Methods and compositions for preventing type 1 diabetes

Abstract

To provide a method for preventing or delaying the onset of clinical type 1 diabetes (T1D).SOLUTION: Providing a non-diabetic subject at risk of T1D, administering to the non-diabetic subject a prophylactically effective amount of anti- CD3 antibodies, and prior to or subsequent to the administering step, determining that the non-diabetic subject has greater than about 5% to greater than about 10% TIGIT + T1D + CD3 + T cells among total KLRG1 + T cells, indicating successful prevention or delay of the onset of clinical CD8.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 037,968, filed June 11, 2020, Taiwan Patent Application No. 110102871, filed January 26, 2021, U.S. Provisional Patent Application No. 63 / 192,242, filed May 24, 2021, and U.S. Patent Application No. 17 / 345,495, filed June 11, 2021, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing The ASCII text file entitled "010904seq.txt", created on June 11, 2021, and having a size of 6,216 bytes, submitted herewith via EFS-Web is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates generally to compositions and methods, and more particularly to the use of anti-CD3 antibodies, to prevent or delay the onset of clinical type 1 diabetes (T1D) in at-risk subjects. [Background technology]

[0004] Type 1 diabetes (T1D) is caused by autoimmune destruction of insulin-producing beta cells in the islets of Langerhans, leading to a dependence on exogenous insulin injections for survival. Approximately 1.6 million Americans have type 1 diabetes, and it remains one of the most common childhood diseases after asthma. Despite improvements in management, individuals most affected by T1D consistently fail to achieve the desired glycemic target. There are persistent concerns regarding the increased risk of both morbidity and mortality for individuals with type 1 diabetes. Two recent studies found a 17.7-year loss in life expectancy for children diagnosed before age 10 and an 11-year and 13-year loss in life expectancy for Scottish men and women diagnosed as adults, respectively.

[0005] In genetically susceptible individuals, T1D progresses through an asymptomatic stage characterized by the emergence of autoantibodies (Stage 1) and then dysglycemia (Stage 2), prior to the development of overt hyperglycemia. In Stage 2, the metabolic response to a glucose load is impaired, but other metabolic indicators, such as glycosylated hemoglobin, are normal, and insulin treatment is not required. These immunological and metabolic features identify individuals at high risk for developing clinical disease with overt hyperglycemia and the need for insulin treatment (Stage 3). Several immune interventions have been shown to slow the decline of beta cell function in recent-onset clinical T1D. One promising treatment is the non-FcR-binding anti-CD3 monoclonal antibody teplizumab, which has been shown in several studies to durably reduce the loss of beta cell function with short-term treatment, with observable effects observed as long as 7 years after diagnosis and treatment. The drug modifies the function of CD8+ T lymphocytes, which are believed to be the key effector cells responsible for driving beta cell death.

[0006] To date, interventions initiated before clinical diagnosis (i.e., at stage 1 or 2) have not altered progression to clinical stage 3 T1D. Thus, there is a need for treatments that prevent or delay the onset of clinical T1D in at-risk individuals. Summary of the Invention

[0007] Methods for preventing or delaying the onset of clinical type 1 diabetes (T1D) include: providing a non-diabetic subject at risk for T1D; administering to a non-diabetic subject a prophylactically effective amount of an anti-CD3 antibody; determining, before or after the administering step, that the non-diabetic subject has greater than about 5% to greater than about 10% TIGIT+KLRG1+CD8+ T cells among total CD3+ T cells, which indicates successful prevention or delay of the onset of clinical T1D; Includes:

[0008] In some embodiments, the non-diabetic subject is a relative of a T1D patient.

[0009] In some embodiments, the method further includes determining that the non-diabetic subject (1) has substantially no antibodies to zinc transporter 8 (ZnT8), (2) is HLA-DR4+, and / or (3) is not HLA-DR3+.

[0010] In some embodiments, the non-diabetic subject has two or more diabetes-associated autoantibodies selected from islet cell antibodies (ICA), insulin autoantibodies (IAA), and antibodies against glutamic acid decarboxylase (GAD), tyrosine phosphatase (IA-2 / ICA512), or ZnT8.

[0011] In some embodiments, the non-diabetic subject has impaired glucose tolerance on an oral glucose tolerance test (OGTT), ie, impaired glucose tolerance on an OGTT is defined as a fasting glucose level of 110-125 mg / dL, or a 2-hour plasma glucose level of 140 mg / dL or greater but less than 200 mg / dL, or an intervening glucose value at 30, 60, or 90 minutes during the OGTT of greater than 200 mg / dL.

[0012] In some embodiments, the non-diabetic subject does not have antibodies to ZnT8.

[0013] In some embodiments, the non-diabetic subject is HLA-DR4+, but not HLA-DR3+.

[0014] In some embodiments, the anti-CD3 antibody is selected from teplizumab, otelixizumab, or foralarumab. In some embodiments, a prophylactically effective amount of the antibody is 10 to 1000 micrograms per square meter (μg / m 2 ) by subcutaneous (SC) injection, intravenous (IV) infusion, or oral administration over a 10-14 day course, preferably 51 μg / m on days 0-3, respectively. 2 , 103 μg / m2 , 207 μg / m 2 , and 413 μg / m 2 IV infusion at 826 μg / m on each of days 4–13 2 A single dose of IV infusion is administered over a 14-day course.

[0015] In some embodiments, a prophylactically effective amount is one that delays the median time to clinical diagnosis of T1D by at least 50%, at least 80%, or at least 90%, or by at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months.

[0016] In some embodiments, determining TIGIT+KLRG1+CD8+ T cells is by flow cytometry.

[0017] In some embodiments, the method further comprises determining a decrease in the percentage of CD8+ T cells expressing the proliferation markers Ki67 and / or CD57.

[0018] A method for predicting responsiveness to anti-CD3 antibodies in preventing or delaying the onset of type 1 diabetes (T1D) includes: providing a non-diabetic subject at risk for T1D; administering to a non-diabetic subject a prophylactically effective amount of an anti-CD3 antibody; determining a C-peptide area under the curve (AUC):glucose AUC ratio, wherein an increase in said ratio indicates responsiveness to the anti-CD3 antibody and / or non-progression of clinical T1D; Includes: [Brief explanation of the drawings]

[0019] [Figure 1A] Figure 1A shows a consort diagram of at-risk individuals enrolled in the teplizumab prophylaxis trial. [Figure 1B]Figure 1B shows that teplizumab treatment was associated with a sustained effect on the exacerbation of type 1 diabetes over 923 days of follow-up. Updated Kaplan-Meier curves were based on 923 days of follow-up (range 74 to 3,119 days). The hazard ratio for the occurrence of type 1 diabetes in participants treated with teplizumab versus placebo was 0.457, p=0.01. The median time to diabetes was 24.4 months (or 27.1 months according to Sims et al., Sci. Transl. Med. 13, eabc8980 (2021), incorporated by reference) and 59.6 months in the placebo and teplizumab treatment groups, respectively. At the end of this period, 7 (22%) and 22 (50%) participants, respectively, were not diagnosed with T1D. [Figure 2] Figures 2A and 2B show the results of OGTT studies over the first 36 months in participants treated with (Figure 2A) teplizumab and (Figure 2B) placebo; each line represents a participant. Symbols indicate the time of OGTT study. Results are shown as follows: black dots = diabetes level / diagnosis, red dots = dysglycemia, and blue dots = normal. [Figure 3A] Figure 3A shows that glycemic improvement in participants treated with teplizumab is associated with maintenance of dysglycemic status. Figure 3A shows OGTT classification for participants in each group over 36 months of follow-up. Data are shown up to 36 months because participants treated with placebo were lost due to a clinical diagnosis of T1D (see Figures 2A-2B for individual participants). [Figure 3B]Figure 3B shows that glycemic improvement in participants treated with teplizumab is associated with maintenance of dysglycemic status. Figure 3B shows a boxplot depicting the median and interquartile range of mean on-study OGTT glucose AUC for participants from the placebo- and teplizumab-treated groups. An ANCOVA model incorporating baseline values, age, and treatment group showed that treatment group had a significant effect in lowering mean on-study glucose AUC (ANCOVA teplizumab effect: 92.8%, p=0.02). [Figure 4] Figure 4 shows the mean on-study hemoglobin A1c levels by treatment group in a boxplot showing the median and interquartile range for the mean on-study hemoglobin A1c AUC for participants from the placebo- and teplizumab-treated groups. An ANCOVA model incorporating baseline values, age, and treatment group showed no significant effect of treatment group (p=0.14). [Figure 5] Figure 5 shows that teplizumab treatment was associated with an increase in mean on-study C-peptide AUC. Figure 5 shows a boxplot depicting the median and interquartile range of mean on-study OGTT C-peptide AUC for participants from the placebo- and teplizumab-treated groups. An ANCOVA model that included baseline C-peptide AUC and age showed that treatment was associated with a higher mean on-study C-peptide AUC (p=0.009). [Figure 6] Figure 6 shows the relationship between mean on-study C-peptide AUC and age and mean on-study glucose AUC. Figure 6 shows a scatter plot of age vs. on-study C-peptide AUC (r=0.44, p=0.0001). [Figure 7]Figure 7 shows C-peptide over time in the two treatment arms over the first year. Log-transformed mean C-peptide AUC is shown. Arrows indicate individual dropout from OGTT monitoring due to the development of diabetes after each time point. The median value for the "pre-baseline" time point was 24. The median number of months before randomization and the median "baseline" time point was 0.85 months before randomization. *P<0.05 for the comparison of 6-month on-treatment C-peptide AUC values ​​in the teplizumab group with baseline and for the comparison of 6-month C-peptide AUC values ​​in the teplizumab group with 6-month C-peptide AUC values ​​in the placebo group. [Figure 8A] Figure 8A shows insulin secretion after treatment with teplizumab or placebo. Estimated slopes for insulin secreted (pmol) during the entire OGTT (Figure 8A) at the pre-enrollment visit and over the first 6 months after treatment with study drug. Median values ​​(and 95% CI for shaded colors) are shown. Statistical analysis is referred to Table 5. [Figure 8B] Figure 8B shows insulin secretion after treatment with teplizumab or placebo. Estimated slopes for insulin secreted (pmol) during hour 1 of the OGTT (Figure 8B) at the pre-enrollment visit and over the first 6 months after treatment with study drug. Medians (and 95% CI for shaded colors) are shown. Statistical analysis is referred to Table 5. [Figure 8C] Figure 8C shows insulin secretion after treatment with teplizumab or placebo. Estimated slopes for insulin secreted (pmol) during hour 2 of the OGTT (Figure 8C) at the pre-enrollment visit and over the first 6 months after treatment with study drug. Medians (and 95% CI for shaded colors) are shown. Statistical analysis is referred to Table 5. [Figure 8D] Figure 8D shows insulin secretion after treatment with teplizumab or placebo. Figure 8D shows representative insulin secretion rates during sequential OGTTs for two teplizumab participants (ages 11 and 12) without a diagnosis of T1D. Colored lines indicate the time of visits relative to study drug administration. [Figure 8E]Figure 8E shows insulin secretion after treatment with teplizumab or placebo. Figure 8E shows representative insulin secretion rates during sequential OGTTs for two teplizumab participants (ages 11 and 12) without a diagnosis of T1D. Colored lines indicate the time of visit in relation to study drug administration. [Figure 8F] Figure 8F shows insulin secretion after treatment with teplizumab or placebo. (Figure 8F) Representative insulin secretion rates during sequential OGTTs for two placebo-treated individuals (both 13 years old) diagnosed with T1D. Colored lines indicate the time of visit in relation to study drug administration. [Figure 8G] Figure 8G shows insulin secretion after treatment with teplizumab or placebo. (Figure 8G) Representative insulin secretion rates during sequential OGTTs for two placebo-treated individuals (both 13 years old) diagnosed with T1D are shown. Colored lines indicate the time of visit in relation to study drug administration. [Figure 9A] Figure 9A shows that teplizumab preserves C-peptide over the course of the study until the peridiagnostic period. For all panels, data from participants treated with teplizumab are shown in blue, and data from participants treated with placebo are shown in maroon. Figure 9A shows the regression line for C-peptide AUC values ​​over the study period of OGTT monitoring from the baseline study visit until diagnosis (teplizumab n=44, placebo n=32). [Figure 9B] Figure 9B shows that teplizumab preserves C-peptide over the course of the study until the peridiagnostic period. For all panels, data from participants treated with teplizumab are shown in blue, and data from participants treated with placebo are shown in maroon. Figure 9B shows the regression line for C-peptide AUC values ​​over the study period of OGTT monitoring from the baseline study visit until diagnosis (teplizumab n=44, placebo n=32). [Figure 9C]Figure 9C shows that teplizumab preserves C-peptide over the course of the study up to the peridiagnostic period. For all panels, data from participants treated with teplizumab are shown in blue, and data from participants treated with placebo are shown in maroon. Figure 9C shows the regression line for C-peptide AUC values ​​over the 6-month period prior to diabetes diagnosis (placebo n=23, teplizumab n=22). [Figure 9D] Figure 9D shows that teplizumab preserves C-peptide over the course of the study up to the peridiagnostic period. For all panels, data from participants treated with teplizumab are shown in blue, and data from participants treated with placebo are shown in maroon. Figure 9D shows the regression line for C-peptide AUC values ​​over the 6-month period prior to diabetes diagnosis (placebo n=23, teplizumab n=22). [Figure 9E] Figure 9E shows that teplizumab preserves C-peptide over the course of the study up to the peridiagnostic period. For all panels, data from participants treated with teplizumab are shown in blue, and data from participants treated with placebo are shown in maroon. Figure 9E shows the slope of the C-peptide AUC for the 6-month period before diagnosis in individuals who developed T1D and for the final 6 months of the study in individuals who remain T1D-free. [Figure 10] Figures 10A and 10B show that C-peptide values ​​were similar between treatment groups at the time of diagnosis. Figure 10A shows the C-peptide AUC / glucose AUC at the time of clinical diagnosis of T1D. Figure 10B shows the C-peptide AUC at the time of clinical diagnosis of T1D. Values ​​were obtained from the first of two consecutive diagnostic OGTTs that were consistent with classification of T1D. [Figure 11A]Figure 11A shows that changes in T cell function are associated with improved metabolic function. Figure 11A shows the change in TIGIT+KLRG1+CD45RO+CD8+ T cells from baseline to 3 months and the change in C-peptide AUC from baseline to 6 months. There was a significant correlation between changes in this cell subset and C-peptide in teplizumab-treated participants (Pearson, r=0.44, p=0.014, n=31) but not in placebo-treated participants (r=0.28, p=0.25, n=18). [Figure 11B] Figure 11B shows that changes in T cell function are associated with improved metabolic function. Figure 11B shows the frequency of double-positive (DP, i.e., TIGIT+KLRG1+) CD8+ memory cells producing IFNγ or TNFα at baseline and 3 months in placebo-treated participants (red dots, n=16) and drug-treated participants (blue dots, n=24). The frequency of IFNγ- and TNFα-producing cells decreased in participants treated with teplizumab (paired T-test, ***p<0.0001). [Figure 11C] Figure 11C shows that changes in T cell function are associated with improved metabolic function. Figure 11C shows the frequency of double-positive (DP, i.e., TIGIT+KLRG1+) CD8+ memory cells producing IFNγ or TNFα at baseline and 3 months for placebo-treated participants (red dots, n=16) and drug-treated participants (blue dots, n=24). The frequency of IFNγ- and TNFα-producing cells decreased in participants treated with teplizumab (paired T-test, ***p<0.0001). [Figure 12] FIG. 12 shows that subjects treated with teplizumab who had the best outcomes had more TIGIT+KLRG1+CD8+ T cells. [Figure 13] FIG. 13 shows that total TIGIT+KLRG1+CD8+ T cells resemble exhausted cells. [Figure 14]FIG. 14 shows that total TIGIT+KLRG1+CD8+ T cells resemble exhausted cells. [Figure 15] FIG. 15 shows that total TIGIT+KLRG1+CD8+ T cells are heterogeneous. [Figure 16] FIG. 16 shows that total TIGIT+KLRG1+CD8+ T cells are heterogeneous. [Figure 17] FIG. 17 shows that TIGIT+KLRG1+ T cells are interspersed across a large memory CD8 landscape in the teplizumab TrialNet Stage 2 study. [Figure 18] FIG. 18 shows that TIGIT+KLRG1+ subsets differ in response and outcome. [Figure 19] FIG. 19 shows that TIGIT+KLRG1+ subsets differ in response and outcome. [Figure 20] FIG. 20 shows that TIGIT+KLRG1+ subsets differ in response and outcome. [Figure 21] FIG. 21 shows that TIGIT+KLRG1+ subsets differ in response and outcome. [Figure 22] Figure 22 shows the OGTT patterns of glucose and C-peptide from 30 to 120 minutes. [Figure 23] FIG. 23 shows that 2D plots of mean glucose and C-peptide values ​​identify distinct longitudinal OGTT patterns between "exacerbators" and "non-exacerbators" within DPT-1. [Figure 24] Figure 24 shows that 2D plots of mean glucose and C-peptide values ​​at 30-120 min OGTT time points show distinctly different patterns at the 3-month visit. [Figure 25] FIG. 25 shows that the 3-month change in the ratio of C-peptide AUC / glucose AUC shows the most significant difference compared to other metabolic measures. [Figure 26] FIG. 1 shows predicted mean teplizumab serum concentration versus time profiles after a 14-day regimen across different body weights. DETAILED DESCRIPTION OF THE INVENTION

[0020] In some embodiments, provided herein are methods for preventing or delaying the onset of clinical type 1 diabetes (T1D), comprising: providing a non-diabetic subject at risk for T1D; administering to the non-diabetic subject a prophylactically effective amount of an anti-CD3 antibody; and determining, before or after the administering step, that the non-diabetic subject has greater than about 5% to greater than about 10% TIGIT+KLRG1+CD8+ T cells among total CD3+ T cells, which indicates successful prevention or delay of the onset of clinical T1D.

[0021] In some embodiments, methods are provided for predicting responsiveness to an anti-CD3 antibody, e.g., teplizumab, in preventing or delaying the onset of T1D. The methods may include providing a non-diabetic subject at risk for T1D, administering to the non-diabetic subject a prophylactically effective amount of an anti-CD3 antibody, e.g., teplizumab, and determining a C-peptide area under the curve (AUC):glucose AUC ratio, where an increase in the ratio indicates responsiveness to the anti-CD3 antibody.

[0022] definition Certain terms are defined herein below: Additional definitions are provided throughout this application.

[0023] As used herein, the articles "a" and "an" refer to one or to more than one, e.g., at least one, of the grammatical object of the article. The use of the word "a" or "an," when used in conjunction with the term "comprising," can mean "one" herein, but is also consistent with the meanings of "one or more," "at least one," and "one or more than one."

[0024] As used herein, "about" and "approximately" generally refer to an acceptable degree of error for the quantity measured given the nature or precision of the measurements. is within 20 percent (%), typically within 10%, and more typically within 5% of a given range of values. The term "substantially" means greater than 50%, preferably greater than 80%, and most preferably greater than 90% or 95%.

[0025] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective component(s) present in a given embodiment, but are open to the inclusion of unspecified elements.

[0026] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the disclosure.

[0027] The term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding any element not recited in the description of that embodiment.

[0028] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0029] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0030] As used herein, the term "prophylactic agent" refers to a CD3 binding molecule, such as teplizumab, that can be used to prevent, treat, manage or ameliorate one or more symptoms of T1D.

[0031] As used herein, the term "development" of a disease associated with type 1 diabetes refers to a patient meeting the criteria established by the American Diabetes Association for the diagnosis of type 1 diabetes (see Mayfield et al., 2006, Am. Fam. Physician 58:1355-1362).

[0032] As used herein, the terms "prevent," "preventing," and "prevention" refer to the prevention of the onset of one or more symptoms of T1D in a subject resulting from the administration of a prophylactic or therapeutic agent.

[0033] As used herein, a "protocol" includes dosing schedules and dosing regimens. A protocol herein is a method of use and includes prophylactic and therapeutic protocols. A "dosing regimen" or "course of treatment" can include administration of a therapeutic or prophylactic agent in several doses over a period of 1 to 20 days.

[0034] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the terms "subject" and "subjects" refer to animals, preferably mammals, including non-primates (e.g., cows, pigs, horses, cats, dogs, rats, and mice) and primates (e.g., monkeys or humans), more preferably humans.

[0035] As used herein, the term "prophylactically effective amount" refers to an amount of teplizumab sufficient to result in a delay or prevention of the onset, recurrence, or development of one or more symptoms of T1D. In some embodiments, a prophylactically effective amount preferably refers to an amount of teplizumab that delays the onset of T1D in a subject by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0036] Various aspects of the disclosure are described in further detail below. Additional definitions are set forth throughout the specification.

[0037] Anti-CD3 antibodies and pharmaceutical compositions The terms "anti-CD3 antibody" and "antibody that binds to CD3" refer to an antibody or antibody fragment that can bind to cluster of differentiation 3 (CD3) with sufficient affinity that the antibody is therefore useful as a prophylactic, diagnostic, and / or therapeutic agent in targeting CD3. In some embodiments, the extent of binding of an anti-CD3 antibody to an unrelated, non-CD3 protein is less than about 10% of the antibody's binding to CD3, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the dissociation constant (Kd) of an antibody that binds to CD3 is <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 Up to M, e.g., 10 -9 M~10 -13In some embodiments, the anti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3 from different species.

[0038] In some embodiments, the anti-CD3 antibody can be ChAglyCD3 (otelixizumab). Otelixizumab is a humanized, Fc-nonbinding anti-CD3 that was first evaluated in a Phase 2 study by the Belgian Diabetes Registry (BDR), then developed by Tolerx, which then partnered with GSK in the Phase 3 DEFEND new-onset T1D trials (NCT00678886, NCT01123083, NCT00763451). Otelixizumab is administered IV as an infusion over 8 days. See, e.g., Wiczling et al., J. Clin. Pharmacol. 50(5) (May 2010) 494-506; Keymeulen et al., N Engl J Med. 2005; 352:2598-608; Keymeulen et al., Diabetologia. 2010; 53:614-23; Hagopian et al., Diabetes. 2013; 62:3901-8; Aronson et al., Diabetes Care. 2014; 37:2746-54; Ambery et al. al., Diabet Med.2014;31:399-402;Bolt et al.,Eur.J.Immunol.lYY3.23: 403-411;Vlasakakis et al.,Br J Clin Pharmacol(2019)85 704-714;Guglielmi et al,Expert Opinion on Biological Therapy,16:6,841-846;Keymulen et al.,N Engl J Med 2005;352:2598-608;Keymulen et al.,BLOOD 2010,VOL 115,No.6;Sprangers et al. al., Immunotherapy(2011) 3(11),1303-1316;Daifotis et al.,Clinical See Immunology (2013) 149, 268-278.

[0039] In some embodiments, the anti-CD3 antibody can be visilizumab (also known as HuM291; Nuvion). Visilizumab is a humanized anti-CD3 monoclonal antibody characterized by a mutant IgG2 isotype, lack of Fcγ receptor binding, and the ability to selectively induce apoptosis in activated T cells. Visilizumab has been evaluated in patients with graft-versus-host disease (NCT00720629; NCT00032279), ulcerative colitis (NCT00267306), and Crohn's disease (NCT00267709). See, for example, Sandborn et al., Gut 59(11) (Nov 2010) 1485-1492, which is incorporated herein by reference.

[0040] In some embodiments, the anti-CD3 antibody can be foralamuab, a fully human anti-CD3 monoclonal antibody being developed for NASH and T2D by Tiziana Life Sciences, PLC (NCT03291249). See, e.g., Ogura et al., Clin. Immunol.2017;183:240-246;Ishikawa et al.,Diabetes.2007;56(8):2103-9;Wu et al.,J See Immunol. 2010;185(6):3401-7.

[0041] In some embodiments, the anti-CD3 antibody may be teplizumab. Teplizumab, also known as hOKT3yl (Ala-Ala) (containing alanine at positions 234 and 235), is an anti-CD3 antibody engineered to alter the function of T lymphocytes that mediate the destruction of insulin-producing beta cells in pancreatic islets. Teplizumab binds to an epitope on the CD3ε chain expressed on mature T cells, thereby altering their function. The sequence and composition of teplizumab are disclosed in U.S. Patent Nos. 6,491,916; 8,663,634; and 9,056,906, each of which is incorporated herein by reference in its entirety. The complete sequences of the light and heavy chains are set forth below. The bolded parts are complementarity-determining regions. Teplizumab light chain (SEQ ID NO: 1): [ka] Teplizumab heavy chain (SEQ ID NO: 2): [ka]

[0042] In some embodiments, pharmaceutical compositions are provided herein. Such compositions comprise a prophylactically effective amount of an anti-CD3 antibody and a pharmaceutically acceptable carrier. In some embodiments, the term "pharmaceutically acceptable" refers to a compound that meets the federally recognized standards for use in animals, and more particularly, in humans. or approved by a state government regulatory agency, or listed in the United States Pharmacopoeia or other commonly recognized pharmacopeia. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like (see, e.g., Handbook of Pharmaceutical Excipients, Arthur H. Kibbe (ed., 2000), incorporated herein by reference in its entirety, Am. Pharmaceutical Association, Washington, DC).

[0043] If desired, the compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral formulations may contain standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions contain a prophylactically or therapeutically effective amount of a prophylactic or therapeutic agent, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to a patient. The formulation should be compatible with the mode of administration. In some embodiments, the pharmaceutical compositions are sterile and in a form suitable for administration to subjects, preferably animal subjects, more preferably mammalian subjects, and most preferably human subjects.

[0044] In some embodiments, it may be desirable to administer the pharmaceutical composition locally to the area in need of treatment, which can be achieved, for example, but not by way of limitation, by local injection or by using an implant, which can be a porous, non-porous, or gelatinous material, including a membrane, such as a silastic membrane, or a fiber. Preferably, when administering an anti-CD3 antibody, care must be taken to use a material to which the anti-CD3 antibody does not absorb.

[0045] In some embodiments, the compositions can be delivered as vesicles, particularly liposomes (Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

[0046] In some embodiments, the compositions can be delivered in a controlled-release or sustained-release system. In some embodiments, a pump can be used to achieve controlled or sustained release (Langer, supra; Sefton, (1987), CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574. In some embodiments, polymeric materials can be used to achieve controlled or sustained release of the antibodies, or fragments thereof, of the invention (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, (1983), J. Macromol. Sci. Rev. Macromol. Chem. 23:61; also, Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al. al., 1989, J. Neurosurg. 71:105); U.S. Patent Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; PCT Publication No. WO 99 / 15154; and PCT Publication No. WO 99 / 20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactic acid (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In some embodiments, the polymers used in sustained release formulations are inert, free of potentially leachable impurities, stable during storage, sterile, and biodegradable. In some embodiments, controlled- or sustained-release systems can be placed in proximity to the therapeutic target, i.e., the lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in See Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984).

[0047] Controlled-release systems are discussed in the review by Langer (1990, Science 249:1527-1533). Any technique known to those of ordinary skill in the art can be used to prepare sustained-release formulations comprising one or more antibodies or fragments thereof of the invention. See, e.g., U.S. Pat. No. 4,526,938; PCT Publication No. WO 91 / 05548; PCT Publication No. WO 96 / 20698; Ning et al., 1996, Radiotherapy & Oncology 39:179-189; Song et al., 1995, PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which is incorporated by reference in its entirety.

[0048] A pharmaceutical composition can be formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral administration, such as intravenous administration, intradermal administration, subcutaneous administration, oral administration, intranasal administration (e.g., inhalation), transdermal (topical) administration, transmucosal administration, and rectal administration. In some embodiments, the composition is formulated according to conventional procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to humans. In some embodiments, the pharmaceutical composition is formulated according to conventional procedures for subcutaneous administration to humans. Generally, compositions for intravenous administration are solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.

[0049] The composition can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion.The preparation for injection can be provided in a unit dosage form, for example, in ampoules or multi-dose containers, and preservatives can be added.The composition can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and can also contain formulatory agents such as suspending agents, stabilizing agents, and / or dispersing agents.Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0050] In some embodiments, the present disclosure provides dosage forms (e.g., in conjunction with a pump or other device for such delivery) that allow for continuous administration of anti-CD3 antibodies over a period of several hours or days, e.g., 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, 24 hours, 30 hours, 36 hours, 4 days, 5 days, 7 days, 10 days, or 14 days. In some embodiments, the present disclosure provides for continuous administration of anti-CD3 antibodies over a period of several hours or days, e.g., 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, 24 hours, 30 hours, 36 hours, 4 days, 5 days, 7 days, 10 days, or 14 days. 2 / day to 826 μg / m 2 Dosage forms are provided that allow for administration over a 24 hour, 30 hour, 36 hour, 4 day, 5 day, 7 day, 10 day or 14 day period, increasing the dose to 1 / day.

[0051] The composition can be formulated as a neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0052] Generally, the components of the compositions disclosed herein are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or a water-free concentrate, in a sealed container such as an ampoule or sachet indicating the quantity of active agent.When the composition is administered by injection, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline.When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0053] In particular, the present disclosure provides an anti-CD3 antibody or pharmaceutical composition thereof that can be packaged in a sealed container, such as an ampoule or sachet, indicating the quantity of the agent. In some embodiments, the anti-CD3 antibody or pharmaceutical composition thereof is supplied as a dry, sterile, lyophilized powder or water-free concentrate in a sealed container, which can be reconstituted with, for example, water or saline to a concentration suitable for administration to a subject. The anti-CD3 antibody or pharmaceutical composition thereof is preferably supplied as a dry, sterile, lyophilized powder in a sealed container in a unit dose of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg. The lyophilized prophylactic or therapeutic agent or pharmaceutical composition herein should be stored in its original container at 2°C to 8°C, and the prophylactic or therapeutic agent or pharmaceutical composition of the present invention should be administered within one week, preferably within five days, 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 5 hours, 3 hours, or 1 hour after reconstitution. In some embodiments, the pharmaceutical composition is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the agent. The liquid form of the composition to be administered is at least 0.25 mg / ml, more preferably at least Preferably, the liquid form is supplied at 0.5 mg / ml, at least 1 mg / ml, at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 75 mg / ml or at least 100 mg / ml. The liquid form should be stored in its original container at a temperature between 2°C and 8°C.

[0054] In some embodiments, the present disclosure provides compositions of the invention packaged in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of anti-CD3 antibody.

[0055] The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack includes, for example, metal or plastic foil, such as a blister pack.

[0056] The amount of the composition of the present invention that is effective in preventing or ameliorating one or more symptoms associated with T1D can be determined by standard clinical techniques. The precise dose to be used in the formulation will also depend on the route of administration and the severity of the condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0057] Methods and Uses In some embodiments, the present disclosure encompasses administering an anti-human CD3 antibody, such as teplizumab, to individuals who are predisposed to developing type 1 diabetes or who have preclinical stage type 1 diabetes, but who do not meet diagnostic criteria established by the American Diabetes Association or the Immunology of Diabetes Society, to prevent or delay the onset of type 1 diabetes and / or prevent or delay the need for insulin administration in such patients. In some embodiments, high-risk factors for identifying predisposed subjects include having a first- or second-degree relative diagnosed with type 1 diabetes, abnormal fasting glucose levels (e.g., at least one determination of a glucose level of 100-125 mg / dL after fasting (8 hours without food)), impaired glucose tolerance in response to a 75g OGTT (e.g., a 75g OGTT), or impaired glucose tolerance in response to a 75g OGTT (e.g., a 75g OGTT). Predisposing factors include at least one determination of a 2-hour glucose level of 140-199 mg / dL in response to an OGTT; HLA type DR3, DR4, or DR7 for Caucasians; HLA type DR3 or DR4 for individuals of African descent; HLA type DR3, DR4, or DR9 for individuals of Japanese descent; exposure to a virus (e.g., Coxsackie B virus, enterovirus, adenovirus, rubella, cytomegalovirus, Epstein-Barr virus); a positive diagnosis of at least one other autoimmune disorder (e.g., thyroid disease, celiac disease) according to art-accepted criteria; and / or detection of autoantibodies, particularly ICA and type 1 diabetes-associated autoantibodies, in serum or other tissues. In some embodiments, subjects identified as having a predisposition to developing type 1 diabetes have at least one of the risk factors described herein and / or known in the art. The present disclosure also encompasses the identification of subjects predisposed to developing type 1 diabetes, wherein said subjects exhibit a combination of two or more, three or more, four or more, or more than five of the risk factors disclosed herein or known in the art.

[0058] Serum autoantibodies associated with type 1 diabetes or a predisposition to developing type 1 diabetes are islet cell autoantibodies (e.g., anti-ICA512 autoantibodies), glutamic acid decarbamylase autoantibodies (e.g., anti-GAD65 autoantibodies), IA2 antibodies, ZnT8 antibodies, and / or anti-insulin autoantibodies. Thus, in a particular example according to this embodiment, the invention provides for the detection of an individual with a detectable autoantibody (e.g., anti-IA2, anti-ICA512, anti-GAD, or anti-insulin autoantibody) associated with a predisposition to developing type 1 diabetes or associated with early stage type 1 diabetes. In some embodiments, the presence of autoantibodies is detected by ELISA, electrochemiluminescence (ECL), radioassay (see, e.g., Yu et al., 1996, J. Clin. Endocrinol. Metab. 81:4264-4267), agglutination PCR (Tsai et al., ACS Central Science 2016 2(3), 139-147), or any other method for immunospecifically detecting antibodies described herein or known to one of skill in the art.

[0059] Before, during and after treatment, β-cell function can be evaluated by the methods described herein or by any method known to those skilled in the art.For example, the Diabetes Control and Complications Trial (DCCT) research group has established monitoring percentage glycosylated hemoglobin (HA1 and HA1c) as the standard for evaluating blood glucose control (DCCT, (1993), N.Engl.J.Med.329:977-986). Alternatively, characterization of daily insulin requirements, C-peptide levels / response, low blood glucose episodes, and / or FPIR can be used as markers of beta cell function or to establish a therapeutic index (see Keymeulen et al., 2005, N. Engl. J. Med. 352:2598-2608; Herold et al., 2005, Diabetes 54:1763-1769; U.S. Patent Application Publication No. 2004 / 0038867 A1; and Greenbaum et al., 2001, Diabetes 50:470-476, respectively). For example, FPIR is calculated as the sum of the 1- and 3-minute insulin values ​​from an IGTT performed according to the Islet Cell Antibody Register User's Study protocol (see, e.g., Bingley et al., 1996, Diabetes 45:1720-1728 and McCulloch et al., 1993, Diabetes Care 16:911-915).

[0060] In some embodiments, the individual predisposed to developing T1D may be a non-diabetic subject who is a relative of a T1D patient. In some embodiments, the non-diabetic subject has two or more diabetes-associated autoantibodies selected from islet cell antibodies (ICA), insulin autoantibodies (IAA), and antibodies against glutamic acid decarboxylase (GAD), tyrosine phosphatase (IA-2 / ICA512), or ZnT8.

[0061] In some embodiments, the non-diabetic subject has impaired glucose tolerance on an oral glucose tolerance test (OGTT), defined as a fasting glucose level of 110-125 mg / dL, or a 2-hour plasma glucose level of 140 mg / dL or greater but less than 200 mg / dL, or a median glucose value at 30, 60, or 90 minutes during the OGTT of greater than 200 mg / dL.

[0062] In some embodiments, non-diabetic subjects who respond to an anti-CD3 antibody, such as teplizumab, do not have antibodies to ZnT8. In some embodiments, such non-diabetic subjects are HLA-DR4+ but not HLA-DR3+. In some embodiments, non-diabetic subjects who respond to such an anti-CD3 antibody, such as teplizumab, demonstrate an increase in the frequency (or relative amount) of TIGIT+KLRG1+CD8+ T cells in peripheral blood mononuclear cells (e.g., by flow cytometry) after administration (e.g., 1 month, 2 months, 3 months, or later or earlier).

[0063] In some embodiments, a prophylactically effective amount is an anti-CD3 antibody, such as teplizumab, administered at a dose of 10 to 1000 micrograms per square meter (μg / m 2 ) by subcutaneous (SC) injection or intravenous (I V) A 10-14 day course of infusions of an anti-CD3 antibody, e.g., teplizumab, at 51 μg / m on days 0-3, respectively. 2 , 103 μg / m 2 , 207 μg / m 2 , and 413 μg / m 2 IV infusion at 826 μg / m on each of days 4–13 2 In some embodiments, a prophylactically effective amount delays the median time to clinical diagnosis of T1D by at least 50%, at least 80%, or at least 90%, or by at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months, or more.

[0064] In some embodiments, a course of dosing with an anti-CD3 antibody, e.g., teplizumab, can be repeated at intervals of 2 months, 4 months, 6 months, 8 months, 9 months, 10 months, 12 months, 15 months, 18 months, 24 months, 30 months, or 36 months. In some embodiments, the efficacy of treatment with an anti-CD3 antibody, e.g., teplizumab, is determined as described herein or as known in the art 2 months, 4 months, 6 months, 9 months, 12 months, 15 months, 18 months, 24 months, 30 months, or 36 months after the previous treatment.

[0065] In some embodiments, a subject is administered one or more unit doses of about 0.5-50 μg / kg, about 0.5-40 μg / kg, about 0.5-30 μg / kg, about 0.5-20 μg / kg, about 0.5-15 μg / kg, about 0.5-10 μg / kg, about 0.5-5 μg / kg, about 1-5 μg / kg, about 1-10 μg / kg, about 20-40 μg / kg, about 20-30 μg / kg, about 22-28 μg / kg, or about 25-26 μg / kg of an anti-CD3 antibody, e.g., teplizumab, to prevent, treat, or ameliorate one or more symptoms of T1D. In some embodiments, a subject is administered about 200 μg / kg, 178 μg / kg, 180 μg / kg, 128 μg / kg, 100 μg / kg, 95 μg / kg, 90 μg / kg, 85 μg / kg, 80 μg / kg, 75 μg / kg, 70 μg / kg, 65 μg / kg, 60 μg / kg, 55 μg / kg, 50 μg / kg, 45 μg / kg, 40 μg / kg, 35 μg / kg, 30 μg / kg, 45 μg / kg, 4 ... In some embodiments, one or more unit doses of 26 μg / kg, 25 μg / kg, 20 μg / kg, 15 μg / kg, 13 μg / kg, 10 μg / kg, 6.5 μg / kg, 5 μg / kg, 3.2 μg / kg, 3 μg / kg, 2.5 μg / kg, 2 μg / kg, 1.6 μg / kg, 1.5 μg / kg, 1 μg / kg, 0.5 μg / kg, 0.25 μg / kg, 0.1 μg / kg, or 0.05 μg / kg of an anti-CD3 antibody, e.g., teplizumab, are administered.

[0066] In some embodiments, the subject is administered a dose of about 5 to 1200 μg / m 2 , preferably 51 to 826 μg / m 2 In some embodiments, the subject is administered one or more doses of 1200 μg / m of an anti-CD3 antibody, e.g., teplizumab, to prevent, treat, slow the progression of, delay the onset of, or reverse one or more symptoms of T1D. 2 , 1150 μg / m 2 , 1100 μg / m 2 , 1050 μg / m 2 , 1000 μg / m 2 , 950 μg / m 2 , 900 μg / m 2 , 850 μg / m 2 , 800 μg / m 2 , 750 μg / m 2 , 700 μg / m 2 , 650 μg / m 2 , 600 μg / m 2 , 550 μg / m 2 , 500 μg / m 2 , 450 μg / m 2 , 400 μg / m 2 , 350 μg / m 2 , 300 μg / m 2 , 250 μg / m 2 , 200 μg / m 2 , 150 μg / m 2 , 100 μg / m 2 , 50 μg / m 2 , 40 μg / m 2 , 30 μg / m 2 , 20 μg / m 2 , 15 μg / m 2 , 10 μg / m 2 , or 5 μg / m 2 of an anti-CD3 antibody, e.g., teplizumab, are administered.

[0067] In some embodiments, the subject is administered a prophylactically effective amount of an anti-CD3 antibody, e.g., teplizumab. Or a treatment regimen comprising multiple doses is administered, wherein the course of treatment is administered for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the treatment regimen comprises administering a prophylactically effective amount of a dose every day, every other day, every third day, every third day, or every fourth day. In some embodiments, the treatment regimen comprises administering a prophylactically effective amount of a dose on Monday, Tuesday, Wednesday, and Thursday of a given week, with no prophylactically effective amount of a dose administered on Friday, Saturday, and Sunday of the same week, until 14, 13, 12, 11, 10, 9, or 8 doses have been administered. In some embodiments, the dose administered is the same on each day of the regimen.

[0068] In some embodiments, the subject is administered a treatment regimen comprising one or more doses of a prophylactically effective amount of an anti-CD3 antibody, e.g., teplizumab, wherein the prophylactically effective amount is 200 μg / kg / day, 175 μg / kg / day, 150 μg / kg / day, 125 μg / kg / day, 100 μg / kg / day, 95 μg / kg / day, 90 μg / kg / day, 85 μg / kg / day, 80 μg / kg / day, 75 μg / kg / day, 70 μg / kg / day, 65 μg / kg / day, 60 μg / kg / day, 55 μg / kg / day, 50 μg / kg / day, 45 μg / kg / day, 40 μg / kg / day, and / or the prophylactically effective amount is 35 μg / kg / day, 30 μg / kg / day, 26 μg / kg / day, 25 μg / kg / day, 20 μg / kg / day, 15 μg / kg / day, 13 μg / kg / day, 10 μg / kg / day, 6.5 μg / kg / day, 5 μg / kg / day, 3.2 μg / kg / day, 3 μg / kg / day, 2.5 μg / kg / day, 2 μg / kg / day, 1.6 μg / kg / day, 1.5 μg / kg / day, 1 μg / kg / day, 0.5 μg / kg / day, 0.25 μg / kg / day, 0.1 μg / kg / day, or 0.05 μg / kg / day; and / or the prophylactically effective amount is 1200 μg / m 2 / day, 1150μg / m 2 / day, 1100μg / m 2 / day, 1050μg / m 2 / day, 1000μg / m 2 / day, 950 μg / m 2 / day, 900μg / m 2 / day, 850 μg / m 2 / day, 800μg / m 2 / day, 750 μg / m 2 / day, 700μg / m 2 / day, 650 μg / m 2 / day, 600μg / m 2 / day, 550μg / m 2 / day, 500μg / m 2 / day, 450μg / m 2 / day, 400μg / m 2 / day, 350μg / m 2 / day, 300μg / m 2 / day, 250μg / m 2 / day, 200μg / m 2 / day, 150μg / m 2 / day, 100μg / m 2 / day, 50μg / m 2 / day, 40μg / m 2 / day, 30μg / m 2 / day, 20 μg / m 2 / day, 15μg / m 2 / day, 10μg / m 2 / day or 5 μg / m 2 / day.

[0069] In some embodiments, 1200 μg / m 2 Below, 1150μg / m 2 Below, 1100μg / m 2 Below, 1050μg / m 2 Below, 1000μg / m 2 Below, 950μg / m 2 Below, 900μg / m 2 Below 850μg / m 2 Below 800μg / m 2 Below, 750μg / m 2 Below, 700μg / m 2 Below, 650μg / m 2 Below, 600μg / m 2 Below, 550μg / m 2 Below, 500μg / m 2 Below, 450μg / m2 Below 400μg / m 2 Below 350μg / m 2 Below 300μg / m 2 Below 250μg / m 2 Below, 200μg / m 2 Below 150μg / m 2 Below, 100μg / m 2 Below, 50μg / m 2 Below, 40μg / m 2 Below, 30μg / m 2 Below, 20μg / m 2 Below, 15μg / m 2 Below, 10μg / m 2 or less than 5 μg / m 2 The following intravenous doses of an anti-CD3 antibody, e.g., teplizumab, are administered over about 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1.5 hours, about 1 hour, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, about 2 minutes, about 1 minute, about 30 seconds, or about 10 seconds. The total dose over the duration of the regimen totals 9000 μg / m 2 Less than 8000 μg / m 2 Less than 7000 μg / m 2 Less than 6000 μg / m 2 Preferably less than 5000 μg / m 2 Less than 4000 μg / m 2 Less than 3000 μg / m 2 Less than 2000 μg / m 2 Less than or equal to 1000 μg / m 2 In some embodiments, the total dose administered in the regimen may be less than 1 00μg / m 2 ~200μg / m 2 , 100 μg / m 2 ~500μg / m 2 , 100 μg / m 2 ~1000μg / m 2 , or 500 μg / m 2 ~1000μg / m 2 is.

[0070] In some embodiments, the dose is escalated over the first quarter, half, or two-thirds of the doses of a treatment regimen (e.g., over the first 2, 3, 4, 5, or 6 days of a 10-, 12-, 14-, 16-, 18-, or 20-day regimen of one dose per day) until a daily prophylactically effective dose of anti-CD3 antibody, e.g., teplizumab, is achieved. In some embodiments, the subject is administered a treatment regimen comprising one or more doses of a prophylactically effective amount of an anti-CD3 antibody, e.g., teplizumab, where the prophylactically effective amount is administered in daily increments of, for example, 0.01 μg / kg, 0.02 μg / kg, 0.04 μg / kg, 0.05 μg / kg, 0.06 μg / kg, 0.08 μg / kg, 0.1 μg / kg, 0.2 μg / kg, 0.25 μg / kg, 0.5 μg / kg, 0.75 μg / kg, 1 μg / kg, 1.5 μg / kg, 2 μg / kg, or , 4 μg / kg increments, 5 μg / kg increments, 10 μg / kg increments, 15 μg / kg increments, 20 μg / kg increments, 25 μg / kg increments, 30 μg / kg increments, 35 μg / kg increments, 40 μg / kg increments, 45 μg / kg increments, 50 μg / kg increments, 55 μg / kg increments, 60 μg / kg increments, 65 μg / kg increments, 70 μg / kg increments, 75 μg / kg increments, 80 μg / kg increments, 85 μg / kg increments, 90 μg / kg increments, 95 μg / kg increments, 100 μg / kg increments, or 125 μg / kg increments; or, for example, 1 μg / m daily 2 Each dose is 5 μg / m 2 Each dose is 10 μg / m 2 Each dose is 15 μg / m 2 Each dose is 20 μg / m 2 Each dose is 30 μg / m 2 Each dose is 40 μg / m 2 Each dose is 50 μg / m 2 Each dose is 60 μg / m 2 Each dose is 70 μg / m 2 Each dose is 80μg / m 2 Each dose is 90μg / m 2 Each dose is 100μg / m 2 Each dose is 150 μg / m 2 Each dose is 200 μg / m 2Each dose is 250 μg / m 2 Each dose is 300 μg / m 2 Each dose is 350 μg / m 2 Each dose is 400 μg / m 2 Each dose is 450 μg / m 2 Each, 500μg / m 2 Each dose is 550 μg / m 2 Each, 600μg / m 2 or 650 μg / m 2 In some embodiments, a subject is administered a treatment regimen comprising one or more doses of a prophylactically effective amount of an anti-CD3 antibody, e.g., teplizumab, where the prophylactically effective amount is increased by 1.25-fold, 1.5-fold, 2-fold, 2.25-fold, 2.5-fold, or 5-fold until a daily prophylactically effective dose of the anti-CD3 antibody, e.g., teplizumab, is achieved.

[0071] In some embodiments, a subject is administered 200 μg / kg or less, preferably 175 μg / kg or less, 150 μg / kg or less, 125 μg / kg or less, 100 μg / kg or less, 95 μg / kg or less, 90 μg / kg or less, 85 μg / kg or less, 80 μg / kg or less, 75 μg / kg or less, 70 μg / kg or less, 65 μg / kg or less, 60 μg / kg or less, 55 μg / kg or less, 50 μg / kg or less to prevent, treat, or ameliorate one or more symptoms of T1D. , 45 μg / kg or less, 40 μg / kg or less, 35 μg / kg or less, 30 μg / kg or less, 25 μg / kg or less, 20 μg / kg or less, 15 μg / kg or less, 10 μg / kg or less, 5 μg / kg or less, 2.5 μg / kg or less, 2 μg / kg or less, 1.5 μg / kg or less, 1 μg / kg or less, 0.5 μg / kg or less, or 0.2 μg / kg or less of an anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralarumab, is administered intramuscularly.

[0072] In some embodiments, a subject is administered 200 μg / kg or less, preferably 175 μg / kg or less, 150 μg / kg or less, 125 μg / kg or less, 100 μg / kg or less, 95 μg / kg or less, 90 μg / kg or less, 85 μg / kg or less, 80 μg / kg or less, 75 μg / kg or less, 70 μg / kg or less, 65 μg / kg or less, 60 μg / kg or less, 55 μg / kg or less, 50 μg / kg or less, 45 μg / kg or less, 40 μg / kg or less, 35 μg / kg or less, 30 μg / kg or less, 25 μg / kg or less, 20 μg / kg or less, 15 μg / kg or less to prevent, treat, or ameliorate one or more symptoms of T1D. In some embodiments, one or more doses of an anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralarumab, at or below 10 μg / kg, 5 μg / kg, 2.5 μg / kg, 2 μg / kg, 1.5 μg / kg, 1 μg / kg, 0.5 μg / kg, or 0.2 μg / kg are administered subcutaneously.

[0073] In some embodiments, a subject is administered 100 μg / kg or less, preferably 95 μg / kg or less, 90 μg / kg or less, 85 μg / kg or less, 80 μg / kg or less, 75 μg / kg or less, 70 μg / kg or less, 65 μg / kg or less, 60 μg / kg or less, 55 μg / kg or less, 50 μg / kg or less, 45 μg / kg or less, 40 μg / kg or less, 3 μg / kg or less, 5 μg / kg or less, 6 μg / kg or less, 7 μg / kg or less, 8 μg / kg or less, 9 μg / kg or less, 10 μg / kg or less, 11 μg / kg or less, 12 μg / kg or less, 13 μg / kg or less, 14 μg / kg or less, 15 μg / kg or less, 16 μg / kg or less, 17 μg / kg or less, 18 μg / kg or less, 19 μg / kg or less, 20 μg / kg or less, 21 μg / kg or less, 22 μg / kg or less, 23 μg / kg or less, 24 μg / kg or less, 25 μg / kg or less, 26 μg / kg or less, 27 μg / kg or less, 28 μg / kg or less, 29 μg / kg or less, 30 μg / kg or less, 31 μg / kg or less, 32 μg / kg or less, 33 μg / kg or less, 34 μg / kg or less, 35 μg / kg or less, 36 μg / kg or less, 37 μg / kg or less, 38 μg / kg or less, 39 μg / kg or less, 40 μg / kg or less, 41 μg / kg One or more doses of 5 μg / kg or less, 30 μg / kg or less, 25 μg / kg or less, 20 μg / kg or less, 15 μg / kg or less, 10 μg / kg or less, 5 μg / kg or less, 2.5 μg / kg or less, 2 μg / kg or less, 1.5 μg / kg or less, 1 μg / kg or less, 0.5 μg / kg or less, or 0.2 μg / kg or less of an anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralaumab, are administered intravenously. In some embodiments, a dose of 100 μg / kg or less, 95 μg / kg or less, 90 μg / kg or less, 85 μg / kg or less, 80 μg / kg or less, 75 μg / kg or less, 70 μg / kg or less, 65 μg / kg or less, 60 μg / kg or less, 55 μg / kg or less, 50 μg / kg or less, 45 μg / kg or less, 40 μg / kg or less, 35 μg / kg or less, 30 μg / kg or less, 25 μg / kg or less, 20 μg / kg or less, 15 μg / kg or less, or a combination of these is used to prevent, treat, or ameliorate one or more symptoms of T1D. An intravenous dose of 10 μg / kg or less, 10 μg / kg or less, 5 μg / kg or less, 2.5 μg / kg or less, 2 μg / kg or less, 1.5 μg / kg or less, 1 μg / kg or less, 0.5 μg / kg or less, or 0.2 μg / kg or less of an anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralarumab, is administered over about 6 hours, about 4 hours, about 2 hours, about 1.5 hours, about 1 hour, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, about 2 minutes, about 1 minute, about 30 seconds, or about 10 seconds.

[0074] In some embodiments, a subject is administered 100 μg / kg or less, preferably 95 μg / kg or less, 90 μg / kg or less, 85 μg / kg or less, 80 μg / kg or less, 75 μg / kg or less, 70 μg / kg or less, 65 μg / kg or less, 60 μg / kg or less, 55 μg / kg or less, 50 μg / kg or less, 45 μg / kg or less, 40 μg / kg or less, 3 μg / kg or less, 5 μg / kg or less, 6 μg / kg or less, 7 μg / kg or less, 8 μg / kg or less, 9 μg / kg or less, 10 μg / kg or less, 11 μg / kg or less, 12 μg / kg or less, 13 μg / kg or less, 14 μg / kg or less, 15 μg / kg or less, 16 μg / kg or less, 17 μg / kg or less, 18 μg / kg or less, 19 μg / kg or less, 20 μg / kg or less, 21 μg / kg or less, 22 μg / kg or less, 23 μg / kg or less, 24 μg / kg or less, 25 μg / kg or less, 26 μg / kg or less, 27 μg / kg or less, 28 μg / kg or less, 29 μg / kg or less, 30 μg / kg or less, 31 μg / kg or less, 32 μg / kg or less, 33 μg / kg or less, 34 μg / kg or less, 35 μg / kg or less, 36 μg / kg or less, 37 μg / kg or less, 38 μg / kg or less, 39 μg / kg or less, 40 μg / kg or less, 41 μg / kg One or more doses of 5 μg / kg or less, 30 μg / kg or less, 25 μg / kg or less, 20 μg / kg or less, 15 μg / kg or less, 10 μg / kg or less, 5 μg / kg or less, 2.5 μg / kg or less, 2 μg / kg or less, 1.5 μg / kg or less, 1 μg / kg or less, 0.5 μg / kg or less, or 0.2 μg / kg or less of an anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralaumab, are administered orally. In some embodiments, a dose of 100 μg / kg or less, 95 μg / kg or less, 90 μg / kg or less, 85 μg / kg or less, 80 μg / kg or less, 75 μg / kg or less, 70 μg / kg or less, 65 μg / kg or less, 60 μg / kg or less, 55 μg / kg or less, 50 μg / kg or less, 45 μg / kg or less, 40 μg / kg or less, 35 μg / kg or less, 30 μg / kg or less, 25 μg / kg or less, 20 μg / kg or less, 15 μg / kg or less, or a combination of these is used to prevent, treat, or ameliorate one or more symptoms of T1D. An oral dose of 0.5 μg / kg or less, 10 μg / kg or less, 5 μg / kg or less, 2.5 μg / kg or less, 2 μg / kg or less, 1.5 μg / kg or less, 1 μg / kg or less, 0.5 μg / kg or less, or 0.2 μg / kg or less of an anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralarumab, is administered over about 6 hours, about 4 hours, about 2 hours, about 1.5 hours, about 1 hour, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, about 2 minutes, about 1 minute, about 30 seconds, or about 10 seconds.

[0075] In some embodiments, where increasing doses are administered over the first few days of a dosing regimen, The dose of Men on day 1 is 5-100 μg / m 2 / day, preferably 51 μg / m 2 / day, gradually increasing to the daily dose listed immediately above by day 3, 4, 5, 6, or 7. For example, subjects may receive approximately 51 μg / m on day 1. 2 / day, and on day 2, approximately 103 μg / m 2 / day, and approximately 207 μg / m on day 3 2 / day, and on day 4, approximately 413 μg / m 2 / day and 826 μg / m on subsequent days of the regimen (e.g., days 5–14). 2 In some embodiments, the subject is administered approximately 227 μg / m on day 1. 2 / day, and on day 2, approximately 459 μg / m 2 / day, approximately 919 μg / m on day 3 and several days thereafter 2 In some embodiments, the subject is administered approximately 284 μg / m on day 1. 2 / day, and approximately 574 μg / m on day 2. 2 / day, approximately 1148 μg / m on day 3 and several days thereafter 2 Administer 1 dose per day.

[0076] In some embodiments, the initial dose is 1 / 4, 1 / 2, or the same amount as the last daily dose of the regimen, but administered in divided doses spaced 6, 8, 10, or 12 hours apart. For example, a 13 μg / kg / day dose is administered in four doses of 3-4 μg / kg, spaced 6 hours apart, to reduce the level of cytokine release caused by antibody administration. In some embodiments, the first 1, 2, 3, 4, or all doses of the regimen are administered intravenously more slowly to reduce the potential for cytokine release and other adverse effects. For example, a 51 μg / m 2The daily dose can be administered over about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, and about 22 hours. In some embodiments, the dose is administered by slow infusion, for example, over a 20-24 hour period. In some embodiments, the dose is infused by pump, preferably with increasing concentrations of antibody administered as the infusion progresses.

[0077] In some embodiments, the above 51 μg / m 2 / day~826μg / m 2 The specified fraction of the dose for the / day regimen is administered in escalating doses. In some embodiments, the fraction is 1 / 10, 1 / 4, 1 / 3, 1 / 2, 2 / 3, or 3 / 4 of the daily dose of the above regimen. Thus, if the fraction is 1 / 10, the daily dose is 5.1 μg / m on day 1. 2 , and 10.3 μg / m on the second day. 2 , and on the third day it was 20.7 g / m 2 , and 41.3 μg / m on the fourth day. 2 , and 82.6 μg / m on days 5–14. 2 If the fraction is 1 / 4, the dose is 12.75 μg / m on day 1. 2 , and 25.25 μg / m on the second day. 2 , and 51 μg / m on the third day. 2 , and 103 μg / m on the fourth day. 2 , and 207 μg / m on days 5–14. 2 If the fraction is 1 / 3, the dose is 17 μg / m on day 1. 2 , and 34.3 μg / m on the second day. 2 , and 69 μg / m on the third day. 2 , and 137.6 μg / m on the fourth day. 2 , and 275.3 μg / m on days 5–14. 2 If the fraction is 1 / 2, the dose is 25.5 μg / m on day 1. 2 , and 51 μg / m on the second day. 2 , and 103 μg / m on the third day. 2 , and 207 μg / m on the fourth day. 2 , and 413 μg / m on days 5–14.2 If the fraction is 2 / 3, the dose is 34 μg / m on day 1. 2 , and 69 μg / m on the second day. 2 , and 137.6 μg / m on the third day. 2 , and 275.3 μg / m on the fourth day. 2 , and 550.1 μg / m on days 5–14. 2 If the fraction is 3 / 4, the dose is 38.3 μg / m on day 1. 2 , and 77.3 μg / m on the second day. 2 , and 155.3 μg / m on the third day. 2 , and 309.8 μg / m on the fourth day. 2 , and 620 μg / m on days 5–14. 2 In some embodiments, the regimen is the same as one of the regimens above, but only for days 1-4, days 1-5, or days 1-6. For example, in some embodiments, the dose is 17 μg / m on day 1; 2 , and 34.3 μg / m on the second day. 2 , and 69 μg / m on the third day. 2 , and 137.6 μg / m on the fourth day. 2 , and 275.3 μg / m on days 5 and 6 2 becomes.

[0078] In some embodiments, an anti-CD3 antibody, such as teplizumab, otelixizumab, or fulvoxambucil Rather than being administered in a single daily dose over multiple days, foralumab is administered by infusion in a continuous manner over 4, 6, 8, 10, 12, 15, 18, 20, 24, 30, or 36 hours. The infusion may be constant, e.g., starting with a low dose for the first 1, 2, 3, 5, 6, or 8 hours of the infusion, and then increasing to a higher dose. Over the course of the infusion, the patient receives a dose equivalent to that administered in the 5-20 day regimen described above. For example, approximately 150 μg / m 2 , 200 μg / m 2 , 250 μg / m 2 , 500 μg / m 2 , 750 μg / m 2 , 1000 μg / m 2, 1500 μg / m 2 , 2000 μg / m 2 , 3000 μg / m 2 , 4000 μg / m 2 , 5000 μg / m 2 , 6000 μg / m 2 , 7000 μg / m 2 , 8000 μg / m 2 , or 9000 μg / m 2 In particular, the speed and duration of the infusion are designed to minimize the level of free anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralumab, in the subject after administration. In some embodiments, the level of free anti-CD3 antibody, e.g., teplizumab, should not exceed 200 ng / ml of free antibody. Furthermore, the infusion is designed to achieve a combination of at least 50%, 60%, 70%, 80%, 90%, 95%, or 100% T cell receptor coating and modulation.

[0079] In some embodiments, an anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralumab, is administered chronically to treat, prevent, slow or delay the onset or progression of, or reverse one or more symptoms of type 1 diabetes. For example, in some embodiments, an anti-CD3 antibody, e.g., teplizumab, is administered at a lower dose once per month, twice per month, three times per month, once per week, or more frequently, either as an alternative to, or to enhance or maintain the efficacy of, the 6-14 day dosing regimen described above. Such a low dose can be as low as 1 μg / m 2 to 100 μg / m 2 Any dose up to, for example, approximately 5 μg / m 2 , 10 μg / m 2 , 15 μg / m 2 , 20 μg / m 2 , 25 μg / m 2 , 30 μg / m 2 , 35 μg / m 2 , 40 μg / m 2 , 45 μg / m 2 , or 50 μg / m 2etc.

[0080] In some embodiments, a subject may be, or may be expected to be, re-dosed sometime after administration of a dosing regimen of an anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralarumab, e.g., based on one or more physiological parameters. Such administration of and / or assessment of the need for such re-dosing may occur 2 months, 4 months, 6 months, 8 months, 9 months, 1 year, 15 months, 18 months, 2 years, 30 months, or 3 years after administration of the dosing regimen, and may include administration of a course of treatment every 6 months, 9 months, 1 year, 15 months, 18 months, 2 years, 30 months, or 3 years indefinitely. [Example]

[0081] [Example 1] Teplizumab improves and stabilizes beta-cell function in seropositive high-risk individuals summary We analyzed the effects of a single 14-day course of teplizumab treatment on metabolic function and immune cells among participants in a randomized controlled trial of nondiabetic relatives at high risk for type 1 diabetes (T1D). In a previously reported extended follow-up study of teplizumab treatment (median 923 days), the median time to diagnosis was 59.6 months and 24.4 months (or 27.1 months according to Sims et al., Sci. Transl. Med. 13, eabc8980 (2021), incorporated herein by reference) for participants treated with teplizumab and placebo, respectively (HR = 0.457, p = 0.01). Only 22% of participants treated with placebo had a 5% or greater risk of developing type 1 diabetes. 0 percent remained diabetes-free. Glucose tolerance, C-peptide area under the curve (AUC), and insulin secretion rate were calculated and analyzed for their relationship to T cell subsets and function. Teplizumab treatment improved beta cell function as reflected by mean on-study C-peptide AUC (1.96 vs. 1.68 pmol / ml; p=0.009) (or 1.94 vs. 1.72 pmol / ml; p=0.006 according to Sims et al., Sci. Transl. Med. 13, eabc8980 (2021), incorporated herein by reference).

[0082] Drug treatment reversed the pre-enrollment decline in insulin secretion and subsequently stabilized the decline in C-peptide AUC seen with placebo treatment. Changes in C-peptide with teplizumab treatment were associated with an increase in partially exhausted memory KLRG1+TIGIT+CD8+ T cells (r=0.44; p=0.014) and reduced IFNγ and TNFα secretion. A single course of teplizumab had a sustained effect on delaying T1D diagnosis and improving beta cell function in high-risk individuals. Changes in CD8+ T-cell subsets indicate that partially exhausted effector cells are associated with clinical response. This is the first study to demonstrate successful modulation of autoimmune diabetes with immunotherapy.

[0083] Introduction Type 1 diabetes (T1D) is an autoimmune disease characterized by T cell-mediated destruction of insulin-producing beta cells within the pancreatic islets of Langerhans. Longitudinal observational studies spanning more than 30 years have documented the progression of this autoimmune disease from the initial appearance of autoantibodies until beta cell function is severely impaired, clinical diagnosis is made, and often ketoacidosis occurs (1-5). T1D is associated with the need for lifelong exogenous insulin administration for survival, increased morbidity and mortality due to immediate (e.g., hypoglycemia) and long-term complications (e.g., vascular, renal, and ocular diseases), as well as reduced survival, impaired vital function, and significant healthcare-related costs (6-9). Therefore, strategies to prevent progression to clinical T1D before incurable beta cell destruction and insulin deficiency are of paramount importance.

[0084] Alterations in beta cell function precede clinical diagnosis of T1D and have been examined in natural history cohorts of individuals identified as at-risk for the disease based on the presence of islet autoantibodies (10-12). Some studies suggest an ongoing, intermittent, progressive decline in beta cell function, which begins several years before clinical diagnosis when glucose tolerance is normal. During this period, signs of ongoing autoimmunity are present. Based on natural history findings, individuals with two or more islet autoantibodies have been classified as T1D stages with further specification according to the level of metabolic dysfunction: stage 1 precedes glucose abnormalities, stage 2 involves glycemic abnormalities during an oral glucose tolerance test (OGTT), and stage 3 involves clinical findings of hyperglycemia (2, 13, 14). However, the relationship between altered beta cell function and clinical disease remains poorly defined. For example, it is known that glucose tolerance, as defined by response to an oral glucose tolerance test (OGTT), can vary between abnormal and normal values ​​within at-risk individuals (15, 16). Furthermore, the clinical diagnosis and the OGTT glucose tolerance classification used to designate beta cell function measured by C-peptide response to metabolic challenge may not be closely related, and many individuals diagnosed using an OGTT have clinically meaningful C-peptide responses (15-18).

[0085] Based on the success from previous trials in stage 3 T1D patients (i.e., after clinical diagnosis) with teplizumab, a non-Fc receptor binding anti-CD3ε monoclonal antibody that demonstrated reduced stimulation of C-peptide responses compared with placebo or control participants ( 19 – 25 ), the TrialNet TN10 trial evaluated laparoscopic evaluation of teplizumab in individuals with stage 2 disease to test whether treatment prevented or delayed clinical diagnosis of T1D. A randomized phase II trial was conducted (26). This time-to-event study found a median delay to diagnosis of 24 months with teplizumab versus placebo and a reduction in the annual rate of diagnosed diabetes from 35.9% to 14.9% (26). This trial was the first to demonstrate the success of immunotherapy in preventing or delaying the diagnosis of T1D (27-31).

[0086] The successful outcome of TrialNet TN10, which used a clinical disease-modifying therapeutic intervention, allowed us to evaluate the effect of treatment on beta cell function and its relationship to immune modification, even when disease progression was clinically silent. To test the hypothesis that immunotherapy improves beta cell function in individuals at risk from TN10, we analyzed metabolic testing and immune response results from the trial. Our data indicate that prolonged treatment leads to a delay in clinical manifestations of T1D. We demonstrate that treatment with a single course of teplizumab reversed the decline in C-peptide production before study entry and improved beta cell responses to oral glucose after treatment compared with placebo. Early insulin secretion also improved with teplizumab, suggesting a qualitative improvement in beta cell function. After the first 3 to 6 months of treatment, C-peptide responses remained stable compared with placebo until a sudden decline in response occurred approximately 6 months before diagnosis in individuals diagnosed with clinical T1D. Improved C-peptide responses were associated with increased frequencies of TIGIT+KLRG1+ memory CD8+ T cells, which exhibited reduced secretion of IFNγ and TNFα, two proinflammatory cytokines associated with beta cell destruction (32). These studies demonstrate that even before clinical diagnosis, treatment with teplizumab can improve metabolic functions associated with modulation of pathological T cell signatures.

[0087] result Teplizumab treatment resulted in sustained delay of T1D during extended follow-up studies:A total of 76 relatives without a clinical diagnosis of T1D but at high risk were enrolled in a teplizumab prophylaxis trial (26). The median age was 13 years (range 8-49 years), and all participants had a 2+ autoantibody test result within 6 months prior to enrollment. We previously reported that 42 participants were diagnosed with T1D after a median follow-up of 742 days (range 74-2683 days). We have since continued to follow study participants for a median time of 923 days (range 74-3119 days) (Figure 1A). Over this extended follow-up period, 25 / 32 (78%) placebo-treated participants and 22 / 44 (50%) teplizumab-treated participants were diagnosed with T1D (Figure 1B) (Cox model adjusted for stratification and age: HR = 0.457, p = 0.01). The median time to diagnosis of T1D was 59.6 and 24.4 months in the teplizumab and placebo treatment groups, respectively (or 27.1 months according to Sims et al., Sci. Transl. Med. 13, eabc8980 (2021), incorporated herein by reference). Ten of the 13 subjects followed for more than 60 months or 5 years were not diagnosed with T1D. Of these individuals, eight were in the teplizumab group and two were in the placebo group.

[0088] Teplizumab treatment improved quantitative OGTT glucose AUC values ​​over the course of the study: To determine how teplizumab treatment affected glucose tolerance, OGTT outcomes were classified as normal, dysglycemic, or diabetic at study entry and matched for the frequency of those outcomes at each study visit over the first 36 months of the study and thereafter (Figure 3A; Figures 2A, 2B). Study participants were recruited based on dysglycemic OGTT test results. At randomization, consistent with known variability in OGTT results, a small number of subjects had normal (n = 3) or diabetic (n = 6) glucose tolerance at that visit. The primary endpoint of the study, a clinical diagnosis of T1D, required two consecutive diabetic OGTTs; therefore, participants continued in the study with a single diabetic OGTT. Three months of teplizumab or placebo treatment At subsequent visits, the frequency of dysglycemic OGTTs decreased and the frequency of normal OGTTs increased in both groups (6.8% to 30.2% with teplizumab treatment, McNemar's test: p=0.009; 15.6% to 36.7% with placebo, McNemar's test: p=0.02). Diabetic OGTTs also increased at this time point in both groups, particularly in the placebo group. Thereafter, the frequencies of normal and dysglycemic OGTTs remained relatively constant in the teplizumab group, and the frequency of diabetic OGTTs increased in both groups, but at a slower rate in participants treated with teplizumab.

[0089] Changes in OGTT classification may miss more subtle effects of treatment on OGTT glucose response. Therefore, we calculated and compared the mean on-study glucose AUC for each individual, correcting for time on study. The mean on-study glucose AUC was higher in those treated with teplizumab versus placebo (mean (IQR) 175 (159, 195) mg / dl vs. 165 (154, 180) mg / dl; ANCOVA teplizumab effect: 92.8%, p = 0.02) (Figure 3B, Table 1). Individual glucose AUC at study entry was a predictor of mean on-study glucose AUC, but values ​​at entry were similar between groups (geometric means for placebo and teplizumab unadjusted groups: 155.5 mg / dl for placebo and 162.2 mg / dl for teplizumab, p = 0.25).

[0090] [Table 1]

[0091] Mean on-study hemoglobin A1c (HbA1c) AUC was also calculated and analyzed. In contrast to glucose, mean on-study HbA1c AUC was not statistically different in those treated with placebo versus teplizumab (mean (IQR) 5.44% (5.29, 5.58) vs. 5.3% (4.99, 5.55); ANCOVA treatment: p=0.14) (Figure 4). Because the frequency of diabetes was higher in the placebo group, the similarity of HbA1c, a measure of chronic glucose exposure, and the higher mean on-study glucose AUC levels in the placebo group were most likely due to acute rather than chronic changes in glucose levels.

[0092] Teplizumab treatment increased C-peptide responses: The mean on-study C-peptide AUC was higher in the teplizumab treatment group compared to placebo (mean (IQR) 1.96 (1.48, 2.61) pmol / ml vs. 1.68 (1.32, 2.11) pmol / ml (p=0.009) (Figure 5, Table 2) (see also Sims et al., 2004, pp. 111-112, 2004, incorporated herein by reference). (1.94 vs. 1.72 pmol / ml; P = 0.006) according to [End Page 110]. To assess the relationship of this endpoint to incident diabetes, mean on-study C-peptide AUC values ​​were compared between participants who did or did not develop T1D during the observation period. For the participating study population, mean on-study C-peptide AUC was higher in individuals who remained diabetes-free compared to individuals who progressed to T1D (mean (IQR) 2.18 (1.52, 2.79) pmol / ml vs. 1.76 (1.30, 2.18) pmol / ml (p = 0.016)). However, within each treatment arm, those diagnosed with diabetes and There was no clear difference in mean C-peptide levels among those who remained diabetes-free: in the placebo arm, 11 / 17 and 10 / 16 individuals in the bottom and top halves of individuals, respectively, were diagnosed with T1D, and in the teplizumab arm, 13 / 22 and 7 / 22 individuals in the bottom and top halves of individuals, respectively (chi-square distribution, p=0.13) (Figure 5).

[0093] [Table 2]

[0094] Baseline C-peptide AUC (p<0.0001) was a significant determinant of mean on-study C-peptide AUC, although baseline values ​​were similar between treatment groups (unadjusted group means 1.95 pmol / ml and 1.99 pmol / ml for placebo and teplizumab (p=0.454)). As previously noted with new-onset T1D in studies of at-risk individuals (33), there was also a direct relationship between participant age and mean C-peptide AUC across both treatment arms and outcomes (Figure 6) (from ANCOVA, r=0.44, p=0.0001). In contrast to the relationship with time to clinical diagnosis ( 26 ), HLA-DR4+, HLA-DR3−, or anti-ZnT8 antibody status showed no significant interaction with mean on-study C-peptide AUC (using Wald test: HLA-DR3 p = 0.71, HLA-DR4 p = 0.27, ZnT8 p = 0.79).

[0095] Teplizumab treatment reverses the decrease in C-peptide AUC during the first 6 months of treatment:Because mean on-study C-peptide AUC may obscure more pronounced between-group differences at individual study time points, we next analyzed the timing of changes in C-peptide AUC relative to treatment and insulin secretion patterns. Because participants were recruited from the TN01 Natural History study, we were able to analyze C-peptide responses to OGTT before enrollment in this study and compare them with post-enrollment values. Geometric group means over the median 2.4 months before randomization and over the 12 months thereafter are shown in Figure 7 and Tables 3-4. There was a decrease in C-peptide AUC before study entry in both groups (pre-baseline and baseline): placebo 1.94 (1.68, 2.23) and 1.83 pmol / ml (1.59, 2.08), teplizumab 2.01 (1.77, 2.28) and 1.89 pmol / ml (1.67, 2.12), with a mean slope of -0.0202 (-0.0471, 0.0201) (n=43). In placebo-treated participants, the decrease in C-peptide persisted at the same rate for the first 6 months after enrollment, with no significant difference in pre- vs. post-treatment slope, even after adjustment for age at enrollment and C-peptide (mean C-peptide AUC over 6 months was 1.62 pmol / ml (1.35, 1.91)). In contrast, there was a significant increase in C-peptide AUC in participants treated with teplizumab 6 months after enrollment (mean C-peptide AUC at 6 months was 2.06 pmol / ml (1.85, 2.29); paired T-test p=0.02). Post-treatment slopes were significantly different between placebo- and teplizumab-treated participants by ANCOVA after adjusting for age and pre-treatment slopes (p=0.002).

[0096] [Table 3]

[0097] [Table 4]

[0098] Both total and early insulin secretion are improved by teplizumab treatment:In addition to the quantitative decrease in C-peptide AUC, studies by our group and others have identified qualitative abnormalities in beta cell secretory kinetics, accompanied by a loss of early insulin secretion, reflecting beta cell dysfunction prior to the onset of T1D (10, 33-36). To determine whether the quantitative improvement in C-peptide AUC was associated with qualitative changes in insulin secretion kinetics, we used a two-compartment model to determine the insulin secretion rate (ISR) during the OGTT and assessed kinetic and total insulin secretion (Figures 8A-8G, Table 5). We compared the OGTT insulin secretory response and the change in the secretory response (slope) over the same period and found a significant improvement in C-peptide AUC in the teplizumab-treated arm. This analysis allowed us to distinguish between early and late secretory responses (i.e., 1st and 2nd hours). The slopes describing the change in total insulin secreted, 1st-hour insulin, and 2nd-hour insulin were similar in both groups prior to study enrollment (p = 0.95). After treatment with teplizumab, there was a significant increase in total insulin secreted during the study in the teplizumab group, which was significantly greater than in the placebo group (p = 0.01, p = 0.0004). Insulin secreted during the first hour decreased in the placebo group but increased in the teplizumab group (p = 0.007). Insulin secretion at the second hour also improved in the teplizumab-treated group (p = 0.03) but not in the placebo group (p = 0.38) (Table 5). These results indicate that there is improvement in insulin secretion, particularly within the first hour of the OGTT, during the first 6 months after teplizumab treatment, suggesting improvement in beta-cell function, although there is a continued deterioration in insulin secretion in placebo-treated participants.

[0099] [Table 5]

[0100] C-peptide preservation is maintained for the last 6 months preceding clinical diagnosis:To determine the duration of these metabolic effects, we analyzed the C-peptide trajectories (least-squares straight lines) throughout the study period or up to 6 months before participants were diagnosed with T1D (Figures 9A and 9B). This analysis revealed that C-peptide AUC continued to decrease in the placebo group, with a median slope significantly less than zero (median, IQR: -0.00382, -0.0107 to 0.000755; Wilcoxon 1-sample: p = 0.04). The loss of C-peptide in the placebo group was even more pronounced at 6 months between the penultimate and final OGTT (mean slope (IQR) was -0.0242 (-0.0469, -0.0041); significantly non-zero (Wilcoxon 1-sample: p = 0.0001)) (Figures 9C and 9E).

[0101] In contrast, by the end of the study period, or 6 months before participants were diagnosed with T1D, the median slope for the teplizumab group was not significantly different from 0 (mean (IQR): -0.000294 (-0.00372, 0.00304), Wilcoxon 1-sample: p=0.63) (Figure 9B), and therefore less C-peptide AUC was lost over time compared to placebo-treated participants (Wilcoxon 2-sample: p=0.04). In teplizumab-treated participants diagnosed with T1D, there was also a decrease in C-peptide AUC in the peri-diagnosis period, but it was less pronounced than in the placebo-treated arm diagnosed with T1D (mean slope (IQR): -0.0112, -0.0818, 0.0107), Wilcoxon 1-sample compared to 0: p=0.09) (Figure 9D) (Wilcoxon 2-sample comparison with placebo and teplizumab slopes: p=0.06) (Figure 9E). Differences in insulin sensitivity between the two treatment arms were not a likely explanation for these findings, as C-peptide AUC / glucose AUC were similar in the teplizumab and placebo groups at the time of T1D diagnosis (p=0.23) (Figures 10A, 10B).

[0102] C-peptide responses correlate with an increase in partially exhausted CD8+ T cells:We hypothesized that the rapid improvement in metabolic response was related to the effects of teplizumab on T cells. We previously described an increase in the frequency of memory CD8+ T cells with teplizumab treatment, suggesting that they were "partially exhausted" by the expression of TIGIT and KLRG1+ (double-positive cells) and that the transcriptional activation / exhaustion signature could be further reduced by TIGIT ligation (23, 25, 26, 37, 38). Therefore, we checked whether their frequency was related to C-peptide AUC during or immediately after the drug treatment period and whether they were functionally exhausted. We observed a significant correlation between the frequency of CD8+KLRG1+TIGIT+ T cells and the fold change in C-peptide AUC at 3, 6, and 18 months (Table 6). Changes in T cell subsets most likely preceded changes in C-peptide; therefore, we also analyzed the fold change in double-positive CD8+ T cells at 3 months and the fold change in C-peptide at 6 months. There was a significant association between these two parameters in drug-treated participants but not in placebo-treated participants (p=0.014) (FIG. 11A).

[0103] [Table 6]

[0104] T cell exhaustion has been associated with decreased cytokine production after activation (39). Therefore, we measured intracellular cytokines after stimulation of PBMCs with anti-CD3 and anti-CD28. Among double-positive CD8+ T cells, the frequencies of IFNγ-producing cells (p<0.0001, p=0.0004) and TNFα-producing cells (p<0.0001 for both) decreased at 3 months (Figures 11B, 11C) and 6 months, respectively, in participants treated with teplizumab, but not in participants treated with placebo. In contrast, the relative proportions of IFNγ- and TNFα-producing cells among double-positive memory CD8+ T cells remained stable in the placebo group at 3- and 6-month follow-up. The fold decrease in the frequency of IFNγ and TNFα among double-positive CD8 memory cells between enrollment and month 3 correlated with the fold improvement in C-peptide between enrollment and month 6 (IFNγ: Pearson r = -0.29, p = 0.164; TNFα: r = -0.39, p = 0.056) (not shown).

[0105] Consideration Natural history cohort studies have described altered metabolic function during progression to T1D in at-risk relatives. Our successful intervention trial with teplizumab in an at-risk population provides a unique opportunity to directly assess how altering immune cells may affect metabolic function and progression to a clinical diagnosis of T1D in at-risk relatives. This extended follow-up study demonstrates that the effects of a single 14-day course of teplizumab treatment persist: the median time to diabetes in the teplizumab group was approximately 5 years compared with approximately 2 years in placebo-treated participants, and 50% of teplizumab-treated participants versus 22% of placebo-treated participants were not diagnosed with T1D. 18 percent of teplizumab-treated participants versus 6 percent of placebo-treated participants were followed for more than 5 years and were not diagnosed. Importantly, this is the first study to demonstrate successful modulation of the progression of beta-cell failure prior to the diagnosis of T1D with immune intervention.

[0106] Although participants enrolled in the study were not diagnosed with T1D, teplizumab treatment improved beta cell function. Teplizumab treatment resulted in lower mean OGTT glucose levels and higher C-peptide responses. There were improvements in total and early insulin secretion rates, identifying functional and quantitative improvements in insulin release. Early insulin secretion, a hallmark of normal beta cell function, was most dramatically altered, indicating an improvement in the impaired "beta cell glucose sensitivity" described in patients who progressed to clinical diabetes (34). Metabolic changes were associated with an increase in the frequency of TIGIT+KLRG1+ memory CD8+ T cells and a decrease in secretion of cytokines (TFNα and IFNγ) associated with T1D pathology, indicating functional T cell exhaustion (23, 25, 26, 37, 38).

[0107] Because the clinical trial was designed as a time-to-event protocol, the variable duration of the study for each participant created challenges in analyzing metabolic responses during the study OGTT. Therefore, mean on-study C-peptide, glucose, and HbA1c AUC were used, which included all of the available data for each participant.

[0108] Although time on study was not a significant determinant of mean C-peptide AUC, there was a time-dependent metabolic effect of drug treatment. Beta cell function declined in participants as they enrolled in the TN10 trial. Indeed, in previous studies, we found that levels of beta cell death were elevated among similar high-risk individuals, and other studies have documented beta cell dysfunction in the peridiagnostic period (35, 40, 41). This metabolic data, along with the relatively short median time to T1D diagnosis in the placebo group, indicates that the screening method utilized identified individuals at very high risk for disease activity and exacerbation. Consistent with preclinical studies, the effect during this period of active disease supports the notion that this intervention may be most effective in the presence of immune cell activation (42). The highest increase in C-peptide occurred immediately after teplizumab treatment and subsequent stabilization of beta cell function, whereas in the placebo group, beta cell function gradually declined over time. Consistent with previous reports, in those who developed clinical diabetes in both treatment arms, there was a steep decline in stimulated C-peptide levels seen approximately 6 months before the onset of T1D ( 40 ).

[0109] Unexpectedly, we found no relationship between mean intra-study glucose AUC and C-peptide AUC. Furthermore, OGTT outcomes varied even within individuals who did and did not develop T1D. Perhaps this variability reflects a population with weak levels of residual insulin. Consistent with this, OGTTs were not uniformly normalized in individuals without diagnosed T1D. Minor changes, such as insulin secretion kinetics or host factors, can alter OGTT outcomes, which are categorically classified based on blood glucose levels, which are related to long-term microvascular complications and not necessarily beta cell function or insulin secretion (43). These clinical outcomes are similar to the effects of anti-CD3 mAb in the NOD model of T1D, where insulin did not return beta cell mass to normal levels but did gradually improve outcomes (44, 45). Further testing with metabolic clamps may improve our analysis of metabolic function; such testing is not warranted in this clinical trial. These findings also suggest that it may be valuable to combine teplizumab with drugs that improve beta-cell function through complementary mechanisms.

[0110] Factors predicting disease 6 months before clinical T1D diagnosis in both the treatment and placebo arms were unclear at this time. The similar association between C-peptide and glucose in the two treatment arms among those diagnosed with T1D suggests that insulin insensitivity was not a contributing factor for diagnosis. Interestingly, even with progression to clinical diabetes, C-peptide declines tended to be less with teplizumab versus placebo, suggesting that the effects of drug treatment on C-peptide may persist even during and after clinical diagnosis. There may also be a waning effect of anti-CD3 antibodies on immune cells, which we previously identified by tracking CD8+ memory double-positive cells (23). Other observations in the field indicate that progression to clinical diabetes is associated with the acquisition of effector T cell function; in this setting, it is possible that the restored effector function may include a waning immune effect of teplizumab or even new or regenerated pathological T cells that repopulate the repertoire after a single course of drug. The median age at the time of treatment in the TN10 trial was 13.9 years, and in younger children, T cell thymic output may be ongoing. Another study of long-term outcomes in patients treated with teplizumab found an increased frequency of programmed cell death protein 1 (PD-1) memory CD8+ T cells in responders compared with non-responders and controls, suggesting that phenotypic and functional changes in the CD8+ memory compartment may occur over time (46). Ongoing work tracking TCR and single-cell analyses may help address these hypotheses and suggest agents that could be used to extend diabetes-free periods, perhaps by blocking pathways necessary for T effector expansion (47).

[0111] Our study has limitations. The number of subjects was relatively small, and the study was designed to detect differences in the occurrence of diabetes, rather than changes in C-peptide AUC, insulin secretion, and immune function. Additionally, the time-to-event design of the original study had some important implications for the analyses included. We did not perform OGTT analyses on most individuals after T1D diagnosis, which limited our ability to compare OGTT data between all members of the placebo and teplizumab groups over the same period, particularly for the placebo group, which showed a more rapid progression to diabetes. The time-to-event design also limited our ability to compare the relationship between metabolic endpoints and T1D progression, because the study included some individuals who did not progress to diabetes and who would eventually develop T1D. Furthermore, given previous results showing that teplizumab treatment preserved C-peptide in patients with recent-onset T1D (19-25), it may be possible to predict that a positive effect on C-peptide would occur among individuals who developed diabetes during this study. Participants from both arms of the study were enrolled in the TrialNet LIFT study, which conducts long-term metabolic studies in participants diagnosed with T1D ( 13 ).

[0112] In summary, we demonstrate a prolonged delay in progression to T1D in at-risk subjects treated with teplizumab. Teplizumab treatment altered the biological course of the disease by enhancing beta cell function, reflected by quantitative and qualitative improvements in insulin secretion. These changes were associated with modulation of memory D8+ T cell frequency and function. The apparent early efficacy of the drug following stabilization of beta cell function suggests that repeated treatment with teplizumab or the addition of other complementary agents at critical points in the clinical course may be valuable in extending the delay or even preventing the diagnosis of T1D. Finally, our findings have implications for other autoimmune diseases by demonstrating how immune intervention can alter pathobiology even before disease diagnosis and result in clinically significant outcomes.

[0113] material and method Study design The design of this phase 2, randomized, placebo-controlled, double-blind trial (NCT01030861) has been previously reported (26). Institutional review board approval was obtained at each participating site. Written informed consent or assent was obtained from participants, their parents, or both prior to study entry. Participants were identified through the TrialNet Pathway to Prevention study (TN01) (14, 48). In that study, OGTTs were performed at approximately 6-month intervals in islet autoantibody-positive individuals (including anti-glutamic acid decarboxylase 65, microinsulin, anti-islet antigen 2, anti-zinc transporter 8, and / or islet cell antibodies). Glucose results from these tests were used to identify eligibility for the anti-CD3 prevention trial (TN01) and were used in this data analysis. Islet autoantibody testing, HLA genotyping, and OGTT testing were performed as previously described (4, 49).

[0114] Briefly, eligibility criteria included age ≥8 years at randomization, a relative history of type 1 diabetes, positive titers for two or more islet autoantibodies, and abnormal glucose levels on an OGTT (fasting plasma glucose 110–125 mg / dL (6.1–6.9 mmol / L), a 2-hour postprandial plasma glucose level of ≥140 mg / dL (7.8 mmol / L) and <200 mg / dL (11.1 mmol / L), or a postprandial glucose level at 30, 60, or 90 minutes after intervention of >200 mg / dL). For participants who did not have hemoglobin A1c available at the baseline visit, values ​​obtained within 3 months prior to treatment were used.

[0115] Participants were randomly assigned to receive teplizumab or saline and treated with a 14-day outpatient course administered as an IV infusion at a clinical research center. Teplizumab was administered at a dose of 51 μg / m on day 0. 2 , 103 μg / m on day 1 2 , 207 μg / m on day 2 2 , 413 μg / m on day 3 2, followed by 826 μg / m on days 4–13. 2 Participants were administered a dose of 100 mg / dL (11.1 mmol / L). OGTTs were performed 3 and 6 months after infusion and every 3 months thereafter. Random screening glucose levels were assessed at 3-month intervals, and OGTTs were performed if the random glucose level was >200 mg / dL (11.1 mmol / L). T1D was diagnosed using ADA criteria during OGTTs but only after serially confirmed diabetic OGTTs. The date of diagnosis was determined as the first of two diagnostic tests (50). Six participants were clinically diagnosed with T1D outside of OGTT monitoring. The original study end date was May 2019. Participants who were not diagnosed with T1D were transferred to the TrialNet Pathway to Prevention Natural History study (TN01) for follow-up OGTT monitoring. Data from follow-up between June 2011 and March 2020 are included in this analysis. Participants who developed T1D were offered enrollment in TrialNet's Long Term Investigational Follow-up (LIFT) study for continued metabolic follow-up.

[0116] metabolic analysis OGTT C-peptide and glucose values ​​were measured using TOSOH and Roche, respectively. Testing was performed by Northwest Lipids Research Laboratories using C-peptide and glucose immunoassays. OGTT results were assigned to the closest study visit (within 3 months of the official time point assignment). OGTT results were classified as normal, dysglycemic, or diabetic based on the above definitions used for study entry. The baseline OGTT was at or immediately prior to randomization.

[0117] Area-under-the-curve (AUC) values ​​for ISR, C-peptide, and glucose were calculated using the trapezoidal rule. The mean AUC values ​​for C-peptide, glucose, and HbA1c during the study were multiplied by the mean AUC values ​​for each OGTT visit and the number of days between visits (as a trapezoidal rule) to calculate the total study AUC, which was then divided by the number of days from the first to the last OGTT (or the confirmatory diabetes OGTT in the case of incident T1D). Insulin secretion rates (ISR) were calculated using Chronobiological Series Analyzer (CSA) software, which uses a two-compartment model for hormone clearance and standard kinetic parameters for C-peptide (51-53). ISR calculations were performed using participants' OGTT C-peptide and glucose values, as well as their age, sex, height, and weight. Insulin secretion was divided into pmol secreted over the 2-hour OGTT or at the first or second hour of the study.

[0118] Flow cytometry analysis Peripheral blood mononuclear cells (PBMCs) were processed and stored at the NIDDK repository. Cryopreserved vials of PBMCs were sent to the INT Core laboratory at Benaroya Research Institute for flow cytometric analysis using the antibody panel shown in Tables 7 and 8. T cell phenotyping was performed on thawed PBMCs, and the frequency of CD45RO+CD8+ T cells that were TIGIT+KLRG1+CD57- was determined as previously described (54). Intracellular cytokine expression was measured 6 hours after stimulation of PBMCs with plate-bound anti-CD3 (1 μg / ml) and soluble anti-CD28 (10 μg / ml) in the presence of equimolar amounts of Golgi-stop. The frequency of TIGIT+KLRG1+CD8+ memory (CD45RA-) T cells producing IFNγ or TNFα was determined at baseline and 3 months.

[0119] [Table 7]

[0120] [Table 8]

[0121] Instrument standardization was performed using 8-peak rainbow calibration beads (Spherotech, Lake Forest, IL) to consistently adjust PMT voltages to the seventh peak mean fluorescence intensity. All samples from the same subject were run on the same day, and an internal control arm from the same subject was run each week. Sample acquisition was performed on an LSR-Fortessa (BD Biosciences) using FACS Diva software as previously described and analyzed using FlowJo software version 9.5 (Tree Star, Ashland, OR) (54). Quadrants were established based on staining controls. Gated populations with <100 events were excluded from analysis.

[0122] statistical analysis The original study was designed as a time-to-event analysis; therefore, participants diagnosed with T1D were not further followed in the study. The effect of teplizumab treatment on the emergence of type 1 diabetes after enrollment was analyzed using a Cox proportional hazards model. For this analysis, metabolic parameters across all study sites included OGTT data from the visit immediately preceding study drug treatment and all OGTT data after study drug treatment (the confirmatory diabetes OGTT for individuals diagnosed with diabetes or the last available OGTT for individuals who remained diabetes-free). The slopes for changes in glucose and C-peptide before and after enrollment were calculated using linear regression analysis of available OGTT visit data for specific intervals. The effect of treatment on each endpoint was determined by fitting results to an ANCOVA model with age, baseline value, and treatment group included as covariates. Wald tests were used to determine whether covariates significantly affected the model.

[0123] The estimated slope of change in insulin secretion rate was also calculated using linear regression models and mixed models for repeated measures at pretreatment (up to 6 months prior to baseline) and at the start of treatment. The change in the pre-treatment and post-treatment slopes was calculated for each subject based on the change from baseline (up to 6 months after baseline). Insulin secretion rates were calculated over the entire 2-hour interval, and specifically for the 1st and 2nd hour intervals of the OGTT. The difference in these slopes before and after treatment was compared using the Wilcoxon signed rank test within and across treatment arms. The difference in these slopes before and after treatment and the percent change were also evaluated using a general linear model to assess the effect of the treatment arm.

[0124] Flow cytometry data were log-transformed for statistical analysis. Pearson correction coefficients were calculated to determine the association between the fold change in C-peptide AUC and the frequency of TIGIT+KLRG1+CD8+ memory T cells. The frequency of TNFγ- or TNFα-producing TIGIT+KLRG1+CD8+ memory T cells was analyzed by paired t-test.

[0125] [Example 2] Anti-CD3 antibody (teplizumab) delays the onset of type 1 diabetes in stage 2 type 1 diabetes As shown in Figures 12-21, in at-risk (stage 2) relatives of T1D patients, a single course of teplizumab (as described in Example 1) increased TIGIT+KLRG1+ exhausted CD8+ T cells, which correlated with delay / prevention of clinical T1D. Surprisingly, no change to clinical T1D was observed in subjects with >10% exhausted CD8+ T cells (% of total CD3+ T cells, i.e., top quartile of exhausted T cells) in the circulation after 3 months of teplizumab treatment (p=0.005). Subjects with more TIGIT+KLRG1+CD8+ T cells after 3 months of treatment with teplizumab responded best.

[0126] These cells may express other markers of exhaustion, such as PD1 and Eomes, and may be partially depleted and produce lower levels of inflammatory cytokines than in placebo-treated patients.

[0127] TIGIT+KLRG1+CD8+ T cells are not homogenous, but instead vary in number and function across individuals. Rather than being uniformly depleted, TIGIT+KLRG1+ cells are a mixture of less and more functional populations.

[0128] We also observed a decrease in CD8+ T cells expressing the proliferation markers Ki67 and CD57 compared to placebo, consistent with a decline in effector T cells and an increase in exhausted T cells. Decreases in Ki67 and CD57 correlated with clinical response (p=0.003 and p=0.006, respectively). CD127+ CD8 T cells were associated with poorer outcomes.

[0129] Thus, in some embodiments, teplizumab can be repeatedly dosed and used in combination to increase the generation / maintenance of exhausted T cells and improve response and outcome.

[0130] In some embodiments, teplizumab responsiveness can be predicted before or immediately after dosing by determining exhausted T cells.

[0131] [Example 3] 2D analysis of glucose and C-peptide demonstrates the efficacy of teplizumab in individuals at risk for T1D after 3 months of treatment As shown in Figures 22-25, in at-risk (stage 2) relatives of T1D patients, a single course of teplizumab (as described in Example 1) increased the C-peptide AUC / glucose AUC ratio for at least 6 months. After 3 months of dosing, teplizumab The teplizumab arm increased (1.5 ± 2.9) versus the placebo arm (0.78 ± 2.7; p = 0.001). This difference persisted 6 months after treatment (p = 0.004). Therefore, this ratio can be used as a new early endpoint (e.g., 3 months) for further testing of teplizumab (retreatment of monotherapy, combination therapy).

[0132] Teplizumab also reduced the Diabetes Prevention Trial Risk Score (DPTRS) (see Sosenko et al., Diabetes Care. 2012 Jul;35(7):1552-1555, incorporated herein by reference). This is consistent with a benefit on C-peptide and blood glucose: +0.56 for placebo and -0.22 for teplizumab (p=0.02). The DPTRS can also be used to help guide re-medication in at-risk individuals.

[0133] [Example 4] Clinical Pharmacokinetics and Pharmacodynamics Mechanism of Action: Teplizumab is a humanized monoclonal antibody that targets the cluster of differentiation 3 (CD3) antigen, which is co-expressed with the T cell receptor (TCR) on the surface of T lymphocytes. Teplizumab's mechanism of action for the proposed indication has not been confirmed but appears to involve weak agonist activity on signaling through the TCR-CD3 complex, which may expand regulatory T cells and reestablish immune tolerance.

[0134] Pharmacokinetics: Figure 26 shows a plot of predicted mean teplizumab concentrations over time using a 14-day intravenous (IV) dosing regimen with a 4-day escalation followed by repeat doses of 826 μg / m on days 5-14. The left panel represents a typical 60 kg male subject, and the right panel represents typical 40 kg and 90 kg male subjects. Dosing based on body surface area (BSA) normalizes exposure across body size.

[0135] Repeated IV infusions resulted in increasing serum teplizumab levels, but steady-state PD was not reached at the end of dosing (day 14 within this dosing regimen). The mean accumulation ratio for the area under the curve (AUC) between days 5 and 14 was 3.4. The predicted mean (±SD) total AUC for the 14-day dosing regimen was 6421 ± 1940 ng·day / mL, with Cmax and Cmin of 826 ± 391 and 418 ± 225 ng / mL, respectively, on day 14.

[0136] Distribution: The central and peripheral volumes of distribution from the population PK analysis were 3.4 L and 6.9 L, respectively.

[0137] Elimination: Teplizumab clearance is not dose-proportional and may be driven by its saturable binding to the CD3 receptor on the surface of T cells. Teplizumab is expected to be degraded into smaller peptide fragments by catabolic pathways. The clearance of teplizumab after a 14-day dosing regimen was estimated to be 2.3 L / day from population PK analysis, with a terminal half-life of approximately 4 days.

[0138] The planned commercial formulation was manufactured at a different facility from the clinical trial product and was not used in the clinical trials submitted to support efficacy and safety. A single-dose PK bridging study evaluating the biocomparability of the commercial and clinical trial formulations was conducted in healthy volunteers. The mean AUC0-inf for the commercial product was less than half (48.5%, 90% CI: 43.6-54.1) of the AUC0-inf for the product used in the pivotal efficacy study. The reason for this difference is likely a faster clearance of the drug from the circulation rather than differences in product strength, as similar concentrations were observed immediately after IV infusion. The Cmax of the marketed product was 94.5% (90% CI; 84.5-106) of the Cmax observed in the clinical trial formulation.

[0139] Modifications and variations of the methods and compositions described herein will be apparent to those skilled in the art without departing from the scope and spirit of the present disclosure. Although the present disclosure has been described in connection with specific embodiments, it should be understood that the present disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the present disclosure are intended to be within the scope of the present disclosure, as expressed by the following claims, and will be understood by those skilled in the relevant art to which the present disclosure pertains.

[0140] Incorporation by Reference All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

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Claims

1. 1. A method for preventing or delaying the onset of clinical type 1 diabetes (T1D), comprising: Providing a non-diabetic subject at risk for T1D; administering to the non-diabetic subject a prophylactically effective amount of an anti-CD3 antibody; determining, before or after the administering step, that the non-diabetic subject has greater than about 5% to greater than about 10% TIGIT+KLRG1+CD8+ T cells among total CD3+ T cells, which indicates successful prevention or delay of the onset of clinical T1D; A method comprising:

2. 10. The method of claim 1, wherein the non-diabetic subject is a relative of a T1D patient.

3. 10. The method of claim 1, further comprising determining that the non-diabetic subject (1) is substantially free of antibodies against zinc transporter 8 (ZnT8), (2) is HLA-DR4+, and / or (3) is not HLA-DR3+.

4. 4. The method of claim 3, wherein the non-diabetic subject has two or more diabetes-associated autoantibodies selected from islet cell antibodies (ICA), insulin autoantibodies (IAA), and antibodies against glutamic acid decarboxylase (GAD), tyrosine phosphatase (IA-2 / ICA512), or ZnT8.

5. 10. The method of claim 1, wherein the non-diabetic subject has impaired glucose tolerance in an oral glucose tolerance test (OGTT).

6. 6. The method of claim 5, wherein the impaired glucose tolerance on OGTT is a fasting glucose level of 110-125 mg / dL, or a 2-hour plasma glucose level of 140 mg / dL or greater but less than 200 mg / dL, or a median glucose value at 30, 60 or 90 minutes during the OGTT of greater than 200 mg / dL.

7. The method of claim 3, wherein the non-diabetic subject does not have antibodies to ZnT8.

8. The method of claim 3, wherein the non-diabetic subject is HLA-DR4+ and not HLA-DR3+.

9. 2. The method of claim 1, wherein the anti-CD3 antibody is selected from teplizumab, otelixizumab, or foralumab.

10. The prophylactically effective amount may comprise administering the anti-CD3 antibody in an amount of 10 to 1000 micrograms per square meter (μg / m ２ ) by subcutaneous (SC) injection or intravenous (IV) infusion or orally for a 10-14 day course, preferably at 51 μg / m on days 0-3, respectively. ２ , 103 μg / m ２ , 207 μg / m ２ , and 413 μg / m ２ IV infusion at 826 μg / m on each of days 4-13 ２ 10. The method of claim 9, comprising a 14-day course of IV infusion of a single dose of

11. 10. The method of claim 9, wherein the prophylactically effective amount delays the median time to clinical diagnosis of T1D by at least 50%, at least 80%, or at least 90%, or by at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months.

12. The step of determining TIGIT KLRG1 CD8 T cells comprises flow cytometry.

12. The method according to any one of claims 1 to 11, wherein the method is by tree.

13. 12. The method of any one of claims 1 to 11, further comprising determining a decrease in the percentage of CD8+ T cells expressing the proliferation markers Ki67 and / or CD57.

14. 1. A method for predicting responsiveness to an anti-CD3 antibody in preventing or delaying the onset of type 1 diabetes (T1D), comprising: Providing a non-diabetic subject at risk for T1D; administering to the non-diabetic subject a prophylactically effective amount of an anti-CD3 antibody; determining the area under the C-peptide curve (AUC):glucose AUC ratio, wherein an increase in said ratio indicates responsiveness to the anti-CD3 antibody and / or non-progression of clinical T1D; A method comprising:

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