A natural goldmine of binding proteins and soluble receptors simplified their translation to blockbuster drugs, all in one decade.

Human urinary proteins are a goldmine of natural proteins a feature that simplifies their translation to biologics. Combining this goldmine together with the ligand-affinity-chromatography (LAC) purification method, proved a winning formula in their isolation. LAC specificity, efficiency, simplicity and inherent indispensability in the search for predictable and unpredictable proteins, is superior to other separation techniques. Unlimited amounts of recombinant cytokines and monoclonal antibodies (mAb) accelerated the "triumph". My approach concluded 35 years of worldwide pursuit for Type I IFN receptor (IFNAR2) and advanced the understanding of the signal transduction of this Type of IFN. TNF, IFNγ and IL-6 as baits enabled the isolation of their corresponding soluble receptors and N-terminal amino acid sequence of the isolated proteins facilitated the cloning of their cell surface counterparts. IL-18, IL-32, and heparanase as the baits yielded the corresponding unpredictable proteins: the antidote IL-18 Binding Protein (IL-18BP), the enzyme Proteinase 3 (PR3) and the hormone Resistin. IFNß proved beneficial in Multiple Sclerosis and is a blockbuster drug, Rebif®. TNF mAbs translated into Remicade® to treat Crohn's disease. Enbrel® based on TBPII is for Rheumatoid Arthritis. Both are blockbusters. Tadekinig alfa™, a recombinant IL-18BP, is in phase III clinical study for inflammatory and autoimmune diseases. Seven years of continuous compassionate use of Tadekinig alfa™ in children born with mutations (NLRC4, XIAP) proved life-saving and is an example of tailored made medicine. IL-18 is a checkpoint biomarker in cancer and IL-18BP is planned recently to target cytokine storms resulting from CAR-T treatment and in COVID 19.

The intercorrelations between blood levels of ferritin, sCD163, and IL-18 in COVID-19 patients and their association to prognosis.

Coronavirus disease 2019 (COVID-19) is associated with immune dysregulation, severe respiratory failure, and multiple organ dysfunction caused by a cytokine storm involving high blood levels of ferritin and IL-18. Furthermore, there is a resemblance between COVID-19 and macrophage activation syndrome (MAS) characterized by high concentrations of soluble CD163 (sCD163) receptor and IL-18. High levels of ferritin, IL-18, and sCD163 receptor are associated with "hyperferritinemic syndrome", a family of diseases that appears to include COVID-19. In this retrospective cohort study, we tested the association and intercorrelations in the serum levels of ferritin, sCD163, and IL-18 and their impact on the prognosis of COVID-19. We analyzed data of 70 hospitalized patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The levels of sCD163, ferritin, and IL-18 were measured and the correlation of these parameters with the respiratory deterioration and overall 30-day survival was assessed. Among the 70 patients, 60 survived 30 days from hospitalization. There were substantial differences between the subjects who were alive following 30 days compared to those who expired. The differences were referring to lymphocyte and leukocyte count, CRP, D-dimer, ferritin, sCD163, and IL-18. Results showed high levels of IL-18 (median, 444 pg/mL in the survival group compared with 916 pg/mL in the mortality group, p-value 8.54 × 10-2), a statistically significant rise in levels of ferritin (median, 484 ng/mL in the survival group compared with 1004 ng/mL in the mortality group p-value, 7.94 × 10-3), and an elevated value of in sCD163 (mean, 559 ng/mL in the survival group compared with 840 ng/mL in the mortality group, p-value 1.68 × 10-2). There was no significant correlation between the rise of ferritin and the levels sCD163 or IL-18. Taken together, sCD163, ferritin, and IL-18 were found to correlate with the severity of COVID-19 infection. Although these markers are associated with COVID-19 and might contribute to the cytokine storm, no intercorrelation was found among them. It cannot be excluded though that the results depend on the timing of sampling, assuming that they play distinct roles in different stages of the disease course. The data represented herein may provide clinical benefit in improving our understanding of the pathological course of the disease. Furthermore, measuring these biomarkers during the disease progression may help target them at the right time and refine the decision-making regarding the requirement for hospitalization.

Heparanase interacts with resistin and augments its activity.

In an attempt to isolate a heparanase receptor, postulated to mediate non-enzymatic functions of the heparanase protein, we utilized human urine collected from healthy volunteers. Affinity chromatography of this rich protein source on immobilized heparanase revealed resistin as a heparanase binding protein. Co-immunoprecipitation and ELISA further confirmed the interaction between heparanase and resistin. Importantly, we found that heparanase potentiates the bioactivity of resistin in its standard bioassay in which monocytic human leukemia cell line, THP1, differentiates into adherent macrophage-like foam cells. It is thus conceivable that this newly identified complex of heparanase and resistin exerts a stimulatory effect also in various inflammatory conditions known to be affected by the two proteins.

Interleukin-18, more than a Th1 cytokine.

Together with IL-12 or IL-15, interleukin-18 (IL-18) plays a major role in the production of interferon-γ from T-cells and natural killer cells; thus, IL-18 is considered to have a major role in the Th1 response. However, without IL-12, IL-18 is proinflammatory in an IFNγ independent manner. IL-18 is a member of the IL-1 family of cytokines and similar to IL-1ß, the cytokine is synthesized as an inactive precursor requiring processing by caspase-1 into an active cytokine. IL-18 is also present as an integral membrane protein but requires caspase-1 for full activity in order to induce IFNγ. Uniquely, unlike IL-1ß, the IL-18 precursor is constitutively present in nearly all cells in healthy humans and animals. The activity of IL-18 is balanced by the presence of a high-affinity, naturally occurring IL-18 binding protein (IL-18BP). In humans, increased disease severity can be associated with an imbalance of IL-18 to IL-18BP such that the levels of free IL-18 are elevated in the circulation. Increasing number of studies have expanded the role of IL-18 in mediating inflammation in animal models of disease using the IL-18BP, IL-18 deficient mice, neutralization of IL-18 or deficiency in the IL-18 receptor alpha chain. A role for IL-18 has been implicated in several autoimmune diseases, myocardial function, emphysema, metabolic syndromes, psoriasis, inflammatory bowel disease, macrophage activation syndrome, sepsis and acute kidney injury, although paradoxically, in some models of disease, IL-18 is protective. The IL-18BP has been used safely in humans and clinical trials of IL-18BP as well as neutralizing anti-IL-18 antibodies are being tested in various diseases.

Interleukin-18 and IL-18 binding protein.

Interleukin-18 (IL-18) is a member of the IL-1 family of cytokines. Similar to IL-1ß, IL-18 is synthesized as an inactive precursor requiring processing by caspase-1 into an active cytokine but unlike IL-1ß, the IL-18 precursor is constitutively present in nearly all cells in healthy humans and animals. The activity of IL-18 is balanced by the presence of a high affinity, naturally occurring IL-18 binding protein (IL-18BP). In humans, increased disease severity can be associated with an imbalance of IL-18 to IL-18BP such that the levels of free IL-18 are elevated in the circulation. Increasing number of studies have expanded the role of IL-18 in mediating inflammation in animal models of disease using the IL-18BP, IL-18-deficient mice, neutralization of IL-18, or deficiency in the IL-18 receptor alpha chain. A role for IL-18 has been implicated in several autoimmune diseases, myocardial function, emphysema, metabolic syndromes, psoriasis, inflammatory bowel disease, hemophagocytic syndromes, macrophage activation syndrome, sepsis, and acute kidney injury, although in some models of disease, IL-18 is protective. IL-18 plays a major role in the production of interferon-γ from T-cells and natural killer cells. The IL-18BP has been used safely in humans and clinical trials of IL-18BP as well as neutralizing anti-IL-18 antibodies are in clinical trials. This review updates the biology of IL-18 as well as its role in human disease.

LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) exhibits a remarkably robust and pantropic infectivity, mediated by its coat protein, VSV-G. Using this property, recombinant forms of VSV and VSV-G-pseudotyped viral vectors are being developed for gene therapy, vaccination, and viral oncolysis and are extensively used for gene transduction in vivo and in vitro. The broad tropism of VSV suggests that it enters cells through a highly ubiquitous receptor, whose identity has so far remained elusive. Here we show that the LDL receptor (LDLR) serves as the major entry port of VSV and of VSV-G-pseudotyped lentiviral vectors in human and mouse cells, whereas other LDLR family members serve as alternative receptors. The widespread expression of LDLR family members accounts for the pantropism of VSV and for the broad applicability of VSV-G-pseudotyped viral vectors for gene transduction.

Protection from inflammatory organ damage in a murine model of hemophagocytic lymphohistiocytosis using treatment with IL-18 binding protein.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition due to the association of an infectious agent with lymphocyte cytotoxicity defects, either of congenital genetic origin in children or presumably acquired in adults. In HLH patients, an excess of lymphocyte or macrophage cytokines, such as IFN-γ and TNFα is present in serum. In animal models of the disease, IFN-γ and TNF-α have been shown to play a central pathogenic role. In humans, unusually high concentrations of IL-18, an inducer of IFN-γ, and TNF-α have been reported, and are associated with an imbalance between IL-18 and its natural inhibitor IL-18 binding protein (IL-18BP) resulting in an excess of free IL-18. Here we studied whether IL-18BP could reduce disease severity in an animal model of HLH. Mouse cytomegalovirus infection in perforin-1 knock-out mice induced a lethal condition similar to human HLH characterized by cytopenia with marked inflammatory lesions in the liver and spleen as well as the presence of hemophagocytosis in bone marrow. IL-18BP treatment decreased hemophagocytosis and reversed liver as well as spleen damage. IL-18BP treatment also reduced both IFN-γ and TNF-α production by CD8(+) T and NK cells, as well as Fas ligand expression on NK cell surface. These data suggest that IL-18BP is beneficial in an animal model of HLH and in combination with anti-infectious therapy may be a promising strategy to treat HLH patients.

Ligand affinity chromatography, an indispensable method for the purification of soluble cytokine receptors and binding proteins.

Ligand affinity chromatography separation is based on unique interaction between the target analyte and a ligand, which is coupled covalently to a resin. It is a simple, rapid, selective, and efficient purification procedure of proteins providing tens of thousands fold purification in one step. The biological activity of the isolated proteins is retained in most cases thus function is revealed concomitantly with the isolation. Prior to the completion of the genome project this method facilitated rapid and reliable cloning of the corresponding gene. Upon completion of this project, a partial protein sequence is enough for retrieving its complete mRNA and hence its complete protein sequence. This method is indispensable for the isolation of both expected (e.g. receptors) but mainly unexpected, unpredicted and very much surprising binding proteins. No other approach would yield the latter. This chapter provides examples for both the expected target proteins, isolated from rich sources of human proteins, as well as the unexpected binding proteins, found by serendipity.

High circulating levels of free interleukin-18 in patients with active SLE in the presence of elevated levels of interleukin-18 binding protein.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies particularly to nuclear antigens and by an abnormal production of proinflammatory cytokines. In the present study, we measured the levels of the proinflammatory cytokine IL-18 and its natural inhibitor, the IL-18 binding protein (IL-18BP), in sera of SLE patients at various stages of the disease. This is the first study to present IL-18BP levels in sera of SLE patients as well as the calculated, biologically active, free IL-18 concentrations that are most probably more relevant to the pathology of SLE. Sera from 48 unselective SLE patients (total of 195 samples) were obtained longitudinally with a mean follow-up period of 11.1 +/- 8.9 years and were compared to sera from 100 healthy volunteers. Circulating levels of IL-18, IL-18BP and free IL-18 in the SLE patients were significantly higher than the levels of healthy controls (5 fold, 6 fold and 3 fold for IL-18, IL-18BP and free IL-18, respectively) and correlated with disease activity as scored by SLEDAI-2K. Furthermore, these levels during active disease (SLEDAI-2K > or = 6) were higher compared to the levels measured in the sera of the same patients during remission or during mild disease (SLEDAI-2K 0-5). The high levels of IL-18 and IL-18BP in sera of active SLE patients suggest their possible role in the pathogenesis and course of the disease. However, despite the elevated levels of IL-18BP during active disease, free IL-18 remained more than 2 fold higher than the levels in healthy controls suggesting a potential benefit of administration of exogenous IL-18BP as a novel therapeutic approach for active SLE.

IL-18 activity in systemic lupus erythematosus.

Interleukin-18 (IL-18) is an inflammation-related cytokine that plays a central role both in innate defense reactions and in Th1 activation and specific immune responses. Increased levels of IL-18 can be detected in biological fluids and organs of individuals affected by several autoimmune pathologies, as well as in autoimmune animal models. In this review, the role of IL-18 in systemic lupus erythematosus (SLE) is critically examined, including its possible role in the pathogenesis of disease. In SLE, increased levels of IL-18 have been found in serum/plasma of affected persons, which positively correlated with disease severity. The possibility that circulating IL-18 levels are predictive of renal damage has been proposed, suggesting that IL-18 may be a prognostic marker of renal involvement useful to identify patients at risk of renal failure. The evaluation of urinary levels of free active IL-18 indeed suggests a correlation with the degree of renal involvement. The possible pathogenic role of IL-18 in lupus has been studied in a mouse model of progressive disease, which makes possible the identification, at the level of the different affected organs, of IL-18 changes preceding disease development and those appearing after disease onset. It can be concluded that IL-18 has a multifaceted role in autoimmune lupus, being apparently involved both in the effector phases of the late organ damage and, in some organs, in the initial pathogenic events. Therapeutic strategies targeting IL-18 in autoimmunity are under development.

Local and systemic interleukin-18 and interleukin-18-binding protein in children with inflammatory bowel disease.

BACKGROUND: Interleukin-18 (IL-18) is increased in the inflamed mucosa of patients with Crohn's disease (CD). The balance between this pleiotropic proinflammatory cytokine and its natural inhibitor, IL-18-binding protein (IL-18BP), may contribute to the pathogenesis of inflammatory bowel disease (IBD). METHODS: Serum and mucosal biopsies were collected from children with IBD, from children with celiac disease, and from controls. Biopsies were maintained in culture for 24 hours, and supernatant was collected. Serum and supernatant IL-18 and IL-18BPa concentrations were measured by immunoassay. Disease activity score (PCDAI) and standard serum inflammatory markers (albumin, platelets, ESR, and CRP) were recorded. RESULTS: Serum IL-18 was greater in children with CD (537 pg/mL) than in controls (335 pg/mL; P < 0.05) but not in children with ulcerative colitis (UC) or IBD type unclassified (IBDU). Mucosal IL-18 was greater in children with CD and UC/IBDU than in controls (P < 0.01). Serum IL-18BPa was increased in children with CD compared with that in controls (3.9 versus 2.6 ng/mL; P < 0.05), but was not elevated in children with UC/IBDU. Furthermore, calculated free-serum IL-18 was elevated in CD, but not UC/IBDU, compared with that in controls (P = 0.001). Total and free-serum IL-18 were elevated in severe CD relative to in mild/moderate disease. CONCLUSIONS: IL-18, produced in the colons of children with IBD, may contribute to local inflammatory changes. Systemic IL-18 level may be a useful indicator of gut inflammation. Furthermore, free IL-18 is greatly elevated in children with CD, suggesting that compensatory increases in IL-18BPa are insufficient. Further exploration of the role of this cytokine in the pathogenesis of IBD is now required.

Free Interleukin (IL)-18 levels, and the impact of IL18 and IL18BP genetic variation, in CHD patients and healthy men.

OBJECTIVE: To investigate free interleukin-18 (fIL-18) levels, and variation within the IL-18 system genes, in heart surgery patients, and healthy men. METHODS AND RESULTS: fIL-18 was calculated from IL-18 and IL-18 binding protein (BP) levels, in 421 healthy men and 196 post-coronary artery bypass graft (CABG) patients. After surgery, fIL-18 peaked at 6 hours (from 117 to 331 pg/mL) but fell to below presurgery levels at 24 hours (99 pg/mL), because of changes in total IL-18 and IL-18BP. fIL-18 24 hours postsurgery was significantly higher in those who suffered a major complication after surgery (125 versus 80 pg/mL, P<0.01). Baseline total IL-18 was also higher in healthy men who went on to suffer an MI over 17 years of prospective study (276 versus 240 pg/mL, P=0.01). Tagging SNPs for IL18 (n=5) and IL18BP (n=3) were determined, in both studies the IL18 HapIII haplotype (frequency 30%) was associated with 36% lower baseline fIL-18 levels before surgery (P<0.01), and 7% lower in healthy men (P=0.04). The frequency of HapIII was lower in CABG patients than in healthy men (20.7 versus 29.8%, P<0.01). CONCLUSIONS: IL-18 levels, which are determined in part by variation in IL18, play a role in CHD development and postsurgery outcome.

The tale of soluble receptors and binding proteins: from bench to bedside.

Our approach of isolating proteins from a rich source of human proteins by ligand-affinity-chromatography enabled rapid and efficient isolation of not only soluble receptors corresponding to cell-associated receptors, but also independent binding-proteins and associated enzymes. No other approach would yield the latter. During the early 80's we prepared the tools and the infrastructure that enabled the subsequent 20 years of achievements. Thus we described eight soluble receptors (R) and binding proteins (BP) for various cytokines including the IL-6R, IFN-gammaR, TNFRI, TNFRII, LDLR, IFN-alpha/betaR, IL-18BP and IL-32BP identified as Proteinase 3. The isolation of the soluble IFN-alpha/beta receptor led to the cloning of its long sought cell surface ligand binding counterpart. We have established the concept that soluble receptors and binding proteins are normal constituents of body fluids in healthy individuals and that the levels of these biomarkers are modulated in various pathological situations. Each of these proteins contributed to basic science, one of them serves as a basis for therapy and some others are in various stages of clinical development.

Antiviral and immunoregulatory activities of IFN-gamma depend on constitutively expressed IL-1alpha.

IFN-gamma induces its immunoregulatory activities by activating genes mainly through the Jak-STAT signaling pathway. Here we show that what was considered to be intrinsic IFN-gamma activities depend largely on the basal level of NF-kappaB, which is maintained by constitutively expressed IL-1alpha. The IL-1 receptor antagonist and antibodies to IL-1alpha, but not to IL-1beta, inhibited the antiviral activity of IFN-gamma by 90%, whereas no inhibition of type I IFN activity was observed. Similarly, the induction of many genes by IFN-gamma, including HLA-DR, ICAM-1, IL-18BP, and genes mediating its antiviral activity, greatly depended on basal IL-1alpha. Furthermore, IFN-gamma induced serum IL-18 binding protein in wild-type mice but not in IL-1alpha/beta double-deficient mice. Thus, constitutively expressed IL-1alpha is critical for numerous IFN-gamma activities.

Proteinase 3 is an IL-32 binding protein.

IL-32, a recently discovered proinflammatory cytokine with four isoforms, induces IL-1beta, TNF-alpha, IL-6, and chemokines. Here, we used ligand (IL-32alpha) affinity chromatography in an attempt to isolate an IL-32alpha soluble receptor or binding protein. Recombinant IL-32alpha was covalently immobilized on agarose, and preparations of concentrated crude human urinary proteins were applied for chromatographic separation. A specific 30-kDa protein eluted from the column during acid washing and was identified by mass spectrometry as proteinase 3 (PR3) and confirmed by N-terminal microsequencing. PR3, a neutrophil granule serine protease, exists in a soluble or membrane form and is the major autoantigen for autoantibodies in the systemic vasculitic disease, Wegener's granulomatosis. The affinity of IL-32alpha to PR3 was determined by surface plasmon resonance. The dissociation constants were 2.65 +/- 0.4 nM for urinary PR3 and 1.2 +/- 0.05 nM for neutrophil-derived PR3. However, irreversible inactivation of PR3 enzymatic activity did not significantly change binding to the cytokine. Nevertheless, limited cleavage of IL-32 yielded products consistent with PR3 enzyme activity. Moreover, after limited cleavage by PR3, IL-32alpha was more active than intact IL-32alpha in inducing macrophage inflammatory protein-2 in mouse macrophages and IL-8 in human peripheral blood mononuclear cells. We suggest that PR3 is a specific IL-32alpha binding protein, independent of its enzymatic activity. However, limited cleavage of IL-32alpha by PR3 enhances activities of the cytokine. Therefore, specific inhibition of PR3 activity to process IL-32 or neutralization of IL-32 by inactive PR3 or its fragments may reduce the consequences of IL-32 in immune regulated diseases.

Severe imbalance of IL-18/IL-18BP in patients with secondary hemophagocytic syndrome.

Hemophagocytic syndrome (HPS) is characterized by an uncontrolled and poorly understood activation of T-helper 1 (Th-1) lymphocytes and macrophages. We studied 20 patients with HPS secondary to infections, autoimmune disease, lymphoma, or cancer and observed that the concentrations of serum interleukin 18 (IL-18), a strong inducer of Th-1 responses, interferon gamma (IFN-gamma) production, and stimulation of macrophages and natural killer (NK) cells were highly increased in HPS but not in control patients. In contrast, concentrations of its natural inhibitor, the IL-18 binding protein (IL-18BP), were only moderately elevated, resulting in a high level of biologically active free IL-18 in HPS (4.6-fold increase compared with controls; P < .001). Free IL-18 but not IL-12 concentrations significantly correlated with clinical status and the biologic markers of HPS such as anemia (P < .001), hypertriglyceridemia, and hyperferritinemia (P < .01) and also with markers of Th-1 lymphocyte or macrophage activation, such as elevated concentrations of IFN-gamma and soluble IL-2 and tumor necrosis factor alpha (TNF-alpha) receptor concentrations. Despite high IL-18 elevation, in vitro NK-cell cytotoxicity was severely impaired in HPS patients, in part due to NK-cell lymphopenia that was observed in a majority of patients but also secondary to an intrinsic NK-cell functional deficiency. We concluded that a severe IL-18/IL-18BP imbalance results in Th-1 lymphocyte and macrophage activation, which escapes control by NK-cell cytotoxicity and may allow for secondary HPS in patients with underlying diseases.