IL-18 Binding Protein Research Articles

Evaluation of serum IL 18 / IL 18 binding protein ratio and their relation with IL- 18 gene polymorphisms in sample of Iraqi type 2 diabetes mellitus patients. A case control study.

To evaluate the ratio of interleukin18 and interleukin18 binding protein in type 2 diabetes mellitus patients, and its relation with the presence of interleukin18-607C/A and interleukin18-137G/C gene polymorphisms and the type of complications. The case-control study was conducted at the endocrinology clinic of the Madinat Al-Imamin Al-Kazemin Teaching Hospital, Iraq, from September 2020 to July 2021, and comprised diagnosed patients of type 2 diabetes mellitus, and healthy controls matched for age and gender. Blood samples were obtained that were processed for serum separation. Serum interleukin18 and interleukin18 binding protein levels were assessed, and part of the sample was used for deoxyribonucleic acid extraction using tetra-primer amplification refractory mutation system polymerase chain reaction with four specific primers for interleukin18-607C/A and interleukin-18-137G/C gene polymorphisms in both the groups. Data was analysed using SPSS 20. Of the 168 subjects, 86(51.2%) were patients; 67(77.9%) females and 19(22.1%) males with mean age 52±8.97 years. There were 82(48.8%) controls; 56(68.3%) females and 26(31.7%) males with mean age 48±9.44 years (p>0.05). Higher serum level of interleukin18 and lower level of interleukin18 binding protein were seen in type 2 diabetes mellitus group compared to the controls (p<0.001). Interleukin18-137G/C and interleukin18-607C/A mutant alleles had odd ratios 3.52 (confidence interval: 1.91- 6.63) and 3.25 (confidence interval: 1.77-5.83), respectively, as risk factors for the occurrence of type 2 diabetes mellitus. There was an association of interleukin18- 137G/C homozygous mutant genotype with the occurrence of retinopathy among type 2 diabetes mellitus patients (p=0.046), but risk allele C was not associated with retinopathy (p>0.05). The presence of interleukin18-137G/C and interleukin18-607C/A gene polymorphism might be considered a risk factor for type 2 diabetes mellitus.

Hyperferritinemia Screening to Aid Identification and Differentiation of Patients with Hyperinflammatory Disorders

High ferritin is an important and sensitive biomarker for the various forms of hemophagocytic lymphohistiocytosis (HLH), a diverse and deadly group of cytokine storm syndromes. Early action to prevent immunopathology in HLH often includes empiric immunomodulation, which can complicate etiologic work-up and prevent collection of early/pre-treatment research samples. To address this, we instituted an alert system at UPMC Children’s Hospital where serum ferritin > 1000 ng/mL triggered real-time chart review, assessment of whether the value reflected “inflammatory hyperferritnemia (IHF)”, and biobanking of remnant samples from consenting IHF patients. We extracted relevant clinical data; periodically measured serum total IL-18, IL-18 binding protein (IL-18BP), and CXCL9; retrospectively classified patients by etiology into infectious, rheumatic, or immune dysregulation; and subjected a subgroup of samples to a 96-analyte biomarker screen. 180 patients were identified, 30.5% of which had IHF. Maximum ferritin levels were significantly higher in patients with IHF than with either hemoglobinopathy or transplant, and highly elevated total IL-18 levels were distinctive to patients with Stills Disease and/or Macrophage Activation Syndrome (MAS). Multi-analyte analysis showed elevation in proteins associated with cytotoxic lymphocytes in all IHF samples when compared to healthy controls and depression of proteins such as ANGPT1 and VEGFR2 in samples from hyperferritinemic sepsis patients relative to non-sepsis controls. This real-time IFH screen proved feasible and efficient, validated prior observations about the specificity of IL-18, enabled early sample collection from a complex population, suggested a unique vascular biomarker signature in hyperferritinemic sepsis, and expanded our understanding of IHF heterogeneity.

IL-18 signaling is regulated by caspase 6/8 and IL-18BP in turbot (Scophthalmus maximus)

Interleukin (IL)-18 is synthesized as a precursor that requires intracellular processing to become functionally active. In human, IL-18 is processed by caspase 1 (CASP1). In teleost, the maturation and signal transduction mechanisms of IL-18 are unknown. We identified two IL-18 variants, IL-18a and IL-18b, in turbot. IL-18a, but not IL-18b, was processed by CASP6/8 cleavage. Mature IL-18a bound specifically to IL-18 receptor (IL-18R) α-expressing cells and induced IL-18Rα—IL-18Rβ association. Bacterial infection promoted IL-18a maturation in a manner that required CASP6 activation and correlated with gasdermin E activation. The mature IL-18a induced proinflammatory cytokine expression and enhanced bacterial clearance. IL-18a-mediated immune response was suppressed by IL-18 binding protein (IL-18BP), which functioned as a decoy receptor for IL-18a. IL-18BP also functioned as a pathogen pattern recognition receptor and directly inhibited pathogen infection. Our findings revealed unique mechanism of IL-18 maturation and conserved mechanism of IL-18 signaling and regulation in turbot, and provided new insights into the regulation and function of IL-18 related immune signaling.

IL-6 and IL-18 cytokine traps in COVID-19

Cytokines are mediators of immunity that regulate inflammation. Intensity of inflammatory process is strongly dependent on the cytokine type and duration of its effect. Interleukin 6 (IL-6) and interleukin 18 (IL-18) play an important role in the initiation and progression of inflammation. Cytokines regulate the inflammatory process in different ways by inducing or inhibiting inflammatory reactions. Functional activity of cytokines is limited by trap molecules whose levels determine initiation of protective or pathological effects of interleukins. Soluble glycoprotein sgp130 functions as a trap for IL-6, while IL-18 is controlled by IL-18 binding protein (IL-18BP). High IL-6 and IL-18 levels were recorded in COVID-19 patients, being associated with unfavorable outcome of the disease. Our objective was to compare sgp130 and IL-18BP levels in patients with different degrees of COVID-19. Retrospective study included 74 COVID-19 patients (40 men and 34 women) aged 63±14 years. The patients were assigned to groups according to severity of lung damage. Group 1 included patients without lung damage; group 2, patients with moderate pneumonia ( 50% lung damage); group 3, patients with severe pneumonia ( 50% lung damage). Plasma levels of cytokines and their trap molecules were determined by quantitative immunoenzyme assay. IL-6 and IL-18 plasma concentrations increased with COVID-19 severity. Ambiguous changes were recorded for their traps. Plasma levels of sgp130 were lower in patients with moderate pneumonia than in patients without lung damage. In patients with severe pneumonia sgp130 plasma concentrations were higher than those in patients with mild pneumonia, being similar to those in patients without lung damage. In contrast to sgp130, IL-18BP levels decreased with COVID-19 severity. Thus, an increase in IL-6 and IL-18 levels parallel to COVID-19 severity is accompanied by ambiguous changes in the levels of their trap molecules. The ratio between the levels of IL-6 and IL-18 and their traps reflects the degree of COVID-19 severity.

Interleukin-18 binding protein regulates mast cell activation and mast cell induced osteoclastogenesis of rheumatoid arthritis.

Mast cell activation induces pathological responses, including increased osteoclastogenesis in rheumatoid arthritis (RA). Interleukin (IL)-18 binding protein (IL-18BP) has anti-inflammatory effects. In this study, we evaluated the effect of IL-18BP on mast cell activation and mast cell induced osteoclastogenesis. Mast cells were activated by IL-33 (100 ng/mL) and cultured with IL-18BP (10, 50, and 100 ng/mL). The proliferation, apoptosis, and necroptosis of mast cells were measured using flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of mast cell enzymes, matrix metalloproteinase (MMP), soluble RANKL (sRANKL), and pro-inflammatory cytokines in the culture media. Monocytes from patients with RA patients (n=5) were cultured with activated mast cells with various concentrations of IL-18BP. TRAP+ multinucleated osteoclasts, bone resorption area, and osteoclast differentiation-related genes were measured. Proliferation of tryptase+chymase+c-kit+FcεR1+ mast cells was suppressed following incubation with IL-18BP (10, 50, and 100 ng/mL). RNA expression levels of tryptase and chymase were reduced by 100 ng/mL IL-18BP. Additionally, the levels of MMP-3/9, IL-17A, IL-6, TNF-α, and sRANKL were significantly inhibited by 100 ng/mL IL-18BP. Annexin V+ and annexin V-PI+ mast cells were reduced following incubation with 100 ng/mL IL-18BP. The addition of IL-33 significantly stimulated mast cell and increased TRAP+ multinucleated cells and bone resorption area, and these effects were suppressed by IL-18BP. The osteoclast-related genes (TRAP, ATP6v0d2, RANK, and cathepsin K) expression were suppressed by IL-18BP. IL-18BP suppressed mast cell activation and mast cell induced osteoclastogenesis. This suggests a potential anti-arthritic role for IL-18BP in patients with RA.

YTHDF2 Regulates Advanced Glycation End Products-Induced Melanogenesis through Inhibiting A20 Expression in Human Dermal Fibroblasts.

Fibroblast A20 suppresses advanced glycation end products (AGEs)-induced melanogenesis by inhibiting NLRP3 inflammasome activation. AGEs repress A20 expression and significantly m6A-methylate A20 mRNA in fibroblasts. YTHDF2 is the most studied m6A reader protein and can accelerate degradation of m6A-modified mRNA. Whether YTHDF2 regulates AGEs-induced A20 expression and pigmentation is unknown. In this study, we confirmed that YTHDF2 inversely regulated AGEs-BSA-inhibited A20 expression but facilitated AGEs-BSA-activated NF-κB signaling and NLRP3 inflammasome in fibroblasts via YTHDF2 knockdown and overexpression experiments. Mechanistically, YTHDF2 bound to m6A-modified A20 mRNA induced by AGEs-BSA and increased its degradation. Moreover, fibroblast YTHDF2 robustly promoted AGEs-BSA-induced IL-18 level in coculture supernatants and melanin content, tyrosinase activity, and expression of microphthalmia-associated transcription factor and tyrosinase in melanocytes, which were significantly blocked by IL-18 binding protein. Further, fibroblast YTHDF2 markedly increased AGEs-BSA-induced epidermal melanin level in cocultured ex vivo skin and MAPKs activation in melanocytes. Importantly, upregulated dermal YTHDF2 expression was negatively correlated with dermal A20 level and positively associated with both epidermal melanin and dermal AGEs content in sun-exposed skin and lesions of melasma and solar lentigo. These findings suggest that fibroblast YTHDF2 positively regulates AGEs-induced melanogenesis mainly via A20/ NF-κB /NLRP3 inflammasome/ IL-18 /MAPKs axis in an m6A-dependent manner and functions in photoaging-induced hyperpigmentation skin disorders.

Hyperferritinemia screening to aid identification and differentiation of patients with hyperinflammatory disorders.

High ferritin is an important and sensitive biomarker for hemophagocytic lymphohistiocytosis (HLH), a diverse and deadly group of cytokine storm syndromes. Early action to prevent immunopathology in HLH often includes empiric immunomodulation, which can complicate etiologic work-up and prevent collection of early/pre-treatment research samples. To address this, we instituted an alert system where serum ferritin > 1000ng/mL triggered real-time chart review, assessment of whether the value reflected "inflammatory hyperferritnemia (IHF)", and biobanking of remnant samples from consenting IHF patients. We extracted relevant clinical data; periodically measured serum total IL-18, IL-18 binding protein (IL-18BP), and CXCL9; retrospectively classified patients by etiology into infectious, rheumatic, or immune dysregulation; and subjected a subgroup of samples to a 96-analyte biomarker screen. 180 patients were identified, 30.5% of which had IHF. Maximum ferritin levels were significantly higher in patients with IHF than with either hemoglobinopathy or transplant, and highly elevated total IL-18 levels were distinctive to patients with Stills Disease and/or Macrophage Activation Syndrome (MAS). Multi-analyte analysis showed elevation in proteins associated with cytotoxic lymphocytes in all IHF samples when compared to healthy controls and depression of proteins such as ANGPT1 and VEGFR2 in samples from hyperferritinemic sepsis patients relative to non-sepsis controls. This single-center, real-time IFH screen proved feasible and efficient, validated prior observations about the specificity of IL-18, enabled early sample collection from a complex population, suggested a unique vascular biomarker signature in hyperferritinemic sepsis, and expanded our understanding of IHF heterogeneity.

Plasmatic Inactive IL-18 Predicts a Worse Overall Survival for Advanced Non-Small-Cell Lung Cancer with Early Metabolic Progression after Immunotherapy Initiation.

This ancillary study includes 195 patients with metastatic non-small-cell lung cancer (NSCLC) treated with ICI in monotherapy, either pembrolizumab or nivolumab. Plasmatic levels of IL-18-related compounds, comprising the inhibitor IL-18 binding protein (IL-18BP), the inactive IL-18 (corresponding to IL-18/IL-18BP complex), and the active free IL-18, were assayed by ELISA. Objective tumoral response was analyzed by 18FDG PET-CT at baseline, 7 weeks, and 3 months post treatment induction, using PERCIST criteria. Plasmatic IL-18BP and total IL-18 levels are increased at baseline in NSCLC patients compared with healthy controls, whereas IL-18/IL-18BP complexes are decreased, and free IL-18 levels remain unchanged. Neither of the IL-18-related compounds allowed to discriminate ICI responding to nonresponding patients. However, inactive IL-18 levels allowed to discriminate patients with a first tumor progression, assessed after 7 weeks of treatment, with worse overall survival. In addition, we showed that neutrophil concentration is also a predictive indicator of patients' outcomes with OS (HR = 2.6, p = 0.0001) and PFS (HR = 2.2, p = 0.001). Plasmatic levels of inactive IL-18, combined with circulating neutrophil concentrations, can effectively distinguish ICI nonresponding patients with better overall survival (OS), potentially guiding rapid decisions for therapeutic intensification.

Phase 1 study of CAR-37 T cells in patients with relapsed or refractory CD37+ lymphoid malignancies

AbstractWe report a first-in-human clinical trial using chimeric antigen receptor (CAR) T cells targeting CD37, an antigen highly expressed in B- and T-cell malignancies. Five patients with relapsed or refractory CD37+ lymphoid malignancies were enrolled and infused with autologous CAR-37 T cells. CAR-37 T cells expanded in the peripheral blood of all patients and, at peak, comprised >94% of the total lymphocytes in 4 of 5 patients. Tumor responses were observed in 4 of 5 patients with 3 complete responses, 1 mixed response, and 1 patient whose disease progressed rapidly and with relative loss of CD37 expression. Three patients experienced prolonged and severe pancytopenia, and in 2 of these patients, efforts to ablate CAR-37 T cells, which were engineered to coexpress truncated epidermal growth factor receptor, with cetuximab were unsuccessful. Hematopoiesis was restored in these 2 patients after allogeneic hematopoietic stem cell transplantation. No other severe, nonhematopoietic toxicities occurred. We investigated the mechanisms of profound pancytopenia and did not observe activation of CAR-37 T cells in response to hematopoietic stem cells in vitro or hematotoxicity in humanized models. Patients with pancytopenia had sustained high levels of interleukin-18 (IL-18) with low levels of IL-18 binding protein in their peripheral blood. IL-18 levels were significantly higher in CAR-37–treated patients than in both cytopenic and noncytopenic cohorts of CAR-19–treated patients. In conclusion, CAR-37 T cells exhibited antitumor activity, with significant CAR expansion and cytokine production. CAR-37 T cells may be an effective therapy in hematologic malignancies as a bridge to hematopoietic stem cell transplant. This trial was registered at www.ClinicalTrials.gov as #NCT04136275.

Acquisition of different transcriptional shear mRNA and biological function of porcine interleukin 18 binding protein in PRRSV infection.

Interleukin-18 binding protein (IL-18BP), a natural regulator molecule of the pro-inflammatory cytokine interleukin-18 (IL-18), plays an important role in regulating the expression of the cellular immunity factor interferon-γ (IFN-γ). In a previous RNA-seq analysis of porcine alveolar macrophages (PAM) infected with the TIM and TJ strains of porcine reproductive and respiratory syndrome virus (PRRSV), we unexpectedly found that the mRNA expression of porcine interleukin 18-binding protein (pIL-18BP) in PAM cells infected with the TJM strain was significantly higher than that infected with the TJ strain. Studies have shown that human interleukin-18 binding protein (hIL-18bp) plays an important role in regulating cellular immunity in the course of the disease. However, there is a research gap on pIL-18BP. At the same time, PRRSV infection in pigs triggers weak cellular immune response problems. To explore the expression and the role of pIL-18BP in the cellular immune response induced by PRRSV, we strived to acquire the pIL-18BP gene from PAM or peripheral blood mononuclear cell (PBMC) with RT-PCR and sequencing. Furthermore, pIL-18BP and pIL-18 were both expressed prokaryotically and eukaryotically. The colocalization and interaction based on recombinant pIL-18BP and pIL-18 on cells were confirmed in vitro. Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in pigs with different PRRSV infection states to interpret the biological function of pIL-18BP in vivo. The results showed there were five shear mutants of pIL-18BP. The mutant with the longest coding region was selected for subsequent functional validation. First, it was demonstrated that TJM-induced pIL-18BP mRNA expression was higher than that of TJ. A direct interaction between pIL-18BP and pIL-18 was confirmed through fluorescence colocalization, bimolecular fluorescent complimentary (BIFC), and co-immunoprecipitation (CO-IP). pIL-18BP also can regulate pIFN-γ mRNA expression. Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in different PRRSV infection states. Surprisingly, both mRNA and protein expression of pIL-18 were suppressed. These findings fill the gap in understanding the roles played by pIL-18BP in PRRSV infection and provide a foundation for further research.IMPORTANCEPRRSV-infected pigs elicit a weak cellular immune response and the mechanisms of cellular immune regulation induced by PRRSV have not yet been fully elucidated. In this study, we investigated the role of pIL-18BP in PRRSV-induced immune response referring to the regulation of human IL-18BP to human interferon-gamma (hIFN-γ). This is expected to be used as a method to enhance the cellular immune response induced by the PRRSV vaccine. Here, we mined five transcripts of the pIL-18BP gene and demonstrated that it interacts with pIL-18 and regulates pIFN-γ mRNA expression. Surprisingly, we also found that both mRNA and protein expression of pIL-18 were suppressed under different PRRSV strains of infection status. These results have led to a renewed understanding of the roles of pIL-18BP and pIL-18 in cellular immunity induced by PRRSV infection, which has important implications for the prevention and control of PRRS.

Structural transitions enable interleukin-18 maturation and signaling

Several interleukin-1 (IL-1) family members, including IL-1β and IL-18, require processing by inflammasome-associated caspases to unleash their activities. Here, we unveil, by cryoelectron microscopy (cryo-EM), two major conformations of the complex between caspase-1 and pro-IL-18. One conformation is similar to the complex of caspase-4 and pro-IL-18, with interactions at both the active site and an exosite (closed conformation), and the other only contains interactions at the active site (open conformation). Thus, pro-IL-18 recruitment and processing by caspase-1 is less dependent on the exosite than the active site, unlike caspase-4. Structure determination by nuclear magnetic resonance uncovers a compact fold of apo pro-IL-18, which is similar to caspase-1-bound pro-IL-18 but distinct from cleaved IL-18. Binding sites for IL-18 receptor and IL-18 binding protein are only formed upon conformational changes after pro-IL-18 cleavage. These studies show how pro-IL-18 is selected as a caspase-1 substrate, and why cleavage is necessary for its inflammatory activity.

Deficiency of IL-22–binding protein enhances the ability of the gut microbiota to protect against enteric pathogens

Interleukin 22 (IL-22) promotes intestinal barrier integrity, stimulating epithelial cells to enact defense mechanisms against enteric infections, including the production of antimicrobial peptides. IL-22 binding protein (IL-22BP) is a soluble decoy encoded by the Il22ra2 gene that decreases IL-22 bioavailability, attenuating IL-22 signaling. The impact of IL-22BP on gut microbiota composition and functioning is poorly understood. We found that Il22ra2-/- mice are better protected against Clostridioides difficile and Citrobacter rodentium infections. This protection relied on IL-22-induced antimicrobial mechanisms before the infection occurred, rather than during the infection itself. Indeed, the gut microbiota of Il22ra2-/- mice mitigated infection of wild-type (WT) mice when transferred via cohousing or by cecal microbiota transplantation. Indicator species analysis of WT and Il22ra2-/- mice with and without cohousing disclosed that IL22BP deficiency yields a gut bacterial composition distinct from that of WT mice. Manipulation of dietary fiber content, measurements of intestinal short-chain fatty acids and oral treatment with acetate disclosed that resistance to C. difficile infection is related to increased production of acetate by Il22ra2-/--associated microbiota. Together, these findings suggest that IL-22BP represents a potential therapeutic target for those at risk for or with already manifest infection with this and perhaps other enteropathogens.

Blood Interleukin-18 (IL-18) and IL-18 Binding Protein (IL-18BP) Levels Following Midline Laparotomy: A Prospective Randomized Study of Patients With Benign Disease and Patients With Cancer.

The acute phase immune response (APR) in midline laparotomy (MLa) patients following surgery has been rarely studied, with no studies assessing the association of blood IL-18 (interleukin-18) and IL-18BP (IL-18 binding protein) values with the numeric rating scale (NRS) pain score following MLa. Blood levels of seven cytokines (CYT) (IL-18, IL-18BP, IL-1ra, IL-6, IL-8, IL-10, IL-1β) and high-sensitivity C-reactive protein (hs-CRP) were measured at three time points; before operation (PRE), immediately after operation (POP1), and 24 h after operation (POP2) in 56 patients with MLa. The satisfaction of the patients at 24 h following MLa (SFS24; 0=fully unsatisfied; 10=fully satisfied) was recorded on a 11-point numeric rating scale. In all patients, the IL-18 and IL-18BP blood levels decreased at POP1 and the drop between the preoperative and POP1 levels in the IL-18 and IL-18BP was highly significant (p<0.001). However, the median IL-18 and IL-18BP blood levels increased significantly at POP2 (p<0.001) with the linear mixed-effect model (LME) showing a statistically significant time effect (p<0.001). The hs-CRP blood levels increased significantly at POP2 with the LME model showing a statistically significant time effect. The preoperative and POP2 IL-18 values were clearly higher in patients with cancer versus benign disease (177/182 vs. 135/126, p=0.039/p=0.013, respectively). Interestingly, in all patients of the study, the median IL-18 versus IL-18BP blood levels correlated at POP1 (r=0.315, p=0.036). A noteworthy discovery of this study is the correlation of IL-18BP with SFS24 (r=0.361, p=0.05), proposing that APR and quality of life are associated in MLa patients.

Interleukin‑18 binding protein: Biological properties and roles in human and animal immune regulation (Review).

IL-18 binding protein (IL-18BP) is a natural regulatory molecule of the proinflammatory cytokine IL-18. It can regulate activity of IL-18 by high affinity binding. The present review aimed to highlight developments, characteristics and functions of IL-18BP. IL-18BP serves biological and anti-pathological roles in treating disease. In humans, it modulates progression of a number of chronic diseases, such as adult-onset Still's disease. The present review summarizes molecular structure, role of IL-18BP in disease and interaction with other proteins in important pathological processes.

Abstract 4076: Interleukin-18 engineered for resistance to IL-18 binding protein (IL-18BP) and half-life extension to enhance its therapeutic potential

Abstract Interleukin-18 (IL-18) possesses a unique combination of innate and acquired immune functions with high potential to be transformative in cancer immunotherapy of solid tumors. In clinical studies, recombinant wild-type IL-18 was limited by rapid upregulation of IL-18 binding protein (IL-18BP), which serves as a negative feedback checkpoint of the IL-18 pathway. As its name implies, IL-18BP tightly binds to IL-18, thereby blocking its ability to activate the IL-18 receptor. We have focused our protein engineering efforts to obtain variants of IL-18 that are resistant to IL-18BP suppression while retaining IL-18 biological activity. To further capitalize on the immunological effects of IL-18, the IL-18BP resistant variants were fused to half-life extension protein scaffolds for enhanced in vivo exposure. Rational protein design and combinatorial approaches were used to generate a pool of IL-18 variants that were screened for potency and resistance to IL-18BP. Several IL-18 variants showed undetectable binding to IL-18BP (up to 1 mM IL-18BP) and displayed a spectrum of potencies relative to recombinant wild-type IL-18. Importantly, these variants retained their full biological activity in the presence of super-physiological levels of IL-18BP, as tested with in vitro cell-based IFN-γ release assays. In vivo pharmacokinetic studies in mice compared several IL-18BP resistant variants fused to a half-life extension scaffold with a non-half-life enhanced IL-18BP resistant variant. The half-lives for the fusion proteins ranged from 15 - 30h, while the non-fused variant showed a half-live of 0.6h. Furthermore, the IL-18 variants displayed a more durable, Th1 immune response compared to a non-half-life enhanced variant. Thus, IL-18 variants with strong resistance to IL-18BP that are fused to a half-life extension protein scaffold displayed enhanced pharmacokinetic and pharmacodynamic properties in preclinical mouse models. These protein engineering modifications will enable us to select an appropriate development candidate with enhanced pharmaceutical properties to achieve the full therapeutic potential of IL-18 to assess in patients with cancer. Citation Format: Jean Chamoun, Clifford DiLea, Kristiana Dreaden, Pinar Gurel, Joshua Heiber, Robert G. Newman, Su-Ping Pearson, Chunhua Wang, Yanchun Zhao, Mark Whitmore. Interleukin-18 engineered for resistance to IL-18 binding protein (IL-18BP) and half-life extension to enhance its therapeutic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4076.

Abstract 4074: Discovery of WTX-518, an IL-18 pro-drug that is conditionally activated within the tumor microenvironment and induces regressions in mouse tumor models

Abstract Systemic administration of proinflammatory cytokines is a promising approach to treat cancer. IL-18 has been shown to promote activation of both innate and adaptive anti-tumor responses in pre-clinical models. Specifically, IL-18 has been shown to impact macrophage suppressive function as well as drive CD4+ T cells towards a TH1 phenotype. However, IL-18 therapies have been hampered by a lack of efficacy due to the suppressive activity of IL-18 binding protein (IL-18BP), which binds to IL-18 and prevents its interaction with its receptor. Several groups have taken the approach of modifying IL-18 to render it resistant to IL-18BP as a potential strategy to enhance anti-tumor activity. To explore the therapeutic benefit of delivering a conditionally-activated IL-18 to the tumor microenvironment, we have developed inducible polypeptides (INDUKINE™ molecules) consisting of mouse IL-18 or IL-18BP resistant IL-18 (BPR IL-18) tethered via protease-sensitive linkers to a high affinity antibody blockade domain, and a half-life extension (HLE) domain which improves exposure in the tumor. IL-18 INDUKINE™ molecules are inactive until reaching the tumor microenvironment, where the linkers are cleaved by intra-tumoral proteases, releasing active IL-18. Intraperitoneal (i.p.) administration of an IL-18BP resistant IL-18 (BPR IL-18) INDUKINE™ molecule led to complete tumor regression in the MC38 tumor model. In contrast, equimolar doses of wild-type IL-18 INDUKINE™ protein were not as efficacious. Moreover, BPR IL-18 INDUKINE treatment led to increased activation and frequencies of NK cells and tumor specific CD8 T cells in MC38 tumors. WTX-518 is a novel INDUKINE™ molecule that is designed to selectively deliver active human BPR IL-18 to the tumor microenvironment. WTX-518 is inducible in vitro, either when tested using a reporter assay or primary immune cells. Importantly, the active BPR IL-18 payload is resistant to IL-18BP in reporter or primary immune cell assays. WTX-518 is also selectively inducible, as incubation ex vivo with various primary human tumors led to the release of active BPR IL-18, while WTX-518 was stable in human serum and after exposure to primary cells from healthy tissues. Pharmacological and efficacy data support continued preclinical development of this innovative and differentiated engineered IL-18 therapy. Citation Format: Kristin R. Morris, Heather Brodkin, Kyriakos Economides, Daniel J. Hicklin, Julie LePrevost, Celesztina Nagy-Domonkos, Christopher Nirschl, Andres Salmeron, Cynthia Seidel-Dugan, Cierra Spencer, Zoe Steuert, William M. Winston. Discovery of WTX-518, an IL-18 pro-drug that is conditionally activated within the tumor microenvironment and induces regressions in mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4074.

Abstract 4073: Decoy-resistant IL-18 enhances checkpoint inhibitor combinations beyond anti-PD-1 in vitro and in vivo

Abstract Background: Interleukin-18 (IL18) is a proinflammatory cytokine that modulates both innate and adaptive immune responses. Historically, wild-type recombinant IL-18 has shown limited anti-tumor efficacy in preclinical models and clinical trials, likely due to upregulation of IL-18 binding protein (IL-18BP), a negative regulator of the IL-18 signaling axis. Accordingly, an engineered IL-18 cytokine capable of interacting with the IL-18 receptor, but resistant to IL-18BP interactions (i.e., “Decoy-Resistant IL-18”, DR-18), has demonstrated enhanced therapeutic potential in mouse tumor models, both as a single agent and in combination with anti-PD-1 [1, 2]. Here we evaluated murine DR-18 in combination with additional checkpoint inhibitors beyond anti-PD-1 in a set of mouse tumor models, and tested human DR-18 activity in vitro/ex vivo for activation of human immune cells and anti-tumor activity. Results: Treatment with mDR-18 elicited strong efficacy as a single agent and combination activity with immune checkpoint inhibitors (ICIs) across diverse syngeneic mouse models of solid tumors. As a single agent, mDR-18 demonstrated robust single agent activity in tumor growth inhibition for MC38 (&amp;gt;80%), CT26 (&amp;gt;60%) and B16-F10 (&amp;gt;55%). Combination treatment of mDR-18 with anti-PD-1, anti-CTLA-4, or anti-LAG-3 increased the efficacy for the MC38 (&amp;gt;85-95%), CT26 models (&amp;gt;80-85%), while the B16-F10 (&amp;gt;60%) showed smaller efficacy enhancement with the combinations. In vitro assays revealed human DR-18 (ST-067) activated immune cells and enhanced A549 tumor cell killing over 72hrs. Furthermore, ex vivo studies of 3D patient-derived tumor spheroids demonstrated that ST-067 impaired growth and increased immune cell infiltration, highlighting the potential of hDR-18 to enhance immune activity within the human tumor microenvironment. Conclusion: These studies expand the breadth of IL-18/checkpoint synergism beyond anti-PD-1 and confirm enhanced human immune response in vitro with the clinical candidate ST-067. Taken together, these findings strengthen the rationale for clinical combination of ST-067 with ICI agents in patients with solid tumors. 1.Zhou T, Damsky W, Weizman OE, et al. IL-18BP is a secreted immune checkpoint and barrier to IL-18 immunotherapy. Nature. 2020;583(7817):609-614. doi:10.1038/s41586-020-2422-62.Minnie SA, Waltner OG, Ensbey KS, et al. Depletion of exhausted alloreactive T cells enables targeting of stem-like memory T cells to generate tumor-specific immunity. Sci Immunol. 2022;7(76):eabo3420. doi:10.1126/sciimmunol.abo3420 Citation Format: Jeffrey N. Lindquist, Kai Qin, Aaron M. Ring, Hirdesh Uppal. Decoy-resistant IL-18 enhances checkpoint inhibitor combinations beyond anti-PD-1 in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4073.

Abstract 4071: The first-in-class PD1-IL18 conjugate BPT567 induces potent anti-tumor immunity by preferentially activating PD1+IL18R+ intratumoral effector T cells in cis

Abstract Antibody-cytokine conjugates leverage orthogonal mechanisms of action (MoA) in one molecule to induce potent antitumor immune responses. PD-1-targeting conjugates are of particular interest since they preferentially target antigen-experienced PD-1+ CD8+ T cells enriched in the tumor microenvironment (TME) while simultaneously blocking the PD-(L)1 pathway and inducing potent cytokine receptor stimulation in the same CD8+ T cell in cis (cis-signaling). Interleukin 18 (IL-18) is a proinflammatory cytokine able to integrate both innate and adaptive immunity resulting in profound anti-tumor immune responses mediated by T effector and NK cells. BPT567 has been generated via chemical conjugation of the anti-PD-1 Ab LipustobartTM to an enhanced human IL-18 variant of increased potency and reduced sensitivity to IL-18 binding protein (IL-18BP). BPT567 is designed to specifically target and activate a subset of tumor-infiltrating PD-1+ IL18R+ CD8+ T effector cells recently described to exhibit superior cytotoxic and proliferative activity (Codarri Deak et al., Nature 2022). Due to its ability to signal in cis, BPT567 shows enhanced potency as well as increased resistance to IL-18BP when analyzing IFNγ release in vitro in PD-1+ cells. Subsequent PD-1 receptor occupancy analyses showed that it is fully sufficient for BPT567 to engage a low number of PD-1 receptors to induce maximum IFNγ release in NK92 cells expressing human PD-1. Strikingly, despite addressing overlapping epitopes in PD-1, even a 200-fold excess of pembrolizumab has no impact on the in vitro potency of BPT567. In vivo, BPT567 exhibits strong anti-tumor efficacy at significantly lower IL-18 doses compared to the combination of an untargeted antibody-IL-18 conjugate and an anti-PD1 Ab further corroborating the importance of cis-signaling as a unique MoA of this PD1-IL18 conjugate. Detailed analyses of tumor-infiltrating immune leukocytes (TIL) revealed that BPT567 triggers a preferential expansion of CD8+ T effector memory cells within the TME. In line with this finding, activity in the MC38 tumor model is dependent on the presence of CD8+ T cells while depletion of other immune cell subsets (e.g. NK cells, CD4+ T cells, macrophages) had no impact on efficacy. Although not required for anti-tumor efficacy, CD4+ T cells are essential for proficient formation of an immunological memory. In an effort to confirm findings generated in murine models in relevant human ex vivo systems, we analyzed the release of cytokines and chemokines in dissociated cells isolated from primary human tumor explants. Also in this human model, BPT567 is able to induce significantly stronger IFNγ release compared to either single agents or the combination of untargeted IL-18 and an anti-PD1 Ab, thus confirming the unique MoA of the PD1-IL18 conjugate triggering superior TIL activation in cis. Citation Format: Kea Martin, Thuy Luu, Jean-Philippe Carralot, Lilian Gremlich, Petra Herzig, Caoimhe Herr, Roy Meoded, Philipp Moosmann, Arnaud Goepfert, Alfred Zippelius, Vijaya Pattabiraman, Bertolt Kreft. The first-in-class PD1-IL18 conjugate BPT567 induces potent anti-tumor immunity by preferentially activating PD1+IL18R+ intratumoral effector T cells in cis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4071.

Chemogenetic inhibition of central amygdala CRF-expressing neurons decreases alcohol intake but not trauma-related behaviors in a rat model of post-traumatic stress and alcohol use disorder

Post-traumatic stress disorder (PTSD) and alcohol use disorder (AUD) are often comorbid. Few treatments exist to reduce comorbid PTSD/AUD. Elucidating the mechanisms underlying their comorbidity could reveal new avenues for therapy. Here, we employed a model of comorbid PTSD/AUD, in which rats were subjected to a stressful shock in a familiar context followed by alcohol drinking. We then examined fear overgeneralization and irritability in these rats. Familiar context stress elevated drinking, increased fear overgeneralization, increased alcohol-related aggressive signs, and elevated peripheral stress hormones. We then examined transcripts of stress- and fear-relevant genes in the central amygdala (CeA), a locus that regulates stress-mediated alcohol drinking. Compared with unstressed rats, stressed rats exhibited increases in CeA transcripts for Crh and Fkbp5 and decreases in transcripts for Bdnf and Il18. Levels of Nr3c1 mRNA, which encodes the glucocorticoid receptor, increased in stressed males but decreased in stressed females. Transcripts of Il18 binding protein (Il18bp), Glp-1r, and genes associated with calcitonin gene-related peptide signaling (Calca, Ramp1, Crlr-1, and Iapp) were unaltered. Crh, but not Crhr1, mRNA was increased by stress; thus, we tested whether inhibiting CeA neurons that express corticotropin-releasing factor (CRF) suppress PTSD/AUD-like behaviors. We used Crh-Cre rats that had received a Cre-dependent vector encoding hM4D(Gi), an inhibitory Designer Receptors Exclusively Activated by Designer Drugs. Chemogenetic inhibition of CeA CRF neurons reduced alcohol intake but not fear overgeneralization or irritability-like behaviors. Our findings suggest that CeA CRF modulates PTSD/AUD comorbidity, and inhibiting CRF neural activity is primarily associated with reducing alcohol drinking but not trauma-related behaviors that are associated with PTSD/AUD.

Ratios between the Levels of IL-18, Free IL-18, and IL-1β-Binding Protein Depending on the Severity and Outcome of COVID-19.

In 89 patients with COVID-19, the ratios between IL-18, free IL-18, and IL-18-binding protein (IL-18BP) were analyzed depending on severity and outcome of the disease. At admission to the hospital, the levels of IL-18 and free IL-18 were significantly higher than 3 months after discharge from the hospital, the levels IL-18BP of being almost the same. In patients with more severe lung injury (computed tomography data), the levels of IL-18 and free IL-18 were higher and IL-18BP levels were lower than in patients with mild and moderate COVID-19. Three months after discharge from the hospital, no differences between these parameters were found. In 9 patients who died in the hospital, free IL-18 levels were significantly higher and IL-18BP levels were lower than in survivors. Thus, high levels of bioactive free IL-18 in combination with low levels of IL-18BP can be indicative of severe inflammatory phase of COVID-19 and the risk of worse clinical outcomes.