Targeting the neonatal fc receptor for antigen delivery using engineered fc fragments.

The development of approaches for Ag delivery to the appropriate subcellular compartments of APCs and the optimization of Ag persistence are both of central relevance for the induction of protective immunity or tolerance. The expression of the neonatal Fc receptor, FcRn, in APCs and its localization to the endosomal system suggest that it might serve as a target for Ag delivery using engineered Fc fragment-epitope fusions. The impact of FcRn binding characteristics of an Fc fragment on in vivo persistence allows this property to also be modulated. We have therefore generated recombinant Fc (mouse IgG1-derived) fusions containing the N-terminal epitope of myelin basic protein that is associated with experimental autoimmune encephalomyelitis in H-2(u) mice. The Fc fragments have distinct binding properties for FcRn that result in differences in intracellular trafficking and in vivo half-lives, allowing the impact of these characteristics on CD4(+) T cell responses to be evaluated. To dissect the relative roles of FcRn and the "classical" FcgammaRs in Ag delivery, analogous aglycosylated Fc-MBP fusions have been generated. We show that engineered Fc fragments with increased affinities for FcRn at pH 6.0-7.4 are more effective in delivering Ag to FcRn-expressing APCs in vitro relative to their lower affinity counterparts. However, higher affinity of the FcRn-Fc interaction at near neutral pH results in decreased in vivo persistence. The trade-off between improved FcRn targeting efficiency and lower half-life becomes apparent during analyses of T cell proliferative responses in mice, particularly when Fc-MBP fusions with both FcRn and FcgammaR binding activity are used.

Identification and quantification of glycerolipids in cotton fibers: reconciliation with metabolic pathway predictions from DNA databases.

The lipid profiles of cotton fiber cells were determined from total lipid extracts of elongating and maturing cotton fiber cells to see whether the membrane lipid composition changed during the phases of rapid cell elongation or secondary cell wall thickening. Total FA content was highest or increased during elongation and was lower or decreased thereafter, likely reflecting the assembly of the expanding cell membranes during elongation and the shift to membrane maintenance (and increase in secondary cell wall content) in maturing fibers. Analysis of lipid extracts by electrospray ionization and tandem MS (ESI-MS/MS) revealed that in elongating fiber cells (7-10 d post-anthesis), the polar lipids-PC, PE, PI, PA, phosphatidylglycerol, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and phosphatidylglycerol-were most abundant. These same glycerolipids were found in similar proportions in maturing fiber cells (21 dpa). Detailed molecular species profiles were determined by ESI-MS/MS for all glycerolipid classes, and ESI-MS/MS results were consistent with lipid profiles determined by HPLC and ELSD. The predominant molecular species of PC, PE, PI, and PA was 34:3 (16:0, 18:3), but 36:6 (18:3,18:3) also was prevalent. Total FA analysis of cotton lipids confirmed that indeed linolenic (18:3) and palmitic (16:0) acids were the most abundant FA in these cell types. Bioinformatics data were mined from cotton fiber expressed sequence tag databases in an attempt to reconcile expression of lipid metabolic enzymes with lipid metabolite data. Together, these data form a foundation for future studies of the functional contribution of lipid metabolism to the development of this unusual and economically important cell type.

Identification and expression of a new delta-12 fatty acid desaturase (FAD2-4) gene in upland cotton and its functional expression in yeast and Arabidopsis thaliana plants.

A cotton (Gossypium hirsutum L.) genomic clone encompassing a 17.9-kb DNA fragment was found to contain a delta-12 fatty acid desaturase gene (designated FAD2-4). The FAD2-4 open reading frame has 1,155bp and is uninterrupted, encoding a conceptual FAD2-4 polypeptide of 384 amino acids that has 98% identity with the cotton FAD2-3 polypeptide. The FAD2-4 gene has a single intron of 2,780 bp in its 5'-untranslated region (5'-UTR). The 3'-flanking regions and 5'-UTR introns in the FAD2-4 and FAD2-3 genes are quite different, indicating that the genes might be paralogs in the cotton genome. Reverse transcriptase (RT)-PCR analysis indicated that the FAD2-4 and FAD2-3 genes were expressed in all tissues examined, including seeds, seedling tissues, young and mature leaves, roots, stems, developing flower buds, and ovule fibers. These constitutive patterns of expression were notably different from that of the FAD2-1 gene, which was restricted to seeds and developing flower buds, or to the expression of a newly-identified FAD2-2 gene isoform, which was barely detectable in roots, hypocotyls, stems, and fibers, but was expressed in all other tissues. The FAD2-4 coding region was expressed in yeast and shown to encode a functional delta-12 desaturase, converting oleic acid (C18:1) to linoleic acid (C18:2) in recombinant yeast cells. In addition, both the FAD2-4 and the FAD2-3 genes were transferred into the Arabidopsis thaliana fad2-1 mutant background where they effectively restored wild type fatty acid composition and growth characteristics. Finally, the cotton FAD2-4 green fluorescent protein (GFP) fusion polypeptide appeared to be localized in the endomembrane system of transgenic Arabidopsis plants in the complemented fad2-1 mutant background, supporting a functional ER location for the cotton FAD2-4 polypeptide in this heterologous plant system. Thus, a new functional member of the FAD2 gene family in cotton has been characterized, indicating a complex regulation of membrane lipid desaturation in this important fiber/oilseed crop.

Divergent activities of an engineered antibody in murine and human systems have implications for therapeutic antibodies.

The MHC class I-related receptor, neonatal Fc receptor (FcRn), plays a central role in regulating the transport and in vivo persistence of immunoglobulin G (IgG). IgG-FcRn interactions can be targeted for engineering to modulate the in vivo longevity and transport of an antibody, and this has implications for the successful application of therapeutic IgGs. Although mice are widely used to preclinically test antibodies, human and mouse FcRn have significant differences in binding specificity. Here we show that an engineered human IgG1 has disparate properties in murine and human systems. The mutant shows improved transport relative to wild-type human IgG1 in assays of human FcRn function but has short in vivo persistence and competitively inhibits FcRn activity in mice. These studies indicate potential limitations of using mice as preclinical models for the analysis of engineered antibodies. Alternative assays are proposed that serve as indicators of the properties of IgGs in humans.