Meenu Wadhwa

“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.

Biosimilars: what clinicians should know

Biosimilar medicinal products (biosimilars) have become a reality in the European Union and will soon be available in the United States. Despite an established legal pathway for biosimilars in the European Union since 2005 and increasing and detailed regulatory guidance on data requirements for their development and licensing, many clinicians, particularly oncologists, are reluctant to consider biosimilars as a treatment option for their patients. Major concerns voiced about biosimilars relate to their pharmaceutical quality, safety (especially immunogenicity), efficacy (particularly in extrapolated indications), and interchangeability with the originator product. In this article, the members and experts of the Working Party on Similar Biologic Medicinal Products of the European Medicines Agency (EMA) address these issues. A clear understanding of the scientific principles of the biosimilar concept and access to unbiased information on licensed biosimilars are important for physicians to make informed and appropriate treatment choices for their patients. This will become even more important with the advent of biosimilar monoclonal antibodies. The issues also highlight the need for improved communication between physicians, learned societies, and regulators.

Biosimilars—why terminology matters

volume 29 number 8 august 2011 nature biotechnology very often don’t have time to devote employees to outside projects, such as IMI. Our editorial attempted to highlight the problem that the innovative agenda of EFPIA members may not be as broad as the innovative agenda put forward by less established and smaller companies who seek to disrupt conventional approaches. For example, it is clear that cells derived from human induced pluripotent stem cells offer considerable potential in drug discovery screens and safety assessment, and this has been demonstrated by the investment by the pharmaceutical industry in these approaches in recent years. But what about the potential of such products as experimental therapies in themselves? Clearly, a focus for many SMEs and academic groups but not a major focus for many major pharmaceutical companies. Perhaps IMI could play a role in moving such unconventional approaches forward, especially if the funding and expertise from EU and EFPIA could be used to help SMEs focus their efforts to address the formidable manufacturing, regulatory and reimbursement issues that cell therapies face before reaching the market. biomarker candidates for drug-induced injury of the kidney, the liver and the vascular system and established a generic strategy to qualify biomarkers4, whereas the eTOX consortium (http://www.e-tox. net/consortium.html), which includes four IT solution SMEs, developed an innovative multi-scale modeling strategy allowing the in silico prediction of drug effects on the heart using electrocardiogram simulations5. In parallel, four education and training projects are running, covering different areas of pharmaceutical sciences, including pharmacovigilance, of direct relevance to industry and regulatory authorities. Therefore, the alarmist and negative description of IMI reported in this journal does not reflect reality. In an era where biopharmaceutical companies rely more and more on noncompetitive research and open collaboration to develop new models for drug development, IMI offers unique opportunities for academic groups and SMEs interested in translating results of their endeavors into innovative therapies. The update of the IMI Scientific Research Agenda has just been completed and will result in a series of even more ambitious projects based on sharing of data and know-how to address major unmet medical needs. The currently running 4th Call for Proposals (Table 1) already contains two ‘Think Big’ projects with a transformational potential: the first aims at developing a European framework for patient-level health information, which will be exploited for investigations on major diseases in adult and pediatric populations; the second will focus on the use of induced pluripotent stem cells derived from patients as innovative tools for drug discovery and safety assessment. The budget of each project will be around €50 (

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

70) million, with equal contributions from the European Commission and companies in EFPIA (the European Federation of Pharmaceutical Industries and Associations), the latter in the form of inkind contributions. More than ever, the European Commission and EFPIA are determined to stimulate industry, including SMEs, and academia to collaborate on large-scale ‘game changing’ IMI projects to foster scientific talents and strengthen the ecosystem of pharmaceutical research across the European Union, for the ultimate benefit of patients.

Cytokines in skin lesions of psoriasis

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

Cytokine levels were compared in aqueous extracts of stratum corneum from psoriatic lesions and normal heel. Samples from heel contained high levels of interleukin-1 alpha (IL-1 alpha) and beta measured in immunoassays, although only the IL-1 alpha was biologically active. No other cytokines could be detected in heel samples. Interleukin-1 (IL-1) levels were dramatically reduced in lesional samples. A neutrophil chemoattractant was found in all lesional extracts, and was demonstrated to be mainly interleukin-8 (IL-8) using a specific neutralizing antiserum. Tumor necrosis factor alpha (TNF-alpha) and beta (TNF-beta), and interferon alpha (IFN-alpha) and gamma (IFN-gamma) were detected in lesional extracts using immunoassays, however, no equivalent biological activities could be detected. Interleukins 2 (IL-2), 4 (IL-4), and 6 (IL-6), granulocyte and granulocyte/macrophage colony stimulating factor (GM-CSF), could not be detected in any samples. IL-8 is therefore the only biologically active cytokine shown in this study to be elevated in psoriatic lesional extracts, and may therefore play a role in the pathogenesis of the disease.

Cytokine levels in platelet concentrates: quantitation by bioassays and immunoassays

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.

Increased IL-2, IL-4 and interferon-gamma (IFN-gamma) in steroid-sensitive nephrotic syndrome.

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.

Detection and measurement of cytokines.

We investigated the production of cytokines by peripheral blood mononuclear cells (PBMC) and serum cytokine concentrations in children with steroid‐sensitive idiopathic nephrotic syndrome (SSNS). PBMC from patients off treatment were collected during remission and relapse and cultured in medium alone or stimulated with calcium ionophore plus phorbol myristate acetate. Control PBMC were taken from healthy age‐matched children. IL‐2 was measured by bioassay, IL‐4 by immunoradiometric assay, and IL‐8 and IFN‐γ by ELISA. After 24 h culture without stimulation, IL‐2, IL‐4 and IFN‐γ were not detectable in the supernatant in any of the children. After stimulation, the supernatant concentrations of IL‐2 (median 172 U/ml at 24 h) and IL‐4 (160pg/ml at 24 h; 210pg/ml at 72 h) were significantly increased in relapse compared with remission (IL‐2 37 U/ml; IL‐4 65pg/ml and 60pg/ml) and controls (IL‐2 69 U/ml; IL‐4 40pg/ml and 40pg/ml) (P <0.05). The concentration of IFN‐γ was not significantly increased in relapse compared with remission and controls (600, 325, and 145 U/ml, respectively, at 72 h). IL‐8 concentrations were similar in relapse, remission and controls with stimulation (median 32, 40 and 40 ng/ml, respectively) and without (30, 17 and 10 ng/ml). IL‐2 was not detectable in serum, but IL‐4, IL‐8 and IFN‐γ were measurable in about half the patients, both in relapse and remission, though were virtually undetectable in controls. We conclude that relapse of SSNS in children is associated with T lymphocyte activation with release of IL‐2, IL‐4 and IFN‐γ.

Cytokines in sera from insulin-dependent diabetic patients at diagnosis.

Accurate and sensitive methods for the measurement and detection of cytokines are an obvious pre-requisite for the study of cytokine biology, biochemistry and the possible involvement of these molecules in pathology. In this review, the various methods available for cytokine measurement and detection (bioassays, immunoassays and other procedures) are described and compared. A critical appraisal of the potential advantages and limitations of the techniques is included.