Universal carrier for targeting molecules to GB3 receptor expressing cells

Abstract

The present invention concerns an universal polypeptidic carrier for targeting directly or indirectly a molecule to Gb3 receptor expressing cells and having the following formula STxB-Z(n)-Cys, wherein: STxB is the Shiga Toxin B subunit or a functional equivalent thereof, Z is an amino-acid devoided of sulfydryl group, n being 0, 1 or a polypeptide, Cys is the amino-acid Cysteine,
and the use thereof for MHC class I and MHC class II presentation of antigens.

Claims

1. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising the nucleotide sequence STxB encoding the Shiga Toxin B subunit or a functional equivalent thereof bearing at its 3 end the codon TGT, or the codon TGC encoding cysteine, wherein the functional equivalent thereof binds to the Gb3 receptor and triggers the internalization of an antigen and its presentation in an MHC class I and MHC class II restricted pathway on the same antigen presenting cell; b) a polynucleotide comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence encoding the Shiga Toxin B subunit or a functional equivalent thereof, bearing at its 3 end the codon TGT or TGC, wherein the functional equivalent thereof binds to the Gb3 receptor and triggers the internalization of an antigen and its presentation in an MHC class I and MHC class II restricted pathway on the same antigen presenting cell and c) a nucleotide sequence complementary to the sequence in a) or b).

2. The polynucleotide according to claim 1, comprising SEQ ID No. 2.

3. A recombinant vector or plasmid, comprising a polynucleotide sequence according to claim 1.

4. A recombinant cell line obtained by transformation with the recombinant vector according to claim 3.

5. The recombinant cell line according to claim 4, which is a prokaryotic cell line.

6. The recombinant cell line according to claim 5, which prokaryotic cell line is E. coli.

7. The recombinant cell line according to claim 6, deposited at the CNCM on Dec. 19, 2000 under accession number I-2604.

8. A method for constructing a recombinant vector according to claim 3 comprising: a) providing a plasmid comprising a STxB sequence; b) applying two PCR amplification steps using two couples of primers AA and BB wherein A and B are complementary to each other and comprise the Cys codon and A and B are outside the STxB sequence; c) isolating the amplified fragments; d) hybridizing the amplified fragments; e) applying a PCR amplification on the hybridized fragments; and f) inserting the amplified fragments into a plasmid.

9. The method according to claim 8, wherein in step f) the fragments are inserted into a SphI and SalI restriction site of the plasmid pSU108.

10. A process for producing an isolated polypeptide comprising: a). culturing a recombinant cell line of claim 4; b) obtaining a periplasmic extract of said cells; and c) purifying said polypeptide.

11. The process according to claim 10 wherein in step a) the cell line is E. coli and in step c) the purification is by anion exchange column chromatography followed by a gel filtration column chromatography.

Description

LEGEND OF THE FIGURES

(1) FIG. 1 represents the protein profile of the final SephaDex 75 column yielding purified STxB-Cys. Fractions 20-25 contain mostly monomeric STxB-Cys (the positions of monomeric and dimeric STxB-Cys are indicated to the right). Molecular weight markers are indicated to the left.

(2) FIG. 2a represents the coupling of Type2 of Pep2 [as defined in example 2] to STxB-Cys, followed by an in vitro antigen presentation assay on D1 dendritic cells, as described in (2). Two different preparations of STxB-Cys coupled to the SL8 peptide, an immunodominant epitope of ovalbumin were used (termed 4A and 9A). Upon fixation, antigen presentation is abolished showing that no extracellular processing occurred.

(3) FIG. 2b represents a control experiment of FIG. 2a, in which it is shown that fixed D1 can still present free SL8 peptide.

(4) FIG. 3 represents another experiment on fixed and non fixed D1 cells using a coupling reaction of Type1 on STxB-SH and Pep1 [as defined in example 2].

(5) FIG. 4 represents the B-subunit dependent presentation of antigenic peptides derived from a coupling of Pep2 to B-Glyc-Cys-KDEL. The use of Fab fragments from an antibody that neutralize STxB binding to Gb3 also abolishes antigen presentation.

(6) FIG. 5a shows that the Gb3 synthesis inhibitor PPMP inhibits B-subunit dependent antigen presentation and FIG. 5b shows that SL8 presentation is not decreased in PPMP treated cells.

(7) FIG. 6 represents reaction scheme for full size Ova coupling to STxB-Cys using the heterobifunctional cross linker MBS. Top: first reaction linking MBS to primary amines of Ova. Middle: second reaction between activated Ova and STxB-Cys. Bottom: Structure of MBS.

(8) FIG. 7 shows western analysis of Ova coupling to STxB-Cys. The upper part of the figure represents an immunoblot using anti-STxB antibody, the lower part an immunoblot using anti-Ova antibody. The intermediates of different steps of the purification procedure are shown. Lane 1: uncoupled proteins (marked by a cross). Lane 2: coupling reaction (coupling product marked by an arrow). Lane 3: eluate of the immunoaffinity column doted with anti-STxB antibody. Lanes 4-7: fractions from the gelfiltration column. Lane 4: fractions 9-10. Lane 5: fractions 11-12. Lane 6: fractions 13-14. Lane 7: fractions 15-19 (free STxB-Cys). Fractions 11-12 contain the bulk of monomeric coupling product. Some material with lower electrophoretic mobility can also be detected, originating likely from dimeric Ova present in the original preparation.

(9) FIG. 8 represents immunofluorescence analysis of STxB-Cys-Ova transport in HeLa cells. The coupling product STxB-Cys-Ova (upper part of the figure) or a mixture of STxB-Cys and Ova (lower part of the figure) were incubated with HeLa cells on ice. After washing, the cells were shifted for 45 min to 37 C., fixed, and stained with the indicated antibodies. Note that when Ova is linked to STxB-Cys (top), the protein is vectorized into the Golgi apparatus, co-stained for the Golgi marker Rab6. If Ova is only mixed with STxB-Cys (bottom), the protein cannot be detected on the cells.

(10) FIG. 9a shows MHC class I and II restricted antigen presentation using STxB-Cys-Ova.sub.329-339 used to sensitize the murine dendritic cell line D1.

(11) FIG. 9b shows MHC class I and II restricted antigen presentation using STxB-Cys-Ova.sub.257-264 and the immunodominant SL8 Ova-derived peptide alone used to sensitize the murine dendritic cell line D1.

EXAMPLE 1

Preparation of the Universal Carrier

(12) a) Construction of a Plasmid Expressing STxB-Cys:

(13) In a preferred embodiment, the plasmid pSU108 (7) was modified to introduce the Cysteine codon tgt at the 3 end of the B-fragment cDNA. PCR primer A: SEQ ID no 3 (5-AGCGAAGTTATTTTTCGTTGTTGACTCAGAATAGCTC-3) and primer A: SEQ ID no 4 (5-GAGCTATTCTGAGTCAACACGAAAAATAACTTC-3) were used with plasmid specific primers ShigaAtpE: SEQ ID no 5 (5-CACTACTACGTTTTAAC-3) and Shiga-fd: SEQ ID no 6 (5-CGGCGCAACTATCGG-3) to produce DNA fragments which, in a second PCR with primers Shiga AtpE and Shiga-fd yielded a fragment that was cloned into the SphI and SalI restriction sites of pSU108. Sequences derived by PCR were verified by dideoxy-sequencing.

(14) b) Protein Purification:

(15) b) 1. Preparation of the Periplasmic Extract was Performed as Follows: Inoculate 125 ml of LB/Amp with 125 l of an overnight culture grown at 30 C., grow over night at 30 C., transfer into 375 ml of LB/Amp at 50 C.; incubate 4 hours at 42 C., centrifuge to pellet cells, wash cells 3 times with 10 mM Tris/HCl, pH 8.0, re-suspend cells in 200 ml of 25% sucrose, 1 mM EDTA, 10 mM Tris/HC1, Ph 8.0; incubate at room temperature for 10 min., centrifuge to pellet cells, re-suspend cells in 200 ml of ice cold water containing a protease inhibitor cocktail; incubate on ice for 10 min., centrifuge; collect supernatant; add 20 mM Tris/HC1, Ph8.0.

(16) b) 2. Purification on Columns:

(17) The periplasmic extract was loaded on a QFF anion exchanger column (pharmacia) and eluted at 230 mM NacI. STxB-Cys containing fractions are pooled, diluted 4-fold and loaded on a Mono Q anion exchanger column (pharmacia), followed by elution at 230 mM NacI. After concentration with microconcentration devices from PallFiltron, the pooled fractions were passed through a Sephadex 75 gel filtration column. Purity was above 95% (FIG. 1).

(18) b) 3. Product Characterization:

(19) The B-fragments of STxB-Cys, purified from Sephadex 75 gel filtration columns, are essentially monomeric (FIG. 1). This is in marked difference to constructions where the Cysteine was added at more than 2 amino acids from the natural C-terminus of the B-fragment. In these cases, neighbouring B-fragments within a pentamer are engaged in disulfide bonds.

EXAMPLE 2

Conditions for Coupling of Activated Peptides to the Universal Carrier

(20) a) Carriers:

(21) Three different carriers have been compared.

(22) 1) STxB-Cys: B-fragment to which a Cys has been added right to its C-terminus. This protein elutes as a monomer from the purification columns.

(23) 2) STxB-Z.sub.2-Cys: carrier with a short spacer (2 amino acids resulting from a cloning cassette) between the C-terminus of the wild type B-fragment and the Cys. The majority of the protein eluted as dimers from the purification columns. These can be separated under reducing conditions, indicating the formation of disulfide bonds between monomers in the pentameric B-subunit complex.

(24) 3) STxB-Glyc-Cys-KDEL: carrier in which the Cys is located between a Glycosylation cassette being 9 amino acid long and a C-terminal KDEL peptide. The majority of the protein eluted as dimers from the purification columns. These can be separated under reducing conditions, indicating the formation of disulfide bonds between monomers in the pentameric B-subunit complex.

(25) b) Test Peptides:

(26) 1) Pep1: a synthetic peptide of 16 amino acids carrying the SL8 antigenic peptide derived from chicken ovalbumin.

(27) 2) Pep2: a synthetic peptide of 24 amino acids as above with, in addition, a His-gag at its C-terminus.

(28) 3) SL8: the antigenic peptide from ovalbumin that can directly exchange with peptides on MHC class I complexes at the plasma membrane of antigen presenting cells.

(29) c) Coupling Conditions:

(30) Under reducing conditions (Type1): Fusion proteins were treated with DTT overnight, then activated peptide (carrying a bromo acetate group at its N-terminus) was added in excess. Conditions used for the first coupling experiments using fusion proteins will mostly dimerize monomers (proteins STxB-Z.sub.2-Cys and STxB-Glyc-Cys-KDEL).

(31) Under non-reducing conditions (Type2): Fusion proteins are directly reacted with the activated peptides.

(32) d) Biochemical and Morphological Controls:

(33) Pep2 carries a His-tag. This has allowed us, using an anti-His antibody, to show the presence of Pep2 on B-subunit by Western blotting, and the B-subunit dependent transport of Pep2 in HeLa cells.

(34) FIG. 2a shows that a dose dependent stimulation of the B3Z CTL hybridoma (measurement of -galactosidase activity) was observed with non-fixed cells, while fixation abolished antigen presentation.

(35) Note that antigen presentation only works on non-fixed cells, indicating that the observed presentation does not result from contaminating free Pep2.

(36) FIG. 2b shows the control experiment of FIG. 2a in which it is shown that fixed D1 cells can still present free SL8 peptide.

(37) In FIG. 3, it appears that in this type of protocol, some free Pep1 appears to co-purify with the fusion protein, since at high doses (200-1000 nM), some presentation was observed on fixed cells. Presentation by SL8 is shown to the right.

(38) In FIG. 4, the coupled protein (lanes 1 and 2) or the SL8 Peptide (lanes 5 and 6) were incubated (lanes 2 and 5) or not (lanes 1 and 4) with anti-B-subunit Fab-fragment derived from the 13C4 antibody which inhibits the binding of the B-subunit to Gb3. Note that the Fab-fragment neutralizes the capacity of the B-subunit to introduce the antigenic peptide into the class I pathway, while the presentation with SL8 is not affected under these conditions. The background signal in this experiment was at 0.3.

(39) In FIG. 5a, D.sub.1 cells were pre-treated with PPMP (see FIG. 3b of (2)) for 3 days. This treatment lead to an important decrease of Gb3 expression at the cell surface, without however eliminating it completely. Under this condition, antigen presentation from a coupling reaction of Pep1 with STxB-Glyc-Cys-KDEL was significantly reduced, indicating that Gb3 is important for the presentation phenomena.

(40) It appears from all these experiments that the coupling under non-reducing to STxB-Cys is surprisingly efficient (in terms of sensitivity; note that, as shown in FIG. 1, only 4 nM of STxB-Cys-Pep2 are necessary to have a response). Thus, the universal carrier STxB-Cys is preferred due to its simplicity in its preparation and to the reproducibility of the coupling.

(41) Hence, the optimal conditions for coupling of activated peptides to STxB-Cys were the following: dialyse STxB-Cys against 20 mM Borate buffer, pH 9.0, 150 mM NaCl, concentrate to 1 mg/ml, dissolve N-terminally activated peptide (activated with bromoacetate anhydride) at 12 mM in DMSO, dilute peptide to 0.2 mM in protein solution, incubate 12 hours at room temperature, dialyse against PBS.

EXAMPLE 3

Characterization of TxB as to its Antigen Presentation Capacity

(42) The following experimental series will help to fully describe the capacity of STxB to function in antigen presentation system.

(43) a) Class I- and Class II-restricted Antigen Presentation:

(44) A peptide carrying class I- and II-restricted antigenic peptides from chicken ovalbumin (BrCH2-CONH-LEQLESIINFEKLTEWSLKISQAVHAAHAEINEAGR (SEQ ID NO:6), sequences 257-264 and 323-339 were coupled to STxB-Cys, and the class I- and class II-restricted presentation of these peptides were assayed using the corresponding T-cell hybridomas.

(45) b) Coupling of Whole Size Proteins.

(46) Our preliminary evidence suggests that chicken ovalbumin can be coupled to STxB-Cys. These experiments have been done with the SPDP heterobifunctional cross-linker. (Carlsson et al., 1978).

(47) A first series of antigen presentation experiments indicated that the ovalbumin protein can be introduced into the endogenous MHC class I-restricted antigen presentation pathway of mouse dendritic cells. SPDP has the inconvenience of being cleavable by serum thiolases. This cross-linker was successfully substituted by MBS which is non-cleavable. Other antigenic proteins (Mart 1 and polypeptides derived from HPV16-E7 and Muc1) are tested to show that the procedure is of universal use.

(48) c) Coupling of Complex Protein Mixtures.

(49) A lysate from the cervix carcinoma-derived cell line Caski is used. This cervix carcinoma cell line, which expresses the HLA-A2 allele at its membrane, also expresses Human papillomavirus derived peptides. E7 is a early transcribed ORF from HPV which is necessary for transformation of primary keratinocytes. Since anti-E7 HLA A2-restricted CTL are elicited in vitro. The efficacy of the coupling of this protein mixture by a presentation assay specific for HLA-A2 E7 derived peptides was tested. As control, a lysate from a HLA-A2-positive cell line which does not express E7 (croft cells or Daudi) was coupled to STxB-lys.

EXAMPLE 4

Application to MHC Class I-restricted Antigen Presentation

(50) The experiment of FIG. 4 shows that STxB-Cys dependent antigen presentation is inhibited when the interaction with Gb.sub.3 is abolished. Here it is found that pre-binding of the Fab fragment of monoclonal Ab against STxB to 0.1 M STxB-Cys, coupled to SL8, inhibited antigen presentation, suggesting that STxB-Cys binding to Gb.sub.3 is necessary for antigen presentation. Similar results were obtained when Gb.sub.3-expression was inhibited with a drug (FIG. 4).

EXAMPLE 5

Reaction Chain for Coupling Ovalbumine to the STxB-Cys

(51) The reaction scheme is shown in FIG. 6.

(52) In a first reaction, the N-hydroxysuccinimide ester moiety of MBS reacts with primary amines on an antigenic target protein, such as the model protein ovalbumin (Ova). The reaction product is purified and then incubated in a second reaction with STxB-Cys leading to coupling via the maleimidobenzoyl moiety.

(53) FIG. 7 shows the SDS-PAGE and Western analysis of a typical coupling reaction involving STxB-Cys and Ova. For coupling, 20 mg/ml of Ova in 100 mM HEPES, pH 7.4, was incubated with 4.5 mM of MBS for 30 min at room temperature. The reaction is passed through a PBS/EDTA 10 mM equilibrated gel filtration column. Eluted Ova is concentrated to 20 mg/ml. 1 volume of STxB-Cys at 3.5 mg/ml in PBS/EDTA is mixed with 1 volume of activated Ova and incubated over night at room temperature.

(54) FIG. 7 shows that within the coupling reaction, bands with lower electrophoretic mobility (labeled with arrows; coupling product) can be detected in addition to uncoupled STxB (upper part) and uncoupled Ova (lower part; uncoupled proteins are labeled with a cross). The reaction product is purified by passage through an immunoaffinity column made with 13C4 anti-STxB monoclonal antibody (lane IP column). Note that free Ova is eliminated. Eluted STxB-Cys (coupled and non-coupled) is then passed through a gel filtration column to separate free STxB (fractions 15-19) from coupled STxB-Cys (fractions 9-14; note that fractions 11-12 contain the bulk of coupled protein; the upper coupling band, which is minor compared to the lower band, probably results from dimeric Ova).

EXAMPLE 6

Intracellular Transport Characteristics of STxB-Cys-Ova Coupling Product

(55) 0.5 M of STxB-Cys-Ova was incubated with HeLa cells on ice. The cells were washed and shifted to 37 C. for 45 min, fixed, and stained for the indicated antibodies. As shown in FIG. 8, when STxB-Cys and Ova were linked by MBS, Ova immunoreactivity could be detected together with STxB immunoreactivity in the Golgi apparatus, stained by Rab6. When both proteins are incubated as separate entities with HeLa cells, only STxB-Cys is transported to the Golgi, while Ova cannot be detected on the cells. These data clearly show that couples STxB-Cys is still transported in the same manner as uncoupled STxB-Cys, and that Ova is vectorized via STxB-Cys.

EXAMPLE 7

The STxB-Cys Allows Both, MHC Class I and II Restricted Presentation of Peptides Derived from Full Size Exogenous Antigenic Proteins

(56) In a first experiment (FIG. 9, left), we have shown that when STxB-Cys-Ova is used to sensitize the murine dendritic cell line D1, a clear increase of the presentation of the MHC class II restricted Ova derived peptide Ova.sub.323-339 is observed, compared to D1 cells pulsed with non-vectorized Ova. Indeed, a significant presentation of Ova.sub.323-339 peptide could be detected with as little as 0.01 nM of STxB-Cys-Ova, whereas 10 nM of full size Ova was required for an efficient presentation of the same peptide. The presentation of the IA.sup.b restricted Ova.sub.323-339 peptide was revealed using the B097.10 hybridoma that produces IL-2 after recognition of this peptide. As a control, we did not observe IL-2 secretion when an irrelevant MHC class II restricted hybridoma was used instead of B097.10 (data not shown).

(57) In a second experiment, we have pulsed the same D1 dendritic H2.sup.b restricted cell line with either Ova alone or with STxB-Cys-Ova. No presentation of the Ova-derived immunodominant SL8 peptide (Ova.sub.257-264) was observed when the D1 cells were sensitized with up to 100 nM of free Ova, while 1-10 nM of STxB-Cys-Ova allowed the presentation of the SL8 peptide, as revealed by the specific B3Z hybridoma that recognize the SL8 peptide in the context of K.sup.b molecules. As a control, it was shown that no activation of an irrelevant hybridoma was observed under the same experimental conditions.

(58) Altogether, these results clearly demonstrate that STxB-Cys targets full size proteins with high efficiency into both, the MHC class I and class II pathways.

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