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1.
Hussam H. Shaheen Bianka Prinz Ming-Tang Chen Tej Pavoor Song Lin Nga Rewa Houston-Cummings Renee Moore Terrance A. Stadheim Dongxing Zha 《PloS one》2013,8(7)
State-of-the-art monoclonal antibody (mAb) discovery methods that utilize surface display techniques in prokaryotic and eukaryotic cells require multiple steps of reformatting and switching of hosts to transition from display to expression. This results in a separation between antibody affinity maturation and full-length mAb production platforms. Here, we report for the first time, a method in Glyco-engineered
Pichia
pastoris
that enables simultaneous surface display and secretion of full-length mAb molecules with human-like N-glycans using the same yeast cell. This paradigm takes advantage of homo-dimerization of the Fc portion of an IgG molecule to a surface-anchored "bait" Fc, which results in targeting functional “half” IgGs to the cell wall of
Pichia
pastoris
without interfering with the secretion of full length mAb. We show the utility of this method in isolating high affinity, well-expressed anti-PCSK9 leads from a designed library that was created by mating yeasts containing either light chain or heavy chain IgG libraries. Coupled with Glyco-engineered
Pichia
pastoris
, this method provides a powerful tool for the discovery and production of therapeutic human mAbs in the same host thus improving drug developability and potentially shortening the discovery time cycle. 相似文献
2.
Ningyan Zhang Liming Liu Calin Dan Dumitru Nga Rewa Houston Cummings Michael Cukan Youwei Jiang Yuan Li Fang Li Teresa Mitchell Muralidhar R Mallem Yangsi Ou Rohan N Patel Kim Vo Hui Wang Irina Burnina Byung-Kwon Choi Hans Huber Terrance A Stadheim Dongxing Zha 《MABS-AUSTIN》2011,3(3):289-298
Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 from Pichia had antagonist activities comparable to trastuzumab. However, Pichia-produced material showed a 5-fold increase in binding affinity to FcγIIIA and significantly enhanced antibody dependent cell-mediated cytotoxicity (ADCC) activity, presumably due to the lack of fucose on N-glycans. In a breast cancer xenograft mouse model, anti-HER2 was comparable to trastuzumab in tumor growth inhibition. Furthermore, comparable pharmacokinetic profiles were observed for anti-HER2 and trastuzumab in both mice and cynomolgus monkeys. We conclude that glycoengineered Pichia provides an alternative production platform for therapeutic mAbs and may be of particular interest for production of antibodies for which ADCC is part of the clinical mechanism of action.Key words: glycoengineered Pichia, anti-HER2, trastuzumab, xenograft, PK, ADCC 相似文献
3.
Li H Sethuraman N Stadheim TA Zha D Prinz B Ballew N Bobrowicz P Choi BK Cook WJ Cukan M Houston-Cummings NR Davidson R Gong B Hamilton SR Hoopes JP Jiang Y Kim N Mansfield R Nett JH Rios S Strawbridge R Wildt S Gerngross TU 《Nature biotechnology》2006,24(2):210-215
As the fastest growing class of therapeutic proteins, monoclonal antibodies (mAbs) represent a major potential drug class. Human antibodies are glycosylated in their native state and all clinically approved mAbs are produced by mammalian cell lines, which secrete mAbs with glycosylation structures that are similar, but not identical, to their human counterparts. Glycosylation of mAbs influences their interaction with immune effector cells that kill antibody-targeted cells. Here we demonstrate that human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of the yeast Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. Glycoengineered P. pastoris provides a general platform for producing recombinant antibodies with human N-glycosylation. 相似文献
4.
Peter J. Blank Galen P. Dively Douglas E. Gill Charles A. Rewa 《The Journal of wildlife management》2011,75(1):116-125
Filter strips are strips of herbaceous vegetation planted along agricultural field margins adjacent to streams or wetlands and are designed to intercept sediment, nutrients, and agrichemicals. Roughly 16,000 ha of filter strips have been established in Maryland through the United States Department of Agriculture's Conservation Reserve Enhancement Program. Filter strips often represent the only uncultivated herbaceous areas on farmland in Maryland and therefore may be important habitat for early-successional bird species. Most filter strips in Maryland are planted to either native warm-season grasses or cool-season grasses and range in width from 10.7 m to 91.4 m. From 2004 to 2007 we studied the breeding and wintering bird communities in filter strips adjacent to wooded edges and non-buffered field edges and the effect that grass type and width of filter strips had on bird community composition. We used 5 bird community metrics (total bird density, species richness, scrub-shrub bird density, grassland bird density, and total avian conservation value), species-specific densities, nest densities, and nest survival estimates to assess the habitat value of filter strips for birds. Breeding and wintering bird community metrics were greater in filter strips than in non-buffered field edges but did not differ between cool-season and warm-season grass filter strips. Most breeding bird community metrics were negatively related to the percent cover of orchardgrass (Dactylis glomerata) in ≥1 yr. Breeding bird density was greater in narrow (<30 m) compared to wide (>60 m) filter strips. Our results suggest that narrow filter strips adjacent to wooded edges can provide habitat for many bird species but that wide filter strips provide better habitat for grassland birds, particularly obligate grassland species. If bird conservation is an objective, avoid planting orchardgrass in filter strips and reduce or eliminate orchardgrass from filter strips through management practices. © 2011 The Wildlife Society. 相似文献
5.
Calcium-mediated changes in peroxidase and O-diphenol oxidase activities of cotton fibres (Gossypium spp.) and its possible relationship to ABA 总被引:3,自引:0,他引:3
A. S. Basra R. S. Sarlach Rewa Dhillon-Grewal C. P. Malik 《Plant Growth Regulation》1992,11(2):159-164
This work is an investigation of the influence of ABA and calcium on soluble and wall-bound activities of peroxidase and O-diphenol oxidase of cotton fibres at the stages of primary and secondary wall development, during incubation of intact fibres for 3h at 28°C. ABA (10 M) caused marked inhibition of enzyme activities in both the fractions, whereas calcium (1 mM) was promotory. The incorporation of 1 mM EGTA (a calcium chelator) and chlorpromazine (10 g cm–3) (a calmodulin antagonist) resulted in decreased enzyme activities suggesting regulation of enzyme synthesis and/or secretion to the cell wall by calcium-calmodulin. As a general trend, the relative effect of Ca2+ on the activity of peroxidase in the wall was much greater than on the soluble activity, but this was not true of O-diphenol oxidase. It is inferred that ABA inhibits enzymic activities by inhibiting calmodulin synthesis or its mobilization to sites of action.Abbreviations ABA
abscisic acid
- EGTA
ethylene glycol-bis( amino-ethyl ether)N,N tetraacetic acid
- DAA
days after anthesis 相似文献
6.
G Rewa P Man C T Kappagoda 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1985,180(3):505-512
High frequency oscillatory ventilation (HFOV) is a new method of artificial ventilation which has been advocated for use in critically ill individuals. It alters the discharge in pulmonary stretch receptors (SAR) from a phasic to a continuous pattern. Since some cardiovascular neurones in the medulla are influenced by the discharge from SAR, experiments were undertaken to determine whether the reflexes from the left atrial (volume) receptors (LAR) were influenced by HFOV. The reflex increases in heart rate and urine flow which result from activation of the (LAR) were examined during both intermittent positive pressure ventilation (IPPV) and HFOV. In five dogs, the increase in heart rate was 23.9 +/- 4.3 and 24.5 +/- 5.4 beats/min during IPPV and HFOV, respectively. In six dogs the response of an increase in urine flow was examined and this response also was not altered by HFOV. It is concluded that the integrity of these reflexes was unaffected by HFOV in the anesthetized dog model. 相似文献
7.
Structural elucidation of an α-1,2-mannosidase resistant oligosaccharide produced in Pichia pastoris
Gomathinayagam S Mitchell T Zartler ER Heiss C Azadi P Zha D Houston-Cummings NR Jiang Y Li F Giaccone E Porambo RJ Anderson CL Sethuraman N Li H Stadheim TA 《Glycobiology》2011,21(12):1606-1615
The N-glycosylation pathway in Pichia pastoris has been humanized by the deletion of genes responsible for fungal-type glycosylation (high mannose) as well as the introduction of heterologous genes capable of forming human-like N-glycosylation. This results in a yeast host that is capable of expressing therapeutic glycoproteins. A thorough investigation was performed to examine whether glycoproteins expressed in glycoengineered P. pastoris strains may contain residual fungal-type high-mannose structures. In a pool of N-linked glycans enzymatically released by protein N-glycosidase from a reporter glycoprotein expressed in a developmental glycoengineered P. pastoris strain, an oligosaccharide with a mass consistent with a Hexose(9)GlcNAc(2) oligosaccharide was identified. When this structure was analyzed by a normal-phase high-performance liquid chromatography (HPLC), its retention time was identical to a Man(9)GlcNAc(2) standard. However, this Hexose(9)GlcNAc(2) oligosaccharide was found to be resistant to α-1,2-mannosidase as well as endomannosidase, which preferentially catabolizes endoplasmic reticulum oligosaccharides containing terminal α-linked glucose. To further characterize this oligosaccharide, we purified the Hexose(9)GlcNAc(2) oligosaccharide by HPLC and analyzed the structure by high-field one-dimensional (1D) and two-dimensional (2D) (1)H NMR (nuclear magnetic resonance) spectroscopy followed by structural elucidation by homonuclear and heteronuclear 1D and 2D (1)H and (13)C NMR spectroscopy. The results of these experiments lead to the identification of an oligosaccharide α-Man-(1 → 2)-β-Man-(1 → 2)-β-Man-(1 → 2)-α-Man-(1 → 2) moiety as part of a tri-antennary structure. The difference in enzymatic reactivity can be attributed to multiple β-linkages on the α-1,3 arm of the Man(9)GlcNAc(2) oligosaccharide. 相似文献
8.
Jiang Y Li F Zha D Potgieter TI Mitchell T Moore R Cukan M Houston-Cummings NR Nylen A Drummond JE McKelvey TW d'Anjou M Stadheim TA Sethuraman N Li H 《Protein expression and purification》2011,76(1):7-14
A robust and scalable purification process was developed to quickly generate antibody of high purity and sufficient quantity from glycoengineered Pichia pastoris fermentation. Protein A affinity chromatography was used to capture the antibody from fermentation supernatant. A pH gradient elution was applied to the Protein A column to prevent antibody precipitation at low pH. Antibody from Protein A chromatography contained some product related impurities, which were the misassembling of cleaved heavy chain, heavy chain and light chain. It also had some process related impurities, including Protein A residues, endotoxin, host cell DNA and proteins. Cation exchange chromatography with optimal NaCl gradient at pH 4.5-6.0 efficiently removed these product and process related impurities. The antibody from glycoengineered P. pastoris was comparable to its commercial counterpart in heterotetramer folding, physical stability and binding affinity. 相似文献
9.
Gene 1 product (gp1) of Bacillus subtilis phage psi29 is known to promote DNA replication of the phage. Although its role in the DNA replication is not clear, gp1 is reported to exhibit multiple characteristics, including RNA binding, cell membrane localization, and self-association. To investigate these characteristics, we undertook the isolation of a series of missense mutants of gene 1 bearing substitutions at various regions. During cloning of gene 1, we found that its expression severely inhibited the growth of its host Escherichia coli cells. In this study, we utilized this growth-inhibition phenomenon to screen a random library muta-genized by error-prone PCR, expecting that mutants which could not inhibit cell growth would be affected in the authentic functions of gp1. Using this approach, we obtained 31 different mutants bearing single amino acid substitutions at 26 positions along the entire length of gp1. As a preliminary analysis of these mutants, we compared the deduced amino acid sequences of gp1s from psi29 and its related phages PZA, B103 and M2. Alignment of these sequences revealed three conserved regions, i.e. a hydrophobic region near the carboxyl terminus (assumed to be involved in the membrane localization and self-association of gp1), coiled-coil motif (essential for self-association), and a region of unknown function near the amino terminus. Interestingly, many of the substitutions in the isolated mutants occurred at strongly conserved residues in these regions and affected characteristic features of the regions (e.g. hydrophobicity of the hydrophobic region). These substitutions are expected to affect authentic functions of gp1, and the mutants will be useful for studies of the structure and functions of gp1. 相似文献
10.
Rewa Kohli Vidula Purohit Latika Karve Vinod Bhalerao Shilpa Karvande Sheela Rangan Srikanth Reddy Ramesh Paranjape Seema Sahay 《PloS one》2012,7(9)