Yogesh Mistry

“Cytokine Storm” in the Phase I Trial of Monoclonal Antibody TGN1412: Better Understanding the Causes to Improve PreClinical Testing of Immunotherapeutics

The CD28-specific mAb TGN1412 rapidly caused a life-threatening “cytokine storm” in all six healthy volunteers in the Phase I clinical trial of this superagonist, signaling a failure of preclinical safety testing. We report novel in vitro procedures in which TGN1412, immobilized in various ways, is presented to human white blood cells in a manner that stimulates the striking release of cytokines and profound lymphocyte proliferation that occurred in vivo in humans. The novel procedures would have predicted the toxicity of this superagonist and are now being applied to emerging immunotherapeutics and to other therapeutics that have the potential to act upon the immune system. Data from these novel procedures, along with data from in vitro and in vivo studies in nonhuman primates, suggest that the dose of TGN1412 given to human volunteers was close to the maximum immunostimulatory dose and that TGN1412 is not a superagonist in nonhuman primates.

Infusion of pharmaceutical-grade natural human C-reactive protein is not proinflammatory in healthy adult human volunteers.

Rationale: Baseline circulating concentrations of C-reactive protein (CRP) are significantly associated with cardiovascular disease risk in general populations. This modest association has been inappropriately conflated with causality, and it has been claimed that CRP is proatherogenic. Most of the known causative factors for atherosclerosis stimulate increased CRP production, but comprehensive genetic epidemiology studies provide no support for a pathogenic role of CRP. The reported proinflammatory effects of human CRP preparations on healthy cells in vitro and in healthy animals in vivo have all been produced by poorly characterized CRP preparations, demonstrably caused by impurities, or elicited by CRP made in recombinant Escherichia coli not by humans. None of the in vitro or animal findings have been reproduced with pure natural human CRP. Nevertheless, the strong proinflammatory effects of infusing recombinant bacterial CRP into humans have still been inappropriately ascribed to CRP. Objective: To investigate the effects of infusion into healthy adult human volunteers of pure natural human CRP. Methods and Results: Comprehensively characterized, pharmaceutical-grade, endotoxin-free, purified CRP, prepared to GMP standard from pooled normal human donor plasma was infused as an intravenous bolus in 7 healthy adult human volunteers at ⩽2 mg/kg to provide circulating CRP concentrations ⩽44 mg/L. No recipient showed any significant clinical, hematologic, coagulation, or biochemical changes, or any increase in proinflammatory cytokines or acute phase proteins. Conclusions: The human CRP molecule itself is not proinflammatory in healthy human adults.

Attenuated fever in rats during late pregnancy is linked to suppressed interleukin-6 production after localized inflammation with turpentine.

An attenuated fever response to pathogens during late pregnancy is a phenomenon that has been described in several mammalian species, and although mechanisms are not completely understood, decreased prostaglandin E2 (PGE2) synthesis has been implicated. Upstream of PGE2, there is evidence to suggest that anti‐inflammatory cytokines such as interleukin‐1 receptor antagonist (IL‐1ra) could play a significant role. In the present study we addressed the role of pro‐inflammatory cytokines during late pregnancy, specifically interleukin‐6 (IL‐6), an important circulating mediator in fever. Turpentine oil (TURP), a very potent pyrogen and activator of IL‐6, was injected into the hind‐limb muscle of rats at the 18th day of pregnancy (GD 18) or in non‐pregnant (NP) age‐matched female controls. As expected, TURP injection induced a highly significant fever in the NP animals, which peaked 11 h post‐injection and lasted for over 24 h. This was accompanied by a significant rise in circulating IL‐6 levels, which correlated with changes in PGE2 synthesizing enzymes expression in the hypothalamus. In complete contrast, TURP‐induced fever was totally absent in GD 18 animals whose body temperature did not deviate from basal values. The lack of response was additionally reflected by the absence of change in IL‐6 concentration and by the significant attenuation of PGE2 synthesizing enzymes expression, which correlated with the suppressed expression of SOCS3, a hypothalamic marker of IL‐6 activity. Contrary to the changes in circulating IL‐6 levels at GD 18, IL‐1ra was induced to levels comparable to those of NP females, suggesting that the influence of this anti‐inflammatory cytokine on the fever response to TURP is at best minimal. These data further confirm the importance of IL‐6 in fever generation and provide evidence that it may be a key component of the attenuated fever response in late pregnancy.

Isolation and characterization of pharmaceutical grade human pentraxins, serum amyloid P component and C‐reactive protein, for clinical use

The human pentraxin proteins, serum amyloid P component (SAP) and C‐reactive protein (CRP) are important in routine clinical diagnosis, SAP for systemic amyloidosis and CRP for monitoring the non‐specific acute phase response. They are also targets for novel therapies currently in development but their roles in health and disease are controversial. Thus, both for clinical use and to rigorously elucidate their functions, structurally and functionally intact, pharmaceutical grade preparations of the natural, authentic proteins are required. We report here the production from normal human donor plasma and the characterization of the first such preparations. Importantly, we demonstrate that, contrary to reports using recombinant proteins and less well characterized preparations, neither CRP nor SAP stimulate the release by human peripheral blood mononuclear cells in vitro of any TNFα, IL‐6 or IL‐8, nor does SAP cause release of IL‐1β or IL‐10. Furthermore neither of our preparations was pro‐inflammatory in mice in vivo.

Inhibition of tissue factor and cytokine release.

The effect of inhibitors of cytokine release and plasma coagulation on lipopolysaccharide (LPS)-induced tissue factor and interleukin-6 (IL-6) was investigated. Dexamethasone, an inhibitor of cytokine production, inhibited LPS-induced tissue factor and IL-6 release by mononuclear cells (MNC), but enhanced IL-1beta-evoked tissue factor activity. Clinical antithrombin (AT) concentrates inhibited, in a dose-dependent manner, tissue factor and IL-6 production by MNC and human umbilical vein endothelial cells (HUVEC). The three AT preparations tested, when compared using the same antithrombin unit, had different potencies. Activated protein C (APC) augmented LPS stimulation of HUVEC and further increased the production of tissue factor and IL-6. The same effect was not observed with MNC; LPS-induced tissue factor and IL-6 release were unaffected by APC. Truncated tissue factor pathway inhibitor (TFPI1-161) inhibited LPS-induced MNC tissue factor and IL-6 production, but was unable to prevent LPS stimulatory activity on HUVEC. These data suggest a complex interaction between the coagulation pathway and the cytokine network.