Generation and Characterization of Mirikizumab, a Humanized Monoclonal Antibody Targeting the p19 Subunit of IL-23.

Interleukin (IL)-23 exists as a heterodimer consisting of p19 and p40 and is a key cytokine for promoting inflammatory responses in a variety of target organs. IL-23 plays a key role in the differentiation and maintenance of T helper 17 cells, and deregulation of IL-23 can result in autoimmune pathologies of the skin, lungs, and gut. This study describes the generation and characterization of mirikizumab (miri), a humanized IgG4 monoclonal antibody directed against the p19 subunit of IL-23. Miri binds human and cynomolgus monkey IL-23 with high affinity and binds rabbit IL-23 weakly but does not bind to rodent IL-23 or the other IL-23 family members IL-12, IL-27, or IL-35. Miri effectively inhibits the interaction of IL-23 with its receptor, and potently blocks IL-23-induced IL-17 production in cell-based assays while preserving the function of IL-12. In both local and systemic in vivo mouse models, miri blocked IL-23-induced keratin mRNA or IL-17 production, respectively. These data provide a comprehensive preclinical characterization of miri, for which efficacy and safety have been demonstrated in human clinical trials for psoriasis, ulcerative colitis, and Crohn's disease. SIGNIFICANCE STATEMENT: This article describes the generation and characterization of mirikizumab, a high affinity, neutralizing IgG4 variant monoclonal antibody that is under development for the treatment of ulcerative colitis and Crohn's disease. Neutralization of interleukin (IL)-23 is achieved by preventing the binding of IL-23 p19 subunit to the IL-23 receptor and does not affect the IL-12 pathway.

ImmUniverse Consortium: Multi-omics integrative approach in personalized medicine for immune-mediated inflammatory diseases.

Immune mediated inflammatory diseases (IMIDs) are a heterogeneous group of debilitating, multifactorial and unrelated conditions featured by a dysregulated immune response leading to destructive chronic inflammation. The immune dysregulation can affect various organ systems: gut (e.g., inflammatory bowel disease), joints (e.g., rheumatoid arthritis), skin (e.g., psoriasis, atopic dermatitis), resulting in significant morbidity, reduced quality of life, increased risk for comorbidities, and premature death. As there are no reliable disease progression and therapy response biomarkers currently available, it is very hard to predict how the disease will develop and which treatments will be effective in a given patient. In addition, a considerable proportion of patients do not respond sufficiently to the treatment. ImmUniverse is a large collaborative consortium of 27 partners funded by the Innovative Medicine Initiative (IMI), which is sponsored by the European Union (Horizon 2020) and in-kind contributions of participating pharmaceutical companies within the European Federation of Pharmaceutical Industries and Associations (EFPIA). ImmUniverse aims to advance our understanding of the molecular mechanisms underlying two immune-mediated diseases, ulcerative colitis (UC) and atopic dermatitis (AD), by pursuing an integrative multi-omics approach. As a consequence of the heterogeneity among IMIDs patients, a comprehensive, evidence-based identification of novel biomarkers is necessary to enable appropriate patient stratification that would account for the inter-individual differences in disease severity, drug efficacy, side effects or prognosis. This would guide clinicians in the management of patients and represent a major step towards personalized medicine. ImmUniverse will combine the existing and novel advanced technologies, including multi-omics, to characterize both the tissue microenvironment and blood. This comprehensive, systems biology-oriented approach will allow for identification and validation of tissue and circulating biomarker signatures as well as mechanistic principles, which will provide information about disease severity and future disease progression. This truly makes the ImmUniverse Consortium an unparalleled approach.

Proteomic characterisations of ulcerative colitis endoscopic biopsies associate with clinically relevant histological measurements of disease severity.

AIMS AND METHODS: Accurate protein measurements using formalin-fixed biopsies are needed to improve disease characterisation. This feasibility study used targeted and global mass spectrometry (MS) to interrogate a spectrum of disease severities using 19 ulcerative colitis (UC) biopsies. RESULTS: Targeted assays for CD8, CD19, CD132 (interleukin-2 receptor subunit gamma/common cytokine receptor gamma chain), FOXP3 (forkhead box P3) and IL17RA (interleukin 17 receptor A) were successful; however, assays for IL17A (interleukin 17A), IL23 (p19) (interleukin 23, alpha subunit p19) and IL23R (interleukin 23 receptor) did not permit target detection. Global proteome analysis (4200 total proteins) was performed to identify pathways associated with UC progression. Positive correlation was observed between histological scores indicating active colitis and neutrophil-related measurements (R2=0.42-0.72); inverse relationships were detected with cell junction targets (R2=0.49-0.71) and ß-catenin (R2=0.51-0.55) attributed to crypt disruption. An exploratory accuracy assessment with Geboes Score and Robarts Histopathology Index cut-offs produced sensitivities/specificities of 72.7%/75.0% and 100.0%/81.8%, respectively. CONCLUSIONS: Pathologist-guided MS assessments provide a complementary approach to histological scoring systems. Additional studies are indicated to verify the utility of this novel approach.

Development of tibulizumab, a tetravalent bispecific antibody targeting BAFF and IL-17A for the treatment of autoimmune disease.

We describe a bispecific dual-antagonist antibody against human B cell activating factor (BAFF) and interleukin 17A (IL-17). An anti-IL-17 single-chain variable fragment (scFv) derived from ixekizumab (Taltz®) was fused via a glycine-rich linker to anti-BAFF tabalumab. The IgG-scFv bound both BAFF and IL-17 simultaneously with identical stoichiometry as the parental mAbs. Stability studies of the initial IgG-scFv revealed chemical degradation and aggregation not observed in either parental antibody. The anti-IL-17 scFv showed a high melting temperature (Tm) by differential scanning calorimetry (73.1°C), but also concentration-dependent, initially reversible, protein self-association. To engineer scFv stability, three parallel approaches were taken: labile complementary-determining region (CDR) residues were replaced by stable, affinity-neutral amino acids, CDR charge distribution was balanced, and a H44-L100 interface disulfide bond was introduced. The Tm of the disulfide-stabilized scFv was largely unperturbed, yet it remained monodispersed at high protein concentration. Fluorescent dye binding titrations indicated reduced solvent exposure of hydrophobic residues and decreased proteolytic susceptibility was observed, both indicative of enhanced conformational stability. Superimposition of the H44-L100 scFv (PDB id: 6NOU) and ixekizumab antigen-binding fragment (PDB id: 6NOV) crystal structures revealed nearly identical orientation of the frameworks and CDR loops. The stabilized bispecific molecule LY3090106 (tibulizumab) potently antagonized both BAFF and IL-17 in cell-based and in vivo mouse models. In cynomolgus monkey, it suppressed B cell development and survival and remained functionally intact in circulation, with a prolonged half-life. In summary, we engineered a potent bispecific antibody targeting two key cytokines involved in human autoimmunity amenable to clinical development.

Interleukin-33 Contributes Toward Loss of Tolerance by Promoting B-Cell-Activating Factor of the Tumor-Necrosis-Factor Family (BAFF)-Dependent Autoantibody Production.

Breaking tolerance is a key event leading to autoimmunity, but the exact mechanisms responsible for this remain uncertain. Here we show that the alarmin IL-33 is able to drive the generation of autoantibodies through induction of the B cell survival factor BAFF. A temporary, short-term increase in IL-33 results in a primary (IgM) response to self-antigens. This transient DNA-specific autoantibody response was dependent on the induction of BAFF. Notably, radiation resistant cells and not myeloid cells, such as neutrophils or dendritic cells were the major source of BAFF and were critical in driving the autoantibody response. Chronic exposure to IL-33 elicited dramatic increases in BAFF levels and resulted in elevated numbers of B and T follicular helper cells as well as germinal center formation. We also observed class-switching from an IgM to an IgG DNA-specific autoantibody response. Collectively, the results provide novel insights into a potential mechanism for breaking immune-tolerance via IL-33-mediated induction of BAFF.

Human mast cells release the migraine-inducing factor pituitary adenylate cyclase-activating polypeptide (PACAP).

Background Many patients with migraines suffer from allergies and vice versa, suggesting a relationship between biological mechanisms of allergy and migraine. It was proposed many years ago that mast cells may be involved in the pathophysiology of migraines. We set out to investigate the relationship between mast cell activation and known neurogenic peptides related to migraine. Methods Cultured human mast cells were assayed for the presence of neuropeptides and their receptors at the RNA and protein level. Immunohistochemistry analyses were performed on tissue resident and cultured mast cells. Mast cell degranulation assays were performed and pituitary adenylate cyclase-activating polypeptide (PACAP) activity was measured with a bioassay. Results We found that cultured and tissue resident human mast cells contain PACAP in cytoplasmic granules. No other neurogenic peptide known to be involved in migraine was detected, nor did mast cells express the receptors for PACAP or other neurogenic peptides. Furthermore, mast cell degranulation through classic IgE-mediated allergic mechanisms led to the release of PACAP. The PACAP released from mast cells was biologically active, as demonstrated using PACAP receptor reporter cell lines. We confirmed existing literature that mast cell degranulation can also be induced by several neurogenic peptides, which also resulted in PACAP release. Conclusion Our data provides a potential biological explanation for the association between allergy and migraine by demonstrating the release of biologically active PACAP from mast cells.

Gsk3 is a metabolic checkpoint regulator in B cells.

B cells predominate in a quiescent state until an antigen is encountered, which results in rapid growth, proliferation and differentiation of the B cells. These distinct cell states are probably accompanied by differing metabolic needs, yet little is known about the metabolic control of B cell fate. Here we show that glycogen synthase kinase 3 (Gsk3) is a metabolic sensor that promotes the survival of naive recirculating B cells by restricting cell mass accumulation. In antigen-driven responses, Gsk3 was selectively required for regulation of B cell size, mitochondrial biogenesis, glycolysis and production of reactive oxygen species (ROS), in a manner mediated by the co-stimulatory receptor CD40. Gsk3 was required to prevent metabolic collapse and ROS-induced apoptosis after glucose became limiting, functioning in part by repressing growth dependent on the myelocytomatosis oncoprotein c-Myc. Notably, we found that Gsk3 was required for the generation and maintenance of germinal center B cells, which require high glycolytic activity to support growth and proliferation in a hypoxic microenvironment.

Elevated levels of Interleukin (IL)-33 induce bone pathology but absence of IL-33 does not negatively impact normal bone homeostasis.

Interleukin (IL)-33 is a member of the IL-1 family. IL-33 effects are mediated through its receptor, ST2 and IL-1RAcP, and its signaling induces the production of a number of pro-inflammatory mediators, including TNFα, IL-1ß, IL-6, and IFN-γ. There are conflicting reports on the role of IL-33 in bone homeostasis, with some demonstrating a bone protective role for IL-33 whilst others show that IL-33 induces inflammatory arthritis with concurrent bone destruction. To better clarify the role IL-33 plays in bone biology in vivo, we studied IL-33 KO mice as well as mice in which the cytokine form of IL-33 was overexpressed. Mid-femur cortical bone mineral density (BMD) and bone strength were similar in the IL-33 KO mice compared to WT animals during the first 8months of life. However, in the absence of IL-33, we observed higher BMD in lumbar vertebrae and distal femur in female mice. In contrast, overexpression of IL-33 resulted in a marked and rapid reduction of bone volume, mineral density and strength. Moreover, this was associated with a robust increase in inflammatory cytokines (including IL-6 and IFN-γ), suggesting the bone pathology could be a direct effect of IL-33 or an indirect effect due to the induction of other mediators. Furthermore, the detrimental bone effects were accompanied by increases in osteoclast number and the bone resorption marker of C-terminal telopeptide collagen-I (CTX-I). Together, these results demonstrate that absence of IL-33 has no negative consequences in normal bone homeostasis while high levels of circulating IL-33 contributes to pathological bone loss.

IgG-Immune Complexes Promote B Cell Memory by Inducing BAFF.

Memory B cell responses are vital for protection against infections but must also be regulated to prevent autoimmunity. Cognate T cell help, somatic hypermutation, and affinity maturation within germinal centers (GCs) are required for high-affinity memory B cell formation; however, the signals that commit GC B cells to the memory pool remain unclear. In this study, we identify a role for IgG-immune complexes (ICs), FcγRs, and BAFF during the formation of memory B cells in mice. We found that early secretion of IgG in response to immunization with a T-dependent Ag leads to IC-FcγR interactions that induce dendritic cells to secrete BAFF, which acts at or upstream of Bcl-6 in activated B cells. Loss of CD16, hematopoietic cell-derived BAFF, or blocking IC:FcγR regions in vivo diminished the expression of Bcl-6, the frequency of GC and memory B cells, and secondary Ab responses. BAFF also contributed to the maintenance and/or expansion of the follicular helper T cell population, although it was dispensable for their formation. Thus, early Ab responses contribute to the optimal formation of B cell memory through IgG-ICs and BAFF. Our work defines a new role for FcγRs in GC and memory B cell responses.

Glucose metabolic trapping in mouse arteries: nonradioactive assay of atherosclerotic plaque inflammation applicable to drug discovery.

BACKGROUND: (18)F-Fluorodeoxyglucose (FDG)-positron emission tomography (PET) imaging of atherosclerosis in the clinic is based on preferential accumulation of radioactive glucose analog in atherosclerotic plaques. FDG-PET is challenging in mouse models due to limited resolution and high cost. We aimed to quantify accumulation of nonradioactive glucose metabolite, FDG-6-phosphate, in the mouse atherosclerotic plaques as a simple alternative to PET imaging. METHODOLOGY/PRINCIPAL FINDINGS: Nonradioactive FDG was injected 30 minutes before euthanasia. Arteries were dissected, and lipids were extracted. The arteries were re-extracted with 50% acetonitrile-50% methanol-0.1% formic acid. A daughter ion of FDG-6-phosphate was quantified using liquid chromatography and mass spectrometry (LC/MS/MS). Thus, both traditional (cholesterol) and novel (FDG-6-phosphate) markers were assayed in the same tissue. FDG-6-phosphate was accumulated in atherosclerotic lesions associated with carotid ligation of the Western diet fed ApoE knockout mice (5.9 times increase compare to unligated carotids, p<0.001). Treatment with the liver X receptor agonist T0901317 significantly (2.1 times, p<0.01) reduced FDG-6-phosphate accumulation 2 weeks after surgery. Anti-atherosclerotic effects were independently confirmed by reduction in lesion size, macrophage number, cholesterol ester accumulation, and macrophage proteolytic activity. CONCLUSIONS/SIGNIFICANCE: Mass spectrometry of FDG-6-phosphate in experimental atherosclerosis is consistent with plaque inflammation and provides potential translational link to the clinical studies utilizing FDG-PET imaging.

Tumor-derived death receptor 6 modulates dendritic cell development.

Studies in murine models of cancer as well as in cancer patients have demonstrated that the immune response to cancer is often compromised. This paradigm is viewed as one of the major mechanisms of tumor escape. Many therapies focus on employing the professional antigen presenting dendritic cells (DC) as a strategy to overcome immune inhibition in cancer patients. Death receptor 6 (DR6) is an orphan member of the tumor necrosis factor receptor superfamily (TNFRSF21). It is overexpressed on many tumor cells and DR6(-/-) mice display altered immunity. We investigated whether DR6 plays a role in tumorigenesis by negatively affecting the generation of anti-tumor activity. We show that DR6 is uniquely cleaved from the cell surface of tumor cell lines by the membrane-associated matrix metalloproteinase (MMP)-14, which is often overexpressed on tumor cells and is associated with malignancy. We also demonstrate that >50% of monocytes differentiating into DC die when the extracellular domain of DR6 is present. In addition, DR6 affects the cell surface phenotype of the resulting immature DC and changes their cytokine production upon stimulation with LPS/IFN-gamma. The effects of DR6 are mostly amended when these immature DC are matured with IL-1beta/TNF-alpha, as measured by cell surface phenotype and their ability to present antigen. These results implicate MMP-14 and DR6 as a mechanism tumor cells can employ to actively escape detection by the immune system by affecting the generation of antigen presenting cells.

Identification of anergic B cells within a wild-type repertoire.

The contribution of anergy to silencing of autoreactive B cells in physiologic settings is unknown. By comparing anergic and nonanergic immunoglobulin-transgenic mouse strains, we defined a set of surface markers that were used for presumptive identification of an anergic B cell cohort within a normal repertoire. Like anergic transgenic B cells, these physiologic anergic cells exhibited high basal intracellular free calcium and did not mobilize calcium, initiate tyrosine phosphorylation, proliferate, upregulate activation markers, or mount an immune response upon antigen-receptor stimulation. Autoreactive B cells were overrepresented in this cohort. On the basis of the frequency and lifespan of these cells, it appears that as many as 50% of newly produced B cells are destined to become anergic. In conclusion, our findings indicate that anergy is probably the primary mechanism by which autoreactive B cells are silenced. Thus maintenance of the unresponsiveness of anergic cells is critical for prevention of autoimmunity.

Enhanced B cell expansion, survival, and humoral responses by targeting death receptor 6.

Targeted disruption of death receptor (DR)6 results in enhanced CD4(+) T cell expansion and T helper cell type 2 differentiation after stimulation. Similar to T cells, DR6 is expressed on resting B cells but is down-regulated upon activation. We examined DR6(-/-) B cell responses both in vitro and in vivo. In vitro, DR6(-/-) B cells undergo increased proliferation in response to anti-immunoglobulin M, anti-CD40, and lipopolysaccharide. This hyperproliferative response was due, at least in part, to both increased cell division and reduced cell apoptosis when compared with wild-type B cells. Consistent with these observations, increased nuclear levels and activity of nuclear factor kappaB transcription factor, c-Rel, and elevated Bcl-x(l) expression were observed in DR6(-/-) B cells upon stimulation. In addition, DR6(-/-) B cells exhibited higher surface levels of CD86 upon activation and were more effective as antigen-presenting cells in an allogeneic T cell proliferation response. DR6(-/-) mice exhibited enhanced germinal center formation and increased titers of immunoglobulins to T-dependent as well as T-independent type I and II antigens. This is the first demonstration of a regulatory role of DR6 in the activation and function of B cells.