Chemical Screen of Modulators for Vaccine Adjuvants

Abstract

The present disclosure provides methods and compositions that can be used to modulate an immune response. Described is a method comprising administering to a subject an effective amount of (a) an adjuvant and (b) one or more of bafetinib (INNO-406), LY3009120, MK-8353 (SCH900353), amodiaquine, zanubrutinib (BGB-3111), Ku55933, tucidinostat, PD318088, WNK463, and mesylate of TRx0237 (LMTX). The method may be for vaccination of a subject or for the treatment or prevention of cancer, graft rejection, graft-versus-host disease, bacterial infection, or viral infection in a subject. The method may be for modulating an immune response in vivo, in vitro, or ex vivo. The method also includes the immune activation of a population of immune cells in vitro or ex vivo. A method for identifying the efficacy of an adjuvant is also described.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 347,438, filed May 31, 2022; U.S. Provisional Patent Application No. 63 / 359,620, filed Jul. 8, 2022; and U.S. Provisional Patent Application No. 63 / 424,201, filed Nov. 10, 2022. Each of U.S. Provisional Patent Application No. 63 / 347,438, U.S. Provisional Patent Application No. 63 / 359,620, and U.S. Provisional Patent Application No. 63 / 424,201 is hereby incorporated by reference in its entirety.

[0002] Description of Research or Development Sponsored by the Federal Government This invention was made with government support under grant numbers 75N93019C00041 - P00004 - 9999 - 1, 75N93019C00041 - P00001 - 9999 - 1, 75N93019C00041 - P00002 - 9999 - 1, 75N93019C00041 - P00003 - 9999 - 1, 75N93019C00041 - P00001 - 9999 - 2, AI124286, AI112194, GM099594, and HDTRA1 - 18 - 1 - 0052 awarded by the National Institutes of Health. The U.S. government has certain rights in this invention.

[0003] 1. Field of the Invention The present invention generally relates to the fields of immunology and immunotherapy. Methods and compositions for enhancing the safety and efficacy of vaccines and immunotherapies are described herein.

Background Art

[0004] 2. Background Activation of Toll - like receptors (TLRs) has been associated with the high immunogenicity and protective effects of vaccines. Incorporating TLR adjuvants into subunit and epitope - based vaccine formulations has significantly improved both antibody and T - cell levels and antigen specificity. 1,2 。By incorporating TLR adjuvants into subunit and epitope - based vaccine formulations, both antibody and T - cell levels and antigen specificity have been significantly improved.3,4 。Currently, many small molecule adjuvants have been discovered 5,6 。However, the tolerability of these in preclinical and clinical studies has restricted the use of many of these compounds, thus necessitating reformulation or redesign 7 。

[0005] Historically, the discovery of adjuvants has been experience-based, but synthetic small molecule adjuvants and modern drug discovery techniques have been applied to optimize adjuvant activity. This has led to the development of a class of adjuvants collectively called small molecule immune potentiators (SMIPs) 8,9 。In this class, imidazoquinolinones that activate toll-like receptor-7 and toll-like receptor-8 (TLR7 / 8), such as imiquimod (R837) and resiquimod (R848), have been extensively studied. Currently, imiquimod is approved for clinical immunotherapy use in topical cream 9-11 。These SMIPs have been shown to induce antigen-specific cellular responses when administered as adjuvants 12-14 。Furthermore, when TLR7 / 8 is activated by resiquimod, IFN-γ, IL-2, and IL-10 are produced, and thus CD8 + T cells and CD4 + Antitumor activity is promoted by APC activation of Th cells 15-17 。However, due to the high bioavailability of compounds structurally related to imidazoquinolinones, unacceptable systemic inflammation levels due to adjuvant toxicity occur, and thus their use is greatly restricted

[0006] Therefore, current vaccination and immunotherapy, especially those involving the use of adjuvants, may pose safety issues, and strategies for enhancing the safety and tolerability of vaccines and cancer immunotherapy are needed in the art

SUMMARY OF THE INVENTION

[0007] The present disclosure provides methods and compositions that can be used to modulate an immune response. A method is described that includes administering to a subject an effective amount of (a) an adjuvant and (b) one or more (or any combination) of bafetinib (INNO-406), LY3009120, MK-8353 (SCH900353), amodiaquine, zanubrutinib (BGB-3111), Ku55933, tucidinostat, PD318088, WNK463, and TRx0237 (LMTX) mesylate. The method may be for vaccination of a subject or for the treatment or prevention of cancer, graft rejection, graft-versus-host disease, bacterial infection, or viral infection in a subject. The method may be for modulating an immune response in vivo, in vitro, or ex vivo. The method also includes immunostimulation of a population of immune cells in vitro or ex vivo. A method for determining the potency of an adjuvant is also described, the method including (a) administering an adjuvant to a population of cells; (b) administering a PRR agonist to the population of cells; and (c) measuring the expression of one or more cytokines from the cells. A pharmaceutical composition is also described that includes (a) an adjuvant and (b) one or more (or a combination) of bafetinib (INNO-406), LY3009120, MK-8353 (SCH900353), amodiaquine, zanubrutinib (BGB-3111), Ku55933, tucidinostat, PD318088, WNK463, and TRx0237 (LMTX) mesylate. It is intended that one, two, three, four, five, six, seven, eight, or nine of bafetinib (INNO-406), LY3009120, MK-8353 (SCH900353), amodiaquine, zanubrutinib (BGB-3111), Ku55933, tucidinostat, PD318088, WNK463, and TRx0237 (LMTX) mesylate may be included in any of the methods or compositions described herein.In some aspects, one, two, three, four, five, six, seven, or eight of bufetinib (INNO-406), LY3009120, MK-8353 (SCH900353), amodiaquine, zanubrutinib (BGB-3111), Ku55933, tucidinostat, PD318088, WNK463, and mesylate of TRx0237 (LMTX) may be excluded.

[0008] The method may include administering an antigen to a subject, and may further include administering an antigen to a subject. The pharmaceutical composition may include an antigen. The antigen may be an antigen associated with cancer, bacteria, or virus. The antigen may be an antigen associated with cancer, bacteria, or virus that the subject has, an antigen associated with cancer, bacteria, or virus that the subject is diagnosed with, and / or an antigen associated with cancer, bacteria, or virus that the subject has symptoms of. The antigen may be an antigen associated with cancer, bacteria, or virus that the subject has a risk of. The antigen may be a bacterial antigen, a viral antigen, or a tumor antigen. The antigen may be an antigen described herein.

[0009] The method may comprise, or consist of, the step of administering an adjuvant and bufetinib (INNO-406). The method may comprise, or consist of, the step of administering an adjuvant, bufetinib (INNO-406), and an antigen. The pharmaceutical composition may comprise, or consist of, an adjuvant and bufetinib (INNO-406). The pharmaceutical composition may comprise, or consist of, an adjuvant, bufetinib (INNO-406), and an antigen. The method may comprise, or consist of, the step of administering an adjuvant and LY3009120. The method may comprise, or consist of, the step of administering an adjuvant, LY3009120, and an antigen. The pharmaceutical composition may comprise, or consist of, an adjuvant and LY3009120. The pharmaceutical composition may comprise, or consist of, an adjuvant, LY3009120, and an antigen. The method may comprise, or consist of, the step of administering an adjuvant and MK-8353 (SCH900353). The method may comprise, or consist of, the step of administering an adjuvant, MK-8353 (SCH900353), and an antigen. The pharmaceutical composition may comprise, or consist of, an adjuvant and MK-8353 (SCH900353). The pharmaceutical composition may comprise, or consist of, an adjuvant, MK-8353 (SCH900353), and an antigen. The method may comprise, or consist of, the step of administering an adjuvant and amodiaquine. The method may comprise, or consist of, the step of administering an adjuvant, amodiaquine, and an antigen. The pharmaceutical composition may comprise, or consist of, an adjuvant and amodiaquine. The pharmaceutical composition may comprise, or consist of, an adjuvant, amodiaquine, and an antigen. The method may comprise, or consist of, the step of administering an adjuvant and zanubrutinib. The method may comprise, or consist of, the step of administering an adjuvant, zanubrutinib, and an antigen. The pharmaceutical composition may comprise, or consist of, an adjuvant and zanubrutinib.The pharmaceutical composition may comprise, or consist of, an adjuvant, zanubrutinib, and an antigen. The method may comprise, or consist of, the step of administering an adjuvant and Ku55933. The method may comprise, or consist of, the step of administering an adjuvant, Ku55933, and an antigen. The pharmaceutical composition may comprise, or consist of, an adjuvant and Ku55933. The pharmaceutical composition may comprise, or consist of, an adjuvant, Ku55933, and an antigen. The method may comprise, or consist of, the step of administering an adjuvant and tucidinostat. The method may comprise, or consist of, the step of administering an adjuvant, tucidinostat, and an antigen. The pharmaceutical composition may comprise, or consist of, an adjuvant and tucidinostat. The pharmaceutical composition may comprise, or consist of, an adjuvant, tucidinostat, and an antigen. The method may comprise, or consist of, the step of administering an adjuvant and PD318088. The method may comprise, or consist of, the step of administering an adjuvant, PD318088, and an antigen. The pharmaceutical composition may comprise, or consist of, an adjuvant and PD318088. The pharmaceutical composition may comprise, or consist of, an adjuvant, PD318088, and an antigen. The method may comprise, or consist of, the step of administering an adjuvant and WNK463. The method may comprise, or consist of, the step of administering an adjuvant, WNK463, and an antigen. The pharmaceutical composition may comprise, or consist of, an adjuvant and WNK463. The pharmaceutical composition may comprise, or consist of, an adjuvant, WNK463, and an antigen. The method may comprise, or consist of, the step of administering an adjuvant and TRx0237 (LMTX) mesylate. The method may comprise, or consist of, the step of administering an adjuvant, TRx0237 (LMTX) mesylate, and an antigen. The pharmaceutical composition may comprise, or consist of, an adjuvant and TRx0237 (LMTX) mesylate.The pharmaceutical composition may comprise, or may consist of, an adjuvant, mesylate of TRx0237 (LMTX), and an antigen.

[0010] The method may include a step of administering an adjuvant to a subject. The pharmaceutical composition may contain an adjuvant. The method may further include a step of administering an additional adjuvant to the subject. The pharmaceutical composition may contain, or consist of, an adjuvant containing one or more of 3’3’-cGAMP, Tri-DAP, MDP, and mIFN-β. The adjuvant may contain, or consist of, a PRR agonist. The PRR agonist may be further defined as a TLR agonist. The TLR agonist may be an agonist for TLR1, TLR2 / 1, TLR2, TLR2 / 6, TLR3, TLR4, TLR5 TLR7, TLR8, TLR7 / 8, TLR9, and / or TLR11. In some aspects, any of these agonists may be excluded. The TLR agonist may be peptidoglycan, triacyl lipoproteins, lipoteichoic acid, peptidoglycan derived from Bacillus subtilis, peptidoglycan derived from E. coli 0111:B4, peptidoglycan derived from E. coli K12, peptidoglycan derived from Staphylococcus aureus, atypical lipopolysaccharide (LPS), leptospirosis LPS, Porphyromonas gingivalis LPS, synthetic diacylated lipoprotein, FSL-1, Pam2CSK4, lipoarabinomannan derived from M. smegmatis, M.Lipomannan derived from Smegmatis; Triacylated lipoproteins, Pam3CSK4, MALP-2 derived from Mycoplasma, MALP-404 derived from Mycoplasma, OspA of Borrelia burgdorferi, Porin derived from Neisseria meningitidis, Porin derived from Haemophilus influenza, Antigen mixture derived from Propionibacterium acnes, Yersinia LcrV, Lipomannan derived from Mycobacterium, Lipomannan derived from Mycobacterium tuberculosis, GPI anchor of Trypanosoma cruzi, Lysophosphatidylserine of Schistosoma mansoni, Lipophosphoglycan (LPG) of Leishmania major, Glycosylphosphatidylinositol (GPI) of Plasmodium falciparum, Zymosan, Antigen mixture derived from Aspergillus fumigatus, Antigen mixture derived from Candida albicans, Antigen mixture derived from Measles hemagglutinin, Double-stranded RNA, Polyadenylic acid - Polyuridylic acid (Poly(A:U)), Polyinosinic acid: Polycytidylic acid (Poly(I:C)), Polyinosinic acid: Polycytidylic acid high molecular weight (Poly(I:C)HMW), Polyinosinic acid: Polycytidylic acid low molecular weight (Poly(I:C)LMW)), LPS derived from Escherichia coli, LPS derived from species of Salmonella, Monophosphoryl lipid A, Flagellin, Flagellin derived from Bacillus subtilis, Flagellin derived from P. aeruginosa, Flagellin derived from S. typhimurium(S.Flagellin derived from Salmonella typhimurium, single-stranded RNA having a 6UUAU repeat sequence, single-stranded RNA homopolymer (naked ss polyU), ssRNA derived from HIV-1 LTR (ssRNA40), ssRNA having a 2GUCCUUCAA repeat sequence (ssRNA-DR)), imidazoquinoline compounds, imiquimod, Imiquimod VacciGrade™, Gardiquimod VacciGrade™, Gardiquimod™, adenine analog CL264, base analog CL307, guanosine analog roquinimex, TL8-506, thiazoloquinoline compound CL075, imidazoquinoline compound CL097, 2Bxy, R848, R848 VacciGrade™, CpG ODN, and / or one or more of Toxoplasma gondii profilin, or may consist of them. In some aspects, any agonist may be excluded. TLR agonists may also include the TLR agonists described herein.

[0011] The TLR agonist may be a TLR7 / 8 agonist. The TLR7 / 8 agonist may be R848. The TLR agonist may be a TLR5 agonist. The TLR5 agonist may be flagellin. The TLR agonist may be a TLR9 agonist. The TLR9 agonist may be a CpG oligonucleotide. The CpG oligonucleotide may include or consist of CpG 1826.

[0012] In the methods of the present disclosure, one or more cytokines include one or more (or any combination thereof) of IL-12p40, IP-10, IL-1β, CCL4, TNF-α, and IFN-β. One or more of the cytokines IL-12p40, IP-10, IL-1β, CCL4, TNF-α, and IFN-β may be excluded.

[0013] The method may further include the step of administering to a subject a further cancer therapy, and may further include the step of administering to a subject a further cancer therapy. The further cancer therapy may include chemotherapy, radiotherapy, immunotherapy, or a combination thereof. The further cancer therapy may include immunotherapy. The subject may be a human subject. The subject may be a non-human primate, an experimental animal, a mammal, a rat, a dog, a pig, a horse, a mouse, a rabbit, a goat, or a cat. The subject may be a subject not diagnosed with cancer. The subject may be a subject diagnosed with cancer. The subject may be a subject previously treated by therapy for cancer. The subject may be in remission. The subject may be a subject confirmed to be resistant to a previous therapy. The pharmaceutical composition may be administered intratumorally to the subject.

[0014] The composition may be administered to the subject by intramucosal administration, intramuscular administration, parenteral administration, or subcutaneous administration. The adjuvant of (a), the compound of (b), and / or the antigen may be administered by intramucosal administration, intramuscular administration, parenteral administration, or subcutaneous administration. The adjuvant of (a), the compound of (b), and / or the antigen may be administered in the same composition or in separate compositions.

[0015] The method may be a method for preventing a disease in a subject. The method may be a method for treating a disease in a subject.

[0016] The method and composition may be combined with vaccine compositions known in the art, such as current influenza vaccines, hepatitis B vaccines, or Covid vaccines. Specific examples include Fluzone and Heplisav, but others are applicable. The vaccine composition may be provided in a separate composition or within the same composition. The vaccine composition may be provided before, after, or substantially simultaneously with the composition of the present disclosure.

[0017] The compounds or compositions disclosed herein may be formulated for mucosal administration, intramuscular administration, parenteral administration, or subcutaneous administration. The composition may further comprise a pharmaceutical excipient.

[0018] Vaccine preparations as active immunogenic components may be prepared as injection solutions, either liquid solutions or suspensions. Solid forms suitable for solutions or suspensions that dissolve in a liquid prior to infection can also be prepared. The preparations may be emulsified and encapsulated in liposomes. The active immunogenic components are often mixed with a pharmaceutically acceptable carrier that is compatible with the active components.

[0019] To maximize delivery of the antigen to the site for maximum (or in some cases minimum) immune response, as long as the target tissue is accessible via any common route, administration of the vaccines according to the present disclosure may be via that route. Administration is generally intralesional injection, intradermal injection, mucosal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, or intravenous injection. Other areas for delivery include oral, nasal, buccal, rectal, vaginal, or topical. The vaccines of the present invention are preferably administered parenterally by injection, for example, subcutaneously or intramuscularly.

[0020] The vaccine can be administered in a manner compatible with the dosage formulation and in an amount effective for prevention and / or treatment. The amount administered will depend on, for example, the ability of the subject's immune system to synthesize antibodies and the degree of protection or treatment desired, for the subject to be treated. A suitable dosage range is on the order of several hundred micrograms of the active ingredient per single vaccine inoculation, in the range of about 0.1 mg to 1000 mg, for example, in the range of about 1 mg to 300 mg, or in the range of about 10 mg to 50 mg. The regimen suitable for the initial dose and booster injections can also vary, but is represented by the initial dose followed by subsequent inoculations or other administrations. The exact amount of active ingredient required for administration will depend on the judgment of the person skilled in the art and may be specific to each subject. The therapeutically effective amount of the nucleic acid molecule or fusion polypeptide of the present invention will depend, inter alia, on the dosing schedule, the unit dose of the antigen administered, whether the vaccine composition is administered in combination with other therapeutic agents, and the immune and health status of the recipient, as will be apparent to those skilled in the art.

[0021] The vaccine may be given in a single-dose schedule or in a multiple-dose schedule. A multiple-dose schedule involves a primary course of vaccine inoculations that includes, for example, one, two, three, four, five, six, seven, eight, nine, or ten separate doses, followed by, for subsequent doses, intervals between subsequent doses necessary to maintain and / or reinforce the immune response, for example, one month, two months, three months, or four months, and, if necessary, several months later for other doses. To maintain a desired level of protective immunity, periodic boosters at intervals of one year, two years, three years, four years, five years, for example, three years, are desirable.

[0022] The vaccine may be provided in one or more "unit doses". A unit dose is defined as containing a predetermined amount of vaccine calculated to produce a desired response in relation to its administration, i.e., the appropriate route and treatment regimen. The amount administered, as well as the specific route and prescription, are within the skill of those in the clinical art. The subject to be treated, particularly the immune system and protection status of the desired subject, may also be evaluated. A unit dose need not be administered in a single injection and may include continuous infusion over a predetermined period. The unit dose of the present invention may conveniently be expressed in mg / kg body weight. The dose of NFkB inhibitor, adjuvant, or antigen is at least 0.05 mg / kg, 0.10 mg / kg, 0.15 mg / kg, 0.20 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, 1,000 mg / kg, or any range derivable therein, at most 0.05 mg / kg, 0.10 mg / kg, 0.15 mg / kg, 0.20 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, 1,000 mg / kg, or any range derivable therein, or about 0.05 mg / kg, 0.10 mg / kg, 0.15 mg / kg, 0.20 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, 1,000 mg / kg, or any range derivable therein It may also be so. Similarly, the amount of vaccine delivered to an individual in vivo may be about 0.2 to about 8.0 mg / kg body weight, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.8 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5.0 mg / kg, 5.5 mg / kg, 6.0 mg / kg, 6.5 mg / kg, 7.0 mg / kg, and 7.5 mg / kg (or any range derivable therefrom). The dosage of the vaccine administered is highly dependent on the body weight and physical condition of the subject being treated as well as the route of administration and the frequency of treatment.

[0023] The method of the present disclosure may include the step of administering one or more compositions two or more times. The compositions may be administered at intervals of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, and / or 14 days, and / or at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, and / or 52 weeks, and / or at intervals of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 36 months, 48 months, 60 months, 72 months, 84 months, or 96 months, and / or at intervals of 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, or 20 years (or at intervals of any range derivable therefrom).

[0024] Kits comprising a composition of the present disclosure and instructions for use are also described.

[0025] The method may further comprise the step of testing a patient for an infectious disease such as a viral infection, or diagnosing a patient with an infectious disease such as a viral infection.

[0026] Any aspect discussed in the context of the composition can be implemented in any aspect of the methods discussed herein.

[0027] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect can also be described in claim language such as "Use of" any compound, composition, or agent discussed herein for achieving or effecting the therapeutic, diagnostic, or physiological purpose or effect described.

[0028] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, while the detailed description and specific examples illustrate preferred embodiments of the present invention, it is to be understood that these are by way of illustration only and that various modifications and changes within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description.

Brief Description of the Drawings

[0029] The following drawings form a part of this specification and are included to further demonstrate certain specific aspects of the present invention. The present invention can be more deeply understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments shown herein.

[0030]

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Mode for Carrying Out the Invention

[0031] Description of Exemplary Embodiments Imidazoquinoline derivatives that activate Toll-like receptor (TLR) 7 / 8 are small molecule immune potentiators (SMIPs) that have potent activity as vaccine adjuvants and as anti-tumor agents. However, these molecules have high bioavailability that results in unacceptable levels of systemic inflammation due to adjuvant toxicity, and thus their use is greatly restricted.

[0032] Small molecule NF-κB inhibitors can be used to enhance cytidine phosphate guanosine (CPG, a TLR 9 agonist) in vaccine formulations. Capsaicin and honokiol, which are small molecule NF-κB inhibitors, have been shown to reduce pro-inflammatory systemic IL-6 and TNF-α levels while maintaining the vaccine protective effect. 19 However, this effect was not observed when in vivo experiments were repeated using R848 as an adjuvant. This is likely due to the high diffusion of small molecule adjuvants and immune potentiators.

[0033] The present disclosure is at least partially based on the design of hybrid molecules in which vanilloids, catechols, and honokiol 19 derivatives are covalently shared with imidazoquinolone derivatives 20 via linkable amine handles to reduce the degree of diffusion. Using in vitro assays, a mini-library of synthetic dimers was screened, and viable candidates were selected for further in vivo experiments. Mice were vaccinated with ovalbumin as a model antigen treated with the synthetic dimer. From the results, these dimers were demonstrated to reduce systemic toxicity to baseline levels while maintaining adjuvant activity in vaccine formulations. Furthermore, the selected dimers enhanced viability while inducing low adjuvant toxicity in a CT26 WT mouse colon cancer tumor model.

[0034] I. Definitions As used herein, the term "adjuvant" refers to a substance that, when administered before, together with, or after an antigen, accelerates, prolongs, and / or enhances the quality and / or strength of the immune response to the antigen as compared to administration of the antigen alone.

[0035] As used herein, the term "vaccine" describes a composition that can be administered to a human or animal to induce an immune system response. This immune system response may result in antibody production or simply result in the activation of certain cells, such as antigen-presenting cells, T lymphocytes, and / or B lymphocytes. A vaccine may be able to produce an immune response in a patient that results in the production of neutralizing antibodies against the antigen provided in the vaccine. A vaccine may be a composition for prophylactic purposes, a composition for therapeutic purposes, or a composition for both purposes.

[0036] As used herein, the term "antigen" refers to any antigen that can be used in a vaccine, whether the vaccine is accompanied by the whole microorganism or a part thereof, and various types (e.g., peptides, proteins, glycoproteins, polysaccharides, glycolipids, lipopeptides, etc.). Thus, the term "antigen" refers to a molecule that can initiate a humoral immune response and / or a cellular immune response in the recipient of the antigen. The antigen may also be a molecule that causes a disease for which vaccination may be a beneficial treatment. The antigen may be a substance, its product, or a synthetic substitute prepared from an agent causing the disease and processed to act as an antigen without inducing the disease, which stimulates the production of antibodies and confers immunity against one or several diseases. The antigen may contain peptides or polypeptides.

[0037] The term "pharmaceutically acceptable carrier" refers to a carrier that does not cause allergic reactions or other adverse effects in the subject to which it is administered. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol or the like, and combinations thereof. Further, if desired, the vaccine may contain trace amounts of auxiliary substances, such as wetting agents or emulsifying agents, and pH buffering agents.

[0038] As used herein, the term "agonist" refers to a molecule that can produce a cellular response when it binds to a receptor. The agonist may be a ligand that binds directly to the receptor. Alternatively, the agonist may bind indirectly to the receptor, for example, by (a) forming a complex with another molecule that binds directly to the receptor, or (b) otherwise modifying another molecule so that another molecule binds directly to the receptor. An agonist may be called an agonist of a specific receptor or receptor family (e.g., a TLR agonist).

[0039] The terms "individual", "subject", and "patient" are used synonymously and may refer to a human or a non-human.

[0040] As used herein, the terms "a" or "an" may mean one or more. When used in the claims herein, the terms "a" or "an" may mean one or more when used in conjunction with the word "comprising".

[0041] The use of the term "or" in the claims is used to mean "and / or" unless explicitly stated to refer only to alternatives or to alternatives that are mutually exclusive, although this disclosure supports a definition that refers only to alternatives and "and / or". As used herein, the term "another" may mean at least a second or more.

[0042] Throughout this application, the term "about" is used to indicate that a value includes the inherent variability of the device, method by which the value is determined, or the variability that exists between the subjects being studied.

[0043] The term "consisting essentially of" may include the recited active ingredients, e.g., the described dimers, and may also include any buffer, pharmaceutical excipient, etc. not described, but excludes any other active ingredients such as other hybrid molecules.

[0044] Antigen As used herein, the term "antigen" refers to a molecule capable of initiating a humoral and / or cellular immune response in a subject. Antigens can be any type of biological molecule, including, for example, simple intermediary metabolites, sugars, lipids, and hormones, as well as macromolecules such as complex carbohydrates, phospholipids, nucleic acids, and proteins. General categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoan and other parasite antigens, tumor antigens, antigens involved in autoimmune diseases, allergies, and transplant rejection, as well as other various miscellaneous antigens. In certain compositions and methods of the present disclosure, the antigen is a peptide.

[0045] The inventors have demonstrated that the hybrid molecules disclosed herein reduce systemic toxicity to baseline levels while maintaining adjuvant activity in vaccine formulations. Antigens useful in the methods and compositions of the present disclosure include, for example, Bacillus anthracis, cancer, chikungunya, dengue (1, 2, 3, 4-dengue), diphtheria, Escherichia coli, Shiga toxin-producing (STEC), Ebola, non-polio enterovirus, enterovirus D68 (EV-D68), gonorrhea, hepatitis A (HepA), hepatitis B (HepB), hepatitis C (HepC), hepatitis D (HepD), hepatitis E (HepE), herpes, herpes zoster, HIV, HPV, influenza, malaria, measles, viral meningitis, bacterial meningitis, mumps, norovirus, pertussis, plague; bubonic, septicemic, pneumonic, pneumococcal disease, poliomyelitis (polio), pustular disease (smallpox, monkey pox, cowpox), Q fever, rabies, Salmonellosis gastroenteritis (Salmonella), severe acute respiratory syndrome, bacterial dysentery gastroenteritis (Shigella), smallpox, tetanus, tuberculosis, varicella (chickenpox), viral hemorrhagic fever (Ebola, Lassa, Marburg), West Nile virus, yellow fever, Yersenia (Yersinia), and antigenic components derived from Zika virus infection. It is contemplated that one or more of the antigens and antigenic components listed in this paragraph may be specifically excluded.

[0046] Further examples of antigens useful in the methods and compositions of the present disclosure are provided below and throughout the present disclosure.

[0047] A. Viral Antigens Examples of viral antigens include retroviral antigens, such as retroviral antigens derived from human immunodeficiency virus (HIV) antigens, such as gene products of the gag, pol, and env genes, Nef protein, reverse transcriptase, and other HIV components; hepatitis virus antigens, such as the S, M, and L proteins of hepatitis B virus, hepatitis B virus, and pre-S antigens, viral components of other hepatitis, such as hepatitis A, B, and C, such as hepatitis C virus RNA; influenza virus antigens, such as hemagglutinin and neuraminidase and other influenza virus components; measles virus antigens, such as the measles virus fusion protein and other measles virus components; rubella virus antigens, such as proteins E1 and E2 and other rubella virus components; rotavirus antigens, such as VP7sc and other rotavirus components; cytomegalovirus antigens, such as envelope glycoprotein B and other cytomegalovirus antigen components; respiratory syncytial viral antigen, such as RSV fusion protein, M2 protein and other respiratory syncytial viral antigen components; herpes simplex virus antigens, such as immediate early proteins, glycoprotein D, and other herpes simplex virus antigen components; varicella-zoster virus antigens, such as gpI, gpII, and other varicella-zoster virus antigen components; Japanese encephalitis virus antigens, such as protein E, M-E, M-E-NS 1, NS 1, NS 1-NS2A, 80%E, and other Japanese encephalitis virus antigen components; rabies virus antigens, such as rabies glycoprotein, rabies nucleoprotein and other rabies virus antigen components, but are not limited thereto. For further examples of viral antigens, see Fundamental Virology, Second Edition, eds. Fields, B. N. and Knipe, D. M. (Raven Press, New York, 1991). It is intended that one or more of the antigens and antigenic components listed in this paragraph may be specifically excluded.

[0048] B. Bacterial antigens Bacterial antigens that can be used in the compositions and methods of the present disclosure include Bordetella pertussis antigens such as pertussis toxin, filamentous hemagglutinin, pertactin, FIM2, FIM3, adenylate cyclase, and other Bordetella pertussis antigen components; Corynebacterium diphtheriae antigens such as diphtheria toxin or toxoid and other Corynebacterium diphtheriae antigen components; Clostridium tetani antigens such as tetanus toxin or toxoid and other Clostridium tetani antigen components; Streptococcus bacterial antigens such as M protein and other Streptococcus bacterial antigen components; Gram-negative bacillus bacterial antigens such as lipopolysaccharide and other Gram-negative bacterial antigen components; Mycobacterium tuberculosis bacterial antigens such as mycolic acid, heat shock protein 65 (HSP65), 30 kDa major secreted protein, antigen 85A, and other mycobacterial antigen components; Helicobacter pylori bacterial antigen components; Streptococcus pneumoniae bacterial antigens such as pneumolysin, Streptococcus pneumoniae capsular polysaccharide, and other Streptococcus pneumoniae bacterial antigen components; Haemophilus influenzae bacterial antigens such as capsular polysaccharide and other Haemophilus influenzae bacterial antigen components; Bacillus anthracis bacterial antigens such as Bacillus anthracis protective antigen and other Bacillus anthracis bacterial antigen components; Rickettsia bacterial antigens such as romps and other Rickettsia bacterial antigen components, but are not limited thereto. Along with the bacterial antigens described herein, antigens of any other bacteria, mycobacteria, mycoplasmas, rickettsias, or chlamydias are also included. It is contemplated that one or more of the antigens and antigenic components listed in this paragraph may be specifically excluded from the methods and compositions of the present disclosure.

[0049] C. Fungal antigens Fungal antigens that can be used in the compositions and methods of the present disclosure include Candida fungal antigen components; Histoplasma fungal antigens, such as heat shock protein 60 (HSP60) and other Histoplasma fungal antigen components; Cryptococcus fungal antigens, such as capsular polysaccharide and other Cryptococcus fungal antigen components; Coccidiodes fungal antigens, such as spherule antigen and other Coccidiodes fungal antigen components; and dermatophyte fungal antigens, such as trichophytin and other Coccidiodes fungal antigen components, but are not limited thereto. It is contemplated that one or more of the antigens and antigenic components listed in this paragraph may be specifically excluded from the methods and compositions of the present disclosure.

[0050] D. Parasite antigens Examples of protozoan and other parasite antigens include Plasmodium falciparum antigens, such as merozoite surface antigen, sporozoite surface antigen, circumsporozoite antigen, gametocyte / gamete surface antigen, blood-stage antigen pf 155 / RESA, and other Plasmodium antigen components; Toxoplasma antigens, such as SAG-1, p30, and other Toxoplasma antigen components; Schistosoma antigens, such as glutathione-S-transferase, paramyosin, and other Schistosoma antigen components; Leishmania amazonensis and other Leishmania antigens, such as gp63, lipophosphoglycan, and related proteins, and other Leishmania antigen components; and Trypanosoma cruzi antigens, such as 75-77 kDa antigen, 56 kDa antigen, and other Trypanosoma antigen components, but are not limited thereto. It is contemplated that one or more of the antigens and antigenic components listed in this paragraph may be specifically excluded from the methods and compositions of the present disclosure.

[0051] E. Tumor antigens Tumor antigens that can be used in the compositions and methods of the present disclosure include, but are not limited to, telomerase components; multidrug resistance proteins, such as P-glycoprotein; MAGE-1, alpha-fetoprotein, carcinoembryonic antigen, mutant p53, immunoglobulins from B-cell malignancies, fusion polypeptides expressed from genes juxtaposed by chromosomal translocations, human chorionic gonadotropin, calcitonin, tyrosinase, papillomavirus antigens, gangliosides or other carbohydrate-containing components of melanoma or other tumor cells. It is contemplated by the present disclosure that antigens derived from any type of tumor cell can be used in the compositions and methods described herein. It is contemplated that one or more of the antigens and antigenic components listed in this paragraph may be specifically excluded in the methods and compositions of the present disclosure.

[0052] F. Antigens associated with autoimmunity Antigens involved in autoimmune diseases, allergies, and graft rejection reactions can be used in the compositions and methods of the present disclosure. For example, the following autoimmune diseases or disorders: diabetes, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's syndrome including keratoconjunctivitis sicca secondary to Sjogren's syndrome, alopecia areata, allergic reactions resulting from arthropod bite reactions, Crohn's disease, aphthous ulcers, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug rashes, reversal reactions of leprosy, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, erythroleukemia, idiopathic thrombocytopenia, relapsing polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Crohn's disease, Graves' ophthalmopathy, sarcoidosis, primary biliary cirrhosis, posterior uveitis, and interstitial pulmonary fibrosis, can be used in the present disclosure. Examples of antigens involved in autoimmune diseases include glutamic acid decarboxylase 65 (GAD65), native DNA, myelin basic protein, myelin proteolipid protein, acetylcholine receptor components, thyroglobulin, and thyroid-stimulating hormone (TSH) receptors. Examples of antigens involved in allergies include pollen antigens, such as Japanese cedar pollen antigen, ragweed pollen antigen, ryegrass pollen antigen, animal-derived antigens, such as dust mite antigen and cat antigen, histocompatibility antigens, as well as penicillin and other therapeutic drugs. Examples of antigens involved in graft rejection reactions include antigenic components of grafts transplanted into graft recipients, such as graft components of the heart, lung, liver, pancreas, kidney, and nerve. The antigen may also be an altered peptide ligand useful in the treatment of autoimmune diseases. It is intended that one or more of the antigens and antigenic components listed in this paragraph be specifically excluded from the methods and compositions of the present disclosure.It is further intended that self-antigens can be specifically excluded in the methods and compositions of the present disclosure.

[0053] Examples of various heterogeneous antigens that can be used in the compositions and methods of the present disclosure include endogenous hormones such as luteinizing hormone, follicle-stimulating hormone, testosterone, growth hormone, prolactin, and other hormones, addictive drugs such as cocaine and heroin, and idiotypic fragments of antigen receptors such as the Fab-containing portion of an anti-leptin receptor antibody.

[0054] II. Adjuvant Aspects of the present disclosure include adjuvants and methods for administering an adjuvant to a subject. The immunogenicity of a particular composition can be enhanced by using a non-specific stimulant of the immune response known as an adjuvant. As used herein, an "adjuvant" refers to a substance that, when administered before, together with, or after an antigen, accelerates, prolongs, and / or enhances the quality and / or strength of the immune response to the antigen as compared to administration of the antigen alone. Adjuvants that can be used according to the aspects include, but are not limited to, IL-1, IL-2, IL-4, IL-7, IL-12, gamma-interferon, GM-CSF, BCG, aluminum hydroxide, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). Other exemplary adjuvants may include complete Freund's adjuvant (a non-specific immune response stimulant containing heat-killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and / or aluminum hydroxide adjuvant.

[0055] In some aspects, the adjuvants of the present disclosure are TLR agonists or pattern recognition receptor (PRR) agonists. The use of TLR agonists as adjuvants is described, for example, in Li et al., TLR Agonists as Adjuvants for Cancer Vaccines. Adv Exp Med Biol. 2017;1024:195-212, which is incorporated herein by reference in its entirety. A PRR agonist describes any molecule that directly or indirectly activates a PRR or stimulates PRR signaling. PRRs include cell surface receptors (e.g., toll-like receptor (TLR) agonists) and intracellular receptors (e.g., RIG-I-like receptors). Examples of PRRs that can be targeted by the agonists of the present disclosure include NOD-like receptors, RIG-I-like receptors, STING receptors, and toll-like receptors. In some embodiments, PRR agonists are disclosed herein, and the PRR agonist is a NOD-like receptor agonist, a RIG-I-like receptor agonist, a STING agonist, or a TLR agonist. In some embodiments, the PRR agonist of the present disclosure is a TLR agonist.

[0056] Aspects of the present disclosure relate to TLR agonists comprising a polymer comprising a TLR agonist or a derivative thereof. A TLR agonist can be any molecule that directly or indirectly activates a TLR and / or stimulates TLR signaling. In some cases, a TLR agonist is a molecule that binds directly to a TLR. In some aspects, an immunomodulator is disclosed that comprises one or more TLR agonists linked by a polypeptide backbone.

[0057] In some embodiments, the TLR agonist is a TLR agonist known in the art and / or described herein. TLR agonists include TLR1 (e.g., peptidoglycan or triacylated lipoproteins), TLR2 (e.g., lipoteichoic acid; peptidoglycan derived from Bacillus subtilis, Escherichia coli 0111:B4, Escherichia coli K12, or Staphylococcus aureus; atypical lipopolysaccharide (LPS), such as leptospiral LPS and Porphyromonas gingivalis LPS; synthetic diacylated lipoproteins, such as FSL-1 or Pam2CSK4; lipoarabinomannan or lipomannan derived from M. smegmatis; triacylated lipoproteins, such as Pam3CSK4; lipoproteins, such as MALP-2 and MALP-404 derived from Mycoplasma; Borrelia burgdorferi OspA; porins derived from Neisseria meningitidis or Haemophilus influenzae; Propionibacterium acnes antigen mixture; Yersinia pestis LcrV; lipomannan derived from Mycobacterium or Mycobacterium tuberculosis; Trypanosoma cruzi GPI anchor; Schistosoma mansoni lysophosphatidylserine; Leishmania major lipophosphoglycan (LPG); Plasmodium falciparum glycophosphatidylinositol (GPI); zymosan; antigen mixture derived from Aspergillus fumigatus or Candida albicans; and measles hemagglutinin), TLR3 (e.g., double-stranded RNA, polyadenylic acid-polyuridylic acid (poly(A:U)); polyinosinic acid:polycytidylic acid (poly(I:C)); polyinosinic acid:polycytidylic acid high molecular weight (poly(I:C)HMW); and polyinosinic acid:polycytidylic acid low molecular weight (poly(I:C)LMW)), TLR4 (e.g., LPS derived from Escherichia coli and Salmonella species), TLR5 (e.g., Bacillus subtilis, P. aeruginosa, or S.Flagellin derived from Chlamydia muridarum), TLR8 (e.g., single-stranded RNA, e.g., ssRNA having a 6UUAU repeat sequence, RNA homopolymer (naked ss polyU), HIV-1 LTR-derived ssRNA (ssRNA40), or ssRNA having a 2GUCCUUCAA repeat sequence (ssRNA-DR)), TLR7 (e.g., imiquimod, an imidazoquinoline compound, Imiquimod VacciGrade™, Gardiquimod VacciGrade™, or Gardiquimod™; adenine analog CL264; base analog CL307; guanosine analog roquinimex; TLR7 / 8 (e.g., thiazoloquinoline compound CL075; imidazoquinoline compounds CL097, 2Bxy, R848, or R848 VacciGrade™), TLR9 (e.g., CpG ODNs); and agonists for TLR11 (e.g., Toxoplasma gondii profilin) may be included. In some embodiments, the TLR agonist is an amphiphilic TLR agonist. In some embodiments, the TLR agonist is a TLR2 / 6 agonist, e.g., Pam2CSK4 or Pam3CSK4. In some embodiments, the TLR agonist is a hydrophobic TLR agonist. In some embodiments, the TLR agonist is a TLR7, TLR8, or TLR7 / 8 agonist, e.g., 2Bxy or an imidazoquinoline. In some aspects, the TLR agonist of the present disclosure is an imidazoquinoline. In certain embodiments, the TLR agonist is a specific agonist listed above. Derivatives of any of the TLR agonists listed above are also contemplated herein. The TLR agonists of the present disclosure include any derivatives of the molecules listed above having TLR agonist activity. It is further contemplated that any TLR agonist disclosed herein may be specifically excluded in the methods and compositions of the present disclosure..

[0058] In a further aspect, the TLR agonist specifically stimulates one type of TLR or two types of TLRs. In some aspects, the linked TLR agonist comprises different types of TLR agonists (e.g., TLR agonists capable of activating different classes of TLRs). Alternatively, the linked TLR agonist may comprise the same type of TLR agonist.

[0059] In one aspect, small molecule compounds suitable for use as TLR agonists are disclosed herein. Examples of small molecule TLR agonists include compounds having a 2-aminopyridine fused to a 5-membered nitrogen-containing heterocycle.Such compounds include, for example, substituted imidazoquinolineamines such as aminoalkyl-substituted imidazoquinolineamines, amide-substituted imidazoquinolineamines, sulfonamide-substituted imidazoquinolineamines, urea-substituted imidazoquinolineamines, aryl ether-substituted imidazoquinolineamines, heterocyclic ether-substituted imidazoquinolineamines, amide ether-substituted imidazoquinolineamines, sulfonamide ether-substituted imidazoquinolineamines, urea-substituted imidazoquinoline ethers, and thioether-substituted imidazoquinolineamines, but are not limited thereto; tetrahydroimidazoquinolineamines such as amide-substituted tetrahydroimidazoquinolineamines, sulfonamide-substituted tetrahydroimidazoquinolineamines, urea-substituted tetrahydroimidazoquinolineamines, aryl ether-substituted tetrahydroimidazoquinolineamines, heterocyclic ether-substituted tetrahydroimidazoquinolineamines, amide ether-substituted tetrahydroimidazoquinolineamines, sulfonamide ether-substituted tetrahydroimidazoquinolineamines, urea-substituted tetrahydroimidazoquinoline ethers, and thioether-substituted tetrahydroimidazoquinolineamines, but are not limited thereto; imidazopyridineamines such as amide-substituted imidazopyridineamines, sulfonamide-substituted imidazopyridineamines, urea-substituted imidazopyridineamines, but are not limited thereto; aryl ether-substituted imidazopyridineamines, heterocyclic ether-substituted imidazopyridineamines, amide ether-substituted imidazopyridineamines, sulfonamide ether-substituted imidazopyridineamines, urea-substituted imidazopyridine ethers, and thioether-substituted imidazopyridineamines; 1,2-bridged imidazoquinolineamines; 6,7-fused cycloalkylimidazopyridineamines; imidazonaphthyridineamines; tetrahydroimidazonaphthyridineamines; oxazolquinolineamines; thiazolquinolineamines; oxazolopyridineamines; thiazolopyridineamines; oxazolonaphthyridineamines; and thiazolonaphthyridineamines are included.

[0060] In certain embodiments, the TLR agonist is an imidazonaphthyridinamine, tetrahydroimidazonaphthyridinamine, oxazoloquinolinamine, thiazoloquinolinamine, oxazolopyridinamine, thiazolopyridinamine, oxazolonaaphthyridinamine, or thiazolonaaphthyridinamine.

[0061] In certain embodiments, the TLR agonist is a sulfonamide-substituted imidazoquinolinamine. In alternative embodiments, the TLR agonist may be a urea-substituted imidazoquinoline ether. In another alternative embodiment, the TLR agonist may be an aminoalkyl-substituted imidazoquinolinamine. In one particular embodiment, the TLR agonist is 4-amino-α,α,2-trimethyl-1H-imidazo[4,5-c]quinolin-1-ethanol. In an alternative particular embodiment, the TLR agonist is N-(2-{2-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl]ethoxy}ethyl)-N-methylmorpholine-4-carboxamide. In another alternative embodiment, the TLR agonist is 1-(2-amino-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-amine. In another alternative embodiment, the TLR agonist is N-[4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide. In yet another alternative embodiment, the TLR agonist is N-[4-(4-amino-2-propyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide.

[0062] In certain embodiments, the TLR agonist may be a substituted imidazoquinolinamine, tetrahydroimidazoquinolinamine, imidazopyridinamine, 1,2-bridged imidazoquinolinamine, 6,7-fused cycloalkylimidazopyridinamine, imidazonaphthyridinamine, tetrahydroimidazonaphthyridinamine, oxazoloquinolinamine, thiazoloquinolinamine, oxazolopyridinamine, thiazolopyridinamine, oxazolonaaphthyridinamine, or thiazolonaaphthyridinamine.

[0063] As used herein, a substituted imidazoquinolineamine refers to an aminoalkyl-substituted imidazoquinolineamine, an amide-substituted imidazoquinolineamine, a sulfonamide-substituted imidazoquinolineamine, a urea-substituted imidazoquinolineamine, an aryl ether-substituted imidazoquinolineamine, a heterocyclic ether-substituted imidazoquinolineamine, an amide ether-substituted imidazoquinolineamine, a sulfonamide ether-substituted imidazoquinolineamine, a urea-substituted imidazoquinoline ether, or a thioether-substituted imidazoquinolineamine.

[0064] III. Pharmaceutical Compositions Administration of the composition is typically via any common route. This includes, but is not limited to, parenteral injection, topical injection, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intranasal injection, or intravenous injection. The vaccine composition may be inhaled (e.g., U.S. Patent No. 6,651,655, which is hereby specifically incorporated by reference). Further formulations suitable for other administration methods include oral formulations. Oral formulations contain commonly used excipients such as, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain from about 10% to about 95%, for example from about 25% to about 70%, of the active ingredient.

[0065] Typically, the composition is administered in a manner compatible with the dosage formulation and in an amount effective for treatment and immunomodulation. The amount administered depends on the subject to be treated. The exact amount of active ingredient required for administration depends on the judgment of the person skilled in the art.

[0066] The method of administration can be widely variable. Any conventional method for administering an antibody can be applied. These are considered to include oral application, such as attached to a solid physiologically acceptable base or dissolved in a physiologically acceptable dispersion, and parenteral application such as by injection. The dosage of the pharmaceutical composition depends on the route of administration and varies according to the size and health status of the subject.

[0067] In many cases, it is desirable to have multiple administrations, at most about 3, 4, 5, 6, 7, 8, 9, 10, or more times, or at least about 3, 4, 5, 6, 7, 8, 9, 10, or more times. The administrations may be at intervals of 2 days to 12 weeks, more usually at intervals of 1 week to 2 weeks. After a course of administration, assays of alloreactive immune responses and T cell activity may be performed.

[0068] The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse reactions, allergic reactions, or other untoward reactions when administered to an animal or human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Its use in immunogenic and therapeutic compositions is contemplated, except where any conventional media or agent is incompatible with the active ingredient. The pharmaceutical compositions of the present disclosure are pharmaceutically acceptable compositions.

[0069] The hybrid molecule can be formulated for parenteral administration, for example, for injection via intravenous, intradermal, intramuscular, subcutaneous routes, or for injection via intraperitoneal route. The composition may be administered by intradermal injection. The composition may be administered by intravenous injection. The composition may be administered by intramuscular injection. The composition of the present disclosure can be prepared as an injection solution either in a liquid solution or a suspension. A solid form suitable for use in preparing a solution or suspension by adding a liquid before injection can also be prepared. The preparation can also be emulsified.

[0070] Pharmaceutical forms suitable for injection use include sterilized aqueous solutions or dispersions; preparations containing sesame oil, peanut oil, or aqueous propylene glycol solutions; and sterilized powders for immediate preparation of sterilized injection solutions or dispersions. In all cases, the form must be sterile and must have sufficient fluidity to be easily injectable. The form must also be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

[0071] The composition may be formulated in a neutral form or in the form of a salt. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of proteins), which are formed using, for example, inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid. Salts formed with free carboxyl groups can also be obtained from, for example, inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine.

[0072] The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycols, etc.), suitable mixtures thereof, and vegetable oils. The activity of microorganisms can be inhibited by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include an isotonic agent, such as sugar or sodium chloride. The long-term absorption of injectable compositions can be achieved by using absorption delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0073] Sterile injectable solutions are prepared by incorporating the required amount of the active ingredient, optionally together with the various other ingredients described above, into a suitable solvent and then filtering and sterilizing. Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and the necessary other ingredients derived from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield powders of the active ingredient and any additional desired ingredients from a previously filtered and sterilized solution.

[0074] An effective amount of a therapeutic or prophylactic composition is determined based on the intended purpose. The terms "unit dose" or "dosage" refer to physically discrete units suitable for use in a subject, each unit containing a predetermined amount of the composition calculated to produce the desired response discussed above in relation to administration, i.e., via a suitable route and regimen. The amount administered depends on the number of treatments and the unit dose, and is influenced by the desired result and / or protection. The exact amount of the composition also depends on the judgment of the practitioner and is specific to each subject. Factors affecting the dosage include the physical and clinical state of the subject, the route of administration, the intended purpose of the treatment (symptom relief versus cure), as well as the efficacy, stability, and toxicity of the particular composition. Once formulated, the solution is administered in a manner compatible with the dosage form and in an amount effective for treatment or prevention. The formulations are readily administered in various dosage forms, such as the injectable solution type described above.

[0075] IV. Treatment Methods As discussed above, the compositions and methods of using these compositions can treat a subject having an infectious disease, cancer, or a related disease, suspected of having an infectious disease, cancer, or a related disease, having one or more symptoms of an infectious disease, cancer, or a related disease, or at risk of developing an infectious disease, cancer, or a related disease (e.g., can prevent an infectious disease, can elicit a strong immune response against an antigen, or can reduce or prevent tumor growth).

[0076] As used herein, the term "immune response" or its equivalent "immunological response" refers to a humoral (antibody-mediated) response, a cellular response (mediated by antigen-specific T cells or their secreted products), or both a humoral response and a cellular response directed against the proteins, peptides, or polypeptides of the invention in a recipient patient. Treatment or therapy may be an active immune response induced by administration of an immunogen or a passive therapy caused by administration of an antibody, antibody-containing material, or primed T cells.

[0077] The presence of a cellular immunological response can be confirmed by a proliferation assay (CD4(+) T cells) or a CTL (cytotoxic T lymphocyte) assay. The relative contributions of the humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating IgG and T cells from immunized syngeneic animals and measuring the protective or therapeutic effect in another subject. The terms "antibody" or "immunoglobulin" as used herein and in the claims are used synonymously.

[0078] Optionally, the antibody or preferably an immunological portion of the antibody may be chemically conjugated or expressed as a fusion protein with another protein. For the purposes of this specification and the appended claims, all such fusion proteins are included within the definition of an antibody or an immunological portion of an antibody.

[0079] The method may include treating or preventing a disease or condition caused by a pathogen. Further, in some examples, treatment includes administration of other agents commonly used against viral infections, such as one or more antiviral or antiretroviral compounds.

[0080] The therapeutic composition is administered in a manner compatible with the dosage formulation and in an amount effective for treatment. The amount administered is dependent on the subject to be treated. The exact amount of active ingredient required to be administered is dependent on the judgment of the skilled person. Appropriate regimes for the initial dose and booster immunizations may also vary, but are represented by the initial dose and subsequent, later doses.

[0081] The method of application may vary widely. Any conventional method for administering a polypeptide therapeutic agent can be applied. These are considered to include oral application, such as attached to a solid physiologically acceptable base or dissolved in a physiologically acceptable dispersion, and parenteral application by injection, etc. The dosage of the composition depends on the route of administration and varies according to the size and health status of the subject.

[0082] In certain cases, it is desirable to have multiple administrations of the composition, such as 2, 3, 4, 5, 6, or more administrations. The administrations may be spaced apart by intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks up to 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks, including all ranges within this.

[0083] Subjects include Approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 445, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 micrograms, mg, μg / kg, or mg / kg (or any range derivable therefrom), At least about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295,300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 445, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 micrograms, mg, μg / kg, or mg / kg (or any range derivable therefrom), or, At most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 445, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 micrograms, mg, μg / kg, or mg / kg (or any range derivable therefrom), a hybrid molecule or composition of may be administered.

[0084] The dosage may be administered as needed, every 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, or 24 hours (or any range derivable therein), or once, twice, three times, four times, five times, six times, seven times, eight times, nine times (or any range derivable therein) per day. Initially, the dosage may be administered before the signs of the condition or after the signs of the condition. The first dosage of the regimen may be administered to the patient 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours (or any range derivable therein), or 1 day, 2 days, 3 days, 4 days, or 5 days (or any range derivable therein) after the patient has experienced or exhibited signs or symptoms of the condition. The patient may be treated over 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more (or any range derivable therein), or until the symptoms of the condition disappear or are alleviated, or until 6 hours, 12 hours, 18 hours, or 24 hours after the symptoms of the infection have disappeared or are alleviated, or 1 day, 2 days, 3 days, 4 days, or 5 days later.

[0085] V. Combination Therapy The administration of the composition and related methods, particularly the composition comprising the composition of the present disclosure, may also be used in combination with the administration of one or more additional therapies.

[0086] The therapy may be used together with an antiviral treatment or an antiretroviral treatment. The therapy may be used together with a cancer treatment (e.g., chemotherapy, cancer immunotherapy, etc.). The therapy may be performed before other drug treatments at intervals ranging from several minutes to several weeks, or after other drug treatments. When other drugs and / or proteins or polynucleotides are applied separately, it is generally ensured that the effective period does not end during the time of each delivery so that the therapeutic composition can still exert an advantageous combined effect on the subject. In such cases, it is intended that both modalities may be administered within about 12 to 24 hours, for example, within about 6 to 12 hours after administering either modality. However, depending on the situation, it may be desirable to significantly extend the administration period. In this case, the period between each administration is several days (2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) to several weeks (1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks).

[0087] The vaccine may be administered as part of a prime / boost strategy. The prime vaccine dose can be administered by any method described herein. The vaccine boost can be administered using a second vaccine, either of the same type or a different type of vaccine. Examples of such different vaccines include naked DNA vaccines or recombinant poxviruses.

[0088] Various combinations of therapies can be used. As an example, where the adjuvant is "A" and the NFkB inhibitor is "B", A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A are exemplified.

[0089] Administration of the composition to a patient / subject follows the general protocol for administering such compounds, taking into account the toxicity of the composition if any. Treatment cycles are expected to be repeated as needed. It is also intended that various standard therapies such as hydration can be applied in combination.

[0090] VI. Cancer Therapy The disclosed method may include the step of administering cancer therapy to a subject or patient. The cancer therapy may include local cancer therapy. The cancer therapy may exclude systemic cancer therapy. The cancer therapy may exclude local therapy. The cancer therapy may include local cancer therapy without the administration of systemic cancer therapy. The cancer therapy may include the step of administering a dimer of the present disclosure. The cancer therapy may include radiation therapy. The cancer therapy may include chemotherapy. The cancer therapy may include immunotherapy which may be checkpoint inhibitor therapy. Any of these cancer therapies can be excluded. Combinations of these therapies can also be administered.

[0091] The term "cancer" as used herein may be used to describe solid tumors, metastatic cancer, or non-metastatic cancer. Cancer may occur in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gingiva, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. The cancer may be stage I cancer. The cancer may be stage II cancer. The cancer may be stage III cancer. The cancer may be stage IV cancer.

[0092] Cancer may specifically be, but is not limited to, cancers of the following histological types: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; carcinoma of giant and spindle cells; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobic carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenocortical carcinoma; endometroid carcinoma; carcinoma of skin appendages; apocrine adenocarcinoma; sebaceous gland carcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; theca cell tumor, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell tumor; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; chromaffin cell tumor; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; fetal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; müllerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma;Mesenchymal tumors, malignant; Brenner tumors, malignant; Phyllodes tumors, malignant; Synovial sarcoma; Mesotheliomas, malignant; Undifferentiated embryonal cell tumors; Embryonal carcinoma; Teratomas, malignant; Ovarian struma ovarii, malignant; Choriocarcinoma; Mesonephromas, malignant; Angiosarcoma; Hemangioendotheliomas, malignant; Kaposi's sarcoma; Pericytic tumors, malignant; Lymphangiosarcoma; Osteosarcoma; Parosteal osteosarcoma; Chondrosarcoma; Chondroblastomas, malignant; Mesenchymal chondrosarcoma; Giant cell tumors of bone; Ewing's sarcoma; Odontogenic tumors, malignant; Ameloblastic odontogenic sarcoma; Ameloblastic epitheliomas, malignant; Ameloblastic fibrosarcomas; Pinealomas, malignant; Chordomas; Gliomas, malignant; Ependymomas; Astrocytomas; Protoplasmic astrocytomas; Fibrous astrocytomas; Astroblastomas; Glioblastomas; Oligodendrogliomas; Oligodendroblastomas; Undifferentiated neuroectodermal; Cerebellar sarcoma; Ganglioneuroblastomas; Neuroblastomas; Retinoblastomas; Olfactory nerve tumors; Meningiomas, malignant; Neurofibrosarcomas; Schwannomas, malignant; Granular cell tumors, malignant; Malignant lymphomas; Hodgkin's disease; Hodgkin's; Lateral granuloma; Malignant lymphomas, small lymphocytic; Malignant lymphomas, diffuse large cell; Malignant lymphomas, follicular; Mycosis fungoides; Other non-Hodgkin lymphomas as specified; Malignant histiocytosis; Multiple myeloma; Mast cell sarcoma; Immunoproliferative small intestinal disease; Leukemias; Lymphocytic leukemias; Plasma cell leukemias; Erythroleukemias; Lymphosarcoma cell leukemias; Myelogenous leukemias; Basophilic leukemias; Eosinophilic leukemias; Monocytic leukemias; Mast cell leukemias; Megakaryoblastoid leukemias; Myelosarcomas; and Hairy cell leukemias.;

[0093] A method for treating cancer arising from the colon is disclosed. The cancer may be colon cancer. The cancer may be colorectal cancer.

[0094] The method may involve determining, administering, or selecting an appropriate cancer "management regimen" and predicting the outcome of the appropriate cancer "management regimen". As used herein, the phrase "management regimen" refers to a management plan that specifies the types of tests, screenings, diagnoses, investigations, care, and treatments (e.g., dosage, schedule, and / or duration of treatment) provided to a subject in need of a "management regimen" (e.g., a subject diagnosed with cancer).

[0095] A. Radiation therapy Radiation therapy such as ionizing radiation may be administered to the subject. As used herein, "ionizing radiation" means radiation including particles or photons that have sufficient energy to cause ionization (acquisition or loss of electrons) or can produce sufficient energy to cause ionization (acquisition or loss of electrons) through nuclear interactions. A preferred non-limiting example of ionizing radiation is x-rays. Means for delivering x-rays to a target tissue or cell are well known in the art.

[0096] Radiation therapy may include external beam radiation therapy, internal radiation therapy, radioimmunotherapy, or intraoperative radiation therapy (IORT). External beam radiation therapy may include three-dimensional conformal radiation therapy (3D-CRT), intensity-modulated radiation therapy (IMRT), proton beam therapy, image-guided radiation therapy (IGRT), or stereotactic radiation therapy. Internal radiation therapy may include interstitial brachytherapy, intracavitary brachytherapy, or intraluminal radiation therapy. Radiation therapy may be administered to the primary tumor.

[0097] The amount of ionizing radiation is greater than 20 Gy and may be administered in a single dose. The amount of ionizing radiation is 18 Gy and may be administered in three doses. The amount of ionizing radiation is at least 0.5 Gy, 1 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy, 10 Gy, 15 Gy, 16 Gy, 17 Gy, 18 Gy, 19 Gy, 20 Gy, 21 Gy, 22 Gy, 23 Gy, 24 Gy, 25 Gy, 26 Gy, 27 Gy, 18 Gy, 19 Gy, 30 Gy, 31 Gy, 32 Gy, 33 Gy, 34 Gy, 35 Gy, 36 Gy, 37 Gy, 38 Gy, 39 Gy, 40 Gy, 41 Gy, 42 Gy, 43 Gy, 44 Gy, 45 Gy, 46 Gy, 47 Gy, 48 Gy, 49 Gy, 50 Gy, 51 Gy, 52 Gy, 53 Gy, 54 Gy, 55 Gy, 56 Gy, 57 Gy, 58 Gy, 59 Gy, or 60 Gy (or any range derivable therein), At most 0.5 Gy, 1 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy, 10 Gy, 15 Gy, 16 Gy, 17 Gy, 18 Gy, 19 Gy, 20 Gy, 21 Gy, 22 Gy, 23 Gy, 24 Gy, 25 Gy, 26 Gy, 27 Gy, 18 Gy, 19 Gy, 30 Gy, 31 Gy, 32 Gy, 33 Gy, 34 Gy, 35 Gy, 36 Gy, 37 Gy, 38 Gy, 39 Gy, 40 Gy, 41 Gy, 42 Gy, 43 Gy, 44 Gy, 45 Gy, 46 Gy, 47 Gy, 48 Gy, 49 Gy, 50 Gy, 51 Gy, 52 Gy, 53 Gy, 54 Gy, 55 Gy, 56 Gy, 57 Gy, 58 Gy, 59 Gy, or 60 Gy (or any range derivable therefrom), or exactly 0.5 Gy, 1 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy, 10 Gy, 15 Gy, 16 Gy, 17 Gy, 18 Gy, 19 Gy, 20 Gy, 21 Gy, 22 Gy, 23 Gy, 24 Gy, 25 Gy, 26 Gy, 27 Gy, 18 Gy, 19 Gy, 30 Gy, 31 Gy, 32 Gy, 33 Gy, 34 Gy, 35 Gy, 36 Gy, 37 Gy, 38 Gy, 39 Gy, 40 Gy, 41 Gy, 42 Gy, 43 Gy, 44 Gy, 45 Gy, 46 Gy, 47 Gy, 48 Gy, 49 Gy, 50 Gy, 51 Gy, 52 Gy, 53 Gy, 54 Gy, 55 Gy, 56 Gy, 57 Gy, 58 Gy, 59 Gy, or 60 Gy (or any range derivable therefrom) may also be used. The ionizing radiation may be administered in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 administrations (or any range derivable therefrom), at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 administrations (or any range derivable therefrom), or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 administrations (or any range derivable therefrom). When multiple doses are administered, the administrations may be spaced apart by about 1 hour, 4 hours, 8 hours, 12 hours, or 24 hours, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days, or 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 14 weeks, or 16 weeks, or any range derivable therefrom.

[0098] The amount of radiation therapy administered to a subject may be indicated by the total dose of radiation therapy, and then the total dose may be administered in divided doses. For example, the total dose may be 50 Gy and each may be administered in 10 divided doses of 5 Gy. The total dose may be 50 to 90 Gy and each may be administered in 20 to 60 divided doses of 2 to 3 Gy. The total dose of radiation is, at least 0.5 Gy, 1 Gy, 2 Gy, 3 Gy, 4 Gy, 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 11 Gy, 12 Gy, 13 Gy, 14 Gy, 15 Gy, 16 Gy, 17 Gy, 18 Gy, 19 Gy, 20 Gy, 21 Gy, 22 Gy, 23 Gy, 24 Gy, 25 Gy, 26 Gy, 27 Gy, 28 Gy, 29 Gy, 30 Gy, 31 Gy, 32 Gy, 33 Gy, 34 Gy, 35 Gy, 36 Gy, 37 Gy, 38 Gy, 39 Gy, 40 Gy, 41 Gy, 42 Gy, 43 Gy, 44 Gy, 45 Gy, 46 Gy, 47 Gy, 48 Gy, 49 Gy, 50 Gy, 51 Gy, 52 Gy, 53 Gy, 54 Gy, 55 Gy, 56 Gy, 57 Gy, 58 Gy, 59 Gy, 60 Gy, 61 Gy, 62 Gy, 63 Gy, 64 Gy, 65 Gy, 66 Gy, 67 Gy, 68 Gy, 69 Gy, 70 Gy, 71 Gy, 72 Gy, 73 Gy, 74 Gy, 75 Gy, 76 Gy, 77 Gy, 78 Gy, 79 Gy, 80 Gy, 81 Gy, 82 Gy, 83 Gy, 84 Gy, 85 Gy, 86 Gy, 87 Gy, 88 Gy, 89 Gy, 90 Gy, 91 Gy, 92 Gy, 93 Gy, 94 Gy, 95 Gy, 96 Gy, 97 Gy, 98 Gy, 99 Gy, 100 Gy, 101 Gy, 102 Gy, 103 Gy, 104 Gy, 105 Gy, 106 Gy, 107 Gy, 108 Gy, 109 Gy, 110 Gy, 111 Gy, 112 Gy, 113 Gy, 114 Gy, 115 Gy, 116 Gy, 117 Gy, 118 Gy, 119 Gy, 120 Gy, 125 Gy, 130 Gy, 135 Gy, 140 Gy, or 150 Gy (or any range derivable therein), At most 0.5 Gy, 1 Gy, 2 Gy, 3 Gy, 4 Gy, 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 11 Gy, 12 Gy, 13 Gy, 14 Gy, 15 Gy, 16 Gy, 17 Gy, 18 Gy, 19 Gy, 20 Gy, 21 Gy, 22 Gy, 23 Gy, 24 Gy, 25 Gy, 26 Gy, 27 Gy, 28 Gy, 29 Gy, 30 Gy, 31 Gy, 32 Gy, 33 Gy, 34 Gy, 35 Gy, 36 Gy, 37 Gy, 38 Gy, 39 Gy, 40 Gy, 41 Gy, 42 Gy, 43 Gy, 44 Gy, 45 Gy, 46 Gy, 47 Gy, 48 Gy, 49 Gy, 50 Gy, 51 Gy, 52 Gy, 53 Gy, 54 Gy, 55 Gy, 56 Gy, 57 Gy, 58 Gy, 59 Gy, 60 Gy, 61 Gy, 62 Gy, 63 Gy, 64 Gy, 65 Gy, 66 Gy, 67 Gy, 68 Gy, 69 Gy, 70 Gy, 71 Gy, 72 Gy, 73 Gy, 74 Gy, 75 Gy, 76 Gy, 77 Gy, 78 Gy, 79 Gy, 80 Gy, 81 Gy, 82 Gy, 83 Gy, 84 Gy, 85 Gy, 86 Gy, 87 Gy, 88 Gy, 89 Gy, 90 Gy, 91 Gy, 92 Gy, 93 Gy, 94 Gy, 95 Gy, 96 Gy, 97 Gy, 98 Gy, 99 Gy, 100 Gy, 101 Gy, 102 Gy, 103 Gy, 104 Gy, 105 Gy, 106 Gy, 107 Gy, 108 Gy, 109 Gy, 110 Gy, 111 Gy, 112 Gy, 113 Gy, 114 Gy, 115 Gy, 116 Gy, 117 Gy, 118 Gy, 119 Gy, 120 Gy, 125 Gy, 130 Gy, 135 Gy, 140 Gy, or 150 Gy (or any range derivable therefrom), or Approximately 0.5 Gy, 1 Gy, 2 Gy, 3 Gy, 4 Gy, 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 11 Gy, 12 Gy, 13 Gy, 14 Gy, 15 Gy, 16 Gy, 17 Gy, 18 Gy, 19 Gy, 20 Gy, 21 Gy, 22 Gy, 23 Gy, 24 Gy, 25 Gy, 26 Gy, 27 Gy, 28 Gy, 29 Gy, 30 Gy, 31 Gy, 32 Gy, 33 Gy, 34 Gy, 35 Gy, 36 Gy, 37 Gy, 38 Gy, 39 Gy, 40 Gy, 41 Gy, 42 Gy, 43 Gy, 44 Gy, 45 Gy, 46 Gy, 47 Gy, 48 Gy, 49 Gy, 50 Gy, 51 Gy, 52 Gy, 53 Gy, 54 Gy, 55 Gy, 56 Gy, 57 Gy, 58 Gy, 59 Gy, 60 Gy, 61 Gy, 62 Gy, 63 Gy, 64 Gy, 65 Gy, 66 Gy, 67 Gy, 68 Gy, 69 Gy, 70 Gy, 71 Gy, 72 Gy, 73 Gy, 74 Gy, 75 Gy, 76 Gy, 77 Gy, 78 Gy, 79 Gy, 80 Gy, 81 Gy, 82 Gy, 83 Gy, 84 Gy, 85 Gy, 86 Gy, 87 Gy, 88 Gy, 89 Gy, 90 Gy, 91 Gy, 92 Gy, 93 Gy, 94 Gy, 95 Gy, 96 Gy, 97 Gy, 98 Gy, 99 Gy, 100 Gy, 101 Gy, 102 Gy, 103 Gy, 104 Gy, 105 Gy, 106 Gy, 107 Gy, 108 Gy, 109 Gy, 110 Gy, 111 Gy, 112 Gy, 113 Gy, 114 Gy, 115 Gy, 116 Gy, 117 Gy, 118 Gy, 119 Gy, 120 Gy, 125 Gy, 130 Gy, 135 Gy, 140 Gy, or 150 Gy (or any range derivable therefrom) It may also be. The total dose is at least 1 Gy, 2 Gy, 3 Gy, 4 Gy, 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 12 Gy, 14 Gy, 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, or 50 Gy (or any range derivable therefrom), at most 1 Gy, 2 Gy, 3 Gy, 4 Gy, 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 12 Gy, 14 Gy, 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, or 50 Gy (or any range derivable therefrom), or exactly 1 Gy, 2 Gy, 3 Gy, 4 Gy, 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 12 Gy, 14 Gy, 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, or 50 Gy (or any range derivable therefrom). At least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 fractional doses (or any range derivable therefrom), At most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 fractionation doses (or any range derivable therefrom), or Exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 fractionation doses (or any range derivable therefrom) It may be administered. In one day, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any range derivable therefrom), at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any range derivable therefrom), or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any range derivable therefrom) of divided doses may be administered. In one week, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any range derivable therefrom), at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any range derivable therefrom), or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any range derivable therefrom) of divided doses may be administered.

[0099] B. Cancer Immunotherapy The method may include the administration of cancer immunotherapy. Cancer immunotherapy (sometimes referred to as immuno-oncology, abbreviated as IO) is the use of the immune system to treat cancer. Immunotherapy can be classified as active, passive, or hybrid (active and passive). These approaches utilize the fact that cancer cells often have molecules on their surface known as tumor-associated antigens (TAAs) that can be detected by the immune system. Tumor-associated antigens (TAAs) are often proteins or other macromolecules (e.g., carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapy enhances existing anti-tumor responses and includes the use of monoclonal antibodies, lymphocytes, and cytokines. Various immunotherapies are known in the art and examples are described below.

[0100] 1. Checkpoint Inhibitors and Combination Treatments The methods and compositions of the present disclosure may include administration of immune checkpoint inhibitors. Examples of immune checkpoint inhibitors are further described below. As used herein, “checkpoint inhibitor therapy” (also referred to as “immune checkpoint inhibition therapy”, “immune checkpoint therapy”, “ICT”, “checkpoint inhibitory immunotherapy”, or “CBI”) refers to a cancer therapy that includes providing one or more immune checkpoint inhibitors to a subject suffering from cancer or suspected of having cancer.

[0101] a. PD-1, PDL1, and PDL2 Inhibitors PD-1 can act in the tumor microenvironment where T cells encounter infections or tumors. Activated T cells upregulate PD-1 and continue to express PD-1 in peripheral tissues. Cytokines such as IFN-γ induce PDL1 expression on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit effector T cell activity in the periphery and prevent excessive damage to tissues during an immune response. The inhibitors of the present disclosure may block one or more functions of PD-1 and / or PDL1 activity.

[0102] Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7-DC, Btdc, and CD273. PD-1, PDL1, and PDL2 may be human PD-1, PDL1, and PDL2.

[0103] A PD-1 inhibitor may be a molecule that inhibits the binding of PD-1 to its ligand binding partner. The PD-1 ligand binding partner may be PDL1 and / or PDL2. A PDL1 inhibitor may be a molecule that inhibits the binding of PDL1 to its binding partner. The PDL1 binding partner may be PD-1 and / or B7-1. A PDL2 inhibitor may be a molecule that inhibits the binding of PDL2 to its binding partner. The PDL2 binding partner may be PD-1. The inhibitor may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art as described in U.S. Patent Application Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all of which are incorporated herein by reference.

[0104] The PD-1 inhibitor may be an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). The anti-PD-1 antibody may be selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. The PD-1 inhibitor may be an immunoadhesin (e.g., an immunoadhesin comprising an extracellular portion of PDL1 or PDL2 or a PD-1 binding portion fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). The PDL1 inhibitor may include AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO (registered trademark), and is an anti-PD-1 antibody described in WO2006 / 12116. Pembrolizumab is also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA (registered trademark), and SCH-900475, and is an anti-PD-1 antibody described in WO2009 / 114335. Pidilizumab is also known as CT-011, hBAT, or hBAT-1, and is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224 is also known as B7-DCIg, and is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. Further PD-1 inhibitors include MEDI0680, also known as AMP-514 and REGN2810.

[0105] The immune checkpoint inhibitor may be a PDL1 inhibitor, e.g., durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, MSB00010118C, avelumab, also known as BMS-936559, or a combination thereof. The immune checkpoint inhibitor may be a PDL2 inhibitor such as rHIgM12B7.

[0106] The inhibitor may include the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. The inhibitor may be the V of nivolumab, pembrolizumab, or pidilizumab HThe CDR1, CDR2, and CDR3 domains of the region, and the V L region may include the CDR1, CDR2, and CDR3 domains. The antibody may be an antibody that competes with the above-described antibody in binding to the same epitope on PD-1, PDL1, or PDL2, and / or binds to the same epitope on PD-1, PDL1, or PDL2 as the above-described antibody. The antibody may have at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (or any range derivable therein) variable region amino acid sequence identity with the above-described antibody.

[0107] b. CTLA-4, B7-1, and B7-2 Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has GenBank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 resembles the T cell co-stimulatory protein CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA-4 is also found in regulatory T cells and may be important for their function. T cell activation via the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. The inhibitors of the present disclosure may block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. The inhibitor may be one that blocks the interaction between CTLA-4 and B7-1. The inhibitor may be one that blocks the interaction between CTLA-4 and B7-2.

[0108] The immune checkpoint inhibitor may be an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0109] An anti-human CTLA-4 antibody (or a V H domain and / or V L domain) suitable for use in the present method can be prepared using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. For example, US8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, also known as tremelimumab; previously ticilimumab), U.S. Patent No. 6,207,156; the anti-CTLA-4 antibodies disclosed in Hurwitz et al., 1998 can be used in the methods disclosed herein. The disclosure of each of the above publications is incorporated herein by reference. Antibodies that compete with any of these antibodies recognized in the art in binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001 / 014424, WO2000 / 037504, and U.S. Patent No. 8,017,114. All are incorporated herein by reference.

[0110] Further anti-CTLA-4 antibodies useful as checkpoint inhibitors in the methods and compositions of the present disclosure are ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO01 / 14424).

[0111] The inhibitor may comprise the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. The inhibitor may be a V of tremelimumab or ipilimumab HThe CDR1, CDR2, and CDR3 domains of the region, and the V L region may include the CDR1, CDR2, and CDR3 domains. The antibody may be an antibody that competes with the above-described antibody in binding to the same epitope on PD-1, B7-1, or B7-2, and / or binds to the same epitope on PD-1, B7-1, or B7-2 as the above-described antibody. The antibody may have at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (or any range derivable therein) variable region amino acid sequence identity with the above-described antibody.

[0112] c.LAG3 Another immune checkpoint that can be targeted in the methods provided herein is lymphocyte-activation gene 3 (LAG3), also known as CD223 and lymphocyte activating 3. The complete mRNA sequence of human LAG3 has GenBank accession number NM_002286. LAG3 is a member of the immunoglobulin superfamily found on the surface of activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. The main ligand of LAG3 is MHC class II, and it has been reported to negatively regulate T cell proliferation, activation, and homeostasis in the same way as CTLA-4 and PD-1, and to play a role in Treg suppression function. LAG3 also helps to maintain CD8+ T cells in an immune tolerance-inducing state and, together with PD-1, helps to maintain CD8 exhaustion during chronic viral infection. LAG3 is also known to be involved in the maturation and activation of dendritic cells. The inhibitors of the present disclosure may block one or more functions of LAG3 activity.

[0113] The immune checkpoint inhibitor may be an anti-LAG3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0114] An anti-human LAG3 antibody (or a V domain and / or V domain derived therefrom) suitable for use in the present method H domain and / or V LThe (domain) can be prepared using methods well known in the art. Alternatively, anti-LAG3 antibodies recognized in the art can be used. For example, anti-LAG3 antibodies can include GSK2837781, IMP321, FS-118, Sym022, TSR-033, MGD013, BI754111, AVA-017, or GSK2831781. US9,505,839 (also known as BMS-986016, relatlimab); US10,711,060 (IMP-701, also known as LAG525); US9,244,059 (IMP731, also known as H5L7BW); US10,344,089 (25F7, also known as LAG3.1); WO2016 / 028672 (MK-4280, also known as 28G-10); WO2017 / 019894 (BAP050); anti-LAG3 antibodies disclosed in Burova E., et al., J. ImmunoTherapy Cancer, 2016; 4(Supp. 1):P195 (REGN3767); Yu, X., et al., mAbs, 2019; 11:6 (LBL-007) can be used in the methods disclosed herein. These and other anti-LAG-3 antibodies useful in the present invention are found, for example, in WO2016 / 028672, WO2017 / 106129, WO2017062888, WO2009 / 044273, WO2018 / 069500, WO2016 / 126858, WO2014 / 179664, WO2016 / 200782, WO2015 / 200119, WO2017 / 019846, WO2017 / 198741, WO2017 / 220555, WO2017 / 220569, WO2018 / 071500, WO2017 / 015560; WO2017 / 025498, WO2017 / 087589, WO2017 / 087901, WO2018 / 083087, WO2017 / 149143, WO2017 / 219995, US2017 / 0260271, WO2017 / 086367, WO2017 / 086419, WO2018 / 034227, and WO2014 / 140180. The disclosure of each of the above publications is incorporated herein by reference.In binding to LAG3, antibodies that compete with any of the antibodies recognized in the art can also be used.

[0115] The inhibitor may comprise the heavy and light chain CDRs or VRs of an anti-LAG3 antibody. The inhibitor may comprise CDR1, CDR2, and CDR3 domains of the V H region of an anti-LAG3 antibody and CDR1, CDR2, and CDR3 domains of the V L region of an anti-LAG3 antibody. The antibody may have at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (or any range derivable therein) variable region amino acid sequence identity with the antibodies described above.

[0116] d.TIM-3 Another immune checkpoint that can be targeted in the methods provided herein is T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), also known as hepatitis A virus cellular receptor 2 (HAVCR2) and CD366. The complete mRNA sequence of human TIM-3 has GenBank accession number NM_032782. TIM-3 is found on the surface of IFNγ-producing CD4+ Th1 and CD8+ Tc1 cells. The extracellular region of TIM-3 consists of a membrane distal single variable immunoglobulin domain (IgV) and a glycosylated mucin domain of varying length located near the membrane. TIM-3 is an immune checkpoint and, together with other inhibitory receptors including PD-1 and LAG3, mediates T cell exhaustion. TIM-3 has also been shown to be a CD4+ Th1-specific cell surface protein that regulates macrophage activation. The inhibitors of the present disclosure can block one or more functions of TIM-3 activity.

[0117] The immune checkpoint inhibitor may be an anti-TIM-3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0118] An anti-human TIM-3 antibody (or a V H domain and / or V L domain) suitable for use in the present method can be prepared using methods well known in the art. Alternatively, anti-TIM-3 antibodies recognized in the art can be used. For example, anti-TIM-3 antibodies including MBG453, TSR-022 (also known as Cobolimab), and LY3321367 can be used in the methods disclosed herein. These and other anti-TIM-3 antibodies useful in the present invention can be found, for example, in US9,605,070, US8,841,418, US2015 / 0218274, and US2016 / 0200815. The disclosure of each of the above publications is incorporated herein by reference. Antibodies that compete with any of these antibodies recognized in the art in binding to TIM-3 can also be used.

[0119] The inhibitor may include the heavy and light chain CDRs or VRs of the anti-TIM-3 antibody. The inhibitor may include the CDR1, CDR2, and CDR3 domains of the V H region of the anti-TIM-3 antibody and the CDR1, CDR2, and CDR3 domains of the V L region of the anti-TIM-3 antibody. The antibody may have at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (or any range or value derivable therein) variable region amino acid sequence identity with the above-described antibodies.

[0120] 2. Activation of costimulatory molecules Immunotherapy may include activators of costimulatory molecules. The activators may include agonists of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, OX40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. The activators include agonist antibodies, polypeptides, compounds, and nucleic acids.

[0121] 3. Dendritic cell therapy Dendritic cell therapy induces an anti-tumor response by presenting tumor antigens to lymphocytes on dendritic cells, activating the lymphocytes, and stimulating the lymphocytes to kill other cells presenting the antigen. Dendritic cells are antigen-presenting cells (APCs) in the mammalian immune system. In cancer treatment, dendritic cells help target cancer antigens. An example of dendritic cell-based cellular cancer therapy is Sipuleucel-T.

[0122] One way to induce dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of proteins corresponding to protein antigens on cancer cells). These peptides are often given in combination with an adjuvant (a highly immunogenic substance) to enhance the immune and anti-tumor responses. Other adjuvants include proteins or other chemical substances that attract and / or activate dendritic cells, such as granulocyte macrophage colony-stimulating factor (GM-CSF).

[0123] Dendritic cells can also be activated in vivo by expressing GM-CSF in tumor cells. This can be achieved by genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with a tumor-regressive virus expressing GM-CSF.

[0124] Another strategy is to remove dendritic cells from a patient's blood and activate them outside the body. Dendritic cells are activated in the presence of tumor antigens. The tumor antigen may be a single type of tumor-specific peptide / protein or a lysate of tumor cells (a solution of broken-down tumor cells). These cells (along with any adjuvant) are injected to induce an immune response.

[0125] Dendritic cell therapy involves the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens are added to the antibodies to induce dendritic cells to mature and can bring about immunity against tumors. Dendritic cell receptors, such as TLR3, TLR7, TLR8, or CD40, have been used as antibody targets.

[0126] 4. CAR-T Cell Therapy Chimeric antigen receptors (CARs, also known as chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors) are engineered receptors that combine new specificities with immune cells to target cancer cells. Typically, these receptors transplant the specificity of monoclonal antibodies into T cells. These receptors are called chimeric because they are fused from parts derived from different sources. CAR-T cell therapy refers to the treatment that uses such transformed cells for cancer therapy.

[0127] The basic principle of CAR-T cell design involves a recombinant receptor that combines antigen-binding function and T cell activation function. A general premise of CAR-T cells is to artificially create T cells that are targeted to markers found on cancer cells. Scientists can take T cells from a person, genetically modify them, and return them to the patient so that the T cells can attack cancer cells. Once the T cells are engineered to become CAR-T cells, they act as a "living drug." CAR-T cells create a link between the extracellular ligand recognition domain and the intracellular signaling molecule, resulting in the activation of T cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is to ensure that only cancer tumor cells are targeted and normal cells are not. The specificity of CAR-T cells is determined by the selection of the molecule to be targeted.

[0128] Example CAR-T therapies include tisagenlecleucel (Kymriah) and axicabtagene ciloleucel (Yescarta).

[0129] 5. Cytokine Therapy Cytokines are proteins produced by many types of cells present within tumors. Cytokines can regulate the immune response. Tumors often utilize cytokines to grow and be able to reduce the immune response. Due to these immunomodulatory effects, it becomes possible to use cytokines as drugs to induce an immune response. Two commonly used cytokines are interferon and interleukin.

[0130] Interferons are produced by the immune system. Usually, interferons are involved in the antiviral response, but they also have uses against cancer. Interferons are classified into three groups: type I (IFNα and IFNβ), type II (IFNγ), and type III (IFNλ).

[0131] Interleukins have a series of immune system effects. IL-2 is an example of interleukin cytokine therapy.

[0132] 6. Adoptive T cell therapy Adoptive T cell therapy is a type of passive immunity by T cell injection (adoptive cell transfer). T cells are found in blood and tissues and are usually activated when foreign pathogens are discovered. Specifically, T cells are activated when the surface receptors of T cells encounter cells showing a part of foreign proteins on cell surface antigens. These can be infected cells or antigen-presenting cells (APCs). These are found in normal tissues and tumor tissues, and in tumor tissues they are known as tumor-infiltrating lymphocytes (TILs). T cells are activated by the presence of APCs such as dendritic cells presenting tumor antigens. These cells can attack tumors, but the environment within the tumor is highly immunosuppressive, so immune-mediated tumor death is blocked.

[0133] Multiple techniques for producing and obtaining tumor-targeted T cells have been developed. T cells specific to tumor antigens can be removed from tumor samples (TILs) or filtered from the blood. Then, activation and culture are performed ex vivo and the results are reinjected. Activation may be performed by gene therapy or by exposing T cells to tumor antigens.

[0134] Cancer treatment is intended to be able to exclude any cancer treatment described herein. The methods and compositions of the present disclosure include patients who have been previously treated for the therapies described herein, patients who are currently being treated for the therapies described herein, or patients who have not been treated for the therapies described herein. The patient may be a patient who has been confirmed to be resistant to the therapies described herein. The patient may be a patient who has been confirmed to be sensitive to the therapies described herein. For example, the patient may be a patient who has been confirmed to be sensitive to immune checkpoint inhibitor therapy based on the determination of whether the patient has pancreatitis or had pancreatitis previously.

[0135] C. Chemotherapy Additional therapies may include chemotherapy. Suitable classes of chemotherapeutic agents include (a) alkylating agents such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil), ethyleneimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chloroozotocin, streptozocin), and triazines (e.g., dacarbazine); (b) antimetabolites such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine), and purine analogs and related materials (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin); (c) natural products such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophyllotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, and mitoxantrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., interferon-α); and (d) various miscellaneous agents such as platinum coordination compounds (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine), and adrenocortical suppressants (e.g., taxol and mitotane). Cisplatin can be particularly suitable as a chemotherapeutic agent.

[0136] Cisplatin has been widely used to treat cancers such as metastatic testicular or ovarian cancer, advanced bladder cancer, head and neck cancer, cervical cancer, lung cancer, or other tumors. Cisplatin is not absorbed orally and thus must be delivered via other routes such as intravenous injection, subcutaneous injection, intratumoral injection, or intraperitoneal injection. Cisplatin may be used alone or in combination with other agents, and is typically administered at a dose of about 15 mg / m² to about 20 mg / m² for 5 days every 3 weeks for a total of 3 courses. 2 ~ about 20 mg / m²2 An effective dosage for clinical use, including

[0137] Other suitable chemotherapeutic agents include microtubule inhibitors such as paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”). The combination of the Egr-1 promoter / TNFα construct delivered via an adenovirus vector and doxorubicin has been confirmed to be effective in overcoming chemotherapy and / or resistance to TNF-α. From this, it is suggested that the combination treatment with the said construct and doxorubicin overcomes resistance to both doxorubicin and TNF-α.

[0138] Nitrogen mustard is another suitable chemotherapeutic agent useful in the methods of the present disclosure. Nitrogen mustard can include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN®) is available from Mead Johnson, and NEOSTAR® is available from Adria, and is another suitable chemotherapeutic agent. Oral dosages suitable for adults include, for example, from about 1 mg / kg / day to about 5 mg / kg / day, and intravenous dosages include, for example, initially from about 40 mg / kg to about 50 mg / kg in divided doses over a period of about 2 days to about 5 days, or about 10 mg / kg to about 15 mg / kg every about 7 days to about 10 days, or about 3 mg / kg to about 5 mg / kg twice a week, or about 1.5 mg / kg / day to about 3 mg / kg / day. The intravenous route is preferred due to gastrointestinal side effects. The drug is also sometimes administered intramuscularly, by infiltration, or into body cavities.

[0139] Additional suitable chemotherapeutic agents include pyrimidine analogs such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluorouracil; 5-FU), and floxuridine (fluorodeoxyuridine; FudR). 5-FU can be administered to a subject at any dosage from about 7.5 to about 1000 mg / m2. Further, the 5-FU dosing schedule can cover various periods, for example, up to 6 weeks, or a period as determined by one of ordinary skill in the art to which this disclosure pertains.

[0140] The amount of chemotherapeutic agent delivered to a patient may be variable. When chemotherapy is administered with a construct, the chemotherapeutic agent can be administered in an amount effective to cause cancer arrest or regression in a host. The chemotherapeutic agent can be administered in an amount anywhere from 1 / 2 to 1 / 10,000 of the chemotherapeutically effective dose of the chemotherapeutic agent. For example, the chemotherapeutic agent can be administered in an amount of about 1 / 20, about 1 / 500, or even about 1 / 5000 of the chemotherapeutically effective dose of the chemotherapeutic agent. The chemotherapeutic agents of the present disclosure can be tested in vivo to determine the desired therapeutic activity in combination with a construct, as well as the effective dosage. For example, such compounds can be tested in suitable animal model systems including, but not limited to, rats, mice, chickens, cows, monkeys, rabbits, etc. prior to testing in humans. In vitro testing may also be used, as described in the examples, to determine the appropriate combinations and dosages.

[0141] D. Hormone Therapy In some aspects, the cancer therapy of the present disclosure is hormone therapy. In certain aspects, prostate cancer therapy includes hormone therapy. A variety of hormone therapies are known in the art and are contemplated herein. Examples of hormone therapies include, but are not limited to, luteinizing hormone-releasing hormone (LHRH) analogs, LHRH antagonists, androgen receptor antagonists, and androgen synthesis inhibitors.

[0142] E. Surgery Approximately 60% of people with cancer undergo some type of surgical procedure, including prophylactic surgery, surgery for diagnosis or staging, curative surgery, and palliative surgery. Curative surgery involves resection in which all or part of the cancer tissue is physically removed, excised, and / or destroyed, and may be used in conjunction with other therapies such as the procedures of this aspect, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least part of a tumor. In addition to tumor resection, surgical procedures include laser surgery, cryosurgery, electrocautery, and microsurgically controlled surgery (Mohs surgery).

[0143] Removing part or all of the cancer cells, tissue, or tumor may create a cavity in the body. The treatment may be performed by perfusing, directly injecting, or topically applying additional anti-cancer therapy to that area. Such treatment may be repeated, for example, daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every 7 days, or every week, every 2 weeks, every 3 weeks, every 4 weeks, and every 5 weeks, or every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or every 12 months. These treatments may also be in various dosages.

[0144] F. Additional Cancer Therapies The therapeutic methods disclosed herein may include one or more additional cancer therapies. The cancer therapies of the present disclosure may include, for example, cryoablative therapy, high-intensity focused ultrasound therapy (also referred to as "high-intensity focused ultrasound"), photodynamic therapy, laser ablation, and / or irreversible electroporation. The cancer therapies of the present disclosure may include one, two, three, four, five, or more distinct therapeutic methods.

[0145] Cancer treatment is intended to exclude any cancer treatment described herein. Further, aspects of the present disclosure include patients who have been previously treated for the therapies described herein, patients who are currently being treated for the therapies described herein, or patients who have not been treated for the therapies described herein. In some aspects, the patient is a patient who has been determined to be resistant to the therapies described herein. In some aspects, the patient is a patient who has been determined to be sensitive to the therapies described herein.

Example

[0146] VII. Example The following examples are included to demonstrate preferred embodiments of the invention. The techniques disclosed in the following examples are those discovered by the inventors to function well in the practice of the invention and are thus to be understood by those skilled in the art as constituting preferred modes for carrying out the invention. However, in view of the present disclosure, it should be understood by those skilled in the art that many changes can be made to the specific embodiments disclosed and that still further, similar or analogous results can be obtained without departing from the spirit and scope of the invention.

[0147] Example 1 - High-Throughput Screening of Immunomodulators for Vaccine Adjuvants A. Materials and Methods 1. Raw Dual Cell Culture RAW-Dual cells were purchased from InvivoGen and cultured with DMEM and 10% FBS / 1% P / S.

[0148] 2. NF-κB and IRF Transcription Factor Screening RAW-Dual cells (InvivoGen) were plated at 50,000 cells / well in 45 μL of DMEM with 5% HI FBS from col 2-23 in clear 384-well plates. The cells were allowed to adhere for 1 hour at room temperature. 50 nL of a 10 mM modulator library was added to the experimental wells (cols 3-22) via a pintool by Janus G3 to a final concentration of 10 μM. After a 1-hour incubation, 5 μL of a PRR agonist was added via a MultiDrop Combi liquid handler (col 3-23). The cells were incubated overnight at 37 °C and 5% CO2. After 20 hours, 12.5 μL of QuantiLuc Plus was plated into opaque white 384-well plates. Then, after adding 5 μL of cell supernatant, luminescence values were measured with a BioTek Synergy NEO2 plate reader. In parallel, 15 μL of 5X concentrated QuantiBlue was added directly to the remaining cell supernatant. Absorbance values were measured at 620 nm at various time intervals.

[0149] 3. Survival rate monitoring The survival rate after overnight addition of the modulator was monitored by monitoring confluence by IncuCyte imaging. Using two sets of parameters, a confluence mask covering all imaged wells was created. If both sets of confluence masks were <70% of the confluence mask of resting cells, the modulator was confirmed to be toxic. The confluence mask was quantified using IncuCyte software. This method was verified with a selected library plate using the conventional CellTiter Glo assay (Promega).

[0150] 4. THP-1 cell culture THP-1 cells were purchased from ATCC and cultured in RPMI 1640 containing 10% biotin-free HI-FBS and 1% P / S. The cells were maintained at 0.2 - 1.0x10 6 cells / mL.

[0151] 5. Cytokine Level Screening THP-1 cells were seeded at 50,000 cells / well in 45 μL of biotin-free RPMI + 5% HI-FBS in clear 384-well plates. After 24 hours of incubation, 50 nL of a 10 mM modulator library was added to the experimental wells (cols 3 - 22) via a pin tool by Janus G3 to a final concentration of 10 μM. After 1 hour, 5 μL of agonist was added via a MultiDrop Combi liquid handler (col 3 - 23). The next day, 5 μL of the supernatant was transferred to a white low-volume ProxiPlate. A 10 μL mixture of prepared acceptor beads (final concentration 10 μg / mL) and biotinylated antibody (final concentration 1 nM) was added to the cell supernatant via a liquid handler. After incubation for 1 hour at RT, 5 μL of donor beads (final concentration 40 μg / mL) was added in the dark. After an additional 1 hour of incubation, the plate was read on a plate reader using AlphaPlex filters for europium (615 nm) and terbium (545 nm) luminescence. Another plate containing a calibration curve of known analytes for interpolation purposes was run daily.

[0152] 6. In Vivo Studies a. Animals All animal procedures were conducted under a protocol approved by the University of Chicago Institutional Animal Care and Use Committee (IACUC). C57 / B6 female mice, 6 - 8 weeks old, were purchased from Jackson laboratory. All vaccinations were administered intramuscularly into the hind limb. Blood was collected from the submandibular vein at the indicated time points.

[0153] VacciGrade ovalbumin and AddaVax were purchased from InvivoGen. VaccigGrade CpG ODN 1826, ultra-high purity flagellin, and VaccigGrade R848 were purchased from InvivoGen. Modulators were purchased through Selleck Chemicals.

[0154] b. Vaccination Mice were lightly anesthetized with isoflurane and injected intramuscularly into the hind limb with a 50 μL injection volume containing the antigen, the adjuvant, and a combination of DMSO / AddaVax. The antigen dosage was as follows: OVA (100 μg). The agonist dosages were as follows: flagellin, 10 μg; R848, 50 μg; CpG, 50 μg. Modulators were added at 1.5 μmol.

[0155] c. Plasma cytokine analysis Blood was collected from the mice at the indicated time points and placed into 0.2 mL heparin-coated collection tubes (VWR Scientific). Plasma was isolated by centrifugation at 1500 xg for 15 minutes at 4°C. Samples were collected and stored at -80°C until use. Plasma was analyzed using Bio-Legend's LegendPlex™ Mouse Inflammation Cytokine Panel (13-Plex) according to the manufacturer's protocol. Samples were analyzed using an ACEA NovoCyte Flow Cytometer. Data were analyzed using the LEGENDplex™ Data Analysis Software Suite and GraphPad Prism.

[0156] d. Antibody quantification The indicated formulation was vaccinated into mice. Blood was collected at the indicated time points and placed into 0.2 mL heparin-coated collection tubes (VWR Scientific). Plasma was isolated by centrifugation at 1500 x g for 15 minutes at 4°C. Samples were collected and stored at -80°C until use. Samples were analyzed according to the specified protocol using an anti-OVA IgG ELISA kit (Chondrex). Antibody levels were analyzed using a Multiskan FC plate reader (Thermo Fisher), and absorbance was measured at 450 nm. Data were analyzed using GraphPad Prism.

[0157] 7. Statistical values and replicates Data were plotted and reported in the text as mean ± SEM. Sample sizes were as indicated for biological replicates in all in vivo and in vitro experiments. If there was a significant difference in the means, the sample size was selected based on preliminary experiments or literature precedents indicating that the number was sufficient to detect the significant difference in the means. P-values were calculated using one-way analysis of variance (ANOVA) and Tukey post hoc test or two-tailed unpaired heteroscedastic t test, as appropriate. All experiments were repeated (sometimes with minor variations for reagents and materials), and the repetitions were successful.

[0158] B. Results 1. Primary screening demonstrates control of NF-κB and IRF transcription factor activities For the purpose of identifying new adjuvants, the inventors conducted a high-throughput screen to examine the NF-κB activity and IRF activity of various levels of innate immune cells after treatment with a combination of immunomodulators and PRR agonists (Figure 1C). To achieve this goal, the inventors selected cell lines that quantitatively report RAW-Dual macrophage-NF-κB activity and IRF activity by secreted alkaline phosphatase (SEAP) and Lucia luciferase under the control of their respective transcription factor promoters (11, 12). For the initial screen, the inventors searched a targeted small molecule library: 246 NF-κB inhibitors and IRF inhibitors and 2,895 pathway-specific inhibitors (Table 1, Figures 7A-7B). Many of the compounds included have been studied previously, and some have even received FDA approval for other therapeutic uses. The inventors hypothesized that this library is likely to modulate desirable immune signaling pathways. The inventors observed that this library modulates 14 PRR agonists centered on toll-like receptors (TLRs) (Table 1) (13). The inventors included this broad range of agonists to further understand trends in modulator activity across similar or distinct PRRs and signaling pathways.

[0159] (Table 1) Table of compounds constituting the primary screen TIFF2025520120000001.tif57128

[0160] To screen this initial library for activity that modulates NF-κB activity and IRF activity, the inventors seeded 50,000 cells into a 384-well plate containing 45 μL of complete medium. The inventors transferred an immunomodulator compound from the source plate to a final concentration of 10 μM. After incubating at 37 °C for 1 hour, one of 14 PRR agonists was added to 5 μL of medium to a desired concentration (Table 2). The cells were incubated with the agonist overnight until the transcriptional factor activity was analyzed. This activity was measured using QUANTI-Blue and QUANTI-Luc, substrates under intellectual property rights for reading absorbance and luminescence. Using an assay based on the supernatant, the inventors simultaneously observed both NF-κB activity and IRF activity from the same well. To ensure consistent and high-quality results, the inventors optimized this screening workflow from biological aspects including cell seeding density, incubation time, agonist concentration, to intricate matters of the assay such as liquid handling, reagent volume, and plate uniformity (Figures 8A - 8C)(14,15).

[0161] (Table 2) Agonists, agonist targets, activated pathways, and working concentrations used in the primary screen TIFF2025520120000002.tif121128

[0162] The inventors' initial screening approach presented a problem unique to this assay optimization and analysis. Most high-throughput screens attempt to maximize or minimize a desired output (16-18). For example, screening for novel TLR4 agonists might attempt to boost activity beyond resting levels. However, in this case, the inventors modulated existing PRR activity and thus compared the modulator + agonist combination to the agonist alone, reporting the modulation as a fold change. The inventors expected to find inhibition of both immune pathways, but were surprised to observe enhanced transcription factor activity. Indeed, the modulator enhanced or inhibited NF-κB and IRF by over 100-fold while maintaining cell viability (Figures 2A-2B). For all 14 agonists studied, the modulator produced significant enhancement or inhibition. This modulation persisted even when using potent agonists with high basal levels of activity. For example, modulation of the STING agonist 3’3’-cGAMP showed a 5-fold increase in IRF activity. This result was surprising as few molecular entities achieved higher STING activation than 3’3’-cGAMP (19). Perhaps most surprisingly, LPS, a TLR4 agonist often considered a standard of strong activity, showed a 10-fold increase in NF-κB activity. This result presented a challenge as screens that produce both inhibition and enhancement require a difficult balance of the assay's dynamic range. This remains an issue across various screening efforts.

[0163] 2. Exploration of the characteristics and trends of modulation across PRR agonists With a large dataset in place, the inventors began studying modulator activity. They were particularly interested in both pathway crosstalk and individual receptor trends. First, the inventors confirmed that the modulator alone showed neither a specific stimulus for NF-κB nor a specific stimulus for IRF, and that only the combination of the modulator and agonist changed the transcription factor activity (Figure 3A). Compounds with significant activation of both pathways were removed from further study, but these were less than 1% of the entire library. When comparing NF-κB activity and IRF transcriptional activity, the inventors observed little correlation between the two. This indicates that these pathways can be studied independently using this assay (Figure 3B). Furthermore, the inventors observed that the modulator acts specifically or generally across multiple PRRs. For example, modulator X can enhance only IRF for TLR4, while other PRR activities remain unaffected. Conversely, modulator Y can enhance IRF for all receptors. To identify each type of regulation, the inventors refer to immunomodulators specific to one or two receptors as "specialists" and modulators that affect all or nearly all receptors as "generalists" (Figure 3C). Additionally, some modulators can be enhancers of one PRR in a specific pathway but inhibitors of another PRR in the same pathway. The inventors observed a broad distribution across each receptor, and some agonists show great statistical significance due to their broad dynamic range. Monitoring the distribution across the entire set of similar PRR targets reveals correlations in these activities. For example, both MPLA and LPS are TLR4 agonists and show similar trends across NF-κB and IRF activities. Pam2CSK4 (TLR1 / 2), Pam3CSK4 (TLR2 / 6), and other NF-κB dominant agonists also have a degree of correlation (Figure 8).

[0164] 3. Narrowing by removal of inactive and undesirable combinations Using the results from these initial screens, the inventors identified high-value compounds and attempted to remove combinations of modulators / agonists that were inactive and toxic. In the inventors' planned secondary screen, assays that require increased time and cost are performed and thus a narrowing of the number of compounds to be studied is needed. First, the inventors designed a high-throughput method to remove toxic modulators by measuring viability. As a proxy for viability, the inventors created a confluence mask using a combination of live cell imaging and digital analysis (Figure 9)(20). By using this method, the inventors were able to monitor toxicity on the same experimental data plate from which they collected IRF and NF-κB data. The inventors determined toxic compounds to be those with a viability < 70% of the viability of their negative unstimulated control. The inventors confirmed the use of confluence as an indicator of cell health and the secondary, dedicated viability assay CellTiter-Glo(21).

[0165] In high-throughput screening, the Z-factor was utilized as a surrogate for the statistical reliability of the assay. Due to the dynamic range of the positive control and the unique pathways of each agonist, a Z-factor score greater than zero was not obtained for certain agonist / transcription factor combinations. After applying the survival mask, the inventors identified the compounds most likely to alter the IRF response and the NF-κB response. The inventors removed certain weak agonists that showed only basal activity based on a low Z-factor cutoff score (Table 3). In this narrowing process, the inventors did not prioritize the enhancement or inhibition of both pathways; instead, they only attempted to remove compounds that had a minimal effect on PRR agonist activity. Therefore, the inventors performed principal component analysis on the dataset to quantitatively compare the levels of dispersion among the compounds (22). This dataset included both NF-κB distributions and IRF distributions from eight agonists for a total of 16 variables. PC1 and PC2 explained 49% of the variation within the inventors' dataset. By selecting only the compounds that varied more than a circle with a radius of approximately 1.75 PCA units centered at the origin, the inventors reduced the number of effective immunomodulators from 3,147 compounds to 720 modulators (Figure 3F). This cutoff was selected based on the screening constraints of the modulators that the inventors could explore in the next screen due to the limited ability to screen for multiplexed cytokines. The inventors created this cutoff with the goal of retaining as many regulatory compounds as possible that the inventors could test in a lower-throughput secondary screen. The PCA-selected compounds successfully removed modulators that did not change the cellular response while retaining the maximum and minimum activities of the primary screen distribution (Figure 15).

[0166] (Table 3) Z-factor analysis of primary screen agonists TIFF2025520120000003.tif162138

[0167] 4. The secondary library shows regulation across the 6 - cytokine panel Cytokines and chemokines are a broad category of secreted proteins that are important for adjuvants in modulating adaptive immunity (23). However, excessive cytokine production by adjuvants can cause direct tissue damage and has been correlated with vaccine tolerance (24, 25). To validate the findings of the primary screening results and explore the effects on downstream immune outcomes, the inventors measured the levels of 6 cytokines and chemokines (IL - 12p40, IP - 10, IL - 1β, CCL4, TNF - α, and IFN - β, Table 4) involved in inflammation and the adaptive response. The inventors measured cytokine expression by AlphaLISA, an assay well - suited for its broad dynamic range, in - situ measurement, and high throughput for a wide range of cytokines. This has been previously used to investigate small - molecule biological inhibitors (26 - 28).

[0168] Since the inventors narrowed down the compounds, they wanted to ensure compatibility with the human immune response. Conveniently, human AlphaLISA had many more available multiplexing options that lowered costs and were much more than mouse-compatible. After the initial screening of cytokines from stimulated human THP-1 monocytes, the inventors found weak expression / measurements from some identified targets including IL-27 and IL-6. The inventors' final cytokine panel consisting of IL-12p40, IP-10, IL-1b, CCL4, TNF-α, and IFN-b was selected for their involvement in vaccine responses, excellent dynamic range, and assay measurement criteria (Table 4)(29). TNF-α, IL-1b, and IL-6 are endogenous pyrogens. This is because there are multiple reports correlating them with fever induction in vaccine tolerance (30). TNF-α was selected because it is a strong correlate of inflammation and the most studied cytokine. IL-1b is produced by activated macrophages, is another mediator of the inflammatory response, and can induce the expression of other interleukins by γδT cells (31). IL-1b, unlike TNF-α, is in part a measure of inflammation beyond the scope of direct NF-κB regulation. This allows the inventors to decipher by which pathway compounds can bias inflammation. Since IFN-b strongly correlates with the IRF pathway results from the inventors' primary screen, the inventors chose to study IFN-b. IFN-b is a well-characterized antiviral type I interferon that induces cells to make IFN-α, i.e., amplifies the interferon response (32). IL-12 / 23p40 activates NK cells and induces the production of IFN-γ (33). IP-10 (CXCL10) is a chemoattractant for T cells and DC cells and is correlated with early signals that induce strong responses in adjuvant studies (34). Finally, CCL4 is a chemoattractant for monocytes and NK cells (35).For example, IP-10 and CCL4 have recently been shown to correlate with positive responses in the BNT162b2 immune response, whereas excessive TNF-α or IL-6 may correlate with tolerance issues (36).

[0169] (Table 4) Measured cytokines. Functions of cytokines selected for the secondary screen in the AlphaPlex study. TIFF2025520120000004.tif71160

[0170] For the secondary assay, there was the same workflow as the primary screen prior to the cytokine assay. Cell supernatants were collected and cytokines were measured in three duplex measurements (Figure 11A). Similarly, the inventors optimized the calibration curve range, crosstalk correction factor, incubation time, and other parameters of the secondary screen (Figures 11B - 11D). As in the inventors' primary screen, the modulators enhance or inhibit cytokine production independently for each agonist across all six cytokines (Figures 4A - 4G, Figure 11E). The distribution within the cytokines varies based on the dynamic range and Z - factor obtained for each cytokine and agonist studied (Table 5). High - throughput screening utilizes the Z - factor as a proxy for the statistical reliability of the assay for the dynamic range of the positive control and the endogenous cytokines secreted for each agonist, and a Z - factor score greater than 0 was not obtained for certain agonist / transcription factor combinations. Since each agonist produced different levels of cytokines, sometimes at the limits of the calibration curve, it was difficult to modify this assay for high - throughput nature. Since the assay beads were multiplexed, diluting individual wells or selecting other AlphaPlex excitation / emission profiles would significantly increase cost and time. This led to some skewing of cytokines with low or high response levels. Most notably, IFN - b with relatively low signal and concentration, and IP - 10 / CCL4 with high signal and concentration. Since the inventors measured the fold - change and further reduced the dimensionality in the analysis, this approach was sufficient to compare the compounds within the inventors' dataset for shortlisting.

[0171] (Table 5) Z - factor analysis of the secondary screen TIFF2025520120000005.tif92169

[0172] Similar to the primary screen, the inventors observed that the modulators alone did not naturally affect or induce cytokine release, but that the combination of agonist and modulator induced a significant increase or decrease in the production of cytokines and chemokines (Figure 12). Changes in cytokine activity did not always correlate with corresponding levels of transcription factor activity for all modulators, but the inventors observed that for the most active compounds, transcription factor activity correlated with increases or decreases in cytokine responses. For example, the most potent NF-κB inhibitor also resulted in the lowest TNF-α levels (Figure 13A). Also notably, modulators can change responses in unique patterns but appear to conserve the pathway to some extent. When comparing agonists with similar profiles such as Pam2CS4K and Pam3CSK4, the inventors observed that they shared similar trends of enhancement and inhibition for each cytokine (Figure 13B).

[0173] To verify that the narrowing down from the primary screen was successful, the inventors compared cytokine regulation between the selected compounds and an equivalent random portion of the original primary screen library. The inventors selected LPS as the agonist for its broad dynamic range. The narrowed secondary library contained significantly more active compounds compared to an equal number of random primary screen compounds (Figure 14). This supports that the use of NF-κB activity and IRF activity are useful narrowing tools.

[0174] 5. Development of a Flexible Quantitative Scoring System for Top Candidate Selection As the number of variables to be considered during the search for a desirable agonist / modulator combination increases, the inventors have attempted to develop a general framework to assist in the final narrowing down of modulators for testing in various in vivo applications. Since the inventors' previous research focused on improving adjuvants for prophylactic vaccines, the inventors developed their first scoring system for identifying candidates for this use and created a "vaccine score" as a quantitative measure.

[0175] After considering the categories and the ranked scoring system, the inventors determined that the best representation of modulator performance requires preserving cytokine changes, but that this needs to be done by normalizing for the dynamic range of each cytokine and agonist (37). Because there are differences in the dynamic range of all six of the inventors' cytokines, the inventors attempted to normalize the data to ensure that the distribution did not bias their results. Thus, the inventors transformed the distribution of each cytokine to a range from -1 to 1 (Figure 5A). Unlike previous screens, the inventors considered increases and decreases in cytokine responses separately when selecting molecules for vaccination studies. For the vaccine score, promising candidates need to increase IFN-β and chemokine production while producing minimal pro-inflammatory cytokines (24, 25). Additionally, the inventors may prioritize the importance of modulating one cytokine over another. To account for each of these issues, the inventors assigned various magnitudes of weighting variables to each cytokine according to the desired regulatory effect (Figure 15A) (38). Since the modulators act on individual receptors with specific responses, the initial results from the vaccine scoring system are "specialist" scores for specific agonist + modulator combinations (Figure 15B). Then, to determine which modulators have more general trends across multiple PRRs, the individual scores were summed to obtain a "generalist" score across all agonists (Figure 5B).

[0176] Based on the results of the vaccine scoring method, a spread between compounds was created that ranged from a maximum of 10 to a minimum of approximately -20. Among the compounds that were ranked highest for the generalist modulator, this scoring system yielded approximately 20 compounds with scores of 6 - 10 from the entire pool of 720 potential compounds. Among these, the inventors included one exemplary compound, buffetinib (Inno-416), to demonstrate the pattern observed for the individual modulator (Figure 5C). Buffetinib was one of the compounds with the largest scores in the inventors' ranking system. Further examination by individual cytokines revealed that the inventors observed a significant enhancement of IP-10 across almost all agonists, and for some agonists, the enhancement of IFN-β and IL-12 was a factor contributing to their increased scores. Similarly, buffetinib decreased the expression of TNF-α across all agonists, with a very large suppression for Pam3 and resiquimod (R848). However, perhaps most importantly, the inventors noticed that this was not equally effective for all agonists in the "generalist" selection. Comparing the enhancement of IP-10 and IFN-β for Pam3, Pam2 vs cGAMP, there was almost a two-digit difference between the enhancing capabilities of these agonists and pathways. This suggests that, depending on the application, a generalist may still have limitations in enhancing specific pathways compared to a specialist modulator. However, for the suppression of inflammatory signals and the resulting tolerance, this example, like many of the modulators the inventors studied, appeared to be broadly general. Since the generalist partially suppressed inflammatory signals across many receptor categories, it is suggested that the generalist may be better applicable for improved tolerance and widespread use in improved vaccination.

[0177] The inventors were able to compare the results of bufetinib to MK-8353, a compound in the dataset with a strong negative vaccine score (Figure 5D). This compound removes IL-12, IFN-β, and IP-10 across almost all of the receptors studied, while enhancing IL-1β and TNF-α up to 100-fold. MK-8353 is not useful as a vaccine adjuvant, but the ability to fundamentally alter secreted cytokines underscores the broad potential of the inventors' modulators. This score is tailored to prophylactic vaccine adjuvants, but compounds within this dataset may be applicable for exploration in additional uses. For example, the mesylate of TRx0237 (LMTX) (Figure 5E) upregulates beneficial anti-tumor cytokines and chemokines and may be worthy of further study in cancer immunotherapy (39, 40). This compound was able to enhance TNF-α up to 36-fold using cGAMP and was shown to enhance IFN-β secretion across all agonists studied.

[0178] The inventors used a general vaccine score to identify lead modulators of interest that they wished to carry forward into preliminary in vivo studies. Although limited by the cytokine panel studied, the simple mathematical nature underlying the inventors' scoring system allows this method to be applied to various areas of interest. Additionally, further screening efforts, such as cell surface marker expression, could be incorporated into this scoring system in the future. The inventors developed a vaccine scoring system, but a similar approach could be adapted for other uses, whether for inflammatory therapeutics or cancer immunotherapy. Control of immune pathways has potential in many immunotherapy spaces.

[0179] 6. Identified candidates show promising results in mouse vaccination studies The inventors selected modulators for testing in a mouse in vivo model using a vaccine score. The inventors repeated the conventional prime-boost vaccination schedule as in their previous preliminary studies using ovalbumin as a model antigen (9,10). To test the generalist nature of the modulators, to these subunit vaccinations, the inventors added as adjuvants a subset of PRR agonists from their preliminary screen: R848 (TLR7 / 8), flagellin (TLR5), and CpG1826 (TLR9). This subset was selected for the previous use in vaccines and for the broad cross section of potential use in both subunits (R848, CpG), and as being close to whole bacterial (flagellin, CpG) vaccine products (41-43). The inventors selected a modulator dosage of 1.5 μmol, guided by their previous experience with low molecular weight and compound solubility limits (10). To improve the formulation of these hydrophobic compounds, the inventors used a 1:1 mixture of DMSO:AddaVax as the vehicle. The inventors monitored inflammatory cytokine levels 1 hour after the first injection and antigen-specific antibody levels at the indicated time points after the boost (Figure 6A). AddaVax has intrinsic adjuvant properties, but the inventors judged its use to be appropriate because (a) this experiment was testing the modulation of existing adjuvants that need to be formulated in these administrations, and (b) by controls of only separate adjuvants (without modulators), the inventors were able to account for the intrinsic variations to these responses.

[0180] The goal was, as before, to discover compounds that (a) enhance the tolerability of the vaccine formulation, as estimated by the inventors using a simple measurement criterion for systemic cytokines 1 hour after injection, and (b) improve the antibody response to vaccination. The inventors began by experimenting with bufetinib, the compound with the highest performance from the generalist scoring system. When this modulator was added, the humoral responses of all three agonists tested increased (Figure 6B). When bufetinib was added, the antibody responses of these adjuvants were improved 2 - 6 - fold compared to these agonist / antigen controls. This increase was fairly consistent across all agonists and persisted over both time points (Figure 16A). However, the inventors did not observe significant cytokine modulation using this modulator (Figure 16B).

[0181] Encouraged by these positive results, the inventors expanded the search to five more high - scoring generalists according to the same vaccination schedule as before (Figure 6C). The inventors observed that for compounds 2 and 3 with respect to the five modulators, in the case of CpG, TNF - α was strongly reduced to near 0 in 1 hour (Figure 6D). This level of reduction was strongly correlated with improvements in clinical scoring, body temperature decrease, and weight loss. This strongly indicates that these compounds may be used to improve CpG tolerability in further applications. Interestingly, there was a similarity between the compounds that reduced inflammatory cytokines in the case of CpG and those that reduced inflammatory cytokines in the case of R848. However, as in the inventors' previous experiments, the modulator was unable to completely remove the inflammation of R848 because R848 diffused more rapidly (9). But compound 5 still reduced the inflammation to half of the original formulation. In the case of flagellin, no compound showed significant cytokine reduction, but compound 5 showed a significant decrease (Figure 17A).

[0182] However, the reduction of TNF - α was mostly not correlated with changes in antibody levels (Figure 17B).

[0183] Surprisingly, and in contrast to our previous studies, the systemic cytokine regulation and antibody response, the two arms of our study, did not appear to be intrinsically related. The modulator that most potently enhanced antigen-specific IgG levels, bufetinib, partially downregulated inflammatory cytokines depending on the adjuvant. Conversely, modulators that altered cytokine levels had minimal impact on the humoral response. However, we identified both a modulator that potently decreased the systemic cytokines of the current adjuvant and a modulator that enhanced the antibody response. After multiple investigations, we identified two classes of lead modulators, namely, a modulator that reduces inflammatory cytokines and a modulator that enhances IgG antibodies.

[0184] 7. Development of a novel mRNA vaccination system; immunomodulators suppress in vitro and in vivo inflammatory responses by mRNA vaccines Experiments were conducted to determine whether the immunomodulator suppresses the inflammatory response of the mRNA vaccine in vitro and in vivo. For the in vitro experiment, various concentrations of the immunomodulator were added to BMDCs 1 hour before adding the mRNA vaccine. Supernatant systemic cytokines were measured at 24 hours (Figures 23A - 23B), and intracellular cytokine staining was performed at 48 hours (Figures 24A - 24B). Both PME - 564 and PME - 2834 significantly reduced the production of TNF - α and IL - 6 at both time points. For the in vivo experiment, C57BL / 6J (n = 4) was immunized intramuscularly with a handmade SARS - CoV - 2 mRNA vaccine (WA - 1 / Omicron, 5 μg / dose) and an immunomodulator (1.5 μmol). The immunomodulator also reduced (although not statistically significant) the production of IL - 6 cytokine (Figure 25A). Vaccination was unable to statistically reduce the production of systemic cytokines (Figure 25A). LNP reduces its stability upon addition of DMSO, and so far, PME - 564 is the most soluble in DMSO and other organic solvents. Further synthetic modifications to PME - 564 for encapsulation within LNP will likely be explored in the future. At the same time, the antibody titers against SARS - CoV - 2 spike were measured 7 days after injection. A large amount of antibodies was found in all vaccinated groups. In particular, there was no difference between the "mRNA vaccine alone" and any of the modulator groups (Figure 25B). Overall, these in vitro and in vivo studies suggest that the immunomodulator may potentially reduce the excessive inflammatory response by the mRNA vaccine without reducing antibody titers.

[0185] All of the methods disclosed and claimed in this specification can be made and executed without undue experimentation in light of this disclosure. Although the compositions and methods of the invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the methods and in the steps or the order of the steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically related agents can be used in place of the agents described herein, and at the same time, it is obvious that the same or similar results can be obtained. All such similar substitutions and modifications as are apparent to those skilled in the art are considered to be within the scope of the spirit, scope, and concept of the invention as defined by the appended claims.

[0186] References The following references, and publications cited elsewhere in this specification, are hereby specifically incorporated by reference to the extent that they show details of exemplary procedures or other details that supplement those described herein. TIFF2025520120000006.tif77160TIFF2025520120000007.tif245160TIFF2025520120000008.tif238160TIFF2025520120000009.tif165160

Claims

Claim 1 An effective amount of (a) an adjuvant and (b) one or more of bafetinib (INNO-406), LY3009120, MK-8353 (SCH900353), amodiaquine, zanubrutinib (BGB-3111), Ku55933, tucidinostat, PD318088, WNK463, and mesylate of TRx0237 (LMTX), or a combination thereof administered to a subject, comprising a method for modulating an immune response. Claim 2 An effective amount of (a) an adjuvant and (b) one or more of bafetinib (INNO-406), LY3009120, MK-8353 (SCH900353), amodiaquine, zanubrutinib (BGB-3111), Ku55933, tucidinostat, PD318088, WNK463, and mesylate of TRx0237 (LMTX), or a combination thereof administered to a population of immune cells, comprising a method for immunopotentiation. Claim 3 An effective amount of (a) an adjuvant and (b) one or more of bafetinib (INNO-406), LY3009120, MK-8353 (SCH900353), amodiaquine, zanubrutinib (BGB-3111), Ku55933, tucidinostat, PD318088, WNK463, and mesylate of TRx0237 (LMTX), or a combination thereof, and (c) an antigen administered to a subject, comprising a method for vaccinating a subject. Claim 4 An effective amount of (a) an adjuvant and (b) one or more of bafetinib (INNO-406), LY3009120, MK-8353 (SCH900353), amodiaquine, zanubrutinib (BGB-3111), Ku55933, tucidinostat, PD318088, WNK463, and mesylate of TRx0237 (LMTX) administered to a subject, comprising a method for preventing or treating cancer, a bacterial infection, or a viral infection. Claim 5 The method according to claim 4, further comprising administering an antigen. Claim 6 The method according to claim 5, wherein the antigen is associated with cancer, bacteria, or a virus. Claim 7 The method according to any one of claims 3 to 6, wherein the antigen is a bacterial antigen, a viral antigen, or a tumor antigen. Claim 8 The method according to any one of claims 1 to 7, wherein (b) comprises or consists of bufetinib (INNO-406). **Claim 9** The method according to any one of claims 1 to 8, wherein (b) comprises or consists of LY3009120. **Claim 10** The method according to any one of claims 1 to 9, wherein (b) comprises or consists of MK-8353 (SCH900353). **Claim 11** The method according to any one of claims 1 to 10, wherein (b) comprises or consists of amodiaquine. **Claim 12** The method according to any one of claims 1 to 11, wherein (b) comprises or consists of zanubrutinib (BGB-3111). **Claim 13** The method according to any one of claims 1 to 12, wherein (b) comprises or consists of Ku55933. **Claim 14** The method according to any one of claims 1 to 13, wherein (b) comprises or consists of tucidinostat. **Claim 15** The method according to any one of claims 1 to 14, wherein (b) comprises or consists of PD318088. **Claim 16** The method according to any one of claims 1 to 15, wherein (b) comprises or consists of WNK463. **Claim 17** The method according to any one of claims 1 to 16, wherein (b) comprises or consists of TRx0237 (LMTX) mesylate. **Claim 18** The method according to any one of claims 1 to 17, wherein the adjuvant comprises 3’3’-cGAMP, Tri-DAP, MDP, and / or mIFN-β. **Claim 19** The method according to any one of claims 1 to 18, wherein the adjuvant comprises a PRR agonist. **Claim 20** The method according to claim 19, wherein the PRR agonist is a TLR agonist. **Claim 21** The method according to claim 20, wherein the TLR agonist is an agonist for TLR1, TLR2 / 1, TLR2, TLR2 / 6, TLR3, TLR4, TLR5, TLR7, TLR8, TLR7 / 8, TLR9, and / or TLR11. **Claim 22** TLR agonists include peptidoglycan, triacyl lipoproteins, lipoteichoic acid, peptidoglycan derived from Bacillus subtilis, peptidoglycan derived from Escherichia coli 0111:B4, peptidoglycan derived from Escherichia coli K12, peptidoglycan derived from Staphylococcus aureus, atypical lipopolysaccharide (LPS), leptospirosis LPS, Porphyromonas gingivalis LPS, synthetic diacylated lipoproteins, FSL-1, Pam2CSK4, lipoarabinomannan derived from M. smegmatis, M.Lipomannan derived from Smegmatis; Triacylated lipoproteins, Pam3CSK4, MALP-2 derived from Mycoplasma, MALP-404 derived from Mycoplasma, OspA of Borrelia burgdorferi, Porin derived from Neisseria meningitidis, Porin derived from Haemophilus influenza, Antigen mixture derived from Propionibacterium acnes, Yersinia LcrV, Lipomannan derived from Mycobacterium, Lipomannan derived from Mycobacterium tuberculosis, GPI anchor of Trypanosoma cruzi, Lysophosphatidylserine of Schistosoma mansoni, Lipophosphoglycan (LPG) of Leishmania major, Glycosylphosphatidylinositol (GPI) of Plasmodium falciparum, Zymosan, Antigen mixture derived from Aspergillus fumigatus, Antigen mixture derived from Candida albicans, Antigen mixture derived from measles hemagglutinin, Double-stranded RNA, Polyadenylic acid - Polyuridylic acid (Poly(A:U)), Polyinosinic acid: Polycytidylic acid (Poly(I:C)), Polyinosinic acid: Polycytidylic acid high molecular weight (Poly(I:C)HMW), Polyinosinic acid: Polycytidylic acid low molecular weight (Poly(I:C)LMW)), LPS derived from Escherichia coli, LPS derived from species of Salmonella, Monophosphoryl lipid A, Flagellin, Flagellin derived from Bacillus subtilis, Flagellin derived from P. aeruginosa, Flagellin derived from S. typhimurium(S.the method according to claim 20 or 21, comprising one or more of, or consisting of, flagellin derived from Salmonella typhimurium, single-stranded RNA having a 6UUAU repeat sequence, single-stranded RNA homopolymer (naked ss poly U), ssRNA derived from HIV-1 LTR (ssRNA40), ssRNA having a 2GUCCUUCAA repeat sequence (ssRNA-DR), imidazoquinoline compounds, imiquimod, Imiquimod VacciGrade™, Gardiquimod VacciGrade™, Gardiquimod™, adenine analog CL264, base analog CL307, guanosine analog roquinimex, TL8-506, thiazoloquinoline compound CL075, imidazoquinoline compound CL097, 2Bxy, R848, R848 VacciGrade™, CpG ODN, and Toxoplasma gondii profilin. **Claim 23** The method according to any one of claims 1 to 21, wherein the TLR agonist is a TLR7 / 8 agonist. **Claim 24** The method according to claim 23, wherein the TLR7 / 8 agonist is R848.

25. The method according to any one of claims 1 to 24, wherein the TLR agonist is a TLR5 agonist.

26. The method according to claim 25, wherein the TLR5 agonist is flagellin.

27. The method according to any one of claims 1 to 26, wherein the TLR agonist is a TLR9 agonist.

28. The method according to claim 27, wherein the TLR9 agonist is a CpG oligonucleotide.

29. The method according to claim 28, wherein the CpG oligonucleotide contains CpG 1826.

30. The method according to any one of claims 1 to 29, further comprising the step of administering an additional adjuvant to the subject.

31. The method according to any one of claims 1 to 30, wherein the subject is a human subject.

32. The method according to any one of claims 1 to 31, wherein (a), (b), and / or the adjuvant are administered by mucosal administration, intramuscular administration, parenteral administration, or subcutaneous administration.

33. The method according to any one of claims 1 to 32, further comprising administering a vaccine composition.

34. The method according to claim 33, wherein the vaccine composition contains an influenza vaccine, a hepatitis B vaccine, or a COVID vaccine.

35. The method according to claim 34, wherein the vaccine composition contains Fluzone or Heplisav.

36. (a) an adjuvant and (b) one or more of bafetinib (INNO-406), LY3009120, MK-8353 (SCH900353), amodiaquine, zanubrutinib (BGB-3111), Ku55933, tucidinostat, PD318088, WNK463, and mesylate of TRx0237 (LMTX), or a combination thereof comprising a pharmaceutical composition.

37. The pharmaceutical composition according to claim 36, further comprising an antigen.

38. The pharmaceutical composition according to claim 37, wherein the antigen contains a bacterial antigen, a viral antigen, or a tumor antigen, or consists of a bacterial antigen, a viral antigen, or a tumor antigen.

39. The pharmaceutical composition according to any one of claims 36 to 38, wherein (b) contains bafetinib (INNO-406) or consists of bafetinib (INNO-406).

40. The pharmaceutical composition according to any one of claims 36 to 39, wherein (b) comprises LY3009120 or consists of LY3009120.

41. The pharmaceutical composition according to any one of claims 36 to 40, wherein (b) comprises MK-8353 (SCH900353) or consists of MK-8353 (SCH900353).

42. The pharmaceutical composition according to any one of claims 36 to 41, wherein (b) comprises amodiaquine or consists of amodiaquine.

43. The pharmaceutical composition according to any one of claims 36 to 42, wherein (b) comprises zanubrutinib (BGB-3111) or consists of zanubrutinib (BGB-3111).

44. The pharmaceutical composition according to any one of claims 36 to 43, wherein (b) comprises Ku55933 or consists of Ku55933.

45. The pharmaceutical composition according to any one of claims 36 to 44, wherein (b) comprises tucidinostat or consists of tucidinostat.

46. The pharmaceutical composition according to any one of claims 36 to 45, wherein (b) comprises PD318088 or consists of PD318088.

47. The pharmaceutical composition according to any one of claims 36 to 46, wherein (b) comprises WNK463 or consists of WNK463.

48. The pharmaceutical composition according to any one of claims 36 to 47, wherein (b) comprises TRx0237 (LMTX) mesylate or consists of TRx0237 (LMTX) mesylate.

49. The pharmaceutical composition according to any one of claims 36 to 48, wherein the adjuvant comprises 3'3'-cGAMP, Tri-DAP, MDP, and / or mIFN-β.

50. The pharmaceutical composition according to any one of claims 36 to 49, wherein the adjuvant comprises a PRR agonist.

51. The pharmaceutical composition according to claim 50, wherein the PRR agonist is a TLR agonist.

52. The pharmaceutical composition according to claim 51, wherein the TLR agonist is an agonist for TLR1, TLR2 / 1, TLR2, TLR2 / 6, TLR3, TLR4, TLR5 TLR7, TLR8, TLR7 / 8, TLR9, and / or TLR11.

53. TLR agonists include peptidoglycan, triacyl lipoproteins, lipoteichoic acid, peptidoglycan derived from Bacillus subtilis, peptidoglycan derived from Escherichia coli 0111:B4, peptidoglycan derived from Escherichia coli K12, peptidoglycan derived from Staphylococcus aureus, atypical lipopolysaccharide (LPS), leptospirosis LPS, Porphyromonas gingivalis LPS, synthetic diacylated lipoprotein, FSL-1, Pam2CSK4, lipoarabinomannan derived from M. smegmatis, lipomannan derived from M. smegmatis; triacylated lipoprotein, Pam3CSK4, MALP-2 derived from Mycoplasma, MALP-404 derived from Mycoplasma, Borrelia burgdorferi OspA, porin derived from Neisseria meningitidis, porin derived from Haemophilus influenzae, Propionibacterium acnes antigen mixture, Yersinia LcrV, lipomannan derived from Mycobacterium genus, lipomannan derived from Mycobacterium tuberculosis, Trypanosoma cruzi GPI anchor, Schistosoma mansoni lysophosphatidylserine, Leishmania amazonensis lipophosphoglycan (LPG), Plasmodium falciparum glycosylphosphatidylinositol (GPI), zymosan, antigen mixture derived from Aspergillus fumigatus, antigen mixture derived from Candida albicans, antigen mixture derived from measles hemagglutinin, double-stranded RNA, polyadenylic acid - polyuridylic acid (poly(A:U)), polyinosinic acid:polycytidylic acid (poly(I:C)), polyinosinic acid:polycytidylic acid high molecular weight (poly(I:C)HMW), polyinosinic acid:polycytidylic acid low molecular weight (poly(I:C)LMW)), LPS derived from Escherichia coli, LPS derived from Salmonella species, monophosphoryl lipid A, flagellin, flagellin derived from Bacillus subtilis, flagellin derived from P. aeruginosa, S.The pharmaceutical composition according to claim 51 or 52, comprising one or more of flagellin derived from Chlamydia muridarum, single-stranded RNA having a 6UUAU repeat sequence, single-stranded RNA homopolymer (naked ss poly U), ssRNA derived from HIV-1 LTR (ssRNA40), ssRNA having a 2GUCCUUCAA repeat sequence (ssRNA-DR), imidazoquinoline compounds, imiquimod, Imiquimod VacciGrade™, Gardiquimod VacciGrade™, Gardiquimod™, adenine analog CL264, base analog CL307, guanosine analog roquinimex, TL8-506, thiazoloquinoline compound CL075, imidazoquinoline compound CL097, 2Bxy, R848, R848 VacciGrade™, CpG ODN, and Toxoplasma gondii profilin, or consisting of the same.

54. The pharmaceutical composition according to any one of claims 36 to 53, wherein the TLR agonist is a TLR7 / 8 agonist.

55. The pharmaceutical composition according to claim 54, wherein the TLR7 / 8 agonist is R848.

56. The pharmaceutical composition according to any one of claims 36 to 55, wherein the TLR agonist is a TLR5 agonist.

57. The pharmaceutical composition according to claim 56, wherein the TLR5 agonist is flagellin.

58. The pharmaceutical composition according to any one of claims 36 to 57, wherein the TLR agonist is a TLR9 agonist.

59. The pharmaceutical composition according to claim 58, wherein the TLR9 agonist is a CpG oligonucleotide.

60. The pharmaceutical composition according to claim 59, wherein the CpG oligonucleotide contains CpG 1826 or is CpG 1826.

61. The pharmaceutical composition according to any one of claims 36 to 60, further comprising the step of administering an additional adjuvant to a subject.

62. The pharmaceutical composition according to any one of claims 36 to 61, comprising an oil-in-water emulsion.

63. The pharmaceutical composition according to any one of claims 36 to 62, based on squalene.

64. The pharmaceutical composition according to any one of claims 36 to 63, comprising AddaVax (trademark).

65. The pharmaceutical composition according to any one of claims 36 to 64, further comprising a vaccine composition.

66. The pharmaceutical composition according to claim 65, wherein the vaccine composition comprises an influenza vaccine, a hepatitis B vaccine, or a COVID vaccine.

67. The pharmaceutical composition according to claim 66, wherein the vaccine composition contains Fluzone or Heplisav or is Fluzone or Heplisav.

68. A method for determining the potency of an adjuvant, the method comprising the following steps: (a) administering the adjuvant to a population of cells; (b) administering a PRR agonist to the population of cells; and (c) measuring the expression of one or more cytokines from the cells.

69. The method according to claim 68, wherein the one or more cytokines comprise one or more of IL-12p40, IP-10, IL-1β, CCL4, TNF-α, and IFN-β.

70. The method according to claim 68, wherein the one or more cytokines comprise IL-12p40, IP-10, IL-1β, CCL4, TNF-α, and IFN-β.

71. The method according to any one of claims 68 to 70, wherein the PRR agonist is a TLR agonist. **Claim 72** The method according to claim 71, wherein the TLR agonist is an agonist for TLR1, TLR2 / 1, TLR2, TLR2 / 6, TLR3, TLR4, TLR5, TLR7, TLR8, TLR7 / 8, TLR9, and / or TLR11. **Claim 73** TLR agonists include peptidoglycan, triacyl lipoproteins, lipoteichoic acid, peptidoglycan derived from Bacillus subtilis, peptidoglycan derived from Escherichia coli 0111:B4, peptidoglycan derived from Escherichia coli K12, peptidoglycan derived from Staphylococcus aureus, atypical lipopolysaccharide (LPS), leptospiral LPS, Porphyromonas gingivalis LPS, synthetic diacylated lipoprotein, FSL-1, Pam2CSK4, lipoarabinomannan derived from M. smegmatis, lipomannan derived from M. smegmatis; triacylated lipoprotein, Pam3CSK4, MALP-2 derived from Mycoplasma, MALP-404 derived from Mycoplasma, Borrelia burgdorferi OspA, porin derived from Neisseria meningitidis, porin derived from Haemophilus influenzae, Propionibacterium acnes antigen mixture, Yersinia LcrV, lipomannan derived from Mycobacterium, lipomannan derived from Mycobacterium tuberculosis, Trypanosoma cruzi GPI anchor, Schistosoma mansoni lysophosphatidylserine, Leishmania tropica lipophosphoglycan (LPG), Plasmodium falciparum glycosylphosphatidylinositol (GPI), zymosan, antigen mixture derived from Aspergillus fumigatus, antigen mixture derived from Candida albicans, antigen mixture derived from measles hemagglutinin, double-stranded RNA, polyadenylic acid-polyuridylic acid (poly(A:U)), polyinosinic acid:polycytidylic acid (poly(I:C)), polyinosinic acid:polycytidylic acid high molecular weight (poly(I:C)HMW), polyinosinic acid:polycytidylic acid low molecular weight (poly(I:C)LMW)), LPS derived from Escherichia coli, LPS derived from Salmonella species, monophosphoryl lipid A, flagellin, flagellin derived from Bacillus subtilis, flagellin derived from P. aeruginosa, S.The method according to claim 71 or 72, comprising one or more of, or consisting of, flagellin derived from typhimurium, single-stranded RNA having a 6UUAU repeat sequence, single-stranded RNA homopolymer (naked ss poly U), HIV-1 LTR-derived ssRNA (ssRNA40), ssRNA having a 2GUCCUUCAA repeat sequence (ssRNA-DR), imidazoquinoline compounds, imiquimod, Imiquimod VacciGrade™, Gardiquimod VacciGrade™, Gardiquimod™, adenine analog CL264, base analog CL307, guanosine analog roquinimex, TL8-506, thiazoloquinoline compound CL075, imidazoquinoline compound CL097, 2Bxy, R848, R848 VacciGrade™, CpG ODN, and toxoplasma gondii profilin. **Claim 74** The method according to claim 71, wherein the TLR agonist is a TLR7 / 8 agonist. **Claim 75** The method according to claim 74, wherein the TLR7 / 8 agonist is R848. **Claim 76** The method according to claim 71, wherein the TLR agonist is a TLR5 agonist. **Claim 77** The method according to claim 76, wherein the TLR5 agonist is flagellin. **Claim 78** The method according to claim 71, wherein the TLR agonist is a TLR9 agonist. **Claim 79** The method according to claim 78, wherein the TLR9 agonist is a CpG oligonucleotide.

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