AU741558B2 - Tgfbeta rii receptor nucleic sequence, coded peptide, and uses - Google Patents

Abstract

The invention concerns a peptide comprising or consisting of a first peptide sequence of not more than 20 consecutive amino acids defined in a second peptide sequence coded by all or part of SEQ ID NO: 42 and different from SEQ ID NO: 1, said peptide being capable of being fixed on at least a class I major histocompatibility complex (MHC I) glycoprotein. The invention also concerns all nucleic sequence coding for such a peptide and the uses thereof in a vector, a host cell and in a pharmaceutical composition for diagnosing, preventing or treating in particular gastric or colon cancers.

Description

WO 99/64588 PCT/FR99/01354 NUCLEIC SEQUENCE FOR THE TGFP RII RECEPTOR, ENCODED PEPTIDE, AND USES The immune response directed against a specific antigen can be divided into two distinct categories, one involving antibodies (immune response of the humoral type), the other the T lymphocytes (immune response of the cellular type). More particularly, both types of response are distinguished by the fact that the antibodies recognize the antigens in their threedimensional form, whereas the T lymphocytes recognize peptide portions of said antigens, combined with glycoproceins encoded by the genes for the major histocompatibility complex (or MHC), in particular the genes for the major histocompatibility complex type I, which are ubiquitously expressed at the surface of cells, or the genes for the major histocompatibility complex type II, which are specifically expressed at the surface of the cells involved in the presentation of antigens (APC).

According to a first aspect, the immune response of the cellular type is characterized in that the T cells of the CD4 type ("helper" T cells), after activation by the antigenic fragments presented by the APCs, produce cytokines which in turn induce the proliferation of APC cells capable of producing said cytokines; the cellular differentiation of the B lymphocytes capable of producing specific antibodies, proliferation of the CD4 T cells and the stimulation of the cytotoxic lymphocytes (CTL).

According to a second aspect of the cellular immune response, the cytotoxic lymphocytes of the CD8' type (CTL) are activated a) after interaction with antigenic peptides attached to and presented by the glycoproteins carried by the ubiquitous cells and encoded by the genes belonging to the MHC I system of the APCs with costimulatory molecules, and b) possibly by the cytokines produced by the CD4+ cells. The CTLs Sthus activated are then capable of destroying the cells 2 expressing said antigenic peptide combined with the MHC I glycoproteins.

The presentation of the antigenic fragments by the MHC I molecules depends on an identified intracellular process (see Groettrup et al., 1996, Immunology Today, 17, 429-435 for a review) during which antigenic peptides which are very short in size (about 7-13 amino acids) are produced by degradation of a more complex polypeptide against which the final immune reaction will be directed (for example a viral protein). These short peptides are then combined with the MHC I molecules to form a trimolecular complex which is transported to the cellular surface so as to present said peptides to the circulating cytotoxic T lymphocytes. It should be noted, in addition, that the specificity of the MHC I molecules toward antigenic peptides varies as a function of the MHC I molecule (HLA-A, HLA-B, and the allele (HLA-A2, HLA-A3, HLA-All, which are considered. Within the same animal species, from one individual to another, there is a high variability of the genes encoding the molecules of the MHC system (in this regard, see in particular George et al., 1995, Immunology Today, 16, 209-212) In the case of cancers, Hellstrom et al., (1969, Adv. Cancer Res. 12, 167-223) have shown that the defence of the organism with respect to tumors is based most particularly on the immune response involving the T lymphocytes, in particular the cytotoxic T lymphocytes. However, it appears that this natural immune reaction is not sufficiently effective to prevent tumor development. That is the reason why it is desirable to have a method of activating the immune response directed against the tumors, and more particularly the response involving the cytotoxic lymphocytes CTLs, so as to have more effective methods of preventing or treating cancerous states.

Tumor cells express specific antigens which are ot present, or which are present in a smaller 3 quantity, in normal cells, and many studies have shown that these antigens can be the targets of antitumor reactions observed, for example, after immunizing the organism with the complete antigenic protein, with peptide fragments of such a protein, after administration of DNA encoding all or part of said protein, in particular after administration of recombinant viral vectors capable of expressing such a tumor-specific antigen.

However, this antitumor vaccinal approach is not always satisfactory and leads for example to limited immune reactions directed solely against immunodominant epitopes or against antigens exhibiting high variability. Likewise, the incorrect presentation of the antigens by the glycoproteins of the MHC system at the surface of the cells does not make it possible to develop an appropriate antitumor immunity in the treated patient.

In order to overcome these problems, some authors have proposed, in the context of such vaccine methods, selecting the antigenic minimal fragments corresponding to the peptide portions capable of being specifically recognized by the cytotoxic T lymphocytes, expressing them in cells so that they combine with the MHC I molecules and are presented at the surface of the cells in order to induce, in the treated patient, a perfectly targeted immune reaction (Toes et al., 1997, Proc. Natl. Acad. Sci., 94, 14660-14665) More particularly, it has been shown that epitopes which are very small in size (varying from 7 to about 13 amino acids) which are expressed by minigenes introduced into a vaccinia virus could induce a cellular type immunization. It has moreover been shown that several minigenes could be jointly expressed by the same vector (this particular construct is called "string of beads"). Such a construct has the advantage of inducing a synergistic CTL type immune reaction (Whitton et al., 1993, J. of Virology, 67, 348-352) 4 The present invention relates to nucleic sequences identified from the nucleic sequence encoding the mutant of the transforming growth factor (TGFP) RII receptor (WO-A-96/31605), the vectors allowing the transfer and the expression of these nucleic sequences in the target cells, the immunoreactive peptides expressed by these sequences, as well as the uses of said nucleic sequences and said peptides.

Colon cancers and gastric cancers are among the most frequent cancers. It is known that a large proportion of these cancers is linked to a dysfunction of the TGFP factor which loses its normal function of inhibiting the growth of different cell types.

There have in fact been observed, in these cases, different nucleic sequences for the TGFP RII receptor which exhibit, relative to the reference sequence, a reading frame shift. The expression of some of these "shifted" nucleic sequences encoding proteins different from the protein naturally encoded by the reference nucleic sequence (called mutant forms of the TGFP RII receptor) causes inactivation of the TGF which is thought to contribute to the development of tumors.

More particularly, in accordance with the document WO-A-96/31605, there has been identified, for some tumors, a mutation in the nucleic sequence for the RII receptor consisting in an addition or suppression of at least one adenine nucleotide in the polyadenine sequence localized at position 709-718 in the reference sequence for said receptor. This "shifted" nucleic sequence encodes a polypeptide which is shorter (161 amino acids) than the polypeptide encoded by the reference sequence, whose sequence differs at the level of the C-terminal end and now comprises the 34 amino acids presented in the sequence SEQ ID NO: 1 defined in the sequence listing at the end of the description.

The applicant has now identified nucleic sequences encoding a peptide, which is defined l/ hereinafter and which constitutes a first subject of 5 the invention. Said peptide according to the invention comprises or consists of a first peptide sequence of at most 20 consecutive amino acids defined in a second peptide sequence encoded by all or part of SEQ ID NO: 42 and different from SEQ ID NO: 1, said peptide being capable of binding to at least one MHC I glycoprotein.

In particular, the first sequence defining the peptide of the invention is chosen from SEQ ID NOS: 2 to 21.

Advantageously, a peptide of the invention meets at least any one of the following characteristics: the first sequence is chosen from SEQ ID NOS: 2 to 6, and said peptide is capable of binding to the MHC I HLA-A2 glycoproteins, the first sequence is chosen from SEQ ID NOS: 7 to 9, and said peptide is capable of binding to the MHC I HLA-A3 glycoproteins, the first sequence is chosen from SEQ ID NOS: 7 to 9, and said peptide is capable of binding to the MHC I HLA-A11 glycoproteins, the first sequence is chosen from SEQ ID NOS: 10 to 17, and said peptide is capable of binding to the MHC I HLA-B8 glycoproteins, the first sequence is chosen from SEQ ID NOS: 18 and 19, and said peptide is capable of binding to the MHC I HLA-B7 glycoproteins, the first sequence is SEQ ID NO: 20, and said peptide is capable of binding to the MHC I glycoproteins, the first sequence is SEQ ID NO: 21, and said peptide is capable of binding to the MHC I HLA-B27 glycoproteins, the first sequence is SEQ ID NO: 2, and said peptide is capable of binding to the MHC I HLA-B62 glycoproteins.

A second subject of the invention consists of a nucleic sequence encoding a peptide of the invention as defined above.

6 "Nucleic sequences" are understood to mean an isolated natural or synthetic, linear or circular, double-stranded or single-stranded DNA and/or RNA fragment designating a defined succession of nucleotides, modified or otherwise, allowing a sequence capable of encoding, in the presence of an appropriate expression system, a peptide according to the present invention, to be defined.

The preferred nucleic sequences are those chosen from SEQ ID NO: 22 to 41 and their complementary sequences.

The present invention also relates to cloning or expression vectors comprising at least one nucleic sequence as described above.

According to particularly advantageous embodiments, said vector comprises at least two different nucleic sequences chosen respectively from the following sequences: the sequences encoding a on the one hand, and the peptide as defined in on the other hand, the sequences encoding a on the one hand, and the peptide as defined in on the other hand, the sequences encoding a on the one hand, and the peptide as defined in on the other hand, the sequences encoding a on the one hand, and the peptide as defined in on the other hand, the sequences encoding a on the one hand, and the peptide as defined in on the other hand, peptide as defined in sequences encoding a or peptide as defined in sequences encoding a or peptide as defined in sequences encoding a or peptide as defined in sequences encoding a or peptide as defined in sequences encoding a or 7 the sequences encoding a peptide as defined in on the one hand, and the sequences encoding a peptide as defined in or on the other hand, and the sequences encoding a peptide as defined in on the one hand, and the sequences encoding a peptide as defined in or on the other hand, and the sequences encoding a peptide as defined in on the one hand, and the sequences encoding a peptide as defined in or on the other hand.

As regards more particularly the expression vectors, these comprise, in addition, elements allowing the expression of said nucleic sequence in a host cell, in particular promoter sequences and/or regulatory sequences which are effective in said cell. By way of example, there may be mentioned the promoters such as the RSV, MPSV, SV40, CMV or 7.5k vaccinia virus promoters, the promoters of the gene encoding muscle creatine kinase, actin or pulmonary surfactant. It is, in addition, possible to choose a promoter sequence specific for a given cellular type, or activable under defined conditions. The literature provides a large amount of information relating to such promoter sequences.

Moreover, the nucleic acids according to the present invention may also be nucleic acids modified such that it is not possible for them to integrate into the genome of the target cell or nucleic acids stabilized with the aid of agents such as, for example, spermine.

The vector in question may be chosen from autonomously replicating viral vectors, such as the vectors produced based on viruses which may be in particular adenoviruses, poxviruses, vaccinia-derived viruses, or herpesviruses, or from the viral vectors allowing chromosomal integration, such as the retroviruses or the AAVs (adenovirus-associated 8 viruses), or alternatively from the nonviral vectors consisting either of a nucleic acid, preferably a DNA in plasmid form, optionally combined with at least one compound promoting its introduction into cells such as, for example, a cationic lipid or cationic polymer, with a solvent or any other compound facilitating the introduction of said nucleic acid into cells. The literature provides a large number of examples of such viral and nonviral vectors.

The invention also relates to host cells comprising at least one nucleic sequence or at least one vector of the invention. More particularly, it relates to prokaryotic cells, yeast cells and animal cells, in particular mammalian cells, transformed with at least one vector as described above.

Another subject of the invention is a pharmaceutical composition or medicament comprising, as active ingredient, at least one nucleic sequence, a vector or a host cell of the invention. This is in particular a vaccine composition which can be used in the context of an immunotherapy via gene transfer.

Administration of such a medicament to a patient makes it possible in particular to induce an immune response based on the activation of the cytotoxic lymphocytes by the peptides encoded by said nucleic sequences.

According to one advantageous case, such a medicament is in particular intended for the treatment of gastric and colon cancers.

For its administration to humans or animals, such a composition comprises a pharmaceutically acceptable carrier.

The invention therefore also relates to the use of a nucleic sequence, of a vector or of a host cell as defined above for the preparation of a medicament intended for the preventive, curative or vaccinal treatment of the human or animal body, and more particularly intended for a gene therapy treatment of Sgastric and colon cancers.

9 The medicament may be administered in injectable form, in particular by the intramuscular, intratracheal, intratumoral, intragastric, intraperitoneal, epidermal, intravenous or intraarterial route, by inhalation, by instillation, by aerosolization, by the topical route or by the oral route.

Another subject of the invention relates to the use of a peptide of the invention for the preparation of a diagnostic, prophylactic or therapeutic composition, in particular a vaccine composition whose administration to a patient is capable of inducing an immune response characterized by a CTL response. The invention therefore also relates to any diagnostic, prophylactic or therapeutic composition comprising at least one peptide of the invention.

By way of examples of diagnostic uses of a peptide of the invention, it is possible to mention the methods consisting in detecting, and optionally quantifying: an antibody directed against said peptide, by bringing a biological sample which is likely to contain said antibody into contact with said peptide, and then detecting the formation of an immune complex between the antibody and the peptide by techniques known to persons skilled in the art, cytotoxic T lymphocytes according to the socalled ELISPOT technique described by Scheibenbogen et al., 1997, Clinical Cancer Research, 3, 221-226, whose content forms an integral part of the present application. Such a determination is particularly advantageous when it is desired to evaluate the effectiveness of a vaccinal approach used on a given patient or to diagnose a potential pathological state in a patient by seeking to demonstrate an immune response which is naturally developed by said patient against the mutated TGF beta RII antigen.

10 The characteristics and advantages of the invention are illustrated in the following examples, and supported by the figures.

Figure 1 represents the effect of the duration of incubation for different peptides on their association with the HLA-A2 molecules, the detection corresponding to a reading at 340/460 nm with the aid of a cytofluorimeter and being expressed in fluorescence unit the legend to the figure is as follows: O positive control peptide X negative control peptide peptide of SEQ ID NO: 4 A peptide of SEQ ID NO: 2 peptide of SEQ ID NO: 0 peptide of SEQ ID NO: 6 peptide of SEQ ID NO: 3 Figure 2 represents the effect of the duration of incubation for different peptides on their association with the HLA-A3 molecules, the detection corresponding to a reading at 340/460 nm with the aid of a cytofluorimeter and being expressed in fluorescence unit the legend to the figure is as follows: o positive control peptide negative control peptide peptide of SEQ ID NO: 7 0 peptide of SEQ ID NO: 8 peptide of SEQ ID NO: 9 Figure 3 represents the effect of the duration of incubation for different peptides on their association with the HLA-B8 molecules, the detection corresponding to a reading at 340/460 nm with the aid of a cytofluorimeter and being expressed in fluorescence unit the legend to the figure is as follows: positive control peptide 11 negative control peptide O peptide of SEQ ID NO: A peptide of SEQ ID NO: 17 0 peptide of SEQ ID NO: 11 X peptide of SEQ ID NO: 12 peptide of SEQ ID NO: 13 O peptide of SEQ ID NO: Figure 4 represents the effect of the dilution of different peptides on their association with the HLA-B7 molecules, the detection corresponding to a reading at 340/460 nm with the aid of a cytofluorimeter and being expressed in fluorescence unit the legend to the figure is as follows: O positive control peptide negative control peptide A peptide of SEQ ID NO: O peptide of SEQ ID NO: 13 Figure 5 represents the effect of the dilution of different peptides on their association with the molecules, the detection corresponding to a reading at 340/460 nm with the aid of a cytofluorimeter and being expressed in fluorescence unit the legend to the figure is as follows: O positive control peptide negative control peptide peptide of SEQ ID NO: 18 A peptide of SEQ ID NO: 19 peptide of SEQ ID NO: 0 peptide of SEQ ID NO: 13 Figure 6 represents the effect of the dilution of the peptide of SEQ ID NO: 21 on its association with the HLA-B27 molecules, the detection corresponding to a reading at 340/460 nm with the aid of a cytofluorimeter and being expressed in fluorescence unit the Slegend to the figure is as follows: peptide of 12 SEQ ID NO: 21 (N 31), positive control peptide and negative control peptide.

O positive control peptide negative control peptide peptide of SEQ ID NO: 21 Figure 7 represents the effect of the duration of incubation of the peptide of SEQ ID NO: 21 on its association with the HLA-B27 molecules, the detection corresponding to a reading at 340/460 nm with the aid of a cytofluorimeter and being expressed in fluorescence unit the legend to the figure is as follows: positive control peptide negative control peptide peptide of SEQ ID NO: 21 Example 1: Detection of the association between the peptides and the histocompatibility molecules 1) Materials Sources of histocompatibility molecules They are currently of two main types: mutant cells and purified histocompatibility molecules.

The mutant cell used is the human T2 cell which is a variant of the Tl line produced by fusion of the CEM T lymphoma and of the B lymphoma 721.174 (Salter and Cresswell, EMBO 1986, 5(5) 943-949). This cell, which lacks peptide transporters, contains heavy chains of class I molecules free of peptides which will be able to accept exogenous peptides.

Class I histocompatibility molecules purified by affinity chromatography from human B cell lines transformed with EBV can also be used. In this case, the endogenous peptides should be removed by a treatment with 1.5 M urea and 12.5 mM sodium hydroxide (pH 11.7) for 1 h at 4 0 C, followed by their removal with a desalting column (PDLO, Pharmacia). The histocompatibility molecules are immediately placed RA. again in the presence of the peptides to be tested in a 13 PBS buffer with 0.05% Tween 20, 2 mM EDTA, 0.1% and 6 mM CHAPS, in the presence of 2 g/ml B2m in order to facilitate reassociation (Gnjatic et al., Eur. J.

Immunol., 1995, 25, 1638-1642).

Peptides The peptides tested have in general 8 to residues, sometimes 11 or 12. They were synthesized by N6osystem (Strasbourg), or by CHIRON mimotopes (Victoria, Australia). They are used at concentrations varying from 100 iM to 0.1 nM.

2) Protocol for the assembly (Connan et al., Eur. J. Immunol., 1994, 24:777; Couillin et al., Eur.

J. Immunol., 1995, 25, 728-732) Aliquots of 8 x 10 5 cells in a volume of 64 pl, distributed into Eppendorf microfuge tubes, are brought into contact with a lysis buffer containing 10 mM PBS, pH 7.5, 1% NP40, protease inhibitors (1 mM PMSF, 100 pM iodoacetamide, 2 pg/ml aprotinin, 10 pM leupeptin, pM pepstatin and 10 pg/ml trypsin inhibitor). The lysis takes place in the presence of the peptides to be tested for 30 min or 1 h at 37 0 C. After removal of the nonsolubilized material by centrifugation at 15,000 revolutions/min at 4°C, the supernatant is supplemented with 140 1l of PBS containing 0.05% Tween 20, 3 mM sodium azide, 1 mM PMSF and 10 mg/ml bovine albumin.

Each sample is incubated for 20 h at 4 0 C in 2 wells of a microtiter plate of the Nunc type, Maxisorb, which have been previously coated with a monoclonal antibody pg/ml in PBS) which recognizes the histocompatibility molecules having a conformation or conformations appropriate for the presentation of the peptides and similar to that(those) present at the surface of the cells. The plate coated with antibodies is saturated beforehand with bovine albumin at 10 mg/ml in PBS-Tween before the introduction of the sample. The second antibody which allows the detection of the assembly of the histocompatibility molecules is directed against B2m. It is coupled either to biotin (NHS-LC biotin, Perce) or to alkaline phosphatase (P- 14 552, Sigma) and is incubated at 2 gg/ml for one hour at 370C. In the case of the use of biotin, an incubation of 45 minutes at 20-25 0 C with extravidin coupled to alkaline phosphatase (E-2636, Sigma) is carried out.

The activity of the alkaline phosphatase is measured using, as substrate, 4-methylumbelliferyl phosphate (M- 8883, Sigma) at 100 iM in 50 mM diethanolamine, pH with 1 mM MgC12. The reading is made at 340/460 nm with the aid of a cytofluorimeter.

3) Stability of the HLA/peptide complexes The stability of the abovementioned complexes was studied because it determines the correct presentation of the antigen and the induction of the T response.

To this effect, either purified HLA or the T2 cell lysate was used.

With the purified HLA, the endogenous peptides were removed [as described in and then it was brought into contact with the peptide to be tested in an Eppendorf tube at 370C, for times which can vary from a few minutes to several days. The next phase of incubation on a 96-well plate [as described in with the anti-HLA antibody is performed for one hour at 370C. The visualization is performed in a conventional manner.

With the T2 cell lysate, all the incubations are also carried out at 370C, after addition of all the protease inhibitors.

The examples below are intended to illustrate the potential applications of the peptides of the invention.

Example 2: Preparation of antibodies Immunization of rabbits and mice The following immunogen was prepared: the peptide SEQ ID NO: 43 coupled to Keyhole Limpet haemocyanin, abbreviated peptide-KLH, as carrier for its use in immunization, and said peptide coupled to human serum albumin, abbreviated peptide-HSA, intended Sfor the ELISA tests.

15 The rabbits were subjected to an injection of peptide-KLH using complete Freund's adjuvant (CFA) and then two injections of the same peptide using incomplete Freund's adjuvant (IFA). The mice were subjected to the same injections following the same protocol, with the exception of a few mice which were immunized by injection of peptide-HSA.

The sera and the hybridoma culture supernatants derived from the animals immunized with the peptide (SEQ ID NO: 43)-HSA were analyzed by ELISA test, using the peptides-KLH, and conversely.

Production of monoclonal antibodies The antibodies directed against the peptide SEQ ID NO: 43 were tested for and analyzed in the mice immunized as described above. The mice immunized with the peptides-HSA possess the highest antibody titer.

The spleen cells of these mice were consequently recovered and fuzed with myeloma cells. The hybridoma culture supernatants were analyzed by ELISA test from the point of view of their reactivity with the peptides of the invention and cells infected with the recombinant vaccinia virus VVTG-8048 (cf. below).

Example 3: Materials and methods for other uses of the peptides of the invention 1) Production of the recombinant vaccinia virus (VVTG-8048) The shifted complete sequence (reading frame shift of of the TGFP RII receptor, called RIIf, was cloned into the plasmid pTG186 using thymidine kinase (TK) for the site of insertion and the p7.5 promoter.

This assembly product was transferred into the vaccinia virus in order to obtain the recombinant virus called VVTG-8048.

The same abovementioned shifted complete sequence was cloned into the plasmid pTG186 at the site of insertion del-3 and promoter p7.5 in order to obtain an assembly product in the MVA system. This product carries both the abovementioned sequence and the IL-2 sequence, which have the same insertion site, but the 16 RIIf sequence is controlled by the p 7.5 promoter and the IL-2 sequence is controlled by the H5R promoter.

2) Western blots BHK cell cultures were infected with VVTG-8048 for 24 hours. The cells were then lysed by sonication.

The culture supernatants as well as the sonication pellets were analyzed by Western blotting.

Extracts of E. coli 8051 and 8067 were enriched with RIIf by passing over resin chelated with nickel (provided by Qiagen) according to the protocol which will be described later.

The two types of extracts were tested by Western blotting under denaturing conditions (in sodium dodecyl sulfate, SDS). The expression of the RII protein was visualized with a rabbit polyclonal antiserum which is commercially available (Santa Cruz, SC1700) and directed against the native form of RII.

This antiserum recognizes, in addition, the epitopes of the mutated molecule.

3) Recombinant protein The recombinant protein corresponding to the RIIf sequence was expressed in E. coli as recombinant protein with either poly-His, or poly-His epitope 5F3 intended for the immunopurification. The insoluble protein fraction is solubilized in 8 M urea. The enrichment of the product was carried out on resin chelated with nichel [sic] (Qiagen). The column was washed with decreasing urea concentrations. The elution was carried out with imidazole in the absence of urea.

4) Expression in mammals The RIIf sequence was fully cloned into pCDNA-3 for transfection into mammalian cells. The Balb/c 3T3, RMA and P815 cells were transfected by electroporation and selected in 1 mg/ml G418. Balb/c 3T3 cells (H-2d) were transfected with CaPO 4 The P815-HT cells (H-2d also) were transfected with CaPO 4 The RMA cells (H-2d) were transfected with lipofectin and with CaP0 4 17 The G418-resistant clones were tested for the expression of the RIIF insert by PCR. Several clones are capable of expressing the RIIF insert.

Example 4: Demonstration in vivo of the immunogenic power of the peptide of SEQ ID NO: The immunogenic power of the peptides of the invention was also studied in vivo. This immunogenic power is evaluated relative to the capacity which these peptides have to induce cytolytic T effectors in transgenic A2/R b mice expressing HLA/A2 (Correale et al., J. Immunol. 1998 Sep 15; 161(6) 3186-94). The peptide of SEQ ID NO: 5 (RLSSCVPVA) was chosen to illustrate this study.

In the first instance, the mice are coinjected with two epitopes: the peptide SEQ ID NO: 5 which binds to the HLA-A2 molecule, and a peptide with "helper" effect, the HBVc peptide 128-140, derived from the hepatitis B virus "core" protein, which is recognized by the mouse TCD4+ lymphocytes. A subcutaneous injection was done at the base of the tail three times with 200 il of an emulsion in incomplete Freund's adjuvant (IFA) containing 50 pig of the peptide of SEQ ID NO: 5 (2 mg/ml in NaC1) and 140 pg of the HBVc peptide 128-140 (2 mg/ml in NaC1).

The experiment was carried out on a group of 6 male mice comprising: 2 mice immunized with 200 gl IFA NaCl HBVc 128-140 (mouse pO), 2 mice immunized with 200 il IFA NaC1 HBVc 128-140 peptide of SEQ ID NO: 5 (mouse and 2 mice immunized with 200 il IFA HBVc peptide 128-140 MART 27-35 peptide as T epitope (Cornier et al., 1997, Cancer J Sci Am; 37-44).

To demonstrate the presence of cytolytic effectors, an additional stimulation is then carried out in vivo (at D11) This stimulation is carried out Swith 3.106 splenocytes/ml and 106 blasts induced by LPS 18 and irradiated. The blasts were prepared at D8 from splenocytes of untreated mice using 1.5. 106 cells/ml of RPMI 10% FCS, 25 pg/ml LPS (1 mg/ml in NaCi), 7 pg/ml of dextran sulfate (7 mg/ml in water). At D11, the blasts are irradiated (4000 rads), washed and suspended at a concentration of 40.106 cells/ml with the peptide of SEQ ID NO: 5 (100 pg/ml) for one hour at 37 0 C. After washing, the concentration of the blasts is adjusted to 10 7 cells/ml and then the blasts are mixed with splenocytes of the immunized mice. The test for cytolytic T effectors is performed by carrying out the CRT test (Chromium release Test, Mariani et al., 1994, J Immunol Methods. 24; 172(2): 173-8).

lysis mouse pO 0 0 mouse pO p5 0 mouse pMART 0 0 mouse pMART pMART 0 mouse p5 0 0 mouse p5 P5 37 The results of the CRT test demonstrate the capacity of the peptide of SEQ ID NO: 5 to induce cytolytic T effectors.

EDITORIAL NOTE NO.40471/99 Sequence listing pages 1 to 10 are to be inserted after the description and before the claims.

SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: TRANSGENE STREET: 11, rue de Molsheim CITY: STRASBOURG COUNTRY: FRANCE POSTAL CODE: 67082 cedex (ii) TITLE OF INVENTION: (iii) NUMBER OF SEQUENCES: 43 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) INFORMATION FOR SEQ ID NO:1 SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 Ser Leu Val Arg Leu Ser Ser Cys Val Pro Val Ala Leu Met Ser Ala 1 5 10 Met Thr Thr Ser Ser Ser Gin Lys Asn Ile Thr Pro Ala Ile Leu Thr 25 Cys cys INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2

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2 Ser Leu Val Arg Leu Ser Ser Cys Val 1 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Ser Leu Val Arg Leu Ser Ser Cys Val Pro Val 1 5 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Arg Leu Ser Ser Cys Val Pro Val 1 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Arg Leu Ser Ser Cys Val Pro Val Ala 1 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Arg Leu Ser Ser Cys Val Pro Val Ala Leu 1 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acid residues TYPE: amino acids 3 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Met Thr Thr Ser Ser Ser Gin Lys 1 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Ala Met Thr Thr Ser Ser Ser Gin Lys 1 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Ser Ala Met Thr Thr Ser Ser Ser Gin Lys 1 5 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Ile Met Lys Glu Lys Lys Ser Leu 1 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Ile Met Lys Glu Lye Lys Ser Leu Val 1 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: 4 LENGTH: 8 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Lys Ser Leu Val Arg Leu Ser Ser 1 INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Lys Ser Leu Val Arg Leu Ser Ser Cys Val 1 5 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Thr Ser Ser Ser Gin Lys Asn Ile 1 INFORMATION FOR SEQ ID NO-: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Ser Ser Ser Gin Lys Asn Ile Thr 1 INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Thr Ser Ser Ser Gin Lys Asn Ile Thr

I

5 INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: Cys Ile Met Lys Glu Lys Lys Ser Leu 1 INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Val Pro Val Ala Leu Met Ser Ala 1 INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: ,Leu Ser Ser Cys Val Pro Val Ala Leu 1 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Val Pro Val Ala Leu Met Ser Ala Met 1 INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

I

-6 Val Arg Leu Ser Ser Cys Val Pro Val 1 INFORMATION FOR SEQ ID NO: 22 SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: AGCCTGGTGA GACTTTCTTC ATGTCTT 27 INFORMATION FOR SEQ ID NO: 23 SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: AGCCTGGTGA GACTTTCTTC ATGTGTTCCT GTA 33 INFORMATION FOR SEQ ID NO: 24 SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: AGACTTTCTT CATGTGTTCC TGTA 24 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AGACTTTCTT CATGTGTTCC TGTAGCT 27 INFORMATION FOR SEQ ID NO: 26 SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: AGACTTTCTT CATGTGTTCC TGTAGCTCTG INFORMATION FOR SEQ ID NO: 27 7 SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: ATGACAACAT CATCTTCTCA GAAG INFORMATION FOR SEQ ID NO: 28 SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: GCAATGACAA CATCATCTTC TCAGAAG INFORMATION FOR SEQ ID NO: 29

SEQUENCE.CHARACTERISTICS:

LENGTH: 30 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: ,n AGTGCAATGA CAACATCATC TTCTCAGAAG INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ IT ATTATGAAGG AAAAAAAAAG CCTG INFORMATION FOR SEQ ID NO: 31 SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ IE ATTATGAAGG AAAAAAAAAG CCTGGTG INFORMATION FOR SEQ ID NO: 32 SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA D NO: SNO: 31: 8 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: AAAAGCCTGG TGAGACTTTC TTCA INFORMATION FOR SEQ ID NO: 33 SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: AAAAGCCTGG TGAGACTTTC TTCATGTGTT INFORMATION FOR SEQ ID NO: 34 SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: ACATCATCTT CTCAGAAGAA TATA INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ II TCATCTTCTC AGAAGAATAT AACA INFORMATION FOR SEQ ID NO: 36 SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ II ACATCATCTT CTCAGAAGAA TATAACA D NO: NO: 36 INFORMATION FOR SEQ ID NO: 37 SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37 TGCATTATGA AGGAAAAAAA AAGCCTG INFORMATION FOR SEQ ID NO: 38 9 SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ II GTTCCTGTAG CTCTGATGAG TGCA INFORMATION FOR SEQ ID NO: 39 SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ IE CTTTCTTCAT CTGTTCCTGT AGCTCTG D NO: 38 NO: 39 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ II GTTCCTGTAG CTCTGATGAG TGCAATG INFORMATION FOR SEQ ID NO: 41 SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ II GTGAGACTTT CTTCATGTCT TCCTGTA D NO: NO: 41 INFORMATION FOR SEQ ID NO: 42 SEQUENCE CHARACTERISTICS: LENGTH: 123 base pairs TYPE: nucleotide (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42 TGCATTATGA AGGAAAAAAA AACCCTGGTG AGACTTTCTT CATGTGTTCC TGTAGCTCTG ATGAGTGCAA TGACAACATC ATCTTCTCAG AAGAATATAA CACCAGCAAT CCTGACTTGT 120 TGC 123 INFORMATION FOR SEQ ID NO: 43 SEQUENCE CHARACTERISTICS: 10 LENGTH: 26 amino acid residues TYPE: amino acids (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: Tyr Lys Lys Pro Val Ala Leu met Ser Ala Met Thr Thr Ser Ser Ser 1 5 10 Gin Lys Asn Ile Thr Pro Ala Ile Leu Thr 20 1

Claims (9)

1. Peptide comprising or consisting of a first peptide sequence of at most 20 consecutive amino acids defined in a second peptide sequence encoded by all or part of SEQ ID NO: 42 and different from SEQ ID NO: 1, said peptide being capable of binding to at least one major histocompatibility complex class I (MHC I) glycoprotein.

2. Peptide according to Claim 1, characterized in that the first sequence is chosen from SEQ ID NOS: 2 to

21. 3. Peptide according to Claim 2, characterized in that the first sequence is chosen from SEQ ID NOS: 2 to 6, said peptide being capable of binding to the MHC I HLA-A2 glycoproteins. 4. Peptide according to Claim 2, characterized in that the first sequence is chosen from SEQ ID NOS: 7 to 9, said peptide being capable of binding to the MHC I HLA-A3 glycoproteins. Peptide according to Claim 2, characterized in that the first sequence is chosen from SEQ ID NOS: 7 to 9, said peptide being capable of binding to the MHC I HLA-All glycoproteins. 6. Peptide according to Claim 2, characterized in that the first sequence is chosen from SEQ ID NOS: to 17, said peptide being capable of binding to the MHC I HLA-B8 glycoproteins. 7. Peptide according to Claim 2, characterized in that the first sequence is chosen from SEQ ID NOS: 18 and 19, said peptide being capable of binding to the MHC I HLA-B7 glycoproteins. 8. Peptide according to Claim 2, characterized in that the first sequence is SEQ ID NO: 20, said peptide being capable of binding to the MHC I glycoproteins. 9. Peptide according to Claim 2, characterized in that the first sequence is SEQ ID NO: 21, said peptide 20 being capable of binding to the MHC I HLA-B27 glycoproteins. Peptide according to Claim 2, characterized in that the first sequence is SEQ ID NO: 2, said peptide being capable of binding to the MHC I HLA-B62 glycoproteins. 11. Nucleic sequence encoding a peptide according to any one of Claims 1 to 12. Sequence according to Claim 11, characterized in that it is chosen from SEQ ID NOS: 22 to 41, and their complementary sequences. 13. Cloning or expression vector comprising at least one nucleic sequence according to Claim 11 or 12, and elements necessary for its expression in a host cell. 14. Vector according to Claim 13, characterized in that it comprises at least two different nucleic sequences chosen respectively: from the sequences encoding a peptide of Claim 3 and the sequences encoding a peptide of any one of Claims 4 to from the sequences encoding a peptide of Claim 4' and the sequences encoding a peptide of any one of Claims 3 and 4 to from the sequences encoding a peptide of Claim and the sequences encoding a peptide of any one of Claims 3, 4 and 6 to from the sequences encoding a peptide of Claim 6 and the sequences encoding a peptide of any one of Claims 3 to 5 and 7 to from the sequences encoding a peptide of Claim 7 and the sequences encoding a peptide of any one of Claims 3 to 6 and 8 to from the sequences encoding a peptide of Claim 8 and the sequences encoding a peptide of any one of Claims 3 to 7, 9 and from the sequences encoding a peptide of Claim 9 and the sequences encoding a peptide of any one of SClaims 3 to 8 and 10, and 21 from the sequences encoding a peptide of Claim and the sequences encoding a peptide of any one of Claims 3 to 9. Vector according to Claim 13 or 14, characterized in that it is chosen from the autonomously replicating viral vectors or the viral vectors allowing chromosomal integration and from the nonviral vectors. 16. Vector according to Claim 15, characterized in that it is a viral vector produced based on an adenovirus, a retrovirus, a poxvirus, the vaccinia virus or a herpesvirus. 17. Vector according to Claim 15, characterized in that it is a nonviral vector consisting of a plasmid optionally combined with at least one compound capable of facilitating the introduction of said plasmid into cells to be transfected. 18. Vector according to Claim 17, characterized in that said compound is chosen from the cationic lipids and the cationic polymers. 19. Host cell comprising at least one nucleic sequence according to Claim 11 or 12, or at least one vector according to any one of Claims 13 to 18. Pharmaceutical composition comprising, as active ingredient, at least one nucleic sequence according to Claim 11 or 12, a vector according to any one of Claims 13 to 18, or a host cell according to Claim 18. 21. Composition according to Claim characterized in that it is intended for the treatment of gastric or colon cancers.

22. Composition according to Claim 20 or 21, characterized in that it comprises a pharmaceutically acceptable carrier allowing its administration to humans or animals.

23. Use of a nucleic sequence according to Claim 11 or 12, of a vector according to any one of Claims 13 to 8 or of a host cell according to Claim 19, for l he preparation of a medicament intended for the 22 preventive, curative or vaccinal treatment of the human or animal body.

24. Use according to Claim 23, characterized in that the medicament is intended for a gene therapy treatment. Use according to Claim 23, characterized in that the medicament is intended for treating gastric or colon cancers.

26. Use according to Claim 22, characterized in that the medicament is intended to be administered in injectable form, in particular by the intramuscular, intratracheal, intratumoral, intragastric, intraperitoneal, epidermal, intravenous or intra- arterial route, by inhalation, by instillation, by aerosolization, by the topical route or by the oral route.

27. Diagnostic, prophylactic or therapeutic composition comprising at least one peptide according to any one of Claims 1 to

28. Use of a peptide according to any one of Claims 1 to 10, for preparing a diagnostic, prophylactic or therapeutic composition intended for detecting, preventing or treating gastric or colon cancers. REPLACEMENT SHEET (RULE 26)