Compositions and methods for degradation-enhanced immunotherapies

Abstract

The present disclosure relates to compositions and methods for inducing an immune response using a degradation-enhanced composition. In particular, the compositions comprise a degradation-enhanced immunogenic construct comprising: (a) an antigen, (b) a ligand that binds a degradation-enhancing target molecule, and (c) a linker connecting the antigen and the ligand, wherein the composition enhances antigen processing, presentation, and immune activation, thereby improving adaptive immune responses.

Description

Docket No. 45501.601COMPOSITIONS AND METHODS FOR DEGRADATION-ENHANCED IMMUNOTHERAPIESThe present application claims priority to United States Provisional Patent Application Serial Number 63 / 704,654. filed October 8, 2024, the disclosure of which is herein incorporated by reference in its entirety.TECHNICAL FIELD

[0001] The present disclosure relates to compositions and methods for inducing an immune response using a degradation-enhanced composition. In particular, the compositions comprise a degradation-enhanced immunogenic construct comprising: (a) an antigen, (b) a ligand that binds a degradation-enhancing target molecule, and (c) a linker connecting the antigen and the ligand, wherein the composition enhances antigen processing, presentation, and immune activation, thereby improving adaptive immune responses.BACKGROUND|0002] Immunotherapies, including prophylactic and therapeutic approaches, aim to modulate the immune system to prevent or treat disease. Prophylactic immunotherapies are designed to prevent the onset of disease by priming the immune system to recognize and respond to specific antigens before exposure. Therapeutic immunotherapies are intended to treat existing conditions by enhancing or redirecting immune responses against diseased cells, such as tumor cells or infected cells. Despite advances in the field, current strategies often face limitations in generating robust, durable, and specific immune responses.

[0003] Many current immunotherapies employ natural or engineered proteins as antigens, produced directly from biological systems. A key rate-limiting step in the effectiveness of these therapies is the proteolytic processing of protein antigens into minimal peptide epitopes, a step that depends on the endogenous proteolysis machinery of antigen-presenting cells. Inefficient proteolysis can restrict the subsequent binding of epitopes to MHC molecules, limit antigen presentation to naive T cells, and reduce the overall adaptive immune response. As a result, the therapeutic or prophylactic potential of such immunotherapies may be compromised. There remains a need for improved immunotherapeutic approaches that overcome these limitations and provide broadly applicable systems and methods to enhance the effectiveness, consistency, and specificity of immune activation for both preventive and therapeutic applications.Docket No. 45501.601SUMMARY

[0004] Aspects of the present disclosure relate to compositions and methods for inducing an immune response using a degradation-enhanced composition. In particular, the compositions comprise a degradation-enhanced immunogenic construct comprising: (a) an antigen, (b) a ligand that binds a degradation-enhancing target molecule, and (c) a linker connecting the antigen and the ligand, wherein the composition enhances antigen processing, presentation, and immune activation, thereby improving adaptive immune responses.

[0005] The immunogens (e g., antigens) used in current vaccine technologies are largely natural proteins or natural proteins in whole or fragment of biological cells. These immunogens are taken up by antigen presenting cells, proteolytically processed by cell endogenous proteasome into minimal antigenic epitopes that bind with MHC molecules, and the resulting MHC / epitope complexes then travel to the surfaces of antigen-presenting cells to present these epitopes to naive T cells, thus generating T cell responses in an immunostimulatory context. A rate-limiting step in these cascades is the proteolytic processing of immunogens by the cellular machinery in antigen presenting cells. Embodiments of the present disclosure improve the step of proteolysis, which is important for efficient antigen presentation and adaptive immunomodulation. The presently disclosed subject provides a novel strategy for enhancing the efficacy of protein-based immunotherapies that rely on proteolytic processing of antigens. These approaches find use for all protein vaccines, protein-containing pathogenic cell vaccines (inactivated pathogens or cancer cells, deactivated pathogens or tumor cells, viral vectors or plasmid vectors or mRNA vectors that encode protein immunogens). Further, these compositions can be used for the prophylaxis and therapy of a wide variety of diseases including, but not limited to, microbial future infections or pre-infection, cancer, as well as autoimmune diseases that involves T cell autoimmunity.

[0006] In some embodiments, the disclosed compositions and methods are designed to facilitate targeted degradation of the antigen in antigen-presenting cells through the recruitment of endogenous degradation machinery, resulting in improved generation of antigenic epitopes and more robust activation of T and B lymphocytes. The present disclosure further encompasses both prophylactic and therapeutic applications of these degradation- enhanced constructs across a wide range of disease settings.

[0007] Embodiments of the present disclosure provide a composition for inducing an immune response comprising: (a) an antigen, (b) a ligand that binds a degradation-enhancing target molecule, and (c) a linker connecting the antigen and the ligand. In some embodiments, the composition is formulated as a conjugate, complex, or fusion construct, wherein the antigenDocket No. 45501.601 is covalently or non-covalently attached to the ligand through the linker to maintain functional integrity and stability of each component.

[0008] In some embodiments, the ligand is an E3 ligase ligand. In some embodiments, the ligand binds to an E3 ubiquitin ligase involved in mediating protein ubiquitination and subsequent proteasomal degradation. In some embodiments, the E3 ligase ligand is a pomalidomide. thalidomide, lenalidomide, or any structural analog, derivative, or functional equivalent thereof capable of binding an E3 ligase complex and facilitating substrate ubiquitination.

[0009] In some embodiments, the linker has a hydrophobicity that modulates antigen presentation and T cell response. In some embodiments, the linker is hydrophilic polyethylene glycol (PEG) linker, a hydrophobic carbon (C) lipid linker, or a variation thereof. In some embodiments, the linker further influences the spatial orientation, flexibility, and accessibility of the antigen-ligand complex, thereby modulating degradation efficiency and immunogenic potency. In some embodiments, the linker has a variable length that modulates antigen presentation and T cell response. In some embodiments, the linker is a C5, C6. C8, PEG1. PEG2, PEG3, PEG4. PEG5. PEG6, or any variation thereof. In some embodiments, the linker is chemically optimized to balance hydrophobic and hydrophilic characteristics, thereby tuning antigen uptake, intracellular trafficking, and proteasomal processing.

[0010] In some embodiments, the degradation-enhancing target molecule is a ligase involved in protein ubiquitination. In some embodiments, the ligase is an E3 ubiquitin ligase.

[0011] In some embodiments, the antigen is a protein, polypeptide, peptide, or fragment thereof derived from a pathogen, a tumor-associated antigen, or an autoantigen. In some embodiments, the antigen may include recombinant proteins, synthetic peptides, or fragments containing one or more T-cell or B-cell epitopes. The antigen may be derived from viral, bacterial, parasitic, cancer-associated, or self-antigens implicated in autoimmune disorders. In some embodiments, multiple antigens or antigenic fragments may be incorporated within a single construct to induce broad or multivalent immune responses.

[0012] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the carrier is selected from the group consisting of buffered aqueous solutions, lipid-based carriers, polymeric nanoparticles, emulsions, or liposomal formulations suitable for in vivo delivery. The carrier may facilitate controlled release, targeted uptake, or enhanced stability of the degradation-enhanced construct. In some embodiments, the composition may be delivered alone or in combination with one or moreDocket No. 45501.601 pharmaceutically acceptable carriers, adjuvants, or delivery systems to facilitate targeted uptake by antigen-presenting cells.

[0013] Embodiments of the present disclosure provide a method of modulating an immune response in a subject, comprising administering to the subject a degradation-enhanced composition. In some embodiments, the method comprises administering an effective amount of the composition to stimulate an antigen-specific immune response, enhance immune memory, or restore immune homeostasis. The method may be performed via subcutaneous, intramuscular, intravenous, or intradermal administration, or through other clinically acceptable routes.

[0014] In some embodiments, the administration of the composition enhances proteolytic processing of the antigen. In some embodiments, the administration of the composition enhances antigen presentation by antigen-presenting cells. In some embodiments, the administration of the composition enhances adaptive immune responses, including one or more of T cell responses and B cell responses. In some embodiments, the disclosed compositions increase both the magnitude and quality of antigen-specific T cell responses, including but not limited to, the induction of cytotoxic CD8+ T cells and helper CD4+ T cells, as well as the generation of memory T cells and high-affinity antibody-producing B cells.

[0015] In some embodiments, the administration of the composition improves the immunomodulatory efficacy relative to an unmodified antigen. In some embodiments, the administration of the composition improves the efficacy of a prophylactic vaccine. In some embodiments, the administration of the composition improves the efficacy of a therapeutic vaccine. In some embodiments, the composition is a protein-based vaccine, a peptide-based vaccine, a viral vector, a nucleic acid vaccine, a protein-containing pathogenic cell vaccine, or a derivative thereof. In some embodiments, the prophylactic vaccine is for the prevention of a microbial infection, a viral infection, cancer, an autoimmune disease or any derivative thereof. In some embodiments, the wherein the therapeutic vaccine is for the treatment of a microbial infection, a viral infection, cancer, an autoimmune disease, or any derivative thereof. In some embodiments, the therapeutic vaccine or immunotherapy is intended for treatment or management of an existing disease condition, including chronic infection, malignancy, or immune dysregulation. Collectively, the presently disclosed compositions and methods provide a versatile platform for enhancing antigen degradation and presentation, thereby improving both the potency and durability of adaptive immune responses across a range of prophylactic and therapeutic applications.Docket No. 45501.601

[0016] Current vaccine technologies have used largely natural proteins or natural or engineered proteins produced directly from biological systems (cells) as immunogens (i.e.. antigens). One rate-limiting step of these immunogens used in current vaccine technologies is the proteolysis of protein immunogens into minimal peptide epitopes, a step that relies on the cell endogenous proteoly sis machinery in antigen presenting cells. Consequently, this ratelimiting step limits the efficiency of MHC binding, antigen presentation to naive T cells, as well as the T cell modulatory efficacy.

[0017] The present disclosure improves the step of proteolysis, which is critical for efficient antigen presentation and adaptive immunomodulation. Specifically, the present technology' provides an enhancement of proteolysis step using a broadly applicable chemical modification.

[0018] Embodiments may be used for the majority of vaccines that involves the proteolytical processing of protein immunogens. Specifically, these vaccines include all protein vaccines, protein-containing pathogenic cell vaccines (inactivated pathogens or cancer cells, deactivated pathogens or tumor cells, viral vectors or plasmid vectors or mRNA vectors that encode protein immunogens); further, these vaccines can be used for the prophylaxis and therapy of a wide variety7of diseases including, but not limited to, microbial future infections or pre-infection, cancer, as well as autoimmune diseases that involves T cell autoimmunity7. This technology can be integrated into current vaccine development and manufacturing technologies.

[0019] The immunogens (e g., antigens) used in current vaccine technologies are largely natural proteins or natural proteins in whole or fragment of biological cells. These immunogens are taken up by antigen presenting cells, proteolytically processed by cell endogenous proteasome into minimal antigenic epitopes that bind with MHC molecules, and the resulting MHC / epitope complexes then travel to the surfaces of antigen-presenting cells to present these epitopes to naive T cells, thus generating T cell responses in an immunostimulatory7context.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The patent or application file contains drawings executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.Docket No. 45501.601

[0021] Having thus described the presently disclosed subj ect matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0022] Figure 1 shows a schematic illustration of the working mechanism of proteolysis- enhanced vaccines as a ty pe of degradation-enhanced vaccines. Ub: ubiquitin; APC: antigen- presenting cells; MHC: major histocompatibility’ complex.[0023J Figure 2 shows a schematic of proteolysis-enhanced antigen constructs for improved antigen processing and immune activation. Top: schematic structure of proteolysis-enhanced vaccines, in which antigens (e.g., immunogens) are modified with one or multiple copies of proteolysis-targeting ligands. The proteolysis-targeting ligands bind to E3 ligase, thereby recruiting E3 ligases in close proximity of antigens, which promotes the ubiquitination of antigens, enhanced proteolytic processing of antigens, enhanced antigen presentation, and eventually enhanced T cell responses as well as B cell responses. The library’ of Rs include an E3 ligase ligand Pomalidomide with a series of different linkers with vary ing lengths and hydrophobicity.

[0024] Figure 3 shows a screening of a library of model antigen OVA-based proteolysis- enhanced vaccine candidates for the ability to elicit T cell responses. (A) Study design in C57BL / 6 mice (n = 5). Vaccines were administered subcutaneously at mouse tail base. All OVA or OVA derivatives were mixed with CpG adjuvant prior to administration. T cell specificity against antigen epitopes were measured using a tetramer staining assay. (B, C) Representative flow cytometry7dot plots of SIINFEKL-specific PBMC CD8 T cells on day 35 post priming immunization (B) and quantification of SIINFEKL-specific CD8 T cells on day 35, 45, and 68 post priming immunization (C). These results idicate that leads of proteolysis- enhanced vaccines promoted the quantity of antigen-specific T cells as shown by the enhanced frequency of OVA-derived epitope SIINFEKL-specific CD8 T cells in PBMCs.

[0025] Figure 4 shows during the screening of a library7of proteolysis-enhanced vaccine candidates using OVA as a model antigen, some leads promoted the quality’ of antigen-specific T cells as shown by their ability to produce functional cytokines. Intracellular staining of TNF- alpha and INF-beta in CD4 T cells and CD8 T cells on Day 35 post priming immunization in C57B1 / 6 mice (6-8 weeks old) as shown above. PBMC T cells were pulsed with OVA, prior to cytokine staining.

[0026] Figure 5 shows OVA-P4 vaccine potentiated the T cell responses relative to OVA vaccine in aged mice. (A) Study timeline. (B) Tetramer staining results showing the fraction of SIINFEKL-specific CD8+ T cells among all PBMC CD8+ T cells. (C) Intracellular cytokineDocket No. 45501.601 staining results showing the IFN-gamma-positive CD8+ T cells among all PBMC CD8+ T cells.

[0027] Figure 6 shows confocal microscopy visualization of intracellular degradation of the OVA antigen, enhanced by conjugation with an E3 ligase ligand, in treated DC2.4 cells. DQ- conjugated ovalbumin (DQ-OVA) is a self-quenched conjugate of OVA that exhibits bright green fluorescence upon proteolytic degradation.

[0028] Figure 7 shows H-2kb-SIINFEKL staining on cultured DC2.4 cells treated with OVA- P4 and controls. OVA or OVA-P4: 5 ug / rnL; treatment: 24 hours.

[0029] Figure 8 shows that vaccination with melanoma TgT-P4 enhances antigen-specific antitumor T cell responses. (A) Experimental design. C57BL / 6 mice w ere treated subcutaneous with PROTAV vaccine with different dose and controls on Day 0 and Day 14. On Day 21 blood were isolated for immune analysis. On Day 42, C57BL / 6 mice were inoculated subcutaneously (s.c.) with 2 x 10A5 B16F10 cells on the right flank. Quantification of the H- 2Kb-SIINFEKL tetramer staining (B); effector T (C); cytokine production with TRP2 restimulation (D), GP100 restimulation (E); (F) Tumor growth curves of mice rechallenged with B16F10 cells on Day 42.

[0030] Figure 9 shows In situ vaccination with TgT-P4 (i.e., TgT-PROTAV) enhanced antitumor immune responses. (A) Experimental design. C57BL / 6 mice were inoculated subcutaneously (s.c.) with 2 x 10A5 B16F10 cells on the right flank. On Day 3. day 9, day 15, day 21 mice were treated subcutaneous with TgT-PROTAV vaccine with different dose and controls. (B) Tumor growth curves of mice. (C-E) Percentages of the gplOO (KVPRNQDWL)- specific CD8+ T cells among total liver CD8+ T cells by H-2Db-KVPRNQDWL tetramer staining in tumor (C), tumor draining lymph nodes (D), and peripheral blood (E).

[0031] Figure 10 shows E7-P4 (i.e., E7-PROTAV) enhanced E7-specific CD8+ T cell responses in mice. (A) Experimental design. C57BL / 6 mice were treated subcutaneous with E7-PROTAV vaccine with different dose and controls on Day 0 and Day 14. On Day 21, blood was isolated for immune analysis. (B-D) Quantification of the H-2Db-RAFIYNIVTF tetramer staining (B); effector T cells (C); and intracellular cytokine production upon E7 peptide restimulation (D).

[0032] Figure 11 shows Tumor growth curves of E7-positive TCI tumors in syngeneic C57BL / 6 mice upon the indicated treatment. CpG and svg3 were used as adjuvants. All peptide antigens, CpG and svg3 were loaded into LNPs, which were administered subcutaneously at mouse tail base. Immune checkpoint inhibitor antibodies were administered intraperitoneally.Docket No. 45501.601DEFINITIONS

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0034] The terms “comprise(s),’' “include(s),” “having,’" “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0035] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2. 6.3, 6.4, 6.5, 6.6. 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0036] The term "‘administration” refers to the act of giving a drug, prodrug, or other agent, or therapeutic treatment (e.g., pharmaceutical compositions of the present disclosure) to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through the mouth (oral), nose (nasal), lungs (e.g., inhalant, intratracheal), skin (transdermal), oral mucosa (buccal), by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.

[0037] The term “protein” is used synonymously with “peptide,” “polypeptide,” or “peptide fragment.” A “purified” polypeptide, protein, peptide, or peptide fragment is substantially free of cellular material or other contaminating proteins from the cell, tissue, or cell-free source from which the amino acid sequence is obtained, or substantially free from chemical precursors or other chemicals when chemically synthesized.

[0038] The terms “treat,"’ “treatment,” and “treating” refer to reducing the amount or severity of a particular condition, disease state, or symptoms thereof, in a subject presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate completeDocket No. 45501.601 treatment (e.g., total elimination of the condition, disease, or symptoms thereof). "Treatment,” encompasses any administration or application of a therapeutic or technique for a disease (e.g., in a mammal, including a human), and includes inhibiting the disease, arresting its development, relieving the disease, causing regression, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process.

[0039] The terms ‘"prevent,” “prevention,” and preventing” refer to reducing the likelihood of a particular condition or disease state from occurring in a subject not presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete or absolute prevention.

[0040] The term "pharmaceutical composition" as used herein means a product comprising an active, compound or a salt thereof together with pharmaceutical excipients such as buffer, preservative and tonicity modifier, said pharmaceutical composition being useful for treating, preventing or reducing the severity of a disease or disorder by administration of said pharmaceutical composition to a person. Thus a pharmaceutical composition is also known in the art as a pharmaceutical formulation.

[0041] The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in the therapeutic compositions is contemplated. Supplementary active ingredients, such as antibiotics, antifungals, antimicrobials, can also be incorporated into the compositions. In addition, various adjuvants such as are commonly used in the art may be included. These and other such compounds are described in the literature, e.g., in the Merck Index, Merck & Company, Rahway. N.J. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (2006); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 11th Ed., The McGraw-Hill Companies.

[0042] The term “subject” as used herein refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, mice, rats, etc.). As used herein, the term “patient” typically refers to a human subject that is being treated for a disease or condition or prophy lactically.

[0043] The term “therapeutically effective amount” refers to the amount of a composition of the present invention that is capable of achieving a therapeutic effect in a subject in need thereof. For example, a therapeutically effective amount of a composition of the presentDocket No. 45501.601 invention can be the amount that is capable of preventing or relieving one or more symptoms associated with a disease or disorder.

[0044] '‘Adjuvant” as used herein may mean any molecule added to the immunogenic compositions described herein to enhance antigenicity of the one or more immunogens.|0045] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular.DETAILED DESCRIPTION

[0046] In some embodiments, the proteolysis-enhanced composition (e.g., vaccines) comprise three parts: an antigen (e.g., immunogen), a ligand that bind a degradation-enhancing target molecule (e.g., proteolysis-enhancing ligases (e.g., E3 ligases)), and linkers that connect the antigen and ligand. Figure 2 shows a schematic structure of an exemplary proteolysis- enhanced vaccine, in which antigens (e.g., immunogens) are modified with one or multiple copies of proteolysis-targeting ligands. The proteolysis-targeting ligands bind to E3 ligase and thereby recruit E3 ligases in close proximity of antigens, which promotes the ubiquitination of antigens, enhanced proteolytic processing of antigens, enhanced antigen presentation, and eventually enhanced T cell responses as well as B cell responses. A library of linkers were designed and screened with varying lengths and hydrophobicity (hydrophilic polyethylene glycol (PEG) linkers and relatively hydrophobic carbon (C) lipid linkers) (Figure 2). Pomalidomide was an E3 ligase ligand. Subsequently, experiments were conducted using the model antigen chicken ovalbumin (OVA) modified with the proteolysis enhancers.

[0047] Using the library of linkers, OVA-based proteolysis-enhanced vaccine candidates were screened for their ability to elicit T cell responses in immunocompetent C57BL / 6 mice (Figure 3A). All OVA or OVA derivatives were mixed with CpG adjuvant in solution and were administered subcutaneously at mouse tail base which allows efficient vaccine retention in draining inguinal and axillary lymph nodes, and uptake into intranodal antigen-presenting cells. Peripheral blood mononuclear cell (PBMC) T cell specificity against antigen epitopes wereDocket No. 45501.601 measured using a tetramer staining assay (Figure 3B and Figure 3C) on day 35, 45, and 68 post priming immunization. Several high performing linkers from the library were identified, including linkers P2 [(PEG)2], P4 [(PEG)4], and P5 [(PEG)s]. These results show that proteolysis-enhanced vaccines promoted the quantity of antigen-specific T cells as shown by the enhanced frequency of OVA-derived epitope SIINFEKL-specific CD8 T cells in PBMCs. Moreover, proteolysis-enhanced vaccines also enhanced the quality of T cells. Specifically, intracellular staining of TNF-alpha and INF-beta were performed in CD4 T cells and CD8 T cells on Day 35 post priming immunization in C57B1 / 6 mice immunized. Relative to control unmodified OVA antigen, the composition of proteolysis-enhanced OVA derivatives enhanced the ability of T cells to produce antitumor and antiviral cytokines TNF-alpha and INF-beta in response to antigen restimulation (Figure 4).

[0048] In certain embodiments, the ligand that binds a degradation-enhancing target molecule comprises an E3 ligase ligand. In some embodiments, the E3 ligase ligand includes, but is not limited to, one of the following: a thalidomide, a lenalidomide, a pomalidomide, an avadomide (CC-122), an iberdomide (CC-220), a mezigdomide (CC-92480), a bicyclic imide, a phenyl glutarirmde (e.g.. dCeMM2 series), and a CelMoD derivative.

[0049] In some embodiments, the linkers employed herein include, but are not limited to, C5- COOH, C6-COOH, C8-COOH, CX-COOH where X is 2 to 16, PEG1-COOH, PEG2-COOH, PEG2-C4-COOH, PEG3-COOH, PEG4-COOH. PEG4-COOH, PEG5-COOH, PEG6-C4- COOH, PEGX-COOH where X is 1 to 10. PEGX-CR-COOH where X is 1 to 10 and R is 2 to 16, an alky linker, a straight-chain aliphatic carboxylic acid linker, a short PEG acid linker, a medium PEG acid linker, a long PEG acid linker, a PEGylated carboxylic acid linker, an aminononanoic acid, an omega- amino carboxylic acid, a thioether PEG acid, an amide-linked PEG acid, and an amino acid linker.

[0050] In other embodiments, the linker employed herein includes one of the following: C3 linker, C4 linker, C5 linker, C6 linker, C7 linker, C8 linker, C9 linker, CIO linker, PEG1 linker, PEG2 linker, PEG3 linker, PEG4 linker, PEG5 linker, PEG6 linker, PEG8 linker, PEG10 linker, P3C3 linker, P4C2 linker, P5C3 linker, P6C4 linker, P2C6 linker, P8C2 linker, PEG- alkyl hybrid linker, s-aminocaproic acid linker. (3-alanine linker, y-aminobutyric acid linker, aminononanoic acid linker, 6-aminohexanoic acid linker, aminooctanoic acid linker, amino- PEG2 linker, amino-PEG3 linker, amino-PEG4 linker, PEG2-COOH linker, PEG3-COOH linker, PEG4-COOH linker, PEG6-COOH linker, PEG8-COOH linker, PEG2-NH2linker, PEG3-NH2 linker, PEG4-NH2 linker, PEG6-NFE linker, alkyl-ether linker, alkyl-thioether linker, alkyl-amide linker, alkyl-urea linker, alkyl-carbamate linker, triethylene glycol linker.Docket No. 45501.601 tetraethylene glycol linker, hexaethylene glycol linker, octaethylene glycol linker, benzy l linker, phenoxyacetic acid linker, phenethyl linker, cyclohexyl linker, cyclohexylmethyl linker, piperazine linker, morpholine linker, triazole linker, click-triazole linker, azido-PEG linker, alkyne-PEG linker, PEG-azide linker, PEG-alkyne linker, maleimide linker, succinimide linker, NHS-ester linker, carbamate linker, urea linker, oxetane linker, dioxane linker, pyrrolidine linker, piperidine linker, short alkyl-acid linker, medium PEG-acid linker, long PEG-acid linker, mixed PEG-alkyl linker, hydrophobic alkyl spacer, and a hydrophilic PEG spacer.

[0051] In particular embodiments, the antigen employed herein (e.g., a human antigen) is a protein selected from the following: an oncogenic kinase, a nuclear receptor, an epigenetic regulator, an oncogenic transcription factor or co-activator, an apoptosis or cell-cycle regulator, an immune or inflammatory antigen, a neurodegeneration or CNS antigen, a viral or infectious disease antigen, and a metabolic or fibrotic disease antigen.

[0052] In certain embodiments, the oncogenic kinase antigen is selected from: Bruton’s tyrosine kinase (BTK). Breakpoint cluster region- Abelson ty rosine kinase fusion (BCR-ABL), Epidermal growth factor receptor (EGFR). Human epidermal growth factor receptor 2 (HER2), Anaplastic lymphoma kinase (ALK), Cyclin-dependent kinase 4 (CDK4), Cyclin-dependent kinase 6 (CDK.6), Cyclin-dependent kinase 9 (CDK9), B-Raf proto-oncogene serine / threonine- protein kinase (BRAF), RAC-alpha serine / threonine-protein kinase (AKT). Proto-oncogene tyrosine-protein kinase Src (SRC), Focal adhesion kinase (FAK), Mitogen-activated protein kinase kinase 1 (MEK1), Mitogen-activated protein kinase kinase 2 (MEK2), Extracellular signal-regulated kinase 1 (ERK1), Extracellular signal-regulated kinase 2 (ERK2), and Fms- like ty rosine kinase 3 (FLT3).

[0053] In certain embodiments, the nuclear receptor antigen is selected from the following: Androgen receptor (AR), Estrogen receptor alpha (ERa), Progesterone receptor (PR), Glucocorticoid receptor (GR), Retinoic acid receptor alpha (RARa), Retinoic acid receptor beta (RAR(3), Retinoic acid receptor gamma (RARy), Retinoid X receptor alpha (RXRa), Retinoid X receptor beta (RXR ), Retinoid X receptor gamma (RXRy), and Vitamin D receptor (VDR).

[0054] In some embodiments, the epigenetic regular antigen is selected from the following: Bromodomain-containing protein 4 (BRD4), Bromodomain-containing protein 2 (BRD2), Bromodomain-containing protein 3 (BRD3), Enhancer of zeste homolog 2 (EZH2), Embry' onic ectoderm development protein (EED), Histone deacetylase 1 (HDAC1), Histone deacetylase 2 (HDAC2), Histone deacetylase 3 (HDAC3), Histone deacetylase 6 (HDAC6), SWI / SNF-Docket No. 45501.601 related matrix-associated actin-dependent regulator of chromatin subfamily A member 2 (SMARCA2), SWI / SNF -related matrix-associated actin-dependent regulator of chromatin subfamily A member 4 (SMARCA4), CREB-binding protein (CBP), and ElA-binding protein p300 (p300).10055] In other embodiments, the Oncogenic Transcription Factors or Co-activator antigen is selected from the following: Myc proto-oncogene protein (c-Myc), Signal transducer and activator of transcription 3 (STAT3), Catenin beta-1 (P-Catenin). Hypoxia-inducible factor 1- alpha (HIF-la), Hypoxia-inducible factor 2-alpha (HIF-2a), Forkhead box protein Ml (FOXM1), Nuclear factor kappa-B subunit p65 (NF-KB), Myb proto-oncogene protein (MYB), Runt-related transcription factor 1 (RUNX1), and GATA-binding protein 3 (GATA3).

[0056] In further embodiments, the apoptosis and Cell-Cycle Regulator antigen is selected from the following: B-cell lymphoma-extra large protein (BCL-XL), B-cell lymphoma 2 (BCL-2), Mouse double minute 2 homolog (MDM2), Cellular tumor antigen p53 (p53), Cyclin DI (CCND1), Cyclin K (CCNK), Weel-like protein kinase (WEE1), Polo-like kinase 1 (PLK1), Aurora kinase A (Aurora A), and Aurora kinase B (Aurora B).

[0057] In certain embodiments, the immune and inflammatory antigens are selected from the following: Interleukin- 1 receptor-associated kinase 4 (IRAK4), Signal transducer and activator of transcription 6 (STAT6), Signal transducer and activator of transcription 5 (STAT5), Interferon regulatory factor 4 (IRF4). Myeloid differentiation primary response protein 88 (MyD88), Toll / interleukin-1 receptor domain-containing adaptor inducing interferon-beta (TRIF), Tyrosine kinase 2 (TYK2), Janus kinase 1 (JAK1), Janus kinase 2 (JAK2), Janus kinase 3 (JAK3), NACHT, LRR and PYD domains-containing protein 3 (NLRP3), Ikaros family zinc finger protein 1 (IKZF1), and Ikaros family zinc finger protein 3 (IKZF3)

[0058] In particular embodiments, the Neurodegeneration and CNS antigen is selected from the following: Microtubule-associated protein tau (Tau), Alpha-synuclein (a-Synuclein), Huntingtin protein (Huntingtin), TAR DNA-binding protein 43 (TDP-43), and Leucine-rich repeat kinase 2 (LRRK2).

[0059] In some embodiments, the viral and infection disease antigen is selected from: HIV trans-activator of transcription protein (HIV Tat), HIV Rev regulatory protein (HIV Rev), Hepatitis B virus X protein (HBx), Hepatitis B surface antigen (HBsAg), SARS-CoV-2 spike glycoprotein (SARS-CoV-2 Spike), SARS-CoV-2 main protease (SARS-CoV-2 Mpro), and SARS-CoV-2 RNA-dependent RNA polymerase (SARS-CoV-2 RdRp).Docket No. 45501.601

[0060] In other embodiments, the metabolic or fibrotic disease antigen is selected from the following: Heat shock protein 90 (HSP90). Transforming growth factor beta receptor 1 (TGF[3R1), Activin receptor-like kinase 5 (ALK5), Peroxisome proliferator-activated receptor gamma (PPARy), NACHT, LRR and PYD domains-containing protein 3 (NLRP3), Kelch-like ECH-associated protein 1 (Keapl), and Nuclear factor ery throid 2-related factor 2 (NRF2).

[0061] The precise effective amount for a human subject will depend upon the severity’ of the subject's disease state, general health, age, weight, gender, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance or response to therapy. Compositions may be administered individually to a patient, or they may be administered in combination with other drugs, hormones, agents, and the like.

[0062] In some embodiments, the composition comprises a carrier. Carriers include, for example, vehicles, excipients, and diluents. It is preferred that the carrier be one which is chemically^ inert to the active agent(s), and one which has little or no detrimental side effects or toxicity7under the conditions of use. Examples of the carriers include soluble carriers such as known buffers which can be physiologically acceptable (e.g., phosphate buffer) as well as solid compositions such as solid-state carriers or latex beads.

[0063] The carriers or diluents used herein may’ be solid carriers or diluents for solid formulations, liquid carriers or diluents for liquid formulations, or mixtures thereof.

[0064] Solid carriers or diluents include, but are not limited to, gums, starches (e g., com starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e g., microcrystalline cellulose), acrylates (e g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[0065] For liquid formulations, pharmaceutically acceptable carriers may be, for example, aqueous or non-aqueous solutions, or suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic / aqueous solutions, cyclodextrins, emulsions or suspensions, including saline and buffered media.

[0066] Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Formulations suitable for parenteral administration include, for example, aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueousDocket No. 45501.601 sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0067] It will be appreciated by one of skill in the art that, in addition to the above-described vaccine compositions, the compositions of the invention can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.

[0068] Intravenous vehicles include, for example, fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

[0069] The choice of carrier will be determined, in part, by the particular antigen-containing compositions, as well as by the particular method used to administer the composition. Accordingly, there are a variety of suitable formulations of the pharmaceutical compositions of the invention. More than one route can be used to administer the compositions of the present invention, and in certain instances, a particular route can provide a more immediate and more effective response than another route.

Claims

Docket No. 45501.601ClaimsWhat is claimed is:

1. A degradation-enhanced composition for inducing an immune response comprising: (a) an antigen, (b) a ligand that binds a degradation-enhancing target molecule, and (c) a linker connecting the antigen and the ligand.

2. The composition of claim 1. wherein the ligand is an E3 ligase ligand.

3. The composition of claim 2, wherein the E3 ligase ligand is a Pomalidomide or any derivative thereof.

4. The composition of claim 1 , wherein the linker has a hydrophobicity that modulates antigen presentation and T cell response.

5. The composition of claim 4, wherein the linker is hydrophilic polyethylene glycol (PEG) linker, a hydrophobic carbon (C) lipid linker, or a variation thereof.

6. The composition of claim 1 , wherein the linker has a variable length that modulates antigen presentation and T cell response.

7. The composition of claim 6, wherein the linker is a C5, C6, C8, PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, or any variation thereof.

8. The composition of claim 1, wherein the degradation-enhancing target molecule is a ligase involved in protein ubiquitination.

9. The composition of claim 8, wherein the ligase is an E3 ubiquitin ligase.

10. The composition of claim 1, wherein the antigen is a protein, polypeptide, peptide, or fragment thereof derived from a pathogen, a tumor-associated antigen, or an autoantigen.

11. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.

12. A method of modulating an immune response in a subject, comprising administering to the subject a degradation-enhanced composition according to any one of claims 1-11.

13. The method of claim 12, wherein administration of the composition enhances proteolytic processing of the antigen.

14. The method of claim 12, wherein the administration of the composition enhances antigen presentation by antigen-presenting cells.Docket No. 45501.60115. The method of claim 12, wherein the administration of the composition enhances adaptive immune responses, including one or more of T cell responses and B cell responses.

16. The method of claim 12, wherein the administration of the composition improves the immunomodulatory efficacy relative to an unmodified antigen.

17. The method of claim 12, wherein the administration of the composition improves the efficacy of a prophylactic vaccine.

18. The method of claim 12, wherein the administration of the composition improves the efficacy of a therapeutic vaccine.

19. The method of claim 17 or 18. wherein the composition is a protein-based vaccine, a peptide-based vaccine, a viral vector, a nucleic acid vaccine, a protein-containing pathogenic cell vaccine, or a derivative thereof.

20. The method of claim 17, wherein the prophylactic vaccine is for the prevention of a microbial infection, a viral infection, cancer, an autoimmune disease or any derivative thereof.

21. The method of claim 18, wherein the therapeutic vaccine is for the treatment of a microbial infection, a viral infection, cancer, an autoimmune disease, or any derivative thereof.

22. Use of a composition comprising any one of claims 1-11.

23. Use of a composition comprising any one of claims 1-11, for inducing an immune response in a subject.

24. Use of a composition comprising any one of claims 1-11, for the prevention of a disease in a subject.

25. Use of a composition comprising any one of claims 1-11, for the treatment of a disease in a subject.

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