EP1179019A2 - Immunogenic peptides derived from mage and the use thereof - Google Patents

Abstract

Peptides derived from proteins of the MAGE family and the use thereof as immunogenic agents in the treatment of tumors, the compositions containing them, a method for inducing a cytotoxic response against tumor cells, a melanoma cell line not expressing histocompatibility antigens and the applications thereof.

Description

IM U OGENIC PEPTIDES AND THE USE THEREOF

The present invention relates to peptides derived from proteins of the MAGE family and to the use thereof as lmmunogenic agents m the prevention and treatment of tumors .

⁵ Over the last few years, the use of tumor-specifIC cytotoxic T Lymphocytes (CTLs) has allowed the isolation of several genes encoding tumor antigens (Van den Eynde, B.J. et al . , Curr. Opm. Immunol., 5:684-693, 1997). According to the pattern of expression m neoplastic and normal tissues,

^■lO these antigens can be classified into four classes, depending on the tumor specificity and clinical relevance. The first class comprises antigens encoded by genes expressed in various tumors of different histotypes but not m normal tissues other than testicular germ cells, such as

¹⁵ MAGE (Melanoma Associated Antigen) , GAGE and BAGE . The second class represents differentiation antigens that are only expressed in melanoma and melanocytes, such as Tyrosmase, MeIan-A/MART-1 , gplOO, TRP-1 and TRP-2. The antigens belonging to the third class are generated by point

20 mutations m genes that are ubiquitously expressed. The fourth class of antigens, which has been defined only recently, is represented by TRP-2 -IΝT2, an antigen shared between melanomas but not expressed in normal cells of the melanocytic lineage.

25 Especially tumor-antigens belonging to the MAGE family have elicited considerable interest, because six of them, MAGE-1, 2, 3, 4, 6 and 12 are expressed selectively by a significant proportion of primary and metastatic tumors including melanomas, lung, bladder, ovarian and breast 0 carcinomas (van der Bruggen, P., et al . , Science, 254:1643- 1647, 1991) .

Nine ammo acid peptides derived from mtracellular processing of MAGE-1 (van der Bruggen et al . , Science

254:1643-1647, 1991) and MAGE-3 proteins, of length from 8 to 10 ammo acids, have been disclosed and suggested as lmmunogenic agents (WO 95/19783). Clinical trials of vaccination, based on the use of antigenic peptides encoded by MAGE-1 and 3 are progress patients affected by melanoma and other neoplastic diseases (Marchand, M. et al . Int. J. Cancer 80 : 219-230, 1999) . To date, MAGE-2, 4 and 6 antigens cannot actually be considered tumor antigens and therefore they are not used as targets for tumor—specific immunotherapy . The reason is that cytotoxic T lymphocytes capable of recognizing a peptide derived from MAGE-2, 4, 6 have not yet been identified.

Limitations to the therapeutic approach based on the use of peptides are limited number of characterized CTL epitopes (i.e. tumor-antigen-peptides and appropriate HLA- class I alleles) and the m vivo generation of antigen-loss variants, able to escape the immune response elicited by a mono-antigenic vaccine (Restifo, N.P., et al . , J. Natl. Cancer. Inst . , 88 : 100-108, 1996). In particular, the development of antigen- loss variants, which is a major cause of failure m immunization protocols, has been found to be due to the selection of tumor cells which fail to express the antigen recognized by tumor-reactive lymphocytes, for example as a consequence of molecular defects resulting m loss or downregulation of HLA class I alleles (Garrido, F., at al . , Immunol. Today, 14 : 491-499, 1993). However, certain circumstances the tumor loses or modify the genes coding for the tumor antigen (Jager, E. et al . Int. J. Cancer, 71: 142-147, 1997) using still a further mechanism to escape immune control .

Usually clinical protocols of tumor-specific vaccination apply only to patients carrying a tumor expressing a well-known tumor antigen and a defined HLA allele. Unfortunately, the large majority of cancer patients do not fulfil these including criteria. Therefore, there is need for identification of new antigenic determinants for use in anti -tumor vaccination protocols, preferably "wide spectrum" ones, namely expressed by a higher number of tumor histotypes, or expressed m multiple form by the same tumor.

It has now been found, and this is a first aspect of the invention, a group of peptides encoded by homologous regions of MAGE 1, 2, 3, 4, 6 and 12 genes, identifying a new HLA-B*3701-restrιcted epitope.

Depending on their origin, the peptides of the invention have been named MAGE-1, 2, 3, 4, 6, ¹²127 -136 ^an^ have the sequences reported SEQ ID No. 1-3. MAGE-1, 2, 3, 6 have the same sequence identified by SEQ ID No 1, whereas MAGE-4 and 12 have the sequences identified by SEQ ID No 2 and 3, respectively.

Cytotoxicity in vi tro assays have proved that cytotoxic T cells recognize the peptides of the invention bound to the HLA-B*3701 histocompatibility allele on various cell lines expressing the different antigens MAGE-1, 2, 3, 4, 6, 12, and are activated upon exposure to them. In these assays, the peptide having sequence SEQ ID No. 1 showed the highest activity and is therefore preferred.

Identification of an epitope common to various antigens of the MAGE family is extremely advantageous m that it allows to at least partly overcome the drawbacks mentioned above. More importantly, the use of such "pluπantigenic" epitope, i.e. shared by more antigens of the MAGE family, allows to overcome the above mentioned loss of antigenicity, as often a given tumor co-expresses more than one MAGE gene, making therefore loss of the immune response induced by the peptides of the invention very unlikely. This allows to increase the number of patients eligible for successful clinical cancer vaccination trials. It should be stressed that to date the immunogenic potential of MAGE-2 and MAGE-6 in humans has never been demonstrated, even though 12% of the ovarian carcinomas were known to express MAGE-2 and/or MAGE-6 in the absence of MAGE-1 and MAGE-3.

The peptides of the invention can be prepared with conventional techniques, preferably synthetically, for example according to the procedure described in Merrifield,

(1986) Science 232:341-347, and Barany and Merrifield, The Peptides, Gross and Meienhofer, eds (N.Y., Academic Press), pp. 1-284 (1979) . The synthesis can be carried out in solution or in solid phase or with an automatized synthesizer (Stewart and Young, Solid Phase Peptide Synthesis, 2 ed., Rockford 111., Pierce Chemical Co. (1984). Alternatively, the peptides can be prepared with recombinant DNA technology or still starting from natural protein precursors containing the desired fragments . The amino acidic residues can be chemically modified, for example by bonding to lipids, glycosylation, conjugations with other peptides, to obtain more favourable characteristics, such as higher affinity for the HLA molecule, higher immunogenicity, higher selectivity of induction of the immune response or higher bioavailability after administration. The peptides can also be conjugated with other epitopes known to induce a T "helper" cellular response through the same or a different class of MHC molecules .

The invention further relates to pharmaceutical compositions containing an effective amount of a peptide herein described. According to a preferred embodiment, said compositions are vaccines, particularly suited for preventive vaccination of patients with predisposition to cancer or in therapeutical vaccination of neoplastic patients. In addition to the active ingredients, the compositions will contain pharmaceutically acceptable excipients. "Effective amount" means an amount sufficient to elicit an effective CTL response against the tumor. Such amount will depend on the peptide used, on the route and type of administration, on the severity of the pathology to be treated and on the general conditions of the patient and will usually range from 100 to 300 μg in 3 or more administrations at 1 month intervals or at closer times. Two subcutaneous inoculations and two intradermal ones are usually effected, at doses corresponding to 4/10 the total dose for each subcutaneous inoculation, and 1/10 at each of the intradermal sites. Inoculations sites are changed at each vaccination, if possible. The techniques for the preparation and use of the vaccines are known to those skilled in the art and are described, for example, in Paul, Fundamental Immunology, Raven Press, New York (1989) or Cryz, S. J., I munotherapy and Vaccines , VCH

Verlagsgesselschaft (1991) . Vaccines are usually prepared in the form of injectable suspensions or solutions, but they can also be used in the form of solid or liposome preparations. The immunogenic ingredients can be mixed with pharmacologically acceptable excipients, such as emulsifiers, buffering agents, and adjuvants enhancing the efficacy of the vaccine. The vaccine can be administered according to a single or multiple dosage scheme. In case of multiple dosage, 1 to 10 separated doses are provided, each containing an antigen amount ranging from 1 μg to 1000 μg, followed by other booster doses at subsequent time intervals, necessary to maintain or strenghten the immune response and, if necessary, a further dose after several months. In each case, the treatment regimen will depend on the response of the treated patient, on its general conditions, and on the progress of the tumor.

In a further aspect, the invention provides a method for inducing a cytotoxic response against tumor cells expressing one or more MAGE antigens, which comprises contacting T lymphocytes with the peptides of the invention in conditions suitable for the activation of cytotoxic T cells. Suitable conditions for obtaining the desired cytotoxic effect comprise direct exposure of T lymphocytes to the peptides in culture or the previous binding of the peptides with the HLA class I molecules, preferably HLA-

B*3701, or the preventive binding of the peptides with antigen presenting cells (APC) expressing said HLA molecules, followed by exposure to T lymphocytes. Suitable

APC cells are peripheral blood autologous mononuclear cells

(PBMC) , or dendritic cells, macrophages and activated B cells. An APC culture is added with the peptide (s) for a time sufficient to bind peptide/APC, and subsequently with a cell population containing T lymphocytes which are thus activated and proliferate. Lymphocytes can be taken from the treated patient and, after activation, reinjected in the same patient. The peptide/APC binding can be increased through preventive "stripping" of the histocompatibility molecules present on APC. Optionally, APC cells can be engineerized to express the HLA-B*3701 histocompatibility allele. Furthermore, the culture medium can contain one or more cytokines contributing to increase the activation of CD8⁺ precursors. Prior to their reinjection into the patient, lymphocytes can be purified for example through an affinity column with a specific ligand.

The invention also relates to a cytotoxic T cell line specifically recognizing a complex consisting of an HLA class I molecule, preferably the HLA-B*3701 molecule, and a peptide selected from the group SEQ ID 1-6. The cytotoxic T line can be obtained by selection, starting from a lymphocyte pool, of cells capable of activating upon exposure to tumor cells containing antigemc determinants herein described.

According to a further aspect, the invention provides cells inducing the immune response, such as APC, dendritic cells etc., engineerized with vectors encoding the peptides of the invention (for example viral or retroviral vectors, such as those derived from adenoviruses or lentiviruses or

MLV) , optionally in the form of fusion proteins with a suitable carrier, to be efficiently expressed in the cell, and then processed and exposed on the cell surface. In this case, the DNA coding for the epitopes disclosed in the present invention, will be inserted, under control of a suitable promoter, for example a viral promoter such as CMV, SV40, if a very high expression level is required, or an inducible promoter such as that controlled by ecdisone, in the suitable expression vector. In this case, the described epitopes are coded by the MAGE-1 cDNA region (GenBank, N. M77481) corresponding to 127-136 amino acids and by the homologous regions for the other antigens of the MAGE family: Aa Amino acid sequence MAGE-1 Nt (cDNA) 127-136 REPVTKAEML 562-590

According to a further aspect, the invention relates to a melanoma cell line, named MSR3-mel and deposited at the CBA — Interlab Cell line Collection — (Genua, Italy) under the N. PD99001, expressing undetectable levels of histocompatibility antigens. As described in further detail in example 1, this cell line can be transfected with cDNA encoding any allele of the histocompatibility system and subsequently transfected or transduced with cDNA encoding for the tumor antigens which it deos not express, or for fragments thereof, or still for viral antigens, and then be used again for inducing in vi tro antigen-specific effectors, or reinjected into the patient according to applications of active immunotherapy in vivo . In a preferred embodiment, the tumor antigens are melanoma antigens and the histocompatibility molecule is HLA class I B*3701. The MSR3- mel line can conveniently be used for the ex-vivo induction or expansion of antigen- specific cytotoxic T lymphocytes for use adoptive immunotherapy protocols and/or for the identification of novel antigenic determinants. Furthermore, the MSR3-mel line genetically modified with genes encoding

HLA molecules and/or tumor antigens can be used as a vaccine . According to a further aspect, the invention relates to antibodies, their fragments or derivatives, directed to the above described peptides. The general methodology for producing antibodies is well known and described for example Kohler and Milstem, Nature 256 (1975) , 494 or J.G.R. Hurrel, Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press Inc., Boco Raron, FL (1982). The antibodies comprise monoclonal (mAb) or polyclonal, preferably monoclonal, and their fragments can be F(ab')2, Fab, Fv or scFv. Figure Legends

Figure 1: Expression of HLA-class I molecules by MSR3 - mel and MSR3-B37. Tumor cells were incubated with mAb W6/32

(anti-HLA-class I) or with an lsotype control, washed, and labeled with goat ant I -mouse Ig antibodies coupled to fluorescem. The analysis was performed before and after HLA-B*3701 transfection of MSR3-mel.

Figure 2: Recognition of a HLA-B*3701-restπcted antigen by CTLs 337. The cytotoxic activity of CTLs 337 was assessed against the autologous melanomas MSR3*mel and MSR3- B37, and against the allogenic melanoma ETl at various E/T ratio .

Figure 3 : Identification of the tumor-antigens recognized by CTLs 337. Cos-7 cells were co-transfected with HLA-B*3701 alone or together with cDNAs encoding gene MAGE- 1, 2, 3, 6 and 12. After 48 h. CTLs 337 were added and the y-IFN released was measured 24 h later as described

Materials and Methods. MSR3-B37 was included as positive control . Figure 4: A) Recognition by CTLs 337 of the peptide

MAGE._{127 136}. MSR3-EBV were incubated with three- fold dilutions of peptide MAGE.₁₂₇ 136' starting from 10 mM and used as target cells m a standard cytotoxicity assay. E/T ratio was fixed at 10:1. The amount of peptide needed for half -maximal lysis is indicated as ED^_Q. B) Binding of peptides 4._{127 136} and M12. l_]_27-i36 ^o HLA-B*3701 evaluated m a competition assay. Competitor peptides included the

^M4 • 127-136 P^eP^{tl e} KELVTKAEML and the ^M12. l₁₂₇__13β peptide

REPFTKAEML. The M3.A1 (i.e. M3.₂₇₁_₂₇₉) peptide unable to bind to the HLA-B*3701 molecule was used as negative control. Lysis without competitor peptides was 52%.

Figure 5: Recognition by CTLs 337 of a MAGE-6 positive melanoma cell line. The HLAB*3701 negative line Me 14932 and the HLA-B*3701 positive line Me 14932-LB37 were pulsed or not with 16 μM of peptide MAGE- 127-136 ^an<^ ^{used as} target cells a standard cytotoxicity assay at the indicated E/T ratios .

Materials and Methods Synthetic Peptides Synthetic peptide were purchased from PRIMM (Milan, Italy). Peptides were ^MAGE-i27-i36 (REPVTKAEML) , encoded by codons 127-136 of MAGE-1, 2, 3 and 6 genes, ^M4-127-136 (KELVTKAEML) and M12.₁₂₇_₁₃₆ (REPFTKAEML), corresponding to ammo acids 127-136 encoded by genes MAGE-4 and MAGE- 12, respectively. Peptides were dissolved to lOmM m DMSO and diluted further m 0,9% NaCI .

Subcloning of HLA-B*3701 Allele.

Total RNA was prepared from MSR3 PBLs by the RNeasy Total RNA Kit (QIAGEN, H lden, Germany) . Single-stranded cDNA synthesis was carried out on 2 μg of total RNA using oligo-dT primer and Moloney murme leukemia virus-derived reverse transcriptase without RNase-H activity (MMLVRT

RNase-H- Superscript; GIBCO BRL, Gaithersburg, MD) according to manufacturer's instructions. cDNA corresponding to 300 ng of total RNA was amplified by PCR using a primer pair suitable for specific amplification and directional cloning of the full length coding region of HLA-B alleles. The 1.1 kb PCR-product was subcloned mto the eukaryotic expression vector pcDNA3.1 (Invitrogen Corporation, Oxon, U.K.). Plasmid clones encoding the HLA-B*3701 or the B*52011 (the HLA-B37 and B5 alleles of patient MSR3 ) were identified using diagnostic restriction enzymes. The HLA-B*3701 gene was then sequenced to verify the correspondence to the published DNA sequence. This plasmid was called pcDNA3.1/B*3701.

Transfection of Melanoma Cell Lines.

Melanoma cell lines were transfected by the calcium phosphate precipitation technique with pcDNA3. l/HLA-B*3701 , and selected in G418. Expression of the transfected HLA- B*3701 molecule stable transfectants was verified by flow cytometry with the HLA-A, B and C specific monoclonal antibody W6/32.

In vitro Induction of Cytotoxic T Lymphocytes line 337 (CTLs 337) .

CTL line 337 was obtained using a protocol previously described by others (Van den Eynde, et al . , Int. J. Cancer., 44 : 634-640, 1989) with slight modifications. Briefly, PBLs from patient MSR3 were separated by Ficoll gradient and cultivated (1 to 2x10 /well) with the autologous, irradiated

MSR3-B37 melanoma cells (0.5 to lxl0⁵/well) m 2 ml of IMDM supplemented with 10% human serum (HS) , glutamme and antibiotics. After three days of culture, 10 U/ml of IL-2

(Chiron, Milan, Italy) and 5 ng/ml of IL-7 (Genzyme Corp.) were added. Lymphocytes were restimulated weekly with

0.5xl0⁵ irradiated MSR3-B37 cells and tested a cytotoxicity assay after three stimulations. After the fifth re-stimulation, 2xl0⁶ LG2-EBV irradiated LG2-EBV were added as feeder cells and IL-2 was increased to 50 U/ml .

Assay for Cytolytic activity and Peptide Binding Studies .

Lytic activity of the cytotoxic T cell lines was tested m a chromium release assay as previously described (Fleischhauer, K. , et al . , Cancer Res., 58 : 2969-2972,

1998). Peptides were tested chromium release assays:

51 Cr-labeled target cells were incubated for 1 h at room temperature m 96-wells microplates with various concentration of the peptide before addition of effector cells at a fixed effector/target ratio. Binding of peptide

^M4 • 127-136 ^{and M1} - 127-136 ^to HLA-B*3701 molecule was studied a competition assay, as previously described

(Herman, J., et al . , Immunogenetics , 43 : 377-384, 1996). As standard peptide, we used peptide ^MAGE.₁₂₇_₁₃g (300 nM) recognized by CTLs 337. CTLs were used at E/T ratio of 30:1.

Production of Subfragments of MAGE-1.

Subfragments of MAGE-1 gene (495 and 1,072 bp) were obtained by digestion of MAGE-1 cDNA with Bglll and EcoRI . After purification on agarose gel, the fragments were cloned mto the pcDNA3.1 plasmid (Invitrogen) . Clones were isolated, plasmid DNA was extracted and transfected mto Cos-7 cells along with the HLA-B*3701 gene.

Transfection of Cos-7 Cells and'V-IFN Release Assay.

Transfection of Cos-7 cells was performed by the DEAE- dextran-chloroqu e method (Coulie, P.G., et al . , Proc . Natl . Acad. Sci . , 91 : 6458-6463, 1994) . Briefly, l,5xl0⁴ Cos-7 cells were transfected with 100 ng of plasmid pcDNA3. l/B*3701 and 100 ng of expression vectors containing the cDNA of one of the following genes: MAGE-1, 2, 3, 4, 6, and 12. Transfected Cos-7 cells were tested in a y-IFN assay after 48 hs : 5000 responder CTLs, at day 5 after stimulation, were added in 150 μl IMDM/ 10% HS supplemented with 25 U/ml IL-2. After 24 hs at 37°C, 100 μl of supernatant were harvested and the "y-IFN concentration was measured using a-V-IFN release kit (Genzyme, MA) according to the manufacturer's recommendations.

Retroviral vector mediated gene transfer of HLA-B*3701 into Me 14932. The retroviral vector B37-CSM, coding for the HLA- B*3701 molecule of patient MSR3 was constructed as previously described (Fleischhauer, K. , et al . , J. Immunol., 259:2513-2521, 1997) . Briefly, the full-length cDNAs coding for the HLA-B*3701 molecule was cloned under the control of the viral LTR, while the truncated form of the human low affinity nerve growth factor receptor (ALNGFR) was driven by the Sv40 promoter. The ecotropic murine fibroblast cell line GP+E86 was transiently transfected with 30 μg of retroviral construct by standard calcium-phosphate method. Infection of the amphotropic murine packaging cell line GP+env Am 12, by supernatant of 48 h cultures of transfected GP+E86 cells, was performed for 4 h in the presence of 8 μg/ml polybrene . Infected packaging cells were immunoselected for ΔLNGFR expression by magnetic beads (Dynabeads M-450, Dynal A.S., Oslo, Norway) coated with the LNGFR- specific monoclonal antibody 20.4 (American Type Culture Collection, Rockville, MD) . Transduction of Me 14932 was performed by cultivation with retrovirus-containing supernatant in the presence of polybrene (8 μg/ml) . Five or six rounds of infection of at least 4 h were performed. Efficiency of infection was evaluated by immunofluorescence analysis with the LNGFRspecific monoclonal antibody 20.4 and with a HLA-Bw4 -specific monoclonal antibody. RT-PCR analysis.

MAGE-1, 2, 3, 4, 6, 12 and S₂ -microglobulin (S₂m) cDNAs were detected by PCR amplification. Reaction mixture contained 5 μl of cDNA suspension, 4 μl of a a 10 mM dNTPs mixture (containing each dNTP at 2.5 mM) , 5 μl of lOx DNA polymerase Buffer (Finnzy es Oy, Espoo, Finland) , 2 U of

DynaZyme DNA polymerase (Finnzymes Oy) and sterile distilled water up to 50 μl of total reaction volume. For oligonucleotide primer sequences and PCR amplification programs see Weynants et al . (Weynants, P., et al . , Int. J.

Cancer, 56 : 826-829, 1994) (MAGE-1, 2 and 3), De Plaen et al . (De Plaen, E., et al . , J. Immunol., 259: 2513-2521,

1997) (MAGE-4, 6 and 12). β2m cDNA was amplified using the sense primer Beta 5' (5'-AAC CAC GTG ACT TTG TCA CAG C-3'), and anti-sense primer Beta 3' (5'-CTG CTC AGA TAC ATC AAA CAT

G-3 ' ) ; PCR amplification was performed for 30 cycles (1 min at 94°C, 30 sec at 56°C and 2 min at 72°C) ; expected length of S2m amplification product is 230 b . RNA integrity was tested by PCR reaction with β-Actin specific oligonucleotide primers (De Smet, C, et al . , Immunogenetics , 39 : 121-129,

1994) . Samples scored positive when a band of the appropriate size was visible on an agarose gel in the presence of ethidium bromide .

The invention will be illustrated in further detail by the following examples.

EXAMPLES

Exa-m.ple 1

MSR3-B37 Induces an Antigen-Specific Immune Response.

The melanoma line MSR3 was established from a cutaneous metastasis resected from patient MSR3. Expression of the HLA class I alleles by the tumor cells, was evaluated by immunofluorescence analysis with the HLA-A, B and C specific mAb W6/32.

The undetectable or barely detectable level of surface molecules observed (Fig. 1) appeared to be inadequate to allow antigen presentation to immune effectors. Indeed, the

MSR3 line failed to induce a cytotoxic response from autologous PBLs . The lack of class I cell surface expression by MSR3-mel was not caused by impaired β2m synthesis, since a β2m-specific mRNA could be detected by RT-PCR analysis.

To determine whether HLA class I antigen expression could he restored, MSR3-mel cells were stably transfected with cDNA encoding the autologous HLA-B*3701 molecule. After G418 selection flow cytometric analysis showed staining of the transfected MSR3-B37 cell line by the W6/32 mAb (Fig. 1) .

To evaluate the presence on the surface of MSR3-B37 line of tumor-specific antigens, the melanoma cells were tested for their ability to induce tumor-specific cytotoxic effectors and for their susceptibility to lysis by these CTLs. Patient's PBLs were in vi tro stimulated by MSR3-B37 as described in Material and Methods . After three rounds of stimulation, the polyclonal cytotoxic T cell line 337 (CTLs 337) specifically lysed MSR3B37 cell line, but not the untransfected MSR3-mel (Fig. 2) . Autologous MSR3-EBV cells and PHA-activated T blasts were not recognized suggesting that the epitopes recognized by these CTLs are melanoma/melanocyte specific. Indeed, in addition to the autologous melanoma cells, CTLs 337 also lysed the HLA- B*3701 positive melanoma line ETl (Fig.2), suggesting that one or more shared melanoma antigens are recognized.

These data indicate that HLA class I expression can be restored by transfection of MSR3 melanoma cells and that the melanoma line transfected with the HLA-B*3701 molecule is able to induce a tumor-specific cytotoxic T cell response. Example 2

Identification of the Antigenic Epitope Recognized by CTLs 337. In order to identify the antigen recognized by CTLs

337, the y-IFN release of CTLs 337 m the presence of Cos-7 cells transfected with plasmid pcDNA3. l/B*3701 was evaluated, along with cDNA encoding six members of the MAGE family (i.e. MAGE-1, 2, 3, 4, 6 and 12), some of which are expressed by both MSR3-mel and ETl. CTLs 337 specifically recognized Cos-7 cells transfected with MAGE-1, 2, 3, and 6, suggesting that the epitope target of CTLs 337 was shared among the four different antigens or that distinct components of the oligoclonal T cell line were recognizing peptides derived from the four MAGE-gene products. A low level of y-IFN was detected m the presence of MAGE-4 and MAGE-12 transfected Cos-7 cells (Fig.3).

To identify the sequence coding for the antigenic peptides recognized by CTLs 337, cDNA encoding MAGE-1 was digested with Bglll and EcoRI obtaining two subfragments of approximately 495 and 1,072 bp . These fragments were cloned mto plasmid pcDNA 3.1 and transfected mto Cos-7 cells along with HLA-B*3701 molecule. The presence of an m frame start codon at 202bp and 707bp m the 495 and 1,072 bp fragments respectively, assured the expression of the two subfragments m the transfected cells. The level of y-IFN released by CTLs 337 m the presence of Cos-7 cells transfected with the 495 bp fragment was comparable to that conferred by the entire MAGE-1 gene, indicating that the antigenic peptide was encoded withm this region. The ammo acid sequence encoded by the 495 bp fragment was screened for peptides carrying the binding motif for HLA-B*3701

(Rammensee, H.G., et al . , Immunogenetics , 41 : 178-228, 1995) . Five peptides carrying aspartate or glutamate m position 2 and isoleucme or leucme m position 9/10 were identified. One of these peptides, REPVTKAEML, was present also m the ammo acid sequences encoded by MAGE-2, MAGE-3 and MAGE-6. This peptide, denominated MAGE . -, ₂₇_₁₃₆ , was used to sensitize the MSR3-EBV line to lysis by CTLs 337 m a titration assay. MSR3-EBV cells were pulsed with peptide

REPVTKAEML and then used as target cells m a cytotoxicity assay (Fig. 4a) . The half -maximal lysis was reached with 90 nM peptide. No lysis of MSR3EBV pulsed with an unrelated peptide able to bind to HLA-B*3701 was observed (Fig. 4b) .

Low levels of -y-IFN ^f≡re released by the CTLs 337 m the presence of Cos-7 cells expressing MAGE-4 and MAGE-12

(Fig. 3) . To verify whether this release could be ascribed to recognition of peptides encoded by codon 127-136 withm MAGE-4, a peptide-bmdmg study was performed, using as targets MSR3-EBV cells pulsed with the two peptides. Peptide ^M4 • 127-136' KELVTKAEML, differs for two ammo acids (lysme versus argmme m position 1 and leucme versus prol e m position 3) from peptide REPVTKAEML, while peptide M12.₁₂₇_ ₁₃₆, REPFTKAEML, differs for only one ammo acid (phenyalanme versus valme m position 4) . The results revealed that the two peptides can bind to HLA-B*3701, since increasing amounts of both were able to inhibit the lysis of MSR3-EBV pulsed with peptide REPVTKAEML but not with an unrelated HLA-A1 -binding peptide (i.e. M3.271-279*¹ (^Fl9- 4b) . However, no recognition of EBV cells pulsed with peptides M4.₁₂₇ _***_₃g and ^M12.₁₂₇_₁₃g was observed.

In conclusion, these data indicate that CTLs 337 are able to recognize a MAGE-1, 2, 3 and 6 peptides endogenously processed.

Example 3

CTL 337 specifically recognizes the products of the MAGE-2 and MAGE-6 genes. To date, no evidence exists m humans of the immunogenicity of the proteins encoded by MAGE-2 and MAGE-6. Indeed, while peptides encoded by MAGE-1, 3, 4, and 12 which bind HLA class I molecules to form antigen complexes recognized by various CTLs have been identified, no peptides encoded by he MAGE-2 or MAGE-6 genes have been identified.

In order to prove that peptide REPVTKAEML could be processed in melanoma cells also from MAGE-2 and MAGE-6 genes and presented to CTLs 337, melanoma cell lines expressing MAGE-2 or MAGE-6 but not other MAGE genes were searched. Unfortunately, expression of MAGE genes in melanomas is strictly correlated and most melanomas express more than one component of the MAGE gene family. Indeed, no melanoma cell lines selectively expressing MAGE-2 could be found, while a melanoma line, Me 14932, selectively expressing MAGE-6 was found.

The Me 14932 melanoma line was assayed by RT-PCR, using specific oligonucleotide primers for each MAGE gene and it proved positive only for MAGE-6, with a low expression level. To determine whether the REPVTKAEML peptide is endogenously processed from the MAGE-6 products and presented by HLA-B*3701, Mel4932 was transduced with a retroviral vector encoding the HLA-B*3701 molecule and a surface marker (ALNGFR) as described in Materials and Methods. The transduced cells, Me 14932-LB37, were immunoselected for A-LNGFR expression by magnetic beads. As indicated by im unofluorescence analysis with HLA-Bw4- specific mAb, the expression at the cell surface of HLA- B*3701 on Me 14932 transduced cells was at least twice lower than that of MSR3-B37 melanoma cells. CTLs 337 were able to recognize the Me 14932 -LB37 line in a cytotoxicity assay and the lysis level increased upon hexogenous addition of peptide REPVTKAEML, whereas pulsed and not pulsed Me 14932 line was not recognised (Fig. 5) . The low lysis levels of Me 14932-LB37 melanoma might be related with the weak expression of MAGE-6 gene or with the weak expression of HLA-B*3701 surface molecules.

To evaluate whether the inclusion of MAGE-2 and MAGE-6 in the list of target antigens for tumor specific immunotherapy could increase the proportion of eligible patients, the expression of MAGE-1, 2, 3 and 6 in fresh tumor samples of various histotypes was analyzed. Melanomas were not analyzed because expression of the different MAGE genes was clearly correlated (Dalerba, P., et al . , Int. J.

Cancer, 77: 200-204, 1998). The results indicate that 12% of the ovarian carcinomas and 5% of colon and breast carcinomas express MAGE-2 and/or MAGE-6 in the absence of MAGE-1 and

MAGE-3 (Table) . On the other hand, in all bladder and lung carcinomas studied the four genes were always co-expressed.

In conclusion, the data reported in this study indicate that MAGE-2 and MAGE-6 can be included in the list of possible target antigens for tumor-specific immunotherapy, increasing the number of patients that could benefit from this therapy.

Table

Expression of MAGE Genes by Fresh Tumor Samples (a)

Only

Histological Types MAGE-1 MAGE- -3 MAGE- -2 MAGE- -6 MAGE-2 or MAGE-6

Lung Carcinomas (n 28) (b) 35 39 32 29 0

Breast Carcinomas (n 20) 30 10 10 15 5 Ovary Carcinomas (n 25) 24 20 32 20 12 Bladder Carcinomas (n 25] 28 28 20 24 0 Colon Carcinomas (n 17) 0 5 5 5 5

^^a' As determined by reverse-PCR analysis. Results are shown as % of RT-PCR-positive tumors k-¹ Number of fresh tumor samples analyzed

BIBLIOGRAPHY

1. Marchand, M., et al . van Baren, N., Weynants, P., Brichard, V., Dreno, B., Tessier, M,-H., Rankin, ^"E., Parmiani, G., Arienti, F., Humblet, Y., Bourlond, A., Vanwijck, R. , Lienard. D., Beauduin, M., Dietrich, P.-H., Traversari, C, Kerger. J. , Masucci, G., Jager, E., De Greve, J., Atzopodien, J., Brasseur, F., Coulie, P. G., van der Bruggen, P., and Boon, T. Regressions observed in metastatic melanoma of patients treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1. Int. J. Cancer, 80: 219-230. 1999.

2. Jager, E., Ringhoffer, M., Altmannsberger , M., Arand, M., Karbach, J., Jager, D., Oesch, F., and Knuth, A. Immunoselection in vivo : independent loss of MHC class I and melanocyte differentiation antigen expression in metastatic melanoma. Int. J. Cancer, 71: 142-147, 1997.

3. Van den Eynde, B.J. and van der Bruggen, P. T cell defined tumor antigens. Curr. Opin. Immunol., 9:684-693, 1997.

4. van der Bruggen, P., Traversari, C, Chomez, P., Lurquin, C, De Plaen, E., Van den Eynde, B., Knuth, A., and Boon, T. A gene encoding an antigen recognised by cytolytic T lymphocytes on a human melanoma. Science, 254:1643-1647, 1991.

5. Van den Eynde, B., Peeters, 0., De Backer, 0., Gaugler, B., Lucas, S., e Boon, T. A new family of genes coding for an antigen recognised by autologous cytolytic T lymphocytes on a human melanoma. J. Exp . Med., 182:689-698, 1995. 6. Boel, P., Wildmann, C, Sensi, M.L., Brasseur, R., Renauld, J.C. Coulie, P., Boon, T. and vam der Bruggen, P. BAGE: a new gene encoding an antigen recognized on human melanomas by cytolytic T lymphocytes. Immunity, 2: 167-175, 1995 .

7. Coulie, P.G., Brichard, V., Van Pel, A., Wolfel, T., Schneider, J. , Traversari , C, Mattei, S., De Plaen, E., Lurwuin, C, Szikora, J.P., Renauld, J.C. and Boon, T. A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes associated with in vivo tumor rejection. Pre. Natl . Acad. Sci . , 91 : 6458-6463, 1994.

8. Weynants, P., Lethe, B., Brasseur, F., Marchand, M., and Boon, T. Expression of MAGE genes by non- small -cell lung carcinomas. Int. J. Cancer, 56 : 826-829, 1994.

9. Restifo, N.P., Marincola, M.F., Kawakami , Y., Taubenberger, J., Yannelli, J. R. , and Rosenberg, S.A. Loss of functional β₂-microglobulin in metastatic melanomas from five patients receiving immunotherapy. J. Natl. Cancer. Inst., 88 : 100-108, 1996.

10. Van den Eynde, B., Hainaut , P., Herin, M. and Knuth, A. Presence on a human melanoma of multiple antigens recognized by autologous CTL . Int. J. Cancer., 44 : 634-640. 1989.

11. Fleischhauer , K. , Gattinoni, L., Dalerba, P., Lauvau, G. Zanaria, E., B., D., van Endert, P. M., Bordignon, C. and

Traversari, C, The DAM gene family encodes a new group of tumor-specific antigens recognized by human leukocyte antigen A2 -restricted cytotoxic T lymphocytes. Cancer Res., 58 : 2969-2972, 1998. 12. Herman, J., van der Bruggen, P., Luescher, I.F., Mandruzzato, S., Rome-ro, P., Thonnard, J. , Fleischhauer, K. , Boon, T., and Coulie, P.G. A peptide encoded by human gene MAGE-3 induces cytolytic T lymphocytes that reco-gnizes tumor cells expressing MAGE-3. Immuogenetics , 43 : 377-384, 1996.

13. Fleischhauer, K. , Tanzarella, S., Russo, V., Sensi, M.L., van der Bruggen, P., Bordignon, C, and Traversari, C, Functional heterogeneity of HLA-A*02 subtypes revealed by presentation of a MAGE-3 -encoded peptide of cytotoxic T cell clones. J. Immunol., 159:2513-2521, 1997.

14. De Plaen, E., Arden, K. , Traversari, C, Functional heterogeneity of HLA-A*02 subtypes revealed by presentation of a MAGE-3 -encoded peptide to cytotoxic T cell clones. J. Immunol., 159 : 2513-2521, 1997.

15. De S et, C, Lurquin, C, van der Bruggen, P., De Plaen, E., Brasseur, F., and Boon, T. Sequence and expression pattern of the human MAGE-2 gene. Immunogenetics , 39 : 121-129, 1994.

16. Rammensee, H.G., Friede, T., and Stefanovic, S. MHC ligands and peptide motifs: first listing. Immunogenetics, 41 : 178-228, 1995.

17. Dalerba, P., Ricci, A., Russo, V., Rigatti, D., Nicotra, M.R. Mottolese, M., Bordignon, C, Natali, P.G., and Traversari, C. High homogeneity of MAGE, BAGE, GAGE, Tyrosinase and Melan-A/MART-1 gene expression in clusters of multiple simultaneous metastases of human melanoma: implication for protocol design of therapeutic antigen- specific vaccination strategies., Int. J. Cancer, 77: 200- 204, 1998.

18. Garrido, F., Cabrera, T., Concha, A., Glew, S., Ruiz- Cabello, F., and Stern, P. L., Natural history of HLA expression during tumor development. Immunol. Today, 14 : 491-499, 1993.

19. Lehmann, F., Marchand, M., Hainaut, P., Pouillart, P., Sastre, X., Ikeda, H., Boon, T., and Coulie, P., Differences in the antigens recognised by cytolytic T cells on two successive metastases of a melanoma patient are consistent with immune selection. Eur . J. Immunol., 25 : 340-347, 1995.

Claims

1. Peptides binding the allele HLA-B*3701 selected from the group consisting of: a) REPVTKAEML b) KELVTKAEML c) REPFTKAEML

2. Monoclonal or polyclonal antibodies raised directed to the peptides of claim 1.

3. Pharmaceutical composition comprising an effective amount of a peptide of claim 1 together with pharmaceutically acceptable excipients.

4. Composition as claimed m claim 3, for use as vaccine.

5. A method for inducing a cytotoxic response against tumor cells expressing a MAGE-1 , 2 , 3 , 4 , 6 , 12 antigen, which comprises contacting T lymphocytes with the peptides of claim 1 m suitable conditions for the activation of the lymphocytes themselves .

6. A method as claimed m claim 5, wherein lymphocytes are directly exposed to the peptides m culture.

7. A method as claimed m claim 5, wherein peptides are previously bound to HLA class I molecules, preferably the HLA-B*3701 allele.

8. Cytotoxic T cell line specifically recognizing a complex consisting of an HLA class I molecule, preferably

HLA-B*3701, and a peptide of claim 1.

9. Melanoma cell line not expressing histocompatibility antigens, deposited at the CBA — Interlab Cell Line Collection - under the N. PD99001.

10. Cell line of claim 9, genetically engmeerized with genes encoding HLA molecules and/or tumor antigens.

11. Cell line as claimed claim 10, wherein said HLA molecule is of class I B*3701, and the tumor antigen is of melanoma .

12. The use of the line of claims 9-11 for inducing and expanding ex-vivo antigen-specific cytotoxic T lymphocytes.

13. The use of the line of claims 10 and 11 for the preparation of a vaccine.

14. The use of the peptides of claim 1 for the preparation of an anti-tumor medicament.

15. The use as claimed in claim 14, wherein said medicament is a vaccine.