Slide 1

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 2

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 3

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 4

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 5

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 6

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 7

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 8

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 9

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 10

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 11

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 12

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 13

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 14

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 15

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 16

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 17

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 18

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 19

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 20

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 21

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 22

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 23

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 24

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 25

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 26

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

www.csir.co.za

Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

www.csir.co.za

Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za


Slide 27

Novel Platforms for Essential Biomolecules:
Expression of Antibodies in Plants.
Presented at the CSIR Research and Innovation Conference

Dr Rachel K Chikwamba
CSIR Biosciences
Research Group Leader (Plant Biotechnology)
28 February 2006

Challenges of conventional protein
expression platforms
Protein based drugs fastest growing class of new drugs for treatment
and prevention of human disease.
But we face these barriers:

• Capacity:
• Insufficient capacity for drugs in the pipeline

• Cost
• Cost of goods
• Capital for manufacturing facilities
• Safety
• Risk of contamination with mammalian pathogens
• Many require needles, which are risky, costly
• Efficacy
Slide 2

© CSIR 2006

www.csir.co.za

Our Goal
To develop a transgenic plant platform for the production
of molecules to prevent and treat infectious disease, at
levels that are relevant for commercial development

Slide 3

© CSIR 2006

www.csir.co.za

Advantages of the plant
expression platform
Plants are the most efficient
producers of proteins on earth
• Plants are scalable bioreactors
• Plants provide cost advantages

Plants cells are similar to human
cells in many ways
• Similar protein synthesis
machinery
• Read the same genetic code
• Assemble, fold and secrete
complex proteins
Slide 4

© CSIR 2006

www.csir.co.za

A Novel Approach to Pharmaceutical
Production
Product Formulation
Identify gene for virus
neutralizing monoclonal
antibody gene

Transform plant cell
with antigen gene

Picture courtesy Charles Arntzen

Picture courtesy Charles Arntzen

Slide 5

© CSIR 2006

www.csir.co.za

Biosafety
• Cognisant of the need to protect the environment and
preserve the sanctity of the food supply, our group will
exercise the highest level of stewardship,

• Where maize is used as a host plant, and in light of its role
as a staple crop in Southern Africa, the work with maize will
be implemented under strict genetic containment.
Biosafety measures will be built into every aspect of the
project, including bulking up of select events under strict
containment and breeding of select events with elite male
sterile lines.

• In the expectation that the results from this study could go
forward eventually to cGMP manufacturing and Phase I and
II clinical trials, all the relevant work will be carried out
under Good Laboratory Practice (GLP).

Slide 6

© CSIR 2006

www.csir.co.za

Target molecules for the CSIR
Transgenic plant platform
Microbicidal
Antibodies

peptides
Subunit
vaccines

Slide 7

© CSIR 2006

www.csir.co.za

Focus on Recombinant Monoclonal
Antibodies (MAbs)
• Inherently stable human
mucosal surface defense
proteins
• High specificity; low toxicity
• Injectable, topical and oral
applications
• Appropriate for chronic
conditions

• Potential long-lasting benefits

High potential as microbicidal molecules
Slide 8

© CSIR 2006

www.csir.co.za

Antibodies are effective drugs for
many indications
Antibody

Slide 9

Indication

Developer

Launch

Reopro

Coronary
restenosis

Centocor

1995

Rituxan

NHL

IDEC

1997

Remicade

RA and Crohn’s

Centocor

1998

Synagis

RSV

MedImmune

1998

Herceptin

Breast Cancer

Genentech

1998

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 as model antibody in
transgenic plants

Rabies: The disease
•acute viral disease of the central
nervous system
•affects humans and other mammals
•If post exposure treatment is not
administered, disease is 100% fatal
• Caused by the caused by a rod- or
bullet-shaped virus in the family
Rhabdoviridae
• patient is vaccinated immediately
administration
of
human
rabies
immune globulin (HRIG) or equine
(ERIG) for passive immunization
in
wound and muscle, followed by active
immunization.
Slide 11

© CSIR 2006

www.csir.co.za

Rabies antibodies: developing the
plant platform
• Rabies is an important disease in the developing
countries, particularly in Asia but also in Africa.

• 10 Million people receive ERIG or HRIG prophylaxis
annually
• Key target disease for Pharmaplanta,

Rabies antibody production in plants to be used
as model for other high value antibody targets

Slide 12

© CSIR 2006

www.csir.co.za

Production of the rabies monoclonal
antibody E559 in transgenic tobacco

Production of Pharmaceuticals in Tobacco
Advantages
• Well established technology for gene
transfer and expression
• High Biomass yield (100,000 kg/ha)
• Prolific seed production
• Good regulatory elements
• Large scale processing infrastructure
• Non food / feed crop

Key disadvantages:
•Requires immediate freezing, drying or other processing
•High Nicotine/alkaloid content making purification expensive

Slide 14

© CSIR 2006

www.csir.co.za

Plasmids encoding antibody chains for tobacco
transformation
Hin dIII (13850)
Sal I (13836)
Bam HI (13830)

NOS terminator
Eco RI (13567)
Sma I (13277)

Heavy Chain

Xma I (13275)

Bgl II (12507)
Xho I (12205)
Kpn I (12204)

Murine Ig Leader Sequence
CaMV35S promoter

Hin dIII (13148)
Sal I (13134)

Nco I (12150)
Bgl II (12142)
Eco RV (12021)

Bam HI (13128)

pRHC14.4.1(35S)

NOS terminator

13922 bp

Eco RI (12865)
Sac I (12818)

Light Chain
Hin dIII (12518)
Xho I (12205)
Kpn I (12204)

Murine Ig leader sequence
Nco I (12150)
Bgl II (12142)
Eco RV (12021)

pRLC15.1.1(35S)

CaMV35S promoter

Slide 15

© CSIR 2006

13220 bp

www.csir.co.za

Tobacco Transformation
Transgenic plants expressing
heavy and light antibody chains
separately

Agrobacterium transformed
tobacco leaf discs

Slide 16

Non-transformed
tobacco leaf
discs
© CSIR 2006

Lines expressing heavy and
light chains transferred to the
green house. Plants with HC
and LC crossed, and seedlings
growing in vitro.
www.csir.co.za

Screening of MAb E559 Expression in
Tissue Culture Plants
ELISA results of HC transformed Tobacco

A total of 35 plants assayed for
heavy chain expression 23
showed expression

0.7
0.6
0.5

OD450

0.4
0.3
0.2
0.1

H4
9

D1

A1

HC

14

8

4

21
HC

HC

HC

HC

H1
1

1
HH

HE
1

-0.1

HF
2

0

Tobacco plants assayed

All data corrected for background
absorbance. New standard acquired
for quantification of expression
levels by ELISA

ELISA results on LC transformed Tobacco
1.5

A total of 42 plants assayed
for heavy chain expression 33
showed expression

OD450

1
0.5

Slide 17

Tobacco plant assayed

L39

L30

LC15

LC12

LC9

LC6

L63

L60

L46

L31

LC3

-0.5

L25

L21

L17

0

© CSIR 2006

www.csir.co.za

Screening of MAb E559 Expression in
Greenhouse Plants

Bottom leaves
Middle leaves

C8
C1
0
H
11
H
C1
3
H
H F1
F2
H 29
F2
14
H
G
1
H
H1
2
H 9
H1
14

Only some of the plants survived
transplantation into greenhouse.
Not all plants showed expression
once moved to the greenhouse.

H

H

3

C7

Top leaves

H

1
0.8
0.6
0.4
0.2
0
-0.2

H

OD450

ELISA results on HC transformed Tobacco in
Greenhouse

Tobacco plant assayed

ELISA results on LC transformed Tobacco plants in
Greenhouse

All data corrected for background
absorbance, three leaves at different
stages assayed per plant. New standard
acquired
for
quantification
of
expression levels by ELISA

0.4

OD450

0.3
Bottom leaves

0.2

Middle leaves

0.1

Top leaves

0

For the LC, plants show consistent
expression between all three leaves

LC
7
LC
11
LC
12
LC
15
LC
17
L2
0
L2
1
L2
2
L3
1
L3
2
L3
9
L4
9

-0.1

Tobacco plant assayed

Slide 18

© CSIR 2006

www.csir.co.za

Future perspectives
1. Evaluation of hybrids expressing heavy and light
chains
2. Breeding programme to develop lines expressing
high levels of functional antibody (target of 0.1% of
total soluble protein)

3. Extraction of functional antibody from crude
extracts
4. Evaluation of efficacy of the plant made antibody
against infective rabies virus in collaboration with
other partners
Slide 19

© CSIR 2006

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Progress on the Expression of the

Rabies MAb E559 in Maize

Production of Pharmaceuticals in maize
Advantages:
• Seed allow for long term storage
• Seed are specialized are protein
accumulating organs
• Dry grain concentrates product
• Simplified processing and purification
Key disadvantages:
• Biosafety issues with pollen dispersal and potential for contamination of
food/feed crops
• Food crop/public perception issues
Proactive biosafety measures:
• All crossing to be done under containment in green houses
• Field bulking facilities also under containment
• In future, color markers, male sterile lines will be employed
Slide 21

© CSIR 2006

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Cloning vectors for maize transformation
Xho I (5174)

TEV leader

Kpn I (5163)

Nco I (141)

Signal Peptide

Sma I (5157)
Pst I (187)

Xma I (5155)

Kpn I (195)

Maize 27kDa gamma zein promoter

Xho I (196)

Light Chain
Hin dIII (509)
Sal I (4030)

Sac I (809)

Pst I (4028)
Hin dIII (4012)

Eco RI (856)

pRLC15.1.1

5178 bp

Nos terminator
Bam HI (1119)
Eco RI (1377)

Signal peptide

TEV leader
Xho I (5876)
Kpn I (5865)

Xho I (196)
Nco I (141)

Sma I (5859)

AP

r

Xma I (5857)

Kpn I (195)

Maize 27kDa gamma zein promoter

Bgl II (498)

Heavy chain
Sal I (4732)
Hin dIII (4714)

Xma I (1266)

pRHC14.4.1

5880 bp

Designed both heavy and light chain
constructs to be transformed into
maize

Nos terminator
Bam HI (1821)
Eco RI (2079)

AP

Slide 22

© CSIR 2006

Sma I (1268)
Eco RI (1558)

www.csir.co.za

r

Minimal cassettes used for transformation
A

XhoI (1363)

Mai ze 2 7 kD a g a m m a ze in p ro m o te r

NotI (2700)

NcoI (1308)

NcoI (1785)

T E V l e ad e r

SalI (19)
HindIII (1)

EcoRI (2707)
N OS te rm i n ato r

BamHI (1853)
H C 5 .1 .1

XhoI (1163)

BamHI (2970)

5.1.1BB Fragment

2975 bp
Xho I (1163)

B

Maize 27kDa gamma zein promoter

Not I (2016)
Eco RI (2023)

TEV leader

Sal I (19)

Nco I (1308)

Hin dIII (1)

Xho I (1363)

LC 6.1.11

NOS terminator

Hin dIII (1676)

Bam HI (2286)

6.1.11BB Fragment

2292 bp

PstI (1384)

C

XhoI (1364)
PstI (1355)

SalI (20)

HindIII (2)

NotI (2719)

NcoI (1309)

EcoRI (2726)

T EV

PstI (18)
Ze i n P rom o te r

For biosafety reasons, only the gene
expression cassettes were used. No
antibiotic markers introgressed into
the transgenic plants.
Mannose
(sugar) selectable marker system
used

N OS te rm i n ato r
H C 1 4.4 .1

XhoI (1164)

BamHI (2989)

14.4.1BBfragment

2993 bp

D

Xh o I (11 64)

Sa cI (19 77)

T EV

Sa lI (20 )
Pst I (18 )
Hin dIII (2)

Slide 23

NotI (20 17)

NcoI (13 09)

Ze in P rom ote r

1 5.1 .1 BBfra gme nt

2 29 1 b p

Eco RI (202 4)

Pst I (13 55)

LC 15.1.1

Xh o I (13 64)

Hin dIII (167 7)

© CSIR 2006

N OS te rm i na tor

Ba m HI (228 7)

www.csir.co.za

Maize transformation
Genes = Heavy Chain
Blast genes with
particle gun into
embryos

Light Chain
d

Mannose (marker)

a
c

e
b

Slide 24

© CSIR 2006

www.csir.co.za

Current Status:

Transgenic maize plants
just recovered

A

B

C

Greenhouse

•Transgenic plants co-bombarded with both HC and LC undergoing
molecular analysis to confirm transgene integration.
•Evaluation of gene expression to be undertaken in maize seed
Slide 25

© CSIR 2006

www.csir.co.za

Future perspectives for the platform
•

New, relevant and high social impact targets

•

New vectors for faster transient expression

•

Custom made plant hosts

Acknowledgements
Scientists

• Therese Lotter (MSc)
• Nosisa Dube (MSc)
• Maretha O’Kennedy (PhD)
Technicians

• Gugu Ngwenya
• Jefferey Mathabe
The Plant Biotechnology Research Group

Slide 27

© CSIR 2006

www.csir.co.za