Immunomodulation for the Treatment of Type 1 Diabetes

Abstract

The present specification provides a composition comprising a functionalized pancreatic extracellular matrix (CP-PAN-ECM) comprising at least one immune checkpoint molecule. The present specification also provides a method for preparing such a composition and a method for using such a composition for the treatment of diabetes.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 543,368 filed on October 10, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to a composition, a method of preparation and a method of use, and a pancreatic extracellular matrix comprising an immune checkpoint molecule. In one aspect, the present disclosure relates to a medical treatment using the disclosed composition and method. Background Technology

[0004] Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease characterized by hyperglycemia resulting from the loss of beta cells. Currently, about 1.25 million Americans suffer from T1DM, and the number of newly diagnosed cases of T1DM is increasing.

[0005] Most T1DM patients maintain blood glucose levels using multiple daily insulin injections or insulin pump therapy. Nevertheless, less than one-third of T1DM patients consistently achieve target blood glucose levels. Despite significant advances in disease management and care, T1DM remains associated with a significantly higher probability of developing acute conditions such as neuropathy, nephropathy, retinopathy, and cardiovascular disease, as well as a high early mortality rate, compared to the general population. Since a substantial number of beta cells remain present during the early symptomatic phase, there is considerable interest in developing novel immunotherapeutic strategies to delay and even reverse early-onset T1DM. This could enable patients to restore metabolic regulation.

[0006] In recent years, there has been increasing interest in developing other therapies to slow and reverse the progression of T1DM in the dysglycemic and early hyperglycemic stages by preserving insulin-producing pancreatic islets and suppressing pancreatic-infiltrated autoreactive T cells.

[0007] In one embodiment, the present disclosure comprises a functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently bonded to the pancreatic extracellular matrix (PAN-ECM). The immune checkpoint molecule may comprise, for example, PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, or CD96. In one embodiment, at least one immune checkpoint molecule is bonded to the PAN-ECM using click chemistry. In one embodiment, at least one immune checkpoint molecule is bound to PAN-ECM via alkyne-azide cyclization addition (SPAAC) chemistry. In one embodiment, at least one immune checkpoint molecule comprises a dibenzocyclooctane moiety. In one embodiment, the dibenzocyclooctane moiety is DBCO, or a derivative thereof, or a conjugate thereof. In one embodiment, the immune checkpoint molecule is a bioconjugate comprising a dendrimer, a linear polymer, a nanoparticle, or an Fc fusion protein.

[0008] In one embodiment, the present disclosure also comprises a composition comprising CP-PAN-ECM as disclosed herein. In one embodiment, the present disclosure comprises a functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently bonded to the pancreatic extracellular matrix (PAN-ECM). In one embodiment, the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pancreatic cell or an immune cell. In one embodiment, the cell is a pancreatic cell, e.g., a beta cell (β cell). In some embodiments, the pancreatic cell or immune cell comprises at least one immune checkpoint molecule covalently bonded to the surface of the pancreatic cell or immune cell. In some embodiments, the immune checkpoint molecule is selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the immune checkpoint molecule is covalently bonded to the surface of pancreatic cells or immune cells by metabolic glycoengineering. In one embodiment, the pharmaceutical composition comprises at least one excipient, or at least one additional therapeutic agent, or a combination thereof. In one embodiment, the composition is intended for use in the prevention or treatment of diabetes mellitus.

[0009] In one embodiment, the present disclosure also comprises a method for preparing a functionalized pancreatic extracellular matrix (CP-PAN-ECM), comprising the steps of: providing a functionalized PAN-ECM; providing a functionalized immune checkpoint molecule; and contacting the functionalized PAN-ECM with the immune checkpoint molecule to form a CP-PAN-ECM using alkyne-azide cyclization addition (SPAAC) chemistry: in one embodiment, the functionalized PAN-ECM comprises an azide moiety. In one embodiment, the functionalized immune checkpoint molecule comprises a dibenzocyclooctine moiety. In one embodiment, the dibenzocyclooctine moiety comprises DBCO, or a derivative thereof, or a conjugate thereof. In one embodiment, the immune checkpoint molecule is a bioconjugate comprising a dendrimer, a linear polymer, a nanoparticle, or an Fc fusion protein.

[0010] In one embodiment, the present disclosure also comprises a method for treating diabetes or reducing the progression of diabetes in a subject, comprising the step of administering a composition as disclosed herein to a subject who requires treatment of diabetes or reduction of the progression of diabetes. In one embodiment, the subject is a mammal. In one embodiment, administration is performed via an intravenous route. In one embodiment, diabetes is autoimmune diabetes.

[0011] In one aspect, the present disclosure comprises a structure of (PAN-ECM) ― (residue of an azide-containing molecule) ― (residue of a cyclooctane) ― (linker 1) ― (residue of a functionalized dendrimer) q ― (residue of an immune checkpoint molecule), wherein q is 1 or 0; and a dash (―) indicates a covalent bond, comprising a functionalized pancreatic extracellular matrix. Brief explanation of the drawing

[0012] Aspects of the present disclosure in which similar reference numerals indicate similar elements are described as examples: Figure 1 is a schematic diagram of the bioengineering of checkpoint-functionalized CP-PAN-ECM (top) and the mechanism of action of subcutaneously injected β cells + CP-PAN-ECM (bottom). Figures 2a to 2d show line graphs representing blood glucose concentrations in diabetic NOD mice after sc injection of PD-L1 (Fig. 2a), HVEM (Fig. 2b), FasL (Fig. 2c), or PD-L1 / HVEM / FasL (Fig. 2d)-functionalized PAN-ECM + β cells (n = 4 mice per group). Fig. 3a shows engineered single- or triple-functionalized PAN ECM + NIT-1 cells (1x10 6 Shows a diagram of the processing timeline for the NOD mouse using ). Figure 3b shows a line graph indicating changes in blood glucose levels in diabetic NOD mice after subcutaneous (sc) injection of unmodified or different monofunctionalized PAN-ECM + NIT-1 cells. Figure 3c shows a line graph indicating fluctuations in blood glucose levels in diabetic NOD mice after sc injection of unmodified or different triple-functionalized PAN-ECM + NIT-1 cells. Figure 4a shows the survival curve of NOD mice after treatment with unmodified or different single-functionalized PAN-ECM + NIT-1 cells. Figure 4b shows the survival curves of NOD mice after treatment with unmodified or different triple-functionalized PAN-ECM + NIT-1 cells. Figure 5a is a line graph showing blood glucose levels in NOD mice after treatment with FasL / GITRL / TGFβ / HVEM-functionalized PAN-ECM + PD-L1-NIT-1 cells. Figure 5b shows the survival curve of NOD mice after treatment with FasL / GITRL / TGFβ / HVEM-functionalized PAN-ECM + PD-L1-NIT-1 cells. Figure 6a shows a fluorescence-activated cell sorting (FACS) plot quantifying the levels of surface PD-L1, HVEM, FasL, and GITRL in different monofunctionalized NIT-1 cells. Figure 6b is a schematic diagram showing the experimental process for detecting the death of engineered NIT-1 cells by pancreatic islet antigen-specific 8.3 CD8+ T cells in the presence of exogenous IGRP peptides. Figure 6c shows a bar graph displaying the percentage of cell lysis of various monofunctionalized NIT-1 cells after incubation with 8.3 T cells proliferated at E:T = 5:1 for 18 hours in the presence of IGRP peptide, as determined by the CytoTox non-radiotoxicity assay (n = 4). Figure 6d shows a bar graph displaying the percentage of cell lysis of HVEM- or PD-L1 or HVEM / PD-L1-functionalized NIT-1 cells after incubation with 8.3 T cells proliferated at E:T = 5:1 for 18 hours in the presence of IGRP peptide as determined by the CytoTox non-radiotoxicity assay (n = 4). Fig. 7a shows engineered PAN ECM + engineered NIT-1 cells (0.5 x 10⁻⁶ 6 This is a diagram showing the processing timeline for a NOD mouse using ). Figure 7b shows a line graph indicating changes in blood glucose levels in diabetic NOD mice after subcutaneous (sc) injection of PD-L1 / HVEM-functionalized PAN-ECM + NIT-1 cells. Figure 7c shows a line graph indicating changes in blood glucose levels in diabetic NOD mice after subcutaneous (sc) injection of HVEM-functionalized PAN-ECM + PD-L1-functionalized NIT-1 cells. Figure 8 is a schematic diagram illustrating a method for inducing differentiation of mouse embryonic stem cells (mESCs) into insulin-secreting pancreatic islet-like clusters (IPCs). Figure 9a shows insulin secretion (μIU / 10⁻¹⁰) under low and high glucose conditions from mESCs, islet cells, and islet-like clusters (IPCs). 4 This is a bar graph displaying cells. Data for each cell type is displayed from left to right, starting with low glucose conditions followed by high glucose conditions. Fig. 9b shows bright-field optical microscopy imaging of dithizone-stained mESCs, typical mouse pancreatic islets, and IPCs. Scale bar = 100 μm. FIG. 9c shows a fluorescence-activated cell sorting (FACS) plot quantifying insulin (647) and glucagon (FITC) in mESCs, pancreatic islet cells, and pancreatic islet-like clusters (IPCs). Figure 10a is a schematic diagram illustrating the functionalization of IPC through alkyne-azide cyclization (SPAAC) following metabolic glycoengineering using DBCO-functionalized PD-L1 and HVEM. Figure 10b shows a fluorescence-activated cell sorting (FACS) plot quantifying PD-L1 and HVEM expression in unmodified IPCs and engineered IPCs functionalized with PD-L1 and HVEM. Figure 10c is a diagram showing a therapeutic treatment schedule using an engineered IPC in allogeneic BABL / c diabetic mice after streptozotocin (STZ) injection to induce diabetes. Figure 10d shows a line graph showing blood glucose levels in allogeneic BABL / c diabetic mice after subcutaneous (sc) injection of PBS, IPC + PAN-ECM, and HVEM / PD-L1-IPC + PAN ECM. Figure 10e shows a fluorescence-activated cell sorting (FACS) plot quantifying the expression of insulin and glucagon in mice treated with IPC + PAN-ECM and HVEM / PD-L1-IPC + PAN ECM on day 30. The drawings do not limit the present disclosure to the specific embodiments disclosed and described herein. The drawings are not necessarily in scale, but instead focus on clearly illustrating the principles of the specific embodiments of the present disclosure. Specific details for implementing the invention

[0013] The following detailed description refers to the accompanying drawings illustrating various aspects of the present disclosure. The drawings and description are intended to provide sufficient detail to enable those skilled in the art to practice the present disclosure. Other components may be used, and modifications may be made without departing from the scope of the present disclosure. Accordingly, the following description should not be construed as limiting.

[0014] In one aspect, the present disclosure is the result of a surprising discovery that pancreatic extracellular matrix (PAN-ECM), when covalently bound to an immune checkpoint molecule and injected into mice, can exhibit potent antigen-specific effector T cell suppression induced by the interaction between PAN-ECM and diabetes-inducing T cells. Deficiencies or mutations in co-inhibitory checkpoints are often associated with the progression of autoimmune diabetes. Introducing co-inhibitory checkpoint molecules into pancreatic islets can attenuate diabetes-inducing T cells. Consequently, the reversal of early-onset hyperglycemia can be achieved. It was previously revealed that pancreatic beta (β) cells containing sufficient co-inhibitory checkpoint molecules can reverse early-onset hyperglycemia in NOD mice by attenuating the activity of diabetes-inducing T cells. In this study, it was found that pancreatic extracellular matrix (PAN-ECM) containing checkpoint molecules can also effectively slow the progression of hyperglycemia and extend overall survival. Nevertheless, there are numerous checkpoint molecules that play a crucial role in immune homeostasis, and therefore, combinations of different checkpoint molecules may have a better reversal effect on novel diabetes. To achieve this goal, PAN-ECM was engineered with different checkpoint molecules, and then an immunogenic pancreatic microenvironment was created by subcutaneously injecting PAN-ECM mixed with β cells near pancreatic lymph nodes.

[0015] I. Terminology

[0016] The phrases and terms used in this specification are for illustrative purposes only and should not be construed as limiting. For example, the use of singular terms is not intended to limit the number of items. Additionally, the use of positional relation terms such as, but not limited to, "top," "bottom," "left," "right," "upper," "lower," "lower," "top," and "side" is for illustrative purposes to provide clarity in specific references to the drawings and is not intended to limit the scope of this disclosure or the appended claims.

[0017] Furthermore, since the present disclosure may allow for many different forms of embodiments, the present disclosure should be regarded as an example of the principles of the present disclosure and is not intended to limit the present disclosure to the specific embodiments presented and described. Any one of the features of the present disclosure may be used individually or in combination with any other features. Any reference to the terms “sunset,” “sunsets,” and / or similar terms in the description means that the features and / or features mentioned are included in at least one embodiment of the description. Any individual reference to the terms “sunset,” “sunsets,” and / or similar terms in the description does not necessarily refer to the same embodiment, nor are they mutually exclusive unless otherwise stated and / or readily apparent to a person skilled in the art from the description. For example, features, structures, processes, steps, operations, etc. described in one embodiment may be included in other embodiments, but are not required to be included. Accordingly, the present disclosure may include various combinations and / or combinations of the embodiments described herein. Furthermore, not all embodiments of the present disclosure as described herein are essential for practice. Likewise, other systems, methods, features, and benefits of the present disclosure will be or will become apparent to those skilled in the art upon review of the drawings and description. All such additional systems, methods, features, and benefits are incorporated into this description, fall within the scope of the present disclosure, and are intended to be incorporated by the claims.

[0018] Any term relating to degree, such as but not limited to “substantially” as used in the description and appended claims, shall be understood to include an exact configuration or a similar but inaccurate configuration. For example, “substantially flat surface” means having an exact flat surface or a similar but inaccurate flat surface. Similarly, terms “about” or “approximately” as used in the description and appended claims shall be understood to include a stated value or a value that is at least three times or one-third of a stated 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, these terms may refer to ± 5% or less, e.g. ± 2% or less, e.g. ± 1% or less, e.g. ± 0.5% or less, e.g. ± 0.2% or less, e.g. ± 0.1% or less, e.g. ± 0.05% or less.

[0019] The terms “comprising” and “having” are used interchangeably in this disclosure. The terms “comprising” and “having” mean that they include, but are not necessarily limited to, those described therein.

[0020] Finally, terms “or” and “and / or” as used herein shall be interpreted as inclusive or as meaning any one or any combination thereof. Accordingly, “A, B or C” or “A, B and / or C” means any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” Exceptions to these definitions will occur only where the combination of elements, functions, steps or actions is, in any way, mutually exclusive.

[0021] II. Composition

[0022] Functionalized pancreatic extracellular matrix

[0023] In one aspect, the present disclosure comprises 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 bonded to the pancreatic extracellular matrix (PAN-ECM). As used herein, the functionalized pancreatic extracellular matrix comprises a separated acellular pancreatic extracellular matrix or a component thereof and further comprises at least one covalently bonded immune checkpoint molecule. In one aspect, the immune checkpoint (CP) molecule is bonded using the functionalized CP and the functionalized PAN-ECM, each comprising a moiety that is compatible and complementary to one another for a click chemical reaction.

[0024] In one solar term, 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 one solar term, the immune checkpoint molecule is PD-L1 or HVEM. In one solar term, the functionalized PAN-ECM contains at least one PD-L1, or at least one HVEM, or both.

[0025] In one case, the immune checkpoint molecule can be a fusion protein, for example, PD-L1 can be PD-L1-Ig.

[0026] PD-L1, programmed death-ligand 1 (Uniprot: Q9NZQ7), is a 40 kDa type 1 transmembrane protein. PD-L1 is a ligand for PD-1. PD-L1 is also known as B7-H1 (B7 homolog 1).

[0027] HVEM (UniProtKB / Swiss-Prot: Q92956) is encoded by the TNFRSF14 gene. The encoded protein functions in signaling pathways that activate inflammatory and suppressive T cell immune responses. It binds to the viral envelope glycoprotein D (gD) of the herpes simplex virus (HSV) and mediates its entry into cells. Alternative splicing generates multiple transcriptomic variants.

[0028] CD86, the T-lymphocyte activating antigen CD86 (Uniprot: P42081), is a type I membrane protein. CD86 is a ligand for CTLA-4 on activated T cells. CD86 (along with CD80) provides the co-stimulatory signals necessary for T cell activation and survival.

[0029] Gal-9, which is galectin 9 (Uniprot: 000182), is a 36 kDa beta-galactoside lectin protein. Gal-9 is a ligand for TIM-3.

[0030] In one aspect, the content described herein relates to a functionalized CP-PAN-ECM comprising the structure (PAN-ECM) ― (residue of an azide-containing molecule) ― (residue of a cyclooctane) ― (linker 1) ― (residue of a functionalized dendrimer) q ― (residue of an immune checkpoint molecule), wherein q is 1 or 0; and dashes indicate covalent bonds. In one aspect, the cyclooctane moiety is a residue from dibenzocyclooctane, e.g., DBCO. When q is 0, the dendrimer is absent, which becomes a DBCO direct bonding strategy. As used herein, the term “residue” or “residue of a chemical moiety” refers to a chemical moiety bonded to a molecule, wherein at least one covalent bond replaces at least one atom of the original chemical moiety through bonding, thereby creating a residue of the chemical moiety within the molecule.

[0031] In another aspect, the content described herein relates to a functionalized CP-PAN-ECM comprising the structure (PAN-ECM) ― (residue of an azide-containing molecule) ― (residue of a cyclooctane) ― (linker 1) ― (residue of a functionalized dendrimer)q ― (immune checkpoint molecule FcIg fusion protein), wherein the dash indicates a covalent bond. In another aspect, the content described herein relates to a functionalized CP-PAN-ECM comprising the structure (PAN-ECM) ― (residue of a cyclooctane-containing molecule) ― (residue of an azide) ― (linker 1) ― (immune checkpoint molecule FcIg fusion protein), wherein the dash indicates a covalent bond. In one aspect, the immune checkpoint molecule / immune checkpoint molecule FcIg fusion protein may be conjugated via amine-NHS ester chemistry or thiol-maleimide chemistry. In another aspect, the present specification relates to a functionalized CP-PAN-ECM comprising the structure (PAN-ECM) ― (residue of an azide-containing molecule) ― (residue of a cyclooctane) ― (nanoparticle) ― (linker such as linker 1) ― (immune checkpoint molecule))y)x, wherein the dash indicates a covalent bond and x and y are as described in the present specification.

[0032] In various aspects, the present disclosure comprises 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 bonded to the pancreatic extracellular matrix (PAN-ECM), for example, the immune checkpoint molecule may be, for example, about two, about three, about four, or about five.

[0033] In some embodiments, the present disclosure comprises a functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises about two immune checkpoint molecules or functional fragments thereof covalently bonded to the pancreatic extracellular matrix (PAN-ECM). In some embodiments, the about two immune checkpoint molecules are selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the CP-PAN-ECM comprises PD-L1 and HVEM covalently bonded to the PAN-ECM.

[0034] In some embodiments, the present disclosure comprises a functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises about three immune checkpoint molecules covalently bonded to the pancreatic extracellular matrix (PAN-ECM), or a functional fragment thereof. In some embodiments, the about three immune checkpoint molecules are selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the CP-PAN-ECM comprises PD-L1, FGL1, and TGFβ covalently bonded to the PAN-ECM. In some solar terms, CP-PAN-ECM comprises FasL, HVEM, and FGL1 covalently coupled to PAN-ECM. In some solar terms, CP-PAN-ECM comprises FasL, TGFβ, and PD-L1 covalently coupled to PAN-ECM. In some solar terms, CP-PAN-ECM comprises FasL, GITRL, and TGFβ covalently coupled to PAN-ECM. In some solar terms, CP-PAN-ECM comprises PD-L1, HVEM, and FasL covalently coupled to PAN-ECM.

[0035] In another embodiment, the present disclosure comprises a functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises about four immune checkpoint molecules covalently bonded to the pancreatic extracellular matrix (PAN-ECM), or a functional fragment thereof. In some embodiments, the about four immune checkpoint molecules are selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some solar terms, CP-PAN-ECM contains FasL, GITRL, TGFβ, and HVEM covalently bonded to PAN-ECM.

[0036] In one case, thiol-maleimide click chemistry can be used to modify PAN-ECM. Generally, free thiol groups on the surface can be created to react with maleimide-functionalized biomolecules through stable thioester bonds to form stable functionalized PAN-ECM. Maleimide-functionalized biomolecules can be prepared by an amine-NHS reaction between the desired biomolecule and an NHS-maleimide crosslinking agent (e.g., sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC)).

[0037] In some embodiments, the contents described herein relate to functionalized CP-PAN-ECM, wherein the residue of the functionalized dendrimer has a structure of ― (dendrimer) ― (linker 2) ― (residue of cyclooctane) ― (residue of azide-containing molecule) ―. In one embodiment, linker 2 is It has the structure, where z is an integer from 0 to 10.

[0038] In a single sun, z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a single sun, z is 3. In a single sun, z is an integer from 0 to 100,000. In a single sun, z is an integer from 0 to 10, 0 to 100, 0 to 1,000, 0 to 5,000, or 0 to 10,000. In a single sun, z is an integer from 10 to 100,000, 100 to 100,000, 1,000 to 100,000, 5,000 to 100,000, or 10,000 to 100,000. In one sun, the functionalized PAN-ECM contains at least one covalently bonded immune checkpoint molecule of about 0.5 pg to about 100 pg per about 1 million functionalized PAN-ECMs. In one sun, the functionalized PAN-ECM comprises at least one covalently bonded immune checkpoint molecule in an amount of 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 per about 1 million functionalized PAN-ECMs. In one sun, the functionalized PAN-ECM contains at least one covalently bonded immune checkpoint molecule of about 0.5 pg, about 1 pg, about 10.0 pg, about 20.0 pg, about 30.0 pg, about 40.0 pg, about 50.0 pg, about 60.0 pg, or about 75.0 pg per about 1 million functionalized PAN-ECMs.The total amount of immune checkpoint molecules can be quantified, for example, by fluorescence spectroscopy (via fluorescently labeled proteins) or quantitative Western blot (e.g., AutoWest).

[0039] In one embodiment, at least one covalently bonded immune checkpoint molecule is an immune checkpoint molecule-functionalized nanoparticle or polymer. In one embodiment, the covalent bond is formed through conjugation to a thiol group on the PAN-ECM.

[0040] In another embodiment, the immune checkpoint moiety further comprises a residue of a dendrimer, linear polymer, nanoparticle, or Fc fusion protein. In one embodiment, the nanoparticle is a dendrimer, liposome, inorganic nanoparticle, or polymer nanoparticle. In one embodiment, 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 one embodiment, the nanoparticle is about 2 nm to about 1000 nm, about 2 nm to about 750 nm, about 2 nm to about 500 nm, about 2 nm to about 250 nm, about 2 nm to about 200 nm, about 2 nm to about 100 nm, or 2 nm to about 50 nm. In one sun, the nanoparticle is about 10 nm to about 1000 nm, about 25 nm to about 1000 nm, about 50 nm to about 1000 nm, about 100 nm to about 1000 nm, about 200 nm to about 1000 nm, about 500 nm to about 1000 nm, or 750 nm to about 1000 nm. In suns, the nanoparticle is about 2 nm, about 5 nm, about 10 nm, about 50 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm. In suns, the nanoparticle is further covalently bonded via a linker to one or more immune checkpoint molecules as described herein. In one sun, the dendrimer is a multivalent dendrimer. In one sun, the multivalent dendrimer is a polyamidoamine dendrimer. In one sun, the nanoparticle is a pegylated nanoparticle (e.g., DBCO-functionalized PEG-PLGA nanoparticle). In the suns, the pegylated nanoparticle has a diameter of less than 200 nm.

[0041] In one embodiment, the polyamidedoamine dendrimer has a molecular weight (MW) of about 500 to about 1,000,000. In one embodiment, the polyamidedoamine dendrimer has a molecular weight of 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.

[0042] In one embodiment, the polyamidedoamine dendrimer has a molecular weight of about 20,000 to about 35,000. In one embodiment, the polyamidedoamine dendrimer has a molecular weight of about 20,000 to about 30,000. In one embodiment, the polyamidedoamine dendrimer has a molecular weight of about 25,000 to about 30,000.

[0043] In one sun, the polyamidedoamine dendrimer has a molecular weight of about 20,000, about 21,000, about 22,000, about 23,000, about 24,000, about 25,000, about 26,000, about 27,000, about 28,000, about 29,000, about 30,000, about 31,000, about 32,000, about 33,000, about 34,000, or about 35,000. In one sun, the polyamidedoamine dendrimer has a molecular weight of about 28,000.

[0044] In one embodiment, the present specification describes an acellular pancreatic extracellular matrix (PAN-ECM), wherein the PAN-ECM is functionalized as described in the present specification and comprises a pancreatic-derived protein. In another embodiment, the acellular pancreatic extracellular matrix is ​​in the form of an injectable. In one embodiment, the acellular pancreatic extracellular matrix is ​​in the form of an injectable that is not a gel. In one embodiment, the acellular pancreatic extracellular matrix is ​​in the form of an injectable that is a gel. In one embodiment, the acellular pancreatic extracellular matrix is ​​in the form of an injectable that is a gel that is not a heat-sensitive hydrogel.

[0045] Pharmaceutical composition

[0046] In one embodiment, the present specification describes a pharmaceutical composition comprising a functionalized pancreatic extracellular matrix (CP-PAN-ECM) containing an immune checkpoint molecule (CP) as described herein, and a pharmaceutically acceptable excipient. In one aspect, the pharmaceutical composition may further comprise a cell. In one aspect, the cell is a pancreatic beta cell.

[0047] In one embodiment, the present specification describes a vaccine comprising a functionalized pancreatic extracellular matrix (CP-PAN-ECM) and a pharmaceutically acceptable liquid vehicle.

[0048] The term "vaccine" refers to a composition capable of inducing an immune response and preventing a subject from contracting or developing a disease or condition, and / or the vaccine may be therapeutic for a subject with a disease or condition.

[0049] "Pharmaceuticalally acceptable excipients" refers to a vehicle for carrying a functionalized extracellular matrix (CP-PAN-ECM) that can be introduced into a subject without serious adverse effects and without adverse effects on the acellular extracellular matrix. That is, "pharmaceuticalally acceptable" refers to any formulation that is safe and provides appropriate delivery of an effective amount of CP-PAN-ECM for use in the methods disclosed herein through at least one desired route of administration. Pharmaceutically acceptable carriers, vehicles, or excipients are well known. Descriptions of suitable pharmaceutically acceptable carriers and factors involved in their selection can be found in various readily available sources, such as, for example, the literature [Remington's Pharmaceutical Sciences, 18th ed., 1990], the full text of which is incorporated herein by reference for all purposes. Such carriers may be suitable for any route of administration (e.g., parenteral, enteral (e.g., oral), or topical application). These pharmaceutical compositions can be buffered, for example, and the pH is maintained at a specific desired value in the range of pH 4.0 to pH 9.0 depending on the stability and route of administration of CP-PAN-ECM.

[0050] Suitable pharmaceutically acceptable carriers include, for example, sterile water, salt solutions such as saline solution, buffer solutions such as glucose, phosphate buffer solution or bicarbonate buffer solution, alcohol, gum arabic, vegetable oil, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates (e.g., lactose, amylose, or starch), magnesium stearate, talc, silica, viscous paraffin, white paraffin, glycerol, alginate, hyaluronic acid, collagen, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc. The pharmaceutical composition or vaccine may also include adjuvants, such as diluents, stabilizers (e.g., sugars and amino acids), preservatives, wetting agents, emulsifiers, pH buffers, viscosity-enhancing additives, lubricants, salts to affect osmotic pressure, buffers, vitamins, coloring agents, flavoring agents, aromatic substances, etc., and these adjuvants do not react adversely with functionalized cells or acellular extracellular matrix.

[0051] For liquid formulations, for example, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, emulsions, or oils. Non-aqueous solvents include, for example, injectable organic esters such as propylene glycol, polyethylene glycol, and ethyl oleate. Aqueous carriers include, for example, water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline solution and a buffer medium. Examples of oils include those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil. Solid carriers / diluents include, for example, gums, starches (e.g., corn starch, gelatinized starch), sugars (e.g., lactose, mannitol, sucrose, or dextrose), cellulose materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethyl acrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[0052] Optionally, sustained-release or directed-release pharmaceutical compositions or vaccines may be formulated. This may be achieved, for example, through the use of liposomes or compositions in which the active compound is protected by a differentially degradable coating (e.g., by microencapsulation, multi-coating, etc.). Such compositions may be formulated for immediate release or sustained-release. It is also possible to freeze-dry the composition and use the resulting lyophilisate (e.g., for the manufacture of injectable products).

[0053] Treatment methods

[0054] In another embodiment, the contents described herein relate to a method for treating diabetes or delaying the onset of diabetes in a subject, comprising the step of administering to the subject a composition containing CP-PAN-ECM as described herein. In one embodiment, the subject receives a pharmaceutical composition or vaccine containing CP-PAN-ECM.

[0055] In some aspects, the present specification provides a method for treating diabetes or reducing the progression of diabetes in a subject requiring treatment of diabetes or reduction of the progression of diabetes, comprising the step of 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 bonded to the pancreatic extracellular matrix (PAN-ECM); and the functionalized pancreatic cells comprise at least one immune checkpoint molecule covalently bonded to the surface of the pancreatic cells.

[0056] In another aspect, the present invention provides a functionalized pancreatic extracellular matrix (CP-PAN-ECM) for use in a method for treating diabetes or reducing the progression of diabetes in a subject requiring treatment of diabetes or reduction of the progression of diabetes, comprising the step of administering an effective amount of CP-PAN-ECM and functionalized pancreatic cells to a subject, wherein CP-PAN-ECM comprises at least one immune checkpoint molecule covalently bonded to the pancreatic extracellular matrix (PAN-ECM); and the functionalized pancreatic cells comprise at least one immune checkpoint molecule covalently bonded to the surface of the pancreatic cells.

[0057] In some embodiments, type 1 diabetes is early-onset type 1 diabetes or early-onset hyperglycemia. In other embodiments, the content described herein relates to a method for reversing early-onset type 1 diabetes in a subject, comprising the step of administering functionalized CP-PAN-ECM or a pharmaceutical composition or vaccine containing it to a subject. In other embodiments, the content described herein relates to a method for reversing early-onset type 1 diabetes in a subject, comprising the step of administering beta cells and functionalized CP-PAN-ECM or a pharmaceutical composition or vaccine containing it to a subject. In embodiments, the content described herein relates to a method for protecting pancreatic beta cells in a subject, comprising the step of administering CP-PAN-ECM or a pharmaceutical composition or vaccine containing it to a subject.

[0058] In one aspect, treatment includes the improvement of existing disease symptoms or prevention of exacerbation, prevention of the occurrence of additional symptoms, improvement of or prevention of the underlying metabolic cause of symptoms, suppression of the disorder or disease, e.g., prevention of the occurrence of the disorder or disease, alleviation of the disorder or disease, induction of regression of the disorder or disease, alleviation of the condition caused by the disease or disorder, or cessation of symptoms of the disease or disorder.

[0059] The terms "treatment" or "treating" refer to both therapeutic treatment and preventive or protective measures aimed at preventing, reducing, or decreasing the severity of autoimmune disease symptoms. Treatment may include directly affecting or curing the autoimmune disease, suppressing the autoimmune disease, inhibiting the autoimmune disease, preventing the autoimmune disease, reducing the severity of the autoimmune disease, delaying the onset of the autoimmune disease, slowing the progression of the autoimmune disease, stabilizing the progression of the autoimmune disease, reducing or improving symptoms associated with the autoimmune disease, or one or more combinations thereof. The term "reducing severity" refers to clinical or subjective determinations regarding the alleviation of signs or symptoms following treatment.

[0060] The term “subject” refers to mammals (e.g., humans) that require treatment for diabetes or are prone to developing diabetes. The term “subject” also refers to mammals (e.g., humans) receiving prophylactic 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 that are healthy in all respects and do not have diabetes or show signs of diabetes.

[0061] As used herein, the term “organism” includes, but is not limited to, humans, non-human primates such as those mentioned above, and any transgenic species thereof, and further includes any living eukaryote.

[0062] The terms “effective dose” or “therapeutic effective dose” refer to an amount of composition sufficient to provide a desired biological outcome. Such outcome may be a reduction and / or alleviation of signs, symptoms, or causes of a disease or medical condition, or any other desired change in the biological system. For example, an “effective dose” for therapeutic use is an amount of composition required to provide a clinically significant change in a disease state, symptom, or medical condition. In any individual case, an appropriate “effective” dose may be determined by a person skilled in the art using conventional experiments. Accordingly, the expression “effective dose” generally refers to an amount of the active substance that produces a therapeutically desired effect. The effective dose or dosage of the composition of the embodiments may be determined by conventional methods, such as modeling, dose escalation, or clinical trials, taking into account conventional factors such as, for example, the pattern or route of administration or drug delivery, the pharmacokinetics of the formulation, the severity and course of the infection, the subject’s health status, condition, and body weight, and the judgment of the treating physician. Exemplary doses are within the range of the active ingredient of about 1 pg to 10 mg per kilogram of the subject's body weight per day. The total dose may be administered as a single or divided unit (e.g., BID, TID, QID). Once the patient's condition improves, the dose may be adjusted for prophylactic or maintenance therapy. For example, the dose or frequency of administration, or both, may be reduced to a level where the desired therapeutic or prophylactic effect is maintained according to the symptoms. Of course, if symptoms have been adequately relieved, treatment may be discontinued. However, the patient may require intermittent treatment over a long period in the event of a recurrence of symptoms. The patient may also require chronic treatment over a long period.

[0063] In certain embodiments, the present disclosure provides a composition formulated for one or more routes of administration. A suitable route of administration may be, for example, parenteral delivery. In some embodiments, the composition formulated herein may be formulated for parenteral delivery. In some embodiments, the composition formulated herein may be formulated for intramuscular, subcutaneous, intramedullary, or intravenous injection.

[0064] In certain embodiments, the compositions of this specification may be administered topically or systemically, for example, by direct local injection of the pharmaceutical composition into a tissue area of ​​the patient. In some embodiments, the pharmaceutical compositions disclosed in this specification may be administered parenterally.

[0065] In certain aspects, the pharmaceutical composition of the present disclosure may be prepared by processes well known in the art, e.g., conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, capturing, or freeze-drying processes.

[0066] In certain aspects, a pharmaceutical composition for use according to the present disclosure may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and adjuvants that facilitate the processing of the active ingredient into a pharmaceutically usable product. The appropriate formulation depends on the selected route of administration. For injection, the active ingredient of the pharmaceutical composition of the present specification may be formulated as an aqueous solution, preferably a physiologically compatible buffer, such as Hank's solution, Ringer's solution, physiological salt buffer, or any combination thereof.

[0067] In certain aspects, the pharmaceutical composition described herein may be formulated in the form of nanoparticles.

[0068] In various embodiments, the treatment method comprises the step of administering an effective amount of monofunctionalized PAN-ECM to a subject requiring monofunctionalized PAN-ECM, wherein the monofunctionalized PAN-ECM comprises an immune checkpoint molecule covalently bonded to the pancreatic extracellular matrix (PAN-ECM). In some embodiments, the immune checkpoint molecule is selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the method further comprises the step of administering an effective amount of functionalized pancreatic cells or immune cells to the subject. In some embodiments, the functionalized pancreatic cell is a functionalized beta cell. In some embodiments, the functionalized pancreatic cell or immune cell comprises one or more immune checkpoint proteins covalently bonded to the surface of the pancreatic cell or immune cell, wherein the immune checkpoint protein comprises a protein selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the functionalized pancreatic cell or immune cell comprises one or more immune checkpoint proteins covalently bonded to the surface of the pancreatic cell or immune cell, wherein the immune checkpoint protein comprises PD-L1.

[0069] In various other embodiments, the functionalized pancreatic cell or immune cell comprises one or more immune checkpoint proteins covalently bonded to the surface of the pancreatic cell or immune cell, wherein the immune checkpoint protein comprises HVEM. In some embodiments, the functionalized pancreatic cell or immune cell comprises one or more immune checkpoint proteins covalently bonded to the surface of the pancreatic cell or immune cell, wherein the immune checkpoint protein comprises FasL. In some embodiments, the functionalized pancreatic cell or immune cell comprises one or more immune checkpoint proteins covalently bonded to the surface of the pancreatic cell or immune cell, wherein the immune checkpoint protein comprises GITRL. In some embodiments, the functionalized pancreatic cell or immune cell comprises two immune checkpoint proteins covalently bonded to the surface of the pancreatic cell or immune cell, wherein the two immune checkpoint proteins comprise HVEM and PD-L1.

[0070] In some embodiments, the method comprises the step of administering an effective amount of monofunctionalized PAN-ECM to a subject requiring monofunctionalized PAN-ECM, wherein the monofunctionalized PAN-ECM comprises PD-L1-functionalized PAN-ECM. In some embodiments, the subject receives a pharmaceutical composition or vaccine comprising PD-L1-PAN-ECM.

[0071] In another embodiment, the method comprises the step of administering an effective amount of monofunctionalized PAN-ECM to a subject requiring monofunctionalized PAN-ECM, wherein the monofunctionalized PAN-ECM comprises HVEM-functionalized PAN-ECM. In some embodiments, the subject receives a pharmaceutical composition or vaccine containing HVEM-PAN-ECM.

[0072] In some embodiments, the method comprises the step of administering an effective amount of monofunctionalized PAN-ECM to a subject requiring monofunctionalized PAN-ECM, wherein the monofunctionalized PAN-ECM comprises FasL-functionalized PAN-ECM. In some embodiments, the subject receives a pharmaceutical composition or vaccine comprising FasL-PAN-ECM.

[0073] In some embodiments, the method comprises the step of administering an effective amount of monofunctionalized PAN-ECM to a subject requiring monofunctionalized PAN-ECM, wherein the monofunctionalized PAN-ECM comprises FGL1-functionalized PAN-ECM. In some embodiments, the subject receives a pharmaceutical composition or vaccine comprising FGL1-PAN-ECM.

[0074] In another embodiment, the method comprises the step of administering an effective amount of monofunctionalized PAN-ECM to a subject requiring monofunctionalized PAN-ECM, wherein the monofunctionalized PAN-ECM comprises PD-L1-functionalized PAN-ECM. In some embodiments, the method further comprises the step of administering an effective amount of functionalized pancreatic cells or immune cells to a subject. In some embodiments, the functionalized pancreatic cells or immune cells comprise one or more immune checkpoint proteins covalently bound to the surface of the pancreatic cells or immune cells, wherein the immune checkpoint proteins comprise proteins selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the functionalized pancreatic cells are functionalized beta cells. In some embodiments, a subject is administered a pharmaceutical composition or vaccine comprising PD-L1-PAN-ECM and functionalized pancreatic cells or immune cells.

[0075] In some embodiments, the method comprises the step of administering an effective amount of monofunctionalized PAN-ECM to a subject requiring monofunctionalized PAN-ECM, wherein the monofunctionalized PAN-ECM comprises HVEM-functionalized PAN-ECM. In some embodiments, the method further comprises the step of administering an effective amount of functionalized pancreatic cells or immune cells to a subject. In some embodiments, the functionalized pancreatic cells or immune cells comprise one or more immune checkpoint proteins covalently bound to the surface of the pancreatic cells or immune cells, wherein the immune checkpoint proteins comprise proteins selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the functionalized pancreatic cells are functionalized beta cells. In some embodiments, a subject is administered a pharmaceutical composition or vaccine comprising HVEM-PAN-ECM and functionalized pancreatic cells or immune cells. In some embodiments, the method comprises the step of administering an effective amount of monofunctionalized PAN-ECM to a subject requiring monofunctionalized PAN-ECM, wherein the monofunctionalized PAN-ECM comprises FasL-functionalized PAN-ECM. In some embodiments, the method further comprises the step of administering an effective amount of functionalized pancreatic cells or immune cells to a subject.In some embodiments, the functionalized pancreatic cells or immune cells comprise one or more immune checkpoint proteins covalently bound to the surface of the pancreatic cells or immune cells, wherein the immune checkpoint proteins comprise proteins selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the functionalized pancreatic cells are functionalized beta cells. In some embodiments, a subject is administered a pharmaceutical composition or vaccine comprising FasL-PAN-ECM and functionalized pancreatic cells or immune cells.

[0076] In some embodiments, the method comprises the step of administering an effective amount of monofunctionalized PAN-ECM to a subject requiring monofunctionalized PAN-ECM, wherein the monofunctionalized PAN-ECM comprises FGL1-functionalized PAN-ECM. In some embodiments, the method further comprises the step of administering an effective amount of functionalized pancreatic cells or immune cells to a subject. In some embodiments, the functionalized pancreatic cells or immune cells comprise one or more immune checkpoint proteins covalently bound to the surface of the pancreatic cells or immune cells, wherein the immune checkpoint proteins comprise proteins selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the functionalized pancreatic cells are functionalized beta cells. In some embodiments, a subject is administered a pharmaceutical composition or vaccine comprising FGL1-PAN-ECM and functionalized pancreatic cells or immune cells.

[0077] In another embodiment, the treatment method comprises the step of administering an effective amount of dual-functionalized PAN-ECM to a subject requiring dual-functionalized PAN-ECM, wherein the dual-functionalized PAN-ECM comprises two immune checkpoint molecules covalently bonded to the pancreatic extracellular matrix (PAN-ECM). In some embodiments, the immune checkpoint molecules are selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the method further comprises the step of administering an effective amount of functionalized pancreatic cells or immune cells to the subject. In some embodiments, the functionalized pancreatic cells are functionalized beta cells. In some embodiments, the functionalized pancreatic cells or immune cells comprise one or more immune checkpoint proteins covalently bonded to the surface of the pancreatic cells or immune cells, wherein the immune checkpoint proteins comprise proteins selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96.

[0078] In some embodiments, the method comprises the step of administering an effective amount of dual-functionalized PAN-ECM to a subject requiring dual-functionalized PAN-ECM, wherein the dual-functionalized PAN-ECM comprises PD-L1-HVEM-functionalized PAN-ECM. In some embodiments, the subject receives a pharmaceutical composition or vaccine comprising PD-L1-HVEM-PAN-ECM.

[0079] In some embodiments, the method comprises the step of administering an effective amount of dual-functionalized PAN-ECM to a subject requiring dual-functionalized PAN-ECM, wherein the dual-functionalized PAN-ECM comprises PD-L1-HVEM-functionalized PAN-ECM. In some embodiments, the method further comprises the step of administering an effective amount of functionalized pancreatic cells or immune cells to a subject. In some embodiments, the functionalized pancreatic cells or immune cells comprise one or more immune checkpoint proteins covalently bound to the surface of the pancreatic cells or immune cells, wherein the immune checkpoint proteins comprise proteins selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the functionalized pancreatic cells are functionalized beta cells. In some embodiments, a subject is administered a pharmaceutical composition or vaccine comprising PD-L1-HVEM-PAN-ECM and functionalized pancreatic cells or immune cells.

[0080] In various embodiments, the treatment method comprises the step of administering an effective amount of triple-functionalized PAN-ECM to a subject requiring triple-functionalized PAN-ECM, wherein the triple-functionalized PAN-ECM comprises three immune checkpoint molecules covalently bonded to the pancreatic extracellular matrix (PAN-ECM). In some embodiments, the immune checkpoint molecules are selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the method further comprises the step of administering an effective amount of functionalized pancreatic cells or immune cells to the subject. In some embodiments, the functionalized pancreatic cells are functionalized beta cells. In some embodiments, the functionalized pancreatic cells or immune cells comprise one or more immune checkpoint proteins covalently bonded to the surface of the pancreatic cells or immune cells, wherein the immune checkpoint proteins comprise proteins selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96.

[0081] In various other embodiments, the method comprises the step of administering an effective amount of triple-functionalized PAN-ECM to a subject requiring triple-functionalized PAN-ECM, wherein the triple-functionalized PAN-ECM comprises PD-L1-FGL1-TGFβ-functionalized PAN-ECM. In some embodiments, the subject receives a pharmaceutical composition or vaccine comprising PD-L1-FGL1-TGFβ-functionalized PAN-ECM.

[0082] In another embodiment, the method comprises the step of administering an effective amount of triple-functionalized PAN-ECM to a subject requiring triple-functionalized PAN-ECM, wherein the triple-functionalized PAN-ECM comprises FasL-HVEM-FGL1-functionalized PAN-ECM. In some embodiments, the subject receives a pharmaceutical composition or vaccine comprising FasL-HVEM-FGL1-functionalized PAN-ECM.

[0083] In some embodiments, the method comprises the step of administering an effective amount of triple-functionalized PAN-ECM to a subject requiring triple-functionalized PAN-ECM, wherein the triple-functionalized PAN-ECM comprises FasL-TGFβ-PD-L1-functionalized PAN-ECM. In some embodiments, the subject receives a pharmaceutical composition or vaccine comprising FasL-TGFβ-PD-L1-functionalized PAN-ECM.

[0084] In some embodiments, the method comprises the step of administering an effective amount of triple-functionalized PAN-ECM to a subject requiring triple-functionalized PAN-ECM, wherein the triple-functionalized PAN-ECM comprises FasL-GITRL-TGFβ-functionalized PAN-ECM. In some embodiments, the subject receives a pharmaceutical composition or vaccine comprising FasL-GITRL-TGFβ-functionalized PAN-ECM.

[0085] In another embodiment, the method comprises the step of administering an effective amount of triple-functionalized PAN-ECM to a subject requiring triple-functionalized PAN-ECM, wherein the triple-functionalized PAN-ECM comprises PD-L1-HVEM-FasL-functionalized PAN-ECM. In some embodiments, the subject receives a pharmaceutical composition or vaccine comprising PD-L1-HVEM-FasL-functionalized PAN-ECM.

[0086] In some embodiments, the method comprises the step of administering an effective amount of triple-functionalized PAN-ECM to a subject requiring triple-functionalized PAN-ECM, wherein the triple-functionalized PAN-ECM comprises PD-L1-FGL1-TGFβ-functionalized PAN-ECM. In some embodiments, the method further comprises the step of administering an effective amount of functionalized pancreatic cells or immune cells to a subject. In some embodiments, the functionalized pancreatic cells or immune cells comprise one or more immune checkpoint proteins covalently bound to the surface of the pancreatic cells or immune cells, wherein the immune checkpoint proteins comprise proteins selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the functionalized pancreatic cells are functionalized beta cells. In some embodiments, a subject is administered a pharmaceutical composition or vaccine comprising PD-L1-FGL1-TGFβ-PAN-ECM and functionalized pancreatic cells or immune cells.

[0087] In various embodiments, the method comprises the step of administering an effective amount of triple-functionalized PAN-ECM to a subject requiring triple-functionalized PAN-ECM, wherein the triple-functionalized PAN-ECM comprises FasL-HVEM-FGL1-functionalized PAN-ECM. In some embodiments, the method further comprises the step of administering an effective amount of functionalized pancreatic cells or immune cells to a subject. In some embodiments, the functionalized pancreatic cells or immune cells comprise one or more immune checkpoint proteins covalently bound to the surface of the pancreatic cells or immune cells, wherein the immune checkpoint proteins comprise proteins selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the functionalized pancreatic cells are functionalized beta cells. In some embodiments, a subject is administered a pharmaceutical composition or vaccine comprising FasL-HVEM-FGL1-functionalized PAN-ECM and functionalized pancreatic cells or immune cells.

[0088] In some embodiments, the method comprises the step of administering an effective amount of triple-functionalized PAN-ECM to a subject requiring triple-functionalized PAN-ECM, wherein the triple-functionalized PAN-ECM comprises FasL-TGFβ-PD-L1-functionalized PAN-ECM. In some embodiments, the method further comprises the step of administering an effective amount of functionalized pancreatic cells or immune cells to a subject. In some embodiments, the functionalized pancreatic cells or immune cells comprise one or more immune checkpoint proteins covalently bound to the surface of the pancreatic cells or immune cells, wherein the immune checkpoint proteins comprise proteins selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the functionalized pancreatic cells are functionalized beta cells. In some embodiments, a subject is administered a pharmaceutical composition or vaccine comprising FasL-TGFβ-PD-L1-PAN-ECM and functionalized pancreatic cells or immune cells.

[0089] In another embodiment, the method comprises the step of administering an effective amount of triple-functionalized PAN-ECM to a subject requiring triple-functionalized PAN-ECM, wherein the triple-functionalized PAN-ECM comprises FasL-GITRL-TGFβ-functionalized PAN-ECM. In some embodiments, the method further comprises the step of administering an effective amount of functionalized pancreatic cells or immune cells to a subject. In some embodiments, the functionalized pancreatic cells or immune cells comprise one or more immune checkpoint proteins covalently bound to the surface of the pancreatic cells or immune cells, wherein the immune checkpoint proteins comprise proteins selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the functionalized pancreatic cells are functionalized beta cells. In some embodiments, a subject is administered a pharmaceutical composition or vaccine comprising FasL-GITRL-TGFβ-PAN-ECM and functionalized pancreatic cells or immune cells.

[0090] In some embodiments, the method comprises the step of administering an effective amount of triple-functionalized PAN-ECM to a subject requiring triple-functionalized PAN-ECM, wherein the triple-functionalized PAN-ECM comprises PD-L1-HVEM-FasL-functionalized PAN-ECM. In some embodiments, the method further comprises the step of administering an effective amount of functionalized pancreatic cells or immune cells to a subject. In some embodiments, the functionalized pancreatic cells or immune cells comprise one or more immune checkpoint proteins covalently bound to the surface of the pancreatic cells or immune cells, wherein the immune checkpoint proteins comprise proteins selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the functionalized pancreatic cells are functionalized beta cells. In some embodiments, a subject is administered a pharmaceutical composition or vaccine comprising PD-L1-HVEM-FasL-PAN-ECM and functionalized pancreatic cells or immune cells.

[0091] In some embodiments, the treatment method comprises the step of administering an effective amount of quadruple-functionalized PAN-ECM to a subject requiring quadruple-functionalized PAN-ECM, wherein the quadruple-functionalized PAN-ECM comprises four immune checkpoint molecules covalently bonded to the pancreatic extracellular matrix (PAN-ECM). In some embodiments, the immune checkpoint molecules are selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the method further comprises the step of administering an effective amount of functionalized pancreatic cells or immune cells to the subject. In some embodiments, the functionalized pancreatic cells are functionalized beta cells. In some embodiments, the functionalized pancreatic cells or immune cells comprise one or more immune checkpoint proteins covalently bonded to the surface of the pancreatic cells or immune cells, wherein the immune checkpoint proteins comprise proteins selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96.

[0092] In another embodiment, the method comprises the step of administering an effective amount of quadruple-functionalized PAN-ECM to a subject requiring quadruple-functionalized PAN-ECM, wherein the quadruple-functionalized PAN-ECM comprises FasL-GITRL-TGFβ-HVEM-functionalized PAN-ECM. In some embodiments, the subject receives a pharmaceutical composition or vaccine comprising FasL-GITRL-TGFβ-HVEM-functionalized PAN-ECM.

[0093] In various other embodiments, the method comprises the step of administering an effective amount of quadruple-functionalized PAN-ECM to a subject requiring quadruple-functionalized PAN-ECM, wherein the quadruple-functionalized PAN-ECM comprises FasL-GITRL-TGFβ-HVEM-functionalized PAN-ECM. In some embodiments, the method further comprises the step of administering an effective amount of functionalized pancreatic cells or immune cells to a subject. In some embodiments, the functionalized pancreatic cells or immune cells comprise one or more immune checkpoint proteins covalently bound to the surface of the pancreatic cells or immune cells, wherein the immune checkpoint proteins comprise proteins selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. In some embodiments, the functionalized pancreatic cells are functionalized beta cells. In some embodiments, a subject is administered a pharmaceutical composition or vaccine comprising FasL-GITRL-TGFβ-HVEM-PAN-ECM and functionalized pancreatic cells or immune cells.

[0094] III. Manufacturing Method

[0095] In one aspect, the present disclosure also includes a method for preparing the disclosed composition. A method for preparing a functionalized pancreatic extracellular matrix (CP-PAN-ECM) may include the steps of providing a functionalized PAN-ECM in which the PAN-ECM is functionalized with a moiety comprising residues suitable for click chemistry; providing a functionalized immune checkpoint molecule in which the immune checkpoint molecule is functionalized with a moiety comprising residues suitable for click chemistry; and contacting the azide-modified PAN-ECM with an immune checkpoint bioconjugate to form a CP-PAN-ECM using alkyne-azide cyclization addition (SPAAC) chemistry. In one aspect, the functionalized PAN-ECM comprises an azide moiety. In one aspect, the functionalized immune checkpoint molecule comprises a dibenzocyclooctane moiety. In one aspect, the immune checkpoint molecule is a bioconjugate further comprising a dendrimer, a linear polymer, a nanoparticle, or an Fc fusion protein. In one embodiment, the dibenzocyclooctane moiety comprises DBCO, or a derivative thereof, or a conjugate thereof. Optionally, a purification step may be performed after any reaction described herein to improve the purity of the disclosed composition.

[0096] In some embodiments, PAN-ECM is produced from acellularized pancreatic tissue. In some embodiments, acellularized pancreatic tissue is produced by decellularizing a pancreas isolated from a mammal. In some embodiments, the mammal is a rodent such as a mouse or rat. In some embodiments, pancreatic tissue is decellularized using a detergent such as Triton-X-100 and an ammonia solution. In some embodiments, pancreatic tissue is decellularized using a mixed solution of 1% Triton-X-100 and 0.1% ammonia solution.

[0097] An exemplary method of manufacturing and using the disclosed CP-PAN-ECM is provided in the embodiments described herein.

[0098] Implementation example

[0099] 1. A functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently bonded to the pancreatic extracellular matrix (PAN-ECM).

[0100] 2. In Embodiment 1, the immune checkpoint molecule comprises PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, or CD96, CP-PAN-ECM.

[0101] 3. In Embodiment 1, CP-PAN-ECM, wherein at least one immune checkpoint molecule is bound to PAN-ECM using click chemistry.

[0102] 4. In Embodiment 3, CP-PAN-ECM, wherein at least one immune checkpoint molecule is bound to PAN-ECM via alkyne-azide cyclization addition (SPAAC) chemistry.

[0103] 5. In Embodiment 4, CP-PAN-ECM, wherein at least one immune checkpoint molecule comprises a dibenzocyclooctine moiety.

[0104] 6. In Embodiment 5, the dibenzocyclooctane moiety is CP-PAN-ECM, which is DBCO, or a derivative thereof, or a conjugate thereof.

[0105] 7. In Embodiment 5, CP-PAN-ECM, wherein the immune checkpoint molecule is a bioconjugate comprising a dendrimer, a linear polymer, a nanoparticle, or an Fc fusion protein.

[0106] 8. A composition comprising CP-PAN-ECM of any one of Embodiments 1 to 7.

[0107] 9. A composition comprising a functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently bonded to the pancreatic extracellular matrix (PAN-ECM).

[0108] 10. In Embodiment 8 or 9, the composition is a pharmaceutical composition.

[0109] 11. A composition in any one of embodiments 8 to 10, wherein the composition further comprises pancreatic cells or immune cells.

[0110] 12. A composition in which, in Embodiment 11, the pancreatic cells are beta cells (β cells).

[0111] 13. A composition in any one of embodiments 8 to 12, further comprising at least one excipient, or at least one additional therapeutic agent, or a combination thereof.

[0112] 14. A composition for use in the prevention or treatment of diabetes mellitus in any one of embodiments 8 to 13.

[0113] 15. A method for preparing a functionalized pancreatic extracellular matrix (CP-PAN-ECM), comprising the following steps (a), (b), and (c): (a) providing a functionalized PAN-ECM; (b) providing a functionalized immune checkpoint molecule; and (c) contacting the functionalized PAN-ECM with a functionalized immune checkpoint bioconjugate to form CP-PAN-ECM using alkyne-azide cyclization addition (SPAAC) chemistry.

[0114] 16. A method according to Embodiment 15, wherein the functionalized PAN-ECM comprises an azide moiety.

[0115] 17. A method according to Embodiment 15, wherein the functionalized immune checkpoint molecule comprises a dibenzocyclooctine moiety.

[0116] 18. Method according to Embodiment 17, wherein the dibenzocyclooctane moiety comprises DBCO, or a derivative thereof, or a conjugate thereof.

[0117] 19. Method of Embodiment 18, wherein the immune checkpoint molecule is a bioconjugate comprising a dendrimer, a linear polymer, a nanoparticle, or an Fc fusion protein.

[0118] 20. A method for treating diabetes or reducing the progression of diabetes in a subject, comprising the step of administering any one of the compositions of Embodiments 8 to 14 to a subject who requires treatment of diabetes or reduction of the progression of diabetes.

[0119] 21. Method in Embodiment 20, wherein the subject is a mammal.

[0120] 22. A method according to Embodiment 21, wherein administration is performed via a parenteral route.

[0121] 23. In the embodiment 21, the diabetes is autoimmune diabetes.

[0122] 24. A functionalized pancreatic extracellular matrix comprising the structure (PAN-ECM) ― (residue of an azide-containing molecule) ― (residue of a cyclooctane) ― (linker 1) ― (residue of a functionalized dendrimer)q ― (residue of an immune checkpoint molecule), wherein q is 0 or 1; and a dash (―) indicates a covalent bond.

[0123] Examples

[0124] The following examples are incorporated to illustrate preferred embodiments of the present disclosure. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques found by the inventors to be fully functional in the practice of the present disclosure and thus may be considered to constitute a preferred method for practicing the present disclosure. However, those skilled in the art should understand that, based on the present disclosure, many variations may be made to the specific embodiments disclosed without departing from the spirit and scope of the present disclosure, and similar or similar results may still be obtained.

[0125] Example 1: Preparation and Decellularization of the Pancreas

[0126] The pancreas of healthy, live rats aged 6 months was stored in vitro at -80°C and frozen for 12 hours. The connective and adipose tissue surrounding the pancreas was peeled off, and approximately 0.5 to 1 cm 3 The tissue was cut into pieces of a suitable size. The pancreatic tissue fragments were rinsed three times with cold PBS for 1 hour each. A mixed solution of 1% Triton-X-100 and 0.1% ammonia solution was prepared by adding Triton-X-100 and ammonia water (Guangdong, China) to PBS. The pancreatic tissue fragments were placed in the Triton-X-100 solution and vibrated at 4°C for 72 hours. The Triton-X-100 solution was replaced three times every 24 hours. Subsequently, to completely remove the detergent, the tissue was rinsed three times at 4°C for 12 hours each with PBS containing pen / strep. The acellularized pancreatic tissue was stored at -80°C.

[0127] Azide-modified PAN-ECM was generated by a carbodiimide / N-hydroxysuccinimide (EDC / NHS) reaction with azide-PEG8-NHS. Before attaching checkpoints to the azide-modified PAN-ECM, DBCO-functionalized immune checkpoint bioconjugates were first prepared via a bioorthogonal click reaction between the checkpoint and the DBCO-dendrimer. Subsequently, checkpoint-functionalized PAN-ECM was prepared using a bioorthogonal click reaction between the DBCO-functionalized immune checkpoint bioconjugate and the azide-functionalized PAN-ECM. The therapeutic efficacy of various subcutaneously (sc) injectable checkpoint-functionalized PAN-ECM + beta cells was evaluated in novel diabetic NOD female mice 2 to 3 days after the onset of diabetes. Additionally, two boosters were administered 2 and 4 weeks after the initial treatment.

[0128] Example 2: Preparation of DBCO-functionalized PAMAM G5

[0129] PAMAM G5 (0.7 mL of 5 wt / wt% PAMAM G5 containing 27.9 mg of dendrimer and 0.97 μmol of 0.97 μmol in methanol) was first dried under nitrogen gas at 20°C for 15 minutes, and then dried under vacuum for an additional 2 hours. DBCO-PEG4-NHS ester (764 μL, 25 mM, 12.3 mg in DMSO) was added to the dried dendrimer film. The mixture was stirred at 20°C (in the dark) for 4 hours. An excess of acetic anhydride (0.5 mL) was added to the reaction mixture and stirred at 20°C (in the dark) for an additional 18 hours. The DBCO-functionalized dendrimer was further purified by equilibrium dialysis for an additional 3 days (6 cycles) in the dark. The purified dendrimer was freeze-dried and stored at -80°C (in the dark) until further study.

[0130] Example 3: Preparation of azide-functionalized PD-L1

[0131] 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 of PD-L1 (0.194 nmol, dissolved in 100 μL PBS) was incubated with an azide-PEG4-NHS solution (10 μL of 3.88 nmol, 0.388 mM azide-PEG4-NHS) at 20°C for 1.5 hours. The functionalized PD-L1 was purified three times using Zebra Spin 7K MWCO Desalting Columns according to the manufacturer's protocol. The concentration of the purified azide-functionalized PD-L1 was quantified by UV-visible spectroscopy.

[0132] Azide-functionalized TGF-β (TGF-β-N3), azide-functionalized HVEM (HVEM-N3), azide-functionalized FasL (FasL-N3), azide-functionalized CD47 (CD47-N3), azide-functionalized GITRL (GITRL-N3), and azide-functionalized Gal-9 (Gal-9-N3) were synthesized using the same method, except that the target degree of function of TGF-β, FasL, CD47, and Gal-9 was 10. Figure 1 provides a schematic diagram of the bioengineering of checkpoint-functionalized PAN-ECM (top) and the mechanism of action of sc-injected beta cells + PAN-ECM (bottom).

[0133] Example 4: Preparation of PD-L1-Dend bioconjugate

[0134] Purified azide-functionalized PD-L1 was quantitatively conjugated to DBCO-functionalized PAMAM G5 at a target molar ratio of 1:1. PD-L1-N3 (10 μg) and DBCO-functionalized PAMAM G5 dendrimers (8 μg, 0.8 μL of dendrimer solution in 10 mg / mL PBS) were mixed and incubated at 4°C for 24 hours. The PD-L1-Dend bioconjugate was used in subsequent studies without further purification.

[0135] TGF-β-Dend, HVEM-Dend, FasL-Dend, CD47-Dend, GITRL-Dend, and Gal-9-Dend bioconjugates were prepared using the same method.

[0136] Example 5: Decoration of Checkpoint Molecularly Modified PAN-ECM

[0137] PAN-ECM (100 mg) was washed twice with PBS and resuspended in PBS at 350 mg / mL. Subsequently, azide-PEG8-NHS (20 μL, 285 mM in DMSO) was added to PAN-ECM and incubated overnight at 4°C. PD-L1-N3 (10 μg) and DBCO-functionalized PAMAM G5 dendrimer (8 μg, 0.8 μL of dendrimer solution in 10 mg / mL PBS) were mixed and incubated at 37°C for 30 minutes. Meanwhile, azide-PAN-ECM was washed three times and resuspended in PBS at 1000 mg / mL. Next, azide-PAN-ECM was 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.

[0138] TGF-β-, HVEM-, FasL-, CD47-, GITRL-, and Gal-9-modified PAN-ECM were synthesized using the same method.

[0139] Example 6. In vivo evaluation of CP-PAN-ECM for reversing early-onset T1D NOD mice

[0140] To expand the list of effective checkpoints that could be targeted, a cohort of NOD / ShiltJ (NOD, female) mice was recruited, and their blood glucose levels were monitored twice weekly. After the onset of diabetes defined by blood glucose levels exceeding 250 mg / dL, mice were administered a combination of checkpoint-modified decellularized pancreatic extracellular matrix (PAN-ECM) and NIT-1 beta cells via subcutaneous injection twice over a 2-week period (Fig. 3a). Initially, mice were administered a single checkpoint-modified ECM to compare the efficacy of various checkpoints (Fig. 3b).

[0141] From this study, several candidates were identified as effective checkpoints to target that help improve overall survival and reverse the onset of diabetes. When novel diabetic NOD female mice were treated with PD-L1, HVEM, and FasL-functionalized PAN-ECM, 50% of the PD-L1-functionalized PAN-ECM treatment group reversed to a normoglycemic state (Fig. 2a). Among these checkpoint groups, HVEM showed the strongest response, delaying the onset of diabetes in 75% of the mice beyond a 100-day monitoring period (Fig. 3b). Approximately half of the HVEM-functionalized PAN-ECM-treated mice (Fig. 2b) and FasL-functionalized PAN-ECM-treated mice (Fig. 2c) remained diabetes-free for 100 days. All groups, including those receiving co-injection of unmodified NIT-1 cells and ECM, had some effect on delaying the onset of diabetes and improving survival, demonstrating a strong baseline for this technology. Next, we investigated whether PAN-ECMs simultaneously modified across all three immune checkpoints could be used as a therapeutic vaccine to reverse early-onset hyperglycemia. The results indicated that, compared to the three monofunctionalized PAN-ECMs, PD-L1 / HVEM / FasL-functionalized PAN-ECMs resulted in more potent and simultaneous inhibition of multiple immune checkpoint pathways, thereby leading to a more effective induction of antigen-specific T cell depletion (Fig. 2d).

[0142] To confirm the potential synergistic effects of these immune checkpoints, selected checkpoint combinations were conjugated to PAN-ECM and injected with NIT-1 cells as described above. The following five groups of checkpoints 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. All treatments including FGL1 showed no improvement compared to unmodified PAN-ECM, as most mice progressed to hyperglycemia within 30 days (Fig. 3c). All other groups exhibited extended survival (Figs. 4a and 4b), and most mice maintained normoglycemic levels during a 100-day monitoring period (Fig. 3c). These groups included various combinations of PD-L1, HVEM, FASL, GITRL, and TGF β, thereby providing a list of potentially synergistic target combinations that help soothe autoimmune attacks of natural pancreatic islets.

[0143] To determine the potential maximum response, a mouse cohort was treated with combinations of all cytokines and checkpoints that demonstrated efficacy in previous studies. Diabetic mice were treated with a combination of HVEM, FAS-L, GITRL, and TGF β-modified ECM combined with doses of PD-L1-modified NIT-1 cells. This treatment showed the most potent response, with 80% of mice maintaining normal blood glucose levels until week 15 and 30% of mice avoiding hypoglycemia for more than 20 weeks (Figs. 5a and 5b).

[0144] Example 7: In vitro evaluation of CP-functionalized NIT-1 cells for anergizing antigen-specific CD8+ T cells

[0145] To test whether anchoring various immune checkpoint molecules to the surface enhances the ability to inhibit the cytotoxic effects of islet antigen-specific T cells, cytotoxicity assays were performed using variously functionalized NIT-1 cells in the presence of islet-specific IGRP peptides (Fig. 6b). Fluorescence-activated cell sorting (FACS) analysis indicated that PD-L1, HVEM, FasL, and GITRL could be successfully decorated on NIT-1 cells by metabolic glycoengineering and bioorthogonal click chemistry (Fig. 6a). With the exception of FasL, the remaining three immune checkpoints significantly inhibited T cell-mediated cytotoxicity against islet cells when modified on the cell surface (Fig. 6c). Interestingly, T cell apoptosis efficiency increased slightly when FasL was anchored to the cell surface, suggesting that FasL could potentially act as a negative checkpoint in diabetes treatment. Therefore, PD-L1 and HVEM can serve as potential checkpoints for reversing diabetes. Subsequent studies on the combined effects of these agents revealed that they significantly reduced T cell-mediated cytotoxicity against pancreatic islet cells compared to the use of individual checkpoints (Fig. 6d).

[0146] Example 8. In vivo administration of CP-ECM and PD-L1-functionalized NIT-1 cells reverses early-onset T1D

[0147] Initial screening indicated that modifying PAN-ECM with PD-L1 / HVEM / FasL resulted in a 75% reversal of diabetes, and one mouse died on day 24 (Fig. 3c). In a follow-up study, PD-L1 was also placed on the surface of NIT-1 cells (Fig. 7a). As shown in Figs. 7b and 7c, separating two checkpoints rather than modifying all checkpoints on PAN-ECM yielded improved results. This suggests that the inhibitory effect of PD-L1 on T cells may depend on antigen presentation.

[0148] Example 9: Diabetes treatment using an in vivo mouse model

[0149] This embodiment identifies a method for developing pancreatic islet-like cells from sources other than NIT-1 cells. Examples of such sources include mouse embryonic stem cells (mESCs, derived from C57BL / 6), which provide a pathway for generating insulin-producing cells through a complex sequence of medium exchanges (Fig. 8). Briefly, embryonic stem cells isolated from C57BL / 6 mice were cultured in N2B27 medium in bacterial culture petri dishes for 3 days. Differentiation of the mESCs was initiated by transferring the cells to a 0.1% gelatin-coated dish in Differentiation Medium I, followed by a 3-day culture period in N2B27 medium supplemented with Y27632. Subsequently, the cells were cultured in Differentiation Medium II for 18 days to induce pancreatic differentiation, resulting in the formation of pancreatic islet-like clusters. Next, dithizone staining was performed to identify and purify pancreatic islet-like clusters from undifferentiated cells. Subsequently, the pancreatic islet-like clusters can be cultured in an insulin-free medium before performing the glucose-stimulated insulin secretion assay described herein.

[0150] The change in insulin secretion fold-change between low and high glucose conditions was not significant between IPCs and mouse pancreatic islet controls (Fig. 9a). Surprisingly, differentiated IPCs showed a uniform distribution of insulin granules among different clusters compared to human pancreatic islets, as indicated by dicyzone staining (Fig. 9b). Furthermore, 88% of differentiated IPCs produced both insulin and glucagon (Fig. 9c), indicating efficient β-cell induction.

[0151] The functionalization of IPCs using HVEM and PD-L1 (engineered IPCs) was achieved by a bioengineering approach involving metabolic glycoengineering followed by bioorthogonal click responses (Fig. 10a). FACS studies confirmed the successful decoration of IPCs using HVEM and PD-L1, in which 100% of the engineered IPCs exhibited both HVEM and PD-L1 positivity (Fig. 10b). The in vivo transplantation of these allogeneic cells was evaluated in a streptozotocin (STZ)-induced diabetic BALB / c mouse model. STZ-induced diabetes was induced by administering 175 mg of STZ per kg of body weight via intraperitoneal injection to 10-week-old female BALB / c mice 5 days prior to IPC and PAN-ECM transplantation. The induction of diabetes was confirmed by measuring blood glucose levels 2 days prior to IPC and PAN-ECM transplantation. IPC and PAN-ECM were implanted under the abdominal skin of recipient BALB / c mice (Fig. 10c). Mice were divided into the following three groups: (i) control mice without STZ treatment, (ii) STZ-treated mice with subcutaneous implantation of IPC and PAN ECM, and (iii) STZ-treated mice with subcutaneous implantation of engineered IPC and PAN ECM decorated with HVEM and PD-L1. Control IPC administered in combination with PAN ECM to diabetic BALB / c mice did not effectively alleviate the STZ-induced diabetic state in BALB / c mice, and all mice progressed to hyperglycemia by day 30 (Fig. 10d). In stark contrast, mice administered engineered IPC decorated with HVEM and PD-L1 in combination with PAN ECM showed significant stabilization of blood glucose levels (Fig. 10d). These results suggest that engineered IPCs can provide significant relief of hyperglycemia and support metabolic homeostasis when administered in combination with PAN ECM.

[0152] Thirty days after transplantation, the composition of the IPC + PAN-ECM graft was analyzed by flow cytometry in exemplary mice from the IPC + PAN-ECM cohort and the engineered IPC + PAN-ECM cohort (see Fig. 10d, arrows indicate mice from which the graft was removed for analysis). Notably, upon removal of the graft from the engineered IPC + PAN-ECM mice, the blood glucose levels of the mice increased rapidly to 600 mg / dL, indicating that the engineered IPC + PAN-ECM graft was the sole and significant source of insulin within the mice (Fig. 10d). To perform this analysis, the IPC + PAN-ECM graft was removed from each mouse and dissociated using GentleMACS. Apoptotic cells were excluded from the analysis using a live / dead kit. Subsequently, flow cytometry was performed by gating for pancreatic islets and checkpoint markers, including insulin and glucagon. The results of this flow cytometry analysis show that engineered IPCs decorated with HVEM and PD-L1 continued to produce glucagon and insulin 30 days after in vivo implantation in the presence of PAN ECM (Fig. 10e).

Claims

Claim 1 A functionalized pancreatic extracellular matrix (CP-PAN-ECM), wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently bonded to a pancreatic extracellular matrix (PAN-ECM). Claim 2 CP-PAN-ECM, wherein the immune checkpoint molecule comprises PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, or CD96. Claim 3 CP-PAN-ECM according to claim 1 or 2, wherein the at least one immune checkpoint molecule is bound to the PAN-ECM using click chemistry. Claim 4 CP-PAN-ECM according to paragraph 3, wherein two, three, or four immune checkpoint molecules are bound to the PAN-ECM using click chemistry. Claim 5 CP-PAN-ECM according to any one of claims 1 to 3, wherein the at least one immune checkpoint molecule is bound to the PAN-ECM through alkyne-azide cyclization addition (SPAAC) chemistry. Claim 6 CP-PAN-ECM according to claim 5, wherein two, three, or four immune checkpoint molecules are bound to the PAN-ECM via alkyne-azide cyclization addition (SPAAC) chemistry. Claim 7 In claim 5, the above-mentioned immune checkpoint molecule comprises a dibenzocyclooctine moiety, CP-PAN-ECM. Claim 8 In claim 7, the dibenzocyclooctane moiety is CP-PAN-ECM, which is DBCO, or a derivative thereof, or a conjugate thereof. Claim 9 In claim 7, the immune checkpoint molecule is a bioconjugate comprising a dendrimer, a linear polymer, a nanoparticle, or an Fc fusion protein, CP-PAN-ECM. Claim 10 A composition comprising CP-PAN-ECM of any one of claims 1 to 9. Claim 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 bonded to the pancreatic extracellular matrix (PAN-ECM). Claim 12 A composition according to claim 11, wherein the CP-PAN-ECM comprises two, three, or four immune checkpoint molecules covalently bonded to the PAN-ECM. Claim 13 A composition according to claim 11 or 12, wherein at least one immune checkpoint molecule is selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. Claim 14 A composition according to any one of claims 11 to 13, wherein the CP-PAN-ECM comprises a dual-functionalized CP-PAN-ECM comprising PD-L1 and HVEM covalently bonded to the PAN-ECM. Claim 15 A composition according to any one of claims 11 to 13, wherein the CP-PAN-ECM comprises a tri-functionalized CP-PAN-ECM comprising 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, covalently bonded to the PAN-ECM. Claim 16 A composition according to any one of claims 11 to 13, wherein the CP-PAN-ECM comprises a quadruple-functionalized CP-PAN-ECM comprising FasL, GITRL, TGFβ, and HVEM covalently bonded to the PAN-ECM. Claim 17 A composition according to any one of claims 10 to 16, wherein the composition is a pharmaceutical composition. Claim 18 A composition according to any one of claims 10 to 17, wherein the composition further comprises pancreatic cells or immune cells. Claim 19 A composition according to claim 18, wherein the pancreatic cell or immune cell comprises at least one immune checkpoint molecule covalently bonded to the surface of the pancreatic cell or immune cell. Claim 20 A composition according to claim 19, wherein the immune checkpoint molecule is selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. Claim 21 In claim 20, the composition wherein the immune checkpoint molecule comprises PD-L1, HVEM, FasL, or GITRL. Claim 22 A composition according to any one of claims 19 to 21, wherein the immune checkpoint molecule is covalently bonded to the surface of the pancreatic cell or immune cell by metabolic glycoengineering. Claim 23 A composition according to any one of claims 19 to 22, wherein the immune checkpoint molecule is covalently bonded to the surface of the pancreatic cell or immune cell using click chemistry. Claim 24 A composition according to any one of claims 19 to 23, wherein the pancreatic cells are beta cells (β cells). Claim 25 A composition according to any one of claims 10 to 24, further comprising at least one excipient, or at least one additional therapeutic agent, or a combination thereof. Claim 26 A composition for use in the prevention or treatment of diabetes mellitus, in any one of claims 10 to 25. Claim 27 A method for preparing a functionalized pancreatic extracellular matrix (CP-PAN-ECM), comprising the following steps (a), (b), and (c): (a) providing a functionalized PAN-ECM; (b) providing a functionalized immune checkpoint molecule; and (c) contacting the functionalized PAN-ECM with the functionalized immune checkpoint bioconjugate to form the CP-PAN-ECM using alkyne-azide cyclization addition (SPAAC) chemistry. Claim 28 In claim 27, the method wherein the functionalized PAN-ECM comprises an azide moiety. Claim 29 In claim 27, the method wherein the functionalized immune checkpoint molecule comprises a dibenzocyclooctine moiety. Claim 30 In claim 29, the method wherein the dibenzocyclooctane moiety comprises DBCO, or a derivative thereof, or a conjugate thereof. Claim 31 A method according to any one of claims 27 to 30, wherein the immune checkpoint molecule is a bioconjugate comprising a dendrimer, a linear polymer, a nanoparticle, or an Fc fusion protein. Claim 32 A method for treating diabetes or reducing the progression of diabetes in a subject, comprising the step of administering a composition of any one of claims 10 to 26 to a subject who requires treatment of diabetes or reduction of the progression of diabetes. Claim 33 In paragraph 32, the above-mentioned subject is a mammal, method. Claim 34 A method according to paragraph 32 or 33, wherein the administration is performed via a parenteral route. Claim 35 A method according to any one of paragraphs 32 to 34, wherein the diabetes is autoimmune diabetes. Claim 36 A method for treating diabetes or reducing the progression of diabetes in a subject requiring treatment of diabetes or reduction of the progression of diabetes, comprising the step of 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 bonded to the pancreatic extracellular matrix (PAN-ECM), and the functionalized pancreatic cells comprise at least one immune checkpoint molecule covalently bonded to the surface of the pancreatic cells. Claim 37 A method according to claim 36, wherein the at least one immune checkpoint molecule covalently bonded to the pancreatic extracellular matrix (PAN-ECM) or the at least one immune checkpoint molecule covalently bonded to the surface of the pancreatic cell is selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. Claim 38 A method according to claim 36 or 37, wherein the at least one immune checkpoint molecule covalently bonded to the pancreatic extracellular matrix (PAN-ECM) or the at least one immune checkpoint molecule covalently bonded to the surface of the pancreatic cell comprises PD-L1, HVEM, FasL, or GITRL. Claim 39 A method according to any one of claims 36 to 38, wherein the functionalized pancreatic cell comprises HVEM and PD-L1 covalently bonded to the surface of the pancreatic cell. Claim 40 A method according to any one of paragraphs 36 to 39, wherein the subject is a mammal. Claim 41 A method according to any one of claims 36 to 40, wherein the administration is performed via a parenteral route. Claim 42 A method according to any one of paragraphs 36 to 41, wherein the diabetes is autoimmune diabetes. Claim 43 A functionalized pancreatic extracellular matrix (CP-PAN-ECM) for use in a method for treating diabetes or reducing the progression of diabetes in said subject, comprising the step of administering an effective amount of functionalized pancreatic extracellular matrix (CP-PAN-ECM) and functionalized pancreatic cells to said subject who requires treatment of diabetes or reduction of the progression of diabetes, wherein the CP-PAN-ECM comprises at least one immune checkpoint molecule covalently bonded to the pancreatic extracellular matrix (PAN-ECM); and said functionalized pancreatic cells comprise at least one immune checkpoint molecule covalently bonded to the surface of said pancreatic cells. Claim 44 A method according to claim 43, wherein the at least one immune checkpoint molecule covalently bonded to the pancreatic extracellular matrix (PAN-ECM) or the at least one immune checkpoint molecule covalently bonded to the surface of the pancreatic cell is selected from the group consisting of PD-L1, HVEM, CD86, Gal-9, PD-L2, TIGIT, TIM-1, TIM-3, TNFR1, VISTA, BTLA, NKG2A, CTLA-4, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, ICOS, NKp30, LAG3, CD137, FasL, GITRL, TGFβ, FGL1, and CD96. Claim 45 A method according to claim 43 or 44, wherein the at least one immune checkpoint molecule covalently bonded to the pancreatic extracellular matrix (PAN-ECM) or the at least one immune checkpoint molecule covalently bonded to the surface of the pancreatic cell comprises PD-L1, HVEM, FasL, or GITRL. Claim 46 A method according to any one of claims 43 to 45, wherein the functionalized pancreatic cell comprises HVEM and PD-L1 covalently bonded to the surface of the pancreatic cell. Claim 47 A method according to any one of paragraphs 43 to 46, wherein the subject is a mammal. Claim 48 A method according to any one of claims 43 to 47, wherein the administration is performed via a parenteral route. Claim 49 A method according to any one of paragraphs 43 to 48, wherein the diabetes is autoimmune diabetes. Claim 50 A functionalized pancreatic extracellular matrix comprising the structure (PAN-ECM) ― (residue of azide-containing molecule) ― (residue of cyclooctane) ― (linker 1) ― (residue of functionalized dendrimer)q ― (residue of immune checkpoint molecule), where q is 0 or 1; and a dash (―) indicates a covalent bond.

Images