The novel calicheamicin-conjugated CD22 antibody inotuzumab ozogamicin (CMC-544) effectively kills primary pediatric acute lymphoblastic leukemia cells.

We investigated whether the newly developed antibody (Ab) -targeted therapy inotuzumab ozogamicin (CMC-544), consisting of a humanized CD22 Ab linked to calicheamicin, is effective in pediatric primary B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells in vitro, and analyzed which parameters determine its efficacy. CMC-544 induced dose-dependent cell kill in the majority of BCP-ALL cells, although IC(50) values varied substantially (median 4.8 ng/ml, range 0.1-1000 ng/ml at 48 h). The efficacy of CMC-544 was highly dependent on calicheamicin sensitivity and CD22/CMC-544 internalization capacity of BCP-ALL cells, but hardly on basal and renewed CD22 expression. Although CD22 expression was essential for uptake of CMC-544, a repetitive loop of CD22 saturation, CD22/CMC-544 internalization and renewed CD22 expression was not required to achieve intracellular threshold levels of calicheamicin sufficient for efficient CMC-544-induced apoptosis in BCP-ALL cells. This is in contrast to studies with the comparable CD33 immunotoxin gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukemia (AML) patients, in which complete and prolonged CD33 saturation was required for apoptosis induction. These data suggest that CMC-544 treatment may result in higher response rates in ALL compared with response rates obtained in AML with Mylotarg, and that therefore clinical studies in ALL, preferably with multiple low CMC-544 dosages, are warranted.

Serum leptin levels during pregnancy in multiple sclerosis.

BACKGROUND: Disease activity in patients with multiple sclerosis (MS) is suppressed during pregnancy, whereas attack frequency increases after delivery. It is yet unclear, which immuno - endocrinological processes mediate these disease fluctuations. Leptin has been identified as a hormone that can influence inflammatory activity. OBJECTIVE: The aim of this study was to investigate whether pregnancy-induced fluctuations of serum leptin levels differed between patients with MS and controls and whether serum leptin levels correlate with periods of enhanced and diminished disease activity. METHODS: Women with MS and healthy women were prospectively followed during and after pregnancy. The MS group could be studied already at a timepoint before pregnancy. Serum leptin and soluble leptin receptor (SLR) levels were measured using enzyme-linked immunosorbent assay. RESULTS: Pre-pregnancy serum leptin levels were (mean +/- SD) 22.9 +/- 12.8 ng/ml in the MS group. These levels increased in the third trimester to 28.5 +/- 15.0 ng/ml (P = 0.007). The third trimester serum leptin levels in healthy women were comparable, 29.4 +/- 19.0 ng/ml. Serum leptin levels after delivery dropped to 18.5 +/- 12.8 ng/ml in women with MS (P < 0.001) and to a lesser extend (22.0 +/- 17.5 ng/ml) in healthy women (P = 0.04). SLR levels showed the same pattern. Remarkably, women with the highest relative decrease in serum leptin levels after delivery had more often a postpartum relapse (P = 0.008). CONCLUSION: In women with MS, leptin increased during late pregnancy. A postdelivery drop in leptin levels was observed in both the MS and control group. The postdelivery drop was associated with the occurrence of postpartum relapse.

Peptidoglycan and peptidoglycan-specific Th1 cells in psoriatic skin lesions.

We have previously demonstrated, in psoriatic skin lesions, the presence of a subset of dermal CD4+ T cells that produce interferon-gamma (IFN-gamma) in response to a mixture of cell wall proteins extracted from group A streptococci. However, the identity of the antigen(s) involved is unknown. To investigate the hypothesis that peptidoglycan (PG), the major constituent of the streptococcal cell wall, acts as a T cell activator in psoriasis, we performed in situ analysis to detect antigen-presenting cells containing PG in lesional versus non-lesional skin, and determined proliferation and IFN-gamma responses of lesional skin T cells. Increased numbers of PG-containing cells were detected in the dermal papillae and cellular infiltrates of guttate and chronic plaque skin lesions compared with normal and non-lesional psoriatic skin. A varying proportion of these were CD68+ macrophages, but the remaining cells did not double stain for either Langerhans' or dendritic cell markers. Psoriatic dermal streptococcal-specific CD4+ T cell lines proliferated and produced IFN-gamma in a self HLA-DR allele-restricted manner in response to streptococcal PG, excluding mitogenic or superantigenic stimulation, but were unresponsive to staphylococcal PG. Similarly, psoriatic staphylococcus-specific T cell lines recognized staphylococcal, but not streptococcal, PG by IFN-gamma production. The presence of PG-containing macrophages in close association with PG-specific CD4+ T cells in lesional skin suggests that PG may be responsible, at least in part, for T cell activation in psoriasis.

A model system for optimising the selection of membrane antigen-specific human antibodies on intact cells using phage antibody display technology.

The functional expression of human antibody fragments on the surface of filamentous bacteriophage, and selection of phage antibodies (PhAbs) with antigens, has provided a powerful tool for generating novel antibodies. Applications of phage antibody display technology have increased over the past decade. Successful isolation of phage antibodies has been reported mostly using purified antigens. Isolation has proven to be more complicated with complex mixtures of antigens, such as intact cells. A given cell type contains thousands of different epitopes, each capable in theory of binding phage antibodies. Often antigens are not known or cannot be purified without disrupting their conformational integrity. To overcome problems involving phage antibody selections on intact cells, we have developed an experimental model system that allows for optimisation and comparison of various selection strategies. The model system comprises labelling of intact cells with the fluorescently labelled phospholipid fluorescein-DHPE. Upon incubation, this phospholipid is readily incorporated in the membrane of any cell type. Labelling intensity is regulated by varying the phospholipid concentration. After optimisation of key steps in the selection procedure, we were able to isolate fluorescein-DHPE specific phage from a synthetic library using intact cells. This model system can be applied to any cell type and we demonstrate that it can be used to efficiently compare and optimise selection strategies.