Planet Biotechnology, Inc. | Date: 2011-03-09
Immunoadhesins active against toxins and pathogens are described, with specific examples directed to immunoadhesins for thwarting pathogens such as anthrax and the common cold. The immunoadhesin-receptor ligand principle can be employed to counter virtually any pathogen, toxicant or toxin, including, e.g., natural and synthetic metabolic poisons.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 3.00M | Year: 2010
DESCRIPTION (provided by applicant): Planet Biotechnology Inc. (PBI) is developing a therapeutic recombinant protein, PBI-220, that neutralizes anthrax toxin in vitro and protects against inhalational anthrax in vivo. PBI uses transgenic plants as an innovative, economical and flexible production technology. Our therapeutic protein, a fusion of the high- affinity human anthrax toxin receptor CMG2 and the Fc portion of IgG, offers broad-spectrum protection against anthrax because it can neutralize not only wild-type anthrax toxin, but toxin forms that have been modified to avoid neutralization by monoclonal antibodies. We believe that PBI-220 may be the 'best in class' therapeutic in development for anthrax. We have demonstrated the in vitro toxin-neutralizing potency of PBI-220, using a standard assay, to be as high as the best anti-PA antibodies under development by Avanir, Elusys or Human Genome Sciences. A dose of PBI-220 as low as 2 mg/kg was sufficient to completely protect rabbits challenged with gt100 LD50 of B. anthracis spores. We have created transgenic tobacco plants that accumulate PBI-220 at commercially useful levels (gt75 mg/kg fresh weight of leaves) and are scaling up our purification procedure. We propose to advance the development of PBI-220 beyond efficacy testing in rabbits to efficacy testing in a non-human primate (NHP) model of inhalational anthrax. These experiments will determine the dose, route of administration and timing of PBI-220 treatment that will provide protection at the maximum delay after spore inhalation, during periods when antibiotic alone becomes ineffective. We will evaluate the pharmacokinetics of PBI-220 in NHP, to help determine appropriate dosing in people. We will conduct pilot toxicology studies in rats and NHP to demonstrate PBI-220's safety. All of these studies are designed to support eventual filing of an Investigational New Drug Application with FDA for PBI-220, and its licensure under the FDA Animal Rule . An important aspect of the product development this grant will support is optimization of our manufacturing process and implementation of a Quality System so that we can produce PBI-220 under current Good Manufacturing Practices. PUBLIC HEALTH RELEVANCE: The American public is vulnerable to a bioterrorist attack using Anthrax (Bacillus anthracis). We believe than an immunoadhesin, comprised of the human anthrax toxin receptor CMG2 and a human IgG or IgA Fc, can provide complete protection against anthrax after the development of symptoms, without the need for vaccination, and would allow the development of the body's own protective antibodies against Bacillus anthracis.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 276.77K | Year: 2013
DESCRIPTION (provided by applicant): Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism by which therapeutic monoclonal antibodies (mAbs) kill tumor cells. However, killing by current therapeutic IgG mAbs is not optimal. InefficientADCC provides a potential escape mechanism for tumors and can be targeted to improve antibody- based cancer therapies. Most of the tumor-directed mAbs used in clinical trials are human IgG1, which can activate complement and/or recruit NK cells for ADCC by binding to Fc RIIIa (CD16). Several groups have shown that IgA triggers potent ADCC by binding to the Fc RI (CD89) and recruiting neutrophils. Our goal is to create a new class of potent anti-tumor antibodies that can activate a wide variety of immune cell types bearing either the Fc RI or Fc?RIIIa. These novel chimeric heavy chains and associated light chains will be expressed in plants to produce antibodies with specific N-glycan structures and enhanced antibody dependent cellular cytotoxicity (ADCC) activity. Structural studies of IgA binding to the Fc?RI and IgG1 binding to the Fc?RIIIa suggest that a chimeric antibody containing both IgG1 and IgA domains, and bound to a tumor cell target, will bind and activate both receptors on immune effectorcells. As proof of concept we will link the Fab domains of an anti-HER2 antibody to a fusion of the Fc ?1 CH1-CH2-CH3 domains and Fc ?2 CH2-CH3 domains. IgG1-only and IgA2-only versions will be constructed as controls. The Fc of IgG1 is expected to conferthree benefits: (a) prolongation of serum half-life via FcRn binding; (b) purification using Protein A; and (c) augmented cellular recruitment and activation of natural killer (NK) cells via Fc? receptor binding. The IgA2 Fc is expected to confer augmented cellular recruitment and activation of polymorphonuclear cells (PMN) via Fc?RI binding. Recent studies have demonstrated the importance of IgG Fc glycosylation for Fc?R binding and maximizing ADCC. In particular, the absence of core ?1-6)-Fucose andthe presence of a bisecting N- acetylglucosamine (GlcNAc) residue each enhance Fc binding to Fc?RIIIa. By co-expressing our recombinant antibodies along with specific glycosyltransferases in a transgenic Nicotiana benthamiana background we will produce antibodies with N-glycan structures that are optimal for participating in ADCC. We will produce six variant antibody forms (IgA, IgG and chimeric IgG/A, each with two different N- glycosylation types), all bearing the same anti-HER-2/neu/c-Erb-B2 Fab region, using our plant expression system and evaluate their ability to direct ADCC in vitro against standard breast cancer cell lines. Our collaborators at the University of Pennsylvania will evaluate the ability of these recombinant antibodies to shrink HER2-expressing syngeneic tumors in transgenic mice expressing both HER2/neu and human Fc?RI. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: Despite significant gains in diagnosis and therapy, cancer remains the second leading cause of death in the US. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism by which therapeutic monoclonal antibodies (mAbs) kill tumor cells. However, killing by current therapeutic IgG mAbs is not optimal. Our goal is to create a new class of potent anti-tumor antibodies that can activate a wider variety of immune cell types and thus enhance ADCC activity.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 282.63K | Year: 2012
DESCRIPTION (provided by applicant): In this project we propose producing, and testing in vivo, a recombinant protein that should sustainably reduce plasma low-density lipoprotein cholesterol (LDL-C). Proprotein convertase subtilisin/kexin type 9 (PCSK9),a protein secreted in the liver, plays a key role in maintaining cholesterol homeostasis by regulating cell-surface levels of the low-density lipoprotein receptor (LDLR), a plasma membrane glycoprotein that removes LDL-C particles from the plasma. Levels of circulating PCSK9 in human populations vary over a gt100- fold range, and there is a positive correlation between PCSK9 levels and levels of LDL-C (and between PCSK9 and coronary artery disease events). Statins are widely prescribed as therapy for hypercholesterolemia (high cholesterol). A therapy that would down-regulate PCSK9 should act as an adjunct to statins in maintaining healthy levels of LDL-C, especially in individuals with familial hypercholesterolemia. A protein fragment containing the epidermal growth factor-like repeat AB (EGF-AB) domains of the LDLR binds to PCSK9 and blocks the ability of PCSK9 to deplete LDLR on hepatocytes. The EGF-AB fragment, however, is unstable in vivo, and thus impractical as a therapeutic. In this project we proposeto produce, and test in vivo, a stable decoy molecule incorporating EGF-AB, which should deplete plasma PCSK9 and sustainably reduce plasma LDL-C. Using a plant expression system we will produce a series of recombinant proteins comprised of EGF- AB variants fused to the Fc of human IgG1. One variant will incorporate the wild-type EGF-AB sequence, but the others will contain single amino acid mutations found in the LDLR of individuals with hypercholesterolemia (and some non-natural mutations). It is expected that some of these variants will bind more tightly to PCSK9 and thus better block its ability to deplete LDLR. The EGF-AB variants will first be assayed for their ability to interfere with the binding of labeled LDLR extracellular domain to PCSK9. The variants with the greatest activity will then be tested in a cellular LDL uptake assay to determine how well they inhibit the LDLR-lowering activity of exogenously added PCSK9. The two best EGF-AB-Fc variants will be tested for their ability to lower LDL-C in transgenic mice expressing elevated levels of human PCSK9. PUBLIC HEALTH RELEVANCE: Coronary artery disease is the leading cause of death worldwide. The primary causal factor in the pathogenesis of coronary artery disease is an elevated plasma level of low-density lipoprotein cholesterol (LDL-C). A receptor decoy based on a protein fragment of the low-density lipoprotein receptor fused to IgG Fc is expected to dramatically lower LDL-C and reduce the risk of coronary artery disease.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 225.00K | Year: 2014
DESCRIPTION (provided by applicant): Middle East respiratory syndrome coronavirus (MERS-CoV) is newly emerging human health threat with a more than 40% case fatality rate. The cell surface protein dipeptidyl peptidase 4 (DPP4) is used by MERS-CoV to enterand infect cells. Soluble recombinant human DPP4 binds the MERS-CoV spike (S) glycoprotein and inhibits MERS-CoV infection of VERO cells, but the concentration required to achieve 50% inhibition is fairly high. The aim of this project is to design, produceand test a superior inhibior of MERS-CoV infection using a fusion of DPP4 and the Fc of human immunoglobulin. We expect DPP4-Fc to have increased potency due to the stoichiometry of DPP4 in the Fc fusion (two DPP4 binding domains per molecule). DPP4-Fc isalso expected to have superior pharmacokinetics, as Fc will confer a long circulating half-life and the ability to be delivered to airway mucosal surfaces, the site of MERS-CoV infection. Unlike antibodies against MERS-CoV, a DPP4-Fc decoy will not
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 219.37K | Year: 2014
DESCRIPTION (provided by applicant): The goal of this research is to develop an economical, safe, effective therapy to reverse the activity of the catalytic component, LF, of the Bacillus anthracis toxin (lethal toxin), within the cytoplasm of the intoxicated cell. Success in achieving this goal will lead to better outcomes than currently seen with antibiotics or antitoxins like raxibacumab or PBI-220, and this could save countless more lives in the event of a bioterrorist attack with B. anthracis. We propose to screen the humanized camelid library to identify high-affinity single chain variable fragment, a nanobody, able to block protease activity of lethal factor (LF). We will then create genetic fusion of the nanobody that blocks LF with another domain of LF, LFN, that had the translocating activity buts is devoid of catalytic activity to enable it to be transported into the cytoplasm of mammalian cells in the presence of PA. Thus, we plan to co-opt the B. anthracis toxin delivery system to deliver,
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 224.13K | Year: 2014
DESCRIPTION (provided by applicant): There is a clear need, in plants, for an efficient and practical system for targeted mutagenesis. Planet Biotechnology's interest is in using the tobacco species Nicotiana benthamiana as a factory for the economical production of human therapeutic proteins, including a recombinant Fc fusion protein for treatment of inhalational anthrax. The CRISPR/Cas system could be used to modify numerous traits that interfere with this plant achieving its full potential as a therapeutic protein production system. This applicatio proposes the first attempt to use the CRISPR/Cas system for site-specific gene modification in plants. We intend to demonstrate a proof-of-concept by mutating fucosyltransferase (or fucosyltransferase and xylosyltransferase) in N. benthamiana, glycosyltransferases responsible for making plant-specific N-glycan residues. We will test two different CRISPR/Cas strategies. The first has been published and shown to work in human, zebrafish and Drosophila cell
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 225.00K | Year: 2014
DESCRIPTION (provided by applicant): Cell culture produced monoclonal antibodies (mAbs) have advantages over polyclonal antibodies derived from human or animal sera, such as reproducibility of production and enhanced safety profile. Although targeting multiple epitopes on cancer cells, viruses or toxins with multiple mAbs in combination has demonstrated superior therapeutic effects compared to mAb monotherapy, oligoclonal mAb therapy is not widely practiced partially because of the cost of separately producing and characterizing multiple mAbs for use in combination. In this proposal we will test a novel idea for producing an oligoclonal mixture of mAbs using a plant expression system. Plant viruses exhibit a phenomenon called superinfection exclusion, whereby a preexisting viral infection prevents a secondary infection with the same or a closely related virus. We propose to express multiple mAbs in a single plant simultaneously, exploiting the superinfection exclusion characteristic of plant virus ex
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 1.58M | Year: 2015
DESCRIPTION provided by applicant Middle East respiratory syndrome coronavirus MERS CoV is a newly emerging human health threat with a case fatality rate MERS CoV uses dipeptidyl peptidase DPP a cell surface protein to enter and infect cells During our Phase I study using a transient plant expression system we produced fusions of human DPP and the human immunoglobulin Fc sequences of IgG IgA and IgA to produce andquot receptor decoysandquot to block cellular infection with MERS CoV We demonstrated that DPP Fc binds to the S domain of MERS CoV S protein and that DPP Fc is a more potent inhibitor of MERS CoV cellular infection than soluble DPP We showed that a DPP Fc fusion based on IgA might possibly be more effective than one based on IgG In addition we demonstrated that binding and virus neutralization could be improved by more than fold by modifying a single amino acid where human DPP and MERS CoV spike protein contact DPP Fc is also expected to have superior pharmacokinetics as Fc will confer a long circulating half life and the ability to be delivered to airway mucosal surfaces the site of MERS CoV infection In a phase II study we will produce new DPP Fc constructs to improve DPP andapos s affinity for MERS CoV spike protein and eliminate DPP andapos s peptidase activity by mutating the active site We will optimize new constructs to improve expression in plants and to produce DPP Fc with human like N linked glycans New DPP Fc fusions will be ranked for MERS CoV spike protein binding by ELISA and tested for the ability to neutralize MERS CoV infection in susceptible cells The best performing DPP Fc variants will be tested for protective efficacy in a mouse MERS model evaluating both intraperitoneal and intranasal routes of administration We will scale up production and purification of our lead molecule to purify grams of DPP Fc which will be used for a pilot week repeat dose safety toxicology study in rats We will evaluate safety and quantify DPP Fc in serum to obtain pharmacokinetic parameters We will also screen for anti DPP Fc antibodies in these studies PUBLIC HEALTH RELEVANCE Middle East respiratory syndrome coronavirus MERS CoV also termed hCoV EMC was first identified in humans in in the Middle East To date known people have contracted MERS in countries resulting in deaths Epidemiology studies suggest human to human transmission of this deadly virus leading to global concern about a MERS pandemic We propose a novel therapeutic a recombinant protein comprised of the extracellular domain of DPP the MERS CoV cellular receptor fused to Fc of a human immunoglobulin e g IgG which could be used as a andquot receptor decoyandquot to block the interaction of MERS CoV with DPP on human cells and thus stop infection
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 236.85K | Year: 2011
DESCRIPTION (provided by applicant): The ultimate goal of the research described in this application is to develop an economical, safe and effective therapeutic that can reverse the paralytic symptoms of botulism. Success in achieving this goal could savecountless lives in the event of a bioterrorist attack by botulinum toxin. The goal of administering an anti-toxin antibody is rapid and complete removal of the toxin from the body. Currently available immunotherapies for botulism (equine antitoxin, human botulism immune globulin, monoclonal antibodies) can remove only toxin molecules that have not yet reached motor neurons. Once the catalytic light chain of the toxin reaches the cytosol of motor neurons, where it cleaves proteins involved in neurotransmission, it is inaccessible to normal antibodies. This is why victims of botulinum neurotoxin poisoning may need supportive care on a respirator for many weeks. We propose using a new single-chain variable fragment, a nanobody, against the catalytic site of thetoxin light chain, along with technology that allows proteins to penetrate into the cytosol of motor neurons, to reverse the paralysis caused by the toxin. We will start with a single-domain variable fragment, isolated from a llama antibody library in thelaboratory of our collaborator, Dr James Marks (UCSF), that inhibits the ability of the toxin light chain to cleave it's protein substrate. We will express fusions of this nanobody to various cell-penetrating peptides, using a plant expression system. Wewill then test the ability of these nanobody fusions to enter primary rat neuron cells and inhibit toxin activity. Finally, we will test the toxicity of the cell-penetrating nanobody in mice. PUBLIC HEALTH RELEVANCE: Botulinum toxin is the deadliestbiological substance known, and would be easy for a terrorist to produce and use to cause large numbers of casualties. This makes developing an effective and economical countermeasure to protect the public a public health priority. The research supportedby this grant will lay the groundwork for using plants to produce a highly protective, safe and economical antitoxin therapy.