NATURAL ANTIBODIES IN PROPHYLAXIS AND THERAPY
20230295281 · 2023-09-21
Inventors
Cpc classification
A61P31/00
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K2039/58
HUMAN NECESSITIES
A61P9/10
HUMAN NECESSITIES
C07K16/00
CHEMISTRY; METALLURGY
A61K2039/507
HUMAN NECESSITIES
C07K2317/24
CHEMISTRY; METALLURGY
A61P25/28
HUMAN NECESSITIES
International classification
A61K45/06
HUMAN NECESSITIES
A61K39/395
HUMAN NECESSITIES
Abstract
Described is a human or humanized natural IgM and/or IgA antibody recognizing oxidized phospholipids and/or oxidation-specific epitopes for use in a method of treating or preventing a disorder or a disease associated with/related to/caused by a natural IgM/IgA antibody deficiency (NAD) in a subject. Moreover, described is a human or humanized natural IgM and/or IgA antibody recognizing oxidized phospholipids and/or oxidation-specific epitopes for use in a method of treating or preventing a disorder or a disease associated with/related to/caused by a natural IgM/IgA antibody deficiency (NAD) in a subject, wherein said natural IgM and/or IgA is derived from IgM and/or IgA enriched plasma pools from healthy individuals. Further, described is a human or humanized natural IgM and/or IgA antibody recognizing oxidized phospholipids and/or oxidation-specific epitopes for use in a method of treating or preventing a disorder or a disease associated with/related to/caused by a natural IgM/IgA antibody deficiency (NAD) in a subject, wherein said antibody is a recombinant human monoclonal natural IgM antibody. Moreover, described is a vaccine comprising a compound that induces the generation of natural IgM and/or IgA antibodies for use in a method of reducing or preventing the clinical signs or disease associated with/related to/caused by natural IgM/IgA antibody deficiency (NAD) in a subject, wherein said vaccine comprises a pharmaceutically acceptable carrier or excipient. Further, described is such a vaccine for use in a method of reducing or preventing the clinical signs or disease associated with/related to/caused by natural IgM/IgA antibody deficiency (NAD) in a subject, wherein said compound induces human natural IgM and/or IgA antibody recognizing oxidized phospholipids and/or oxidation-specific epitopes.
Claims
1. A method of treating or preventing a disorder or disease associated with/related to/caused by a natural IgM/IgA antibody deficiency (NAD) in a subject, comprising administration of a composition comprising an effective amount of at least one human or humanized natural IgM and/or IgA antibody recognizing oxidized phospholipids and/or oxidation-specific epitopes, wherein said disorder or disease is selected from the group consisting of a natural antibody deficient infectious disease, inflammatory disease, neurodegenerative disease, metabolic disease, autoimmune disease, and cardiovascular disease, wherein each said human natural IgM and/or IgA antibody is from a subgroup of the total IgM and/or IgA repertoire of at least one source, said subgroup essentially consisting of antibodies recognizing oxidized phospholipids and/or oxidation-specific epitopes, and wherein said composition contains more than about 35% said human or humanized natural IgM and/or IgA antibody recognizing oxidized phospholipids and/or oxidation-specific epitopes.
2. (canceled)
3. (canceled)
4. The method of claim 1, wherein said source is at least one healthy individual and said subgroup of IgM and/or IgA antibodies is derived from IgM and/or IgA enriched plasma pools from said at least one healthy individuals, said subgroup essentially consisting of antibodies recognizing oxidized phospholipids and/or oxidation-specific epitopes.
5. (canceled)
6. The method of claim 1, wherein said disorder or a disease associated with/related to/caused by natural IgM/IgA antibody deficiency (NAD) is the virus infection disease COVID-19 caused by the β-Coronavirus SARS-CoV2 or is long COVID-19.
7. The method of claim 1, wherein said human or humanized natural IgM and/or IgA antibody is capable of inhibiting the cell-to-cell spread of a virus from an infected cell to an adjacent non-infected cell.
8. The method of claim 1, wherein said human or humanized natural IgM and/or IgA antibody has an anti-inflammatory activity.
9. The method of claim 1, wherein said disorder or disease associated with natural IgM/IgA antibody deficiency (NAD) is an inflammatory disease or a virus infection disease.
10. The method of claim 9, wherein said inflammatory disease is selected from at least one of the group consisting of infectious diseases mediated by respiratory viruses, infectious diseases caused by bacterial infections mediated by gram positive or gram negative pathogens, infectious diseases caused by fungi, infectious diseases caused by parasites, sterile inflammatory diseases, metabolic disorders, neurodegenerative diseases, and autoimmune diseases.
11. The method of claim 9, wherein said virus infection disease is selected from the group consisting of infections by coronaviruses, influenza viruses, parainfluenza viruses, respiratory syncytial viruses (RSV), rhinoviruses, adenoviruses, enteroviruses, human metapneumoviruses, and herpesviruses.
12. The method of claim 1, wherein said composition is a pharmaceutical composition comprising said effective amount of said human or humanized natural IgM and/or IgA antibody recognizing oxidized phospholipids and/or oxidation-specific epitopes and at least one pharmaceutically acceptable excipient.
13. (canceled)
14. (canceled)
15. The method of claim 1, wherein said human or humanized natural IgM and/or IgA antibody recognizes and binds to at least one of phosphorylcholine exposed by oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine, to oxidized cardiolipin, to oxidized phosphatidylserine, malondialdehyde-modified proteins, 4-hydroxynonenal-modified proteins, 2-(ω-carboxyethyl)-pyrrole-modified proteins, and oligomeric amyloid-β peptide.
16. The method of claim 1, wherein said composition comprises at least one human or humanized natural IgM and/or IgA antibody recognizing oxidized phospholipids and/or oxidation-specific epitopes wherein said antibody comprises the complementarity determining regions V.sub.HCDR1 comprising SEQ ID NO: 1, V.sub.HCDR2 comprising SEQ ID NO: 2, V.sub.HCDR3 comprising SEQ ID NO: 3, V.sub.LCDR1 comprising SEQ ID NO: 4, V.sub.LCDR2 comprising SEQ ID NO: 5, and V.sub.LCDR3 comprising SEQ ID NO:6, wherein said antibody recognizes and binds to at least one of phosphorylcholine exposed by oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine, to oxidized cardiolipin, to oxidized phosphatidylserine, malondialdehyde-modified proteins, 4-hydroxynonenal-modified proteins, 2-(ω-carboxyethyl)-pyrrole-modified proteins, and oligomeric amyloid-β peptide; or wherein said antibody comprises the complementarity determining regions V.sub.HCDR1 comprising SEQ ID NO: 9, V.sub.HCDR2 comprising SEQ ID NO: 10, V.sub.HCDR3 comprising SEQ ID NO: 11, V.sub.LCDR1 comprising SEQ ID NO: 12, V.sub.LCDR2 comprising SEQ ID NO: 13, and V.sub.LCDR3 comprising SEQ ID NO:14, wherein said antibody recognizes and binds to at least one of phosphorylcholine exposed by oxidized phosphatidylcholine and/or oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine, oxidized cardiolipin, oxidized phosphatidylserine, malondialdehyde-modified proteins, 4-hydroxynonenal-modified proteins, 2-(ω-carboxyethyl)-pyrrole-modified proteins, and oligomeric amyloid-β peptide.
17-28. (canceled)
29. The method of claim 1, wherein said at least one human or humanized natural IgM and/or IgA antibody recognizing oxidized phospholipids and/or oxidation-specific epitopes wherein said is administered in combination with at least one of: (a) at least one of an inhibitor/antagonist of the Angiotensin-Converting-Enzyme (ACE), an inhibitor/antagonist of the Angiotensin-II-type 1 receptor (AT1R), a compound that modulates the expression of the ACE2 receptor, Ang(1-7), AT2R agonists, and MAS-receptor agonists, (b) at least one of a compound inhibiting/antagonizing/neutralizing ligands of Receptor of Advanced Glycation Endproducts (RAGE), and an inhibitor/antagonist of RAGE, and (c) at least one of Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and/of a compound that increases the phagocytic activity of alveolar macrophages (AM), optionally azithromycin.
30. The method of claim 1, wherein said at least one human or humanized natural IgM and/or IgA antibody recognizing oxidized phospholipids and/or oxidation-specific epitopes wherein said is administered in combination with an antiviral compound.
31. The method of claim 1, wherein said at least one human or humanized natural IgM and/or IgA antibody recognizing oxidized phospholipids and/or oxidation-specific epitopes wherein said is administered in combination with an immunomodulator.
32-39. (canceled)
40. The method of claim 8, wherein said anti-inflammatory activity is selected from at least one of reducing the accumulation of free oxidized phospholipids, reducing the accumulation of free oxidized phospholipids in infected lungs, clearing cellular debris in lung tissue, stimulating IL-10 and/or TGFβ secretion, and neutralizing of pro-inflammatory immune responses triggered by cytokines.
41. The method of 10, wherein said infectious diseases mediated by respiratory viruses comprise at least one disease selected from COVID19, long COVID-19, influenza, MERS-COV and SARS-COV, said sterile inflammatory diseases comprise at least one disease selected from cardiovascular diseases, atherosclerosis, coronary heart disease, heart attack, and stroke, said metabolic disorders comprise diabetes mellitus, said neurodegenerative diseases comprise Alzheimer's Disease, and said autoimmune diseases comprise at least one disease selected from Systemic Lupus Erythematodes and Multiple Sclerosis.
42. The method of claim 11, wherein said coronaviruses are selected from at least one of SARS-CoV, SARS-CoV-2, and MERS, and said herpesviruses are selected from at least one of HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6, HHV-7, and HHV-8.
43. The method of claim 30, wherein said antiviral compound is selected from at least one of remdesivir, favipiravir, camostat mesylate, nafamostat mesylate, umifenovir, and stronger neo-minophagen C.
44. The method of claim 31, wherein said immunomodulator is selected from at least one of anti-PD-1, anti-PD-L1, anti-CD40 (agonist), CD40-Ligand, anti-GM-CSF, anti-CSF-1R, anti-CTLA-4, an antibody that binds to at least one cytokine, an antibody that binds to IL-6, an antibody that binds to an IL-6-specific receptor, anti-IL-6R, an antibody that binds to at least one chemokine, anti-CCL2, anti-CCL5, an antibody that binds to a specific chemokine receptor, anti-CCR2, antiCCR5, synthetic molecules that bind to chemokine receptors, Maraviroc CCR5 receptor inhibitor, and dexamethason.
Description
[0508] Other aspects and advantages of the invention will be described in the following examples, which are given for purposes of illustration and not by way of limitation. Each publication, patent, patent application or other document cited in this application is hereby incorporated by reference in its entirety.
[0509]
[0510] Five patients presenting with deteriorating COVID-19 pneumonia were treated with Pentaglobin®“P” indicates days on which patients were treated with Pentaglobin® (closed circles). Clinical parameters IL-6 (A), CRP (B), PCT (C) or mean daily blood pCO.sub.2 (error bars are standard deviations) (D) were measured over the time. Presence (+) or absence (−) of SARS-CoV-2 in bronchoalveolar lavages (BAL) was monitored.
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[0512] Illustration of mechanisms leading to the formation of ROS and the accumulation of oxPL and OSE (
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[0514] HD: Healthy donor serum; COV: COVID-19 serum; Positive Control (provided in Kallestad HEp2 Kit).
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EXAMPLES
Example 1: Demonstration of the Binding of Generated nIgMs to Oxidized Lipids
[0525] Commercially available ELISAs (e.g. MDA-BSA coated plate ELISA (DEIACP15) or anti ox-LDL ELISA (DEIA081J) from Creative Diagnostics) are used to evaluate the binding of generated OSE-specific IgM and/or IgA antibodies to oxidized phospholipids. Avanti Lipid Snoopers® ELISA test strips coated with oxidized phosphatidycholine are used to analyze binding of IgM and/or IgA antibodies. IgM and/or IgA antibodies against oxidized cardiolipin or oxidized phosphatidylserine are measured using an ELISA method described in (Frostegard, Su et al., 2014, PLoS One, Vol. 9 (12)).
[0526] The binding to oxidized lipids is further investigated by inducing cell-apoptosis in cultured cells and staining with the generated OSE-specific IgM and/or IgA antibodies, Pentaglobin® or sera from BCG/Pneumovax-vaccinated individuals. Flow cytometry analysis is used to specifically look at apoptotic cells (e.g. marker annexin V) and binding of nIgMs is monitored by appropriate commercially available anti-IgM and anti-IgA secondary antibodies.
Example 2: Demonstration of the Binding of OSE-Specific IgM and/or IgA Antibodies to Virus Infected Cells
[0527] Virus infection of cells leads to a plethora of events to ensure virus replication, including changes in the lipid composition on cellular membranes. A variety of viruses for example induces flipping of phosphatidyl-serines towards the exterior of the cell membrane, where they are prone for oxidation, normally a process that takes place during cell apoptosis and reflecting an ‘eat-me’ signal for phagocytes. The induced change in the lipid composition on cell membranes is incorporated into viral membranes during virus budding. Hence, some viruses use this as a mask to improve uptake by phagocytic cells, that recognize the virus as an apoptotic body, which can serve for better infection and virus spread. The changes in the lipid composition in viral membranes, but also viral surface glycoproteins directly may influence nIgM binding.
[0528] The binding of produced monoclonal OSE-specific IgM and/or IgA antibodies to Herpes Simplex Virus Type 1 or 2 infected cells or to cells expressing viral surface glycoproteins (e.g. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) glycoprotein S, Human Immunodeficiency virus (HIV-1) Env, Vesicular stomatitis virus (VSV) glycoprotein G, HSV-1/2 glycoproteins gB, gD, gH, gL, gI or gE) is tested. In these assays, affinity of binding (Kd) is determined for cells that express the respective viral glycoproteins and cells that do not express the protein as negative control.
Example 3: The Demonstration of the Direct Antiviral Activity of Pentaglobin®, OSE-Specific IgM and/or IgA Antibodies and Serum from BCG/Pneumovax-Vaccinated Individuals In Vitro and In Vivo
[0529] Different in vitro and in vivo models are used to address the direct antiviral effects of Pentaglobin®, monoclonal OSE-specific IgM and/or IgA antibodies and serum from BCG/Pneumovax-vaccinated individuals on virus infection and spread.
[0530] Prototype viruses from diverse virus families are investigated. These include Herpesviridae (HSV-1/2), Rabdoviridae (recombinant VSV with GFP reporter, rVSVdeltaG-GFP), as well as Murine Leukemia Virus (MLV) and HIV-1-based vectors pseudotyped with different gylcoproteins including VSV-G, HIV-1 Env, or SARS-CoV-2 glycoprotein S.
[0531] It is conceivable that enveloped viruses contain oxidized phospholipids and OSE in their membrane that can be bound by OSE-specific IgM and/or IgA antibodies. Direct effects on cell-free virus infection are tested by incubating serial dilutions of pre-BCG/Pneumovax-vaccination and post-vaccination serum, monoclonal OSE-specific IgM and/or IgA antibodies or Pentaglobin® with viruses or viral vectors and subsequent infection of appropriate target cell lines, e.g. the African green monkey cell line Vero or human embryonic kidney cell line HEK293T. Depending on the virus or vector that is used, infection is measured either by plaque formation (HSV-1 or HSV-2) or by reporter genes delivery through virus or viral vector infection (e.g. GFP or Luciferase).
[0532] For HSV-1 and HSV-2 effects of pre-/and post-BCG/Pneumovax vaccination sera, Pentaglobin® and monoclonal OSE-specific IgM and/or IgA antibodies on virus cell-to-cell spread are measured by infecting target cells with virus and then applying serial dilutions of the aforementioned drugs containing OSE-specific IgM and/or IgA antibodies.
[0533] Immunodeficient as well as immunocompetent mice are infected intravaginally with HSV-1 or HSV-2 and inject either before infection (prophylactic approach) or after infection (therapeutic approach) different doses of monoclonal OSE-specific IgM and/or IgA antibodies, Pentaglobin® or sera derived from BCG/Pneumovax-vaccinated individuals.
[0534] Survival and lesion development are monitored over time. Viral replication is measured via quantitative PCR. In these experiments Pentaglobin® serves as positive control, since due to the high seroprevalence of HSV it will likely contain virus-neutralizing IgGs.
[0535] Natural IgMs are also known to protect from primary cutaneous infections with HSV-1 (Deshpande, Kumaraguru et al., 2000, Cell Immunol, Vol. 202 (2)). It is investigated whether intravenous prophylactic or therapeutic injection of OSE-specific IgM and/or IgA antibodies or sera from BCG/Pneumovax-vaccinated individuals protects from cutaneous lesions induced by HSV-1 infection.
[0536] It is also tested whether topical treatment with these OSE-specific IgM and/or IgA antibody-containing compounds prevents lesion development after primary cutaneous HSV-1 infection. Since HSV-1 seroprevalence in the human population is approximately 80% we are exclusively using sera from HSV-1/2 seronegative (naïve) individuals.
Example 4: Analyzing of the Binding of nIgMs to Specific Leukocyte Surface Proteins to Prevent Induction of Proinflammatory Responses
[0537] (Lobo, Schlegel et al., 2008, J Immunol, Vol. 180 (3)) (Lobo, 2016, Front Immunol, Vol. 7)) demonstrated that IgM-ALA (leukocyte-binding nIgMs) have a positive role in recipients of heart and kidney transplants. Low or no levels of IgM-ALA was associated with increased inflammation, host vs. graft disease and transplant loss. It was demonstrated that IgM-ALA bind to (i) certain co-stimulatory receptors, that is, CD4, CD86, CD40, and PD1 and (ii) chemokine receptors. However, these polyreactive IgM-ALA autoantibodies manifest some form of specificity as they do not randomly bind to glycoproteins on other cell receptors, that is, CD8, CD80, CD40L, PDL1, CD28, CD1d, and HLA receptors. It was also shown that IgM-ALA bind to TcR, CD3, and CD45.
[0538] It is investigated by ELISAs and by flow cytometric assays whether identified and selected monoclonal nIgMs bind aforementioned co-stimulatory receptors of leukocytes. Prevention of the induction of proinflammatory responses by these nIgMs can be monitored by incubation of primary blood monocytes or purified leukocyte populations with nIgMs under stimulating conditions followed by multiplex analysis of secreted cytokines using established flow cytometry-based methods (Biolegend).
Example 5: Anti-Inflammatory Effects of Pentaglobin® or Monoclonal OSE-Specific IgM and/or IgA Antibodies or Sera from BCG/Pneumovax-Vaccinated Individuals on Acute Respiratory Disease Syndrome (ARDS) In Vivo
[0539] SARS-CoV-2 infection causes the development of Coronavirus disease (COVID-19), which can present with subclinical or mild symptoms as well as with severe symptoms reflecting an acute respiratory disease syndrome (ARDS). Two preclinical mouse models of ARDS that arise from direct lung injury have been described (D'Alessio, 2018, Methods Mol Biol, Vol. 1809)). These use intratracheal instillation of LPS (sterile inflammation) or Streptococcus pneumoniae to mimic human pneumonia. The models are chosen because they are highly reproducible, elicit robust neutrophilic alveolitis and disruption of the alveolar-capillary membrane, are easily titratable (degrees of pulmonary inflammation), and allow for evaluation of both the early and resolution phases of acute lung injury (ALI).
[0540] ARDS mice are treated with Pentaglobin® injection, or injection with monoclonal nIgMs or sera from BCG/Pneumovax-vaccinated individuals or non-vaccinated control sera. The response to treatment, i.e. alleviation of ARDS is monitored.
Example 6: Generation of OSE-Specific IgM and/or IgA Antibodies Libraries from BCG/Pneumovax-Vaccinated SARS-CoV-2 Seropositive Individuals with Mild/No Symptoms During SARS-CoV-2 Infection
[0541] It is demonstrated that BCG/Pneumovax-vaccinated individuals have a benefit in fighting severe infections with immunologically unseen pathogens due to increased levels of natural antibodies.
[0542] It was shown, that the repertoire of natural IgM changes with age in both level of expression as well as diversity (Rodriguez-Zhurbenko, Quach et al., 2019, Front Immunol, Vol. 10)). The conclusion from this observation is that the nIgM repertoire from young individuals may have enhanced anti-pathogenic characteristics.
[0543] In order to test this, natural IgM and/or IgA antibody phage-display libraries are generated from young BCG/Pneumovax-vaccinated SARS-CoV-2 seropositive individuals that showed only mild or no symptoms during the SARS-CoV-2 infection phase. From these natural antibody libraries several monoclonal IgM and/or IgA antibodies are cloned that show characteristic low affinity polyreactive binding properties to natural antibody-specific antigens and are able to bind to viral glycoproteins and show virus-neutralizing activities. The monoclonal IgM and/or IgA antibodies are tested individually and in combination (pools) to test for enhanced virus-neutralizing activity when used as a pool.
Example 7. Population-Wide Analysis of OSE-Specific IgM and/or IgA Antibody Serum Concentration and Correlation with BCG/Pneumovax-Vaccination Status
[0544] To demonstrate that BCG/Pneumovax vaccine induces higher levels of protecting nIgMs nIgM concentrations are analyzed and compared in sera from individuals that were not BCG and/or Pneumovax-vaccinated with sera from BCG and/or Pneumovax-vaccinated individuals. OSE-specific IgM and/or IgA serum levels are determined by ELISA methods described above. Pooled sera from vaccinated and not-vaccinated individuals are compared for virus-neutralizing activities in the viral assays described above.
Example 8. Defining the In Vivo Effects of Pentaglobin®, Monoclonal OSE-Specific IgM and/or IgA and Sera from BCG-Pneumovax-Vaccinated Individuals on Atherosclerosis
[0545] The two most frequently used models of mouse atherosclerosis are the apoE−/− model and the Idlr−/− model. It is tested whether injection of Pentaglobin®, monoclonal nIgMs or sera from BCG/Pneumovax-vaccinated individuals reduces atherosclerosis in these two models.
Example 9. SARS-CoV-2 Infected Lung Cells have Increased Levels of oxPL
[0546] As explained above, pathogens such as SARS-CoV-2, SARS-CoV or H5N1 influenza virus trigger increased rates of lipid peroxidation in cellular membranes, which is the production of oxidized phospholipids (oxPL) that occurs as a result of oxidative damage. It is postulated that OxPL is generated by SARS-CoV-2 infected cells as a result of cellular stress responses.
[0547] In fact, the accumulation of oxPL was detected on the surface of SARS-CoV-2 infected lung adenocarcinoma cells (Calu-3) as determined by stronger staining intensities of infected cells with the murine natural IgM E06, which detects the phosphorylcholine headgroup of oxPL, but not of native PL (
Example 10. Increased Oxidative Stress in Sera from COVID-19 Patients
[0548] Lipids containing polyunsaturated fatty acids are particularly susceptible to an oxidative attack, typically by reactive oxygen species (ROS), resulting in a chain reaction with the production of oxPL and end products such as malondialdehyde (MDA) that additionally contribute to the pathology of pathogen-induced inflammation in the infected tissue. OxPL and aldehydes such as MDA are not exclusively localized to the tissue where they were formed by ROS and cellular stress responses, but can also enter the circulation after they are released by cells under oxidative stress.
[0549] Since an increased formation of oxPL by SARS-CoV-2 infected cells was detected (see above), it is postulated that degradation products such as MDA may be elevated in sera of COVID-19 patients.
[0550] Indeed, significantly elevated concentrations of MDA were found in the sera derived from hospitalized COVID-19 patients and high MDA serum concentrations discriminated patients suffering from severe disease from patients with mild disease or healthy donors (
Example 11. Significantly Elevated Serum Levels of oxLDL in COVID-19 Patients
[0551] Human low-density lipoprotein (LDL) is one of the key lipid-protein complexes in blood and is a crucial component of metabolism responsible for the transport of lipids throughout the body. OxPL and aldehydes such as MDA in circulation can induce oxidative modifications of circulating LDL and other lipoproteins (Parthasarathy et al., 2010, Methods Mol Biol, Vol. 610 (403)).
[0552] Oxidation of LDL is a complex process during which both the lipids and proteins undergo oxidative changes and form complex products. For example, the peroxidised lipids decompose generating aldehydes such as MDA that covalently modify amino groups of lysine residues in apolipoprotein-B100 (apoB-100) of LDL. This not only generates Schiff's bases that modify charges on the amino acids, but also results in proteolysis of the apoB-100 protein as well as in both intra- and intermolecular crosslinks between proteolyzed apoB-100, resulting in excessive alteration of the protein composition and structure. Therefore, as opposed to native LDL, oxidized LDL (oxLDL) particles possess a variety of novel antigenic determinants that are recognized by receptors of innate and adaptive immunity, and cells of the vascular wall, thereby playing a key pathogenic role in cardiovascular diseases (CVD). In CVD, the proinflammatory functions of oxLDL are mediated by dendritic cells and macrophages that bind oxLDL with high affinity via scavenger receptors, leading to uncontrolled uptake of oxLDL and conversion of macrophages to foam cells, the defining characteristic of fatty streak and atherosclerotic lesions. Also, T cell activation has been linked to modified LDL since peptides derived from oxLDL have been shown to be recognized by T cells (Stemme et al., 1995, Proc Natl Acad Sci, Vol. 92 (3893)).
[0553] The surprising finding of the present invention that COVID-19 patients with severe disease contain high concentrations of MDA in their serum (
[0554] In line with this, significantly elevated levels of oxLDL were detected in sera derived from COVID-19 patients as compared to sera from healthy donors (
[0555] Thus, the present results demonstrate three novel and surprising findings: [0556] 1) high amounts of oxPL- and OSE-bearing structures such as SARS-CoV-2-infected cells and cellular debris are formed in lungs and possibly other infected tissues in COVID-19 patients; [0557] 2) aldehydes such as MDA are released into serum of COVID-19 patients, e.g., by SARS-CoV-2 infected lung cells, where they induce oxidative modifications to circulating lipoproteins such as LDL; and [0558] 3) circulating oxidized LDL particles in COVID-19 patients show requirements for high-affinity binding to scavenger receptors expressed by innate immune cells and, therefore, possess the potential to trigger systemic proinflammatory responses in a similar way as described for CVD when not cleared efficiently.
Example 12. Elevated Anti-oxLDL IgG and IgA Autoantibodies in Sera from Patients with Severe COVID-19
[0559] It is known from diseases associated with increased oxLDL levels, such as atherosclerosis and other CVD, diabetes mellitus, systemic lupus erythematosus or rheumatoid arthritis, that up to 90% of oxLDL are complexed by autoantibodies generating oxLDL-immune complexes, which levels often correlate with severity of the disease.
[0560] The autoantibodies that are complexed with oxLDL under disease conditions are predominantly of the IgG1 and IgG3 isotypes, which are both proartherogenic and can exert proinflammatory responses through their interaction with Fc-gamma receptors expressed by innate immune cells such as macrophages (Mironova et al.; Arterioscler Thromb Vasc Biol.; 1996 February; 16(2):222-9) (Virella et al., 2003, Clin Diagn Lab Immunol, Vol. 10 (499)). Thereby, oxLDL-IgG immune complexes induce stronger proinflammatory responses as compared to free oxLDL because the immune complexes engage Fc-gamma receptors in addition to scavenger receptors. Fc-gamma receptor-mediated NLRP3 inflammasome activation contributes to the secretion of proinflammatory cytokines, e.g. IL-1b and IL-6, from innate immune cells in response to oxLDL-IgG immune complexes (Rhoads et. al; J Immunol; 2017 Mar. 1; 198(5):2105-2114).
[0561] Since COVID-19 patients with severe disease surprisingly showed highly elevated level of oxLDL in their sera compared to patients with mild disease or healthy donors, it was tested if these patients also contained increased titers of anti-oxLDL IgG autoantibodies that potentially form proinflammatory immune complexes.
[0562] In full support of the concept presented herein, significantly elevated levels of anti-oxLDL IgG autoantibodies in sera from COVID-19 patients compared to sera from healthy donors, and these levels correlated with the severity of the disease (
[0563] Since a fraction of serum IgA antibodies, particularly of the IgA2 subtype, can exert proinflammatory responses via binding of IgA2-containing immune complexes to macrophages and neutrophils expressing the Fc-alpha receptor, anti-oxLDL IgA autoantibody levels were also compared between COVID-19 patients with severe disease, with mild disease and healthy donors. Interestingly, it has been found that anti-oxLDL IgA autoantibody levels were specifically and significantly increased in patients with severe compared to mild forms of COVID-19 or healthy donors (
[0564] These surprising findings support a role for pathogenic oxLDL-IgG and oxLDL-IgA immune complexes in severe COVID-19 patients, where they trigger systemic hyperinflammation responses.
Example 13. OxPL-Specific Monoclonal Antibodies Compete with IgG from COVID-19 Sera for Binding to oxLDL
[0565] OxLDL particles display a large variety of immunogenic determinants that have not yet been defined in detail and that can be bound by antibodies.
[0566] To show that anti-oxLDL antibodies in sera from COVID-19 patients indeed bind to oxPL, an oxPL-masking assay was performed using two monoclonal mouse antibodies that bind to distinct classes of oxPL:
[0567] IgM antibody E06 binds to the phosphorylcholine headgroup exposed by oxidized phosphatidylcholine (oxPC), whereas IgM antibody 509 binds to oxidized phosphatidylethanolamine (oxPE) but not to its native non-oxidized counterpart (Bochkov et al., 2016, Biomark Med., Vol. 10 (8), 797-810).
[0568] ELISA plates were coated with oxLDL and the coated wells were preincubated with these monoclonal antibodies alone or in combination to block oxPC and/or oxPE exposed by oxLDL. Then the COVID-19 serum samples were added and IgG binding to oxLDL was detected by HRP-conjugated anti-human IgG secondary antibody.
[0569] The data revealed that blocking oxPC or oxPE alone on oxLDL by preincubation with one of these two monoclonal antibodies did not lead to detectable inhibition of IgG binding to oxLDL.
[0570] However, when both classes of oxPL were blocked simultaneously, a significant inhibition of binding of IgG antibodies in COVID-19 sera to oxLDL particles by ˜20% on average was observed (
Example 14. COVID-19 Sera Contain Elevated IgG and IgA Antibodies to Oxidation-Specific Epitopes
[0571] OxPL such as oxPC and oxPE can be predominantly found on OxLDL particles in the early phase of oxidation, whereas end products of lipid peroxidation such as Malondialdehyde or 4-Hydroxynonenal dominate on LDL particles with an advanced oxidation state.
[0572] To show that anti-oxLDL antibodies in sera from COVID-19 patients bind to different types of OSE exposed by oxLDL particles, ELISA plates were coated with phosphorylcholine (PC), Malondialdehyde (MDA) and 4-Hydroxynonenal (HNE), representing early and late-stage oxidation-specific epitopes. Coated wells were incubated with sera derived from hospitalized COVID-19 patients with severe disease or with sera from healthy donors, and antibody binding was detected by isotype-specific HRP-conjugated secondary antibodies.
[0573] It was found that sera from healthy donors contained IgG and IgA antibodies that bound to PC, whereas levels of anti-MDA and anti-HNE IgG and IgA antibodies were low or undetectable, respectively (
[0574] Since PC is the immunodominant determinant of pneumococcal cell-wall polysaccharides, anti-PC IgG and IgA antibodies in healthy donors may represent previous pneumococcal infection or vaccination histories. However, and in sharp contrast, highly significantly elevated levels of IgG and IgA antibodies were detected that bound to MDA and HNE in sera from severe COVID-19 patients compared to sera from healthy donors (
[0575] This novel finding supports the following mechanism that contributes to the pathogenesis of severe COVID-19: [0576] 1) oxPL and OSE are formed in COVID-19 patients; [0577] 2) oxPL and OSE may not be cleared efficiently in a population of COVID-19 patients, e.g., because of a natural antibody deficiency, leading to accumulation of oxPL and OSE in SARS-CoV-2 infected cells, apoptotic cells and lipoproteins, which then trigger autoimmune responses and generation of IgG and IgA autoantibodies; [0578] 3) anti-oxPL and OSE IgG and IgA autoantibodies bind to oxPL- and OSE-bearing structures, e.g., on LDL particles or infected cells, and mediate proinflammatory immune responses by simultaneously engaging scavenger receptors and Fc-receptors expressed by innate immune cells, thereby driving the hyperinflammation state observed in severely ill COVID-19 patients.
Example 15. An IgM Fraction from Pentaglobin Binds to Apoptotic Cells Displaying oxPL
[0579] As shown above, COVID-19 patients develop oxidative stress and an increased exposure of oxPL in the membranes of SARS-CoV-2 infected cells and circulating lipoprotein particles was found. These structures possess the ability to induce IgG and IgA autoantibody responses and it is postulated that they contribute to the pathogenesis of severe COVID-19 if not cleared efficiently.
[0580] In mice, natural IgM antibodies and other soluble pattern recognition receptors (PRRs) of innate immunity bind to oxPL- and OSE-bearing structures and thereby facilitate their safe and anti-inflammatory clearance.
[0581] Therefore, the novel concept is herewith supported that treatment of severely ill COVID-19 patients with oxPL-specific natural IgM antibodies neutralize the proinflammatory effects of oxPL, facilitate their safe clearance and thereby prevents the induction of destructive autoimmune responses.
[0582] Pentaglobin is a human immunoglobulin infusion preparation enriched for IgM and IgA antibodies and is approved to treat patients with severe bacterial infections and sepsis, and immunodeficient patients that lack endogenous immunoglobulins. Since the immunoglobulins in Pentaglobin constitute of pooled serum antibodies obtained from thousands of healthy human donors, it has been found herein that particularly the IgM pool, and to a lesser extend the IgA and IgG fractions, of Pentaglobin contain natural antibodies recognizing different types of oxPL and OSE.
[0583] To test for the presence of oxPL-specific IgM antibodies, Pentaglobin was used to stain human cells under oxidative stress exposing different types of oxPL and OSE in their plasma membrane and IgM binding was detected by fluorescently labelled secondary antibodies.
[0584] Oxidative stress was induced by incubation of cells with different concentrations of H.sub.2O.sub.2, an agent that potently initiates the lipid peroxidation reaction, and formation of oxPL was monitored by staining of treated cells with the mouse natural IgM E06 recognizing the phosphorylcholine headgroup exposed by oxPL. Indeed, most H.sub.2O.sub.2-treated cells stained positive with E06 and this was dependent on induction of apoptosis, indicating that treated cells displayed a huge amount of oxPL on their surface (
Example 16. An IgM Fraction from Pentaglobin® Binds to SARS-CoV-2 Infected Cells
[0585] As shown above, it has been found that lung adenocarcinoma cells (Calu-3) infected with SARS-CoV-2 display large amounts of oxPL in their membranes that were bound by the mouse natural IgM antibody E06 (
[0586] To test if Pentaglobin contains antibodies that bind to SARS-CoV-2 infected lung cells, e.g., to oxPL exposed in the plasma membrane of infected cells, infected Calu-3 cells were stained with Pentaglobin and IgM binding was detected by incubation with fluorescently labelled anti-human IgM secondary antibody. Indeed, the data suggest that Pentaglobin contains an IgM fraction that can bind to infected cells and that most of these antibodies likely bind to oxPL presented on the plasma membrane (
Example 17. Pentaglobin® Contains Antibodies that Bind to oxLDL
[0587] OxPL are not exclusively exposed by membranes of apoptotic cells but can be present also on circulating lipoproteins such as LDL, where they constitute the major pathogenic component of oxLDL.
[0588] In fact, high amounts of oxPL were found in the plasma membrane of SARS-CoV-2 infected cells and in circulating oxLDL in sera from COVID-19 patients. Since it has surprisingly been found herein that Pentaglobin contains natural IgM antibodies that bind to apoptotic cells displaying high amounts of oxPL, it was tested whether Pentaglobin contains antibodies that also bind to oxLDL. To test this, ELISA plates were coated with oxLDL, the coated wells were incubated with different concentrations of Pentaglobin®, and antibody binding was detected by labelled isotype-specific secondary antibodies.
[0589] In fact, it has been found that Pentaglobin contains antibodies that bind to oxLDL in a concentration-dependent manner (
[0590] For uninfected mice it was shown that ˜80% of the IgM pool and ˜50% of serum IgA are derived from B1 cells, hence IgM and IgA represent the most common isotypes of natural antibodies (Meyer-Bahlburg, 2015, Ann N Y Acad Sci, Vol. 1362, 122-31). Therefore, this supports that most of oxLDL-binding IgM and IgA antibodies within Pentaglobin represent human natural antibodies.
Example 18. Pentaglobin® Contains Antibodies that Bind to Oxidation-Specific Epitopes
[0591] To show that anti-oxLDL antibodies in Pentaglobin indeed bind to OSE exposed by oxidized LDL particles, ELISA plates were coated with different classes of OSE including Phosphorylcholine (PC), Malondialdehyde (MDA) and 4-Hydroxynonenal (HNE), which are well-described targets for natural antibodies.
[0592] Coated wells were incubated with 4 consecutive dilutions of Pentaglobin and antibody binding was detected by HRP-conjugated secondary antibodies. The results showed that Pentaglobin® indeed contains antibodies that bind to all classes of OSE tested, and that anti-PC antibodies constitute the most prominent OSE-binding fraction, followed by anti-HNE and lower level of anti-MDA antibodies.
[0593] Interestingly, when an IgM-specific secondary antibody was used to specifically detect IgM binding, we found a similar binding pattern to all classes of OSE tested, indicating that OSE-binding antibodies in Pentaglobin® primarily belong to the IgM pool (
[0594] In support of this, it has been shown that pneumococcal immunization of mice induced high level of PC-specific natural IgM antibodies, which conferred protection from atherosclerosis due to molecular mimicry between S. pneumoniae and oxLDL (Binder, 2003, Nat. Med., Vol. 9, 736-43).
[0595] Taken together, these results show that Pentaglobin contains natural antibodies primarily of the IgM isotype that bind to different classes of OSE and we suggest that these antibodies may confer protection from oxPL-induced proinflammatory responses in severe COVID-19 patients.
Example 19. Pentaglobin® Contains Antibodies that Block Binding of IgG and IgA from COVID-19 Sera to oxLDL
[0596] The above findings indicate that the severity of COVID-19 is accompanied by the development of oxPL- and OSE-specific IgG and IgA autoantibodies that form immune complexes with oxidatively modified particles such as circulating lipoproteins, which represent potent drivers of the hyperinflammation state observed in severely ill COVID-19 patients.
[0597] The above data support the novel concept that neutralization of oxPL and OSE by natural IgM antibodies neutralize their proinflammatory potential in COVID-19 patients by multiple mechanisms: [0598] 1) natural IgM block binding of oxidatively modified particles such as oxLDL to scavenger receptors on innate immune cells; [0599] 2) natural IgM block binding of IgG and IgA autoantibodies to oxidatively modified particles such as oxLDL and thereby prevent formation of proinflammatory IgG- and IgA-containing immune complexes; [0600] 3) natural IgM facilitate the safe and anti-inflammatory clearance of structures exposing oxPL such as cellular debris and thereby prevent the accumulation of oxPL and OSE and the development of IgG and IgA autoantibodies.
[0601] The above surprising findings that neutralization of two classes of oxPL on oxLDL by monoclonal mouse IgM antibodies significantly inhibited binding of IgG autoantibodies from COVID-19 sera to oxLDL, and that Pentaglobin contains a significant fraction of oxPL-specific human natural antibodies primarily in its IgM pool, it is supported that Pentaglobin can inhibit binding of IgG and IgA autoantibodies present in COVID-19 sera to oxLDL similarly as the oxPL-specific mouse monoclonal IgM antibodies did.
[0602] In fact, preincubation of oxLDL-coated wells with Pentaglobin significantly inhibited the binding of autoantibodies from sera of COVID-19 patients to oxLDL by >20%, and this effect was evident for both IgG and IgA autoantibodies (
[0603] These novel findings support the application of Pentaglobin® to treat severely ill COVID-19 patients because of its surprising properties to neutralize different classes of oxPL and OSE and to prevent the formation of immune complexes containing IgG and IgA autoantibodies, thereby showing potential to ameliorate the hyperinflammatory immune responses and to contribute to an rapid improvement of the clinical condition.
Example 20. Clinical NAD Modulating Intervention Strategy for COVID-19
[0604] COVID-19 is a disease with extraordinarily high medical need in terms of both treating and preventing the disease. We anticipate NAD modulation as valuable intervention strategy for both treatment and prevention of COVID-19.
[0605] 1. Clinical Treatment Trials [0606] a. In a small study with up to 10 patients with severe COVID-19 infection who require mechanistic ventilation or according to medical assessment will require mechanistic ventilation within 1-4 hours due to rapid pulmonary deterioration the natural antibody containing formulation Pentaglobin® is administered at 5 m L/kg at 28 mL/h for 3 consecutive days. [0607] b. Subsequently, a controlled clinical phase II study with up to 50 patients at two centers is commenced. In this trial, effects of potential NAD modulating activity of Pentaglobin® is systematically assessed and correlated with key clinical outcome data. [0608] c. Subsequently, a large phase III multinational multicenter trial is implemented. Moreover, novel natural antibody enriched plasma formulations (i.e. from female donors <25 years of age) are developed for subsequent approval trials, preferably already at a time point with first available interims data from the phase III study. [0609] d. Subsequently, recombinant monoclonal natural antibodies are simultaneously developed for replacement of NAD modulating Pentaglobin® for treating COVID-19 and other NAD associated diseases.
[0610] 2. Clinical Prevention Trials [0611] Preclinical data indicate potential NAD modulating effects for currently available vaccines such as BCG or Pneumovax. [0612] Following successful preclinical confirmation of this in vitro and in relevant animal models natural antibody inducing vaccination trials with respective compounds are initiated. Since for some of these compounds (e.g. BCG) protection from acquiring severe COVID19 disease has been postulated on the basis of epidemiological data. However, induction of natural antibodies as the potential causative reason for protection has thus far not been shown. Consequently, NAD modulation is systematically analyzed in explorative parts of these trials and also correlated with clinical outcome data.
Example 21. Clinical Treatment of COVID-19 with Pentaglobin®
[0613] 5 patients with very severe COVID-19 course of disease and in whom 4 out of 5 required invasive mechanical ventilation were administered with Pentaglobin.
[0614] Patient 1:
[0615] 59 year old male (#1).
[0616] Comorbidities: M. Bechterew (morphine-dependent chronical pain patient), osteoporosis, hypertension.
[0617] History: On Mar. 28, 2020, presentation at local hospital with abdominal pain and rapid impairment of general condition, positive SARS-CoV-2 swab on the same day. Rapid development of respiratory insufficiency, in the course deterioration and requirement for intubation and invasive CPAP ventilation on April 4. Transfer to ICU UKHD on April 14, at time of admission requirement for catecholamine (noradrenaline). In CT Thorax from April 14, signs of typical COVID-19 pneumonia, suspicious of pre-existing lung fibrosis. On April 16, administration of 10 g Pentaglobin, initiation of Aciclovir therapy after tested positive for HSV-1 in BAL. Cardiopulmonal stabilization, thereafter, change to BIPAP ventilation, tracheostoma. On April 22, spontaneous breathing attempt, not yet well tolerated w/ tachypnea, and pathological breathing mechanics.
[0618] Patient 2:
[0619] 80 year old male (#2).
[0620] Comorbidities: M. Bechterew, coronary 3 heart disease, acute myocardial infarction due to RCA occlusion, acute cervical vertebra 7 fracture after collapse.
[0621] History: Respiratory infection for 1 week, non-responsive to antibiotics. On April 2, patient was found unconscious at home with head laceration, alarming of emergency physician, at arrival, vigilance significantly reduced yet responsive, SO2 87% at 12 l O2/min, transport to hospital emergency room. Diagnostics: swab Sars-2 positive, CT scan: central lung arterial embolism, typical signs of COVID-19 lung disease. Elevated temperature 38.5° C. In ECG, signs of myocardial infarction (RCA), dilatation and stenting of RCA and recanalization successful, intubation for intervention required, catecholamine dependency. In the course, significant impairment of pulmonary situation, in CT scan on April 12, significant impairment of COVID-19 lung infiltrates with beginning consolidation. Between April 13 and Apr. 15, 2020, administration of 22.5 g Pentaglobin daily. On April 16, temporary impairment of retention parameters yet improved diuresis (hemodialysis initiated), thereafter slow clinical stabilization of kidney and pulmonary situation, on April 20, intermittent CPAP w/slow PEEP reduction over the next days, attempts to intermittently pause assisted ventilation successful, oxygenation not yet satisfactory. On April 22, three subsequent negative tests for Sars-2, thus transfer to cardiologic ICU for further stabilization of the cardiopulmonary situation and planning subsequent surgery of the vertebra fracture.
[0622] Patient 3:
[0623] 62 year old female (#3).
[0624] Comorbidities: Adipositas, Klippel-Trenaunay syndrome.
[0625] History: On Mar. 29, 2020, presentation at local hospital with fever and dyspnea for 5 days and impairment of general condition, positive SARS-CoV-2 swab on the same day. Rapid development of respiratory insufficiency, in the course deterioration and requirement for intubation and invasive BIPAP ventilation on April 8. Transfer to another local hospital ICU (Schwäbisch Hall) and subsequent transfer to ICU UKHD on April 13, temporarily low dose catecholoamine requirement. In CT Thorax from April 13, signs of typical COVID-19 pneumonia. Detection of free floating thrombus in V. jug., full dose heparinization. On April 14 and 15, administration of 10 g Pentaglobin on each day. Rapid cardiopulmonal improvement thereafter, weaning and extubation without any complications. Initiation of Aciclovir therapy after tested positive for HSV-1 in tracheal fluid on April 19. On April 20, SARS-CoV-2 negative swab. Re-transport to local hospital with 1 l/min O2 via nose.
[0626] Patient 4:
[0627] 76 year old male (#4).
[0628] Comorbidities: Diabetes mellitus II insulin dependent, hypertension, dyslipidemia, severe coronary 3 heart disease with bradycardiac atrial fibrillation, NSTEMI w/ high grade LCX stenosis and RCA occlusion.
[0629] History: Starting with fever, dyspnea, rapid impairment of general condition since April 3, patient presented at local hospital on April 13. SARS-CoV2 tested positive the day before, exposition by family member. Rapid impairment of cardiopulmonary situation and requirement for intubation and invasive intubation starting April 14. Subsequent development of increased troponin levels and acute renal failure. Transfer to ICU UKHD on April 15. Immediate coronary angiography revealed occluded RCA with sufficient collaterals and proximal LCX Stenosis, PTCA and LCX stenting successfully performed in the same session, catecholamines and hemodialysis required. CT scan on April 19, revealed typical signs of COVID-19 lung disease, suspicion of aortic ulcus of unknown origin. In the course, significant impairment of pulmonary situation, in CT scan on April 12, significant impairment of COVID-19 lung infiltrates with beginning consolidation. On April 16, patient received 10 g Pentaglobin. Development of bradycardiac atrial fibrillation episodes, improvement after clonidine administration stop. Cardiopulmonary stabilization. On April 19, administration of another 10 g Pentaglobin. Last fever episode on April 20, at 38.8° C., reduction of sedation. Since April 21, weaning attempt, since April 22, stabilized cardiopulmonary condition.
[0630] Patient 5:
[0631] 62 year old male (#5).
[0632] Comorbidities: None.
[0633] History: On Mar. 29, 2020 anamnestic contact to COVID-19 positive person. On April 1, dry cough, fever, chills and impairment of general condition, presentation at local hospital, tested positive in SARS-CoV-2 swab on April 13. Subsequently, continuous increase of O2 demand and development of respiratory insufficiency. On April 15, transport to IMC unit UKHD due to respiratory deterioration. At UKHD, throat swab for SARS-CoV-2 negative. In CT Thorax from the same day signs of typical COVID-19 pneumonia. From April 15 to April 21, highflow oxygenation therapy (HFOT, Optiflow®). On April 16, administration of steroids and 10 g Pentaglobin. In the further course, stabilization of the pulmonary situation allowing for stepwise reduction of apparative oxygenation and termination of HFOT on April 21. Sputum tested for SARS-CoV-2 negative on April 17. Notably, TNT remained stably high without clinical symptoms and alterations in ECG. Re-transport to local hospital in good condition with 1 l/min O2 via nose.
[0634] The following Tables show the clinical data determined for Patient 1, Patient 2, Patient 3, Patient 4 and Patient 5, respectively, over the course of time.
TABLE-US-00002 Patient #1 (age 59, male) Symptoms x x x x x x x x x Hospitalisation x x x x x x x x x Invasive ventilation x x x x x x x x x Anti-infectives administered Pip/Taz/Caspo Pip/Taz/ Pip/Taz/Caspo Meropenem/ Meropenem/ Meropenem/ Meropenem/ Meropenem/ Meropenem/ Meropenem/Vanco Caspo Meropenem/Vanco Vanco Vanco Vanco Vanco Vanco Vanco Aciclovir with HSV Aciclovir Aciclovir Aciclovir Aciclovir Aciclovir Aciclovir Aciclovir Immunomodulators administered Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Prednisolon 180 mg Prednisolon Pentaglobin 10 g Pentaglobin SARS-COV-2 result negative negative negative negative Laboratory Days Day 25 Day 26 Day 27 Day 28 Day 29 Day 30 Day 31 Day 32 Day 33 results* Test Unit Normal value Apr. 14, 2020 Apr. 15, 2020 Apr. 16, 2020 Apr. 17, 2020 Apr. 18, 2020 Apr. 19, 2020 Apr. 20, 2020 Apr. 21, 2020 Apr. 22, 2020 Clinical Sodium mmol/l 135-146 149 149 155 151 155 154 151 148 151 chemistry Potassium mmol/l 3.4-4.8 4.90 4.30 4.66 4.23 4.13 4.94 4.59 4.53 4.50 Creatinine mg/dl 10.6-1.2 1.36 1.79 2.20 2.27 1.81 1.40 1.24 0.99 0.92 GFR using CKD-EPI >60 56.6 40.6 31.6 30.4 40.0 54.6 63.2 83.0 90.7 Urea mg/dl <45 74 95 111 138 153 128 115 90 77 Creatine kinase (CK) U/l <190 633 NA 392 190 181 118 164 122 128 Troponin T (TNT) pg/ml <14 56 77 66 53 58 62 61 54 88 Lactate dehydrogenase U/l <317 655 516 485 422 428 437 555 482 528 (LDH) GOT/AST U/l <46 141 118 123 66 68 93 103 75 60 GPT/ALT U/l <50 48 44 55 36 38 63 71 59 46 Gamma-glutamyltransferase U/l <60 85 65 80 64 78 121 109 87 70 (GGT) Iron μmol/l 14-32 7.6 1.7 2.6 6.8 1.6 1.2 2.0 2.0 2.9 Triglycerides mg/dl <150 NA NA 220 183 317 229 189 147 127 Albumin g/l 30-50 30.2 28.8 28.5 30.7 32.3 28.9 30.6 27.4 26.2 C-reactive protein (CRP) mg/l <5 315.7 324.5 398.9 270.9 127.7 126.2 174.9 175.9 155.6 Hematology Leucocytes /nl 4-10 18.14 15.94 17.80 10.80 8.49 8.64 12.29 13.47 13.55 Neutrophil granulocytes % 50-80 1 88.1 89.0 90.6 82.0 74.9 70.6 68.1 NA (automated) 80.3 Lymphocytes (automated) % 25-40 14.7 7.5 6.8 4.9 9.3 12.8 15.6 17.8 NA Eosinophil granulocytes % 2-4 1.3 0.2 0.7 0.3 4.7 6.9 7.3 7.0 NA (automated) Lymphocytes (absolute) /nl 1.0-4.8 2.67 1.20 1.21 0.53 0.79 1.11 1.92 2.40 1.90 Coagulation D-Dimer mg/l <0.5 17.10 6.65 3.46 5.30 7.76 9.32 10.44 11.20 12.58 IgG g/l 7.0-16.0 NA 13.15 12.31 11.54 10.46 9.31 NA NA NA IgA g/l 0.7-4.0 NA 11.76 11.16 8.81 8.40 7.70 NA NA NA IgN g/l 0.4-2.3 NA 2.02 1.88 1.49 1.44 1.30 NA INA NA Transferrin g/l 2.0-3.6 0.62 0.82 0.60 0.55 0.90 0.86 0.90 0.91 0.88 Transferrin saturation % 16-45 49 8 17 49 7 6 9 9 13 Ferritin μg/l 30-300 1794 3276 2197 1766 1581 1121 1460 1378 1405 Procalcitonin (PCT), sensitive ng/ml <0.05 1.16 16.75 19.75 18.51 10.38 4.51 2.07 1.10 0.68 POCT pH value (POCT) 7.37-7.45 7.08 7.27 7.08 7.46 7.47 7.49 7.48 7.49 7.48 Carbon dioxide partial pressure mmHg 35-45 NA 64.6 101.3 49.6 36.2 36.3 47.1 52.9 37.3 (pCO2) (POCT) Oxygen partial pressure (pO2) mmHg >arterial 103.4 76 65 57 77 65 61 61 64 59 (POCT) minus (0.42 times age in years) Base Excess, standard mmol/L −2-+3 −7.5 −3.2/4.8 −4.5/6.9 5.6 3.4 4.2 4.1 −6.6/6.5 3.5 Lactate (BGA) mg/dl <16 15.1 17.7 19.5 18.9 18.6 12.4 13.9 12.5 8.7 CSO2 % 91.6 91.6 86.4 94.7 92.1 89.8 91.8 91.7 91.0 Blood collection method arterial arteria arterial arterial arterial arterial arteria arterial arterial Proteins sCD25 U/ml <900 NA NA 4847 NA NA NA NA NA NA Soluble transferrin receptor mg/l 2.2-5.0 NA NA NA 2.2 2.9 3.3 3.7 4.3 NA (sTFR) Ferritin index 3.2 NA NA NA 0.68 0.91 1.08 1.17 1.37 NA Interleukin 6 pg/ml 350.0 157.0 270.0 <2.0 37.3 89.0 75.6 50.5 NA *Please note that in case lab testing was performed several times a day, the documented result is the worst result from that day. NA = not available
TABLE-US-00003 Patient #2 (age 80, male) Symptoms x x x x x x x Hospitalisation x x x x x x x Invasive ventilation x x x x x x x Anti-infectives administered Pip/Taz/Caspo Pip/Taz Pip/Taz Pip/Taz Pip/Taz Pip/Taz Pip/Taz Pip/Taz Moxiflox Caspo Caspo Caspo Caspo Meropenem Aciclovir with HSV Immunomodulators administered Maraviroc 2 × 75 mg Pentaglobin 22.5 g SARS-COV-2 result positive positive positive Symptoms x x x x x x x Hospitalisation x x x x x x x Invasive ventilation x x x x x x x Anti-infectives administered Pip/Taz/Caspo Pip/Taz Caspo Pip/Taz Pip/Taz Pip/Taz Pip/Taz Caspo Caspo Caspo Meropenem Aciclovir with HSV Immunomodulators administered Maraviroc 2 × 75 mg Maraviroc Pentaglobin 22.5 g Pentaglobin Pentaglobin Pentaglobin SARS-COV-2 result positive positive Symptoms x x x x x x x Hospitalisation x x x x x x x Invasive ventilation x x x x x x x Anti-infectives administered Pip/Taz/Caspo Pip/Taz Pip/Taz Pip/Taz Meropenem Meropenem Meropenem Meropenem Moxiflox Caspo Caspo Caspo Caspo Caspo Caspo Caspo Meropenem Meropenem Aciclovir Aciclovir Aciclovir Aciclovir with HSV Immunomodulators administered Maraviroc 2 × 75 mg Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Pentaglobin 22.5 g SARS-COV-2 result positive positive negative negative negative Laboratory Days Day 11 Day 12 Day 13 Day 14 Day 15 Day 16 Day 17 results* Test Unit Normal value Apr. 2, 2020 Apr. 3, 2020 Apr. 4, 2020 Apr. 5, 2020 Apr. 6, 2020 Apr. 7, 2020 Apr. 8, 2020 Clinical Sodium mmol/l 135-146 138 142 146 147 150 151 150 chemistry Potassium mmol/l 3.4-4.8 5.03 4.62 4.82 5.87 5.15 1.78 4.57 Creatinine mg/dl 0.6-1.2 1.18 1.76 2.26 3.07 4.12 5.38 5.25 GFR using >60 57.9 35.7 26.4 18.2 12.8 9.3 9.5 CKD-EPI Urea mg/dl <45 37 44 55 68 91 135 140 Creatine kinase (CK) U/l <190 1353 NA NA NA NA NA 79 Troponin T (TNT) pg/ml <14 4000 6837 651 637 5936 5950 NA Lactate U/l <317 NA 658 580 595 434 451 350 dehydrogenase (LDH) GOT/AST U/l <46 107 254 155 95 57 47 40 GPT/ALT U/l 50 70 83 62 49 43 37 29 Gamma-glutamyl- U/l 60 69 58 50 43 39 54 60 transferase (GGT) Iron μmol/l 14-32 NA NA 1.3 0.8 1.5 7.0 4.9 Triglycerides mg/dl <150 NA NA NA NA NA NA 132 Albumin g/l 30-50 37.5 35.2 34.3 31.4 31.5 28.7 28.4 C-reactive mg/l 5 130.3 151.1 273.3 291.0 304.0 238.9 155.9 protein (CRP) Hematology Leucocytes /nl 14-10 13.66 11.37 10.38 11.52 11.90 8.21 7.82 Neutrophil % 50-80 93.8 84.3 88.8 89.7 90.5 91.5 88.6 granulocytes (automated) Lymphocytes % 25-40 2.0 8.8 5.2 4.4 3.3 3.2 4.0 (automated) Eosinophil % 2-4 0.1 0.2 0.8 1.0 1.0 1.8 2.3 granulocytes (automated) Lymphocytes /nl 1.0-4.8 0.27 1.00 0.54 0.51 0.39 0.25 0.31 (absolute) Coagulation D-Dimer mg/l <0.5 32.16 NA 3.18 2.35 2.06 2.95 3.62 IgG g/l 7.0-16.0 NA NA NA NA NA NA NA IgA g/l 0.7-4.0 VA VA NA NA NA NA NA IgM g/l 0.4-2.3 NA NA NA NA NA NA NA Transferrin 2.0-3.6 1.60 1.49 1.06 0.95 0.79 0.75 0.75 Transferrin saturation % 16-45 NA NA 5 3 8 37 26 Ferritin μg/l 30-300 178 207 198 220 214 221 205 Procalcitonin (PCT), ng/ml <0.05 0.18 0.42 0.68 0.50 0.63 0.98 1.08 sensitive POCT pH value (POCT 7.37-7.45 7.08 7.20 7.18 7.19 7.22 7.23 7.25 Carbon dioxide mmHg 35-45 73.3 55.2 58.1 60.7 55.1 57.2 53.1 partial pressure (pCO2) (POCT) Oxygen partial mmHg >arterial 103.4 73 82 69 76 65 46 65 pressure (pO2) minus (0.42 times (POCT) age in years) Base Excess, standard mmol/L −2-+3 −9.8 −7.9 −7.2 −6.5 −8.0 −4.9 −4.6 Lactate (BGA) mg/dl <16 13.5 15.0 13.1 12.5 13.0 13.3 10.5 cSO2 % 91.0 94.8 91.0 93.0 89.7 75.1 91.0 Blood collection arterial arterial arterial arterial arterial arterial arterial method Proteins sCD25 U/ml <900 NA NA NA NA NA NA NA Soluble transferrin mg/l 2.2-5.0 NA VA NA NA NA NA NA receptor (sTFR) Ferritin index 13.2 NA NA NA NA NA NA NA Interleukin 6 pg/ml 122.0 NA NA 365.0 297.0 180.0 96.2 Laboratory Days Day 18 Day 19 Day 20 Day 21 Day 22 Day 23 Day 24 results* Test Unit Normal value Apr. 9, 2020 Apr. 10, 2020 Apr. 11, 2020 Apr. 12, 2020 Apr. 13, 2020 Apr. 14, 2020 Apr. 15, 2020 Clinical Sodium mmol/l 135-146 1.53 151 152 148 147 146 149 chemistry Potassium mmol/l 3.4-4.8 4.64 4.22 4.30 4.96 6.19 4.39 4.63 Creatinine mg/dl 0.6-1.2 5.14 5.02 4.95 4.86 4.94 5.09 4.87 GFR using >60 9.8 10.1 10.2 10.5 10.3 9.9 10.4 CKD-EPI Urea mg/dl <45 143 149 1.59 168 169 205 227 Creatine kinase (CK) U/l <190 NA NA NA NA 78 NA 1058 Troponin T (TNT) pg/ml <14 4020 3649 2796 2582 NA 767 522 Lactate U/l <317 360 338 325 383 339 377 305 dehydrogenase (LDH) GOT/AST U/l <46 43 41 46 64 66 167 125 GPT/ALT U/l 50 28 27 27 29 32 48 50 Gamma-glutamyl- U/l 60 75 75 85 106 107 107 101 transferase (GGT) Iron μmol/l 14-32 4.2 3.5 3.7 4.5 2.7 7.0 11.2 Triglycerides mg/dl <150 NA NA 136 NA 138 NA 123 Albumin g/l 30-50 30.6 30.9 31.2 29.9 34.5 31.5 29.6 C-reactive mg/l 5 146.6 161.6 174.7 185.9 196.6 151.9 NA protein (CRP) Hematology Leucocytes /nl 14-10 8.76 8.68 10.46 9.43 12.34 12.10 12.29 Neutrophil % 50-80 85.5 85.8 83.6 79.8 88.9 90.6 NA granulocytes (automated) Lymphocytes % 25-40 4.9 4.4 6.4 9.6 5.6 4.8 NA (automated) Eosinophil % 2-4 1.9 2.1 1.9 1.7 0.9 0.1 NA granulocytes (automated) Lymphocytes /nl 1.0-4.8 0.43 0.38 0.67 0.91 0.55 0.58 NA (absolute) Coagulation D-Dimer mg/l <0.5 4.31 3.00 2.41 2.92 3.1 3.75 NA IgG g/l 7.0-16.0 NA NA NA NA NA NA NA IgA g/l 0.7-4.0 NA NA NA NA NA NA NA IgM g/l 0.4-2.3 NA NA NA NA NA NA NA Transferrin 2.0-3.6 0.74 0.87 1.03 1.06 1.09 1.22 1.51 Transferrin saturation % 16-45 23 16 14 17 10 23 30 Ferritin μg/l 30-300 195 174 172 181 209 253 189 Procalcitonin (PCT), ng/ml <0.05 0.83 0.76 0.65 0.56 0.55 0.46 0.38 sensitive POCT pH value (POCT 7.37-7.45 7.20 7.29 7.45 7.22 7.23 7.32 7.30 Carbon dioxide mmHg 35-45 68.1 55.2 47.3 68.9 66.7 51.9 49.6 partial pressure (pCO2) (POCT) Oxygen partial mmHg >arterial 103.4 64 64 64 68 76 69 72 pressure (pO2) minus (0.42 times (POCT) age in years) Base Excess, standard mmol/L −2-+3 3.2 2.8 5.0 3.5 2.4 −2.2 −1.0 Lactate (BGA) mg/dl <16 11.2 12.2 12.6 11.4 15.7 15.0 11.8 cSO2 % 90.1 90.4 91.8 88.7 93.3 92.3 93.5 Blood collection arterial arterial arterial arterial arterial arterial arterial method Proteins sCD25 U/ml <900 NA NA NA NA NA NA NA Soluble transferrin mg/l 2.2-5.0 NA NA NA NA NA NA NA receptor (sTFR) Ferritin index 13.2 NA NA NA NA NA NA NA Interleukin 6 pg/ml 92.9 100.0 68.2 40.0 12.3 <2 NA Laboratory Days Day 25 Day 26 Day 27 Day 28 Day 29 Day 30 Day 31 results* Test Unit Normal value Apr. 16, 2020 Apr. 17, 2020 Apr. 18, 2020 Apr. 19, 2020 Apr. 20, 2020 Apr. 21, 2020 Apr. 22, 2020 Clinical Sodium mmol/l 135-146 151 152 149 151 151 149 148 chemistry Potassium mmol/l 3.4-4.8 4.70 4.59 5.07 5.16 4.55 4.98 5.33 Creatinine mg/dl 0.6-1.2 4.93 3.97 2.79 3.02 2.71 2.56 2.49 GFR using >60 10.3 13.4 20.5 18.6 21.2 22.7 23.5 CKD-EPI Urea mg/dl <45 242 194 127 136 142 145 145 Creatine kinase (CK) U/l <190 480 294 185 181 205 115 114 Troponin T (TNT) pg/ml <14 560 542 343 244 169 139 135 Lactate U/l <317 367 368 410 366 433 307 326 dehydrogenase (LDH) GOT/AST U/l <46 80 67 66 64 82 76 83 GPT/ALT U/l 50 41 39 41 41 54 54 55 Gamma-glutamyl- U/l 60 101 124 123 2 125 121 124 transferase (GGT) Iron μmol/l 14-32 7.6 4.7 4.5 5.3 11.5 5.8 5.3 Triglycerides mg/dl <150 215 150 156 123 116 115 103 Albumin g/l 30-50 28.9 30.4 30.5 31.0 33.3 31.1 31.6 C-reactive mg/l 5 18.4 120.5 160.3 134.9 101.9 75.4 79.9 protein (CRP) Hematology Leucocytes /nl 14-10 8.83 10.76 13.55 14.80 13.87 13.41 13.65 Neutrophil % 50-80 82.4 81.6 82.8 82.51 80.2 79.5 82.5 granulocytes (automated) Lymphocytes % 25-40 7.9 9.3 8.4 NA 9.5 9.2 7.8 (automated) Eosinophil % 2-4 .0 3.0 3.4 3.8 4.9 4.6 4.0 granulocytes (automated) Lymphocytes /nl 1.0-4.8 0.70 1.00 1.14 1.27 1.32 1.23 1.06 (absolute) Coagulation D-Dimer mg/l <0.5 5.35 5.31 3.79 4.81 5.94 6.92 7.05 IgG g/l 7.0-16.0 NA NA NA 18.16 NA NA NA IgA g/l 0.7-4.0 NA NA NA 6.62 NA NA NA IgM g/l 0.4-2.3 NA NA NA 1.74 NA NA NA Transferrin 2.0-3.6 1.67 1.59 1.49 1.38 1.42 1.59 1.57 Transferrin saturation % 16-45 18 12 12 15 32 15 13 Ferritin μg/l 30-300 143 134 157 163 201 244 277 Procalcitonin (PCT), ng/ml <0.05 0.29 0.35 0.25 0.24 0.23 0.26 0.29 sensitive POCT pH value (POCT 7.37-7.45 7.33 7.36 7.39 7.36 7.30 7.46 7.38 Carbon dioxide mmHg 35-45 52.5 35.0 31.1 31.9 30.7 27.5 30.7 partial pressure (pCO2) (POCT) Oxygen partial mmHg >arterial 103.4 69 42 63 71 62 74 64 pressure (pO2) minus (0.42 times (POCT) age in years) Base Excess, standard mmol/L −2-+3 −3.5 −2.3 −4.6 −6.7 −7.0 −7.4 −5.1 Lactate (BGA) mg/dl <16 12.9 14.2 14.6 13.3 14.0 15.9 10.6 cSO2 % 92.0 71.1 91.3 92.9 89.7 94.6 92.8 Blood collection arterial arterial arterial arterial arterial arterial arterial method Proteins sCD25 U/ml <900 NA NA NA NA NA NA NA Soluble transferrin mg/l 2.2-5.0 4.6 4.4 5.7 5.9 5.8 6.1 NA receptor (sTFR) Ferritin index 13.2 2.13 2.07 2.60 2.67 2.52 2.56 NA Interleukin 6 pg/ml 23.4 45.6 19.0 11.8 7.4 14.4 NA *Please note that in case lab testing was performed several times a day, the documented result is the worst result from that day. NA = not available
TABLE-US-00004 Patient #3 (age 62, female) Symptoms x x x x x x x x x x Hospitalisation x x x x x x x x x x Invasive ventilation x x x x x x Anti-infectives administered Pip/Taz/Caspo Pip/Taz Pip/Taz Pip/Taz/ Pip/Taz/ Pip/Taz/ Pip/Taz/ Pip/Taz/ Pip/Taz/ Pip/Taz/ Pip/Taz/ Aciclovir with HSV Caspo Caspo Caspo Caspo Caspo Caspo Caspo Caspo Aciclovir Aciclovir Aciclovir Aciclovir Immunomodulators administered Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Prednisolon 100 mg Prednisolon Pentaglobin 10 g Pentaglobin Pentaglobin SARS-COV-2 result positive negative negative Laboratory Days Day 21 Day 22 Day 23 Day 24 Day 25 Day 26 Day 27 Day 28 Day 29 Day 30 results* Test Unit Normal value Apr. 13, 2020 Apr. 14, 2020 Apr. 15, 2020 Apr. 16, 2020 Apr. 17, 2020 Apr. 18, 2020 Apr. 19, 2020 Apr. 20, 2020 Apr. 21, 2020 Apr. 22, 2020 Clinical Sodium mmol/l 135-146 150 150 155 154 149 145 140 140 138 NA chemistry Potassium mmol/l 3.4-4.8 4.55 5.55 4.63 4.68 4.46 4.81 4.72 4.45 4.49 NA Creatinine mg/dl 0.6-1.2 1.15 1.01 0.84 0.72 0.70 0.62 0.51 0.53 0.48 NA GFR using CKD-EPI >60 51.1 59.8 74.7 90.0 93.2 97.0 103.4 102.1 105.5 NA Urea mg/dl <45 85 79 77 58 36 24 17 26 21 NA Creatine kinase (CK) U/l <190 159 133 184 81 45 51 49 40 28 NA Troponin T (TNT) pg/ml <14 24 20 27 27 23 22 14 20 21 NA Lactate dehydrogenase U/l <317 517 530 349 363 388 382 475 280 349 NA (LDH) GOT/AST U/l <46 107 81 59 61 64 53 58 41 50 NA GPT/ALT U/l <50 92 83 55 57 57 58 60 52 52 NA Gamma- U/l <60 308 297 215 205 208 191 181 168 167 NA glutamyltransferase (GGT) Iron μmol/1 14-32 4.2 4.6 7.2 5.5 4.3 5.2 7.3 9.4 9.8 NA Triglycerides mg/dl <150 NA 176 193 198 167 190 221 176 200 NA Albumin g/l 30-50 32.5 34.0 28.0 30.2 29.9 29.2 30.6 33.0 33.8 NA C-reactive protein (CRP) mg/l <5 89.3 90.7 41.6 29.6 32.5 24.6 19.2 15.8 14.4 NA Hematology Leucocytes /nl 4-10 22.97 17.59 6.79 6.94 7.27 7.41 8.09 7.30 7.36 NA Neutrophil granulocytes % 50-80 73.7 80.6 85.2 79.0 76.2 73.2 77.5 74.9 73.0 NA (automated) Lymphocytes (automated) % 25-40 16.4 4.4 8.0 12.5 13.6 15.5 10.9 14.6 15.9 NA Eosinophil granulocytes % 2-4 5.2 0.3 0.3 3.2 4.0 4.8 6.1 4.1 3.8 NA (automated) Lymphocytes (absolute) /nl 1.0-4.8 2.88 0.77 0.54 0.87 0.99 1.15 0.88 1.07 1.17 NA Coagulation D-Dimer mg/l <0.5 10.99 5.31 3.11 4.66 5.56 7.94 8.71 5.77 6.58 NA IgG g/l 7.0-16.0 VA NA NA NA NA 13.81 13.39 NA NA NA IgA g/l 0.7-4.0 NA NA NA NA NA 2.47 2.39 NA NA NA IgM g/l 0.4-2.3 NA NA NA NA NA 1.21 1.20 NA NA NA Transferrin g/l 2.0-3.6 0.90 0.97 1.06 1.10 1.00 1.10 1.08 1.49 1.49 NA Transferrin saturation % 16-45 19 19 34 20 17 19 27 25 26 NA Ferritin μg/l 30-300 828 1181 500 357 365 343 360 305 379 NA Procalcitonin (PCT), ng/ml <0.05 0.10 2.62 2.70 1.07 0.43 0.26 0.15 0.13 0.09 NA sensitive POCT pH value (POCT) 7.37-7.45 6.90 6.97 7.51 7.49 7.48 7.47 7.45 7.48 7.48 NA Carbon dioxide partial mmHg 35-45 172.9 158.8 51.8 48.7 44.1 31.8 33.4 35.2 35.4 NA pressure (pCO2) (POCT) Oxygen partial pressure mmHg >arterial 103.4 77 68 67 75 68 82 77 69 84 NA (pO2) (POCT) minus (0.42 times age in years) Base Excess, standard mmol/L −2-+3 −4.0 11.4 12.1 8.1 6.4 −3.3 −0.7 −0.8 1.2 NA Lactate (BGA) mg/dl <16 16.5 10.7 11.6 7.3 7.9 6.7 7.4 7.3 7.0 NA cSO2 % 89.2 93.4 94.8 95.8 94.3 96.4 95.3 94.2 96.8 NA Blood collection method arterial arterial arterial arterial arterial arterial arterial arterial arterial NA Proteins sCD25 U/ml <900 NA NA NA NA NA NA NA NA NA NA Soluble transferrin receptor mg/l 2.2-5.0 NA NA 2.9 2.9 3.2 4.3 4.5 4.4 4.5 NA (sTFR) Ferritin index 3.2 NA NA 1.11 1.14 1.25 1.70 1.76 1.77 1.75 NA Interleukin 6 pg/ml 23.3 NA <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 NA *Please note that in case lab testing was performed several times a day, the documented result is the worst result from that day. NA = not available
TABLE-US-00005 Patient #4 (age 76, male) Symptoms x x x x x x x x Hospitalisation x x x x x x x x Invasive ventilation x x x x x x x x Anti-infectives administered Pip/Taz/Caspo Pip/Taz/ Pip/Taz/Caspo Meropenem/ Meropenem/Vanco Meropenem/Vanco Meropenem/Vanco Meropenem/ Meropenem/ Meropenem/Vanco Caspo Meropenem/ Vanco Vanco Vanco Vanco Immunomodulators administered Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Prednisolon 80 mg Prednisolon Pentaglobin 10 g Pentaglobin Pentaglobin SARS-COV-2 positive positive result Days Day 13 Day 14 Day 15 Day 16 Day 17 Day 18 Day 19 Day 20 Laboratory Test Unit Normal value Apr. 15, 2020 Apr. 16, 2020 Apr. 17, 2020 Apr. 18, 2020 Apr. 19, 2020 Apr. 20, 2020 Apr. 21, 2020 Apr. 22, 2020 Clinical Sodium mmol/l 135-146 142 142 141 142 139 139 139 139 chemistry Potassium mmol/l 3.4-4.8 4.60 5.07 4.90 5.61 5.19 4.82 5.08 4.43 Creatinine mg/dl 0.6-1.2 5.37 5.25 6.33 6.54 5.64 6.13 6.35 5.25 GFR using CKD-EPI >60 9.5 9.8 7.8 7.5 9.0 8.1 7.8 9.8 Urea mg/dl <45 148 119 134 133 122 140 126 115 Creatine kinase (CK) U/l <190 2207 1786 1182 689 1172 738 1013 563 Troponin T (TNT) pg/ml <14 881 993 809 990 830 975 857 658 Lactate dehydrogenase U/l <317 935 771 576 605 541 439 405 404 (LDH) GOT/AST U/l <46 93 83 68 57 60 55 82 106 GPT/ALT U/l <50 37 31 28 26 26 27 32 45 Gamma-glutamyltransferase U/l <60 26 26 32 26 26 24 30 35 (GGT) Iron μmol/l 14-32 1.4 1.7 2.5 2.7 2.7 1.8 1.8 2.6 Triglycerides mg/dl <150 167 139 142 177 133 116 97 96 Albumin g/l 30-50 30.9 29.7 29.7 27.6 29.7 29.2 28.4 25.8 C-reactive protein (CRP) mg/l <5 335.8 395.2 349.3 182.7 208.9 217.7 198.8 148.2 Hematology Leucocytes /nl 4-10 15.57 10.38 7.10 6.60 2.67 5.57 5.06 3.78 Neutrophil granulocytes % 50-80 93.4 87.3 91.0 82.8 84.5 88.9 85.8 80.4 (automated) 93.4 Lymphocytes (automated) % 25-40 3.6 8.1 4.3 6.3 8.4 6.3 7.0 9.2 Eosinophil granulocytes % 2-4 0.3 0.6 0.1 0.8 1.9 0.8 1.2 1.8 (automated) Lymphocytes (absolute) /nl 1.0-4.8 0.56 0.84 0.31 0.42 0.22 0.35 0.35 0.35 Coagulation D-Dimer mg/l <0.5 1.95 1.67 1.88 2.16 4.58 2.74 4.85 9.57 IgG g/l 7.0-16.0 NA 5.48 NA 6.71 NA NA NA NA IgA g/l 0.7-4.0 NA 2.96 NA 3.00 NA NA NA NA IgM g/l 0.4-2.3 NA 0.67 NA 0.76 NA NA NA NA Transferrin g/l 2.0-3.6 1.14 0.94 0.79 0.96 0.95 1.03 1.06 1.08 Transferrin saturation % 16-45 5 7 13 11 11 7 7 7 10 Ferritin μg/l 30-300 295 301 245 237 179 203 202 205 Procalcitonin (PCT), ng/ml <0.05 2.60 4.32 4.83 3.34 2.53 1.85 1.45 0.99 sensitive POCT pH value (POCT) 7.37-7.45 7.13 7.26 7.19 7.26 7.29 7.16 7.20 7.34 Carbon dioxide partial mmHg 35-45 73.8 54.5 61.3 50.6 48.9 65.2 59.6 35.8 pressure (pCO2) (POCT) Oxygen partial pressure mmHg >arterial 103.4 80 78 74 73 83 71 60 82 (pO2) (POCT) minus (0.42 times age in years) Base Excess, standard mmol/L −2-+3 −5.9 −7.1 −5.6 −5.6 −4.9 −7.4 −6.0 −3.9 Lactate (BGA) mg/dl <16 18.8 16.2 11.8 14.4 12.5 9.2 10.9 8.5 CSO2 % 94.5 92.9 91.4 92.7 95.2 91.2 89.6 94.4 Blood collection method arterial arterial arterial arterial arterial arterial arterial arterial Proteins sCD25 U/ml <900 1294 NA NA NA Soluble transferrin receptor mg/l 2.2-5.0 NA 4.7 4.4 4.6 4.9 5.0 5.1 (sTFR) Ferritin index 3.2 NA 1.90 1.84 1.94 2.18 2.17 2.21 Interleukin 6 pg/ml 789.0 482.0 25.9 169.0 1603.0 362.0 121.0 246.0 *Please note that in case lab testing was performed several times a day, the documented result is the worst result from that day. NA = not available
TABLE-US-00006 Patient #5 (age 62, male) Symptoms x x x x x x x x Hospitalisation x x x x x x x x Invasive ventilation Anti-infectives administered Azithromycin 4d (amb.) Meropenem Meropenem Meropenem Meropenem Meropenem Meropenem Meropenem Caspofungin (not all dates available) Unacid 5d (amb.) Caspofungin Caspofungin Caspofungin Caspofungin Caspofungin Caspofungin Caspofungin Meropenem Meropenem Caspofungin Immunomodulators administered Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Maraviroc Prednisolon 80 mg Prednisolon Pentaglobin 10 g Pentaglobin SARS-COV-2 result negative negative negative negative Days Day 15 Day 16 Day 17 Day 18 Day 19 Day 20 Day 21 Day 22 Laboratory results* Test Unit Normal value Apr. 15, 2020 Apr. 16, 2020 Apr. 17, 2020 Apr. 18, 2020 Apr. 19, 2020 Apr. 20, 2020 Apr. 21, 2020 Apr. 22, 2020 Clinical chemistry Sodium mmol/l 135-146 129 130 134 133 130 130 131 132 Potassium mmol/l 3.4-4.8 4.45 4.57 3.17 3.53 2.37 4.50 3.53 3.56 Creatinine mg/dl 0.6-1.2 0.52 0.49 0.49 0.53 0.48 0.58 0.55 0.51 GFR using CKD-EPI >60 114.3 117.1 117.1 113.4 118.1 109.3 111.7 115.2 Urea mg/dl <45 18 20 32 23 19 23 20 20 Creatine kinase (CK) U/l <190 298 248 128 157 127 100 92 67 Troponin T (TNT) pg/ml <14 54 53 39 50 50 56 55 53 Lactate dehydrogenase (LDH) U/l <317 598 414 366 384 360 350 318 360 GOT/AST U/l <46 150 91 82 77 51 40 41 37 GPT/ALT U/l <50 208 167 143 131 98 89 80 72 Gamma-glutamyltransferase U/l <60 139 121 105 114 94 87 82 (GGT) Iron μmol/l 14-32 2.9 3.9 11.6 5.8 4.1 4.2 4.3 4.8 Triglycerides mg/dl <150 92 100 112 125 118 140 148 162 Albumin g/l 30-50 31.8 30.7 27.9 28.3 28.4 32.4 32.4 31.0 C-reactive protein (CRP) mg/l <5 198.5 184.4 103.4 47.6 32.9 37.5 28.5 19.2 Hematology Leucocytes /nl 4-10 8.03 6.38 6.29 5.66 6.02 6.96 6.17 5.12 Neutrophil granulocytes % 50-80 84.7 88.8 79.9 81.2 82.6 79.1 80.2 74.6 (automated) Lymphocytes (automated) % 25-40 5.9 6.3 9.5 9.3 9.3 11.0 9.7 13.5 Eosinophil granulocytes % 2-4 0.6 0.2 0.2 0.5 0.9 1.1 1.3 1.6 (automated) Lymphocytes (absolute) /nl 1.0-4.8 0.47 0.40 0.60 0.53 0.58 0.77 0.60 0.69 Coagulation D-Dimer mg/l <0.5 5.08 4.88 4.25 3.92 3.25 2.38 2.09 1.68 IgG g/l 7.0-16.0 NA NA NA NA NA NA NA NA IgA g/l 0.7-4.0 NA NA NA NA NA NA NA NA IgM g/l 0.4-2.3 NA NA NA NA NA NA NA NA Transferrin g/l 2.0-3.6 0.89 1.02 0.92 1.06 1.08 1.22 1.30 1.33 Transferrin saturation % 16-45 13 15 50 22 15 14 13 14 Ferritin μg/l 30-300 2261 1976 2176 1597 1375 1295 1486 1480 Procalcitonin (PCT), sensitive ng/ml <0.05 0.25 0.24 0.19 0.12 0.08 0.07 0.06 0.05 POCT pH value (POCT) 7.37-7.45 7.47 7.48 7.52 7.55 7.52 7.55 7.56 7.49 Carbon dioxide partial pressure mmHg 35-45 30.4 30.8 29.5 24.6 18.9 24.8 25.4 28.2 (pCO2) (POCT) Oxygen partial pressure (pO2) mmHg >arterial 103.4 minus 38 74 61 60 69 63 66 62 (POCT) (0.42 times age in years) Base Excess, standard mmol/L −2-+3 −1.1 −4.0 2.4 2.9 −7.1 −2.9 −0.7 −3.2 Lactate (BGA) mg/dl <16 13.9 16.6 16.3 13.9 10.4 14.8 14.7 15.5 CSO2 73.0 93.9 90.9 91.7 93.3 91.4 93.5 91.9 Blood collection method arterial arterial arterial arterial arterial arterial arterial arterial Proteins sCD25 U/ml <900 824 NA INA NA NA NA NA NA Soluble transferrin receptor mg/l 2.2-5.0 NA 2.4 1.9 2.5 2.7 3 .2 3.5 3.5 (sTFR) Ferritin index 3.2 NA 0.73 0.57 0.78 0.85 1.03 1.10 1.10 Interleukin 6 pg/ml 118.0 12.5 4.4 20.9 21.5 12.4 5.6 3.9 *Please note that in case lab testing was performed several times a day, the documented result is the worst result from that day. NA = not available
[0635]
[0636] In summary, it has been shown that 5 patients with very severe COVID-19 course of disease and in whom 4 out of 5 required invasive mechanical ventilation that administration of Pentaglobin improved the clinical conditions in almost all cases.
[0637] Notably, because Pentaglobin was not given in the context of a controlled clinical trial administered doses varied from 10 g on one single day up to 22 g daily for 3 consecutive days. In total only one patient has received the approved dose for treatment of bacterial sepsis of 5 mL/kg for 3 consecutive days. Nevertheless, in most patients Interleukin-6 (IL-6) and other inflammatory markers dropped in a highly significant manner along with significant improvement of the clinical condition and most notably ventilation parameters.
[0638] Notably, all patients have received the CCR 5 inhibitor Maraviroc as additional experimental therapeutic. Although improvement of inflammatory parameters may at least in part be also attributed to Maraviroc there is clear evidence for a Maraviroc-independent temporal relationship between Pentaglobin administration, reduction of inflammation (11-6), and clinical improvement. Since also pro-inflammatory macrophage mediated effects of Maraviroc have been shown previously it is also possible that Maraviroc at least in some cases may even antagonize anti-inflammatory Pentaglobin effects.
[0639] The data of this pre-study collectively indicate significant improvement of severe COVID-19 disease and thereby confirm the concept of successful treatment of NAD disease. A controlled clinical study confirming the therapeutic principle mediated by Pentaglobin is warranted.
Example 22. Detection of Anti-Nuclear Autoimmunantibodies in COVID-19 Patient Sera
[0640] HEp2 Slides (Kallestad), which are commonly used to detect anti-nuclear autoimmune antibodies (ANA) in serum, were incubated with sera derived from three patients with severe COVID-19 (COV #6, COV #7, COV #8) treated in the intensive care unit or with sera from four healthy donors (HD #1, HD #2, HD #3, HD #4). The results are shown in
[0641] As shown in
[0642] A solution containing ANA included in the test kit was used as positive control. The total IgG concentration of each undiluted serum is indicated below.
[0643] The data show significantly stronger staining signal with sera from COVID-19 patients, indicating the presence of ANA in these sera. In contrast, healthy donor control sera showed little to no staining, indicating the absence of ANA.
[0644] These data support that the lack of natural antibodies (nABs) can result in the development of autoimmune antibodies during severe COVID-19 courses. The presence of these autoimmune antibodies provides evidence for recurring or long-lasting COVID-19 disease symptoms, supporting that sufficient levels of natural antibodies, provision of monoclonal natural IgMs or IgAs, or preparations enriched for natural antibodies (e.g. Pentaglobin®) in terms of the present invention can prevent the generation or reduce the levels of autoimmune antibodies.
Summary of Examples 1 to 22
[0645] The above data presented herein further support the model underlying the present invention and can be explained by the unusual feature of lung pathogens such as SARS-CoV-2, SARS, or H5N1 influenza virus, to trigger excessive formation of oxPL and OSE according to the mechanisms explained herein.
[0646] Viral replication and, consequently, accumulation of oxPL and OSE in lungs of infected patients trigger immune responses involving recruitment and local activation of monocytes, T cells and B cells, including those that produce protective virus-specific IgG and oxPL-specific IgM antibodies. Some of the B-cell clones that produce IgM antibodies toward oxPL and OSE, may become erroneously stimulated to undergo isotype class-switching from IgM to IgA or IgG, e.g., by presentation of antigenic oxidation-derived peptides or viral peptides to T cells. Such class-switched B cells then produce IgA or IgG autoantibodies that bind to oxPL and OSE displayed by many different oxidatively modified structures including apoptotic cells and oxLDL. Indeed, significantly elevated levels of IgG and IgA autoantibodies in the sera of severe COVID-19 patients that potently bound to oxLDL were found, and these autoantibodies likely form oxLDL-IgG- and oxLDL-IgA-immune complexes. OxPL-specific natural IgM antibodies protect from proinflammatory IgG and IgA autoantibodies in different ways, e.g. by blocking oxPL-binding to scavenger receptors, preventing the formation of pathogenic IgG- and IgA-immune complexes, and by facilitating the safe clearance of oxPL-exposing structures. However, in conditions when the balance between the formation and the neutralization of oxPL are disrupted, e.g., when individuals with low levels of endogenous natural IgM and possibly IgA1 antibodies become infected with SARS-CoV-2, the production of proinflammatory isotypes such as IgG and IgA2 autoantibodies get out of control and newly formed IgG- and IgA2-containing immune complexes of oxLDL eventually deposit at distinct sites in the body, e.g. in vascular walls, joints or glomerular capillary walls, where they potently trigger inflammatory responses through scavenger receptor-, Fc-gamma- and Fc-alpha-receptor-mediated activation of dendritic cells, macrophages and neutrophils.
[0647] Such autoimmune immune responses become the main driver of the systemic hyperinflammation state observed in the late phase of severe COVID-19 when no virus can be detected anymore, and in the long-term eventually culminate in Lupus-like autoimmune manifestations such as arthritis, vascular damage, acute kidney injury, induction of a procoagulant state and multiorgan damage, and possibly contribute to a phenomena known as Long-COVID. Therefore, individuals exhibiting reduced levels of oxPL- and OSE-specific natural IgM and possibly IgA1 antibodies, which otherwise would neutralize the proinflammatory functions of oxPL-exposing structures, are particularly prone to develop multiorgan hyperinflammation phenomena induced by immune complexes oxPL-IgG or oxPL-IgA2.
[0648] This concept further supports that treating COVID-19 patients with severe disease, or Long-COVID patients experiencing ongoing proinflammatory autoimmune conditions, with IgM antibodies, IgG2 or IgG4 antibodies, IgG antibodies carrying modifications to erase Fc-effector functions, or antigen-binding fragments thereof, recognizing oxPL and OSE, leads to significant reduction of the hyperinflammatory state by neutralizing oxPL- and OSE-exposing structures, thereby preventing the formation of pathogenic oxPL-IgG and oxPL-IgA containing immune complexes and facilitating their safe clearance.
Example 23. Two Monoclonal Antibodies that Bind to Different Danger-Associated Molecular Pattern (DAMPs), Including OSE and DNA
[0649] Two monoclonal antibodies that bind to different danger-associated molecular pattern (DAMPs), including OSE and DNA are characterized herein.
[0650] These antibodies are structurally described above with reference to SEQ ID NOs: 1 to 6 (corresponding to “Clone 1”) and SEQ ID NOs: 9 to 14 (corresponding to “Clone 2”), respectively (as well as with reference to SEQ ID NOs: 7 to 8 (corresponding to “Clone 1”) and SEQ ID NOs: 15 to 16 (corresponding to “Clone 2”), respectively).
[0651] The antibodies are of the IgM isotype and were isolated from single cell-sorted human B cells exhibiting the phenotype of CD5.sup.posCD20.sup.posCD27.sup.posCD43.sup.posCD70.sup.neg.
[0652] Human B cells exhibiting this phenotype were described to constitute the human counterpart of mouse B1 cells (Griffin, Holodick et al., 2011, J Exp Med, Vol. 208 (1)). Therefore, the two monoclonal antibodies disclosed herein possess characteristics of natural antibodies.
[0653] To test for the specificities of the monoclonal antibodies isolated by the method described herein, these antibodies were expressed as fully human IgM molecules and their antigen binding properties were tested. Two clones were identified that bound to at least two antigens that are well described targets for mouse and human natural antibodies. The following table summarizes the results of the binding assay for the two clones disclosed herein.
TABLE-US-00007 MDA- MDA- PC- OxLDL LDL LDL BSA BSA BSA DNA Clone 1 + − + + + − + Clone 2 + − − − − − +
[0654] Each clone of mouse or human monoclonal antibodies with characteristics of natural antibodies known in the art showed fine specificity for a clearly definable epitope.
[0655] For instance, some of the OSE-specific monoclonal IgM antibodies isolated from apoE-deficient mice (including clone E06 used in some of the experiments presented here) showed unique binding specificities for the phosphorylcholine (PC) headgroup exposed by oxLDL, oxPL such as 2-(5-oxovaleryl) phosphatidylcholine (POVPC), PC-protein adducts, or PC-containing polysaccharides, but not to MDA, while other clones specifically bound MDA-modified LDL and MDA-protein adducts, but not to PC epitopes (Shaw et al., 2000, J Clin Invest, Vol. 105(12)).
[0656] The mouse monoclonal IgM antibody 509 used in some experiments presented here above showed specificity toward oxidized phosphatidylethanolamine (PE), but not to oxidized phosphatidylcholine, oxidized phosphatidylserine, oxidized phosphatidic acid, or their native non-oxidized counterparts (Bochkov et al., 2016, Biomark Med., Vol. 10 (8)).
[0657] Monoclonal antibody LA25 showed exclusive specificity toward the OSE Malondialdehyde-acetaldehyde (MAA), but not to the structurally related OSE MDA (WO/2018/049083, PCT/US2017/050566).
[0658] It is, therefore, surprising that the monoclonal antibodies of “Clone 1” and “Clone 2” characterized above bind to at least two epitopes of DAMPs including oxidized LDL, MDA-proteins adducts, PC-protein adducts, and DNA.
[0659] Each of this epitope has been described to be implicated in chronic and acute proinflammatory diseases and to be targets for natural antibodies.
[0660] Because of this unique feature, the monoclonal antibodies described herein are particularly suitable to be used to treat patients suffering from inflammatory conditions associated with a natural antibody deficiency, as for instance acute pathogen-induced inflammation, acute lung injury, atherosclerosis, and many other conditions. In such patients, oxidative stress and innate immune responses generate multiple forms of DAMPs including oxPL, degradation products such as MDA, and DNA derived from apoptotic cells or neutrophil extracellular traps (NETs), all of which possess strong proinflammatory effects when not cleared efficiently from circulation, e.g. by natural antibodies. It is therefore desirable that monoclonal antibodies used to treat such patients recognize and neutralize as many DAMPs and OSE as possible to achieve anti-inflammatory and beneficial effects, and we demonstrated herein that only the combination of both monoclonal antibodies E06 and 509 showed significant inhibition of binding of autoreactive IgG antibodies in sera from COVID-19 patients to oxLDL. The unique characteristics of the monoclonal antibodies disclosed herein, therefore, provide important advantages over monospecific natural antibodies know in the art.