MHC class i antigen-presenting nanoparticles and methods of using same

Abstract

Disclosed herein are antigen-presenting nanoparticles (APNs) presenting self-epitopes via MHC class I molecules. The APNs encapsulate one or more encapsulated components which induce cell death which are delivered to autoreactive immune cells (e.g., autoreactive T cells) that recognize the self-epitopes, thereby killing the autoreactive immune cells. These APNs can be used to treat various autoimmune diseases and disorders, including type 1 diabetes.

Description

MHC CLASS I ANTIGEN-PRESENTING NANOPARTICLES AND METHODS OF USING SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No.63 / 712,845, filed October 28, 2024, which is incorporated by reference herein in its entirety.GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under CA280832 awarded by the National Institutes of Health. The Government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] The sequence listing submitted on October 28, 2025, as an. XML file entitled “10034-398W01._ST26.xml” created on October 28, 2025, and having a file size of 31,345 bytes is hereby incorporated by reference pursuant to 37 C. F. R. § 1.52(e)(5).BACKGROUND

[0004] Autoreactive T cells are a subset of T cells that recognize self-antigens due to a failure in central or peripheral tolerance mechanisms. During thymic development, T cells undergo positive and negative selection; however, incomplete deletion or inadequate anergy induction can result in the escape of autoreactive T cells. These autoreactive T cells can contribute to autoimmune disorders by targeting host tissues through antigen-specific cytotoxicity or proinflammatory cytokine secretion. Regulatory T cell dysfunction or impaired checkpoint signaling (e.g., CTLA-4, PD-1 pathways) further exacerbates their activity, underscoring their role in the etiology of diseases like type 1 diabetes, multiple sclerosis, and systemic lupus erythematosus.

[0005] Type 1 diabetes (T1D) is an autoimmune disease in which autoreactive cytotoxic T cells attack and destroy the insulin-producing cells within the pancreatic islets of Langerhans. As of 2024, the only FDA-approved disease-modifying therapy for T1D is an Fc receptornonbinding «CD3 monoclonal antibody (teplizumab) to delay the onset of clinical T1D by modifying the phenotype and activity of CD8 T cells, including autoreactive CD8+ T cells. While effective, aCD3 monoclonal antibodies broadly modulate T cell immunity, thereby making subjects vulnerable to serious side effects, such as cytokine release syndrome, and opportunistic infections.

[0006] Accordingly, there exists a need for improved treatments for eliminating autoreactive T cells, particularly for the treatment of autoimmune disorders like type 1 diabetes. These needs and others are at least partially satisfied by the present disclosure.SUMMARY

[0007] Disclosed herein are antigen-presenting nanoparticles (APNs) which target and destroy autoreactive T cells. The APNs present self-epitopes on their surface, which would be recognized and bound only by autoreactive T cells (i.e., thereby preventing off target delivery of the APNs to normal T cells). Upon binding to an autoreactive T cell, the APN delivers to the cell one or more encapsulated components which induce cell death, which kills the cell. These APNs enable targeted and efficient elimination of autoreactive T cells, which can be particularly useful for the treatment, prevention, and / or management of autoimmune diseases such as type 1 diabetes.

[0008] In one aspect, disclosed herein are antigen-presenting nanoparticles (APNs) (such as, for example, a lipid nanoparticle, a liposome, or a polymeric nanoparticle), comprising: a major histocompatibility complex (MHC) class I comprising a self-epitope or a mimotope thereof (such, as, for example, self-epitope or mimotope thereof comprising NRP-V7, IGRP, InsulinA, InsulinB, GAD65, chromogranin A, p31, p79, or any fragments thereof, including, but not limited to KYNKANVFL (SEQ ID NO: 14), VYLKTNVFL (SEQ ID NO: 15), LYLVCGERV (SEQ ID NO: 16), or any variants thereof), wherein the MHC class I is presented on a surface of the APN; one or more encapsulated components which induce cell death. In some aspects, the one or more encapsulated components comprise a nucleic acid encoding an enzyme or fragment thereof which induces cell death (such as, for example, caspase, BIM, BID, granzyme B, PUMA, or gasdermin D). In some aspects, the one or more encapsulated components comprise one or more toxins (such as, for example, diphtheria toxin, pseudomonas exotoxin A, ricin, shiga toxin, or ribosome-inactivating proteins).

[0009] Also disclosed herein are APNs of any preceding aspect wherein the APN comprises at least one ionizable lipid (including, but not limited to cKK-E12, SM102, MC3, Lipid-5, Alc-0315, LP01, Lipid A9, or any combination thereof), cholesterol, phospholipid (including, but not limited to DSPC, DOPE, or any combination thereof), PEGylated lipid (including, but not limited to ALC-0159, DMG-PEG, DSPE-PEG, PEG14-2000), or a combination thereof. In some aspects, the APN comprises from about 30 mol% to about 60 mol% of the ionizable lipid; from about 35 mol% to about 50 mol% cholesterol; from about 5 mol% to about 20 mol% of the phospholipid; and / or from about 0.5 mol% to about 5 mol% ofthe PEGylated lipid. In some aspects, the ratio of PEGylated lipid to total lipids is from about 0.01:1 to about 0.05:1. In some aspects, the ratio of MHC class I to total lipids is from about 0.3:1 to about 6:1.

[0010] In one aspect, disclosed herein are methods of killing an autoreactive immune cell (such as, for example, a CD8+ T cell including, but not limited to human CD8+ T cells), the method comprising exposing the APN of any preceding aspect to said autoreactive immune cell. In some aspects, the APN is administered to a subject having an autoimmune disease or disorder.

[0011] Also disclosed herein are methods of treating, decreasing, reducing, inhibiting, ameliorating and / or preventing diabetes and / or onset of hyperglycemia in a subject, the method comprising administering to the subject the APN of any preceding aspect, wherein the selfepitope or mimotope thereof comprises NRP-V7, IGRP, InsulinA, InsulinB, GAD65, chromogranin A, p31, p79, or any fragments thereof (such, as, for example, KYNKANVFL (SEQ ID NO: 14), VYLKTNVFL (SEQ ID NO: 15), LYLVCGERV (SEQ ID NO: 16), or any variants thereof).

[0012] In some aspects, the APN is administered in a concentration of from about 0.1 mg / kg to about 5 mg / kg. In some aspects, the APN is repeatedly administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times at an interval of about every 5 days.

[0013] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims.BRIEF DESCRIPTION OF DRAWINGS

[0014] FIGURE 1 depicts an overall schematic of APN compositions and application for in vivo depletion of autoreactive β-islet specific T cells.

[0015] FIGURES 2A-2C depict that incorporation of UV-labile amino acid into Kd-restricted peptide enables UV-mediated ligand exchange. FIG. 2A shows representative flow plots analyzing tetramer staining from conventional refolded pMHC-tetramers and UV exchanged pMHC-tetramers. FIGS. 2B-2C show NOD8.3 splenocytes stained with conventionally refolded and UV-exchanged pMHC-tetramers. The total % CD8+Tet+ was quantified (FIG. 2B) as well as the mean fluorescence intensity (MFI) (FIG. 2C). One-way analysis of variance (ANOVA) with Tukey post-test and correction for multiple comparisons; ns= not significant, **P<0.01, ****P<0.0001. All data are means ± SD; n=3 independent wells.

[0016] FIGURES 3A-3G depict in vitro transfection of NOD8.3 T cells with cognate APNs. FIG. 3A is a schematic showing APN delivery of diverse mRNA cargo to cognate T cells. FIGS.3B-3G show activated NOD8.3 CD8 T cells transfected in vitro with PBS, Kd / Ctrl (noncognate) APNs, or Kd / NRP-V7 (cognate) APNs. After 24 hours, transfection readout was measured. APNs carrying secreted nLuc were analyzed via IVIS (FIG. 3B) and quantified (FIG. 3C). FIGS. 3D-3E show representative flow plots (FIG. 3D) and frequency bar plot (FIG. 3E) of intracellular BFP. FIGS. 3F-3G show representative flow plots (FIG. 3F) and frequency bar plot (FIG.3G) of surface-bound VHH expression. One-way analysis of variance (ANOVA) and Tukey post-test and correction for multiple comparisons; ****P<0.0001. All data are means ± SD; n=3 independent wells.

[0017] FIGURES 4A-4B depict screening and identification of potent proapoptotic mRNA constructs. FIG. 4A shows quantification of mouse CD8 T cell death 24 hours after electroporation with mRNA encoding for the specified proapoptotic protein. FIG. 4B shows quantification of cell viability after in vitro transfection of mouse CD8 T cells with LNPs encapsulated with VHH or Casp6 mRNA. Two-way ANOVA with Sidak post-test and correction for multiple comparisons; means ± SD, n=3 independent wells ****P<0.0001.

[0018] FIGURES 5A-5C depict that Kd / NRP-V7 Casp6 APNs deplete adoptively transferred NOD8.3 T cells and maintain total T cell homeostasis. FIG. 5A shows a timeline for T1D model development and treatment, and a schematic showing APNs selectively target NOD8.3 T cells T cells in vivo and delivery of Casp6 mRNA triggers apoptosis. FIG.5B shows quantification of % NOD8.3 T cells in peripheral blood, pancreatic LN (pLN), and spleen after treatment. FIG. 5C shows total CD8 T cells in the peripheral blood, pLN, and spleen after treatment. Organs isolated with less than 1% viable cells after processing were excluded from analysis. One-way ANOVA with Tukey ’s post-test and correction for multiple comparisons, n=4-6 biological replicates, ns = not significant; *, **P < 0.01, ***P < 0.001, ****P<0.0001.

[0019] FIGURES 6A-6C depict that APNs prevent the onset of hyperglycemia in murine ACT model of T1D. FIG. 6A is timeline and schematic showing treatment strategies; high dose aCD3 was administered at 2.5 mg / kg and APNs at 0.1 mg / kg at the specified timepoints. APNs prevent hyperglycemia by selectively depleting autoreactive NOD8.3 T cells and sparing β-islet cell function. FIG. 6B shows blood glucose traces of mice after receiving respective treatments; light-blue shaded region underneath dashed line represents healthy blood glucose levels (<250 mg / dL). FIG. 6C shows the survival curve of mice; survival refers to living mice with blood glucose levels <250 mg / dL. Log-rank (Mantel-Cox) test, n=4-6 biological replicates, NS = not significant.

[0020] FIGURES 7A-7C depict that APNs durably prevent the onset of hyperglycemia. FIG. 7A is a timeline and schematic showing treatment strategies; low dose aCD3 and APNs were administered at 0.1 mg / kg at the specified timepoints. FIG. 7B shows long term blood glucose traces of Kd / NRP-V7 Casp6 APNs treated mice. FIG. 7C shows the survival curve of mice; survival refers to living mice with blood glucose levels <250 mg / dL. Log-rank (Mantel-Cox) test, n=8-16 biological replicates.

[0021] FIGURES 8A-8B depict that APNs do not cause off-target toxicity in the liver and kidney. FIG. 8A shows blood serum biochemical analysis of organ function; alanine transaminase (ALT); aspartate aminotransferase (AST); blood urea nitrogen (BUN). One-way ANOVA with Tukey’s post-test and correction for multiple comparisons; means ± SD, n=7-8 biological replicates, ns = not significant, **P<0.01, ***P<0.001. FIG. 8B shows quantification of % body weight change after treatment with Kd / NRP-V7 Casp6 APNs. Dashed line represents initial body weight and light-blue shaded region shows healthy body weight range.

[0022] FIGURES 9A-9D depict long-term monitoring of glucose levels after treatment with NRP-V7 Casp6 APNs. FIG. 9A shows overall schematic of experiment timeline and dosing strategy. FIG. 9B shows the quantification of body weight change over duration of experiment. The dotted line represents the initial body weight. FIG. 9C shows the blood glucose traces of mice. The dotted line represents when mice are considered diabetic at a glucose measurement of 250mg / dL. FIG. 9D shows long-term survival curve of the mice. Survival refers to alive mice with blood glucose levels <250mg / dL.

[0023] FIGURES 10A-10C depict increased dosing regimen preventing the onset of hyperglycemia at O. lmg / kg, 0.25mg / kg, and 0.5mg / kg. FIG. 10A shows a schematic outlining the dosing regiment: mice receive 5 daily doses of the APNs at either 0.1, 0.25, or 0.5 mg / kg after infusion of NOD8.3 T cells. FIG. 10B shows the blood glucose levels of mice after the designated treatment of FIG. 10A. Light blue shaded area represents healthy mice with blood glucose levels under 250mg / dL. FIG. 10C shows the survival curve of mice after the designated treatment of FIG. 10A. Survival refers to alive mice with blood glucose levels <250 mg / dL.

[0024] FIGURES 11A-11B depict NRP-V7 Cas6 APNs being well tolerated at 0.5mg / kg with 5 daily injections. FIG. 11A shows the quantification of body weight change over the duration of the study. Dashed black line indicates initial body weight. FIG. 11B shows the blood serum biochemical analysis of organ function; alanine transaminase (ALT); aspartate aminotransferase (ALT); creatinine, and total protein. One-way ANOVA with Tukey’s post-test and correction for multiple comparisons; means ± SD, n = 4-5 biological replicates, ns = not significant.DETAILED DESCRIPTION

[0025] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.DEFINITIONS

[0026] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0027] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.

[0028] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.

[0029] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can beexpressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0030] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0031] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0032] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10%’ variation unless otherwise indicated orinferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0033] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material. For example, an “effective amount” of a monomer refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g. desired antioxidant release rate or viscoelasticity. The specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors including the amount and type of monomer, amount and type of polymer, e.g., acrylamide, amount of antioxidant, and desired release kinetics.

[0034] As used herein, the term “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular' subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts. In the case of treating a particular disease or condition, in some instances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.

[0035] For example, it is well within the skill of the ait to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. Thedosage can be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a subject may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0036] A response to a therapeutically effective dose of a disclosed drug delivery composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. The amount of a treatment may be varied for example by increasing or decreasing the amount of a disclosed compound and / or pharmaceutical composition, by changing the disclosed compound and / or pharmaceutical composition administered, by changing the route of administration, by changing the dosage riming and so on. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0037] As used herein, the term “prophylactically effective amount” refers to an amount effective for preventing onset or initiation of a disease or condition.

[0038] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0039] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0040] As used interchangeably herein, “subject,” “individual,” or “subject” can refer to a vertebrate organism, such as a mammal (e.g. human). "Subject" can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.

[0041] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof. The effect can be therapeutic in terms of a partial or completecure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein can include any treatment of a disease disorder in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and / or its symptoms or conditions. The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and / or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0042] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and / or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0043] As used herein, “therapeutic” can refer to treating, healing, and / or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.

[0044] The terms “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand).

[0045] Reference also is made herein to peptides, polypeptides, proteins and compositions comprising peptides, polypeptides, and proteins. As used herein, a polypeptide and / or protein is defined as a polymer of amino acids, typically of length>100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). A peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110).

[0046] A “functional fragment” as referred to herein comprises a portion of a polypeptide which retains its functional ability.

[0047] As disclosed herein, exemplary peptides, polypeptides, proteins may comprise, consist essentially of, or consist of any reference amino acid sequence disclosed herein, or variants of the peptides, polypeptides, and proteins may comprise, consist essentially of, or consist of an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any amino acid sequence disclosed herein. Variant peptides, polypeptides, and proteins may include peptides, polypeptides, and proteins having one or more amino acid substitutions, deletions, additions and / or amino acid insertions relative to a reference peptide, polypeptide, or protein. Also disclosed are nucleic acid molecules that encode the disclosed peptides, polypeptides, and proteins (e.g., polynucleotides that encode any of the peptides, polypeptides, and proteins disclosed herein and variants thereof).

[0048] The term “amino acid,” includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, P-alanine, p- Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N -Methyllysine, 2,4-Diaminobutyric acid, N-Methylvaline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid, Norleucine, 2,3-Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.

[0049] The peptides, polypeptides, and proteins disclosed herein may be modified to include non-amino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N-terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4-dimethylglutaric acid), amidation (e.g., C -terminal amidation via addition of an amide orsubstituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C-terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation ( thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N -terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine).

[0050] Variants comprising deletions relative to a reference amino acid sequence or nucleotide sequence are contemplated herein. A “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides relative to a reference sequence. A deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues or nucleotides. A deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C -terminal truncation or both of a reference polypeptide or a 5 '-terminal or 3 '-terminal truncation or both of a reference polynucleotide).

[0051] Variants comprising a fragment of a reference amino acid sequence or nucleotide sequence are contemplated herein. A “fragment” is a portion of an amino acid sequence or a nucleotide sequence which is identical in sequence to but shorter in length than the reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one nucleotide / amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. Fragments may be preferentially selected from certain regions of a molecule, for example the N-terminal region and / or the C -terminal region of apolypeptide or the 5'-terminal region and / or the 3' terminal region of a polynucleotide. The term “at least a fragment” encompasses the full length polynucleotide or full length polypeptide.

[0052] Variants comprising insertions or additions relative to a reference sequence are contemplated herein. The words “insertion” and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues or nucleotides.

[0053] Fusion proteins and fusion polynucleotides also are contemplated herein. A “fusion protein” refers to a protein formed by the fusion of at least one peptide, polypeptide, protein or variant thereof as disclosed herein to at least one molecule of a heterologous peptide, polypeptide, protein or variant thereof. The heterologous protein(s) may be fused at the N-terminus, the C -terminus, or both termini. A fusion protein comprises at least a fragment or variant of the heterologous protein(s) that are fused with one another, preferably by genetic fusion (i.e., the fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a first heterologous protein is joined in-frame with a polynucleotide encoding all or a portion of a second heterologous protein ). The heterologous protein(s), once part of the fusion protein, may each be referred to herein as a “portion”, “region” or “moiety” of the fusion protein.

[0054] A fusion polynucleotide refers to the fusion of the nucleotide sequence of a first polynucleotide to the nucleotide sequence of a second heterologous polynucleotide (e.g., the 3' end of a first polynucleotide to a 5' end of the second polynucleotide). Where the first and second polynucleotides encode proteins, the fusion may be such that the encoded proteins are in-frame and results in a fusion protein. The first and second polynucleotide may be fused such that the first and second polynucleotide are operably linked (e.g., as a promoter and a gene expressed by the promoter as discussed below).

[0055] A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

[0056] A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for BiotechnologyInformation's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.

[0057] Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.

[0058] “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0059] A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. vol. 1 3, Cold Spring Harbor Press, Plainview N. Y. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.

[0060] “Transformation” describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed cells” includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmidor as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RN A for limited periods of time.

[0061] “Substantially isolated or purified” nucleic acid or amino acid sequences are contemplated herein. The term “substantially isolated or purified” refers to nucleic acid or amino acid sequences that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.ANTIGEN-PRESENTING NANOPARTICLES

[0062] In some aspects, disclosed herein is an antigen-presenting nanoparticle (APN) including: a major histocompatibility complex (MHC) class I including a self-epitope or a mimotope thereof, wherein the MHC class I is presented on a surface of the APN; and one or more encapsulated components which induce cell death.

[0063] In some aspects, the MHC class I can be human MHC class I. This can limit the immunogenicity of the APNs, particularly when used for repeat administrations.

[0064] As used herein, the term “epitope” or refers to the portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed from contiguous amino acids and / or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Linear epitope is an epitope formed from contiguous amino acids on the linear sequence of amino acids. A linear epitope is typically retained upon protein denaturing. Conformational or structural epitope is an epitope composed of amino acid residues that are not contiguous and thus made up of separated parts of the linear sequence of amino acids that are brought into proximity to one another by folding of the molecule, such as through secondary, tertiary, and / or quaternary structures. A conformational or structural epitope is typically lost upon protein denaturation. In some embodiments, an epitope can include at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Thus, an epitope as used herein encompasses a defined epitope in which an antibody binds only portions of the defined epitope.

[0065] Similarly, a “self-epitope” as described herein refers to the epitope of a self-antigen, where the term “self-antigen” refers to an antigen that originates from within an animal, tissue, or cell. In some aspects, a self-antigen can include an endogenous antigen.

[0066] Self-antigens are typically tolerated by the immune system, as developing T cells undergo positive and negative selection to eliminate any autoreactive T cells which have high affinity for self-antigens. However, due to the incomplete representation of self-antigens in the thymus and the stochastic nature of TCR gene rearrangement, some autoreactive T cells escapedeletion. Peripheral tolerance mechanisms, such as anergy, deletion, and suppression by regulatory T cells are critical in preventing these cells from initiating autoimmunity. Dysregulation or failure of these tolerance checkpoints can lead to the activation of autoreactive T cells, resulting in the breakdown of self-tolerance and the development of autoimmune pathologies. By presenting a self-epitope or mimotope thereof, in some aspects, the disclosed APNs can be recognized by and deliver their contents to autoreactive immune cells which have escaped deletion.

[0067] As used herein, the term “mimotope” refers to a molecule which is a mimic of an epitope. The mimotope may also act as a competitor for the epitope of which it is a mimic in in vitro assays (e.g. ELISA assays) and preferably binds to the same antigen-binding region of an antibody which binds immunospecifically to an epitope of a desired antigen. The mimotope may elicit an immunological response in a host that is reactive to the antigen of which it is a mimic.

[0068] In some aspects, the self-epitope or mimotope thereof can include NRP-V7, IGRP, InsulinA, InsulinB, GAD65, chromogranin A, p31, p79, or any fragments thereof.

[0069] In some aspects, the self-epitope or mimotope thereof can include KYNKANVFL (SEQ ID NO: 14), VYLKTNVFL (SEQ ID NO: 15), LYLVCGERV (SEQ ID NO: 16), or any variants thereof.

[0070] As used herein, a component which “induces cell death” refers to any moiety which participates in initiating or promoting cell death through various pathways, including, but not limited to, apoptosis, necrosis, autophagy, pyroptosis, or other forms of programmed or nonprogrammed cell death. Such components can participate directly in cell death pathways (e.g., enzymes which are involved in cell death pathways) or indirectly lead to the activation or cell death pathways by, for example, inducing cellular- stress or damage (e.g., toxins). Upon delivery of the disclosed APNs to an autoreactive immune cell, any encapsulated components which induce cell death can trigger the death of the autoreactive cell, thereby preventing or minimizing autoimmune responses in a host.

[0071] In some aspects, the one or more encapsulated components can include a nucleic acid encoding an enzyme or fragment thereof which induces cell death. In some aspects, the enzyme or fragment thereof can be caspase (e.g., caspase 6, caspase 9). Caspases (cysteineaspartic proteases) are a family of intracellular cysteine proteases that play a central role in the execution of apoptosis and inflammation. They are synthesized as inactive zymogens (procaspases) and become activated through proteolytic cleavage at specific aspartate residues. Caspases are broadly classified into initiator caspases (e.g., caspase-8, -9) and effector caspases(e.g., caspase-3, -6, -7), where initiators activate downstream effector caspases to orchestrate the cleavage of key cellular substrates, ultimately leading to controlled cell dismantling. In other aspects, the enzyme or fragment thereof can be BIM, BID, granzyme B, PUMA, or gasdermin D. In some aspects, the enzyme or fragment thereof can include one or more modifications, for example, to increase its expression or functionality. For example, in some aspects, the enzyme or fragment thereof can be modified to be constitutively active.

[0072] In some aspects, the nucleic acid encoding an enzyme or fragment thereof which induces cell death can include about 80% similarity or more (e.g., about 81% similarity or more, about 82% similarity or more, about 83% similarity or more, about 84% similarity or more, about 85% similarity or more, about 86% similarity or more, about 87% similarity or more, about 88% similarity or more, about 89% similarity or more, about 90% similarity or more, about 91% similarity or more, about 92% similarity or more, about 93% similarity or more, about 94% similarity or more, about 95% similarity or more, about 96% similarity or more, about 97% similarity or more, about 98% similarity or more, about 99% similarity or more, about 100% similarity or more) to any one of SEQ ID NOS: 6-13.

[0073] In some aspects, the one or more encapsulated components can include one or more toxins which, for example, can induce cell death. Example toxins include, but are not limited to, diphtheria toxin, pseudomonas exotoxin A (PE, PE38, PE40), ricin A chain, ricin holotoxin, shiga toxin 1 A subunit, shiga toxin 2 A subunit, shiga-like toxin (Stx), abrin A chain, abrin holotoxin, modeccin, gelonin, saporin, trichosanthin, a-sarcin, mitogillin, bouganin, volkensin, ebulin 1, cholera toxin (CTX), Escherichia coli heat-labile toxin (LT), pertussis toxin (PTX), Clostridium botulinum neurotoxins (BoNT / A-G), tetanus toxin (TT), anthrax lethal toxin (PA + LF), anthrax edema toxin (PA + EF), Staphylococcal enterotoxin B (SEB), Staphylococcal a-toxin, streptolysin O, cytolysin A (ClyA), exotoxin T (ExoT), exotoxin S (ExoS), ExoU (phospholipase toxin), listeriolysin O (LLO), adenylate cyclase toxin (CyaA, Bordetella pertussis), Clostridium perfringens epsilon toxin, Clostridium perfringens a-toxin (phospholipase C), Pasteurella multocida toxin (PMT), Vibrio vulnificus RTX toxin, hemolysin BL (Bacillus cereus), and cytotoxic necrotizing factor (CNF1), and ribosome-inactivating proteins.

[0074] In some aspects, the APN can be a lipid nanoparticle, a liposome, or a polymeric nanoparticle. As used herein, “lipid nanoparticles” refer to particles having at least one dimension on the order of nanometers (e.g., 1-1000 nm) and including one or more lipids. In the context of the present invention, a lipid nanoparticle typically serves to transport a desired nucleic acid to a target cell or tissue. The process of incorporation of a desired nucleic acid intoa lipid nanoparticle is often referred to as “loading”. The lipids and the nucleic acid can create a self-assembled structure via counterion interactions. The purpose of incorporating a nucleic acid into a transfer vehicle, such as a lipid nanoparticle, is often to protect the nucleic acid from an environment which may contain enzymes or chemicals that degrade nucleic acids and / or systems or receptors that cause the rapid excretion of the nucleic acids. Accordingly, in some aspects, a suitable delivery vehicle is capable of enhancing the stability of the nucleic acid contained therein and / or facilitate the delivery of nucleic acid to the target cell or tissue.

[0075] In some aspects, the APN can include at least one ionizable lipid, cholesterol, phospholipid, PEGylated lipid, or a combination thereof.

[0076] As used herein, the term “ionizable lipid” refers to a lipid, e.g., cationic lipid or anionic lipid, having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will be understood by one of ordinary skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Generally, ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7. In some aspects, the ionizable lipid can include cKK-E12, SM102, MC3, Lipid-5, Alc-0315, LP01, Lipid A9, or any combination thereof.

[0077] In some aspects, the nanoparticle can include at least about 30 mol% (e.g., at least about 32. mol%, at least about 34 mol%, at least about 36 mol%, at least about 38 mol%, at least about 40 mol%, at least about 42 mol%, at least about 44 mol%, at least about 46 mol%, at least about 48 mol%, at least about 50 mol%, at least about 52 mol%, at least about 54 mol%, at least about 56 mol%, at least about 58 mol%, at least about 60 mol%) of the ionizable lipid. In some aspects, the nanoparticle can include up to about 60 mol% (e.g., up to about 58 mol%, up to about 56 mol%, up to about 54 mol%, up to about 52 mol%, up to about 50 mol%, up to about 48 mol%, up to about 46 mol%, up to about 44 mol%, up to about 42 mol%, up to about 40 mol%, up to about 38 mol%, up to about 36 mol%, up to about 34 mol%, up to about 32 mol%, up to about 30 mol%) of the ionizable lipid.

[0078] It is considered that the nanoparticle can include an amount of the ionizable lipid ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the nanoparticle can include from about 30 mol% to about 60 mol% (e.g., from about 32 mol% to about 58 mol%, from about 34 mol% to about 56 mol%, from about 36 mol% to about 54 mol%, from about 38 mol% to about 52mol%, from about 40 mol% to about 50 mol%, from about 42 mol% to about 48 mol%, from about 44 mol% to about 46 mol%, from about 30 mol% to about 46 mol%, from about 32 mol% to about 44 mol%, from about 34 mol% to about 42 mol%, from about 36 mol% to about 40 mol%, from about 44 mol% to about 60 mol%, from about 46 mol% to about 58 mol%, from about 48 mol% to about 56 mol%, from about 50 mol% to about 54 mol%) of the ionizable lipid.

[0079] In some aspects, the nanoparticle can include at least about 35 mol% (e.g., at least about 36 mol%, at least about 37 mol%, at least about 38 mol%, at least about 39 mol%, at least about 40 mol%, at least about 41 mol%, at least about 42 mol%, at least about 43 mol%, at least about 44 mol%, at least about 45 mol%, at least about 46 mol%, at least about 47 mol%, at least about 48 mol%, at least about 49 mol%, at least about 50 mol%) of the cholesterol. In some aspects, the nanoparticle can include up to about 50 mol% (e.g., up to about 49 mol%, up to about 48 mol%, up to about 47 mol%, up to about 46 mol%, up to about 45 mol%, up to about 44 mol%, up to about 43 mol%, up to about 42 mol%, up to about 41 mol%, up to about 40 mol%, up to about 39 mol%, up to about 38 mol%, up to about 37 mol%, up to about 36 mol%, up to about 35 mol%) of the cholesterol.

[0080] It is considered that the nanoparticle can include an amount of the cholesterol ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the nanoparticle can include from about 35 mol% to about 50 mol% (e.g., from about 36 mol% to about 49 mol%, from about 37 mol% to about 48 mol%, from about 38 mol% to about 47 mol%, from about 39 mol% to about 46 mol%, from about 40 mol% to about 45 mol%, from about 41 mol% to about 44 mol%, from about 42 mol% to about 43 mol%, from about 35 mol% to about 43 mol%, from about 36 mol% to about 42 mol%, from about 37 mol% to about 41 mol%, from about 38 mol% to about 40 mol%, from about 42 mol% to about 50 mol%, from about 43 mol% to about 49 mol%, from about 44 mol% to about 48 mol%, from about 45 mol% to about 47 mol%) of the cholesterol.

[0081] In some aspects, the phospholipid can include DSPC, DOPE, or any combination thereof.

[0082] In some aspects, the nanoparticle can include at least about 5 mol% (e.g., at least about 6 mol%, at least about 7 mol%, at least about 8 mol%, at least about 9 mol%, at least about 10 mol%, at least about 11 mol%, at least about 12 mol%, at least about 13 mol%, at least about 14 mol%, at least about 15 mol%, at least about 16 mol%, at least about 17 mol%, at least about 18 mol%, at least about 19 mol%, at least about 20 mol%) of the phospholipid. In some aspects, the nanoparticle can include up to about 20 mol% (e.g., up to about 19 mol%,up to about 18 mol%, up to about 17 mol%, up to about 16 mol%, up to about 15 mol%, up to about 14 mol%, up to about 13 mol%, up to about 12 mol%, up to about 11 mol%, up to about 10 mol%, up to about 9 mol%, up to about 8 mol%, up to about 7 mol%, up to about 6 mol%, up to about 5 mol%) of the phospholipid.

[0083] It is considered that the nanoparticle can include an amount of the phospholipid ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the nanoparticle can include from about 5 mol% to about 20 mol% (e.g., from about 6 mol% to about 19 mol%, from about 7 mol% to about 18 mol%, from about 8 mol% to about 17 mol%, from about 9 mol% to about 16 mol%, from about 10 mol% to about 15 mol%, from about 11 mol% to about 14 mol%, from about 12 mol% to about 13 mol%, from about 5 mol% to about 13 mol%, from about 6 mol% to about 12 mol%, from about 7 mol% to about 11 mol%, from about 8 mol% to about 10 mol%, from about 12 mol% to about 20 mol%, from about 13 mol% to about 19 mol%, from about 14 mol% to about 18 mol%, from about 15 mol% to about 17 mol%) of the phospholipid.

[0084] In some aspects, the PEGylated lipid can include ALC-0159, DMG-PEG, DSPE-PEG, PEG14-2000, or any combination thereof.

[0085] In some aspects, the nanoparticle can include at least about 0.5 mol% (e.g., at least about 1 mol%, at least about 1.5 mol%, at least about 2 mol%, at least about 2.5 mol%, at least about 3 mol%, at least about 3.5 mol%, at least about 4 mol%, at least about 4.5 mol%, at least about 5 mol%) of the PEGylated lipid. In some aspects, the nanoparticle can include up to about 5 mol% (e.g., up to about 4.5 mol%, up to about 4 mol%, up to about 3.5 mol%, up to about 3 mol%, up to about 2.5 mol%, up to about 2 mol%, up to about 1.5 mol%, up to about 1 mol%, up to about 0.5 mol%) of the PEGylated lipid.

[0086] It is considered that the nanoparticle can include an amount of the PEGylated lipid ranging from any of the minimum values described above. For example, in some aspects, the nanoparticle can include from about 0.5 mol% to about 5 mol% (e.g. from about 1 mol% to about 4.5 mol%, from about 1.5 mol% to about 4 mol%, from about 2 mol% to about 3.5 mol%, from about 2.5 mol% to about 3 mol%, from about 0.5 mol% to about 3 mol%, from about 1 mol% to about 2.5 mol%, from about 1.5 mol% to about 2 mol%, from about 2.5 mol% to about 5 mol%, from about 3 mol% to about 4.5 mol%, from about 3.5 mol% to about 4 mol%) of the PEGylated lipid.

[0087] In some aspects, the ratio of PEGylated lipid to total lipids can be at least about 0.01:1 (e.g., at least about 0.015:1, at least about 0.02:1, at least about 0.025:1, at least about 0.03: 1, at least about 0.035: 1, at least about 0.04: 1, at least about 0.045: 1, at least about 0.05: 1).In some aspects, the ratio of PEGylated lipid to total lipids can be up to about 0.05:1 (e.g., up to about 0.045:1, up to about 0.04:1, up to about 0.035:1, up to about 0.03:1, up to about 0.025:1, up to about 0.02:1, up to about 0.015:1, up to about 0.01:1 )

[0088] It is considered that the ratio of PEGylated lipid to total lipids can range from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the ratio of PEGylated lipid to total lipids can be from about 0.01:1 to about 0.05:1 (e.g., from about 0.015:1 to about 0.045:1, from about 0.02:1 to about 0.04:1, from about 0.025:1 to about 0.035:1, from about 0.01:1 to about 0.03:1, from about 0.015:1 to about 0.025:1, from about 0.03:1 to about 0.05:1, from about 0.035:1 to about 0.045:1).

[0089] In some aspects, the ratio of MHC class I to total lipids can be about 0.3:1 or more (e.g., about 0.4:1 or more, about 0.5:1 or more, about 0.75:1 or more, about 1:1 or more, about 1.25:1 or more, about 1.5:1 or more, about 1.75:1 or more, about 2:1 or more, about 2.25:1 or more, about 2.5:1 or more, about 3:1 or more, about 3.5:1 or more, about 4:1 or more, about 4.5:1 or more, about 5: 1 or more, about 5.5:1 or more, about 6: 1 or more). In some aspects, the ratio of MHC class I to total lipids can be about 6:1 or less (e.g., about 5.5:1 or less, about 5:1 or less, about 4.5:1 or less, about 4: 1 or less, about 3.5:1 or less, about 3:1 or less, about 2.5:1 or less, about 2.25:1 or less, about 2:1 or less, about 1.75:1 or less, about 1.5:1 or less, about 1.25:1 or less, about 1:1 or less, about 0.75:1 or less, about 0.5:1 or less, about 0.4:1 or less, about 0.3:1 or less).

[0090] It is considered that the ratio of MHC class I to total lipids can range from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the ratio of MHC class I to total lipids can be from about 0.3:1 to about 6:1 (e.g., from about 0.4:1 to about 5.5:1, from about 0.5:1 to about 5:1, from about 0.75:1 to about 4.5:1, from about 1:1 to about 4:1, from about 1.25:1 to about 3.5:1, from about 1.5:1 to about 3:1, from about 1.75:1 to about 2.5:1, from about 2:1 to about 2.25:1, from about 0.3:1 to about 2.25:1, from about 0.4:1 to about 2:1, from about 0.5:1 to about 1.75:1, from about 0.75:1 to about 1.5:1, from about 1:1 to about 1.25:1, from about 2:1 to about 6:1, from about 2.25:1 to about 5.5:1, from about 2.5:1 to about 5:1, from about 3:1 to about 4.5:1, from about 3.5:1 to about 4:1).METHODS

[0091] In some aspects, disclosed herein is a method of killing an autoreactive immune cell, the method including exposing any of the disclosed APNs to said autoreactive immune cell.

[0092] In some aspects, the autoreactive immune cell can be a CD8+ T cell. In some aspects, the autoreactive immune cell can be a human immune cell. In some aspects, the method can be used to treat, inhibit, reduce, decrease, ameliorate, and / or prevent an autoimmune disease or disorder in a human subject. In some aspects, the APN can be administered to a subject having an autoimmune disease or disorder. Examples of autoimmune diseases or disorders include, but are not limited to, thrombocytopenia, chronic immune thrombocytopenia, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, pemphigus vulgaris, diabetes mellitus (e.g., juvenile diabetes or type 1 diabetes), Henoch-Schonlein purpura, post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitis obliterans, Sjogren's syndrome, primary biliary-cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis / dermatomyositis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis / polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, or fibrosing alveolitis.

[0093] In some aspects, also disclosed herein is a method of treating, inhibiting, reducing, decreasing, ameliorating and / or preventing diabetes (e.g., type 1 diabetes) in a subject, the method including administering to the subject an antigen-presenting nanoparticle (APN) including: a major histocompatibility complex (MHC) class I presented on a surface of the APN and including NRP-V7, IGRP, InsulinA, InsulinB, GAD65, chromogranin A, p31, p79, or any fragments thereof; and one or more encapsulated components which induce cell death.

[0094] Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disorder characterized by the immune-mediated destruction of insulin -producing P-cells in the pancreatic islets of Langerhans. This process is primarily driven by autoreactive T lymphocytes, particularly CD8+ T cells, which recognize P-cell-specific antigens and initiate an inflammatory response. The destruction of -cells leads to an absolute deficiency of endogenous insulin, a hormone critical for glucose homeostasis. The autoimmune response may also involve autoantibodies targeting islet cell antigens such as insulin, glutamic acid decarboxylase (GAD65), and islet antigen-2 (IA-2), which serve as biomarkers for disease prediction and diagnosis.

[0095] In some aspects, the self-epitope or mimotope thereof can include KYNKANVFL (SEQ ID NO: 14), VYLKTNVFL (SEQ ID NO: 15), LYLVCGERV (SEQ ID NO: 16), or any variants thereof. In some aspects, the APN can include any of the APNs disclosed herein.

[0096] In some aspects, the APN can be administered in a concentration of about 0.1 mg / kg or more (e.g., about 0.2 mg / kg or more, about 0.3 mg / kg or more, about 0.4 mg / kg or more, about 0.5 mg / kg or more, about 0.75 mg / kg or more, about 1 mg / kg or more, about 1.25 mg / kg or more, about 1.5 mg / kg or more, about 1.75 mg / kg or more, about 2 mg / kg or more, about 2.25 mg / kg or more, about 2.5 mg / kg or more, about 3 mg / kg or more, about 3.5 mg / kg or more, about 4 mg / kg or more, about 4.5 mg / kg or more, about 5 mg / kg or more). In some aspects, the APN can be administered in a concentration of about 5 mg / kg or less (e.g., about 4.5 mg / kg or less, about 4 mg / kg or less, about 3.5 mg / kg or less, about 3 mg / kg or less, about 2.5 mg / kg or less, about 2.25 mg / kg or less, about 2 mg / kg or less, about 1.75 mg / kg or less, about 1.5 mg / kg or less, about 1.25 mg / kg or less, about 1 mg / kg or less, about 0.75 mg / kg or less, about 0.5 mg / kg or less, about 0.4 mg / kg or less, about 0.3 mg / kg or less, about 0.2 mg / kg or less, about 0.1 mg / kg or less).

[0097] It is considered that the APN can be administered in a concentration ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the APN can be administered in a concentration of from about 0.1 mg / kg to about 5 mg / kg (e.g., from about 0.2 mg / kg to about 4.5 mg / kg, from about 0.3 mg / kg to about 4 mg / kg, from about 0.4 mg / kg to about 3.5 mg / kg, from about 0.5 mg / kg to about 3 mg / kg, from about 0.75 mg / kg to about 2.5 mg / kg, from about 1 mg / kg to about 2.25 mg / kg, from about 1.25 mg / kg to about 2 mg / kg, from about 1.5 mg / kg to about 1.75 mg / kg, from about 0.1 mg / kg to about 1.75 mg / kg, from about 0.2 mg / kg to about 1.5 mg / kg, from about 0.3 mg / kg to about 1.25 mg / kg, from about 0.4 mg / kg to about 1 mg / kg, from about 0.5 mg / kg to about 0.75 mg / kg, from about 1.5 mg / kg to about 5 mg / kg, from about 1.75 mg / kg to about 4.5 mg / kg, from about 2 mg / kg to about 4 mg / kg, from about 2.25 mg / kg to about 3.5 mg / kg, from about 2.5 mg / kg to about 3 mg / kg).

[0098] In some aspects, the APN can be repeatedly administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times at an interval of about every 5 days (e.g., about every 1 day, about every 2 days, about every 3 days, about every 4 days, about every 5 days, about every 6 days, about every 7 days, about every 8 days, about every 9 days, about every 10 days, about every 2 weeks, about every 3 weeks, about every 4 weeks).

[0099] The autoimmune destruction of pancreatic P-cells in type 1 diabetes results in an absolute deficiency of insulin, a hormone essential for facilitating the uptake of glucose intocells, particularly in muscle and adipose tissue. Without insulin, glucose cannot efficiently enter these cells and instead accumulates in the bloodstream, leading to persistent hyperglycemia. Additionally, insulin normally suppresses hepatic gluconeogenesis and glycogenolysis; in its absence, the liver continues to produce and release glucose unchecked, further exacerbating elevated blood glucose levels. The combined effects of impaired peripheral glucose uptake and unrestrained hepatic glucose output create a metabolic state characterized by chronic hyperglycemia, which is the hallmark of type 1 diabetes and the primary contributor to its acute complications, such as diabetic ketoacidosis (DKA), and longterm vascular damage. Accordingly, in some aspects, the method can be used to prevent onset of hyperglycemia for up to about 30 days or more (e.g., about 35 days or more, about 40 days or more, about 45 days or more, about 50 days or more, about 55 days or more, about 60 days or more, about 65 days or more, about 70 days or more, about 75 days or more, about 80 days or more, about 85 days or more, about 90 days or more).

[0100] In some aspects, the method can further include administering one or more additional treatments for diabetes, for example, insulin and / or teplizumab.EXAMPLESExample 1: Methods and compositions for in vivo depletion of auto reactive T cells to prevent type 1 diabetes

[0101] To improve the treatment precision for T1D, selective depletion of autoreactive P cell-specific T cells has been explored using peptide major histocompatibility complex (MHC) tetramers conjugated with toxin, lentiviral vectors encoding pro-apoptotic caspase 9 transgene, and engineered T cells targeting autoreactive T cells. While promising, the translation potential of a bacterial-derived streptavidin used in pMHC tetramers and lentiviral vectors could be limited by their immunogenicity which can trigger unintended immune responses and reduce efficacy upon repeat dosing. Furthermore, broad subject access to ex vivo engineered T cells is hindered by the complex and resource-intensive manufacturing process. Accordingly, there remains a need for an off-the-shelf therapeutic platform that permits repeated administration without inducing strong immune responses against foreign protein components (e.g., streptavidin) or triggering anti-viral immunity. Antigen-presenting nanoparticles (APNs) address this need by employing clinically validated lipid nanoparticles that are surface decorated with MHC proteins that are endogenous to the host, thereby enabling safe and durable depletion of autoreactive T cells.

[0102] Autoreactive antigen-specific T cells recognize their cognate target cells through T cell receptors (TCRs), which interact with specific self-peptide antigens that are presented onMHC molecules on the surface of the target cells. Mimicking this endogenous Ag-specific TCR-MHC interaction, a study was conducted which developed a delivery system, Ag-presenting lipid nanoparticles (APNs), which can deliver mRNA to engineer the cell fate and functions of antigen-specific T cell subsets in vivo (FIG. 1). The APNs are designed to deplete autoreactive P cell-specific T cells in vivo by selective delivery of pro-apoptotic caspase 6 (Casp6) mRNA to autoreactive P-islet specific T cells. The data below demonstrate that APNs eliminate autoreactive p cell-specific T cells while minimizing indiscriminate T cell modulation and avoiding off-target toxicity in liver and kidney. Moreover, the ability of APNs to preferentially deliver Casp6 to P-islet specific CD8 T cells prevents the onset of hyperglycemia in an aggressive adoptive transfer murine model of T1D. Considering the critical role of autoreactive cytotoxic CD8 T cells in other autoimmune diseases, such as multiple sclerosis, it is anticipated that APNs may be applicable to other cytotoxic T cell-driven autoimmune diseases.

[0103] High throughput ligand exchange by UV light to target p-islet autoreactive T cells: High-throughput refolding of class I peptide major histocompatibility complex (pMHCI) molecules can be achieved through UV-mediated ligand exchange, in which a sacrificial peptide is initially refolded to stabilize the pMHCI structure and then rapidly degrades when exposed to UV light, allowing peptides of interest to replace it in the binding groove. UV-labile peptides have been engineered to fit a variety of human heavy chain alleles (HLA-A1, -A3, -Al l, -B7) and murine heavy chain alleles (H2-Db, -Kb, -Ld). Here, the study engineered UV-labile peptides for the murine heavy chain H2-Kdallele by incorporation of a non-natural, U V-sensitive amino acid (abbreviated “J” in the amino acid sequence) into a well-established H2-Kd-restricted influenza peptide (SEQ ID NOs: 1-4). To test the efficacy of the Kd-restricted UV-labile peptides, the study used the TCR-transgenic NOD8.3 mouse model, in which every CD8 T cell expresses a TCR that recognizes the NRP-V7 peptide (KYNKANVFL, SEQ ID NO: 23) displayed in the KdMHC, and stained NOD8.3 splenocytes with conventionally refolded and UV-exchanged pMHCI-tetramers. The study observed a comparable percentage of NRP-V7+ CD8 T cells and mean fluorescent intensity of samples stained with conventionally refolded and UV-exchanged pMHCI-tetramers, indicating the UV-labile peptide facilitates efficient exchange of Kd-restricted peptides (FIGS.2A-2B). Next, the study sought to test the staining ability of UV-exchanged pMHCI-tetramers to identify rare endogenous T cells that recognize three p-islet antigens associated in T1D in the polyclonal non-obese diabetic (NOD) mouse model: NRP-V7, IGRP (VYEKTNVFL, SEQ ID NO: 24) and InsulinB (LYLVCGERV, SEQ ID NO: 25). After peptide-pulsed expansion of NODsplenocytes, the study stained cells with UV-exchanged pMHCI-tetramers, benchmarked against conventionally refolded pMHCI-tetramers. It was found that UV-exchanged pMHCI-tetramers showed comparable tetramer + T cell percentages as conventionally refolded pMHCI-tetramers (FIG. 2C). This approach can expedite the synthesis of Kd-pMHCI molecules with various T-cell targeting specificities, while also minimizing the necessary labor and resources.

[0104] Antigen-specific transfection of β-islet autoreactive T cells in vitro:. The study formulated APNs using a post-insertion method, where lipid-conjugated pMHCI molecules are mixed with preformed lipid nanoparticles (LNPs) similar in composition to the FDA-approved COVID-19 vaccines14. Briefly, the LNP core includes ALC-0315, cholesterol, DSPC, and ALC-0319 at a molar ratio of 46.3:42.7:9.4: 1.6, and the APNs are formulated with a MHC:lipid molar ratio from 0.3:1 to 6:1. The pMHCI molecules are engineered to express a free cysteine on the C-terminus of the KdMHC heavy chain (SEQ ID NO: 5), which is used to conjugate a lipid tail through thiol-maleimide chemistry. To test the ability of APNs to transfect therapeutically relevant autoreactive T cells, the study used transgenic NOD8.3 mice whose CD8+ T cells express a TCR that specifically recognizes NRP-V7, a peptide mimotope derived from the T ID-associated antigen IGRP. In the LNP core of APNs, the study encapsulated mRNA encoding one of three reporters — secreted nLuc, fluorescent protein mTagBFP, or membrane-bound VHH nanobody -to mimic functional proteins that are secreted, retained intracellularly, or cell-membrane bound (FIG. 3A). The study showed that Kd / NRP-V7 APNs transfected NOD8.3 CD8 T cells with all three mRNA constructs, whereas non-cognate APN treated groups showed minimal transfection (FIGS. 3B-3G). These data collectively support the activity of APNs in transfecting NOD8.3 CD8 T cells with mRNA encoding for proteins localized to unique cellular compartments in an antigen-specific manner.

[0105] Engineering pro-apoptotic mRNA to efficiently deplete mouse CDS T cells: To leverage the antigen- specific targeting of APNs for depletion of autoreactive cells, the study first identified a pro-apoptotic mRNA sequence that can effectively induce apoptosis in mouse CD8 T cells. To do this, the study designed and synthesized mRNA constructs encoding pro-apoptotic peptides (BIM (SEQ ID NO: 6), BID (SEQ ID NO: 7)) or proteins (granzyme B (SEQ ID NO: 8), Casp9 (SEQ ID NO: 9), as well as human and mouse Casp6 (SEQ ID NO: 10, SEQ ID NO: 11)). Endogenous pro-apoptotic proteins exist in a catalytically inactive form and require other proteases to cleave them to become active. To bypass this process, previous work has engineered human endogenous caspases to be constitutively active either through rearrangement of protein subunits (hCasp615) or engineering dimerization interfaces to encourage spontaneous dimerization necessary for activation (hCasp916). Using the sameprinciples, the study engineered constitutively active murine Casp6 and Casp9 to deplete primary mouse T cells and showed that Casp6 led to the highest cell death via electroporation (FIG. 4A) and retained its activity when delivered via LNP (FIG. 4B).

[0106] APNs selectively deplete P-isIet autoreactive T cells after adoptive cell transfer: To test the ability of APNs to deplete therapeutically relevant T cells, the study used an aggressive adoptive transfer model of T1D by adoptively transferring peptide-pulsed NOD8.3 T cells into host wildtype NOD mice to induce hyperglycemia. The study treated these mice with cognate APNs encapsulated with Casp6 (Kd / NRP-V7 Casp6 APNs) to deplete these autoreactive T cells (FIG.5A). It was found that cell death was mRNA- and pMHC -dependent, as cognate APNs loaded with non-relevant mRNA (Kd / NRP-V7 VHH APN) and non-cognate APNs loaded with Casp6 (Kd / Ctrl Casp6 APN) depleted less NOD8.3 T cells in the blood, spleen, and pLN, compared to Kd / NRP-V7 Casp6 APNs (FIG. 5B). As a positive control, the study also compared APN treatment to Fc-nonbinding aCD3 (aCD3) treatment by following preclinical dosing schemes of 2.5 mg / kg doses daily for five days in a row17. It was found that treatment with this αCD3 can deplete NOD8.3 T cells (FIG. 5B), but was not antigen-specific, leading to decreased total CD8 T cell percentages in the blood, spleen, and pLN, while Kd / NRP-V7 Casp6 APNs preserved the total CD8 T cell percentages (FIG.5C). Collectively, these data show that APNs can selectively deliver Casp6 to deplete autoreactive T cells, while avoiding the reduction of total CD8 T cell percentages in the major organs and tissues.

[0107] APNs prevent the onset of hyperglycemia after depletion of p-islet autoreactive T cells: To assess the therapeutic relevance of APN-mediated depletion of autoreactive T cells, the study tracked the blood glucose levels of host NOD mice after adoptive cell transfer of peptide-pulsed NOD8.3 T cells and treatment with APNs (FIG. 6A). The study showed that mice treated with Kd / NRP-V7 VHH APNs and Kd / Ctrl Casp6 APNs had comparable high blood glucose levels as untreated mice (PBS) (> 2.50 mg / dL is considered diabetic) (FIGS.6B-6C). However, mice treated with Kd / NRP-V7 Casp6 APNs maintained healthy blood glucose levels like the aCD3 treated mice and healthy mice (<250 mg / dL), furthering demonstrating the necessity for both cognate pMHC for T cell uptake and relevant mRNA for translation of Casp6 protein to induce cell death (FIGS. 6B-6C).

[0108] Prevention of hyperglycemia after APN treatment is durable: The study next wanted to test the durability of APN treatment with Kd / NRP-V7 Casp6 APNs compared to aCD3 when dosed similarly. The study matched the dosing strategy of aCD3 to APN treatment, administering 0.1 mg / kg on day 1 and day 4 after ACT of NOD8.3 T cells (FIG. 7A). The study tracked blood glucose levels of APN treated mice up to day 30 and blood glucose levelsremained stable (<2.50 mg / dL) (FIG. 7B), while all <xCD3 treated mice were hyperglycemic (>250 mg / dL) by day 14 (FIG. 7C). These data indicate that Kd / NRP-V7 Casp6 APNs can effectively and persistently prevent the onset of hyperglycemia up to 30 days post-treatment, whereas aCD3 at the same dose leaves mice vulnerable to hyperglycemia two weeks posttreatment.

[0109] APNs are well tolerated at the administered dose: Due the inherent cytotoxic nature of the mRNA being delivered in the Kd / NRP-V7 Casp6 APNs, the study sought to analyze how the APNs were tolerated in the mice. The study evaluated the compatibility of APNs using biochemical markers of liver function in the blood serum of mice, such as aspartate aminotransferase (AST) and alanine transaminase (ALT), and saw no significance between APN treated mice and healthy mice (FIG. 8A). The study also analyzed biochemical makers to evaluate toxicity to kidneys, such as total blood serum protein, blood urea nitrogen, creatinine, phosphorous, and calcium, and saw equivalent levels in the blood serum of APN treated mice and healthy mice (FIG. 8A). Additionally, the study did not observe any decline in body weight of all APN treated mice over 30 days (FIG. 8B). Taken together, these data demonstrate that Kd / NRP-V7 Casp6 APNs did not cause off-target toxicity in liver and kidney.Example 2: Additional methods and compositions for in vivo depletion of autoreactive T cells

[0110] Treatment with with Kd / NRP-V7 Casp6 APNs leads to durable control of hyperglycemia for over 300 days: Using an acute model of type 1 diabetes, a study treated mice following the dosing scheme described in FIG. 9A. Briefly, after adoptive cell transfer of NOD8.3 T cells, the study treated NOD wildtype mice with injections of Kd / NRP-V7 Casp6 APNs one day and four days after, for a total of two doses each at 0.1 mg / kg. As a positive control, the study followed the preclinical dosing scheme of Fc-nonbinding aCD3 antibodies at a dose of 2.5 mg / kg for a total of 5 daily injections after ACT of the NOD8.3 T cells. The study monitored the blood glucose levels and quantified the change in body weight of the mice that received the ACT of NOD8.3 T cells and were treated with either PBS, aCD3 antibodies, or Kd / NRP-V7 Casp6 APNs (FIGS.9B-9C). The study also tracked the body weight and blood glucose levels of NOD wildtype mice that did not receive any ACT of NOD8.3 T cells, which spontaneously develop diabetes with median female incidence at 18 weeks. It was observed that treatment with Kd / NRP-V7 Casp6 APNs can maintain normal glycemia levels in host mice up to 300 days after transfer of NOD8.3 T cells.

[0111] Dose escalation of NRP-V7 Casp6 APNs prevents the onset of hyperglycemia and is well tolerated at the given dose: To determine the safety profile of the Kd / NRP-V7Casp6 APNs, the study treated mice with increased doses, both in frequency and mass, to determine the efficacy and tolerability of treatment. Following the dosing scheme outlined in FIG. 10A, the study treated NOD wildtype mice that received ACT of NOD8.3 T cells for 5 daily doses of APNs, either at a dose of 0.1, 0.25, or 0.5 mg / kg. It was observed that mice treated with Kd / NRP-V7 Casp6 APNs at all 3 doses were able to maintain normal blood glucose levels after treatment (FIG. 10B) and survived up to 30 days after treatment (FIG.10C). Notably, mice treated with non-cognate APNs (Ctrl Casp6 APNs), did not survive treatment at the highest dose, highlighting the activity of the Casp6 mRNA as well as the necessity for targeted delivery to be well tolerated. The study monitored the body weight of mice treated with the highest dosing regimen (0.5 mg / kg) of the Kd / NRP-V7 Casp6 APNs and did not observe any decrease in body weight over the study duration (FIG. 11A). Additionally, the study analyzed the blood serum of these mice treated with 0.5 mg / kg of Kd / NRP-V7 Casp6 APN and did not observe any change in biochemical markers of organ function, such as alanine transaminase (ALT), aspartate aminotransferase (ALT), creatinine, and total protein (FIG.11B).

[0112] The following patents, applications and publications as listed below and throughout this document are hereby incorporated by reference in their entirety herein.Reference List1. Long, S. A. et al. Partial exhaustion of CD8 T cells and clinical response to teplizumab in new-onset type 1 diabetes. Science Immunology 1, eaai7793 (2016).2. Herold, K. C. et al. An anti-CD3 antibody, teplizumab, in relatives at risk for type 1 diabetes. New England Journal of Medicine 381, 603-613 (2019).3. Gaston, R. S. et al. OKT3 first-dose reaction: association with T cell subsets and cytokine release. Kidney international 39, 141-148 (1991).4. Keymeulen, B. et al. Transient Epstein-Barr virus reactivation in CD3 monoclonal antibody-treated patients. Blood, The Journal of the American Society of Hematology 115, 1145-1155 (2010).5. Junker, A., et al. Epstein-Barr virus infections following 0KT3 treatment. Transplantation 47, 574-575 (1989).6. Hess, P. R. et al. Selective deletion of antigen-specific CD8+ T cells by MHC class I tetramers coupled to the type 1 ribosome-inactivating protein saporin. Blood 109, 3300-3307 (2007).7. Goldberg, S. D. et al. A strategy for selective deletion of autoimmunity-related T cells by pMHC-targeted delivery. Pharmaceutics 13, 1669 (2021).8. Guo, X.-z. J. & Elledge, S. J. V-CARMA: A tool for the detection and modification of antigen-specific T cells. Proceedings of the National Academy of Sciences 119, e2116277119 (2022).9. Fishman, S. et al. Adoptive transfer of mRNA-transfected T cells redirected against diabetogenic CD8 T cells can prevent diabetes. Molecular Therapy 25, 456-464 (2017). 10. Bakker, A. H. et al. Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1,-A3,-A11, and-B7. Proceedings of the National Academy of Sciences 105, 3825-3830 (2008).11. Frickel, E.-M. et al. Parasite stage-specific recognition of endogenous Toxoplasma gondii -derived CD8+ T cell epitopes. The Journal of infectious diseases 198, 1625-1633 (2008).12. Grotenbreg, G. M. et al. Discovery of CD8+ T cell epitopes in Chlamydia trachomatis infection through use of caged class I MHC tetramers. Proceedings of the National Academy of Sciences 105, 3831-3836 (2008).13. Toebes, M. et al. Design and use of conditional MHC class I ligands. Nature medicine 12, 246-251 (2006).14. Polack, F. P. et al. Safety and efficacy of the BNT162b2 mRNA Covid- 19 vaccine. New England journal of medicine 383, 2603-2615 (2020).15. Srinivasula, S. M. et al. Generation of constitutively active recombinant caspases-3 and-6 by rearrangement of their subunits. Journal of Biological Chemistry 273, 10107-10111 (1998).16. Chao, Y. et al. Engineering a dimeric caspase-9: a re-evaluation of the induced proximity model for caspase activation. PLoS biology 3, e183 (2005).17. Chatenoud, L. et al. Anti-CD3 antibody induces long-term remission of overt autoimmunity in nonobese diabetic mice. Proceedings of the National Academy of Sciences 91, 123-127 (1994).SEQUENCESSEQ ID 1: UV-labile peptide sequences for murine MHC heavy chain H2-KdTYQJTRALVSEQ ID 2: UV-labile peptide sequences for murine MHC heavy chain H2-KdTYQRTJALVSEQ ID 3: UV-labile peptide sequences for murine MHC heavy chain H2-KdTYQRTRJLVSEQ ID 4: UV-labile peptide sequences for murine MHC heavy chain H2-KdTYQRTRAJVSEQ ID 5: Amino acid sequence of murine MHC heavy chain H2-Kdengineered with C-terminal cysteine MGPHSLRYFVTAVSRPGLGEPRFIAVGYVDDTQFVRFDSDADNPRFEPRAPWMEQE GPEYWEEQTQRAKSDEQWFRVSLRTAQRYYNQSKGGSHTFQRMFGCDVGSDWRLL RGY QQFA YDGRD YIALNEDLK T W TA ADTA Al 1'1 RRKWEQ AGD AEY YRAYLEGEC V EWT, RRYI. EI. GNETLLRTDSPKAHVTYHPRSQVDVTT. RCWALGFYPADITLTWQLNG EDLTQDMELVETRPAGDGTFQKWAAVVVPLGKEQNYTCHVHHKGLPEPLTLRWKL PPSGSCSEQ ID 6: BIM mRNA sequence GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGCU GCGACCCGAAAUCCGGAUCGCUCAGGAGCUCCGAAGAAUUGGCGAUGAGUUUA ACGAGUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCU CCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGGCGGC CGCSEQ ID 7: BID mRNA sequence GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGA AGAGAUCAUUCAUAAUAUCGCACGACACCUUGCACAAAUAGGCGAUGAAAUG GAUCACUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGGCGG CCGCSEQ ID 8: Mouse granzyme B mRNA sequence GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGAA AAUACUUUUGCUCCUCCUUACUCUUAGUUUGGCCAGUAGAACAAAAGCCGGAG AGAUAAUAGGUGGUCACGAGGUCAAACCCCACUCACGCCCUUACAUGGCCCUC UUGUCAAUAAAAGACCAGCAACCCGAGGCAAUAUGUGGUGGAUUUCUGAUCC GGG A AG A UUUCGUUU UGAC UGC UGC UC A UUGUG AGGGAUCC A UC AUG AAUGU AACACUCGGAGCACAUAACAUCAAAGAGCAGGAGAAAACUCAGCAAGUGAUU CCAAUGGUCAAGUGCAUACCUCAUCCAGAUUACAACCCCAAAACAUUUUCUAA UGAUAUUAUGCUGUUGAAAUUGAAGUCUAAGGCAAAACGAACAAGGGCAGUG CGCCCCCUUAACUUGCCUAGACGCAACGUUAAUGUUAAACCAGGUGACGUUUG CUAUGUUGCAGGGUGGGGAAGAAUGGCACCAAUGGGCAAGUAUUCAAAUACU CUCCAAGAGGUGGAGUUGACAGUCCAGAAAGAUAGAGAGUGCGAAUCCUACU UUAAGAACAGAUAUAACAAAACCAACCAGAUAUGCGCUGGCGAUCCAAAGAC AAAGAGAGCAUCCUUCCGGGGCGAUUCUGGGGGACCCUUGGUGUGUAAAAAG GUAGCCGCAGGAAUUGUUAGUUACGGCUAUAAGGACGGGUCACCUCCCCGCGC UUUCACAAAGGUAUCAUCAUUCCUUUCCUGGAUAAAAAAGACCAUGAAGUCA AGCUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCC C UUGC ACC UGUACC UC UUGGUCUUUGAAUAAAGCCUGAG UAGGAAGGCGGCCG CSEQ ID 9: Dimeric mouse caspase 9 mRNA sequence GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGA UGAAGCAGAUCGGC AACUGCUGAGGAGGUGCCGCGUUCGGCUGGUGAGUGAG CUUCAAGUCGCCGAACUCUGGGACGCACUCCUCAGUAGGGAACUGUUCACAAG GGACAUGAUCGAGGAUAUUCAACAAGCAGGCAGUGGGUCUCGACGCGACCAA GCCAGACAACUGGUCACCGACCUUGAAACUCGCGGUCGGCAAGCCCUUCCCCU GUUCAUAAGUUGCCUUGAAGACACAGGACAAGGAACUCUCGCAUCACUGCUUC AGUCAGGCCGCCAGGCAGCCAAACAAGAUCCAGAGGCUGUCAAACCACUUGAC CAUCUUGUGCCUGUGGUGCUUGGGCCAAUGGGUCUCACUGCCAAGGAACAGCG AGUCGUCAAGCUUGAUCCUUCCCAACCUGCCGUAGGCAAUCUUACACCCGUGG UGCUUGGCCCAGAGGAACUCUGGCCCGCACGGCUUAAACCUGAGGUCCUUCGGCCCGAAACUCCUCGACCAGUGGAUAUAGGUAGUGGUGGUGCACACGAUGUGU GUGUACCAGGAAAAAUCCGCGGCCACGCAGAUAUGGCAUAUACUCUUGAUAGC GAUCCUUGUGGCCACUGUCUCAUAAUUAAUAAUGUAAAUUUUUGUCCAUCCU CUGGUCUGGGGACUAGGACCGGAAGCAACCUGGAUCGAGAUAAGCUCGAACAC CGGUUCCGGUGGCUGCGAUUCAUGGUGGAAGUUAAGAACGAUCUGACAGCUA AAAAGAUGGUGACCGCAUUGAUGGAAAUGGCACAUCGAAACCACAGAGCCCUC GAUUGUUUCGUAGUGGUCAUUCUGUCUCAUGGCUGUCAAGCAUCUCACCUCCA AUUUCCCGGGGCUGUAUACGGGACUGACGGGUGCAGCGUGUCAAUCGAGAAA AUAGUCAAUAUUUUCAAUGGUUCCGGUUGCCCAUCUCUUGGCGGAAAGCCCAA GUUGUUUUUCAUACAAGCUUGCGGCGGGGAGCAGAAAGACCAUGGAUUCGAA GUGGCUUGUACUUCAAGUCAGGGCAGAACUCUCGACUCUGAUAGCGAACCAGA UGCUGUCCCAUAUCAAGAGGGCCCACGCCCUCUGGAUCAGCUGGAUGCUGUCA GUUCACUCCCAACCCCAAGUGAUAUUCUUGUUUCCUACAGCACCUUCCCAGGC UUUGUAUCUUGGCGGGAUAAGAAAAGCGGUAGCUGGUACAUAGAAACACUCG AUGGAAUACUGGAACAGUGGGCACGGUCCGAAGACCUUCAGUCUCUCCUGUUG CGCGUAGCAAACGCUGUUAGCGCAAAAGGAACCUACAAACAGAUACCAUGCAU CGUAUCAAUGUUGCGCAAGAAACUCUUCUUCAAGACUAGUUAAGCUGCCUUCU GCGGGGC UUGCC UUCUGGCC AUGCCCUUC UUC UCUCCCUUGCACCUGU ACCUC UUGGUC UUUG A AUA AAGCCUG AGU AGGA AGGCGGCCGCSEQ ID 10: Reverse human caspase 6 mRNA sequenceGGG A AA f A AGAGA GA A A AGA A GAG T A AG A AGA A AT A f’AAG AGCC ACC A'l GGT A GAAATAGATGCAGCCTCCGTTTACACGCTGCCTGCTGGAGCTGACTTCCTCATGT GTTACTCTGTTGCAGAAGGATATTATTCTCACCGGGAAACTGTGAACGGCTCATG GTACATTCAAGATTTGTGTGAGATGTTGGGAAAATATGGCTCCTCCTTAGAGTTC AC AGA ACTCCTC AC ACTGGTG A AC AGGA AAGTTTCTC A GCGCCGA GTGGACTTTT GCAAAGACCCAAGTGCAATTGGAAAGAAGCAGGTTCCCTGTTTTGCCTCAATGCT AACTAAAAAGCTGCATTTCTITCCAAAATCTAATCTCGAGCACCACCACCACCAC CACGTTGAAATTGATGGGGGATCCCCC ATGAGC TCGGCCTCGGGGCTCCGCAGG GGGCACCCGGCAGGTGGGGAAGAAAACATGACAGAAACAGATGCCTTCTATAA AAGAGAAATGTTTGATCCGGCAGAAAAGTACAAAATGGACCACAGGAGGAGAG GAATTGCTTTAATCTTCAATCATGAGAGGTTCTTTTGGCACTTAACACTGCCAGA AAGGCGGGGCACCTGCGCAGATAGAGACAATCTTACCCGCAGGTTTTCAGATCT AGGATTTGAAGTGAAATGCTTTAATGATCTTAAAGCAGAAGAACTACTGCTCAAAATTCATGAGGTGTCAACTGTTAGCCACGCAGATGCCGATTGCTTTGTG'rGTGTCTrCCTGAGCCATGGCGAAGGCAATCACATITATGCATATGATGCTAAAATCGAAA TTCAGACATTAACTGGCTTGTTCAAAGGAGACAAGTGTCACAGCCTGGTTGGAA AACCCAAGATAn ATCATCCAGGCATGTCGGGGAAACCAGCACGATGTGCCAG TCATrCCTn’GGATGTAGTAGAlTGATAATAGGCTGGAGCCTCGGTGGCCATGCT TCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCC GTGGTCTTTGAATAAAGTCTGAGTGGGCGGCSEQ ID 11: Reverse mouse caspase 6 mRNA sequence GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGU CGAGAUCGAUGCCGCUUCCGUUUACACACUUCCUGCUGGAGCUGACUUCCUUA UGUGUUAUUCAGUUGCAGAAGGAUAUUACAGUCAUAGAGAGACCGUCAAUGG GAGCUGGUACAUCCAGGACUUGUGUGAAAUGUUGGCACGAUAUGGAAGCUCC CUGGAAUUCACAGAGCUUCUUACACUGGUGAACAGGAAAGUGAGCCAACGAA GAGU AGAC UUC UGUAAAGACCCUGACGC AAUAGGUAAGAAAC AGGUACC AUG CUUUGCUAGCAUGUUGACAAAAAAGCUGCACUUCUGUCCAAAGCCUUCAAAAU UGGAACAUCACCAUCACCACCACGUGGAGAUUGAUGGAGGUUCUCCUGGUGGA GGCGGUUCCGGAGGUGGGGGGAGUGGCGGUGGAGGUUCUGGUGGAGGUGGUA GCAUGACUGAAACCGAUGGAUUCUAUAAGUCCCGCGAGGUUUUUGAUCCAGCC GAGCAAUACAAAAUGGAUCACAAACGGAGAGGCGUUGCACUCAUUUUUAACC ACGAGCGCUUUUUCUGGCAUCUCACACUGCCAGAAAGACGGGGCACAAAUGCC GACCGCGACA AUUUGAC AAGGAGAUUCUCCGACCUCGGUUUCGAGGUCA AGUG UUUCAACGAUUUGAGAGCCGAAGAACUCCUUCUCAAGAUCCAUGAGGUCAGCA CCAGUUCUCAUAUCGAUGCCGAUUGUUUCAUCUGCGUUUUUUUGAGCCACGGA GAAGGCAAUCAUGUGUACGCAUACGACGCAAAAAUCGAGAUCCAAACCCUGAC UGGCCUGUUUAAGGGUGACAAGUGUCAGUCUCUUGUCGGGAAGCCAAAAAUU UUCAUUAUACAAGCCUGUAGAGGAUCCCAACACGACGUACCAGUCGUUCCACU GGACAUGGUGGAUUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCU UCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGG AAGGCGGCCGCSEQ ID NO: 12 Human granzyme B mRNA sequence GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAA GAGCTCTCTGCTGATGAAATCAGACGGCGGAGATTGGCGAGGCTCGCAGGCGGTCAGACGTCCCAGCCCACCACTCCACTGACCTCTCCCCAGCGCGAGAATCCTCCTG GCCCACCAATCGCCGCTTCAGCACCAGGGCCGTCACAATCCCTTGGGCTGAACGT ACACAATATGACACCTGCCACCAGCCCGATTGGTGCCAGCGGCGTTGCACACAG ATCTCAGAGTAGCGAGGGAGTITCATCTCTGTCAAGCTCACCCAGCAACTCACTT GAGACACAGTCTCAAAGTCTTAGCCGCTCCCAGTCTATGGACATTGATGGTGTGA GCTGTGAAAAGTCAATGTCCCAAGTGGATGTGGATTCCGGAATTGAGAACATGG AAGTTGACGAGAACGACCGCAGGGAGAAGAGATCTCTGTCAGACAAAGAGCCA AGCAGTGGTCCTGAGGTATCTGAAGAGCAAGCCCTTCAATTGGTGTGTAAGATTT TCCGCGTGTCATGGAAGGACCGCGATCGGGACGTCATC1 CI GTCATCTCTCTC TGCTCAGTTCAAACAGAATCCCAAGGAAGTGTTTAGCGACTTCAAAGATCTGATT GGCCAGATCCTTATGGAAGTGCTCATGATGAGCACCCAGACACGGGACGAGAAC CCCTTTGCCTCTCTCACTGCTACATCCCAGCCGATAGCCGCTGCCGCCCGGTCCCC AGATAGGAACCTTCTGCTGAACACGGGGTCCAATCCCGGGACATCCCCCATGTTC TGCTCAGTGGCGAGCTTCGGAGCTAGCAGTCTCTCCAGCCTTTATGAAAGTTCAC CCGCTCCAACCCCATCAl CTGGAGCAGCGTGCCAGTGATGGGGCCTrCn GGC AAGCCCCTCCCGGGCCGCTTCCCAGCTGGCAGTGCCGAGTACACCTCTGTCCCCT CACAGCGCGGCTTCCGGAACAGCCGCCGGGTCCCAGCCCTCCTCACCACGGTAT AGACClTATACAGTGACCCATCCATGGGCrAGCAGTGGAGTTAGCA'rCCrGTCAA GCrCTCCCAGCCCACCTGCCCTrGCATCC'rCCCCACAGGCAG'rTCCTGCGAGCrC AAGTCGCCAACGCCCATCCAGTACAGGCCCTCCCCTGCCACCGGCTAGTCCCTCT GCTACCTCTAGGCGGCCTI’CTrCTCTGAGAATAAGCCCnrTCTGGGAGCGTCCG GGGGGGCTTCAAATTGGGACAGCTATAGCGACCATTTCACTATCGAGACATGCA AAGAGACCGATATGTTGAACTACCTCATCGAATGCTTTGACCGGGTGGGGATCG AGGAGAAAAAGGCCCCAAAGATGTGCTCTCAGCCAGCCGTGAGTCAACTTCTGA GCAACATTAGGTCCCAATGCATTTCTCACACCGCACTCG'rCClGCAGGGCTCAC'r GACGCAGCCAAGGTCACTCCAGCAGCCCAGCTTCCTGGTGCCCTACATGCTGTGT AGAAACTTGCCCTACGGGTTCATTCAGGAGCTGGTGCGAACCACGCATCAGGAC GAGGAGGTCTTCAAACAGArCrrCATACCAATTCTCCAGGGGCrGGCTCITGC'rG CTAAAGAGTGCAGCCTGGACAGCGACTACTTCAAGTATCCCCTGATGGCCCTCGG AGAACTGTGTGAGACCAAGTTTGGAAAGACACACCCCGTCTGTAATCTGGTAGC TTCTCTGCGCCTGTGGCTCCCTAAGTCCCTGTCTCCAGGCTGCGGACGAGAACTC CAGCGCCTGAGn’ATCTGGGGGCATrCTI’CTCCTI’CAGCGTGrnGCGGAGGATG ATGTCAAGGTGGTGGAAAAATATTTCTCCGGACCAGCTATAACCCTTGAGAACA CTCGCGTTGTATCACAGAGTTTGCAGCACTACCTGGAGCTTGGTAGACAGGAGCTGT1 AAGATTCTGCACTCCATCCTCCTGAACGGCGAGACCAGGGAGGCGGCTCTC AGTTACATGGCCGCTGTTGTGAACGCCAATATGAAGAAAGCACAGATGCAAACA GACGATAGGCTTGTCTCCACCGATGGATTTATGCTGAATTTTCTGTGGGTTCTCCA ACAGCTGTCCACCAAAATTAAACTTGAGACCGTTGACCCAACTTACATCTrTCAC CCTCGCTGTAGGATCACACTGCCGAACGACGAGACGAGGGTAAACGCGACCATG GAGGATGTTAATGACTGGCTGACTGAACTTTACGGCGACCAGCCACCTTTCAGCG AACCTAAGTTTCCAACTGAGTGTTTTTTCCTTACTCTGCATGCCCATCACCTGTCA ATCCTGCCAAGCTGCCGGAGGTATATCCGCCGCCTGCGAGCCATACGCGAACTG AATAGAACAGTCGAGGATCTGAAGAATAACGAATCTCAGTGGAAAGATTCACCT CTGGCCACACGGCATAGGGAGATGCTGAAAAGATGCAAAACACAACTGAAAAA AC1TGTGAGGTGCAAGGCATGCGCCGACGCTGGGCTGCTGGACGAATCCTTTCTT CGGAGGTGCCTCAATTTTTACGGCCTCTTGATCCAGCTGCTGCTTCGAATACTGG ATCCGGCCTATCCTGACATTACCCTCCCTCTGAACAGTGACGTCCCTAAGGTGTT TGCAGCGCTGCCTGAATTCTACGTGGAGGACGTGGCCGAGTTCCTGTTTTTTATA GTGCAGTACAGCCCACAGGCTCTCTACGAACCCTGTACCCAGGATATCGTGATGT TCCTGGTCGTAATGCTGTGCAATCAGAATTATATACGGAATCCATACTTGGTGGC AAAGCTTGTTGAGGTGATGTTCATGACCAATCCAGCCGTGCAGCCTCGCACACAG AAGTTCTTTGAGATGATAGAGAACCACCCTTTGAGCACTAAACTCTTGGTGCCTA GCCTCATGAAATn ATACTGACGTCGAACATACCGGCGCAACCAGCGAATTTTA CGATAAGTTTACTATTCGGTACCATATAAGCACTATCTTCAAGTCCCTCTGGCAG AATATrGCACACCATGGTACClTrATGGAGGAGl CAAl C'rGGTAAACAGTnG TGCGATACATCAACATGCTTATCAATGACACAACGTTTCTCCTCGACGAGAGCCT CGAATCACTTAAGCGAATACATGAGGTGCAGGAGGAGATGAAGAACAAAGAGC AATGGGATCAGCTCC: CAGAGATCAGCAGCAGGC(: GGCAATCTCAACTGGCTC AGGACGAGCGGG'rGTCCCGATClTATCTGGCCCTCGCCACAGAGACCG'rAGACATGTTCC AT ATTCTT ACC AAGC AGGTGC A A A A ACC AIT1 "CTTAGGCC AGA GCTGGG ACCACGCCTGGCTGCTATGCTGAATTTCAACCTCX^AGCAGCTTTGTGGGCCGAAA TGTAGGGACCTGAAGGTAGAAAATCCCGAGAAGTACGGC1XTGAACCCAAAAAG TTGCTGGACCAGCTGACAGATATATATCTCCAACTGGACTGCGCTCGATTCGCAA AAGCCATTGCTGACGATCAGCGCAGCTACTCTAAGGAACTGTTTGAGGAGGTCA TTAGTAAGATGAGAAAGGCTGGTATCAAGTCCACTATAGCAATCGAGAAGTTCA AACrGCTGGCAGAGAAAGrGGAGGAGATrGTGGCCAAAAATGCCAGGGCCGAA ATAGACTATTCCGACGCCCCAGATGAGTTTCGCGACCCGTTGATGGACACACTTA TGACTGACCCCGTGAGGTTGCCTTCTGGCACAATTATGGACAGATCAATAATTCTGAGACACCTGCTGAACAGCCCAACAGATCC1TTCAACAGGCAGACTITGACGGA ATCAATGCTGGAACCCGTGCCCGAGClTAAAGAGCAGATrCAGGCATGGATGCG AGAGAAACAGAATTCCGACCACTAAGCTGCCTTCTGCGGGGCTTGCCTTCTGGCC ATGCCCTTCITCTCTCCCITGCACCTGTACCTCTTGG'rCTTTGAATAAAGCCTGAG TAGGAAGGCGGCCGCSEQ ID NO: 13 Dimeric human caspase 9 mRNA sequence GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGA CGAAGCUGAUAGACGCCUUCUUCGGAGGUGCAGACUUCGCCUCGUGGAGGAAC UGCAAGUAGAUCAGCUGUGGGACGCCCUCCUUAGCCGGGAACUGUUCCGGCCA CACAUGAUUGAGGAUAUCCAGAGGGCUGGGUCAGGAAGCAGGAGAGAUCAGG CC AG AC AGCUC AUU A UCGA UCUGG AGAC AC GAGGC AGCC AGGC UC UGCC AC UC UUCAUUUCCUGCCUUGAAGACACUGGCCAGGAUAUGCUGGCCUCAUUCCUGAG AACUAACCGACAAGCUGCGAAGCUGUCCAAGCCCACAUUGGAGAAUCUGACCC CAGUAGUCCUUCGGCCUGAGAUCAGGAAACCUGAAGUGCUCCGGCCAGAGACC CCUCGGCCGGUUGACAUCGGAUCAGGGGGCUUUGGCGACGUUGGCGCUCUGGA AUCACUCAGAGGUAACGCAGACCUGGCGUACAUCCUCUCUAUGGAGCCUUGCG GACACUGCUUGAUCAUCAAUAAUGUGAACUUCUGUCGCGAAAGUGGCUUGCG AACUAGAACAGGCAGCAAUAUCGACUGCGAAAAGCUCAGACGGCGGUUUUCA AGCCUGCACUUUAUGGUGGAGGUGAAGGGUGACCUGACUGCCAAGAAAAUGG UUCUCGCUCUCCUGGAGCUGGCACAGCAGGACCAUGGCGCUCUGGACUGUUGU GUGGUGGUGAUUCUUAGCC AUGGGUGUC AGGCCUCCCACCUGCAGUUCCC AGG CGCGGUGUACGGUACGGAUGGCUGUCCCGUCAGCGUGGAAAAGAUAGUCAAC AUUUUUAACGGGACAUCUUGCCCAAGCCUCGGGGGUAAGCCCAAGCUGUUCUU UAUUCAGGCCUGCGGCGGUGAGCAGAAGGACCACGGUUUCGAAGUGGCCUCUA CAUCUCCUGAAGAUGAGUCACCUGGAAGCAACCCUGAGCCUGAUGCCACCCCA UUUCAGGAGGGUUUGAGGACCUUUGACCAAUUGGACGCGAUCAGUUCUCUGC CUACACCAAGCGACAUUUUUGUUAGUUACAGUACAUUUCCUGGGUUCGUGAG C UGGAG AGACCC A A AGUC AGGGUC AUGGUA UGUGG AGAC GCUC GAC GAC A UC UUCGAGCAGUGGGCUCAUAGCGAAGACCUGCAGUCUCUCCUUCUGAGAGUCGC AAAUGCUGUGUCUGUGAAAGGCAUCUAUAAGCAGAUGCCAUGCAUCGUUUCC AUGCUGAGGAAGAAGCUCUUCUUCAAGACGUCCUAAGCUGCCUUCUGCGGGGC UUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCU UUGAAUAAAGCCUGAGUAGGAAGGCGGCCGC

Claims

CLAIMS1. An antigen-presenting nanoparticle (APN), comprising:a major histocompatibility complex (MHC) class I comprising a self-epitope or a mimotope thereof, wherein the MHC class I is presented on a surface of the APN; and one or more encapsulated components which induce cell death.

2. The APN of claim 1, wherein the one or more encapsulated components comprise a nucleic acid encoding an enzyme or fragment thereof which induces cell death.

3. The APN of claim 2, wherein the enzyme or fragment thereof is caspase, BIM, BID, granzyme B, PUMA, or gasdermin D.

4. The APN of claim 3, wherein the nucleic acid encoding an enzyme or fragment thereof which induces cell death comprises about 80% similarity or more to any one of SEQ ID NOS: 6-13.

5. The APN of any one of claims 1-4, wherein the one or more encapsulated components comprise one or more toxins.

6. The APN of any one of claims 1-5, wherein the self-epitope or mimotope thereof comprises NRP-V7, IGRP, InsulinA, InsulinB, GAD65, chromogranin A, p31, p79, or any fragments thereof.

7. The APN of claim 6, wherein the self-epitope or mimotope thereof comprises KYNKANVFL (SEQ ID NO: 14), VYLKTNVFI., (SEQ ID NO: 15), LYLVCGERV (SEQ ID NO: 16), or any variants thereof.

8. The APN of any one of claims 1-7, wherein the APN is a lipid nanoparticle, a liposome, or a polymeric nanoparticle.

9. The APN of claim 8, wherein the APN comprises at least one ionizable lipid, cholesterol, phospholipid, PEGylated lipid, or a combination thereof.

10. The APN of claim 9, wherein the at least one ionizable lipid comprises cKK-E12, SM102, MC3, Lipid-5, Alc-0315, LP01, Lipid A9, or any combination thereof.

11. The APN of any one of claims 9-10, wherein the APN comprises from about 30 mol% to about 60 mol% of the ionizable lipid.

12. The APN of any one of claims 9-11, wherein the APN comprises from about 35 mol% to about 50 mol% cholesterol.

13. The APN of any one of claims 9-12, wherein the at least one phospholipid comprises DSPC, DOPE, or any combination thereof.

14. The APN of any one of claims 9-13, wherein the APN comprises from about 5 mol% to about 20 mol% of the phospholipid.

15. The APN of any one of claims 9-14, wherein the at least one PEGylated lipid comprises ALC-0159, DMG-PEG, DSPE-PEG, PEG14-2000, or any combination thereof.

16. The APN of any one of claims 9-15, wherein the APN comprises from about 0.5 mol% to about 5 mol% of the PEGylated lipid.

17. The APN of any one of claims 9-16, wherein a ratio of PEGylated lipid to total lipids is from about 0.01: 1 to about 0.0: 1.

18. The APN of any one of claims 9-17, wherein the ratio of MHC class 1 to total lipids is from about 0.3:1 to about 6:1.

19. A method of killing an autoreactive immune cell, the method comprising exposing the APN of any one of claims 1-18 to said autoreactive immune cell.

20. The method of claim 19, wherein the autoreactive immune cell is a CD8+ T cell.

21. The method of any one of claims 19-20, wherein the autoreactive immune cell is a human immune cell.

22. The method of any one of claims 19-21, wherein the APN is administered to a subject having an autoimmune disease or disorder.

23. A method of treating and / or preventing diabetes in a subject, the method comprising administering to the subject the APN of any one of claims 1-18, wherein the self-epitope or niiniotope thereof comprises NRP-V7, IGRP, InsulinA, InsulinB, GAD65, chromogranin A, p31, p79, or any fragments thereof.

24. The method of claim 23, wherein the APN is administered in a concentration of from about 0.1 mg / kg to about 5 mg / kg.

25. The method of any one of claims 23-24, wherein the APN is repeatedly administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times at an interval of about every 5 days.

26. The method of any one of claims 23-25, wherein the method is used to prevent onset of hyperglycemia for up to about 30 days or more.

27. The method of any one of claims 23-26, wherein the self-epitope or mimotope thereof comprises KYNKANVFL (SEQ ID NO: 14), VYLKTNVFL (SEQ ID NO: 15), LYLVCGERV (SEQ ID NO: 16), or any variants thereof.

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