1. Guy A. Cardineau, Ph.D.Higher Accumulation ofF1-V Recombinant FusionProtein in Plants AfterInduction of Protein BodyFormationDirector, Centro de AgrobiotecnologíaDepartamento Agrobiotecnología y AgronegociosTecnológico de Monterrey, Campus MonterreyASASU Centennial Professor, EmeritusResearch Professor, Emeritus & Faculty FellowCenter for Infectious Disease and VaccinologyThe Biodesign Institute,The School of Life Sciences andThe Sandra Day O’Connor College of LawArizona State University
2. Biotechnology Drug Approvals 1982-2008While the number of approved biotech-based productsapproved per year is variable, the trend is upward.Biotechnology drugs appear the fastest-growing sector fordrug development, and it is predicted that biotech drugs willcomprise over 50% of all drug approvals by 2015 and morethan 75% by 2025. These predictions are supported by theexpected benefits of increased understanding of drug targetsand the molecular and genetic bases of disease, as well asthe declining conventional small-molecule drug pipelines inmost major pharma companies. BioWorld Today Sept 1,2009The table to the left represents informationfrom an article published in BioWorld Today inlate August 2009, written by Michael Harris,2late August 2009, written by Michael Harris,Executive Editor, about the top 25 biotechdrugs currently on the market. The dataprovided includes revenues for each of thesebiotech drugs in 2008 (>$70B US), the dateeach drug product was first approved by theFDA and when patents protecting each drugare due to expire. It should be kept in mindthat one feature of all these drugs is that theyhave been approved for more than onendication; Harris reports that GenentechsAvastin is being tested in more than 450clinical trials for treating more than 30 differenttypes of cancer. It should also be kept in mindthat 7 of the 25 "biotech" drugs are smallmolecules, and another 6 are antibodies.
3. Historically, Plants Have Been Routinely Used toProduce Pharmaceuticals, NaturallyGlobal over-the-counter sales of plant-derived drugs are estimatedat $40 billion per yearWell established regulatory systems are in place for these productsEstimated one-quarter of the prescription drugs sold in theUS, Canada and Europe contain active ingredients derived fromplantsTens of thousands of plants are used for medicinal purposesWell established regulatory systems are in place for these productsDrug/Chemical Action/Clinical Use Plant SourceCocaine Local anaesthetic Erythroxylum cocaCodeine Analgesic Papaver somniferumDigitalin, Digitoxin Cardiotonic Digitalis purpureaQuinine Antimalarial Cinchona ledgerianaTaxol Antitumor agent Taxus brevifoliaVinblastine, Vincristine Antitumor, Antileukemic Catharanthus roseusSUMMARY from Large Scale Biology, Inc.
4. • Hormones and immune modulators• Monoclonal antibodies - IgG• Subunit vaccines• EnzymesClasses of New Protein Drug ProductsClasses of New Protein Drug ProductsProduction Systems in UseProduction Systems in UseProduction Systems in UseProduction Systems in Use• Bacterial fermentation• Mammalian cells in fermentation• Yeast• Insect cells (GSK’s cervical cancer vaccine; 2005/6)• Green plants – Stable and Transient Transformation,Whole Plants and Plant CellsOne approved product in the market in plant cells• Bacterial fermentation• Mammalian cells in fermentation• Yeast• Insect cells (GSK’s cervical cancer vaccine; 2005/6)• Green plants – Stable and Transient Transformation,Whole Plants and Plant CellsOne approved product in the market in plant cells
6. Dow AgroSciences/ASU collaborationdeveloped a Newcastles Disease Virussubunit vaccine in tobacco NT1 cells.United States Patent 7,132,291, Cardineau, et al., November 7, 2006 (Canadian counterpart CA 2524293)Vectors and cells for preparing immunoprotective compositions derived from transgenic plantsAbstractThe invention is drawn towards vectors and methods useful for preparing genetically transformed plant cells that expressimmunogens from pathogenic organisms which are used to produce immunoprotective particles useful in vaccine preparations. Theinvention includes plant optimized genes that encode the HN protein of Newcastle Disease Virus. The invention also relates tomethods of producing an antigen in a transgenic plant.
7. WHY ORALLY DELIVEREDPLANT-MADE VACCINES?Plant-derived vaccines are cost-effective andstable at room temperature.Plants provide both an encapsulated antigenand an oral delivery system that stimulatesthe mucosal immune system resulting in bothsecretory and circulating antibodies.The mucosal immune system is the first lineof defense against most pathogens.Oral vaccines are potentially safer, require noneedles and may not require trained medicalpersonnel to administer.Several Phase I Human Clinical Trials withplant-made vaccines have been run resultingin positive immune responses.
8. WHY INCREASE F1-V FUSION PROTEINACCUMULATION IN PLANTS?Our primary objective is to produce plant-derived heatstable vaccines that can be delivered orally.We have been using F1-V, a fusion between twoantigens from the plague bacterium Yersiniapestis, as our model antigen in productionimprovement studies.pestis, as our model antigen in productionimprovement studies.We are assessing parameters that affect expressionof F1-V fusion protein in plants and plant cells to beused as both a production and delivery system ofvaccines and potentially other biopharma proteins.High antigen accumulation is required to compensatefor partial proteolysis in the gut upon oral delivery.
9. Protein accumulation in plant tissues reflects abalance between protein synthesis and degradation• To date, most efforts have focused on increasing proteinsynthesis.– enhanced transgene expression can be obtained by optimizingregulatory elements including stronger promoters, transcriptionalenhancers, translational enhancers, alternative polyadenylation signals,using synthetic genes with codons that have been optimized for geneexpression in target plants, overcoming RNAi and silencing• Unfortunately, high transgene expression does not alwaysguarantee high levels of recombinant protein accumulationsince proteins may be expressed successfully butsubsequently degraded.• It has been demonstrated that post-synthesis and/or post-secretion instability and degradation are critical factorscontributing to low foreign protein yield.
10. 250003000035000preboostpostboostANIMAL TRIALS: PRIME-BOOST STRATEGYPRIME: s.c. 15 µgbacterially derivedF1-VBOOST: 2 g non-transgenictomato (n = 5) on daysBOOST: 2 g F1-V transgenictomato (n = 6) on days21, 28, 35 (300 ug) and 42(1200 ug)[Ug/ml]250300350preboostpostboost[Ug/ml]05000100001500020000250003000035000F1-specific IgG1 V -specific IgG1preboostpostboost[Ug/ml]F1-specific IgG1 V-specific IgG1050100150200250300350F1-specific IgG2 V-specific IgG2preboostpostboost[Ug/ml]F1-specific IgG1 V-specific IgG105000100001500020000F1-specific IgG1 V -specific IgG1Combined F1-V and V-specific IgG1 titerscorrelate with protection in mouse model(Williamson et. al., Clin. Exp.Immunol., 1999, 116; 107-114.)tomato (n = 5) on days21, 28, 35 and 42)F1-specific IgG1 V-specific IgG1050100150200F1-specific IgG2 V-specific IgG2F1-specific IgG2a V-specific IgG2aCHALLENGE (s.c. 20 LD50 Y. pestis)01020304050607080901000 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25Days post-infection%survivalTGWTCONTROLSChallenge of thevaccinated micewith s.c. Y. pestisAlvarez & CardineauBiotechnology Advances2010, 28 (1): 184-196%ofsurvivalDays post-infectionCHALLENGE (s.c. 20 LD50 Y. pestis)
12. Protein accumulation in plant tissues reflects abalance between protein synthesis and degradation• There are several possible sites and mechanisms of foreignprotein degradation in plants. Cytoplasmic proteases contributesignificantly to product losses within plant cells.• Proteolytic degradation of foreign proteins can be minimized bytargeting synthesis to the endoplasmic reticulum (ER) rather thanthe cytosol, but this doesn’t always work.the cytosol, but this doesn’t always work.– ER retention of soluble transport-competent proteins is inducible by thecarboxy-terminal retention/retrieval signal KDEL or HDEL, which isrecognized by a receptor located in the Golgi complex.– Upon binding, the receptor retrieves C-terminal tagged proteins back into theER. Localization within the ER via the addition of KDEL or HDEL increasesthe accumulation of foreign proteins in transgenic plants.– However, the ER retention via KDEL is mediated by a KDEL receptor.When the receptor is saturated with KDEL ligands, the KDEL-taggedrecombinant protein either secretes or is transported to the lytic vacuole
13. Protein accumulation in plant tissues reflects abalance between protein synthesis and degradation• Some KDEL-tagged recombinant protein can be also misfoldedand delivered for degradation through an ER-dependentmechanism named ‘‘unfolded protein response’’ or UPR, whichfunctions for both endogenous or heterologous proteins• The K/HDEL system is common to all eukaryotes, but plants canuse a different ER localization system in seeds consisting ofspecialized organelles called protein bodies (PB), which stablyspecialized organelles called protein bodies (PB), which stablyaccumulate seed storage proteins within the ER.• The maize 27 kD γ-zein seed protein is not secreted even thoughit bears an N-terminal signal sequence and lacks a canonicalKDEL/HDEL ER-retention signal; it is able to form ER-localizedPB not only in maize endosperm but also when expressed instorage or vegetative tissues of transgenic Nicotiana tabacum,Hordeum vulgare and Arabidopsis thaliana plants, respectively.• PB formation can lead to higher protein accumulation in the ERpossibly because of the exclusion from the normal ER turnover
14. The Rules of Science
15. WHAT IS ZERA®®®®?Cereal grains have evolved to store large amounts of proteins:γ-Zein is the major storage protein in maize.Zera® (γ-Zein ER-accumulating domain) is the N-terminalproline-rich domain of γ-zein that is sufficient to induce theassembly of protein bodies.Zera® adopts an extended helix conformation where polarZera® adopts an extended helix conformation where polarresidues (histidines) are located on one side of the helix andhydrophobic residues (leucines and valines) on the oppositeside of the helix.This conformation provides high solubility in aqueous mediaand the ability to self-assemble both in hydrophobic andhydrophilic environments.
16. The Zera® domain retains its ability to developprotein bodies after being fused with an exogenousprotein of interest.Zera® contains two targeting signals:ZERA®®®® PROTEIN BODIESOrganelles surrounded by a membrane derived from the ER.Organelles surrounded by a membrane derived from the ER.Zera® contains two targeting signals:1- A signal peptide that internalizes Zera® fusionprotein inside the ER2- The Zera® domain itself that oligomerizes coatingthe ER membrane and inducing the protein bodyformation.
18. The benefits of Zera®®®® induced protein bodies(PBs)Zera® fusion proteins inside PBs escape the ER degradationpathway allowing higher accumulation rates.The accumulation of the Zera® fusion proteins in PBs alsoprotect the plant cell from toxic proteins.protect the plant cell from toxic proteins.Post-translational modifications of Zera® fusion proteins insidePBs: ER classical processing (N-glycosylation). Absence ofGolgi complex glyco-modifications.The easy isolation of the protein body-like organelles makesthem an extraordinary enrichment tool.
21. NT1 TRANSFORMATION:Zera®®®®-F1-V vs. F1-V3-week oldSelection of thehealthiest NT1 calli3-week oldcalliLiquid culture ofNT1 cellsFreeze-driedNT1 cell culture.Selection of the elitelines by Western-blot
29. CONCLUSIONSThe F1-V fusion protein accumulation in NT1 cells andalfalfa was at least 3X higher using Zera® technology.The accumulation of F1-V in ER-derived PB-like structuresinduced by Zera® was confirmed by EM.The regeneration of alfalfa or NT1 calli expressing Zera®-F1-V was delayed compared to F1-V likely due to the PB-like formation.These results confirm the potential of Zera® technology asa strategy to increase value-added proteins in plants.
39. AcknowledgementsBoyce ThompsonBoyce ThompsonBoyce ThompsonBoyce ThompsonInstituteInstituteInstituteInstituteDanDanDanDan KlessigKlessigKlessigKlessigJoyce Van EckJoyce Van EckJoyce Van EckJoyce Van EckTishTishTishTish KeenKeenKeenKeenXiurenXiurenXiurenXiuren ZhangZhangZhangZhangWendyWendyWendyWendy VonhofVonhofVonhofVonhofJasonJasonJasonJason EibnerEibnerEibnerEibnerNoreneNoreneNoreneNorene BuehnerBuehnerBuehnerBuehnerBryan MaloneyBryan MaloneyBryan MaloneyBryan MaloneyArizona State University >>Arizona State University >>Arizona State University >>Arizona State University >> Lucrecia AlvarezAmanda Walmsley >Federico MartinDwayne Kirk >Emel TopalYuguang Jin Heidi PinyerdJacki Kilbourne Jason CrisantesAaron Hicks Manuela RiganoDavid Julovich Michael EwingJulia Pinkhasov Angela RojasEric Chandler Amber GustinLuca Santi Deborah PauleyHugh Mason Jilliane MillerBenchmarkBenchmarkBenchmarkBenchmark BiolabsBiolabsBiolabsBiolabsMatt FantonTim MillerDow AgroSciencesDow AgroSciencesDow AgroSciencesDow AgroSciencesSteve WebbSteve WebbSteve WebbSteve WebbChuck MihaliakChuck MihaliakChuck MihaliakChuck MihaliakJennifer RiceJennifer RiceJennifer RiceJennifer RiceButch MercerButch MercerButch MercerButch MercerUniversity of ArizonaChieri KubotaRyo Matsudo4040Hugh Mason Jilliane MillerCharles Arntzen Andrew KoonsEssential Sponsors and CollaboratorsEssential Sponsors and CollaboratorsEssential Sponsors and CollaboratorsEssential Sponsors and CollaboratorsArizona State UniversityArizona State UniversityArizona State UniversityArizona State University BiodesignBiodesignBiodesignBiodesign Institute at ASUInstitute at ASUInstitute at ASUInstitute at ASUCornell U. Dept. of Food ScienceCornell U. Dept. of Food ScienceCornell U. Dept. of Food ScienceCornell U. Dept. of Food Science DowDowDowDow AgroSciencesAgroSciencesAgroSciencesAgroSciencesBenchmarkBenchmarkBenchmarkBenchmark BiolabsBiolabsBiolabsBiolabs US Department of DefenseUS Department of DefenseUS Department of DefenseUS Department of DefenseUniversity of ArizonaUniversity of ArizonaUniversity of ArizonaUniversity of Arizona Science Foundation ArizonaScience Foundation ArizonaScience Foundation ArizonaScience Foundation ArizonaTecnologicoTecnologicoTecnologicoTecnologico de Monterreyde Monterreyde Monterreyde Monterrey FondosFondosFondosFondos ZHZHZHZHFEMSAFEMSAFEMSAFEMSAButch MercerButch MercerButch MercerButch MercerTec de MonterreyTec de MonterreyTec de MonterreyTec de MonterreyTec de MonterreyTec de MonterreyTec de MonterreyTec de MonterreyIsrael RamirezIsrael RamirezIsrael RamirezIsrael RamirezIsrael RamirezIsrael RamirezIsrael RamirezIsrael Ramirez Cecy GarciaCecy GarciaCecy GarciaCecy GarciaCecy GarciaCecy GarciaCecy GarciaCecy Garcia Andrea MartinezAndrea MartinezAndrea MartinezAndrea MartinezAndrea MartinezAndrea MartinezAndrea MartinezAndrea Martinez Jose Manuel AguilarJose Manuel AguilarJose Manuel AguilarJose Manuel AguilarJose Manuel AguilarJose Manuel AguilarJose Manuel AguilarJose Manuel AguilarValeria LobosValeria LobosValeria LobosValeria LobosValeria LobosValeria LobosValeria LobosValeria Lobos Veronica RochaVeronica RochaVeronica RochaVeronica RochaVeronica RochaVeronica RochaVeronica RochaVeronica Rocha Federico LopezFederico LopezFederico LopezFederico LopezFederico LopezFederico LopezFederico LopezFederico Lopez Sergio GarciaSergio GarciaSergio GarciaSergio GarciaSergio GarciaSergio GarciaSergio GarciaSergio Garcia EchauriEchauriEchauriEchauriEchauriEchauriEchauriEchauriCarlosCarlosCarlosCarlosCarlosCarlosCarlosCarlos OrigelOrigelOrigelOrigelOrigelOrigelOrigelOrigel Javier GarciaJavier GarciaJavier GarciaJavier GarciaJavier GarciaJavier GarciaJavier GarciaJavier Garcia Jesus HernandezJesus HernandezJesus HernandezJesus HernandezJesus HernandezJesus HernandezJesus HernandezJesus Hernandez Ricardo Camilo ChavezRicardo Camilo ChavezRicardo Camilo ChavezRicardo Camilo ChavezRicardo Camilo ChavezRicardo Camilo ChavezRicardo Camilo ChavezRicardo Camilo ChavezPaulina CalderonPaulina CalderonPaulina CalderonPaulina CalderonPaulina CalderonPaulina CalderonPaulina CalderonPaulina Calderon Cristina MoralesCristina MoralesCristina MoralesCristina MoralesCristina MoralesCristina MoralesCristina MoralesCristina Morales JoharisJoharisJoharisJoharisJoharisJoharisJoharisJoharis SalgadoSalgadoSalgadoSalgadoSalgadoSalgadoSalgadoSalgado Gonzalo MendozaGonzalo MendozaGonzalo MendozaGonzalo MendozaGonzalo MendozaGonzalo MendozaGonzalo MendozaGonzalo MendozaMiguel Angel OrtizMiguel Angel OrtizMiguel Angel OrtizMiguel Angel OrtizMiguel Angel OrtizMiguel Angel OrtizMiguel Angel OrtizMiguel Angel Ortiz Cesar OrtizCesar OrtizCesar OrtizCesar OrtizCesar OrtizCesar OrtizCesar OrtizCesar Ortiz Axel GomezAxel GomezAxel GomezAxel GomezAxel GomezAxel GomezAxel GomezAxel Gomez MiguelMiguelMiguelMiguelMiguelMiguelMiguelMiguel SuasteguiSuasteguiSuasteguiSuasteguiSuasteguiSuasteguiSuasteguiSuastegui