Products and methods for the assessment and treatment of sepsis and immunological and inflammatory conditions

Abstract

The invention relates to pharmaceutical compositions and methods of treating or preventing immunological or inflammatory conditions, such as sepsis or septic shock. Also provided are methods of predicting the likelihood of a subject experiencing an immunological or inflammatory condition, such as sepsis or septic shock.

Description

PRODUCTS AND METHODS FOR THE ASSESSMENT AND TREATMENT OF SEPSIS AND IMMUNOLOGICAL AND INFLAMMATORY CONDITIONSPriority claim

[0001] This application claims priority from Australian provisional application AU 2024903705 filed 12 November 2024, the entire contents of which are incorporated by way of this reference.Technical Field

[0002] The present disclosure relates to pharmaceutical compositions and methods of treating or preventing immunological or inflammatory conditions, such as sepsis or septic shock. Also provided are methods of predicting the likelihood of a subject experiencing an immunological or inflammatory condition, such as sepsis or septic shock.Background of Invention

[0003] Sepsis is a severe and systemic inflammatory response to infection resulting from dysregulation of a subject's immune response which leads to a "cytokine storm" and resultant tissue damage. Sepsis can progress to septic shock, a severe form of sepsis characterised by refractory hypotension and often resulting in organ failure and death. Mortality in septic shock is still overwhelming high at around 25-40%, with little change in this rate over the past 3 decades.

[0004] The mainstay of current therapy is supportive management of blood pressure with inotropes, combined with antimicrobials, ventilation and renal replacement therapy. Concurrently, corticosteroids (such as hydrocortisone) are indicated in patients displaying persistent hypotension despite fluid resuscitation and inotropic support, in an attempt to regulate the systemic inflammatory response. However, despite extensive use, meta-analysis shows little benefit in corticosteroid administration.

[0005] Whilst corticosteroids have immunomodulatory actions which theoretically should help kerb the inflammatory response, the lack of improvements in mortality indicatessuboptimal biological activity in the context of systemic inflammation. Further, indiscriminate corticosteroid exposure (in particular glucocorticoid exposure) can lead to problems such as hyperglycaemia, myopathy, neurocognitive disturbance and susceptibility to secondary infections.

[0006] Accordingly, there is an unmet need for therapies to control immunological conditions, particularly inflammatory conditions such as sepsis and septic shock. In addition, there remains a need to predict which patients suffering from inflammatory conditions such as sepsis or septic shock will likely have an adverse outcome, or a clinical pathology, to allow early and appropriate intervention.Summary of Invention

[0007] The Inventors of the present invention have shown, for the first time, that administration of corticosteroid binding globulin (CBG) to subjects suffering from the lifethreatening inflammatory condition sepsis can help rescue subjects that would otherwise succumb to the condition and died.

[0008] Over the last three decades, there has been little improvement in the survival rate of patients suffering from sepsis and septic shock. While corticosteroids are currently indicated for patients suffering from sepsis, and septic shock, extensive analysis of clinical data demonstrates only weak evidence of their efficacy. Accordingly, it appears that manipulating cortisol levels and function in patients may not be an effective pathway for treating sepsis or septic shock. Therefore, it is surprising that the administration of CBG has a significant effect in animal models of sepsis and septic shock. Importantly, this treatment provides an opportunity to improve the outcome of a highly lethal condition.

[0009] Accordingly, in an aspect, the present invention provides a method of treating or preventing sepsis, or treating, preventing or delaying a clinical pathology of sepsis, the method comprising administering to a patient an effective amount of CBG. In preferred embodiments of this invention, the method treats or prevents septic shock, or treats, prevents or delays a clinical pathology of septic shock.

[0010] Septic shock requires admission to the intensive care unit (ICU) of a hospital due to the lethal nature of the condition and the need for extensive medical intervention. Specifically, septic shock can result in numerous clinical pathologies and interventions including: death, intubation and mechanical ventilation, vasopressor therapy, inotrope therapy, renal replacement therapy (dialysis), Extracorporeal Membrane Oxygenation (ECMO) therapy, antimicrobial therapy, vascular injury, capillary leakage, hypotension, hyperthermia, tachycardia, tachypnea, lactic acidosis, hypoxia, cyanosis, ileus, hyperglycemia, CNS dysfunction, sequential organ failure, multiorgan dysfunction syndrome (MODS), lung dysfunction, cardiac dysfunction, kidney dysfunction, and liver dysfunction. Accordingly, in some embodiments, the method of the present invention treats or prevents a clinical pathology of sepsis or septic shock selected from one or more of: mortality, time in septic shock, time in first septic shock episode, time to septic shock onset, number of septic shock episodes, intubation time, requirement for mechanical ventilation, requirement for vasopressor therapy, requirement for inotrope therapy, requirement for renal replacement therapy, requirement for Extracorporeal membrane oxygenation (ECMO) therapy, requirement for antimicrobial therapy, vascular injury, capillary leakage, hypotension, hyperthermia, tachycardia, tachypnea, lactic acidosis, hypoxia, cyanosis, ileus, hyperglycemia, CNS dysfunction, sequential organ failure, multiorgan dysfunction syndrome (MODS), lung dysfunction, cardiac dysfunction, kidney dysfunction, and liver dysfunction.

[0011] Various means are known in the art for defining sepsis and septic shock and a person skilled in the art can define if a patient is suffering from sepsis oris in septic shock based on their professional expertise and skill. However, in some embodiments, sepsis or septic shock is measured by one or more prognostic markers and wherein the method modulates the one or more prognostic markers, wherein the markers are selected from: increased serum lactate, reduced bicarbonate, increased pH, elevated serum creatinine, reduced eGFR, reduced serum albumin, increased bilirubin, reduced platelet count, elevated international normalised ratio, prolonged prothrombin time, elevated D-Dimer, elevated C-reactive protein, elevated interleukin-6, elevated interleukin-8, reduced interleukin-10, elevated monocyte chemoattractant protein 1, elevated presepsin, elevated pro-adrenomedullin, elevated procalcitonin, elevated cystatin-C, elevated troponin, elevated alanine transaminase, elevatedaspartate transaminase, elevated tumour necrosis factor alpha, total CBG less than 200 nmol / L, or Sequential Organ Failure Assessment (SOFA) score.

[0012] Ultimately, the inflammation associated with sepsis and septic shock can result in dysfunction and failure of multiple organs, leading to MODS. Therefore, the method of the present invention can treat or prevent MODS.

[0013] The Inventors of the present invention have also found that specific glycoforms of CBG are associated with better outcomes in patients with sepsis or in septic shock. While not wanting to be bound by theory, it is proposed that this is due to specific glycoforms acting as more effective carriers of cortisol (and corticosteroids) and therefore allowing transport of cortisol to required location where free cortisol can be released. This may ultimately improve the biological activity of cortisol and other corticosteroids. These specific glycoforms include a triantennary glycan at asparagine (Asn) 347. Accordingly, in some embodiments, the administered CBG may comprises a CBG glycoform having a triantennary glycan at asparagine (Asn) 347.

[0014] As CBG is a carrier of cortisol and other corticosteroids (particularly glucocorticoids), aspects of the invention also provide a method of modulating the biological activity of a corticosteroid in a subject, the method comprising administering an effective amount of CBG. Preferably, the CBG includes a triantennary glycan at Asn 347.

[0015] Further provided, in an aspect of the invention, is a method of treating a corticosteroid-associated condition, the method comprising administering to a subject an effective amount of a corticosteroid-binding globulin (CBG). Preferably, the CBG comprises a CBG glycoform having a triantennary glycan at asparagine (Asn) 347.

[0016] It is proposed the that the efficacy of CBG administration is its ability to help reduce inflammation in the subject, thereby dampening the inappropriately controlled inflammatory response seen in sepsis and septic shock. This proposed mechanism of action may be useful in other inflammatory conditions and therefore aspects of the present invention provide a method of treating or preventing an immunological or inflammatory condition, the method comprising administering to a patient an effective amount of CBG. Preferably, the CBGcomprises a CBG glycoform having a triantennary glycan at asparagine (Asn) 347. The immunological or inflammatory condition may be a chronic immunological or inflammatory condition. Alternatively, the immunological or inflammatory condition may be an acute immunological or inflammatory condition.

[0017] The present inventors have demonstrated that triantennary trisia lylated (TS3) and triantennary trisia lylated core-fucosylated (TS3F) glycans at Asn347 are specifically relevant to the outcome of a patient suffering from sepsis or septic shock. Therefore, in embodiments of the above aspects of the invention comprising a triantennary glycoform at Asn347, it is preferrable that the triantennary glycan comprises TS3 and / or TS3F.

[0018] One useful source of CBG is from the blood of animals, including humans. Therefore, in embodiments of the above aspects of the invention the CBG is serum or plasma isolated. Preferably, although not essentially, the serum or plasma isolated CBG is enriched for a CBG glycoform having a triantennary glycan at Asn347, for example enriched for TS3 and / or TS3F.

[0019] Alternatively, it may be desirable to produce or use recombinant CBG. Therefore, in embodiments of the above aspects of the invention the CBG is recombinant. Preferably, although not essentially, the recombinant CBG is enriched for a CBG glycoform having a triantennary glycan at Asn347, for example enriched for TS3 and / or TS3F. This may be achieved by producing such enriched CBG in specific, or modified cell lines, such as mammalian or human cell lines, of maybe achieved by enriching the recombinantly produced CBG after or during isolation.

[0020] Appropriated dosing schedules can be determined by a person skilled in the art. However, in some embodiments the effective amount of the CBG is administered in a dosage schedule comprising at least two doses. Alternatively, the dosing may be a continuous or prolonged infusion. Envisaged routes of administration include subcutaneous injection or intravenous injection. However, other routes are envisaged.

[0021] Due to the proposed ability of CBG to improve the biological activity of corticosteroids, aspects of the present invention provide a combination therapy comprisingadministration of a corticosteroid in combination with the CBG. Preferred corticosteroids are indicated for sepsis or septic shock or treating immunological or inflammatory conditions. These include one or more of: betamethasone, budesonide, ciclesonide, corticosterone, corticosterone acetate, fludrocortisone, hydrocortisone, methylprednisolone, dexamethasone, prednisolone or prednisone. Preferably, the corticosteroid is a glucocorticoid.

[0022] As the concentration of CBG is associated with the outcome of immunological or inflammatory conditions, such as sepsis and septic shock, the methods of the present invention may comprise a step of quantifying the concentration of total CBG or a glycoform of CBG in a biological sample from the patient. This may be performed before commencing treatment or may be during or after treatment. When quantifying CBG, the method may include looking at specific glycoforms of CBG, preferably glycoforms of CBG including a triantennary glycan at asparagine (Asn) 347, preferably TS3 and / or TS3F.

[0023] In view of the potential of CBG for therapeutic treatments of conditions (such as those disclosed herein) aspects of the present invention provide a pharmaceutical composition comprising isolated CBG, a recombinant CBG, or an in vitro produced CBG. Preferably, this CBG comprises a triantennary glycan at Asn347 of CBG or is enriched for CBG having a triantennary glycan at Asn347, preferably TS3 and / or TS3F. A preferred relative abundance of CBG in the pharmaceutical composition is about 42% or more of the CBG having a triantennary glycan at Asn347. Preferably the CBG is human, or a recombinant form of human CBG, but in some embodiments the isolated or recombinant CBG comprising a triantennary glycan at Asn347 is a non-human CBG.

[0024] CBG can be isolated from blood products including fractions of serum or plasma. These fractions may be enriched for a triantennary glycan at Asn347. In some embodiments, the serum or plasma isolated CBG is obtained by Cohn fractionation.

[0025] Alternatively, in some embodiments the CBG is recombinant CBG. Preferably the recombinant CBG will have an effective amount of triantennary glycan at Asn347, preferably TS3 and / or TS3F. In some embodiments, the relative abundance of CBG in the recombinant CBG is about 42% or more of the CBG having a triantennary glycan at Asn347.

[0026] As will be understood, the host cell will affect the post-translation modification of any produced CBG. Given the importance of the glycosylation of Asn 347, the cell line will preferably be one that ensures the appropriate CBG glycosylation profile, such as those described herein. In some embodiments, the recombinant CBG is produced by a mammalian, preferably a human, cell line.

[0027] Due to the association of CBG glycoforms with the outcome of sepsis and septic shock, the present invention provides a method of predicting the likelihood of a patient experiencing sepsis or a clinical pathology of sepsis, the method comprising the steps of: analysing an isolated biological sample from the patient for a concentration of a glycoform of CBG, wherein the glycoform of CBG has a triantennary glycan at asparagine (Asn) 347, wherein the concentration of the glycoform of CBG is indicative of the risk of sepsis or a clinical pathology of sepsis.

[0028] Specifically, in some embodiments, the concentration of the glycoform of CBG is inversely correlated to the risk of sepsis or a clinical pathology of sepsis. Specifically, a decrease in the concentration of the glycoform of CBG is indicative of a higher likelihood of sepsis, septic shock or a clinical pathology of sepsis or septic shock (such as those defined herein), while an increase concentration of the glycoform of CBG is indicative of a lower likelihood of sepsis, septic shock or a clinical pathology of sepsis or septic shock

[0029] As detailed above, a clinical pathology of sepsis is septic shock and therefore, in preferred embodiments, the above method predicts the likelihood of a patient experiencing septic shock or a clinical pathology of septic shock. Clinical pathologies of sepsis and septic shock that may be predicted by the method include: mortality, time in septic shock, time in first septic shock episode, time to septic shock onset, number of septic shock episodes, intubation time, requirement for mechanical ventilation, requirement for vasopressor therapy, requirement for inotrope therapy, requirement for renal replacement therapy, requirement for Extracorporeal membrane oxygenation (ECMO) therapy, requirement for antimicrobial therapy, vascular injury, capillary leakage, hypotension, hyperthermia, tachycardia, tachypnea, lactic acidosis, hypoxia, cyanosis, ileus, hyperglycemia, CNS dysfunction, sequential organ failure, multiorgan dysfunction syndrome (MODS), lung dysfunction, cardiac dysfunction,kidney dysfunction, and liver dysfunction. Preferably, the method predicts the likelihood of mortality of the patient and / or the likelihood of multiorgan dysfunction syndrome.

[0030] Specifically envisaged triantennary glycans at Asn347 are TS3 and / or TS3F.

[0031] In some embodiments of the method of prediction, the glycoform of CBG is a TS3F Asn347 glycoform and method predicts one or more of: the likelihood of the patient requiring mechanical ventilation, the duration of mechanical ventilation of the patient, or the sepsis severity of the patient.

[0032] To evaluate relative changes of CBG and glycoforms of CBG, the method of prediction may comprise comparing the concentration of the glycoform of CBG to a reference standard.

[0033] Some specifically envisaged ranges of serum concentration of the glycoform of CBG, having a triantennary glycan at Asn347, are about 60 nmol / L or less, of about 55 nmol / L or less, or about 50 nmol / L or less, or about 45 nmol / L or less, or about 40 nmol / L or less, or about 35 nmol / L or less, or about 30 nmol / L or less, or about 25 nmol / L or less, being indicative of a higher likelihood of sepsis or a clinical pathology of sepsis in a patient.

[0034] In some embodiments, a serum concentration of about 45 nmol / L or more of the glycoform of CBG, having a triantennary trisia lylated (TS3) glycan at Asn347, indicates a higher likelihood of survival.

[0035] In some embodiments, a serum concentration of about 30 nmol / L or less of the glycoform of CBG, having a triantennary trisia lylated (TS3) glycan at Asn347, indicates a higher likelihood of mortality.

[0036] In some embodiments, a serum concentration of a triantennary trisia lylated (TS3F) glycan at Asn347 of about 53 nmol / L or more indicates a lower likelihood of the patient requiring mechanical ventilation.

[0037] In some embodiments, a serum concentration of triantennary trisia lylated (TS3F) glycan at Asn347 of about 38 nmol / L or less indicates a higher likelihood of the patient requiring mechanical ventilation.

[0038] Means for measuring CBG include LC-MS / MS.

[0039] In embodiments of the present invention, the method of treating or preventing sepsis, or treating, preventing or delaying a clinical pathology of sepsis described herein includes performing the method of predicting the likelihood of a patient experiencing sepsis or a clinical pathology of sepsis as described herein.

[0040] In aspects of the present invention, there is provided a method of predicting the response of a subject to administration of a corticosteroid, comprises analysing an isolated biological sample from the subject for a concentration of a glycoform of corticosteroid-binding globulin (CBG). Preferably, a glycoform of CBG having a triantennary glycan at asparagine (Asn) 347.

[0041] In embodiments of the present invention, the method of treating or preventing sepsis, or treating, preventing or delaying a clinical pathology of sepsis described herein comprises administering the pharmaceutical composition as described herein.

[0042] Where the invention calls for quantification of CBG, or glycoforms of CBG, the biological sample may be a blood sample, serum sample or plasma sample.

[0043] Also provided in aspects of the invention is the pharmaceutical composition as described herein for use in the treatment or prevention of an immunological or inflammatory condition or sepsis, or for the treatment, prevention or delay of a clinical pathology of an immunological or inflammatory condition or sepsis.

[0044] Further provided, in aspects of the invention, is the pharmaceutical composition as described herein for use in the treatment or prevention of septic shock, or for use in the treatment, prevention or delay of a clinical pathology of septic shock, or for use in modulating the biological activity of a corticosteroid in a subject, or for use in a combination therapy with a corticosteroid in the treatment or prevention of an immunological or inflammatory condition or sepsis, or for use in the treatment, prevention or delay of a clinical pathology of an immunological or inflammatory condition or sepsis, or for use in a combination therapy with a corticosteroid (such as those disclosed herein) in the treatment or prevention of septic shock, or for use in the treatment, prevention or delay of a clinical pathology of septic shock.Brief Description of Drawings

[0045] Figure 1 shows the relative abundance of four asparagine (Asn) 347 glycoforms of corticosteroid binding globulin (CBG) in septic shock patients. The four glycoforms are biantennary disialylated (BS2), biantennary disialylated core-fucosylated (BS2F), triantennary trisialylated (TS3) and triantennary trisialylated core-fucosylated (TS3F) glycan.

[0046] Figure 2 shows the concentration of CBG having a triantennary trisialylated (TS3) glycan at Asn347 in septic shock patients in ICU who survived or succumbed to sepsis. Dotted line represents mean ± SEM. Septic shock non-survivors (LHC) had a mean concentration of 29.74 nmol / L and septic shock survivors (RHC) had a mean concentration of 45.16 nmol / L.

[0047] Figure 3A shows the concentrations of trianternnary trisialylated core-fucosylated (TS3F) Asn347 glycoforms of CBG and ICU mechanical ventilation requirement (Mean + / -SEM). Patients not ventilated (LHC) had a mean concentration of CBG of 52.6 nmol / L. Patients ventilated (RHC) had a mean concentration of CBG of 37.9 nmol / L. Figure 3B shows a negative correlation between the duration (hours) of ventilation and the concentration of TS3F Asn347 glycoform (r = Pearson's correlation coefficient).

[0048] Figure 4 shows septic shock severity (as indicated by SOFA scores) on day 1 of ICU admission and concentrations of CBG Asn347 glycoforms (r = Pearson's correlation coefficient).

[0049] Figure 5 shows correlation between Asn347 glycoforms and the percentage of neutrophil elastase (NE) cleaved CBG relative to total CBG in serum (%laCBG) (r = Pearson's correlation coefficient).

[0050] Figure 6 shows changes in the absolute concentrations of each CBG Asn347 glycoform from day 1 to the last day of ICU admission in septic shock patients.

[0051] Figure 7 shows the change in relative abundance of CBG Asn347 glycoforms from day 1 to the last day of ICU admission in septic shock patients (categorised as recovery, death or 7 days post admission).

[0052] Figure 8 shows that CBG therapy improves survival and increases circulating CBG levels. Figure 8A shows Kaplan-Meier survival over 96 hours in control CLP and CLP-CBG miceadministered different dosages of CBG (6, 30 hr administered vehicle, n=24), low- (2.5mg / kg at 6 hr; n=6), mid- (6 hr 3.5mg / kg at 6 hr and 2.5mg / kg at 30 hr; n=12) and high-dose CBG (6, 30 hr administered 5mg / kg; n=10). Figure 8B shows plasma CBG in control CLP mice and CLP-CBG mice administered low-, mid-, or high-dose CBG. Vertical dotted lines mark times of vehicle (control CLP) or CBG administration (CLP-CBG). n=10 control CLP, n=10 CLP-CBG mice. Light grey boxes represent the diurnal range of plasma CBG in the 48 hours preceding CLP. Vertical dotted lines indicate the time of administration of vehicle or CBG. Data are mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 (CLP-CBG vs. control CLP).

[0053] Figure 9 shows CBG therapy improves plasma lactate levels and decreases morbidity in CLP-sepsis. Figure 9A shows plasma lactate over 96 hours (shaded boxes represent the diurnal range of lactate in the 48 hours preceding CLP). Figure 9B shows changes in body weight over 96 hours. Figure 9C shows changes in cumulative disease index (CDI, i.e., unoperated mice score zero) over 96 hours; n=10 control CLP, n=10 CLP-CBG mice. Vertical dotted lines mark times of treatment with vehicle (control CLP, black) or CBG administration (CLP-CBG, grey). Data are mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 (CLP-CBG vs. control CLP).

[0054] Figure 10 shows CBG therapy improves haemodynamic recovery in CLP-sepsis. Figure 10 A shows mean arterial blood pressure (MAP) average in the control-CLP treatment group and CLP-CBG survivors treatment group over 96 hr. Figure 10B shows individual MAP records in CLP non-survivors (in-cage death, n=2 or early euthanasia, n=24 prior to 96 hr); n=10 control CLP, n=10 CLP-CBG. Vertical dotted lines mark times of vehicle (control CLP, black) or CBG administration (CLP-CBG, grey). Shaded region represents the averaged diurnal MAP range in the 48 hours before CLP.

[0055] Figure 11 shows CBG therapy improves thermoregulation in CLP-sepsis. Figure 11A shows the core temperature (°C) average in control-CLP and CLP-CBG survivors over 96 hours. Figure 11B shows individual temperature records in CLP non-survivors (in-cage death, n=2 or early euthanasia, n=24 prior to 96 hr); n=10 control CLP, n=10 CLP-CBG. Vertical dotted lines mark times of vehicle (control CLP, black) or CBG administration (CLP-CBG, grey). Shaded region represents averaged diurnal temperature range in the 48 hours before CLP.

[0056] Figure 12 shows CBG therapy attenuates total and free plasma free corticosterone in CLP-sepsis. Time course series plasma profiles of CBG (Figure 12 A, same as Figure 8A "Mid" CBG treatment), total concentration ("T-Cort" - Figure 12 B), and free corticosterone ("F-Cort" - Figure 12C) in control-CLP and CLP-CBG mice (each n=10 per timepoint) from 6 to 96 hr post-CLP. Vertical dotted lines mark times of vehicle (control CLP, black) or CBG administration (CLP-CBG, grey). Shaded region highlights averaged diurnal CBG, total or free corticosterone concentrations in age-matched, unoperated mice (n=6 each at 0, 6, 12, and 24 hr). Data are mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 (CLP-CBG vs control CLP).

[0057] Figure 13 shows CBG therapy transiently attenuates pro-inflammatory cytokines and results in a sustained elevation of anti-inflammatory cytokines in CLP-sepsis. Figures 13A to G provided a time course series of plasma profiles of interleukin (IL)-ip, IL-6, IL-12, macrophage inflammatory protein 2 (MIP-2), tumour necrosis factor a (TNFa), IL-10, and interferon (IFN)-pi (Figures 13A to G, respectively) in control and CBG treated CLP mice (each n=10 per timepoint) from 6 to 96 hours post-CLP. Vertical dotted lines mark times of vehicle (control CLP, black) or CBG administration (CLP-CBG, blue). Shaded region highlights averaged diurnal IL-ip, IL-6, IL-12, MIP-2 orTNFa concentrations in age-matched, unoperated mice. Data are mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 (CLP-CBG vs control CLP).

[0058] Figure 14 shows CBG therapy stabilises plasma albumin and lowers plasma lactate in CLP-sepsis. Figures 14 A and B provide a time course series plasma profiles of albumin (Figure 14A) and lactate (Figure 14B) in control treated and CBG treated CLP mice (each n=10 per timepoint) from 6 to 96 hours post-CLP. Vertical dotted lines mark times of vehicle (control CLP, black) or CBG administration (CLP-CBG, blue). Shaded region indicates averaged diurnal albumin or lactate concentration in age-matched, unoperated mice. Data are mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 (CLP-CBG vs control CLP).

[0059] Figure 15 shows a time course series plasma profiles of cystatin-C (Figure 15A), troponin-l (Figure 15B), alanine aminotransferase (ALT) (Figure 15C), and aspartate aminotransferase (AST - Figure 15D) in control CLP and CLP-CBG mice (each n=10 per timepoint) from 6 to 96 hr post-CLP. Vertical dotted lines mark times of vehicle (control CLP, black) or CBG administration (CLP-CBG, blue); Light grey region highlights averaged diurnalcystatin-C, troponin-l, ALT or AST concentration in age-matched, unoperated mice. Data are mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 (CLP-CBG vs control. CLP).Detailed Description

[0060] Before describing the present invention in detail, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and the invention is not intended to be limited to the detailed embodiments.

[0061] The present disclosure relates to a method of treating or preventing sepsis, or treating, preventing or delaying a clinical pathology of sepsis, the method comprising administering to a patient an effective amount of a corticosteroid-binding globulin (CBG).

[0062] As used herein, the terms "treating", "treat" or "treatment" include intervening (for example administering a therapeutic agent) to thereby reduce or eliminate at least one symptom of a specified disease or to slow progression of the disease.

[0063] As used herein, the term "preventing", "prevent" or "prevention" includes providing prophylaxis with respect to occurrence or recurrence of a specified disease. An individual may be predisposed to or at risk of developing the disease or relapse but has not yet been diagnosed with the disease or the relapse.

[0064] An "effective amount" refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired result. For example, the desired result may be a therapeutic or preventative result. An effective amount can be provided in one or more administrations. In some embodiments of the present disclosure, the term "effective amount" is meant an amount necessary to effect treatment or prevention of a disease as described herein. In some embodiments of the present disclosure, the term "effective amount" is meant an amount necessary to treat or prevent sepsis or septic shock. The effective amount may vary according to the disease to be treated or factor to be altered and according to the weight, age, racial background, sex, health and / or physical condition and other factors relevant to the subject being treated. Typically, the effective amount will fall within a relatively broad range (e.g., a "dosage" range) that can be determined through routine trial and experimentation by a medical practitioner. Accordingly, this term is not to be construed to limit the disclosure toa specific quantity. The effective amount can be administered in a single dose or in a dose repeated once or several times over a treatment period and / or in combination with another therapeutic agent.

[0065] As used herein a "patient" of "subject" includes a living human or non-human subject that is receiving medical care or that should receive medical care due to a disease or condition. This includes subjects with no defined illness or observable symptoms of a specified disease or condition who are being investigated for signs of pathology. In some embodiments, and in the context of sepsis or septic shock, the patient or subject has a risk factor for sepsis or septic shock, preferably a diagnosed or suspected infection. Additionally, the patient or subject may have one or more factors including a fever or low temperature (hyper- or hypothermia), confusion, difficulty breathing or rapid breathing (tachypnoea), clammy and sweaty skin, extreme or excessive body pain or discomfort, high heart rate (tachycardia), weak pulse or low blood pressure (hypotension), lethargy, listlessness, or low urine output, or any other diagnostic factor defined herein.

[0066] Corticosteroid-binding globulin (CBG) is a heavily glycosylated 50-60 kDa protein belonging to the serpin family and is the primary binding molecule for cortisol. Corticosteroid-binding globulin (UniProt accession number P08185, also known as Serpin A6 and Transcortin) is encoded by the SERPINA6 gene (HGNC accession number 1540) and, in humans, is a secreted 405 amino acid long glycoprotein. It is the major systemic transporter protein for glucocorticoids and progestins in vertebrates and binds aldosterone, and 11-deoxycorticosterone. It is primarily produced by the liver and is up-regulated by estrogens and down-regulated by steroids. When bound to CBG, cortisol is maintained in a biologically inactive state. As such, CBG provides a reservoir from which cortisol can be released when required.

[0067] Eighty percent of circulating cortisol is bound to CBG, while approximately 10-15% is bound to serum albumin, and the remaining cortisol is free and active cortisol. The affinity of CBG for cortisol, and its resultant ability to bind and transport cortisol, is altered by a number of factors such as temperature, pH and cleavage by enzymes. Accordingly, spatiotemporal distribution and tissue availability of cortisol can be modified by local variations in theseparameters. This allows for CBG to bind cortisol under homeostatic conditions and release free cortisol in response to physiological or pathological signals, such as inflammation. As a result, CBG is a critical component in the in vivo biological activity of cortisol and importantly transportation and release of cortisol at target tissues and sites of inflammation.

[0068] CBG binds cortisol at a 1:1 molar ratio with high affinity (Ka 76 x 106l / mol). Surface plasmon resonance (SPR) studies have demonstrated that a temperature increase from 37°C to 39°C and a reduction in pH from 7.4 to 7.0, results in a 3.5-fold reduction in the affinity of CBG for cortisol, increasing KD from 214.4 to 740.1 l / mol. Additionally, cleavage of CBG by neutrophil elastase (NE) at a region called reactive centre loop (RCL) also results in a 9-fold reduction in cortisol binding affinity via a reversable structural alteration. Moreover, production of CBG is reduced by cytokines including TNFa, IL-1 and IL-6. All of these factors reduce the affinity of CBG for cortisol and as such are conducive to delivery of free cortisol in response to local or systemic inflammation where there are localised increases in temperature, reductions of pH, inflammatory cytokines and NE producing neutrophils.

[0069] As demonstrated in the Examples, the Inventors have shown for the first time that CBG administration can prevent negative clinical outcomes in inflammatory conditions, such as septic shock. This implements CBG as an active modulator of inflammation and offers new treatments for a range of immunological or inflammatory conditions, including life threatening conditions such as sepsis and septic shock.

[0070] Corticosteroid binging globulin has six conserved N-glycosylation sites, of which five are occupied on average, increasing its molecular weight from 40 kDa to 50-60 kDa. CBG N-glycosylation overall displays primarily bi- and tri-antennary branching and terminate with a2,3-linked neuraminic acid (NeuAc) residues. Of these sites, the present inventors have identified that N-glycosylation at asparagine (Asn) 347 appears to correlate with outcomes in response to inflammatory disorders, such as sepsis and septic shock.

[0071] Without wishing to be bound by theory, the Asn347 site lies within the reactive centre loop (RCL) of the CBG glycoprotein, only three amino acids away from the neutrophil elastase (NE) cleavage site. Accordingly, glycosylation of Asn374 can modulate cleavage of the RCL by NE and therefore influence cortisol binding to CBG and consequently biological activityof cortisol. Furthermore, while this is one likely mechanism for CBG glycosylation affecting biological activity of cortisol, (and as shown in the Examples herein) NE cleavage of CBG (as defined by %laCBG) is low. Therefore, the Inventors hypothesize that glycosylation at Asn347 can reduce the affinity of CBG for cortisol by a currently unknown mechanism, independent of NE cleavage.

[0072] The Inventors have demonstrated that the abundance of CBG as well as specific glycosylated forms of CBG (namely triantennary glycosylation of Asn347) provide prognostic markers for immunological or inflammatory conditions such as sepsis and septic shock. As such, the Inventors propose that quantification of glycoforms of CBG can inform clinical treatment of immunological or inflammatory conditions. Moreover, the Inventors propose that in addition to utilising CBG to treat immunological or inflammatory conditions, using composition comprising glycoforms of CBG comprising a triantennary glycosylation at Asn347 would lead to improved clinical outcomes for patient suffering from immunological or inflammatory conditions, in particular patients suffering from sepsis and septic shock.

[0073] The skilled person would understand that "sepsis" is an acute, systemic and lifethreatening organ dysfunction caused by a dysregulated inflammatory response to an infection such as a bacterial or viral infection.

[0074] Diagnostic criteria for sepsis is known in the art, including systemic inflammatory response syndrome (SIRS) criteria, vital signs, signs of infection, quick Sequential Organ Failure Score (qSOFA), or Sequential Organ Failure Assessment (SOFA) criteria, National Early Warning Score (NEWS), Modified Early Warning Score (MEWS), and blood lactate (see Evans L, Rhodes A, Alhazzani W, et al. Surviving sepsis campaign: international guidelines for management of sepsis and septic shock 2021. Intensive Care Med. 2021;47(ll):1181-1247. doi:10.1007 / s00134-021-06506-y).

[0075] Organ dysfunction, defined as an increase of 2 points or more in the Sequential Organ Failure Assessment (SOFA) score, in combination with an infection, results in an overall mortality rate of 10% in sepsis affected patients.

[0076] "Septic shock" as used herein is a severe form of sepsis exhibited by a sepsis patient for a period of time. Septic shock can be defined by the co-existence of persistent hypotension requiring vasopressors to maintain mean arterial pressure >65 mmHg; and serum lactate >2 mmol / L. With these criteria, the hospital mortality rate is in excess of 40%.

[0077] Sepsis or septic shock can result in one or more clinical pathologies. A clinical pathology is considered to be any adverse outcome that arises as a result of sepsis or septic shock. Some clinical outcomes are acute and resolve upon recovery while others are chronic. Various clinical pathologies of sepsis and septic shock are known in the art (see Mahapatra S, Heffner AC. Septic Shock. [Updated 2023 Jun 12]. In: StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2024 Jan; and Singer M, Deutschman CS, Seymour CW, et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA.2016;315(8):801-810). In some embodiments the clinical pathology is selected from one or more of the following: mortality, time in septic shock, time in first septic shock episode, time to septic shock onset, number of septic shock episodes, intubation time, requirement for vasopressor therapy, requirement for inotrope therapy, requirement for renal replacement therapy, requirement for Extracorporeal membrane oxygenation (ECMO) therapy, requirement for antimicrobial therapy, vascular injury, capillary leakage, hypotension, hyperthermia, tachycardia, tachypnea, lactic acidosis, hypoxia, cyanosis, ileus, hyperglycemia, CNS dysfunction, sequential organ failure, multiorgan dysfunction syndrome (MODS), lung dysfunction, cardiac dysfunction, kidney dysfunction, and liver dysfunction.

[0078] In some embodiments, the clinical pathology in a treated subject or treated subject group is improved by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% relative to an untreated subject, or untreated subject group.

[0079] It is to be understood that a reduction in the requirement for a specific therapy or treatment includes a reduction in the time of administering the therapy (for example a treatment or prevention of the requirement for mechanical ventilation or renal replacementtherapy includes a reduction in the duration of mechanical ventilation or renal replacement therapy, or ECMO), a reduction in the number of patients in a population treated with CBG requiring the therapy, or a reduction in the extent of the therapy (for example a reduced dose of a vasopressor or inotrope).

[0080] In some embodiments, the mortality rate is reduced in a treated patient population having had CBG administered, compared to an untreated, control, patient population. In some embodiments, the mortality rate can be reduced by up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, or up to about 10% as compared to a population not treated in accordance with the invention. In some embodiments, the mortality rate of a patient population suffering from sepsis and having had administration of CBG is about 1% or less, about 2% or less, about 3% or less, about 4% or less, about 5% or less, about 6% or less, about 7% or less, about 8% or less, about 9% or less, or about 10% or less. In some embodiments, the mortality rate of a patient population suffering from septic shock and having had administration of CBG is about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, or about 40% or less.

[0081] In some embodiments, mortality rate, of the patient population is measured during a time post CBG administration. In some embodiments, the time post-administration of CBG time is up to about 96 hours, up to about 72 hours, up to about 48 hours or up to about 24 hours.

[0082] In some embodiments, the time in septic shock is defined as the time spent in a hypotensive event. In some embodiments, the hypotensive event is defined by the time period between an initial drop in Mean Arterial Pressure (MAP) and a subsequent return to a control MAP or the prior MAP.

[0083] In some embodiments, the duration of time spent in a first, second or further septic shock is reduced in a treated patient having had CBG administered compared to an untreated patient without having had CBG administered. In some embodiments, the duration of time spent in a first septic shock by a patient is reduced by up to about 30%, up to about 35%, up to about 40%, up to about 45%, up to about 50%, up to about 55%, or up to about 60%. In someembodiments, the duration of time spent in a first septic shock of patients not treated with CBG is between about 5 and about 9 hours, between about 6 and about 8 hours, or about 7 hours. In some embodiments, the duration of time spent in a first septic shock episode in a treated patient is between about 1.5 and about 5.5 hours, between about 2.5 and about 4.5 hours or about 3.5 hours, or is less than 5.5 hours, or is less than 5 hours, or is less than 4.5 hours, or is less than 4 hours, or is less than 3.5 hours, or is less than 3 hours, or is less than 2.5 hours, or is less than 2 hours, or is less than 1.5 hours, or is less than 1 hour, or less than 30 minutes.

[0084] In some embodiments, the cumulative time spent in septic shock (i.e., the sum of a first, second and / or further septic shock episode) is reduced in a treated patient or patient population having had CBG administered compared to an untreated patient or patient population without having had CBG administration. In some embodiments, the cumulative time spent in septic shock is reduced by up to about 30%, up to about 35%, up to about 40%, up to about 45%, up to about 50%, up to about 55%, or up to about 60%.

[0085] In some embodiments, the time to first septic shock is in a treated patient or patient population is increased by about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 110% or more, about 120% or more, about 130% or more, about 140% or more, about 150% or more, about 160% or more, about 170% or more, about 180% or more, about 190% or more, or about 200% or more compared to an untreated patient or patient population.

[0086] In some embodiments, the number of septic shock episodes are reduced in a treated patient population by an average of about 0.5 events or more, about 1 event or more, about 1.5 events or more, about 2 events or more, or about 2.5 events or more compared to an untreated patient population. In some embodiments, the treated patients have, on average, less than about 0.5 events, less than about 1 event, less than about 1.5 events, less than about 2 events, less than about 2.5 events, or less than about 3 events.

[0087] In some embodiments, the method treats or prevents multiorgan dysfunction syndrome (MODS). MODS is an acute condition that can be brought about during sepsis orseptic shock where two or more organs do not function in a typical manner. The condition often requires immediate medical attention and ICU admission. The organs commonly affected include lungs, heart, brain, kidneys, liver, or blood. In some embodiments, the rate of organ failure, or multiple organ failure, in a treated subject group is improved by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% relative to an untreated subject group.

[0088] In some embodiments, sepsis or septic shock can be measured by one or more prognostic indicators and the method modulates the one or more prognostic indicator. In some embodiments, the prognostic indicator is selected from: increased serum lactate, reduced bicarbonate, increased pH, elevated serum creatinine, reduced eGFR, reduced serum albumin, increased bilirubin, reduced platelet count, elevated international normalised ratio, prolonged prothrombin time, elevated D-Dimer, elevated C-reactive protein, elevated interleukin-6, elevated interleukin-8, reduced interleukin-10, elevated monocyte chemoattractant protein 1, elevated presepsin, elevated pro-adrenomedullin, elevated procalcitonin, elevated cystatin-C, elevated troponin, elevated alanine transaminase, elevated aspartate transaminase, elevated tumour necrosis factor alpha, total CBG less than 200 nmol / L, or Sequential Organ Failure Assessment (SOFA) score.

[0089] SOFA score is a numerical scoring system, ranging from 0 to 24 (with scores of 0 to 4 for six organs / systems, being; respiratory, coagulation, liver, circulatory, central nervous system, and renal), used to predict clinical outcomes in patents. Higher scores are indicative of an increased likelihood of a patient with an infection developing sepsis and / or septic shock. In addition, higher scores are indicative of a patient requiring ICU treatment, requiring ventilation and / or ultimate mortality. The scoring system assesses several of the organs as well as other identifiers in a patient including blood pressure, blood indicators (for example platelet levels), neurologic indicators (for example the Glasgow Coma Score), respiratory performance (PaCh / FiCh mm Hg), liver performance (bilirubin mg / dL) and / or kidney performance (creatinine mg / dL) (see Vincent JL, Moreno R, Takala J, Willatts S, De Mendonca A, Bruining H,et al. The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction / failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine. Intensive Care Med. 1996;22:707-710). An increase in SOFA score in the first 96 hrs after hospital admission indicates at least a 50% chance of mortality while no change in score indicates approximately a one in three chance of mortality, and a decreased score indicates approximately a one in four change of mortality. Accordingly, in some embodiments, the method of prevention or treatment causes the score to remain unchanged over a period of 24 hours, 48 hours, 72 hours or 96 hours post treatment. In some embodiments, the method of prevention or treatment causes the score to reduce over a period of 24 hours, 48 hours, 72 hours or 96 hours post treatment.

[0090] In some embodiments, SOFA score is reduced in a treated patient or patient population compared to an untreated patient or patient population. In some embodiments, the SOFA score is reduced by an average of about 2.0 or more, about 4.0 or more, about 6.0 or more, about 8.0 or more, about 10.0 or more, about 12.0 or more, about 14.0 or more, about 16.0 or more, about 18.0 or more, about 20.0 or more, or about 22.0 or more in a treated subject group compared to an untreated subject group. In some embodiments, the SOFA score is reduced by about 2.0 or more, about 4.0 or more, about 6.0 or more, about 8.0 or more, about 10.0 or more, about 12.0 or more, about 14.0 or more, about 16.0 or more, about 18.0 or more, about 20.0 or more, or about 22.0 or more in a subject post-treatment compared to prior to treatment. In preferred embodiments, the SOFA score is reduced by an average of about 1.0 or more, about 2.0 or more, about 3.0 or more, about 4.0 or more, about 5.0 or more, about 6.0 or more, about 7.0 or more, about 8.0 or more, about 9.0 or more, about 10.0 or more, about 11.0 or more, or about 12.0 or more in a treated subject group compared to an untreated subject group. In preferred embodiments, the SOFA score is reduced by about 1.0 or more, about 2.0 or more, about 3.0 or more, about 4.0 or more, about 5.0 or more, about 6.0 or more, about 7.0 or more, about 8.0 or more, about 9.0 or more, about 10.0 or more, about 11.0 or more, or about 12.0 or more in a subject post-treatment compared to prior to treatment.

[0091] In some embodiments of the method of treating or preventing sepsis or septic shock, or treating, preventing or delaying a clinical pathology of sepsis or septic shock, themethod comprising administering to a patient an effective amount of a CBG, wherein the CBG comprises a CBG glycoform having a triantennary glycan at Asn347. In some embodiments, the triantennary glycan at Asn347 is selected from triantennary trisialylated (TS3) and / or triantennary trisialylated core-fucosylated (TS3F) glycan.

[0092] The present disclosure also relates to a method of modulating the biological activity of a corticosteroid in a subject, the method comprising administering an effective amount of a corticosteroid-binding globulin (CBG). In some forms, the CBG includes a triantennary glycan at Asn347. Without wishing to be bound by theory, due to the effect on binding affinity of the corticosteroid caused by the triantennary glycan at Asn347, the Inventors expect the biological activity of corticosteroid to be modulated by the CBG. Currently, in the absence of a pathology such as a mutated form or CBG, it is not considered that CBG supplementation could be beneficial in improving corticosteroid biological activity. However, the Inventors have shown that CBG supplementation can treat immunological or inflammatory conditions, with the proposed mechanism of action being improved biological activity of corticosteroids.

[0093] In some embodiments, the corticosteroid is endogenous. In some embodiments, the corticosteroid is exogenous and administered to the patient. In some preferred embodiments, the corticosteroid can be selected from: betamethasone, budesonide, ciclesonide, corticosterone, corticosterone acetate, fludrocortisone, hydrocortisone, methylprednisolone, dexamethasone, prednisolone, prednisone or mixtures thereof. In some embodiments, the corticosteroid is a glucocorticoid.

[0094] The present invention also provides a method of treating a corticosteroid-associated condition, the method comprising administering to a patient an effective amount of a corticosteroid-binding globulin (CBG). In preferred embodiments, the CBG comprises a CBG glycoform having a triantennary glycan at Asn347, for example TS3 and / or TS3F.

[0095] A corticosteroid-associated condition is any condition in which a deficiency in corticosteroid is implicated in the pathology of the condition, or in which corticosteroid administration is indicated. In some embodiments the indicated corticosteroid is a glucocorticoid.

[0096] The present invention also provides a method of treating or preventing an immunological or inflammatory condition, the method comprising administering to a patient an effective amount of a corticosteroid-binding globulin (CBG). In some embodiments, the CBG comprises a CBG glycoform having a triantennary glycan at Asn347, for example TS3 and / or TS3F.

[0097] An "immunological condition" is defined as any condition or pathology in a subject that results from the action of the immune system. Typically, immunological conditions can result from an aberrant or unregulated immune response to a stimulus. Such stimuli may be an exogenous antigen or may be an endogenous antigen or an aberrant response to a "self" antigen.

[0098] An "inflammatory condition" is an immune response defined by an increase in local or systemic immune activity. Inflammatory conditions can be defined by a variety of factors including elevated immune cell numbers (locally and / or systemically), secretion or one or more inflammatory cytokines including interleukin (IL) 1, IL-lb, IL-2, IL-12, IL-18, Tumour Necrosis Factor alpha (TNF-a), Interferon gamma (IFN-g), and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), as well as biomarkers such as C-reactive protein, procalcitonin and calprotectin (see Germolec DR, Shipkowski KA, Frawley RP, Evans E. Markers of inflammation. In: DeWitt J, Rockwell C, Bowman C, eds. Immunotoxicity Testing. Methods in Molecular Biology. Vol 1803. New York, NY: Humana Press; 2018).

[0099] In some embodiments, the immunological or inflammatory condition can be acute, chronic, localized, or systemic (for example a cytokine storm). Acute inflammation is initiated following a specific injury or immunological challenge such as an infection, and results in triggering the release of soluble mediators such as cytokines, acute phase proteins, and chemokines. If inflammation does not quickly resolve it transitions to subacute inflammation. After persistent inflammation for six weeks or more, it transitions to chronic inflammation.

[0100] Sepsis and septic shock are specific forms of acute systemic inflammation mediated by an inappropriately controlled "cytokine storm" resulting from immune system hyperactivity.

[0101] In some embodiments, the CBG used in the methods of treatment and prevention is isolated. CBG can be isolated from biological sources such as blood, serum or plasma. The blood, serum or plasma can be derived from a suitable animal. The skilled person will understand that isolation can be performed using understood means disclosed in the art.

[0102] The terms "isolation" and "enrichment" when used in reference to CBG, or glycoforms of CBG, relates to an increase in the concentration of CBG, or the specified glycoform of CBG, relative to before the process of isolation or enrichment.

[0103] In some embodiments, CBG can be isolated by immunoprecipitation using suitable antibodies (for example the 12G2 monoclonal antibody). In some embodiments, CBG may be isolated or enriched from biological samples (such as serum or plasma) using fractionation techniques such as sample displacement chromatography, affinity chromatography and Cohn Fractionation (see Srajer Gajdosik M, Clifton J, Josie D. Sample displacement chromatography as a method for purification of proteins and peptides from complex mixtures. J Chromatogr A.2012;1239:1-9; Rosner W, Bradlow HL. Purification of corticosteroid-binding globulin from human plasma by affinity chromatography. J Clin Endocrinol Metab. 1971;33(2):193-198.; and Burnouf T. An overview of plasma fractionation. Ann Blood 2018;3:33). In some embodiments, CBG is enriched of isolated from pooled plasma samples. In preferred embodiments, the CBG is isolated or enriched by Cohn fractionation.

[0104] In some embodiment, the CBG is recombinant. Preferably, the recombinant CBG is human CBG. Preferably, the recombinant CBG is produced in mammalian cells, such as a human cell line. However, alternative cell lines such as insect cell lines, or prokaryotic cell lines may be suitable. Due to the importance of glycosylation, the selection of the appropriate cell line may influence the efficacy and potency of the recombinant CBG for treating inflammatory or immunological conditions such as sepsis and septic shock. Therefore, the types and abundance of glycoforms of GBC may be assessed in the recombinant CBG, with a particular focus on triantennary forms of Asn347 (preferably TS3 and / or Ts3F).

[0105] In some embodiments, the isolated or recombinant CBG can be enriched for a CBG glycoform having a triantennary glycan at Asn347, for example TS3 and / or TS3F. Such enrichment will result in an elevation of the specific glycoforms within a sample. Thisenrichment may be relative to the concentration of any one or more of the glycoforms prior to the enrichment process. In some preferred forms, the ratio of CBG glycoforms having a triantennary glycan at Asn347 is increased relative to other glycoforms of CBG in the sample, for example biantennary glycans at Asn347 (such as BS2 and BS2F). This can be achieved by either selecting for the desired glycoforms or depleting a sample of undesired glycoforms. Such enrichment may be as a result of isolation or purification of the GBC (for example from cells or cell supernatant or serum or plasma) or may be performed after isolation or purification.

[0106] Enrichment can be measured by methods disclosed herein and as understood in the art. The Inventors expect that any increase in absolute values of the triantennary glycan at Asn347 (for example TS3 and / orTS3F) is likely to improve the ability of the enriched / isolated CBG to bind to corticosteroids thereby leading to favourable clinical outcomes when administered.

[0107] Enrichment of specific glycoforms can be measured by mean relative abundance and as described herein. The mean relative abundance of the triantennary glycan at Asn347 prior to enrichment can be from about 10% to about 70%, from about 20% to about 60% or from about 30% to about 50%. The mean relative abundance of the TS3 glycoform can be from about 5% to about 35%, from about 10% to about 30% or from about 15% to about 25%. For example, the mean relative abundance of the TS3F glycoform can be from about 5% to about 35%, from about 10% to about 30% or from about 15% to about 25%. In some embodiments, the mean relative abundance is from about 10%, or from about 15%, or from about 20%, or from about 25%, or from about 30%, or from about 35%, or from about 40%, or from about 45%, or from about 50%, or from about 55%, or from about 60%, or from about 65%, or from about 70%, or from about 75%, or from about 80%, or from about 85%, or from about 90%, or from about 95%.

[0108] Appropriate dosing schedules and regimens can be determined based on standard clinical trials. However, in some embodiments, the effective amount of the CBG is administered in a dosage schedule comprising at least two doses or is continuous infusion. In some embodiments, CBG is administered in a dosage schedule comparing at least two doses. In some embodiments, CBG is administered in a dosage schedule comparing at least three doses. Insome embodiments, CBG is administered in a dosage schedule comparing at least four doses. In some embodiments, CBG is administered in a dosage schedule comparing at least five doses. The doses can be administered at time intervals to provide the most effective clinical outcome.

[0109] In some embodiments, the CBG is administered at a dose of between approximately 30 to 180 mg (approximately 420 pg / kg to 2.5 mg / kgfor a 70kg person). In some embodiments, the CBG is administered at a dose of between approximately 50 and 160 mg (approximately 714 pg / kg to 2.3 mg / kg for a 70kg person). In some embodiments, the CBG is administered at a dose of between approximately 70 and 140 mg (approximately 1 mg / kg to 2 mg / kg for a 70kg person).

[0110] In some embodiments, CBG can be administered by understood suitable modes of administration. For example, injection by intravenous, intramuscular, subcutaneous, intraosseous or intradermal routes.

[0111] The present disclosure also relates to a combination therapy comprising administration of a corticosteroid in combination with the CBG. The corticosteroid can be selected from the following betamethasone, budesonide, ciclesonide, corticosterone, corticosterone acetate, fludrocortisone, hydrocortisone, methylprednisolone, dexamethasone, prednisolone, prednisone or mixtures thereof. In some embodiments, the corticosteroid is a glucocorticoid.

[0112] A "combination therapy" as defined herein relates to the administration of the two components, namely CBG and a corticosteroid, in a dosing schedule or form that ensures that the two components have a working inter-relationship and interact in vivo. This may be achieved by simultaneous or sequential administration of the two components. Further, it may be achieved by separate administration of the two components so long as the first administered component is active at the time of administration of the second component. The determination of such timing is within the purview of a person skilled in the art based on knowledge of the pharmacokinetics of the components.

[0113] The skilled person would understand an in vivo interaction between the corticosteroid and CBG can be expected to occur during the combination therapy. Forexample, and without wishing to be bound by theory, at least due to the modulation of biological activity of the corticosteroid by CBG. The combination therapy can comprise simultaneous, sequential and / or separate administration of the corticosteroid and the CBG.

[0114] In some embodiments, the method is informed by the concentration of CBG, or glycoforms of CBG, within a patient, with a decrease in CBG below a reference standard indicating supplementation of CBG or a CBG glycoform. Accordingly, in some embodiments, the method comprises a step of quantifying the concentration of total CBG or a glycoform of CBG in a biological sample from the patient. In some embodiments, the glycoform of CBG comprises a triantennary glycan at Asn347. In some embodiments, the triantennary glycan at Asn347 is TS3 and / or TS3F. Total CBG, or glycoforms thereof, can be measured by understood methods in the art. For example, a CBG rapid flow test.

[0115] The present disclosure also relates to a pharmaceutical composition comprising isolated corticosteroid-binding globulin (CBG), a recombinant CBG, or an in vitro produced CBG. In some embodiments, the CBG comprises a triantennary glycan at Asn347 of CBG. In some embodiments, the triantennary glycan is core-fucosylated or trisialylated. In some embodiments, the pharmaceutical composition comprises CBG enriched for CBG glycoforms having a triantennary glycan at Asn347, preferably TS3 and / or TS3F. As detailed above, this enrichment may be defined as an enrichment relative to prior to the enrichment process or may be an enrichment of specifically desired glycoforms relative to other glycoforms or prior to the enrichment process.

[0116] In some embodiments, the pharmaceutical composition or recombinant CBG comprises a relative abundance of about 30% or more, or about 32% or more, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, or 60% of CBG having a triantennary glycan at Asn347

[0117] In some embodiments, the isolated CBG is a non-human CBG. For example, the CBG can be derived from non-human animals including mammals such as pigs, horses or avians such as chickens.

[0118] In some embodiments, the isolated CBG comprises a serum or plasma fraction of CBG. In some embodiments, the serum or plasma fraction of CBG is enriched for a triantennary glycan at Asn347. Serum or plasma fractionation can be conducted by understood methods in the art and as discussed herein, for example by Cohn fractionation and / or chromatography.

[0119] In some embodiments, the pharmaceutical composition can comprise a corticosteroid. Suitable corticosteroids may be selected from: betamethasone, budesonide, ciclesonide, corticosterone, corticosterone acetate, fludrocortisone, hydrocortisone, methylprednisolone, dexamethasone, prednisolone, prednisone or mixtures thereof. In some embodiments, the corticosteroid is a glucocorticoid.

[0120] The pharmaceutical composition can comprise additional components selected from a solvent, an excipient, a stabilizer, an adjuvant, a buffer, or mixtures thereof.

[0121] In some embodiments, the pharmaceutical composition as described herein can be for use in the treatment or prevention of sepsis, or for the treatment, prevention or delay of a clinical pathology of sepsis. In some embodiments, the pharmaceutical composition for use can be for use in the treatment or prevention of septic shock, or for use in the treatment, prevention or delay of a clinical pathology of septic shock. In some embodiments, the pharmaceutical composition for use can be for use in the treatment or prevention of septic shock, or for use in the treatment, prevention or delay of a clinical pathology of septic shock. In some embodiments, the pharmaceutical composition as described herein can be for use in the treatment or prevention of an immunological or inflammatory condition, or for the treatment, prevention or delay of a clinical pathology of immunological or inflammatory condition. In some embodiments, the pharmaceutical composition as described herein is for use in modulating the biological activity of a corticosteroid in a subject. In some embodiments, the pharmaceutical composition as described herein can be for use in a combination therapy with a corticosteroid in the treatment or prevention of sepsis, or for the treatment, prevention or delay of a clinical pathology of sepsis. In some embodiments, the pharmaceutical composition as described herein can be for use in a combination therapy with a corticosteroid in the treatment or prevention of septic shock, or for the treatment, prevention or delay of a clinical pathology of septic shock. In some embodiments, the pharmaceutical composition asdescribed herein can be for use in a combination therapy with a corticosteroid in the treatment or prevention of an immunological or inflammatory condition, orforthe treatment, prevention or delay of a clinical pathology of an immunological or inflammatory condition.

[0122] In some embodiments, the corticosteroid is one or more of: betamethasone, dexamethasone, fludrocortisone, ciclesonide, corticosterone, corticosterone acetate, hydrocortisone, methylprednisolone, prednisolone or prednisone.

[0123] In some embodiments, the present disclosure relates to a method of predicting the likelihood of a patient experiencing sepsis or septic shock, or a clinical pathology of sepsis or septic shock. The method comprises the steps of analysing an isolated biological sample from the patient for a concentration of a glycoform of CBG. In some forms, the glycoform of CBG has a triantennary glycan at Asn347.

[0124] The Inventors have shown for the first time that the concentration of the glycoforms of CBG within the isolated biological sample can be indicative of the risk of sepsis or septic shock, or a clinical pathology of sepsis or septic shock. Specifically, it has been shown that the concentration of glycoforms of CBG with a triantennary glycan at Asn347 are indicative of the risk or sepsis or septic shock, or a clinical pathology of sepsis or septic shock. Preferably, the glycan at Asn347 is TS3 and / or TS3F. In some embodiments, the absolute concentration of the glycoform of CBG is inversely correlated to the risk of sepsis or a clinical pathology of sepsis. The skilled person would understand that higherabsolute concentration of glycoforms of CBG, such as a triantennary glycan at Asn347 (preferably TS3 or TS3F), is correlated with, or indicates, a reduced risk of experiencing severe sepsis or a clinical pathology of sepsis. Conversely, lower absolute concentration of the glycoforms of CBG is correlated with, or indicates, an increased risk of sepsis.

[0125] Methods are known in the art for determining appropriate thresholds for diagnostic and prognostic markers. Such methods can be used in clinical studies to determine appropriate thresholds and parameters for determining if a patient will be likely or unlikely to experience sepsis, septic shock or a clinical pathology of sepsis or septic shock based on the concentration of a glycoform of CBG. For example, following quantification of the glycoforms of CBG in relevant samples, the concentration in the samples can be compared to referencestandards by any appropriate statistical analysis. Methods of statistical analysis are known in the art, and the appropriate method will be determined based on many factors including (but not limited to) the number of samples collected, the number of markers being analysed and the nature of the samples (i.e., paired or unpaired). Appropriate statistical analysis techniques include negative binomial models, pair-wise comparison and Bayesian approaches, and receiver operating characteristics (ROCs). Programs and statistical frameworks for performing such analysis include edgeR, DESeq, baySeq, EBSeq, limma-voom, QPROT and maSigPro.

[0126] Methods for performing statistical analysis are provided in: Yunshun Chen et al. (2021), edgeR: differential analysis of sequence read count data User's Guide; Bergemann, T.L., and Wilson, J. (2011), Proportion statistics to detect differentially expressed genes: a comparison with log-ratio statistics. BMC Bioinformatics 12, 228; Hardcastle, TJ. and Kelly, K.A. (2010) baySeq: Empirical Bayesian methods for identifying differential expression in sequence count data. BMC Bioinformatics 11, 422; Leng N. et al., (2013), EBSeq: an empirical Bayes hierarchical model for inference in RNA-seq experiments, Bioinformatics, 29, 8, p.1035-1043; Law, C.W., et al. (2014), Voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol 15, R29 (2014); Conesa A. et al. (2006), maSigPro: a method to identify significantly differential expression profiles in time-course microarray experiments, Bioinformatics, 22, 9, p. 1096-1102; Parodi, S et al. (2008), Not proper ROC curves as new tool for the analysis of differentially expressed genes in microarray experiments. BMC Bioinformatics 9, 410; and Gordon S., et al. (2015) QPROT: Statistical method for testing differential expression using protein-level intensity data in label-free quantitative proteomics. J Proteomics. 3;129:121-126.

[0127] In some embodiments, the isolated biological sample is a blood sample, serum sample or plasma sample.

[0128] Various methods for detecting the glycosylation status of proteins (glycomic analysis) and quantification are known in the art from both isolated glycoproteins and complex mixtures of glycoproteins (see Rudd PM, Karlsson NG, Khoo KH, et al. Glycomics and glycoproteomics. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology.4th ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2022: Chapter 51). Theseinclude the isolation and quantification techniques described herein. One exemplary method provided herein includes purification of the glycoform of CBG by immunoprecipitation using the 12G2 monoclonal antibody. Absolute concentrations of non-glycosylated and deglycosylated RCL peptides, and each Asn347 glycoform were calculated using pre-existing total CBG concentration measurement via 12G2 immunoassay for each corresponding sample (see the examples herein).

[0129] In some embodiments, the clinical pathology of septic shock predicted by the method is selected from one or more of: mortality, time in septic shock, time in first septic shock episode, time to septic shock onset, number of septic shock episodes, intubation time, requirement for mechanical ventilation, requirement for vasopressor therapy, requirement for inotrope therapy, requirement for renal replacement therapy, requirement for Extracorporeal membrane oxygenation (ECMO) therapy, requirement for antimicrobial therapy, vascular injury, capillary leakage, hypotension, hyperthermia, tachycardia, tachypnea, lactic acidosis, hypoxia, cyanosis, ileus, hyperglycemia, CNS dysfunction, sequential organ failure, multiorgan dysfunction syndrome, lung dysfunction, cardiac dysfunction, kidney dysfunction, and liver dysfunction.

[0130] In some embodiments, the method predicts the likelihood of multiorgan dysfunction syndrome.

[0131] In some embodiments, the method predicts the likelihood of mortality of the patient.

[0132] In some embodiments, a serum concentration of the TS3 glycoforms of CBG below about 30 nmol / L indicates a lower likelihood of patient survival of sepsis or septic shock. In some embodiments, a serum concentration of the TS3 glycoforms of CBG below about 45 nmol / L indicates a lower likelihood of patient survival of sepsis or septic shock.

[0133] In some embodiments, a serum concentration of the TS3 glycoforms of CBG above about 30 nmol / L indicates a higher likelihood of patient survival of sepsis or septic shock. In some embodiments, a serum concentration of the TS3 glycoforms of CBG above about 45 nmol / L indicates a higher likelihood of patient survival of sepsis or septic shock.

[0134] In some embodiments, a serum concentration of the glycoform of the CBG having a triantennary glycan at Asn347 of about 55 nmol / L or less, or about 54 nmol / L or less, or about 53 nmol / L or less, or about 52 nmol / L or less, or about 51 nmol / L or less, or about 50 nmol / L or less, or about 49 nmol / L or less, or about 48 nmol / L or less, or about 47 nmol / L or less, or about 46 nmol / L or less, or about 45 nmol / L or less, or about 44 nmol / L or less, or about 43 nmol / L or less, or about 42 nmol / L or less, or about 41 nmol / L or less, or about 40 nmol / L or less, or about 39 nmol / L or less, or about 38 nmol / L or less, or about 37 nmol / L or less, or about 36 nmol / L or less, or about 35 nmol / L or less, or about 34 nmol / L or less, or about 33 nmol / L or less, or about 32 nmol / L or less, or about 31 nmol / L or less, or about 30 nmol / L or less, or about 29 nmol / L or less, or about 28 nmol / L or less, or about 27 nmol / L or less, or about 26 nmol / L or less, or about 25 nmol / L or less, or about 24 nmol / L or less, or about 23 nmol / L or less, or about 22 nmol / L or less, or about 21 nmol / L or less, or about 20 nmol / L or less is indicative of a higher likelihood of a patient experiencing sepsis, septic shock or a clinical pathology of sepsis or septic shock. The above diagnostic serum concentrations may apply to either TS3 and / or TS3F, preferably TS3 alone.

[0135] Conversely, in some embodiments, a serum concentration of about 20 nmol / L or more, about 21 nmol / L or more, about 22 nmol / L or more, about 23 nmol / L or more, about 24 nmol / L or more, about 25 nmol / L or more, or about 26 nmol / L or more, or about 27 nmol / L or more, or about 28 nmol / L or more, or about 29 nmol / L or more, or about 30 nmol / L or more, or about 31 nmol / L or more, or about 32 nmol / L or more, or about 33 nmol / L or more, or about 34 nmol / L or more, or about 35 nmol / L or more, or about 36 nmol / L or more, or about 37 nmol / L or more, or about 38 nmol / L or more, or about 39 nmol / L or more, or about 40 nmol / L or more, or about 41 nmol / L or more, or about 42 nmol / L or more, or about 43 nmol / L or more, or about 44 nmol / L or more, or about 45 nmol / L or more, or about 46 nmol / L or more, or about 47 nmol / L or more, or about 48 nmol / L or more, or about 49 nmol / L or more, or about 50 nmol / L or more, or about 51 nmol / L or more, or about 52 nmol / L or more, or about 53 nmol / L or more, or about 54 nmol / L or more, or about 55 nmol / L or more, or about 56 nmol / L or more, or about 57 nmol / L or more, or about 58 nmol / L or more, or about 59 nmol / L or more, or about 60 nmol / L or more of a glycoform of CBG having a triantennary glycan at Asn347, indicates a lower likelihood of a patient experiencing sepsis or septic shock,or a clinical pathology of sepsis or septic shock, or indicates a higher likelihood of survival. The above diagnostic serum concentrations may apply to either TS3 and / or TS3F, preferably TS3 alone.

[0136] In some embodiments, the absolute serum concentration of CBG having a triantennary glycan at Asn347 (for example the concentration of TS3 and TS3F combined) above a defined serum concentration indicates a lower likelihood or a patient experiencing sepsis or septic shock, or a clinical pathology of sepsis or septic shock, or indicates a higher likelihood of survival of sepsis or septic shock. In such embodiments, the serum concentration may be above about 40 nmol / L, or above about 42 nmol / L, or above about 44 nmol / L, or above about 46 nmol / L, or above about 48 nmol / L, or above about 50 nmol / L, or above about 52 nmol / L, or above about 54 nmol / L, or above about 56 nmol / L, or above about 58 nmol / L, or above about 60 nmol / L, or above about 62 nmol / L, or above about 64 nmol / L, or above about 66 nmol / L, or above about 68 nmol / L, or above about 70 nmol / L, or above about 72 nmol / L, or above about 74 nmol / L, or above about 76 nmol / L, or above about 78 nmol / L, or above about 80 nmol / L, or above about 82 nmol / L, or above about 84 nmol / L, or above about 86 nmol / L, or above about 88 nmol / L, or above about 90 nmol / L, or above about 92 nmol / L, or above about 94 nmol / L, or above about 96 nmol / L, or above about 98 nmol / L, or above about 100 nmol / L, or above about 102 nmol / L, or above about 104 nmol / L, or above about 106 nmol / L, or above about 108 nmol / L, or above about 110 nmol / L, or above about 112 nmol / L, or above about 114 nmol / L, or above about 116 nmol / L.

[0137] In some embodiments, the absolute serum concentration of CBG having a triantennary glycan at Asn347 (for example, the concentration of TS3 and TS3F combined) below a defined serum concentration indicates a higher likelihood of a patient experiencing sepsis or septic shock, or a clinical pathology of sepsis or septic shock, or indicates a lower likelihood of survival or sepsis or septic shock. In such embodiments, the serum concentration may be below about 116 nmol / L, or below about 114 nmol / L, or below about 112 nmol / L, or below about 110 nmol / L, or below about 108 nmol / L, or below about 106 nmol / L, or below about 104 nmol / L, or below about 102 nmol / L, or below about 100 nmol / L, or below about 98 nmol / L, or below about 96 nmol / L, or below about 94 nmol / L, or below about 92 nmol / L, or below about 90 nmol / L, or below about 88 nmol / L, or below about 86 nmol / L, or below about84 nmol / L, or below about 82 nmol / L, or below about 80 nmol / L, or below about 78 nmol / L, or below about 76 nmol / L, or below about 74 nmol / L, or below about 72 nmol / L, or below about 70 nmol / L, or below about 68 nmol / L, or below about 66 nmol / L, or below about 64 nmol / L, or below about 62 nmol / L, or below about 60 nmol / L, or below about 58 nmol / L, or below about 56 nmol / L, or below about 54 nmol / L, or below about 52 nmol / L, or below about 50 nmol / L, or below about 48 nmol / L, or below about 46 nmol / L, or below about 44 nmol / L, or below about 42 nmol / L, or below about 40 nmol / L.

[0138] In some embodiments, the method predicts the likelihood of the patient requiring mechanical ventilation.

[0139] In some embodiments, a serum concentration of a TS3F glycan at Asn347 of about 53 nmol / L or more indicates a lower likelihood of the patient requiring mechanical ventilation. In some embodiments, a serum concentration of a TS3F glycan at Asn347 of about 38 nmol / L or more indicates a lower likelihood of the patient requiring mechanical ventilation.

[0140] In some embodiments, a serum concentration of a TS3F glycan at Asn347 of about 53 nmol / L or less indicates a higher likelihood of the patient requiring mechanical ventilation. In some embodiments, a serum concentration of a TS3F glycan at Asn347 of about 38 nmol / L or less indicates a higher likelihood of the patient requiring mechanical ventilation.

[0141] In some embodiments, a serum concentration of the glycoform of the CBG having a triantennary glycan at Asn347 of about 63 nmol / L or less, about 62 nmol / L or less, about 61 nmol / L or less, about 60 nmol / L or less, or about 59 nmol / L or less, or about 58 nmol / L or less, or about 57 nmol / L or less, or about 56 nmol / L or less, or about 55 nmol / L or less, or about 54 nmol / L or less, or about 53 nmol / L or less, or about 52 nmol / L or less, or about 51 nmol / L or less, or about 50 nmol / L or less, or about 49 nmol / L or less, or about 48 nmol / L or less, or about 47 nmol / L or less, or about 46 nmol / L or less, or about 45 nmol / L or less, or about 44 nmol / L or less, or about 43 nmol / L or less, or about 42 nmol / L or less, or about 41 nmol / L or less, or about 40 nmol / L or less, or about 39 nmol / L or less, or about 38 nmol / L or less, or about 37 nmol / L or less, or about 36 nmol / L or less, or about 35 nmol / L or less, or about 34 nmol / L or less, or about 33 nmol / L or less, or about 32 nmol / L or less, or about 31 nmol / L or less, or about 30 nmol / L or less, or about 29 nmol / L or less, or about 28 nmol / L is indicative ofa higher likelihood of a patient requiring mechanical ventilation. In some forms of the above embodiments, the diagnostic serum concentration is the concentration of TS3 and / or TS3F, preferably TS3F alone.

[0142] Conversely, in some embodiments, a serum concentration of about 28 nmol / L or more, or about 29 nmol / L or more, or about 30 nmol / L or more, or about 31 nmol / L or more, or about 32 nmol / L or more, or about 33 nmol / L or more, or about 34 nmol / L or more, or about 35 nmol / L or more, or about 36 nmol / L or more, or about 37 nmol / L or more, or about 38 nmol / L or more, or about 39 nmol / L or more, or about 40 nmol / L or more, or about 41 nmol / L or more, or about 42 nmol / L or more, or about 43 nmol / L or more, or about 44 nmol / L or more, or about 45 nmol / L or more, or about 46 nmol / L or more, or about 47 nmol / L or more, or about 48 nmol / L or more, or about 49 nmol / L or more, or about 50 nmol / L or more, or about 51 nmol / L or more, or about 52 nmol / L or more, or about 53 nmol / L or more, or about 54 nmol / L or more, or about 55 nmol / L or more, or about 56 nmol / L or more, or about 57 nmol / L or more, or about 58 nmol / L or more, or about 59 nmol / L or more, or about 60 nmol / L or more, or about 61 nmol / L or more, or about 62 nmol / L or more, or about 63 nmol / L or more of a glycoform of CBG having a triantennary glycan at Asn347, indicates a lower likelihood of a patient requiring mechanical ventilation. In some forms of the above embodiments, the diagnostic serum concentration is the concentration of TS3 and / or TS3F, preferably TS3F alone.

[0143] In some embodiments, the absolute serum concentration of CBG having a triantennary glycan at Asn347 (for example the concentration of TS3 and TS3F combined) above a defined serum concentration indicates a lower likelihood of a patient requiring mechanical ventilation. In such embodiments, the serum concentration may be above about 56 nmol / L, or above about 58 nmol / L, or above about 60 nmol / L, or above about 62 nmol / L, or above about 64 nmol / L, or above about 66 nmol / L, or above about 68 nmol / L, or above about 70 nmol / L, or above about 72 nmol / L, or above about 74 nmol / L, or above about 76 nmol / L, or above about 78 nmol / L, or above about 80 nmol / L, or above about 82 nmol / L, or above about 84 nmol / L, or above about 86 nmol / L, or above about 88 nmol / L, or above about 90 nmol / L, or above about 92 nmol / L, or above about 94 nmol / L, or above about 96 nmol / L, or above about 98 nmol / L, or above about 100 nmol / L, or above about 102 nmol / L, or aboveabout 104 nmol / L, or above about 106 nmol / L, or above about 108 nmol / L, or above about 110 nmol / L, or above about 112 nmol / L, or above about 114 nmol / L, or above about 116 nmol / L, or above about 118 nmol / L, or above about 120 nmol / L, or above about 124 nmol / L, or above about 128 nmol / L, or above about 132 nmol / L

[0144] In some embodiments, the absolute serum concentration of CBG having a triantennary glycan at Asn347 (for example, the concentration of TS3 and TS3F combined) below a defined serum concentration indicates a higher likelihood of a patient requiring mechanical ventilation. In such embodiments, the serum concentration may be below about 132 nmol / L, or below about 128 nmol / L, or below about 124 nmol / L, about 120 nmol / L, or below about 118 nmol / L, or below about 116 nmol / L, or below about 114 nmol / L, or below about 112 nmol / L, or below about 110 nmol / L, or below about 108 nmol / L, or below about 106 nmol / L, or below about 104 nmol / L, or below about 102 nmol / L, or below about 100 nmol / L, or below about 98 nmol / L, or below about 96 nmol / L, or below about 94 nmol / L, or below about 92 nmol / L, or below about 90 nmol / L, or below about 88 nmol / L, or below about 86 nmol / L, or below about 84 nmol / L, or below about 82 nmol / L, or below about 80 nmol / L, or below about 78 nmol / L, or below about 76 nmol / L, or below about 74 nmol / L, or below about 72 nmol / L, or below about 70 nmol / L, or below about 68 nmol / L, or below about 66 nmol / L, or below about 64 nmol / L, or below about 62 nmol / L, or below about 60 nmol / L, or below about 58 nmol / L, or below about 56 nmol / L.

[0145] To determine which prognostic group a subject belongs to, or has a higher likelihood of belonging to, the quantity or concentration of one or more of the glycoforms of CBG having a triantennary glycan at Asn347 may be assess in a biological sample from the patient and compared to one or more reference standards. Accordingly, in some embodiments, the method further comprises comparing the concentration of a glycoform of CBG in the isolated biological sample to a reference standard.

[0146] Such reference standards provide a benchmark which allows for the assessment of the relative quantity or concentration of a glycoform of CBG in the biological sample, thereby allowing the determination of which prognostic group a patient belongs to. Typically, thereference standard is pre-determined and based on obtained information of a population or individual.

[0147] In some embodiments, the reference standard is based on the quantity or concentration of one or more of the glycoforms of CBG in a population of patients that died from sepsis or septic shock. In some embodiments, the reference standard is based on the quantity or concentration of one or more of the glycoforms of CBG in a population of patients that survived sepsis or septic shock. In some embodiments, the reference standard is based on the quantity or concentration of one or more of the glycoforms of CBG in a population of patients that had severe sepsis or septic shock. In some embodiments, the reference standard is based on the quantity or concentration of one or more of the glycoforms of CBG in a population of patients that had mild sepsis or septic shock.

[0148] In some embodiments, the reference standard is based on the quantity or concentration of one or more of the glycoforms of CBG in a population of patients that have an immunological or inflammatory condition, or poor response to corticosteroids. In some embodiments, the reference standard is based on the quantity or concentration of one or more of the glycoforms of CBG in a population of patients that do not have an immunological or inflammatory condition or have a good response to corticosteroids. Such reference standards may be used for methods described herein relating the immunological or inflammatory conditions, or for assessing the likelihood of subject responding to corticosteroid administration.

[0149] In some embodiments, the reference standard is the median quantity or concentration of the one or more glycoforms of CBG in one of the above identified populations. In some embodiments, the reference standard is the mean quantity or concentration of the one or more glycoforms of CBG in one of the above identified populations.

[0150] In some embodiments the reference standard may indicate the 5th percentile, 10th percentile, 15th percentile, 20th percentile, 25th percentile, 30th percentile, 35th percentile, 40th percentile, 45th percentile, 50th percentile, 55th percentile, 60th percentile, 65th percentile, 70th percentile, 75th percentile, 80th percentile, 85th percentile, 90thpercentile, 95th percentile in a population that died from sepsis or septic shock. In such embodiments, a concentration below the reference standard may indicate a poor prognosis.

[0151] In some embodiments the reference standard may indicate the 5th percentile, 10th percentile, 15th percentile, 20th percentile, 25th percentile, 30th percentile, 35th percentile, 40th percentile, 45th percentile, 50th percentile, 55th percentile, 60th percentile, 65th percentile, 70th percentile, 75th percentile, 80th percentile, 85th percentile, 90th percentile, 95th percentile in a population that survived sepsis or septic shock. In such embodiments, a concentration above the reference standard may indicate a good prognosis.

[0152] In some embodiments, the reference standard is based on the quantity or concentration of one or more of the glycoforms of CBG in a subject when admitted to hospital, or prior to treatment.

[0153] The skilled person will appreciate that the reference standard can be multifactorial and may vary based on patient factors such as age, sex, genetic predispositions, or diagnostic or prognostic measures of sepsis or septic shock, for example. Accordingly, the reference standard may take these factors into consideration and therefore be different for different sub-groups of patients. In addition, the reference standard can vary significantly if the patent population comprises a significant sub-population suffering a specific disease (for example bacterial infection) or a comorbidity. A person skilled in the art may take this into account when applying the method of determining the likelihood of a patient experiencing sepsis or septic shock, or a clinical pathology of sepsis or septic shock. Such factors may be considered when determining the reference standard, for example in the context of a clinical trial.

[0154] The determination of the method of predicting the likelihood of a patient experiencing sepsis or septic shock may not be binary (i.e., high or low likelihood) but may be on a gradient, may allocate a patient to a percentile or quartile, or may include additional categories, such as intermediate risk. Accordingly, the reference standard may provide multiple values to allow allocations into multiple groups or may be an algorithm or formula which provides an output along a continuum or takes into account multiple factors.

[0155] The skilled person will appreciate that the reference standard can be calculated from, and adjusted for, any specific patient population or sub-population.

[0156] In some embodiments, if the serum concentration of the glycoform of the CBG having a triantennary glycan at Asn347 in the isolated biological sample from a patient is lower than the reference standard, this indicates a higher likelihood of mortality from sepsis or septic shock.

[0157] In some embodiments, if the serum concentration of the glycoform of the CBG having a triantennary glycan at Asn347 in the isolated biological sample from a patient is higher than the reference standard, this indicates a higher likelihood of survival from sepsis or septic shock.

[0158] As indicated herein, various means for quantifying CBG and glycoforms of CBG are known in the art. However, in some preferred embodiments, the concentration of the glycoform of CBG in the isolated biological sample is measured by chromatograph and mass spectrometry, such as LC-MS / MS or ELISA. In some embodiments, the method for measuring CBG comprises purifying CBG by immunoprecipitation, for example using 12G2 monoclonal antibodies.

[0001] In aspects, the method of predicting the likelihood of a subject experiencing sepsis or a clinical pathology of sepsis can be performed on a computing system. In some embodiments of these aspects, the method comprises using computer software executable by a processor to process data representative of the concentration of CBG or a glycoform of CBG, such as CBG having a triantennary glycan as Asn347. In some embodiments, the processed data is also representative of the concentration TS3 or TS3F triantennary glycosylation at Asn347. The software, when executed by a processor, can compare the expression of the markers to a control standard to provide a determination of the likelihood of the subject experiencing sepsis or septic shock, or a clinical pathology of sepsis or septic shock. Suitable reference standards are described herein.

[0002] Such a computer system can allow for automated comparison of input concentration data of one or more of CBG or a glycoform thereof to the reference standards and provide a determined likelihood that a subject will experience sepsis or septic shock, or a clinical pathology of sepsis or septic shock.

[0003] Further provided by the present invention is a system for determining if a subject is likely to experience sepsis or septic shock or a clinical pathology of sepsis or septic shock, the system comprising: a means for detecting the concentration of CBG or a glycoform of CBG (as detailed herein) in a biological sample (preferably a blood, serum or plasma sample) from the subject; a processor; memory; and software resident in the memory accessible to the processor, the software comprising a series of instructions executable by the processor to process data from the means to detect the concentration of CBG or a glycoform thereof in the sample from the subject, and optionally compare it to a reference standard, to thereby determine the likelihood of the subject experiencing sepsis or a clinical pathology of sepsis.

[0004] In some embodiments of the system, the means for detecting CBG or a glycoform of CBG is ELISA or LC-MS / MS. The system may include means for determining or analysing such assays.

[0159] The present disclosure relates to a method of predicting the response of a patient to administration of a corticosteroid, the method comprising analysing an isolated biological sample from the subject for a concentration of a glycoform of CBG, wherein the glycoform of CBG has a triantennary glycan at Asn347. In embodiments of this method, a lower concentration of the glycoform indicates a lower likelihood of responding to administration of the corticosteroid. Accordingly, the dosing, schedule or treatment regimen can be adjusted based on the results of performing this method. Alternatively, a combination therapy comprising a corticosteroid and CBG (or CBG enriched for a glycoform of CBG having a triantennary glycan at Asn347, preferably TSF or TS3F) can be administered if a subject has a low concentration of CBG (for example a concentration below a reference standard described herein).

[0160] In some embodiments, the present disclosure provides a method of treating or preventing sepsis, or treating, preventing or delaying a clinical pathology of sepsis as described herein, including performing the method of predicting the likelihood of a patient experiencing sepsis or a clinical pathology of sepsis as described herein.

[0161] In some embodiments, the methods as described herein comprises administering the pharmaceutical composition as described herein. For example, if the patient has an immunological or inflammatory condition, or sepsis or is in septic shock or is at risk of sepsis or septic shock. In some embodiments, the immunological or inflammatory condition is achronic immunological or inflammatory condition or an acute immunological or inflammatory condition.

[0162] In some embodiments, the present disclosure also provides a use of CBG, or a CBG comprising a triantennary glycan at Asn347, in the manufacture of a medicament for treating or preventing sepsis, or treating, preventing or delaying a clinical pathology of sepsis, or an immunological or inflammatory condition, as described herein. In some embodiments, the CBG can be isolated from serum or plasma. In some embodiments the CBG is enriched for a glycoform having a triantennary trisialylated (TS3) glycan at Asn347. In some embodiments, the triantennary trisialylated (TS3) glycan at Asn347 is TS3 or TS3F. In some embodiments the medicament can be manufactured from the pharmaceutical composition as described herein. In some embodiments the medicament can be used for treating or preventing sepsis, or treating, preventing or delaying a clinical pathology of an immunological or inflammatory condition, or sepsis or septic shock or if a patient is at risk of sepsis or septic shock. In some embodiments, the immunological or inflammatory condition is a chronic immunological or inflammatory condition or an acute immunological or inflammatory condition.

[0163] There is also provided a use of a pharmaceutical composition as described herein for the manufacture of a medicament for modulating the biological activity of a corticosteroid in a subject. In some embodiments, there is provided a use of pharmaceutical composition as described herein for use the manufacturer of a medicament for a combination therapy with a corticosteroid in the treatment or prevention of sepsis, or for the treatment, prevention or delay of a clinical pathology of sepsis or for the treatment or prevention of an immunological or inflammatory condition. In some embodiments, there is provided a use of pharmaceutical composition as described herein and a corticosteroid for the manufacturer of a medicament of a combination therapy for the treatment or prevention of sepsis, or for the treatment, prevention or delay of a clinical pathology of sepsis or for the treatment or prevention of an immunological or inflammatory condition.

[0164] In some embodiments, there is provided a use of a pharmaceutical composition as described herein fora combination therapy with a corticosteroid for the treatment, prevention or delay of sepsis or septic shock, or for the treatment, prevention or delay of a clinicalpathology of sepsis or septic shock. In some embodiments, there is provided a use of a pharmaceutical composition as described herein for a combination therapy with a corticosteroid for the treatment, prevention or delay of an immunological or inflammatory condition, or for the treatment, prevention or delay of a corticosteroid-associated disease. In some embodiments, the corticosteroid is one or more of: betamethasone, dexamethasone, fludrocortisone, ciclesonide, corticosterone, corticosterone acetate, hydrocortisone, methylprednisolone, prednisolone or prednisone.

[0165] The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.

[0166] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.

[0167] Future patent applications may be filed on the basis of, or claiming priority from, the present application. It is to be understood that the following claims are not intended to limit the scope of what may be claimed in any such future application(s). Features may be added to or omitted from the claims at a later date so as to further define or re-define the claimed invention.

[0168] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context or the understanding of a skilled addressee. The use of any and all examples, or exemplary language (e.g., "such as", "i.e.", "for example"), is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention, unless otherwise claimed or stated.No language in the specification should be construed as indicating any non-claimed element as essential.

[0169] Referenced documents, publications and patents are to be included in their entirety by way of reference. The teachings and disclosures in such documents, publications and patents are therefore considered to form part of the disclosure of this specification.

[0170] Examples

[0171] The present disclosure is further described by the following examples. It is to be understood that the following examples are for the purpose of describing particular embodiments only, and they are not intended to be limiting with respect to the above description.

[0172] Example 1 - Defining optimal corticosteroid binding globulin glycosylation patterns at the reactive centre loop for cortisol tissue delivery in human septic shock.

[0173] As set out below, the Inventors have shown that the glycoprofile of corticosteroid binding globulin (CBG), particularly at the site of the reactive centre loop (RCL), is associated with patient outcomes in sepsis and sepsis shock. Accordingly, assessment of glycoforms of CBG in patients can predict the likelihood of the patient experiencing sepsis or septic shock, or a clinical pathology of sepsis or septic shock.

[0174] Materials and methods

[0175] Study Population

[0176] The study population consisted of 135 septic shock patients in the intensive care unit (ICU) or The Royal Adelaide Hospital, age >18 years and receiving IV norepinephrine. Septic shock was defined as the presence of a clinically or microbiologically documented infection; >2 points on the Sequential [sepsis- related] Organ Failure Assessment (SOFA) score; receiving norepinephrine for at least 6 hours to maintain a mean arterial pressure > 65 mmHg. Exclusion criteria included pregnancy; expectation of death within 24 hours; receipt of norepinephrine infusions for >48 hours; known conditions that alter cortisol secretion, including the use of systemic glucocorticoids for >3 months and disorders of the HPA axis.Serum was collected throughout duration of ICU admission. Serum from the last day of ICU admission (either due to recovery and / or discharge, or death, up to a maximum of 7 days) was analysed for CBG glycoform profile. In addition, serum samples from 8 healthy individuals, 4 male and 4 female, were also obtained for analysis and glycoprofiling.

[0177] CBG purification and generation of CBG RCL glyco- / peptides

[0178] CBG was immunoprecipitated from serum, using mouse 12G2 mAb (RRID: AB_2632404), produced and purified in-house from 12G2 hybridoma cell culture. 12G2 mAb was covalently bound to Dynabeads™ M-280 Tosylactivated (ThermoFisher Scientific) via 22-hour incubation at 38 °C in 0.1M borate buffer. One mg of 12G2 mAb coupled magnetic beads were incubated with 125 pl of serum, with subsequent elution using 0.1 M glycine, neutralisation with 500mM ammonium bicarbonate and vacuum centrifugation for drying.

[0179] CBG was then reconstituted in 8 M urea with reduction and alkylation using 5 mM dithiothreitol (DTT) and 20 mM iodoacetamide (IAA), respectively. This was transferred to S-TrapTM mini spin columns (ProtiFi) with the addition of phosphoric acid and wash / binding buffer and centrifuged to trap proteins, then washed 3 times with wash buffer. The trapped proteins were trypsin digested overnight, and the resulting glyco- / peptides were eluted by addition of triethylammonium bicarbonate (TEAB), formic acid and acetonitrile (ACN) into the S-trap column. The eluted fractions were combined and dried in a vacuum concentrator.

[0180] Mass spectrometric assessment of CBG RCL glycosylation

[0181] An aliquot of each glyco- / peptide sample was reconstituted in 0.1% formic acid and loaded on to a trap column custom packed with ReproSil-Pur C18-AQ 5 pm resin (Dr Maisch, Germany) operated by UltiMate 3000 RSLCnano HPLC system (ThermoFisher Scientific). The mobile phases were 99.9% ACN in 0.1% formic acid (B) and aqueous 0.1% formic acid (A). The nanoLC was connected to Q-Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific) operating in positive ion polarity mode. MS / MS in parallel reaction monitoring mode (PRM) was used for glycoform profiling. For CBG RCL Asn347 glycosylation site occupancy, aliquots of glyco- / peptides were de- glycosylated via PNGase F (10 U in 20pl 50mM ammonium bicarbonate with subsequent LC-MS / MS analysis as above, in DDA m ode.To establish Asn347 site occupancy, the area under the curve (AUC) of de-glycosylated RCL peptide and non-glycosylated RCL peptide were measured. As de-glycosylation by PNGase F deamidates Asparagine (N) to aspartic acid (D) residues, CBG RCL peptides which were not glycosylated at the Asn347 site, were distinguishable from those that were glycosylated but subsequently de-glycosylated with PNGase F. Asn347 site occupancy was calculated as a percentage of de-glycosylated RCL peptide over total RCL peptide. Relative abundance of each Asn347 glycoforms was also obtained using AUC measurements.

[0182] Absolute concentrations of non-glycosylated and glycosylated RCL peptides, and each Asn347 glycoform were calculated using the above MS data obtained from AUC measurements, and pre-existing data on total CBG concentration measurement via 12G2 immunoassay for each corresponding sample.

[0183] Results

[0184] Asn347 glycosylation site occupancy

[0185] The Asn347 glycosylation site occupancy ranged between 57.0% to 95.9% (mean 76.6%, standard deviation= 7.0%). There was no significant correlation between Asn347 site occupancy and total CBG concentration as measured by 12G2 ELISA (Pearson's correlation coefficient (r)= -0.169, p= 0.052).

[0186] Glycosylation profiling at the Asn347 revealed four glycans that were consistently identified across the study population; biantennary disia lylated (BS2), biantennary disia ly lated core-fucosylated (BS2F), triantennary trisialylated (TS3) and triantennary trisialylated core-fucosylated (TS3F) glycans, with mean relative abundance of 47.9%, 10.2%, 20.5% and 21.1%, respectively (figure 1).

[0187] TS3 glycoform and ICU mortality

[0188] As can be seen in figure 2, significant association was observed between TS3 Asn347 glycoforms and ICU mortality. Absolute concentrations of the TS3 glycoforms were lower in septic shock non-survivors compared to septic shock survivors (mean 29.74 vs 45.16 nmol / L, p=0.007). No such association with mortality was seen with other glycoforms; TS2(96.8 vs 103.0 nmol / L, p=0.580), TS2F (20.9 vs 22.8 nmol / L, p=0.612) and TS3F (43.3 vs 44.4 nmol / L, p=0.859).

[0189] TS3F glycoform and ventilation

[0190] As can be seen in figure 3A, higher concentrations of TS3F glycoforms were associated with more favourable clinical outcome measures. Those who were intubated and ventilated during ICU admission had lower mean concentration of TS3F An347 glycoform CBG compared to those who didn't require mechanical ventilation (37.9 vs 52.6 nmol / L, p=0.002). There was also a weak negative correlation between concentration of TS3F Asn346 glycoforms and duration of ventilation (r= -0.185, p= 0.0363 - figure 3B). No such correlation was seen with other glycoforms.

[0191] CBG glycoform and sepsis severity

[0192] As shown in figure 4, septic shock severity (defined by total SOFA score) on Day 1 of ICU admission showed negative correlation with TS3F glycoform concentration (r= - 0.335, p <0.001). There was also a negative correlation with same day 1 SOFA subcomponents: SOFA liver (r= -0.253, p=0.004); SOFA respiratory (r= -0.309, p <0.001); SOFA coagulation (r= -0.205, p=0.020) [data not shown]. No association was seen between day 1 total SOFA score and any of the other Asn347 glycoforms; BS2 (r=0.033, p=0.709), BS2F (r= -0.100, p=0.263), TS3 (r= -0.072, p= 0.420).

[0193] CBG affinity form measurement

[0194] For the 8 healthy samples, aliquots of the above trypsin and PNGase F digested peptides were reconstituted in 40% ACN and 0.2% formic acid before CBG internal standards were added. For quantification of laCBG, heavy labelled peptides of the N- terminal fragment of RCL truncated at the NE cleavage site (AVLQLNEEGV*DTAGSTGV) was used. An unrelated heavy labelled peptide upstream from RCL and separate from known glycosylation sites (ITQDAQL*K) was used as a surrogate measure of total CBG. Heavy peptides were labelled via 13C at the designated leucine (L*), isoleucine(l*) and or valine (V*), and were purchased from Mimotopes (Melbourne, Australia). Standard curves were generated from the heavy labelled peptides and used as internal standards for quantification.

[0195] The targeted LC-MS / MS method was performed using an ACQUITY UPLC M-Class system (Waters, Milford, MA, USA) connected to a ZenoTOF 7600 mass spectrometer with an OptiFlow Turbo V ion source (SCIEX, Singapore, Singapore). Samples (4 pL containing 20% ACN and 0.1% formic acid) were loaded onto a Waters nanoEase M / Z HSS T3 reversed phase C18 column heated to 50°C. Mobile phases A and B were 0.1% formic acid and 0.1% formic acid in ACN, respectively. A Zeno multiple reaction monitoring (MRM) scheme was applied, with the precursor and fragment mass for ITQDAQLK set at 458.76 m / z > 702.38 m / z, respectively; and for AVLQLNEEGVDTAGSTGV, the precursor and fragment mass were 880.44 m / z > 1053.55 m / z, respectively. Isotopically labelled peptides were used as internal standards and the area of AVLQLNEEGVDTAGSTGV / I TQDAQLK was used to calculate %laCBG.

[0196] CBG affinity form analysis of healthy samples

[0197] As shown in figure 5, mean % laCBG, calculated as (laCBG / total CBG) xl00% of the 8 healthy samples was 0.041% (range 0.020-0.083%). This contrasts with the % laCBG in day 1 septic shock patients measured by our group previously, showing mean % laCBG of 0.23% (range 0.07-0.74%).

[0198] The percentage of laCBG data from day 1 septic shock patients, was also correlated with day 1 septic shock serum glycoform analysis. This showed a weak, yet statistically significant negative correlation between % laCBG and concentration of CBG glycosylated at the Asn347 site (r= - 0.184, p= 0.045). Moreover, a significant negative correlation between % laCBG and concentration of CBG with TS3 glycan at Asn347 (r =-0.190, p= 0.040) and a stronger negative correlation with CBG with TS3F glycan at Asn347 site (r= -0.252, p= 0.006) (figure 5). Of note, day 1 TS3 and TS3F concentrations were found to confer mortality and morbidity benefit. No significant correlation was seen between % laCBG and concentration and BS2F (r= -0.63, p= 0.503) glycans at Asn347.

[0199] CBG glycoform analysis of the last day sepsis samples

[0200] Greater concentrations of TS3 and TS3F Asn347 glycoform of CBG are associated with lower mortality and sepsis severity, respectively, in septic shock at day 1 of ICU admission. To better understand the changes in composition of glycoforms throughout the septic shockdisease course and effect on clinical outcome, the Inventors performed glycosylation profiling on serum from the last day of ICU admission (whether due to recovery and / or discharge, or death in ICU, up to a maximum of seven days). Asn347 site glycosylation occupancy reduced from day 1 to the last day of ICU admission in both survivors (76.5% vs. 60.1%, p<0.001) and non-survivors (76.6% vs. 59.5%, p<0.001), and there was no difference between survivors and non-survivors at these time points (data not shown).

[0201] As shown in figure 6, the absolute concentration of TS3 glycoforms showed the most pronounced reduction from day 1 to the last day of ICU admission in all patients (42.27 vs 25.87 nmol / L, p<0.0001), followed by TS3F (44.21 vs 34.08 nmol / L, p-0.0019) and BS2 (101.8 vs. 88.4 nmol / L, p=0.0267). Meanwhile, the concentration of the BS2F glycoform remained unchanged from day 1 to the last day of ICU admission (22.40 vs 22.39 nmol / L, p=0.993) and there was a trend for higher TS3 in survivors (27.19 vs 19.96 nmol / L, p=0.07).

[0202] As shown in figure 7, higher relative abundance of TS3 glycoform was associated with ICU survival 16.7% vs 13.3% (p=0.0034), whereas on the contrary, higher relative concentration of BS2F glycoform 16.8% vs 12.3% (p=0.0044) was associated with ICU mortality.

[0203] The absolute concentration of TS3 Asn347 glycoform on the last day was negatively associated with the last day total SOFA score (Pearson's coefficient r= -0.181, p=0.047); the last day SOFA CVS score (r= -0.277, p=0.002) and the last day SOFA CNS score (r=-0.190, p=0.037). Absolute concentration of TS3F Asn347 glycoform on the last day of ICU admission, showed a negative correlation with the last day total SOFA score (r=-0.209, p=0.022 - data not shown). No clinical correlation was seen with BS2 or BS2F glycoforms and SOFA scores.

[0204] The above work demonstrates that TS3 glycoforms, shown herein to be associated with septic shock survival, reduced the most during septic shock, whilst BS2F generally remains unchanged. Higher absolute concentrations of TS3 and TS3F on the earliest of the last day of ICU admission or day 7 post ICU admission is associated with milder septic shock severity, which is in line with the day 1 findings.

[0205] Overall, this data shows that maintaining higher levels of CBG glycoforms having a triantennary glycan as Asn347 (particularly TS3 and TS3F) may have a beneficial effect in protecting against adverse outcomes during sepsis and septic shock episodes.

[0206] Example 2- CBG therapy in a CLP murine model of septic shock

[0207] As set out below, the Inventors have shown for the first time that a significant reduction in mortality in a preclinical model of septic shock can be achieved by administering CBG to subjects. Current, pharmacological treatments for targeting inflammation in septic shock, such as corticosteroid treatment, offer little to no clinical benefit. Accordingly, these data provide a method for treating or preventing septic shock, as well as clinical pathologies of septic shock. As such, the findings of these studies provide a potential breakthrough in the treatment of a highly fatal condition.

[0208] Materials and methods

[0209] Animals

[0210] Adult (10-12 weeks old) male C57BL / 6 mice (n=182 bred in SAHMRI Bioresources were housed individually in a 12:12 hr light-dark cycle under constant temperature (22 ± 0.5° C) and humidity (40-60%) with ad libitum access to water and a standard chow diet (13kJ / g: 24% from protein, 18% from fat, 58% from carbohydrates; #2918, Teklad Global Diet, USA).

[0211] Surgical procedures

[0212] Carotid telemetry

[0213] 10-12 weeks old male C57BL / 6 mice were fasted and anaesthetised before the catheter of an HD-X10 telemetry (#270-0171-002X; Data Science International, USA) was placed and secured 1-2 mm into the mouse's aortic arch. Mice recovered under strictly controlled conditions for 4 days.

[0214] High-grade caecal ligation and puncture

[0215] Mice were fasted and anaesthetised before ligation of the caecum 17-18 mm from the caecal tip. The caecum was punctured through-and-through twice with a 21g needle toallow extrusion of faeces, before the caecum was returned, and the external incision closed. Mice were subsequently provided analgesia (buprenorphine) and antibiotic treatment (enrofloxacin) before being subjected to post-operative care including analgesia and wellbeing monitoring for four days.

[0216] Survival studies

[0217] Initial dose-finding studies were performed using mouse recombinant CBG protein (His Tag, Sino Biological, Beijing, China; W50314-M08H) to establish a regimen that normalised plasma CBG concentrations in CLP mice to levels in healthy, unoperated controls (n=28, Figures 8A and 8B).

[0218] To assess the therapeutic effects of CBG on sepsis progression, morbidity, and mortality, a refined dose strategy was implemented in CLP mice equipped with telemetry. An optimal (Mid) CBG dose of 3.5 mg / kg was administered via intravenous (tail vein) injection at the 6-hour median onset of physiological decline, marked by hypotension and initial CBG decline, followed by a second dose of 2.5 mg / kg at 30 hr (n=12). Control CLP mice received volume-matched vehicle (200 mM mannitol and 100 mM trehalose in 0.9% (w / v) sterile saline) via tail vein injection (150 pL at 6 hr, 100 pL at 30 hr, n=24). All mice received postoperative care with tail vein blood sampling (20 pL) 48 hr prior to CLP, and at 12, 30, 54, and 78 hr post-CLP, used for CBG and lactate analyses.

[0219] Surviving mice at day 4 were fasted and anaesthetised before terminal cardiac bloods collected. Blood from mice that met early euthanasia criteria were also collected, but without fasting.

[0220] Post-operative care regimen

[0221] Mice were monitored and scored for body condition, real-time severe hypothermia (below 30°C) or hyperthermia (above 38°C), weight loss, reduced mobility, diarrhoea, abdominal distension, laboured breathing, or loss of righting reflex for a validated cumulative disease index (CDI) score (see Otero-Anton E, Gonzalez-Quintela A, Lopez-Soto A, Lopez-Ben S, Llovo J, Perez LF. Cecal ligation and puncture as a model of sepsis in the rat: influence of the puncture size on mortality, bacteraemia, endotoxemia and tumour necrosis factor alpha levels.Eur Surg Res. 2001;33(2):77-9) every 8 hr (0700, 1500, 2300) for 4 days. The presence of laboured breathing or loss of righting reflex was the threshold for early euthanasia. Mice received analgesia three times daily by subcutaneous injection.

[0222] Time-course studies

[0223] A longitudinal assessment of CBG's effects on sepsis progression and plasma biomarker profiles was undertaken in CLP mice equipped with telemetry and dosed with optimal CBG, as above. CLP mice were randomised to administration of intravenous (tail vein) CBG (n=40) or vehicle ( n=40), then humanly killed at 12, 24, 48, and 96 hr post-CLP (n=10 per timepoint) with 2 hr fasting and blood collection as above. A subset of mice was humanely killed at 6 hr post-CLP to serve as pre-intervention controls.

[0224] To establish reference plasma biomarker baseline concentrations and diurnal variation, we measured biomarkers in age-matched unoperated male C57BL / 6 mice (n=6 per timepoint) at 0, 6, 12, and 24 hr. Sham-operated controls were not used, as these show only transient, physiologically ranged increases in these biomarkers.

[0225] Plasma analyses

[0226] Plasma concentrations of total corticosterone, albumin, tissue hypoxia marker lactate, and organ damage markers of cardiac troponin-l, renal cystatin-C, liver alanine transaminase, and aspartate aminotransferase were determined using commercial ELISA kits. Free corticosterone was calculated using Coolen's equation. Plasma cytokines were determined via a customised MILLIPLEX® Mouse High Sensitivity T Cell Magnetic Bead Panel for TNFa, IL-6, IL-10, IL-12, macrophage inflammatory protein-2 (MIP-2), and interferon-|31 (I FN-pi). All assays were conducted as per the manufacturer's instructions.

[0227] Total plasma CBG was quantified using a custom in-house sandwich ELISA. Briefly, 96-well plates were coated with 100 pL of rabbit polyclonal anti-CBG antibody (#50314, Sino Biological, Beijing, China) diluted 1:1000 in coating buffer (1.9 g Na2CO3, 2.9 g NaHCO3 in 1 L of distilled water, pH adjusted to 9.6) and incubated for 18 hr at 4°C with shaking (200 rpm). After washing (3x) in phosphate-buffered saline with 0.05% Tween 20 (PBS-T), wells were blocked with PBS-T containing 1% gelatin for 30 min, followed by another wash. Mouse plasmasamples (100 pL), diluted 1:1000 in PBS-T with 0.1% gelatin, were then added and incubated for 2 hr at room temperature. The plate was washed (6x) and 100 pL of biotinylated rabbit polyclonal anti-CBG antibody (1:500 in PBS-T with 0.1% gelatin; Sino Biological) was added per well for 1 hr at RT. After washes (6x), 95 pL of streptavidin alkaline phosphatase diluted 1:1000 in PBS-T with 0.1% gelatin was added and incubated for 30 min at RT. After a final wash, 100 pL of QUANTI-Blue™ (#rep-qbs, InvivoGen, San Diego, CA, USA) was added per well for 1 hr with intermittent shaking. Absorbance was then measured at 640 nm using a BioTek Synergy HTX Multimode reader™.

[0228] Statistical analyses

[0229] Survival in CLP mice was evaluated using a 4-day Kaplan-Meier curve. All data were assessed for normality via the Shapiro-Wilk test. Weight change, GDI score, haemodynamics, and plasma biomarkers were compared between surviving and early euthanasia mice using a univariate analysis of variance in SPSS v29.0 (IBM Corporation, NY, USA), with time included as a between-mice effect. CDI score, weight changes, and plasma time course data were analysed using a two-way ANOVA with Tukey's post hoc analysis using GraphPad Prism vlO.O (Dotmatics, Boston, MA, USA).

[0230] Results

[0231] Survival series (tail bleeds; longitudinal)

[0232] The impact of CBG therapy on mortality, plasma CBG and lactate concentrations, weight, and CDI scores is detailed in Figures 8A and 8B and Figures 9A to 9C.

[0233] Figure 8A and Figure 8B shows the mortality rate and serum concentration of CBG following different doses of CBG in CLP mice ("Low" - single dose 63 pg (2.5 mg / kg), "Mid" -double dose 94 pg + 63 pg (3.5 mg / kg + 2.5 mg / kg), and "High" - 126 pg + 126 pg dose (5 mg / kg + 5 mg / kg)). Mortality was as low as 16.6% in mice treated with "Mid" CBG doses, compared to control mice mortality of 58.3% (Figure 6B). Treatment with CBG demonstrated a reduction in mortality in all treatment groups with up to 71.5% reduction with CBG administration (double dose at 3.5mg / kg + 2.5mg / kg at 6 hr and 30 hr, respectively). Further, as shown in Figure 8A, following CLP, plasma CBG concentration declined below the diurnal mean (shadedzone) from 12 hr (45%, p < 0.001) to 96 hr (61%, p < 0.001) with a nadir at 54 hr (63%) in control mice. Intravenous CBG administration at 6 and 30 hr post-CLP maintained plasma CBG within the pre-CLP diurnal range in CLP-CBG mice, except for an increase at 12 hr in both the Mid and High treatment groups, and 54 and 78 hours in the High treatment group, Figure 8A.

[0234] In view of the result for different treatments, the Mid dose (3.5 mg / kg at the 6-hour median onset of physiological decline followed by a second dose of 2.5 mg / kg at 30 hr) was used as the treatment dose (termed "CLP-CBG") for the remaining results presented herein.

[0235] Across both cohorts, lower plasma CBG was consistently associated with early euthanasia (p < 0.001 - Table 1). Plasma lactate increased steadily in control CLP mice from 54 hr (30%, p = 0.002) to 96 hr (69%, p < 0.001) relative to the diurnal mean, while CLP-CBG mice maintained plasma lactate concentrations close to the diurnal range of unoperated mice at all time points (Figure 9A); irrespective of treatment, mice requiring early euthanasia exhibited higher plasma lactate (29%, p < 0.001; Table 1).Table 1: Plasma biomarkers for CLP-sepsis survivors and non-survivors.< < < < < < < < < < < < < < <<<0236] pl = 96 hr adjusted CLP-CBG survivors vs control CBG survivors (each n=10), p2 = 96 hr adjusted control CBG survivors ( n=10) vs non-survivors (n=14).

[0237] Body weight decreased in control CLP mice from 32 hr (7.5%, p = 0.008) to 96 hr (11.3%, p < 0.001) relative to pre-operative levels, while CLP-CBG mice had decreased weight from 40 hr (8.1%, p = 0.002) to 72 hr post-CLP (7.9%, p = 0.003) before recovery at 96 hr (treatment effect, p < 0.001; Figure 9B). Weight loss was similar in surviving mice and mice requiring early euthanasia in their respective groups. Cumulative disease index score peaked at 24 hr (~4) in control CLP mice before declining to stabilise through to 96 hr (~3). The CDI score in CLP-CBG mice showed an earlier (16 hr), lower peak (~3.5), and then declined further (to ~2) from 24 to 96 hr (24 hr p = 0.003; 96 hr p = 0.024), indicating a CBG effect to lower CDI(treatment effect p < 0.001; Figure 9C). Cumulative disease index was higher in all CLP nonsurvivors compared to survivors (2.6-fold, p < 0.001).

[0238] Blood pressure

[0239] A hallmark of septic shock is hypotensive episode characterised by periods of low blood pressure (<65 mmHg in humans). Therefore, the influence of CBG administration on the blood pressure of mice was assessed. Isolated episodes of hypotension, defined as a 40 mmHg decrease in MAP from the pre-CLP diurnal mean within a 10-minute period, were seen in all CLP mice in the period from 3.8 to 7.1 hr post CLP, with onset at 4.9 ± 0.4 hr (prior to invention at 6 hrs). In control CLP survivors, the first hypotensive episode lasted for 7.5 ± 1.5 hr, reaching a nadir of 61 ± 4 mmHg (Figure 10A), but was longer in control CLP non-survivors, lasting 11.6 ± 1.1 hr with a nadir of 59 ± 6 mmHg (p = 0.042). In contrast, CLP-CBG survivors experienced a shorter first episode lasting 3.4 ± 0.3 hr, less than that in control CLP survivors (p = 0.005) and non-survivors (p < 0.001), despite a similar MAP nadir (62 ± 3 mmHg, Figure 10A). Of the CLP control mice that experienced episodes of hypotension, half (12 / 24) had two or more episodes, which were lethal. The other half (12 / 24) had a single episode, and most survived (10 / 12). In contrast, only three CLP-CBG mice (3 / 12) exhibited multiple episodes of hypotension, which were lethal in two mice; the remaining CLP-CBG mice (9 / 12) had a single episode, and all survived.

[0240] As indicated herein, septic shock is typically defined as hypotension concurrent with an elevation in plasma lactate 20% above the diurnal mean in unoperated mice. This developed in 58% (14 / 24) of control CLP mice, proving uniformly lethal. The onset of septic shock varied, at 13.5 ± 0.9 hr (2 mice), 39.3 ± 1.3 hr (8 mice), and 64.5 ± 0.8 hr (4 mice) and lasted 6.7 ± 0.8 hr until early euthanasia, with a final MAP of 53 ± 3 mmHg (Figure 10B). In contrast, none of the CLP-CBG mice developed septic shock, and although 2 / 12 underwent early euthanasia at 56 hr coincident with elevated lactate, their final MAP remained above the threshold of hypotension (93, 96 mmHg, Figure 10B).

[0241] Temperature

[0242] Figure 11A provides real-time calibrated core temperature profiles of CLP mice (both treated and untreated). Mild hypothermia between 35.5 and 32°C was observed in all CLP mice at 4.2 ± 0.3 hr post-CLP (prior to any intervention). This lasted 6.8 ± 0.9 hr in control CLP survivors and 10.3 ± 2.4 hr in non-survivors, with a nadir of 33.7 ± 0.4°C and 32.6 ± 0.5°C, respectively. In contrast, mild hypothermia in CLP-CBG survivors lasted 3.9 ± 0.6 hr with a nadir of 34.3 ± 0.3°C and was shorter than that in control CLP survivors (p = 0.015) and non-survivors (p < 0.001), indicating a significant CBG effect (p = 0.046). For both CLP-CBG non-survivors, mild hypothermia lasted 4.5 and 6.3 hr, with nadirs of 35.1 and 32.3°C, respectively.

[0243] An onset of hypothermia (below 32°C) was observed in all control CLP non-survivors at 3.9 hr (1 / 14), 15.9 hr (1 / 14), 33.5 hr (2 / 14), 41.0 ± 0.5 hr (6 / 14), 54.6 hr (1 / 14), and 66.1 hr (3 / 12), with most (10 / 14) beginning 1.9 ± 0.6 hr prior to the septic shock-related MAP decline, then progressing to severe hypothermia, below 30°C, with a final temperature of 28.4 ± 0.5°C at early euthanasia (Figure 11B). The remaining control CLP non-survivors (4 / 14) exhibited an earlier temperature decline starting 9.9 ± 1.1 hr before the septic shock-related MAP decline, then progressed to severe hypothermia with a final temperature of 27.9 ± 1.3°C at early euthanasia. Even without septic shock, both CLP-CBG non-survivors exhibited a similar temperature decline at 38.9 and 37.5 hr post-CLP, resulting in final temperatures of 30.7 and 29.8°C at early euthanasia (Figure 11B).

[0244] Time course series (cardiac bloods)

[0245] CBG and corticosterone

[0246] Figure 12 illustrates the profile of plasma CBG, total, and free corticosterone in CLP mice treated with CBG (CLP-CBG) or control CLP mice. As shown in Figure 8 (CBG Mid treatment), and reiterated in Figure 12A for reference, plasma CBG concentration declined in control CLP mice, prior to any intervention, from 6 hr to 96 hr with a nadir at 48 hr relative to the diurnal CBG mean in unoperated mice. CBG therapy maintained plasma CBG concentrations within or above this diurnal range.

[0247] Plasma total corticosterone increased from 6 hr in control CLP mice (2.2-fold, p = 0.002) prior to any intervention, peaked at 12 hr (2.8-fold, p < 0.001), then declined butremained elevated to 96 hr relative to the diurnal total corticosterone mean in unoperated mice (1.6-fold, p = 0.032; Figure 12B). In contrast, CLP-CBG mice exhibited a 56% decline in total corticosterone at 12 hr compared to control CLP mice (p < 0.001), then normalised within the diurnal range of unoperated mice beyond 24 hr, with a trend for CBG treatment effect (p = 0.059; Figure 12B). Plasma total corticosterone concentrations were higher in control CLP non-survivors compared to survivors (69%, p < 0.001; Table 1 above).

[0248] Plasma free corticosterone increased at 6 hr in control CLP mice (6.3-fold, p < 0.001), prior to any intervention, peaked at 12 hr (8.2-fold, p < 0.001), then declined but remained elevated at, and beyond, 24 hr relative to the diurnal free corticosterone mean in unoperated mice (3.5-fold, p = 0.002; Figure 12C). In contrast, free corticosterone was lower in CLP-CBG mice compared to control CLP mice beyond 12 hr (74%, p < 0.001), then normalised within the diurnal range of unoperated mice with a significant CBG treatment effect (p < 0.001; Figure 12C). Free corticosterone concentrations were higher in all CLP non-survivors compared to survivors (149%, p < 0.001; Table 1 above).

[0249] Cytokines

[0250] Plasma cytokine profiles, detailed in Figure 13, were markedly elevated in control CLP mice. Prior to any intervention I L-ip, IL-6, IL-12, MIP-2, TNFa, IL-10, and I FN-01 increased at 6 hr by 61-fold, 84-fold, 37-fold, 21-fold, 9.7-fold, 45-fold, and 8.8-fold, respectively, relative to their diurnal mean concentration in unoperated mice (all p < 0.001). These peaked at 12 hr, with an increase of 71-fold for IL-1 , 178-fold for IL-6, 164-fold for IL-12, 40-fold for MIP-2, 12-fold for TNFa, 70-fold for IL-10, and 17-fold for IFN-01 (all p < 0.001). Subsequently, IL-10, IL-6, IL-12, MIP-2, TNFa, and IL-10 declined, but remained elevated to 96 hr (20-fold, p = 0.003; 94-fold, p < 0.001; 57-fold, p = 0.002; 16-fold, p < 0.001; 6.1-fold, p = 0.026, respectively). In contrast, IFN-01 concentrations normalised to the diurnal IFN-01 range in unoperated mice by 96 hr.

[0251] CBG-treated CLP-CBG mice exhibited a transient, marked reduction in plasma concentrations of IL-10 (36%), IL-6 (48%), IL-12 (44%), MIP-2 (58%), and TNFa (40%) at 12 hr relative to control CLP mice (all p < 0.001), followed by an increase at 24 hr. IL-6 concentrations in CLP-CBG mice fell below those in control CLP mice at 48 and 96 hr (46% and 44%,respectively; p < 0.001), whereas IL-1 , IL-12, MIP-2, and TNFa returned to concentrations observed in control CLP mice at these time points (Figure 13). In stark contrast, IL-10 and IFN-pi peaked higher in CLP-CBG mice at 12 hr than in control CLP mice (by 33% and 32%, respectively, both p < 0.001) then despite a later decline, these anti-inflammatory cytokines remained markedly elevated in CLP-CBG mice to 96 hr relative to control CLP mice (46% and 87%, respectively, both p < 0.001; Figure 13). Significant CBG treatment effects were observed for IL-10 (p = 0.048), IL-6 (p = 0.033), IL-10 (p < 0.001), IL-12 (p = 0.037), and I FN-10 (p < 0.001), while IL-13 (71%), IL-6 (91%), IL-10 (96%), IL-12 (1.4-fold), TNFa (1.7-fold), and I FN-10 (18-fold) were higher in control CLP non-survivors compared to survivors (p < 0.001 for all; Table 1 above).

[0252] Systemic damage markers

[0253] Figure 14 illustrates plasma albumin and lactate profiles in CBG treated and control treated CLP mice. Concentrations of both remained within or close to the diurnal ranges of unoperated mice (shaded areas) at 6 hr, prior to any intervention. Albumin concentrations then decreased in control CLP mice after 24 hr, with a 48 hr nadir (45%, p = 0.0032; Figure 14A), while lactate concentrations increased from 48 hr through to 96 hr (45%, p < 0.001; Figure 14B). Conversely, CLP-CBG mice maintained albumin and lactate concentrations within the diurnal ranges of unoperated mice throughout the study period (CBG treatment effect, p = 0.003, p = 0.046, respectively). Furthermore, control CLP non-survivors exhibited higher plasma lactate and lower albumin concentrations compared to survivors (both p < 0.001; Table 1 above).

[0254] Organ damage markers

[0255] Figure 15 illustrates plasma cystatin-C, troponin-l, ALT, and AST profiles in CBG treated and control treated CLP mice. These markers increased at 6 hr, prior to any intervention, in control CLP mice (2.1-fold for cystatin-C, 6.3-fold for troponin-l, 1.7-fold for ALT, and 2.9-fold for AST; all p < 0.001) and peaked at 12 hr (increases of 3.2-fold, 14.6-fold, 3.5-fold, and 2.8-fold, respectively; all p < 0.001) relative to diurnal mean concentrations in unoperated mice (shaded areas) . Cystatin-C remained elevated to 96 hr (2.8-fold, p < 0.001; Figure 15A), while troponin-l increased progressively to 96 hrs in control CLP mice (50.7-fold,p < 0.001; Figure 15B). ALT peaked at 48 hr with a 3.3-fold increase (p < 0.001; Figure 15C) before declining in control CLP mice. However, ALT remained elevated at 96 hr (1.3-fold, p = 0.045; Figure 10C). AST concentrations returned to the diurnal range by 96 hr (Figure 15D).CBG therapy markedly attenuated the rise in plasma cystatin-C and troponin-l concentrations seen in control CLP. CLP-CBG mice demonstrated lower cystatin-C at 12 hr and beyond as lower troponin-l at 12 hr and beyond 48 hr (respective CBG treatment effect, p < 0.001 and p = 0.031) with peak differences at 96 hr (43% and 55%; Figure 15A, B). Plasma ALT concentrations in CLP-CBG mice also decreased transiently following CBG administration at 12 hr (59%, p < 0.001) and 48 hr (60%, p < 0.001), despite similar concentrations in control CLP and CLP-CBG mice at 24 and 96 hr (Figure 15C). In contrast, plasma AST was only transiently decreased at 12 hr in CLP-CBG mice (43%, p = 0.002), then paralleled the response in control CLP mice (Figure 15D). Control CLP non-survivors exhibited higher plasma troponin-l (20%, p = 0.046) and AST (1.6-fold, p = 0.044) concentrations compared to survivors (Table 1, above).

[0256] The above work demonstrates that CBG administration (including administering CBG containing triantennary glycans at Asn347, particularly TS3 and TS3F) can rescue subjects from fatal shock and attenuate the physiological response to sepsis. This provides, a significant clinical intervention which substantially reduces fatality caused by sepsis and septic shock.

[0257] Qualifications

[0258] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0259] Any embodiment of the present disclosure herein shall be taken to apply mutatis mutandis to any other embodiments of the disclosure unless specifically stated otherwise.

[0260] Although the present disclosure has been described with reference to particular embodiments, it will be appreciated that the disclosure may be embodied in many other forms. It will also be appreciated that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosureincludes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

[0261] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art.

[0262] It is to be noted that, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context already dictates otherwise.

[0263] Where a range of values is expressed, it will be clearly understood that this range encompasses the upper and lower limits of the range, and all numerical values or sub-ranges in between these limits as if each numerical value and sub-range is explicitly recited. The statement "about X% to Y%" has the same meaning as "about X% to about Y%," unless indicated otherwise.

[0264] The term "about" as used in the specification means approximately or nearly and in the context of a numerical value or range set forth herein is meant to encompass variations of + / - 10% or less, + / - 5% or less, + / - 1% or less, or + / - 0.1% or less of and from the numerical value or range recited or claimed.

[0265] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0266] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in the field in any country.

[0267] Any subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure orthe claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.

[0268] Future patent applications may be filed on the basis of the present application, for example by claiming priority from the present application, by claiming a divisional status and / or by claiming a continuation status. It is to be understood that the following claims are provided by way of example only and are not intended to limit the scope of what may be claimed in any such future application. Nor should the claims be considered to limit the understanding of (or exclude other understandings of) the present disclosure.

Claims

CLAIMS:

1. A method of treating or preventing sepsis, or treating, preventing or delaying a clinical pathology of sepsis, the method comprising administering to a patient an effective amount of corticosteroid-binding globulin (CBG).

2. The method according to claim 1, wherein the method treats or prevents septic shock, or treats, prevents or delays a clinical pathology of septic shock.

3. The method according to claim 2, wherein the clinical pathology of septic shock is selected from one or more of: mortality, time in septic shock, time in first septic shock episode, time to septic shock onset, number of septic shock episodes, intubation time, requirement for mechanical ventilation, requirement for vasopressor therapy, requirement for inotrope therapy, requirement for renal replacement therapy, requirement for Extracorporeal Membrane Oxygenation (ECMO) therapy, requirement for antimicrobial therapy, vascular injury, capillary leakage, hypotension, hyperthermia, tachycardia, tachypnea, lactic acidosis, hypoxia, cyanosis, ileus, hyperglycemia, CNS dysfunction, sequential organ failure, multiorgan dysfunction syndrome, lung dysfunction, cardiac dysfunction, kidney dysfunction, and liver dysfunction.

4. The method according to any one of claims 1 to 3, wherein sepsis or septic shock is measured by one or more prognostic markers and wherein the method modulates the one or more prognostic markers, wherein the markers are selected from: increased serum lactate, reduced bicarbonate, increased pH, elevated serum creatinine, reduced eGFR, reduced serum albumin, increased bilirubin, reduced platelet count, elevated international normalised ratio, prolonged prothrombin time, elevated D-Dimer, elevated C-reactive protein, elevated interleukin-6, elevated interleukin-8, reduced interleukin-10, elevated monocyte chemoattractant protein 1, elevated presepsin, elevated pro-adrenomedullin, elevated procalcitonin, elevated cystatin-C, elevated troponin, elevated alanine transaminase, elevated aspartatetransaminase, elevated tumour necrosis factor alpha, total CBG less than 200 nmol / L, or Sequential Organ Failure Assessment (SOFA) score.

5. The method according to claim 2 or claim 3, wherein the method treats or prevents multiorgan dysfunction syndrome (MODS).

6. The method according to any one of claims 1 to 5, wherein the CBG comprises a CBG glycoform having a triantennary glycan at asparagine (Asn) 347.

7. A method of modulating the biological activity of a corticosteroid in a subject, the method comprising administering an effective amount of a corticosteroid-binding globulin (CBG), wherein the CBG includes a triantennary glycan at asparagine (Asn) 347.

8. A method of treating a corticosteroid-associated condition, the method comprising administering to a subject an effective amount of a corticosteroid-binding globulin (CBG), wherein the CBG comprises a CBG glycoform having a triantennary glycan at asparagine (Asn) 347.

9. A method of treating or preventing an immunological or inflammatory condition, the method comprising administering to a patient an effective amount of a corticosteroid-binding globulin (CBG), wherein the CBG comprises a CBG glycoform having a triantennary glycan at asparagine (Asn) 347.

10. The method according to claim 9, wherein the immunological or inflammatory condition is a chronic immunological or inflammatory condition.

11. The method according to claim 9, wherein the immunological or inflammatory condition is an acute immunological or inflammatory condition.

12. The method according to any one of claims 6 to 11, wherein the triantennary glycan comprises triantennary trisialylated (TS3) and / or triantennary trisialylated core- fucosylated (TS3F) glycan.

13. The method according to any one of claims 1 to 12, wherein the CBG is serum or plasma isolated, or is a recombinant CBG.

14. The method according to claim 13, wherein the serum or plasma isolated CBG or the recombinant CBG is enriched for a CBG glycoform having a triantennary glycan at Asn347.

15. The method according to claim 14, wherein the triantennary glycan comprises triantennary trisialylated (TS3) and / or triantennary trisialylated core-fucosylated (TS3F).

16. The method according to any one of claims 1 to 15, wherein the effective amount of the CBG is administered in a dosage schedule comprising at least two doses or is continuous infusion.

17. The method according to any one of claims 1 to 16, wherein the CBG is administered by subcutaneous injection or intravenous injection.

18. The method according to any one of claims 1 to 17, wherein the method comprises a combination therapy comprising administration of a corticosteroid in combination with the CBG.

19. The method according claims 18, wherein the corticosteroid is one or more of betamethasone, budesonide, ciclesonide, corticosterone, corticosterone acetate, fludrocortisone, hydrocortisone, methylprednisolone, dexamethasone, prednisolone or prednisone.

20. The method according to claim 18, wherein the corticosteroid is a glucocorticoid.

21. The method according to any one of claims 1 to 20, wherein the method comprises a step of quantifying the concentration of total CBG or a glycoform of CBG in a biological sample from the patient.

22. The method according to claim 21, wherein the glycoform of CBG includes a triantennary glycan at asparagine (Asn) 347.

23. The method according to claim 22, wherein the triantennary glycan is selected from: triantennary trisialylated (TS3) and triantennary trisialylated core-fucosylated (TS3F).

24. A pharmaceutical composition comprising isolated corticosteroid-binding globulin (CBG), a recombinant CBG, or an in vitro produced CBG, comprising a triantennary glycan at asparagine (Asn) 347 of CBG.

25. The pharmaceutical composition according to claim 24, wherein the isolated or recombinant CBG is enriched for CBG having a triantennary glycan at Asn347.

26. The pharmaceutical composition according to claim 24 or claim 25, wherein the pharmaceutical composition comprises a relative abundance of about 42% or more of CBG having a triantennary glycan at Asn347.

27. The pharmaceutical composition according to any one of claims 24 to 26, wherein the isolated CBG comprises a serum or plasma fraction of CBG enriched for a triantennary glycan at Asn347.

28. The pharmaceutical composition according to any one of claims 24 to 27, wherein the triantennary glycan is trisialylated (TS3).

29. The pharmaceutical composition according to any one of claims 24 to 28, wherein the triantennary glycan is core-fucosylated (TS3F).

30. The pharmaceutical composition according to any one of claims 24 to 29, wherein the isolated CBG is isolated from serum or plasma.

31. The pharmaceutical composition according to claim 30, wherein the serum or plasma isolated CBG is obtained by cohn fractionation.

32. The pharmaceutical composition according to any one of claims 24 to 26, wherein the recombinant CBG comprising a triantennary glycan at Asn347 is produced from a mammalian cell line.

33. A method of predicting the likelihood of a patient experiencing sepsis or a clinical pathology of sepsis, the method comprising the steps of:analysing a biological sample from the patient for a concentration of a glycoform of corticosteroid-binding globulin (CBG), wherein the glycoform of CBG has a triantennary glycan at asparagine (Asn) 347,wherein the concentration of the glycoform of CBG is indicative of the risk of sepsis or a clinical pathology of sepsis.

34. The method according to claim 33, wherein the concentration of the glycoform of CBG is inversely correlated to the risk of sepsis or a clinical pathology of sepsis.

35. The method according to claim 33 or claim 34, wherein the sepsis results in septic shock or a clinical pathology of septic shock.

36. The method according to claim 35, wherein the clinical pathology of septic shock is selected from one or more of: mortality, time in septic shock, time in first septic shock episode, time to septic shock onset, number of septic shock episodes, intubation time, requirement for mechanical ventilation, requirement for vasopressor therapy, requirement for inotrope therapy, requirement for renal replacement therapy, requirement for Extracorporeal membrane oxygenation (ECMO) therapy, requirement for antimicrobial therapy, vascular injury, capillary leakage, hypotension, hyperthermia, tachycardia, tachypnea, lactic acidosis, hypoxia, cyanosis, ileus, hyperglycemia, CNS dysfunction, sequential organ failure, multiorgan dysfunction syndrome, lung dysfunction, cardiac dysfunction, kidney dysfunction, and liver dysfunction.

37. The method according to any one of claims 33 to 36, wherein the method predicts the likelihood of mortality of the patient.

38. The method according to any one of claims 33 to 37, wherein the method predicts the likelihood of multiorgan dysfunction syndrome (MODS).

39. The method according to any one of claims 33 to 38, wherein the triantennary glycan at Asn347 is selected from: triantennary trisia lylated (TS3) and / or triantennary trisialylated core-fucosylated (TS3F).

40. The method according to any one of claims 33 to 39, wherein the glycoform of CBG is a TS3F Asn347 glycoform and method predicts one or more of: the likelihood of the patient requiring mechanical ventilation, the duration of mechanical ventilation of the patient, or the sepsis severity of the patient.

41. The method according to any one of claims 33 to 40, wherein the method further comprises comparing the concentration of the glycoform of CBG to a reference standard.

42. The method according to any one of claims 33 to 41, wherein a serum concentration of the glycoform of CBG, having a triantennary glycan at Asn347, of about 60 nmol / L or less, of about 55 nmol / L or less, or about 50 nmol / L or less, or about 45 nmol / L or less, or about 40 nmol / L or less, or about 35 nmol / L or less, or about 30 nmol / L or less, or about 25 nmol / L or less is indicative of a higher likelihood of sepsis or a clinical pathology of sepsis.

43. The method according to any one of claims 33 to 42, wherein a serum concentration of about 45 nmol / L or more of the glycoform of CBG, having a triantennary trisialylated (TS3) glycan at Asn347, indicates a higher likelihood of survival.

44. The method according to any one of claims 33 to 43, wherein a serum concentration of about 30 nmol / L or less of the glycoform of CBG, having a triantennary trisialylated (TS3) glycan at Asn347, indicates a higher likelihood of mortality.

45. The method according to any one of claims 33 to 44, wherein a serum concentration of a triantennary trisialylated (TS3F) glycan at Asn347 of about 53 nmol / L or more indicates a lower likelihood of the patient requiring mechanical ventilation.

46. The method according to any one of claims 33 to 44, wherein a serum concentration of triantennary trisia lylated (TS3F) glycan at Asn347 of about 38 nmol / L or less indicates a higher likelihood of the patient requiring mechanical ventilation.

47. The method according to any one of claims 33 to 46, wherein the concentration of the glycoform of CBG is measured by LC-MS / MS.

48. A method of treating or preventing sepsis, or treating, preventing or delaying a clinical pathology of sepsis, according to any one of claims 1 to 23, including performing the method of predicting the likelihood of a patient experiencing sepsis or a clinical pathology of sepsis according any one of claims 33 to 47.

49. A method of predicting the response of a subject to administration of a corticosteroid, the method comprising analysing a biological sample from the subject for a concentration of a glycoform of corticosteroid-binding globulin (CBG), wherein the glycoform of CBG has a triantennary glycan at asparagine (Asn) 347.

50. The method according to any one of claims 1 to 23 comprising administering the pharmaceutical composition according to any one of claims 24 to 32.

51. The method according to any one of claims 21 to 23, 33 to 47, or 49, wherein the biological sample is a blood sample, serum sample or plasma sample.

52. The pharmaceutical composition according to any one of claims 24 to 32 for use in the treatment or prevention of an immunological or inflammatory condition or sepsis, or for the treatment, prevention or delay of a clinical pathology of an immunological or inflammatory condition or sepsis.

53. The pharmaceutical composition according to any one of claims 24 to 32 for use in the treatment or prevention of septic shock, or for use in the treatment, prevention or delay of a clinical pathology of septic shock.

54. The pharmaceutical composition according to any one of claims 24 to 32 for use in modulating the biological activity of a corticosteroid in a subject.

55. The pharmaceutical composition according to any one of claims 24 to 32 for use in a combination therapy with a corticosteroid in the treatment or prevention of an immunological or inflammatory condition or sepsis, or for use in the treatment, prevention or delay of a clinical pathology of an immunological or inflammatory condition or sepsis.

56. The pharmaceutical composition for use according to any one of claims 24 to 32 for use in a combination therapy with a corticosteroid in the treatment or prevention of septic shock, or for use in the treatment, prevention or delay of a clinical pathology of septic shock.

57. The pharmaceutical composition for use according to claim 55 or claim 56, wherein the corticosteroid is one or more of: betamethasone, dexamethasone, fludrocortisone, ciclesonide, corticosterone, corticosterone acetate, hydrocortisone, methylprednisolone, prednisolone or prednisone.

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