Molecular Pharming: a renewable resource for bioproduction
of high value therapeutic proteins.
Acosta, Walter; Ayala, Jorge; Dolan, Maureen; Cramer, Carole.
Arkansas Biosciences Institute at Arkansas State University, P.O. Box 639, State University, AR 72467.
Plant-based bioproduction of RTB and human iduronidase fusion proteins.
Abstract
Producing therapeutic proteins in plants has many economic and environmental advantages. Most complex human recombinant proteins for
pharmaceutical applications are currently produced in mammalian cell culture systems, which have issues of safety, cost, and scale. Plant-based
bioproduction of human pharmaceutical proteins addresses these issues and provides a “green” renewable source of safe bioactive human therapeutics.
Our research is focused on integrating the advantages of plant-based protein bioproduction with novel strategies to deliver the pharmaceutical protein to
target cells and compartment within the cells. The delivery of bioactive molecules into cells and organelles remains a challenge for many therapeutic
applications including enzyme replacement therapeutics (ERTs) for lysosomal storage disorders (LSD). The plant lectin RTB, mediates endocytotic uptake
into mammalian cells and trafficking to lysosomes or ER of associated proteins or ‘payloads”. In order to test the potential of RTB to deliver lysosomal
ERTs, RTB (plant protein) was genetically fused to human L-alpha-iduronidase (IDUA), the enzyme that is deficient in Hurler Disease patients. Gene
constructs encoding RTB:IDUA were developed and expressed in leaves of Nicotiana benthamiana plants using an Agrobacterium-mediated transient
expression system. The recombinant RTB:IDUA fusion products were purified and characterized for RTB lectin activity, human IDUA enzyme activity, and
the ability to direct uptake into human cells. In order to determine whether RTB effectively delivers bioactive IDUA enzyme to the site of disease substrate
accumulation, Hurler patient fibroblast were treated with RTB:IDUA and analyzed for glycosaminoglycan (GAG) content compared with non treated cells.
Results of these studies support strategies to use plant-based bioproduction of these RTB:human enzyme therapeutics to provide replacement enzymes
that address key delivery and targeting issues as well as providing a safe, energy efficient and scalable commercial production platform.
Why plants?
Currently, patients are treated by enzyme replacement therapy that uses recombinant enzymes expressed in Chinese hamster ovary (CHO) cells. Plant
production systems offer certain advantages for ERT like :
• Safety: unlike mammalian cell culture systems, plant cells do not support growth of human pathogens
• Generation of bioactive human lysosomal enzymes : plants have all the machinery to perform the post-translational modification needed for correct
functioning of the protein.
• Capitalization cost: The investment for plant bioproduction system is much lower than conventional mammalian cell bioreactors.
• Scalability: The availability of adequate product supply to patient populations is currently limited by bioreactor capacity; scale-up of plant
bioproduction is simple and cost effective.
Our goal is to address these plant-bioproduction advantages though integration of novel delivery technologies for ERT based on the RTB lectin
The RTB lectin – a potential carrier for lysosomal ERTs
RTB is the non-toxic carbohydrate-binding subunit of ricin
• Plant-based expression of recombinant RTB (rRTB) alone yields selective lectin binding activity without the toxicity of
RTA, the ribosome inactivating subunit.
RTB enters cells by multiple routes
• RTB binds to galactose/galactosamine-containing glycoproteins and glycolipids, triggering absorptive endocytosis into
many cell types
• RTB has mannose-terminated glycans and enters cells via high-mannose or MMR receptors
• RTB delivers transmucosally
RTB trafficking
RTB traffics via multiple pathways within the cell
• The majority of RTB is delivered to endosomal/lysosomal compartments.
• RTB mediates transcytosis and traverses multiple cell layers
• A small proportion of RTB moves retrograde to ER. (This is the route used to delivery the RTA
toxin to the cytosol for cells exposed to active ricin toxin.)
ERT
RTB
Harvest plant
leaves
48-72-96 hrs
Purify
protein
Western blot analysis of plant leaves crude extracts using
IDUA monoclonal IDUA-specific antibody. Plants were
infiltrated with Agrobacterium containing an empty
vector as a negative control (pBibKan). IDUAHIS and RTB:
IDUAHIS are well expressed in plant transient expression
system providing product of the expected sizes (78 KD
and 112 kD respectively).
Western blot analysis of plant crude extracts using
monoclonal IDUA-specific antibody. Plants were infiltrated
with Agrobacterium containing an empty vector as a
negative control (pBibKan). Expression of the RTB: IDUAHIS
fusion is significantly higher using the codon optimized
version (RTB:Opt IDUA) compared to the native sequence
(RTB:Wt IDUA).
RTB retains lectin activity when fused to IDUA
• RTB is a galactose/galactosamine lectin that can be efficiently purified using
lactose-affinity chromatography. Recovery of RTB:IDUAHIS by lactose affinity was
used to demonstrate RTB lectin activity.
• Crude cell-free extracts of tobacco leaves transiently expressing RTB:IDUAHIS
were incubated with a lactose resin. Column washing removed unbound proteins
and bound proteins were eluted using a high concentration D-galactose solution.
1
2
3
4
150 kD
100 kD
RTB: IDUAHIS protein purification by lactose affinity column.
Western blot analysis using IDUA monoclonal IDUA specific antibody. Fractions were collected
during purification for SDS analysis.
1. Plant crude extract from leaves before loading the column
2. Flow through after incubation
3. Elution 1
4. Elution 2
IDUA shows enzymatic activity when fused to RTB
• Enzymatic activity of α-L-iduronidase activity was determined in a fluorometric enzyme assay measuring
4-MU released by hydrolysis of the fluorogenic substrate 4-methylumbelliferyl-alpha-L-iduronide (4-MUI).
• Samples were based on volume in crude extract – differences reflect expression levels but assay
demonstrates both IDUA and RTB:IDUA are active.
• Activity was undetected in extracts from leaves infiltrated with an “empty” pBIB-Kan vector used as a
negative control.
RTB:IDUAHIS mediates uptake into mammalian cells
• RTB:IDUAHIS was purified using lactose-affinity and nickel-affinity
chromatography.
• Purified product was fluorescently labeled with NHS-fluorescein.
• Human epithelial cell (HT-29) were incubated with the fluorescent
dyed protein. After 120 min of incubation, clear punctuate
structures are observed indicating internalization and accumulation
in endosomal compartments.
RTB:IDUAHIS
T=120 min
HT-29 epithelial cells incubated with fluorescent
dyed RTB: IDUAHIS
cRTB
T=120 min
HT-29 epithelial cells incubated with fluorescent
dyed RTB
Correction of Hurler cells following incubation with RTB:IDUAHIS fusion protein.
Plant-based bioproduction of RTB and human iduronidase fusion proteins.
Gene constructs
• RTB sequence was fused to the N-terminus of the mature IDUA. Plant signal peptide (Patatin) was used to direct the protein through the secretory pathway.
Preliminary data shows that IDUA expression in plants is relatively low. Sequence was codon optimized for expression in tobacco
Wt IDUA cDNA
A. tumefaciens
Transient expression
• Gene constructs were inserted in the T-DNA region of a plant transformation pBIB-Kan vector.
• Agrobacterium tumefaciens (LBA4404) was transformed with pBIB-Kan vector containing each construct.
• Nicotiana benthamiana plants were vacuum infiltrated with a culture containing the transformed Agrobacterium.
• Plant tissue was harvested 72 hours after infiltration.
Opt IDUA cDNA
Hurler patient
•Proper turnover of glycosaminoglycans (GAGs) is ensured by lysosomal degradation mediated by
specific acid hydrolyses like IDUA. IDUA deficiency leads to build up of GAG within the lysosomes. GAG
content was measured based on the absorbance at 490nm after complexing with sulphated GAG with
dimethyl-methylene blue (DMB) .
•Hurler fibroblast and normal fibroblast (Coriell Institute) were grown to confluence in media
supplemented with 10 ng/ml of the different proteins. Fibroblasts were harvested, washed and stored
at -20. Pellets were dried, weighed, and resuspended in a buffer and digested overnight at 65°C with
papain (0.3%).
•The GAG concentration was determined in supernatants by measuring the absorbance at 490nm
after addition of DMB dye solution using bovine heparan sulfate as standard.
Non-treated fibroblast
Treated Hurler fibroblast
Storage correction in cells of patients suffering MPS I
• Commercial RTB (cRTB; Vector Labs) was used to test the effect of RTB molecule in GAG
concentration (negative control).
• RTB:IDUAHIS was purified using lactose column affinity. Protein concentration was
quantified using 4-MUI with a rhIDUA standard curve.
• Extracts from plants, infiltrated with an empty vector (PBibKan), were purified using the
same purification protocol and extracts were used as negative control.
• Hurler fibroblast were treated with recombinant human alpha-L-IDUA (rhIDUA ) from
murine myeloma cells (R&D Systems) as a positive control.
RTB and RTB:ERT fusions are efficiently produced in plant-based bioproduction platforms providing cost-efficient bioproduction and scalability. Other innovations supported include:
Production
Mammalian (CHO)
cell
Plant
Purification
Standard plus
Viral removal
Lactose affinity
Delivery Method
Modify sugars
post-production
To target lysosomes
RTB delivers!
Administration
I.V every
two weeks
Nasal Spray
Recombinant
enzymes
produced in CHO
cells
RTB:IDUA
produced in
plants
New administration routes for ERT therapy. RTB mediates trans-mucosal delivery greatly expanding patient treatment options (e.g., inhalational administration).
Significant expansion of repertoire of cell types receiving ERT. RTB has high affinity for cell types important in LSD pathology using both lectin-mediated and high-mannose receptor routes. This technology would
expand cell types and tissues receiving therapeutic doses compared to current treatments limited to cells with significant surface MMR or mannose-6P receptors (M6PR).
Efficient delivery to lysosomes. The majority of endocytosed RTB naturally traffics to lysosomes eliminating the need for ERT glycan modifications for efficient lysosome delivery.
Safe plant-based bioproduction of lysosomal enzymes. Plant cells cannot support human viruses eliminating human health and commercial risks of adventitious viral contamination that has recently plagued CHObased imiglucerase production leading to production shut-down and unmet patient needs.
Toxin-free RTB production. The delivery potential of RTB has been recognized by others but purification from natural plant sources carries the potential of residual ricin toxicity. We developed plant-based systems
for high-level production of recombinant RTB and RTB fusions that eliminate any issues of toxicity.
Poster presented at the 22nd National NSF EPSCoR Conference . Research was funded by an ASU Arkansas Biosciences Institute grant to C. Cramer and M. Dolan and a Molecular Bioscience Program fellowship to W. Acosta