NO175188B - - Google Patents

Abstract

A method of introducing a peptide into a cytosol by binding the peptide to a bacterial or plant toxin, or to a mutant thereof. A process for the preparation of a vaccine by binding a. peptide to a bacterial or plant toxin, or to a mutant thereof, to translocate the peptide into the cytosol for subsequent presentation on the cell surface using class I MHC antigens to elicit a class I restricted immune response and to expand the relevant population of CD8T lymphocytes. Vaccines prepared by the method and their application against viruses, intracellular bacteria and parasites, and against malignant conditions.

Description

Field of the invention

The present invention is directed to a method for the production of a peptide conjugate with the ability to penetrate into the cell cytosol, and in particular to a new principle in the production of vaccines against viruses, intracellular parasites and bacteria, and against malignant cells.

The background of the invention

In protection against pathogenic organisms and in their removal, antigen presentation by class I major histocompatibility antigens (MHC) plays an important role. Cytotoxic T-lymphocytes recognize cells that express foreign or unusual antigens on their surface and destroy the cells, which is important for clearing an infection. The same mechanism works in the removal of malignant cells. Antigen presentation by class I MHC requires that the antigen to be presented be present in the cytosol or in the endoplasmic reticulum (Germain, R.N. Nature 322, 687-689 (1986)). Polypeptides added from the outside therefore do not normally trigger a class I response. If the antigen is artificially introduced into the cytosol, however, presentation by MHC class I can occur (Moore, M.W., Carbone, F.R. & Bevan, M.J. Cell 54, 777-785

(1988)). The usual way to immunize against such structures today is to use weakened live viruses that are able to enter cells and replicate so that the relevant peptides are formed in the cells and can be presented on the cell surface. In this way, the population of the relevant cytotoxic CD8<+-> cells is expanded, and after later exposure to the corresponding virulent virus strain, the organism has an immune protection. The problems with this method are partly due to the fact that the attenuated viruses can sometimes revert to virulence, and partly to the problems of making attenuated viruses in many cases. Suitable and non-innocuous methods of introducing foreign peptides into the cytosol, such as viral antigens, could therefore be useful for vaccine purposes to expand the relevant population of CD8<+> MHC class I-restricted cytotoxic T lymphocytes.

The only established examples of external proteins that enter the cytosol are certain bacterial and plant toxins, such as diphtheria toxin, Pseudomonas aeruglnosa exotoxin A, ricin, abrin, viscumin, modeccin, Shigella toxin, cholera toxin, pertussis toxin (Olsnes, S. & Sandvig, K. I: "Immunotoxins" (A.E. Frankel, ed.), Kluwer Academic Publishers, Boston 1988, pp. 39-73; Olsnes, S. & Sandvig, K. I: "Receptor-mediated endocytosis" (I. Pastan & M.C. Willingham, eds.), Plenum Publ. Corp., 1985, pp. 195-234). Toxins of this group enter the cytosol where they carry out enzymatic reactions that are harmful to the cell or the organism. By means of genetic manipulations it is possible to form toxin molecules which have very low toxicity (Barbieri, J.T. & Collier, R.J. Infect. Immun. 55, 1647-1651 (1987)). If the toxins were able to introduce additional peptide material into cells, such non-toxic mutants could be useful for vaccine purposes by introducing antigenic peptides that can be presented by class I MHC antigens into the cytosol (Cerundolo et al. Nature 345, 449 (1990)). Such antigen sequences can be obtained from a number of viruses, bacteria and parasites, and it is also possible to extract such structures from certain malignant cells.

The purpose of the present invention is to provide a method for the production of a peptide conjugate with the ability to penetrate into the cell cytosol, so that a mechanism is obtained for the translocation of antigenic peptide sequences into the cytosol in a safe manner, in order, among other things, to expand the population of cytotoxic T-lymphocytes that are able to react with the corresponding antigen. Although the mechanism of action of the various toxins mentioned above is in principle the same, it has been worked out most extensively for the case of diphtheria toxin. This is the toxin that has been used in most investigations in connection with this invention.

Summary of the invention

We show here that an essentially non-toxic mutant of diphtheria toxin is able to translocate oligopeptide bound to its N-terminus into the cytosol. The peptides that we have examined are sufficiently different in sequence to conclude that a large number of different peptides can be introduced into the cells in the same way.

The present invention thus relates to a method for the production of a peptide conjugate with the ability to penetrate into the cell cytosol, which is characterized by the fact that the gene that codes for the peptide is bound by recombinant DNA technology to the gene that codes for the A part of a bacterial or plant holotoxin , or for a mutant thereof, which has been altered by mutation so that it has lost its toxicity without having lost the ability to penetrate the cell cytosol and bring additional peptide material into the cytosol, after which the peptide-toxin compound is expressed.

According to a particularly preferred embodiment of the invention, a vaccine is produced by a method characterized by the fact that the gene that codes for a peptide is linked by recombinant DNA technology to the gene that codes for the A part of a bacterial or plant holotoxin, or for a mutant thereof , which has been altered by mutation so that it has lost its toxicity without having lost the ability to penetrate the cell cytosol and bring additional peptide material into the cytosol, after which the peptide-toxin compound with the ability to penetrate the cell cytosol is expressed, as that the peptide can be translocated into the cytosol for subsequent presentation on the cell surface by means of class I MHC antigens to trigger a class I limited immune response and expand the relevant population of CD8<+->T lymphocytes. Vaccines produced using the above-mentioned method can be used against viruses, intracellular bacteria and parasites, and against malignant conditions.

Figure texts

Figure 1. N-terminal extensions of diphtheria toxin.

A. The coding region of the diphtheria toxin gene carrying a triple mutation that changes Glu<148> to Ser, and where Gly<1> was replaced with initiator-Met placed behind a T3 promoter, resulting in pBD-1S (McGill, S. , Stenmark, H., Sandvig, K.

& Olsnes, S.: EMBO J. 8, 2843-2848 (1989)). To obtain pB-B3-D1, pBD-1 was cleaved with NcoI, and an oligonucleotide encoding the oligopeptide MGVDEYNEMPMPVN (referred to as B3) was inserted. pGD-2 encodes diphtheria toxin with its native signal sequence MSRKLFASILIGALLGIGAPPSAHA (referred to as ss), following an SP6 promoter. The plasmid was obtained by dividing pGD-1 (McGill, S., Stenmark, H., Sandvig, K. & Olsnes, S.: EMBO J. 8, 2843-2848 (1989)) with HindIII and Pstl, removing the overhangs with S^nuclease and religase, whereby pGD-2 is formed.

B. The genes were transcribed in vitro and the mRNAs obtained were translated in rabbit reticulocyte lysate systems in the presence of [<35>S]methionine (McGill, S., Stenmark, H., Sandvig, K. & Olsnes, S.: EMBO J. 8, 2843-2848 (1989)). To remove reducing agents and allow disulfide bridges to form, the translation mixture was dialyzed overnight against PBS (0.14 M NaCl, 10 mM Na phosphate, pH 7.4), and then for 4 h against Hepes medium (Dul-becco -modified Eagle's medium where the bicarbonate had been replaced with 20 mM Hepes, pH 7.4). An aliquot of each sample was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) under reducing conditions (Olsnes, S. & Eiklid, K. J. Biol. Chem. 255, 284-289 (1980)). In some cases, the translation product was treated with protein A-Sepharose previously incubated with rabbit anti-B3 antiserum (lanes 3 and 4) or antiricin (lane 5). The adsorbed material was analyzed by SDS-PAGE. DT, translation product from pBD-1; B3-DT, translation product from pB-B3-D1; ss-DT, translation product from pGD-2.

Figure 2. Translocation to cytosol of A-fragment with N-terminally added B3-oligopeptide. pBD-1 and pB-B3-D1 were transcribed and translated in vitro. The corresponding translation products (DT and B3-DT) were added to Vero cells grown as monolayers in 24-well microtiter plates and maintained at 24 °C for 20 minutes in the presence of 10 µM monensin (McGill, S., Stenmark, H. , Sandvig, K. & Olsnes, S.: EMBO J. 8, 2843-2848

(1989)). The cells were washed twice with Hepes medium and then treated with 0.4 µg/ml TPCK (N-tosyl-L-phenylalanine-chloromethyl ketone)-treated trypsin in Hepes medium containing 10 µM monensin for 5 minutes at 20" C. The cells were washed and exposed to Hepes medium, pH 4.8, containing 10 mM Na-gluconate to increase buffering capacity at the low pH.After 2 minutes at 37°C, the cells were washed with Hepes medium, pH 7, 4, and then treated with 3 mg/ml pronase in Hepes medium, pH 7.4, containing 10 pM monensin for 5 minutes at 37° C. The cells detached from the plastic by the treatment were recovered by centrifugation and washed once with Hepes medium containing 1 mM NEM (N-ethylmaleimide) and 1 mM PMSF (phenyl-methylsulfonyl fluoride). In some cases (lanes 1-3 and 8-10) the cells were lysed with "Triton X-100" in the phosphate buffer saline solution containing 1 mM PMSF and 1 mM NEM, nuclei were removed by centrifugation and the protein in the supernatant fraction precipitated with 10% (w/v) trichloroacetic acid e or precipitated immunologically with anti-B3 antibodies adsorbed to protein A-"Sepharose". In other cases (lanes 4-7), the cells were treated with 50 µg/ml saponin in PBS containing 1 mM PMSF and 1 mM NEM to release translocated A fragment, and then both the proteins in the pellet and in the supernatant fractions were precipitated with trichloroacetic acid. In all cases, the precipitated material was analyzed by SDS-PAGE (13.5% gel) under non-reducing conditions. Figure 3. Translocation to cytosol of diphtheria toxin with signal sequence. Lanes 1-4: <125>I-labeled native toxin (wt-DT, lane 1) and in vitro translated pGD-2 ([35S]methionine-labeled toxin with signal sequence, ss-DT) were bound to Vero cells and "nicked" on the cells (fields 1 and 2). In field 3, the cells were treated as in field 2, except that six times more translation product was used, and the cells were then exposed to pH 4.8 and pronase as in Figure 2. The cells were lysed with "Triton X-100" and cores removed. The supernatants were incubated with protein A-Sepharose which had been pre-incubated with rabbit anti-diphtheria toxin serum. The adsorbed material was analyzed by reducing (lanes 1 and 2) or non-reducing (lanes 3 and 4) SDS-PAGE (10% gel). In field 4, the pronase-treated cells were treated with 50 µg/ml saponin, and the material released into the medium was analyzed directly. Lanes 5-12: Translation product from pBD-1 (DT) and pGD-2 (ss-DT) was ligated to Vero cells, "nicked", exposed to pH 4.8 and then treated with pronase. The lysed cells were either analyzed by non-reducing SDS-PAGE (15% gel) directly (lanes 5-8), or they were treated with saponin, and the membrane pellets (lanes 9 and 10) and the supernatant fractions (lanes 11 and 12) were analyzed separately.

Detailed description

Diphtheria toxin is synthesized by pathogenic strains of Corynebacterium diphtheriae as a single-chain polypeptide. The protein is easily split ("nicked") in a trypsin-sensitive site, whereby two disulfide-bonded fragments, A and B, are obtained (Pappenheimer, A.M., Jr. Rnnu. Rev. Biochem. 46, 69-94

(1977)).

The B-fragment (37 kD) binds to cell surface receptors, while the A-fragment (21 kD) is an enzyme that is translocated to the cytosol where it inactivates elongation factor 2 by ADP-ribosylation and thereby blocks protein synthesis (Van Ness, B.G., Hovard, J.B. & Bodley, J. W. J. Biol. Chem. 255, 10710-10716 (1980)). The translocation that normally occurs across the boundary membrane of endosomes is triggered by the low pH in the acidic vesicles (Draper, R.K. & Simon, M.I. J. Cell Biol. 87, 849-854 (1980); Sandvig, K. & Olsnes, S. J. Cell Biol. 87 , 828-832 (1980)). When cells with surface-bound toxin are exposed to acidic medium, translocation occurs from the cell surface (Sandvig, K. & Olsnes, S. J. Biol. Chem. 256, 9068-9076

(1981)). In the presented examples, we have used this artificial system because it makes it possible to distinguish between translocated and non-translocated material (Moskaug, 3. 0., Sandvig, K. & Olsnes, S. J. Biol. Chem. 262, 10339-10345 ( 1987); Moskaug, J.Ø., Sandvig, K. & Olsnes, S.J. Biol. Chem. 263, 2518-2525

(1988)).

To avoid a toxic effect on the cells with the diphtheria toxin vector, a mutant toxin containing a triple mutation that changes Glu<148>, located in the toxin's enzymatic active site, to Ser was used (Barbieri, J. T. & Collier, R. J. Infect. Immun .55, 1647-1651 (1987)). The modified toxin has greatly reduced toxicity.

Examples

Two variants of the mutated toxin were used, one without (pBD-1) (McGill, S., Stenmark, H., Sandvig, K. & Olsnes, S.: EMBO J. 8, 2843-2848 (1989)) , and one with (pGD-2), the natural 25-amino acid signal sequence (Figure 1A). In one case, a foreign oligopeptide, called B3, was bound to the N-terminus of the toxin, resulting in the plasmid pB-B3-D1.

The constructs placed behind T3 or SP6 RNA polymerase promoters were transcribed and translated in vitro (McGill, S., Stenmark, H., Sandvig, K. & Olsnes, S.: EMBO J. 8, 2843-2848 ( 1989)). In each case, a major band corresponding to the full-length protein and only traces of lower molecular weight material was obtained (Figure 1B). Toxin with signal sequence (lane 7) or with B3 (lane 1) migrated as expected slightly slower than toxin as such (lanes 2 and 6). Moreover, toxin with B3 was selectively precipitated with anti-B3 (lane 4), but not with a control serum (lane 5). Toxin without B3 was not precipitated with anti-B3 (lane 3).

The dialyzed translation products were bound to Vero cells, "nicked" on the cells with low concentrations of trypsin, and then the cells were exposed to pH 4.8. Under these conditions, part of the bound toxin was translocated to the cytosol and was thereby protected against pronase added to the medium (Moskaug, 3. 0., Sandvig, K. & Olsnes, S. J. Biol. Chem. 263, 2518-2525 (1988)) . In the case of diphtheria toxin as such, under these conditions two fragments (molecular weights 21 kD and 25 kD) were protected (Figure 2, lane 1) corresponding to the whole A fragment (21 kD) and part of the B fragment (25 kD of a total of 37 kD). The interfragment disulfide was reduced, obviously after exposure to the cytosol (Moskaug, 3. 0., Sandvig, K. & Olsnes, S. J. Biol. Chem. 262, 10339-10345 (1987)).

Example 1

When the same experiment was performed with toxin containing B3, two major fragments (25 kD and 22.5 kD) were protected in addition to small amounts of the 21 kD fragment (lane 2). The latter probably constitutes the A fragment where B3 had been cleaved off. When exposure to low pH was omitted, no fragments were protected (lane 3). The 22.5 kD fragment was precipitated with anti-B3 (lane 9) but not with preimmune serum (lane 10). Protected A fragment without the oligopeptide was not precipitated with anti-B3 (lane 8). The apparent greater amount of protected A fragment with B3 is due to more incorporated radioactivity as B3 contains three methionines and the A fragment alone 5.

When cells with translocated diphtheria toxin are treated with a low concentration of saponin that allows cytoplasmic marker enzymes to leak into the cells without dissolving the membranes, the translocated A fragment is released into the medium, while the B fragment-derived 25 kD polypeptide remains bound to the membrane fraction (Moskaug, 3. 0., Sandvig, K. & Olsnes, S. J. Biol. Chem. 263, 2518-2525 (1988); Moskaug, 3. 0., Sletten, K., Sandvig, K. & Olsnes, S. J. Biol. Chem. 264, 15709-15713

(1989); Moskaug, 3. 0., Sandvig, K. & Olsnes, S. J. Biol. Chem. 264, 11367-11372 (1989)). This indicates that the translocated A fragment is free in the cytosol, while the 25 kD polypeptide is inserted into the membrane. Also, most of the A-fragment containing B3 was released with saponin (lane 7) in the same way as normal A-fragment (lane 6), while the 25 kD fragment was bound to the membranes (lanes 4 and 5). It therefore appears that diphtheria toxin is able to translocate B3 (14 amino acids) into the cytosol.

Example 2

To test whether a larger oligopeptide could also be translocated, toxin carrying its normal signal sequence (25 amino acids) was chosen. As shown in Figure 3, lane 2, this protein was "nicked" by trypsin into a 23.5 kD A fragment and into a 37 kD B fragment. (In this experiment, the toxin was only partially "nicked". Partially "nicked" <125>I-labeled natural toxin is shown for comparison in panel 1. ) When the toxin with signal sequence was bound to cells, "nicked" and then exposed to pH 4.8, two fragments (23.5 kD and 25 kD) were protected against pronase (lane 8). Protected A fragment with uncleaved signal sequence is also shown in lane 3, where the material was precipitated with an anti-diphtheria toxin serum that binds the entire toxin, the A fragment as well as the entire B fragment (see lanes 1 and 2), but not the 25 kD fragment . When the pronase-treated cells were treated with saponin, the extended A fragment was released into the medium (lanes 4 and 12), while the 25 kD fragment remained in the membrane fraction (lane 10).

Claims (4)

1. Method for producing a peptide conjugate with the ability to penetrate the cell cytosol, characterized in that the gene that codes for the peptide is linked by recombinant DNA technology to the gene that codes for the A part of a bacterial or plant holotoxin, or for a mutant thereof, which has been altered by mutation so that it has lost its toxicity without having lost the ability to penetrate the cell cytosol and bring additional peptide material into the cytosol, after which the peptide-toxin compound is expressed. 2. Method according to claim 1 for the production of a vaccine, characterized in that the gene that codes for a peptide is linked by recombinant DNA technology to the gene that codes for the A part of a bacterial or plant holotoxin, or for a mutant thereof, which has been changed by mutation so that it has lost its toxicity without to have lost the ability to penetrate the cell cytosol and bring additional peptide material into the cytosol, after which the cell cytosol-penetrating peptide-toxin compound is expressed, allowing the peptide to be translocated into the cytosol for subsequent presentation on the cell surface by class I MHC antigens to trigger a class I limited immune response and expand the relevant population of CD8<+->T lymphocytes. 3. Method according to claim 1 or 2, characterized in that a non-toxic mutant of diphtheria holotoxin or a related holotoxin is used, such as ricin, abrin, modeccin, viscumin, volkensin, Pseudomonas aeruginosa exotoxin A, Siiigella toxin, anthrax -toxin, cholera toxin, E. coil heat-labile toxin or pertussis toxin. 4. Method according to claims 1-3, characterized in that a non-toxic mutant of diphtheria holotoxin is used.