Biological molecules are increasingly becoming a part of the therapeutics portfolio that has been either recently approved for marketing or those that are in the pipeline of several biotech and ...pharmaceutical companies. This is largely based on their ability to be highly specific relative to small molecules. However, by virtue of being a large protein, and having a complex structure with structural variability arising from production using recombinant gene technology in cell lines, such therapeutics run the risk of being recognized as foreign by a host immune system. In the context of immune-mediated adverse effects that have been documented to biological drugs thus far, including infusion reactions, and the evolving therapeutic platforms in the pipeline that engineer different functional modules in a biotherapeutic, it is critical to understand the interplay of the adaptive and innate immune responses, the pathophysiology of immunogenicity to biological drugs in instances where there have been immune-mediated adverse clinical sequelae and address technical approaches for their laboratory evaluation. The current paradigm in immunogenicity evaluation has a tiered approach to the detection and characterization of anti-drug antibodies (ADAs) elicited in vivo to a biotherapeutic; alongside with the structural, biophysical, and molecular information of the therapeutic, these analytical assessments form the core of the immunogenicity risk assessment. However, many of the immune-mediated adverse effects attributed to ADAs require the formation of a drug/ADA immune complex (IC) intermediate that can have a variety of downstream effects. This review will focus on the activation of potential immunopathological pathways arising as a consequence of circulating as well as cell surface bound drug bearing ICs, risk factors that are intrinsic either to the therapeutic molecule or to the host that might predispose to IC-mediated effects, and review the recent literature on prevalence and intensity of established examples of type II and III hypersensitivity reactions that follow the administration of a biotherapeutic. Additionally, we propose methods for the study of immune parameters specific to the biology of ICs that could be of use in conjunction with the detection of ADAs in circulation.
Abstract Rheumatoid arthritis (RA) is a multifactorial and polygenic immune-mediated disease, the pathogenesis of which involves different cell types. T and B lymphocytes, macrophages, endothelial ...cells, fibroblasts and osteoclasts have all been implicated in mediating the production of autoantibodies, proinflammatory cytokines and ultimately bone erosions. Cytotoxic T lymphocyte-associated antigen 4 immunoglobulin fusion protein (CTLA-4-Ig, abatacept) is a unique biologic agent targeting the co-stimulatory molecules CD80/CD86, and is indicated for the treatment of moderate-to-severe RA in patients who have had an inadequate response to one or more disease-modifying anti-rheumatic drugs, including methotrexate or anti-tumor necrosis factor agents. There is a growing body of evidence that, through selective modulation of the CD80/CD86 co-stimulatory molecules expressed by a variety of activated cell types, CTLA-4-Ig may inhibit the pathogenic RA process at several levels, both directly and indirectly. Here, we provide an overview of recent mechanistic studies of the action of CTLA-4-Ig on different cell types involved in mediating inflammation and joint damage in RA.
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This volume covers many topics in the field of T-cell costimulation. The need for such a volume is testament to the growth of the field. From its beginning as a concept in the 1980s, we have now ...progressed to the point where many molecules now have functionally defined roles in T-cell costimulation. In addition, the field has progressed 'from bench to bedside'. Abatacept cytotoxic T-lymphocyte antigen-4 (CTLA-4)-immunoglobulin (Ig) (CTLA-4-Ig), an inhibitor of CD28-mediated T-cell costimulation, was approved for the treatment of moderate-to-severe rheumatoid arthritis in 2006 by the Food and Drug Administration and in 2007 by the European Medicines Agency. This chapter first presents a personal historical perspective on the early basic studies on the elucidation of the CD28/B7 T-cell costimulatory pathway and the discovery of CTLA-4-Ig. We next present an overview of studies of CTLA-4-Ig in preclinical animal studies. The material discussed in these first two sections is selective rather than exhaustive; their purpose is to provide context for the final section, a summary of human clinical studies performed with abatacept.
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Agents targeting the PD1-PDL1 axis have transformed cancer therapy. Factors that influence clinical response to PD1-PDL1 inhibitors include tumor mutational burden, immune infiltration of the tumor, ...and local PDL1 expression. To identify peripheral correlates of the anti-tumor immune response in the absence of checkpoint blockade, we performed a retrospective study of circulating T cell subpopulations and matched tumor gene expression in melanoma and non-small cell lung cancer (NSCLC) patients. Notably, both melanoma and NSCLC patients whose tumors exhibited increased inflammatory gene transcripts presented high CD4
and CD8
central memory T cell (CM) to effector T cell (Eff) ratios in blood. Consequently, we evaluated CM/Eff T cell ratios in a second cohort of NSCLC. The data showed that high CM/Eff T cell ratios correlated with increased tumor PDL1 expression. Furthermore, of the 22 patients within this NSCLC cohort who received nivolumab, those with high CM/Eff T cell ratios, had longer progression-free survival (PFS) (median survival: 91 vs. 215 days). These findings show that by providing a window into the state of the immune system, peripheral T cell subpopulations inform about the state of the anti-tumor immune response and identify potential blood biomarkers of clinical response to checkpoint inhibitors in melanoma and NSCLC.
In giant cell arteritis, vessel-wall infiltrating CD4 T cells and macrophages form tissue-destructive granulomatous infiltrates, and the artery responds with a maladaptive response to injury, leading ...to intramural neoangiogenesis, intimal hyperplasia, and luminal occlusion. Lesion-residing T cells receive local signals, which represent potential therapeutic targets.
The authors examined how CD28 signaling affects vasculitis induction and maintenance, and which pathogenic processes rely on CD28-mediated T-cell activation.
Vasculitis was induced by transferring peripheral blood mononuclear cells from giant cell arteritis patients into immunodeficient NSG mice engrafted with human arteries. Human artery–NSG chimeras were treated with anti-CD28 domain antibody or control antibody. Treatment effects and immunosuppressive mechanisms were examined in vivo and in vitro applying tissue transcriptome analysis, immunohistochemistry, flow cytometry, and immunometabolic analysis.
Blocking CD28-dependent signaling markedly reduced tissue-infiltrating T cells and effectively suppressed vasculitis. Mechanistic studies implicated CD28 in activating AKT signaling, T-cell proliferation and differentiation of IFN-γ and IL-21–producing effector T cells. Blocking CD28 was immunosuppressive by disrupting T-cell metabolic fitness; specifically, the ability to utilize glucose. Expression of the glucose transporter Glut1 and of glycolytic enzymes as well as mitochondrial oxygen consumption were all highly sensitive to CD28 blockade. Also, induction and maintenance of CD4+CD103+ tissue-resident memory T cells, needed to replenish the vasculitic infiltrates, depended on CD28 signaling. CD28 blockade effectively suppressed vasculitis-associated remodeling of the vessel wall.
CD28 stimulation provides a metabolic signal required for pathogenic effector functions in medium and large vessel vasculitis. Disease-associated glycolytic activity in wall-residing T-cell populations can be therapeutically targeted by blocking CD28 signaling.
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Checkpoint inhibitors target the inhibitory receptors expressed by tumor-infiltrating T cells in order to reinvigorate an anti-tumor immune response. Therefore, understanding T cell composition and ...phenotype in human tumors is crucial. We analyzed by flow cytometry tumor-infiltrating lymphocytes (TILs) from two independent cohorts of patients with different cancer types, including RCC, lung, and colon cancer. In healthy donors, peripheral T cells are usually either CD4+ or CD8+ with a small percentage of CD4+ CD8+ DP cells (<5%). Compared to several other cancer types, including lung, and colorectal cancers, TILs from about a third of RCC patients showed an increased proportion of DP CD4+CD8+ T cells (>5%, reaching 30-50% of T cells in some patients). These DP T cells have an effector memory phenotype and express CD38, 4-1BB, and HLA-DR, suggesting antigen-driven expansion. In fact, TCR sequencing analysis revealed a high degree of clonality in DP T cells. Additionally, there were high levels of PD-1 and TIM-3 expression on DP T cells, which correlated with higher expression of PD-1 and TIM-3 in conventional single positive CD8 T cells from the same patients. These results suggest that DP T cells could be dysfunctional tumor-specific T cells with the potential to be reactivated by checkpoint inhibitors.
Targeting the CD28-CD80/86 pathway with an anti-CD28 antagonist is a promising alternative to current therapies for autoimmunity. However, attempts at generating conventional anti-CD28 mAbs lacking ...stimulatory activity has been challenging. In this study, we describe anti-human CD28 receptor antagonist domain Abs (dAbs) that are specific for human CD28. These dAbs are potent inhibitors of T cell activation, with an EC50 of 35 ± 14 ng/ml for inhibition of proliferation. The EC50 of 53 ± 11 ng/ml in an ex vivo CD28 receptor occupancy assay corresponds with in vitro functional activity, suggesting a direct correlation. The anti-CD28 dAb is equipotent in the inhibition of CD80- and CD86-mediated T cell proliferation and does not interfere with CTLA-4-mediated downmodulation of CD86 expression on APCs. The anti-CD28 dAbs are monomeric and do not demonstrate any evidence of agonism or costimulatory activity. In cynomolgus monkeys, the anti-CD28 dAb demonstrated pharmacodynamic activity, as measured by the inhibition of a T cell-dependent Ab response, without evidence of T cell depletion or cytokine release. Furthermore, there was a strong correlation between systemic exposure, duration, and extent of CD28 receptor occupancy, and pharmacodynamic activity. Taken together, these data support clinical evaluation of this novel anti-CD28 dAb for the treatment of autoimmune diseases.
Current clinical anti-CD40 biologic agents include both antagonist molecules for the treatment of autoimmune diseases and agonist molecules for immuno-oncology, yet the relationship between CD40 ...epitope and these opposing biological outcomes is not well defined. This report describes the identification of potent antagonist domain antibodies (dAbs) that bind to a novel human CD40-specific epitope that is divergent in the CD40 of nonhuman primates. A similarly selected anti-cynomolgus CD40 dAb recognizing the homologous epitope is also a potent antagonist. Mutagenesis, biochemical, and X-ray crystallography studies demonstrate that the epitope is distinct from that of CD40 agonists. Both the human-specific and cynomolgus-specific molecules remain pure antagonists even when formatted as bivalent Fc-fusion proteins, making this an attractive therapeutic format for targeting hCD40 in autoimmune indications.
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•Can agonist and antagonist activities be separated for CD40 therapeutic agents?•dAbs identified with unique human-specific antagonist epitope.•X-ray crystal structures show molecular details of dAb and antibody epitopes.•Cynomolgus-specific surrogate dAb molecules generated, also potent antagonist•dAbs targeted to unique epitope are functional antagonists as bivalent Fc-fusion.
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Objective
To determine at the phenotypic, functional, and transcriptional levels whether abatacept, a CTLA‐4Ig molecule that binds with high affinity to CD80/86 on antigen‐presenting cells (APCs) and ...is used to treat rheumatoid arthritis, induces a state of immunologic tolerance in T cells and dendritic cells in mice.
Methods
We investigated the capacity of abatacept to regulate the development of antigen‐specific immunologic tolerance in vivo using murine models of priming and tolerance to generate highly purified antigen‐specific T cell populations and CD11c+ APCs. These were combined with detailed immunologic and full genome transcriptional analyses.
Results
We found that abatacept inhibited T cell activation, but did not render T cells anergic or lead to the generation of Treg cells. However, it induced a sustained inhibition of T cell activation due to the inability of these cells to progress through the cell cycle following T cell receptor stimulation. We also observed that this state was accompanied by an inhibition of dendritic cell activation due to their reduced licensing by T cells.
Conclusion
This study provides detailed insight into the mode of action of abatacept, demonstrating that its effectiveness is not due to the induction of T cell tolerance, but rather to a sustained inhibition of T cell activation that results in reduced functionality of APCs, with significant implications for its clinical application.
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Objective
To develop an objective, readily measurable pharmacodynamic biomarker of glucocorticoid (GC) activity.
Methods
Genes modulated by prednisolone were identified from in vitro studies using ...peripheral blood mononuclear cells from normal healthy volunteers. Using the criteria of a >2‐fold change relative to vehicle controls and an adjusted P value cutoff of less than 0.05, 64 up‐regulated and 18 down‐regulated genes were identified. A composite score of the up‐regulated genes was generated using a single‐sample gene set enrichment analysis algorithm.
Results
GC gene signature expression was significantly elevated in peripheral blood leukocytes from normal healthy volunteers following oral administration of prednisolone. Expression of the signature increased in a dose‐dependent manner, peaked at 4 hours postadministration, and returned to baseline levels by 48 hours after dosing. Lower expression was detected in normal healthy volunteers who received a partial GC receptor agonist, which is consistent with the reduced transactivation potential of this compound. In cohorts of patients with systemic lupus erythematosus and patients with rheumatoid arthritis, expression of the GC signature was negatively correlated with the percentages of peripheral blood lymphocytes and positively correlated with peripheral blood neutrophil counts, which is consistent with the known biology of the GC receptor. Expression of the signature largely agreed with reported GC use in these populations, although there was significant interpatient variability within the dose cohorts.
Conclusion
The GC gene signature identified in this study represents a pharmacodynamic marker of GC exposure.
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