Peptide carrier fusion proteins as allergy vaccines
09844591 · 2017-12-19
Assignee
Inventors
- Katarzyna Niespodziana (Vienna, AT)
- Margarete Focke-Tejkl (Vienna, AT)
- Susanne Vrtala (Vienna, AT)
- Srinita Banerjee (Vienna, AT)
- Kuan-Wei Chen (Vienna, AT)
- Milena Weber (Vienna, AT)
- Rudolf Valenta (Theresienfeld, AT)
- Katharina Marth (Vienna, AT)
Cpc classification
A61K2039/64
HUMAN NECESSITIES
C12N2730/10122
CHEMISTRY; METALLURGY
C12N2730/10134
CHEMISTRY; METALLURGY
A61K39/292
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to a polypeptide comprising at least three peptide fragments consisting of 10 to 50 consecutive amino acid residues of at least one wild-type allergen fused to the N- and C-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide.
Claims
1. A nucleic acid molecule encoding a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16 and SEQ ID No. 17.
2. A vector, comprising the nucleic acid molecule of claim 1.
3. The vector of claim 2, which is an expression vector.
4. The vector of claim 2, which is a bacterial, fungal, insect, viral or mammalian vector.
5. A host cell, comprising the nucleic acid molecule of claim 1.
6. A host cell, comprising the vector of claim 2.
7. A method for treating a grass pollen allergy in a human or animal subject in need thereof, the method comprising administering an effective amount of the nucleic acid molecule of claim 1 to the subject.
Description
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
(1) The present invention is further illustrated by the following figures and examples, however, without being restricted thereto.
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EXAMPLES
Example 1: Construction of Expression Plasmid for HBV_Phlp1_4xP5 (BM321)
(49) The synthetic BM321 gene were assembled from synthetic oligo-nucleotides and/or PCR products and was cloned into an appropriate standard vector (pMK-RQkanR). The plasmid was purified from a transformed E. coli K12 strain (DH10B-T1R) and concentration was determined by UV spectroscopy. The final synthetic and codon-optimized BM321 DNA-sequence was further cloned into the expression vector pET28b(+) using appropriate restriction sites (NcoI site at the 5′-end and EcoRI at the 3′-end). The plasmid DNA was purified from transformed E. coli K12 DH10B (dam+dcm+) and concentration determined by UV spectroscopy. The final construct was verified by sequencing of the insert. A summary of plasmid data and a plasmid map of final expression vector “pBM-321” is shown below.
(50) Summary of BM321 sequence cloned into final expression vector pET-28b(+).
(51) TABLE-US-00004 Alias name Gene Plasmid Plasmid Restriction Sequence sequence size size name sites BM321 HBV_Phlp1_4xP5 882 bp 6153 bp pBM- NcoI/ 321 EcoRI
Example 2: Transformation of Expression Plasmid into Expression Host for HBV_Phlp1_4xP5 (BM321)
(52) Chemically competent E. coli BL21(DE3) cells were transformed with the expression plasmid by heat shock method. Transformed cells were plated on LB-agar-plates consisting of 0.5% sodium chloride 1% soy peptone, 0.5% yeast extract, 1.5% agar and 50 μg/mL kanamycin for selection. Cells on LB plates were grown by over-night cultivation at 37° C. Single colonies of transformed BL21(DE3) E. coli cells were isolated, cultured in LB-medium and screened for growth and expression of BM321. The best performing clone was selected for the further establishment of a Master Cell Bank.
Example 3: Preparation of a Master Cell Bank for HBV_Phlp1_4xP5 (BM321)
(53) An aliquot of the selected clone was used for inoculation of 150 mL culture medium (composition: 0.5% sodium chloride, 1% soy peptone, 0.5% yeast extract, 50 μg/mL kanamycin). The Master Cell Bank (MCB) culture was incubated at 37° C. under constant agitation at 200 rpm until the culture reached an optical density of OD.sub.600=1-2. Glycerol was added in order to obtain a final glycerol concentration of 15% v/v and the MCB was aliquoted into 1 mL vials and stored in an ultra deep freezer at −75±10° C.
Example 4: High Cell Density Fed-Batch Fermentation of HBV_PhlP1_4xP5 (BM321)
(54) Synthetic culture medium (100 mL, pH=6.8, salts and trace elements, 10 g/L glucose as carbon source) was inoculated with 1 mL of Master Cell Bank (E. coli BL21(DE3)/pBM321) and cultured in a shake flask (37° C., 200 rpm) until an optical density target value of OD=1 was reached. A 22 L stainless steel fermenter was used to perform the fed-batch fermentation. For automatic and reproducible feed control, a recipe was programmed allowing to pre-define specific growth-rate, feed rate, duration of batch-phase and duration of exponential feed-phase. In order to increase the oxygen transfer rate of the fermenter, back-pressure was controlled and set to 1 bar. The fermenter was in-situ sterilized with the synthetic culture medium as mentioned above and the fermentation was started by inoculation with preculture. After depletion of glucose, the exponential feeding phase was started in order to maintain a specific growth rate of μ=0.25 h.sup.−1. At an OD=45, the expression of recombinant BM321 was induced by the bolus addition of IPTG (0.8 mM final concentration). The culture was harvested at OD.sub.600=73. BM321 product titer obtained from the fed-batch fermentation was 1.2 g per L culture broth. Afterwards, the bacterial culture broth was cooled down to ≦20° C. and centrifuged at 7,000 rpm (5,500 g) at 4° C. for 15 min. Wet cell biomass was aliquoted and stored at −75° C.
Example 5: Cell Disruption and Clarification
(55) For cell disruption, 748 gram biomass from Example 6 were thawed and subdivided into aliquots á 125 gram and resuspended in a homogenization buffer (20 mM Tris, 1 mM EDTA, 0.1% Triton X-100, pH 11.0) under mechanical agitation at room temperature for 30 min. For cell disruption, a freeze/thaw procedure was applied by freezing −75° C. and subsequent thawing, followed by mechanical homogenisation. The pH of the homogenate was adjusted to pH=10.0. The crude cell homogenate was subjected to a centrifugation step at 7,000 rpm (5,500 g) at 4° C. for 30 min. The supernatants were subjected to precipitation with PEI (polyethyleneimine) under mechanical agitation. Insoluble matters were separated by a subsequent centrifugation step. The clarified supernatants were subjected to the following chromatography step.
Example 6: Chromatographic Purification of HBV_Phlp1_4xP5 (BM321)
(56) A total of 1840 mL of the PEI precipitation supernatant from the clarification step as described in Example 7 were loaded on a 5×30 cm Q-Sepharose FF column and equilibrated with buffer A (TrisHCl, EDTA). Unbound material was removed by washing with buffer A, followed by a wash with buffer C (1 sodium phosphate, EDTA, pH 7.0). Elution of the product fraction was accomplished by a linear gradient elution with 0-100% BM32 buffer E (sodium phosphate, EDTA, NaCl pH 7.0) in BM32 buffer C. Selection of product-containing fractions for pooling was performed according to SDS-PAGE analysis, by densitometric evaluation of fraction purity and by product band intensity.
(57) The pooled fractions from the capture step were adjusted to a conductivity of 115 mS/cm by the addition of 2.5 M sodium chloride, and this feedstock was loaded on a Phenyl Sepharose HP column equilibrated with buffer D (sodium phosphate, EDTA, NaCl pH 7.0). Unbound material was removed by washing with buffer D. Elution of the product fraction was accomplished by a gradient elution from 40-100% buffer C (sodium phosphate, EDTA, pH 7.0) in buffer D. Selection of product-containing fractions for pooling was performed according to SDS-PAGE analysis, by densitometric evaluation of fraction purity and by product band intensity.
The pooled fractions from the intermediate step were adjusted to a conductivity of 80 mS/cm by the addition of 2.5 M sodium chloride, and this feedstock was loaded on a Toyopearl Butyl 650-S column equilibrated with a mixture buffer F (sodium phosphate, EDTA, NaCl pH 7.0). Unbound material was removed by a gradient wash with 80-0% BM32 buffer F in buffer C (sodium phosphate, EDTA, pH 7.0). Elution of the fraction was accomplished by a gradient elution from 0-1 buffer G (sodium phosphate, EDTA, isopropanol, pH 7.0) in buffer C. Selection of product-containing fractions for pooling was performed according to SDS-PAGE analysis, by densitometric evaluation of fraction purity and by product band intensity.
Example 7: Manufacturing of HBV_Phlp2_4xP3 (BM322), HBV_Phlp5_V2 (BM325), and HBV_Phlp6_4xP1 (BM326)
(58) For expression and manufacturing of the recombinant molecules according to the invention, namely HBV_Phlp2_4xP3 (BM322), HBV_Phlp5_V2 (BM325), and HBV_Phlp6_4xP1 (BM326), the same, similar or comparable methods and procedures as described in Example 1, Example 2, Example 3, Example 4, Example 5 and Example 6 were applied.
Example 8: Preparation of an Injectable Formulation Consisting of a Mixture of HBV_PhlP1_4xP5 (BM321); HBV_PhlP2_4xP3 (BM322), HBV_PhlP5_V2 (BM325), and HBV_PhlP6_4xP1 (BM326)
(59) Each of the recombinant purified proteins was dissolved in an isotonic buffer containing 0.9% sodium chloride and 2 mM sodium phosphate and to each protein solution an appropriate amount of aluminium hydroxide was added. A mixture containing equal parts of the four resulting suspensions was prepared and aliquoted under sterile condition into sealed vials. The injectable formulation obtained by this procedure contained 0.4 mg/mL of each HBV_PhlP1_4xP5; HBV_PhlP2_4xP3, HBV_PhlP5_V2 and HBV_PhlP6_4xP1.
Example 9: Preparation of his-Tagged HBV_Betv1_4xPA
(60) The gene coding for fusion proteins consisting of PreS fused with Bet v 1-derived peptide PA twice at the N- and C-terminus (i.e. 4PA-PreS) was synthesized by ATG:biosynthetics, Merzhausen, Germany and inserted into the NdeI/XhoI sites of the vector pET-17b (Novagen, Germany). The DNA sequences were confirmed by means of automated sequencing of both DNA strands (Microsynth, Balgach, Switzerland).
(61) The fusion protein was expressed in E coli strain BL21 (DE3; Stratagene, La Jolla, Calif.). Cells were grown in Luria Bertani-medium containing 50 μg/mL kanamycin to an OD of 0.6. Protein expression was induced by adding isopropyl-β-D-thiogalactopyranoside to a final concentration of 1 mmol/L over night at 37° C. Cells were harvested by centrifugation at 3500 rpm for 10 minutes. The protein product was mainly detected in the inclusion body fraction. It was solubilized in 6M GuHCl, 100 mM NaH2PO4, 10 mM TRIS, pH 8.0 over night. The homogenate was centrifuged at 14,000 g for 18 minutes. Supernatants of were incubated with 2 mL of a previously equilibrated Ni-NTA resin for 4 hours (Qiagen, Hilden, Germany) and the suspensions were subsequently loaded onto a column, washed with 2 column volumes of washing buffer (8 mol/L urea, 100 mmol/L NaH2PO4, and 10 mmol/LTris-HCl [pH=6.1]), and eluted with the same buffer (pH=3.5). The purified protein was dialyzed against water.
(62) The purity of recombinant proteins was analyzed by Coomassie-stained SDS-PAGE (12.5%) under reducing conditions.
(63) The identity of the fusion protein was confirmed by the means of dot blot using monoclonal antibodies, specific for Bet v 1-derived peptides P2′ (mAb2) and P4′ (mAb12) and PreS-specific rabbit antibodies as well as corresponding rabbit preimmune IgGs. One μg of PreS fusion proteins, PreS and HSA (control) have been immobilized on nitrocellulose and were incubated with monoclonal as well as rabbit sera diluted 1:1000 have at 4° C. Bound antibodies were detected with iodine .sup.125-labelled rabbit anti-mouse IgG (mAb2, mAb12) or .sup.125I-goat anti-rabbit IgG (rabbit anti-PreS, rabbit preimmune) (Perkin-Elmer, Waltham, Mass.) diluted 1:500 for 2 hours and visualized by autoradiography. Furthermore ELISA plates (Maxisorp, Nunc, Denmark) were coated with 2 μg of PreS fusion protein and PreS, diluted in 0.1 mol/L carbonate buffer, pH 9.6 washed with PBS containing 0.05% vol/vol Tween 20 (PBST) 3 times and blocked for 2 hours with 1% BSA-PBST. Subsequently plates were incubated with mAb2, mAb12, anti-PreS rabbit serum and rabbit anti-Bet v 1 antibodies in a dilution of 1:5000 (dilution buffer: 0.5% wt/vol BSA in PBST) overnight at 4° C. After washing 5 times, bound IgG antibodies have been detected with a HRP-labelled sheep anti-mouse antibody (for mAb2, mAb12) or HRP-labelled donkey anti-rabbit antibody (rabbit sera) (both GE Healthcare, Uppsala, Sweden) and colour reaction was developed.
Example 10: Preparation of his-Tagged HBV_Betv1_2 xPA2xPB (BM31)
(64) Genes coding for fusion protein consisting of PreS fused twice with Bet v 1-derived peptides at the N- and C-terminus 2xPA2xPB-PreS) was synthesized by GenScript Piscataway, N.J., USA, 2PAPB-Pres) and inserted into the NdeI/XhoI sites of the vector pET-17b (Novagen, Germany). The DNA sequences were confirmed by means of automated sequencing of both DNA strands (Microsynth, Switzerland).
(65) The recombinant PreS fusion proteins was expressed in E coli strain BL21 (DE3; Stratagene, Calif.). Cells were grown in Luria Bertani-medium containing 50 μg/mL kanamycin to an OD of 0.6. Protein expression was induced by adding isopropyl-β-D-thiogalactopyranoside to a final concentration of 1 mmol/L over night at 37° C. Cells were harvested by centrifugation at 3500 rpm for 10 minutes. Proteins were mainly detected in the inclusion body fraction. The resulting protein was solubilized in 6M GuHCl, 100 mM NaH2PO4, 10 mM TRIS, pH 8.0 over night. The homogenate was centrifuged at 14,000 g for 18 minutes. Supernatants of were incubated with 2 mL of a previously equilibrated Ni-NTA resin for 4 hours (Qiagen, Hilden, Germany) and the suspensions were subsequently loaded onto a column, washed with 2 column volumes of washing buffer (8 mol/L urea, 100 mmol/L NaH2PO4, and 10 mmol/LTris-HCl [pH=6.1]), and eluted with the same buffer (pH=3.5). Protein was dialyzed against 10 mM NaH2PO4.
(66) The purity of recombinant proteins was analyzed by Coomassie-stained SDS-PAGE (12.5%) under reducing conditions. The identity of the fusion proteins was confirmed by the means of dot blot using monoclonal antibodies, specific for Bet v 1-derived peptides P2′ (mAb2) and P4′ (mAb12) and PreS-specific rabbit antibodies as well as corresponding rabbit preimmune IgGs. One μg of PreS fusion protein, PreS and HSA (control) have been immobilized on nitrocellulose and were incubated with monoclonal as well as rabbit sera diluted 1:1000 have at 4° C. Bound antibodies were detected with iodine 125-labelled rabbit anti-mouse IgG (mAb2, mAb12) or 125I-goat anti-rabbit IgG (rabbit anti-PreS, rabbit preimmune) (Perkin-Elmer, Waltham, Mass.) diluted 1:500 for 2 hours and visualized by autoradiography. Furthermore ELISA plates (Maxisorp, Nunc, Rosklide, Denmark) were coated with 2 μg of PreS fusion protein and PreS, diluted in 0.1 mol/L carbonate buffer, pH 9.6 washed with PBS containing 0.05% vol/vol Tween 20 (PBST) 3 times and blocked for 2 hours with 1% BSA-PBST. Subsequently plates were incubated with mAb2, mAb12, anti-PreS rabbit serum and rabbit anti-Bet v 1 antibodies in a dilution of 1:5000 (dilution buffer: 0.5% wt/vol BSA in PBST) overnight at 4° C. After washing 5 times, bound IgG antibodies have been detected with a HRP-labelled sheep anti-mouse antibody (for mAb2, mAb12) or HRP-labelled donkey anti-rabbit antibody (rabbit sera) (both GE Healthcare, Uppsala, Sweden) and colour reaction was developed.
Example 11: Detection of IgE Reactivity of Fusion Protein HBV_Phlp1_4xP5 (BM3212
(67) IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for HBV_Phlp1_4xP5 (BM321) as shown in
Example 12: Detection of IgE Reactivity of Fusion Protein HBV_Phlp2_4xP3 (BM322)
(68) IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for HBV_Phlp2_4xP3 (BM321) as shown in
Example 13: Detection of IgE Reactivity of Fusion Protein HBV_Phlp5_V2 (BM3252
(69) IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for HBV_Phlp5_V2 (BM325) as shown in
Example 14: Detection of IgE Reactivity of Fusion Protein HBV_Phlp6_4xP1 (BM326)
(70) IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for HBV_Phlp1_4xP1 (BM326) as shown in
Example 15: Detection of IgE Reactivity of Fusion Protein HBV_etV1_4xPA und HBV_Betv1_2 xPA2xPB (BM31)
(71) IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for both fusion proteins as shown in
Example 16: Rabbit Anti-r89P5 Antibodies Block Patient's IgE-Binding to rPhl p 1
(72) To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to rPhl p 1, ELISA plates were coated with 1 μg/ml rPhl p 1, washed and blocked. The plates were preincubated with 1:100-diluted rabbit anti-peptide (HBV_Phlp1_4xP5, KLHP5), a rabbit anti rPhl p 1 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from Phl p 1-allergic patients (1:3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1:1000) and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: 100-OD.sub.i/OD.sub.P×100.
(73) OD.sub.i and OD.sub.P represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 1 shows the capacity of anti-Phl p 1 peptide antibodies to inhibit the binding of 13 allergic patients' IgE to complete rPhl p 1. Anti-fusion protein sera blocked the IgE-binding to the same extent as sera against rPhl p 1 and KLHP5. Table 2 shows the inhibition (in %) of all 13 patients.
(74) TABLE-US-00005 TABLE 1 % inhibition of 13 patients' IgE-binding to rPhl p 1 after incubation with rabbit anti-rPhl p 1, anti-HBV_Phlp1_4xP5 and anti-KLHP5 antisera % inhibition patient rPhl p 1 HBV_Phlp1_4xP5 KLHP5 1 83.63 86.11 85.17 2 88.74 95.69 93.85 3 95.66 96.80 98.42 4 97.43 97.72 96.29 5 92.77 90.84 88.45 6 93.56 91.93 90.07 7 95.00 94.56 96.84 8 85.25 89.10 90.05 9 97.07 104.72 93.73 10 91.55 103.02 95.47 11 98.85 102.43 100.49 12 94.01 92.12 93.91 13 87.75 59.62 42.98 Mean 92.41 92.59 89.67
Example 17: Rabbit Anti-HBV_Phlp2_4xP3 Antibodies Block Patient's IgE-Binding to rPhl p 2
(75) To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to rPhl p 2, ELISA plates were coated with 1 μg/ml rPhl p 2, washed and blocked. The plates were preincubated with 1:100-diluted rabbit anti-peptide (HBV_Phlp2_4xP3, KLHP3), a rabbit anti rPhl p 2 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from Phl p 2-allergic patients (1:3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1:1000) and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: 100−OD.sub.i/OD.sub.P×100.
(76) OD.sub.i and OD.sub.P represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 2 shows the capacity of anti-Phl p 2 peptide antibodies to inhibit the binding of 19 allergic patients' IgE to complete rPhl p 2. Anti-fusion protein sera blocked the IgE-binding to the same extent as sera against rPhl p 2 and KLHP3. Table 2 shows the inhibition (in %) of all 19 patients.
(77) TABLE-US-00006 TABLE 2 % inhibition of 19 patients' IgE-binding to rPhl p 2 after incubation with rabbit anti-rPhl p 1, anti-HBV_Phlp2_4xP3 and anti-KLHP3 antisera % inhibition patient rPhl p 2 HBV_Phlp2_4xP3 KLHP3 1 98.24 81.36 2 97.50 83.90 3 96.46 98.57 90.58 4 98.31 86.77 5 96.46 81.17 6 99.43 72.45 9 91.25 91.38 90.44 8 95.78 54.49 9 98.60 87.55 10 95.45 82.68 11 91.36 96.70 78.21 12 98.47 90.21 13 97.67 93.20 14 96.57 85.64 15 97.00 91.35 16 93.73 98.06 83.62 17 95.55 76.27 18 95.91 86.49 19 95.90 83.99 Mean 93.20 97.19 83.18
Example 18: Rabbit Anti-HBV_Phlp5_V2 Antibodies Block Patient's IgE-Binding to rPhl p 5
(78) To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to rPhl p 5, ELISA plates were coated with 1 μg/ml rPhl p 5, washed and blocked. The plates were preincubated with 1:100-diluted rabbit anti-peptide (HBV_Phl p2_V2), a rabbit anti rPhl p 5 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from Phl p 5-allergic patients (1:3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1:1000) and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: 100−OD.sub.i/OD.sub.P×100.
(79) OD.sub.i and OD.sub.P represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 3 shows the capacity of anti-Phl p 5 peptide antibodies to inhibit the binding of 16 allergic patients' IgE to complete rPhl p 5. Anti-fusion protein sera blocked the IgE-binding to the same extent as sera against rPhl p 5 and better than KLH peptide mix. Table 3 shows the inhibition (in %) of all 16 patients.
(80) TABLE-US-00007 TABLE 3 % inhibition of 13 patients' IgE-binding to rPhl p 5 after incubation with rabbit anti-rPhl p 1, anti-HBV_Phlp5_V2 and anti-KLH peptide mix antisera % inhibition patient rPhl p 5 HBV_Phlp5_V2 KLHPmix 1 99.00 96.69 91.74 2 94.57 94.15 68.42 3 98.98 95.88 85.74 4 97.39 88.38 80.23 5 98.95 93.74 62.33 6 98.52 93.36 78.82 9 97.22 91.35 79.94 8 96.02 89.70 80.14 9 97.09 88.48 61.11 10 99.30 84.03 92.92 11 99.50 94.09 86.46 12 95.45 88.97 81.31 13 96.22 93.34 60.87 14 90.86 94.80 83.02 15 98.45 94.15 83.60 16 94.68 92.46 91.77 Mean 97.01 92.10 79.28
Example 19: Rabbit Anti-HBV_Phlp6_4xP1 Antibodies Block Patient's IgE-Binding to rPhl p 6
(81) To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to rPhl p 6, ELISA plates were coated with 1 μg/ml rPhl p 6, washed and blocked. The plates were preincubated with diluted rabbit anti-peptide (HBV_Phlp6_4xP1, KLHP1), a rabbit anti rPhl p 6 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from Phl p 6-allergic patients (1:3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1:1000) and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: 100−OD.sub.i/OD.sub.P×100. OD.sub.i and OD.sub.P represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 4 shows the capacity of anti-Phl p 6 peptide antibodies to inhibit the binding of 21 allergic patients' IgE to complete rPhl p 6. Anti-fusion protein sera blocked the IgE-binding to the same extent as sera against rPhl p 6 and KLHP1. Table 4 shows the inhibition (in %) of all 21 patients.
(82) TABLE-US-00008 TABLE 4 % inhibition of 21 patients' IgE-binding to rPhl p 6 after incubation with rabbit anti-rPhl p 6, anti-HBV_Phlp6_4xP1 and anti-KLHP1 antisera % inhibition patient rPhl p 6 HBV_Phlp6_4xP1 KLHP1 1 96.52 95.96 95.64 2 88.26 91.20 88.06 3 95.07 95.39 94.10 4 82.77 83.74 81.98 5 96.71 96.35 95.20 6 95.46 93.38 92.83 7 90.52 88.07 86.06 8 86.69 85.14 83.08 9 89.09 91.56 89.00 10 97.05 96.48 97.42 11 86.97 89.19 84.95 12 37.22 49.14 44.90 13 75.97 79.19 75.85 14 91.05 92.13 87.93 15 89.01 88.25 85.82 16 92.46 91.82 91.30 17 78.99 84.13 77.93 18 47.25 67.02 67.825 19 93.84 86.62 79.841 20 58.42 56.69 71.388 21 39.92 56.69 67.797 Mean 81.39 83.36 82.81
Example 20: IgE Reactivity of PreS Fusion Proteins Determined by Dot Blot and ELISA
(83) Purified rBet v 1, recombinant fusion proteins 4xPA-PreS, 2xPA2xPB-PreS were tested for their IgE reactivity by RAST-based, non-denaturing dot blot assays. Two μg of the purified proteins and, for control purposes, HSA were dotted onto nitrocellulose membrane strips (Schleicher & Schuell, Dassel, Germany).
(84) Nitrocellulose strips were blocked in buffer A (Vrtala, J Clin Invest, 1997) and incubated with sera from birch pollen allergic patients (n=50), sera from non-allergic persons (n=3) diluted 1:10, buffer control and positive control (1:1000 diluted rabbit anti-rBet v 1 antiserum). Bound IgE antibodies were detected with .sup.125I-labelled anti-human IgE antibodies (BSM Diagnostica, Vienna, Austria), bound rabbit antibodies with a .sup.125I-labeled goat anti-rabbit antiserum (Perkin-Elmer) and visualized by autoradiography (Valenta et al., 1992). Additionally, ELISA plates were coated with rBet v 1 and the purified PreS fusion proteins (5 μg/mL). After washing and blocking as described above, plates were incubated with sera of birch pollen allergic patients (n=21) and three non-allergic control sera diluted 1:5. Bound IgE was detected by purified mouse anti human IgE (BD Pharmingen) diluted 1:1000 overnight and visualized with HRP-labelled sheep anti mouse IgG (GE Healthcare) diluted 1:2000. After washing, colour reaction was determined as described above.
Example 21: Allergen-Induced Upregulation of CD203c of Allergic Patients' Basophils
(85) Heparinized blood samples were obtained from birch allergic patients after informed consent was given and were incubated with increasing concentrations of rBet v 1, 4PA-PreS, 2PAPB-PreS ranging from 0.001 to 1 mg/mL, a monoclonal anti-IgE antibody (Immunotech, Marseille, France) as positive control, or PBS (negative control) for 15 min (37° C.). CD 203c expression was determined as previously described.
Example 22: Lymphoproliferative Responses and Cytokine Induction in PBMC from Birch Pollen Allergic Patients
(86) PBMCs from birch pollen allergic patients (n=6) have been isolated by Ficoll (Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation. Subsequently PBMCs were resuspended in AIM V medium (Life Technologies, Grand Island, N.Y.) to a final concentration of 2×10.sup.5 cells/well and stimulated with decreasing antigen doses (equimolar amounts of 5 μg/well rBet v 1, PA, PB, PreS, 2PA-PreS, 2PB-PreS, 4PA-PreS, 2PAPB-PreS), with medium alone (negative control) or with IL-2 (4 IE/well) (positive control). After 6 days, proliferative responses were measured by [.sup.3H] thymidine incorporation and are expressed as stimulation indices (SI).
(87) Furthermore cytokine production of 17 different cytokines (i.e. IL-113, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IFN-γ, TNF-α, G-CSF, GM-CSF, MIP-1β, MCP-1) has been measured after 6 days of stimulation with Bio-plex Pro Human Cytokine 17-Plex Panel (Bio-Rad Laboratories) according the manufacturer's instructions. Briefly, the undiluted supernatants were mixed with anti-cytokine/chemokine mouse monoclonal antibodies coupled to different beads as capture antibodies (Bio-Rad). An 8-point standard curve was used to achieve low-end sensitivity. After washing, anti-cytokine biotinylated detection antibody was added. The reaction was visualized by adding Streptavidin-labelled Phycoerythrin (PE) and assay buffer. The samples were analyzed on a Luminex 100 instrument (Biosource, Nivelles, Belgium) and the data were acquired using the Bio-Plex Manager 6.0 software. All samples were analyzed in one run. Results are shown in
Example 23: Analysis of Rabbit Sera Immunized with rBet v 1 and PreS Fusion Proteins for their Recognition of rBet v 1, Bet v 1 Homologous Allergens and Bet v 1-Derived Peptides by ELISA
(88) ELISA plates (Maxisorp, Nunc) were coated either with 1 μg/ml rBet v 1 or homologous allergens in alder (rAln g 1), hazel (rCor a 1), apple (rMal dl) and additionally with several Bet v 1-derived peptides in a concentration of 1 μg/ml overnight at 4° C. After washing and blocking as described above sera from rabbits immunized with rBet v 1 and the PreS fusion proteins conjugated to alum or CFA, were incubated in serial 1:2 dilutions ranging from 1:500 to 1:1 280 000 and in a concentration of 1:1000. Bound rabbit IgG was detected with HRP-labelled donkey anti-rabbit antibodies (GE Healthcare) and colour reaction was determined as described above.
Example 24: Inhibition of Allergic Patients' IgE Binding to rBet v 1
(89) An inhibition ELISA was used to study the inhibition of the binding of birch pollen allergic patients' IgE to rBet v 1. ELISA plates were coated with rBet v 1 in a concentration of 1 μg/m at 4° C. overnight. After washing and blocking plates were pre-incubated with rabbit sera directed against the PreS fusion protein 2PAPB-PreS and anti-Bet v 1 rabbit serum in a dilution of 1:80 and 1:160 in comparison with rabbit preimmune sera overnight at 4° C. After an additional washing step sera of birch pollen allergic patients diluted 1:5 were added overnight at 4° C. and bound human IgE were detected with a 1:1000 diluted alkaline phosphatase-conjugated mouse monoclonal anti human IgE antibody (BD Pharmingen). The percentage of inhibition of IgE binding to rBet v 1 after pre-incubation with 2PAPB-PreS rabbit antisera and Bet v 1 rabbit antisera was calculated as follows: percent inhibition=100−(OD.sup.i×100/OD.sup.p). OD.sup.p and OD.sup.i represent the extinctions after pre-incubation with specific rabbit IgG (OD.sup.i) or preimmune sera (OD.sup.p), respectively. (
Example 25: Use of a Vaccine Formulation Comprising a Mixture of 4 Hypoallergenic Fusion Proteins for the Treatment of Grass Pollen Allergy in Grass Pollen Allergic Human Individuals
(90) An injectable formulation of hypoallergenic fusion proteins SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, and SEQ ID No. 17 with aluminum hydroxide was prepared as described in example 8. In the course of a clinical study, the vaccine was administered 3 times subcutaneously to 69 grass pollen allergic human subjects. (
(91) Vaccination with the vaccine formulation led to a robust IgG immune response. Induction of allergen-specific IgG following s.c. injection of the 3 different dose levels of the vaccine and placebo was determined by ELISA in the sera collected from the study participants before and after treatment with 3 s.c. injections of the vaccine formulation. (
(92) For this purpose, ELISA plates (Nunc Maxisorp, Roskilde, Denmark) were coated with 5 μg/ml of the antigens Phl p 1, Phl p 2, Phl p 5, and Phl p 6 or human serum albumin (HSA) as control over night at 4° C. After washing with PBS containing 0.5% Tween 20 (PT) and blocking with 2% w/v BSA in PT, plates were subsequently incubated with 1:10 to 1:100 diluted sera from patients, serum from a non-atopic individual or buffer alone in triplicates overnight at 4° C. Bound IgE antibodies were detected with HRP-coupled anti-human IgE antibodies diluted in PT, 0.5% w/v BSA. The colour development was performed by addition of staining solution ABTS (2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt; Sigma-Aldrich, St. Louis, Mo., USA) (100 μl/well). The optical density was measured using an ELISA Reader at 405 nm. The results of IgG assessments are shown in
(93) The vaccine did not provoke any relevant T-cell reactivity towards the hypoallergenic fusion proteins present in the vaccine formulation as determined by in-vitro T-cell proliferation assay (
(94) T-cell proliferation assays were performed using the following procedure: Peripheral blood mononuclear cells (PBMC) were isolated from heparinised blood samples of the grass pollen allergic patients by Ficoll (Amersham Pharmacia Biotech, Little Chalfont, UK) density gradient centrifugation. PBMC (2×10.sup.5) were then cultured in triplicates in 96-well plates (Nunclone; Nalge Nunc International, Roskilde, Denmark) in 200 μl serum-free Ultra Culture medium (BioWhittaker, Rockland, Me.) supplemented with 2 mM L-glutamin (SIGMA, St. Louis, Mo.), 50 μM b-mercaptoethanol (SIGMA) and 0.1 mg gentamicin per ml (SIGMA) at 37° C. and 5% CO.sub.2 in a humidified atmosphere. Cells were stimulated with a mixture containing 0.25 μg of each polypeptide component of the vaccine and for comparison an equimolar concentrations of grasspollen extract or for control purposes with 4 U Interleukin-2 per well (Boehringer Mannheim, Germany) or medium alone. After 6 d culture 0.5 μCi per well [3H]thymidine (Amersham Pharmacia Biotech) was added and 16 h thereafter incorporated radioactivity was measured by liquid scintillation counting using a microbeta scintilllation counter (Wallac ADL, Freiburg, Germany). Mean cpm were calculated from the triplicates and stimulation indices (SI) were calculated as the quotient of the cpm obtained by antigen or interleukin-2 stimulation and the unstimulated control. Results of proliferation assays are shown in
(95) Treatment with the vaccine induced IgG antibodies with the capability to modulate the allergen-specific T-cell response as demonstrated by a reduced proliferative response upon stimulation with grass pollen allergens in the presence of treatment-induced IgG. (
(96) For this purpose, T-cell proliferation assays were performed with PBMCs isolated from study participants after treatment as described above with the exception that the stimulation was done with a mixture of the 4 grass pollen allergens Phl p 1, Phl p 2, Phl p5, and Phl p 6 (0.25 μg per allergen) together with serum collected from the same participant before and after the treatment. The experimental set-up and results are shown in
(97) Reduction of nasal allergy symptoms induced by provocation in a pollen chamber and reduction of skin reactivity as determined by titrated skin prick testing was observed in patients having received 3 injections containing either 20 μg or 40 μg of each of the 4 polypeptides while there was no reduction in those parameters after treatment with doses of 10 μg of each polypeptide. (see
Example 26: Selection of Peptides Derived from House Dust Mite Allergen Der p 2 and Design of PreS Fusion Proteins Using Those Peptides
(98) The 5 non IgE binding Der p 2 derived peptides—Der p2 Pep1 (SEQ ID No. 96), Der p2 Pep2 (SEQ ID No. 97), Der p2 Pep3 (SEQ ID No. 98), Der p2 Pep4 (SEQ ID No. 99), and Der p2 Pep5 (SEQ ID No. 100)—were screened with respect to their IgE binding properties (dot blot assay) their potential to induce Der p 2 specific T-cell reactions, and (T-cell proliferation assay) their ability to induce Der p 2-specific antibodies with the capacity to block human patient's IgE to Der p 2. (inhibition ELISA using rabbit anti-peptide IgG)
(99) For that purpose, each of the peptides was chemically coupled to KLH. KLH and chemical coupling of the peptides was used in this screening experiment because it is an easy-to-use and well established and straight forward model system allowing initial comparison of the different peptides.
(100) IgE binding of the Der p 2 derived peptides in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from 26 house dust mite allergic patients were incubated with dotted KLH-conjugated peptides and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for any of the 5 peptides as shown below.
(101) To identify peptides which induce a low lymphoproliferative response in PBMC from house dust mite allergic patients PBMCs isolated from 10 patients were stimulated with the 5 Der p 2 derived peptides alone, the KLH-conjugated peptides, and wild-type Der p 2 for comparison.
(102) PBMCs from all 10 patient were stimulated by the wild-type Der p 2, and there was no or only very low proliferation upon stimulation with Der p2 Pep1, Der p2 Pep2, and Der p2 Pep4. Stimulation with Der p2 Pep3 and Der p2 Pep5 however, resulted in significant proliferation of the PBMCs in 4 out of 10 and 3 out of 10 cases, respectively, indicating that peptides 3 and 5 contain important T-cell epitopes.
(103) To identify the ability of the peptides to induce blocking IgG, rabbits were immunized with the 5 individual KLH-peptide conjugates. Subsequently, the ability of peptide-induced rabbit IgG to inhibit the binding of allergic patients' IgE antibodies to rDer p 2 was investigated by ELISA. ELISA plates were coated with 1 μg/ml rDer p 2, washed and blocked. The plates were preincubated with 1:100-diluted rabbit anti-peptide (KLH-P1, KLH-P2, KLH-P3, KLH-P4, and KLH-P5), a rabbit anti rDer p 2 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from house dust mite allergic, Der p 2 sensitized patients (1:3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1:1000) and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: 100−ODi/ODP×100.
(104) TABLE-US-00009 TABLE 5 Inhibition capacity of anti-Der p 2-peptide antibodies to inhibit the binding of 20 allergic patients' IgE to complete rDer p 2. Anti- KLH-peptide sera induced by peptides 2, 3, and 4 blocked the IgE-binding to the same extent as sera against wild-type Der p 2. Table 5 shows the inhibition (in %) of all 20 patients. Patient Peptide Peptide Peptide Peptide Peptide Der # 1 2 3 4 5 p 2 1 50.63 74.41 78.36 75.50 1.07 78.26 2 49.61 77.15 82.95 77.85 4.16 82.74 3 64.73 87.41 92.13 89.25 0.00 93.34 4 37.98 72.24 81.08 75.60 2.48 84.25 5 0.00 43.56 50.52 47.28 0.00 56.70 6 54.12 80.63 82.64 80.94 1.10 83.21 7 51.43 79.64 92.08 83.25 16.16 93.51 8 42.93 71.02 79.55 75.44 0.83 78.35 9 30.33 58.36 50.94 56.49 7.76 57.03 10 38.46 66.79 71.20 71.25 0.00 69.06 11 48.15 74.60 83.13 78.97 5.59 83.56 12 46.06 68.54 74.05 71.32 10.05 76.46 13 44.71 73.62 87.29 77.19 4.97 84.34 14 39.20 63.55 53.94 65.30 0.00 66.20 15 43.62 71.82 89.94 74.54 0.51 94.39 16 38.09 69.94 84.08 72.45 1.29 86.83 17 43.63 74.16 87.12 78.50 2.98 89.10 18 29.09 73.75 89.97 77.59 1.38 90.66 19 40.44 56.77 62.09 62.30 0.00 66.16 20 20.89 60.85 70.76 63.16 2.69 74.98 mean 40.71 69.94 77.69 72.71 3.15 79.46
(105) TABLE-US-00010 TABLE 6 Decision matrix for selection of peptides. Peptides 2 and 4 meet all requirements of peptide fragments of the present invention. peptide induces IgG which peptide is peptide induces inhibit binding non-IgE no or only low of human IgE Peptide binding T-cell reactivity to Der p 2 suitable? Der p2 Pep1 ✓ ✓ X no Der p2 Pep2 ✓ ✓ ✓ yes Der p2 Pep3 ✓ X ✓ no Der p2 Pep4 ✓ ✓ ✓ yes Der p2 Pep5 ✓ X X no
Example 27: Selection of Der p 1 Derived Hypoallergenic Peptides
(106) The ability of Der p 1 derived peptides to induce IgE-blocking IgG antibodies was determined using rabbit-anti-peptide KLH antisera and sera from 6 house dust mite allergic patients in an inhibition ELISA as described in example 26 with the exception that the ELISA plates were coated with wild-type Der p 1 instead of Der p 2.
(107) TABLE-US-00011 TABLE 7 Inhibition capacity of anti-Der p 1- peptide antibodies to inhibit the binding of 6 allergic patients' IgE to complete Der p 1. Anti-KLH-peptide sera induced by peptides 1, 2, and 8 were found to block the IgE-binding to a similar extent as sera against wild-type Der p 1. Table 7 shows the inhibition (in %) of 6 patients. Patient I Patient II Patient III Patient IV Patient V Patient VI mean der p 1 72.9 91.3 80 90.8 87.5 89.7 85.4 peptide 1 50 68.4 65.5 87.7 77.4 85.1 72.4 peptide 2 47.8 73.4 66.1 83.2 72.6 82.5 70.9 peptide 3 22.5 28.2 22.1 35.5 26.4 27.6 27.1 peptide 4 24.4 42.4 33.4 46.5 33.2 42 37.0 peptide 5 22.7 31.4 23.3 38.4 30.4 31.5 29.6 peptide 6 1.9 12.8 3.6 5.6 4.2 5.4 5.6 peptide 7 30 51.8 43.5 67.4 52.1 59.6 50.7 peplide 8 41.1 65.8 52.8 76 66.2 73.9 62.6