Combination therapy with immunomodulator, DYRK1A inhibitor and GLP1R agonist for treatment of type 1 diabetes

Abstract

Disclosed herein are methods of treating a condition associated with insufficiency of insulin secretion in a subject by administering a bispecific tyrosine phosphorylation modulating kinase 1A (DYRK1A) inhibitor, glucagon-like peptide-1 receptor (GLP1R) to a subject in need of treatment of a condition associated with insufficiency of insulin secretion level The present invention relates to a method for treating a condition associated with insufficiency of insulin secretion in a subject, comprising administering to the subject a condition associated with insufficiency of insulin secretion, an agonist and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., an anti-CD3 antibody), where the administration is performed under conditions effective to reverse loss of beta cell quality and function in the subject to treat the condition associated with insufficiency of insulin secretion in the subject. Compositions and methods of increasing beta cell quality and function in a population of pancreatic beta cells are also disclosed.

Description

[0001] This application claims the benefit of priority of U.S. Provisional Application Serial No. 63 / 347,977, filed on June 1, 2022, which is hereby incorporated by reference in its entirety.

[0002] This invention was made with government support under Grant No. DK105015 awarded by the National Institute of Health. The government has certain rights in the invention. Technical Field

[0003] Methods and compositions are described for treating conditions in a subject related to insufficient insulin secretion. Background Art

[0004] 1.6 million people in the United States and 20 million people worldwide have type 1 diabetes (“T1D”). Approximately twice as many are family members at high risk of developing T1D. T1D is caused by autoreactive host immune cells (T cells and other immune system cells) that destroy and kill insulin-producing β-cells in the pancreas. Although the etiology of T1D is not fully understood, it involves a combination of genetic, environmental, and infectious pathogen factors. There is currently no known effective therapy to delay, prevent, or reverse β-cell loss and progression to diabetes in people with T1D. Immunomodulatory therapies are making progress, which can block or delay immune system dysfunction in people with early T1D who still have an adequate supply of β-cells, but these therapies do not restore the lost β-cells in people with established T1D. Although β-cell loss in T1D has prompted attempts to replace the lost β-cells through pancreatic transplantation, islet transplantation, and stem cell-derived human β-cell transplantation, these methods are neither scalable nor cost-effective for millions of people with T1D.

[0005] Failure of self-tolerance leads to autoimmune destruction of pancreatic β-cells and T1D. A therapy that can simultaneously achieve the following could provide a treatment for curing T1D: 1) regulate T cell activation; 2) protect β-cells from known drivers of T1D-related β-cell dysfunction and destruction, such as pro-inflammatory cytokines and ER stress; and 3) induce β-cell regeneration.

[0006] Recently, it has been shown that in streptozotocin-induced diabetic mice transplanted with human islets, three months of combination therapy with the DYRK1A inhibitor harmine and the GLP1R agonist exendin-4 can increase human β-cell mass by 6-7 fold (Rosselot et al., “In Vivo Human β-Cell Mass Expansion with the Combination of Harmine and Exendin-4: Quantification and Visualization by iDISCO+ 3D Imaging”, biorxiv (2021)). If translated to T1D patients, this significant increase in human β-cell mass could be sufficient to normalize blood glucose levels. However, T1D-related autoimmunity will likely continue to destroy newly regenerated β-cells. Interestingly, several immunomodulatory interventions have been shown to delay the decline of β-cell function in patients with new-onset T1D (Atkinson et al., “The Challenge of Modulating β-Cell Autoimmunity in Type 1 Diabetes”, Lancet Diabetes Endocrinol. 7:52-64 (2019)). One such promising therapy is the humanized, non-mitogenic aglycosylated anti-CD3 monoclonal antibody teplizumab. This anti-CD3 antibody reduces the loss of β-cell function in patients with new-onset type 1 diabetes up to seven years after 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", The New England Journal of Medicine 352:2598-608(2005); Sherry et al., "Teplizumab for Treatment of Type 1 Diabetes (Protégé Study): 1-Year Results from a Randomised, Placebo-Controlled Trial", The 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 Protégé Trial", Diabetes 62:3901-8(2013); Herold et al., "An Anti-CD3 Antibody, Teplizumab, In Relatives at Risk for Type 1 Diabetes", The New England Journal of Medicine 381:603-613(2019)). The antibody can modify autoreactive CD8+ T lymphocytes, which are the important effector cells that kill β-cells in T1D. Subsequent reports have shown that CD3-specific monoclonal antibodies can enhance the function and / or proliferation of regulatory T cells, thereby exerting dominant inhibition on autoreactive T cells. Although this is an exciting advancement in T1D treatment, further improvements are still needed in several aspects. First, the method only delays the onset of T1D but does not cure T1D; second, the effect on subjects with established T1D is limited, which may reflect the fact that the β-cell mass in these patients is reduced and β-cells are unable to replicate or regenerate; third, there may be potential associated risks with the use of high doses of the antibody, which are related to lymphoproliferative diseases in young patients.

[0007] The present disclosure aims to overcome the deficiencies in the prior art. Summary of the Invention

[0008] One aspect of the present disclosure relates to a method of treating a condition in a subject related to insufficient insulin secretion. The method involves administering a dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody to a subject in need of treatment for a condition related to insufficient insulin secretion levels, optionally wherein the immunomodulatory monoclonal antibody is an anti-CD3 antibody, wherein the administration is carried out under conditions effective to reverse the loss of β-cell mass and function in the subject to treat the condition in the subject related to insufficient insulin secretion.

[0009] Another aspect of the present disclosure 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 and / or an immunosuppressant (e.g., an anti-CD3 antibody).

[0010] A further aspect of the present disclosure relates to a method of increasing β-cell mass and function in a pancreatic β-cell population. The method involves 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 immunosuppressant (e.g., an 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 an example of the present disclosure, it was hypothesized that treatment with a combination of an anti-CD3 antibody and the β-cell regenerative drugs harmine and exendin-4 would reverse T1D. To test this hypothesis, a mouse model (NOD female mice) that spontaneously developed T1D was treated continuously with an anti-CD3 monoclonal antibody for three days, followed by continuous delivery of harmine plus exendin-4 for eight weeks. Surprisingly, it was found that this treatment combination completely reversed T1D in 100% of the mice. These mice showed reduced insulitis in the pancreas, enhanced β-cell proliferation and mass, reduced activated T cells (Th1+ cells), and increased regulatory T cells.

[0012] The examples described herein demonstrate for the first time that anti-CD3 immunomodulatory treatment (e.g., at a low dose) and the harmine + exendin-4 β-cell regeneration and anti-apoptosis combination treatment increase immune tolerance, enhance β-cell proliferation and mass, and protect β-cells from cytokines and ER stress, and these effects together result in the reversal of early-onset T1D in a T1D mouse model.

[0013] The present disclosure relates to a therapeutically and economically effective combination of immunomodulation and β-cell regeneration therapies, which can be applied to millions of people with diagnosed long-term T1D and recently onset T1D. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figures 1A - 1H Shows the protective effect of harmine plus exendin-4 on human β-cells in vitro after treatment with two typical inducers of β-cell death associated with T1D (pro-inflammatory cytokines and ER stress). Figure 1A Is a graph showing the results of human islet cells in cultures treated with cytokines (CKS) and with or without 10 μM harmine (H), 10 nM exendin-4 (E), or both (H+E) for 24 hours. N = 7 different human islet preparations were analyzed. Figure 1B Shows representative images of these experiments. Figure 1C Is a graph showing the results of human islet cells treated with 500 nM thapsigargin (TH) and with or without E, H, or E+H for 24 hours. N = 5 different human islet preparations were analyzed. P values are indicated in the figure; each point represents a different human islet preparation. Figures 1D - 1H Is a graph showing Figure 1A Single-cell RNA sequencing (scRNA-seq) analysis of human islets treated with cytokines and H, E, and H+E as in Figure 1D . Gene set enrichment analysis (GSEA) of human β-cells in the scRNA-seq study revealed increased pro-inflammatory ( Figure 1E ), intrinsic and extrinsic apoptosis ( Figure 1F ), human leukocyte antigen (HLA) class I molecule ( Figure 1G ), chemokine CXCL9-11 ( Figure 1H ) and interferon regulatory factor 1-9 (

[0015] Figures 2A - 2F Shows that treatment with harmine and exendin-4 after administration of anti-CD3 can reverse diabetes in NOD diabetic mice. Figures 2A - 2FFigure showing blood glucose levels in mice before and after spontaneous onset of diabetes and then treated with: 5 μg IgG / mouse / day for three days, and 3 mg / kg / day harmine (H) and 0.1 mg / kg / day exendin-4 (E), or vehicle (H2O), for eight weeks (N = 6 - 8 mice)( Figure 2A ); 5 μg anti-CD3 / mouse / day for three days, and 3 mg / kg / day harmine (H) or 0.1 mg / kg / day exendin-4 (E), for eight weeks (N = 5 - 6( Figure 2B ); 5 μg anti-CD3 / day for three days, and 3 mg / kg / day harmine (H) and 0.1 mg / kg / day exendin-4 (E), or vehicle (H2O), for eight weeks (N = 19 mice / group)( Figure 2C ). An alzet minipump (pump) was implanted every four weeks. Figures 2D - 2F Figure showing Figure 2A ( Figure 2D ), Figure 2B ( Figure 2E ) and Figure 2C ( Figure 2F ) the percentage of diabetic mice after treatment as shown. Diabetes was defined as blood glucose above 250 mg / dl.

[0016] Figures 3A - 3J show the results of the immune profile in splenocytes from NOD mice treated with anti-CD3 and harmine plus exendin-4. Figure 3A is a figure showing the total number of CD45 + cells. Figure 3B is a figure showing the ratio of CD4 / CD8 T cells. Figure 3C is a figure showing CD44 / CD62L (naive, memory, and effector) CD8 + cells. Figures 3D - 3E are figures showing the decrease in activated (interferon-γ, IFNγ + cells, Th1) CD4 + and CD8 + lymphocytes with anti-CD3 and H+E treatment. Figures 3F - 3G are figures showing the increase in FoxP3 + CD25 + (Treg) cells in the spleens of mice treated with anti-CD3 and H+E. Figure 3H is a figure representing the circulating TNFα levels from Figure 2C representative mice (N = 5). Figure 3I is a figure showing the CXCR3 expression levels in CD4 Figure 2C and CD8 + cells from + representative mice (N = 3). Figure 3J Figure showing in as Figure 2CCD4 in NOD mice (N = 3 - 7 mice) treated with anti - CD3 for three days and then with vehicle (water) or H + E for 2 weeks as in + and CD8 + levels of T - cell exhaustion markers PD1, TIGIT, TOX, and EOMES in cells. *P < 0.05; **P < 0.01.

[0017] Figures 4A - 4E Shows the results of pancreatic analysis of NOD mice treated with anti - CD3 and harmine (H) plus exendin - 4 (E). Figure 4A Shows Figure 2C Representative images of hematoxylin and eosin staining of the pancreas from mice treated as in Figure 4B Is a graph showing the quantification of isletitis scores in the islets of these mice (N = 19 / group). Score 0, no isletitis; score 1, peripancreatic isletitis; score 2, mild isletitis; score 3, severe isletitis; score 4, nearly 100% isletitis. Figure 4C Shows flow cytometry analysis of islets from 3 mice of these groups to detect CD45 + cells (immune cells). Figure 4D Is a graph showing Figure 2C Quantification of Ki67 + (red) / insulin + (green) cells / DAPI (blue, nucleus) (β - cell proliferation) and TUNEL (green), insulin (red), and DAPI (blue, nucleus) (β - cell death) in pancreatic sections from mice treated as in + / insulin + cells (upper panel) or TUNEL + insulin + cells (lower panel). Graphs of quantification for these mice are shown on the right. Figure 4E Is a graph showing the quantification of β - cell mass (mg). N = 6 - 19 mice / group. *P < 0.05; **P < 0.01: ***P < 0.001; ****P < 0.00001. Detailed Description

[0018] Disclosed are methods and compositions for treating conditions associated with insufficient insulin secretion in a subject.

[0019] One aspect of the present disclosure relates to a method of treating a subject having a condition associated with insufficient insulin secretion. The method involves administering to a subject in need of treatment for a condition associated with insufficient insulin secretion levels 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 β-cell mass and function in the subject to treat the subject's condition associated with insufficient insulin secretion.

[0020] DYRK1A inhibitors and GLP1R agonists suitable for practicing the methods of the present disclosure are described in the following international publications: International Publication No. WO 2018 / 081401 by Stewart et al., International Publication No. 2019 / 100062 by DeVita et al., International Publication No. WO 2019 / 183245 by Kumar et al., International Publication No. 2019 / 100062 by DeVita et al., International Publication No. WO 2019 / 136320 by Stewart et al., International Publication No. WO 2020 / 142485 by DeVita et al., International Publication No. WO 2020 / 142486 by DeVita et al., and International Publication No. WO 2021 / 263129 by DeVita et al., which are hereby incorporated by reference in their entirety.

[0021] Several DYRK1A inhibitors from natural sources as well as small molecule drug discovery programs have been identified and characterized and can be used to practice the methods disclosed herein. For example, suitable DYRK1A inhibitors include, but are not limited to, harmine; INDY (having the chemical structure

[0022]

[0023] as described by Wang et al., “A High-Throughput Chemical Screen Reveals That Harmine-Mediated Inhibition of DYRK1A Increases Human Pancreatic Beta Cell Replication”, Nature Medicine 21:383-388 (2015), which is hereby incorporated by reference in its entirety); leucettine having the chemical structure

[0024]

[0025] As described in 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. 55:9312-30 (2012), which is hereby incorporated by reference in its entirety); 5-iodotubercidin (5-IT); GNF4877; harmaline analogs; CC-401; thiazodiazine kinase inhibitors; and the like. Other suitable DYRK1A inhibitors include, but are not limited to, GNF7156 and GNF6324 (see Shen et al., “Inhibition of DYRK1A and GSK3B Induces Human Beta Cell Proliferation,” Nat. Commun. 6:8372 (2015), which is hereby incorporated by reference in its entirety). Combinations of DYRK1A inhibitors can be used in practicing the methods of the present disclosure or forming the compositions of the present disclosure.Among all DYRK1A inhibitors, harmine and its analogues (β-carbolines) are the most frequently studied and remain the most potent and orally bioavailable class of inhibitors to date (Becker et al., “Activation, Regulation, and Inhibition of DYRK1A”, FEBS J. 278(2):246-256 (2011) and Smith et al., “Recent Advances in the Design, Synthesis, and Biological Evaluation of Selective DYRK1A Inhibitors: A New Avenue for a Disease Modifying Treatment of Alzheimer's?”, ACS Chem. Neurosci. 3(11):857-872 (2012), which are hereby incorporated by reference in their entirety).

[0026] In addition to harmine, 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), which are hereby incorporated by reference in their entireties), leucettines (Tahtouh et al., “Selectivity, Cocrystal Structures, and Neuroprotective Properties of the Protein Kinase Inhibitor Family Leucettines 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), which is hereby incorporated by reference in its entirety), quinalizarine (Cozza et al., "Quinalizarin as a Potent, Selective and Cell - permeable Inhibitor of Protein Kinase CK2", Journal of Biological Chemistry 421(3):387 - 395(2009), which is hereby incorporated by reference in its entirety), 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), which is hereby incorporated by reference in its entirety), benzocoumarin (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), which is hereby incorporated by reference in its entirety) 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), which is hereby incorporated by reference in its entirety) are other natural products that have been shown to inhibit DYRK1A and other kinases.

[0027] Among other scaffolds identified in small molecule drug discovery efforts, INDY (Ogawa et al., "Development of a Novel Selective Inhibitor of the Down Syndrome-Related Kinase Dyrk1A", Nature Communications 1: article number 86 (2010), which is hereby incorporated by reference in its entirety), DANDY (Gourdain et al., "Development of DANDYs, New 3,5-Diaryl-7-Azaindoles Demonstrating Potent DYRK1A Kinase Inhibitory Activity", Journal of Medicinal Chemistry 56(23):9569-9585 (2013), which is hereby incorporated by reference in its entirety) and FINDY (Kii et al., "Selective Inhibition of the Kinase DYRK1A by Targeting its Folding Process", Nature Communications 7:11391 (2016), which is hereby incorporated by reference in its entirety), pyrazolidine-dione (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 is hereby incorporated by reference in its entirety), amino-quinazoline (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)", Bioorganic & Medicinal Chemistry Letters 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", Journal of Medicinal Chemistry 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", Journal of Medicinal Chemistry 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 entireties), 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), which is hereby incorporated by reference in its entirety), chromenoidoles (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), which is hereby incorporated by reference in its entirety), 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",The Journal of Neurochemistry (J. Neurochem.) 133(3):440-451 (2015), which is hereby incorporated by reference in its entirety, and 5-iodotubercidin (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", Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci.) 109(10):3915-3920 (2012), which are hereby incorporated by reference in their entirety) show effective DYRK1A activity with varying degrees of kinase selectivity.,

[0028] 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", Journal of Medicinal Chemistry 63:2958–2973 (2020), which is hereby incorporated by reference in its entirety) and those described in Liu et al., "DYRK1A Inhibitors for Disease Therapy: Current Status and Perspectives", European Journal of Medicinal Chemistry 229:114062 (2022), which is hereby incorporated by reference in its entirety.

[0029] Suitable thiazide kinase inhibitors include, for example but not limited to, thiazide kinase inhibitors described in the following international publications: 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), which are hereby incorporated by reference.

[0030] As described above, glucagon-like peptide-1 receptor agonists mimic the action of the incretin hormone GLP-1, which is released from the intestine in response to food intake. Their actions include increasing insulin secretion, decreasing glucagon release, increasing satiety, and slowing gastric emptying.

[0031] GLP1R agonists suitable for practicing the methods disclosed herein are described in PCT Publication No. WO 2019 / 136320 to Stewart et al., which PCT publication is hereby incorporated by reference in its entirety, and the GLP1R agonists 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 the Gila monster (Heloderma suspectum) (lizard). Liraglutide is an acylated analog of GLP-1 that self-associates into a heptameric structure, thereby delaying absorption from the subcutaneous injection site. Taspoglutide has 3% homology with native GLP-1 and is completely resistant to DPP-4 degradation. Lixisenatide is a human GLP1R agonist. Albiglutide is a long-acting GLP-1 mimetic that is resistant to DPP-4 degradation. Dulaglutide is a long-acting GLP1 analog. Semaglutide is a GLP1R agonist approved for T2D. Clinically available GLP1R agonists include, for example, exenatide, liraglutide, albiglutide, dulaglutide, lixisenatide, semaglutide.

[0032] In some embodiments, the GLP1R agonist is selected from the group consisting of exendin-4, GLP1(7-36), liraglutide, lixisenatide, semaglutide, tirzepatide (also known as Mounjaro), and combinations thereof.

[0033] Other suitable GLP1 agonists 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 their derivatives, e.g., 7-(4-chlorophenyl)-1,3-dimethyl-5,5-bis(trifluoromethyl)-5,8-dihydropyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (see, e.g., 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", Journal of Medicinal Chemistry 60:1611-1616 (2017), which is hereby incorporated by reference in its entirety).

[0034] Other suitable GLP-1 agonists include positive allosteric modulators (“PAMs”) of the GLP-1R, such as (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, which PCT publication is hereby incorporated by reference in its entirety).;

[0035] Other suitable GLP1 agonists include, but are not limited to, chimeric peptides such as dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonists (i.e., tirzepatide). See, e.g., Frias et al., “Tirzepatide Versus Semaglutide Once Weekly in Patients with Type 2 Diabetes,” The New England Journal of Medicine 385:503–515 (2021), which is hereby incorporated by reference in its entirety. Both GIP and GLP1 are GPCRs that act through cAMP. Thus, in some embodiments, the GLP1 agonist increases cAMP in the β-cell population (e.g., human β-cells).

[0036] Glucagon-like peptide-1 receptor agonists mimic the action of the incretin hormone GLP-1, which is released from the gut in response to food intake. Their actions include increasing insulin secretion, reducing glucagon release, increasing satiety, and slowing gastric emptying. An alternative way to enhance GLP-1 concentration in the blood is to prevent its degradation by the enzyme DPP4. GLP-1 receptor agonists and DPP4 inhibitors are 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).

[0037] Thus, as a supplement or alternative to GLP1 agonists, the methods and compositions according to the present disclosure may include dipeptidyl peptidase IV (DPP4) inhibitors. Exemplary suitable DPP4 inhibitors include, but are not limited to, sitagliptin, vildagliptin, saxagliptin, alogliptin, teneligliptin, and anagliptin.

[0038] In practicing the methods disclosed herein, the immunomodulatory monoclonal antibody can be an anti-CD3 antibody. Suitable anti-CD3 antibodies can include any antibody that targets or is capable of specifically binding to the CD3 receptor on the surface of T cells (typically CD3 on human T cells, particularly human CD3ε (CD3E)). Anti-CD3 antibodies include, but are not limited to, teplizumab, otelixizumab, and visilizumab. Another non-limiting example of a suitable anti-CD3 antibody is OKT3, also known as muromonab, UHCTI clone, also known as T3 and CD3E. OKT3 is a murine anti-CD3 antibody (DrugBank accession number DB00075, which is hereby incorporated by reference in its entirety); Abz287a is a humanized version of OKT3, and Abz494 to Abz498 are pH-dependent antibodies. The sequence of Abz287a is found in GenBank accession number ALJ79286 and described in Pegu et al., "Activation and Lysis of Human CD4 Cells Latently Infected with HIV-1", Nature Communications 6:8447 (2015), which is hereby incorporated by reference in its entirety.

[0039] Other suitable immunomodulatory monoclonal antibodies include, but are not limited to, anti-TNF-α 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. See, e.g., 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 Endocrinol. 9:212–224 (2021) and International Publication No. WO 2014 / 004857, which are hereby incorporated by reference in their entirety).

[0040] Identification of suitable immunomodulatory monoclonal antibody versions and fragments (e.g., anti-CD3 antibody versions and fragments) that can be used to practice the methods of the present disclosure can be accomplished by methods and techniques well recognized and known in the art, such as by histidine substitution via a phage display library or by histidine substitution from a combinatorial histidine substitution library via yeast surface display.

[0041] As used herein, the terms "antibody" or "immunoglobulin" are used in the broadest sense and specifically cover intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, provided that they exhibit the desired biological activity. The intact antibodies or whole antibodies can be classified into different classes according to the amino acid sequence of the constant region of the intact antibody or whole antibody. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0042] "Antibody fragments" according to the present disclosure include a portion of an intact antibody, such as including its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), and Fc fragments, bifunctional antibodies, linear antibodies, single-chain antibody molecules; bispecific and multispecific antibodies formed from antibody fragments.

[0043] In some embodiments, the monovalent antibody fragment for an antibody according to the present disclosure is scFv or Fab.

[0044] An "intact" or "whole" antibody according to the present disclosure is an antibody that includes an antigen-binding variable region as well as the light-chain constant domain (CL) and the heavy-chain constant domains CH1, CH2, and CH3.

[0045] The "Fc" region of an antibody according to the present disclosure generally includes CH2, CH3, and the hinge region of the major classes of IgG1 or IgG2 antibodies. The hinge region is a group of about 15 amino acid residues that combines the CH1 region and the CH2-CH3 region.

[0046] The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain and has only one antigen-binding site.

[0047] The "Fab'" fragment differs from the Fab fragment by the addition of several residues at the carboxyl terminus of the heavy-chain CH1 domain that includes one or more cysteine residues from the antibody hinge region.

[0048] An "F(ab')2" antibody according to the present disclosure is produced as a pair of Fab' fragments that have a hinge cysteine between them.

[0049] A "single-chain Fv" or "scFv" antibody fragment according to the present disclosure comprises the VH and VL domains of an antibody, wherein 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, which enables the scFv to form the desired structure for antigen binding.

[0050] The "variable domain" of an antibody according to the present disclosure comprises framework regions (usually FR1 to FR4) and CDR domains (usually CDR1, CDR2, and CDR3), which are referred to as "hypervariable regions".

[0051] As used herein, the term "hypervariable region" or "CDR" refers to the amino acid residues of an antibody responsible for antigen binding. Hypervariable regions generally include amino acid residues from the "complementary determining region" or "CDR" (e.g., residues 24 - 34 (L1), 50 - 56 (L2), and 89 - 97 (L3) in the light chain variable domain and residues 31 - 35 (H1), 50 - 65 (H2), and 95 - 102 (H3) in the heavy chain variable domain).

[0052] Unless otherwise indicated, amino acid positions within an antibody molecule according to the present disclosure are numbered according to Kabat.

[0053] "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.

[0054] An "antibody variant" according to the present disclosure comprises an antibody having a modified amino acid sequence compared to a parental antibody but having the same or altered binding affinity for a target antigen. Antibody variants differ from the parental antibody by the substitution, deletion, or addition of one or more amino acid residues at specific positions within the variable domain (including CDR domains) and / or constant region of the antibody in order to modify certain properties of the antibody, such as binding affinity and / or receptor function, e.g., ADCC, FcRn binding, etc. An antibody with a histidine mutation according to the present disclosure without further modification is not designated as an "antibody variant" according to the present disclosure. Compared to the parental antibody, an antibody variant according to the present disclosure exhibits a sequence homology of 80 - 99%, or in some embodiments 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%, depending on the specific positions of the amino acid residues to be substituted, deleted, or added.

[0055] The term "cytokine" is a general term for proteins released by one cell population that act as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monokines, and conventional polypeptide hormones such as vascular endothelial growth factor (VEGF); integrin thrombopoietin (TPO); nerve growth factors such as NGF.β; platelet growth factor; transforming growth factor (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; 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.

[0056] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered to contain a human antibody constant domain and a non-human variable domain that has been modified to have a high level of sequence homology with a human variable domain. This can be achieved by transplanting six non-human antibody CDRs (which together form the antigen-binding site) onto a homologous human receptor framework region (FR) (see PCT Publication No. WO 92 / 22653 and European Patent No. 0629240, which are hereby incorporated by reference in their entirety). To fully reconstruct 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 (backmutations). Structural homology modeling can assist 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 non-human CDR sequences, predominantly human framework regions (optionally including one or more amino acid backmutations to non-human amino acid sequences), and a fully human constant region.

[0057] As used herein, the term "human antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. A human antibody may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The human monoclonal antibodies of the present disclosure can be produced by a variety of techniques, including conventional monoclonal antibody methods, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, "Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity", Nature 256:495 (1975), which is hereby incorporated by reference in its entirety. Although somatic cell hybridization procedures can be used, in principle, other techniques for producing monoclonal antibodies can be employed, e.g., viral or oncogenic transformation of B lymphocytes, or phage display techniques using human antibody gene libraries. An animal system suitable for the preparation of hybridomas secreting human monoclonal antibodies is the murine system. The production of hybridomas in mice is a very well-established procedure. Immunization protocols and techniques for isolating immune spleen cells for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. Thus, human monoclonal antibodies can be produced using, for example, transgenic or transchromosomic mice or rats that carry a portion of the human immune system rather than a murine or rat system. Thus, in some embodiments, a human antibody is obtained from a transgenic animal (such as a mouse or rat) that carries human germline immunoglobulin sequences rather than animal immunoglobulin sequences. In such embodiments, the antibody originates from the human germline immunoglobulin sequences introduced into the animal, but the final antibody sequence is the result of further modification of the human germline immunoglobulin sequences by somatic hypermutation and affinity maturation by the endogenous animal antibody mechanism (see, e.g., Mendez et al., "Functional Transplant of Megabase Human Immunoglobulin Loci Recapitulates Human Antibody Response in Mice", 15:146-56 (1997), which is hereby incorporated by reference in its entirety).

[0058] In some embodiments, an immunomodulatory monoclonal antibody (e.g., an anti-CD3 antibody) is administered at a low dose. As used herein, a "low dose" with respect to the administration of an immunomodulatory monoclonal antibody (e.g., an anti-CD3 antibody) is a sub-optimal dose, or a dose that is lower than the dose at which an immunomodulatory monoclonal antibody (e.g., an anti-CD3 antibody) is administered as a treatment for a condition in a subject that is associated with insufficient endogenous insulin secretion (i.e., without any other concomitant agents). In other words, due to the effects of the concomitant administration of a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor and a glucagon-like peptide-1 receptor (GLP1R) agonist, 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 the dose typically administered for the treatment of T1D. In Bresson et al., "Anti-CD3 and NasalProinsulin Combination Therapy Enhances Remission from Recent-OnsetAutoimmune Diabetes by Inducing Tregs", J. Clin. Invest. 116(5):1371-1381 (2006) ("Bresson"), which is hereby incorporated by reference in its entirety, the ability of an anti-CD3 antibody to reverse T1D was tested at different doses in NOD mice (see Bresson, Figure 1E ). Bresson selected 40 μg / day as the "sub-optimal" dose, which resulted in reversal of diabetes in only 30% of the mice, as their goal was to test whether combination with another drug (in this case nasal insulin) could further improve reversal of diabetes in these mice. In the examples discussed in the disclosure hereinbelow, a "sub-optimal" dose of anti-CD3 was used, and its combination with harmine plus exenatide (a 39 amino acid polypeptide that is a synthetic version of exendin-4) treatment reversed diabetes in 100% of the 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 over a 14-day course of escalating intravenous teplizumab, where the total cumulative dose is approximately 9034 mg / m 2 , and is further defined as (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 5C14, 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 less than 1 / 2 or 1 / 4 of the dose provided during the 14-day course of escalating doses of intravenous tilizumab.

[0059] Also contemplated is a method of treating a subject for a condition associated with insufficient insulin secretion. The method involves administering to a subject in need of treatment for a 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 β-cell mass and function in the subject to treat the subject's condition associated with insufficient insulin secretion.

[0060] Suitable DYRK1A inhibitors and GLP1R agonists are described in detail above.

[0061] Suitable immunosuppressants include, but are not limited to, immunomodulatory monoclonal antibodies (e.g., anti-CD3 antibodies) and tacrolimus, rapamycin, mycophenylate mofetil, and glucocorticoids such as prednisone, cortisone, and dexamethasone.

[0062] As used herein, a condition associated with an insufficient level of insulin secretion refers to a condition in which the plasma level of insulin produced by a subject is lower than the plasma level required to maintain a normal glucose level in the blood, such that the subject suffering from the condition associated with insufficient insulin secretion becomes hyperglycemic. In such a case, the level of insulin secreted by the pancreatic β-cells of the affected subject is insufficient to maintain a normal concentration of glucose in the blood (i.e., euglycemia).

[0063] One of the conditions associated with insufficient insulin secretion levels is insulin resistance. Insulin resistance is a condition in which the cells of a subject become less sensitive to the glucose-lowering effects of insulin. Insulin resistance in muscle and fat cells reduces glucose uptake (and thus, the local storage of glucose as glycogen and triglycerides), while insulin resistance in liver cells causes reduced glycogen synthesis and storage and fails to inhibit glucose production and release into the bloodstream. Insulin resistance generally refers to a reduced glucose-lowering effect of insulin. However, other functions of insulin may also be affected. For example, insulin resistance in fat cells reduces the normal effect of insulin on lipids and results in reduced uptake of circulating lipids and increased hydrolysis of stored triglycerides. Increased mobilization of stored lipids in these cells raises free fatty acids in the plasma. Elevated blood fatty acid concentrations, reduced muscle glucose uptake, and increased liver glucose production all contribute to an elevated blood glucose level. If insulin resistance is present, the pancreas needs to secrete more insulin. If this compensatory increase does not occur, the blood glucose concentration increases and type II diabetes develops.

[0064] One of the conditions associated with insufficient insulin secretion levels is diabetes. Diabetes can be classified into two major groups of diseases: type I (“T1D”) and type II (“T2D”).

[0065] In some embodiments, the condition associated with insufficient insulin secretion levels is type 1A diabetes or immune-mediated diabetes. In some embodiments, the condition associated with insufficient insulin secretion levels is type 1B diabetes or idiopathic diabetes.

[0066] The term “diabetes” as used herein also refers to a group of metabolic diseases in which a patient has elevated blood glucose levels, 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), and several forms of monogenic diabetes.

[0067] In certain embodiments, a subject has or is receiving treatment for one or more of the following: 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 receiving treatment for type I diabetes. Alternatively, the subject has or is receiving treatment for type II diabetes.

[0068] In some embodiments, the subject has long-standing T1D. In some embodiments, the subject has recently onset T1D. For a description of long-standing T1D and recently onset T1D, see, e.g., 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.

[0069] In some embodiments, the subject has a disease or disorder associated with a mutant and / or aberrant expression or function of DYRK1A. According to such embodiments, the subject may have Down's syndrome. Down's syndrome is associated with an increased incidence of autoimmune diseases such as an increased risk and prevalence of type 1 diabetes.

[0070] The treatment methods described herein can effectively treat subjects with insufficient insulin secretion levels, for example, by increasing immune tolerance in the subject, enhancing β-cell proliferation in the subject, protecting β-cells in the subject, increasing β-cell mass in the subject, or any combination thereof.

[0071] In some embodiments, the condition associated with insufficient insulin secretion levels is metabolic syndrome. Metabolic syndrome is commonly used to define a cluster of abnormalities associated with an increased risk of developing type II diabetes and atherosclerotic vascular disease. Associated conditions and symptoms include, but are not limited to, fasting hyperglycemia (type II diabetes or impaired fasting glucose, impaired glucose tolerance, or insulin resistance); hypertension; central obesity (also known as visceral obesity, android obesity, or apple-shaped obesity), i.e., overweight with fat mainly deposited around the waist; reduced HDL cholesterol; and elevated triglycerides.

[0072] In some embodiments, the condition associated with insufficient insulin secretion levels is metabolic syndrome or insulin resistance, and the methods described herein are implemented to treat subjects having or being treated for metabolic syndrome or insulin resistance.

[0073] Other conditions that may be associated with insufficient insulin secretion levels include, but are not limited to, hyperuricemia, progression of fatty liver (especially in concomitant obesity) to non-alcoholic fatty liver disease, polycystic ovary syndrome (in females), and acanthosis nigricans.

[0074] The related conditions can also be treated according to the treatment methods disclosed herein, including but not limited to any diseases associated with blood or plasma glucose levels outside the normal range, such as hyperglycemia. Thus, the term "related conditions" includes impaired glucose tolerance ("IGT"), impaired fasting glucose ("IFG"), insulin resistance, metabolic syndrome, postprandial hyperglycemia, and overweight / obesity. Such related conditions are also characterized by abnormal blood and / or plasma insulin levels.

[0075] The methods described herein can be implemented to treat a subject having a condition associated with β-cell failure or defect. Such conditions include but are not limited to type 1 diabetes (T1D), type 2 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 involves a condition caused by the use of drugs toxic to β-cells (e.g., steroids, antidepressants, second-generation antipsychotics, and immunosuppressants). Exemplary immunosuppressive drugs include but are not limited to members of the cortisone family (e.g., prednisone and dexamethasone), rapamycin / sirolimus, everolimus, and calcineurin inhibitors (e.g., FK-506 / tacrolimus).

[0076] Other conditions associated with β-cell deficiency include but are not limited to hypoglycemia unawareness, brittle insulin-dependent diabetes, pancreatectomy, partial pancreatectomy, pancreatic transplantation, pancreatic islet allotransplantation, pancreatic islet autotransplantation, and pancreatic islet xenotransplantation.

[0077] As used herein, hypoglycemia unawareness is a complication of diabetes in which the patient does not perceive a sharp drop in blood glucose because the sharp drop in blood glucose fails to trigger the secretion of epinephrine, the secretion of which produces the characteristic symptoms of hyperglycemia (e.g., palpitations, sweating, anxiety), the characteristic symptoms being used to warn the patient of the drop in blood glucose.

[0078] Pancreas transplantation can be performed alone, after kidney transplantation, or in combination with kidney transplantation. For example, in patients with severe and potentially life-threatening complications due to asymptomatic hypoglycemia and persistent, unstable insulin-dependent diabetes despite optimal medical management, pancreas transplantation alone may be considered medically necessary. Pancreas transplantation can be performed after kidney transplantation in patients with insulin-dependent diabetes. In patients with uremic insulin-dependent diabetes, pancreas transplantation can be performed in combination with kidney transplantation. After primary pancreas transplantation failure, pancreas re-transplantation may be considered.

[0079] As used herein, islet transplantation is a procedure in which only the pancreatic islets of Langerhans, which contain pancreatic endocrine cells (including insulin-producing β-cells and glucagon-producing α-cells), are isolated and transplanted into a patient. Islet allotransplantation occurs when islets of Langerhans are isolated from one or more human donor pancreases. Islet cells can also be derived from human embryonic stem cells or induced pluripotent stem cells. Islet xenotransplantation occurs when islets of Langerhans are isolated from one or more non-human pancreases (e.g., porcine pancreas or primate pancreas). Islet autotransplantation occurs when islets of Langerhans are isolated from the pancreas of a patient who has undergone pancreatectomy (e.g., for chronic pancreatitis caused by gallstones, drugs, and / or familial genetic causes) and are returned to the same patient by infusion into the portal vein, by laparoscopic omentum, by endoscopic gastric wall, or by subcutaneous infusion through a small incision. Like pancreas transplantation, islet transplantation can be performed alone, after kidney transplantation, or in combination with kidney transplantation. For example, islet transplantation can occur alone to restore asymptomatic hypoglycemia, 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 hereby incorporated by reference in its entirety).

[0080] Islet transplantation can occur in combination with total pancreatectomy. For example, islet transplantation can be performed after total pancreatectomy to prevent or improve surgically induced diabetes by preserving beta cell function (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), which is hereby incorporated by reference in its entirety). Thus, islet transplantation can provide sustained long-term insulin independence.

[0081] In some embodiments, islet transplantation can occur in combination with the administration of immunosuppressive agents. Suitable immunosuppressive agents include, but are not limited to, daclizumab (Zenapax; Roche), low-dose rapamycin (sirolimus), and FK506 (tacrolimus) (Van Belle et al., "Immunosuppression in Islet Transplantation", J. Clin. Invest. 118(5):1625-1628 (2008), which is hereby incorporated by reference in its entirety).

[0082] In some embodiments, islet transplantation occurs in the context of an encapsulation device to protect the transplanted islet cells from the host's autoimmune response while allowing glucose and nutrients to reach the transplanted islet cells.

[0083] The methods described herein can be implemented to regenerate pancreatic β-cells in a patient to enhance pancreas, islet allotransplantation, islet autotransplantation, and islet xenotransplantation. For example, the methods of the present disclosure can be used to prevent or improve surgically induced diabetes by preserving β-cell function, restore asymptomatic hypoglycemia, provide glycemic control, and / or protect the patient from severe hypoglycemic events. Accordingly, other aspects of the present disclosure relate to methods of regenerating pancreatic β-cells in a transplanted patient. Such methods involve administering to the transplanted patient a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an anti-CD3 antibody, wherein the administration is carried out under conditions effective to reverse the loss of β-cell mass and function in the subject to treat the transplanted patient.

[0084] The methods can be implemented to treat a subject at risk of developing type II diabetes. A patient at risk of developing type II diabetes may have prediabetes / metabolic syndrome.

[0085] A patient at risk of developing type II diabetes may have been treated with psychoactive drugs, including but not limited to selective serotonin reuptake inhibitors (SSRI) for depression, obsessive-compulsive disorder (“OCD”), etc.

[0086] The subject can be a mammalian subject, such as a human subject. Suitable human subjects include but are not limited to children, adults, and elderly subjects with β-cell and / or insulin deficiency.

[0087] The subject can also be non-human, such as cows, sheep, pigs, cats, horses, rodents, dogs, rabbits, etc.

[0088] Administering to the subject 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) can increase the number of proliferating pancreatic β-cells in the subject by at least about 4%, 5%, 6%, 7%, 8%, 9%, 10% or more.

[0089] Administering to the subject 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) can increase the number of proliferating pancreatic β-cells in the subject by about 4 - 10% / day, or about 4 - 6% / day, 5 - 7% / day, 6 - 9% / day, or 7 - 10% / day.

[0090] 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., an anti-CD3 antibody) to a subject can increase the number of proliferative pancreatic β-cells in the subject by about 6-10% per day.

[0091] 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., an anti-CD3 antibody) to a subject can increase glucose-stimulated insulin secretion in the pancreatic β-cells of the subject (e.g., as compared to a subject who has not been 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., an anti-CD3 antibody)).

[0092] Administration of a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an anti-CD3 antibody to a subject can be effected by administration of a single composition comprising all three of a DYRK1A inhibitor, a GLP1R agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., an anti-CD3 antibody). Alternatively, administration 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., an anti-CD3 antibody) to a subject can be effected sequentially. For example, an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., an anti-CD3 antibody) can be administered to the subject first and then a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor (or a composition comprising a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor) and then a glucagon-like peptide-1 receptor (GLP1R) agonist (or a composition comprising a glucagon-like peptide-1 receptor (GLP1R) agonist), or after administration of an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., an anti-CD3 antibody), then a glucagon-like peptide-1 receptor (GLP1R) agonist (or a composition thereof) and then a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor (or a composition thereof), or in yet another embodiment, after administration of an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., an anti-CD3 antibody), then a combination therapy of a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor (or a composition comprising a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor) and a glucagon-like peptide-1 receptor (GLP1R) agonist (or a composition comprising a glucagon-like peptide-1 receptor (GLP1R) agonist).

[0093] Administration of any one or a combination of dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitors, glucagon-like peptide-1 receptor (GLP1R) agonists, and immunomodulatory monoclonal antibodies and / or immunosuppressive agents (e.g., anti-CD3 antibodies) can be performed multiple times a day, daily, weekly, twice weekly, monthly, bi-monthly, annually, semi-annually, or at any amount of time therebetween. The immunomodulatory monoclonal antibodies and / or immunosuppressive agents (e.g., anti-CD3 antibodies), DYRK1A inhibitors, and glucagon-like peptide-1 receptor (GLP1R) agonists can be administered at the same or different frequencies of administration. In some embodiments, administration of any one or more of dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitors, glucagon-like peptide-1 receptor (GLP1R) agonists, and immunomodulatory monoclonal antibodies and / or immunosuppressive agents (e.g., anti-CD3 antibodies) is performed acutely or chronically. For example, administration can be performed chronically over a period of 1 year, 2 years, 3 years, 4 years, or longer. In some embodiments, administration is performed infrequently.

[0094] As used herein, the term "treating" means prophylactic treatment, ameliorative treatment, or curative treatment. In other words, a treatment method can be implemented to prevent a subject from developing a condition associated with insufficient insulin secretion or to prevent a subject's condition associated with insufficient insulin secretion from worsening. Alternatively, a treatment method is implemented to improve a subject's condition 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 levels as judged by a qualified healthcare professional).

[0095] In some embodiments, "treating" is implemented to reverse β-cell mass and function loss in a subject with T1D. In some embodiments, "treating" is implemented to prevent the progression of T1D in a subject with recently onset T1D. In other words, the methods of the present disclosure can be implemented to restore the loss of β-cell mass and function in a subject who has lost β-cell mass and function due to a condition associated with insufficient insulin secretion (such as from T1D). In some embodiments, "treating" is implemented to prevent the progression of T1D in a subject at risk of developing T1D.

[0096] The term "treating" means correcting, reducing the rate of change, or alleviating impaired glucose homeostasis in a subject. Glucose levels in the blood fluctuate throughout the day. Blood glucose levels are typically lower in the morning, before the first meal of the day, and rise within a few hours after a meal. Thus, the term "treating" includes controlling a subject's blood glucose level by increasing or decreasing the subject's blood glucose level. This can depend on many factors, including the subject's condition and / or the specific time of day, as blood glucose levels fluctuate throughout the day.

[0097] "Treatment" means a transient or persistent reduction in the blood glucose level in a subject suffering from diabetes or a related disorder. The term "treatment" can also mean an improvement in insulin release in the subject (e.g., by pancreatic beta cells).

[0098] It may be desirable to regulate the blood glucose level in a subject to normalize or adjust the blood or plasma glucose level in a subject having an abnormal level (i.e., a level that is lower or higher than a known reference value, median, or average of a corresponding subject with normal glucose homeostasis). The treatment methods of the present disclosure can be implemented to achieve such an effect.

[0099] In practicing the treatment methods of the present disclosure, administering to a subject 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) can involve administering a pharmaceutical composition comprising a dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor or a glucagon-like peptide-1 receptor (GLP1R) agonist or an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., an anti-CD3 antibody) or all three of them in a therapeutically effective amount, which means an amount of the DYRK1A inhibitor, GLP1R agonist, and immunomodulatory monoclonal antibody and / or immunosuppressant (e.g., an anti-CD3 antibody) that is effective in treating the condition and / or disorder in the subject. Such amounts typically vary depending on many factors within the knowledge of a person of ordinary skill in the art. These factors include, but are not limited to, the general health, age, weight, height, general physical condition, medical history of the particular subject, the particular compound used, the carrier in which the compound is formulated, the route of administration selected for the compound, the length or duration of the treatment, and the nature and severity of the condition being treated.

[0100] Administration typically involves administering a pharmaceutically acceptable dosage form, which means a dosage form of the compounds described herein and includes, for example, tablets, pills, powders, elixirs, syrups, liquid preparations (including suspensions, sprays, inhalation tablets, lozenges, emulsions, solutions, granules, capsules, and suppositories), and liquid preparations for injection (including liposomal preparations). Techniques and formulations are generally found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition, which is hereby incorporated by reference in its entirety.

[0101] In the practice of the treatment methods, DYRK1A inhibitors, GLP1R agonists, and / or anti-CD3 antibodies can be contained in any suitable carrier substance in any appropriate amount. The DYRK1A inhibitors, GLP1R agonists, and anti-CD3 antibodies can be present in an amount of up to 99% by weight of the total weight of the composition. The composition can be provided in dosage forms suitable for oral, parenteral (e.g., intravenous, intramuscular), rectal, dermal, nasal, vaginal, inhalational, transdermal (patch), or ophthalmic administration routes. Accordingly, the composition can be in the form of, for example, tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels (including hydrogels), pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injections, implants, sprays, or aerosols.

[0102] 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 time period after administration.

[0103] Controlled-release formulations include (i) formulations that produce a substantially constant drug concentration in the body over an extended period of time; (ii) formulations that produce a substantially constant drug concentration in the body over an extended period of time after a predetermined lag time; (iii) formulations that maintain the drug action over a predetermined period of time by maintaining a relatively constant and effective drug level in the body while minimizing the undesirable side effects associated with fluctuations in the plasma level of the active drug substance; (iv) formulations that localize the drug action by, for example, spatially placing the controlled-release composition adjacent to or within the diseased tissue or organ; (v) formulations that target the drug action by delivering the drug to specific target cell types using a carrier or chemical derivative.

[0104] Administration of DYRK1A inhibitors, GLP1R agonists, and immunomodulatory monoclonal antibodies and / or immunosuppressants (e.g., anti-CD3 antibody) in the form of controlled-release formulations may be preferred in cases where the drug has (i) a narrow therapeutic index (i.e., a small difference between the plasma concentration that causes harmful side effects or toxic reactions and the plasma concentration that causes a therapeutic effect; 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 such that frequent dosing is required during the day to maintain the plasma level at a therapeutic level.

[0105] DYRK1A inhibitors, GLP1R agonists, and immunomodulatory monoclonal antibodies and / or immunosuppressants (e.g., anti-CD3 antibodies) can be used enterally or parenterally. The agent to be administered can be orally administered in an amount of about 0.1 mg / day to 1,000 mg / day. For parenteral, sublingual, intranasal, or intrathecal administration, the compounds according to the present disclosure can be used in an amount of about 0.5 mg / day to about 100 mg / day; for depot administration and implants, the compounds according to the present disclosure can be used in an amount of about 0.5 mg / day to about 50 mg / day; for topical administration, the compounds according to the present disclosure can be used in an amount of about 0.5 mg / day to about 200 mg / day; for rectal administration, the compounds according to the present disclosure can be used in an amount of about 0.5 mg to about 500 mg. In some embodiments, for oral administration, the therapeutically effective amount is about 1 mg / day to about 100 mg / day; and for parenteral administration, the therapeutically effective amount is about 5 mg to about 50 mg per day. In some embodiments, the therapeutically effective amount for oral administration is about 5 mg / day to about 50 mg / day.

[0106] The daily dose of the active ingredient can be expected to be about 0.001 to about 1,000 milligrams per kilogram of body weight, with a preferred dose being about 0.1 to about 30 mg / kg. The daily oral dose of the compound can vary from about 0.01 mg to 1,000 mg, 0.1 mg to 100 mg, or 10 mg to 500 mg. The daily dose can be administered as a single dose or divided doses, and additionally, when indicated, the upper limit can also be exceeded.

[0107] Any one of a variety of strategies can be employed to achieve controlled release, where the release rate exceeds the metabolic rate of the therapeutic agent under discussion. Controlled release can be obtained by appropriately selecting various formulation parameters and ingredients, including, for example, various types of controlled release compositions and coatings. Thus, the drug is formulated into a pharmaceutical composition together with a suitable excipient, and the pharmaceutical composition releases the drug in a controlled manner upon administration (tablet or capsule compositions in single or multiple units, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes). Thus, administration can be carried out via the nose, orally, topically, transdermally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by intracavitary or intravesical instillation, intravitreally, intraarterially, intralesionally, or by application to mucous membranes. The compounds can be administered alone or together with a suitable pharmaceutical carrier and can be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions. In certain embodiments, administration is carried out via the nose, orally, transdermally, parenterally, subcutaneously, intravenously, intramuscularly, or intraperitoneally.

[0108] In some embodiments, administration is performed using an infusion pump to provide, for example, rate-controlled infusion, periodic infusion, and / or bolus dose infusion. The infusion pump can be a stationary infusion pump or a mobile infusion pump. Stationary infusion pumps are mainly used at the patient's bedside. Mobile infusion pumps are relatively small, at least substantially self-contained devices that are used to introduce drugs and other infusible substances (e.g., insulin) into a selected subject. Some mobile infusion pumps are configured to be worn on a belt, carried in a clothing pocket, or otherwise supported within some holder (collectively referred to as "pocket pumps"). Other infusion pumps are configured to adhere to the skin in a patch-like manner (referred to as "patch pumps"). Infusion pumps can be used to intravenously or subcutaneously introduce (or "infuse") drugs on a continuous or even continuous basis outside of a clinical setting, for example. Infusion pumps greatly reduce the frequency of subcutaneous access events such as needle-based injections. In some embodiments, the infusion pump is a subcutaneous or intravenous infusion pump. For example, the infusion pump can be a mobile subcutaneous insulin infusion pump.

[0109] Another aspect of the present disclosure 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).

[0110] Suitable DYRK1A inhibitors are as described above and include, for example, harmine, INDY, leucettines-41, 5-iodotubercidin (5-IT), GNF4877, CC-401, kinase inhibitors, and derivatives thereof.

[0111] Suitable GLP1R agonists are as described above and include, for example, exendin-4, liraglutide, lixisenatide, semaglutide, and derivatives thereof.

[0112] Suitable immunomodulatory monoclonal antibodies have been described in detail above. Suitable anti-CD3 antibodies are as described above and include, for example, tilizumab.

[0113] Compositions comprising a dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunosuppressant are also contemplated. Suitable immunosuppressants have been described in detail above.

[0114] The composition can further comprise a carrier. Suitable carriers are as described above. The carrier can be a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers are as described above.

[0115] Another aspect of the present disclosure relates to a method of increasing β-cell mass and function in a pancreatic β-cell population. The method involves 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, wherein the contacting is carried out under conditions effective to increase β-cell mass and function in the pancreatic β-cell population.

[0116] In practicing this method and other methods described herein, the pancreatic β-cells can be mammalian cells. Mammalian cells include cells from, for example, mice, hamsters, rats, cows, sheep, pigs, goats, horses, monkeys, dogs (e.g., domestic dog (Canis familiaris)), cats, rabbits, guinea pigs, and primates (including humans). For example, the cells can be human pancreatic β-cells.

[0117] In some embodiments, the "pancreatic β-cells" are primary human pancreatic β-cells.

[0118] In some embodiments, this method and other methods described herein are carried out ex vivo or in vivo. When carried out ex vivo, a cell population can be provided by obtaining cells from a 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, suitable and non-limiting media can be based on commercially available media such as RPMI 1640 from Invitrogen.

[0119] 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 increases or is induced. Other methods include testing whether β-cell specific transcription factors are expressed, detecting β-cell specific gene products by RNA quantitative PCR, transplanting candidate cells into diabetic mice and subsequently testing the physiological response after the transplantation, and analyzing the cells by electron microscopy.

[0120] In practicing the methods described herein, a pancreatic β-cell population 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.

[0121] In some embodiments, contacting the 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 anti-CD3 antibody is carried out with harmine, exendin-4, and teplizumab.

[0122] 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., an anti-CD3 antibody) can be carried out with a single composition comprising each of a DYRK1A inhibitor, a GLP1R agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., an anti-CD3 antibody). Alternatively, 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., an anti-CD3 antibody) can be carried out sequentially. For example, a pancreatic β cell population can first be contacted with an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., an anti-CD3 antibody) and then with a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor (or a composition comprising a dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) inhibitor) and a glucagon-like peptide-1 receptor (GLP1R) agonist (or a composition comprising a glucagon-like peptide-1 receptor (GLP1R) agonist) (together or separately).

[0123] In practicing the methods described herein, 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., an anti-CD3 antibody) can be carried out multiple times a day, daily, weekly, twice a week, monthly, bi-monthly, annually, semi-annually, or any amount of time in between. The DYRK1A inhibitor, the glucagon-like peptide-1 receptor (GLP1R) agonist, and the immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., an anti-CD3 antibody) can be administered at different frequencies of administration. Contacting a pancreatic β cell population with a DYRK1A inhibitor, a GLP1R agonist, and an immunomodulatory monoclonal antibody and / or an immunosuppressant (e.g., an anti-CD3 antibody) can be carried out acutely or chronically. For example, the contact can be carried out chronically over a period of 1 year, 2 years, 3 years, 4 years, or longer. In some embodiments, the administration is carried out infrequently.

[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) increases β-cell function and / or increases insulin sensitivity in the population 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 treatment-induced β-cell function (such as β-cell proliferation) are well known in the art and include, for example, hyperglycemic clamp, intravenous glucose tolerance test (IVGTT), graded glucose infusion, glucose-potentiated 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), which is hereby incorporated by reference in its entirety). Methods for measuring changes in treatment-induced insulin sensitivity 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), which is hereby incorporated by reference in its entirety).

[0125] 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) increases the number of proliferative pancreatic β-cells in the population 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.

[0126] 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 an immunosuppressant (e.g., an anti-CD3 antibody) increases the human β-cell mass by at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, 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. In some embodiments, contacting a pancreatic β-cell population (e.g., a human 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., an anti-CD3 antibody) increases β-cell survival compared to when the pancreatic β-cell population is not contacted.

[0127] 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 an immunosuppressant (e.g., an anti-CD3 antibody) increases the transdifferentiation of non-β-cells (e.g., α-cells, δ-cells, PP-cells, and / or duct cells) into β-cells compared to when the pancreatic β-cell population is not contacted.

[0128] 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 an immunosuppressant (e.g., an anti-CD3 antibody) increases the number of proliferative pancreatic β-cells in the population by about 4 - 10% / day, or about 4 - 6% / day, 5 - 7% / day, 6 - 9% / day, or about 7 - 10% / day.

[0129] 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 an immunosuppressant (e.g., an anti-CD3 antibody) increases the number of proliferative pancreatic β-cells in the population by about 6 - 10% / day.

[0130] When compared to contact with a dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, or an immunomodulatory monoclonal antibody and / or immunosuppressant (e.g., anti-CD3 antibody), the method of contacting a pancreatic β-cell population with a DYRK1A inhibitor, a GLP1R agonist, and an immunomodulatory monoclonal antibody and / or immunosuppressant (e.g., anti-CD3 antibody) can be carried out under conditions that are effective to cause a synergistic increase in cell proliferation in the pancreatic β-cell population, which particularly means an increase in the number of proliferative pancreatic β-cells in the population.

[0131] 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. Additionally, the contact can induce β-cell differentiation and increase glucose-stimulated insulin secretion.

[0132] The method can also be carried out to enhance cell survival in a pancreatic β-cell population (in addition to reversing and / or restoring β-cell mass and function). For example, the method can be carried out to enhance cell survival of a treated pancreatic β-cell population relative to an untreated pancreatic β-cell population. Alternatively, the method can be carried out to reduce cell death or apoptosis of a contacted pancreatic β-cell population relative to a non-contacted pancreatic β-cell population.

[0133] Examples

[0134] Example 1 - Materials and Methods

[0135] In vitro analysis of human β-cell death

[0136] As previously described for dispersed human islet cells (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, after washing human islets twice with PBS, 200 μl of pre-warmed Accutase (Corning) was added and the tube was incubated at 37°C for 10 minutes. Then, complete RPMI medium (RPMI containing 5 mM D-glucose, 10% FBS, 100 units / ml penicillin, and 100 μg / ml streptomycin) was added, the sample was centrifuged at 1000 rpm for three minutes, the pellet was washed with PBS and centrifuged again at 1000 rpm for three minutes. The pellet was resuspended in complete RPMI medium and 50,000 cells / well were plated onto 12 mm glass coverslips placed in 24-well plates and incubated at 37°C and 5% CO2 for 24 hours.Next, the following were added to the wells: saline, cytokines (50 units / mL IL-1β, 1,000 units / mL TNF-α, and 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), and 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). The cells were incubated at 37 °C and 5% CO2 for 24 h. Then, the cells were fixed in 4% paraformaldehyde and TUNEL (cell death marker), and insulin staining was performed using the DeadEnd Fluorescent TUNEL System (Promega), anti-proinsulin / C-peptide antibody (DSHB), and DAPI for nuclear detection. A minimum of 1,500 β-cells were examined per coverslip.

[0137] Single-cell RNA sequencing (scRNA-seq) of human islets

[0138] Human islets treated with cytokines and harmine (H), exendin-4 (E), and harmine + exendin-4 (E+H) (as described above) for 6 hours were subjected to Sc-RNAseq analysis at 37 °C and 5% CO2. After treatment, the human islets were washed twice with PBS and centrifuged at 300 rpm for three minutes. After removing the 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, the samples were centrifuged at 1000 rpm for three minutes, and the pellet was washed with PBS. The cells were then resuspended in binding buffer (Miltenyi Biotec) and dead cell removal beads, the tube was incubated at room temperature for 15 minutes, and the cell suspension was applied to a dead cell removal column (Miltenyi Biotec), which was attached to a MACS separator. Subsequently, the centrifuged effluent was collected and resuspended in PBS containing 200 μl of 2% BSA and 200 U / ml of RNase inhibitor, the cells were mixed with AOPI (Nexcelon Bioscience) at a 1:1 ratio, and the cell concentration was checked in a Countess 3 automated cell counter (Thermo-Fisher).

[0139] Cell samples were prepared according to the user guide of the 10X Genomics Single Cell 3' V3.1 kit, processed with a 10X Genomics Chromium Controller for partitioning and barcoding, and then a cDNA library was generated. The total cell concentration analyzed by the Countess 3 was then sequenced by the NovaSeq 6000 system (Illumina). The FASTQ files were downloaded from the sequencing facility and aligned using Cell Ranger V.6.1.1 with the Single Cell 3' V3 chemistry on the 10X Cloud pipeline. After generating the 10X, h5 format files, the data was analyzed using the Seurat software package V.4.0 on the R language platform. After creating the scRNA-seq data, ambient mRNA adjustment was performed using SoupX (20% contamination estimate). Cells with less than 500 gene counts, less than 250 gene variances, less than 0.8 log 10 genes / UMI, and a mitochondrial gene ratio greater than 20% were filtered out. Then, doublets were algorithmically removed using the DoubletFinder software package (20% estimate).

[0140] 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. Then, cell type identities were assigned based on the normalized gene expression levels with reference to canonical pancreatic cell type genes. To discover changes in apoptotic and pro-inflammatory pathways in different treatments, single-cell level gene set enrichment analysis was performed in the β cell population using the escape package that accesses the entire Molecular Signatures Database (v.7.0). Whole C2 library enrichment was performed by chemical and genetic perturbations and canonical pathways containing four well-known databases (Biocarta, KEGG, Reactome, and Wikipathways). After calculating the enrichment score for each single cell, the enrichment scores were added to the metadata for analysis and visualization by dot plots.

[0141] Treat early-onset type 1 diabetes (T1D) non-obese diabetic (NOD) mice in vivo with anti-CD3 and harmine plus exendin-4 (H+E).

[0142] Twelve- to sixteen-week-old NOD / LtJ (NOD) female mice (The Jackson Laboratory) were housed under specific pathogen-free conditions. Non-fasting blood glucose was measured once a week using a portable glucometer (AlphaTRAK 2; Abbott Laboratories); mice were considered diabetic when blood glucose >250 mg / dL in three consecutive measurements over three consecutive days (Lu et al., "Dextran Sulfate Protects Pancreatic β-Cells, Reduces Autoimmunity, and Improves Type 1 Diabetes," Diabetes 69:1692-1707 (2020)). Then, early-onset diabetic mice were injected intravenously once a day for 3 days with 5 μg of IgG or anti-CD3 antibody (non-Fc-binding monoclonal anti-CD3ε F(ab')2 obtained from Bio X Cell, https: / / bxcell.com / product / m-CD3e-fab2-fragments / ). After the third injection, a minipump was implanted in the interscapular region of the mice for continuous delivery of harmine, exendin-4, or harmine plus exendin-4 (Rosselot et al., "In Vivo Human β-Cell Mass Expansion with a Combination of Harmine and Exendin-4: Quantification and Visualization by iDISCO+ 3D Imaging," biorxiv (2021), which is hereby incorporated by reference in its entirety). Briefly, harmine and exendin-4 were dissolved in water and loaded onto Alzet (Cupertino, CA) model 1004 miniosmotic pumps at concentrations of 27 mg / ml and 1 mg / ml, respectively, to allow subcutaneous delivery of harmine and exendin-4 at continuous rates of 3 mg / kg / day and 0.1 mg / kg / day, respectively, for one month. For the two-month treatment, the pump was replaced with a new pump and fresh harmine and exendin-4 at 28 days. Control pumps contained water. After pump implantation, non-fasting blood glucose was measured weekly as described above, and the percentage of diabetic mice was calculated. Animal studies were conducted with the approval of the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee and in accordance with the guidelines established by it.

[0143] Immunophenotypic analysis of splenocytes from non-obese diabetic (NOD) mice treated with anti-CD3 and harmine and exendin-4 (H+E)

[0144] At the end of the eight-week treatment, spleens were harvested, ground, and made into cell suspensions after lysing red blood cells and filtering (Lu et al., "Dextran sulfate protects pancreatic β-cells, reduces autoimmunity, and improves 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 soluble anti-CD3 and 2 μg / mL soluble anti-CD28 (BioLegend) for 16 hours. Surface and intracellular staining of T cells for flow cytometry was achieved 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 ZombieNIR Fixable Viability Kit (BioLegend). Cells were analyzed in an Attune Nxt flow cytometer (Thermo Fisher Scientific).

[0145] Histomorphometric analysis of pancreas obtained from non-obese diabetic (NOD) mice treated with anti-CD3 and H+E

[0146] At the end of the eight-week treatment, pancreases were harvested and fixed overnight in neutral-buffered formalin at room temperature. The pancreases were then paraffin-embedded and sectioned, and β-cell mass in three non-consecutive insulin and hematoxylin-stained sections per mouse was measured using ImageJ (National Institutes of Health). Sections were also stained for Ki67 (Thermo Fisher Scientific) 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 assessment 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 improves type 1 diabetes", Diabetes 69:1692-1707 (2020), which is hereby incorporated by reference in its entirety).

[0147] Statistical analysis

[0148] Data presented as bar graphs, scatter plots, and dot plots show mean ± SEM. Statistical significance analysis was performed using one-way ANOVA (Tukey's post hoc test) or Student's t-test, as appropriate, for comparison between groups. P < 0.05 was considered statistically significant.

[0149] Example 2 - Protection of Human β-Cells from Inducers of Cell Death In Vitro by the Combination of Harmine and Exendin-4

[0150] Proinflammatory cytokines and ER stress are established inducers of β-cell death in type 1 diabetes (T1D) (Lu et al., "Dextran Sulfate Protects Pancreatic β-Cells, Reduces Autoimmunity, and Improves Type 1 Diabetes," Diabetes 69:1692 - 1707 (2020), which is hereby incorporated by reference in its entirety). Thus, the first question posed was whether harmine (H), exendin-4 (E), or the combination (H + E) might have a protective effect on human β-cells when treated in vitro with proinflammatory cytokines or the ER stress inducer thapsigargin. As Figures 1A - 1C shown, 10 μM harmine (H) and 10 nM exendin-4 (E) together significantly reduced cytokine-( Figures 1A - 1B ) or 500 nM thapsigargin-( Figure 1C )-induced human β-cell death compared to treatment with only 10 μM harmine or 10 nM exendin-4, which provided only a non-significant partial protection against cell death.

[0151] Next, single-cell RNA sequencing (scRNA-seq) analysis was performed on human islets treated with cytokines and harmine (H) and / or exendin-4 (E) as described above. It was found that proinflammatory-( Figure 1D ), intrinsic and extrinsic apoptosis-( Figure 1E ), human leukocyte antigen (HLA) class I molecule-( Figure 1F ), chemokine CXCL9 - 11-( Figure 1G ), and interferon regulatory factor 1 - 9-( Figure 1H ) signaling pathways were upregulated in β-cells treated with cytokines, and treatment with the combination of harmine + exendin-4 (H + E) significantly downregulated the expression of these pathways to near-normal levels-( Figures 1D - 1H ). Thus, treatment with harmine + exendin-4 (H + E) appears to provide protection for β-cells in the T1D environment.

[0152] Example 3 - Complete reversal of early-onset type 1 diabetes (T1D) in non-obese diabetic (NOD) mice by consecutive administration of harmine + exendin-4 after transient treatment with anti-CD3

[0153] Based on the pro-survival and regenerative effects of harmine + exendin-4 in human β cells ( Figures 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., “In Vivo Human β Cell Mass Expansion with a Combination of Harmine and Exendin-4: Quantification and Visualization by iDISCO+ 3D Imaging,” biorxiv (2021), which are hereby incorporated by reference in their entirety), and 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: Identification of Responding Subgroups by Metabolic and Immunological Characteristics at Baseline,” Diabetes 62:3766-74 (2013); Herold et al., “Anti-CD3 Monoclonal Antibodies in New-Onset Type 1 Diabetes,” N. Engl. J. Med. 346:1692-8 (2002); Keymeulen et al., “Insulin Requirements after CD3 Antibody Therapy in New-Onset Type 1 Diabetes,” N. Engl. J. Med. 352:2598-608 (2005); Sherry et al., “Teplizumab (Protégé Study) for the Treatment of Type 1 Diabetes: 1-Year Results from a Randomized Placebo-Controlled Trial,” Lancet 378:487-97 (2011); Hagopian et al., “Teplizumab Preserves C-Peptide in Recently Onset Type 1 Diabetes: Two-Year Results from the Randomized Placebo-Controlled Protégé Trial,” Diabetes 62:3901-8 (2013); and Herold et al., “The Anti-CD3 Antibody Teplizumab in Relatives at Risk for Type 1 Diabetes,” N. Engl. J. Med. 381:603-613 (2019), which are hereby incorporated by reference in their entirety), it was tested whether the combination of harmine + exendin-4 (H+E) with anti-CD3 could reverse diabetes in early-onset diabetic NOD mice.

[0154] Mice spontaneously developed diabetes at 12 - 16 weeks of age (blood glucose > 250 mg / dl for three consecutive measurements), at which time the mice were injected intravenously once daily with 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 / ) for 3 consecutive days. Notably, this non - FcR - binding monoclonal anti - CD3 induces apoptosis of antigen - activated T cells in vivo by allowing persistent expression of the TCR and continuous signaling. However, importantly, it has been shown that Foxp3+ Tregs are resistant to CD3 antibody - mediated depletion. After the third injection, an Alzet minipump was implanted for continuous delivery of harmine, exendin - 4, harmine and exendin - 4, or water for four weeks.

[0155] After four weeks, the minipump was replaced with a new minipump (Rosselot et al., “In Vivo Human β - Cell Mass Expansion with a Combination of Harmine and Exendin - 4: Quantification and Visualization by iDISCO+ 3D Imaging”, biorxiv (2021), which is hereby incorporated by reference in its entirety), for a second four - week period. As Figure 2C shown, until two months after the intervention, the harmine + exendin - 4 treatment had reduced blood glucose levels to below 250 mg / dl, while diabetic mice treated with vehicle remained diabetic during the eight - week follow - up period. Notably, 95% of the mice treated with anti - CD3 and H+E maintained normal blood glucose from the second to the eighth week ( Figure 2F ). In contrast, only 40% of the mice treated with anti - CD3 and vehicle maintained normal blood glucose at the eighth week after the start of treatment ( Figure 2F ). Notably, during the eight - week follow - up period, treating mice with 5 μg anti - CD3 / mouse / day for three days followed by 3 mg / kg / day harmine or 0.1 mg / kg / day exendin - 4 did not reduce blood glucose levels to below 250 mg / dl ( Figure 2B ); from the 3rd to the 8th week of the eight - week follow - up period, 70% of the mice treated with anti - CD3 for three days followed by harmine remained diabetic ( Figure 2E ); and from the 4th to the 7th week of the eight - week follow - up period, 40% of the mice treated with anti - CD3 for three days followed by exendin - 4 remained diabetic, and 60% of the mice remained diabetic at the 8th week of the eight - week follow - up period ( Figure 2E)。When mice were treated with 5 μg IgG / mouse / day for three days followed by (i) 3 mg / kg / day harmine and 0.1 mg / kg / day exendin-4 or (ii) vehicle (H2O) for eight weeks ( Figure 2A , Figure 2D ), no significant differences in blood glucose levels were observed, where 70 - 100% of the mice remained diabetic in both groups during the eight-week follow-up period.

[0156] Example 4 - Immunophenotypic analysis of splenocytes in early-onset type 1 diabetes (T1D) non-obese diabetic (NOD) mice treated with anti-CD3 and H+E

[0157] In an ongoing study, analysis of immune cell populations in the blood, spleen, and pancreatic lymph nodes of early-onset T1D NOD mice treated with anti-CD3 and harmine + exendin-4 (H+E) has been initiated. Analysis of splenocytes at the end of the study (week 8) showed that the total number of immune cells (CD45 + , naive, memory, and effector CD4 + and CD8 + T lymphocytes) did not change significantly in either treatment group (Figures 3A - 3C). Analysis of specific immune cell populations revealed that treatment with harmine + exendin-4 (H+E) significantly reduced the number of activated CD4 + and CD8 + T cells (Th1) (Figures 3D - 3E) and significantly increased the number of FoxP3 + CD25 + regulatory T cells (Treg) by more than 50% (Figures 3F - 3G). This indicates induction of immune tolerance (reduced T cell activation and enhanced regulatory T cells) after treatment with the combination of anti-CD3 and harmine + exendin-4 (H+E). In a study in which NOD mice were treated with anti-CD3 / day for three days and 3 mg / kg / day harmine (H) and 0.1 mg / kg / day exendin-4 (E) or vehicle (H2O) for eight weeks, circulating TNFα levels (Figure 3H) and CXCR3 + CD8 + (Figure 3I, top) and CXCR3 + CD4 + cells (Figure 3I, bottom) were lower in mice treated with harmine + exendin-4 (H+E) than in those treated with vehicle, as were circulating TNFα levels and CXCR3 + CD8 + and CXCR3 + CD4 +cells, and compared to NOD mice treated with anti-CD3 for three days and then with vehicle (water) for 2 weeks, CD4 in NOD mice treated with anti-CD3 for three days and then with harmine + exendin-4 (H+E) for 2 weeks + and CD8 + cells had higher T cell exhaustion markers PD1, TIGIT, TOX, and EOMES ( Figure 3J ).

[0158] Example 5 - Analysis of pancreas from NOD mice treated with anti-CD3 and H+E

[0159] Pancreas sections obtained eight weeks after treatment with anti-CD3 and harmine + exendin-4 (H+E) or vehicle ( Figures 4A - 4B ) were stained with hematoxylin and eosin. It was evident that islets from vehicle-treated mice contained the expected insulitis, and this expected insulitis was reduced in mice treated with anti-CD3 and harmine + exendin-4 (H+E). Isletitis scores of islets in these pancreases revealed that compared to vehicle-treated mice, mice treated with anti-CD3 and harmine + exendin-4 (H+E) had more islets with scores of 0 to 2 (no insulitis to mild insulitis), while the number of islets with strong and severe insulitis (score 3 - 4) was less ( Figure 4B ). Thus, anti-CD3 + harmine + exendin-4 (H+E) treatment alleviated islet inflammation in NOD diabetic mice. Flow cytometry analysis of islets from treated mice showed that CD45 + cells (immune cells) were fewer in islets of mice treated with anti-CD3 + harmine + exendin-4 (H+E) compared to vehicle-treated mice ( Figure 4C ).

[0160] Next, β-cell proliferation, β-cell death, and β-cell mass in these pancreases were analyzed. As Figures 4D - 4E shown, Ki67 + / insulin + cells were significantly increased in mice treated with anti-CD3 and harmine + exendin-4 (H+E) compared to vehicle-treated mice, while TUNEL + / insulin + cells were significantly decreased, indicating increased β-cell proliferation and decreased β-cell death. Additionally, analysis of the total β-cell mass in these pancreases showed that anti-CD3 and harmine + exendin-4 (H+E) treatment doubled the number of β-cells compared to vehicle-treated animals.

[0161] Conclusion

[0162] Overall, the studies described in these examples demonstrate for the first time that transient immunomodulatory treatment with anti-CD3 followed by treatment with the harmine + exendin-4 (H+E) combination increases immune tolerance, enhances β-cell proliferation, protects β-cells and increases β-cell mass, and these effects together lead to reversal of early-onset T1D in NOD mice.

[0163] Although the preferred embodiments have been described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, etc. can be made without departing from the spirit of the invention, and thus these are considered to be within the scope of the invention as defined in the following claims.

Claims

1. A method of treating a condition in a subject associated with insufficient insulin secretion, the method comprising: administering to a subject in need of treatment for a condition associated with insufficient insulin secretion level a dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, a glucagon-like peptide-1 receptor (GLP1R) agonist, and an immunomodulatory monoclonal antibody, optionally wherein the immunomodulatory monoclonal antibody is an anti-CD3 antibody; wherein the administering is carried out under conditions effective to reverse the loss of β-cell mass and function in the subject to treat the condition in the subject associated with insufficient insulin secretion.

2. The method according to claim 1, wherein treating one or more of the following in the subject: 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.

3. The method according to claim 2, wherein treating type I diabetes in the subject.

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

5. The method according to any one of claims 1 to 3, wherein the subject has recently-onset type 1 diabetes.

6. The method according to any one of claims 1 to 5, wherein the administering increases immune tolerance in the subject, enhances β-cell proliferation in the subject, protects β-cells in the subject, increases β-cell mass in the subject, and combinations thereof.

7. The method according to any one of claims 1 to 6, wherein the DYRK1A inhibitor is harmine.

8. The method according to any one of claims 1 to 7, wherein the GLP1R agonist is exendin-4.

9. The method according to any one of claims 1 to 8, wherein the anti-CD3 antibody is teplizumab.

10. The method according to any one of claims 1 to 9, wherein the administering is carried out with harmine, exendin-4, and teplizumab.

11. The method according to any one of claims 1 to 10, wherein the administering is carried out sequentially with each of the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitor, the glucagon-like peptide-1 receptor (GLP1R) agonist, and the anti-CD3 antibody.

12. The method according to any one of claims 1 to 11, wherein the administering is carried out by first administering the anti-CD3 antibody.

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

14. The method according to any one of claims 1 to 13, wherein the anti-CD3 antibody is administered at a low dose.

15. The method according to any one of claims 1 to 14, wherein the administration is by nasal, oral, transdermal, parenteral, subcutaneous, intravenous, intramuscular or intraperitoneal routes.

16. The method according to any one of claims 1 to 15, wherein the subject is a mammalian subject.

17. The method according to any one of claims 1 to 16, wherein the subject is a human subject.

18. 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, optionally wherein the immunomodulatory monoclonal antibody is an anti-CD3 antibody.

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

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

21. The composition according to any one of claims 18 to 20, wherein the DYRK1A inhibitor is harmine.

22. The composition according to any one of claims 18 to 21, wherein the GLP1R agonist is exendin-4.

23. The method according to any one of claims 18 to 22, wherein the anti-CD3 antibody is tilizumab.

24. A method of increasing β-cell mass and function in a pancreatic β-cell population, the method comprising: 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, optionally wherein the immunomodulatory monoclonal antibody is an anti-CD3 antibody, wherein the contact is carried out under conditions effective to increase β-cell mass and function in the pancreatic β-cell population.

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 carried out 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 any one of claims 24 to 27, wherein the GLP1R agonist is exendin-4.

29. The method according to any one of claims 24 to 28, wherein the anti-CD3 antibody is tilizumab.

30. The method according to any one of claims 24 to 29, wherein the pancreatic β-cells are primary human pancreatic β-cells.

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