Combination therapy of immunomodulatory drugs, DYRK1A inhibitors, and GLP1R agonist agents in the treatment of type 1 diabetes

Abstract

Disclosed herein are dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitors, glucagon-like peptide-1 receptor (GLP1R) agonist agents, and immunomodulatory monoclonal antibodies and / or immunosuppressive agents (e.g., anti-CD3 antibodies) for a subject in need of treatment of a condition associated with insufficient levels of insulin secretion, wherein the administration is carried out under conditions effective to reverse loss of β-cell mass and function in the subject for treating the subject for a condition associated with insufficient insulin secretion. Also disclosed are compositions and methods for increasing β-cell mass and function of a pancreatic β-cell population.

Description

Technical Field

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 347,977, filed Jun. 1, 2022, the entire contents of which are incorporated herein by reference.

[0002] This invention was made with government support under grant DK105015 from the National Institutes of Health. The government has certain rights in this invention.

[0003] Field The present invention relates to methods and compositions for treating subjects with conditions associated with insulin secretory insufficiency.

Background Art

[0004] In the United States, 1.6 million people and worldwide 20 million people suffer from type 1 diabetes (hereinafter “T1D”). Approximately twice that number are in families at high risk of developing T1D. T1D results from autoreactive host immune cells (T cells and other immune system cells) damaging and killing pancreatic insulin-producing β-cells. Its etiology is unknown, but a combination of genetic, environmental, and infectious factors is involved. In T1D patients, no effective treatment methods are known that can delay, prevent, or reverse the decrease in β-cells and the progression to diabetes. For people in the early stages of T1D where β-cell supply is still sufficient, immunomodulatory therapies that block or delay immune system dysfunction have advanced, but for people with established T1D, these therapies cannot restore lost β-cells. Such β-cell loss in T1D has led to attempts to replace lost β-cells through pancreatic transplantation, islet transplantation, and transplantation of stem cell-derived human β-cells, but these are not scalable or cost-effective approaches for the millions of T1D patients.

[0005] When self-tolerance fails, autoimmune destruction of pancreatic β-cells occurs, leading to T1D. A treatment method that can simultaneously perform the following three actions: 1) regulating the activation of T cells, 2) protecting β-cells from known promoters of β-cell dysfunction and destruction associated with T1D, such as inflammatory cytokines and endoplasmic reticulum stress, and 3) inducing β-cell regeneration, may provide a treatment method for curing T1D.

[0006] Recently, it has been shown that in streptozotocin-induced diabetic mice transplanted with human islets, co-administration of the DYRK1A inhibitor harmine and the GLP1R agonist exendin-4 for 3 months results in a 6- to 7-fold increase in human β-cell mass (Rosselot et al., “Human Beta Cell Mass Expansion In Vivo with a Harmine and Exendin-4 Combination: Quantification and Visualization by iDISCO+ 3D Imaging,” biorxiv (2021)). If applied to T1D patients, this substantial increase in human β-cell mass could be sufficient to normalize blood glucose levels. However, the autoimmunity associated with T1D is highly likely to continue destroying newly regenerated β-cells. Interestingly, in recently diagnosed T1D patients, several immunomodulatory interventions have been shown to delay the decline in β-cell function (Atkinson et al., “The Challenge of Modulating β-Cell Autoimmunity in Type 1 Diabetes,” Lancet Diabetes Endocrinol. 7:52-64 (2019)). One such promising treatment is teplizumab, a humanized non-immunogenic deglycosylated anti-CD3 monoclonal antibody. This anti-CD3 antibody suppresses the decline in β-cell function in newly diagnosed type 1 diabetes patients for up to 7 years from the initial diagnosis (Herold et al., “Teplizumab (anti-CD3 mAb) Treatment Preserves C-Peptide Responses in Patients with New-Onset Type 1 Diabetes In a Randomized Controlled Trial: Metabolic and Immunologic Features at Baseline Identify a Subgroup of Responders,” Diabetes 62:3766-74 (2013); Herold et al., “Anti-CD3 Monoclonal Antibody in New-Onset Type 1 Diabetes Mellitus,” N. Engl. J. Med. 346:1692-8 (2002); Keymeulen et al., “Insulin Needs After CD3-Antibody Therapy in New-Onset Type 1 Diabetes,” N. Engl. J. Med. 352:2598-608 (2005); Sherry et al., “Teplizumab for Treatment of Type 1 Diabetes (Protege Study): 1-Year Results from a Randomised, Placebo-Controlled Trial,” Lancet 378:487-97 (2011); Hagopian et al., “Teplizumab Preserves C-Peptide in Recent-Onset Type 1 Diabetes: Two-Year Results from the Randomized, Placebo-Controlled Protege Trial,” Diabetes 62:3901-8 (2013); Herold et al., “An Anti-CD3 Antibody, Teplizumab, In Relatives at Risk for Type 1 Diabetes,” N. Engl. J. Med.381:603-613 (2019)). This antibody modifies autoreactive CD8+ T lymphocytes, which are important effector cells that kill β cells in T1D. According to subsequent reports, CD3-specific monoclonal antibodies have been shown to enhance the function and / or proliferation of regulatory T cells, which exert dominant suppression over autoreactive T cells. This is a stimulating advance in the treatment of T1D, but further improvements are needed in several areas. First, this method only delays the onset of T1D and does not cure T1D. Second, the effects on established T1D patients are limited, probably reflecting the reduced β-cell mass and the inability of β cells to replicate or regenerate in these patients. Third, the use of high doses of this antibody is associated with potential risks related to lymphoproliferative disorders in young patients.

[0007] The present invention aims to overcome the drawbacks of the art.

Summary of the Invention

[0008] One aspect of the present invention is directed to a method for treating a subject with a medical condition associated with insufficient insulin secretion. This method involves administering to a subject in need of treatment for a medical condition associated with insufficient levels of insulin secretion a dual-specific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody, and optionally, the immunomodulatory monoclonal antibody is an anti-CD3 antibody, and the administration may be carried out under conditions effective to reverse the loss of β-cell mass and function in the subject for treating the subject with a condition associated with insulin secretory deficiency.

[0009] Another aspect of the present invention relates to a composition comprising a dual-specific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., an anti-CD3 antibody).

[0010] A further aspect of the present invention relates to a method of increasing β-cell mass and function in a pancreatic β-cell population. The method comprises contacting a pancreatic β-cell population with a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and a low dose of an immunomodulatory monoclonal antibody and / or an immunosuppressive agent (e.g., anti-CD3 antibody), wherein the contacting is carried out under conditions effective to increase β-cell mass and function in the pancreatic β-cell population.

[0011] In the examples herein, the hypothesis was put forward that combination treatment of anti-CD3 antibody with halmcin and exendin-4, which are β-cell regenerating agents, would reverse T1D. To test this hypothesis, anti-CD3 monoclonal antibody was administered to NOD female mice, which are a T1D spontaneous onset model mouse, for 3 consecutive days, and then halmcin and exendin-4 were administered for 8 consecutive weeks. Surprisingly, 100% of the mice completely recovered from T1D by this combination treatment method. These mice had reduced pancreatic insensitivity, increased β-cell proliferation and mass, decreased activated T cells (Th1+ cells), and increased regulatory T cells.

[0012] The examples described herein demonstrate for the first time that a combination of anti-CD3 immunomodulatory treatment (e.g., low dose) and halmcin + exendin-4 β-cell regeneration and anti-apoptosis combination treatment increases immune tolerance, increases β-cell proliferation and mass, protects β-cells from cytokines and ER stress, and that the effects collectively lead to reversal of early-onset T1D in a mouse model of T1D.

[0013] The present disclosure relates to a combination of an immunomodulatory therapy and a β-cell regeneration therapy that is therapeutically and economically effective and applicable to millions of established long-term T1D patients and recently diagnosed T1D patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0014]

Figure 1

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Figure 4

Mode for Carrying Out the Invention

[0015] Detailed Description This specification describes methods and compositions for treating subjects with conditions associated with insulin secretory insufficiency.

[0016] One aspect of the present invention is directed to a method of treating a subject with a medical condition associated with insufficient insulin secretion. The method involves administering to a subject in need of treatment for a medical condition associated with insufficient levels of insulin secretion a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody (e.g., an anti-CD3 antibody), wherein the administration is carried out under conditions effective to reverse the loss of pancreatic beta cell mass and function in the subject for treating the subject with a condition associated with insulin secretory deficiency.

[0017] Suitable DYRK1A inhibitors and GLP1R agonists for practicing the methods of the present invention are described in International Publication WO 2018 / 081401 to Stewart et al., International Publication WO2019 / 100062 to DeVita et al., International Publication WO 2019 / 183245 to Kumar et al., International Publication WO 2019 / 100062 to DeVita et al., International Publication WO 2019 / 136320 to Stewart et al., International Publication WO 2020 / 142485 to DeVita et al., International Publication WO 2020 / 142486 to DeVita et al., and International Publication WO 2021 / 263129 to DeVita et al., which are hereby incorporated by reference in their entireties.

[0018] Several DYRK1A inhibitors derived from natural products as well as small molecule drug discovery programs have been identified and characterized and can be used in practicing the methods disclosed herein. For example, suitable DYRK1A inhibitors include, but are not limited to, harmine; INDY (having the following chemical structure):

Chemical Structure

[0019] Apart from halmint, EGCg, and other flavan-3-ols (Guedj et al., “Green Tea Polyphenols Rescue of Brain Defects Induced by Overexpression of DYRK1A,” PLoS One 4(2):e4606 (2009) and Bain et al., “The Specificities of Protein Kinase Inhibitors: An Update,” Biochem. J. 371(1):199-204 (2003), the entire contents of which are incorporated herein by reference), leucettines (Tahtouh et al., “Selectivity, Cocrystal Structures, and Neuroprotective Properties of Leucettines, a Family of Protein Kinase Inhibitors Derived from the Marine Sponge Alkaloid Leucettamine B,” J. Med. Chem. 55(21):9312-9330 (2012) and Naert et al., “Leucettine L41, a DYRK1A-preferential DYRKs / CLKs Inhibitor, Prevents Memory Impairments and Neurotoxicity Induced by Oligomeric Aβ25-35 Peptide Administration in Mice,” Eur. Neuropsychopharmacol. 25(11):2170-2182 (2015), the entire contents of which are incorporated herein by reference), quinalizarin (Cozza et al., “Quinalizarin as a Potent, Selective and Cell-permeable Inhibitor of Protein Kinase CK2,” Biochem. J.421(3):387 - 395 (2009), the entire content of which is incorporated herein by reference), peltogynoids Acanilol A and B (Ahmadu et al, “Two New Peltogynoids from Acacia nilotica Delile with Kinase Inhibitory Activity,” Planta Med. 76(5):458 - 460 (2010), the entire content of which is incorporated herein by reference), benzocoumarins (dNBC) (Sarno et al., “Structural Features Underlying the Selectivity of the Kinase Inhibitors NBC and dNBC: Role of a Nitro Group that Discriminates Between CK2 and DYRK1A,” Cell. Mol. Life Sci. 69(3):449 - 460 (2012), the entire content of which is incorporated herein by reference), and indolocarbazoles (staurosporine, rebeccamycin, and their analogs) (Sanchez et al., “Generation of Potent and Selective Kinase Inhibitors by Combinatorial Biosynthesis of Glycosylated Indolocarbazoles,” Chem. Commun. 27:4118 - 4120 (2009), the entire content of which is incorporated herein by reference) are other natural products that have been shown to inhibit DYRK1A and other kinases.

[0020] Other findings identified from attempts at small molecule drug discovery include INDY (Ogawa et al., “Development of a Novel Selective Inhibitor of the Down Syndrome-Related Kinase Dyrk1A,” Nat. Commun. 1: Article Number 86 (2010), the entire content of which is incorporated herein by reference), DANDY (Gourdain et al., “Development of DANDYs, New 3,5-Diaryl-7-Azaindoles Demonstrating Potent DYRK1A Kinase Inhibitory Activity,” J. Med. Chem. 56(23):9569-9585 (2013), the entire content of which is incorporated herein by reference), and FINDY (Kii et al., “Selective Inhibition of the Kinase DYRK1A by Targeting its Folding Process,” Nat. Commun. 7:11391 (2016), the entire content of which is incorporated herein by reference), pyrazolidinediones (Koo et al., “QSAR Analysis of Pyrazolidine-3,5-Diones Derivatives as Dyrk1A Inhibitors,” Bioorg. Med. Chem. Lett. 19(8):2324-2328 (2009); Kim et al., “Putative Therapeutic Agents for the Learning and Memory Deficits of People with Down Syndrome,” Bioorg. Med. Chem. Lett.16(14):3772 - 3776 (2006), which are hereby incorporated by reference in their entirety, aminokynazolines (Rosenthal et al., “Potent and Selective Small Molecule Inhibitors of Specific Isoforms of Cdc2-Like Kinases (Clk) and Dual Specificity Tyrosine-Phosphorylation-Regulated Kinases (Dyrk),” Bioorg. Med. Chem. Lett. 21(10):3152 - 3158 (2011), which is hereby incorporated by reference in its entirety), meriolins (Giraud et al., “Synthesis, Protein Kinase Inhibitory Potencies, and In Vitro Antiproliferative Activities of Meridianin Derivatives,” J. Med. Chem. 54(13):4474 - 4489 (2011); Echalier et al., “Meriolins (3-(Pyrimidin-4-yl)-7-Azaindoles): Synthesis, Kinase Inhibitory Activity, Cellular Effects, and Structure of a CDK2 / Cyclin A / Meriolin Complex,” J. Med. Chem. 51(4):737 - 751 (2008); and, Akue-Gedu et al., “Synthesis and Biological Activities of Aminopyrimidyl-Indoles Structurally Related to Meridianins,” Bioorg. Med. Chem. 17(13):4420 - 4424 (2009), which are hereby incorporated by reference in their entirety), pyridines and pyrazines (Kassis et al., “Synthesis and Biological Evaluation of New 3-(6-hydroxyindol-2-yl)-5-(Phenyl) Pyridine or Pyrazine V-Shaped Molecules as Kinase Inhibitors and Cytotoxic Agents,” Eur. J. Med. Chem. 46(11):5416-5434 (2011), the entire content of which is incorporated herein by reference), chromenolides (Neagoie et al., “Synthesis of Chromeno[3,4-b]indoles as Lamellarin D Analogues: A Novel DYRK1A Inhibitor Class,” Eur. J. Med. Chem. 49:379-396 (2012), the entire content of which is incorporated herein by reference), 11H-indolo[3,2-c]quinoline-6-carboxylic acid, thiazolo[5,4-f]quinazoline (EHT 5372) (Foucourt et al., “Design and Synthesis of Thiazolo[5,4-f]quinazolines as DYRK1A Inhibitors, Part I.,” Molecules 19(10):15546-15571 (2014) and Coutadeur et al., “A Novel DYRK1A (Dual Specificity Tyrosine Phosphorylation-Regulated Kinase 1A) Inhibitor for the Treatment of Alzheimer’s Disease: Effect on Tau and Amyloid Pathologies In Vitro,” J. Neurochem. 133(3):440-451 (2015), which are incorporated herein by reference in their entireties), and 5-iodotubercidins (Dirice et al., “Inhibition of DYRK1A Stimulates Human Beta Cell Proliferation,” Diabetes 65(6):1660-1671 (2016) and Annes et al., “Adenosine Kinase Inhibition Selectively Promotes Rodent and Porcine Islet β-cell Replication,” Proc. Natl. Acad. Sci. 109(10):3915-3920 (2012) (the entire contents of which are incorporated herein by reference) show potent DYRK1A activity with varying degrees of kinase selectivity.

[0021] Additional suitable DYRK1A inhibitors include, but are not limited to, GNF2133 (Liu et al., “Selective DYRK1A Inhibitor for the Treatment of Type 1 Diabetes: Discovery of 6-Azaindole Derivative GNF2133,” J. Med. Chem. 63:2958-2973 (2020), the entire contents of which are incorporated herein by reference) and those described in Liu et al., “DYRK1A Inhibitors for Disease Therapy: Current Status and Perspectives,” Eur. J. Med. Chem. 229:114062 (2022) (which is incorporated herein by reference in its entirety).

[0022] Suitable thiazidine kinase inhibitors include, for example, but are not limited to, those described in International Publication No. WO 2019 / 100062 by DeVita et al. and International Publication No. WO 2019 / 136320 by Stewart et al. (see Tables 1 and 2) (the entire contents of which are incorporated herein by reference).

[0023] As described above, glucagon-like peptide-1 receptor agonist agents mimic the actions of the incretin hormone GLP-1, which is released from the intestine in response to food intake. These actions include an increase in insulin secretion, a decrease in glucagon release, an increase in satiety, and a delay in gastric emptying.

[0024] Suitable GLP1R agonist agents for practicing the methods disclosed herein are described in Stewart et al.'s PCT published patent application WO 2019 / 136320, the entire contents of which are incorporated herein by reference, and include, but are not limited to, exenatide, liraglutide, exenatide LAR, taspoglutide, lixisenatide, albiglutide, dulaglutide, and semaglutide. Exenatide and exenatide LAR are synthetic exendin-4 analogs obtained from the saliva of lizards. Liraglutide is an acylated analog of GLP-1 that self-associates into a heptameric structure that delays absorption from the subcutaneous injection site. Taspoglutide has 3% homology with native GLP-1 and is not completely subject to degradation by DPP-4. Lixisenatide is a human GLP1R agonist agent. Albiglutide is a long-acting GLP-1 mimetic that is less susceptible to degradation by DPP-4. Dulaglutide is a long-acting GLP1 analog. Semaglutide is a GLP1R agonist agent approved as a treatment for T2D. Clinically available GLP1R agonist agents include, for example, exenatide, liraglutide, albiglutide, dulaglutide, lixisenatide, semaglutide, and the like.

[0025] In some embodiments, the GLP1R agonist agent is selected from the group consisting of exendin-4, GLP1(7-36), liraglutide, lixisenatide, semaglutide, tizepatide (also known as mounjaro), and combinations thereof.

[0026] Additional suitable GLP1 agonist agents include, but are not limited to, disubstituted-7-aryl-5,5-bis(trifluoromethyl)-5,8-dihydropyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione compounds and derivatives thereof, such as 7-(4-chlorophenyl)-1,3-dimethyl-5,5-bis(trifluoromethyl)-5,8-dihydropyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (see, for example, Nance et al., “Discovery of a Novel Series of Orally Bioavailable and CNS Penetrant Glucagon-like Peptide-1 Receptor (GLP-1R) Noncompetitive Antagonists Based on a 1,3-Disubstituted-7-aryl 5,5-bis(trifluoromethyl)-5,8-dihydropyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione Core,” J. Med. Chem. 60:1611-1616 (2017), which is hereby incorporated by reference in its entirety).

[0027] Even more suitable GLP1 agonist agents include positive allosteric modulators of GLP1R (“PAMS”), (S)-2-cyclopentyl-N-((1-isopropylpyrrolidin-2-yl)methyl)-10-methyl-1-oxo-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; (R)-2-cyclopentyl-N-((1-isopropylpyrrolidin-2-yl)methyl)-10-methyl-1-oxo-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; 2-cyclopentyl-N-(((S)-1-isopropylpyrrolidin-2-yl)methyl)-10-methyl-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-4-carboxamide; N-(((S)-1-isopropylpyrrolidin-2-yl)methyl)-10-methyl-1-oxo-2-((S)-tetrahydrofuran-3-yl)-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; N-(((R)-1-isopropylpyrrolidin-2-yl)methyl)-10-methyl-1-oxo-2-((S)-tetrahydrofuran-3-yl)-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; (S)-2-cyclopentyl-8-fluoro-N-((1-isopropylpyrrolidin-2-yl)methyl)-10-methyl-1-oxo-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; (R)-2-cyclopentyl-8-fluoro-N-((1-isopropylpyrrolidin-2-yl)methyl)-10-methyl-1-oxo-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; (R)-2-cyclopentyl-N-(((S)-1-isopropylpyrrolidin-2-yl)methyl)-10-methyl-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-4-carboxamide; (S)-2-cyclopentyl-N-(((S)-1-isopropylpyrrolidin-2-yl)methyl)-10-methyl-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-4-carboxamide;(S)-10-Chloro-2-cyclopentyl-N-((1-isopropylpyrrolidin-2-yl)methyl)-1-oxo-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; (R)-10-Chloro-2-cyclopentyl-N-((1-isopropylpyrrolidin-2-yl)methyl)-1-oxo-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; (S)-10-Bromo-2-cyclopentyl-N-((1-isopropylpyrrolidin-2-yl)methyl)-1-oxo-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; (R)-10-Bromo-2-cyclopentyl-N-((1-isopropylpyrrolidin-2-yl)methyl)-1-oxo-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; (R)-N-((1-isopropylpyrrolidin-2-yl)methyl)-10-methyl-1-oxo-2-phenyl-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; (S)-10-Cyano-2-cyclopentyl-N-((1-isopropylpyrrolidin-2-yl)methyl)-1-oxo-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; (S)-2-Cyclopentyl-N-((1-isopropylpyrrolidin-2-yl)methyl)-1-oxo-10-vinyl-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; (S)-N-((1-isopropylpyrrolidin-2-yl)methyl)-10-methyl-2-(1-methyl-1H-pyrazol-4-yl)-1-oxo-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; (R)-N-((1-isopropylpyrrolidin-2-yl)methyl)-10-methyl-2-(1-methyl-1H-pyrazol-4-yl)-1-oxo-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; (S)-N-((1-isopropylpyrrolidin-2-yl)methyl)-10-methyl-1-oxo-2-(pyridin-3-yl)-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide;(R)-N-((1-Isopropylpyrrolidin-2-yl)methyl)-10-methyl-1-oxo-2-(pyridin-3-yl)-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; N-(azetidin-2-ylmethyl)-2-cyclopentyl-10-methyl-1-oxo-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; and 2-cyclopentyl-N-((1-isopropylazetidin-2-yl)methyl)-10-methyl-1-oxo-1,2-dihydropyrazino[1,2-a]indole-4-carboxamide; or a pharmaceutically acceptable salt thereof (see PCT Publication No. WO 2017 / 117556, the entire contents of which are incorporated herein by reference).;

[0028] Further suitable GLP1 agonist agents include, but are not limited to, chimeric peptides such as dual receptor agonist agents of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) (i.e., tirzepatide). See, for example, Frias et al., “Tirzepatide Versus Semaglutide Once Weekly in Patients with Type 2 Diabetes,” N. Engl. J. Med. 385:503-515 (2021) (the entire contents of which are incorporated herein by reference). Both GIP and GLP1 are GPCRs that act via cAMP. Thus, in some embodiments, the GLP1 agonist agent increases cAMP in a population of β-cells (e.g., human β-cells).

[0029] Glucagon-like peptide-1 receptor agonist agents mimic the actions of the incretin hormone GLP-1, which is released from the intestine in response to food intake. These actions include an increase in insulin secretion, a decrease in glucagon release, an increase in satiety, and a delay in gastric emptying. Another approach to increasing blood GLP-1 levels is to prevent the breakdown of GLP-1 by the enzyme DPP4.GLP-1 receptor agonists and DPP-4 inhibitors are among the most widely used drugs for the treatment of type 2 diabetes (Campbell et al., “Pharmacology, Physiology and Mechanisms of Incretin Hormone Action,” Cell Metab. 17:819-37 (2013); Guo X-H., “The Value of Short- and Long-Acting Glucagon-Like Peptide Agonists in the Management of Type 2 Diabetes Mellitus: Experience with Exenatide,” Curr. Med. Res. Opinion 32(1):61-76 (2016); Deacon et al., “Dipeptidyl Peptidase-4 Inhibitors for the Treatment of Type 2 Diabetes: Comparison, Efficacy and Safety,” Expert Opinion on Pharmacotherapy 14:2047-58 (2013); Lovshin, “Glucagon-Like Peptide-1 Receptor Agonists: A Class Update for Treating Type 2 Diabetes,” Can. J. Diabetes 41:524-35 (2017); and Yang et al., “Lixisenatide Accelerates Restoration of Normoglycemia and Improves Human Beta Cell Function and Survival in Diabetic Immunodeficient NOD-scid IL2rg(null) RIP-DTR Mice Engrafted With Human Islets,” Diabetes Metab. Syndr. Obes. 8:387-98 (2015), which are hereby incorporated by reference in their entirety).

[0030] Thus, in addition to or instead of a GLP1 agonist agent, the methods and compositions of the present invention may include a dipeptidyl peptidase IV (DDP4) inhibitor. Exemplary suitable DDP4 inhibitors include, but are not limited to, sitagliptin, vildagliptin, saxagliptin, alogliptin, teneligliptin, anagliptin, and the like.

[0031] When practicing the methods described herein, the immunomodulatory monoclonal antibody may be an anti-CD3 antibody. Suitable anti-CD3 antibodies include those that are directed against or specifically bind to the CD3 receptor on the surface of T cells, generally human CD3 on human T cells, particularly human CD3ε (CD3E). Anti-CD3 antibodies include, but are not limited to, teprotumumab, otelixizumab, and besilesomab. Another non-limiting example of a suitable anti-CD3 antibody is OKT3, also known as muromonab, the UHCT1 clone, also known as T3, and CD3E. OKT3 is a mouse anti-CD3 antibody (DrugBank accession number DB00075, the entire content of which is incorporated herein by reference), Abz287a is a humanized antibody of OKT3, and Abz494 to Abz498 are pH-dependent antibodies. The sequence of Abz287a was found in GenBank accession number ALJ79286 and is described in Pegu et al., “Activation and Lysis of Human CD4 Cells Latently Infected with HIV-1,” Nat. Commun. 6:8447 (2015), the entire content of which is incorporated herein by reference.

[0032] Suitable additional immunomodulatory monoclonal antibodies include, but are not limited to, anti-TNFα antibodies (e.g., infliximab, etanercept, adalimumab, golimumab, certolizumab pegol), anti-IL1 antibodies (e.g., canakinumab), anti-CTLA-4 antibodies (abatacept), anti-thymocyte globulin antibodies (e.g., anti-thymocyte globulin), anti-CD6 antibodies (e.g., itolizumab), anti-CD20 antibodies (e.g., rituximab), anti-interleukin-21 antibodies, etc. For example, see Li et al., “Drugs for Autoimmune Inflammatory Diseases: From Small Molecule Compounds to Anti-TNF Biologics,” Front. Pharmacol. 8:460 (2017); von Herrath et al., “Anti-Interleukin-21 Antibody and Liraglutide for the Preservation of β-Cell function in Adults with Recent-Onset Type 1 Diabetes: A Randomised, Double-Blind, Placebo-Controlled, Phase 2 Trial,” Lancet Diabetes Endrocrinol. 9:212-224 (2021) and International Publication WO 2014 / 004857 (the entire contents of which are incorporated herein by reference).

[0033] The identification of suitable immunomodulatory monoclonal antibody versions and fragments (e.g., anti-CD3 antibody versions and fragments) useful for practicing the methods of the present invention can be achieved by known methods and techniques established in the art, such as histidine substitution via phage display libraries, or histidine substitution from combinatorial histidine substitution libraries by yeast surface display.

[0034] As used herein, the terms “antibody” or “immunoglobulin” are used in the broadest sense and specifically include intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. Intact antibodies or whole antibodies are classified into different classes based on the amino acid sequence of their constant regions. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0035] As used herein, “antibody fragment” includes a portion of an intact antibody that includes its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab’, F(ab’)2, Fv, single-chain Fv (scFv), Fc fragment, diabody, linear antibody, single-chain antibody molecule, bispecific and multispecific antibodies formed from antibody fragments, and the like.

[0036] In some embodiments, the monovalent antibody fragment of the antibodies described herein is scFv or Fab.

[0037] As used herein, “whole” antibody or “complete” antibody refers to an antibody that includes an antigen-binding variable region as well as light chain constant domain (CL) and heavy chain constant domains (CH1, CH2, and CH3).

[0038] The “Fc” region of the antibodies described herein generally includes the CH2, CH3, and hinge regions of the major class of IgG1 or IgG2 antibodies. The hinge region is a group of about 15 amino acid residues that connects the CH1 region to the CH2-CH3 region.

[0039] The “Fab” fragment also includes the constant domain of the light chain and the first constant domain (CH1) of the heavy chain and has only one antigen-binding site.

[0040] The "Fab" fragment is different from the Fab fragment in that several residues containing one or more cysteine residues from the antibody hinge region are added to the carboxy terminus of the heavy chain CH1 domain.

[0041] The "F(ab’)2" antibodies described herein are produced as pairs of Fab’ fragments with hinge cysteines in between.

[0042] The "single-chain FV" or "scFv" antibody fragments described herein contain the VH and VL domains of the antibody, and these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain that enables the scFv to form the desired structure for antigen binding.

[0043] The "variable domain" of the antibodies described herein includes framework regions (usually FR1 - FR4) and CDR domains shown as "hypervariable regions" (usually CDR1, CDR2, and CDR3).

[0044] As used herein, the term "hypervariable region" or "CDR" means the amino acid residues of the antibody that are responsible for antigen binding. Hypervariable regions generally include the amino acid residues of "complementary determining regions" or "CDRs" (e.g., residues 24 - 34 (L1), 50 - 56 (L2), and 89 - 97 (L3) of the light chain variable domain, and residues 31 - 35 (H1), 50 - 65 (H2), and 95 - 102 (H3) of the heavy chain variable domain).

[0045] Unless otherwise specified, the amino acid positions within the antibody molecules described herein are numbered according to Kabat.

[0046] "Framework region" or "FR" residues are variable domain residues other than the hypervariable region residues as defined herein.

[0047] The "antibody variant" described in this specification includes antibodies that have an amino acid sequence modified compared to the parental antibody, but whose binding affinity for the target antigen is the same or has changed. Antibody variants differ from the parental antibody by substitution, deletion, or addition of one or more amino acid residues at specific positions within the variable domain, including the CDR domains of the antibody, and / or the constant region, in order to alter certain properties of the antibody, such as binding affinity and / or receptor function, such as ADCC, FcRn binding, etc. The histidine mutant antibodies described in this specification without further mutations are not referred to as "antibody variants" herein. The antibody variants described in this specification exhibit 80-99% sequence homology compared to the parental antibody, depending on the specific positions of the amino acid residues that are substituted, deleted, or added, and in some embodiments 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence homology.

[0048] The term "cytokine" refers to a general term for proteins that are released from a certain cell population and act on other cells as intercellular mediators. Examples of such cytokines include lymphokines, monokines, and conventional polypeptide hormones such as vascular endothelial growth factor (VEGF); integrin thrombopoietin (TPO); nerve growth factors such as NGFβ; platelet growth factors; transforming growth factors (TGF) such as TGFα and TGFβ; erythropoietin (EPO); interferons such as IFNα, IFNβ, and IFNγ; colony-stimulating factors such as M-CSF, GM-CSF, and G-CSF; and interleukins such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, and TNF-α or TNF-β.

[0049] As used herein, the term "humanized antibody" means a genetically engineered non-human antibody that includes a non-human variable domain modified to include a high level of sequence homology with human antibody constant domains and human variable domains. This can be achieved by grafting the six non-human antibody CDRs that form the antigen-binding site into homologous human acceptor framework regions (FRs) (see PCT Publication WO 92 / 22653 and European Patent No. 0629240, the entire contents of which are incorporated herein by reference). In order to fully reproduce the binding affinity and specificity of the parental antibody, it may be necessary to substitute framework residues from the parental antibody (i.e., the non-human antibody) into the human framework region (back mutations). Structural homology modeling can be useful in identifying amino acid residues in the framework region that are important for the binding properties of the antibody. Thus, a humanized antibody can include a predominantly human framework region, which may include non-human CDR sequences and, optionally, one or more amino acid back mutations to non-human amino acid sequences, and a fully human constant region.

[0050] As used herein, the term “human antibody” means an antibody having variable and constant regions derived from immunoglobulin sequences of the human germ line. A human antibody may include amino acid residues not encoded by the immunoglobulin sequences of the human germ line (e.g., mutations introduced by in vitro random mutagenesis or site-directed mutagenesis, or somatic mutations in vivo). However, the term “human antibody” as used herein is not intended to include antibodies in which CDR sequences derived from the germ line of other mammalian species such as mice are grafted onto human framework sequences. The human monoclonal antibodies of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methodologies, such as the standard somatic cell hybridization techniques of Kohler and Milstein, “Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity,” Nature 256: 495 (1975) (the entire contents of which are incorporated herein by reference). The somatic cell hybridization method may be used, but in principle, other techniques for producing monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes, or phage display techniques using libraries of human antibody genes, can also be employed. A suitable animal system for preparing hybridomas that secrete human monoclonal antibodies is the mouse system. Hybridoma production in mice is a well-established procedure. Immune protocols and techniques for separating immune spleen cells for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known. Thus, human monoclonal antibodies can be prepared, for example, using transgenic mice or rats having a part of the human immune system, rather than mouse or rat systems. Thus, in some embodiments, human antibodies are obtained from transgenic animals such as mice or rats having human germ line immunoglobulin sequences instead of animal immunoglobulin sequences.In such embodiments, the antibody is derived from human germline immunoglobulin sequences introduced into an animal, but the final antibody sequences are the result of further modification of the human germline immunoglobulin sequences by somatic hypermutations and affinity maturation by the endogenous animal antibody machinery (see, e.g., Mendez et al., “Functional Transplant of Megabase Human Immunoglobulin Loci Recapitulates Human Antibody Response in Mice,” Nat. Genet. 15:146-56 (1997), the entire content of which is incorporated herein by reference).

[0051] In some embodiments, an immunomodulatory monoclonal antibody (e.g., an anti-CD3 antibody) is administered at a low dose. As used herein, “low dose” with respect to the administration of an immunomodulatory monoclonal antibody (e.g., an anti-CD3 antibody) refers to a dose below the optimal dose, or a dose lower than when the immunomodulatory monoclonal antibody (e.g., an anti-CD3 antibody) is administered as a treatment to a subject for a condition associated with insufficient insulin secretion by itself (i.e., in the absence of other companion drugs). In other words, an immunomodulatory monoclonal antibody (e.g., an anti-CD3 antibody) administered according to the methods described herein can be administered at a lower dose than is typically administered for the treatment of T1D due to the effect of the concomitant administration of a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1 receptor (GLP1R) agonist. In Bresson et al., “Anti-CD3 and Nasal Proinsulin Combination Therapy Enhances Remission from Recent-Onset Autoimmune Diabetes by Inducing Tregs,” J. Clin. Invest. 116(5):1371-1381 (2006) (hereinafter “Bresson”) (the entire contents of which are incorporated herein by reference), the ability of an anti-CD3 antibody to reverse T1D in NOD mice was tested at different doses (see Bresson, Figure 1E). Bresson selected 40 μg / day as a “sub-optimal” dose that resulted in remission of diabetes in only 30% of the mice, as the goal was to test whether the combination with another drug (in this case nasal insulin) further improved the remission of diabetes in these mice. In the examples described later herein, using a “sub-optimal” dose of anti-CD3, in combination with halmide + exenatide (a 39-amino acid peptide that is a synthetic version of exendin-4), diabetes was reversed in 100% of diabetic NOD mice. In some embodiments, the sub-optimal dose of an anti-CD3 antibody for a human patient is less than the dose provided in a 14-day course of escalating intravenous administration of teplizumab, and the total cumulative dose is about 9034 mg / m 2) and is further defined as follows (Day 1, 51 mg / m 2 ; Day 2, 103 mg / m 2 ; Day 3, 206 mg / m 2 ; Day 4, 413 mg / m 2 ; Day 5, 826 mg / m 2 ; median cumulative dose 11.6 mg; interquartile range 5.7 mg). Thus, in some embodiments, the dose of the anti-CD3 antibody is 1 / 2 or less, or 1 / 4 or less, of the dose provided in a 14-day escalating course by intravenous administration of teprotumumab.

[0052] Also contemplated is a method of treating a medical condition associated with insulin secretory insufficiency. The method comprises administering to a subject in need of treatment of a medical condition associated with an insufficient level of insulin secretion a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunosuppressant, wherein the administration is carried out under conditions effective to reverse the loss of pancreatic β-cell mass and function in the subject for treating the subject of the medical condition associated with insufficient insulin secretion.

[0053] Suitable DYRK1A inhibitors and GLP1R agonists are detailed above.

[0054] Suitable immunosuppressants include immunomodulatory monoclonal antibodies (e.g., anti-CD3 antibody), as well as tacrolimus, rapamycin, mycophenolate mofetil, and glucocorticoids such as prednisone, cortisone, dexamethasone, but are not limited thereto.

[0055] As used herein, a medical condition associated with insufficient insulin secretion means a state in which a subject secretes less insulin than the plasma level of insulin required for the subject to maintain a normal blood glucose level such that a subject with a medical condition associated with insufficient insulin secretion becomes hyperglycemic. In such a state, the pancreatic β-cells of the affected individual do not secrete a sufficient amount of insulin to keep the blood glucose concentration normal (i.e., maintain normoglycemia).

[0056] One of the conditions associated with insufficient insulin secretion is insulin resistance. Insulin resistance is a state in which the target cells have a reduced sensitivity to the glucose-lowering effect of insulin. Insulin resistance in muscle and fat cells reduces glucose uptake (and thus locally stores glucose as glycogen and triglycerides). On the other hand, insulin resistance in hepatocytes reduces glycogen synthesis and storage and fails to suppress glucose production and release into the blood. Usually, insulin resistance means a reduction in the glucose-lowering effect of insulin. However, other functions of insulin can also be affected. For example, insulin resistance in adipocytes reduces the normal action of insulin on lipids, decreases the uptake of circulating lipids, and increases the hydrolysis of stored triglycerides. When the mobilization of lipids accumulated in these cells increases, the free fatty acids in the plasma increase. The increase in blood fatty acid concentration, the decrease in glucose uptake into muscle, and the increase in glucose production in the liver all contribute to an increase in blood glucose levels. When insulin resistance is present, more insulin needs to be secreted from the pancreas. If this compensatory increase does not occur, the blood glucose concentration rises and type II diabetes develops.

[0057] One of the conditions of insufficient insulin secretion is diabetes. Diabetes can be broadly divided into two types of diseases: type I ("T1D") and type II ("T2D").

[0058] In some embodiments, the condition associated with an insufficient level of insulin secretion is type 1A diabetes or immune-mediated diabetes. In some embodiments, the condition associated with an insufficient level of insulin secretion is type 1B diabetes or idiopathic diabetes.

[0059] As used herein, the term “diabetes” also refers to a group of metabolic diseases in which patients exhibit hyperglycemia, including type I diabetes, type II diabetes, gestational diabetes, congenital diabetes, maturity-onset diabetes of the young (MODY), cystic fibrosis-related diabetes, hemochromatosis-related diabetes, drug-induced diabetes (e.g., steroid diabetes, etc.), and some forms of monogenic diabetes.

[0060] In certain aspects, the subject has or is being treated for one or more of type I diabetes (T1D), type IA diabetes, type 1B diabetes, type II diabetes (T2D), gestational diabetes, congenital diabetes, maturity-onset diabetes of the young (MODY), cystic fibrosis-related diabetes, hemochromatosis-related diabetes, drug-induced diabetes, or monogenic diabetes. For example, the subject has or is being treated for type I diabetes. Alternatively, the subject has or is being treated for type II diabetes.

[0061] In some aspects, the subject has long-term T1D. In one aspect, the subject has recently-onset T1D. For descriptions of long-term T1D and recently-onset T1D, see, for example, Coppieters et al., “Demonstration of Islet-Autoreactive CD8 T Cells in Insulitic Lesions from Recent Onset and Long-Term Type 1 Diabetes Patients,” J. Exp. Med. 209:51-60 (2012), which is hereby incorporated by reference in its entirety.

[0062] In some aspects, the subject has a disease or disorder associated with mutations and / or abnormal expression or function of DYRK1A. According to such aspects, the subject may have Down syndrome. Down syndrome is associated with an increased incidence of autoimmune diseases, such as an increased risk and prevalence of type 1 diabetes.

[0063] The treatment methods described herein are effective for treating a subject having an insufficient level of insulin secretion, for example, by increasing immune tolerance in the subject, enhancing β-cell proliferation in the subject, protecting β-cells in the subject, increasing the amount of β-cells in the subject, or any combination thereof.

[0064] In some embodiments, the medical conditions associated with insufficient levels of insulin secretion are metabolic syndrome. Metabolic syndrome is generally used to define a cluster of abnormalities associated with an increased risk of developing type II diabetes and atherosclerotic vascular disease. Associated medical conditions and symptoms include, but are not limited to, fasting hyperglycemia (type II diabetes, or impaired fasting glucose, impaired glucose tolerance, insulin resistance), hypertension, central obesity (also known as visceral fat, android fat, apple-shaped fat), i.e., overweight with fat deposition mainly around the waist, decreased HDL cholesterol, elevated triglycerides, etc.

[0065] In some embodiments, the medical conditions associated with insufficient levels of insulin secretion are metabolic syndrome or insulin resistance, and the methods described herein are implemented to treat a subject having or being treated for metabolic syndrome or insulin resistance.

[0066] Other medical conditions that may be associated with insufficient insulin secretion include, but are not limited to, hyperuricemia, fatty liver progressing to non-alcoholic fatty liver disease (especially when associated with obesity), polycystic ovary syndrome (in females), and acanthosis nigricans.

[0067] Diseases associated with blood or plasma glucose levels outside the normal range, such as hyperglycemia, but not limited to these, can be treated according to the treatment methods described herein. As a result, the term "associated diseases" includes impaired glucose tolerance ("IGT"), impaired fasting glucose ("IFG"), insulin resistance, metabolic syndrome, postprandial hyperglycemia, overweight / obesity. Such associated diseases are also characterized by abnormal insulin concentrations in the blood and / or plasma.

[0068] The methods described herein can be implemented to treat subjects having conditions associated with β-cell insufficiency or deficiency. Such conditions include, but are not limited to, type I diabetes (T1D), type II diabetes (T2D), gestational diabetes, congenital diabetes, maturity-onset diabetes of the young (MODY), cystic fibrosis-related diabetes, hemochromatosis-related diabetes, drug-induced diabetes, or monogenic diabetes. Drug-induced diabetes refers to diabetes caused by the use of drugs toxic to β-cells (such as steroids, antidepressants, second-generation antipsychotics, immunosuppressants, etc.). Exemplary immunosuppressants include, but are not limited to, corticosteroids (e.g., prednisone, dexamethasone), rapamycin / sirolimus, everolimus, calcineurin inhibitors (e.g., FK-506 / tacrolimus), etc.

[0069] Other conditions associated with β-cell deficiency include, but are not limited to, hypoglycemia unawareness, brittle insulin-dependent diabetes, pancreatectomy, partial pancreatectomy, pancreatic transplantation, islet allotransplantation, islet autotransplantation, islet xenotransplantation, etc.

[0070] As used herein, hypoglycemia unawareness is a complication of diabetes in which patients are unable to notice a profound drop in blood glucose because they cannot induce the secretion of epinephrine, which plays a role in warning patients of a drop in blood glucose, to produce the characteristic symptoms of hyperglycemia (palpitations, sweating, anxiety, etc.).

[0071] Pancreas transplantation is performed alone, after kidney transplantation, or in combination with kidney transplantation. For example, in patients with severe physical disabilities and life-threatening complications due to unstable insulin-dependent diabetes that persists despite the absence of hypoglycemic awareness and optimal medical management, pancreas transplantation alone may be considered medically necessary. In patients with insulin-dependent diabetes, pancreas transplantation may be performed after kidney transplantation. In patients with insulin-dependent diabetes with uremia, pancreas transplantation may be used in combination with kidney transplantation. Pancreas re-transplantation may be considered after failure of the primary pancreas transplantation.

[0072] As used herein, islet transplantation is a method of isolating only the islets of Langerhans containing endocrine cells of the pancreas, including β cells that produce insulin and α cells that produce glucagon, and transplanting them into a patient. Islet transplantation is performed by isolating islets of Langerhans from one or more human donor pancreases. The islet cells may be derived from human embryonic stem cells or induced pluripotent stem cells. Islet xenotransplantation is performed when islets of Langerhans are isolated from one or more non-human pancreases (e.g., porcine pancreas or primate pancreas). Islet autotransplantation is performed when islets of Langerhans are isolated from the pancreas of a patient who has undergone pancreatectomy (e.g., due to chronic pancreatitis caused by gallstones, drugs, and / or familial genetic causes) and returned to the same patient by injection into the portal vein, injection into the omentum by laparoscopy, injection into the gastric wall by endoscopy, or subcutaneous transplantation by minilaparotomy. Similar to pancreas transplantation, islet transplantation can be performed alone, after kidney transplantation, or in combination with kidney transplantation. For example, islet transplantation can be performed alone to restore hypoglycemic awareness, provide glycemic control, and / or protect the patient from severe hypoglycemic events (Hering et al., “Phase 3 Trial of Transplantation of Human Islets in Type 1 Diabetes Complicated by Severe Hypoglycemia,” Diabetes Care 39(7):1230-1240 (2016), which is incorporated herein by reference in its entirety).

[0073] Islet transplantation may be performed in combination with total pancreatectomy. For example, islet transplantation may be performed after total pancreatectomy to prevent or improve surgically induced diabetes by preserving β-cell function (see Johnston et al., “Factors Associated With Islet Yield and Insulin Independence After Total Pancreatectomy and Islet Cell Autotransplantation in Patients With Chronic Pancreatitis Utilizing Off-site Islet Isolation: Cleveland Clinic Experience,” J. Chem. Endocrinol. Metab. 100(5):1765-1770 (2015). The entire content is incorporated herein by reference). Thus, islet transplantation can result in long-term sustained insulin independence.

[0074]

[0075] In some embodiments, islet transplantation is performed in an encapsulation device that protects the transplanted islet cells from the host's autoimmune response while allowing glucose and nutrients to reach the transplanted islet cells.

[0076] ​The methods described herein can be implemented to enhance pancreatic, islet allotransplantation, islet autotransplantation, and islet xenotransplantation by regenerating a patient's pancreatic β-cells. For example, the methods described herein can be used to prevent or ameliorate surgically induced diabetes by maintaining β-cell function, restore hypoglycemia awareness, provide glycemic control, and / or protect the patient from severe hypoglycemic events. Accordingly, other aspects described herein relate to methods of regenerating pancreatic β-cells in transplant patients. Such methods include administering to a transplant patient a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist agent, and an anti-CD3 antibody, wherein said administration is carried out under conditions effective to reverse loss of β-cell mass and function in the subject being treated.

[0077] The method can be implemented to treat a subject at risk of developing type II diabetes. A patient at risk of developing type II diabetes may be a pre-diabetic / metabolic syndrome.

[0078] A patient at risk of developing type II diabetes may be under treatment with a psychotropic agent such as a selective serotonin reuptake inhibitor (SSRI) due to, for example, depression, obsessive-compulsive disorder (OCD), etc.

[0079] The subject is a mammalian subject, such as a human subject. Suitable human subjects include, but are not limited to, children, adults, and the elderly having β-cell and / or insulin deficiency.

[0080] The subject may also be a non-human animal, such as a cow, pig, cat, horse, mouse, dog, rabbit, etc.

[0081] By administering a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody) to a subject, the number of proliferating pancreatic β-cells in the subject can be increased by at least about 4%, 5%, 6%, 7%, 8%, 9%, 10% or more.

[0082] By administering a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody) to a subject, the number of proliferating pancreatic β-cells in the subject can be increased by about 4-10% per day, or about 4-6% per day, about 5-7% per day, about 6-9% per day, or about 7-10% per day.

[0083] When a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody) are administered to a subject, the number of proliferating pancreatic β-cells in the subject can be increased by about 6-10% per day.

[0084] By administering a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody) to a subject, (e.g., as compared to a subject not administered a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody)), glucose-stimulated insulin secretion in the pancreatic β-cells of the subject can be increased.

[0085] Administration to a subject of a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an anti-CD3 antibody can be effected by administering a single composition comprising the DYRK1A inhibitor, the GLP1R agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressive agent (e.g., an anti-CD3 antibody). Alternatively, administration to a subject of a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, an immunomodulatory monoclonal antibody and / or an immunosuppressive agent (e.g., an anti-CD3 antibody) can also be carried out sequentially. For example, first an immunomodulatory monoclonal antibody and / or an immunosuppressive agent (e.g., an anti-CD3 antibody), then a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor (or a composition comprising the DYRK1A inhibitor), then a glucagon-like peptide-1 receptor (GLP1R) agonist (or a composition comprising the GLP1R agonist) are administered, or, after administering an immunomodulatory monoclonal antibody and / or an immunosuppressive agent (e.g., an anti-CD3 antibody), a glucagon-like peptide-1 receptor (GLP1R) agonist (or its composition) is administered, then a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor (or its composition) is administered, or, in yet another embodiment, after administering an immunomodulatory monoclonal antibody and / or an immunosuppressive agent (e.g., an anti-CD3 antibody), a combination therapy of a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor (or a composition comprising the DYRK1A inhibitor) and a glucagon-like peptide-1 receptor (GLP1R) agonist (or a composition comprising the GLP1R agonist) is carried out.

[0086] Administration of any one or a combination of a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody) can be carried out multiple times daily, once a day, once a week, twice a week, once a month, once every two months, once a year, once every six months, or at any interval therebetween. The immunomodulatory monoclonal antibody and / or immunosuppressant (e.g., anti-CD3 antibody), DYRK1A inhibitor, and glucagon-like peptide-1 receptor (GLP1R) agonist may be administered at the same dosing frequency or at different dosing frequencies. In some embodiments, administration of any one or more of the dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, glucagon-like peptide-1 receptor (GLP1R) agonist, and immunomodulatory monoclonal antibody and / or immunosuppressant (e.g., anti-CD3 antibody) is carried out acutely or chronically. For example, administration can be carried out chronically over a period of one year, two years, three years, four years or more. In some embodiments, administration is infrequent.

[0087] As used herein, the term “treating” means prophylactic treatment, ameliorative treatment or curative treatment. In other words, a treatment method can be carried out to prevent a subject from developing a condition associated with insufficient insulin secretion or to prevent the condition of a subject associated with insufficient insulin secretion from worsening. Alternatively, a treatment method is carried out to ameliorate the condition of a subject associated with insufficient insulin secretion or to completely cure the condition (i.e., such that the subject no longer has a condition associated with insufficient insulin secretion as determined by a qualified medical professional).

[0088] In one aspect, the "treatment" is performed to restore the reduced β-cell mass and function in T1D patients. In some aspects, the "treatment" is performed to prevent the progression of T1D in subjects having recently developed T1D. In other words, the methods described herein can be implemented to restore the loss of β-cell mass and function in subjects who have lost β-cell mass and function due to a medical condition associated with insufficient insulin secretion such as T1D. In some aspects, the "treatment" is performed to prevent the progression of T1D in subjects at risk of developing T1D.

[0089] The term "treating" means correcting, reducing the rate of change of, or alleviating a subject's glucose homeostasis disorder. Glucose concentrations in the blood vary throughout the day. Glucose values are typically low in the morning, before the first meal of the day, and increase for several hours after a meal. As a result, the term "treating" includes controlling a subject's blood glucose value by increasing or decreasing the subject's blood glucose value. Since blood glucose values vary throughout the day, this can depend on many factors such as the subject's condition and / or a particular time of day.

[0090] "Treatment" means regulating a temporary or sustained decrease in the blood glucose value of a subject having diabetes or a related disease. The term "treatment" also means improving the insulin release (e.g., release by pancreatic β-cells) of a subject.

[0091] It is desirable to adjust the blood glucose level of a subject to normalize or regulate the blood or plasma glucose level of the subject having an abnormal value (i.e., a level below or above the known reference value, median, or average value of a corresponding subject having normal glucose homeostasis). The treatment methods described herein can be implemented to obtain such an effect.

[0092] When implementing the treatment method described in this specification, administering a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody) to a subject may include administering a DYRK1A inhibitor or a GLP1R agonist, or an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody), or all three of these, to the subject in a pharmaceutically effective composition, which means an amount effective to treat the described medical condition and / or disorder in the subject for the DYRK1A inhibitor, the GLP1R agonist, and the immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody). Such amounts generally vary depending on many factors that are well understood by those of ordinary skill in the art. These include the general health status, age, weight, height, general physical condition, medical history of the particular subject, the particular compound being used, as well as the carrier in which it is formulated, and the route of administration selected therefor, the duration or period of treatment, and the nature and severity of the condition being treated, but are not limited thereto.

[0093] Administration typically includes administration in a pharmaceutically acceptable dosage form, which means a dosage form of the compounds described in this specification, and includes, for example, tablets, dragees, powders, elixirs, syrups, liquid formulations including suspensions, sprays, inhalable tablets, troches, emulsions, solutions, granules, capsules, suppositories, and liquid injection formulations including liposome formulations. The techniques and formulations are generally described in the latest edition of Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.

[0094] In the practice of the treatment method, the DYRK1A inhibitor, GLP1R agonist, and / or anti-CD3 antibody may be contained in any suitable carrier substance in any suitable amount. The DYRK1A inhibitor, GLP1R agonist, and anti-CD3 antibody can be present in an amount up to 99% by weight based on the total weight of the composition. The composition can be provided in a dosage form suitable for oral, parenteral (e.g., intravenous, intramuscular), rectal, dermal, nasal, vaginal, inhalation, transdermal (patch), or ocular administration routes. Thus, the composition can be, for example, in the form of tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, patches, drainage agents, osmotic delivery devices, suppositories, enemas, injections, implants, sprays, or aerosols.

[0095] The pharmaceutical composition can be formulated to release the active DYRK1A inhibitor, GLP1R agonist, and immunomodulatory monoclonal antibody and / or immunosuppressant (e.g., anti-CD3 antibody) substantially immediately after administration or at any predetermined time or period after administration.

[0096] Controlled-release formulations include (i) formulations that produce a substantially constant concentration of the drug(s) in the body over a long period of time, (ii) formulations that produce a substantially constant concentration of the drug(s) in the body over a long period of time after a predetermined lag time; (iii) formulations that maintain the action of the drug(s) for a predetermined period by minimizing the undesirable side effects associated with fluctuations in the plasma levels of the active drug substance while maintaining a relatively constant effective drug level in the body; (iv) formulations that localize the action of the drug(s) by, for example, spatially placing the controlled-release composition adjacent to or within the diseased tissue or organ; and, (v) formulations that target the action of the drug(s) by using a carrier or chemical derivative to deliver the drug to a specific target cell type.

[0097] Administering a DYRK1A inhibitor, a GLP1R agonist, an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., an anti-CD3 antibody) in the form of a controlled release formulation is useful when the drug has (i) a narrow therapeutic index (i.e., generally, the therapeutic index (“TI”) is defined as the ratio of the median lethal dose (LD 50 ) to the median effective dose (ED 50 ), (ii) a narrow absorption window in the gastrointestinal tract, or (iii) a very short biological half-life and needs to be administered frequently throughout the day to maintain plasma concentrations at therapeutic levels.

[0098] DYRK1A inhibitors, GLP1R agonists, immunomodulatory monoclonal antibodies and / or immunosuppressants (e.g., anti-CD3 antibodies) can be used enterally or parenterally. Agents for oral administration can be administered in an amount of about 0.1 mg to 1,000 mg per day. In the case of parenteral administration, sublingual administration, nasal administration, or intrathecal administration, the compounds described herein are about 0.5 to about 100 mg / day; in the case of depot administration and implants, about 0.5 mg / day to about 50 mg / day; in the case of topical administration, about 0.5 mg / day to about 200 mg / day; and in the case of rectal administration, they can be used in an amount of about 0.5 mg to about 500 mg. In some embodiments, the therapeutically effective amount for oral administration is from about 1 mg / day to about 100 mg / day; and the therapeutically effective amount for parenteral administration is from about 5 mg / day to about 50 mg / day. In some embodiments, the therapeutically effective amount for oral administration is from about 5 mg / day to about 50 mg / day.

[0099] The daily dosage of the active ingredient is expected to be about 0.001 to about 1000 milligrams per kilogram of body weight, and the preferred dosage is about 0.1 to about 30 mg / kg. The oral dosage per day can be varied from about 0.01 mg to 1000 mg, 0.1 mg to 100 mg, or 10 mg to 500 mg of the compound per day. The daily dosage can be a single dose or divided doses, and furthermore, if judged appropriate, the upper limit can also be exceeded.

[0100] To obtain controlled release such that the release rate exceeds the metabolic rate of the therapeutic agent in question, any of a number of strategies can be pursued. Release control can be achieved by appropriate selection of various formulation parameters and components, including, for example, various types of controlled release compositions and coatings. Thus, the drug is formulated with suitable excipients into a pharmaceutical composition (single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches and liposomes) that releases the drug in a controlled manner upon administration. Accordingly, administration can be effected by nasal, oral, topical, transdermal, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, intracavitary or intravesical administration, intraocular administration, intraarterial administration, intravesical administration, or application to a mucosa. The compounds can be administered alone or with a suitable pharmaceutical carrier and can be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, emulsions, etc. In certain embodiments, administration is effected nasally, orally, transdermally, parenterally, subcutaneously, intravenously, intramuscularly, or intraperitoneally.

[0101] In certain embodiments, administration is effected using an infusion pump, for example, to provide rate-controlled infusion, intermittent infusion, and / or bolus infusion. The infusion pump may be a stationary infusion pump or a portable infusion pump. A stationary infusion pump is mainly used at the patient's bedside. A portable infusion pump is a relatively small, at least substantially self-contained device used to introduce drugs and other infusible substances (e.g., insulin) into a selected subject. Some portable infusion pumps are configured to be worn on a belt, carried in a pocket of clothing, or supported within some type of holder (collectively referred to as a "pocket pump"). Other infusion pumps are configured to adhere to the skin in a patch-like manner ("patch pump"). Infusion pumps can be used, for example, outside of a clinical environment to introduce (or "inject") pharmaceuticals continuously or intermittently intravenously or subcutaneously. Infusion pumps significantly reduce the frequency of subcutaneous access such as that using needles. In certain embodiments, the infusion pump is a subcutaneous or intravenous infusion pump. For example, the infusion pump may be a portable subcutaneous insulin infusion pump.

[0102] Another aspect described herein relates to a composition comprising a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody (e.g., an anti-CD3 antibody).

[0103] Suitable DYRK1A inhibitors are as described above and include, for example, harmine, INDY, rocetin-41, 5-iodotubercidin (5-IT), GNF4877, CC-401, kinase inhibitors, and derivatives thereof.

[0104] Suitable GLP1R agonists are as described above and include, for example, exendin-4, liraglutide, lixisenatide, semaglutide, and derivatives thereof.

[0105] Suitable immunomodulatory monoclonal antibodies are as described above. Suitable anti-CD3 antibodies are as described above and include, for example, teprilizumab.

[0106] Also contemplated are compositions comprising a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunosuppressive agent. Suitable immunosuppressive agents were detailed above.

[0107] The composition may further comprise a carrier. Suitable carriers are as described above. The carrier may be a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers are as described above.

[0108] A further aspect described herein relates to a method of increasing β-cell mass and function in a pancreatic β-cell population. The method comprises contacting a pancreatic β-cell population with a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and a low dose of an anti-CD3 antibody, said contacting being carried out under conditions effective to increase the β-cell mass and function of the pancreatic β-cell population.

[0109] When carrying out this and other methods described herein, the pancreatic β-cells may be mammalian cells. Mammalian cells include cells of primates such as, for example, mouse, hamster, rat, bovine, ovine, porcine, caprine, equine, monkey, dog (e.g., domestic dog), cat, rabbit, guinea pig, and human. For example, the cells are human pancreatic β-cells.

[0110] In one embodiment, the "pancreatic β-cells" are primary human pancreatic β-cells.

[0111] In some embodiments, this and other methods described herein are performed ex vivo or in vivo. When performed ex vivo, a cell population can be provided by harvesting cells from the pancreas and culturing the cells in a liquid medium suitable for in vitro or ex vivo culture of mammalian cells, particularly human cells. For example, but not limited to, commercially available media such as RPMI1640 from Invitrogen can be used as a suitable and non-limiting medium.

[0112] Methods for determining whether cells have a pancreatic β-cell phenotype are known in the art and include, but are not limited to, incubating the cells with glucose and testing whether insulin expression in the cells is increased or induced. Other methods include examining whether β-cell specific transcription factors are expressed, detecting β-cell specific gene products using RNA quantitative PCR, transplanting candidate cells into diabetic mice, examining the subsequent physiological responses after transplantation, and analyzing the cells by electron microscopy.

[0113] When practicing the methods described herein, a population of pancreatic β-cells is contacted with a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an anti-CD3 antibody.

[0114] In some embodiments, contacting a population of pancreatic β-cells with a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an anti-CD3 antibody is performed with harmine, exendin-4, and teprotumumab.

[0115] Contacting a pancreatic β-cell population with a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody) can be carried out using a single composition containing each of the DYRK1A inhibitor, the GLP1R agonist, and the immunomodulatory monoclonal antibody and / or the immunosuppressant (e.g., anti-CD3 antibody). Alternatively, the pancreatic β-cell population may be contacted successively with a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody). For example, the population of pancreatic β-cells is first contacted with an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody), a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor (or a composition containing the dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor), and a glucagon-like peptide-1 receptor (GLP1R) agonist (or a composition containing the glucagon-like peptide-1 receptor (GLP1R) agonist) (either together or separately).

[0116] When practicing the methods described herein, contacting a population of pancreatic β-cells with a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody) can be done multiple times a day, once a day, once a week, twice a week, once a month, once every two months, once a year, once every six months, or any number of times in between. The DYRK1A inhibitor, the glucagon-like peptide-1 receptor (GLP1R) agonist, and the immunomodulatory monoclonal antibody and / or immunosuppressant (e.g., anti-CD3 antibody) can be administered at different frequencies of administration. The contact between the DYRK1A inhibitor, GLP1R, immunomodulatory monoclonal antibody and / or immunosuppressant (e.g., anti-CD3 antibody) agonist and the pancreatic β-cell population can occur acutely or chronically. For example, the contact occurs chronically over a period of 1 year, 2 years, 3 years, 4 years, or more. In some embodiments, the administration is irregular.

[0117] In some embodiments, contacting a population of pancreatic β-cells with a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., an anti-CD3 antibody) increases β-cell function by at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, or more. Methods for measuring changes in β-cell function (such as β-cell proliferation) due to treatment are well known in the art and include, for example, hyperglycemic clamp, intravenous glucose tolerance test (IVGTT), stepwise glucose infusion, glucose-enhanced arginine stimulation, oral glucose tolerance test (OGTT) or mixed meal tolerance test (MMTT), and fasting measurements (see, e.g., Hannon et al., “A Review of Methods for Measuring β-Cell function: Design Considerations from the Restoring Insulin Secretion (RISE) Consortium,” Diabetes Obes. Metab. 20(1):14-24 (2018), the entire content of which is incorporated herein by reference). Methods for measuring changes in insulin sensitivity due to treatment are well known in the art and include, for example, hyperinsulinemic-euglycemic clamp, hyperglycemic clamp-derived insulin sensitivity, IVGTT-minimal model-derived insulin sensitivity (see, e.g., Hannon et al., “A Review of Methods for Measuring β-Cell function: Design Considerations from the Restoring Insulin Secretion (RISE) Consortium,” Diabetes Obes. Metab. 20(1):14-24 (2018), the entire content of which is incorporated herein by reference).

[0118] In some embodiments, contacting a population of pancreatic β cells with a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody) results in at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, or more increase in the number of proliferating pancreatic β cells in the population.

[0119] In some embodiments, contacting a population of pancreatic β cells with a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody) results in at least about 100%, at least about 110%, at least about 130%, at least about 140%, at least about 150%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600%, at least about 650%, at least about 700%, or more increase in human β cell mass. In some embodiments, contacting a population of pancreatic β cells (e.g., human β cells) with a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody) results in an extended survival period of the β cells as compared to not contacting the pancreatic β cell population.

[0120] In some embodiments, when a population of pancreatic β-cells is contacted with a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody), the differentiation of non-β cells (e.g., alpha cells, delta cells, PP cells, and / or duct cells) into β-cells is promoted as compared to when not contacted with the pancreatic β-cell population.

[0121] In some embodiments, when a population of pancreatic β-cells is contacted with a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody), the number of proliferating pancreatic β-cells in the population is increased by about 4-10%, or about 4-6%, 5-7%, 6-9%, or 7-10% per day.

[0122] In some embodiments, when a population of pancreatic β-cells is contacted with a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody), the number of proliferating pancreatic β-cells in the population increases by about 6-10% per day.

[0123] The method of contacting a population of pancreatic β-cells with a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody) can be carried out under conditions effective to cause a synergistic increase in cell proliferation in the population of pancreatic β-cells, which means, inter alia, that the number of proliferating pancreatic β-cells in the population increases as compared to when the cells are contacted with a DYRK1A inhibitor, a GLP1R agonist, or an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., anti-CD3 antibody).

[0124] In some embodiments, contacting a pancreatic β-cell population with a dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody and / or immunosuppressant (e.g., anti-CD3 antibody) does not induce β-cell death or DNA damage in the cell population. Further, the contact can induce β-cell differentiation and increase glucose-stimulated insulin secretion.

[0125] The method can also be carried out to increase the cell viability of a pancreatic β-cell population (in addition to restoring and / or recovering β-cell mass and function). For example, the method can be carried out to increase the cell viability of a treated pancreatic β-cell population compared to an untreated pancreatic β-cell population. Alternatively, the method can also be carried out to reduce cell death or apoptosis of the contacted pancreatic β-cell population compared to a non-contacted pancreatic β-cell population.

Examples

[0126] Example 1 Materials and Methods Analysis of Human β-Cell Death In Vitro Human pancreatic islets were dispersed as previously described (Vasavada et al., “Tissue-Specific Deletion of the Retinoblastoma Protein in the Pancreatic β-Cell Has Limited Effects on β-Cell Replication, Mass, and Function,” Diabetes 56:57-64 (2007), which is hereby incorporated by reference in its entirety). Briefly, human islets were washed twice with PBS, then 200 μl of pre-warmed Accutase (Corning) was added and the tubes were incubated at 37 °C for 10 minutes. Thereafter, complete RPMI medium (RPMI containing 5 mM D-glucose, 10% FBS, 100 units / ml penicillin, 100 μg / ml streptomycin) was added, centrifuged at 1000 rpm for 3 minutes, the pellet was washed with PBS and then centrifuged again at 1000 rpm for 3 minutes. The pellet was resuspended in complete RPMI medium, 50,000 cells / well were seeded onto 12 mm glass coverslips, placed in 24-well plates and cultured at 37 °C, 5% CO2 for 24 hours.Next, the following were added: phosphate-buffered saline, cytokines (50 units / mL IL-1β, 1,000 units / mL TNF-α, 1,000 units / mL IFN-γ) (R&D Systems) or 500 nM thapsigargin (ER stress inducer, Sigma-Aldrich) (Mellado-Gil et al., “Disruption of Hepatocyte Growth Factor / c-Met Signaling Enhances Pancreatic Beta-Cell Death and Accelerates the Onset of Diabetes,” Diabetes 60:525-36 (2011); Lu et al., “Dextran Sulfate Protects Pancreatic β-Cells, Reduces Autoimmunity, and Ameliorates Type 1 Diabetes,” Diabetes 69:1692-1707 (2020), which are hereby incorporated by reference in their entirety), together with 10 nM exendin-4 (MedChemExpress) (E), 10 μM harmine-HCl (Robert DeVita Lab, ISMMS) (H), or a combination of exendin-4 and harmine (E+H) were added to the wells. Cells were cultured at 37°C, 5% CO2 for 24 hours. Thereafter, the cells were fixed with 4% paraformaldehyde and subjected to TUNEL (cell death marker) staining and insulin staining using the DeadEnd Fluorescent TUNEL System (Promega), anti-proinsulin / C-peptide antibody (DSHB), and DAPI for nuclear detection. At least 1,500 β-cells per coverslip were examined.

[0127] Human pancreatic islet single-cell RNA sequencing (scRNA-seq) Sc-RNAseq analysis was performed on human islets treated with cytokines and halmide (H), exendin-4 (E), halmide + exendin-4 (E+H) (as above) at 37°C and 5% CO2 for 6 hours. After treatment, the human islets were washed twice with PBS and centrifuged at 300 rpm for 3 minutes. After removing PBS, 200 μl of pre-warmed Accutase was added, and the islets were incubated at 37°C for 10 minutes. Then, complete RPMI medium was added, and centrifuged at 1000 rpm for 3 minutes, and the pellet was washed with PBS. Then, the cells were resuspended in binding buffer (Miltenyi Biotec) and dead cell removal beads, incubated at room temperature for 15 minutes, and the cell suspension was applied to a dead cell removal column (Miltenyi Biotec) attached to a MACS separator. Then, the centrifuged effluent was collected, resuspended by adding 200 μl of 2% BSA and 200 U / ml of RNase inhibitor in PBS, the cells were mixed with AOPI (Nexcelon Bioscience) at a ratio of 1:1, and the cell concentration was confirmed with a Countess 3 automatic cell counter (Thermo-Fisher).

[0128] Cell samples were prepared according to the user guide of the 10X Genomics Single Cell 3’ V3.1 reagent kit, partitioned and barcoded with a 10X Genomic Chromium controller, and then a cDNA library was created. The total cell concentration analyzed by Countess 3 was sequenced on a NovaSeq 6000 system (Illumina). FASTQ files were downloaded from the sequencing facility and aligned using Cell Ranger V.6.1.1 with single cell 3’ V3 chemistry in the 10X clouds pipeline. After the 10X, h5 format files were generated, the data was analyzed on the R language platform using the Seurat package V.4.0. After the scRNA-seq data was created, natural mRNA was adjusted using SoupX (estimated contamination of 20%). The number of genes was less than 500, the number of gene species was less than 250, and the number of genes per UMI was 0.8 log10 Subsequently, cells with a mitochondrial gene ratio of 20% or more were excluded using a filter. Then, the Doubltfinder package (20% estimation) was used to algorithmically remove duplicates.

[0129] After evaluating the data quality control parameters as described above, integrated scRNA-data was created using the SCTransform function of Seurat without assigning method parameters. Next, cell types were assigned identities with reference to pancreatic cell type genes according to the normalized gene expression levels. To find changes in apoptosis and inflammation-promoting pathways in different treatment methods, gene set enrichment analysis at the single-cell level of the β-cell population was performed using the Escape package that provides access to the entire Molecular Signature Database (v.7.0). Enrichment of the entire C2 library was performed using chemical and genetic perturbations and canonical pathways including four well-known databases (Biocarta, KEGG, Reactome, Wikipathways). After calculating the enrichment score for each single cell, it was added to the metadata for dot plot-based analysis and visualization.

[0130] In vivo treatment of early-onset type 1 diabetes (T1D) non-obese diabetic (NOD) mice with anti-CD3 drug and harmine + exendin-4 (H+E) NOD / LtJ (NOD) female mice (The Jackson Laboratory) at 12 - 16 weeks of age were housed under specific pathogen - free conditions. Non - fasting blood glucose levels were measured once a week using a portable glucometer (AlphaTrak 2; Abbott Laboratories). Mice with blood glucose levels exceeding 250 mg / dL in three consecutive days of measurement were defined as diabetic (Lu et al., “Dextran Sulfate Protects Pancreatic β - Cells, Reduces Autoimmunity, and Ameliorates Type 1 Diabetes,” Diabetes 69:1692 - 1707 (2020)). Next, early - onset diabetic mice were intravenously (iv) administered 5 μg of IgG or anti - CD3 antibody (non - Fc - binding monoclonal anti - CD3εF(ab’)2 obtained from Bio X Cell, https: / / bxcell.com / product / m - CD3e - fab2 - fragments / ) once a day for 3 days. After the third injection, a mini - pump was implanted in the interscapular region of the mice to continuously deliver harmine, exendin - 4, or harmine + exendin - 4 (Rosselot et al., “Human Beta Cell Mass Expansion In Vivo with a Harmine and Exendin - 4 Combination: Quantification and Visulaization by iDISCO+ 3D Imaging,” biorxiv (2021), the entire content of which is incorporated herein by reference). Briefly, harmine and exendin - 4 were dissolved in water and loaded into an Alzet (Cupertino, CA) model 1004 mini - osmotic pump at concentrations of 27 mg / ml and 1 mg / ml, respectively, enabling subcutaneous administration of harmine and exendin - 4 at continuous rates of 3 mg / kg / day and 0.1 mg / kg / day for 1 month. For the 2 - month treatment, the pump was replaced with a new pump and fresh harmine and exendin - 4 on the 28th day. The control pump contained water. After pump implantation, non - fasting blood glucose was measured weekly as described above, and the proportion of diabetic mice was calculated.Animal experiments were conducted with the approval of the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee and in accordance with its guidelines.

[0131] Immune cell phenotype analysis of splenocytes from non-obese diabetic (NOD) mice administered with anti-CD3 drug, harmine, and exendin-4 (H+E) After 8 weeks of treatment, spleens were harvested, crushed, and cell suspensions were prepared after erythrocyte lysis and filtration (Lu et al., “Dextran Sulfate Protects Pancreatic β-Cells, Reduces Autoimmunity, and Ameliorates Type 1 Diabetes,” Diabetes 69:1692-1707 (2020), which is hereby incorporated by reference in its entirety). Cells (10 6 cells / ml) were treated with 2 μg / mL of soluble anti-CD3 and 2 μg / mL of soluble anti-CD28 (BioLegend) for 16 hours. Surface and intracellular staining of T cells for flow cytometry was performed using APC anti-mouse CD45 (BioLegend), anti-CD44-PE (BioLegend), anti-mouse CD62L-Brilliant violet 605 (BioLegend), anti-CD8-FITC (eBioscience), anti-CD4-Pacific Blue (BioLegend), anti-IFN-γ-phycoerythrin (eBioscience), anti-CD25-PerCP-Cy5.5 (BioLegend), and anti-FoxP3-phycoerythrin (eBioscience). Live / dead cells were identified using the Zombie NIR fixable viability dye kit (BioLegend). Cells were analyzed using an Attune Nxt flow cytometer (Thermo-Fisher).

[0132] Histomorphological analysis of pancreas collected from non-obese diabetic (NOD) mice treated with anti-CD3 and H+E After 8 weeks of treatment, the pancreas was excised and fixed overnight at room temperature in neutral buffered formalin. The pancreas was then paraffin-embedded and sectioned, and the β-cell mass was measured in three non-consecutive sections per mouse stained with insulin and hematoxylin using ImageJ (National Institutes of Health). Sections were also stained with Ki67 (Thermo-Fisher) or TUNEL (cell death, see above) and insulin (guinea pig anti-insulin antibody, Abcam) to detect β-cell proliferation and death. Sections were also stained with hematoxylin and eosin for pathological evaluation of insulitis, which was calculated as the percentage of islets per mouse at each stage of insulitis (Lu et al., “Dextran Sulfate Protects Pancreatic β-Cells, Reduces Autoimmunity, and Ameliorates Type 1 Diabetes,” Diabetes 69:1692-1707 (2020), which is incorporated herein by reference in its entirety).

[0133] Statistical analysis Data shown in bar graphs, scatter plots, and dot plots are presented as mean ± SEM. Statistical significance analysis was performed using one-way analysis of variance (Tukey's post hoc test) or Student's t-test when appropriate for comparison between groups. P < 0.05 was considered statistically significant.

[0134] Example 2 Combination of harmine and exendin-4 protects human β-cells from in vitro cell death-inducing factors Inflammatory cytokines and endoplasmic reticulum stress have been established as inducers of β-cell death in type 1 diabetes (T1D) (Lu et al., “Dextran Sulfate Protects Pancreatic β-Cells, Reduces Autoimmunity, and Ameliorates Type 1 Diabetes,” Diabetes 69:1692-1707 (2020), which is incorporated herein by reference in its entirety). Therefore, first, it was examined whether halmin (H), exendin-4 (E), or a combination thereof (H+E) has a protective effect on human β-cells treated in vitro with inflammatory cytokines or the endoplasmic reticulum stress inducer thapsigargin. As shown in FIGS. 1A-1C, the combination of 10 μM halmin (H) and 10 nM exendin-4 (E) significantly reduced human β-cell death induced by cytokines (FIGS. 1A-1B) or 500 nM thapsigargin (FIG. 1C) compared to treatment with 10 μM halmin or 10 nM exendin-4. However, it only provided a non-significant partial protection against cell death.

[0135] Next, single-cell RNA sequencing (scRNA-seq) analysis of human pancreatic islets treated with cytokines and halmin (H) and / or exendin-4 (E) was performed. The signaling pathways of pro-inflammation (FIG. 1D), intrinsic apoptosis and extrinsic apoptosis (FIG. 1E), human leukocyte antigen (HLA) class I molecules (FIG. 1F), chemokine CXCL9-11 (FIG. 1G), and interferon regulatory factors 1-9 (FIG. 1H) were upregulated in β-cells treated with cytokines, and the expression of these pathways was significantly downregulated and approached normal levels by the combined treatment with halmin+exendin-4 (H+E) (FIGS. 1D-1H). Thus, treatment with halmin+exendin-4 (H+E) appears to protect β-cells in the T1D environment.

[0136] Example 3 Sustained administration of harmine + exendin-4 after single administration of anti-CD3 drug completely restores early-onset type 1 diabetes (TID) in non-obese diabetic (NOD) mice Survival and regenerative effects of harmine + exendin-4 in human β cells (Figure 1A-1H; Ackeifi et al., “GLP-1 Receptor Agonists Synergize with DYRK1A Inhibitors to Potentiate Functional Human β Cell Regeneration,” Sci. Trans. Med. 12:eaaw9996 (2020) and Rosselot et al., “Human Beta Cell Mass Expansion In Vivo with a Harmine and Exendin-4 Combination: Quantification and Visulaization by iDISCO+ 3D Imaging,” biorxiv (2021), which are hereby incorporated by reference in their entirety), as well as the promising but partial efficacy of anti-CD3 antibodies for the treatment of early-onset type 1 diabetes (T1D) (Herold et al., “Teplizumab (anti-CD3 mAb) Treatment Preserves C-Peptide Responses in Patients with New-Onset Type 1 Diabetes in a Randomized Controlled Trial: Metabolic and Immunologic Features at Baseline Identify a Subgroup of Responders,” Diabetes 62:3766-74 (2013); Herold et al., “Anti-CD3 Monoclonal Antibody in New-Onset Type 1 Diabetes Mellitus,” N. Engl. J. Med. 346:1692-8 (2002); Keymeulen et al., “Insulin Needs After CD3-Antibody Therapy in New-Onset Type 1 Diabetes,” N. Engl. J. Med. 352:2598-608 (2005); Sherry et al., "Teplizumab for Treatment of Type 1 Diabetes (Protege Study): 1-Year Results from a Randomised, Placebo-Controlled Trial," Lancet 378:487-97 (2011); Hagopian et al., "Teplizumab Preserves C-Peptide in Recent-Onset Type 1 Diabetes: Two-Year Results from the Randomized, Placebo-Controlled Protege Trial," Diabetes 62:3901-8 (2013); and Herold et al., "An Anti-CD3 Antibody, Teplizumab, In Relatives at Risk for Type 1 Diabetes," N. Engl. J. Med. 381:603-613 (2019) (the entire contents of which are incorporated herein by reference), the combination of halmintide + exendin-4 (H+E) and anti-CD3 was tested to see if it could reverse diabetes in newly onset diabetic NOD mice.

[0137] Mice spontaneously developed diabetes (blood glucose levels exceeding 250 mg / dl three times in a row) at 12 - 16 weeks of age, and at that time, IgG or anti-CD3 antibody (non-Fc-binding monoclonal anti-CD3εF(ab’)2 obtained from Bio X Cell, https: / / bxcell.com / product / m-CD3e-fab2-fragments / ) was administered intravenously (iv) at 5 μg once a day for 3 days. Notably, this non-FcR-binding monoclonal anti-CD3 induces apoptosis of antigen-activated T cells in vivo by enabling the continuous expression and signaling of the TCR. However, importantly, it has been shown that Foxp3+ Tregs are resistant to depletion by CD3 antibodies. After the third injection, an Alzet mini pump was implanted, and halmintide, exendin-4, halmintide and exendin-4, or water was continuously administered for 4 weeks.

[0138] After 4 weeks, the mini-pumps were replaced with new ones (Rosselot et al., “Human Beta Cell Mass Expansion In Vivo with a Harmine and Exendin-4 Combination: Quantification and Visulaization by iDISCO+ 3D Imaging,” biorxiv (2021), the entire content of which is incorporated herein by reference), and the second 4-week period was conducted. As shown in Figure 2C, by 2 months after the intervention, harmine + exendin-4 administration reduced blood glucose levels to less than 250 mg / dl, while vehicle-administered diabetic mice remained diabetic throughout the 8-week follow-up period. Notably, 95% of the mice treated with anti-CD3 antibody and H+E maintained hypoglycemia from week 2 to week 8 (Figure 2F). In contrast, only 40% of the mice treated with anti-CD3 and vehicle maintained hypoglycemia even at week 8 after the start of treatment (Figure 2F). Notably, mice administered 5 μg / day of anti-CD3 per mouse for 3 days followed by 3 mg / kg / day of harmine or 0.1 mg / kg / day of exendin-4 did not reduce blood glucose levels to less than 250 mg / dl during the 8-week follow-up period (Figure 2B). 70% of the mice administered harmine after 3 days of anti-CD3 administration remained diabetic from week 3 to week 8 of the 8-week follow-up period (Figure 2E). Also, 40% of the mice administered exendin-4 after 3 days of anti-CD3 administration remained diabetic from week 4 to week 7 of the 8-week follow-up period, and 60% of the mice remained diabetic at week 8 of the 8-week follow-up period (Figure 2E). When (i) 3 mg / kg / day of harmine and 0.1 mg / kg / day of exendin-4, or (ii) vehicle (H2O) were administered for 8 weeks after administering 5 μg / day of IgG per mouse for 3 days (Figure 2A, Figure 2D), no significant difference in blood glucose levels was observed, and 70 - 100% of the mice in both groups remained diabetic throughout the 8-week follow-up period.

[0139] Example 4 Immunophenotypic analysis of splenocytes administered anti-CD3 and H+E to early-onset type 1 diabetes (T1D) non-obese diabetic (NOD) mice In ongoing experiments, analysis of immune cell populations in the blood, spleen, and pancreatic lymph nodes of early-onset T1D NOD mice administered anti-CD3 and harmine + exendin-4 (H+E) has begun. Analysis of splenocytes at the end of the experiment (week 8) showed no significant changes in the total number of immune cells (CD45+, naive, memory, effector CD4 + and CD8 + T lymphocytes) between the two treatment groups (Figure 3A-3C). Analysis of specific immune cell populations revealed that administration of harmine + exendin-4 (H+E) significantly decreased the number of activated CD4 + and CD8 + T cells (Th1) (Figure 3D-3E), and significantly increased the number of FoxP3 + CD25 + regulatory T cells (Treg) by over 50% (Figure 3F-3G). This suggests that combination therapy with anti-CD3 and harmine + exendin-4 (H+E) induces immune tolerance (decreased activation of T cells and enhanced regulatory T cells). In an experiment where NOD mice were treated with anti-CD3 for 3 days, harmine at 3 mg / kg / day (H) and exendin-4 at 0.1 mg / kg / day (E) or vehicle (H2O) for 8 weeks, circulating TNFα levels (Figure 3H), as well as CXCR3 + CD8 + cells (Figure 3I, top) and CXCR3 + CD4 + cells (Figure 3I, bottom) were lower in harmine + exendin-4 (H+E)-treated mice than in vehicle-treated mice. 3I, bottom) cells were lower in harmine + exendin-4 (H+E)-treated mice than in vehicle-treated mice, but the T cell exhaustion markers PD1, TIGIT, TOX, and EOMES were higher in CD4 + cells and CD8 + cells of NOD mice treated with harmine + exendin-4 (H+E) for 2 weeks after treatment with anti-CD3 for 3 days compared to NOD mice treated with vehicle (water) for 2 weeks after treatment with anti-CD3 for 3 days (Figure 3J).

[0140] Example 5: Analysis of the pancreas of NOD mice administered with anti-CD3 and H+E Hematoxylin and eosin staining of pancreatic sections obtained 8 weeks after treatment with anti-CD3 and halmintide + exendin-4 (H+E) or vehicle was performed (Figures 4A-4B). Pancreatic islets in vehicle-administered mice had the expected insulitis, and it was immediately apparent that this insulitis was reduced in mice administered with anti-CD3 and halmintide + exendin-4 (H+E). The insulitis scores of these islets showed that anti-CD3 and halmintide + exendin-4 (H+E)-administered mice had more islets with scores of 0-2 (no insulitis to mild insulitis) and fewer islets with strong to severe insulitis (scores of 3-4) compared to vehicle-administered mice (Figure 4B). Thus, administration of anti-CD3 + halmintide + exendin-4 (H+E) reduces islet inflammation in NOD diabetic mice. Flow cytometry analysis of the islets of treated mice showed that the number of CD45 + cells (immune cells) was lower in the islets of anti-CD3 + halmintide + exendin-4 (H+E)-treated mice compared to vehicle-treated mice (Figure 4C).

[0141] Next, β-cell proliferation, β-cell death, and β-cell mass were analyzed in these pancreases. As shown in Figures 4D-4E, in anti-CD3 and halmintide + exendin-4 (H+E)-administered mice, Ki67 + / insulin + cells were significantly increased, while TUNEL + / insulin + cells were significantly decreased, suggesting an increase in β-cell proliferation and a decrease in β-cell death. Furthermore, analysis of the total β-cell mass in these pancreases showed that administration of anti-CD3 and halmintide + exendin-4 (H+E) doubled the number of β-cells compared to vehicle-administered animals.

[0142] Conclusion Collectively, the tests described in these examples have demonstrated for the first time that temporary immunomodulatory treatment with anti- and subsequent combined treatment with harmine + exendin-4 (H+E) increases immune tolerance, enhances β-cell proliferation, protects β-cells, and increases β-cell mass.

[0143] Although preferred embodiments have been described and illustrated in detail herein, it will be apparent to those skilled in the relevant art that various changes, additions, substitutions, etc. can be made without departing from the spirit of the invention. Therefore, these are considered to be within the scope of the invention as defined in the appended claims.

Claims

1. A method for treating conditions related to insulin deficiency, For patients requiring treatment of conditions associated with insufficient insulin secretion, administer a bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody, if necessary, an anti-CD3 antibody. This includes, where the administration is carried out under conditions effective in reversing the loss of β-cell volume and function in a subject to treat a condition associated with insulin secretion deficiency. method.

2. The method according to claim 1, wherein the subject is treated for one or more of the following conditions: type 1 diabetes ("T1D"), type 2 diabetes ("T2D"), gestational diabetes, congenital diabetes, late-onset diabetes ("MODY"), cystic fibrosis-related diabetes, hemochromatosis-related diabetes, drug-induced diabetes, or solitary diabetes.

3. The method according to claim 2, wherein the subject is undergoing treatment for type 1 diabetes.

4. The method according to any one of claims 1 to 3, wherein the subject is long-term type 1 diabetes.

5. The method according to any one of claims 1 to 3, wherein the subject is a recently developed type 1 diabetes.

6. The method according to claim 1, wherein the administration is to increase immune tolerance in the subject, enhance β-cell proliferation in the subject, protect β-cells in the subject, increase the β-cell volume in the subject, or a combination thereof.

7. The method according to claim 1, wherein the DYRK1A inhibitor is harmine.

8. The method according to claim 1, wherein the GLP1R agonist is exendin-4.

9. The method according to claim 1, wherein the anti-CD3 antibody is teprizumab.

10. The method according to claim 1, wherein the administration is carried out using harmine, exendin-4, and teprizumab.

11. The method according to claim 1, wherein the administration is carried out sequentially with a bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an anti-CD3 antibody, respectively.

12. The method according to claim 1, wherein the administration is carried out by first administering an anti-CD3 antibody.

13. The method according to claim 12, wherein the administration of the anti-CD3 antibody is followed by treatment with a bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1 receptor (GLP1R) agonist.

14. The method according to claim 1, wherein an anti-CD3 antibody is administered in a low dose.

15. The method according to claim 1, wherein the administration is performed via nasal, oral, transdermal, non-enteral, subcutaneous, intravenous, intramuscular, or intraperitoneal cavity.

16. The method according to claim 1, wherein the target is a mammal.

17. The method according to claim 1, wherein the subject is a human subject.

18. Bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitors; Glucagon-like peptide-1 receptor (GLP1R) agonists; and Immunomodulatory monoclonal antibodies A composition comprising, if necessary, an immunomodulatory monoclonal antibody which is an anti-CD3 antibody.

19. The composition according to claim 18, further comprising a carrier.

20. The composition according to claim 18 or 19, wherein the carrier is a pharmaceutically acceptable carrier.

21. The composition according to claim 18, wherein the DYRK1A inhibitor is harmine.

22. The composition according to claim 18, wherein the GLP1R agonist is exendin-4.

23. The composition according to claim 18, wherein the anti-CD3 antibody is teprizumab.

24. A method for increasing the volume and function of β-cells in a pancreatic β-cell population, The method comprises contacting a pancreatic β-cell population with a bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and a low dose of an immunomodulatory monoclonal antibody, wherein the immunomodulatory monoclonal antibody is an anti-CD3 antibody, and the contact is carried out under conditions effective in increasing β-cell volume and function in the pancreatic β-cell population. method.

25. The method according to claim 24, wherein the method is carried out ex vivo.

26. The method according to claim 24, wherein the method is performed in vivo.

27. The method according to any one of claims 24 to 26, wherein the DYRK1A inhibitor is harmine.

28. The method according to claim 24, wherein the GLP1R agonist is exendin-4.

29. The method according to claim 24, wherein the anti-CD3 antibody is teprizumab.

30. The method according to claim 24, wherein the pancreatic β-cells are primary human pancreatic β-cells.

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