Dataset Information

Species-Specific Chromosome Engineering Greatly Improves Fully Human Polyclonal Antibody Production Profile in Cattle.

ABSTRACT: Large-scale production of fully human IgG (hIgG) or human polyclonal antibodies (hpAbs) by transgenic animals could be useful for human therapy. However, production level of hpAbs in transgenic animals is generally very low, probably due to the fact that evolutionarily unique interspecies-incompatible genomic sequences between human and non-human host species may impede high production of fully hIgG in the non-human environment. To address this issue, we performed species-specific human artificial chromosome (HAC) engineering and tested these engineered HAC in cattle. Our previous study has demonstrated that site-specific genomic chimerization of pre-B cell receptor/B cell receptor (pre-BCR/BCR) components on HAC vectors significantly improves human IgG expression in cattle where the endogenous bovine immunoglobulin genes were knocked out. In this report, hIgG1 class switch regulatory elements were subjected to site-specific genomic chimerization on HAC vectors to further enhance hIgG expression and improve hIgG subclass distribution in cattle. These species-specific modifications in a chromosome scale resulted in much higher production levels of fully hIgG of up to 15 g/L in sera or plasma, the highest ever reported for a transgenic animal system. Transchromosomic (Tc) cattle containing engineered HAC vectors generated hpAbs with high titers against human-origin antigens following immunization. This study clearly demonstrates that species-specific sequence differences in pre-BCR/BCR components and IgG1 class switch regulatory elements between human and bovine are indeed functionally distinct across the two species, and therefore, are responsible for low production of fully hIgG in our early versions of Tc cattle. The high production levels of fully hIgG with hIgG1 subclass dominancy in a large farm animal species achieved here is an important milestone towards broad therapeutic applications of hpAbs.

SUBMITTER: Matsushita H

PROVIDER: S-EPMC4479556 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Species-Specific Chromosome Engineering Greatly Improves Fully Human Polyclonal Antibody Production Profile in Cattle.

Matsushita Hiroaki H Sano Akiko A Wu Hua H Wang Zhongde Z Jiao Jin-An JA Kasinathan Poothappillai P Sullivan Eddie J EJ Kuroiwa Yoshimi Y

PloS one 20150624 6

Large-scale production of fully human IgG (hIgG) or human polyclonal antibodies (hpAbs) by transgenic animals could be useful for human therapy. However, production level of hpAbs in transgenic animals is generally very low, probably due to the fact that evolutionarily unique interspecies-incompatible genomic sequences between human and non-human host species may impede high production of fully hIgG in the non-human environment. To address this issue, we performed species-specific human artifici ...[more]

PMID: 26107496

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Project description:We have performed a systematic variability study of polyclonal antibody catalysis by using five rabbits immunized with the same hapten. Important results from this work are the following. (1) Similarities were observed in the catalytic polyclonal antibodies derived from all five rabbits. Four of the five rabbits produced polyclonal samples that were nearly the same in terms of catalytic activity, whereas the fifth rabbit, designated as rabbit 2, displayed a somewhat higher level of catalytic activity. The catalytic activities (as kcat/kuncat) of these polyclonal samples were similar to that from the best murine monoclonal antibody that had been previously elicited by the same hapten. (2) Titre was not an accurate indicator of polyclonal antibody catalytic activity. (3) A mathematical analysis to describe a distribution of Michaelis-Menten catalysts was performed to help interpret our results. (4) Kinetic analysis indicated that the binding parameters of the different samples were remarkably homogeneous, because one or two components were all that were required to fit the on-rate and off-rate data satisfactorily. Interestingly, the most active catalytic polyclonal sample, that from rabbit 2, displayed the slowest off-rate (so slow it could not be measured) and thus the highest overall affinity. (5) Catalytic analysis of eluted fractions of antibody from a substrate column indicated that each polyclonal sample was also relatively homogeneous in terms of catalytic parameters. The main conclusion of our study is that for this hapten-animal system, the overall catalytic immune response is relatively consistent at two levels. Consistent catalytic activity was observed between the polyclonal samples elicited in the different animals, and the elicited hapten-specific polyclonal antibodies were relatively homogeneous in terms of binding and catalytic parameters within each immunized animal. The observed similarities of the catalytic activity in the different animals is surprising, because the immune response is based on specific binding of antibodies to hapten. There is no known selective pressure to maintain consistent levels of catalytic activity. Our results can therefore be interpreted as providing evidence that for this hapten there is a fixed relationship between hapten structure and catalytic activity and/or consistent genetic factors that dominate the catalytic immune response.

Project description:Our aim was to simultaneously investigate the gut bacteria typical characteristic and conduct rumen metabolites profiling of high production dairy cows when compared to low-production dairy cows. The bacterial differences in rumen fluid and feces were identified by 16S rDNA gene sequencing. The metabolite differences were identified by metabolomics profiling with liquid chromatography mass spectrometry (LC-MS). The results indicated that the high-production dairy cows presented a lower rumen bacterial richness and species evenness when compared to low-production dairy cows. At the phylum level, the high-production cows increased the abundance of Proteobacteria and decreased the abundance of Bacteroidetes, SR1, Verrucomicrobia, Euryarchaeota, Planctomycetes, Synergistetes, and Chloroflexi significantly (p < 0.05). At the genus level, the rumen fluid of the high-production group was significantly enriched for Butyrivibrio, Lachnospira, and Dialister (p < 0.05). Meanwhile, rumen fluid of high-production group was depleted for Prevotella, Succiniclasticum, Ruminococcu, Coprococcus,YRC22, CF231, 02d06, Anaeroplasma, Selenomonas, and Ruminobacter significantly (p < 0.05). A total of 92 discriminant metabolites were identified between high-production cows and low-production cows. Compared to rumen fluid of low-production dairy cows, 10 differential metabolites were found up-regulated in rumen fluid of high-production dairy cows, including 6alpha-Fluoropregn-4-ene-3,20-dione, 3-Octaprenyl-4-hydroxybenzoate, disopyramide, compound III(S), 1,2-Dimyristyl-sn-glycerol, 7,10,13,16-Docosatetraenoic acid, ferrous lactate, 6-Deoxyerythronolide B, vitamin D2, L-Olivosyl-oleandolide. The remaining differential metabolites were found down-regulated obviously in high-production cows. Metabolic pathway analyses indicated that most increased abundances of rumen fluid metabolites of high-yield cows were related to metabolic pathways involving biosynthesis of unsaturated fatty acids, steroid biosynthesis, ubiquinone and other terpenoid-quinone biosynthesis. Most down-regulated metabolic pathways were relevant to nucleotide metabolism, energy metabolism, lipid metabolism and biosynthesis of some antibiotics.

Dataset Information

Species-Specific Chromosome Engineering Greatly Improves Fully Human Polyclonal Antibody Production Profile in Cattle.

Publications

Species-Specific Chromosome Engineering Greatly Improves Fully Human Polyclonal Antibody Production Profile in Cattle.

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