Use of albumin for regulating immune cells

Abstract

The present invention relates to the use of albumin for use in regulating immune cells in a subject in need thereof. The present invention relates particularly to a composition comprising human albumin for use in regulating immune cells in a subject having systemic inflammatory response syndrome.

Description

[0001] Use of albumin for regulating immune cells

[0002] The present invention relates to the use of albumin in regulating immune cells in a subject in need thereof. The present invention relates particularly to a composition comprising human albumin for use in regulating immune cells in a subject having systemic inflammatory response syndrome.

[0003] The immune system is a conglomerate of cells and molecules that cooperate to protect human beings from infectious agents. The immune system also provides human beings with a surveillance mechanism to continuously monitor the integrity of cells or tissues. By this surveillance, changes in major histocompatibility complex proteins or viral proteins on the cell membrane that signal cancer development or intracellular viral infection are recognized. The immune system functions under two major principles: (1) recognition of foreign (non-self) material by highly sensitive plasma membrane or intracellular receptors present in the immune cells, which results in expression of hundreds of genes regulating the inflammatory response and proliferation of the immune cells; (2) elimination of microorganisms by a diverse repertoire of mechanisms, including unspecific anti- microbial proteins synthesized by the liver (i.e. the complement system which kills bacteria either by direct lysis or activating phagocytosis by the immune cells); the release by granulocytes, T-cytotoxic lymphocytes (Tc cells) and natural killer (NK) cells of intracellular cytotoxic granules that contain a battery of enzymes and poreforming proteins (perforins) able to kill microorganisms, phagocytosis (granulocytes and macrophages), and specific antibodies [B-lymphocytes (B -cells)]. Activated Tc and NK cells present Fas-ligands that interact with Fas-receptors on the target cells (cancer or virus infected cells) leading to cell-death by apoptosis. Antibodies, complement, and granulocytes give protection against most extracellular organisms, whereas macrophages, Tc-cells and NK cells are involved in the surveillance process and elimination of tumoral cells and virally infected cells. Each immune system works complementarily.

[0004] The innate immune system serves as a rapid reaction force that deploys within minutes a range of non-specific (but highly effective) weapons to eradicate the infectious agent or to keep the infection contained. Cells from the innate immune system use conserved non-specific pattern-recognition receptors (PRRs) that very reliably recognize common pathogen- associated molecular pattern (PAMPs) or damage associated molecular patterns (DAMPs) released during tissue damage. The recognition of PAMPs and DAMPs and the primary immune response is a simple process and occurs within minutes. The innate immune system is composed by granulocytes (neutrophils, basophils, and eosinophils), macrophages (tissue cells derived for the circulating monocytes) and dendritic cells (specialized cells in presenting antigens to T, B and NK cells for the immune response). The NK cells, although being lymphoid cells, are also part of the innate immune system.

[0005] The association of systemic inflammation, immunosuppression, high prevalence of secondary bacterial infections and multiorgan system failure identifies a syndrome commonly observed in critically ill patients, including patients with acutely decompensated cirrhosis, acute liver failure, severe sepsis (including septic shock), acute pancreatitis and other intraabdominal inflammatory processes, polytraumatism, or severe burns.

[0006] Acutely decompensated cirrhosis, as defined by the recent development of ascites, hepatic encephalopathy, gastrointestinal hemorrhage or any combination of these,1 2has two severe forms including acute-on-chronic liver failure (ACLF)1’2and also pre-ACLF.34ACLF defines patients who present single or multiple organ failure.1 2Among patients with acutely decompensated cirrhosis, immune activation is more marked in those with acute-on-chronic liver failure (ACLF), which is a syndrome characterized by intense systemic inflammation, multiorgan dysfunction / failure and extremely high prevalence of bacterial infections (Moreau et al., 2013, Gastroenterology, 144: 1426-37, 1437. e1-9; Arroyo, et al., 2020, N Engl J Med, 382:2137-2145) than among those without ACLF (Weiss et al., 2021 , Frontiers in Immunol, vol. 12, art. 699563). Pre-ACLF defines a group of patients with acutely decompensated cirrhosis admitted to hospital without ACLF but who develop ACLF during hospitalization or within a period of 3-months after admission.34Recent, still unpublished, investigations have shown that pre-ACLF consists in two different phenotypes: “early” pre- ACLF, which defines patients who develop ACLF within the index hospitalization, and “delayed” pre-ACLF, which defines patients who develop ACLF between discharge from the index hospitalization and the end of the 3-month follow-up period. Studies in patients with ACLF have shown that these patients exhibit intense systemic inflammation (indicated by leukocytosis,1 2neutrophilia,5elevated blood levels of cytokines,6bioactive lipids7and C- reactive protein1 2), immunosuppression (indicated by decreased responses of specific monocyte subsets to bacterial products;8-10decreased neutrophil capacity to kill microbes;5and lymphopenia involving T cells, B cells and NK cells5), and a high incidence of secondary infections,11’12which are deadly complications.11Patients hospitalized with “early” pre-ACLF also exhibit intense systemic inflammation, a feature which is moderate in patients with “slowly progressive” pre-ACLF. To date, there are no therapies against immune-cell deregulation in these patients.

[0007] Albumin is commonly used in patients with cirrhosis, including patients treated by large-volume paracentesis,13and patients with spontaneous bacterial peritonitis (SBP),14or hepatorenal syndrome-acute kidney injury (HRS-AKI).15The effects of albumin are usually attributed to plasma volume expansion.11Recent studies, however, proposed that albumin may affect particular immune cell functions,16’17findings that may explain the lower incidence rate of SBP and other infections reported among patients receiving long-term albumin administration in comparison to patients receiving standard medical therapy.18

[0008] Furthermore, although there is no established therapy against immunosuppression to date, recent studies suggests that intravenous albumin, beyond its plasma expander properties, may reduce the severity of systemic inflammation (Fernandez et al., 2018, Gut, 67:1870-1880; Casulleras et al., 2020, Sci Transl Med, 12(566):eaax5135).

[0009] Brief summary of the disclosure

[0010] The effects of intravenous albumin on lymphopenia and defective neutrophil anti-microbial functions that characterize patients with acute-on-chronic liver failure (ACLF) are unknown.

[0011] The invention is based on the surprising finding that treatment with human albumin affects different subsets of immune cells (specifically neutrophils, CD4+ T cells, dendritic cells and / or monocytes) in a subject. Compositions comprising human albumin therefore have beneficial utility in regulating immune cells (specifically neutrophils, CD4+ T cells, dendritic cells and / or monocytes) in a subject in need thereof. The invention is particularly useful in the context of subjects in whom immune cells are compromised (specifically neutrophils, CD4+ T cells, dendritic cells and / or monocytes), and / or subjects that would benefit from an increase in immune cell function (specifically neutrophil function, CD4+ T cell function, dendritic cell function and / or monocyte function) e.g. to prevent microbial infections.

[0012] As described in the examples included herein, the inventors investigated forty-nine patients admitted for severe acutely decompensated cirrhosis without ACLF with the use of wholeblood RNA sequencing (RNA-seq) on admission and after a median period of 15 days once they had developed ACLF. Such patients were selected because they follow a steady systemic inflammation course. Thirty patients had received albumin during the progression to ACLF but not the 19 others. Furthermore, in additional patients with acutely decompensated cirrhosis, the inventors performed single-cell RNA-seq (scRNA-seq) in peripheral blood mononuclear cells (PBMCs) exposed ex vivo to albumin or vehicle for 2 hours, and also assessed the antimicrobial capacity of neutrophils exposed ex vivo to albumin.

[0013] Analysis of whole-blood RNA-seq data revealed that patients who had received albumin exhibited specific upregulation of signatures related to B cells, plasma cells and immunoglobulins; CD4 T cells; myeloid cells; mismatch repair, cell cycle and mitosis; and transcription factors such as c-Myc and E2F family members. The use of scRNA-seq to analyze patients’ PBMCs exposed ex vivo to albumin showed increases in signatures related to B cells, myeloid cells and CD4 T cells. Furthermore, the inventors demonstrated that neutrophils exposed ex vivo to albumin exhibited increased degranulation responses and enhanced phagocytosis.

[0014] Advantageously, the inventors therefore showed that in patients with severe acutely decompensated cirrhosis, albumin promotes the expansion of the B-cell compartment and also of the CD4 T-cell compartment, acts on mononuclear myeloid cells and resets neutrophil antimicrobial functions to normal.

[0015] Accordingly, a composition comprising human albumin for use in regulating immune cells in a subject having systemic inflammatory response syndrome is provided herein, wherein:

[0016] (a) the composition comprising human albumin is for regulating neutrophil function, wherein the human albumin is for administration to the subject in a dose sufficient to increase neutrophil anti-microbial function; and / or

[0017] (b) the composition comprising human albumin is for regulating CD4+ T cells, wherein the human albumin is for administration to the patient in a dose sufficient to increase the level of central memory ITGB1+ CD4+ T cells and / or decrease the level of activated CD4+ memory T cells; and / or

[0018] (c) the composition comprising human albumin is for regulating dendritic cells, wherein the human albumin is for administration to the subject in a dose sufficient to increase the level of plasmacytoid dendritic cells; and / or

[0019] (d) the composition comprising human albumin is for regulating monocyte cells, wherein the human albumin is for administration to the subject in a dose sufficient to increase the level of intermediate HAVCR2+ monocytes.

[0020] Suitably, the increase in neutrophil anti-microbial function may be an increase in neutrophil degranulation and / or neutrophil phagocytosis.

[0021] Suitably, the human albumin may be for administration to the subject in a dose sufficient to upregulate the expression of a neutrophil gene selected from the group consisting of: CD177, OLFM4, PRG2, MPO, BPI, RETN, LCN2, CEACAM8, and MCEMP1 , ora combination thereof.

[0022] Suitably, the systemic inflammatory response syndrome may be related to a disease selected from the group consisting of: decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer.

[0023] Suitably, the subject may be a sepsis patient or a patient with a liver condition.

[0024] Suitably, the subject may have a liver condition selected from the group consisting of: decompensated cirrhosis, pre-acute on chronic liver failure, and acute on chronic liver failure.

[0025] A composition comprising human albumin for the treatment of defective immune cell function in a subject in need thereof is also provided, wherein the defective immune cell function is selected from the group consisting of:

[0026] (a) defective neutrophil function;

[0027] (b) defective central memory ITGB1+ CD4+ T cell function and / or activated CD4+ memory T cell function;

[0028] (c) defective plasmacytoid dendritic cell function; and

[0029] (d) defective intermediate HAVCR2+ monocyte function.

[0030] Suitably, the treatment may increase neutrophil anti-microbial function in the subject, optionally wherein the treatment may increase neutrophil degranulation and / or neutrophil phagocytosis.

[0031] Suitably, the human albumin may be for administration to the subject in a dose sufficient to upregulate the expression of a neutrophil gene selected from the group consisting of: CD177, OLFM4, PRG2, MPO, BPI, RETN, LCN2, CEACAM8, and MCEMP1 , ora combination thereof.

[0032] Suitably, said defective immune cell function may be related to a disease selected from the group consisting of: systemic inflammatory response syndrome, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemiareperfusion syndrome, inflammation diseases, and cancer.

[0033] Suitably, the human albumin may be for administration by an administration route selected from the group consisting of: intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intrapulmonary, intranasal, oral, rectal, and combinations thereof.

[0034] Suitably, the human albumin may be for administration at multiple intervals as part of a multiple-dosage regimen. Suitably, the multiple dosing regimen may comprise multiple portion doses administered at equally spaced intervals of from about 1 day to about 30 days; or the multiple dosing regimen may comprise multiple portion doses administered at unequally spaced intervals of from about 1 day to about 30 days; or the multiple dosing regimen may comprise two or more administrations up to a total cumulative dose.

[0035] Suitably, the human albumin may be for administration at individual doses of about 5 g / interval to about 500 g / interval as part of a multiple-dosage regimen; or the human albumin may be for administration at individual doses of about 20 g / interval to about 200 g / interval as part of a multiple-dosage regimen.

[0036] Suitably, the dosing interval may be every 15 days, or less.

[0037] Suitably, the human albumin may be human plasma-derived albumin or recombinant human albumin.

[0038] Suitably, the concentration of human albumin may be between 4 % and 25 % (w / v); or wherein the concentration of human albumin is about 20 % (w / v).

[0039] As described herein, human albumin affects different subsets of immune cells (specifically neutrophils, CD4+ T cells, dendritic cells and / or monocytes) in a subject. It is therefore a therapeutic agent in the compositions described herein. The term “human albumin” may therefore be used interchangeably with the term “composition comprising human albumin” throughout.

[0040] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0041] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0042] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0043] Various aspects of the invention are described in further detail below.

[0044] Brief description of the Figures Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0045] Figure 1. Whole-blood gene signatures associated with albumin treatment. (A) Euler plot shows differentially expressed genes (DEGs) between time 2 (T2) and time 1 (T1) for the albumin group and the no-albumin group. DEGs were defined by an absolute fold-change (FC) greater than 1.5 and P <0.05. (B) Volcano plots show differential expression effect-size (Iog2 FC) between T2 and T1 plotted against significance (-Iog10 P), for the albumin group (top) and no-albumin group (bottom). In both Volcano plots, gray points indicate genes with no significant difference in expression between T2 and T 1 (with absolute FC of less than 1 .5 and P>0.05, i.e., -Iog10 P <1.3). Greyscale points indicate DEGs, either upregulated or downregulated genes. (C) Differential expression effect-size (Iog2 FC) between T2 and T1 in the albumin group compared with corresponding differential effect-size in the no-albumin group. Only genes included in the Gene Ontology gene set “GOCCJmmunoglobulin Complex” are shown here. DEGs with concordant sign were considered as shared. (D) Violin plots of RNA-seq inferred signatures at T1 and T2 for plasmablasts, NK cells, and T cells determined with the use of the SingleR R software, in the albumin and the no-albumin groups. Baseline values in healthy subjects are also shown. White rhomboid symbols indicate medians. The P values are from Kruskal-Wallis tests followed by Mann-Whitney II tests. (E) Heat maps of 32 differentially expressed (DE) blood transcription modules (BTMs) between T2 and T1 that were specific for either albumin group or no-albumin group. Thirty-one DE BTMs were specific for the albumin group whereas only one DE BTM (“endoplasmic reticulum (M37.2)”) was specific for the no-albumin group. BTMs are hierarchically clustered based on QuSAGE activity scores obtained in the albumin group. Asterisks denote P <0.05. Greyscale represents QuSAGE activity score (-0.4 to 0.4).

[0046] Figure 2. Single-cell RNA sequencing identifies specific immune-cell changes in peripheral blood mononuclear cells (PBMCs) exposed ex vivo to albumin. This figure has been designed with results obtained in PBMCs from patients with acutely decompensated cirrhosis. (A) Uniform manifold approximation and projection (UMAP) of 1946 patients’ B lymphocytes exposed to albumin and vehicle, colored by cell types. (B) Overlay of albumin and vehicle exposure on the B lymphocyte UMAP. (C) Box plots for the abundance of B lymphocyte populations that significantly changed after albumin exposure. (D) UMAP of 21819 patients’ myeloid cells exposed to albumin and vehicle, indicated by cell populations. (E) Overlay of albumin and vehicle exposure on the myeloid cell UMAP. (F) Box plots for the abundance of myeloid cell types that significantly changed after albumin exposure (plasmacytoid dendritic cells; pDCs). (G) UMAP of 12692 patients’ CD4 T cells exposed to albumin and vehicle, colored by cell types. (H) Overlay of albumin and vehicle exposure on CD4 T cell LIMAP. (I) Box plots for the abundance of CD4 T cells that significantly changed after albumin exposure. (J) Left, Representative density plot showing the signature score for the BTM “mitotic cell cycle in stimulated CD4 T cells (M4.11)” on the CD4 T cells LIMAP across vehicle and albumin conditions. Right, Comparison of log(signature score) for the BTM 4.11 between albumin and vehicle on the CD4 T cell compartment. Statistical analysis for BTM signature was performed using a Wilcoxon signed-rank sum test; p-value significance is indicated. All comparison of cell type abundance between albumin and vehicle were tested for significance by paired Wilcoxon signed-rank sum test, and significant adjusted p-values are indicated.

[0047] Figure 3. Effects of albumin on the host defense function of neutrophils from patients with acutely decompensated cirrhosis. Panels A and B have been designed using functional assays in freshly isolated peripheral neutrophils from 6 patients with acutely decompensated cirrhosis and 5 age-matched healthy subjects. (A) Neutrophils, from both patients with acutely decompensated cirrhosis and healthy subjects, were incubated with cell medium (vehicle), human serum albumin (15 mg / ml) or recombinant human albumin (15 mg / ml) for 2 hours at 37°C in a 5% CO2 incubator. Neutrophil degranulation was assessed by measuring the MPO enzymatic activity in the cell supernatants (B) Phagocytic capacity assessed by incubating (for 60 min) neutrophils, from both patients with acutely decompensated cirrhosis and healthy subjects, with FITC-conjugated zymosan bioparticles alone or in the presence of human serum albumin and recombinant albumin (15 mg / mL) and compared to vehicle control.

[0048] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

[0049] Various aspects of the invention are described in further detail below.

[0050] Detailed Description

[0051] The invention is based on the surprising finding that treatment with human albumin affects different subsets of immune cells (specifically neutrophils, CD4+ T cells, dendritic cells and / or monocytes) in a subject.

[0052] After treatment with albumin, immune cell gene signatures in patients with acutely decompensated cirrhosis (with pre-ACLF who developed ACLF) (“albumin group”) were compared to equivalent patients that were not given albumin (“no-albumin group”). A striking number of similarities between the transcriptional characteristics of pre-ACLF and ACLF patients was identified. In addition, differential expression of several gene modules in the albumin group was observed. Specifically, patients treated with albumin had upregulation of several genes that are markers of activated low-density neutrophils. Furthermore, significant increases in the abundance of intermediate monocytes HAVCR2+ and in plasmacytoid DCs were observed in the albumin group (Figs. 2E and 2F). Finally, albumin was shown to change the profile of the CD4+ T-cell compartment (Fig. 2H), with a significant decrease in the abundance of activated memory CD4+ T cells and an increase in central memory ITGB1 + CD4+ T cells (Fig. 2I).

[0053] The prevalence of bacterial infections at admission in patients with acutely decompensated cirrhosis is very high. Among the uninfected patients at admission, a significant proportion develop bacterial infection during hospitalization due to a severe impairment of their defensive mechanisms against microbes. The results of the present study indicate that albumin can rescue both the defective neutrophil antimicrobial functions and the depleted lymphocyte compartment in the most severe forms of acutely decompensated cirrhosis.

[0054] Regulating neutrophils

[0055] The inventors have shown herein that treatment with human albumin specifically upregulates nine major neutrophil genes CD177, OLFM4, PRG2, MPO, BPI, RETN, LCN2, CEACAM8, MCEMP1) (Fig. 1 B) in a subject. In addition, it is shown herein that neutrophils exposed ex vivo to human albumin surprisingly exhibit increased anti-microbial activity, including increased degranulation responses and enhanced phagocytosis (Fig. 3). The data provided herein indicate that human albumin can regulate neutrophil function (in particular increasing neutrophil anti-microbial function) in a subject.

[0056] Accordingly, a composition comprising human albumin for use in regulating neutrophil function in a subject is provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase neutrophil anti-microbial function (e.g. wherein the increase in neutrophil anti-microbial function is an increase in neutrophil degranulation and / or neutrophil phagocytosis). Such compositions may be used to prevent infections and / or dampen pathogen-associated molecular pattern (PAMP)-induced systemic inflammation in the subject. A composition comprising human albumin for preventing infection and / or dampening pathogen-associated molecular pattern (PAMP)-induced systemic inflammation in a subject is therefore also provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase neutrophil anti- microbial function (e.g. wherein the increase in neutrophil anti-microbial function is an increase in neutrophil degranulation and / or neutrophil phagocytosis ).

[0057] In one example, a composition comprising human albumin for preventing microbial infections and / or dampening pathogen-associated molecular pattern (PAMP)-induced systemic inflammation in a subject is provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase neutrophil antimicrobial function (e.g. wherein the increase in neutrophil anti-microbial function is an increase in neutrophil degranulation and / or neutrophil phagocytosis ).

[0058] Examples of suitable microbial infections include bacterial infections, such as spontaneous bacterial peritonitis (SBP). SBP is one of the most common infections in patients with cirrhosis. Pathogens most commonly involved in SBP are Enterobacteriaceae and non-enterococcal streptococci, with enterococci increasingly involved in nosocomial episodes. Among other infections, the most common are urinary tract infections (UTIs), pneumonia, primary / spontaneous bloodstream infections (BSIs) and skin and soft tissue infections. Pathogens most frequently involved in UTIs are Enterobacteriaceae and enterococci, while those most frequently involved in primary BSI are Enterobacteriaceae and staphylococci. Other less common infections are cholangitis, endocarditis, catheter-related bloodstream infections, Clostridium difficile enterocolitis and secondary peritonitis. See Fernandez et al, Management of bacterial and fungal infections in cirrhosis: The MDRO challenge, Journal of Hepatology, Volume 75, Supplement 1 , July 2021 , Pages S101-S117. These examples of suitable microbial infections apply to all relevant aspects described herein.

[0059] The compositions provided herein are particularly useful when administered to a subject having systemic inflammatory response syndrome (SIRS). Subjects having SIRS typically have defective neutrophil anti-microbial function. Advantageously, the compositions provided herein can be used to increase neutrophil anti-microbial function (e.g. wherein the increase in neutrophil anti-microbial function is an increase in neutrophil degranulation and / or neutrophil phagocytosis ) in these subjects and therefore can be used to prevent infections and / or dampen pathogen-associated molecular pattern (PAMP)-induced systemic inflammation in subjects having SIRS specifically. Accordingly, a composition comprising human albumin for use in regulating neutrophil function in a subject having SIRS is provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase neutrophil anti-microbial function (e.g. wherein the increase in neutrophil antimicrobial function is an increase in neutrophil degranulation and / or neutrophil phagocytosis ). The compositions provided herein may therefore be for treating SIRS in a subject by regulating their neutrophil function. A composition comprising human albumin for preventing infection and / or dampening pathogen-associated molecular pattern (PAMP)-induced systemic inflammation in subjects having SIRS is also provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase neutrophil antimicrobial function (e.g. wherein the increase in neutrophil anti-microbial function is an increase in neutrophil degranulation and / or neutrophil phagocytosis ). The compositions provided herein may therefore be for treating SIRS in a subject by preventing infection and / or dampening their pathogen-associated molecular pattern (PAMP)-induced systemic inflammation.

[0060] Examples of subjects having SIRS are described elsewhere herein. For example, the SIRS may be related to a disease selected from the group consisting of: decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatism, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer. In other words, the subject having SIRS may also have a disease selected from the group listed above.

[0061] In one example, the subject having SIRS is a subject having decompensated cirrhosis. For example, the subject having SIRS may be a subject having pre-ACLF decompensated cirrhosis (e.g. early pre-ACLF decompensated cirrhosis) or ACLF decompensated cirrhosis. In one example, the pre-ACLF decompensated cirrhosis is early pre-ACLF decompensated cirrhosis.

[0062] A composition comprising human albumin for use in regulating neutrophil function in a subject having decompensated cirrhosis (e.g. pre-ACLF decompensated cirrhosis or ACLF decompensated cirrhosis) is also provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase neutrophil antimicrobial function (e.g. wherein the increase in neutrophil anti-microbial function is an increase in neutrophil degranulation and / or neutrophil phagocytosis). The compositions provided herein may therefore be for treating decompensated cirrhosis (e.g. pre-ACLF decompensated cirrhosis or ACLF decompensated cirrhosis) in a subject by regulating their neutrophil function. Furthermore, a composition comprising human albumin for preventing infection and / or dampening pathogen-associated molecular pattern (PAMP)-induced systemic inflammation in a subject having decompensated cirrhosis (e.g. pre-ACLF decompensated cirrhosis or ACLF decompensated cirrhosis) is also provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase neutrophil anti- microbial function (e.g. wherein the increase in neutrophil anti-microbial function is an increase in neutrophil degranulation and / or neutrophil phagocytosis ). The compositions provided herein may therefore be for treating decompensated cirrhosis (e.g. pre-ACLF decompensated cirrhosis or ACLF decompensated cirrhosis) in a subject by preventing infection and / or dampening their pathogen-associated molecular pattern (PAMP)-induced systemic inflammation.

[0063] The compositions provided herein are also useful when administered to a subject having defective neutrophil function (e.g. defective neutrophil anti-microbial function (e.g. wherein the defective neutrophil anti-microbial function may be defective neutrophil degranulation and / or defective neutrophil phagocytosis)). Advantageously, the compositions provided herein can be used to increase neutrophil anti-microbial function (e.g. wherein the increase in neutrophil anti-microbial function is an increase in neutrophil degranulation and / or neutrophil phagocytosis) in these subjects and therefore can be used to prevent infections and / or dampen pathogen-associated molecular pattern (PAMP)-induced systemic inflammation in subjects having defective neutrophil function specifically. Accordingly, a composition comprising human albumin for the treatment of defective neutrophil function in a subject in need thereof is provided herein. In this example, the human albumin is for administration / is administered to the subject in a dose sufficient to increase neutrophil anti-microbial function (e.g. wherein the increase in neutrophil anti-microbial function is an increase in neutrophil degranulation and / or neutrophil phagocytosis). A composition comprising human albumin for preventing infection and / or dampening pathogen-associated molecular pattern (PAMP)- induced systemic inflammation in subjects with defective neutrophil function is also provided herein. In this example, the human albumin is for administration / is administered to the subject in a dose sufficient to increase neutrophil anti-microbial function (e.g. wherein the increase in neutrophil anti-microbial function is an increase in neutrophil degranulation and / or neutrophil phagocytosis).

[0064] Examples of subjects having defective neutrophil function can readily be identified by a person of skill in the art. Defects of neutrophil function may manifest as defects of the various biological processes that are involved in the bactericidal activity of neutrophils and include defects of arrest on the endothelium or adherence, defects of oxidative burst, defects of extracellular bactericidal mechanisms such as the release of neutrophil extracellular traps or NETS, defects of neutrophil degranulation and / or defects of neutrophil phagocytosis.

[0065] For example, the defective neutrophil function may be related to a disease selected from the group consisting of: SIRS, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatism, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heatshock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer. Accordingly, defective neutrophil function may be present in a subject having a disease selected from the group consisting of: SIRS, decompensated cirrhosis, acute liver failure, preacute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatism, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer. The compositions comprising human albumin described herein may therefore be used to treat defective neutrophil function in a disease selected from the group consisting of: SIRS, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatism, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heatshock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer (in other words, the compositions may be used to treat the defective neutrophil function aspect of the recited disease(s)). A composition comprising human albumin for the treatment of a disease selected from the group consisting of: SIRS, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatism, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer is therefore also provided herein, wherein the composition is for regulating neutrophil function (e.g. thereby treating defective neutrophil function).

[0066] In one example, the subject having defective neutrophil function is a subject having decompensated cirrhosis. For example, the subject having defective neutrophil function may be a subject having pre-ACLF decompensated cirrhosis (e.g. early pre-ACLF decompensated cirrhosis) or ACLF decompensated cirrhosis. In one example, the pre-ACLF decompensated cirrhosis is early pre-ACLF decompensated cirrhosis.

[0067] In one example, the compositions provided herein comprise human albumin, wherein the human albumin is for administration to the subject in a dose sufficient to upregulate the expression of a neutrophil gene selected from the group consisting of: CD177, OLFM4, PRG2, MPO, BPI, RETN, LCN2, CEACAM8, and MCEMP1 , or a combination thereof. This example applies to all suitable aspects provided herein, including when the composition comprising human albumin is for use in regulating immune cells in a subject (e.g. a subject having SIRS and / or a subject having decompensated cirrhosis), or when the composition human albumin is for the treatment of defective neutrophil function in a subject in need thereof. CD177 (NCBI Gene ID: 57126; UniProtKB / Swiss-Prot: Q8N6Q3) encodes a glycosylphosphatidylinositol (GPI)-linked cell surface glycoprotein that plays a role in neutrophil activation. The protein can bind platelet endothelial cell adhesion molecule-1 and function in neutrophil transmigration. Mutations in this gene are associated with myeloproliferative diseases. Over-expression of this gene has been found in patients with polycythemia rubra vera. Autoantibodies against the protein may result in pulmonary transfusion reactions, and it may be involved in Wegener's granulomatosis.

[0068] Olfactomedin 4 (OLFM4 - NCBI Gene ID: 10562; UniProtKB / Swiss-Prot: Q6LIX06) is a glycoprotein predominantly expressed in myeloid cells and in gastrointestinal tissues. OLFM4 is stored in specific granules of human neutrophils where it defines a subset of neutrophils ranging from 5-40% OLFM4 positive neutrophils. OLFM4 has been proposed to inhibit cathepsin C, a cysteine protease essential for activation of serine proteases.

[0069] Proteoglycan 2 (PRG2) gene (NCBI Gene ID: 5553; UniProtKB / Swiss-Prot: P13727, also known as pro eosinophil major basic protein) encodes the predominant constituent of the crystalline core of the eosinophil granule. High levels of the proform of PRG2 protein are also present in placenta and pregnancy serum, where it exists as a complex with several other proteins including pregnancy-associated plasma protein A (PAPPA), angiotensinogen (AGT), and C3dg. PRG2 protein may be involved in antiparasitic defence mechanisms as a cytotoxin and helminthotoxin, and in immune hypersensitivity reactions. PRG2 protein contains a peptide that displays potent antimicrobial activity against Gram-positive bacteria, Gramnegative bacteria, and fungi. It is directly implicated in epithelial cell damage, exfoliation, and bronchospasm in allergic diseases.

[0070] Myeloperoxidase (MPO - NCBI Gene ID: 4353; UniProtKB / Swiss-Prot: P05164) is a heme protein synthesized during myeloid differentiation that constitutes the major component of neutrophil azurophilic granules. Produced as a single chain precursor, myeloperoxidase is subsequently cleaved into a light and heavy chain. The mature myeloperoxidase is a tetramer composed of 2 light chains and 2 heavy chains. MPO produces hypohalous acids central to the microbicidal activity of neutrophils.

[0071] Bactericidal permeability increasing protein (BPI - NCBI Gene ID: 671 ; UniProtKB / Swiss-Prot: P17213) encodes a lipopolysaccharide binding protein. It is associated with human neutrophil granules and has antimicrobial activity against gram-negative organisms. BPI is a potent antibacterial protein of neutrophils with bactericidal and LPS-neutralizing activities and may also be involved in stimulation of phagocytosis of Gram-negative bacteria by neutrophils by means of promotion of complement activation. Resistin (RETN - NCBI Gene ID: 56729; UniProtKB / Swiss-Prot: Q9HD89) belongs to the family defined by the mouse resistin-like genes. The characteristic feature of this family is the C-terminal stretch of 10 cysteine residues with identical spacing. RETN protein has an antimicrobial role in skin, displaying antibacterial activity against both Gram positive and Gram negative bacteria. RETN promotes neutrophil pro-inflammatory activation and neutrophil extracellular trap formation.

[0072] Lipocalcin 2 (LCN2 - NCBI Gene ID: 3934; UniProtKB / Swiss-Prot: P80188) encodes a protein that belongs to the lipocalin family. Members of this family transport small hydrophobic molecules such as lipids, steroid hormones and retinoids. LCN2 protein is a neutrophil gelatinase-associated lipocalin and plays a role in innate immunity by limiting bacterial growth as a result of sequestering iron-containing siderophores. The presence LCN2 protein in blood and urine is an early biomarker of acute kidney injury. LCN2 is thought to be involved in multiple cellular processes, including maintenance of skin homeostasis, and suppression of invasiveness and metastasis. Mice lacking this gene are more susceptible to bacterial infection than wild type mice.

[0073] CEA cell adhesion molecule 8 (CEACAM8 - NCBI Gene ID: 1088; UniProtKB / Swiss-Prot: P31997) is expressed exclusively on granulocytes and encodes a cell surface glycoprotein that plays a role in cell adhesion in a calcium-independent manner. CEACAM8 mediates heterophilic cell adhesion with other carcinoembryonic antigen-related cell adhesion molecules, such as CEACAM6. In addition, heterophilic interactions with CEACAM8 occurs in activated neutrophils.

[0074] Mast cell expressed membrane protein 1 (MCEMP1 - NCBI Gene ID: 199675; UniProtKB / Swiss-Prot: Q8IX19) is a type II transmembrane protein primarily expressed in myeloid lineage immune cells, such as lung-resident mast cells and alveolar macrophages. MCEMP1 is one of the top inducible genes in many inflammatory diseases, such as asthma, idiopathic pulmonary fibrosis, cancer, sepsis, and stroke.

[0075] As used herein, “regulating neutrophil function” refers to controlling neutrophil function. Neutrophil function may be controlled by increasing or decreasing neutrophil activity (e.g. by stimulating existing neutrophils, or by increasing the overall quantity of neutrophils that are present). In the context of the invention, it is desirable to regulate neutrophil function by increasing neutrophil anti-microbial function (also referred to neutrophil anti-microbial activity herein). Increased neutrophil anti-microbial function may also be referred to as enhanced neutrophil anti-microbial function. Neutrophils are known to perform several anti-microbial functions in vivo. Neutrophil antimicrobial functions include neutrophil degranulation and neutrophil phagocytosis. An increase in neutrophil anti-microbial function, as used herein, may refer to an increase in: neutrophil degranulation and / or neutrophil phagocytosis. In some examples, an increase in neutrophil anti-microbial function refers to an increase in a combination of these features i.e. an increase in neutrophil degranulation and neutrophil phagocytosis.

[0076] Methods for determining if there is an increase in neutrophil anti-microbial function are well known. Examples of such methods are provided in the examples section below.

[0077] During the host defense response, neutrophils release myeloperoxidase (MPO), an essential anti-microbial protein localized mainly to the azurophil or primary neutrophil granules. Thus, an increase in neutrophil degranulation can be quantified by measurement of MPO activity. An increase in neutrophil degranulation may also be referred to as enhanced neutrophil degranulation herein.

[0078] Phagocytosis can be assessed by determining the ingestion of fluorescent-labeled zymosan particles by neutrophils. This method can be used to determine if there is an increase in neutrophil phagocytosis. An increase in neutrophil phagocytosis may also be referred to as enhanced neutrophil phagocytosis herein.

[0079] Corresponding methods for regulating neutrophil function (and / or preventing infection and / or dampening pathogen-associated molecular pattern (PAMP)-induced systemic inflammation) in a subject are also provided, wherein the composition comprising human albumin is administered to the subject in a dose sufficient to increase neutrophil anti-microbial function (e.g. wherein the increase in neutrophil anti-microbial function is an increase in neutrophil degranulation and / or neutrophil phagocytosis ). Corresponding methods for treating defective neutrophil function in a subject in need thereof are also provided.

[0080] Regulating CD4+ T cells

[0081] The inventors have shown herein that treatment with albumin specifically upregulates the blood transcription module (BTM) “mitotic cell cycle in stimulated CD4 T cells (M4.11)” in a subject (Fig. 1 E), indicating activation of transcription in CD4+ T cells. In addition, it is shown herein that administration of albumin changed the profile of the CD4+ T-cell compartment in a subject (Fig. 2H), with a significant decrease in the abundance of activated memory CD4+ T cells and an increase in central memory ITGB1+ CD4+ T cells (Fig. 2I). The data provided herein indicate that albumin can regulate the population of CD4+ T cells (in particular by increasing the level of central memory ITGB1+ CD4+ T cells and decreasing the level of activated CD4+ memory T cells) in a subject, and alters transcription in CD4+ T cells, thereby altering CD4+ T cell function.

[0082] Accordingly, a composition comprising human albumin for use in regulating CD4+ T cells in a subject is provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of central memory ITGB1+ CD4+ T cells and / or decrease the level of activated CD4+ memory T cells. Such compositions may be used to regulate CD4+ T cell function in the subject. Central memory ITGB1+ CD4+ T cells have been recently shown as having cytotoxic properties (JI 2021 , 207: 2966-2975), suggesting that these cells are involved in host resistance against infections. Accordingly, the compositions provided herein may be used to prevent infection in the subject. A composition comprising human albumin for preventing infection in a subject is therefore also provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of central memory ITGB1+ CD4+ T cells and / or decrease the level of activated CD4+ memory T cells.

[0083] The compositions provided herein are particularly useful when administered to a subject having systemic inflammatory response syndrome (SIRS). Subjects having SIRS may have defective CD4+ T cell function (particularly a deficient level of central memory ITGB1+ CD4+ T cells and / or an excess level of activated CD4+ memory T cells). Advantageously, the compositions provided herein can be used to increase the level of central memory ITGB1 + CD4+ T cells and / or decrease the level of activated CD4+ memory T cells in these subjects and therefore can be used to prevent infection in subjects having SIRS specifically. Accordingly, a composition comprising human albumin for use in regulating CD4+ T cells (e.g. regulating CD4+ T cell function) in a subject having SIRS is provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of central memory ITGB1+ CD4+ T cells and / or decrease the level of activated CD4+ memory T cells. The compositions provided herein may therefore be for treating SIRS in a subject by regulating their CD4+ T cells (e.g. regulating CD4+ T cell function). A composition comprising human albumin for preventing infection in subjects having SIRS is also provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient increase the level of central memory ITGB1+ CD4+ T cells and / or decrease the level of activated CD4+ memory T cells. The compositions provided herein may therefore be for treating SIRS in a subject by preventing infection.

[0084] Examples of subjects having SIRS are described elsewhere herein. These examples apply equally to this aspect. In one example, the subject having SIRS is a subject having decompensated cirrhosis. For example, the subject having SIRS may be a subject having pre-ACLF decompensated cirrhosis (e.g. early pre-ACLF decompensated cirrhosis) or ACLF decompensated cirrhosis. In one example, the pre-ACLF decompensated cirrhosis is early pre-ACLF decompensated cirrhosis.

[0085] A composition comprising human albumin for use in regulating CD4+ T cells (e.g. regulating CD4+ T cell function) in a subject having decompensated cirrhosis (e.g. pre-ACLF decompensated cirrhosis or ACLF decompensated cirrhosis) is also provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of central memory ITGB1+ CD4+ T cells and / or decrease the level of activated CD4+ memory T cells. The compositions provided herein may therefore be for treating decompensated cirrhosis (e.g. pre-ACLF decompensated cirrhosis or ACLF decompensated cirrhosis) in a subject by regulating their CD4+ T cells (e.g. regulating CD4+ T cell function). Furthermore, a composition comprising human albumin for preventing infection in a subject having decompensated cirrhosis (e.g. pre-ACLF decompensated cirrhosis or ACLF decompensated cirrhosis) is also provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of central memory ITGB1+ CD4+ T cells and / or decrease the level of activated CD4+ memory T cells. The compositions provided herein may therefore be for treating decompensated cirrhosis (e.g. pre-ACLF decompensated cirrhosis or ACLF decompensated cirrhosis) in a subject by preventing infection.

[0086] The compositions provided herein are also useful when administered to a subject having defective CD4+ T cell function (e.g. defective central memory ITGB1+ CD4+ T cell function and / or activated CD4+ memory T cell function). Advantageously, the compositions provided herein can be used to increase the level of central memory ITGB1+ CD4+ T cells and / or decrease the level of activated CD4+ memory T cells in these subjects and therefore can be used to prevent infection in subjects having defective CD4+ T cell function specifically. Accordingly, a composition comprising human albumin for the treatment of defective CD4+ T cell function in a subject in need thereof is provided herein. In this example, the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of central memory ITGB1+ CD4+ T cells and / or decrease the level of activated CD4+ memory T cells. A composition comprising human albumin for preventing infection in subjects with defective CD4+ T cell function is also provided herein. In this example, the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of central memory ITGB1+ CD4+ T cells and / or decrease the level of activated CD4+ memory T cells.

[0087] Examples of subjects having defective CD4+ T cell function can readily be identified by a person of skill in the art. For example, the defective CD4+ T cell function may be related to a disease selected from the group consisting of: SIRS, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer. Accordingly, defective CD4+ T cell function may be present in a subject having a disease selected from the group consisting of: SIRS, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer. The compositions comprising human albumin described herein may therefore be used to treat defective CD4+ T cell function in a disease selected from the group consisting of: SIRS, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatism, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer (in other words, the compositions may be used to treat the defective CD4+ T cell function aspect of the recited disease(s)).

[0088] A composition comprising human albumin for the treatment of a disease selected from the group consisting of: SIRS, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer is therefore also provided herein, wherein the composition is for regulating CD4+ T cell function (e.g. thereby treating defective CD4+ T cell function).

[0089] In one example, the subject having defective CD4+ T cell function is a subject having decompensated cirrhosis. For example, the subject having defective CD4+ T cell function may be a subject having pre-ACLF decompensated cirrhosis (e.g. early pre-ACLF decompensated cirrhosis) or ACLF decompensated cirrhosis. In one example, the pre-ACLF decompensated cirrhosis is early pre-ACLF decompensated cirrhosis. As used herein, “regulating CD4+ T cells” refers to controlling the CD4+ T cells. This encompasses e.g. controlling the number of CD4+ T cells, controlling the CD4+ T cell population, controlling CD4+ T cell function. CD4+ T cell function may be controlled by increasing or decreasing CD4+ T cell activity, for example by stimulating a desired CD4+ T cell subset (to increase the desired T cell activity of these cells), and / or by increasing the (relative) abundance of the desired cells (to increase the number of cells with the desired T cell activity). Similarly, CD4+ T cell function may be controlled by decreasing CD4+ T cell activity, for example by de-activating an undesired CD4+ T cell subset (to decrease the desired T cell activity of these cells), and / or by decreasing the (relative) abundance of the undesired cells (to decrease the number of cells with the undesired T cell activity). In the context of the invention, it is desirable to regulate CD4+ T cell function by increasing the level of central memory ITGB1+ CD4+ T cells and / or decreasing the level of activated CD4+ memory T cells.

[0090] The term “level” as used herein refers to the amount of the specified cells (e.g. central memory ITGB1+ CD4+ T cells or activated CD4+ memory T cells). The level may be an absolute amount of the cells and / or a relative amount of the specified cells. The relative amount of the specified cells may be relative to other cells in the subject, or in a sample from the subject. Said “other cells” may be for example all white blood cells, or a specific cell subpopulation, such as CD4+ T cells. The relative amount may be for example a percentage, fraction, or ratio. In the context of a relative amount, it will be appreciated that the relative amount of the specified cells may be altered (for example increased or decreased) with or without altering the absolute amount of the specified cells. By the same token, the absolute amount of the specified cells may be altered (increased or decreased), with or without altering the relative amount.

[0091] Methods for determining if there is an increase in the level of central memory ITGB1+ CD4+ T cells and / or a decrease in activated CD4+ memory T cells are well known. Examples of such methods are provided in the examples section below.

[0092] Corresponding methods for regulating CD4+ T cell function (and / or preventing infection) in a subject are also provided, wherein the composition comprising human albumin is administered to the subject in a dose sufficient to increase the level of central memory ITGB1+ CD4+ T cells and / or decrease the level of activated CD4+ memory T cells. Corresponding methods for treating defective CD4+ T cell function in a subject in need thereof are also provided.

[0093] Regulating dendritic cells

[0094] The inventors have shown herein that treatment with albumin specifically increases the abundance of plasmacytoid dendritic cells in a subject (Fig. 1 E and 2D-2F). The data provided herein indicate that albumin can regulate dendritic cells (in particular by increasing the level of plasmacytoid dendritic cells), and alters transcription in dendritic cells, thereby altering dendritic cell function.

[0095] Accordingly, a composition comprising human albumin for use in regulating dendritic cells (DCs) in a subject is provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of plasmacytoid dendritic cells (pDCs). Such compositions may be used to regulate dendritic cell function in the subject. Plasmacytoid DCs are DCs specialized in antiviral responses mediated by interferons. Accordingly, the compositions provided herein may be used to prevent viral infection in the subject. A composition comprising human albumin for preventing viral infection in a subject is therefore also provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of plasmacytoid dendritic cells.

[0096] The compositions provided herein are particularly useful when administered to a subject having systemic inflammatory response syndrome (SIRS). Subjects having SIRS may have defective dendritic cell function (particularly a deficient level of plasmacytoid dendritic cells). Advantageously, the compositions provided herein can be used to increase the level of plasmacytoid dendritic cells in these subjects and therefore can be used to prevent viral infection in subjects having SIRS specifically. Accordingly, a composition comprising human albumin for use in regulating dendritic cells (e.g. regulating dendritic cell function) in a subject having SIRS is provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of plasmacytoid dendritic cells. The compositions provided herein may therefore be for treating SIRS in a subject by regulating their dendritic cells (e.g. regulating dendritic cell function). A composition comprising human albumin for preventing viral infection in subjects having SIRS is also provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient increase the level of plasmacytoid dendritic cells. The compositions provided herein may therefore be for treating SIRS in a subject by preventing viral infection.

[0097] Examples of subjects having SIRS are described elsewhere herein. These examples apply equally to this aspect.

[0098] In one example, the subject having SIRS is a subject having decompensated cirrhosis. For example, the subject having SIRS may be a subject having pre-ACLF decompensated cirrhosis (e.g. early pre-ACLF decompensated cirrhosis) or ACLF decompensated cirrhosis. In one example, the pre-ACLF decompensated cirrhosis is early pre-ACLF decompensated cirrhosis.

[0099] A composition comprising human albumin for use in regulating dendritic cells (e.g. regulating dendritic cell function) in a subject having decompensated cirrhosis (e.g. pre-ACLF decompensated cirrhosis or ACLF decompensated cirrhosis) is also provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of plasmacytoid dendritic cells. The compositions provided herein may therefore be for treating decompensated cirrhosis (e.g. pre-ACLF decompensated cirrhosis or ACLF decompensated cirrhosis) in a subject by regulating their dendritic cells (e.g. regulating dendritic cell function). Furthermore, a composition comprising human albumin for preventing viral infection in a subject having decompensated cirrhosis (e.g. pre-ACLF decompensated cirrhosis or ACLF decompensated cirrhosis) is also provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of plasmacytoid dendritic cells. The compositions provided herein may therefore be for treating decompensated cirrhosis (e.g. pre-ACLF decompensated cirrhosis or ACLF decompensated cirrhosis) in a subject by preventing viral infection.

[0100] The compositions provided herein are also useful when administered to a subject having defective dendritic cell function (e.g. defective plasmacytoid dendritic cell function). Advantageously, the compositions provided herein can be used to increase the level of plasmacytoid dendritic cells in these subjects and therefore can be used to prevent viral infection in subjects having defective dendritic cell function specifically. Accordingly, a composition comprising human albumin for the treatment of defective dendritic cell function in a subject in need thereof is provided herein. In this example, the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of plasmacytoid dendritic cells. A composition comprising human albumin for preventing viral infection in subjects with defective dendritic cell function is also provided herein. In this example, the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of plasmacytoid dendritic cells.

[0101] Examples of subjects having defective dendritic cell function can readily be identified by a person of skill in the art. For example, the defective dendritic cell function may be related to a disease selected from the group consisting of: SIRS, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer. Accordingly, defective dendritic cell function may be present in a subject having a disease selected from the group consisting of: SIRS, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer. The compositions comprising human albumin described herein may therefore be used to treat defective dendritic cell function in a disease selected from the group consisting of: SIRS, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatism, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer (in other words, the compositions may be used to treat the defective dendritic cell function aspect of the recited disease(s)).

[0102] A composition comprising human albumin for the treatment of a disease selected from the group consisting of: SIRS, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer is therefore also provided herein, wherein the composition is for regulating dendritic cell function (e.g. thereby treating defective dendritic cell function).

[0103] In one example, the subject having defective dendritic cell function is a subject having decompensated cirrhosis. For example, the subject having defective dendritic cell function may be a subject having pre-ACLF decompensated cirrhosis (e.g. early pre-ACLF decompensated cirrhosis) or ACLF decompensated cirrhosis. In one example, the pre-ACLF decompensated cirrhosis is early pre-ACLF decompensated cirrhosis.

[0104] As used herein, “regulating dendritic cells” refers to controlling the dendritic cells. This encompasses e.g. controlling the number of dendritic cells, controlling the dendritic cell population, controlling dendritic cell function. Dendritic cell function may be controlled by increasing or decreasing dendritic cell activity, for example by stimulating a desired dendritic cell subset (to increase the desired dendritic cell activity of these cells), and / or by increasing the (relative) abundance of the desired cells (to increase the number of cells with the desired dendritic cell activity). In the context of the invention, it is desirable to regulate dendritic cell function by increasing the level of plasmacytoid dendritic cells. The term “level” as used herein refers to the amount of the specified cells (e.g. plasmacytoid DCs). The level may be an absolute amount of the cells and / or a relative amount of the specified cells. The relative amount of the specified cells may be relative to other cells in the subject, or in a sample from the subject. Said “other cells” may be for example all white blood cells, or a specific cell subpopulation, such as dendritic cells. The relative amount may be for example a percentage, fraction, or ratio. In the context of a relative amount, it will be appreciated that the relative amount of the specified cells may be altered (for example increased or decreased) with or without altering the absolute amount of the specified cells. By the same token, the absolute amount of the specified cells may be altered (increased or decreased), with or without altering the relative amount.

[0105] Methods for determining if there is an increase in the level of plasmacytoid dendritic cells are well known. Examples of such methods are provided in the examples section below.

[0106] Plasmacytoid dendritic cells (pDCs) have a unique role in linking innate and adaptive immunity. They have a lymphoid shape and a plasma cell morphology with an extensive endoplasmic reticulum, multiple mitochondria, and a small Golgi apparatus. pDCs have poor antigen presentation ability, in particular for exogenous antigens, but can acquire antigen- presenting cell function following activation with the expression of co-stimulatory molecules that can instruct T cells toward specific functional subsets. Once activated by TLRs, the expression of co-stimulatory molecules is induced in all pDCs, which confers T cell priming properties to these cells. Although pDCs can be activated through different cell surface receptors and cytosolic nucleic acid sensors, the sensing of nucleic acids through TLR7 and TLR9 seems to be the dominant mode of activation of these cells with respect to IFN production. Signalling through these two TLRs leads to the rapid and massive production of all type I and type III IFNs, which triggers the induction of IFN-stimulated genes (ISGs), many of them with antiviral properties. Because of these properties, it is postulated that the key function of pDCs is to act as antiviral cells. The activation of pDCs and the production of IFN- 1 can be important to the antiviral response and can promote tissue repair. However, the chronic or long-term persistent activation of these cells, which can be seen in autoimmunity and persistent viral infections, can lead or contribute to impaired immunity and disease progression.

[0107] Therefore, as used herein “defective pDCs” or “defective pDCs function” may refer to pDCs that are chronically activated or persistently activated. Defective pDCs function also refers to pDCs that have reduced production or expression of IFN molecules. In some examples, defective pDCs may refer to pDCs that overproduce IFN molecules, such as IFN-I as in the case of systemic lupus erythematosus and psoriasis. This may lead to abnormal production of T cells. In some examples, defective pDCs may produce immunosuppressive mediators such as OX40L and ICOSL that may at least partially inhibit other immune cells.

[0108] Markers of pDCs that are dysfunctional may be detected by determining the levels of cytokines such as IFN molecules. Other markers of pDCs dysfunction may be elevated expression of Tim-3.

[0109] Corresponding methods for regulating dendritic cell function (and / or preventing viral infection) in a subject are also provided, wherein the composition comprising human albumin is administered to the subject in a dose sufficient to increase the level of plasmacytoid dendritic cells. Corresponding methods for treating defective dendritic cell function in a subject in need thereof are also provided.

[0110] Regulating monocytes

[0111] The inventors have shown herein that treatment with albumin specifically increases the abundance of intermediate HAVCR2+ monocytes in a subject (Fig. 2D-2F). The data provided herein indicate that albumin can regulate monocytes (in particular by increasing the level of intermediate HAVCR2+ monocytes), and alters transcription in monocytes, thereby altering monocyte cell function.

[0112] Accordingly, a composition comprising human albumin for use in regulating monocytes in a subject is provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of intermediate HAVCR2+ monocytes. Such compositions may be used to regulate monocyte function in the subject. Intermediate HAVCR2+ monocytes are monocytes that dampen the inflammatory responses from other immune cells (e.g. classical monocytes, CD4 T cells) (Front. Immunol 2016 ;7, 229). HAVCR2 (alias Tim 3) is an immune checkpoint molecule. Accordingly, the compositions provided herein may be used to dampen an inflammatory response in the subject. A composition comprising human albumin for dampening an inflammatory response in a subject is therefore also provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of intermediate HAVCR2+ monocytes.

[0113] The compositions provided herein are particularly useful when administered to a subject having systemic inflammatory response syndrome (SIRS). Subjects having SIRS may have defective monocyte function (particularly a deficient level of intermediate HAVCR2+ monocytes). Advantageously, the compositions provided herein can be used to increase the level of intermediate HAVCR2+ monocytes in these subjects and therefore can be used to dampen an inflammatory response in subjects having SIRS specifically. Accordingly, a composition comprising human albumin for use in regulating monocytes (e.g. regulating monocyte function) in a subject having SIRS is provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of intermediate HAVCR2+ monocytes. The compositions provided herein may therefore be for treating SIRS in a subject by regulating their monocytes (e.g. regulating monocyte function). A composition comprising human albumin for dampening an inflammatory response in subjects having SIRS is also provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient increase the level of intermediate HAVCR2+ monocytes. The compositions provided herein may therefore be for treating SIRS in a subject by dampening an inflammatory response.

[0114] Examples of subjects having SIRS are described elsewhere herein. These examples apply equally to this aspect.

[0115] In one example, the subject having SIRS is a subject having decompensated cirrhosis. For example, the subject having SIRS may be a subject having pre-ACLF decompensated cirrhosis (e.g. early pre-ACLF decompensated cirrhosis) or ACLF decompensated cirrhosis. In one example, the pre-ACLF decompensated cirrhosis is early pre-ACLF decompensated cirrhosis.

[0116] A composition comprising human albumin for use in regulating monocytes (e.g. regulating monocyte function) in a subject having decompensated cirrhosis (e.g. pre-ACLF decompensated cirrhosis or ACLF decompensated cirrhosis) is also provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of intermediate HAVCR2+ monocytes. The compositions provided herein may therefore be for treating decompensated cirrhosis (e.g. pre-ACLF decompensated cirrhosis or ACLF decompensated cirrhosis) in a subject by regulating their monocytes (e.g. regulating monocyte function). Furthermore, a composition comprising human albumin for dampening an inflammatory response in a subject having decompensated cirrhosis (e.g. pre- ACLF decompensated cirrhosis or ACLF decompensated cirrhosis) is also provided herein, wherein the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of intermediate HAVCR2+ monocytes. The compositions provided herein may therefore be for treating decompensated cirrhosis (e.g. pre-ACLF decompensated cirrhosis or ACLF decompensated cirrhosis) in a subject by dampening an inflammatory response.

[0117] The compositions provided herein are also useful when administered to a subject having defective monocyte function (e.g. defective intermediate HAVCR2+ monocyte function). Advantageously, the compositions provided herein can be used to increase the level of intermediate HAVCR2+ monocytes in these subjects and therefore can be used to dampen an inflammatory response in subjects having defective monocyte function specifically. Accordingly, a composition comprising human albumin for the treatment of defective monocyte function in a subject in need thereof is provided herein. In this example, the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of intermediate HAVCR2+ monocytes. A composition comprising human albumin for dampening an inflammatory response in subjects with defective monocyte function is also provided herein. In this example, the human albumin is for administration / is administered to the subject in a dose sufficient to increase the level of intermediate HAVCR2+ monocytes.

[0118] Examples of subjects having defective monocyte function can readily be identified by a person of skill in the art. For example, the defective monocyte function may be related to a disease selected from the group consisting of: SIRS, decompensated cirrhosis, acute liver failure, preacute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer. Accordingly, defective monocyte function may be present in a subject having a disease selected from the group consisting of: SIRS, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer. The compositions comprising human albumin described herein may therefore be used to treat defective monocyte function in a disease selected from the group consisting of: SIRS, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatism, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemiareperfusion syndrome, inflammation diseases, and cancer (in other words, the compositions may be used to treat the defective monocyte function aspect of the recited disease(s)).

[0119] A composition comprising human albumin for the treatment of a disease selected from the group consisting of: SIRS, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer is therefore also provided herein, wherein the composition is for regulating monocyte function (e.g. thereby treating defective monocyte function).

[0120] In one example, the subject having defective monocyte function is a subject having decompensated cirrhosis. For example, the subject having defective monocyte function may be a subject having pre-ACLF decompensated cirrhosis (e.g. early pre-ACLF decompensated cirrhosis) or ACLF decompensated cirrhosis. In one example, the pre-ACLF decompensated cirrhosis is early pre-ACLF decompensated cirrhosis.

[0121] As used herein, “regulating monocytes” refers to controlling the monocytes. This encompasses e.g. controlling the number of monocytes, controlling the monocyte cell population, controlling monocyte function. Monocyte function may be controlled by increasing or decreasing monocyte cell activity, for example by stimulating a desired monocyte cell subset (to increase the desired monocyte activity of these cells), and / or by increasing the (relative) abundance of the desired cells (to increase the number of cells with the desired monocyte cell activity). In the context of the invention, it is desirable to regulate monocyte function by increasing the level of intermediate HAVCR2+ monocytes.

[0122] The term “level” as used herein refers to the amount of the specified cells (e.g. intermediate HAVCR2+ monocytes). The level may be an absolute amount of the cells and / or a relative amount of the specified cells. The relative amount of the specified cells may be relative to other cells in the subject, or in a sample from the subject. Said “other cells” may be for example all white blood cells, or a specific cell subpopulation, such as monocyte cells. The relative amount may be for example a percentage, fraction, or ratio. In the context of a relative amount, it will be appreciated that the relative amount of the specified cells may be altered (for example increased or decreased) with or without altering the absolute amount of the specified cells. By the same token, the absolute amount of the specified cells may be altered (increased or decreased), with or without altering the relative amount.

[0123] Methods for determining if there is an increase in the level of intermediate HAVCR2+ monocytes are well known. Examples of such methods are provided in the examples section below.

[0124] Corresponding methods for regulating monocyte function (and / or dampening an inflammatory response in a subject are also provided, wherein the composition comprising human albumin is administered to the subject in a dose sufficient to increase the level of intermediate HAVCR2+ monocytes. Corresponding methods for treating defective monocyte function in a subject in need thereof are also provided. Composition

[0125] The compositions provided herein comprise human albumin.

[0126] It will be understood by those skilled in the art that reference to “albumin” herein encompasses a protein having the same and / or very similar tertiary structure as human serum albumin (HSA) or HSA domains and has similar properties of HSA or the relevant domains. The term albumin includes variants, and / or derivatives such as fusions and / or conjugations of an albumin or of an albumin variant. The term "variant" means a polypeptide derived from a parent albumin comprising an alteration, i.e. , a substitution, insertion, and / or deletion, at one or more (several) positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding 1-3 amino acids adjacent to an amino acid occupying a position. The altered polypeptide (variant) can be obtained through human intervention by modification of the polynucleotide sequence encoding the naturally occurring (wild type) albumin.

[0127] In a preferred embodiment of the invention the albumin is human albumin, preferably human albumin purified from human plasma (also referred to as human plasma-derived albumin, human serum albumin, or serum albumin herein). Examples, of commercially available human serum albumin include Albutein®, and Plasbumin® (Grifols). In some examples, the albumin is a recombinant human albumin. Several examples of recombinant human albumin are known in the art, including: commercially available recombinant human albumins such as Recombumin® (Albumedix), Cellastim S® (InVitria), and Albagen® (Albumin Bioscience) as well as human albumin expressed in plants such as rice, and yeast such as Saccharomyces cerevisiae and Pichia pastoris.

[0128] As used herein, the term “recombinant” refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids. In some embodiments, the albumin is a recombinant albumin, such as recombinant human albumin.

[0129] As used herein, the term “human plasma-derived” refers to a biomolecule, e.g., a gene or protein, which are obtained from a standard of pooled human plasma from donors. In some embodiments, the term human plasma-derived is used to refer a human plasma-derived albumin.

[0130] The compositions described herein are for administration to a subject. They may therefore be referred to as pharmaceutical compositions. These compositions may include additional active agents (in addition to the human albumin described herein e.g. serum albumin or recombinant human albumin), provided that the human albumin is present at a level (amount, concentration, or dose) that the enables the recited function. In other words, the compositions described herein comprise a level (amount, concentration or dose) of human albumin that is in a dose sufficient to regulate the immune cells in the subject in the recited manner.

[0131] A pharmaceutical composition may comprise the human albumin described herein along with a pharmaceutically acceptable excipient, adjuvant, diluent and / or carrier.

[0132] Compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents or compounds.

[0133] As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the human albumin without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0134] Excipients are natural or synthetic substances formulated alongside an active ingredient (e.g. the human albumin as provided herein), included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art. By way of example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.

[0135] Adjuvants are pharmacological and / or immunological agents that modify the effect of other agents in a formulation. Pharmaceutically acceptable adjuvants are well known in the art. A suitable adjuvant is therefore easily identifiable by one of ordinary skill in the art. Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.

[0136] Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art.

[0137] As used herein the term “subject” refers to an individual, e.g., a human, having or at risk of having a specified condition, disorder or symptom. The subject may be a patient i.e. a subject in need of treatment in accordance with the invention. The subject may have received treatment for the condition, disorder or symptom. Alternatively, the subject has not been treated prior to treatment in accordance with the present invention.

[0138] The compositions described herein can be administered to the subject by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be by an administration route selected from the group consisting of: intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intrapulmonary, intranasal, oral, rectal, and combinations thereof.

[0139] The compositions described herein may be in any form suitable for the above modes of administration. Suitable dosages of the compositions described herein may be identified by a person of skill in the art.

[0140] The compositions described herein are for administration in an effective amount. An “effective amount” is an amount that alone, or together with further doses, produces the desired (therapeutic or non-therapeutic) response. The effective amount to be used will depend, for example, upon the therapeutic (or non-therapeutic) objectives, the route of administration, and the condition of the patient / subject. For example, the suitable dosage of the composition of the invention for a given patient / subject will be determined by the attending physician (or person administering the composition), taking into consideration various factors known to modify the action of the composition of the invention for example severity and type of haematological malignancy, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. The dosages and schedules may be varied according to the particular condition, disorder or symptom the overall condition of the patient / subject. The pharmaceutical compositions described herein are advantageously presented in unit dosage form.

[0141] In one example, the human albumin (e.g. serum albumin or recombinant human albumin) is for administration at multiple intervals as part of a multiple-dosage regimen. Suitable multipledosage regimens may be identified by a person of skill in the art. For example, the multiple dosing regimen may comprise multiple portion doses administered at equally spaced intervals of from about 1 day to about 30 days. In one example, the multiple dosing regimen may comprise multiple portion doses administered at unequally spaced intervals of from about 1 day to about 30 days. In one example, the multiple dosing regimen may comprise two or more administrations up to a total cumulative dose. In some examples, the human albumin may be administered at equally or unequally spaced intervals of from about 1 day to 6 days.

[0142] A suitable level (amount, concentration or dose) of human albumin is present within the compositions described herein. In the context of any of the dosage regimens described herein, the human albumin in the composition may be for administration at individual doses of about 5 g / interval to about 500 g / interval when part of a multiple-dosage regimen (e.g. when part of any of the multiple-dosage regimens described herein specifically). For example, the human albumin in the composition may be for administration at individual doses of about 20 g / interval to about 200 g / interval when as part of a multiple-dosage regimen (e.g. when part of any of the multiple-dosage regimens described herein specifically). For example, 40g / interval. For example, 40g / day.

[0143] In a specific example, a multiple dosing regimen may comprise multiple portion doses of the composition described herein administered at equally spaced intervals of from about 1 day to about 30 days, wherein the human albumin in the composition is for administration at individual doses of about 5 g / interval to about 500 g / interval (e.g. about 20 g / interval to about 200 g / interval). In another specific example, a multiple dosing regimen may comprise multiple portion doses of the composition described herein administered at unequally spaced intervals of from about 1 day to about 30 days, wherein the human albumin in the composition is for administration at individual doses of about 5 g / interval to about 500 g / interval (e.g. about 20 g / interval to about 200 g / interval). In another specific example, a multiple dosing regimen may comprise two or more administrations of the composition described herein up to a total cumulative dose, wherein the human albumin in the composition is for administration at individual doses of about 5 g / interval to about 500 g / interval (e.g. about 20 g / interval to about 200 g / interval). In some examples, the multiple dosing regimen may include administration of about 40g / interval of human albumin. In some examples, the multiple dosing regimen may include administration of about 40g / day of human albumin. In some examples, the dosing regimen may include administration of about 40g / interval of human albumin over a period of 2 to 6 days. For example, the dosing regimen may include administration of about 40g / day of human albumin over a period of 2 to 6 days. In some examples, the dosing regimen may include administration of about 40g / day of human albumin over a median period of 2.5 days.

[0144] In each of these examples, the dosing interval may be every 15 days, or less. For example, the dosing interval may be every 10 days, or less.

[0145] In a specific example, the compositions described herein comprise human albumin at a concentration of between about 4 % and 25 % (w / v) (e.g. about 20% (w / v)). In other words, the doses and dosage regimens provided above may be achieved using a composition comprising human albumin, wherein the concentration of human albumin is between about 4 % and 25 % (w / v) (e.g. about 20% (w / v)).

[0146] The compositions described herein are particularly useful for treating subjects. The term “treatment” or “treating” means any treatment of a disease or disorder in a subject, such as a mammal, including: preventing or protecting against the disease or disorder, that is, causing the clinical symptoms not to develop; inhibiting the disease or disorder, that is, arresting or suppressing the development of clinical symptoms; and / or relieving the disease or disorder that is, causing the regression of clinical symptoms. In some examples, the term treatment is used for the treatment of defective immune cell function. In some embodiments, the terms treatment is used for the treatment of a disease selected from the group consisting of: systemic inflammatory response syndrome, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer.

[0147] It will be understood by those skilled in the art that in human medicine, it is not always possible to distinguish between “preventing” and “suppressing” since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events. Therefore, as used herein the term “prophylaxis” is intended as an element of “treatment” to encompass both “preventing” and “suppressing” as defined herein.

[0148] As used herein, systemic inflammatory response syndrome (SIRS) refers to an inflammatory state resulting from an infectious or non-infectious result. SIRS is a serious condition related to systemic inflammation, organ dysfunction, and organ failure. It is a subset of cytokine storm, in which there is abnormal regulation of various cytokines. SIRS is also closely related to sepsis, in which patients satisfy criteria for SIRS and have a suspected or proven infection. Subjects with SIRS can be identified using the criteria below:

[0149] In adults, manifestations of SIRS include, but are not limited to:

[0150] (i) Body temperature less than 36 °C (96.8 °F) or greater than 38 °C (100.4 °F)

[0151] (ii) Heart rate greater than 90 beats per minute

[0152] (iii) Tachypnea (high respiratory rate), with greater than 20 breaths per minute; or, an arterial partial pressure of carbon dioxide less than 4.3 kPa (32 mmHg)

[0153] (iv) White blood cell count less than 4000 cells / mm3(4 x 109 cells / L) or greater than 12,000 cells / mm3(12 x 109 cells / L); or the presence of greater than 10% immature neutrophils (band forms). Band forms greater than 3% is called bandemia or a "leftshift".

[0154] When two or more of these criteria are met with or without evidence of infection, patients may be diagnosed with "SIRS". Patients with SIRS and acute organ dysfunction may be termed "severe SIRS".

[0155] In children, the SIRS criteria are modified as follows:

[0156] (i) Heart rate is greater than 2 standard deviations above normal for age in the absence of stimuli such as pain and drug administration, or unexplained persistent elevation for greater than 30 minutes to 4 hours. In infants, also includes heart rate less than 10th percentile for age in the absence of vagal stimuli, beta-blockers, or congenital heart disease or unexplained persistent depression for greater than 30 minutes.

[0157] (ii) Body temperature obtained orally, rectally, from Foley catheter probe, or from central venous catheter probe less than 36 °C or greater than 38.5 °C.

[0158] (iii) Respiratory rate greater than 2 standard deviations above normal for age or the requirement for mechanical ventilation not related to neuromuscular disease or the administration of anesthesia.

[0159] (iv) White blood cell count elevated or depressed for age not related to chemotherapy, or greater than 10% bands plus other immature forms.

[0160] Temperature or white blood cell count must be abnormal to qualify as SIRS in paediatric patients.

[0161] SIRS may be related to the subject having a disease selected from the group consisting of: decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure (pre-ACLF), acute on chronic liver failure (ACLF), severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer. Accordingly, SIRS may be present in a subject having a disease selected from the group consisting of: decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure (pre-ACLF), acute on chronic liver failure (ACLF), severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer. The compositions comprising human albumin described herein may therefore be used to treat SIRS in a disease selected from the group consisting of: decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure (pre-ACLF), acute on chronic liver failure (ACLF), severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer (in other words, the compositions may be used to treat the SIRS aspect of the recited disease(s)). Methods for identifying whether or not a subject has one or more of these diseases are well known.

[0162] In one example, the subject is a sepsis patient or a patient with a liver condition. Accordingly, the subject having SIRS may have SIRS that is related to sepsis or a liver condition. Examples of sepsis patients include patients with severe sepsis or septic shock. Examples, of patients with a liver condition include patients with decompensated cirrhosis, acute liver failure, pre- acute on chronic liver failure (pre-ACLF), and / or acute on chronic liver failure (ACLF).

[0163] In one example, the subject has decompensated cirrhosis. Decompensated cirrhosis is defined as an acute deterioration in liver function in a patient with cirrhosis and is characterised by recent development of ascites, hepatic encephalopathy, gastrointestinal hemorrhage or any combination of these. Decompensated cirrhosis is also referred to as “acute decompensated cirrhosis”, “acutely decompensated cirrhosis” or “decompensated liver disease” herein, and these terms can be used interchangeably.

[0164] Among subjects with acutely decompensated cirrhosis, immune activation is more marked in those with acute-on-chronic liver failure (ACLF), a syndrome characterized by intense systemic inflammation, single multiorgan dysfunction / failure and extremely high prevalence of bacterial infections (Moreau et al., 2013, Gastroenterology, 144: 1426-37, 1437. e1-9; Arroyo, et al., 2020, N Engl J Med, 382:2137-2145) than among those without ACLF (Weiss et al., 2021 , Frontiers in Immunol, vol. 12, art. 699563). Moreover, the PREDICT study has recently identified a subset of subjects with severe systemic inflammation but without ACLF who are at high risk of developing ACLF and to die during hospitalization or within few weeks after discharge (Trebicka et al., 2020, J Hepatol, 73:842-854). This subset of subjects is classified as subjects having pre-ACLF acutely decompensated cirrhosis.

[0165] Pre-ACLF defines a group of patients with acutely decompensated cirrhosis admitted to hospital without ACLF but who develop ACLF during hospitalization or within a period of 3- months after admission. pre-ACLF can be split into two different phenotypes: “early” pre- ACLF, which defines patients who develop ACLF within the index hospitalization, and “delayed” pre-ACLF, which defines patients who develop ACLF between discharge from the index hospitalization and the end of the 3-month follow-up period. Patients hospitalized with “early” pre-ACLF exhibit intense systemic inflammation, a feature which is moderate in patients with “slowly progressive” pre-ACLF.

[0166] A subject having acutely decompensated cirrhosis with ACLF is also referred to as an ACLF subject or an ACLF patient herein. A subject having pre-ACLF acutely decompensated cirrhosis is also referred to as a pre-ACLF subject or a pre-ACLF patient herein.

[0167] In one example, the subject is a ACLF subject or a pre-ACLF subject. In other words, the subject may have ACLF decompensated cirrhosis (also referred to as ACLF acutely decompensated cirrhosis herein) or pre-ACLF decompensated cirrhosis (also referred to as pre-ACLF acutely decompensated cirrhosis herein). In one example, the pre-ACLF decompensated cirrhosis is early pre-ACLF decompensated cirrhosis.

[0168] In one example, the subject has acute liver failure. Acute liver failure is characterized by acute liver injury, hepatic encephalopathy (altered mental status), and an elevated prothrombin time / international normalized ratio (INR). It has also been referred to as fulminant hepatic failure, acute hepatic necrosis, fulminant hepatic necrosis, and fulminant hepatitis.

[0169] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0170] Aspects of the invention are demonstrated by the following non-limiting examples.

[0171] EXAMPLES

[0172] EXAMPLE 1 - Effects of albumin treatment on blood immune cell gene signatures in patients with acutely decompensated cirrhosis (effects of albumin on blood immune cells)

[0173] Background

[0174] Assessing genomewide RNA expression in blood is a valuable tool to investigate simultaneously signatures of different types of circulating immune cells.5’19'21The inventors now report the results of the Discovery study which is an analysis of whole-blood RNA-seq data obtained twice among 49 patients of the PREDICT study; first, when these patients were at the pre-ACLF stage (on admission) and second when they had progressed to the ACLF stage, a few days after admission during the index hospitalization. All patients therefore had an “early” form of pre-ACLF. Because 30 patients had received albumin during the progression to ACLF whereas the remaining 19 did not, the Discovery study was a first approach to capture the in vivo effects of albumin on gene signatures related to blood immune cells. Both the longitudinal design and the well-established similarity of the intensity of systemic inflammation of “early” pre-ACLF as compared with ACLF should minimize the interindividual variability of gene expression that would otherwise make difficult to capture the in vivo effects of albumin on blood immune cells.

[0175] Surprisingly, the Discovery study uncovered that patients treated with albumin had specific increases in gene signatures primarily related to B cells and plasma cells, and neutrophils. These findings prompted the inventors to conduct additional studies which included new patients with acutely decompensated cirrhosis to further investigate the ex vivo effects of albumin on cells that compose freshly isolated peripheral-blood mononuclear cells (PBMCs) and on freshly isolated blood neutrophils.

[0176] Methods

[0177] Patients and Study Design

[0178] All patients (i.e., 64 patients) had acutely decompensated cirrhosis without ACLF at entry. Each patient provided written informed consent to participate. Each study described below had been previously approved by the Ethics Committee of the Hospital Clinic, Barcelona, Spain.

[0179] Discovery study

[0180] The inventors first considered 49 patients of the PREDICT study3who were carefully selected according to the following criteria: i) All patients had “early” pre-ACLF at enrollment (time 1 [T1 ]) because they developed ACLF during the index hospitalization (at time 2 [T2]; data not shown). For simplicity, the inventors hereafter used the term “pre-ACLF” instead of that of “early pre-ACLF”. The inventors assumed that investigating patients exhibiting the most severe forms acutely decompensated cirrhosis with the use of longitudinal data would limit the effect of interindividual variability on the assessment of albumin effects, ii) In all patients, whole-blood RNA-seq data were obtained at T1 and, as prespecified in the protocol, at T2. iii) None of the 49 patients had received albumin before T1 . iv) Thirty out of the 49 patients had received albumin between T 1 and T2 and composed the albumin group whereas the 19 others remained free of albumin between T1 and T2 and composed the no-albumin group (data not shown). Of note, the inventors also obtained results of clinical blood counts and whole-blood RNA-seq from 10 age-matched healthy subjects (HS) (data not shown). The inventors analyzed longitudinal whole-blood RNA-seq data from the 49 patients with two objectives. The first objective was to assess whether transcriptomic characteristics in the 49 patients studied at the pre-ACLF stage were different or not from those in the same patients when they have progressed to ACLF, irrespective of albumin administration. Indeed, unlike whole-blood transcriptome characteristics of ACLF that have been previously described,5those of pre- ACLF have not yet been investigated. The second and major objective was to assess whether albumin may affect RNA signatures of circulating immune cells through the analyses of blood transcriptome in the albumin group and no-albumin group.

[0181] Additional studies

[0182] Results of the Discovery study led the inventors to enroll 9 new patients with acutely decompensated cirrhosis whose peripheral blood mononuclear cells (PBMCs) were freshly isolated to investigate the ex vivo effects of albumin on cells that compose PBMCs with the use of single-cell RNA-seq (scRNA-seq). In addition, neutrophils from an additional set of 6 new patients were freshly isolated to study the ex vivo effects of albumin on major neutrophil antimicrobial functions.

[0183] RNA Preparation for Whole-Blood RNA-seq

[0184] As previously described,5 19RNA was isolated from blood stored in Tempus tubes using the Tempus™ Spin RNA Isolation Kit (reference 43802 Applied Biosystems, Foster City, CA). Importantly, using the HUGO Genome Nomenclature Committee, which is a resource for approved human gene nomenclature, the inventors designed a comprehensive protocol that included not only protein-coding genes, but also genes among other gene locus types, such as immunoglobulin genes, and T-cell receptor genes, resulting in a total of 14,615 genes for analysis. Genes brought by sexual chromosomes and mitochondrial DNA were not included in the analysis.

[0185] Whole-Blood RNA-Seq Analysis

[0186] Assessing differential gene expression.

[0187] Differentially expressed genes (DEGs) were defined by the simultaneous presence of 2 criteria, i.e., an absolute fold-change (FC) greater than 1.5 and moderated P value of less than 0.05.

[0188] Assessing immune-cell signatures with the use of SingleR R software

[0189] The SingleR software22was used to compare whole-blood RNA-seq data from the study participants with a reference dataset containing 114 human bulk RNA-seq samples of sorted immune-cell populations from 4 healthy subjects (GSE107011).23This reference dataset contained 114 human RNA-seq samples annotated to 10 main immune-cell types. The dataset (GSE107011) also contained samples that were additionally annotated to 29 fine immune-cell types. The SingleR pipeline compared the whole-blood RNA-seq data, first with the 10 main immune-cell types of reference and then with the 29 fine immune-cell types.

[0190] Analyzing gene-set enrichment

[0191] The Quantitative Set Analysis for Gene Expression (QuSAGE) method, as implemented in the QuSAGE package,24was used to conduct gene-set enrichment analysis using the 346 blood transcription modules (BTMs) as gene sets. BTMs are a set of gene modules developed through large-scale network integration of publicly available human blood transcriptomes.20QuSAGE provides an activity score for each gene set in each comparison (e.g., pre-ACLF vs healthy subjects; T2 vs T1 for the albumin group). A BTM was identified as differentially expressed when the QuSAGE activity score of this BTM had a false discovery rate (FDR) <0.05 or P <0.05, where appropriate.

[0192] Analyzing shared genes and shared BTMs

[0193] A DEG or a differentially expressed BTM was defined as shared between 2 comparisons when the DEG or differentially expressed BTM were concordant in sign. ScRNA-seq in PBMCs

[0194] Peripheral venous blood (8 ml) was collected by venipuncture into EDTA-coated sterile pyrogen-free tubes (Becton Dickinson, East Rutherford, NJ) from 9 patients and 4 age- matched healthy subjects. PBMCs were immediately isolated as previously described.25PBMCs were seeded at a density of 1.5x106cells / ml and incubated with either albumin (Albutein®, Grifols, Barcelona, Spain) (15 mg / ml) or vehicle (culture medium) for 2 hours at 37°C in a 5% CO2 incubator. At the end of the culture, cells were rapidly (within 30 min) transferred to ice at the Single Cell Genomics platform from the CNAG.

[0195] Experiments in Neutrophils

[0196] Neutrophils were isolated from peripheral venous blood (20 ml) obtained from 6 patients and 5 age-matched healthy subjects using the Ficoll-Hypaque method. Isolated neutrophils were immediately used for different experiments that assessed major anti-microbial functions of neutrophils such as degranulation assay and phagocytosis assay.

[0197] Results

[0198] Assessing Characteristics of Pre-ACLF at T1 and ACLF at T2 in the 49 Patients of the Discovery Cohort

[0199] Clinical characteristics.

[0200] The inventors first characterized all patients at T1 (pre-ACLF) and at T2, when they had progressed to ACLF. Characteristics were similar in terms of standard laboratory values (including clinical differential blood counts for neutrophils, monocytes, and lymphocytes; data not shown), and blood levels of inflammatory markers data not shown. Of note, in both pre- ACLF and ACLF the blood levels of inflammatory markers were elevated as compared with corresponding levels in healthy subjects (data not shown). The only two differences were the presence of organ failures in ACLF at T2 but not in pre-ACLF at T1 , as expected and the higher white-cell count in ACLF than in pre-ACLF (data not shown). Compared with healthy subjects, pre-ACLF and as expected ACLF5were characterized by leukocytosis, neutrophilia, and lymphopenia (data not shown). Of note, the inventors found that clinical blood counts and RNA-seq-inferred blood counts were strongly positively correlated (data not shown), findings indicating that RNA-seq analysis of whole-blood RNA was able to provide information that correlated with criterion-standard clinical measurements of blood counts.

[0201] Next, the inventors considered characteristics at T1 and T2 in patients of the albumin group and in those of the no-albumin group. In the albumin group, the median time between T1 and T2 for blood collections for RNA-seq was 11.5 days (interquartile range [IQR], 6.2 to 18.7). The median duration of albumin treatment was 2.5 days (IQR, 2 to 6) and the albumin dose was 40.0 g per day (IQR, 22.5 to 57.5). The time between the last albumin administration and subsequent blood collection for RNA-seq was 1.5 days (IQR, 0.0 to 2.7), indicating that the inventors explored patients soon after albumin administration. Albumin was given for standard medical indications . In the no-albumin group the median time between T1 and T2 was 29 days (IQR, 16 to 52).

[0202] In both the albumin group and no-albumin group, progression from pre-ACLF (T1) to ACLF (T2) was associated with significant increases in the MELD score. However, except for serum creatinine which increased at T2, there were no significant within-group changes regarding the rest of longitudinally collected standard laboratory data and circulating levels of 14 inflammatory mediators (Table 1). However, there were some between-group differences. The delay between T1 and T2 was shorter in the albumin group than the no-albumin group. Blood levels of C-reactive protein and interleukin-6 were higher in the albumin group, both at T1 and T2. A higher percentage of patients in the albumin group than patients in the no-albumin group died by 28 days and 90 days, this difference being expected differences considering the higher prevalence of SBP and HRS-AKI in the albumin group.

[0203] Transcriptional characteristics of all patients at pre-ACLF stage (T1) and ACLF stage (T2)

[0204] Analyzing DEGs in 2 comparisons, pre-ACLF vs healthy subjects and ACLF vs healthy subjects, the inventors observed, as expected, strong similarities between the 2 disease’s stages. Thus, the number of DEGs was high in the two comparisons, i.e., there were 4615 DEGs in pre-ACLF relative to healthy subjects and 4529 in ACLF relative to healthy subjects. Of the 4615 DEGs associated with pre-ACLF, 3929 (85%) overlapped with the DEGs assigned to ACLF (data not shown). Moreover, when the inventors compared effect-size changes (Iog2 fold-change [FC]) in pre-ACLF vs healthy subjects with ACLF vs healthy subjects, the inventors observed highly concordant magnitude of changes between the two signatures (data not shown).

[0205] Next, the inventors used QuSAGE to analyze the differential expression of blood gene modules (BTMs). BTMs are gene sets related to blood cells (e.g., plasma cells, immunoglobulins (M156.1)) or biological functions (e.g., muscle contraction, SRF targets (M 195)). Among the 258 annotated BTMs,20the total number of differentially expressed (DE) BTMs (FDR <0.05) was 190 in pre-ACLF vs healthy subjects and 186 in ACLF vs healthy subjects (data not shown;), findings that indicated extensive changes in the blood transcription module space in both pre-ACLF and ACLF. Strikingly, only 20 BTMs had specific differential expression relative to healthy subjects, either in pre-ACLF (12 modules) or ACLF (8 modules), whereas 179 DE BTMs were shared, i.e., DE modules with a concordant sign in the two comparisons Table SX). Of these shared DE gene modules, 108 were upregulated and 71 downregulated. The 20 top gene modules for shared upregulated BTMs and 20 top gene modules for shared downregulated BTMs were noted (data not shown). Shared upregulated BTMs were related to innate immunity, including those relating to TLR and inflammatory signaling, interferon-alpha response, and innate immune cells such as neutrophils, monocytes, and dendritic cells. Shared downregulated BTMs were related to T cells, NK cells, and antigen presentation. These results were consistent with those obtained when analyzing gene signatures for the 10 main immune cell types and the 29 fine immune cell types with the use of SingleR R software. Collectively, the analyses of whole-blood RNA-seq data with the use of QuSAGE and SingleR R software provided consistent results that highlighted the similarity of blood transcriptional module landscape in pre-ACLF and ACLF; both disease’s stages being characterized by simultaneity between increases in gene signatures related to innate immune cells and decreases in gene signatures related to adaptive immune cells.

[0206] Whole-blood Gene Signatures Associated with Albumin Treatment

[0207] The inventors studied differential gene expression between T2 vs T1 for the albumin group and no-albumin group and found that gene signatures differed between the two groups. Indeed, 269 DEGs (92 up- and 177 downregulated genes) were specific for the albumin group whereas 103 DEGs (64 up- and 39 downregulated genes) were specific for the no-albumin group; only 36 DEGs with concordant sign were shared by the 2 groups (Fig. 1 A). Identification of DEGs in Volcano plots (Fig. 1 B) illustrated the group-specificity of both upregulated genes and downregulated genes. For example, a broad variety of immunoglobulin genes (see below) and several major neutrophil genes (CD177, OLFM4, PRG2, MPO, BPI, RETN, LCN2, CEACAM8, MCEMPT) were present among upregulated genes at T2 vs T1 in the albumin group (Fig. 1 B, top), but not among those that were upregulated in the no-albumin group (Fig. 1 B, bottom).

[0208] Because analysis of DEGs (Fig. 1 B) drew attention on immunoglobulin genes, the inventors compared the effect-size changes (Iog2 fold change) between T2 and T1 within each group, for each of the 125 genes (including 120 immunoglobulin genes) that are included in the Gene Ontology gene set entitled “GOCC Immunoglobulin Complex”. The inventors found that effectsize changes were greater in the albumin group than the no-albumin, the number of upregulated immunoglobulin genes being 33 vs only 6, respectively (Fig. 1C). Genes coding for constant regions of immunoglobulin heavy chains (JGHM, IGHG2, IGHG3, IGHG4, IGHA2) were specifically upregulated in the albumin group. These findings are consistent with the results of SingleR analysis which showed increases in gene signatures for plasmablasts that were specific for the albumin group (Fig. 1 D, top). Immunoglobulin genes that were specifically upregulated in the albumin group also included genes for constant regions of immunoglobulin light chains kappa ( / G C), and lambda (IGLC1), genes for V regions of the variable domain of immunoglobulin heavy chains, and genes for V regions of the variable domain of light chains kappa and lambda. The 3 genes that were specifically upregulated in the no-albumin group were genes coding for V regions of the variable domain of immunoglobulin heavy chains (Fig. 10). Together these findings indicated an extensive upregulation of genes coding for immunoglobulins that was specific for patients who had received albumin.

[0209] Next, the inventors applied QuSAGE to identify differentially expressed BTMs between T2 and T 1 , within each group. The inventors first observed that the number of differentially expressed BTMs was greater in the albumin group than in the no-albumin group (36 vs 6, respectively;). Only 1 differentially expressed BTM was specific for the no-albumin group; this BTM was upregulated and related to endoplasmic reticulum (Fig. 1 E). In sharp contrast, 31 differentially expressed BTMs (13 down, 18 up) were specific for the albumin group (Fig. 1 E). Downregulated modules were related to either erythropoiesis, cytoskeleton or cell junction (Fig. 1 E). Among upregulated BTMs specific for the albumin group, there were modules related to innate immunity and inflammation including those relating to activated dendritic cells; complement and other receptors in DCs; proinflammatory cytokines and chemokines (Fig. 1 E). Upregulated BTMs specific for the albumin group also comprised modules related to B cells (including those relating to enriched in B cells, plasma cells and immunoglobulins), and modules related to mismatch repair, cell cycle, and mitosis. Finally, BTMs related to transcriptional networks of transcription factors (TFs) such as c-Myc and members of the E2F family were specifically upregulated in the albumin group (Fig. 1 E), findings that indicated an increased activity of these TFs in blood from patients who had received albumin. Because c- Myc and members of the E2F family are regulators of cell cycle, increases in their activity may explain the induction of gene modules related to cell cycle and mitosis observed in blood of patients who had received albumin.

[0210] Of note, RNA-seq-inferred signatures for T cells and NK cells (Figs. 1 D) as well as gene modules related to these cells, whose downregulation is a hallmark of ACLF (and pre-ACLF; data not shown), remained all downregulated among patients treated with albumin. Nevertheless, the inventors observed that a BTM entitled “mitotic cell cycle in stimulated CD4 T cells (M4.11)” was specifically upregulated among patients treated with albumin (Figure 1 E) , a finding that suggests some activation of transcription in CD4 T cells of these patients. Consistent with this, the inventors found no overlap between member genes of the BTM related to mitotic cell cycle in stimulated CD4 T cells (M4.11) and genes used for analysis of the signature of CD4 T cells with the use of SingleR R software. Collectively, the results revealed that blood from patients who had received albumin was characterized by increased signatures related to immune cells such as B cells, plasma cells, some innate immune cells and perhaps CD4 T cells, associated with increased signatures of mitosis, cell cycle, and signatures witnessing increased activities of TFs of the E2F family.

[0211] ScRNA-seq of human PBMCs to Investigate Ex Vivo Effects of Albumin

[0212] Results obtained through the analysis of whole-blood RNA-seq led the inventors to investigate the ex vivo effects of albumin on human PBMCs using scRNA-seq. Fresh PBMCs from 9 patients with acutely decompensated cirrhosis and 4 healthy controls were exposed ex vivo to albumin (15 mg / ml) and vehicle during 2 hours before conducting the scRNA-seq experiments.

[0213] The inventors jointly analyzed 66064 human PBMCs from patients and healthy subjects, which were clustered into 3 major immune cell lineages, including B lymphocytes, myeloid cells and T cells. The inventors further subclustered each lineage using canonical gene markers to annotate the different cell types.

[0214] The inventors analyzed 3786 B lymphocytes (1946 and 1840 cells from patients and healthy subjects, respectively), identifying 10 main populations. Results from patients’ cells are shown in Figs. 2A. Of note, the inventors described two subpopulations that acquired a transitional- like B-cell profile characterized by a higher expression of CD79B, IGHD and TCL1A (Transitional 1 B cell) and CD55 (Transitional 2 B cell). The inventors observed that patients’ cells exposed to albumin had significant increases in the abundance of cells expressing transitional-like B cell signatures and decreased naive B cells (Figs. 2B and 2C), but no changes in the other B-lymphocyte populations. The inventors further assessed 26898 myeloid cells (21677 and 5053 cells from patients and healthy subjects, respectively), which clustered into 13 different cell populations 7 clusters related to monocytes, 3 related to dendritic cells (DCs) and other 3 including myeloid-derived suppressor cells and granulocytemonocyte progenitors. Results obtained for patients’ cells are shown in Figs. 2D. The inventors observed that patients’ cells exposed to albumin had significant increases in the abundance of intermediate monocytes HAVCR2+and in plasmacytoid DCs (Figs. 2E and 2F), while the abundance of the other myeloid cell populations did not change.

[0215] Based on the differential expressed BTMs found for the T-cell compartment, the inventors also analyzed 35380 T lymphocytes, including 21731 CD4 T cells (12692 and 9039 cells from patients and healthy subjects, respectively), 10072 CD8 T cells (3483 and 6589 cells from patients and healthy subjects, respectively) and 3577 unconventional T cells (1292 and 2285 cells from patients and healthy subjects, respectively). CD4 T cells were clustered into 10 different cell populations. Results obtained for patients’ cells are shown in Figs. 2G. The inventors observed that exposure to albumin in patients changed the profile of the CD4 T-cell compartment (Fig. 2H), with a significant decrease in the abundance of activated memory CD4 T cells and an increase in central memory ITGB1+CD4 T cells (Fig. 2I). CD8 T cells and unconventional T cells were clustered into 13 different cell populations, but no significant changes on cell populations were found after albumin exposure.

[0216] One of the specifically upregulated BTMs in patients who had received albumin was the BTM entitled “mitotic cell cycle in stimulated CD4 T cells (M4.11)” (Fig. 1 E). To validate the M4.11 signature on scRNA-seq, the inventors computed the gene signature score for this BTM across all CD4 T cells using the llcell package.26The M4.11 BTM signature showed a different distribution pattern on CD4 T cells between albumin and vehicle conditions, exhibiting a significantly higher cell density on albumin-treated cells relative to vehicle (Fig. 2J).

[0217] In addition, the analysis of PBMCs from healthy subjects showed effects of albumin similar to those in patients for both B-lymphocyte and myeloid-cell compartments.

[0218] Investigating Ex Vivo Effects of Albumin on Neutrophils

[0219] Analysis of whole-blood RNA-seq data in the albumin group and the no-albumin group drew the inventors’ attention to neutrophils. Indeed, pairwise comparison of whole-blood RNA-seq data between T2 and T1 in the albumin group and the no-albumin group revealed an upregulation of 9 major neutrophil genes that was specific for patients who had received albumin (Fig. 1 B). These findings, therefore, led the inventors to design ex vivo studies in freshly isolated neutrophils. Since neutrophils provide the first-line of defense against most pathogens and they express fewer genes than any other leukocyte, the study focused on analyzing the effect of albumin on neutrophil function.

[0220] During the host defense response, neutrophils release myeloperoxidase (MPO), an essential anti-microbial protein localized mainly to the azurophil or primary neutrophil granules.27Thus, neutrophil degranulation can be quantified by measurement of MPO activity. Fig. 3A shows augmented MPO activity in the supernatant of neutrophils from both patients with acutely decompensated cirrhosis and healthy subject incubated with either serum albumin or recombinant human albumin.

[0221] Phagocytosis is another defensive function against pathogens that can be assessed by determining the ingestion of fluorescent-labeled zymosan particles by neutrophils. Fig 3B shows increased zymosan phagocytosis by neutrophils from both patients with acutely decompensated cirrhosis and healthy subjects incubated with either serum albumin or recombinant human albumin.

[0222] Discussion

[0223] This study (the Discovery study) is the first to use longitudinal genomics to explore the mechanism of action of albumin in patients with acutely decompensated cirrhosis. The design of this study, particularly in those aspects related to the selection of candidates, and to albumin treatment, was done after considering aspects already known and some still unpublished. Patients hospitalized with ACLF, who are known to present an extremely dynamic clinical course, improving, following a steady course, or worsening within few days after admission in parallel with marked changes in the magnitude of systemic inflammation,2were clearly poor candidates for the study, which aimed to assess the effect of albumin treatment on immunecell transcriptomics. Three other phenotypes of acutely decompensated cirrhosis, including “unstable” and “stable” decompensated cirrhosis and “slowly progressive” pre-ACLF, were also not considered because they associate with moderate systemic inflammation which may further decrease during hospitalization, and the inventors were interested to include only patients with intense systemic inflammation to better assess potential changes on immunecell gene expression induced by albumin. For this reason, the inventors concentrated on patients with “early” pre-ACLF, in whom the magnitude of systemic inflammation is similar to that seen in ACLF. The inventors decided to include only patients who had received albumin treatment for well-established indications (paracentesis treatment, prevention of HRS-AKI associated with SBP, or treatment of HRS-AKI)13despite the fact that this would introduce the bias of higher severity in patients receiving albumin treatment. The inventors did not select patients receiving albumin within one month prior to T1 , those with a too long interval between the 2 assessments of whole-blood RNA-seq and those with a delay >10 days between the last dose of albumin and T2. Finally, although albumin dosage and duration of treatment varied according to indications, the inventors decided to analyze data in all patients irrespective of albumin dosage. Of the 1273 patients enrolled in the PREDICT study, 49 patients fulfilled all the selection criteria and were, therefore, investigated in the present Discovery study.

[0224] The inventors first compared blood transcriptome obtained at each time point in all patients with blood transcriptome obtained in healthy subjects. The inventors observed that pre-ACLF and ACLF were characterized by increases in gene signatures related to innate immune cells (including those relating to neutrophils, monocytes, and dendritic cells) and decreases in gene signatures related to lymphocytes (including T cells, B cells and NK cells). These findings indicate simultaneous activation of innate immunity mediated by myeloid cells and depletion of the lymphocyte compartment in blood from patients with cirrhosis and intense systemic inflammation. The similarity of pre-ACLF and ACLF with respect to transcriptional characteristics as well as most clinical characteristics, standard laboratory values, and levels of blood cytokines, confirmed the inventors’ assumption that the study patients would be in a relatively steady state of systemic inflammation during the study period. Such a steady state was a prerequisite to avoid confounders to hidden the in vivo effects of albumin on blood immune cells.

[0225] The major findings of the Discovery study were provided by the comparison of blood transcriptome at T2 vs T1 in the 30 patients who had received albumin and the 19 patients who had not received albumin. The inventors observed that patients treated with albumin exhibited gene signatures that were not observed in patients who had not received this treatment. Thus, patients treated with albumin were characterized by specific increases in signatures related to adaptive immune cells (including those relating to B cells, plasma cells and immunoglobulins, CD4 T cells) and to innate mononuclear myeloid cells. In addition, patients treated with albumin had specific increases in signatures related to cell cycle, mitosis and TFs such as c-Myc and E2F family members. Importantly, results obtained with scRNA- seq in patients’ PBMCs exposed ex vivo to albumin showed that albumin per se caused changes in adaptive and innate immune cells. Together these findings suggest that albumin administration triggered signals that caused an expansion of the B-cell compartment and also of the CD4 T-cell compartment while activating some mononuclear myeloid cells.

[0226] Another major finding of the present studies was the activation of neutrophils by albumin. Patients treated with albumin had upregulation of several genes that are markers of activated low-density neutrophils. Furthermore, the ex vivo experiments strikingly demonstrated that patients’ neutrophils exposed to albumin exhibited increased degranulation responses and enhanced phagocytosis. Therefore, albumin restores neutrophil-mediated defensive mechanisms that are known to be severely impaired in acutely decompensated cirrhosis.528

[0227] The prevalence of bacterial infections at admission in patients with acutely decompensated cirrhosis is very high, 37.3% in patients with ACLF11and 25.1% in patients without ACLF.34Among the uninfected patients at admission, 46% of those who presented with ACLF11and 18% of those who presented without ACLF4developed bacterial infection during hospitalization. These data indicate severe impairment of the defensive mechanisms against microbes. The existence of intestinal bacterial translocation on top of both the profound impairment of the innate immune cells (illustrated by defective antimicrobial functions of neutrophils and monocytes) and depletion of the lymphocyte compartment (including T cells, B cells and NK cells), likely explain such a high risk of infections in patients with acutely decompensated cirrhosis.29The results of the present study strongly suggest that albumin can rescue both the defective neutrophil antimicrobial functions and the depleted lymphocyte compartment in the most severe forms of acutely decompensated cirrhosis.

[0228] An interesting observation of the ex vivo studies presented herein was that the responses to albumin in lymphocytes (scRNA-seq experiments) and neutrophils responses (experiments assessing degranulation and phagocytosis), occurred within less than 2 hours, indicating that the signal brought by the albumin molecule was very rapidly transduced in the target cells.

[0229] In conclusion, in patients with severe acutely decompensated cirrhosis, the inventors have advantageously shown that albumin promotes the expansion of the B-cell compartment and also of the CD4 T-cell compartment, acts on mononuclear myeloid cells and resets neutrophil antimicrobial functions to normal. Advantageously, clinical benefits such as preventing infections and dampening of PAMP-induced systemic inflammation in patients with decompensated cirrhosis may therefore be achieved when human albumin is administered to patients with decompensated cirrhosis.

[0230] Abbreviations

[0231] ACLF, acute-on-chronic liver failure; AKI, acute kidney injury; FDR, false discovery rate; HRS, hepatorenal syndrome; NK, natural killer; PAMP, pathogen-associated molecular pattern; PBMC, peripheral blood mononuclear cell; QuSAGE, Quantitative Set Analysis for Gene Expression; RNA-seq, RNA sequencing; scRNA-seq, single-cell RNA-seq; LIMAP, Uniform Manifold Approximation and Projection.

[0232] Table 1. Characteristics of patients at time 1 (T1) and time 2 (T2) enrolled in the albumin and no-albumin groups Liver failure 6(20) 7(23) 1 4(21) 6(32) 0.71

[0233] Kidney failure 0 (0) 23 (77) <0.01 0(0) 13(68) <0.01

[0234] Circulatory failure 0(0) 10(33) <0.01 1 (5) 1 (5) 1

[0235] Cerebral failure 0(0) 6(20) <0.01 0(0) 0 (0)b1

[0236] Coagulation failure 0 (0) 4 (13) 0.12 0 (0) 4 (22) 0.10

[0237] Respiratoryfailure 0(0) 9(30) <0.01 0(0) 0 (0)b1

[0238] ACLF grade - no. (%)“

[0239] Gradel 0(0) 13(43) <0.01 0(0) 13(72) <0.01

[0240] Grade 2-3 0 (0) 17 (57) <0.01 0(0) 5(28) 0.05

[0241] Precipitating events

[0242] Infection as precipitant 14 (47) 20 (67) 0.19 5 (26) 7 (37) 0.73 atT1 orT2-n (%)

[0243] Alcohol-related 14 (50) 14 (47) 1 7 (39) 7 (37) 1 hepatitis as precipitant at T1 orT2-n (%)

[0244] Laboratory data

[0245] INR - median (IQR) 1.6(1.4-17) 1.6(1.5-2.0) 0.08 1.5 (1.2-1.8) 1.4(1.3-2.2) 0.48

[0246] Median total bilirubin 37(2.0-9.0) 3.6(2.4-11.3) 0.83 3.2(1.3-9.5) 5.0(1.0-13.5) 0.99

[0247] (IQR)-mgZL

[0248] Median serum 1.2(1.0-1.6) 27(1.9-3.4) <0.01 1.4(1.1-1.6) 2.1 (1.4-2.4)b0.01 creatinine (IQR) - mg / dL

[0249] Serum sodium - 131±5.8 133±-8.4 0.31 134.0±6.9 132±6.9 0.53 mmol / L

[0250] Median serum albumin 2.6 (2.2-3.2J 3.0 (2.3-3.5J 0.21 27(2.5-3.4) 3.0(2.8-3.1) 0.36

[0251] (IQR) - g / dl

[0252] Median white-cell count 8.3(7.0-10.3) 11.9 (6.2- 0.06 6.6 (4.6-S.6) 8.1 (5.8-12.4) 0.12

[0253] (IQR) -x107mm313.7)

[0254] Median absolute 0.9 (0.6-1.3) 1.1 (07-1.6) 0.25 1.1 (0.8-1.5) 1.2 (0.8-1.8) 0.54 lymphocyte count (IQR) - x103 / mm3

[0255] Median absolute 0.8 (0.6-1.1) 1.0 (0.6-1.3) 0.1 0.6 (0.3-1.0) 07(0.6-0.9) 0.48 monocyte count (IQR) - x103 / mm3 Median absolute 6.1 (4.2-77) 8.9 (4.2-11.8) 0.12 4.0 (3.4-6.4) 4.5 (3.0-7.8) 0.73 neutrophil count (IQR) - x103 / mm3

[0256] Median C-reactive 31.3 (19.7- 33.5 (19.4- 0.5 13.3 (7.4- 16.1 (11.4- 0.69 protein (IQR) - mg / L 54.0) 90.4) 25.0)b21.8)b

[0257] Median blood levels of protein mediators of inflammation (IQR) - pg / ml

[0258] Eotaxin 66.0 (43.9- 86.3 (58.6- 71.0 (43.7- 64.3 (50.1-

[0259] 97.1 ) 104.1 ) 0.1 121.0) 121.9) 1

[0260] G-CSF 18.8 (4.4- 22.5 (9.0- 23.9 (7.6- 16.9 (3.6-

[0261] 43.2) 73.8) 0.59 99.6) 70.9) 0.75

[0262] Interferon-a2 10.0 (2.2- 16.8 (2.4- 12.8 (7.6- 17.6 (8.2-

[0263] 24.8) 33.4) 0.43 22.0) 22.7) 0.5

[0264] Interferon-Y 33.3 (7.0- 23.8 (12.1- 19.2 (7.2- 25.9 (7.9-

[0265] 86.0) 97.2) 0.93 98.7) 45.8) 0.84 lnterleukin-1a 4.3 (17-7.2) 2.0 (0.8-6.1 ) 0.3 2.5 (1.1-3.3) 2.0 (0.3-3.9) 0.71 lnterleukin-1 p 5.4 (3.1-10.8) 4.7 (2.3-13.0) 0.93 5.2 (1.8-97) 5.2 (1.9-87) 0.89 lnterleukin-6 24.7 (13.6- 52.5 (24.3- 9.3 (6.8- 16.9 (8.8-

[0266] 46.5) 168.9) 0.16 18.1 )b23.1 )b0.1 lnterleukin-8 12.6 (4.0-

[0267] 5.5 (3.5-10.7) 18.1 ) 0.19 4.3 (2.6-13.0) 4.3 (1.0-9.0) 0.6

[0268] Interleukin-10 10.0 (3.9- 13.1 (3.8-

[0269] 30.4) 26.4) 0.97 2.4 (1 ,2-3.6)b47 (3.2-5.8) 0.08

[0270] IP-10 (CXCL10) 231 (123- 231 (169- 214 (166- 237 (141-

[0271] 374) 615) 0.34 482) 487) 1

[0272] MCP-1 179 (147- 349 (230- 176 (158- 198.5 (100-

[0273] 257) 533) <0.01 279) 281 )b0.8

[0274] MIP-1a (CCL3) 11.6 (8.4- 13.3 (4.5-

[0275] 23.5) 15.2 (9-28.9) 0.38 8.5 (2-18) 19.4) 0.41

[0276] MIP-1 (CCL4) 15.7 (11.7- 19.9 (13.8- 15.9 (12- 15.2 (11.7-

[0277] 19.7) 30.6) 0.08 22.7) 19.6) 0.7

[0278] Tumor necrosis factor 30.8 (18.1- 35.3 (24- 37.6 (18.8- 35.6 (20.8-

[0279] 42) 66.6) 0.31 47.2) 49.3) 0.89

[0280] Additional Materials and Methods

[0281] Neutrophils were isolated from peripheral venous blood (20 ml) obtained from 6 patients with acutely decompensated cirrhosis (AD) and 5 age-matched healthy subjects (HS) using the

[0282] Ficoll-Hypaque method. Briefly, patients with acutely decompensated cirrhosis were enrolled in the liver intensive care unit from Hospital Clinic (Barcelona, Spain). Blood from four healthy donors was obtained through an agreement with Hospital Clinic blood bank. Peripheral venous blood (8 ml) was obtained by venopuncture and collected into EDTA-coated sterile pyrogen- free tubes (Becton Dickinson. Grenoble. France). Blood samples were centrifuged at 200 g for 10 min to collect plasma. Sedimented cells were diluted with DPBS- / - up to a volume of 20 ml. Diluted blood was layered over 13.3 ml of Ficoll-Hypaque and centrifuged at 500 g for 25 min with the break-off. After separation of the PBMC layer in the supernatant, neutrophils were collected from the pellet and incubated with pre-warmed ammonium-chloride-potassium lysis buffer for 10 min at room temperature to remove red blood cells and then centrifuged at 400 g for 5 min. The red blood lysis procedure was repeated twice, and the resultant pellet was washed with DPBS--. Isolated neutrophils were resuspended in RPMI 1640 medium containing penicillin (100 U / rnL), streptomycin (100 U / rnL) and L-glutamine (4 mM) without fetal bovine serum (FBS) and used for the following neutrophil function assays.

[0283] Degranulation assay. After 30 minutes of resting, neutrophils were seeded at a density of 3x106cells / mL and incubated with either albumin, recombinant human albumin (both at 15 mg / ml) or vehicle control in the absence or presence of phorbol 12-myristate 13-acetate (100 nM) for 2 hours at 37°C in a 5% CO2 incubator. At the end of the incubation period, supernatants were collected to measure degranulation using the Neutrophil Myeloperoxidase Activity Assay Kit (Cayman Chemical, Ann Arbor, Ml). Briefly, 25 pl of neutrophil supernatant together with 25 pl of assay buffer were added to each well of the experimental plate. Then, 50 pl of 3,3',5,5'-tetramethylbenzidine (TMB), a substrate for horseradish peroxidase, was added to each well and absorbance was measured at minute one and minute five after the addition of TMB in a microplate reader (Infinite M PLEX Monochromator, TECAN, Mannedorf, Switzerland). The assay was performed at room temperature.

[0284] Phagocytosis assay. After 30 minutes of resting, neutrophils were seeded at a density of 5x105cells / mL and incubated with either albumin, recombinant human albumin (both at 15 mg / ml) or vehicle control for 2 hours at 37°C in a 5% CO2 incubator. Thereafter, 50 pL of opsonized fluorescein conjugate zymosan bioparticles (Thermo Fisher Scientific) was added to each well (ratio cells / bioparticles, 1 :10) with a final volume of 200 pL and incubated at 37°C for 60 min. Cells were then washed with sterile DPBS-- and 100 pL trypan blue solution (diluted 1 / 10 in sterile DPBS--) was added to quench fluorescence of extracellular bioparticles. Plates were finally centrifuged for 5 min at 400 g at room temperature and excess trypan blue was carefully aspirated. The fluorescent intensity of each well was read in a microplate reader (FLUOstar Optima, Ortenberg, Germany).

[0285] The results indicate that albumin can rescue defective neutrophil antimicrobial function. Advantageously, clinical benefits such as preventing infections and dampening of pathogen- associated molecular pattern (PAMP)-induced systemic inflammation in patients with decompensated cirrhosis may therefore be achieved when human albumin is administered to patients with decompensated cirrhosis.

[0286] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0287] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0288] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0289] The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0290] References

[0291] 1 . Moreau R, Jalan R, Gines P, et al. Acute-on-chronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis. Gastroenterology 2013;144:1426-37. e1-9

[0292] 2. Arroyo V, Moreau R, Jalan R. Acute-on-Chronic Liver Failure. N Engl J Med 2020;382:2137-45.

[0293] 3. Trebicka J, Fernandez J, Papp M, et al. The PREDICT study uncovers three clinical courses in acutely decompensated cirrhosis with distinct pathophysiology. J Hepatol 2020;73:842-854.

[0294] 4. Trebicka J, Fernandez J, Papp M, et al. PREDICT identifies precipitating events associated with the clinical course of acutely decompensated cirrhosis. J Hepatol 2021 ;74:1097-1108.

[0295] 5. Weiss E, de la Grange P, Defaye M, et al. Characterization of Blood Immune Cells in Patients With Decompensated Cirrhosis Including ACLF. Front Immunol 2021 ;11 :619039.e1-9.

[0296] 6. Claria J, Stauber RE, Coenraad MJ, et al. Systemic inflammation in decompensated cirrhosis: characterization and role in acute-on-chronic liver failure. Hepatology 2016;64:1249-1264.

[0297] 7. Lopez-Vicario C, Checa A, Urdangarin A, et al. Targeted lipidomics reveals extensive changes in circulating lipid mediators in patients with acutely decompensated cirrhosis. J Hepatol 2020;73:817-828.

[0298] 8. O'Brien AJ, Fullerton JN, Massey KA, et al. Immunosuppression in acutely decompensated cirrhosis is mediated by prostaglandin E2. Nat Med 2014;20:518-523.

[0299] 9. Bernsmeier C, Pop OT, Singanayagam A, et al. Patients with acute-on-chronic liver failure have increased numbers of regulatory immune cells expressing the receptor tyrosine kinase MERTK. Gastroenterology 2015;148:603-15. e14.

[0300] 10. Korf H, du Plessis J, van Pelt J, et al. Inhibition of glutamine synthetase in monocytes from patients with acute-on-chronic liver failure resuscitates their antibacterial and inflammatory capacity. Gut 2019;68:1872-1883.

[0301] 11 . Fernandez J, Acevedo J, Wiest R, et al. Bacterial and fungal infections in acute-on-chronic liver failure: prevalence, characteristics and impact on prognosis. Gut 2018;67:1870-80.

[0302] 12. Bajaj JS, O’Leary JG, Reddy KR, et al. Survival in infection-related acute-on-chronic liver failure is defined by extrahepatic organ failures. Hepatology 2014;60:250-256.

[0303] 13. European Association for the Study of the Liver. EASL Clinical Practice Guidelines for the management of patients with decompensated cirrhosis. J Hepatol 2018;69:406-60.

[0304] 14. Sort P, Navasa M, Arroyo V, et al. Effect of intravenous albumin on renal impairment and mortality in patients with cirrhosis and spontaneous bacterial peritonitis. N Engl J Med 1999;341 :403-9.

[0305] 15. Wong F, Pappas SC, Curry MP, et al. Terlipressin plus Albumin for the Treatment of Type 1 Hepatorenal Syndrome. N Engl J Med 2021 ;384:818-28.

[0306] 16. Fernandez J, Claria J, Amoros A, et al. Effects of Albumin Treatment on Systemic and Portal Hemodynamics and Systemic Inflammation in Patients With Decompensated Cirrhosis. Gastroenterology 2019;157:149-62.

[0307] 17. Casulleras M, Flores-Costa R, Duran-Guell M, et al. Albumin internalizes and inhibits endosomal TLR signaling in leukocytes from patients with decompensated cirrhosis. Sci Transl Med 2020;12(566):eaax5135.

[0308] 18. Caraceni P, Riggio O, Angeli P, at al. Long-term albumin administration in decompensated cirrhosis (ANSWER): an open-label randomised trial. Lancet 2018;391(10138):2417-2429.

[0309] 19. Banchereau R, Hong S, Cantarel B, et al. Personalized Immunomonitoring Uncovers Molecular Networks that Stratify Lupus Patients. Cell 2016;165:551-565.

[0310] 20. Li S, Rouphael N, Duraisingham S, et al. Molecular signatures of antibody responses derived from a systems biology study of five human vaccines. Nat Immunol 2014;15:195- 204.

[0311] 21. Hagan T, Gerritsen B, Tomalin LE, et al. Transcriptional atlas of the human immune response to 13 vaccines reveals a common predictor of vaccine-induced antibody responses. Nat Immunol 2022;23:1788-1798. 22. Aran D, Looney AP, Liu L, et al. Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage. Nat Immunol 2019;20:163-72.

[0312] 23. Monaco G, Lee B, Xu W, et al. RNA-Seq Signatures Normalized by mRNA Abundance Allow Absolute Deconvolution of Human Immune Cell Types. Cell Rep 2019;26:1627- 4O.e7.

[0313] 24. Yaari G, Bolen CR, Thakar J, et al. Quantitative set analysis for gene expression: a method to quantify gene set differential expression including gene-gene correlations. Nucleic Acids Res 2013;41 :e170.

[0314] 25. Gandoura S, Weiss E, Rautou PE, et al. Gene- and exon-expression profiling reveals an extensive LPS-induced response in immune cells in patients with cirrhosis. J Hepatol 2013;58:936-948.

[0315] 26. Andreatta M, Carmona SJ. UCell: Robust and scalable single-cell gene signature scoring. Comput Struct Biotechnol J 2021 ;19:3796-3798.

[0316] 27. Amulic B, Cazalet C, Hayes GL, et al. Neutrophil function: from mechanisms to disease. Annu Rev Immunol. 2012;30:459-89.

[0317] 28. Bernsmeier C, van der Merwe S, Perianin A. Innate immune cells in cirrhosis. _J Hepatol 2020;73:186-201.

[0318] 29. Arroyo V, Moreau R, Kamath PS, et al. Acute-on-chronic liver failure in cirrhosis. Nat Rev Dis Primers 2016;2:16041.

[0319] 30. Ip WK, Medzhitov R .Macrophages monitor tissue osmolarity and induce inflammatory response through NLRP3 and NLRC4 inflammasome activation. Nat Commu. 2015;6:6931.

[0320] 31. Jiang W, Le J, Wang PY, et al. Extracellular Acidity Reprograms Macrophage Metabolism and Innate Responsiveness. J Immunol 2021 ;206:3021-3031.

[0321] 32. Murase M, Kawasaki T, Hakozaki R, et al. Intravesicular Acidification Regulates Lipopolysaccharide Inflammation and Tolerance through TLR4 Trafficking. J Immunol 2018;200:2798-2808.

[0322] 33. Vidarsson G, Stemerding AM, Stapleton NM, et al. FcRn: an IgG receptor on phagocytes with a novel role in phagocytosis. Blood 2006;108:3573-9.

[0323] 34. Pyzik M, Rath T, Lencer Wl, et al. FcRn: The Architect Behind the Immune and Nonimmune Functions of IgG and Albumin. J Immunol 2015;194:4595-603. 35. Zhao R, Dai H, Arias RJ, et al. Direct activation of the proton channel by albumin leads to human sperm capacitation and sustained release of inflammatory mediators by neutrophils. Nat Commun 2021 ; 12:3855.

[0324] Numbered Paragraphs

[0325] Paragraph 1. A composition comprising human albumin for use in the treatment of defective immune cell function in a subject having systemic inflammatory response syndrome, wherein:

[0326] (a) the composition comprising human albumin is for treating defective neutrophil function, wherein the human albumin is for administration to the subject in a dose sufficient to increase neutrophil anti-microbial function; and / or

[0327] (b) the composition comprising human albumin is for treating defective CD4+ T cell function, wherein the human albumin is for administration to the patient in a dose sufficient to increase the level of central memory ITGB1+ CD4+ T cells and / or decrease the level of activated CD4+ memory T cells; and / or

[0328] (c) the composition comprising human albumin is for treating defective dendritic cell function, wherein the human albumin is for administration to the subject in a dose sufficient to increase the level of plasmacytoid dendritic cells; and / or

[0329] (d) the composition comprising human albumin is for treating defective monocyte cell function, wherein the human albumin is for administration to the subject in a dose sufficient to increase the level of intermediate HAVCR2+ monocytes.

[0330] Paragraph 2. The composition for use according to paragraph 1(a), wherein the increase in neutrophil anti-microbial function is an increase in neutrophil degranulation and / or neutrophil phagocytosis.

[0331] Paragraph 3. The composition for use according to paragraph 1(a) or paragraph 2, wherein the human albumin is for administration to the subject in a dose sufficient to upregulate the expression of a neutrophil gene selected from the group consisting of: CD177, OLFM4, PRG2, MPO, BPI, RETN, LCN2, CEACAM8, and MCEMP1 , or a combination thereof.

[0332] Paragraph 4. The composition for use according to any one of the preceding paragraphs, wherein the systemic inflammatory response syndrome is related to a disease selected from the group consisting of: decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer.

[0333] Paragraph 5. The composition for use according to any one of the preceding paragraphs, wherein the subject is a sepsis patient or a patient with a liver condition.

[0334] Paragraph 6. The composition for use according to any one of the preceding paragraphs, wherein the subject has a liver condition selected from the group consisting of: decompensated cirrhosis, pre-acute on chronic liver failure, and acute on chronic liver failure.

[0335] Paragraph 7. A composition comprising human albumin for use in the treatment of defective immune cell function in a subject in need thereof, wherein the defective immune cell function is selected from the group consisting of:

[0336] (a) defective neutrophil function;

[0337] (b) defective central memory ITGB1+ CD4+ T cell function and / or activated CD4+ memory T cell function;

[0338] (c) defective plasmacytoid dendritic cell function; and

[0339] (d) defective intermediate HAVCR2+ monocyte function; wherein said defective immune cell function is related to a disease selected from the group consisting of: systemic inflammatory response syndrome, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer.

[0340] Paragraph 8. The composition for use according to any one of the preceding paragraphs, wherein the human albumin is for administration by an administration route selected from the group consisting of: intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intrapulmonary, intranasal, oral, rectal, and combinations thereof.

[0341] Paragraph 9. The composition for use according to any one of the preceding paragraphs, wherein the human albumin is for administration at multiple intervals as part of a multipledosage regimen.

[0342] Paragraph 10. The composition for use according to paragraph 9, wherein: the multiple dosing regimen comprises multiple portion doses administered at equally spaced intervals of from about 1 day to about 30 days; or the multiple dosing regimen comprises multiple portion doses administered at unequally spaced intervals of from about 1 day to about 30 days; or the multiple dosing regimen comprises two or more administrations up to a total cumulative dose. Paragraph 11. The composition for use according to any one of paragraphs 9 to 10, wherein: the human albumin is for administration at individual doses of about 5 g / interval to about 500 g / interval as part of a multiple-dosage regimen; or the human albumin is for administration at individual doses of about 20 g / interval to about 200 g / interval as part of a multiple-dosage regimen. Paragraph 12. The composition for use according to any one of paragraphs 9 to 11 , wherein the dosing interval is every 15 days, or less.

[0343] Paragraph 13. The composition for use according to any one of the preceding paragraphs, wherein the human albumin is human plasma-derived albumin or recombinant human albumin. Paragraph 14. The composition for use according to any one of the preceding paragraphs, wherein the concentration of human albumin is between 4 % and 25 % (w / v); or wherein the concentration of human albumin is about 20 % (w / v).

Claims

Claims1. A composition comprising human albumin for use in regulating immune cells in a subject having systemic inflammatory response syndrome, wherein:(a) the composition comprising human albumin is for regulating neutrophil function, wherein the human albumin is for administration to the subject in a dose sufficient to increase neutrophil anti-microbial function; and / or(b) the composition comprising human albumin is for regulating CD4+ T cells, wherein the human albumin is for administration to the patient in a dose sufficient to increase the level of central memory ITGB1+ CD4+ T cells and / or decrease the level of activated CD4+ memory T cells; and / or(c) the composition comprising human albumin is for regulating dendritic cells, wherein the human albumin is for administration to the subject in a dose sufficient to increase the level of plasmacytoid dendritic cells; and / or(d) the composition comprising human albumin is for regulating monocyte cells, wherein the human albumin is for administration to the subject in a dose sufficient to increase the level of intermediate HAVCR2+ monocytes.

2. The composition for use according to claim 1(a), wherein the increase in neutrophil antimicrobial function is an increase in neutrophil degranulation and / or neutrophil phagocytosis.

3. The composition for use according to claim 1(a) or claim 2, wherein the human albumin is for administration to the subject in a dose sufficient to upregulate the expression of a neutrophil gene selected from the group consisting of: CD177, OLFM4, PRG2, MPO, BPI, RETN, LCN2, CEACAM8, and MCEMP1 , or a combination thereof.

4. The composition for use according to any one of the preceding claims, wherein the systemic inflammatory response syndrome is related to a disease selected from the group consisting of: decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heat-shock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer.

5. The composition for use according to any one of the preceding claims, wherein the subject is a sepsis patient or a patient with a liver condition.

6. The composition for use according to any one of the preceding claims, wherein the subject has a liver condition selected from the group consisting of: decompensated cirrhosis, preacute on chronic liver failure, and acute on chronic liver failure.

7. A composition comprising human albumin for the treatment of defective immune cell function in a subject in need thereof, wherein the defective immune cell function is selected from the group consisting of:(a) defective neutrophil function;(b) defective central memory ITGB1+ CD4+ T cell function and / or activated CD4+ memory T cell function;(c) defective plasmacytoid dendritic cell function; and(d) defective intermediate HAVCR2+ monocyte function.

8. The composition for use according to claim 7, wherein said defective immune cell function is related to a disease selected from the group consisting of: systemic inflammatory response syndrome, decompensated cirrhosis, acute liver failure, pre-acute on chronic liver failure, acute on chronic liver failure, severe sepsis, septic shock, severe polytraumatic conditions, severe burns, acute pancreatitis, severe acute pancreatitis, hemophagocytic syndrome, heatshock syndrome, acute ischemia-reperfusion syndrome, inflammation diseases, and cancer.

9. The composition for use according to any one of the preceding claims, wherein the human albumin is for administration by an administration route selected from the group consisting of: intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intrapulmonary, intranasal, oral, rectal, and combinations thereof.

10. The composition for use according to any one of the preceding claims, wherein the human albumin is for administration at multiple intervals as part of a multiple-dosage regimen.11 . The composition for use according to claim 10, wherein: the multiple dosing regimen comprises multiple portion doses administered at equally spaced intervals of from about 1 day to about 30 days; or the multiple dosing regimen comprises multiple portion doses administered at unequally spaced intervals of from about 1 day to about 30 days; or the multiple dosing regimen comprises two or more administrations up to a total cumulative dose.

12. The composition for use according to any one of claims 10 to 11 , wherein: the human albumin is for administration at individual doses of about 5 g / interval to about 500 g / interval as part of a multiple-dosage regimen; or the human albumin is for administration at individual doses of about 20 g / interval to about 200 g / interval as part of a multiple-dosage regimen.

13. The composition for use according to any one of claims 10 to 12, wherein the dosing interval is every 15 days, or less.

14. The composition for use according to any one of the preceding claims, wherein the human albumin is human plasma-derived albumin or recombinant human albumin.

15. The composition for use according to any one of the preceding claims, wherein the concentration of human albumin is between 4 % and 25 % (w / v); or wherein the concentration of human albumin is about 20 % (w / v).

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