Treatment of type 1 diabetes (T1D) with tolerance-enhancing nanoparticles

Abstract

Type 1 diabetes (T1D) places a very heavy burden on the body, and unfortunately, there is no treatment for T1D. [Solution] This disclosure relates to a method for treating type 1 diabetes (T1D) using tolerance-modifying immunomodulatory nanoparticles encapsulating T1D-related antigens.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 504,271, filed on 25 May 2023, and U.S. Provisional Patent Application No. 63 / 566,571, filed on 18 March 2024, each of which is incorporated herein by reference in its entirety.

[0002] Integration by referencing electronically submitted documents The entirety of which is incorporated by reference is a computer-readable nucleotide / amino acid sequence listing submitted concurrently with this specification and identified below as: filename: 59248_seqlisting.xml, size: 23,766 bytes, creation date: May 24, 2024.

[0003] This disclosure relates to a method for treating type 1 diabetes (T1D) using tolerance-modifying immunomodulatory nanoparticles encapsulating T1D-related antigens. [Background technology]

[0004] Type 1 diabetes mellitus (T1D), also known as type 1 diabetes mellitus (T1DM), is an autoimmune disease characterized by T and B cell responses to proteins expressed due to insulin-secreting pancreatic β-cells, inflammatory lesions within the pancreatic islets (insular pancreatitis), and β-cell loss.

[0005] T1D affects approximately 1.6 million people in the United States and approximately 4.7 million people worldwide. 1 The incidence of T1D is highest in children, but T1D can occur at any age. There appears to be no gender difference in the overall incidence of childhood diabetes; the male-female ratio seems to be the same. 2 .

[0006] Autoimmune activity in T1D primarily targets β-cell proteins. Inflammatory lesions within the pancreatic islets result in patients who cannot effectively stabilize proper glucose metabolism.

[0007] The burden of T1D is very high, and there is currently no cure for T1D. The current standard of treatment for T1D relies on exogenous insulin replacement to manage blood glucose levels in combination with diet and exercise modifications. The newly approved drug teplizumab can delay the onset of T1D by an average of two years, but it does not address the underlying cause of T1D, namely CD8 + T cells, CD4 + It does not cure or treat activated autoimmune T cells that mediate T cell-mediated inflammation or persistent pancreatic β-cell destruction through autoantibodies. [Overview of the project]

[0008] Tolerant immunomodulatory particles (TIMPs) containing one or more antigens have been previously described for the treatment of immune-mediated disorders (e.g., autoimmune diseases and allergies) through the induction of antigen-specific immune tolerance (WO2013 / 192532 and WO2015 / 023796, incorporated herein by reference). In several preclinical models of autoimmune diseases and allergies, TIMPs have demonstrated efficacy in inducing T-cell tolerance. Induction of antigen-specific tolerance to T1D autoantigens using TIMPs encapsulating T1D-related antigens (TIMP-T1D) can improve, or potentially cure, T1D.

[0009] Provided herein are methods for treating type 1 diabetes mellitus (T1D) in subjects requiring treatment for T1D, comprising administering TIMP-T1D to the subject, wherein TIMP-T1D is administered in a dose determined based on the subject's body weight. In various embodiments, TIMP-T1D is administered in doses of 0.01 to 12 mg / kg (including all values ​​and ranges between these values). In various embodiments, TIMP-T1D is administered in fixed doses of 1 mg to 800 mg (including all values ​​and ranges between these values). In various embodiments, the disclosure provides herein are methods for treating type 1 diabetes mellitus (T1D) in subjects, comprising administering tolerant immunomodulatory particles (TIMP-T1D) encapsulating one or more T1D-related antigens, wherein TIMP-T1D is administered at dose levels of 1 mg to 800 mg. In various embodiments, TIMP-T1D is administered in doses of approximately 0.01 to 12 mg / kg, approximately 0.05 to 10 mg / kg, approximately 0.01 to 5 mg / kg, approximately 0.1 to 10 mg / kg, approximately 1 to 8 mg / kg, approximately 1.5 to 10 mg / kg, approximately 2 to 12 mg / kg, approximately 2 to 10 mg / kg, approximately 3 to 10 mg / kg, approximately 4 to 10 mg / kg, approximately 4 to 12 mg / kg, or approximately 5 to 12 mg / kg. In various embodiments, TIMP-T1D is administered in doses of approximately 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5 mg / kg, 6 mg / kg, 8.0 mg / kg, 10 mg / kg, or 12 mg / kg. In various embodiments, TIMP-T1D is administered in fixed doses of approximately 1 mg, 2 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, or 800 mg.

[0010] In various embodiments, the method further includes administering at least one booster dose of TIMP-T1D. In various embodiments, the booster dose of TIMP-T1D is administered as a single dose or in multiple doses after a loading dose of TIMP-T1D. In various embodiments, the booster dose is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every four months, once every five months, once every six months, or once per year. In various embodiments, TIMP-T1D is administered as two loading doses one week apart, followed by at least one booster dose administered as a single dose once a month, once every two months, or once every three months. In various embodiments, TIMP-T1D is administered in two loading doses at one-week intervals, followed by at least one booster dose as a single dose every month, every two or three months, every four months, every five months, every six months, or once a year. In various embodiments, TIMP-T1D is administered in two loading doses at one-week intervals, followed by a booster dose as a single dose every three months. In various embodiments, one, two, three, four, or five booster doses are administered to the subject.

[0011] In various embodiments, the booster dose of TIMP-T1D is administered in doses of 0.01 to 12 mg / kg (including all values ​​and ranges between these values). In various embodiments, the booster dose of TIMP-T1D is approximately 0.01 to 12 mg / kg, approximately 0.05 to 10 mg / kg, approximately 0.01 to 5 mg / kg, approximately 0.1 to 10 mg / kg, approximately 1 to 8 mg / kg, approximately 1.5 to 10 mg / kg, approximately 2 to 12 mg / kg, approximately 2 to 10 mg / kg, approximately 3 to 10 mg / kg, approximately 4 to 10 mg / kg, approximately 4 to 12 mg / kg It is administered in doses of g, or approximately 5 to 12 mg / kg, or approximately 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5 mg / kg, 6 mg / kg, 8.0 mg / kg, 10 mg / kg, or 12 mg / kg. In various embodiments, the booster dose of TIMP-T1D is administered in doses of approximately 1 mg, 2 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, or 800 mg.

[0012] In various embodiments, subjects requiring it have been diagnosed with T1D within 1 week to 7 years prior to administration of TIMP-T1D. In various embodiments, subjects requiring it have been diagnosed within 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, or 7 years (including all ranges and values ​​within that range).

[0013] In various embodiments, the subject requiring it has recently developed T1D. In various embodiments, the subject requiring it has stage 3 T1D. In various embodiments, the subject requiring it has ≥1 autoantibody specific to pancreatic β-cell protein, GAD65, IA-2, ZnT8, insulin, or pancreatic islet cell cytoplasmic autoantibodies. In various embodiments, the subject requiring it has an autoantibody level <50,000 IU / mL. In various embodiments, the subject requiring it has a fasting plasma glucose (FPG) level ≥126 mg / dL (7.0 mmol / L). In various embodiments, the subject requiring it has random plasma glucose ≥200 mg / dL (11.1 mmol / L) with typical symptoms of hyperglycemia or hyperglycemic emergency. In various embodiments, the subject requiring it has a peak-stimulated C peptide of ≥0.2 nmol / L during a mixed food challenge test. In various embodiments, the subject requiring it has a fasting C peptide level of ≥0.12 nmol / L (≥0.36 ng / mL). In a particular embodiment, the subject requiring it has a fasting C peptide level of ≥0.12 to ≤1.5 nmol / L (≥0.36 to ≤4.5 ng / mL). In a particular embodiment, the subject requiring it has an HbA1c level of ≥5.6%. In various embodiments, the percentage of those requiring it is approximately 5.6% to 9.5% (approximately 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%). The subject has an HbA1c level of %, 9.0%, 9.1%, 9.2%, 9.3%, 9.4%, or 9.5% (including all ranges and values ​​within that range). In certain embodiments, the subject requiring it has an HbA1c level of approximately ≤12%. In various embodiments, the subject requiring it is receiving a stable insulin dose or insulin administration regimen for 1 week to 1 month (1 week, 2 weeks, 3 weeks, 4 weeks (including values ​​within that range)).In various embodiments, the subject requiring it has at least 20% β-cell function before administration of the first dose. In various embodiments, the subject requiring it is approximately 7–75 years old (approximately 7, 8, 9, 10, 11, 12, 13…72, 73, 74, or 75 years old (including all ranges and values ​​within that range)).

[0014] In various embodiments, administering a booster dose may be used to evaluate the following: changes in the area under the curve (AUC) of the stimulated C-peptide response over the first two hours of a 2 or 4-hour MMTT, maintenance of C-peptide levels, changes in maximum C-peptide during the MMTT, changes in C-peptide measured at 30, 60, 90, and 120 minutes during the MMTT, changes in fasting C-peptide, proinsulin to C-peptide ratio, changes in antigen-specific T cells, changes in activated antigen-specific T cells, changes in antigen-specific Tregs, changes in HbA1c levels, hypoglycemic events, or Changes in the number of severe hypoglycemic events, changes in the proportion of hypoglycemic events, changes in the use / intake of exogenous insulin, changes in the time spent within the target blood glucose range of 70-180 mg / dL (3.9-10.0 mmol / L), changes in the time spent within the target blood glucose range of 70-110 mg / dL (3.9-6.1 mmol / L), changes in insulin dose-adjusted HbA1c (IDAA1c), insulin sensitivity score (ISS), changes in blood glucose variability / variability, percentage of patients with IDAA1c ≤9, and spikes exceeding 0.2 nmol / L (0.6 ng / mL) at 15 months. The following factors were measured: the percentage of patients with the most acutely elevated C peptide level, the percentage of patients with a stimulated 90-minute C peptide level greater than 0.2 nmol / L (0.6 ng / mL) at 15 months, changes in body weight and body mass index, serum autoantibody concentrations (number and titer), serum autoantibody isotype concentrations (IgG1, IgG2, IgG3, IgG4), and secretion of cytokines IL-1, IL-2, IL-5, IL-13, IL-10, IL-17, interferon (IFN)γ, and tumor necrosis factor (TNF)α by PBMCs upon stimulation with T1D-related antigens. The determination is based on one or more of the following: serum concentrations of cytokines IL-1, IL-2, IL-5, IL-13, IL-10, IL-17, IFNγ, and TNFα; secretion of cytokines IL-1, IL-2, IL-5, IL-13, IL-10, IL-17, IFNγ, and TNFα by PBMCs upon stimulation with anti-CD3 and anti-CD28; proliferation of PBMCs upon stimulation with T1D-related antigens; circulating methylated and unmethylated insulin DNA levels; and circulating hormone levels (e.g., glucagon, incretin, adiponectin).

[0015] In various embodiments, the subjects requiring it are approximately 7 to 17 years old. In various embodiments, the subjects requiring it are approximately 12 to 35 years old. In various embodiments, the subjects requiring it are approximately 18 to 35 years old (including all ranges and values ​​within that range). In various embodiments, the subjects requiring it are approximately 18 to 75 years old. In various embodiments, the subjects requiring it have an HLA genotype selected from the group consisting of HLA-DQ, HLA-DR, HLA-DP, or HLA-B. In various embodiments, the subjects requiring it have an HLA genotype selected from the group consisting of DR3, DR4, or DQ8. In various embodiments, the subject requiring it has an HLA genotype selected from the group consisting of DRB1*03:01-DQA1*05:01-DQB1*02:01 (abbreviated as "DR3"), DRB1*04:01 / 02 / 04 / 05 / 08-DQA1*03:01-DQB1*03:02 / 04 (or DQB1*02; abbreviated as "DR4"), DR3-DR4, DPB1*03:01, DPB1*02:02, B*39:06, A*02:01, A*24:02, B*18:01, or C*03:03.

[0016] In various embodiments, TIMP-T1D encapsulates one or more T1D-related antigens or antigenic epitopes. In various embodiments, the T1D-related antigens include insulin (preproinsulin (PPI)), tyrosine phosphatase-like autoantigen or insulinoma antigen-2 (IA2; ICA512, PTPRN), IA-2β (Fogrin, PTPRN2), glutamate decarboxylase 65-kDa isoform (GAD65), glutamate decarboxylase 67-kDa isoform (GAD67), islet cell antigen-69 (ICA69), and islet-specific glucose-6-phosphatase catalytic subunits. The following are selected from the group consisting of iodine-related proteins (IGRP), zinc transporter 8 (ZnT8), chromogranin A, 38kDa granular antigen, peripherin, islet amyloid peptide (ppIAPP), carboxypeptidase H / E, heat shock protein 60 (HSP60), heat shock protein 70 family (e.g., HSPA1A, HSPA1B, HSPA1L, HSPA2, HSPA5, HSPA6, HSPA8, HSPA9, and other family members), sulfatides, or vasostatin-1.

[0017] In various embodiments, T1D-related antigens are listed in Table 1. In various embodiments, T1D-related antigens are listed in Table 2. In various embodiments, T1D-related antigens are selected from the group consisting of SEQ ID NOs: 1 to 17. In various embodiments, TIMP-T1D encapsulates T1D-related antigens including PPI, ZnT8, GAD65, and IGRP.

[0018] In various embodiments, TIMP-T1D encapsulates T1D-related antigens including SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. In various embodiments, TIMP-T1D encapsulates T1D-related antigens including SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 16. In various embodiments, the ZnT8 protein lacks aspartic acid (D), the last amino acid in SEQ ID NO: 4, and has the sequence described in SEQ ID NO: 16 (ZnT8 variant). [Table 1]

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

[0019] In various embodiments, TIMP-T1D is administered as a single dose or multiple doses. In various embodiments, TIMP-T1D is administered as two doses at one-week intervals. In various embodiments, TIMP-T1D is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year.

[0020] In various embodiments, TIMP-T1D comprises poly(coglycolic acid lactate) (PLGA) particles encapsulating one or more T1D antigens and a suitable buffer or excipient. In various embodiments, TIMP-T1D particles are surface-functionalized. In various embodiments, TIMP-T1D particles are surface-functionalized by carboxylation. In various embodiments, TIMP-T1D particles have a negative zeta potential. In various embodiments, the negative zeta potential of TIMP-T1D particles is approximately -100mV to approximately 0mV. In various embodiments, the zeta potential of the particles is approximately -100mV to approximately -25mV, approximately -100 to approximately -30mV, approximately -80mV to approximately -30mV, approximately -75mV to approximately -30mV, approximately -70mV to approximately -30mV, approximately -75 to approximately -35mV, approximately -70 to approximately -25mV, approximately -60mV to approximately -30mV, approximately -60mV to approximately -35mV, or approximately -50mV to approximately -30mV. In various embodiments, the zeta potential is approximately -25mV, -30mV, -35mV, -40mV, -45mV, -50mV, -55mV, -60mV, -65mV, -70mV, -75mV, -80mV, -85mV, -90mV, -95mV, or -100mV (including all ranges and values ​​within that range).

[0021] In various embodiments, the size, or diameter, of the TIMP-T1D particles is 0.05 μm to approximately 10 μm. In various embodiments, the diameter of the TIMP-T1D particles is 0.1 μm to approximately 10 μm. In various embodiments, the diameter of the TIMP-T1D particles is 0.1 μm to approximately 5 μm. In various embodiments, the diameter of the TIMP-T1D particles is 0.1 μm to approximately 3 μm. In various embodiments, the diameter of the TIMP-T1D particles is 0.3 μm to approximately 5 μm. In various embodiments, the diameter of the TIMP-T1D particles is approximately 0.3 μm to approximately 3 μm. In various embodiments, the diameter of the TIMP-T1D particles is approximately 0.3 μm to approximately 1 μm. In various embodiments, the diameter of the TIMP-T1D particles is approximately 0.4 μm to approximately 1 μm. In various embodiments, TIMP-T1D particles have diameters of approximately 100-10000 nm, approximately 100-5000 nm, approximately 100-3000 nm, approximately 100-2000 nm, approximately 300-5000 nm, approximately 300-3000 nm, approximately 300-1000 nm, approximately 300-800 nm, approximately 400-800 nm, or approximately 200-700 nm. In various embodiments, TIMP-T1D particles have diameters of approximately 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm. In various embodiments, the diameter of the negatively charged particles is 400 nm to 800 nm. In various embodiments, the particles have a size of approximately 400 to approximately 800 nm and a zeta potential of -30 mV to -80 mV. In various embodiments, the polydispersity index (PDI) or heterogeneity index with respect to particle size is 0.01 to 1.0 (e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1, including all values ​​within this range).

[0022] In various embodiments, the particles have a homogeneous size distribution. In various embodiments, the particles have a homogeneous size distribution, and at least 90% of the particles have diameters of 0.05 μm to about 10 μm, 0.1 μm to about 10 μm, 0.1 μm to about 5 μm, 0.1 μm to about 3 μm, 0.3 μm to about 5 μm, or 0.3 μm to about 3 μm. In various embodiments, the particles have a homogeneous size distribution, and at least 90% of the particles have diameters of about 100 to 10000 nm, about 100 to 5000 nm, about 100 to 3000 nm, about 100 to 2000 nm, about 300 to 5000 nm, about 300 to 3000 nm, about 300 to 1000 nm, about 300 to 800 nm, about 400 to 800 nm, or about 200 to 700 nm. In various embodiments, the TIMP-T1D particles have diameters of approximately 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm. In various embodiments, the particles have a homogeneous size distribution, with at least 50% of the particles having diameters of approximately 0.05 μm to approximately 10 μm, approximately 0.1 μm to approximately 10 μm, approximately 0.1 μm to approximately 5 μm, approximately 0.1 μm to approximately 3 μm, approximately 0.3 μm to approximately 5 μm, and approximately 0.3 μm to approximately 3 μm. In various embodiments, the particles have a homogeneous size distribution, and at least 50% of the particles have a diameter of approximately 100-10000 nm, approximately 100-5000 nm, approximately 100-3000 nm, approximately 100-2000 nm, approximately 300-5000 nm, approximately 300-3000 nm, approximately 300-1000 nm, approximately 300-800 nm, approximately 400-800 nm, or approximately 200-700 nm. In various embodiments, the TIMP-T1D particles have a diameter of approximately 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm. In various embodiments, the particles have a homogeneous size distribution, with at least 10% of the particles having diameters of approximately 0.05 μm to 10 μm, 0.1 μm to 10 μm, 0.1 μm to 5 μm, 0.1 μm to 3 μm, 0.3 μm to 5 μm, and 0.3 μm to 3 μm.In various embodiments, the particles have a homogeneous size distribution, and at least 10% of the particles have a diameter of approximately 100-10000 nm, approximately 100-5000 nm, approximately 100-3000 nm, approximately 100-2000 nm, approximately 300-5000 nm, approximately 300-3000 nm, approximately 300-1000 nm, approximately 300-800 nm, approximately 400-800 nm, or approximately 200-700 nm. In various embodiments, the TIMP-T1D particles have a diameter of approximately 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm.

[0023] In various embodiments, TIMP-T1D particles useful for this method or composition encapsulate T1D-related antigens including PPI, ZnT8, GAD65, and IGRP, have a negative zeta potential of -30mV to -80mV, and have a diameter of 400nm to 800nm.

[0024] In various embodiments, TIMP-T1D encapsulates T1D-related antigens including SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, has a negative zeta potential of -30mV to -80mV, and has a diameter of 400nm to 800nm. In various embodiments, TIMP-T1D encapsulates T1D-related antigens including SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, has a negative zeta potential of -30mV to -80mV, and has a diameter of 400nm to 800nm. In various embodiments, TIMP-T1D encapsulates T1D-related antigens including SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 16, has a negative zeta potential of -30mV to -80mV, and has a diameter of 400nm to 800nm.

[0025] In various embodiments, TIMP-T1D is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, lymphatically, or orally.

[0026] In various embodiments, TIMP-T1D is administered at concentrations ranging from 0.005 mg / mL to 50 mg / mL (including all values ​​and ranges between these values). In various embodiments, TIMP-T1D is administered at concentrations of approximately 0.05 mg / mL, approximately 0.1 mg / mL, approximately 0.5 mg / mL, approximately 1 mg / mL, approximately 2 mg / mL, approximately 3 mg / mL, approximately 3.25 mg / mL, approximately 3.5 mg / mL, approximately 4 mg / mL, approximately 5 mg / mL, approximately 6 mg / mL, approximately 7 mg / mL, approximately 8 mg / mL, approximately 9 mg / mL, approximately 10 mg / mL, approximately 11 mg / mL, approximately 12.5 mg / mL, approximately 15 mg / mL, approximately 17.5 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 30 mg / mL, approximately 40 mg / mL, or approximately 50 mg / mL. In various embodiments, TIMP-T1D is administered via intravenous infusion lasting approximately 1, 2, 3, 4, 5, 6, 7, or 8 hours.

[0027] In various embodiments, administering TIMP-T1D to a subject in need alleviates one or more symptoms of T1D. In various embodiments, symptoms of T1D include increased or frequent urination, increased thirst, increased hunger, extreme fatigue, blurred vision, muscle loss, slow-healing cuts or bruises, coronary artery disease with chest pain (angina), heart attack, stroke, atherosclerosis, hypertension, nerve injury (neuropathy), kidney injury (nephropathy), diabetic retinopathy, diabetic ketoacidosis, foot injuries, increased bacterial and viral infections in the skin and mouth, periodontal disease, miscarriage, stillbirth, The group is selected from those comprising pregnancy complications such as birth defects, exogenous insulin use, hypoglycemic events, elevated HbA1c, impaired β-cell function, decreased C-peptide levels, reduced time within the target glucose range of 70-110 mg / dL (3.9-6.1 mM / L), i.e., hyperglycemia, reduced time within the target glucose range of 70-180 mg / dL (3.9-10.0 mM / L), microvascular complications, and weight loss.

[0028] In various embodiments, administering TIMP-T1D to a subject in need reduces the duration and severity of an autoimmune or inflammatory immune response to one or more T1D antigens. In various embodiments, the inflammatory immune response is a T-cell response, a B-cell response, a Th1 response, a myeloid cell response, and / or an antibody response. In various embodiments, the efficacy of TIMP-T1D in alleviating one or more symptoms of T1D and / or reducing the duration and severity of an inflammatory immune response to one or more T1D antigens is determined by assays of one or more biological samples from the subject. In various embodiments, the biological samples are selected from the group consisting of whole blood, peripheral blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, and / or bone marrow biopsy.

[0029] In various embodiments, administration of TIMP-T1D reduces the level of antigen-specific T cells compared to baseline measurements of the subject and / or placebo control. In various embodiments, administration of TIMP-T1D reduces the level of antigen-specific CD4+ T cells compared to baseline measurements of the subject and / or placebo control. In various embodiments, administration of TIMP-T1D reduces the level of antigen-specific CD8+ T cells compared to baseline measurements of the subject and / or placebo control. In various embodiments, administration of TIMP-T1D stabilizes the level of antigen-specific T cells compared to baseline measurements of the subject and / or placebo control. In various embodiments, administration of TIMP-T1D stabilizes the level of antigen-specific CD4+ and / or CD8+ T cells compared to baseline measurements of the subject and / or placebo control.

[0030] In various embodiments, administration of TIMP-T1D to a subject reduces the level of activated antigen-specific T cells relative to the subject's baseline measurement and / or placebo control. In various embodiments, administration of TIMP-T1D to a subject reduces the level of activated antigen-specific CD4+ and / or CD8+ T cells relative to the subject's baseline measurement and / or placebo control. In various embodiments, administration of TIMP-T1D to a subject stabilizes the level of activated antigen-specific T cells relative to the subject's baseline measurement and / or placebo control. In various embodiments, administration of TIMP-T1D to a subject stabilizes the level of activated antigen-specific CD4+ and / or CD8+ T cells relative to the subject's baseline measurement and / or placebo control.

[0031] In various embodiments, administration of TIMP-T1D increases the level of antigen-specific regulatory T (Treg) cells compared to baseline measurements of the subject and / or placebo control. In various embodiments, administration of TIMP-T1D increases the level of antigen-specific CD4+ Tregs compared to baseline measurements of the subject and / or placebo control. In various embodiments, administration of TIMP-T1D increases the level of antigen-specific CD8+ Tregs compared to baseline measurements of the subject and / or placebo control.

[0032] In various embodiments, administering TIMP-T1D to a subject stabilizes C-peptide levels relative to the subject's baseline measurements and / or placebo control. In various embodiments, administering TIMP-T1D to a subject increases C-peptide levels relative to the subject's baseline measurements and / or placebo control.

[0033] In various embodiments, administration of TIMP-T1D to a subject stabilizes the area under the curve (AUC) of the stimulated C-peptide response in a mixed-food challenge test (MMTT) relative to the subject's baseline measurements and / or placebo control. In various embodiments, administration of TIMP-T1D to a subject slows the decline in the area under the curve (AUC) of the stimulated C-peptide response in a mixed-food challenge test (MMTT) relative to the subject's baseline measurements and / or placebo control. In various embodiments, administration of TIMP-T1D to a subject increases the area under the curve (AUC) of the stimulated C-peptide response in a mixed-food challenge test (MMTT) relative to the subject's baseline measurements and / or placebo control. In various embodiments, administration of TIMP-T1D to a subject stabilizes or increases C-peptide levels in a glucagon stimulation test. In various embodiments, administration of TIMP-T1D to a subject stabilizes or increases the change in fasting C-peptide levels in a glucagon stimulation test.

[0034] In various embodiments, administration of TIMP-T1D to a subject reduces HbA1c levels relative to the subject's baseline measurement and / or placebo control. In various embodiments, administration of TIMP-T1D to a subject stabilizes HbA1c levels relative to the subject's baseline measurement and / or placebo control. In various embodiments, administration of TIMP-T1D to a subject stabilizes or reduces insulin dose adjustment A1c (IDAA1c). In various embodiments, administration of TIMP-T1D to a subject stabilizes or reduces insulin dose adjustment A1c (IDAA1c) to ≤9. In various embodiments, administration of TIMP-T1D to a subject alters the insulin sensitivity score (ISS).

[0035] In various embodiments, administration of TIMP-T1D to a subject reduces or stabilizes blood glucose variability / variability. In various embodiments, administration of TIMP-T1D to a subject increases the time the subject is within the blood glucose target range of 70-110 mg / dL (3.9-6.1 mmol / L) compared to the subject's baseline measurements and / or placebo control. In various embodiments, administration of TIMP-T1D to a subject stabilizes the time the subject is within the blood glucose target range of 70-110 mg / dL (3.9-6.1 mmol / L) compared to the subject's baseline measurements and / or placebo control. In various embodiments, the blood glucose range is measured by continuous glucose monitoring. In various embodiments, administration of TIMP-T1D to a subject increases the time the subject is within the blood glucose target range of 70-180 mg / dL (3.9-10.0 mM / L) compared to the subject's baseline measurements and / or placebo control. In various embodiments, administering TIMP-T1D to a subject stabilizes the subject's time within the target glucose range of 70–180 mg / dL (3.9–10.0 mM / L) relative to the subject's baseline measurements and / or placebo control. In various embodiments, the glucose range is measured by continuous glucose monitoring.

[0036] In various embodiments, administration of TIMP-T1D to a subject reduces exogenous insulin use relative to the subject's baseline measurements and / or placebo control. In various embodiments, administration of TIMP-T1D to a subject stabilizes exogenous insulin use relative to the subject's baseline measurements and / or placebo control.

[0037] In various embodiments, administering TIMP-T1D to a subject reduces hypoglycemic events compared to the subject's baseline measurements and / or placebo control. In various embodiments, administering TIMP-T1D to a subject stabilizes hypoglycemic events compared to the subject's baseline measurements and / or placebo control.

[0038] In various embodiments, administration of TIMP-T1D to a subject reduces blood glucose levels relative to the subject's baseline measurements and / or placebo control. In various embodiments, administration of TIMP-T1D to a subject stabilizes blood glucose levels relative to the subject's baseline measurements and / or placebo control.

[0039] In various embodiments, the Disclosure provides a method for treating T1D or T1D-related symptoms in subjects requiring treatment of T1D or T1D-related symptoms, comprising administering a composition comprising TIMP-T1D alone or in combination with one or more therapeutic agents useful for treating T1D to the subject. In various embodiments, combination therapeutic agents useful for treating T1D are selected from the group consisting of insulin, insulin pumps, antihypertensive drugs, aspirin, cholesterol-lowering drugs, artificial pancreases, anti-IL-21 antibodies, B lymphocyte depletion (e.g., rituximab), anti-CD3 antibodies (e.g., teplizumab), adoptive transfer of Tregs (e.g., autologous Tregs, umbilical cord-derived Tregs, polyclonal Tregs), anti-thymocyte globulins, sodium-glucose cotransporter-2 (SGLT2) inhibitors (e.g., dapagliflozin, sotagliflozin), stem cell recruitment (e.g., plerixafor), β-cell transplantation, islet transplantation, stem cell transplantation, and incretin therapy (e.g., liraglutide). In various embodiments, insulin is selected from the group consisting of insulin glargine, ultra-rapid-acting insulin (e.g., insulin aspart, insulin lispro, insulin glulisine), short-acting insulin (ACTRAPID®, HUMULIN S®, INSUMAN RAPID®), long-acting insulin (LEVEMIR®, ABASAGLAR®, LANTUS®, TOUJEO®, TRESIBA®), or intermediate-acting insulin (INSULATARD®, INSUMAN BASAL®).

[0040] In various embodiments, the combination therapy induces Tregs. In various embodiments, the therapy increases the frequency and / or number of Tregs. In various embodiments, the therapy is an IL-2 therapy that induces Tregs. In various embodiments, the IL-2 therapy is a low-dose IL-2, an IL-2 mutain engineered to proliferate Tregs, an IL-2 variant engineered to proliferate Tregs, an IL-2 molecule engineered to be selective for high-affinity IL-2 receptors, a PEGylated IL-2, an IL-2 complex, or an IL-2 / CD25 fusion protein.

[0041] In various embodiments, one or more therapeutic agents are administered before, in conjunction with, or subsequently to the administration of TIMP-T1D. In various embodiments, the therapeutic agent is administered 0.5 to 23 hours before the administration of TIMP-T1D. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 days before the administration of TIMP-T1D. In various embodiments, the therapeutic agent is administered 1, 2, 3, or 4 weeks before the administration of TIMP-T1D. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months before the administration of TIMP-T1D. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years before the administration of TIMP-T1D. In various embodiments, the therapeutic agent is administered 0.5 to 24 hours after the administration of TIMP-T1D. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-T1D. In various embodiments, the therapeutic agent is administered 1, 2, 3, or 4 weeks after administration of TIMP-T1D. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration of TIMP-T1D. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years after administration of TIMP-T1D.

[0042] Compositions comprising TIMP-T1D as described herein for use in the treatment of type 1 diabetes are also intended. In various embodiments, this disclosure provides the use of compositions comprising TIMP-T1D as described herein in the preparation of pharmaceuticals for the treatment of type 1 diabetes.

[0043] Each feature or embodiment or combination described herein is a non-limiting, exemplary example of any aspect of the Invention, and is understood to mean that such an example can be combined with any other feature or embodiment or combination described herein. For example, where a feature is described in terms such as “one embodiment,” “several embodiments,” “a particular embodiment,” “further embodiments,” “a particular exemplary embodiment,” and / or “another embodiment,” each of these types of embodiments is a non-limiting example of a feature intended to be combined with any other feature or combination of features described herein, and it is not necessary to enumerate all possible combinations. Such features or combinations of features are applicable to any aspect of the Invention. Where examples of values ​​falling within a range are disclosed, any of these examples are contemplated as possible endpoints of the range, any and all numerical values ​​between such endpoints are contemplated, and any and all combinations of upper and lower endpoints are assumed. [Brief explanation of the drawing]

[0044] [Figure 1] This is the event schedule for clinical trials evaluating the safety and efficacy of CNP-103. [Figure 2A-2B]Preclinical administration of CNP-T1D with booster doses in NOD mice. Diabetic NOD mice were treated with two loading doses of CNP-T1D, followed by booster doses every four weeks. Treatment with CNP-T1D significantly inhibited T1D expression in glycemic NOD mice, with further inhibition by booster doses (Figure 2A). Treatment with either two or five doses of 2.5 mg of CNP-T1D significantly improved protection from T1D in glycemic NOD mice compared to control NOD mice treated with physiological saline (Figure 2B) (***p<0.0005). [Figure 3] Reversal of blood glucose abnormalities (blood glucose >150 mg / dL and <250 mg / dL) was more pronounced in NOD mice receiving a five-dose booster schedule compared to mice receiving only two loading doses. Comparison of CNP-T1D between the 2.5 mg dose, the two-dose dose, and the five-dose dose was statistically significant (*p=0.0183). [Figure 4] This is the event schedule for a clinical trial evaluating the safety and efficacy of a three-dose regimen of CNP-103. [Modes for carrying out the invention]

[0045] There is a need for therapeutic agents to address the immune imbalance in T1D, leading to improved disease symptoms and outcomes without the risk of toxic side effects. This disclosure provides a methodology for monitoring the induction and maintenance of immune tolerance in subjects with T1D after receiving immunotherapy.

[0046] The headings in this specification are for the convenience of the reader and are not intended to limit the scope of the invention. Additional aspects, embodiments, and variations of the invention will be apparent from the detailed description and / or the drawings and / or claims.

[0047] definition Unless otherwise stated, the following terms used in this application, including in this specification and the claims, have the definitions given below.

[0048] As used herein and in the appended claims, the indefinite articles "a" and "an" and the definite article "the" include plural and singular referents unless the context clearly indicates otherwise.

[0049] The terms “approximately” or “about” mean a tolerance for a particular value as determined by those skilled in the art, which depends in part on how the value is measured or determined. In certain embodiments, the terms “approximately” or “about” mean within 1, 2, 3, or 4 standard deviations. In certain embodiments, the terms “approximately” or “about” mean within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Whenever the terms “approximately” or “about” precede the first number in a series of two or more numbers, it is understood that the terms “approximately” or “about” apply to each of the numbers in that series.

[0050] As used herein, “particles” refers to any non-tissue-derived composition, which may be spheres or sphere-like entities, beads, or liposomes. The terms “particles,” “immunomodulated particles,” “carrier particles,” and “beads” may be used interchangeably depending on the context. Additionally, the term “particles” may be used to encompass beads and spheres.

[0051] As used herein, “negatively charged particles” refers to particles that have been modified to have an effective surface charge of less than zero.

[0052] "Carboxylated particles," "carboxylated beads," or "carboxylated spheres" include any particles modified to contain carboxyl groups on their surface. In some embodiments, the addition of carboxyl groups enhances phagocytic / monocyte uptake of particles from circulation through interaction with scavenger receptors such as MARCO. Carboxylation of particles can be achieved using any compound to which carboxyl groups are added.

[0053] As used herein, the terms “Th cell” or “helper T cell” refer to CD4 + Refers to a cell. CD4 + T cells assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and memory B cells, as well as the activation of cytotoxic T cells and macrophages. T cells are activated when peptide antigens are presented by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs).

[0054] As used herein, the term “Th1 cells” refers to a subset of Th cells that produce pro-inflammatory mediators. Th1 cells play a role in host defense against pathogens by secreting cytokines to stimulate the immune response and, in part, by mediating the recruitment of neutrophils and macrophages to infected tissue. Th1 cells secrete cytokines including IFN-γ, IL-2, IL-10, and TNFα / β to coordinate defense against intracellular pathogens such as viruses and certain bacteria.

[0055] "Polypeptide" and "protein" refer to polymers composed of amino acid residues, associated naturally occurring structural variants, and naturally non-natural analogs of their synthesis, linked via peptide bonds or peptide bond isostears. Synthetic polypeptides can be synthesized, for example, using automated polypeptide synthesizers. The terms "polypeptide" and "protein" are not limited to the minimum length of the product. The term "protein" typically refers to a large polypeptide. The term "peptide" typically refers to a short polypeptide. Thus, peptides, oligopeptides, dimers, multimers, etc., are included in the definition. Both full-length proteins and their fragments are included by definition. The terms "polypeptide" and "protein" also include post-expression modifications of polypeptides or proteins, such as glycosylation, acetylation, phosphorylation, etc. Furthermore, for the purposes of this disclosure, "polypeptide" may include "modifications" to the native sequence, such as deletions, additions, substitutions (which may include substitutions with any of the 20 amino acids that may be essentially conserved or commonly present in human proteins, or any other naturally occurring, non-natural, or atypical amino acids), and chemical modifications (e.g., addition or substitution with peptide mimes). These modifications may be intentional, by site-directed mutagenesis, or by chemical modification of amino acids to remove or attach chemical moieties, or they may be accidental, by mutations occurring via protein-producing host cells, or by errors resulting from PCR amplification prior to host cell transfection.

[0056] As used herein, “antigenic moiety” or “antigen” refers to any portion recognized by the host’s immune system, such as a peptide. Examples of antigenic moieties include, but are not limited to, autoantigens, allergens, enzymes, and / or bacterial or viral proteins, peptides, drugs, or components.

[0057] "Pharmacologically acceptable carriers" refer to any of the standard pharmaceutical carriers, buffers, etc., such as phosphate-buffered saline, a 5% aqueous solution of dextrose, and emulsions (e.g., oil / water or water / oil emulsions). Non-limiting examples of excipients include adjuvants, binders, fillers, diluents, disintegrants, emulsifiers, wetting agents, lubricants, flow enhancers, sweeteners, flavoring agents, and colorants. Preferred pharmaceutical carriers, excipients, and diluents are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995). Preferred pharmaceutical carriers depend on the intended mode of administration of the activator. Typical modes of administration include enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal injection; or topical, transdermal, or transmucosal administration).

[0058] "Pharmacologically acceptable" means a material that is not biologically or otherwise undesirable, that is, a material that can be administered to an individual without causing any undesirable biological effect, or without harmful interaction with any of the components of a composition containing it, or with any components present on or within the individual's body.

[0059] As used herein, the term “subject” encompasses both mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; domesticated animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals such as rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds and fish. The term does not indicate a specific age or sex.

[0060] The term "epitope" refers to any portion of a molecule that can be recognized and bound by a selective binder at one or more antigen-binding regions. Epitopes typically consist of chemically active surface groupings of a molecule, such as amino acids or carbohydrate side chains, and possess specific three-dimensional structural and charge characteristics. Epitopes as used herein may be continuous or discontinuous. Furthermore, an epitope may be a mimotope in that it contains the same or similar three-dimensional structure and / or amino acid sequence as the epitope used to generate an antibody, but contains none or only some of the amino acid residues found in the target used to stimulate the antibody immune response. As used herein, a mimotope is not considered a different antigen from the epitope bound by the selective binder, and the selective binder recognizes the same three-dimensional structure and / or amino acid sequence of the epitope and mimotope.

[0061] The term “symptom” is used herein to mean any physical or observable sign of a disorder, whether or not the disorder is a general characteristic of that disorder. The term “symptom” can mean all such signs or any subset thereof.

[0062] The term "therapeutic dose" is used herein to indicate the amount of the antigen-specific composition of this disclosure that is effective in improving or alleviating the symptoms or signs of the disease being treated.

[0063] As used herein in relation to methods, the terms “to treat,” “to be treated,” “to treat,” and “treatment” mean eliminating, reducing, inhibiting, or improving one or more clinical symptoms, signs, or progression of an event, disease, or condition, whether temporary or permanent, partial or complete. Such treatment does not need to be absolute to be useful. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction of the extent of the disease, stabilization (i.e., non-exacerbating) of the disease, delay or slowing of disease progression, improvement or reduction of the disease condition, and remission (whether partial or complete), whether detectable or undetectable. “Treatment” may also mean extending survival compared to the expected survival without treatment. Those requiring treatment include those who already have a condition or disability, those who are prone to developing a condition or disability, or those for whom a condition or disability should be prevented.

[0064] Hypoglycemia, or "typical" hypoglycemia, can be defined as a blood glucose level of ≥54 mg / dL (3.0 mmol / L) but <70 mg / dL (3.9 mmol / L). Severe hypoglycemia, a clinically significant hypoglycemic event, is defined as a blood glucose level <54 mg / dL.

[0065] Hyperglycemia can be defined as a blood glucose level >180 mg / dL but ≤250 mg / dL (>10 mmol / L but ≤13.9 mmol / L). Severe hyperglycemia is >250 mg / dL. Typical symptoms include a plasma glucose level of ≥200 mg / dL (11.1 mmol / L). Hyperglycemia can also be defined as a fasting plasma glucose level >110 mg / dL.

[0066] Insulin dose-adjusted HbA1c (IDAA1c) can be calculated as HbA1c (%) + 4 times the insulin dose (in units per kilogram per 24 hours).

[0067] Insulin sensitivity score: 4:64725-0:02032

number

number

[0068] particle Particle size and charge are important for tolerance induction. While particles vary in size and charge based on the antigen encapsulated within them, generally, the particles described herein are effective in inducing tolerance when they are approximately 100 nanometers to approximately 1500 nanometers in diameter and have a negative charge of 0 to approximately -100 mV. In various embodiments, particles have a diameter of 400 to 800 nanometers and a charge of approximately -25 mV to -70 mV. In various embodiments, particles have a diameter of 400 to 800 nanometers and a charge of approximately -30 mV to -80 mV. Average particle size and particle charge may be slightly modified during the freeze-drying process; therefore, both post-synthesis average and post-freeze-drying average are described. As used herein, the terms “post-synthesis size” and “post-synthesis charge” refer to the size and charge of particles before freeze-drying. The terms “freeze-drying size” and “freeze-drying charge” refer to the size and charge of particles after freeze-drying.

[0069] In some embodiments, the particles are nonmetallic. In these embodiments, the particles may be formed from polymers. In preferred embodiments, the particles are biodegradable in solids. In this embodiment, the particles can be supplied in solids in multiple doses without particle accumulation in solids. Examples of preferred particles include polystyrene particles, PGA particles, PLA particles, PLGA particles, PLURIONICS-stabilized polypropylene sulfide particles, polypropylene sulfone particles, poly(ethylene glycol)-block-poly(propylene sulfide) copolymer particles, and diamond particles. In some embodiments, liposomes encapsulate polynucleotides encoding T1D-related antigens. In various embodiments, the polynucleotides consist of DNA, RNA, mRNA, or circular RNA.

[0070] Preferably, the particle surface is composed of a material that minimizes nonspecific or undesirable biological interactions. Interactions between the particle surface and the interstitial space can be factors that play a role in lymphatic uptake. The particle surface may be coated with a material that prevents or reduces nonspecific interactions. Stereostabilization by coating the particles with a hydrophilic layer such as poly(ethylene glycol) (PEG) and its copolymers, e.g., PLURONICS® (containing the copolymer of poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol)) can reduce nonspecific interactions with interstitial proteins, as demonstrated by improved lymphatic uptake after subcutaneous injection. All of these facts demonstrate the relevance of the physical properties of the particles from the perspective of lymphatic uptake. Biodegradable polymers may be used to produce all or part of the polymer and / or particles and / or layers. Biodegradable polymers may be degraded, for example, as a result of the reaction of functional groups with water in solution. As used herein, the term “degradation” refers to becoming soluble, either by a reduction in molecular weight or by the conversion of hydrophobic groups to hydrophilic groups. Polymers containing ester groups, such as polylactides and polyglycolides, are generally subjected to spontaneous hydrolysis.

[0071] The particles disclosed herein may also contain additional components. For example, the carrier may have a contrast agent incorporated into or conjugated to the carrier. An example of a currently commercially available carrier nanosphere with a contrast agent is the Kodak X-sight nanosphere. Inorganic quantum confinement luminescent nanocrystals, known as quantum dots (QDs), have emerged as ideal donors for FRET applications: their high quantum yield and tunable size-dependent Stokes shift allow them to emit different sizes from blue to infrared when excited by a single ultraviolet wavelength. (Bruchez, et al., Science, 1998, 281, 2013; Niemeyer, CM Angew. Chem. Int. Ed. 2003, 42, 5796; Waggoner, A. Methods Enzymol. 1995, 246, 362; Brus, LEJ Chem. Phys. 1993, 79, 5566). Quantum dots, such as hybrid organic / inorganic quantum dots based on a class of polymers known as dendrimers, can be used in biological labeling, imaging, and optical biosensing systems (Lemon, et al., J.Am.Chem.Soc.2000, 122, 12886). Unlike the conventional synthesis of inorganic quantum dots, the synthesis of these hybrid quantum dot nanoparticles does not require high temperatures or highly toxic, unstable reagents (Etienne, et al., Appl.Phys.Lett.87, 181913, 2005).

[0072] The particles can be formed from a wide range of materials. Preferably, the particles are composed of materials suitable for biological use. For example, the particles may consist of glass, silica, polyesters of hydroxycarboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxycarboxylic acids and dicarboxylic acids. More generally, the carrier particles may consist of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked, polyesters of alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxyhydroxy acids, or linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked, polyanhydrides of alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxydicarboxylic acids. Additionally, the carrier particles may be quantum dots or may consist of quantum dots, such as quantum dot polystyrene particles (Joumaa et al. (2006) Langmuir 22:1810-6). Carrier particles containing mixtures of ester and anhydride bonds (e.g., copolymers of glycolic acid and sebacic acid) may also be used. For example, carrier particles may include materials containing polyglycolic acid polymer (PGA), polylactic acid polymer (PLA), polysebacic acid polymer (PSA), poly(lactic-co-glycol) acid copolymer (PLGA or PLG; terms are interchangeable), poly(lactic-co-sebacic) acid copolymer (PLSA), poly(glycol-co-sebacic) acid copolymer (PGSA), polypropylene sulfide polymer, poly(caprolactone), chitosan, etc. Other biocompatible, biodegradable polymers useful in the present invention include polymers or copolymers of caprolactone, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates, and biodegradable urethanes, as well as copolymers of these having linear or branched, substituted or unsubstituted alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy or dicarboxylic acid.In addition, biologically important amino acids having reactive side chain groups, such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or their enantiomers, may be included in copolymers with any of the aforementioned materials to provide reactive groups and conjugate moieties for conjugation to antigen peptides and proteins. Suitable biodegradable materials for the present invention include diamond, PLA, PGA, polypropylene sulfide, and PLGA polymers. Biocompatible but non-biodegradable materials may also be used for the carrier particles of the present invention. For example, non-biodegradable polymers of acrylates, ethylene-vinyl acetate, acyl-substituted cellulose acetate, non-degradable urethanes, styrene, vinyl chloride, vinyl fluoride, vinylimidazole, chlorosulfonated olefins, ethylene oxide, vinyl alcohol, TEFLON® (DuPont, Wilmington, Del.), and nylon may be used.

[0073] In certain embodiments, the particles are copolymers having a molar ratio of about 80:20 to about 100:0 or 20:80 to 100:0. Preferred copolymer ratios for current tolerant immunomodulated particles may be 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0. In certain embodiments, the particles are PLURONICS-stabilized polypropylene sulfide particles, polyglycolic acid particles (PGA), polylactic acid particles (PLA), or poly(lactic acid-coglycolic acid) particles, or carboxylated polyglycolic acid particles (PGA), carboxylated polylactic acid particles (PLA), or carboxylated poly(lactic acid-coglycolic acid) particles. In certain embodiments, the particles have copolymer ratios of polylactic acid / polyglycolic acid 80:20, polylactic acid / polyglycolic acid 90:10, or polylactic acid / polyglycolic acid 50:50. In various embodiments, the particles are poly(lactic acid-coglycolic acid) particles with a copolymer ratio of approximately 50:50 polylactic acid:polyglycolic acid.

[0074] The particles are intended to further contain surfactants. Surfactants can be anionic, cationic, zwitterionic, or nonionic. Poloxamer and poloxamine family surfactants are commonly used in particle synthesis. Possible surfactants include, but are not limited to, PEG, Tween-80, gelatin, dextran, Pluronic L-63, polyvinyl alcohol (PVA), polyacrylic acid (PAA), methylcellulose, lecithin, didodecyldimethylammonium bromide (DMAB), and poly(ethylene-alto-maleic acid) (PEMA). Additionally, biodegradable and biocompatible surfactants include, but are not limited to, vitamin E TPGS (Da-tocopheryl polyethylene glycol 1000 succinate), polyamino acids (e.g., polymers of lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or their enantiomers), and sulfate polymers. In certain embodiments, two surfactants are used. For example, when particles are produced by a double emulsion method, the two surfactants may include a hydrophobic surfactant for the first emulsion and a hydrophobic surfactant for the second emulsion. In certain embodiments, the polypeptide antigen is encapsulated in the particles by a single emulsion process. In further embodiments, the polypeptide antigen is more hydrophobic. Sometimes, the double emulsion process results in the formation of larger particles, which can lead to leakage of hydrophilic active components and low capture efficiency. Coalitioning and Ostwald maturation are two mechanisms that can destabilize double emulsion droplets, while diffusion of hydrophilic active components through the organic phase is the main mechanism that causes low levels of captured active components. In some embodiments, reducing the nanoparticle size may be beneficial. One strategy to achieve this is to apply a second strong shear rate. The leakage effect can be reduced by using high polymer concentrations and high polymer molecular weights, which involves increasing the viscosity of the internal aqueous phase and increasing the surfactant molecular weight.In certain embodiments, the particles encapsulating the antigen are produced by nanoprecipitation, coprecipitation, inert gas condensation, sputtering, microemulsion, sol-gel methods, layer-by-layer techniques, or ion gelation. Several methods for producing nanoparticles are described in the literature and are incorporated herein by reference.

[0075] antigen An antigen refers to a modest portion of a molecule, such as a polypeptide or peptide sequence, the three-dimensional structure of a polypeptide or peptide, or a polysaccharide or polynucleotide that can be recognized by host immune cells. Antigen-specificity refers to the ability of the target host cell to recognize and generate an immune response to the antigen alone or to a molecule very similar to the antigen, as well as to an epitope or mimotope.

[0076] "Anergy," "tolerance," or "antigen-specific tolerance" refers to the insensitivity or reprogramming of T cells to T cell receptor-mediated stimulation. Such reprogramming is generally antigen-specific and persists after cessation of exposure to the antigenic peptide. This reprogramming leads to the induction of regulatory T cells, Tr1 cells, and T cell anergy. For example, insensitivity in T cells is characterized by a lack of effector cytokine production, lack of proliferation, or lack of activation. Reprogramming occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD3-mediated signal) in the absence of a second signal (costimulatory signal) or in the presence of an inhibitory signal (negative costimulatory or regulatory cytokine). Under these conditions, re-exposure of cells to the same antigen (even if re-exposure occurs in the presence of a costimulatory molecule) leads to failure of cytokine production and subsequent failure of proliferation. Thus, failure of cytokine production prevents proliferation. However, anergic T cells can proliferate when cultured with (e.g., IL-2).

[0077] The tolerance therapies described herein are intended to be antigen-specific. For example, a TIMP administered as a tolerance therapy encapsulates one or more antigens related to the tolerance therapy and the associated disease or condition being treated. The TIMP used in tolerance therapy is intended to contain one or more T1D antigens. One or more T1D antigens may be a whole protein, polypeptide, or peptide containing a T1D antigen epitope.

[0078] In certain embodiments, one, two, three, or more antigens or antigenic peptides are used in the TIMP. In certain embodiments, one or more T1D antigens are encapsulated in the TIMP by covalent bonding to the inner surface of a particle (see, for example, U.S. Patent Publication 2019 / 0282707, incorporated herein by reference). In certain embodiments, sequences of two or more T1D antigens are intended to be linked within a fusion protein and encapsulated within the TIMP described herein. A method for constructing a TIMP having a bound epitope is described in U.S. Patent Publication 2019 / 0365656, incorporated herein by reference.

[0079] How to use Type 1 diabetes (also known as T1D or type 1 diabetes mellitus) is a T-cell-driven autoimmune disease targeting the beta cells of the pancreas, where a combination of T-cell and B-cell activation leads to islet beta cell loss. The prevalence of T1D in the United States is estimated at approximately 1.6 million people. This disease represents a significant unmet medical need, and TIMP-T1D presents an opportunity to positively impact those affected by this condition.

[0080] The most common cause of death in patients with T1D is complications of the disease. Acute or sudden complications include hypoglycemia and ketoacidosis. Chronic complications include heart disease, stroke, neuropathy, nephropathy, the need for amputation of the toes, feet, or legs, and diabetic retinopathy. The current standard of treatment for T1D is insulin therapy, which can lead to hypoglycemia, hyperglycemia, and diabetic ketoacidosis.

[0081] Currently, clinically tested treatments for T1D fall into these categories: anti-IL-21 antibodies, islet and stem cell transplantation, peptide therapy, and immunosuppressants such as rituximab and abatacept. However, these classes of therapies do not address the underlying causes of the pathology, which are driven by antigen-specific autoimmune attacks against the pancreas.

[0082] A method for treating T1D in a subject is provided herein, comprising administering TIMP-T1D to the subject, wherein TIMP-T1D is administered at a dose level determined based on the subject's body weight. It is also intended that TIMP-T1D may be administered at a fixed dose regardless of the subject's body weight. In various embodiments, a method for treating T1D in a subject is intended, comprising administering TIMP-T1D to the subject, wherein TIMP-T1D is administered at a dose of 0.01 to 12 mg / kg (including all values ​​and ranges between these values) or at a fixed dose of 1 mg to 800 mg (including all values ​​and ranges between these values) based on the subject's body weight. Furthermore, the Specified Provisions Provide a method for reducing the inflammatory immune response to the T1D antigen in subjects suffering from T1D, comprising administering TIMP-T1D to the subject, wherein TIMP-T1D is administered in a dose of 0.01 to 12 mg / kg (including all values ​​and ranges between these values) or in a fixed dose of 1 mg to 800 mg (including all values ​​and ranges between these values) based on the subject's body weight.

[0083] TIMP-T1D is also intended to be administered in doses of approximately 0.01 to 12 mg / kg, 0.05 to 10 mg / kg, 0.01 to 5 mg / kg, 0.1 to 10 mg / kg, 1 to 8 mg / kg, 1.5 to 10 mg / kg, 2 to 12 mg / kg, 2 to 10 mg / kg, 3 to 10 mg / kg, 4 to 10 mg / kg, 4 to 12 mg / kg, or 5 to 12 mg / kg. TIMP-T1D is administered at random doses of approximately 0.01 mg / kg, 0.02 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 6 mg / kg, 8.0 mg / kg, 10 mg / kg, or 12 mg / kg. Alternatively, TIMP-T1D may be administered in doses of approximately 1 mg, 2 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, or 800 mg. In another embodiment, TIMP-T1D is administered at concentrations of approximately 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 3.25 mg / mL, 3.5 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12.5 mg / mL, 15 mg / mL, 17.5 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, or 50 mg / mL, with the range being 0.005 mg / mL to 50 mg / mL (including all values ​​and ranges between these values).

[0084] TIMP-T1D is intended to be administered as a single dose or in multiple doses. In various embodiments, TIMP-T1D is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, once a year, once every two years, once every three years, once every four years, once every five years, once every six years, once every seven years, once every eight years, once every nine years, or once every ten years. In a particular embodiment, TIMP-T1D is administered in two doses, one week apart.

[0085] In various embodiments, the method further includes administering at least one booster dose of TIMP-T1D. In various embodiments, the booster dose of TIMP-T1D is administered as a single dose or multiple doses after a loading dose of TIMP-T1D. In various embodiments, the booster dose is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every four months, once every five months, once every six months, or once per year. In various embodiments, TIMP-T1D is administered as two loading doses one week apart, followed by a booster dose administered as a single dose every three months. In various embodiments, one, two, three, four, or five booster doses are administered to the subject.

[0086] In various embodiments, the booster dose of TIMP-T1D is administered in doses of 0.01 to 12 mg / kg (including all values ​​and ranges between these values). In various embodiments, the booster dose of TIMP-T1D is approximately 0.01 to 12 mg / kg, approximately 0.05 to 10 mg / kg, approximately 0.01 to 5 mg / kg, approximately 0.1 to 10 mg / kg, approximately 1 to 8 mg / kg, approximately 1.5 to 10 mg / kg, approximately 2 to 12 mg / kg, approximately 2 to 10 mg / kg, approximately 3 to 10 mg / kg, approximately 4 to 10 mg / kg, approximately 4 to 12 mg / kg It is administered in doses of g, or approximately 5 to 12 mg / kg, or approximately 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5 mg / kg, 6 mg / kg, 8.0 mg / kg, 10 mg / kg, or 12 mg / kg. In various embodiments, the booster dose of TIMP-T1D is administered in doses of approximately 1 mg, 2 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, or 800 mg.

[0087] In various embodiments, TIMP-T1D is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, lymphatically, or orally. When TIMP-T1D is administered intravenously, it is intended to be administered via intravenous infusion lasting approximately 1, 2, 3, 4, 5, 6, 7, or 8 hours.

[0088] The TIMP-T1D therapy is intended to alleviate, reduce, or improve one or more syndromes or symptoms of T1D. Symptoms of T1D include increased or frequent urination, increased thirst in the throat, increased hunger, extreme fatigue, blurred vision, muscle loss, slow-healing cuts or bruises, coronary artery disease (angina) with chest pain, heart attack, stroke, atherosclerosis, high blood pressure, nerve damage (neuropathy), kidney damage (nephropathy), diabetic retinopathy, diabetic ketoacidosis, foot injuries, increased bacterial and viral infections in the skin and mouth, periodontal disease, miscarriage, stillbirth, and pregnancy complications such as congenital defects, exogenous insulin use, hypoglycemic events, increased HbA1c, deterioration of β-cell function, decreased C-peptide levels, decreased time within the blood glucose target range of 70-110 mg / dL (3.9-6.1 mmol / L) glucose, decreased time within the blood glucose target range of 70-180 (3.9-10 mmol / L) glucose, i.e., hyperglycemia, and weight loss, but are not limited thereto.

[0089] The TIMP-T1D therapy is also intended to reduce, shorten, or improve the duration and severity of the autoimmune response or inflammatory immune response against one or more T1D antigens in the subject. Autoimmune responses or inflammatory immune responses include T-cell responses, B-cell responses, Th1 responses, myeloid cell responses, and / or antibody responses. In various embodiments, administration of TIMP-T1D induces immune anergy. In various embodiments, administration results in apoptosis of macrophages or monocytes in the subject. In various embodiments, administration of TIMP-T1D reduces the pancreatic islet stress response, reduces ineffective control of effector T cells, reduces pancreatic islet / β-cell damage, reduces epigenetic scarring, reduces immune effector amplification, reduces metabolic dysfunction, or reduces immune infiltration into the pancreatic islets. In various embodiments, administration of TIMP-T1D induces pancreatic islet cell / β-cell regeneration, increases β-cell mass / number, induces T r 1 cells, induces T reg :T effIt increases the ratio, induces bystander tolerance, induces infectious tolerance, induces binding inhibition, induces T cell exhaustion, induces T cell apoptosis, induces T cell deletion, or induces genotype-independent tolerance. In various embodiments, administration of TIMP-T1D induces effector T cell retention / sequesterance, regulatory APC, IL-10, IL-35, TGF-β, CTLA-4 expression, ICOS expression, GITR expression, PD-L1 / L2 expression, PD-1 expression, FasL marker, Foxp3, or PGE2. In various embodiments, administration of TIMP-T1D decreases IL-6, IL-12, IL-23, IL-1β, TNF, type 1 interferon, CD80 expression, CD86 expression, CD40 / 40L signaling, or T-bet expression. In various embodiments, the efficacy of TIMP-T1D in alleviating one or more symptoms of T1D and / or reducing the duration and severity of the inflammatory immune response to one or more T1D antigens is determined by assays of one or more biological samples derived from the subject as described herein.

[0090] Screening method The induction and maintenance of immune tolerance are monitored in subjects affected by T1D, who are treated or are intended to be treated with antigen-specific tolerance therapy, including TIMP containing the T1D antigen described herein.

[0091] Methods for screening cell types, cytokines, or other measures of tolerance from subjects receiving tolerance therapy as described herein are known in the art. Methods for assessing tolerance are performed using techniques such as flow cytometry, mass cytometry (CyTOF), ELISA, ELISPOT, in vitro or ex vivo cell stimulation assays (including, but not limited to, cell proliferation assays and macrophage stimulation assays), autoantibody measurement, or immunoglobulin (Ig) serotyping, for example, by the ImmunoCap assay.

[0092] In various embodiments, the immune tolerance status of a subject is determined by assays of one or more biological samples from the subject. Biological samples include whole blood, peripheral blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, and / or bone marrow biopsy. In various embodiments, the assays of biological samples include analyzing the levels, and / or presence or absence, of cell surface proteins, extracellular proteins, intracellular proteins, nucleic acids, metabolites, enzymes, and / or combinations thereof that are associated with disease or disorder.

[0093] Cells assayed from biological samples include immune cells, non-immune cells, and / or combinations thereof. Immune cells include innate immune cells, adaptive immune cells, and / or combinations thereof. Innate immune cells assayed from biological samples are antigen-presenting cells (APCs). Exemplary innate immune cells assayed from biological samples include monocytes, macrophages, neutrophils, granulocytes, dendritic cells, mast cells, eosinophils, basophils, and / or combinations thereof. Adaptive immune cells assayed from biological samples include effector immune cells such as CD4+ T cells, CD8+ T cells, B cells, NK cells, NK-T cells, and / or combinations thereof. In various embodiments, T cells are Th1 cells, Th2a cells, Treg cells, and Tr1 cells.

[0094] In certain embodiments, the cells assayed from a biological sample are epithelial cells, stromal cells, endothelial cells, fibroblasts, pericytes, adipocytes, mesenchymal stem cells, hematopoietic stem cells, hematopoietic progenitor cells, hepatocytes, hepatic sinusoidal endothelial cells (LSEC), and / or Kupffer cells.

[0095] The immune tolerance state of a subject and one aspect of its immune signature are determined by analyzing one or more proteins from one or more biological samples from the subject. In various embodiments, the proteins are cytokines and / or chemokines. In various embodiments, the proteins are cell signaling proteins. In various embodiments, the cytokines and chemokines are IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12p70, IL-13, IL-14, IL-15, IL-16, IL-17, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL -26, IL-27, IL-27b, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, CCL1, CCL2, CCL3, CCL4, CCL5, CC L6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL 23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2(MCP-1), CXCL3(MIP-1α, CXCL4(MIP-1β, CXCL5(RANTES), CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, GM-CSF, IFN-α, The protein is selected from the group consisting of IFN-β, IFN-γ, TNF-α, TGF-β1, TGF-β2, TGF-β3, soluble CD14, and / or combinations thereof. In various embodiments, the protein is a protease. In various embodiments, the protease is an aspartate protease, a cysteine ​​protease, a metalloprotease, a serine protease, and / or a threonine protease.In various embodiments, the following are selected from the group consisting of ADAM1, ADAM2, ADAM7, ADAM8, ADAM9, ADAM10, ADAM11, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAM20, ADAM21, ADAM22, ADAM23, ADAM28, ADAM29, ADAM30, ADAM33, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28. In various embodiments, the apoptosis-related protein is selected from the group consisting of P53, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, BCL-2, BCL-XL, MCL-1, CED-9, A1, BFL1, BAX, BAK, DIVA, BCL-XS, BIK, BIM, BAD, BID, and EGL-1. Several methods for assaying proteins from biological samples are described in the literature, including enzyme-linked immunosorbent assay (ELISA), Western blotting, and mass spectrometry. In various embodiments, the protein is one or more immunoglobulins (Ig). In various embodiments, Ig is selected from the group consisting of IgA, IgD, IgE, IgM, and / or their variants. In various embodiments, immunoglobulins are antigen-specific. Several methods for detecting immunoglobulins from biological samples are described in the literature, including ELISA and ImmunoCap.

[0096] The immune tolerance state and one aspect of the immune signature of a target can be determined by analyzing one or more cell surface proteins from a biological sample. In various embodiments, the cell surface proteins include CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32b, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD45RA, CD47, CD45RA, CD45RO, CD48, CD52, CD55, CD56, CD58, C D61, CD66b, CD69, CD70, CD72, CD79, CD68, CD84, CD86, CD93, CD94, CD95, CRACC, BLAME, BCMA, CD103, CD107, CD112, CD120a, CD120b, CD123, CD125, CD127, CD134, CD135, CD1 40a, CD141, CD154, CD155, CD160, CD161, CD163, CD172a, XCR1, CD203c, CD204, CD206, CD207CD226, CD244, CD267, CD268, CD269, CD355, CD358, CRTH2, NKG2A, NKG2B, NKG2C,NKG2D、NKG2E、NKG2F、NKG2H、KIR2DL1、KIR2DL2、KIR2DL3、KIR2DL5A、KIR2DL5B、KIR3DL1、KIR3DL2、KIR3DL3、KIR3DL4、KIR2DS1、KI R2DS2、KIR2DS3、KIR2DS4、KIR2DS5、DAP12、KIR3DS、NKp44、NKp46、TCR、BCR、インテグリン、FcβεRI、MHC-I、MHC-II、IL-1R、IL-2Rα、IL-2Rβ 、IL-2Rγ、IL-3Rα、CSF2RB、IL-4R、IL-5Rα、CSF2RB、IL-6Rα、gp130、IL-7Rα、IL-9R、IL-10R、IL-12Rβ1、IL-12Rβ2、IL-13Rα1、IL-13R α1 α2、IL-22Rβ、IL-28RA、PD-1、PD-1H、BTLA、CTLA-4、PD-L1、PD-L2、2B4、B7-1、B7-2、B7-H1、B7-H4、B7-DC、DR3、LIGHT、LAIR、LTα1β2、 LTβR、TIM-1、TIM-3、TIM-4、TIGIT、LAG-3、ICOS、ICOS-L、SLAM、SLAMF2、OX-40、OX-40L、GITR、GITRL、TL1A、HVEM、41-BB、41BB-L、TL- 1A、TRAF1、TRAF2、TRAF3、TRAF5、BAFF、BAFF-R、APRIL、TRAIL、RANK、AITR、TRAMP、CCR1、CCR2、CCR3、CCR4、CCR5、CCR6、CCR7、CCR8、C CR9、CCR10、CCR11,CXCR1、CXCR2、CXCR3、CXCR4、CXCR5、CXCR6、CXCR7、CLECL9a、DC-SIGN、IGSF4A、SIGLEC、EGFR、PDGFR、VEGFR、FAP,Examples include α-SMA, FAS, FAS-L, FC, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM-1, MICA, MICB, UL16, ULBP1, ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULT1, RAE1α, β, γ, δ, and ε, H60a, H60b, H60c, GPR15, ST2, and / or combinations thereof. Integrins include α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8, and / or combinations thereof. TCRs include α, β, γ, δ, ε, ζ chain, and / or combinations thereof. Several methods for assaying cell surface protein expression, including flow cytometry and mass cytometry (CyTOF), have been described in the literature.

[0097] In various embodiments, the immune tolerance status and immune signature of the subject are determined by analyzing the liver function of the subject. In various embodiments, the liver function of the subject is determined by assays of total cholesterol, triglycerides, LDL cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (GGT), alkaline phosphatase (ALP), albumin, total protein, total bilirubin, globulin, creatine kinase (CK), and lactate dehydrogenase (LDH).

[0098] The immune tolerance state and one aspect of the immune signature of the subject are determined by analyzing one or more metabolites from a biological sample. In various embodiments, the metabolites are inflammatory metabolites. In various embodiments, the metabolites are anti-inflammatory metabolites. Examples of inflammatory metabolites in various embodiments include acids, lipids, sugars, amino acids, lactates, trimethylamine N-oxide, O-acetylcreatine, L-carnitine, choline, succinate, glutamine, fatty acids, cholesterol, 3-hydroxybutyrate, 3'-sialyl lactose, arachidonic acid, prostaglandins (G2 and H2), PGD2, PGE2, PGF2a, PGI2, TXA2, leukotrienes (A4, B4, C4, D4, E4), kynurenine, 3-hydroxykynurenine, lipoxin A4, and lipoxin B4. In various embodiments, examples of anti-inflammatory metabolites include 2-amino-3-carboxymucone-6-semialdehyde, picolinic acid, anthranilic acid, 3-hydroxyanthranilic acid, glutaryl co-A, NAD+, quinolinic acid, arginine, butyrate, and adenosine.

[0099] A list of human metabolites that can be assayed from biological samples can be found in literature including (Psychogios et al., 2011), (Wishart et al., HMDB: the Human Metabolome Database. Nucleic Acids Res. 2007 Jan;35 (Database issue): D521-6, 2007), and the Human Metabolome Database (HMDB), which are incorporated herein by reference.

[0100] In certain embodiments, the tolerance status of the subject is determined by analyzing nucleic acids from a biological sample. In various embodiments, nucleic acids include, but are not limited to, single-stranded DNA, double-stranded DNA, mRNA, rRNA, tRNA, siRNA, miRNA, long non-coding RNA (long ncRNA, lncRNA), non-coding RNA (ncRNA), and mitochondrial RNA. In various embodiments, the miRNA is miR-21, miR-34, miR-155, miR-146, miR-200, miR-429, miR-122, miR-451, miR-29, miR-7, miR-124, m iR-23, miR-98, miR-590, miR-26, miR-101, miR-326, miR-15, miR-31, miR-149, miR-150, miR-9, miR-30, miR-25, miR-126 , miR-144, miR-16, miR-192, miR-194, miR-27, miR-152, miR-148, miR-339, miR-425, miR-103a-3p, miR-155-5p, miR-200 a-3p, miR-210-3p, hsa-miR-1-3p, hsa-miR-200b-3p, hsa-miR-9-5p, and hsa-miR-1200, miR-204-5P, miR-497, miR-21-3p, miR-424, miR-150-5p, miR-342, miR-491, miR-507, miR-517a-3p, miR-517B, miR-4691, miR-448, miR-370, miR-1236, miR -1267, miR-555, miR-93, miR-146a, miR-181, miR-30a-5p, miR-181a, miR-24, miR-210, miR-27a, miR-29a, miR-27b, miR-2 00a, miR-26a, miR-454-3p, miR-144-5p, miR-345-5p, miR-125a-3p, miR-502-3p, miR-500a-5p, miR-324-5p, miR-140-5p , miR-192-5p, miR-331-3p, miR-377-3p, miR-221-3p, miR-182-5p, miR-103a-2-5p, miR-183-5p, let-7e-5p, miR-18a-5p,Selected from the group consisting of miR-324-3p, miR-1468, miR-214-5p, miR-23b-3p, miR-720, miR-636, miR-630, miR-490-5p, miR-154-3p, miR-675-3p, miR-100-5p, miR-639, hsa-miR-146a-5p, hsa-miR-194-5p, hsa-miR-197-3p, hsa-miR-301a-3p, miR-197-3p, miR-122-5p, miR-125b-5p, miR-136-5p, miR-34a-5p, miR-342-3p, miR-320b, miR-28-5p, miR-151a-3p, miR-181a-5p, miR-151a-5p, miR-423-5p, miR-199a-3p, miR-126-3p, miR-652-3p, miR-148b-3p, miR-27b-3p, miR-107, miR-22-3p, miR-590-5p, let-7c-5p, miR-30d-5p, miR-106b-5p, miR-142-3p, miR-17-5p, let-7g-5p, miR-32-5p, miR-22-5p, miR-16-2-3p, miR-93-5p, miR-140-3p, miR-19a-3p, miR-19b-3p, miR-16-5p, miR-30e-5p, miR-363-3p, miR-222-3p, miR-144-3p, miR-24-3p, miR-1225-5p, miR-320c, miR-224, miR-148a, miR-26 miR-375, miR-302d-3p, miR-378e, miR-570-3p, miR-574-5p, miR-579, or miR-25-3p.

[0101] In various embodiments, the immune tolerance state of a target is determined by assaying gene expression from a biological sample. In various embodiments, the immune tolerance state is determined by assaying gene expression related to immune function, antibodies, xenobiotic response, metabolism, apoptosis, cell death, necrosis, ferroptosis, autophagy, cell migration, endocytosis, phagocytosis, phagocytosis, tight junction regulation, cell adhesion, differentiation, and / or combinations thereof. In various embodiments, the immune tolerance state is determined by assaying gene expression related to immunosuppression. In various embodiments, the immune tolerance state is determined by assaying gene expression related to immune activation. In various embodiments, the immune tolerance state is determined by assaying gene expression related to regulatory function. In various embodiments, nucleic acid analysis is used to generate an immune tolerance signature. Several methodologies for high-throughput gene expression analysis, including RNA sequencing (RNA-seq), single-cell RNA sequencing (scRNA-seq), exome sequencing, and microarray-based analysis, are described in the literature.

[0102] Biological samples are assayed after in vivo and / or ex vivo stimulation with one or more stimuli, such as an antigen and one or more activators, as optional. The T cells, B cells, and immunoglobulins used in the assay are intended to be antigen-specific. Exemplary T cells include effector memory T cells, antigen-specific T cells, activated antigen-specific T cells, Th1 cells, Th17 cells, T follicular helper (TFH) cells, TH0 cells, or other antigen-specific T cells. B cells include effector B cells, memory B cells, plasma cells, and Breg cells. In certain embodiments, T cells are identified based on the expression of proteins listed in Table 3. [Table 3]

[0103] In various embodiments, the immune tolerance status of the subject is determined by obtaining one or more samples, such as whole blood, from the subject before the first dose of TIMP-T1D (day 1), 7 days after the second dose (day 15), and then on days 60, 76, 90, 120, 166, 180, 256, 270, 346, and 360. The whole blood can then be processed to isolate peripheral blood mononuclear cells (PBMCs), basophils, neutrophils, plasma, and serum for downstream analysis. Assays of cells isolated from one or more samples are collected from the subject and analyzed using methods such as those described below.

[0104] In various embodiments, the immune tolerance status of the subject determined before administration of TIMP-T1D serves as a baseline. In various embodiments, the baseline of the subject is determined from assays of one or more biological samples 1, 2, 3, 4, 5, 6, or 7 days prior to administration of TIMP-T1D. In various embodiments, the baseline of the subject is determined from assays of one or more biological samples 1, 2, 3, or 4 weeks prior to administration of TIMP-T1D. In various embodiments, the baseline of the subject is determined from assays of one or more biological samples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months prior to administration of TIMP-T1D.

[0105] In various embodiments, the immune tolerance status of the subject is determined after administration of TIMP-T1D. In various embodiments, the immune tolerance status of the subject is determined from assays of one or more biological samples 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-T1D. In various embodiments, the status of the subject is determined from assays of one or more biological samples 1, 2, 3, or 4 weeks after administration of TIMP-T1D. In various embodiments, the status of the subject is determined from assays of one or more biological samples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration of TIMP-T1D. In various embodiments, the status of the subject is determined from assays of one or more biological samples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years after administration of TIMP-T1D. In various embodiments, the immune tolerance status of the subject determined after administration of TIMP-T1D is compared to baseline. In various embodiments, the immune tolerance status of subjects determined after administration of TIMP-T1D is compared to that of placebo controls.

[0106] The target immune tolerance signature is generated using one or more of the following parameters, assayed from one or more biological samples obtained from the target and stimulated in vivo and / or ex vivo: A. The proportion of effector T cells in the total T cell population. B. The proportion of Treg cells in the total T cell population, C. The proportion of effector B cells in the total B cell population. D. Levels of specific IgG and / or IgM, E. Levels of inflammatory cytokines and chemokines, F. Levels of anti-inflammatory cytokines and chemokines, G. Liver enzyme levels H. Levels of inflammatory metabolites, and I. Levels of anti-inflammatory metabolites.

[0107] The immune tolerance signature indicates maintained immune tolerance if 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the parameters listed in (a) to (i) above indicate maintained immune tolerance. In various embodiments, the immune tolerance signature indicates maintained immune tolerance if at least 2 out of 9 of the parameters listed in (a) to (i) above indicate maintained immune tolerance. In various embodiments, if 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the parameters listed in (a) to (i) above indicate maintained immune tolerance, the subject is determined not to require treatment with TIMP. In various embodiments, if at least 3 out of 9 of the parameters listed in (a) to (i) above indicate maintained immune tolerance, the subject is determined not to require treatment with TIMP.

[0108] The immune tolerance signature of a subject generated using one or more parameters described herein indicates weakened and / or absent immune tolerance before or after treatment with TIMP-T1D if:

[0109] a. The proportion of effector T cells in the total T cell population is between 0.01% and 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all values ​​and ranges between these values)) relative to baseline measurements of the subject and / or placebo control, and / or

[0110] b. The proportion of Treg cells in the total T cell population is between 0.01% and 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all values ​​and ranges between these values)) relative to baseline measurements of the subject and / or placebo control, and / or

[0111] c. The proportion of effector B cells in the total B cell population is between 0.01% and 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all values ​​and ranges between these values)) relative to baseline measurements of the subject and / or placebo control, and / or

[0112] d. IgG and / or IgM levels were approximately 0.01% to 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 9%) compared to baseline measurements of the subject and / or placebo control. It increases to 0%, approximately 95%, or approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)), and / or

[0113] e. Inflammatory cytokine / chemokine levels were approximately 0.01% to 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%) compared to baseline measurements of the subject and / or placebo control. It increases to 90%, approximately 95%, or approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)), and / or

[0114] f. Levels of anti-inflammatory cytokines and chemokines were approximately 0.01% to 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 9%) compared to baseline measurements in the subject and / or placebo control. Decreases to 0%, approximately 95%, or approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)), and / or

[0115] g. Liver enzyme levels were approximately 0.01% to 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%) compared to baseline measurements of the subject and / or placebo control. It increases to 95%, or approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)), and / or

[0116] h. Levels of inflammatory metabolites were approximately 0.01% to 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%) compared to baseline measurements in the subject and / or compared to placebo control. It increases to approximately 95%, or approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)), and / or

[0117] i. The levels of anti-inflammatory metabolites should be approximately 0.01% to 100% (e.g., approximately 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%) compared to baseline measurements in the subject and / or placebo control. It decreases to approximately 95%, or approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or to approximately 2-1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

[0118] The efficacy of TIMP-T1D in alleviating one or more symptoms of T1D and / or reducing the duration and severity of autoimmune or inflammatory immune responses to the T1D antigen is determined by assays of one or more biological samples derived from the subject. Biological samples include whole blood, peripheral blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, and / or bone marrow biopsy. In various embodiments, assays of biological samples may involve analyzing the levels, and / or presence or absence, of cell surface proteins, extracellular proteins, intracellular proteins, nucleic acids, metabolites, enzymes, and / or combinations thereof.

[0119] In various embodiments, the efficacy of TIMP-T1D in alleviating one or more symptoms of T1D and / or reducing the duration and severity of the inflammatory immune response to the T1D antigen is determined based on assays of cells derived from one or more subject-derived biological samples before and after treatment with TIMP-T1D. In various embodiments, the cells are immune cells, non-immune cells, and / or combinations thereof. In various embodiments, immune cells include innate immune cells, adaptive immune cells, and / or combinations thereof. Innate immune cells assayed from biological samples are antigen-presenting cells (APCs). Exemplary innate immune cells assayed from biological samples include monocytes, macrophages, neutrophils, granulocytes, dendritic cells, mast cells, eosinophils, basophils, and / or combinations thereof. Adaptive immune cells assayed from biological samples include effector immune cells such as CD4+ T cells, CD8+ T cells, B cells, NK cells, NK-T cells, and / or combinations thereof. In various embodiments, T cells are Th1 cells, Th2a cells, Treg cells, and Tr1 cells.

[0120] In certain embodiments, the cells assayed from a biological sample are epithelial cells, stromal cells, endothelial cells, fibroblasts, pericytes, adipocytes, mesenchymal stem cells, hematopoietic stem cells, hematopoietic progenitor cells, hepatocytes, hepatic sinusoidal endothelial cells (LSEC), and / or Kupffer cells.

[0121] In various embodiments, the efficacy of TIMP-T1D in alleviating one or more symptoms of T1D and / or reducing the duration and severity of the inflammatory immune response to one or more T1D antigens is determined based on assays of cell surface proteins derived from one or more subject-derived biological samples before and after treatment with TIMP-T1D. In various embodiments, the cell surface proteins include CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32b, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD45RA, CD47, CD45RA, CD45RO, CD48, CD52, CD55, CD56, CD58, CD61, CD66b, CD69, CD70, CD72, CD79, CD68, CD84, CD86, CD93, CD94, CD95, CRACC, BLAME, BCMA, CD103, CD107, CD112, CD120a, CD120b, CD123, CD125, CD127, CD134, CD135, CD140a, C D141, CD154, CD155, CD160, CD161, CD163, CD172a, XCR1, CD203c, CD204, CD206, CD207CD226, CD244, CD267, CD268, CD269, CD355, CD358, CRTH2, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR 3DL2, KIR3DL3, KIR3DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS, NKp44, NKp46, TCR, BCR, integrin, FcβεRI , MHC-I, MHC-II, IL-1R, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-3Rα, CSF2RB, IL-4R, IL-5Rα, CSF2RB, IL-6Rα, gp130, IL-7Rα, IL-9R, IL-10R,IL-12Rβ1, IL-12Rβ2, IL-13Rα1, IL-13Rα2, IL-15Rα, IL-21R, IL23R, IL-27Rα, IL-31Rα, OSMR, CSF-1R, cell surface IL-15, IL-10Rα, IL-10Rβ, IL-20Rα, IL-20Rβ, IL-22Rα1, IL-22Rα2, IL-22Rβ, IL-28RA, PD-1, PD-1H, BTLA, CTLA-4, PD-L1, P D-L2, 2B4, B7-1, B7-2, B7-H1, B7-H4, B7-DC, DR3, LIGHT, LAIR, LTα1β2, LTβR, TIM-1, TIM-3, TIM-4, TIGIT, LAG-3, I COS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL1A, HVEM, 41-BB, 41BB-L, TL-1A, TRAF1, TRAF2, TRAF3, TR AF5, BAFF, BAFF-R, APRIL, TRAIL, RANK, AITR, TRAMP, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CLECL9a, DC-SIGN, IGSF4A, SIGLEC, EGFR, PDGFR, VEGFR, F The group is selected from AP, α-SMA, FAS, FAS-L, FC, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM-1, MICA, MICB, UL16, ULBP1, ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULT1, RAE1α, β, γ, δ, and ε, H60a, H60b, H60c, GPR15, ST2, and / or combinations thereof. The integrins include α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8, and / or combinations thereof. TCRs include α, β, γ, δ, ε, ζ chains and / or combinations thereof. Several methods for assaying cell surface protein expression, including flow cytometry and mass cytometry (CyTOF), are described in the literature.

[0122] In various embodiments, treatment with TIMP-T1D increases the expression of inflammatory cell surface proteins by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%) compared to baseline values ​​of the target and / or placebo control. Reduce to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

[0123] In various embodiments, treatment with TIMP-T1D increases the expression of anti-inflammatory cell surface proteins by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%) compared to baseline values ​​of the target and / or placebo control. Increase by 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or by approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)).

[0124] In various embodiments, the efficacy of TIMP-T1D in alleviating one or more symptoms of T1D and / or reducing the duration and severity of the inflammatory immune response to the T1D antigen is determined based on assays of proteins derived from one or more subject-derived biological samples before and after treatment with TIMP-T1D. In various embodiments, the proteins are cytokines and / or chemokines. In various embodiments, the proteins are cell signaling proteins. In various embodiments, the cytokines and chemokines are IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12p70, IL-13, IL-14, IL-15, IL-16, IL-17, IL-17, IL-18, IL-20, IL- 21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-27b, IL-28, IL-29, IL-30, IL-31, IL-32, I L-33, IL-35, IL-36, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL1 2. Selected from the group consisting of CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2 (MCP-1), CXCL3 (MIP-1α), CXCL4 (MIP-1β), CXCL5 (RANTES), CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, GM-CSF, IFN-α, IFN-β, IFN-γ, TNF-α, TGF-β1, TGF-β2, TGF-β3, and / or combinations thereof.

[0125] In various embodiments, the protein is a protease. In various embodiments, the protease is an aspartate protease, a cysteine ​​protease, a metalloprotease, a serine protease, and / or a threonine protease. In various embodiments, the following are selected from the group consisting of ADAM1, ADAM2, ADAM7, ADAM8, ADAM9, ADAM10, ADAM11, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAM20, ADAM21, ADAM22, ADAM23, ADAM28, ADAM29, ADAM30, ADAM33, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28. In various embodiments, the apoptosis-related protein is selected from the group consisting of P53, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, BCL-2, BCL-XL, MCL-1, CED-9, A1, BFL1, BAX, BAK, DIVA, BCL-XS, BIK, BIM, BAD, BID, and EGL-1. Several methods for assaying proteins from biological samples are described in the literature, including enzyme-linked immunosorbent assay (ELISA), Western blotting, and mass spectrometry. In various embodiments, the protein is one or more immunoglobulins (Ig). In various embodiments, Ig is selected from the group consisting of IgA, IgD, IgE, IgM, and / or their variants. In various embodiments, immunoglobulins are antigen-specific. Several methods for detecting immunoglobulins from biological samples are described in the literature, including ELISA and ImmunoCap.

[0126] In various embodiments, treatment with TIMP-T1D reduces inflammatory protein levels to 5%–100% (e.g., approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (of these values) relative to baseline measurements of the target and / or placebo control. Reduce to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)). In various embodiments, treatment with TIMP-T1D reduces anti-inflammatory protein levels to 5%–100% (e.g., approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) relative to baseline levels of the target and / or placebo control. Increase by 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or by approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)).

[0127] In various embodiments, the efficacy of TIMP-T1D in alleviating one or more symptoms of T1D and / or reducing the duration and severity of the inflammatory immune response to the T1D antigen is determined based on assays of metabolites derived from one or more subject-derived biological samples before and after treatment with TIMP-T1D. In various embodiments, the metabolites are inflammatory metabolites. In various embodiments, the metabolites are anti-inflammatory metabolites. In various embodiments, examples of inflammatory metabolites include acids, lipids, sugars, amino acids, lactates, trimethylamine N-oxide, O-acetylcreatine, L-carnitine, choline, succinate, glutamine, fatty acids, cholesterol, 3-hydroxybutyrate, 3'-sialyl lactose, arachidonic acid, prostaglandins (G2 and H2), PGD2, PGE2, PGF2a, PGI2, TXA2, leukotrienes (A4, B4, C4, D4, E4), kynurenine, 3-hydroxykynurenine, lipoxin A4, and lipoxin B4. In various embodiments, examples of anti-inflammatory metabolites include 2-amino-3-carboxymucone-6-semialdehyde, picolinic acid, anthranilic acid, 3-hydroxyanthranilic acid, glutaryl co-A, NAD+, quinolinic acid, arginine, butyrate, and adenosine. In various embodiments, treatment with TIMP-T1D reduces the levels of inflammatory metabolites to 5%–100% (e.g., approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (of these values) relative to baseline measurements of the target and / or placebo control. Reduce to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).In various embodiments, treatment with TIMP-T1D raises the levels of anti-inflammatory metabolites to 5%–100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (these) of baseline measurements and / or placebo controls. Increase by 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or by approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)).

[0128] In various embodiments, the effectiveness of TIMP-T1D in alleviating one or more symptoms of T1D and / or reducing the duration and severity of the inflammatory immune response to the T1D antigen is determined based on the following evaluations before and after TIMP-T1D administration: A. Change from baseline in area under the curve (AUC) of the stimulated C peptide response over the first two hours of a 4-hour MMTT. B. Maintenance of C-peptide from baseline C. Changes from baseline in antigen-specific T cells D. Changes from baseline in activated antigen-specific T cells E. Changes from baseline in antigen-specific Tregs F. Change from baseline in HbA1c levels G. Change from baseline in the number of hypoglycemic events H. Change from baseline in insulin use 1. Change from baseline in the time spent within the target blood glucose range of 70-110 mg / dL (3.9-10.0 mmol / L).

[0129] In various embodiments, the efficacy of TIMP-T1D is determined based on one or more of the following evaluations before and after TIMP-T1D administration. In various embodiments, a booster dose is administered as needed, so as to be determined based on one or more of the following evaluations before and after TIMP-T1D administration: A. Changes in area under the curve (AUC) of the stimulated C peptide response over the first two hours of a 2 or 4-hour MMTT. Maintaining BC peptide levels. Changes in the maximum C-peptide during C.MMTT Changes in C-peptide measured at 30, 60, 90, and 120 minutes during D.MMTT. E. Changes in C-peptide during fasting F. Proinsulin to C-peptide ratio G. Changes in antigen-specific T cells. H. Changes in activated antigen-specific T cells. I. Changes in antigen-specific Tregs. Changes in J.HbA1c levels. K. Changes in the number of hypoglycemic events or severe hypoglycemic events L. Changes in the rate of hypoglycemic events M. Changes in the use / intake of exogenous insulin. N. Changes in the time spent within the blood glucose target range of 70-180 mg / dL (3.9-10.0 mmol / L) O. Changes in HbA1c (IDAA1c) due to insulin dose adjustment P. Insulin Sensitivity Score (ISS) Q. Blood glucose variability / changes in variability Percentage of patients with IDAA1c R ≤ 9 S. Percentage of patients with a maximum stimulation C-peptide level greater than 0.2 nmol / L (0.6 ng / mL) at 15 months of age. T. Percentage of patients with a stimulated 90-minute C peptide level greater than 0.2 nmol / L (0.6 ng / mL) at 15 months of age. U. Changes in body weight and body mass index V. Concentration of serum autoantibodies (number and titer). W. Concentration of serum autoantibody isotypes (IgG1, IgG2, IgG3, IgG4). Secretion of cytokines IL-1, IL-2, IL-5, IL-13, IL-10, IL-17, interferon (IFN)γ, and tumor necrosis factor (TNF)α by PBMCs upon stimulation with X.T1D-associated antigens. Y. Serum concentrations of cytokines IL-1, IL-2, IL-5, IL-13, IL-10, IL-17, IFNγ, and TNFα Z. Secretion of cytokines IL-1, IL-2, IL-5, IL-13, IL-10, IL-17, interferon (IFN)γ, and tumor necrosis factor (TNF)α by PBMCs stimulated with anti-CD3 and anti-CD28 chemotherapeutic agents. Proliferation of PBMCs upon stimulation with AA.T1D-related antigens BB. Circulating methylated and unmethylated insulin DNA levels CC. Circulating hormone levels (e.g., glucagon, incretin, adiponectin)

[0130] In various embodiments, treatment with TIMP-T1D stabilizes the AUC of the stimulated C-peptide response over the first two hours of a 4-hour MMTT compared to baseline measurements of the subject and / or placebo control. In various embodiments, treatment with TIMP-T1D stabilizes the AUC of the stimulated C-peptide response over the first two hours of a 4-hour MMTT compared to baseline measurements of the subject and / or placebo control by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or Increase by approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or by approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)).

[0131] In various embodiments, treatment with TIMP-T1D stabilizes C-peptide levels relative to baseline measurements of the subject and / or placebo control. In various embodiments, treatment with TIMP-T1D stabilizes C-peptide levels relative to baseline measurements of the subject and / or placebo control by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (of these values). Increase by 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or by approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)).

[0132] In various embodiments, treatment with TIMP-T1D stabilizes antigen-specific T cells relative to baseline values ​​of the subject and / or placebo control. In various embodiments, treatment with TIMP-T1D stabilizes antigen-specific T cells relative to baseline values ​​of the subject and / or placebo control, ranging from 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (between these values) Reduce to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

[0133] In various embodiments, treatment with TIMP-T1D stabilizes activated antigen-specific T cells relative to baseline values ​​of the subject and / or placebo control. In various embodiments, treatment with TIMP-T1D stabilizes activated antigen-specific T cells relative to baseline values ​​of the subject and / or placebo control by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (of these values) Reduce to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

[0134] In various embodiments, treatment with TIMP-T1D stabilizes antigen-specific Treg cells relative to baseline values ​​of the subject and / or placebo control. In various embodiments, treatment with TIMP-T1D stabilizes antigen-specific Treg cells relative to baseline values ​​of the subject and / or placebo control, ranging from 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (these) Increase by 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or by approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)).

[0135] In various embodiments, treatment with TIMP-T1D stabilizes HbA1c levels relative to the baseline measurement of the subject and / or relative to the placebo control. In various embodiments, treatment with TIMP-T1D stabilizes HbA1c levels relative to the baseline measurement of the subject and / or relative to the placebo control by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (between these values). It reduces the HbA1c level to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or to about 2-1 / 100 (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)). In various embodiments, treatment with TIMP-T1D reduces the absolute HbA1c level to less than 140 mg / dL (or 6.5% HbA1c). In various embodiments, treatment with TIMP-T1D reduces absolute HbA1c levels from 137 mg / dL (6.4% HbA1c) to 117 mg / dL (5.7% HbA1c). In various embodiments, treatment with TIMP-T1D reduces absolute HbA1c levels to less than 117 mg / dL (5.7% HbA1c).

[0136] In various embodiments, treatment with TIMP-T1D stabilizes hypoglycemic events relative to baseline measurements of the subject and / or placebo control. In various embodiments, treatment with TIMP-T1D stabilizes hypoglycemic events relative to baseline measurements of the subject and / or placebo control by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (all between these values). Reduce to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

[0137] In various embodiments, treatment with TIMP-T1D stabilizes exogenous insulin use relative to baseline values ​​of the subject and / or placebo control. In various embodiments, treatment with TIMP-T1D stabilizes exogenous insulin use relative to baseline values ​​of the subject and / or placebo control by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (between these values) Reduce to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

[0138] In various embodiments, treatment with TIMP-T1D stabilizes the time spent within the target blood glucose range of 70-180 mg / dL (3.9-10.0 mmol / L) relative to the baseline measurement of the subject and / or the placebo control. In various embodiments, treatment with TIMP-T1D stabilizes the time spent within the target blood glucose range of 70-180 mg / dL (3.9-10.0 mmol / L) relative to the baseline measurement of the subject and / or the placebo control by 5%-100% (e.g., approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%). Alternatively, it may be increased to approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)). In various embodiments, the time spent within the blood glucose range is measured by continuous glucose monitoring.

[0139] In various embodiments, treatment with TIMP-T1D stabilizes the time spent within the target blood glucose range of 70-110 mg / dL (3.9-6.1 mmol / L) relative to the baseline measurement of the subject and / or placebo control. In various embodiments, treatment with TIMP-T1D stabilizes the time spent within the target blood glucose range of 70-180 mg / dL (3.9-6.1 mmol / L) relative to the baseline measurement of the subject and / or placebo control by 5%-100% (e.g., approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%). Alternatively, it may be increased to approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)). In various embodiments, the time spent within the blood glucose range is measured by continuous glucose monitoring.

[0140] In various embodiments, treatment with TIMP-T1D stabilizes C-peptide levels measured at 30, 60, 90, and 120 minutes during MMTT compared to baseline measurements of the subject and / or placebo control. In various embodiments, treatment with TIMP-T1D stabilizes C-peptide levels measured at 30, 60, 90, and 120 minutes during MMTT compared to baseline measurements of the subject and / or placebo control by 5% to 100% (e.g., approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or... Increase by approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or by approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)).

[0141] In various embodiments, treatment with TIMP-T1D stabilizes fasting C peptide relative to baseline values ​​of the subject and / or placebo control. In various embodiments, treatment with TIMP-T1D stabilizes fasting C peptide relative to baseline values ​​of the subject and / or placebo control by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (these values) Increase by 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or by approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)).

[0142] In various embodiments, treatment with TIMP-T1D stabilizes the proinsulin-to-C peptide ratio relative to the baseline measurement of the subject and / or relative to the placebo control. In various embodiments, treatment with TIMP-T1D stabilizes the proinsulin-to-C peptide ratio relative to the baseline measurement of the subject and / or relative to the placebo control by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (these values) Reduce to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

[0143] In various embodiments, treatment with TIMP-T1D stabilizes insulin dose-adjusted HbA1c (IDAA1c) relative to the baseline measurement of the subject and / or relative to the placebo control. In various embodiments, treatment with TIMP-T1D stabilizes IDAA1c relative to the baseline measurement of the subject and / or relative to the placebo control by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (all values ​​between these values). Reduce to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

[0144] In various embodiments, treatment with TIMP-T1D stabilizes the insulin sensitivity score (ISS) relative to the baseline measurement of the subject and / or relative to the placebo control. In various embodiments, treatment with TIMP-T1D stabilizes the ISS relative to the baseline measurement of the subject and / or relative to the placebo control by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (all of these values). Reduce to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

[0145] In various embodiments, treatment with TIMP-T1D stabilizes blood glucose variability / fluctuations relative to the baseline measurement of the subject and / or relative to the placebo control. In various embodiments, treatment with TIMP-T1D stabilizes blood glucose variability / fluctuations relative to the baseline measurement of the subject and / or relative to the placebo control by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (between these values). Reduce to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

[0146] In various embodiments, treatment with TIMP-T1D stabilizes the proportion of patients with IDAA1c ≤9 compared to placebo control. In various embodiments, treatment with TIMP-T1D stabilizes the proportion of patients with IDAA1c ≤9 compared to baseline measurements of the subject and / or placebo control, ranging from 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%). Increase by 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or by approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)).

[0147] In various embodiments, treatment with TIMP-T1D stabilizes the percentage of patients with stimulated maximal C peptide levels above 0.2 nmol / L compared to placebo controls. In various embodiments, treatment with TIMP-T1D stabilizes the percentage of patients with stimulated maximal C peptide levels above 0.2 nmol / L compared to baseline measurements of subjects and / or placebo controls, ranging from 5% to 100% (e.g., approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.). Increase by approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or by approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)).

[0148] In various embodiments, treatment with TIMP-T1D stabilizes the secretion of cytokines IL-1, IL-2, IL-5, IL-13, IL-10, IL-17, interferon (IFN)γ, and tumor necrosis factor (TNF)α from PBMCs upon stimulation with T1D-associated antigens, compared to baseline measurements of the subject and / or placebo control. In various embodiments, TIMP-T1D treatment stimulates the secretion of cytokines IL-1, IL-2, IL-5, IL-13, IL-10, IL-17, interferon (IFN)γ, and tumor necrosis factor (TNF)α from PBMCs to T1D-associated antigens, increasing by 5% to 100% (e.g., approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%) from baseline levels in the target population and / or placebo control population. Change it to approximately 75%, 80%, 85%, 90%, 95%, or 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)).

[0149] In various embodiments, treatment with TIMP-T1D stabilizes serum concentrations of cytokines IL-1, IL-2, IL-5, IL-13, IL-10, IL-17, IFNγ, and TNFα relative to baseline values ​​of the subject and / or placebo control. In various embodiments, treatment with TIMP-T1D stabilizes serum concentrations of cytokines IL-1, IL-2, IL-5, IL-13, IL-10, IL-17, IFNγ, and TNFα relative to baseline values ​​of the subject and / or placebo control by 5% to 100% (e.g., approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%). Change it to %, approximately 95%, or approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)).

[0150] In various embodiments, treatment with TIMP-T1D stabilizes the secretion of cytokines IL-1, IL-2, IL-5, IL-13, IL-10, IL-17, interferon (IFN)γ, and tumor necrosis factor (TNF)α from PBMCs stimulated with anti-CD3 and anti-CD28, relative to baseline measurements of the subject and / or placebo control. In various embodiments, treatment with TIMP-T1D increases the secretion of cytokines IL-1, IL-2, IL-5, IL-13, IL-10, IL-17, interferon (IFN)γ, and tumor necrosis factor (TNF)α from PBMCs upon stimulation with anti-CD3 and anti-CD28 compared to baseline values ​​of the subject and / or placebo control, by 5% to 100% (e.g., approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%). Change it to %, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, or approximately 100% (including all values ​​and ranges between these values)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)).

[0151] In various embodiments, treatment with TIMP-T1D stabilizes PBMC proliferation upon stimulation with T1D-associated antigens relative to baseline measurements of the subject and / or placebo control. In various embodiments, treatment with TIMP-T1D stabilizes PBMC proliferation upon stimulation with T1D-associated antigens relative to baseline measurements of the subject and / or placebo control by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%). (Including all values ​​and ranges between these values), change to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or by approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)).

[0152] In various embodiments, treatment with TIMP-T1D stabilizes circulating methylated or unmethylated insulin DNA levels relative to baseline measurements of the subject and / or placebo control. In various embodiments, treatment with TIMP-T1D stabilizes circulating unmethylated insulin DNA levels relative to baseline measurements of the subject and / or placebo control by 5% to 100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%). Reduce to 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2 to 1 / 100 (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 1 / 100 (including all values ​​and ranges between these values)).

[0153] In various embodiments, treatment with TIMP-T1D stabilizes circulating hormone levels (e.g., glucagon, incretin, adiponectin) relative to baseline measurements of the subject and / or placebo control. In various embodiments, treatment with TIMP-T1D stabilizes circulating hormone levels (e.g., glucagon, incretin, adiponectin) relative to baseline measurements of the subject and / or placebo control by 5% to 100% (e.g., approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, or The value is changed to approximately 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, or approximately 2-100 times (for example, approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges between these values)).

[0154] The effectiveness of TIMP-T1D in improving one or more symptoms of T1D is determined by one or more of the following parameters assayed from one or more biological samples obtained from subjects: A. The proportion of effector T cells in the total T cell population. B. The proportion of Treg cells in the total T cell population, C. The proportion of effector B cells in the total B cell population. D. Levels of specific IgG and / or IgM, E. Levels of inflammatory cytokines and chemokines, F. Levels of anti-inflammatory cytokines and chemokines, G. Liver enzyme levels H. Levels of inflammatory metabolites, and I. Levels of anti-inflammatory metabolites.

[0155] In various embodiments, the effectiveness of TIMP-T1D in improving one or more symptoms of T1D is determined by the results of assays of one, two, three, four, five, six, seven, eight, or nine parameters listed above (a) to (i).

[0156] In various embodiments, the efficacy of TIMP-T1D in improving one or more symptoms of T1D and / or reducing the duration and severity of the inflammatory response to the T1D antigen is determined based on the use of alternative T1D therapy. In various embodiments, administration of TIMP-T1D reduces the use of alternative T1D therapy. In various embodiments, administration of TIMP-T1D stabilizes the use of alternative T1D therapy. In various embodiments, therapies are selected from the group consisting of insulin, insulin pumps, antihypertensive drugs, aspirin, cholesterol-lowering drugs, artificial pancreases, anti-IL-21 antibodies, B lymphocyte depletion (e.g., rituximab), anti-CD3 antibodies (e.g., teplizumab), adoptive transfer of Tregs (e.g., autologous Tregs, umbilical cord-derived Tregs, polyclonal Tregs), anti-thymocyte globulin, sodium-glucose cotransporter-2 (SGLT2) inhibitors (e.g., dapagliflozin, sotagliflozin), stem cell recruitment (e.g., plerixafor), β-cell transplantation, islet transplantation, stem cell transplantation, and incretin therapy (e.g., liraglutide). In various embodiments, insulin is selected from the group consisting of insulin glargine, ultra-rapid-acting insulin (e.g., insulin aspart, insulin lispro, insulin glulisine), short-acting insulin (ACTRAPID®, HUMULIN S®, INSUMAN RAPID®), long-acting insulin (LEVEMIR®, ABASAGLAR®, LANTUS®, TOUJEO®, TRESIBA®), or intermediate-acting insulin (INSULATARD®, INSUMAN BASAL®).

[0157] In various embodiments, administration of TIMP-T1D reduces the use of alternative T1D therapy by 1% to 100% (e.g., about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (including all values ​​and ranges between these values)), 10 to 95%, 15 to 90%, 20 to 85%, 25 to 75%, 30 to 70%, 35 to 65%, 40 to 60%, 45 to 55%, or 50% compared to baseline measurements of the subject and / or placebo control.

[0158] In various embodiments, the use of alternative T1D therapy is reduced or eliminated 1, 2, 3, 4, 5, 6, or 7 days after administration of TIMP-T1D. In various embodiments, the use of alternative T1D therapy is reduced or eliminated 1, 2, 3, or 4 weeks after administration of TIMP-T1D. In various embodiments, the use of alternative T1D therapy is reduced or eliminated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration of TIMP-T1D. In various embodiments, the use of alternative T1D therapy is reduced or eliminated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years after administration of TIMP-T1D.

[0159] Cells assayed from biological samples include immune cells, non-immune cells, and / or combinations thereof. Immune cells include innate immune cells, adaptive immune cells, and / or combinations thereof. Innate immune cells assayed from biological samples are antigen-presenting cells (APCs). Exemplary innate immune cells assayed from biological samples include monocytes, macrophages, neutrophils, granulocytes, dendritic cells, mast cells, eosinophils, basophils, and / or combinations thereof. Adaptive immune cells assayed from biological samples include effector immune cells such as CD4+ T cells, CD8+ T cells, B cells, NK cells, NK-T cells, and / or combinations thereof. In various embodiments, T cells are Th1 cells, Th2a cells, Treg cells, and Tr1 cells.

[0160] In certain embodiments, the cells assayed from a biological sample are epithelial cells, stromal cells, endothelial cells, fibroblasts, pericytes, adipocytes, mesenchymal stem cells, hematopoietic stem cells, hematopoietic progenitor cells, hepatocytes, hepatic sinusoidal endothelial cells (LSEC), and / or Kupffer cells.

[0161] Combination therapy The simultaneous administration of two therapeutic agents does not require that the drugs be administered simultaneously or via the same route, as long as there is an overlap in the periods during which the drugs exert their therapeutic effects. Prior, simultaneous, or consecutive administrations are intended, as are administrations on different days or weeks.

[0162] TIMP-T1D and the concomitant therapy are intended to be administered concurrently or simultaneously, in the same formulation or in separate formulations. It is further intended that TIMP-T1D and the concomitant therapy be administered in separate formulations, concurrently, with concurrent administration meaning that the drugs are administered within 30 minutes to 12 hours of each other.

[0163] In another embodiment, TIMP-T1D and the concomitant therapeutic agent are administered before the administration of the other composition. Pre-administration refers to administering TIMP-T1D and the concomitant therapeutic agent within a period ranging from one week before treatment with the other therapy to 30 minutes before administration of the other therapy.

[0164] In another embodiment, TIMP-T1D and the T1D therapeutic agent are administered following the administration of the other composition. Subsequent administration refers to the administration of TIMP-T1D and the T1D therapeutic agent within one week after treatment with the other therapy, or within 30 minutes after the administration of the other therapy.

[0165] It is further intended that other adjunctive or alternative therapies may be administered as needed.

[0166] In various embodiments, TIMP-T1D is administered alone or in combination with a T1D therapeutic agent. In various embodiments, the therapeutic agent is administered before, concurrently with, or subsequently to the administration of TIMP-T1D.

[0167] In various embodiments, the combination therapy is selected from the group consisting of insulin, insulin pumps, antihypertensive drugs, aspirin, cholesterol-lowering drugs, artificial pancreas, anti-IL-21 antibodies, B lymphocyte depletion (e.g., rituximab), anti-CD3 antibodies (e.g., teplizumab), adoptive transfer of Tregs (e.g., autologous Tregs, umbilical cord-derived Tregs, polyclonal Tregs), anti-thymocyte globulin, sodium-glucose cotransporter-2 (SGLT2) inhibitors (e.g., dapagliflozin, sotagliflozin), stem cell recruitment (e.g., plerixafor), β-cell transplantation, islet transplantation, stem cell transplantation, and incretin therapy (e.g., liraglutide). In various embodiments, insulin is selected from the group consisting of insulin glargine, ultra-rapid-acting insulin (e.g., insulin aspart, insulin lispro, insulin glulisine), short-acting insulin (ACTRAPID®, HUMULIN S®, INSUMAN RAPID®), long-acting insulin (LEVEMIR®, ABASAGLAR®, LANTUS®, TOUJEO®, TRESIBA®), or intermediate-acting insulin (INSULATARD®, INSUMAN BASAL®).

[0168] In various embodiments, the combination therapy induces Tregs. In various embodiments, the combination therapy increases the frequency and / or number of Tregs. In various embodiments, the combination therapy is an IL-2 therapy that induces Tregs. In various embodiments, the IL-2 therapy is a low-dose IL-2, an IL-2 mutain engineered to proliferate Tregs, an IL-2 variant engineered to proliferate Tregs, an IL-2 molecule engineered to be selective for high-affinity IL-2 receptors, a PEGylated IL-2, an IL-2 complex, or an IL-2 / CD25 fusion protein.

[0169] In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 days before the administration of TIMP-T1D. In various embodiments, the therapeutic agent is administered 1, 2, 3, or 4 weeks before the administration of TIMP-T1D. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months before the administration of TIMP-T1D. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years before the administration of TIMP-T1D. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 days after the administration of TIMP-T1D. In various embodiments, the therapeutic agent is administered 1, 2, 3, or 4 weeks after the administration of TIMP-T1D. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the administration of TIMP-T1D.

[0170] Pharmaceutical preparations The pharmaceutical compositions of this disclosure containing TIMP-T1D as an active ingredient may contain pharmaceutically acceptable carriers or additives depending on the route of administration. Examples of such carriers or additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylic acid, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, acacia gum, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, and pharmaceutically acceptable surfactants. The additives used are selected, as necessary, from the above or combinations thereof, depending on the dosage form of this disclosure, but are not limited to these.

[0171] The formulation of the pharmaceutical composition will vary depending on the chosen route of administration (e.g., solution, emulsion). A suitable composition containing the therapeutic agent to be administered can be prepared in a physiologically acceptable vehicle or carrier. Regarding solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions, or suspensions containing physiological saline and a buffer medium. Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or fixative oil. Intravenous vehicles may include various additives, preservatives, or liquids, nutrients, or electrolyte supplements.

[0172] Various aqueous carriers, such as sterile phosphate-buffered saline, bacteriostatic water, water, buffer water, 0.4% saline, and 0.3% glycine, may contain other proteins for enhanced stability, such as albumin, lipoproteins, and globulins, which have undergone mild chemical modifications.

[0173] Therapeutic formulations of inhibitors are prepared for storage by mixing the inhibitor of the desired purity in the form of a lyophilized formulation or aqueous solution with an optional physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the dose and concentration used and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than approximately 10 residues) polypeptides; serum albumin, gelatin The material comprises proteins such as tin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, algin, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0174] Preparations used for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filtration membrane.

[0175] The aqueous suspension may contain the active compound mixed with excipients suitable for the preparation of the aqueous suspension. Such excipients may be suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and acacia gum, and dispersing or wetting agents may be naturally occurring phosphatides, such as lecithin, or condensate products of alkylene oxide and fatty acids, such as polyoxyethylene stearate, or condensate products of ethylene oxide and long-chain aliphatic alcohols, such as heptadecaethyl-enoxycetanol, or condensate products of ethylene oxide and partial esters derived from fatty acids and hexitol, such as polyoxyethylene sorbitol monostearate, or condensate products of ethylene oxide and partial esters derived from fatty acids and hexitol anhydride, such as polyethylene sorbitan monostearate. The aqueous suspension may also contain one or more preservatives, such as ethyl or n-propyl, p-hydroxybenzoate.

[0176] The TIMP-T1D described herein can be freeze-dried for storage and reconstituted in a suitable carrier before use.

[0177] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the modified particles are mixed with at least one inert, pharmaceutically acceptable excipient or carrier, e.g., sodium citrate or dicalcium phosphate, and / or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarders such as paraffin; f) absorption enhancers such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl acid, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also include a buffer.

[0178] kit In additional embodiments, the Disclosure includes a kit comprising one or more compounds or compositions packaged in a manner that facilitates their use in carrying out the methods of the Disclosure. In one embodiment, such a kit comprises a compound or composition described herein (e.g., a composition comprising TIMP alone or in combination with a second agent), is packaged in a container such as a sealed bottle or vessel, and has a label affixed to the container or included in the package that describes the use of the compound or composition in carrying out the method. Preferably, the compound or composition is packaged in unit dosage form. The kit may further include a suitable device for administering the composition according to a specific route of administration or for carrying out a screening assay. Preferably, the kit includes a label that describes the use of the inhibitor composition.

[0179] In further embodiments, the Disclosure provides a product or unit dosage form comprising (a) a composition of a substance comprising TIMP-T1D as described herein, (b) a container comprising the composition, and (c) a label affixed to the container or a document included in the container, with reference to the use of TIMP-T1D in the treatment of T1D as described herein.

[0180] Additional aspects and details of this disclosure will be apparent from the following examples, which are intended to be illustrative rather than restrictive. [Examples]

[0181] Example 1: Phase 1b / 2a trial of TIMP-T1D in patients with T1D A phase 1b / 2a double-blind, randomized, placebo-controlled trial will be conducted to evaluate the safety, tolerability, pharmacodynamics (PD), and efficacy of TIMP-T1D particles (CNP-103). CNP-103 consists of PLGA nanoparticles encapsulating T1D antigens including PPI, ZnT8, IGRP, and GAD65. CNP-103 particles have an average diameter of 400–800 nm and a negative zeta potential of -30 mV–-80 mV. CNP-103 will be supplied as a lyophilized formulation. CNP-103 will be reconstituted in sterile water for injection and diluted in sterile saline (0.9% sodium chloride, USP) before administration. Participants aged 18–35 years with recently diagnosed stage 3 type 1 diabetes mellitus (T1D) will be screened up to 14 days prior to enrollment in the trial. Note: If a subject is currently receiving standard treatment, they may continue their current standard treatment throughout the study, at the discretion of the principal investigator, regardless of the treatment group (CNP-103 or placebo). Screening will be completed according to the event schedule (Figure 1). Subjects who meet all inclusion criteria and do not meet exclusion criteria after completing their screening visit will be enrolled in this study. Subjects will then be randomized on day 1 in a 1:1 ratio to the then-available dose-level cohort and will receive two separate intravenous doses of either CNP-103 or placebo on days 1 and 8. The study drug will be administered by IV infusion over approximately 3-4 hours using a stepped infusion rate. Subjects will be medically observed in the clinic for acute adverse events for 4 hours after infusion on days 1 and 8. Participants will be discharged 4 hours after infusion if all scheduled assessments are completed on the day of visit, vital signs (sitting or supine blood pressure, heart rate, and body temperature) measured 4 hours after infusion are within the expected range, and no other health concerns are raised by the principal investigator. After administration, participants will return to the clinic for immunosafety clinical tests, PD measurements, reporting of hypoglycemic events and insulin unit use, evaluation of adverse events, and medication changes according to the event schedule (Figure 1). Participants will undergo a mixed-food challenge test (MMTT) at the screening day, day 90, day 180, and day 360 of the study. On the screening day, day 76, day 166, day 256, and day 346, patients will have an implanted glucose sensor for continuous glucose monitoring.Glucose data (blood glucose target) will be read from the sensor on the screening day, day 1, day 76, day 90, day 166, day 180, day 256, day 270, day 346, and day 360. Participants will return to the clinic for the final evaluation of MMTT, safety clinical laboratory data collection, PD measurement, hypoglycemic events and insulin unit use reporting, and AEs and medication changes, and to conclude their trial visits.

[0182] The trial will enroll up to three cohorts (6 subjects per cohort) at repeated dose escalation levels in the dose-level evaluation phase, followed by an expansion phase at the safe and tolerable doses identified in the dose-level evaluation phase. Subjects will be randomized in a 1:1 ratio to receive either CNP-103 or placebo (0.9% sodium chloride injection, USP) as a 200 mL intravenous infusion on days 1 and 8. The planned dose levels are as follows: ● Cohort 1: 150 mg ● Cohort 2: 350 mg ● Cohort 3: 550 mg CNP-103 is administered using the following stepwise infusion rates: ● For the first 15 minutes, 20 mL / hour, ●For the next 15 minutes, 40 mL / hour, ●The remaining infusion time is 80 mL / hour. Medication administration to subjects within Cohort 1 will be isolated for at least 48 hours. After all subjects in a dose cohort have completed their 15-day outpatient visit (7 days after the second dose), a DMC will be convened to review all available safety data and determine whether it is acceptable to proceed to the next dose-escalation cohort, whether cohort expansion is justified, or whether any other clinical recommendations should be made. An interim analysis will be performed after all subjects in a dose cohort have completed 180 days.

[0183] Participation criteria 1. Subjects who are willing and able to provide written informed consent and privacy language approved by the Institutional Review Board (IRB) in accordance with domestic regulations. 2. Men, and women who are not pregnant or breastfeeding, including those aged 18-35. 3. Diagnosis of T1D within 6 months prior to trial enrollment, ≥1 autoantibody and according to at least one of the American Diabetes Association [ADA] criteria: ● Fasting plasma glucose (FPG) level ≥ 126 mg / dL (7.0 mmol / L) ● Random plasma glucose levels ≥ 200 mg / dL (11.1 mmol / L) in patients with typical symptoms of hyperglycemia or hyperglycemic emergency. 4. Patients with a peak stimulated C peptide level >0.2 nmol / L as measured by a mixed food challenge test (MMTT). 5. The study included women who are unable to become pregnant (e.g., those who have undergone surgical sterilization or have not had a menstrual period for one year). 6. A woman of potential pregnancy who agrees not to become pregnant during the trial, has a negative pregnancy test at the time of her screening visit, and agrees to one of the following: a. Continue using two highly effective forms of contraception from the start of the initial screening until day 360 throughout the entire trial. b. Continue practicing abstinence from the start of the initial screening until day 360 of the entire trial. 7. This program is for women who agree not to breastfeed from the start of the initial screening until day 360 throughout the entire trial. 8. This program is for women who agree not to donate eggs from the start of the initial screening until day 360 of the entire trial. 9. Men who have a spouse or a partner of potential pregnancy and who have agreed that they and their spouse or partner will proactice an effective form of contraception, as discussed by the study physician or study staff, from the start of screening until day 360 throughout the entire study.

[0184] Exclusion Criteria 1. Those who have used the following medications: ●T-cell depletion immunotherapy (e.g., teprizumab) within 12 months prior to the first dose of medication. ●T-cell modification immunotherapy within 6 months prior to the first dose (e.g., abatacept, etanercept, ustekinumab) ● The exclusion of additional immunomodulations is at the discretion of the medical monitor and the principal investigator at the trial site. 2. Subjects with any complex medical problems that interfere with blood collection or monitoring. 3. Subjects with a body mass index (BMI) below the 5th percentile and above the 95th percentile, according to age and sex. 4. Patients with a history of tuberculosis or a positive PPD skin test result. 5. Individuals who received the vaccine within the following time frame: ● Live vaccine (other than intranasal influenza) administered within 28 days prior to screening. ● Any subunit vaccine administered within 14 days prior to screening. 6. Any COVID-19 vaccine administered within 14 days prior to screening. Subjects who have received their first dose of any COVID-19 vaccine are exempt from screening in this study until 14 days have passed since their last dose of the vaccine, if applicable. 7. Known or suspected acute infection, including COVID-19, at the time of screening or within two weeks prior to screening. After confirming a recent COVID-19 infection, a negative polymerase chain reaction test is required before randomization. Any scheduled vaccination prior to day 8.15. 9. Subjects who have used systemic steroids within 3 months prior to screening. 10. Subjects whose laboratory test results at the time of screening or before the investigational drug administration are outside the normal limits and who are considered clinically significant by the principal investigator. 11. Subjects who have positive test results for hepatitis B surface antigen (HBsAg), hepatitis C virus (HCV) antibody, or human immunodeficiency virus (HIV) antigen / antibody, as determined at the time of screening. 12. Subjects with a history of immune disorders other than T1D (including autoimmune diseases) or currently active immune disorders (unless, after discussion with the medical monitor, the subject is deemed eligible to participate in this study). 13. Subjects with a history of or current active disease requiring immunosuppressants (including azathioprine, prednisone, prednisolone, budesonide, cyclosporine, tacrolimus, methotrexate, or mycophenolate mofetil) (unless, after consultation with the medical monitor, the subject is deemed eligible to participate in this study). 14. Subjects with a clinical history of severe cardiovascular disease within the past 12 months. 15. Subjects with complications of malignant tumors or a medical history thereof. 16. In the opinion of the principal investigator, subjects who are unable to follow the trial procedures. 17. Patients who received an investigational therapy other than CNP-103 within 28 days prior to screening or within 5 half-lives, whichever is longer. 18. In the opinion of the principal investigator, any subject who has any known active condition that would make them unsuitable for participation in the study. 19. Known susceptibility of CNP-103 to any component.

[0185] The primary objective of this study is to evaluate the safety and tolerability of CNP-103. General safety assessment: ● Frequency of adverse events (AEs) and serious adverse events (SAEs), MedDRA23.0 (CTCAE v.5.0) or latest version Immunosafety assessment: ●Serum cytokines (IL-1β, TNF-α, IL-6, MCP-1, MIP-1α, IFN-γ, IL-4, IL-10)

[0186] A secondary or exploratory objective of this study is to evaluate the efficacy of CNP-103. ● Change from baseline in the area under the curve (AUC) of the stimulated C-peptide response over the first two hours of the 4-hour MMTT between CNP-103 and placebo at days 180 and 360. ● Maintenance of C-peptide from baseline between CNP-103 and placebo at days 180 and 360. ● Changes from baseline in antigen-specific T cells between CNP-103 and placebo on day 15 ● Changes from baseline in activated antigen-specific T cells between CNP-103 and placebo on day 15 ● Changes from baseline in antigen-specific Tregs between CNP-103 and placebo on day 15 ● Changes from baseline in HbA1c levels between CNP-103 and placebo at days 180 and 360 ● Change from baseline in the number of hypoglycemic events between CNP-103 and placebo at days 180 and 360. ● Changes from baseline in insulin use between CNP-103 and placebo at days 180 and 360. ● Change from baseline in time within the blood glucose target range of 70-180 mg / dL (3.9-10.0 mmol / L) [measured by continuous glucose monitoring] between CNP-103 and placebo at days 180 and 360.

[0187] Example 2: Booster administration in a preclinical NOD mouse study Preclinical administration of CNP-T1D in NOD mice with booster doses. CNP-T1D contains surface-functionalized carboxylated PLGA nanoparticles, 400-800 nm in diameter, encapsulated recombinant insulin with a voltage of -80 mV to -30 mV, GAD65, and chromogranin A. NOD mice with dysglycemic abnormalities and blood glucose levels of 150-250 mg / dL were divided into three groups. One group was treated with two loading doses of CNP-T1D at weeks 16 and 17. The second group was treated with two loading doses of CNP-T1D at weeks 16 and 17, followed by booster doses every four weeks (weeks 21, 25, and 29), and then finally discontinued. The third group was a control group treated with saline. Treatment of mice with two doses of CNP-T1D significantly protected them from T1D expression, with enhanced protection observed by including a booster dose, compared to both doses with saline (Figure 2A).

[0188] Dysglycemic NOD mice with blood glucose levels of 150-250 mg / dL were divided into three groups. Group 1 was treated with two loading doses of 2.5 mg of CNP-T1D at weeks 16 and 17. Group 2 was treated with two loading doses of 2.5 mg of CNP-T1D at weeks 16 and 17, followed by booster doses of 2.5 mg every four weeks (weeks 21, 25, and 29), and finally discontinued at week 30. Group 3 was a control group treated with saline.

[0189] Treatment of mice with either two or five doses of 2.5 mg of CNP-T1D significantly improved protection against T1D progression in dysglycemic NOD mice compared to control NOD mice treated with physiological saline (***p<0.0005) (Figures 2A and 2B). However, reversal of dysglycemic abnormalities, i.e., reduction of blood glucose levels to normal blood glucose levels (<150 mg / dL), was more pronounced in NOD mice receiving a five-dose booster regimen compared to mice receiving two loading doses. The comparison of CNP-T1D between the 2.5 mg dose, the two-dose dose, and the five-dose dose was significant (*p=0.0183) (Figure 3). These results indicate that TIMP-T1D is effective in inducing tolerance and treating the symptoms of type 1 diabetes in NOD mice.

[0190] Example 3: Phase 1b / 2a trial of TIMP-T1D in patients with T1D using a 3-dose regimen. This study is a Phase 1b / 2a human first-in-hospital (FIH) clinical trial to evaluate the safety, tolerability, pharmacodynamics (PD), and efficacy of CNP-103. The study consists of an escalation phase and an expansion phase with 28 and 180 day study days for screening. Upon completion of the study, all subjects will have the opportunity to roll over to a long-term follow-up study. CNP-103 consists of PLGA (50:50 acid-terminated) nanoparticles encapsulating T1D antigens including PPI, ZnT8, IGRP, and GAD65. CNP-103 particles have an average diameter of 400–800 nm and a negative zeta potential of -30 mV–-80 mV. Subjects aged 18–35 years with recently diagnosed stage 3 type 1 diabetes (T1D) will be screened up to 28 days prior to enrollment in the study. Note: If a subject is currently receiving standard treatment, they may continue their current standard treatment throughout the study, regardless of the treatment group (CNP-103 or placebo), at the discretion of the principal investigator. Screening will be completed according to the event schedule (Figure 4). Subjects who meet all inclusion criteria and do not meet exclusion criteria after completing their screening visit will be enrolled in this study. Subjects will then be randomized on day 1 to the then-available dose-level cohort in a 2:1 ratio and will receive three separate intravenous doses of either CNP-103 or placebo on days 1, 8, and 90. The study drug will be administered by IV infusion over approximately 3-4 hours using a stepped infusion rate. Subjects will be medically observed in the clinic for acute adverse events for 4 hours after infusion on days 1, 8, and 90. Participants will be discharged 4 hours after their visit if all scheduled assessments are completed, their vital signs (seated or supine blood pressure, heart rate, and body temperature) measured 4 hours after infusion are within the expected range, and the principal investigator does not raise any other health concerns. Participants will return to the clinic for immunosafety clinical tests, PD measurement, continuous glucose monitoring, reporting of hypoglycemic events and insulin unit use, evaluation of adverse events, and medication changes according to the event schedule (Figure 4). Participants will undergo mixed-food challenge tests (MMTT) at their trial visits on the screen day, day 90, and day 180.

[0191] The subjects will return to the clinic for the final evaluation of MMTT, safety clinical laboratory data collection, PD measurement, continuous glucose monitoring, hypoglycemic events and insulin unit use reporting, and AEs and medication changes, as well as for the conclusion of their trial visits.

[0192] The trial will enroll up to three cohorts (6 subjects per cohort) at repeated dose escalation levels in the escalation phase, followed by an expansion phase at the safe and tolerable doses identified in the escalation phase. Subjects will be randomized in a 2:1 ratio to receive either CNP-103 or placebo (0.9% sodium chloride injection, USP) as a 200 mL intravenous infusion on days 1, 8, and 90. The planned dose levels are as follows: ● Cohort 1: 100 mg ● Cohort 2: 300 mg ● Cohort 3: 600 mg CNP-103 is administered using the following stepwise infusion rates: ● For the first 15 minutes, 20 mL / hour, ●For the next 15 minutes, 40 mL / hour, ●The remaining infusion time is 80 mL / hour. Medication administration to subjects within Cohort 1 will be isolated for at least 48 hours. After all subjects in a dose cohort have completed their 15-day outpatient visit (7 days after the second dose), a DMC will be convened to review all available safety data and determine whether it is acceptable to proceed to the next dose-escalation cohort, whether cohort expansion is justified, or whether any other clinical recommendations should be made.

[0193] Participation criteria ●Those who are willing and able to provide written informed consent and privacy language approved by the Institutional Review Board (IRB) in accordance with domestic regulations. ● This includes men and women who are not pregnant or breastfeeding, aged 18-35. ● A diagnosis of T1D within 180 days prior to trial enrollment, with ≥1 autoantibody and at least one of the American Diabetes Association (ADA) criteria: ○Fasting plasma glucose (FPG) level ≥ 126 mg / dL (7.0 mmol / L) ○ Random plasma glucose ≥ 200 mg / dL (11.1 mmol / L) in patients with typical symptoms of hyperglycemia or hyperglycemic emergency. ● Patients with a peak stimulated C peptide level >0.2 nmol / L as measured by a mixed food challenge test (MMTT). ●For subjects taking any medication used to treat symptoms of T1D (e.g., corticosteroids), the subject must have been taking a stable dose for at least three months prior to enrollment and must agree not to increase their dose throughout the 180-day trial unless reviewed and approved by the medical monitor and the principal investigator. ●This program is for women of childbearing potential who agree not to become pregnant during the trial, have a negative pregnancy test result at the time of their screening visit, and agree to one of the following: ○Continuous use of two highly effective forms of contraception from the start of initial screening throughout the entire trial. ○Continue practicing abstinence from the start of the initial screening throughout the entire examination. ●This study is for women who agree not to breastfeed from the start of the initial screening until the end of the trial. ●This trial is for women who agree not to donate eggs from the start of the initial screening throughout the entire trial. ● Men who have a spouse or a partner who may become pregnant, and who agree that they and their spouse or partner will practice an effective form of contraception, as discussed by the study physician or study staff from the start of screening through the entire study.

[0194] The primary objective of this study is to evaluate the safety and tolerability of CNP-103. General safety assessment: ● Frequency of adverse events (AEs) and serious adverse events (SAEs), MedDRA23.0 (CTCAE v.5.0) or latest version Immunosafety assessment: ●Serum cytokines (IL-1β, TNF-α, IL-6, MCP-1, MIP-1α, IFN-γ, IL-4, IL-10)

[0195] A secondary or exploratory objective of this study is to evaluate the efficacy of CNP-103. ● Change from baseline in the area under the curve (AUC) of the stimulated C-peptide response over the first two hours of the 2-hour MMTT between CNP-103 and placebo at days 180 and 360. ● Maintenance of C-peptide from baseline between the CNP-103 and placebo treatment groups at days 180 and 360. ● Changes from baseline in antigen-specific T cells between CNP-103 and placebo at days 60 and 180. ● Changes from baseline in activated antigen-specific T cells between CNP-103 and placebo at days 60 and 180. ● Changes from baseline in antigen-specific Tregs between CNP-103 and placebo at days 60 and 180. ● Changes from baseline in HbA1c levels between CNP-103 and placebo at days 180 and 360 ● Change from baseline in the number of severe hypoglycemic events between CNP-103 and placebo at days 180 and 360. ● Changes from baseline in insulin use between CNP-103 and placebo at days 180 and 360. ● Change from baseline in the time spent within the blood glucose target range of 70-180 mg / dL (3.9-10.0 mmol / L) [measured by continuous glucose monitoring] between CNP-103 and placebo at days 180 and 360.

[0196] It is understood that all embodiments of the Disclosure described herein may be optionally combined with one or more other embodiments described herein. All patent and non-patent documents referenced herein are incorporated herein by reference in their entirety.

[0197] Therefore, it should be understood that the present invention is not limited to any specific embodiment disclosed, but is intended to encompass the spirit and scope of the invention as defined by the appended claims, all modifications within the above description, and / or shown in the appended drawings. Accordingly, only limitations that appear in the appended claims should be included in this disclosure.

[0198] References 1.Gepts,W.Islet changes suggesting a possible immune aetiology of human diabetes mellitus.Acta Endocrinol.Suppl.1976;205,95-106. 2. Gregory, GA et al. Glolab incidence, prevelance, and mortality of type 1 diabetes in 2021 with projection to 2040: a modeling study. The Lancet Diabetes & Endocrinology. 2022;10:P741-760.

Claims

1. A method for treating type 1 diabetes mellitus (T1D) in a subject, comprising administering to the subject a tolerance-modified immunomodulatory particle (TIMP-T1D) encapsulating one or more T1D-related antigens, wherein the TIMP-T1D is administered in a dose level of 1 mg to 800 mg.

2. The method according to claim 1, wherein TIMP-T1D is administered in a fixed dose of 1 mg, 2 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, or 800 mg.

3. The method according to claim 1 or 2, wherein TIMP-T1D is administered at a dose level of approximately 0.01 mg / kg to 12 mg / kg.

4. The method according to any one of claims 1 to 3, wherein the T1D-related antigen comprises insulin (preproinsulin (PPI)), tyrosine phosphatase-like autoantigen or insulinoma antigen-2 (IA2; ICA512, PTPRN), IA-2β (fogrin, PTPRN2), GAD65, GAD67, islet cell antigen-69 (ICA69), IGRP, ZnT8, chromogranin A, 38kDa granule antigen, periferin, islet amyloid peptide (ppIAPP), carboxypeptidase H / E, heat shock protein 60 (HSP60), heat shock protein 70 (HSP70) family, sulfatide, and vasostatin-1.

5. The method according to any one of claims 1 to 4, wherein the T1D-related antigen is one of those listed in Table 1.

6. The method according to any one of claims 1 to 5, wherein the T1D-related antigen is selected from the group consisting of SEQ ID NOs: 1 to 17.

7. The method according to any one of claims 1 to 6, wherein the T1D-related antigen comprises PPI, ZnT8, IGRP, and GAD65.

8. The method according to claim 7, wherein the T1D-related antigen includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:

16.

9. The method according to any one of the prior claims, wherein the TIMP-T1D particles have an average diameter of 100 nm to 1500 nm.

10. The method according to any one of the prior claims, wherein the TIMP-T1D particles have an average diameter of 400 nm to 800 nm.

11. The method according to any one of the prior claims, wherein the TIMP-T1D particles have a negative zeta potential.

12. The method according to any one of the prior claims, wherein the particles have a negative zeta potential of -30 mV to -100 mV.

13. The method according to any one of the prior claims, wherein TIMP-T1D is administered at a concentration of 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 3.25 mg / mL, 3.5 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12.5 mg / mL, 15 mg / mL, 17.5 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, or 50 mg / mL.

14. The method according to any one of the prior claims, wherein TIMP-T1D is administered at a dose level of 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 6 mg / kg, 8.0 mg / kg, 10 mg / kg, or 12 mg / kg.

15. The method according to any one of the prior claims, wherein TIMP-T1D is administered in a single dose or multiple doses.

16. The method according to any one of the prior claims, wherein TIMP-T1D is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year.

17. The method according to any one of the prior claims, wherein TIMP-T1D is administered in two doses at one-week intervals.

18. The method according to any one of the prior claims, wherein TIMP-T1D is administered in two loading doses at one-week intervals, followed by a booster dose administered as a single dose once every three months.

19. The method according to any one of the prior claims, wherein TIMP-T1D is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intralymphatically, intranasally, by inhalation, or orally.

20. The method according to any one of the prior claims, wherein administration to a patient with TIMP-T1D reduces or improves one or more symptoms of T1D.

21. The method according to claim 20, wherein one or more of the symptoms of T1D are selected from the group consisting of increased or frequent urination, increased thirst, increased hunger, extreme fatigue, blurred vision, muscle loss, slow-healing cuts or bruises, coronary artery disease with chest pain, heart attack, stroke, atherosclerosis, hypertension, nerve damage, kidney damage, diabetic retinopathy, diabetic ketoacidosis, foot injury, increased intracutaneous and oral bacterial and viral infections, periodontal disease, pregnancy complications such as miscarriage, stillbirth, and birth defects, exogenous insulin use, hypoglycemic events, elevated HbA1c, impaired β-cell function, decreased C-peptide levels, decreased time within the blood glucose target range of 70–110 mg / dL (3.9–6.1 mmol / L), i.e., hyperglycemia, and weight loss.

22. The method according to any one of the prior claims, wherein administration to TIMP-T1D reduces the duration and / or severity of an autoimmune response or inflammatory immune response to the T1D antigen.

23. The method according to claim 22, wherein the autoimmune response or inflammatory immune response is a T cell, B cell, or myeloid cell response.

24. The method according to claim 22 or 23, wherein the inflammatory immune response is assayed from one or more biological samples obtained from the subject.

25. The method according to claim 24, wherein the biological sample is selected from the group consisting of whole blood, peripheral blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, and / or bone marrow biopsy.

26. The method according to any one of claims 1 to 25, wherein administration of TIMP-T1D induces immune anergy.

27. The method according to any one of claims 1 to 25, wherein administration of TIMP-T1D induces apoptosis of macrophages or monocytes in the subject.

28. The method according to any one of claims 1 to 25, wherein administration of TIMP-T1D reduces the islet stress response, reduces ineffective regulation of effector T cells, reduces islet / β-cell damage, reduces epigenetic scarring, reduces immune effector amplification, reduces metabolic dysfunction, or reduces immune infiltration into the islets.

29. The method according to any one of claims 1 to 25, wherein administration of TIMP-T1D induces islet cell / β-cell regeneration, increases β-cell mass / number, induces Tr1 cells, increases the Treg:Teff ratio, induces bystander tolerance, induces infectious tolerance, induces binding inhibition, induces T-cell exhaustion, induces T-cell apoptosis, induces T-cell deletion, or induces genotype-independent tolerance.

30. The method according to any one of claims 1 to 25, wherein administration of TIMP-T1D induces retention / sequesteration of effector T cells and induces regulatory APC, IL-10, IL-35, TGF-β, CTLA-4 expression, ICOS expression, GITR expression, PD-L1 / L2 expression, PD-1 expression, FasL marker, or PGE2.

31. The method according to any one of claims 1 to 25, wherein administration of TIMP-T1D reduces IL-6, IL-12, IL-23, IL-1β, TNF, type 1 interferon, CD80 expression, CD86 expression, CD40 expression, or T-bet expression.

32. The method according to any one of the prior claims, wherein administration to TIMP-T1D reduces or stabilizes the level of activated antigen-specific T cells.

33. The method according to claim 32, wherein administration to TIMP-T1D reduces or stabilizes the level of activated antigen-specific CD4+ and / or CD8+ T cells.

34. The method according to claim 32 or 33, wherein administration of TIMP-T1D reduces the level of the activated antigen-specific T cells by 5% to 100% or 2 to 1 / 100th compared to the baseline measurement of the subject and / or a placebo control.

35. The method according to any one of the prior claims, wherein administration to TIMP-T1D reduces or stabilizes the level of antigen-specific T cells.

36. The method according to claim 35, wherein administration to TIMP-T1D reduces or stabilizes the level of antigen-specific CD4+ and / or CD8+ T cells.

37. The method according to claim 35 or 36, wherein administration of TIMP-T1D reduces the level of antigen-specific T cells by 5% to 100% or 2 to 1 / 100th compared to the baseline measurement of the subject and / or a placebo control.

38. The method according to any one of the prior claims, wherein administration to TIMP-T1D increases the level of antigen-specific Treg cells.

39. The method according to claim 38, wherein administration of TIMP-T1D increases the level of antigen-specific Treg cells by 5% to 100% or 2 to 100 times compared to the baseline measurement of the subject and / or placebo control.

40. The method according to any one of the prior claims, wherein administration to TIMP-T1D stabilizes or increases the area under the curve (AUC) of the stimulated C-peptide response.

41. The method according to claim 40, wherein administration of TIMP-T1D increases the area under the curve (AUC) of the stimulated C-peptide response by 5% to 100% or 2 to 100 times compared to the baseline measurement of the subject and / or the placebo control.

42. The method according to any one of the prior claims, wherein administration to TIMP-T1D stabilizes or increases C-peptide levels.

43. The method according to claim 42, wherein administration of TIMP-T1D increases the C-peptide level by 5% to 100% or about 2 to 100 times compared to the baseline measurement of the subject and / or compared to a placebo control.

44. The method according to any one of the prior claims, wherein administration to TIMP-T1D stabilizes or reduces HbA1c levels.

45. The method according to claim 44, wherein administration of TIMP-T1D reduces the HbA1c level to less than 140 mg / dL (or 6.5% HbA1c), between 137 mg / dL (6.4% HbA1c) and 117 mg / dL (5.7% HbA1c), or less than 117 mg / dL (5.7% HbA1c).

46. The method according to claim 44 or 45, wherein administration of TIMP-T1D reduces the HbA1c level by 5% to 100% or about 2 to 1 / 100 compared to the baseline measurement of the subject and / or a placebo control.

47. The method according to any one of the prior claims, wherein administration to TIMP-T1D stabilizes or reduces hypoglycemic events.

48. The method according to claim 47, wherein administration to TIMP-T1D reduces hypoglycemic events by 5% to 100% or 2 to 100 times.

49. The method according to any one of the prior claims, wherein administration to TIMP-T1D stabilizes or reduces insulin use.

50. The method according to claim 49, wherein administration to TIMP-T1D reduces insulin use by 5% to 100% or 2 to 100 times.

51. The method according to any one of the prior claims, wherein administration to TIMP-T1D stabilizes or increases the time the blood glucose is within the target range of 70–110 mg / dL (3.9–6.110.0 mmol / L).

52. The method according to claim 51, wherein administration to TIMP-T1D increases the time spent within the blood glucose target range of 70-110 mg / dL (3.9-6.110.0 mmol / L) by 5% to 100% or 2 to 100 times.

53. The method according to claim 51 or 52, wherein the time spent within the blood glucose range is measured by continuous glucose monitoring.

54. The method according to any one of the prior claims, wherein administering TIMP-T1D stabilizes or reduces the use of alternative therapies.

55. The method according to claim 54, wherein the therapy is selected from the group consisting of insulin, insulin pump, antihypertensive drugs, aspirin, cholesterol-lowering drugs, artificial pancreas, anti-IL-21 antibody, B lymphocyte depletion, anti-CD3 antibody, Treg adoptive transfer, anti-thymocyte globulin, sodium-glucose cotransporter-2 (SGLT2) inhibitor, stem cell mobilization, β-cell transplantation, islet transplantation, stem cell transplantation, and incretin therapy.

56. The method according to claim 54 or 55, wherein administration of TIMP-T1D reduces the use of alternative T1D therapy in the subjects by 1% to 100%.

57. The method according to any one of the prior claims, wherein TIMP-T1D is administered in combination with a T1D therapeutic agent.

58. The method according to claim 57, wherein the combination therapy is selected from the group consisting of insulin, insulin pump, antihypertensive drugs, aspirin, cholesterol-lowering drugs, artificial pancreas, anti-IL-21 antibody, B lymphocyte depletion, anti-CD3 antibody, Treg adoptive transfer, anti-thymocyte globulin, sodium-glucose cotransporter-2 (SGLT2) inhibitor, stem cell mobilization, β-cell transplantation, islet transplantation, stem cell transplantation, and incretin therapy.

59. The method according to claim 58, wherein the insulin is selected from the group consisting of insulin glargine, ultra-rapid-acting insulin, short-acting insulin, long-acting insulin, or intermediate-acting insulin.

60. The method according to claim 57, wherein the concomitant therapeutic agent increases the frequency and / or number of Tregs.

61. The method according to claim 60, wherein the concomitant therapeutic agent is IL-2 therapy that induces Treg.

62. The method according to claim 61, wherein the combination therapy agent is low-dose IL-2, IL-2 mutein engineered to proliferate Treg, IL-2 variant engineered to proliferate Treg, IL-2 molecule engineered to be selective for high-affinity IL-2 receptor, PEGylated IL-2, IL-2 complex, or IL-2 / CD25 fusion protein.

63. The method according to any one of claims 57 to 62, wherein the therapeutic agent is administered before, in conjunction with, or subsequently to the administration of TIMP-T1D.

64. Administering a booster dose of TIMP-T1D can be used to evaluate the following: changes in the area under the curve (AUC) of the stimulated C-peptide response over the first two hours of a 2 or 4-hour MMTT, maintenance of C-peptide levels, changes in maximum C-peptide during the MMTT, changes in C-peptide measured at 30, 60, 90, and 120 minutes during the MMTT, changes in fasting C-peptide, proinsulin to C-peptide ratio, changes in antigen-specific T cells, changes in activated antigen-specific T cells, changes in antigen-specific T-cells, changes in HbA1c levels, and hypoglycemia. Changes in the number of hypoglycemic or severe hypoglycemic events, changes in the proportion of hypoglycemic events, changes in the use / intake of exogenous insulin, changes in the time spent within the blood glucose target range of 70-180 mg / dL (3.9-10.0 mmol / L), changes in the time spent within the blood glucose target range of 70-110 mg / dL (3.9-6.1 mmol / L), changes in insulin dose-adjusted HbA1c (IDAA1c), insulin sensitivity score (ISS), changes in blood glucose variability / variability, percentage of patients with IDAA1c ≤9, and 0.2 nmol / L (0.6 ng) at 15 months. Percentage of patients with a maximum stimulated C peptide level greater than 0.2 nmol / L (0.6 ng / mL), percentage of patients with a 90-minute stimulated C peptide level greater than 0.2 nmol / L (0.6 ng / mL) at 15 months, changes in body weight and body mass index, serum autoantibody concentrations, serum autoantibody isotype concentrations (IgG1, IgG2, IgG3, IgG4), cytokines IL-1, IL-2, IL-5, IL-13, IL-10, IL-17, interferon (IFN) γ, tumor necrosis factor (TNF) α by PBMCs upon stimulation with T1D-related antigens The method according to any one of the prior claims, determined based on one or more of the following: secretion, serum concentrations of cytokines IL-1, IL-2, IL-5, IL-13, IL-10, IL-17, IFNγ, and TNFα; secretion of cytokines IL-1, IL-2, IL-5, IL-13, IL-10, IL-17, IFNγ, and TNFα by PBMCs upon stimulation with anti-CD3 and anti-CD28; proliferation of PBMCs upon stimulation with T1D-related antigens; circulating methylated and unmethylated insulin DNA levels; or circulating hormone levels.

65. The method according to any one of the prior claims, wherein the subject has recently developed T1D.

66. The method according to any one of the prior claims, wherein the subject has a stage T1D of stage 3.

67. The aforementioned subjects have autoantibodies specific to pancreatic β-cell protein ≥ 1, autoantibodies against GAD65, IA-2, ZnT8, insulin, or pancreatic islet cell cytoplasmic protein autoantibodies, autoantibody levels < 50,000 IU / mL, fasting plasma glucose (FPG) levels ≥ 126 mg / dL (7.0 mmol / L), random plasma glucose ≥ 200 mg / dL (11.1 mmol / L) with typical symptoms of hyperglycemia or hyperglycemic emergency, peak-stimulated C peptide ≥ 0.2 nmol / L during a mixed food challenge test, fasting C peptide levels ≥ 0.12 nmol / L (≥ 0.36 ng / mL), and The method according to any one of the prior claims, wherein the patient has a ventricular C-peptide level of ≥0.12 to ≤1.5 nmol / L (≥0.36 to ≤4.5 ng / mL), an HbA1c level of ≥5.6%, an HbA1c level of approximately ≤12%, has been receiving a stable insulin dose or insulin regimen for 1 week to 1 month, has at least 20% β-cell function before administration of the first dose, is approximately 7 to 75 years old, has an HLA genotype selected from the group consisting of HLA-DQ, HLA-DR, HLA-DP, or HLA-B, and has HLA genotype DR3, DR4, or DR3-DR4.

68. A composition comprising tolerance-modified immunomodulatory particles (TIMP-T1D) encapsulating one or more T1D-related antigens for use in treating type 1 diabetes mellitus (T1D) in a subject, wherein the TIMP-T1D is administered in dose levels of 1 mg to 800 mg.

69. Use of a composition comprising tolerance-modified immunomodulatory particles (TIMP-T1D) encapsulating one or more T1D-related antigens in the preparation of a pharmaceutical for the treatment of type 1 diabetes in a subject, wherein the TIMP-T1D is administered in a dose level of 1 mg to 800 mg.

70. The composition or use according to claim 68 or 69, wherein TIMP-T1D is administered at a dose level of approximately 0.01 mg / kg to 12 mg / kg.

71. The composition or use according to any one of claims 68 to 70, wherein the T1D-related antigen comprises insulin (preproinsulin (PPI)), tyrosine phosphatase-like autoantigen or insulinoma antigen-2 (IA2; ICA512, PTPRN), IA-2β (Fogrin, PTPRN2), GAD65, GAD67, islet cell antigen-69 (ICA69), IGRP, ZnT8, chromogranin A, 38kDa granule antigen, peripherin, islet amyloid peptide (ppIAPP), carboxypeptidase H / E, heat shock protein 60 (HSP60), heat shock protein 70 (HSP70) family, sulfatide, and vasostatin-1.

72. The composition or use according to any one of claims 68 to 71, wherein the T1D-related antigen is selected from the group consisting of SEQ ID NOs: 1 to 17.

73. The composition or use according to any one of claims 68 to 72, wherein the T1D-related antigen comprises PPI, ZnT8, IGRP, and GAD65.

74. The composition or use according to claim 73, wherein the T1D-related antigen comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:

16.

75. The composition or use according to any one of claims 68 to 74, wherein the TIMP-T1D particles have an average diameter of 100 nm to 1500 nm.

76. The composition or use according to any one of claims 68 to 75, wherein the TIMP-T1D particles have an average diameter of 400 nm to 800 nm.

77. The composition or use according to any one of claims 68 to 76, wherein the TIMP-T1D particles have a negative zeta potential.

78. The composition or use according to any one of claims 68 to 77, wherein the particles have a negative zeta potential of -30 mV to -100 mV.

79. The composition or use according to any one of claims 69 to 78, wherein the T1D-related antigen comprises PPI, ZnT8, IGRP, and GAD65.

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