Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2000-01-18
2003-09-02
Caputa, Anthony C. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C514S002600, C424S184100
Reexamination Certificate
active
06613882
ABSTRACT:
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 Gb
3
receptor or soluble analogues of the receptor. By way of example, phage libraries expressing random peptide sequences for selection on affinity columns comprising Gb
3
or after hybridisation with soluble radioactive Gb
3
analogues can be used. The glycolopid Gb
3
has been identified as being the cellular receptor of the Shiga toxin (Lingwood (1993), Adv. Lipid Res., 25: 189-211). Gb
3
is expressed by cells which are sensitive to the toxin and internalisation of the toxin would be permitted by an interaction with Gb
3
. The present inventors have demonstrated that in HeLa cells in which expression of the Gb
3
receptor has been inhibited (FIG.
1
A), 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 (FIG.
1
B).
This hypothesis, whereby in the absence of the Gb
3
receptor, the B fragment is no longer transported to the biosynthesis system or secretion system, has been confirmed by biochemical experiments (FIG.
2
).
The inventors have demonstrated that in the presence of an inhibitor of Gb
3
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 Gb
3
receptor synthesis is not inhibited (−PPNP), a much smaller proportion of internalised B fragment becomes TCA soluble. It can thus be concl
Goud Bruno
Johannes Ludger
Caputa Anthony C.
DeConti, Jr. Giulio A.
Institut Curie and Centre National de la Recherche Scientifique
Lahive & Cockfield LLP
Soroos Cynthia M.
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