Patent 6,613,882

The invention pertains to chimeric polypeptides of the formula: B--X wherein B represents the B fragment of Shiga toxin or a functional equivalent thereof, and X represents one or more polypeptides of therapeutic significance. Compositions for therapeutic use comprising the polypeptide B--X are also included.

Description of the Invention

The invention relates to means and to their use for intracellular transport of proteins or polypeptides, also to the membrane presentation of certain epitopes.

Retrograde transport can be defined as the movement of molecules from the cell membrane to the endoplasmic reticulum (ER), passing if necessary via the Golgi apparatus. This mechanism has been demonstrated for certain classes of proteins of the endoplasmic reticulum carrying the tetrapeptide KDEL (SEQ ID NO:8) at the carboxy terminal (or HDEL (SEQ ID NO:9) in starch). A great deal of biochemical and morphological evidence indicates that those proteins leave the endoplasmic reticulum, reach the Golgi apparatus in which modifications are made to their carbohydrate chain and are then redirected to the endoplasmic reticulum. The tetrapeptide KDEL (SEQ ID NO:8) is a retention signal which traps the peptide or protein to which it is attached in the endoplasmic reticulum, such trapping taking place by interaction at a receptor protein for the KDEL (SEQ ID NO: 8) motif described by Lewis M. J. et al in Nature, 348 (6297): 162: 3, Nov. 8, 1990.

Other evidence for the existence of intracellular retrograde transport arises from a study of certain bacterial toxins which enter the cytosol of eukaryotic cells after passing into the endoplasmic reticulum (Pelham et al (1992) Trends cell. Biol., 2: 183-185). A particular example which has been studied is that of the Shiga toxin from Shigella dysenteriae, also E. coli Shiga-like toxins. Such toxins are composed of two polypeptide chains; one (the A fragment) is the toxic fragment and carries a deadenylase activity which inhibits protein synthesis by acting on the 28S ribosomal RNA, while the other sub-unit (the B fragment) enables the toxin to bind to the target (O'Brien et al (1992), Curr. Top. Microbiol. Immunol. 180: 65-94). Electron microscope studies have shown that Shiga toxin can be detected in the ER of A431, Vero, and Daudi cells in particular (Sandvig et al, 1992 and 1994; KHINE, 1994). Further, treating cells with a fungal metabolite which cause the loss of the Golgi apparatus structure (brefeldin A) protects the cells against Shiga toxin thus suggesting that they traverse the Golgi apparatus before reaching the ER. Finally, Kim et al (1996) have confirmed that the B fragment of the toxin is localised in the Golgi apparatus.

The following references demonstrate the state of the art as regards retrograde transport, in particular transport of the B fragment of Shiga toxin in the ER: Sandvig et al (1992), Nature 358:510-512; Sandvig et al (1994) J. Cell. Biol 126: 53-64; Kim et al (1996) J. Cell. Biol 134: 1387-1399.

Intracellular transport is defined as the ensemble of exchanges between the different cellular compartments.

The authors of the present application have observed that the B fragment is not only moved towards the ER, but also to the nucleus of hematopoietic lines, in particular dendritic cells and macrophages.

The authors have shown that those cells, incubated in the presence of two micromoles of BGly-KDEL (SEQ ID NO:8) fragment, as described below, for 3 hours then fixed, have a reactivity with specific antibodies against the toxin in the nucleus and even in the nucleole of such cells (unpublished results) which clearly indicate the existence of intracellular transport of that fragment.

The present invention results from observations on intracellular transport of the B fragment of Shiga toxin (B fragment) and uses its routing properties to construct a chimeric polypeptide sequence containing:

either a peptide or a polypeptide of therapeutic significance bound to said fragment or any functional equivalent thereof;

or a polynucleotide sequence carrying a sequence the expression of which is desired. The B fragment and the polynucleotide sequence are coupled using any technique which is known to the skilled person and in particular that described by Allinquant B. et al in the Journal of Cell Biology 1288 (5): 919-27 (1995).

In addition to covalent coupling of DNA molecules or other molecules to the B fragment, coupling can be via a strong non covalent interaction. To this end and by way of example, the cDNA of the B fragment is fused with that of streptavidin or with any other avidin derivative using known methods (Johannes et al (1987), J. Biol. Chem., 272: 19554-19561).

The protein resulting from fusion (B-streptavidin) can react with biotinylated DNA obtained by PCR using biotinylated primers, or with any other biotinylated substance. The resulting complex is bound to target cells and should be transported like the intracellular B fragment.

A further coupling method employs site-specific biotinylation of the B fragment. To this end, the cDNA of the B fragment is fused with cDNA coding for the BirA enzyme recognition site (Boer et al (1995), J. Bacterial, 177: 2572-2575; Saou et al (1996) Gene, 169: 59-64). After in vitro biotinylation, the B fragment is bound to other biotinylated molecules (such as cDNA, see above) via streptavidin or any other tetravalent avidine derivative.

The term "functional equivalent" means any sequence derived from the B fragment by mutation, deletion or addition, and with the same routing properties as the B fragment.

More precisely, a functional equivalent can be constituted by any fragment with the same retrograde transport properties and even intracellular transport to the nucleus as those described for the B fragment. Examples which can be cited are the B fragment of verotoxin described in the Proceedings of the National Academy of Sciences of the United States of America, 84 (13): 4364-8 1987, July, or the B fragment from ricin described by Lamb F. I. Et al in the European Journal of Biochemistry, 148(2): 265-70 (1995). After describing the particular transport properties of such fragments, the skilled person will be able to select the fragment which would be the best candidate as a vector for routing any sequence in any cellular compartment.

Thus the present invention encompasses the use of the B fragment of Shiga toxin or any other sub unit of bacterial toxins which would have comparable activities, in particular routing properties analogous to those of fragment B, including polypeptides miming the Shiga toxin B fragment. These polypeptides, and in general these functional equivalents, can be identified by screening methods which have in common the principle of detecting the interaction between random peptide sequences and the Gb₃ receptor or soluble analogues of the receptor. By way of example, phage libraries expressing random peptide sequences for selection on affinity columns comprising Gb₃ or after hybridisation with soluble radioactive Gb₃ analogues can be used. The glycolopid Gb₃ has been identified as being the cellular receptor of the Shiga toxin (Lingwood (1993), Adv. Lipid Res., 25: 189-211). Gb₃ is expressed by cells which are sensitive to the toxin and internalisation of the toxin would be permitted by an interaction with Gb₃. The present inventors have demonstrated that in HeLa cells in which expression of the Gb₃ receptor has been inhibited, the internalised B fragment is not transported into the Golgi apparatus but is accumulated in vesicular structures in the cytoplasm, principally represented by lysosomes. In the control cells, the B fragment is transported to the Golgi apparatus.

This hypothesis, whereby in the absence of the Gb₃ receptor, the B fragment is no longer transported to the biosynthesis system or secretion system, has been confirmed by biochemical experiments.

The inventors have demonstrated that in the presence of an inhibitor of Gb₃ receptor synthesis, PPNP (+PPNP), up to 50% of the internalised B fragment is degraded in the form of TCA-soluble material, which conforms to a transport activity towards a subsequent degradation compartment such as an endosomal or lysosomal compartment. When Gb₃ receptor synthesis is not inhibited (-PPNP), a much smaller proportion of internalised B fragment becomes TCA soluble. It can thus be concluded that the presence of the Gb₃ receptor is necessary for addressing the B fragment to specific compartments, which tends to favour the fact the main factor in the activity of the B fragment is its binding to the Gb₃ receptor.

The present invention provides chimeric polypeptide sequences, said sequences comprising at least: the Shiga toxin B fragment or a functional equivalent thereof the carboxy-terminal end of which has bound to it one or more X polypeptides with the following formula:

B--X, wherein:

B represents the B fragment of a toxin such as the Shiga toxin, the sequence of which has been described by N. G. Seidah et al (1986), J. Biol. Chem. 261: 13928-31, and in Strockbine et al (1988), J. Bact. 170: 1116-22, or a functional equivalent thereof, or from verotoxin or from ricin (references supra);

X represents one or more polypeptides the upper limit to the total length of which being that of compatibility with retrograde or intracellular transport.

The present invention also provides chimeric molecules with the following structure:

BX'

where B has the same meaning as above and X' represents a nucleotide sequence coding for a peptide sequence X the expression of which is desired, in particular an antigen epitope.

The chimeric molecules of the invention can also comprise:

a) modification sites such as an N-glycosylation site constituted by about 20 amino acids, phosphorylation sites or any sequence necessary for any maturation of the molecule;

b) a retention signal of the tetrapeptide KDEL type (Lys-Asp-Glu-Leu) (SEQ ID NO: 8) which, when it is bound to the carboxy-terminal end of resident ER proteins, causes retention after maturation of the proteins by passage into the Golgi apparatus. A discourse on the role of the retention signal in protein maturation has been provided by M. J. Lewis et al, (1992), cell, 68:

More generally, the chimeric polypeptide sequences can comprise:

any sequence necessary for maturation of the protein in a suitable cellular system;

any sequence necessary for recognition of a given cell type by the chimeric molecule, thus enabling selectivity of action and penetration into the cell cytoplasm.

The common factor between all chimeric sequences with structure B--X or B--X' is that they contain the B fragment or a functional equivalent thereof.

The chimeric molecules of the invention enable X sequences or the expression product of X' to be routed in the ER. When X is bound to the B fragment, retrograde transport also occurs via the Golgi apparatus and probably via the endosomes. Further, under certain conditions, the molecules of the invention can undergo maturation leading to a membrane presentation of certain epitopes contained in the chimeric polypeptide sequence.

The term "maturation" means any process which, from a given polypeptide, leads to the emergence of peptides which themselves can be presented in a cellular compartment including the cytoplasm. Maturation can occur either by enzymatic clipping in the endoplasmic reticulum, or by transport into the cytoplasm in which the polypeptide is cleaved then the peptides obtained are again transported in the endoplasmic reticulum.

Molecules of the class I major histocompatibility complex (c1 I MHC) can become charged with polypeptide molecules of interest X or X' after such cleavage and be presented on the cellular membranes.

When the chimeric molecule of the invention consists of coupling a B fragment or its equivalent with a polynucleotide molecule or an expression vector comprising a sequence the expression of which is desired, after transcription in the nucleus then translation in the cytoplasm, the polypeptide which is synthesised can undergo the same steps of cleavage, maturation and intracellular transport as that described above for a polypeptide chimeric sequence.

Chimeric polypeptide molecules in accordance with the invention can constitute an active principle in a therapeutic composition for immunotherapy by a mechanism which is close to biological processes regarding antigen presentation suitable for development of the immune reaction. The X fragment thus represents one or more epitopes for which membrane presentation is desired at the cell surface. The size of the X fragment is limited only by the intracellular transit capacity of the chimeric molecules under consideration.

This approach can be envisaged both for an anti-infectious or an anti-cancer immunotherapy and for constituting an antigenic bait in certain autoimmune diseases.

Any type of antigen presented by c1 I MHC is a good candidate for selecting simple or chimeric epitopes which form part of the constructions of the invention. Examples which can be cited are:

a) Human epitopes derived from melanoma cell proteins:

BAGE from tyrosinase (Boel, P et al (1995), Immunite 2, 167-75);

GAGE from gp75 (Van den Eynde, B. et al (1995), J. Exp. Med. 182, 689-98;

tyrosinase (Brichard V. et al (1993), J. Exp. Med. 178, 489-95);

p15 from A/MART-1 melanoma (Coulie P. G. et al (1994), J. Exp. Med. 180, 35-42; Kawakami Y. et al (1994), J. Exp. Med. 180, 347-52);

MAGE-1 and -3 from .beta.-catenin (De Plaen E. et al (1994), Immunogenetics 40, 369-9; Traversari C et al (1992), J. Exp. Med. 176, 1453-7.

b) Human enitopes derived from virus proteins involved in cancer development:

Peptides derived from E6 and E7 proteins of HPV 16 (Feltkamp M. C. et al (1993), Eur. J. Immunol. 23, 2242-9; Davis H. L. et al (1995), Hum. Gene Ther. 6, 1447-56);

Peptides derived from the Hbs protein of HBV (Rehermann B. et al (1995), J. Exp. Med. 181, 1047-58);

Peptides derived from proteins from EBV (Murray R. J. et al (1992), J. Exp. Med. 176, 157-68);

Peptide derived from cytomegalovirus.

c) Human epitopes derived from oncogenes:

p21ras (Peace D. J. (1993), J. Immunother 14, 1104; Ciemik, I. F. et al (1995) Hybridoma 14, 139-42);

p53 (Gnjatic S. (1995), Eur. J. Immunol. 25, 1638-42).

d) Epitopes of interest in autoimmune diseases:

These epitopes can be selected from those described by Chiez, R. M. et al (1994) in Immunol. Today 15, 155-60.

e) Epitopes of interest in infectious diseases:

Examples of such epitopes which can be cited are those described by Furukawa K. et al (1994) in J. Clin. Invest. 94, 1830-9.

In the constructions of the invention, X or the expression product of X' can also represent a polypeptide sequence which can restore an intracellular transport function which has been perturbed by whatever cause. As an example, a biological molecule can be trapped in the ER due to a modification by mutation, deletion or addition of a sequence, having the effect of blocking maturation or transit of that molecule. This is the case, for example, with mutated CFTR (.DELTA.F508) where binding to a chaperone molecule such as calnexin is modified such that its release is prevented or retarded, thus preventing intracellular transit. This mutation is the cause of cystic fibrosis. Introducing a non mutated replica into the endoplasmic reticulum could displace N-glycosylated chains of the CFTR (.DELTA.F508) glycoprotein from the interaction site with calnexin, with the result that protein transport to the plasma membrane is renewed, and the epithelial cells of the lung function normally.

The invention also provides nucleic acid constructions, in particular DNA or cDNA comprising a sequence of nucleotides coding for the chimeric protein the structure and variations of which have been defined above. More particularly, the invention provides expression vectors or plasmids carrying the above constructions and capable of being expressed in bacterial cultures. By way of example, the expression vector can be the pSU108 plasmid described by G. F. Su et al (1992), Infect. Immun. 60: 3345-59.

More particularly, the invention provides constructions comprising:

the sequence coding for the B fragment;

a sequence coding for one or more polypeptides the expression of which is desired. These may be epitopes the membrane expression of which is desired at the cell surface; they may also be polypeptides which can retain proteins in the Golgi apparatus; finally, they may be polypeptides which can restore a disturbed intracellular transport function.

The construction can also comprise any nucleic acid sequence coding for a polypeptide the presence of which enables proper intracellular transport in cells intended to be treated by the molecules of the invention. In particular, it may be:

a sequence coding for an N-glycosylation signal;

a sequence coding for the KDEL (SEQ ID NO: 8) retention signal.

The polynucleotide constriction of the invention is under the control of a promoter, preferably a strong promoter which can produce the correct degree of expression in bacteria into which it has been transfected.

The invention also provides transfected bacteria comprising these constructions, and capable of producing the chimeric polypeptides or proteins of the invention.

The host cells treated by the molecules of the invention also form part of the invention; they may be any type of cell, in particular:

those which can be treated in vivo such as immune system cells which are active in triggering cellular immunity, such as dendritic cells, macrophages or B lymphocytes;

those which can be treated in situ such as epithelial cells for use in restoring functions which have been altered either by a genetic defect or by a metabolic perturbation; cancer cells.

In general, the chimeric molecules of the invention allow a novel therapeutic method to be postulated which can overcome the problems linked to viral vectors or retroviral vectors which are normally used to integrate and express exogenous molecules in animal cells. The therapeutic method which derives from the molecules of the invention consists of directly treating the cells of a patient, either ex vivo or by direct stereotaxic application with the chimeric polypeptide sequences, or by conventional mucosal treatment methods such as aerosols.

The invention concerns the use of chimeric polypeptide or polynucleotide sequences coding for the polypeptides of the Livention in the production of therapeutic compositions in which particular polypeptides are expressed in the membranes of target cells. These polypeptides are advantageously epitopes against which the development of an immunological reaction is desired which are then presented on the surface of the immune system cells, in particular dendritic cells, macrophages or B lymphocytes. The B fragment of the Shiga toxin acts as an epitope vector enabling cells presenting antigens to be programmed.

The present invention concerns an immunotherapeutic method consisting of increasing cellular immunity as the result of the presence of an undesirable antigen in an organism, said method consisting of causing key cells of the immune system, such as dendritic cells and macrophages, to express particular epitopes. The treatment method of the invention is aimed at triggering immunity to cellular and humoral mediation by charging the c1 I or c1 II MHC molecules with the epitopes of interest, after restriction in the target cells. This leads to activation of cytotoxic T cells against the antigen which it is desired to eliminate.

The epitopes presented through the constructions of the invention originate from viral, parasitic or bacterial antigens or from any cell, organite, or micro-organism the elimination of which is desired, such as cancer cells or infected cells. The epitopes can also act as bait enabling "self" molecules recognised as foreign antigens in autoimmune diseases to be replaced by the epitopes of the invention, thus slowing down or reducing the immune reaction.

Examples of these epitopes have been cited above in the description of the chimeric polypeptide sequences.

The invention also concerns the use of the chimeric molecules of the invention in the manufacture of therapeutic compositions in which the particular polypeptides which it is desired to express can restore intracellular transit of a protein the altered structure of which leads to it being trapped in the ER and to an expression deficit. This is the case for membrane expression proteins which undergo intracellular maturation, including glycosylations, sulphatations, folding etc.

A particular example is that of mutated CFTR (.DELTA.F508) wherein the attachment of a chaperone molecule such as calnexin is modified following modification of the protein; this leads to the molecule being trapped, causing cystic fibrosis, leading to a general insufficiency of exocrin secretions, in particular in the pancreas and lungs.

The present invention concerns a therapeutic treatment method for diseases having an origin in a fault in protein secretion; the method consists of directly administering the chimeric polypeptides or administering the genetic information to the cells of patients in the form of plasmids carrying exogenic sequences coding for a peptide or polypeptide which can restore the deficient cellular function.

This restoration can result either in supplementation of the deficient function by the polypeptide X or competition between the mutated protein and the polypeptide synthesised from the exogenic sequence for binding with a specific molecule or receptor of the cellular machinery. A particular example is the treatment of the mutant cited above, causing cystic fibrosis, by administering a vector carrying a sequence coding for the attachment site for the CFTR protein with its chaperone molecule or by direct administration of the chimeric polypeptide.

The constructions of the invention endow the human or animal health world with a novel therapeutic means for treating diseases caused by a deficit in intracellular transit or for increasing or inducing a membrane presentation of a molecule, a polypeptide or an epitope of interest.

Claim 1 of 12 Claims

What is claimed is:

1. A chimeric polypeptide of the formula:

B--X

wherein

B represents the B fragment of Shiga toxin or a functional equivalent thereof, and

X represents one or more polypeptides of therapeutic significance, wherein said polypeptides are compatible with retrograde transport mediated by B to ensure processing or correct addressing of X.

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