WO2000050071A1 - PROTEIN OmpA OF KLEBSIELLA PNEUMONIAE ASSOCIATED WITH THE HCG HORMONE OR A COMPOUND INVOLVED IN CELL PROLIFERATION OR FERTILITY - Google Patents

Abstract

The invention concerns the use of a mixture or complex comprising an enterobacterium membrane protein OmpA, in particular of Klebsiella pneumoniae, associated with an immunogen selected among the beta hCG, a compound involved in tumour cell proliferation or fertility, or with one of their fragments, for preparing a pharmaceutical composition for enhancing the response against said immunogen. The invention further concerns a pharmaceutical composition comprising said mixture or complex in particular for preventing and for treating tumours, or for treating fertility.

Description

 KLEBSIELLA PNEUMONIAE OMPA PROTEIN ASSOCIATED WITH HORMONE HCG OR A COMPOUND INVOLVED IN TUMOR CELL PROLIFERATION OR FERTILITY

The invention relates to the use of a mixture or complex comprising an OobA membrane protein of enterobacterium, in particular of Klebsiella pneumoniae, associated with an immunogen chosen from βhCG, a compound involved in the proliferation of tumor cells or in fertility. , or a fragment thereof, for the preparation of a pharmaceutical composition intended to increase the immune response against said immunogen. The invention further comprises a pharmaceutical composition comprising said mixture or complex, intended in particular for the prevention and treatment of tumors, or for the treatment of fertility.

Vaccination is an effective way to prevent or reduce viral or bacterial infections. The success of vaccination campaigns in this area has made it possible to extend the concept of vaccine in the field of autoimmune diseases and cancer.

Human gonadotropin chorionic hormone (hCG) is a dimeric hormone which belongs to the super family of growth factors with a cysteine core (TGFβ, NGF, PDGFβ) and to the family of gonadotropins. This family includes on the one hand the pituitary hormones LH (luteinizing hormone or interstitial cell stimulating hormone) and FSH (follicle stimulating hormone or follitropin) responsible for the stimulation of testicular and ovarian functions. On the other hand, hormones of placental origin, among which the hCG synthesized during pregnancy and whose physiological role is to improve the growth of the functional corpus luteus.

All these hormones, despite the diversity of their physiological functions, are close in structure and consist of two chains or subunits α or β glycosylated at specific sites and linked by disulfide bridges. Within a given species, the sequence of the α subunit is fundamentally identical for all of these hormones. The hormonal activity of these αβ hybrids is dictated by the particularity of the β subunit present (see Figure 1).

In addition to its placental origin, a pituitary localization has been shown for hCG or hCG-like molecules. This pituitary source (Hoermann et al. J. Endocrinal Metab. 1990; 71: 179-186) could be responsible for the very low concentrations of "hCG like" material present in the serum of healthy individuals of both sexes (Odell et al. N Engl J. Med 1987; 317: 1688-1691; Odell et al. J. Clin. Endocrinol. Metab. 1990; 71: 1318-1321).

Furthermore, hCG and its fragments appear to be products of the malignant transformation. Indeed, while benign neoplasms do not express, or do not produce, this kind of material, several authors have shown that malignant tumors, but also embryonic and fetal cells, express hCG in vivo and in vitro. It has been observed that immunoreactivity to hCG is present in 10 to 50% of patients with different non-trophoblastic tumors including tumors of the endometrium, ovary, pancreas, stomach, colon, the liver, lung, breast, kidneys, bladder and certain lymphomas (Braunstein et al. Ann. Intern. Med. 1973; 78: 39-45; Kuida et al. Arch. Pathol. Lab. Med. 1988 ; 112: 282-285).

In tumor cells, hCG, its subunits or its fragments can be associated with the membrane, thus constituting an insoluble pool of non-extractable sialoglycoproteins without destruction of the membrane. They can also constitute a soluble hydrophilic pool secreted by cells and present in the blood and urine.

It should be noted that in the case of certain tumor types such as the lung or the bladder, the existence of βhCG activity is correlated with the stage of advancement of the tumor (Yoshimura et al. Cancer 1994; 73: 2745-2752 ; Norman et al. Clinical Endocrinol. 1985; 23: 25-34; Olivier et al. Molecular Biotherapy 1988; 1: 43-47).

All of these data indicate that immunological targeting of βhCG or its fragments may be of particular interest in the treatment of certain types of cancer. Preclinical studies in mice (Acevedo et al. Cancer Detection and Prevention 1987; suppl. 1: 477-486) or in rats (Kellen et al. Cancer 1982; 49: 2300-2304) have respectively shown efficacy of DT-CTP37 or TT-βhCG conjugates on the growth of a lung tumor (Lewis LL / 2 carcinoma) or on the appearance of breast tumor metastases (R3230).

The use of a carrier protein called P40, derived from the outer membrane of type A (or OmpA for "Outer Membrane protein typeA" of Klebsiella pneumoniae has been described in patent applications WO 95/27787 and WO 96/14415 as making it possible to increase the immune response, in particular of humoral type, when this was associated with an immunogen derived from a viral protein of the respiratory syncytial virus (NRS). Surprisingly, the authors of the present invention have demonstrated that the association between an enterobacterium protein OmpA and a compound involved in the proliferation of tumor cells or in fertility could not only induce an antibody response specific for the compound and high from the second injection with low concentrations of the associated products injected, but that moreover this association led to a significant decrease in tumor mass in the treated animals, greater than the reduction obtained with a carrier compound such as diphtheria toxin.

Thus, the present invention relates to the use of an enterobacterium protein OmpA or a fragment thereof, associated with an immunogen chosen from cytokines or their receptor, growth factors or their receptor, hormones or their receptor and / or tumor-specific markers, to one of their fragments, or to one of their analogs, for the preparation of a pharmaceutical composition intended to improve the immunological response against said immunogen, in particular in the absence of adjuvant of l 'immunity. Among the cytokines, growth factors and / or hormones, there may be mentioned in particular, but not limited to:

- cytokines and / or growth factors as well as their receptor involved in tumor cell proliferation and / or tumor specific markers such as for example: G-CSF, IL-6 receptor, IGF-1, IGF-2, EGF, FGF, NEGF, CEA, PSA; - growth hormones or hormones involved in fertility, such as β-hCG; as well as any peptide whose amino acid sequence has at least 80% homology with the sequences of said cytokines, growth factors and / or hormones according to the invention.

In the present invention, the term “protein” will also be understood to denote the peptides or polypeptides and the term “OmpA” (for “Outer Membrane Protein”), the proteins of the outer membrane of type A.

The term “fragment of an OmpA protein” is intended to denote in particular any fragment of the amino acid sequence included in the amino acid sequence of the OmpA protein which, when associated with an immunogen, is capable of generating or increasing an immune response, in particular a humoral response, directed against said immunogen and comprising at least 5 amino acids, preferably at least 10 amino acids or more preferably at least 15 amino acids.

The term “fragment of a cytokine, of a growth factor or of a hormone” is intended to denote in particular any fragment of amino acid sequence included in the amino acid sequence of the cytokine, of growth factor or of the hormone which, alone or combined with an immunity adjuvant, is capable of inducing an immune response, in particular humoral, directed against said fragment, comprising at least 5 amino acids, preferably at least 10 amino acids, or more preferred at least 15 amino acids of the amino acid sequence of said cytokine, said growth factor or said hormone.

The expression “analog of an immunogen of the cytokine, growth factor or hormone type” is intended to denote in particular in the present description a compound having a structural analogy with said immunogen, capable of inducing an immunological response directed against said immunogen in a previously organism. immunized with said analog.

In a particular embodiment, the invention comprises the use of an enterobacterium OmpA protein or of a fragment thereof according to the invention, characterized in that said enterobacterium OmpA protein, or one of its fragments, is obtained by an extraction process 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. We can cite for example, but not limited to the extraction process described by Haeuw JF 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 according to the invention, characterized in that said enterobacterium OmpA protein, or the one of its fragments is obtained recombinantly.

The methods for preparing recombinant proteins are today well known to those skilled in the art and will not be developed in the present description, however, reference may be made to the method described in the examples. Among the cells which can be used for the production of these recombinant proteins, mention must of course be made of bacterial cells (Olins PO and Lee SC, 1993, Recent advances in heterologous gene expression in E. coli. Curr. Op. Biotechnology 4: 520-525 ), but also yeast cells (Buckholz RG, 1993, Yeast Systems for the Expression of Heterologous Gene Products. Curr. Op. Biotechnology 4: 538-542), as well as animal cells, in particular cell cultures mammals (Edwards CP and Aruffo A., 1993, Current applications of COS cell based transient expression Systems. Curr. Op. Biotechnology 4: 558-563) but also insect cells in which methods can be used using example of baculo viruses (Luckow NA, 1993, Baculovirus Systems for the expression of human gene products. Curr. Op. Biotechnology 4: 564-572).

Most preferably, the use according to the invention is characterized in that said enterobacterium is Klebsiella pneumoniae. In particular, the invention relates to the use according to the invention, characterized in that the amino acid sequence of said OmpA protein of Klebsiella pneumoniae, or one of its fragments, comprises: a) the sequence d amino acids of sequence SEQ ID Ν ° 2; b) the amino acid sequence of a sequence having a homology of at least 80, preferably 90%, 95% and 99%, with the sequence SEQ ID No. 2; or c) the amino acid sequence of a fragment of at least 5 amino acids, preferably at least 10 amino acids, or more preferably at least 15 amino acids, of a sequence as defined in a).

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

By “percentage of identity” between two nucleic acid or amino acid sequences within the meaning of the present invention is meant a percentage of identical nucleotides or amino acid residues 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 traditionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by "comparison window" to identify and compare the regions. sequence similarity locale. The optimal alignment of the sequences for comparison can be achieved, besides manually, by means of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2: 482], using the local homology algorithm of Neddleman and Wunsch (1970) [J. Mol. Biol. 48: 443], using the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85: 2444], using computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by BLAST comparison software N or BLAST P).

The percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences per comparison window in which the region of the nucleic acid or amino acid sequence to be compared. may include additions or deletions from the reference sequence for optimal alignment between these two sequences. The percentage of 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, by dividing this number of identical positions by the total number of positions in the comparison window. and multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences.

For example, we could use the BLAST program, "BLAST 2 sequences", available on the website htφ: //www.ncbi.nlm.nih.gov/gorf/bl2.html, the parameters used being those given by default (in particular for the parameters “open gap penaltie”: 5, and “extenxion gap penaltie”: 2; the matrix chosen being for example the matrix “BLOSUM 62” proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly by the program.

Thus, using these sequence comparison software, such as in particular the "BLAST version 2" software with the substitution matrices and their associated parameters by default, those skilled in the art will be able to easily determine the sequences having a identity percentage of at least 80% after optimal alignment according to the present invention.

The present invention also relates to the use according to the invention, characterized in that said immunogen is coupled by covalent bond, in particular by chemical coupling, with said protein OmpA or one of its fragments.

The invention further comprises the use according to the invention, characterized in that said immunogen is chosen from peptides.

In a particular embodiment, the use according to the invention is characterized in that one or more binding elements are introduced into said OmpA protein, or one of its fragments, and / or into said immunogen to facilitate the chemical coupling, preferably, said connecting element introduced is an amino acid.

According to the invention, it is possible to introduce one or more binding elements, in particular amino acids to facilitate the coupling reactions between the OmpA protein, or one of its fragments, and said immunogen. The covalent coupling between the OmpA protein, or one of its fragments, and said immunogen according to the invention can be carried out at the N- or C-terminus of the OmpA protein, or one of its fragments. The bifunctional reagents allowing this coupling will be determined according to the end of the OmpA protein, or one of its fragments, chosen to effect the coupling and to the nature of said immunogen to be coupled.

In another particular embodiment, the use according to the invention is characterized in that said hybrid protein obtained after coupling between said immunogen and said OmpA protein, or one of its fragments, is prepared by genetic recombination. The conjugates resulting from a coupling to said OmpA protein, or one of its fragments, can be prepared by genetic recombination. The chimeric or hybrid protein (conjugate) can be produced by recombinant DNA techniques by insertion or addition to the DNA sequence coding for said OmpA protein, or one of its fragments, of a sequence coding for said immunogen protein in nature.

The methods for synthesizing hybrid molecules include the methods used in genetic engineering to construct hybrid polynucleotides encoding the desired polypeptide sequences. One can, for example, advantageously refer to the technique for obtaining genes coding for fusion proteins described by D.N. Goeddel (Gene expression technology, Methods in Enzymology, vol. 185, 3-187, 1990).

In another aspect, the invention relates to the use according to the invention, characterized in that the pharmaceutical composition contains a vector comprising a nucleic construct coding for said hybrid protein or a host cell transformed by said vector capable of expressing said protein hybrid.

In a particular embodiment of the invention, said nucleic construct contained in said vector comprises a nucleic sequence chosen from the sequence SEQ ID Ν ° 1, one of its fragments having at least 15 consecutive nucleotides of the sequence SEQ ID N ° 1, or a sequence having a homology of at least 80%, preferably 90%, 95% and 99%, with one of said sequences. The present invention particularly relates to the use according to the invention, characterized in that the immunogen is the hormone βhCG, or one of its fragments, in particular the peptide of amino acid sequence SEQ ID No. 3: TCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ . A subject of the invention is also the use according to the invention, characterized in that the immunogen is a cytokine or a growth factor involved in the proliferation of tumor cells.

A subject of the invention is also the use according to the invention, for the preparation of a pharmaceutical composition of the vaccine type intended for preventing or treating cancers such as cancer of the pancreas, colon, prostate, the bladder, lung or any other tumor.

In another aspect, the invention relates to the use according to the invention, characterized in that the immunogen is a hormone, or one of its fragments, involved in fertility. Thus, the present invention comprises the use according to the invention for the preparation of a pharmaceutical composition of the vaccine type intended for contraception.

The invention also relates to a pharmaceutical composition characterized in that it comprises an enterobacterium OmpA protein or one of its fragments, in particular of Klebsiella pneumoniae, associated with an immunogen chosen from cytokines, growth factors and / or hormones, or a fragment thereof.

The invention further relates to a pharmaceutical composition characterized in that it comprises a nucleic construct coding for an enterobacterium OmpA protein, or one of its fragments, in particular of Klebsiella pneumoniae, associated with a nucleic construct coding for an immunogen peptide chosen from cytokines, growth factors and / or hormones, or a fragment thereof.

In a preferred embodiment of the invention, the immunogen is the hormone βhCG, one of its fragments or analogous fragments, in particular the peptide of amino acid sequence SEQ ID No. 3. Of course, and in the same way as described above for the uses according to the invention, said pharmaceutical composition may comprise said OmpA protein, or one of its fragments, and said associated immunogen in identical forms such as mixed, coupled, in the form of recombinant hybrid proteins or a host cell capable of expressing said hybrid protein.

The invention also includes the use according to the invention, or pharmaceutical composition according to the invention, characterized in that said pharmaceutical composition is conveyed in a form making it possible to improve its stability and / or its immunogenicity, preferably in vivo. Preferably, said vehicle is a liposome, a viral vector containing a nucleic construct coding for said OmpA protein, or one of its fragments, said immunogen, or for said hybrid protein, or a transformed host cell capable of expressing said protein. OmpA, or a fragment thereof, said immunogen, or said hybrid protein. Finally, the invention comprises the use or pharmaceutical composition according to the invention, characterized in that said pharmaceutical composition comprises an immunity adjuvant, mixed or coupled to said OmpA protein, to said immunogen, or to one of their fragments, or to said hybrid protein.

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

Fi, gure 1: Schematic representation of the heterodimeric structure of hCG. Figure 2: Mass spectrum (ES-MS) of the purified peptide [β-hCG (109-145)]. Figures 3A and 3B: SDS-PAGE electrophoresis analysis (10% polyacrylamide gel) of the conjugates rP40-CTP37 (Figure 3A) and DT-CTP37 (Figure 3B) (rP40 for recombinant P40 protein). Figure 3A:

- track 1: molecular weight standard,

- track 2: rP40, - track 3: rP40 activated using MCS,

- lane 4: rP40-CTP37 conjugate before dialysis, - track 5: rP40-CTP37 conjugate after dialysis,

- lane 6: rP40-CTP37 conjugate after purification and concentration. Figure 3B:

- lane 1: molecular weight standard, - lane 2: DT,

- track 3: DT dialyzed,

- track 4: DT activated using MCS,

- track 5: DT-CTP37 conjugate before dialysis,

- lane 6: DT-CTP37 conjugate after dialysis, - lane 7: DT-CTP37 conjugate after purification and concentration.

Figure 4: Anti-CTP37 humoral response induced after injection of variable doses of rP40-CTP37 in C57 / B16 mice.

Figure 5: Anti-CTP37 humoral response induced after injection of 200 μg of rP40-CTP37 conjugate in the absence or presence of an adjuvant in C57 / B16 mice. Figure 6: Comparison of the anti-CTP37 IgG response in presensitized or non-presensitized mice with the bacterium Klebsiella pneumoniae. Figures 7A and 7B: Evaluation of the anti-tumor activity of the rP40-CTP37 conjugate in C57 / B16 mice not presensitized (Figure 7A) or presensitized (Figure 7B) with Klebsiella pneunoniae before treatment and grafting of the tumor. EXAMPLE 1 Cloning of the rP40 Gene

The gene coding for the P40 protein of Klebsiella pneumoniae was obtained by PCR amplification from the genomic DNA of Klebsiella pneumoniae IP 1145 (Nguyen et al., Gene, 1998). The gene fragment coding for the P40 protein is inserted into various expression vectors, in particular a vector under the control of the promoter of the Trp operon. The nucleotide sequence and the peptide sequence of rP40 (rP40 for recombinant P40 protein) are represented respectively by the sequence SEQ ID No. 1 and SEQ ID No. 2 from the list of sequences below. An E. coli K12 producing strain was transformed with an expression vector pvaLP40. The recombinant protein P40, here called rP40, is produced in the form of inclusion bodies with a high yield (> 10%, g of proteins / g of dry biomass). This example is only an illustration of the expression of rP40, but it can be extended to other bacterial strains as well as other expression vectors.

EXAMPLE 2 Fermentation Process for rP40 Fusion Proteins

Inoculate with the E. coli strain in an Erlenmeyer flask containing 250 ml of TSB medium (Tryptic Soy Broth, Difco) containing Ampicillin (100 μg / ml, Sigma) and Tetracycline (8 μg / ml, Sigma). recombinant described above. Incubate overnight at 37 ° C. and then 200 ml of this culture are used to seed 2 liters of culture medium in a fermenter (Biolafitte, France). Quite conventionally, the culture medium can be composed of chemical agents, supplemented with vitamins, yeast extracts, known to have a high density growth of bacterial cells.

The parameters controlled during fermentation are: pH, agitation, temperature, oxygenation rate, feeding of combined Glycerol or Glucose sources. In general, the pH is regulated at 7.0, the temperature is fixed at 37 ° C. Growth is controlled by supplying glycerol (87%) at a constant flow rate (12 ml / h) to obtain the voltage signal for dissolved oxygen at 30%. When the turbidity of the culture (measured at 580 nm) reaches the value of 80 (after approximately 24 hours of culture), the production of the proteins is triggered by addition of indole acrylic acid (IAA) to the final concentration of 25 mg / 1. About four hours after induction, the cells are harvested by centrifugation. The quantity of wet biomass obtained is around 200 gr.

EXAMPLE 3 Method of Extraction and Purification of the rP40 Protein Extraction of the rP40

After centrifuging the culture broth (4,000 rpm, 10 min, 4 ° C), the cells are resuspended in 25 mM Tris-HCl buffer pH 8.5. The insolubles or inclusion bodies are obtained after treatment with lysozyme (0.5 g / liter, 1 hour at room temperature / gentle stirring). The inclusion body pellet obtained by centrifugation (15 min at 10,000 g at 4 ° C) is taken up in a 25 mM Tris-HCl buffer at pH 8.5 and 5 mM MgCl 2, then centrifuged (15 min at 10,000 g). The inclusion bodies are dissolved at 37 ° C. for 2 hours in a 25 mM Tris-HCl buffer, pH 8.5, containing 7 M urea (denaturing agent) and 10 mM Dithiothreitol (reduction of disulfide bridges). Centrifugation (15 min at 10,000 g) eliminates the insoluble particles. It is then resuspended in 13 X volumes of 25 mM Tris-HCl buffer pH 8.5 containing NaCl (8.76 g / 1) and Zwittergent 3-14 (0.1%, w / v). The solution is left overnight at room temperature with gentle stirring in contact with air (promotes renaturation of the protein by dilution and reoxidation of the disulphide bridges). Purification of the rP40 protein a) Anion exchange chromatography step.

After a further centrifugation, the solution is dialyzed against a 25 mM Tris-HCl buffer pH 8.5 containing 0.1% Zwittergent 3-14 (100 X volumes of buffer) overnight at 4 ° C. The dialysate is deposited on a column containing a support of the strong anion exchanger type (Biorad Macro Prep High Q gel) balanced in the buffer described above at a linear flow rate of 15 cm / h. Proteins are detected at 280 nm. The rP40 protein is eluted, with a linear flow rate of 60 cm / h, for a concentration of 0.2 M NaCl in the 25 mM Tris-HCl buffer, pH 8.5; 0.1% Zwittergent 3-14. b) Cation exchange chromatography step.

The fractions containing the rP40 protein are combined and concentrated by ultrafiltration using an Amicon shaking cell system used with a Diaflo membrane of the YM10 type (cutoff threshold 10 kDa) for volumes of the order of 100 ml. , or using a Minitan Millipore tangential flow filtration system used with membrane plates with a cutoff threshold

10 kDa for higher volumes. The fraction thus concentrated is dialyzed overnight at 4 ° C. against a 20 mM citrate buffer, pH 3.0, with 0.1% of Zwittergent 3-14.

The dialysate is deposited on 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, at 0.1% of Zwittergent 3-14. The rP40 protein is eluted (speed 61 cm / h) for a 0.7 M NaCl concentration. Electrophoretic profiles show a degree of purity of the order of 95%. The state of the protein is followed by SDS-PAGE. Depending on its denatured or native form, the P40 protein extracted from the membrane of Klebsiella pneumoniae has a characteristic electrophoretic (migration) behavior. The native form (β-sheet structure) has a lower molecular weight than the denatured form (α-helical structure) under the action of a denaturing agent, such as urea or guanidine hydrochloride, or by heating to 100 ° C. in the presence of SDS). The rP40 protein is not correctly renatured at the end of renaturation, whether this is carried out in the absence or in the presence of 0.1%; (w / v) Zwittergent 3-14. On the other hand, total renaturation is obtained after dialysis against a 25 mM Tris / HCl buffer pH 8.5 containing 0.1% (w / v) Zwittergent 3-14. However, it should be noted that this renaturation is only obtained when the dilution step and the treatment at ambient temperature are carried out themselves in the presence of Zwittergent 3-14 (negative results in the absence of detergent). Example 4 Preparation of the CTP37 Fragment Derived from βhCG The CTP37 peptide is a peptide of 37 amino acids which corresponds to the C-terminal fragment 109-145 of βhCG (human chorionic gonadotropin β) of sequence SEQ ID No. 3:

T ¹⁰⁹ CDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ ¹⁴⁵ . It is obtained by chemical synthesis on solid phase and it is intended for chemical conjugation using a heterobifunctional coupling reagent on different proteins carrying antigens (protein P40 and diphtheria toxoid (DT)). Contrary to usage, this peptide does not require the addition of an additional cysteine for the unequivocal coupling insofar as the Cys ¹¹⁰ present on the N-terminal side can be used for this purpose. The peptide was synthesized using an automatic solid phase peptide synthesizer from the C side to the N-terminal side (FMOC chemistry on the scale of 0.1; 0.25 or 1.0 mmol). The synthesis of the CTP37 peptide is carried out from a preloaded glutamine on an HMP type resin, which allows after cleavage to obtain a free acid function on the C-terminal side or a Rink amide MHBA type resin, which allows after cleavage to obtain an amide function on the C-terminal side. The reactive functions of the side chains of the amino acids used, are protected by groups compatible with FMOC chemistry [Cys (Trt); Arg (Pmc); Asn (Trt); Gln (Trt); Lys (Boc); Ser (tBu); Thr (tBu)]. A coupling cycle takes place as follows: deprotection of the N-terminal amino function of the first amino acid using piperidine, activation of the acid function of the second amino acid to be coupled using HBTU / NMP and coupling.

The sequence of CTP37 has the particularity of containing 10 prolines out of 37 amino acids. Prolines are known to form bends in the polypeptide sequences and thus to strongly influence the secondary structure of these sequences (EP Rock et al. Am. J. Reprod. Imm., 1996, 35, 156-162). From the synthesis point of view, this implies particular difficulties. To overcome this difficulty in particular at the level of the 3 p _ro ¹²⁴ ' ¹²⁵ - ¹²⁶ successive, we have introduced three double coupling steps for Pro ¹²⁴ , Ala ¹²³ and Lys ^{122 in} order to optimize the quality of the crude peptide. Furthermore, all the coupling cycles are followed by a “capping” step using acetic anhydride in order also to improve the quality of the crude peptide and thus to facilitate the purification.

At the end of the synthesis, the peptide is cleaved from the resin and the side chains are deprotected by reaction with a water / TFA mixture.

The crude peptide is purified by semi-preparative reverse phase HPLC. The homogeneity of the purified peptide is verified by reverse phase HPLC and by capillary electrophoresis (FZCE). The theoretical structure is confirmed by comparison of the compatibility of the mass measured by mass spectrometry of the ES-MS type, with the mass calculated from the theoretical amino acid sequence to the nearest dalton (FIG. 2). Characteristics of the CTP37 peptide - Synthesis: Theoretical weight of peptide-resin at the end of synthesis: 650 mg Weight measured after drying with nitrogen: 642 mg

Mass of crude peptide cleaved after lyophilization: 352 mg (75% of net amount of peptide, ie 77% of yield). - Purification:

Mass of purified peptide after lyophilization: 269 mg (75% of net quantity or 58% of yield).

- Characterization: Homogeneity of the purified peptide:> 99% (RP-HPLC; UV 210 nm)

91% (FZCE / Microcoat ™; UN 200 nm).

- Mass spectrometry (ES-MS):

Calculated mass (C ₁₆₇ H ₂₆₄ Ν ₄₆ O ₅₈ S): 3876.28 Da

Measured mass: 3876.00 Da ± 0.06 - Abbreviations:

DT: Diphtheria toxoid; ES-MS: Electrospray - Mass spectrometry; FMOC: fluorenylmethoxycarbonyl; FZCE: Free Zone Capillary Electrophoresis; HBTU: 2- (1H-Benzotriazole-1-yl) -1, 1,3,3-tetramethyluronium hexafluorophosphate; HF: hydrofluoric acid; HMPA: 4-hydroxymethylphenoxyacetic acid (resin); NMP: N-methyl-2-pyrrolidone; MBHA: p-methylbenzydrylamine (resin); Pmc: 2,2,5,7,8-Pentamethylchroman-6-sulfonyl; RP-HPLC: Reverse Phase - High Performance Liquid Chromatography; tBOC: t-butyloxycarbonyl; tBu: tertiary Butyl; TFA: trifluoroacetic acid; Trt: trityl; A: ultra-violet.

Example 5 Synthesis, Purification and Characterization of rP40-CTP37 Conjugates and

DT-CTP37

Synthesis and purification of the rP40-CTP37 conjugate

The coupling of the peptide is carried out using the reagent MCS, or 6-maleimidocaproic acyl Ν-hydroxysuccinimide ester (Lee et al, 1980, Mol. Immunol. 17, 749-756). This heterobifunctional reagent allows activation of the Lysine residues of the protein, then coupling of the peptide by the free thiol group of the Cysteine residue carried by the CTP37 peptide (Cys in position 2).

Initially, the rP40 protein is dialyzed against a 0.1 M sodium phosphate buffer pH 6.6 containing 0.1% Zwittergent 3-14 overnight at + 4 ° C. The dialyzed protein is then activated using the MCS reagent added at the right of 340 μg per mg of rP40, then the whole is placed under stirring, for 2 hours, at room temperature and in the dark.

The activated rP40 is desalted by molecular sieving chromatography on Pharmacia ™ Sephadex G75 support. Elution of the protein is carried out using a 0.1 M sodium phosphate buffer pH 6.6 containing 10 mM EDTA and 0.1%

Zwittergent 3-14. The fractions containing the activated rP40 are combined.

For coupling, the activated protein solution is added to the lyophilized peptide at a rate of 1 mg of peptide / mg of rP40. After saturation under a stream of nitrogen, the whole is stirred overnight at room temperature and in the dark.

After dialysis against a 0.1 M sodium phosphate buffer pH 6.6 containing

0.1% Zwittergent 3-14 for 24 hours at + 4 ° C, the unbound CTP37 peptide is removed by molecular sieve chromatography on a support column

Pharmacia ™ Sephadex G50 eluted by the aforementioned buffer. The fractions containing the rP40-CTP37 conjugate are combined, and the solution is concentrated by ultrafiltration using an Amicon concentration cell on a YM10 filter (cutoff threshold 10 kDa).

Synthesis and purification of the DT-CTP37 conjugate

After dialysis against a 0.1 M sodium phosphate buffer pH 6.6 overnight at + 4 ° C, the DT protein (DT for Diphtheria Toxoid) (SBL Vaccine AB, lot n ° U235) is activated using the MCS reagent added at a rate of 416 μg per mg of DT.

The whole is then placed under stirring for 1 hour at room temperature and in the dark.

The activated protein is desalted by molecular sieve chromatography on Pharmacia ™ Sephadex G75 support. The elution is carried out using a 0.1 M sodium phosphate buffer pH 6.6 containing 10 mM EDTA. The fractions containing the

DT activated are gathered.

For coupling, the activated protein in solution is added to the lyophilized peptide in an amount of 1.25 mg of peptide / mg of DT. After saturation under a stream of nitrogen, the whole is stirred overnight at room temperature and in the dark. After dialysis against a 0.1 M sodium phosphate buffer pH 6.6 for 24 hours at + 4 ° C, the unbound CTP37 peptide is removed by molecular sieve chromatography on a Pharmacia ™ Sephadex G50 support column eluted with the same buffer . The fractions containing the DT-CTP37 conjugate are combined, and the solution is concentrated by ultrafiltration using an Amicon concentration cell on a YM10 filter (cutoff threshold 10 kDa). Characterization of the rP40-CTP37 and DT-CTP37 conjugates

The rP40-CTP37 and DT-CTP37 conjugates are analyzed by SDS-PAGE electrophoresis (Figures 3 A and 3B). A determination of the amino acids released after hydrolysis (6N HCl) is carried out by HPLC (Waters Pico Tag method) after derivatization using the PITC reagent. This assay makes it possible to determine the protein concentration of the conjugate solutions, and to precisely define the number of CTP37 peptides fixed per mole of rP40 or DT protein as well as the concentration of fixed peptide.

All the results obtained are collated in Table I below:

Table I: Characterization of the rP40-CTP37 and DT-CTP37 conjugates

Example 6 Immunogenicity of the adjuvanted rP40-CTP37 conjugates

Increasing concentrations of rP40-CTP37 conjugate are administered subcutaneously at the base of the neck in batches of 5 six-week-old C57B1 / 6 mice in the presence of an adjuvant mixture composed of N-acetylmuramyl-L-alanyl-D - isoglutamine and squalene mannide monooleate. The mice receive four immunizations, respectively on D0, D7, D14, and D22. The humoral response developed by these animals against the peptide CTP37 coupled to the carrier protein rP40 is evaluated, 7, 14, 22 and 35 days after the first immunization. The animals are also punctured on D0 in order to check their seronegativity towards the peptide CTP37 and one in mice injected with a mixture of PBS (phosphate buffered saline) and adjuvant is added as a negative control.

The results presented in FIG. 4 show that rP40 behaves like a carrier protein with respect to CTP37 against which it induces a specific and high antibody response from the lowest concentrations of conjugate injected. The response seems to be almost maximum from the second injection, regardless of the dose. The antibody level reaches its maximum for a concentration of 100 μg of conjugate.

Example 7: Immunogenicity of the non-adjuvanted rP40-CTP37 conjugates The rP40-CTP37 conjugate was also tested at the highest dose in the absence of adjuvant (FIG. 5).

The data presented in FIG. 5 show that in the absence of adjuvant the humoral response reaches, after 4 immunizations, values of the same order as those observed in the presence of adjuvant. These results highlight the efficacy of the rP40 carrier in inducing a humoral response against a protein antigen in the absence of an adjuvant.

Example 8 Immunogenicity of the Conjugates After Sensitization by Klebsiella Pneumoniae

Since the rP40 protein is isolated from the bacterium Klebsiella pneumoniae which is a pathogen of the human respiratory tract, the whole population has antibody titres against rP40. In order to mimic the human situation, mice

C57B1 / 6 are presensitized twice 7 days apart with Kp, intranasally before being immunized, subcutaneously, with a dose of 200 μg of rP40-CTP37 conjugate. The results presented in FIG. 6 show that a presensitization of mice with Klebsiella pneumoniae, which results in the existence in these animals of an anti-carrier titer of the order of 3 log ₁₀ , has no influence on the intensity of the response to CTP37. These results suggest an absence of epitopic suppression when rP40 is used as a carrier unlike data published with other carriers such as TT (Kaliyaperumal A. et al. Eur. J.

Immunol. 1995; 25: 3375-3380). EXAMPLE 9 Tumor regression induced by rP40-βhCG conjugates

In order to evaluate the effectiveness of a vaccination with the rP40-CTP37 conjugate on the evolution of tumor growth in vivo, of a line positive for βhCG, the Lewis carcinoma model, in the mouse C57B1 / 6 , was chosen after verification, by immunostaining techniques, of the positivity of the cells injected into the animal for βhCG.

For this experiment, the mice are immunized on D0, D7, D14 and D21 with 200 μg of rP40-CTP37 conjugate. After verification of the anti-CTP37 antibody titer on D35, the mice receive, on D39, 10 ⁶ LL / 2 cells by subcutaneous injection. 17 days after implantation of the cells, the mice are sacrificed and the tumors weighed individually. A DT-CTP37 conjugate is used as a reference control.

The two experiments presented in FIGS. 7A and 7B show that the induction of an immune response against CTP37 by injection of rP40-CTP37 leads to a significant reduction in the tumor mass in the treated animals. This response is specifically linked to the targeting of a βhCG activity of the tumor cell since the mice immunized with P40 alone in the presence of the adjuvant mixture do not exhibit a significant reduction in the tumor mass at the end of the experiment. Presensitization of mice by two intranasal administrations of Klebsiella pneunoniae has no influence on the impact of treatment with rP40-CTP37. These results are in agreement with the observations made above (FIG. 6) concerning the similarity of the anti-CTP37 IgG titers in mice presensitized or not by Klebsiella pneunoniae.

On the other hand, compared to the PBS control with adjuvant, the rP40-CTP37 conjugate is more effective than the DT-CTP37 conjugate.

Claims

1. Use of an enterobacterium protein OmpA or a fragment thereof, associated with an immunogen chosen from cytokines or their receptor, growth factors or their receptor, hormones or their receptor and / or specific markers of a tumor, to a fragment thereof, or to one of their analogs, for the preparation of a pharmaceutical composition intended to improve the immunological response against said immunogen. 2. Use according to claim 1, characterized in that said enterobacterium protein OmpA, or one of its fragments, is obtained by an extraction process from a culture of said enterobacterium. 3. Use according to claim 1, characterized in that said enterobacterium protein OmpA, or one of its fragments, is obtained by recombinant route. 4. Use according to one of claims 1 to 3, characterized in that said enterobacterium is Klebsiella pneumoniae. 5. Use according to claim 4, characterized in that the amino acid sequence of said OmpA protein, or one of its fragments, comprises: a) the amino acid sequence of sequence SEQ ID No. 2; b) the amino acid sequence of a sequence having at least one homology 80% with the sequence SEQ ID No. 2; or c) the amino acid sequence of a fragment of at least 5 consecutive amino acids of a sequence as defined in a) or b). 6. Use according to one of claims 1 to 5, characterized in that said immunogen is coupled or mixed with said protein OmpA or one of its fragments. 7. Use according to claim 6, characterized in that said immunogen is coupled by covalent bond with said OmpA protein or one of its fragments. 8. Use according to claim 7, characterized in that the coupling by covalent bond is a coupling produced by chemical synthesis. 9. Use according to one of claims 1 to 7, characterized in that said immunogen is chosen from peptides. 10. Use according to claims 8 and 9, characterized in that one or more binding elements are introduced into said OmpA protein, or one of its fragments, and / or into said immunogen to facilitate climatic coupling. 11. Use according to claim 10, characterized in that said connecting element introduced is an amino acid. 12. Use according to claim 9, characterized in that the hybrid protein obtained after coupling between said peptide immunogen and said OmpA protein, or one of its fragments, is prepared by genetic recombination. 13. Use according to claim 12, characterized in that the pharmaceutical composition contains a vector comprising a nucleic construct coding for said hybrid protein or a host cell transformed by said vector capable of expressing said hybrid protein. 14. Use according to claim 13, characterized in that said nucleic construct comprises a nucleic sequence chosen from the sequence SEQ ID N ° 1, one of its fragments having at least 15 consecutive nucleotides of the sequence SEQ ID N ° 1, or a sequence having a homology of at least 80% with one of said sequences. 15. Use according to one of claims 1 to 14, characterized in that the immunogen is the hormone βhCG, or one of its fragments. 16. Use according to claim 15, characterized in that the immunogen is the peptide of amino acid sequence SEQ ID No. 3: TCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ. 17. Use according to one of claims 1 to 14, characterized in that the immunogen is a cytokine or a growth factor involved in the proliferation of tumor cells. 18. Use according to one of claims 15 to 17, for the preparation of a pharmaceutical composition of the vaccine type intended to prevent or treat cancers. 19. Use according to one of claims 1 to 16, characterized in that the immunogen is a hormone, or one of its fragments, involved in fertility. 20. Use according to claim 19, for the preparation of a pharmaceutical composition of the vaccine type intended for contraception. 21. Pharmaceutical composition, characterized in that it comprises an enterobacterium protein OmpA or one of its fragments, in particular of Klebsiella pneumoniae, associated with an immunogen chosen from cytokines, growth factors and / or hormones, or one of their fragments. 22. Pharmaceutical composition characterized in that it comprises a nucleic construct coding for an enterobacterium OmpA protein, or one of its fragments, in particular of Klebsiella pneumoniae, associated with a nucleic construct coding for a peptide immunogen chosen from cytokines , growth factors and / or hormones, or a fragment thereof. 23. Pharmaceutical composition according to claim 21 or 22, characterized in that the immunogen is the hormone βhCG, or one of its fragments. 24. Pharmaceutical composition according to claim 23, characterized in that the immunogen is the peptide of amino acid sequence SEQ ID No. 3: TCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ. 25. Use according to one of claims 1 to 20, or pharmaceutical composition according to one of claims 21 to 24, characterized in that said pharmaceutical composition is conveyed in a form making it possible to improve its stability and / or its immunogenicity. 26. Use or pharmaceutical composition according to claim 25, characterized in that said vehicle is a liposome, a viral vector containing a nucleic construct coding for said OmpA protein, or one of its fragments, said immunogen, or for said hybrid protein , or a transformed host cell capable of expressing said OmpA protein, or one of its fragments, said immunogen, or said hybrid protein. 27. Use or pharmaceutical composition according to any one of the preceding claims, characterized in that the said pharmaceutical composition comprises an immunity adjuvant, mixed or coupled to said OmpA protein, to said immunogen, or to one of their fragments, or to said hybrid protein.