AU776903B2 - Use of an OmpA enterobacterium protein associated with the elagigiltv peptide for treating melanomas - Google Patents

Abstract

The invention concerns the use of an enterobacterium membrane protein OmpA, in particular of Klebsiella pneumoniae, associated with an antigen or a hapten for preparing a pharmaceutical composition designed to generate or enhance a cytotoxic T response directed against a tumor cell. The invention also concerns the use of said compounds for preventing or treating infection or cancer, in particular cancers associated with a tumoral antigen such as melanoma, and pharmaceutical compositions comprising some of said compounds.

Description

WO 00/48629 PCT/FROO/00394 USE OF AN OmpA ENTEROBACTERIUM PROTEIN ASSOCIATED WITH THE ELAGIGILTV PEPTIDE, FOR TREATING MELANOMAS The invention relates to the use of an enterobacterium, in particular Klebsiella pneumoniae, OmpA membrane protein, associated with an antigen or a hapten, for preparing a pharmaceutical composition intended to generate or increase a cytotoxic T response directed against an infectious agent or a tumor cell. The invention comprises the use of these compounds for preventing and treating infection or cancer, in particular cancers associated with a tumor antigen, such as melanomas, and also for pharmaceutical compositions comprising some of these compounds.

Vaccination is an effective means of preventing or reducing viral or bacterial infections. The success of vaccination campaigns in these domains has made it possible to extend the vaccine concept, until now used in the domain of infectology, to the domains of cancer and of autoimmune diseases. Vaccinal antigens administered alone to the host are often not immunogenic enough to induce an immune response and must, therefore, be associated with an adjuvant or coupled to a carrier protein in order to induce (or increase) the immunogenicity. Under these conditions, only an immune response of the humoral type can be induced. Now, in the context of antiviral therapy, the generation of cytotoxic T lymphocytes (CTLs) capable of recognizing and destroying the virus is of great importance (Bachmann et al., 1994, Eur. J. Immunol., 24, 2228-2236; Borrow 1997, J. Virol. Hepat., 4, 16-24), as attested by many studies showing, in vivo, the protective role of responses directed against viral epitopes (Arvin AM, 1992, J. Inf. Dis., 166, S 35-S41; Koszinowski et al., 1987 Immunol. Lett., 16, 185-192).

The importance of CTL responses has also been greatly documented in antitumor responses, in particular those directed against melanoma cells (review in Rivoltini et 2 directed against melanoma cells (review in Rivoltini et al., 1998, Crit. Rev. Immunol. 18, 55-63). The CTL epitope(s) (peptide sequences which interact with class I molecules and are presented to CD8+ T lymphocytes) have been defined for several antigens.

However, the difficulty lies in generating CTLs in vivo, due to the weak immunogenicity of these peptides (Melief, 1992, Adv. Cancer Res., 58, 143-175; Nandaz and Sercaz, 1995, Cell, 82, 13-17).

Research is consequently directed toward identifying novel adjuvants, or of an antigen delivery system, making it possible to induce CTLs. Due to their effectiveness in presenting antigens and in stimulating the immune system, dendritic cells, for example, have been used to generate antiviral CTL responses (Ludewig B et al., 1998, J. Virol., 72, 3812-3818; Brossard P.

et al., 1997, J. Immunol., 158, 3270-3276) or anticancer CTL responses (Nestle F.O. et al., 1998, Nat. Med., 4, 328-332). The approaches have consisted in loading the dendritic cells ex vivo, with the antigen of interest (peptides or cell lysate) and reimplanting these cells into the patient. Other approaches consist in transfecting, ex vivo, the dendritic cells with the gene encoding the antigen of interest and in reinjecting these transfected cells (Gilboa E. et al., 1998, Cancer Immunol. Immunother., 46, 82-87). These approaches have been used successfully in mice and recently in humans (Hsu F.J.

et al., 1996, Nat. Med., 2, 52-58), but nevertheless remain complex since the cells must be treated ex vivo (transformation of the cells or internalization of the antigens) and transplanted into the host organism.

Similarly, the use of viral-type particles (Layton G.T.

et al., 1993, J. Immunol., 151, 1097-1107) or of incomplete Freund's adjuvant (IFA) (Valmori et al., Eur. J. Immunol., 1994, 24, 1458-1462) makes it possible to generate CTL responses. However, antiviral 3 corresponding to CTL epitopes and in the presence of such an adjuvant may lead to a state of specific tolerance, which may, in certain cases, produce the opposite effect to that desired, i.e. a decrease in the immune response (Toes et al., Proc. Nat. Acad. Sci.

USA, 1996, 93, 7855-7860).

Thus, there exists, today, a great need for a compound which, when associated with a molecule, in particular an antigen or hapten, is capable of generating CTLs directed against said molecule. Such a compound could, in particular, be used for preparing a vaccinal composition intended to induce immune protection of the antiviral, antibacterial, antifungal, antiparasitic or antitumor CTL type.

Surprisingly, it has been demonstrated that an outer membrane protein of a gram-negative bacterium, in particular an enterobacterium OmpA protein such as the Klebsiella pneumoniae P40 protein (protein described in WO 95/27787 and WO 96/14415), has the property of eliciting a CTL response against a molecule which is covalently or noncovalently associated with it, preferably without having to add another adjuvant.

Thus, the present invention relates to the use of an enterobacterium OmpA protein, of a fragment thereof or of a nucleic acid sequence encoding said OmpA protein or a fragment thereof, for preparing a pharmaceutical composition intended to generate or increase a cytotoxic T response against an infectious agent or a tumor cell, in vitro or in vivo, preferably in vivo, and also for preparing a pharmaceutical composition intended to generate or increase said cytotoxic T response.

In the present invention, the term "protein" is intended to denote both peptides or polypeptides and 4 the term "OmpA" (for "outer membrane protein") is intended to denote outer membrane proteins of the A type.

The expression "fragment of an OmpA protein" is intended to denote, in particular, any fragment of amino acid sequence included in the amino acid sequence of the OmpA protein which, when it is associated with an antigen or hapten specific for an infectious agent or for a tumor cell, is capable of generating or increasing a cytotoxic T response directed against said infectious agent or said tumor cell, said fragment of the OmpA protein comprising at least 5 amino acids, preferably at least 10 amino acids or more preferably at least 15 amino acids.

The expression "antigen or hapten specific for an infectious agent or for a tumor cell" is intended to denote, in particular, any compound expressed by an infectious agent, such as a virus, a bacterium, a yeast, a fungus or a parasite, or by a tumor cell, or a structural analog thereof, which, alone or in association with an adjuvant of immunity, is capable of inducing an immune response specific for said infectious agent or for said tumor cell.

In the present description, the expression "analog of an antigen or hapten" is intended to denote, in particular, a compound having structural similarity with said antigen or hapten, capable of inducing an immunological response directed against said antigen or hapten in an organism immunized beforehand with said similar compound.

A subject of the invention is also the use as claimed in the invention, characterized in that said pharmaceutical composition also comprises, associated with said enterobacterium OmpA protein, an antigen or a 5 hapten specific for said infectious agent or for said tumor cell.

Preferably, the invention comprises the use as claimed in the invention, characterized in that said infectious agent is a viral particle, a bacterium, a yeast, a fungus or a parasite.

In a particular embodiment, the invention comprises the use of an enterobacterium OmpA protein, or of a fragment thereof, as claimed in the invention, characterized in that said enterobacterium OmpA protein, or a fragment thereof, is obtained using a method of extraction from a culture of said enterobacterium.

The methods for extracting bacterial membrane proteins are known to those skilled in the art and will not be developed in the present description. Mention may, for example, be made, but without being limited thereto, of the extraction method described by Haeuw J.H. et al.

(Eur. J. Biochem, 255, 446-454, 1998) In another preferred embodiment, the invention also comprises the use of an enterobacterium OmpA protein, or of a fragment thereof, as claimed in the invention, characterized in that said enterobacterium OmpA protein, or a fragment thereof, is obtained via the recombinant route.

The methods for preparing the recombinant proteins are, today, well known to those skilled in the art and will not be developed in the present description; reference may however be made to the method described in the examples. Among the cells which may be used for producing these recombinant proteins, mention should, of course, be made of bacterial cells (Olins P.O. and Lee 1993, Recent advances in heterologous gene expression in coli. Curr. Op. Biotechnology 6 4:520-525), and also yeast cells (Buckholz 1993, Yeast Systems for the Expression of Heterologous Gene Products. Curr. Op. Biotechnology 4:538-542), as well as animal cells, in particular mammalian cell cultures (Edwards C.P. and Aruffo 1993, Current applications of COS cell based transient expression systems. Curr.

Op. Biotechnology 4:558-563), and also insect cells in which methods may be used which implement, for example, baculoviruses (Luckow 1993, Baculovirus systems for the expression of human gene products. Curr. Op.

Biotechnology 4:564-572).

Entirely preferably, the use as claimed in the invention is characterized in that the said enterobacterium is Klebsiella pneumoniae.

In particular, the invention relates to the use as claimed in the invention, characterized in that the amino acid sequence of said Klebsiella pneumoniae OmpA protein, or a fragment thereof, comprises: a) the amino acid sequence of sequence SEQ ID No. 2; b) the amino acid sequence of a sequence having at least 80%, preferably 90% and 95% homology, after optimal alignment, with the sequence SEQ ID No. 2; or c) the amino acid sequence of a fragment of at least amino acids of a sequence as defined in a).

The expression "nucleic acid or amino acid sequence with at least 80% homology, after optimal alignment, with a given nucleic acid or amino acid sequence" is intended to denote a sequence which, after optimal alignment with said given sequence, comprises a percentage identity of at least 80% with said given sequence.

For the purposes of the present invention, the term "percentage identity" between two nucleic acid or amino 7 acid sequences is intended to denote the percentage of nucleotides or of amino acid residues which are identical between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length. Sequence comparisons between two nucleic acid or amino acid sequences are conventionally carried out by comparing these sequences after having aligned them optimally, said comparison being carried out by segment or by "window of comparison" in order to identify and compare local regions of sequence similarity. The optimal alignment of the sequences for comparison may be produced, other than manually, by means of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2:482], by means of the local homology algorithm of Neddleman and Wunsch (1970) Mol. Biol. 48:443], by means of the similarity search method of Pearson and Lipman (1988) [Proc. Natl.

Acad. Sci. USA 85:2444], by means of computer software which uses these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or with BLAST N or BLAST P comparison software).

The percentage identity between two nucleic acid or amino acid sequences is determined by comparing these two sequences which are optimally aligned by the window of comparison in which the region of the nucleic acid or amino acid sequence to be compared may comprise additions or deletions with respect to the reference sequence for optimal alignment between these two sequences. The percentage identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, dividing this number of identical positions by the total number of positions in the window of comparison and multiplying the result 8 obtained by 100 in order to obtain the percentage identity between these two sequences.

Use may, for example, be made of the BLAST program "BLAST 2 sequences", which is available on the site http://www.ncbi.nlm.nih.gov/gorf/bl2.html, the parameters used being those given by default (in particular for the "open gap penalty" parameter: 5, and the "extension gap penalty" parameter: 2; the matrix chosen being, for example, the "BLOSUM 62" matrix provided by the program), the percentage identity between the two sequences to be compared being calculated directly by the program.

Among said sequences having at least 80% homology with the reference OmpA sequence, preference is given to the sequences of, or encoding, peptides capable of inducing CTL activity directed specifically against the antigen or hapten which is associated with it, such as the CTL activity measured using the standard techniques described in the examples hereinafter.

The invention also comprises the use as claimed in the invention, characterized in that said antigen or hapten is chosen from proteins, lipopeptides, polysaccharides, oligosaccharides, nucleic acids, lipids or any compound capable of specifically directing the CTL response against said infectious antigen or said tumor cell.

A subject of the present invention is also the use as claimed in the invention, characterized in that said antigen or hapten is coupled to or mixed with said OmpA protein or a fragment thereof.

The invention also comprises the use as claimed in the invention, characterized in that said antigen or hapten is coupled by covalent attachment, in particular by chemical coupling, with said OmpA protein or a fragment thereof.

9 In a particular embodiment, the use as claimed in the invention is characterized in that one or more attachment elements is(are) introduced into said OmpA protein, or a fragment thereof, and/or into said antigen or hapten, in order to facilitate the chemical coupling; preferably, said attachment element introduced is an amino acid.

As claimed in the invention, it is possible to introduce one or more attachment elements, in particular amino acids, in order to facilitate the coupling reactions between the OmpA protein, or a fragment thereof, and said antigen or hapten. The covalent coupling between the OmpA protein, or a fragment thereof, and said antigen or hapten as claimed in the invention may be carried out at the N- or C-terminal end of the OmpA protein or a fragment thereof. The difunctional reagents which enable this coupling will be determined as a function of the end of the OmpA protein, or a fragment thereof, which is chosen for carrying out the coupling, and of the nature of said antigen or hapten to be coupled.

In another particular embodiment, the use as claimed in the invention is characterized in that the coupling between said antigen or hapten and said OmpA protein, or a fragment thereof, is produced by genetic recombination, when said antigen or hapten is peptide in nature.

The conjugates derived from coupling to said OmpA protein, or a fragment thereof, may be prepared by genetic recombination. The chimeric or hybrid protein (conjugate) may be produced using recombinant DNA techniques, by inserting or adding a sequence encoding said antigen or hapten which is peptide in nature into the DNA sequence encoding said OmpA protein or a fragment thereof.

10 The methods for synthesizing the hybrid molecules encompass the methods used in genetic engineering for constructing hybrid polynucleotides encoding desired polypeptide sequences. Advantageously, reference may, for example, be made to the technique for obtaining genes encoding fusion proteins, described by D.V. Goeddel (Gene expression technology, Methods in Enzymology, Vol. 185, 3-187, 1990).

In another aspect, the invention relates to the use as claimed in the invention, characterized in that the pharmaceutical composition comprises a nucleic acid construct encoding said hybrid protein, or comprises a vector containing a nucleic acid construct encoding said hybrid protein or a transformed host cell containing said nucleic acid construct, which is capable of expressing said hybrid protein.

The invention also comprises the use as claimed in the invention, for preparing a pharmaceutical composition intended to eliminate infectious agents or inhibit tumor growth.

Preferably, the use as claimed in the invention relates to the preparation of a pharmaceutical composition intended to prevent or treat infectious diseases or cancers, preferably cancers associated with a tumor antigen.

Among cancers in which the tumors express an associated tumor antigen, and which may be prevented or treated with the uses as claimed in the present invention, mention may be made, in particular, but without being limited thereto, of: breast cancer, lung cancer, colon cancer and gastric carcinoma (Kawashima et al., 1999, Cancer Res.

59:431-5); 11 mesothelioma, osteosarcoma, brain cancers (Xie et al., 1999, J. Natl. Cancer. Inst. 91:169-75); melanoma (Zheuten et al., 1998, Bratilsl. Lek. Listy 99:426-34); cystic adinoma of the pancreas (Hammel et al., 1998, Eur. J. gastroenterol. Hepatol. 10:345-8); colorectal cancer (Ogura et al., 1998, Anticancer Res. 18:3669-75); renal cell carcinoma (Jantzer et al., 1998, Cancer Res. 58:3078-86); and cancer of the ovary and of the cervix (Sonoda et al., 1996, Cancer. 77:1501-9).

A subject of the invention is in particular the use of an enterobacterium OmpA protein, or of a fragment thereof, as claimed in the invention, for preparing a pharmaceutical immunization composition intended to prevent or treat an infectious disease, in particular of viral, bacterial, fungal or parasitic origin, or a cancer, preferably associated with a tumor antigen, in particular melanomas.

The invention also comprises the use as claimed in the invention, characterized in that said pharmaceutical composition is vehicled in a form which makes it possible to improve its stability and/or its immunogenicity, in particular in the form of a liposome.

Preferably, the invention comprises the use as claimed in the invention, characterized in that said vehicle is a viral vector containing a nucleic acid construct encoding said OmpA protein or a fragment thereof, said antigen or hapten, or said hybrid protein, or a transformed host cell capable of expressing said OmpA protein or a fragment thereof, said antigen or hapten, or said hybrid protein.

12 The invention also comprises the use as claimed in the invention, characterized in that said nucleic acid construct, or the nucleic acid construct contained in said vector or said transformed host cell, comprises a nucleic acid sequence chosen from the sequence SEQ ID No. 1, a fragment thereof having at least consecutive nucleotides, preferably 30 consecutive nucleotides, of the sequence SEQ ID No. 1, or a sequence having at least 80% homology, after optimal alignment, with one of said sequences.

Preferably, a subject of the present invention is the use of an enterobacterium OmpA protein, or of a fragment thereof, associated with an antigen or a hapten, for preparing a pharmaceutical composition intended to generate a cytotoxic T response directed against a tumor cell, as claimed in the present invention, characterized in that said antigen or hapten is the peptide of sequence SEQ ID No. 3: ELAGIGILTV and in that the cytotoxic T response is directed against melanoma cells.

Also preferably, a subject of the present invention is the use of an enterobacterium OmpA protein, or a fragment thereof, associated with the peptide of sequence ELAGIGILTV, as claimed in the present invention, for preparing a pharmaceutical composition intended to treat or prevent malignant melanomas.

In another aspect, a subject of the invention is a pharmaceutical composition as defined above, in particular: a pharmaceutical composition, characterized in that it comprises an enterobacterium OmpA protein, or a fragment thereof, associated, by mixing or by covalent coupling, with the peptide of sequence ELAGIGILTV; or a pharmaceutical composition, characterized in that it comprises a nucleic acid construct containing a 13 nucleic acid encoding an enterobacterium OmpA protein, or a fragment thereof, and a nucleic acid encoding the peptide of sequence ELAGIGILTV.

As defined above for the use as claimed in the invention, said pharmaceutical composition as claimed in the invention may, for example, comprise an enterobacterium OmpA protein, or a fragment thereof, coupled, by covalent attachment, to the peptide of sequence ELAGIGILTV by chemical synthesis, using recombinant OmpA or OmpA obtained via an extraction method, or coupled by genetic recombination.

As also defined above for the use as claimed in the invention, said pharmaceutical composition as claimed in the invention may, for example, comprise a vector comprising a nucleic acid construct containing a nucleic acid encoding an enterobacterium OmpA protein, or a fragment thereof, and/or a nucleic acid encoding the peptide of sequence ELAGIGILTV, or alternatively a transformed cell capable of expressing an enterobacterium OmpA protein, or a fragment thereof, and/or the peptide of sequence ELAGIGILTV.

A subject of the invention is also a pharmaceutical composition, characterized in that it comprises the Klebsiella pneumoniae OmpA protein of sequence SEQ ID No. 2, a protein, the sequence of which has at least homology, after optimal alignment, with the sequence SEQ ID No. 2, or a fragment of at least amino acids of said OmpA protein of sequence SEQ ID No. 2, associated, by mixing or by coupling, with the peptide of sequence ELAGIGILTV.

A subject of the present invention is also a pharmaceutical composition, characterized in that it comprises a nucleic acid construct containing a nucleic acid encoding the Klebsiella pneumoniae OmpA protein of sequence SEQ ID No. 2, a protein, the sequence of which 14 has at least 80% homology, after optimal alignment, with the sequence SEQ ID No. 2, or a fragment of at least 5 amino acids of said OmpA protein of sequence SEQ ID No. 2, and a nucleic acid encoding the peptide of sequence ELAGIGILTV.

According to the present invention, said compositions will be vehicled in a form which makes it possible to improve its stability and/or its immunogenicity, such as in the form of a liposome, or of a viral vector or of a transformed host cell capable of expressing said OmpA protein, or a fragment thereof, and said peptide of sequence ELAGIGILTV.

According to the present invention, said compositions will preferably be contained in a pharmaceutically acceptable medium.

For the purposes of the present invention, the pharmaceutically acceptable medium is the medium in which the compounds of the invention are administered, preferably a medium which can be injected into humans.

It may consist of water, of an aqueous saline solution or of an aqueous solution based on dextrose and/or on glycerol.

According to the present invention, said compositions may also contain a detergent.

The compositions as claimed in the invention may also contain a detergent, and in particular any type of pharmaceutically acceptable surfactant, such as for example anionic, cationic, nonionic or amphoteric surfactants. Use is preferably made of the detergents Zwittergent 3-12 and octylglucopyrannoside, and even more preferably Zwittergent 3-14.

0Q4514155 The invention also comprises the compositions as claimed in the invention, characterized in that they contain no other adjuvant for inducing a CTL response.

Preferably, said pharmaceutical composition as claimed in the invention contains no adjuvant of immunity, besides the enterobacterium OmpA protein, or a fragment thereof, or a nucleic acid encoding the enterobacterium OmpA protein, or a fragment thereof, characteristic of the pharmaceutical compositions of the invention.

As used herein, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude other additives, components, integers or steps.

The legends to the figures and examples which follow are intended to illustrate the invention without in any way limiting the scope thereof.

Legends to the figures: Figures 1A, 1B, 1C and 1D: Measurement of the anti-MELAN-A and anti- TRP-2 CTL activity of effector cells After immunization with 50 pg of hELA mixed with 3 jig of rP40 (figure 1A), [ig of hELA mixed with 300 lag of rP40 (figure 1B), 50 [ig of hELA coupled to (figure 1C) or 50 plg of the TRP-2 peptide mixed with 30 pg of rP40 (figure 1D), the draining lymph mode cells are stimulated with EL-4 A2/Kb cells (figures 20 1A, 1B and 1C) or EL-4 cells (figure 1D) which had been irradiated and prepulsed ::..with 1 pM of the relevant peptide, before being evaluated for their capacity to kill :e target cells which may (rectangle) or may not (diamond) have been prepulsed with

S

the relevant peptide.

The X-axes of the points of figures 1A to 1D correspond to the ratio of the 25 effector T cells (active lymphocytes) mixed together with the target cells (EL-4 A2/Kb or EL-4).

Figures 2A, 2B, 2C and 2D: Measurement of the anti-MELAN-A CTL activity of effector cells in the presence 16 of the rP40 protein compared to the CTL activity obtained with standard immunization protocol.

After immunization with hELA (50 rig) alone (ELA, figure 2A), hELA mixed with 300 4g of rP40 (ELA figure 2B), hELA coupled to 300 pg of rP40 figure 2C) or hELA mixed with 50 4g of P30 peptide adjuvanted with IFA (ELA IFA TT, figure 2D) (IFA for Incomplete Freund's Adjuvant and TT for Tetanus Toxoid), the draining lymph node cells are stimulated in vitro for two weeks with EL-4 A2/Kb cells which have been irradiated and prepulsed with 1 pM of the relevant peptide, before being evaluated for their capacity to kill EL-4 A2/Kb target cells which may (rectangle) or may not (diamond) have been prepulsed with the hELA peptide.

Example 1: Cloning of the gene encoding the Klebsiella pneumoniae P40 protein The gene encoding the P40 protein was obtained by PCR amplification using the genomic DNA of Klebsiella pneumoniae IP 1145 (Nguyen et col., Gene, 1998). The gene fragment encoding this gene is inserted into various expression vectors under the control of various promoters, in particular that of the Trp operon. The nucleotide sequence and the peptide sequence of the protein are represented by the sequences SEQ ID No. 1 and SEQ ID No. 2 hereinafter. An E. coli K12 producer strain was transformed with a pvaLP40 expression vector. The recombinant P40 protein (named rP40) is produced, in the form of inclusion bodies, with a considerable yield 10% in g of proteins/g of dry biomass).

This example is merely an illustration of the expression of the rP40 protein, this illustration possible being extended to other bacterial strains and to other expression vectors.

17 Example 2: Method for fermentation of rP40 fusion proteins An Erlenmeyer flask containing 250 ml of TSB (Tryptic Soy Broth, Difco) medium containing ampicilline (100 pg/ml, Sigma) and tetracyclin (8 pg/ml, Sigma) is inoculated with the transformed E. coli strain described above. After overnight incubation at 37 0

C,

200 ml of this culture are used to seed 2 liters of culture medium in a fermenter (Biolaffite, France). In a quite conventional way, the culture medium may be composed of chemical agents supplemented with vitamins and/or yeast extracts, which are known to promote high density bacterial cell growth.

The parameters controlled during the fermentation are: pH, stirring, temperature, level of oxygenation and supply of combined sources (glycerol or glucose). In general, the pH is regulated at 7.0 and the temperature is fixed at 37 0 C. The growth is controlled by supplying glycerol at a constant rate (12 ml/h) in order to maintain the dissolved oxygen tension signal at When the turbidity of the culture (measured at 580 nm) reaches the value of 80 (after culturing for approximately 24 hours), the protein production is treated by adding indole acrylic acid (IAA) at the final concentration of 25 mg/l. Approximately 4 hours after induction, the cells are harvested by centrifugation. The amount of wet biomass obtained is approximately 200 g.

Example 3: Method for extracting and purifying the protein Extracting the After centrifugation of the culture broth (4000 rpm, min, the cells are resuspended in a 25 mM Tris-HCl buffer, pH 8.5. The insoluble components, or inclusion bodies, are obtained after treatment with 18 lysozyme (0.5 g/liter, 1 hour at room temperature with gentle stirring). The inclusion body pellet obtained by centrifugation (15 min at 10,000 g at 4 0 C) is taken up in a 25 mM Tris-HCl buffer at pH 8.5 containing 5 mM MgC1 2 and then centrifuged (15 min at 10,000 g).

The inclusion bodies are solubilized at 37 0 C for 2 hours in a 25 mM Tris-HCl buffer, pH 8.5, containing 7 M urea (denaturing agent) and 10 mM of dithiothreitol (reduction of disulfide bridges). Centrifugation min at 10,000 g) makes it possible to eliminate the insoluble particles.

This is then followed by resuspension in 13 volumes of 25 mM Tris-HCl buffer, pH 8.5, containing NaCl (8.76 g/l) and Zwittergent 3-14 The solution is left overnight at room temperature with gentle stirring in contact with the air (to promote renaturation of the protein by dilution and reoxidation of the disulfide bridges).

Purifying the rP40 protein Anion exchange chromatography step After a further centrifugation, the solution is dialyzed against a 25 mM Tris-HCl buffer, pH containing 0.1% Zwittergent 3-14 (100 volumes of buffer) overnight at 4 0

C.

The dialyzate is loaded on to a column containing a support of the strong anion exchanger type (Biorad Macro Prop High Q gel) equilibrated in the buffer described above, at a linear flow rate of 15 cm/h. The proteins are detected at 280 nm. The rP40 protein is eluted, with a linear flow rate of 60 cm/h, for an NaC1 concentration of 0.2 M in the 25 mM Tris-HC1 buffer, pH 8.5: 0.1% Zwittergent 3-14.

19 Cation exchange chromatography step The fractions containing the rP40 protein are pooled and concentrated by ultrafiltration with the aid of an Amicon cell system with stirring, used with a Diaflo membrane (10 kDa cut-off threshold), for volumes of about 100 ml, or with the aid of a Millipore Minitan tangential flow filtration system, used with membrane plates having a 10 kDa cut-off threshold, for larger volumes. The fraction thus concentrated is dialyzed overnight at 4°C against a 20 mM citrate buffer, pH 3.0, containing 0.1% of Zwittergent 3-14.

The dialyzate is loaded on to a column containing a support of the strong cation exchanger type (Biorad Macro Prep High S gel) equilibrated in the 20 mM citrate buffer, pH 3.0, containing 0.1% of Zwittergent 3-14. The rP40 protein is eluted (rate 61 cm/h) for a 0.7 M NaCl concentration. The electrophoretic profiles show about a 95% degree of purity. The condition of the protein is monitored by SDS-PAGE. The P40 protein, extracted from the Klebsiella pneumoniae membrane, has a characteristic electrophoretic (migration) behavior depending on whether it is in denatured or native form.

The native form (p-sheet structure) in fact has a lower molecular mass than the form which is denatured (ahelical structure) by the action of a denaturing agent, such as urea or guanidine hydrochloride, or by heating at 100 0 C in the presence of SDS. The rP40 protein is not properly renatured at the end of renaturation, regardless of whether the latter is carried out in the presence or absence of 0.1% Zwittergent 3-14. On the other hand, total renaturation is obtained after dialysis against a 25 mM Tris/HCl buffer, pH containing 0.1% Zwittergent 3-14. However, it should be noted that this renaturation is only obtained when the dilution step and treatment at room temperature are, themselves, carried out in the 20 presence of Zwittergent 3-14 (negative results in the absence of detergent).

Example 4: Generation of CTLs The antitumor CTL responses directed against melanoma cells were defined for several antigens. These antigens are included in one of three categories: a) rejection antigen specific for melanoma, such as those of the MAGE family (review by van der Bruggen et al., Science 254:1643); b) antigens resulting from the mutation of normal proteins. This group includes MUM-1 (Coulie et al., Proc. Natl. Acad. Sci. USA 92:7976-7980 (1995)); CDK4 (Wolfel et al., Science 296:1281-1284 (1995)) and HLA- A2 (Brandel et al., J. Exp. Med. 183:2501-2508 (1996)); c) differentiation antigens expressed by melanomas and melanocytes. This group includes tyrosinase (Wolfel et al., Eur. J. Immunol. 4:759 (1994) and Brichard et al., Eur. J. Immunol. 26:224 (1996)); gp 100 (Kang et al., J. Immunol. 155:1343 (1995), Cox et al., Science 264:716 (1994), and Kawakami et al., J. Immunol.

155:3961 (1995)); gp75 (Wang et al., J. Exp. Med.

183:1131 (1996)), and Mart-1/MelanA (see US patent 5,620,886).

Of all these antigens, Mart-1/MelanA appears to be the best candidate for use in immunotherapy, this being for several reasons. Firstly, this antigen was identified on the basis of the CTL response, in vivo, of the lymphocytes infiltrating the melanoma and not that, in vitro, of the peripheral blood cells, which would suggest greater relevance of this antigen in the natural response, in vivo, against melanoma (Kawakami et al., J. Exp. Med. 180:347 (1994)). In addition, Mart-1/MelanA is expressed on all melanomas examined, which makes it a preferred target for intervention by immunotherapy. Finally, peptides derived from 21 Mart-1/MelanA are capable of inducing a specific CTL response in patients with melanoma expressing the HLA-A2 histocompatibility antigen (Rivoltini et al., J.

Immunol. 154:2257 (1995); Valmori et al., J. Immunol.

160:1750 (1998)).

HLA-A2 is the most common allele expressed in Caucasians. The CTL epitopes of Mart-1/MelanA have been defined for this allele. The antigenic peptide recognized by the majority of human CTL lines comprises amino acids 27-35 AAGIGILTV (Kawakami et al., J. Exp.

Med. 180:347 (1994)). In addition, studies on the affinity of binding with HLA-A*0201 and recognition by CTL clones have demonstrated that the optimum peptide for these two functions is the 26-35 decapeptide EAAGIGILTV (Romero et al., J. Immunol. 159:2366 (1997)). However, it appears that these peptides are weakly immunogenic in vitro (Valmori et al., J.

Immunol. 160:1750 (1998)) and in vivo (Jaeger et al., Int. J. Cancer 66:162 (1996)).

When comparing the amino acid sequence of the T epitopes of Mart-1/MelanA with the peptide motifs of A*0201 (Rammensee et al., Immunogenetics 41:178 (1995)), it appears that the 26-35 and 27-35 peptides have nondominant anchoring residues at position 2 and therefore weakly bind the HLA-A*0201 molecule (Kawakami et al., J. Immunol. 154:3961 (1995)), which might explain their weak immunogenicity. The international patent application published under the number WO 98/58951 describes an analog to the 26-35 peptide, in which the alanine at position 2 has been replaced with a leucine (sequence which will be named ELA).

The hELA peptide, used in the experiments below, is the subject of patent application WO 98/58951 which is the property of the Institut Ludwig de Recherche sur le Cancer [Ludwig Cancer Research Institute]. hELA is an analog of the 26-35 decapeptide (EAAGIGILTV) of 22 Melan-A/MART-1, which is a protein expressed on melanocytes and melanomas. Although the 26-35 decapeptide of Melan-A/MART-1 is capable of binding to the HLA-A0201 molecule (Romero et al., 1997, J.

Immunol. 159, 2366-2374), it is weakly immunogenic in vitro and in vivo (Valmori et al., 1998, J. Immunol.

160, 1750-1758). The hELA analog was generated by substituting the second amino acid of the 26-35 decapeptide of Melan-A/MART-1 (an alanine) with a leucine. The result of this substitution, which is based on analysis of the residues required for anchoring the peptides to the HLA-A0201 molecule, is more effective recognition by the CTLs of patients with melanoma and better immunogenicity in vitro (Valmori et al., 1998, J. Immunol. 160, 1750-1758).

HLA-A*0201/Kb (A2/Kb) transgenic mice of the strain C57B1/6 x BDA/2 (Vitiello et al., 1991, J. Exp. Med., 173, 1007-1015) were used in this study to test ELA.

The class I MHC molecule expressed in these mice is a chimeric molecule made from the al and a2 domains of the human HLA-A0201 molecule (the most common allotype found) and from the a3 domain of the murine Kb molecule.

The TRP-2 peptide of sequence SEQ ID No. 4 is an octapeptide corresponding to amino acids 181-188 (VYDFFVWL) of tyrosinase-related protein 2 (TRP-2).

TRP-2 is expressed in melanocytes and melanomas. It has been demonstrated that this antigen induces CTL responses which protect against melanoma in C57BL/6 (H-2Kb) mice (Bloom et al., 1997, J. Exp. Med. 185, 453-459).

A: Generation of anti-Melan-A and anti-TRP-2 CTLs after immunization with rP40 mixed with a peptide which is an analog to Melan-A or TRP-2 Experimental protocol 23 A2/Kb mice received, by subcutaneous injection at the base of the tail: 50 pg of ELA mixed with 3 or 300 pg of 50 pg of ELA covalently coupled to 300 pg of C57BL/6 mice received, by subcutaneous injection into the base of the tail: 50 pg of the TRP-2 peptide (181-188) mixed with 300 pg of Generation of cytotoxic effector cells days after immunization, the mice are sacrificed and the lymphocytes from the draining lymph nodes are recovered in order to be stimulated, in vitro, with the relevant peptide.

These lymphocytes (4 to 5 x 106) are cultured in a 24 well plate in DMEM plus 10 mM HEPES, 10% FCS and 50 pM P-2-mercaptoethanol, with 2 to 5 x 105 EL-4 A2/Kb cells or EL4 cells which have been irradiated kRads) and prepulsed for 1 h at 37 0 C with 1 .M of the relevant peptide. After two weekly stimulations, the cells are assayed for their cytotoxic activity.

Measurement of cytotoxic activity The EL-4 A2/Kb cells or EL4 cells are incubated for 1 h with 51 Cr in the presence or absence of the relevant peptide, washed and then coincubated with the effector cells at various ratios, in a 96-well plate in a volume of 200 il for 4 to 6 h at 37 0 C. The cells are then centrifuged and the 5Cr release is measured in 100 pl of supernatant. The percentage of specific lysis is calculated as follows: specific lysis (experimental release spontaneous release) (total release spontaneous release) X 100.

Results 24 As shown in figures 1A to 1D, the immunization of mice with an optimal dose of rP40 (300 pg) in a mixture with hELA (figure 1B) or TRP-2 (figure ID) induces a strong specific CTL response. Such a response is also observed after immunization with rP40 coupled to hELA (figure 1C). On the other hand, the immunization with the peptide alone or rP40 alone (results not shown) or with the hELA peptide in a mixture with a suboptimal dose of rP40 (3 ig) does not induce any CTL activity (figure 1A) These results demonstrate that the molecule mixed with or coupled to immunogenic peptides makes it possible to induce a specific CTL response in vivo, this being without the addition of adjuvant.

B: Generation of anti-Melan-A CTLs after immunization with rP40 mixed with a peptide which is an analog to Melan-A, compared to a standard immunization protocol Experimental protocol A2/Kb mice received: 50 pl of IFA (incomplete Freud's adjuvant) by subcutaneous injection at the base of the tail, then, 3 weeks later, 50 pg of hELA in the presence of 50 ig of a helper-T p30 peptide derived from Tetanus Toxoid (TT) (Panina-Bordignon et al., Eur. J. Immunol., 1989, 19, 2237) adjuvanted with IFA. This protocol has been described for generating anti-peptide CTLs (Valmori et al., Eur. J. Immunol., 1994, 24, 1458) and is used as a positive control.

50 ng of hELA alone or 300 pg of rP40 mixed with or coupled to 50 ng of hELA.

Generation of cytotoxic effector cells 10 days after the final immunization, the mice are sacrificed and the lymphocytes from the draining lymph nodes are recovered in order to be stimulated, in vitro, with the relevant peptide.

004514155 These lymphocytes (4 to 5 x 106) are cultured in a 24-well plate in DMEM plus 10 mM HEPES, 10% FCS and 50 pM p-2-mercaptoethanol, with 2 to 5 x 105 EL-4 A2/Kb cells (murine cells transfected with the HLA-A* 0201/Kb gene) which have been irradiated (10 kRads) and prepulsed for 1 h at 370C with 1 tM of the relevant peptide.

After one, two or three weekly stimulations, the cells are assayed for their cytotoxic activity.

The cytotoxic activity is measured according to the method described above.

Results After immunization with nonadjuvanted rP40 coupled to hELA, an anti-hELA CTL activity comparable to that observed after immunization with hELA is measured (cf. figures 2C and 2D). Similarly, the rP40 hELA peptide mixture, itself also nonadjuvanted, generates CTLs in a way which is similar to that obtained with a conventional protocol for generating CTLs (cf. figures 2B and 2D).

No CTL activity was detected after immunization with the peptide alone (cf.

figure 2A) or the rP40 protein alone (not shown), regardless of the day on which the effector cells were stimulated.

Reference to any prior art in the specification is not, and should not be 20 taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction.

part of the common general knowledge in Australia or any other jurisdiction.

1 SEQUENCE LISTING <110> PIERRE FABRE MEDICAMENT <120> USE OF AN OmpA ENTEROBACTERIUM PROTEIN ASSOCIATED WITH THE ELAGIGILTV PEPTIDE, FOR TREATING MELANOMAS.

<130> D18441 <150> FR 99 01917 <151> 1999-02-17 <160> 4 <170> PatentIn Ver. 2.1 <210> <211> <212> <213> <220> <221> <222> <220> <221> <222> <220> <221> <222> 1 1035

ADN

Klebsiella pneumoniae exon (1)..(1032) intron (1033)..(1035)

CDS

(1)..(1032) <400> 1 atg aaa gca att ttc gta ctg aat gcg gct ccg aaa gat aac Met Lys Ala Ile Phe Val Leu Asn Ala Ala Pro Lys Asp Asn acc tgg Thr Trp tat gca ggt Tyr Ala Gly tac ggt aac Tyr Gly Asn ggt Gly aaa ctg ggt tgg Lys Leu Gly Trp tcc Ser 25 cag tat cac gac Gin Tyr His Asp acc ggt ttc Thr Gly Phe aac gat cag Asn Asp Gin ggt ttc cag aac Gly Phe Gin Asn aac ggt ccg acc Asn Gly Pro Thr ctt ggt Leu Gly get ggt gcg ttc Ala Gly Ala Phe ggt tac cag gtt Gly Tyr Gin Val ccg tac ctc ggt Pro Tyr Leu Gly ttc Phe gaa atg ggt tat Glu Met Gly Tyr gac Asp 70 tgg ctg ggc cgt Trp Leu Gly Arg atg Met 75 gca tat aaa ggc Ala Tyr Lys Gly agc Ser gtt gac aac ggt Val Asp Asn Gly ttc aaa gct cag ggc gtt cag ctg Phe Lys Ala Gin Gly Val Gin Leu acc gct aaa Thr Ala Lys ctg ggt tac ccg atc act gac gat ctg gac ate tac acc cgt ctg ggc Leu Gly Tyr Pro Ile Thr Asp Asp Leu Asp Ile Tyr Thr Arg Leu Gly -2- 100 110 ggc atg gtt Gly Met Val 115 tgg cgc gct gac Trp Arg Ala Asp aaa ggc aac tac Lys Gly Asn Tyr tct acc ggc Ser Thr Gly gtt tcc Val Ser 130 cgt agc gaa cac Arg Ser Glu His gac Asp 135 act ggc gtt tcc Thr Gly Val Ser cca Pro 140 gta ttt gct ggc Val Phe Ala Gly ggc Gi y 145 gta gag tgg gct Val Glu Trp, Ala gtt Val1 150 act cgt gac atc Thr Arg Asp Ile gct Al a 155 acc cgt ctg gaa Thr Arg Leu Giu ta c Tyr 160 432 480 528 cag tgg gtt aac Gin Trp Val Asn atc ggc gac gcg Ile Gly Asp Ala ggc Gi y 170 act gtg ggt acc Thr Val Gly Thr cgt cct Arg Pro 175 gat aac ggc atg ctg agc ctg ggc gtt Asp Asn Gly Met Leu Ser Leu Gly Val 180 185 tcc tac cgc ttc Ser Tyr Arg Phe ggt cag gaa Gly Gin Glu 190 ccg gaa gtg Pro Glu Val 576 gat gct gca Asp Ala Ala 195 ccg gtt gtt gct Pro Val Val Ala ccg Pro 200 gct ccg gct ccg Ala Pro Ala Pro qct Al a 205 gct acc Ala Thr 210 aag cac ttc acc Lys His Phe Thr ctg Leu 215 aag tct gac gtt Lys Ser Asp Val ttc aac ttc aac Phe Asn Phe Asn gct acc ctg aaa Ala Thr Leu Lys ccg Pro 230 gaa ggt cag cag Glu Gly Gin Gin gct Al a 235 ctg gat cag ctg Leu Asp Gin Leu tac Tyr 240 act cag ctg agc Thr Gin Leu Ser aac As n 245 atg gat ccg aaa Met Asp Pro Lys ga c Asp 250 ggt tcc gct gtt Gly Ser Ala Val gtt ctg Val Leu 255 ggc tac acc Gly Tyr Thr gag aaa cgt Glu Lys Arg 275 gac Asp 260 cgc atc ggt tcc Arg Ile Gly Ser gaa Glu 265 gct tac aac cag Ala Tyr Asn Gin cag ctg tct Gin Leu Ser 270 aaa ggc atc Lys Gly Ile gct cag tcc gtt Ala Gin Ser Val gtt Val1 280 gac tac ctg gtt Asp Tyr Leu Val gct Ala 285 ccg gct Pro Ala 290 ggc aaa atc tcc Gly Lys Ile Ser gct Al a 295 cgc ggc atg ggt Arg Gly Met Gly gaa Giu 300 tcc aac ccg gtt Ser Asn Pro Val act Th r 305 ggc aac acc tgt Gly Asn Thr Cys aac gtg aaa gct Asn Val Lys Ala gct gcc ctg atc Ala Ala Leu Ile 912 960 1008 tgc ctg gct ccg Cys Leu Ala Pro ga t Asp 325 cgt cgt gta gag Arg Arg Val Giu atc Ilie 330 gaa gtt aaa ggc Giu Val Lys Gly tac aaa Tyr Lys 335 gaa gtt gta Glu Val Val act Th r 340 cag ccg gcg ggt taa Gin Pro Ala Giy 1035 3 <210> 2 <211> 344 <212> PRT <213> Klebsiella pneumoniae <400> 2 Met Lys Ala Ile Phe Val Leu 1 Tyr Tyr Leu Phe Val Leu Gly Val Gly 145 Gin Asp Asp Ala Lys 225 Thr Gly Ala Gly Gly Glu Asp Gly Met Ser 130 Val Trp Asn Ala Thr 210 Ala Gin Tyr Gly Asn Ala Met Asn Tyr Val 115 Arg Glu Val Gly Ala 195 Lys Thr Leu Thr Gly Gly Gly Gly Gly Pro 100 Trp Ser Trp Asn Met 180 Pro His Leu Ser Asp 260 5 Lys Phe Ala Tyr Ala Ile Arg Glu Ala Asn 165 Leu Val Phe Lys Asn 245 Arg Leu Gin Phe Asp 70 Phe Thr Ala His Val 150 Ile Ser Val Thr Pro 230 Met Ile Gly Asn Gly 55 Trp Lys Asp Asp Asp 135 Thr Gly Leu Ala Leu 215 Glu Asp Gly Asn Ala Trp Ser 25 Asn Asn 40 Gly Tyr Leu Gly Ala Gin Asp Leu 105 Ser Lys 120 Thr Gly Arg Asp Asp Ala Gly Val 185 Pro Ala 200 Lys Ser Gly Gin Pro Lys Ser Glu 265 Ala 10 Gin Gly Gin Arg Gly 90 Asp Gly Val Ile Gly 170 Ser Pro Asp Gin Asp 250 Ala Pro Tyr Pro Val Met 75 Val Ile Asn Ser Ala 155 Thr Tyr Ala Val Ala 235 Gly Tyr Lys Asp His Asp Thr Arg Asn Pro Ala Tyr Gin Leu Tyr Thr Tyr Ala 125 Pro Val 140 Thr Arg Val Gly Arg Phe Pro Ala 205 Leu Phe 220 Leu Asp Ser Ala Asn Gin Asn Thr Asn Tyr Lys Thr Arg 110 Ser Phe Leu Thr Gly 190 Pro Asn Gin Val Gin 270 Thr Trp Gly Phe Asp Gin Leu Gly Gly Ser Ala Lys Leu Gly Thr Gly Ala Gly Glu Tyr 160 Arg Pro 175 Gln Glu Glu Val Phe Asn Leu Tyr 240 Val Leu 255 Leu Ser Glu Lys Arg Ala Gin Ser Val 275 Val Asp Tyr Leu Val Ala Lys Gly Ile 280 285 4 Pro Ala Gly Lys Ile Ser Ala Arg Gly Met Gly Glu Ser Asn Pro Val 290 295 300 Thr Gly Asn Thr Cys Asp Asn Val Lys Ala Arg Ala Ala Leu lie Asp 305 310 315 320 Cys Leu Ala Pro Asp Arg Arg Val Glu Ile Glu Val Lys Gly Tyr Lys 325 330 335 Glu Val Val Thr Gin Pro Ala Gly 340 <210> 3 <211> <212> PRT <213> Homo sapiens <220> <223> Peptide derived from the Mart-1/MelanA antigen expressed by melanoma cells.

<400> 3 Glu Leu Ala Gly Ile Gly Ile Leu Thr Val 1 5 <210> 4 <211> 8 <212> PRT <213> Homo sapiens <220> <223> Derivative of tyrosine-related protein 2 (TRP-2).

<400> 4 Val Tyr Asp Phe Phe Val Trp Leu 1

Claims (23)

1. The use of an enterobacterium OmpA protein, or of a fragment thereof, associated with the peptide of sequence SEQ ID No. 3 ELAGIGILTV, for preparing a pharmaceutical composition intended to generate a cytotoxic T response directed against melanoma cells.

2. The use of an enterobacterium OmpA protein, or of a fragment thereof, associated with the peptide of sequence SEQ ID No. 3, as claimed in claim 1, for preparing a pharmaceutical composition intended for treating or preventing malignant melanomas.

3. The use as claimed in claim 1 or 2, characterized in that said enterobacterium OmpA protein, or a fragment thereof, is obtained using a method of extraction from a culture of said enterobacterium.

4. The use as claimed in claim 1 or 2, characterized in that said enterobacterium OmpA protein, or a fragment thereof, is obtained via the recombinant route. The use as claimed in one of claims 1 to 4, characterized in that said enterobacterium is Klebsiella pneumoniae.

6. The use as claimed in claim 5, characterized in that the amino acid sequence of said OmpA protein, or a fragment thereof, comprises: a) the amino acid sequence of sequence SEQ ID No. 2; b) the amino acid sequence of a sequence having at least 80% homology with the sequence SEQ ID No. 2; or 27 c) the amino acid sequence of a fragment of at least 5 amino acids of a sequence as defined in a)

7. The use as claimed in one of claims 1 to 6, characterized in that said peptide of sequence SEQ ID No. 3 is coupled to or mixed with said OmpA protein or a fragment thereof.

8. The use as claimed in claim 6, characterized in that said peptide of sequence SEQ ID No. 3 is coupled, by covalent attachment, with said OmpA protein or a fragment thereof.

9. The use as claimed in claim 8, characterized in that the coupling by covalent attachment is coupling produced by chemical synthesis. The use as claimed in claim 9, characterized in that one or more attachment elements is(are) introduced into said OmpA protein, or a fragment thereof, and/or into said peptide of sequence SEQ ID No. 3, in order to facilitate the chemical coupling.

11. The use as claimed in claim 10, characterized in that said attachment element introduced is an amino acid.

12. The use as claimed in claim 8, characterized in that the hybrid protein resulting from the coupling between said peptide of sequence SEQ ID No. 3 and said OmpA protein, or a fragment thereof, is obtained by genetic recombination.

13. The use as claimed in claim 12, characterized in that the pharmaceutical composition comprises a nucleic acid construct encoding said hybrid protein. 28

14. The use as claimed in claim 13, characterized in that said nucleic acid construct is contained in a vector, or in a transformed host cell capable of expressing said hybrid protein. The use as claimed in one of claims 1 to 14, for preparing a pharmaceutical composition which can be administered by the subcutaneous or intradermal route.

16. The use as claimed in one of claims 1 to characterized in that said pharmaceutical composition is vehicled in a form which makes it possible to improve its stability and/or its immunogenicity.

17. A pharmaceutical composition as defined in any one of claims 1 to 16.

18. The pharmaceutical composition as claimed in claim 17, characterized in that it comprises the Klebsiella pneumoniae OmpA protein of sequence SEQ ID No. 2, a protein, the sequence of which has at least 80% homology with the sequence SEQ ID No. 2, or a fragment of at least 5 amino acids of said OmpA protein of sequence SEQ ID No. 2, associated, by mixing or by coupling, with the peptide of sequence SEQ ID No. 3.

19. A pharmaceutical composition, characterized in that it comprises a nucleic acid construct containing a nucleic acid encoding the Klebsiella pneumoniae OmpA protein of sequence SEQ ID No. 2, a protein, the sequence of which has at least homology with sequence SEQ ID No. 2, or a fragment of at least 5 amino acids of said OmpA protein of sequence SEQ ID No. 2, and a nucleic acid encoding the peptide of sequence SEQ ID No. 3. 004514155 29 The composition as claimed in one of claims 17 to 19, characterized in that said pharmaceutical composition is vehicled in a form which makes it impossible to improve its stability and/or its immunogenicity.

21. The composition as claimed in claim 20, characterized in that said vehicle is a liposome, or a viral vector or a transformed host cell capable of expressing said OmpA protein, or a fragment thereof, and said peptide of sequence SEQ ID No. 3.

22. The composition as claimed in one of claims 17 to 21, characterized in that said composition is contained in a pharmaceutically acceptable medium.

23. The composition as claimed in one of claims 17 to 22, characterized in that said composition also contains a detergent.

24. The composition as claimed in one of claims 17 to 23, without any other adjuvant for inducing a CTL response. A method of generating a cytotoxic T response directed against melanoma cells comprising use of an enterobacterium OmpA protein, or a fragment thereof, associated with the peptide of Sequence ID No. 3, ELAGIGILTV.

26. A method of treating or preventing malignant melanomas comprising use of an enterobacterium OmpA protein, or a fragment thereof, associated with the peptide of Sequence ID No. 3. ELAGIGILTV.

27. The use of an enterobacterium OmpA protein according to any one of 20 claims 1 to 15, substantially as hereinbefore described with reference to any one of the examples.

28. A pharmaceutical composition substantially as hereinbefore described with reference to any one of the examples. Pierre Fabre Medicament 25 By their registered patent attorneys: Freehills Carter Smith Beadle 19 July 2004