WO2024137136A1 - Inhibitors of il-15 and their use in treating or preventing sepsis, severe sepsis and septic shock - Google Patents

Abstract

The present disclosure is directed to peptoids and peptoid multimers that inhibit IL-15 and their use in treating sepsis and septic shock.

Description

DESCRIPTION

INHIBITORS OF IL-15 AND THEIR USE IN TREATING OR PREVENTING SEPSIS, SEVERE SEPSIS AND SEPTIC SHOCK

PRIORITY CLAIM

This application claims benefit of priority to U.S. Serial No. 63/476,737, filed December 22, 2022, the entire contents of which are hereby incorporated by reference.

BACKGROUND

1. Field of the Disclosure

The present disclosure relates generally to the fields of biology, medicine, and immunology. More particular, the disclosure relates to peptoids and peptoid multimers for use in inhibiting IL- 15 and treating sepsis, severe sepsis and septic shock.

2. Background

Sepsis, defined as “organ dysfunction caused by a dysregulated host response to infection” by the Third International Consensus (Sepsis-3) (Singer et al., 2016), is a lifethreatening condition. In septic shock, cardiovascular function is severely deteriorated (with the persisting hypotension requiring vasopressors to maintain a mean arterial pressure of 65 mm Hg or higher and a serum lactate level greater than 2 mmol/L (18 mg/dL) despite adequate volume resuscitation), leading to mortality rate as high as 60% (Rudd et al., 2020). The treatment of sepsis is mostly supportive care (fluid resuscitation, respiratory support, and vasopressors) and antibiotics. To date, there is only one FDA-approved drug with specific indication for sepsis: Giapreza (Angiotensin II). Another FDA-approved Xigris (drotrecogin alfa, activated protein C) was withdrawn from the market in 2011 after a major study showed no efficacy for the treatment of sepsis. Thus, new therapeutics are in urgent need for sepsis and septic shock.

SUBSTITUTE SHEET (RULE 26) SUMMARY

Thus, in accordance with the present disclosure, there is provided a peptoid of the following structure:

or a multimer thereof. The multimer may be dimer, a trimer or a tetramer. The multimer may comprise peptoids linked through their N-terminus, their C-terminus, or through a mid-chain connection. The multimer may comprise peptoids linked through a straight chain lower alkyl or through a peptide or distinct peptoid. Multiple copies of said peptoid or peptoid multimer may be located on the surface of a particle, such as a bead. The multimer may have a structure selected from the group consisting of:

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SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

4

SUBSTITUTE SHEET (RULE 26) IFRA3D3

10

SUBSTITUTE SHEET (RULE 26) IFRA3D0TAQ1

6

SUBSTITUTE SHEET (RULE 26) IFRA3DOTAT1

In another embodiment, there is provided a method of inhibiting IL- 15 signaling in a subject having or at risk of having sepsis or septic shock comprising administering to said subject a peptoid of the following structure:

or a multimer thereof. The multimer may be dimer, a trimer or a tetramer. The multimer may comprise peptoids linked through their N-terminus, their C-terminus, or through a mid-chain

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SUBSTITUTE SHEET (RULE 26) connection. The multimer may comprise peptoids linked through a straight chain lower alkyl or through a peptide or distinct peptoid. Multiple copies of said peptoid or peptoid multimer may be located on the surface of a particle, such as a bead.

The subject may be a human, such as a neonate, a pediatric patient, a teenager, an adult or a patient over about 60 years of age. The subject may be a non-human mammal. Administering may be chronic, such as daily, weekly, monthly, every other month, every three months, every four months, every five months, every six months, every nine months or every year. The method may further comprise administering to said subject a second therapy, such as activated protein C, antibiotics, intravenous fluids, blood products, vasopressors, steroids or other palliative care. The multimer may have a structure selected from the group consisting of:

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26) IFRA3D2

IFRA3D3

io

SUBSTITUTE SHEET (RULE 26) IFRA3D0TAQ1

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SUBSTITUTE SHEET (RULE 26) IFRA3DOTAD1

The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number.

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SUBSTITUTE SHEET (RULE 26) It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

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SUBSTITUTE SHEET (RULE 26) BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1. Chemical structure of IFRA3 tetramer designated IFRA3Q1.

FIGS. 2A-B. IFRA3Q1 inhibits LPS-induced septic shock. A. C57BL/6 mice (20 months old) were treated with 325 pg LPS i.p.. IFRA3Q1 or saline was given 24 hours before LPS and the same time with LPS. B. The numbers of NK cells, NKT cells, memory CD8+ T cells, and naive T cells in spleen were analyzed by flow cytometry. * P < 0.05, ** P < 0.01

FIGS. 3A-B. IFRA3Q1 protects mice from Pseudomonas -induced septic shock. (FIG. 3A) C57BL/6 mice were infected with non-lethal 5xl0⁶ CFUs of P. aeruginosa. IFRA3Q1 was given at 24 hours before infection (200 pg, i.p.) and the same time with infection (100 pg, i.p.). MSS was recorded as described in literature. *** P < 0.001. (FIG. 3B) C57BL/6 mice were infected with lethal dose IxlO⁷ CFUs of P. aeruginosa. IFRA3Q1 was given at 4 and 24 hours after infection (200 pg, i.p.). Survival rates were plotted as Kaplan-Meier survival graph. Five mice per group.

FIG. 4. Chemical structures of IFRA3 and multimer derivatives.

FIG. 5. Chemical structures of additional multimers.

SUBSTITUTE SHEET (RULE 26) DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As discussed above, there is only one FDA-approved drug with specific indication for sepsis and additional therapeutics are urgently needed for sepsis and septic shock. In this disclosure, the inventors report that a peptoid interleukin- 15 (IL-15) receptor antagonist, IFRA3Q1, shows activity that can provide for the treatment of sepsis and septic shock. These and other aspects of the disclosure are set out in detail below.

I. Sepsis and Septic Shock

Sepsis, formerly known as septicemia or blood poisoning, is a life-threatening condition that arises when the body's response to infection causes injury to its own tissues and organs. This initial stage is followed by suppression of the immune system. Common signs and symptoms include fever, increased heart rate, increased breathing rate, and confusion. There may also be symptoms related to a specific infection, such as a cough with pneumonia, or painful urination with a kidney infection. The very young, old, and people with a weakened immune system may have no symptoms of a specific infection, and the body temperature may be low or normal instead of having a fever. Severe sepsis causes poor organ function or blood flow. The presence of low blood pressure, high blood lactate, or low urine output may suggest poor blood flow. Septic shock is low blood pressure due to sepsis that does not improve after fluid replacement.

Sepsis is caused by many organisms including bacteria, viruses and fungi. Common locations for the primary infection include the lungs, brain, urinary tract, skin, and abdominal organs. Risk factors include being very young or old, a weakened immune system from conditions such as cancer or diabetes, major trauma, and burns. Previously, a sepsis diagnosis required the presence of at least two systemic inflammatory response syndrome (SIRS) criteria in the setting of presumed infection. In 2016, a shortened sequential organ failure assessment score (SOFA score), known as the quick SOFA score (qSOFA), replaced the SIRS system of diagnosis. qSOFA criteria for sepsis include at least two of the following three: increased breathing rate, change in the level of consciousness, and low blood pressure. Sepsis guidelines recommend obtaining blood cultures before starting antibiotics; however, the diagnosis does not require the blood to be infected. Medical imaging is helpful when looking for the possible location of the infection. Other potential causes of similar signs and symptoms include anaphylaxis, adrenal insufficiency, low blood volume, heart failure, and pulmonary embolism.

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SUBSTITUTE SHEET (RULE 26) Sepsis requires immediate treatment with intravenous fluids and antimicrobials. Ongoing care often continues in an intensive care unit. If an adequate trial of fluid replacement is not enough to maintain blood pressure, then the use of medications that raise blood pressure becomes necessary. Mechanical ventilation and dialysis may be needed to support the function of the lungs and kidneys, respectively. A central venous catheter and an arterial catheter may be placed for access to the bloodstream and to guide treatment. Other helpful measurements include cardiac output and superior vena cava oxygen saturation. People with sepsis need preventive measures for deep vein thrombosis, stress ulcers, and pressure ulcers unless other conditions prevent such interventions. Some people might benefit from tight control of blood sugar levels with insulin. The use of corticosteroids is controversial, with some reviews finding benefit, and others not.

Disease severity partly determines the outcome. The risk of death from sepsis is as high as 30%, while for severe sepsis it is as high as 50%, and septic shock 80%. Sepsis affected about 49 million people in 2017, with 11 million deaths (1 in 5 deaths worldwide). In the developed world, approximately 0.2 to 3 people per 1000 are affected by sepsis yearly, resulting in about a million cases per year in the United States. Rates of disease have been increasing. Some data indicate that sepsis is more common among males than females, however, other data show a greater prevalence of the disease among women. Descriptions of sepsis date back to the time of Hippocrates.

In addition to symptoms related to the actual cause, people with sepsis may have a fever, low body temperature, rapid breathing, a fast heart rate, confusion, and edema. Early signs include a rapid heart rate, decreased urination, and high blood sugar. Signs of established sepsis include confusion, metabolic acidosis (which may be accompanied by a faster breathing rate that leads to respiratory alkalosis), low blood pressure due to decreased systemic vascular resistance, higher cardiac output, and disorders in blood-clotting that may lead to organ failure. Fever is the most common presenting symptom in sepsis, but fever may be absent in some people such as the elderly or those who are immunocompromised.

The drop in blood pressure seen in sepsis can cause lightheadedness and is part of the criteria for septic shock. Oxidative stress is observed in septic shock, with circulating levels of copper and vitamin C being decreased.

Infections leading to sepsis are usually bacterial but may be fungal, parasitic or viral. Gram-positive bacteria were the primary cause of sepsis before the introduction of antibiotics in the 1950s. After the introduction of antibiotics, gram-negative bacteria became the predominant cause of sepsis from the 1960s to the 1980s. After the 1980s, gram-positive

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SUBSTITUTE SHEET (RULE 26) bacteria, most commonly staphylococci, are thought to cause more than 50% of cases of sepsis. Other commonly implicated bacteria include Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella species. Fungal sepsis accounts for approximately 5% of severe sepsis and septic shock cases; the most common cause of fungal sepsis is an infection by Candida species of yeast, a frequent hospital- acquired infection. The most common causes for parasitic sepsis are Plasmodium (which leads to malaria), Schistosoma and Echinococcus.

The most common sites of infection resulting in severe sepsis are the lungs, the abdomen, and the urinary tract. Typically, 50% of all sepsis cases start as an infection in the lungs. In one-third to one-half of cases, the source of infection is unclear.

Sepsis is caused by a combination of factors related to the particular invading pathogen(s) and to the status of the immune system of the host. The early phase of sepsis characterized by excessive inflammation (sometimes resulting in a cytokine storm) may be followed by a prolonged period of decreased functioning of the immune system. Either of these phases may prove fatal. On the other hand, systemic inflammatory response syndrome (SIRS) occurs in people without the presence of infection, for example, in those with burns, polytrauma, or the initial state in pancreatitis and chemical pneumonitis. However, sepsis also causes similar response to SIRS.

Bacterial virulence factors, such as glycocalyx and various adhesins, allow colonization, immune evasion, and establishment of disease in the host. Sepsis caused by gramnegative bacteria is thought to be largely due to a response by the host to the lipid A component of lipopolysaccharide, also called endotoxin. Sepsis caused by gram-positive bacteria may result from an immunological response to cell wall lipoteichoic acid. Bacterial exotoxins that act as superantigens also may cause sepsis. Superantigens simultaneously bind major histocompatibility complex and T-cell receptors in the absence of antigen presentation. This forced receptor interaction induces the production of pro-inflammatory chemical signals (cytokines) by T-cells.

There are a number of microbial factors that may cause the typical septic inflammatory cascade. An invading pathogen is recognized by its pathogen-associated molecular patterns (PAMPs). Examples of PAMPs include lipopolysaccharides and flagellin in gramnegative bacteria, muramyl dipeptide in the peptidoglycan of the gram-positive bacterial cell wall, and CpG bacterial DNA. These PAMPs are recognized by the pattern recognition receptors (PRRs) of the innate immune system, which may be membrane -bound or cytosolic. There are four families of PRRs: the toll-like receptors, the C-type lectin receptors,

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SUBSTITUTE SHEET (RULE 26) the NOD-like receptors, and the RIG-I-like receptors. Invariably, the association of a PAMP and a PRR will cause a series of intracellular signaling cascades. Consequentially, transcription factors such as nuclear factor-kappa B and activator protein- 1, will up-regulate the expression of pro-inflammatory and anti-inflammatory cytokines.

Upon detection of microbial antigens, the host systemic immune system is activated. Immune cells not only recognize pathogen-associated molecular patterns but also damage- associated molecular patterns from damaged tissues. An uncontrolled immune response is then activated because leukocytes are not recruited to the specific site of infection, but instead they are recruited all over the body. Then, an immunosuppression state ensues when the proinflammatory T helper cell 1 (TH1) is shifted to TH2, mediated by interleukin 10, which is known as “compensatory anti-inflammatory response syndrome.” The apoptosis (cell death) of lymphocytes further worsens the immunosuppression. Neutrophils, monocytes, macrophages, dendritic cells, CD4+ T cells, and B cells all undergo apoptosis, whereas regulatory T cells are more apoptosis resistant. Subsequently, multiple organ failure ensues because tissues are unable to use oxygen efficiently due to inhibition of cytochrome c oxidase.

Inflammatory responses cause multiple organ dysfunction syndrome through various mechanisms as described below. Increased permeability of the lung vessels causes leaking of fluids into alveoli, which results in pulmonary edema and acute respiratory distress syndrome (ARDS). Impaired utilization of oxygen in the liver impairs bile salt transport, causing jaundice (yellowish discoloration of the skin). In kidneys, inadequate oxygenation results in tubular epithelial cell injury (of the cells lining the kidney tubules), and thus causes acute kidney injury (AKI). Meanwhile, in the heart, impaired calcium transport, and low production of adenosine triphosphate (ATP), can cause myocardial depression, reducing cardiac contractility and causing heart failure. In the gastrointestinal tract, increased permeability of the mucosa alters the microflora, causing mucosal bleeding and paralytic ileus. In the central nervous system, direct damage of the brain cells and disturbances of neurotransmissions causes altered mental status. Cytokines such as tumor necrosis factor, interleukin 1, and interleukin 6 may activate procoagulation factors in the cells lining blood vessels, leading to endothelial damage. The damaged endothelial surface inhibits anticoagulant properties as well as increases antifibrinolysis, which may lead to intravascular clotting, the formation of blood clots in small blood vessels, and multiple organ failure.

The low blood pressure seen in those with sepsis is the result of various processes, including excessive production of chemicals that dilate blood vessels such as nitric oxide, a deficiency of chemicals that constrict blood vessels such as vasopressin, and activation

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SUBSTITUTE SHEET (RULE 26) of ATP-sensitive potassium channels. In those with severe sepsis and septic shock, this sequence of events leads to a type of circulatory shock known as distributive shock.

Early diagnosis is necessary to properly manage sepsis, as the initiation of rapid therapy is key to reducing deaths from severe sepsis. Some hospitals use alerts generated from electronic health records to bring attention to potential cases as early as possible.

Within the first three hours of suspected sepsis, diagnostic studies should include white blood cell counts, measuring serum lactate, and obtaining appropriate cultures before starting antibiotics, so long as this does not delay their use by more than 45 minutes. To identify the causative organism(s), at least two sets of blood cultures using bottles with media for aerobic and anaerobic organisms are necessary. At least one should be drawn through the skin and one through each vascular access device (such as an IV catheter) that has been in place more than 48 hours. Bacteria are present in the blood in only about 30% of cases. Another possible method of detection is by polymerase chain reaction. If other sources of infection are suspected, cultures of these sources, such as urine, cerebrospinal fluid, wounds, or respiratory secretions, also should be obtained, as long as this does not delay the use of antibiotics.

Within six hours, if blood pressure remains low despite initial fluid resuscitation of 30 mL/kg, or if initial lactate is > four mmol/L (36 mg/dL), central venous pressure and central venous oxygen saturation should be measured. Lactate should be re-measured if the initial lactate was elevated. Evidence for point of care lactate measurement over usual methods of measurement, however, is poor.

Within twelve hours, it is essential to diagnose or exclude any source of infection that would require emergent source control, such as a necrotizing soft tissue infection, an infection causing inflammation of the abdominal cavity lining, an infection of the bile duct, or an intestinal infarction. A pierced internal organ (free air on an abdominal X-ray or CT scan), an abnormal chest X-ray consistent with pneumonia (with focal opacification), or petechiae, purpura, or purpura fulminans may indicate the presence of an infection.

IL IL-15 and IL-15Ra

A. IL-15

Interleukin- 15 (IL- 15) is a cytokine with structural similarity to Interleukin-2 (IL-2). Like IL-2, IL- 15 binds to and signals through a complex composed of IL-2/IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 is secreted by mononuclear phagocytes (and some other cells) following infection by virus(es). This cytokine induces cell proliferation of natural killer cells; cells of the innate immune system whose

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SUBSTITUTE SHEET (RULE 26) principal role is to kill virally infected cells. It has also been reported to play a role in celiac disease and non-alcoholic fatty liver disease.

IL- 15 was discovered in 1994 by two different laboratories and characterized as T cell growth factor. Together with Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-7 (IL-7), Interleukin-9 (IL-9), granulocyte colony-stimulating factor (G-CSF), and granulocytemacrophage colony-stimulating factor (GM-CSF), IL-15 belongs to the four a-helix bundle family of cytokine.

IL- 15 is constitutively expressed by a large number of cell types and tissues, including monocytes, macrophages, dendritic cells (DC), keratinocytes, fibroblasts, myocyte and nerve cells. As a pleiotropic cytokine, it plays an important role in innate and adaptive immunity.

IL- 15 is 14-15 kDa glycoprotein encoded by the 34 kb region of chromosome 4q31 in humans, and at the central region of chromosome 8 in mice. The human IL- 15 gene comprises nine exons (1 - 8 and 4 A) and eight introns, four of which (exons 5 through 8) code for the mature protein.

Two alternatively spliced transcript variants of this gene encoding the same protein have been reported. The originally identified isoform, with long signal peptide of 48 amino acids (IL- 15 LSP) consisted of a 316 bp 5 ’ -untranslated region (UTR), 486 bp coding sequence and the C-terminus 400 bp 3’-UTR region. The other isoform (IL-15 SSP) has a short signal peptide of 21 amino acids encoded by exons 4 A and 5. Both isoforms shared 11 amino acids between signal sequences of the N-terminus. Although both isoforms produce the same mature protein, they differ in their cellular trafficking. IL- 15 LSP isoform was identified in Golgi apparatus [GC], early endosomes and in the endoplasmic reticulum (ER). It exists in two forms, secreted and membrane-bound particularly on dendritic cells. On the other hand, IL- 15 SSP isoform is not secreted and it appears to be restricted to the cytoplasm and nucleus where plays an important role in the regulation of cell cycle.

It has been demonstrated that two isoforms of IL- 15 mRNA are generated by alternatively splicing in mice. The isoform which had an alternative exon 5 containing another 3’ splicing site, exhibited a high translational efficiency, and the product lack hydrophobic domains in the signal sequence of the N-terminus. This suggests that the protein derived from this isoform is located intracellularly. The other isoform with normal exon 5, which is generated by integral splicing of the alternative exon 5, may be released extracellularly.

Although IL- 15 mRNA can be found in many cells and tissues including mast cells, cancer cells or fibroblasts, this cytokine is produced as a mature protein mainly by dendritic cells, monocytes and macrophages. This discrepancy between the wide appearance of IL-15

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SUBSTITUTE SHEET (RULE 26) mRNA and limited production of protein might be explained by the presence of the twelve in humans and five in mice upstream initiating codons, which can repress translation of IL- 15 mRNA. Translational inactive mRNA is stored within the cell and can be induced upon specific signal. Expression of IL-15 can be stimulated by cytokine such as GM-CSF, double-strand mRNA, unmethylated CpG oligonucleotides, lipopolysaccharide (LPS) through Toll-like receptors (TLR), interferon gamma (IFN-y) or after infection of monocytes herpes virus, Mycobacterium tuberculosis and Candida albicans.

IL- 15 regulates the activation and proliferation of T and natural killer (NK) cells. Survival signals that maintain memory T cells in the absence of antigen are provided by IL- 15. This cytokine is also implicated in NK cell development. In rodent lymphocytes, IL- 15 prevents apoptosis by inducing BCL2Ll/BCL-x(L), an inhibitor of the apoptosis pathway. In humans with celiac disease IL- 15 similarly suppresses apoptosis in T-lymphocytes by inducing Bcl-2 and/or Bcl-xL.

A hematopoietin receptor, the IL- 15 receptor, that binds IL- 15 propagates its function. Some subunits of the IL- 15 receptor are shared in common with the receptor for a structurally related cytokine called Interleukin 2 (IL-2) allowing both cytokines to compete for and negatively regulate each other’s activity. CD8+ memory T cell number is controlled by a balance between IL-15 and IL-2. When IL-15 binds its receptor, IAK kinase, STAT3, STAT5, and STAT6 transcription factors are activated to elicit downstream signaling events.

IL-15 and its receptor subunit alpha (IL-15Ra) are also produced by skeletal muscle in response to different exercise doses (myokine), playing significant roles in visceral (intraabdominal or interstitial) fat reduction and myofibrillar protein synthesis (hypertrophy).

B. IL-15Ra

Interleukin 15 receptor, alpha subunit is a subunit of the interleukin 15 receptor that in humans is encoded by the IL15RA gene. The IL-15 receptor is composed of three subunits: IL- 15R alpha, CD122, and CD132. Two of these subunits, CD122 and CD132, are shared with the receptor for IL-2, but IL-2 receptor has an additional subunit (CD25). The shared subunits contain the cytoplasmic motifs required for signal transduction, and this forms the basis of many overlapping biological activities of IL15 and IL2, although in vivo the two cytokines have separate biological effects. This may be due to effects of the respective alpha chains, which are unique to each receptor, the kinetics and affinity of cytokine-cytokine receptor binding, or due to the availability and concentration of each cytokine.

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SUBSTITUTE SHEET (RULE 26) IL-15Ra specifically binds IL 15 with very high affinity and is capable of binding IL- 15 independently of other subunits. It is suggested that this property allows IL- 15 to be produced by one cell, endocytosed by another cell, and then presented to a third-party cell.

This receptor is reported to enhance cell proliferation and expression of apoptosis inhibitor BCL2L1/BCL2-XL and BCL2. Multiple alternatively spliced transcript variants of this gene have been reported. The full-length sequences of only two variants encoding distinct isoforms are available.

Several isoforms of the IL-15Ra protein have been detected. These isoforms can either result from alternative splicing of the mRNA encoding for the receptor or by shedding of the extra cellular domain of the receptor protein.

III. Peptoid Compositions

Peptoids, or poly-N-substituted glycines, are a class of peptidomimetics whose side chains are appended to the nitrogen atom of the peptide backbone, rather than to the a-carbons (as they are in amino acids). In peptoids, the side chain is connected to the nitrogen of the peptide backbone, instead of the a-carbon as in peptides. Notably, peptoids lack the amide hydrogen which is responsible for many of the secondary structure elements in peptides and proteins.

Following the sub-monomer protocol originally created by Ron Zuckermann, each residue is installed in two steps: acylation and displacement. In the acylation step a haloacetic acid, typically bromoacetic acid activated by diisopropylcarbodiimide reacts with the amine of the previous residue. In the displacement step (a classical SN2 reaction), an amine displaces the halide to form the N-substituted glycine residue. The submonomer approach allows the use of any commercially available or synthetically accessible amine with great potential for combinatorial chemistry.

Like D-Peptides and 0 peptides, peptoids are completely resistant to proteolysis, and are therefore advantageous for therapeutic applications where proteolysis is a major issue. Since secondary structure in peptoids does not involve hydrogen bonding, it is not typically denatured by solvent, temperature, or chemical denaturants such as urea (see details below).

Notably, since the amino portion of the amino acid results from the use of any amine, thousands of commercially available amines can be used to generate unprecedented chemical diversity at each position at costs far lower than would be required for similar peptides or peptidomimetics. To date, at least 230 different amines have been used as side chains in peptoids.

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SUBSTITUTE SHEET (RULE 26) Peptoid oligomers are known to be conformationally unstable, due to the flexibility of the main-chain methylene groups and the absence of stabilizing hydrogen bond interactions along the backbone. Nevertheless, through the choice of appropriate side chains it is possible to form specific steric or electronic interactions that favour the formation of stable secondary structures like helices, especially peptoids with C-a-branched side chains are known to adopt structure analogous to polyproline I helix. Different strategies have been employed to predict and characterize peptoid secondary structure, with the ultimate goal of developing fully folded peptoid protein structures. The cis/trans amide bond isomerization still leads to a conformational heterogeneity which doesn’t allow for the formation of homogeneous peptoid foldamers. Nonetheless scientists were able to find trans-inducer N-Aryl side chains promoting polyproline type II helix, and strong cis-inducer such as bulky naphtylethyl and tert-butyl side chains. It was also found that n^n* interactions can modulate the ratio of cis/trans amide bond conformers, until reaching a complete control of the cis conformer in the peptoid backbone using a functionalizable triazolium side chain.

Peptoid and peptoid multimers of the present disclosure are shown in FIGS. 4-6.

IV. Treatment/Prevention of Sepsis/Severe Sepsis/Septic Shock

A. Formulation and Administration

The present disclosure provides pharmaceutical compositions comprising peptoids and peptoid multimers. Such compositions comprise a prophylactically or therapeutically effective amount of a peptoids and peptoid multimers, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice,

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SUBSTITUTE SHEET (RULE 26) flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the peptoid or peptoid multimer, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, or delivered by mechanical ventilation.

Generally, the ingredients of compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compositions of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The inventors suggest dosing ranges at 0.08 mg/kg to 2 mg/kg based on previous reports (Nair & Jacob, 2016). Additional dosing values include 0.05-5 mg/kg, 0.5-3 mg/kg, 0.1-2 mg/kg, and 0.5-1 mg/kg, including 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9. 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 mg/kg.

B. Combination Therapy

One general approach to treat disease is to combine multiple therapies as a way of increasing their efficacy. In the context of the present disclosure, the inventors propose that

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SUBSTITUTE SHEET (RULE 26) the peptoid and peptoid multimer therapy can be used successfully in conjunction with another therapeutic or regimen to treat or prevent sepsis or septic shock.

Using the methods and compositions of the present disclosure, one would generally contact a subject with the peptoids and peptoid multimers of the present disclosure and another therapy. These therapies would be provided in a combined amount effective to address one or more symptom or underlying cause of disease. This process may involve administering both agents/therapies at the same time. This may be achieved by administering a single composition or pharmacological formulation that includes both therapies, or by using two distinct compositions or formulations, at the same time, wherein one composition includes the peptoids and peptoid multimers of the present disclosure and the other therapy.

Alternatively, one treatment may precede or follow the other therapy by intervals ranging from minutes to weeks. In embodiments where the therapies are applied separately, one would generally ensure that a significant period of time did not expire between each delivery, such that the therapies would still be able to exert an advantageously combined effect on the subject. In such instances, it is contemplated that one would contact the cell with both modalities within about 12-24 hours of each other, within about 6-12 hours of each other, or with a delay time of only about 12 hours. In some situations, it may be desirable to extend the time period for treatment significantly; however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

It also is conceivable that more than one administration of either the peptoid/peptoid multimer or the other therapy will be desired. Various combinations may be employed, where the peptoid or peptoid multimer is “A” and the other therapy is “B,” as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

Other combinations are contemplated. Again, to achieve a therapeutic goal, both therapies are delivered to a subject in a combined amount effective to achieve that goal. Specific combination therapy strategies include combining the peptoid/peptoid multimer therapy with the following: activated protein C, antibiotics, intravenous fluids, blood products, vasopressors, steroids or other palliative care.

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SUBSTITUTE SHEET (RULE 26) V. Kits

In still further embodiments, the present disclosure concerns kits for use with the methods described above. The kits will thus comprise, in suitable container means, one or more peptoids and/or peptoid multimers that inhibit IL- 15, and optionally other reagents. The components of the kits may be packaged either in aqueous media or in lyophilized form.

The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the peptoid or multimer may be placed, or preferably, suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the peptoid, peptoid multimer, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

VI. Examples

The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

Example 1

The inventors have created a peptoid inhibitor of the IL- 15 receptor, IFRA3Q1 (chemical structure shown in FIG. 1), and demonstrated its efficacy in treating rheumatoid arthritis (Cho et al., 2022). Using an endotoxin shock model, they determined the effects of IFRA3Q1 in sepsis. C57BL/6 mice were administered with 375 mg/mouse ultrapure LPS-EB (InvivoGen) via intraperitoneal injection. As shown in FIG. 2 A, all mice in the control group (LPS + saline) died from septic shock within 24 hours. In contrast, 87.5% of the mice survived after treatment with two i.p. doses of IFRA3Q1 (200 pg per mouse at 24 hours before LPS and 100 pg per mouse at the same time of LPS administration). In addition, IFRA3Q1 significantly reduced the numbers of NK cells, NKT cells, memory CD8+ T cells, and naive T cells in the septic mice (FIG. 2B).

Pseudomonas aeruginosa (PA) is a common cause of infection and sepsis in hospitalized patients and is associated with higher mortality than other bacteria (Thaden et al.,

26

SUBSTITUTE SHEET (RULE 26) 2017). The emergence of multidrug resistant Pseudomonas strains is a severe problem in hospitals. The acute lung infection Pseudomonas aeruginosa mouse model is clinically relevant.

To determine the efficacy of IFRA3Q1 in sepsis caused by infection with P. aeruginosa the inventors inoculated bacteria into the trachea of the anesthetized mice (under laryngoscopic guidance) to induce pneumonia. Severe pneumonia caused severe sepsis symptoms and septic shock. First, mice were infected with a non-lethal 5 x 10⁶ CFUs of PA. Mice were treated with IFRA3Q1 (200 pg per mouse, i.p.) at 24 hours before bacterial infection. On the day of bacterial infection, mice were given the 2^nd dose of IFRA3Q1 (200 pg/mouse or 5 mg/kg) by i.p. administration. They assessed the sepsis score using the published Murine Sepsis Score (MSS) system (Mai et al., 2018), which involves observing six components: appearance, level of consciousness, activity, response to stimulus, eyes, respiratory quality (scoring 0-3 for each component, with the maximum score for each mouse is 18). The results in FIG. 3 A show that IFRA3Q1 is very effective in suppressing septic shock in this model. Strikingly, IFRA3Q1 (200 pg per mouse) was able to rescue mice dying from a lethal P. aeruginosa infection of 1 x 10⁷ CFUs (IFRA3Q1 was administered at 4 hr after P. aeruginosa infection, at which point the mice were severely ill and showing no movement when provoked) (FIG. 3B). This effective rescue by IFRA3Q1 is surprising as the survival mice were fully recovered and completely healthy on day 4 after infection, despite never being treated with antibiotics. Apparently, IFRA3Q1 was able to tone down the overactive immune system (in sepsis) to just the right level, which not only prevented death caused by the cytokine storm, but also gave the mice a chance to eliminate the large CFUs of P. aeruginosa using their still functional immune system.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to

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SUBSTITUTE SHEET (RULE 26) those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

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SUBSTITUTE SHEET (RULE 26) VII. REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

Remington’s Pharmaceutical Sciences, 15th Ed., 3:624-652, 1990.

Shukla et al. , European J. Med. Chem. 137: 1-10, 2017.

Singer, M. et al., JAMA 315, 801-810 (2016).

Rudd, K.E. et al., Lancet 395, 200-211 (2020).

Waldmann, T.A., Miljkovic, M.D. & Conlon, K.C., The Journal of experimental medicine 217

(2020).

Tagaya, Y., Bamford, R.N., DeFilippis, A.P. & Waldmann, T.A., Immunity 4, 329-336 (1996).

Fehniger, T.A. & Caligiuri, M.A., Blood 97, 14-32 (2001).

Guo, Y. et al., J Immunol 198, 1320-1333 (2017).

Cho, K.B. et al., Clin Transl Immunology 11, el432 (2022).

Thaden, J.T. et al., Antimicrob Agents Chemother 61 (2017).

Mai, S.H.C. et al., Intensive Care Med Exp 6, 20 (2018).

Nair AB, Jacob S., J Basic Clin Pharm. 2016 Mar;7(2):27-31. doi: 10.4103/0976-0105.177703.

SUBSTITUTE SHEET (RULE 26)

Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting IL- 15 signaling in a subject having or at risk of sepsis or septic shock comprising administering to said subject a peptoid of the following structure:

or a multimer thereof.

2. The method of claim 1, wherein said peptoid multimer is a dimer.

3. The method of claim 1, wherein said peptoid multimer is a trimer.

4. The method of claim 1 , wherein said peptoid multimer is a tetramer.

5. The method of any one of claims 1-4, wherein said multimer comprises said peptoid linked through their N-terminus or C-terminus.

6. The method of any one of claims 1-4, wherein said multimer comprises said peptoid linked through a mid-chain linkage.

7. The method of any one of claims 1-6, wherein said multimer comprises said peptoid linked through a straight chain lower alkyl.

8. The method of any one of claims 1-6, wherein said multimer comprises said peptoid linked through a peptide or peptoid.

9. The method of any one of claims 1-8, wherein said peptoid or peptoid multimer is linked to a particle, such as a bead.

SUBSTITUTE SHEET (RULE 26)

10. The method of claim 1, wherein multiple copies of said peptoid are located on the surface of a particle, such as a bead.

11. The method of any one of claims 1-10, wherein the subject is a human.

12. The method of claim 11, wherein said subject is selected from a neonate, a pediatric patient, a teenager, an adult or a patient over about 60 years of age.

13. The method of any one of claims 1-10, wherein the subject is a non-human mammal.

14. The method of any one of claims 1-13, wherein the administering is chronic, such as daily, weekly, monthly, every other month, every three months, every four months, every five months, every six months, every nine months or every year.

15. The method of any one of claims 1-14, further comprising administering to said subject a second therapy.

16. The method of claim 15, wherein said second therapy is activated protein C, antibiotics, intravenous fluids, blood products, vasopressors, steroids or other palliative care.

17. The method of claim 1, wherein the multimer has a structure selected from the group consisting of:

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

IFRA3D2

SUBSTITUTE SHEET (RULE 26) IFRA3D3

SUBSTITUTE SHEET (RULE 26) IFRA3D0TAQ1

SUBSTITUTE SHEET (RULE 26) IFRA3DOTAT1

, or

IFRA3D0TAD1

SUBSTITUTE SHEET (RULE 26)