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.

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.

Production

Modify sugarspost-productionTo target lysosomes

RTB delivers!

Purification

Standard plus Viral removal

Lactose affinity

I.V every two weeks

Nasal Spray

Delivery Method Administration

Mammalian (CHO) cell

Plant

Recombinant enzymes produced in CHO cells

RTB:IDUAproduced in plants

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

RTB and RTB:ERT fusions are efficiently produced in plant-based bioproduction platforms providing cost-efficient bioproduction and scalability. Other innovations supported include:

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 CHO-

based 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.

Abstract

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.)

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.

ERTRTB

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

The RTB lectin – a potential carrier for lysosomal ERTs

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

Why plants?

Plant-based bioproduction of RTB and human iduronidase fusion proteins.

RTB retains lectin activity when fused to IDUA

IDUA shows enzymatic activity when fused to RTB

• 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.

• 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 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 column2. Flow through after incubation3. Elution 14. Elution 2

1 2 3 4150 kD

100 kD

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.

HT-29 epithelial cells incubated with fluorescent dyed RTB

HT-29 epithelial cells incubated with fluorescent dyed RTB: IDUAHIS

T=120 min T=120 minRTB:IDUAHIS cRTB

Wt IDUA cDNA Opt IDUA cDNA

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.

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).

Harvest plant leaves

48-72-96 hrs

A. tumefaciensA. tumefaciensA. tumefaciensA. tumefaciens

Purify protein

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).

Plant-based bioproduction of RTB and human iduronidase fusion proteins.

Correction of Hurler cells following incubation with RTB:IDUAHIS fusion protein.•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

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

Hurler patient

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.