Recombinant Der P 2 expressed in Pichia Pastoris as a “natural-like” allergen for immunotherapy and diagnostic purposes

Abstract

The present invention concerns a method for producing a recombinant Dermatophagoides pteronyssinus 2 protein (rDer p 2), comprising the steps of cultivating a Pichia pastoris yeast strain previously transformed with a rDer p 2 coding sequence, and isolating the rDer p 2 protein from said Pichia pastoris yeast strain. The invention also relates to compositions and kits comprising the rDer p 2 protein for therapeutic or diagnostic use.

Claims

1. A method for producing a recombinant Dermatophagoides pteronyssinus 2 (rDer p 2) protein selected from the group consisting of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, which comprises only the three disulfide bonds respectively between amino acids C8 and C119, between amino acids C21 and C27, and between amino acids C73 and C78, by reference to the amino acid positions as shown in sequence SEQ ID NO:1, said method comprising: a) cultivating in a buffered methanol medium a Pichia pastoris yeast strain previously transformed with a rDer p 2 encoding sequence preceded by a secretion signal; and b) isolating the rDer p 2 protein from said Pichia pastoris yeast strain cultivated in step a), wherein isolating the rDer p 2 protein in step b) comprises ion exchange chromatography; wherein said method does not comprise precipitation of the rDer p 2 protein, and wherein the rDer p 2 protein has the same conformation as natural Der p 2.

2. The method according to claim 1, wherein said conformation is assessed using one or more methods selected from the group consisting of (i) superimposing the circular dichroism spectrum of said rDer p 2 in the far-UV spectral region on the circular dichroism spectrum of natural Der p 2; (ii) determining that said rDer p 2 comprises 40-60% beta-sheet when analyzed by crystallography or circular dichroism; (iii) determining that said rDer p 2 comprises 1-10% alpha helix when analyzed by crystallography, or 30-40% alpha helix when analyzed by circular dichroism; (iv) determining that the percentage of beta-sheet of said rDer p 2 does not differ by more than 20% from the percentage of beta-sheet of the natural Der p 2 when both the rDer p 2 and natural Der p 2 are analyzed either by crystallography or circular dichroism; and (v) determining that the percentages of beta-sheet and alpha-helix said rDer p 2 do not differ by more than 20% from the percentage of beta-sheet and alpha helix, respectively, of the natural Der p 2 when both the rDer p 2 and natural Der p 2 are analysed either by crystallography or circular dichroism.

3. The method according to claim 1, wherein the rDer p 2 encoding sequence is a nucleotide sequence encoding a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

4. The method according to claim 1, wherein isolating the rDer p 2 protein in step b) comprises ion exchange chromatography, followed by dialysis and concentration.

5. The method according to claim 1, wherein said rDer p 2 protein isolated from the Pichia pastoris yeast strain is further formulated into a pharmaceutical composition.

6. A method for producing a recombinant Dermatophagoides pteronyssinus 2 (rDer p 2) protein selected from the group consisting of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, which comprises only the three disulfide bonds respectively between amino acids C8 and C119, between amino acids C21 and C27, and between amino acids C73 and C78, by reference to the amino acid positions as shown in sequence SEQ ID NO:1, said method comprising: a) cultivating in a buffered methanol medium a Pichia pastoris yeast strain previously transformed with a rDer p 2 encoding sequence preceded by a secretion signal; and b) isolating the rDer p 2 protein from said Pichia pastoris yeast strain cultivated in step a), wherein isolating the rDer p 2 protein in step b) comprises ion exchange chromatography; wherein said method does not comprise precipitation of the rDer p 2 protein, wherein the rDer p 2 protein has the same conformation as natural Der p 2, and wherein said conformation is assessed using one or more methods selected from the group consisting of (i) superimposing the circular dichroism spectrum of said rDer p 2 in the far-UV spectral region on the circular dichroism spectrum of natural Der p 2; (ii) determining that said rDer p 2 comprises 40-60% beta-sheet when analyzed by crystallography or circular dichroism; (iii) determining that said rDer p 2 comprises 1-10% alpha helix when analyzed by crystallography, or 30-40% alpha helix when analyzed by circular dichroism; (iv) determining that the percentage of beta-sheet of said rDer p 2 does not differ by more than 20% from the percentage of beta-sheet of the natural Der p 2 when both the rDer p 2 and natural Der p 2 are analyzed either by crystallography or circular dichroism; and (v) determining that the percentages of beta-sheet and alpha-helix said rDer p 2 do not differ by more than 20% from the percentage of beta-sheet and alpha helix, respectively, of the natural Der p 2 when both the rDer p 2 and natural Der p 2 are analysed either by crystallography or circular dichroism.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Monoclonal antibodies and IgE reactivity

(2) Western blot analyses were performed on purified recombinant Der p 2 (rDer p 2) and natural Der p 2 (nDer p 2) with the Anti-DpX (A) and Kori 221B (B) monoclonal antibodies directed against the natural protein and with an IgE pool made sera obtained from House Dust Mites allergic patients (C). 100 ng of protein were loaded for each allergen.

(3) FIG. 2. Basophil activation

(4) Basophils from 8 HDM allergic donors were exposed to varying amounts of natural Der p 2 (nDer p 2) or recombinant Der p 2 (rDer p 2). The expression of the CD203c activation marker was determined by cytofluorometry among CRTH2 cells. Data are represented as mean percentage values of CD203c.sup.high basophils obtained from the 8 independent experiments.

(5) FIG. 3. Circular dichroism analysis

(6) Recombinant Der p 2 (rDer p 2) expressed either constitutively (E9590; 0.36 M) or after induction with methanol (E9250 ; 0.28 M) in 150 mM NaCl 10 mM sodium acetate pH5 buffer were subjected to circular dichroism. Natural purified Der p 2 (nDer p 2) (77.9 M) in PBS buffer was used as a control.

(7) FIG. 4. Secondary structure analysis by circular dichroism spectroscopy

(8) (A) The higher-order structure of natural and recombinant Der p 2 molecules (300 g/mL) was examined by circular dichroism using a Jasco J-815 CD spectrophotometer (Jasco, Bouguenais, France) against a buffer blank (10 mM sodium acetate, 150 mM NaCl, pH 5.0). Eight independent spectral scans were recorded at 20 C. in the 200-260 nm wavelength range, in 1 mm cuvettes with a scanning speed of 100 nm/min, a 1 nm bandwidth and a 0.2 nm data pitch, and averaged. Thermal stability of nDer p 2 (B) and rDer p 2 (C) was assessed by measuring CD spectra after heating the proteins with 10 C. incremental steps and cooling back to 20 C. Resulting spectra were obtained as the average of 3 scans.

(9) FIG. 5. Sub-lingual immuno therapy (SLIT) with rDerp2 decreases airway hyper-responsiveness in nDerp2 sensitized animals

(10) After 8 weeks of SLIT with either PBS or 50 g rDerp2, mice were re-exposed to allergen extracts for 2 consecutive days. Airway hyper-responsiveness (AHR) to methacholine was measured by whole body plethysmography 24 hrs after the last challenge and expressed as Penh index. Results are expressed as mean valuesSEM, with n=6 mice per group. **p<0.01 in comparison to PBS-treated group. ns: non-statistically different from healthy mice.

(11) FIG. 6. SLIT with rDerp2 reduces lung eosinophilia

(12) Differential cells counts were performed after May-Grunwald Giemsa staining of bronchoalveolar lavage (BAL) cytospins. Results are expressed as mean valuesSEM, with n=6 mice per group. *p<0.05 in comparison to PBS-treated group.

(13) FIG. 7. SLIT with rDerp2 diminishes nDerp2-specific Th2 T cell responses

(14) Lung cells were recovered from PBS and rDerp2-treated groups were recovered and restimulated in vitro with 10 g purified nDerp2 for 72 hrs. IL-13 and IL-5 cytokine levels were measured by cytometric bead array (CBA) in culture supernatants. Results are expressed as mean valuesSEM, with n=6 mice per group. *p<0.05 in comparison to PBS-treated group.

(15) FIG. 8. Basophil activation

(16) Basophils from 10 HDM allergic donors were exposed to varying amounts of natural Der p 2 (nDer p 2) or recombinant Der p 2 (rDer p 2). The percentage of activated basophils was determined by cytofluorometry among CRTH2 cells.

BRIEF DESCRIPTION OF THE SEQUENCES

(17) SEQ ID NO: 1 shows the sequence of the 2.0101 isoform of Der p 2.

(18) SEQ ID NO: 2 shows the sequence of the M76V mutant of Der p 2.

(19) SEQ ID NO: 3 shows the sequence of the M111L mutant of Der p 2.

(20) SEQ ID NO: 4 shows the sequence of the M76V-M111L mutant of Der p 2.

EXAMPLES

Example 1

Materials and Methods

(21) Cloning and Expression in P. pastoris

(22) DNA sequences coding Der p 2.0101 were cloned in pPICZ and pGAPZ (Invitrogen) in phase with the factor sequence signal at the XhoI and NotI sites. Constructs were checked by DNA sequencing. The protein was expressed in methanotrophic P. pastoris GS115 or X33 yeast strains, according to the manufacturer's instructions (Invitrogen). pPICZ-Der p 2 and pGAPZ-Der p 2 plasmids were first respectively digested with SacI or AvrII and used to transform yeast cells by electroporation. Positive clones were selected on YPD zeocine (100 g/ml) plates and construct integration was confirmed by PCR. Selected clones were inoculated in buffered glycerol complex medium (BMGY pH6) and grown overnight at 30 C. up to a 2-6 OD600 nm. Cells were then diluted in buffered methanol complex medium (BMMY pH6) to an initial OD600 nm of 1 and further grown for 1 to 6 days at 30 C. Culture supernatants were harvested after centrifugation and stored at 20 C. until use. Alternatively, for constitutive expression, cells were grown in YPD for 5 to 6 days at 30 C. Culture supernatants were collected by centrifugation and stored at 20 C. until use.

(23) Der p 2 was purified by weak anionic exchange chromatography onto a SP XL column (GE Healthcare). Briefly, supernatants were thawed and dialysed against 10 mM NaCl 10 mM sodium acetate pH5 binding buffer and applied onto a 20 ml CV SP XL. Der p 2 was eluted with a gradient of 500 mM NaCl 10 mM sodium acetate pH5 buffer. Collected fractions were pooled and concentrated onto an Amicon Cell, using a 5 kDa cut-off membrane (Millipore).

(24) SDS-PAGE and Western Blot Analysis

(25) Proteins were separated by SDS-PAGE using Nupage 4-12% Bis-Tris acrylamide gels with MES buffer under non-reducing conditions and then transferred onto nitrocellulose membranes, as per the manufacturer's instructions (Invitrogen). Apparent molecular masses were estimated using the SeeBlue Plus2 ladder (Invitrogen). Membranes were treated with Qentix western blot signal enhancer (Pierce) prior to blocking non specific sites with TBS buffer containing 1% non fat milk (BioRad). Specific antibodies were added overnight in TBS buffer supplemented with 0.2% non fat milk and 0.1% Tween 20. Detection was performed with the anti-Dpx (0.2 g/ml; Indoor Biotechnologies Limited) and Kori 221B (0.2 g/ml; Stallergenes) mouse monoclonal antibodies directed to natural Der p 2 or a serum pool from HDM allergic patients (1:30). Secondary antibodies included as appropriate, either a HRP conjugated sheep anti-mouse IgG (1:1000; Sigma-Aldrich), a mouse anti goat/sheep IgG (1:5000; Sigma-Aldrich) or a rabbit anti-human IgE (1:1000; Dako) followed by an HRP conjugated goat anti-rabbit IgG (1:10000; Calbiochem). Detection was performed using the SuperSignal West Pico chemiluminescent substrate (Pierce) and a Chemismart 2000 CCD camera (Vilbert Lourmat).

(26) Circular Dichroism

(27) CD analyses were performed on a Jasco-810 spectropolarimeter (Jasco). Recombinant Der p 2 (rDer p 2) molecules produced in P. pastoris were purified by IEX chromatography and stored in 150 mM NaCl 10 mM sodium acetate pH5 buffer. The rDer p 2 concentration in E9250 (methanol induction) and E9590 (constitutive expression) batches was 0.28 M and 0.36 M respectively. Natural purified Der p 2 (nDer p 2) in PBS buffer (77.9 M) was purchased from Indoor Biotechnologies Limited (Warminster, United Kingdom). Spectra were recorded as the average of 10 scans between 195 to 250 nm using 0.1 cm path length cells with the base-line established with buffer spectra.

(28) Basophil Activation Test

(29) Basophil activation was measured by flow cytometry (FC500; Beckman Coulter) using the Allergenicity kit (Beckman Coulter) (Burtin et al., 2009). Briefly, heparinised whole blood samples were obtained from ten HDM allergic patients and incubated in absence or presence of various concentrations of natural or recombinant Der p 2 at 37 C. for 15 minutes. Once excluding T lymphocytes (CD3.sup.+ cells), activated basophils were detected by dual staining with anti-CRTH2 and anti-CD203c antibodies. Whole blood cells incubated with either PBS or anti-IgE antibodies were used as negative and positive controls, respectively.

(30) Mass Spectrometry Analyses

(31) RP-RSLC-UV-MS Analysis

(32) Der p 2 was applied onto a HypersilGold C18 (2.1200 mm, 1.9 m) column (ThermoScientific) and separated using a 0-100% gradient of acetonitrile containing 0.1% formic acid (both from JT Baker) gradient over 15 minutes. Eluents were then injected in a Maxis Mass spectrometer (Bruker Daltonics).

(33) RP-RSLC-UV-MS/MS Peptidic Map

(34) Samples were alkylated for 30 minutes with 15 mM iodoacetamide in a 75 mM ammonium bicarbonate, then digested with trypsin (Sigma) in 75 mM ammonium bicarbonate using a 1/25 (w/w) trypsin/Der p 2 ratio. After 16 h digestion at 37 C., the reaction was stopped by adding TFA to 0.5%. Digests were separated on an Acquity C18 (BEH130, 1002.1 mm, 1.7 m) column (Waters) using a RSLC Ultimate 3000 chromatographic apparatus (Dionex). Peptidic fragments were eluted using a 0-100% 0.1% formic acid (JT baker) gradient over a 55 minutes period before injection in a Maxis mass spectrometer (Bruker Daltonics).

Example 2

Results

(35) In order to develop vaccines against common house dust mite allergies, the inventors have expressed the Der p 2 allergen, either constitutively or after induction with methanol, as a secreted molecule in the yeast P. pastoris. The molecule is well recognised by anti-Der p 2 specific monoclonal antibodies and by IgEs from HDM allergic patients (FIG. 1). Furthermore, recombinant Der p 2 is able to trigger basophil activation in a comparable manner to the natural counterpart (FIG. 2 and FIG. 8).

(36) The inventors further analysed the conformation of the recombinant molecule in comparison with natural Der p 2. The mass of rDer p 2 calculated by mass spectrometry for two independent batches was in good agreement with the theoretical mass (less than 8 ppm difference). In particular, it indicated that all cysteines were engaged in disulfides bonds. Since the latter are key elements to stabilize the 3-D structure of the molecule and thus impacting its allergenicity (Smith and Chapman, 1996), the inventors mapped disulfide bridges by LC-MS/MS. As shown in table 1, contrary to rDer p 2 refolded out of E. coli inclusion bodies, rDer p 2 expressed in P. pastoris exhibits only the expected three disulfide bonds (Cys8-Cys119, Cys21-Cys27, Cys73-Cys78).

(37) TABLE-US-00001 TABLE 1 disulfide bridges mapping rDer p 2 rDer p 2 nDer p 2 E. coli P. pastoris Expected disulfide bridges Cys8-Cys119 Yes Yes Yes Cys21-Cys27 Yes Yes Yes Cys73-Cys78 Yes Yes Yes Unexpected disulfide bridges e.g. Cys8-Cys78 No Yes No Cys21-Cys119 No Yes No Cys73-Cys119 No Yes No Disulfide bridges were assigned by LC-MS/MS. The 6 cysteines residues are paired in 3 bonds (Cys8-Cys119, Cys21-Cys27 and Cys73-Cys78) (Derewenda et al., 2002; Mueller et al., 1997). Unexpected bridges observed in the recombinant molecule produces in E. coli are mispairings occurring during in vitro refolding.

(38) Recombinant and natural Der p 2 were subsequently submitted to circular dichroism to compare secondary structure contents. The spectra obtained for both molecules indicate that the recombinant protein is folded in a comparable manner to the natural protein (FIG. 3 and FIG. 4). Moreover, both natural and recombinant Der p 2 were found to display thermal stability, as assessed by circular dichroism after heating the proteins with 10 C. incremental steps and cooling back to 20 C. (FIG. 4). Finally, the percentages of beta-sheet and alpha helix were found to be similar within natural and recombinant Der p 2 using the algorithm of Yang, as shown in Table 2.

(39) TABLE-US-00002 TABLE 2 percentages of beta-sheet and alpha-helix in natural and recombinant Der p 2 Natural Der p 2 Recombinant Der p 2 % alpha-helix 34.2 36.1 % beta-sheet 50.6 59.6 The percentages of beta-sheet and alpha helix within natural and recombinant Der p 2 were determined using the algorithm of Yang for analysis of circular dichroism spectrum (Yang, J. T., C. S. Wu, et al. (1986).

(40) Finally, the inventors showed that rDer p 2 can desensitised nDerp 2-allergic mice as judged by a decreased airway hyper-responsiveness, reduced lung eosinophilia and lower nDerp2-specific Th2 T cell responses in nDerp2 sensitized animals after treatment (FIGS. 5-7).

(41) Collectively, our results indicate that recombinant expression of Der p 2 in the yeast P. pastoris yields a protein with both conserved immunoreactivity and natural-like conformation. These results are unexpected since attempts to produce in P. pastoris a Der p 2 molecule similar to its natural counterpart have failed as of today, yielding at best a molecule with partial folding (Tanyaratsrisakul et al., 2009). The inventors conclude that a rDer p 2 molecule expression in P. pastoris is suitable for immunotherapy and diagnostic purposes.

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