Expression vectors for cell-surface expression of polypeptides comprising a transmembrane domain of glycophorin A

Abstract

The present invention provides expression vectors for cell-surface expression of polypeptides comprising a transmembrane domain of glycophorin A.

Claims

1. A nucleic acid expression vector for cell-surface expression of proteins comprising in order a polynucleotide sequence comprising a sequence encoding a secretion signal peptide, a cloning site for inserting a polynucleotide sequence encoding a protein to be expressed and a polynucleotide sequence comprising a sequence encoding a transmembrane domain of glycophorin, wherein the vector comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

2. The nucleic acid vector of claim 1, wherein the protein to be expressed is a membrane associated protein.

3. A eukaryotic cell comprising the vector of claim 1.

4. The eukaryotic cell of claim 3, wherein the eukaryotic cell is a mammalian cell.

5. The eukaryotic cell of claim 3, wherein the eukaryotic cell is an HEK cell.

6. A method for expressing a protein suitable for antibody generation, the method comprising culturing the cell of claim 3 under conditions suitable for expressing the protein.

7. A method for the generation of monoclonal antibodies against a specific protein comprising the steps: a) immunization of a non-human animal with cells expressing on its cell surfaces the specific protein using the vector of claim 1, b) isolating spleen cells of the non-human animals of step a), c) fusing the spleen cells of step b) with myeloma cells to generate B cell hybridomas and d) identification of B cell hybridomas expressing antibodies directed against the specific protein.

8. The method of claim 7, wherein the non-human animal is a mouse or hamster.

Description

SHORT DESCRIPTION OF FIGURES

(1) FIG. 1A shows the primary structure of human ABCA1 (Seq. Id. No. 7), rat TMEM27 (Seq. Id. No. 9) and P. falciparum PFF0620c (Seq. Id. No. 11) proteins used in the examples.

(2) The domains used for the constructs described are marked with the diagonal lines with the amino acids at the N and C termini indicated;

(3) FIG. 1B shows schematic diagrams of the expressed protein constructs derived from the vectors described in the examples. The extracellular domains are equivalent to the ones shown in FIG. 1A;

(4) FIG. 2 shows a Westerm blot using anti-FLAG M2-HRP conjugated antibody (Sigma) of total cell lysates from HEK293 cells transfected with pANITA2-ABCA1 and pANITA2-TMEM27. Strong expression with bands at appropriate molecular weights is seen;

(5) FIG. 3A shows cell-surface expression of PFF0620C on stably transfected HEK cells. Fluorescence (column 2 & 3) and differential interference contrast micrographs (column 1) of non-transfected HEK cells (line 1) and HEK cells displaying PFF0620C (line 2). Cells were grown on chamber-slides and stained without fixation with anti-FLAG antibody and FITC-labelled anti-mouse IgG antibodies. Nuclei were stained with DAPI;

(6) FIG. 3B shows extracellular localisation of PFF0620C on stably transfected HEK cells. Fluorescence (line 1 & 3) and differential interference contrast micrograph (line 2 & 4) of PFF0620C-HEK cells after staining with anti-FLAG (left column) or anti-6His antibodies (right column) and FITC-labelled anti-mouse IgG antibodies. With the anti-FLAG antibody living cells and methanol-fixed cells were stained, whereas the anti-His antibody only stained methanol-fixed cells, indicating intracellular localisation of the His-tag and extracellular localisation of the FLAG-tag together with the P. falciparum derived protein domain;

(7) FIG. 4 shows the results of a screening of antibodies for binding to transfected cells. In a second step, all wells positive for IgG production were screened for antibody binding to transfected cells by IFA (immuno fluorescence assay). Transfected and non-transfected HEK cells spotted onto multiwell glass-slides were stained with individual hybridoma supernatants and analysed by fluorescence microscopy;

(8) FIG. 5 shows a western blot analysis of the reactivity of generated monoclonal antibodies with the recombinant P. falciparum proteins. Specificity of representative monoclonal antibodies for the corresponding recombinant proteins is demonstrated by Western-blot analysis. Lysates of PFF0620c- (line 1), control pANITA2 constructs containing unrelated proteins (lines 2 & 3) and non-transfected HEK cells (line 4) were probed with anti-6His mAb and an anti PFF0620cmAb generated as described, respectively.

(9) FIG. 6 shows that PFD1130w-specific monoclonal antibodies inhibit parasite growth in vivo.

EXAMPLES

(10) Expression Proteins on the Cell Surface of Mammalian Cells.

(11) The P. falciparum ORF PFF0620c, human ABCA1 extracellular domain and rat TMEM27 extracellular domain were expressed on the cell surface of HEK cells using the expression plasmids pANITA2-PFF0620C; pANITA2-ABCA1 or pANITA2-TMEM27 respectively. To ensure high levels of expression on the cell surface, the genes were modified in several ways (FIG. 1): i. the endogenous sequences were codon-optimised for expression in mammalian cells and only predicted extracelluar domains were used; ii. the endogenous secretion signal sequences were replaced by the secretion signal sequence of bee-venom melittin; iii. for membrane anchoring the transmembrane domain encoding sequence of mouse glycophorin A was used instead of the predicted GPI-attachment signal sequence or predicted transmembrane domains; iv. to allow expression analysis, a FLAG tag was inserted N-terminally of the transmembrane domain and a 6His tag was placed at the C-terminus. The two tags were positioned just before and after the transmembrane domain to facilitate verification of the extracellular localisation of the recombinantly expressed antigens.

(12) HEK-derived cell lines expressing P. falciparum PFF0620c, human ABCA1 extracellular domain and rat TMEM27 extracellular domain were established by stable transfection.

(13) To obtain highly expressing cell lines, transfectants were separated into high-expressing cell-pools by fluorescent-activated-cell-sorting after surface staining with anti-FLAG antibodies. The mean fluorescence intensity of the cells gated for sorting into the high-expressing cell pool was 2.1-4.3 times higher than that of all transfectants.

(14) Human ABCA1 and rat TMEM27-expressing cell lines were tested for expression by Western blot analysis, showing a high level of expression of a protein with the expected molecular weight. (FIG. 2) Cell surface expression of the P. falciparum PFF0620c protein was shown by immunofluoresence analysis with anti-FLAG antibody yielding strong signals on living cells. (FIG. 3) In contrast, staining with anti-6His antibody gave strong signals only on methanol fixed cells but not on living cells (FIG. 3B). These results verified that PFF0620c is expressed and anchored in the cell wall with the FLAG-tag lying extracellularly and the His-tag lying intracellularly.

(15) Development of Malaria Antigen Specific Antibodies in Mice Immunised with Transfected HEK Cells

(16) The high-expressing cell pool of PFF0620c-HEKwas used to immunise NMRI mice. Mice received intravenous injections of 10.sup.6 cells on three consecutive days and another suite of three daily injections two weeks later. Development of serum antibody titres was analysed by flow cytometry comparing immune-staining of the transfectant with that of non-transfected HEK cells. The fluorescence intensity observed with the transfectant was fourfold higher than that of non-transfected control HEK cells. This indicated that the mice had mounted an antibody response against the malaria antigen expressed on the surface of the transfected HEK cells.

(17) Spleen cells of mice immunised with the transfected HEK cells were fused with PAI myeloma cells to generate B cell hybridoma. Fused cells were distributed in microtitre culture plate wells. To identify hybridoma cells that produce PFF0620c-specific antibodies a two-step screening procedure was used that completely obviates the requirement for purified recombinant proteins. First all culture wells were tested for IgG production by ELISA. Between 18 and 29%, of the tested wells were positive. In a second step all wells positive for IgG production were screened for antibody binding to transfected cells by IFA. Transfected and non-transfected HEK cells spotted onto multiwell glass-slides were stained with individual hybridoma supernatants and analysed by fluorescence microscopy (FIG. 4). Non-transfected HEK cells served as a negative control for each sample. Numerous clones positive on the transfected cells were also positive on non-transfected cells. However, the fusion yielded also numerous wells containing antibodies strongly reactive with the transfectant but not reactive with untransfected HEK cells. All other antibodies were specific for the transfected cells used for immunisation and did not stain control transfectants. From wells of this category, 17 hybridoma clones were derived by recloning from the PFF0620c-fusion.

(18) The specificity of the monoclonal antibodies was further confirmed by Western blot analysis (FIG. 5). 16 of the mAbs stained the corresponding recombinant protein in the lysate of the transfectant used for immunisation, but not in lysates of control transfected or untransfected HEK cells.

(19) PFD1130w-Specific Monoclonal Antibodies Inhibit Parasite Growth In Vivo

(20) We evaluated the in vivo parasite inhibitory activity of anti-PFD1130w mAbs in a P. falciparum SCID mouse model. The anti-PFD1130w mAbs were produced using the same methods and vectors that were used for the generation of the mAbs against P. falciparum PFF0620c (see methods section below). This model uses non-myelodepleted NOD-scid IL2Rnull mice engrafted with human erythrocytes in order to allow the growth of P. falciparum. Groups of three mice with a parasitemia of 0.580.14% were injected once with 2.5 mg anti-PFD1130w c12 mAb, 0.5 mg anti-PFD1130w c12 mAb or 2.5 mg isotype/subclass control mAb per mouse, respectively. Parasitemia of all mice was monitored for the next six days. While the parasitemia in mice that had received PBS only or the control mAb increased continuously, reaching 11.30.8% after six days, parasitemia of mice that received 0.5 mg anti-PFD1130w c12 mAb increased to a much lower extent, reaching 5.61.3% after six days. Parasitemia of mice receiving 2.5 mg anti-PFD1130w c12 mAb stayed low till the end of the experiment (1.40.3% on day 6). The difference in parasitemia after 6 days compared to the negative control group was highly significant (two-sided t-test; P<0.0001) (FIG. 6).

(21) The fact that anti-PFD1130w mAbs inhibit parasite growth in vivo indicates the power of the described entirely cell-based technology to generate mAbs that bind the endogenous protein in its native context.

(22) Methods

(23) Construction of Plasmids and Transformation

(24) A double-stranded oligonucleotide encoding the secretion signal sequence of bee-venom melittin was ligated to NheI digested pcDNA3.1(+) (Invitrogen) resulting in plasmid pcDNA3.1_BVM, with a single NheI site retained 3 of the signal sequence. A mouse glycophorin cytoplasmic and transmembrane domain cDNA was obtained by rtPCR (Invitrogen SuperScript III First Strand Synthesis kit and Roche Expand High Fidelity PCR System) using RNA extracted from bone marrow as a template. The resulting PCR amplicon being cloned into a pCR2.1 cloning vector. Primers to mouse glycophorin contained a 5 NotI site and 3 histidine tag followed by a stop codon and EagI site. The glycophorin-6His fragment was excised with EagI and ligated to NotI-digested pcDNA3.1_BVM resulting in plasmid pcDNA3.1_BVM_GP with the pcDNA3.1 NotI site preserved at the 5 end of the glycophorin sequence. To create the finished expression vector (pANITA2) a double-stranded oligonucleotide was ligated into NotI-digested pcDNA3.1_BVM_GP encoding a Flag-tag flanked by short linker sequences and resulting in a unique NotI site to the 5 side of the Flag-tag.

(25) Rat TMEM27 extracellular domain (aa 15-130 of Seq. Id. No. 9); a predicted extracellular domain of P. falciparum gene PFF0620C (aa 21-353 of Seq. Id. No. 11) and human ABCA1 N-terminal extracellular domain (aa 43-640 of Seq. Id. No. 7) cDNA sequences were synthesised with optimisation of codon usage to give high expression in mammalian cell culture. The genes were ligated into the unique NheI and NotI sites of the pANITA2 vector and the sequence of the vectors confirmed by DNA sequencing. The resulting plasmids are hereafter referred to as pA-NITA2-TMEM27; pANITA2-PFF0620C or pANITA2-ABCA1 respectively.

(26) In pANITA3.1 and pANITA3.3, the native pcDNA3.1 XbaI and XhoI sites were also removed by site-directed mutagenesis. The features of the multiple cloning sites and fusion-protein-coding sequences are shown in the table 1 below, with numbering from the insert start.

(27) Armenian hamster glycophorin sequence was determined by PCR-cloning and nucleotide sequencing using the Chinese hamster glycophorin sequence as a guide for primer design and cDNA generated from Armenian hamster bone-marrow RNA preparations. The following sequences are depicted in table 1: pANITA2 with Kozak sequence=Seq. Id. No. 15, pANITA3.1=Seq. Id. No. 13 and pANITA3.3=Seq. Id. No. 14.

(28) TABLE-US-00001 TABLE 1 Comparison of expression vectors Vector element pANITA2 pANITA3.1 pANITA3.3 Kozak sequence 1-12 1-12 1-12 Bee venom melittin signal 9-72 9-72 9-72 sequence Unique NheI restriction site 70-75 70-75 70-75 Unique KpnI restriction site 82-87 82-87 82-87 Unique BamHI restriction site 94-99 94-99 94-99 Unique EcoRI restriction site 106-111 106-111 106-111 Unique EcoRV restriction site 112-117 112-117 112-117 Unique XbaI restriction site 118-123 118-123 Unique NotI restriction site 124-131 124-131 124-131 Flag tag/Enterokinase 133-156 133-156 133-156 cleavage site Unique HindIII restriction 154-159 154-159 site Mouse glycophorin membrane 172-369 163-369 anchor Armenian hamster glycophorin 178-375 membrane anchor 6-His tag 382-399 382-399 388-405 Stop codons 400-405 400-405 406-411

(29) Establishment of HEK 293 Cell Lines Stably Expressing PFF0620C, TMEM27 or ABCA1 Domains.

(30) 293 HEK cells were transfected with pANITA2-TMEM27; pANITA2-PFF0620C or pA-NITA2-ABCA1 using JetPEI (PolyPlus) transfection reagent following the manufacturer's protocol. Antibiotic selection was started 48 h after transfection. The selection medium containing 500 ug/ml of Geneticin (Gibco) was exchanged every 3-4 days. After non-antibiotic resistant cells had died off and resistant cells started growing normally, a high-expressing pool was generated by FACS. Cells were dissociated with enzyme-free dissociation buffer (Cell dissociation buffer enzyme-free Hanks'-based, Gibco), washed with blocking buffer (PBS containing 3% BSA). The cells were then incubated with 200 l of 100 g/ml anti-FLAG mAb=FLAG-27 diluted in blocking buffer for 15 min on ice. The cells were then washed with blocking buffer and incubated with 200 l of 100 g/ml FITC-conjugated goat anti-mouse IgG antibodies (RAM/IgG(H+L)/FITC, Nordic Immunological Laboratories) diluted in blocking buffer for 15 min on ice. After a final wash the labelled cells were analysed and sorted using a BD FACSAria running FACSDiva software. All analyses were performed using appropriate scatter gates to exclude cellular debris and aggregates. Gating settings were set to collect highly labelled cells. Post-sorting, the cells were collected in culture medium with 20% FCS and plated in 35 mm wells.

(31) Immunofluorescence Staining of Living HEK Cells

(32) For immunofluorescence staining of live HEK cells chamber slides (4-well chamber-slide, Lab-Tek, Nunc) were used. Wells were coated with 100 mg/l poly-D-lysine in H.sub.2O in a humid box at room temperature over night. After washing the wells three times with sterile H.sub.2O, 40,000 cells were seeded per well. Three days later the immunostaining was performed by incubating the wells with 500 l of an appropriate mAb diluted in serum-free culture medium for 30 min on ice. After washing two times with serum-free culture medium 500 l of 100 g/ml FITC-conjugated goat anti-mouse IgG antibodies (RAM/IgG(H+L)/FITC, Nordic Immunological Laboratories) diluted in serum-free culture medium were added to the wells and incubated for 30 min on ice. Finally, the wells were rinsed twice with serum-free culture medium and once with DPBS (Dulbecco's Phosphate-Buffered Saline containing calcium, Gibco). The slides were mounted with mounting solution containing DAPI (ProLong Gold antifade reagent with DAPI, Invitrogen) and covered with a coverslip Stainings were assessed as described above.

(33) Immunisation of Mice

(34) NMRI mice were immunised by intravenous injections of 10.sup.6 stably transfected HEK cells. Cells were thawed, washed and resuspended in 0.9% NaCl. Injections were accomplished on three consecutive days and after two weeks again on three consecutive days. After the boost, blood was collected and the serum was tested for the presence of anti-PFF0620C antibodies by IFA using stably transfected 293 HEK cells.

(35) Animals with serum strongly reactive with expressing cells were selected for fusion. These received a final injection of 10.sup.6 cells two and one day before the fusion. Mice were sacrificed and the spleen was removed. Spleen cells were harvested by trituration under sterile conditions and fused with the myeloma cell partner (PAI mouse myeloma cells, derived from P-3X63-Ag8) using polyethylene glycol 1500 (Roche Diagnostics). The fusion mix was plated into multiwell plates and hybridomas were selected by growing in HAT medium supplemented with culture supernatant of mouse macrophages P388. Wells were screened for specific IgG production between 2-3 weeks post-fusion by ELISA and IFA as described below. Cells from wells positive in initial screens were cloned by limiting dilution to obtain monoclonal populations.

(36) IgG ELISA Screen

(37) Maxisorp plates (Nunc) were coated overnight at 4 C. in a humid box with 100 l of 5 g/ml goat anti-mouse IgG (-chain specific) mAb (Sigma) diluted in PBS. After two washings with PBS containing 0.05% Tween-20, wells were blocked with blocking buffer (50 mM Tris, 140 mM NaCl, 5 mM EDTA, 0.05% NONidet P40, 0.25% gelatine, 1% BSA) for 1 h at 37 C. and afterwards washed two times. 50 l hybridoma supernatants were added to the wells and incubated for 1 h at 37 C. After washing 4 times, plates were incubated with 50 l horseradish peroxidase-conjugated goat anti-mouse IgG (-chain specific) (Sigma) diluted 1:1000 in blocking buffer for 1 h at room-temperature in a humid box in the dark. After washing 4 times, TMB peroxidase substrate solution was added and the colour change monitored.

(38) Antibody Production and Characterisation

(39) Identification of antibody isotypes was performed using a Mouse Monoclonal Antibody Isotyping Kit (ISO2, Sigma). For large-scale mAb production hybridoma cell lines were cultured in 500 ml roller-bottles (Corning). MAbs were purified by affinity chromatography using protein A or protein G Sepharose.

(40) DNA and Protein Sequences

(41) TABLE-US-00002 Seq. Gene/Protein Id. name Species Description No. Glycophorin A Mouse Transmembrane + 1 cytoplasmic domain of glycophorin A Melittin Bee Secretion signal of bee 2 venom melittin Flag tag Flag tag 3 His tag His tag 4 Expression Expression vector 5 vector sequence comprising pANITA2 secretion signal of bee venom (without melittin, cloning site for a Kozak protein to be expressed sequence) and transmembrane domain of mouse glycophorin A ABCA1 Human DNA encoding human 6 ABCA1 protein ABCA1 Human ABCA1 protein 7 TMEM27 Rat DNA encoding rat 8 TMEM27 TMEM27 Rat TMEM27 protein 9 PFF0620C Plasmodium DNA encoding 3D7 10 falciparum protein PFF0620C Plasmodium 3D7 protein 11 falciparum Glycophorin A Armenian Transmembrane + 12 hamster cytoplasmic domain of glycophorin A Expression Expression vector 13 vector sequence comprising pANITA3.1 secretion signal of bee venom melittin, cloning site for a protein to be expressed and transmembrane domain of mouse glycophorin A Expression Expression vector 14 vector sequence comprising pANITA3.3 secretion signal of bee venom melittin, cloning site for a protein to be expressed and transmembrane domain of Armenian hamster glycophorin A Expression Expression vector 15 vector sequence comprising pANITA2 with secretion signal of bee venom Kozak sequence melittin, cloning site for a (nt 1-12) protein to be expressed and transmembrane domain of mouse glycophorin A

(42) Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.