Patent ID: 12216128

DETAILED DESCRIPTION

The typical standard of care cascade for a child who has experienced a febrile seizure followed by additional seizures or status epilepticus generally involves first administering benzodiazepines and then, if no response, occurs, either levetiracetam or fosphenytoin. Failure to respond to either of these drugs typically is followed by administration of valproic acid and intravenous (IV) phenobarbital. Failure to respond to these drugs generally escalates to midazolam infusion with increasing dosage, and finally, failure to respond to midazolam results in induction of a barbiturate coma. This entire protocol usually occurs within the first hour after presentation, and represents the standard of care for febrile status epilepticus that has remained essentially unchanged for decades. A child who fails the initial steps of this protocol, in the absence of any other clear etiology (e.g., stroke or trauma) typically undergoes a lumbar puncture for collection of CSF. Blood samples collected upon presentation and again at the time of the lumbar puncture can be submitted for determination of an infectious cause immediately prior to initiation of prophylactic antibiotics. The samples also may be submitted at this point for clinical testing using an autoimmune epilepsy battery.

Anakinra (also known as Kinaret) is recombinantly-produced human IL-1RA, and is a drug that can be used to treat seizure disorders in some patients. For example, some children with FIRES respond to anakinra, as described elsewhere (Kenney-Jung et al.,Ann. Neurol.80(6):939-945, 2016). However, many children presenting with FIRES do not respond to anakinra. Likewise, many children who present with seizure disorders that do not fulfill the criteria for FIRES (a much larger population of NICU and PICU cases) may respond to drugs like anakinra, but to date there is no clear path to identifying patients who are likely to respond.

This document provides materials and methods for identifying subjects (e.g., humans or non-human mammals) who are likely to respond to anakinra treatment or other 1L-1RA replacement or supplementation therapies, or other treatments that can attenuate IL-1R inflammatory signaling (e.g., compounds that modulate or antagonize the IL-1β pathway), as well as materials and methods for identifying subjects who are not likely to respond to treatment with such therapies. In some embodiments of the methods provided herein, testing for IL-1RA function and/or IL1RN expression (e.g., to determine the IL-1RA isoform expression pattern) may occur when a CSF sample is tested using an autoimmune epilepsy battery as described above.

The methods provided herein can include assaying a biological sample from a subject to determine the level of IL-1RA protein expression, the level of IL1RN mRNA expression, or the level of IL-1RA activity. Useful biological samples can include, without limitation, serum, serum microvesicles, cerebrospinal fluid (CSF), and cells such as neutrophils, monocytes, peripheral blood mononuclear cells (PBMC), or fibroblasts. Subjects who can be evaluated and treated according to the methods described herein can be humans or non-human mammals (e.g., laboratory animals such as mice and rats; in some cases, such rodents can serve as models for seizure disorders).

In some embodiments, the level of IL-1RA function can be used to guide decisions regarding the use of anakinra or other therapies that can increase IL-1RA function in acutely ill pediatric patients suffering from seizure disorders such as FIRES, DIRA, PASS, or MRE. Other therapies that may be useful in the methods provided herein include, without limitation, one or more of the following: anakinra (available from Swedish Orphan Biovitrum), EBI-005 (chimeric IL-1RA; Eleven Biotherapeutics), MEDI-8968 (IL1R1 blocker; Medlmmune), rilonacept (solIL1R; Regeneron), canakinumab (anti-IL-1β; Novartis), gevokizumab (anti-IL-1β; Novartis), LY2189102 (anti-IL-1β; Eli Lilly), P2D7KK (anti-IL-1β), MABp1 (anti-IL1α;)(Biotech), GSK1070806 (anti-IL-18; GlaxoSmithKline), sIL1RII (IL-1β sink), HuMAX-IL8 (anti-IL-8; Genmab), HuMab-10F8 (anti-IL-8; Cormorant Pharmaceuticals), CMPX-1023 (IL-1 alphabody; Copmlix NV), VX765 (inflammasome inhibitor; Vertex Pharmaceuticals), VX740 (inflammasome inhibitor; Vertex Pharmaceuticals), MCC950 (inflammasome inhibitor; Pfizer), beta-hydroxybutyrate (inflammasome inhibitor; Accera), glibenclamide (P2X7 inhibitor; Sanofi-Aventis), AZD9056 (P2X7 inhibitor; AstraZeneca), CE-2245354 (P2X7 inhibitor), GSK1482169 (P2X7 inhibitor; GlaxoSmithKline), pralnacasan (caspase-1 inhibitor; Vertex Pharmaceuticals), DF2156A (CXCR2 inhibitor; Dompe Farmaceutici S.p.A), and sc-rAAV2.5IL-1RA (IL-1RA gene therapy).

Various techniques can be used to assess the level of IL-1RA expression and activity in a subject. For example, the methods provided herein can include measuring the functional activity of IL-1RA, IL-1β, and/or IL-18 (a member of the IL1 family) in serum or CSF using immunological methods (e.g., ELISA) or cytokine bead array assays. In some cases, ex vivo cell-based assays can be used. The IL-1 family cytokines are among the most potently proinflammatory innate immune proteins, and their signaling therefore is tightly regulated by a variety of factors. IL-18 and IL-1β are produced as preforms that are activated only when cleaved by the inflammasome. IL-1β and IL-la bind to the surface decoy receptor (IL-1RII) with higher affinity than the active receptor (IL-1RI). Conversely, IL-1RA binds with higher affinity to IL-1RI, without causing signal transduction, and it competitively blocks binding of IL-1β and IL-la. In addition, soluble decoy forms of IL-1RI and IL-1RII are secreted, and can bind and sequester IL-la and IL-β to prevent signaling. A critical failure in any one of these regulatory components could lead to elevated IL-1R signaling, increasing the chance of a false negative result by single parameter diagnostic measures. Therefore, a cell-based assay can be a useful functional measure of overall IL-1R signaling. For example, as described in the Examples herein, defective IL-1RA activity can be detected in a cell-based reporter assay that tests the ability to block a concentration curve of IL-1β (essentially establishing an IC50for the patient's IL-1RA molecule). Since some subjects who do not respond to anakinra have normal IL-1RA function, this assay may be a useful diagnostic tool for guiding therapy decisions, by rapidly identifying a functional DIRA-like condition.

In some embodiments, methods that include measuring the stimulated release of inflammatory cytokines from neutrophils, monocytes, or PBMCs acutely isolated from subjects with seizure disorders, even after in vitro stimulation periods as short as about 3 hours, can be used to provide insight into inflammatory status and guide therapy decisions. Because the IL-1 family of cytokines is extremely labile in body fluids, detection in patient-derived biospecimens such as serum, plasma, or CSF using traditional techniques such as ELISA or other antibody-based assay methods can be challenging. Determining that a subject has elevated release of IL-1β in neutrophils, monocytes, or PBMCs stimulated with LPS or TNFα, for example, may support the introduction of IL-1RA boosting or IL-1β-targeting therapies.

Neutrophils and monocytes are principal sources of IL-1β. Seizures can be triggered, maintained, or propagated by neutrophil-derived IL-1β in response to diverse stimuli such as peripheral infection, or due to genetic or epigenetic predisposition to neutrophil hyper-responsiveness. In some assay methods, therefore, neutrophils, monocytes, or PBMCs can be acutely isolated from a patient blood sample and primed by exposure to LPS (or another toll-like receptor agonist, or a biological or chemical compound that can drive neutrophil activation) prior to stimulation with a dose range of ATP or another stimulant (e.g., an ATP derivative, a peptide agonists such as N-formylmethionyl-leucyl-phenylalanine (fMLP), or another biological or chemical compound that can drive neutrophil effector function). Under such conditions, neutrophils or monocytes can release IL-1β in an amount that can be readily measured in culture supernatants using methods such as ELISA or cytokine bead array assays. The dose-response curve, normalized to number of neutrophils or monocytes, can be used to calculate a half-maximal effective concentration (EC50) for ATP stimulated IL-1β release from the patient's cells. By comparison to the EC50from corresponding healthy controls, a patient can be categorized as “normal” (e.g., when the patient's EC50is within one standard deviation of the healthy control EC50, less than a z-score or effect size of 1.0, indicating an IL-1β response similar to 84% of the healthy population), “abnormal” (e.g., when the patient's EC50is within 1 to 2 standard deviations above the healthy control EC50, less than a z-score or effect size of 2.0, indicating an IL-1β response similar to 98% of the healthy population), or “hyper-responsive” (e.g., when the patient's EC50is greater than 2 standard deviations above the healthy control EC50, greater than a z-score or effect size of 2.0, indicating an IL-1β response that exceeds that of 98% of the healthy population). Characterization of a patient as “hyper-responsive” can justify administration of drugs such as anakinra to reduce or ameliorate seizures, while characterization as “normal” or “abnormal” can indicate that the patient is not a good candidate for IL-1RA or IL-1β based therapy, and that a different approach should be used.

It is noted that, in addition to IL-1β, inflammatory responses that may be indicative of inflammamodulatory therapy can include other ligands of the IL-1 receptor, such as IL-la and IL-1RA, as well as members of the IL-1 superfamily such as IL-18, IL-33, IL-36, IL-37, and IL-38. Thus, these markers also can be measured to assess whether a subject can be identified as likely to respond to immunomodulatory therapy such as anakinra. Further, alternative effect size ranges may be employed as evidence of anakinra efficacy builds; likewise, alternative effect size ranges may be warranted for different age groups, such as neonates, infants, children, adolescents, young adults, adults, and the elderly. For patients with periodic seizure syndromes, assessment of neutrophil hyper-responsiveness can be used during remission phase to predict impending relapse. For example, the IL-1β release EC50can be measured regularly over an interval of time, and a shift in the EC50indicating increased neutrophil responsiveness can be used to initiate therapy aimed at preventing relapse. Alternative readouts of neutrophil hyper-responsiveness can include flow cytometric assessment of surface markers that indicate activation status, such as increased CD66b, increased CD88, or decreased CD62L. The degree of activation can be measured by establishing a mean fluorescence intensity (MFI) for these surface markers on neutrophils or monocytes from healthy subjects and comparing the MFI from seizure patients.

In some cases, neutrophils, monocytes, or PBMCs acutely isolated from a subject can be primed and stimulated, and treated with a candidate drug to evaluate the cellular response to the drug. For example, neutrophils from a FIRES patient can be primed (e.g., with LPS), treated with anakinra or another drug (e.g., another inflammamodulatory drug), stimulated (e.g., with ATP), and then evaluated for release of IL-1β or IL-1R ligand or IL-1 superfamily member. By comparing the release with and without the test drug, the likelihood of the patient's response to the drug can be determined.

In some cases, an IL-1RA functional test can be employed to generate an antagonism index for the subjects endogenous IL-1RA, both in serum and CSF (although serum alone may be adequate if necessary, based on availability of material). The antagonism index can be compared to a control range of antagonistic function measured in healthy subjects, such that the healthy control range can be used to determine a threshold for a “normal” antagonism index. Values below the threshold can result in identification of the subject as likely to respond to recombinant IL-1RA (anakinra) as a therapeutic intervention. For example, a child with 50% of the functional antagonism of “normal” IL-1RA may not be likely to benefit substantially from anakinra, but a child with 10% or 1% of the functional antagonism may show a profound benefit from supplementation with the recombinant antagonist. Thus, the in vitro antagonism index can provide a diagnostic marker for use of a drug such as anakinra or adjunct therapies. The use of such diagnostic strategies can accelerate the time to use of anakinra or another IL-1RA based therapy, thereby reducing the brain injury that can accrue with time in status epilepticus and/or drug-induced coma. The diagnostic methods provided herein also can aide in guiding therapy decisions away from the IL-1β pathway when the antagonism index is normal, accelerating the use of alternative agents targeting other inflammatory pathways.

In some embodiments, methods for determining IL-1RA activity based on an antagonism index can include measuring binding displacement of fluorescently conjugated IL-1β to derivatized beads (also fluorescent, but in another channel) having IL-1R covalently conjugated to their surface. In some cases, for example, binding displacement assays can include immunoprecipitating endogenous IL-1β in a patient's sample on beads conjugated with anti-IL-1β antibodies. The IL-1β-depleted sample can be incubated with IL-1R beads in the presence of labeled IL-1β at EC90(enough IL-1β to saturate 90% of the binding sites). A displacement index then can be calculated for the patient and compared to healthy control ranges. Such methods also can be translated into a chip-based system using absorbance or fluorescence and IL-1R bound to the detector surface, again measuring displacement of labeled IL-1β.

Somewhat similar to the above methods, another strategy for assessing IL-1RA function can include measuring displacement of IL-1β binding from a molecularly imprinted polymer (MIP). Such methods can utilize, for example, an electrically conductive MIP that binds IL-1β (mimicking IL-1R binding), where the MIP-IL-1β complex serves a substrate for screening patient IL-1RA displacement. In some cases, the MIP-based methods can include a fluorescent assay instead of an electrical assay, or can be bead-based via flow cytometry.

In some embodiments of the methods provided herein, portions of IL-1 family genes in a subject can be sequenced (e.g., using rapid RNA-seq based targeted exon sequencing or another suitable method) to determine whether the subject has IL-1 polymorphisms that may be associated with functional impairment or altered expression of key isoforms. As described in the Examples, for example, a pediatric patient with deficient IL-1RA function showed elevated levels of IL-1RA protein in CSF, indicating that absolute expression of IL-1RA is insufficient to determine a deficiency in function. Sequencing analysis in this patient revealed markedly reduced expression of the principal intracellular isoforms of IL-1RA (isoforms 2 and 3, which are the acute response isoforms of the protein), which were verified by protein analysis of lysates from the patient's PBMCs. Thus, sequence-based methods also can be used in the methods provided herein for identifying subjects as being likely or not likely to respond to IL-1RA related therapies. In some cases, sequencing methods can be used to establish a pattern of mutations within intronic regions that is consistent with possible splice site acceptor-donor defects. Alternatively or at the same time, RNAseq or ChIPseq can be used to characterize isoform expression patterns and/or splicing defects. In the index case described in the Examples herein, a chain of possible SNPs has been identified in the intron between exons 3 and 4, within a possible splice controller region near the start of exon 4. Several of these SNPs have not been previously characterized or published.

In some cases, surface plasmon resonance (SPR) can be used to measure binding affinity of patient-specific IL-1RA, since some functional deficiencies can result from reduced binding affinity or receptor on-off times. SPR can provide a reasonably high-throughput, highly quantitative method for assaying IL-1RA antagonism to detect binding on IL-1R either adhered to a detector surface or expressed on membranes that have been disrupted into sheets and adhered to the detector surface. This method may be used with a chip-based sensor or diagnostic SPR device, for example. As with the HEK-based functional assay, a binding index (e.g., affinity) can be calculated and compared to healthy control ranges.

In some embodiments, an assay for determining IL-1RA function can include using a commercially available sensing technology such as the Octet series offered by ForteBio. This can involve using a bio-layer interferometry that is similar in concept to SPR and can have similar sensitivity but a much higher throughput.

As described herein, this document provides methods that can be used to identify subjects with seizure disorders as being likely to respond to treatment with a therapy that can reduce IL-1R inflammatory signaling (e.g., anakinra). In some cases, the methods can include determining that a subject with a seizure disorder has decreased IL-1RA function as compared to a control level of IL-1RA function (e.g., the level of IL-1RA function in one or more normal subjects who do not have a seizure disorder). It is to be understood that a control level of IL-1RA function, as used herein, typically is determined using the same method used to determine the level of IL-1RA function in the subject having the seizure disorder. This document discloses multiple methods that can be used to assess IL-1RA function; any of these can be used to identify a subject as being likely to respond to treatment with anakinra or another therapeutic that reduces IL-1R inflammatory signaling. As used herein, a “decrease” in in IL-1RA function refers is a level of IL-1RA function that is lower (e.g., at least 5% lower, at least 10% lower, at least 25% lower, at least 50% lower, at least 75% lower, at least 90% lower, or at least 95% lower) than a corresponding control level of IL-1RA function. In some cases, a decrease in IL-1RA function can be a decrease in functional IL-1RA antagonism below a predetermined threshold level of functional IL-1RA antagonism. The threshold can be, for example, a level of functional IL-1RA antagonism that is 50% or less (e.g., 25% or less, 10% or less, or 5% or less) than a corresponding control level of functional IL-1RA antagonism.

In some cases, however, as described herein, a reduction in IL-1RA function can be indicated by an increased inflammatory response in a subject. The inflammatory response can be increased by at least 5% (e.g., at least 10%, at least 25%, or at least 50%) as compared to a corresponding control inflammatory response observed for subjects who do not have the seizure disorder. In some embodiments, in increased inflammatory response can be at least two (e.g., at least 2.5, at least 3, or at least 4) standard deviations higher than the corresponding control inflammatory response. Further, in some cases, the methods provided herein can include measuring the relative amounts of IL-1RA isoforms in a sample from a subject with a seizure disorder, and determining that the subject has a ratio of soluble IL-1RA (including isoforms 1 and 4/5) to intracellular IL-1RA (including isoforms 2 and 3) that is increased relative to a control ratio of soluble IL-1RA:intracellular IL-1RA. The ratio can be increased by at least 5% (e.g., at least 10%, at least 25%, or at least 50%) as compared to a corresponding control ratio of soluble IL-1RA:intracellular IL-1RA.

Once a subject with a seizure disorder is identified as being likely to respond to treatment that attenuates IL-1R inflammatory signaling, using the methods disclosed herein, the subject can be treated with, for example, anakinra or another IL-1RA replacement therapy, an IL-1RA supplementation therapy, or another therapy that reduces inflammatory signaling via IL-1R.

The methods described herein also can be used to identify a subject (e.g., a human or a non-human mammal) as being at risk for experiencing a seizure disorder. For example, IL-1RA function can be assessed in the subject using a method as described herein, and if the level of IL-1RA function is decreased as compared to a corresponding control level of IL-1RA function, the subject can be identified as having an increased likelihood of experiencing a seizure disorder.

In addition, the methods provided herein can be used as part of a diagnostic protocol for identifying subjects who may respond to IL-1RA-based therapies or other therapies for treatment of seizure disorders. As depicted inFIG.9, for example, IL-1RA function can be assessed using one or more of the methods described herein and, based on the assessment, a treatment strategy can be indicated.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1—Materials and Methods

Patient samples: Samples from patients with normal pressure hydrocephalus (NPH), focal epilepsy, Landau-Kleffner Syndrome/electrical status epilepticus in sleep (ESES), or FIRES were obtained from the Mayo Clinic Neuroimmunology Laboratory. Healthy control serum or PBMCs were obtained directly from consenting volunteers.

IL-1RA enzyme-linked immunosorbent assay: The human IL-1RA/IL1F3 Quantikine ELISA kit (R&D Systems) was used according to the manufacturer's instructions. Serum and CSF samples were diluted (1:2-1:5000 in assay diluent) and duplicate samples were measured against a standard curve (31.3-2000 pg/mL in duplicate). After chromogen development, absorbance was measured on a Spectramax M3 multi-mode microplate reader (Molecular Devices). Absorbances falling outside the range of the standard curve were reanalyzed at different dilutions.

Cytometric bead assay: The BD CBA human inflammatory cytokine Kit, BD CBA human chemokine kit, and BD CBA human enhanced sensitivity IL-1β flex set (BD Biosciences) were used according to the manufacturer's instructions. Clarified cell supernatants were diluted 1:2-1:10 in assay diluent and were measured against a standard curve. Briefly, supernatants were incubated in the dark with capture beads and detection reagent for 2-3 hours at room temperature. Beads were washed and then acquired on a BD Accuri C6 (BD Biosciences) flow cytometer equipped with a 488 nm laser (filter set: 533/30, 585/40, 670LP) and a 640 nm laser (filter set: 675/25, 780/60). Data were analyzed in FCAP Array Software (BD Biosciences).

HEK-Blue IL1R cells: Human embryonic kidney (HEK) cells expressing murine and human IL-1 receptor proteins, as well as expressing secreted embryonic alkaline phosphatase under NF-kB/AP1 transcriptional control (HEK-Blue IL1R, InvivoGen), were maintained in DMEM with 10% FBS, 2 mM glutamine, 50 U/mL penicillin, 50 μg/ml streptomycin, 100 μg/ml Normocin, 200 μg/mL hygromicin B, 1 μg/mL puromycin, and 100 μg/ml Zeocin. Heat inactivated human serum tested negative for background alkaline phosphatase activity. In initial experiments, HEK-Blue IL1R cells were treated for 24 hours with 0-100 ng/mL recombinant human IL-1β (Peprotech) in the presence of 0-200 ng/mL anakinra. In subsequent experiments, cells were treated with 32 pg/mL IL-1β for all conditions. After 3 hours, saturating concentrations (10 μg/mL) of anakinra were added to the cells to block further IL-1R signaling, and endpoint supernatants were collected at 24 hours. Supernatants (20-40 μL) were mixed with prewarmed QUANTI-Blue reagent (160 μL, InvivoGen) and incubated at 37° C. in a Spectramax M3 multi-mode microplate reader (Molecular Devices). Absorbance at 655 nm was recorded every 5 minutes for 3 hours via kinetic read. The basal absorbance of untreated HEK-Blue IL-1R cell supernatant was subtracted from all samples to normalize for non-specific signal.

Real-time polymerase chain reaction: All lysate samples were stored at −80° C. in 1% β-mercaptoethanol in Buffer RLT Plus (Qiagen) prior to disruption and homogenization with QIAShredder columns (Qiagen), and RNA isolation using the RNeasy Plus Micro Kit (Qiagen). RNA concentration was estimated with a NanoDrop spectrophotometer (ThermoFisher). The Transcriptor First Strand cDNA Synthesis kit (Roche) was used to synthesize cDNA from RNA samples using oligo-dT primers to target mRNA. Equal amounts of sample template RNA were used for each cDNA reaction. The reactions were placed in a thermal block cycler and incubated at 55° C. for 45 minutes, and then inactivated by heating at 85° C. for 5 minutes. Samples were diluted with PCR grade water (1:10-1:50) and stored at −20° C. RT-PCR was performed according to the protocol outlined with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and each sample was run in triplicate. Briefly, 20 μL reactions were prepared by adding 10 μL of SsoAdvanced universal SYBR Green 2× master, 2 μL each of 5 forward and 5 μM reverse primers (total 4 μL), 1 μL nuclease-free water, and 5 μl sample template containing 2-10 ng cDNA. Samples were run on a Bio-Rad CFX Connect system for 50 cycles with the following protocol: 15 seconds at 95° C., 45 seconds at 55° C., and 5 seconds at 65° C. Primers (TABLE 1) were selected using Primer BLAST (NCBI) to have melt temperatures around 60° C. Melt curve analysis was used to determine specificity of each reaction. Data were exported and analyzed in excel using the Pfaffl method to determine relative quantitation based on an estimated amplification efficiency of 95%. Expression across all samples was normalized to the GAPDH housekeeping gene.

IL-1RN Sanger sequencing: IL-1RN gene segment amplicons were generated from FIRES patient DNA by PCR using Phusion High-Fidelity DNA polymerase (New England Biosciences) and primers targeting ˜4000 bp segments of IL-1RN (TABLE 2). Thermocycling conditions followed manufacturer recommendations, with a 2 minute elongation step for each of 35 cycles. Amplicons were extracted from agarose gels following electrophoresis using the Qiaquick Gel Extraction Kit (Qiagen). Targeted long read Sanger sequencing was performed on extracted amplicons by the Mayo Clinic Gene Expression Core facility and by Genewiz using amplicon specific sequencing primers (TABLE 2). Trace data were analyzed and aligned with Mutation Surveyor software and confirmed variants were compared to NG 021240.1 RefSeqGen cited variants (available at www.ncbi.nlm.nih.gov/gene/3557).

Statistical analyses: α=0.05 and β=0.2 were established a priori. Post hoc power analysis was performed for all experiments and significance was only considered when power was ≥0.8. Normality was determined by the Shapiro-Wilk test or the Kolmogorov-Smirnov test. For multiple comparisons, one-way analysis of variance (ANOVA) or non-parametric (Kruskal-Wallis) tests were performed where appropriate. Reported P values were corrected for multiple comparisons (Holm-Sidak correction for ANOVA; Dunn's correction for Kruskal-Wallis). Unpaired two-tailed Student's t-tests were used for comparisons made between two groups. Curran-Everett guidelines were followed.

Example 2—Elevated IL-1RA in Serum and IL-1β and IL-1RA in CSF of FIRES Patients

To determine whether there was a diminished level of endogenous IL-1RA in FIRES patient serum or CSF, IL-1RA levels were measured by enzyme-linked immunosorbent assay (ELISA) in FIRES patient serum and CSF before and after initiation of anakinra treatment, as well as in serum from healthy controls, CSF from normal pressure hydrocephalus patients, and serum and CSF from patients with other seizure disorders. Surprisingly, it was observed that prior to anakinra therapy, IL-1RA (FIG.1A) and IL-1β (FIG.1B) were marginally elevated in FIRES serum as compared to healthy controls. IL-1RA was strongly elevated in FIRES CSF compared to normal pressure hydrocephalus controls and patients with ESES or focal epilepsy (FIG.1C). CSF IL-1β levels, which were only rarely detectable in healthy controls, were also elevated in among FIRES patients (FIG.1D). Serum levels of IL-1RA did not differ between FIRES patients and other chronic seizure patients (FIG.1A). In several cases, IL-1RA levels in CSF exceeded serum levels in FIRES, suggesting predominant CNS production. This finding also was consistent with reports of elevated IL-1RA following seizure activity (Lehtimaki et al.,Neuroimmunomodulation17(1):19-22, 2010). Following anakinra treatment, both serum and CSF levels of IL-1RA were further increased, reflecting detection of the exogenous IL-1RA.

Example 3—A Sensitive Cell-Based Assay for Measuring Serum IL-1R Signaling Activity

Given that both IL-1β and IL-1RA were elevated in FIRES patient serum, experiments were conducted to determine whether the elevation translated into a change in overall IL-1R signaling activity. To develop a sensitive functional measurement of IL-1R signaling activity, a cell based assay using HEK-Blue IL1R cells was developed and optimized. These cells express human IL-1R and respond to IL-1R signaling-induced NFκB activity by producing secreted embryonic alkaline phosphatase (SEAP), which dose-dependently results in increased absorbance of 655 nm wavelength light upon incubation with Quanti-Blue substrate (FIG.2A). Further, this effect can be competitively blocked by co-treatment with recombinant IL-1RA (FIG.2B). Titration showed that dose of 32 pg/mL IL-1β was most sensitive to blockade by IL-1RA while still retaining sufficient signal-to-noise ratio to limit intra-assay variability (FIG.2C). Quenching HEK-Blue IL1R cells two hours after treatment, by applying excess IL-1RA, further increased sensitivity of the assay (FIG.2D).

Example 4—Elevated IL1R Signaling Activity of FIRES Patient Serum

The assay described above was used to measure the endogenous IL-1R signaling activity of serum from healthy controls, and from patients with focal epilepsy, ESES, or FIRES (FIG.3A). Both FIRES and ESES patients exhibited elevated serum IL-1R activity relative to healthy controls or patients with focal epilepsy, who did not differ from controls. Next, studies were conducted to measure how patient serum modified IL-1R activity in response to a known concentration of IL-1β (FIG.3B). Surprisingly, FIRES and ESES patient serum synergized with exogenous IL-1β, resulting in a difference in IL-1R activity between cells treated with IL-1β plus FIRES patient serum and cells treated with IL-1β plus healthy control serum that was greater than the difference measured between cells treated with FIRES patient serum alone and healthy control serum alone. Given that the measured concentration of serum IL-1β in these patients was far below the 32 pg/mL used in the assay, the multiplicative increase in IL-1R activity suggested that other serum factors may be driving IL-1R activity, or that a larger reservoir of the serum IL-1β remained undetected by ELISA—possibly because it may be present in microvesicles as described elsewhere (Lopez-Castejon and Brough,Cytokine Growth Factor Rev22(4):189-195, 2011). Indeed, serum IL-1β levels did not strongly correlate with measured IL-1R activity across all patients (FIG.3C). Additionally, contrary to expectation, IL-1RA levels correlated with assayed IL-1R activity (FIG.3D), suggesting that serum factors may be affecting the inhibitory capacity of IL-1RA and causing it to function as an IL-1R agonist rather than an IL-1R antagonist in this assay. This notion was rejected in follow up studies confirming that, in all conditions, the measured increase in IL-1R activity was at least partially suppressed by the addition of exogenous rIL-1RA (FIG.3E). Serum-elicited IL-1R signaling activity was not completely suppressed by exogenous IL-1RA, indicating that a portion of serum IL-1R ligands may have bound to IL-1R in compartments that were not accessible to exogenously added rIL-1RA. This would be consistent with the presence of IL-1β in serum microvesicles. These findings suggested that, while informative, serum measures of IL-1β and IL-1RA alone or together may represent an incomplete picture of serum IL-1R activity, which may be more accurately gauged by functional cell based assays.

Example 5—Reduced Functional Blockade of IL-1R Signaling by Endogenous IL-1RA in an Anakinra-Responsive FIRES Patient

It is likely that the concentration of IL-1RA at the site of production in the brain interstitial space—and by extension, its relevant functional activity—far exceeds the levels detected in serum or CSF. IL-1RA is a downstream transcriptional target of IL-1R signaling and a principle mechanism of negative feedback. Therefore, elevated levels of CSF IL-1RA may represent either a normal protective response to terminate CNS IL-1R signaling in the acute setting, or the continuation of a failed termination of IL-1R signaling in the chronic setting. The fact that the levels of IL-1RA detected in the CSF of an anakinra-responsive FIRES patient represent concentrations of IL-1RA that are sufficient to cause suppression of IL-1R signaling (see, e.g., McIntyre et al.,J Exp Med173(4):931-939, 1991; Greenfeder et al.,J Biol Chem270(38):22460-22466, 1995; and Hou et al.,Proc Natl Acad Sci USA110(10):3913-3918, 2013) suggested the possibility of a dysfunctional IL-1RA protein and failed IL-1R signaling termination.

To investigate this possibility, the antagonistic activity of FIRES patient CSF-derived IL-1RA was assessed using an in vitro HEK-Blue IL1R cell-based assay. HEK-Blue IL1R cells cultured in media containing 50% control CSF or artificial CSF supplemented with recombinant IL-1RA did not show inhibition of IL-1R signaling, compared to HEK-Blue IL1R cells cultured in complete media (FIGS.4A,4B, and4C). However, dialyzing CSF against DMEM/F12 using a 100 Dalton molecular weight cut off cellulose acetate membrane restored anakinra-mediated inhibition (FIG.4D). Thus, HEK-Blue IL1R cells were treated with 32 pg/mL IL-1β in the presence of either 50% dialyzed FIRES patient CSF containing 65 pM patient IL-1RA or 50% dialyzed CSF derived from NPH patients that was supplemented with recombinant IL-1RA prior to dialysis to reach concentrations of IL-1RA equimolar to that seen in FIRES patient CSF. The percent inhibition of IL-1R-induced SEAP was then compared in cells treated with 50% CSF containing 65 pM FIRES patient IL-1RA vs. 50% CSF containing 65 pM anakinra. Surprisingly, FIRES patient CSF-derived IL-1RA was significantly less effective than recombinant IL-1RA (anakinra) at suppressing HEK IL-1R signaling (FIG.5A). In contrast, treatment with NPH CSF supplemented with 65 pM anakinra showed significantly less IL-1R signaling in IL-1β treated HEK cells compared to unsupplemented NPH CSF (FIG.5B). Importantly, FIRES patient CSF alone did not drive HEK IL-1R signaling (FIG.5C), suggesting that the observed lack of IL-1R inhibition was not due to a concomitant increase in ligation of IL-1R by CSF derived agonists.

Example 6—Multiple Non-Coding Polymorphisms in FIRES Patient IL1RN Gene

Sanger sequencing was performed on overlapping 4000 bp amplicons spanning the IL1RN gene in FIRES patient DNA, but no variants were detected within the translated portion of the coding sequences. Several novel and known variants were detected within the intronic sequences and in the untranslated portion of exon 6. TABLE 1 lists each variant, along with its genomic location and associated risks for variants that were previously reported.

Example 7—Marked Reduction in Expression of Intracellular Isoforms of IL1RN in FIRES Patient PBMCs

To determine whether there was aberrant expression of IL-1RA in FIRES, control and FIRES patient peripheral blood mononuclear cells (PBMCs) were isolated and treated with lipopolysaccharide (LPS) for 6 to 24 hours to ensure maximal IL-1RA production. In separate experiments, protein and RNA were isolated from cell lysates and collected supernatants. Secreted IL-1RA (isoforms 1 and 4/5) in cell supernatants and intracellular IL-1RA (isoforms 2 and 3) were measured by ELISA. Total IL1RN mRNA expression was determined by real time polymerase chain reaction (RTPCR). No differences were observed in the level of secreted IL-1RA between FIRES and control PBMCs (FIG.6A), but the levels of intracellular IL-1RA were significantly reduced in FIRES PBMCs in both untreated and LPS-treated conditions (FIG.6B). This corresponded with significantly reduced IL1RN mRNA levels in FIRES PBMCs relative to controls (FIG.6C). IL-1RA is translated from five distinct isoforms that give rise to two secreted and two intracellular variants (the isoform 4 and 5 mRNA sequences only differ upstream of their translational start site). Importantly, the intracellular forms can be secreted in response to ATP and other triggers (see, Jeong et al.,Mediators Inflamm2016:7984853; Wilson et al.,J Immunol173(2):1202-1208, 2004; and Evans et al.,Cytokine33(5):274-280, 2006). Thus, to determine whether the reduced intracellular levels of IL-1RA were due to aberrant expression of IL1RN isoforms in FIRES, expression of secreted (isoforms 1 and 4/5) and intracellular (isoforms 2 and 3) IL1RN gene products were analyzed by RTPCR. While IL1RN isoform 1 was somewhat increased following LPS stimulation in FIRES PBMCs relative to controls (FIG.7A), this effect was small relative to the >10 fold reduced expression of isoform 2 (FIG.7B) and isoform 3 (FIG.7C) in FIRES PBMCs relative to control PBMCs. Differences between FIRES and control PBMCs in the total expression of isoforms 4 and 5 were not observed (FIG.7D). The profound abrogation of expression of IL1RN isoforms 2 and 3 in FIRES patient PBMCS meant that these isoforms represented only 20% and 25%, respectively, of IL1RN expression in untreated conditions, jumping to a mere 25% and 35% of IL1RN expression after LPS treatment (FIG.7E). In contrast, in control PBMCs these isoforms were the dominant IL1RN transcripts, together representing >95% of all expressed isoforms in both untreated and LPS treated conditions (FIG.7F).

A representative amino acid sequence for isoform 1 of human IL-1RA is

(SEQ ID NO: 42)MEICRGLRSHLITLLLFLFHSETICRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE.

A representative amino acid sequence for isoform 1 of human IL-1RA is

(SEQ ID NO: 43MALETICRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIEGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE.

A representative amino acid sequence for isoform 3 of human IL-1RA is

(SEQ ID NO: 44)MALADLYEEGGGGGGEGEDNADSKETICRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE.

A representative amino acid sequence for isoforms 4 and 5 of human IL-1RA is

(SEQ ID NO: 45)MQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE.

TABLE 1IL1RN gene polymorphisms in FIRES patientLocation(GRCh38.p7)LocationVariation IDVariantAssociated Risks/Notes113120414intron 2—G > GT113120473intron 2—T > A113121042intron 2—dup TTC113121075intron 2rs200489291del CTCrs1553468945 = deletion at this site113121134intron 2—C > A113121077intron 2rs759842341del CTT113121138intron 2rs377086A > G113121321intron 2—A > AT113121720intron 2rs3213448G > Aincreased IL1Ra levels113122472intron 2rs4251991T > G113122715intron 2rs2853628C > G113122916intron 2rs4251993G > A113123788intron 2rs315935G > A113124298intron 2—T > TA113126106intron 2rs4252001A > G113126625intron 2rs3087262G > C113126824intron 2rs439154A > Gchildhood IgA nephropathy113128424intron 3—ins T113128430intron 3—C > G113128443intron 3—T > G113128472intron 3rs3181052G > Aincreased risk of osteoarthritis progression113128773intron 3rs1794066A > Gincreased risk of osteoarthritis progression113128807intron 3rs1794067A > Gincreased risk of asthma113129906intron 4rs2071459C > Tincreased risk of small bowelneuroendocrine tumor113130873intron 4—T > Ars910001631 = T > C at this site113130874intron 4—ins CC113130875intron 4—G > T113130888intron 4rs448341A > GGVHD risk113130893intron 5rs434792C > T113130905intron 4—G > A113130906intron 4rs020465621A > G113130909intron 4—del TG113130910intron 4—del TG113131853intron 5rs315955G > Ccardiovascular disease113132211intron 5rs315954A > G113132426intron 6rs315953C > T113132727exon 6rs315952T > Csterile multifocal osteomyelitis withperiostitis and pustulosis,increased risk of acute coronary syndrome,systemic lupus erythematosus, andosteoarthritis severity1131330093′ UTRrs315951C > Gsterile multifocal osteomyelitis withperiostitis and pustulosis, increased risk ofulcerative colitis113133462—T > TG113133680—G > GA

TABLE 2IL1RN PrimersIL1RN RTPCR primersDirectionSequence (SEQ ID NO:IL1RN VNTR FForwardCTC AGC AAC ACT CCT AT (1)IL1RN VNTR RReverseTCC TGG TCT GCA GGT AA (2)IL1RN_1FForwardAAC TCT GGG CCC GCA ATG (3)IL1RN_2FForwardTGA CTC AAA GGA GAC GAT CTG (4)IL1RN_3FForwardCAT GGC TTT AGA GAC GAT CTG C (5)IL1RN_4-5FForwardTCA AAG CCA AGA AGG CAA GAG (6)IL1RN_all isoforms FForwardCAA GAT GCA AGC CTT CAG AAT C (7)IL1RN_common RReverseTCT GGT CTC ATC ACC AGA CT (8)IL1RN PCR primersDirectionSequenceIL1RN FWD Set 1ForwardCAA ACC CTA ACT CAA TCC CAA AT (9)IL1RN REV Set 1ReverseAGG CAT TTT CAA GAT TTT ATT GTA AAA C (10)IL1RN_1 FWD Set 1ForwardGGG CAG CTC CAC CCT GG (11)IL1RN_1 REV Set 1ReverseGTC CTG CCA AGT AGC CAA GTT AAT (12)IL1RN FWD Set 9ForwardCAA GCT GGA TGC CAA CAT TTC (13)IL1RN REV Set 9ReverseGCC CTC AAA GGA AGA CAC TAT T (14)IL1RN FWD Set 12ForwardTGC TAG CTG CCT TCT CTT TC (15)IL1RN REV Set 12ReverseGTG ACC AAG GGT CTG GAT TT (16)IL1RN FWD Set 14ForwardCAT GGT GAA ACC CTG TCT CTA T (17)IL1RN REV Set 14ReverseGCC CAG CCC ATA ATC TAC TT (18)IL1RN FWD Set 16ForwardCTG TGG GTG TAT GAG TGA CAA G (19)IL1RN REV Set 16ReverseGGA CTC TGG GAC CTA GGT TTA T (20)IL1RN FWD Set 11ForwardAAC TCC AGC CAT CCT GAA TAA (21)IL1RN REV Set 11ReverseCCG TGT GAC CTT GAA CAA ATC (22)IL1RN FWD Set 15ForwardCAT TCT CCT TTC TGG GTC TTA CT (23)IL1RN REV Set 15ReverseGAG TGC AGT GGA GCA ATC TA (24)IL1RN FWD Set 5ForwardCGG GAT GGA CCC TGT TAT T (25)IL1RN REV Set 5ReverseGGT TCA GGC TAC TCT GTC TAT G (26)IL1RN SequencingDirectionSequenceIL1RN_14920FForwardACC AAT ATG CCT GAC GAA GG (27)IL1RN_15240FForwardTCT GCA TTC AGG ATC AAA CCC (28)IL1RN_15787FForwardAGA AGT TTC TCA GCT CCC AAG G (29)IL1RN_14578FForwardATA AAC CTA GGT CCC AGA GTC C (30)IL1RN_12921FForwardGAC ATC ACA TGG AAC ATC C (31)IL1RN_9415FForwardCAG GAA CAG TAG GGA GTT TGG (32)IL1RN_2043FForwardGGT GAG AAC AGA GGG TAA AGG (33)IL1RN_66FForwardGCT CAG TTG AGT TAG AGT CTG G (34)IL1RN_11430FForwardCTC AGA TGG GAA GCA AGT AAG G (35)IL1RN_13355RReverseGGA ACA GAA CTA CCC AGC TAA TC (36)IL1RN_2965RReverseCCC ACA CTA CAG TCC TAA A (37)IL1RN_10549RReverseTCG GCC CAG ACA AAC ATA AA (38)IL1RN_513RReverseGGC TCA GTG CCA CAT TCT ATT A (39)IL1RN_15561RReverseGAG TCC AGA TTA TGG AAG TG (40)IL1RN_15484RReverseATC TCC AAA TGA AGG GCT CTC (41)

Example 8—Cytokine Production in Patient Neutrophils in Response to ATP Stimulation

Blood was collected from a 10 year old girl with periodic autoinflammatory disorder of unknown etiology during disease remission. Neutrophils were enriched by density gradient centrifugation and then either primed with 100 ng/mL LPS for 90 minutes or left unprimed for the same amount of time. After priming, the cells were stimulated with a dose range of ATP (0-5 mM) for 45 minutes. At the end of stimulation, cells were pelleted and supernatants were collected and clarified. Cytokines (TNFα, IL-6, IL-1β, and IL-8) were measured using a multiplexed fluorescent bead array and quantified by comparison to standard curves. Levels of cytokines were compared in supernatants from LPS-primed and unprimed neutrophils before (prestim) and after ATP stimulation. Notably, LPS priming drove release of TNFα, IL-6, and IL-8, but did not directly induce IL-1β release (FIG.8A). The response for these factors can serve as an addition marker of neutrophil responsivity. Supernatants from unprimed or primed neutrophils stimulated with different concentrations of ATP were then assessed for IL-1β release. While unprimed cells showed no response to ATP at any concentration (FIG.8B), the primed cells exhibited robust IL-1β production and release at higher ATP concentrations. A 3-parameter sigmoidal curve was fit to the ATP response data, and the second differential of this curve was used to determine the EC50for ATP.

Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.