Download Development of novel and high value products in green algae

Development of novel and high value products in green algae
Edible algae: Production of biologics to
improve gut health
Efficient production of biologic molecules
1978
1950s
Bacteria (e. coli)
Swine pancreas
Insulin
Insulin
Use microbial organisms to efficiently and economically produce biologic
molecules important to human or animal health
How can we harness
those advantages to
produce new biologic
products?
Where does
Algae have an
advantage?
Algae
biologic
molecules?
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Biologics are expensive with existing systems
Expensive Traditional Platforms
• Production in traditional protein expression
platforms (requiring purification) is too
expensive
Host
Coli
Yeast
CHO
• Algae offer a scalable, low-cost alternative
Insect
Algae
Low cost
production
No Purification
required?
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Algae are edible
• They can serve as a delivery vehicle for
biologics in the mammalian gut
• Purification cost are eliminated
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Global health: Triton’s products enable treatment of
disease through food products
Diarrhea is the largest public health problem in the
world
•
Leading cause of mortality, with 1.5 million deaths annually;
primarily in developing countries
Botanicals
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Botanicals address these
diseases
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Consumers or animals will
eat enhanced foods for
nutritional and health
benefits
Major issue for young children:
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33% of children under the age of five experience extreme
diarrhea at least once per year
750,000 children die each year from diarrheal disease
Especially an issue for women do not breastfeed; infant
formula ingredients do not address diarrhea
Animal Health
•
Dependency on antibiotics to reduce diarrhea
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Products: Triton has a pipeline of products
Botanicals
Highlights of current products
MAA
Osteopontin
• An active GI biologic, tested and proven in human and animals to
prevent GI disease and improve digestive function
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A phosphorylated biologic that naturally occurs in mammalian milk
Helps to increase bone fracture resistance
Prevents calcification
Plays a major role in neonatal immune development
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Why osteopontin as a health and nutrition product?
Low levels of
OPN in infant
formula and
bonvine milk
Scack et al. 2009 Considerable variation in the concentration of osteopontin in human milk, bovine milk and infant formula
Osteopontin (OPN): Phospho-protein with 36
phosphates on human osteopontin and 28
phosphates on bovine osteopontin
Chatterton et al. Anti-inflammatory mechanisms of bioactive milk proteins in the intestine of newborns. 2013
Human milk contains
significantly more OPN
than either bovine milk
or infant formula
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Osteopontin’s role in neonatal gut protection and
development
Function of Th-1 cells
• Enhance macrophage phagocytosis of
bacteria and protozoa
• Maximize the proliferation of CD8 T-cells
- Improve viral clearance
Arla Foods
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Expression and accumulation of osteopontin in
chloroplast of green algae
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Lane 1: Negative Control
Lane 2: Chloroplast ostoepontin 1
Lane 3: Chloroplast osteopontin 2
Lane 4: Chloroplast osteopontin 3
Lane 5: Chloroplast osteopontin 4
Lane 6: Chloroplast osteopontin 5
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Phosphorylation of osteopontin by chloroplast
Mass spec identification of Osteopontin Phosphorylation Sites
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Algae expressed Osteopontin is biologically active
CD4 Th1 cell
activation
%CD4 T cells
in culture
Osteopontin mediated Cell
Adhesion
Algal
OPN
None
nM algal osteopontin
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Trtion Conclusions
• Osteopontin is currently isolated from whey protein (30 metric tons of whey
to get 1kg of osteopontin >$24,000)
 To get 1kg of osteopontin from algae a company would only need to
produce 100kg of biomass
• Algae biotechnology opens up new opportunities in health and nutrition
• Advances made by algae cultivators can be applied to algal strains
producing high value biologics
• Trition is constantly seeking new partners, collaborators and opportunities
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Production of advance next generation targeted
antibody therapies
Chloroplasts as a unique biochemical environment
for protein production
• Chloroplasts house the photosynthetic apparatus
Maul et al. 2002
• Contains a single cup shaped chloroplast that comprises 4060% of the entire cell
• Sequenced genomes
• Has ribosomes and translation factors that resemble bacteria
• Chloroplast are a distinct biochemical compartment
Nield et al. 2011
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Chloroplasts contain protein disulfide isomerases
that are necessary for complex protein assembly
FD(red)
TR(red)
SH
PSI
SH
FD(ox)
Light
Translation-ON
cPDI
cPABP
HS
5’
SH
AUG
3’
TR(ox)
S
cPDI
PSII
Translation-Off
cPABP
S
S
S
5’
AUG
Pi
3’
eH2O
Kinase
[ADP]
Adapted from Kim and Mayfield 1997
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Chloroplasts contain chaperones that are necessary
for complex protein assembly
Schroda 2004 Photos. Res.
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in silico analysis of the C. reinhardtii genome showed
that chloroplasts contain a complete set of complex
chaperones including: Hsp100, HSp90, Hsp70, HSP40,
Cpn60, Cpn10/20, and SHSP
• Some chaperones appear to be of eukaryotic origin
 Hsp90, Hsp70
• Some appear to be of prokaryotic origin but contain
distinct domains
 Cpn60 (GroEL), Cpn10/20 (GroEs)
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The Unique biochemical environment of chloroplast
allow them to produce complex antibody toxin fusions
Antibody
Domain
Toxin
Domain
Becker and Benhar 2012
• The prokaryotic-like translational apparatus allows them to produce toxins capable of killing eukaryotic cells
• Complex chaperones and PDIs allow chloroplast to fold and assemble complex multiple domain proteins
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Algal chloroplasts can accumulate and assemble
antibody toxin fusions
Reduced
Non-reduced
Lane 1: wt total soluble
Lane 2: α-CD22scFV-Antibody
Lane 3: α-CD22scFV-Exotoxin A
Lane 4: α-CD22scFV-HCH23-Exotoxin
Tran et al. PNAS 2013
αFlag
αFlag
• Immunotoxins containing exotoxin A accumulate as
soluble molecules in algal chloroplasts
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Algal chloroplasts produced antibody toxins can
specifically target and kill cancer cells
Tran et al. PNAS 2013
Only targeted cancer cells are killed while normal healthy cells remained
unharmed
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Algal chloroplasts allow for the rapid production of
large numbers of antibody toxin fusions
Week
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Antibody genes
designed
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Antibody genes
ordered
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Antibody genes
weaponized
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Algae
transformed
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Transgeneic
strains
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Expression
confirmed
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Purification
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Cell Viability
Assay
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Dozens of antibodies can be weaponized in months for cancer therapies vs.
a few in years using traditional methods
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Coming of Age: Algae Chloroplast Expression Platform
1989:
First Algae
Chloroplast
Transformation
1997:
Identification of Protein
Disulfide Isomerase in
Chloroplast
2002:
Sequenced
Chloroplast
Genome
2004:
Identification of
Prokaryotic like elongation
factors in chloroplast
2004:
Chloroplast
Chaperones Identified
2007:
Chloroplast
ribosomes resemble
those in bacteria
2007:
High levels of protein
accumulation in
chloroplast
2012:
Chloroplast produce
antibody toxin fusions
2009
Chloroplast
Assembly of human
antibody
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Closing Remarks
• Chloroplast are capable of producing complex multiple domain proteins
• Chloroplast can produce a class of cancer therapies that no other
expression platform can
• Verdant has produced a large library of antibody toxin fusions
• Expression levels are still low (ug/L to mg/L)
• Verdant will focus on developing novel methodologies for increasing
recombinant protein accumulation and recovery
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Acknowledgements
Management Team
Dr. Stephen Mayfield
Dr. Jason Pyle
Nick Hofmesiter
Daniel Sachs
Funding Sources
NSF
NIH
California Energy commission
Scientific Team & Collaborators
Dr. Beth Rasala
Mike Mayfield
Austin Hallgren
Dr. Bill Juilien
Dr. Livia seno Ferreira Camargo
Dr. John Chang
Dr. Soo Ngoi
Dr. Jack Bui
Dr. Zhouxin Shen
Dr. Steve Briggs