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.

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
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
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
Background of milk allergy and selection of milk allergen specific antibodies

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
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
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
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
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
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
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
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
Barley-produced BLG specific antibody, D1 scFv

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. 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
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
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
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
11/11/2014
Transgenic barley plants expressing BLG specific antibody, D1 scFv

20. 20
11/11/2014
Transgenic barley plants expressing BLG specific antibody, D1 scFv

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
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
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. ACKNOWLEDGEMENTS:
Vanga Siva Reddy Sadhu Leelavathi
Marja-Leena Laukkanen
Sirpa Jylhä
Kristiina Takkinen
Kirsi-Marja Oksman-Caldentey

25. TECHNOLOGY FOR BUSINESS