Materials, methods and systems for providing enhanced immunity

Abstract

An immunogenic composition that includes an adjuvant and a soluble protein. The adjuvant may be a nanoparticle, such as a zinc chitosan nanoparticle. The soluble protein may comprise a tag, such as a His tag. A method for inducing an immune response in a subject in need thereof, breaking an immune tolerance in a subject in need thereof, and / or for active immunization to prevent a disease in a subject by administering the immunogenic composition. A system for inducing an immune response in a subject in need thereof, breaking an immune tolerance in a subject in need thereof, and / or for active immunization to prevent a disease in a subject that includes the immunogenic composition and a delivery system.

Description

MATERIALS, METHODS AND SYSTEMS FOR PROVIDING ENHANCED IMMUNITYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Applications 63 / 655,427 and 63 / 655,449, both filed on June 3, 2024, which are hereby incorporated by reference in their entirety.FIELD

[0002] Materials, methods, and systems for generating an enhanced immune response and breaking immune tolerance in a mammal, including the field of novel adjuvant and delivery platforms.BACKGROUND

[0003] A challenge in vaccine development has been generating an enhanced immune response and breaking immune tolerance. There remains a need for novel materials, methods, and systems for making effective vaccines.SUMMARY

[0004] The inventors of the present invention appreciate that the identification of novel adjuvants and delivery platforms for generating an enhanced immune response and breaking immune tolerance is a challenge that was not previously met.

[0005] Provided are materials, methods and systems for using zinc chitosan nanoparticles as an antigen delivery platform or adjuvant. As an adjuvant / delivery platform, zinc chitosan allows not only slow release of immunogens but also an adjuvant / delivery platform that is free of oil or agents that exhibit chaotropic property that could potentially disrupt the epitope structure. Chitosan is a cationic polysaccharide with repeating units of P-(l-4)-linked-D-glucosamine and N-acetyl-D- glucosamine, derived by the partial deacetylation of chitin. It is non-toxic, biocompatible and biodegradable.

[0006] Provided is an immunogenic composition comprising (a) an adjuvant means and (b) a soluble protein. In some embodiments, the adjuvant means comprises a nanoparticle comprising zinc chitosan.

[0007] Also provided is an immunogenic composition comprising (a) an adjuvant comprising a nanoparticle comprising zinc chitosan and (b) a soluble protein.

[0008] In any of the compositions, the soluble protein may comprise a tag. In further embodiments, the tag may be a His tag.

[0009] In any of the compositions, the soluble protein is HER3, IL-11, CD45, CD33, CDH17, HER2, LAG3, CDHR5, CD117, TROP2, VG9, PDL-1, TfR, NKp46, ACE2, C7, CD79B, IL-la, IL- 1P, GUCY2C, ENPP3, TNFR2, IL-27R, FcRH5, EMR2, KLK2, TMEFF2, CD3, VP-17-Fab, VP-17- Ab, C3, C5, TL1A, IL- 13, or a combination thereof.

[0010] In any of the compositions, the nanoparticle is about 150 nm to about 250 nm in size.

[0011] In any of the compositions, the immunogenic composition further comprises a pharmaceutically acceptable carrier.

[0012] Provided are methods for inducing an immune response in a subject, breaking an immune tolerance in a subject, and / or active immunization to prevent a disease in a subject. The method(s) generally comprise administering to the subject any of the immunogenic compositions disclosed herein.

[0013] In any of the methods, the subject is a mammal, for example a human, a simian, a mouse, a rat, or a llama.

[0014] Provided are systems for inducing an immune response in a subject, breaking an immune tolerance in a subject, and / or active immunization to prevent a disease in a subject. The system(s) may comprise any of the immunogenic compositions disclosed herein.

[0015] In any of the systems, a delivery system may be included. In embodiments of the system(s), the delivery system may comprise a syringe. In some embodiments, the immunogenic composition is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration. In some embodiments, the subject is a mammal, for example a human, a simian, a mouse, a rat, or a llama.DESCRIPTION OF THE FIGURES

[0016] FIG. 1 shows a schematic representation of a zinc chitosan nanoparticle.

[0017] FIGS. 2A-2C illustrate representative bar graphs for immunogenicity. FIG. 2A: Sigma (Sigma Adjuvant System - S6322-1VL) vs Zinc Nanoparticles (NP) for immunization with CD3 and KLK2 (Kallikrein-2) in AlivaMab mice (Ablexis™). FIG. 2B: CL413 vs Zinc Nanoparticles vs C / IFA for immunization with IL-ip, VP-17-Fab or VP-17-Ab in AlivaMab mice (Ablexis™). FIG.2C: C / IFA vs Zinc Nanoparticles for immunization with EMR2 (EGF-like module-containing mucin-like receptor-like 2) in Llama.DETAILED DESCRIPTION

[0018] While the general inventive concepts are susceptible of embodiment in many forms, there are shown in the drawings, and will be described herein in detail, specific embodiments thereof with the understanding that the present disclosure is to be considered an exemplification of the principles of the general inventive concepts. Accordingly, the general inventive concepts are not intended to be limited to the specific embodiments illustrated herein.

[0019] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0020] The articles “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “a” cell means one cell or more than one cell.

[0021] “About,” as used herein, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±5%, preferably ±1%, and still more preferably ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.

[0022] The term “adjuvant,” as used herein, refers to a pharmacological or immunological agent that modifies the effect of other agents (e.g., vaccines) while having few if any direct effects when given by itself. They are often included in vaccines to enhance the recipient’s immune response to a supplied antigen while keeping the injected foreign material at a minimum.

[0023] As used herein, “administering” generally refers to a method of giving a dosage of a pharmaceutical composition (e.g., an immunogenic composition, for example a vaccine) to a subject. The immunogenic compositions utilized in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, by gavage, in cremes, or in lipid compositions. The preferred method of administration can vary depending on various factors (e.g., the components of theimmunogenic composition being administered and the severity of the condition being treated).

[0024] As used herein, “subject” means any animal, preferably a mammal, most preferably a human, to whom will be or has been treated by or subjected to a method according to an embodiment of the invention. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, simians, monkeys, llamas, humans, etc., more preferably a human.

[0025] The term “immunogenic composition,” as used herein, means a material used to provoke an immune response and may confer immunity after administration of the immunogenic composition to a subject.

[0026] As used herein, the term “enhanced” when used with respect to an immune response, means stimulating, evoking, increasing, improving, or augmenting any response of a mammal's immune system. The immune response may be a cellular response (i.e. cell-mediated, such as cytotoxic T lymphocyte mediated) or a humoral response (i.e. antibody mediated response), and may be a primary or secondary immune response. Examples of enhancement of immune response include increased CD4+ or CD8+ T cell activity and generation of cytolytic T cells. The enhancement of immune response can be assessed using a number of in vitro or in vivo measurements known to those skilled in the art, including, but not limited to, cytotoxic T lymphocyte assays, release of cytokines (for example IL-2 production), regression of tumors, survival of tumor bearing animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity.

[0027] As used herein, the term “CD4+ or CD8+ T cell activity” refers to a T cell immune response that is characterized by observing a high proportion of immunogen-specific CD4+ T cells or CD8+ T cells within the population of total responding T cells following vaccination. The total immunogenspecific T-cell response can be determined by an IFN-gamma Enzyme-Linked ImmunoSpot (ELISPOT) assay. The immunogen-specific CD4+ or CD8+ T cell immune response can be determined by an intracellular cytokine staining (ICS) assay.

[0028] The immunogenic compositions disclosed herein may enable the breaking of immune tolerance. As used herein, the term “immune tolerance” is the state where the immune system becomes unresponsive to specific substances or tissues that would normally trigger an immune response. This unresponsiveness prevents the immune system from attacking the body's own cells or tissues, or “self-antigens”. This entrained limitation on immune function against such self-antigensprotects the host against adverse autoimmune reactions. It also presents a major obstacle for presently available immunotherapy protocols, such as cancer immunotherapy protocols.

[0029] The terms “antibody” and “antibodies,” as used herein, are meant in a broad sense and include immunoglobulin molecules including polyclonal antibodies, monoclonal antibodies including murine, human, human-adapted, humanized and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, and single chain antibodies.

[0030] Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further subclassified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (K) and lambda (A), based on the amino acid sequences of their constant domains.

[0031] The term "antibody fragments" refers to a portion of an immunoglobulin molecule that retains the heavy chain and / or the light chain antigen binding site, such as heavy chain complementarity determining regions (HCDR) 1, 2 and 3, light chain complementarity determining regions (LCDR) 1, 2 and 3, a heavy chain variable region (VH), or a light chain variable region (VL). Antibody fragments include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a domain antibody (dAb) fragment, which consists of a VH domain. VH and VL domains can be engineered and linked together via a synthetic linker to form various types of single chain antibody designs where the VH / VL domains pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in PCT Inti. Publ. Nos. W01998 / 44001, WO1988 / 01649, WO1994 / 13804, and W01992 / 01047. These antibody fragments are obtained using well known techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are full length antibodies.

[0032] The phrase "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an isolatedantibody specifically binding CD38 is substantially free of antibodies that specifically bind antigens other than human CD38). An isolated antibody that specifically binds CD38, however, can have cross-reactivity to other antigens, such as orthologs of human CD38, such as Macaca fascicularis (cynomolgus monkey) CD38. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0033] "Humanized antibody" refers to an antibody in which the antigen binding sites are derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Humanized antibodies may include substitutions in the framework regions so that the framework may not be an exact copy of expressed human immunoglobulin or germline gene sequences.

[0034] "Human antibody" refers to an antibody having heavy and light chain variable regions in which both the framework and the antigen binding sites are derived from sequences of human origin. If the antibody contains a constant region, the constant region also is derived from sequences of human origin. A human antibody comprises heavy or light chain variable regions that are "derived from" sequences of human origin wherein the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such systems include human immunoglobulin gene libraries displayed on phage, and transgenic non- human animals such as mice carrying human immunoglobulin loci as described herein. A human antibody may also contain amino acid differences when compared to the human germline or rearranged immunoglobulin sequences due to, for example naturally occurring somatic mutations or intentional introduction of substitutions in the framework or antigen binding sites. Typically, a human antibody is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical in amino acid sequence to an amino acid sequence encoded by a human germline or rearranged immunoglobulin gene.

[0035] Isolated humanized antibodies may be synthetic. Human antibodies, while derived from human immunoglobulin sequences, may be generated using systems such as phage display incorporating synthetic CDRs and / or synthetic frameworks, or can be subjected to in vitro mutagenesis to improve antibody properties, resulting in antibodies that do not naturally exist within the human antibody germline repertoire in vivo.

[0036] The term “recombinant antibody,” as used herein, includes all antibodies that are prepared,expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the antibody, antibodies isolated from a recombinant, combinatorial antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences, sequences, or antibodies that are generated in vitro using Fab arm exchange such as bispecific antibodies.

[0037] The term “monoclonal antibody,” as used herein, refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope, or in a case of a bispecific monoclonal antibody, a dual binding specificity to two distinct epitopes.

[0038] The term “epitope,” as used herein, means a portion of an antigen to which an antibody specifically binds. Epitopes usually consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope can be composed of contiguous and / or discontiguous amino acids that form a conformational spatial unit. For a discontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3-dimensional space through the folding of the protein molecule.

[0039] The terms “His tag” or “6xHis,” as used herein, refer to a stretch of amino acids comprising multiple histidine residues. Typically, the His tag can bind to metal ions, for example, Zn2+, Ni2+, Co2+, or Cu2+ions. Optionally, the His tag comprises 2, 3, 4, 5, 6, 7, 8 or more histidine residues. In some embodiments, the His tag is fused to the N- or C-terminus of a protein; alternatively, it can be fused at any suitable location within the protein. In specific embodiments, the His tag is fused to the N- or C-terminal of the soluble protein, such as any of the soluble proteins disclosed herein.

[0040] As used herein, the term “soluble protein” refers to proteins and polypeptides which are in solution and not precipitated. Usually, the soluble form of the protein is identical or very similar to the native, natural and active form of the protein

[0041] In some embodiments of any of the immunogenic compositions or methods described herein, a range is intended to comprise every integer or fraction or value within the range.

[0042] Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of’ and / or “consisting essentially of’ such features.

[0043] To date the identification of novel adjuvants and delivery platforms for generating an enhanced immune response and breaking immune tolerance was a challenge that was not previously met. Provided are immunogenic compositions, methods and systems that do not have the same limitations as previously described methods.A. IMMUNOGENIC COMPOSITIONS

[0044] Provided is an immunogenic composition comprising (a) an adjuvant and (b) a soluble protein. The adjuvant generally comprises a nanoparticle, such as a nanoparticle comprising zinc chitosan.

[0045] In some embodiments, the immunogenic composition comprises (a) an adjuvant comprising a zinc chitosan nanoparticle and (b) a soluble protein.Soluble Protein

[0046] In some embodiments, the soluble protein is a fusion protein. In some embodiments, the soluble protein is fused to an antibody or antibody fragment.

[0047] In some embodiments, the soluble protein comprises a tag. In further embodiments, the tag is a His tag, such as a 6xHis.

[0048] In some embodiments, the soluble protein is HER3, IL-11, CD45, CD33, CDH17, HER2, LAG3, CDHR5, CD117, TROP2, VG9, PDL-1, TfR, NKp46, ACE2, C7, CD79B, IL-la, IL-1 , GUCY2C, ENPP3, TNER2, IL-27R, EcRH5, EMR2, KLK2, TMEEE2, CD3, V0-17-Eab, V0-17-Ab, C3, C5, TL1A, IL-13, or a combination thereof. Eurthermore, solubilizable material(s), such as protein, non-protein, oligopeptide, and other non-amino acid based soluble materials, etc. are suitable for use with the present invention.Zinc Chitosan

[0049] Chitosan is a cationic polysaccharide consisting of repeating units of 0-(l— >4)-2-acetamido- d-glucose (A-acetyl-d-glucosamine, GlcNAc) and 0-(l— »4)-2-amino-d-glucose (d-glucosamine, GlcN) . It is non-toxic, biocompatible, and biodegradable.Formula I

[0050] Chitosan (Formula I) is the principal N-deacetylated derivative of chitin (a P-1,4 linked polymer of GlcNAc; Formula II), although the N-deacetylation is almost never complete. The source of the chitin is usually crab shells or shrimp shells, but chitin and thus chitosan can be obtained from the shells of other crustaceans, such as lobsters, as well as from fish scales and many other types of organisms (insects and fungi).Formula II

[0051] Different grades of chitosan can be distinguished dependent upon the deacetylation grade.The deacetylation grade of chitosan may range from 40 % up to 100 %. The deacetylation grade of chitosan according to the present disclosure may be more than 50 %, or 60 %, or 70%, or 80 %, or more than 90 %. The deacetylation grade of chitosan according to the present disclosure may be less than 100 %, such as less than 90 %, or 80 %, or 70%, or less than 60 %. Any combination of upper and lower values may define a range, such that the deacetylation grade of chitosan according to the present disclosure may be 60 to 90 %, or 70 to 90 %, or 70 to 80 %, and the like.

[0052] The molecular weight of chitosan generally ranges between 50 kDa to 2,000 kDa and depends on the number of subunits (e.g., n=30 to n=1500) and the level of deacylation. As a measure for the molecular weight, the viscosity of a 1 % solution in 1 % acetic acid in water is used. The molecular weight of chitosan according to the present disclosure may result in a viscosity ranging from 10 to 300 cP, such as from 20 to 300 cP, 15 to 250 cP, or from 20 to 100 cP.

[0053] The pH range in which chitosan is soluble depends upon the deacetylation grade of the chitosan. The less the deacetylation grade of the chitosan the higher the pH needed to solubilize the chitosan (i.e., higher degrees of deacetylation are required for improved solubility in water).

[0054] The pH of the immunogenic composition according to the present invention may range from 3.0 to 10 depending on the form of chitosan selected, the presentation of the His tag on the protein,and the binding affinity of the zinc to the 6xHis on the soluble protein (i.e., the zinc of the zinc chitosan). Typically, the pH range of the composition is from 5.0 to 9.0, such as 6.0 to 9.0, or 7.0 to 9.0, or 7.0 to 8.5, or 7.5 to 8.5, or even 7.5 to 8.0.

[0055] Chitosan can be generated as a metal ion complex with zinc (Zn2+). Proteins with 6xHis residues can be bound to zinc chitosan. In some embodiments, the proteins have a C-terminal 6xHis tag. Surprisingly, the inventors unexpectedly found zinc chitosan can function as an antigen depot that allows slow release of antigens for extended stimulation of the immune system.

[0056] In some embodiments, a chitosan solution and a zinc acetate solution are mixed to form zinc chitosan. In some embodiments, the chitosan solution is 2% chitosan solution. In some embodiments, the zinc acetate solution is 0.2M zinc acetate.

[0057] In some embodiments, the zinc chitosan is sonicated to form nanoparticles.

[0058] The amount of chitosan or zinc chitosan in the composition ranges from 0.001 % to 10 % (w / w), such as from 0.005 % to 5% (w / w), or 0.01 % to 1.0 % (w / w).

[0059] In some embodiments, the nanoparticles are about 100 nm to about 300 nm in size. A range is intended to comprise every integer or fraction or value within the range. As example, the nanoparticles may be at least 100 nm in size, e.g., diameter, such as at least 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, or 240 nm. The nanoparticles may be not more than 300 nm in size, e.g., diameter, such as not more than290 nm, 280 nm, 270 nm, 260 nm, 250 nm, 240 nm, 230 nm, 220 nm, 210 nm, 200 nm, 190 nm, 180 nm, 170 nm, 160 nm, 150 nm, 140 nm, 130 nm, 120 nm, or 110 nm. The nanoparticles may have a size range defined by any lower and upper size indicated herein, such as 150 nm to 250 nm.Pharmaceutically Acceptable Carrier

[0060] In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier. “Pharmaceutically acceptable” means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the subjects to which they are administered, i.e., are generally safe, non-toxic, and neither biologically nor otherwise undesirable. A pharmaceutically acceptable carrier may be as simple as water, but can also include, for example, a solution of physiological salt concentration or saline. A pharmaceutically acceptable carrier can be, or may include, stabilizers, diluents, and buffers. Suitable stabilizers may include, for example, SPGA (Sucrose, Phosphate, Glutamate, Albumin), glucose, trehalose,mannitol, carbohydrates (such as dried milk, serum albumin, or casein) or degradation products thereof, surfactants, polyvinylpyrrolidone (PVP) or polyvinyl alcohol (PVA), or polyphosphates. Diluents include water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol). Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington’s Pharmaceutical Sciences, 18thedition, A.R. Gennaro, Ed., Mack Publishing Company (1990); Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and E. Hovgaard, Eds., Taylor & Francis (2000); and Handbook of Pharmaceutical Excipients, 3rdedition, A. Kibbe, Ed., Pharmaceutical Press (2000)).B. METHODS

[0061] Provided is a method for inducing an immune response in a subject. The method generally comprises administering to the subject any of the immunogenic compositions disclosed herein.

[0062] Also provided is a method for breaking an immune tolerance in a subject. The method generally comprises administering to the subject any of the immunogenic compositions disclosed herein.

[0063] Further yet provided is a method for active immunization to prevent a disease in a subject. The method generally comprises administering to the subject the immunogenic composition of any one of the preceding embodiments.

[0064] In some embodiments of these methods, the subject is a mammal, for example a human, a simian, a mouse, a rat, or a llama.

[0065] The enhanced immune response generated by the method may comprise an enhanced antibody response against the antigenic protein in the human subject. For example, such a response can be characterized by the presence of a high proportion of responders, such as more than 50%, 60%, 70%, 80%, 90%, or 100% of subjects tested.

[0066] The enhanced immune response generated by the method may comprise an enhanced CD 8+ T cell response against the antigenic protein in the human subject, such as a response characterized by the presence of a high proportion of CD8+ responders, such as more than 50%, 60%, 70%, 80%, 90%, or 100% of subjects tested as determined by an ICS assay, with a median total cytokine response of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or more.

[0067] The enhanced CD8+ T cell response may comprise an increase or induction of polyfunctional CD8+ T cells specific to the antigenic protein. Such polyfunctional CD8+ T cells express more than one cytokine, such as two or more of IFN-gamma, IL-2 and TNF-alpha.

[0068] The enhanced immune response generated by the method may comprise an enhanced CD4+ T cell response against the antigenic protein in the human subject, such as a response characterized by the presence of a high proportion of CD4+ responders, such as more than 50%, 60%, 70%, 80%, 90%, or 100% of subjects tested as determined by an ICS assay, with a median total cytokine response of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or more.

[0069] The enhanced CD4+ T cell response may comprise an increase or induction of polyfunctional CD4+ T cells specific to the antigenic protein. Such polyfunctional CD4+ T cells express more than one cytokine, such as two or more of IFN-gamma, IL-2 and TNF-alpha.

[0070] The enhanced immune response may comprise any or all of an enhanced CD4+ T cell response, an enhanced antibody response, and an enhanced CD8+ T cell response, against the antigenic protein in the human subject.

[0071] An enhanced antibody response may be assessed by an of: a higher antibody titer in a subject (e.g., a greater concentration of antibodies circulating in the subject’s blood), an increase in the antibody avidity / affinity to their target antigen or enhanced neutralization of the target antigen, faster antibody production, an extended antibody response (e.g., longer antibody half-life in the subject), and / or by improvement in the antibody effector functions, such as improved complement activation or increased antibody-dependent cellular cytotoxicity. Also useful for evaluating the immune response is examination of germinal center formation via meso scale discovery (MSD) assays or enzyme-linked immunosorbent sssays (ELISA), and employing flow cytometry.

[0072] Breakage of an immune tolerance may be measured by assessing the levels of B cells that produce autoantibodies against specific antigens, such as self-antigens. Such levels may be measured using an ELISA or immunofluorescence to detect the presence of specific antigens, such as nuclear proteins, autoantibodies against cyclic citrullinated peptides (CCP), or rheumatoid factors. Breakage of an immune tolerance may also be measured by simple blood tests for immunoglobulin levels.C. SYSTEMS

[0073] Provided is a system for inducing an immune response in a subject, comprising the immunogenic composition of any one of the preceding embodiments. In some embodiments, the system for inducing an immune response in a subject further comprises a delivery system. In furtherembodiments, the delivery system comprises a syringe.

[0074] In some embodiments, the immunogenic composition is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.

[0075] Provided is a system for active immunization to prevent a disease in a subject, the system comprising the immunogenic composition of any one of the preceding embodiments. In some embodiments, the system for active immunization to prevent a disease in a subject further comprises a delivery system. In some embodiments, the delivery system is a syringe. In further embodiments, the immunogenic composition is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.

[0076] In some embodiments, the subject is a mammal, for example a human, a simian, a mouse, a rat, or a llama.D. KITS

[0077] Provided is a kit comprising: a container comprising an immunogenic composition comprising (a) an adjuvant comprising a nanoparticle comprising zinc chitosan; and (b) a soluble protein. In some embodiments, the kit further comprises instructions for use thereof.

[0078] In some embodiments, the kit further comprises a delivery system. In some embodiments, the delivery system is a syringe. In further embodiments, the immunogenic composition is to be administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.E. ASPECTS OF THE DISCLOSURE

[0079] The present disclosure provides an immunogenic composition comprising (a) an adjuvant means and (b) a soluble protein. The adjuvant means may comprise zinc chitosan. The zinc chitosan may be in the form of a nanoparticle, such as a nanoparticle having a size of 100 nm to 300 nm, such as 150 nm to 250 nm.

[0080] The soluble protein may comprise a tag, such as a tag capable of binding to, or complexing with, a metal ion such as Zn2+. The soluble protein may comprise a His tag, such as attached at the N- or C- terminus. The soluble protein may comprise a C-terminal His tag, such as a 6xHis.

[0081] The soluble protein may be HER3, IL-11, CD45, CD33, CDH17, HER2, LAG3, CDHR5,CD117, TR0P2, VG9, PDL-1, TfR, NKp46, ACE2, C7, CD79B, IL-la, IL-1 , GUCY2C, ENPP3, TNFR2, IL-27R, FcRH5, EMR2, KLK2, TMEFF2, CD3, V0-17-Fab, V0-17-Ab, C3, C5, TL1A, IL- 13, or any combination thereof.

[0082] The immunogenic composition may comprise a pharmaceutically acceptable carrier.

[0083] The present disclosure provides methods for inducing an immune response in a subject, breaking an immune tolerance in a subject, and / or active immunization to prevent a disease in a subject.

[0084] These methods may comprise providing a means for inducing the immune response, breaking the immune tolerance, and / or the active immunization in the subject, and administering the means to the subject. The means generally comprise any of the immunogenic compositions disclosed herein, wherein the means may be administered via a syringe. The subject may be a mammal, for example a human, a simian, a mouse, a rat, or a llama. The administration may be via a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.

[0085] These methods may comprise administering to the subject any of the immunogenic compositions disclosed herein. The subject may be a mammal, for example a human, a simian, a mouse, a rat, or a llama. The administration may be via a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.

[0086] The present disclosure provides systems for inducing an immune response in a subject, breaking an immune tolerance in a subject, and / or active immunization to prevent a disease in a subject.

[0087] These systems may comprise a first means for inducing the immune response, breaking the immune tolerance, and / or the active immunization in the subject, and a second means for delivering the first means to the subject. The first means generally comprise any of the immunogenic compositions disclosed herein. The second means generally comprise a syringe. The subject may be a mammal, for example a human, a simian, a mouse, a rat, or a llama. The administration may be via a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.

[0088] These systems may comprise any of the immunogenic compositions disclosed herein and a delivery system. An exemplary delivery system comprises a syringe.

[0089] The present disclosure provides a kit for practicing any of the methods disclosed herein.

[0090] The kits may include a container comprising means for inducing the immune response, breaking the immune tolerance, and / or the active immunization in the subject, and a delivery system. An exemplary delivery system comprises a syringe. The means generally comprise any of the immunogenic compositions disclosed herein, wherein the means may be administered via a syringe.

[0091] The kits may include a container comprising any of the immunogenic compositions disclosed herein and a delivery system. An exemplary delivery system comprises a syringe.

[0092] The systems and kits may further include an insert, such as at least one written instruction sheet for use of the system or kit.EXAMPLESExample 1: Method for preparing zinc chitosan nanoparticlesMaterials

[0093] 2% Acetic Acid in dH?O: 245 ml of Ultrapure distilled water was transferred into a new flask. 5 ml of glacial Acetic Acid was added to yield a 2% Acetic Acid solution.

[0094] 2% Chitosan in 2% Acetic Acid: 4g of Chitosan (SIGMA Life Science, C3646 or 448869, Chitosan from shrimp shells; Mw about 150kDa). was added to a flask. 200 ml of 2% Acetic Acid were added to the flask and mixed gently overnight. The 2% Chitosan Solution in 2% Acetic Acid (“2% Chitosan Solution”) was autoclaved.

[0095] 0.2M Zinc Acetate: 21.95g of Zinc Acetate were added to a flask and dlLO was added in. The solution was mixed and filtered through 0.2 pm filter.Method

[0096] The ratio for preparation of the zinc chitosan nanoparticles was 4 ml of 2% chitosan solution to 6 ml of 0.2 M zinc acetate. For 40 ml total preparation volume, 24 ml of 0.2 M zinc acetate was added to a 50 ml conical vial and 16 ml of a 1% chitosan solution was added (i.e., 1:1 dilution of the 2% chitosan solution in dfLO.). The preparation was mixed for 3 hours.

[0097] Two aliquots of 900 pl each of 10N NaOH were added to the preparation to adjust the pH to 12.0-12.5, and the mixture was sonicated for one minute 5 times (Branson Sonifier 450 at 70% amplitude). Once the nanoparticles were formed (FIG. 1), they were resuspended in PBS (pH 8)

[0098] The tubes were centrifuged for 3 minutes at lOOOxg to collect the zinc chitosan nanoparticles trapped in solution (precipitate). The liquid phase was decanted. 25-30 ml of pH 7.4 dPBS was added to each tube and vortexed well to wash the particles. The remaining portion of each vial was filled to the 50 ml line with pH 7.4 dPBS and centrifuged again for 3 minutes at lOOOxg.

[0099] A second wash was performed and the dPBS was decanted. Then pH 8.0 PBS was added to the halfway mark, vortexed and topped off to the 50 mL with pH 8.0 PBS.

[0100] To determine the concentration of zinc chitosan nanoparticles, a zinc chitosan preparation tube was centrifuged for 5 minutes at lOOOxg to pellet the particles and aid in decanting the supernatant. The tube containing the pellet was weighed, and the weight of an empty tube was subtracted to determine the mass of zinc chitosan in the tube.

[0101] The volume of pH 8.0 PBS to add back to the tube in order to achieve a determined concentration of zinc chitosan appropriate for downstream use was determined (i.e. 200-500 mg / ml).Example 2: Method for producing soluble his-tagged proteins

[0102] In specific embodiments, the soluble protein comprises a His-tag, such as a 6xHis. In some embodiments, the his-tagged soluble protein comprises a His-tagged antigen, such as at the C- terminal. The His-tag may be added via cloning into vectors or using CRISPR-cas9 gene editing to tag the protein. Expression systems and purification methods that are well known in the art may be used to produce and purify the His-tagged soluble antigen.Example 3: Method for incorporating soluble protein in the zinc chitosan nanoparticles

[0103] His-tagged antigens were loaded onto zinc chitosan by mixing antigens to zinc chitosan in a 1: 1000 w / w. Zinc chitosan was measured according to the dose, e.g., lOpg per mouse per injection for AlivaMab mice (Ablexis™), spun in centrifuge at 3000 xg for 5 mins. The PBS was decanted, leaving a zinc chitosan-soluble protein pellet. A pre-calculated volume of PBS was added, and a solution of the zinc chitosan-soluble protein and PBS was formed by rocking / mixing for 3 hours at room temperature. From the solution, lOpg per mouse per injection was used (AlivaMab mice).Example 4: Method for immunization using zinc chitosan nanoparticles

[0104] Mice were immunized on a weekly basis via intrapleural injection route. 10 pg of the antigen / nanoparticles mix obtained as described in the Example 2, in a total volume of 100 pl of PBS were injected per mouse. Serum samples were collected for antibody titer evaluations.Example 5: Method of testing for an enhanced immune response

[0105] Testing for an enhanced immune response typically involves assessing the levels and activity of key immune cells and molecules. This can be achieved using blood tests, tissue samples, and in vitro assays, such as assays that measure antibody levels, T cell responses, and cytokine production.

[0106] FIGS. 2A-2C illustrate representative bar graphs for immunogenicity.

[0107] FIGS. 2A and 2B illustrate immunogenicity measured using ELISA. FIG. 2A illustrates injection with a sigma adjuvant system (S6322-1VL) vs zinc nanoparticles (NP) for immunization with CD3 and KLK2 (Kallikrein-2) in AlivaMab mice (Ablexis™). FIG. 2B illustrates injection with CL413 vs zinc nanoparticles vs C / IFA for immunization with IL-ip, VP-17-Fab or VP-17-Ab in AlivaMab mice (Ablexis™). The endpoint titer is defined as the reciprocal of the highest analyte dilution that gives a reading above the cutoff (i.e., previous dilution to the one that doesn't have a signal).

[0108] FIG. 2C illustrates immunogenicity measured via flow cytometry, showing injection of C / IFA (naive) vs zinc nanoparticles for immunization with EMR2 (EGF-like module-containing mucin-like receptor-like 2) in Elama (EMR2 v. EMR2-nanoparticles).

[0109] An intracellular cytokine staining (ICS) assay is a functional immunology assay that uses flow cytometry to assess cytokine production by individual cells. It involves stimulating immune cells with specific antigens or mitogens, inducing cytokine production, and then fixing and permeabilizing the cells to allow intracellular staining of the cytokines. This technique is used to determine which cells are producing specific cytokines and to assess the strength of an immune response.

[0110] Peripheral blood mononuclear cells are tested for the presence of polyfunctional CD4+ or CD8+ T cells by ICS. After short term (5 hour) stimulation with an immunogenic composition of the present disclosure, cells are surface stained for phenotypic markers such as CD3, CD4, CD8, CD27, CD45RA, fixed, permeabilized and stained further for the presence of intracellular cytokines such as INF-y, TNF-a and IE-2. Flow cytometry analysis using Flow-Jo software is used to detect single / multiple cytokine secreting central memory (CD27+CD45RA-), CD4+ or CD8+ T cells at baseline, and post-vaccination time points. Cytokine release may also be measured by other means known in the art, e.g., using Western blot, EEISA, or immunohistochemical assays to detect the presence of released cytokines in a sample containing CD4+ and / or CD8+ T-cells.

[0111] All publications and patents referred to herein are incorporated by reference. Various modifications and variations of the described subject matter will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to these embodiments. Indeed, various modifications for carrying out the invention are obvious to those skilled in the art and are intended to be within the scope of the following claims.

Claims

CLAIMSWhat is claimed is:

1. An immunogenic composition comprising:(a) an adjuvant comprising a nanoparticle comprising zinc chitosan; and(b) a soluble protein.

2. The immunogenic composition of claim 1, wherein the soluble protein comprises a tag.

3. The immunogenic composition of claim 2, wherein the tag is a His tag.

4. The immunogenic composition of claim 2, wherein the tag is a C-terminal His tag.

5. The immunogenic composition of claim 1, wherein the soluble protein is HER3, IL-11,CD45, CD33, CDH17, HER2, LAG3, CDHR5, CD117, TROP2, VG9, PDL-1, TfR, NKp46, ACE2, C7, CD79B, IL- la, IL- 10, GUCY2C, ENPP3, TNFR2, IL-27R, FcRH5, EMR2, KLK2, TMEFF2, CD3, V0-17-Fab, V0-17-Ab, C3, C5, TL1A, IL-13, or a combination thereof.

6. The immunogenic composition of claim 1, wherein the nanoparticle is about 150 nm to about 250 nm in size.

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

8. An immunogenic composition comprising:(a) an adjuvant means; and(b) a soluble protein.

9. The immunogenic composition of claim 8, wherein the soluble protein comprises a tag.

10. The immunogenic composition of claim 9, wherein the tag is a His tag.

11. The immunogenic composition of claim 9, wherein the tag is a C-terminal His tag.

12. The immunogenic composition of claim 8, wherein the soluble protein is HER3, IL-11,CD45, CD33, CDH17, HER2, LAG3, CDHR5, CD117, TROP2, VG9, PDL-1, TfR, NKp46, ACE2, C7, CD79B, IL- la, IL- 10, GUCY2C, ENPP3, TNFR2, IL-27R, FcRH5, EMR2,KLK2, TMEFF2, CD3, V0-17-Fab, V0-17-Ab, C3, C5, TL1A, IL-13, or a combination thereof.

13. The immunogenic composition of claim 8, wherein the adjuvant means comprises a nanoparticle comprising zinc chitosan.

14. The immunogenic composition of claim 13, wherein the nanoparticle is about 150 nm to about 250 nm in size.

15. The immunogenic composition of claim 8, further comprising a pharmaceutically acceptable carrier.

16. A method for inducing an immune response in a subject, the method comprising administering to the subject the immunogenic composition of any one of claims 1-15.

17. A method for breaking an immune tolerance in a subject, the method comprising administering to the subject the immunogenic composition of any one of claims 1-15.

18. A method for active immunization to prevent a disease in a subject, the method comprising administering to the subject the immunogenic composition of any one of claims 1-15.

19. A method for inducing an immune response in a subject, the method comprising: providing a means for inducing the immune response in the subject; and administering the means to the subject.

20. A method for breaking an immune tolerance in a subject, the method comprising: providing a means for breaking the immune tolerance in the subject; and administering the means to the subject.

21. A method for active immunization to prevent a disease in a subject, the method comprising: providing a means for active immunization to prevent the disease in the subject; and administering the means to the subject.

22. The method according to any one of claims 19-21, wherein the means comprises the immunogenic composition according to any one of claims 1-7.

23. The method according to any one of claims 16-21, wherein the subject is a mammal, such as a human, a simian, a mouse, a rat, or a llama.

24. A system for inducing an immune response in a subject, comprising the immunogenic composition of any one of claims 1-15, and a delivery system.

25. A system for breaking an immune tolerance in a subject, comprising the immunogenic composition of any one of claims 1-15, and a delivery system.

26. A system for active immunization to prevent a disease in a subject, comprising the immunogenic composition of any one of claims 1-15, and a delivery system.

27. The system according to any one of claims 24-26, wherein the delivery system comprises a syringe.

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