PER18 Antibody

What is the biological relationship between IL-18 and IL-18BP?

Interleukin-18 (IL-18) functions as an immunoregulatory cytokine that potently induces T helper 1 and cytotoxic responses in immune cells. Its activity is tightly regulated by IL-18 binding protein (IL-18BP), which acts as a natural decoy receptor. IL-18BP forms high-affinity complexes with IL-18, effectively blocking IL-18 from binding to its cognate receptors on cell surfaces. This regulatory mechanism is critical for maintaining immune homeostasis, as unregulated IL-18 activity can drive excessive inflammation. Under normal physiological conditions, circulating IL-18BP is present at high concentrations in both humans and mice, sequestering excess IL-18 and preventing inappropriate immune activation . The binding affinity between IL-18 and IL-18BP is remarkably high, with a dissociation constant (Kd) of approximately 100 pM .

How does a disbalance between IL-18 and IL-18BP affect inflammatory conditions?

A disbalance between IL-18 and IL-18BP levels, particularly when characterized by excessive IL-18 signaling, can lead to systemic inflammation and inflammatory disorders. In experimental models, IL-18BP knockout (KO) mice exhibit exacerbated severity of CpG-induced macrophage activation syndrome (MAS), demonstrating the crucial regulatory role of IL-18BP . In specific clinical conditions such as Still's disease, macrophage activation syndrome, and NLRC4 gain-of-function mutations, extraordinarily elevated levels of IL-18 can overwhelm the binding capacity of IL-18BP, resulting in the presence of free, biologically active IL-18 that drives pathological inflammation . This mechanism highlights why measuring the ratio of free IL-18 to IL-18BP, rather than total IL-18 alone, provides more meaningful insights into the inflammatory status in various diseases.

What are the basic approaches to studying IL-18/IL-18BP interactions?

Several fundamental approaches are employed to study IL-18/IL-18BP interactions in research settings. These include: (1) Sandwich ELISA techniques to detect and quantify IL-18BP, (2) Co-immunoprecipitation assays to analyze IL-18:IL-18BP complex formation, (3) Cell-based bioassays using IL-18 receptor-expressing cell lines to measure functional IL-18 activity, (4) Biolayer interferometry to determine binding affinity between antibodies and IL-18BP, and (5) In vivo models of inflammation that are IL-18-dependent, such as CpG-induced macrophage activation syndrome . These approaches allow researchers to characterize both the biochemical properties of the IL-18:IL-18BP interaction and its functional consequences in biological systems.

How are monoclonal antibodies against murine IL-18BP developed?

The development of monoclonal antibodies against mouse IL-18BP follows a multi-step process as demonstrated in recent research. Initially, rabbits are immunized with mouse IL-18BP protein to elicit an immune response. Following immunization, B lymphocytes are isolated from the blood of these rabbits at various time points. These B cells are then sorted into single cells using fluorescence-activated cell sorting (FACS) and cultured for approximately 7 days. The supernatants from these B-cell cultures are tested using direct ELISA in a 384-microtiterplate format to identify antibodies specifically targeting mouse IL-18BP. Positive samples containing anti-IL-18BP antibodies are selected for further processing. Plasmids encoding unique antibody sequences are created and utilized for the production of recombinant rabbit antibodies. In one study, this approach yielded ten antibody candidates that were subsequently tested using sandwich ELISA and functional assays with RAW 264.7 cells stably expressing mouse IL-18R .

What methods are used to evaluate the functional activity of anti-IL-18BP antibodies?

Functional characterization of anti-IL-18BP antibodies employs several complementary approaches. A primary method involves using stably transfected cell lines expressing IL-18Rα/β (such as modified RAW 264.7 cells) that produce TNFα in response to IL-18 stimulation. When IL-18BP is added to this system, it inhibits IL-18-induced TNFα production in a dose-dependent manner. To evaluate antibody function, researchers add anti-IL-18BP antibodies at different molar ratios relative to IL-18BP and measure changes in TNFα production. Neutralizing antibodies reverse the inhibitory effect of IL-18BP, while non-neutralizing antibodies have no impact. Additionally, co-immunoprecipitation experiments with tagged IL-18 and IL-18BP can determine whether antibodies interfere with IL-18BP:IL-18 complex formation. Some antibodies can co-immunoprecipitate both IL-18BP and IL-18, indicating they don't disrupt the complex, while others precipitate only IL-18BP, suggesting they prevent or disrupt IL-18:IL-18BP interactions .

How can researchers distinguish between neutralizing and non-neutralizing anti-IL-18BP antibodies?

Distinguishing between neutralizing and non-neutralizing anti-IL-18BP antibodies requires functional testing rather than simple binding assays. While both types of antibodies may bind to IL-18BP with similar affinity, they differ fundamentally in their effect on IL-18BP function. A comprehensive approach to this differentiation includes: (1) IL-18 bioassays using IL-18R-expressing cells to determine if antibodies reverse IL-18BP's inhibitory effect on IL-18 signaling, (2) Co-immunoprecipitation studies to assess whether antibodies disrupt preformed IL-18:IL-18BP complexes, (3) Free IL-18 detection assays to determine if antibodies can release IL-18 from IL-18BP in biological samples, and (4) Timing experiments where antibodies are added at different intervals after IL-18:IL-18BP complex formation to evaluate their ability to reverse established inhibition. For example, in studies with antibodies 441 and 445, both bound to IL-18BP with comparable affinity, but only antibody 445 demonstrated neutralizing activity by preventing IL-18BP from inhibiting IL-18 signaling and by releasing IL-18 from preformed complexes .

How do anti-IL-18BP antibodies impact experimental models of systemic inflammation?

Anti-IL-18BP antibodies with neutralizing activity can significantly alter the course of experimental models of systemic inflammation, particularly those dependent on IL-18 signaling. In the CpG-induced macrophage activation syndrome (MAS) model, administration of the neutralizing anti-IL-18BP antibody 445 to wild-type mice significantly aggravated disease manifestations compared to the non-neutralizing antibody 441. Specific exacerbated parameters included: more pronounced weight loss, greater splenomegaly, worsened anemia, elevated spleen mRNA levels of inflammation-related genes (Ifng, Cxcl9, Ciita, and Il18bp), and increased serum levels of inflammatory markers (IFN-γ, CXCL9, and ferritin). Flow cytometry analysis revealed that neutralizing IL-18BP also altered immune cell populations, with higher numbers of splenic macrophages and lower numbers of neutrophils, T cells (both CD4+ and CD8+), and NK cells in the spleen, alongside higher percentages of circulating total dendritic cells and plasmacytoid dendritic cells . These findings mirror the phenotype observed in IL-18BP knockout mice, confirming that neutralizing antibodies effectively create a functional IL-18BP deficiency.

What are the mechanisms by which neutralizing anti-IL-18BP antibodies modify IL-18 activity?

Neutralizing anti-IL-18BP antibodies can modify IL-18 activity through at least two distinct mechanisms. First, they can prevent the initial formation of IL-18:IL-18BP complexes by binding to IL-18BP in a manner that interferes with the IL-18 binding site. Second, and perhaps more significantly, certain neutralizing antibodies like clone 445 can actively disrupt already-formed IL-18:IL-18BP complexes, effectively releasing biologically active IL-18. This second mechanism was demonstrated in experiments where the neutralizing antibody 445 was added at different time points (0h, 0.5h, or 2h) after IL-18 and IL-18BP were combined. Even when added 2 hours after complex formation, antibody 445 reversed the inhibitory activity of IL-18BP, whereas the non-neutralizing antibody 441 had no effect regardless of timing . This ability to release IL-18 from established complexes was further confirmed by showing that antibody 445 enabled the detection of free IL-18 in wild-type mouse serum samples spiked with recombinant IL-18, whereas these samples typically show undetectable free IL-18 due to sequestration by endogenous IL-18BP .

How can researchers verify the specificity of anti-IL-18BP antibody effects in experimental models?

Verifying the specificity of anti-IL-18BP antibodies requires multiple control experiments to rule out off-target effects. A comprehensive approach includes: (1) Administering the antibodies to naïve (non-disease) wild-type mice to confirm they don't induce inflammatory changes in the absence of an inflammatory stimulus, (2) Comparing the effects of non-neutralizing antibodies to vehicle controls (e.g., PBS) to ensure they don't exert unexpected activities, (3) Testing the antibodies in gene knockout models lacking either IL-18 or IL-18BP to confirm that observed effects depend on these specific molecules, and (4) Performing detailed immunophenotyping to identify any unexpected cellular effects. In published research, when neutralizing (445) and non-neutralizing (441) antibodies were administered to naïve wild-type mice, IL-18 knockout mice, or IL-18BP knockout mice, no differences in inflammatory parameters were observed between treatment groups, confirming specificity. Additionally, the non-neutralizing antibody 441 produced results indistinguishable from PBS treatment in wild-type mice with CpG-induced MAS .

What cell-based assays are optimal for evaluating IL-18 and IL-18BP functional interactions?

The optimal cell-based assay for evaluating IL-18 and IL-18BP functional interactions utilizes cells stably expressing both chains of the IL-18 receptor (IL-18Rα/β). In published research, RAW 264.7 macrophage cells have been successfully transfected to constitutively express these receptors, creating a reliable and sensitive system for detecting IL-18 activity. These transfected cells produce TNFα in response to IL-18 stimulation, providing a quantifiable readout. The specificity of the assay is confirmed by showing that control cells transfected with an empty vector do not respond to IL-18 but maintain responsiveness to other stimuli like LPS. In the assay, cells are typically stimulated with a fixed concentration of IL-18 (e.g., 5 ng/mL) and varying concentrations of IL-18BP to establish dose-dependent inhibition curves. When testing anti-IL-18BP antibodies, a fixed concentration of IL-18BP known to inhibit most of IL-18's activity is used, and antibodies are added at different molar ratios. The produced TNFα is measured as the functional readout, with neutralizing antibodies increasing TNFα production by preventing IL-18BP's inhibitory effect .

What technical considerations are important when developing sandwich ELISAs for IL-18BP detection?

Developing effective sandwich ELISAs for IL-18BP detection requires several key technical considerations. First, selection of appropriate capture antibodies is crucial—they must efficiently bind IL-18BP without interfering with critical epitopes needed for detection. In the reported research, multiple monoclonal antibodies were tested as capture antibodies, with all except one (clone 443) successfully capturing mouse IL-18BPd . Second, careful validation is necessary to ensure specificity for the target protein and minimal cross-reactivity. Notably, one antibody (clone 447) recognized both mouse IL-18BPd and human IL-18BPa, indicating potential cross-species reactivity that could be advantageous or problematic depending on the research context . Third, complementary techniques such as pulldown experiments should be performed to confirm ELISA results, as was done in the reported research where antibodies that captured IL-18BP in ELISA also showed binding in pulldown assays. Fourth, assay sensitivity and dynamic range must be optimized through careful titration of antibody concentrations and detection systems. Finally, proper controls including recombinant proteins and samples from gene knockout animals should be incorporated to confirm assay specificity.

How should researchers approach biolayer interferometry experiments to determine antibody-IL-18BP binding kinetics?

Biolayer interferometry (BLI) provides valuable data on antibody-antigen binding kinetics and should be approached methodically to obtain reliable results. When determining antibody-IL-18BP binding kinetics, researchers should first immobilize either the antibody or IL-18BP (typically the antibody) onto appropriate biosensor tips. The immobilization should be optimized to achieve sufficient loading without overcrowding that could affect binding measurements. After establishing stable baselines, association kinetics are measured by exposing the immobilized molecule to varying concentrations of its binding partner in solution. Dissociation kinetics are then measured by transferring the biosensor to buffer without the binding partner. Multiple concentrations of the analyte should be tested to ensure the reliability of calculated parameters. Data analysis should fit the curves to appropriate binding models (typically 1:1 binding) to derive association rate constants (ka), dissociation rate constants (kd), and equilibrium dissociation constants (KD). In the reported research, BLI showed that both antibodies 441 and 445 had comparable IL-18BP binding affinity in the low nanomolar range despite their different functional effects , highlighting the importance of complementing binding studies with functional assays.

How can discrepancies between antibody binding affinity and functional effects be explained?

Discrepancies between antibody binding affinity and functional effects are common in immunological research and can be explained by several mechanisms. In the case of anti-IL-18BP antibodies, studies revealed that antibodies 441 and 445 bound to IL-18BP with similar affinity (comparable KD values in the low nanomolar range) yet had dramatically different functional effects . This apparent contradiction occurs because binding affinity measures only the strength of interaction between an antibody and its target, not the functional consequences of that binding. The key explanation lies in epitope specificity—antibodies that bind to different regions (epitopes) on IL-18BP can have vastly different effects on its function. Antibodies that bind to or near the IL-18 interaction site on IL-18BP (like antibody 445) can interfere with IL-18BP's ability to capture IL-18, while antibodies binding to non-critical regions (like antibody 441) allow IL-18BP to maintain its inhibitory function. Additionally, some antibodies may induce conformational changes in IL-18BP that alter its ability to bind IL-18, even if they don't directly compete for the same binding site. These findings emphasize the importance of complementing binding studies with functional characterization when developing and selecting antibodies for research or therapeutic applications.

What are the implications of IL-18BP neutralization for therapeutic targeting of the IL-18 pathway?

The implications of IL-18BP neutralization for therapeutic targeting of the IL-18 pathway are multifaceted and context-dependent. On one hand, neutralizing IL-18BP can significantly exacerbate inflammatory conditions dependent on IL-18 signaling, as demonstrated by the aggravation of CpG-induced macrophage activation syndrome when neutralizing antibody 445 was administered . This provides a cautionary note regarding potential adverse effects of therapies that might inadvertently inhibit IL-18BP function. On the other hand, controlled neutralization of IL-18BP could have beneficial applications in contexts where enhanced IL-18 activity is desirable, such as in anti-tumor immunity and certain infectious disease settings. The research notes that "targeting IL-18BP can have promising effects to enhance immune responses against pathogens and cancer" . This therapeutic potential exists because IL-18 promotes Th1 and cytotoxic responses that are beneficial for eliminating intracellular pathogens and cancer cells. Development of tools like the characterized neutralizing antibody 445 provides valuable research reagents for exploring these potential therapeutic applications. Additionally, the described methodology for developing and characterizing neutralizing versus non-neutralizing antibodies provides a valuable framework for future development of more selective modulators of the IL-18 pathway.

What are the key experimental findings regarding antibody-mediated neutralization of IL-18BP?

The key experimental findings regarding antibody-mediated neutralization of IL-18BP provide significant insights into both methodology and biological consequences. First, among ten developed monoclonal antibodies (clones 441-450), nine successfully bound mouse IL-18BP, but their functional effects varied dramatically. While antibody 441 did not interfere with IL-18BP's inhibitory function, others like 445, 447, and 449 showed potent neutralizing activity even at low molar ratios (1:1 antibody:IL-18BP) . Second, neutralizing antibodies could not only prevent IL-18BP from binding to IL-18 but could also disrupt preformed IL-18:IL-18BP complexes, releasing biologically active IL-18. This was demonstrated by antibody 445's ability to reverse IL-18BP inhibition even when added two hours after IL-18BP and IL-18 were combined . Third, the binding affinity to IL-18BP did not predict functional activity, as both neutralizing (445) and non-neutralizing (441) antibodies bound IL-18BP with similar affinity in the low nanomolar range . Fourth, in vivo studies showed that neutralizing antibody 445 significantly aggravated CpG-induced macrophage activation syndrome in wild-type mice compared to non-neutralizing antibody 441, mirroring the phenotype seen in IL-18BP knockout mice . Finally, multiple control experiments in naïve mice and various knockout models confirmed the specificity of these effects.

How do different anti-IL-18BP antibody clones compare in terms of functional characteristics?

The functional characteristics of different anti-IL-18BP antibody clones showed remarkable variation despite similar binding to IL-18BP. Among the ten monoclonal antibodies tested (clones 441-450), distinct functional patterns emerged:

*IC50 values could not be extrapolated due to high inhibitory activity at the concentrations used
**Also recognizes human IL-18BPa

Based on these characteristics, antibody 445 was selected as the prototype neutralizing antibody for detailed studies, while 441 served as the non-neutralizing control . This comparison highlights the importance of comprehensive functional characterization beyond simple binding assays when developing antibodies for research applications.

What changes in immune cell populations occur following neutralization of IL-18BP in inflammatory models?

Neutralization of IL-18BP in inflammatory models induces significant alterations in immune cell populations across multiple compartments. In the CpG-induced macrophage activation syndrome model, administration of the neutralizing antibody 445 to wild-type mice resulted in distinct changes compared to mice receiving the non-neutralizing antibody 441. In the spleen, neutralizing IL-18BP led to increased numbers of macrophages, reflecting enhanced myeloid cell responses. Concurrently, there were decreased numbers of neutrophils, total T cells (both CD4+ and CD8+ subsets), and natural killer (NK) cells . Analysis of circulating immune cells revealed a different pattern with higher percentages of total dendritic cells (DCs) and plasmacytoid DCs (pDCs), alongside lower percentages of Ly6C+ monocytes and total and CD4+ T cells . These alterations in immune cell distribution likely contribute to the exacerbated inflammatory phenotype observed when IL-18BP is neutralized, particularly the enhanced production of IFN-γ and IFN-γ-induced proteins like CXCL9. Importantly, these changes were specific to IL-18BP neutralization, as they were not observed when comparing the effects of antibodies 441 and 445 in IL-18 knockout or IL-18BP knockout mice , confirming the IL-18:IL-18BP axis as the primary driver of these immunological shifts.

What unresolved questions remain about IL-18BP neutralization in different disease contexts?

Several unresolved questions remain regarding IL-18BP neutralization across different disease contexts. First, while neutralizing IL-18BP exacerbates experimental macrophage activation syndrome , its effects in other inflammatory models require investigation—particularly in conditions where IL-18 plays different or context-dependent roles. Second, the temporal aspects of IL-18BP neutralization need exploration—determining whether short-term versus sustained neutralization produces different immunological outcomes could inform potential therapeutic applications. Third, the precise molecular mechanisms by which neutralizing antibodies like clone 445 disrupt preformed IL-18:IL-18BP complexes remain unclear and could offer insights into protein-protein interaction dynamics. Fourth, the potential utility of IL-18BP neutralization for enhancing anti-tumor immunity or responses to certain infections, as suggested in the literature , requires systematic investigation in appropriate models. Fifth, species differences in IL-18:IL-18BP biology might affect translational relevance, necessitating comparative studies between murine and human systems. Finally, the potential for IL-18BP neutralization to synergize with or antagonize other immunomodulatory approaches remains largely unexplored but could reveal important combination strategies for research or therapeutic purposes.