Methods of treating or preventing infection-related immune conditions using compositions comprising IgM

Abstract

The present application relates to methods of treating or preventing infection-related immune conditions using compositions comprising IgM. In particular, the present application provides a method for treating immune complications resulting from infection in a patient in need thereof, characterized in that the method comprises administering a composition comprising IgM. In some embodiments, the treatment of immune complications is carried out by immunomodulation. Also provided is a method for immunomodulating an infection in a patient in need thereof, characterized in that the method comprises administering a composition comprising IgM. The present application also provides a composition comprising IgM for use in the treatment of sepsis and a composition comprising IgM for use in the treatment of immune complications resulting from infection. In some embodiments, the above-mentioned infection or sepsis results from E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Clostridium difficile, Clostridium botulinum, or a combination thereof.

Description

[0001] This application is a divisional application of the application filed on July 29, 2016, with application number 201610616759.1 and invention title "Method for treating or preventing infection-related immune status using a composition containing IgM". Technical Field

[0002] This invention relates to the treatment or prevention of immune symptoms or immune conditions by administering a composition containing IgM. Background of the Invention Microbial species can become extremely harmful to an infected patient if the individual cannot clear the infection. The infection can also become septic, spreading from the infected organ into the bloodstream. These septic infections have a poor outcome for the patient.

[0003] It has been well characterized that plasma-derived IgM can bind endotoxins and prevent endotoxin-mediated toxicity in patients. This is attributed to the inherent ability or preference of IgM to bind to glycans, thereby preventing their effects. Typically, these toxic effects of endotoxins are a response to bacterial death or lysis induced by antibiotics or the patient's immune system.

[0004] In addition, based on the interaction of IgM with specific hallmarks of autoimmune diseases or atherosclerosis, such as autoantibodies, it has been proposed to use IgM compositions or IgM-enriched compositions for the treatment of said diseases (Hurez, V. et al., “Pooled Normal Human Polyspecific IgM Contains Neutralizing Anti-Idioitypes to IgG Autoantibodies of Autoimmune Patients and Protects from Exaperimental Autoimmune Disease”, Blood, 1997, Vol. 90, No. 10, 4004-4013; and Cesena, HY., “Immune-modulation by polyclonal IgM treatment reduces atherosclerosis in hypercholesterolemic apoE - / - mice”, Atherosclerosis, 2012, Vol. 220, 59-65).

[0005] Septic status and other complications of microbial infections are often associated with overstimulation of the immune system, and due to the excessive scale and rapid evolution of microbes, they are often impossible to control. Therefore, there remains a need for effective treatments to reduce, suppress, or prevent overstimulation of the immune system. Invention Overview Embodiments of the present invention provide a composition comprising, consisting primarily of, or consisting of plasma-derived IgM and, optionally, one or more excipients in the form of a drug carrier.

[0006] In another embodiment, the present invention provides an IgM-containing composition for use in the treatment of sepsis.

[0007] In another embodiment, the present invention provides an IgM-containing composition for use in the treatment of immune complications arising from infection.

[0008] In another embodiment, the present invention provides a method for treating immune complications arising from infection in patients who require it, characterized in that the method comprises administering a composition comprising IgM.

[0009] In another embodiment, the present invention also provides a method for infection in patients for whom immune modulation is required, characterized in that the method comprises administering a composition comprising IgM.

[0010] This application provides the following: 1. A method for treating immune complications arising from infection in patients who require it, characterized in that the method comprises administering a composition comprising IgM.

[0011] 2. The method according to Project 1, characterized in that the infection is caused by *Escherichia coli* (…). Escherichia coli ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa Staphylococcus aureus Staphylococcus aureus Klebsiella pneumoniae ( Klebsiella pneumoniae Streptococcus pneumoniae () Streptococcus pneumoniae Clostridium difficile ( Clostridium difficile ), Clostridium botulinum ( Clostridium botulinum (or a combination thereof)

[0012] 3. The method according to Project 1, characterized in that the treatment of immune complications is carried out through immune modulation.

[0013] 4. The method according to Project 3, characterized in that immune regulation is carried out by inhibiting NF-κB induction, by inhibiting the proliferation of peripheral blood mononuclear cells, or a combination thereof.

[0014] 5. The method according to Project 1, characterized in that the composition used has an IgM purity of at least 90% (w / v).

[0015] 6. The method according to item 5, characterized in that the composition has an IgM purity of 95% (w / v).

[0016] 7. The method according to Project 1, characterized in that the dosage of the composition to be administered is from 75 mg IgM / kg patient to 1 g IgM / kg patient.

[0017] 8. The method according to item 7, characterized in that the composition is applied at least once a week.

[0018] 9. The method according to Project 1, characterized in that the IgM is recombinant, plasma-derived, cell culture-derived, transgenic, or chemically synthesized.

[0019] 10. The method according to item 9, characterized in that the IgM is plasma-derived IgM isolated from a suitable plasma fraction.

[0020] 11. The method according to item 1, characterized in that the composition containing IgM is applied alone.

[0021] 12. The method according to Project 1, characterized in that the IgM-containing composition is administered together with one or more other compositions or molecules selected from: anti-inflammatory molecules, small molecule antibiotics, molecules that are essentially antimicrobial agents, natural or synthetic peptide antimicrobial agents, proteins with antimicrobial properties, other immunomodulators, or combinations thereof.

[0022] 13. The method according to item 12, wherein the other composition or molecule is vancomycin, meropenem, lactoferrin, or a combination thereof.

[0023] 14. A method for treating an infection in a patient for whom immune regulation is required, characterized in that the method comprises administering a composition comprising IgM.

[0024] 15. The method according to item 14, characterized in that immune modulation is used to treat or prevent infection or infection-related symptoms or conditions.

[0025] 16. The method according to item 14, characterized in that the infection is caused by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Clostridium difficile, Clostridium botulinum, or a combination thereof.

[0026] 17. The method according to Project 14, characterized in that immune regulation is carried out by inhibiting NF-κB induction, by inhibiting the proliferation of peripheral blood mononuclear cells, or a combination thereof.

[0027] 18. The method according to item 14, characterized in that the composition used has an IgM purity of at least 90% (w / v).

[0028] 19. The method according to item 18, characterized in that the composition has 95% (w / v) IgM purity.

[0029] 20. The method according to item 14, characterized in that the dosage of the composition to be administered is from 75 mg IgM / kg patient to 1 g IgM / kg patient.

[0030] 21. The method according to item 20, characterized in that the composition is applied at least once a week.

[0031] 22. The method according to Item 14, characterized in that the IgM is recombinant, plasma-derived, cell culture-derived, transgenic, or chemically synthesized.

[0032] 23. The method according to item 22, characterized in that the IgM is plasma-derived IgM isolated from a suitable plasma fraction.

[0033] 24. The method according to item 14, characterized in that the composition containing IgM is applied alone.

[0034] 25. The method according to item 14, characterized in that the IgM-containing composition is administered together with one or more other compositions or molecules selected from: anti-inflammatory molecules, small molecule antibiotics, molecules that are essentially antimicrobial agents, natural or synthetic peptide antimicrobial agents, proteins with antimicrobial properties, other immunomodulators, or combinations thereof.

[0035] 26. The method according to item 25, wherein the other composition or molecule is vancomycin, meropenem, lactoferrin, or a combination thereof.

[0036] 27. An IgM-containing composition for use in the treatment of sepsis.

[0037] 28. The composition for use according to item 27, characterized in that the sepsis is caused by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Clostridium difficile, Clostridium botulinum, or a combination thereof.

[0038] 29. The composition for use according to item 27, characterized in that the treatment of sepsis is carried out through immune modulation.

[0039] 30. The composition for use according to item 29, characterized in that immune modulation is achieved by inhibiting NF-κB induction, by inhibiting the proliferation of peripheral blood mononuclear cells, or a combination thereof.

[0040] 31. The composition for use according to item 27, characterized in that the composition used has an IgM purity of at least 90% (w / v).

[0041] 32. The composition for use according to item 31, characterized in that the composition has an IgM purity of 95% (w / v).

[0042] 33. The composition for use according to item 27, characterized in that the dose to be administered is from 75 mg IgM / kg patient to 1 g IgM / kg patient.

[0043] 34. The composition for use according to item 33, characterized in that the composition is applied at least once a week.

[0044] 35. The composition for use according to item 27, characterized in that the IgM is recombinant, plasma-derived, cell culture-derived, transgenic, or chemically synthesized.

[0045] 36. The composition for use according to item 35, characterized in that the IgM is plasma-derived IgM isolated from a suitable plasma fraction.

[0046] 37. The composition for use according to item 27, characterized in that the composition containing IgM is applied alone.

[0047] 38. The composition for use according to item 27, characterized in that the IgM-containing composition is administered together with one or more other compositions or molecules selected from: anti-inflammatory molecules, small molecule antibiotics, molecules that are essentially antimicrobial agents, natural or synthetic peptide antimicrobial agents, proteins with antimicrobial properties, other immunomodulators, or combinations thereof.

[0048] 39. The composition for use according to item 38, characterized in that the other composition or molecule is vancomycin, meropenem, lactoferrin or a combination thereof.

[0049] 40. An IgM-containing composition for use in the treatment of immune complications arising from infection.

[0050] 41. The composition for use according to item 40, characterized in that the infection is caused by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Clostridium difficile, Clostridium botulinum, or a combination thereof.

[0051] 42. The composition for use according to item 40, characterized in that the treatment of immune complications is carried out through immune modulation.

[0052] 43. The composition for use according to item 42, characterized in that immune modulation is achieved by inhibiting NF-κB induction, by inhibiting the proliferation of peripheral blood mononuclear cells, or a combination thereof.

[0053] 44. The composition for use according to item 40, characterized in that the composition has an IgM purity of at least 90% (w / v).

[0054] 45. The composition for use according to item 44, characterized in that the composition has an IgM purity of 95% (w / v).

[0055] 46. ​​The composition for use according to item 40, characterized in that the dose to be administered is from 75 mg IgM / kg patient to 1 g IgM / kg patient.

[0056] 47. The composition for use according to item 46, characterized in that the composition is applied at least once a week.

[0057] 48. The composition for use according to item 40, characterized in that the IgM is recombinant, plasma-derived, cell culture-derived, transgenic, or chemically synthesized.

[0058] 49. The composition for use according to item 48, characterized in that the IgM is plasma-derived IgM isolated from a suitable plasma fraction.

[0059] 50. The composition for use according to item 40, characterized in that the composition containing IgM is applied alone.

[0060] 51. The composition for use according to item 40, characterized in that the IgM-containing composition is administered together with one or more other compositions or molecules selected from: anti-inflammatory molecules, small molecule antibiotics, molecules that are essentially antimicrobial agents, natural or synthetic peptide antimicrobial agents, proteins having antimicrobial properties, other immunomodulators, or combinations thereof.

[0061] 52. The composition for use according to item 51, characterized in that the other composition or molecule is vancomycin, meropenem, lactoferrin or a combination thereof. Brief description of the attached diagram For better understanding, the invention is described in more detail below with reference to the accompanying drawings, which are presented by way of example, and to illustrative examples that are not intended to limit the invention.

[0062] Figure 1A and Figure 1B This demonstrates the evaluation of IgM against Escherichia coli (E. coli). Escherichia coli The results were obtained regarding the effect of LPS-induced NF-κB activation in THP-1 cells carrying stable NF-κB reporter granules. Figure 1A and Figure 1B In both figures, the y-axis shows a fold increase in NF-κB activity as measured by the NF-κB SEAP reporter, which is proportional to the induction of NF-κB activity. Furthermore, in both figures ( Figure 1A and Figure 1B In the diagram, multiple processing groups are shown on the x-axis. The standard error is indicated on each bar.

[0063] Figure 2 Clostridium difficile (shown) Clostridium difficile Results obtained by inducing NF-κB with toxoid A or toxoid B in THP-1 cells carrying stable NF-κB reporter particles. The fold increase in NF-κB activity, as measured by the NF-κB SEAP reporter, is indicated on the y-axis and is directly proportional to the induction of NF-κB activity. The concentration of toxoid A or toxoid B, in μg / mL, is shown on the x-axis.

[0064] Figure 3 The results show the effects of inhibiting NF-κB activation induced by Clostridium difficile toxoid B in THP-1 cells carrying stable NF-κB reporter particles. The fold increase in NF-κB activity, as measured by the NF-κB SEAP reporter, is indicated on the y-axis and is proportional to the induction of NF-κB activity. Multiple treatment groups are shown on the x-axis. Standard errors are indicated on the individual bars.

[0065] Figure 4A and Figure 4B It shows inhibition of Klebsiella pneumoniae ( Klebsiella pneumoniae ) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa The results of NF-κB activation and IL-8 secretion induced in THP-1 cells carrying stable NF-κB reporter granules. Figure 4A In the diagram, the y-axis shows the fold increase in NF-κB activity as measured by the NF-κB SEAP reporter, which is proportional to the induction of NF-κB activity; and the x-axis shows multiple treatment groups. Figure 4B In the diagram, the y-axis shows the concentration of IL-8 secreted into the culture supernatant; and the x-axis shows the multiple treatment groups. Figure 4B In the figure, “UD” indicates that IL-8 was undetectable in the untreated control group. In both figures, the standard error is indicated on each bar.

[0066] Figure 5 Results of experiments on the blockade of IgM-mediated proliferation of peripheral blood mononuclear cells (PBMCs), particularly T cells, induced by phorbol 12-myristate 13-acetate (PMA) / ionomycin are shown. The fold increase in relative proliferation compared to the control group, as measured by Cell Titer Glow, is indicated on the y-axis and is proportional to the induction of cell proliferation. Multiple treatment groups are shown on the x-axis. Standard deviations are indicated on the individual bars. Invention Details Following extensive and detailed experiments and studies, the inventors have discovered that the IgM composition exhibits immunomodulatory activity (e.g., by reducing NF-κB activity to induce and / or reduce the proliferation of peripheral blood mononuclear cells), which makes the composition suitable for treating conditions associated with overstimulation of the immune system, such as overstimulation of the immune system associated with sepsis.

[0067] Embodiments of the present invention provide a composition comprising, consisting primarily of, or consisting of plasma-derived IgM and optionally one or more excipients in the form of a drug carrier. According to a particular embodiment, one or more excipients and / or the drug carrier are synthetic, i.e., not naturally occurring.

[0068] As used herein, the term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or medium administered with the plasma-derived IgM of the present invention. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents. Salt solutions and solutions containing hydrated dextran and glycerol can also be used as liquid carriers, particularly for injectable solutions. Depending on the specific embodiment, the pharmaceutically acceptable carrier is synthetic (i.e., the carrier is not naturally occurring).

[0069] Non-limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, glycerol, propylene glycol, water, ethanol, etc. Excipients may also include wetting agents or emulsifiers or pH buffers such as acetates, citrates, or phosphates; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for tonicity adjustment such as sodium chloride or dextrose. According to a particular embodiment, one or more excipients are synthetic (i.e., the excipients are not naturally occurring).

[0070] Embodiments of the present invention also relate to a method for immunomodulating the response to microbial infection independently of an endotoxin-mediated immune response.

[0071] Therefore, in a first embodiment, the present invention provides an IgM-containing composition for use in the treatment of sepsis.

[0072] In a second embodiment, the present invention provides an IgM-containing composition for use in the treatment of immune complications arising from infection.

[0073] In another embodiment, the present invention provides a method for treating immune complications arising from infection in patients who require it, characterized in that the method comprises administering a composition comprising IgM.

[0074] Furthermore, the present invention provides a method for infection in patients requiring immune modulation, characterized in that the method comprises administering a composition comprising IgM.

[0075] As used herein, “hypercytokinemia” and its plural form refer to an immune complication characterized by the production of excessive pro-inflammatory cytokines due to a positive feedback loop between cytokines and leukocytes, including T cells and macrophages.

[0076] As used in this article, "hypercoagulopathy" and its plural form refer to an immune complication caused by the overactivation of blood clotting factors and their products from exogenous and intrinsic pathways of certain blood cell components, such as platelets and microorganisms.

[0077] In one embodiment, the present invention provides an IgM-containing composition for use in the treatment of sepsis. According to a particular embodiment, the composition comprises, is primarily composed of, or consists of plasma-derived IgM and optionally one or more excipients in the form of a drug carrier.

[0078] Sepsis can be caused by one or more bacteria (Gram-positive or Gram-negative) and one or more fungi (e.g., Aspergillus flavus). Aspergillus flavus Sepsis is caused by one or more viruses or combinations thereof. In a preferred embodiment, sepsis is caused by one or more bacteria, more preferably by *Escherichia coli*, *Pseudomonas aeruginosa*, or *Staphylococcus aureus*. Staphylococcus aureus Klebsiella pneumoniae, Streptococcus pneumoniae Streptococcus pneumoniae Clostridium difficile, Clostridium botulinum ( Clostridium botulinum (or a combination thereof)

[0079] Treatment of sepsis or septic conditions is carried out by treating immune complications, such as pro-inflammatory responses or overactivation of the immune system, and subsequently by immunomodulation. This immunomodulation is preferably independent of endotoxin-mediated immune responses and can be applied by a variety of methods. In a preferred embodiment, immunomodulation is carried out by inhibiting NF-κB induction. In another embodiment, immunomodulation is carried out by inhibiting the proliferation of peripheral blood mononuclear cells, preferably T cells. In the most preferred embodiment, immunomodulation is applied by both of the methods mentioned above.

[0080] Preferably, the composition has an IgM purity of at least 90% (w / v), more preferably at least 95% IgM purity. In the most preferred embodiment, the composition has an IgM purity of 95% (w / v).

[0081] The dosage of the composition to be administered is preferably from 75 mg IgM / kg to 1 g IgM / kg, more preferably from 75 mg IgM / kg to 600 mg IgM / kg, and most preferably from 75 mg IgM / kg to 300 mg IgM / kg. Furthermore, the composition is preferably provided or administered at least once a week, every other day, three times a week, or daily.

[0082] In addition, the IgM may be recombinant, plasma-derived, cell culture-derived, transgenic, or chemically synthesized.

[0083] If IgM is recombinant, it can be obtained using any technique known in the fields of protein expression, production, and purification. For example, the IgM nucleic acid sequence can be inserted into any vector suitable for expression in a selected host cell, such as bacteria (Escherichia coli, Bacillus subtilis, etc.). Bacilus subtilis Salmonella typhimurium ( Salmonella typhimurium ), Pseudomonas spp. Pseudomonas Streptomyces ( Streptomyces ) and Staphylococcus spp. Staphylococcus )), yeast (saccharomyces genus ( Saccharomyces ), Pichia pastoris ( Pichia ) or Kluyveromyces ( Kluyveromyces )), insect cells (silkworm ( Bombyx mori ), cabbage moth ( Mamestra brassicae Fall armyworm ( Spodoptera frugiperda ), Powdered Noctuid moth ( Trichoplusia ni ) or fruit fly with black belly ( Drosophila melanogaster () or mammalian cells (HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS-1), human hepatocellular carcinoma cells (e.g., Hep G2), 293 cells transformed with human adenovirus, mouse L-929 cells, HaK hamster cell line, murine 3T3 cells derived from Swiss mice, Balb-c or NIH mice, CV-1 cell line, cell lines derived from in vitro cultures of primary tissues or primary explants).

[0084] Cell culture-derived IgM can be produced by any method or procedure known in the art, such as hybridoma methods.

[0085] Transgenic and chemically synthesized IgM can be produced by any method or procedure known in the art.

[0086] In a preferred embodiment, the IgM is plasma-derived IgM isolated from a suitable plasma fraction, such as the washes from two ANX column procedures following the collection of IgG as described in U.S. Patent 6,307,028, or the fraction further purified by negative selection affinity chromatography or size exclusion chromatography.

[0087] Regarding the plasma-derived IgM, it may optionally be isolated or purified to increase the relative amount of specific antibodies against a given exotoxin, secreted protein, microbial species, or combination thereof.

[0088] According to a specific implementation plan, the composition containing IgM is envisioned to be applied alone.

[0089] In another embodiment, the IgM-containing composition is administered together with one or more other compositions or molecules. The other compositions or molecules are preferably selected from anti-inflammatory molecules, antibiotics (e.g., small molecule antibiotics, molecules that are essentially antimicrobial agents, natural or synthetic peptide antimicrobial agents, and / or proteins with antimicrobial properties), other immunomodulators, or combinations thereof. In a preferred embodiment, the other compositions or molecules are vancomycin, meropenem, lactoferrin, or combinations thereof. Administration of the IgM-containing composition together with one or more other compositions or molecules as mentioned above includes simultaneous administration of all the provided compositions or molecules or one after another.

[0090] Compositions containing IgM can be used to treat sepsis (as mentioned above) and to prevent both the disease.

[0091] In another embodiment, the present invention discloses an IgM-containing composition for use in the treatment of immune complications arising from infection.

[0092] The infection may be caused by one or more bacteria (Gram-positive or Gram-negative), one or more fungi (e.g., Aspergillus flavus), one or more viruses, or combinations thereof. In a preferred embodiment, the infection is caused by one or more bacteria, more preferably by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Clostridium difficile, Clostridium botulinum, or combinations thereof.

[0093] Treatment of immune complications, such as pro-inflammatory responses or overactivation of the immune system, is achieved through immunomodulation. This immunomodulation is preferably independent of endotoxin-mediated immune responses and can be applied by a variety of methods. In a preferred embodiment, immunomodulation is achieved by inhibiting NF-κB induction. In another embodiment, immunomodulation is achieved by inhibiting the proliferation of peripheral blood mononuclear cells, preferably T cells. In the most preferred embodiment, immunomodulation is applied by both of the methods mentioned above.

[0094] The composition preferably has an IgM purity of at least 90% (w / v), more preferably at least 95% (w / v). In the most preferred embodiment, the composition used comprises 95% (w / v) IgM purity.

[0095] The dosage of the composition to be administered is preferably from 75 mg IgM / kg to 1 g IgM / kg, more preferably from 75 mg IgM / kg to 600 mg IgM / kg, and most preferably from 75 mg IgM / kg to 300 mg IgM / kg. Furthermore, the composition is preferably provided or administered at least once a week, every other day, three times a week, or daily.

[0096] In addition, the IgM may be recombinant, plasma-derived, cell culture-derived, transgenic, or chemically synthesized.

[0097] If the IgM is recombinant, it can be obtained using any technique known in the field of protein expression, production, and purification. For example, the IgM nucleic acid sequence can be inserted into any vector suitable for expression in a selected host cell, such as bacteria (Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, and Staphylococcus), yeast (Saccharomyces, Pichia pastoris, or Kluyveromyces), insect cells (Bombyx mori, Cabbage looper, Fall armyworm, White spot moth, or Drosophila melanogaster), or mammalian cells (HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS-1), human hepatocellular carcinoma cells (e.g., Hep G2), 293 cells transformed with human adenovirus, mouse L-929 cells, HaK hamster cell line, mouse 3T3 cells derived from Swiss mice, Balb-c or NIH mice, CV-1 cell line, cell lines derived from in vitro cultures of primary tissues or primary explants).

[0098] Cell culture-derived IgM can be produced by any method or procedure known in the art, such as hybridoma methods.

[0099] Transgenic and chemically synthesized IgM can be produced by any method or procedure known in the art.

[0100] In a preferred embodiment, the IgM is plasma-derived IgM isolated from a suitable plasma fraction, such as the washes from two ANX column procedures following the collection of IgG as described in U.S. Patent 6,307,028, or the fraction further purified by negative-selective affinity chromatography or size exclusion chromatography.

[0101] In another preferred embodiment, the immune complications resulting from the infection are hypercytokineemia, hypercoagulability, or organ failure.

[0102] Regarding the plasma-derived IgM, it may optionally be isolated or purified to increase the relative amount of specific antibodies against a given exotoxin, secreted protein, microbial species, or combination thereof.

[0103] According to a specific implementation plan, the composition containing IgM is envisioned to be applied alone.

[0104] In another embodiment, the IgM-containing composition is administered together with one or more other compositions or molecules. The other compositions or molecules are preferably selected from anti-inflammatory molecules, antibiotics (e.g., small molecule antibiotics, molecules that are essentially antimicrobial agents, natural or synthetic peptide antimicrobial agents, and / or proteins with antimicrobial properties), other immunomodulators, or combinations thereof. In a preferred embodiment, the other compositions or molecules are vancomycin, meropenem, lactoferrin, or combinations thereof. Administration of the IgM-containing composition together with one or more other compositions or molecules as mentioned above includes simultaneous administration of all the provided compositions or molecules or one after another.

[0105] Compositions containing IgM can be used to treat immune complications arising from infection (as mentioned above) and to prevent both.

[0106] In another embodiment, the present invention relates to a method for treating immune complications arising from infection in patients who require it, characterized in that the method comprises administering a composition comprising IgM.

[0107] The infection may be caused by one or more bacteria (Gram-positive or Gram-negative), one or more fungi (e.g., Aspergillus flavus), one or more viruses, or combinations thereof. In a preferred embodiment, the infection is caused by one or more bacteria, more preferably by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Clostridium difficile, Clostridium botulinum, or combinations thereof.

[0108] Treatment of immune complications, such as pro-inflammatory responses or overactivation of the immune system, is achieved through immunomodulation. This immunomodulation is preferably independent of endotoxin-mediated immune responses and can be applied by a variety of methods. In a preferred embodiment, immunomodulation is achieved by inhibiting NF-κB induction. In another embodiment, immunomodulation is achieved by inhibiting the proliferation of peripheral blood mononuclear cells, preferably T cells. In the most preferred embodiment, immunomodulation is applied by both of the methods mentioned above.

[0109] The composition preferably has an IgM purity of at least 90% (w / v), more preferably at least 95% (w / v). In the most preferred embodiment, the composition used comprises 95% (w / v) IgM purity.

[0110] The dosage of the composition to be administered is preferably from 75 mg IgM / kg to 1 g IgM / kg, more preferably from 75 mg IgM / kg to 600 mg IgM / kg, and most preferably from 75 mg IgM / kg to 300 mg IgM / kg. Furthermore, the composition is preferably provided or administered at least once a week, every other day, three times a week, or daily.

[0111] In addition, the IgM may be recombinant, plasma-derived, cell culture-derived, transgenic, or chemically synthesized.

[0112] If the IgM is recombinant, it can be obtained using any technique known in the field of protein expression, production, and purification. For example, the IgM nucleic acid sequence can be inserted into any vector suitable for expression in a selected host cell, such as bacteria (Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, and Staphylococcus), yeast (Saccharomyces, Pichia pastoris, or Kluyveromyces), insect cells (Bombyx mori, Cabbage looper, Fall armyworm, White spot moth, or Drosophila melanogaster), or mammalian cells (HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS-1), human hepatocellular carcinoma cells (e.g., Hep G2), 293 cells transformed with human adenovirus, mouse L-929 cells, HaK hamster cell line, mouse 3T3 cells derived from Swiss mice, Balb-c or NIH mice, CV-1 cell line, cell lines derived from in vitro cultures of primary tissues or primary explants).

[0113] Cell culture-derived IgM can be produced by any method or procedure known in the art, such as hybridoma methods.

[0114] Transgenic and chemically synthesized IgM can be produced by any method or procedure known in the art.

[0115] In a preferred embodiment, the IgM is plasma-derived IgM isolated from a suitable plasma fraction, such as washes from two ANX column procedures following the collection of IgG as described in U.S. Patent 6,307,028, or the fraction further purified by negative selection affinity chromatography or size exclusion chromatography.

[0116] Regarding the plasma-derived IgM, it may optionally be isolated or purified to increase the relative amount of specific antibodies against a given exotoxin, secreted protein, microbial species, or combination thereof.

[0117] According to a specific implementation plan, the composition containing IgM is envisioned to be applied alone.

[0118] In another embodiment, the IgM-containing composition is administered together with one or more other compositions or molecules. The other compositions or molecules are preferably selected from anti-inflammatory molecules, antibiotics (e.g., small molecule antibiotics, molecules that are essentially antimicrobial agents, natural or synthetic peptide antimicrobial agents, and / or proteins with antimicrobial properties), other immunomodulators, or combinations thereof. In a preferred embodiment, the other compositions or molecules are vancomycin, meropenem, lactoferrin, or combinations thereof. Administration of the IgM-containing composition together with one or more other compositions or molecules as mentioned above includes simultaneous administration of all the provided compositions or molecules or one after another.

[0119] The method of the present invention can be used to treat immune complications arising from infection (as mentioned above) and to prevent both said immune complications.

[0120] Finally, the present invention also discloses a method for infection in patients requiring immune modulation, characterized in that the method comprises administering a composition containing IgM.

[0121] The immune modulation can be used to treat or prevent infection or infection-related symptoms or conditions.

[0122] The infection may be caused by one or more bacteria (Gram-positive or Gram-negative), one or more fungi (e.g., Aspergillus flavus), one or more viruses, or combinations thereof. In a preferred embodiment, the infection is caused by one or more bacteria, more preferably by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Clostridium difficile, Clostridium botulinum, or combinations thereof.

[0123] The immunomodulation allows for the treatment of immune complications, such as pro-inflammatory responses or overactivation of the immune system, and is preferably independent of endotoxin-mediated immune responses, and can be applied by a variety of methods. In a preferred embodiment, immunomodulation is performed by inhibiting NF-κB induction. In another embodiment, immunomodulation is performed by inhibiting the proliferation of peripheral blood mononuclear cells, preferably T cells. In the most preferred embodiment, immunomodulation is applied by both of the methods mentioned above.

[0124] The composition preferably has an IgM purity of at least 90% (w / v), more preferably at least 95% (w / v). In the most preferred embodiment, the composition used comprises 95% (w / v) IgM purity.

[0125] The dosage of the composition to be administered is preferably from 75 mg IgM / kg to 1 g IgM / kg, more preferably from 75 mg IgM / kg to 600 mg IgM / kg, and most preferably from 75 mg IgM / kg to 300 mg IgM / kg. Furthermore, the composition is preferably provided or administered at least once a week, every other day, three times a week, or daily.

[0126] In addition, the IgM may be recombinant, plasma-derived, cell culture-derived, transgenic, or chemically synthesized.

[0127] If the IgM is recombinant, it can be obtained using any technique known in the field of protein expression, production, and purification. For example, the IgM nucleic acid sequence can be inserted into any vector suitable for expression in a selected host cell, such as bacteria (Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, and Staphylococcus), yeast (Saccharomyces, Pichia pastoris, or Kluyveromyces), insect cells (Bombyx mori, Cabbage looper, Fall armyworm, White spot moth, or Drosophila melanogaster), or mammalian cells (HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS-1), human hepatocellular carcinoma cells (e.g., Hep G2), 293 cells transformed with human adenovirus, mouse L-929 cells, HaK hamster cell line, mouse 3T3 cells derived from Swiss mice, Balb-c or NIH mice, CV-1 cell line, cell lines derived from in vitro cultures of primary tissues or primary explants).

[0128] Cell culture-derived IgM can be produced by any method or procedure known in the art, such as hybridoma methods.

[0129] Transgenic and chemically synthesized IgM can be produced by any method or procedure known in the art.

[0130] In a preferred embodiment, the IgM is plasma-derived IgM isolated from a suitable plasma fraction, such as the washes from two ANX column procedures following the collection of IgG as described in U.S. Patent 6,307,028, or the fraction further purified by negative-selective affinity chromatography or size exclusion chromatography.

[0131] Regarding the plasma-derived IgM, it may optionally be isolated or purified to increase the relative amount of specific antibodies against a given exotoxin, secreted protein, microbial species, or combination thereof.

[0132] According to a specific implementation plan, the composition containing IgM is envisioned to be applied alone.

[0133] In another embodiment, the IgM-containing composition is administered together with one or more other compositions or molecules. The other compositions or molecules are preferably selected from anti-inflammatory molecules, antibiotics (e.g., small molecule antibiotics, molecules that are essentially antimicrobial agents, natural or synthetic peptide antimicrobial agents, and / or proteins with antimicrobial properties), other immunomodulators, or combinations thereof. In a preferred embodiment, the other compositions or molecules are vancomycin, meropenem, lactoferrin, or combinations thereof. Administration of the IgM-containing composition together with one or more other compositions or molecules as mentioned above includes simultaneous administration of all the provided compositions or molecules or one after another.

[0134] The embodiments described herein are intended to be exemplary of the invention and not to limit it. Those skilled in the art will understand that modifications can be made to the embodiments and examples of this disclosure without departing from the scope of this disclosure.

[0135] The term "comprising" and its variations are used above to describe embodiments of the invention. However, it is intended by the inventors that in any embodiment described herein, the term "comprising" may be replaced by "consisting of" and "consisting essentially of" without departing from the scope of the invention. Unless otherwise specified, all values ​​provided herein include and include up to and all given start and end points.

[0136] The following examples further illustrate the implementation of the present invention and should be understood as illustrative rather than limiting the invention.

[0137] Example Example 1. Analysis of the effect of IgM on NF-κB activity induced by lipopolysaccharide (LPS) endotoxin of Escherichia coli.

[0138] As a method for monitoring the activation of the NF-κB signaling pathway, the experiment was performed using the human THP-1 monocyte cell line carrying a stable plasmid (Invivogen, San Diego, CA USA) with an NF-κB-dependent promoter that drives a secretory alkaline phosphatase (SEAP) reporter.

[0139] Culture the cells in proliferation medium according to the manufacturer's recommendations.

[0140] This experiment used both LPS derived from *E. coli* strain O111:B4 and LPS derived from *E. coli* strain K12. The THP-1 cells mentioned above were treated with 100 ng / mL of *E. coli* LPS derived from strain O111:B4 (see [link to THP-1 cell analysis]). Figure 1A ) or 1 ng / mL of Escherichia coli LPS derived from strain K12 (see Figure 1B Processing time is 24 hours.

[0141] When using IgM, LPS is pre-incubated with purified IgM (95% (w / v) or higher purity) before cell treatment.

[0142] For NF-κB activity assay, 1x10 5 Cells were seeded in each well of a 96-well plate. The assay medium was the same as the proliferation medium, except that the assay medium contained reduced serum to reduce the non-specific effects of proteins derived from fetal bovine serum (FBS). Cells were treated with IgM (at least 95% (w / v) pure) to a final concentration of 2.5 mg / mL as described above.

[0143] NF-κB activity was determined by evaluating the level of secreted alkaline phosphatase (SEAP) in the supernatant, which was measured using a Quanti-Blue assay (Invivogen, San Diego, CA, USA) according to the manufacturer's instructions. The relative cell number at the time of NF-κB assay was determined using a Cell Titer Glow assay (PromegaCorp. Madison, WI, USA) according to the manufacturer's instructions. Figure 1A and Figure 1BThe relative NF-κB activity shown was calculated as follows: the NF-κB assay value was normalized to the relative cell number determined by the Cell Titer Glow assay, and then the experimental control was normalized to a value of 1.

[0144] Surprisingly, it was observed that, in all the tests and mentioned above, purified IgM had no significant effect on the activation of LPS-mediated NF-κB signaling (see [link to relevant documentation]). Figure 1A and Figure 1B ).

[0145] Example 2. Analysis of the effect of IgM on NF-κB activity induced by Clostridium difficile toxoid B.

[0146] It is known that Clostridium difficile toxins induce cytokine secretion in monocytes, including IL-8 and TNF-α (Linevsky, JK., et al., "IL-8 release and neutrophil activation by Clostridium difficile toxin-exposed human monocytes," Am J Physiol Gastrointest Liver Physiol, 1997, 273, G1333-G1340; and Sun, X., et al., "Essential role of the glucose transferase activity in..."). Clostridium difficile (Toxin-induced secretion of TNF-α by macrophages, Microbial Pathogenesis, 2009, 16, 298-305). It is also known that each of these cytokines is induced upon NF-κB activation.

[0147] Therefore, it was tested whether Clostridium difficile toxoid B could induce NF-κB signaling in human THP-1 cells carrying stably integrated NF-κB reporter plasmids (Invivogen, San Diego, CA USA), as mentioned above. Cells were cultured in proliferation medium according to the manufacturer's recommendations.

[0148] Because the toxin itself is cytotoxic, inactivated toxoids are used to avoid complex interpretation of results. For NF-κB activity assays, 2 x 10⁻⁶ toxoids are prepared before treatment. 5Cells were seeded in each well of a 96-well plate for 24 hours. The cells were then treated with several concentrations of Clostridium difficile toxoid A (List Biological Labs, Inc., Campbell, CA USA) or toxoid B (List Biological Labs, Inc., Campbell, CA USA). NF-κB activity was determined by evaluating the level of secretory alkaline phosphatase (SEAP) in the supernatant, measured using a Quanti-Blue assay (Invivogen, San Diego, CA USA) according to the manufacturer's instructions. The relative cell number at the time of NF-κB assay was determined using a Cell Titer Glow assay (Promega Corp. Madison, WI, USA) according to the manufacturer's instructions. Figure 2 The relative NF-κB activity shown was calculated as follows: the NF-κB assay value was normalized to the relative cell number determined by the Cell Titer Glow assay, and then the experimental control was normalized to a value of 1.

[0149] Although toxoid A does not induce NF-κB signaling in these cells, toxoid B does induce NF-κB reporter signaling in these cells (see [link to original text]). Figure 2 ).

[0150] The ability of a solution of BSA (bovine serum albumin) to inhibit NF-κB activity was evaluated compared to that of a control IgM solution (90%-95% pure IgM).

[0151] Cells were treated with 9.25 μg / mL Clostridium difficile toxoid B for 24 hours. Alternatively, in cases of treatment with a BSA or IgM combination, toxoid B was pre-incubated with either the BSA or a solution of IgM (90%–95% w / v purity) before cell treatment.

[0152] The assay medium was the same as the proliferation medium, except that the assay medium contained 0.025 µM vitamin D3. For the NF-κB activity assay, 2 x 10⁻⁶ ppm of vitamin D3 was added before treatment. 5Cells were seeded in each well of a 96-well plate for 24 hours. Cells were then treated with either 3 mg / mL IgM (90%–95% w / v IgM purity) or BSA, as described above. NF-κB activity was determined by evaluating the level of secretory alkaline phosphatase (SEAP) in the supernatant, measured using a Quanti-Blue assay (Invivogen, San Diego, CA, USA) according to the manufacturer's instructions. The relative cell number at the time of NF-κB assay was determined using a Cell Titer Glow assay (Promega Corp. Madison, WI, USA) according to the manufacturer's instructions. Figure 3 The relative NF-κB activity shown was calculated as follows: the NF-κB assay value was normalized to the relative cell number determined by the Cell Titer Glow assay, and then the experimental control was normalized to a value of 1.

[0153] BSA has no effect on the NF-κB activation of toxoid B, while the aforementioned IgM solution weakens the effect of toxoid B (see...). Figure 3 ).

[0154] Example 3. Analysis of the effects of IgM on NF-κB activity induction and IL-8 secretion induced by Klebsiella pneumoniae and Pseudomonas aeruginosa.

[0155] Since IgM cannot attenuate NF-κB signaling mediated by E. coli cell wall endotoxin LPS (see Example 1), the effects of purified IgM on undamaged intact bacteria (with cell wall components such as LPS) were evaluated.

[0156] Treatment with live microbial cultures can overtake and interfere with the results of mammalian cell culture assays. Therefore, intact bacteria (Klebsiella pneumoniae and Pseudomonas aeruginosa) are fixed in formaldehyde to kill the bacteria while preserving cell integrity.

[0157] THP-1 cells carrying stable NF-κB reporter particles, as mentioned above, were treated with formaldehyde-killed Klebsiella pneumoniae and Pseudomonas aeruginosa for 24 hours.

[0158] When using IgM, the aforementioned microorganisms are co-treated with purified IgM during the treatment process.

[0159] For NF-κB activity assay, 1x10 5Cells were seeded in each well of a 96-well plate. The assay medium was the same as the proliferation medium, except that the assay medium contained reduced serum to reduce the non-specific effects of FBS-derived proteins. Cells were treated with a final concentration of 2.5 mg / mL IgM (at least 95% pure IgM) as described in the accompanying illustration. NF-κB activity was determined by evaluating the level of secreted alkaline phosphatase (SEAP) in the supernatant, which was measured using a Quanti-Blue assay (Invivogen, San Diego, CA, USA) according to the manufacturer's instructions. The relative cell number at the time of NF-κB assay was determined using a Cell Titer Glow assay (Promega Corp. Madison, WI, USA) according to the manufacturer's instructions. The relative NF-κB activity shown was calculated by normalizing the NF-κB assay value against the relative cell number determined by the Cell Titer Glow assay, and then normalizing the experimental controls to a value of 1.

[0160] Both types of formaldehyde-treated bacteria were able to effectively induce the NF-κB pathway in the cell systems or models used in Examples 1 and 2 (i.e., human THP-1 cells carrying stably integrated NF-κB reporter particles) (see Example 1). Figure 4A ).

[0161] Surprisingly, co-treatment with either formaldehyde-treated Klebsiella pneumoniae or Pseudomonas aeruginosa and purified IgM showed a reduction in NF-κB signaling.

[0162] Activation of NF-κB signaling induces the secretion of pro-inflammatory cytokines, a fact well-known in the art. IL-8 is an NF-κB-responsive pro-inflammatory cytokine well-characterized in humans. Therefore, IL-8 secretion in the culture supernatant of the aforementioned cell cultures was evaluated to observe whether this IL-8 secretion also responded to bacteria (Klebsiella pneumoniae or Pseudomonas aeruginosa) and IgM (see [link to relevant documentation]). Figure 4B The IL-8 cytokine secreted into the supernatant was measured using the AlphaLISA IL-8 Immunoassay Kit (Perkin-Elmer, Waltham, MA USA) according to the manufacturer's operating protocol.

[0163] Similar to NF-κB activation, formaldehyde-fixed bacteria alone induce IL-8 secretion, while co-treatment with the bacteria and IgM showed reduced IL-8 secretion compared to cultures treated alone (see [link to original text]). Figure 4B ).

[0164] Example 4. Analysis of the effect of IgM on the proliferation of peripheral blood mononuclear cells.

[0165] Because IgM exhibits significant effects on the NF-κB pro-inflammatory pathway, the question of whether purified IgM has a more general effect on immune function was also evaluated. The proliferation of blood lymphocytes, particularly T cells, stimulated by antigens is one of the most fundamental pro-inflammatory immune responses.

[0166] Phorbolol 12-myristate 13-acetate (PMA) and iomycin (a very common proliferation stimulant) were used to induce the proliferation of peripheral blood mononuclear cells, or more precisely, blood T-lymphocytes or T cells.

[0167] Human peripheral blood mononuclear cells were cultured in RPMI containing 10% heat-inactivated human serum. For proliferation assays, 3 x 10⁶ cells were cultured in the medium. 5 One cell was seeded in each well of a 96-well plate.

[0168] T cells were treated with 10 ng / mL PMA and 0.5 μM iomycin for 3 days. When using IgM, cells were treated or co-treated with purified IgM (at a final concentration of about 5 mg / mL and a purity of at least 95%), depending on the situation.

[0169] Cell titers for different groups were measured using a Cell Titer Glow (Promega Corp. Madison, WI, USA) (according to the manufacturer’s instructions) and based on measurements of induction of cell proliferation as a function of ATP content.

[0170] A significant increase in proliferation was observed in peripheral blood mononuclear cells 3 days after PMA / ionomycin stimulation (see [link]). Figure 5 Compared to untreated control cells and compared to PMA / ionomycin treatment, IgM treatment reduced the proliferation of peripheral blood mononuclear cells in the presence or absence of PMA / ionomycin. This observation is highly surprising given that PMA / ionomycin is a very potent and extremely nonspecific mitogen. This suggests that IgM itself plays a regulatory role in the immune response.

Claims

1. A method for treating immune complications arising from infection in patients who require it, characterized in that... The method includes applying a composition containing IgM.

2. The method according to claim 1, characterized in that, The infection was caused by Escherichia coli (E. coli) Escherichia coli ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa Staphylococcus aureus Staphylococcus aureus Klebsiella pneumoniae ( Klebsiella pneumoniae Streptococcus pneumoniae () Streptococcus pneumoniae Clostridium difficile ( Clostridium difficile ), Clostridium botulinum ( Clostridium botulinum (or a combination thereof) 3. The method according to claim 1, characterized in that, Treatment of immune complications is achieved through immune modulation.

4. The method according to claim 3, characterized in that, Immunomodulation occurs by inhibiting NF-κB induction, by inhibiting the proliferation of peripheral blood mononuclear cells, or a combination thereof.

5. The method according to claim 1, characterized in that, The composition used has an IgM purity of at least 90% (w / v).

6. The method according to claim 5, characterized in that, The composition has an IgM purity of 95% (w / v).

7. The method according to claim 1, characterized in that, The dosage of the composition to be administered is from 75 mg IgM / kg to 1 g IgM / kg.

8. The method according to claim 7, characterized in that, The composition is applied at least once a week.

9. The method according to claim 1, characterized in that, IgM can be recombinant, plasma-derived, cell culture-derived, genetically modified, or chemically synthesized.

10. The method according to claim 9, characterized in that, IgM is plasma-derived IgM isolated from appropriate plasma fractions.

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