Cardineau guy pep talk 11, jan 12,2012

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Text of Cardineau guy pep talk 11, jan 12,2012

  • Guy A. Cardineau, Ph.D.

    Higher Accumulation ofF1-V Recombinant Fusion Protein in Plants After Induction of Protein Body Formation

    Director, Centro de AgrobiotecnologaDepartamento Agrobiotecnologa y AgronegociosTecnolgico de Monterrey, Campus Monterrey

    ASASU Centennial Professor, EmeritusResearch Professor, Emeritus & Faculty FellowCenter for Infectious Disease and Vaccinology

    The Biodesign Institute,The School of Life Sciences and

    The Sandra Day OConnor College of LawArizona State University

  • Biotechnology Drug Approvals 1982-2008While the number of approved biotech-based products approved per year is variable, the trend is upward. Biotechnology drugs appear the fastest-growing sector for drug development, and it is predicted that biotech drugs will comprise over 50% of all drug approvals by 2015 and more than 75% by 2025. These predictions are supported by the expected benefits of increased understanding of drug targets and the molecular and genetic bases of disease, as well as the declining conventional small-molecule drug pipelines in most major pharma companies. BioWorld Today Sept 1,2009

    The table to the left represents information from an article published in BioWorld Today in late August 2009, written by Michael Harris,

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    late August 2009, written by Michael Harris, Executive Editor, about the top 25 biotech drugs currently on the market. The data provided includes revenues for each of these biotech drugs in 2008 (>$70B US), the date each drug product was first approved by the FDA and when patents protecting each drug are due to expire. It should be kept in mind that one feature of all these drugs is that they have been approved for more than one ndication; Harris reports that Genentech's Avastin is being tested in more than 450 clinical trials for treating more than 30 different types of cancer. It should also be kept in mind that 7 of the 25 "biotech" drugs are small molecules, and another 6 are antibodies.

  • Historically, Plants Have Been Routinely Used to Produce Pharmaceuticals, Naturally

    Global over-the-counter sales of plant-derived drugs are estimated at $40 billion per yearWell established regulatory systems are in place for these products

    Estimated one-quarter of the prescription drugs sold in the US, Canada and Europe contain active ingredients derived from plants

    Tens of thousands of plants are used for medicinal purposes

    Well established regulatory systems are in place for these products

    Drug/Chemical Action/Clinical Use Plant Source Cocaine Local anaesthetic Erythroxylum coca Codeine Analgesic Papaver somniferum Digitalin, Digitoxin Cardiotonic Digitalis purpurea Quinine Antimalarial Cinchona ledgeriana Taxol Antitumor agent Taxus brevifolia Vinblastine, Vincristine Antitumor, Antileukemic Catharanthus roseus

    SUMMARY from Large Scale Biology, Inc.

  • Hormones and immune modulators Monoclonal antibodies - IgG Subunit vaccines Enzymes

    Classes of New Protein Drug ProductsClasses of New Protein Drug Products

    Production Systems in UseProduction Systems in UseProduction Systems in UseProduction Systems in Use Bacterial fermentation Mammalian cells in fermentation Yeast Insect cells (GSKs 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 (GSKs cervical cancer vaccine; 2005/6) Green plants Stable and Transient Transformation,

    Whole Plants and Plant CellsOne approved product in the market in plant cells

  • Early Patent Filings on Plant Made

    Pharmaceuticals

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    Original Concepts of Therapeutic Protein,Vaccine Antigen, and Antibody Expression in Plants

  • Dow AgroSciences/ASU collaborationdeveloped a Newcastles Disease Virus subunit 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 plants AbstractThe invention is drawn towards vectors and methods useful for preparing genetically transformed plant cells that express immunogens 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 to methods of producing an antigen in a transgenic plant.

  • WHY ORALLY DELIVERED PLANT-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 with plant-made vaccines have been run resulting in positive immune responses.

  • WHY INCREASE F1-V FUSION PROTEIN ACCUMULATION IN PLANTS?

    Our primary objective is to produce plant-derived heat stable vaccines that can be delivered orally.

    We have been using F1-V, a fusion between two antigens from the plague bacterium Yersinia pestis, as our model antigen in production improvement studies.pestis, as our model antigen in production improvement studies.

    We are assessing parameters that affect expression of F1-V fusion protein in plants and plant cells to be used as both a production and delivery system of vaccines and potentially other biopharma proteins.

    High antigen accumulation is required to compensate for partial proteolysis in the gut upon oral delivery.

  • Protein accumulation in plant tissues reflects a balance between protein synthesis and degradation To date, most efforts have focused on increasing protein

    synthesis. enhanced transgene expression can be obtained by optimizing

    regulatory elements including stronger promoters, transcriptional enhancers, translational enhancers, alternative polyadenylation signals, using synthetic genes with codons that have been optimized for gene expression in target plants, overcoming RNAi and silencing

    Unfortunately, high transgene expression does not always guarantee high levels of recombinant protein accumulation since proteins may be expressed successfully but subsequently degraded.

    It has been demonstrated that post-synthesis and/or post-secretion instability and degradation are critical factors contributing to low foreign protein yield.

  • 25000

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    preboos tpos tboos t

    ANIMAL TRIALS: PRIME-BOOST STRATEGY

    PRIME: s.c. 15 g bacterially derived F1-V

    BOOST: 2 g non-transgenic tomato (n = 5) on days

    BOOST: 2 g F1-V transgenic tomato (n = 6) on days 21, 28, 35 (300 ug) and 42 (1200 ug)

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    F1-spec if ic IgG1 V -spec if ic IgG1

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    F1-specific IgG1 V-specific IgG10

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    Combined 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 days 21, 28, 35 and 42)

    F1-specific IgG1 V-specific IgG10

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    F1-specif ic IgG2 V-specif ic IgG2F1-specific IgG2a V-specific IgG2a

    CHALLENGE (s.c. 20 LD50 Y. pestis)

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    TGWT CONTROLS

    Challenge of the vaccinated mice

    with s.c. Y. pestisAlvarez & Cardineau

    Biotechnology Advances 2010, 28 (1): 184-196

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  • Protein accumulation in plant tissues reflects a balance between protein synthesis and degradation

    There are several possible sites and mechanisms of foreign protein degradation in plants. Cytoplasmic proteases contribute significantly to product losses within plant cells.

    Proteolytic degradation of foreign proteins can be minimized by targeting synthesis to the endoplasmic reticulum (ER) rather than the cytosol, but this doesnt always work. the cytosol, but this doesnt always work. ER retention of soluble transport-competent proteins is inducible by the

    carboxy-terminal retention/retrieval signal KDEL or HDEL, which is recognized by a receptor located in the Golgi complex.

    Upon binding, the receptor retrieves C-terminal tagged proteins back into the ER. Localization within the ER via the addition of KDEL or HDEL increases the accumulation of foreign proteins in transgenic plants.

    However, the ER retention via KDEL is mediated by a K