HERV-k-derived antigens as shared tumor antigens for Anti-cancer vaccine

Abstract

To provide a composition or vaccine comprising at least one peptide, or an expression vector that induces expression of the at least one peptide in vivo, the peptide consisting of, or comprising, shared HERV-K derived antigens, and a pharmaceutically acceptable vehicle or excipient.SOLUTION: The present invention provides a composition comprising Cytotoxic T Lymphocytes (CTLs) of a patient treated with such a peptide, or comprising T-cell Receptor (TCR) engineered T cells recognizing such a peptide.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the identification of epitopes or neoepitopes derived from the HERV-K antigen that are shared by some tumor subtypes and can be used for diagnosis, prognosis and immune monitoring, as well as antigenic compositions, immunogenic compositions, anti-cancer vaccines or T-cell based immunotherapy. Thus, the present invention relates to the field of cancer and immunotherapy, to the development of peptide-based antigenic immunogenic compositions that contain one or more, preferably several of these epitopes and are useful in diagnosis, prognosis and immune monitoring, as well as in the treatment and prevention of cancer, and to the development of immunogenic compositions and vaccines that contain one or more, preferably several of these epitopes for the treatment and prevention of cancer. Alternatively, the composition of the present invention comprises one or more vectors that carry out or cause the expression of said peptides in vivo, for example the vectors can be DNA or RNA vectors, or bacterial or viral vectors. [Background technology]

[0002] Human endogenous retroviruses (HERVs) represent 8% of the human genome. They probably correspond to remnants of ancient germline infections of exogenous retroviruses. Most HERV genes are nonfunctional due to DNA recombination, mutations, and deletions, but some produce functional proteins such as group-specific antigens (Gag), polymerase with reverse transcriptase (Pol), and envelope (Env) surface units. HERV expression is suppressed in normal cells by epigenetic mechanisms.

[0003] HERVs have strong immunogenic properties associated with "viral mimicry" and their expression is increased in some solid tumors by demethylation. HERVs are thought to represent possible pathogenic agents in carcinogenesis that may act by insertional mutagenesis or by participating in chromosomal aberrations. Some HERV proteins, such as HERV-K Rec and Np9, are also putative oncogenes.

[0004] The expression of HERVs in cancer is associated with a variety of effects on the immune system: -Immunomodulation via the immunosuppressive domain of the Env unit, - Activation of natural immunity by HERV dsRNA (induction of innate type I interferon signaling), -Induction of adaptive immune responses against HERV antigens. Summary of the Invention

[0005] Thus, according to the present invention, there is provided an isolated or purified peptide, the peptide consisting of or comprising an epitope consisting of a sequence selected from the group consisting of FLQFKTWWI (SEQ ID NO: 1), RLIPYDWEI (SEQ ID NO: 2), KLIDCYTFL (SEQ ID NO: 3), YLSFIKILL (SEQ ID NO: 4), AMIPKDWPL (SEQ ID NO: 5), YAMSNLFSI (SEQ ID NO: 6), and / or SMDDQLNQL (SEQ ID NO: 7), as well as a composition comprising at least one such peptide, or an expression vector directing the expression of at least one such peptide in vivo. The composition may further comprise a suitable liquid, buffer, or vehicle. In the composition for use in human therapy, the composition further comprises a pharma- ceutically acceptable vehicle, carrier, or excipient.

[0006] In particular, there is provided an immunogenic composition comprising at least one peptide consisting of or comprising an epitope consisting of a sequence selected from the group consisting of FLQFKTWWI (SEQ ID NO:1), RLIPYDWEI (SEQ ID NO:2), KLIDCYTFL (SEQ ID NO:3), YLSFIKILL (SEQ ID NO:4), AMIPKDWPL (SEQ ID NO:5), YAMSNLFSI (SEQ ID NO:6), and / or SMDDQLNQL (SEQ ID NO:7), or an expression vector directing the expression of said at least one peptide in vivo, and a pharma- ceutically acceptable vehicle, carrier, or excipient.

[0007] The present invention provides a vaccine or anti-cancer vaccine comprising at least one peptide, the peptide consisting of or comprising an epitope consisting of a sequence selected from the group consisting of FLQFKTWWI (SEQ ID NO:1), RLIPYDWEI (SEQ ID NO:2), KLIDCYTFL (SEQ ID NO:3), YLSFIKILL (SEQ ID NO:4), AMIPKDWPL (SEQ ID NO:5), YAMSNLFSI (SEQ ID NO:6), and / or SMDDQLNQL (SEQ ID NO:7), or an expression vector directing the expression of the at least one peptide in vivo, and a pharma- ceutically acceptable vehicle, carrier, or excipient.

[0008] The composition according to the invention may in particular comprise: 2, 3, 4, 5, 6 or 7 peptides having 9 to 100 amino acid residues, each peptide comprising at least one, in particular one, of the epitopes of the sequences SEQ ID NO: 1 to 7, and each peptide comprising at least one different epitope with respect to the other peptides, or one or more expression vectors directing the expression of said 2, 3, 4, 5, 6 or 7 peptides in vivo, or At least one peptide having 9 to 100 amino acid residues and containing 2, 3, 4, 5, 6, or 7 epitopes of the sequences of SEQ ID NOs: 1 to 7, or an expression vector that induces the expression of the at least one peptide in vivo.

[0009] In one embodiment, the composition according to the invention may in particular comprise: 2, 3, 4, 5, 6 or 7 peptides having 9 to 100 amino acid residues, wherein one peptide comprises an epitope of the sequence of SEQ ID NO: 1 or 6 and at least one other peptide comprises at least one other epitope of the sequence of SEQ ID NO: 1 to 7, and each peptide comprises at least one different epitope with respect to the other peptides, or At least one peptide having 9 to 100 amino acid residues and containing 2, 3, 4, 5, 6, or 7 epitopes of sequences of SEQ ID NOs: 1 to 7, including epitopes of sequences of SEQ ID NOs: 1 or 6 (particularly both), or an expression vector that induces the expression of said at least one peptide in vivo.

[0010] As described hereinafter, the composition according to the invention may include the following embodiments (although other embodiments are also evident from the rest of the description): - the composition comprises 2, 3, 4, 5, 6 or 7 peptides of sequences SEQ ID NO: 1 to 7, or the composition comprises one or more expression vectors comprising the in vivo expression of these peptides, in one embodiment the peptides of sequences SEQ ID NO: 1 or 6 or both peptides are present or expressed, - the composition comprises a peptide of 9 to 100 amino acid residues and at least a peptide of sequence SEQ ID NO: 1 to 7, the peptide may comprise 2, 3, 4, 5, 6 or 7 of the disclosed peptides, in one embodiment the composition may comprise a gag epitope and / or a pol epitope, or at least two or three of these gag and / or pol epitopes, as disclosed herein, or the composition comprises one or more expression vectors comprising in vivo expression of this or these peptides, in one embodiment the peptide contained or expressed comprises a peptide of sequence SEQ ID NO: 1 or 6, or both peptides, - said composition comprises 2, 3, 4, 5, 6 or 7 peptides having 9 to 100 amino acid residues, each peptide comprising at least one, preferably one, epitope of the sequences SEQ ID NO: 1 to 7, and each peptide comprising at least one different epitope with respect to the other peptides, or said composition comprises one or more expression vectors comprising the in vivo expression of these peptides, in one embodiment a peptide of the sequences SEQ ID NO: 1 or 6 or both of these peptides are present or expressed, - each of the peptides of 9 to 100 amino acid residues contained or expressed contains a specific epitope (different from other peptides in the composition) of one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7; - the peptide(s) contained or expressed comprise 9 to 50 amino acid residues of HERV gag or pol, including at least one of the peptides of sequences SEQ ID NO: 1 to 7; - the composition comprises 1, 2, 3, 4, 5, 6 or 7 peptides selected from the group consisting of peptides of SEQ ID NO: 8 to 14, or - the composition comprises one or more expression vectors comprising in vivo expression of this or these peptides, in one embodiment a peptide of sequence SEQ ID NO: 8 or 13 or both peptides are present or expressed.

[0011] The epitopes of the sequences of SEQ ID NO: 4, 2, and 1 are derived from HERV-K gag. The epitopes of the sequences of SEQ ID NO: 5, 3, 6, and 7 are derived from HERV-K pol. These are MHC class I HLA-A2 epitopes. In one embodiment, the composition comprises or expresses one, two, or three HERV-K gag epitopes. In one embodiment, the composition comprises or expresses one, two, three, or four HERV-K pol epitopes.

[0012] In the context of said composition, an immunogenic composition or vaccine according to the invention, said comprised or expressed peptide may comprise 9 to 100, in particular 9 to 70, or 9 to 50, 40, 30, 25, 20 contiguous residues, preferably these residues are derived from the HERV-K gag and / or pol, more preferably the native consensus HERV-K gag and / or pol sequence comprising at least one of said epitopes. The peptide may be less than 50 residues in length, such as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 or 45 residues.

[0013] The composition, i.e., the immunogenic composition or vaccine of the present invention (hereinafter referred to as "composition" unless otherwise specified), may comprise two or more peptides of HERV-K, particularly derived from gag and / or pol, or an expression vector that induces the in vivo expression of two or more of these peptides, particularly derived from gag and / or pol, or each of several (two or more) expression vectors that induce the in vivo expression of different peptides (among these peptides) of HERV-K, particularly derived from gag and / or pol.

[0014] In one embodiment, the peptide (having 9 or more residues) is a native HERV-K fragment that includes a 9 amino acid long (9-mer) epitope and adjacent amino acids at the N-terminus and / or C-terminus to form a peptide of defined length, hi one embodiment, the peptide includes two or more HERV-K epitopes (e.g., 2, 3, 4, 5, 6, or 7 of the disclosed epitopes).

[0015] In one embodiment, the peptide (having 9 or more residues) is a native HERV-K fragment that includes a 9 amino acid long epitope, amino acids adjacent to the N-terminus and / or C-terminus, and additional foreign amino acids to form a peptide of defined length, hi one embodiment, the peptide includes two or more HERV-K epitopes (e.g., 2, 3, 4, 5, 6, or 7 of the disclosed epitopes).

[0016] In another embodiment, all or part of the peptide sequence of the peptide contained or expressed in the composition is foreign to HERV-K. In this embodiment, the peptide can easily contain more than two HERV-K epitopes, for example, 2, 3, 4, 5, 6, or 7 of the disclosed epitopes. The length of the peptide is appropriate to include the number of epitopes and possible additional amino acids. Thus, the peptide can be 9 or 10 to 69 peptides long or longer.

[0017] Preferably, the composition comprises, or the vector directs the expression of, 2, 3, 4, 5, 6 or 7 different peptides, each peptide comprising or consisting of a different epitope consisting of a sequence selected from the group consisting of FLQFKTWWI (SEQ ID NO: 1), RLIPYDWEI (SEQ ID NO: 2), KLIDCYTFL (SEQ ID NO: 3), YLSFIKILL (SEQ ID NO: 4), AMIPKDWPL (SEQ ID NO: 5), YAMSNLFSI (SEQ ID NO: 6), SMDDQLNQL (SEQ ID NO: 7).

[0018] In one embodiment, the composition comprises or the vector directs the expression of at least one peptide comprising or consisting of at least two distinct epitopes consisting of sequences selected from the group consisting of FLQFKTWWI (SEQ ID NO: 1), RLIPYDWEI (SEQ ID NO: 2), KLIDCYTFL (SEQ ID NO: 3), YLSFIKILL (SEQ ID NO: 4), AMIPKDWPL (SEQ ID NO: 5), YAMSNLFSI (SEQ ID NO: 6), SMDDQLNQL (SEQ ID NO: 7). In one embodiment, the peptide comprises 2, 3, 4, 5, 6, or 7 of said epitopes and the expression vector directs the expression of a peptide comprising 2, 3, 4, 5, 6, or 7 of said epitopes.

[0019] There are several solutions to represent different epitopes in a composition or expression product in vivo in the same patient: the composition may comprise, or the vector directs the expression of, one or more peptides comprising one or more of the different epitopes of the group FLQFKTWWI (SEQ ID NO: 1), RLIPYDWEI (SEQ ID NO: 2), KLIDCYTFL (SEQ ID NO: 3), YLSFIKILL (SEQ ID NO: 4), AMIPKDWPL (SEQ ID NO: 5), YAMSNLFSI (SEQ ID NO: 6), SMDDQLNQL (SEQ ID NO: 7), such that 2, 3, 4, 5, 6 or 7 of said epitopes are present or expressed in the composition.

[0020] When speaking of an expression vector directing the expression of several peptides according to the invention, it refers to a composition comprising vectors directing the expression of several peptides (wherein the peptides may comprise one or more epitopes of said group, e.g. 2, 3, 4, 5, 6, 7 of said epitopes), or at least two expression vectors, each of several vectors directing the expression of at least one peptide. In one embodiment, said composition comprises one vector or several vectors, said vectors directing the expression of one or more peptides and 2, 3, 4, 5, 6 or 7 of said epitopes.

[0021] An example of an isolated or purified 29 amino acid long (29-mer) peptide containing an epitope of the invention and other gag or pol amino acid residues is the following: KSKIKSKYAS YLSFIKILL KRGGVKVSTK (SEQ ID NO: 8), TLLDSIAHGH RLIPYDWEI LAKSSLSPSQ (SEQ ID NO: 9), LAKSSLSPSQ FLQFKTWWI DGVQEQVRRN (SEQ ID NO: 10), GPLQPGLPSP AMIPKDWPL LIIIDLKDCF (SEQ ID NO: 11), KLIDCYTFL QAEVANAGLAIASDKIQTST (SEQ ID NO: 12), WIRPTLGIPT YAMSNLFSI LRGDSDLNSK (SEQ ID NO: 13), and RDVETALIKY SMDDQLNQL FNLLQQTVRK (sequence number 14). These isolated or purified 29 amino acid long peptides or fragments, or 28-10 amino acid residues, comprising the 9 amino acid long epitope, in isolated or purified form, respectively, are objects of the present invention. The length of said peptides may be less than 29 residues, such as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 residues of the sequences of SEQ ID NOs: 8-14, comprising the 9 amino acid long epitope. Interestingly, the amino acid sequence added to said epitope may comprise other potential CD4 and / or CD8T epitopes, as in the case of the peptides of SEQ ID NOs: 8-14. The present invention provides peptides comprising an epitope as disclosed herein and "further amino acids" at the C-terminus and / or N-terminus. These further amino acids may be gag or pol sequences as disclosed herein with the sequences of SEQ ID NOs: 1-16. However, the invention encompasses amino acid variations at the level of these "additional amino acids" within these gag / pol sequences. Thus, the invention encompasses those sequences including those of SEQ ID NOs: 1-16, where the additional amino acid sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (%) to those gag / pol sequences.

[0022] In one embodiment, a composition of the invention comprises or expresses a peptide of SEQ ID NO:8, SEQ ID NO:13, or both peptides of SEQ ID NO:8 and SEQ ID NO:13, and one, two, three, four, or five other peptides of SEQ ID NO:8-14.

[0023] In one embodiment, the composition further comprises an adjuvant.

[0024] The peptide RLIPYDWEI (SEQ ID NO: 2) in isolated or purified form is an object of the present invention.

[0025] The peptide KLIDCYTFL (SEQ ID NO: 3) in isolated or purified form is an object of the present invention.

[0026] The peptide YLSFIKILL (SEQ ID NO: 4) in isolated or purified form is an object of the present invention.

[0027] The peptide AMIPKDWPL (SEQ ID NO:5) in isolated or purified form is an object of the present invention.

[0028] The peptide YAMSNLFSI (SEQ ID NO: 6) in isolated or purified form is an object of the present invention.

[0029] The peptide SMDDQLNQL (SEQ ID NO: 7) in isolated or purified form is an object of the present invention.

[0030] Peptides of 10 to 100 amino acids comprising at least one of said peptides of SEQ ID NOs: 1 to 7 in isolated or purified form are an object of the present invention.

[0031] Peptides comprising 2, 3, 4, 5, 6 or 7 of the epitopes FLQFKTWWI (SEQ ID NO: 1), RLIPYDWEI (SEQ ID NO: 2), KLIDCYTFL (SEQ ID NO: 3), YLSFIKILL (SEQ ID NO: 4), AMIPKDWPL (SEQ ID NO: 5), YAMSNLFSI (SEQ ID NO: 6) and SMDDQLNQL (SEQ ID NO: 7) in isolated or purified form are an object of the present invention. Said peptides preferably consist of FLQFKTWWI (SEQ ID NO: 1) and / or YAMSNLFSI (SEQ ID NO: 6).

[0032] In one embodiment, the peptide may comprise 9-100, in particular 9-70, or 9-50, 40, 30, 25, 20, or even 10-30, 12-25, preferably 14-18, such as 14, 15, 16, 17, or 18 contiguous residues of a HERV-K gag and / or pol, more preferably a native consensus HERV-K gag and / or pol sequence comprising at least one of the above epitopes, wherein the amino acids other than the corresponding epitopes of the sequences of SEQ ID NOs: 1-7 are native amino acids present in the HERV-K gag or pol as disclosed herein, and extend 5', 3', or 5' and 3' of said epitope. Said peptides may be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, or 45 residues in length, typically less than 50 residues, such as 14, 15, 16, 17, or 18 residues. Another object of the invention is an expression vector comprising a nucleic acid encoding amino acids selected from the group consisting of FLQFKTWWI (SEQ ID NO: 1), RLIPYDWEI (SEQ ID NO: 2), KLIDCYTFL (SEQ ID NO: 3), YLSFIKILL (SEQ ID NO: 4), AMIPKDWPL (SEQ ID NO: 5), YAMSNLFSI (SEQ ID NO: 6), SMDDQLNQL (SEQ ID NO: 7) and at least two of said sequences (in particular 2, 3, 4, 5, 6, or 7 of them) and the necessary elements to lead to the in vivo expression of said nucleic acid (polynucleotide) in a patient.

[0033] An example of a HERVK-gag polypeptide containing three gag epitopes: MGQTKSKIKSKYA SYLSFIKILL KRGGVKVSTKNLIKLFQIIEQFCPWFPEQGTLDLKDWSQKETEGLHCEYVAEPVMAQSTQNVDYNQLQEVIYPETLKLEESKPRGTSPLPAGQVPVTLQPQKQVKENKTQP PVAYQYWPPAELQYRPPPESQYGYPGMPPAPQGRAPYPQPPTRRLNPTAPPSRQGSKLHEIAQEGEPPTVEARYKSFSIKKLKDMKEGVKQYGPNSPYMRTLLDSIAHGHR LIPYDWEI LAKSSLSPSQ FLQFKTWWIDGVQEQVRRNRAANPPVNIDADQLLGIGQNWSTISQQALMQNEAIEQVRAICLRAWEKIQDPSKEPYPDFVARLQDVAQKSIADEKARKVIVELMAYENANPECQSAIKPLKGKVPAGSDVISEYVKACDGIGGAMHKAMLMAQAITGVVLGGQVRTFGRKCYNCGQIGHLKKNCPVLNKQNITIQATTTGREPPDLCNEQRGQPQAPQQTGAFPIQPFVPQGFQGQQPPLSQVFQGISQLPQYNNCPPP (SEQ ID NO: 15).

[0034] An example of a HERVK-pol polypeptide containing four pol epitopes: NKSRKRRNRESLLGAATVEPPKPIPLTWKTEKPVWVNQWPLPKQKLEALHLLANEQLEKGHIEPSFSPWNSPVFVIQKKSGKWRMLTDLRAVNAVIQPMGPLQPGLPSP AMIPKDWPL IIIDLKDCFFTIPLAEQDCEKFAFTIPAINNKEPATRFQWKVLPQGMLNSPTICQTFVGRALQPVREKFSDCYIIHCIDDILCAAETKD KLIDCYTFL QAEVANAGLAIASDKIQTSTPFHYLGMQIENRKIKPQKIEIRKDTLKTLNDFQKLLGDINWIRPTLGIPT YAMSNLFSI LRGDSDLNSKRMLTPEATKEIKLVEEKIQSAQINRIDPLAPLQLLIFATAHSPTGIIIQNTDLVEWSFLPHSTVKTFTLYLDQIATLIGQTRLRIIKLCGNDPDKIVVLTKEQVRQAFI NSGAWKIGLANFVGIIDNHYPKTKIFQFLKLTTWILPKITRREPLENALTVFTDGSSNGKAAYTGPKERVIKTPYQSAQRAELVAVITVLQDFDQPINIISDSAYVVQATRDVETALIKY SMDDQLNQLFNLLQQTVRKRNFPFYITHIRAHTNLPGPLTKANEQADLLVSSALIKAQELHALTHVNAAGLKNKFDVTWKQAKDIVQHCTQCQVLHLPTQEAGVNPRGLCPNALWQMDVTHVPSFGRLSYVHVTVDTYSHFIWATCQSTSHVKKHLLSCFAVMGVPEKIKTDNGPGYCSKAFQKFLSQWKISHTTGIPYNSQGQAIVERTNRTLKTQLVKQKEGGDSKCTTPQMQLNLALYTLNFLNIYRNQTTTSAEQHLTGKKSPGENQLPVWIPTRHLKFYNEPIRDAKKSTSA (sequence number 16).

[0035] Isolated or purified polypeptides of SEQ ID NO:15 and 16 are defined herein and are the subject of the present invention, as are compositions, immunogenic compositions and anti-cancer vaccines comprising or expressing the polypeptides of SEQ ID NO:15 and / or SEQ ID NO:16.

[0036] In one embodiment, the expression vector comprises a nucleic acid encoding a peptide of 9-100, in particular 9-70, or 9-50, 40, 30, 25, 20 amino acids (the encoded peptide may be less than 50 residues in length, such as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, or 45 residues), and an epitope selected from the group: FLQFKTWWI (SEQ ID NO: 1), RLIPYDWEI (SEQ ID NO: 2), KLIDCYTFL (SEQ ID NO: 3), YLSFIKILL (SEQ ID NO: 4), AMIPKDWPL (SEQ ID NO: 5), YAMSNLFSI (SEQ ID NO: 6), SMDDQLNQL (SEQ ID NO: 7), and elements required for in vivo expression of the nucleic acid (polynucleotide) in a patient. As above, the vector may comprise a nucleic acid sequence such that the vector directs the expression of two, three, four, five, six or seven epitope-containing peptides or directs the expression of a peptide that includes two, three, four, five, six or seven of said epitopes. Also as above, the vector may comprise a nucleic acid encoding a peptide that includes one or several of these epitopes, and amino acid residues in addition to the relevant epitopes, which additional residues may be derived from pol or gag or may be exogenous to gag or pol.

[0037] The expression construct or vector may be a bacterial expression construct, a DNA or RNA expression construct, or a non-viral expression construct, such as a viral expression construct. The expression construct may be located in an antigen-presenting cell. The construct may result in integration of the expression construct into the genome of the cell.

[0038] The present invention also relates to these compositions for use in the treatment of cancer.The cancers concerned in this use may be selected in particular (but not limited to) from the following cancers: breast cancer, including triple-negative breast cancer, ovarian cancer, melanoma, sarcoma, teratocarcinoma, bladder cancer, lung cancer (non-small cell lung cancer and small cell lung cancer), head and neck cancer, colorectal cancer, glioblastoma, and leukemia, for example breast cancer, including triple-negative breast cancer, ovarian cancer, melanoma, sarcoma, teratocarcinoma, bladder cancer, and leukemia.

[0039] Said use may be aimed at activating a T cell response, a B cell response, or both, in a patient, for example a breast cancer patient.

[0040] The present invention also relates to the use of one or several 9 amino acid long epitopes as disclosed herein, one or several peptides as disclosed herein, one or several expression vectors as disclosed herein, or a composition as disclosed herein for the manufacture of an immunogenic composition or vaccine for treating cancer as disclosed herein.

[0041] Another object of the present invention is a method for treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of an immunogenic composition or vaccine as disclosed herein. The composition may comprise one or more peptides, or one or more expression vectors or constructs, as described above. The method may comprise administering the vaccine two or more times. The daily therapeutically effective amount of peptide administered (total amount of peptide according to the present invention) may be in the range of 0.01 mg to 10 mg, in the range of 0.025 mg to 5.0 mg, or in the range of 0.025 mg to 1.0 mg.

[0042] Another object of the present invention is a method for treating cancer in a patient, comprising: (a) contacting cytotoxic T lymphocytes (CTLs) of a patient in need of cancer treatment with a composition or immunogenic composition according to the present invention; and (b) administering a therapeutically effective amount of the CTLs of step (a) to the patient. The method may further comprise expanding the CTLs by ex vivo or in vivo methods prior to administration. The contacting may comprise providing antigen-presenting cells loaded with the peptide of the present invention or expressing the peptide or polypeptide from an expression construct. A therapeutically effective amount of CTL cells required to provide a therapeutic effect is about 0.1×10 6 per kilogram of body weight of the subject. 4 ~Approx. 5×10 9The method may comprise performing step (b) two or more times.

[0043] The present invention also relates to a method for preparing cytotoxic T lymphocytes (CTLs) of a patient in need of cancer treatment, comprising contacting the CTLs with a composition or an immunogenic composition according to the invention, and optionally expanding said CTLs ex vivo. Contacting may comprise providing antigen-presenting cells loaded with a peptide of the invention, or expressing said peptide or polypeptide from an expression construct.

[0044] A composition comprising such CTLs in a pharmaceutical vehicle, prepared as described above, is also an object of the present invention.

[0045] Another object of the present invention are T cell receptor (TCR) modified T cells that recognize the epitope peptides from the present invention and compositions comprising such T cells in a pharmaceutical vehicle. The process of preparing these T cells is known to the skilled person. It can be as follows (and the process is also an object of the present invention): (i) TCR α and β chains are isolated from T cells that recognize the epitope peptides of the present invention and inserted into a vector (such as a lentivirus or retrovirus); (ii) T cells isolated from the peripheral blood of a patient or donor are modified with such a vector (such as a lentivirus or retrovirus) to code for the TCR αβ sequence of interest; (iii) These modified T cells are then expanded in vitro to obtain sufficient numbers for therapy and administered to a patient. Of note, the TCR sequence may be altered to optimize TCR affinity. Methods of using these T cells, for example for the treatment of cancer, are another object of the present invention and include administering an efficient amount of those T cells to a subject in need thereof. A therapeutically effective amount of T cells required to provide a therapeutic effect is about 0.1×10 per kilogram of body weight of said subject. 4 ~Approx. 5×10 9 The cell may be a single cell.

[0046] In one embodiment, the cancer is triple-negative breast cancer, other breast cancer, ovarian cancer, melanoma, sarcoma, teratocarcinoma, bladder cancer, lung cancer (non-small cell lung cancer and small cell lung cancer), head and neck cancer, colorectal cancer, glioblastoma, and leukemia, e.g., breast cancer, including triple-negative breast cancer, ovarian cancer, melanoma, sarcoma, teratocarcinoma, bladder cancer, and leukemia.

[0047] Antigens prepared as disclosed herein or containing the epitopes also generate anti-HERV-K antibodies and + It can be used to detect the presence of anti-HERV-K antibodies in cancer patients.

[0048] The compositions comprising the epitopes according to the invention and at least one antigenic peptide may be used in diagnostic, prognostic or immunomonitoring methods. In particular, the present invention also relates to a method for immunomonitoring of immune responses in patients. The induction of an antitumor adaptive response after immunotherapy (vaccines using said epitopes or compositions of the invention, or any other immunotherapy inducing an adaptive antitumor T cell response) is evaluated by measuring a specific T cell response against the HERV epitopes of the invention. The measurement may be performed directly or after ex vivo stimulation with the peptides according to the invention, for example by using multimers comprising the epitopes according to the invention. The measurement of the T cell response may also be performed after ex vivo stimulation with the peptides of the invention by detecting specific T cell activation using fluorescence activated cell sorting (FACS) analysis, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT) or other methods.

[0049] In one embodiment, the biological sample is blood, circulating cells from a tumor or a blood derivative comprising lymphocytes. Preferably, the method comprises determining that a fraction of lymphocytes in the blood are capable of specifically recognizing and / or specifically reactivating a peptide of interest upon in vitro stimulation.

[0050] Those skilled in the art will know various assays to determine whether an immune response against a tumor-associated peptide has been generated. The term "immune response" includes both cellular and humoral immune responses. Various B-lymphocyte and T-lymphocyte assays are well known, such as cytotoxic T-lymphocyte (CTL) assays such as ELISA, chromium release assays, proliferation assays using peripheral blood lymphocytes (PBL), tetramer assays, cytokine production assays, etc. See Benjamini et al. (1991), which is incorporated herein by reference. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] We localized HERV sequences to the human genome and developed an RNAseq analysis of HERVs. 84 breast cancers (42 triple-negative breast cancers (TNBC) and 42 ER cancers) from the public database were analyzed. + Using RNAseq data from TNBC (all TNBC subtypes), we compared this expression with RNAseq from normal breast tissue samples (51 from peritumoral regions and 5 from reduction mammoplasty samples). Nineteen HERVs were specifically overexpressed in TNBC, the majority of which belong to the HERV-K family.

[0052] Multicomponent analysis showed that HERVs can be used to characterize triple-negative subtypes. HERV expression was associated with elevated OCT4 (POU5F1) and reduced levels of TRIM28 in TNBC, two factors that positively and negatively regulate HERV transcription, respectively. A relationship with the EMT signature was also observed, which may be related to the stemness function of TNBC. Interestingly, HERV expression was significantly correlated with the transcriptomic signature of T cells and cytotoxic lymphocytes. This may be explained by the presence of type I interferon (IFN) responses and antigen-presenting cell signatures. The effector T cell signature was counterbalanced by an immune-modulatory signature (including negative immune checkpoints and IDO1 / 2) and suppressor cells (including regulatory T cells and MDSCs).

[0053] Polymorphism of HERVs is often considered the main obstacle to characterize T cell responses to specific HERV antigens or to use them in cancer vaccination strategies. Based on the specific expression of a limited number of HERVs that characterize TNBC, we hypothesized that it may be possible to identify common regions within Gag and Pol proteins shared among different HERVs expressed in TNBC and determine the T cell epitopes present in these domains. We effectively found common regions of Gag and Pol of several HERV-Ks overexpressed in TNBC and containing intact ORFs of each protein. Interestingly, these shared domains include several regions enriched for potential strong epitope binders of the most frequent MHC class I and II alleles using various epitope prediction tools (including NetMHC I and II).

[0054] Nine amino acid long peptides corresponding to predicted HLA-A2 epitopes were synthesized and used in an in vitro protocol consisting of stimulating peripheral blood mononuclear cells (PBMCs) to induce a specific response against the peptide of interest. + The presence of T cells was assessed by multimer staining, and functional responses (IFN-γ production and degranulation) were further assessed against T2 cells pulsed with cognate peptides, demonstrating that CD8 + Specific activation of T cells was observed. Furthermore, HERV-specific CD8 + T cell cytotoxicity was determined by CD8 + This was demonstrated using T cells, confirming the functional antitumor properties of T cells generated by these peptides.

[0055] Considering the enhanced HERV expression in tumor cells and the results obtained, the following conclusions can be made: -HERVs are preferentially expressed in tumors, and 19 HERV subtypes characterize triple-negative breast cancer (TNBC), most of which belong to the HERV-K family. - Common sequences containing T cell epitopes are found among these 19 HERV subtypes. - The following seven 9 amino acid long peptides were identified as strong HLA-A2 binders and induced specific CD8+ binding with specific cytotoxic responses against cognate peptide-pulsed T2 cells or HERV-expressing tumor cell lines: + The following antibodies were able to induce T cell responses: FLQFKTWWI (SEQ ID NO: 1), RLIPYDWEI (SEQ ID NO: 2), KLIDCYTFL (SEQ ID NO: 3), YLSFIKILL (SEQ ID NO: 4), AMIPKDWPL (SEQ ID NO: 5), YAMSNLFSI (SEQ ID NO: 6), SMDDQLNQL (SEQ ID NO: 7). -HERV products thus represent shared tumor antigens capable of inducing functional T cell responses. HERV-derived tumor antigens can be used for the development of cancer vaccines and for monitoring adaptive immune responses.

[0056] definition The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components which normally accompany the material as found in its native state. Thus, isolated peptides according to the present invention are preferably free of materials normally associated with the peptides in their in situ environment.

[0057] "Major histocompatibility complex" or "MHC" is a cluster of genes that play a role in controlling cellular interactions involved in physiological immune responses. In humans, the MHC complex is also known as the HLA complex. For a detailed description of the MHC complex and the HLA complex.

[0058] "Human leukocyte antigens" or "HLA" are human class I or class II major histocompatibility complex (MHC) proteins.

[0059] The phrases "pharmacologically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans. The preparation of aqueous compositions containing proteins as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, with conventional pharma-ceutically acceptable vehicles, excipients, or carriers, and may also be prepared in solid forms suitable for dissolution or suspension in liquid prior to injection.

[0060] As used herein, "vehicle, excipient, carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutical active substances is well known in the art.

[0061] "Immunogenic peptide" means that the peptide, when presented to a patient's immune system, is capable of eliciting a humoral and / or cellular immune response, which response is immunogenic, but not necessarily protective. This applies to "immunogenic compositions."

[0062] "Immunogenic response" refers to a CTL and / or HTL response against an antigen derived from an infectious pathogen or a tumor antigen. The immune response may also include an antibody response promoted by stimulation of helper T cells.

[0063] In particular, the immunogenic compositions are directed to CD8 antigens that are directed against HERV peptides present in the compositions and / or against HERV peptides or polypeptides that contain similar epitopes expressed on tumor cells. + It can induce in vivo activation of T cells.

[0064] A "vaccine composition" or "vaccine peptide" means that when administered to a patient and presented to the patient's immune system, the composition or peptide, respectively, is capable of inducing a humoral and / or cellular immune response, which immune response is protective.

[0065] "Protective immune response" refers to a CTL and / or HTL response against antigens derived from infectious agents or tumor antigens, which prevents or at least partially arrests disease symptoms or progression. The immune response may also include an antibody response promoted by stimulation of helper T cells.

[0066] By "immunogenic" is meant that a peptide or epitope is capable of eliciting a humoral and / or cellular immune response when present in a patient, particularly in the patient's blood, tissues, or organs.

[0067] A composition or vector "induces expression" means that it comprises one or more expression vectors comprising a nucleic acid, DNA or RNA, encoding a peptide. The vector may in particular be an RNA vector, a DNA vector or a plasmid, a viral vector or a bacterial vector. Depending on the nature of the vector and as is well known to those skilled in the art, there may or may not be integration of the expression cassette into the host cell genome. The expression vector or expression cassette may further comprise elements necessary for the in vivo expression of the nucleic acid (polynucleotide) in the patient. As a minimum, it consists of an initiation codon (ATG), a stop codon and a promoter, as well as a polyadenylation sequence for certain vectors, such as plasmids and non-poxvirus viral vectors. The ATG is located 5' of the reading frame and the stop codon is located 3'. As is well known, other elements allowing expression to be controlled may be present, such as enhancer sequences, stabilizing sequences and signal sequences allowing secretion of the peptide.

[0068] The protein or peptide may be produced by any technique known to those of skill in the art, including expression of the protein, polypeptide, or peptide by standard molecular biology techniques, isolation of the protein or peptide from a natural source, or chemical synthesis of the protein or peptide. Synthetic peptides are usually about 35 residues in length, which is the approximate upper length limit of the automated peptide synthesizers available from Applied Biosystems (Foster City, Calif.). Longer peptides may also be prepared, for example, by recombinant means.

[0069] A "peptide epitope" or "epitope" is a peptide that contains an allele-specific motif or supermotif such that the peptide binds to an HLA molecule and induces a CTL and / or HTL response. Thus, an immunogenic or vaccinal peptide of the invention that contains at least one "peptide epitope" can bind to the appropriate HLA molecule and subsequently induce a cytotoxic or helper T cell response against the antigen from which the immunogenic or vaccinal peptide is derived.

[0070] It is contemplated that the peptides of the present invention may further use amino acid sequence variants, such as substitution, insertion, or deletion variants. Deletion variants are missing one or more residues of the native protein. Insertion variants usually involve the addition of material at a non-terminal point of the polypeptide. Substitutions are changes to existing amino acids. These sequence variants may produce truncations, point mutations, and frameshift mutations. As known to those skilled in the art, synthetic peptides may be produced by these mutations.

[0071] It will be understood that amino acid sequence variants may contain additional residues, such as additional N-terminal or C-terminal amino acids, but will still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the above criteria, including maintenance of biological activity.

[0072] The following is a discussion based on changing the amino acids of proteins, such as the peptides or proteins of the present invention, to produce mutated, truncated, or modified proteins. For example, certain amino acids can be substituted with other amino acids in tumor-associated peptides or proteins to produce greater CTL immune responses. Because it is the interaction ability and properties of a protein that define its biological functional activity, certain amino acid substitutions can be made in the protein sequence and its underlying nucleic acid coding sequence, thereby producing mutated, truncated, or modified proteins.

[0073] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic index in conferring interactive biological function on a protein is generally understood in the art. It is recognized that the relative hydrophilic and hydrophobic properties of amino acids contribute to the secondary structure of the resulting protein, which in turn defines the interactions of the protein with other molecules, e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0074] It is also understood in the art that substitutions of like amino acids can be made effectively based on hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, describes that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of adjacent amino acids, correlates with the biological properties of the protein. The following hydrophilicity values ​​have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartic acid (+3.0±1), glutamic acid (+3.0±1), asparagine (+0.2), and glutamine (+0.2); hydrophilic non-ionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2). , and threonine (-0.4); sulfur-containing amino acids: cysteine ​​(-1.0) and methionine (-1.3); hydrophobic non-aromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5±1), alanine (-0.5), and glycine (0); hydrophobic aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).

[0075] It is understood that an amino acid can be substituted for another having a similar hydrophilicity to produce a biologically or immunologically modified protein. In such changes, substitutions of amino acids whose hydrophilicity values ​​are within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0076] As outlined above, amino acid substitutions are typically based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account the various characteristics discussed above are well known to those of skill in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0077] Other components In other embodiments of the invention, the composition may include additional immunostimulatory agents or nucleic acids encoding such agents. Immunostimulatory agents include, but are not limited to, additional antigens, immunomodulatory agents, antigen-presenting cells, or adjuvants. In other embodiments, one or more additional agents are covalently bound to the peptide. Other immune enhancing compounds, such as polysaccharides, including chitosan, are also contemplated for use with the compositions of the invention. Multiple (two or more) epitopes or peptides may be crosslinked (e.g., polymerized) to each other.

[0078] The use of small peptides for immunization or vaccination may also typically require the conjugation of the peptide to a carrier peptide, polypeptide, or protein that confers immunogenicity or strongest immunogenicity to the final product or target peptide or epitope. Thus, in one embodiment of the present invention, each peptide selected from FLQFKTWWI (SEQ ID NO: 1), RLIPYDWEI (SEQ ID NO: 2), KLIDCYTFL (SEQ ID NO: 3), YLSFIKILL (SEQ ID NO: 4), AMIPKDWPL (SEQ ID NO: 5), YAMSNLFSI (SEQ ID NO: 6), and SMDDQLNQL (SEQ ID NO: 7) is conjugated or conjugated via a peptide bond to a peptide, polypeptide, or protein (additional amino acid residues) that confers immunogenicity or strongest immunogenicity to the conjugation product or peptide of the present invention.

[0079] In one embodiment, the peptide or epitope of FLQFKTWWI (SEQ ID NO: 1), RLIPYDWEI (SEQ ID NO: 2), KLIDCYTFL (SEQ ID NO: 3), YLSFIKILL (SEQ ID NO: 4), AMIPKDWPL (SEQ ID NO: 5), YAMSNLFSI (SEQ ID NO: 6), and / or SMDDQLNQL (SEQ ID NO: 7) is present in a longer peptide or polypeptide, in particular a HERV-K peptide or polypeptide, preferably a naturally occurring longer peptide or polypeptide that contains the epitope in HERV-K from which the epitope is derived. Thus, longer sequences are presented as examples in the sequences of SEQ ID NOs: 8 to 14. In variants, several epitopes are part of the same long peptide. It is also encompassed by the present invention to conjugate those longer peptides to peptides, polypeptides, or proteins that confer immunogenicity or strongest immunogenicity to the conjugated final product.

[0080] In an immunogenic composition or vaccine according to the invention, the peptide contained therein or expressed by the vector is immunogenic or capable of inducing a protective immune response.

[0081] When the composition or peptide or epitope is used for diagnostic or assay purposes, such as immune monitoring, the peptide or epitope may be antigenic. Thus, in one embodiment of the composition, the peptide or epitope of FLQFKTWWI (SEQ ID NO: 1), RLIPYDWEI (SEQ ID NO: 2), KLIDCYTFL (SEQ ID NO: 3), YLSFIKILL (SEQ ID NO: 4), AMIPKDWPL (SEQ ID NO: 5), YAMSNLFSI (SEQ ID NO: 6), and / or SMDDQLNQL (SEQ ID NO: 7), or a peptide or epitope comprising such an epitope, is antigenic. The antigenic peptide or epitope may be in unbound form (consisting of epitope sequences SEQ ID NO: 1-7) or may be bound to a peptide or polypeptide moiety, as disclosed herein.

[0082] Those skilled in the art will know various assays to determine whether an immune response against a tumor-associated peptide has been generated. The term "immune response" includes both cellular and humoral immune responses. Various B-lymphocyte and T-lymphocyte assays are well known, such as cytotoxic T-lymphocyte (CTL) assays such as ELISA, chromium release assays, proliferation assays using peripheral blood lymphocytes (PBL), tetramer assays, cytokine production assays, etc. See Benjamini et al. (1991), which is incorporated herein by reference.

[0083] Adjuvants As is also well known in the art, the immunogenicity of a particular immunogen composition may be enhanced by the use of non-specific stimulators of immune response known as adjuvants. Some adjuvants affect the way antigens are presented. For example, precipitation of protein antigens with alum increases the immune response. Emulsification of antigens also extends the period of antigen presentation. Suitable molecular adjuvants include all acceptable immune stimulating compounds, such as cytokines, toxins, or synthetic compositions.

[0084] Exemplary and often preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of immune response that contains killed tuberculosis bacteria), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other adjuvants that can be used include IL-1, IL-2, IL-4, IL-7, IL-12, interferon, GM-CSF, BCG, aluminum hydroxide, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene / Tween 80 emulsion, is also contemplated. MHC antigens can also be used.

[0085] In one aspect, the adjuvant effect is achieved by the use of agents such as alum, used in about 0.05 to about 0.1% solution in phosphate buffered saline. Alternatively, the antigen is made as a mixture with a synthetic polymer of sugar (Carbopol®), used as a solution of about 0.25%. The adjuvant effect can also result in aggregation of the antigen in the vaccine by heat treatment for 30 seconds to 2 minutes at temperatures ranging from about 70°C to about 101°C, respectively. Aggregation by reactivation with pepsin-treated (Fab) antibodies against albumin, mixtures with bacterial cells such as Cryptosporidium parvum (C. parvum), endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsions in physiologically acceptable oil vehicles such as mannide monooleate (Aracel A), emulsions containing a 20% solution of perfluorocarbon (Fluosol-DA®) used as a block substitute can also be employed.

[0086] Some adjuvants, such as certain organic molecules derived from bacteria, act on the host rather than on the antigen. An example is the bacterial peptidoglycan muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine [MDP]). MDP stimulates macrophages but also appears to stimulate B cells directly.

[0087] In certain embodiments, hemocyanins and hemoerythrins may also be used in the present invention, in which the use of hemocyanin from keyhole limpets (KLH) is preferred, although hemocyanins and hemoerythrins from other mollusks and arthropods may also be used.

[0088] Various polysaccharide adjuvants may also be used. For example, the use of various pneumococcal polysaccharide adjuvants on the antibody response in mice has been described. Polyamine species of polysaccharides such as chitin and chitosan, including deacetylated chitin, are particularly preferred.

[0089] Another group of adjuvants is the muramyl dipeptide (MDP, N-acetylmuramyl-L-alanyl-D-isoglutamine) group of bacterial peptidoglycans. Derivatives of muramyl dipeptide, such as the amino acid derivative threonyl-MDP, and the fatty acid derivative MTPPE, are also contemplated. U.S. Patent No. 4,950,645 describes lipophilic disaccharide-tripeptide derivatives of muramyl dipeptide for use in artificial liposomes formed from phosphatidylcholine and phosphatidylglycerol.

[0090] BCG (Bacillus Calmette-Guerin, an attenuated strain of Mycobacterium) and BCG cell wall skeleton (CWS) can also be used as adjuvants, with or without trehalose dimycolate. Trehalose dimycolate itself may also be used. BCG is an important clinical tool due to its immune stimulating properties.

[0091] Amphipathic surfactants, such as saponin and derivatives such as QS21 (Cambridge Biotech), form yet another group of adjuvants for use with the immunogens of the invention. Non-ionic block copolymer surfactants may also be used. Oligonucleotides are another useful group of adjuvants. Quil-A® and lentinan are other adjuvants that may be used in certain embodiments of the invention.

[0092] Another group of adjuvants are detoxified endotoxins, such as the purified detoxified endotoxin of US Pat. No. 4,866,034.

[0093] Those skilled in the art are aware of various types of adjuvants that can be conjugated to cellular vaccines according to the present invention, including alkyl lysophospholipids (ALP), BCG, and biotin (including biotinylated derivatives), among others. Particular adjuvants that are particularly contemplated for use are the teichoic acids from gram cells. These include lipoteichoic acid (LTA), ribitol teichoic acid (RTA), and glycerol teichoic acid (GTA). The active forms of their synthetic counterparts may also be used in the context of the present invention.

[0094] Adjuvants can be encoded by nucleic acids (e.g., DNA or RNA). It is contemplated that such adjuvants can also be encoded in a nucleic acid (e.g., an expression vector) encoding the antigen, or in a separate vector or other construct. Nucleic acids encoding adjuvants can also be delivered directly, for example with lipids or liposomes. An example of such an adjuvant is poly-ICLC.

[0095] Expression vector The peptides according to the invention may be produced in vivo in the patient's body.

[0096] The immunogenic composition or vaccine may include RNA or DNA encoding one or more of the peptides as described above, such that the peptides are produced in situ. The RNA or DNA may be present in any of a variety of delivery systems known to those skilled in the art, including nucleic acid expression systems (nude DNA or plasmids, RNA vectors), bacterial or viral expression systems. Suitable nucleic acid expression systems include the necessary RNA or DNA sequences for expression in the patient, such as appropriate promoters and termination signals. Bacterial delivery systems involve the administration of bacteria (such as Bacillus Calmette-Guerin) that induce the expression of an immunogenic portion of a polypeptide on its cell surface. In a preferred embodiment, the RNA or DNA may be introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may include the use of a non-pathogenic (defective), replication-competent virus. Techniques for incorporating RNA or DNA into such expression systems are well known to those skilled in the art. The DNA may be "naked" as described, for example, in Ulmer et al., Science 259:1745-1749 (1993) and reviewed in Cohen, Science 259:1691-1692 (1993). Uptake of naked DNA can be increased by coating the DNA onto biodegradable beads that are efficiently transported into cells.

[0097] Preferred vectors include DNA vectors, RNA vectors, viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, poxviruses such as vaccinia virus and attenuated poxviruses (such as Modified Vaccinia Virus Ankara (MVA), NYVAC, ALVAC, TROVAC), other viral vectors such as Sindbis virus, cytomegalovirus, herpes simplex virus, and bacterial vectors.

[0098] The term "expression" is used according to the present invention in its most general sense and includes the production of RNA and / or peptides or polypeptides, for example by transcription and / or translation. With respect to RNA, the terms "expression" or "translation" particularly relate to the production of peptides or polypeptides. Also included is partial expression of a nucleic acid. Furthermore, expression can be transient or stable.

[0099] There are several ways in which an expression vector can be introduced into a cell. In certain embodiments of the present invention, the expression vector comprises a modified vector derived from a virus or a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, integrate into the genome of the host cell, and stably and efficiently express viral genes makes them promising candidates for introducing foreign genes into mammalian cells. The first viruses used as gene vectors were DNA viruses, including papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) and adenoviruses.

[0100] A particular method for delivery of nucleic acids involves the use of adenoviral expression vectors. Adenoviral vectors are known to have a low ability to integrate into genomic DNA, but this feature is offset by the high efficiency of gene transfer afforded by these vectors. "Adenoviral expression vector" is meant to include a construct that contains sufficient adenoviral sequences to (a) support packaging of the construct and (b) ultimately express the tissue- or cell-specific construct cloned therein. With knowledge of the genetic organization or adenovirus, a 36 kb linear double-stranded DNA virus, it is possible to replace large pieces of adenoviral DNA with foreign sequences up to 7 kb in size.

[0101] Nucleic acid can be introduced into cells using adenovirus-assisted transfection. Improved transfection efficiency has been reported in cell systems using adenovirus-linked systems (Kelleher and Vos, 1994;Cotten et al., 1992;Curiel, 1994). Adeno-associated virus (AAV) is a promising vector system for use in the vaccines of the present invention. AAV has a broad host range of infection. Details regarding the generation and use of rAAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368, each of which is incorporated herein by reference.

[0102] Retroviruses are promising gene delivery vectors for vaccines because they can integrate genes into the host genome, transfer large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in specialized cell lines. To construct a retroviral vector, a nucleic acid (e.g., encoding an antigen of interest) is inserted into the viral genome in place of a specific viral sequence to produce a replication-deficient virus. To generate virions, a packaging cell line is constructed that contains the gag, pol, and env genes but does not contain the LTRs and packaging components. When a recombinant plasmid containing cDNA along with retroviral LTRs and packaging sequences is introduced into a specialized cell line (e.g., by calcium phosphate precipitation), the packaging sequences allow the RNA transcripts of the recombinant plasmid to be packaged into viral particles that are secreted into the medium. The medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer.

[0103] Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (see, for example, U.S. Patent Nos. 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency viruses (HIV-1, HIV-2) and simian immunodeficiency viruses (SIV). Lentiviral vectors are generated by multiple attenuation of HIV pathogenicity genes, e.g., the env, vif, vpr, vpu, and nef genes are deleted, making the vector biologically safe.

[0104] Recombinant lentiviral vectors can infect non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentiviruses capable of infecting non-dividing cells, in which suitable host cells are transfected with two or more vectors carrying packaging functions, i.e., gag, pol, and env, and rev and tat, are described in U.S. Patent No. 5,994,136, which is incorporated herein by reference. To target a receptor on a specific cell type, the recombinant virus can be targeted by conjugating the envelope protein with an antibody or a specific ligand. By inserting the sequence of interest (including the regulatory region) into the viral vector together with another gene that codes for a ligand of a receptor on a specific target cell, for example, the vector is target-specific.

[0105] Immunogenic Composition or Vaccine Administration To use the methods and compositions of the invention to kill cells, inhibit cell proliferation, inhibit metastasis, reduce tumor or tissue size, or otherwise reverse or reduce the malignant phenotype of tumor cells, one of skill in the art would typically administer (or express) a peptide of the invention to induce T cells capable of recognizing and killing the targeted cancer cells. The route of administration will, of course, depend on the location and nature of the lesion, and may include, for example, intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration.

[0106] Intratumoral injection, or injection into the tumor vasculature, is particularly contemplated for discrete, solid, accessible tumors. Localized, regional, or systemic administration may also be appropriate. For tumors larger than 4 cm, the administration volume is about 4-10 mL (preferably 10 mL), while for tumors smaller than 4 cm, a volume of about 1-3 mL is used (preferably 3 mL). Multiple injections delivered as a single dose include a volume of about 0.1 to about 0.5 mL. Viral particles may be advantageously contacted by multiple injections into the tumor spaced about 1 cm apart.

[0107] In the case of surgical intervention, the present invention may be used preoperatively to target inoperable tumors for resection. Alternatively, the present invention may be used during surgery and / or thereafter to treat residual or metastatic disease. For example, a resected tumor bed may be injected or perfused with a formulation comprising a tumor-associated peptide, polypeptide, or construct encoding the same. Perfusion may be continued after resection, for example, by leaving a catheter implanted at the surgical site. Periodic postoperative treatment is also envisioned.

[0108] Continuous administration may also be applied where appropriate, for example, when a tumor has been resected and the tumor bed treated to eliminate residual microscopic disease.

[0109] Delivery by syringe or catheter is preferred. Such continuous perfusion may occur for a period of about 1-2 hours, about 2-6 hours, about 6-12 hours, about 12-24 hours, about 1-2 days, about 1-2 weeks or more after the start of treatment. Typically, the dose of the therapeutic composition by continuous perfusion will be equivalent to the dose given by single or multiple injections, adjusted over the period during which perfusion occurs. It is further contemplated that the therapeutic composition of the present invention can be administered using limb perfusion, particularly in the treatment of melanoma and sarcoma.

[0110] Treatment regimens also vary and may often depend on the type of tumor, the location of the tumor, the progression of the disease, and the health and age of the patient. Obviously, certain types of tumors will require more aggressive treatment, but at the same time, certain patients may not be able to tolerate more challenging protocols. The clinician will be best suited to make such decisions based on the known efficacy and toxicity (if any) of the therapeutic formulation.

[0111] An effective amount of a pharmaceutical immunogenic or vaccine composition is generally defined as an amount sufficient to detectably and repeatedly ameliorate, reduce, minimize, or limit the extent of a disease or condition or its symptoms. A stricter definition may apply to a vaccine composition, including eliminating, eradicating, or curing the disease.

[0112] In certain embodiments, the tumor being treated may not be resectable, at least initially. Treatment with a therapeutic viral construct may increase the resectability of the tumor, either by shrinkage at the tumor margins or by removal of certain particularly invasive portions. After treatment, resection may become possible. Additional treatment after resection may help eliminate microscopic residual disease at the tumor site.

[0113] A typical treatment regimen for either the primary tumor or the tumor bed following resection will include multiple administrations. The daily therapeutically effective amount of peptide administered (total amount of peptide according to the invention) may be in the range of 0.01 mg to 10 mg, particularly in the range of 0.025 mg to 5.0 mg, or in the range of 0.025 mg to 1.0 mg.

[0114] The treatment may include various "unit doses." A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount and the particular route and formulation administered are within the skill of one of ordinary skill in the clinical arts and may vary depending on the nature of the composition as either a peptide composition or an expression vector composition. A unit dose need not be administered as a single injection, but may include continuous infusion over a set period of time. A unit dose of the present invention may be conveniently described in terms of plaque forming units (pfu) of the viral construct. A unit dose may be greater than 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , or 10 13 Alternatively, depending on the vector type and achievable titer, the unit dose may be 1-100, 10-50, 100-1000, or up to about 1 x 10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , or 1 × 10 15 The above infectious viral particles (vp) are delivered to the patient or to the patient's cells. [Brief description of the drawings]

[0115] [Figure 1] Figure 1A shows the correlation between the expression of 19 HERV subtypes overexpressed in TNBC and immune signatures, with correlations between antigen presentation, cytotoxicity, and immune regulation pathways, with positive correlations indicated by dots (light gray or dark gray). Figure 1B shows the correlation between HERV-K10 expression and T cell signatures as an example. [Diagram 2] Figure 2A shows representative plots of dextramer staining of CD8+ T cells with and without stimulation with CMV pp65 peptide (upper quadrants, respectively) and with and without stimulation with HERV peptide P5 (SEQ ID NO:5) (lower quadrants, respectively). Figure 2B shows the percentage of dextramer-positive CD8+ T cells on day 12 in PBMCs of several donors with and without stimulation with specific HERV peptides (P1-P7: SEQ ID NO:1-7) or controls. [Diagram 3] Figure 3A shows representative plots of IFN-γ production by CD8+ T cells after contact with T2 cells pulsed with negative and positive controls (upper quadrants) and HERV peptides P1, P2, and P3 (SEQ ID NOs: 1, 2, and 3) (lower quadrants). Figure 3B shows the percentage of IFN-γ positive CD8+ T cells on day 12 with and without stimulation with specific HERV peptides (P1, P2, and P3: SEQ ID NOs: 1, 2, and 3) or controls on PBMCs of several donors. [Figure 4] Figure 4A shows representative plots of dextramer staining of CD8+ T cells with and without stimulation with CMV pp65 peptide (upper quadrants, respectively) and with and without stimulation with HERV peptide P1 (SEQ ID NO: 1) (lower quadrants, respectively). Figure 4B shows the fold change ratio of the percentage of dextramer-positive specific CD8+ T cells on day 12 in peptide-stimulated vs. unstimulated (P1-P7: SEQ ID NO: 1-7) PBMCs of several donors. Figure 4C shows representative histograms of the number of IFN-γ+ and Granzyme-β+ spots after 12 days of stimulation and after 24 hours of contact with cognate peptide-pulsed T2 cells. [Diagram 5]FIG. 5A shows representative plots of IFN-γ production by CD8+ T cells after contact with T2 cells pulsed with negative and positive controls (upper quadrants) and HERV peptides P1, P2, and P3 (SEQ ID NOs: 1-3) (lower quadrants) in donor a. FIG. 5A (continued) shows representative plots of IFN-γ production by CD8+ T cells after contact with T2 cells pulsed with negative and positive controls (upper quadrants) and HERV peptides P4, P5, P6, and P7 (SEQ ID NOs: 4-7) (lower quadrants) in donor b. FIG. 5B shows the percentage of IFN-γ positive CD8+ T cells on day 12 with and without stimulation with specific HERV peptides (P1-P7: SEQ ID NOs: 1-7) or controls in PBMCs of several donors. [Figure 6] Figure 6A shows a representative plot of dextramer staining of P1 (SEQ ID NO: 1) after sorting and expansion of specific P1 CD8+ T cells (right quadrant) and their negative counterparts (left quadrant). Figure 6B shows a representative histogram of the number of IFN-γ+ and granzyme-β+ spots after 24 hours of culture of P1 (SEQ ID NO: 1)-specific CD8+ T cells with T2 cells pulsed with P1 or negative control (no peptide loading). Figure 6C shows a representative curve of real-time cell death quantification when MDA-MB-231 cell lines (pulsed or not with peptide of interest) are co-cultured with P1 (SEQ ID NO: 1)-specific CD8+ T cells or their negative counterparts. Figure 6D shows a representative histogram of the percentage of intracellular staining of IFN-γ (PE) of P1 (SEQ ID NO: 1)-specific CD8+ T cells (black) versus their negative counterparts (non-specific CD8+ T cells, white) after 6 hours of co-culture with MDA-MB-231 cell lines pulsed or not with the peptide of interest. Addition of HLA-A2 blocking antibody was used as a control. [Figure 7]FIG. 7A shows a representative schema of dextramer-positive tumor infiltrating lymphocytes (TILs) (black quadrants) found in CD8+ cells grown from TNBC pieces. P1-P7 (SEQ ID NOs: 1-7) represent dextramers of peptides 1-7, and TNBC1-TNBC4 represent four different patients. FIG. 7B shows a representative schema of dextramer-positive TILs (black quadrants) found in CD8+ cells grown from ovarian tumor pieces. P1-P7 (SEQ ID NOs: 1-7) represent dextramers of peptides 1-7, and Ovarian1-Ovarian3 represent three different patients.

[0116] The invention will now be described by way of non-limiting examples with reference to the drawings, in which: EXAMPLES

[0117] Identification of HERV sequences DNA sequences of HERVs from different families (including HERV-K, HERV-H, HERV-W, HERV-E, and ERV3) were extracted from the Genbank database. Using BLAST, these sequences were localized on the human genome (GRCH37) with at least 98% similarity and maintaining gap-free positions with at least 85% of the query sequence. Thus, 66 functional HERV sequences were identified.

[0118] Analysis of HERV sequences in TNBC HERV expression was analyzed in an existing database of 84 breast cancer samples, including 42 TNBCs. Comparisons were made with 56 normal breast samples (51 peritumor and 5 reduction mammoplasty samples). RNA was extracted from fresh tumor biopsy tissues subjected to DNAse treatment and polyA selection. Functional HERV sequences were concordant with RNAseq data if showing sufficient quality (RNA integrity number >6.5).

[0119] A multicomponent analysis of 66 human endogenous retrovirus (HERV) subtypes was performed. 42 HERVs were expressed. 19 HERVs were expressed in normal tissues and ER. +Specifically characterize triple-negative breast cancer (TNBC) in subtypes (HERVK_10, 17, 22, 7, 6, 21, 25, 11, 20, 16, 23, 1, and 5, HERVH_4 and 7, and HERV3_1).

[0120] Peptide Selection and Synthesis Common regions of Gag and Pol shared among 19 overexpressed HERVs were identified after alignment of the reads on the reference genome. Various epitope prediction tools (NetMHC I and II) were used to identify potential strong epitope binders of the most frequent MHCI and II alleles. Among them, the following seven predicted 9 amino acid long strong binders of HLA-A*0201 were selected and synthesized: four Gag peptides and three Pol peptides (JPT Peptide Technologies, Berlin, Germany). Peptide identity was confirmed by mass spectrometry by the vendor. Purity of >95% was expected and determined by high performance liquid chromatography. Lyophilized peptides were dissolved in deionized water with less than 5% DMSO, aliquoted and stored at -20 °C until use.

[0121] 29 amino acid long GMX peptides (SEQ ID NOs: 8-14) containing strong binders of 9 amino acid long peptides of class I MHC (SEQ ID NOs: 1-7) and lateral sequences of class II motifs (10 amino acid long (10-mer) on each side, except for peptide SEQ ID NO: 12, with sequence SEQ ID NO: 5 at the C-terminus) were identified and analyzed for synthesis.

[0122] Bioinformatics analysis of the correlation between HERV expression and T cell signatures To assess the immune characteristics of the tumors, various signatures were used: T cells for control analysis, CD8 +MCP counter signatures of T cells, NK cells, cytotoxic lymphocytes, as well as fibroblasts, neutrophils, and endothelial cells (see http: / / cit.ligue-cancer.net / ?p=1338&lang=en); the ESTIMATE package for ImmuneScore and StromalScore (http: / / bioinformatics.mdanderson.org / estimate / index.html); immunophenograms of effector cells, immunomodulators (immune checkpoints), and suppressor cells (regulatory T cells and MDSC) (Immunophenogram15). Specific genes (OCT4, TRIM28, SETDB1, etc.) are also evaluated, as are EMT signatures (SSGSEA and Jean-Paul Thierry signatures) and signatures specifically related to tumor subtypes. Correlations between these signatures and HERVs are analyzed using traditional statistical methods. Figure 1A shows the significant correlation between 19 HERVs and specific immune signatures, such as antigen presentation-related, cytotoxicity, or immunomodulatory signatures. As an example, the expression of HERV-K10 is strongly correlated with the T cell signature of breast cancer (Figure 1B).

[0123] specific CD8 + HERV + PBMC culture for stimulation PBMCs from HLA-A2 donors were cultured for 12 days in AIM-V medium (Thermo Fisher Scientific) supplemented with 5% AB human serum (pool of 5 donors from EFS Lyon Hospital, filtered) and 5 μg / mL R848 (InvivoGen, San Diego, USA) and 10 μg / mL Poly-IC (InvivoGen) and 20 UI / mL IL-2 (PROLEUKIN® aldesleukin, Prometheus, Vevey, Switzerland) enriched with the peptide of interest (10 μM). Cultures were performed in U-bottom 96-well plates at 1.5 × 10 cells per well. 5Cells were cultured in 20 wells for each peptide condition. 100 μL of medium was replaced and enriched with R848, Poly-IC, IL-2, and peptide of interest (peptides with sequences of SEQ ID NOs: 1-7) to achieve the same final concentrations on day 3. IL-2 and peptide of interest were added on day 6, and IL-2 alone was added on day 10. The positive control was cultured with 0.1 μg / mL PP65 (JPT Peptide Technologies) in the Dextramer® experiment. PP65 is presented by MHC class I and binds to CD8 + This is a CMV peptide that specifically stimulates T cells. In the IFN-γ experiments, 0.4 μg / mL of CEF peptide (Mabtech) was used. This is a CMV peptide that specifically stimulates CD8 + It is composed of a pool of 23 MHC class I-restricted viral peptides from human CMV, EBV, and influenza viruses that stimulate T cells to preferentially synthesize IFN-γ.

[0124] Dextramers assay and screening On day 12, cells from the same conditions were pooled together in polypropylene tubes, washed with 2 mL FACS buffer and resuspended in FACS buffer. Conditions were stained with 10 μL of the corresponding dextramer (Immudex, Copenhagen, Denmark) for 15 min at room temperature in the dark. Viability was assessed using the Zombie NIR™ (near infrared) Fixable Viability kit (Zombie NIR™, BioLegend, Paris, France) at 1 / 400. Anti-CD3 (BV421, Biolegend) and anti-CD8 (FITC, Beckman Coulter, Brea, CA, USA) antibodies were then added to each condition (1 / 10 for assay in Figure 2 and 1 / 25 for Figure 4) and left for 20 min at 4°C in the dark, after which the cells were washed twice with 2 mL FACS buffer and resuspended in 350 μL FACS buffer until analysis. Analysis was performed using multimer-specific HERV CD8 + Identification of T cells was performed using FACS Fortessa™ (BD).

[0125] The results in Figure 2A show the dextramer-stained populations in non-specific (left panel) and specific (right panel) peptide-pulsed PBMCs. In the top panel, CD8 + Up to 72% of T cells were positive after stimulation with the positive control pp65 peptide from CMV, compared to 2.02% in unstimulated PBMCs (indicating the possible presence of memory T cells against CMV from previous infection). Interestingly, PBMCs stimulated with HERV peptides (e.g., SEQ ID NO: 5 in the lower panel) showed a high percentage of dextramer-positive CD8 T cells, compared to 0.04% in the unstimulated condition. + For T cells, the figure was 34.2%. The results obtained for four different donors are summarized in Figure 2B.

[0126] Results in Figure 4A show the populations stained by dextramer in non-specific (left panel) and specific (right panel) peptide-pulsed PBMCs. In the top panel, up to 23.40% of CD8+ T cells were positive after stimulation with the positive control pp65 peptide from CMV, compared to 3.63% in unstimulated PBMCs (indicating the possible presence of memory T cells against CMV due to previous infection). Interestingly, PBMCs stimulated with HERV peptides (e.g., SEQ ID NO: 1 in the bottom panel) showed a significantly higher number of dextramer-positive CD8+ T cells compared to 0.091% in the unstimulated condition. + The results for 12 different donors are summarized in FIG. 4B. P1, P4, and P6 (e.g., SEQ ID NOs: 1, 4, and 6) showed dextramer-positive CD8 + There was a significant increase in T cells, with a slight increase in P2 and P3 (eg, SEQ ID NOs: 2 and 3).

[0127] ELISPOT assays for IFN-γ and granzyme-β were performed using peptide-stimulated PBMCs after 12 days of culture (Figure 2C). 5The cells were co-cultured with T2 cells specific for the peptide of interest at a ratio of 10:1. ELISPOT was performed after 24 hours and showed IFN-γ and granzyme-β spots specific for P1 and P6 (e.g., SEQ ID NOs: 1 and 6).

[0128] Cytotoxicity assay using Th2 cells On day 12 of PBMC culture, T2 cells were washed with RPMI and resuspended in AIM-V medium (Thermo Fisher Scientific). T2 (SD cell line) is a lymphoblastoid cell line that is deficient in the transporter associated with antigen processing (TAP) protein and therefore cannot present endogenous peptides on MHC class I, but can be used to monitor CTL responses to exogenous antigens of interest in a non-competitive environment. T2 cells were pulsed with HERV peptides by first adding 10 μg / mL of the corresponding peptide to 2 M of T2 cells for 2 h at 37 °C. PBMCs were pooled, counted, and added to new U-bottom 96-well plates in co-culture with the corresponding T2 cells at a concentration of 1:5, respectively.

[0129] After 4 hours of incubation at 37°C, the co-cultured cells were washed and pooled in V-bottom well plates according to staining conditions in FACS buffer. Zombie NIR™ (BioLegend) was used at 1 / 400 to assess viability. Anti-CD3 (PerCP, BioLegend) and anti-CD8 (FITC, Beckman Coulter) antibodies were added at 1 / 10 per condition and left at 4°C for 25 minutes. Cells were washed again and then fixed using Fixation / Permeabilization Solution Kit (Invitrogen, Carlsbad, CA, USA) for 15 minutes at room temperature according to the manufacturer's instructions. Cells were washed twice with FACS buffer and kept at 4°C.

[0130] On day 13, cells were permeabilized with Permeabilization Solution Kit (Invitrogen) for 5 min at room temperature and anti-IFN-γ (PE, BioLegend) antibody was added at 1 / 20 in solution for another 25 min at 4°C. Cells were washed twice and resuspended in 350 μL of FACS buffer before FACS analysis. Analysis was performed on a FACS Fortessa™ (BD) to investigate specific cytotoxicity and degranulation on T2 cells expressing HERV sequences.

[0131] The results in FIG. 3A show the stimulated CD8 T cells pulsed with non-pulsed T2 cells (P0: upper left panel), with different peptides (P(-): upper center panel), or with each cognate peptide (positive control P(+): HERV peptides of SEQ ID NOs: 1, 2, and 3 in upper / right panels and lower panel). + The results show specific IFN-γ production for T2 cells expressing HERV peptides. The results for five different donors are summarized in Figure 3B. CD8 + Approximately 10% (median) of T cells and CD8 + This shows that approximately 5% (median) of T cells produce IFN-γ.

[0132] The results in Figure 5A show the stimulated CD8 T cells pulsed with non-pulsed T2 cells (P0: top left panel, both quadrants of donor a and donor b), with different peptides (negative control P(-): top center panel, both quadrants of donor a and donor b), or with each cognate peptide (positive control P(+): top / right panels, HERV peptides with SEQ ID NOs: 1-3 in the bottom panel of donor a and HERV peptides with SEQ ID NOs: 4-7 in the bottom panel of donor b). + The results show specific IFN-γ production for T2 cells expressing HERV peptides. The results for 12 different donors are summarized in Figure 5B. A significant number of CD8 T cells stimulated with P1 (SEQ ID NO: 1) were positively stimulated with P1 (SEQ ID NO: 1). + T cells and CD8 stimulated with P2 and P3 (SEQ ID NOs: 2 and 3) + T cells and CD8 stimulated with moderate numbers of P6 (SEQ ID NO: 6)+ 1 shows IFN-γ production in T cells.

[0133] P1-specific CD8 + T cell cytotoxicity The dextramer-stained cells were sorted using FACS Aria (BD), and peptide-specific CD8 + T cells were separated from their non-specific counterparts. Both fractions were collected and grown separately on feeder cells in round-bottom 96-well plates for 14 days. The purity of the specific versus non-specific fractions was assessed on day 14 (Figure 6A), with >90% dextramer-positive CD8 + T cells, which accounted for less than 5% of the negative fraction. These cells were used in cytotoxicity experiments.

[0134] Selected and expanded CD8 + T cells were assessed for cytotoxic potential by ELISPOT assay (Cellular Technology Limited (CTL)). 4 P1-specific CD8 + T cells were co-cultured with T2 cells pre-pulsed with peptide P1. The number of spots counted was the percentage of P1-specific CD8 T cells against target cells pulsed with the cognate peptide compared to the negative control. + The experiments show the production of IFN-γ and granzyme-β by T cells (approximately 800 spots for both cytokines) (FIG. 6B). These experiments demonstrate that the cells are specific and functional.

[0135] In silico analysis of HERV expression performed in HMEC cell lines (HLA-A2 human mammary epithelial cells) and MDA-MB-231 cell lines (HLA-A2 triple-negative breast cancer cell line) showed overexpression of HERVs in the MDA-MB-231 cell line compared to HMEC.

[0136] The HLA-A2 TNBC cell line, MDA-MB-231, expresses P1-specific CD8 + It was used as a target for real-time analysis of T cell-induced cell death. 3MDA-MB-231 cells were pulsed with or without peptide P1 and allowed to adhere to a 96-well plate. + T cells or their negative counterparts (controls) were added to the wells. Co-culture was performed in the presence of Cytotox Green Dye (Essen BioScience), which enters cells when cell membrane integrity is compromised and increases fluorescence 100-1000-fold when bound to deoxyribonucleic acid (DNA). Co-culture of MDA-MB-231 with P1-specific T cells shows a marked increase in the kinetics of cell death compared to the negative counterpart. As expected, when target MDA-MB-231 cells were pulsed with P1 and co-cultured with P1-specific T cells, a further increase in cell death was observed (probably due to an increase in the number of HLA-peptide 1 complexes on the target cells) (Figure 6C).

[0137] In addition, P1-specific CD8 + After 6 h of co-culture between T cells or their negative counterparts and the MDA-MB-231 HLA-A2 TNBC cell line, intracellular staining of IFN-γ was performed by FACS (Figure 6D). The results show that when P1-specific cells are co-cultured, the percentage of IFN-γ-producing cells is increased compared to co-culture with the negative counterpart. This percentage is slightly increased when MDA-MB-231 are pre-pulsed with P1. Using an anti-HLA-A2 specific blocking antibody, this effect can be reversed, demonstrating its specificity.

[0138] Taken together, these experiments demonstrated that P1-specific CD8 + We demonstrate that T cells function by specifically recognizing target cells presenting the cognate peptide (in this experiment, T2 cells) and specifically recognizing and killing tumor cells that endogenously express the HERV-derived antigen (in this experiment, the MDA-MB-231 TNBC cell line).

[0139] Dextramerstaining of tumor infiltrating lymphocytes (TIL) from TNBC and ovarian cancer The tumor was divided into small pieces and digested with collagenase IV and DNAse for 45 min. The resulting cells were resuspended in RPMI with 5% human serum and distributed into 96-well plates (5.10 per well). 4 Anti-CD3 / CD28 microbeads (Miltenyi Biotec) were added to the wells at a ratio of 1:4 to the cells in the presence of IL-2 (final concentration 100 IU / mL). TILs were cultured for 14 days with medium changes on days 5, 7, 9, and 12, and the cell number was maintained at 0.5 × 10 6 Adjusted to cells / mL.

[0140] On day 14, dextramer staining was performed for the seven peptides of interest (P1-P7: SEQ ID NOs: 1-7) (see the dextramer assay paragraph) to detect dextramer-specific CD8 + T cells were identified by FACS analysis in TNBC (Figure 7A) and ovarian cancer (Figure 7B). The results showed that CD8 + We show that CD8 T cells specific for P1 and P6 are found in the tumors, with some variation between samples. This confirms the in vivo immunogenicity of the peptides, indicating that these peptides can be naturally processed and induce a detectable immune response during tumor development. + T cells are more frequently presented in tumors and are the peptides chosen as the basis for a vaccine or immunogenic composition. These results also support the combination of several peptides to provide a vaccine or immunogenic composition that can be used in patients with unknown status regarding this reactivity, preferably including P1 and / or P6.

Claims

[Claim 1] The invention described in this specification.

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