Field of the invention

The invention broadly relates to the treatment or prevention of neonatal sepsis.

Cross-reference to earlier application

This application claims priority from Australian provisional application no. 2021900585, the entire contents of which are hereby incorporated by reference in their entirety.

Background of the invention

Neonatal infection represents a significant cause of global childhood mortality, accounting for approximately 1.6 million deaths annually. Sepsis represents the most severe and significant contributor to this death toll. Decades of effort have unfortunately resulted in limited progress in diagnostic accuracy, clinical management or in reducing mortality rates for neonatal sepsis. Importantly, a causative microbial pathogen is only identified in less than one quarter of sepsis cases. Therefore, it is likely that for the time being, progress in broadly reducing neonatal sepsis-related mortality will only be achieved through the development of pathogen agnostic, and host-centric treatment modalities.

Sepsis occurs when the body has an overwhelming immune response to infection triggering inflammatory responses throughout the body. This inflammation can trigger a cascade of changes that can damage multiple organ systems, causing them to fail. If sepsis progresses to septic shock, blood pressure drops dramatically, which may lead to death. Anyone can develop sepsis, but it's most common and most dangerous in older adults, young children or those with weakened immune systems. Early treatment of sepsis, usually with antibiotics and large amounts of intravenous fluids, may improve chances for survival.

Sepsis is also a major cause of death during the first few weeks of life, and approximately 13-15% of all neonatal deaths are the result of neonatal sepsis. The mortality rate of neonatal sepsis can be as high as 50% for infants who are not treated or when treatment is not begun quickly. As soon as sepsis is suspected, neonates are typically started on a course of antibiotics until culture results are returned. Unfortunately, antibiotics do not work on sepsis caused by viral infection or by infection by antibiotic resistant bacteria.

Neonatal sepsis is sepsis in babies less than 4 weeks old, typically caused by bacteria, but sometimes by viruses. Neonates are particularly susceptible to the consequences of infection. Serious consequences of neonatal infection include sepsis as well as meningitis, but all organs can be affected.

Medications available for treatment of neonatal sepsis include antibiotics, and vasopressors. Treatment with antibiotics begins immediately usually even before the infectious agent is identified. Initially the patient receives broad-spectrum antibiotics, which are effective against a wide variety of bacteria. Usually, such antibiotics are administered intravenously (IV). If blood pressure remains too low even after receiving intravenous fluids, a patient may be given a vasopressor medication, which constricts blood vessels and helps to maintain blood pressure. Other medications that may be administered include low doses of corticosteroids to reduce the host inflammatory response to infection, insulin to help maintain stable blood sugar levels, and painkillers or sedatives.

Despite advances in understanding the pathophysiology of neonatal sepsis, there is still a scarcity of effective treatment or prophylactic options for sepsis in neonates.

Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

Summary of the invention

In one aspect, the present invention provides a method of treating or preventing neonatal sepsis in a subject in need thereof, comprising administering to the subject a Tie-2 agonist, thereby treating or preventing the neonatal sepsis in the subject.

In one aspect, the present invention provides a method of treating or preventing neonatal sepsis in a subject in need thereof, comprising administering to the subject Angiopoietin-1 (Ang-1) or an Ang-1 functional equivalent, analog or variant thereof, thereby treating or preventing the neonatal sepsis in the subject.

In another aspect, the present invention provides a method of treating or preventing neonatal sepsis in a subject in need thereof, comprising administering to said subject L-Arginine (L-Arg), thereby treating or preventing the neonatal sepsis in the subject.

In another aspect, the present invention provides a method of treating or preventing neonatal sepsis in a subject in need thereof, comprising administering to said subject arachidonic acid (AA), thereby treating or preventing the neonatal sepsis in the subject.

In another aspect, the present invention provides a method of treating or preventing neonatal sepsis in a subject in need thereof, comprising administering to said subject an angiopoietin-2 (Ang-2) antagonist, thereby treating or preventing the neonatal sepsis in the subject.

In another aspect, the present invention provides a method of treating or preventing neonatal sepsis in a subject in need thereof, comprising administering one or more of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or an Ang-1 functional equivalent, analog or variant thereof, L-Arginine (L-Arg), arachidonic acid (AA), and Angiopoietin-2 (Ang-2) antagonist, thereby treating or preventing the neonatal sepsis in the subject.

In any aspect of the invention, one or more of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or an Ang-1 functional equivalent, analog or variant thereof, L-Arginine (L-Arg), arachidonic acid (AA), and Angiopoietin-2 (Ang-2) antagonist may be administered simultaneously or sequentially. Preferably, in separate dosage forms. Alternatively, in one dosage form.

In any aspect of the invention, Angiopoietin-2 (Ang-2) antagonist is a purified antibody, or antigen-binding fragment thereof, that specifically binds Ang-2. Preferably, in some embodiments of the invention said antibody is Angy-2-1, or any Ang-2 antibody described herein. Preferably, said antibody is a chimeric, humanised or fully human antibody. In any aspect, the Tie-2 agonist is Ang-1 or an Ang-1 functional equivalent, analog or variant thereof.

In any aspect of the invention, the subject is a neonate. The subject may be less than 28 days old, preferably less than 20 days old, preferably less than 15 days old, preferably less than 10 days old, preferably less than 5 days old, preferably less than 3 days old, preferably less than 1 day old. The subject is, or about, 28 days old; or, or about 20 days old; is, or about, 15 days old; is, or above, 10 days old; is, or about, 5 days old; is, or about, 3 days old; or is, or about, 1 day old.

In any aspect of the invention, the method further comprises a step of diagnosing the subject with neonatal sepsis.

In another aspect, the present invention provides a method of treating or preventing neonatal sepsis in a subject in need thereof, the method comprising:

- diagnosing the subject with neonatal sepsis; and

- administering one or more of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or an Ang-1 functional equivalent, analog or variant thereof, L-Arginine (L-Arg), arachidonic acid (AA) or Angiopoietin-2 (Ang-2) antagonist; thereby treating or preventing the neonatal sepsis in the subject.

In another aspect, the present invention provides a method of diagnosing a neonate susceptible to treatment with one or more of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or an Ang-1 functional equivalent, analog or variant thereof, L-Arginine (L-Arg), arachidonic acid (AA) or Angiopoietin-2 (Ang-2) antagonist comprising:

- diagnosing the subject with neonatal sepsis; and

- administering one or more of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or an Ang-1 functional equivalent, analog or variant thereof, L-Arginine (L-Arg), arachidonic acid (AA) and Angiopoietin-2 (Ang-2) antagonist. In another aspect, the present invention provides a use of a Tie-2 agonist in the manufacture of a medicament for treating or preventing neonatal sepsis in a subject in need thereof.

In another aspect, the present invention provides a use of Angiopoietin-1 (Ang-1) or an Ang-1 functional equivalent, analog or variant thereof in the manufacture of a medicament for treating or preventing neonatal sepsis in a subject in need thereof.

In another aspect, the present invention provides a use of L-Arginine (L-Arg) in the manufacture of a medicament or food supplement for treating or preventing neonatal sepsis in a subject in need thereof.

In another aspect, the present invention provides a use of arachidonic acid (AA) in the manufacture of a medicament or food supplement for treating or preventing neonatal sepsis in a subject in need thereof.

In another aspect, the present invention provides a use of Angiopoietin-2 (Ang-2) antagonist in the manufacture of a medicament for treating or preventing neonatal sepsis in a subject in need thereof.

In another aspect, the present invention provides a use of one or more of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or an Ang-1 functional equivalent, analog or variant thereof, L-Arginine (L-Arg), arachidonic acid (AA) and Angiopoietin-2 (Ang-2) antagonist in the manufacture of a medicament or food supplement for treating or preventing neonatal sepsis in a subject in need thereof.

In another aspect, the present invention provides a Tie-2 agonist for use in treating or preventing neonatal sepsis in a subject in need thereof.

In another aspect, the present invention provides Angiopoietin-1 (Ang-1) or an Ang-1 functional equivalent, analog or variant thereof for use in treating or preventing neonatal sepsis in a subject in need thereof.

In another aspect, the present invention provides L-Arginine (L-Arg) for use in treating or preventing neonatal sepsis in a subject in need thereof. In another aspect, the present invention provides arachidonic acid (AA) for use in treating or preventing neonatal sepsis in a subject in need thereof.

In another aspect, the present invention provides an Angiopoietin-2 (Ang-2) antagonist for use in treating or preventing neonatal sepsis in a subject in need thereof.

In another aspect, the present invention provides one or more of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or an Ang-1 functional equivalent, analog or variant thereof, L- Arginine (L-Arg), arachidonic acid (AA) or Angiopoietin-2 (Ang-2) antagonist for use in treating or preventing neonatal sepsis in a subject in need thereof.

In any aspect of the invention, one or more of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or an Ang-1 functional equivalent, analog or variant thereof, L-Arginine (L-Arg), arachidonic acid (AA), or Angiopoietin-2 (Ang-2) antagonist is administered or formulated as a composition that further comprises a pharmaceutically or physiologically acceptable excipient, diluent or carrier. In one embodiment, the composition consists essentially of or consists of, one or more of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or an Ang-1 functional equivalent, analog or variant thereof, L-Arginine (L-Arg), arachidonic acid (AA) or Angiopoietin-2 (Ang-2) antagonist and a pharmaceutically or physiologically acceptable excipient, diluent or carrier. In another embodiment, in a composition comprising L-Arg, the only amino acid present in the composition is L-Arg. In another embodiment, in a composition comprising L-Arg, the active agent present in the composition is L-Arg. In another embodiment, in a composition comprising Ang-1, the active agent present in the composition is Ang-1. In another embodiment, in a composition comprising arachidonic acid (AA), the active agent present in the composition is arachidonic acid (AA). In another embodiment, in a composition comprising Angiopoietin-2 (Ang-2) antagonist, the active agent present in the composition is Angiopoietin-2 (Ang-2) antagonist.

In another aspect, the present invention provides a composition comprising, consisting essentially of or consists of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or an Ang-1 functional equivalent, analog or variant thereof, L-Arginine (L-Arg), arachidonic acid (AA), and a pharmaceutically or physiologically acceptable excipient, diluent or carrier. In any aspect of the invention, the Angiopoietin-1 (Ang-1) comprises a sequence as referred to in any one of the GenBank Accession Numbers described herewith, preferably, Q15389 (SEQ ID NO: 1). Further, in any aspect of the invention, the Angiopoietin-1 (Ang-1) is encoded by a nucleic acid sequence comprising a sequence as referred to in any one of the GenBank Accession Numbers described herewith.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

Figure 1. Whole blood, liver and spleen transcriptomics comparing likely survivors and non-survivors of murine neonatal sepsis. (A) Unsupervised clustering of normalized RNA-seq data using principal component analyses in transcriptomic data collected from liver, spleen and blood (n=7 control, 9 CS-Survive, 10 CS-Die). (B) Heat- map exhibiting gene expression patterns in blood (left), liver (middle) and spleen (right) from healthy control, predicted survivor and predicted non-survivor mice. (C) Differentially expressed genes in predicted survivor and non-survivor mice relative to healthy control mice. (D) Top 5 most enriched GO terms (by adjusted p-value) of differentially expressed genes in predicted survivors and non-survivors in the liver, blood, and spleen.

Figure 2. Exogenous arachidonic acid improves outcome, minimizes oxidative stress, and maintains the angiopoietin-1 / angiopoietin-2 ratio during murine neonatal sepsis. (A) Expression of genes captured in the ‘eicosanoid metabolic processes’ GO term in the blood (top panel) and ‘Arachidonic acid metabolic processes’ GO term in the liver (bottom panel). (B) Overall survival of cecal slurry (CS) challenged mice with (top line, CS+AA) or without prophylactic AA treatment (bottom line, CS) 4-6 hours prior to challenge (p=0.003, log rank test). (C) ROS (O2 ) accumulation in the liver of CS challenged mice 8 hours after challenge as determined by relative fluorescence intensity of dihydroethidium normalized to untreated controls (p<0.001, Wilcoxon rank- sum test, n=10 for each group). (D) Serum angiopoietin-1 (Ang1) and angiopoietin-2 levels (Ang 2) in control, challenged (CS) and challenged / AA treated mice (AA+CS) as determined by ELISA (paired Wilcoxon rank-sum; n=6 for each group).

Figure 3. The Ang/TIE-2 signalling axis modulates vascular permeability and outcome during CS challenge. (A) Overall survival of CS challenged mice after prophylactic administration of exogenous Ang-1 (top line) or Ang-2 (bottom line); or saline control (middle line)(p = 0.0019, log-rank) or (B) prophylactic administration of anti-Ang-2 blocking antibody (Anti-Ang2 is top line; saline control is bottom line)(p = 0.0077, log-rank). * denotes p<0.05, **p<0.01 , ***p<0.001, ****p<0.0001.

Figure 4. Nitric oxide regulates functionality of the Ang/Tie signalling axis. (A) Overall survival of CS challenged mice with (bottom line) or without (top line) concurrent administration of universal NOS inhibitor L-NAME (p=0.0013; log rank test). (B) Serum Ang-1 and Ang-2 levels as determined by ELISA in mice CS challenged mice with or without concurrent administration of L-NAME (paired Wilcoxon rank-sum; n=5 mice per group). (C) Accumulation of ROS (O2 ^· ) in the liver of CS treated mice with or without concurrent administration of L-NAME, as measured by dihydroethidium staining (paired Wilcoxon rank-sum; n=8 control, 6 CS, 6 L-NAME). (D) Overall survival of CS challenged mice treated with L-NAME alone (bottom line) or in combination with AA (AA +L-NAME, top line) administered 4-6 hours prior to CS challenge (p=0.34; log rank). (E) Overall survival of an LD50 CS challenged mice (at LD50) treated with L-NAME alone (bottom line) or in combination with Ang1 (top line) 4-6 hours prior to challenge (p<0.0001 ; log rank). (F) Survival of CS challenged mice (at LD90) treated with saline only (bottom line), L-Arginine (third line from the top) , Ang-1 (second line from the top), or combined L-Arginine and Ang-1 together (top line); 1-hour post-challenge (p<0.0001; log rank test). * denotes p<0.05, **p<0.01 , ***p<0.001, ****p<0.0001.

Figure 5. AA metabolism and Ang/Tie signalling is associated with human neonatal sepsis. (A) qPCR analysis of ALOX15 (left), PTGS2 (middle) and CYP2J2 (right) in a cohort of septic neonates from Malawi (n=14 cases and 16 controls; Wilcoxon rank-sum). (B) Plasma ANG-1 (left panel) and ANG-2 (right panel) levels in neonates in infants with any LOS (n = 22 infants with n = 31 samplings) or and no LOS (n = 40 infants with n = 43 episodes) as measured by ELISA (Wilcoxon rank-sum). (C) Plasma ANG-1/ANG-2 ratio in cases vs. control neonates as measured by ELISA (Wilcoxon rank-sum). (D) Plasma sPLA2 levels in cases vs. control neonates as measured by ELISA (Wilcoxon rank-sum). (E) Pearson correlation showing relationship between plasma sPLA2 levels and plasma Ang-1 levels (left-panel), plasma Ang-2 levels (middle panel) and plasma ANG-1/ANG-2 ratio (right panel) for all neonates (cases and controls) within our cohort. * denotes p<0.05, **p<0.01, ***p<0.001,

****p<0.0001.

Figure 6. Schematic for sample inclusion / exclusion criteria for sPLA-2/Ang human cohort.

Figure 7. Genes driving enrichment of the ‘Angiogenesis’ GO term in the spleen.

Detailed description of the embodiments

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

All of the patents and publications referred to herein are incorporated by reference in their entirety. For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

The general chemical terms used in the formulae herein have their usual meaning.

The inventors have developed methods for the treatment or prevention for neonatal sepsis. The inventors have demonstrated a clear association between AA supplementation and reduced dysregulation of the Ang/Tie signalling axis in neonatal sepsis. The results derived from the murine CS challenge model also suggest that nitric oxide (NO) is a necessary mediator of AA induced protection. The results suggest that NO is required as an intermediary between AA and its impact on the Ang/Tie signalling axis. These findings have been utilised to develop a therapeutic intervention for neonatal sepsis by the administration of a Tie-2 agonist, Angiopietin-1 (Ang-1) or an Ang-1 functional equivalent, analog or variant thereof alone, L-Arginine alone or the combined administration of Angiopoietin-1 protein or an Ang-1 functional equivalent, analog or variant thereof and L-Arginine.

Further, the inventors have reported a direct link between AA metabolism and the regulation of this signalling pathway during inflammatory insult. The inventors have also demonstration that plasma sPLA2 levels are positively correlated with Ang-2 and negatively correlated with the Ang-1/Ang-2 ratio. Moreover, these findings support the notion that influencing the Ang-1/Ang-2 ratio and plasma sPLA-2 concentrations holds therapeutic and/or preventive potential in humans.

Without being bound by any theory or mode of action it is believed that this effect is mediated through the AA induced reduction in ROS accumulation post-CS challenge, ROS accumulation which is typically responsible for inducing the release of Ang-2 from WP bodies and initiating a positive feedback loop promoting endothelial cell proliferation, vascular remodelling and integrity. L-Arginine is converted to nitric oxide by endothelial nitric oxide synthase. The downstream effects of nitric oxide in combination with Angiopoietin-1 work to stabilize/protect the vascular endothelium during periods of infection or inflammation. This may help prevent the vascular and tissue destruction which leads to organ failure - a key physiological end point in sepsis leading to mortality. Taken together the inventors have developed therapeutic or prophylactic interventions which include administering one or more of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or an Ang-1 functional equivalent, analog or variant thereof, L-Arginine (L-Arg), arachidonic acid (AA) or Angiopoietin-2 (Ang-2) antagonist, preferably of Angiopietin-1 (Ang-1) alone, L-Arginine alone, combined administration of Angiopoietin-1 protein and L-Arginine, combined administration of L-Arginine and AA, or the combined administration of L-Arginine, AA and Ang-1.

Tie-2

Tie 2 is an angiopoietin receptor, also known as CD202B, TIE-2, TEK, TEK tyrosine receptor kinase, or VMCM1. As used herein, the term “Tie-2 agonist” is intended to refer to a molecule that can stimulate, enhance, activate, increase or upregulate Tie 2 receptor activity, as measured by any method, technique, signal, detector or indicator that is known in the art to be indicative of Tie 2 receptor activity. Non-limiting examples of such indicators of Tie 2 activity include Tie 2 phosphorylation for example of human Tie 2 at amino acid residue Y992, Y814, Y1100 S1119, Y1102, Y1108, or Y1106; phosphorylation of one or more of MAPK, AKT and eNOS; recruitment of docking protein Dok-R (also referred to as p56 ^Dok2 or FRIP) to Tie 2; stimulation of Tie 2 oligomerization, clustering and/or localization at the cell membrane; and/or increased Tie 2 expression in a tissue of interest.

A Tie-2 agonist may be a small molecule, nucleic acid, peptide, antibody or antibody fragment, polypeptide, or a functional equivalent, analog or variant thereof (See, for example, Khan et al. 2021 Ang2 inhibitors and Tie2 activators: potential therapeutics in perioperative treatment of early stage cancer. EMBO Molecular Medicine. 13:e08253; incorporated by reference).

A Tie-2 agonist may bind to Tie 2. In some embodiments, the Tie-2 agonist binds to the extracellular domain (ECD) of Tie 2, wherein the Tie 2 ECD includes three fibronectin type III (Fn) domains (Fn1, Fn2, Fn3), an Ig domain, and an epidermal growth factor-like domain. In some embodiments, the Tie-2 agonist is Angiopoietin-1 (Ang-1) or a functional equivalent, analog or variant thereof. In some embodiments, the Tie-2 agonist is an Ang-1 variant, for example COMP-Ang1, Bow-Ang1, CMP-Ang1, or MAT-Ang1. In some embodiments, the Tie-2 agonist is a tyrosine kinase inhibitor of Tie 2; non-limiting examples include Regorafenib, Pexmetinib, Altiratinib, Rebastinib.

The Tie-2 agonist may bind to a protein or proteins upstream or downstream of Tie 2 to activate Tie2. Upstream or downstream proteins include but are not limited to Tie 1, Ang1, Ang2, VEGF (including VEGF-A), VEGFR1, VEGFR2, VEGFR3, p38, BRAF, RAD1, PDGFRs, FGFRs, and/or MAPK. In some embodiments, the Tie 2 agonist is Cabozantinib. In some embodiments, the Tie-2 agonist is a small-molecule inhibitor of VE-PTP (or HRTRb) which is a protein tyrosine phosphatase that dephosphorylates Tie 2, for example AKB-8778.

In other embodiments, the Tie-2 agonist is an antibody or antibody fragment that binds to a protein upstream or downstream of Tie-2 to activate Tie2. For example, the Tie-2 agonist may be an antibody or antibody fragment that binds to Ang1 and/or Ang2. Further non-limiting examples of Tie-2 agonists include Ang2-binding Tie2-activating antibody (ABTAA) or AB-Tie1-39 (Tie1-binding human antibody).

Angiopoietin-1

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), which were originally described as mediators of developmental angiogenesis are both peptide ligands that bind the Tie-2 receptor tyrosine kinase found primarily on endothelial cells (ECs). Ang-1 and Ang-2 are thought to function as a competitive agonist/antagonist pair for Tie-2 receptor signaling although this dichotomous action appears to be context, dose, and duration specific (Maisonpierre et al., Science 277: 55-60 (1997), Teichert-Kuliszewska et al., Cardiovasc. Res. 49: 659-670 (2001), Saharinen et al., J. Cell Biol. 169: 239-243 (2005)).

The term “angiopoietin-1” or “Ang-1” when used herein is a protein and polypeptide having an amino acid sequence which is substantially identical to a native angiopoietin-1 amino acid sequence of which are biologically active in that they are capable of binding to the Tie receptor or cross-reacting with an anti-angiopoietin-1 antibody raised against angiopoietin-1. Some examples of angiopoietin-1 polypeptides useful in the present invention include [noted by their National Center for Biotechnology Information (NCBI) GenBank database Accession Number]: AAB50557; AAB50558; AAC61872; AAC78246; AAC78245; NP-001137; AAG34113; AAK14992; AAK31330; AAL13077; AAK83347; NP-033770; 035460; 008538; 018920; Q15389 (SEQ ID NO:1); XP— 204878; NP-647451; NP-445998; BAC10290; AAM92271 ; AAM81745; AAH29406; BAB91325; and NP— 571888 . In a preferred embodiment, the angiopoietin- 1 polypeptide used in the present invention is as shown by SEQ ID NO: 1.

The amino acid sequence of SEQ ID NO:1 is as follows:

MTVFLSFAFLAAILTHIGCSNQRRSPENSGRRYNRIQHGQCAYTFILPEHDGNC

RESTTDQYNTNALQRDAPHVEPDFSSQKLQHLEHVMENYTQWLQKLENYIVEN

MKSEMAQIQQNAVQNHTATMLEIGTSLLSQTAEQTRKLTDVETQVLNQTSRLEI

QLLENSLSTYKLEKQLLQQTNEILKIHEKNSLLEHKILEMEGKHKEELDTLKEEKE

NLQGLVTRQTYIIQELEKQLNRATTNNSVLQKQQLELMDTVHNLVNLCTKEGVL

LKGGKREEEKPFRDCADVYQAGFNKSGIYTIYINNMPEPKKVFCNMDVNGGGW

TVIQHREDGSLDFQRGWKEYKMGFGNPSGEYWLGNEFIFAITSQRQYMLRIEL

M DWEG N RAY SQYD R F H I G N EKQ N Y RLYLKG HTGT AG KQSS LI LH GA D FSTKDA

DNDNCMCKCALMLTGGWWFDACGPSNLNGMFYTAGQNHGKLNGIKWHYFK

GPSYSLRSTT MMIRPLDF

A skilled artisan recognizes that angiopoietin-1 comprises a modular structure comprising a receptor-binding domain, a dimerization motif, and a superclustering motif able to form varable-sized multimers. In specific embodiments, a C-terminal domain (similar to fibrinogen or fibrinogen-like domain) of Ang1 is required for receptor binding, a central coiled-coil domain is capable of dimerizing the C-terminal domains, and a short N-terminal region forms ring-like structures that supercluster dimers into variable sized multimers. In specific embodiments of the present invention, a therapeutic agent comprising angiopoietin-1, wherein the angiopoietin-1 comprises one or more of said domains/motifs, is utilized. A skilled artisan recognizes that the beginning of the coiled- coil and F (fibrinogen or fibrinogen-like) domains are defined by the amino acid sequences FSSQKLQH (SEQ ID NO:2) and FRDCADVY (SEQ ID NO:3), respectively. A skilled artisan recognizes that dimerization and/or multimerization and/or oligomerization of angiopietin-1, preferably to form high-order oligomers, may mediate improved Tie 2 activation compared to monomeric Ang-1.

Ang-1 is a secreted protein that is approximately 55 kDa in size and the glycosylated forms can be approximately 70 kDa. Ang-1 nucleic acid molecules encode an Ang-1 polypeptide that preferably has substantial identity to the amino acid sequence set forth in SEQ ID NO: 1. Ang-1 can also include fragments, derivatives, or analogs of Ang-1 that preferably retain at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more Ang-1 biological activity (e.g., binding to the Tie-2 receptor). Ang-1 polypeptides may be isolated from a variety of sources, such as from mammalian tissue or cells or from another source, or alternatively prepared by recombinant or synthetic methods familiar to those skilled in the art. The term “Ang-1” also encompasses modifications to the polypeptide, fragments, derivatives, analogs, functional equivalents, and variants of the Ang-1 polypeptide.

The term “Ang-1 biological activity” or “Ang-1 functional equivalent” includes analogs of angiopoietin-1 molecules which exhibit at least some biological activity in common with native angiopoietin-1. Such analogs include, but are not limited to, truncated angiopoietin-1 polypeptides and angiopoietin-1 polypeptides having fewer amino acids than native angiopoietin-1. Furthermore, those skilled in the art of mutagenesis will appreciate that homologs to the mouse angiopoietin-1 gene, including human homologs, which homologs are as yet undisclosed or undiscovered, may be used in the methods and compositions disclosed herein. These analogs may include the following activities: binding to the Tie-2 receptor, activation of the Tie-2 receptor, induction of Tie-2 phosphorylation, pro-angiogenic or anti-angiogenic activity depending on the environment (Stoeltzing et at, Cancer Res. 63:3370-3377 (2003)), activation of p190RhoGAP, activation of Rac1, downregulation or inhibition of RhoA GTPase or Rho kinase activity, inhibition of vascular permeability, promotion of tumor angiogenesis and tumor vessel plasticity, promotion of endothelial cell survival, anti-inflammatory activity, reduction in expression of inflammatory molecules (e.g., ICAM1) and blood vessel development. Assays for Ang-1 biological activity are known in the art or described herein and include Tie-2 receptor binding assays, Tie-2 receptor activation assays, Tie- 2 phosphorylation assays, in vitro and in vivo angiogenesis assays, and vascular permeability assays.

The term “angiopoietin-1 gene” or “angiopoietin-1 polynucleotide” refers to any DNA sequence that is substantially identical to a DNA sequence encoding an angiopoietin-1 gene product as defined above. The term also refers to RNA, or antisense sequences compatible with such DNA sequences. A “angiopoietin-1 gene” may also comprise any combination of associated control sequences.

Some examples of angiopoietin-1 polynucleotides useful in the present invention include (noted by their National Center for Biotechnology Information's GenBank database Accession Number): U83508; U83509; AF093573; AF030376; AF032923; NM_001146; AF311727; AF233227; AF345932; AY052399; AF379602; NM_009640; NM_004673; NM_053546; NM_139290; XM_204878; AB080023; AY124380;

AY121504; BC029406; AB084454; AB084284; and NIVM31813.

In one embodiment, the Ang-1 functional equivalent, analog or variant thereof is COMP-Ang-1 (also known as Comp-Ang1), wherein the N-terminal portion of Ang1 is replaced with the short coiled-coil domain of cartilage oligomeric matrix protein (COMP) (See, for example, Cho et al. 2004 Proc Natl Acad Sci ;101(15):5547-52). In another embodiment, the the Ang-1 functional equivalent, analog or variant thereof is Bow- Ang1, wherein four Ang1 fibrinogen-like domains are fused to dimerized Fc domain (See, for example, Khan et al. 2021 EMBO Molecular Medicine. 13:e08253).

In one embodiment, the angiopoietin-1 (Ang-1) polypeptide is provided in the form of a prodrug.

In one embodiment, the Ang-1 is delivered intravenously.

Angiopoietin-2

As used herein, the term “angiopoietin-2” or “Ang-2”, unless specified as being from a non-human species (e.g., “mouse Ang-2,” “monkey Ang-2,” etc.), refers to human Ang-2 or a biologically active fragment thereof (e.g., a fragment of the Ang-2 protein which is capable of inducing angiogenesis in vitro or in vivo).

Antibodies that specifically bind to Ang-2, have a high affinity for Ang-2 and/or neutralize or prevent Ang-2 activity and the use of such antibodies in the therapeutic method are included in the invention. Examples of Ang-2 antibodies including Angy-2-1 (Adipogen: #AG-27B-0016PF), L1-7(N), 2Xcon4, L-10 (N) and AB536 (Oliner et al., (2004) Cancer Cell 6:507-516), anti-Ang-2 antibodies from Research Diagnostics Inc., (e.g., catalog nos. RDI-ANGIOP2XabR, RDI-ANG218NabG, and RDI-MANGIOP2abrx) and from AbCam Inc. (e.g. catalog nos. Ab18518, Ab8452, and Ab10601). L1-7(N) is an example of an antibody with high affinity for Ang-2. The ICso for L1-7(N) was 0.071 nM for mouse Ang-2 as compared to >100 nM for Ang-1.

Antibodies that specifically bind to Tie-2 and selectively inhibit binding of Ang-2 but not Ang-1 to the Tie-2 receptor are also useful in the therapeutic methods of the invention.

The term “antibody”, as used herein, is intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1 , CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (Ci_1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-Ang-2 antibody (or antigen-binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

The term “antibody,” as used herein, also includes antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR)). Other engineered molecules, such as diabodies, triabodies, tetrabodies and minibodies, are also encompassed within the expression “antigen-binding fragment,” as used herein.

An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the Vhi and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-Vi_or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) VH-CH1 ; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (V) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1 ; (ix) VL-CH2; (X) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH ₂ -CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using routine techniques available in the art.

Those skilled in the art will be aware that protein scaffolds can bind to antigens with affinity and specificity, which exceeds that of antibodies and therefore can substitute for antibodies for many applications. Such protein scaffolds include, but are not limited to affibodies and their two-helix variants, affilins, affimers, alphabodies, anticalins, affimers, fynomers, kunitz domains, knottins, fibronectins, DARPins, i-bodies, nanobodies and monobodies.

The constant region of an antibody is important in the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody may be selected on the basis of whether it is desirable for the antibody to mediate cytotoxicity.

The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The term “recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the Vhiand VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline Vhiand VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These forms have been extremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human lgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using a human lgG1 hinge. The instant invention encompasses antibodies having one or more mutations in the hinge, CH2 or CH3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form. An “isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds human Ang-2 or a human Ang-2 fragment is substantially free of antibodies that specifically bind antigens other than human Ang-2). The term “specifically binds,” or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by a Koof about 1 ^c 10 ^-8 M or less. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. An isolated antibody that specifically binds human Ang-2 may, however, have cross-reactivity to other antigens, such as Ang-2 molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

A “neutralizing” or “blocking” antibody, as used herein, is intended to refer to an antibody whose binding to Ang-2 blocks the interaction between Ang-2 and its receptor (Tie-2) and/or results in inhibition of at least one biological function of Ang-2. The inhibition caused by an Ang-2 neutralizing or blocking antibody need not be complete so long as it is detectable using an appropriate assay. Exemplary assays for detecting Ang-2 inhibition are described elsewhere herein.

The fully-human anti-Ang-2 antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present invention includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are back-mutated to the corresponding germline residue(s) or to a conservative amino acid substitution (natural or non-natural) of the corresponding germline residue(s) (such sequence changes are referred to herein as “germline back-mutations”). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline back-mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the germline sequence. In other embodiments, only certain residues are mutated back to the germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. Furthermore, the antibodies of the present invention may contain any combination of two or more germline back- mutations within the framework and/or CDR regions, i.e., wherein certain individual residues are mutated back to the germline sequence while certain other residues that differ from the germline sequence are maintained. Once obtained, antibodies and antigen-binding fragments that contain one or more germline back-mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.

The present invention also includes anti-Ang-2 antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-Ang-2 antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein.

The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore™ system (Biacore Life Sciences division of GE Healthcare, Piscataway, N.J.).

The term “KD”, as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction. The term “epitope” refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule or protein scaffold known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstance, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

As applied to polypeptides, the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul etal. (1997) Nucleic Acids Res. 25:3389-402.

The term “Angiopoietin-2 (Ang-2) antagonist” used herein refers to any compound that can antagonise the binding of Ang-2 to its receptor. Non-limiting examples of Ang-2 antagonists include any synthetic or natural polypeptide, nucleic acid, or small molecule compound that can decrease the levels of Ang-2 or reduce or block Ang-2 signalling either by affecting Ang-2 directly or by affecting downstream effector molecules of Ang-2 signalling pathways.

The present invention features therapeutic nucleic acids that can be used to decrease the level of Ang-2 for treatment or prevention of neonatal sepsis. Such therapeutic nucleic acids include anti-sense nucleobase oligomers or small RNAs to downregulate expression of Ang-2 mRNA directly.

L-Arginine

The terms “Arginine” or “Arg” or “L-Arg” or “L-Arginine” are used interchangeably and reference herein this disclosure to “L-Arg” may be replaced with all biochemical equivalents (i.e. salts, precursors such as L-citrulline, and its basic form of L-arginine), preferably those that act as a substrates of NOS with resulting increase in the production of NO. For example, L-lysine may be a biological equivalent of L-arginine. Other bio-equivalents of L-arginine may include arginase inhibitors, citrulline, ornithine, and hydralazine. As used herein a “biological equivalent” is an agent or composition, or combination thereof, which has similar biological function or effect as the agent or composition to which it is being deemed equivalent.

In vitro and in vivo studies have suggested that endotoxin-induced loss of vascular responsiveness is due to activation of NO which is synthesized from L-arginine and can be blocked by NO synthase inhibitors, L-arginine analogues, such as N-nitro-L- arginine methyl ester (L-NAME).

In one embodiment, the L-Arg is provided in the form of a prodrug.

In one embodiment, L-Arg is provided in the form of a food supplement, and therefore delivered orally.

In any embodiment, the L-Arg is a biochemical or biological equivalent of L-Arg as defined herein. Arachidonic acid (AA)

The terms “Arachidonic acid” or “AA” or “ARA” are used interchangeably and reference herein this disclosure to “AA” may be replaced with all biochemical equivalents (i.e. salts, precursors). Arachidonic acid is a polyunsaturated fatty acid. It is considered an "essential" fatty acid. Arachidonic acid is vital to the operation of the prostaglandin system. Prostaglandins are part of a class of substances called eicosanoids. Eicosanoids influence numerous metabolic activities including platelet aggregation (blood clotting), inflammation, hemorrhages, vasoconstriction and vasodilation, blood pressure, and immune function. The eicosanoids contain twenty carbons and include the prostaglandins (PG), prostacyclins (PGI2), thromboxanes (TX), leukotrienes (LT), and hydroxy acids.

Eicosanoids are lipid-signalling molecules primarily generated from arachidonic acid (AA), which is released by phospholipase A2 enzymes from membrane phospholipids. The subsequent enzymatic metabolization via cyclooxygenase (COX), cytochrome P450, and lipoxygenase pathways or via nonenzymatic peroxidation mediates the generation of a broad spectrum of eicosanoids. As proinflammatory molecules (prostaglandin [PG] H2), chemoattractants (leukotriene B4), platelet (PLT) aggregation factors, and contractors of smooth muscle cells (thromboxane A2), modifiers of vascular permeability (leukotrienes) and potent vasodilators (PGE2 and PGI2), these cell-derived mediators are involved in the pathogenesis of inflammatory diseases and sepsis. Two prostaglandins arachidonic acid is the substrate to are PGE2 and PGF2a.

In one embodiment, the AA is provided in the form of a prodrug.

In one embodiment, the AA is provided in the form of a food supplement, and therefore delivered orally.

In any embodiment, the AA is a biochemical or biological equivalent of AA as defined herein. Proteins and polypeptides

"Isolated," describes the various polypeptides disclosed herein, means the polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non- proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated protein includes polypeptide in situ within recombinant cells, since at least one component of the polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

A "fragment" is a portion of a polypeptide of the present invention that retains substantially similar functional activity or substantially the same biological function or activity as the polypeptide, which can be determined using assays described herein.

“Percent (%) amino acid sequence identity” or “percent (%) identical” with respect to a polypeptide sequence, i.e. a polypeptide of the invention defined herein, is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide of the invention, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms (non-limiting examples described below) needed to achieve maximal alignment over the full-length of the sequences being compared.

When amino acid sequences are aligned, the percent amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain percent amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as: percent amino acid sequence identity = X/Y100, where X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not equal the percent amino acid sequence identity of B to A.

In calculating percent identity, typically exact matches are counted. The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et ai (1990) J. Mol. Biol. 215:403. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et ai (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et ai (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. Alignment may also be performed manually by inspection. Another non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the ClustalW algorithm (Higgins et ai (1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, CA). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed. A non-limiting examples of a software program useful for analysis of ClustalW alignments is GENEDOC™ or JalView (http://www.jalview.org/). GENEDOC™ allows assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11- 17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA). When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

The polypeptide desirably comprises an amino end and a carboxyl end. The polypeptide can comprise D-amino acids, L-amino acids or a mixture of D- and L-amino acids. The D-form of the amino acids, however, is particularly preferred since a polypeptide comprised of D-amino acids is expected to have a greater retention of its biological activity in vivo.

The polypeptide can be prepared by any of a number of conventional techniques. The polypeptide can be isolated or purified from a naturally occurring source or from a recombinant source. Recombinant production is preferred. For instance, in the case of recombinant polypeptides, a DNA fragment encoding a desired peptide can be subcloned into an appropriate vector using well-known molecular genetic techniques (see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, 1982); Sambrook etai, Molecular Cloning A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, 1989). The fragment can be transcribed and the polypeptide subsequently translated in vitro. Commercially available kits also can be employed (e.g., such as manufactured by Clontech, Palo Alto, Calif.; Amersham Pharmacia Biotech Inc., Piscataway, N.J.; InVitrogen, Carlsbad, Calif., and the like). The polymerase chain reaction optionally can be employed in the manipulation of nucleic acids.

The term "conservative substitution" as used herein, refers to the replacement of an amino acid present in the native sequence in the peptide with a naturally or non- naturally occurring amino acid or a peptidomimetic having similar steric properties. Where the side-chain of the native amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non- naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side- chain of the replaced amino acid). Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that may be considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

As naturally occurring amino acids are typically grouped according to their properties, conservative substitutions by naturally occurring amino acids can be determined bearing in mind the fact that replacement of charged amino acids by sterically similar non-charged amino acids are considered as conservative substitutions. For producing conservative substitutions by non-naturally occurring amino acids it is also possible to use amino acid analogs (synthetic amino acids) well known in the art. A peptidomimetic of the naturally occurring amino acid is well documented in the literature known to the skilled person and non-natural or unnatural amino acids are described further below. When affecting conservative substitutions the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.

The phrase "non-conservative substitution" or a “non-conservative residue” as used herein refers to replacement of the amino acid as present in the parent sequence by another naturally or non-naturally occurring amino acid, having different electrochemical and/or steric properties. Thus, the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted. Examples of non-conservative substitutions of this type include the substitution of phenylalanine or cyclohexylmethyl glycine for alanine, isoleucine for glycine, or -NH-CH[(-CH2)5-COOH]-CO- for aspartic acid. Non-conservative substitution includes any mutation that is not considered conservative.

A non-conservative amino acid substitution can result from changes in: (a) the structure of the amino acid backbone in the area of the substitution; (b) the charge or hydrophobicity of the amino acid; or (c) the bulk of an amino acid side chain. Substitutions generally expected to produce the greatest changes in protein properties are those in which: (a) a hydrophilic residue is substituted for (or by) a hydrophobic residue; (b) a proline is substituted for (or by) any other residue; (c) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine; or (d) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl.

Alterations of the native amino acid sequence to produce mutant polypeptides, such as by insertion, deletion and/or substitution, can be done by a variety of means known to those skilled in the art. For instance, site-specific mutations can be introduced by ligating into an expression vector a synthesized oligonucleotide comprising the modified site. Alternately, oligonucleotide-directed site-specific mutagenesis procedures can be used, such as disclosed in Walder et al., Gene 42: 133 (1986); Bauer et al., Gene 37: 73 (1985); Craik, Biotechniques, 12-19 (January 1995); and U.S. Pat. Nos. 4,518,584 and 4,737,462. A preferred means for introducing mutations is the QuikChange Site-Directed Mutagenesis Kit (Stratagene, LaJolla, Calif.).

Any appropriate expression vector (e.g., as described in Pouwels et al., Cloning Vectors: A Laboratory Manual (Elsevier, N.Y.: 1985)) and corresponding suitable host can be employed for production of recombinant polypeptides. Expression hosts include, but are not limited to, bacterial species within the genera Escherichia, Bacillus, Pseudomonas, Salmonella, mammalian or insect host cell systems including baculovirus systems (e.g., as described by Luckow et al., Bio/Technology 6: 47 (1988)), and established cell lines such as the COS-7, C127, 3T3, CHO, HeLa, and BHK cell lines, and the like. The skilled person is aware that the choice of expression host has ramifications for the type of polypeptide produced. For instance, the glycosylation of polypeptides produced in yeast or mammalian cells (e.g., COS-7 cells) will differ from that of polypeptides produced in bacterial cells, such as Escherichia coli.

Alternately, a polypeptide of the invention can be synthesized using standard peptide synthesizing techniques well-known to those of ordinary skill in the art (e.g., as summarized in Bodanszky, Principles of Peptide Synthesis (Springer-Verlag,

Heidelberg: 1984)). In particular, the polypeptide can be synthesized using the procedure of solid-phase synthesis (see, e.g., Merrifield, J. Am. Chem. Soc. 85: 2149- 54 (1963); Barany et al., Int. J. Peptide Protein Res. 30: 705-739 (1987); and U.S. Pat. No. 5,424,398). If desired, this can be done using an automated peptide synthesizer. Removal of the t-butyloxycarbonyl (t-BOC) or 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid blocking groups and separation of the polypeptide from the resin can be accomplished by, for example, acid treatment at reduced temperature. The polypeptide- containing mixture can then be extracted, for instance, with dimethyl ether, to remove non-peptidic organic compounds, and the synthesized polypeptide can be extracted from the resin powder (e.g., with about 25% w/v acetic acid). Following the synthesis of the polypeptide, further purification (e.g., using high performance liquid chromatography (HPLC)) optionally can be done in order to eliminate any incomplete polypeptides or free amino acids. Amino acid and/or HPLC analysis can be performed on the synthesized polypeptide to validate its identity. For other applications according to the invention, it may be preferable to produce the polypeptide as part of a larger fusion protein, such as by the methods described herein or other genetic means, or as part of a larger conjugate, such as through physical or chemical conjugation, as known to those of ordinary skill in the art and described herein.

A polypeptide of the invention may also be modified by, conjugated or fused to another moiety to facilitate purification, or increasing the in vivo half-life of the polypeptides, or for use in immunoassays using methods known in the art. For example, a polypeptide of the invention may be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.

A “peptidomimetic” is a synthetic chemical compound that has substantially the same structure and/or functional characteristics of a polypeptide of the invention, the latter being described further herein. A peptidomimetic generally contains at least one residue that is not naturally synthesised. Non-natural components of peptidomimetic compounds may be according to one or more of: a) residue linkage groups other than the natural amide bond ('peptide bond') linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e , to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.

Peptidomimetics can be synthesized using a variety of procedures and methodologies described in the scientific and patent literatures, e.g., Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley & Sons, Inc., NY, al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1:114-119;

Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996) Methods Enzymot.267:220-234

Modifications contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during polypeptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the polypeptides of the invention. Any modification, including post-translational modification that reduces the capacity of the molecule to form a dimer is contemplated herein. An example includes modification incorporated by click chemistry as known in the art. Exemplary modifications include PEGylation and glycosylation.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBhU; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBhU.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal. The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy- 5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4- amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids contemplated herein is shown in Table 2.

Table 2

Non-conventional Code Non-conventional Code amino acid amino acid a-aminobutyric acid Abu L-N-methylalanine Nmala a-amino-a-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine Dgln L-N-methylnorvaline Nmnva

D-glutamic acid Dglu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine Dile L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine Nle

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr a-methyl-aminoisobutyrate Maib

D-valine Dval a-methyl-y-aminobutyrate Mgabu

D-a-methylalanine Dmala a-methylcyclohexylalanine M chexa

D-a-methylarginine Dmarg a-methylcylcopentylalanine Mcpen

D-a-methylasparagine Dmasn a-methyl-a-napthylalanine Manap

D-a-methylaspartate Dmasp a-methylpenicillamine Mpen

D-a-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu

D-a-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-a-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-a-methylisoleucine Dmile N-amino-a-methylbutyrate Nmaabu

D-a-methylleucine Dmleu a-napthylalanine Anap

D-a-methyllysine Dmlys N-benzylglycine Nphe D-a-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln

D-a-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-a-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu

D-a-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-a-methylserine Dmser N-cyclobutylglycine Ncbut

D-a-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-a-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-a-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-a-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe

D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-Y-aminobutyrate Nmgabu

N-methylcyclohexylalanineNmchexa D-N-methylmethionine Dnmmet D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine Nala D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser

N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtrp N-(1 -methylethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methyl-a-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen g-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr

L-/-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-a- ethylalanine Mala

L-a-methylarginine Marg L-a-methylasparagine Masn L-a-methylaspartate Masp L-a-methyl-f-butylglycine Mtbug

L-a-methylcysteine Mcys L-methylethylglycine Metg

L-a-methylglutamine Mgln L-a-methylglutamate Mglu

L-a-methylhistidine Mhis L-a-methylhomophenylalanine Mhphe

L-a-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L-a-methylleucine Mleu L-a-methyllysine Mlys

L-a-methylmethionine Mmet L-a-methylnorleucine Mnle

L-a-methylnorvaline Mnva L-a-methylornithine Morn

L-a-methylphenylalanine Mphe L-a-methylproline Mpro

L-a-methylserine Mser L-a-methylthreonine Mthr

L-a-methyltryptophan Mtrp L-a-methyltyrosine Mtyr

L-a-methylvaline Mval L-N-methylhomophenylalanine Nmhphe

N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine

1-carboxy-1-(2,2-diphenyl-Nmbc ethylamino)cyclopropane

Crosslinkers can be used, for example, to stabilise 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N- hydroxysuccinimide and another group specific-reactive moiety.

Nucleic acids

Nucleic acid molecules that encode any of the polypeptides of the invention are also within the scope of the invention. The nucleic acids are useful, for example, in making the polypeptides of the present invention and as therapeutic agents. They may be administered to cells in culture or in vivo and may include a secretory signal that directs or facilitates secretion of the polypeptide of the invention from the cell. Also within the scope of the invention are expression vectors and host cells that contain or include nucleic acids of the invention (described further below). While the nucleic acids of the invention may be referred to as “isolated,” by definition, the polypeptides of the invention are not wild-type polypeptides and, as such, would not be encoded by naturally occurring nucleic acids. Thus, while the polypeptides and nucleic acids of the present invention may be “purified,” “substantially purified,” “isolated,” “recombinant” or “synthetic” they need not be so in order to be distinguished from naturally occurring materials.

An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide encoding nucleic acid. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule includes nucleic acid molecules contained in cells that ordinarily express Ang-1 where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.

The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide of the invention may be provided in isolated or purified form. A nucleic acid sequence which “encodes” a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. For the purposes of the invention, such nucleic acid sequences can include, but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

Polynucleotides of the invention can be synthesised according to methods well known in the art, as described by way of example in Sambrook et al (1989, Molecular Cloning — a laboratory manual; Cold Spring Harbor Press). The polynucleotide molecules of the present invention may be provided in the form of an expression cassette which includes control sequences operably linked to the inserted sequence, thus allowing for expression of the polypeptide of the invention in vivo in a targeted subject. These expression cassettes, in turn, are typically provided within vectors (e.g., plasmids or recombinant viral vectors) which are suitable for use as reagents for nucleic acid immunization. Such an expression cassette may be administered directly to a host subject. Alternatively, a vector comprising a polynucleotide of the invention may be administered to a host subject. Preferably the polynucleotide is prepared and/or administered using a genetic vector. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a polypeptide of the invention.

The present invention thus includes expression vectors that comprise such polynucleotide sequences. Thus, the present invention provides a vector for use in preventing or treating an inflammatory disease or condition comprising a polynucleotide sequence which encodes a polypeptide of the invention and optionally one or more further polynucleotide sequences which encode different polypeptides as defined herein.

Furthermore, it will be appreciated that the compositions and products of the invention may comprise a mixture of polypeptides and polynucleotides. Accordingly, the invention provides a composition or product as defined herein, wherein in place of any one of the polypeptide is a polynucleotide capable of expressing said polypeptide.

Expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for expression of a peptide of the invention. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al.

Thus, a polypeptide of the invention may be provided by delivering such a vector to a cell and allowing transcription from the vector to occur. Preferably, a polynucleotide of the invention or for use in the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.

“Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given regulatory sequence, such as a promoter, operably linked to a nucleic acid sequence is capable of effecting the expression of that sequence when the proper enzymes are present. The promoter need not be contiguous with the sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the nucleic acid sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.

A number of expression systems have been described in the art, each of which typically consists of a vector containing a gene or nucleotide sequence of interest operably linked to expression control sequences. These control sequences include transcriptional promoter sequences and transcriptional start and termination sequences. The vectors of the invention may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. A “plasmid” is a vector in the form of an extra-chromosomal genetic element. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell. The vectors may also be adapted to be used in vivo, for example to allow in vivo expression of the polypeptide.

A “promoter” is a nucleotide sequence which initiates and regulates transcription of a polypeptide-encoding polynucleotide. Promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. It is intended that the term “promoter” or “control element” includes full-length promoter regions and functional (e.g., controls transcription or translation) segments of these regions.

A polynucleotide, expression cassette or vector according to the present invention may additionally comprise a signal peptide sequence. The signal peptide sequence is generally inserted in operable linkage with the promoter such that the signal peptide is expressed and facilitates secretion of a polypeptide encoded by coding sequence also in operable linkage with the promoter.

Typically a signal peptide sequence encodes a peptide of 10 to 30 amino acids for example 15 to 20 amino acids. Often the amino acids are predominantly hydrophobic. In a typical situation, a signal peptide targets a growing polypeptide chain bearing the signal peptide to the endoplasmic reticulum of the expressing cell. The signal peptide is cleaved off in the endoplasmic reticulum, allowing for secretion of the polypeptide via the Golgi apparatus. Thus, a peptide of the invention may be provided to an individual by expression from cells within the individual, and secretion from those cells.

The phrase “therapeutically effective amount” generally refers to an amount of one or more polypeptides or polynucleotides of the invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.

The term “small RNA” is meant any RNA molecule, either single-stranded or double-stranded” that is at least 15 nucleotides, preferably, 17, 18, 19, 20, 21, 22, 23,

24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35, nucleotides in length and even up to 50 or 100 nucleotides in length (inclusive of all integers in between). Preferably, the small RNA is capable of mediating RNAi. As used herein the phrase “mediates RNAi” refers to the ability to distinguish which RNAs are to be degraded by the RNAi machinery or process. Included within the term small RNA are “small interfering RNAs” and “microRNA.” In general, microRNAs (miRNAs) are small (e.g., 17-26 nucleotides), single-stranded noncoding RNAs that are processed from approximately 70 nucleotide hairpin precursor RNAs by Dicer. Small interfering RNAs (siRNAs) are of a similar size and are also noncoding, however, siRNAs are processed from long dsRNAs and are usually double stranded. siRNAs can also include short hairpin RNAs in which both strands of an siRNA duplex are included within a single RNA molecule. Small RNAs can be used to describe both types of RNA. These terms include double-stranded RNA, single-stranded RNA, isolated RNA (partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA), as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the small RNA or internally (at one or more nucleotides of the RNA). Nucleotides in the RNA molecules of the present invention can also comprise non standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides. See “nucleobase oligomers” above for additional modifications to the nucleic acid molecule. In a preferred embodiment, the RNA molecules contain a 3' hydroxyl group.

Treatment and prevention of neonatal sepsis

The term “sepsis” is a disorder or state characterized by a source of infection, proven, for example, by a positive blood culture for a source of infection (or inferred on clinical grounds) accompanied by two or more of the following: a heart rate greater than 90 beats per minute; a body temperature less than 36° C. or 96.8° F. or greater than 38° C. or 100.4° F.; hyperventilation (high respiratory rate) greater than 20 breaths per minute or on blood gas a P _a Co2 less than 32 mm Hg; and a white blood cell count <4000 cells/mm ³ or >12000 cells/mm ³ (<4*10 ⁹ or >12x10 ⁹ cells/L), or greater than 10% band forms (immature white blood cells).

The term “neonatal sepsis” is disorder or state characterized by early onset (day of life 0-3) or late onset (LOS) (day of life 4 or later). Newborns with early-onset sepsis, 85% present within 24 hours (median age of onset 6 hours), 5% present at 24-48 hours and a smaller percentage present within 48-72 hours. Onset is most rapid in premature neonates. In particular, the term “early onset sepsis” is often associated with acquisition of microorganisms from the mother. Infection can occur via hematogenous, transplacental spread from an infected mother, or more commonly, via ascending infection from the cervix of the mother. The term “therapeutic amount” is meant an amount that when administered to a subject suffering from any of the disorders of the invention (e.g., neonatal sepsis) is sufficient to cause a qualitative or quantitative reduction in the symptoms associated with the sepsis, for example, as described below.

The term “reduce or inhibit” describes the ability to cause an overall decrease preferably of 20% or greater, more preferably of 50% or greater, and most preferably of 75%, 80%, 85%, 90%, 95%, or greater. For therapeutic applications, reduce or inhibit can refer to the symptoms of the disorder being treated or the presence or extent of sepsis in neonates. Symptoms of the disorder include impairment in the inability to oxygenate the blood as well as impairment in the ability to ventilate the lungs (as with mechanical ventilation). These impairments generally mandate intensification of ventilation strategies (e.g. introduction of greater positive pressure to inflate stiff lungs). Other clinical endpoints that may be reduced or inhibited include the following: overall survival, days of ICU care required, long-term oxygen requirement, requirement for pulmonary bypass procedures such as ECMO (extracorporeal membrane oxygenation), development of pneumothorax, requirement for immunomodulatory therapies, such as glucocorticoids, development of chronic patterns of injury as a result of severe ARDS such as bronchiolitis obliterans and pulmonary fibrosis. For diagnostic or monitoring applications, reduce or inhibit can refer to a decrease in the level of protein or nucleic acid, detected by the aforementioned assays (see “expression”).

The term “treating” is meant administering a compound or a pharmaceutical composition for prophylactic and/or therapeutic purposes or administering treatment to a subject already suffering from a disease to improve the subject's condition or to a subject who is at risk of developing a disease. By “treating neonatal sepsis” is meant that the disease and the symptoms associated with the disease are alleviated, reduced, cured, or placed in a state of remission. More specifically, a Tie-2 agonist, Ang-1 or a functional equivalent, analog or variant thereof, L-Arg, AA, or Ang-2 antagonist or a combination thereof, is generally provided in a therapeutically effective amount to achieve any one or more of the following: reduce mortality, reduce vascular leakage, restore the integrity of vessel walls, prevent requirement for mechanical ventilation, reduce organ damage, increase in arterial blood pressure, increase in cardiac output, decreased systemic vascular resistance, decrease in the number of vasopressor medications necessary to maintain tissue perfusion, reduction in edema-bedside clinical assessment, increased urine output, decreased weight gain upon administration of intravenous fluids, increase in oxygenation of blood — increased Pa02/Fi02, increased oxygen saturation (Sp02), decreased positive end-expiratory pressure (PEEP) needed to ventilate lungs adequately, fall in respiratory rate, decrease in time to discontinuing mechanical ventilation, decrease in number of ICU days required, and decrease in time to resolution of shock.

By “preventing” is meant prophylactic treatment of a subject who is not yet ill, but who is susceptible to, or otherwise at risk of, developing a particular disease. Preferably, a subject is determined to be at risk of developing any type of sepsis in neonates, using the diagnostic methods known in the art or described herein. For example, risk of developing neonatal sepsis could be determined bygestational age at birth (i.e. prematurity) or birthweight, in that the lower the gestational age at birth or the lower the birthweight, the higher the risk for neonatal sepsis. Approximately 10% of all births in Australia or the United States are premature (before 37 gestational weeks). Neonates at highest risk (the most premature and/or those with lowest birthweight), can readily be identified merely on their personal medical history and offered this prophylaxis as they stand to benefit the most from a prophylactic approach.

In the case of a neonate already diagnosed with (definite sepsis) or suspected to suffer from sepsis (probable or possible serious bacterial infection or very severe disease), “preventing” can refer to the prevention of worsening of the subsequent clinical course. In areas of the world with minimal diagnostic support, this ‘diagnosis’ is often solely based on clinical impression where newborns that appear to have difficulty breathing (e.g. chest indrawing; take breaths over 60 per minute), or are lethargic (move only when stimulated or not move at all), or have convulsions, or are not able to feed well, or have a high (>38°C) or low (<35.5°C) body temperature are considered to have possible serious bacterial infection or very severe disease (See for example, Integrated Management of Childhood Illness: management of the sick young infant aged up to 2 months. IMCI chart booklet. Geneva: World Health Organization; 2019). Where possible, such clinical impression of possible serious bacterial infection or very severe disease can be supplemented with laboratory assays such as blood culture, white blood cell counts (under 5,000 or over 15,000 WBC per microliter), C-reactive protein and other acute phase reactants, including some that reflect inflammation and vascular integrity (See Leligodowicz et al. Risk-stratification of febrile African children at risk of sepsis using sTREM-1 as basis for a rapid triage test. Nat Commun. 2021 Nov 25;12(1):6832).

For example, in the case of a patient already diagnosed with sepsis, “preventing” can refer to the prevention of severe sepsis in neonates, lung failure, or death.

The term “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

In circumstances where the subject is at risk of neonatal sepsis then the subject may be given one or more of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or a functional equivalent, analog or variant thereof, L-Arginine (L-Arg), arachidonic acid (AA) or Angiopoietin-2 (Ang-2) antagonist prophylactically. In other words, before the onset of sepsis or of any suspicion/moderate to severe risk of sepsis (for example indication of possible serious bacterial infection or very severe disease) in a subject at risk, the neonate may be provided with one or more of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or a functional equivalent, analog or variant thereof, L-Arginine (L-Arg), arachidonic acid (AA) or Angiopoietin-2 (Ang-2) antagonist in a form and via a route of administration as described herein. In circumstances where the subject is diagnosed as suffering from neonatal sepsis or there is suspicion/ moderate to severe risk of sepsis then the subject may be given one or more of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or a functional equivalent, analog or variant thereof, L-Arginine (L-Arg), arachidonic acid (AA) or Angiopoietin-2 (Ang-2) antagonist therapeutically. In other words, in the presence of clinical signs or symptoms suspicious of sepsis, the neonate may be provided with one or more of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or a functional equivalent, analog or variant thereof, L-Arginine (L-Arg), arachidonic acid (AA) or Angiopoietin-2 (Ang-2) antagonist in a form and via a route of administration as described herein.

Dosage

It will be well within the purview of the person skilled in the art to determine an appropriate dosage of a Tie-2 agonist, Ang-1 or a functional equivalent, analog or variant thereof (preferably a recombinant Angiopoietin 1 protein), L-Arginine or arachidonic acid (AA) as described herein for the purpose of treating or preventing neonatal sepsis.

The present methods contemplate the provision of a range of dosages of a Tie-2 agonist, Angiopoietin-1 or a functional equivalent, analog or variant thereof (preferably an Ang-1 protein), and/or L-Arginine and/or arachidonic acid (AA). It will be appreciated that the dose may vary depending on the mode of administration of the Tie-2 agonist, Angiopoietin-1 or a functional equivalent, analog or variant thereof (preferably Ang-1 protein), and/or L-Arginine and/or arachidonic acid (AA), the form in which it is provided (e.g., as an oral dosage form, injection or dietary agent) and the intended site of action of the Tie-2 agonist, Angiopoietin-1 or a functional equivalent, analog or variant thereof (preferably Ang-1 protein), and/or L-Arginine and/or arachidonic acid (AA), and the age as well as clinical condition of the recipient.

Preferably, where the method involves the administration of an oral dosage form for delivery of the Tie-2 agonist, Angiopoietin-1 or a functional equivalent, analog or variant thereof (preferably an Ang-1 protein), and/or L-Arginine and/or arachidonic acid (AA) into the large intestine of an individual, the dose is approximately 0.01 mg/kg to 100 mg/kg per day, preferably 0.1 mg/kg to 100 mg/kg per day, more preferably 1 mg/kg to 50 mg/kg. In a most preferred embodiment, the daily dose of any one of Angiopoietin-1 protein, and/or L-Arginine and/or arachidonic acid (AA) is 2 mg/kg to 10 mg/kg, including 3, 4, 5, 6, 7, 8, 9 mg/kg, and 10 mg/kg.

In circumstances involves the administration of the composition via intravenous delivery of the Tie-2 agonist, Angiopoietin-1 or a functional equivalent, analog or variant thereof (preferably an Ang-1 protein), and/or L-Arginine and/or arachidonic acid (AA) into blood of an individual, the dose is approximately 0.01 mg/kg to 100 mg/kg per day, preferably 0.1 mg/kg to 100 mg/kg per day, more preferably 1 mg/kg to 50 mg/kg. In a most preferred embodiment, the daily dose of any one of the Tie-2 agonist, preferably a Angiopoietin-1 or a functional equivalent, analog or variant thereof (preferably an Ang-1 protein), and/or L-Arginine and/or arachidonic acid (AA) is 2 mg/kg to 10 mg/kg, including 3, 4, 5, 6, 7, 8, 9 mg/kg, and 10 mg/kg. Administration

The skilled person will be familiar with appropriate administration of a Tie-2 agonist, Angiopoietin-1 or a functional equivalent, analog or variant thereof (preferably Ang-1 protein), and/or L-Arginine and/or arachidonic acid (AA) described herein. It will be appreciated that a Tie-2 agonist, Angiopoietin-1 or a functional equivalent, analog or variant thereof, and/or L-Arginine and/or arachidonic acid (AA) can be administered separately to individual to treat neonatal sepsis.

The pharmaceutical compositions of the invention may be administered by any suitable means, for example, orally, such as in the form of tablets, capsules, granules or powders; sublingually; buccally; parenterally, such as by subcutaneous, intravenous, intramuscular, intraperitoneal or intracisternal injection or infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions); nasally such as by inhalation spray; topically, such as in the form of a cream or ointment; or rectally such as in the form of suppositories or enemas; in dosage unit formulations containing non toxic, pharmaceutically acceptable vehicles or diluents. They may, for example, be administered in a form suitable for immediate release or extended release, for example, by the use of devices such as subcutaneous implants, encapsulated spheroids or osmotic pumps.

In an alternative embodiment, the methods of the present invention involve the administration of a Tie-2 agonist, Angiopoietin-1 or a functional equivalent, analog or variant thereof, and/or L-Arginine and/or arachidonic acid (AA) via intravenous administration.

In a preferred embodiment, the present invention includes a method of preventing or treating an neonatal sepsis in an individual in need thereof, comprising administering to the individual, a first pharmaceutical dosage form comprising a Tie-2 agonist, preferably a Angiopoietin-1 or a functional equivalent, analog or variant thereof, and/or a second pharmaceutical dosage form comprising L-Arginine, and/or a third pharmaceutical dosage form comprising arachidonic acid (AA) wherein the first pharmaceutical dosage form is adapted for intravenous injection, the second pharmaceutical dosage form is adapted for intravenous injection and the third pharmaceutical dosage form is adapted for intravenous injection. In a preferred embodiment, the present invention includes a method of preventing or treating an neonatal sepsis in an individual in need thereof, comprising administering to the individual, a first pharmaceutical dosage form comprising a Tie-2 agonist, preferably Angiopoietin-1 or a functional equivalent, analog or variant thereof, and/or a second pharmaceutical dosage form comprising L-Arginine, and/or a third pharmaceutical dosage form comprising arachidonic acid (AA) wherein the first pharmaceutical dosage form is adapted for intravenous injection, the second pharmaceutical dosage form is adapted for oral administration and the third pharmaceutical dosage form is adapted for oral administration.

In yet an alternative embodiment, the present invention includes a method of preventing or treating an neonatal sepsis in an individual in need thereof, comprising administering to the individual, a first pharmaceutical dosage form comprising a Tie-2 agonist, preferably Angiopoietin-1 or a functional equivalent, analog or variant thereof, and a second pharmaceutical dosage form comprising L-Arginine, wherein the first pharmaceutical dosage form is adapted for parenteral injection and the second pharmaceutical dosage form is adapted for oral administration.

Alternatively, the invention provides a method of preventing or neonatal sepsis in an individual in need thereof, comprising administering to the individual, a pharmaceutical dosage form comprising a Tie-2 agonist, preferably Angiopoietin-1 or a functional equivalent, analog or variant thereof and L-Arginine. The pharmaceutical dosage form may be adapted for parenteral injection (including intravenous or subcutaneous injection), or oral administration.

Combination therapy

The present invention contemplates the use of the combination of a Tie-2 agonist, Angiopoietin-1 or a functional equivalent, analog or variant thereof (preferably an Ang-1 protein), L-Arginine and/or arachidonic acid (AA) to treat or prevent neonatal sepsis. For example, a combination of:

• a Tie-2 agonist, preferably Angiopoietin-1 or a functional equivalent, analog or variant thereof, and L-Arginine;

• a Tie-2 agonist, preferably Angiopoietin-1 or a functional equivalent, analog or variant thereof, and AA; • L-Arginine and AA; or

• a Tie-2 agonist, preferably Angiopoietin-1 or a functional equivalent, analog or variant thereof, L-Arginine and AA.

In any embodiment, the Ang-1 is an Ang-1 protein.

In another embodiment, any combination referred to herein further comprises an Ang-2 antagonist, for example any antagonist described herein.

In another embodiment, an Ang-2 antagonist may comprise an antibody, an antibody fragment or a protein scaffold, as described above.

For example, the methods of the present invention include the prior, simultaneous or sequential provision to an individual of Angiopoietin-1 protein and L- Arginine.

Further, the present invention also contemplates the use of a Tie-2 agonist, preferably an Angiopoietin-1 or a functional equivalent, analog or variant thereof, L- Arginine and/or arachidonic acid (AA), in combination with other compositions or methods for preventing or treating neonatal sepsis.

In one embodiment, the one or more of one or more of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or a functional equivalent, analog or variant thereof, L-Arginine (L-Arg), arachidonic acid (AA), and Angiopoietin-2 (Ang-2) antagonist is in the form of a prodrug.

In another embodiment, the one or more of one or more of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or a functional equivalent, analog or variant thereof, L-Arginine (L-Arg), arachidonic acid (AA), and Angiopoietin-2 (Ang-2) antagonist is in the form of a bispecific drug with an additional agent, which releases its constituent agents upon administration to the subject. In a further embodiment, the additional agent is one or more of one or more of a Tie-2 agonist, Angiopoietin-1 (Ang-1) or a functional equivalent, analog or variant thereof, L-Arginine (L-Arg), arachidonic acid (AA), and Angiopoietin-2 (Ang-2) antagonist.

Examples Example 1: Materials and Methods

Mice and Monitoring

All animal work conducted was approved by the Animal Ethics Committee at Telethon Kids Institute. Specific pathogen-free mouse breading pairs for C57BL/6J mice were bought in from The Jackson Laboratory and bred in-house. To generate neonatal mice, paired matings were established weekly, females were isolated from males after being visually identified as pregnant and then monitored daily thereafter to ensure an accurate date of birth was determined for all experimental mice. All experimental mice were monitored as previously described (Brook et al. (2019) A Controlled Mouse Model for Neonatal Polymicrobial Sepsis. J Vis Exp.). Briefly, mice were monitored every 8 to 12 hours for the first 2 days post challenge and then daily thereafter. Humane end-point was determined using a righting reflex and mobility score as previously described (Brook et al. (2019) A Controlled Mouse Model for Neonatal Polymicrobial Sepsis. J Vis Exp.).

Murine Sepsis Challenge

The neonatal sepsis model was utilized as previously described (Brook et al. (2019) A Controlled Mouse Model for Neonatal Polymicrobial Sepsis. J Vis Exp.). In short, caecal slurry (CS) was obtained from adult male caeca and resuspended in dextrose 5% water at a concentration of 100mg/mL and then passed through a 70mhi filter. Aliquots were frozen at -80°C until challenge. Neonatal mice at DOL6-8 were challenged via intraperitoneal injection at a weight adjusted dose of 1mg/g body weight. Litter- to- litter variation was accounted for by having a balanced number of treated and control mice in each litter.

Transcriptomics and statistical analyses

All statistical analysis and figure generation was performed in R v4.0.2. RNA samples with <1 million reads after globin removal were excluded from analysis. Normalization and generation of DE gene lists was done with DESeq2 v1.28.1 - genes were considered to be differentially expressed with a Benjamini-Hochberg adjusted p- value of <0.05 and a FC value of ± 1.5. GO term and pathway enrichment analysis used clusterProfiler v3.16.0 (Yu et al. 2012, clusterProfiler: an R package for comparing biological themes among gene clusters. Omi. A J. Integr. Biol. 16, 284-287).

Survival Experiments

For prophylactic arachidonic acid (AA) experiments, 10mg of AA (Fisher Scientific: #ICN19462510) was diluted in 30mI_ of corn oil which was administered 4-6 prior to CS challenge via oral gavage. For exogenous Ang-1 and Ang-2 experiments, 500ng of Ang-1 or Ang-2 (R&D Systems: #9936-AN and #7186-AN respectively) was administered via intraperitoneal injection 1-hour prior to CS challenge. For anti- Angiopoietin-2 antibody treatment, 50mg of antibody (Adipogen: #AG-27B-0016PF) was administered concurrently with CS challenge via intraperitoneal injection. For L-NAME experiments, 1C^g of L-NAME (Abeam: #120136) reconstituted in distilled water was injected via intraperitoneal injection concurrently with CS challenge. Finally, for L- Arginine experiments, 5mg of L-Arginine (Cayman Chemical: #23703) reconstituted in distilled water was administered via intraperitoneal injection 4-6 hours prior to CS challenge. The schedules described above were maintained for all combinational interventions described.

ROS Quantification and Immunohistochemistry

For ROS detection, mice were treated according to the schedule described above. 8 hours post CS challenge we administered dihydroethidium (DHE) (Cayman Chemical: #12013) reconstituted in DMSO at a concentration of 10mg/g body weight via intraperitoneal injection. After a 20-minute incubation period, mice were euthanized, and livers were excised and immediately drop fixed in 4% paraformaldehyde (Thermo Scientific: #AAJ19943K2) for 48 hours. Livers were then snap frozen, sectioned and DAPI counterstained. DHE was visualized using fluorescence microscopy utilizing a standard ethidium bromide filter. Relative ROS quantification was performed using ImageJ as previously described (Emrich et al. 2019 Anatomically specific reactive oxygen species production participates in Marfan syndrome aneurysm formation. J. Cell. Mol. Med. 23, 7000-7009). Briefly, a DAPI counter-mask was created to isolate nuclei, background fluorescence was subtracted, and MFI of DHE was calculated. All results are normalized relative to batch-matched unchallenged controls that were imaged under identical parameters. For immunohistochemistry experiments, mice were treated according to the above described schedule. Eight hours post CS challenge mice were euthanized, livers and lungs were removed and drop fixed in 4% paraformaldehyde for 48 hours. Tissues were then paraffin embedded and sectioned, endogenous peroxidase activity was blocked by incubating sections in 3% H2O2 solution in methanol. After washing with PBS, sections were incubated in 10mM citrate buffer followed by blocking buffer (10% FBS in PBS) and subsequently stained with anti-CD31 and anti-ZO-1 antibodies. For electron microscopy mice were treated according to the above described schedule and subsequently euthanized 8 hours after CS challenge. Livers and lungs were excised, sectioned and immediately fixed in 4% glutaraldehyde.

Murine Angiopoietin 1 and 2 ELISA

Mice were treated according to the above described schedule. Blood was drawn 2- or 4-hours post-CS challenge via cardiac puncture. Blood was allowed to clot at room temperature for 15 minutes at room tempura prior to centrifugation to isolate serum. Mouse Ang-1 (Sapphire Biosciences: #LS-F2956) and Ang-2 (R&D Systems: #MANG20) ELISAs were performed according to manufacturer instructions.

Human study (sPLA-2/Ang) participants and whole blood sampling

A total of 87 infants were enrolled in a prospective, observational study that was approved by the institutional ethics review board at King Edward Memorial Hospital (RGS0000000862). Blood plasma samples were collected near the time of blood culture sampling for suspected late-onset sepsis (LOS) (± median 1.2 (IQR 25 ^th -75 ^th 0.0-7.5) hours). Plasma from 156 suspected episodes of LOS was stored at -80°C until analysis; episodes with insufficient sample (n=76) were excluded.

‘Any LOS’ was defined as a positive blood culture and/or a CRP of > 20 mg/L within 72 hours of culture sampling (As per Strunk et al. 2021 Impaired Cytokine Responses to Live Staphylococcus epidermidis in Preterm Infants Precede Gram positive, Late-onset Sepsis. Clin. Infect. Dis. 72, 271-278; Klingenberg et al. 2018 Culture-negative early-onset neonatal sepsis - at the crossroad between efficient sepsis care and antimicrobial stewardship. Frontiers in Pediatrics vol. 6285). Suspected sepsis episodes with a negative blood culture and 2 - 4 serial CRP < 20 mg/L within 72 hours of culture sampling were defined as ‘no LOS’. Infants were assigned an overall LOS classification based on the most severe outcome of all episodes.

Three suspected LOS episodes with a positive Gram-positive coagulase-negative staphylococci blood culture, 2 - 4 serial CRP < 20 mg/L within 72 hours of blood culture sampling, and the absence of sustained clinical features of LOS were classified as blood culture contaminants were excluded, as were episodes of necrotising enterocolitis (n=3).

The basic demographics of the 62 extremely premature infants included in the analysis were similar between the any LOS (n=22) and no LOS infants (n=40), except any LOS infants were older at the time of blood culture sampling (Figure 6). A total of seventy-four episodes of suspected LOS were included in the analysis (any LOS n=31, no LOS n=43), with 10 infants having more than one episode. Two infants in the LOS group died from multi-organ failure septicaemia and ten infants (any LOS n=5; no LOS n=5) died from causes unrelated to LOS; all data collected prior to death were included in the analysis. Of the LOS episodes, 42% (13/31) had a positive culture. Gram-positive bacteria accounted for 54% (7 cases); predominantly CoNS (4 cases) followed by two cases of Staphylococcus aureus and one case of Streptococcus gallolyticus. Gram negative infections (6 cases; 46%) were mainly caused by Escherichia coli (5 cases) and one case of Klebsiella pneumoniae. The clinical characteristics of infants with positive blood culture (n=10; episodes n=13) and negative blood culture LOS (n=12; episodes n=18) were similar with the exception of an elevated maximum CRP within 72 hours of blood culture in infants with positive blood culture LOS (Table 3).

Table 3. Characteristics of sPLA-2/Ang LOS cohort.

Human sPLA-2, Anq-1 and Anq-2 ELISA

Plasma sPLA-2 Type 11 A, Ang-1 and Ang-2 was measured by ELISA kit (Cayman Chemical: #501380; R&D Systems: #DANG10, #DANG20, respectively), as per manufacturer instructions. Samples were run in duplicate with absorbance read at 450nm in real time using a spectrophotometer. sPLA-2 concentration, in pg/mL, were generated from a seven-point four-parameter logistic standard curve.

Malawi cohort - qPCR Gene expression data in infants with sepsis was generated from samples obtained from young infants under three months of age with suspected sepsis prospectively enrolled at the Kamuzu Central Hospital (KCH) in Lilongwe, Malawi (Popescu et al. 2020 Whole blood genome-wide transcriptome profiling and metagenomics next-generation sequencing in young infants with suspected sepsis in low-and middle-income countries: A study protocol. Gates Open Res. 4, 139). In the parent study, whole blood was collected from infants at the time of blood culture sampling in RNALater (Ambion/lnvitrogen RNALater tubes, catalog number AM7022), and stored at -80°C for batch analyses. After thawing on ice, samples were centrifuged at 14.000 rpm for 4 minutes. Plasma was used for metabolomics analyses, whereas cell pellets were used for gene expression analyses. RNA was extracted from cell pellets using the RiboPure RNA Purification Kit (catalog number AM1928). Quantification and quality assessment of total RNA was performed on an Agilent 2100 Bioanalyzer. PCR was subsequently carried out for the genes of interest. The samples from this cohort were collected for the described purpose, under ethics approval from the National Health Sciences Research Committee (#17/8/1819) and the University of British Columbia Children’s & Women’s Research Ethics Board (#H16-02639). Expression of ALOX15, PTGS2, CYP2J2, and TUBB (housekeeping gene) were compared for 16 control samples and 14 sepsis samples of the neonatal cohort by qPCR. qPCR primers were designed to amplify transcripts such that either the forward or reverse primer bridged an exon-exon junction (see table below). Primers were designed using Primer-BLAST (Ye et al. 2012, Primer-BLAST: a tool to design target- specific primers for polymerase chain reaction. BMC Bioinformatics 13, 134) for the primary transcript. If more than one transcript was of interest, common exons were chosen for the primer pairs. The product size was set between 100-250bp and the primer set had to span an exon-exon junction (otherwise, separate exons were used for the primer pair). All primer pairs were specific to the transcripts of interest. The location of the primers were verified using the CCDS of the specific gene and the Reverse Complement tool (Stothard et al. 2000, The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences. Biotechniques 28).

Exemplary qPCR primers include:

Example 2: Gene expression patterns differ between ‘predicted survive’ and ‘predicted die’ mice

To uncover biological mechanisms central to determining the outcome of neonatal sepsis, the inventors performed RNA-seq analysis in a neonatal sepsis cecal slurry (CS) model. Whole blood, liver and spleen tissue were harvested from 7-8 day old mice following CS or sham challenge. All CS challenged mice were classified into likely survivors or non-survivors using a gradient boosting machine learning model trained on previous data (Brook et al., (2019) Robust health-score based survival prediction for a neonatal mouse model of polymicrobial sepsis. PLoS One 14, e0218714). Briefly, a series of machine learning models were trained on a cohort of 222 CS-challenged pups with known outcomes and tested on a random selection of 74 pups. The final gradient boosting machine model was able to accurately classify pups as likely survivors or likely non-survivors (Table 4). Table 4. Confusion matrix showing accuracy of the Gradient Boosting Machine model when applied to test set of 74 mice

Using monitoring data collected at 18 and 24-hours post-challenge (weight, righting reflex and mobility), a Gradient Boosting machine learning model was able to distinguish survivors and non-survivors with an accuracy score of 0.85; 33/39 and 30/35 nonsurvivors were correctly classified.

Unsupervised principal component analyses on the normalized RNA-seq data revealed a clear separation not only between challenged and un-challenged mice but also within likely survivors and non-survivors (Figure. 1A). This delineation was evident on a per sample basis and collectively demonstrated that those challenged mice predicted to survive exhibited expression patterns more closely resembling unchallenged mice than did those of mice challenged and predicted not to survive (Figure. 1B). This was further exemplified when assessing the overall pattern of differentially expressed genes between predicted survivors and non-survivors in relation to healthy control mice (Figure. 1C) which revealed that the magnitude of gene expression changes are almost universally more severe in predicted non-survivors. GO functional enrichment analysis of the differentially expressed genes between survivors and non-survivors revealed a consistent pattern of change across the blood, liver, and spleen. The inventors found broad, patterns related to the induced immune response. Further amongst the most enriched terms were those related to eicosanoids in the blood, AA in the liver and vasculature development/circulatory system processes in the spleen (Figure. 1D).

Example 3: Exogenous arachidonic acid improves outcome in a murine sepsis model

Further analysis revealed that the GO term enrichment of eicosanoid processes (in the blood) and AA processes (in the liver) in the neonatal sepsis model was driven by genes spread across several of the AA metabolic pathways (Figure. 2A). While the eicosanoid metabolic process term was also significantly enriched in the spleen, the most significantly enriched GO terms in the spleen were broadly associated with angiogenesis / vascular integrity (Figure 7). Considering the broad scope of the AA- related genes driving the observed GO enrichment, and the overall prominence of these signatures across all three tissues in the differentiation between predicted survivors and non-survivors, it was considered that AA levels likely plays an important role in determining outcome of neonatal sepsis. To further assess this, AA was administered (2mg) by oral gavage 4-6 hours prior to CS challenge. Surprisingly, exogenous AA significantly improved survival when compared to vehicle (corn oil) control (Figure. 2B).

The inflammatory response inherent to sepsis results in pathophysiological production and accumulation of reactive oxygen species (ROS). It is widely accepted that oxidative stress-induced injury plays an important role in the development of organ failure. Of the numerous effects of ROS on cells, one of the best described is the oxidation of membrane fatty acids, which has deleterious effects on cell signalling and protein function. Induction of Phospholipase A2 enzymes, including sPLA2, which initiates AA release, is believed to be a critical protective mechanism against pathophysiological oxidative stress. AA itself has been implicated in the defence against oxidative stress via the induction of cellular antioxidants.

The inventors then investigated the mechanism by which AA induces protection from CS challenge and whether it is associated with a reduction in oxidative stress. To assess this, the inventors administered AA 4-6 prior to CS challenge and subsequently utilized dihydroethidium to measure ROS (O2 ^· ) accumulation in the liver 8 hours post challenge. AA administration induced a significant reduction in ROS accumulation relative to CS challenge only mice, in fact, prophylactic administration of AA reduced ROS accumulation in the liver to levels similar to that of untreated control mice (Figure. 2C).

In light of the enrichment of GO term associated with vascular processes and angiogenesis, the protective effect of AA terminally mediated by angiogenesis regulating proteins whose induction and/or function is modulated by oxidative stress was investigated. The inventors assessed the dynamics of Ang-1/Ang-2 levels in their CS model, as well as the impact of AA administration on this pathway. Enzyme-linked immunosorbent assays (ELISA) was used to assess serum levels of Ang-1 and Ang-2 post CS challenge. CS challenge induced a significant increase in serum Ang-2 and corresponding decrease in Ang-1 levels relative to unchallenged controls (Figure. 2D). Prophylactic administration of AA 4-6 hours prior to CS challenge completely abrogated the CS induced increase in serum Ang-2 levels and significantly reduced the magnitude of the CS induced reduction in Ang-1 (Figure. 2D).

Example 4: Ang/TIE-2 signalling axis affects vascular stability and outcome in murine sepsis model

To test the impact of Angiopoietin/TIE-2 signalling axis within neonatal sepsis, the inventors first administered exogenous Ang-1 and Ang-2 one hour prior to CS challenge. While the addition of Ang-2 had little effect on survival, prophylactic Ang-1 administration significantly increased survival in CS challenged mice (Figure. 3A).

To confirm that modulation of the Ang-1/Ang-2 ratio impacts outcome during neonatal sepsis, the inventors administered an antagonistic monoclonal anti-Ang-2 antibody concurrently with CS challenge. Similar to exogenous Ang-1, neutralization of Ang-2 significantly improved survival in the neonatal sepsis model (Figure. 3A - bottom). Supporting these findings that the Ang/TIE-2 signalling axis and its impact on vascular homeostasis acts as the terminal effector of AA induced protection, the inventors observed significant differences in endothelial integrity and vascular permeability across treatment groups.

Example 5: Nitric oxide is a critical secondary regulator of AA-Angiopoietin dynamics after CS challenge

Nitric oxide (NO) is a critical regulator of vascular homeostasis. Of its numerous biological activities, NO regulates the expression of Ang-1 and the release of Ang-2 from WP (Weibel-Palade) storage. Moreover, it has also been demonstrated that endothelial nitric oxide synthase (eNOS) derived NO is required for Ang-1 induced promotion of vascular stability. The inventors then investigated whether NO bioavailability is a secondary mediator of both AA and Ang-1 induced protection from neonatal sepsis. Firstly, the inventors assessed the impact of the universal NOS inhibitor L-Nitroarginine methyl ester (L-NAME) administered 1 hour prior to CS challenge. Administration of L-NAME significantly worsened survival (Figure. 4A). The inventors measured the effect of L-NAME on serum Ang-1 and Ang-2 levels using ELISA. Interestingly, while CS challenge in the context of L-NAME induced the expected decrease in serum Ang-1 levels, there was no corresponding increase in Ang- 2 levels (Figure. 4B). The addition of L-NAME also resulted in significantly reduced accumulation of ROS in the livers of CS challenged mice as measured by dihydroethidium (Figure. 4C), suggesting that oxidative stress is a required precursor for release of Ang-2 from WP bodies during neonatal sepsis.

To determine the relevance of NO in the AA induced protective mechanism, the inventors assessed the capacity of prophylactic AA to mitigate the negative impact of L- NAME on survival. Administration of AA had no effect on the survival of L-NAME treated CS challenged mice (Figure. 4D) indicating that NO is a necessary component of the AA induced protective mechanism. Conversely, administration of Ang-1 to L-NAME treated mice prior to CS challenge reduced mortality (Figure. 4E). Considering the demonstrated impact of NO in the neonatal sepsis model, the inventors aimed to assess how increasing NO bioavailability prior to CS challenge would impact survival. L- arginine (L-Arg) is the substrate which is converted to NO by NOS. Administration of L- Arg prior to CS challenge, either alone or especially in combination with Ang-1 significantly improved survival (Figure 4F).

Example 6: Arachidonic acid metabolism and Ang/Tie signalling is central to human neonatal sepsis

To assess the relevance of both AA metabolism and the Ang/Tie signalling axis in human neonatal sepsis, the inventors determined the expression of genes central to the primary AA metabolic pathways in Malawian young infants with sepsis. Relative to uninfected controls, infants with clinical sepsis exhibited significantly reduced expression of ALOX15, PTGS2 and CYP2J2, implicating all main AA metabolic pathways in sepsis (Fig. 5A). The dysregulation of multiple AA metabolism genes in newborn infants with sepsis suggests wide-spread perturbation of AA metabolism in septic infants, potentially originating upstream of the genes measured.

The inventors next measured plasma sPLA-2, ANG-1 and ANG-2 levels in neonates with probable or confirmed sepsis. The primary function of secretory phospholipase A2 (sPLA2) is AA mobilization through the hydrolyzation of phospholipids. To assess the relevance of AA metabolism in human neonatal sepsis, the inventors measured the plasma concentration of sPLA2 in probable or confirmed neonatal sepsis cases and controls. Plasma sPLA2 levels were significantly elevated in neonatal late-onset sepsis (LOS; onset >72 hours of age) cases relative to healthy age- matched controls (Figure. 5B).

To correlate dysregulation in AA metabolism with changes in the Ang/TIE signalling axis the inventors measured the levels of ANG-1 and ANG-2. Comparing probable or confirmed sepsis cases with non-sepsis controls the inventors observed that plasma ANG-1 levels were significantly lower, and ANG-2 levels significantly higher in cases relative to controls (Figure. 5C). Fittingly, the plasma ANG-1/ANG-2 ratio was significantly reduced in cases versus controls (Figure. 5D).

Results from the murine CS challenge model suggested that modulating AA levels through the administration of exogenous AA had a demonstrable impact on serum ANG-1/ANG-2 levels in the context of CS challenge. The inventors assessed the relationship between plasma sPLA-2, ANG-1 and ANG-2 levels in the tested neonatal sepsis cohort irrespective of case-control status. ANG-2 concentration and the ANG- 1/ANG-2 ratio were both significantly positively and negatively correlated with sPLA2, respectively (Figure. 5E), demonstrating the association between AA metabolism and the ANG-1/ANG-2 axis of vascular integrity. By contrast, the correlation between plasma ANG-1 and sPLA2 levels only trends towards a significant negative correlation.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.