Interleukin-21 (IL-21), a member of the common cytokine receptor γ chain (γc) family, is secreted by CD4+ T cells and natural killer T cells and induces effector function through interactions with the IL-21 receptor (IL-21R)/γc complex expressed on both immune and non-immune cells. Numerous studies suggest that IL-21 plays a significant role in autoimmune disorders. Therapeutic intervention to disrupt the IL-21/IL-21R/γc interaction and inhibit subsequent downstream signal transduction could offer a treatment paradigm for these diseases. Potent neutralizing antibodies reported in the literature were generated after extensive immunizations with human IL-21 alone and in combination with various adjuvants. To circumvent the laborious method of antibody generation while targeting a conserved functional epitope, we designed a novel alternating-antigen immunization strategy utilizing both human and cynomolgus monkey (cyno) IL-21. Despite the high degree of homology between human and cyno IL-21, our alternating-immunization strategy elicited higher antibody titers and more potent neutralizing hybridomas in mice than did the immunization with human IL-21 antigen alone. The lead hybridoma clone was humanized by grafting the murine complementarity-determining regions onto human germline framework templates, using a unique rational design. The final humanized and engineered antibody, MEDI7169, encodes only one murine residue at the variable heavy/light-chain interface, retains the sub-picomolar affinity for IL-21, specifically inhibits IL-21/IL-21R-mediated signaling events and is currently under clinical development as a potential therapeutic agent for autoimmune diseases. This study provides experimental evidence of the immune system's potential to recognize and respond to shared epitopes of antigens from distinct species, and presents a generally applicable, novel method for the rapid generation of exceptional therapeutic antibodies using the hybridoma platform.

Immunofibroblasts have been described within tertiary lymphoid structures (TLS) that regulate lymphocyte aggregation at sites of chronic inflammation. Here we report, for the first time, an immunoregulatory property of this population, dependent on inducible T-cell co-stimulator ligand and its ligand (ICOS/ICOS-L). During inflammation, immunofibroblasts, alongside other antigen presenting cells, like dendritic cells (DCs), upregulate ICOSL, binding incoming ICOS + T cells and inducing LTα3 production that, in turn, drives the chemokine production required for TLS assembly via TNFRI/II engagement. Pharmacological or genetic blocking of ICOS/ICOS-L interaction results in defective LTα expression, abrogating both lymphoid chemokine production and TLS formation. These data provide evidence of a previously unknown function for ICOSL-ICOS interaction, unveil a novel immunomodulatory function for immunofibroblasts, and reveal a key regulatory function of LTα3, both as biomarker of TLS establishment and as first driver of TLS formation and maintenance in mice and humans.

Interactions between ICOS and ICOS ligand (ICOSL) are essential for the development of T follicular helper (Tfh) cells and thus the formation and maintenance of GC reactions. Given the conflicting reports on the requirement of other CD4(+) T-cell populations for ICOS signals, we have employed a range of in vivo approaches to dissect requirements for ICOS signals in mice during an endogenous CD4(+) T-cell response and contrasted this with CD28 signals. Genetic absence of ICOSL only modestly reduced the total number of antigen-specific CD4(+) T cells at the peak of the primary response, but resulted in a severely diminished number of both T central memory and T effector memory cells. Treatment with blocking anti-ICOS mAb during the primary response recapitulated these effects and caused a more substantial reduction than blocking CD28 signals with CTLA4Ig. During the memory phase of the response further signals through ICOS or CD28 were not required for survival. However, upon secondary challenge only Tfh cell expansion remained heavily ICOS-dependent, while CD28 signals were required for optimal expansion of all subsets. These data demonstrate the importance of ICOS signals specifically for memory CD4(+) T-cell formation, while highlighting the potential of therapeutically targeting this pathway.

Small cell lung cancer (SCLC) is an aggressive disease with poor survival. A few sequencing studies performed on limited number of samples have revealed potential disease-driving genes in SCLC, however, much still remains unknown, particularly in the Asian patient population. Here we conducted whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients. Dysregulation of tumor suppressor genes TP53 and RB1 was observed in 82% and 62% of SCLC patients, respectively, and more than half of the SCLC patients (62%) harbored TP53 and RB1 mutation and/or copy number loss. Additionally, Serine/Arginine Splicing Factor 1 (SRSF1) DNA copy number gain and mRNA over-expression was strongly associated with poor survival using both discovery and validation patient cohorts. Functional studies in vitro and in vivo demonstrate that SRSF1 is important for tumorigenicity of SCLC and may play a key role in DNA repair and chemo-sensitivity. These results strongly support SRSF1 as a prognostic biomarker in SCLC and provide a rationale for personalized therapy in SCLC.

Systemic sclerosis (SSc) is a clinically heterogeneous, life-threatening disease characterized by fibrosis, microvasculopathy, and autoimmunity. Extensive nonclinical and clinical data implicate B cells in the pathogenesis of SSc. MEDI-551 is an investigational humanized monoclonal antibody that targets the B cell surface antigen CD19 and mediates antibody-dependent, cell-mediated cytotoxicity of B cells. This clinical study evaluated the safety and tolerability, pharmacokinetics, and pharmacodynamics of MEDI-551 in subjects with SSc.

Background: Innate lymphoid cells (ILCs) have now been identified within most tissues of the body and current evidence indicates that this family of cells play a fundamental role in maintaining tissue homeostasis. However, few studies have compared the ILC populations between several tissues. Methods: We sought to generate a comprehensive characterisation of the ILC populations in different tissues of C57BL/6 WT and genetically modified mice targeting costimulatory pathways, using transcription factor expression to define specific groups. Results: Consistent with studies individually describing the ILC composition in different tissues, our analysis revealed different ILC groups dominate the ILC population in different tissues. Additionally, we observed a population of  IL-7Rα +Id2 + cells lacking expression of lineage markers but also lacking expression of GATA-3, RORgt or T-bet. This population was most evident in ear skin where it outnumbered the defined ILC groups, however, further experiments demonstrated that detection of these cells was influenced by how the tissue was digested, raising concerns as to its real nature. Since both ILC2 and ILC3 express ICOS, we then investigated the requirement for ICOS:ICOSL interactions in the homeostasis of ILC populations at these sites. Surprisingly, no significant differences were detected in the number of ILC1, ILC2 or ILC3 between WT and ICOSL -/- mice in any tissue, indicating that this pathway is not required for ILC homeostasis at these sites. These data were compared with CD80 -/-CD86 -/- mice given evidence of CD28 expression by some ILC and ILC crosstalk with activated T cells. Notably, the absence of CD28 ligands resulted in a significant increase in ILC2 and ILC3 numbers in the intestine. Conclusions: Together, these data provide new insight into ILC composition in different tissues in both WT and genetically modified mice where key costimulatory pathways are genetically deleted, providing a useful resource for further research into ILC biology.

Vesicular stomatitis virus encoding the IFNβ transgene (VSV-IFNβ) is a mediator of potent oncolytic activity and is undergoing clinical evaluation for the treatment of solid tumors. Emerging preclinical and clinical data suggest treatment of tumors with oncolytic viruses may sensitize tumors to checkpoint inhibitors and increase the anti-tumor immune response. New generations of immuno-oncology molecules including T cell agonists are entering clinical development and could be hypothesized to enhance the activity of oncolytic viruses, including VSV-IFNβ. Here, we show that VSV-IFNβ exhibits multiple mechanisms of action, including direct cell killing, stimulation of an innate immune response, recruitment of CD8 T cells, and depletion of T regulatory cells. Moreover, VSV-IFNβ promotes the establishment of a CD8 T cell response to endogenous tumor antigens. Our data demonstrate a significant enhancement of anti-tumor function for VSV-IFNβ when combined with checkpoint inhibitors, but not OX40 agonists. While the addition of checkpoint inhibitors to VSV-IFNβ generated robust tumor growth inhibition, it resulted in no increase in viral replication, transgene expression, or immunophenotypic changes beyond treatment with VSV-IFNβ alone. We hypothesize that tumor-specific T cells generated by VSV-IFNβ retain activity due to a lack of immune exhaustion when checkpoint inhibitors were used.

Recent studies have demonstrated the importance of CD47 in protecting malignant B cells from antibody dependent cellular phagocytosis (ADCP). Combined treatment of anti-CD47 and -CD20 antibodies synergistically augment elimination of tumor B cells in xenograft mouse models. This has led to the development of novel reagents that can potentially enhance killing of malignant B cells in patients. B cell depleting therapy is also a promising treatment for autoimmune patients. In the current study, we aimed to investigate whether or not CD47 protects non-malignant B cells from ADCP. We show that CD47 is expressed on all B cells in mice, with the highest level on plasma cells in bone marrow and spleen. Although its expression is dispensable for B cell development in mice, CD47 on B cells limits antibody mediated phagocytosis. B cell depletion following in vivo anti-CD19 treatment is more efficient in CD47-/- mice than in wild type mice. In vitro, both naïve and activated B cells from CD47-/- mice are more sensitive to ADCP than wild type B cells. Lastly, we show in an ADCP assay that blocking CD47 can enhance anti-CD19 antibody mediated phagocytosis of wild type B cells. These results suggest that in addition to its already demonstrated benefit in cancer, targeting CD47 may be used as an adjunct in combination with B cell depletion antibodies for treatment of autoimmune diseases.

Immune complexes (ICs) play a critical role in the pathology of autoimmune diseases. The aim of this study was to generate and characterise a first-in-class anti-FcγRIIA antibody (Ab) VIB9600 (previously known as MEDI9600) that blocks IgG immune complex-mediated cellular activation for clinical development.

Continuous support from follicular CD4(+) T helper (Tfh) cells drives germinal center (GC) responses, which last for several weeks to produce high affinity memory B cells and plasma cells. In autoimmune Sle1 and NZB/W F1 mice, elevated numbers of Tfh cells persist, promoting the expansion of self-reactive B cells. Expansion of circulating Tfh like cells have also been described in several autoimmune diseases. Although, the signals required for Tfh differentiation have now been well described, the mechanisms that sustain the maintenance of fully differentiated Tfh are less understood. Recent data demonstrate a role for GC B cells for Tfh maintenance after protein immunization.

MEDI9447 is a human monoclonal antibody that is specific for the ectoenzyme CD73 and currently undergoing Phase I clinical trials. Here we show that MEDI9447 is a potent inhibitor of CD73 ectonucleotidase activity, with wide ranging immune regulatory consequences. MEDI9447 results in relief from adenosine monophosphate (AMP)-mediated lymphocyte suppression in vitro and inhibition of mouse syngeneic tumor growth in vivo. In contrast with other cancer immunotherapy agents such as checkpoint inhibitors or T-cell agonists, MEDI9447 drives changes in both myeloid and lymphoid infiltrating leukocyte populations within the tumor microenvironment of mouse models. Changes include significant alterations in a number of tumor micro-environmental subpopulations including increases in CD8(+) effector cells and activated macrophages. Furthermore, these changes correlate directly with responder and non-responder subpopulations within animal studies using syngeneic tumors. Combination data showing additive activity between MEDI9447 and anti-PD-1 antibodies using human cells in vitro and mouse tumor models further demonstrate the potential value of relieving adenosine-mediated immunosuppression. Based on these data, a Phase I study to test the safety, tolerability, and clinical activity of MEDI9447 in cancer patients was initiated (NCT02503774).

Release of endogenous damage associated molecular patterns (DAMPs), including members of the S100 family, are associated with infection, cellular stress, tissue damage and cancer. The extracellular functions of this family of calcium binding proteins, particularly S100A8, S100A9 and S100A12, are being delineated. They appear to mediate their functions via receptor for advanced glycation endproducts (RAGE) or TLR4, but there remains considerable uncertainty over the relative physiological roles of these DAMPs and their pattern recognition receptors. In this study, we surveyed the capacity of S100 proteins to induce proinflammatory cytokines and cell migration, and the contribution RAGE and TLR4 to mediate these responses in vitro. Using adenoviral delivery of murine S100A9, we also examined the potential for S100A9 homodimers to trigger lung inflammation in vivo. S100A8, S100A9 and S100A12, but not the S100A8/A9 heterodimer, induced modest levels of TLR4-mediated cytokine production from human PBMC. In contrast, for most S100s including S100A9, RAGE blockade inhibited S100-mediated cell migration of THP1 cells and major leukocyte populations, whereas TLR4-blockade had no effect. Intranasal administration of murine S100A9 adenovirus induced a specific, time-dependent predominately macrophage infiltration that coincided with elevated S100A9 levels and proinflammatory cytokines in the BAL fluid. Inflammatory cytokines were markedly ablated in the TLR4-defective mice, but unexpectedly the loss of TLR4 signaling or RAGE-deficiency did not appreciably impact the S100A9-mediated lung pathology or the inflammatory cell infiltrate in the alveolar space. These data demonstrate that physiological levels of S100A9 homodimers can trigger an inflammatory response in vivo, and despite the capacity of RAGE and TLR4 blockade to inhibit responses in vitro, the response is predominately independent of both these receptors.

Ectopic follicles are non-encapsulated organized lymphoid structures that form at sites of inflammation and presumably contribute to the activation and differentiation of cells with autoreactive potential within target tissues. As such, directed targeting of ectopic follicles in settings of autoimmunity may provide a means to specifically inhibit the activation of autoreactive cells without impairing protective immune responses ongoing in peripheral lymphoid tissues. NOD·H2h4 mice are a non-diabetic strain of NOD mice which develop a Sjögren's syndrome-like disease which includes the formation of ectopic follicles in the salivary gland and characteristic Sjögren's autoantibodies. The goal of these studies was to better characterize the formation of ectopic follicles in this model and to explore their contribution to autoimmunity. Our studies show that by 8 weeks of age, young NOD·H2h4 mice spontaneously develop an abundance of splenic germinal centers, prior to the emergence of lymphocyte infiltration in the salivary gland tissue. Ectopic follicle formation in the salivary gland begins to appear in these mice between 12 and 16 weeks of age. Interestingly, anti-Ro and anti-La autoantibodies precede the development of ectopic follicles in young NOD·H2h4 mice. In contrast, production of anti-dsDNA antibodies is delayed and largely coincides with the formation of ectopic follicles in these mice. These data suggest that tertiary lymphoid structures may arise from the trafficking of activated T and B cells to sites of inflammation in non-lymphoid tissues. Furthermore, local presentation of autoantigens may then promote the expansion of autoreactive cells with specificities distinct from those generated in the splenic micro-environment.

A hallmark of autoimmunity in murine models of lupus is the formation of germinal centers (GCs) in lymphoid tissues where self-reactive B cells expand and differentiate. In the host response to foreign antigens, follicular dendritic cells (FDCs) maintain GCs through the uptake and cycling of complement-opsonized immune complexes. Here, we examined whether FDCs retain self-antigens and the impact of this process in autoantibody secretion in lupus. We found that FDCs took up and retained self-immune complexes composed of ribonucleotide proteins, autoantibody, and complement. This uptake, mediated through CD21, triggered endosomal TLR7 and led to the secretion of interferon (IFN) α via an IRF5-dependent pathway. Blocking of FDC secretion of IFN-α restored B cell tolerance and reduced the amount of GCs and pathogenic autoantibody. Thus, FDCs are a critical source of the IFN-α driving autoimmunity in this lupus model. This pathway is conserved in humans, suggesting that it may be a viable therapeutic target in systemic lupus erythematosus.

Checkpoint inhibitors have revolutionized cancer treatment. However, only a minority of patients respond to these immunotherapies. Here, we report that blocking the inhibitory NKG2A receptor enhances tumor immunity by promoting both natural killer (NK) and CD8+ T cell effector functions in mice and humans. Monalizumab, a humanized anti-NKG2A antibody, enhanced NK cell activity against various tumor cells and rescued CD8+ T cell function in combination with PD-x axis blockade. Monalizumab also stimulated NK cell activity against antibody-coated target cells. Interim results of a phase II trial of monalizumab plus cetuximab in previously treated squamous cell carcinoma of the head and neck showed a 31% objective response rate. Most common adverse events were fatigue (17%), pyrexia (13%), and headache (10%). NKG2A targeting with monalizumab is thus a novel checkpoint inhibitory mechanism promoting anti-tumor immunity by enhancing the activity of both T and NK cells, which may complement first-generation immunotherapies against cancer.

ADAM17 (a disintegrin and metalloproteinase 17)/TACE (TNFα converting enzyme) has emerged as a potential therapeutic target in colorectal cancer (CRC) and other cancers, due in part to its role in regulating various tumor cell surface proteins and growth factors and cytokines in the tumor microenvironment. The emergence of MEDI3622, a highly potent and specific antibody-based ADAM17 inhibitor, has allowed testing of the concept that targeting ADAM17 may be an important new therapeutic approach for CRC patients. We demonstrate that MEDI3622 is highly efficacious on tumor growth in multiple human CRC PDX models, resulting in improved survival of animals bearing tumor xenografts. MEDI3622 was further found to impact Notch pathway activity and tumor-initiating cells. The promising preclinical activity seen here supports further clinical investigation of this treatment approach to improve therapeutic outcome for patients diagnosed with metastatic CRC, including patients with KRAS-mutant tumors for whom other therapeutic options are currently limited.

Immunoglobulin E (IgE) plays a key role in allergic asthma and is a clinically validated target for monoclonal antibodies. Therapeutic anti-IgE antibodies block the interaction between IgE and the Fc epsilon (Fcε) receptor, which eliminates or minimizes the allergic phenotype but does not typically curtail the ongoing production of IgE by B cells. We generated high-affinity anti-IgE antibodies (MEDI4212) that have the potential to both neutralize soluble IgE and eliminate IgE-expressing B-cells through antibody-dependent cell-mediated cytotoxicity. MEDI4212 variants were generated that contain mutations in the Fc region of the antibody or alterations in fucosylation in order to enhance the antibody's affinity for FcγRIIIa. All MEDI4212 variants bound to human IgE with affinities comparable to the wild-type (WT) antibody. Each variant was shown to inhibit the interaction between IgE and FcεRI, which translated into potent inhibition of FcγRI-mediated function responses. Importantly, all variants bound similarly to IgE at the surface of membrane IgE expressing cells. However, MEDI4212 variants demonstrated enhanced affinity for FcγRIIIa including the polymorphic variants at position 158. The improvement in FcγRIIIa binding led to increased effector function in cell based assays using both engineered cell lines and class switched human IgE B cells. Through its superior suppression of IgE, we anticipate that effector function enhanced MEDI4212 may be able to neutralize high levels of soluble IgE and provide increased long-term benefit by eliminating the IgE expressing B cells before they differentiate and become IgE secreting plasma cells.

Respiratory syncytial virus (RSV), a common respiratory pathogen in infants and the older population, causes pulmonary inflammation and airway occlusion that leads to impairment of lung function. Here, we have established a role for receptor for advanced glycation end products (RAGE) in RSV infection. RAGE-deficient (ager(-/-)) mice were protected from RSV-induced weight loss and inflammation. This protection correlated with an early increase in type I interferons, later decreases in proinflammatory cytokines, and a reduction in viral load. To assess the contribution of soluble RAGE (sRAGE) to RSV-induced disease, wild-type and ager(-/-) mice were given doses of sRAGE following RSV infection. Of interest, sRAGE treatment prevented RSV-induced weight loss and neutrophilic inflammation to a degree similar to that observed in ager(-/-) mice. Our work further elucidates the roles of RAGE in the pathogenesis of respiratory infections and highlights the opposing roles of membrane and sRAGE in modulating the host response to RSV infection.

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection. Infection is critically dependent on the RSV fusion (F) protein, which mediates fusion between the viral envelope and airway epithelial cells. The F protein is also expressed on infected cells and is responsible for fusion of infected cells with adjacent cells, resulting in the formation of multinucleate syncytia. The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that is constitutively highly expressed by type I alveolar epithelial cells. Here, we report that RAGE protected HEK cells from RSV-induced cell death and reduced viral titres in vitro. RAGE appeared to interact directly with the F protein, but, rather than inhibiting RSV entry into host cells, virus replication and budding, membrane-expressed RAGE or soluble RAGE blocked F-protein-mediated syncytium formation and sloughing. These data indicate that RAGE may contribute to protecting the lower airways from RSV by inhibiting the formation of syncytia, viral spread, epithelial damage and airway obstruction.

Canonically, immunoglobulin E (IgE) mediates allergic immune responses by triggering mast cells and basophils to release histamine and type 2 helper cytokines. Here we found that in human systemic lupus erythematosus (SLE), IgE antibodies specific for double-stranded DNA (dsDNA) activated plasmacytoid dendritic cells (pDCs), a type of cell of the immune system linked to viral defense, which led to the secretion of substantial amounts of interferon-α (IFN-α). The concentration of dsDNA-specific IgE found in patient serum correlated with disease severity and greatly potentiated pDC function by triggering phagocytosis via the high-affinity FcɛRI receptor for IgE, followed by Toll-like receptor 9 (TLR9)-mediated sensing of DNA in phagosomes. Our findings expand the known pathogenic mechanisms of IgE-mediated inflammation beyond those found in allergy and demonstrate that IgE can trigger interferon responses capable of exacerbating self-destructive autoimmune responses.