WO2015153715A1 - Ciblage de l'interleukine-3 (il-3) dans le sepsis - Google Patents

Ciblage de l'interleukine-3 (il-3) dans le sepsis Download PDF

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WO2015153715A1
WO2015153715A1 PCT/US2015/023798 US2015023798W WO2015153715A1 WO 2015153715 A1 WO2015153715 A1 WO 2015153715A1 US 2015023798 W US2015023798 W US 2015023798W WO 2015153715 A1 WO2015153715 A1 WO 2015153715A1
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sample
level
sepsis
subject
septic shock
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PCT/US2015/023798
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Filip K. SWIRSKI
Georg F. Weber
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The General Hospital Corporation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • IL-3 interleukin 3
  • Sepsis is a frequently fatal condition characterized by an uncontrolled and harmful host reaction to microbial infection. Despite the prevalence and severity of sepsis, we lack a fundamental grasp of its pathophysiology.
  • the present invention is based, at least in part, on the discovery that the cytokine interleukin (IL)-3 potentiates inflammation in sepsis.
  • IL cytokine interleukin
  • IRA innate response activator B cells
  • IL-3 deficiency protects mice against sepsis.
  • high plasma IL-3 levels associate with high mortality even after adjusting for prognostic indicators.
  • the methods include obtaining a sample comprising serum from a subject; determining a level of interleukin-3 (IL-3) in the sample; comparing the level of IL-3 in the sample to a reference level; and diagnosing a subject with a level in the sample above the reference level as having sepsis, severe sepsis, or septic shock.
  • IL-3 interleukin-3
  • the methods include obtaining a sample comprising serum from a subject; determining a level of interleukin-3 (IL-3) in the sample; comparing the level of IL-3 in the sample to a reference level; and predicting that a subject with a level in the sample above the reference level has an increased risk of mortality.
  • IL-3 interleukin-3
  • the methods include selecting the subject for administration of an IL-3 inhibitor, and optionally administering the IL-3 inhibitor to the subject.
  • the methods include obtaining a first sample comprising serum from a subject; determining a level of interleukin-3 (IL-3) in the first sample; obtaining a second sample comprising serum from the subject at a later time; determining a level of interleukin-3 (IL-3) in the second sample; and comparing the level of IL-3 in the first sample to the level of IL-3 in the second sample, wherein a subject whose level of IL-3 in the first sample is greater than the level in the second sample has a worsened condition or increased creased risk of mortality at the second time point as compared to the first time point, and a subject whose level of IL-3 in the first sample is less than the level in the second sample has an improved condition or decreased risk of mortality at the second time point as compared to the first time point.
  • IL-3 interleukin-3
  • the treatment is administration of an IL-3 inhibitor. If the subject has a worsened condition or increased creased risk of mortality at the second time point, the methods can include changing treatment or providing a more aggressive treatment (e.g., a higher dose where appropriate).
  • the methods include obtaining a first sample comprising serum from a subject; determining a level of interleukin-3 (IL-3) in the first sample; administering the treatment to the subject; obtaining a second sample comprising serum from the subject at a later time; determining a level of interleukin-3 (IL-3) in the second sample; and comparing the level of IL-3 in the first sample to the level of IL-3 in the second sample, wherein the presence of a level of IL-3 in the first sample that is greater than the level in the second sample indicates that the treatment has not been effective, and a level of IL-3 in the first sample that is less than the level in the second sample indicates that the treatment has been effective.
  • the treatment is administration of an IL-3 inhibitor. If the treatment has not been effective, the methods can include changing treatment or providing a more aggressive treatment (e.g., a
  • determining a level of interleukin-3 (IL-3) in the sample comprises contacting the sample with an anti-IL-3 antibody and detecting binding of the antibody to IL-3 in the sample, and optionally quantitating the level of binding.
  • an immunoassay, ELIZA, or lateral flow test kit is used.
  • the methods include administering to the subject a therapeutically effective amount of an IL-3 inhibitor.
  • the methods include obtaining a sample comprising serum from a subject; determining a level of interleukin-3 (IL-3) in the sample; comparing the level of IL-3 in the sample to a reference level; and selecting a subject with a level in the sample above the reference level for administration of the IL-3 inhibitor.
  • IL-3 interleukin-3
  • the methods described herein include obtaining a sample comprising blood from a subject;
  • the subject has a level of circulating leukocytes above 10 G/l.
  • the IL-3 inhibitor is an anti-IL-3 antibody or an anti-IL-3 receptor alpha chain (IL-3Ra) antibody.
  • the anti-IL-3Ra antibody is 7G3 or CSL362.
  • kits comprising an anti-IL-3 antibody for use in the methods described herein.
  • IL-3 is detrimental in experimental sepsis. Comparison of IL-3 ⁇ and Balb/c (WT) mice during experimental sepsis using the cecal ligation and puncture (CLP) model.
  • IL-3 induces emergency hematopoiesis and potentiates the cytokine storm in sepsis.
  • Figs. 3 A- J. IRA B cells are major sources of IL-3 in sepsis.
  • B Identification of IL-3 producing cells in the spleen 4 d after CLP.
  • D Western blot showing IL-3 expression by B cells and non-B cells sorted from the spleen and thymus Id after CLP.
  • G Immunofluorescence microscopy of spleen tissue in the steady state and 1 day after CLP.
  • H Co-staining of representative IL-3 + cells with IgM.
  • I Adoptive transfer of 1.5 x 10 6 peritoneal Bl B cells from GFP + mice into WT mice subjected to CLP at the time of cell transfer.
  • IL-3 is an independent early predictor for outcome in human sepsis.
  • A Total leukocyte number in non-septic people and in septic patients at the time of sepsis onset (0) and 1, 4, 7, 14, and 28 days later.
  • C IL-3 plasma levels in healthy people and in patients at sepsis onset and 1 day later.
  • D Correlation of IL-3 plasma levels with total blood monocytes, and with CD14 + and CD16 + blood monocytes in septic patients with measurable IL-3 plasma levels.
  • E Kaplan-Meier analysis showing survival of patients in the RAMMSES and SEPIL-3 studies with IL-3 at >89.4 pg/ml (top quintile, measured within 1 day after sepsis onset) vs. patients with IL-3 ⁇ 89.4 pg/ml.
  • IL-3 has no effects on myeloid production of inflammatory cytokines.
  • B
  • Intracellular IL- ⁇ , IL-6 and TNF-a staining gated on splenic monocytes, neutrophils, and other cells in WT and IL-3-/- mice 1 day after LPS.
  • the grey histogram denotes isotype staining. Error bars indicate means ⁇ SEM. Significance was assessed by Mann- Whitney test (A).
  • Figs. 8A-D Leukocyte flux after CLP.
  • B Gating strategy for identifying basophils and mast cells.
  • Figs. 9A-D IL-3 potentiates septic shock.
  • Fig. 10 HSPC gating strategy. Flow cytometry plots identifying Lin-c-kit+ hematopoietic stem and progenitor cells (HSPC), common myeloid progenitors (CMP), megakaryocyte and erythrocyte progenitors (MEP), granulocyte and macrophage progenitors (GMP), and macrophage and dendritic cell progenitors (MDP) in the bone marrow.
  • IL-3 -producing B cells are IRA B cells.
  • A Identification of IRA B cells as GM-CSF-producing IgM+ CD 19+ B220+ MHCII+ B cells.
  • IL-3 and GM-CSF produced by IRA B cells have distinct functions.
  • B Sorted Bla B cells from WT and IL-3-/- mice were placed into culture and stimulated with LPS for 2 d. The cells were then stained to detect intracellular IgM levels. Data show that IL-3-/- Bla cells augment intracellular IgM levels at similar levels compared to WT cells. Cells producing IgM at high levels are termed IgM(ic)high.
  • Fig. 14 Characterization of IL-3 producing cells after CLP.
  • Immunofluorescence microscopy in the splenic red pulp identifies IL-3+ B cells as CD19+ and CD3- c-kit- CD90.2- CD49b-. Representatives of >100 cells examined are shown.
  • Figs. 16A-B Association of IL-3 plasma levels with survival in the
  • Fig. 17 Association of IL-3 plasma levels with survival in the SEPIL-3 cohort. Kaplan-Meier analysis showing survival of patients in the SEPIL-3 cohort with IL-3 at >89.4 pg/ml (top quintile, measured within 1 day after sepsis onset) vs. patients with IL-3 ⁇ 87.4 pg/ml.
  • B cells are sources of IL-3 in the human spleen.
  • A Flow cytometry plot showing IgGl-PE isotype control in human splenocytes.
  • B
  • peritoneal Bla cells are activated by microbial pathogens and give rise to IL-3+ B cells in the red pulp of the spleen.
  • IL-3 acts on HSPC to promote the emergency generation of inflammatory leukocytes that are released into the circulation. This leads to an uncontrolled cytokine storm, multi-organ failure, septic shock, and death.
  • Septic shock is the presence of infection associated with a systemic inflammatory response that results in physiologic alterations at the capillary endothelial level, manifesting as a drop in blood pressure.
  • Septic shock can be caused by any type of bacteria, as well as some fungi and viruses.
  • septic shock also occurs in people who suffer other illnesses, including diabetes, immune system disorders such as AIDS, diseases of the genitourinary, biliary, or intestinal tracts, cardiovascular disease (e.g. mesenterial infarction), leukemia, or lymphoma, or who have indwelling long-term catheters, recent surgeries, or use of steroids or antibiotics.
  • Outward symptoms of septic shock include, e.g., reduced urine output (e.g., oliguria or anuria), cool, pale extremities; high or very low temperature, chills;
  • reduced urine output e.g., oliguria or anuria
  • Blood cultures may not become positive for several days after the blood has been taken, or for several days after the shock has developed. See, e.g., Vincent, "Septic Shock.” In: Fink et al, eds. Textbook of Critical Care. 5th ed. Philadelphia, Pa: Saunders Elsevier; 2005: chap 147; Jones and Kline, "Shock.” In: Marx, ed. Rosen's Emergency Medicine: Concepts and Clinical Practice. 6th ed. Philadelphia, Pa: Mosby Elsevier; 2006: chap 4;
  • SIRS Systemic Inflammatory Response Syndrome
  • sepsis is defined by the same criteria as SIRS, plus the presence of a documented infection or a suspected infection (a pathological process induced by a micro-organism) plus some of the following: general parameters (Fever (core temperature >38.3°C), Hypothermia (core temperature ⁇ 36°C, Heart rate >90 bpm or >2 SD above the normal value for age, Tachypnea: >30 bpm, Altered mental status, Significant edema or positive fluid balance (>20 ml/kg over 24 h), and/or Hyperglycemia (plasma glucose >110 mg/dl or 7.7 mM/1) in the absence of diabetes); Inflammatory parameters (Leukocytosis (white blood cell count >12,000/ ⁇ 1), Leukopenia (white blood cell count ⁇ 4,000/ ⁇ 1), Normal white blood cell count with >10% immature forms, Plasma C reactive protein
  • Hyperbilirubinemia plasma total bilirubin >4 mg/dl or 70 mmol/1); and/or Tissue perfusion parameters (Hyperlactatemia (>3 mmol/1) and/or Decreased capillary refill or mottling). See, e.g., Levy et al, Crit Care Med (2003) 31 : 1250-1256; Levy et al, Intensive Care Med (2003) 29:530-538.
  • Severe sepsis is defined by the criteria of sepsis plus organ dysfunction, hypoperfusion, or hypotension. Hypoperfusion and perfusion abnormalities can include, but are not limited to, lactic acidosis, oliguria, or an acute alteration in mental status. See, e.g., Levy et al, Crit Care Med (2003) 31 : 1250-1256; Levy et al, Intensive Care Med (2003) 29:530-538.
  • Septic shock is a state of acute circulatory failure characterized by persistent arterial hypotension unexplained by other causes, and severe sepsis plus hypotension (a systolic arterial pressure below 90 mmHg (in children, less than 2 SD below normal for age); mean arterial pressure lower than 60, or a reduction in systolic blood pressure of more than 40 mmHg from baseline, despite adequate fluid volume resuscitation.
  • Perfusion abnormalities that can include, but are not limited to, lactic acidosis, oliguria, or an acute alteration in mental status.
  • identification of septic patients can be performed based on the criteria of the International Sepsis Definitions Conference (See, e.g., Levy et al., Crit Care Med (2003) 31 : 1250-1256; Levy et al, Intensive Care Med (2003) 29:530-538. ).
  • IL-3 protein levels As demonstrated herein, the presence of sepsis, severe sepsis, or septic shock is associated with a rise in IL-3 protein levels.
  • the present results support the use of levels of IL-3 as a biomarker for sepsis, severe sepsis, or septic shock diagnosis and for prognosis and successful drug treatment of sepsis, severe sepsis, or septic shock (e.g., and a resulting reduction in risk of death from sepsis, severe sepsis, or septic shock).
  • Individuals considered at risk for septic shock may benefit particularly from the methods described herein, primarily because once an elevated level of IL-3 is detected, e.g., in a subject who is at risk for septic shock, early treatment can begin before there is any clinical evidence of septic shock.
  • Individuals "at risk” include, e.g., individuals suffering from any condition described above, e.g., sepsis or severe sepsis, or having another factor that may put a patient at risk for severe infection, e.g., a chronic or hereditary disorder (e.g., SCID) or because of immune-suppressive medical treatments (e.g., chemotherapy or steroids), or a planned or unplanned trauma or surgical intervention.
  • a person suffering from an infection can be diagnosed according to the methods described herein and treated before full-blown septic shock occurs.
  • a patient can be identified as at risk for septic shock by any method known in the art, e.g., by a physician or other medical personnel.
  • IL-3 levels of IL-3 vary among healthy individuals; thus a healthy person who has normally high baseline levels of IL-3 may be at an increased risk for developing sepsis. When such people are about to undergo a surgical intervention, a prophylactic IL-3 inhibitor may be administered.
  • the methods of diagnosis described herein are performed in conjunction with a standard septic shock workup, e.g., including laboratory and other tests (e.g., as described above, plus complete blood count (CBC); prothrombin time and/or activated partial thromboplastin time; urine output rate; arterial blood gases (ABG) (levels reflect acid-base and perfusion status); and lactate and base deficit (used in some centers to indicate the degree of metabolic debt;
  • a standard septic shock workup e.g., including laboratory and other tests (e.g., as described above, plus complete blood count (CBC); prothrombin time and/or activated partial thromboplastin time; urine output rate; arterial blood gases (ABG) (levels reflect acid-base and perfusion status); and lactate and base deficit (used in some centers to indicate the degree of metabolic debt;
  • Imaging studies e.g., (standard radiography, computed tomography, ultrasonography, and directed angiography), an ECG, or tissue oximetry can also be used.
  • Methods for diagnosing sepsis, severe sepsis, or septic shock, or risk of mortality from sepsis, severe sepsis, or septic shock include determining a level of IL-3 in the subject to obtain a IL-3 value, and comparing the value to an appropriate reference value, e.g., a value that represents a threshold level, above which the subject can be diagnosed as having sepsis, severe sepsis, or septic shock, or as having an increased risk of mortality from sepsis, severe sepsis, or septic shock.
  • an appropriate reference value e.g., a value that represents a threshold level
  • the reference can also be a range of values, e.g., that indicate severity of sepsis, severe sepsis, or septic shock in the subject or likelihood of death from sepsis, severe sepsis, or septic shock.
  • a suitable reference value can be determined by methods known in the art.
  • the methods include obtaining a sample from a subject, and evaluating the presence and/or level of IL-3 in the sample, and comparing the presence and/or level with one or more references, e.g., a control reference that represents a normal level of IL-3, e.g., a level in an unaffected subject, and/or a disease reference that represents a level of IL-3 associated with sepsis, severe sepsis, or septic shock, e.g., a level in a subject having sepsis, severe sepsis, or septic shock.
  • a control reference that represents a normal level of IL-3, e.g., a level in an unaffected subject
  • a disease reference that represents a level of IL-3 associated with sepsis, severe sepsis, or septic shock, e.g., a level in a subject having sepsis, severe sepsis, or septic shock.
  • the presence and/or level of IL-3 is comparable to the presence and/or level of the protein(s) in the disease reference, and the subject has one or more symptoms associated with sepsis, severe sepsis, or septic shock, then the subject has sepsis, severe sepsis, or septic shock.
  • the subject has one or more symptoms associated with sepsis, severe sepsis, or septic shock
  • sepsis can be diagnosed in a subject who has SIRS and elevated IL-3 levels (i.e., levels above a disease reference level)
  • sepsis can be diagnosed in a subject who has elevated IL-3 levels and SIRS, plus organ dysfunction, hypoperfusion, or hypotension.
  • IL-3 levels indicates a diagnosis of septic shock.
  • the subject has no overt signs or symptoms of septic shock, but the presence and/or level of one or more of the proteins evaluated is comparable to the presence and/or level of the protein(s) in the disease reference, then the subject has an increased risk of developing septic shock.
  • a treatment e.g., as known in the art or as described herein, can be administered. The efficacy of the treatment can be monitored using the methods described herein.
  • levels of IL-3 are correlated with severity of disease; thus, the methods can include comparing the level of IL-3 in a sample from a subject to a reference level or a range of reference levels that represent (are correlated with) differing degrees of severity, thereby determining the severity of disease in a subject.
  • the levels of IL-3 in a subject is determined at sepsis onset, e.g., within 12-48, within 12-24, or within 24-48 hours of the onset of clinically diagnosed sepsis as defined above.
  • Patients with levels of IL-3 above the reference level can be selected for aggressive treatment, e.g., aggressive standard treatment, and/or for treatment using an IL-3 inhibitor.
  • a reference level can be a risk reference level, i.e., a level that is correlated with an increased risk of mortality, complications, or length of hospital stay, or can represent risk of progressing to a more severe form of disease, e.g., from SIRS, sepsis or severe sepsis to septic shock.
  • IL-3 IL-3 above a risk reference level indicates that the subject has an increased risk of mortality, complications, or length of hospital stay, or can represent risk of progressing to a more severe form of disease (e.g., developing septic shock), and should be treated accordingly (e.g., with a treatment known in the art and/or described herein).
  • Complications can include multiple organ dysfunction (e.g., respiratory failure, renal failure, hepatic damage, and/or cardiac dysfunction), secondary infections, and prolonged need for supportive care (e.g., dialysis, ventilator, and/or cardiac drugs), resulting in longer ICU and hospital stay, more interventions/procedures, increased costs, and worse long-term disability (i.e., poor functional status), thus the level of IL- 3 above a risk reference level can also represent an increased risk of any of these complications or sequelae.
  • organ dysfunction e.g., respiratory failure, renal failure, hepatic damage, and/or cardiac dysfunction
  • supportive care e.g., dialysis, ventilator, and/or cardiac drugs
  • the methods include obtaining a sample from a subject, and evaluating the presence and/or level of IL-3 in the sample, and comparing the presence and/or level with one or more references, e.g., a control reference that represents a normal level of IL-3, e.g., a level in a subject who has no or a low/normal risk of death from sepsis, severe sepsis, or septic shock, and/or a disease reference that represents a level of IL-3 associated with a high risk of death from sepsis, severe sepsis, or septic shock, e.g., a level in a cohort of subjects having sepsis, severe sepsis, or septic shock who die from the sepsis, severe sepsis, or septic shock or related causes.
  • a control reference that represents a normal level of IL-3, e.g., a level in a subject who has no or a low/normal risk of death from sepsis
  • the risk of death can be risk within a certain time period, e.g., 1 month, 3 weeks, 2 weeks, 1 week, 5 days, 3 days, 2 days, or 1 day, or a range of time periods, e.g., risk of death within the subsequent 1-21 days, 1-14 days, 1-7 days, 1-5 days, 1-3 days, or 24-28 hours.
  • the risk of death is the likelihood that the subject will die from the sepsis, severe sepsis, or septic shock or a directly related cause (e.g., multiple organ dysfunction (e.g., respiratory failure, renal failure, hepatic damage, and/or cardiac dysfunction) or secondary infections).
  • Suitable reference values can be determined using methods known in the art, e.g., using standard clinical trial methodology and statistical analysis.
  • the reference values can have any relevant form.
  • the reference comprises a predetermined value for a meaningful level of IL-3, e.g., a control reference level that represents a normal level of IL-3, e.g., a level in an unaffected subject or a subject who is not at risk of developing sepsis, severe sepsis, or septic shock, and/or a disease reference that represents a level of the proteins associated with sepsis, severe sepsis, or septic shock.
  • the predetermined level can be a single cut-off (threshold) value, such as a median or mean, or a level that defines the boundaries of an upper or lower quartile, tertile, or other segment of a clinical trial population that is determined to be statistically different from the other segments. It can be a range of cut-off (or threshold) values, such as a confidence interval. It can be established based upon comparative groups, such as where association with risk of developing disease or presence of disease in one defined group is a fold higher, or lower, (e.g.,
  • n-quantiles i.e., n regularly spaced intervals
  • the reference level is 70 pg/ml, 75 pg/ml, 80 pg/ml, 85 pg/ml, 90 pg/ml, 95 pg/ml, or 100 pg/ml plasma, and the presence of a level of IL-3 higher than the reference level (e.g., above 90 pg/ml plasma) are selected for and optionally administered an IL-3 inhibitor;
  • the reference level can be equivalent to 90 pg/ml measured as shown in the Examples herein, as the results show that patients with IL-3 plasma levels higher than 90pg/ml plasma have reduced survival/increased risk of mortality from sepsis, severe sepsis, or septic shock or directly related causes.
  • the predetermined level is a level or occurrence in the same subject, e.g., at a different time point, e.g., an earlier time point.
  • a control reference subject does not have a disorder described herein (e.g. sepsis, severe sepsis, or septic shock).
  • a disorder described herein e.g. sepsis, severe sepsis, or septic shock.
  • the control subject is septic, and in other cases it may be desirable that a control subject is healthy.
  • the control subject has severe sepsis or septic shock, and in other cases it may be desirable that a control subject does not have severe sepsis or septic shock.
  • a disease reference subject is one who has (or has an increased risk of developing) sepsis, severe sepsis, or septic shock.
  • An increased risk is defined as a risk above the risk of subjects in the general population. For example, a subject with an increased risk of mortality is more likely to die than the population of subjects with sepsis, severe sepsis, or septic shock as a whole.
  • the level of IL-3 in a subject being less than or equal to a reference level of IL-3 is indicative of a likely good outcome (e.g., a positive prognosis, increased likelihood of survival and/or recovery).
  • the level of IL-3 in a subject being greater than or equal to the reference level of IL-3 is indicative of the presence of sepsis, severe sepsis, or septic shock or increased risk of mortality from sepsis, severe sepsis, or septic shock.
  • the amount by which the level in the subject is less than or greater than the reference level is sufficient to distinguish a subject from a control subject, and optionally is a statistically significantly less than or greater than the level in a control subject.
  • the "being equal" refers to being approximately equal (e.g., not statistically different).
  • the predetermined value can depend upon the particular population of subjects (e.g., human subjects) selected. For example, an apparently healthy population will have a different 'normal' range of levels of IL-3 than will a population of subjects which have, are likely to have, or are at greater risk to have, sepsis, severe sepsis, or septic shock. Accordingly, the predetermined values selected may take into account the category (e.g., sex, age, health, risk, presence of other diseases) in which a subject (e.g., human subject) falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art. In characterizing likelihood, or risk, numerous predetermined values can be established.
  • category e.g., sex, age, health, risk, presence of other diseases
  • the clinical course of sepsis in a subject can be characterized by at least two phases: a first inflammatory phase, and a second immunosuppressive phase.
  • the present methods include determining whether a subject is in the inflammatory or immunosuppressive phase, and selecting subjects who are in the inflammatory phase for treatment using an IL-3 inhibitor.
  • the methods can include determining levels of circulating leukocytes, e.g., circulating monocytes and neutrophils.
  • Levels of circulating leukocytes, and/or of circulating monocytes and/or neutrophils can be determined using methods known in the art; for example, these cells can be differentiated in a blood screening test (e.g., differentiated leukocyte screening), and compared to a reference level of circulating leukocytes, and/or of circulating monocytes and/or neutrophils (such reference levels can be determined using methods known in the art, e.g., as described herein).
  • normal values for leukocytes are between 5-10 G/1; thus, in some embodiments the methods include determining that the subject has a level of circulating leukocytes of above 10 G/1, and selecting and optionally administering an inhibitor of IL-3 to the subject.
  • septic patients When septic patients are within a normal range (e.g., 5-10 G/1) treatment could be stopped. Alternatively or in addition, if a subject develops nosocomial secondary infections or reactivation of viral infections (Hotchkiss R.S. et al, Nat. Med. 2009 May;15(5):496-7), the treatment can be stopped.
  • the methods described herein can include determining a level of IL-3 in a sample from a subject, e.g., a sample comprising a biological fluid.
  • the sample is or includes serum (e.g., whole blood, serum, or plasma), cerebrospinal fluid, urine, saliva, peritoneal fluid, or a portion or subfraction thereof.
  • the sample is or includes serum or a portion or subfraction thereof.
  • the sample is or includes urine or a portion or subfraction thereof.
  • the presence and/or level of IL-3 can be evaluated using methods known in the art, e.g., using quantitative immunoassay methods such as enzyme linked immunosorbent assays (ELISAs),
  • the methods include contacting an agent that selectively binds to the IL-3 protein (such as an antibody or antigen-binding portion thereof) with a sample, to evaluate the level of protein in the sample.
  • the antibody bears a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or an antigen-binding fragment thereof (e.g., Fab or F(ab')2) can be used.
  • detectable substance encompasses direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reactivity with a detectable substance.
  • detectable substances include chemiluminescent, fluorescent, radioactive, or colorimetric labels.
  • detectable substances can include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 1, 35 S or 3 H.
  • high throughput methods e.g., protein or gene chips as are known in the art (see, e.g., Ch. 12, “Genomics,” in Griffiths et al, Eds. Modern genetic Analysis, 1999,W. H. Freeman and Company; Ekins and Chu, Trends in Biotechnology, 1999;17:217-218; MacBeath and Schreiber, Science 2000,
  • kits that include a reagent comprising at least one (e.g., at least two, three, four, or five) anti-IL-3 antibody or antigen-binding fragments thereof for use in a method described herein.
  • Kits are generally comprised of the following major elements: packaging, reagents comprising binding compositions as described above, optionally a control, and instructions.
  • Packaging can be a box-like structure for holding a vial (or number of vials) containing said binding compositions, a vial (or number of vials) containing a control, and instructions for use in a method described herein. Individuals skilled in the art can readily modify the packaging to suit individual needs.
  • kits provided herein can contain at least one (e.g., at least two, three, or four) anti-IL-3 antibodies or antigen-binding fragments described herein.
  • a kit can contain at least one (e.g., at least two, three, four, or five) antibody or antigen-binding fragments thereof.
  • the antibody can be coupled to a detectable or imaging agent.
  • agents are well known in the art and include paramagnetic agents, bio luminescent or fluorescent labels (e.g., GFP, FITC, rhodamine, or Texas Red), radioactive isotopes, and colorimetric/enzymatic agents (e.g., HRP, B-galactosidase).
  • the antibody is coupled to a paramagnetic agent, e.g., a paramagnetic nanoparticle, e.g., cross-linked iron oxide (CLIO) nanoparticles; see, e.g., US 20110046004; Josephson et al, Bioconjug.
  • a paramagnetic agent e.g., a paramagnetic nanoparticle, e.g., cross-linked iron oxide (CLIO) nanoparticles
  • the kit can be designed for use in a chemiluminescent microparticle immunoassay (CMIA), such as the ARCHITECT assays from Abbot Diagnostics (Abbott Park, IL), and thus can contain paramagnetic microparticles coated with anti-BNP antibodies, and paramagnetic microparticles coated with anti- IL-3 antibodies.
  • CMIA chemiluminescent microparticle immunoassay
  • these microparticles are contacted with a sample, and the IL-3 present in the sample can bind to the coated microparticles.
  • the sample can be split into at least two aliquots, and each type of microparticle can be contacted with a separate aliquot.
  • anti-IL-3 acridinium-labeled conjugate can be added to create a reaction mixture in the second step.
  • pre-trigger and trigger solutions are added to the reaction mixture.
  • the resulting chemiluminescent reaction is measured, e.g., using the ARCHITECT i System optics (Abbot Diagnostics, Abbott Park, Illinois). A direct relationship exists between the amount of IL-3 in the sample and the chemiluminescence detected.
  • a kit as provided herein contains at least one anti-IL-3 antibody or antigen-binding fragment, and one or more solid phase immunoassay components for detecting IL-3 via solid phase analysis.
  • Solid phase immunoassays employ a solid support to which one member of a ligand-receptor pair, e.g., an antibody or antigen-binding fragment thereof, is bound.
  • solid supports include plates, tubes, beads of polystyrene, and various porous materials such as, e.g., nylon, nitrocellulose, cellulose acetate, and glass fibers. See e.g., U.S. Pat. Nos. 4,703,017; 4,743,560; and 5,073,484.
  • a kit comprises components for a solid phase immunoassay, in which a solid phase-bound antibody or antigen-binding fragment thereof (e.g., an anti-IL-3 antibody or antigen- binding fragment thereof) is contacted with a sample containing an analyte of interest (e.g., IL-3), after which the solid phase is washed to remove unbound material.
  • a solid phase-bound antibody or antigen-binding fragment thereof e.g., an anti-IL-3 antibody or antigen- binding fragment thereof
  • an analyte of interest e.g., IL-3
  • a kit contains components for a flow-through solid phase immunoassay.
  • Flow-through solid phase immunoassays obviate the need for incubation and washing steps associated with other types of solid phase
  • an antibody (specific to a target antigen analyte) is bound to a porous membrane or filter to which a liquid sample is added. As the liquid flows through the membrane, target analyte binds to the antibody. The addition of sample is followed by addition of labeled antibody. The visual detection of labeled antibody provides an indication of the presence of target antigen analyte in the sample.
  • U.S. Pat. No. 5,229,073 describes a semiquantitative competitive immunoassay lateral flow method that employs a plurality of capture zones or lines containing immobilized antibodies for measuring plasma lipoprotein levels.
  • other methods of detection can be used, e.g., colorimetric assays, radioimmunoassays, or chemiluminescent assays.
  • Sandwich assays can be used as well, e.g., using two monoclonal antibodies, one labeled with iodine 125 and the other adsorbed onto beads, e.g., as used in the IRMA-BNP2 kit from CISBIO International (France) and the ShionoRIA BNP or ANP kits
  • Kits as provided herein can be used in accordance with any of the methods (e.g., diagnostic and prognostic methods) described herein.
  • kits containing at least one anti-IL-3 antibody or antigen-binding fragment thereof described herein can be used to determine the level of IL-3 in a sample.
  • kits containing at least one anti-IL-3 antibody or antigen-binding fragment thereof can be used to determine a IL-3 reference level.
  • the kit is provided as an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the IL-3 antibodies are used in a lateral flow test strip, or immunochromatographic assays, for in vitro diagnostic testing.
  • Lateral flow tests are commonly known in the art and are typically used for medical diagnostics for home testing, point-of-care testing, or laboratory use. Briefly, a lateral flow test as described herein can be used to detect the presence, absence, or semi-quantitative levels of IL-3 in a sample.
  • An exemplary lateral flow test includes four components, a sample pad where the biological sample (e.g., blood) is added; a conjugate pad (e.g., a fiberglass pad) that contains an antibody (e.g., anti-IL-3) conjugated to detectable particles (e.g., visually detectable particles such as colloidal gold or colored latex particles, or otherwise detectable particles such as a fluorophore or chromophore) that mix with the sample and bind to a target analyte (e.g., IL-3); a reaction membrane (e.g., nitrocellulose) where a second anti-IL-3 antibody can be present in one or more defined lines or zones (referred to herein as "stripes") to immobilize the analyte- antibody color conjugated complex; and an absorbent pad (e.g., made of filter paper) designed to draw the sample across the test strip by capillary action.
  • a conjugate pad e.g., a fiberglass pad
  • an antibody e
  • a lateral flow test would work as follows: the sample (e.g., blood serum, blood plasma, or whole blood (collected in a heparinized capillary tube)) is loaded on the sample pad and through capillary or wick action the sample moves to the conjugate pad where the polyclonal anti-IL-3 IgG color particle conjugated antibodies bind to any IL-3 present in the sample forming a IL-3-mAb conjugate.
  • the sample e.g., blood serum, blood plasma, or whole blood (collected in a heparinized capillary tube)
  • the sample e.g., blood serum, blood plasma, or whole blood (collected in a heparinized capillary tube)
  • the conjugate pad e.g., blood serum, blood plasma, or whole blood (collected in a heparinized capillary tube)
  • the IL-3-mAb conjugates move to the reaction membrane where the conjugates are immobilized by stripes of monoclonal anti-IL-3 IgM antibodies, where, if IL-3 is present in the sample, the color conjugated complexes cause a colored line to appear on the test after a predetermined time period (e.g., about 5 to 30 minutes, e.g., 10 to 25 minutes, e.g., 10 minutes).
  • a predetermined time period e.g., about 5 to 30 minutes, e.g., 10 to 25 minutes, e.g., 10 minutes.
  • the lateral flow test strip is contained in a reusable or disposable plastic housing or cassette.
  • the lateral flow test strip can include a sample pad onto which the biological sample is dispensed or applied.
  • the sample pad can be placed over the conjugate pad, and can be configured to allow a part of the sample to pass through on to the conjugate pad.
  • the sample pad can be configured to allow blood plasma to diffuse through while blocking other constituents of the blood, e.g., to remove red cells, white cells and platelets, allowing the plasma to diffuse onto the nitrocellulose.
  • the sample pad can be referred to as a plasma separation pad.
  • a plasma separation membrane such as the Vivid plasma separation membrane manufactured by Pall Corporation can be used as the sample or plasma separation pad.
  • the reaction membrane includes a control stripe region with an antibody that binds to the polyclonal anti-IL-3 IgG antibody on the conjugate pad, e.g., that binds to the Fc end of the anti-IL-3 antibody.
  • a visible signal on the control stripe indicates that the sample diffused through the whole test even if a positive IL-3 signal is not seen on the test.
  • the lateral flow test strips described herein can be used to easily and rapidly measure IL-3 levels in the body from a small biological sample (e.g., a blood drop).
  • a small biological sample e.g., a blood drop.
  • microfluidic e.g., "lab-on-a-chip,” “micro-a-fluidic chips”
  • Such devices have been successfully used for microfluidic flow cytometry, continuous size-based separation, and
  • chromatographic separation In particular, such devices can be used for the isolation of specific biological particles such as specific proteins (e.g., IL-3) from complex mixtures such as serum (e.g., whole blood, serum, or plasma), cerebrospinal fluid, urine, saliva, or peritoneal fluid.
  • specific biological particles such as specific proteins (e.g., IL-3)
  • complex mixtures such as serum (e.g., whole blood, serum, or plasma), cerebrospinal fluid, urine, saliva, or peritoneal fluid.
  • a variety of approaches may be used to separate IL- 3 proteins from a heterogeneous sample.
  • some techniques can use functionalized materials to capture IL-3 using functionalized surfaces that bind to the target cell population.
  • the functionalized materials can include surface-bound capture moieties such as antibodies or other specific binding molecules, such as aptamers, as are known in the art.
  • microfluidic chip technology may be used in diagnostic and prognostic devices for use in the methods described herein.
  • micro fluidics devices comprising IL-3 binding moieties, e.g., anti-IL-3 antibodies or antigen-binding fragments thereof. Treating Septic Shock
  • the methods described herein can include the administration of an effective amount of an IL-3 inhibitor for the treatment of sepsis, severe sepsis, or septic shock.
  • the methods can include identifying a subject who has sepsis, severe sepsis, or septic shock, optionally using levels of IL-3 to select a treatment for subject, e.g., the administration of an effective amount of a pharmaceutical agent for the treatment of septic shock, e.g., an IL-3 inhibitor, with or without a standard treatment for septic shock such as an antimicrobial agent.
  • the methods can administering a treatment for septic shock to a subject as having a level of IL-3 above a reference level.
  • the methods can include identifying a subject who has sepsis, severe sepsis, or septic shock, e.g., who is within 24-48 hours of onset of sepsis, severe sepsis, or septic shock, and administering of an effective amount of a pharmaceutical agent for the treatment of septic shock, e.g., an IL-3 inhibitor, with or without a standard treatment for septic shock such as an antimicrobial agent.
  • a pharmaceutical agent for the treatment of septic shock e.g., an IL-3 inhibitor
  • the methods can include identifying a subject who has sepsis, severe sepsis, or septic shock, and who has levels of circulating leukocytes above a reference level, and administering of an effective amount of a pharmaceutical agent for the treatment of septic shock, e.g., an IL-3 inhibitor, with or without a standard treatment for septic shock such as an antimicrobial agent.
  • a pharmaceutical agent for the treatment of septic shock e.g., an IL-3 inhibitor
  • effective amount and "effective to treat,” as used herein, refer to an amount that is effective within the context of its administration for causing an intended effect or physiological outcome. Effective amounts in the present context include, for example, amounts that reduce injury to a specific organ(s) effected by septic shock, or generally improve the patient's prognosis following septic shock.
  • treat(ment) is used herein to describe delaying the onset of, inhibiting, or alleviating the detrimental effects of a condition, e.g., organ injury/failure associated with or caused by septic shock.
  • the methods can include determining a level of IL-3 in the subject, and administering a treatment to the subject if the level of IL-3 is above a preselected reference level or threshold.
  • the methods can include determining that a subject is in the
  • the treatment can include administration of an inhibitor of IL-3, with or without a standard treatment for septic shock such as an antimicrobial agent.
  • inhibitors of IL-3 are known in the art, and include agents such as antibodies that bind to IL-3 itself and inhibitors of the IL-3 receptor (CD 123).
  • agents such as antibodies that bind to IL-3 itself and inhibitors of the IL-3 receptor (CD 123).
  • CD 123 inhibitors of the IL-3 receptor
  • antibody refers to an immunoglobulin molecule or an antigen-binding portion thereof.
  • antigen-binding portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments, which retain the ability to bind antigen.
  • the antibody can be polyclonal, monoclonal, recombinant, chimeric, de-immunized or humanized, fully human, non-human, (e.g., murine), or single chain antibody.
  • the antibody has effector function and can fix complement.
  • the antibody has reduced or no ability to bind an Fc receptor.
  • the antibody can be an isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.
  • Methods for making antibodies and fragments thereof are known in the art, see, e.g., Harlow et. al, editors, Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies: Principles and Practice, (N.Y. Academic Press 1983); Howard and Kaser, Making and Using Antibodies: A Practical Handbook (CRC Press; 1st edition, Dec 13, 2006);
  • the sequence of the human IL-3 is at NCBI Reference Sequence:
  • the IL-3 receptor is a heterodimer that shares a beta chain (cytokine receptor common subunit beta precursor, NCBI Reference Sequence: NP 000386.1) in common with the receptors for IL5 and GMCSF, with a ligand-specific alpha chain (IL-3RA). There are 2 isoforms.
  • the sequence of human interleukin 3 receptor, alpha, isoform 1 precursor, is at NCBI Reference Sequence: NP 002174.1, as follows:
  • NCBI Reference Sequence NP 001254642.1, as follows:
  • an IL-3 receptor antibody used in the present methods is made using, or binds to, an antigenic portion of the IL-3 binding region.
  • Exemplary monoclonal antibodies include the anti IL-3 Receptor Alpha chain monoclonal antibody 7G3 (WO1997024373; US6,177,078; Sun et al, Blood. 1996 Jan l;87(l):83-92); CSL360 (WO 2009/070844); and CSL362 (CSL
  • Exemplary monoclonal antibodies include several anti IL-3 antibodies that are commercially available, for example from Hytest (4IL-35-4E6 and 4IL-35-2F2); R&D System Clones 4806 and 4815 (Catalog No. MAB203 and No. MAB603); LifeTechnologies (AHC0832 andAHC0939); Lifespan Biosciences (LS-C86928); and from BDBiosciences Clones BVD3-1F9 and BVD8-3G11; as well as F14-570 and F14-746 (J. Immunol. 1991, 146:893-898; Abrams and Pearce, J. Immunol. 140(1): 131-137 (1988)) and the antibodies described in Jones and Ziltener, Blood.
  • the antibodies useful in the methods described herein also block IL-3 signaling, e.g., neutralizing antibodies.
  • Standard treatments for septic shock include fluid resuscitation and the transfusion of fluids (e.g., with isotonic crystalloids and/or colloids, titrated to a selected central venous pressure (CVP) goal between 8 and 12 mm Hg or signs of volume overload (dyspnea, pulmonary rales, or evidence of pulmonary edema on a chest radiograph), blood and/or blood products; mechanical ventilation;
  • CVP central venous pressure
  • drugs to treat low blood pressure e.g., administration of inotropics or vasopressors
  • infection e.g., antimicrobial, antiviral, or antifungal therapy
  • blood clotting e.g., Activated Protein C (APC)
  • oxygen as needed; and surgical excision or drainage of the infected tissues, if possible.
  • APC Activated Protein C
  • the treatments include pharmacological blockade of high-mobility group Bl protein (HMGB1), macrophage migration inhibitory factor (MIF), the complement split product, C5a, and apoptosis inhibitors.
  • HMGB1 high-mobility group Bl protein
  • MIF macrophage migration inhibitory factor
  • C5a the complement split product
  • apoptosis inhibitors See, e.g., Dellinger et al, Crit Care Med. Jan 2008; 36(l):296-327, which is incorporated herein by reference in its entirety, and especially for teachings relating to treatment of septic shock.
  • Aggressive treatment for septic shock patients can include hemodialysis when renal failure occurs, mechanical ventilation and nova lung when ARDS occurs and the oxygenation of the patient is at risk, surgical procedures to remove the infectious focus, interventional radiology to evacuate the infectious focus. See also the guidelines from the surviving sepsis campaign (Dellinger et al, Critical Care
  • the treatment can optionally include administration of an antibiotic or antimicrobial agent.
  • antibiotics classes of antibiotics that can be used in the methods described herein include penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, quinolones, tetracyclines,
  • the methods described herein can include using levels of IL-3 to monitor the effectiveness of a treatment for septic shock, e.g., the administration of an effective amount of a pharmaceutical agent for the treatment of septic shock.
  • levels of IL-3 can be determined over time, and the change in levels is indicative of whether the treatment is effective: a decrease in IL-3 serum levels over time indicates that the treatment is effective, while no change or an increase indicates that the treatment is not effective.
  • the methods can also be used to monitor changes in risk of progression to a more severe form of disease, e.g., from sepsis or severe sepsis to septic shock; multiple serum levels of IL-3 can be determined over time, and the change in levels is indicative of whether the treatment is effective in reducing risk: a decrease in IL-3 serum levels over time indicates that the treatment is effective in reducing risk of progression, while no change or an increase indicates that the treatment is not effective in reducing risk of progression.
  • a decrease in IL-3 serum levels over time indicates that the treatment is effective in reducing risk of progression, while no change or an increase indicates that the treatment is not effective in reducing risk of progression.
  • mice (WT), C57BL/6 (WT), and CByJ.B6-Tg(UBC-GFP)30Scha/J (GFP + ) female mice (from Jackson Laboratories) were used in this study.
  • IL-3 deficient mice H-3 ⁇ A
  • GM-CSF-deficient mice Q 2 ⁇ A
  • mice were 8-12 weeks of age at the time of sacrifice. All protocols were approved by the Animal Review Committee at Massachusetts General Hospital.
  • mice were administered 25 ⁇ g of LPS (Sigma), by i.p. injections in PBS.
  • Cecal ligation and puncture (CLP) This rodent model of sepsis was carried out as previously described (9). In brief, the peritoneal cavity was opened during isoflurane anesthesia, and the cecum was exteriorized and ligated at different points distal of the ileo-cecal valve using a non-absorbable 7-0 suture. To induce high-grade CLP -60-80% of the cecum was ligated; to induce mid-grade CLP -30-50% of the cecum was ligated.
  • Bl cells, Bla cells, and GMP were FACS sorted from the peritoneum or bone marrow of WT, IL-3 ⁇ ⁇ , or naive GFP + mice.
  • Cells were injected into the peritoneum or the tail vein of recipient mice as indicated.
  • IL-3 injection IL-3 ⁇ mice were injected with 5 ⁇ g recombinant IL-3 (R&D Systems), twice, in 50 ⁇ PBS or 50 ⁇ PBS alone into the tail vein 30 min and 12 h after CLP.
  • WT animals were injected with an IL-3 complex, as previously described (35).
  • IL-3 (10 ⁇ g; R&D Systems) was mixed with anti-IL-3 Ab (5 ⁇ g; MP2-8F8, BD Pharmingen) at RT for 1 min and the complex (in 200 ⁇ saline) was injected into each mouse into the tail vein at the beginning of the experiment. Mice were sacrificed at 24 h.
  • Anti-CD 123 injection 200 ⁇ g anti-CD 123 or 200 ⁇ g IgGl isotype control (Biolegend) in 200 ⁇ PBS were injected into the tail vein of WT mice 1, 6, and 24 h after CLP was performed. Mice were sacrificed at 24 h (cytokine analysis) or 48 h (cell analysis).
  • Phagocyte depletion To deplete neutrophils and monocytes, 200 ⁇ g of anti-Ly6G (isotype 1 A8, Biolegend) antibody were injected into the tail vein 48 h before the CLP and 200 ⁇ of clodronate liposome were injected into the tail vein 24 h and 6 h before CLP; control mice were injected with 200 ⁇ g IgG2a isotope control
  • Blood pressure measurement The blood pressure of WT and mice was measured by using a tail-cuff plethysmograph according to the manufacturer's instructions. Mice were placed on a 37°C heated plate and measurements were performed 5 times/each animal. Per animal the mean systolic value was then calculated.
  • Peripheral blood for flow cytometric analysis was collected by aortic puncture, using heparin as the anticoagulant. Erythrocytes were lysed using RBC Lysis Buffer (BioLegend). Total white blood cell count was determined by preparing a 1 :10 dilution of (undiluted) peripheral blood obtained from the orbital sinus using heparin-coated capillary tubes in RBC Lysis Buffer
  • Liver, lung, thymus, lymph node tissue were cut into small pieces and subjected to enzymatic digestion with 450 U/ml collagenase I, 125 U/ml collagenase XI, 60 U/ml DNase I and 60 U/ml hyaluronidase (Sigma-Aldrich, St. Louis, MO) for 1 h at 37°C while shaking at 750 rpm.
  • Total viable cell numbers were obtained using Trypan Blue (Cellgro, Mediatech, Inc, VA). To determine total bone marrow cellularity, one femur and one tibia were estimated to represent 7% of total marrow (36).
  • Cells were seeded at a density of 50,000 cells/ ⁇ in 24-well flat- bottom, or 96-well round-bottom plates (Corning) and cultured 24 or 96 h in medium. Where indicated, LPS was added at 1 ⁇ g/ml and rIL-3 was added at 20 ng/ml in PBS. To determine IgM production, serosal Bla cells were obtained from Balb/C (WT), C57BL/6 (WT), Csf2- f - and IL-3 '- mice. Cells were sorted on a BD FACSAria II (BD Biosciences) and cultured at 37°C for 48h in B cell medium. Where indicated, LPS (Sigma) was added at a dose of 10 ⁇ g/mL.
  • Mouse anti- CD43-FITC, S7 (BD Biosciences); anti-Ly6C-FITC, AL-21 (BD Biosciences); anti- Ly6G-FITC, 1A8 (BD Biosciences); anti-CD l lb-FITC, Ml/70 (BD Biosciences); anti-CD3e-FITC, 145-2C11 (BD Biosciences); anti-CD4-FITC, RM4-5 (BD
  • Cytofix/Cytoperm Plus Kit (BD Biosciences) according to the manufacturer's instructions. Intracytoplasmatic IgM staining was done as previously described (19). Briefly, cells were stained for 30 min with a primary IgM antibody (Percp.Cy5.5 channel) in a high concentration (1 :200) to ensure saturation of surface IgM together with additional surface antibodies in normal concentration (1 :700). After cell membrane permeabilization using Cytofix/Cytoperm Plus Kit (BD Biosciences) intracytoplasmatic IgM was performed using the secondary IgM antibody (APC channel) in a lower concentration (1 :350). Cells were defined as: (i) Monocytes (Ly6C higMow CD 115 + CD 1 lb + MHCirCD 11 C -F4/80 low/int Linr (mouse) or
  • mice The lungs, livers and spleens from Balb/c control mice and IL-3 ⁇ mice were harvested in steady state or 1 day after CLP and embedded in a 2- methylbutane bath (Sigma- Aldrich) on dry ice. The lungs were filled with a mixture of O.C.T. compound and PBS (1 : 1) through the tracheas prior to harvesting. Serial 6 ⁇ thick fresh-frozen sections were prepared and stained with hematoxylin and eosin (H&E) for overall histological analysis.
  • H&E hematoxylin and eosin
  • spleen sections were incubated with anti-IL-3 biotin (MP2-8F8, BioLegend), anti-IgM-FITC (11/41, BD Biosciences), anti- CDl lb-FITC (Ml/70, BD Biosciences), anti-CD 19-FITC (1D3, BD Biosciences), anti-CD3e-FITC (145-2C11, BD Biosciences), anti-CD 117-FITC (c-kit 2B8, BD Biosciences), anti-CD90.2-Alexa Four 488 (30-H12, BioLegend), anti-CD49b-Alexa Fluor (HMa2, BioLegend), anti-CD 1 lb- APC (Ml/70, BD Biosciences).
  • APC anti-CD 1 lb- APC
  • biotinylated secondary antibody Vector Laboratories, Inc.
  • streptavidin-Alexa Fluor 594 Invitrogen
  • the slides were coverslipped using a mounting medium with DAPI (Vector Laboratories, Inc.) to identify the nuclei. Images were captured using a motorized fluorescence
  • spleen sections were incubated with anti-IgM-FITC (G20-127, BD Pharmingen, 1/50), anti-CD 19-FITC (HIB19, BD Pharmingen, 1/50), anti-IL-3-PE (BVD3-1F9, BD Pharmingen, 1/25), or IgGl-PE isotype control (1/25) (R3-34, BD Pharmingen, 1/25) overnight at 4°C. After washing, counterstaining was performed with DAPI and slides were coverslipped (lOmin at RT). After mounting, spleen sections were imaged with Axiovert 200 Inverted Fluorescence Microscope and Axiovision image processing software (Zeiss, Germany).
  • the enumeration of IL-3 producing IgM + B cells in human spleens was conducted by blinded analysis of 6 field-of- views at 20 x magnification. The average amount of IL-3 producing IgM + B cells per field-of-view is presented.
  • ELISA IL- ⁇ , IL-3, IL-6, and TNF-a ELISA was performed with R&D ELISA kits according to the manufacturer's instructions on peritoneal lavage fluid, serum and cell culture supernatants.
  • Protein assay Total protein from the bronchoalveolar lavage (BAL) fluid was measured using the Bio-Rad Protein Assay according to the manufacturer's instructions.
  • AST and ALT AST and ALT were measured in plasma with Sigma kits according to the manufacturer's instructions.
  • Phagocytosis assay PHrodoTM labelled Escherichia coli particles (Invitrogen) were used following the manufacturer's instructions. Steady state peritoneal cells from control and IL-3 ⁇ mice were seeded at 3 x 10 5 cells/well in a 96 well plate. Cells were allowed to seed 1 h at 37°C, the medium was then removed and replaced by medium with or without E. coli particles (1 mg/mL) and cells were incubated at 37°C or 4°C (negative control) for 2h. Cells were then retrieved and stained for flow cytometry. Phagocytosis rate was determined by the percentage of PHrodo/PE + peritoneal macrophages.
  • Multivariate logistic regression analyses were used to evaluate the input of IL-3 on the prediction of death at 28 days, and to adjust for potential confounders.
  • IL-3 contributes to leukocyte production, proliferation, and survival (1-4).
  • Myeloid cells such as monocytes and neutrophils produce IL- ⁇ , IL-6, and tumor necrosis factor (TNF)a, the three inflammatory hallmark cytokines constituting the cytokine storm during septic shock (5-7).
  • TNF tumor necrosis factor
  • IL-3 ⁇ mice have normal blood monocyte and neutrophil profiles (Figs. 5A-G) (8) and thus do not require IL-3 for myelopoiesis in the steady state.
  • IL-3 ⁇ mice were protected from sepsis, as seen in lower mortality rates, even after antibiotic treatment (Fig. 1A).
  • IL-3 ⁇ mice had better clinical scores, body temperatures (Fig. IB), and blood pressure (Fig. 1C), and their recovery associated with efficient microbial clearance, indicating that the absence of IL-3 did not compromise bactericidal activity or recognition (Fig. ID and 6).
  • Example 2
  • TNFa (Fig. IF). Phagocytic leukocytes were major sources of IL- ⁇ , IL-6, and TNFa, as phagocyte depletion with clodronate liposomes and anti-Ly-6G prior to CLP abolished the cytokine storm (Fig. 7A). However, IL-3-mediated cytokine induction was indirect: both WT and IL-3 1' neutrophils and monocytes contained similar intracellular reservoirs of the 3 cytokines (Fig. 7B). Analyzing other leukocytes showed IL-3 -dependent differences in T and B cell numbers after CLP (Fig. 8A), but no differences in basophils, mast cells (10-12) (figs. 8B, C), or histamine (Fig.
  • IL-3 promotes hematopoiesis by acting on its receptor, a heterodimer that consists of the IL-3-specific a chain (CD123) and the common ⁇ chain (CD131) (4).
  • Lin c-kit + hematopoietic stem and progenitor cells including megakaryocyte and erythrocyte progenitors (MEP), common myeloid progenitors (CMP), granulocyte and macrophage progenitors (GMP), and macrophage and dendritic progenitors (MDP), expressed CD 123 at the same level in both WT and lL-3 ⁇ ' ⁇ mice (Figs. 2A and 10).
  • Lin bone marrow cells containing predominantly HSPC
  • Lin bone marrow cells containing predominantly HSPC
  • IL-3 but not LPS
  • IL-3 increased cell expansion and generated myeloid cells well above numbers initially placed into culture
  • IL-3 alone modestly affected IL- ⁇ , IL-6, and TNFa production
  • combined IL-3 and LPS exacerbated the response (Fig. 2E).
  • the data suggest that IL-3 is responsible for the cytokine storm, albeit indirectly, by generating a large pool of cells that, upon recognizing bacterial components, produce cytokines in larger quantities.
  • IL-3 can trigger severe sepsis in vivo, whether it can do so alone or in combination with infection, and whether it relies on its specific receptor.
  • rIL-3 recombinant (r)IL-3 to otherwise healthy WT mice; (ii) anti-CD123 to WT mice subjected to CLP; and (iii) rIL-3 to H-J 7 mice subjected to CLP.
  • rIL-3 augmented GMP in the bone marrow and leukocyte numbers in the blood of healthy WT mice to levels akin to those in WT mice subjected to CLP (Fig. 2F). Despite this increase, rIL-3 per se did not induce a cytokine storm in the absence of infection (Fig.
  • Activated T cells (16) and thymic epithelial cells (17) produce IL-3 in the steady state, but the cytokine's source in sepsis is unknown.
  • mR A profiling identified the spleen, thymus, and lymph nodes as hubs of basal IL-3 expression.
  • IL-3 mRNA progressively increased in the spleen, followed by the thymus and lymph nodes, with no signal in the bone marrow, lung, liver, peritoneum, or duodenum (Fig. 3A).
  • Fig. 3B, C By flow cytometry and Western blots (Fig. 3D), IL-3 + cells were CD19 + B cells.
  • IL-3 levels increased in serum after CLP (Fig. 3E) but to a lesser extent in spenectomized mice (Fig. 3E).
  • Example 6 Example 6
  • Identifying B cells as sources of IL-3 prompted testing whether IL-3- producing B cells resemble IRA B cells (Fig. 12A), whose GM-CSF product protects against sepsis and pneumonia via polyreactive IgM (18, 19). Phenotypic profiling showed that splenic IL-3 producers were IgM high CD23 low CD19 + CD138 high CD43 + VLA4 + (Fig. 3F and Fig. 12B), as well as CD5 int LFA1 + CD284 + CD1 lb low/ - (Fig. 12C). This phenotype matches that of IRA B cells (18-20). The remaining, non-B IL- 3-positive cells in spleen and thymus were CD4 + T cells, CD8 + T cells, and non-T, non-B cells (Fig. 12D). Example 7.
  • IL-3 and GM-CSF which are two IRA B cell products
  • the growth factors are not interdependent: in response to CLP, the spleens of Csf2 ⁇ f ⁇ mice accumulated IL-3 -producing IRA B cells whereas IL-3 ⁇ mice accumulated GM-CSF-producing IRA B cells (Fig. 13A).
  • IL-3 was not essential to IgM production (Fig. 13B, C).
  • GM-CSF was dispensable to emergency myelopoiesis (Fig. 13D, E). The IL-3 -producing IRA B cells were readily visualized by
  • IRA B cells can both protect against and aggravate sepsis, depending on the particular growth factor they produce.
  • Peritoneal Bl cells relocate to the spleen after peritoneal LPS challenge (21) and differentiate to IRA B cells (18).
  • peritoneal LPS challenge 21) and differentiates IRA B cells (18).
  • IL-3 + B cells arise similarly, we transferred Bl cells from the peritoneum of naive GFP + mice into the peritoneum of WT mice.
  • Two days after peritoneal LPS challenge IL-3 + (Fig. 31) and GM-CSF + B cells (Fig. 15) accumulated in the spleen, indicating peritoneal B cell relocation, splenic accumulation, and IRA B cell differentiation.
  • Table 2 Patients' characteristics separated by IL-3 levels ⁇ RAMMSES-trial).
  • IL-3 induces basophil expansion in vivo by directing granulocyte-monocyte progenitors to differentiate into basophil lineage-restricted progenitors in the bone marrow and by increasing the number of basophil/mast cell progenitors in the spleen., J Immunol 182, 2835 (2009).

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Abstract

L'invention concerne des méthodes de diagnostic et de traitement du sepsis, ainsi que de réduction du risque de développer un sepsis, basées sur la mesure ou le ciblage de l'interleukine 3 (IL-3). La présente invention est basée, au moins en partie, sur la découverte du rôle de la cytokine interleukine (IL)-3 comme amplificateur de l'inflammation lors d'un sepsis. Ainsi, l'invention concerne des méthodes pour diagnostiquer un syndrome septique, un sepsis sévère ou un choc septique chez un patient. L'invention concerne également des méthodes de prédiction du risque de mortalité chez un p atteint d'un syndrome septique, d'un sepsis sévère ou d'un choc septique. L'invention concerne également des méthodes de surveillance de l'état d'un patient atteint d'un syndrome septique, d'un sepsis sévère ou d'un choc septique, ou de surveillance du risque de mortalité chez un tel patient. En outre, l'invention concerne des méthodes permettant de déterminer l'efficacité d'un traitement du syndrome septique, du sepsis sévère ou du choc septique.
PCT/US2015/023798 2014-04-01 2015-04-01 Ciblage de l'interleukine-3 (il-3) dans le sepsis WO2015153715A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005048823A2 (fr) * 2003-11-17 2005-06-02 Janssen Pharmaceutica N.V. Modelisation d'une reponse inflammatoire systemique a une infection
WO2007109674A2 (fr) * 2006-03-20 2007-09-27 Senesco Technologies, Inc. Utilisation d'arnsi eif-5a spécifique de l'apoptose pour la régulation négative de l'expression de cytokines pro-inflammatoires afin de traiter la septicémie
US8029982B2 (en) * 2004-01-20 2011-10-04 Alere San Diego, Inc. Biomarkers for sepsis
US20140017261A1 (en) * 2011-02-03 2014-01-16 Mark Totoritis Selection and treatment of subjects
US20140080135A1 (en) * 2006-12-20 2014-03-20 Myriad Genetics, Inc. Ilcs based pattern recognition of sepsis

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005048823A2 (fr) * 2003-11-17 2005-06-02 Janssen Pharmaceutica N.V. Modelisation d'une reponse inflammatoire systemique a une infection
US8029982B2 (en) * 2004-01-20 2011-10-04 Alere San Diego, Inc. Biomarkers for sepsis
WO2007109674A2 (fr) * 2006-03-20 2007-09-27 Senesco Technologies, Inc. Utilisation d'arnsi eif-5a spécifique de l'apoptose pour la régulation négative de l'expression de cytokines pro-inflammatoires afin de traiter la septicémie
US20140080135A1 (en) * 2006-12-20 2014-03-20 Myriad Genetics, Inc. Ilcs based pattern recognition of sepsis
US20140017261A1 (en) * 2011-02-03 2014-01-16 Mark Totoritis Selection and treatment of subjects

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HASSLACHER ET AL.: "Levosimendan inhibits release of reactive oxygen species in polymorphonuclear leukocytes in vitro and in patients with acute heart failure and septic shock: a prospective observational study", CRITICAL CARE, vol. 15, 12 July 2011 (2011-07-12), pages 1 - 10, XP021109793 *
WEBER ET AL.: "Interleukin-3 amplifies acute inflammation and is a potential therapeutic target in sepsis", SCEINCE, vol. 347, no. Iss. 6227, 13 March 2015 (2015-03-13), pages 1260 - 1265, XP055229899 *
WONG ET AL.: "Testing the Prognostic Accuracy of the Updated Pediatric Sepsis Biomarker Risk Model", PLOS ONE, vol. 09, no. Iss. 01, 29 January 2014 (2014-01-29), pages 1 - 6, XP055229902 *

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