies, cerebrospinal fluid, nasopharyngeal and oropharyngeal wash, sputum, endotracheal aspirate, bronchoalveolar lavage or other biological or histological samples obtainable from said subject, preferably the sample is a cancer sample isolated from said subject, in particular a histological cancer sample.

[0210] Under histological sample according to the present invention for example a sample of isolated cancer cells is meant such as a portion of lymphoid tissue. For the preparation of said histological samples any one of the histological techniques, known to the person skilled in the art, could be used.

[0211] Suitable labels for the affinity reagents, e.g. in the form of an antibody, or secondary antibody have been disclosed supra, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, [beta]-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol.

[0212] The diagnostic and / or prognostic methods according to any embodiments herein disclosed can also include a further step of comparing the expression levels determined and / or quantified in step i) with a reference value. Preferably, a detected higher expression level than the reference value will show a negative diagnosis and / or a prognosis, whereas a lower value will show a positive diagnosis and / or prognosis, namely a positive survival outcome. According to an aspect of the invention, said reference value can be derived from an analysis performed on a biological sample of an individual affected by a cancer or by an analysis performed on a biological sample of a healthy subject.

[0213] In case of a method to monitor the development of cancer, an adequate reference value can be a value corresponding to the expression level of the surface antigen, detected in a biological sample obtained from the same subject in different moments during the therapy and / or during the period or disease.

[0214] Under the expression “determining and / or quantifying the expression levels” according to the present description, “detecting the presence” and / or “quantifying” the expression levels of a surface antigen of HERV according to any of the variants disclosed in the present specification in the sample obtained from the subject, in relative or absolute terms, according to any one of the methods available to the person skilled in the art is meant; according to an aspect of the invention, in the herein described methods the detection and / or quantification of the expression levels can be performed by means of the execution of an in vitro test.

[0215] Not limiting examples of in vitro tests suitable to be used for the detection of the presence and / or for the quantification of the expression levels of a HERV surface antigen, include, for example, an immunoassay, an aptamer-based assay, a histological or cytological assay, a colorimetric assay, an assay of protein activity, flow cytometry, RT-PCT, and / or a mRNA expression level assay, preferably include flow cytometry analysis.

[0216] Such assay can be performed, for example, by means of experiments of RT-PCR or “chain reaction of inverse polymerase”, followed by experiments of mRNA sequencing. The extraction of mRNA from said biological sample obtained from a subject can be performed according to any one of the techniques known to the person skilled in the art, for example by using reagents and commercial kits.

[0217] All types of the above indicated in vitro tests are known to the person skilled in the art who, knowing the surface antigen that has to be detected and / or quantified and on which sample said test has to be performed, could select the most suitable protocol.

[0218] An immunoassay suitable for the detection of the presence and / or for the quantification of the expression levels of the HERV surface antigens according to the present invention, is an immunoassay providing the use of at least an affinity reagent such as an antibody, nanobody or a fragment thereof according to any of the variants disclosed in the present specification and in the claims.

[0219] According to one preferred embodiment, step i) of said in vitro or ex vivo method for the diagnosis and / or prognosis and / or to monitor the development of a cancer, comprises the following steps: ii) Contacting an affinity reagent or an antigen-binding portion thereof according to any of the embodiments disclosed in the present specification and in the claims with the sample of said subject; iii) Detecting the binding of said affinity reagent or antigen-binding portion thereof with said surface antigen of said human endogenous retrovirus (HERV).

[0220] Preferably, in a method according to any of the embodiments disclosed herein, the affinity reagent or antigen-binding portion thereof is used that specifically binds to an envelope peptide or protein of a virus belonging to the K family of HERVs, preferably of at least one virus belonging to the HML-2 subgroup of the HERV-K family, even more preferably of HERV-K113. More preferably, the affinity reagent is the nanobody having the aminoacidic sequence set forth in SEQ ID NO. 5.

[0221] In one embodiment, said affinity reagent is directly labelled with a detectable label. In another embodiment, a first affinity reagent is used that is unlabelled and a second antibody or other molecule that can bind the selected affinity reagent is labelled. As is well known to one of skill in the art, a second antibody is chosen that is able to specifically bind the particular species and class of the first affinity reagent.

[0222] For example, if the selected affinity reagent is a human IgG, then the secondary antibody could be an anti-human-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially, e.g., from Pierce Chemical Co.

[0223] The diagnostic and / or prognostic methods according to any embodiments herein disclosed may be followed by a further step of the administration in the positive subject of an affinity reagent, interfering agent or pharmaceutical composition thereof according to any of the variants herein disclosed.

[0224] In another aspect the invention further provides a diagnostic and / or prognostic kit. In particular the invention provides a diagnostic or prognostic kit comprising as a specific reagent an affinity reagent or an antigen-binding portion thereof according to any of the embodiments disclosed herein. The kit is intended in particular for use in a method for determining and / or quantifying, in a sample from a subject, particularly a cancer sample isolated from said subject, the expression levels of a surface antigen of a HERV according to any of the variants illustrated in the present specification and in the claims, preferably the expression levels of the envelope protein of at least one virus belonging to the HML-2 subgroup of the HERV-K family, even more preferably of HERV-K113.

[0225] As previously mentioned, a diagnostic or prognostic kit according to the invention can also include instructions for use in a diagnostic method, as well as packaging material such as, but not limited to, ice, dry ice, styrofoam, foam, plastic, cellophane, shrink wrap, bubble wrap, cardboard, and starch peanuts. In one embodiment, the kit includes one or more affinity reagents or a composition comprising it and a diagnostic agent that can be used in a method described above.

[0226] In an embodiment the kit according to the invention will include also control means which could be used for example to compare the expression levels of HERV surface antigens with reference values obtained from a suitable control, for example controls which have known expression levels of said surface antigens. Such control means includes for example a biological sample obtained from a healthy subject or from a sick subject, wherein the expression level of said HERV surface antigens was previously measured.

[0227] The kit according to any one of the herein described embodiments could further include auxiliary components, such as buffers, fluorophores, chromophores, diluting agents and / or use instructions.

[0228] The invention further provides the in vitro or ex vivo use of a kit according to any of the embodiments herein disclosed for the diagnosis and / or prognosis and / or to monitor the development of a cancer in a subject in need thereof, preferably of a lymphoma selected from diffuse large B-cell lymphoma (DLBCL), such as ABC-DLBCL, GCB-DLBCL, mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), chronic lymphocytic leukemia (CLL), Burkitt lymphoma (BL) and primary mediastinal B-cell lymphoma (PMBCL), more preferably DLBCL.

[0229] ***

[0230] The following experimental section is provided solely by way of illustration and not limitation and does not intend to restrict the scope of the invention as defined in the appended claims. The claims are an integral part of the description.

[0231] EXAMPLES

[0232] 1. Development of a humanized nanobody (FF-01) against endogenous retroviruses to target cancer cells.

[0233] Due to the need to define novel therapeutics for DLBCL patients, using an open-source pipeline, an ad hoc analysis has been performed to specifically quantify the RNA expression of viral genes in DLBCL clinical specimens and healthy people.

[0234] Materials and methods

[0235] CELL LINES

[0236] Cell lines were cultured according to the recommended conditions, as previously reported (Spriano F, Chung EYL, Gaudio E, et al. The ETS Inhibitors YK-4-279 and TK-216 Are Novel Antilymphoma Agents. Clin Cancer Res. 2019;25(16):5167-5176; Tarantelli C, Gaudio E, Arribas AJ, et al. PQR309 Is a Novel Dual PI3K / mT0R Inhibitor with Preclinical Antitumor Activity in Lymphomas as a Single Agent and in Combination Therapy. Clin Cancer Res. 2018;24(l): 120-129).

[0237] Media were supplemented with fetal bovine serum (FBS, 10%) plus 0.1% Penicillin- Streptomycin 100X (Euroclone, ECB3001D). Cell line's identity was confirmed by short tandem repeat (STR) DNA fingerprinting using the Promega GenePrint 10 System kit (B9510)13. Cells were tested to confirm mycoplasma negativity using the My coAlert Mycoplasma Detection Kit (Lonza, Visp, Switzerland).

[0238] RNA EXTRACTION AND ENVELOPE QUANTIFICATION

[0239] Total RNA was extracted with TRIzol Reagent (Thermo Fisher Scientific, Waltham, Massachusetts, US). DNAse treatment was performed (DNAse, Sigma-Aldrich, St. Louis, Missouri, US). Reverse transcription and quantitative real-time PCR were performed with the QuantiFast SYBR Green PCR Kit (Qiagen, Hilden, Germany). Since the endogenous retrovirus envelope is composed by only one exon a negative control without retro transcription, to infare the DNA contamination, was applied.

[0240] Primer used for HERV envelope q-PCR are described by Grbger et al. (Groger V, Wieland L, Naumann M, et al. Formation of HERV-K and HERV-Fcl Envelope Family Members is Suppressed on Transcriptional and Translational Level. IntJMol Sci. 2020;21(21)).

[0241] GAPDH was used as negative control. HERVK-1 13 PCR detection

[0242] The strategy described by Moyes et al. was used (Moyes DL, Martin A, Sawcer S, et al. The distribution of the endogenous retroviruses HERV-K113 and HERV-K115 in health and disease. Genomics. 2005;86(3):337-34T). Briefly, three different PCR reactions were performed. A first one (mix 1) amplifies the pre-integration sites in the absence of any viral integration. The other two mixes amplify only in the presence of the viral insertion. Homozygous samples lacking a viral allele would give a band only for mix 1, while homozygous individuals who have the viral allele would show bands only in mix 2 and 3. Heterozygous sample would have a band in all three reactions.

[0243] Flow cytometry

[0244] Around 500’000 cells for each condition were harvested, washed, and incubated with primary antibody 30’ in ice. Similarly, cells were incubated with secondary antibody for 30’ in ice. Flow cytometry was performed with BD FACSCanto™ II (BD biosciences, California). Analysis was performed with FlowJo software.

[0245] Anti -HER V-envel ope Ab (MBS9216561) from MyBioSource; Isotype control Ab ( ab37415) from abeam .

[0246] FACS buffer: PBS, 1% BSA, 0.1% sodium azide.

[0247] Immunoblotting

[0248] Protein extraction, separation, and immunoblotting were performed as previously described (Spriano F, Sartori G, Tarantelli C, et al. Pharmacologic screen identifies active combinations with BET inhibitors and LRRK2 as a novel putative target in lymphoma. EJHaem. 2022;3(3):764-774). The following antibodies were used: anti-HERVK envelope MBS9216561) from MyBioSource, anti-GAPDH (FF26A) from eBioscience, secondary mouse (NA93 IV) and rabbit (NA934V) antibodies from GE healthcare. Data were analyzed with Fusion solo software (Vilberg, France).

[0249] Peptide prediction

[0250] The HERV-K113 sequence as well as the prediction about the localization of protein domains and putative glycosylation points were retrieved from Uniprot (https: / / www.uniprot.org / uniprotkb / Q902F9 / entry). The protein model was obtained from alphafold . The dedicated database accessible at https: / / alphafold.ebi.ac.uk. Custom peptides were purchased from GeneScript.

[0251] SPR experiments

[0252] SPR experiments were run on a Biacore8000K instruments. Streptavidin was immobilized on the CM5 surface by a standard amino coupling. Streptavidin was used to immobilize peptl and pept2 by the biotin moiety bounds to the peptide n-terminal ends. Five increasing concentrations of the nanobodies 6.25,12.5, 25, 50 and 100 nM were injected using a singlecycle kinetics setting. 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% Tween-20 was used as running buffer. Subsequently, curve fitting was executed using Biacore Insight Evaluation Software (version 5.0.18). The fitting successfully passed the quality control assessment integrated within the software.

[0253] ELISA

[0254] 96 well plates were coated with NeutrAvidin (2pg / ml; Thermo Fisher, Massachusetts, US) overnight at room temperature (RT). Plates were washed and subsequently coated with biotinylated peptides, 1 hour at RT. Plates were then washed, blocked and incubate 1 hour with plasma (1 :50 dilution). Peroxidase-conjugated anti-human igG (Sigma-Aldrich, St. Louis, Missouri, US) was added (1 hour, RT). Finally, substrate (Pierce™ TMB Substrate Kit, Thermo Fisher Scientific, Massachusetts, US) was administered. Absorbase (450nm) was red with BioTek Cytation 3 (Agilent, California, US). Absorbance results above 0.2 were considered positive.

[0255] Antibody-dependent cell-mediated cytotoxicity

[0256] Antibody-dependent cell-mediated cytotoxicity (ADCC) was performed with the ADCC Reporter Bioassay, Core Kit (Promega, Wisconsin, Stati Uniti) following manufacturer’s protocols. Effector to Target cells ratio was 3 to 1 (45’000 Effector and 15’000 target cells). FF-01 was compared to a non-binding nanobody (FF-00) or to isotype control antibody. ADCC induction was evaluated after 6 or 14 hours of coculture.

[0257] Internalization For the internalization experiment the Incucyte Fabfluor-pH Antibody labeling reagent (Sartorius, Germany) was used following manufacturer’s protocols. 96 well plates were coated with poly-L-Omithine. 30’000 cells for each well in 50 pl were seeded. Cells were allowed to adhere to the plate bottom for at least one hour and subsequently incubated with 50 pls containing the antibodies (1 pg / ml) and the Fabfluor antibody at 1 :6 molar ratio. Plates were insert in the live cell imaging Incucyte and images were taken every 45 minutes. Analysis was performed with the internal Incucyte analysis software as red area divided on phase confluence. Normalized on time 0.

[0258] Envelope downregulation

[0259] For transient knock down, the Amaxa 4D Nucleofector system (Lonza, CH) was used to introduce HERV-K envelope siRNAs. SiRNA design was done using Sfold sotware and the tool provided by Darmacon. 2 different siRNAs were designed and tested plus a nontargeting siRNA as control and BLOCK-iT Fluorescent Oligo, for electroporation efficiency control. Protocols were followed according to the SG Cell Line 4D -Nucleofector X Kit L (Lonza). In brief, 2 * 106 cells were prepared and resuspended in 100 pl SG solution with 800 pmol siRNA. Efficiency was confirmed 48 hours after nucleofection, thanks to BLOCK-iT control, by flow cytometry. Cell proliferation was evaluated periodically until 72 hours and protein downregulation after 72 hours from the starting of the experiment was determined.

[0260] Bioinformatic analysis

[0261] The analysis was started by downloading the fastq files from scratch for EGAD00001003783 and GSE71702 datasets. The datasets were processed separately, including quality control, mapping, and gene qualification using Gencode v22. The aligned BAM files were then inputted into Telescope (vl.0.3) (Bendall ML, de Mulder M, Iniguez LP, et al. Telescope: Characterization of the retrotranscriptome by accurate estimation of transposable element expression. PLoS Comput Biol. 2019; 15(9):el006453\ a tool for retrotranscriptomic profiling. Telescope utilizes the expectation maximization algorithm to identify transposable elements (TEs) at specific genomic loci. Next, the raw counts matrices of the two datasets were merged and the HERV counts were filtered. Only features with more than 5 observations within a minimum sample threshold were retained. To normalize the counts, the TMM algorithm in edgeR was employed (Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26(l): 139-140), and any batch effects were removed using the removeBatchEffect function of limma (Law CW, Chen Y, Shi , Smyth GK. voom: Precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 2014;15(2):R29). For visualization purposes, an MDSPlot was conducted on the log2 transformed counts per million (lepm) and utilized the plotMDS function in limma. To cluster the DLBCL samples, the k-means algorithm was employed with k=4 in the stats R package (version 3.6.2. Additionally, Fisher's exact test was used to test each cluster against the cell-of-origin. To compare the HERV clusters to the previous DLBCL cell-of-origin classification, alluvial plots were generated using ggalluvial (vO.12.3). For survival analysis, the survival R package (v3.1-12) was utilized and conducted group-level comparisons using the log-rank test. Kaplan-Meier survival plots were created using survminer (vO.4.9) and ggplot2 (v3.3.6). All analyses were performed using Bash, R (v4.2.2), and the BioConductor package manager (vl.30.19).

[0262] Results

[0263] Transposable elements are expressed and can subcluster diffuse large B cell lymphoma patients.

[0264] Thanks to the computational software tool Telescope, the expression of transposable elements (TE) comprising endogenous retroviral elements in RNA-seq data from two publicly available datasets (EGAD00001003783 and GSE71702) of patients with diffuse large B-cell lymphoma was assessed. In parallel normal specimens were also analyzed. After batch effect correction, an unsupervised multidimensional scaling (MDS) plot was performed. The expression of TE was able to clearly separate tumoral from normal samples (Figure 1A). These data demonstrate that tumoral and normal specimens have distinct TE and HERV expression profiles. Focusing on tumor samples only, we wonder whether we could identify specific signature. MDS plot was also able to well separate ABC and GCB- DLBCL (Figure IB).

[0265] Performing k-means algorithm (k=4) four different clusters (Cluster 1 / 2 / 3 / 4) were identified in which DLBCL could be separated based on TE and HERV expression (Figure 1C).

[0266] ABC-DLBCL were principally enriched in cluster 3, while cluster 1, cluster 2 and cluster 4 belonged mainly to the GCB DLBCL class with different numbers of ABC and unclassified patients (Figure ID). The four clusters presented also different prognosis. Cluster 3 showed the worst prognosis followed by cluster 2 and cluster 1, with cluster 4 showing the best prognosis (Figure IE). As discussed in the background section, the HERV-K (HML-2) family has an intact open reading frame. Looking for differences in RNA expression between tumoral and normal specimens, many HERV-K elements were identified significantly upregulated in tumoral compared to normal (Figure 2A). Among the different HERV-K family members, KI 13 showed to be one of the more differentially expressed (Figure 2B).

[0267] Endogenous retroviruses in cell lines.

[0268] A similar analysis was performed in approximately 50 cell lines comprising ABC-DLBCL, GCB-DLBCL, mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), chronic lymphocytic leukemia (CLL), Burkitt lymphoma (BL) and primary mediastinal B-cell lymphoma (PMBCL). Similarly of what observed in DLBCL patients, MDS plot we were able to separate the different subtypes of lymphoma based on TE and HERV expression (Figure 3).

[0269] Based on this information and the identification of expression of endogenous retroviruses in lymphoma patients, its expression in DLBCL lymphoma cell lines was investigated. The primary focus was on the envelope (env) gene of HERVs and its expression was discovered in all the DLBCL cell lines tested at different degrees (Figure 4A). In addition to RNA expression, it was also demonstrated that the RNA can be translated to protein in multiple DLBCL cell lines (Figure4B).

[0270] Importantly, we demonstrate that human endogenous retroviruses envelope proteins can not only be expressed but are also exposed on the cell surface. Among the cell lines tested, RCK8 exhibited the highest expression of HERVK-env (Figure 5).

[0271] As HERV-K113 represents one of the most studied human endogenous retroviruses, our investigation aimed to the genomic insertion of the KI 13 in DLBCL cell lines. On this purpose, we used the methodology outlined by Moyes et al. (Figure 6) (Moyes DL, Martin A, Sawcer S, et al. The distribution of the endogenous retroviruses HERV-K113 and HERV- K115 in health and disease. Genomics. 2005;86(3):337-34T). Among the cell lines tested, we demonstrated an heterozygous genomic insertion of HERV-K113 in the DLBCL cell line RCK8 (Figure 7), which also exhibited the highest surface envelope expression. Conversely, the remaining DLBCL cell lines did not present any HERV-K113 genomic insertion (Figure 8).

[0272] Notably, it is worth mentioning that while RCK8 is the cell line with the highest expression, other cell lines showed detectable protein expression by WB and at less extent by FACS. These findings suggest the presence of others HERV-K family members other than KI 13 that can be re-expressed in tumors. Due to the presence of KI 13 insertion and the highest level of surface envelope expression, RCK8 was used as positive control in further experiments.

[0273] Envelope of Endogenous retroviruses sustain growth of lymphoma cell lines.

[0274] The role of endogenous retroviruses in cancer remains poorly understood. While they have been reported to be upregulated in many tumors, whether this upregulation is a cause of cancer or the effect of an epigenetic reshaping of the cells is still a subject of debate.

[0275] As described above, we demonstrate the expression of HERV-K both at RNA and protein level in lymphoma patients and cell lines. To understand whether this expression is happening because of an epigenetic reshaping of cells, without a real involvement in cellular growth, we decided to silence the expression of the envelope of HERV-K. For this purpose, we used two different siRNAs designed to target different regions of the HERV-K envelope (Figure 9).

[0276] We obtained a nucleofection efficiency higher than 90% and both siRNAs showed a downregulation of the target higher than 50% (Figure 10A / B). The downregulation of the envelope of endogenous retroviruses lead to a decrease in cell proliferation, with approximately 50% less living cells number after 72hours in siRNA-treated cells compared to cells treated with a negative control siRNA (Figure 10C). The impact was more pronounced in decreasing the rate of cell proliferation rather than inducing cell death, as demonstrated by only a slight decrease in the percentage of dead cells after envelope downregulation (Figure 10D). These data prove the importance of endogenous retroviruses in sustaining tumoral cell growth.

[0277] Identification of envelope surface epitopes

[0278] Having demonstrated the presence of HERV envelope on the surface of lymphoma cells, it becomes evident the possible use of them as novel tumor antigen and potentially targetable by antibodies. To date, no atomistic structures HERV-K113 are deposited into the protein data bank (PDB), therefore the epitopes prediction was conducted by analyzing the structural model generated by alphafold, a recently released Al-based tool for protein structure prediction, deposited into the EMBL database EMBL (https: / / alphafold.ebi.ac.uk).

[0279] The model (Figure 11A / B) includes two main regions, one predicted to be transmembrane or cytosolic (TM) and one to be located into the extracellular surface (SU), in agreement with reported data. SU and TM are posttranslational cleaved and are attached by noncovalent interactions or by a labile interchain disulfide bond.

[0280] By focusing on the extracellular region, a careful examination was conducted to identify epitopes with high potential for antibody binding. Through this comprehensive analysis, a set of four peptides (peptide 2 / 3 / 4Z5) was selected as optimal candidates for the subsequent nanobody selection process using phage display screening. In particular, the selection of the peptides was carried out considering their accessibility in the context of the protein structure, the absence of putative glycosylation sites as well as the absence of free cysteines.

[0281] Serum of lymphoma patients contains antibodies targeting endogenous retroviruses.

[0282] Since previous work have shown the presence of antibodies in serum samples of patients with Systemic Lupus Erythematosus, and breast cancer, we checked whether lymphoma patients could have anti-HERV envelope antibodies confirming the immunogenicity of the identified peptides. We tested serum samples of around 160 patients, by ELISA, for the presence of antibodies recognizing the 4 peptides. Among the 160 patients 72 showed positivity for peptide 2, 89 for peptide 3, while 54 and 29 for peptide 4 and 5 respectively, using a cut-off for positivity of 0.2 (Figure 12).

[0283] The data were validated in a second dose-response experiment, confirming the presence of antibodies targeting the envelope of endogenous retroviruses in lymphoma patients (Figure 13). This data showed us that the selected peptides have immunogenic properties and that peptide 2 and 3 showed higher immunogenicity.

[0284] Identified patients with anti-HERV antibodies could serve as a future source of therapeutic human antibodies.

[0285] Antibodies targeting endogenous retroviruses induce immune response. All four peptides were produced and used in a phage display screening to identify potential camelid nanobodies capable of recognizing one or more of the selected peptides.

[0286] Thanks to the screening we identified two nanobodies that we called FF-00 and FF-01. Both showed specificity of binding to peptide 3. The binding affinities of the nanobodies identified during the screening process were validated using surface plasmon resonance (SPR) experiments. Notably, among the identified nanobodies, only one exhibited a strong interaction with peptide 3 (FF-01), as confirmed by the SPR results (Figure 14A). This nanobody demonstrated a remarkable binding affinity, with a dissociation constant (Kd) of 142 nM. In contrast, the remaining nanobody (FF-00) displayed weak or negligible affinity towards the target peptides. FF-00 and FF-01 were humanized with the expression with IgGl human Fc.

[0287] In accordance with SPR data and similarly to the rabbit commercial antibody, FF-01 was able to strongly bind to env protein on the cellular surface, while FF-00 showed only a mild or null binding (Figure 14B).

[0288] Sequence of peptide 3 is conserved among different HERV-K family members (Figure 15). This makes the antibody potentially capable of binding to and recognizing different members of the family.

[0289] We tested the possible direct anti-lymphoma activity of the two antibodies, but we did not see any activity that was superior in treated cell lines to the negative control buffer only.

[0290] One of the first mechanisms by which antibodies can exploit anti-tumoral activity is the antibody dependent cellular cytotoxicity (ADCC). FF-01 showed capability to induce ADCC at 1 pg / ml in the high env expressor cell line and not in the others. On the contrary FF-00 and a second negative control antibody did not show any sign of ADCC induction (Figure 16).

[0291] Finally, as proof of principle for a possible antibody drug conjugate, we analyze the internalization properties of the antibodies. In line with the strength of binding of the two antibodies, FF-01 was nicely internalized in high env expressor cell line but also in the low expressor U2932 but not in the negative cell line TOLEDO. To a lesser extent FF-00 was internalized only in RCK8 high expressor cell line but with a milder signal compared to FF- 01 (Figure 17A / B).

[0292] Conclusions We have demonstrated the presence and expression of endogenous retroviruses in lymphoma patients and cell lines. With an ad-hoc analysis we have identified the expression of endogenous retroviruses in lymphoma patients using reads that are usually discarded from normal RNA-seq data. We were able to separate them in 4 different clusters with different prognosis and recapitulate ABC vs GCB differences. Human endogenous retroviruses (HERV) were differentially expressed in lymphoma patients compared to normal samples and especially from the family K (HML-2) were overexpressed in tumoral samples compared to normal samples.

[0293] In lymphoma cell lines we have confirmed the RNA expression of HERVs, more specifically the envelope gene, and demonstrate that the RNA expression could be also translated to protein. The envelope protein can be transported on membrane and extracellularly exposed. Among the HERVs of the family K, K-113 is one of the more recent ones with an intact ORF. We identified one cell line, RCK8, among the panel of DLBCL cell lines tested with the K-113 heterozygous insertion, while the others were negative. Despite the presence of K-113 only in RCK8, some of the other negative cell lines showed HERVs RNA and protein expression at different degrees, suggesting that K- 113 is not the only player. Previous studies show the involvement of endogenous retroviruses in the growth of cancers. We have demonstrated, by silencing the env RNA expression, that HERV-K env sustains the growth of lymphoma cells. Cell lines with downregulation of env showed a decreased cellular proliferation compared to normal cells. Since the extracellular presentation of the env protein by some lymphoma cell lines, thanks to computational methods, we identified putative extracellular epitopes potentially bound by antibodies. We identified 4 epitopes called 2, 3, 4 and 5. Peptides 2 and 3 were partially overlapping between each other and with the sequence of the commercial rabbit antibody used previously for FACS analysis, confirming their capabilities to be recognized by antibodies. We performed a phage display screening where we identified 2 camelid nanobodies able to recognize peptide 3. The two nanobodies were called FF-00 and FF-01. Surface plasmon resonance experiments confirmed the binding of FF-00 and FF-01 specifically to peptide 3 with a strong binding for FF-01 and very weak for FF-00 that will be used as negative control in future experiments. Nanobodies were coupled with human Fc IgGl producing a human heavy chain antibody. One of the key advantages of heavy chain antibodies is the smaller size that allows them to penetrate tissues and tumors more rapidly and deeply than mAbs. FF-01 demonstrates to bind also to the env full protein when extracellularly expressed by cell lines and at less extent FF-00 too.

[0294] Recently, antibodies targeting endogenous retroviruses have been shown to promote lung cancer immunotherapy. With the aim of targeting HERVs we tested FF-00 and FF-01 for the capability of having a direct anti-lymphoma activity. Neither antibody showed any direct antitumoral activity. On the contrary, FF-01 exhibited the ability to induce antibodydependent cellular cytotoxicity in line with previous findings in lung cancer. Additionally, both FF-01 and, to a less extent, FF-00 demonstrated internalization in cell lines expressing the envelope protein on their surface. Finally, we checked whether lymphoma patients could already express antibodies able to recognize endogenous retroviruses. We identified patients with antibodies able to recognize the envelope protein. On the contrary we did not identify any antibody in normal samples. These patients could be a source of fully humanized antibodies.

[0295] Sequence Listing in the description

[0296] SEQ ID NO. 1 - sequence of the epitope “peptide 2”

[0297] KFRPKGKPCPKEIPKESKNTEVLV

[0298] SEQ ID NO. 2 - sequence of the epitope “peptide 3”

[0299] YLQDFSYQRSLKFRPKGKP

[0300] SEQ ID NO. 3 - sequence of the epitope “peptide 4”

[0301] LDKHKHKKLQSFY

[0302] SEQ ID NO. 4 - sequence of the epitope “peptide 5”

[0303] GNIVIKPDSQTITSENSRLL

[0304] SEQ ID NO 5 Aminoacidic sequence of the antibody herein identified as FF-01

[0305] MAEVQLQESGGGFVQAGGSLRLSCAASGSNVHVQSMGWFRQAPGKEREFVAAIS MPPSSNSYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTATYYCATFRISQRYY EAEYWGQGTQ VT VS S AAA

[0306] SEQ ID NO. 6 - Aminoacidic sequence of the envelope protein of HERV-K112

[0307] MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQMKLPSTKKAEPPTWAQL KKLTQLATKYLENTKVTQTPESMLLAALMIVSMVVSLPMPAGAAAANYTYWAY VPFPPLIRAVTWMDNPIEIYVNDSVWVPGPTDDCCPAKPEEEGMMINISIGYRYPPI CLGRAPGCLMPAVQNWLVEVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSL

[0308] KFRPKGKPCPKEIPKESKNTEVLVWEECVANSAVILQNNEFGTLIDWAPRGQFYHN CSGQTQSCPSAQVSPAVDSDLTESLDKHKHKKLQSFYPWEWGEKGISTARPKIISP VSGPEHPELWRLTVASHHIRIWSGNQTLETRDRKPFYTIDLNSSLTVPLQSCVKPPY MLVVGNIVIKPDSQTITCENCRLLTCIDSTFNWQHRILLVRAREGVWIPVSMDRPW EASPSVHILTEVLKGVLNRSKRFIFTLIAVIMGLIAVTATAAVAGVALHSSVQSVNF VNDWQNNSTRLWNSQSSIDQKLANQINDLRQTVIWMGDRLMSLEHRFQLQCDW NTSDFCITPQIYNESEHHWDMVRCHLQGREDNLTLDISKLKEQIFEASKAHLNLVP GTEAIAGVADGLANLNTVTWVKTIGSTTIINLILILVCLFCLLLVYRCTQQLRRDSD HRERAMMTMVVLSKRKGGNVGKSKRDQIVTVSV

[0309] SEQ ID NO. 7 - sequence of the epitope “peptide 3” in K101_22ql 1.21 YLQDFPYQRSLKFRPKGKP SEQ ID NO. 8 - sequence of the epitope “peptide 3” in KC19_19ql 1 YLQDFSYQRSFKFRPKGKP

[0310] SEQ ID NO. 9 - sequence of the epitope “peptide 3” in K18_lq23.3 CLQDF S YQRSLKFRPKGKT

[0311] SEQ ID NO. 10 - sequence of the epitope “peptide 3” in K42_12q24.33 NLQDSSYQRSLKFRPKGKP

[0312] SEQ ID NO. 11 - sense siRNA2 (5p^3p)

[0313] GCAGCUAACUAUACCUACUUU

[0314] SEQ ID NO. 12 - sense siRNA3 (5p^3p)

[0315] CGAGGUCAAUUCUACCACAUU

[0316] SEQ ID NO. 13 - antisense siRNA2 (5p- 3p)

[0317] AGUAGGUAUAGUUAGCUGCUU

[0318] SEQ ID NO. 14 - antisense siRNA3 (5p- 3p)

[0319] UGUGGUAGAAUUGACCUCGUU

Claims

CLAIMS1. An affinity reagent capable of specifically binding to a surface antigen of a virus belonging to human endogenous retroviruses (HERV), for use in a prophylactic and / or therapeutic treatment of a cancer in a subject in need thereof.

2. The affinity reagent for use according to claim 1, wherein said virus belongs to the type- K human endogenous retrovirus family (HERV-K).

3. The affinity reagent for use according to claim 2, wherein said virus belongs to one of the following subgroups of the HERV-K family: HML-1, HML-2, HML-3, HML-4, HML-5, HML-6, HML-7, HML-8, HML-9, and HML-10.

4. The affinity reagent for use according to claims 2 or 3, wherein said virus is selected among the following viruses belonging to the HML-2 subgroup of the HERV-K family: HERV-K113, HERV-K115, HERV- K102, HERV-K50A, HERV-K107, HERV-K108, HERV-K 109, HERV-K36, HERV-K41, HERV-K101, HERV-C19, HERV-K33, HERV- K18, HERV-K42, and HERV-K103, more preferably is HERV-K113.

5. The affinity reagent for use according to any one of claims 1 to 4, wherein said surface antigen is the envelope protein of said HERV virus, preferably is the envelope protein having the aminoacidic sequence set forth in SEQ ID NO. 6.

6. The affinity reagent for use according to any one of claims 1 to 5, wherein said affinity reagent or antigen-binding portion thereof binds to an epitope comprising or consisting of a peptide selected from: peptide 2 having the sequence set forth in SEQ ID No. 1, peptide 3 having the sequence set forth in SEQ ID No. 2, peptide 3 having the sequence set forth in SEQ ID No. 7, peptide 3 having the sequence set forth in SEQ ID No. 8, peptide 3 having the sequence set forth in SEQ ID No. 9, peptide 3 having the sequence set forth in SEQ ID No. 10, peptide 4 having the sequence set forth in SEQ ID No. 3, and peptide 5 having the sequence set forth in SEQ ID No. 4.

7. The affinity reagent for use according to any one of claims 1 to 6, which is selected from a monoclonal antibody, a multi-specific antibody, a bi-specific antibody, a three-specific antibody, a scFV, a diabody, a triabody, a tetrabody, a minibody, a linear antibody, a chelating recombinant antibody, a tribody, a bibody, an intrabody, a nanobody, an antibody-drug conjugate, a bindingdomain immunoglobulin fusion protein, a fusion antibody, an immunoadhesin, or an antigen binding fragment thereof, preferably is a nanobody having the aminoacidic sequence set forth in SEQ ID No. 5.

8. The affinity reagent for use according to any one of claims 1 to 7, wherein said cancer is a lymphoma, in particular is selected from diffuse large B-cell lymphoma (DLBCL), such as ABC- DLBCL, GCB-DLBCL, mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), chronic lymphocytic leukemia (CLL), Burkitt lymphoma (BL) and primary mediastinal B- cell lymphoma (PMBCL), preferably is DLBCL.

9. An interfering agent capable of preventing and / or inhibiting the expression or activity of a target gene of a human endogenous retrovirus (HERV), preferably of a target gene encoding for a surface antigen of HERV-K113, more preferably wherein said surface antigen is the envelope protein of said HERV, for use in a prophylactic and / or therapeutic treatment of a cancer in a subject in need thereof, preferably wherein said cancer is a lymphoma, more preferably wherein said cancer is a diffuse large B-cell lymphoma (DLBCL).

10. The agent according to claim 9, wherein said agent is an antisense oligonucleotide, a small interfering RNA (siRNA), an aptamer, or a ribozyme, preferably wherein said agent is a siRNA.I L A pharmaceutical composition comprising one or more affinity reagents as defined in any one of the claims from 1 to 8, and / or one or more agents as defined in claims 9 or 10, and a pharmaceutically acceptable carrier.

12. The composition according to claim 11, for use in the prevention and / or treatment of a cancer in a subject in need thereof, preferably wherein said cancer is a lymphoma, more preferably wherein said cancer is a diffuse large B-cell lymphoma (DLBCL).

13. In vitro use of a surface antigen of a human endogenous retrovirus (HERV), preferably a surface antigen of HERV-K113, as a biomarker for the diagnosis and / or prognosis and / or to monitor the development of a cancer in a subject, in particular wherein said cancer is a lymphoma, more preferably wherein said cancer is a diffuse large B-cell lymphoma (DLBCL).

14. An in vitro method for the diagnosis and / or prognosis and / or to monitor the development of a cancer in a subject comprising the following step: i) determining and / or quantifying the expression levels of a surface antigen of a human endogenous retrovirus (HERV), preferably a surface antigen of HERV-K113, in a sample isolated from said subj ect; preferably wherein said cancer is a lymphoma, more preferably wherein said cancer is a diffuse large B-cell lymphoma (DLBCL).

15. A diagnostic or prognostic kit comprising as a specific reagent an affinity reagent as defined in any one of the claims from 1 to 8, said kit being intended in particular for use in a method for determining and / or quantifying, in a sample isolated from a subject, the expression levels of a surface antigen of a human endogenous retrovirus (HERV), preferably a surface antigen of HERV-K113.

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