SUPERANTIGEN POST-EXPOSURE THERAPY

Abstract

The present invention relates to a post-exposure therapeutic against superantigen-mediated disease in a subject.

Claims

1. A pharmaceutical composition for use in the post-exposure treatment of superantigen (SAg)-mediated disease in a subject, wherein the pharmaceutical composition comprises an active ingredient which is capable of binding an antigen presenting cell B7 receptor.

2. A pharmaceutical composition according to claim 1 administered as a single dose.

3. A pharmaceutical composition according to claim 1, wherein the active ingredient is selected from CTLA4Ig or derivatives thereof.

4. A pharmaceutical composition according to claim 3, wherein the active ingredient is CTLA4Ig.

5. A pharmaceutical composition according to claim 1, wherein the SAg is Staphylococcus Enterotoxin B.

6. A pharmaceutical composition according to claim 1, wherein the SAg-mediated disease is sepsis, toxic shock syndrome, infective endocarditis or necrotizing fasciitis.

7. A pharmaceutical composition according to claim 1, to be administered at least 3 hours post-exposure to SAg.

8. A pharmaceutical composition according to claim 1, to be administered at least 8 hours post-exposure to SAg.

9. A pharmaceutical composition according to claim 1, to be administered at least 144 hours post-exposure to SAg.

10. An agent capable of binding an antigen presenting cell B7 receptor for use in the post-exposure treatment of SAg-mediated disease.

11. An agent according to claim 10 administered as a single dose.

12. An agent according to claim 10, wherein the agent is CTLA4Ig, and derivatives thereof.

13. A method for monitoring subject responsiveness to post-exposure treatment of SAg-mediated disease with a pharmaceutical composition according to claim 1, the method comprising the steps of: measuring the concentration of at least one of IFN- and IL-6 in a first and a second biological sample, wherein the first biological sample is from a subject pre-treatment, and the second biological sample is post-treatment; comparing the pre- and post-treatment concentration of each respective biomarker; and wherein a respective biomarker concentration lower in the post-treatment biological sample, relative to the pre-treatment biological sample, is a positive indicator of subject responsiveness to post-exposure treatment.

Description

[0050] The present invention will now be described with reference to the following non-limiting examples and figures in which:

[0051] FIG. 1 is a graph showing in vivo efficacy of CTLA4Ig, administered 3 hr post SEB exposure, to mitigate SEB-induced weight loss in a sub-lethal murine model. Weight change is represented as mean percent change of body weight as compared to mouse weights on day 1 prior to SEB exposure. Graph represents mean with error bars for 95% Cl.

[0052] FIG. 2 is a graph showing in vivo efficacy of CTLA4Ig, administered 8 hr post SEB exposure, to mitigate SEB-induced weight loss in a sub-lethal murine model. Data represents two replicate in vivo studies. Weight change is represented as mean percent change of body weight as compared to mouse weights on day 1 prior to SEB exposure. Graph represents mean with error bars for 95% Cl.

[0053] FIG. 3 is a graph showing SEB-induced clinical signs of the SEB positive control group. Graph represents percentage of mice presenting severe, moderate, mild or no clinical signs plotted for each time point.

[0054] FIG. 4 is a graph showing SEB-induced clinical signs of the SEB treated with CTLA4Ig therapy group. Graph represents percentage of mice presenting severe, moderate, mild or no clinical signs plotted for each time point.

[0055] FIG. 5 is a graph showing SEB-induced clinical signs of the PBS negative control group. Graph represents percentage of mice presenting severe, moderate, mild or no clinical signs plotted for each time point.

[0056] FIG. 6 is a graph showing lung pathology scores from the SEB positive control group, SEB treated with CTLA4Ig therapy group and PBS negative control group. Scores from a minimum of 6 animals from the SEB positive control, PBS negative control and SEB treated with CTLA4Ig therapy group were measured at 3, 6 and 14 days. Graphs represent median with interquartile range.

[0057] FIG. 7 is a graph showing organ to body weigh percent weight of lung in the SEB positive control group, SEB treated with CTLA4Ig therapy group and PBS negative control group. Weights were determined at 3, 6 and 14 days post SEB exposure. Graphs depict mean organ values with 95% Cl.

[0058] FIG. 8 is a graph showing organ to body weigh percent weight of liver in the SEB positive control group, SEB treated with CTLA4Ig therapy group and PBS negative control group. Weights were determined at 3, 6 and 14 days post SEB exposure. Graphs depict mean organ values with 95% Cl.

[0059] FIG. 9 is a graph showing organ to body weigh percent weight of spleen in the SEB positive control group, SEB treated with CTLA4Ig therapy group and PBS negative control group. Weights were determined at 3, 6 and 14 days post SEB exposure. Graphs depict mean organ values with 95% Cl.

[0060] FIG. 10 is a graph showing plasma concentrations of CXCL1 in the SEB positive control group, SEB treated with CTLA4Ig therapy group and PBS negative control group. Graphs depict mean cytokine values with 95% Cl.

[0061] FIG. 11 is a graph showing plasma concentrations of IL-1 in the SEB positive control group, SEB treated with CTLA4Ig therapy group and PBS negative control group. Graphs depict mean cytokine values with 95% Cl.

[0062] FIG. 12 is a graph showing plasma concentrations of TNF- in the SEB positive control group, SEB treated with CTLA4Ig therapy group and PBS negative control group. Graphs depict mean cytokine values with 95% Cl.

[0063] FIG. 13 is a graph showing plasma concentrations of IL-2 in the SEB positive control group, SEB treated with CTLA4Ig therapy group and PBS negative control group. Graphs depict mean cytokine values with 95% Cl.

[0064] FIG. 14 is a graph showing plasma concentrations of IFN- in the SEB positive control group, SEB treated with CTLA4Ig therapy group and PBS negative control group. Graphs depict mean cytokine values with 95% Cl.

[0065] FIG. 15 is a graph showing plasma concentrations of IL-6 in the SEB positive control group, SEB treated with CTLA4Ig therapy group and PBS negative control group. Graphs depict mean cytokine values with 95% Cl.

[0066] FIG. 16 is a graph showing plasma concentrations of IL-10 in the SEB positive control group, SEB treated with CTLA4Ig therapy group and PBS negative control group. Graphs depict mean cytokine values with 95% Cl.

[0067] FIG. 17 is a graph showing the effect of CTLA4Ig on SEB-induced murine splenocyte proliferation. Means and 95% Cl are represented for each treatment.

[0068] FIG. 18 is a graph showing the effect of CTLA4Ig on SEB-induced murine splenocyte cytotoxicity. Cytotoxicity is presented as mean fluorescence intensity (MFI) of dead cells using a MultiTox-Fluor Multiplex cytotoxicity assay. Means and 95% Cl are represented for each treatment.

[0069] FIG. 19 is a graph showing the effect of CTLA4Ig on CCL2 expression by SEB-treated splenocytes. Means and 95% Cl are represented for each treatment.

[0070] FIG. 20 is a graph showing the effect of CTLA4Ig on IL-1 expression by SEB-treated splenocytes. Means and 95% Cl are represented for each treatment.

[0071] FIG. 21 is a graph showing the effect of CTLA4Ig on IL-2 expression by SEB-treated splenocytes. Means and 95% Cl are represented for each treatment.

EXAMPLE 1

[0072] Animal Conditions

[0073] Age-matched male Balb/C mice purchased from a designated supplier (42-49 days old; Charles River Laboratories Ltd, Margate, Kent, UK) were used for all animal studies. The mice were housed in a Home Office designated establishment in rooms maintained at 21 C. +/2 C. on a 12/12-hour dawn to dusk cycle. Humidity was maintained at 55% +/10% with airflow of 15-18 changes/hour. Mice were kept in polycarbonate shoebox-type cages with steel cage tops and corncob bedding (International Product Supplies, Wellingborough, UK). Mice were fed a standard pelleted Teklad TRM 19% protein irradiated diet (Harlan Teklad, Bicester, UK) and given fresh water daily, ad libitum. All animals were housed according to the 1986 Scientific Procedures Act, under is appropriate ethically approved licenses from the UK Home Office.

[0074] Toxin & Therapeutics

[0075] SEB toxin was obtained from the Health Protection Agency (Porton Down, Wiltshire, UK). SEB toxin was prepared for use in all in vitro and in vivo studies at the correct concentration using 0.9% phosphate-buffered saline. CTLA4Ig (abatacept; Orencia) (250 mg) was obtained from Bristol Myers Squibb.

[0076] In Vivo SEB Sub-Lethal (Incapacitation) Model

[0077] In vivo studies were carried out using CTLA4Ig to provide confirmation of the inhibition of SEB-induced toxicity in mice. Balb/C mice were randomly assigned to treatment or control groups and weighed on day 0 of the experiment. Intranasal administration of the SEB toxin was performed under light anaesthesia with recovery induced using Halothane (5% in the presence of 4 Lmin.sup.1 oxygen). SEB toxin used for intranasal administration was prepared as a 0.25 g/g dose in Dubbecco's Phosphate Buffered Saline (PBS) and given as a total dose of 50 l split between the two nares. All studies were carried out according to the Home Office Animal Licence which clearly defined humane endpoints including the loss of 30% weight loss or more and reduced mobility in the presence of severe clinical signs.

[0078] In a first study to assess efficacy of CTLA4Ig to mitigate SEB-induced weight loss, 3 groups of 6 mice were dosed as follows: SEB via the intranasal route and PBS via the intravenous route (SEB positive control groups); PBS via the intranasal and intravenous route (PBS negative control group); or SEB via the intranasal route and CTLA4Ig intravenously (10 mgkg.sup.1) (SEB treated with CTLA4Ig therapy group). Intravenous PBS or CTLA4Ig was administered 3 hr post SEB intranasal challenge. Mice were weighed daily for 14 days following SEB exposure. Statistical difference between the positive control group and CTLA4Ig therapy group was determined by a mixed linear model.

[0079] In a second study to assess efficacy of CTLA4Ig to mitigate SEB-induced weight loss, 3 groups of 1.8 mice were dosed as follows: SEB via the intranasal route and PBS via the intravenous route (SEB positive control groups).; PBS via the intranasal and intravenous route (PBS negative control group); or SEB via the intranasal route and CTLA4Ig intravenously (10 mgkg.sup.1) (SEB treated with CTLA4Ig therapy group). Intravenous PBS or CTLA4Ig was administered 8 hr post SEB intranasal challenge. Mice were weighed daily for 14 days following SEB exposure. Data represents two replicate in vivo studies. Statistical difference between the positive control group and CTLA4Ig therapy group was determined by a mixed linear model.

[0080] During both studies, the efficacy of CTLA4Ig to mitigate SEB-induced clinical signs was investigated. Data represents two replicate in vivo studies. Mice were scored twice daily for 14 days following SEB exposure, using the following scoring system for signs of SEB intoxication: 0=normal mouse; 1=slight piloerection; 2=medium piloerection; and 3=severe piloerection. The degree of abdominal pinching was also scored as mild, medium or severe. Clinical signs of intoxication were monitored according to the scoring criteria set out in Table 1. Statistical difference between the SEB positive control group and CTLA4Ig therapy group was determined by chi-squared analysis of the total number of observations for each clinical signs category.

TABLE-US-00001 TABLE 1 Scoring of clinical signs and mobility in the mouse. Severity assessment of signs Score Visible signs and mobility of intoxication 0 None + normal mobility Nil 1 Mild piloerection + normal mobility Mild 2 Medium piloerection + normal Moderate mobility 3 Severe piloerection + normal Severe mobility 4 Severe piloerection + unwilling to Severe move/reduced mobility 5 (humane end Severe piloerection + unable to Severe point) move

[0081] Summary of results: FIG. 1-5 show that CTLA4Ig treatment at 3 hr and 8 hr post SEB exposure prevents SEB-induced weight loss and clinical signs. SEB positive control group had a significant drop in weight loss (for example>15% around day 5). However, in the SEB treated with CTLA4Ig therapy group and the PSB negative control group no weigh loss was observed and indeed similar levels of weight gain demonstrated. This indicates that the CTLA4Ig completely mitigates the substantial weight loss and clinical signs induced by SEB exposure.

[0082] Pathological Assessment of Tissues

[0083] Animals were culled using cervical dislocation at either 3, 6 or 14 days following SEB intoxication. The necropsy of lung, kidney and spleen were recorded as a crude indicator for presence of atrophy, hyperplasia or tissue oedema and weighed. Lungs were examined for signs of gross pathology and the lung wet weight data recorded. The gross lung pathology severity was assessed and scored also using an increasing scale of severity from 0 (no pathology) to 4 (severe pathology) using the pre-determined scoring system set out in Table 2. Organ to body weight results, expressed as percentages of animal total body weigh at the time of culling, were assessed using a Two-way ANOVA with a Bonfferoni post test to compare the PBS negative control group and the SEB treated with CTLA4Ig therapy group to the SEB positive control group.

TABLE-US-00002 TABLE 2 Scoring of gross lung pathology. Score Description 0 Normal Generally pale pink colouration No hyperinflation If present only few (<5) white foci 1 Mild Pale patches of congestion Few minor focal haemorrhages Possible hyperinflation 2 Moderate Overall general congestion Some hyperinflation Froth exuding from cut trachea 3 Severe Haemorrhagic congestion on few lobes Hyper inflated Some consolidation may be evident 4 Very severe Haemorrhagic congestion evident in all lung lobes with extensive consolidation evident

[0084] Summary of results: FIG. 6 shows that SEB exposure increased lung pathology scores at day 3 and 6. At day 6, all animals in the SEB positive control group had visible inflammatory-induced pathology of the lungs. Animals that received CTLA4Ig had reduced lung pathology scores throughout the study as compared to the SEB positive control group. Only one animal showed mild pathology of the lungs at day 6 in the SEB treated with CTLA4Ig therapy group. In addition to reduced lung pathology scores, lung to body weigh percentage was significantly lower in the SEB treated with CTLA4Ig therapy group as compared to the SEB positive control group at day 6 (FIG. 7).

[0085] Importantly, the lung weights of the SEB treated with CTLA4Ig therapy group were similar to the PBS negative control group. The results suggest that lung odema is mitigated in the therapy group when exposed to SEB as compared to animals receiving SEB and not the therapy. Visibly pathology scores and lung weights suggest that CTLA4Ig prevents SAg-induced immunopathology of the lungs.

[0086] Significant differences were observed in liver weight at day 6 between the SEB positive control group verses the SEB treated with CTLA4Ig therapy group and the PBS negative control group (FIG. 8), which may have implications in the systemic response to SAg. There were no significant changes in the weight of the spleens across all groups (FIG. 9).

[0087] CBA for Plasma and Tissues

[0088] At days 3, 6 and 14 days post SEB exposure, whole blood from 6 mice per study group was sampled into EDTA tubes. Samples were rolled for 5 min at room temperature prior to being centrifuged at 1000 g for 15 min at 4 C. Plasma supernatant was subsequently removed and stored prior to luminex analysis. Samples were diluted 1:4, according to manufacturer's instructions, prior to analysis with luminex performance assay for murine CCL2, GM-CSF, IFN-, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A, CXCL1, CXCL2, TNF- and VEGF (R&D Systems, USA). Assays were, performed as per manufacturer's instructions and analysed on Bio-Rad Bio-Plex analyser 200. For analysis, 5PL curves were used to fit standard curves with automated optimisation. Cytokine levels were compared using a Two-way ANOVA with a Bonfferoni post test comparing the PBS negative control and the SEB treated with CTLA4Ig therapy group to the SEB positive control group.

[0089] Summary of results: Pro-inflammatory cytokines/chemokines (FIG. 10, CXCL1; FIG. 11, IL-1; FIG. 12, TNF-) were significantly increased in the SEB positive control group as compared to the PBS negative control group. For CXCL1 and IL-13, these differences were only observed at day 3. CTLA4Ig therapy significantly reduced CXCL1 and IL-1 at 3 days post-exposure, suggesting that the therapy prevented the systemic response regarding these pro-inflammatory mediators. Interestingly, whilst TNF- was raised in the SEB positive control group at 3, 6 and 14 days, CTLA4Ig administration did not significantly reduce TNF- levels. This would suggest, contrary to current understanding, that TNF- is not an important mediator to the clinical outcome of subjects exposed to SAgs as weight loss and clinical signs remain unaffected in the SEB treated with CTLA4Ig therapy group. Importantly, this would suggest that TNF- is not a good biomarker for determining efficacy of B7 receptor antagonists for SEB-mediated disease, or potentially other disease states where this therapy is used.

[0090] In contrast, at day 3, levels of IFN- (FIG. 14) and IL-6 (FIG. 15) were significantly raised in the SEB positive control group but were reduced almost to baseline levels when CTLA4Ig was administered. This would suggest that IFN- and IL-6, and not TNF-, are suitable biomarkers to monitor subject responsiveness to use of CTLA4Ig in the post-exposure treatment of SAg intoxication. This further indicates that IFN- and IL-6 are potentially appropriate biomarkers to evaluate subject responsiveness to CTLA4Ig, as well as other pharmaceutical composition active ingredients or agents capable of binding to B7 family receptors, in other disease states.

[0091] IL-2 (FIG. 13) was shown to be significantly raised in the SEB positive control group as compared to the PBS negative control group. While significant reduction to IL-2 was not seen for the SEB treated with CTLA4Ig therapy group, there was a trend in reduced IL-2 levels which suggests that CTLA4Ig may reduce T-cell proliferation.

[0092] At day 3, IL-10 (FIG. 16) was raised in the SEB positive control group as compared to the PBS negative control group. However, on days 6 and 14, the IL-10 response was equivalent to the PBS negative control group. Interestingly, there was a trend for increased IL-10 production by the SEB treated with CTLA4Ig therapy group at days 6 and 14 as compared to the SEB positive control group and PBS negative control group. This would suggest that CTLA4Ig allows the anti-inflammatory response to be induced after SEB exposure in order to reduce inflammation.

[0093] Splenocyte Isolation

[0094] Spleens were aseptically isolated from Balb/C mice, placed in 10 m cell strainer and splenocytes pushed through the strainer into a sterile petri dish using the handle from a sterile cell scraper or sterile 10 ml syringe. The splenocyte cell suspension was added to a 50 ml falcon tube, brought up to a final volume of 30 ml with RPMI-1640 media containing 15% (v/v) fetal calf serum (Sigma-Aldrich, Poole, Dorset, UK), 1% (v/v) Penicillin/Streptomycin solution and 1% (v/v) L-Glutamine (Sigma-Aldrich, Poole, Dorset, UK) and centrifuged for 10 minutes at 1100 rpm. After centrifugation, the supernatant was discarded and the red blood cells lysed by adding 3 ml of red blood cell lysing buffer (Sigma, Dorset UK), mixing gently for 1 min. Sterile RPMI-1640 medium was added until the cell suspension final,volume was 30 ml, and the resulting solution was centrifuged for 10 min at 1100 rpm. After this centrifugation, the supernatant was discarded and 4 ml of sterile RPMI-1640 medium added. The pellet was re-suspended and the cells quantified using a Neubauer haemocytometer. The final suspension was then prepared to 1.010.sup.6 cells/ml in RPMI-1640 medium.

[0095] Proliferation MTT and SEB Cytotoxicity Assays

[0096] Isolated splenocytes were treated with PBS (negative control), SEB (positive control) or with SEB and CTLA4Ig and, after 24 hrs, cell proliferation and cell viability was measured using a modified MTT assay. This assay measured these parameters via the reduction of the active yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide. Cells were plated at a seeding density of 110.sup.6 cells per well and incubated for 48 hr. 10 l of the MTT reagent was added and the plate incubated for 4 hr. 100 l detergent reagent was then added and the plates re-incubated for a further 3 hr. The plates were then read at 570 nms. For each of the 5 experiments the proliferation response of CTLA4Ig-treated cells were normalised against the negative control and the positive SEB control (0% and 100% respectively). Optical density was analysed using a Repeated Measure ANOVA with Dunnett's post test comparing the SEB treated with CTLA4Ig group to SEB positive control group and splenocyte only negative control group.

[0097] The ability of CTLA4Ig to inhibit the cytotoxic effect of SEB was investigated using a Promega MultiTox-Flour Multiplex Cytotoxicity Assay according to the manufacturer's instructions (Promega, USA). Cell toxicity assays were performed on 4 separate occasions: splenocytes only; splenocytes treated with CTLA4Ig; splenocytes exposed to SEB; and splenocytes exposed to SEB and treated with CTLA4Ig. Briefly, splenocytes were seeded into wells of 96-well flat bottomed cell culture plates at a density of 110.sup.6 cells per well (B. E. Thompson Supplies, Andover, UK). These cells were then exposed to 1.2510.sup.8M or 1.610.sup.9M SEB in culture medium. Dead cell number were determined using fluorescence at 485Ex/535Em 3 hr after addition of the 5 MultiFlour reagent prepared according to the manufacturers guidance. Con A was also used as a positive proliferation control with a mean value of 366% increase in proliferation as compared to SEB (data not shown). Optical density was analysed using a Repeated Measure ANOVA with Dunnett's post test.

[0098] Summary of results: SEB characteristically causes a dose dependant proliferation of T-cells. Thus, primary cultures of isolated mouse splenocytes, which contain a high proportion of T-cells, were used as to measure SEB activity. CTLA4Ig significantly reduced splenocyte proliferation in response to SEB exposure across all concentration of CTLA4Ig (10-0.3125 g/ml) (FIG. 17). The reduction in SEB-induced proliferation was in a dose-responsive manner and at the highest concentration reduced SEB proliferation to less than 40%. The reduction in proliferation was not due to therapeutic toxicity as CTLA4Ig-treated cells showed no cytotoxicity compared to splenocyte exposed to PBS only (FIG. 18).

[0099] ELISA for Cytokines IL-1, IL-2 and MIP-1 Determination

[0100] Quantitative Quantikine ELISAs were performed to determine the effect of CTLA4Ig on CCL2, IL-1, and IL-2 expression by murine splenocytes following SEB exposure. ELISA plates pre-coated for mouse CCL2, IL-1 , IL-2 or MIP-1 were supplied by Quantikine. Standards, controls and samples were pipetted into the wells of the ELISA plate as per the manufacturer's instructions. Following removal of the supernatant from the plates used in the cytotoxicity study, the means of five experimental replicates was determined. CCL2, IL-1 and IL-2 levels expressed by splenocytes was analysed using a Repeated Measure ANOVA with Dunnett's post test comparing a range of CTLA4Ig treatment concentrations to SEB positive controls.

[0101] Summary of results: The SEB-induced expression of CCL2 (FIG. 19), IL-1 (FIG. 20) and IL-2 (FIG. 21) was significantly reduced when CTLA4Ig was administered to splenocytes at various concentrations. Importantly, the reduction of CCL2 and IL-1 suggests that CTLA4Ig intervention reduced SEB-induced inflammation. Additionally, the reduction observed for IL-2 suggests that CTLA4Ig reduces IL-2-induced T-cell proliferation when splenocytes are exposed to SEB.