CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application Serial No. 63/122,697, filed December 8, 2020, entitled “Perforin Inhibitors and Uses Thereof,” the contents of which are incorporated by reference in their entirety for all purposes.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

[0002] This invention was made with government support under DPI DA038017 and R01 AI148080 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0003] While vaccines are routinely used and developed for a variety of purposes, including infectious and noninfectious disease states, a challenge in the art is obtaining vaccines capable of eliciting a desired immune response. While adjuvants are known and commonly used, there is a need in the art for methods and/or compositions that may be used to improve vaccine efficacy, including, for example, improving a vaccine’s ability to prevent or limit the severity of disease caused by infectious agents, or for use in the treatment of tumors or other diseases/conditions (see, for example, https://pubmed.ncbi.nlm.nih.gov/26302599/). The instant disclosure seeks to address one or more of the aforementioned needs in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0004] This application file may contain at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0005] FIG 1. Perforin inhibitor (PRF-I) enhance NP-specific antibody production 6 days post NP-KLH immunization. A) Experimental scheme of acute vaccine model using NP-

KLH immunization. 8-10 weeks old male WT C57BL/6 were either depleted of NK cells using 25μg of anti-NKl.l (PK136) antibody or mouse IgG2a isotype antibody treated 24 hours prior to alum adjuvanted NP-KLH immunization or mice were treated with single injection 35mg/kg PRF-I or treated once on the same day of NP-KLH immunization or from day 0 through day 2 post NP-KLH immunization. B) NP-specific IgG were quantified using ELISA. PRF-I treatment may be applied one or more times between days 0 and 3 post vaccine and achieve a similarly effective result.

|0006] FIG 2. Inhibitor of perforin (PRF-I) (“Compound 1”, https://mcule.com/MCULE- 4157904124/) blocks (A) NK-92 cell triggering of granzyme-B luciferase reporter in K562 cells, (B) primary human NK-cell redirected lysis induction of caspase-3/7 luciferase reporter in P815 cells bound by anti-CD16 antibody, (C) KHYG1 NK-cell killing ( ⁵¹ Cr release) of Jurkat T cells.

[0007] FIG 3. Overlapping perforin-dependent NK-cell suppression of Tfh differentiation of LCMV- specific S MARTA CD4 T cells in spleen of control of NK-depleted (ANK) C57BL/6 (WT) or Prf1 ^k _o mice at day 3 of LCMV infection.

[0008] FIG 4. PRF-I (Compound 16) suppresses in vivo NK-cell activity and enhances vaccine responses. (A) C57BL/6 or Prf1 ^k _o mice were i.p. injected once with 35 mg/kg PRF-I or anti-NKl.l antibody (ANK), then infused with a ~1:1 mix of CFSE ^low β 2m ^KO cells and CFSE ^hlgh wild-type cells. After 24 hours, survival of donor cells was quantified by flow cytometry and killing % determined relative to control Prf1 ^k _o recipients. (B, C) C57BL/6 or Prf1 ^k _o mice (3-5/group) were immunized i.p. with NP-KLH in alum, with or without daily application of PRF-I on days -1 to 3. At day 7, (B) T _FH (CXCR5+ PD-1+ CD4+ T cells) or germinal center B cells (CD19+ B220+ IgD ^low GL7+) were quantified by flow cytometry in spleen and draining LN. p values determined by ANOVA.

BRIEF SUMMARY

[0009] The instant disclosure describes methods and compositions useful for improving a vaccine’s ability to prevent or limit the severity of disease caused by infectious agents, or for the treatment of tumors or other diseases/conditions using vaccine compositions. DETAILED DESCRIPTION

[0010] DEFINITIONS

[0011] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0012] As one of skill in the art will appreciate, an “immune response” is a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. An immune response can be a B cell response, which results in the production of specific antibodies, such as antigen specific neutralizing antibodies. An immune response can also be a T cell response, such as a CD4+ response or a CD8+ response. In some cases, the response is specific for a particular antigen (that is, an “antigen-specific response”). A “protective immune response” is an immune response that inhibits a detrimental function or activity of a pathogen, reduces infection by a pathogen, or decreases symptoms (including death) that result from infection by the pathogen. A protective immune response can be measured, for example, by the inhibition of viral replication or plaque formation in a plaque reduction assay or ELISA-neutralization assay, or by measuring resistance to pathogen challenge in vivo.

[0013] Enhancing an immune response in a subject provides a meaningful clinical benefit to the subject. Such benefit may be, e.g., preventing, ameliorating, treating, inhibiting, and/or reducing one of more pathological conditions associated with a viral infection or related sequelae, in a subject. In further aspects, the enhancement of an immune response may be in association with preventing disease or symptoms associated with bacteria, fungi, toxins (venom) drugs, tumors, and the like, more particularly, enhancement of any vaccine composition for any therapeutic use in which enhancing an immune response is desired. The disclosed methods may be considered therapeutic methods or preventative or prophylactic methods, wherein such terms are not intended to encompass “100%” prevention or prophylactic treatment, but rather, substantially preventative or prophylactic with respect to a given disease, symptom, or infection.

[0014] As used herein, the terms, “induce,” “enhance,” “immune enhancing,” “enhancement of immunity,” “modulator of immune responses to antigen,” and like terms encompass any increase in immunity, and any measure of immunity, including enhancement of cellular and/or humoral immunity and/or by protective efficacy of an antigen, in a subject. In one aspect, both humoral and cellular immune responses may be enhanced in a subject using the disclosed methods and compositions.

|0015] As understood herein, a “subject”, a “patient”, a “subject in need thereof’ and like terms are interchangeable and may include humans and non-humans who may benefit from the methods and compositions of the instant disclosure. In one aspect, the subject may be a human subject. The subject may already be infected with an infectious agent, or may be at risk of infection.

|0016] As used herein, an “immunogenic composition” and like terms encompass compositions that may be administered to a subject in need thereof (e.g., human or animal) in order to enhance an immune response. Exemplary immunogenic compositions may comprise, for example, whole purified virus or antigenic subunits, e.g., polypeptides thereof, or antigenic epitopes. Such compositions may further comprise an effective amount of one or more additional agents, e.g., one or more pharmaceutical excipients, carriers, and/or adjuvants.

[0017] As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0018] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system.

For example, “about” may mean within 1 standard deviation, or more than 1 standard deviation. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0020] As used herein, the term “effective amount” means an amount of a composition sufficient to show a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[0020] The active agent may form salts, which are also within the scope of the invention. Reference to a compound of the active agent herein is understood to include reference to salts thereof, unless otherwise indicated. The term “salt(s)”, as employed herein, denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases. In addition, when an active agent contains both a basic moiety, such as, but not limited to an amine or a pyridine or imidazole ring, and an acidic moiety, such as, but not limited to a carboxylic acid, zwitterions (“inner salts”) may be formed and are included within the term “salt(s)” as used herein. Pharmaceutically acceptable (e.g., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful, e.g., in isolation or purification steps, which may be employed during preparation. Salts of the compounds of the active agent may be formed, for example, by reacting a compound of the active agent with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization. When the compounds are in the forms of salts, they may comprise pharmaceutically acceptable salts. Such salts may include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p- aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids, sulphates, nitrates, phosphates, perchlorates, borates, acetates, benzoates, hydroxynaphthoates, glycerophosphates, ketoglutarates and the like. Examples of metal salts include lithium, sodium, potassium, magnesium salts and the like. Examples of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts and the like. Examples of organic bases include lysine, arginine, guanidine, diethanolamine, choline and the like.

[0021] Pathogens like HIV, SARS-CoV-2, Mycobacterium tuberculosis, and Plasmodium falciparum continue to pose serious threats to global human health, in large part due to the lack of efficacious vaccines to prevent these infections ¹ . Yet further, vaccines may be used to induced immune responses against toxins, drugs, pathogens, tumors, lipids, proteins, sugars, and the like, which may pose a health threat. Intrinsic immunoregulatory mechanisms that limit development of protective immune responses after immunization represent a major roadblock to advancement of successful vaccines. For example, antibodies capable of broadly neutralizing a highly mutable virus like HIV develop in only a small proportion of infected individuals years after acquisition of virus ^{2, 3} . To date, no HIV vaccine regimen tested in animals or humans can reliably elicit these broadly neutralizing antibodies (bnAb) ⁴ . During virus infection, intrinsic regulatory mechanisms determine the balance between efficient viral clearance and potentially harmful inflammation by controlling the strength, duration, and character of immune responses. These same regulatory mechanisms can limit the quality and magnitude of antiviral immunological memory elicited by vaccines. Therefore, development of translational means to relieve regulatory control of immunity during immunization holds great promise for amplifying the efficacy of emerging vaccines to prevent infections with viruses that are major causes of human diseases, including HIV and SARS-CoV-2, among many others.

[0022] Perforin is a glycoprotein stored in cytoplasmic granules of NK cells and T cells that polymerizes to form pores in the target cell membrane, resulting in target cell death through a combination of osmotic lysis and delivery of apoptosis-inducing granzymes. Thus, perforin is a key mediator of NK-cell killing of tumors and virus-infected cells. Therefore, most translational studies to date focus on means of enhancing NK cell-mediated release of perforin to augment immune-mediated elimination of viruses and tumors. Natural killer (NK) cells suppress T and B cell responses during infection and immunization. In mice, Applicant has shown that early, perforin-dependent killing of activated T cells by NK cells reduces the magnitude and quality of immunization-induced T-cell memory, germinal center B-cells, long-lived antibody- secreting cells, and affinity maturation of neutralizing antibody responses (Rydyznski, 2015 & 2018). Human NK cells also show interindividual variability in their capacity to suppress generation of neutralizing antibodies specific for HIV (Bradley, 2018) or yellow fever virus (Muyanja, 2014).

[0023] Natural killer (NK) cells recently emerged as potent regulators of immune responses during infection and immunization ^5-9 . Classically valued for their ability to kill tumor and virus-infected cells ^{10, 11} , NK cells can also kill otherwise healthy host cells ^12-17 — including activated CD4 T cells ^{18, 19} — to substantially modulate the magnitude and quality of adaptive immunity ^5-7 . Depletion of NK cells or perforin-deficiency non- additively enhance vaccine- induced T- and B-cell responses (Rydyznski, 2015 & 2018). Applicant demonstrated that activity of NK cells determines immune exhaustion, host survival, and viral persistence in the context of chronic lymphocytic choriomeningitis virus (LCMV) infection of mice ^{18, 20} . Applicant discovered that a similar perforin-dependent mechanism of NK-cell-mediated suppression constrains the generation of long-lived antiviral memory T cells and neutralizing antibodies ²¹ . Mechanistically, the suppression of humoral immunity by NK cells involves perforin-dependent reductions in the magnitude of follicular helper T cell (Tfh) and germinal center (GC) B cell responses ²¹ . NK-cell and perforin-mediated inhibition of GC responses restricted somatic mutation and affinity maturation of immunoglobulins after administration of protein-based vaccines ²² . These results are consistent with evidence of NK-cell inhibition of vaccine- (yellow fever vaccine 17D) or virus- (HIV) elicited neutralizing antibody responses in humans ^{23, 24} .

[0024] Based on work in NK-cell-depleted or perforin-deficient (Prf1 ^KO ) mice, Applicant has found that NK cell-derived perforin is essential in immunoregulatory lysis of activated CD4 T cells, suppression of Tfh differentiation, impairment of germinal center responses, inhibition of somatic hypermutation, and constraint of affinity maturation. Applicant proposes that inhibition of perforin-dependent, NK cell-mediated killing of T cells via the administration of perforin inhibitors may be used to remove the immunological roadblock and augment vaccine-elicited Tfh and germinal center B cell responses. It is likewise proposed that perforin inhibition may result in increased numbers of, and functionally enhanced CD4 and CD8 memory T cells, as well as increased numbers of antibody- secreting B cells in the bone marrow months after infection and increased amounts of antibodies with improved activity. Further, increased affinity maturation of immunoglobulin in B cells may also result from perforin inhibition.

[0025] Disclosed herein are inhibitors of perforin as vaccine adjuvants and methods for the use thereof. The methods and compositions described herein may be used, for example, to induce or enhance an immune response in a subject in need thereof. Such administration may treat, prevent and/or ameliorate one or more pathological conditions associated with infection in the subject. In one aspect, the disclosed compositions and methods may be used to increase the amount of broadly neutralizing antibodies (“bnAbs”) or the rate of development of bnAbs in an individual receiving the disclosed composition, and/or further, may trigger antibodies with non-neutralizing functions (i.e., ADCC).

[0026] In one aspect, a composition comprising a vaccine and a perforin inhibitor is disclosed. In certain aspects, the perforin inhibitor (PRF-I) may have the structure (referred to herein as “Compound 16”), or a pharmaceutically acceptable salt thereof. The vaccine may be one as typically understood by one of ordinary skill in the art, that is, an agent that induces an immune response in a mammal. In certain aspects, the vaccine composition may induce an immune response more particularly in a human subject.

[0027] In one aspect, the vaccine may be selected from a whole pathogen vaccine, a subunit vaccine, a chimeric vaccine, and combinations thereof. In other aspects, the vaccine may be a subunit vaccine selected from an acellular vaccine, a polysaccharide vaccine, a conjugate vaccine, a toxoid vaccine, a recombinant protein vaccine, a virus-like particle (VLP), and a nanoparticle vaccine. In certain aspects, the vaccine may be a nucleic acid vaccine selected from a DNA plasmid vaccine, an mRNA vaccine, and a recombinant vector vaccine. In yet further aspects, the vaccine may be an inactivated vaccine, a live-attenuated vaccine, or combinations thereof.

[0028] The composition may take a variety of forms. In one aspect, the vaccine composition may be provided in a lyophilized state, which may be reconstituted prior to administration. The composition may be provided in a sterile saline solution, and in certain aspects, may further comprise an adjuvant, as described herein.

[0029] The vaccine used with the disclosed PRF-I compounds may be effective against a variety of infectious agents, for example, coronavirus (SARS-COV-2) infection, influenza infection, and/or one or more viruses selected from Measles, mumps, rubella (MMR combined vaccine), varicella (chickenpox), Influenza (nasal spray), Rotavirus, Polio (IPV), Hepatitis A, Diphtheria, tetanus, Hepatitis B, Influenza (injection), Haemophilus influenza type b (Hib), Pertussis (part of DTaP combined immunization), Pneumococcal, Meningococcal, Zoster (shingles), Yellow fever, rabies, human papillomavirus (HPV), and combinations thereof. In further aspects, the vaccine may be one which is not directed to an infectious agent, but rather, a vaccine effective against one or more of a toxin, an allergen, a cancer, a tumor, a fungi, and combinations thereof. In a yet further aspect, the vaccine used with the disclosed PRF-I compounds may be one which is directed to a drug of addiction, in which the vaccine composition is administered for the purpose of preventing or treating a drug addiction.

[0030] In a further aspect, the perforin inhibitor may comprise a carrier molecule, such as, for example, a cyclodextrin molecule, which may be used to improve perforin inhibitor stability. Such molecules are known in the art and the implementation of such molecules will be withing the skill of one of ordinary skill in the art.

[0031] In a further aspect, a method of treating an individual using a vaccine composition as disclosed herein is described. In this aspect, a method of enhancing the immunogenicity of a vaccine composition is disclosed. The method may comprise administering to an individual in need thereof a perforin inhibitor having the structure of Compound 16 or a pharmaceutically acceptable salt thereof; and b) a vaccine as described herein. The perforin inhibitor may be administered before, after, or during administration of the vaccine, and the vaccine and/or said perforin inhibitor may be administered at a time point selected from prior to, during, or after disease onset or infection.

[0032] In one aspect, the perforin inhibitor may be in a composition that is co-administered with a vaccine composition. As used herein, “co-administration” includes administration of unit dosages of the compounds disclosed herein before or after administration of unit dosages of one or more additional vaccine compositions, for example, administration of the compound disclosed herein within seconds, minutes, hours, or a day, or days of the administration of one or more additional vaccine composition. For example, a unit dose of a compound of the present disclosure may be administered first, followed within seconds or minutes by administration of a unit dose of one or more vaccine composition. Alternatively, a unit dose of one or more vaccine composition may be administered first, followed by administration of a unit dose of a compound of the present disclosure within seconds or minutes. A unit dose of a compound of the present disclosure may be administered first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of one or more vaccine composition. In other aspects, a unit dose of one or more vaccine composition may be administered first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of a compound of the present disclosure. Co-administration of a compound disclosed herein with one or more vaccine composition generally refers to simultaneous or sequential administration of a compound disclosed herein and one or more vaccine composition, such that therapeutically effective amounts of each agent are present in the body of the patient. In one aspect, the co-administration is such that the perforin inhibitor is administered in the same composition as the vaccine. In one aspect, the co-administration is such that the perforin inhibitor is administered in the same device as the vaccine composition. In one aspect, the co-administration is such that the perforin inhibitor is administered at the same site as the vaccine composition, and within the same 24 hour period as the vaccine composition. In one aspect, the administration of the PRF-I is a single injection on the day of immunization.

|0033] In a further aspect, the vaccine composition may comprise the disclosed compounds, the disclosed compound being a perforin inhibitor. In this aspect, the composition may be a single composition that comprises both the vaccine composition and a perforin inhibitor as disclosed herein. Suitable concentrations of the perforin inhibitor may be determined by one of ordinary skill in the art, and may be present in an amount effective to obtain the desired effect.

[0034] In certain aspects, the perforin inhibitor may be administered at a dose of from about 15 to about 150 mg/kg, or from about 25 to 125 mg/kg, or from about 30 to about 100 mg/kg, or from about 35 to about 75 mg/kg.

[0035] In one aspect, the perforin inhibitor and vaccine composition may be administered at least twice, or at least three times, or at least four times to said individual.

[0036] The disclosed composition may take a variety of different forms suited for a desired route of administration, and include, for example, dosage forms selected from liquid preparations, suspensions, parenteral preparations, subcutaneous preparations, intradermal preparations, intramuscular preparations, intraperitoneal preparations, intravenous preparations, and intranasal preparations. The composition may be, for example, administered in the form of a sterile isotonic aqueous solutions, a suspension, an emulsion, or a viscous composition. In some aspects, the composition may be administered to a subject as an injectable, for example, as an injectable for delivery by intramuscular, intravenous, subcutaneous, or transdermal injection. In further aspects, the administration may utilize an oral formulation, wherein the composition may be in a dosage form selected from a solution, a powder, a suspension, a tablet, a pill, a capsule, a caplet, a sustained release formulation, a time-release formulation or combinations thereof. The composition may be buffered to a selected pH. In one aspect, compositions may be administered to a subject as an injectable, including but not limited to injectable compositions for delivery by intramuscular, intravenous, subcutaneous, or transdermal injection.

[0037] It is contemplated herein that the disclosed compositions may be administered to a subject by a variety of routes according to conventional methods, including but not limited to parenteral (e.g., by intracistemal injection and infusion techniques), intradermal, transmembranal, transdermal (including topical), intramuscular, intraperitoneal, intravenous, intra-arterial, intralesional, subcutaneous, oral, and intranasal (e.g., inhalation) routes of administration. Administration may also be by continuous infusion or bolus injection.

[0038] One or more additional adjuvants known in the art may be included in the disclosed compositions. Adjuvants suitable for use with the disclosed methods and compositions are familiar to one of skill in the art and are available from a variety of commercial vendors. These may include, for example, glycolipids, chemokines, compounds that induce the production of cytokines and chemokines, interferons, inert carriers (such as alum, bentonite, latex, and acrylic particles), pluronic block polymers, depot formers, surface active materials (such as saponin, lysolecithin, retinal, liposomes, and pluronic polymer formulations), macrophage stimulators (such as bacterial lipopolysaccharide), alternate pathway complement activators (such as insulin, zymosan, endotoxin, and levamisole), non-ionic surfactants, poly(oxyethylene)-poly(oxypropylene) tri-block copolymers, trehalose dimycolate (TDM), cell wall skeleton (CWS), complete Freund's adjuvant, incomplete Freund's adjuvant, macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), 3-O-deacylated MPL, CpG oligonucleotides, polyoxyethylene ethers, polyoxyethylene esters, aluminum, Poly[di(carboxylatophenoxy)phosphazene] (PCPP), monophosphoryl lipid A, QS-21, cholera toxin and formyl methionyl peptide, and combinations thereof.

[0039] In one aspect, the disclosed compositions may be administered orally. Oral formulations may include a variety of dosage forms, e.g., solutions, powders, suspensions, tablets, pills, capsules, caplets, sustained release formulations, or preparations which are time-released or which have a liquid filling, e.g., gelatin covered liquid, whereby the gelatin is dissolved in the stomach for delivery to the gut. Such formulations may include a variety of pharmaceutically acceptable excipients described herein, including but not limited to mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate.

[0040] In one aspect, compositions for oral administration may be a liquid formulation. Such formulations may comprise a pharmaceutically acceptable thickening agent which can create a composition with enhanced viscosity which facilitates mucosal delivery of the immunogen, e.g., by providing extended contact with the lining of the stomach. Such viscous compositions may be made by one of skill in the art employing conventional methods and employing pharmaceutical excipients and reagents, e.g., methylcellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, and carbomer.

[0041] Other dosage forms suitable for nasal or respiratory (mucosal) administration, e.g., in the form of a squeeze spray dispenser, pump dispenser or aerosol dispenser, are contemplated herein. Dosage forms suitable for rectal or vaginal delivery are also contemplated herein. The disclosed compositions may also be lyophilized and may be delivered to a subject with or without rehydration using conventional methods.

[0042] As discussed above, the viral vaccines and other pharmaceutical compositions disclosed herein may be formulated by one of skill in the art using a variety of pharmaceutical excipients, carriers, diluents, etc. familiar to one of skill in the art using art recognized methods. Such vaccines and compositions may be administered to a subject alone, e.g., as individual dosage forms, or administered in combination in the form of an immunogenic composition.

[0043] In addition to the foregoing, as understood herein, the methods may comprise administering the compositions to a subject according to various regimens familiar to one of skill in the art, i.e., in an amount and in a manner and for a time sufficient to provide a clinically meaningful benefit to the subject. Suitable administration regimens may be determined by one of skill in the art according to conventional methods. For example, it is contemplated herein that an effective amount of a disclosed composition may be administered to a subject as a single dose, a series of multiple doses administered over a period of days, or a single dose followed by a boosting dose thereafter, e.g., several years later. A “prime boost” schedule may be employed if deemed desirable. The term “dose” or “dosage” as used herein refers to physically discrete units suitable for administration to a subject, each dosage containing a predetermined quantity of a vaccine as an active pharmaceutical ingredients calculated to produce a desired response on the immune system of the subject. In one aspect, active agents provided herein may be administered in an dosage form selected from intravenous or subcutaneous unit dosage form, oral, parenteral, intravenous, and subcutaneous. In some aspects, compositions provided herein may be formulated into liquid preparations for, e.g., oral administration. Suitable forms include suspensions, syrups, elixirs, and the like. In some aspects, unit dosage forms for oral administration include tablets and capsules. Unit dosage forms configured for administration once a day; however, in certain aspects it may be desirable to configure the unit dosage form for administration twice a day, or more.

[0044] In one aspect, the disclosed compositions may be formulated to be isotonic with the blood or other body fluid of the recipient. The isotonicity of the compositions may be attained using sodium tartrate, propylene glycol or other inorganic or organic solutes. An example includes sodium chloride. Buffering agents may be employed, such as acetic acid and salts, citric acid and salts, boric acid and salts, and phosphoric acid and salts. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like.

[0045] A pharmaceutically acceptable preservative may be employed to increase the shelf life of the pharmaceutical compositions. Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed. A suitable concentration of the preservative is typically from about 0.02% to about 2% based on the total weight of the composition, although larger or smaller amounts may be desirable depending upon the agent selected. Reducing agents, as described above, may be advantageously used to maintain good shelf life of the formulation. [0046] In one aspect, active agents provided herein may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, or the like, and may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Such preparations may include complexing agents, metal ions, polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, and the like, liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. The presence of such additional components may influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application, such that the characteristics of the carrier are tailored to the selected route of administration.

[0047] Controlled release formulations may be employed wherein the active agent or analog(s) thereof is incorporated into an inert matrix that permits release by either diffusion or leaching mechanisms. Slowly degenerating matrices may also be incorporated into the formulation. Other delivery systems may include timed release, delayed release, or sustained release delivery systems.

[0048] Coatings may be used, for example, nonenteric materials such as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols, or enteric materials such as phthalic acid esters. Dyestuffs or pigments may be added for identification or to characterize different combinations of active agent doses.

[0049] When administered orally in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added to the active ingredient(s). Physiological saline solution, dextrose, or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol are also suitable liquid carriers. The pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive or arachis oil, a mineral oil such as liquid paraffin, or a mixture thereof. Suitable emulsifying agents include naturally-occurring gums such as gum acacia and gum tragamayth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsions may also contain sweetening and flavoring agents.

[0050] Pulmonary delivery of the active agent may also be employed. The active agent may be delivered to the lungs while inhaling and traverses across the lung epithelial lining to the blood stream. A wide range of mechanical devices designed for pulmonary delivery of therapeutic products may be employed, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. These devices employ formulations suitable for the dispensing of active agent. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants, and/or carriers useful in therapy.

[0051] The active ingredients may be prepared for pulmonary delivery in particulate form with an average particle size of from 0.1 um or less to 10 um or more, for example, from about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 um to about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5 um. Pharmaceutically acceptable carriers for pulmonary delivery of active agent include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations may include DPPC, DOPE, DSPC, and DOPC. Natural or synthetic surfactants may be used, including polyethylene glycol and dextrans, such as cyclodextran. Bile salts and other related enhancers, as well as cellulose and cellulose derivatives, and amino acids may also be used. Liposomes, microcapsules, microspheres, inclusion complexes, and other types of carriers may also be employed.

[0052] Pharmaceutical formulations suitable for use with a nebulizer, either jet or ultrasonic, typically comprise the active agent dissolved or suspended in water at a concentration of about 0.01 or less to 100 mg or more of active agent per mL of solution, for example, from about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg to about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 mg per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the active agent caused by atomization of the solution in forming the aerosol.

[0053] Formulations for use with a metered-dose inhaler device generally comprise a finely divided powder containing the active ingredients suspended in a propellant with the aid of a surfactant. The propellant may include conventional propellants, such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, and hydrocarbons. Example propellants include trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, 1, 1,1,2- tetrafluoroethane, and combinations thereof. Suitable surfactants include sorbitan trioleate, soya lecithin, and oleic acid.

[0054] Formulations for dispensing from a powder inhaler device typically comprise a finely divided dry powder containing active agent, optionally including a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in an amount that facilitates dispersal of the powder from the device, typically from about 1 wt. % or less to 99 wt. % or more of the formulation, for example, from about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 wt. % to about 55, 60, 65, 70, 75, 80, 85, or 90 wt. % of the formulation.

[0055] In some aspects, the active agents provided herein may be provided to an administering physician or other health care professional in the form of a kit. The kit is a package which houses a container which contains the active agent(s) in a suitable pharmaceutical composition, and instructions for administering the pharmaceutical composition to a subject. The kit may optionally also contain one or more additional therapeutic agents currently employed for treating a disease state as described herein. For example, a kit containing one or more compositions comprising active agents provided herein in combination with one or more additional active agents may be provided, or separate pharmaceutical compositions containing an active agent as provided herein and additional therapeutic agents may be provided. The kit may also contain separate doses of an active agent provided herein for serial or sequential administration. The kit may optionally contain one or more diagnostic tools and instructions for use. The kit may contain suitable delivery devices, e.g., syringes, and the like, along with instructions for administering the active agent(s) and any other therapeutic agent. The kit may optionally contain instructions for storage, reconstitution (if applicable), and administration of any or all therapeutic agents included. The kits may include a plurality of containers reflecting the number of administrations to be given to a subject.

EXAMPLES

[0056] The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus may be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0057] Potent, soluble, and non-toxic substituted arylsulphonamide compounds as inhibitors of perforin and uses thereof are disclosed here. These compounds inhibit in vivo pathophysiologic activity of perforin in a mouse model of fulminant viral hepatitis. Applicant assessed the capacity of perforin inhibitors to prevent perforin-dependent delivery of granzymes into the cytoplasm and subsequent activation of proapoptotic caspases using target cells stably transfected with biosensors of granzyme or caspase activity. Treatment of human NK-92 NK-cell line with 300 pM 2,4-difhioro-N-(2-chloro-5-(5-(2-methyl-l-oxoisoindolin- 5-yl)thiophen-2-yl)pyridine-3-yl)benzenesulphonamide (Compound 11, having the structure:

(perforin-inhibitor, PRF-I) prevented granzyme B-dependent cleavage-induced activation of the VGPD luciferase biosensor in K562 target cells is shown in Fig. 2A. Likewise, PRF-I treatment of primary human blood NK cells inhibited caspase-3/7 DEVD biosensor activation in a redirected lysis assay against anti-CD16 antibody-bound P815 cells (Fig. 2B). Application of PRF-I to human KHYG1 NK-cell line co-cultures with Jurkat T cells (20:1 effector: target ratio) resulted in dose-dependent inhibition of Jurkat cell death (Fig. 2C). These results support use of PRF-I to transiently inhibit perforin function in vivo during immunization.

[0058] A successful vaccine must orchestrate optimal follicular helper T cell (Tfh) and germinal center B cell responses to facilitate efficient affinity maturation of HIV-specific immunoglobulins. NK cells and perforin redundantly contribute to apop to tic elimination of activated CD4 T cells and curtailed Tfh differentiation during the first 3 days of infection (Fig. 3). Applicant hypothesized that in vivo administration of PRF-I will prevent NK-cell killing of activated CD4 T cells and thereby augment Tfh responses.

[0059] FIG 4, panel A shows that PRF-I (Compound 16) blocks -50% of perforin dependent killing of targets “missing self’ (no class I MHC in absence of β 2M) in the in vivo setting. In fact, a single injection of small molecule inhibitors of perforin (PRF-I) ⁹⁸ reduced in vivo NK- cell elimination of β 2M ^KO targets by >50% (Fig. 4A). PRF-I application during the first three days of NP-KLH:alum immunization enhanced Tfh (Fig. 4B) and germinal center B-cell (Fig. 4C) responses >2-fold to similar levels as those in Prfl ^KO mice. These results suggest that PRF-I can alleviate perforin-dependent NK-cell suppression of vaccine-elicited adaptive immune responses.

[0060] Materials and methods:

[0061] Mice

[0062] 8-10 weeks old male C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and 8-10 weeks old in-house bred male Prf1-/- mice were used for the experiments. Mice were housed under specific pathogen- free conditions and experiments were performed using the ethical guidelines approved by the Institutional Animal Use and Care Committees of Cincinnati Children’s Hospital Medical Center.

[0063] In vivo NK-cell depletion

[0064] NK cells were depleted in mice using one intraperitoneal injection of 25 pg per mouse anti-NKl.l monoclonal antibody (PK136) a day prior to either in vivo NK cell cytotoxicity assay or NP-KLH immunization. 25 μg of mouse IgG2a (Cl.18.4) produced by Bio-X-Cell (West Lebanon, NH) was used as isotype control.

[0065] Preparation of Test Compound 16

[0066] Test compound 16 stock was prepared by dissolving 1OOmg/ml in 10% DMSO.

35mg/kg of test compound 16 were prepared by incubating with 20% of (2-Hydroxypropyl)- (3-cyclodextrin (H5784, Sigma) in a rotator at 4°C for an hour and mice were treated intraperitoneally. Mice were treated with compound 16 daily once day -1 through day -3 for NP-KLH mouse model and treated day 0 & day 1 for in vivo NK cell cytotoxicity assay. Mice were either treated with Compound 16 daily once day 0 through day 2 or treated once on day 0 as stated in the experiments.

[0067] Test compound 16 (“Compound 16”) has the following structure:

[0068] NP-KLH Immunization

[0069] 4-hydroxy-3-nitrophenylacetyl conjugated to keyhole limpet hemocyanin (NP-KLH) was purchased from Biosearch Technologies (Petaluma, CA). Img/ml of NP-KLH was incubated with alum (Imject alum, Thermo Fisher) in a 1:1 ratio on a rotator for an hour at room temperature. 1OOμg of alum-adsorbed NP-KLH were given once intraperitoneally in a volume of 200 μL on day 0.

[0070] In vivo NK cell cytotoxicity assay

[0071] Single cell suspensions were prepared from spleens of β2m ^ko ond WT C57BL/6.β2m ^ko cells (low CFSE) and WT cells (high CFSE) labelled at 37°C for 10 minutes. These cells were mixed in an equal proportion adoptively transferred into WT mice or NK cell depleted or Compound 16 treated mice. Spleens were harvested from recipient mice 16 hours post cell transfer and CFSE labelled cells were traced using flow cytometry. High CFSE WT cells transferred into WT recipient mice served as internal control.

[0072] Enzyme-linked immunosorbent assays (ELISA)

[0073] Quantification of NP-specific IgG, were performed by coating NP (20) conjugated to BSA in PBS to 96-well high binding plates (Corning) overnight at 4 °C. Plates were then blocked with PBS 2% BSA for 1 hour at room temperature, washed and loaded with serially diluted serum for a 2 hour incubation. Plates were washed again and incubated for 1 h with IgGl conjugated to horseradish peroxidase (Invitrogen), followed by washing and development with TMB -substrate solution (Invitrogen) for 15 minutes and stopped using (2N H2SO4). Absorbance were measured at 450nm and values were graphed as relative absorbance.

[0074] Antibodies and Flow cytometry analysis

[0075] Single-cell suspensions were prepared from spleens and LNs by pressing through 70μm cell strainer and red blood cells were lysed in ACK lysis buffer for 5 minutes at 37°C. Lysed cells were washed, pelleted and suspended in flow cytometry buffer (IX HBSS, 2mM EDTA, 5% heat-inactivated fetal bovine serum). Cells were suspended in IX PBS and Live/dead staining was performed using Zombie NIR (1:1000 diluted in lx PBS buffer) at room temperature for 4 minutes. Cells were washed and surface stained with antibodies prepared in flow cytometry buffer containing FACS block (anti CD16/32, clone 2.4G2, Tonbo Biosciences) for 30 minutes at 4°C. Cells were washed, fixed in BD Cytofix buffer for 4 minutes and resuspended in flow cytometry buffer. The following antibodies/reagents were used for flow cytometry: Zombie NIRTM (1:1000), anti-CD3 BUV395 (clone 145-2C11, BD Biosciences), anti-CD4 BV605 (clone GK1.5 Biolegend), anti-B220 BUV737 (clone RA3- 6B2 BD Biosciences), anti-CD19 BV605 (clone 6D5 Biolegend), anti-CD95 PE-Cy7 (clone Jo.2 BD Biosciences), anti-GL-7 ef450 (clone GL7 eBiosciences), anti-IgD FITC (clone 11- 26c.2a Biolegend), anti- CD44 BV786 (clone IM-7 Biolegend), anti-PD-1 ef450 (clone J43 eBiosciences), anti-CXCR5 PE-Cy7 (clone L138D7 Biolegend), anti-DX5 PE (clone Rl-2 Biolegend), anti-NKl.l BV711 (clone PK136 Biolegend), anti-NKp46 APC (clone 29A1.4 Biolegend), anti-IgGl APC (clone RMG1-1 Biolegend). Stained cells were analyzed on a BD

Fortessa 2 flow cytometer and data were processed using FlowJo software (Treestar).

[0076] Synthesis of Compound 16

[0077] Compounds 1 and 4 were synthesized according to procedures outlined as detailed below:

ASTRAZENECA AS: ASTRAZENECA UK LWE&, FORD. Rhowt: KINCHIN, Etobesh: MATHER, AMw, METE, Anionic; MWCHiP. tan; STANiER, AfKifew Geeltey

W02GW154677, 2€-H,A1

[0078]

[Journal of Medicinal Chemistry, 20' 8, vol. 61. # 14. p. 6087 - 61091

[0079]

[0080] Compound 2. Potassium carbonate (176 mg) in water (2 mL) was added to 2- thiopheneboronic acid pinacol ester (132 mg, 0.63 mmol), 3-bromo-6-methyl-5H- pyrrolo[3,4- b]pyridin-7(6H)-one (1) (144 mg, 0.63 mmol) and 1,1 bis-(tert- butylphosphino)ferrocene palladium dichloride (50 mg) in degassed acetonitrile (12 mL) at

200C under nitrogen. The resulting solution was stirred at 800C for 90 min. The reaction mixture was cooled and filtered and the filtrate diluted with ethyl acetate, and washed with water. The aqueous was then extracted with dichloromethane and the combined organics dried over magnesium sulfate, filtered and evaporated to afford crude product. The crude product was purified by chromatography on silica eluting with methanol I ethyl acetate (15:85 to 25:75). Pure fractions were evaporated to dryness to afford the compound 2 (108 mg, 75% yield).

[0081] Compound 3. Compound 2 (48 mg, 0.21 mmol) and acetic acid (3 mL) were charged with a single-neck round-bottom flask under ambient conditions. After the compound was completely dissolved, liquid bromine (Br2) (40 mg, 0.25 mmol) in dichloromethane (1 mL) was added. The reaction was kept stirring at room temperature for 1 h. Then the reaction mixture was extracted with dichloromethane and washed with saturated NaHCCL solution and brine. The organic extract was dried over sodium sulfate and collected under reduced pressure. The crude material was purified by column chromatography on silica gel to obtain white solid 3 (45 mg), 71.4% yield.

[0082] 1H NMR (400 MHz, DMSO-d6) 3.10 (s, 3H), 4.49 (s, 2H), 7.35 (d, J = 2.4 Hz, 1H), 7.61 (d, J = 2.4 Hz, 1H), 8.25 (s, 1H), 8.97 (s, 1H).

[0083] Target Compound (Compound 16). Potassium carbonate (176 mg) in water (2 mL) was added to the pinacol ester (4) (268 mg, 0.63 mmol), compound 3 (195 mg, 0.63 mmol) and 1,1 bis-(tert-butylphosphino)ferrocene palladium dichloride (50 mg) in degassed acetonitrile (12 mL) at 200C under nitrogen. The resulting solution was stirred at 800C for 90 min. The reaction mixture was cooled and filtered and the filtrate diluted with ethyl acetate, and washed with water. The aqueous was then extracted with dichloromethane and the combined organics dried over magnesium sulfate, filtered and evaporated to afford crude product. The crude product was purified by chromatography on silica eluting with methanol I ethyl acetate (15:85 to 25:75). Pure fractions were evaporated to dryness to afford the target compound (253 mg, 76% yield). 1H NMR (400 MHz, DMSO-d6) 3.10 (s, 3H), 3.76 (s, 3H) 4.49 (s, 2H), 7.09 (t, J = 6.4 Hz, 1H), 7.18 (t, J = 7.2 Hz, 1H), 7.22 (d, J = 2.8 Hz, 1H), 7.43 (s, 1H), 7.68 (s, 1H), 7.71 (d, J = 2.8 Hz, 1H), 7.86 (q, J = 6.0 Hz, 1H), 8.25 (s, 1H), 9.01 (s, 1H).

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[00199] All percentages and ratios are calculated by weight unless otherwise indicated.

[00200] All percentages and ratios are calculated based on the total composition unless otherwise indicated.

[00201] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[00202] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.”

[00203] Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. All accessioned information (e.g., as identified by PUB MED, PUBCHEM, NCBI, UNIPROT, or EBI accession numbers) and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[00204] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.