Redox-dependent plasticity of oxMIF facilitates its interaction with CD74 and therapeutic antibodies

Zhang L., Zhang H., Agborbesong E., Zhou J.X., Li X.

AbstractPrimary cilia are microtubule-based organelles that play important roles in development and tissue homeostasis. Macrophage migration inhibitory factor (MIF) has long been recognized as a secreted cytokine in the pathogenesis of various human diseases, including cancer and autosomal dominant polycystic kidney disease (ADPKD). Unlike other cytokines, unique functional characteristics of intracellular MIF have emerged. In this study, we show that MIF is localized and formed a ring like structure at the proximal end of centrioles, where it regulates cilia biogenesis through affecting 1) the recruitment of TTBK2 to basal body and the removal of CP110 from mother centriole, 2) the accumulation of CEP290 at centriolar satellites, and 3) the trafficking of intraflagellar transport (IFT) related proteins. We also show that MIF functions as a novel transcriptional factor to regulate the expression of genes related to ciliogenesis via binding on the promotors of those genes. MIF also binds chromatin and regulates transcription of genes involved in diverse homeostatic signaling pathways. We identify phosphatidylinositol-5-phosphate 4-kinase type 2 alpha (PIP4K2a) as an upstream regulator of MIF, which interacts with and phosphorylates MIF at S91 to increase its interaction with 14-3-3ζ, resulting in its nuclear translocation and transcription regulation. This study suggests that MIF is a key player in cilia biogenesis and a novel transcriptional regulator in homeostasis, which forward our understanding of how MIF is able to carry out several nonoverlapping functions.

Ferhat M., Mangano K., Mirkina I., Mayer J., Rossmueller G., Schinagl A., Kerschbaumer R., Nicoletti F., Thiele M., Landlinger C.

Macrophage Migration Inhibitory Factor (MIF) is a pleiotropic inflammatory cytokine that emerged as a pivotal regulator in the pathogenesis of several autoimmune diseases including rheumatoid arthritis (RA). MIF occurs in two immunologically distinct conformational isoforms, indicated as reduced (redMIF) and oxidized MIF (oxMIF) where the latter exerts disease-related activities. In this study we demonstrate the presence of circulating oxMIF in RA patients and investigate the in vivo effects of an oxMIF-neutralizing antibody in a murine model of RA. By advanced antibody engineering we generated the fully human anti-oxMIF antibody ON104 with abolished effector functions. The therapeutic potential of ON104 was tested in a model of Collagen-Induced Arthritis (CIA) in DBA/1j mice. At disease onset, the mice received ON104 twice a week for three weeks. Clinical symptoms were assessed daily, and histological examinations of the joints were performed at the end of the study. Antibody ON104, specifically targeting human and murine oxMIF, is highly affine and does not elicit effector functions in vitro. The treatment of CIA mice with ON104 profoundly modulated disease progression with marked amelioration of clinical signs of arthritis that was associated with reduced synovial and cartilage damage and reduced F4/80-positive macrophages in the joints. These data prove that oxMIF is a relevant target in a well-known model of human RA and its specific neutralization by the antibody ON104 ameliorates clinical and histological signs of the disease in the so-treated mice. Thus, ON104 represents a new and promising treatment option for RA and possibly other autoimmune diseases.

Ebert S., Zang L., Ismail N., Otabil M., Fröhlich A., Egea V., Ács S., Hoeberg M., Berres M., Weber C., Moreira J.M., Ries C., Bernhagen J., El Bounkari O.

Tissue inhibitor of metalloproteinases-1 (TIMP-1), an important regulator of matrix metalloproteinases (MMPs), has recently been shown to interact with CD74, a receptor for macrophage migration inhibitory factor (MIF). However, the biological effects mediated by TIMP-1 through CD74 remain largely unexplored. Using sequence alignment and in silico protein–protein docking analysis, we demonstrated that TIMP-1 shares residues with both MIF and MIF-2, crucial for CD74 binding, but not for CXCR4. Subcellular colocalization, immunoprecipitation, and internalization experiments supported these findings, demonstrating that TIMP-1 interacts with surface-expressed CD74, resulting in its internalization in a dose-dependent manner, as well as with a soluble CD74 ectodomain fragment (sCD74). This prompted us to study the effects of the TIMP-1–CD74 axis on monocytes and vascular smooth muscle cells (VSCMs) to assess its impact on vascular inflammation. A phospho-kinase array revealed the activation of serine/threonine kinases by TIMP-1 in THP-1 pre-monocytes, in particular AKT. Similarly, TIMP-1 dose-dependently triggered the phosphorylation of AKT and ERK1/2 in primary human monocytes. Importantly, Transwell migration, 3D-based Chemotaxis, and flow adhesion assays demonstrated that TIMP-1 engagement of CD74 strongly promotes the recruitment response of primary human monocytes, while live cell imaging studies revealed a profound activating effect on VSMC proliferation. Finally, re-analysis of scRNA-seq data highlighted the expression patterns of TIMP-1 and CD74 in human atherosclerotic lesions, thus, together with our experimental data, indicating a role for the TIMP-1–CD74 axis in vascular inflammation and atherosclerosis.

Rossmueller G., Mirkina I., Maurer B., Hoeld V., Mayer J., Thiele M., Kerschbaumer R.J., Schinagl A.

Abstract High levels of macrophage migration inhibitory factor (MIF) in patients with cancer are associated with poor prognosis. Its redox-dependent conformational isoform, termed oxidized MIF (oxMIF), is a promising tumor target due to its selective occurrence in tumor lesions and at inflammatory sites. A first-generation anti-oxMIF mAb, imalumab, was investigated in clinical trials in patients with advanced solid tumors, where it was well tolerated and showed signs of efficacy. However, imalumab has a short half-life in humans, increased aggregation propensity, and an unfavorable pharmacokinetic profile. Here, we aimed to optimize imalumab by improving its physicochemical characteristics and boosting its effector functions. Point mutations introduced into the variable regions reduced hydrophobicity and the antibodies’ aggregation potential, and increased plasma half-life and tumor accumulation in vivo, while retaining affinity and specificity to oxMIF. The introduction of mutations into the Fc region known to increase antibody-dependent cellular cytotoxicity resulted in enhanced effector functions of the novel antibodies in vitro, whereas reduced cytokine release from human peripheral blood mononuclear cells in the absence of target antigen by the engineered anti-oxMIF mAb ON203 versus imalumab reveals a favorable in vitro safety profile. In vivo, ON203 mAb demonstrated superior efficacy over imalumab in both prophylactic and established prostate cancer (PC3) mouse xenograft models. In summary, our data highlight the potential of the second-generation anti-oxMIF mAb ON203 as a promising immunotherapy for patients with solid tumors, warranting clinical evaluation.

Thiele M., Donnelly S.C., Mitchell R.A.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with a pleiotropic spectrum of biological functions implicated in the pathogenesis of cancer and inflammatory diseases. MIF is constitutively present in several cell types and non-lymphoid tissues and is secreted after acute stress or inflammation. MIF triggers the release of proinflammatory cytokines, overrides the anti-inflammatory effects of glucocorticoids, and exerts chemokine function, resulting in increased migration and recruitment of leukocytes into inflamed tissue. Despite this, MIF is a challenging target for therapeutic intervention because of its ubiquitous nature and presence in the circulation and tissue of healthy individuals. Oxidized MIF (oxMIF) is an immunologically distinct disease-related structural isoform found in the plasma and tissues of patients with inflammatory diseases and in solid tumor tissues. MIF converts to oxMIF in an oxidizing, inflammatory environment. This review discusses the biology and activity of MIF and the potential for autoimmune disease and cancer modification by targeting oxMIF. Anti-oxMIF antibodies reduce cancer cell invasion/migration, angiogenesis, proinflammatory cytokine production, and ERK and AKT activation. Anti-oxMIF antibodies also elicit apoptosis and alter immune cell function and/or migration. When co-administered with a glucocorticoid, anti-oxMIF antibodies produced a synergistic response in inflammatory models. Anti-oxMIF antibodies therefore counterregulate biological activities attributed to MIF. oxMIF expression has been observed in inflammatory diseases (eg, sepsis, psoriasis, asthma, inflammatory bowel disease, and systemic lupus erythematosus) and oxMIF has been detected in ovarian, colorectal, lung, and pancreatic cancers. In contrast to MIF, oxMIF is specifically detected in plasma and/or tissues of diseased patients, but not in healthy individuals. Therefore, as a druggable isoform of MIF, oxMIF represents a potential new therapeutic target in inflammatory diseases and cancer. Fully human, monoclonal anti-oxMIF antibodies have been shown to selectively bind oxMIF in preclinical and phase I studies; however, additional clinical assessments are necessary to validate their use as either a monotherapy or in combination with standard-of-care regimens (ie, immunomodulatory agents/checkpoint inhibitors, anti-angiogenic drugs, chemotherapeutics, and glucocorticoids).

Valadez-Cosmes P., Raftopoulou S., Mihalic Z.N., Marsche G., Kargl J.

Myeloperoxidase is a heme-peroxidase which makes up approximately 5% of the total dry cell weight of neutrophils where it is predominantly found in the primary (azurophilic) granules. Other cell types, such as monocytes and certain macrophage subpopulations also contain myeloperoxidase, but to a much lesser extent. Initially, the function of myeloperoxidase had been mainly associated with its ability as a catalyzer of reactive oxidants that help to clear pathogens. However, over the past years non-canonical functions of myeloperoxidase have been described both in health and disease. Attention has been specially focused on inflammatory diseases, in which an exacerbate infiltration of leukocytes can favor a poorly-controlled production and release of myeloperoxidase and its oxidants. There is compelling evidence that myeloperoxidase derived oxidants contribute to tissue damage and the development and propagation of acute and chronic vascular inflammation. Recently, neutrophils have attracted much attention within the large diversity of innate immune cells that are part of the tumor microenvironment. In particular, neutrophil-derived myeloperoxidase may play an important role in cancer development and progression. This review article aims to provide a comprehensive overview of the roles of myeloperoxidase in the development and progression of cancer. We propose future research approaches and explore prospects of inhibiting myeloperoxidase as a strategy to fight against cancer.

Skeens E., Gadzuk-Shea M., Shah D., Bhandari V., Schweppe D.K., Berlow R.B., Lisi G.P.

Macrophage migration inhibitory factor (MIF) is a multifunctional immunoregulatory protein that is a key player in the innate immune response. Given its overexpression at sites of inflammation and in diseases marked by increasingly oxidative environments, a comprehensive understanding of how cellular redox conditions impact the structure and function of MIF is necessary. We used NMR spectroscopy and mass spectrometry to investigate biophysical signatures of MIF under varied solution redox conditions. Our results indicate that the MIF structure is modified and becomes increasingly dynamic in an oxidative environment, which may be a means to alter the MIF conformation and functional response in a redox-dependent manner. We identified latent allosteric sites within MIF through mutational analysis of redox-sensitive residues, revealing that a loss of redox-responsive residues attenuates CD74 receptor activation. Leveraging sites of redox sensitivity as targets for structure-based drug design therefore reveals an avenue to modulate MIF function in its "disease state."

Herrero-Cervera A., Soehnlein O., Kenne E.

Chronic inflammation is a component of many disease conditions that affect a large group of individuals worldwide. Chronic inflammation is characterized by persistent, low-grade inflammation and is increased in the aging population. Neutrophils are normally the first responders to acute inflammation and contribute to the resolution of inflammation. However, in chronic inflammation, the role of neutrophils is less well understood and has been described as either beneficial or detrimental, causing tissue damage and enhancing the immune response. Emerging evidence suggests that neutrophils are important players in several chronic diseases, such as atherosclerosis, diabetes mellitus, nonalcoholic fatty liver disease and autoimmune disorders. This review will highlight the interaction of neutrophils with other cells in the context of chronic inflammation, the contribution of neutrophils to selected chronic inflammatory diseases, and possible future therapeutic strategies.

Chen E., Reiss K., Shah D., Manjula R., Allen B., Murphy E.L., Murphy J.W., Batista V.S., Bhandari V., Lolis E.J., Lisi G.P.

The macrophage migration inhibitory factor (MIF) family of cytokines contains multiple ligand-binding sites and mediates immunomodulatory processes through an undefined mechanism(s). Previously, we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel. Despite structural and functional similarity, the MIF homolog D-dopachrome tautomerase (also called MIF-2) has low sequence identity (35%), prompting the question of whether this dynamic regulatory network is conserved. Here, we establish the structural basis of an allosteric site in MIF-2, showing with solution NMR that dynamic communication is preserved in MIF-2 despite differences in the primary sequence. X-ray crystallography and NMR detail the structural consequences of perturbing residues in this pathway, which include conformational changes surrounding the allosteric site, despite global preservation of the MIF-2 fold. Molecular simulations reveal MIF-2 to contain a comparable hydrogen bond network to that of MIF, which was previously hypothesized to influence catalytic activity by modulating the strength of allosteric coupling. Disruption of the allosteric relay by mutagenesis also attenuates MIF-2 enzymatic activity in vitro and the activation of the cluster of differentiation 74 receptor in vivo, highlighting a conserved point of control for nonoverlapping functions in the MIF superfamily.

Cloutier M., Fortin J., Thibodeau J.

Invariant chain (CD74, Ii) is a multifunctional protein expressed in antigen presenting cells. It assists the ER exit of various cargos and serves as a receptor for the macrophage migration inhibitory factor. The newly translated Ii chains trimerize, a structural feature that is not readily understood in the context of its MHCII chaperoning function. Two segments of Ii, the luminal C-terminal region (TRIM) and the transmembrane domain (TM), have been shown to participate in the trimerization process but their relative importance and impact on the assembly with MHCII molecules remains debated. Here, we addressed the requirement of these domains in the trimerization of human Ii as well as in the oligomerization with MHCII molecules. We used site-directed mutagenesis to generate series of Ii and DR mutants. These were transiently transfected in HEK293T cells to test their cell surface expression and analyse their interactions by co-immunoprecipitations. Our results showed that the TRIM domain is not essential for Ii trimerization nor for intracellular trafficking with MHCII molecules. We also gathered evidence that in the absence of TM, TRIM allows the formation of multi-subunit complexes with HLA-DR. Similarly, in the absence of TRIM, Ii can assemble into high-order structures with MHCII molecules. Altogether, our data show that trimerization of Ii through either TM or TRIM sustains nonameric complex formation with MHCII molecules.

Pai D., Gruber M., Pfaehler S., Bredthauer A., Lehle K., Trabold B.

Chemotaxis and the formation of suicidal neutrophil extracellular traps (suicidal NETosis) are key functions of polymorphonuclear cells (PMNs). Neutrophil extracellular traps in particular are known to be significantly involved in the severity of inflammatory and immunological disorders such as rheumatoid arthritis and Crohn’s disease. Therefore, detailed knowledge of PMNs is essential for analyzing the mechanisms involved in, and developing new therapies for, such diseases. To date, no standard method to analyze these cell activities has been established. This study used in vitro live cell imaging to simultaneously observe and analyze PMN functions. To demonstrate this, the effects of phorbol-12-myristat-13-acetat (PMA, 0.1-10 nM), N-formylmethionine-leucyl-phenylalanine (fMLP, 10 nM), and protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) on PMN chemotaxis and suicidal NETosis were studied. PMA (1 nM-10 nM) resulted in significant concentration-dependent behavior in chemotaxis and an earlier onset of maximum oxidative burst and NET formation of up to 44%. When adding H7, PMA-triggered PMN functions were reduced, demonstrating that all three functions rely mostly on protein kinase C (PKC) activity, while PKC is not essential for fMLP-induced PMN activity. Thus, the method here described can be used to objectively quantify PMN functions and, especially through the regulation of the PKC pathway, could be useful in further clinical studies of immunological disorders.

Pantouris G., Khurana L., Ma A., Skeens E., Reiss K., Batista V.S., Lisi G.P., Lolis E.J.

In proteins with multiple functions, such as macrophage migration inhibitory factor (MIF), the study of its intramolecular dynamic network can offer a unique opportunity to understand how a single protein is able to carry out several nonoverlapping functions. A dynamic mechanism that controls the MIF-induced activation of CD74 was recently discovered. In this study, the regulation of tautomerase activity was explored. The catalytic base Pro1 is found to form dynamic communications with the same allosteric node that regulates CD74 activation. Signal transmission between the allosteric and catalytic sites take place through intramolecular aromatic interactions and a hydrogen bond network that involves residues and water molecules of the MIF solvent channel. Once thought to be a consequence of trimerization, a regulatory function for the solvent channel is now defined. These results provide mechanistic insights into the regulation of catalytic activity and the role of solvent channel water molecules in MIF catalysis.

Davies M.J., Hawkins C.L.

Significance: The release of myeloperoxidase (MPO) by activated leukocytes is critical in innate immune responses. MPO produces hypochlorous acid (HOCl) and other strong oxidants, which kill bacteria and other invading pathogens. However, MPO also drives the development of numerous chronic inflammatory pathologies, including atherosclerosis, neurodegenerative disease, lung disease, arthritis, cancer, and kidney disease, which are globally responsible for significant patient mortality and morbidity. Recent Advances: The development of imaging approaches to precisely identify the localization of MPO and the molecular targets of HOCl in vivo is an important advance, as typically the involvement of MPO in inflammatory disease has been inferred by its presence, together with the detection of biomarkers of HOCl, in biological fluids or diseased tissues. This will provide valuable information in regard to the cell types responsible for releasing MPO in vivo, together with new insight into potential therapeutic opportunities. Critical Issues: Although there is little doubt as to the value of MPO inhibition as a protective strategy to mitigate tissue damage during chronic inflammation in experimental models, the impact of long-term inhibition of MPO as a therapeutic strategy for human disease remains uncertain, in light of the potential effects on innate immunity. Future Directions: The development of more targeted MPO inhibitors or a treatment regimen designed to reduce MPO-associated host tissue damage without compromising pathogen killing by the innate immune system is therefore an important future direction. Similarly, a partial MPO inhibition strategy may be sufficient to maintain adequate bacterial activity while decreasing the propagation of inflammatory pathologies.

Hawkins C.

Abstract Myeloperoxidase (MPO) is a mammalian heme peroxidase released by activated immune cells, which forms chemical oxidants, including hypochlorous acid (HOCl), to kill bacteria and other invading pathogens. In addition to this important role in the innate immune system, there is significant evidence from numerous chronic inflammatory pathologies for the elevated production of HOCl and associated oxidative modification of proteins and damage to host tissue. Proteins are major targets for HOCl in biological systems, owing to their abundance and the high reactivity of several amino acid side-chains with this oxidant. As such, there is significant interest in understanding the molecular mechanisms involved in HOCl-mediated protein damage and defining the consequences of these reactions. Exposure of proteins to HOCl results in a wide range of oxidative modifications and the formation of chlorinated products, which alter protein structure and enzyme activity, and impact the function of biological systems. This review describes the reactivity of HOCl with proteins, including the specific pathways involved in side-chain modification, backbone fragmentation and aggregation, and outlines examples of some of the biological consequences of these reactions, particularly in relation to the development of chronic inflammatory disease.

Hawkins C.L., Davies M.J.

Exposure of biological molecules to oxidants is inevitable and therefore commonplace. Oxidative stress in cells arises from both external agents and endogenous processes that generate reactive species, either purposely (e.g. during pathogen killing or enzymatic reactions) or accidentally (e.g. exposure to radiation, pollutants, drugs, or chemicals). As proteins are highly abundant and react rapidly with many oxidants, they are highly susceptible to, and major targets of, oxidative damage. This can result in changes to protein structure, function, and turnover and to loss or (occasional) gain of activity. Accumulation of oxidatively-modified proteins, due to either increased generation or decreased removal, has been associated with both aging and multiple diseases. Different oxidants generate a broad, and sometimes characteristic, spectrum of post-translational modifications. The kinetics (rates) of damage formation also vary dramatically. There is a pressing need for reliable and robust methods that can detect, identify, and quantify the products formed on amino acids, peptides, and proteins, especially in complex systems. This review summarizes several advances in our understanding of this complex chemistry and highlights methods that are available to detect oxidative modifications—at the amino acid, peptide, or protein level—and their nature, quantity, and position within a peptide sequence. Although considerable progress has been made in the development and application of new techniques, it is clear that further development is required to fully assess the relative importance of protein oxidation and to determine whether an oxidation is a cause, or merely a consequence, of injurious processes.

Rossmueller G., Mirkina I., Thiele M., Puchol Tarazona A., Rueker F., Kerschbaumer R.J., Schinagl A.

Background: Rigorous assessment of antibody developability is crucial for optimizing lead candidates before progressing to clinical studies. Recent advances in predictive tools for protein structures, surface properties, stability, and immunogenicity have streamlined the development of new biologics. However, accurate prediction of the impact of single amino acid substitutions on antibody structures remains challenging, due to the diversity of complementarity-determining regions (CDRs), particularly CDR3s. Methods: In this study, we combined in silico tools with in vitro assessments to engineer improved antibodies against the oxidized isoform of the macrophage migration inhibitory factor (oxMIF), building on the first generation anti-oxMIF antibody imalumab. Results: We identified hydrophobic hotspots conferring increased self-interaction and aggregation propensity on imalumab, which unravels its unusually short half-life in humans. By introducing mutations into the variable regions, we addressed these liabilities. Structural prediction tools and molecular dynamics simulations guided the selection of mutations, which were then experimentally validated. The lead candidate antibody, C0083, demonstrated reduced hydrophobicity and self-interaction due to the restructuring of its heavy chain CDR3 loop. Despite these structural changes, C0083 retained target specificity and binding affinity to oxMIF. Conclusions: Altogether, this study shows that a small number of well-selected mutations was sufficient to substantially improve the biophysicochemical properties of imalumab.