Novel melanoma antigen peptide and uses thereof

专利摘要:
The present invention relates to a melanoma antigen peptide comprising the amino acids sequence selected in the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or a function-conservative variant thereof. Moreover the invention also relates to a melanoma antigen peptide according to the invention for use in the prevention or the treatment of melanoma in patient.
公开号:AU2012380679A1
申请号:U2012380679
申请日:2012-05-22
公开日:2014-10-30
发明作者:Mathilde BOBINET；Nathalie Labarriere；Francois Lang；Anne ROGEL
申请人:Centre National de la Recherche Scientifique CNRS；Universite dAngers；Universite de Nantes；Institut National de la Sante et de la Recherche Medicale INSERM；
IPC主号:C07K14-47

专利说明:
WO 2013/175256 PCT/IB2012/001310 NOVEL MELANOMA ANTIGEN PEPTIDE AND USES THEREOF FIELD OF THE INVENTION: 5 The present invention relates to a melanoma antigen peptide comprising the amino acids sequence selected in the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or a function-conservative variant thereof. Moreover the invention also relates to a melanoma antigen peptide according to the invention for use in 10 the prevention or the treatment of melanoma in patient. BACKGROUND OF THE INVENTION: In antitumor immune responses, CD8 cytotoxic T lymphocytes (CTL) have been 15 identified as the most powerful effector cells (Vesely MD et al., 2011). As a consequence, most previous anti-cancer vaccines use class I HLA-restricted peptides derived from tumor antigens in order to stimulate CTL responses. However, the clinical impact of peptide-based cancer vaccines remains still modest, even if a recent gp1OO-derived peptide vaccination was shown to increase patient survival in melanoma (Rosenberg SA et al., 2004 and 20 Schwartzentruber DJ et al., 2011). In addition to a variety of immune suppressive mechanisms originating from the tumor itself, suboptimal design of vaccines used so far may explain this failure. In particular, short epitopic peptides, could induce vanishing CTL responses or tolerance towards targeted antigens (Bijker MS et al., 2007 and Toes RE et al., 1996). In the meanwhile, CD4 helper T cells have gained interest in anti-tumor immunity and 25 immunotherapy. Indeed, tumor-reactive CD4+ T helper 1 T cells (Thl) produce several cytokines (such as IFN-y, TNF-a and IL-2) essential for the induction of cell-mediated immunity against tumors. One widely accepted model demonstrates the ability of CD4+ T cells to 'license' dendritic cells (DCs) for efficient CD8+ T cell priming through the interaction of costimulatory receptors (Bennett SR et al., 1998 and Smith CM et al., 2004). 30 The cytokines secreted by CD4+ Th1 cells also exert direct antitumor and antiangiogenic effects. Furthermore, it has been demonstrated in a mouse model that only tumor-reactive CD4+ T cells have been found to ensure efficient effector CTLs recruitment at the tumor site. In a clinical standpoint, a high density of tumor-infiltrating CD4+ Th1 cells has been recently shown as a good prognostic marker in colorectal cancer patients emphasizing the role of these WO 2013/175256 PCT/IB2012/001310 -2 cells in cancer immunosurveillance. In melanoma, tumor-reactive CD4 T cells have also been associated with a good clinical outcome (Robbins PF et al., 2002), and more recently the same group showed that tumor specific CD4 T cells were present in at least 20% of metastatic melanomas, and suggested that the infusion of TIL populations containing CD4 specific T 5 cells could enhance the efficacy of adoptive cell therapy (Friedman KM et al., 2012). In the same line of thought, it has been demonstrated in a melanoma patient that the adoptive cell transfer of CD4 T cells specific for NYESO-1 antigen induces durable clinical remission and led to endogenous responses against non-targeted tumor antigens, suggesting the stimulation of immune responses by transferred CD4 T cells (Hunder NN et al., 2008). 10 In the field of peptide vaccination, it has been documented twenty years ago, in a mouse model that the generation of a strong CD8 response against a LCMV-derived peptide depended on the presence of CD4 helper T cells (Fayolle C et al., 1991). These results have been more recently confirmed in a clinical setting by the use of synthetic long peptides (SLP) in colorectal cancer, using P53 derived SLP (Speetjens FM et al., 2009), in vulvar 15 intraepithelial neoplasia (Kenter GG et al., 2009) and cervical cancer patients (Welters MJ et al., 2008) using HPV16-derived SLP. In the case of vulvar neoplasia, clinical responses appeared to be correlated with the induction of strong HPV16 specific immune responses. Synthetic long peptides containing immunogenic CD8 and CD4 tumor epitopes are therefore attractive tools to implement therapeutic cancer vaccine. 20 One of the main issues in the field of long peptide vaccination in solid tumors is to identify immunogenic long peptides derived from relevant tumor associated antigens. Target antigens should be widely expressed, and able to induce robust CD8 and CD4 anti-tumor T cell responses. In melanoma, the Melan-A antigen fulfills these requirements and the inventors recently reported the efficiency of a Melan-A modified SLP, to cross-prime human 25 tumor-reactive T cells (Chauvin JM et al., 2012). Another attractive target for melanoma vaccination would be the MELOE-1 antigen (46 amino acids), specifically overexpressed in melanoma. Indeed, the inventors previously reported that the infusion of tumor infiltrating lymphocytes (TIL) specific for the MELOE-1 antigen was associated with a prolonged relapse-free survival for HLA-A2 melanoma patients who received TIL therapy (Godet Y et 30 al., 2008). Furthermore, they documented the presence of a large and tumor reactive CD8 T cell repertoire in HLA-A2 melanoma patients (Godet Y et al., 2010) and the presence of two class II epitopes in the vicinity of the class I epitope, located at the C-terminal end of the polypeptide (Rogel A et al., 2011).
WO 2013/175256 PCT/IB2012/001310 -3 Despites these results, the identification of additional melanoma antigens with a documented immunogenic potential remains a major issue to address for melanoma immunotherapy. 5 SUMMARY OF THE INVENTION: In this study, the inventors find new epitopes located all along the MELOE- 1 sequence and characterized by their T helper profile of CD4 T cell response. Thus, the invention relates to a melanoma antigen peptide comprising the amino acids 10 sequence selected in the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or a function-conservative variant thereof Moreover the invention also relates to a melanoma antigen peptide according to the invention for use in the prevention or the treatment of melanoma in patient. 15 DETAILED DESCRIPTION OF THE INVENTION: Definitions: Throughout the specification, several terms are employed and are defined in the following paragraphs. 20 As used herein, the term "peptide" refers to an amino acid sequence having less than 50 amino acids. As used herein, the term "peptide" encompasses amino acid sequences having less than 50 amino acids, less than 40 amino acids, less than 30 amino acids, less than 25 amino acids, less than 20 amino acids, less than 15 amino acids or less than 10 amino 25 acids. Melanoma antigen peptides of the invention are described in table A. SEQ ID number Sequences Nomenclatures used in the patent application Peptide SEQ ID NO:1 SCVGYPDEATSREQFLPSEC MELOE-1 2
-
21 or 2-21 Peptide SEQ ID NO:2 VGYPDEATSREQFLPS MELOE-1 4
.
1 9 or 4-19 Peptide SEQ ID NO:3 VGYPDEATSREQFL MELOE-1 41 7 or 4-17 Peptide SEQ ID NO:4 GYPDEATSREQFLPS MELOE-1 5
_
1 9 or 5-19 Peptide SEQ ID NO:5 PDEATSREQFLPS MELOE-1 7
.
1 9 or 7-19 WO 2013/175256 PCT/IB2012/001310 -4 Peptide SEQ ID NO:6 PWHPSERISSTLNDECWPASL MELOE-1 26 -4 6 or 26-46 Peptide SEQ ID NO:7 RISSTLNDECWPAS MELOE-1 32 -4 5 or 32-45 Peptide SEQ ID NO:8 RISSTLNDECWPA MELOE-1 32 -4 4 or 32-44 Peptide SEQ ID NO:9 ERISSTLNDECWPA MELOE-1 3 1-4 4 or 31-44 Peptide SEQ ID NO:10 TSREQFLPSEGAACPPWHPS MELOE-IIt- 30 or 11-30 Peptide SEQ ID NO:11 REQFLPSEGAACPPW MELOE-1 1 3- 27 or 13-27 Peptide SEQ ID NO:12 EQFLPSEGAACPPW MELOE-1 1 4- 27 or 14-27 Peptide SEQ ID NO:13 QFLPSEGAACPPW MELOE-1 15
-
27 or 15-27 Peptide SEQ ID NO:14 TSREQFLPSEGAA MELOE-1 11
-
23 or 11-23 Peptide SEQ ID NO:15 SREQFLPSEGAAC MELOE-1 1 2- 24 or 12-24 Peptide SEQ ID NO:16 PSEGAACPPWHPSERISSTL MELOE-1 1
-
37 or 18-37 Peptide SEQ ID NO:17 AACPPWHPSERISSTLNDECWPASL MELOE-1 224 6 or 22-46 Peptide SEQ ID NO:18 CPPWHPSERISSTL MELOE-1 24
-
3 7 or 24-37 Peptide SEQ ID NO:19 CPPWHPSERISST MELOE-1 24
-
36 or 24-36 Peptide SEQ ID NO:20 MSCVGYPDEATSREQFLPSEGAACPPW MELOE-1 1
-
46 or 1-46 HPSERISSTLNDECWPASL Peptide SEQ ID NO:21 GHGHSYTTAEELAGIGILTVILGVL Melan-A 16
-
40 L Table A: melanoma antigen peptides of the invention As used herein, the term "antibody" refers to a protein capable of specifically binding 5 an antigen, typically and preferably by binding an epitope or antigenic determinant or said antigen. The term "antibody" also includes recombinant proteins comprising the binding domains, as well as variants and fragments of antibodies. Examples of fragments of antibodies include Fv, Fab, Fab', F(ab')2, dsFv, scFv, sc(Fv)2, diabodies and multispecific antibodies formed from antibody fragments. 10 "Function-conservative variants" as used herein refer to those in which a given amino acid residue in a protein or enzyme has been changed (inserted, deleted or substituted) without altering the overall conformation and function of the polypeptide. Such variants include protein having amino acid alterations such as deletions, insertions and/or substitutions. A "deletion" refers to the absence of one or more amino acids in the protein. An 15 "insertion" refers to the addition of one or more of amino acids in the protein. A "substitution" refers to the replacement of one or more amino acids by another amino acid residue in the protein. Typically, a given amino acid is replaced by an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, WO 2013/175256 PCT/IB2012/001310 -5 basic, hydrophobic, aromatic, and the like). Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70 % to 99 % as determined according to an alignment scheme such as by the Cluster Method, 5 wherein similarity is based on the MEGALIGN algorithm. A "function-conservative variant" also includes a polypeptide which has at least 60 % amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75 %, more preferably at least 85%, still preferably at least 90 %, and even more preferably at least 95%, and which has the same or substantially similar properties or functions as the native or parent protein to which it is 10 compared. Two amino acid sequences are "substantially homologous" or "substantially similar" when greater than 80 %, preferably greater than 85 %, preferably greater than 90 % of the amino acids are identical, or greater than about 90 %, preferably greater than 95 %, are similar (functionally identical) over the whole length of the shorter sequence. Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG 15 (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any of sequence comparison algorithms such as BLAST, FASTA, etc. The term "Major Histocompatibility Complex" (MHC) is a generic designation meant to encompass the histo-compatibility antigen systems described in different species including 20 the human leucocyte antigens (HLA). The term "melanoma" as used herein includes, but is not limited to, all types of melanocytes cancers at all stages of progression like metastatic melanocyte cancer. The term "treating" a disorder or a condition refers to reversing, alleviating or inhibiting the process of one or more symptoms of such disorder or condition. The term 25 "preventing" a disorder or condition refers to preventing one or more symptoms of such disorder or condition. As used herein, the term "patient" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably a patient according to the invention is a human. A "therapeutically effective amount" as used herein is intended for a minimal amount 30 of active agent which is necessary to impart therapeutic benefit to a patient. For example, a "therapeutically effective amount of the active agent" to a patient is an amount of the active agent that induces, ameliorates or causes an improvement in the pathological symptoms, disease progression, or physical conditions associated with the disease affecting the patient.
WO 2013/175256 PCT/IB2012/001310 -6 The term "adjuvant" as used herein refers to a compound or a mixture that may be non-immunogenic when administered in the host alone, but that augments the host's immune response to an antigen when administered conjointly with that antigen. 5 Peptide, fusion protein and uses thereof A first object of the invention relates to a melanoma antigen peptide comprising the amino acids sequence selected in the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or a function-conservative variant thereof 10 In one embodiment, the melanoma antigen peptide has the sequence SEQ ID NO:5. Melanoma antigen peptides of the invention, are generated from the peptide MELOE 1 (SEQ ID NO:20) as described in the patent application WO 2010 026165 and in table A. 15 In one embodiment, the melanoma antigen peptide of the invention is not the peptide MELOE-1 (SEQ ID NO:20). In one embodiment of the invention, by "antigen peptide" is meant a peptide capable of binding to HLA molecule and causing a cellular or humoral response in a patient. 20 In a preferred embodiment of the invention, said antigen peptide may comprise a specific motif such that the polypeptide binds an HLA molecule and induces a CTL response. In another preferred embodiment of the invention, said antigen peptide may comprise a specific motif such that the polypeptide binds an HLA molecule and induces a helper T cell response. 25 In one embodiment of the invention, said melanoma antigen peptides as described here above are HLA-DQ1*0201 restricted. In one embodiment of the invention, said melanoma antigen peptides as described here above are HLA-DQ 1*0202 restricted. 30 In one embodiment of the invention, said antigen peptide is an amino acid sequence of less than 50 amino acids long that comprises the amino acid motif SEQ ID NO: 1, 2, 3, 4 or 5 as defined here above.
WO 2013/175256 PCT/IB2012/001310 -7 In another embodiment of the invention, said antigen peptide is an amino acid sequence of less than 45 amino acids long that comprises the amino acid motif SEQ ID NO: 1, 2, 3, 4 or 5 as defined here above. In another embodiment of the invention, said antigen peptide is an amino acid 5 sequence of less than 40 amino acids long that comprises the amino acid motif SEQ ID NO: 1, 2, 3, 4 or 5 as defined here above. In another embodiment of the invention, said antigen peptide is an amino acid sequence of less than 30 amino acids long that comprises the amino acid motif SEQ ID NO: 1, 2, 3, 4 or 5 as defined here above. 10 In another embodiment of the invention, said antigen peptide is an amino acid sequence of less than 25 amino acids long that comprises the amino acid motif SEQ ID NO: 1, 2, 3, 4 or 5 as defined here above. In one embodiment, the antigen peptide according to the invention comprises at least 15 60% identity over said SEQ ID NO: 1, 2, 3, 4 or 5 even more preferably at least 700%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% and is still able to bind to HLA molecule and causing a cellular or humoral response in a patient. In another embodiment, the antigen peptide according to the invention consists in the 20 amino acid sequence as set forth in SEQ ID NO: 1, 2, 3, 4 or 5 or a variant thereof comprising at least 60%, preferably at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with SEQ ID NO: 1 and is still able to bind to HLA molecule and causing a cellular or humoral response in a patient. 25 The invention also encompasses peptides that are function-conservative variants of antigen peptides comprising SEQ ID NO: 1, 2, 3, 4 or 5 as described here above. Typically, the invention encompasses peptides substantially identical to antigen peptides comprising SEQ ID NO: 1, 2, 3, 4 or 5 in which one or more residues have been 30 conservatively substituted with a functionally similar residue and which displays the functional aspects of the antigen peptides comprising SEQ ID NO: 1, 2, 3, 4 or 5 as described here above, i.e. being still able to bind to an HLA molecule in substantially the same way as a peptide consisting of the given amino acid sequence.
WO 2013/175256 PCT/IB2012/001310 Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one 5 basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid or another. The term "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue. "Chemical derivative" refers to a patient peptide having one or more residues chemically derivatized by reaction of a functional side group. 10 Examples of such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl 15 derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im benzylhistidine. Chemical derivatives also include peptides which contain one or more naturally-occurring amino acid derivatives of the twenty standard amino acids. For examples: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for 20 serine; and ornithine may be substituted for lysine. According to the invention, the antigen peptides of the invention can be obtained by synthesizing the peptides according to the method for peptide synthesis known in the art. 25 In another embodiment, the antigen peptides of the invention may be incorporated into polytopes or fusion proteins. Two or more peptides of the invention can be joined together directly, or via the use of flanking sequences. Thompson et al., Proc. Natl. Acad. Sci. USA 92 (13): 5845- 5849 (1995), teaches the direct linkage of relevant epitopic sequences. The use of polytopes or fusion proteins as vaccines is well known. See, e. g. Gilbert et al., Nat. 30 Biotechnol. 15 (12): 1280-1284 (1997); Thomson et al. , supra; Thomson et al., J. Immunol. 157 (2): 822- 826 (1996); Tam et al., J. Exp. Med. 171 (1) : 299-306 (1990), all of which are incorporated by reference. The Tam et al. reference in particular shows that polytopes or fusion proteins, when used in a mouse model, are useful in generating both antibody and protective immunity.
WO 2013/175256 PCT/IB2012/001310 -9 Thus, the invention also relates to a fusion protein comprising a melanoma antigen peptide according to the invention and a melanoma antigen peptide comprising the amino acids motif: - TX2NDECWPX9 (SEQ ID NO: 22) 5 wherein X2 is leucine, methionine, valine, isoleucine or glutamine and X9 is alanine, valine or leucine. In one embodiment of the invention, said second melanoma antigen peptide is selected in the group consisting of peptides having the sequence SEQ ID NO: 23 to SEQ ID NO: 37 as described below. 10 Peptide SEQ ID NO 23 X2 = L X 9 = A TLNDECWPA Peptide SEQ ID NO 24 X2 = M X 9 = A TMNDECWPA Peptide SEQ ID NO 25 X2 = V X 9 = A TVNDECWPA Peptide SEQ ID NO 26 X2 = I X 9 = A TINDECWPA Peptide SEQ ID NO 27 X2 = Q X 9 = A TQNDECWPA Peptide SEQ ID NO 28 X2 = L X 9 = V TLNDECWPV Peptide SEQID NO 29 X2= M X 9 = V TMNDECWPV Peptide SEQ ID NO 30 X2 = V X 9 = V TVNDECWPV Peptide SEQ ID NO 31 X2 = I X 9 = V TINDECWPV Peptide SEQ ID NO 32 X2= Q X 9 = V TQNDECWPV Peptide SEQ ID NO 33 X2 = L X 9 = L TLNDECWPL Peptide SEQ ID NO 34 X2 = M X 9 = L TMNDECWPL Peptide SEQ ID NO 35 X2 = V X 9 = L TVNDECWPL Peptide SEQ ID NO 36 X2 = I X 9 = L TINDECWPL Peptide SEQ ID NO 37 X2 = Q X 9 = L TQNDECWPL In another embodiment, the melanoma antigen peptide according to the invention or the fusion protein according to the invention may be use in the prevention or the treatment of melanoma in patient. 15 In one embodiment, said patient is genotyped with HLA-DQ 1*0201 or HLA DQI *0202 alleles.
WO 2013/175256 PCT/IB2012/001310 - 10 Nucleic acids, vectors, recombinant host cells and uses thereof Another object of the invention relates to a nucleic acid sequence encoding a melanoma antigen peptide according to the invention or a fusion protein according to the 5 invention. Another object of the invention relates to an expression vector comprising a nucleic acid sequence encoding a melanoma antigen peptide according to the invention or a fusion protein according to the invention. In one embodiment of the invention, said expression vector comprises the nucleic acid 10 sequence corresponding to a melanoma antigen peptide having the sequence SEQ ID NO:1 to SEQ ID NO: 5. According to the invention, expression vectors suitable for use in the invention may comprise at least one expression control element operationally linked to the nucleic acid 15 sequence. The expression control elements are inserted in the vector to control and regulate the expression of the nucleic acid sequence. Examples of expression control elements include, but are not limited to, lac system, operator and promoter regions of phage lambda, yeast promoters and promoters derived from polyoma, adenovirus, retrovirus, lentivirus or SV40. Additional preferred or required operational elements include, but are not limited to, leader 20 sequence, termination codons, polyadenylation signals and any other sequences necessary or preferred for the appropriate transcription and subsequent translation of the nucleic acid sequence in the host system. It will be understood by one skilled in the art that the correct combination of required or preferred expression control elements will depend on the host system chosen. It will further be understood that the expression vector should contain 25 additional elements necessary for the transfer and subsequent replication of the expression vector containing the nucleic acid sequence in the host system. Examples of such elements include, but are not limited to, origins of replication and selectable markers. It will further be understood by one skilled in the art that such vectors are easily constructed using conventional methods or commercially available. 30 Another object of the invention is a host cell comprising an expression vector as described here above. According to the invention, examples of host cells that may be used are eukaryote cells, such as animal, plant, insect and yeast cells and prokaryotes cells, such as E. coli. The WO 2013/175256 PCT/IB2012/001310 - 11 means by which the vector carrying the gene may be introduced into the cells include, but are not limited to, microinjection, electroporation, transduction, or transfection using DEAE dextran, lipofection, calcium phosphate or other procedures known to one skilled in the art. In a preferred embodiment, eukaryotic expression vectors that function in eukaryotic 5 cells are used. Examples of such vectors include, but are not limited to, viral vectors such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus; lentivirus, bacterial expression vectors, plasmids, such as pcDNA3 or the baculovirus transfer vectors. Preferred eukaryotic cell lines include, but are not limited to, COS cells, CHO cells, HeLa cells, NIH/3T3 cells, 293 cells (ATCC# CRL1573), T2 cells, 10 dendritic cells, or monocytes. In one embodiment, the nucleic acid sequence according to the invention or the expression vector according to the invention or the host cell according to the invention may be use in the prevention or the treatment of melanoma in patient. 15 Antibodies and uses thereof Another object of the invention relates to an antibody or fragment thereof that binds to 20 the melanoma antigen peptide according to invention. In one embodiment of the invention, said antibody or fragment thereof binds to melanoma antigen peptide having the sequence SEQ ID NO: I to SEQ ID NO: 5. In one embodiment of the invention, said antibody is monoclonal. In another embodiment of the invention, said antibody is polyclonal. 25 Such antibodies may be easily prepared, for example, according to the method described in "Antibodies: A laboratory manual", Lane H. D. et al. eds, Cold Spring Harbor Laboratory Press, New York, 1989 or Antibody Engineering: Methods and Protocols, 2003, Benny K. Lo. 30 MHC/peptide multimer Another object of the invention relates to a MHC/peptide multimer comprising a melanoma antigen peptide as described here above. According to the invention, said WO 2013/175256 PCT/IB2012/001310 - 12 MHC/peptide multimer include, but are not limited to, a MHC/peptide dimer, trimer, tetramer or pentamer. In one embodiment of the invention, said MHC/peptide multimer is a HLA-class II/peptide multimer. 5 In another embodiment of the invention, said MHC/peptide multimer is a HLA DQPI1*0201 II/melanoma antigen peptide multimer or a HLA-DQ 1*0202/melanoma antigen peptide multimer. Methods for obtaining MHC/peptide tetramers are described in W096/26962 and WOO 1/18053, which are incorporated by reference. 10 In one embodiment of the invention, said MHC/peptide multimer can be used to visualise T cell populations that are specific for the complex HLA-DQP 1*0201/ melanoma antigen peptide or HLA-DQ 1*0202/melanoma antigen peptide multimer as described here above. In another embodiment of the invention, said MHC/peptide multimer can be used for 15 the detection and/or isolation by screening (in flow cytometry or by immunomagnetic screening) of T cell population that are specific for a complex HLA/ melanoma antigen peptide as described here above. In another embodiment of the invention, said HLA-DQ 1*0201/ melanoma antigen peptide or HLA-DQ 1I*0202/melanoma antigen peptide multimer can be used for the 20 detection and/or isolation by screening (in flow cytometry or by immunomagnetic screening) of T cell population that are specific for a complex HLA-DQp1*0201/ melanoma antigen peptide or HLA-DQp1 *0202/melanoma antigen peptide multimer as described here above. Another object of the invention is beads coated with MHC/peptide multimers as described here above. 25 Immunizing composition and uses thereof Another object of the invention relates to an immunising composition comprising: 30 (a) at least one melanoma antigen peptide as described here above or (b) at least one fusion protein as described here above, or (c) at least one nucleic acid sequence as described here above, or (d) at least one expression vector as described here above, or (e) at least one host cell as described here above, or WO 2013/175256 PCT/IB2012/001310 - 13 (f) at least one antibody as described here above. In one embodiment, said immunising composition comprises a melanoma antigen peptide having a sequence SEQ ID NO: I to SEQ ID NO: 5. 5 The prophylactic administration of the immunizing composition of the invention should serve to prevent or attenuate melanoma in a mammal. In a preferred embodiment mammals, preferably human, at high risk for melanoma are prophylactically treated with the immunising composition of the invention. Examples of such mammals include, but are not limited to, humans with a family history of melanoma. 10 When provided therapeutically, the immunising composition of the invention is provided to enhance the patient's own immune response to the melanoma antigen present on the melanoma or metastatic melanoma. In one embodiment of the invention, the peptides of the invention may be conjugated with lipoprotein or administered in liposomal form or with adjuvant. 15 In one embodiment, said immunising composition is a pharmaceutical composition. In such embodiment, said immunising composition, for human use, comprises at least one antigen peptide as described here above or at least one antibody as described here above, together with one or more pharmaceutically acceptable carriers and, optionally, other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible 20 with the other ingredients of the composition and not deleterious to the recipient thereof. The immunising compositions may conveniently be presented in unit dosage form and may be prepared by any method well-known in the pharmaceutical art. Immunising compositions suitable for intravenous, intradermal, intramuscular, subcutaneous, or intraperitoneal administration conveniently comprise sterile aqueous 25 solutions of the active agent with solutions which are preferably isotonic with the blood of the recipient. Such compositions may be conveniently prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride (e.g. 0.1-2.0M), glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile. These may be 30 present in unit or multi-dose containers, for example, sealed ampoules or vials. The immunising compositions of the invention may incorporate a stabilizer. Illustrative stabilizers are polyethylene glycol, proteins, saccharides, amino acids, inorganic acids, and organic acids which may be used either on their own or as admixtures. These stabilizers are preferably incorporated in an amount of 0.11-10,000 parts by weight per part WO 2013/175256 PCT/IB2012/001310 - 14 by weight of active agent. If two or more stabilizers are to be used, their total amount is preferably within the range specified above. These stabilizers are used in aqueous solutions at the appropriate concentration and pH. The specific osmotic pressure of such aqueous solutions is generally in the range of 0.1-3.0 osmoles, preferably in the range of 0.8-1.2. The 5 pH of the aqueous solution is adjusted to be within the range of 5.0-9.0, preferably within the range of 6-8. Additional pharmaceutical methods may be employed to control the duration of action. Controlled release preparations may be achieved through the use of polymer to complex or absorb the peptides of the invention. The controlled delivery may be exercised by selecting 10 appropriate macromolecules (for example polyester, polyamirio acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled-release preparations is to incorporate the antigen peptides of the invention into particles of a 15 polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylaceiate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxy methylcellulose or gelatin-microcapsules and poly(methylmethacylate) microcapsules, 20 respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions. When oral preparations are desired, the compositions may be combined with typical carriers, such as lactose, sucrose, starch, talc magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate or gum arabic among others. 25 Immunisation of a patient with the immunising composition of the invention can be conducted by conventional methods, for example, in the presence of conventional adjuvants. Examples of conventional adjuvant include, but are not limited to, metal salts, oil in water emulsions, Toll like receptors agonists, saponins, lipid A, alkyl glucosaminide phosphate, Freund's adjuvant, keyhole limpet haemocyanin (KLH), mannan, BCG, alum, cytokines such 30 as IL-I, IL-2, macrophage colony stimulating factor, and tumor necrosis factor; and other substances that act as immunostimulating agents such as muramyl peptides or bacterial cell wall components, toxins, toxoids and TLR ligands. The immunising composition can be administered by any route appropriate for antibody production and/or T cell activation such as intravenous, intraperitoneal, WO 2013/175256 PCT/IB2012/001310 - 15 intramuscular, subcutaneous, and the like. The immunising composition may be administered once or at periodic intervals until a significant titre of anti-Nectin4 immune cells or anti Nectin4 antibody is produced. The presence of anti-Nectin4 immune cells may be assessed by measuring the frequency of precursor CTL (cytoxic T-lymphocytes) against the antigen 5 peptides of the invention prior to and after immunization by specific tetramer labelling or by a CTL precursor analysis assay. The antibody may be detected in the serum using an immunoassay. Antibodies directed to the antigens of the invention can also be used directly as anti 10 melanoma agents. To prepare antibodies, a host animal may be immunized using melanoma antigen peptide as described here above. The host serum or plasma is collected following an appropriate time to provide a composition comprising antibodies reactive to said antigen peptides. The gamma globulin fraction or the IgG antibodies can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those 15 skilled in the art. The antibodies are substantially free of many of the adverse side effects which may be associated with other anti-cancer agents such as chemotherapy. The immunising composition of the invention comprising antibodies as described here above can be made even more compatible with the host system by minimizing potential adverse immune system responses. This is accomplished by removing all or a portion of the 20 Fe portion of a foreign species antibody or using an antibody of the same species as the host patient, for example, the use of antibodies from human/human hybridomas. Humanized antibodies (i.e., nonimmunogenic in a human) may be produced, for example, by replacing an immunogenic portion of an antibody with a corresponding, but nonimmunogenic portion (i.e., chimeric antibodies). Such chimeric antibodies may contain the reactive or antigen binding 25 portion of an antibody from one species and the Fc portion of an antibody (nonimmunogenic) from a different species. Examples of chimeric antibodies, include but are not limited to, nonhuman mammal-human chimeras, rodent-human chimeras, murine-human and rat-human chimeras. Methods for obtaining said antibodies, chimeric antibodies and humanized chimeric 30 antibodies are well-known in the art. The immunising composition comprising the antibodies of the invention can also be used as a means of enhancing the immune response. The antibodies can be administered in amounts similar to those used for other therapeutic administrations of antibody. For example, pooled gamma globulin is administered at a range of about 1mg to about 100mg per patient.
WO 2013/175256 PCT/IB2012/001310 - 16 Thus, antibodies reactive with the antigen peptides of the invention can be passively administered alone or in conjunction with other anti-cancer therapies to a mammal afflicted with cancer. Examples of anti-cancer therapies include, but are not limited to, chemotherapy, radiation therapy, adoptive immunotherapy therapy with TIL. 5 The antibodies or chimeric antibodies described herein may also be coupled to toxin molecules, radioisotopes and drugs by conventional methods. Examples of toxins to which the antibodies may be coupled to included, but are not limited to, ricin or diphtheria toxin. Examples of drugs or chemotherapeutic agents include, but are not limited to, cyclophosphamide or doxorubicin. Examples of radioisotopes, include, but are not limited to, 10 1311. Antibodies covalently conjugated to the aforementioned agents can be used in cancer immunotherapy for treating melanoma. If the patient to be immunized is already afflicted with cancer or metastatic cancer, the immunising composition of the invention can be administered in conjunction with other 15 therapeutic treatments. Examples of other therapeutic treatments includes, but are not limited to, adoptive T cell immunotherapy, coadministration of cytokines or other therapeutic drugs for cancer. The dose of antigen peptide of the invention to be administered to a patient may be 20 adjusted as appropriate depending on, for example, the disease to be treated, the age and the body weight of said patient. Ranges of antigen peptides of the invention that may be administered are about 0.001 to about 100 mg per patient, preferred doses are about 0.01 to about 10mg per patient. The immunising composition of the invention may be evaluated first in animal 25 models, initially rodents, and in nonhuman primates and finally in humans. The safety of the immunization procedures is determined by looking for the effect of immunization on the general health of the immunized animal (weight change, fever, appetite behavior etc.) and looking for pathological changes on autopsies. After initial testing in animals, cancer patients can be tested. Conventional methods would be used to evaluate the immune response of the 30 patient to determine the efficiency of the immunising composition. Another object of the invention relates to an immunising composition as described above for use in the prevention or treatment of melanoma in a patient in need thereof WO 2013/175256 PCT/IB2012/001310 - 17 In another embodiment, said patient is genotyped with HLA-DQ3 *0201 or HLA DQ 1*0202 alleles. Antigen presenting cell 5 Another object of the invention is an antigen presenting cell comprising a complex HLA antigen and a melanoma antigen peptide of the invention. In one embodiment of the invention, said complex HLA antigen is a HLA DQPI1*0201 or HLA-DQ$31*0202 antigen. 10 In one embodiment of the invention, said antigen presenting cell is derived from the patient to be treated. The term "antigen presenting cell" (APCs) refers to any cell that expresses an HLA antigen capable of presenting the antigen peptide of the invention on its surface. Dendritic cells, which are reported to have an especially high antigen-presenting ability, are preferred. 15 In another embodiment, artificial APCs may also be used such mammalian cells (fibroblast, endothelial cells, keratinocytes), or cell lines. In order to prepare such APCs of the invention, cells having an antigen-presenting ability are isolated from the patient to be treated, and pulsed ex vivo with at least one antigen peptide of the invention to form a complex with the HLA-DQj1*0201 or HLA-DQj1*0202 20 antigen. In case dendritic cells are used, the APC of the invention can be prepared as follows. Lymphocytes are isolated from peripheral blood of the patient to be treated by Ficoll method; adherent cells are separated from non-adherent cells; the adherent cells are then cultured in the presence of GM-CSF and IL-4 to induce dendritic cells; and the dendritic cells are pulsed 25 by culturing with at least one antigen peptide of the invention to obtain the APCs of the invention. The dendritic cells should be exposed to the antigen peptide for sufficient time to allow the antigens to be internalized and presented on the dendritic cells surface. The resulting dendritic cells can then be re-administrated to the patient to be treated. Such methods are described in W093/208185 and EP0563485, which are incorporated by 30 reference. Another object of the invention is a composition for active immunotherapy comprising antigen presenting cells comprising a complex HLA antigen and a melanoma antigen peptide of the invention.
WO 2013/175256 PCT/IB2012/001310 - 18 In one embodiment of the invention, said antigen presenting cells comprise a complex HLA-DQj1*0201 or HLA-DQ31*0202 antigen and a melanoma antigen peptide of the invention. Said APCs may be preferably contained in physiological saline, phosphate buffered 5 saline (PBS), culture medium, or the like. Administration may be achieved, for example, intravenously, hypodermically, or intradermally. Lymphocytes T and uses thereof 10 Another object of the invention relates to a T lymphocyte that recognizes specifically the melanoma antigen peptide of the invention or a fusion protein of the invention. In one embodiment of the invention, said T lymphocyte is a T lymphocyte helper. In another embodiment of the invention, said T lymphocyte is HLA-DQf1*0201 or 15 HLA-DQj 1*0202 restricted. In another embodiment of the invention, said T lymphocyte is a T cell clone. In another embodiment, said T lymphocyte is a genetically modified T lymphocyte that expresses a TCR that recognizes specifically the melanoma antigen peptide of the invention. 20 Another object of the invention is a composition for adoptive therapy comprising said T lymphocytes as described here above that recognizes specifically the melanoma antigen peptide of the invention or a fusion protein of the invention. In the case of melanoma, it has been observed that an adoptive immunotherapy 25 wherein intratumoral T cell infiltrate taken from the patient to be treated are cultured ex vivo in large quantities, and then returned into the patient achieves a therapeutic gain. It is preferred that the T cells are contained in physiological saline, phosphate buffered saline (PBS), culture medium, or the like in order to their stable maintain. Administration may be achieved, for example, intravenously or intra-tumoraly. By returning the T cells that 30 recognizes specifically the antigen peptide of the invention into the patient's body, the toxicity of said T cells, or the stimulation of CD8 cytotoxic T cells by said cells towards tumor cells is enhanced in the patient who is positive for the melanoma antigen peptide of the invention. The tumor cells are destroyed and thereby the treatment of tumor is achieved.
WO 2013/175256 PCT/IB2012/001310 - 19 Examples of where T-lymphocytes can be isolated, include but are not limited to, peripheral blood cells lymphocytes (PBL), lymph nodes, or tumor infiltrating lymphocytes (TIL). Such lymphocytes can be isolated from tumor or peripheral blood of the individual to 5 be treated by methods known in the art and cultured in vitro. Lymphocytes are cultured in media such as RPMI or RPMI 1640 for 2-5 weeks, preferably for 2-3 weeks. Viability is assessed by trypan blue dye exclusion assay. The lymphocytes are exposed to the antigen peptide of the invention for all of the culture duration. In a preferred embodiment the lymphocytes are exposed to the melanoma antigen 10 peptide of the invention at a concentration of about 1 to about 10 micrograms(pg)/ml for all the duration of lymphocyte culture. Cytokines may be added to the lymphocyte culture such as IL-2. The melanoma antigen peptide of the invention may be added to the culture in presence of antigen presenting cells such as dendritic cells or allogeneic irradiated cancer cell 15 line cells. After being sensitized to the peptide, the T-lymphocytes are administered to the patient in need of such treatment. Examples of how these sensitized T-cells can be administered to the mammal include but are not limited to, intravenously, intraperitoneally or intralesionally. Parameters that may 20 be assessed to determine the efficacy of these sensitized T-lymphocytes include, but are not limited to, production of immune cells in the patient being treated or tumor regression. Conventional methods are used to assess these parameters. Such treatment can be given in conjunction with cytokines or gene modified cells (Rosenberg, S.A. et al. (1992) Human Gene Therapy, 3: 75-90; Rosenberg, S.A. et al. (1992) Human Gene Therapy, 3: 57-73). 25 Another object of the invention is a method for producing T lymphocytes that recognize specifically a melanoma antigen peptide of the invention, said method comprising the steps of: (a) stimulating peripheral blood mononuclear cells (PBMCs) or tumor infiltrating 30 lymphocytes (TIL) obtained from a patient with at least one melanoma antigen peptide of the invention or a fusion protein of the invention, (b) enriching the population of T lymphocytes specific for the melanoma antigen peptide(s) used in (a), WO 2013/175256 PCT/IB2012/001310 - 20 (c) optionally cloning said population of T lymphocytes specific for the melanoma antigen peptide(s) used in (a). Enrichment and/or cloning may be carried out by using an MHC/peptide multimer as described here above. Cloning may also be carried out by conventional methods. 5 In one embodiment of the invention, the T lymphocytes that recognize specifically a melanoma antigen peptide of the invention are HLA-DQj1*0201 or HLA-DQ1*0202 restricted. In such embodiment, enrichment and/or cloning may be carried out by using an HLA-DQj 1*0201 or HLA-DQ 1*0202 /peptide multimer as described here above. 10 Stimulation of PBMCs may be carried out with at least one melanoma antigen peptide of the invention alone, or presented by an antigen presenting cell such as dendritic cells or allogeneic irradiated cancer cell line cells. Typically, cytokines such as IL-2 may also be added to the culture. 15 Another object of the invention is a composition for adoptive therapy that comprises lymphocytes that recognizes specifically the melanoma antigen peptide of the invention for preventing or treating melanoma in a patient in need thereof, wherein said T lymphocytes are to be re-administrated to the patient. In one embodiment, said lymphocytes that recognizes specifically the antigen peptide 20 of the invention are HLA-DQj1*0201 or HLA-DQj31*0202 restricted. The invention also relates to a method for preventing or treating melanoma in a patient in need thereof, comprising administering a therapeutically effective amount of (a) at least one melanoma antigen peptide as described here above or 25 (b) at least one fusion protein as described here above, or (c) at least one nucleic acid sequence as described here above, or (d) at least one expression vector as described here above, or (e) at least one host cell as described here above, or (f) at least one antibody as described here above. 30 The invention also relates to a method for preventing or treating melanoma in a patient in need thereof, comprising administering a therapeutically effective amount of T lymphocytes that recognizes specifically the melanoma antigen peptide of the invention. In one embodiment, said T lymphocytes are HLA-DQ3 *0201 or HLA-DQ3 *0202 restricted.
WO 2013/175256 PCT/IB2012/001310 - 21 The invention also relates to a method for preventing or treating melanoma in a patient in need thereof, comprising administering a therapeutically effective amount of antigen presenting cells comprising a complex HLA antigen and a melanoma antigen peptide of the invention. In one embodiment, said complex HLA/peptide is a complex HLA-DQj1*0201 or 5 HLA-DQj 1*0202 / antigen peptide of the invention. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the 10 scope of the present invention. FIGURES: Figure 1: (A) Percentages of TNF-a producing CD4 T cells among positive 15 microcultures. Fourteen days after PBMC stimulation with MELOE-1 whole polypeptide (2 46), microcultures were re-stimulated with each indicated peptide during 5 hours. TNF-at production was then assessed by a double labeling TNF-a -CD4. Results were analyzed with a non-parametric test (Kruskal-Wallis) followed by a Dunns post-test. (B) Frequency of microcultures containing CD4 T cells specific for MELOE-1 peptides (assessed by TNF-a 20 intracellular staining after re-stimulation with the 4 indicated peptides). 624 microcultures (from 7 healthy donors) were analyzed by a contingency table followed by a Fisher exact test. Figure 2: (A) Percentages of cytokine producing CD4 T cells among positive microcultures. Fourteen days after PBMC stimulation with MELOE-1 whole polypeptide (2 25 46), microcultures were re-stimulated with each indicated peptide during 5 hours. IFN-y (left panel) and IL4 (right panel) production was then assessed by a triple labeling IFN-y -IL4 CD4. Results were analyzed with a non-parametric test (Kruskal-Wallis) followed by a Dunns post-test. (B) Frequency of microcultures containing CD4 T cells specific for MELOE-1 peptides (assessed by IFN-y or IL4 intracellular staining after re-stimulation with the 3 30 indicated peptides). 576 microcultures (from 10 melanoma patients) were analyzed by a contingency table followed by a Fisher exact test.
WO 2013/175256 PCT/IB2012/001310 - 22 Figure 3: HLA-restricting element of MELOE-1 specific T cell clones and reactivity against HLA-matched melanoma cell lines. The HLA restriction of MELOE- 1 specific T cell clones was first assessed using anti-HLA blocking antibodies (upper panel). T cell clones were stimulated for 5 hours in the presence of brefeldin A (10 pig/mL) either with peptide 5 alone (10 ptM) in an autopresentation assay and in presence or not of blocking antibodies at a concentration of 12.5 pg/mL. HLA restriction was confirmed with HLA-matched B-EBV cell lines pulsed 2 hrs with the cognate peptide, at a ratio 1: 2 (middle panel). Reactivity of each T cell clone against HLA-class II expressing melanoma cells was assessed, in presence or not of exogeneous peptide (lower panel). After 5 hours of stimulation, cells were then stained with 10 APC-conjugated anti-CD4 mAb, fixed with 4% paraformaldehyde, labeled with PE conjugated anti-TNF-a mAb and analyzed by flow cytometry. Figure 4: Class II epitopes are naturally processed from MELOE-1 whole antigen. Autologous DC, were loaded (before or after fixation) with MELOE-1 2 46 (1 pM) or, as a 15 negative control, with Melan-A16-40L peptide (1 1 iM), and matured. T cell clones were then stimulated with DC at a ratio 1: 1, during 5h in presence of Brefeldin A, then stained with APC-conjugated anti-CD3 mAb, fixed with 4% paraformaldehyde, labeled with PE conjugated anti-TNF-a mAb and analyzed by flow cytometry. Histograms illustrated the % of TNF-a producing cells among CD3 positive T cells. 20 Figure 5: Minimal peptides recognized by MELOE-1 specific CD4 T cell clones. MELOE-1 specific CD4 T cell clones were incubated with various concentrations of the indicated peptides during 5 hours in presence of Brefeldin A. TNF-U production was assessed by intracellular labeling with an anti-TNF-a specific antibody. The core peptide sequence is 25 indicated in bold on each figure panel, and black circles illustrate the best fitting peptide. 30 WO 2013/175256 PCT/IB2012/001310 - 23 Peptide Sequences Seq ID No MELOE-1 MSCVGYPDEATSREQFLPSEGAACPPWHPSERISSTLNDECWPASL 20 MELOE-1 2
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21 SCVGYPDEATSREQFLPSEG I MELOE-1 11
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30 TSREQFLPSEGAACPPWHPS 10 MELOE-1 1 8
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37 PSEGAACPPWHPSERISSTL 16 MELOE-1 26
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46 PWHPSERISSTLNDECWPASL 6 MELOE-1 22
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46 AACPPWHPSERISSTLNDECWPASL 17 Table I: MELOE-1 and MELOE-1 derived peptide sequences. All the peptides were purchased from Millegen company (France), with a purity > 85%. In bold are indicated the DR-11 (SEQ ID NO:18) and the DQ-6 (SEQ ID NO:8) 5 overlapping epitopes already described, and in italics is indicated the HLA-A2 restricted class I epitope (TLNDECWPA, SEQ ID NO:23)). Microcultures containing MELOE-1 specific CD4+ T cells (TNF-a p oduction) Donor MELOE-1 2
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21 MELOE-1 1 1 - MELOE-1 1 8 - MELOE-1 26 - Class 11 HLA 30 37 46 _______ ___ 9/96 13/96 6/96 5/96 DPB1*0902/1501 HD9 (1.4% t0.6) (2.4% t 1.5) (1.4% ±0.6) (1.5% 0.5) DRB1*1104/1201 6/96 26/96 7/96 10/96 DPB1*0401/0301 HD17 (3.2% 1.9) (8.2% t 6.5) (3% ± 0.7) (5.2%± 4.1) DQB1*0301/0603 __________ __________DRI*l 1101/1301 1/96 2/96 2/96 DPB1*0401/1101 HD22 (1%) (3.9% ± 3.3) 0/96 (1% i 0.1) DQB1*0202 DRB 1*0701 3/96 46/96 DPB1*0401/0101 HD24 (1.3% 0.8) (2% ± 1.4) 0/96 0/96 DQB1*0501/0602 I _DRB1*0101/1501 3/96 3/96 15/96 DPB1*0401/0402 HD25 (0.6% ±0.1) (1.2% t 0.7) 0/96 (1.1% ±0.9) DQB1*0201 ____________________ DR31*0301 5/96 DPB1 NA HD27 0/96 (5% 3.4) 0/96 0/96 DQB1*501/0201 (5I ±34) DRB1*0101/0301 30/48 14/48 0/48 DPB1 NA HD28 (2.7%±1.2) (2.4% ±0.-9) 0/96 DQNA IDRB1*0301 WO 2013/175256 PCT/IB2012/001310 - 24 Table II: Assessment of MELOE-1 CD4 T cell responses in PBMC from healthy donors. PBMC from healthy donors were stimulated with 10 ptM of MELOE-1. After 14 days, 5 the presence of CD4 T cells specific for the different regions of MELOE-1 was assessed by restimulating cells with MELOE-1 2 -21, MELOE-1n- 3 0 , MELOE-1 18
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37 and MELOE-1 2 6
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4 6 peptides, followed by CD4 / TNF-a double staining and flow cytometry analysis. Between brackets is indicated the mean % of TNF-a producing CD4 T cells, in positive microcultures. NA: not available. 10 Microcultures containing MELOE-1 specific CD4+ T cells Th1 responses (IFN-y positive Th2 responses (IL4 positive microcultures) microcultures) MELOE- MIELOE- MELOE-1 22 _ MELOE- MELOE- MELOE 12-21 111-30 46 12-21 111-30 122-46 Pt#1 1/48 10/48 11/48 0/48 1/48 4/48 (0.7%) (1.4%+0.7) (4.8%±7.2) (1%) (0.8%0.3) Pt#2 4/96 16/96 2/96 0/96 0/96 0/96 (0.8% 0.2) (1.6%z1.2) (0.6% O0.02) Pt#3 DPB1*0201/ 2001 10/96 5/96 4/96 DQB1*0303/ (1.7%2.9) (16.2%±33. (1.9%61.3) 0/96 0/96 0/96 0501 2) DRB1*0101/ 0701 Pt#4 0/48 (09/0.5) 0/48 (070 0/48 0/48 Pt05 1/48 6/48 0/48 2/48 0/48 1/48 (0.6%) (1.5%1.1) (0.7%0.1) (0.8%) Ptf6 0/48 5/48 0/48 0/48 0/48 0/48 _________(1.5%±+1.9) Pt47 0/48 0/48 (29±35) 0/48 0/48 (1.50o00.7) 2./+48 1/48 1/48 Ptf8 0/48 (2.9(.00.04 0/48 0/48 (0.6%) 1/48 9/48 3/48 Pt#9 0/48 0/48 0/48 (0.6%) (0.6%±0.09 (0.7%±0.15 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _) ) Table III: Assessment of MELOE- 1 CD4 T cell responses in PBMC from melanoma patients. PBMC from melanoma patients were stimulated with 10 pM of MELOE-1. After 14 15 days, the presence of CD4 T cells specific for the different regions of MELOE-1 was assessed WO 2013/175256 PCT/IB2012/001310 - 25 by restimulating cells with MELOE-1 2
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2 1 , MELOE-1 1 1 3 0 MlE-1- 4 .g.. and MELOE-1 2 2
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4 6 peptides, followed by CD4/IFN-y double staining for the detection of Th1 responses, and by CD4/IL4 double staining for Th2 responses. Between brackets is indicated the mean % of cytokine producing CD4 T cells, in positive microcultures. 5 M,EL-OE-1 22
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1 specific CD4 T cell clone (C2 D QI 1*0202) CDR3 beta chain Cytokine profile V beta chain VB2.1 TNF 1'1 CDR3 beta CSA SPDTHWGTDTQ YFG IFN high J beta chain JB 2.3 IL2 high GM-CSF high 1L4 high IL5w IL13 high IL10 neg
MELOE-I
1 especific CD4 I cell clonel A5 -DRB1 1101 V beta chain ND TNF high CDR3 beta ND IFN high J beta chain ND 1L2 11gh GM-CSF high 1L4 lo IL5 neg IL13 neg IL10 neg MELOE-1 1 1 30 specific CD4 T cell clone (5F9-DRF31*0101) V beta chain ND TNF ig CDR3 beta ND IFN high J beta chain ND IL2 GM-CSF high IL4 high IL5 neg IL13 high ___ ___ ___ __ ___ ___ ___ __ ___ ___ __ ILIO neg MELOE- 1 6 4 6 sp-ecific CD4 T cell clone (4E ___________DQP31 *020 1) V beta chain VB2.1 TNF high CDR3 beta CSA SGRRKFYEQ YFG IFN "9" J beta chain JB 2.7 1L2 high GM-CSF igh IL4 high IL5 ' IL13 high IL10 neg WO 2013/175256 PCT/IB2012/001310 - 26 Table IV: TCR characterization and cytokine profile of MELOE-1 specific CD4 T cell clones CD4 T cell clones were stimulated for 5 hours in the presence of brefeldin A (10 gg/mL) either with the cognate peptide (10 ptM) in an autopresentation assay. After 5 hours of stimulation, cells were stained with APC-conjugated anti-CD4 mAb, fixed with 4% paraformaldehyde, labeled with PE 5 conjugated anti-cytokine mAb and analyzed by flow cytometry. EXAMPLE: 10 Material & Methods Cells Blood samples from healthy subjects and melanoma patients were respectively obtained from Etablissement Franqais du Sang, Nantes, France and from the department of 15 onco-dermatology, Nantes Hospital, France. Melanoma and B-EBV cell-lines were maintained in RPMI 1640 (GIBCO) containing 10% fetal calf serum (FCS). Lymphocytes were grown in RPMI 1640 8% human serum (HS) with 50 or 75 IU/ml of recombinant interleukin-2 (IL-2, Chiron, France) and 2nM of L-Glutamin. For experiments using dendritic cells (DC), RPMI supplemented with 20mg/mL of human albumin (LFB 20 BIOMEDICAMENTS, France) was used to avoid peptide degradation by serum proteases. Reagents Antibodies were purchased from BD Biosciences-France or from Miltenyi Biotec, France. Purified cytokines were purchased from CellGenix, Germany. The different peptides 25 (Millegen, France, purity >85%) used in this study are described in Table I. HLA A*0201/MELOE-136_ 44 monomers were generated by the recombinant protein facility of our institute (SFR 26). Dendritic cells generation and loading 30 Monocytes were purified from PBMC of healthy donors by a CD14-enrichment kit, according the recommendations of the supplier (Stem Cell, France). Immature dendritic cells (iDC) were generated by culturing monocytes in RPMI supplemented with 20mg/mL of human albumin, 1000 lU/mL of GM-CSF and 200 lU/mL of IL-4 for 5 days. Then, iDC were pulsed with the whole MELOE-1 (1pM) protein or the modified Melan-A 16 4o A27L as WO 2013/175256 PCT/IB2012/001310 - 27 negative control (1 M) and matured with 20 ng/mL of TNF-a and 50 ptg/mL of PolyI:C for 4 hours at 37 0 C. Finally they were fixed for 1 minute with PBS/0.015% glutaraldehyde. Alternatively, iDC were first matured, fixed and then pulsed with antigens at the same concentration. 5 Stimulation of MELOE-1 specific T cells PBMC from healthy donors or melanoma patients (2.105 cells/well) were cultured for 14 days with 10 gM of MELOE-1 whole antigen (46 amino acids) in RPMI medium supplemented with 8% HS, 50 IU/ml of rIL-2 (Chiron, France) and L-Glutamin, in 96-well 10 multiplates. Microcultures were then restimulated individually with each overlapping peptide (MELOE-12-21, MELOE-111_0, MELOE-11837, MELOE-126_46 or MELOE-122_46 for melanoma patients) in the presence of 10 gg/mL brefeldin A for 5 hours and the percentage of CD4+ specific T cells was assessed by TNF-a, IFN-y or IL-4 intracellular staining. A negative control without peptide was included in all experiments. 15 Alternatively, MELOE-1 specific CD4+ T cell clones were stimulated by autologous MELOE- 1 loaded and matured DC at a 1:1 ratio. T cell cloning and TCR characterization Polyclonal cultures containing specific CD4+ T cells were cloned by limiting dilution 20 as previously described (Gervois N. et al., 2000). After 2 weeks, each clone was checked for peptide specificity by TNF production assay. For TCR sequencing, RNA from 5.106 T cell clones was extracted with RNable reagent (Eurobio, France) according to the supplier's instructions. Reverse transcriptions, PCR amplifications and sequencing were performed as described (Davodeau F. et al, 2001). We used the TCR nomenclature established by Arden et 25 al. (Arden et al., 1995). TNF production assay CD4+ T cell clones were cultured for 5 hours at 37 0 C in the presence of the recognized 20-mer peptide. Culture supernatants were harvested and TNF was measured in a biological 30 assay using cytotoxicity against WEHI 164 clone 13 (Espevik T. et al., 1986). Cytokine intracellular staining WO 2013/175256 PCT/IB2012/001310 - 28 Lymphocytes were stimulated for 5 hours in the presence of brefeldin A (10 jig/mL) either with peptide alone (10 pM) in an autopresentation assay or with B-EBV or HLA-class II expressing melanoma cells pulsed 2 hours with the cognate peptide, at a ratio 1: 2. In some experiments, blocking mAb against HLA-DP (clone B7.21 from Dr Charron, UMR940, 5 Paris), HLA-DQ (clone SPVL3, Beckman Coulter) or HLA-DR (clone L243, BD Biosciences) were added at a concentration of 12,5ptg/ml. Cells were then stained with APC conjugated anti-CD4 mAb, fixed with 4% paraformaldehyde, labeled with PE-conjugated anti-cytokine mAb and analyzed by flow cytometry. 10 Statistical analyses Statistical analyses were done with GraphPad Prism@ software. Bar graphs were used to compare frequencies of T cells specific for MELOE-1-derived peptides, in all donors and patients and were analyzed by a contingency table followed by a Fisher exact test. Scatter-dot graphs were made to compare the percentage of TNFa positive cells among positive 15 microcultures and were analyzed with a non-parametric test (Kruskal-Wallis followed by a Dunns post-test). RESULTS 20 Frequency and distribution of MELOE-1 specific CD4 responses in healthy donor's PBMC stimulated with MELOE-1 antigen Our purpose was to look for the existence of class II helper epitopes all along MELOE-1 sequence (SEQ ID NO: 20), in order to document the immunogenicity of the 25 different regions of this melanoma antigen. We stimulated 2. 107 PBMC from seven healthy donors with MELOE-1 whole antigen and tested, after a 14-day culture period the presence of CD4 T cells specific for each region of the protein. Microcultures were screened for TNFa production by CD4+ T cells, after restimulation with four MELOE-1 derived overlapping peptides (Table I), in an autopresentation assay. As shown in table II, all donors exhibited 30 CD4 responses against at least 1 out of 4 overlapping peptides. Responses against the N terminal region of MELOE-1 (2-21) were detected in 6/7 donors, with rather low frequencies (from 1 to 9% of positive microcultures containing between 0.6 to 5.6% of TNFU producing CD4 T cells), unless in HD28 healthy donor, who exhibited 62% of positive microcultures.
WO 2013/175256 PCT/IB2012/001310 - 29 The region 11-30 appears especially immunogenic, with CD4 specific responses detected in each tested donor (from 2 to 48% of positive microcultures containing between 0.7 to 24 % of TNFa producing CD4 T cells), and with very high frequencies in three donors (HD 17, HD24 and HD28). On the contrary, the central region 18-37, containing an already described DRI 1 5 restricted epitope (24-37) located just at the end of this 20-mer peptide (Rogel et al., 2011), induced specific responses in microcultures deriving from only 2/7 donors (HD9 and HD17, both expressing the DR 11 element). In these two donors, we detected 6 and 7% of positive microcultures, containing between 0.6 to 3.7 % of TNFa producing CD4 T cells. Finally, the C-terminal region (26-46), containing an already described DQ6-restricted epitope (Rogel et 10 al., 2011), was recognized by stimulated microcultures from 4 out of 7 donors (all do not expressing the DQ6 element), with frequencies ranging from 2 to 16% of microcultures containing between 0.5 and 16% of TNFa producing CD4 T cells. Overall, the frequency of MELOE-11130 positive microcultures was significantly higher than frequency of microcultures specific for the three other regions of MELOE-1 (Figure 1B) (p<0.0001). The 15 two terminal regions (2-21 and 26-46) were equivalent in terms of frequencies of positive microcultures, and these two regions induced significantly more responses than the central region 18-37 (Figure 1A). Nonetheless, the mean fractions of CD4 reactive T cells induced in positive microcultures were not significantly different from a region to another (Figure 1B). 20 Frequency, distribution and Th profile of MELOE-1 specific CD4 responses in melanoma patient's PBMC stimulated with MELOE-1 antigen In order to confirm the immunogenicity of each MELOE-1 regions in melanoma patients, we stimulated melanoma patients PBMC with the MELOE-1 whole protein, and tested the reactivity of stimulated lymphocytes towards the three most immunogenic regions: 25 2-21, 11-30, and 22-46. For this study, instead of challenging microcultures with the 18-37 peptide, that appeared poorly immunogenic, we extended the C-terminus region from 26-46 to 22-46, in order to also detect responses to our previously described HLA-DRll-restricted epitope (24-37). Indeed, the location of this epitope just at the end of the 18-37 peptide could be deleterious for the detection of specific responses in additional DR contexts, and we 30 previously showed that CD4 T cells specific for MELOE-1 2 4
-
3 6 epitope were efficiently induced by 22-46 peptide stimulation (Rogel et al., 2011). We tested the induction of CD4 specific responses from MELOE-1 stimulated PBMC of 10 melanoma patients. We documented CD4 responses specific for the central region of MELOE-1 (11-30) for 7/9 WO 2013/175256 PCT/IB2012/001310 - 30 patients, whereas responses specific for MELOE-1 2
-
2 1 and MELOE-1 2 2
_
4 6 were respectively detected in 4/9 and 5/9 patients (Table III). These responses were mainly Th1 responses (IFN g production) while less frequent Th2 responses specific for the three regions of MELOE-1 were detected in 3/9 patients for MELOE-1 2
-
21 , 2/9 patients for MELOE-1 11 30 and 5/9 5 patients for MELOE-1 2 2
_
4 6 (Table III). Considering the various regions of MELOE-1, Th1 responses specific for the N-term region of MELOE-1 (2-21) were significantly less frequent than those specific for the central region (p<0.0001) and the C-term region (p=0,0001), with respectively 3.3%, 10.8% and 9.6% of 576 tested microculutures (Figure 2A). Concerning Th2 responses, much less frequent, the C-term region appeared to induce more frequently the 10 growth of IL-4 producing CD4 T cells than the two other regions (Figure 2A). As observed for healthy donors, even if the frequencies were different, the mean fraction of reactive T cells (Th1 and Th2) induced in positive microcultures were not significantly different from a region to another (Figure 2B). In summary, stimulation of patient's PBMC with MELOE-1 induced Th1 responses specific for diverse epitopes located all along the protein sequence, 15 and among the different regions, the central region (11-30) and the C-term region (22-46) appeared to be especially immunogenic in term of frequency of responses. Production and characterization of CD4 T cell clones specific for the different regions of MELOE-1 20 In order to formally characterize the recognized epitopes, we derived CD4 T cell clones specific for each region of MELOE-1 by limiting dilution, from microcultures of healthy donors or melanoma patients, containing at least 0.5% of specific CD4 T cells. We succeeded to derive CD4 specific T cell clones from HD17, HD22, HD25 and Pt#3 microcultures, which were reactive against MELOE-1 2
-
21 (HD22), MELOE-1 1 1 30 (HD17 and 25 Pt3) and MELOE-1 26
_
4 6 (HD25). From each cloning experiment, we obtained between one and ten reactive CD4 T cell clones, that turned out to be the same clonotype after CDR38 sequencing (Table IV). A single CD4 T cell clone for each specificity was used for further experiments. The HLA-restriction was determined for each T cell clone, first by using HLA class II blocking monoclonal antibodies (Figure 3, upper panel), and further by testing the 30 recognition of HLA-matched B-EBV cell lines loaded with each recognized long peptide (Figure 3 middle panel). The two T cell clones named 9C12 and 4E2, derived from HD22 and HD25 and specific for MELOE-1 2
-
21 and MELOE-1 2 6
_
46 were restricted by the HLA DQB1*0202 and the DQB1*0201 molecules respectively. As, these two donors were homozygous for the HLA-DQ locus, a single HLA-DQ matched B-EBV cell line was tested WO 2013/175256 PCT/IB2012/001310 - 31 to confirm the HLA restriction of these two CD4 T cell clones. As shown on figure 3 (upper panel), the two other T cell clones 1A5 and 5F9 recognized the 11-30 region of MELOE-1, in a HLA-DR context. The use of HLA-matched B-EBV cell lines allowed to precise that 1A5 T cell clone was restricted by the HLA-DRB1 *1101 molecule and the 5F9 T cell clone by the 5 HLA-DR31*0101 molecule. We further tested the reactivity of these CD4 T cell clones against HLA-matched melanoma cell lines positive for meloe expression, by qPCR analysis. All the melanoma cell lines tested expressed HLA-DQ and DR at the cell surface. All the T cell clones were reactive against HLA-matched melanoma cell lines when loaded with the cognate peptide (Figure3, 10 lower panel, black bars). The two DQ2-restricted T cell clones were reactive against peptide loaded DQB1*0201 and 0202 melanoma cell lines. In absence of peptide, only the 4E2 DQ 31*0201-restricted T cell clone was able to recognize unloaded M77 melanoma cell line (also DQB1*0201), but not the DQB1*0202 melanoma cell line, M88. Similarly, the DRI-restricted T cell clone 5F9 also recognized one of the DR31*0101 melanoma cell line, in the absence of 15 exogenous peptide (M10 1). We also documented the T helper profile of each T cell clone, by stimulating the CD4 T cell clones with the cognate peptide, and analyzing cytokine production. All the clones expressed Th1 cytokines (TNFa, IFNy, IL2 and GM-CSF). On the contrary, these clones differ in their expression of Th2 cytokines. Indeed, the two DQ2 restricted T cell clones 20 (9C12 and 4E2) and the DRi restricted one (5F9) also strongly express two Th2 cytokines (IL4, and IL13), whereas the DR 1-restricted T cell clone only weakly expressed IL4 (Table IV). None of the CD4 T cell expresses IL10 or IL5 at a significant level. Processing of the recognized epitopes from autologous DC loaded with MELOE-1 25 antigen Initial PBMC stimulation was carried out with MELOE-1 whole antigen, and thus we assume that CD4 T cell responses were generated against peptides naturally processed by monocytes. Nonetheless, we could not formally exclude that the 14-day culture period artificially generated shorter class II epitopes that elicited CD4 T cell responses. Thus, it 30 remained crucial to assess that all these new epitopes were naturally processed by autologous dendritic cells loaded with MELOE-1 whole protein, in serum-free medium. To this end, we loaded autologous iDC with 1 gM of MELOE-1 antigen in serum-free medium, in presence of maturating agents, and fixed these DC before stimulation of the CD4 specific T cell clones. In these conditions, the four T cell clones were reactive against MELOE-1 loaded autologous WO 2013/175256 PCT/IB2012/001310 - 32 DC (Figure 4, left panel), whereas we could not detect any reactivity of CD4 T cell clones to DC loaded with an irrelevant long peptide, synthesized in the same conditions (Melan-A 16 40L). As an additional control, we loaded autologous DC with MELOE-1 after DC fixation, 5 and we could observe only a weak recognition by specific CD4 T cell clones, indicating that only a small fraction of the protein had been externally degraded into shorter peptides (Figure 4, right panel). Thus, the four new epitopes identified by PBMC stimulation, are naturally processed from MELOE-1 antigen. 10 Characterization of the minimal recognized epitopes Our T cell clones were reactive against 20-mer peptides that are probably not the exact peptides naturally processed. In order to formally identify the minimal recognized epitopes, we tested shorter peptides derived from each of the MELOE-1 recognized regions, chosen on the basis of the core peptide sequence supposed to be recognized by the T cell clones 15 (indicated in bold on figure 5). Three shorter peptides were better recognized by the DQ2-restricted 9C12 T cell clone, the shortest one being MELOE-1 7
_
1 9 (13-mer), recognized with an EC50 of 100 nM. The deletion of the two amino acids in C-term strongly reduces T cell clone recognition. The other DQ2-restricted T cell clone (4E2) better recognized a 14-mer peptide (31-44) also with 20 an EC50 of 100 nM, and also recognized to a lower extent the 32-44 epitope (Figure 5) previously described in the HLA-DQB1*0603 context (Rogel et al., 2011). Concerning the DR-restricted T cell clones, optimal shorter peptides were 13-mer peptides, MELOE- 1i5 27 for the DRI31*1101 restricted T cell clone 1A5 (EC50=100 nM), and MELOE-1 1 1-23 or MELOE 112-24 for the DRB1*0101 restricted one (Figure 5). Nonetheless all of these clones recognized 25 a series of shortened peptides, thus we cannot formally assess that the shortest ones will be the exact peptides naturally presented on class II molecules. 30 REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.
WO 2013/175256 PCT/IB2012/001310 - 33 Arden B, Clark SP, Kabelitz D, and Mak TW Human T-cell receptor variable gene segment families. Immunogenetics 1995; 42: 455-500. Bennett SR, Carbone FR, Karamalis F, Flavell RA, Miller JF, and Heath WR Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 1998; 393: 478-80. 5 Bijker MS, van den Eeden SJ, Franken KL, Melief CJ, Offringa R, and van der Burg SH CD8+ CTL priming by exact peptide epitopes in incomplete Freund's adjuvant induces a vanishing CTL response, whereas long peptides induce sustained CTL reactivity. J Immunol 2007; 179: 5033-40. Chauvin JM, Larrieu P, Sarrabayrouse G, Prevost-Blondel A, Lengagne R, 10 Desfrancois J, et al. HLA Anchor Optimization of the Melan-A-HLA-A2 Epitope within a Long Peptide Is Required for Efficient Cross-Priming of Human Tumor-Reactive T Cells. J Immunol 2012; 188: 2102-10. Davodeau F, Difilippantonio M, Roldan E, Malissen M, Casanova JL, Couedel C, et al. The tight interallelic positional coincidence that distinguishes T-cell receptor Jalpha usage 15 does not result from homologous chromosomal pairing during ValphaJalpha rearrangement. EMBO J 2001; 20: 4717-29. Espevik T and Nissen-Meyer J A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes. J Immunol Methods 1986; 95: 99-105. 20 Gervois N, Labarriere N, Le Guiner S, Pandolfino MC, Fonteneau JF, Guilloux Y, et al. High avidity melanoma-reactive cytotoxic T lymphocytes are efficiently induced from peripheral blood lymphocytes on stimulation by peptide-pulsed melanoma cells. Clin Cancer Res 2000; 6: 1459-67. Godet Y, Desfrancois J, Vignard V, Schadendorf D, Khammari A, Dreno B, et al. 25 Frequent occurrence of high affinity T cells against MELOE-1 makes this antigen an attractive target for melanoma immunotherapy. Eur J Immunol 2010; 40: 1786-94. Godet Y, Moreau-Aubry A, Guilloux Y, Vignard V, Khammari A, Dreno B, et al. MELOE-1 is a new antigen overexpressed in melanomas and involved in adoptive T cell transfer efficiency. J Exp Med 2008; 205: 2673-82. 30 Hunder NN, Wallen H, Cao J, Hendricks DW, Reilly JZ, Rodmyre R, et al. Treatment of metastatic melanoma with autologous CD4+ T cells against NY-ESO-1. N Engl J Med 2008; 358: 2698-703. Fayolle C, Deriaud E, and Leclerc C In vivo induction of cytotoxic T cell response by a free synthetic peptide requires CD4+ T cell help. J Immunol 1991; 147: 4069-73.
WO 2013/175256 PCT/IB2012/001310 - 34 Friedman KM, Prieto PA, Devillier LE, Gross CA, Yang JC, Wunderlich JR, et al. Tumor-specific CD4+ Melanoma Tumor-infiltrating Lymphocytes. J Immunother 2012. Kenter GG, Welters MJ, Valentijn AR, Lowik MJ, Berends-van der Meer DM, Vloon AP, et al. Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N 5 Engl J Med 2009; 361: 1838-47. Robbins PF, El-Gamil M, Li YF, Zeng G, Dudley M, and Rosenberg SA Multiple HLA class II-restricted melanocyte differentiation antigens are recognized by tumor infiltrating lymphocytes from a patient with melanoma. J Immunol 2002; 169: 6036-47. Rogel A, Vignard V, Bobinet M, Labarriere N, and Lang F A long peptide from 10 MELOE-1 contains multiple HLA class II T cell epitopes in addition to the HLA-A*0201 epitope: an attractive candidate for melanoma vaccination. Cancer Immunol Immunother 2011; 60: 327-37. Rosenberg SA, Yang JC, and Restifo NP Cancer immunotherapy: moving beyond current vaccines. Nat Med 2004; 10: 909-15. 15 Schwartzentruber DJ, Lawson DH, Richards JM, Conry RM, Miller DM, Treisman J, et al. gplOO peptide vaccine and interleukin-2 in patients with advanced melanoma. N Engl J Med 2011; 364: 2119-27. Smith CM, Wilson NS, Waithman J, Villadangos JA, Carbone FR, Heath WR, et al. Cognate CD4(+) T cell licensing of dendritic cells in CD8(+) T cell immunity. Nat Immunol 20 2004; 5: 1143-8. Speetjens FM, Kuppen PJ, Welters MJ, Essahsah F, Voet van den Brink AM, Lantrua MG, et al. Induction of p53-specific immunity by a p53 synthetic long peptide vaccine in patients treated for metastatic colorectal cancer. Clin Cancer Res 2009; 15: 1086-95. Toes RE, Offringa R, Blom RJ, Melief CJ, and Kast WM Peptide vaccination can lead 25 to enhanced tumor growth through specific T-cell tolerance induction. Proc Natl Acad Sci U S A 1996; 93: 7855-60. Vesely MD, Kershaw MH, Schreiber RD, and Smyth MJ Natural innate and adaptive immunity to cancer. Annu Rev Immunol 2011; 29: 235-71. Welters MJ, Kenter GG, Piersma SJ, Vloon AP, Lowik MJ, Berends-van der Meer 30 DM, et al. Induction of tumor-specific CD4+ and CD8+ T-cell immunity in cervical cancer patients by a human papillomavirus type 16 E6 and E7 long peptides vaccine. Clin Cancer Res 2008; 14: 178-87.

权利要求:
Claims (14)
[1] 1. A melanoma antigen peptide comprising the amino acids sequence selected in the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or 5 SEQ ID NO:5 or a function-conservative variant thereof
[2] 2. A fusion protein comprising a melanoma antigen peptide according to claim 1 and a melanoma antigen peptide comprising the amino acids motif: - TX 2 NDECWPX 9 (SEQ ID NO: 23) wherein X 2 is leucine, methionine, valine, isoleucine or glutamine and X 9 is alanine, 10 valine or leucine.
[3] 3. A nucleic acid sequence encoding a melanoma antigen peptide according to claim 1 or a fusion protein according to claim 2.
[4] 4. An expression vector comprising a nucleic acid sequence according to claim 3.
[5] 5. A host cell comprising an expression vector according to claim 4. 15
[6] 6. An antibody or fragment thereof that binds to a melanoma antigen peptide according to claim 1.
[7] 7. A melanoma antigen peptide according to claim 1 or a fusion protein according to claim 2 or a nucleic acid sequence according to claim 3 or an expression vector according to claim 4 or a host cell according to claim 5 for use in the prevention or the 20 treatment of melanoma in patient.
[8] 8. A melanoma antigen peptide or a fusion protein according to claim 7 wherein the patient is genotyped with HLA-DQ31*0201 or HLA-DQ 1*0202 alleles.
[9] 9. An immunising composition comprising: (a) at least one melanoma antigen peptide according claim 1 or 25 (b) at least one fusion protein according to claim 2, or (c) at least one nucleic acid sequence according to claim 3 or WO 2013/175256 PCT/IB2012/001310 - 36 (d) at least one expression vector according to claim 4, or (e) at least one host cell according to claim 5, or (f) at least one antibody according to claim 6.
[10] 10. The immunising composition according to claim 9 for use in the prevention or 5 treatment of a melanoma in a patient in need thereof
[11] 11. A T lymphocyte that recognizes specifically a melanoma antigen peptide according to claim 1 or a fusion protein according to claim 2.
[12] 12. A composition for adoptive therapy comprising T lymphocytes according to claim 11.
[13] 13. The composition for adoptive therapy according to claim 12 for preventing or treating 10 melanoma in a patient in need thereof, wherein said T lymphocytes that recognize specifically a melanoma antigen peptide or a fusion protein are to be re-administrated to the patient.
[14] 14. A method for producing said T lymphocytes according to claim 11, said method comprising the steps of: 15 (a) stimulating PBMCs or tumor infiltrating lymphocytes obtained from a patient with at least one melanoma antigen peptide according to claim 1 or a fusion protein according to claim 2, (b) enriching the population of T lymphocytes specific for the melanoma antigen peptide(s) used in (a), 20 (c) optionally cloning said population of T lymphocytes specific for the melanoma antigen peptide(s) used in (a).

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AU687733B2|1992-04-01|1998-03-05|Rockefeller University, The|Method for in vitro proliferation of dendritic cell precursors and their use to produce immunogens|

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WO2013175258A1|2012-05-22|2013-11-28|Inserm |Novel melanoma antigen peptide and uses thereof|WO2022008634A1|2020-07-10|2022-01-13|Humanitas Mirasole S.P.A.|Tumor-associated peptides and uses thereof|

法律状态:
2015-02-12| DA3| Amendments made section 104|Free format text: THE NATURE OF THE AMENDMENT IS: AMEND THE NAME OF THE INVENTOR TO READ LABARRIERE, NATHALIE; LANG, FRANCOIS; BOBINET, MATHILDE AND ROGEL, ANNE |

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2017-11-30| MK5| Application lapsed section 142(2)(e) - patent request and compl. specification not accepted|

优先权:

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申请号 | 申请日 | 专利标题

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PCT/IB2012/001310|WO2013175256A1|2012-05-22|2012-05-22|Novel melanoma antigen peptide and uses thereof|

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