Compositions and methods for treating diseases associated with ricin intoxication

Abstract

The invention concerns a combination therapy for prophylaxis, or treatment of diseases resulting from ricin toxin poisoning comprising the administration of an anti-ricin antibody and a DNase.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING DISEASES ASSOCIATED WITH RICIN INTOXICATION

[0002] TECHNOLOGICAL FIELD

[0003] This invention generally relates to treating ricin-toxin poisoning by a combination of anti-ricin antibody and DNase.

[0004] BACKGROUND ART

[0005] References considered to be relevant as background to the presently disclosed subject matter are listed below:

[0006] Falach, R. et al (2018) Generation of highly efficient equine-derived antibodies for post-exposure treatment of ricin intoxications by vaccination with monomerized ricin. Toxins, 10, 466.

[0007] Falach, R. et al. (2020) Post-exposure anti -ricin treatment protects swine against lethal systemic and pulmonary exposures. Toxins, 12(6), 354.

[0008] Farrera, C. at al. (2013) Macrophage clearance of neutrophil extracellular traps is a silent process. J. Immunol. 191, 2647-2656

[0009] Gal, Y. et al (2014) Antibody / doxycycline combined therapy for pulmonary ricinosis: Attenuation of inflammation improves survival of ricin-intoxicated mice. Toxicol. Rep. 1, 496-504.

[0010] Gal, Y. et al. (2017) Treatments for pulmonary ricin intoxication: current aspects and future prospects. Toxins 9(10), 311Guo, R.F. at al. (2001) Regulatory effects of eotaxin on acute lung inflammatory injury. J. Immunol. 111(8), 5208-5218

[0011] Guo, Y. et al. (2015) Platelet-derived WNT antagonist Dickkopf-1 is implicated in ICAM-l / VCAM-l-mediated neutrophilic acute lung inflammation. Blood 126(19), 2220-2229

[0012] Izuhara, K. at al. (2016) Roles of periostin in respiratory disorders. Am.

[0013] J Respir. Crit. Care Med. 193(9), 949-956

[0014] Kovtun, A. et al. (2018) Neutrophils in Tissue trauma of the skin, bone, and lungs: two sides of the same coin. J. Immunol. Res. 1-12

[0015] Laukova, L. et al (2020) Deoxyribonucleases and their applications in biomedicine. Biomolecules 10(7): 1036

[0016] Lugano, R. et al. (2023) CD93 maintains endothelial function by limiting the phosphorylation and turnover of VE-Cadherin. FASEB J. 37(4), e22894

[0017] Lin and Liu, S.Y. (1986) Studies on the antitumor lectins isolated from the seeds of Ricinus communis (castor bean) Toxicon 24, 757-765

[0018] Lindau, D. et al. (2013) Primary blood neutrophils express a functional cell surface toll-like receptor 9. Eur. J. Immunol. 43, 2101-2113

[0019] Mutua, V. and Gershwin, L.J. (2020) A review of neutrophil extracellular traps (NETs) in disease: potential anti-NETs therapeutics. Clinical Reviews in Allergy and Immunology, https: / / doi.org / 10.1007 / sl2016-020-08804-7.

[0020] Narasaraju, T. et al. (2011) Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis. Am. J. Pathol. 179, 199-Noy-Porat, T., et al. (2016) Isolation of Anti -Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates. Toxins, 8(3), 64.

[0021] Poli, V. et al. (2023) Neutrophil intrinsic and extrinsic regulation of NETosis in health and disease. Trends Immunol. 31, 280-293

[0022] Sapoznikov, A. etal. (2015) Diverse profiles of ricin-cell interactions in the lung following intranasal exposure to ricin. Toxins 2015, 7: 4817-4831

[0023] Sapoznikov, A. etal. (2019) Early disruption of the alveolar-capillary barrier in a ricin-induced ARDS mouse model: Neutrophil-dependent and -independent impairment of junction proteins. Am. J. Physiol. Lung Cell. Mol. Physiol. 316(1), L255-L268

[0024] Shak et al., (1990) Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum PNAS 87:9188-9192

[0025] Silva, I.A.N. et al. (2022) A Semi-quantitative scoring system for green histopathological evaluation of large animal models of acute lung injury. Bio-protocol 12(16), e4493

[0026] Wang, Y. et al. (2009) Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J. Cell Biol. 184, 205-21

[0027] Wright, T.K. et al. (2016) Neutrophil extracellular traps are associated with inflammation in chronic airway disease: NETs in chronic airway disease. Respirology

[0028]

[0029] 467-475

[0030] US10,233,256

[0031] US20230416707

[0032] Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

[0033] BACKGROUNDNeutrophils eliminate pathogens by phagocytosis, degranulation (release of the neutrophil granule content by exocytosis), and by releasing neutrophil extracellular traps (NETs). To keep tissue homeostasis, the neutrophil reaction should be proportional to the threat, such that the invading pathogen is effectively eliminated without causing damage to the host (Poli, 2023).

[0034] The NETosis process, in which NETs are produced and released from neutrophils, is induced by a variety of pathogens, however, it also occurs in sterile inflammation. During sterile inflammation in a tissue, the neutrophils are recruited and play an important role in removing cellular debris to resume homeostasis in the tissue. However, in many cases, neutrophils react in an uncontrolled manner and the excessive NETosis leads to tissue damage (Kovtun, 2018). TLR9 receptors, expressed on the surface of activated neutrophiles, recognize cell-free DNA released from damaged cells (Lindau, 2013). The TLR9 activation serves as the primary event causing NETs formation and release from the neutrophils. Production of reactive oxygen species (ROS), which among others activate protein-arginine deiminase 4 (PAD4) that is a key enzyme in the induction of neutrophil extracellular traps (NETs) and mediates the citrullination of core histones H3, H4, and H2A by converting amine groups into ketones. This process results in the loss of the positive charge, facilitating chromatin decondensation. Additionally, PAD4 promotes chromatin decondensation by reducing the affinity between DNA and histones (Wang, 2009). The expansion of DNA due to the decondensation process causes rupture of the nuclear membrane, leading to the release of nuclear DNA into the cytoplasm.

[0035] Chromatin nets that are present in NETs, destruct the capillary alveolar barrier, damage the epithelial cells and cause over permeability of blood vessels (Narasaraju, 2011). The NETosis process contributes to the pathogenesis of inflammatory lung diseases, such as cystic fibrosis, chronic obstructive pulmonary disease (COPD) and allergic asthma (Wright 2016; Poli 2023).

[0036] Host DNases were identified as having a major role in the removal of NETs from an affected tissue. In addition, preprocessing of NETs by DNase I facilitated their clearance by macrophages (Farrera 2013).

[0037] Ricin is a toxic glycoprotein found in the seeds of the plant Ricinus communis. Increased permeability of the alveolar-capillary barrier and influx of protein-rich fluid into the interstitial and alveolar space are hallmark features of pulmonary ricinintoxication. These processes lead to the development of pulmonary edema, clinically manifesting as acute respiratory distress syndrome (ARDS), respiratory failure, and ultimately, death (Gal, 2017). Sapoznikov etal. (2019) have shown that massive amounts of neutrophils, which are recruited to the lungs following ricin exposure, secrete metalloproteinases that cause destruction of various types of junction proteins such as adherens, tight junction and gap junction proteins. These proteins connect the cells in the tissue and are vital to the integrity of the tissue. Destruction of junction proteins between blood vessel endothelial cells and lung epithelial cells causes alveolar-capillary hyper permeability leading to severe edema and suffocation. Anti ricin antibody treatment administered 24 hours post-exposure to ricin reduces lung permeability. Specific depletion of neutrophils or inhibition of metalloproteinases activity in mice intoxicated with ricin reduced the damage caused to the junction proteins, relieved lung edema and significantly postponed the time to death of the intoxicated mice (Sapoznikov, 2019).

[0038] Various antibodies directed against ricin toxin were developed, see for example US10,233,256.

[0039] The principal therapeutic use of human DNase has been to reduce the viscoelasticity of pulmonary secretions (including mucus) in diseases such as pneumonia and cystic fibrosis (CF), by hydrolyzing high molecular weight DNA that is present in such secretions, thereby aiding in the clearing of respiratory airways (Shak et al., 1990). Mucus also contributes to the morbidity of chronic bronchitis, asthmatic bronchitis, bronchiectasis, emphysema, acute and chronic sinusitis, and even the common cold. The pulmonary secretions of people having such diseases are complex materials that include mucus glycoproteins, mucopolysaccharides, proteases, actin, and DNA.

[0040] US20230416707 discloses a PEGylated long-acting DNase protein.

[0041] GENERAL DESCRIPTION

[0042] In one of its aspect the present invention provides a method of prophylaxis, or treatment of a disease or disorder resulting from ricin toxin poisoning comprising administering to a subject in need thereof a therapeutically effective amount of an antiricin antibody, or an antigen binding fragment thereof which binds to ricin toxin, and a DNase.

[0043] In another aspect, the present invention provides DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricintoxin, in prophylaxis, or treatment of a disease or disorder resulting from ricin toxin poisoning, in a subject in need thereof.

[0044] In a further aspect, the present invention provides a kit comprising:

[0045] (a) an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, or a pharmaceutical composition comprising same, and (b) a DNase, or a pharmaceutical composition comprising same, and optionally (c) instructions for use of the anti-ricin antibody and the DNase in combination for prophylaxis, or treatment of a disease or disorder resulting from ricin toxin poisoning.

[0046] In one embodiment, the DNase is DNase I.

[0047] In one embodiment, DNase l is a long-acting PEGylated DNase I.

[0048] In one embodiment, the PEGylated DNase I comprises or has an amino acid sequence having at least 90% homology with the amino acid sequence set forth in SEQ ID NO: 1.

[0049] In one embodiment, said anti-ricin antibody is an isolated anti-ricin monoclonal antibody or an isolated anti-ricin polyclonal antibody.

[0050] In some embodiments, said monoclonal antibody is a chimeric, a humanized or a human anti-ricin monoclonal antibody.

[0051] In some embodiments, said antigen binding fragment that binds to ricin toxin is selected from the group consisting of Fv, single chain Fv (scFv), Fab, F(ab)2' and any combination thereof.

[0052] In one embodiment, said anti-ricin antibody is a neutralizing antibody.

[0053] In some embodiments, said anti-ricin monoclonal antibody or antigen-binding fragment thereof which binds to ricin toxin, is one or more of:

[0054] (a) a monoclonal antibody comprising a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 9, CDRH2 denoted by SEQ ID NO. 10, CDRH3 denoted by SEQ ID NO. 11, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 12, a CDRL2 denoted by SEQ ID NO. 13, and a CDRL3 denoted by SEQ ID NO.

[0055] 14, or a variant thereof (also referred to as MH77);

[0056] (b) a monoclonal antibody, comprising the CDRH1 denoted by SEQ ID NO. 15, CDRH2 denoted by SEQ ID NO. 16, CDRH3 denoted by SEQ ID NO. 17, and CDRL1 denoted by SEQ ID NO. 18, a CDRL2 denoted by SEQ ID NO.19, and a CDRL3 denoted by SEQ ID NO. 20, or a variant thereof (also referred to as MH1); or

[0057] (c) a monoclonal antibody comprising the CDRH1 denoted by SEQ ID NO. 21, CDRH2 denoted by SEQ ID NO. 22, CDRH3 denoted by SEQ ID NO. 23, and CDRL1 denoted by SEQ ID NO. 24, a CDRL2 denoted by SEQ ID NO.

[0058] 25, and a CDRL3 denoted by SEQ ID NO. 26, or a variant thereof (also referred to MH75).

[0059] In some embodiments, said anti-ricin monoclonal antibody or antigen-binding fragment thereof which binds to ricin toxin, comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 27 (MH77), SEQ ID NO. 29 (MH1), or SEQ ID NO. 31 (MH75), and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 28 (MH77), SEQ ID NO. 30 (MH1), or SEQ ID NO. 32 (MH75).

[0060] In some embodiments, said anti-ricin monoclonal antibody or antigen-binding fragment thereof which binds to ricin toxin, is one or more of:

[0061] (a) a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 3 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 4 or a variant thereof;

[0062] (b) a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 5 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 6 or a variant thereof; or

[0063] (c) a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 7 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 8 or a variant thereof.

[0064] In one embodiment, said anti-ricin antibody and said DNase are administered to said subject prior to exposure to ricin toxin.

[0065] In one embodiment, said anti-ricin antibody and said DNase are administered to said subject after exposure to ricin toxin.In one embodiment, said anti-ricin antibody and said DNase are administered to said subject immediately after exposure to ricin toxin or between about 1 to about 96 hours after exposure to ricin toxin.

[0066] In one embodiment, said anti-ricin antibody and said DNase are administered to said subject about 24 hours after exposure to ricin toxin.

[0067] In one embodiment, said anti-ricin antibody and said DNase are administered to said subject simultaneously.

[0068] In one embodiment, said anti-ricin antibody and said DNase are administered to said subject sequentially.

[0069] In one embodiment, said anti-ricin antibody or said DNase are each independently administered to said subject as a single dose or as multiple doses.

[0070] In one embodiment, said anti-ricin antibody is administered to said subject as a single dose, and said DNase is administered to said subject in multiple doses.

[0071] In one embodiment, said anti-ricin antibody and said DNase are each independently administered to said subject by an administration route selected from a group consisting of intraperitoneal (ip), intravenous (iv), oral, sub-cutaneous, inhalation and intranasal administration.

[0072] In one embodiment, said method further comprises administering to the subject in need thereof an additional therapeutic agent (e.g., an anti-ricin agent, an antiinflammatory agent, an immunomodulatory agent an inhibitor of neutrophil recruitment (e.g., an anti-eotaxin antibody, or an Ly6G antibody), a NETosis inhibitor (e.g., a protein arginine deiminase 4 (PAD4) inhibitor), a neutrophil elastase (NE) or gasdermin D inhibitor, and / or an inhibitor of alveolar-capillary barrier disruption).

[0073] In one embodiment, said disease or disorder resulting from ricin toxin poisoning is selected from a group consisting of lung inflammation (pneumonia), ARDS, respiratory failure, gastrointestinal disorders (e.g., vomiting, diarrhea), dehydration, nausea, hemorrhages, anuria, cramps, fever, tachycardia, cardiac failure, vascular collapse and shock.

[0074] BRIEF DESCRIPTION OF THE DRAWINGS

[0075] To better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of a non-limiting example only, with reference to the accompanying drawings, in which:Fig. 1A is a Western blot showing levels of PAD4 (74kDa) in BALF taken from control mice (sham) and from mice at different time points (24h, 48h and 72h) post exposure to ricin. Mw - molecular weight ladder displays 75kDa band.

[0076] Fig. IB is a graph showing results of optical densitometry (band quantitation was done using the ImageJ software) of the Western blot bands for PAD4 shown in Fig. 1 A. The values are presented as average ± SEM (n=5-10 mice in each group, each point in the graph represents a single mouse), *p<0.05, ***p<0.001 compared to mice that were not intoxicated with ricin, n.s. - not significant.

[0077] Fig. 1C is a Western blot showing levels of CitH3 (15kDa) in BALF taken from control mice (sham) and from mice at different time points (24h, 48h and 72h) post exposure to ricin. Mw - molecular weight ladder which displays bands corresponding to 20, 15 and lOkDa from top to bottom.

[0078] Fig. ID is a graph showing results of optical densitometry for CitH3 of the Western blot bands for CitH3 shown in Fig. 1C. The values are presented as average ± SEM (n=5-10 mice in each group, each point in the graph represents a single mouse), *p<0.05, ***p<0.001 compared to mice that were not intoxicated with ricin, n.s. - not significant.

[0079] Fig. IE is a graph showing results of a commercial ELISA assay for CitH3 showing amounts (ng / ml) of citrullinated H3 (CitH3). The values are presented as average ± SEM (n=5-10 mice in each group, each point in the graph represents a single mouse), **p<0.01, ***p<0.001 compared to mice that were not intoxicated with ricin.

[0080] Fig- 2 is a graph showing quantification of NETosis by counting the number of neutrophils undergoing NET release per microscopic field. Five different fields were imaged per sample, and the number of NET -releasing neutrophils was counted in each field. Each data point represents an individual neutrophil undergoing NETosis within a given field.

[0081] Fig. 3A-C are graphs showing quantification of mean fluorescence intensity (MFI) for Ly6B (A), extracellular DNA (Sytox Green) (B), and citH3 (C) in lung sections at different time points post-exposure. Data are presented as mean ± SEM (n = 5 per group). For each lung section, five images were taken, and MFI was measured in three different fields per image. Each data point represents the MFI of a specific field within- lo an image. Statistical significance: *p < O.O5, **p < 0.01, ***p < 0.001 compared to nonexposed control mice, (n.s., not significant).

[0082] Fig. 4A is a schematic representation of the design of the experiment. Mice were intranasally exposed to 9.6 pg / kg (2LDso) ricin. At 24 hours post-exposure, mice received either an intravenous (i.v.) injection of an anti-ricin polyclonal antibody (RR003, 100 pl, single dose) or intraperitoneal (i.p.) administration of PRX-119 (5 mg / kg), which was continued daily until the end of the experiment. A group of mice received combined treatment of anti -ricin antibody and PRX-119. Body weight and survival were monitored for 14 days post-exposure.

[0083] Fig. 4B-4C are graphs showing changes in body weight over the course of the experiment in different treatment groups (4B), and survival rates of mice in the different treatment groups throughout the study (4C). Groups: ricin-exposed mice (n = 15), ricin + anti-ricin antibody (n = 15), ricin + anti-ricin antibody + PRX-119 (n = 40), and ricin + PRX-119 (n = 5). Statistical significance: *p < 0.05, comparing antibody-only treatment to combination treatment with antibody and PRX-119.

[0084] Fig.5A-C are graphs showing extracellular DNA levels (cell-free DNA) in BALF of mice following ricin exposure and different treatments. (A) Quantification of extracellular DNA levels in BALF of control and ricin-exposed mice at different time points post-exposure. (B) Quantification of extracellular DNA levels in BALF of ricin-exposed mice at 72 hours post-exposure, following different treatment regimens. (C) Quantification of extracellular DNA levels in BALF at 96- and 120-hours post-exposure in mice treated with anti-ricin antibody or with combination of anti-ricin-antibody and PRX-119. Each data point represents an individual mouse. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001 compared to untreated toxin-exposed mice in (A); comparison between antibody-only and combination treatment in (C). (n.s., not significant).

[0085] Fig.6A and 6D are images showing Western blot analysis of citH3 (A) and PAD4 (D) in BALF collected from control mice and mice intranasally exposed to 9.6 pg / kg (2LDso) ricin. Twenty-four hours post-exposure, mice received either an i.v. injection of an anti-ricin antibody (RR003, 100 pl, single dose) alone or in combination with i.p. administration of PRX-119 (5 mg / kg), which was continued daily until the end of the experiment. BALF was collected at 48 hours post-exposure, and protein levels were analyzed.Fig. 6B and 6E are graphs showing densitometric quantification of citH3 (B) and PAD4 (E) bands using ImageJ software.

[0086] Fig. 6C is a graph showing quantification of citH3 levels in BALF using a commercial ELISA assay. In 6B, 6C and 6E data are presented as mean ± SEM (n = 5-7 per group, each data point represents an individual mouse). Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001. (n.s., not significant).

[0087] Fig. 7A-C are graphs showing quantification of mean fluorescence intensity (MFI) for Ly6B (7A), extracellular DNA (Sytox Green) (7B), and citH3 (7C), representing NETosis intensity in lung sections at different time points post-exposure. Data are presented as mean ± SEM (n = 5 per group). For each lung section, five images were taken, and MFI was measured in three different fields per image. Each data point represents the MFI of a specific field within an image. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001. (n.s., not significant).

[0088] Fig. 8A-H are representative hematoxylin and eosin (H&E) stained lung sections from mice intranasally exposed to 9.6pg / kg (2LDso) ricin. Twenty-four hours postexposure, mice received either an i.v. injection of an anti-ricin antibody (RR003, 100 pl, single dose) alone or in combination with i.p. administration of PRX-119 (5 mg / kg), which was continued daily until the end of the experiment. Lungs were harvested 24 hours after treatment and processed for histological evaluation. Figs. 8A, 8C, 8E, and 8G are presented at x2 magnification. Figs. 8B, 8D, 8F, and 8H are presented at xlO magnification.

[0089] Fig. 81 is a graph showing quantification of lung histological injury score, assessed based on six different parameters across five randomly selected fields per lung section. Data are presented as mean ± SEM (n = 5 per group). Each data point represents the average histological injury score calculated from five different lung fields in each of the five analyzed lungs per group. Statistical significance: ***p < 0.001. (n.s., not significant).

[0090] Fig- 9 is a graph showing alveolar-capillary barrier permeability in mice following ricin exposure and treatment with anti-ricin antibody and PRX-119. Mice were intranasally exposed to 9.6 pg / kg (2LDso) ricin. Twenty-four hours post-exposure, mice received either an i.v. injection of an anti -ricin antibody (RR003, 100 pl, single dose) alone or in combination with i.p. administration of PRX-119 (5 mg / kg), which was continued daily until the end of the experiment. At 72 hours post-exposure, Evans Bluedye (EBD) was administered i.v. to assess alveolar-capillary barrier permeability. One hour after injection lungs were harvested, and dye concentration was quantified as a measure of vascular leakage (EBD extravasation). Data are presented as mean ± SEM (n = 5 per group), with each data point representing an individual mouse. Statistical significance: **p < 0.01, ***p < 0.001. (n.s., not significant).

[0091] Fig. 10A-D are graphs showing changes in protein levels in BALF of mice following ricin exposure and treatment with anti-ricin antibody and PRX-119. Mice were intranasally exposed to 9.6 pg / kg (2LDso) ricin. Twenty-four hours post-exposure, mice received either an i.v. injection of an anti -ricin antibody (RR003, 100 pl, single dose) alone or in combination with i.p. administration of PRX-119 (5 mg / kg), which was continued daily until the end of the experiment. BALF was collected at 72 hours postexposure, and protein levels were quantified using Luminex assay. Protein measurements included: (A) Eotaxin, (B) CD93, (C) Dkk-1, and (D) Periostin. Data are presented as mean ± SEM (n = 5 per group), with each data point representing an individual mouse, (n.s., not significant).

[0092] Fig. 11A is a schematic representation of the design of the experiment. Mice were exposed to 9.6 pg / kg (2LD50) ricin via intranasal administration. At 24 hours postexposure, mice received an intravenous (iv) injection of an anti-ricin monoclonal antibody (MH77, 300 pg, single dose). A group of mice received combined treatment of anti-ricin antibody and intraperitoneal (ip) administration of PRX-119 (5 mg / kg) which was continued daily until the end of the experiment. Body weight and survival were monitored for 14 days post-exposure.

[0093] Fig. 11B-11C are graphs showing changes in body weight over the course of the experiment in different treatment groups (B), and survival rates of mice in the different treatment groups throughout the study (C). Groups: ricin-exposed mice (n = 6), ricin + anti-ricin monoclonal antibody (n = 20) and ricin + anti-ricin monoclonal antibody + PRX-119 (n = 32). Statistical significance: *p < 0.05, comparing antibody-only treatment to combination treatment with antibody and PRX-119.

[0094] DETAILED DESCRIPTION OF EMBODIMENTS

[0095] The present disclosure is based on the surprising finding that a combination of two therapeutic agents, a NET degrading agent (i.e., a DNase) and an anti-ricin antibody, resulted in an improvement in lung disease and a significant increase in survival of miceexposed to ricin toxin. As shown in the Examples below, ricin intoxication increased NETosis in the lungs, DNase I inhibited the NETosis process, and the combined treatment showed superior effects on the survival of mice exposed to ricin as compared to the use of each of these agents alone. The combined effect can be considered as physiological synergy, namely the therapeutic agents act on different biological systems but their combination leads to a combined beneficial effect.

[0096] Therefore, the present disclosure provides a method of prophylaxis or treatment of a disease or disorder resulting from ricin toxin poisoning, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-ricin antibody, or any active fragment thereof, and a DNase.

[0097] In another aspect, the present invention provides DNase for use in combination with an anti-ricin antibody in prophylaxis, or treatment of a disease or disorder resulting from ricin toxin poisoning.

[0098] By the term "prophylaxis" as herein defined it is meant to provide preventive or prophylactic treatment, namely acting in a protective manner, to defend against or prevent the detrimental effects of ricin poisoning, namely administration before exposure to ricin toxin as a preventive measure given to a subject at risk of being poisoned by or exposed to ricin toxin.

[0099] The terms "treatment" , "treating", "treat" or forms thereof as used herein, mean preventing, ameliorating or delaying the onset of one or more clinical indications of poisoning or disease activity resulting from exposure to ricin toxin in a subject that was exposed to ricin toxin.

[0100] The term "ricin toxin" refers to a glycoprotein found in the seeds of Ricinus communis (R. communis, also known as castor oil plant). These seeds are also referred to herein as "castor beans". Ricin toxin is composed of two polypeptide chains, A and B, joined together by a disulfide bond. Being a powerful toxin, it may be used as a terrorism agent.

[0101] The terms “ricin toxin poisoning” and “ricin toxin intoxication” generally refer to the plethora of physiological responses arising in a subject exposed to ricin toxin either by inhalation, ingestion, parenteral exposure (e.g., intramuscular, intravascular, or intraperitoneal exposure), or eye contact.The term “disease or disorder resulting from ricin toxin poisoning” encompasses, but is not limited to lung inflammation (pneumonia), ARDS, respiratory failure, gastrointestinal disorders (e.g., vomiting, diarrhea), dehydration, nausea, hemorrhages, anuria, cramps, fever, tachycardia, cardiac failure, vascular collapse or shock.

[0102] Administration according to the present invention may be performed by any of the following routes: oral administration, intravenous, intramuscular, intraperitoneal, intrathecal or subcutaneous injection; intrarectal administration; intranasal administration (e.g., by inhalation), ocular administration or topical administration.

[0103] In specific embodiments, administration of the anti-ricin antibody according to the present invention is performed intravenously. In other embodiments the administration of DNase I according to the present invention is performed by intraperitoneal administration.

[0104] The term "subject in need thereof' as herein defined means warm-blooded animals, such as for example rats, mice, dogs, cats, guinea pigs, primates and humans at risk of being exposed to ricin toxin or anyone who has come in contact with ricin toxin, for example mail handlers, military personnel, laboratory workers and people who may have been exposed to ricin toxin during a bio-terror event.

[0105] The “therapeutically effective amount for purposes herein defined is determined by such considerations as are known in the art to cure, arrest or at least alleviate or ameliorate the medical conditions associated with ricin poisoning. For any preparation used in the methods of the invention, the dosage or the therapeutically effective amount can be estimated initially based on animal models known in the art, for example the mouse intoxication model demonstrated in the Examples below.

[0106] The term “DNase” (deoxyribonuclease) encompasses several types of proteins classified as DNase I (EC 3.1.21.1), DNase I LI, DNase I L2, DNase I L3, DNase II (EC 3.1.22.1), DNase Ila, DNasellb, and L-DNase II, based on their biochemical properties and enzymatic activities (Laukova et al., 2020).

[0107] In one embodiment, DNase is human DNase I.

[0108] In another embodiment, the DNase is modified human DNase I, wherein the modified DNase I is attached to at least two, or at least three, or at least four poly (alkylene glycol) moi eties, or is attached to from 2 to 7 poly (alkylene glycol) moi eties Furthermore, the modified DNase l is a long-acting, namely has a prolong pharmacokinetic profile ascompared with the unmodified form of DNase I. In an embodiment, at least one of the alkylene glycol moieties are polyethylene glycol moieties. Thus, in an embodiment, DNase I, is PEGylated DNase I.

[0109] In a specific embodiment, DNase I is the PEGylated long-acting DNase PRX-119 (as described in US20230416707, incorporated herein by reference). PRX-119 harbors enhanced stability, and extended duration in the bloodstream.

[0110] In a specific embodiment the PEGylated DNase I protein comprises or has the amino acid sequence set forth in SEQ ID NO: 1 :

[0111] LKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGK LLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCE PCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQE KWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHC AYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK

[0112] In another specific embodiment, the PEGylated DNase I, is encoded by a nucleic acid sequence comprising or having the nucleic acid sequence as set forth in SEQ ID NO:2:

[0113] atgattgtgctttctgtgggatctgcttcttcttctccaattgtggtggtgttctctgtggctcttcttcttttctacttctctga gacttctcttggccttaaaatcgctgctttcaacatccaaactttcggagagactaagatgtctaacgctactcttgtgtcctacatc gttcagattctctccagatacgatattgctcttgttcaggaagttagggattctcaccttactgctgtgggaaagcttcttgataacct caatcaggatgctccagatacttaccactacgttgtgtctgaaccacttggaagaaactcctacaaagagcgttacctctttgttta ccgtccagatcaagtttctgctgtggattcctactactacgatgatggatgtgagccatgcggaaacgatactttcaatagagag ccagctatcgttcgttttttcagtaggttcactgaagttcgtgagtttgctattgtgccacttcatgctgctccaggtgatgctgttgct gagattgatgctctctacgatgtgtaccttgatgttcaagagaagtggggattggaggatgttatgctcatgggagatttcaatgc tggatgctcttatgttaggccatctcagtggtcatctattaggctttggacttccccaactttccaatggcttatcccagattccgctg atacaactgctactccaactcattgtgcttacgataggattgtggtggctggaatgcttcttagaggtgctgttgttccagattctgc tctcccattcaatttccaagctgcttacggactttctgatcaacttgctcaggctatttctgatcactacccagttgaggtgatgttga agtgatga

[0114] The underlined nucleic acids encode the signal peptide which is not included in the mature DNase polypeptide

[0115] DNase in accordance with the invention also encompasses variants of the DNase polypeptide. By the term “variant” it is meant DNase polypeptides with sequences of amino acids or encoding nucleotides that are different from the sequences specificallyidentified herein, namely, in which one or more amino acid residues or nucleotides are deleted, substituted or added.

[0116] It should be appreciated that by the term "added ", as used herein it is meant any addition(s) of amino acid residues to the sequence described herein. For example, the variant DNase proteins of the invention may be extended at their N-terminus and / or C-terminus with various amino acid residues.

[0117] Variants also encompass various amino acid substitutions. An amino acid ^substitution is the result of replacing one amino acid with another amino acid which has similar or different structural and / or chemical properties. Amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0118] Variants further encompass conservative amino acid substitutions. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include the amino acids aspartic acid and glutamic acid.

[0119] Each of the following eight groups contains other exemplary amino acids that are conservative substitutions for one another:

[0120] 1) Alanine (A), Glycine (G);

[0121] 2) Aspartic acid (D), Glutamic acid (E);

[0122] 3) Asparagine (N), Glutamine (Q);

[0123] 4) Arginine (R), Lysine (K);

[0124] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0125] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0126] 7) Serine (S), Threonine (T); and

[0127] 8) Cysteine (C), Methionine (M).

[0128] Conservative nucleic acid substitutions are nucleic acid substitutions resulting in conservative amino acid substitutions as defined above.As used herein, the term “ amino acid" or “ amino acid residue refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

[0129] Variant sequences refer to amino acid or nucleic acids sequences that may be characterized by the percentage of the identity of their amino acid or nucleotide sequences, respectively, with the amino acid or nucleotide sequences described herein (namely the amino acid or nucleotide sequences of DNase I herein described).

[0130] Therefore, in some embodiments variant sequences as herein defined refer to amino acid sequences of DNase I, each having a sequence of amino acids with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity when compared to the sequence of the DNase I (SEQ ID NO: 1) described herein.

[0131] In some embodiments variant sequences as herein defined refer to nucleic acid sequences that encode the DNase I protein, having a sequence of nucleotides with at least 70% or 75% of sequence identity, around 80% or 85% of sequence identity, around 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity when compared to the nucleic acid sequence encoding DNase I (SEQ ID NO: 2) described herein.

[0132] The term “anti-ricin antibody refers to a polypeptide encoded by an immunoglobulin gene or functional fragments thereof that specifically binds an antigen, namely ricin toxin.

[0133] The anti-ricin antibody in accordance with the invention may be a monoclonal anti-ricin antibody or a polyclonal anti -ricin antibody (e.g., a plasma preparation from a vaccinated animal).

[0134] The terms monoclonal antibody , monoclonal anti-ricin antibody or "antiricin mAb as herein defined refer to a population of substantially homogenous antibodies, i.e., the individual antibodies comprising the population are identical except for possibly naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are directed against a single antigenic site (epitope).

[0135] Monoclonal antibodies may be prepared and purified by any method known in the art. For example, monoclonal antibodies may be prepared from B cells taken from the spleen or lymph nodes of immunized animals (e.g. rats, mice, monkeys, or horses), by fusion with immortalized B cells under conditions which favor the growth of hybrid cells or synthesized by cloning the genes that encode the antibody's variable regions into expression systems, such as bacteria, yeast, or mammalian cells. Monoclonal antibodiesmay be also recombinant or synthetic antibodies created by computational design (e.g., using artificial intelligence (Al) models).

[0136] Immunization of animals may be carried out by any method known to the skilled person, for example by immunizing horses, or monkeys. In a non-limiting example, immunized monkeys are sacrificed, and samples are taken from their blood and lymphatic nodes to isolate mRNA that will be used for variable heavy and variable light (VH / VL) chain amplification and further used for example for constructing a phage display library, to select active antibodies. Based on the results obtained from a phage display library, full length antibodies are produced, as known in the art.

[0137] Purification of monoclonal antibodies may be performed using any method known in the art, for example by affinity chromatography, namely, by using an affinity column to which a specific epitope (or antigen) is conjugated. Alternatively, purification of antibodies may be based on using protein A column chromatography, as described below.

[0138] An exemplary antibody structural unit comprises a tetramer, as known in the art. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light chain" and one “heavy chain The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen (or epitope) recognition.

[0139] Thus, the terms “heavy chain variable region (VH) and “light chain variable region (VL) refer to these heavy and light chains, respectively. More specifically, the variable region is subdivided into hypervariable and framework (FR) regions. Hypervariable regions have a high ratio of different amino acids in a given position, relative to the most common amino acid in that position. Four FR regions which have more stable amino acids sequences separate the hypervariable regions. The hypervariable regions directly contact a portion of the antigen's surface. For this reason, hypervariable regions are herein referred to as "complementarity determining regions", or "CDRs", the CDRs are positioned either at the heavy chain of the antibody ("a heavy chain complementarity determining region") or at the light chain of the antibody (a "light chain complementarity determining region").

[0140] From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to asCDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located.

[0141] Thus, the complementarity determining regions CDRH1, CDRH2 and CDRH3 refer to the three complementarity determining regions starting from the N-terminus of the antibody’s heavy chain (also referred to herein as heavy chain complementarity determining region) and the complementarity determining regions CDRL1, CDRL2 and CDRL3 refer to the three complementarity determining regions starting from the N-terminus of the antibody’s light chain (also referred to herein as light chain complementarity determining region).

[0142] In an embodiment, the monoclonal antibodies in accordance with the invention are isolated, neutralizing antibodies, namely antibodies capable of neutralizing the activity of the ricin toxin, or any antigen-binding fragment thereof which binds to ricin toxin.

[0143] In some embodiments the isolated monoclonal antibody according to the invention is the antibody also referred to herein as "MH77', namely a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 3 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 4 or a variant thereof.

[0144] In some embodiments the isolated monoclonal antibody according to the invention is the antibody also referred to herein as "MH1", namely a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 5 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 6 or a variant thereof.

[0145] In yet further embodiments the isolated monoclonal antibody according to the invention is also referred to herein as "MH75", namely a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 7 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 8 or a variant thereof.

[0146] MH77 and MH75 bind to the RTB subunit of ricin.

[0147] MH1 binds to the RTA subunit of ricin.

[0148] The amino acid sequences of the variable region of the heavy and the light chains of the antibodies MH77, MH1, and MH75 are shown in Table 1 below. The complementarity determining regions (CDRs) are shown in grey boxes.Table 1 Amino acid sequences of the antibodies' heavy and light chains

[0149]

[0150] In specific embodiments, the isolated monoclonal antibody or any antigenbinding fragment thereof which binds to ricin toxin, is one or more of

[0151] a. a monoclonal antibody comprising a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 9, CDRH2 denoted by SEQ ID NO. 10, CDRH3 denoted by SEQ ID NO. 11, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 12, a CDRL2 denoted by SEQ ID NO. 13, and a CDRL3 denoted by SEQ ID NO. 14, or a variant thereof (also referred to as MH77);

[0152] b. a monoclonal antibody, comprising the CDRH1 denoted by SEQ ID NO.

[0153] 15, CDRH2 denoted by SEQ ID NO. 16, CDRH3 denoted by SEQ ID NO.

[0154] 17, and CDRL1 denoted by SEQ ID NO. 18, a CDRL2 denoted by SEQID NO. 19, and a CDRL3 denoted by SEQ ID NO. 20, or a variant thereof (also referred to as MH1); or

[0155] c. a monoclonal antibody comprising the CDRH1 denoted by SEQ ID NO.

[0156] 21, CDRH2 denoted by SEQ ID NO. 22, CDRH3 denoted by SEQ ID NO.

[0157] 23, and CDRL1 denoted by SEQ ID NO. 24, a CDRL2 denoted by SEQ ID NO. 25, and a CDRL3 denoted by SEQ ID NO. 26, or a variant thereof (also referred to MH75).

[0158] The CDRs of the antibodies referred to herein are presented in Table 2.

[0159] Table 2 Amino acid sequences of the antibodies heavy and light chain CDRs

[0160]

[0161] By the term “variant” it is meant sequences of amino acids or nucleotides that are different from the sequences specifically identified herein, namely, in which one or more amino acid residues or nucleotides are deleted, substituted or added.

[0162] The nucleic acid sequences of the heavy and the light chains of the antibodies are shown in Table 3 below.

[0163] Table 3 Nucleic acid sequences of the antibodies heavy and light chains

[0164]

[0165]

[0166] In some embodiment variant sequences as herein defined refer to nucleic acid sequences that encode the heavy and light chain variable regions, each having a sequence of nucleotides with at least 70% or 75% of sequence identity, around 80% or 85% of sequence identity, around 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity when compared to the sequences of the heavy and light chain variable regions described herein.

[0167] In some embodiments the isolated monoclonal antibody or any antigen-binding fragment thereof according to the invention is wherein said antibody comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 27 (MH77), SEQ ID NO. 29 (MH1), or SEQ ID NO. 31 (MH75), and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 28 (MH77), SEQ ID NO. 30 (MH1), or SEQ ID NO. 32 (MH75).

[0168] In further embodiments the isolated monoclonal antibody or any antigen-binding fragment thereof according to the invention is wherein the heavy chain variable region of the antibody is encoded by a nucleic acid sequence which is at least 70% identical to thenucleic acid sequence denoted by SEQ ID NO. 27 and wherein the light chain variable region of the antibody is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 28 (MH77).

[0169] In other embodiments the isolated monoclonal antibody or any antigen-binding fragment thereof according to the invention is wherein the heavy chain variable region of the antibody is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 29 and wherein the light chain variable region of the antibody is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 30 (MH1).

[0170] In some embodiments the isolated monoclonal antibody or any antigen-binding fragment thereof according to the invention is wherein the heavy chain variable region of the antibody is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 31 and wherein the light chain variable region of the antibody is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 32 (MH75).

[0171] In one embodiment, the method of the invention comprises administration of one or more anti-ricin monoclonal antibodies (e.g., the antibodies denoted MH77, MH75, and MH1) or antigen binding fragments thereof which bind to ricin toxin, and a human DNase I.

[0172] In a specific non-limiting embodiment, the method of the invention comprises administration of the anti-ricin antibody denoted as MH77, or antigen binding fragment thereof which binds to ricin toxin, which comprises a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 9, CDRH2 denoted by SEQ ID NO. 10, CDRH3 denoted by SEQ ID NO. 11, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 12, a CDRL2 denoted by SEQ ID NO. 13, and a CDRL3 denoted by SEQ ID NO. 14, and a PEGylated DNase I comprising or having an amino acid sequence having at least 90% homology with the amino acid sequence set forth in SEQ ID NO: 1.

[0173] In a specific non-limiting embodiment, the method of the invention comprises administration of a polyclonal anti-ricin antibody, and a PEGylated DNase I comprising or having an amino acid sequence having at least 90% homology with the amino acid sequence set forth in SEQ ID NO: 1.In another non-limiting example, anti-ricin antibodies are generated in horses e.g., as described in Falach et al (2018). Briefly, plasmapheresis is conducted once in three months utilizing a veterinary plasmapheresis instrument (e.g., using a plasma collection system, PCS-2, Haemonetics Corporation, Braintree, MA, USA) and hyperimmune plasma are stored at -20 °C until further use. Anti-ricin F(ab’)2 preparations are generated by pepsin (1200 U / mL pepsin A from porcine stomach mucosa, e.g., from Sigma, Steinhaim, Germany) cleavage of the Fc fragments, purification of the F(ab’)2 fragments and concentration of the antitoxin solution.

[0174] In the process of anti-ricin antibody preparation, for the vaccination of animals, the ricin toxin may be prepared using any method known to the skilled person, for example by extraction from seeds of R. communis essentially as described in Gal et al (2014). Briefly, seeds are homogenized in 5% acetic acid / phosphate buffer (ISfeHPC , pH7.4), the homogenate is centrifuged and the clarified supernatant containing the toxin is subjected to ammonium sulfate precipitation (60% saturation). The precipitate is dissolved in phosphate buffered saline (PBS) and dialyzed extensively against the same buffer. The toxin preparation appears on a Coomassie Blue-stained non-reducing 10% polyacrylamide gel as 2 major bands of molecular weight, approximately 65kDa (= ricin toxin, -80%) and 120 kDa (= ricinus communis agglutinin (RCA), -20%). Protein concentration can be determined by 280 nm absorption in a Nanodrop device (e.g., by Thermo Fisher Scientific, Waltham, MA, USA).

[0175] In an embodiment, a monomeric form of the toxin is prepared, for example as described in Falach et al (2018).

[0176] Purification of monoclonal antibodies may be performed using any method known in the art, for example by affinity chromatography, namely, by using an affinity column to which a specific epitope (or antigen) is conjugated. Alternatively, purification of antibodies may be based on using protein A column chromatography.

[0177] The efficacy of binding of antibodies or antigen-binding fragments thereof to ricin-toxin may be determined by using any method known in the art, for example using ELISA or BIAcore analysis.

[0178] The isolated monoclonal antibody according to the invention may be a non-human antibody (e.g., an equine or monkey antibody), a chimeric, a humanized or a human antibody.The term "chimeric" antibodies as herein defined refers to antibodies in which a portion of the heavy and / or light chain is derived from a particular species, while the remainder of the chain(s) is derived from another species, as well as fragments of such antibodies, which exhibit the same biological activity (namely biding to the ricin toxin). Chimeric antibodies may be prepared by any method known in the art.

[0179] It is appreciated that "humanized" forms of non-human antibodies are antibodies that contain a human-derived immunoglobulin framework with minimal sequences derived from non-human immunoglobulin at the CDRs and optionally at additional relevant positions. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, horse or nonhuman primate having the desired specificity, affinity, and activity.

[0180] The term "human antibody" as used herein refers to an antibody that possesses an amino acid sequence corresponding to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies.

[0181] Preparation of humanized and human antibodies is well known in the art.

[0182] The present invention further encompasses any antigen-binding fragments of the anti-ricin antibody of the invention. Such antigen-binding fragments may be for example Fab and F(ab')2, which are capable of binding antigen. Such fragments may be produced by any method known in the art, for example by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).

[0183] Therefore, in some embodiments the anti-ricin antibody fragment according to the invention is selected from the group consisting of Fv, single chain Fv (scFv), Fab, F(ab)2(and any combination thereof.

[0184] In some embodiments the anti-ricin antibody according to the invention is a neutralizing antibody.

[0185] The term “Neutralizing antibody'’'’ as herein defined refers to an antibody which is capable of blocking, preventing or at least reducing the biological activity of ricin toxin, for example, blocking, preventing or at least reducing its ability to inactivate the 28 S ribosomal subunit.

[0186] The ability of the antibody to neutralize the toxicity of ricin toxin may be monitored by any method known in the art, e.g., using a viability assay which follows thesurvival of cells that were exposed to ricin toxin and incubated in the presence of the antiricin antibody, for example, but not limited to, in a Hela toxicity cell-based assay.

[0187] In some embodiments the method according to the invention further comprises administering to a subject in need thereof an additional therapeutic agent, e.g., an antiricin agent, an anti-inflammatory or immunomodulatory agent, an inhibitor of neutrophil recruitment (e.g., an anti-eotaxin or Ly6G antibody), a NETosis inhibitor (i.e., an agent preventing NET formation, for example, but not limited to, a protein arginine deiminase 4 (PAD4) inhibitor, see Mutua and Gershwin (2020) for a list of potential anti-NETs therapeutics), neutrophil elastase (NE) or gasdermin D inhibitor and an inhibitor of alveolar-capillary barrier disruption.

[0188] The term "additional anti-ricin agent" as herein defined refers to any agent known in the art for the treatment of ricin poisoning. In some embodiments the additional anti-ricin agent in accordance with the invention is a sugar analogue or low-density lipoprotein receptor-related protein-1 (LRP1) receptor antagonist or inhibitor (that prevents ricin binding to its target), and / or an inhibitor of the catalytic subunit of ricin toxin (for example azidothymidine).

[0189] According to the invention, the anti-ricin antibody or any antigen-binding fragment thereof, and the DNase, as herein defined (or pharmaceutical compositions comprising same) may be administered to the subject prior to or after exposure to ricin toxin.

[0190] In some embodiments the anti-ricin antibody or any antigen-binding fragment thereof, and the DNase, as herein defined (or pharmaceutical compositions comprising same) are administered to the subject immediately after exposure to ricin toxin or between about 1 to about 96 hours after exposure to ricin toxin.

[0191] As used herein the term "immediately" encompasses the instant time frame following detection of ricin poisoning, e.g. minutes after detection.

[0192] By way of a non-limiting example, the anti-ricin antibody or any antigen-binding fragment thereof, and the DNase, as herein defined (or pharmaceutical compositions comprising same) are administered to the subject about 1, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, or about 96 hours after exposure to ricin toxin, for example, but not limited to, 24 hours after exposure.The anti-ricin antibody or any antigen-binding fragment thereof, and the DNase, as herein defined (or pharmaceutical compositions comprising same) may be administered to the subject simultaneously or sequentially.

[0193] In some further embodiments the anti-ricin antibody or any antigen-binding fragment thereof, and the DNase, as herein defined (or pharmaceutical compositions comprising same) are each independently administered to the subject as a single dose or as multiple doses.

[0194] In an embodiment, said anti-ricin antibody or any antigen-binding fragment thereof is administered to the subject as a single dose, and the DNase is administered to the subject in multiple doses. The dosing regimen, i.e., the number of administered doses can be defined by the physician based on the pace of the patient’s recovery, thus treatment may be administered to the patient until disease resolution.

[0195] The anti-ricin antibody and the DNase in accordance with the invention may be provided in the same pharmaceutical composition or in separate pharmaceutical compositions.

[0196] Thus, a pharmaceutical composition'’'’ in accordance with the invention generally comprises an anti-ricin antibody or any antigen-binding fragment thereof as herein defined, and / or a DNase as herein defined and a buffering agent, an agent which adjusts the osmolarity of the composition and optionally, one or more pharmaceutically acceptable carriers, excipients and / or diluents as known in the art.

[0197] As used herein the term pharmaceutically acceptable carrier, excipient or diluent includes any solvents, dispersion media, coatings, antibacterial and antifungal agents and the like, as known in the art. The carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject. Except as any conventional media or agent is incompatible with the active ingredient, its use in the pharmaceutical composition is contemplated.

[0198] In some further embodiments the pharmaceutical compositions according to the invention further comprise an additional therapeutic agent (for example, but not limited to, an anti-ricin agent, an anti-inflammatory agent, an inhibitor of neutrophil recruitment (e.g., an anti-eotaxin antibody), a NETosis inhibitor (i.e., an agent preventing NETformation, for example, but not limited to, a protein arginine deiminase 4 (PAD4) inhibitor, see Mutua and Gershwin (2020) for a list of potential anti-NETs therapeutics), neutrophil elastase (NE) inhibitor and an inhibitor of alveolar-capillary barrier disruption.

[0199] The present invention further provides a kit comprising an anti-ricin antibody, or a pharmaceutical composition comprising same, and a DNase, or a pharmaceutical composition comprising same, together with instructions for use of these elements in combination for prophylaxis, or treatment of a disease or disorder resulting from ricin toxin poisoning.

[0200] The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range.

[0201] EXAMPLES

[0202] Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present disclosure to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the claimed invention in any way.

[0203] Standard molecular biology protocols known in the art not specifically described herein are generally followed essentially as in Sambrook & Russell, 2001.

[0204] Standard medicinal chemistry methods known in the art not specifically described herein are generally followed essentially in the series “Comprehensive Medicinal Chemistry” by various authors and editors, published by Pergamon Press.

[0205] Experimental procedures

[0206] Animals

[0207] CD1 outbred mice (females weighing 27-32g) were purchased from Charles River Laboratories (England) and were kept in an animal house. The mice were fed with a standard diet and kept in a dedicated facility at least 4-8 days prior to commencement of the experiment, under a 12-hour light regime. The animals had free access to food and water. The experimental protocols were approved by the Ethics Committee of the Biological Institute.Preparation of the ricin toxin

[0208] Crude ricin was prepared from seeds of Ricinus communis (R communis) as previously described (Lin and Liu, 1986). Briefly, seeds were homogenized in a Waring blender in 5% acetic acid / phosphate buffer (ISfeHPCU, pH 7.4). The homogenate was centrifuged and the supernatant containing the toxin was subjected to ammonium sulphate precipitation (60% saturation). The precipitate was dissolved in PBS and dialyzed against the same buffer. The crude ricin preparation was loaded onto a Coomassie blue stained non-reducing 10% polyacrylamide gel visualized as two bands one with molecular weight of 65kDa (the ricin toxin ~ 80%) and the second band with a molecular weight of about 120kDa (Ricinus agglutinin, ~ 20%). Protein concentration was determined by absorbance at 280nm (E=203,000 M^cm’^in a Nano-Drop spectrophotometer (Thermo Scientific, Waltham, MA, USA).

[0209] Administration of ricin toxin / anti-ricin antibody / PRX-119

[0210] Mice were anesthetized by intraperitoneal injection of 0.2ml of a combination of ketamine (Ketaset, lOOmg / ml, Fort Dodge, USA) and xylazine (Chanazine 2%, 20mg / ml, Chanelle, Ireland) at a ratio of 1ml ketamine, 0.5ml xylazine, and 9 ml PBS. Ricin was administered intranasally at a dose of 9.6 pg / kg (2LDso), at a volume of 25 pl per nostril.

[0211] Polyclonal antibody treatment was given once by intravenous administration (100 pl), 24 hours after exposure to ricin. The antibody preparation was an F(ab)2 antibody fragment, purified from hyperimmune plasma of horses vaccinated with ricin (RR003, a purified F(ab)2 antibody fragment, with a titer of 1716 Neutralizing Israeli Units) (Falach et al, 2020).

[0212] Monoclonal MH77 antibody treatment was given once by intravenous administration (300 pg), 24 hours after exposure to ricin. The MH77 antibody was developed by immunizing a rhesus macaque with a ricin subunit-based vaccine, followed by the isolation of the antibody from an immune scFv phage-display library. It was subsequently reformatted into a full-length chimeric IgG consisting of macaque variable regions and human constant regions. As a K isotype antibody, MH77 specifically targets the ricin B subunit (RTB) at a distinct, non-overlapping epitope (Noy-Porat et al, 2016).

[0213] PRX-119 (Long-Acting DNase I) was administered intraperitoneally 5mg / kg at a volume of 100 pl. Treatment commenced 24 hours after exposure to ricin and was administered once a day for 14 days.Protein quantitation using Western blot (WB)

[0214] Bronchoalveolar lavage fluid (BALF) was taken from healthy mice or from mice subjected to ricin poisoning, with or without treatment with anti-ricin antibodies and / or PRX-119. The BALF was taken after 24, 48 or 72 hours from the ricin administration. Levels of the proteins citrullinated Histone 3 (CitH3), and PALM were measured in the BALF samples. For obtaining BALF, mice were anesthetized, the trachea was exposed, a canula was inserted and the lung was washed with PBS containing 1% protease inhibitors (Sigma) at a volume of 1ml. The wash fluid (lOOOpl) was centrifuged, the supernatant collected, and the protein concentration was determined using Bradford reagent. 20 pl batches of BALF from each sample were separated on NuPage 4-12% BisTris SDS-PAGE Gel (Invitrogen_NP0335BOX) and transferred to an iBlot Transfer Stack Nitrocellulose PVDF membrane (Invitrogen, IB01001). The membranes were blocked for one hour in room temperature in TBST solution (0.15MNaCl, 0.05% Tween-20, 0.0 IM Tris-HCl pH 8) containing 5% Skim Milk powder (Difco Skim Milk, BD 232100). The incubation with rabbit primary antibody against each of the tested proteins (anti-citH3, ab5103; anti-PAD4, ab214810, Abeam) was performed for 16 hours at 4°C while shaking. After washing with TBST, the membranes were incubated while shaking for one hour at room temperature with goat anti-rabbit IRDye 680RD secondary antibody (LI-COR, 926-68071) diluted 1:10,000 in TBST solution with 5% skim milk powder. After washing, the bands were visualized using ODYSSEY CLx (LI-COR) imager. The intensity of each band was quantified using ImageJ software.

[0215] In addition, the levels of citH3 in BALF were quantified using ELISA citrullinated histone H3 (Cayman Chemicals 501620 Clone 11D3).

[0216] Quantifying cell-free DNA in BALF

[0217] Cell-free DNA quantification was performed using Quant-iT™ PicoGreen™ dsDNA (Invitrogen) kit in accordance with manufacturer’s instructions. Briefly, a calibration curve was prepared including from 0 to lOOOng / ml DNA in XI 0 intervals. lOOpl volumes of BALF were transferred into black 96 well plates (Greiner) and an equal volume of PicoGreen dsDNA reagent was added to each well. Fluorescent reads of the DNA were measured in a Microplate reader (Gen 5 3.04), excitation and emission at 485nm and 538nm, respectively.Quantifying NETs in neutrophils isolated from lungs

[0218] The mice were sacrificed, and their lungs were taken and incubated in a PBS Ca2+Mg2+solution containing 4mg Collagenase D (Roche) for two hours at 37°C. Following the incubation the specimen was pipetted for a long time to separate the cells, and the cell suspension was transferred through a 0.7 p filter. Neutrophils were isolated using a Histopaque gradient. Briefly, 3ml Histopaque-1119 (Sigma) was inserted into 15ml tubes and on top of it 3ml Histopaque- 1077 (Sigma). Above the gradient, 6ml of lung cells suspended in PBS containing 5% BSA were added cautiously. The tubes were centrifuged at 700g for 30 min at room temperature without brake and the neutrophils which resided in the interface between the gradient layers, were carefully collected. Next, to obtain a clean neutrophil fraction, the cells were sorted using MACS based on isolating neutrophils by negative selection of cells that did not bind to a column (Miltenyi Biotec, 130-097-658). The isolated cells were counted, resuspended in Opti-MEM medium containing 2% heat-inactivated FBS and spread l-2xl06cells / well in a 24 well plate containing poly-L-Lysine coated glass coverslips. Subsequent preparations and NETs staining were performed as described above with respect to staining BALF neutrophils.

[0219] Quantifying NETosis in lung tissue in vivo

[0220] Lungs obtained from the sacrificed mice were perfused with 10ml PBS and incubated with 4% paraformaldehyde for two weeks at room temperature. Subsequently, blocks of paraffin were prepared. 5pM sections were placed on slides and were deparaffmized. Antigens exposure was performed by incubation with Target Retrieval Solution (DAKO) at 95°C for 30 min. After blocking with 5% BSA in PBS, the slides were incubated with the primary antibodies anti-citH3, ab5103, and anti-Ly6B, ab53457 (Abeam) over night at 4°C. Subsequently, the slides were incubated for 1 hour at room temperature with fluorescent secondary antibodies Alexa Fluor 594-coupled goat anti-rat IgG (Abeam, abl50160) and Atto550 goat anti-rabbit IgG (Rockland, 61 l-154-122s). Nuclear staining was performed with Prolong® Gold antifade reagent containing DAPI (Molecular probes). Analysis of the staining was performed using a confocal microscope LSM 710 (Zeiss, Germany) with the following lasers argon multiline (458 / 488 / 514 nm), diode 405nm, DPSS 561 nm and helium-neon 633 nm. Analysis of the mean fluorescence intensity (MFI) of the staining was done using Zen 2008 (Zeiss, Germany).Histology

[0221] To examine the lung morphology, tissue sections were stained with Hematoxylin and Eosin (H&E). The stained sections were photographed using 3D HISTECH Panoramic Midi II slide viewer (Budapest, Hungary), and analysis of the staining was performed using Case Viewer 2.4.0 software (Budapest, Hungary). Semi-quantitative analysis of histological injury scores in the mice’ lungs was performed according to various parameters known in the art (Silva, et al. (2022) Bio-protocol 12(16), e4493). Scoring was performed by providing a score of 0 to 8 (0 meaning no phenomenon and 8 meaning a severe phenomenon) according to the following parameters inflammatory cells, hyaline membrane, thickening of alveolar wall, enhanced injury, vessel congestion, and atelectasis. Lungs were obtained from 5 mice in each treatment group. The calculated score was an average of the 5 different fields for each of the tested parameters.

[0222] Testing lung tissue permeability

[0223] Mice were injected intravenously with Evans Blue (EB, Sigma) dye at a concentration of 7.5mg / ml at a dose of 50mg / kg. After 60 min the mice were anesthetized and perfused by making an incision in the left ventricle and washing with 5ml PBS through the right chamber. The lungs were removed, and EB was extracted by incubation with 0.5ml Formamide (Sigma) at 60°C for 24 hours. Optical density of the dye in the extraction solution was measured at 620 nm in a Spectramax ABS Plus (Molecular Devices) spectrophotometer.

[0224] Statistical analysis

[0225] All statistical analyses were conducted with GraphPad Prism software (version 5.01, GraphPad Software Inc., La Jolla, CA, USA, 2007). Data were first tested for normality using the Shapiro-Wilk test. Simple comparisons were performed using the unpaired two-tailed Student’s t-test. For multiple comparisons, one-way or two-way analysis of variance (ANOVA) tests followed by Tukey’s or Bonferroni multiple comparisons test were applied, respectively. Survival data were analyzed using Kaplan-Meier survival curves, and differences between groups were assessed with the Log-rank (Mantel-Cox) test. Data are presented as means ± SEM. Differences were considered significant at p < 0.05.EXAMPLE 1

[0226] NETosis in the lungs of mice following pulmonary exposure to ricin

[0227] Mice were intranasally exposed to ricin (9.6 pg / kg (2LDso) and bronchoalveolar lavage fluid (BALF) was collected at 24, 48, and 72 hours post-exposure. The BALF was analyzed for the presence of peptidyl arginine deiminase 4 (PALM), an enzyme catalyzing histone citrullination essential for chromatin decondensation in NETosis, and citrullinated histone H3 (citH3). Protein levels in BALF were assessed employing Western blot. Results revealed a significant increase in PALM levels in BALF at 24 hours postexposure, reaching peak levels at 48 hours, followed by a decline at 72 hours (Figure 1 A, IB). CitH3 levels peaked at 48 hours and declined at 72 hours (Figure 1C, ID) consistent with ELISA measurements (Figure IE). The presence of PAD4 and citrullinated histones in BALF indicate that NETosis occurred in the lungs following exposure to ricin.

[0228] EXAMPLE 2

[0229] NETosis in Lung-Resident Neutrophils

[0230] NETosis in neutrophils isolated and purified directly from lung tissue was next examined. The neutrophils were isolated from the lungs of control mice or mice intranasally exposed to 9.6 pg / kg (2LDso) ricin. Neutrophils were enriched using a gradient and isolated by magnetic sorting at 48 hours post-exposure, the time point of peak NETosis, as was shown in Figure 1. Isolated neutrophils were stained for extracellular DNA and citH3, NET structures appeared as long chromatin threads extending from neutrophils and were predominantly observed in neutrophils isolated from toxin-exposed mice. To ensure that the observed NETosis was not an artifact resulting from neutrophil activation during the isolation process, NETosis was quantified, and the number of NET-forming cells was compared between neutrophils isolated from control mice and those from ricin-intoxicated mice 48 hours post-exposure. Quantification of NETosis confirmed significantly higher rates in neutrophils from exposed mice compared to controls (Figure 2).

[0231] EXAMPLE 3

[0232] NETosis in lung tissue in vivoTo confirm NETosis within lung tissue, mice were exposed to ricin, and lungs were harvested at 24-, 48-, and 72-hours post-exposure for immunohistochemical analysis. This approach allowed the visualization of the presence and localization of key NETosis markers within the lung parenchyma over time. Lung tissue sections from naive control mice and mice intranasally exposed to 9.6 pg / kg (2LDso) ricin at different time points were stained for neutrophils, extracellular DNA, and citH3. NETosis was indicated by the presence of extracellular chromatin colocalized with citH3 staining, increasing with time post-exposure. Control lungs contained few neutrophils (3 A). At 24 hours postexposure, neutrophil recruitment was evident, but NETosis had not yet occurred (Figure 3A-C). After 48 hours, numerous neutrophils exhibited colocalized extracellular DNA and citH3 staining, indicative of NETosis (Figure 3 A-C). At 72 hours, NETosis remained extensive but was slightly reduced compared to 48 hours (Figure 3 A-C).

[0233] These findings are in line with the Western blot results (Figure 1). About 72 hours post-exposure to ricin, mice generally begin to succumb, and their overall well-being deteriorates significantly. Without wishing to be bound by theory, the apparent "decline" in NETosis at 72 hours compared to 48 hours is likely due to the severe physiological deterioration and impending collapse of the animals rather than a true reduction in the process.

[0234] EXAMPLE 4

[0235] Treatment of ricin intoxicated mice with a NET-degrading agent (PRX-119) and an anti-ricin antibody

[0236] Mice were treated at 24 hours post-exposure with PRX-119, with an anti-ricin antibody, or with a combination of both agents. Weight loss and survival were monitored for 14 days (Figure 4A). Mice treated with PRX-119 alone exhibited weight loss similar to untreated mice exposed to ricin until they succumb, whereas those receiving antibody treatment alone lost -30% of body weight before beginning of recovery from day 9. Combination treatment reduced weight loss to 20%, with earlier recovery beginning on day 6 (Figure 4B). While PRX-119 alone did not improve the survival, combination treatment increased survival to 73%, significantly higher than the 40% survival observed with antibody treatment alone (Figure 4C).

[0237] EXAMPLE 5The effect of a PRX-119 and an anti-ricin antibody treatment on NETosis process and its components

[0238] Extracellular DNA in the lungs may originate from fragmented DNA of dead cells that perished due to the direct catalytic effect of ricin or from acute inflammation, as well as from NET release by neutrophils. First, the levels of extracellular DNA in the BALF of mice were examined at different time points following ricin intoxication. DNA levels were significantly elevated at 24 to 72 hours post-exposure (Figure 5A).

[0239] Next, the changes in extracellular DNA levels following treatment were examined. BALF of intranasally ricin-intoxicated mice, as well as of intoxicated mice treated with an anti-ricin antibody or PRX-119 as a standalone treatment, or a combination of anti-ricin antibody and PRX-119, was collected 72 hours post-exposure to ricin. The results indicated that, in contrast to antibody treatment alone, which did not affect the high levels of extracellular DNA, PRX-119 treatment alone significantly reduced extracellular DNA levels in the lungs. Similarly, the combined treatment with both the antibody and PRX-119 led to a significant decrease in extracellular DNA concentration (Figure 5B).

[0240] It is important to note that despite the significant reduction in extracellular DNA levels following PRX-119 treatment, DNA levels remained relatively high in the lungs even 72 hours post-exposure. Therefore, DNA levels were also measured at later points, i.e., 96- and 120-hour time points post-exposure. These measurements revealed that at later time points, extracellular DNA levels continued to decline significantly following the combined treatment, compared to treatment with the anti -toxin antibody alone (Figure 5C).

[0241] Next, the protein levels of key components involved in the NETosis process were analyzed, using Western Blot to assess their trends following treatment.

[0242] Western blot analysis showed a reduction in citH3 levels following antibody treatment alone, with an even greater decrease following combination therapy, reaching near-baseline levels (Figure 6A-6B). The levels of citH3 were also quantified using a commercial ELISA assay, revealing a reduction in citH3 following antibody treatment and an even more significant decrease after the combined antibody and PRX-119 treatment (Figure 6C). PAD4 levels followed a similar trend, though combination therapy did not significantly outperform antibody treatment alone (Figure 6D-6E). These results demonstrated that the combined treatment with anti -toxin antibody and PRX-119 therapytargeting extracellular DNA degradation significantly reduced NETosis and nearly abolished the process.

[0243] Next, the resolution of the NETosis process in treated mice was monitored using immunohistochemical staining of lung tissue. Ricin intoxicated mice (intranasally exposed to 9.6pg / kg (2LDso) ricin) were treated with anti-ricin antibody (iv injection of an anti-ricin antibody (RR003, 100 pl, single dose)) or with combined treatment antibody and PRX-119 (5 mg / kg ip) at 24 hours post-exposure which was continued daily until the end of the experiment. Immunohistochemical analysis of lung sections from naive control mice and ricin intoxicated mice was performed 48 hours post-exposure. Lung sections were stained, with neutrophil (Ly6B) and NETosis (citH3 and extracellular DNA) markers. The analysis showed extensive NETosis in ricin exposed untreated mice, characterized by a high number of neutrophils positive for extracellular DNA and citH3. Moderate reduction in NETosis was observed following antibody treatment, and nearcomplete resolution of NETosis in the combination therapy group (Figure 7A-C).

[0244] EXAMPLE 6

[0245] The impact of PRX-119 and an anti-ricin antibody treatment on lung pathology To evaluate the impact of treatment on lung pathology, histological analysis was performed on lung tissue sections from intoxicated and treated mice. Histological examination using hematoxylin and eosin (H&E) staining of the lungs from intoxicated mice revealed severely edematous, non-aerated lungs, heavily infiltrated with inflammatory cells and displaying increased tissue density. Focusing on perivascular regions, significant neutrophil infiltration was observed, as well as marked expansion of these areas, and the presence of proteinaceous exudates. Pulmonary blood vessels were congested with red blood cells, surrounded by perivascular and peribronchiolar edema, and showed evidence of cellular debris indicative of cell death. Within the alveoli, prominent neutrophilic infiltration, cell death, and edema were evident. The lungs exhibited extensive vascular leakage and fibrin accumulation within the alveolar airspaces (Figure 8C-8D). These pathological changes contrasted sharply with the normal, well-aerated lung structure observed in non-intoxicated mice, which displayed an absence of neutrophil infiltration, intact bronchiolar and alveolar architecture, and the presence of alveolar macrophages (Figure 8A-8B). It was observed that after antibody treatment alone, the lungs remained severely damaged, with poor aeration, inflammatorycell infiltration, and edematous foci (Figure 8E-8F). In contrast, mice that received the combined treatment with an anti-toxin antibody and extracellular DNA-targeting therapy exhibited a marked reduction in these pathological features. They were better aerated, with only minimal inflammatory foci, well-preserved alveolar architecture, and very limited perivascular infiltration and appeared more like those of control mice (Figure 8G-8H). A semi-quantitative assessment of the histological injury score in the lungs of mice, based on established criteria from the literature (Silva, 2022), revealed that the score in intoxicated mice receiving the combined treatment was significantly lower than that of mice treated with the anti-ricin antibody alone (42±14% vs. 59±9%, respectively). 72 hours post-intoxication, the injury score in untreated mice was 65 ±7% (Figure 81).

[0246] To evaluate lung permeability, mice were intoxicated with ricin, with some receiving either antibody treatment alone or the combined treatment. Seventy -two hours post-exposure, lung permeability was assessed using the Evans Blue Extravasation Assay (EBD). The results showed that while the increased permeability following ricin intoxication was partially reduced by antibody treatment, the combined treatment significantly improved barrier integrity compared to antibody treatment alone (Figure 9). These findings demonstrate that the combined treatment effectively mitigates the morphological and histological lung damage following pulmonary ricin exposure and significantly reduces the disruption of the alveolar-capillary barrier.

[0247] EXAMPLE 7

[0248] Effects of PRX-119 and an anti-ricin antibody treatment on various analytes and markers associated with lung repair

[0249] Using multiplex analysis, which allows precise measurement of multiple analytes within a single sample, a broad panel of factors involved in various pro- and antiinflammatory processes that influence lung cell repair was assembled. These include factors associated with epithelial and endothelial cells, correction of vascular leak, reduction of pulmonary edema, fibrosis-related proteins, and markers of lung remodeling following treatment. After assembling the panel, the expression levels of these analytes in BALF samples from four groups were compared. The groups were: control, ricin-exposed untreated, ricin-exposed and treated with an anti-ricin antibody, and ricin-exposed mice treated with the combination of anti-ricin antibody and PRX-119. Several analytes were identified as significantly altered following combination treatment. Theproteins eotaxin and CD93 were found to be significantly lower in mice treated with combination therapy, whereas no substantial change was observed in the antibody-only treatment group compared to untreated ricin-exposed mice (Figure 10A, 10B). Eotaxin plays a role in neutrophil recruitment to the lungs (Guo, 2001) and CD93 is crucial for maintaining endothelial barrier integrity, promoting angiogenesis, and regulating VEGF levels. Additionally, it maintains cytoskeletal stability in endothelial cells, ensuring the stability of junction proteins that connect endothelial cells (Lugano, 2023). Another protein, Dkk-1 was markedly elevated following ricin exposure, with even higher levels detected in mice that received antibody-only treatment. However, combination therapy significantly reduced Dkk-1 levels (Figure 10C). Dkk-1 is secreted by platelets and promotes neutrophil infiltration into injured lung tissue by increasing the expression of ICAM-1 and VCAM-1, facilitating neutrophil extravasation through the alveolar-capillary barrier and exacerbating barrier permeability (Guo, 2015). Similarly, to Dkk-1, periostin levels were markedly elevated in ricin-exposed mice, and treatment with the antibody alone did not significantly reduce them. In contrast, the combination therapy substantially decreased periostin levels (Figure 10D). Periostin is secreted by fibroblasts, endothelial, and epithelial cells in response to lung epithelial injury and is implicated in pathological fibrosis. High periostin levels are correlated with impaired pulmonary function and reduced survival rates (Izuhara, 2016). These findings show that combination therapy reduced the levels of Eotaxin, CD93, Dkk-1, and Periostin, proteins which are associated with neutrophil recruitment and alveolar-capillary barrier disruption through direct or indirect effects on lung endothelial and epithelial cells. The reduction in these proteins indicates that combination therapy with anti-ricin antibody and PRX-119 promotes lung repair, restores alveolar-capillary barrier integrity, inhibits neutrophil infiltration, and mitigates pulmonary edema, ultimately leading to improved pulmonary outcomes following ricin-induced lung injury.

[0250] EXAMPLE 8

[0251] Treatment of ricin intoxicated mice with a NET-degrading agent (PRX-119) and an anti-ricin monoclonal antibody

[0252] Mice were treated at 24 hours post-exposure with PRX-119, with an anti-ricin monoclonal antibody (MH-77), or with a combination of both agents. Weight loss and survival were monitored for 14 days (Figure 11 A). Ricin-intoxicated, untreated micecontinued to lose weight until they succumbed. Mice that received antibody treatment alone lost around 30% of body weight before beginning of recovery from day 9. Combination treatment reduced weight loss to approximately 25%, with earlier recovery beginning on day 6 (Figure 11B). Combination treatment increased survival to 50%, significantly higher than the 10% survival observed with antibody treatment alone (Figure 11C).

Claims

CLAIMS:

1. A method of prophylaxis, or treatment of a disease or disorder resulting from ricin toxin poisoning comprising administering to a subject in need thereof a therapeutically effective amount of an anti-ricin antibody, or an antigen binding fragment thereof which binds to ricin toxin, and a DNase.

2. The method of claim 1 wherein said DNase is DNase I.

3. The method of claim 2, wherein said DNase l is a long-acting PEGylated DNase I.

4. The method of claim 3, wherein the PEGylated DNase I comprises or has an amino acid sequence having at least 90% homology with the amino acid sequence set forth in SEQ IDNO: 1.

5. The method of any one of the preceding claims wherein said anti-ricin antibody is an isolated anti-ricin monoclonal antibody or an isolated anti-ricin polyclonal antibody.

6. The method of claim 5 wherein said monoclonal antibody is a chimeric, a humanized or a human anti-ricin monoclonal antibody.

7. The method of any one of the preceding claims, wherein said antigen binding fragment that binds to ricin toxin is selected from the group consisting of Fv, single chain Fv (scFv), Fab, F(ab)2' and any combination thereof.

8. The method of any one of the preceding claims, wherein said anti-ricin antibody is a neutralizing antibody.

9. The method of any one of claims 5 to 8 wherein said anti-ricin monoclonal antibody or antigen-binding fragment thereof which binds to ricin toxin, is one or more of:(a) a monoclonal antibody comprising a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 9, CDRH2 denoted by SEQ ID NO. 10, CDRH3 denoted by SEQ ID NO. 11, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 12, a CDRL2 denoted by SEQ ID NO. 13, and a CDRL3 denoted by SEQ ID NO.14, or a variant thereof (also referred to as MH77);(b) a monoclonal antibody, comprising the CDRH1 denoted by SEQ ID NO. 15, CDRH2 denoted by SEQ ID NO. 16, CDRH3 denoted by SEQ ID NO. 17, and CDRL1 denoted by SEQ ID NO. 18, a CDRL2 denoted by SEQ ID NO.19, and a CDRL3 denoted by SEQ ID NO. 20, or a variant thereof (also referred to as MH1); or(c) a monoclonal antibody comprising the CDRH1 denoted by SEQ ID NO. 21, CDRH2 denoted by SEQ ID NO. 22, CDRH3 denoted by SEQ ID NO. 23, and CDRL1 denoted by SEQ ID NO. 24, a CDRL2 denoted by SEQ ID NO.25, and a CDRL3 denoted by SEQ ID NO. 26, or a variant thereof (also referred to MH75).

10. The method of any one of claims 5 to 9 wherein said anti-ricin monoclonal antibody or antigen-binding fragment thereof which binds to ricin toxin, comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 27 (MH77), SEQ ID NO. 29 (MH1), or SEQ ID NO. 31 (MH75), and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 28 (MH77), SEQ ID NO. 30 (MH1), or SEQ ID NO. 32 (MH75).

11. The method of any one of claims 5 to 10 wherein said anti -ricin monoclonal antibody or antigen-binding fragment thereof which binds to ricin toxin, is one or more of:(a) a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 3 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 4 or a variant thereof;(b) a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 5 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 6 or a variant thereof; or(c) a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 7 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 8 or a variant thereof.

12. The method of any one of the preceding claims, wherein said anti-ricin antibody and said DNase are administered to said subject prior to exposure to ricin toxin.

13. The method of any one of claims 1 to 11, wherein said anti -ricin antibody and said DNase are administered to said subject after exposure to ricin toxin.

14. The method of claim 13, wherein said anti -ricin antibody and said DNase are administered to said subject immediately after exposure to ricin toxin or between about 1 to about 96 hours after exposure to ricin toxin.

15. The method of claim 14, wherein said anti-ricin antibody and said DNase are administered to said subject about 24 hours after exposure to ricin toxin.

16. The method of any one of the preceding claims, wherein said anti-ricin antibody and said DNase are administered to said subject simultaneously.

17. The method of any one of claims 1 to 15, wherein said anti -ricin antibody and said DNase are administered to said subject sequentially.

18. The method of any one of the preceding claims wherein said anti-ricin antibody or said DNase are each independently administered to said subject as a single dose or as multiple doses.

19. The method of claim 18 wherein said anti -ricin antibody is administered to said subject as a single dose, and said DNase is administered to said subject in multiple doses.

20. The method according to any one of the preceding claims wherein said anti-ricin antibody and said DNase are each independently administered to said subject by an administration route selected from a group consisting of intraperitoneal (ip), intravenous (iv), oral, sub-cutaneous, inhalation and intranasal administration.

21. The method of any one of the preceding claims, wherein said method further comprises administering to the subject in need thereof an additional therapeutic agent (e.g., an anti-ricin agent, an anti-inflammatory agent, an immunomodulatory agent an inhibitor of neutrophil recruitment (e.g., an anti-eotaxin antibody, or an Ly6G antibody), a NETosis inhibitor (e.g., a protein arginine deiminase 4 (PAD4) inhibitor), a neutrophil elastase (NE) or gasdermin D inhibitor, and / or an inhibitor of alveolar-capillary barrier disruption).

22. The method of any one of the preceding claims wherein said disease or disorder resulting from ricin toxin poisoning is selected from a group consisting of lung inflammation (pneumonia), ARDS, respiratory failure, gastrointestinal disorders (e.g., vomiting, diarrhea), dehydration, nausea, hemorrhages, anuria, cramps, fever, tachycardia, cardiac failure, vascular collapse and shock.

23. DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, in prophylaxis, or treatment of a disease or disorder resulting from ricin toxin poisoning, in a subject in need thereof.

24. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of claim 23 wherein said DNase is DNase I.

25. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of claim 24, wherein the DNase I is a long-acting PEGylated DNase I.

26. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of claim 25, wherein the PEGylated DNase I comprises or has an amino acid sequence having at least 90% homology with the amino acid sequence set forth in SEQ ID NO: 1.

27. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of any one of claims 23 to 26 wherein said anti-ricin antibody is an isolated anti-ricin monoclonal antibody or an isolated antiricin polyclonal antibody.

28. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of claim 27 wherein said monoclonal antibody is a chimeric, a humanized or a human anti-ricin monoclonal antibody.

29. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of any one of claims 23 to 28, wherein said antigen binding fragment which binds to ricin toxin is selected from the group consisting of Fv, single chain Fv (scFv), Fab, F(ab)2' and any combination thereof.

30. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of any one of claims 23 to 29, wherein said anti-ricin antibody is a neutralizing antibody.

31. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin of any one of claims 27 to 30 wherein said anti-ricin monoclonal antibody or antigen-binding fragment thereof which binds to ricin toxin, is one or more of:(a) a monoclonal antibody comprising a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 9, CDRH2 denotedby SEQ ID NO. 10, CDRH3 denoted by SEQ ID NO. 11, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 12, a CDRL2 denoted by SEQ ID NO. 13, and a CDRL3 denoted by SEQ ID NO.14, or a variant thereof (also referred to as MH77);(b) a monoclonal antibody, comprising the CDRH1 denoted by SEQ ID NO. 15, CDRH2 denoted by SEQ ID NO. 16, CDRH3 denoted by SEQ ID NO. 17, and CDRL1 denoted by SEQ ID NO. 18, a CDRL2 denoted by SEQ ID NO.19, and a CDRL3 denoted by SEQ ID NO. 20, or a variant thereof (also referred to as MH1); or(c) a monoclonal antibody comprising the CDRH1 denoted by SEQ ID NO. 21, CDRH2 denoted by SEQ ID NO. 22, CDRH3 denoted by SEQ ID NO. 23, and CDRL1 denoted by SEQ ID NO. 24, a CDRL2 denoted by SEQ ID NO.25, and a CDRL3 denoted by SEQ ID NO. 26, or a variant thereof (also referred to MH75).

32. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of any one of claims 27 to 31 wherein said anti-ricin monoclonal antibody or antigen-binding fragment thereof which binds to ricin toxin, comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence denoted by SEQ ID NO. 27 (MH77), SEQ ID NO. 29 (MH1), or SEQ ID NO. 31 (MH75), and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 28 (MH77), SEQ ID NO. 30 (MH1), or SEQ ID NO. 32 (MH75).

33. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin of any one of claims 27 to 32, wherein said anti-ricin monoclonal antibody or antigen-binding fragment thereof which binds to ricin toxin, is one or more of:(a) a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 3 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 4 or a variant thereof;(b) a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 5 or a variant thereof and alight chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 6 or a variant thereof; or(c) a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 7 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 8 or a variant thereof.

34. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of any one of claims 23 to 33, wherein said anti-ricin antibody and said DNase are administered to said subject prior to exposure to ricin toxin.

35. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of any one of claims 23 to 33, wherein said anti -ricin antibody and said DNase are administered to said subject after exposure to ricin toxin.

36. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of claim 35, wherein said anti-ricin antibody and said DNase are administered to said subject immediately after exposure to ricin toxin or between about 1 to about 96 hours after exposure to ricin toxin.

37. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of claim 36, wherein said anti-ricin antibody and said DNase are administered to said subject about 24 hours after exposure to ricin toxin.

38. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of any one of claims 23 to 37, wherein said anti -ricin antibody and said DNase are administered to said subject simultaneously.

39. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of any one of claims 23 to 37, wherein said anti -ricin antibody and said DNase are administered to said subject sequentially.

40. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of any one of claims 23 to 39 whereinsaid anti -ricin antibody or said DNase are each independently administered to said subject as a single dose or as multiple doses.

41. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of claim 40 wherein said anti-ricin antibody is administered to said subject as a single dose, and said DNase is administered to said subject in multiple doses.

42. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of any one of claims 23 to 41 wherein said anti-ricin antibody and said DNase are each independently administered to said subject by an administration route selected from a group consisting of intraperitoneal (ip), intravenous (iv), oral, sub-cutaneous, inhalation and intranasal administration.

43. The DNase for use in combination with an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, of any one of claims 23 to 42, wherein said DNase and said anti-ricin antibody are administered with an additional therapeutic agent (e.g., an anti-ricin agent, an anti-inflammatory agent, an immunomodulatory agent an inhibitor of neutrophil recruitment (e.g., an anti-eotaxin antibody, or an Ly6G antibody), a NETosis inhibitor (e.g., a protein arginine deiminase 4 (PAD4) inhibitor), a neutrophil elastase (NE) or gasdermin D inhibitor, and / or an inhibitor of alveolar-capillary barrier disruption).

44. The DNase for use in combination with an anti-ricin antibody of any one of claims 23 to 43 wherein said disease or disorder resulting from ricin toxin poisoning is selected from a group consisting of lung inflammation (pneumonia), ARDS, respiratory failure, gastrointestinal disorders (e.g., vomiting, diarrhea), dehydration, nausea, hemorrhages, anuria, cramps, fever, tachycardia, cardiac failure, vascular collapse and shock.

45. A kit comprising:(a) an anti-ricin antibody or an antigen binding fragment thereof which binds to ricin toxin, or a pharmaceutical composition comprising same, and (b) a DNase, or a pharmaceutical composition comprising same, and optionally (c) instructions for use of the anti-ricin antibody and the DNase in combination for prophylaxis, or treatment of a disease or disorder resulting from ricin toxin poisoning.

46. The kit of claim 45 wherein said DNase is DNase I.

47. The kit of claim 46, wherein the DNase lis a long-acting PEGylated DNase I.

48. The kit of claim 47, wherein the PEGylated DNase I comprises or has an amino acid sequence having at least 90% homology with the amino acid sequence set forth in SEQ ID NO: 1.

49. The kit of any one of claims 45 to 48, wherein said anti-ricin antibody is an isolated anti-ricin monoclonal antibody or an isolated anti-ricin polyclonal antibody.

50. The kit of claim 43 wherein said monoclonal antibody or an antigen binding fragment thereof which binds to ricin toxin, is a chimeric, a humanized or a human antiricin monoclonal antibody.

51. The kit of any one of claims 45 to 50, wherein said antigen binding fragment which binds to ricin toxin is selected from the group consisting of Fv, single chain Fv (scFv), Fab, F(ab)2(and any combination thereof.

52. The kit of any one of claims 45 to 51 wherein said anti-ricin monoclonal antibody or antigen-binding fragment thereof which binds to ricin toxin, is one or more of:(a) a monoclonal antibody comprising a heavy chain complementarity determining region (CDRH) 1 denoted by SEQ ID NO. 9, CDRH2 denoted by SEQ ID NO. 10, CDRH3 denoted by SEQ ID NO. 11, and the light chain complementarity determining region (CDRL) 1 denoted by SEQ ID NO. 12, a CDRL2 denoted by SEQ ID NO. 13, and a CDRL3 denoted by SEQ ID NO.14, or a variant thereof (also referred to as MH77);(b) a monoclonal antibody, comprising the CDRH1 denoted by SEQ ID NO. 15, CDRH2 denoted by SEQ ID NO. 16, CDRH3 denoted by SEQ ID NO. 17, and CDRL1 denoted by SEQ ID NO. 18, a CDRL2 denoted by SEQ ID NO.19, and a CDRL3 denoted by SEQ ID NO. 20, or a variant thereof (also referred to as MH1); or(c) a monoclonal antibody comprising the CDRH1 denoted by SEQ ID NO. 21, CDRH2 denoted by SEQ ID NO. 22, CDRH3 denoted by SEQ ID NO. 23, and CDRL1 denoted by SEQ ID NO. 24, a CDRL2 denoted by SEQ ID NO.25, and a CDRL3 denoted by SEQ ID NO. 26, or a variant thereof (also referred to MH75).

53. The kit of any one of claims 45 to 52 wherein said anti-ricin monoclonal antibody or antigen-binding fragment thereof which binds to ricin toxin, comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to the nucleic acidsequence denoted by SEQ ID NO. 27 (MH77), SEQ ID NO. 29 (MH1), or SEQ ID NO.31 (MH75), and wherein said light chain variable region is encoded by a nucleic acid sequence which is at least 70% identical to SEQ ID NO. 28 (MH77), SEQ ID NO. 30 (MH1), or SEQ ID NO. 32 (MH75).

54. The kit of any one of claims 45 to 53, wherein said anti-ricin monoclonal antibody or antigen-binding fragment thereof which binds to ricin toxin, is one or more of:(a) a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 3 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 4 or a variant thereof;(b) a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 5 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 6 or a variant thereof; or(c) a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 7 or a variant thereof and a light chain variable region comprising the amino acid sequence denoted by SEQ ID NO. 8 or a variant thereof.

55. The kit of any one of claims 45 to 54, wherein said kit further comprises an additional therapeutic agent (e.g., an anti-ricin agent, an anti-inflammatory agent, an immunomodulatory agent an inhibitor of neutrophil recruitment (e.g., an anti-eotaxin antibody, or an Ly6G antibody), a NETosis inhibitor (e.g., a protein arginine deiminase 4 (PAD4) inhibitor), a neutrophil elastase (NE) or gasdermin D inhibitor, and / or an inhibitor of alveolar-capillary barrier disruption).

56. The kit of any one of claims 45 to 55 wherein said disease or disorder resulting from ricin toxin poisoning is selected from a group consisting of lung inflammation (pneumonia), ARDS, respiratory failure, gastrointestinal disorders (e.g., vomiting, diarrhea), dehydration, nausea, hemorrhages, anuria, cramps, fever, tachycardia, cardiac failure, vascular collapse and shock.

Images