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: 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 II | Award Amount: 797.96K | Year: 2010
DESCRIPTION (provided by applicant): Planet Biotechnology Inc. (PBI) has pioneered methods for the production of functional therapeutic antibodies and immunoadhesins in transgenic tobacco. For reasons that are unclear, the yield of antibodies in transgenic plants has generally been low, with most published literature citing maximum expression of IgG in transgenic tobacco well below 30 mg/kg. We have designed and investigated the expression in plants of human IgG1 variants using DNA sequences in which there are over- or under-represented codon pairs. By utilizing an IgG heavy chain constant region containing many over-represented codon pairs we were able to significantly enhance transient expression of a number of antibodies (up to 35-fold higher than with our codon usage optimized sequence). Furthermore, we have generated stable tobacco plants expressing up to 515 mg IgG/kg fresh weight. Evidence suggests that the more over-represented a codon pair is, the more slowly it is translated. Local translational pausing might provide a time delay, enabling sequential folding of defined portions of nascent polypeptides, which may prevent protein misfolding and aggregation, and reduce degradation. We will test the hypothesis that translation is attenuated at over-represented codon pairs, and that translational pauses at particular positions are necessary for efficient co-translational folding and assembly of immunoglobulins in plants. To determine whether the expression benefit of the presence of many over-represented codon pairs is particular to IgG heavy chain in plants, or whether it is more widely applicable, sequences will be designed and synthesized for a small protein (J chain) and a larger multi-domain protein (IgA2 heavy chain). Furthermore, the IgG1 heavy chain, IgA2 heavy chain and J Chain sequences will be designed for expression in tobacco, in which the positions of over-represented codon pairs in the expression host are harmonized with those in the native host. The expression-competence of these sequences will be established using a tobacco transient expression system, after which stable transgenic tobacco plants will be generated. Specific transcript accumulation levels will be measured by real-time PCR and compared to protein accumulation levels. Endogenous BiP mRNA levels (a molecular marker of the unfolded protein response) will be quantified by real- time PCR in tobacco plants expressing the genes described above. 35S-pulse-chase labeling assays will determine the influence of over- or under-represented codon pairs on protein translation and turnover rates of the genes described within the grant. If successful, the result will be a general platform technology for the quick, easy, scalable production of therapeutic human antibodies in large quantities from plants (and possibly other eukaryotic expression hosts). PUBLIC HEALTH RELEVANCE: Planet is developing tobacco-produced recombinant proteins against several diseases including: intoxication by Botulinum Neurotoxin A (BoNT/A), the most potent toxin known and a bio-terror agent, the common cold, dental caries and intoxication by Anthrax toxin, also a bio-terror agent. All of these product-candidates could benefit from a technology that would increase expression in plants, which would result in decreases in production and purification costs, making therapeutic antibodies more affordable and widely available.
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: | 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,