Myelopoiesis of acute inflammation: lessons from TGN1412-induced cytokine storm.

TGN1412, a superagonist monoclonal antibody targeting CD28, caused cytokine storm in six healthy volunteers in a first-in-man study in 2006. Despite clinical improvement and termination of the cytokine release syndrome within days, anemia persisted in all patients with hemoglobin reaching baseline levels as much as 6 months later. Granulocytic dysplasia continued for 20 days in association with increased expression of CD69 and IL-4, but reduced IL-10; with resolution, this profile reversed to higher IL-10 expression and counter-balanced circannual cycling of IL-4 and IL-10 thereafter over 7 months. Along with immune cell subset and cytokine correlates monitored over 2 years, these observations offer unique insights into the expected changes in myelopoiesis and natural resolution in otherwise healthy young individuals in response to acute inflammation and cytokine storm in the absence of concomitant infection or comorbidity.

Are all cytokine storms the same?

Cytokine release syndrome (CRS) is the result of massive pro-inflammatory cytokine release and imbalance in the absence of adequate immunomodulation from signals such as interleukin (IL)-10, resulting in ongoing inflammation, tissue damage and death if left uncontrolled. Although CRS can result from different pro-inflammatory insults, the treatments proposed are similar, regardless of the phase of response. SARS-CoV-2 causes COVID-19, and CRS has been a defining feature of severe disease. Common approaches to treating CRS in other conditions are now applied to COVID-19 and, although some patients respond, it begs the following questions: (1) are all cytokine storms the same regardless of initiating insult, (2) can treatments be considered equally for all CRS events at any phase of the response, (3) can CRS be predicted based on dynamic acute biomarkers and, (4) should patients with CRS undergo long-term monitoring for secondary effects? The aim of this commentary is not to provide a review of COVID-19 pathophysiology or of cytokine storm, but rather to establish a foundation which could act as a platform to inform treatment approaches to CRS, regardless of cause, and the short- and long-term follow-up which may be necessary for affected patients.

Immune reconstitution and clinical recovery following anti-CD28 antibody (TGN1412)-induced cytokine storm.

Cytokine storm can result from cancer immunotherapy or certain infections, including COVID-19. Though short-term immune-related adverse events are routinely described, longer-term immune consequences and sequential immune monitoring are not as well defined. In 2006, six healthy volunteers received TGN1412, a CD28 superagonist antibody, in a first-in-man clinical trial and suffered from cytokine storm. After the initial cytokine release, antibody effect-specific immune monitoring started on Day + 10 and consisted mainly of evaluation of dendritic cell and T-cell subsets and 15 serum cytokines at 21 time-points over 2 years. All patients developed problems with concentration and memory; three patients were diagnosed with mild-to-moderate depression. Mild neutropenia and autoantibody production was observed intermittently. One patient suffered from peripheral dry gangrene, required amputations, and had persistent Raynaud's phenomenon. Gastrointestinal irritability was noted in three patients and coincided with elevated γδT-cells. One had pruritus associated with elevated IgE levels, also found in three other asymptomatic patients. Dendritic cells, initially undetectable, rose to normal within a month. Naïve CD8+ T-cells were maintained at high levels, whereas naïve CD4+ and memory CD4+ and CD8+ T-cells started high but declined over 2 years. T-regulatory cells cycled circannually and were normal in number. Cytokine dysregulation was especially noted in one patient with systemic symptoms. Over a 2-year follow-up, cognitive deficits were observed in all patients following TGN1412 infusion. Some also had signs or symptoms of psychological, mucosal or immune dysregulation. These observations may discern immunopathology, treatment targets, and long-term monitoring strategies for other patients undergoing immunotherapy or with cytokine storm.

Patients with gastrointestinal irritability after TGN1412-induced cytokine storm displayed selective expansion of gut-homing αß and γδT cells.

Following infusion of the anti-CD28 superagonist monoclonal antibody TGN1412, three of six previously healthy, young male recipients developed gastrointestinal irritability associated with increased expression of 'gut-homing' integrin ß7 on peripheral blood αßT cells. This subset of patients with intestinal symptoms also displayed a striking and persistent expansion of putative Vδ2+ γδT cells in the circulation which declined over a 2-year period following drug infusion, concordant with subsiding gut symptoms. These data demonstrate that TGN1412-induced gastrointestinal symptoms were associated with dysregulation of the 'gut-homing' pool of blood αß and γδT cells, induced directly by the antibody and/or arising from the subsequent cytokine storm.

Human Fcγ receptors compete for TGN1412 binding that determines the antibody's effector function.

The first-in-human clinical trial of the CD28-specific monoclonal antibody (mAb) TGN1412 resulted in a life-threatening cytokine release syndrome. Although TGN1412 was designed as IgG4, known for weak Fc:Fcγ receptor (FcγR) interactions, these interactions contributed to TGN1412-induced T-cell activation. Using cell lines (TFs) expressing human FcγRI, -IIa, -IIb, or -III, we show that TGN1412 and TGN1412 as IgG1 and IgG2 are bound by FcγRs as it can be deduced from literature. However, upon coculture of TGN1412-decorated T cells with TFs or human primary blood cells, we observed that binding capacities by FcγRs do not correlate with the strength of the mediated effector function. FcγRIIa and FcγRIIb, showing no or very minor binding to TGN1412, mediated strongest T cell proliferation, while high-affinity FcγRI, exhibiting strong TGN1412 binding, mediated hardly any T-cell proliferation. These findings are of biological relevance because we show that FcγRI binds TGN1412, thus prevents binding to FcγRIIa or FcγRIIb, and consequently disables T-cell proliferation. In line with this, FcγRI- FcγRII+ but not FcγRI+ FcγRII+ monocytes mediate TGN1412-induced T-cell proliferation. Collectively, by using TGN1412 as example, our results indicate that binding of monomeric IgG subclasses does not predict the FcγR-mediated effector function, which has major implications for the design of therapeutic mAbs.

Different players generate positive responses in two in vitro cytokine assay formats with aqueous and immobilized TGN1412 analog.

To detect potential risk of severe cytokine release syndrome, in vitro assay formats with human cells have been developed. The two major testing platforms are a combination of whole blood with aqueous-phase test articles (whole blood cytokine assay, WBCA) and peripheral blood mononuclear cells with solid-phase articles (PBMC assay). Significant induction of cytokines was seen in both assays after treatment with a widely used control agent, TGN1412 or its analog CD28SA, but the WBCA cytokine profile differed from what was expected from clinical experience. In the WBCA, potential risk of CD28SA was detected by elevation of IL-8 whereas IL-2, a key cytokine after stimulation of CD28, was not induced in approximately 40% of donor samples. Therefore, further mechanistic understanding of the different responses in the in vitro assay was needed. In this study of donor samples treated with CD28SA, we compared the induction of cytokines and identified the cytokine-producing cells in the two assays. IL-2 was markedly elevated in all the donors in the PBMC assay but only in 1 of 3 donors in the WBCA. IL-8, the most sensitive biomarker in the WBCA, was produced by monocytes and granulocytes. T cells, the most relevant player in the PBMC assay with CD28SA, did not contribute to the positive response seen in two donors in the WBCA, which suggests that different players caused the positive cytokine responses to CD28SA in the two assays.

An autologous endothelial cell:peripheral blood mononuclear cell assay that detects cytokine storm responses to biologics.

There is an urgent unmet need for human tissue bioassays to predict cytokine storm responses to biologics. Current bioassays that detect cytokine storm responses in vitro rely on endothelial cells, usually from umbilical veins or cell lines, cocultured with freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy adult volunteers. These assays therefore comprise cells from 2 separate donors and carry the disadvantage of mismatched tissues and lack the advantage of personalized medicine. Current assays also do not fully delineate mild (such as Campath) and severe (such as TGN1412) cytokine storm-inducing drugs. Here, we report a novel bioassay where endothelial cells grown from stem cells in the peripheral blood (blood outgrowth endothelial cells) and PBMCs from the same donor can be used to create an autologous coculture bioassay that responds by releasing a plethora of cytokines to authentic TGN1412 but only modestly to Campath and not to control antibodies such as Herceptin, Avastin, and Arzerra. This assay performed better than the traditional mixed donor assay in terms of cytokine release to TGN1412 and, thus, we suggest provides significant advancement and a definitive system by which biologics can be tested and paves the way for personalized medicine.

Cytokine release assays: current practices and future directions.

As a result of the CD28 superagonist biotherapeutic monoclonal antibody (TGN 1412) "cytokine storm" incident, cytokine release assays (CRA) have become hazard identification and prospective risk assessment tools for screening novel biotherapeutics directed against targets having a potential risk for eliciting adverse pro-inflammatory clinical infusion reactions. Different laboratories may have different strategies, assay formats, and approaches to the reporting, interpretation, and use of data for either decision making or risk assessment. Additionally, many independent contract research organizations (CROs), academic and government laboratories are involved in some aspect of CRA work. As a result, while some pharmaceutical companies are providing CRA data as part of the regulatory submissions when necessary, technical and regulatory practices are still evolving to provide data predictive of cytokine release in humans and that are relevant to safety. This manuscript provides an overview of different approaches employed by the pharmaceutical industry and CROs, for the use and application of CRA based upon a survey and post survey follow up conducted by ILSI-Health and Environmental Sciences Institute (HESI) Immunotoxicology Committee CRA Working Group. Also discussed is ongoing research in the academic sector, the regulatory environment, current limitations of the assays, and future directions and recommendations for cytokine release assays.

Severity of the TGN1412 trial disaster cytokine storm correlated with IL-2 release.

AIM: To determine if cytokine release with a solid phase assay is predictive of adverse responses for a range of therapeutic mAbs. METHODS: Cytokine ELISAs and a multi-array system were used to compare responses generated by different therapeutic mAbs using a solid phase assay. Flow cytometry was employed to determine the cellular source of those cytokines. RESULTS: Only TGN1412 and muromonab-CD3 stimulated CD4+ T-cell mediated cytokine release characterized by significant (all P < 0.0001) IFNγ, TNFα, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17 and IL-22 release, comparable with T-cell mitogen. Significantly greater (P < 0.0001) IL-2 release with TGN1412 (2894-6051 pg ml⁻¹) compared with muromonab-CD3 (62-262 pg ml⁻¹) differentiated otherwise comparable cytokine responses. Likewise, TGN1412 stimulated significantly more (P = 0.0001) IL-2 producing CD4+ T-cells than muromonab-CD3 and induced Th1, Th2, Th17 and Th22 subsets that co-release this cytokine. Significant TNFα release was observed with bevacizumab (P = 0.0001), trastuzumab (P = 0.0031) and alemtuzumab (P = 0.0177), but no significant IL-2 release. TGN1412 and muromonab-CD3 caused pro-inflammatory cytokine release despite significantly (both P < 0.0001) increasing numbers of T-cells with a regulatory phenotype. CONCLUSIONS: The severity of the adverse response to TGN1412 compared with muromonab-CD3 and other therapeutic mAbs correlates with the level of IL-2 release.

The blood endothelial cell chamber - An innovative system to study immune responses in drug development.

The risk for adverse immune-mediated reactions, associated with the administration of certain immunotherapeutic agents, should be mitigated early. Infusion reactions to monoclonal antibodies and other biopharmaceuticals, known as cytokine release syndrome, can arise from the release of cytokines via the drug target cell, as well as the recruitment of immune effector cells. While several in vitro cytokine release assays have been proposed up to date, many of them lack important blood components, required for this response to occur. The blood endothelial cell chamber model is an in vitro assay, composed of freshly drawn human whole blood and cultured human primary endothelial cells. Herein, its potential to study the compatibility of immunotherapeutics with the human immune system was studied by evaluating three commercially available monoclonal antibodies and bacterial endotoxin lipopolysaccharide. We demonstrate that the anti-CD28 antibody TGN1412 displayed an adaptive cytokine release profile and a distinct IL-2 response, accompanied with increased CD3+ cell recruitment. Alemtuzumab exhibited a clear cytokine response with a mixed adaptive/innate source (IFNγ, TNFα and IL-6). Its immunosuppressive nature is observed in depleted CD3+ cells. Cetuximab, associated with low infusion reactions, showed a very low or absent stimulatory effect on proinflammatory cytokines. In contrast, bacterial endotoxin demonstrated a clear innate cytokine response, defined by TNFα, IL-6 and IL-1ß release, accompanied with a strong recruitment of CD14+CD16+ cells. Therefore, the blood endothelial cell chamber model is presented as a valuable in vitro tool to investigate therapeutic monoclonal antibodies with respect to cytokine release and vascular immune cell recruitment.

18th world congress of basic and clinical pharmacology: thought-provoking lectures on drug safety issues.

Introduction: Pharmacology of the Future for Science, Drug Development and Therapeutics was the leitmotif which guided the presentations at the 18th World Congress of Basic and Clinical Pharmacology held in Kyoto in July 2018 (WPC2018).Areas covered: Of the 380 invited lectures, this report reviews the opening presentation on immune checkpoint inhibitors and three talks dealing with drug safety issues (irreproducibility of nonclinical data, clinical Phase 1 catastrophes by TGN1214 and BIA-102,474-101 in healthy volunteers, and Phase I sentinel dosing to reduce risk to clinical study participants).Expert opinion: The nonclinical safety assessment of drug candidates preceding clinical investigations requires the adoption of more human predictable biological assays and a careful and critical analysis of all available knowledge on a candidate to ensure the safety of clinical trial participants.

TGN-1412 and BIA-2474 Trials with Tragic End: Lessons Learnt To Make Clinical Trials Safer.

BACKGROUND: Globally, there have been tremendous efforts by regulatory authorities to make clinical trials safer by making stringent clinical trial regulations. Despite this, we witnessed several tragic events. TGN1412 and BIA 10-2474 phase I trials are infamous trails in which healthy volunteers either succumbed to severe adverse effects or faced irreversible impairments of the test drug. Such afflictions in clinical trials are not only turbulent to the image of pharmaceutical industry but it also conveys dispiriting message for clinical trial participants. OBJECTIVE: To make clinical trials safer for participants, some regulatory changes are warranted. METHODS: Some stipulated measures to improve safety of clinical trial participants include inclusion of patients instead of healthy volunteers in phase I clinical trials, all compounds which are used for first time in humans should be considered as high risk compounds, amendments in first in human clinical trial design to N of 1 randomized control trial in place of 6+2 design with the sequential dosing of subjects both within and between cohorts and the individual patient pharmacokinetic and pharmacodynamic data should be used to calculate sequential dosing. Besides these, there should be appropriate process for systematic risk assessment involving the use of statistical techniques to select pertinent risk factors with high predictive values of studies or sites that may be procumbent to non-compliance. CONCLUSION: Inclusion of above mentioned measures in clinical trials are bound to make them safer and may help in pacifying the insecurity that has emerged among humans to participate in clinical trials.

Self-Recognition Sensitizes Mouse and Human Regulatory T Cells to Low-Dose CD28 Superagonist Stimulation.

In rodents, low doses of CD28-specific superagonistic monoclonal antibodies (CD28 superagonists, CD28SA) selectively activate regulatory T cells (Treg). This observation has recently been extended to humans, suggesting an option for the treatment of autoimmune and inflammatory diseases. However, a mechanistic explanation for this phenomenon is still lacking. Given that CD28SA amplify T cell receptor (TCR) signals, we tested the hypothesis that the weak tonic TCR signals received by conventional CD4+ T cells (Tconv) in the absence of cognate antigen require more CD28 signaling input for full activation than the stronger TCR signals received by self-reactive Treg. We report that in vitro, the response of mouse Treg and Tconv to CD28SA strongly depends on MHC class II expression by antigen-presenting cells. To separate the effect of tonic TCR signals from self-peptide recognition, we compared the response of wild-type Treg and Tconv to low and high CD28SA doses upon transfer into wild-type or H-2M knockout mice, which lack a self-peptide repertoire. We found that the superior response of Treg to low CD28SA doses was lost in the absence of self-peptide presentation. We also tested if potentially pathogenic autoreactive Tconv would benefit from self-recognition-induced sensitivity to CD28SA stimulation by transferring TCR transgenic OVA-specific Tconv into OVA-expressing mice and found that low-dose CD28SA application inhibited, rather than supported, their expansion, presumably due to the massive concomitant activation of Treg. Finally, we report that also in the in vitro response of human peripheral blood mononuclear cells to CD28SA, HLA II blockade interferes with the expansion of Treg by low-dose CD28SA stimulation. These results provide a rational basis for the further development of low-dose CD28SA therapy for the improvement of Treg activity.

The rise and fall of the CD28 superagonist TGN1412 and its return as TAB08: a personal account.

Two decades ago, we discovered 'superagonistic' monoclonal antibodies specific for the CD28 molecule which are able to polyclonally activate T cells, in particular regulatory T cells, and are therapeutically active in many rodent models of autoimmunity, inflammation, transplantation, and tissue repair. A phase I trial of the human CD28 superagonist TGN1412 failed in 2006 due to an unexpected cytokine release syndrome, but after it became clear that dose-reduction allows to preferentially address regulatory T cells also in humans, clinical development was resumed under the name TAB08. Here, I recount the story of CD28 superagonist development from a personal perspective with an emphasis on the dramatic events during and after the 2006 phase I trial, the reasons for the failure of preclinical research to warn of the impending cytokine storm, and on the research which allowed resumption of clinical development.

Cytokine release assays for the prediction of therapeutic mAb safety in first-in man trials--Whole blood cytokine release assays are poorly predictive for TGN1412 cytokine storm.

The therapeutic monoclonal antibody (mAb) TGN1412 (anti-CD28 superagonist) caused near-fatal cytokine release syndrome (CRS) in all six volunteers during a phase-I clinical trial. Several cytokine release assays (CRAs) with reported predictivity for TGN1412-induced CRS have since been developed for the preclinical safety testing of new therapeutic mAbs. The whole blood (WB) CRA is the most widely used, but its sensitivity for TGN1412-like cytokine release was recently criticized. In a comparative study, using group size required for 90% power with 5% significance as a measure of sensitivity, we found that WB and 10% (v/v) WB CRAs were the least sensitive for TGN1412 as these required the largest group sizes (n = 52 and 79, respectively). In contrast, the peripheral blood mononuclear cell (PBMC) solid phase (SP) CRA was the most sensitive for TGN1412 as it required the smallest group size (n = 4). Similarly, the PBMC SP CRA was more sensitive than the WB CRA for muromonab-CD3 (anti-CD3) which stimulates TGN1412-like cytokine release (n = 4 and 4519, respectively). Conversely, the WB CRA was far more sensitive than the PBMC SP CRA for alemtuzumab (anti-CD52) which stimulates FcγRI-mediated cytokine release (n = 8 and 180, respectively). Investigation of potential factors contributing to the different sensitivities revealed that removal of red blood cells (RBCs) from WB permitted PBMC-like TGN1412 responses in a SP CRA, which in turn could be inhibited by the addition of the RBC membrane protein glycophorin A (GYPA); this observation likely underlies, at least in part, the poor sensitivity of WB CRA for TGN1412. The use of PBMC SP CRA for the detection of TGN1412-like cytokine release is recommended in conjunction with adequately powered group sizes for dependable preclinical safety testing of new therapeutic mAbs.

CD28 co-stimulation in T-cell homeostasis: a recent perspective.

T-cells play a key role within the adaptive immune system mediating cellular immunity and orchestrating the immune response as a whole. Their activation requires not only recognition of antigen/major histocompatibility complexes by the T-cell receptor but in addition co-stimulation via the CD28 molecule through binding to CD80, CD86, or as recently discovered, inducible co-stimulator ligand expressed by antigen-presenting cells. Apart from tight control of the co-stimulatory signal by the T-cell receptor complex, expression of the inhibitory receptor cytotoxic T-lymphocyte antigen-4 (CTLA-4) sharing its ligands with CD28 is required to avoid inappropriate or prolonged T-cell activation. CD4(+) Foxp3(+) regulatory T (Treg) cells, which are crucial inhibitors of autoimmunity, add another level of complexity in that they differ from conventional non-regulatory CD4(+) T-cells by strongly depending on CD28 signaling for their generation and homeostasis. Moreover, CTLA-4 is constitutively expressed by Treg cells where it serves as a key mediator of suppression, while conventional CD4(+) T-cells express CTLA-4 only after activation. Here, we discuss recent insights into the molecular events underlying CD28-mediated co-stimulation, its impact on gene regulation, and the differential role of CD28 expression on Treg cells versus conventional CD4(+) and CD8(+) T-cells. Moreover, we summarize the exciting therapeutic options which have arisen from our current understanding of T-cell co-stimulation. Some of these have already been translated into the clinic, while others are expected to follow soon due to promising preclinical results. In particular, we discuss the failed 2006 trial of the CD28 superagonist TGN1412, and the return of this potent T-cell activator to clinical development.

Failure to upregulate cell surface PD-1 is associated with dysregulated stimulation of T cells by TGN1412-like CD28 superagonist.

The CD28 superagonist (CD28SA) TGN1412 was administered to humans as an agent that can selectively activate and expand regulatory T cells but resulted in uncontrolled T cell activation accompanied by cytokine storm. The molecular mechanisms that underlie this uncontrolled T cell activation are unclear. Physiological activation of T cells leads to upregulation of not only activation molecules but also inhibitory receptors such as PD-1. We hypothesized that the uncontrolled activation of CD28SA-stimulated T cells is due to both the enhanced expression of activation molecules and the lack of or reduced inhibitory signals. In this study, we show that anti-CD3 antibody-stimulated human T cells undergo time-limited controlled DNA synthesis, proliferation and interleukin-2 secretion, accompanied by PD-1 expression. In contrast, CD28SA-activated T cells demonstrate uncontrolled activation parameters including enhanced expression of LFA-1 and CCR5 but fail to express PD-1 on the cell surface. We demonstrate the functional relevance of the lack of PD-1 mediated regulatory mechanism in CD28SA-stimulated T cells. Our findings provide a molecular explanation for the dysregulated activation of CD28SA-stimulated T cells and also highlight the potential for the use of differential expression of PD-1 as a biomarker of safety for T cell immunostimulatory biologics.

TGN1412: From Discovery to Disaster.

After a drug is confirmed as safe and efficacious in preclinical studies, it is tested in healthy human volunteers for first in man trials. In 2006, a phase I clinical study was conducted for a CD28 superagonist antibody TGN1412 in six human volunteers. After very first infusion of a dose 500 times smaller than that found safe in animal studies, all six human volunteers faced life-threatening conditions involving multiorgan failure for which they were moved to intensive care unit. After this particular incident, a lot was changed over how first in man trials are approved by regulatory authorities and the way clinical trials are conducted. This review primarily deals with preclinical studies conducted by TeGenero, results of which encouraged them to test the antibody on human subjects, reasons why this drug failed in human trial and aftermath of this drug trial. In addition, another drug-Fialuridine which failed in phase 2 clinical trial leading to death of five human subjects is briefly reviewed.