Conjugates of small molecule immunopotentiators as adjuvants of NFKB activators with enhanced therapeutic effects and reduced toxicity

Abstract

The present disclosure relates to immunomodulatory compositions and methods of use thereof for enhancing response to antigens (e.g., in vaccines), immunotherapies (e.g., cancer immunotherapies), or other immunostimuli. The present disclosure describes immunomodulators having reduced toxicity and increased immune response compared to existing adjuvants. Also disclosed are methods for increasing the immune response to vaccine antigens, cancer immunotherapeutic agents, or other immunostimulatory agents. The present disclosure describes dimeric and polymeric immunomodulators comprising one or more than one pattern recognition receptor (PRR) agonist moiety and one or more than one NF-[kappa] B inhibitor moiety.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 278,011 filed on November 10, 2021 and U.S. Provisional Patent Application No. 63 / 343,176 filed on May 18, 2022, both of which are incorporated herein by reference in their entirety.

[0003] Government Support Statement

[0004] This invention was made with government support under Grants AI112194, AI124286, GM099594, and 75N93019C00041 from the National Institutes of Health. The government has certain rights in this invention. Background Art 1. Technical Field

[0005] The present invention relates generally to the fields of immunology and immunotherapy. Methods and compositions for improving the safety and effectiveness of vaccines and immunotherapies are described herein. 2. Background technology

[0007] Toll-like receptor (TLR) activation is associated with high immunogenicity and protective effects of vaccines 1,2 The addition of TLR adjuvants to subunit and epitope-based vaccine formulations can greatly enhance antibody and T cell levels as well as antigen specificity. 3,4 At present, many small molecule adjuvants have been discovered. 5,6 However, their tolerability in preclinical and clinical studies has limited the use of many of these compounds, necessitating reformulation or redesign. 7 .

[0008] Historically, adjuvant discovery has been empirical, but with the advent of synthetic small molecule adjuvants, modern drug discovery techniques have been applied to optimize adjuvant properties. This has led to the development of a class of adjuvants collectively referred to as small molecule immunopotentiators (SMIPs). 8,9 In this class, imidazoquinolinones that activate Toll-like receptors-7 and -8 (TLR7 / 8) such as imiquimod (R837) and resiquimod (R848) have been extensively studied. Imiquimod is currently approved for clinical immunotherapy in the form of a topical cream. 9-11 The study showed that these SMIPs can elicit antigen-specific cellular responses when administered as adjuvants 12-14 In addition, resiquimod activated TLR7 / 8 to produce antitumor activity, which was mediated by APC activation of CD8 + T cells and CD4 +Promoted by Th cells, which is due to the production of IFN-γ, IL-2, and IL-10, thereby enhancing proliferation 15-17 However, the high bioavailability of imidazoquinolinones and structurally related compounds leads to unacceptable levels of systemic inflammation due to adjuvant toxicity, thus greatly limiting their usefulness.

[0009] Therefore, current vaccination and immunotherapy approaches, especially those using adjuvants, may raise safety issues, and there is a need in the art for strategies to improve the safety and tolerability of vaccines and cancer immunotherapy approaches. Summary of the invention

[0010] Immunomodulators capable of enhancing the immunogenicity and tolerability of protein subunit vaccines are described herein. Synthetic poly-TLR7 / 8a-dopamine (p(TLR7 / 8a-dopa)) conjugates are described herein that reduce toxicity markers and enhance immune responses. As described herein, in the context of antigen vaccination, p(TLR7 / 8a-dopa) produces stronger humoral and cellular immunity than peptides lacking immunomodulators. The present disclosure describes and demonstrates strategies for using immunomodulators to modify existing responses and adjust activity to ideal levels without the need to develop new agonists. The present disclosure also describes the synthesis and use of novel hybrid molecules comprising a TLR 7 / 8 agonist portion and an NF-κB inhibitor portion. Small molecule NF-κB inhibitors can reduce systemic toxicity by reducing proinflammatory systemic IL-6 and TNF-α levels in vaccine formulations. Conjugating NF-κB inhibitory small molecules to TLR 7 / 8 agonist adjuvants limits their systemic spread and alters responses elicited by immune cells. Safety and tolerability can be improved without affecting adjuvantity and anti-tumor activity.

[0011] The present disclosure relates to methods for vaccinating a subject, comprising administering to the subject a hybrid molecule comprising a TLR 7 / 8 agonist portion and a NF-κB inhibitor portion. Methods for treating or preventing cancer are also described, comprising administering to the subject a hybrid molecule comprising a TLR 7 / 8 agonist portion and a NF-κB inhibitor portion. Pharmaceutical compositions comprising a hybrid molecule comprising a TLR 7 / 8 agonist portion and a NF-κB inhibitor portion are also provided.

[0012] The method includes a method of enhancing the immune response of a subject, which includes administering an immunomodulator comprising one or more TLR7 / 8 agonist moieties and one or more NF-κB inhibitor moieties. The TLR7 / 8 agonist moiety and / or one or more NF-κB inhibitor moieties may be attached to a polypeptide backbone. The immunomodulator of the present disclosure may include (a) two or more (e.g., 2, 3, 4, 5, 6 or more) TLR7 / 8 agonist moieties (e.g., imidazoquinoline or a derivative thereof) attached to a polypeptide backbone; and (b) two or more (e.g., 2, 3, 4, 5, 6 or more) NF-κB inhibitor moieties (e.g., dopamine or a derivative thereof) attached to a polypeptide backbone, each moiety being connected by a linker. Figure 7 A schematic diagram of this class of immunomodulators is provided. Figure 8B and Fig. 9 Exemplary immunomodulators of the present disclosure are provided.

[0013] Described herein are compounds, molecules, polymers, polypeptides, immunomodulators, monomeric immunomodulators, polymeric immunomodulators, PRR agonists, TLR agonists, TLR7 / 8 agonists, NF-κB inhibitors, methods for synthesizing immunomodulators, methods for immune activation, methods for TLR activation, methods for immunomodulation, methods due to NF-κB inhibition, pharmaceutical compositions, vaccination methods, methods for enhancing an immune response to an antigen, methods for treating cancer, and methods for preventing cancer.

[0014] The compounds of the present disclosure include polymers, polypeptides, immunomodulators (including monomeric and polymeric immunomodulators), TLR agonists (including TLR7 / 8 agonists) and NF-κB inhibitors. The compounds of the present disclosure may contain at least 1, 2, 3 or more than 3 of the following substances: a TLR agonist portion (e.g., a TLR7 / 8 agonist portion), a NK-kB inhibitor portion, a linker, an amino acid and a polypeptide backbone. Any one or more than one of these components may be excluded from the compounds of the present disclosure.

[0015] The method disclosed herein includes a therapeutic method, a disease prevention method, a vaccination method, a synthesis method, an immune activation method, and a cell activation method. The method disclosed herein may include at least one, two, three, or more than three of the following steps: administering an immunomodulator, administering a polymer immunomodulator, administering p(TLR7 / 8a-dopa), administering a vaccine, administering an antigen, producing a pharmaceutical composition comprising an antigen and an immunomodulator, diagnosing an object with cancer, diagnosing an object with a viral infection, diagnosing an object with an autoimmune disease, and administering cancer therapy. Any one or more than one of these steps may be excluded from the method disclosed herein.

[0016] The pharmaceutical composition of the present invention includes a composition for vaccination, a composition for disease treatment, a composition for disease prevention, a composition for immune activation and a composition for immunosuppression. The pharmaceutical composition of the present invention may contain at least one, two, three or more than three of the following substances: an immunomodulator, a polymer immunomodulator, p(TLR7 / 8a-dopa), an NF-κB inhibitor, a TLR agonist, a TLR7 / 8 agonist, an antigen, a bacterial antigen, a viral antigen, a tumor antigen, a peptide, an excipient, a carrier and a salt.

[0017] Provided herein are compounds of formula (I)

[0018]

[0019] wherein L is a linker; and

[0020] wherein n is an integer from 0 to 10, and m is an integer from 0 to 10.

[0021] The linker can be a bond, -NH-, -CH 2 -, -C(O)-, -C(O)N-, -alkyl-C(O)-, -alkenylC(O)-, -alkyl-NC(O)-, -alkenyl-NC(O)-, -C(O)NC(O)-, -C(O)NH(CH 2 ) z NC(O)-, -alkylC(O)NH(CH 2 ) z NC(O)-, or -alkenyl-C(O)NH(CH 2 ) z NC(O)-, wherein z is an integer from 1 to 10. The linker may be a DBCO linker. The linker may include

[0022] Compounds can also be defined as

[0023]

[0024] wherein n is an integer from 0 to 10, and m is an integer from 0 to 10.

[0025] N may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. m may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 1. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 2. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 3. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 4. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 5. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 6. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 7. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 8. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 9. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 1. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 2. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 3. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 4. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 5. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 6. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 7. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 8. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 9. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 10. n may be 5 and m is 4.

[0026] Compounds can also be defined as

[0027]

[0028] Also disclosed herein are compounds of formula (II)

[0029]

[0030] in:

[0031] A is the NF-κB inhibitor part;

[0032] B is an amino acid or an amino acid analog;

[0033] L is the connector;

[0034] C is an amino acid or an amino acid analog;

[0035] D is a TLR agonist moiety; and

[0036] wherein n is an integer from 1 to 10, and m is an integer from 1 to 10.

[0037] n can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. m can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The linker can be a bond, -NH-, -CH 2 -, -C(O)-, -C(O)N-, -alkyl-C(O)-, -alkenylC(O)-, -alkyl-NC(O)-, -alkenyl-NC(O)-, -C(O)NC(O)-, -C(O)NH(CH 2 ) z NC(O)-, -alkylC(O)NH(CH 2 ) z NC(O)-, or -alkenyl-C(O)NH(CH 2 ) z NC(O)-, wherein z is an integer from 1 to 10. The linker may be a DBCO linker. The linker may include a area.

[0038] The NF-κB inhibitor portion may be or may include a NF-κB inhibitor in Table 1 or a portion thereof having NF-κB inhibitor activity. The NF-κB inhibitor portion may be ferulic acid or a derivative thereof. The NF-κB inhibitor portion may be vanillin or a derivative thereof. The NF-κB inhibitor portion may be honokiol or a derivative thereof. The NF-κB inhibitor portion may be dopamine or a derivative thereof.

[0039] B may be an amino acid or an analog thereof in Table 2. B may be an amino acid or an analog thereof in Table 3. B may be glutamic acid or an analog thereof. C may be an amino acid or an analog thereof in Table 2. C may be an amino acid or an analog thereof in Table 3. C may be glycine or an analog thereof.

[0040] Compounds can also be defined as:

[0041]

[0042] wherein n is an integer from 0 to 10, and m is an integer from 0 to 10.

[0043] Compounds can also be defined as:

[0044]

[0045] wherein n is an integer from 0 to 10, and m is an integer from 0 to 10.

[0046] n may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. m may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 1. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 2. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 3. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 4. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 5. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 6. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 7. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 8. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 9. When m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n may be 1. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 2. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 3. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 4. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 5. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 6. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 7. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 8. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 9. When n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m may be 10. n may be 5 and m is 4.

[0047] Compounds can also be defined as:

[0048]

[0049] Also disclosed are methods of immune activation, comprising administering a compound disclosed herein (e.g., a polymer immunomodulator) to an immune cell population. The immune cell population may include T cells. The immune cell population may include macrophages. The method may be an in vitro method. The method may be an in vivo method.

[0050] Also disclosed herein is a method for vaccinating a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a compound disclosed herein (e.g., a polymer immunomodulator). The method may include or may also include administering an antigen to the subject. The pharmaceutical composition may include or may also include an antigen. The method may be used to prevent a disease in the subject. The method may be used to treat a disease in the subject. The subject may be a subject to which an adjuvant has been previously administered. The subject may be a subject that has an adverse reaction to an adjuvant.

[0051] Also disclosed herein is a method for treating or preventing cancer, comprising administering to a subject an effective amount of a pharmaceutical composition comprising a compound disclosed herein (e.g., a polymer immunomodulator). The method may include or may also include administering other cancer therapies to the subject. Other cancer therapies may include chemotherapy, radiotherapy, immunotherapy, or a combination thereof. Other cancer therapies may include immunotherapy, such as administering a checkpoint inhibitor. The subject may be a subject that has not yet been diagnosed with cancer. The subject may be a subject that has been diagnosed with cancer. The subject may be a subject that has previously been treated for cancer with a previous therapy. The subject may be determined to be resistant to a previous therapy. The pharmaceutical composition may be administered intratumorally.

[0052] Also disclosed are pharmaceutical compositions comprising (a) a compound disclosed herein (eg, a polymeric immunomodulator) and (b) an antigen.

[0053] Antigens can be bacterial antigens. Various bacterial antigens are recognized in the art and are contemplated herein. Antigens can be viral antigens. Various viral antigens are recognized in the art and are contemplated herein. Certain examples include, for example, dengue antigens (e.g., capsid proteins or portions thereof of dengue serotype-2 (DENV-2C)), HIV antigens (e.g., gp120 or portions thereof), influenza antigens, and SARS-CoV-2 antigens (e.g., spike proteins or portions thereof). Antigens can be tumor antigens. Various tumor antigens are recognized in the art and are contemplated herein. Tumor antigens can be antigens that are specifically or preferentially expressed by tumor cells compared to healthy cells, including novel antigens that can be found by methods disclosed in the fields of cancer biology and immunology.

[0054] The present disclosure also describes compounds of formula (I):

[0055]

[0056] Where R 1 H, C 1 -C 12 Alkyl, C 5 -C 12 Cycloalkyl, C 4-C 12 Heterocycloalkyl, C 6 -C 10 Aryl and C 4 -C 10 Heteroaryl, where R 1 Optionally substituted with one or more than one Y; R 2 and R 3 Independently H, C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 2 -C 12 Alkynyl, C 1 -C 12 Heteroalkyl, C 2 -C 12 Heteroalkenyl or C 2 -C 12 heteroalkynyl, or together form a 5- to 6-membered carbocyclic or heterocyclic ring, wherein the 5- to 6-membered carbocyclic or heterocyclic ring is optionally substituted with one or more than one Y; X is a bond, -N-, -C(O)-, -alkyl-C(O)-, -alkenyl-C(O)-, -C(O)N-, -NC(O)-, -alkyl-NC(O)-, alkenyl-NC(O)-, -C(O)N-, -C(O)NC(O)-, -C(O)NH(CH 2 ) z NC(O)-, -alkyl-C(O)NH(CH 2 ) z NC(O)-, or -alkenyl-C(O)NH(CH 2 ) z NC(O)-; z is an integer from 1 to 10; R A is attached to one or more ring atoms, and R A are independently hydrogen, hydroxyl, C 1 -C 12 Alkoxy, C 2 -C 12 Alkenyloxy, C 2 -C 12 Y is substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NH 2 、NO 2 NR 4 R 5 、NC(O)R 5 、OC(O)NR 4 R 5 、OC(O)OR4 、C(O)R 4 、C(O)OR 4 , SH, SR 4 , OR 4 , where R 4 and R 5 is independently substituted or unsubstituted C 1 -C 6 Alkyl. 1 Can be C 1 -C 12 Alkyl. The alkyl group may be n-butyl. 2 and R 3 Together they may form a benzene ring. The benzene ring may be an unsubstituted benzene ring. X may be an amide. X may include one or more than one of an alkyl group, an alkenyl group, an amide group, and a urea group. R A It may be an unsubstituted phenyl group. A The phenyl group may be substituted.

[0057] The compound of formula (I) can also be defined as one of the following compounds:

[0058]

[0059]

[0060] Also provided are compounds of formula (II):

[0061] ABC (II)

[0062] Wherein A is a TLR agonist, B is a linker, and C is a NF-κB inhibitor. The TLR agonist may be a TLR 7 / 8 agonist. The TLR7 / 8 agonist may be an imidazoquinolinone or a derivative thereof. The linker may be a bond, -NH-, -CH 2 -, -C(O)-, -C(O)N-, -alkyl-C(O)-, -alkenyl-C(O)-, -alkyl-NC(O)-, -alkenyl-NC(O)-, -C(O)NC(O)-, -C(O)NH(CH 2 ) z NC(O)-, -alkylC(O)NH(CH 2 ) z NC(O)-, or -alkenyl-C(O)NH(CH 2 ) zNC(O)-, wherein z is an integer from 1 to 10. The linker can be oriented in any direction, for example, the amide linker can be connected to the TLR agonist through the amide amine or the amide carbon. The NF-κB inhibitor can be ferulic acid or a derivative thereof. The NF-κB inhibitor can be vanillin or a derivative thereof. The NF-κB inhibitor can be dopamine or a derivative thereof. The NF-κB inhibitor can be honokiol or a derivative thereof. The compound can also be defined as one of the following compounds:

[0063]

[0064]

[0065] Also described are methods for vaccinating a subject. The method may include administering to the subject an effective amount of a pharmaceutical composition comprising a compound of formula (I) or a compound of formula (II). The method may also include administering to the subject an antigen. The antigen may be a bacterial antigen. The antigen may be a viral antigen. The antigen may be a dengue antigen. The dengue antigen may include a capsid protein of dengue serotype-2 (DENV-2C). The antigen may be an HIV antigen. The HIV antigen may include gp120. The antigen may be a SARS-CoV-2 antigen. The SARS-CoV-2 antigen may be a SARS-CoV-2 spike protein or a portion thereof. The antigen may be a tumor antigen. The subject may be a human subject. The method may be used to prevent a disease in a subject. The method may be used to treat a disease in a subject. The subject may be a subject to which an adjuvant has been previously administered. The subject may be a subject that has an adverse reaction to an adjuvant.

[0066] Also described are methods for treating or preventing cancer, comprising administering to a subject an effective amount of a pharmaceutical composition comprising a compound of formula (I) or a compound of formula (II). The method may include or may also include administering to the subject other cancer therapies. Other cancer therapies may include chemotherapy, radiotherapy, immunotherapy, or a combination thereof. Other cancer therapies may include immunotherapy. The subject may be a human subject. The subject may be a non-human primate, a laboratory 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 that has not yet been diagnosed with cancer. The subject may be a subject that has been diagnosed with cancer. The subject may be a subject that has previously been treated for cancer with a previous therapy. The subject may be a subject that is determined to be resistant to a previous therapy. The pharmaceutical composition may be administered to the subject intratumorally.

[0067] Also described are pharmaceutical compositions comprising a compound of formula (I) or a compound of formula (II). The pharmaceutical composition may also comprise an antigen. The antigen may be a bacterial antigen. The antigen may be a viral antigen. The antigen may be a dengue antigen. The dengue antigen may include a capsid protein of dengue serotype-2 (DENV-2C). The antigen may be an HIV antigen. The HIV antigen may include gp120. The antigen may include a SARS-CoV-2 antigen. The SARS-CoV-2 antigen may include a SARS-CoV-2 spike protein or a portion thereof. The antigen may include an influenza antigen. The antigen may include a tumor antigen.

[0068] The compound of formula (I) or the compound of formula (II) can be administered via intramucosal, intramuscular, parenteral or subcutaneous administration. The compound of formula (I) or the compound of formula (II) can be administered via the administration routes described herein. The compound of formula (I) or the compound of formula (II) can be administered before the administration of the antigen. The compound of formula (I) or the compound of formula (II) can be administered after the administration of the antigen. The compound of formula (I) or the compound of formula (II) can be administered at least or at most 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 10 hours or 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 or 14 days (or any range derivable therefrom) before or after the administration of the antigen. The compound of formula (I) or formula (II) and the antigen can be administered simultaneously. The compound of formula (I) or formula (II) and the antigen can be administered topically to the same part of the subject. The compound of formula (I) or (II) and the antigen can be administered to the subject in the same composition. The compound of formula (I) or (II) can be administered in a different composition from the antigen. The subject can be a subject that has an adverse reaction to a previously administered adjuvant or vaccine. Adverse reactions can include systemic inflammation.

[0069] At least 12 mg of a compound of Formula (I) or Formula (II) can be administered to a subject. At least, up to, or about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, The compound of formula (I) or formula (II) can be administered to a subject at 0.2 mg / kg.At least, up to, or about 0.01 mg / kg, 0.05 mg / kg, 0.07 mg / kg, 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.3 mg / kg, 0.35 mg / kg, 0.4 mg / kg, 0.45 mg / kg, 0.5 mg / kg, 0.55 mg / kg, 0.6 mg / kg, 0.65 mg / kg, 0.75 mg / kg, 0.80 mg / kg, 0.90 mg / kg, 100 mg / kg, 120 mg / kg, 140 mg / kg, 150 mg / kg, 160 mg / kg, 170 mg / kg, 180 mg / kg, 190 mg / kg, 200 mg / kg, 210 mg / kg, 220 mg / kg, 23 7mg / kg, 0.75mg / kg, 0.8mg / kg, 0.85mg / kg, 0.9mg / kg, 1mg / kg, 1.5mg / kg, 2mg / kg, 2.5mg / kg, 3mg / kg, 4mg / kg, 5mg / kg, 6mg / kg, 7mg / kg, 8mg / kg, 9mg / kg, 10mg / kg, 11mg / kg, 12mg / kg, 13mg / kg, 14m 47 mg / kg, 48 mg / kg, 49 mg / kg or 50 mg / kg (or any range derivable therebetween) of a compound of formula (I) or (II). The amount of the compound of Formula (I) or Formula (II) administered to a human or non-human primate subject may correspond to a dose equal to or greater than 50 micrograms administered to a mouse.The amount of a compound of Formula (I) or (II) administered to a human or non-human primate subject can correspond to greater than or less than 1 microgram, 2 micrograms, 3 micrograms, 4 micrograms, 5 micrograms, 6 micrograms, 7 micrograms, 8 micrograms, 9 micrograms, 10 micrograms, 20 micrograms, 30 micrograms, 40 micrograms, 50 micrograms, 60 micrograms, 70 micrograms, 80 micrograms, 90 micrograms, 100 micrograms, 125 micrograms, 150 micrograms, 175 micrograms, 200 micrograms, 225 micrograms, 250 micrograms, 300 micrograms, 400 micrograms, 500 micrograms, 600 micrograms, 700 micrograms, 800 micrograms, 900 micrograms, 1000 micrograms, 1250 micrograms, 1500 micrograms, 1750 micrograms, 2000 micrograms, 2250 micrograms, 250 ... or 900 μg (or any range derivable therebetween).

[0070] The subject can be a human. The subject can be a non-human primate, mouse, goat, rabbit, dog, horse, or sheep.

[0071] The method can be used to prevent a disease in a subject. The method can be used to treat a disease in a subject.

[0072] The compounds or compositions disclosed herein can be formulated for intramucosal, intramuscular, parenteral or subcutaneous administration. The composition may further comprise a pharmaceutical excipient.

[0073] The methods and compositions of the present disclosure can be used to reduce systemic inflammation, such as inflammation associated with vaccination and / or vaccines containing adjuvants. The methods of the present disclosure can reduce adjuvant-induced inflammation while also increasing adaptive immune responses. The methods of the present disclosure can reduce one or both of IL-6 and TNF-α. The methods and compositions of the present disclosure can be used to enhance antigen presentation and T cell activation, and / or increase antibody titers. The methods and compositions of the present disclosure can also enhance epitope selectivity, shift epitope selectivity, and / or provide vaccines for generating broad-spectrum antibody responses.

[0074] Vaccine preparations as active immunogenic ingredients can be prepared as injections in the form of liquid solutions or suspensions; they can also be prepared in solid form suitable for dissolution or suspension in liquids before infection. The preparations can be emulsified or encapsulated in liposomes. The active immunogenic ingredients are usually mixed with a carrier that is pharmaceutically acceptable and compatible with the active ingredient.

[0075] Administration of vaccines according to the present disclosure can be carried out by any common route, as long as the target tissue can be reached by this route so as to deliver the antigen to the site to the greatest extent possible to obtain the maximum (or in some cases the minimum) immune response. Administration is usually carried out by in situ, intradermal, mucosal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Other delivery areas include: oral, nasal, buccal, rectal, vaginal or local. The vaccine of the present invention is preferably administered parenterally by injection, such as by subcutaneous or intramuscular injection.

[0076] Vaccines can be administered in a manner compatible with dosage formulations, and in an amount effective for prevention and / or treatment. The amount to be administered depends on the object to be treated, including, for example, the ability of the object's immune system to synthesize antibodies, and the expected degree of protection or treatment. Suitable dosage ranges are on the order of hundreds of micrograms of active ingredients for each vaccination, ranging from about 0.1 mg to 1000 mg, such as about 1 mg to 300 mg, or about 10 mg to 50 mg. The appropriate regimen for initial administration and booster injection is also variable, but is represented by subsequent inoculation or other administrations followed by initial administration. The precise amount of active ingredient to be administered depends on the judgment of the practitioner and can be specific to each object. It will be apparent to those skilled in the art that the therapeutically effective amount of the nucleic acid molecule or fusion polypeptide of the present invention will depend on the administration regimen, the unit dose of the antigen administered, whether the vaccine composition is administered in combination with other therapeutic agents, and the immune status and health status of the recipient.

[0077] Vaccines can be administered in a single dose regimen or in a multiple dose regimen. A multiple dose regimen is a regimen in which the main course of vaccination may include, for example, 1 to 10 separate doses, followed by other doses at subsequent intervals required for maintaining and / or strengthening the immune response, for example, a second dose is administered at 1 month to 4 months, and if necessary, subsequent one or more than one dose is administered a few months later. It is expected that regular booster immunizations are administered every 1 year to 5 years (usually 3 years) to maintain the desired protective immunity level.

[0078] The vaccine can be provided in one or more than one "unit dose". A unit dose is defined as a vaccine containing a predetermined amount, which is calculated to produce an expected response related to its administration (i.e., an appropriate route and treatment regimen). The dosage, specific route and formulation are all within the capabilities of technicians in the clinical field. The subject to be treated, in particular the state of the subject's immune system and the expected protection can also be evaluated. The unit dose does not need to be administered as a single injection, but can include continuous infusion within a set time period. The unit dose of the present invention can be conveniently described in mg / kg body weight. The dosage of the NFкB inhibitor, adjuvant or antigen can be at least, at most 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, 1000 mg / kg or any range derivable therebetween. Similarly, the amount of the vaccine delivered can be from about 0.2 mg / kg body weight to about 8.0 mg / kg body weight. 0.1mg / kg, 0.2mg / kg, 0.3mg / kg, 0.4mg / kg, 0.5mg / kg, 0.8mg / kg, 1.0mg / kg, 1.5mg / kg, 2.0mg / kg, 2.5mg / kg, 3.0mg / kg, 4.0mg / kg, 5.0mg / kg, 5.5mg / kg, 6.0mg / kg, 6.5mg / kg, 7.0mg / kg and 7.5mg / kg (or any range derivable therefrom) of the vaccine can be delivered to an individual in vivo. The dosage of the vaccine depends largely on the body weight and physical condition of the subject being treated as well as the route of administration and the frequency of treatment.

[0079] The methods of the present disclosure may comprise two or more than two administrations of one or more than one composition. It is contemplated that the compositions can 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, or 14 days, or 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 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 60, 72, 84, or 96 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years (or any range derivable therein).

[0080] Also described are kits comprising the disclosed compositions and instructions for use.

[0081] The method also includes testing the patient for an infection, such as a viral infection, or diagnosing the patient for an infection, such as a viral infection.

[0082] Any embodiment discussed in the context of antibodies can be implemented in any method embodiment discussed herein.

[0083] Any method related to a therapeutic, diagnostic, or physiological purpose or effect may also be described using "use" claim language, such as the "use" of any compound, composition, or agent discussed herein in achieving or carrying out said therapeutic, diagnostic, or physiological purpose or effect.

[0084] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by referring to one or more of these drawings in conjunction with the detailed description of specific embodiments provided herein.

[0086] Figure 1 Innate immune activation measured by NF-κB in RAW-Blue 264.7 macrophage assay is shown. Cells were incubated with each compound at 37°C for 18 h. The supernatant was removed, incubated with QUANTIBlue for 1 h, and the absorbance at 620 nm was measured. Error bars represent standard deviation (SD). Samples were repeated three times. Statistical differences were determined by two-tailed t-test (n=3 per group).

[0087] FIG. 2A to FIG. 2B The co-stimulatory molecule CD40 ( Figure 2A ) and CD86( Figure 2B ) measured immune activation. Cells were incubated with each compound at 37°C for 18 h. Error bars represent SD. Samples were repeated three times. Statistical differences were determined by two-tailed t-test (n=3 per group). Each experiment was repeated three times with similar results.

[0088] Figure 3 Shown is the innate immune activation measured by the concentration of TNF-α in the supernatant of BMDM after exposure to TLR7 / 8 ligands such as pTLR7 / 8_mod, pTLR7 / 8 or mTLR7 / 8 for 12h. Cells were incubated with each compound at 37°C for 18h. The supernatant was removed and analyzed using ELISA, measuring the absorbance at 450nm. Error bars represent SD. Samples were repeated three times. Statistical differences were determined by two-tailed t-test (n=3 per group).

[0089] FIG. 4A to FIG. 4C Figure 2 shows the in vivo IL-23 expression in C57BL / 6 mice 24 h after injection as measured by cytokine bead assay (CBA). Figure 4A )、IL6( Figure 4B ) and MCP-1( Figure 4C ) serum levels. C57BL / 6 mice were subcutaneously immunized with alum containing only 100 μg OVA or with 10 μg TLR7 / 8 ligands formulated as p(TLR7 / 8)+Alum, p(TLR7 / 8_mod)+Alum, p(TLR7 / 8)+Pmod+Alum on days 0 and 14. All symbols represent individual mice. Statistical differences were determined by two-tailed t-test (n=5 per group).

[0090] Figure 5Serum anti-OVA IgG titers of C57BL / 6 mice (n=5 per group) immunized subcutaneously with 100 μg of alum containing OVA alone or 10 μg of TLR7 / 8 ligand formulated as p(TLR7 / 8)+Alum, p(TLR7 / 8_mod)+Alum, p(TLR7 / 8)+Pmod+Alum at days 0 and 14 are shown. Serum antibody titers were analyzed by ELISA at week 3. Statistical differences were determined by two-tailed t-tests.

[0091] FIG. 6A to FIG. 6C IL-6 ( Fig. 6A )、IL-2( Figure 6B ) and TNF-α( Figure 6C ) levels. C57BL / 6 mice (n=5 per group) were subcutaneously immunized with 100 μg of OVA in alum or 10 μg of TLR7 / 8 ligands formulated as p(TLR7 / 8)+alum, p(TLR7 / 8_mod)+alum, p(TLR7 / 8)+Pmod+alum on days 0 and 14. Two weeks after the second immunization, dLN cells were collected and stimulated with OVA again for 48 h, and the supernatants were analyzed for cytokines by CBA. Statistical differences were determined by two-tailed t-tests.

[0092] Figure 7 A schematic diagram of a polymeric immunomodulator of the present disclosure is shown. The modulator comprises repeating dopamine units (left; circles) and repeating imidazoquinoline units (right; squares), each attached to a polypeptide backbone and connected via a linker.

[0093] FIG. 8A to FIG. 8B A schematic diagram of the synthesis scheme of the polymer immunomodulator-p(TLR7 / 8a_dopa) disclosed in the present invention is shown.

[0094] Fig. 9 The structure of p(TLR7 / 8a_dopa) is shown.

[0095] Figures 10A to 10B, (A) Traditional SMIP adjuvant NF-κB pathway. Activation of TLR7 / 8 with agonists results in downstream proinflammatory responses and responses associated with adaptive immunity (antigen presentation). (B) SMIP activation of the NF-κB pathway using the TLR7 / 8 agonist / NF-κB inhibitor dimer disclosed herein. Activation of TLR7 / 8 by dimers also results in agonist activation of the NF-kB pathway, while the tethered enhancer inhibits specific parts of the pathway, thereby reducing proinflammatory immune responses and enhancing or altering adaptive immune responses.

[0096] Figure 11A to Fig. 11C, (A) TLR 7 / 8 small molecule enhancer (SMIP) imidazoquinolinone. (B) NF-κB small molecule inhibitor. (C) Synthetic TLR 7 / 8 agonist / NF-κB inhibitor dimers 1-6, 12 and 13; intermediate compounds 7-11.

[0097] Figure 12A to Fig. 12C In vitro assays to measure TLR 7 / 8 NF-κB activation and enhancement of synthetic dimers are shown. (A) Immune activation was measured by NF-κB stimulated RAW-Blue activation after 24 h incubation with 500 nM of compound at 37°C. (B) IL-6 expression (ELISA). (C) Cell surface protein expression (FACS) was measured after 8 h incubation with bone marrow derived macrophages (BMDC). Compounds were measured at 200 nm.

[0098] Figures 13A to 13B, (A) Intracellular reactive oxygen species (ROS) measured by incubating RAW macrophages with CM-H2DCFDA (6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester), and fluorescence measured using flow cytometry. (B) Nitrite levels in the supernatant of RAW macrophages incubated with 500 nM compound for 16 h measured using Griess reagent.

[0099] Figures 14A to 14B, (A) COX-2 protein expression in lysates of RAW macrophages incubated with 500 nM compound for 16 h. Samples were repeated three times. (B) Graphical representation of COX-2 expression. Statistically significant differences were observed compared to TLR 7 / 8 SMIP by one-way ANOVA, *p≤0.05, ****p≤0.0001.

[0100] Figures 15A to 15C In vivo analysis of TLR 7 / 8 agonist / NF-κB inhibitor dimers. (A) Overview of in vivo vaccination investigation. (B) Serum IL-6 levels were measured 1 hour after injection. IL-6 levels of the linked compound were not detected. (C) Serum TNF-α levels were measured 1 hour after injection. TNF-α levels of the linked compound were not detected.

[0101] Figure 16A to Fig. 16C Serum anti-OVA antibodies were measured on day 28. (A) Serum anti-OVA Ig. (B) Serum anti-OVA IgG. (C) Serum anti-OVA IgA. Compared with the PBS control group, there was a statistically significant difference by one-way ANOVA, *p≤0.05, ****p≤0.0001.

[0102] Figure 17A to Figure 17C, (A) Overview of in vivo tumor model experiments. (B) Systemic IL-6 levels in serum measured 2 hours after intratumoral injection of compounds. (C) Systemic TNF-α levels in serum measured 2 hours after intratumoral injection of compounds.

[0103] Figure 18A to Figure 18C, (A) Hematological analysis of peripheral blood leukocytes (WBC) 24 hours after intratumoral injection of compound. (B) Hematological analysis of peripheral blood lymphocytes 24 hours after intratumoral injection of compound. (C) Viability graph. By one-way ANOVA, compared with PBS, there is a statistically significant difference, *p≤0.05, ****p≤0.0001.

[0104] Figures 19A to 19B, in vivo analysis using R848 and NF-κB inhibitors. Cytokine levels in serum measured 1 hour after injection. (A) IL-6 levels were not significantly reduced compared to R848. (B) TNF-α levels were not significantly reduced compared to R848.

[0105] Fig. 20 , MTT assay shows the viability of cells treated with agonist and agonist dimer. At the assayed concentrations, the viability of the cells was comparable to that of resting cells.

[0106] Figures 21A-21B, (A) SINFEKL MHC-specific tetramers in ex vivo spleens at day 28 post-inoculation. (B) Neonatal splenocyte proliferation assay.

[0107] Fig. 22 , Overlay of results from six in vivo tumor model analyses using peritumoral injections of SMIP modulator dimers into a subcutaneous CT-26 tumor model. Agonists were injected when tumor size was approximately 75 cc, followed by three additional injections every four days.

[0108] Figures 23A to 23F, analysis of six in vivo tumor models using peritumoral injection of SMIP modulator dimers into a subcutaneous CT-26 tumor model. (A) PBS control group. (B) TLR 7 / 8 SMIP. (C) TLR 7 / 8 agonist resiquimod (R-848). (D) TLR 7 / 8 agonist 3M-052. (E) Compound 1. (F) Compound 2. DETAILED DESCRIPTION

[0109] Imidazoquinolinone derivatives that activate Toll-like receptors (TLR) 7 / 8 are small molecule immunopotentiators (SMIPs) with potent activity as vaccine adjuvants and antitumor agents. However, these molecules have high bioavailability, which can lead to unacceptable levels of systemic inflammation due to adjuvant toxicity, greatly limiting their use.

[0110] Small molecule NF-κB inhibitors can be used to boost cytidine monophosphate guanosine (CPG, a TLR 9 agonist) in vaccine formulations. Small molecule NF-κB inhibitors capsaicin and honokiol have been shown to reduce proinflammatory systemic IL-6 and TNF-α levels while maintaining vaccine protection. 19 However, this effect was not observed when the in vivo experiments were repeated using R848 as an adjuvant. This may be due to the high diffusivity of small molecule adjuvants and immunopotentiators.

[0111] The present disclosure is based, at least in part, on the design of hybrid molecules in which an imidazoquinolinone derivative 20 Through the conjugated amine handle (amine handle) with vanillic acid, catechol and honokiol 19 The derivatives were covalently linked to reduce the extent 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 synthetic dimers. The results showed that these dimers reduced systemic toxicity to baseline levels while maintaining adjuvant properties in the vaccine formulation. In addition, the selected dimers improved survival in the CT26WT mouse colon cancer tumor model while inducing low adjuvant toxicity.

[0112] I. Definitions

[0113] As used herein, the term "adjuvant" refers to a substance that accelerates, prolongs and / or enhances the quality and / or intensity of the immune response to an antigen when administered before, with, or after administration of the antigen, compared to administration of the antigen alone.

[0114] NFκB (also known as "NF-κB" or "NFkB") refers to the protein Nuclear Factor κB (encoded by the gene NFKB1).

[0115] As used herein, the term "vaccine" is intended to mean a composition that can be administered to a human or animal to induce an immune system response; the immune system response can result in the production of antibodies or simply the activation of certain cells, such as antigen presenting cells, T lymphocytes and / or B lymphocytes. A vaccine is capable of producing an immune response that results in the patient producing neutralizing antibodies to the antigens provided in the vaccine. A vaccine can be a composition for preventive purposes or therapeutic purposes or both.

[0116] As used herein, the term "antigen" refers to any antigen that can be used in a vaccine, whether it involves a complete microorganism or a part thereof, and various types: (e.g., peptides, proteins, glycoproteins, polysaccharides, glycolipids, lipopeptides, etc.). Therefore, the term "antigen" refers to a molecule that can induce a humoral and / or cellular immune response in an antigen recipient. An antigen can be a molecule that causes a disease, and vaccination can be beneficial for treating the disease. An antigen can include a substance that is used to stimulate the production of antibodies and provide immunity against one or more than one disease, which is prepared from the pathogen of the disease, its product or a synthetic substitute, and is treated as an antigen without inducing the disease. An antigen can include a peptide or a polypeptide.

[0117] As used herein, the term "dimer" refers to a bifunctional molecule. The dimer disclosed herein may include a TLR receptor binding portion and a NF-κB binding portion. The terms "dimer", "hybrid molecule", "adjuvant dimer", "dimer adjuvant", "dimer agonist" and "agonist dimer" are used interchangeably herein.

[0118] The term "pharmaceutically acceptable carrier" refers to a carrier that does not cause allergic reactions or other adverse reactions in the subject to which it is administered. Suitable pharmaceutically acceptable carriers include, for example, one or more than one of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc. and combinations thereof. In addition, if necessary, the vaccine may contain a small amount of auxiliary substances, such as a wetting agent or emulsifier and a pH buffer.

[0119] As used herein, the term "agonist" refers to a molecule that can produce a cellular response after binding to a receptor. An agonist can be a ligand that binds directly to a receptor. Alternatively, an agonist can bind to a receptor indirectly, for example, by (a) forming a complex with another molecule that binds directly to the receptor, or (b) causing modification of another molecule so that the other molecule binds directly to the receptor. An agonist can refer to an agonist of a specific receptor or family of receptors (e.g., a TLR agonist).

[0120] "Individual," "subject," and "patient" are used interchangeably and may refer to either a human or a non-human.

[0121] The term "aryl" includes aryl not substituted with heteroatoms, aryl substituted with heteroatoms, Cn-aryl not substituted with heteroatoms, Cn-aryl substituted with heteroatoms, heteroaryl, heterocyclic aryl, carbocyclic aryl, biaryl and monovalent groups derived from polycyclic condensed hydrocarbons (PAH). The term "Cn-aryl not substituted with heteroatoms" refers to a group having a single carbon atom as a point of attachment, wherein the carbon atom is part of an aromatic ring structure containing only carbon atoms, and also having a total of n carbon atoms, 5 or more than 5 hydrogen atoms and no heteroatoms. For example, C6-C10-aryl not substituted with heteroatoms has 6 to 10 carbon atoms. Non-limiting examples of aryl not substituted with heteroatoms include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C 6 H 4 CH 2 CH 3 , -C 6 H 4 CH 2 CH 2 CH 3 , -C 6 H 4 CH(CH 3 ) 2 , -C 6 H 4 CH(CH 2 ) 2 , -C 6 H 3 (CH 3 )CH 2 CH 3 , -C 6 H 4 CH=CH 2 , -C 6 H 4 CH=CHCH 3 , -C 6 H 4 C≡CH, -C 6 H 4 C≡CCH 3 , naphthyl and groups derived from biphenyl. The term "heteroatom-substituted Cn-aryl" refers to a group having a single aromatic carbon atom or a single aromatic heteroatom as a point of attachment, a total of n carbon atoms, at least one hydrogen atom and at least one heteroatom, wherein each heteroatom is independently selected from N, O, F, Cl, Br, I, Si, P and S. For example, a heteroatom-substituted C1-C10-heteroaryl has 1 to 10 carbon atoms. Non-limiting examples of heteroatom-substituted aryl include the following groups: -C 6 H 4 F, -C 6 H4 Cl、-C 6 H 4 Br、-C 6 H 4 I、-C 6 H 4 OH、-C 6 H 4 OCH 3 、-C 6 H 4 OCH 2 CH 3 、-C 6 H 4 OC(O)CH 3 、-C 6 H 4 NH 2 、-C 6 H 4 NHCH 3 、-C 6 H 4 N(CH 3 ) 2 、-C 6 H 4 CH 2 OH、-C 6 H 4 CH 2 OC(O)CH 3 、-C 6 H 4 CH 2 NH 2 、-C 6 H 4 CF 3 、-C 6 H 4 CN、-C 6 H 4 CHO、-C 6 H 4 CHO、-C 6 H 4 C(O)CH 3 、-C 6 H 4 C(O)C 6 H 5 、-C 6 H 4 CO 2 H、-C 6 H 4 CO 2 CH 3 、-C 6 H 4 CONH 2 、-C6 H 4 CONHCH 3 , -C 6 H 4 CON(CH 3 ) 2 In some embodiments, aryl groups are substituted with 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, 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, 101, 102, 114, 115, 126, 130, 131, 132, 133, 134, 135, 136, 137, 138, 149, 150

[0122] The term "alkoxy" includes straight chain alkoxy, branched chain alkoxy, cycloalkoxy, cyclic alkoxy, alkoxy without heteroatom substitution, alkoxy with heteroatom substitution, C n -alkoxy and heteroatom-substituted C n -alkoxy. In certain embodiments, lower alkoxy is contemplated. The term "lower alkoxy" refers to an alkoxy group of 1 to 6 carbon atoms (i.e., 1, 2, 3, 4, 5, or 6 carbon atoms). The term "unsubstituted C n "-alkoxy" refers to a group having the structure -OR, wherein R is a C substituted with no heteroatom as that term is defined above. n -alkyl. Alkoxy groups not substituted by heteroatoms include: -OCH 3 、-OCH 2 CH 3 、-OCH 2 CH 2 CH 3 、-OCH(CH 3 ) 2 and -OCH(CH 2 ) 2 The term "heteroatom-substituted C n "-alkoxy" refers to a group having the structure -OR, wherein R is a C substituted with a heteroatom as defined above. n -alkyl. For example, -OCH 2 CF 3 It is an alkoxy group substituted by a heteroatom.

[0123] The term "alkenyl" includes straight and branched hydrocarbon groups containing one double bond and having 2 to 6 carbon atoms, for example, ethenyl, 2-propenyl (allyl), 3-butenyl, 2-pentenyl, 3-pentenyl, 3-methyl-2-butenyl, etc. The term "alkenyloxy" refers to an alkenyl ether radical, wherein alkenyl is as defined above.

[0124] The term "allyl" refers to a group H2 C=CH─CH 2 The term "ether" refers to a hydrocarbon group connected to another hydrocarbon group through an oxygen. Thus, an ether substituent of a hydrocarbon group may be hydrocarbon-O-. An ether may be symmetrical or asymmetrical. Examples of ethers include, but are not limited to, vinyl ethers and allyl ethers. The term "vinyl" refers to a molecular moiety that contains a carbon-carbon double bond.

[0125] Various groups described herein, including hydroxy, aryl, alkenyloxy, alkoxy and alkenyl, may be optionally substituted with one or more than one substituent. Non-limiting examples of substituents include halogen, nitro, cyano, hydroxy, amino, sulfhydryl, formyl, carboxyl, carbamoyl, alkyl, heteroalkyl, alkoxy, alkylthio, alkylamino, (alkyl) 2 amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0126] As used in the specification herein, an element without a quantifier preceding it can mean one or more than one. As used in the claims herein, when used in conjunction with the word "comprising", an element without a quantifier preceding it can mean one or more than one.

[0127] The term "or" used in the claims is used to mean "and / or" unless explicitly indicated to refer to only alternatives or the alternatives are mutually exclusive, although the present disclosure supports definitions referring to only alternatives and "and / or." As used herein, "another" may mean at least a second or more than two.

[0128] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device or method being employed to determine the value, or the variation that exists between study subjects.

[0129] The term "consisting essentially of" can include the listed active ingredients, such as the listed dimers, and any unlisted buffers, pharmaceutical excipients, etc., but excludes any other active ingredients, such as other hybrid molecules.

[0130] II. Immunomodulators

[0131] The methods and compositions disclosed herein relate to immunomodulators and methods of use thereof. As used herein, "immunomodulator" refers to any molecule capable of stimulating an innate immune response in a subject. Immunomodulators include molecules capable of activating pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) in immune cells. The present disclosure includes polymer immunomodulators comprising multiple TLR agonist portions and multiple NF-κB inhibitor portions. As disclosed herein, such polymer molecules are capable of activating TLRs while limiting the release of proinflammatory cytokines, thereby improving safety and tolerability without affecting adjuvant and / or anti-tumor activity. The number of TLR agonist portions and NF-κB inhibitor portions on a particular polymer immunomodulator can be adjusted to adjust the expected level of immune activation.

[0132] Disclosed is a polymeric immunomodulator constructed on a polypeptide backbone, which comprises two or more repeating TLR agonist moieties and two or more repeating NF-κB inhibitor moieties. Figure 7 Schematic diagrams of such polymeric immunomodulators are provided. The polymeric immunomodulators of the present disclosure may comprise a plurality of different TLR agonist moieties and / or repeating units of the same TLR agonist moiety. "TLR agonist moiety" describes a portion of a molecule or compound that has TLR agonist activity. The polymeric immunomodulators of the present disclosure may comprise a plurality of different NF-κB inhibitor moieties and / or repeating units of the same NF-κB inhibitor moiety. "NF-κB inhibitor moiety" describes a portion of a molecule or compound that has NF-κB inhibitor activity.

[0133] The polymer immunomodulator may include a first region and a second region, wherein the first region includes two or more TLR agonist portions attached to a polypeptide backbone, and the second region includes two or more NF-κB inhibitor portions attached to a polypeptide backbone. The polypeptide backbone of the first region may include all identical amino acid subunits (e.g., repeated glutamic acid units), or a plurality of different amino acid subunits. The polypeptide backbone of the second region may include all identical amino acid subunits (e.g., repeated glycine units), or a plurality of different amino acid subunits. The first region may include at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 TLR agonist portions. The first region may include exactly 2, 3, 4, 5, 6, 7, or 8 TLR agonist portions. The second region may include at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 NF-κB inhibitor moieties. The second region may include exactly 2, 3, 4, 5, 6, 7, or 8 NF-κB inhibitor moieties. The polymer immunomodulator of the present disclosure may include the same number of TLR agonist moieties as the NF-κB inhibitor moieties, or may include different numbers of TLR agonist moieties and NF-κB inhibitor moieties. For example, the immunomodulator may include 3 TLR agonist moieties and 3 NF-κB inhibitor moieties, or the immunomodulator may include 3 TLR agonist moieties and 5 NF-κB inhibitor moieties.

[0134] The immunomodulator may comprise 3 TLR agonist portions and 2, 3, 4, 5, 6, 7 or 8 NF-κB inhibitor portions. The immunomodulator may comprise 4 TLR agonist portions and 2, 3, 4, 5, 6, 7 or 8 NF-κB inhibitor portions. The immunomodulator may comprise 5 TLR agonist portions and 2, 3, 4, 5, 6, 7 or 8 NF-κB inhibitor portions. The immunomodulator may comprise 6 TLR agonist portions and 2, 3, 4, 5, 6, 7 or 8 NF-κB inhibitor portions. The immunomodulator may comprise 7 TLR agonist portions and 2, 3, 4, 5, 6, 7 or 8 NF-κB inhibitor portions. The immunomodulator may comprise 8 TLR agonist moieties and 2, 3, 4, 5, 6, 7 or 8 NF-κB inhibitor moieties.

[0135] The first region and the second region can be connected by a linker. A variety of linkers are known in the art and are contemplated herein. The first region and the second region can be connected by a triazole.

[0136] The polymeric immunomodulator may comprise a polypeptide backbone, wherein each TLR agonist moiety and / or NF-κB inhibitor moiety is attached to the side chain of each amino acid of the polypeptide backbone. For example, the polymeric immunomodulator of the present disclosure comprises a polyglutamic acid region, wherein each glutamic acid residue is attached to a TLR agonist moiety (e.g., a TLR 7 / 8 agonist such as an imidazoquinoline) via a side chain. Figure 8B and Fig. 9 Non-limiting examples of such polymeric immunomodulators are provided.

[0137] Disclosed are immunomodulators having formula (I):

[0138]

[0139] wherein L is a linker; and

[0140] wherein n is an integer from 1 to 10, and m is an integer from 1 to 10.

[0141] n can be at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. n can be at least 2. n can be 5. m can be at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. m can be at least 2. m can be 5. n can be equal to m. n can not be equal to m. An immunomodulator can also be defined as:

[0142]

[0143] Wherein n is an integer from 1 to 10, and m is an integer from 1 to 10. Both n and m may be 5. An immunomodulator may also be defined as:

[0144]

[0145] Disclosed are immunomodulators having formula (II):

[0146]

[0147] in:

[0148] A is the NF-κB inhibitor part;

[0149] B is an amino acid;

[0150] L is the connector;

[0151] C is an amino acid; and

[0152] D is the TLR agonist part,

[0153] wherein n is an integer from 1 to 10, and m is an integer from 1 to 10.

[0154] The TLR agonist can be a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and / or TLR9 agonist. The TLR agonist can be a TLR7 / 8 agonist, such as an imidazoquinolinone or a derivative thereof having TLR7 / 8 agonist activity. The NF-κB inhibitor can be ferulic acid or a derivative thereof having NF-κB inhibitor activity. The NF-κB inhibitor can be dopamine or a derivative thereof having NF-κB inhibitor activity. The NF-κB inhibitor can be vanillin or a derivative thereof having NF-κB inhibitor activity. The NF-κB inhibitor can be honokiol or a derivative thereof having NF-κB inhibitor activity.

[0155] B and C can be the same amino acid. B and C can be different amino acids. B can be an amino acid in Table 2. B can be an amino acid in Table 3. B can be a glutamic acid residue attached to the NF-κB inhibitor portion. C can be an amino acid in Table 2. C can be an amino acid in Table 3. C can be a glutamic acid residue attached to the TLR agonist portion.

[0156] n may be at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. n may be at least 2. n may be 5. m may be at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. m may be at least 2. m may be 4. n may be equal to m. n may not be equal to m. An immunomodulator is also defined as:

[0157]

[0158] Wherein n is an integer from 1 to 10 and m is an integer from 1 to 10. n is 5 and m may be 5. An immunomodulator may also be defined as:

[0159]

[0160] III. NF-κB Inhibitors

[0161] The present disclosure includes compositions and methods comprising or directed to one or more than one NF-κB inhibitor or derivatives thereof. As used herein, an NF-κB inhibitor describes any molecule capable of inhibiting NF-κB expression or activity, whose activity includes activating or promoting the transcription of one or more than one gene. As used herein, an "NF-κB enhancer" describes an NF-κB inhibitor or derivative thereof that is capable of enhancing the activity of a TLR agonist. Table 1 provides examples of NF-κB inhibitors that can be used in the methods and compositions of the present disclosure.

[0162] Table 1

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185] One or more than one of the NF-κB inhibitors in Table 1 may be specifically excluded from the methods and compositions of the present disclosure.

[0186] IV. Antigens

[0187] As used herein, the term "antigen" refers to a molecule against which a subject can elicit a humoral and / or cellular immune response. Antigens can be any type of biomolecule, such as simple intermediate metabolites, carbohydrates, lipids and hormones, and macromolecules, such as complex carbohydrates, phospholipids, nucleic acids, proteins. Common antigen classes include, but are not limited to, viral antigens, pathogen antigens, fungal antigens, protozoan and other parasitic antigens, tumor antigens, antigens involved in autoimmune diseases, allergies and transplant rejection, and other miscellaneous antigens. In certain compositions and methods of the present disclosure, the antigen is a peptide.

[0188] The inventors have demonstrated that the hybrid molecules disclosed herein reduce systemic toxicity to baseline levels while maintaining adjuvant properties of vaccine formulations. Antigens useful in the methods and compositions of the present disclosure include, for example, antigenic components from anthrax, cancer, chikungunya, dengue (1,2,3,4-dengue), diphtheria, Escherichia coli, Shigella toxin-producing (STEC), Ebola, non-polio enteroviruses, enterovirus D68 (EV-D68), gonorrhea, hepatitis A (Hep A), hepatitis B (Hep B), hepatitis C (Hep C), hepatitis D (Hep D), hepatitis E (Hep E), herpes, herpes zoster, HIV, HPV, influenza, malaria, measles, viral meningitis, bacterial meningitis, mumps, norovirus, pertussis, plague; bubonic plague, septicemia, pneumonia, pneumococcal disease, poliomyelitis (Polio), pustular rash diseases (smallpox, monkeypox, cowpox), Q fever, rabies, salmonella gastroenteritis (Salmonella), severe acute respiratory syndrome, shigellosis (Shigella), smallpox, tetanus, tuberculosis, varicella (Chickenpox), viral hemorrhagic fevers (Ebola, Lassa, Marburg), West Nile virus, yellow fever, Yersinia and Zika virus infection. It is expected that one or more of the antigens and antigenic components listed in this paragraph are specifically excluded.

[0189] Further examples of antigens that can be used in the methods and compositions of the present disclosure are provided below and throughout the disclosure.

[0190] A. Viral antigens

[0191] Examples of viral antigens include, but are not limited to, retroviral antigens, such as retroviral antigens from human immunodeficiency virus (HIV) antigens, such as gene products of gag, pol and env genes, Nef protein, reverse transcriptase and other HIV components; hepatitis virus antigens, such as S, M, L proteins of hepatitis B virus, pre-S antigen of hepatitis B virus, other hepatitis, such as hepatitis A, B, C, viral components such as hepatitis C virus RNA; influenza virus antigens, such as hemagglutinin and neuraminidase and other influenza virus components; measles virus antigens, such as measles virus fusion protein and other measles virus components; rubella virus antigens, such as Such as protein E1, 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 virus antigens, such as RSV fusion protein, M2 protein and other respiratory syncytial virus components; herpes simplex virus antigens, such as immediate early protein, 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, ME, ME-NS1, NS1, NS1-NS2A, 80% E, and other Japanese encephalitis virus antigen components; rabies virus antigens, such as rabies glycoprotein, rabies virus nucleoprotein and other rabies virus antigen components. For other examples of viral antigens, see Fundamental Virology, Second Edition, e's.Fields, BNand Knipe, DM (Raven Press, New York, 1991). It is intended that one or more than one of the antigens and antigenic components listed in this paragraph are specifically excluded.

[0192] B. Bacterial antigens

[0193] Bacterial antigens that can be used in the compositions and methods of the present disclosure include, but are not limited to, pertussis bacterial antigens, such as pertussis toxin, filamentous hemagglutinin, pertussis adhesin, FIM2, FIM3, adenylate cyclase and other pertussis bacterial antigen components; diphtheria bacterial antigens, such as diphtheria toxin or toxoid and other diphtheria bacterial antigen components; tetanus bacterial antigens, such as tetanus toxin or toxoid and other tetanus bacterial antigen components; Streptococcus bacterial antigens, such as M protein and other Streptococcus bacterial antigen components; Gram-negative bacilli 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), 30kDa major secretory protein, antigen 85A and other mycobacterial antigen components; Helicobacter pylori bacterial antigen components; Pneumococcal bacterial antigens, such as pneumolysin, pneumococcal capsular polysaccharide and other pneumococcal bacterial antigen components; Haemophilus influenzae bacterial antigens, such as capsular polysaccharide and other Haemophilus influenzae bacterial antigen components; Anthrax bacterial antigens, such as anthrax protective antigen and other anthrax bacterial antigen components; Rickettsial bacterial antigens, such as romps and other Rickettsial bacterial antigen components. The bacterial antigens described herein also include any other bacterial, mycobacterial, mycoplasma, rickettsial or chlamydial antigens. It is expected that one or more than one antigen and antigen components listed in this paragraph are explicitly excluded from the methods and compositions of the present disclosure.

[0194] C. Fungal antigens

[0195] Fungal antigens that can be used in the compositions and methods of the present disclosure include, but are not limited to, Candida fungal antigen components; Histoplasma antigens, such as heat shock protein 60 (HSP60) and other Histoplasma antigen components; Cryptococcal fungal antigens, such as capsular polysaccharides and other Cryptococcal fungal antigen components; Coccidioides fungal antigens, such as microglobules and other Coccidioides fungal antigen components; and Tinea fungal antigens, such as trichophyton and other coccidioides fungal antigen components. It is contemplated that one or more than one antigen and antigen components listed in this paragraph are specifically excluded from the methods and compositions of the present disclosure.

[0196] D. Parasite antigens

[0197] Examples of protozoan and other parasite antigens include, but are not limited to, Plasmodium falciparum antigens, such as merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte / gamete surface antigens, blood stage antigens pf 1 55 / 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 major and other Leishmania antigens, such as gp63, lipophosphoglycosides and their related proteins and other Leishmania antigen components; and Trypanosoma cruzi antigens, such as 75kDa to 77kDa antigen, 56kDa antigen and other trypanosoma antigen components. It is contemplated that one or more of the antigens and antigen components listed in this paragraph are specifically excluded from the methods and compositions of the present disclosure.

[0198] E. Tumor antigens

[0199] 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 of B-cell derived malignancies, fusion polypeptides expressed by genes juxtaposed by chromosomal translocations, human chorionic gonadotropin, calcitonin, tyrosinase, papillomavirus antigens, gangliosides, or other carbohydrate-containing components of melanoma or other tumor cells. The present disclosure contemplates that antigens 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 are specifically excluded from the methods and compositions of the present disclosure.

[0200] F. Antigens associated with autoimmunity

[0201] Antigens involved in autoimmune diseases, allergies, and transplant rejection can be used in the compositions and methods of the present disclosure. For example, antigens involved in any one or more of the following autoimmune diseases or conditions can be used in the present disclosure: 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, Sjögren's syndrome, including keratoconjunctivitis sicca secondary to Sjögren's syndrome, alopecia areata, allergic reactions caused by arthropod bites, Crohn's disease, aphthous ulcers, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis Inflammation, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruption, leprosy reversal reaction, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, simple red cell anemia, idiopathic thrombocytopenia, 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. Examples of antigens involved in autoimmune diseases include glutamic acid decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelin proteolipids, acetylcholine receptor components, thyroglobulin and thyroid stimulating hormone (TSH) receptor. Examples of antigens involved in allergic reactions include pollen antigens, such as Japanese cedar pollen antigens, ragweed pollen antigens, ryegrass pollen antigens, animal-derived antigens such as dust mite antigens and cat antigens, tissue compatibility antigens, and penicillin, as well as other therapeutic drugs. Examples of antigens involved in transplant rejection include antigenic components of the transplant to be transplanted into the transplant recipient, such as heart, lung, liver, pancreas, kidney and neural transplant components. Antigens can also be altered peptide ligands for the treatment of autoimmune diseases. It is expected that one or more of the antigens and antigenic components listed in this paragraph are explicitly excluded from the methods and compositions of the present disclosure. It is further expected that autoantigens are explicitly excluded from the methods and compositions of the present disclosure.

[0202] Examples of mixed 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.

[0203] V. PRR and TLR agonists

[0204] Certain aspects of the present disclosure relate to pattern recognition receptor (PRR) agonists. 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 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, wherein 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.

[0205] Aspects of the present disclosure relate to TLR agonists, including polymers comprising TLR agonists or derivatives thereof. A TLR agonist can be any molecule that directly or indirectly activates TLR and / or stimulates TLR signaling. In some cases, a TLR agonist is a molecule that directly binds to TLR. In some aspects, an immunomodulator comprising one or more than one TLR agonist connected by a polypeptide backbone is disclosed.

[0206] In some embodiments, the TLR agonist is an agonist known in the art and / or described herein. TLR agonists can include agonists directed against TLR1 (e.g., peptidoglycan or triacyl lipoprotein), TLR2 (e.g., lipoteichoic acid; peptidoglycan from Bacillus subtilis, Escherichia coli 0111:B4, Escherichia coli K12, or Staphylococcus aureus; atypical lipopolysaccharides (LPS) such as Leptospirosis LPS and Porphyromonas gingivalis LPS; synthetic diacylated lipoproteins such as FSL-1 or Pam 2 CSK 4 ; Lipoarabinomannan or lipomannan from Mycobacterium smegmatis; triacylated lipoproteins such as Pam 3 CSK 4; lipoproteins from mycoplasmas such as MALP-2 and MALP-404; Borrelia burgdorferi OspA; porins from Neisseria meningitidis or Haemophilus influenzae; Propionibacterium acnes antigen mixture; Yersinia LcrV; lipomannan from mycobacteria or Mycobacterium tuberculosis; Trypanosoma cruzi GPI anchor; Schistosoma mansoni lysophosphatidylserine; Leishmania major lipophosphoglycan (LPG); Plasmodium falciparum glycosylphosphatidylinositol (GPI); zymosan; antigen mixture from Aspergillus fumigatus or Candida albicans; and measles hemagglutinin), TLR3 (e.g., double-stranded RNA, poly adenylic acid-polyuridine (Poly (A:U)); polyinosinic acid-polycytidylic acid (Poly (I:C)); polyinosinic acid-polycytidylic acid high molecular weight (Poly (I:C) HMW); and low molecular weight polyinosinic acid (Poly (I:C) LMW)), TLR4 (e.g., LPS from Escherichia coli and Salmonella); TLR5 (e.g., flagellin from Bacillus subtilis, Pseudomonas aeruginosa, or Salmonella typhimurium), TLR8 (e.g., single-stranded RNA such as ssRNA with 6UUAU repeats, RNA homopolymers (ssPolyU naked), HIV-1 LTR-derived ssRNA (ssRNA40), or ssRNA with 2 GUCCUUCAA repeats (ssRNA-DR)), TLR7 (e.g., imidazoquinoline compounds Imiquimod, Imiquimod VacciGrade TM Gardiquimod VacciGrade TM or Gardiquimod TM ; adenine analog CL264; base analog CL307; guanosine analog loxoribine; TLR7 / 8 (e.g., thiazoquinoline compound CL075; imidazoquinoline compound CL097, 2Bxy, R848, or R848VacciGrade TM ), TLR9 (e.g., CpG ODN); and TLR11 (e.g., Toxoplasma gondii inhibitory protein). In some embodiments, the TLR agonist is an amphipathic TLR agonist. In some embodiments, the TLR agonist is a TLR 2 / 6 agonist, such as Pam 2 CSK 4 or Pam 3 CSK 4. In some embodiments, the TLR agonist is a hydrophobic TLR agonist. In some embodiments, the TLR agonist is a TLR 7, TLR 8 or TLR 7 / 8 agonist, such as 2Bxy or imidazoquinoline. In some aspects, the TLR agonist of the present disclosure is an imidazoquinoline. In certain aspects, the TLR agonist is a specific agonist listed above. Derivatives of any of the above-mentioned TLR agonists are also contemplated herein, and the TLR agonists of the present disclosure include any derivatives of the above-mentioned molecules having TLR agonist activity.

[0207] In other embodiments, the TLR agonist is an agonist that specifically excites one TLR or two TLRs. In some embodiments, the TLR agonist connected includes different types of TLR agonists (e.g., TLR agonists that can activate different types of TLRs). Alternatively, the TLR agonist connected can include the same type of TLR agonist.

[0208] In some embodiments, disclosed herein are small molecule compounds suitable for use as TLR agonists. Examples of small molecule TLR agonists include compounds having a 2-aminopyridine fused to a five-membered nitrogen-containing heterocycle. Such compounds include, for example, imidazoquinolineamines, including but not limited to 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; tetrahydroimidazoquinolineamines, including but not limited to amide-substituted tetrahydroimidazoquinolineamines, sulfonamide-substituted tetrahydroimidazoquinolineamines, urea-substituted tetrahydroimidazoquinolineamines, aryl ether-substituted tetrahydroimidazoquinolineamines, heterocyclic ether-substituted imidazoquinolineamines; substituted tetrahydroimidazoquinolineamines, amide ether substituted tetrahydroimidazoquinolineamines, sulfonamide ether substituted tetrahydroimidazoquinolineamines, urea substituted tetrahydroimidazoquinoline ethers, and thioether substituted tetrahydroimidazoquinolineamines; imidazopyridineamines, including but not limited to amide substituted imidazopyridineamines, sulfonamide substituted imidazopyridineamines, urea substituted imidazopyridineamines; 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 cycloalkyl imidazopyridineamines; imidazonaphthyridinamines; tetrahydroimidazonaphthyridinamines; Azoquinolineamine; Thiazoloquinolineamine; Azolopyridineamine; Thiazolopyridineamine; oxazolonaphthyridinamines; and thiazolonaphthyridinamines.

[0209] In certain embodiments, the TLR agonist is an imidazonaphthyridinamine, a tetrahydroimidazonaphthyridinamine, Azoquinoline amine, thiazoquinoline amine, Azolopyridineamine, thiazolopyridineamine, Azolacetylide or thiazolacetylide.

[0210] In certain embodiments, the TLR agonist is an imidazoquinoline amine substituted with sulfonamide. In other embodiments, the TLR agonist may be an imidazoquinoline ether substituted with urea. In another other embodiment, the TLR agonist may be an imidazoquinoline amine substituted with aminoalkyl. In a specific embodiment, the TLR agonist is 4-amino-α, α, 2-trimethyl-1H-imidazo[4,5-c]quinoline-1-ethanol. In other specific embodiments, the TLR agonist is N-(2-{2-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5c]quinoline-1-yl]ethoxy}ethyl)-N-methylmorpholine-4-carboxamide. In other specific embodiments, the TLR agonist is l-(2-amino-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5c]quinoline-4-amine. In another other embodiment, the TLR agonist is N-[4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide. In yet another other embodiment, the TLR agonist is N-[4-(4-amino-2-propyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide.

[0211] In certain embodiments, the TLR agonist can be a substituted imidazoquinoline amine, a tetrahydroimidazoquinoline amine, an imidazopyridine amine, a 1,2-bridged imidazoquinoline amine, a 6,7-fused cycloalkylimidazopyridine amine, an imidazonaphthyridinamine, a tetrahydroimidazonaphthyridinamine, Azoquinoline amine, thiazoquinoline amine, Azopyridine amine, thiazopyridine amine, Azolenaphthyridine or thiazolenaphthyridine.

[0212] As used herein, substituted imidazoquinolineamine refers to aminoalkyl-substituted imidazoquinolineamine, amide-substituted imidazoquinolineamine, sulfonamide-substituted imidazoquinolineamine, urea-substituted imidazoquinolineamine, aryl ether-substituted imidazoquinolineamine, heterocyclic ether-substituted imidazoquinolineamine, amide ether-substituted imidazoquinolineamine, sulfonamide ether-substituted imidazoquinolineamine, urea-substituted imidazoquinolineether, or thioether-substituted imidazoquinolineamine.

[0213] VI. Amino Acids

[0214] The term "amino acid" refers to natural amino acids, unnatural amino acids and amino acid analogs, both in their D and L stereoisomers, unless otherwise indicated if their structure permits such stereoisomeric forms. An amino acid may have, for example, the following formula:

[0215] wherein R and R′ are each independently selected from hydrogen, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aryl, optionally substituted heteroaryl, and R d is hydrogen or an amino protecting group. The amino acids covered by the above two formulas include, but are not limited to, natural α-amino acids such as D- and L-isomers of the 20 common naturally occurring α-amino acids found in polypeptides and proteins (e.g., A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V, as shown in Table 2 below), non-natural α-amino acids (examples of which are shown in Table 3 below), natural β-amino acids (e.g., β-alanine) and non-natural β-amino acids.

[0216] Table 2 - Natural amino acids

[0217]

[0218]

[0219] Table 3 - Unnatural Amino Acids

[0220]

[0221]

[0222] Many unnatural amino acids are known, any of which may be included in the polypeptides, polypeptide backbones, polymers and compounds of the present invention. For example, see S. Hunt, The Non-Protein Amino Acids: In Chemistry and Biochemistry of the Amino Acids, edited by GC Barrett, Chapman and Hall, 1985; the entire text of which is incorporated by reference in its entirety. Some examples of unnatural amino acids are 4-hydroxyproline, desmosine, γ-aminobutyric acid, β-cyanoalanine, norvaline, 4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine, 1-amino-cyclopropylcarboxylic acid, 1-amino-2-phenyl-cyclopropylcarboxylic acid, 1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentylcarboxylic acid, 3-amino-cyclohexylcarboxylic acid, 4-piperidineacetic acid, 4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminopimelic acid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-substituted phenylalanine, meta-substituted phenylalanine, and para-substituted phenylalanine (e.g., with —C(═O)C 6 H 5 ;—CF 3 ;—CN; -halogen; —NO 2 ;—CH 3 substituted), disubstituted phenylalanine, substituted tyrosine (e.g., further substituted with —C(═O)C 6 H 5 ;—CF 3 ;—CN; -halogen; —NO 2 ;—CH 3 Replacement) and statins.

[0223] Certain non-natural amino acids can be included in polypeptide chains for peptide stapling or suturing. These non-natural amino acids include terminal unsaturation, such as double or triple bonds. Exemplary amino acids with terminal olefin unsaturation include, but are not limited to: -(CH 2 ) g —S—(CH 2 ) g CH═CH 2 ; —(CH 2 ) g —O—(CH 2 ) g CH═CH 2 ;—(CH 2 ) g —NH—(CH 2 )g CH═CH 2 ; —(CH 2 ) g —(C═O)—S—(CH 2 ) g CH═CH 2 ;—(CH 2 ) g —(C═O)—O—(CH 2 ) g CH═CH 2 ;—(CH 2 ) g —(C═O)—NH—(CH 2 ) g CH═CH 2 ;—CH 2 CH 2 CH 2 CH 2 —NH—(CH 2 ) g CH═CH 2 ;—(C 6 H 5 )-pO—(CH 2 ) g CH═CH 2 ;—CH(CH 3 )—O—(CH 2 ) g CH═CH 2 ;—CH 2 CH(—O—CH═CH 2 )(CH 3 ); -Histidine-N((CH 2 ) g CH═CH 2 ); -tryptophan-N((CH 2 ) g CH═CH 2 ) and (CH 2 ) g+1 (CH═CH 2 ), wherein g is independently at each occurrence from 0 to 10 (inclusive). Specific amino acids having terminal unsaturation are further described and depicted herein.

[0224] The term "amino acid analog" refers to a natural or unnatural amino acid in which one or more of the C-terminal carboxyl group, the N-terminal amino group, and the side chain functional group has been chemically blocked, reversibly or irreversibly, or otherwise modified to another functional group. For example, aspartic acid-(β-methyl ester) is an amino acid analog of aspartic acid; N-ethylglycine is an amino acid analog of glycine; or alanine carboxamide is an amino acid analog of alanine. Other amino acid analogs include methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine ​​sulfoxide, and S(carboxymethyl)-cysteine ​​sulfone.

[0225] VII. Pharmaceutical Compositions

[0226] The present disclosure relates to TLR agonists, including dimers comprising TLR agonists or derivatives thereof. A TLR agonist can be any molecule that directly or indirectly activates TLR and / or stimulates TLR signaling. In some cases, a TLR agonist is a molecule that directly binds to TLR. In some cases, a TLR agonist of the present disclosure is connected by, for example, polyethylene glycol (PEG) or other molecular linkers. A TLR agonist can be formulated into nanoparticles, optionally formulated with one or more than one co-assembly agent (e.g., a functionalized polymer).

[0227] TLR agonists can be agonists known in the art and / or described herein. TLR agonists can include agonists directed against TLR1 (e.g., peptidoglycan or triacyl lipoprotein), TLR2 (e.g., lipoteichoic acid; peptidoglycan from Bacillus subtilis, Escherichia coli 0111:B4, Escherichia coli K12, or Staphylococcus aureus; atypical lipopolysaccharides (LPS) such as Leptospirosis LPS and Porphyromonas gingivalis LPS; synthetic diacylated lipoproteins such as FSL-1 or Pam 2 CSK 4 ; Lipoarabinomannan or lipomannan from Mycobacterium smegmatis; triacylated lipoproteins such as Pam 3 CSK 4; lipoproteins from mycoplasmas such as MALP-2 and MALP-404; Borrelia burgdorferi OspA; porins from Neisseria meningitidis or Haemophilus influenzae; Propionibacterium acnes antigen mixture; Yersinia LcrV; lipomannan from mycobacteria or Mycobacterium tuberculosis; Trypanosoma cruzi GPI anchor; Schistosoma mansoni lysophosphatidylserine; Leishmania major lipophosphoglycan (LPG); Plasmodium falciparum glycosylphosphatidylinositol (GPI); zymosan; antigen mixture from Aspergillus fumigatus or Candida albicans; and measles hemagglutinin), TLR3 (e.g., double-stranded RNA, poly adenylic acid-polyuridine (Poly (A:U)); polyinosinic acid-polycytidylic acid (Poly (I:C)); polyinosinic acid-polycytidylic acid high molecular weight (Poly (I:C) HMW); and low molecular weight polyinosinic acid (Poly (I:C) LMW)), TLR4 (e.g., LPS from Escherichia coli and Salmonella); TLR5 (e.g., flagellin from Bacillus subtilis, Pseudomonas aeruginosa, or Salmonella typhimurium), TLR8 (e.g., single-stranded RNA such as ssRNA with 6UUAU repeats, RNA homopolymers (ssPolyU naked), HIV-1 LTR-derived ssRNA (ssRNA40), or ssRNA with 2 GUCCUUCAA repeats (ssRNA-DR)), TLR7 (e.g., imidazoquinoline compounds Imiquimod, Imiquimod VacciGrade TM Gardiquimod VacciGrade TM or Gardiquimod TM ; adenine analog CL264; base analog CL307; guanosine analog loxoribine; TLR7 / 8 (e.g., thiazoquinoline compound CL075; imidazoquinoline compound CL097, 2Bxy, R848, or R848VacciGrade TM ), TLR9 (e.g., CpG ODN); and TLR11 (e.g., Toxoplasma gondii inhibitory protein). The TLR agonist can be an amphipathic TLR agonist. The TLR agonist can be a TLR 2 / 6 agonist, such as Pam 2 CSK 4 or Pam 3 CSK 4. The TLR agonist can be a hydrophobic TLR agonist. The TLR agonist can be a TLR 7, TLR 8 or TLR 7 / 8 agonist, such as 2Bxy. The TLR agonist can be a specific agonist listed above. The TLR agonist can be an agonist that specifically excites one TLR or two TLRs. The connected TLR agonists can include different types of TLR agonists (e.g., TLR agonists that can activate different classes of TLRs). For example, the connected TLR agonists can include a TLR 2 / 6 agonist and a TLR 7 agonist covalently linked by a molecular linker. Alternatively, the connected TLR agonists can include TLR agonists of the same type.

[0228] Disclosed herein are small molecule compounds suitable for use as TLR agonists. Examples of small molecule TLR agonists include compounds having a 2-aminopyridine fused to a five-membered nitrogen-containing heterocycle. Such compounds include, for example, imidazoquinolineamines, including but not limited to 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; tetrahydroimidazoquinolineamines, including but not limited to amide-substituted tetrahydroimidazoquinolineamines, sulfonamide-substituted tetrahydroimidazoquinolineamines, urea-substituted tetrahydroimidazoquinolineamines, aryl ether-substituted tetrahydroimidazoquinolineamines, heterocyclic ether-substituted imidazoquinolineamines; substituted tetrahydroimidazoquinolineamines, amide ether substituted tetrahydroimidazoquinolineamines, sulfonamide ether substituted tetrahydroimidazoquinolineamines, urea substituted tetrahydroimidazoquinoline ethers, and thioether substituted tetrahydroimidazoquinolineamines; imidazopyridineamines, including but not limited to amide substituted imidazopyridineamines, sulfonamide substituted imidazopyridineamines, urea substituted imidazopyridineamines; 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 cycloalkyl imidazopyridineamines; imidazonaphthyridinamines; tetrahydroimidazonaphthyridinamines; Azoquinolineamine; Thiazoloquinolineamine; Azolopyridineamine; Thiazolopyridineamine; oxazolonaphthyridinamines; and thiazolonaphthyridinamines.

[0229] The TLR agonist can be imidazonaphthyridine amine, tetrahydroimidazonaphthyridine amine, Azoquinoline amine, thiazoquinoline amine, Azolopyridineamine, thiazolopyridineamine, Azolacetylide or thiazolacetylide.

[0230] The TLR agonist may be an imidazoquinoline amine substituted with a sulfonamide. The TLR agonist may be an imidazoquinoline ether substituted with a urea. The TLR agonist may be an imidazoquinoline amine substituted with an aminoalkyl. The TLR agonist may be 4-amino-α,α,2-trimethyl-1H-imidazo[4,5-c]quinoline-1-ethanol. The TLR agonist may be N-(2-{2-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5c]quinolin-1-yl]ethoxy}ethyl)-N-methylmorpholine-4-carboxamide. The TLR agonist may be l-(2-amino-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5c]quinolin-4-amine. The TLR agonist may be N-[4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide. The TLR agonist may be N-[4-(4-amino-2-propyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide.

[0231] The TLR agonist can be a substituted imidazoquinoline amine, a tetrahydroimidazoquinoline amine, an imidazopyridine amine, a 1,2-bridged imidazoquinoline amine, a 6,7-fused cycloalkylimidazopyridine amine, an imidazonaphthyridine amine, a tetrahydroimidazonaphthyridine amine, Azoquinoline amine, thiazoquinoline amine, Azopyridine amine, thiazopyridine amine, Azolenaphthyridine or thiazolenaphthyridine.

[0232] As used herein, substituted imidazoquinolineamine refers to aminoalkyl-substituted imidazoquinolineamine, amide-substituted imidazoquinolineamine, sulfonamide-substituted imidazoquinolineamine, urea-substituted imidazoquinolineamine, aryl ether-substituted imidazoquinolineamine, heterocyclic ether-substituted imidazoquinolineamine, amide ether-substituted imidazoquinolineamine, sulfonamide ether-substituted imidazoquinolineamine, urea-substituted imidazoquinolineether, or thioether-substituted imidazoquinolineamine.

[0233] VIII. Pharmaceutical Compositions

[0234] The administration of the composition is generally carried out via various common routes. This includes, but is not limited to, parenteral, in situ, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal or intravenous injection. The vaccine composition can be inhaled (e.g., U.S. Patent No. 6,651,655, which is specifically incorporated by introduction). Other formulations suitable for other modes of administration include oral formulations. Oral formulations include commonly used excipients, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions are in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations or powders, and contain about 10% to about 95%, for example, about 25% to about 70% of active ingredients.

[0235] Typically, the composition is administered in a manner compatible with the dosage formulation and in an amount that is therapeutically effective and immunomodifying. The amount administered depends on the subject to be treated. The exact amount of active ingredient that needs to be administered depends on the physician's judgment.

[0236] The mode of administration can vary widely. Any conventional method of administering the antibody is applicable. It is believed that these include oral administration of a physiologically acceptable solid matrix or a physiologically acceptable dispersion, parenteral administration, administration by injection, etc. The dosage of the pharmaceutical composition will depend on the route of administration and will vary according to the size and health status of the subject.

[0237] In many cases, multiple administrations of up to about or at least about 3, 4, 5, 6, 7, 8, 9, 10 or more are required. Administration can be at intervals of 2 days to 12 weeks, more typically at intervals of 1 to 2 weeks. The administration process can be followed by determination of alloreactive immune responses and T cell activity.

[0238] The phrase "pharmaceutically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to animals or humans. As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Such media and their use for pharmaceutically active substances are known in the art. Unless any conventional media or agents are incompatible with the active ingredient, their use in immunogenic and therapeutic compositions is contemplated.

[0239] The hybrid molecule can be formulated for parenteral administration, for example, for injection via intravenous, intradermal, intramuscular, subcutaneous or even intraperitoneal routes. The composition can be administered via intradermal injection. The composition can be administered via intravenous injection. The composition can be administered via intramuscular injection. The compositions of the present disclosure can be prepared as injections, as liquid solutions or suspensions; solid forms suitable for preparing solutions or suspensions after adding a liquid prior to injection can also be prepared; the preparations can also be emulsified.

[0240] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; preparations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid for easy injection. It should 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.

[0241] The composition can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (salts formed with free amino groups of proteins) and are formed with inorganic acids such as hydrochloric acid or phosphoric acid or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0242] Carrier can also be a solvent or dispersion medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), its suitable mixture and vegetable oil. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc., can all play the role of preventing microorganisms. In many cases, isotonic agents, such as sugar or sodium chloride, will preferably be included. The extended absorption of injectable compositions can be achieved by using delayed absorption agents in the compositions, such as aluminum monostearate and gelatin.

[0243] Sterile injectable solutions are prepared by incorporating the required amount of active ingredient with the various other ingredients listed above (as required) in an appropriate solvent and then filtering and sterilizing. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile carrier comprising a basic dispersion medium and those other ingredients listed above. In the case of sterile powders for the preparation of sterile injections, preferred preparation methods are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any other desired ingredients from its previously sterile filtered solution.

[0244] The effective amount of the therapeutic or preventive composition is determined based on the intended target. The term "unit dose" or "dose" refers to a physical discrete unit suitable for an object, each unit containing a predetermined amount of the composition, which is calculated to produce the desired response discussed above with respect to its administration (i.e., suitable route and regimen). The amount of administration according to the number of treatments and the unit dose depends on the desired result and / or protection. The precise amount of the composition also depends on the physician's judgment and varies from one individual to another. Factors affecting the dosage include the physical and clinical state of the object, the route of administration, the intended target of treatment (symptom relief and cure), and the efficacy, stability, and toxicity of the specific composition. During preparation, the solution will be administered in a manner compatible with the dosage formulation and in an amount effective for treatment or prevention. The preparation is easily administered in a variety of dosage forms such as the type of the above-mentioned injection solution.

[0245] IX. Treatment Methods

[0246] As described above, compositions and methods using these compositions can treat a subject having, suspected of having, or at risk of developing an infection, cancer, or a related disease (e.g., preventing infection, eliciting a strong immune response to an antigen, or reducing or preventing tumor proliferation).

[0247] As used herein, the phrase "immune response" or its equivalent "immunological response" refers to a humoral (antibody-mediated), cellular (mediated by antigen-specific T cells or their secretory products) or both humoral and cellular response to a protein, peptide or polypeptide of the invention in a recipient patient. Treatment or therapy can be an active immune response induced by the administration of an immunogen, or a passive therapy produced by the administration of antibodies, antibody-containing materials or triggered T cells.

[0248] The presence of a cell-mediated immune response can be determined by proliferation assays (CD4(+) T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contribution of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by isolating IgG and T cells, respectively, from the immunized homologous animal and measuring the protective or therapeutic effect of a second subject. As used herein and in the claims, the terms "antibody" or "immunoglobulin" are used interchangeably.

[0249] Optionally, the antibody or preferably the immunological portion of an antibody can be chemically conjugated to other proteins or expressed as a fusion protein. For the purposes of this specification and the appended claims, all such fusion proteins are included in the definition of an antibody or the immunological portion of an antibody.

[0250] The method may include treating or preventing a disease or condition caused by a pathogen. In addition, in some embodiments, treatment includes administering other agents commonly used to combat viral infections, such as one or more antiviral or antiretroviral compounds.

[0251] The therapeutic composition is administered in a manner compatible with the dosage form and in an amount that is therapeutically effective. The amount administered depends on the subject to be treated. The exact amount of active ingredient that needs to be administered depends on the judgment of the physician. The appropriate regimen for initial administration and booster is also variable, but typically an initial administration is performed first, followed by subsequent administrations.

[0252] The mode of administration can vary widely. Any conventional method for administering the therapeutic polypeptide is applicable. It is believed that these include oral administration of a physiologically acceptable solid matrix or a physiologically acceptable dispersion, parenteral administration, administration by injection, etc. The dosage of the composition will depend on the route of administration and will vary according to the size and health status of the subject.

[0253] In some cases, it will be necessary to administer the composition multiple times, for example, 2, 3, 4, 5, 6 or more than 6 times. Administration can be at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks to 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks, including all ranges therebetween.

[0254] A subject may be administered about, at least about, or at most about the following micrograms, mg, μg / kg, or mg / kg (or any range derivable therein) of a hybrid molecule or composition: 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, 4.10, 4.20, 4.30, 4.40, 4.50, 4.60, 4.70, 4.80, 4.90, 4.11, 4.12, 4.13, 4.14, 4.15 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 2, 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。、

[0255] Doses can be administered as needed based on or 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 or 24 hours (or any range derivable therein) or once, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times (or any range derivable therein) per day. Doses can be administered first before or after the onset of symptoms. The first dose regimen can be administered at the following times after the patient experiences or exhibits signs or symptoms of the condition: 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, 3 days, 4 days or 5 days (or any range derivable therein). Patients may receive treatment for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days (or any range derivable therein) or until symptoms of the condition disappear or are alleviated or 6, 12, 18 or 24 hours after symptoms of infection disappear or are alleviated or 1, 2, 3, 4 or 5 days later.

[0256] X. Combination Therapy

[0257] The administration of the compositions and related methods, particularly compositions comprising hybrid molecules, can also be used in combination with the administration of one or more than one other therapy.

[0258] Treatment can be used in combination with antiviral or antiretroviral therapy. Treatment can be used in combination with anticancer therapy (e.g., chemotherapy, cancer immunotherapy, etc.). Treatment can be spaced a few minutes to a few weeks before or after other agents are treated. When other agents and / or proteins or polynucleotides are used alone, it is usually ensured that there is no long period of time between each delivery time, so that the therapeutic composition can still play a favorable combination effect on the object. In this case, it is expected that two modes can be used within about 12 to 24 hours of each other, for example, within about 6 to 12 hours of each other. In some cases, it may be necessary to significantly extend the application time, however, wherein a few days (2 days, 3 days, 4 days, 5 days, 6 days or 7 days) to a few weeks (1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks) between each management are invalid.

[0259] The vaccine can be administered as part of a prime / boost immunization strategy. The prime vaccine dose can be administered in any of the methods described herein. The vaccine booster can be administered by administering a second vaccine using the same or a different type of vaccine. Examples of such different vaccines include naked DNA vaccines or recombinant poxviruses.

[0260] Various combination therapies can be used, for example, the adjuvant is "A" and the NFkB inhibitor is "B":

[0261]

[0262] Administration of the composition to a patient / subject will follow the general regimen for administration of such compounds, and will take into account the toxicity, if any, of the composition. It is expected that treatment cycles will be repeated as needed. It is also expected that various standard therapies such as hydration may be combined therewith.

[0263] XI. Cancer Therapy

[0264] The disclosed methods may include administering a cancer therapy to a subject or patient. Cancer therapy may include local cancer therapy. Cancer therapy may not include systemic cancer therapy. Cancer therapy may not include local cancer therapy. Cancer therapy may include local cancer therapy without administering systemic cancer therapy. Cancer therapy may include administering a dimer of the present disclosure. Cancer therapy may include radiation therapy. Cancer therapy may include chemotherapy. Cancer therapy may include immunotherapy, which may be a checkpoint inhibitor therapy. These cancer therapies may also be excluded. These therapies may also be administered in combination.

[0265] The term "cancer" as used herein can be used to describe solid tumors, metastatic cancers, or non-metastatic cancers. Cancer can originate from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testicles, tongue, or uterus. Cancer can be a Phase I cancer. Cancer can be a Phase II cancer. Cancer can be a Phase III cancer. Cancer can be a Phase IV cancer.

[0266] Cancer may specifically be of the following histologic types, although it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; 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; mixed hepatocellular and cholangiocellular carcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; bronchioloalveolar carcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic carcinoma; oxophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary follicular adenocarcinoma; nonencapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrial carcinoma; skin appendage carcinoma; apocrine carcinoma; sebaceous gland carcinoma; cerumen gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous cell carcinoma; adenocarcinoma / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; theca cell tumor, malignant; granulosa cell tumor, malignant; fibroblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; angiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrohistiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mixed tumor of the Müllerian duct; Wilms' tumor; hepatoblastoma; carcinosarcoma; stromal tumor, malignant; Brenner's tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; goiter of the ovary, malignant; choriocarcinoma; mesonephroblastoma, malignant; hemangioendothelioma; hemangioendothelioma, malignant; cardiomyopathy Posey's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumors, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astrocytoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumors; meningioma, malignant; neurofibrosarcoma;Schwannoma, malignant; Granulosa cell tumor, malignant; Lymphoma, malignant; Hodgkin's disease; Hodgkin's lymphoma; Paragranuloma; Lymphoma, small lymphocytic; Lymphoma, large cell, diffuse; Lymphoma, follicular; Mycosis fungoides; Other specified non-Hodgkin's lymphomas; Malignant histiocytosis; Multiple myeloma; Mast cell sarcoma; Immunoproliferative small intestinal disease; Leukemia; Lymphoid leukemia; Plasma cell leukemia; Erythroleukemia; Lymphosarcoma cell leukemia; Myeloid leukemia; Basophilic leukemia; Eosinophilic leukemia; Monocytic leukemia; Mast cell leukemia; Megakaryocyte leukemia; Myeloid sarcoma; and Hairy cell leukemia.

[0267] The disclosed method is for treating cancer originating in the colon. The cancer may be colon cancer. The cancer may be colorectal cancer.

[0268] The method may involve determining, administering or selecting an appropriate cancer "management regimen" and predicting the outcome thereof. As used herein, the phrase "management regimen" refers to a management plan provided to a subject in need thereof (e.g., a subject diagnosed with cancer) that specifies the type of examination, screening, diagnosis, monitoring, care, and treatment (e.g., dosage, treatment regimen, and / or duration of treatment).

[0269] A. Radiation therapy

[0270] Radiotherapy, such as ionizing radiation, can be administered to a subject. As used herein, "ionizing radiation" refers to radiation containing particles or photons that have sufficient energy or can generate sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons). A preferred non-limiting example of ionizing radiation is x-radiation. Means for delivering x-rays to target tissues or cells are well known in the art.

[0271] Radiotherapy can include external beam therapy, internal beam therapy, radioimmunotherapy or intraoperative radiotherapy (IORT). External beam therapy can include three-dimensional conformal radiotherapy (3D-CRT), emphasized radiation therapy (IMRT), proton beam therapy, image-guided radiotherapy (IGRT) or stereotactic radiotherapy. Internal beam therapy can include interstitial brachytherapy, intracavitary brachytherapy or intraductal brachytherapy. Radiotherapy can be applied to primary tumors.

[0272] The amount of ionizing radiation is greater than 20 Gy and is administered in one dose. The amount of ionizing radiation may be 18 Gy and is administered in three doses. The amount of ionizing radiation may be at least, at most, 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 The ionizing radiation is administered in at least, at most or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses (or any range derivable therein). When more than one dose is administered, each dose may be separated 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 therein.

[0273] The amount of radiotherapy administered to a subject can be expressed as a total dose of radiotherapy, which is then administered in fractionated doses. For example, a total dose can be 50 Gy, administered in 10 fractionated doses of 5 Gy each. A total dose can be 50 Gy to 90 Gy, administered in 20 to 60 fractionated doses of 2 Gy to 3 Gy each. The total dose of radiation can be at least, at most, or about 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 2Gy, 33Gy, 34Gy, 35Gy, 36Gy, 37Gy, 38Gy, 39Gy, 40Gy, 41Gy, 42Gy, 43Gy, 44Gy, 45Gy, 46Gy, 47Gy, 48Gy, 4 9Gy, 50Gy, 51Gy, 52Gy, 53Gy, 54Gy, 55Gy, 56Gy, 57Gy, 58Gy, 59Gy, 60Gy, 61Gy, 62Gy, 63Gy, 64Gy, 65Gy, 6 6Gy, 67Gy, 68Gy, 69Gy, 70Gy, 71Gy, 72Gy, 73Gy, 74Gy, 75Gy, 76Gy, 77Gy, 78Gy, 79Gy, 80Gy, 81Gy, 82Gy, 83Gy, 84Gy, 85Gy, 86Gy, 87Gy, 88Gy, 89Gy, 90Gy, 91Gy, 92Gy, 93Gy, 94Gy, 95Gy, 96Gy, 97Gy, 98Gy, 99Gy, Gy, 130 Gy, 135 Gy, 140 Gy or 150 Gy (or any range derivable therein). The total dose may be administered in fractionated doses of at least, at most, 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 therein) each.The invention can be administered at least, at most, 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 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, At least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times (or any range derivable therein) divided doses may be administered daily. At least, at most, 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 times (or any range derivable therein) divided doses may be administered weekly.

[0274] B. Cancer Immunotherapy

[0275] The method may include administering 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 divided into active, passive or hybrid (active and passive). These methods take advantage of the fact that cancer cells often have molecules on their surface that can be detected by the immune system, called tumor-associated antigens (TAAs); they are typically proteins or other macromolecules (such as 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.

[0276] 1. Checkpoint inhibitors and combination therapy

[0277] The methods and compositions of the present disclosure may include the administration of immune checkpoint inhibitors, examples of which are further described below. As disclosed herein, "checkpoint inhibitor therapy" (also referred to as "immune checkpoint blockade therapy," "immune checkpoint therapy," "ICT," "checkpoint blockade immunotherapy," or "CBI") refers to a cancer therapy that includes providing one or more than one immune checkpoint inhibitor to a subject suffering from or suspected of having cancer.

[0278] a. PD-1, PDL1, and PDL2 inhibitors

[0279] PD-1 can play a role in the tumor microenvironment where T cells encounter infection or tumor. Activated T cells upregulate PD-1 and continue to express it in peripheral tissues. Cytokines such as IFN-γ induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of peripheral effector T cells and prevent excessive damage to tissues during immune responses. The inhibitors of the present invention can block one or more than one function of PD-1 and / or PDL1 activity.

[0280] Other names for "PD-1" include CD279 and SLEB2. Other names for "PDL1" include B7-H1, B7-4, CD274, and B7-H. Other names for "PDL2" include B7-DC, Btdc, and CD273. PD-1, PDL1, and PDL2 may be human PD-1, PDL1, and PDL2.

[0281] PD-1 inhibitors can be molecules that inhibit PD-1 from binding to its ligand binding partner. PD-1 ligand binding partners can be PDL1 and / or PDL2. PDL1 inhibitors can be molecules that inhibit PDL1 from binding to its binding partner. PDL1 binding partners can be PD-1 and / or B7-1. PDL2 inhibitors can be molecules that inhibit PDL2 from binding to its binding partner. PDL2 binding partners can be PD-1. Inhibitors can be antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, or oligopeptides. Exemplary antibodies are described in U.S. Patents Nos. 8735553, 8354509, and 8008449, all of which are incorporated herein by reference. Other PD-1 inhibitors for the methods and compositions provided herein are known in the art, such as described in U.S. Patent Applications US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all of which are incorporated herein by reference.

[0282] The PD-1 inhibitor can be an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). The anti-PD-1 antibody is selected from nivolumab, pembrolizumab, and pidilizumab. The PD-1 inhibitor can be an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). The PDL1 inhibitor can comprise AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and is an anti-PD-1 antibody described in WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, and SCH-900475, are anti-PD-1 antibodies described in WO 2009 / 114335. Pidilizumab, also known as CT-011, hBAT or hBAT-1, is an anti-PD-1 antibody described in WO 2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO 2010 / 027827 and WO2011 / 066342. Other PD-1 inhibitors include MEDI0680 (also known as AMP-514) and REGN2810.

[0283] The immune checkpoint inhibitor can be a PDL1 inhibitor, such as durvalumab (also known as MEDI4736), atezolizumab (also known as MPDL3280A), avelumab (also known as MSB00010118C), MDX-1105, BMS-936559, or a combination thereof. The immune checkpoint inhibitor can be a PDL2 inhibitor, such as rHIgM12B7.

[0284] The inhibitor may comprise the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. The inhibitor may comprise the V H The CDR1, CDR2, and CDR3 domains of the α-terminal region, and the V L The antibody may compete for binding and / or bind to the same epitope on PD-1, PDL1 or PDL2 as the above-mentioned antibodies. 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-mentioned antibodies.

[0285] b. CTLA-4, B7-1 and B7-2

[0286] 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 is retrieved in Genbank as L15006. CTLA-4 is present on the surface of T cells and acts as a "close" 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, expressed on the surface of helper T cells, and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell co-stimulatory protein CD28, both of which bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA-4 has also been found in regulatory T cells and may be important for their function. Activation of T cells by T cell receptors and CD28 leads to increased expression of CTLA-4, which is an inhibitory receptor for B7 molecules. The inhibitors disclosed herein can block one or more than one function of CTLA-4, B7-1 and / or B7-2 activity. The inhibitor can be an inhibitor that blocks the interaction of CTLA-4 and B7-1. The inhibitor can be an inhibitor that blocks the interaction of CTLA-4 and B7-2.

[0287] The immune checkpoint inhibitor can 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.

[0288] Anti-human CTLA-4 antibodies (or V derived therefrom) suitable for use in the methods of the present disclosure H and / or V L Domain) can be produced by methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. For example, anti-CTLA-4 antibodies are disclosed in: US 8119129, WO 01 / 14424, WO 98 / 42752, WO 00 / 37504 (CP675,206, also known as tremelimumab, formerly known as ticilimumab), U.S. Patent No. 6207156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the above publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO 2001 / 014424, WO 2000 / 037504, and U.S. Patent No. 8017114; all of these publications are incorporated herein by reference.

[0289] Another anti-CTLA-4 antibody used as a checkpoint inhibitor in the methods and compositions of the present disclosure is ipilimumab (also known as 10D1, MDX010, MDX101, and ) or antigen-binding fragments and variants thereof (see, e.g., WO 01 / 14424).

[0290] The inhibitor may comprise the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. The inhibitor may comprise the V H The CDR1, CDR2 and CDR3 domains of the region, and the V L The antibody may compete for binding and / or bind to the same epitope on PD-1, B7-1 or B7-2 as the above-mentioned antibodies. 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-mentioned antibodies.

[0291] c.LAG3

[0292] Another immune checkpoint that can be targeted in the methods provided herein is lymphocyte activation gene 3 (LAG3), also known as CD223 and lymphocyte activation gene 3. The complete mRNA sequence of human LAG3 is accessed at Genbank as NM_002286. LAG3 is a member of the immunoglobulin superfamily and is 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, which negatively regulates cell proliferation, activation, and homeostasis of T cells in a manner similar to CTLA-4 and PD-1, and is reported to play a role in Treg suppressive function. LAG3 also helps maintain CD8+T cells in a tolerant state, and together with PD-1, helps maintain CD8 exhaustion during chronic viral infection. It is well known that LAG3 is also involved in the maturation and activation of dendritic cells. The inhibitors of the present invention can block one or more than one function of LAG3 activity.

[0293] The immune checkpoint inhibitor can 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.

[0294] Anti-human LAG3 antibodies (or V antibodies derived therefrom) suitable for use in the methods of the present disclosure H and / or V LDomains) can be produced by methods well known in the art. Alternatively, anti-LAG3 antibodies recognized in the art can be used. For example, anti-LAG3 antibodies may include: GSK2837781, IMP321, FS-118, Sym022, TSR-033, MGD013, BI754111, AVA-017 or GSK2831781. Anti-LAG3 antibodies disclosed in: US 9505839 (BMS-986016, also known as relalizumab); US10711060 (IMP-701, also known as LA 525); US 9244059 (IMP731, also known as H5L7BW); US 10344089 (25F7, also known as LAG3.1); WO 2016 / 028672 (MK-4280, also known as 28G-10); WO 2017 / 019894 (BAP050); 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 claimed invention can be found, for example, in WO 2016 / 028672, WO 2017 / 106129, WO 2017062888, WO 2009 / 044273, WO 2018 / 069500, WO 2016 / 126858, WO 2014 / 179664, WO 2016 / 200782, WO 2015 / 200119, WO 2017 / 019846, WO 2017 / 198741, WO 2017 / 220555, WO 2017 / 220569, WO 2018 / 071500, WO 2017 / 015560; WO 2017 / 025498, WO 2017 / 087589, WO 2017 / 087901, WO 2018 / 083087, WO 2017 / 149143, WO 2017 / 219995, US 2017 / 0260271, WO 2017 / 086367, WO 2017 / 086419, WO 2018 / 034227 and WO 2014 / 140180. The teachings of each of the above publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to LAG3 can also be used.

[0295] The inhibitor may comprise the heavy and light chain CDRs or VRs of an anti-LAG3 antibody. The inhibitor may comprise the V H The CDR1, CDR2 and CDR3 domains of the region, and the VL 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 antibodies.

[0296] d.TIM-3

[0297] Another immune checkpoint that can be targeted in the methods provided herein is T cell immunoglobulin and mucin domain-3 (TIM-3), also known as hepatitis A virus cell receptor 2 (HAVCR2) and CD366. The complete mRNA sequence of human TIM-3 is retrieved in Genbank as NM_032782. TIM-3 is found on the surface of CD4+Th1 and CD8+Tc1 cells that produce IFNγ. The extracellular region of TIM-3 consists of a single variable immunoglobulin domain (IgV) at the distal end of the membrane and a glycosylated mucin domain of variable length located near the membrane. TIM-3 is an immune checkpoint that mediates T cell exhaustion together with other inhibitory receptors including PD-1 and LAG3. TIM-3 has also been shown to be a CD4+Th1-specific cell surface protein that regulates macrophage activation. The inhibitor disclosed herein can block one or more than one function of TIM-3 activity.

[0298] The immune checkpoint inhibitor can 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.

[0299] Anti-human TIM-3 antibodies (or V derived therefrom) suitable for use in the methods of the present disclosure H and / or V L Domains) can be produced 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 include: MBG453, TSR-022 (also known as Copplimab) and LY3321367, which can be used in the methods disclosed herein. These and other anti-TIM-3 antibodies useful in the claimed invention can be found, for example, in: US 9605070, US 8841418, US2015 / 0218274 and US2016 / 0200815. The teachings of each of the above publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to TIM-3 can also be used.

[0300] The inhibitor may comprise the heavy chain and light chain CDRs or VRs of the anti-TIM-3 antibody. The inhibitor may comprise the V H The CDR1, CDR2 and CDR3 domains of the region, and the VL 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 antibodies.

[0301] 2. Activation of co-stimulatory molecules

[0302] Immunotherapy can include activators of costimulatory molecules. Activators can include B7-1 (CD80), B7-2 (CD86), CD28, ICOS, OX40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and agonists of combinations thereof. Activators include agonist antibodies, polypeptides, compounds, and nucleic acids.

[0303] 3. Dendritic cell therapy

[0304] Dendritic cell therapy stimulates an anti-tumor response by having dendritic cells present tumor antigens to lymphocytes, which activate the dendritic cells, triggering them to kill other cells presenting the antigen. Dendritic cells are antigen presenting cells in the mammalian immune system. In cancer treatment, they aid in cancer antigen targeting. An example of a dendritic cell-based cellular cancer therapy is Provenge-T.

[0305] One way to induce dendritic cells to present tumor antigens is by vaccinating them with autologous tumor lysates or short peptides (small parts of proteins that correspond to protein antigens on cancer cells). These peptides are often combined with adjuvants (highly immunogenic substances) to enhance immune and anti-tumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte macrophage colony-stimulating factor (GM-CSF).

[0306] Dendritic cells can also be activated in vivo by causing tumor cells to express GM-CSF. This can be achieved by genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.

[0307] Another strategy is to remove dendritic cells from the patient's blood and activate them in vitro. Dendritic cells are activated in the presence of tumor antigens, which can be a single tumor-specific peptide / protein or a tumor cell lysate (a solution of broken down tumor cells). These cells (and optionally an adjuvant) are injected and an immune response is stimulated.

[0308] Dendritic cell therapy involves the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibodies, and the dendritic cells can be induced to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8, or CD40 have been used as antibody targets.

[0309] 4. CAR-T cell therapy

[0310] Chimeric antigen receptors (CARs, also called chimeric immunoreceptors, chimeric T-cell receptors, or artificial T-cell receptors) are engineered receptors that bind to immune cells with new specificities to target cancer cells. Typically, these receptors transplant the specificity of a monoclonal antibody onto a T cell. These receptors are called chimeras because they are made by fusing parts from different sources. CAR-T cell therapy refers to treatments that use such transformed cells for cancer treatment.

[0311] The basic principle of CAR-T cell design involves recombinant receptors that combine antigen binding and T cell activation functions. The general premise of CAR-T cells is to artificially generate T cells that target markers found on cancer cells. Scientists can remove T cells from the human body, genetically modify them, and then put them back into the patient to allow them to attack cancer cells. After the T cell is modified into a CAR-T cell, it becomes a "living drug." CAR-T cells establish a connection between an extracellular ligand recognition domain and an intracellular signaling molecule, which in turn activates the T cell. 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 how to ensure that only cancerous tumor cells are targeted and normal cells are not targeted. The specificity of CAR-T cells is determined by the choice of targeting molecule.

[0312] Examples of CAR-T therapies include Kymriah and Yescarta.

[0313] 5. Cytokine therapy

[0314] Cytokines are proteins produced by many types of cells present within tumors. They can modulate immune responses. Tumors often use them to grow and reduce immune responses. These immunomodulatory effects allow them to be used as drugs to stimulate immune responses. Two commonly used cytokines are interferons and interleukins.

[0315] Interferons are produced by the immune system. They are usually involved in antiviral responses, but are also used against cancer. There are three types of them: type I (IFNα and IFNβ), type II (IFNγ), and type III (IFNλ).

[0316] Interleukins have a range of effects on the immune system. IL-2 is an example of an interleukin cytokine therapy.

[0317] 6. Adoptive T cell therapy

[0318] Adoptive T cell therapy is a form of passive immunization through the infusion of T cells (adoptive cell transfer). They are present in the blood and tissues and are usually activated when they detect foreign pathogens. Specifically, T cells are activated when their surface receptors encounter cells that display part of a foreign protein on their surface antigens. These cells can be infected cells or antigen presenting cells (APCs). They are found in normal tissues and tumor tissues, where they are called tumor infiltrating lymphocytes (TILs). They are activated by APCs such as dendritic cells that present tumor antigens. Although these cells can attack tumors, the environment within the tumor is highly immunosuppressive to prevent immune-mediated tumor death.

[0319] A variety of methods have been developed to generate and obtain tumor-targeted T cells. Tumor antigen-specific T cells can be taken from tumor samples (TIL) or filtered from the blood. Subsequent activation and cultivation are performed in vitro, and the results are re-injected into the body. Activation can be performed by gene therapy and / or by exposing T cells to tumor antigens.

[0320] It is expected that cancer therapy can exclude any cancer therapy described herein. The methods and compositions disclosed herein include patients who have previously received therapy treatment described herein, patients who are currently receiving therapy treatment described herein, or patients who have not yet received therapy treatment described herein. The patient can be a patient determined to be resistant to therapy described herein. The patient can be a patient determined to be sensitive to therapy described herein. For example, the patient may be a patient determined to be sensitive to immune checkpoint inhibitor treatment based on the determination that the patient has or has previously suffered from pancreatitis.

[0321] C. Chemotherapy

[0322] Other therapies may include chemotherapy. Suitable classes of chemotherapeutic agents include: (a) alkylating agents, such as nitrogen mustards (e.g., dichloromethane, cyclophosphamide, ifosfamide, melphalan, chlorambucil), ethyleneimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorzoxazone, streptozotocin), and triazines (e.g., dicarbazine), (b) antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, fluorouracil, cytarabine, azapyrimidines), and purine analogs and related substances (e.g., oxazolidinone). 6-mercaptopurine, 6-thioguanine, pentostatin), (c) natural products, such as vinca alkaloids (such as vinblastine, vincristine), epipodophyllotoxins (such as etoposide, teniposide), antibiotics (such as actinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitoxantrone), enzymes (such as L-asparaginase) and biological response modifiers (such as interferon-α) and (d) other agents, such as platinum complexes (such as cisplatin, carboplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as methylbenzalazine) and adrenocortical hormone inhibitors (such as paclitaxel and mitotane). Cisplatin can be used as a particularly suitable chemotherapeutic agent.

[0323] 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 cannot be absorbed orally and must therefore be delivered by other routes, such as intravenous, subcutaneous, intratumoral, or intraperitoneal injection. Considering that cisplatin can be used alone or in combination with other drugs, effective doses used in clinical applications include about 15 mg / m 2 About 20 mg / m 2 , administered for 5 days every three weeks, for a total of three courses.

[0324] Other suitable chemotherapeutic agents include anti-microtubule agents such as paclitaxel ("Taxol") and doxorubicin hydrochloride ("Adriamycin"). The combination of the Egr-1 promoter / TNFα construct and doxorubicin delivered by an adenoviral vector was determined to be effective in overcoming resistance to chemotherapy and / or TNF-α, indicating that combined treatment with the construct and doxorubicin overcomes resistance to doxorubicin and TNF-α.

[0325] Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the present disclosure. Nitrogen mustards may include, but are not limited to, dichloromethyl diethylamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide Available from Mead Johnson, and (available from Adria) is another suitable chemotherapeutic agent. Suitable oral dosages for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, and intravenous dosages include, for example, initial divided doses of about 40 mg / kg to about 50 mg / kg for about 2 days to about 5 days, or about 10 mg / kg to about 15 mg / kg for about every 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. Due to adverse gastrointestinal effects, intravenous routes are preferred. The drug can also be administered intramuscularly, infiltrated, or into the body cavity sometimes.

[0326] Other 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 at about 7.5 mg / m 2 About 1000 mg / m 2 In addition, the 5-FU dosage schedule can be various time periods, such as up to six weeks, or determined by a person of ordinary skill in the art to which the present disclosure belongs.

[0327] The amount of the chemotherapeutic agent delivered to the patient can be variable. When chemotherapy is used together with the construct, the chemotherapeutic agent can be applied in an amount that effectively causes the cessation or regression of cancer in the host. The chemotherapeutic agent can be applied in an amount lower than 1 / 2 to 1 / 10000 of the chemotherapeutic effective dose of the chemotherapeutic agent. For example, the chemotherapeutic agent can be applied in an amount lower than about 1 / 20, about 1 / 500 times or even about 1 / 5000 of the chemotherapeutic effective dose of the chemotherapeutic agent. The desired therapeutic activity of the chemotherapeutic agent of the present disclosure can be tested in vivo with the construct, and the effective dose can be determined. For example, these compounds can be tested in a suitable animal model system before human trials, including but not limited to rats, mice, chickens, cattle, monkeys, rabbits, etc. In vitro tests can also be used to determine suitable combinations and dosages, as described in the embodiments.

[0328] D. Hormone therapy

[0329] In some aspects, the cancer therapy of the present disclosure is hormone therapy. In particular aspects, prostate cancer treatment includes hormone therapy. Various hormone therapies are known in the art and are contemplated herein. Examples of hormone therapy include, but are not limited to, luteinizing hormone releasing hormone (LHRH) analogs, LHRH antagonists, androgen receptor antagonists, and androgen synthesis inhibitors.

[0330] E.Surgery

[0331] Approximately 60% of cancer patients will undergo some type of surgery, including preventive, diagnostic or staging, therapeutic, and conservative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and may be used in conjunction with other therapies, such as treatment of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscope-controlled surgery (Mohs surgery).

[0332] After removing part or all of the cancer cells, tissues or tumors, a cavity can be formed in the body. Treatment can be completed by perfusion, direct injection or local application of other anticancer therapies. This treatment can be repeated, for example, every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, or every 1 week, 2 weeks, 3 weeks, 4 weeks and 5 weeks, or every 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months. These treatments can also have different dosages.

[0333] F. Other cancer treatments

[0334] The treatment methods disclosed herein may include one or more than one other cancer therapy. The cancer therapy disclosed herein may include, for example, cryoablation therapy, high intensity ultrasound (also referred to as "high intensity focused ultrasound"), photodynamic therapy, laser ablation, and / or irreversible electroporation. The cancer therapy disclosed herein may include 1, 2, 3, 4, 5, or more than 5 different treatment methods.

[0335] It is contemplated that cancer therapy may exclude any cancer therapy described herein. In addition, aspects of the present disclosure include patients who have previously been treated with a therapy described herein, patients who are currently being treated with a therapy described herein, or patients who have not yet been treated with a therapy described herein. In some aspects, the patient is a patient who is determined to be resistant to a therapy described herein. In some aspects, the patient is a patient who is determined to be sensitive to a therapy described herein.

[0336] XII. Immunostimulants

[0337] Certain aspects of the present disclosure relate to immunostimulants and methods of use. As used herein, the term "immunostimulant" refers to a compound that can stimulate an immune response in a subject, and may include an adjuvant.

[0338] In some aspects, the immunostimulant is an agent that does not constitute a specific antigen but can enhance the intensity and duration of the immune response to the antigen. Such immunostimulants can include, but are not limited to, stimulators of pattern recognition receptors (e.g., Toll-like receptors, RIG-1, and NOD-like receptors), mineral salts such as alum, alum bound to monophosphoryl lipids (MPL) of enterobacteria (e.g., Escherichia coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri), or particularly alum bound to MPL (ASO4), alum bound to MPL A of the above bacteria alone, saponins such as QS-21, Quil-A, ISCOMs, ISOCAMITX, emulsions such as MF59, Montanide, ISA 51, and ISA 720, AS02 (QS21 + squalene + MPL.), liposomes and liposome preparations such as AS01, synthetic or specially prepared microparticles and microcarriers such as outer membrane vesicles (OMV) of bacterial origin of Neisseria gonorrhoeae, Chlamydia trachomatis, etc., or chitosan particles, reservoir forming agents such as Pluronic block copolymers, specifically modified or prepared peptides such as muramyl dipeptide, aminoalkyl glucosamine 4-phosphate such as RC529, or proteins such as bacterial toxins or toxin fragments. Exemplary immunostimulants include polymeric immunomodulators described herein.

[0339] XIII. Examples

[0340] The following examples are included to illustrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention, and therefore can be considered to constitute certain modes of practice of the present invention. However, in light of this disclosure, it will be appreciated by those skilled in the art that many changes may be made in the disclosed specific embodiments, and similar or similar results may still be obtained without departing from the spirit and scope of the present invention.

[0341] Example 1 - Synthesis of polymeric TLR 7 / 8 modulator adjuvants and their use for immune activation and vaccination enhancement

[0342] Oligomeric peptides with repeating TLR7 / 8a(imidazoquinoline) and dopamine units were generated (“pTLR7 / 8a_mod” or “p(TLR7 / 8a_dopa)”; general structure see Figure 7 , an overview of the synthesis scheme can be found in FIG. 8A to FIG. 8B , chemical structure see Fig. 9 p(TLR7 / 8a_dopa) was analyzed in various in vitro and in vivo assays to assess immune activation and vaccine efficacy enhancement.

[0343] A. Materials and Methods

[0344] Peptides: Peptides and modified peptides were synthesized by standard solid phase peptide synthesis using a microwave Liberty blue peptide synthesizer and purified (>90%) by high performance liquid chromatography.

[0345] TLR-7 / 8 agonists: Imidazoquinoline-based TLR-7 / 8 agonists were produced as described previously (see J. Med. Chem. 2010, 53, 11, 4450-4465, which is incorporated herein by reference in its entirety).

[0346] pTLR7 / 8a: The imidazoquinoline-based TLR-7 / 8a was modified with a C6 linker using NHS ester chemistry. The agonist was then loaded onto the glutamate pentamer using HATU coupling chemistry. This peptide was modified with a DBCO handle.

[0347] pdopa: Fmoc-DOPA (acetonide)-OH was purchased from Sigma and the pentamer of DOPA was prepared using solid-state peptide synthesis. This peptide was modified with an azide handle.

[0348] p(TLR7 / 8a_dopa): pTLR7 / 8a and pdopa were dissolved in DMF and mixed in a test tube. The test tube was shaken at room temperature for 1 h. The final product was purified using reverse phase HPLC.

[0349] BMDC activation: BMDCs were prepared from C57Bl / 6 mice as described previously (see Chem.ci., 2021, 12, 6646-6651, which is incorporated herein by reference in its entirety) and used on day 7. For BMDC activation studies, 2×10 5 Cells were seeded in round-bottom 96-well plates (Fisher Scientific) in RPMI containing 10% FBS and 2% penicillin / streptomycin (Life Technologies) and treated with different concentrations of mTLR7 / 8a (imidazoquinoline), mTLR7 / 8a and dopamine mixture, or p (TLR7 / 8a_dopa), and then incubated at 37° C. After 24 h, samples were collected and cytokine concentrations in the culture medium were measured by ELISA according to the manufacturer's instructions.

[0350] RAW264.7 macrophages (RAW-Blue) NF-κB assay: RAW-Blue cells (passages 5-15) were seeded at a density of 100,000 cells / well in 96-well plates containing 180 μL DMEM with 10% heat-inactivated FBS (HI-FBS) and selective antibiotics. Cells were treated with agonists for 20 h at 37°C and 5% CO2. NF-κB activity was measured by QUANTI-Blue (InvivoGen) analysis, and the absorbance at 620 nm was measured using a Multiskan FC microplate reader (Thermo Scientific).

[0351] Flow cytometry for upregulation of cell surface markers: BMDCs were plated at 1 × 10 6 Cells / mL were seeded in untreated 12-well plates and incubated with agonists in culture medium at 37°C and 5% CO2 for 18 h. Cells were released from the plate by vigorous pipetting and then centrifuged at 2500 RPM for 10 minutes at 4°C. The cell pellet was resuspended in cold FACS buffer (PBS, 10% FBS and 0.1% sodium azide) buffer (300 μL) and incubated with CD16 / 32 FcR blocking antibody (1.0 μg / 1×10 6 Cells) were incubated on ice for 15 minutes. The cell suspension was precipitated and the supernatant was removed. Next, the cell pellet was resuspended in cold FACS buffer (100 μL) and stained for CD86 and CD40 for 30 minutes in the dark. The samples were then washed twice with 300 μL FACS buffer. The precipitated cells were resuspended in cold FACS buffer (200 μL) and stored on ice until loaded onto a flow cytometer for analysis.

[0352] OVA immunization studies: C57Bl / 6 mice were vaccinated subcutaneously near the tail with 100 μg of OVA formulated as OVA / p(TLR7 / 8a_dopa) / alum, OVA / alum, OVA / mTLR7 / 8a / alum, OVA / (dopa+mTLR7 / 8) / alum on days 0 and 14. Formulations containing TLR7 / 8 ligands contained molar equivalents of monomeric TLR7 or polymeric TLR7 / 8 ligands (25 nanomoles / mouse). Blood was collected on day 21, serum was separated by centrifugation and stored at -20°C. Antibody levels against OVA in serum were analyzed using ELISA. dLNs from each animal were collected and processed. Lymph node cells were restimulated for 2 days by cytokine microsphere assay (CBA) to measure secreted cytokines.

[0353] B. Results

[0354] Bone marrow-derived macrophages (BMDM) were treated with pTLR7 / 8_mod, pTLR7 / 8, mTLR7 / 8, or phosphate-buffered saline (PBS). Figure 1 Increased NF-κB levels after treatment are shown. FIG. 2A to FIG. 2B CD40 ( Figure 2A ) and CD86( Figure 2B After treatment with pTLR7 / 8_mod, the expression levels of both molecules increased to the greatest extent. Figure 3 Shown are the TNF-α concentrations measured in the supernatants of treated BMDMs. Compared to pTLR7 / 8 and mTLR7 / 8, pTLR7 / 8_mod treatment resulted in the smallest increase in TNF-α levels.

[0355] C57B1 / 6 mice were vaccinated with ovalbumin antigen ("OVA") in combination with alum alone, or with pTLR7 / 8_mod, mTLR7 / 8, or mTLR7 / 8+dopamine (mTLR7 / 8+mod). FIG. 4A to FIG. 4C IL-23 ( Figure 4A )、IL-6( Figure 4B ) and MCP-1( Figure 4C ). Vaccination with pTLR7 / 8_mod showed the lowest levels of all three cytokines. Figure 5 Serum titers of anti-OVA antibodies in mice are shown. The highest anti-OVA antibodies were measured in mice vaccinated with pTLR7 / 8_mod. FIG. 6A to FIG. 6C The production of IL-23 by cells isolated from mouse draining lymph nodes is shown. Fig. 6A )、IL-6( Figure 6B ) and TNF-α( Figure 6C ) and re-stimulated with OVA for 48 h. Cells from mice vaccinated with pTLR7 / 8_mod showed the highest levels of all three cytokines.

[0356] Example 2 - General Materials and Methods

[0357] Reagents and solvents were purchased from commercial sources and used without further purification. Vanillin, dopamine hydrochloride, 4-hydroxy-3-methoxybenzylamine hydrochloride, and ferulic acid were all commercially available. APC anti-mouse CD40, PE anti-mouse CD11c, and purified anti-mouse CD16 / 32 were purchased from BioLegend. COX-2 (mouse) polyclonal antibody was purchased from Cayman Chemical. GAPDH (14C 10) rabbit monoclonal antibody and iNOS (D6B6S) rabbit monoclonal antibody were purchased from Cell Signaling Technology. The proteins were analyzed using a Bruker Avance III HD 500 11.7 Tesla NMR (500 MHz). 1 H and 13 C NMR spectra were characterized. NMR spectra were analyzed using MestreNova software. Couplings in the spectra are expressed in Hertz, and the abbreviations for multiplicities are s = singlet, d = doublet, t = triplet, dd = doublet of a doublet, and m = multiplet, if applicable. Mass spectrometry was performed on an Agilent 6224 TOF-MS. A Phenomenex Luna C 18 or C 8 Prep (150 × 21.2250 mm, 5 μm particle size) column, Gilson 333 / 334 pump system and GX-271 liquid handler system, preparative reverse phase HPLC purification at a flow rate of 21.2 mL / min. Preparative HPLC was performed with UV detection (214 nm, 254 nm and 260 nm). Flow cytometry data were collected using a NovoCyte benchtop flow cytometer. Absorbance measurements were performed on a Multiskan FC microplate reader (Thermo Scientific). Data were analyzed using one-way ANOVA in Graph Pad Prism software. All values ​​are reported as mean ± SD. Female C57 / BL6 mice and male Balb / c mice were purchased from Jackson Laboratories and equilibrated for at least 48 h before use. For all experiments, mice were 6 to 10 weeks old. All animal studies and mouse maintenance were approved by the Institutional Animal Care and Use (IACUC #2012-3048).

[0358] Example 3 - NF-κB analysis of RAW264.7 macrophages (RAW-Blue):

[0359] RAW-Blue cells (InvivoGen) were cultured according to the manufacturer's instructions. Cells were grown in complete medium consisting of Dulbecco's modified Eagle's medium (DMEM) and 4.5 g / L glucose (Life Technologies), 2 mM L-glutamine, 10000 U / mL penicillin, 10 mg / mL streptomycin, 25 μg / mL amphotericin B, and supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific). RAW-Blue cells (generations 5-15) were seeded in 96-well plates containing 10% heat-inactivated FBS (HI-FBS) and selective antibiotics at a density of 100,000 cells / well. Cells were treated with agonists and agonist dimers and LPS controls (50 ng / mL) at 37°C and 5% CO2 for 20 h. NF-κB activity was measured by QUANTI-Blue (InvivoGen) assay, and the absorbance at 620 nm was measured using a Multiskan FC microplate reader (ThermoScientific).

[0360] Example 4 - Collection and culture of bone marrow-derived dendritic cells

[0361] Bone marrow-derived dendritic cells (BMDCs) were collected from female C57Bl / 6 mice. Femurs were removed from mice under sterile conditions, and the bone marrow was extracted into PBS buffer. The cell suspension was centrifuged at 300RCF for 10 minutes at room temperature to pellet the cells. ACK lysis buffer (3 mL, Lonza) was added to the cell pellet and incubated for 2 minutes at room temperature (RT). PBS buffer (13 mL) was then added to the cell suspension, and the cell solution was centrifuged at 300RCF for 10 minutes at room temperature. Next, the cell pellet was resuspended in BMDC complete medium (RPMI1640, 10% heat-inactivated FBS, 20 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 2 mM L-glutamine (Life Technologies), 10000 U / mL penicillin, 10 mg / mL streptomycin, 25 μg / mL amphotericin B, and 50 μM β-mercaptoethanol). The cells were then plated at 1×10 6 Cells / mL were seeded in 10 mL of complete medium in a 100 mm dish and incubated at 37 °C in a CO atmosphere. 2 Incubate in an incubator. On day 3, add 10 mL of fresh BMDC medium to each dish. On day 6, release BMDCs and culture at 1 × 10 6 Cells / mL were seeded into untreated 12-well plates for cell surface marker activation and cytokine secretion experiments.

[0362] Example 5 - Immunoblotting analysis of pathway proteins

[0363] RAW264.7 macrophages (InvivoGen) cultured in complete medium were cultured at 1×10 6 Cells / mL were seeded in 6-well plates and allowed to adhere for 12 h in a 37°C CO2 incubator. Cells were then treated with agonists, agonist dimers, and LPS control for 16 h. Treated cells were washed and scraped into cold phosphate-buffered saline (PBS) and centrifuged at 400 × g for 5 min at 4°C. The cell pellet was suspended in 5% paraformaldehyde containing a protease inhibitor cocktail (cOmplete TM ULTRA Tablets, Sigma) in triple detergent lysis buffer (10 mL) followed by centrifugation to generate whole cell lysates. TM Lysates were quantified by BCA protein analysis kit. 50 μg of total protein was separated by 4% to 15% SDS-PAGE and blotted onto PVDF membrane (Bio-Rad). The membrane was probed with COX-2 monoclonal antibody (Cayman Chemicals, MI) at a dilution of 1:1000 and GAPDH (14C10) at a dilution of 1:1000 and rabbit monoclonal antibody and iNOS (D6B6S) rabbit monoclonal antibody at a dilution of 1:500, and probed with 1:10000 dilution of ... Visualization was performed with 800CW (Abcam) and imaging was performed on an Azure Biosystems Imager. Densitometric analysis was performed using ImageJ.

[0364] Example 6 - Flow cytometry for cell surface marker upregulation and cytokine secretion analysis

[0365] BMDC was added at 1×10 6 Cells were seeded at a density of 100 cells / mL in untreated 12-well plates and incubated with agonists and agonist dimers in culture medium at 37°C and 5% CO. 2 Incubate for 8 h under 4% FACS conditions. Release the cells from the plate by vigorous pipetting and then centrifuge at 2500 RPM for 10 minutes at 4°C. Save the cell culture medium for IL-6 cytokine quantification using ELISA (BioLegend). Resuspend the cell pellet in cold FACS buffer (PBS, 10% FBS and 0.1% sodium azide) (300 μL) and incubate with CD16 / 32 FcR blocking antibody (1.0 μg / 1×10 6The cells were incubated on ice for 15 min. The cell suspension was pelleted and the supernatant removed. Next, the cell pellet was resuspended in cold FACS buffer (100 μL) and stained with PE-CD11c (1.0 μg / 1×10 6 cells) and APC CD40 (1.0 μg / 1×10 6 Cells) were incubated on ice in the dark for 30 minutes. The samples were then washed twice with 300 μL FACS buffer. The precipitated cells were resuspended in cold FACS buffer (200 μL) and kept on ice until loaded on a flow cytometer for analysis.

[0366] Example 7 - Vaccination in mice

[0367] Female C57 / BL6 mice were briefly anesthetized with isoflurane and injected intramuscularly in the right hind leg with 50 μL of ovalbumin (100 μg), adjuvant, adjuvant dimer, vanillin and dopamine (0.07 μM) and a PBS vehicle control group.

[0368] Example 8 - Plasma cytokine analysis and antibody quantification

[0369] One hour after vaccination, mouse blood was collected from the submandibular vein into a 0.2 mL heparin-coated collection tube (VWR Scientific). Serum was separated by allowing the blood to coagulate at room temperature for 30 minutes and centrifuging at 2000 × g for 10 minutes. The supernatant was collected and stored at -80°C until use. Mouse inflammatory cytokine detection kit or LEGENDplex was used according to the manufacturer's protocol. TM Serum was analyzed using the Mouse Inflammation Panel (Biolegend). For antibody quantification, mouse blood was collected by cardiac puncture into 0.2 mL heparin-coated collection tubes (VWR Scientific) 28 days after vaccination. Serum was separated by allowing the blood to clot for 30 minutes at room temperature and centrifuged at 2000×g for 10 minutes. Serum was analyzed using quantitative anti-ovalbumin total Ig's, IgA, and IgG ELISA kits (Alpha Diagnostic International) according to the manufacturer's protocol.

[0370] Example 9 - Tumor Research

[0371] Dissolve 0.2×10 6CT-26 cells were injected subcutaneously into the abdomen of 6-week-old Balb / c mice (10 per group). Tumor size was monitored every other day. Tumor volume was measured using the formula V=1 / 2×L×W×W. Treatment began when the tumor reached a size of approximately 75mm3 (day 11). Various preparations (20 nanomoles of each agonist or PBS) were injected around the tumor every 4 days (days 15, 19, and 23). When the tumor reached 20mm in any linear dimension, the mice were euthanized. Five mice per group were used for blood analysis. Blood was collected two days after the first injection for hematological toxicity analysis, and blood was collected two hours after the second injection for systemic cytokine analysis.

[0372] Example 10 - Nitric Oxide Analysis

[0373] RAW264.7 macrophages (InvivoGen) cultured in complete medium were cultured at 1×10 6 Cells / mL were seeded in 12-well plates and incubated with agonists and agonist dimers at 37°C in CO 2 Treat in an incubator for 16 hours. Collect the cell supernatant and measure the amount of nitrite in the culture medium as an indicator of NO production. Use Griess reagent (2.5% phosphoric acid solution containing 1% sulfonamide and 0.1% naphthylethylenediamine dihydrochloride) to measure the content of nitrite, a stable metabolite of NO. Mix 100 μL of cell culture medium with 100 μL of Griess reagent. After incubation at room temperature for 10 minutes, measure the absorbance at 540 nm with an enzyme reader. Calculate the nitrite content based on the sodium nitrite standard curve.

[0374] Example 11 - Reactive Oxygen Species Measurement

[0375] RAW264.7 macrophages (InvivoGen) cultured in complete medium were cultured at 1×10 6 Cells / mL were seeded in 12-well plates and incubated with agonists and agonist dimers at 37°C in CO 2 The cells were then removed from the plate and centrifuged at 2500 RPM for 10 minutes at room temperature, and the supernatant was aspirated. The cells were then washed twice with PBS (200 μL), and the cell pellet was resuspended in Hanks balanced salt solution (HBSS) containing CM-H2DCFDA (1 uM). Next, the cells were incubated at 37°C and 5% CO 2 Incubate for 30 minutes under 4% CO 2 conditions. After incubation, wash cells twice with cold PBS. Fluorescence was measured on the FL-1 (FITC) channel using flow cytometry.

[0376] Example 12 - Proliferation Assay

[0377] Proliferation analysis was performed as previously reported. Splenocytes were isolated from C57BL / 6 mice and cultured at 5 × 10 4 The splenocytes were then inoculated in 96-well plates with 3 μM agonist and agonist dimer at 37°C in CO 2 Incubate in an incubator for 48 h. Add 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (20 μl, 5 mg / ml, PBS solution) 4 h before the end of incubation. Dissolve the purple crystals in sterile DMSO and incubate for 5 minutes to ensure complete dissolution. Measure the absorbance at 590 nm using a Multiskan FC microplate reader (ThermoScientific). The proliferation rate is calculated as follows: Abs (sample) / Abs (PBS) × 100%.

[0378] Example 13 - Cell viability (MTT) assay

[0379] RAW264.7 macrophages were incubated with agonists and agonist dimers for 16 h before cell viability assays. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was dissolved in PBS to a final concentration of 5 mg / mL and then sterile filtered. The treated cells were resuspended in fresh RPMI medium containing 10% FBS at a concentration of 1 × 10 5 cells / mL and then seeded in a 96-well plate. 10 μL of MTT solution was added to these cells and then incubated at 37°C and 5% CO 2 Incubate for 3 h under 4% paraformaldehyde (pH 7.0) conditions. When purple crystals appear, remove 75 μL of supernatant. Dissolve the purple crystals in sterile DMSO and incubate for 5 minutes to ensure complete dissolution. Measure the absorbance at 590 nm using a Multiskan FC microplate reader (Thermo Scientific). % viability is calculated as follows: Abs(sample)-Abs(blank) / Abs(resting cells)-Abs(blank)×100%.

[0380] Example 14 - Synthesis of imidazoquinolinone-immunoenhancer dimer

[0381] Various small molecule NF-κB inhibitors were screened in vitro and in vivo in vaccine formulations. From this screening, vanillin and honokiol derivatives were identified as the most potent small molecule immunopotentiators 19 These anti-inflammatory and antioxidant molecules have been extensively studied in the literature and modulate NF-κB either by directly inhibiting the canonical NF-κB pathway or by scavenging pro-inflammatory mediators such as nitric oxide and other ROIs. 21–23When a mixture of R848 and capsaicin or honokiol enhancers were used in the vaccine formulation for in vivo experiments, high systemic cytokines were observed 1 h after vaccination (Figures 10A-10B). 20 A series of dimers were designed and synthesized by conjugation with vanillin, catechol, and honokiol-based moieties ( FIG. 11 ).

[0382] Example 15 - In vitro analysis of synthetic dimers

[0383] Next, an in vitro screen was designed to test the synthesized dimers and identify promising candidates for further development in an in vivo model. The overall activity of the compounds was measured using a RAW macrophage NF-κB-SEAP reporter cell line. The activity of the dimers was reduced compared to an equimolar mixture of an imidazoquinolinone agonist and an agonist and an NF-κB inhibitor (Figure 12A). However, the reduced activity may be due to the disruption of cellular uptake or receptor binding by molecular conjugation, thereby reducing the activity of the parent compound. To further elucidate the activity of the dimers, proinflammatory cytokine and cell surface protein expression assays were performed on mouse bone marrow-derived macrophages (BMDCs). After incubating the parent SMIP and dimers with BMDCs for 8 h, dimer compounds 1 to 4 reduced the level of secreted IL-6 to almost baseline levels. Compared to the parent SMIP, compounds 5 and 6 did not significantly change IL-6 levels. An equimolar mixture of SMIP and a small molecule NF-κB inhibitor also did not reduce the level of secreted IL6 (Figure 12B). In a similar BMDC assay, BMDC were stained for cell surface expression of CD40, a well-characterized co-stimulatory molecule that plays a role in adaptive immunity. 24 The expression levels were quantified using flow cytometry. In this experiment, CD40 expression levels remained unchanged for most compounds, with compound 1 expressing slightly lower levels ( Fig. 12C ). This assay was used to screen for dimers that could reduce proinflammatory cytokines while maintaining or improving cell surface protein expression. This suggests that the hybrid molecule is modulating the NF-κB response of the imidazoquinolinone, rather than just inhibiting activity.

[0384] In addition to inhibiting upstream events in the NF-κB pathway, small molecule NF-κB inhibitors incorporated into adjuvant dimers have previously been described in the literature as downstream inhibitors of proinflammatory mediators such as nitric oxide and reactive oxygen species (ROS). 23. The dimer adjuvants were then examined for similar effects on immune cells. To investigate the effects of the dimers on oxidative stress, RAW macrophages were incubated with the compounds for 16 h, and intracellular ROS levels were measured using the ROS-responsive fluorescent dye CM-H2DCFDA, and fluorescence was quantified using flow cytometry. Here, intracellular ROS was reduced by almost half for all dimer agonists, indicating that these compounds are ROS scavengers (Figure 13A). In a similar experiment, RAW macrophages were incubated with the dimer agonists for 16 h, and the levels of the nitric oxide metabolite nitrite in the cell supernatant were measured using Griess reagent. Nitrite levels were quantified by measuring absorbance using a microplate reader. This analysis showed that the dimer adjuvant reduced NO levels to baseline levels compared to the parent SMIP (Figure 13B), indicating that the dimer is a potent inhibitor of nitric oxide by scavenging or inhibiting inducible nitric oxide synthase (iNOS).

[0385] Finally, the inhibitory effect of the dimer agonists on cyclooxygenase-2 (COX-2) was studied. COX-2 is a proinflammatory marker that is associated with the activation of the NFκB and MAPK pathways. After incubation of RAW macrophages with the dimer agonists for 16 h, the cells were lysed and the proteins were separated using SDS-PAGE. The proteins were then transferred to a membrane and the protein levels were probed using an anti-COX-2 antibody. The relative expression of COX-2 protein was low for compounds 1 to 4 and 6, indicating that these compounds slightly inhibited the COX-2 pathway (Figures 14A to 14B).

[0386] Example 16 - In vivo analysis

[0387] Next, in vivo studies were performed to understand how these dimers perform in vaccine formulations. An overview of these studies is shown in Figure 15A. Using ovalbumin as a model, antigenic mice were vaccinated with the most promising dimers (2, 3) screened in vitro. Intramuscular injections (im) were performed with 100 μg ova, 70 nanomoles of imidazoquinolinone, dimers, and 50 μl PBS containing an equimolar mixture of imidazoquinolinone and NF-κB inhibitor. One hour after injection, mouse sera were collected and systemic levels of IL-6 (Figure 15B) and TNF-α (Figure 15C) were quantified. The dimers reduced these systemic cytokines to background levels comparable to PBS, vanillin, and catechol controls. On day 28, mice were sacrificed, sera were collected, and anti-OVA antibodies (Figures 16A to Fig. 16C ). Total Ig levels were observed and comparable Ig levels were observed in mice treated with TLR7 / 8 adjuvant compared to PBS and vanillin and catechol controls. Comparison of specific anti-OVA IgG (Figure 16B) and IgA antibodies ( Fig. 16C ), the levels of these antibodies induced by compound 2 were statistically higher.

[0388] Since TLR7 / 8 activation has been previously associated with enhanced CD8+ T cell function 17 , so the activity of the dimer adjuvant was studied and the activity of the parent SMIP adjuvant was measured. On day 28 of the OVA in vivo vaccination experiment, the spleens of the mice were harvested and single-cell splenocyte suspensions were prepared. The cells were then incubated with SINFEKLMHC-specific tetramers and analyzed using flow cytometry. There was no increase in the activity of the agonist dimer compared to the vehicle control. The parent SMIP adjuvant was also only slightly higher than the PBS / OVA control (Figure 21A). This is not unexpected, as others have shown that this route of administration, mixing OVA and adjuvant in PBS and injecting intramuscularly, can enhance antibody titers but not T cell immunity. In a separate analysis, splenocyte proliferation was examined with newborn splenocytes, and cells were incubated with dimer agonists and parent SMIP for 48h. The dimer agonist promoted lymphocyte proliferation to a level comparable to that of the parent SMIP (Figure 21B).

[0389] Example 17 - Local antitumor activity of dimer agonists

[0390] The antitumor activity of the dimer was then examined. Previous studies have shown that intratumoral adjuvant administration effectively reduces tumor proliferation by enhancing T cell antitumor activity. Specifically, TLR7 / 8 activation leads to enhanced activation of innate immune cells and propagates through increased secretion of IFNα, IL-12, and IFNγ cytokines. 17,25 For most compounds used for this purpose, such as resiquomot (R848), systemic adjuvant toxicity is always a disadvantage. Since the present dimer agonist platform reduces systemic cytokines, these compounds were subsequently tested on tumor models (Figure 17A, Fig. 22 and Figures 23A to 23F). In this in vivo model, the CT26 tumor model was used and adjuvant, PBS control, and adjuvant dimer were administered. In addition, resiqumod (R848) and the resiquimod derivative 3M-052 were included as controls. 3M-502 has been used in some clinical studies because it can be formulated into liposomes or emulsions, thereby reducing the diffusion rate due to the formation of a reservoir. 25 However, in this study, all adjuvants were formulated in PBS.

[0391] Tumors were established 11 days later, and SMIP adjuvants and adjuvant dimers were administered via intratumoral injection. At 2h and 24h after these injections, serum and blood were collected to detect systemic cytokines in serum, and blood was subjected to hematological analysis to better quantify adjuvant toxicity. Similar to the OVA vaccination model, the baseline levels of IL-6 and TNF-α measured in serum were induced by dimer adjuvants. In contrast, parental SMIP and R848 induced high levels of these proinflammatory cytokines, while 3M-052 was comparable to vehicle controls and dimer adjuvants. Unexpectedly, when compared to the remaining dimer adjuvants tested, compound 1 induced slightly elevated IL-6 (Figure 17B) and TNF-α (Figure 17C) levels. In addition, hematological analysis of blood showed that molecules that induced higher systemic inflammatory cytokines resulted in lower WBC (Figure 18A) and lymphocyte (Figure 18B) counts compared to vehicle controls, indicating that these molecules can cause adjuvant toxicity when introduced into tumors.

[0392] The adjuvant dimer was observed to be well tolerated, and tumor proliferation was then monitored (Figure 18C). After 32 days, all mice treated with the vehicle had to be euthanized due to tumor proliferation. Among the dimer adjuvants tested, two dimer molecules 1 and molecule 2 were able to improve survival, with compound 1 providing 80% survival on day 35, higher than the parent adjuvants 3M-052 and R848. Slightly elevated levels of IL-6 and TNF-α were observed in the serum of mice injected with compound 1, suggesting that local inflammation may be required for successful immunotherapy adjuvant candidates. Immune checkpoint inhibitors may be included in dimer adjuvant formulations to enhance dimers, making treatment effective and tolerable.

[0393] Example 18 - Synthesis Method

[0394] Compound 1( Fig. 11C ) was synthesized as follows. In a solution of 7 (19 mg, 0.078 mol) and triethylamine (0.01 mL, 0.078 mol), 1 mL of DMF containing 12 (26 mg, 0.052 mol) was added. The mixture was stirred at room temperature under argon for 12 h. The reaction mixture was purified using preparative HPLC to give a white solid product (15 mg, 40% yield)

[0395] 1H NMR (400MHz, DMSO) δ13.47 (s, 1H), 7.95 (t, J=8.3Hz, 2H), 7.80 (d, J=8.0Hz, 1H), 7.62 (t, J=74Hz, 1H), 7.38 (t, J=7.3Hz, 1 H), 7.30 (d, J=15.7Hz, 1H), 7.21 (d, J=8.2Hz, 2H), 7.12 (d, J=1.7Hz, 1H), 702 (d, J=8.1Hz, 2H), 6.98 (dd, J=8.2, 1.8Hz, 1H) , 6.79 (d, J=81Hz, 1H), 6.42 (d, J=15.7Hz, 1H), 5.93 (s, 2H), 4.15 (s, 2H), 3.80 (s, 4H), 3.14 (dd, J=12.6, 6.5Hz, 2H), 298 ( dd, J=16.6, 9.0Hz, 4H), l.73 (m, J=15.3, 7.6Hz, 2H), 1.57-1.47 (m, 2H), 1.39 (m, J=14.7, 7.4Hz, 2H), 0.87 (t, J=74Hz, 3H). 13 C NMR (100MHz, DMSO) δ165.8, 158.9, 158.6, 158.5, 157.4, 149.2, 148.7, 148.3, 141.2, 139.3, 135.8, 134.3, 134.1, 129.9, 128.0, 126. 9, 125.9, 125.2, 125.1, 122.0, 119.4, 119.0, 116.0, 114.9, 112.9, 111.1, 55.9, 43.9, 36.7, 30.7, 29.7, 29.4, 26.6, 22.9, 22.2, 14.1.

[0396] HRMS (ESI) m / z calculated as C 36 H 41 N 7 O 4 , [M+H] + 635.322; measured value: 636.3294.

[0397] The synthesis of compound 2 is as follows. In a solution of 8 (20 mg, 0.066 mol) and triethylamine (0.01 mL, 0.078 mol), 1 mL of DMF containing 12 (26 mg, 0.052 mol) was added. The mixture was stirred at room temperature for 12 h. The reaction mixture was purified using preparative HPLC to give a white solid product (18 mg, 45% yield)

[0398] 1H NMR (400MHz, DMSO) δ13.51 (s, 1H), 8.15 (t, J = 5.8Hz, 1H), 7.95 (d, J = 8.2Hz, 1H), 7.79 (d, J = 8.0Hz, 1H), 7.62 (t, J = 7.4Hz, 1H), 7.3 7 (t, J=7.4Hz, 1H), 7.18 (t, J=7.3Hz, 2H), 7.01 (d, J=8.1Hz, 2H), 6.79 (d, J=1.7Hz, 1H), 6.69 (d, J=8.0Hz, 1H), 6.62 (dd, J=8.0, 1.8 Hz, 1H), 5.93 (s, 2H), 4.13 (d, J=5.5Hz, 4H), 3.72 (s, 5H), 2.95 (dd, J=15.7, 8.0Hz, 4H), 2.08 (dd, J=9.4, 5.3Hz, 2H), 1.72 (m, J=15 .3, 7.6Hz, 2H), 1.49 (m, J=15.0, 7.5Hz, 2H), 1.35 (m, J=22.4, 14.9, 7.2Hz, 4H), 1.20 (m, J=14.7, 7.3Hz, 2H), 0.87 (t, J=7.4Hz, 3H). 13 C NMR (100MHz, DMSO) δ172.3, 159.0, 158.7, 158.4, 157.4, 149.3, 147.9, 145.8, 141.2, 135.8, 134.4, 134.1, 130.9, 129.8, 128.0, 125 .8, 125.2, 125.1, 122.0, 120.1, 118.9, 115.6, 112.9, 112.1, 55.9, 48.6, 42.9, 42.3, 358, 30.2, 29.7, 26.7, 26.5, 25.6, 22.2, 14.1.

[0399] HRMS (ESI) m / z calculated as C 37 H 45 N 7 O 4 , [M+H] + 651.3533; measured value: 652.3610.

[0400] The synthesis of compound 3 is as follows. Dopamine hydrochloride (20 mg, 0.105 mmol) and triethylamine (0.027 mL, 0.105 mmol) solution was added to 1 mL DMF containing 12 (26 mg, 0.052 mmol). Stir at room temperature for 12 h. The mixture was stirred at room temperature for 12 h. The reaction mixture was purified using preparative HPLC to give a white solid product (9.3 mg, 33% yield)

[0401] 1 H NMR (400MHz, DMSO) δ13.53 (s, 1H), 7.95 (d, J = 8.3Hz, 1H), 7.79 (d, J = 8.0Hz, 1H), 7.62 (t, J = 7.8Hz , 1H), 7.37 (t, J=7.4Hz, 1H), 7.18 (t, J=6.6Hz, 2H), 701 (d, J=81Hz, 2H), 6.61 (d, J=7.9Hz, 1H), 6. 55 (d, J=1.9Hz, 1H), 6.40 (dd, J=8.0, 1.9Hz, 1H), 5.93 (s, 2H), 4.13 (s, 2H), 3.11 (t, J=7.0Hz, 2H) , 3.00-2.89 (m, 2H), 1.73 (m, J=15.3, 7.6Hz, 2H), 1.37 (m, J=146, 7.4Hz, 2H), 0.87 (t, J=7.3Hz, 3H) 13 C NMR (100MHz, DMSO) δ159.1, 158.7, 158.3, 157.4, 149.3, 145.5, 143.9, 141.1, 136.3, 135.8, 134.3, 134.06, 132.28, 130.83, 129.89, 128.06, 1 25.90, 125.23, 125.13, 122.96, 122.04, 119.67, 118.96, 117.83, 116. 4, 115.9, 114.9, 112.9, 48.7, 42.8, 41.7, 36.0, 29.7, 26.7, 22.2, 14.2.

[0402] HRMS (ESI) m / z calculated as C 31 H 34 N 6 O 4 , [M+H] + 554.2642; measured value: 555.2714.

[0403] The synthesis of compound 4 is as follows. Dissolve imidazoquinoline in 5 mL DMF. 3A solution of (30 mg, 0.084 mmol) and 9 (20 mg, 0.093 mmol) was stirred at room temperature under argon, to which triethylamine (0.015 mL, 1.5 eq., 0.15 mmol) and 1[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate (HATU) (41 mg, 1.1 eq., containing 0.12 mmol in 0.2 mL DMF) were added. The mixture was stirred at room temperature for 12 h. The reaction mixture was loaded onto a silica gel column and purified using column chromatography with DCM / MeOH 9:1 to give an off-white powder. (30 mg, 55% yield)

[0404] 1 H NMR (400MHz, DMSO) δ13.55 (s, 1H), 9.57 (s, 1H), 9.15 (t, J=5.9Hz, 1H), 8.09 (s, 1H), 7.97 (d, J=8.3Hz, 1H), 7. 86-7.72 (m, 3H), 7.62 (t, J=7.7Hz, 1H), 7.53 (t, J=7.7Hz, 1H), 7.37 (t, J=7.7Hz, 1H), 7.31 (d, J=8.4Hz, 2H), 7 .26 (d, J=7.9Hz, 1H), 7.12 (d, J=7.8Hz, 1H), 7.07 (dd, J=11.9, 5.1Hz, 3H), 6.80 (dd, J=8.0, 2.1Hz, 1H), 5.95 ( s, 2H), 4.47 (d, J=5.7Hz, 2H), 3.01-2.90 (m, 2H), 1.79-1.64 (m, 2H), 1.44-1.31 (m, 2H), 0.87 (t, J=7.3Hz, 3H). 13 C NMR (100MHz, DMSO) δ166.5, 158.7, 158.4, 157.8, 149.5, 141.4, 140.8, 139.7, 135.8, 135.2, 134.4, 130.4, 129.8, 129.4, 12 8.9, 126.8, 126.4, 125.7, 125.1, 122.2, 119.4, 118.3, 115.2, 114.1, 112.9, 48.7, 42.6, 35.5, 32.3, 29.7, 26.6, 22.2, 14.1.

[0405] HRMS (ESI) m / z calculated as C 35 H 33 N 5 O 2 , [M+H] + 555.2623; measured value: 556.2718.

[0406] The synthesis of compound 5 is as follows. A solution of imidazoquinoline (30 mg, 0.083 mmol) and 10 (35 mg, 0.15 mmol) dissolved in 5 mL of DMF was stirred at room temperature under argon, and triethylamine (0.015 mL, 1.5 eq., 0.15 mmol) and HATU (50 mg, 1.1 eq., containing 0.13 mmol of 0.2 mL DMF) were added thereto. The mixture was stirred at room temperature for 12 h. The reaction mixture was loaded onto a silica gel column and purified by column chromatography using DCM / MeOH 9:1 to give an off-white powder. The product was further purified using preparative HPLC (20 mg, 55% yield)

[0407] 1 H NMR (400MHz, DMSO) δ12.53 (s, 1H), 9.57 (d, J=7.0Hz, 1H), 9.33 (t, J=5.9Hz, 2H), 8.02-7.85 (m , 4H), 7.78 (dd, J=11.6, 8.2Hz, 3H), 7.58 (t, J=7.7Hz, 2H), 7.29 (m, J=15.8, 7.8Hz, 9H), 7.14- 6.96 (m, 13H), 6.85-6.74 (m, 3H), 6.52 (s, 3H), 5.93 (s, 4H), 448 (d, J=58Hz, 4H), 3.02-2.89 (m , 5H), 1.72 (m, J=15.3, 7.5Hz, 5H), 1.36 (m, J=17.0, 8.5Hz, 2H), 0.85 (dd, J=12.4, 5.0Hz, 3H). 13 C NMR (100MHz, DMSO) δ169.4, 161.0, 158.4, 157.4, 149.4, 145.9, 139.2, 135.8, 135.0, 134.6, 134.8, 129.9, 129.8, 128.6, 128.5, 128 .6, 126.3, 126.1, 125.2, 125.1, 122.0, 118.9, 118.3, 116.8, 116.2, 115.4, 114.4, 113.4, 112.9, 48.7, 42.4, 29.7, 26.7, 22.2, 14.1

[0408] HRMS (ESI) m / z calculated as C 35 H 33 N 5 O 3 , [M+H] + 571.2565; measured value: 572.2640.

[0409] The synthesis of compound 6 is as follows. A solution of imidazoquinoline (ref) (30 mg, 0.084 mmol) and 11 (20 mg, 0.093 mmol) dissolved in 5 mL of DMF was stirred at room temperature under argon, and triethylamine (0.015 mL, 1.5 eq., 0.15 mmol) and HATU (41 mg, 1.1 eq., containing 0.12 mmol of 0.2 mL DMF) were added thereto. The mixture was stirred at room temperature for 12 h. The reaction mixture was loaded onto a silica gel column and purified by column chromatography using DCM / MeOH 9:1 to give an off-white powder. The product was further purified using preparative HPLC (33.3 mg, 75% yield)

[0410] 1 H NMR (400MHz, DMSO) δ13.48 (s, 1H), 12.48 (s, 1H), 9.46 (d, J = 6.1Hz, 2H), 8.07 (d, J = 2.2Hz, 2H), 7.96 (d, J = 8.1Hz, 2H), 7.78 (d, J=8.2Hz, 2H), 7.62 (dd, J=12.5, 4.1Hz, 2H), 7.44 (dd, J=11.3, 5.3Hz, 3H), 7.35 (dd, J=14.0, 6.9Hz, 3 H), 7.31 (d, J=82Hz, 2H), 7.05 (d, J=8.2Hz, 2H), 6.91 (dd, J=10.1, 5.4Hz, 4H), 6.84-6.77 (m, 9H), 594 (s, 2H), 4.49 (d, J=5.7Hz, 2H), 3.00-2.89 (m, 2H), 1.71 (m, J=15.3, 7.5Hz, 2H), 1.37 (m, J=14.6, 7.3Hz, 2H), 0.90-0.78 (m, 3H). 13 C NMR (100MHz, DMSO) δ169.1, 160.7, 158.3, 157.4, 149.4, 146.0, 140.7, 139.1, 135.8, 134.6, 134.8, 130.5, 129.9, 128.9, 128.4, 126.1, 125.2, 125.1, 122.0, 118.9, 118.3, 117.9, 117.6, 115.8, 115.8, 114.6, 113.9, 112.9, 48.7, 42.4, 29.7, 26.7, 22.2, 14.1

[0411] HRMS (ESI) m / z calculated as C 35 H 33 N 5 O 3 , [M+H]+ 571.2583; measured value: 572.2659.

[0412] The synthesis of compound 7 is as follows. To a solution of 5 mL DMF containing trans-ferulic acid (200 mg, 1.03 mmol, 1.0 equiv) and 3-azidopropylamine (0.15 mL, 1.5 mmol, 1.5 equiv) was added triethylamine (0.14 mL, 1.02 mmol, 1.0 equiv) and HATU (390 mg, 1.02 mmol, 1.0 equiv), and the solution was stirred at RT for 12 h under argon. The reaction mixture was extracted into ethyl acetate (10 mL x 3) and the solvent was evaporated in vacuo. The crude product obtained in the organic layer was dissolved in 5 mL MeOH / H2O4:1, tris(2-carboxyethyl)phosphine hydrochloride (295 mg, 1.03 mmol) was added to the solution, and the mixture was stirred at RT for 12 h. The reaction mixture was loaded on a silica gel column and purified by column chromatography using DCM / MeOH 9:1 to obtain an amorphous solid (80 mg, 67% yield).

[0413] 1 H NMR (400MHz, DMSO) δ7.33 (d, J=15.7Hz, 1H), 7.13 (d, J=1.7Hz, 1H), 6.99 (dd, J=8.2, 1.7Hz, 1H), 6.79 (d, J=8.1Hz , 1H), 6.45 (d, J=15.7Hz, 1H), 3.80 (s, 3H), 3.24 (q, J=6.5Hz, 2H), 2.81 (m, J=12.4, 6.1Hz, 2H), 1.78-1.68 (m, 2H). 13 C NMR (100MHz, DMSO) δ166.4, 148.8, 148.3, 139.7, 126.7, 122.10, 119.0, 116.1, 111.2, 55.9, 37.3, 36.2, 28.0.

[0414] HRMS (ESI) m / z calculated as C 13 H 18 N 2 O 3 ; [M+Na] + 250.1317; measured value: 274.1242.

[0415] The synthesis of compound 8 is as follows. To a solution of 1 mL DMF containing 4-hydroxy-3-methoxybenzylamine hydrochloride (75 mg, 0.40 mmol, 1.0 equiv) and 6-azidohexanoic acid NHS ester (100 mg, 0.40 mmol, 1.0 equiv) was added triethylamine (0.06 mL, 0.43 mmol, 1.2 equiv), and the solution was stirred at RT for 12 h under argon. The reaction mixture was dissolved in 5 mL MeOH / H2O4:1, tris(2-carboxyethyl)phosphine hydrochloride (120 mg, 0.40 mmol) was added to the solution, and the mixture was stirred at RT for 12 h. The reaction mixture was loaded on a silica gel column and purified by column chromatography using DCM / MeOH 9:1 to give an amorphous solid (58 mg, 50% yield).

[0416] 1 H NMR (400MHz, DMSO) δ681 (d, J=1.7Hz, 1H), 670 (d, J=8.0Hz, 1H), 6.63 (dd, J=8.0, 1.8Hz, 1H), 4.15 (d, J =5.8Hz, 2H), 3.74(s, 3H), 2.84-2.70(m, 2H), 2.17-2.06(m, 2H), 1.59-1.44(m, 4H), 1.36-1.23(m, 2H) 13 C NMR (100MHz, DMSO) δ172.2, 147.9, 145.8, 130.9, 120.2, 115.6, 112.2, 56.0, 42.3, 35.6, 27.3, 25.9, 25.3.

[0417] HRMS (ESI) m / z calculated as C 14 H 22 N 2 O 3 , [M+H] + 266.163; measured value: 267.1705.

[0418] The synthesis of compound 9 is as follows. A mixture of 3-hydroxyphenylboronic acid (220 mg, 1.59 mmol), 3-iodobenzoic acid (200 mg, 0.81 mmol), potassium carbonate (400 mg, 2.89 mmol) and Pd / C (10%) in 20 mL of H2O was refluxed at 80°C for 4 h. The solution was acidified with 1 M HCl, extracted with ethyl acetate, and washed with brine. The solvent was evaporated in vacuo. The compound was purified by column chromatography to give a white powder product (120 mg, 69% yield)

[0419] 1HNMR (400MHz, DMSO) δ8.13 (t, J=1.6Hz, 1H), 7.96-7.91 (m, 1H), 7.88-7.82 (m, 1H), 758 (dd , J=9.6, 5.8Hz, 1H), 7.28 (t, J=7.9Hz, 1H), 7.13-7.05 (m, 2H), 6.81 (dd, J=7.8, 20Hz, 1H). 13 C NMR (100MHz, DMSO) δ167.7, 158.4, 141.1, 141.1, 131.9, 131.4, 130.6, 129.8, 128.6, 127.6, 117.9, 115.4, 113.9.

[0420] HRMS (ESI) m / z calculated as C 13 H 10 O 3 , [M+H] + 214.063; measured value: 215.0661.

[0421] The synthesis of compound 10 is as follows. A mixture of 3-hydroxyphenylboronic acid (220 mg, 1.59 mmol), 2-hydroxy-4-iodobenzoic acid (210 mg, 0.79 mmol), potassium carbonate (400 mg, 2.89 mmol) and Pd / C (10%) in 20 mL of H2O was refluxed at 80°C for 4 h. The solution was acidified with 1 M HCl, extracted with ethyl acetate, and washed with brine. The solvent was evaporated in vacuo. The compound was purified by column chromatography to give the product as a white powder (126 mg, 68% yield).

[0422] 1 H NMR (400MHz, DMSO) δ7.28 (t, J=7.9Hz, 1H), 7.15 (m, J=14.2, 10.4, 5.0Hz, 3H), 7.05 (dd, J=5.2, 3.2Hz, 1H), 6.86-6.80 (m, 1H) 13 C NMR (100MHz, DMSO) δ172.2, 161.8, 158.3, 158.1, 147.8, 140.5, 131.3, 130.5, 118.1, 116.0, 115.0, 114.1, 112.3.

[0423] HRMS (ESI) m / z calculated as C 13 H 10 O 4 , [M+H] + 230.0579; measured value: 231.0579.

[0424] For the synthesis of compound 11, a mixture of 4-hydroxyphenylboronic acid (220 mg, 1.59 mmol), 3-iodobenzoic acid (200 mg, 0.79 mmol), potassium carbonate (400 mg, 2.89 mmol) and Pd / C (10%) in 20 mL of H2O was refluxed at 80°C for 4 h. The solution was acidified with 1 M HCl, extracted with ethyl acetate, and washed with brine. The solvent was evaporated in vacuo. The compound was purified by column chromatography to give the product as a white powder (75 mg, 41% yield)

[0425] 1 H NMR (400MHz, DMSO) δ7.93 (t, J=5.3Hz, 1H), 7.74 (dd, J=8.6, 2.5Hz, 1H), 7.43 (m, J=7.7, 4.8, 2.4Hz, 3H), 7.01 (d, J=8.6Hz, 1H), 6.86-6.81 (m, 3H) 13 C NMR (100MHz, DMSO) δ172.3, 160.2, 157.2, 133.8, 132.0, 130.3, 127.7, 127.6, 127.5, 118.1, 116.2, 116.2, 1136.

[0426] HRMS (ESI) m / z calculated as C 13 H 10 O 4 , [M+H] + 230.0579; measured value: 231.0545.

[0427] The synthesis of compound 12 is as follows. N, N'-disuccinimidyl carbonate (60 mg, 0.235 mmol) was added to a solution of imidazoquinoline (60 mg, 0.167 mmol) and triethylamine (0.023, 0.167 mmol). The reaction mixture was stirred at room temperature for 6 h. The reaction mixture was loaded on a silica gel column and purified by column chromatography using DCM / MeOH 9:1 to obtain a low melting point transparent solid (40 mg, 60% yield)

[0428] 1H NMR (500MHz, DMSO) δ13.90 (s, 1H), 8.85 (t, J=6.0Hz, 1H), 7.96 (d, J=8.3Hz, 1H), 7.79 (d, J=8 .3Hz, 1H), 7.69-7.55 (m, 1H), 7.36 (dd, J=21.9, 14.0Hz, 1H), 7.22 (dd, J=162, 8.0Hz, 2H), 7.0 5 (dd, J=22.6, 7.6Hz, 2H), 5.95 (s, 2H), 4.23 (d, J=3.6Hz, 2H), 2.96 (t, J=7.7Hz, 2H), 275 (s, 4 H), 1.73 (m, J=15.2, 7.7Hz, 2H), 1.38 (m, J=14.6, 7.4Hz, 2H), 0.87 (dd, J=7.8, 6.9Hz, 3H).13C NMR (125MHz, DMSO) δ171.3, 157.5, 152.6, 148.7, 146.0, 138.3, 135.9, 134.9, 129.9, 128 .2, 126.2, 125.2, 125.1, 122.5, 118.9, 112.8, 48.7, 44.4, 29.6, 26.6, 25.7, 22.2, 14.1.

[0429] HRMS (ESI) m / z calculated as C 27 H 28 N 6 O 4 , [M+H]+; 500.2172; found: 501.2261.

[0430] ***

[0431] All methods disclosed and claimed herein can be made and implemented according to the present disclosure without undue experimentation. Although the compositions and methods of the present invention have been described in accordance with preferred embodiments, it is obvious to those skilled in the art that changes can be implemented in the methods and in the steps or the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the present invention. More specifically, it is obvious that specific reagents related to both chemical and physiological aspects can replace the reagents described herein while achieving the same or similar results. All such similar substitutions and changes that are obvious to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0432] References

[0433] The following references and documents cited elsewhere herein, to the extent they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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Claims

1. A compound of formula (I) wherein L is a linker; and wherein n is an integer from 1 to 10, and m is an integer from 0 to 10.

2. The compound according to claim 1, wherein n is at least 2.

3. The compound according to claim 1, wherein n is 5.

4. The compound according to any one of claims 1 to 3, wherein m is at least 1.

5. The compound according to any one of claims 1 to 3, wherein m is 4.

6. The compound according to any one of claims 1 to 5, wherein the linker is a bond, -NH-, -CH2-, -C(O)-, -C(O)N-, -alkyl-C(O)-, -alkenyl-C(O)-, -alkyl-NC(O)-, -alkenyl-NC(O)-, -C(O)NC(O)-, -C(O)NH(CH2) z NC(O)-、-alkyl-C(O)NH(CH2) z NC(O)-, or -alkenyl-C(O)NH(CH2) z NC(O)-, wherein z is an integer from 1 to 10.

7. A compound according to any one of claims 1 to 5, wherein the linker comprises 8. The compound according to any one of claims 1 to 7, wherein the compound is further defined as the following compound:

9. The compound according to any one of claims 1 to 8, wherein the compound is further defined as the following compound:

10. A compound of formula (II) in: A is the NF-κB inhibitor part; B is an amino acid; L is the connector; C is an amino acid; D is a TLR agonist moiety; and wherein n is an integer from 1 to 10, and m is an integer from 1 to 10. The compound according to claim 10 , wherein n is at least 2. The compound according to claim 10 , wherein n is 5.

13. The compound according to any one of claims 10 to 12, wherein m is at least 2.

14. The compound according to any one of claims 10 to 12, wherein m is 5.

15. The compound of any one of claims 10 to 15, wherein the TLR agonist moiety is a TLR7 / 8 agonist.

16. The compound according to claim 15, wherein the TLR7 / 8 agonist is an imidazoquinolinone or a derivative thereof.

17. A compound according to any one of claims 10 to 16, wherein the linker is a bond, -NH-, -CH2-, -C(O)-, -C(O)N-, -alkyl-C(O)-, -alkenyl-C(O)-, -alkyl-NC(O)-, -alkenyl-NC(O)-, -C(O)NC(O)-, -C(O)NH(CH2) z NC(O)-、-alkyl-C(O)NH(CH2) z NC(O)-, or -alkenyl-C(O)NH(CH2) z NC(O)-, wherein z is an integer from 1 to 10.

18. A compound according to any one of claims 10 to 16, wherein the linker comprises 19. The compound according to any one of claims 10 to 18, wherein the NF-κB inhibitor moiety is ferulic acid or a derivative thereof.

20. The compound of any one of claims 10 to 18, wherein the NF-κB inhibitor moiety is vanillin or a derivative thereof.

21. The compound according to any one of claims 10 to 18, wherein the NF-κB inhibitor moiety is honokiol or a derivative thereof.

22. The compound of any one of claims 10 to 18, wherein the NF-κB inhibitor moiety is dopamine or a derivative thereof.

23. The compound according to any one of claims 10 to 22, wherein B is glutamic acid.

24. The compound according to any one of claims 10 to 23, wherein C is glycine.

25. The compound according to any one of claims 10 to 24, wherein the compound is further defined as the following compound: wherein L is a linker; and wherein n is an integer from 1 to 10, and m is an integer from 0 to 10.

26. The compound according to any one of claims 10 to 24, wherein the compound is further defined as the following compound: wherein n is an integer from 1 to 10, and m is an integer from 0 to 10.

27. The compound according to any one of claims 10 to 26, wherein the compound is further defined as the following compound:

28. A compound having the formula wherein n is an integer from 1 to 10, and m is an integer from 2 to 10.

29. The compound of claim 28, wherein the compound is further defined as:

30. A method for immune activation, comprising administering a compound according to any one of claims 1 to 29 to an immune cell population.

31. The method of claim 30, wherein the immune cell population comprises T cells.

32. The method of claim 30, wherein the immune cell population comprises macrophages.

33. The method according to any one of claims 30 to 32, wherein the method is an in vitro method.

34. The method according to any one of claims 30 to 32, wherein the method is an in vivo method.

35. A method for vaccinating a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a compound according to any one of claims 1 to 27.

36. The method of claim 35, further comprising administering an antigen to the subject.

37. A method according to claim 36, wherein the antigen is a bacterial antigen.

38. The method according to claim 36, wherein the antigen is a viral antigen.

39. The method according to claim 36, wherein the antigen is a dengue antigen.

40. The method according to claim 39, wherein the dengue antigen is a capsid protein of dengue serotype-2 (DENV-2C) or a portion thereof.

41. The method according to claim 36, wherein the antigen is an HIV antigen.

42. The method of claim 41, wherein the HIV antigen is gp120 or a portion thereof.

43. The method according to claim 36, wherein the antigen is a SARS-CoV-2 antigen.

44. The method of claim 43, wherein the SARS-CoV-2 antigen is a SARS-CoV-2 spike protein or a portion thereof.

45. The method according to claim 36, wherein the antigen is a tumor antigen.

46. ​​The method of any one of claims 35 to 45, wherein the method is used to prevent a disease in a subject.

47. The method of any one of claims 35 to 45, wherein the method is used to treat a disease in a subject.

48. The method of any one of claims 35 to 47, wherein the subject has previously been administered an adjuvant.

49. The method of any one of claims 35 to 47, wherein the subject has developed an adverse reaction to an adjuvant.

50. A method for treating or preventing cancer, the method comprising administering to a subject an effective amount of a pharmaceutical composition comprising a compound according to any one of claims 1 to 24.

51. The method of claim 50, further comprising administering to the subject an additional cancer therapy.

52. The method of claim 51, wherein the other cancer therapy comprises chemotherapy, radiation therapy, immunotherapy, or a combination thereof.

53. The method of claim 52, wherein the other cancer therapy comprises immunotherapy.

54. The method of claim 53, wherein the other cancer therapy is a checkpoint inhibitor therapy.

55. The method of any one of claims 50 to 54, wherein the subject has not been diagnosed with cancer.

56. The method of any one of claims 50 to 54, wherein the subject has been diagnosed with cancer.

57. The method of any one of claims 50 to 56, wherein the subject has been previously treated for cancer with a prior therapy.

58. The method of any one of claims 50 to 56, wherein the subject is determined to be resistant to a prior therapy.

59. The method of any one of claims 50 to 58, wherein the pharmaceutical composition is administered to the subject intratumorally.

60. A pharmaceutical composition comprising: (a) a compound according to any one of claims 1 to 29; and (b) Antigen.

61. The pharmaceutical composition of claim 60, wherein the antigen is a bacterial antigen.

62. The pharmaceutical composition of claim 60, wherein the antigen is a viral antigen.

63. The pharmaceutical composition of claim 60, wherein the antigen is a dengue antigen.

64. The pharmaceutical composition of claim 63, wherein the dengue antigen is a capsid protein of dengue serotype-2 (DENV-2C) or a portion thereof.

65. The pharmaceutical composition of claim 60, wherein the antigen is an HIV antigen.

66. The pharmaceutical composition of claim 65, wherein the HIV antigen is gp120 or a portion thereof.

67. A pharmaceutical composition according to claim 60, wherein the antigen is a SARS-CoV-2 antigen.

68. A pharmaceutical composition according to claim 67, wherein the SARS-CoV-2 antigen is a SARS-CoV-2 spike protein or a portion thereof.

69. The pharmaceutical composition of claim 60, wherein the antigen is a tumor antigen.

70. A pharmaceutical composition according to any one of claims 60 to 69, further comprising a pharmaceutically acceptable carrier.

71. A compound of formula (I): in R1 is H, C1-C 12 Alkyl, C5-C 12 Cycloalkyl, C4-C 12 Heterocycloalkyl, C6-C 10 Aryl and C4-C 10 heteroaryl, wherein R1 is optionally substituted with one or more than one Y; R2 and R3 are independently H, Cl-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C1-C 12 Heteroalkyl, C2-C 12 Heteroalkenyl or C2-C 12 heteroalkynyl, or together form a 5- to 6-membered carbocyclic or heterocyclic ring, wherein the 5- to 6-membered carbocyclic or heterocyclic ring is optionally substituted with one or more than one Y; X is a bond, -N-, -C(O)-, -alkyl-C(O)-, -alkenyl-C(O)-, -C(O)N-, -NC(O)-, -alkyl-NC(O)-, -alkenyl-NC(O)-, -C(O)N-, -C(O)NC(O)-, -C(O)NH(CH2) z NC(O)-、-alkyl-C(O)NH(CH2) z NC(O)-, or -alkenyl-C(O)NH(CH2) z NC(O)-, wherein z is an integer from 1 to 10; R A is attached to one or more ring atoms, and R A are independently hydrogen, hydroxyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 alkenyl, or substituted or unsubstituted phenyl, and Y is substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NH2, NO2, NR4R5, NC(O)R5, OC(O)NR4R5, OC(O)OR4, C(O)R4, C(O)OR4, SH, SR4, OR4, wherein R4 and R5 are independently substituted or unsubstituted C1-C6 alkyl.

72. The compound according to claim 71, wherein R1 is C1-C 12 alkyl.

73. The compound of claim 72, wherein the alkyl group is n-butyl.

74. according to the compound described in any one of claims 71 to 73, wherein R2 and R3 together form a benzene ring.

75. The compound of claim 74, wherein the benzene ring is an unsubstituted benzene ring.

76. A compound according to any one of claims 71 to 75, wherein X is an amide.

77. A compound according to any one of claims 71 to 75, wherein X comprises one or more than one of an alkyl group, an alkenyl group, an amide group and a urea group.

78. A compound according to any one of claims 71 to 77, wherein R A It is an unsubstituted phenyl group.

79. according to the compound described in any one of claims 71 to 77, wherein R A is a substituted phenyl group.

80. The compound of claim 71, wherein the compound is further defined as one of the following compounds:

81. A compound of formula (II): ABC(II) in: A is a TLR agonist; B is a connector; and C is an NF-κB inhibitor.

82. The compound of claim 81, wherein the TLR agonist is a TLR 7 / 8 agonist.

83. The compound of claim 82, wherein the TLR7 / 8 agonist is an imidazoquinolinone or a derivative thereof.

84. according to the compound described in any one of claims 81 to 83, wherein the linker is a bond, -NH-, -CH2-, -C(O)-, -C(O)N-, -alkyl-C(O)-, -alkenyl-C(O)-, -alkyl-NC(O)-, -alkenyl-NC(O)-, -C(O)NC(O)-, -C(O)NH(CH2) z NC(O)-、-alkyl-C(O)NH(CH2) z NC(O)-, or -alkenyl-C(O)NH(CH2) z NC(O)-, wherein z is an integer from 1 to 10.

85. The compound of any one of claims 81 to 84, wherein the NF-κB inhibitor is ferulic acid or a derivative thereof.

86. The compound of any one of claims 81 to 84, wherein the NF-κB inhibitor is vanillin or a derivative thereof.

87. The compound of any one of claims 81 to 84, wherein the NF-κB inhibitor is dopamine or a derivative thereof.

88. The compound according to any one of claims 81 to 84, wherein the NF-κB inhibitor is honokiol or a derivative thereof.

89. A compound according to any one of claims 81 to 88, wherein the compound is further defined as one of the following compounds:

90. A method for vaccinating a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising (a) a compound according to any one of claims 71 to 80 or (b) a compound according to any one of claims 81 to 89.

91. The method of claim 90, further comprising administering an antigen to the subject.

92. The method according to claim 91, wherein the antigen is a bacterial antigen.

93. The method according to claim 91, wherein the antigen is a viral antigen.

94. The method according to claim 91, wherein the antigen is a dengue antigen.

95. The method of claim 94, wherein the dengue antigen comprises a capsid protein of dengue serotype-2 (DENV-2C).

96. The method according to claim 91, wherein the antigen is an HIV antigen.

97. The method of claim 96, wherein the HIV antigen comprises gp120.

98. The method according to claim 91, wherein the antigen is a SARS-CoV-2 antigen.

99. The method according to claim 98, wherein the SARS-CoV-2 antigen is the SARS-CoV-2 spike protein or a portion thereof.

100. The method according to claim 91, wherein the antigen is a tumor antigen.

101. The method of any one of claims 91 to 100, wherein the subject is a human subject.

102. The method of any one of claims 91 to 101, wherein the method is used to prevent a disease in a subject.

103. The method of any one of claims 91 to 101, wherein the method is used to treat a disease in a subject.

104. The method of any one of claims 91 to 103, wherein the subject has previously been administered an adjuvant.

105. The method of claim 104, wherein the subject has had an adverse reaction to an adjuvant.

106. A method for treating or preventing cancer, the method comprising administering to a subject an effective amount of a pharmaceutical composition comprising (a) a compound according to any one of claims 71 to 80 or (b) a compound according to any one of claims 81 to 89.

107. The method of claim 106, further comprising administering to the subject an additional cancer therapy.

108. The method of claim 107, wherein the other cancer therapy comprises chemotherapy, radiation therapy, immunotherapy, or a combination thereof.

109. The method of claim 107, wherein the other cancer therapy comprises immunotherapy.

110. The method of claim 107, wherein the other cancer therapy comprises a checkpoint inhibitor therapy.

111. The method of any one of claims 106 to 110, wherein the subject is a human subject.

112. The method of any one of claims 106 to 111, wherein the subject has not been diagnosed with cancer.

113. The method of any one of claims 106 to 111, wherein the subject has been diagnosed with cancer.

114. The method of any one of claims 106 to 113, wherein the subject has been previously treated for cancer with a prior therapy.

115. The method of claim 114, wherein the subject is determined to be resistant to prior therapy.

116. The method of any one of claims 106 to 115, wherein the pharmaceutical composition is administered to the subject intratumorally.

117. A pharmaceutical composition comprising: a compound according to any one of claims 71 to 80 or a compound according to any one of claims 81 to 89; and an antigen.

118. The pharmaceutical composition of claim 117, wherein the antigen is a bacterial antigen.

119. The pharmaceutical composition of claim 117, wherein the antigen is a viral antigen.

120. The pharmaceutical composition of claim 117, wherein the antigen is a dengue antigen.

121. The pharmaceutical composition of claim 120, wherein the dengue antigen comprises a capsid protein of dengue serotype-2 (DENV-2C).

122. The pharmaceutical composition of claim 117, wherein the antigen is an HIV antigen.

123. The pharmaceutical composition of claim 117, wherein the HIV antigen comprises gp120.

124. The pharmaceutical composition of claim 117, wherein the antigen is a SARS-CoV-2 antigen.

125. The pharmaceutical composition of claim 124, wherein the SARS-CoV-2 antigen is a SARS-CoV-2 spike protein or a portion thereof.

126. The pharmaceutical composition of claim 117, wherein the antigen is an influenza antigen.

127. The pharmaceutical composition of claim 117, wherein the antigen is a tumor antigen.

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