Immune modulation for the treatment of type 1 diabetes

Abstract

Provided herein are compositions comprising a functionalized pancreatic extracellular matrix (CP-PAN-ECM) comprising at least one immune checkpoint molecule. Also provided herein are methods of making these compositions and using these compositions for the treatment of diabetes.

Description

IMM UNE MODULATION FOR THE TREATMENT OF TYPE 1 DIABETESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 543,368, filed October 10, 2023, the contents of which are hereby incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] The present disclosure relates to compositions, methods of making and using, pancreatic extracellular matrix comprising immune checkpoint molecules. In one aspect, the disclosure relates to medical treatments using the disclosed composition and methods.BACKGROUND OF THE INVENTION

[0003] Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease that is characterized by hyperglycemia resulting from beta cell loss. Today, approximately 1.25 million Americans have T1DM, with the number of newly diagnosed T1DM cases increasing.

[0004] Most T1DM patients maintain their blood glucose levels using multiple insulin injections per day or through insulin-pump therapy. Still, less than a third of the T1DM patients consistently achieve their target blood glucose levels. Despite major advances in disease management and care, T1DM remains associated with a considerably higher probability that patients will develop acute diseases like neuropathy, nephropathy, retinopathy, and cardiovascular disease, along with a higher rate of premature death than in the general population. There is considerable interest in the development of new immunotherapy strategies for delaying and even reversing early-onset T1DM because a substantial mass of b cells is still present at the early-symptomatic stages. This can allow the patient to regain metabolic control.

[0005] In recent years, there has been increasing interest in developing other treatments to slow and reverse the progression of T1DM at the dysglycemia and early hyperglycemia stages by preserving insulin-producing islets and suppressing pancreas- infiltrated autoreactive T cells.SUMMARY

[0006] In an aspect, the current disclosure encompasses a functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently attached to a pancreatic extracellular matrix (PAN-ECM). An immune checkpoint molecule may comprise, for example PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFP, FGL1, or CD96. In an aspect, the at least one immune checkpoint molecule is attached to the PAN-ECM using click chemistry. In an aspect, the at least one immune checkpoint molecule is attached to the PAN-ECM via an alkyne-azide cycloaddition (SPAAC) chemistry. In an aspect, the at least one immune checkpoint molecule comprises a dibenzocyclooctyne moiety. In an aspect, the dibenzocycloctyne moiety is DBCO, or a derivative thereof, or a conjugate thereof. In an aspect, the immune checkpoint molecule is a bioconjugate comprising a dendrimer, a linear polymer, a nanoparticle, or a Fc fusion protein.

[0007] In an aspect, the current disclosure also encompasses a composition comprising a CP-PAN-ECM as disclosed herein. In an aspect, the current disclosure encompasses a functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN- ECM comprises at least one immune checkpoint molecule covalently attached to a pancreatic extracellular matrix (PAN-ECM). In an aspect, the composition is a pharmaceutical composition. In an aspect, the composition further comprises a pancreatic or an immune cell. In an aspect, the cell is a pancreatic cell for example a beta cell (P cells). In some aspects, the pancreatic or an immune cell comprises at least one immune checkpoint molecule covalently attached to the surface of the pancreatic or immune cell. In some aspects, the immune checkpoint molecule is selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1, and CD96. In some aspects, the immune checkpoint molecule is covalently attached to the surface of the pancreatic or immune cell by metabolic glycoengineering. In an aspect, the pharmaceutical composition comprises at least one excipient, or at least one additional therapeutic agent or combinations thereof. In an aspect, the composition is for use in the prevention or treatment of diabetes.

[0008] In an aspect, the current disclosure also encompasses a method of making a functionalized pancreatic extracellular matrix (CP-PAN-ECM), comprising: providing a functionalized PAN-ECM; providing a functionalized immune checkpoint molecule; contacting the functionalized PAN-ECM with the immune checkpoint molecule to form the CP-PAN-ECM using an alkyne-azide cycloaddition (SPAAC) chemistry. In an aspect, the functionalized PAN-ECM comprises an azide moiety. In an aspect, the functionalized immune checkpoint molecule comprises a dibenzocycloctyne moiety. In an aspect, the dibenzocycloctyne moiety comprises a DBCO, a or a derivative thereof, or a conjugate thereof. In an aspect, the immune checkpoint molecule is a bioconjugate comprising a dendrimer, a linear polymer, a nanoparticle, or a Fc fusion protein.

[0009] In an aspect, the current disclosure also encompasses a method of treating or reducing the progression of diabetes in a subject in need thereof, comprising administering to the subject a composition as disclosed herein. In an aspect, the subject is a mammal. In an aspect, the administering is done via the intravenous route. In an aspect, the diabetes is an autoimmune diabetes.

[0010] In an aspect, the current disclosure encompasses a functionalized pancreatic extracellular matrix comprising a structure: (PAN-ECM) — (a residue of an azide- containing molecule) — (a residue of a cyclooctyne) — (a linker 1) — (a residue of a functionalized dendrimer)q — (a residue of an immune checkpoint molecule), wherein, q is one or zero; and, the dash represents a covalent bond.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Aspects of the present disclosure are illustrated by way of example in which like reference numerals indicate similar elements and in which:

[0012] FIG. l is a schematic illustration of the bioengineering of checkpoint- functionalized CP-PAN-ECM (top) and the mechanism of action of the s.c. -injected P cells plus CP-PAN-ECM (bottom).

[0013] FIGS. 2A-2D shows line graphs displaying blood glucose concentrations of diabetic NOD mice after s.c. injection of the PD-L1 (FIG. 2A), HVEM (FIG. 2B), FasL (FIG. 2C), or PD-Ll / HVEM / FasL (FIG. 2D) -functionalized PAN-ECM plus p cells, (n = 4 mice per group).

[0014] FIG. 3A shows a schematic diagram of the treatment timeline for NOD mice with engineered mono- or trifunctionalized-PAN ECM plus NIT-1 cells (I MO6).

[0015] FIG. 3B shows line graphs displaying blood sugar level fluctuations of diabetic NOD mice after subcutaneous (s.c.) injection of the unmodified or different monofunctionalized-PAN-ECM plus NIT-1 cells.

[0016] FIG. 3C shows line graphs displaying blood sugar level fluctuations of diabetic NOD mice after s.c. injection of the unmodified or different trifunctionalized-PAN-ECM plus NIT-1 cells.

[0017] FIG. 4A displays survival curves of NOD mice after treatment with unmodified or different monofunctionalized-PAN-ECM plus NIT-1 cells.

[0018] FIG. 4B shows survival curves of NOD mice after treatment with unmodified or different trifunctionalized-PAN-ECM plus NIT-1 cells.

[0019] FIG. 5A is a line graph showing blood glucose levels of NOD mice after treatment with FasL / GITRL / TGFp / HVEM-functionalized PAN-ECM plus PD-L1-NIT-1 cells.

[0020] FIG. 5B shows survival curves of NOD mice after treatment with FasL / GITRL / TGFp / HVEM-functionalized PAN-ECM plus PD-L1-NIT-1 cells.

[0021] FIG. 6A displays fluorescence activated cell sorting (FACS) plots quantifying the level of surface PD-L1, HVEM, FasL and GITRL of different mono-functionalized NIT-1 cells.

[0022] FIG. 6B is a schematic diagram displaying an experimental workflow to detect the killing of Engineered NIT-1 cells by islet antigen specific 8.3 CD8+ T cells in the presence of exogenous IGRP peptide.

[0023] FIG. 6C shows bar graphs displaying the percentage of cytolysis of different mono-functionalized NIT-1 cells after incubation with expanded 8.3 T cells at E:T = 5: 1 in the presence of the IGRP peptide for 18 h, as determined by the CytoTox nonradioactive cytotoxicity assay (n = 4).

[0024] FIG. 6D shows bar graphs displaying the percentage of cytolysis of the HVEM- or PD-L1 or HVEM / PD-L1 -functionalized NIT-1 cells after incubation with expanded 8.3 T cells at E:T = 5: 1 in the presence of the IGRP peptide for 18 h, as determined by the CytoTox nonradioactive cytotoxicity assay (n = 4).

[0025] FIG. 7A is a schematic diagram displaying the treatment timeline for NOD mice with Engineered PAN ECM plus Engineered NIT-1 cells (0.5 * 106).

[0026] FIG. 7B shows line graphs displaying blood sugar level fluctuations of diabetic NOD mice after subcutaneous (s.c.) injection of the PD-Ll / HVEM-functionalized PAN- ECM plus NIT-1 cells.

[0027] FIG. 7C shows line graphs displaying blood sugar level fluctuations of diabetic NOD mice after subcutaneous (s.c.) injection of the HVEM-functionalized PAN-ECM plus PD-L1 -functionalized NIT-1 cells.

[0028] FIG. 8 is a schematic diagram displaying a method to direct differentiation of mouse embryonic stem cells (mESCs) into insulin-secreting islet-like clusters (IPCs).

[0029] FIG. 9A is a bar graph displaying insulin secretion (pIU / 104cells) in low glucose and high glucose conditions from mESCs, islet cells, and islet-like clusters (IPCs). Data for each cell type is displayed, from left to right, as the low glucose condition followed by the high glucose condition.

[0030] FIG. 9B shows brightfield light microscopy imaging of dithizone-stained mESCs, a typical mouse islet and IPCs. Scale bar = 100 pm.

[0031] FIG. 9C displays fluorescence activated cell sorting (FACS) plots quantifying insulin (647) and glucagon (FITC) in mESCs, islet cells, and islet-like clusters (IPCs).

[0032] FIG. 10A is a schematic diagram displaying the functionalization of IPCs through metabolic glycoengineering followed by alkyne-azide cycloaddition (SPAAC) with DBCO-functionalized PD-L1 and HVEM.

[0033] FIG. 10B displays fluorescence activated cell sorting (FACS) plots quantifying PD-L1 and HVEM expression in unmodified IPCs and engineered IPCs functionalized with PD-L1 and HVEM.

[0034] FIG. 10C is a schematic diagram displaying a therapeutic treatment schedule with Engineered IPCs in allogeneic BABL / c diabetic mice following injection with Streptozotocin (STZ) to induce diabetes.

[0035] FIG. 10D displays line graphs showing blood glucose levels in allogeneic BABL / c diabetic mice after subcutaneous (s.c.) injection of PBS, IPCs + PAN-ECM and HVEM / PD-Ll-IPCs + PAN ECM.

[0036] FIG. 10E displays fluorescence activated cell sorting (FACS) plots quantifying the expression of insulin and glucagon from mice treated with IPCs + PAN-ECM and HVEM / PD-Ll-IPCs + PAN ECM on day 30.

[0037] The drawing figures do not limit the present disclosure to the specific aspects disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed on clearly illustrating principles of certain aspects of the present disclosure.DETAILED DESCRIPTION

[0038] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects and aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.

[0039] In an aspect, the present disclosure is a result of the surprising discovery that pancreatic extracellular matrix (PAN-ECM) when covalently linked to immune checkpoint molecules and injected into mice can produce strong antigen-specific effector T cell suppression evoked by the interaction of PAN-ECM with diabetogenic T cells. The deficiency or mutation of co-inhibitory checkpoints is often associated with the autoimmune diabetes progression. Introducing some co-inhibitory checkpoint molecules to the islet can attenuate diabetogenic T cells. Thus, reversal of early onset hyperglycemia could be achieved. It was previously found that pancreatic beta (P) cells containing sufficient co-inhibitory checkpoint molecules can reverse early-onset hyperglycemia in NOD mice by attenuating the activity of diabetogenic T cells. In this work, it was found that the checkpoint molecules-bearing pancreatic extracellular matrix (PAN-ECM) can also effectively slow the progression of hyperglycemia and prolong the overall survival. Nevertheless, there are many checkpoint molecules that play an important role in immune homeostasis, and then a combination of different checkpoint molecules may have a better reversal effect on new-onset diabetes. To achieve this goal, PAN-ECM were engineered with different checkpoint molecules, and then an immunogenic pancreas microenvironment was created by subcutaneously injecting the PAN-ECM mixed with P cells proximal to the pancreatic lymph nodes.I. Terminology

[0040] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present disclosure or the appended claims.

[0041] Further, as the present disclosure is susceptible to aspects of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present disclosure and not intended to limit the present disclosure to the specific aspects shown and described. Any one of the features of the present disclosure may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and / or the like in the description mean that the feature and / or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect,” “aspects,” and / or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present disclosure may include a variety of combinations and / or integrations of the aspects described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present disclosure will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be encompassed by the claims.

[0042] Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater orone third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1%, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1%, such as less than or equal to ± 0.05%.

[0043] The terms "comprising," "including" and "having" are used interchangeably in this disclosure. The terms "comprising," "including" and "having" mean to include, but not necessarily be limited to the things so described.

[0044] Lastly, the terms “or” and “and / or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B or C” or “A, B and / or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.II. CompositionsFunctionalized pancreatic extracellular matrix

[0045] In an aspect, the current disclosure encompasses a functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule, or a functional fragment thereof, covalently attached to a pancreatic extracellular matrix (PAN-ECM). As used herein a functionalized pancreatic extracellular matrix comprises isolated acellular pancreatic extracellular matrix or components thereof, further comprising at least one covalently attached immune checkpoint molecules. In an aspect, the immune checkpoint (CP) molecule is attached using functionalized CP and functionalized PAN-ECM, each comprising a moiety suitable and complementary to each other for a click chemistry reaction.

[0046] In an aspect, the immune checkpoint molecule is PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7- H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGF|3, FGL1, or CD96. In one aspect, the immune checkpoint molecule is PD-L1, or HVEM. In one aspect, the functionalized PAN-ECM comprises at least one PD-L1, or at least one HVEM, or both.

[0047] In an aspect, the immune checkpoint molecule can be a fusion protein, for example, PD- LI can be a PD-Ll-Ig.

[0048] PD-L1, Programmed death-ligand 1 (Uniprot: Q9NZQ7), is a 40kDa type 1 transmembrane protein. PD-L1 is a ligand for PD-1. PD-L1 is also known as B7-H1 (B7 homolog 1).

[0049] HVEM (UniProtKB / Swiss-Prot: Q92956) is encoded by the gene TNFRSF14. The encoded protein functions in signal transduction pathways that activate inflammatory and inhibitory T-cell immune response. It binds herpes simplex virus (HSV) viral envelope glycoprotein D (gD), mediating its entry into cells. Alternative splicing results in multiple transcript variants

[0050] CD86, T-lymphocyte activation antigen CD86 (Uniprot: P42081), is a type I membrane protein. CD86 is a ligand for CTLA-4 in activated T cells. CD86 (along with CD80) provides costimulatory signals necessary for T-cell activation and survival.

[0051] Gal-9, Galectin 9 (Uniprot: 000182) is a 36 kDa beta-galactoside lectin protein. Gal-9 is a ligand for TIM-3.

[0052] In an aspect, the subject matter described herein is directed to a functionalized CP-PAN-ECM comprising a structure: (PAN-ECM) — (a residue of an azide-containing molecule) — (a residue of a cyclooctyne) — (a linker 1) — (a residue of a functionalized dendrimer)q — (a residue of an immune checkpoint molecule), wherein, q is one or zero; and, the dash represents a covalent bond. In an aspect the cyclooctyne moiety is a residue from a dibenzocyclooctyne for example DBCO. When q is zero, the dendrimer is absent which results in the DBCO direct conjugation strategy. As used herein, the term “residue” or “residue of’ a chemical moiety refers to a chemical moiety that is bound to a molecule, whereby through the binding, at least one covalent bond has replaced at least one atom of the original chemical moiety, resulting in a residue of the chemical moiety in the molecule.

[0053] In another aspect, the subject matter described herein is directed to a functionalized CP-PAN-ECM comprising a structure: (PAN-ECM) — (a residue of an azide-containing molecule) — (a residue of a cyclooctyne) — (a linker 1) — (a residue of a functionalized dendrimer)q — (immune checkpoint molecule Fclg fusion protein), wherein, the dash represents a covalent bond. In another aspect, the subject matter described herein is directed to a functionalized CP-PAN-ECM comprising a structure:(PAN-ECM) — (a residue of a cycoloctyne-containing molecule) — (a residue of a azide) — (a linker 1) — (immune checkpoint molecule Fclg fusion protein), wherein, the dash represents a covalent bond. In aspects, the immune checkpoint molecule / immune checkpoint molecule Fclg fusion protein can be conjugate via amine-NHS ester chemistry, or thiol -maleimide chemistry. In another aspect, the subject matter described herein is directed to a functionalized CP-PAN-ECM comprising a structure: (PAN-ECM) — (a residue of an azide-containing molecule) — (a residue of a cycoloctyne) — (a nanoparticle) — ((a linker, such as linker 1) — (immune checkpoint molecule))y)x, wherein, the dash represents a covalent bond and x and y are as described herein.

[0054] In various aspects, the current disclosure encompasses a functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule, or a functional fragment thereof, covalently attached to a pancreatic extracellular matrix (PAN-ECM), such as, for example, about 2, about 3, about 4, or about 5 immune checkpoint molecules.

[0055] In some aspects, the current disclosure encompasses a functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises about two immune checkpoint molecules, or functional fragments thereof, covalently attached to a pancreatic extracellular matrix (PAN-ECM). In some aspects, the about two immune checkpoint molecules are selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7- H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFP, FGL1, and CD96. In some aspects, the CP-PAN-ECM comprises PD-L1 and HVEM covalently attached to a PAN-ECM.

[0056] In some aspects, the current disclosure encompasses a functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises about three immune checkpoint molecules, or functional fragments thereof, covalently attached to a pancreatic extracellular matrix (PAN-ECM). In some aspects, the about three immune checkpoint molecules are selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7- H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFP, FGL1, and CD96. In some aspects, the CP-PAN-ECM comprises PD-L1, FGL1, and TGFP covalently attached to a PAN-ECM. In some aspects, the CP-PAN-ECMcomprises FasL, HVEM, and FGL1 covalently attached to a PAN -ECM. In some aspects, the CP-PAN-ECM comprises FasL, TGFp, and PD-L1 covalently attached to a PAN- ECM. In some aspects, the CP-PAN-ECM comprises FasL, GITRL, and TGFp covalently attached to a PAN-ECM. In some aspects, the CP-PAN-ECM comprises PD- Ll, HVEM, and FasL covalently attached to a PAN-ECM.

[0057] In other aspects, the current disclosure encompasses a functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises about four immune checkpoint molecules, or functional fragments thereof, covalently attached to a pancreatic extracellular matrix (PAN-ECM). In some aspects, the about four immune checkpoint molecules are selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7- H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFP, FGL1, and CD96. In some aspects, the CP-PAN-ECM comprises FasL, GITRL, TGFp, and HVEM covalently attached to a PAN-ECM.

[0058] In aspects, thiol-maleimide click chemistry can be used to modify the PAN- ECM. Generally, free thiol groups on the surface can be made to react with maleimide- functionalized biomolecule through stable thioester bond to form stable functionalized PAN-ECMs. Maleimide-functionalized biomolecules can be prepared by amine-NHS reaction between desired biomolecule and NHS-maleimide crosslinker (e.g, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate (sulfo-SMCC)).

[0059] In some aspects, the subject matter described herein is directed to a functionalized CP- PAN-ECM, wherein the residue of a functionalized dendrimer has the structure: — (dendrimer) — (a linker 2) — (a residue of a cyclooctyne) — (a residue of an azide-containing molecule) — . In one aspect, the linker 2 has the structure:wherein, z is an integer from 0 to 10.

[0060] In aspects, z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In aspects, z is 3. In one aspect, z is an integer from 0 to 100,000. In one aspect, z is an integer from 0 to 10, 0 to 100, 0 to 1,000, 0 to 5,000, or 0 to 10,000. In one aspect, z is an integer from 10 to 100,000, 100 to 100,000, 1,000 to 100,000, 5,000 to 100,000, or 10,000 to 100,000. In one aspect, the functionalized PAN-ECM comprises from about 0.5 pg to about 100 pg of the at least one covalently attached immune checkpoint molecule per about 1 million functionalizedPAN-ECMs . In one aspect, the functionalized PAN-ECM comprises from about 0.5 pg to about 100.0 pg, about 0.5 pg to about 75.0 pg, about 1 pg to about 60.0 pg, about 1 pg to about 50.0 pg, about 10 pg to about 50.0 pg, about 20 pg to about 50.0 pg, about 30 pg to about 50.0 pg, about 40 pg to about 50.0 pg, about 0.5 pg to about 40.0 pg, about 0.5 pg to about 30.0 pg, about 0.5 pg to about 20.0 pg, or about 0.5 pg to about 10.0 pg of the at least one covalently attached immune checkpoint molecule per about 1 million functionalized PAN-ECMs. In one aspect, the functionalized PAN-ECM comprises from about 0.5 pg, about 1 pg, about 10.0 pg, about 20.0 pg, about 30.0 pg, about 40.0 pg, about 50.0 pg, about 60.0 pg, or about 75.0 pg of at least one covalently attached immune checkpoint molecule per about 1 million functionalized PAN-ECMs. The total amount of immune checkpoint molecule can be quantified, for example, by fluorescence spectroscopy (via fluorescence labeled protein) or quantitative Western blot (e.g Auto We st).

[0061] In aspects, the at least one covalently attached immune checkpoint molecule is an immune checkpoint molecule - functionalized nanoparticle or polymer. In aspects, the covalent attachment is via conjugating to thiol groups on PAN-ECMs.

[0062] In another aspect, the immune checkpoint moiety further comprises a residue of a dendrimer, a linear polymer, a nanoparticle, or a Fc fusion protein. In one aspect, the nanoparticle is a dendrimer, a liposome, an inorganic nanoparticle, or a polymeric nanoparticle. In one aspect, the nanoparticle is about 2 nm to about 10 nm, about 10 nm to about 100 nm, or about 100 nm to about 1000 nm. In aspects, the nanoparticle is about 2 nm to about 1000 nm, about 2 nm to about 750 nm, about 2 nm to about 500 nm, about 2 nm to about 250 nm, about 2 nm to about 200 nm, about 2 nm to about 100 nm, or 2 nm to about 50 nm. In aspects, the nanoparticle is about 10 nm to about 1000 nm, about 25 nm to about 1000 nm, about 50 nm to about 1000 nm, about 100 nm to about 1000 nm, about 200 nm to about 1000 nm, about 500 nm to about 1000 nm, or 750 nm to about 1000 nm. In aspects, the nanoparticle is about 2 nm, about 5 nm, about 10 nm, about 50 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm. In aspects, the nanoparticle is further covalently attached through a linker to one or more immune checkpoint molecules as described herein. In one aspect, the dendrimer is a multivalent dendrimer. In one aspect, the multivalent dendrimer is a polyamidoamine dendrimer. In aspects, the nanoparticle is a pegylated nanoparticle (e.g., DBCO-functionalized PEG-PLGA nanoparticle). In aspects, the pegylated nanoparticle is less than 200 nm in diameter.

[0063] In one aspect, the polyamidoamine dendrimer has a MW of from about 500 to about 1,000,000. In one aspect, the polyamidoamine dendrimer has a MW of from about 1000 to about 1,000,000, about 5000 to about 1,000,000, about 10,000 to about 1,000,000, about 15,000 to about 1,000,000, about 20,000 to about 1,000,000, about 500 to about 100,000, about 500 to about 50,000, or about 500 to about 35,000.

[0064] In one aspect, the polyamidoamine dendrimer has a MW of from about 20,000 to about 35,000. In one aspect, the polyamidoamine dendrimer has a MW of from about 20,000 to about 30,000. In one aspect, the polyamidoamine dendrimer has a MW of from about 25,000 to about 30,000.

[0065] In one aspect, the polyamidoamine dendrimer has a MW of about 20,000, about 21,000, about 22,000, about 23,000, about 24,000, about 25,000, about 26,000, about 27,000, about 28,000, about 29,000, about 30,000, about 31,000, about 32,000, about 33,000, about 34,000, or about 35,000. In one aspect, the polyamidoamine dendrimer has a MW of about 28,000.

[0066] In one aspect, described herein is an acellular pancreatic extracellular matrix (PAN-ECM), wherein the PAN-ECM is functionalized as described herein and comprising pancreatic derived-proteins. In another embodiment, the acellular pancreatic extracellular matrix is in the form of an injectable. In one embodiment, the acellular pancreatic extracellular matrix is in the form of an injectable that is not a gel. In one embodiment, the acellular pancreatic extracellular matrix is in the form of an injectable that is a gel. In one embodiment, the acellular pancreatic extracellular matrix is in the form of an injectable that is a gel that is not a thermal responsive hydrogel.Pharmaceutical composition

[0067] In one embodiment, described herein is a pharmaceutical composition comprising a functionalized pancreatic extracellular matrix (CP -PAN-ECM) comprising an immune checkpoint molecule (CP) as described herein, and a pharmaceutically acceptable excipient. In an aspect, the pharmaceutical composition may further comprise cells. In one aspect, the cell is a pancreatic beta cell.

[0068] In one embodiment, described herein is a vaccine comprising a functionalized pancreatic extracellular matrix (CP-PAN-ECM), and a pharmaceutically acceptable liquid vehicle.

[0069] The term “vaccine” refers to a composition that elicits an immune response and that may prevent a subject from contracting or developing a disease or condition and / or a vaccine may be therapeutic to a subject having a disease or condition.

[0070] A “pharmaceutically acceptable excipient” refers to a vehicle for containing a functionalized extracellular matrix (CP-PAN-ECM) that can be introduced into a subject without significant adverse effects and without having deleterious effects on acellular extracellular matrix. That is, “pharmaceutically acceptable” refers to any formulation which is safe, and provides the appropriate delivery for the desired route of administration of an effective amount of at least one CP-PAN-ECM for use in the methods disclosed herein. Pharmaceutically acceptable carriers or vehicles or excipients are well known. Descriptions of suitable pharmaceutically acceptable carriers, and factors involved in their selection, are found in a variety of readily available sources such as, for example, Remington ’s Pharmaceutical Sciences, 18th ed., 1990, herein incorporated by reference in its entirety for all purposes. Such carriers can be suitable for any route of administration (e.g., parenteral, enteral (e.g., oral), or topical application). Such pharmaceutical compositions can be buffered, for example, wherein the pH is maintained at a particular desired value, ranging from pH 4.0 to pH 9.0, in accordance with the stability of the CP-PAN-ECM and route of administration.

[0071] Suitable pharmaceutically acceptable carriers include, for example, sterile water, salt solutions such as saline, glucose, buffered solutions such as phosphate buffered solutions or bicarbonate buffered solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates (e.g., lactose, amylose or starch), magnesium stearate, talc, silicic acid, viscous paraffin, white paraffin, glycerol, alginates, hyaluronic acid, collagen, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, and the like. Pharmaceutical compositions or vaccines may also include auxiliary agents including, for example, diluents, stabilizers (e.g., sugars and amino acids), preservatives, wetting agents, emulsifiers, pH buffering agents, viscosity enhancing additives, lubricants, salts for influencing osmotic pressure, buffers, vitamins,coloring, flavoring, aromatic substances, and the like which do not deleteriously react with the functionalized cell or the acellular extracellular matrix.

[0072] For liquid formulations, for example, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, emulsions, or oils. Non-aqueous solvents include, for example, propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils include those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil. Solid carriers / diluents include, for example, a gum, a starch (e.g., corn starch, pregeletanized starch), a sugar (e.g., lactose, mannitol, sucrose, or dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g., polymethyl acrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[0073] Optionally, sustained or directed release pharmaceutical compositions or vaccines can be formulated. This can be accomplished, for example, through use of liposomes or compositions wherein the active compound is protected with differentially degradable coatings (e.g., by microencapsulation, multiple coatings, and so forth). Such compositions may be formulated for immediate or slow release. It is also possible to freeze-dry the compositions and use the lyophilisates obtained (e.g., for the preparation of products for injection).Therapeutic methods

[0074] In another embodiment, the subject matter described herein is directed to a method of treating or delaying onset of diabetes in a subject, comprising administering to the subject, a composition comprising CP -PAN -ECM as described herein. In one embodiment, the subject is administered a pharmaceutical composition or a vaccine comprising the CP-PAN-ECM.

[0075] In some aspects, provided herein is a method of treating or reducing the progression of diabetes in a subject in need thereof, comprising administering a functionalized pancreatic extracellular matrix (CP-PAN-ECM) and a functionalized pancreatic cell, wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently attached to a pancreatic extracellular matrix (PAN-ECM); andwherein the functionalized pancreatic cell comprises at least one immune checkpoint molecule covalently attached to the surface of the pancreatic cell.

[0076] In other aspects, provided herein is a functionalized pancreatic extracellular matrix (CP-PAN-ECM) for use in a method of treating or reducing the progression of diabetes in a subject in need thereof comprising administering to the individual an effective amount of the CP-PAN-ECM and a functionalized pancreatic cell, wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently attached to a pancreatic extracellular matrix (PAN-ECM); and wherein the functionalized pancreatic cell comprises at least one immune checkpoint molecule covalently attached to the surface of the pancreatic cell.

[0077] In some embodiments, the type 1 diabetes is early-onset type 1 diabetes or early-onset hyperglycemia. In another embodiment, the subject matter described herein is directed to a method of reversing early-onset type 1 diabetes in a subject comprising administering to the subject, a functionalized CP-PAN-ECM or a pharmaceutical composition or a vaccine comprising the same. In another embodiment, the subject matter described herein is directed to a method of reversing early-onset type 1 diabetes in a subject comprising administering to the subject, a beta cell and a functionalized CP- PAN-ECM or a pharmaceutical composition or a vaccine comprising the same. In embodiments, the subject matter described herein is directed to a method of protecting pancreatic beta cells in a subject comprising administering to the subject, a CP-PAN- ECM or a pharmaceutical composition or a vaccine comprising the same.

[0078] In an aspect, treatment includes ameliorating or preventing the worsening of existing disease symptoms, preventing additional symptoms from occurring, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disorder or disease, e.g., arresting the development of the disorder or disease, relieving the disorder or disease, causing regression of the disorder or disease, relieving a condition caused by the disease or disorder, or stopping the symptoms of the disease or disorder.

[0079] The term “treat” or “treating” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or lessen or reducing the severity of the symptoms of the autoimmune disease. Treating may include one or more of directly affecting or curing, suppressing, inhibiting, preventing, reducingthe severity of, delaying the onset of, slowing the progression of, stabilizing the progression of, reducing / ameliorating symptoms associated with the autoimmune disease, or a combination thereof. The term “reducing the severity” refers to clinical or subjective determination of a lessening of an indication or symptom after treatment.

[0080] The term “subject” refers to a mammal (e.g., a human) in need of therapy for, or susceptible to developing, diabetes. The term subject also refers to a mammal (e.g., a human) that receives either prophylactic or therapeutic treatment. The subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, mice, non-human mammals, and humans. The term “subject” does not necessarily exclude an individual that is healthy in all respects and does not have or show signs of diabetes.

[0081] As used herein, the term “organism” includes, but is not limited to, a human, a non-human primate, such as those mentioned above, and any transgenic species thereof, and further includes any living eukaryote.

[0082] The terms “effective amount” or “therapeutically effective amount” refer to a sufficient amount of the composition to provide the desired biological result. That result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease or medical condition, or any other desired alteration of a biological system. For example, an “effective amount” for therapeutic use is the amount of a composition that is required to provide a clinically relevant change in a disease state, symptom, or medical condition. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. Thus, the expression “effective amount” generally refers to the quantity for which the active substance has a therapeutically desired effect. Effective amounts or doses of the compositions of the embodiments may be ascertained by routine methods, such as modeling, dose escalation, or clinical trials, taking into account routine factors, e.g., the mode or route of administration or drug delivery, the pharmacokinetics of the agent, the severity and course of the infection, the subject's health status, condition, and weight, and the judgment of the treating physician. An exemplary dose is in the range of about 1 pg to 10 mg of active agent per kilogram of subject's body weight per day. The total dosage may be given in single or divided dosage units (e.g., BID, TID, QID). Once improvement of the patient's disease has occurred, the dose may be adjusted for preventative or maintenance treatment. For example, the dosage or the frequency of administration, orboth, may be reduced as a function of the symptoms, to a level at which the desired therapeutic or prophylactic effect is maintained. Of course, if symptoms have been alleviated to an appropriate level, treatment may cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of symptoms. Patients may also require chronic treatment on a long-term basis.

[0083] In certain aspects, the present disclosure provides compositions formulated for one or more routes of administration. Suitable routes of administration may, for example, parenteral delivery. In some aspects, compositions herein formulated can be formulated for parenteral delivery. In some aspects, compositions herein formulated can be formulated intramuscular, subcutaneous, intramedullary, or intravenous injections.

[0084] In certain aspects, one may administer a composition herein in a local or systemic manner, for example, via local injection of the pharmaceutical composition directly into a tissue region of a patient. In some aspects, a pharmaceutical composition disclosed herein can be administered parenterally.

[0085] In certain aspects, pharmaceutical compositions of the present disclosure may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0086] In certain aspects, pharmaceutical compositions for use in accordance with the present disclosure thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the active ingredients of a pharmaceutical composition herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, physiological salt buffer, or any combination thereof.

[0087] In certain aspects, pharmaceutical compositions described herein may be formulated in the form of a nanoparticle.

[0088] In various aspects, the method of treatment comprises administering an effective amount of a monofunctionalized-PAN-ECM to a subject in need thereof, wherein the monofunctionalized-PAN-ECM comprises an immune checkpoint molecule covalentlyattached to a pancreatic extracellular matrix (PAN-ECM). In some aspects, the immune checkpoint molecule is selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7- H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFP, FGL1, and CD96. In some aspects, the method further comprises administering to the subject an effective amount of functionalized pancreatic or immune cell. In some aspects the functionalized pancreatic cell is a functionalized a beta cell. In some aspects the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises a protein selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFP, FGL1, and CD96. In some aspects the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises PD-L1.

[0089] In various other aspects, the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises HVEM. In some aspects, the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises FasL. In some aspects, the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises GITRL. In some aspects, the functionalized pancreatic or immune cell comprises two immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the two immune checkpoint protein comprise HVEM and PD-L1.

[0090] In some aspects, the method comprises administering an effective amount of a monofunctionalized-PAN-ECM to a subject in need thereof, wherein the monofunctionalized-PAN-ECM comprises a PD-Ll-functionalized-PAN-ECM. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the PD-L1 -PAN-ECM.

[0091] In another aspect, the method comprises administering an effective amount of a monofunctionalized-PAN-ECM to a subject in need thereof, wherein the monofunctionalized-PAN-ECM comprises a HVEM-functionalized-PAN-ECM. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the HVEM-PAN-ECM.

[0092] In some aspects, the method comprises administering an effective amount of a monofunctionalized-PAN-ECM to a subject in need thereof, wherein the monofunctionalized-PAN-ECM comprises a FasL-functionalized-PAN-ECM. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the FasL-PAN-ECM.

[0093] In some aspects, the method comprises administering an effective amount of a monofunctionalized-PAN-ECM to a subject in need thereof, wherein the monofunctionalized-PAN-ECM comprises a FGLl-functionalized-PAN-ECM. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the FGL1-PAN-ECM.

[0094] In another aspect, the method comprises administering an effective amount of a monofunctionalized-PAN-ECM to a subject in need thereof, wherein the monofunctionalized-PAN-ECM comprises a PD-Ll-functionalized-PAN-ECM. In some aspects, the method further comprises administering to the subject an effective amount of functionalized pancreatic or immune cell. In some aspects the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises a protein selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7- H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGF|3, FGL1, and CD96. In some aspects, the functionalized pancreatic cell is a functionalized a beta cell. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the PD-L1-PAN-ECM and functionalized pancreatic or immune cell.

[0095] In some aspects, the method comprises administering an effective amount of a monofunctionalized-PAN-ECM to a subject in need thereof, wherein the monofunctionalized-PAN-ECM comprises a HVEM-functionalized-PAN-ECM. In some aspects, the method further comprises administering to the subject an effective amount offunctionalized pancreatic or immune cell. In some aspects the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises a protein selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7- H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1, and CD96. In some aspects, the functionalized pancreatic cell is a functionalized a beta cell. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the HVEM-PAN-ECM and the functionalized pancreatic or immune cell. In some aspects, the method comprises administering an effective amount of a monofunctionalized-PAN-ECM to a subject in need thereof, wherein the monofunctionalized-PAN-ECM comprises a FasL-functionalized-PAN-ECM. In some aspects, the method further comprises administering to the subject an effective amount of functionalized pancreatic or immune cell. In some aspects the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises a protein selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7- H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1, and CD96. In some aspects, the functionalized pancreatic cell is a functionalized a beta cell. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the FasL-PAN-ECM and the functionalized pancreatic or immune cell.

[0096] In some aspects, the method comprises administering an effective amount of a monofunctionalized-PAN-ECM to a subject in need thereof, wherein the monofunctionalized-PAN-ECM comprises a FGLl-functionalized-PAN-ECM. In some aspects, the method further comprises administering to the subject an effective amount of functionalized pancreatic or immune cell. In some aspects the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises a protein selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7- H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1,and CD96. In some aspects, the functionalized pancreatic cell is a functionalized a beta cell. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the FGL1-PAN-ECM and the functionalized pancreatic or immune cell.

[0097] In another aspect, the method of treatment comprises administering an effective amount of a bi-functionalized-PAN-ECM to a subject in need thereof, wherein the bi- functionalized-PAN-ECM comprises two immune checkpoint molecules covalently attached to a pancreatic extracellular matrix (PAN-ECM). In some aspects, the immune checkpoint molecules are selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7- H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFP, FGL1, and CD96. In some aspects, the method further comprises administering to the subject an effective amount of functionalized pancreatic or immune cell. In some aspects the functionalized pancreatic cell is a functionalized a beta cell. In some aspects the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises a protein selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1, and CD96.

[0098] In some aspects, the method comprises administering an effective amount of a bi-functionalized-PAN-ECM to a subject in need thereof, wherein the bi-functionalized- PAN-ECM comprises a PD-Ll-HVEM-functionalized-PAN-ECM. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the PD- L 1 -HVEM-P AN-ECM.

[0099] In some aspects, the method comprises administering an effective amount of a bi-functionalized-PAN-ECM to a subject in need thereof, wherein the bi-functionalized- PAN-ECM comprises a PD-Ll-HVEM-functionalized-P AN-ECM. In some aspects, the method further comprises administering to the subject an effective amount of functionalized pancreatic or immune cell. In some aspects the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint proteincomprises a protein selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7- H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1, and CD96. In some aspects, the functionalized pancreatic cell is a functionalized a beta cell. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the PD-L1-HVEM-PAN-ECM and functionalized pancreatic or immune cell.

[0100] In various aspects, the method of treatment comprises administering an effective amount of a tri-functionalized-PAN-ECM to a subject in need thereof, wherein the tri- functionalized-PAN-ECM comprises three immune checkpoint molecules covalently attached to a pancreatic extracellular matrix (PAN-ECM). In some aspects, the immune checkpoint molecules are selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7- H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFP, FGL1, and CD96. In some aspects, the method further comprises administering to the subject an effective amount of functionalized pancreatic or immune cell. In some aspects the functionalized pancreatic cell is a functionalized a beta cell. In some aspects the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises a protein selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1, and CD96.

[0101] In various other aspects, the method comprises administering an effective amount of a tri-functionalized-PAN-ECM to a subject in need thereof, wherein the tri- functionalized-PAN-ECM comprises a PD-Ll-FGLl-TGFP-functionalized-PAN-ECM. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the PD-Ll-FGLl-TGFP-functionalized-PAN-ECM.

[0102] In another aspect, the method comprises administering an effective amount of a tri-functionalized-PAN-ECM to a subject in need thereof, wherein the tri-functionalized- PAN-ECM comprises a FasL-HVEM-FGLl-functionalized-PAN-ECM. In some aspects,the subject is administered a pharmaceutical composition or a vaccine comprising the FasL-HVEM-FGL 1 -functionalized-P AN-ECM.

[0103] In some aspects, the method comprises administering an effective amount of a tri-functionalized-PAN-ECM to a subject in need thereof, wherein the tri-functionalized- PAN-ECM comprises a FasL-TGFP-PD-Ll-functionalized-PAN-ECM. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the FasL-TGFP-PD-Ll-functionalized-PAN-ECM.

[0104] In some aspects, the method comprises administering an effective amount of a tri-functionalized-PAN-ECM to a subject in need thereof, wherein the tri-functionalized- PAN-ECM comprises a FasL-GITRL-TGFP-functionalized-PAN-ECM. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the FasL-GITRL-TGFP-functionalized-PAN-ECM.

[0105] In another aspect, the method comprises administering an effective amount of a tri-functionalized-PAN-ECM to a subject in need thereof, wherein the tri-functionalized- PAN-ECM comprises a PD-Ll-HVEM-FasL-functionalized-PAN-ECM. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the PD-Ll-HVEM-FasL-functionalized-PAN-ECM.

[0106] In some aspects, the method comprises administering an effective amount of a tri-functionalized-PAN-ECM to a subject in need thereof, wherein the tri-functionalized- PAN-ECM comprises a PD-Ll-FGLl-TGFP-functionalized-PAN-ECM. In some aspects, the method further comprises administering to the subject an effective amount of functionalized pancreatic or immune cell. In some aspects the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises a protein selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7- H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1, and CD96. In some aspects, the functionalized pancreatic cell is a functionalized a beta cell. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the PD-L1-FGL1-TGFP-PAN-ECM and functionalized pancreatic or immune cell.

[0107] In various aspects, the method comprises administering an effective amount of a tri-functionalized-PAN-ECM to a subject in need thereof, wherein the tri-functionalized- PAN-ECM comprises a FasL-HVEM-FGLl-functionalized-PAN-ECM. In some aspects, the method further comprises administering to the subject an effective amount of functionalized pancreatic or immune cell. In some aspects the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises a protein selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7- H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1, and CD96. In some aspects, the functionalized pancreatic cell is a functionalized a beta cell. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the FasL-HVEM-FGLl-functionalized-PAN-ECM and functionalized pancreatic or immune cell.

[0108] In some aspects, the method comprises administering an effective amount of a tri-functionalized-PAN-ECM to a subject in need thereof, wherein the tri-functionalized- PAN-ECM comprises a FasL-TGFP-PD-Ll-functionalized-PAN-ECM. In some aspects, the method further comprises administering to the subject an effective amount of functionalized pancreatic or immune cell. In some aspects the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises a protein selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7- H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1, and CD96. In some aspects, the functionalized pancreatic cell is a functionalized a beta cell. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the FasL-TGFP-PD-Ll-PAN-ECM and functionalized pancreatic or immune cell.

[0109] In another aspect, the method comprises administering an effective amount of a tri-functionalized-PAN-ECM to a subject in need thereof, wherein the tri-functionalized- PAN-ECM comprises a FasL-GITRL-TGFP-functionalized-PAN-ECM. In some aspects, the method further comprises administering to the subject an effective amount of functionalized pancreatic or immune cell. In some aspects the functionalized pancreaticor immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises a protein selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7- H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1, and CD96. In some aspects, the functionalized pancreatic cell is a functionalized a beta cell. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the FasL-GITRL-TGFP-PAN-ECM and functionalized pancreatic or immune cell.

[0110] In some aspects, the method comprises administering an effective amount of a tri-functionalized-PAN-ECM to a subject in need thereof, wherein the tri-functionalized- PAN-ECM comprises a PD-Ll-HVEM-FasL-functionalized-PAN-ECM. In some aspects, the method further comprises administering to the subject an effective amount of functionalized pancreatic or immune cell. In some aspects the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises a protein selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7- H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1, and CD96. In some aspects, the functionalized pancreatic cell is a functionalized a beta cell. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the PD-Ll-HVEM-FasL-PAN-ECM and functionalized pancreatic or immune cell.

[0111] In some aspects, the method of treatment comprises administering an effective amount of a tetra-functionalized-PAN-ECM to a subject in need thereof, wherein the tetra-functionalized-PAN-ECM comprises four immune checkpoint molecules covalently attached to a pancreatic extracellular matrix (PAN-ECM). In some aspects, the immune checkpoint molecules are selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7- H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFP, FGL1, and CD96. In some aspects, the method further comprises administering to the subject an effective amount of functionalized pancreatic or immune cell. In some aspects the functionalized pancreatic cell is a functionalized a beta cell. In some aspectsthe functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises a protein selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1, and CD96.

[0112] In another aspect, the method comprises administering an effective amount of a tetra-functionalized-PAN-ECM to a subject in need thereof, wherein the tetra- functionalized-PAN-ECM comprises a FasL-GITRL-TGFP-HVEM-functionalized-PAN- ECM. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the FasL-GITRL-TGFP-HVEM-functionalized-PAN-ECM.

[0113] In various other aspects, the method comprises administering an effective amount of a tetra-functionalized-PAN-ECM to a subject in need thereof, wherein the tetra-functionalized-PAN-ECM comprises a FasL-GITRL-TGFP-HVEM-functionalized- PAN-ECM. In some aspects, the method further comprises administering to the subject an effective amount of functionalized pancreatic or immune cell. In some aspects the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently attached to the pancreatic of immune cell surface, wherein the immune checkpoint protein comprises a protein selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFP, FGL1, and CD96. In some aspects, the functionalized pancreatic cell is a functionalized a beta cell. In some aspects, the subject is administered a pharmaceutical composition or a vaccine comprising the FasL-GITRL- TGFP-HVEM-PAN-ECM and functionalized pancreatic or immune cell.III. Method of Making

[0114] In an aspect, the current disclosure also encompasses method of making the disclosed compositions. A method of making a functionalized pancreatic extracellular matrix (CP-PAN-ECM), may comprise providing a functionalized PAN-ECM, wherein the PAN-ECM is functionalized with a moiety comprising a residue suitable for click chemistry, providing a functionalized immune checkpoint molecule, wherein the immune checkpoint is functionalized with a moiety comprising a residue suitable for clickchemistry, and contacting the azide-modified PAN-ECM with the immune checkpoint bioconjugate to form the CP -PAN-ECM using a alkyne-azide cycloaddition (SPAAC) chemistry. In an aspect, the functionalized PAN-ECM comprises an azide moiety. In an aspect, the functionalized immune checkpoint molecule comprises a dibenzocycloctyne moiety. In an aspect, the immune checkpoint molecule is a bioconjugate further comprising a dendrimer, a linear polymer, a nanoparticle, or a Fc fusion protein. In an aspect the dibenzocycloctyne moiety comprises a DBCO, a or a derivative thereof, or a conjugate thereof. Optionally, purification steps may be carried out following any reaction described herein to improve the purity of the disclosed compositions.

[0115] In some aspects, the PAN-ECM is produced from an acellularized tissue of a pancreas. In some aspects, the acellularized tissue of a pancreas is generated by decellularizing a pancreas isolated from a mammal. In some aspects, the mammal is a rodent such as a mouse or a rat. In some aspects, the pancreas tissue is decellularized using a detergent, such as for example Triton-X-100, and an ammonia water solution. In some aspects, the pancreas tissue is decellularized using a mixed solution of 1% Triton- X-100 / 0.1% ammonia water.

[0116] An exemplary method of making and using a disclosed CP-PAN-ECM in provided in the examples provided herein.EMBODIMENTS1. A functioned pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN- ECM comprises at least one immune check point molecule covalently attached to a pancreatic extracellular matrix (PAN-ECM).2. The CP-PAN-ECM of embodiment 1, wherein the immune checkpoint molecule comprises PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, or CD96.3. The CP-PAN-ECM of embodiment 1, wherein the at least one immune check point molecule is attached to the PAN-ECM using click chemistry.The CP-PAN-ECM of embodiment 3, wherein the at least one immune check point molecule is attached to the PAN-ECM via an alkyne-azide cycloaddition (SPAAC) chemistry. The CP-PAN-ECM of embodiment 4, wherein the at least one immune check point molecule comprises a dibenzocyclooctyne moiety. The CP-PAN-ECM of embodiment 5, wherein the dibenzocycloctyne moiety is DBCO, or a derivative thereof, or a conjugate thereof. The CP-PAN-ECM of embodiment 5, wherein the immune checkpoint molecule is a bioconjugate comprising a dendrimer, a linear polymer, a nanoparticle, or a Fc fusion protein. A composition comprising a CP-PAN-ECM of any one of embodiments 1-7. A composition comprising a functioned pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises at least one immune check point molecule covalently attached to a pancreatic extracellular matrix (PAN-ECM). The composition of embodiments 8 or 9, wherein the composition is a pharmaceutical composition. The composition of any one of embodiments 8-10, wherein the composition further comprises a pancreatic or an immune cell. The composition of embodiment 11, wherein the pancreatic cell is a beta cell (P cells). The composition of any one of embodiments 8-12, further comprising at least one excipient, or at least one additional therapeutic agent or combinations thereof. The composition of any one of embodiments 8-13, for use in the prevention or treatment of diabetes. A method of making a functioned pancreatic extracellular matrix (CP-PAN-ECM), comprising: a. providing a functionalized PAN-ECM;b. providing a functionalized immune checkpoint molecule; and c. contacting the functionalized PAN-ECM with the functionalized immune checkpoint bioconjugate to form the CP -PAN-ECM using an alkyne-azide cycloaddition (SPAAC) chemistry. The method of embodiment 15, wherein the functionalized PAN-ECM comprises an azide moiety. The method of embodiment 15, wherein the functionalized immune checkpoint molecule comprises a dibenzocycloctyne moiety. The method of embodiment 17, wherein the dibenzocycloctyne moiety comprises a DBCO, a or a derivative thereof, or a conjugate thereof. The method of embodiment 18, wherein the immune checkpoint molecule is a bioconjugate comprising a dendrimer, a linear polymer, a nanoparticle, or a Fc fusion protein. A method of treating or reducing the progression of diabetes in a subject in need thereof, comprising administering to the subject a composition of any one of embodiments 8-14. The method of embodiment 20, wherein the subject is a mammal. The method of embodiment 21, wherein the administering is done via the parenteral route. The method of embodiment 21, wherein the diabetes is autoimmune diabetes. A functioned pancreatic extracellular matrix comprising a structure: (PAN-ECM) — (a residue of an azide-containing molecule) — (a residue of a cyclooctyne) — (a linker 1) — (a residue of a functionalized dendrimer)q — (a residue of an immune checkpoint molecule), wherein, q is one or zero; and, the dash represents a covalent bond.Examples

[0117] The following examples are included to demonstrate preferred aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Example 1: Preparation and Decellularization of the Pancreas

[0118] The pancreas of a 6-month-old healthy living rat was stored at -80 °C in vitro and frozen for 12 h. The connective tissue and adipose tissue around the pancreas were peeled off and cut into pieces with a size of about 0.5-1 cm3. Pancreatic tissue fragments were rinsed with cold PBS, 1 h each three times. Triton-X-100 and ammonia water (Guangdong, China) were added into PBS to prepare a mixed solution of 1% Triton-X- 100 / 0.1% ammonia water. The pancreatic tissue fragments were added into Triton-X-100 solution and vibrated for 72 h at 4 °C. The Triton-X-100 solution was replaced every 24 h three times. Then, rinsing was done with PBS containing pen / strep three times at 4 °C for 12 h each time to thoroughly remove the detergent. The acellular tissue of the pancreas was stored at -80 °C.

[0119] Azide-modified PAN-ECM was generated by carbodiimide / N- hydroxy succinimide (EDC / NHS) reaction with Azide-PEG8-NHS. Before attaching checkpoints to Azide-modified PAN-ECM, we firstly prepared DBCO-functionalized checkpoints bioconjugates via bioorthogonal click reaction between checkpoints and DBCO-Dendrimer. Then checkpoint-functionalized PAN-ECM was prepared using bioorthogonal click reactions between DBCO-functionalized checkpoint bioconjugates and Azide-functionalized PAN-ECM. The therapeutic efficiency of different subcutaneous (s.c.) injectable checkpoint-functionalized PAN-ECM plus P cells was evaluated in newly diabetic NOD female mice 2-3 days after diabetes onset. In addition, two boosters were administered 2 weeks and 4 weeks after the initial treatment.Example 2: Preparation of DBCO-functionalized PAMAM G5

[0120] PAMAM G5 (0.7 mL of 5 wt / wt% PAMAM G5 in methanol containing 27.9 mg dendrimer, 0.97 pmol) was first dried over nitrogen gas at 20 °C for 15 min, before drying in vacuum for another 2 h. DBCO-PEG4-NHS ester (764 pL, 25 mM in DMSO, 12.3 mg) was added to the dried dendrimer film. The mixture was stirred at 20 °C (in the dark) for 4 hours. An excess amount of acetic anhydride (0. 5 mL) was added to the reaction mixture and stirred at 20 °C (in the dark) for another 18 hours. The DBCO- functionalized dendrimer was further purified through equilibrium dialysis in the dark for another three days (six cycles). The purified dendrimer was freeze-dried and stored at -80 °C (in the dark) before further studies.Example 3: Preparation of azide-functionalized PD-L1

[0121] Azide-functionalized PD-L1 (PD-L1-N3) was synthesized via an amine-NHS ester coupling reaction. The target degree of functionalization was 20. For a typical functionalization, 10 pg (0.194 nmol, dissolved in 100 pL PBS) of PD-L1 was incubated with the azide-PEG4-NHS solution (3.88 nmol, 10 pL of 0.388 mM of azide-PEG4- NHS) at 20 °C for 1.5 hours. The functionalized PD-L1 was purified three times via Zebra Spin 7K MWCO Desalting Columns according to the manufacturer’s protocol. The concentration of the purified azide-functionalized PD-L1 was quantified via UV- visible spectroscopy.

[0122] Azide-functionalized TGF-P (TGF-P-N3), azide-functionalized HVEM (HVEM- N3), azide-functionalized FasL (FasL-N3), azide-functionalized CD47 (CD47-N3), azide-functionalized GITRL (GITRL-N3), and azide-functionalized Gal-9 (Gal-9-N3) were synthesized via the same method, except the target degree of TGF-P, FasL, CD47 and Gal-9 was 10. FIG. 1 provides a schematic of the bioengineering of Checkpoint- functionalized PAN-ECM (top) and the mechanism of action of the s.c. -injected P cells plus PAN-ECM (bottom).Example 4: Preparation of PD-Ll-Dend bioconjugates

[0123] Purified azide-functionalized PD-L1 was quantitatively conjugated to DBCO- functionalized PAMAM G5 at a target molar ratio of 1 : 1. PD-L1-N3 (10 pg) and DBCO- functionalized PAMAM G5 of dendrimer (8 pg, 0.8 pL of 10 mg / mL of dendrimersolution in PBS) were mixed and incubated at 4 °C for 24 h. The PD-Ll-Dend bioconjugate was used for subsequent studies without further purification.

[0124] TGF-P-Dend, HVEM-Dend, FasL-Dend, CD47-Dend, GITRL-Dend and Gal-9- Dend bioconjugates were prepared via the same method.Example 5: Decoration of checkpoint molecules-modified PAN-ECM

[0125] PAN-ECM (100 mg) was washed with PBS twice, and then resuspended at 350 mg / mL in PBS. Subsequently, Azide-PEG8-NHS (20 pL, 285 mM in DMSO) was added to the PAN-ECM and incubated at 4 °C overnight. PD-L1-N3 (10 pg) and DBCO- functionalized PAMAM G5 of dendrimer (8 pg, 0.8 pL of 10 mg / mL of dendrimer solution in PBS) were mixed and incubated at 37 °C for 30 minutes. Meanwhile, Azide- PAN-ECM was washed for 3 times and resuspend at 1000 mg / mL in PBS. Next, Azide- PAN-ECM was added to PD-L1-N3 and dendrimer reaction solution and incubated at 4 °C overnight. The PD-L1 -modified PAN-ECM was washed twice and resuspended in PBS.

[0126] TGF-P-, HVEM-, FasL-, CD47-, GITRL- and Gal-9-modified PAN-ECM were synthesized via the same method.Example 6. In vivo evaluations of CP-PAN-ECM to reverse early-onset T1D NOD mice

[0127] To expand the list of viable checkpoints that can be targeted, a cohort of NOD / ShiltJ (NOD, female) mice were obtained, and their blood glucose levels were monitored twice weekly. After diabetes onset, defined as a blood glucose level over 250 mg / dL, mice were dosed with a combination of a checkpoint modified decellularized pancreas extracellular matrix (PAN-ECM) and NIT-1 beta cells injected subcutaneously twice over a two-week period (FIG. 3A). Initially, mice were dosed with single checkpoint modified ECM to compare the efficacy of the different checkpoints (FIG. 3B)

[0128] From this study, multiple candidates were identified as viable checkpoints to target that helped improve the overall survival and reverse the onset diabetes. When newly diabetic NOD female mice were treated with PD-L1, HVEM, and FasL- functionalized PAN-ECM, 50% of the PD-L1 -functionalized PAN-ECM treatment group reversed to normoglycemia (FIG. 2A). From this group of checkpoints, HVEM was shown to have the strongest response delaying the onset of diabetes beyond the 100-daymonitoring period for 75% of the mice (FIG. 3B). About half of the HVEM- functionalized (FIG. 2B) and FasL-functionalized PAN-ECM-treated mice (FIG. 2C) were diabetic-free for 100 days. All groups, including the co-inj ection of unmodified NIT-1 cells and ECM, had some impact on delaying diabetes onset and improving survivability demonstrating the strong baseline for this technology. Next it was investigated if all three checkpoints simultaneously modified to PAN-ECM could be used as a therapeutic vaccine to reverse early-onset hyperglycemia. The results demonstrated that, in comparison to three monofunctionalized PAN-ECM, PD- Ll / HVEM / FasL-functionalized PAN-ECM resulted in a more potent and concomitant suppression of a multitude of immune checkpoint pathways, thereby eliciting a more effective induction of antigen-specific T cell exhaustion (FIG. 2D).

[0129] To identify potential synergistic effects of these checkpoints, select checkpoint combinations were conjugated to PAN-ECM and injected with NIT-1 cells as described above. Five groups of checkpoints were tested, i) PD-L1 + FGL1 + TGFP, ii) FAS-L + HVEM + FGL1, iii) FASL + TGF p+ PD-L1, iv) FASL + GITRL + TGF p, and v) PD- L1 + HVEM + FASL. All treatments that included FGL1 saw no improvement over unmodified PAN-ECM, as most mice progressed to hyperglycemia within 30 days (FIG. 3C). All other groups demonstrated prolonged survival (FIGS. 4A, and 4B), with most mice maintaining normoglycemia through the 100-day monitoring period (FIG. 3C). These groups comprised different combinations of PD-L1, HVEM, FASL, GITRL, and TGF P, thereby providing a list of potentially synergistic combinations of targets to help quiet the autoimmune attack of natural islets.

[0130] To determine a potential maximum response, a cohort of mice were treated with a combination of all the cytokines and checkpoints that showed efficacy in the previous studies. Diabetic mice received a treatment composed of ECM modified with HVEM, FAS-L, GITRL and TGF P combined with a dose of NIT-1 cells modified with PD-L1. This treatment saw the strongest response, with 80% of mice maintaining normoglycemia levels until week 15, while 30% of mice avoided hypoglycemia for over 20 weeks (FIG. 5A, and FIG. 5B).Example 7: In vitro evaluation of CP-functionalized NIT-1 cells to anergize antigen-specificCD8+ T cells

[0131] To test whether anchoring different immune checkpoint molecules to a surface enhances their ability to inhibit the cytotoxic effects of islet antigen-specific T cells, cytotoxicity assays were performed using variously functionalized NIT-1 cells in the presence of the islet-specific IGRP peptide (FIG. 6B). Fluorescence activated cell sorting (FACS) analysis demonstrated that PD-L1, HVEM, FasL, and GITRL can be successfully decorated to NIT-1 cells by metabolic glycoengineering and bioorthogonal click chemistry (FIG. 6A). Apart from FasL, the other three checkpoints significantly inhibited T cell-mediated cytotoxicity against islet cells when modified on the cell surface (FIG. 6C). Interestingly, when FasL was anchored to the cell surface, there was a slight increase in T cell killing efficiency, suggesting that FasL may potentially act as a negative checkpoint in diabetes treatment. PD-L1 and HVEM could therefore serve as potential checkpoints for reversing diabetes. Subsequent studies of their combined effects indeed revealed that they significantly reduced T cell-mediated cytotoxicity against islet cells compared to the use of individual checkpoints (FIG. 6D).Example 8. In vivo administration of CP-ECM plus PD-Ll-functionalized NIT-1 cells reverses early-onset T1D

[0132] Initial screening demonstrated that modifying PAN-ECM with PD- Ll / HVEM / FasL resulted in a 75% reversal of diabetes, one mouse died on day 24 (FIG. 3C). In the subsequent research, PD-L1 was also placed on the surface of NIT-1 cells (FIG. 7A). As shown in FIG. 7B and FIG. 7C, separating the two checkpoints, rather than modifying all of them on PAN-ECM, yielded improved results. This suggests that the inhibitory effect of PD-L1 on T cells may depend on antigen presentation.Example 9: Treatment of diabetes using an in vivo mouse model

[0133] This example identifies methods to develop islet like cells from sources other than NIT-1 cells. An example of such a source comprises mouse embryonic stem cells (mESCs, C57BL / 6 derived), which provide a route to generate insulin producing cells through a complex sequence of media changes (FIG. 8). Briefly, embryonic stem cells isolated from C57BL / 6 mice were cultured in bacteriological petri dishes for three days in N2B27 medium. Differentiation of the mESCs was initiated by transferring the cellsonto 0.1% gelatin coated dishes in Differentiation Medium I, followed by a three-day culture period in N2B27 medium supplemented with Y27632. Pancreatic differentiation was then induced by culturing the cells for 18 days in Differentiation medium II, resulting in the generation of islet-like clusters. Dithizone staining was then performed to identify and purify islet-like clusters from undifferentiated cells. The islet-like clusters could then be cultured in insulin free medium prior to performing the glucose stimulated insulin secretion analysis assays described herein.

[0134] The insulin secretion fold-change between low and high glucose conditions was not significant between IPCs and mouse islet controls (FIG. 9A). Strikingly, the differentiated IPCs displayed uniform insulin granule distribution between different clusters relative to human islets as indicated by dithizone staining (FIG. 9B). In addition, 88% of the differentiated IPCs produced both insulin and glucagon (FIG. 9C), indicating an efficient induction of P-cells.

[0135] Functionalization of IPCs with HVEM and PD-L1 (engineered IPCs) was achieved by a bioengineering approach comprising metabolic glycoengineering followed by a bioorthogonal click reaction (FIG. 10A). FACS studies confirmed the successful decoration of IPCs with HVEM and PD-L1, where 100% of engineered IPCs displayed both HVEM and PD-L1 positivity (FIG. 10B). Transplantation of these allogeneic cells in vivo was assessed on the streptozotocin (STZ)-induced diabetic BABL / c mouse model. STZ induction of diabetes was achieved by administering 175 mg / kg body weight of STZ via intraperitoneal injection in 10-week-old female BALB / c mice five days prior to IPC and PAN-ECM transplantation. Induction of diabetes was confirmed by measuring blood glucose levels two days prior to IPC and PAN-ECM transplantation. IPCs and PAN-ECM were implanted under the skin of the abdomen of the recipient BALB / c mouse (FIG. 10C). Mice were distributed into 3 groups, (i) control mice without STZ treatment, (ii) STZ treated mice implanted subcutaneously with IPCs and PAN ECM, and (iii) STZ treated mice implanted subcutaneously with engineered IPCs decorated with HVEM and PD-L1 and PAN ECM. Control IPCs administered to diabetic BALB / c mice in combination with PAN ECM did not effectively mitigate the diabetic condition induced by STZ in BALB / c mice, with all mice progressing to hyperglycemia by day 30 (FIG. 10D). In stark contrast, mice which were administered engineered IPCs decorated with HVEM and PD-L1 in combination with PAN ECM resulted in significant stabilization of blood glucose levels (FIG. 10D). These resultssuggest that engineered IPCs may provide significant mitigation of hyperglycemia and support metabolic homeostasis when administered in combination with PAN ECM.

[0136] The composition of the IPC + PAN ECM implants were analyzed via flow cytometry 30 days post transplantation in an exemplary mouse from the IPCs + PAN- ECM cohort and the engineered IPCs + PAN ECM cohort (see FIG. 10D, arrows indicate the mouse in which the implant was removed for analysis). Notably, upon removal of the implant from the engineered IPCs + PAN ECM mouse, the blood glucose level of that mouse rapidly increased to 600 mg / dL, indicating that the engineered IPC + PAN ECM implant was the only significant insulin source within the mouse (FIG. 10D). To conduct this analysis, the IPC + PAN ECM implants were removed from each mouse and dissociated with GentleMACS. Dead cells were excluded from analysis via a Live / dead kit. Flow cytometry analysis was then conducted by gating for islet and checkpoint markers comprising insulin and glucagon. Results from this flow cytometry analysis show that the engineered IPCs decorated with HVEM and PD-L1 continued to produce glucagon and insulin following 30 days of implantation in vivo in the presence of PAN ECM (FIG. 10E).

Claims

CLAIMSWhat is claimed is:

1. A functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP- PAN-ECM comprises at least one immune checkpoint molecule covalently attached to a pancreatic extracellular matrix (PAN-ECM).

2. The CP-PAN-ECM of claim 1, wherein the immune checkpoint molecule comprises PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1, or CD96.

3. The CP-PAN-ECM of claim 1 or claim 2, wherein the at least one immune checkpoint molecule is attached to the PAN-ECM using click chemistry.

4. The CP-PAN-ECM of claim 3, wherein two, three, or four immune checkpoint molecules are attached to the PAN-ECM using click chemistry.

5. The CP-PAN-ECM of any one of claims 1-3, wherein the at least one immune checkpoint molecule is attached to the PAN-ECM via an alkyne-azide cycloaddition (SPAAC) chemistry.

6. The CP-PAN-ECM of claim 5, wherein two, three, or four immune checkpoint molecules are attached to the PAN-ECM via alkyne-azide cycloaddition (SPAAC) chemistry.

7. The CP-PAN-ECM of claim 5, wherein the at least one immune checkpoint molecule comprises a dibenzocyclooctyne moiety.

8. The CP-PAN-ECM of claim 7, wherein the dibenzocycloctyne moiety is DBCO, or a derivative thereof, or a conjugate thereof.

9. The CP-PAN-ECM of claim 7, wherein the immune checkpoint molecule is a bioconjugate comprising a dendrimer, a linear polymer, a nanoparticle, or a Fc fusion protein.

10. A composition comprising a CP-PAN-ECM of any one of claims 1-9.

11. A composition comprising a functionalized pancreatic extracellular matrix (CP-PAN- ECM), wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently attached to a pancreatic extracellular matrix (PAN-ECM).

12. The composition of claim 11, wherein the CP-PAN-ECM comprises two, three, or four immune checkpoint molecules covalently attached to the PAN-ECM.

13. The composition of claim 11 or claim 12, wherein the at least one immune checkpoint molecule is selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1, and CD96.

14. The composition of any one of claims 11-13, wherein the CP-PAN-ECM comprises a bi-functionalized CP-PAN-ECM comprising PD-L1 and HVEM covalently attached to the PAN-ECM.

15. The composition of any one of claims 11-13, wherein the CP-PAN-ECM comprises a tri-functionalized CP-PAN-ECM comprising i) PD-L1, FGL1, and TGFP, ii) FasL, HVEM, and FGL1, iii) FasL, TGFp, and PD-L1, iv) FasL, GITRL, and TGFp, or v) PD-L1, HVEM, and FasL covalently attached to the PAN-ECM.

16. The composition of any one of claims 11-13, wherein the CP-PAN-ECM comprises a tetra-functionalized CP-PAN-ECM comprising FasL, GITRL, TGFP, and HVEM covalently attached to the PAN-ECM.

17. The composition of any one of claims 10-16, wherein the composition is a pharmaceutical composition.

18. The composition of any one of claims 10-17, wherein the composition further comprises a pancreatic or an immune cell.

19. The composition of claim 18, wherein the pancreatic or immune cell comprises at least one immune checkpoint molecule covalently attached to the surface of the pancreatic or immune cell.

20. The composition of claim 19, wherein the immune checkpoint molecule is selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3,TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1, and CD96.

21. The composition of claim 20, wherein the immune checkpoint molecule comprises PD-L1, HVEM, FasL, or GITRL.

22. The composition of any one of claims 19-21, wherein the immune checkpoint molecule is covalently attached to the surface of the pancreatic or immune cell by metabolic glycoengineering.

23. The composition of any one of claims 19-22, wherein the immune checkpoint molecule is covalently attached to the surface of the pancreatic or immune cell using click chemistry.

24. The composition of any one of claims 19-23, wherein the pancreatic cell is a beta cell (P cells).

25. The composition of any one of claims 10-24, further comprising at least one excipient, or at least one additional therapeutic agent or combinations thereof.

26. The composition of any one of claims 10-25, for use in the prevention or treatment of diabetes.

27. A method of making a functionalized pancreatic extracellular matrix (CP-PAN- ECM), comprising: a. providing a functionalized PAN-ECM; b. providing a functionalized immune checkpoint molecule; and c. contacting the functionalized PAN-ECM with the functionalized immune checkpoint bioconjugate to form the CP -PAN-ECM using an alkyne-azide cycloaddition (SPAAC) chemistry.

28. The method of claim 27, wherein the functionalized PAN-ECM comprises an azide moiety.

29. The method of claim 27, wherein the functionalized immune checkpoint molecule comprises a dibenzocycloctyne moiety.

30. The method of claim 29, wherein the dib enzocy cl octyne moiety comprises a DBCO, a or a derivative thereof, or a conjugate thereof.

31. The method of any one of claims 27-30, wherein the immune checkpoint molecule is a bioconjugate comprising a dendrimer, a linear polymer, a nanoparticle, or a Fc fusion protein.

32. A method of treating or reducing the progression of diabetes in a subject in need thereof, comprising administering to the subject a composition of any one of claims 10-26.

33. The method of claim 32, wherein the subject is a mammal.

34. The method of claim 32 or claim 33, wherein the administering is done via a parenteral route.

35. The method of any one of claims 32-34, wherein the diabetes is autoimmune diabetes.

36. A method of treating or reducing the progression of diabetes in a subject in need thereof, comprising administering a functionalized pancreatic extracellular matrix (CP-PAN- ECM) and a functionalized pancreatic cell, wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently attached to a pancreatic extracellular matrix (PAN-ECM); and wherein the functionalized pancreatic cell comprises at least one immune checkpoint molecule covalently attached to the surface of the pancreatic cell.

37. The method of claim 36, wherein the at least one immune checkpoint molecule covalently attached to a pancreatic extracellular matrix (PAN-ECM) or the at least one immune checkpoint molecule covalently attached to the surface of the pancreatic cell is selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1, and CD96.

38. The method of claim 36 or claim 37, wherein the at least one immune checkpoint molecule covalently attached to the pancreatic extracellular matrix (PAN-ECM) or the at least one immune checkpoint molecule covalently attached to the surface of the pancreatic cell comprises PD-L1, HVEM, FasL, or GITRL.

39. The method of any one of claims 36-38, wherein the functionalized pancreatic cell comprises HVEM and PD-L1 covalently attached to the surface of the pancreatic cell.

40. The method of any one of claims 36-39, wherein the subject is a mammal.

41. The method of any one of claims 36-40, wherein the administering is done via a parenteral route.

42. The method of any one of claims 36-41, wherein the diabetes is autoimmune diabetes.

43. A functionalized pancreatic extracellular matrix (CP-PAN-ECM) for use in a method of treating or reducing the progression of diabetes in a subject in need thereof comprising administering to the individual an effective amount of the CP-PAN-ECM and a functionalized pancreatic cell, wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently attached to a pancreatic extracellular matrix (PAN-ECM); and wherein the functionalized pancreatic cell comprises at least one immune checkpoint molecule covalently attached to the surface of the pancreatic cell.

44. The method of claim 43, wherein the at least one immune checkpoint molecule covalently attached to the pancreatic extracellular matrix (PAN-ECM) or the at least one immune checkpoint molecule covalently attached to the surface of the pancreatic cell is selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD- L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7- H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFp, FGL1, and CD96.

45. The method of claim 43 or claim 44, wherein the at least one immune checkpoint molecule covalently attached to the pancreatic extracellular matrix (PAN-ECM) or the at least one immune checkpoint molecule covalently attached to the surface of the pancreatic cell comprises PD-L1, HVEM, FasL, or GITRL.

46. The method of any one of claims 43-45, wherein the functionalized pancreatic cell comprises HVEM and PD-L1 covalently attached to the surface of the pancreatic cell.

47. The method of any one of claims 43-46, wherein the subject is a mammal.

48. The method of any one of claims 43-47, wherein the administering is done via a parenteral route.

49. The method of any one of claims 43-48, wherein the diabetes is autoimmune diabetes.

50. A functionalized pancreatic extracellular matrix comprising a structure: (PAN-ECM)— (a residue of an azide-containing molecule) — (a residue of a cyclooctyne) — (a linker 1)— (a residue of a functionalized dendrimer)q — (a residue of an immune checkpoint molecule), wherein, q is one or zero; and, the dash represents a covalent bond.

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