In 2006, a life-threatening 'cytokine storm', not predicted by pre-clinical safety testing, rapidly occurred in all six healthy volunteers during the phase I clinical trial of the CD28 superagonist monoclonal antibody (mAb) TGN1412. To date, no unequivocal explanation for the failure of TGN1412 to stimulate profound cytokine release in vitro or in vivo in species used for pre-clinical safety testing has been established. Here, we have identified a species difference almost certainly responsible for this disparate immunopharmacology. EXPERIMENTAL APPROACHPolychromatic flow cytometry and intracellular cytokine staining were employed to dissect the in vitro immunopharmacology of TGN1412 and other therapeutic mAbs at the cellular level to identify differences between humans and species used for pre-clinical safety testing. KEY RESULTSIn vitro IL-2 and IFN-g release from CD4+ effector memory T-cells were key indicators of a TGN1412-type response. This mechanism of cytokine release differed from that of other therapeutic mAbs, which can cause adverse reactions, because these other mAbs stimulate cytokine release primarily from natural killer cells. In contrast to humans, CD28 is not expressed on the CD4+ effector memory T-cells of all species used for pre-clinical safety testing, so cannot be stimulated by TGN1412. CONCLUSIONS AND IMPLICATIONSIt is likely that activation of CD4+ effector memory T-cells by TGN1412 was responsible for the cytokine storm. Lack of CD28 expression on the CD4+ effector memory T-cells of species used for pre-clinical safety testing of TGN1412 offers an explanation for the failure to predict a 'cytokine storm' in humans.

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Cyclic AMP-dependent expression of the steroidogenic acute regulatory (StAR) protein is thought to be the controlling step for steroid production, but the mechanisms through which external signals are translated into increased transcription of the StAR gene are unknown. We demonstrate that cyclic AMP-induced steroid synthesis is dependent upon the phosphorylation and activation of ERKs and that ERK activation results in enhanced phosphorylation of SF-1 and increased steroid production through increased transcription of the StAR gene. Adenylate cyclase activation with forskolin (FSK) caused a time-dependent increase in ERK activity and translocation from cytoplasm to nucleus, which correlated with an increase in StAR mRNA levels, StAR protein accumulation, and steroidogenesis. Similarly, ERK inhibition led to a reduction in the levels of FSKstimulated StAR mRNA, StAR protein, and steroid secretion. These effects were attributed to the finding that ERK activity is required for SF-1 phosphorylation, a transcription factor required for the regulation of StAR gene transcription. This conclusion was supported by our demonstration of an ERK-dependent increase in the binding of SF-1 from FSK-treated Y1 nuclei to three consensus double-stranded DNA sequences from the StAR promoter region. These observations suggest that the activation of ERK2/1 by increasing cAMP is an obligatory and regulated stage in the stimulation of steroid synthesis by cyclic AMP-generating stimuli.

In this report, we describe the development of a mini-array system suitable for high-throughput quantification of proteins. This mini-array is a multiplexed, sandwich-type ELISA that measures the concentration of seven different human cytokines--TNF-alpha, IFN alpha, IFN gamma, IL-1 alpha, IL-1 beta, IL-6, and IL-10--from a single sample in each well of a 96-well plate. The mini-array is produced by spotting monoclonal antibodies (mAbs) in a 3 x 3 pattern in the bottom of the wells of 96-well polystyrene plates. Cytokines that are captured by the arrayed mAbs are detected by using biotinylated mAbs, followed by the addition of a streptavidin-horseradish peroxidase (HRP) conjugate and a chemiluminescent substrate. The light produced from the HRP-catalyzed oxidation of the substrate is measured at each spot in the array by imaging the entire plate with a commercially available CCD camera. Here, we demonstrate that these 96-well-plate format mini-arrays have performance characteristics that make them suitable for the high-throughput screening of anti-inflammatory compounds.

Protein-tyrosine kinase and protein-tyrosine phosphatase (PTPase) activities are essential for T-cell antigen receptor-mediated signalng. To assess the functional consequences of alteration of the levels of tyrosine phosphorylation in normal human T cells, the effects of vanadate and hydrogen peroxide were studied. In combination, these agents induced tyrosine phosphorylation of cellular substrates, elevated cytosolic free calcium, and induced interleukin 2 receptor (IL-2R) a chain expression but not IL-2 secretion. However, anti-CD28 antibody in combination with vanadate and hydrogen peroxide induced IL-2 secretion, consistent with the requirement for a costimulatory signal in the induction of this gene. The effects of vanadate and hydrogen peroxide were enhanced in the absence of the T-cell PTPase, CD45. Thus, acute pharmacologic manipulation of the level of tyrosine phosphorylation in normal T cells correlates with partial, but not full, activation of these cells; in concert with a costimulatory signal provided by perturbation of the CD28 molecule, the complete program of activation is initiated. These agents should prove useful in dissecting signaling pathways involved in the regulation of genes critical to the immune response.In contrast to the growth factor class of receptors that display intrinsic protein-tyrosine kinase (PTK) activity, many immunologically relevant, oligomeric receptors including the T-cell antigen receptor (TCR) and Fc receptors lack intrinsic kinase activity. Nonetheless, stimulation of these receptors results in protein-tyrosine phosphorylation of cellular substrates (reviewed in refs. 1-10), and pharmacologic inhibition of tyrosine kinases prevents both the early biochemical and late functional events that occur after receptor stimulation (11)(12)(13)(14). Thus, PTK activity appears to be necessary for T-cell activation, but is PTK function alone sufficient to initiate any or all of the events involved in this process? Expression of PTKs in T-cell lines can enhance TCRmediated interleukin 2 (IL-2) induction or in the case of v-src can induce constitutive but suboptimal IL-2 secretion (15)(16)(17). However, the limitation oftransfection studies is that the consequences of acute alteration of the levels of tyrosine phosphorylation in normal T cells cannot be addressed.Vanadate is a well-documented inhibitor of proteintyrosine phosphatases (PlPases), but its effects on intact cells are variable (18)(19)(20). By contrast, the combination of vanadate and H202 generates the compound pervanadate, the efficacy of which has been demonstrated to be far greater than that of vanadate (21)(22)(23) (24). Two-color fluorescence measurements were performed on a FACScan IV flow cytometer (Becton Dickinson) to assess purity. The murine thymoma cell line, BW5147.3, and its CD45-counterpart were grown in RPMI 1640 medium supplemented with glutamine, antibiotics, and 10% fetal bovine serum.Anti-phosphotyrosine [anti-Tyr(P)l Immunobtig. Stock solutions of H202 and sodium orthovanadate, Na3VO4, wer...