Clinical Relevance of Anti-TNF Antibody Trough Levels and Anti-Drug Antibodies in Treating Inflammatory Bowel Disease Patients.

BACKGROUND AND AIMS: The majority of patients treated with anti-tumor necrosis factor (TNF) therapy develop anti-drug antibodies (ADAs), which might result in loss of treatment efficacy. Strict guidelines on measuring trough levels (TLs) and ADA in clinical routine do not exist. To provide real-world data, we took advantage of our tertiary inflammatory bowel disease (IBD) center patient cohort and determined indicators for therapeutic drug monitoring (TDM) and actual consequences in patient care. METHODS: We retrospectively collected clinical data of 104 IBD patients treated with infliximab or adalimumab in our IBD clinic. Patients with TL and ADA measurements between June 2015 and February 2018 were included. RESULTS: The main reason for determining TL was increased clinical disease. Subtherapeutic TLs were found in 33 patients, therapeutic TLs in 33 patients, and supratherapeutic TLs in 38 patients. Adjustments in anti-TNF therapy occurred more frequently (p = 0.01) in patients with subtherapeutic TL (24 of 33 patients; 73%) as compared to patients with therapeutic and supratherapeutic TLs (26 of 71 patients; 37%). No correlation could be found between TL and disease activity (p = 0.16). Presence of ADA was found in 16 patients, correlated with the development of infusion reactions (OR: 10.6, RR: 5.4, CI: 2.9-38.6), and was associated with subtherapeutic TL in 15 patients (93.8%). Treatment adaptations were based on TL and/or ADA presence in 36 of 63 patients. CONCLUSIONS: TDM showed significant treatment adaptations in patients with subtherapeutic TL. Conversely, in patients with therapeutic and supratherapeutic TLs, reasons for adaptations were based on considerations other than TL, such as clinical disease activity. Further studies should focus on decision-making in patients presenting with supratherapeutic TL in remission.

Systemic and mucosal antibody responses specific to SARS-CoV-2 during mild versus severe COVID-19.

BACKGROUND: Whereas severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody tests are increasingly being used to estimate the prevalence of SARS-CoV-2 infection, the determinants of these antibody responses remain unclear. OBJECTIVES: Our aim was to evaluate systemic and mucosal antibody responses toward SARS-CoV-2 in mild versus severe coronavirus disease 2019 (COVID-19) cases. METHODS: Using immunoassays specific for SARS-CoV-2 spike proteins, we determined SARS-CoV-2-specific IgA and IgG in sera and mucosal fluids of 2 cohorts, including SARS-CoV-2 PCR-positive patients (n = 64) and PCR-positive and PCR-negtive health care workers (n = 109). RESULTS: SARS-CoV-2-specific serum IgA titers in patients with mild COVID-19 were often transiently positive, whereas serum IgG titers remained negative or became positive 12 to 14 days after symptom onset. Conversely, patients with severe COVID-19 showed a highly significant increase of SARS-CoV-2-specific serum IgA and IgG titers after symptom onset. Very high titers of SARS-CoV-2-specific serum IgA were correlated with severe acute respiratory distress syndrome. Interestingly, some health care workers with negative SARS-CoV-2-specific serum antibody titers showed SARS-CoV-2-specific IgA in mucosal fluids with virus-neutralizing capacity in some cases. SARS-CoV-2-specific IgA titers in nasal fluids were inversely correlated with age. CONCLUSIONS: Systemic antibody production against SARS-CoV-2 develops mainly in patients with severe COVID-19, with very high IgA titers seen in patients with severe acute respiratory distress syndrome, whereas mild disease may be associated with transient production of SARS-CoV-2-specific antibodies but may stimulate mucosal SARS-CoV-2-specific IgA secretion.

Systematic Review of Safety and Efficacy of Rituximab in Treating Immune-Mediated Disorders.

Background: During the past years biologic agents (also termed biologicals or biologics) have become a crucial treatment option in immunological diseases. Numerous articles have been published on biologicals, which complicates the decision making process on the use of the most appropriate biologic for a given immune-mediated disease. This systematic review is the first of a series of articles assessing the safety and efficacy of B cell-targeting biologics for the treatment of immune-mediated diseases. Objective: To evaluate rituximab's safety and efficacy for the treatment of immune-mediated disorders compared to placebo, conventional treatment, or other biologics. Methods: The PRISMA checklist guided the reporting of the data. We searched the PubMed database between 4 October 2016 and 26 July 2018 concentrating on immune-mediated disorders. Results: The literature search identified 19,665 articles. After screening titles and abstracts against the inclusion and exclusion criteria and assessing full texts, 105 articles were finally included in a narrative synthesis. Conclusions: Rituximab is both safe and effective for the treatment of acquired angioedema with C1-inhibitor deficiency, ANCA-associated vasculitis, autoimmune hemolytic anemia, Behçet's disease, bullous pemphigoid, Castleman's disease, cryoglobulinemia, Goodpasture's disease, IgG4-related disease, immune thrombocytopenia, juvenile idiopathic arthritis, relapsing-remitting multiple sclerosis, myasthenia gravis, nephrotic syndrome, neuromyelitis optica, pemphigus, rheumatoid arthritis, spondyloarthropathy, and systemic sclerosis. Conversely, rituximab failed to show an effect for antiphospholipid syndrome, autoimmune hepatitis, IgA nephropathy, inflammatory myositis, primary-progressive multiple sclerosis, systemic lupus erythematosus, and ulcerative colitis. Finally, mixed results were reported for membranous nephropathy, primary Sjögren's syndrome and Graves' disease, therefore warranting better quality trials with larger patient numbers.

Staphylococcus aureus PSM peptides induce tolerogenic dendritic cells upon treatment with ligands of extracellular and intracellular TLRs.

Dendritic cells (DCs) are key players of the immune system and thus a target for immune evasion by pathogens. We recently showed that the virulence factor phenol-soluble modulin (PSM) produced by community-associated methicillin-resistant Staphylococcus aureus strains induces tolerogenic DCs upon Toll-like receptor (TLR) 2 activation via the p38-CREB-IL-10 pathway. Here, we addressed the question whether this tolerogenic phenotype of DCs induced by PSMs is specific for TLR2 activation. Therefore, bone marrow-derived DCs were treated with various ligands for extracellular and intracellular TLRs simultaneously with PSMα3. We show that PSMα3 modulates antigen uptake, maturation and cytokine production of DCs activated by TLR1/2, TLR2/6, TLR4, TLR7, and TLR9. Pre-incubation of DCs with a p38 MAP kinase inhibitor prevented the PSMα3-induced IL-10 secretion, as well as MHC class II up-regulation upon TLR activation. In consequence, the tolerogenic DCs induced by PSMα3 in response to several TLR ligands promoted priming of regulatory T cells. Thus, PSMs could be useful as inducers of tolerogenic DCs upon TLR ligand stimulation for therapeutic applications.

Expression of inhibitory receptors on intratumoral T cells modulates the activity of a T cell-bispecific antibody targeting folate receptor.

T-cell bispecific antibodies (TCBs) are a novel therapeutic tool designed to selectively recruit T-cells to tumor cells and simultaneously activate them. However, it is currently unknown whether the dysfunctional state of T-cells, embedded into the tumor microenvironment, imprints on the therapeutic activity of TCBs. We performed a comprehensive analysis of activation and effector functions of tumor-infiltrating T-cells (TILs) in different tumor types, upon stimulation by a TCB targeting folate receptor 1 and CD3 (FolR1-TCB). We observed a considerable heterogeneity in T-cell activation, cytokine production and tumor cell killing upon exposure to FolR1-TCB among different FolR1-expressing tumors. Of note, tumors presenting with a high frequency of PD-1hi TILs displayed significantly impaired tumor cell killing and T-cell function. Further characterization of additional T-cell inhibitory receptors revealed that PD-1hi TILs defined a T-cell subset with particularly high levels of multiple inhibitory receptors compared with PD-1int and PD-1neg T-cells. PD-1 blockade could restore cytokine secretion but not cytotoxicity of TILs in a subset of patients with scarce PD-1hi expressing cells; in contrast, patients with abundance of PD-1hi expressing T-cells did not benefit from PD-1 blockade. Our data highlight that FolR1-TCB is a promising novel immunotherapeutic treatment option which is capable of activating intratumoral T-cells in different carcinomas. However, its therapeutic efficacy may be substantially hampered by a pre-existing dysfunctional state of T-cells, reflected by abundance of intratumoral PD-1hi T-cells. These findings present a rationale for combinatorial approaches of TCBs with other therapeutic strategies targeting T-cell dysfunction.

PSM Peptides of Staphylococcus aureus Activate the p38-CREB Pathway in Dendritic Cells, Thereby Modulating Cytokine Production and T Cell Priming.

The challenging human pathogen Staphylococcus aureus has highly efficient immune evasion strategies for causing a wide range of diseases, from skin and soft tissue to life-threatening infections. Phenol-soluble modulin (PSM) peptides are major pathogenicity factors of community-associated methicillin-resistant S. aureus strains. In previous work, we demonstrated that PSMs in combination with TLR2 ligand from S. aureus induce tolerogenic dendritic cells (DCs) characterized by the production of high amounts of IL-10, but no proinflammatory cytokines. This in turn promotes the activation of regulatory T cells while impairing Th1 response; however, the signaling pathways modulated by PSMs remain elusive. In this study, we analyzed the effects of PSMs on signaling pathway modulation downstream of TLR2. TLR2 stimulation in combination with PSMα3 led to increased and prolonged phosphorylation of NF-κB, ERK, p38, and CREB in mouse bone marrow-derived DCs compared with single TLR2 activation. Furthermore, inhibition of p38 and downstream MSK1 prevented IL-10 production, which in turn reduced the capacity of DCs to activate regulatory T cells. Interestingly, the modulation of the signaling pathways by PSMs was independent of the known receptor for PSMs, as shown by experiments with DCs lacking the formyl peptide receptor 2. Instead, PSMs penetrate the cell membrane most likely by transient pore formation. Moreover, colocalization of PSMs and p38 was observed near the plasma membrane in the cytosol, indicating a direct interaction. Thus, PSMs from S. aureus directly modulate the signaling pathway p38-CREB in DCs, thereby impairing cytokine production and in consequence T cell priming to increase the tolerance toward the pathogen.

Modulation of APC Function and Anti-Tumor Immunity by Anti-Cancer Drugs.

Professional antigen-presenting cells (APCs), such as dendritic cells (DCs), are central to the initiation and regulation of anti-cancer immunity. However, in the immunosuppressive environment within a tumor APCs may antagonize anti-tumor immunity by inducing regulatory T cells (Tregs) or anergy of effector T cells due to lack of efficient costimulation. Hence, in an optimal setting, anti-cancer drugs have the power to reduce tumor size and thereby may induce the release of tumor antigens and, at the same time, modulate APC function toward efficient priming of antigen-specific effector T cells. Selected cytotoxic agents may revert APC dysfunction either by directly maturing DCs or through induction of immunogenic tumor cell death. Furthermore, specific cytotoxic agents may support adaptive immunity by selectively depleting regulatory subsets, such as Tregs or myeloid-derived suppressor cells. Perspectively, this will allow developing effective combination strategies with novel immunotherapies to exert complementary pressure on tumors via direct toxicity as well as immune activation. We, here, review our current knowledge on the capacity of anti-cancer drugs to modulate APC functions to promote durable anti-cancer immune responses.

Progression of Lung Cancer Is Associated with Increased Dysfunction of T Cells Defined by Coexpression of Multiple Inhibitory Receptors.

Dysfunctional T cells present in malignant lesions are characterized by a sustained and highly diverse expression of inhibitory receptors, also referred to as immune checkpoints. Yet, their relative functional significance in different cancer types remains incompletely understood. In this study, we provide a comprehensive characterization of the diversity and expression patterns of inhibitory receptors on tumor-infiltrating T cells from patients with non-small cell lung cancer. In spite of the large heterogeneity observed in the amount of PD-1, Tim-3, CTLA-4, LAG-3, and BTLA expressed on intratumoral CD8(+) T cells from 32 patients, a clear correlation was established between increased expression of these inhibitory coreceptors and progression of the disease. Notably, the latter was accompanied by a progressively impaired capacity of T cells to respond to polyclonal activation. Coexpression of several inhibitory receptors was gradually acquired, with early PD-1 and late LAG-3/BTLA expression. PD-1 blockade was able to restore T-cell function only in a subset of patients. A high percentage of PD-1(hi) T cells was correlated with poor restoration of T-cell function upon PD-1 blockade. Of note, PD-1(hi) expression marked a particularly dysfunctional T-cell subset characterized by coexpression of multiple inhibitory receptors and thus may assist in identifying patients likely to respond to inhibitory receptor-specific antibodies. Overall, these data may provide a framework for future personalized T-cell-based therapies aiming at restoration of tumor-infiltrating lymphocyte effector functions.

Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment.

CD40 stimulation on antigen-presenting cells (APC) allows direct activation of CD8(+) cytotoxic T cells, independent of CD4⁺ T-cell help. Agonistic anti-CD40 antibodies have been demonstrated to induce beneficial antitumor T-cell responses in mouse models of cancer and early clinical trials. We report here that anti-CD40 treatment induces programmed death ligand-1 (PD-L1) upregulation on tumor-infiltrating monocytes and macrophages, which was strictly dependent on T cells and IFNγ. PD-L1 expression could be counteracted by coadministration of antibodies blocking the PD-1 (programmed death-1)/PD-L1 axis as shown for T cells from tumor models and human donors. The combined treatment was highly synergistic and induced complete tumor rejection in about 50% of mice bearing MC-38 colon and EMT-6 breast tumors. Mechanistically, this was reflected by a strong increase of IFNγ and granzyme-B production in intratumoral CD8⁺ T cells. Concomitant CTLA-4 blockade further improved rejection of established tumors in mice. This study uncovers a novel mechanism of acquired resistance upon agonistic CD40 stimulation and proposes that the concomitant blockade of the PD-1/PD-L1 axis is a viable therapeutic strategy to optimize clinical outcomes.

The microtubule-depolymerizing agent ansamitocin P3 programs dendritic cells toward enhanced anti-tumor immunity.

In addition to direct tumor cell cytotoxicity, chemotherapy can mediate tumor reduction through immune modulation of the tumor microenvironment to promote anti-tumor immunity. Mature dendritic cells (DCs) play key roles in priming robust immune responses in tumor-bearing hosts. Here, we screened a panel of 21 anticancer agents with defined molecular targets for their ability to induce direct maturation of DCs. We identified ansamitocin P3, a microtubule-depolymerizing agent, as a potent inducer of phenotypic and functional maturation of DCs. Exposure of both murine spleen-derived and human monocyte-derived DCs to ansamitocin P3 triggered up-regulation of maturation markers and production of pro-inflammatory cytokines, resulting in an enhanced T cell stimulatory capacity. Local administration of ansamitocin P3 induced maturation of skin Langerhans cells in vivo and promoted antigen uptake and extensive homing of tumor-resident DCs to tumor-draining lymph nodes. When used as an adjuvant in a specific vaccination approach, ansamitocin P3 dramatically increased activation of antigen-specific T cells. Finally, we demonstrate that ansamitocin P3, due to its immunomodulatory properties, acts in synergy with antibody-mediated blockade of the T cell inhibitory receptors PD-1 and CTLA-4. The combination treatment was most effective and induced durable growth inhibition of established tumors. Mechanistically, we observed a reduced regulatory T cell frequency and improved T cell effector function at the tumor site. Taken together, our study unravels an immune-based anti-tumor mechanism exploited by microtubule-depolymerizing agents, including ansamitocin P3, and paves the way for future clinical trials combining this class of agents with immunotherapy.

Microtubule-depolymerizing agents used in antibody-drug conjugates induce antitumor immunity by stimulation of dendritic cells.

Antibody-drug conjugates (ADC) are emerging as powerful treatment strategies with outstanding target-specificity and high therapeutic activity in patients with cancer. Brentuximab vedotin represents a first-in-class ADC directed against CD30(+) malignancies. We hypothesized that its sustained clinical responses could be related to the stimulation of an anticancer immune response. In this study, we demonstrate that the dolastatin family of microtubule inhibitors, from which the cytotoxic component of brentuximab vedotin is derived, comprises potent inducers of phenotypic and functional dendritic cell (DC) maturation. In addition to the direct cytotoxic effect on tumor cells, dolastatins efficiently promoted antigen uptake and migration of tumor-resident DCs to the tumor-draining lymph nodes. Exposure of murine and human DCs to dolastatins significantly increased their capacity to prime T cells. Underlining the requirement of an intact host immune system for the full therapeutic benefit of dolastatins, the antitumor effect was far less pronounced in immunocompromised mice. We observed substantial therapeutic synergies when combining dolastatins with tumor antigen-specific vaccination or blockade of the PD-1-PD-L1 and CTLA-4 coinhibitory pathways. Ultimately, treatment with ADCs using dolastatins induces DC homing and activates cellular antitumor immune responses in patients. Our data reveal a novel mechanism of action for dolastatins and provide a strong rationale for clinical treatment regimens combining dolastatin-based therapies, such as brentuximab vedotin, with immune-based therapies.

Staphylococcus aureus phenol-soluble modulin peptides modulate dendritic cell functions and increase in vitro priming of regulatory T cells.

The major human pathogen Staphylococcus aureus has very efficient strategies to subvert the human immune system. Virulence of the emerging community-associated methicillin-resistant S. aureus depends on phenol-soluble modulin (PSM) peptide toxins, which are known to attract and lyse neutrophils. However, their influences on other immune cells remain elusive. In this study, we analyzed the impact of PSMs on dendritic cells (DCs) playing an essential role in linking innate and adaptive immunity. In human neutrophils, PSMs exert their function by binding to the formyl peptide receptor (FPR) 2. We show that mouse DCs express the FPR2 homolog mFPR2 as well as its paralog mFPR1 and that PSMs are chemoattractants for DCs at noncytotoxic concentrations. PSMs reduced clathrin-mediated endocytosis and inhibited TLR2 ligand-induced secretion of the proinflammatory cytokines TNF, IL-12, and IL-6, while inducing IL-10 secretion by DCs. As a consequence, treatment with PSMs impaired the capacity of DCs to induce activation and proliferation of CD4(+) T cells, characterized by reduced Th1 but increased frequency of FOXP3(+) regulatory T cells. These regulatory T cells secreted high amounts of IL-10, and their suppression capacity was dependent on IL-10 and TGF-ß. Interestingly, the induction of tolerogenic DCs by PSMs appeared to be independent of mFPRs, as shown by experiments with mice lacking mFPR2 (mFPR2(-/-)) and the cognate G protein (p110γ(-/-)). Thus, PSMs from highly virulent pathogens affect DC functions, thereby modulating the adaptive immune response and probably increasing the tolerance toward the pathogen.

Exploring the topology of the Gid complex, the E3 ubiquitin ligase involved in catabolite-induced degradation of gluconeogenic enzymes.

In the yeast Saccharomyces cerevisiae, key regulatory enzymes of gluconeogenesis such as fructose-1,6-bisphosphatase are degraded via the ubiquitin proteasome system when cells are replenished with glucose. Polyubiquitination is carried out by the Gid complex, a multisubunit ubiquitin ligase that consists of seven different Gid (glucose-induced degradation-deficient) proteins. Under gluconeogenic conditions the E3 ligase is composed of six subunits (Gid1/Vid30, Gid2/Rmd5, Gid5/Vid28, Gid7, Gid8, and Gid9/Fyv10). Upon the addition of glucose the regulatory subunit Gid4/Vid24 appears, binds to the Gid complex, and triggers ubiquitination of fructose-1,6-bisphosphatase. All seven proteins are essential for this process; however, nothing is known about the arrangement of the subunits in the complex. Interestingly, each Gid protein possesses several remarkable motifs (e.g. SPRY, LisH, CTLH domains) that may play a role in protein-protein interaction. We, therefore, generated altered versions of individual Gid proteins by deleting or mutating these domains and performed co-immunoprecipitation experiments to analyze the interaction between distinct subunits. Thus, we were able to create an initial model of the topology of this unusual E3 ubiquitin ligase.