Swaminathan Sethu

Challenges and approaches for the development of safer immunomodulatory biologics.

Immunomodulatory biologics, which render their therapeutic effects by modulating or harnessing immune responses, have proven their therapeutic utility in several complex conditions including cancer and autoimmune diseases. However, unwanted adverse reactions — including serious infections, malignancy, cytokine release syndrome, anaphylaxis and hypersensitivity as well as immunogenicity — pose a challenge to the development of new (and safer) immunomodulatory biologics. In this article, we assess the safety issues associated with immunomodulatory biologics and discuss the current approaches for predicting and mitigating adverse reactions associated with their use. We also outline how these approaches can inform the development of safer immunomodulatory biologics.

Immunogenicity to Biologics: Mechanisms, Prediction and Reduction

Currently, there is a significant rise in the development and clinical use of a unique class of pharmaceuticals termed as Biopharmaceuticals or Biologics, in the management of a range of disease conditions with, remarkable therapeutic benefits. However, there is an equally growing concern regarding development of adverse effects like immunogenicity in the form of anti-drug antibodies (ADA) production and hypersensitivity. Immunogenicity to biologics represents a significant hurdle in the continuing therapy of patients in a number of disease settings. Efforts focussed on the identification of factors that contribute towards the onset of immunogenic response to biologics have led to reductions in the incidence of immunogenicity. An in-depth understanding of the cellular and molecular mechanism underpinning immunogenic responses will likely improve the safety profile of biologics. This review addresses the mechanistic basis of ADA generation to biologics, with emphasis on the role of antigen processing and presentation in this process. The article also addresses the potential contribution of complement system in augmenting or modulating this response. Identifying specific factors that influences processing and presentation of biologic-derived antigens in different genotype and disease background may offer additional options for intervention in the immunogenic process and consequently, the management of immunogenicity to biologics.

Loss of Transcription Factor Nuclear Factor-Erythroid 2 (NF-E2) p45-related Factor-2 (Nrf2) Leads to Dysregulation of Immune Functions, Redox Homeostasis, and Intracellular Signaling in Dendritic Cells

Background: Nrf2 has been implicated in regulating immune cell signaling and function. Results: Nrf2-deficient murine DCs exhibit enhanced maturation phenotype, increased ROS levels with dysregulation of antigen uptake capabilities, and altered intracellular signaling. Conclusion: Nrf2 regulates DC intracellular redox and immune function. Significance: Defining the role of Nrf2 in DC biology underpins development of potential Nrf2 targeted immunotherapeutics. Dendritic cells (DCs) are critical mediators of immunity and immune tolerance by orchestrating multiple aspects of T cell activation and function. Immature DCs (iDCs) expressing low levels of co-stimulatory receptors are highly efficient at antigen capture but are poor activators of T cells. Maturation of DCs is associated with increased expression of co-stimulatory molecules. Co-stimulatory receptor gene expression is regulated by intracellular redox, NF-κB, and MAPK pathways and by histone deacetylase (HDAC) activity. The transcription factor, Nrf2, is important for maintaining intracellular glutathione (GSH) levels and redox homeostasis and has been implicated in modulating DC co-stimulatory receptor expression. It is unclear whether Nrf2 mediates this effect by GSH-dependent mechanisms and whether it influences DC signaling pathways. Using bone marrow-derived iDCs from Nrf2+/+ and Nrf2−/− mice, we demonstrate that Nrf2−/− iDCs have lower basal GSH levels, enhanced co-stimulatory receptor expression, impaired phagocytic functions, and increased antigen-specific CD8 T cell stimulation capacity. Interestingly, lowering GSH levels in Nrf2+/+ iDCs did not recapitulate the Nrf2−/− iDC phenotype. Loss of Nrf2 resulted in elevated basal levels of reactive oxygen species but did not affect basal NF-κB activity or p38 MAPK phosphorylation. Using pharmacological inhibitors, we demonstrate that enhanced co-stimulatory receptor phenotype of Nrf2−/− iDC does not require ERK activity but is dependent on HDAC activity, indicating a potential interaction between Nrf2 function and HDAC. These results suggest that Nrf2 activity is required to counter rises in intracellular reactive oxygen species and to regulate pathways that control DC co-stimulatory receptor expression.

Safety pharmacology--current and emerging concepts.

Safety pharmacology (SP) is an essential part of the drug development process that aims to identify and predict adverse effects prior to clinical trials. SP studies are described in the International Conference on Harmonisation (ICH) S7A and S7B guidelines. The core battery and supplemental SP studies evaluate effects of a new chemical entity (NCE) at both anticipated therapeutic and supra-therapeutic exposures on major organ systems, including cardiovascular, central nervous, respiratory, renal and gastrointestinal. This review outlines the current practices and emerging concepts in SP studies including frontloading, parallel assessment of core battery studies, use of non-standard species, biomarkers, and combining toxicology and SP assessments. Integration of the newer approaches to routine SP studies may significantly enhance the scope of SP by refining and providing mechanistic insight to potential adverse effects associated with test compounds.

Concise Review: Workshop Review: Understanding and Assessing the Risks of Stem Cell-Based Therapies

The field of stem cell therapeutics is moving ever closer to widespread application in the clinic. However, despite the undoubted potential held by these therapies, the balance between risk and benefit remains difficult to predict. As in any new field, a lack of previous application in man and gaps in the underlying science mean that regulators and investigators continue to look for a balance between minimizing potential risk and ensuring therapies are not needlessly kept from patients. Here, we attempt to identify the important safety issues, assessing the current advances in scientific knowledge and how they may translate to clinical therapeutic strategies in the identification and management of these risks. We also investigate the tools and techniques currently available to researchers during preclinical and clinical development of stem cell products, their utility and limitations, and how these tools may be strategically used in the development of these therapies. We conclude that ensuring safety through cutting‐edge science and robust assays, coupled with regular and open discussions between regulators and academic/industrial investigators, is likely to prove the most fruitful route to ensuring the safest possible development of new products.

Nuclear factor-erythroid 2 (NF-E2) p45-related factor-2 (Nrf2) modulates dendritic cell immune function through regulation of p38 MAPK-cAMP-responsive element binding protein/activating transcription factor 1 signaling.

Background: Nrf2 is required for normal dendritic cell immune functions. Results: Loss of Nrf2 alters DC function and results in hyperphosphorylation of CREB/ATF1 transcription factors that are responsive to p38 MAPK inhibition. Conclusion: The p38 MAPK-CREB/ATF1 axis contributes to Nrf2-mediated regulation of DC function. Significance: Defining the relevance of p38-CREB/ATF1 in Nrf2 signaling expands understanding of DC biology. Nrf2 is a redox-responsive transcription factor that has been implicated in the regulation of DC immune function. Loss of Nrf2 results in increased co-stimulatory molecule expression, enhanced T cell stimulatory capacity, and increased reactive oxygen species (ROS) levels in murine immature DCs (iDCs). It is unknown whether altered immune function of Nrf2-deficient DCs (Nrf2−/− iDCs) is due to elevated ROS levels. Furthermore, it is unclear which intracellular signaling pathways are involved in Nrf2-mediated regulation of DC function. Using antioxidant vitamins to reset ROS levels in Nrf2−/− iDCs, we show that elevated ROS is not responsible for the altered phenotype and function of these DCs. Pharmacological inhibitors were used to explore the role of key MAPKs in mediating the altered phenotype and function in Nrf2−/− iDCs. We demonstrate that the increased co-stimulatory molecule expression (MHC II and CD86) and antigen-specific T cell activation capacity observed in Nrf2−/− iDCs was reversed by inhibition of p38 MAPK but not JNK. Importantly, we provide evidence for increased phosphorylation of cAMP-responsive element binding protein (CREB) and activating transcription factor 1 (ATF1), transcription factors that are downstream of p38 MAPK. The increased phosphorylation of CREB/ATF1 in Nrf2−/− iDCs was sensitive to p38 MAPK inhibition. We also show data to implicate heme oxygenase-1 as a potential molecular link between Nrf2 and CREB/ATF1. These results indicate that dysregulation of p38 MAPK-CREB/ATF1 signaling axis underlies the altered function and phenotype in Nrf2-deficient DCs. Our findings provide new insights into the mechanisms by which Nrf2 mediates regulation of DC function.

Heme Oxygenase-1 Regulates Dendritic Cell Function through Modulation of p38 MAPK-CREB/ATF1 Signaling*

Background: HO-1 contributes to redox homeostasis and regulation of immature dendritic cell (DC) phenotype. Results: HO-1 inhibition results in increased ROS, activation of p38 MAPK-CREB/ATF1 pathway, and dysregulation of DC phenotype and function. Conclusion: HO-1 influences DC function through effects on p38 MAPK-CREB/ATF1 signaling pathway. Significance: This study provides new insights into the molecular pathways influenced by HO-1 in DCs. Dendritic cells (DCs) are critical for the initiation of immune responses including activation of CD8 T cells. Intracellular reactive oxygen species (ROS) levels influence DC maturation and function. Intracellular heme, a product of catabolism of heme-containing metalloproteins, is a key inducer of ROS. Intracellular heme levels are regulated by heme oxygenase-1 (HO-1), which catalyzes the degradation of heme. Heme oxygenase-1 has been implicated in regulating DC maturation; however, its role in other DC functions is unclear. Furthermore, the signaling pathways modulated by HO-1 in DCs are unknown. In this study, we demonstrate that inhibition of HO-1 activity in murine bone marrow-derived immature DCs (iDCs) resulted in DCs with raised intracellular ROS levels, a mature phenotype, impaired phagocytic and endocytic function, and increased capacity to stimulate antigen-specific CD8 T cells. Interestingly, our results reveal that the increased ROS levels following HO-1 inhibition did not underlie the changes in phenotype and functions observed in these iDCs. Importantly, we show that the p38 mitogen-activated protein kinase (p38 MAPK), cAMP-responsive element binding protein (CREB), and activating transcription factor 1 (ATF1) pathway is involved in the mediation of the phenotypic and functional changes arising from HO-1 inhibition. Furthermore, up-regulation of HO-1 activity rendered iDCs refractory to lipopolysaccharide-induced activation of p38 MAPK-CREB/ATF1 pathway and DC maturation. Finally, we demonstrate that treatment of iDC with the HO-1 substrate, heme, recapitulates the effects that result from HO-1 inhibition. Based on these results, we conclude that HO-1 regulates DC maturation and function by modulating the p38 MAPK-CREB/ATF1 signaling axis.

T Cell Co-inhibitory Receptors-Functions and Signalling Mechanisms

T cell activation is a central event in the adaptive immune response and essentially begins with the recognition of an antigenic peptide in the context of a major histocompatibility complex (MHC) on an antigen-presenting cell by the T cell receptor (TCR). The process of T cell activation consists of an orchestration of various functional modules such as actin polymerization, cell surface receptor patterning, calcium fluxing, immunological synapse formation, enhanced adhesion and gene transcription. These modules are mediated by a number of signalling proteins through inducible phosphorylation, enzyme activation and protein-protein & protein-lipid interactions. This complex and dynamic interplay of signalling events governs the decisions the T cell makes in terms of gene expression, proliferation, differentiation, survival and migration. These outcomes are influenced by the magnitude, duration and context of the activation signals. The activation signals have the potential to be modulated by a family of receptors termed, co-inhibitory receptors that include PD-1, LAG-3, TIM-3 and CTLA-4. Co-inhibitory receptors modulate signalling by utilising mechanisms such as ectodomain competition with counter receptors and by the use of intracellular mediators such as protein phosphatases. Co-inhibitory receptors can act as threshold-setters, modulators, check-points and feedback mechanisms that can potentially fine tune the quality and magnitude of the T cell immune response. Given the key roles of these receptors in modulating the immune response, they are increasingly being targeted for immune intervention in a variety of disease settings. This review discusses current understanding of the role of co-inhibitory receptors in influencing T cell signalling.

Immunoglobulin G1 and immunoglobulin G4 antibodies in multiple sclerosis patients treated with IFNβ interact with the endogenous cytokine and activate complement

A subset of patients with relapsing-remitting multiple sclerosis (RRMS) on therapy with interferon beta (IFNβ) develop neutralising anti-drug antibodies (ADA) resulting in reduced, or loss of, therapeutic efficacy. The aims were to characterise the relative contributions of anti-IFNβ antibody isotypes to drug neutralising activity, ability of these antibodies to cross-react with endogenous IFNβ, to form immune complexes and activate complement. IFNβ-specific ADA were measured in plasma from RRMS patients treated with IFNβ1a (Rebif®). Neutralisation of endogenous and therapeutic IFNβ by ADA was determined by IFNβ bioassay. IFNβ-ADA profile was predominantly comprised of IgG1 and IgG4 antibody isotypes. The contribution of IgG4-ADA towards neutralising activity was found to be minimal. Neutralising IFNβ-ADA blocks endogenous IFNβ activity. ADA interaction with therapeutic IFNβ results in immune complex formation and complement activation. In summary, IgG1 and IgG4 IFNβ-ADA have the ability to neutralise therapeutic and endogenous protein and to activate complement.

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.