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Molecular farming - Recombinant barley-produced antibody for the detection of the major bovine milk allergen, β-lactoglobu...
2 
11/11/2014 
Molecular farming - Plants as production host 
Häkkinen & Ritala 2010. Medicinal compounds produced in plan...
3 
Molecular farming in barley @ VTT 
T0 plant T1 plant T2 plant 
T1 seeds T2 seeds T3 seeds 
Etc. 
Isolation and culture ...
4 
Background of milk allergy and selection of milk allergen specific antibodies
5 
BLG is one of the major allergens in cow's milk 
Cow´s milk allergy is most prevalent in early childhood, with report...
6 
Approximately 75 % of the children with reported milk allergy tolerate heated milk in an oral food challenge 
Heated ...
7 
Member of the lipocalin family comprising proteins with transport functions 
Possible physiological role as binding a...
8 
Cloning of IgE antibodies from a milk allergic person 
 Validated patient blood samples (HUCH) used for the cloning of...
9 
Selection of IgE antibodies 
Recombinant IgE antibodies specific to cow's milk allergen, b-lactoglobulin (BLG) 
Two dif...
10 
0 
20 
40 
60 
80 
100 
120 
0,0001 0,001 0,01 0,1 1 10 
relative A405 (%) 
native 
denaturated 
D1 Fab 
0 
20 
40 
60...
11 
30 kD - 
20 kD - 
D1 Fab A3 Fab 
Immunoprecipitation of BLG from cow's milk 
s1- casein - 
BLG - 
0' 15' 30' 60' BLG ...
12 
The 3D structure of allergen-IgE immunocomplex 
Niemi et al. 2007. Structure, 15:1413-1421 
Niemi et al. 2008. Acta Cr...
13 
Barley-produced BLG specific antibody, D1 scFv
14 
Construction of barley expression vectors 
Use 
Constructs 
Stable expression in barley cell cultures after nuclear tr...
11/11/2014 15 
Agrobacterium mediated 
transformation and selection 
rounds 
Transgenic callus clones 
Transformation of b...
16 
11/11/2014 
Barley cell culture clones expressing BLG specific antibody, D1 scFv 
D1 scFv accumulation in the five be...
17 
11/11/2014 
Barley cell culture clone expressing BLG specific antibody, D1 scFv 
0.8 – 1.2 mg D1scFv / kg (FW) 
Stab...
18 
11/11/2014 
Transgenic barley plants expressing BLG specific antibody, D1 scFv 
Construct 
Number of infected embryos ...
19 
11/11/2014 
Transgenic barley plants expressing BLG specific antibody, D1 scFv
20 
11/11/2014 
Transgenic barley plants expressing BLG specific antibody, D1 scFv
21 
11/11/2014 
Purification of BLG specific antibody, D1 scFv, from barley grains 
After IMAC purification: 
1.LMW 
2.Gra...
22 
Immunoprecipitation of BLG from cow's milk using barley-produced D1 scFv 
1.LMW 
2.Commercial BLG 
3.Purified D1 scFv ...
23 
Conclusions 
 Barley-based production system for BLG-specific antibody D1scFv established 
 Barley cell cultures ser...
ACKNOWLEDGEMENTS: 
Vanga Siva Reddy Sadhu Leelavathi 
Marja-Leena Laukkanen 
Sirpa Jylhä 
Kristiina Takkinen 
Kirsi-Marja ...
TECHNOLOGY FOR BUSINESS
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2014 11 07_pegs_europe_lisbon_ritala

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Cow´s milk allergy is a common food allergy especially among infants and young children. The major allergen in cow´s milk is beta-lactoglobulin (BLG) and BLG-specific antibodies are needed for diagnostic, therapeutic, food processing and quality verification applications. At VTT we have established a IgE antibody library from a milk allergic person and identified high-affinity antibodies against native and heat denaturated BLG. As an alternative production system, we have used barley as a production host. Proof-of-concept was gained with expressing the BLG-specific antibody in barley cell culture. Best production levels in barley grains were obtained with glutelin-specific promoter combined to ER targeting and retention signals. The barley-produced BLG-specific antibody was successfully purifed with affinity-based chromatography and the functionality of barley-produced BLG-specific antibody was verified by ELISA and immunoprecipitation assays. The developed barley-based expression system clearly demonstrated its potential for application in the processing of dairy milk products as well as in detecting allergens from foods possibly contaminated by cow´s milk.

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2014 11 07_pegs_europe_lisbon_ritala

  1. 1. Molecular farming - Recombinant barley-produced antibody for the detection of the major bovine milk allergen, β-lactoglobulin PEGS Europe, Lisbon, 6-7.11.2014 Anneli Ritala VTT Technical Research Centre of Finland
  2. 2. 2 11/11/2014 Molecular farming - Plants as production host Häkkinen & Ritala 2010. Medicinal compounds produced in plant cell factories. In: Medicinal Plant Biotechnology. Arora, R. (ed.). CAB International. Oxfordshire, 13-35.
  3. 3. 3 Molecular farming in barley @ VTT T0 plant T1 plant T2 plant T1 seeds T2 seeds T3 seeds Etc. Isolation and culture of microspores Regeneration of DH-plants DH0 plant DH1 seeds DH2 seeds DH1 plant Etc. Bombardment or Agrobacterium-infection of immature embryos Four or three rounds of selection Regeneration PCR-analyses of regenerants = 2nd stage: Stable expression in barley cell culture 3rd stage: Stable expression in barley seeds 4th stage: Stable expression in doubled haploid barley seeds 1st stage: Transient expression in barley cell culture (Pokko) Ritala et al. 2008 Prot. Expr. Purif. 59:274–281; Eskelin et al. 2009 Plant Biotech J. 7:657-672
  4. 4. 4 Background of milk allergy and selection of milk allergen specific antibodies
  5. 5. 5 BLG is one of the major allergens in cow's milk Cow´s milk allergy is most prevalent in early childhood, with reported incidences between 2-6%, and decreases toward adulthood to occurrence of 0.1–0.5% b-lactoglobulin (BLG) The treatment of milk allergy has mainly involved avoidance of the allergen and symptom-specific treatment of allergic reactions Milk allergic patients can be divided into two groups: Heated milk-tolerant Heated milk-reactive
  6. 6. 6 Approximately 75 % of the children with reported milk allergy tolerate heated milk in an oral food challenge Heated milk-tolerant patients usually develop less severe reactions when exposed to unheated milk i.e. indicating a milder food allergy phenotype The incorporation of heat-denatured milk proteins into the diet accelerated the development of unheated milk tolerance  These immunotherapeutic effects might therefore be gained via diet modifications. Thus it would be useful to be able to process low BLG content milk products which could be safely included to diet of milk allergic patients  BLG-specific antibodies are needed for diagnostic, therapeutic, food processing and quality verification applications Some research findings and needs related to BLG allergy
  7. 7. 7 Member of the lipocalin family comprising proteins with transport functions Possible physiological role as binding and transporting retinol A and B variants based on sequence differences --> Forms an AB heterodimer in cow's milk (up to 4 g/l) Mw 18 kD (162 aa) with two disulphide bridges and one free cysteine, not glycosylated Sakai et al. Protein Sci. 2000 9:1719-1729 b-lactoglobulin (Bos d 5, BLG)
  8. 8. 8 Cloning of IgE antibodies from a milk allergic person  Validated patient blood samples (HUCH) used for the cloning of IgE responses  Permission of the Ethical Committee of HUCH granted for the cloning of the IgE responses CL CL CL CL VL CL Lymphocytes AAAA AAAA AAAA AAAA cDNA synthesis PCR amplification VH CH1 CH2 CH3 TTTT 5' Primer 3' Primer VL CL 5' Primer TTTT 3' Primer Isolation of mRNAs Cloning into phagemid vector ~10 6 Ab phages VL VL VL VL ~10 6 Ab genes VL VH VH VL Serum IgE levels and RAST of the different milk allergic patients RAST (CAP) kU/l Patient S-IgE (kU/l) milk BLG casein 1 237 3.4 3.7 1.4 2 1206 6.2 7.2 2.4 3 775 14.9 11.6 16.8 Jylhä et al. 2009. JIM, 350:63–70 Laukkanen et al. 2003, JIM, 278:271-281.
  9. 9. 9 Selection of IgE antibodies Recombinant IgE antibodies specific to cow's milk allergen, b-lactoglobulin (BLG) Two different selection strategies Selection with the immobilised BLG Unspecific elution with low pH Isolation of antibodies recognising the heat- treated BLG Selection with the soluble, biotinylated BLG Specific elution with soluble BLG Isolation of antibodies recognizing the native BLG Anti-BLG scFv D1/H2 Anti-BLG scFv A3/G1 Fab fragment human IgG CH1 and CL regions VH CH1 CL VL VH VL scFv IgE VH and  or  VL Production and purification of D1 and A3 Fab fragments Enrichment of bound phages Jylhä et al. 2009. JIM, 350:63–70
  10. 10. 10 0 20 40 60 80 100 120 0,0001 0,001 0,01 0,1 1 10 relative A405 (%) native denaturated D1 Fab 0 20 40 60 80 100 120 0,0001 0,001 0,01 0,1 1 10 relative A405 (%) native denaturated A3 Fab Competitive ELISAs BLG (μM) BLG (μM) Native Denatured (15', 95oC) Native Denatured (15', 95oC) Binding properties of the IgE antibodies Anti-BLG D1 Fab fragment 0 100 200 300 400 500 0 200 400 600 800 1000 time(s) RU 1,23nM 2,47nM 4,94nM 9,88nM 19,75nM 39,5nM 79nM Fab fragment KD (M) x10-9 D1 Fab 1.30 ± 0.66 A3 Fab 142 ± 88 The values are averages ± SD of three (D1) or two (A3) independent runs with seven (D1) or six (A3) different Fab concentrations. The binding kinetics by BIAcore
  11. 11. 11 30 kD - 20 kD - D1 Fab A3 Fab Immunoprecipitation of BLG from cow's milk s1- casein - BLG - 0' 15' 30' 60' BLG D1 Fab +95oC ctrs 0' 15' 30' 60' BLG A3 Fab +95oC ctrs 0 0,5 1 1,5 2 2,5 1 0,1 0,01 0,001 0,0001 0,00001 0,000001 BLG (g/l) A405 D1 Sandwich ELISA Detection limit 1-10 μg / l BLG detection from cow's milk * Immobilisation of the biotinylated D1 Fab fragment onto SA-coated wells * Addition of different amounts of BLG * Detection using D1 scFv-myc and AP-conjugated anti-myc Ab
  12. 12. 12 The 3D structure of allergen-IgE immunocomplex Niemi et al. 2007. Structure, 15:1413-1421 Niemi et al. 2008. Acta Crystallographica Section F64:25-28 Rouvinen et al. 2010. PLoS One, 5:1-9
  13. 13. 13 Barley-produced BLG specific antibody, D1 scFv
  14. 14. 14 Construction of barley expression vectors Use Constructs Stable expression in barley cell cultures after nuclear transformation UBI-I-ss-D1scFvb-His6-HDEL with hygromycin selection marker (pD1b02) Production of transgenic barley plants by nuclear transformation UBI-I-ss-D1scFvb-His6-HDEL with hygromycin selection marker (pD1b02) GLUB1-ss-D1scFvb-His6-HDEL with hygromycin selection marker (pD1b03) GLUB1-ss-D1scFvb-His6 with hygromycin selection marker (pD1b07) Maize UBIQUITIN promoter and first intron (UBI-I) or Rice endosperm-specific GLUTELIN promoter (GLUB1) Arabidopsis basic chitinase signal sequence ss D1scFv_b Single chain variable fragment of b-lactoglobulin-specific IgE D1 antibody, b refers to codon usage optimization for barley, His6 to Histidine tag and HDEL to ER retention signal D1scFv_b_His6_(HDEL) ss Hyg selection marker P P Ritala et al. 2014. Transgenic Res. 23:477-487
  15. 15. 11/11/2014 15 Agrobacterium mediated transformation and selection rounds Transgenic callus clones Transformation of barley to express BLG specific antibody, D1scFv Isolation of immature barley embryos Transgenic barley plants expressing D1scFv Transgenic barley cell cultures expressing D1scFv
  16. 16. 16 11/11/2014 Barley cell culture clones expressing BLG specific antibody, D1 scFv D1 scFv accumulation in the five best producing barley cell culture lines assayed by ELISA
  17. 17. 17 11/11/2014 Barley cell culture clone expressing BLG specific antibody, D1 scFv 0.8 – 1.2 mg D1scFv / kg (FW) Stable production over 21d Best harvesting time at 14d Biomass (g, FW) D1 scFv mg/kg (FW) D1 scFv μg / shake flask (V=25 ml) Time (d) WT D1scFv clone
  18. 18. 18 11/11/2014 Transgenic barley plants expressing BLG specific antibody, D1 scFv Construct Number of infected embryos Proportion of embryos giving transgenic T0 plants (% of all infected embryos) Number of transgenic T0 plants UBI-I-ss-D1scFvb-His6-HDEL 75a 5 (6.6) 29b GLUB1-ss-D1scFvb-His6-HDEL 150 8 (5.3) 24 GLUB1-ss-D1scFvb-His6 150 15 (10.0) 46b 3 different 375 28 (7.5) 99 aOnly part of the calli was plated on regeneration. The main focus was in the production of transgenic cell culture lines bOne plant died before seed-set A half-grain analytical procedure for the non-embryo part of the grain was developed in order to screen the D1scFv accumulation levels The embryo parts can be stored at +14°C for a month. Thus only the embryo halves from best lines were propagated to generate the next generation plants
  19. 19. 19 11/11/2014 Transgenic barley plants expressing BLG specific antibody, D1 scFv
  20. 20. 20 11/11/2014 Transgenic barley plants expressing BLG specific antibody, D1 scFv
  21. 21. 21 11/11/2014 Purification of BLG specific antibody, D1 scFv, from barley grains After IMAC purification: 1.LMW 2.Grain extract 3.Flow through 4.Wash buffer 1 5.Wash buffer 2 6.50 mM imidazole elution 7.75 mM imidazole elution 8.100 mM imidazole elution 9.200 mM imidazole elution 10.500 mM imidazole elution. After SEC: 1.Sample subjected to SEC 2.Flow through 3.LMW 4.- 10. Different fractions from SEC D1 scFv D1 scFv Grain pool of 50 g - Accumulation level of 55 mg D1scFv / kg seeds Purified barley-produced D1scFv functional in ELISA
  22. 22. 22 Immunoprecipitation of BLG from cow's milk using barley-produced D1 scFv 1.LMW 2.Commercial BLG 3.Purified D1 scFv 4.Native milk sample with D1 scFv-Co-Sepharose matrix 5.Milk sample after the heat treatment with D1 scFv-Co- Sepharose matrix 6.No milk sample with D1 scFv-Co-Sepharose matrix BLG
  23. 23. 23 Conclusions  Barley-based production system for BLG-specific antibody D1scFv established  Barley cell cultures served as a proof-of-concept  Reasonable production levels in barley grains with glutelin-specific promoter combined with ER targeting and retention signals  Successful purification with affinity-based chromatography  Functionality of barley-produced BLG-specific antibody D1scFv verified Ritala et al. 2014. Transgenic Res. 23:477-487
  24. 24. ACKNOWLEDGEMENTS: Vanga Siva Reddy Sadhu Leelavathi Marja-Leena Laukkanen Sirpa Jylhä Kristiina Takkinen Kirsi-Marja Oksman-Caldentey
  25. 25. TECHNOLOGY FOR BUSINESS

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