WO1995010299A1

WO1995010299A1 - COMPOSITION FOR TREATMENT BY IMMUNOTHERAPY OF A CANCER SECRETING hCG OR hCG FRAGMENTS

Info

Publication number: WO1995010299A1
Application number: PCT/FR1994/001177
Authority: WO
Inventors: Dominique Bellet; Frédéric TROALEN; Nathalie Rouas-Freiss; Jean-Michel Bidart
Original assignee: Laboratoire L. Lafon
Priority date: 1993-10-08
Filing date: 1994-10-07
Publication date: 1995-04-20
Also published as: AU7941394A; FR2710845B1; FR2710845A1

Abstract

A composition for treatment by immunotherapy of cancers secreting hCG, free sub-units thereof, or fragments of these sub-units, comprises an effective amount of one of these free hCG sub-units or a polypeptide containing at least a part of the primary sequence of hCG and corresponding to an antigen responsible for an immunization state.

Description

Composition intended for the iriotherapy of a cancer secreting hC.3 or hCG fragments

The present invention relates to a method for the treatment of cancer secreting hCG or fragments of hCG and means for carrying out this method. human gonadotropic chorionic hormone

(hCG) is an oligomeric glycoprotein consisting of the non-covalent association of two subunits α (hCG-α) and β (hCG-β). It belongs to the family of gonadothyreotropic hormones also comprising folliculotropic hormone (FSH), luteotropic hormone (LH) and thyreotropic hormone (TSH). HCG is secreted physiologically by the trophoblast from the start of pregnancy and stimulates the corpus luteum to maintain the synthesis of steroid hormones essential for implantation of the embryo. Furthermore, hCG and / or its subunits are also secreted by a majority of trophoblastic tumors and certain non-trophoblastic tumors, thus constituting biological markers which can help in the diagnosis or prognosis and in the monitoring of these tumors (Bellet and al. Rev. Prat. 40: 1677-1990).

The amino acid sequence of h'hCG has been described by Morgan F. J. et al. The amino acid sequence of Human chorionic gonadotropin, J. Biol. Chem. 250, 5247, 1975.

In the normal population, very low serum levels of dimeric hCG are found in men (<0.05 ng / ml corresponding to the detection limit of the method used) and in premenopausal women (<0 , 1 ng / ml). HCG is mainly detectable in 30% of women over 45 (0.1 to 1 ng / ml). Several studies suggest that the hCG present in the serum of healthy subjects would be produced by pituitary cells. HCG-α is detectable in healthy men (<1 ng / ml) and women over 45 years of age who have not yet had a child (<3 ng / ml). In contrast, hCG-β is not detectable in the normal population. In the case of trophoblastic tumors, it has been shown that benign (hydatidiform moles) and malignant (choriocarcinomas) placental tumors are characterized by an important serum hCG-β / hCG ratio: the percentage of free hCG which is 0, 05 to 1% in normal pregnancies amounts to 1-5% in the case of moles and exceeds 5% in choriocarcinomas. This hCG-β / hCG serum ratio is used as a control for the development of testicular tumors: a percentage of hCG-β becoming less than 5% is often characteristic of a favorable development of the tumor during treatment. This ratio will rise quickly in the event of an unfavorable development or a relapse.

In the case of non-trophoblastic tumors, Marcillac et al. Cancer Res. 52: 3901, 1992 have shown that hCG-β, not detectable in healthy individuals, is found in many patients with certain non-trophoblastic tumors (tumors of the bladder, pancreas, cervix, tumors without primitive found, endometrial and lung tumors). The subject of the present invention is a method of treatment of a cancer secreting hCG or its free subunits or fragments of these subunits, which consists in

- Identify in the serum of a patient using a set of polypeptides covering at least a part of the primary sequence of hCG, a state of immunization with respect to one of these polypeptides. administering to the patient an effective amount of at least one polypeptide corresponding to an antigen responsible for the state of immunization. In the context of the present invention, the polypeptides are preferably formed by a chain of 15 to 40 amino acids corresponding to a part of the primary sequence of hCG. The term “set of polypeptides covering at least a part of the primary sequence of hCG” denotes a set of polypeptides as defined above which correspond to successive sequences of the hCG or of its fragments or better which correspond to sequences overlapping.

One can for example use a set of polypeptides corresponding to the following sequences: hCG-α 1-20

15-35 30-50

45-65

60-80

75-92 hCG-β 1-20 15-35

30-50 45-65 60-80 75-95 90-110

105-125 120-145 Preferably, at least one set of polypeptides will be used which covers the primary sequence of the β subunit of hCG.

By "polypeptide corresponding to an antigen responsible for the immunization state is meant a polypeptide which has the same sequence as one of the polypep¬ tides responsible for the immunization state or a sequence resulting from the combination of sequers. these at less partial of the polypeptides responsible for the state of immunization.

The state of immunization with respect to one of the polypeptides can in particular be identified by a test for stimulation of the proliferation of T lymphocytes by the polypeptides.

It is also possible to demonstrate the possible presence of serum antibodies specific for hCG or its fragments. These antibodies can be demonstrated in particular by an immunoenzymatic method such as an ELISA method.

A subject of the invention is also a method of treatment of a cancer secreting hCG, hCG-β or fragments of this subunit which consists in administering to a patient an effective amount of hCG-β.

In this case, it is not necessary to determine for each patient the peptide recognized by its T lymphocytes. This peptide will be produced in vivo by degradation of hCG-β in the antigen presenting cells.

The present invention also relates to a composition intended for the immunotherapy of cancers secreting hCG, its free subunits or fragments of these subunits, comprising an effective dose of one of the free subunits of the hCG or a polypeptide comprising at least part of the primary sequence of hCG and corresponding to an antigen responsible for an immunization state. In a particular embodiment of the invention, the composition comprises an effective dose of the β subunit of hCG.

In another particular embodiment of the invention, the composition comprises an effective dose of a polypeptide corresponding to a part of the primary sequence of the hCG β subunit.

For therapy, the poly¬ peptides can be used in free form or in a more immunogenic form, in particular a form in which the peptide is coupled to a polylysine matrix according to the MAP technique (Posnett et al. J. Biol. Chem. 263, 17, 1988).

In addition, for therapy, the free subunits of hCG or the polypeptide are advantageously administered with an adjuvanting agent of immunity. As examples of adjuvants of immunity, mention may be made of muramyldipeptide and SAF-1 (Allison et al., J. Immunol. Methods 45, 157, 1986).

It is also possible to administer the free subunits of the hCG or the polypeptide coupled to nanospheres or to nanoparticles such as those described in EP 0 240 424.

In the case of the treatment of certain cancers and in particular in the case of bladder cancer, it is also possible to administer concomitantly or in a mixture of BCG (Bacillus Calmette Guérin) constituted by living attenuated strains of tuberculous bacillus (see including D. Lamm et al, New England Journal of Medicine 325, 17, 1205, 1991).

As a variant, it is possible to administer a recombinant BCG expressing a subunit of the hCG or of the polypeptide comprising at least part of the primary sequence of the hCG.

Such a recombinant BCG has advantages: a single immunization is sufficient to obtain an optimal immune response. Indeed, the expression of foreign protein antigens persists as long as the recombinant BCG is present. Thus, this continuous expression makes it possible to develop lasting immunity. This is a significant advantage over if the quantities of polypeptides or proteins injected at each immunization are defined and limited.

It should be remembered that BCG is an intracellular bacterium with a macrophagic location. This characteristic gives the recombinant BCG the advantage of delivering foreign antigens within one of the most effective cells as antigen-presenting cells (APC). Macrophages will thus effectively present foreign antigens in association with MHC class II molecules and allow the stimulation of specific helper T cells which will send the signals necessary for the establishment of cytotoxic and antibody responses. This form of administration allows the simultaneous expression of several antigens from different pathogens.

In the case of a composition for the treatment of bladder cancer, recombinant BCG has an additional advantage. Indeed, studies have shown that BCG is internalized by tumor cells in the bladder. Therefore, recombinant BCG allows the expression of an hCG or polypeptide subunit by the tumor cell, thus amplifying the mechanisms of presentation of these antigens to T lymphocytes.

The recombinant BCG obtained can express the β subunit of hCG or fragments thereof as a fusion protein on the surface of the mycobacterial cell, or express the hCG subunit in the cytoplasm in the form of a fusion protein or alone and possibly secreting these.

Recombinant BCG can be obtained by inserting into an expression vector integrating into the BCG genome, for example a Myco- shuttle vector. bacterium / Escherichia Coli, the DNA sequence coding for the hCG β subunit or a fragment thereof, optionally associated with a DNA sequence coding for a mycobacterium membrane lipoprotein in the case where the we want to obtain a fusion protein.

The plasmid also comprises an appropriate promoter, for example a mycobacterial promoter in the case where the hCG subunit is expressed in the form of a fusion protein associated with a membrane lipoprotein or a promoter suitable for transcription of the DNA of the hCG β subunit when it is desired to express the β subunit of hCG alone. In the case where it is sought to obtain a secreted protein, an appropriate signal sequence is also inserted into the plasmid, for example the signal sequence coding for the 19 kDa antigen or the 38 kDa antigen of Mycobacterium tuberculosis . The vector also advantageously comprises a ribosomal binding site as well as a cassette containing the sequences for the mycobacterial replication of the plasmid as well as a DNA cassette containing the sequences for the replication in Escherichia coli of the plasmid, in the case where the vector is a Mycobacterium / Escherichia coli shuttle plasmid.

The vector advantageously comprises an operon for resistance to HgCl ₂ for the selection of transformants.

Suitable plasmids may for example consist of plasmids as described in WO 93/07897, in which the DNA coding for the surface protein of Borrelia iurgdorferi has been replaced by the DNA coding for the β subunit of 1'hCG or a fragment of it.

The genes encoding the hCG subunits are known. The α subunit is encoded by a single gene located on chromosome 6 and capable of being expressed at the pituitary and placental levels (Fiddes et al., J. Mol. Appl. Broom. 1, 3, 1981).

The synthesis of the β subunit is dependent on a multigenic group comprising 7 genes (Talmadge et al., Nucl. Acids Res. 12, 8415, 1984). nucleotic sequence analysis shows that three of these genes (1, 3, 5) have an identical structure, four others of these genes (2, 6, 7 and 8) encode a variant at codon 117 with substitution of alanine in place of an aspartic acid. A study of gene expression (Bo et al., J. Biol. Chem., 267, 3179, 1992) shows that genes 1, 3 and 5 are expressed by the normal placenta.

It is believed that in the tumherogenesis processes involving non-trophoblastic cells the expression of several genes would lead to both the secretion of the normal protein and of a protein mutated to 117.

In the present invention, therefore, the term β subunit denotes both the normal protein and the protein mutated at 117.

In practice, immunotherapy can be carried out with doses of hCG subunits, of polypeptides or of recombinant BCG expressing them from 1 to 100 mg administered at the level of the tumor. The vaccine can be administered in the case of bladder cancer, as already practiced in the case of immunotherapy with BCG, that is to say by intravesical instillation one week after resection of the tumor. , then this administration is repeated every week for 6 weeks and additional intravesical administrations are carried out 3, 6, 12, 18 and 24 months later. Percutaneous or intramuscular administration is also possible.

For other types of cancer, intradermal administrations can be used. Description of the figures.

Fig. 1 shows the proliferation of lymphocytes from a patient with bladder cancer in the presence of different polypeptides. Fig. 2 shows the proliferation of lymphocytes from two patients with bladder cancer in the presence of hCG and hCG-β.

Fig. 3 shows the proliferation of lymphocytes from a patient with bladder cancer in the presence of different polypeptides.

Fig. 4 is a diagram of the plasmid pENlOO.

Fig. 5 is a diagram of the plasmid pEN101.

Fig. 6 is an analysis of the Bam HI P85-βhCG junction. Fig. 7 is an immunoblot of crude bacterial extracts, lane 1 corresponding to M Smegmatis (pENlOl), lane 2 with BCG (pENlOl), lane 3 with untransformed BCG and lane 4 with β-hCG.

A report of diagnostic tests will be given below.

1) Isolation of mononuclear cells (PBMC) by ficoll gradient:

Heparin whole blood from patients with bladder cancer is placed on a ficoll-hypaque solution (1.077 g / 1) in a conical tube. The tube is centrifuged for 30 min at 900 g, 18-20 ° C. The layer corresponding to the mononuclear cells is recovered using a pipette. The cells are washed in HANKs buffer.

2) Lmphocytic proliferation test

The PBMCs are cultured (2 × 10 ⁵ cells per well) in RPMI 1640 supplemented with 10% human serum AB, 2 mM L-glutamine, 1000 IU / ml of penicillin and 100 mg / ml of streptomycin, in the presence of polypeptide at different concentrations. The cells are then incubated at 37 ° C with 5% C0 ₂ for 5 days. A μCi of tritiated thymidine is then added to each well. The cells are collected 18 hours later and the incorporation of the radioactivity is analyzed by spectrometry in a scintillation liquid using a β counter.

We have given in FIG. 1 the proliferation results of lymphocytes from a patient A with bladder cancer in the presence

- culture medium (M)

- hCG and its α and β subunits

- different polypeptides belonging to the α and β subunits. The results demonstrate a significant proliferation with the polypeptide corresponding to the sequence 95-115 of the β subunit of hCG as well as with the peptide corresponding to the sequence 15-59 of the α subunit of the hCG. The 95-115 hCG-β and 26-59 hCG-α polypeptides can be used for immunotherapy in the patient tested.

We have given in Figs. 2A and 2B the results of PBMC proliferation tests of two patients B and C (respectively Fig. 2A and 2B) in the presence:

- culture medium

- hCG

- hCG-β at a dose of 3 μM, for 4 days. Patient B responds against hCG-β and patient C responds against hCG.

We have given in FIG. 3 the results of PBMC proliferation tests of patient B in the presence of different polypeptides covering the whole of the β subunit of hCG (50 μM). The results show that for patient B the polypeptide corresponding to the β (20-47) region of hCG is the most effective in inducing proliferation of T cells. In addition, other tumors (pancreatic and testicular) have been studied. The T cells of a patient with testicular cancer responded against hCG-β and more precisely against the peptide hCG-β (20-47). These results show that hCG-β or peptides corresponding to part of the hCG-β sequence are immunogenic in patients with a hCG-β-producing tumor and can thus be used to develop immunotherapy antitumor. Conversely, no response against hCG, its subunits or peptides of similar structure was observed in 21 pregnant women.

The preparation of recombinant BCGs expressing β-hCG will be described below.

In order to express and secrete the β subunit of hCG, the reading phase (ORF) of this gene was merged downstream of the expression-export signals of the BCG 85A antigen (Content et al. (2)). For this purpose, an expression plasmid pENlOO (FIG. 4) was prepared, based on the plasmid pRR3 (Ranes et al., (5)) in which the transcription, translation and sequence signals have been cloned. 85A gene signal (indicated by P85A), on the one hand, and the HgCl ₂ resistance operon (indicated by HgCl ₂ R and containing 4 genes indicated by the arrows), on the other hand. The origin of replication for mycobacteria (indicated by the arrow ori) comes from the plasmid PAL5000 (Labidi et al. (3)) and the origin of replication for Escher ± chia coli is a ColEl. This shuttle plasmid is therefore capable of replicating in mycobacteria and in E. coll.

This vector also contains the kanamycin resistance gene (indicated by KanR). For ease of cloning, the kanamycin resistance gene was placed downstream of the 85A expression signals. The Bam HI cloning sites at position 5897 in FIG. 4 overlapping the 85A and Kpn I cleavage site located downstream of the cassette (at position 4624) allow the unidirectional replacement of the KanR cassette by a sequence of interest , in this case the reading phase of the β-hCG gene.

Furthermore, two oligonucleotides were synthesized to allow amplification of the β-hCG gene by PCR ("Polymerase chain reaction") so that it is in phase with the export sequence 85A. These oligonucleotides contain one a Bam HI site and the other a Kpn I site (indicated in bold in the sequence of the oligonucleotides) so as to create a Bam HI and Kpn I site at the 5 'and 3' ends respectively of the fragment d Amplified DNA.

The oligonucleotides have the following sequence:

5'CAT GGG GAT CCC AAG GAG CCG CTT CG3 '5'CGG GGT ACC TTT ATT GTG GGA GGA TCG GG3'.

After amplification of the coding part of the β-hCG gene using these oligonucleotides and resequencing of the amplified fragment, no mutation linked to the amplification was detected. After amplification of the β-hCG fragment, the plasmid pENlOO was restricted by Bam HI and Kpn I and was used for the cloning of the β-hCG fragment previously digested with Bam HI and Kpn I. After ligation and transformation of E . coli, the resulting plasmid was named pEN101 (Fig. 5). The site cloning junction Bam HI was verified and confirmed by sequencing using the oligonucleotide with the following sequence:

5'CAT TGA TGG GGC GGC ACC GT3 '.

The results of this experiment are shown in Figure 6 and confirm the phase cloning of the reading frame of the signal peptide of the 85A antigen and that of the β-hCG.

The vector pEN101 was then used to transform Mycobacterium smegmatis and MycoJbacteriiππ bovis BCG. The selection of transformants was carried out on 12 mg / 1 HgCl ₂ . After transformation, the ycobacteria resistant to 12 mg / 1 HgCl ₂ were analyzed by the electroduction technique (Baulard et al. (1)) in order to verify the presence of the expected plasmid in the strains.

The expression of β-hCG in mycobacteria was analyzed by the immunoblotting technique on crude extracts of bacteria, using an antibody specifically directed against β-hCG. The untransformed or recombinant mycobacteria (containing the expression plasmid pEN101) were cultivated in Sauton medium (Sauton (6)) in the presence of HgCl ₂ harvested in the exponential growth phase and lysed by sonication. The various extracts were then deposited on polyacrylamide-sodium dodecyl sulfate gel (SDS-PAGE) under conditions described by Laemmli (4). After electrophoresis, the samples were transferred onto a nitrocellulose membrane as described by Towbin et al. (7). The membrane was incubated first with the anti-βhCG FB12 monoclonal antibody (5 μg / ml), then with a mouse anti-immunoglobulin antibody conjugated with alkaline phosphatase (dilution 1/7000). The immunoblot (Fig. 7) shows the presence of two immunoreactive bands with FB12 both in BCG (pENlOl) and in M. smegmatis (pENlOl) and the absence of these bands in the in the negative control (BCG not transformed). The lower band probably coincides with the mature β-hCG, after cleavage of the signal peptide 85A. The higher molecular weight band probably corresponds to the preprotein with an uncleaved signal peptide. The two protein forms migrate faster than the purified antigen (FIG. 7, lane 4), because the latter is naturally glycolysed, which increases its molecular weight. This demonstrates the expression of the β-hCG protein in mycobacteria, in this case M. smeçrmatis and M. bovis BCG by the plasmid pEN101.

BIBLIOGRAPHICAL REFERENCES

1 - Baulard, A., C. Jourdan, A. Mercenier, and C. Locht. 1992. Rapid mycobacterial plasmid analysis by electroduction between Mycohacterxu ssp. and Escherichia coli. Nucleic Acids Res., 20: 4105.

2 - Content, J., A. de la Cuvellerie, L. De Wit, V. Vincent-Levy-Frébault, J. Ooms, and J. De Bruyn. 1991. The genes coding for the antigen 85 complexes of Myco¬ bacterium tubercolosis and Mycohacterium bovis BCG are members of a gene family: cloning, sequence determination, and genomic organization of the gene coding for antigen 856C of M. tuberculosis. Infect. Immun. 59: 3205-3212.

3 - Labidi, A., H.L. David, and D. Roulland-Dussoix. 1985. Restriction endonuclease mapping and cloning of

Mycobacterium fortuitum var. fortuitum plasmid pAL5000. Ann. Inst. Pastor Microbiol. 136: 209-215.

4 - Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680-685.

5 - Ranes, M.G., J. Rauzier, M. Lagranderie, M. Gheorghiu, and B. Gicquel. 1990. Functional analysis of pAL5000, a plasmid from Mycohacterxum fortuitum: construction of a "mini" MycoJbacterium-Escherxchxa coli shuttle vector. J. Bacteriol. 172: 2793-2797.

6 - Sauton, M.B. 1912. On the mineral nutrition of the Tuberculous Bacillus. C.R. Acad. Sci. 155: 860-861.

7 - Towbei, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76: 4350-4354.

Claims

1. Composition intended for the immunotherapy of cancers secreting hCG, its free subunits or fragments of these subunits, comprising an effective dose of one of the free subunits of hCG.

2. Composition according to claim 1, comprising an effective dose of the β subunit of hCG.

3. Composition according to any one of claims 1 or 2, further comprising BCG.

4. Composition according to claim 2, in which the β subunit is a mutated protein having an alanine residue at 117.

5. Use of one of the free sub-units of hCG for the manufacture of a composition intended for the immunotherapy of cancers secreting hCG, its free sub-units or fragments of these sub-units.

6. Recombinant BCG expressing a hCG subunit or a polypeptide comprising at least part of the primary sequence of hCG.

7. Recombinant BCG according to claim 6, expressing the β subunit of hCG.

8. Recombinant BCG according to claim 6, expressing a polypeptide corresponding to a part of the primary sequence of the β subunit of hCG.

9. Composition intended for the immunotherapy of cancers secreting hCG, its free subunits or fragments of its subunits comprising an effective dose of a recombinant BCG according to claim 6.

10. Composition intended for the immunotherapy of cancers secreting β-hCG or fragments of β-hCG comprising an effective dose of a recombinant BCG according to claim 7.