Immune modulation for the treatment of type 1 diabetes

Abstract

Provided herein is a composition comprising a functionalized pancreatic extracellular matrix (CP-PAN-ECM) comprising at least one immune checkpoint molecule. Also provided herein is a method of making the composition, and a method of treating diabetes using the composition.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 543,368, filed October 10, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to compositions of pancreatic extracellular matrix comprising immune checkpoint molecules, methods of their preparation, and methods of use. In one aspect, this disclosure relates to medical treatment using the disclosed compositions and methods. Background Technology

[0003] Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease characterized by high blood sugar due to the loss of beta cells. Currently, approximately 1.25 million Americans have T1DM, and the number of newly diagnosed cases is increasing.

[0004] Most patients with type 1 diabetes mellitus (T1DM) maintain their blood glucose levels through multiple daily insulin injections or insulin pump therapy. However, less than one-third of these patients consistently achieve their target blood glucose levels. Despite significant advances in disease management and care, patients with type 1 diabetes still face a higher probability of developing acute conditions such as neuropathy, nephropathy, retinopathy, and cardiovascular disease compared to the general population, and have a higher mortality rate. Because a large number of beta cells remain in the early symptom stages, the development of novel immunotherapeutic strategies to delay or even reverse early-onset T1DM is of great interest. This holds promise for helping patients regain metabolic control.

[0005] In recent years, there has been increasing interest in developing alternative treatments that could delay and reverse the progression of type 1 diabetes mellitus (T1DM) during periods of abnormal glucose metabolism and early hyperglycemia by protecting insulin-producing islets of Langerhans and suppressing autoreactive T cells infiltrating the pancreas. Summary of the Invention

[0006] In one aspect, this disclosure covers a functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently linked to the pancreatic extracellular matrix (PAN-ECM). The immune checkpoint molecule may include, 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, TGFβ, FGL1, or CD96. In one aspect, the at least one immune checkpoint molecule is linked to the PAN-ECM via click chemistry. In another aspect, the at least one immune checkpoint molecule is linked to the PAN-ECM via alkyne-azidocycloaddition (SPAAC). In one aspect, the at least one immune checkpoint molecule comprises a dibenzocyclooctyne moiety. In another aspect, the dibenzocyclooctyne moiety is DBCO, a derivative thereof, or a conjugate thereof. In another aspect, the immune checkpoint molecule is a bioconjugate comprising a dendritic macromolecule, a linear polymer, nanoparticles, or an Fc fusion protein.

[0007] In one aspect, this disclosure also covers a composition comprising a CP-PAN-ECM as described herein. In one aspect, this disclosure covers a functionalized pancreatic extracellular matrix (CP-PAN-ECM) wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently linked to the pancreatic extracellular matrix (PAN-ECM). In one aspect, the composition is a pharmaceutical composition. In one aspect, the composition further comprises pancreatic cells or immune cells. In one aspect, the cell is a pancreatic cell, such as a β cell. In some aspects, the pancreatic cell or immune cell comprises an immune checkpoint molecule covalently linked to the surface of the pancreatic cell or immune cell. In some aspects, the immune checkpoint molecule is selected from 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, TGFβ, FGL1, and CD96. In some aspects, the immune checkpoint molecule is covalently linked to the surface of pancreatic cells or immune cells via metabolic glycoengineering. In one aspect, the pharmaceutical composition comprises at least one excipient, or at least one additional therapeutic agent, or a combination thereof. In one aspect, the composition is used for the prevention or treatment of diabetes.

[0008] In one aspect, this disclosure also covers a method for preparing a functionalized pancreatic extracellular matrix (CP-PAN-ECM), comprising: providing a functionalized PAN-ECM; providing a functionalized immune checkpoint molecule; and contacting the functionalized PAN-ECM with the immune checkpoint molecule using alkyne-azidocycloaddition (SPAAC) chemistry to form CP-PAN-ECM. In one embodiment, the functionalized PAN-ECM comprises an azide moiety. In one aspect, the functionalized immune checkpoint molecule comprises a dibenzocyclooctyne moiety. In one aspect, the dibenzocyclooctyne moiety comprises DBCO, a derivative thereof, or a conjugate thereof. In one aspect, the immune checkpoint molecule is a bioconjugate comprising a dendritic macromolecule, a linear polymer, nanoparticles, or an Fc fusion protein.

[0009] In one embodiment, this disclosure also covers a method for treating diabetes in a subject of need or delaying its progression, the method comprising administering the composition disclosed herein to the subject. In one aspect, the subject is a mammal. In one aspect, the administration is via an intravenous route. In one aspect, the diabetes is autoimmune diabetes.

[0010] In one aspect, this disclosure also covers a functionalized pancreatic extracellular matrix comprising the following structure: (PAN-ECM) — (residues of an azide-containing molecule) — (cyclooctyne residues) — (linker 1) — (functionalized dendritic macromolecule residues) q — (immune checkpoint molecule residues), wherein q is 1 or 0; and the hyphens represent covalent bonds. Attached Figure Description

[0011] Various aspects of this disclosure are illustrated by way of example, wherein the same reference numerals denote similar elements, wherein: Figure 1 The diagram above shows the bioengineering of checkpoint functionalized CP-PAN-ECM and the mechanism of action of subcutaneously injected β cells with CP-PAN-ECM (bottom).

[0012] Figures 2A-2D The graph shows the subcutaneous injection of PD-L1 (…) in diabetic NOD mice. Figure 2A ), HVEM ( Figure 2B ), FasL ( Figure 2C ) or PD-L1 / HVEM / FasL ( Figure 2D Blood glucose concentration after functionalization of PAN-ECM and addition of β cells. (n=4 mice per group).

[0013] Figure 3A This demonstrates the use of engineered monofunctional or trifunctional PAN-ECM plus NIT-1 cells (1×10⁻⁶). 6 A timeline diagram illustrating the treatment of NOD mice.

[0014] Figure 3B Line graphs showing the fluctuations in blood glucose levels in diabetic NOD mice after subcutaneous injection of unmodified PAN-ECM or different monofunctional PAN-ECM plus NIT-1 cells are displayed.

[0015] Figure 3C Line graphs showing the fluctuations in blood glucose levels in diabetic NOD mice after subcutaneous injection of unmodified PAN-ECM or different trifunctional PAN-ECM plus NIT-1 cells are presented.

[0016] Figure 4A Survival curves of NOD mice treated with unmodified PAN-ECM or different monofunctional PAN-ECM plus NIT-1 cells are shown.

[0017] Figure 4B Survival curves of NOD mice treated with unmodified PAN-ECM or different trifunctional PAN-ECM plus NIT-1 cells are shown.

[0018] Figure 5A The graph is a line graph showing the blood glucose levels in NOD mice after treatment with FasL / GITRL / TGFβ / HVEM-functionalized PAN-ECM and PD-L1-NIT-1 cells.

[0019] Figure 5B The survival curves of NOD mice after treatment with FasL / GITRL / TGFβ / HVEM-functionalized PAN-ECM plus PD-L1-NIT-1 cells are shown.

[0020] Figure 6A Fluorescence activated cell sorting (FACS) maps were displayed, and the expression levels of PD-L1, HVEM, FasL and GITRL on the surface of different monofunctional NIT-1 cells were quantified.

[0021] Figure 6B This is a schematic diagram illustrating the experimental procedure for detecting the killing effect of islet antigen-specific 8.3 CD8+ T cells on engineered NIT-1 cells in the presence of exogenous IGRP peptide.

[0022] Figure 6C This is a bar chart showing the percentage of cell lysis after 18 hours of incubation of different monofunctional NIT-1 cells and expanded 8.3T cells at an E:T ratio of 5:1 in the presence of IGRP peptide. The data were determined by CytoTox non-radioactive cytotoxicity assay (n=4).

[0023] Figure 6DThis is a bar chart showing the percentage of cell lysis after 18 hours of incubation of HVEM, PD-L1, or HVEM / PD-L1 functionalized NIT-1 cells with expanded 8.3T cells at an E:T ratio of 5:1 in the presence of IGRP peptide. The data were determined by CytoTox non-radioactive cytotoxicity assay (n=4).

[0024] Figure 7A To demonstrate that NOD mice received engineered PANECM and engineered NIT-1 cells (0.5 × 10⁻⁶), 6 A schematic diagram of the treatment timeline.

[0025] Figure 7B A line graph showing the fluctuations in blood glucose levels in diabetic NOD mice after subcutaneous injection of PD-L1 / HVEM-functionalized PAN-ECM plus NIT-1 cells.

[0026] Figure 7C A line graph showing the fluctuations in blood glucose levels in diabetic NOD mice after subcutaneous injection of HVEM-functionalized PAN-ECM and PD-L1-functionalized NIT-1 cells.

[0027] Figure 8 This is a schematic diagram illustrating a method for directing the differentiation of mouse embryonic stem cells (mESCs) into insulin-secreting islet-like cell clusters (IPCs).

[0028] Figure 9A This is a bar chart showing the insulin secretion (µIU / 10) of mESCs, islet cells, and islet-like cell clusters (IPCs) under hypoglycemic and hyperglycemic conditions. 4 (cells). Data for each cell type are shown from left to right as results under low glucose conditions, followed by results under high glucose conditions.

[0029] Figure 9B Bright-field microscopy images of dithizone-stained mESCs, typical mouse islets, and IPCs are shown. Scale bar = 100 μm.

[0030] Figure 9C Fluorescence activated cell sorting (FACS) atlases were displayed, and insulin (647) and glucagon (FITC) in mESCs, islet cells and IPCs were quantified.

[0031] Figure 10A This is a schematic diagram showing the functionalization of IPCs through metabolic sugar engineering, followed by the alkyne-azidocycloaddition reaction (SPAAC) using DBCO-functionalized PD-L1 and HVEM.

[0032] Figure 10BFluorescence activated cell sorting (FACS) maps were displayed, and the expression levels of PD-L1 and HVEM in unmodified IPCs and engineered IPCs functionalized with PD-L1 and HVEM were quantified.

[0033] Figure 10C This is a schematic diagram illustrating a treatment regimen using engineered IPCs after allogeneic BABL / c mice have been induced to have diabetes by injection of streptozotocin (STZ).

[0034] Figure 10D Line graphs showing blood glucose levels in allogeneic BABL / c diabetic mice after subcutaneous injection of PBS, IPCs+PAN-ECM, and HVEM / PD-L1-IPCs+PAN-ECM.

[0035] Figure 10E Fluorescence activated cell sorting (FACS) atlases were displayed, and the expression levels of insulin and glucagon in mice treated with IPCs+PAN-ECM and HVEM / PD-L1-IPCs+PAN-ECM were quantified on day 30.

[0036] The accompanying drawings are not intended to limit this disclosure to the specific aspects disclosed and described herein. The drawings are not necessarily drawn to scale, but are intended to clearly illustrate certain principles of this disclosure. Detailed Implementation

[0037] The following detailed description refers to the accompanying drawings, which illustrate various aspects of this disclosure. The drawings and description are intended to describe various aspects of this disclosure in sufficient detail to enable those skilled in the art to implement it. Changes may be made using other components without departing from the scope of this disclosure. Therefore, the following description should not be construed as limiting.

[0038] In one respect, this disclosure stems from an unexpected discovery: when pancreatic extracellular matrix (PAN-ECM) is covalently linked to immune checkpoint molecules and injected into mice, the interaction between PAN-ECM and diabetic T cells produces a strong antigen-specific effector T cell suppression effect. The loss or mutation of co-inhibitory checkpoints is commonly associated with the progression of autoimmune diabetes. Introducing certain co-inhibitory checkpoint molecules into the islets can suppress diabetic T cells, thereby reversing early-onset hyperglycemia. Previous studies have found that pancreatic β cells containing sufficient co-inhibitory checkpoint molecules can reverse early-onset hyperglycemia in NOD mice by inhibiting the activity of diabetic T cells. This study found that pancreatic extracellular matrix (PAN-ECM) carrying checkpoint molecules can also effectively delay the progression of hyperglycemia and prolong overall survival. However, many checkpoint molecules play important roles in immune homeostasis; therefore, combinations of different checkpoint molecules may have a better reversal effect on new-onset diabetes. To achieve this goal, PAN-ECM was engineered using different checkpoint molecules, and then PAN-ECM mixed with β cells was subcutaneously injected into the vicinity of pancreatic lymph nodes, thereby constructing an immunogenic pancreatic microenvironment.

[0039] I. Terminology The wording and terminology used herein are for descriptive purposes only and should not be considered restrictive. For example, the use of singular terms such as "a" is not intended to limit the number of items. Furthermore, the use of directional terms such as, but not limited to, "top," "bottom," "left," "right," "above," "below," "downward," "upward," and "side" is for the purpose of clearly illustrating the description in conjunction with the accompanying drawings and is not intended to limit the scope of this disclosure or the appended claims.

[0040] Furthermore, since this disclosure may present various aspects in many different forms, it should be considered as an example of the principles of this disclosure and not intended to limit it to the specific aspects shown and described. Any feature of this disclosure may be used alone or in combination with any other feature. References to the terms "aspect," "multiple aspects," and / or similar terms in this specification mean that one or more features mentioned are included in at least one aspect of the specification. Individual references to the terms "aspect," "multiple aspects," and / or similar terms in the specification do not necessarily refer to the same aspect, and these aspects are not mutually exclusive unless otherwise stated and / or readily understood by a person skilled in the art from the specification. For example, a feature, structure, method, step, action, or similar content described in one aspect may also be included in other aspects, but is not necessarily included. Therefore, this disclosure may include various combinations and / or integrations of the aspects described herein. Furthermore, as stated herein, not all aspects of this disclosure are necessary conditions for its implementation. Similarly, other systems, methods, features, and advantages of this disclosure will become apparent or obvious to those skilled in the art upon review of the accompanying drawings and this specification. All such additional systems, methods, features, and advantages are intended to be included in this specification, fall within the scope of this disclosure, and are covered by the claims.

[0041] Any degree terms used in the specification and appended claims, such as, but not limited to, “substantially,” should be understood to include precise or similar but not precise configurations. For example, “substantially planar surface” refers to a surface having a precise planar shape or a similar but not precise planar shape. Similarly, terms such as “about” or “approximately” used in the specification and appended claims should be understood to include the stated value or a value three times or one-third of that value. For example, “about 3 mm” includes all values ​​from 1 mm to 9 mm, and “about 50 degrees” includes all values ​​from 16.6 degrees to 150 degrees. For example, they may 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%.

[0042] In this disclosure, the terms “comprising,” “including,” and “having” are used interchangeably. The terms “comprising,” “including,” and “having” mean including, but not limited to, the described content.

[0043] Finally, the use of "or" and "and / or" in this document should be interpreted as inclusive, or meaning any one or any combination thereof. Therefore, "A, B, or C" or "A, B, and / or C" refers to any of the following: "A", "B", or "C"; "A and B"; "A and C"; "B and C"; "A, B, and C". This definition does not apply only when the combination of elements, functions, steps, or behaviors is inherently mutually exclusive to some extent.

[0044] II. Composition Functionalized pancreatic extracellular matrix In one aspect, this disclosure covers 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 linked to the pancreatic extracellular matrix (PAN-ECM). As used herein, the functionalized pancreatic extracellular matrix comprises isolated cell-free pancreatic extracellular matrix or a component thereof, and further comprises at least one immune checkpoint molecule covalently linked. In one aspect, the immune checkpoint (CP) molecule is linked via a functionalized CP and a functionalized PAN-ECM, each comprising a suitable and complementary portion for click chemistry.

[0045] In one 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, TGFβ, FGL1, or CD96. In another aspect, the immune checkpoint molecule is PD-L1 or HVEM. In another aspect, the functionalized PAN-ECM contains at least one PD-L1, or at least one HVEM, or both.

[0046] In one respect, immune checkpoint molecules can be fusion proteins; for example, PD-L1 can be PD-L1-Ig.

[0047] PD-L1, programmed death ligand 1 (Uniprot: Q9NZQ7), is a 40 kDa type I transmembrane protein. PD-L1 is a ligand for PD-1. PD-L1 is also known as B7-H1 (B7 homolog 1).

[0048] HVEM (UniProtKB / Swiss-Prot: Q92956) is encoded by the TNFRSF14 gene. This encoded protein is involved in signal transduction pathways that activate inflammatory and suppressive T-cell immune responses. It binds to the envelope glycoprotein D (gD) of herpes simplex virus (HSV), mediating its entry into cells. Alternative splicing leads to the production of multiple transcriptomorphs.

[0049] CD86, or 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 the co-stimulatory signal necessary for T cell activation and survival.

[0050] Gal-9, or galactosyl lectin 9 (Uniprot:000182), is a 36 kDa β-galactosyl lectin protein. Gal-9 is a ligand for TIM-3.

[0051] In one aspect, the subject matter described herein relates to a functionalized CP-PAN-ECM comprising the following structure: (PAN-ECM) — (residue of an azide-containing molecule) — (cyclooctyne residue) — (linker 1) — (functionalized dendritic macromolecule residue) q — (immune checkpoint molecule residue), where q is 1 or 0; and the hyphen represents a covalent bond. In one aspect, the cyclooctyne moiety is a residue of dibenzocyclooctyne (e.g., DBCO). When q is zero, the dendritic macromolecule is absent, thereby forming a direct DBCO coupling strategy. As used herein, the term "residue" or "chemical group residue" refers to a chemical moiety bound to a molecule by which at least one covalent bond replaces at least one atom in the original chemical moiety, thereby forming a residue of that chemical moiety in the molecule.

[0052] In another aspect, the subject matter described herein relates to a functionalized CP-PAN-ECM comprising the following structure: (PAN-ECM) — (residues of an azide-containing molecule) — (cyclooctyne residues) — (linker 1) — (functionalized dendritic macromolecule residues) q — (immune checkpoint molecule Fclg fusion protein), wherein hyphens represent covalent bonds. In some aspects, the immune checkpoint molecule / immune checkpoint molecule Fclg fusion protein can be coupled via amine-NHS ester chemistry or thiol-maleimide chemistry. In another aspect, the subject matter described herein relates to a functionalized CP-PAN-ECM comprising the following structure: (PAN-ECM) — (azide-containing molecular residue) — (cyclooctyne residue) — (nanoparticle) — ((linker, e.g., linker 1) — (immune checkpoint molecule))y)x, wherein the hyphens represent covalent bonds, and x and y are as described herein.

[0053] In various aspects, this disclosure covers a functionalized pancreatic extracellular matrix (CP-PAN-ECM) wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule (e.g., about 2, about 3, about 4, or about 5 immune checkpoint molecules) or a functional fragment thereof covalently linked to the pancreatic extracellular matrix (PAN-ECM).

[0054] In some aspects, this disclosure covers a functionalized pancreatic extracellular matrix (CP-PAN-ECM) comprising about two immune checkpoint molecules or functional fragments thereof covalently linked to the pancreatic extracellular matrix (PAN-ECM). In some aspects, the about two immune checkpoint molecules are selected from 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, TGFβ, FGL1, and CD96. In some aspects, the CP-PAN-ECM comprises PD-L1 and HVEM covalently linked to the PAN-ECM.

[0055] In some aspects, this disclosure covers a functionalized pancreatic extracellular matrix (CP-PAN-ECM) wherein the CP-PAN-ECM comprises about three immune checkpoint molecules or functional fragments thereof covalently linked to the pancreatic extracellular matrix (PAN-ECM). In some aspects, the about three immune checkpoint molecules are selected from 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, TGFβ, FGL1, and CD96. In some aspects, the CP-PAN-ECM comprises PD-L1, FGL1, and TGFβ covalently linked to the PAN-ECM. In some aspects, the CP-PAN-ECM includes FasL, HVEM, and FGL1 covalently linked to the PAN-ECM. In some aspects, the CP-PAN-ECM includes FasL, TGFβ, and PD-L1 covalently linked to the PAN-ECM. In some aspects, the CP-PAN-ECM includes FasL, GITRL, and TGFβ covalently linked to the PAN-ECM. In some aspects, the CP-PAN-ECM includes PD-L1, HVEM, and FasL covalently linked to the PAN-ECM.

[0056] In other aspects, this disclosure covers a functionalized pancreatic extracellular matrix (CP-PAN-ECM) comprising about four immune checkpoint molecules or functional fragments thereof covalently linked to the pancreatic extracellular matrix (PAN-ECM). In some aspects, the about four immune checkpoint molecules are selected from 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, TGFβ, FGL1, and CD96. In some aspects, the CP-PAN-ECM comprises FasL, GITRL, TGFβ, and HVEM covalently linked to the PAN-ECM.

[0057] In some respects, PAN-ECMs can be modified using thiol-maleimide click chemistry. Typically, free thiol groups on the surface react with maleimide-functionalized biomolecules via stable thioester bonds to form stable functionalized PAN-ECMs. Maleimide-functionalized biomolecules can be prepared via an amine-NHS reaction between the target biomolecule and an NHS-maleimide crosslinking agent (e.g., sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (sulfo-SMCC)).

[0058] In some respects, the subject matter described herein relates to a functionalized CP-PAN-ECM, wherein the residues of the functionalized dendritic macromolecule have the following structure: —(dendritic macromolecule)—(linker 2)—(cyclooctyne residue)—(residue of an azide-containing molecule)—. In one respect, linker 2 has the following structure: , where z is an integer from 0 to 10.

[0059] In some aspects, z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some aspects, z is 3. In one aspect, z is an integer between 0 and 100,000. In one embodiment, z is an integer between 0 and 10, 0 and 100, 0 and 1000, 0 and 5000, or 0 and 10000. In one embodiment, z is an integer between 10 and 100,000, 100 and 100,000, 1000 and 100,000, 5000 and 100,000, or 10,000 and 100,000. In one aspect, each approximately 1 million functionalized PAN-ECM contains approximately 0.5 pg to approximately 100 pg of at least one immune checkpoint molecule covalently linked. In one aspect, each approximately 1 million functionalized PAN-ECM comprises 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 at least one immune checkpoint molecule. In one aspect, each approximately 1 million functionalized PAN-ECM contains approximately 0.5 pg, approximately 1 pg, approximately 10.0 pg, approximately 20.0 pg, approximately 30.0 pg, approximately 40.0 pg, approximately 50.0 pg, approximately 60.0 pg, or approximately 75.0 pg of at least one immune checkpoint molecule covalently linked. The total amount of immune checkpoint molecules can be quantified by fluorescence spectroscopy (using fluorescently labeled proteins) or quantitative Western blot (e.g., AutoWest).

[0060] In some respects, the at least one covalently linked immune checkpoint molecule is an immune checkpoint molecule-functionalized nanoparticle or polymer. In some respects, the covalent link is achieved through coupling with a thiol group on the PAN-ECM.

[0061] In another aspect, the immune checkpoint portion also includes residues of dendritic macromolecules, linear polymers, nanoparticles, or Fc fusion proteins. In one aspect, the nanoparticle is a dendritic macromolecule, liposome, inorganic nanoparticle, or polymer 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 some 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 about 2 nm to about 50 nm. In some aspects, the nanoparticles are 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 some aspects, the nanoparticles are 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 some embodiments, the nanoparticles are also covalently linked to one or more immune checkpoint molecules described herein via linkers. In one aspect, the dendritic macromolecule is a multivalent dendritic macromolecule. In one aspect, the multivalent dendritic macromolecule is a polyamidoamine dendritic macromolecule. In some respects, the nanoparticle is a polyethylene glycol-modified nanoparticle (e.g., DBCO-functionalized PEG-PLGA nanoparticles). In other respects, the diameter of the polyethylene glycol-modified nanoparticle is less than 200 nm.

[0062] In one aspect, the MW of the polyamide-amine dendritic macromolecule is about 500 to about 1,000,000. In another aspect, the MW of the polyamide-amine dendritic macromolecule is about 1,000 to about 1,000,000, about 5,000 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.

[0063] In one aspect, the MW of the polyamide-amine dendritic macromolecule is from about 20,000 to about 35,000. In another aspect, the MW of the polyamide-amine dendritic molecule is from about 20,000 to about 30,000. In yet another aspect, the MW of the polyamide-amine dendritic molecule is from about 25,000 to about 30,000.

[0064] In one aspect, the MW of the polyamide-amine dendritic molecule is 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 another aspect, the MW of the polyamide-amine dendritic macromolecule is about 28,000.

[0065] In one aspect, this document describes a cell-free pancreatic extracellular matrix (PAN-ECM) wherein the PAN-ECM is functionalized as described herein and contains proteins derived from the pancreas. In another embodiment, the cell-free pancreatic extracellular matrix is ​​in an injectable form. In one embodiment, the cell-free pancreatic extracellular matrix is ​​in a non-gel injectable form. In one embodiment, the cell-free pancreatic extracellular matrix is ​​in a gel injectable form. In one embodiment, the cell-free pancreatic extracellular matrix is ​​in a gel injectable form and is not a thermoresponsive hydrogel.

[0066] Pharmaceutical Composition In one embodiment, a pharmaceutical composition is described herein comprising a functionalized pancreatic extracellular matrix (CP-PAN-ECM) containing immune checkpoint molecules (CP) as described herein, and pharmaceutically acceptable excipients. In one aspect, the pharmaceutical composition may also comprise cells. In one aspect, the cells are pancreatic β cells.

[0067] In one embodiment, this document describes a vaccine comprising a functionalized pancreatic extracellular matrix (CP-PAN-ECM) and a pharmaceutically acceptable liquid carrier.

[0068] The term "vaccine" refers to a composition that can induce an immune response to prevent a subject from contracting or developing a disease or condition, and / or a vaccine has a therapeutic effect on a subject suffering from a disease or condition.

[0069] "Pharmaceutically acceptable excipient" refers to a carrier for containing a functionalized extracellular matrix (CP-PAN-ECM) that can be introduced into a subject without producing significant adverse effects and without harmful effects on cell-free extracellular matrix. In other words, "pharmaceutically acceptable" means any safe formulation capable of delivering at least one CP-PAN-ECM at an effective dose via the intended route of administration for use in the methods of this disclosure. Pharmaceutically acceptable carriers, delivery agents, or excipients are well known. Descriptions of suitable pharmaceutically acceptable carriers and the factors involved in their selection can be found in a variety of readily available sources, such as Remington's Pharmaceutical Sciences, 18th edition (1990), the entire contents of which are incorporated herein by reference for all purposes. Such carriers are suitable for any route of administration (e.g., parenteral, enteral (e.g., oral), or topical). Such pharmaceutical compositions may be buffered, for example, wherein the pH is maintained within a specific desired range, from pH 4.0 to pH 9.0, depending on the stability of the CP-PAN-ECM and the route of administration.

[0070] Suitable pharmaceutically acceptable carriers include, for example, sterile water, saline solutions (such as physiological saline), glucose, buffer solutions (such as phosphate buffer or bicarbonate buffer), alcohols, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, sugars (such as lactose, amylose, or starch), magnesium stearate, talc, silica, viscous paraffin, white paraffin, glycerol, alginate, hyaluronic acid, collagen, aromatic oils, monoglycerides and diglycerides of fatty acids, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc. Pharmaceutical compositions or vaccines may also contain excipients such as diluents, stabilizers (such as sugars and amino acids), preservatives, wetting agents, emulsifiers, pH buffers, thickeners, lubricants, salts for adjusting osmotic pressure, buffer solutions, vitamins, colorants, flavoring agents, aromatic substances, etc., and these components do not react harmfully with functionalized cells or cell-free extracellular matrix.

[0071] For liquid formulations, 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, alcohol / aqueous solutions, emulsions, or suspensions, including physiological saline and buffer media. Examples of oils include petroleum-derived, animal-, plant-, or synthetic oils, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and cod liver oil. Solid carriers / diluents include, for example, gums, starches (such as corn starch, pregelatinized starch), sugars (such as lactose, mannitol, sucrose, or glucose), cellulose materials (such as microcrystalline cellulose), acrylates (such as polymethyl methacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[0072] Optionally, sustained-release or targeted-release pharmaceutical compositions or vaccines can be prepared. For example, this can be achieved using liposomes or compositions protected by differentially degradable coatings of the active compound (e.g., microencapsulation, multilayer coating, etc.). Such compositions can be formulated as immediate-release or sustained-release. The compositions can also be lyophilized, and the resulting lyophilized powder can be used (e.g., for the preparation of injectable formulations).

[0073] Treatment In another embodiment, the subject matter described herein relates to a method for treating or delaying the onset of diabetes in a subject, the method comprising administering to the subject a composition comprising the CP-PAN-ECM described herein. In one embodiment, a pharmaceutical composition or vaccine comprising CP-PAN-ECM is administered to the subject.

[0074] In some aspects, the present invention provides a method for treating or delaying the progression of diabetes in a subject of need, the method comprising administering a functionalized pancreatic extracellular matrix (CP-PAN-ECM) and functionalized pancreatic cells, wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently linked to the pancreatic extracellular matrix (PAN-ECM); and wherein the functionalized pancreatic cells comprise at least one immune checkpoint molecule covalently linked to the surface of the pancreatic cells.

[0075] In other respects, the present invention provides a method for treating diabetes in a subject of need or delaying its progression using a functionalized pancreatic extracellular matrix (CP-PAN-ECM), the method comprising administering to an individual an effective amount of CP-PAN-ECM and functionalized pancreatic cells, wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently linked to the pancreatic extracellular matrix (PAN-ECM); and the functionalized pancreatic cells comprise at least one immune checkpoint molecule covalently linked to the surface of the pancreatic cells.

[0076] In some embodiments, type 1 diabetes is early-onset type 1 diabetes or early-onset hyperglycemia. In another embodiment, the subject matter herein relates to a method for reversing early-onset type 1 diabetes in a subject, the method comprising administering a functionalized CP-PAN-ECM or a pharmaceutical composition or vaccine containing thereof to the subject. In another embodiment, the subject matter herein relates to a method for reversing early-onset type 1 diabetes in a subject, the method comprising administering β-cells and a functionalized CP-PAN-ECM, or a pharmaceutical composition or vaccine containing thereof to the subject. In some embodiments, the subject matter herein relates to a method for protecting pancreatic β-cells in a subject, the method comprising administering a CP-PAN-ECM, or a pharmaceutical composition or vaccine containing thereof to the subject.

[0077] In one respect, treatment includes improving or preventing the worsening of existing disease symptoms, preventing the occurrence of additional symptoms, improving or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition (e.g., preventing the development of the disease or condition), alleviating the disease or condition, promoting the resolution of the disease or condition, relieving the condition caused by the disease or condition, or terminating the symptoms of the disease or condition.

[0078] The term "treatment" refers to both therapeutic and preventative measures aimed at preventing, alleviating, or reducing the severity of symptoms of autoimmune diseases. Treatment may include one or more of the following: directly affecting or curing an autoimmune disease; suppressing an autoimmune disease; blocking an autoimmune disease; preventing an autoimmune disease; reducing the severity of an autoimmune disease; delaying the onset of an autoimmune disease; slowing the progression of an autoimmune disease; stabilizing the progression of an autoimmune disease; reducing / alleviating symptoms associated with an autoimmune disease; or any combination thereof. The term "reducing severity" refers to a clinically or subjectively determined reduction in symptoms or signs after treatment.

[0079] The term "subject" refers to a mammal (e.g., a human) that requires or is susceptible to diabetes treatment. The term "subject" also refers to a mammal (e.g., a human) receiving preventative or therapeutic treatment. Subjects may include dogs, cats, pigs, cattle, sheep, goats, horses, rats, mice, non-human mammals, and humans. The term "subject" does not necessarily exclude individuals who are otherwise healthy and do not have diabetes or show signs of diabetes.

[0080] As used herein, the term “organism” includes, but is not limited to, humans, the aforementioned non-human primates and any genetically modified species thereof, and also includes any living eukaryote.

[0081] The term "effective amount" or "therapeutic effective amount" refers to an amount of composition sufficient to produce the desired biological outcome. This outcome can be a reduction and / or alleviation of signs, symptoms, or causes of a disease or condition, or any other anticipated change in a biological system. For example, an "effective amount" for treatment refers to an amount of composition required to produce a clinically relevant change in a disease state, symptom, or condition. In any specific case, a person skilled in the art can determine an appropriate "effective" amount through routine experiments. Therefore, the term "effective amount" generally refers to an amount of active substance that produces the desired therapeutic effect. The effective amount or dosage of the composition in each embodiment can be determined by conventional methods, such as modeling, dose escalation, or clinical trials, taking into account conventional factors such as the manner or route of administration or drug delivery, the pharmacokinetics of the drug, the severity and duration of the infection, the subject's health status, physical condition, and weight, and the judgment of the attending physician. Exemplary dosage ranges are from approximately 1 pg to 10 mg of active ingredient per kilogram of subject body weight per day. The total dose can be administered as a single dose or in divided doses (e.g., twice, three, or four times daily). Once the patient's condition improves, the dosage can be adjusted for prophylactic or maintenance therapy. For example, the dosage or frequency of administration (or both) may be reduced depending on the symptoms until the desired therapeutic or preventative effect is maintained. Of course, treatment can be discontinued once symptoms have subsided to an appropriate level. However, if symptoms recur, patients may require long-term intermittent treatment. Patients may also require long-term chronic treatment.

[0082] In some aspects, this disclosure provides compositions formulated for use in one or more routes of administration. Suitable routes of administration may include, for example, parenteral administration. In some aspects, the compositions formulated in this disclosure are formulated for parenteral administration. In some aspects, the compositions formulated in this disclosure are formulated for intramuscular, subcutaneous, intramedullary, or intravenous injection.

[0083] In some respects, the compositions herein may be administered in a local or systemic manner, for example by direct local injection of the pharmaceutical composition into a patient's tissue area. In some respects, the pharmaceutical compositions disclosed herein may be administered via a parenteral route.

[0084] In some respects, the pharmaceutical compositions disclosed herein can be prepared by methods known in the art, such as conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, embedding, or lyophilizing processes.

[0085] In some respects, the pharmaceutical compositions applicable according to this disclosure can be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and adjuvants that facilitate the formulation of the active ingredient into a pharmaceutically usable formulation. The appropriate formulation depends on the chosen route of administration. For injection, the active ingredient of the pharmaceutical compositions herein can be formulated as an aqueous solution, preferably in a physiologically compatible buffer solution, such as Hank's solution, Ringer's solution, physiological saline buffer, or any combination thereof.

[0086] In some respects, the pharmaceutical compositions described herein can be formulated into nanoparticle form.

[0087] In several aspects, the treatment method includes administering an effective amount of a monofunctional PAN-ECM to a subject in need, wherein the monofunctional PAN-ECM comprises an immune checkpoint molecule covalently linked to the pancreatic extracellular matrix (PAN-ECM). In some aspects, the immune checkpoint molecule is selected from 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, TGFβ, FGL1, and CD96. In some aspects, the method also includes administering an effective amount of functional pancreatic cells or immune cells to the subject. In some aspects, the functional pancreatic cells are functional β cells. In some aspects, the functionalized pancreatic cell or immune cell includes one or more immune checkpoint proteins covalently linked to the surface of the pancreatic cell or immune cell, wherein the immune checkpoint protein includes a protein selected from 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, TGFβ, FGL1, and CD96. In some aspects, the functionalized pancreatic cell or immune cell includes one or more immune checkpoint proteins covalently linked to the surface of the pancreatic cell or immune cell, wherein the immune checkpoint protein includes PD-L1.

[0088] In other aspects, the functional pancreas or immune cell includes one or more immune checkpoint proteins covalently linked to the surface of the pancreas or immune cell, wherein the immune checkpoint protein includes HVEM. In some aspects, the functional pancreas or immune cell includes one or more immune checkpoint proteins covalently linked to the surface of the pancreas or immune cell, wherein the immune checkpoint protein includes FasL. In some aspects, the functional pancreas or immune cell includes one or more immune checkpoint proteins covalently linked to the surface of the pancreas or immune cell, wherein the immune checkpoint protein includes GITRL. In some aspects, the functional pancreas or immune cell includes two immune checkpoint proteins covalently linked to the surface of the pancreas or immune cell, wherein the two immune checkpoint proteins include HVEM and PD-L1.

[0089] In some aspects, the method includes administering an effective amount of a monofunctional PAN-ECM to a subject in need, wherein the monofunctional PAN-ECM comprises a PD-L1-functional PAN-ECM. In some aspects, the method involves administering a pharmaceutical composition or vaccine comprising a PD-L1-PAN-ECM to the subject.

[0090] In another aspect, the method includes administering an effective amount of a monofunctional PAN-ECM, wherein the monofunctional PAN-ECM comprises an HVEM-functionalized PAN-ECM, to a subject in need. In some aspects, a pharmaceutical composition or vaccine comprising HVEM-PAN-ECM is administered to the subject.

[0091] In some aspects, the method includes administering an effective amount of a monofunctional PAN-ECM, wherein the monofunctional PAN-ECM comprises FasL-functionalized PAN-ECM, to a subject in need. In other aspects, the method involves administering a pharmaceutical composition or vaccine comprising FasL-PAN-ECM to the subject.

[0092] In some aspects, the method includes administering an effective amount of a monofunctional PAN-ECM to a subject in need, wherein the monofunctional PAN-ECM comprises an FGL1-functionalized PAN-ECM. In some aspects, the method involves administering a pharmaceutical composition or vaccine comprising an FGL1-PAN-ECM to the subject.

[0093] In another aspect, the method includes administering an effective amount of a monofunctional PAN-ECM to a subject in need, wherein the monofunctional PAN-ECM comprises a PD-L1-functionalized PAN-ECM. In some aspects, the method further includes administering an effective amount of functionalized pancreatic cells or immune cells to the subject. In some aspects, the functionalized pancreatic or immune cells comprise one or more immune checkpoint proteins covalently linked to the surface of the pancreatic or immune cells, wherein the immune checkpoint proteins comprise proteins selected from 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, TGFβ, FGL1, and CD96. In some aspects, the functionalized pancreatic cells are functionalized β cells. In some respects, a drug composition or vaccine comprising PD-L1-PAN-ECM and functionalized pancreatic cells or immune cells is administered to the subject.

[0094] In some aspects, the method includes administering an effective amount of a monofunctional PAN-ECM to a subject in need, wherein the monofunctional PAN-ECM comprises an HVEM-functionalized PAN-ECM. In some aspects, the method further includes administering an effective amount of functionalized pancreatic cells or immune cells to the subject. In some aspects, the functionalized pancreatic or immune cells comprise one or more immune checkpoint proteins covalently linked to the surface of the pancreatic or immune cells, wherein the immune checkpoint proteins comprise proteins selected from 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, TGFβ, FGL1, and CD96. In some aspects, the functionalized pancreatic cells are functionalized β cells. In some aspects, the method involves administering to a subject a pharmaceutical composition or vaccine comprising HVEM-PAN-ECM and functionalized pancreatic cells or immune cells. In some aspects, the method includes administering to a subject in need an effective amount of a monofunctionalized PAN-ECM comprising FasL-functionalized PAN-ECM. In some aspects, the method further includes administering to the subject an effective amount of functionalized pancreatic cells or immune cells. In some aspects, the functionalized pancreatic or immune cell comprises one or more immune checkpoint proteins covalently linked to the surface of the pancreatic or immune cell, wherein the immune checkpoint protein comprises a protein selected from 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, TGFβ, FGL1, and CD96. In some aspects, the functionalized pancreatic cell is a functionalized β cell. In some aspects, a pharmaceutical composition or vaccine comprising FasL-PAN-ECM and functionalized pancreatic or immune cells is administered to a subject.

[0095] In some aspects, the method includes administering an effective amount of a monofunctional PAN-ECM to a subject in need, wherein the monofunctional PAN-ECM comprises an FGL1-functionalized PAN-ECM. In some aspects, the method further includes administering an effective amount of functionalized pancreatic cells or immune cells to the subject. In some aspects, the functionalized pancreatic or immune cells comprise one or more immune checkpoint proteins covalently linked to the surface of the pancreatic or immune cells, wherein the immune checkpoint proteins comprise proteins selected from 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, TGFβ, FGL1, and CD96. In some aspects, the functionalized pancreatic cells are functionalized β cells. In some respects, subjects are given a pharmaceutical composition or vaccine containing FGL1-PAN-ECM and functionalized pancreatic cells or immune cells.

[0096] In another aspect, the treatment method includes administering an effective amount of a bifunctional PAN-ECM to a subject in need, wherein the bifunctional PAN-ECM comprises two immune checkpoint molecules covalently linked to the pancreatic extracellular matrix (PAN-ECM). In some aspects, the immune checkpoint molecules are selected from 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, TGFβ, FGL1, and CD96. In some aspects, the method also includes administering an effective amount of functional pancreatic cells or immune cells to the subject. In some aspects, the functional pancreatic cells are functional β cells. In some respects, the functionalized pancreatic cell or immune cell contains one or more immune checkpoint proteins covalently linked to the surface of the pancreatic cell or immune cell, wherein the immune checkpoint protein comprises a protein selected from 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, TGFβ, FGL1, and CD96.

[0097] In some aspects, the method includes administering an effective amount of a bifunctional PAN-ECM to a subject in need, wherein the bifunctional PAN-ECM comprises a PD-L1-HVEM-functionalized PAN-ECM. In some aspects, the method involves administering a pharmaceutical composition or vaccine comprising PD-L1-HVEM-PAN-ECM to the subject.

[0098] In some aspects, the method includes administering an effective amount of a bifunctional PAN-ECM to a subject in need, wherein the bifunctional PAN-ECM comprises a PD-L1-HVEM-functionalized PAN-ECM. In some aspects, the method further includes administering an effective amount of functionalized pancreatic cells or immune cells to the subject. In some aspects, the functionalized pancreatic or immune cells comprise one or more immune checkpoint proteins covalently linked to the surface of the pancreatic or immune cells, wherein the immune checkpoint proteins comprise proteins selected from 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, TGFβ, FGL1, and CD96. In some aspects, the functionalized pancreatic cells are functionalized β cells. In some respects, subjects are given a pharmaceutical composition or vaccine containing PD-L1-HVEM-PAN-ECM and functionalized pancreatic cells or immune cells.

[0099] In several aspects, the treatment method includes administering an effective amount of a trifunctional PAN-ECM to a subject in need, wherein the trifunctional PAN-ECM comprises three immune checkpoint molecules covalently linked to the pancreatic extracellular matrix (PAN-ECM). In some aspects, the immune checkpoint molecules are selected from 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, TGFβ, FGL1, and CD96. In some aspects, the method also includes administering an effective amount of functional pancreatic cells or immune cells to the subject. In some aspects, the functional pancreatic cells are functional β cells. In some respects, the functionalized pancreatic cell or immune cell contains one or more immune checkpoint proteins covalently linked to the surface of the pancreatic cell or immune cell, wherein the immune checkpoint protein comprises a protein selected from 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, TGFβ, FGL1, and CD96.

[0100] In various other aspects, the method includes administering an effective amount of a trifunctional PAN-ECM to a subject in need, wherein the trifunctional PAN-ECM comprises a PD-L1-FGL1-TGFβ-functionalized PAN-ECM. In some aspects, the method involves administering a pharmaceutical composition or vaccine comprising a PD-L1-FGL1-TGFβ-functionalized PAN-ECM to the subject.

[0101] In another aspect, the method includes administering an effective amount of a trifunctional PAN-ECM to a subject in need, wherein the trifunctional PAN-ECM comprises a FasL-HVEM-FGL1 functionalized PAN-ECM. In some aspects, the method involves administering a pharmaceutical composition or vaccine comprising a FasL-HVEM-FGL1 functionalized PAN-ECM to the subject.

[0102] In some aspects, the method includes administering an effective amount of a trifunctional PAN-ECM to a subject in need, wherein the trifunctional PAN-ECM comprises a FasL-TGFβ-PD-L1 functionalized PAN-ECM. In some aspects, the subject is administered a pharmaceutical composition or vaccine comprising the FasL-TGFβ-PD-L1 functionalized PAN-ECM.

[0103] In some aspects, the method includes administering an effective amount of a trifunctional PAN-ECM to a subject in need, wherein the trifunctional PAN-ECM comprises a FasL-GITRL-TGFβ-functionalized PAN-ECM. In some aspects, the method involves administering to the subject a pharmaceutical composition or vaccine comprising the FasL-GITRL-TGFβ-functionalized PAN-ECM.

[0104] In another aspect, the method includes administering an effective amount of a trifunctional PAN-ECM to a subject in need, wherein the trifunctional PAN-ECM comprises a PD-L1-HVEM-FasL-functionalized PAN-ECM. In some aspects, the method involves administering a pharmaceutical composition or vaccine comprising a PD-L1-HVEM-FasL-functionalized PAN-ECM to the subject.

[0105] In some aspects, the method includes administering an effective amount of a trifunctional PAN-ECM to a subject in need, wherein the trifunctional PAN-ECM comprises a PD-L1-FGL1-TGFβ-functionalized PAN-ECM. In some aspects, the method further includes administering an effective amount of functionalized pancreatic cells or immune cells to the subject. In some aspects, the functionalized pancreatic or immune cells comprise one or more immune checkpoint proteins covalently linked to the surface of the pancreatic or immune cells, wherein the immune checkpoint proteins comprise a protein selected from 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, TGFβ, FGL1, and CD96. In some respects, the functional pancreatic cells are functional β cells. In some respects, a pharmaceutical composition or vaccine comprising PD-L1-FGL1-TGFβ-PAN-ECM and functional pancreatic cells or immune cells is administered to the subject.

[0106] In various aspects, the method includes administering an effective amount of a trifunctional PAN-ECM to a subject in need, wherein the trifunctional PAN-ECM comprises a FasL-HVEM-FGL1 functionalized PAN-ECM. In some aspects, the method further includes administering an effective amount of functionalized pancreatic cells or immune cells to the subject. In some aspects, the functionalized pancreatic or immune cells comprise one or more immune checkpoint proteins covalently linked to the surface of the pancreatic or immune cells, wherein the immune checkpoint proteins comprise proteins selected from 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, TGFβ, FGL1, and CD96. In some respects, the functional pancreatic cells are functional β cells. In some respects, a pharmaceutical composition or vaccine comprising FasL-HVEM-FGL1 functionalized PAN-ECM and functionalized pancreatic cells or immune cells is administered to the subject.

[0107] In some aspects, the method includes administering an effective dose of a trifunctional PAN-ECM to a subject in need, wherein the trifunctional PAN-ECM comprises a FasL-TGFβ-PD-L1 functionalized PAN-ECM. In some aspects, the method further includes administering an effective amount of functionalized pancreatic cells or immune cells to the subject. In some aspects, the functionalized pancreatic or immune cells comprise one or more immune checkpoint proteins covalently linked to the surface of the pancreatic or immune cells, wherein the immune checkpoint proteins comprise a protein selected from 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, TGFβ, FGL1, and CD96. In some respects, the functional pancreatic cells are functional β cells. In some respects, a pharmaceutical composition or vaccine comprising FasL-TGFβ-PD-L1-PAN-ECM and functional pancreatic cells or immune cells is administered to the subject.

[0108] In another aspect, the method includes administering an effective amount of a trifunctional PAN-ECM to a subject in need, wherein the trifunctional PAN-ECM comprises a FasL-GITRL-TGFβ functionalized PAN-ECM. In some aspects, the method further includes administering an effective amount of functionalized pancreatic cells or immune cells to the subject. In some aspects, the functionalized pancreatic or immune cells comprise one or more immune checkpoint proteins covalently linked to the surface of the pancreatic or immune cells, wherein the immune checkpoint proteins comprise proteins selected from 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, TGFβ, FGL1, and CD96. In some respects, the functional pancreatic cells are functional β cells. In some respects, a pharmaceutical composition or vaccine comprising FasL-GITRL-TGFβ-PAN-ECM and functional pancreatic cells or immune cells is administered to the subject.

[0109] In some aspects, the method includes administering an effective amount of a trifunctional PAN-ECM to a subject in need, wherein the trifunctional PAN-ECM comprises a PD-L1-HVEM-FasL functionalized PAN-ECM. In some aspects, the method further includes administering an effective amount of functionalized pancreatic cells or immune cells to the subject. In some aspects, the functionalized pancreatic or immune cells comprise one or more immune checkpoint proteins covalently linked to the surface of the pancreas or immune cells, wherein the immune checkpoint proteins comprise a protein selected from 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, TGFβ, FGL1, and CD96. In some respects, the functional pancreatic cells are functional β cells. In some respects, a pharmaceutical composition or vaccine comprising PD-L1-HVEM-FasL-PAN-ECM and functional pancreatic cells or immune cells is administered to the subject.

[0110] In some aspects, the treatment method includes administering an effective dose of a tetrafunctional PAN-ECM to a subject in need, wherein the tetrafunctional PAN-ECM comprises four immune checkpoint molecules covalently linked to the pancreatic extracellular matrix (PAN-ECM). In some aspects, the immune checkpoint molecules are selected from 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, TGFβ, FGL1, and CD96. In some aspects, the method also includes administering an effective amount of functional pancreatic cells or immune cells to the subject. In some aspects, the functional pancreatic cells are functional β cells. In some respects, the functionalized pancreatic cell or immune cell contains one or more immune checkpoint proteins covalently linked to the surface of the pancreatic cell or immune cell, wherein the immune checkpoint protein comprises a protein selected from 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, TGFβ, FGL1, and CD96.

[0111] In another aspect, the method includes administering an effective amount of a tetrafunctional PAN-ECM to a subject in need, wherein the tetrafunctional PAN-ECM comprises a FasL-GITRL-TGFβ-HVEM-functionalized PAN-ECM. In some aspects, the method involves administering a pharmaceutical composition or vaccine comprising a FasL-GITRL-TGFβ-HVEM-functionalized PAN-ECM to the subject.

[0112] In various other aspects, the method includes administering an effective amount of a tetrafunctional PAN-ECM to a subject in need, wherein the tetrafunctional PAN-ECM comprises a FasL-GITRL-TGFβ-HVEM-functionalized PAN-ECM. In some aspects, the method further includes administering an effective amount of functionalized pancreatic cells or immune cells to the subject. In some aspects, the functionalized pancreatic or immune cells comprise one or more immune checkpoint proteins covalently linked to the surface of the pancreatic or immune cells, wherein the immune checkpoint proteins comprise proteins selected from 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, TGFβ, FGL1, and CD96. In some respects, the functional pancreatic cells are functional β cells. In some respects, a pharmaceutical composition or vaccine comprising FasL-GITRL-TGFβ-HVEM-PAN-ECM and functional pancreatic cells or immune cells is administered to the subject.

[0113] III. Preparation Method In one aspect, this disclosure also covers a method for preparing the disclosed compositions. A method for preparing a functionalized pancreatic extracellular matrix (CP-PAN-ECM) may include providing a functionalized PAN-ECM, wherein the PAN-ECM is functionalized to have a moiety of residues suitable for click chemistry; providing a functionalized immune checkpoint molecule, wherein the immune checkpoint is functionalized to have a moiety of residues suitable for click chemistry; and contacting the azide-modified PAN-ECM with an immune checkpoint bioconjugate to form CP-PAN-ECM using an alkyne-azidocycloaddition (SPAAC) chemistry. In one aspect, the functionalized PAN-ECM comprises an azide moiety. In one aspect, the functionalized immune checkpoint molecule comprises a dibenzocyclooctyne moiety. In one aspect, the immune checkpoint molecule is a bioconjugate that further comprises a dendritic macromolecule, a linear polymer, nanoparticles, or an Fc fusion protein. In one aspect, the dibenzocyclooctyne moiety comprises DBCO, a derivative thereof, or a conjugate thereof. Optionally, a purification step may be performed after any of the reactions described herein to improve the purity of the disclosed compositions.

[0114] In some respects, PAN-ECM is prepared from decellularized pancreatic tissue. In some respects, the decellularized pancreatic tissue is prepared by decellularizing pancreatic tissue isolated from a mammal. In some respects, the mammal is a rodent, such as a mouse or rat. In some respects, the pancreatic tissue is decellularized using a detergent (e.g., Triton-X-100) and an aqueous ammonia solution. In some respects, the pancreatic tissue is decellularized using a mixed solution of 1% Triton-X-100 and 0.1% ammonia.

[0115] The examples provided in this article illustrate an exemplary method for preparing and using the disclosed CP-PAN-ECM.

[0116] Implementation 1. A functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently linked to the pancreatic extracellular matrix (PAN-ECM).

[0117] 2. The CP-PAN-ECM according to Embodiment 1, wherein the immune checkpoint molecule includes 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.

[0118] 3. The CP-PAN-ECM according to Embodiment 1, wherein the at least one immune checkpoint molecule is linked to the PAN-ECM via click chemistry.

[0119] 4. The CP-PAN-ECM according to embodiment 3, wherein the at least one immune checkpoint molecule is chemically linked to the PAN-ECM via an acetylation-azidocycloaddition (SPAAC) reaction.

[0120] 5. The CP-PAN-ECM according to Embodiment 4, wherein the at least one immune checkpoint molecule comprises a dibenzocyclooctylene moiety.

[0121] 6. The CP-PAN-ECM according to Embodiment 5, wherein the dibenzocyclooctyne moiety is DBCO, or a derivative thereof, or a coupling thereof.

[0122] 7. The CP-PAN-ECM according to Embodiment 5, wherein the immune checkpoint molecule is a bioconjugate comprising a dendritic macromolecule, a linear polymer, nanoparticles, or an Fc fusion protein.

[0123] 8. A composition comprising the CP-PAN-ECM according to any one of embodiments 1-7.

[0124] 9. A composition comprising functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently linked to the pancreatic extracellular matrix (PAN-ECM).

[0125] 10. The composition according to embodiment 8 or 9, wherein the composition is a pharmaceutical composition.

[0126] 11. The composition according to any one of embodiments 8-10, wherein the composition further comprises pancreatic cells or immune cells.

[0127] 12. The composition according to embodiment 11, wherein the pancreatic cells are β cells.

[0128] 13. The composition according to any one of embodiments 8-12 further comprises at least one excipient, or at least one additional therapeutic agent, or a combination thereof.

[0129] 14. The composition according to any one of embodiments 8-13 is used for the prevention or treatment of diabetes.

[0130] 15. A method for preparing functionalized pancreatic extracellular matrix (CP-PAN-ECM), comprising: a. Provide functionalized PAN-ECM; b. Provide functionalized immune checkpoint molecules; and c. Using acetylation-azidocycloaddition (SPAAC) chemistry, functionalized PAN-ECM is brought into contact with functionalized immune checkpoint bioconjugates to form CP-PAN-ECM.

[0131] 16. The method according to embodiment 15, wherein the functionalized PAN-ECM comprises an azide portion.

[0132] 17. The method according to embodiment 15, wherein the functionalized immune checkpoint molecule comprises a dibenzocyclooctylene moiety.

[0133] 18. The method according to embodiment 17, wherein the dibenzocyclooctyne moiety comprises DBCO, or a derivative thereof, or a coupling thereof.

[0134] 19. The method according to embodiment 18, wherein the immune checkpoint molecule is a bioconjugate comprising a dendritic macromolecule, a linear polymer, nanoparticles, or an Fc fusion protein.

[0135] 20. A method for treating diabetes in a subject in need or delaying its progression, comprising administering to the subject the composition of any one of embodiments 8-14.

[0136] 21. The method according to embodiment 20, wherein the subject is a mammal.

[0137] 22. The method according to embodiment 21, wherein the administration is performed via a parenteral route.

[0138] 23. The method according to embodiment 21, wherein the diabetes is autoimmune diabetes.

[0139] 24. A functionalized pancreatic extracellular matrix comprising the following structure: (PAN-ECM) — (residues of an azide-containing molecule) — (cyclooctyne residues) — (linker 1) — (residues of a functionalized dendritic macromolecule) q — (residues of an immune checkpoint molecule), wherein q is 1 or 0; and the hyphen represents a covalent bond.

[0140] Example The following embodiments are intended to illustrate preferred aspects of this disclosure. Those skilled in the art should understand that the techniques disclosed in the following embodiments are techniques discovered by the inventors that perform well in implementation of this disclosure, and therefore can be considered preferred embodiments thereof. However, those skilled in the art should understand that many modifications can be made to the specific aspects disclosed based on this disclosure, while still obtaining the same or similar results without departing from the spirit and scope of this disclosure.

[0141] Example 1: Preparation and decellularization of the pancreas The pancreas of 6-month-old healthy live rats was frozen in vitro at -80°C for 12 hours. The connective tissue and adipose tissue surrounding the pancreas were dissected, and the pancreas was cut into pieces approximately 0.5-1 cm in size. 3 The pancreatic tissue blocks were prepared in blocks of varying sizes. The blocks were rinsed three times with cold PBS for 1 hour each time. A 1% Triton-X-100 / 0.1% ammonia solution was prepared by adding Triton-X-100 and ammonia (from Guangdong, China) to the PBS. The pancreatic tissue blocks were then added to the Triton-X-100 solution and shaken at 4°C for 72 hours. The Triton-X-100 solution was changed every 24 hours for a total of three times. Subsequently, the blocks were rinsed three times with PBS containing penicillin / streptomycin at 4°C for 12 hours each time to thoroughly remove the detergent. Cell-free pancreatic tissue was stored at -80°C.

[0142] Azide-modified PAN-ECM was prepared by reacting carbodiimide / N-hydroxysuccinimide (EDC / NHS) with azide-PEG8-NHS. Before attaching checkpoints to the azide-modified PAN-ECM, DBCO-functionalized checkpoint bioconjugates were first prepared via a bioorthogonal click reaction between the checkpoints and DBCO-dendritic macromolecules. Subsequently, checkpoint-functionalized PAN-ECM was prepared using a bioorthogonal click reaction between the DBCO-functionalized checkpoint bioconjugates and the azide-functionalized PAN-ECM. The therapeutic effects of different subcutaneously injected types of checkpoint-functionalized PAN-ECM plus β-cell therapy were evaluated in newly diagnosed diabetic (NOD) female mice 2-3 days after the onset of diabetes. Furthermore, two booster treatments were administered at 2 and 4 weeks after the initial treatment.

[0143] Example 2: Preparation of DBCO-functionalized PAMAMG5 First, PAMAMG5 (0.7 mL of 5 wt% PAMAMG5 methanol solution containing 27.9 mg of dendritic macromolecules, 0.97 μmol) was dried under nitrogen at 20 °C for 15 min, followed by further drying under vacuum for 2 h. DBCO-PEG4-NHS ester (764 μL, 25 mM, dissolved in DMSO, 12.3 mg) was added to the dried dendritic macromolecule film. The mixture was stirred at 20 °C (protected from light) for 4 h. An excess of anhydrous acetic anhydride (0.5 mL) was added to the reaction mixture, and stirring was continued at 20 °C (protected from light) for 18 h. The DBCO-functionalized dendritic macromolecules were further purified by equilibration dialysis under light conditions for three days (six cycles). The purified dendritic macromolecules were lyophilized and stored at -80 °C under light conditions for subsequent studies.

[0144] Example 3: Preparation of azide-functionalized PD-L1 Azide-functionalized PD-L1 (PD-L1-N3) was synthesized via an amine-NHS ester coupling reaction. The target degree of functionalization was 20. For typical functionalization, 10 μg (0.194 nmol, dissolved in 100 μL PBS) of PD-L1 was incubated with an azide-PEG4-NHS solution (3.88 nmol, 10 μL of 0.388 mM azide-PEG4-NHS) at 20 °C for 1.5 h. The functionalized PD-L1 was purified three times using a Zebra Spin 7K MWCO desalting column according to the manufacturer's protocol. The concentration of the purified azide-functionalized PD-L1 was quantified by UV-Vis spectroscopy.

[0145] Azide-functionalized TGF-β (TGF-β-N3), azido-functionalized HVEM (HVEM-N3), azido-functionalized FasL (FasL-N3), azido-functionalized CD47 (CD47-N3), azido-functionalized GITRL (GITRL-N3), and azido-functionalized Gal-9 (Gal-9-N3) were all synthesized using the same method, but the target functionalization degree for TGF-β, FasL, CD47, and Gal-9 was 10. Figure 1 A bioengineering diagram of checkpoint-functionalized PAN-ECM is provided (top image), as well as a diagram illustrating the mechanism of action of subcutaneous injection of β cells with PAN-ECM (bottom image).

[0146] Example 4: Preparation of PD-L1-Dend bioconjugate Purified azide-functionalized PD-L1 was quantitatively conjugated with DBCO-functionalized PAMAMMG5 at a target molar ratio of 1:1. PD-L1-N3 (10 μg) was mixed with DBCO-functionalized PAMAMMG5 dendritic macromolecules (8 μg, 0.8 μL of 10 mg / mL PBS dendritic macromolecule solution) and incubated at 4 °C for 24 h. The resulting PD-L1-Dend bioconjugate was ready for subsequent studies without further purification.

[0147] The TGF-β-Dend, HVEM-Dend, FasL-Dend, CD47-Dend, GITRL-Dend, and Gal-9-Dend bioconjugates were all prepared using the same method.

[0148] Example 5: Preparation of PAN-ECM modified with checkpoint molecules PAN-ECM (100 mg) was washed twice with PBS and then resuspended in PBS at a concentration of 350 mg / mL. Azide-PEG8-NHS (20 µL, 285 mM, dissolved in DMSO) was then added to PAN-ECM and incubated overnight at 4 °C. PD-L1-N3 (10 μg) was mixed with DBCO-functionalized PAMAMG5 dendrimer (8 μg, 0.8 μL of 10 mg / mL PBS dendrimer solution) and incubated at 37 °C for 30 min. Simultaneously, azide-PAN-ECM was washed three times and resuspended in PBS at a concentration of 1000 mg / mL. Azide-PAN-ECM was then added to the PD-L1-N3 and dendrimer reaction solution and incubated overnight at 4 °C. PD-L1-modified PAN-ECM was washed twice and resuspended in PBS.

[0149] TGF-β, HVEM, FasL, CD47, GITRL, and Gal-9-functionalized PAN-ECM were all synthesized using the same method.

[0150] Example 6: In vivo evaluation of CP-PAN-ECM reversing early-onset type 1 diabetes in NOD mice To expand the list of effective checkpoints that can be targeted, a batch of NOD / ShiltJ (NOD, female) mice were obtained, and their blood glucose levels were monitored twice weekly. After the onset of diabetes (defined as a blood glucose level exceeding 250 mg / dL), the mice received a combination of checkpoint-modified decellularized pancreatic extracellular matrix (PAN-ECM) and NIT-1β cells, administered subcutaneously twice over two weeks. Figure 3A Initially, mice were injected with ECM modified at a single checkpoint to compare the efficacy of different checkpoints. Figure 3B ).

[0151] This study identified several viable targeted checkpoint candidates that could help improve overall survival and reverse the onset of diabetes. When newly diagnosed 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 recovered to normal blood glucose levels. Figure 2A In this set of checkpoints, HVEM showed the strongest response, delaying the onset of diabetes in 75% of mice to after the 100-day monitoring period. Figure 3B Approximately half accepted HVEM functionalization. Figure 2B ) and FasL functionalization ( Figure 2C Mice treated with PAN-ECM did not develop diabetes within 100 days. All groups (including the group receiving co-injection of unmodified NIT-1 cells and ECM) showed some effect in delaying the onset of diabetes and improving survival, demonstrating the solid foundation of this technology. Next, the study explored whether simultaneously modifying PAN-ECM with three checkpoints could serve as a therapeutic vaccine to reverse early hyperglycemia. The results showed that, compared with the three single-function PAN-ECMs, the PD-L1 / HVEM / FasL-functionalized PAN-ECM was more potent and simultaneously inhibited multiple immune checkpoint pathways, thereby more effectively inducing antigen-specific T cell exhaustion. Figure 2D ).

[0152] To determine the potential synergistic effect of these checkpoints, selected checkpoint combinations were combined with PAN-ECM and co-injected with NIT-1 cells as described above. Five checkpoint combinations were tested: i) PD-L1+FGL1+TGFβ, ii) FAS-L+HVEM+FGL1, iii) FASL+TGFβ+PD-L1, iv) FASL+GITRL+TGFβ, and v) PD-L1+HVEM+FASL. None of the FGL1-containing treatment regimens showed superiority over unmodified PAN-ECM, and most mice developed hyperglycemia within 30 days. Figure 3C All other groups showed prolonged survival. Figure 4A and 4B Most of the mice maintained normal blood glucose levels during the 100-day monitoring period. Figure 3C These groups consist of different combinations of PD-L1, HVEM, FASL, GITRL, and TGFβ, thus providing a range of potential synergistic target combinations that can help suppress autoimmune attacks on natural pancreatic islets.

[0153] To determine the potential maximum response, a group of mice underwent combination therapy using all the cytokines and immune checkpoint molecules that had shown efficacy in previous studies. Diabetic mice received the following treatment: extracellular matrix modified with HVEM, FasL, GITRL, and TGFβ, combined with a specific dose of PD-L1 modified NIT-1 cells. This treatment regimen produced the strongest response, with 80% of mice maintaining normal glycemic levels until week 15, and 30% of mice remaining hypoglycemic for more than 20 weeks. Figure 5A and Figure 5B ).

[0154] Example 7: In vitro evaluation of CP-functionalized NIT-1 cells inducing antigen-specific CD8+ T cell dysfunction To test whether anchoring different immune checkpoint molecules to the surface could enhance their ability to inhibit islet antigen-specific T cell cytotoxicity, cytotoxicity assays were performed using various functionalized NIT-1 cells in the presence of islet-specific IGRP peptide. Figure 6B Fluorescence-activated cell sorting (FACS) analysis showed that PD-L1, HVEM, FasL, and GITRL could be successfully modified onto the surface of NIT-1 cells through metabolic glycoengineering and bioorthogonal click chemistry. Figure 6A Except for FasL, the other three checkpoint molecules, after being modified onto the cell surface, all significantly inhibited T cell-mediated cytotoxicity against pancreatic islet cells. Figure 6CInterestingly, when FasL anchored to the cell surface, T cell killing efficiency increased slightly, suggesting that FasL may act as a negative checkpoint in diabetes treatment. Therefore, PD-L1 and HVEM could serve as potential checkpoints for reversing diabetes. Subsequent studies on their combined effects did indeed show that, compared to using checkpoints alone, they significantly reduced T cell-mediated cytotoxicity against pancreatic islet cells. Figure 6D ).

[0155] Example 8: In vivo administration of CP-ECM plus PD-L1-functionalized NIT-1 cells can reverse early-onset type 1 diabetes. Preliminary screening showed that PD-L1 / HVEM / FasL modification of PAN-ECM could reverse diabetes in 75% of mice, with one mouse dying on day 24. Figure 3C In subsequent studies, PD-L1 was also placed on the surface of NIT-1 cells. Figure 7A ).like Figure 7B and Figure 7C As shown, separating these two checkpoints, rather than modifying them entirely on the PAN-ECM, yields better results. This suggests that the inhibitory effect of PD-L1 on T cells may depend on antigen presentation.

[0156] Example 9: Treatment of diabetes using an in vivo mouse model This embodiment confirms a method for developing islet-like cells from sources other than NIT-1 cells. An example of such a source is mouse embryonic stem cells (mESCs, derived from C57BL / 6), which provide a pathway for generating insulin-producing cells through a series of complex culture medium changes. Figure 8 In summary, embryonic stem cells isolated from C57BL / 6 mice were cultured for three days in N2B27 medium in bacterial culture dishes. Differentiation of mESCs was initiated by transferring cells to culture dishes coated with 0.1% gelatin and placing them in differentiation medium I, followed by three days of culture in N2B27 medium supplemented with Y27632. Subsequently, cells were induced to differentiate into pancreatic cells by culturing in differentiation medium II for 18 days, thus forming islet-like cell clusters. Dithizone staining was then performed to identify and purify the islet-like cell clusters from undifferentiated cells. These islet-like cell clusters were then cultured in insulin-free medium, followed by the glucose-stimulated insulin secretion assays described herein.

[0157] Under both low and high glucose conditions, there was no significant difference in the fold change in insulin secretion between IPCs and the mouse islet control group. Figure 9A Notably, dithizone staining revealed that, compared to human islets, differentiated IPCs exhibited a more uniform distribution of insulin granules across different cell clusters. Figure 9BFurthermore, 88% of differentiated IPCs simultaneously secrete insulin and glucagon (…). Figure 9C This indicates that β cells were effectively induced.

[0158] HVEM and PD-L1 (engineered IPCs) were functionalized onto IPCs using a bioengineering method, which included metabolic sugar engineering followed by a bioorthogonal click reaction. Figure 10A FACS studies confirmed that IPCs were successfully functionalized with HVEM and PD-L1, with 100% of the engineered IPCs showing positivity for both HVEM and PD-L1. Figure 10B The in vivo transplantation of these allogeneic cells was evaluated in a streptozotocin (STZ)-induced diabetic BALB / c mouse model. Diabetes was induced in 10-week-old female BALB / c mice by intraperitoneal injection of 175 mg / kg body weight of STZ five days prior to IPCs and PAN-ECM transplantation. Successful induction of diabetes was confirmed by measuring blood glucose levels two days prior to IPCs and PAN-ECM transplantation. IPCs and PAN-ECM were implanted subcutaneously into the abdomen of recipient BALB / c mice. Figure 10C Mice were divided into three groups: (i) a control group that did not receive STZ treatment; (ii) mice that received STZ treatment and subcutaneously transplanted IPCs and PAN-ECM; and (iii) mice that received STZ treatment and subcutaneously transplanted engineered IPCs modified with HVEM and PD-L1 and PAN-ECM. Administration of control IPCs and PAN-ECM to diabetic BALB / c mice failed to effectively alleviate STZ-induced diabetes, and all mice progressed to hyperglycemia by day 30. Figure 10D In stark contrast, mice that received engineered IPCs modified with HVEM and PD-L1 in combination with PAN ECM showed significantly stable blood glucose levels. Figure 10D These results suggest that the combined use of engineered IPCs and PANECM may significantly reduce hyperglycemia and maintain metabolic homeostasis.

[0159] On day 30 post-transplantation, the composition of the IPCs+PAN-ECM implant in one exemplary mouse from both the IPCs+PAN-ECM cohort and the engineered IPCs+PAN-ECM cohort was analyzed by flow cytometry (see [link to article]). Figure 10D The mouse indicated by the arrow is the one from which the implant was removed for analysis. Notably, after the graft was removed from the engineered IPCs+PAN-ECM mouse, the mouse's blood glucose level rapidly increased to 600 mg / dL, indicating that the engineered IPCs+PAN-ECM graft was the only significant source of insulin in this mouse. Figure 10DTo perform this analysis, IPCs+PAN-ECM grafts were removed from each mouse and dissociated using GentleMACS. Dead cells were excluded from the analysis using a live / dead assay kit. Flow cytometry analysis was then performed using gating to analyze pancreatic islet markers, including insulin and glucagon. Flow cytometry results showed that, in the presence of PAN ECM, HVEM and PD-L1 modified engineered IPCs continued to produce glucagon and insulin 30 days after in vivo implantation. Figure 10E ).

Claims

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

2. The CP-PAN-ECM according to claim 1, wherein the immune checkpoint molecules comprise 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, TGFβ, FGL1, or CD96.

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

4. The CP-PAN-ECM according to claim 3, wherein two, three, or four immune checkpoint molecules are linked to the PAN-ECM via click chemistry.

5. The CP-PAN-ECM according to any one of claims 1 to 3, wherein the at least one immune checkpoint molecule is chemically linked to the PAN-ECM via an alkyne-azidocycloaddition (SPAAC).

6. The CP-PAN-ECM according to claim 5, wherein two, three, or four immune checkpoint molecules are chemically linked to the PAN-ECM via an alkyne-azidocycloaddition (SPAAC) reaction.

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

8. The CP-PAN-ECM according to claim 7, wherein the dibenzocyclooctyne moiety is DBCO, or a derivative thereof, or a coupling thereof.

9. The CP-PAN-ECM according to claim 7, wherein the immune checkpoint molecule is a bioconjugate comprising a dendritic macromolecule, a linear polymer, nanoparticles, or an Fc fusion protein.

10. A composition comprising the CP-PAN-ECM according to any one of claims 1 to 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 linked to the 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 linked to the PAN-ECM.

13. The composition according to claim 11 or 12, wherein the at least one immune checkpoint molecule is selected from 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, TGFβ, FGL1, and CD96.

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

15. The composition according to any one of claims 11-13, wherein the CP-PAN-ECM comprises a trifunctionalized CP-PAN-ECM, the trifunctionalized CP-PAN-ECM comprising elements covalently linked to the PAN-ECM: i) PD-L1, FGL1, and TGFβ, ii) FasL, HVEM, and FGL1, iii) FasL, TGFβ, and PD-L1, iv) FasL, GITRL and TGFβ, or v) PD-L1, HVEM and FasL.

16. The composition according to any one of claims 11-13, wherein the CP-PAN-ECM comprises a tetrafunctionalized CP-PAN-ECM, the tetrafunctionalized CP-PAN-ECM comprising FasL, GITRL, TGFβ and HVEM covalently linked to the PAN-ECM.

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

18. The composition according to any one of claims 10-17, wherein the composition further comprises pancreatic cells or immune cells.

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

20. The composition of claim 19, wherein the immune checkpoint molecule is selected from 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, TGFβ, FGL1, and CD96.

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

22. The composition according to any one of claims 19 to 21, wherein the immune checkpoint molecule is covalently linked to the surface of the pancreatic cells or immune cells via metabolic glycoengineering.

23. The composition according to any one of claims 19 to 22, wherein the immune checkpoint molecule is covalently linked to the surface of the pancreatic cells or immune cells via click chemocovalent bonding.

24. The composition according to any one of claims 19 to 23, wherein the pancreatic cells are β cells.

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

26. The composition according to any one of claims 10 to 25, for the prevention or treatment of diabetes.

27. A method for preparing functionalized pancreatic extracellular matrix (CP-PAN-ECM), comprising: a. Provides functionalized pancreatic extracellular matrix (PAN-ECM); b. Provide functionalized immune checkpoint molecules; as well as c. Using acetylation-azidocycloaddition (SPAAC) chemistry, functionalized PAN-ECM is brought into contact with functionalized immune checkpoint bioconjugates to form CP-PAN-ECM.

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

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

30. The method of claim 29, wherein the dibenzocyclooctyne moiety comprises DBCO, or a derivative thereof, or a coupling thereof.

31. The method according to any one of claims 27-30, wherein the immune checkpoint molecule is a bioconjugate comprising a dendritic macromolecule, a linear polymer, nanoparticles, or an Fc fusion protein.

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

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

34. The method according to claim 32 or 33, wherein the administration is performed via a parenteral route.

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

36. A method for treating diabetes in a subject of need or delaying its progression, comprising administering functionalized pancreatic extracellular matrix (CP-PAN-ECM) and functionalized pancreatic cells. in, The CP-PAN-ECM comprises at least one immune checkpoint molecule covalently linked to the pancreatic extracellular matrix (PAN-ECM); and The functionalized pancreatic cells contain at least one immune checkpoint molecule covalently linked to the surface of the pancreatic cells.

37. The method of claim 36, wherein the at least one immune checkpoint molecule covalently linked to the pancreatic extracellular matrix (PAN-ECM) or the at least one immune checkpoint molecule covalently linked to the surface of pancreatic cells is selected from 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, TGFβ, FGL1, and CD96.

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

39. The method according to any one of claims 36 to 38, wherein the functionalized pancreatic cells comprise HVEM and PD-L1 covalently linked to the surface of the pancreatic cells.

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

41. The method according to any one of claims 36-40, wherein the administration is performed via a parenteral route.

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

43. A method for treating diabetes in a subject of need or delaying its progression using a functionalized pancreatic extracellular matrix (CP-PAN-ECM), said method comprising administering to an individual an effective amount of CP-PAN-ECM and functionalized pancreatic cells. in, The CP-PAN-ECM comprises at least one immune checkpoint molecule covalently linked to the pancreatic extracellular matrix (PAN-ECM); and The functionalized pancreatic cells contain at least one immune checkpoint molecule covalently linked to the surface of the pancreatic cells.

44. The method according to claim 43, wherein, The at least one immune checkpoint molecule covalently linked to the pancreatic extracellular matrix (PAN-ECM) or the at least one immune checkpoint molecule covalently linked to the surface of pancreatic cells is selected from 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, TGFβ, FGL1, and CD96.

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

46. ​​The method according to any one of claims 43 to 45, wherein the functionalized pancreatic cells comprise HVEM and PD-L1 covalently linked to the surface of the pancreatic cells.

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

48. The method according to any one of claims 43-47, wherein the administration is performed via a parenteral route.

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

50. A functionalized pancreatic extracellular matrix comprising the following structure: (PAN-ECM) — (residues of an azide-containing molecule) — (cyclooctyne residues) — (linker 1) — (residues of a functionalized dendritic macromolecule) q — (residues of an immune checkpoint molecule), wherein, q is 1 or 0; and the hyphen indicates a covalent bond.

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