Plant Biotechnology for Human Health: Production of protein ...

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1 Δεκ 2012 (πριν από 4 χρόνια και 11 μήνες)

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Nobuyuki Matoba, Ph.D.
Plant Biotechnology for Human Health:
Production of protein pharmaceuticals in plants
1.
Plant-made pharmaceuticals (PMPs) – a brief overview.
2.
Developing PMPs against the global HIV/AIDS epidemic.
Seminar Agenda
Hormones
Cytokines
Enzymes
Monoclonal Abs
Subunit Vaccines
PMPs
A category of recombinant protein-based
pharmaceuticals produced in plants.
Our research is focused on developing
plant-made pharmaceuticals (PMPs)
Protein Pharmaceuticals (biologics, biologicals)

Highly specific, potent bioactivities.

Produced in recombinant systems.


E. coli
(insulin, growth hormones)

Yeast (HBV and HPV vaccines)

Insect cells (HPV vaccine)

Mammalian cells (monoclonal Abs)

Very expensive with limited supply capacity.
Why use plants?
Plant Expression Systems

Eukaryotes.

Low production cost.

Safe.

Large scale-up capacity.
Plants can offer a global-scale supply of
low-cost protein pharmaceuticals.
1989
Hiatt, et al. “Production of Antibodies in
Transgenic Plants.”
Nature
342: 76-78.
Milestones of PMP Research
1989
Hiatt, et al. “Production of Antibodies in
Transgenic Plants.”
Nature
342: 76-78.
1992
Mason, et al. “Expression of hepatitis B
surface antigen in transgenic plants.”
PNAS

89:11745-49.
Milestones of PMP Research
1989
Hiatt, et al. “Production of Antibodies in
Transgenic Plants.”
Nature
342: 76-78.
1992
Mason, et al. “Expression of hepatitis B
surface antigen in transgenic plants.”
PNAS

89:11745-49.
Milestones of PMP Research
1998
Tacket, et al. “Immunogenicity in
humans of a recombinant bacterial antigen
delivered in a transgenic potato.”
Nat Med

4:607-609.
2006
Dow AgroSciences’ Newcastle disease
vaccine, world’s first licensed plant-made
pharmaceutical.
h"p://www.protalix.com/
Several PMPs are reaching the market
2009
Protalix’s plant-cell expressed
recombinant glucocerebrosidase for the
treatment of Gaucher disease completes
Phase III clinical trial in December.
Technologies for protein expression in plants
1. Transgenic plants: the conventional method.
DNA
Agrobacterium
tumefaciens
2 dpi
4 dpi
6 dpi
I. Infectious virus-based systems.
2. Plant virus vectors (TMV, PVX, geminivirus, etc).
TMV RNA
DNA
2 dpi
4 dpi
6 dpi
I. Infectious virus-based systems.
2. Plant virus vectors (TMV, PVX, geminivirus, etc).
TMV RNA
DNA
DNA
6 dpi
Agrobacterium
tumefaciens
II. Deconstructed virus-based systems (e.g. magnICON
®
system).
Comparison at Cellular Level
Viral Vector
Gene Expression
New Gene
Chromosome
Transgenic
Gene Expression
Transformation
VIRAL VECTOR RNA
Infection
Proteins
mRNA
Transcription
Proteins
Translation

High expression

Rapid

Limited host range

Broad host range

Easy scale-up

Time consuming

Modest expression
Developing  Plant-­‐based  
Microbicides  and  Vaccines  against  HIV
A  global  view  of  HIV  infec;on
33  million  people  
[
30–36  million
]
 living  with  HIV,  2007
2008  Report  on  the  Global  AIDS  Epidemic
by  The  United  Na<ons  Joint  Programme  on  HIV/AIDS  
(UNAIDS)  &  The  World  Health  Organiza<on  (WHO)
Challenges in HIV/AIDS Prevention


HIV infects, suppresses and destroys immune cells.


Hyper-variability of HIV.


Correlates of protective immunity remain undefined.


>95% of new infections occur in developing countries: HIV
microbicide/vaccine must be inexpensive.
Source: AIDS Vaccine Blueprint 2006, IAVI
*Work  done  at  Arizona  State  University  Biodesign  Ins<tute  in  collabora<on  with  Dr.  Morgan  
Bomsel  at  the  Ins<tute  Cochin  in  Paris,  France.
gp120
gp41
HIV
Developing a vaccine blocking HIV mucosal transmission
*Work  done  at  Arizona  State  University  Biodesign  Ins<tute  in  collabora<on  with  Dr.  Morgan  
Bomsel  at  the  Ins<tute  Cochin  in  Paris,  France.
gp120
gp41
HIV
Infected
cell
Target  cell
 
GalCer  
Epithelial  cell
Developing a vaccine blocking HIV mucosal transmission
*Work  done  at  Arizona  State  University  Biodesign  Ins<tute  in  collabora<on  with  Dr.  Morgan  
Bomsel  at  the  Ins<tute  Cochin  in  Paris,  France.
gp120
gp41
HIV
Infected
cell
Target  cell
 
GalCer  
Epithelial  cell
Developing a vaccine blocking HIV mucosal transmission
*Work  done  at  Arizona  State  University  Biodesign  Ins<tute  in  collabora<on  with  Dr.  Morgan  
Bomsel  at  the  Ins<tute  Cochin  in  Paris,  France.
gp120
gp41
HIV
MPR
Membrane  Proximal  Region
 of  gp41
 

Highly conserved

Binds to GalCer, mediating epithelial transcytosis

Highly-exposed, persistently seronegative individuals
have transcytosis-blocking S-IgAs
Infected
cell
Target  cell
 
GalCer  
Epithelial  cell
Developing a vaccine blocking HIV mucosal transmission
Goal:
Create a mucosal vaccine inducing Abs to gp41 MPR.
Obstacle:
MPR is poorly immunogenic.
Hypothesis:
Fusion of MPR to a highly immunogenic antigen
will overcome the immunogenicity obstacle.
Hypothesis:
Fusion of MPR to a highly immunogenic antigen
will overcome the immunogenicity obstacle.
Cholera toxin B subunit (CTB)

Non-toxic

Pentamer

Binds to mucosal epithelial cell receptor (GM1-ganglioside)

Potent mucosal immunogen
HIV-1 gp41 MPR
CTB
A
B
BALB/c ♀
Immunization of mice with CTB-MPR
i.n.
CTB 10µg
CTB-MPR 14µg
CTB-MPR+CT 14+1µg
Weeks
i.n.
i.n.
i.n.
i.n.
i.n.
i.p.
MPR (1µg)
13
12
11
10
9
8
7
6
5
4
3
2
1
0
i.p.
CTB-MPR (3.5µg)
CTB
CTB-MPR
CTBMPR+CT
0
0.35
0.7
0
0.1
0.2
0
0.15
0.3
Serum IgG
Fecal IgA
Vaginal IgA
10
11
13
10
11
13
10
11
13
*
*
**
*
**
**
*
P
<0.05
**
P
<0.01
OD490
Weeks
Matoba et al. PNAS 101:13584-13589 (2004)
Matoba et al. Vaccine 24: 5047-5055 (2006)
*Proof  of  principle  studies  with  
E.  coli
-­‐produced  CTB-­‐MPR
HIV-­‐1-­‐infected  human  
PBMC
Human  epithelial  cell  
monolayer  with  <ght  
junc<ons
HIV
Effects of anti-MPR Abs in a human tight epithelial model
*Proof  of  principle  studies  with  
E.  coli
-­‐produced  CTB-­‐MPR
Abs
HIV-­‐1-­‐infected  human  
PBMC
Human  epithelial  cell  
monolayer  with  <ght  
junc<ons
HIV
Effects of anti-MPR Abs in a human tight epithelial model
*Proof  of  principle  studies  with  
E.  coli
-­‐produced  CTB-­‐MPR
Abs
HIV-­‐1-­‐infected  human  
PBMC
Human  epithelial  cell  
monolayer  with  <ght  
junc<ons
Transcytosis  (%  of  control)
0
50
100
0
50
100
0
50
100
Serum  Ig
Fecal  Ig
Vaginal  Ig
CTB
CTB-­‐MPR+CT
Control

Mouse anti-MPR Abs blocked transcytosis of a primary HIV-1 (D clade).
Matoba  et  al.  PNAS  101:13584-­‐13589  (2004)
Matoba  et  al.  Vaccine  24:  5047-­‐5055  (2006)
HIV
Effects of anti-MPR Abs in a human tight epithelial model
*Proof  of  principle  studies  with  
E.  coli
-­‐produced  CTB-­‐MPR

Also, rabbit anti-MPR Abs blocked B clade HIV-1 transcytosis
Matoba  et  al.  Curr  HIV  Research  (2008)
Abs
HIV-­‐1-­‐infected  human  
PBMC
Human  epithelial  cell  
monolayer  with  <ght  
junc<ons
Transcytosis  (%  of  control)
0
50
100
0
50
100
0
50
100
Serum  Ig
Fecal  Ig
Vaginal  Ig
CTB
CTB-­‐MPR+CT
Control

Mouse anti-MPR Abs blocked transcytosis of a primary HIV-1 (D clade).
Matoba  et  al.  PNAS  101:13584-­‐13589  (2004)
Matoba  et  al.  Vaccine  24:  5047-­‐5055  (2006)
HIV
Effects of anti-MPR Abs in a human tight epithelial model
*Proof  of  principle  studies  with  
E.  coli
-­‐produced  CTB-­‐MPR
R
L
NOS
nptII
Ag7
CTB-­‐MPR
VSP  terminator
TEV
CaMV35S  Promoter
Agrobacterium  
LBA4404
Shoot      
selec;on
Tissue    
culture  
Greenhouse  
produc;on
Nico9ana  benthamiana
Tomato  (TA234)
Callus      
induc;on
Constructing transgenic plants expressing CTB-MPR
CTB
CTB-MPR
CTB-MPR
KDa
E. coli
Wt
N. benthamiana
Non-­‐denaturing  condi;ons
65%  explants  transformed,  in  which  60%  
showed  expression  of  GM1-­‐binding  CTB-­‐MPR.  
130
73
54
35
24
16
130
73
54
35
24
16
Matoba  et  al.  Plant  Biotech  J  (2009)
Tg
Wt
Kb
1.6
.65
4.0
3.0
MfeI
+ +
PstI
+ +
SacI
+ +
1. Southern blot
2. Northern blot
3. Western blot
1.0
.6
.3
Kb
Tg
Wt
Anti-MPR
Anti-CTB
Analysis of transgenic
N. benthamiana
CTB
CTB-MPR
CTB-MPR
KDa
E. coli
Wt
N. benthamiana
Non-­‐denaturing  condi;ons
Transgenic plants can express pentameric, GM1-binding CTB-MPR.
65%  explants  transformed,  in  which  60%  
showed  expression  of  GM1-­‐binding  CTB-­‐MPR.  
130
73
54
35
24
16
130
73
54
35
24
16
Matoba  et  al.  Plant  Biotech  J  (2009)
Tg
Wt
Kb
1.6
.65
4.0
3.0
MfeI
+ +
PstI
+ +
SacI
+ +
1. Southern blot
2. Northern blot
3. Western blot
1.0
.6
.3
Kb
Tg
Wt
Anti-MPR
Anti-CTB
Analysis of transgenic
N. benthamiana
N.  benthamiana  leaves
Debris
Clear  lysate
Metal/Galactose  Affinity  Frac;on
Green  juice
Extract  with  0.5%  CHAPS
Metal  affinity
column
Galactose  
column
Metal  affinity
column
Dialyze  to  remove  CHAPS
Pentameric  CTB-­‐MPR
Purification of
N. benthamiana
-expressed CTB-MPR
Week
i.n.
i.n.
i.n.
i.n.
13
12
11
10
9
8
7
6
5
4
3
2
1
0
i.p.  
Liposome-­‐CTB-­‐MPR  (3  µg)  
i.n.
Liposome-­‐CTB-­‐MPR  (35  µg)  +  1  µg  CT  
BALB/c  
˂
Matoba  et  al.  Plant  Biotech  J  (2009)
Immunization of mice with
N. benthamiana
-expressed CTB-MPR
Week
i.n.
i.n.
i.n.
i.n.
13
12
11
10
9
8
7
6
5
4
3
2
1
0
i.p.  
Liposome-­‐CTB-­‐MPR  (3  µg)  
i.n.
Liposome-­‐CTB-­‐MPR  (35  µg)  +  1  µg  CT  
BALB/c  
˂
10
0
10
2
10
4
10
6
Endpoint  ;ter
10
0
10
1
10
2
Endpoint  ;ter
Serum  an;-­‐
MPR  IgG
Vaginal  an;-­‐
MPR  IgA
Plant-­‐made  CTB-­‐MPR  
induced  an;-­‐MPR  Abs
Matoba  et  al.  Plant  Biotech  J  (2009)
Immunization of mice with
N. benthamiana
-expressed CTB-MPR
.72kb
     Tg                          Wt
SacI
~10kb
>12kb
EcoRI
MfeI
XhoI
+
+
+
+
+
+
+
+

 Southern  blot
0.7
CTB-­‐MPR
Kb

 Northern  blot
18S  rRNA
Matoba  et  al.  in  prepara<on
Wt
Tg
Expression of CTB-MPR in tomato
.72kb
     Tg                          Wt
SacI
~10kb
>12kb
EcoRI
MfeI
XhoI
+
+
+
+
+
+
+
+

 Southern  blot
0.7
CTB-­‐MPR
Kb

 Northern  blot
18S  rRNA
Matoba  et  al.  in  prepara<on
Wt
Tg
0.5
%  Total  Extracted  Protein
0

 GM1-­‐binding  CTB-­‐MPR  level  
?
Expression of CTB-MPR in tomato
.72kb
     Tg                          Wt
SacI
~10kb
>12kb
EcoRI
MfeI
XhoI
+
+
+
+
+
+
+
+

 Southern  blot
100 g of green tomato contained
~1 mg of CTB-MPR
0.7
CTB-­‐MPR
Kb

 Northern  blot
18S  rRNA
Matoba  et  al.  in  prepara<on
Wt
Tg
0.5
%  Total  Extracted  Protein
0

 GM1-­‐binding  CTB-­‐MPR  level  
?
Expression of CTB-MPR in tomato
CTB-MPR Yield
(Fully assembled, pentameric)
Production
System
0.5~1.5 mg/L culture
(≥98% purity)
~5 mg/Kg leaf
(~95% purity);
20 mg/Kg leaf
(Crude extract)
10 mg/Kg green fruit in
crude extract
E. coli
Transgenic
N. benthamiana
Transgenic
Tomato
(green fruits)
Freeze-dried green tomato expressing CTB-MPR
may offer a heat-stable oral vaccine against HIV.
1.
Immunogenicity: use of strong adjuvant, prime-boost
strategy with different MPR-based immunogens, etc.
2.
Testing
in vivo
activity.
The MPR-based vaccine project: challenges ahead
Viral
membrane
HIV
We are focusing on two broadly neutralizing anti-HIV
proteins as potential microbicides.
Developing microbicides blocking HIV
mucosal transmission
1.  Ac;nohivin,  
an    ac;nomycete-­‐
derived  lec;n.
Viral
membrane
HIV
We are focusing on two broadly neutralizing anti-HIV
proteins as potential microbicides.
Developing microbicides blocking HIV
mucosal transmission
1.  Ac;nohivin,  
an    ac;nomycete-­‐
derived  lec;n.
2.  Monoclonal  IgA  
isolated  from  exposed  
seronega;ve  individual.
Viral
membrane
HIV
We are focusing on two broadly neutralizing anti-HIV
proteins as potential microbicides.
Developing microbicides blocking HIV
mucosal transmission
Actinohivin

Isolated from the actinomycete
K97-0003 strain

114aa, ~12.5 KDa

3 sugar-binding sites

Selectively binds to a cluster of
high-Man glycans on gp120

Mid nanomolar anti-HIV activity


Candidate HIV-1 microbicide
The Actinohivin Project
Images  from  
Dr.  Tanaka
Collabora<on  with  Dr.  Haruo  Tanaka  at  Iwaki  Meisei  Univ.  and  KIIM  Pharm  Lab,  Inc.
Goal:
Establish a plant-based expression system for actinohivin,
which can facilitate detailed molecular analysis and design,
as well as economical large-scale production.
Actinohivin gene
A. tumefaciens
Vacuum infiltration
TMV
DNA
Decon TMV
DNA vector
Expression of actinohivin in
N. benthamiana
using
the magnICON deconstructed TMV system
29
30
Expression of rAH in
N. benthamiana
plants using the
magnICON deconstructed TMV system.
250
150
100
75
50
37
25
20
15
10
0dpi
5dpi
kDa
Expression of rAH in
N. benthamiana
plants using the
magnICON deconstructed TMV system.
250
150
100
75
50
37
25
20
15
10
0dpi
5dpi
250
150
100
75
50
37
25
20
15
10
5dpi
0dpi
kDa
Expression of rAH in
N. benthamiana
plants using the
magnICON deconstructed TMV system.
250
150
100
75
50
37
25
20
15
10
0dpi
5dpi
250
150
100
75
50
37
25
20
15
10
5dpi
0dpi
kDa
100
200
0
Total
Gp120-
binding
mg/kg leaf
300
Expression of rAH in
N. benthamiana
plants using the
magnICON deconstructed TMV system.
250
150
100
75
50
37
25
20
15
10
0dpi
5dpi
250
150
100
75
50
37
25
20
15
10
5dpi
0dpi
kDa
100
200
0
Total
Gp120-
binding
mg/kg leaf
300
Expression of rAH in
N. benthamiana
plants using the
magnICON deconstructed TMV system.
250
150
100
75
50
37
25
20
15
10
0dpi
5dpi
250
150
100
75
50
37
25
20
15
10
5dpi
0dpi
kDa
Aher  dialysis
100
200
0
Total
Gp120-
binding
mg/kg leaf
300
Reporter gene syncytium formation assay
using Env- and CD4-expressing HeLa cells
Fusion
Chiba et al. J. Antibiot 54:818-826 (2001).
*
p
<0.05, **
p
<0.01; two-way ANOVA with Bonferroni post-tests
0
20
40
60
80
100
120
No
sample
1:48
1:192
Extract dilution
1:96
*
1:24
**
1:6
**
1:12
*
% Syncytium formation
Plant-expressed rAH retains anti-HIV activity.
Control leaf extract
AH-expressing
leaf extract
Analysis of plant-expressed rAH in syncytium formation assay
The actinohivin project: current endeavors
1.
Preventing tissue necrosis.
2.
Reducing aggregation.

3.
Detailed molecular analysis of plant-made AH.
4.
Protein engineering for potent AH analogues.
Heavy chain
Light
chain
Fc region
Arizona State University
Charles Arntzen
Tsafrir Mor
Michelle Pickel
Jennifer Hurt
Institut Cochin
Morgane Bomsel
Iwaki Meisei University
Haruo Tanaka
Atsushi Takahashi
Funding Supports

NIH grants

R21 AI052761

U19 AI62150

R03 AI073157

JSPS Fellowships

OCRP startup funds

UofL School of Medicine
Basic Grant
University of Louisville
Brian Barnett
Hillary Conway
Adam Husk
David Grunewald
Nancy Pettibone
Kenneth Palmer
Keith Davis
Acknowledgement
ICON Genetics
Yuri Gleba