2014 11 07_pegs_europe_lisbon_ritala

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

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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.

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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

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Background of milk

allergy and selection of

milk allergen specific

antibodies

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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

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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

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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)

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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.

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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

0

20

40

60

80

100

120

0,0001 0,001 0,01 0,1 1 10

rela

tive

A4

05 (

%)

native

denaturated

D1 Fab

0

20

40

60

80

100

120

0,0001 0,001 0,01 0,1 1 10

rela

tive

A4

05

(%

)

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

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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

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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

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Barley-produced BLG

specific antibody, D1

scFv

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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

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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

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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

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Barley cell culture clone expressing BLG


0.8 – 1.2 mg D1scFv / kg (FW)

Stable production over 21d

Best harvesting time at 14d

Bio

ma

ss

(g

, F

W)

D1 s

cF

v m

g/k

g (

FW

) D

1 s

cF

v µ

g /

sh

ak

e f

las

k (

V=

25

ml)

Time (d)

Time (d)

WT

D1scFv clone

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Transgenic barley plants expressing BLG


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

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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




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

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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

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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

ACKNOWLEDGEMENTS:

Vanga Siva Reddy

Sadhu Leelavathi

Marja-Leena Laukkanen

Sirpa Jylhä

Kristiina Takkinen

Kirsi-Marja Oksman-Caldentey

TECHNOLOGY FOR BUSINESS

2014 11 07_pegs_europe_lisbon_ritala

Science

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