Paula Dilger

“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.

Anti-cytokine autoantibodies in autoimmunity: preponderance of neutralizing autoantibodies against interferon-alpha, interferon-omega and interleukin-12 in patients with thymoma and/or myasthenia gravis

We have screened for spontaneous anticytokine autoantibodies in patients with infections, neoplasms and autoimmune diseases, because of their increasingly reported co‐occurrence. We tested for both binding and neutralizing autoantibodies to a range of human cytokines, including interleukin‐1alpha (IL‐1α), IL‐1β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, IL‐12, IL‐18, interferon‐alpha2 (IFN‐α2), IFN‐ω, IFN‐β, IFN‐γ, tumour necrosis factor alpha (TNF‐α), transforming growth factor beta‐1 (TGF‐β1) and granulocyte‐macrophage colony stimulating factor (GM‐CSF), in plasmas or sera. With two notable exceptions described below, we found only occasional, mostly low‐titre, non‐neutralizing antibodies, mainly to GM‐CSF; also to IL‐10 in pemphigoid. Strikingly, however, high‐titre, mainly IgG, autoantibodies to IFN‐α2, IFN‐ω and IL‐12 were common at diagnosis in patients with late‐onset myasthenia gravis (LOMG+), thymoma (T) but no MG (TMG–) and especially with both thymoma and MG together (TMG+). The antibodies recognized other closely related type I IFN‐α subtypes, but rarely the distantly related type I IFN‐β, and never (detectably) the unrelated type II IFN‐γ. Antibodies to IL‐12 showed a similar distribution to those against IFN‐α2, although prevalences were slightly lower; correlations between individual titres against each were so modest that they appear to be entirely different specificities. Neither showed any obvious correlations with clinical parameters including thymoma histology and HLA type, but they did increase sharply if the tumours recurred. These antibodies neutralized their respective cytokine in bioassays in vitro; although they persisted for years severe infections were surprisingly uncommon, despite the immunosuppressive therapy also used in most cases. These findings must hold valuable clues to autoimmunizing mechanisms in paraneoplastic autoimmunity.

Strategies for detection, measurement and characterization of unwanted antibodies induced by therapeutic biologicals

An important aspect of evaluating the safety of therapeutic biologicals is the assessment of the unwanted immunogenicity of such biologicals in recipients. Properly planned immunogenicity studies with appropriately devised strategies are critical if valid conclusions concerning the unwanted immunogenicity are to be derived. Such studies need to be conducted using carefully selected and validated procedures. Several techniques are available for detection and measurement of immunogenicity including immunoassays, radioimmunoprecipitation assays (RIPAs), surface plasmon resonance (SPR) and bioassays. A combination of methods for characterization of the induced antibodies is usually necessary for a detailed understanding of the type(s) of antibodies generated against a therapeutic product. This review considers the benefits and limitations of the various techniques available for antibody detection and outlines a strategy for the assessment of unwanted immunogenicity of therapeutic products.

Cytokine levels in platelet concentrates: quantitation by bioassays and immunoassays

Some adverse reactions to the transfusion of platelet concentrates (PCs) cannot be attributed to antibodies against blood cells or to subclinical microbial agents. It has been suggested that leucocyte‐derived inflammatory cytokines such as interleukin (IL)‐1, IL‐6 and tumour necrosis factor (TNF) may contribute to a large number of unexplained non‐antibody‐mediated adverse reactions. Three types of PCs, containing different levels of leucocytes, are currently produced. Filtration is used on demand to further reduce leucocyte contamination of these components. We have monitored the plasma of PCs prepared by the platelet‐rich plasma method (PRP), the buffy‐coat method or by apheresis for IL‐6, IL‐1, transforming growth factor‐beta (TGF‐β), TNF and interferon γ (IFNγ). Biologically active IL‐6 increased in stored PRP‐PCs from a mean of 140 pg/ml on day 1 to 2395 pg/ml on day 5/6. Elevated levels of IL‐8, as detected by immunoassay, were evident in PRP‐PCs during routine storage under blood bank conditions. Small amounts of immunoreactive IL‐1 with only minimal biological activity were present in some PRP‐PCs by day 5/6. No significant increase in the levels of IL‐8, IL‐6 or IL‐1 were seen in buffy‐coat PCs during storage for 5/6 d. For apheresis PCs, an increase in IL‐8 content, but not in IL‐6 over 6 d was observed. In all three types of PCs, elevated amounts of both bioactive and immunoreactive TGFβ were present, but there was no evidence of any biologically active or immunoreactive TNFα. Pre‐storage filtration of PRP‐PCs for depletion of leucocytes prevented the increase in IL‐8 and IL‐6 levels of these PCs. Our results show that leucocyte reduction by buffy‐coat method reduces cytokine levels to a comparable level to filtered or apheresis PCs, containing low levels of leucocytes, but use of these PCs in minimizing the severity and incidence of reactions in recipients will require clinical evaluation. This is the first comprehensive and comparative study which, on the basis of biological activity of cytokines, directly indicates that the mode of platelet production grossly influences the levels of cytokines.

Inhibition of interleuk:in-2 secretion by factor VIII concentrates: a possible cause of immunosuppression in haemophiliacs

Summary The inhibitory effect of factor VIII concentrate products on IL‐2 secretion by human T‐cells was investigated. The six products used widely in the lJ.K. showed very different activities varying from almost total inhibition to no significant effect. There appeared to be no obvious relationship between inhibitory activity and protein composition but factor VIII itself was not responsible for the effect as affinity purified products were entirely non‐inhibitory. The two wetheated products were most inhibitory whereas dry‐heated products were less inhibitory or non‐inhibitory. However, a wet‐heated version of a non‐inhibitory dry‐heated product was also non‐inhibitory, suggesting that the composition of the concentrate rather than anti‐viral treatment is important for immunosuppressive activity. A product treated by the solvent/detergent procedure showed considerable inhibitory activity. Immunoglobulin and albumin products did not inhibit IL‐2 secretion to any significant extent, but factor IX concentrates were inhibitory. We suggest that inhibition of IL‐2 secretion by factor VIII concentrates may be related to the immunosuppression observed in haemophiliacs treated with high dose factor VIII products and that our results should be considered by clinicians and manufacturers of factor VIII products.

Cytokine accumulation in stored red cell concentrates: effect of buffy-coat removal and leucoreduction.

The accumulation of cytokines in stored red blood cell concentrates (RCCs) has been implicated as a potential cause of transfusion reactions associated with the use of such products. At present, it is unclear whether there is any link between residual leukocyte and/or platelet content with cytokine levels in various RCCs. In this study, we have therefore assessed cytokine levels of leukocyte (e.g., IL8) and platelet (e.g., RANTES, TGF-beta1) origin in supernatants of RCCs prepared by the plasma reduced method or by depletion of the buffy coat. We have also assessed whether the Duffy antigen receptor (DARC, a promiscuous receptor for some chemokines) has any role in the diminution of cytokine levels in stored blood components by comparing cytokine levels in stored plasma reduced RCCs derived from both DARC +ve and DARC -ve individuals. In addition, comparison of filtered and non-filtered products of the same origin has also been conducted. Results showed that supernatants from DARC -ve concentrates contained higher levels of IL-8 up to days 14/15 of storage compared with DARC +ve RCCs. However, at later time points, similar levels of IL-8 were observed in RCCs regardless of their Duffy receptor status. For TGF-beta1 and RANTES, no significant difference in the levels of these cytokines was detected between DARC +ve and DARC -ve concentrates. Removal of leukocytes and platelets by conventional leukocyte filtration significantly reduced the accumulation of cytokines. Buffy coat reduced RCCs contained minimal amounts of IL-8 and TGF-beta1 but no RANTES. We conclude therefore, that the levels of cytokines in the supernatants of RCCs stored at 4 degrees C are related mainly to their leucocyte and platelet content.

Cytokines in WBC-reduced apheresis PCs during storage: a comparison of two WBC-reduction methods.

BACKGROUND: Several studies have suggested that cytokine accumulation during storage of platelet concentrates (PCs) may mediate nonhemolytic febrile transfusion reactions and that a reduction in WBC numbers prevents the generation of cytokines. Despite efforts to minimize WBC contamination in apheresis PCs, high numbers of WBCs and increased cytokine levels may still occur, depending on the quality of the apheresis device employed.

In vitro function of platelet concentrates prepared after filtration of whole blood or buffy coat pools

Background/method  Data on the quality of platelet concentrates (PC) produced by the buffy coat method and stored beyond 5 days in plasma are limited. We therefore evaluated the quality of PCs prepared by leucocyte depletion of whole blood (Terumo WBSP, n = 10) or a buffy coat pool (Pall Autostop, n = 10), and stored for 7 days in plasma by assessing platelet parameters and markers of platelet activation.

Treatment of colitis with a commensal gut bacterium engineered to secrete human TGF-β1 under the control of dietary xylan 1.

Background: While cytokine therapy and the use of immunosuppressive cytokines such as transforming growth factor‐&bgr; (TGF‐&bgr;) offer great potential for the treatment of inflammatory bowel disease (IBD), issues concerning formulation, stability in vivo, delivery to target tissues, and potential toxicity need to be addressed. In consideration of these problems we engineered the human commensal bacterium Bacteroides ovatus for the controlled in situ delivery of TGF‐&bgr;1 and treatment of colitis. Methods: Sequence encoding the human tgf‐&bgr;1 gene was cloned downstream of the xylanase promoter in the xylan operon of B. ovatus by homologous recombination. Resulting recombinants (BO‐TGF) were tested for TGF‐&bgr; production in the presence and absence of polysaccharide xylan in vitro and in vivo, and used to treat experimental murine colitis. Clinical and pathological scores were used to assess the effectiveness of therapy. Colonic inflammatory markers including inflammatory cytokine expression were assessed by colorimetric assay and real‐time polymerase chain reaction (PCR). Results: BO‐TGF secreted high levels of biologically active dimeric TGF‐&bgr; in vitro and in vivo in a xylan‐controlled manner. Administration of xylan in drinking water to BO‐TGF‐treated mice resulted in a significant clinical improvement of colitis, accelerating healing of damaged colonic epithelium, reducing inflammatory cell infiltration, reducing expression of proinflammatory cytokines, and promoting production of mucin‐rich goblet cells in colonic crypts. These beneficial effects are comparable and in most cases superior to that achieved by conventional steroid therapy. Conclusions: This novel drug delivery system has potential for the targeted and controlled delivery of TGF‐&bgr;1 and other immunotherapeutic agents for the long‐term management of various bowel disorders. (Inflamm Bowel Dis 2010;)

Cytokine levels as performance indicators for white blood cell reduction of platelet concentrates.

Background and Objectives With the implementation of universal white blood cell (WBC) reduction in the UK, in‐process WBC‐reduction filters for pooled buffy coat (BC)‐derived platelet concentrates (PCs) and apheresis methods are used routinely for the production of WBC‐reduced PCs. While these strategies meet the specification for WBC reduction (< 5 × 106 WBCs/unit), the products from these processes may differ depending on the process employed and its performance. The aim of this study was therefore to investigate whether PCs prepared using various WBC‐reduction processes are sufficiently depleted of WBCs to limit cytokine accumulation during storage and to assess if cytokine levels detected in platelet products can serve as indicators of acceptable platelet activation as a result of the WBC‐reduction process.