Immunomodulation by LFA3TIP, an LFA-3/IgG1 fusion protein: cell line dependent glycosylation effects on pharmacokinetics and pharmacodynamic markers.

LFA3TIP, a fusion protein comprised of the first extracellular domain of LFA-3 fused to the hinge, CH2, and CH3 domains of human IgG1 inhibits responses of human and non-human primate T cells in vitro. In seeking to optimize the expression efficiency to prepare large quantities of LFA3TIP for primate studies, the protein was produced in both the CHO (Chinese hamster ovary) and murine NS-0 myeloma cell lines. Although LFA3TIP derived from these cell lines performs identically in vitro in CD2 receptor binding and T cell assays, examination of a pharmacodynamic marker-the reduction in CD2+ lymphocyte numbers-following the administration of equal doses of NS-0 or CHO derived LFA3TIP to baboons, suggested that the effect of the NS-0 derived material was less sustained. Pharmacokinetic analysis of the materials in baboons and mice shows that LFA3TIP produced by NS-0 cells is rapidly cleared from circulation relative to the product derived from CHO cells. The disparate clearance profiles correlate with distinct glycosylation patterns, with LFA3TIP derived from NS-0 cells being less extensively sialylated than that from CHO cells due in part to alpha-galactosyl capping of selected lactosamine moieties in the N-linked glycans of NS-0 derived LFA3TIP. Moreover, enzymatic desialylation of CHO derived LFA3TIP results in a glycoprotein with an evanescent serum profile when administered to mice and baboons. These results correlate the extent of N-acetylneuraminic acid capping with the clearance rates of LFA3TIP derived from the two distinct cell lines, and underscore the importance of evaluating glycosylation dependent PK parameters when choosing production cell lines for recombinant immunotherapeutics.

Intracellular lytic enzyme systems and their use for disruption of Escherichia coli.

This article focusses on lytic enzyme systems available in E. coli and their potential use for cellular disruption. In the systems described here the genetic information for lysis would be carried within the microbial host, either integrated or naturally occurring on chromosomal DNA, or on extrachromosomal elements such as plasmids. Each microbe would carry complete information for endogenous enzymatic lysis, and lysis would occur in a controlled manner after being triggered by an external factor such as temperature or inducer addition. The lytic systems explored in this review include the autolytic enzymes, colicin lytic enzymes, and bacteriophage lytic enzymes from phage phiX174, T4, lambda, MS2 and Q beta. Many of the colicin lytic enzymes and all of the bacteriophage lytic enzymes described here have been cloned, and in some instances examined as cellular disruption methods. None of the E. coli autolytic enzymes have been cloned, but information pertinent for use as a disruption method is described.

Nitrosation by alkyl nitrites. Catalysis by inorganic salts.

Isobutyl nitrite is an effective nitrosating agent at acidic, neutral and basic pH in the presence of species arising from phosphate ion. The reaction is first-order in isobutyl nitrite and amine. In the reaction of isobutyl nitrite with sulfanilamide, the pH dependence reflects the change in concentration of the various protonated forms of phosphate, with H3PO4 and H2PO4- most strongly affecting the rate. In the reaction of isobutyl nitrite with dipropylamine, the pH dependence also reflects the change in the concentration of unprotonated amine.

Differential expression of steroid sulphatase locus on active and inactive human X chromosome.

The X chromosome in mammalian somatic cells is subject to unique regulation--usually genes on a single X chromosome are expressed while those on other X chromosomes are inactivated. The X-locus for steroid sulphatase (STS; EC 3.1.6.2), the microsomal enzyme that catalyses the hydrolysis of various 3 beta-hydroxysteroid sulphates, is exceptional because it seems to escape inactivation. Evidence for this comes from fibroblast clones in females heterozygous for mutations that result in a severe deficiency of this enzyme in affected males; all clones from these heterozygotes have STS activity, and enzyme-deficient clones that are expected if the locus were subject to inactivation, have not been found. Further evidence that the STS locus escapes inactivation is that the human inactive X chromosomes contributes STS activity to mouse-human hybrid cells. On the basis of these hybrid studies the STS locus has been mapped to the distal half of the short arm (p22-pter) of the human X chromosome. Although the STS locus on both X chromosomes in human female cells is expressed, quantitative measurements of STS activity in males and females do not accurately reflect the sex differences in number of X chromosomes (Table 1). The ratio of mean values for normal females to that of normal males is greater than 1:1 but less than the ratio of 2:1 expected if STS loci on all X chromosomes were equally expressed. The incomplete dosage effect suggests that the STS locus on the inactive X chromosome might not be fully expressed. To test this hypothesis, we examine two heterozygotes for X-linked STS deficiency who were also heterozygous for the common electrophoretic variants of glucose-6-phosphate dehydrogenase (G6PD A and B). Studies of fibroblast clones from these females provide evidence, presented here, for differential expression of STS loci on the active and inactive X chromosome.