WO2015131245A1

WO2015131245A1 - Sepsis treatment

Info

Publication number: WO2015131245A1
Application number: PCT/AU2015/050085
Authority: WO
Inventors: Hamsa Puthalakath; Marcel DOERFLINGER
Original assignee: La Trobe University
Current assignee: La Trobe University
Priority date: 2014-03-03
Filing date: 2015-03-03
Publication date: 2015-09-11
Anticipated expiration: 2016-09-03

Abstract

The present disclosure relates to methods for preventing and/or treating immunosuppression in a subject in need thereof by administration of an inhibitor of BiP which binds to a secreted form of BiP. In particular, the methods are useful for treatment of immunosuppression characterised by sepsis induced leukopenia. Diagnostic methods based on detection of BiP protein are also provided.

Description

SEPSIS TREATMENT

Incorporation by reference

All documents cited or referenced herein, and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference in their entirety.

This application claims priority from AU 2014900694, the entire contents of which are herein incorporated by reference.

Field of the Invention

Background of the Invention

In the United States alone, approximately 750,000 individuals develop sepsis, and approximately 30% succumb to this disorder annually. Sepsis is the 10th leading cause of death, and the annual financial burden incurred is in excess of $16 billion (Angus DC et al, (2001 ) Crit Care Med 29:1303-1310). In Australia, there has been a four-fold increase in sepsis incidents between 1997 and 2005, owing to an ageing population (Peake S, (2007) Critical Care 1 1 (Suppl 2):P732). Sepsis is defined as the host inflammatory response to severe, life- threatening infection with the presence of organ dysfunction. The host immune response to sepsis can be divided into two stages, a hyper-inflammatory phase and a hypo-inflammatory phase. During the hyper-inflammatory phase, activated immune cells (mostly the innate immune system) produce copious amounts of inflammatory cytokines, which can lead to multiple organ failure. However, improved treatment protocols have resulted in most patients surviving this stage and entering a protracted immune suppressive phase (Hotchkiss RS et al, (2013) Nat Rev Immunol 13:862-874). This phase is characterized by extensive apoptosis in the cells of the adaptive immune system, i.e., B cells, and T cells (Ayala A et al, (1996) Blood 87:4261 -4275; Efron PA et al, (2004) Shock 21 :566-571 ; Hotchkiss RS et al, (1999) Crit Care Med 27:1230-1251 ) leading to prolonged lymphopenia where the patients are susceptible to nosocomial infections. The lymphopenia also leads to the patient's inability to fight of the initial infection or results in the activation a latent infection, ultimately leading to death.

Experimental drug therapies for sepsis are at cross roads with more than 30 drug trials failing in the last 25 years. In January 201 1 , Japan's Eisai announced that it would delay seeking regulatory approval for the company's anti-Toll-like receptor 4 (TLR4) compound after the drug, known as Eritoran, failed to show any benefit in a pivotal 2,000-person trial. Later that year (October 201 1 ), Eli Lilly famously withdrew Xigris (activated protein C) from the market, following the negative results of the 1 ,700-person PROWESSSHOCK trial. With the withdrawal of Xigris, critical-care physicians now have no drugs specifically approved to treat severe sepsis. Many in the field are also eagerly awaiting the results of a 300-person trial testing AstraZeneca's CytoFab, an antibody directed against tumor necrosis factor-alpha (TNFa), a prime mediator of inflammation. Previous experimental TN Fa-targeting monoclonal antibodies have failed to show an effect on mortality rates in patients with sepsis and therefore, the chances of TNFa based therapies being successful are slim. Similarly, the fate of Talactoferrin alfa (an immunomodulatory lactoferrin, Agennix, Germany) is in limbo after more participants died in the treatment arm compared to the placebo arm of the study. Failure of these trials of different anti-inflammatory agents is inconsistent with the hypothesis that inflammation is a key driving mechanism.

The current thinking in the field is that methods for identifying when patients have entered the immunosuppressive phase of sepsis and for detecting particular defects in immunity will enable the application of potent new immunotherapies. Accordingly, there is a need in the art for improved therapies for the treatment of sepsis.

Summary of the Disclosure

Out of all sepsis related deaths, only 15% of deaths occur in intensive care units (ICUs) due to multiple organ failure and the remaining (more than 80%) occur during the prolonged lymphopenia stage where the patients succumb to infections. Lymphocyte apoptosis plays a central role in the pathophysiology of sepsis, however the cause of lymphocyte apoptosis is unknown.

The present inventors sought to identify the factor(s) that lead to apoptosis of lymphocytes and antigen presenting cells in sepsis and hypothesised that activated macrophages and other antigen presenting cells secrete a factor that can induce a systemic lymphocyte death.

The present inventors identified a secreted form of the endoplasmic reticulum (ER) resident chaperone BiP (immunoglobulin binding protein; GRP78) that appears to be responsible for mediating apoptosis in sepsis. In particular, BiP appears to be responsible for mediating apoptosis of leukocytes in the immune suppressive phase of sepsis.

In one embodiment, the present disclosure provides a method for preventing and/or treating immunosuppression in a subject in need thereof, comprising administering to the subject an inhibitor of BiP (GRP78) which binds to, or specifically binds to a secreted form of BiP (GRP78).

In one example, the immunosuppression is characterised by apoptosis of leukocytes. In another example, the immunosuppression is characterised by apoptosis of lymphocytes and antigen presenting cells. In yet another example, the immunosuppression is characterised by apoptosis of T and B lymphocytes. In another example, the inhibitor of BiP inhibits or prevents Bim-induced apoptosis of lymphocytes and/or antigen-presenting cells (APC) by the secreted form of BiP.

In one example, the immunosuppression is characterised by sepsis induced leukopenia.

In one example, the subject being treated is in the immune suppression or hypoinflammatory phase of sepsis characterised by leukopenia and/or lymphopenia. Methods for diagnosing leukopenia or lymphopenia will be familiar to persons skilled in the art. For example, leukopenia or lymphopenia can be readily determined in a subject by measuring the subjects white blood cell count and comparing the value to the normal range. In one example leukopenia or lymphopenia is determined by measuring the white blood cell count according to standard pathology criteria. In yet another example, the subject has a white blood cell count less than 4,000-4,500 white blood cells per microliter of blood. In another example, the subject has a neutrophil count less than 500 cell per microliter of blood. In another example, the subject has a lymphocyte count less than 1 ,500 cells per microliter of blood. In another example, the subject is a paediatric subject having a lymphocyte count less than 3,000 cells per microliter of blood.

In another embodiment, the present disclosure provides a method of inhibiting and/or preventing apoptosis of leukocytes in a subject in need thereof, comprising administering to the subject an inhibitor of BiP (GRP78) which binds to, or specifically binds to a secreted form of BiP (GRP78).

In one example, the leukocytes comprise lymphocytes and antigen presenting cells (APCs). In a further example, the lymphocytes are T and/or B cells. In one example, the subject in need thereof is a sepsis subject.

In one example, the method inhibits or prevents apoptosis of T cells, B cells and NKT cells by the secreted form of BiP. In another example, the method inhibits or prevents apoptosis of T and/or B cells by the secreted form of BiP. In a further example, the inhibitor of BiP inhibits or prevents Bim-induced apoptosis of lymphocytes and/or antigen-presenting cells (APC) by the secreted form of BiP.

In one example, the inhibitor of BiP according to any method described herein is a binding agent which binds to, or specifically binds to secreted BiP (GRP78). In a further example, the secreted BiP is human BiP. In yet a further example, the inhibitor binds to, or specifically binds to a human BiP protein comprising the sequence according to SEQ ID NO:1 . In another example, the inhibitor of BiP binds to, or specifically binds to a sequence encoded by the sequence according to SEQ ID NO:2. In yet a further example, the inhibitor binds to, or specifically binds to a BiP protein comprising the sequence according to SEQ ID NO:3. In yet another example, the binding agent binds to, or specifically binds to a human BiP protein comprising the sequence according to SEQ ID NO:1 or a sequence at least 95%, at least 97%, at least 98%, or at least 99% identical thereto. In a further example, the binding agent binds to a human BiP protein comprising the sequence according to SEQ ID NO:3.

In yet another example, the secreted BiP protein is produced by activated macrophages. In yet a further example, the BiP protein is capable of preventing induction of Bim. In one example, the BiP protein is capable of preventing induction of Bim transcription. While not wishing to be bound by theory, it is postulated that BiP binds to receptor which is present on T and/or B cells which produces a cascade of events within the cell resulting in induction of Bim and expression of Bim protein leading to apoptosis of the cell.

In another embodiment, the present disclosure provides a method of preventing and/or treating sepsis in a subject in need thereof, comprising administering to the subject an inhibitor of BiP (GRP78) which binds to, or specifically binds to a secreted form of BiP (GRP78). In another example, the method prevents and/or treats sepsis induced leukopenia in the subject.

In another embodiment, the present disclosure provides for use of an inhibitor of the secreted form of BiP (GRP78) for preventing and/or treating immunosuppression in a subject in need thereof.

In another embodiment, the present disclosure provides for use of an inhibitor of the secreted form of BiP for preventing and/or treating apoptosis of leukocytes in a subject in need thereof. In one example, the leukocytes are lymphocytes and/or antigen presenting cells (APC).

In another embodiment, the present disclosure provides for use of an inhibitor of the secreted form of BiP (GRP78) for preventing and/or treating sepsis in a subject in need thereof.

In one example, the inhibitor according to any use described herein, binds to, or specifically binds to a secreted form of BiP (GRP78).

In a particular example, the methods and/or uses described herein prevent and/or treat sepsis induced leukopenia in the subject. In another embodiment, the present disclosure provides for an inhibitor to a secreted form of BiP for use in preventing and/or treating immunosuppression in a subject in need thereof.

In another embodiment, the present disclosure provides for an inhibitor of a secreted form of BiP for use in preventing and/or treating apoptosis of leukocytes in a subject in need thereof. In a further example, the leukocytes comprise lymphocytes and antigen presenting cells (APCs). In a further example, the lymphocytes are T and/or B cells.

In another embodiment, the present disclosure provides for an inhibitor of a secreted form of BiP for use in preventing and/or treating sepsis in a subject in need thereof.

In a particular example, the inhibitors described herein prevent and/or treat sepsis induced leukopenia in the subject.

In a particular example, the binding agent according to the methods or uses described herein is an inhibitor of BiP (GRP78). In another example, the inhibitor is an inhibitor of human BiP. In yet another example, the inhibitor binds to, or specifically binds to secreted BiP. In yet another example, the inhibitor of BiP binds to, or specifically binds to a human BiP protein comprising the sequence according to SEQ ID NO:1 or a sequence at least 95%, at least 97%, at least 98%, or at least 99% identical thereto. In a further example, the inhibitor of BiP binds to a human BiP protein comprising the sequence according to SEQ ID NO:3.

The 'subject in need thereof according to the present disclosure is one which has been diagnosed with sepsis, or is suspected of having, or at risk of acquiring sepsis (also referred to herein as a 'sepsis subject'). In another example, the subject exhibits one or more symptoms characteristic of organ dysfunction. In a further example, the subject has, is suspected of having or is at risk of acquiring sepsis-induced leukopenia and/or lymphopenia. Diagnosis of sepsis in a subject is generally made according to art-known criteria and is explained in further detail below.

In a further example, the subject is hospitalised. In yet another example, the subject is in intensive care. Subjects that may be at risk of acquiring sepsis may be selected from, but not limited, elderly subjects, surgical subjects, burns subjects, trauma subjects, cancer subjects, immunocompromised subjects (e.g. AIDS), subjects with acute respiratory distress syndrome (ARDS) or subjects with a hospital-acquired secondary (nosocomial) infection. In a further example, the subject does not have cancer. In yet another example, the subject does not have fungal sepsis. In yet another example, the subject does not have an existing infection. In another example, the subject does not have a heart disorder (e.g. myocardial infarction). In a yet further example, the subject does not have an autoimmune disease, for example rheumatoid arthritis. In another example, the subject does not have a disorder associated with weight gain such as diabetes or obesity. The subject to be treated according to the methods of the disclosure may have acquired sepsis through any means, for example, a bacterial infection, (either Gram-negative or Gram-positive) or by other pathogens such as fungi, viruses, and parasites and non-infective small stimuli such as superantigens. In one example, the subject does not have fungal sepsis. In a further example, the subject has a gram positive bacterial infection. In another example, the subject has a gram negative bacterial infection.

A diagnosis of sepsis in a subject may proceed according to art known criteria. One or more of the following may be symptomatic of a subject with sepsis, including presence of acute inflammation present throughout the entire body, frequently associated with fever and elevated white blood cell count (leucocytosis) or low white blood cell count and lower than average temperature, and vomiting. The subject may also be exhibiting one or more symptoms resulting from the host's immune response to the infection resulting in hemodynamic consequences and damage to organs. This host response has been termed "systemic inflammatory response syndrome (SIRS)" and is characterised by an elevated heart rate (above 90 beats per minute), high respiratory rate (above 20 breaths per minute or a partial pressure of carbon dioxide in the blood of less than 32), abnormal white blood cell count (above 12,000 cells/mm³, lower than 4,000 cells/mm³, or greater than 10% band forms (immature white blood cells)) and elevated or lowered body temperature i.e. under 36^SC or over 38^SC. Sepsis can be differentiated by SIRS by the presence of a known or suspected pathogen. For example, SIRS and a positive blood culture for a pathogen indicates the presence of sepsis. However, in many cases of sepsis no specific pathogen is identified.

The term "sepsis" as used herein will be understand as encompassing all the various forms of sepsis, for example, as derived according to the American College of Chest Physicians and the Society of Critical Care Medicine. Accordingly, the subject according to the present disclosure may be diagnosed as having SIRS, sepsis, severe sepsis or septic shock with the proviso that the subject also exhibits leukopenia or is at risk of developing leukopenia.

The different levels of sepsis include SIRS as described above, sepsis, severe sepsis and septic shock. Sepsis is defined as SIRS in response to a confirmed infectious process. The infection may be suspected or proven (by culture, stain, or polymerase chain reaction), or a clinical syndrome pathognomonic for infection. Specific evidence for infection includes WBC in normally sterile fluid (such as urine or cerebrospinal fluid (CSF)), evidence of perforated viscus (free air on abdominal x-ray or CT scan, signs of acute peritonitis), abnormal chest x-ray consistent with pneumonia or petechiae, purpure, or purpure fulminans. Severe sepsis is defined as sepsis with organ dysfunction, hypoperfusion or hypotension. Septic shock is defined as sepsis with refractory arterial hypotension or hypoperfusion abnormalities in spite of adequate fluid resuscitation. Signs of systemic hypoperfusion may be either end organ dysfunction or serum lactate greater than 4 mmol/dL. Other signs include oliguria and altered mental status. Subjects are defined as having septic shock if they have sepsis plus hypotension after aggressive fluid resuscitation (typically upwards of 6 litres or 40 ml/kg of crystalloid.

The subject may be diagnosed as having end-organ dysfunction, examples of which include lungs (acute lung injury or acute respiratory distress syndrome); brain (encephalopathy, ischemia, haemorrhage, microthrombi, mictoabscesses, multifocal necrotizing leukoencephalopathy); liver (disruption of protein synthetic function or disruption of metabolic function); kidney (oliguria and anuria, electrolyte abnormalities, volume overload); heart (systolic and diastolic failure, cellular damage); cardiovascular dysfunction (after fluid resuscitation with at least 40 ml/kg of crystalloid; hypotentsion, vasopressor requirement, acidosis, oliguria, core to peripheral temperature difference > 3^SC); respiratory dysfunction (in the absence of cyanotic heart disease or known chronic lung disease); neurologic dysfunction; hematologic dysfunction (platelet count <80,000/mm³ or 50% drop from maximum in chronically thrombocytopenic patients; disseminated intravascular coagulation); renal dysfunction; or hepatic dysfunction. With the proviso that the subject is lymphopenic, any of the above forms of sepsis may be treated according to the methods of the present disclosure.

The present inventors are the first to demonstrate a pro-apoptotic function for BiP (GRP78) which exists in a secreted form. This is contrary to the well understood function of BiP as protecting cells from undergoing apoptosis, as described in, for example Luo S et al., (2006) Mol Cell Biol. 26(15):5688-97; Liu et al., (2013) Clinical Cancer Res. 19:6802-1 1 ; Zhang et al, (2013) PLoS One 8(1 1 ):e8007. In particular, the present inventors have determined that, unlike the form of BiP which is recombinantly expressed from bacterial cells and which does not have the property of killing target cells, activated macrophages are capable of producing a secreted form of BiP which possesses the ability to cause systemic lymphocyte death. It is thought that binding of BiP to target cells (for example, T, B and APCs) activates a process causing the induction of Bim in those cells and consequently apoptosis of the target cells.

The term 'secreted' or 'secreted form' of BiP as referred to herein is meant the 78kDa protein or BiP (GRP78) protein which is extracellular or circulating and not the form which is present in the endoplasmic reticulum (ER). Furthermore, the 78kDa protein or BiP (GRP78) according to the present disclosure is not membrane bound or located within, or bound to the plasma membrane. By "circulating" it is meant that BiP (GRP78) can be detected in a body fluid into which the protein is secreted, for example the blood, plasma, urine, lymph or saliva. In one example, the 78kDa protein is a member of HSP70 family of proteins. In another example the 78kD protein is produced by activated macrophages. In another example, the 78kDa protein is human BiP (GPR78). In another example, the inhibitor of BiP (78 kDa protein) is capable of preventing induction of Bim and hence lymphocyte apoptosis.

In another embodiment, the present disclosure also provides a method for detecting a secreted form of BiP (GRP) in a biological sample obtained from a subject, the method comprising contacting the sample with one or more binding agents such that a complex forms and detecting the complex, wherein detection of the complex is indicative of a secreted form of BiP (GRP78) in the sample.

In one example, the biological sample is obtained from a subject who has, or is suspected of having, or at risk of acquiring sepsis. In one example, the biological sample is obtained from a subject who has, or is suspected of having sepsis-induced leukopenia. The method may be performed in vivo or in vitro on a biological sample which has been obtained from the subject.

In another embodiment, the present disclosure also provides a method for diagnosing and/or prognosing sepsis in a subject, the method comprising:

(i) obtaining a biological sample from a subject suspected of having sepsis;

(ii) contacting the sample with a binding agent(s) which binds to, or specifically binds to, a secreted BiP (GRP78);

(iii) detecting binding of the binding agent(s) to BiP (GRP78);

wherein a higher level of BiP compared with a control subject who does not have sepsis is diagnostic or prognostic of sepsis.

In one example, a level of BiP which is at least 2-fold greater compared with a control subject who does not have sepsis is diagnostic or prognostic of sepsis.

In one example, the method further comprises:

(iv) quantifying the level of BiP in the biological sample wherein a level of greater than 1 .5 pg/ml of secreted BiP in the biological sample is diagnostic or prognostic of sepsis. In another example, a level in the range of 1 .5 to 4 pg/ml secreted BiP in the biological sample is diagnostic or prognostic of sepsis. In yet another example, the level of secreted BiP is at least 2-fold, or at least 3-fold greater compared to the level of secreted BiP in a control subject.

By "suspected of having sepsis" it is meant a subject which presents with one or more symptoms indicative of sepsis as described herein including, but not limited to leukopenia, neutropenia or lymphopenia.

In a further example, the detection step is carried out using a sandwich ELISA format. In a further example, the method comprises immobilising a first binding agent (capture agent) which binds to a secreted from of BiP to the surface of an ELISA plate, contacting the first binding agent with the biological sample as described herein, and detecting binding using a second binding agent (detection agent) which binds to the same secreted form of BiP as the first binding agent, wherein the second binding agent is conjugated to a detectable label. Examples of detectable labels include various enzymes (e.g. biotin/streptavidin), prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, electron dense labels, labels for MRI and radioactive materials.

In a further example, the first and second binding agents are obtained by immunising an animal (such as a mouse or rat) with a recombinant human BiP comprising the sequence set forth in SEQ ID NO:4.

In one example, the diagnostic method further comprises administering to the subject an inhibitor of a secreted form of BiP (GRP78).

In one example, the biological sample is selected from serum, plasma, urine, lymph or saliva.

In another embodiment, the present disclosure also provides a method for preventing and/or treating apoptosis of leukocytes, comprising administering to a subject in need of such treatment, and who has previously been diagnosed with, or determined to have, a higher level of BiP compared with a control subject, an effective amount of an inhibitor of a secreted form of BiP (GRP78). Choice of appropriate control subjects will be apparent to persons skilled in the art.

In one example, the leukocytes comprise lymphocytes and antigen presenting cells (APCs). In a further example, the lymphocytes are T and/or B cells. In one example, the subject in need thereof is a sepsis subject. In one example, the subject who has previously been diagnosed with sepsis does not have cancer.

In another embodiment, the present disclosure also provides a method of diagnosing a subject having sepsis as being effectively treated by administration of an inhibitor of a secreted form of BiP (GRP78), comprising:

(i) performing an assay on a biological sample obtained from a subject whereby the level of secreted BiP (GRP78) is determined, said subject having been administered an inhibitor of BiP (GRP78) prior to said sample being obtained;

(ii) diagnosing the subject as being effectively treated with administration of the inhibitor of BiP (GRP78) if the level BiP obtained in said step of performing is less than the level of BiP in a subject having sepsis.

In one example, wherein the level of BiP according to step (ii) is less than 1 .5 pg/ml. In one example, the level of BiP obtained in said step of performing is less than 1 pg/ml. In another example, the level of BiP obtained in said step of performing is at least 3-fold less, or at least 2-fold less compared to the level of BiP obtained in the absence of administration with the inhibitor of BiP. In one example, the assay is a sandwich ELISA assay comprising immobilising a first binding agent which binds to a secreted form of BiP to the surface of an ELISA plate, contacting the first binding agent (capture agent) with the biological sample, and detecting binding using a second binding agent (detection agent) which binds to the same secreted form of BiP as the first binding agent, wherein the second binding agent is conjugated to a detectable label.

In another example, the step of diagnosing is obtained by measuring the level of BiP using a sandwich ELISA assay. In order to quantify the level of secreted BiP protein, a series of labelled standards based on dilutions of recombinant BiP protein (SEQ ID NO:4) were prepared and compared to detected labelled BiP in the sample. Methods of detection and quantification in ELISA involve enzymes such as horse radish peroxidase (HRP) or alkaline phosphate (ALP) which are conjugated to the second binding agent (detection agent). An appropriate substrate is then added, e.g. pNPP (p-nitrophenyl phosphate), hydrogen peroxide, TMB (3,3',5,5'-tetramethylbenzidine), ODP (o-phenylenediamine dihydrochloride) or ABTS (2,2'-azino-di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt) and the resulting reaction measured by measuring absorbance at the appropriate wavelength on a colourimeter.

In one example, the second binding agent (detection agent) is conjugated to HRP and

TMB added as substrate as described in the examples herein.

The first and second binding agents may be commercially available antibodies, for example as described elsewhere herein or may be generated as described in the examples herein.

The diagnostic methods may be performed in any biological sample in which the secreted form or circulating form of BiP (GRP78) is present. In one example, the biological sample is selected from serum, plasma, urine, lymph or saliva. In one example, the biological sample is plasma.

In another embodiment, the present disclosure provides a process for producing a diagnostic agent for diagnosing sepsis, comprising the steps of:

(i) providing a binding agent(s) which bind to or specifically bind to BiP (GRP78);

(ii) validating whether the binding agent(s) according to (i) bind to secreted BiP (GRP78);

(iii) selecting binding agent(s) which bind to the secreted BiP (GRP78); and

(iv) conjugating a detectable label to the binding agent(s) selected in (iii). The present disclosure also provides a process for producing a diagnostic agent for diagnosing sepsis, comprising the steps of:

(i) immunising an animal with human BiP protein (immunogen) according to SEQ ID

NO:5;

(ii) fusing splenic B cells harvested from the immunised animal with a myeloma cell line;

(iii) culturing the fusions for production of hybridomas; and

(iii) screening the hybridomas for binding to the human BiP immunogen.

In one example, the animal to be immunised is a rat, hamster or mouse. In a further example, the BiP protein is a recombinantly produced hexa His tagged human BiP protein according to SEQ ID NO:4. In one example, the myeloma cell line is Sp2/0.

Suitable binding agents, whether for therapeutic use or reagent use in the methods described herein are described elsewhere in this document. In one example, the binding agent according to the present disclosure may be an immunoglobulin molecule or antigen binding fragment thereof. Such molecules will include an antigen binding site. In one example, the immunoglobulin is an antibody. The antibody may be a monoclonal or a polyclonal antibody. In one example, the antibody is a naked antibody. In one example, the antibody or antigen binding fragment thereof is chimeric, de-immunised, humanised, human or primatised.

In one example, the antibody is human. The antibody may bind to a linear or a conformational epitope. In one example the antibody comprises at least a VH and a VL, wherein the VH and VL bind to form a Fv comprising an antigen binding domain. The skilled artisan will understand that that the antigen binding domain comprises the binding site of an antibody. Examples of antigen-binding fragments according to the present disclosure include, for example:

(i) a single chain Fv fragment (scFv);

(ii) a dimeric scFv (di-scFv); or

(iii) at least one of (i) and/or (ii) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and/or CH3.

In one example, the VL and VH are in separate polypeptide chains

For example, the protein is:

(i) a diabody;

(ii) a triabody;

(iii) a tetrabody;

(iv) a Fab;

(v) a F(ab')2;

(vi) a Fv; or (vii) one of (i) to (vi) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and/or CH3.

As referred to herein, a binding agent that 'binds to BiP' provides literal support for a binding agent, including an antibody or antigen binding fragment thereof that 'binds specifically to BiP'. In one example, the antibody or antigen binding fragment thereof specifically binds to human BiP. Mouse BiP and human BiP share a very high degree of sequence identity, about 99% in the mature protein (i.e. absent the signal sequence) and as described in Kozutsumi Y et al., (1989) J Cell Sci Suppl. 1 1 :1 15-37. In one example, the binding agent is cross-reactive with mouse BiP.

In another example, the 'inhibitor of BiP' is an antagonist of BiP (GRP78). Such antagonists may include antibodies or small molecule agents. In another example the inhibitor of BiP is an anti-BiP (anti-GRP78) antibody. In yet another example the inhibitor of BiP blocks binding of BiP to its receptor.

In one example, a BiP inhibitor according to the present disclosure can be generated by immunising an animal (e.g. mouse, rat or hamster) with a human BiP protein according to SEQ ID NO:1 or a sequence at least 95%, at least 97%, at least 98% or at least 99% identical thereto. In another example, the animal can be immunised with the sequence according to SEQ ID NO:3. The functional activity can be assessed by methods known in the art, including the assay methods described herein in the examples.

Without wishing to be bound by theory, the central region (ATPase domain) of heat shock proteins is highly homologous. Therefore, it would be preferable that the inhibitor of BiP does not bind to this region in order to minimise cross-reactivity with other heat shock proteins.

In one example, the inhibitor of BiP inhibits or reduces the activity of a 78kDa protein or BiP (GRP78). In another example, the inhibitor of BiP interferes with the ability of BiP (GRP78) to bind to misfolded proteins.

In one example, the inhibitor of BiP as described herein is capable of increasing the number of lymphocytes and/or antigen presenting cells (APC) in circulation when administered to a subject.

In one example, administration of the inhibitor of BiP results in a 2-fold, 3-fold, or 5-fold increase in the number of circulating lymphocytes (B and/or T cells) and/or APCs. In one example, the lymphocytes (B and/or T cells) and/or APCs are increased between about 2 and 48 hours, between about 4 and 30 hours, between about 5 and 24 hours, between about 10 and 20 hours, or between about 24 and 48 hours.

In one example, the inhibitor of BiP is administered to a subject at a dose of between 0.05m/kg-30mg/kg, preferably between 0.1 mg/kg-10mg/kg, e.g., administered at a dose of 0.1 mg/kg or 1 mg/kg or 2mg/kg or 5 mg/kg or 10mg/kg. Administration of the inhibitor of BiP may be according to art-known methods. In one example, the administration is intravenous. In another example, the administration is oral.

In another example, the inhibitor of BiP is capable of reducing at least one symptom of immunosuppression and/or sepsis in the subject.

In one example, the inhibitor of a secreted form of BiP is conjugated to another compound, for example, a detectable label or a compound that extends the half-life of the inhibitor such as polyethylene glycol or an albumin binding protein.

In one example, the binding agent of the disclosure or inhibitor of BiP of the disclosure binds to secreted BiP (GRP78) with an affinity of at least about 1 0nM, 5nM, 3nM, 1 nM, 0.5nM, 0.3nM or 0.2 nM. In one example, the binding is assessed using a biosensor, e.g., by surface plasmon resonance.

Antibody based inhibitors of BiP which are suitable for use in the treatment of subjects according to the present disclosure can be assayed according to the assay methods described herein. For example, the inhibitor of BiP is assayed by testing for its ability to block apoptosis of mouse embryo fibroblasts (MEF), in the presence of supernatant from activated macrophage cultures as described herein.

Typically, the binding agent or inhibitor of BiP described herein will be in isolated or purified form.

The present disclosure also comprises a kit for detecting a secreted form of BiP in a biological sample comprising first and second binding agents as described herein which bind to human BiP, together with a set of standards comprising human BiP protein according to SEQ ID NO:4, and further comprising instructions for use in performing a sandwich ELISA assay.

Brief Description of the Drawings

Figure 1 : Activated macrophages kill lymphocytes in a Bim-dependent fashion.

A) Schematic diagram of the experiment. Bone marrow macrophages isolated from C57B/6 mice were treated with or without 100 ng/mL LPS. After 24 h B) thymic T cells and C) splenic B cells from wild type and Bim^{_ "} mice were treated with the supernatant from activated macrophages, or conditioned media from non-activated macrophages as the control, and apoptosis measured with the Annexin V - PI assay.

Figure 2: Activated macrophage supernatant induces Bim expression and Bim-dependent apoptosis in Mouse embryonic fibroblasts (MEFs).

A) Schematic diagram of the experiment. B) MEFs were treated with activated supernatant from RAW264.7 macrophages treated with 100 ng/ml LPS and apoptosis assessed by Annexin V - PI assay. C) shows Bim and Puma mRNA expression assessed by droplet-digital PCR and D) shows Bim protein expression assessed by Western Blot using BaxBak double knockout cells which do not undergo apoptosis.

Figure 3: The four-step purification strategy for the apoptotic factor from activated macrophage supernatant.

A) The four-step purification strategy for the apoptotic factor from activated macrophage supernatant: anion exchange followed by gel filtration, heparin sepharose and melon gel separation. Arrows in the elution profiles and the histograms indicate the active fractions. B) At each stage of the purification process fractions were assayed for apoptotic activity using MEFs as the target cell from WT or Bim knockout mice and C) Bim protein expression was assessed in BaxBak double knock-out cells (MEFs) by western blot. D) The final pooled sample (0.2 and 0.4 M elution fractions) from the Melon Gel was separated by SDS page. Five bands were excised from the SDS-PAGE and subjected to mass spectrometry analysis. Bands correspond to 1 ) BiP 2) BSA/L-Plastin 3) BiP 4) CD14 and 5) TCTP/TNFa.

Figure 4: Secreted form of BiP from activated macrophages induces apoptosis in the target cells.

A) Immuno depletion of the macrophage supernatant with anti-BiP antibodies prevents MEFs from undergoing apoptosis. B) In comparison to TLR4^'A and MyD88^~'^~ mice, the activated macrophage supernatant from TRIF^A mice are unable to induce apoptosis in target cells and C) TRIF macrophages do not secrete BiP into the medium. D) BiP expression in macrophages is reduced with treatment of the retroviral vector clones 9 and 1 1 . The activated supernatant from clones 9 and 1 1 are E) unable to induce Bim expression or F) apoptosis in target cells. Figure 5: Secreted form of BiP from activated macrophages induces apoptosis in the target cells.

The chemical chaperone TUDCA can A) block BiP secretion from activated macrophages, and

B) block apoptosis in vitro. C) shows Bim expression in the presence and absence of TUDCA. D) shows TUDCA can block apoptosis in vivo in mice undergoing sepsis.

Figure 6 Detection of human BiP protein in mouse plasma by ELISA

A) shows fold induction of BiP in the mouse plasma following injection with PBS (sham) or cecal slurry. B) shows level of secreted BiP in pg/mL between sham injected mice and mice injected with cecal slurry. C) shows the percentage of live cells (thymocytes) from sham injected (n=3) versus cecal slurry injected (n=8) mice. D) and E) show total cell counts measured in thymus (T cells) and spleen (B cells) of sham compared to cecal slurry injected mice at 20hr post injection.

Key to sequence listing

SEQ ID NO:1 : protein sequence of human BiP

SEQ ID NO:2: cDNA sequence of human BiP

SEQ ID NO:3: sequence of truncated form of secreted BiP protein

SEQ ID NO:4: sequence of recombinant hexa His-tagged human Bi Detailed Description

General

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes 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 or any two or more of said steps or features.

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.

Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise.

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 (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991 ), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1 -4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

The description and definitions of immunoglobulins, antibodies and fragments thereof herein may be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 , Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J. Mol Biol. 196:901 -91 7, 1987, Chothia et al. Nature 342, 877-883, 1989 and/or or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

Throughout this specification the word "comprise", or variations such as "comprises" or

"comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Selected Definitions

For the purpose of nomenclature only and not limitation, an exemplary sequence of a human BiP (78kDa glucose related protein (GRP78)) is set out in UniPRot/Swiss-Prot Reference Sequence P1 1 021 .

In a particular example, the term 'BiP (GRP78)' as used herein is intended to mean human BiP protein. In a further example, the human BiP protein is that according to SEQ ID NO:1 of the present disclosure. Secreted BiP is intended to refer to embodiments of the BiP protein which lacks the signal sequence corresponding to at least the first 18 amino acids of the BiP protein. Secreted BiP also includes embodiments where the protein is present in a truncated form, for example the sequence according to SEQ ID NO:3.

The term 'Bim' as used herein is understood as also referring to Bcl-2-like protein 1 1 which is encoded by the Bcl2L1 1 gene. It is an intracellular protein. The protein belongs to the Bcl-2 protein family and contains a Bcl-2 homology domain (BH3). In a particular example, Bim is human Bim. The term Bim is also understood as including its various isoforms: BimL, BimEL, BimS, Bim-alpha1 , Bim-alpha2 and Bim-alpah3. The protein is an initiator/facilitator of apoptosis in a wide variety of physiological settings. The term "immunosuppression" is understood to mean a suppression of the immune system that results in the subject's inability to fight infection. Immunosuppression may occur as a consequence of the subject's disorder or as a consequence of medication being taken by the subject. In one example, the immunosuppression is characterised by a reduction in white blood cells, measured by white blood cell count.

As used herein, the term 'apoptosis' refers to the process of programmed cell death caused by a series of biochemical events that lead to characteristic cell changes, including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation. Apoptosis is a mechanism of cell death that is distinct from necrosis.

The term 'leukocytes' as used herein is intended to refer to white blood cells. White blood cells will be understood as including neutrophils, eosinophils, basophils, lymphocytes (T cells, B cells and NKT cells) and monocytes.

The term 'lymphocyte' as used herein is intended to refer to natural killer cells (NKT cells), T cells and B cells.

The term 'antigen presenting cells (APCs)' as used herein is understood as referring to any cell that is capable of internalising and presenting an antigen to T cells. Such cells include, but are not limited to dendritic cells, macrophages, fibroblasts, and certain epithelial cells.

As used herein, the term 'leukopenia' (also referred to as leukocytopenia or leucopenia) refers to a decrease in the number of white blood cells (leukocytes) found in the blood or circulation. The term 'leukopenia' also encompasses 'neutropenia' which refers to a decrease in the number of circulating neutrophils. A decrease in white blood cell count can be determined by methods in the art and as described elsewhere herein. As a rough guide, the normal reference interval of a white blood cell count in an adult subject is between 4.0 and 10.5 x 10³/μΙ_.

As used herein the term 'lymphopenia' (also referred to as "lymphocytopenia" or

"lymphocytic leucopenia") refers to an abnormally low level of lymphocytes in the circulating blood or in peripheral circulation. The peripheral circulation of all types of lymphocytes or subpopulations of lymphocytes for example, T lymphocytopenia (low T lymphocytes), B lymphocytopenia (low B lymphocytes) and NK lymphocytopenia (low natural killer cells), may be depleted or abnormally low in a patient suffering from lymphopenia. A normal lymphocyte count for adults is usually between 1 ,000 and 4,800 lymphocytes per μΙ_ blood and between 3,000 to 9,500 lymphocytes per μΙ_ of blood in children. Quantitatively, lymphopenia can be described by various cut-offs. In some examples, a patient is suffering from lymphopenia when their circulating blood total lymphocyte count falls below about 600/mm3. In some examples, an adult patient suffering from lymphopenia has less than about 1000/μΙ_ total circulating lymphocytes in the blood or for children less than circulating lymphocytes 3,000/μΙ_. By 'sepsis induced leukopenia' it is meant a decrease in white blood cells (leukocytes) resulting from infection of the blood stream. Sepsis occurs when chemicals released into the blood stream to fight infection trigger inflammatory changes throughout the body and immunosuppression. The term 'sepsis induced leukopenia' is also intended to encompass sepsis induced lymphopenia.

As used herein the term 'reduces at least one symptom of sepsis' refers to a qualitative or quantitative reduction in detectable symptoms, including but not limited to a detectable impact on the rate of recovery from disease or the rate of disease progression or severity

As used herein, the term 'at risk of acquiring sepsis' in reference to a subject is understood as referring to a subject predisposed to the development of sepsis by virtue of the subject's medical status, including, but not limited to such factors as infection, trauma (e.g. abdominal perforation), surgery, and invasive procedures (e.g. placement of a catheter etc.). infection and the like.

The term 'diagnosed with sepsis' refers to a subject demonstrating one or more symptoms of sepsis and referred to herein. Methods of diagnosing sepsis, for example by blood culture, or other methods as described herein are known in the art.

As used herein, the term 'binds' in reference to the interaction of a protein (e.g. binding agent or inhibitor of BiP) or an antigen binding site thereof with an antigen means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody binds to epitope "A", the presence of a molecule containing epitope "A" (or free, unlabeled "A"), in a reaction containing labeled "A" and the protein, will reduce the amount of labeled "A" bound to the antibody.

As used herein, the term 'specifically binds' or 'binds specifically' shall be taken to mean that a protein of the disclosure (e.g. binding agent or inhibitor of BiP) reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular antigen (e.g. BiP) or cell expressing same than it does with alternative antigens or cells. For example, a protein binds to BiP (GRP78) with materially greater affinity (e.g., 20 fold or 40 fold or 60 fold or 80 fold to 100 fold or 150 fold or 200 fold) than it does to other antigens commonly recognized by polyreactive natural antibodies (i.e., by naturally occurring antibodies known to bind a variety of antigens naturally found in humans). The level of binding may be detected using biosensor analysis (e.g. Biacore) in which the BiP (GRP78) binding agent or BiP inhibitor is immobilized and contacted with the antigen (e.g. BiP).

The term "isolated" as used herein refers to a binding agent or BiP inhibitor, antibody or antigen binding fragment thereof, that by virtue of its origin or source of derivation is not associated with naturally-associated components that accompany it in its native state; is substantially free of other proteins from the same source. Where the binding agent or BiP inhibitor, antibody or antigen binding fragment thereof is a protein, it may be rendered substantially free of naturally associated components or substantially purified by isolation, using protein purification techniques known in the art. By 'substantially purified' is meant the protein is substantially free of contaminating agents, e.g., at least about 70% or 75% or 80% or 85% or

90% or 95% or 96% or 97% or 98% or 99% free of contaminating agents.

The term 'recombinant' shall be understood to mean the product of artificial genetic recombination. Accordingly, in the context of a recombinant protein comprising an antibody antigen binding domain, this term does not encompass an antibody naturally-occurring within a subject's body that is the product of natural recombination that occurs during B cell maturation.

However, if such an antibody is isolated, it is to be considered an isolated protein comprising an antibody antigen binding domain. Similarly, if nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein comprising an antibody antigen binding domain. A recombinant protein also encompasses a protein expressed by artificial recombinant means when it is within a cell, tissue or subject, e.g., in which it is expressed.

As used herein, the terms 'preventing', 'prevent' or 'prevention' include administering a binding agent of the disclosure to thereby stop or hinder the development of at least one symptom of sepsis. The term also encompasses treatment to prevent or hinder relapse in a subject. The term also encompasses administering a binding agent of the disclosure to thereby stop or hinder apoptosis of neutrophils and/or lymphocytes.

As used herein, the terms 'treating', 'treat' or 'treatment' include administering a therapeutically effective amount of a binding agent or BiP inhibitor of the disclosure to thereby reduce or eliminate at least one symptom of sepsis. The term also encompasses administering a binding agent or BiP inhibitor of the disclosure to reduce or eliminate apoptosis of neutrophils and/or lymphocytes.

The term 'sample' or 'biological sample' as used herein is understood as referring to any suitable material in which the presence of secreted BiP (GRP78) can be detected. Preferably the sample is obtained from the subject so that the detection of the presence of BiP may be performed in vitro. Alternatively, the presence of BiP may be detected in vivo in the subject. The sample can be obtained directly from the source or following at least one step of purification. The sample can be prepared in any convenient medium which does not interfere with the method of the disclosure. The sample may be selected from blood, plasma, serum, saliva, lymph or urine. Pre-treatment may involve, for example, preparing plasma from blood. As used herein, the term 'subject' shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and non- human primates. For example, the subject is a human.

By 'sepsis subject' it is meant a subject who has been diagnosed with sepsis, or is suspected of having, or at risk of acquiring sepsis.

The skilled artisan will be aware that an 'antibody' is generally considered to be a protein that comprises a variable region made up of a plurality of polypeptide chains, e.g., a polypeptide comprising a VL and a polypeptide comprising a VH. An antibody also generally comprises constant domains, some of which can be arranged into a constant region, which includes a constant fragment or fragment crystallizable (Fc), in the case of a heavy chain. A VH and a VL interact to form a Fv comprising an antigen binding region that is capable of specifically binding to one or a few closely related antigens. Generally, a light chain from mammals is either a κ light chain or a λ light chain and a heavy chain from mammals is α, δ, ε, γ, or μ. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass. The term 'antibody' also encompasses humanized antibodies, primatized antibodies, human antibodies and chimeric antibodies. Unless specifically stated otherwise, the term 'antibody' is understood as referring to a "full- length antibody" meaning an antibody is its substantially intact form, as opposed to an antigen binding fragment of an antibody. Specifically, whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

As used herein, the term 'antigen binding site' shall be taken to mean a structure formed by a protein that is capable of binding or specifically binding to an antigen. The antigen binding site need not be a series of contiguous amino acids, or even amino acids in a single polypeptide chain. For example, in a Fv produced from two different polypeptide chains the antigen binding site is made up of a series of amino acids of a VL and a VH that interact with the antigen and that are generally, however not always in the one or more of the CDRs in each variable region. In some examples, an antigen binding site is a VH or a VL or a Fv.

Determination of Blood Counts

Leukopenia, neutropenia or lymphopenia can be determined in a subject by measuring the subjects white blood cell count. Determination of the white blood cell count can be carried out on untreated or treated (for example, where the red blood cells are lysed) blood samples. Typically a phlebotomist will collect the blood sample by drawing the blood into a test tube containing an anti-coagulant (e.g. EDTA or citrate) to prevent it from clotting. Alternatively, the plasma fraction may be separated from the blood sample and analysed. A complete blood count can be performed using an automated analyser. Since measurement units and reference ranges can vary between countries, accordingly, the results must be interpreted using the units and references ranges from the laboratory that produced the results. When interpreting the results of the subject's white blood cell count, it is also necessary that the physician factor in the subject's disease and/or other medication that the subject is taking which may artificially result in a lower white blood cell count. For example, a lower white blood cell count may be a result of bone marrow failure or deficiency, cancer, lupus, disease of the liver or spleen or severe bacterial infection. Drugs that have been known to lower white blood cell count include antibiotics, anticonvulsants, antithyroid drugs, capropril, chemotherapy drugs, chlorpromazine, diuretics, histamine-2 blockers, sulphonamides, quinidine, terbinafine and ticlopidine. Additionally, the subject's white blood cell count results should be interpreted in light of any medication that the subject is taking that may artificially increase white blood cell count as this may mask the extent of leukopenia in the subject. Drugs that are known to increase white blood cell count include beta adrenergic agonists, corticosteroids, epinephrine, G-CSF, heparin or lithium.

BiP Protein

Glucose related proteins (GRP) are molecular chaperones localized to endoplasmic reticulum (ER). GRP78 is a 78kD glucose-regulated protein and is also known as BiP (immunoglobulin binding protein) or HSPA5 (heat shock 70kD protein 5) that is localized to the endoplasmic reticulum (ER) and is involved in the folding and assembly of proteins in the ER coordinating the unfolded protein response (UPR). The protein is encoded by the HSPA5 gene. GRP78 is upregulated in conditions of stress for example ER stress, glucose starvation, hypoxia and the presence of toxic agents. Although GRP78 is a known resident ER protein it can exist as a transmembrane protein and in cancer can be localized the plasma membrane.

BiP is a HSP70 molecular chaperone that binds newly synthesised proteins as they are translocated into the ER, and maintains them in a state competent for subsequent folding and oligomerisation. BiP is also an essential component of the translocation machinery, as well as playing a role in retrograde transport across the ER membrane of aberrant proteins destined for degradation by the proteasome.

When the nucleotide binding domain of GRP78 interacts with ATP, its substrate binding domain can interact with unfolded/misfolded protein. Subsequent ATP hydrolysis acts to strengthen the interaction between GRP78 and the unfolded/misfolded protein. Under these conditions, protein disulphide isomerase (PDI) can then work to promote disulphide reduction, rearrangement and reoxidation until the correct protein conformation is achieved. ADP/ATP exchange ends the interaction of GRP78 with the protein and thus PDI's work is halted, as well. Once the correct protein structure is achieved, it is no longer a candidate for GRP78 binding.

Without wishing to be bound by theory, the inventors hypothesise, based on the presence of two BiP bands observed on SDS-PAGE (Figure 3D), that secreted BiP may also exist in a truncated form and that this form may be more potent in inducing apoptosis.

BiP (GRP78) binding agents and inhibitors

The binding agent or inhibitor of BiP according to the present disclosure may be selected from an antibody or antigen binding fragment thereof, small molecule or compound or inhibitor peptide or polypeptide. Such inhibitors may be referred to an antagonists of BiP (GRP78). The binding agent or inhibitor of BiP according to the present disclosure is capable of binding to, or specifically binding to a secreted form of BiP (GRP78) protein. More particularly, the binding agent or inhibitor of BiP is capable of binding to a secreted form of BiP protein according to SEQ ID NO:1 or SEQ ID NO:3.

The inhibitor of BiP according to the present disclosure is capable of directly or indirectly inhibiting, reducing or preventing apoptosis through interfering with the ability of BiP (GRP78) to bind to or interact with target cells (e.g. T, B and/or APCs).

In one example, the inhibitor is an inhibitor of BiP activity. In another example, the inhibitor is an antagonist. BiP (GRP78) antagonists also include antibodies, soluble domains of GRP78 and polypeptides that interact with GRP78 to prevent GRP78 activity. Antagonists can be prepared by methods known in the art. Typical agents for inhibiting or reducing (e.g. antagonistic) activity of BiP (GRP78) include mutant/variant GRP polypeptides or fragments and small organic or inorganic molecules.

Inhibitors of BiP (GRP78) include inhibitory peptides or polypeptides. The term

"peptide" as used herein is understood to mean two or more amino acids linked by a peptide bond. The term "fragment" as used herein is understood to mean a portion of a full length polypeptide or protein (e.g. antibody protein). Inhibitory peptides include chimeric peptides with GRP78 binding motifs. Inhibitory mutants include dominant negative mutants of BiP (GRP78). Such mutants can be generated, for example, by site directed mutagenesis or random mutagenesis.

Binding agents/inhibitors that inhibit BiP (GRP78) include antibodies with antagonistic or inhibitory properties. Antibodies that binds to GRP78 are commercially available and can be tested for their ability to bind secreted BiP using animal models. In addition to intact immunoglobulin molecules, fragments, chimeras or polymers of immunoglobulin molecules are also useful in the methods taught herein, as long as they are chosen for their ability to inhibit BiP (GRP78) according to the disclosed methods.

Inhibitors of BiP (GRP78) also include genistein, (-)-epigallocatechin gallate (EGCG), salicyclic acid from plants, bacterial AB₅ subtilase cytoxin or versipelostatin.

In one example, the BiP (GRP78) inhibitor is tauroursodeoxycholic acic (TUDCA).

In one example, the BiP (GRP78) inhibitor is 4-phenylbutyric acid.

An example of a BiP inhibitor that may be suitable in the methods of the present disclosure is humanised MAb 159 which is described in Liu R et al., (2013) Clin Cancer Res 19(24):6802-681 1 .

The BiP inhibitor is preferably provided in a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient.

In another example, a second therapeutic agent may also be administered to the subject. Examples of second therapeutic agents may include, but are not limited to antibiotics (for example, but not limited to flucloxacillin, gentamicin, cephazolin, benzyl penicillin, ceftriaxone, moxifloxacin, lincomycin, clindamycin, piperacilin with tazobactam, cefepine, azithromycin and/or vancomycin), anti-C5aR antibody (as described in US 7,455,837), corticosteroids, vasopressor medication, TNF-alpha therapies, TLR4 compounds, complement or recombinant protein C. Antibody generation

An anti-BiP (anti-GRP78) inhibitor monoclonal antibody according to the present disclosure may be acquired by methods known in the art. In one example, the monoclonal antibody of the present disclosure is derived from a mammalian animal. The monoclonal antibodies derived from mammalian animals include, for example, those which are produced by hybridomas and those produced from host cells that have been transformed with expression vector harbouring an antibody gene by genetic engineering techniques.

Hybridomas which produce monoclonal antibody can typically be constructed using the BiP (GRP78) protein as a sensitizing antigen to effect immunization in accordance with a conventional immunization method. Immune cells obtained from the immunized animal are then fused to known parent cells by a conventional cell fusion method to yield hybridomas. From the produced hybridomas, cells that produce the desired antibody are screened by a conventional screening method so as to select hybridomas producing the anti-BiP protein.

Monoclonal antibody preparation can typically be performed as described below. First, the BiP (GRP78) protein to be used as a sensitizing antigen for antibody acquisition can be acquired by expressing the BiP (GRP78) gene. A gene sequence coding for BiP (GRP78) is inserted into a known expression vector to transform suitable host cells and the intended human BiP (GRP78) protein can be purified from the transformed host cells or the culture supernatant according to known methods. A purified native BiP (GRP78) protein can also be used.

Purification can be performed by a plurality of conventional chromatographic techniques, for example hydroxyl apatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being an exemplary purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γΙ, γ2, or γ4 heavy chains. Protein G is recommended for all mouse isotypes and for human γ3. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered. In addition, a fusion protein of a desired partial polypeptide of the BiP (GRP78) protein with a different polypeptide may be used as an immunogen. To produce the fused protein that serves as an immunogen, Fc fragments of an antibody, peptide tags and the like may be used. To construct a vector that expresses the fused protein, genes that code for two or more desired polypeptide fragments may be fused in frame and the fused genes inserted into an expression vector. The method of preparing fusion proteins is described in Sambrook, J. et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press, 1989.

The purified BiP (GRP78) protein can be used as a sensitizing antigen to immunize mammals. Partial peptides of BiP (GRP78) can also be used as an sensitizing antigen. For example, the following peptides can serve as sensitizing antigens: (i) a peptide acquired by chemical synthesis from the amino acid sequence of human BiP (GRP78); (ii) a peptide acquired by incorporating part of the human BiP (GRP78) gene into an expression vector and expressing the same; and/or (iii) a peptide acquired by decomposing the human BiP (GRP78) protein with a proteolytic enzyme.

The region and size of the BiP (GRP78) to be used as the partial peptide are by no means limited. The peptide that serves as a sensitizing antigen is preferably composed of at least three, for example five or six, amino acid residues. More specifically, a peptide of 8-50 residues, preferably 10-30 residues, can be used as a sensitizing antigen. In general, rodents are preferred animals to be immunized. Specifically, mouse, rat, hamster or rabbit may be used as an animal to be immunized. Other animals that may be immunized include monkeys.

The animals mentioned above can be immunized with the sensitizing antigen in accordance with known methods. An exemplary general method comprises immunizing a mammal by intraperitoneal or subcutaneous injection of the sensitizing antigen. Specifically, the sensitizing antigen is administered to the mammal several times every 4 to 21 days. The sensitizing antigen is used for immunization after it is diluted to a suitable dilution ratio with PBS (phosphate-buffered saline), physiological saline or the like. If desired, the sensitizing antigen may be administered together with an adjuvant. For example, the sensitizing antigen may be mixed with a Freund's complete adjuvant, which may be emulsified to make a desired sensitizing antigen. In addition, a suitable carrier may be used in immunization with the sensitizing antigen. Particularly in the case where a partial peptide having a small molecular weight is used as the sensitizing antigen, immunization is preferably done by binding the sensitizing antigen peptide to a carrier protein such as albumin or keyhole limpet hemocyanine. After immunization the immune cells are collected from the mammal and subjected to cell fusion. Spleen cells can especially be used as preferred immune cells.

The cells to be fused with the immune cells are mammalian myeloma cells. Myeloma cells are preferably furnished with a selection marker suitable for screening. The selection marker refers to a phenotype that can survive (or cannot survive) under particular culture conditions. Known selection markers include hypoxanthine-guanine-phosphoribosyl transferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells having HGPRT or TK deficiency have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity). In a HAT selective medium, cells having HAT sensitivity are incapable of DNA synthesis and will die; however, if they are fused with normal cells, they can continue the synthesis of DNA by making use of the salvage circuit in the normal cell and hence will proliferate in the HAT selective medium.

A variety of myeloma cells suitable for cell fusion are known. Myeloma cells that can be used include: P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U.1 {Current

Topics in Microbiology and Immunology (1978) 81 , 1 -7), NS-1 (Kohler. G. and Milstein, C. Eur.

J. Immunol. (1976) 6, 51 1 -519), MPC-1 1 (Margulies. D. H. et al., Cell (1976) 8, 405-415),

SP2/0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, S. F. Et al., J.

Immunol. Methods (1980) 35, 1 -21 ), S1 94 (Trowbridge, I. S. J. Exp. Med. (1978) 148, 313- 323), and R21 0 (Galfre, G. et al., Nature (1 979) 277, 131 -133). Cell fusion of the above-mentioned immune cells and myeloma cells can be performed in accordance with known methods, such as the method of Kohler and Milstein (Kohler. G. and Milstein, C, Methods Enzymol. (1981 ) 73, 3-46).

In cell fusion, specified amounts of the above-mentioned immune cells and myeloma cells are mixed well in the culture medium and then mixed with a pre-warmed (37° C) PEG solution to form the desired fused cells (hybridomas). In the cell fusion method, PEG with an average molecular weight of from about 1000 to about 6000 can be added at concentrations typically ranging from 30 to 60% (w/v). Subsequently, procedures of sequentially adding suitable culture media centrifuging them and removing the supernatant are repeated to thereby remove the cell fusion promoting agents and the like that are not preferred for the growth of hybridomas.

The hybridomas thus obtained can be selected by employing a selective culture medium in accordance with the selection marker possessed by the myeloma used in cell fusion. For instance, cells having HGPRT or TK deficiency can be selected by culturing them in a HAT culture medium (i.e., containing hypoxanthine, aminopterin, and thymidine). In the case of using HAT sensitive myeloma cells in cell fusion, the cells that successfully fused to normal cells can be selectively grown in the HAT culture medium. The fused cells are continuously cultured using this HAT culture medium for a sufficient time that cells (nonfused cells) other than the desired hybridomas will die. Specifically, the desired hybridomas can be selected by culturing for a period which typically ranges from several days to several weeks. Subsequently, a conventional method of limiting dilution is implemented to thereby enable the screening and a single cell cloning of hybridomas that produce the desired antibody. Alternatively, antibodies can be constructed by the method described in WO 03/104453.

Screening and cloning of the desired antibodies can advantageously be implemented by screening methods based on known antigen-antibody reactions. For instance, the antigen is bound to a carrier such as beads made of polystyrene or otherwise or a commercial 96-well microtiter plate and reacted with the culture supernatant of hybridomas. Subsequently, the carrier is washed and thereafter reacted with enzyme-labelled secondary antibodies or the like. If the culture supernatant contains the desired antibodies that react with the sensitizing antigen, the secondary antibodies indirectly bind to the carrier via the desired antibodies. Finally, the secondary antibodies indirectly binding to the carrier are detected to thereby determine whether the desired antibodies are present in the culture supernatant. As a result, hybridomas that produce the desired antibodies having the ability to bind to the antigen can be cloned by limiting dilution method. In this case, antigens that can preferably be used include not only the one that was used in immunization but also the BiP (GRP78) protein which is substantially of the same nature. Aside from the method of obtaining the above-mentioned hybridomas by immunizing animals other than humans with the antigen, human lymphocytes may be sensitized with the antigen to obtain the desired antibodies. Specifically, human lymphocytes are sensitized in vitro with the BiP (GRP78) protein. Subsequently, the immunosensitized lymphocytes are fused to a suitable fusion partner. An exemplary fusion partner that can be used is myeloma cells that derive from humans and which are capable of permanent division. The anti-BiP (anti-GRP78) antibody obtained by this method is a human antibody having an activity for binding to the BiP (GRP78) protein.

Further, by administering the antigen BiP (GRP78) protein to a transgenic animal having the full repertoire of human antibody genes, the anti-BiP (anti-GRP78) human antibody can also be obtained. The antibody producing cells in the immunized animal can be immortalized by such treatments as cell fusion with a suitable fusion partner and infection with Epstein-Barr virus or the like. From the thus obtained immortal cells, a human antibody against the BiP (GRP78) protein may be isolated (see WO 94/25585, WO 93/12227, WO 92/0391 8, and WO 94/02602). Further, the immortalized cells may be cloned to achieve cloning of cells that produce an antibody having the desired reaction specificity.

The hybridomas may be cultured in accordance with an ordinary method and the desired monoclonal antibodies may be obtained from the culture supernatant. Alternatively, the hybridomas may be administered to a compatible mammal and allowed to proliferate, yielding monoclonal antibodies in the ascites. The former method is suitable for obtaining antibodies of high purity.

It is also possible to use antibodies that are encoded by the antibody gene cloned from the antibody producing cells. The cloned antibody gene may be incorporated into a suitable vector which is then introduced into a host cell so that it is expressed as an antibody. Methods for isolating the antibody gene, introducing it into a vector, and transforming host cells have already been established (see, for example, Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192, 767-775).

Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991 ); Marks et al., J. Mol. Biol., 222:581 (1 991 )).

Detection and screening of antibodies

It is preferred that the binding agent or inhibitor of BiP (e.g. antibody) of the present disclosure binds specifically to BiP (GRP78). Specifically, a subject antibody is reacted with BiP (GRP78) immobilized on a carrier, which is then treated with a labeled antibody that recognizes the antibody. Suitable supports used in assays include synthetic polymer supports, such as polypropylene, polystyrene, substituted polystyrene, e.g., aminated or carboxylated polystyrene, polyacrylamides, polyamides, polyvinylchloride, glass beads, agarose, or nitrocellulose. Detection of the labelled antibody on the carrier after washing provides a proof for the binding of the subject antibody to BiP (GRP78). Labels that can be utilized include enzymatically active proteins such as peroxidase and β-galactosidase, and fluorescent substances such as FITC. To evaluate the binding activity of the antibody, fixed specimens of BiP (GRP78) expressing cells can also be utilized.

An applicable method of screening antibodies using the binding activity as an index is panning that utilizes a phage vector. Genes coding for the variable regions of heavy and light chains may be ligated by a suitable linker sequence to make a single-chain Fv (scFv). If the gene coding for scFv is inserted into a phage vector, phages can be obtained that have scFv expressed on the surface. These phages are brought into contact with the desired antigen and the phages that have bound to the antigen are recovered, whereupon one can recover the DNA that codes for scFv having the desired binding activity. By repeating this procedure as necessary, the scFv having the desired binding activity can be enriched. For example, US6521404, US5969108 and US7049135 describe the isolation of murine and/or human antibodies, using phage libraries. Subsequent publications describe the production of high affinity (nM range).

After cDNA coding for the V region of the desired anti-BiP (anti-GRP78) antibody is obtained, this cDNA is digested with restriction enzymes that recognize those restriction sites which have been inserted at both ends of the cDNA. The digested cDNA that codes for the V region of the anti-BiP (ant-GRP78) antibody may be inserted into a suitable expression vector to thereby provide an antibody-expressing vector.

An expression vector is provided that harbors DNA coding for a desired antibody's constant region (C region) and a sequence to be recognized by a restriction enzyme that digests the aforementioned V region gene may be located on the 5' side of the vector. The two genes are digested by the same combination of restriction enzymes and fused together in frame to construct a chimeric antibody-expressing vector. To produce the anti-BiP (anti- GRP78) antibody of the present disclosure, the antibody gene may be incorporated into an expression vector in such a way that it will be expressed under control by an expression- regulatory region. The expression-regulatory region for expressing the antibody may include an enhancer or a promoter. Subsequently, suitable host cells are transformed with this expression vector to yield recombinant cells expressing the DNA coding for the anti-BiP (anti-GRP78) antibody.

The antibodies expressed and produced as described above can be purified by the methods used to purify ordinary proteins and they may be used either singly or in suitable combinations. For example, an affinity column such as a protein A column, a chromatographic column, a filter, ultrafiltration, salting out, dialysis, etc. may appropriately be selected and combined to separate and purify the antibodies (Antibodies— A Laboratory Manual, Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).

Additionally, ability of an inhibitor of BiP to block apoptosis can be assayed according to methods described herein in the examples.

Recombinant antibody production

The binding agents (e.g. antibodies) of the present disclosure can also be produced recombinantly, using techniques and materials.

For example, DNA encoding an antibody of the disclosure or a polypepide comprising an antigen binding site of an antibody, e.g., a Fab fragment is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). A hybridoma cell serves as a source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al, (1993) Curr. Opinion in Immunol., 5: 256-262; and Pluckthun, (1992) Immunol. Revs., 130:151 -188. Molecular cloning techniques to achieve these ends are known in the art and described, for example in Ausubel et al. (1988) or Sambrook et al. (1989). A wide variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel: Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif; Sambrook et al., (1989); and Ausubel et al., eds., (1988). Methods of producing recombinant immunoglobulins are also known in the art. See US6331415; and US5585089.

For recombinant production of an antibody, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody is readily isolated or synthesized using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to DNAs encoding the heavy and light chains of the antibody). Many vectors are available. Exemplary vectors are described herein. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding an antibody or protein of the present disclosure (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. The skilled artisan will be aware of suitable sequences for expression of an antibody. For example, exemplary signal sequences include prokaryotic secretion signals (e.g., alkaline phosphatase, penicillinase, Ipp, or heat- stable enterotoxin II), yeast secretion signals (e.g., invertase leader, a factor leader, or acid phosphatase leader) or mammalian secretion signals (e.g., herpes simplex gD signal or an immunoglobulin signal). Exemplary promoters include those active in prokaryotes (e.g., phoA promoter , β-lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter), and those active in mammalian cells (e.g., cytomegalovirus immediate early promoter (CMV), the human elongation factor 1 -a promoter (EF1 ), the small nuclear RNA promoters (U1 a and U1 b), a- myosin heavy chain promoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late promoter, β-actin promoter; hybrid regulatory element comprising a CMV enhancer/ β-actin promoter or an immunoglobulin promoter or active fragment thereof.

Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryotic, yeast, or higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces. One E. coli cloning host is E. coli 294 (ATCC 31 ,446), although other strains such as E. coli B, E. coli X 1776 (ATCC 31 ,537), and E. coli W31 10 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Pichia pastoris (EP 183,070); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibody are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori (silkworm) have been identified. Examples of useful mammalian host cell lines are monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1 651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al. (1977) ; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells (CHO) ; mouse Sertoli cells (TM4); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1 587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51 ); TRI cells (Mather et al. (1982); MRC 5 cells; FS4 cells; and PER.C6™ (Crucell NV).

The host cells used to produce the antibody of this disclosure may be cultured in a variety of media. Commercially available media such as Ham's FIO (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in US4767704; US4657866; US4927762; US4560655; US5122469; WO90/03430; WO87/00195; may be used as culture media for the host cells.

Chimeric Antibodies

In one example a binding agent of the disclosure is a chimeric antibody. The term '"chimeric antibody" refers to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species (e.g., murine, such as mouse) or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species (e.g., primate, such as human) or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US4816397, US481 6567; and US5807715).

Typically chimeric antibodies utilize rodent or rabbit variable regions and human constant regions, in order to produce an antibody with predominantly human domains. The production of chimeric antibodies is known in the art, and may be achieved by standard means (as described, e.g., US4816397, US4816567; and US5807715).

The variable region or variable domain of an antibody comprises the amino acid sequences of complementarity determining regions (CDRs; i.e., CDRI , CDR2, and CDR3), and Framework Regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. CDRs and FRs may be defined according to Kabat (1987 and 1991 ) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD) or Chothia and Lesk (1987) J. Mol Biol., 196: 901 - 917 or any other known technique or combination thereof. The constant region of an antibody is the portion of the antibody molecule which confers effector functions. The heavy chain constant region can be selected from any of the five isotypes: alpha, delta, epsilon, gamma or mu. Further, heavy chains of various subclasses (such as the IgG subclasses of heavy chains) are responsible for different effector functions and thus, by choosing the desired heavy chain constant region, antibodies with desired effector function can be produced. Exemplary heavy chain constant regions are gamma 1 (lgG1 ), gamma 2 (lgG2), gamma 3 (lgG3) and gamma 4 (lgG4). Light chain constant regions can be of the kappa or lambda type, such as of the kappa type.

Complementarity determining regions (syn. CDRs; i.e., CDR1 , CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain the presence of which are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1 , CDR2 and CDR3. Each complementarity determining region may comprise amino acid residues from a "complementarity determining region" as defined by Kabat et al. (1 987 and 1991 ) and/or those residues from a "hypervariable loop" Chothia and Lesk (1987) or any other known technique or combination thereof.

Framework regions (FR) are those variable domain residues other than the CDR residues.

Humanized and Human antibodies

The binding agent of the present disclosure may be a humanized antibody or human antibody.

The term "humanized antibody" shall be understood to refer to a chimeric molecule, generally prepared using recombinant techniques, having an epitope binding site derived from an antibody from a non-human species and the remaining antibody structure of the molecule based upon the structure and/or sequence of a human antibody. The antigen-binding site comprises the complementarity determining regions (CDRs) from the non-human antibody grafted onto appropriate framework regions in the variable domains of a human antibody and the remaining regions from a human antibody. Antigen binding sites may be wild type or modified by one or more amino acid substitutions. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies, antibody chains or polypeptides comprising antigen binding domains thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human antibody. In some instances, Fv framework residues of the human antibody are replaced by corresponding non- human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non- human antibody and all or substantially all of the FR regions are those of a human antibody consensus sequence. The humanized antibody optimally also will comprise at least a portion of an antibody constant region (Fc), typically that of a human antibody.

Methods for humanizing non-human antibodies are known in the art. Humanization can be essentially performed following the method of US6548640, US5585089, US6054297 or US5859205. Other methods for humanizing an antibody are not excluded.

The term "human antibody" as used herein in connection with antibody molecules and binding proteins refers to antibodies having variable (e.g. VH, VL, CDR and FR regions) and constant antibody regions derived from or corresponding to sequences found in humans, e.g. in the human germline or somatic cells. The "human" antibodies can include amino acid residues not encoded by human sequences, e.g. mutations introduced by random or site directed mutations in vitro (in particular mutations which involve conservative substitutions or mutations in a small number of residues of the antibody, e.g. in 1 , 2, 3, 4 or 5 of the residues of the antibody, e.g. in 1 , 2, 3, 4 or 5 of the residues making up one or more of the CDRs of the antibody). These "human antibodies" do not actually need to be produced by a human, rather, they can be produced using recombinant means and/or isolated from a transgenic animal (e.g., mouse) comprising nucleic acid encoding human antibody constant and/or variable regions (e.g., as described above).

Human antibodies can also be produced using various techniques known in the art, including phage display libraries (e.g., as described in US5885793).

Completely human antibodies which recognize a selected epitope can also be generated using a technique referred to as "guided selection." In this approach a selected non- human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope (US5565332).

Purification of recombinant antibodies

When using recombinant techniques, the antibody can be produced intracellular^, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al. (1992) Bio/Technology, 10: 163-167 describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants. Peptide and Polypeptide binding agents

In another example of the disclosure, the binding agent or inhibitor of BiP is a peptide. For example, the peptide is derived from a ligand of a cell surface marker or protein as described herein according to any example of the disclosure.

Alternatively, a ligand is a peptide isolated from a random peptide library. To identify a suitable ligand, a random peptide library is generated and screened as described in US5733731 , US5591 646 and US5834318. Generally, such libraries are generated from short random oligonucleotides that are expressed either in vitro or in vivo and displayed in such a way to facilitate screening of the library to identify a peptide that, is capable of specifically binding to a protein or peptide of interest. Methods of display include, phage display, retroviral display, bacterial surface display, bacterial flagellar display, bacterial spore display, yeast surface display, mammalian surface display, and methods of in vitro display including, mRNA display, ribosome display and covalent display.

A peptide that is capable of binding a protein or peptide of interest is identified by a number of methods known in the art, such as, for example, standard affinity purification methods as described, for example in Scopes, (1994) "Protein purification: principles and practice", Third Edition, Springer Verlag; purification using FACS analysis as described in US645563, or purification using biosensor technology as described in Gilligan et al, (2002) Anal Chem., 74: 2041 -2047. Small Molecules

A chemical small molecule library is also clearly contemplated for the identification of ligands that specifically bind to BiP (GRP78) according to any example of the disclosure. Chemical small molecule libraries are available commercially or alternatively may be generated using methods known in the art, such as, for example, those described in US5463564.

Detectably labelled compounds

In one example, a binding agent as described herein according to any example of the disclosure comprises one or more detectable markers to facilitate detection and/or isolation. For example, the compound comprises a fluorescent label such as, for example, fluorescein (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-l,3- diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, 4'-6-diamidino-2- phenylinodole (DAPI), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, fluorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine (5,6- tetramethyl rhodamine). The absorption and emission maxima, respectively, for some of these fluorescent compounds are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm : 672 nm), Cy5.5 (682 nm ; 703 nm) and Cy7 (755 nm ; 778 nm).

Alternatively, or in addition, the binding agent as described herein according to any example of the disclosure is labelled with, for example, a fluorescent semiconductor nanocrystal (as described, for example, in US630661 0).

Alternatively, or in addition, the binding agent is labelled with, for example, a magnetic or paramagnetic compound, such as, iron, steel, nickel, cobalt, rare earth materials, neodymium-iron-boron, ferrous-chromium-cobalt, nickel-ferrous, cobalt- platinum, or strontium ferrite.

Detection of BiP (GRP78) for diagnosis

The present disclosure also provides methods for detection and diagnosis of sepsis or sepsis-induced leukopenia, using an anti-BiP (anti-GRP78) binding agent of the disclosure. Diagnosis according to the disclosure may be performed either in vitro or in vivo.

Binding to BiP (GRP78) by the binding agent of the disclosure can be determined using any of a variety of techniques known to the skilled artisan such as, for example, a technique selected from the group consisting of, immunohistochemistry, immunofluorescence, an immunoblot, a Western blot, a dot blot, an enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay, fluorescence resonance energy transfer (FRET), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- tof-MS), electrospray ionization (ESI-MS) (including tandem mass spectrometry, e.g. LC-ESI- MS/MS and MALDI-tof/tof-MS), biosensor technology, evanescent fiber-optics technology or protein chip technology.

In one example the assay used to determine the amount or level of secreted BiP (GRP78) is a semi-quantitative method.

In another example the assay used to determine the amount or level of secreted BiP (GRP78) is a quantitative method.

For example, the protein is detected with an immunoassay, e.g., using an assay selected from the group consisting of, immunohistochemistry, immunofluorescence, ELISA, fluorescence-linked immunosorbent assay (FLISA) Western blotting, RIA, a biosensor assay, a protein chip assay and an immunostaining assay (e.g. immunofluorescence).

Standard solid-phase ELISA or FLISA formats are particularly useful in determining the concentration of a protein from a variety of samples. In one form such an assay involves immobilizing a biological sample onto a solid matrix, such as, for example a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e.g. a glass slide).

A binding agent (e.g., an antibody) that specifically binds to BiP (GRp78) is brought into direct contact with the immobilized biological sample, and forms a direct bond with any of its target protein present in said sample. This binding agent is generally labelled with a detectable label, such as, for example, a fluorescent label (e.g. FITC or Texas Red) or a fluorescent semiconductor nanocrystal (as described in US630661 0) in the case of a FLISA or an enzyme (e.g. horseradish peroxidase (HRP), alkaline phosphatase (AP) or β-galactosidase) in the case of an ELISA, or alternatively a second labeled antibody can be used that binds to the first antibody. Following washing to remove any unbound antibody the label is detected either directly, in the case of a fluorescent label, or through the addition of a substrate, such as for example hydrogen peroxide, TMB, or toluidine, or 5-bromo-4-chloro-3-indol-beta-D- galaotopyranoside (x-gal) in the case of an enzymatic label. Such ELISA- or FLISA-based systems are particularly suitable for quantification of the amount of a protein in a sample, by calibrating the detection system against known amounts of a protein standard to which the antibody binds, such as for example, an isolated and/or recombinant polypeptide or immunogenic fragment thereof or epitope thereof.

In another form, an ELISA or FLISA comprises of immobilizing a binding agent (e.g., an antibody) on a solid matrix, such as, for example, a membrane, a polystyrene or polycarbonate microwell, a polystyrene or polycarbonate dipstick or a glass support. A sample is then brought into physical relation with the binding agent, and the protein to which the compound binds is bound or 'captured'. The bound protein is then detected using a second labelled compound that binds to a different protein or a different site in the same protein. Alternatively, a third labelled antibody can be used that binds the second (detecting) antibody.

It will be apparent to the skilled person that the assay formats described herein are amenable to high throughput formats, such as, for example, automation of screening processes or a microarray format as described in Mendoza et al. (1999) Biotechniques 27: 778-788. Furthermore, variations of the above-described assay will be apparent to those skilled in the art, such as, for example, a competitive ELISA.

Biosensor devices generally employ an electrode surface in combination with current or impedance measuring elements to be integrated into a device in combination with the assay substrate (such as that described in US5567301 ). A binding agent specifically binds to a protein or is incorporated onto the surface of a biosensor device and a biological sample contacted to said device. A change in the detected current or impedance by the biosensor device indicates protein binding to said antibody. Some forms of biosensors known in the art also rely on surface plasmon resonance to detect protein interactions, whereby a change in the surface plasmon resonance surface of reflection is indicative of a protein binding to a ligand or antibody (US5485277 and US5492840).

Biosensors are of particular use in high throughput analysis due to the ease of adapting such systems to micro- or nano-scales. Furthermore, such systems are conveniently adapted to incorporate several detection reagents, allowing for multiplexing of diagnostic reagents in a single biosensor unit. This permits the simultaneous detection of several proteins or peptides in a small amount of body fluids.

Evanescent biosensors are also useful as they do not require the pretreatment of a biological sample prior to detection of a protein of interest. An evanescent biosensor generally relies upon light of a predetermined wavelength interacting with a fluorescent molecule, such as for example, a fluorescent antibody attached near the probe's surface, to emit fluorescence at a different wavelength upon binding of the target polypeptide to the compound.

Micro- or nano-cantilever biosensors are also useful as they do not require the use of a detectable label. A cantilever biosensor utilizes a binding agent capable of specifically detecting the analyte of interest that is bound to the surface of a deflectable arm of a micro- or nano-cantilever. Upon binding of the analyte of interest (e.g. a marker within a polypeptide) the deflectable arm of the cantilever is deflected in a vertical direction (i.e. upwards or downwards). The change in the deflection of the deflectable arm is then detected by any of a variety of methods, such as, for example, atomic force microscopy, a change in oscillation of the deflectable arm or a change in pizoresistivity. Exemplary micro-cantilever sensors are described in US20030010097.

To produce protein chips, the proteins, peptides, polypeptides, antibodies or ligands that are able to bind specific antibodies or proteins of interest are bound to a solid support such as for example glass, polycarbonate, polytetrafluoroethylene, polystyrene, silicon oxide, metal or silicon nitride. This immobilization is either direct (e.g. by covalent linkage, such as, for example, Schiff's base formation, disulfide linkage, or amide or urea bond formation) or indirect. Methods of generating a protein chip are known in the art and are described in for example US20020136821 , US20020192654, US2002010261 7 and US6391625. To bind a protein to a solid support it is often necessary to treat the solid support so as to create chemically reactive groups on the surface, such as, for example, with an aldehyde-containing silane reagent. Alternatively, an antibody or ligand may be captured on a microfabricated polyacrylamide gel pad and accelerated into the gel using microelectrophoresis as described in, Arenkov et al. (2000) Anal. Biochem. 278:123-131 . In this regard, the present disclosure also provides a protein chip comprising a binding agent capable of binding to BiP (GRP78). In one example, the binding agent is an antibody or antigen binding fragment thereof. Alternatively, flow cytometry can be used to detect the presence of BiP (GRP78) in a sample (e.g. biological sample). For example, the presence of BiP (GRP78) can be detected and quantified by combining the sample with an anti-BiP (anti-GRP78) binding agent which is labelled, for example with FITC and performing flow cytometry with a flow cytometer (e.g. FACSCalibur, FACSAria, FACSVantage or FACSArray) and analysing the fluorescence intensity data with CELL QUEST Software or the like.

Assays for determining inhibition of apoptosis

Various assays may be employed to determine whether the binding agent of the present disclosure is capable of supressing or blocking apoptosis in target cells, for example T cells, B cells and/or antigen presenting cells. One such assay is as described and exemplified herein in which the binding agent can be tested for its ability to block apoptosis of mouse embryonic fibroblasts when supernatant from activated macrophages is added to the cells.

A method for evaluating apoptosis in vitro may employ the use of Annexin-Pi assay or the use of [³H]-thymidine incorporation assay. Other methods that may be employed include a dye exclusion method (e.g. trypan blue) or propidium iodide. Another method is the MTT method which utilises the ability of viable cells to convert the tetrazolium salt MTT (3-(4, 5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to a blue formazan product. Aside from MTT, reagents such as MTS, XTT, WST-1 and WST-8 can be used.

A sepsis mouse model may be used as a method of in vivo evaluation or apoptosis suppression or blocking ability. For example, the binding agent may be tested for its ability to treat or prevent sepsis in a mouse model of sepsis as described in a review by Mitchell Fink (2014) Virulence 5:1 :143-153 Landes Bioscience. Pharmaceutical Compositions

Binding agents, inhibitors and compositions of the present disclosure suitable for treating and/or preventing sepsis, in particular sepsis-induce leukopenia are useful for parenteral, topical, oral, or local administration, aerosol administration, or transdermal administration, for prophylactic or for therapeutic treatment. Accordingly, in some examples, the compositions comprise an effective amount of the inhibitor of BiP or a therapeutically effective amount of the inhibitor of BiP or a prophylactically effective amount of the inhibitor of BiP.

As used herein, the term "effective amount" shall be taken to mean a sufficient quantity of a binding agent or composition containing the inhibitor of BiP to bind to the target protein in vivo and to reduce or inhibit and/or prevent sepsis or sepsis-induced leukopenia in vivo, compared to the same level in a subject prior to administration and/or compared to a subject of the same species to which the compound has not been administered. For example, the term "effective amount" means a sufficient quantity of the inhibitor of BiP to reduce, prevent or ameliorate sepsis in a subject. The skilled artisan will be aware that such an amount will vary depending on, for example, the specific inhibitor of BiP administered and/or the particular subject and/or the type or severity or level of sepsis. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity, e.g., weight or amount of inhibitor of BiP; rather the present disclosure encompasses any amount of the inhibitor of BiP sufficient to achieve the stated result in a subject.

As used herein, the term "therapeutically effective amount" shall be taken to mean a sufficient quantity of an inhibitor of BiP to reduce or inhibit one or more symptoms of sepsis to a level that is below that observed and accepted as clinically diagnostic or clinically characteristic of that disease. The skilled artisan will be aware that such an amount will vary depending on, for example, the inhibitor of BiP administered and/or the particular subject and/or the type or severity or level of sepsis. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity, e.g., weight or amount of inhibitor of BiP rather the present disclosure encompasses any amount of the inhibitor of BiP sufficient to achieve the stated result in a subject.

As used herein, the term "prophylactically effective amount" shall be taken to mean a sufficient quantity of an inhibitor of BiP to prevent or inhibit or delay the onset of one or more detectable symptoms of sepsis. The skilled artisan will be aware that such an amount will vary depending on, for example, the specific inhibitor of BiP administered and/or the particular subject and/or the type or severity or level of disease and/or predisposition (genetic or otherwise) to the disease. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity, e.g., weight or amount of inhibitor of BiP, rather the present disclosure encompasses any amount of the inhibitor of BiP sufficient to achieve the stated result in a subject.

The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and lozenges. It is recognized that the pharmaceutical compositions of this disclosure, when administered orally, must be protected from digestion. This is typically accomplished either by complexing the inhibitor of BiP with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the compound in an appropriately resistant carrier such as a liposome. Means of protecting proteins from digestion are known in the art.

The pharmaceutical compositions of this disclosure are particularly useful for parenteral administration, such as intravenous administration or administration into a body cavity or lumen of an organ or joint. The compositions for administration will commonly comprise a solution of the binding agent of the present disclosure dissolved in a pharmaceutically acceptable carrier, for example an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of binding agents of the present disclosure in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as mixed oils and ethyl oleate may also be used. Liposomes may also be used as carriers. The vehicles may contain minor amounts of additives that enhance isotonicity and chemical stability, e.g., buffers and preservatives.

The inhibitor of BiP of the present disclosure can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, transdermal, or other such routes, including peristaltic administration and direct instillation into a tumor disease site (intracavity administration). The preparation of an aqueous composition that contains the inhibitor of BiP of the present disclosure as an active ingredient will be known to those of skill in the art.

Suitable pharmaceutical compositions in accordance with the disclosure will generally include an amount of the inhibitor of BiP of the present disclosure admixed with an acceptable pharmaceutical diluent or excipient, such as a sterile aqueous solution, to give a range of final concentrations, depending on the intended use. The techniques of preparation are generally known in the art as exemplified by Remington's Pharmaceutical Sciences, 1 6th Ed. Mack Publishing Company, 1980, incorporated herein by reference.

Upon formulation, an inhibitor of BiP of the present disclosure will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically/prophylactically effective. Formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but other pharmaceutically acceptable forms are also contemplated, e.g., tablets, pills, capsules or other solids for oral administration, suppositories, pessaries, nasal solutions or sprays, aerosols, inhalants, liposomal forms and the like. Pharmaceutical "slow release" capsules or compositions may also be used. Slow release formulations are generally designed to give a constant drug level over an extended period and may be used to deliver an inhibitor of BiP of the present disclosure. WO2002/080967 describes compositions and methods for administering aerosolized compositions.

Suitable dosages of the inhibitor of BiP of the present disclosure will vary depending on the specific binding agent, the condition to be treated and/or the subject being treated. It is within the ability of a skilled physician to determine a suitable dosage, e.g., by commencing with a sub-optimal dosage and incrementally modifying the dosage to determine an optimal or useful dosage. Alternatively, to determine an appropriate dosage for treatment/prophylaxis, data from the cell culture assays or animal studies are used, wherein a suitable dose is within a range of circulating concentrations that include the ED50 of the active binding agent with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. A therapeutically/prophylactically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the inhibitor of BiP which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

EXAMPLES Methods

Droplet Digital PCR

Total RNA was isolated using TRIZOL® (Cat#15596-026, Invitrogen, Carlsbad, USA). Complementary DNA (cDNA) was synthesised from 2.5 μg of total RNA using the Superscript III RT-PCR system (Cat# 18080-051 Invitrogen). cDNA samples were tested in triplicate on a QX200TM Droplet Digital™ System using EvaGreen® ddPCRTM Supermix (Bio-Rad, NSW Australia).

Digital PCR as performed as described in Kiselinova et al, PLoS One 2014 Jan 21 ;9(1 ):e85999.

Western Blot

Cells were lysed in ONYX lysis buffer (20 mM Tris-HCI (pH 7.4), 135 mM NaCI, 1 .5 mM MgCI2, 1 mM EDTA, 10% glycerol, and 1 % Triton X-100; supplemented with Complete inhibitor™ (GE Healthcare). Proteins were separated on 12% SDS-PAGE before transferring to PVDF membrane. Post-transfer, the membranes were blocked in 5% skim-milk powder prepared in phosphate-buffered saline and probed with antibodies against Bim (3c5, in house antibody) or Bip (mouse monoclonal, Cat no 610979, BD Transduction Laboratories) in 1 :1000 fold dilution. The bound antibodies were detected with HRPO conjugated anti-RAT (Cat no. NA935V, GE Healthcare) or antii-MOUSE (Cat no. A9044, Sigma). The signals were detected using ECL Chemiluminescent reagent (Cat no RPN2106, GE Healthcare).

Mouse macrophage samples

Murine RAW264.7 macrophages or murine bone marrow derived macrophages were activated with 100 ng/mL LPS for 24 hours. Supernatants harvested from the cells were concentrated 10x with a 10kDa Millipore cut off spin filter.

Mice

Wild-type C57BL/6 mice, Bim knock-out mice (Bim^{" _}) and BaxBak double knockout (BaxBak^{" _}) mice were obtained from the Walter and Eliza Hall Institute of Medical Research. Relevant cell populations were obtained according to methods known in the art.

Cecal Ligation and Puncture (CLP)-induced sepsis

To investigate the molecular mechanisms of sepsis, the most frequently used model is the cecal ligation and puncture (CLP) model in rodents. In this model, surgically induced puncture of the cecum leads to the leakage of abdominal contents into the peritoneal cavity resulting in bacterial infection of the blood and septicaemia.

Prior to anaesthesia, mice were injected with Buprenex as analgesic to manage post-operative pain and distress (1 .0ml of 0.015 mg/ml). The mice were then anaesthetised using gaseous anaesthetics (2-3% isoflurane) and the anaesthetic level monitored by assessing skin and mucous membrane colour and pedal reflex (response to stimuli).

For cecal ligation, the belly of each mouse was shaved and betadine applied. A mid-line incision to the mouse's left side was then made. The cecum was removed and tied off beyond the ileal-cecal junction. The stool was then forced down to the distal end of the cecum keeping the pressure on to make puncture(s). The cecum was then punctured with an appropriately gauged needle and the puncture site squeezed until the stool was extruded. The cecum was then wiped claims and replaced into the body cavity of the mouse and the muscle closed with a continuous stitch. The skin was then sealed with glue or metallic clips. The mice were resuscitated by injecting with pre-warmed normal saline (37^SC; 5ml per 100 g body weight) subcutaneously. Mice were then returned to cages at the end of the surgical procedure in a temperature controlled room (22^SC) with 12hr light and dark cycles and monitored every 6-8 hours.

Preparation of mouse plasma samples

Mouse plasma samples were obtained by injecting female wild-type (WT) C57BL/6 aged 10 weeks with 2gm/kg cecal slurry to induce poly-microbial sepsis produced by Nobel's nonsurgical method of sepsis induction described above or sham injection (phosphate buffered saline, PBS). Mice were euthanized 20hr later with C0₂. The mice were then immediately heart bled with a 1 mL 23G needle previously rinsed with 80uL (10% of expected blood volume) 0.5M EDTA pH 8.0 to prevent blood coagulation. The blood samples were spun for 10min with 3000rpm at 4 °C to separate plasma and the plasma frozen immediately at -20 °C.

Antigen

C57BL/6 mice (n=1 ) or Sprague-Dawley rats (n=1 ) were immunised with recombinant human BiP (100 μg in 100 mL; SEQ ID NO:4) or PBS mixed with 100 μΙ of Complete Freund's adjuvant for initial immunisation. Mice received two more boosters of the same dose (in complete Freund's adjuvant) at 4 weekly intervals. Mice then received a final booster in incomplete adjuvant a week before the spleen was harvested. All injections were delivered subcutaneously. Hvbridoma fusion

Sp2/0 cells were harvested and a vale cell count performed. Cells were required to be healthy, actively dividing and in log phase with viability >95%. Sp2/0 cells were cultured in HSFM/10% FCS and split 1 :2 or 1 :3 the day prior to the fusion. The mouse or rat used for the fusion was boosted 3-4 days before and on the day of harvest the spleen is placed into 1 0 mL of sterile medium. A single cell suspension was obtained the spleen by teasing out the cells and keeping the membrane intact. The cells were added to a 50 ml tube containing warm DME and washed x2 with DME at 1500 rpm for 5 mins. The Sp2/0 cells were washed twice with warm DME to ensure all traces of serum were removed and 1 x 10⁸ cells were used for fusing one mouse spleen or one third of a rat spleen. The Sp2/0 cells and spleen cells combined in a 50 ml tube in DME and centrifuged at 2100 rpm for 5 minutes. The DME was then removed ensuring the pellet remained intact. The tube was then placed in a heat block and 1 ml of warmed PEG was added dropwise over one minute while stirring gently continuously. A further 1 ml of warm DME is added over another minute while gently stirring followed by another 1 ml of warm DME. 20 ml of DME is then added over 5 mins while maintaining gentle stirring followed by centrifugation at 1500 rpm for 5 minutes. The supernatant is then removed and cells resuspended in the appropriate volume of medium (routinely one mouse spleen or one third of rat spleen is plated out to 10 x 96 well flat bottom plates). Cells were plated out at 0.2 ml per well and then incubated at 37^SC in 8% C0₂. Every 3-4 days the spent media is removed and gently replaced with fresh medium containing HAT. Approximately 10-12 days post fusion, the wells are examined for growth of hybridomas. When hybridomas are at optimal density, the supernatants were removed for screening.

Screening of hybridomas by ELISA

ELISA's for screening were performed in 96 well format. Wells were coated with 100 μΙ_ of the antigen (recombinant human BiP at 100 μg/mL) at 4 ^SC overnight. Wells were blocked with 1 % BSA in PBS for couple of hours. The hybridoma supernatnats were added to these wells and incubated at room temperature for one hour. After 3 washes in 100 μΙ_ PBS-Tween (0.05% tween 20), anti mouse or anti Rat- HRPO conjugated antibodies (1 :1 000 dilution) were added and incubated for one hour at room temperature. Excess antibodies were washed with 3X PBS tween and colour was developed using TMB substrate. Reaction was stopped with the addition of 1 .0M Hcl and colour intensity was measured by a colorimeter.

Induction of polymicrobial sepsis

Nobel Life Sciences has developed a robust and reproducible preclinical model of polymicrobial sepsis. In this model, a cecal slurry is used to induce septic peritonitis resulting in systemic bacteremia, organ infection and eventual systemic release of cytokines. Because it is polymicrobial involving both gram positive and gram negative bacteria, a wide range of pattern recognition receptors such as Toll-like receptors are activated on a variety of immune cells, mimicking sepsis.

10-week old C57B/6 mice were injected with 2gm/kg body weight (in 100 μΙ_ volume) of cecal slurry prepared in 5% dextrose intraperitoneally (Noble's non surgical model). 100 μΙ_ of PBS was used in control mice. Mice were culled 20 hours post injection and blood samples were collected by heart puncture with a 23-gauge needle pre-rinsed with 80 μΙ_ of 0.5M EDTA to prevent coagulation. Blood samples were spun immediately to separate the plasma and the plasma samples were frozen immediately at -20 or -80^SC.

Detection of secreted BiP in mouse plasma blood samples by sandwich ELISA

Nunc Immuno™ 96 well ELISA™ plates were coated with 100uL (100 ug/mL) of mouse monoclonal anti-human BiP antibody (clone 2D9; La Trobe University, generated in-house) in coating buffer (PBS) and incubated for 2h at 37 °C or at 4 °C overnight. Wells were washed 3x with 200uL washing buffer (PBS + 0.05% Tween20), blocked with 200uL blocking buffer (PBS +1 % BSA) and incubated for 1 h at 37 °C. After washing the plate 3x with 200uL washing buffer, 100uL of control or sepsis plasma was added to these wells and the plate incubated for at 37 °C for 45 mins.

The wells were then washed 3x with 200uL washing buffer and then incubated with 1 00uL of 1 ug/mL dilution of horse radish peroxidase (HRP) conjugated rat monoclonal anti-human BiP (clone RD4 La Trobe University, generated in-house) antibody in blocking buffer for 45min at 37 °C. Plates were then washed 3x with 200uL washing buffer per well and 100uL of tetramethylbenzidine (TMB) reagent was added to each well. Colour was developed for 15- 20mins at RT and the reaction stopped with 100uL stopping buffer (1 M HCI). Absorbance readings of each well were taken using a plate colourimeter at 450nm.

Known standard concentrations of the recombinant BiP was used in the same ELISA to prepare a standard curve to quantitate the levels of BiP in the serum.

Anti-BiP antibodies

For the assay described in Figure 4, the antibody was a mouse antibody to human BiP/GRP78 amino acids 525-628 of human BiP available from BD Transduction Laboratories™, catalog number 610978 (BD Biosciences). Recombinant human BiP

The sequence of the hexa His tagged human BiP that was used for generating 2D9 and RD4 in the diagnostic/detection assay is the sequence according to SEQ ID NO:4. BiP knockdown using retroviral vectors

Knockdown of BiP (GRP78) was performed as described in catalog No. SC-44303V from Santa Cruz Biotechnology.

Example 1 Apoptosis of T and B cells is mediated by Bim

In order to identify the factor responsible for lymphocyte apoptosis, the inventors set up an assay as shown in Figure 1 A. Bone marrow derived macrophages were isolated from C57BL/6 mice (either wild-type or Bim^{_ "} mice) according to standard methods, then plated into culture dishes and allowed to adhere to the surface of the culture dishes. The macrophages were cultured in the presence or absence of 100 ng/ml lipopolysaccharide (LPS) which induces activation of macrophages. After 24 hours, thymic T cells and splenic B cells harvested from wild type and Bim^{_ "} mice were treated for 24 hours with supernatant derived from the LPS- stimulated macrophage cultures. Conditioned medium from non-LPS-stimulated macrophages was used as a control.

Apoptosis of T and B lymphocytes was determined after 24 hours using the Annexin V- PI assay according to previously described methods (Koopman G et al, (1994) Blood 84:1415- 1420).

Figure 1 B shows the percentage of live thymocytes exposed to untreated macrophage supernatant (WT UT), wild-type thymocytes exposed to activated macrophage supernatant (Wt Mo Sup), Bim^{_ "} thymocytes exposed to untreated macrophage supernatant (Bim^{" _} UT) and Bim^{_ "} thymocytes exposed to activated macrophage supernatant (Bim^{" _} Mo Sup). Apoptosis was observed in thymocytes (T cells) that had been exposed to supernatant derived from activated macrophages whereas no apoptosis of Bim^{_ "} T cells was observed when these cells were exposed to supernatant from activated macrophages indicating that apoptosis was dependent on Bim (apoptotic factor) which was being induced by something present in the supernatant of activated macrophages.

Figure 1 C shows the percentage of live splenic B cells exposed to untreated macrophage supernatant (WT UT), wild-type splenic B cells exposed to activated macrophage supernatant (Wt Mo Sup), Bim^{_ "} splenic B cells exposed to untreated macrophage supernatant (Bim^{" _} UT) and Bim^{_ "} splenic B cells exposed to activated macrophage supernatant (Bim^{" _} Mo Sup). Apoptosis was observed in splenocytes (B cells) that had been exposed to supernatant derived from activated macrophages whereas similarly no apoptosis of Bim^{_ "} B cells was observed when these cells were exposed to supernatant from activated macrophages indicating that apoptosis was dependent on Bim (apoptotic factor) which was being induced by something present in the supernatant of activated macrophages.

Accordingly, as demonstrated in Figure 1 B (T cells) and 1 C (B cells), activated macrophages could cause apoptosis of T and B cells in a manner that was dependent on Bim (apoptotic factor) and this was statistically significant.

Example 2 Activated macrophage supernatant induces Bim expression on a translational and transcriptional level and Bim-dependent apoptosis in mouse embryo fibroblasts (MEFs)

The inventors developed an assay system as shown in Figure 2A that could be scaled up and which was not reliant upon the isolation of primary macrophages and lymphocytes. For this purpose the RAW264.7 macrophage cell line was used as the source of activated supernatant and mouse embryonic fibroblasts (MEFs) as the target cells enabling the assay to be scaled up (Figure 2A).

Like Bim^{_ "} T and B cells shown earlier, Bim^{_ "} MEFs do not undergo apoptosis in response in LPS-activated macrophage supernatant enabling induction of Bim mRNA and protein to be observed.

Similar to the bone marrow derived macrophages, RAW264.7 cells could induce apoptosis in lymphocytes (data not shown), but more interestingly, they could induce apoptosis in MEFs in a Bim-dependent fashion. Figure 2B shows measurement of apoptosis (as a percentage of live cells) in wild type and Bim^{_ "} MEFs, that were treated with supernatant from RAW264.7 cells to demonstrate that what is being purified is inducing Bim mediated apoptosis.

MEFs from BaxBak double knock-out mice (BaxBak ' ) were also treated with activated supernatant from RAW264.7 cells and induction of Bim expression at the mRNA level was confirmed by droplet digital PCR as shown in Figure 2C. BaxBak double knockout cells are useful targets since these cells do not undergo apoptosis and are ideal for studying Bim induction.

Figure 2C shows absolute gene expression of Bim and Puma (measured as fold induction). Puma is another pro-apoptotic gene whose expression is regulated by the tumour suppressor p53 and was used as a control. As shown in Figure 2C, transcriptional induction was specific to Bim and the activity of the supernatant is not inducing another BH3-only gene Puma.

Figure 2D shows expression at a protein level as determined by Western blot. The left panel shows protein expression of BaxBak double knock-out MEFs exposed to supernatant from untreated bone marrow macrophages, and BaxBak double knock-out MEFs exposed to supernatant from bone marrow macropahes treated with LPS for 6hr and 24hr. The right panel shows BaxBak double knock-out MEFs exposed to supernatant from untreated RAW264.7 macrophages and BaxBak double knock-out MEFs exposed to supernatant from RAW264.7 macrophages activated with LPS for 24 hours.

Example 3 Purification of the macrophage derived lymphocyte apoptotic factor

In order to purify the macrophage apoptotic inducing factor, the inventors generated 2 litres of LPS-activated supernatant from RAW264.7 macrophages. Supernatant was concentrated through an Amicon™ filtration device with 50 kD cut off (activity remains in the retentate). This was followed by a four-step purification protocol as shown in Figure 3A in which the supernatant was subjected to anion exchange Sepharose Q (Cat no. 17-1 179-01 , GE Healthcare), followed by gel filtration Sephacryl (Cat no. 17-0584-01 , GE Healthcare), followed by heparin sepharose (Cat no, 17-0406-01 , GE Healthcare) and then melon gel (Cat no. 45206, Thermo Scientific) according to manufactures' instructions.

At each stage of the purification, fractions were assayed for apoptotic activity using

MEFs (either wild-type) or Bim^{_ "} as the target cells by measuring the percentage of live cells (Figure 3B) and protein expression by Western blot (Figure 3C). The pooled final sample i.e. 0.2 and 0.4 M elution fractions from Melon Gel™ column, were separated on SDS-PAGE and five bands were excised and subjected to mass spectrometry analysis (Matrix-assisted laser desorption/ionization ; MALDI-TOF/TOF) as shown in Figure 3D.

The mass spectrometry data (Table 1 ) revealed five major proteins in the final fraction in the following order of abundance: L-plastin, BiP (GRP78), monocyte differentiation antigen

CD14, Translationally controlled tumor protein (TCTP) and Tumor necrosis factor alpha (TNF- alpha).

Table 1 Mass spectrometry analysis of purified active fraction

The presence of these proteins was confirmed in the published database of macrophage secretome (secreted molecules) as described in Eichelbaum K et al, (2012) Nat Biotechnol 30:984-990; Meissner F et al, (2013) Science 340:475-478. L-plastin was ruled out as the candidate by Cecal Ligation and Puncture (CLP)-induced sepsis in L-plastin knockout mice. Lymphocyte apoptosis in L-plastin knockout mice was not statistically significant to WT control mice (data not shown) using the inventor's assay.

Tumour necrosis factor alpha (TNF-a) was ruled out as a candidate for three reasons. 1 . The discrepancies in molecular weights i.e. molecular weight of 51 kD of trimeric TNF-alpha versus > 65 kD apparent molecular weight of the factor from gel filtration chromatography.

2. Addition of pure TNF-alpha to macrophage supernatant (without LPS) did not induce Bim nor did it elicit any apoptotic response in MEFs; and

3. TNFR1^7", TNFR2^"'^" double knock out MEFs (MEFs without any TNF-alpha receptors) induced Bim and underwent apoptosis in response to the macrophage supernatant treatment (data not shown).

This left three candidates, namely binding immunoglobulin protein (BiP (GRP78)), cluster of differentiation 14 (CD14) and translationally controlled tumour protein (TCTP). The second most abundant protein in the active fraction was BiP (or Immunoglobulin Binding Protein)/GRP78. It is normally an ER resident protein, which binds misfolded proteins with high affinity (Gething MJ (1999) Seminars in cell & developmental biology vol 10:465-472). BiP/GRP78 also localises to the plasma membrane in stem cells and tumour cells. However, it is present in the secretome of activated macrophages (Eichelbaum K et al, (2012) Nat Biotechnol 30:984-990; Meissner F et al, (2013) Science 340:475-478) suggesting that it also exists in a secreted form.

CD14 is known to be an LPS co-receptor with a molecular weight of 50kDa. This was smaller than the expected size of the active protein (>65kDa) and thus was ruled out. Furthermore, TCTP has a molecular weight of 25-30kDa which again is smaller than the expected size of the active protein.

Therefore BiP (GRP78) appeared to be the candidate that was inducing Bim mediated apoptosis of lymphocytes and MEFs. Accordingly, contrary to the known role of BiP as protecting cells from apoptosis, the inventors found that BiP was functioning in a pro-apoptotic manner by mediating apoptosis via Bim. Example 4 BiP mediated apoptotic induction in lymphocytes

BiP mediated apoptotic induction in lymphocytes was assessed and confirmed by four different methods:

1) Immuno-depletion of the activated macrophage supernatant with anti-BiP antibodies:

Treating the activated macrophage supernatant with anti-BiP antibody (BD Biosciences) rescued MEFs from undergoing apoptosis (Figure 4A). The isotype control antibody (lgG2a control) had a marginal effect on apoptosis, which is likely attributable to non-specific binding of BiP to immunoglobulins (BiP was originally identified as an 'Immunoglobulin Binding Protein'). Nevertheless, the percentage of live cells was statistically different in the presence of anti-BiP antibody versus isotype control.

2) Use of Toll-like receptor (TLR) pathway knock-out mice macrophages:

Toll-like receptors play a critical role in the early innate immune response to invading pathogens by sensing microorganisms. Of these, TLR4 is the main receptor for LPS. MyD88 is the downstream adaptor for all TLRs except TLR3. TRIF is a downstream adaptor for TLR3, TLR4 and TLR9 as demonstrated in the below schematic.

The inventors used LPS activated supernatant from wild-type (WT), TLR4 ^", MyD88^{~ ~} and

TRIF^{_ "} macrophages to examine their effect on the target cells (MEFs). After 24 hours the percentage of live cells was determined by Annexin V PI assay as previously described. As shown in Figure 4B, TLR4^{_ "} and MyD88^{_ "} had an intermediate effect on apoptosis, TRIF^{_ "} macrophage supernatant completely lacked the ability to induce apoptosis in the target cells. Consistent with this observation, TRIF^{_ "} macrophages did not secrete BiP into the medium upon LPS activation (Figure 4C, Western blot).

3) shRNA knock down of BiP in macrophages:

BiP expression in macrophages was knocked-down using retroviral vectors according to standard methods as described elsewhere herein. As shown in Figure 4D, clones 9 and 1 1 had reduced levels of BiP compared with other clones and the control (Cont.). The activated supernatant from these clones failed to induce Bim in the target cells (Figure 4E, middle) and had lost the ability to induce apoptosis (Figure 4F, graph).

The inventors activated the wild type and the BiP knockdown clones with LPS and conducted a comparative quantitative mass spectrometric analysis of the secretome. There was no significant difference in the amounts of secretion of TNF-alpha, IL1 , IL6, CXCL5, CXCL10, CD14 and L-Plastin (data not shown) suggesting that BiP knock down did not lead to global down regulation of protein secretion. Unlike wild type macrophages, the BiP knock down macrophage lines did not undergo apoptosis upon LPS treatment, suggesting an autocrine role for secreted BiP in inducing apoptosis (data not shown).

4) Use of chemical chaperone TUDCA:

Tauroursodeoxycholic acid (TUDCA) is an ambiphilic bile acid. It acts as a chemical chaperone to alleviate ER stress, improve insulin sensitivity and normalize hyperglycemia in obese and diabetic mice (Ozcan U YE et al , (2006) Science 313:1 137-1 140). Since there are reports of cells undergoing systemic ER stress during endotoxemia (Hiramatsu N et al, (2006) Nucleic acids research 34:e93), the inventors tested if treating LPS-activated wild-type macrophages with TUDCA would alter the ability of BiP to mediate apoptosis of target cells (MEFs).

Addition of TUDCA (500μg/mL) together with LPS (100 ng/ml) to the wild-type C57BL/6 macrophages for 24 hours resulted in a significant reduction of BiP secretion and complete rescue of the target cells from apoptosis as shown in Figure 5 A and 5B. Figure 5C shows Bim transcriptional expression in BaxBak double knock-out MEFs.

Similarly, in a cecal slurry-induced mouse sepsis model, TUDCA injection (8g/kg body weight) led to a significant reduction in thymocyte cell death (Figure 5D).

Example 5 Detection of BiP in blood samples

From the inventors results, it appears that activated macrophages secrete a soluble form of BiP (GRP78) and this secreted BiP induces lymphocyte apoptosis systemically. Therefore, if the hypothesis is correct, it would be expected that plasma samples from sepsis patients should have detectable levels of BiP (GRP78).

To test this, the inventors developed a sandwich ELISA for detecting BiP in mouse plasma. Mice (n=10) were injected with 2 gm/mL cecal slurry prepared using Nobel's surgical model described herein or sham injected with PBS (n=4 mice). Fold induction of BiP in the serum is shown in Figure 6A. Figure 6B shows quantity of secreted BiP in pg/mL between sham injected mice and mice injected with cecal slurry. Figure 6C shows the percentage of live cells (thymocytes) from sham injected (n=3) versus cecal slurry injected (n=8) mice. Figure 6D and E show total cell counts measured in thymus (T cells) and spleen (B cells) of sham compared to cecal slurry injected mice at 20hr post injection. As expected, total cells and the percentage of live cells was decreased in cecal slurry injected mice compared with sham injected mice consistent with cells undergoing apoptosis.

Human plasma samples will be obtained from a repository of patients diagnosed with sepsis and tested using the sandwich ELISA. The amount of BiP will be quantitated by ELISA in plasma samples to derive a correlation between the level of BiP (GRP78) and lymphocyte count as per the mouse studies. To detect a correlation (r=0.6) using a two-sided test (alpha=0.05 and beta=0.2) 20 samples will be required.

Claims

CLAIMS:

1 . A method for preventing and/or treating immunosuppression in a subject in need thereof, comprising administering to the subject an inhibitor of BiP (GRP78) which binds to, or specifically binds to a secreted form of BiP (GRP78).

2. The method according to claim 1 , wherein the immunosuppression is characterised by sepsis induced leukopenia.

3. A method of inhibiting and/or preventing apoptosis of leukocytes in a subject in need thereof, comprising administering to the subject an inhibitor of BiP (GRP78) which binds to, or specifically binds to a secreted form of BiP (GRP78).

4. The method according to claim 3, wherein the leukocytes comprise lymphocytes and antigen presenting cells (APCs).

5. The method according to any preceding claim, wherein the method inhibits or prevents apoptosis of T and/or B cells by the secreted form of BiP.

6. The method according to any preceding claim wherein the wherein subject has been diagnosed with sepsis, or is suspected of having, or is at risk of acquiring sepsis.

7. A method of preventing and/or treating sepsis in a subject in need thereof, comprising administering to the subject an inhibitor of BiP (GRP78) which binds to, or specifically binds to a secreted form of BiP (GRP78).

8. Use of an inhibitor of the secreted form of BiP (GRP78) for preventing and/or treating immunosuppression in a subject in need thereof.

9. Use of an inhibitor of the secreted form of BiP (GRP78) for preventing and/or treating apoptosis of leukocytes in a subject in need thereof.

10. Use of an inhibitor of the secreted form of BiP (GRP78) for preventing and/or treating sepsis in a subject in need thereof.

1 1 . Use according to any one of claims 8 to 10, wherein the leukocytes comprise lymphocytes and antigen presenting cells (APCs).

12. A method according to any one of claims 1 to 8, or a use according to any one of claims 9 to 1 1 , wherein the inhibitor of BiP binds to a human BiP protein comprising:

(i) the sequence according to SEQ ID NO:1 ;

(ii) a sequence at least 95% identical to SEQ ID NO:1 ; or

(iii) the sequence according to SEQ ID NO:3.

13. A method for diagnosing and/or prognosing sepsis in a subject, the method comprising:

(i) obtaining a biological sample from a subject suspected of having sepsis;

(ii) contacting the sample with a binding agent(s) which bind to, or specifically bind to, a secreted form of BiP (GRP78);

(iii) detecting binding of the binding agent(s) to BiP (GRP78);

14. The method according to claim 13, wherein a level of BiP which is at least 2-fold greater compared with a control subject who does not have sepsis is diagnostic or prognostic of sepsis.

15. The method according to claim 13, further comprising:

(iv) quantifying the level of BiP in the biological sample wherein a level of greater than 1 .5 pg/ml of secreted BiP in the biological sample is diagnostic or prognostic of sepsis.

16. The method according to any one of claims 13 to 15, wherein the detection step is carried out using a sandwich ELISA format.

17. The method according to any one of claims 13 to 16, further comprising administering to the subject an inhibitor of a secreted form of BiP (GRP78).

18. The method according to any one of claims 13 to 17 wherein the biological sample is selected from serum, plasma, urine, lymph or saliva.

19. A method for preventing and/or treating apoptosis of leukocytes, comprising administering to a subject in need of such treatment, and who has previously been diagnosed with a higher level of BiP compared with a control subject, an effective amount of an inhibitor of a secreted form of BiP (GRP78).

20. A method of diagnosing a subject having sepsis as being effectively treated by administration of an inhibitor of a secreted form of BiP (GRP78), comprising:

21 . The method according to claim 20, wherein the level of BiP according to step (ii) is less than 1 .5 pg/ml.

22. The method according to claim 20 or 21 , wherein the assay is a sandwich ELISA assay comprising immobilising a first binding agent which binds to a secreted form of BiP to the surface of an ELISA plate, contacting the first binding agent (capture agent) with the biological sample, and detecting binding using a second binding agent (detection agent) which binds to the same secreted form of BiP, wherein the second binding agent is conjugated to a detectable label.

23. A kit for detecting a secreted form of BiP in a biological sample comprising first and second binding agents which bind to human BiP, together with a set of standards comprising human BiP protein according to SEQ ID NO:4, and further comprising instructions for use in performing a sandwich ELISA assay.