Methods and compositions for preventing type 1 diabetes

Abstract

To provide a treatment that prevents or delays the onset of clinical type 1 diabetes (T1D) in individuals at high onset risk for T1D.SOLUTION: Provided herein, in one aspect, is a method of preventing or delaying the onset of clinical type 1 diabetes (T1D), comprising: providing a non-diabetic subject who is at risk for T1D; 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+; and administering a prophylactically effective amount of an anti-CD3 antibody to the non-diabetic subject.SELECTED DRAWING: None

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 847,466, filed May 14, 2019, and U.S. Utility Application No. 15 / 931,685, filed May 14, 2020, the entire disclosures of which are incorporated herein by reference.

[0002] [Sequence table] An ASCII text file titled "010701seq.txt", measuring 6,083 bytes in size, created on May 14, 2020, submitted via EFS-Web on May 14, 2020, is hereby incorporated by reference in its entirety. [Field of the Invention]

[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 subjects at risk of developing T1D. [Background technology]

[0004] Type 1 diabetes (T1D) is caused by autoimmune destruction of insulin-producing β-cells in the islets of Langerhans, leading to a dependency on exogenous insulin injections for survival. Approximately 1.6 million Americans have type 1 diabetes, and type 1 diabetes remains one of the most common childhood diseases after asthma. 1 Despite improvements in care, individuals most affected by T1D consistently fail to achieve their desired glycemic goals. 2 There are continuing concerns regarding the increased risk of both morbidity and mortality for individuals with type 1 diabetes. Two recent studies found a 17.7-year reduction in survival for children diagnosed before age 10 years, and 11 and 13 years of reduced survival for Scottish men and women diagnosed as adults, respectively. 3,4.

[0005] In genetically susceptible individuals, T1D progresses through an asymptomatic stage characterized first by the appearance of autoantibodies (stage 1) and then glycemic abnormalities (stage 2) prior to overt hyperglycemia. In stage 2, the metabolic response to a glucose load is impaired, but other metabolic indices, e.g., glycosylated hemoglobin, are normal and insulin treatment is not required. 5 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 delay the decline of β-cell function when tested in early clinical T1D. 6 One promising treatment is the non-FcR-binding anti-CD3 monoclonal antibody teplizumab, which has been shown in several studies to durably reduce β-cell function loss after short-term treatment, with observable effects as long as 7 years after diagnosis and treatment. 7~11 The drug modifies the function of CD8+ T lymphocytes, which are thought to be key effector cells in initiating β-cell death. 12,13 .

[0006] To date, there are no interventions initiated prior to clinical diagnosis (i.e., stages 1 or 2) that alter progression to clinical stage 3 T1D. Thus, there is a need for treatments that prevent or delay the onset of clinical T1D in individuals at high risk of developing clinical T1D. Summary of the Invention

[0007] In one embodiment, there is provided a method of preventing or delaying the onset of clinical type 1 diabetes (T1D), comprising: Providing a non-diabetic subject at risk for T1D; Determining that a non-diabetic subject (1) is substantially free of antibodies to zinc transporter 8 (ZnT8), (2) is HLA-DR4+, and / or (3) is not HLA-DR3+; administering to the non-diabetic subject a prophylactically effective amount of an anti-CD3 antibody; A method is provided that includes:

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

[0009] In some embodiments, detection of autoantibodies associated with T1D is performed by point-of-care (POC) screening methods in the general population or in relatives of T1D patients. These POC methods can be qualitative rapid lateral flow tests.

[0010] In some embodiments, the non-diabetic subject has an infection with Coxsackie B virus (CVB) and / or other beta cell tropic virus(es). In some embodiments, the subject infected with a beta cell tropic virus has HLA-DR4 and is highly responsive to teplizumab.

[0011] In various embodiments, the non-diabetic subject has impaired glucose tolerance on an oral glucose tolerance test (OGTT), defined as a fasting glucose level between 110-125 mg / dL, or a 2-hour plasma value between 140 mg / dL and <200 mg / dL, or an intermediate glucose value at 30, 60, or 90 minutes during the OGTT of >200 mg / dL.

[0012] In some embodiments, the non-diabetic subject does not have antibodies to ZnT8. In certain embodiments, the non-diabetic subject is HLA-DR4+ and not HLA-DR3+.

[0013] In one embodiment, the anti-CD3 antibody is teplizumab.

[0014] In various embodiments, a prophylactically effective amount is a dose of 10-1000 micrograms per square meter (μg / m 2 ) of an anti-CD3 antibody, e.g., teplizumab, for a total dose of 6-15 milligrams of anti-CD3 / teplizumab. 2 ) by subcutaneous (SC) injection or intravenous (IV) infusion for 10 to 14 days, preferably with teplizumab at 51 μg / m on days 0 to 3, respectively. 2 , 103 μg / m 2 , 207 μg / m 2 , and 413 μg / m 2 IV infusion at 826 μg / m on days 4–13 2 In certain embodiments, the prophylactically effective amount of an anti-CD3 antibody, such as teplizumab, 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 longer.

[0015] In some embodiments, the anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralumab, is administered via an SC pump, embedded in a slow release biomaterial, or intravenously. In other embodiments, the anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralumab, is embedded in a biomaterial in which the beta cell precursors or beta cells are encapsulated, or is provided parenterally in conjunction with the beta cell precursors or beta cells.

[0016] In some embodiments, the anti-CD3 antibody, eg, teplizumab, otelixizumab, or foralumab, is administered in combination with other pharmacological agents, such as metabolic agents, B-cell inhibitors, or other immunomodulatory agents.

[0017] In some embodiments, the anti-CD3 antibody, eg, teplizumab, otelixizumab, or foralumab, is administered in conjunction with an antigen-specific immunotherapy and / or a vaccine.

[0018] In some embodiments, the method further comprises determining the frequency of TIGIT+KLRG1+CD8+ T cells in peripheral blood mononuclear cells of the non-diabetic subject by flow cytometry, where an increase in frequency following administration of an anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralumab, indicates responsiveness to the anti-CD3 antibody, e.g., teplizumab.

[0019] Another embodiment is a method of predicting responsiveness to an anti-CD3 antibody, such as teplizumab, otelixizumab, or foralumab, in preventing or delaying the onset of type 1 diabetes (T1D), comprising: Providing a non-diabetic subject at risk for T1D; administering to a non-diabetic subject a prophylactically effective amount of teplizumab, otelixizumab or foralumab; determining the frequency of TIGIT+KLRG1+CD8+ T cells in peripheral blood mononuclear cells of the non-diabetic subject by flow cytometry, where an increased frequency indicates responsiveness to an anti-CD3 antibody, such as teplizumab, otelixizumab or foralumab; The present invention relates to a method comprising the steps of:

[0020] In some embodiments, the method may further include determining that the non-diabetic subject (1) is substantially free of antibodies to zinc transporter 8 (ZnT8), (2) is HLA-DR4+, and / or (3) is not HLA-DR3+.

[0021] It will be apparent to one skilled in the art that the aspects and / or embodiments described herein may be combined. [Brief description of the drawings]

[0022] [Figure 1]Figure 1. Participant screening, enrollment, and follow-up. A total of 112 subjects were screened for eligibility at TrialNet sites (see Appendix for a list of study sites). Seventy-six of the subjects were randomized to the drug or placebo arm. Subjects were infused with study drug at 1 of 14 TrialNet sites and followed as per study protocol at 1 of 33 sites. All randomized subjects are included in the analysis. [Figure 2A] Figure 1. Effect of treatment with teplizumab on the occurrence of T1D. Kaplan-Meier estimates of the proportion of diabetes-free participants. The overall hazard ratio was 0.437 (95%CI: 0.229, 0.832) (p=0.006, one-sided, Cox model). The median time to T1D was 48.4 mos for the teplizumab group and 24.4 mos for the placebo group. The insert shows the total number of subjects with T1D at the end of the study and the total number of diabetes-free subjects. [Figure 2B] Figure 1. Effect of treatment with teplizumab on the incidence of T1D. Incidence of type 1 diabetes by treatment group and cumulative and interval hazard ratios (95% confidence intervals) by study year (*The number of participants in each treatment arm with incident T1D during the year interval is shown. In addition, cumulative HRs and HRs for each year interval were calculated. †The Mantel-Haenszel method was applied to time-to-event data for both chi-square tests and hazard ratio estimates34. Euro symbol, likelihood ratio test and hazard ratio estimates and 95% CI from Cox models). [Figure 2C] Figure 1: Effect of treatment with teplizumab on the development of T1D. Absolute lymphocyte counts (ALC) in the study groups. [Figure 3A]Immunologic impact and subgroup analysis of response to teplizumab. Frequency of CD8+KLRG1+TIGIT+CD57- T cells in subjects treated with teplizumab and in subjects treated with placebo (*p<0.05, participants treated with teplizumab vs. participants treated with placebo). Mean values ​​± 95%CI are shown. Comparisons were performed using ANCOVA at each time point and corrected for baseline values. Furthermore, there was a significant increase in the frequency of these cells in participants treated with teplizumab at 3 mos (p=0.009) and 6 mos (p=0.007), but not in placebo-treated participants (paired t-test). [Figure 3B] Figure 1 shows the immunological impact and subgroup analysis of response to teplizumab. The ladder plot shows the hazard ratio for each of the baseline participant characteristics shown. Absence of anti-ZnT8 antibodies (p=0.004, HR: 0.031 for negative, 0.657 for positive), presence of HLA-DR4 (p=0.004, HR: 1.47 for negative, 0.201 for positive), and absence of HLA-DR3 (p=0.01, HR: 0.181 for negative, 0.907 for positive) significantly influenced the hazard ratio. [Figure 3C-E] 3A-3D show immunological impact and subgroup analysis of response to teplizumab. Incidence of diabetes in patients with or without anti-ZnT8 antibodies at baseline (FIG. 3C), or positive or negative for HLA-DR3 (FIG. 3D) or HLA-DR4 (FIG. 3E) are shown. [Figure 4] 1 is a table showing the monoclonal antibodies used in flow cytometry. [Diagram 5] 1 is a graph showing enrolment in the study. [Figure 6]FACS contour plots showing staining for TIGIT (Y-axis) versus KLRG1 (X-axis). Electronic gates were placed on live CD8+CD57- T cells. Expression of KLRG1 and TIGIT in peripheral blood cells from three subjects treated with teplizumab (top row) and three subjects treated with placebo is shown. Numbers refer to the percentage of total cells gated within each quadrant. Quadrants were placed based on staining controls. [Figure 7A-B] Graphs showing the frequency of T cell subsets in treatment groups. The frequencies of CD4+ Treg (FIG. 7A), CD4+ CD127lo Foxp3+, and CD8+ TIGIT-KLRG1-CD57- T cells (FIG. 7B) at study visits are shown. When compared by ANCOVA for each time point and corrected for baseline values, the differences in both cell subsets between teplizumab and placebo, and from baseline to post-treatment, for each treatment arm were not statistically significant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present disclosure provides the surprising discovery that, in some embodiments, non-diabetic subjects who respond to treatment with an anti-CD3 antibody, e.g., teplizumab, do not have antibodies to ZnT8. In certain embodiments, such non-diabetic subjects are HLA-DR4+ and not HLA-DR3+. Unexpectedly, such non-diabetic subjects who respond to treatment with an anti-CD3 antibody have antibodies against ZnT8 after administration of teplizumab (e.g., 1 month, 2 months, 3 months, or more). After or before that, an increase in the frequency (or relative amount) of TIGIT+KLRG1+CD8+ T cells in peripheral blood mononuclear cells (eg, by flow cytometry) is demonstrated.

[0024] In some embodiments, provided herein is a method of preventing or delaying the onset of clinical type 1 diabetes (T1D), comprising providing a non-diabetic subject at risk of T1D; 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+; and administering to the non-diabetic subject a prophylactically effective amount of an anti-CD3 antibody, e.g., teplizumab.

[0025] In certain embodiments, a method for predicting responsiveness to an anti-CD3 antibody, such as teplizumab, in preventing or delaying the onset of T1D is provided. The method may include providing a non-diabetic subject at risk of T1D, administering to the non-diabetic subject a prophylactically effective amount of an anti-CD3 antibody, such as teplizumab, and determining the frequency of TIGIT+KLRG1+CD8+ T cells in peripheral blood mononuclear cells of the non-diabetic subject by flow cytometry, where an increased frequency indicates responsiveness to the anti-CD3 antibody, such as teplizumab.

[0026] [Definition] Certain terms are defined herein below: Additional definitions are provided throughout this application.

[0027] As used herein, the articles "a" and "an" refer to one or 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 meaning of "one or more," "at least one," and "one or more than one."

[0028] As used herein, "about" and "approximately" generally refer to an acceptable degree of error for the measured quantity given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given value range. The term "substantially" refers to greater than 50%, preferably greater than 80%, and most preferably greater than 90% or 95%.

[0029] 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 non-specified elements.

[0030] As used herein, the term "consisting essentially of" refers to those elements required in 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.

[0031] The term "consisting of" refers to compositions, methods, and their respective components described herein, excluding any element not recited in the description of that embodiment.

[0032] 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.

[0033] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2linear antibodies; single-chain antibody molecules (eg, scFv); and multispecific antibodies formed from antibody fragments.

[0034] 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.

[0035] 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).

[0036] 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.

[0037] 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 1-20 days.

[0038] 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.

[0039] As used herein, the term "prophylactically effective amount" refers to an amount of teplizumab sufficient to result in delay or prevention of the onset, recurrence or onset 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%, at least 95%.

[0040] Various aspects of the disclosure are described in further detail below. Additional definitions are set forth throughout the specification.

[0041] <Anti-CD3 antibody and pharmaceutical composition> The terms "anti-CD3 antibody" and "antibody that binds to CD3" refer to cluster of differentiation 3 (C3C). "anti-CD3" refers to an antibody or antibody fragment capable of binding to CD3 with sufficient affinity that the antibody is therefore useful as a prophylactic, diagnostic and / or therapeutic agent in targeting CD3. In one embodiment, the extent of binding of an anti-CD3 antibody to an unrelated protein that is not CD3 is less than about 10% of the binding of the antibody to CD3, as measured, for example, by radioimmunoassay (RIA). In certain 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, for example, 10 -9 M~10 -13 In certain embodiments, the anti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3 from different species.

[0042] In one embodiment, the anti-CD3 antibody can be ChAglyCD3 (otelixizumab). Otelixizumab is a humanized Fc-free 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 study (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., Diabetes. 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.,Immunotherapy(2011)3(11),1303-1316;Daifotis et al. al., Clinical Immunology (2013) 149, 268-278.

[0043] In another embodiment, the anti-CD3 antibody can be vicilizumab (also called HuM291; Nuvion). Vicilizumab is a humanized anti-CD3 monoclonal antibody characterized by a mutant IgG2 isotype, lack of binding to Fcγ receptors, and the ability to selectively induce apoptosis in activated T cells. Vicilizumab has been evaluated in patients with graft-versus-host disease (NCT00720629; NCT00032279) as well as 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.

[0044] In another embodiment, 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 ... Please see 010;185(6):3401-7.

[0045] In another embodiment, the anti-CD3 antibody can be teplizumab. Teplizumab is also known as hOKT3yl(Ala-Ala) (containing alanine at positions 234 and 235) and is an anti-CD3 antibody engineered to change the function of T lymphocytes that mediate the destruction of insulin-producing β cells of pancreatic islets. Teplizumab binds to an epitope on the CD3s 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 listed below. The bolded parts are complementarity determining regions. Teplizumab light chain (SEQ ID NO: 1): JPEG2025072504000001.jpg1296 Teplizumab heavy chain (SEQ ID NO:2): JPEG2025072504000002.jpg2496

[0046] In some embodiments, pharmaceutical compositions are provided herein. Such compositions include a prophylactically effective amount of an anti-CD3 antibody and a pharmaceutically acceptable carrier. In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in animals, more particularly in humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias. 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).

[0047] 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, for example, 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 EW Martin. Such compositions contain a prophylactically or therapeutically effective amount of a prophylactic or therapeutic agent, preferably in purified form, and in a form suitable for administration to a patient. The pharmaceutical composition is preferably in a suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject.

[0048] In certain embodiments, it may be desirable to administer the pharmaceutical composition locally to the area in need of treatment, which can be achieved, for example and without limitation, by local injection or by using an implant, which can be a membrane, such as a silastic membrane, or a porous, non-porous, or gelatinous material, including fibrous materials. Preferably, when administering an anti-CD3 antibody, care must be taken to use a material to which the anti-CD3 antibody does not absorb.

[0049] In another embodiment, the compositions can be delivered as vesicles, in particular 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.).

[0050] In yet another embodiment, the composition can be delivered in a controlled or sustained release system. In one embodiment, a pump can be used to achieve the controlled or sustained release (see 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 another embodiment, a polymeric material can be used to achieve the controlled or sustained release of the antibody or fragment thereof of the invention (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Delivery System, vol. 11, no. 1, pp. 1171-1175). Bioavailability,Drug Product Design and (See, for example, U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 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 preferred embodiments, the polymers used in sustained release formulations are inert, free of potentially leachable impurities, stable in storage, sterile, and biodegradable. In yet another embodiment, the controlled or sustained release system can be placed in the vicinity of the therapeutic target, i.e., the lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, et al., J. Pharmacol. 1999, 143:131-135, 1999). in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984).

[0051] 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 containing 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.

[0052] A pharmaceutical composition can be formulated to suit 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 certain embodiments, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to humans. In preferred embodiments, the pharmaceutical composition is formulated according to routine procedures for subcutaneous administration to humans. In general, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the site of injection.

[0053] 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 unit dosage form, for example, in ampoules or in multi-dose containers, with added preservatives. The composition can take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and can contain formulatory agents such as suspending, stabilizing 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.

[0054] In certain embodiments, the present disclosure provides dosage forms (e.g., in conjunction with a pump or other device for such delivery) that allow for the administration of anti-CD3 antibodies continuously 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 other specific embodiments, the present disclosure provides for the administration of anti-CD3 antibodies in a continuous manner, with doses increasing continuously, e.g., up to 51 μg / m 2 / day to 826 μg / m 2 Dosage forms are provided that allow administration over a period of 24 hours, 30 hours, 36 hours, 4 days, 5 days, 7 days, 10 days or 14 days, increasing the dose to 100 mg / day.

[0055] The compositions can be formulated as neutral or salt forms. 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. can be.

[0056] In general, the components of the compositions disclosed herein are supplied either separately or mixed together in unit dosage form, for example as dry lyophilized powder or 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 injection 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.

[0057] 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 agent. In one embodiment, the anti-CD3 antibody or pharmaceutical composition thereof is provided as a dry, sterile, lyophilized powder or water-free concentrate in a sealed container, which can be reconstituted to a suitable concentration for administration to a subject, for example, with water or saline. The anti-CD3 antibody or pharmaceutical composition thereof is preferably provided 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 pharmaceutical composition herein should be stored in its original container at between 2° C. and 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, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour after reconstitution. In an alternative embodiment, the pharmaceutical composition is supplied in liquid form in a sealed container indicating the quantity and concentration of the drug. The liquid form of the composition to be administered is preferably supplied in a sealed container at at least 0.25 mg / ml, more preferably at least 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 / kg, 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 between 2°C and 8°C.

[0058] In certain 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.

[0059] The compositions can, if desired, be presented in a pack or dispenser device which can 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.

[0060] 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 exact dose to be used in the formulation also depends on the route of administration and the severity of the condition, and should be determined 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.

[0061] Methods and Use In certain embodiments, the present disclosure provides for the administration of an anti-cancer drug, such as teplizumab, to individuals who are predisposed to developing type 1 diabetes or have preclinical stages of type 1 diabetes, but who do not meet the 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 to prevent or delay the need for administration of insulin to such patients. In certain embodiments, high risk factors for identifying a predisposed subject include having a first or second degree relative diagnosed with type 1 diabetes, abnormal fasting glucose levels (e.g., at least one determination of glucose level after fasting (no food for 8 hours) of 100-125 mg / dl), impaired glucose tolerance in response to a 75 g OGTT ... These include at least one determination of a 2-hour glucose level in response to an OGTT of 140-199 mg / dl, HLA type DR3, DR4, or DR7 for Caucasians, HLA type DR3 or DR4 for Africans, HLA type DR3, DR4, or DR9 for Japanese, 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 in serum or other tissues, particularly ICA and type 1 diabetes-associated autoantibodies. In certain 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 having a predisposition to developing type 1 diabetes, wherein the subject exhibits 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.

[0062] Serum autoantibodies associated with type 1 diabetes or a predisposition to developing type 1 diabetes are pancreatic 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 encompasses treating an individual with a detectable autoantibody associated with a predisposition to developing type 1 diabetes or associated with early stage type 1 diabetes (e.g., anti-IA2, anti-ICA512, anti-GAD or anti-insulin autoantibodies), where the individual has not been diagnosed with type 1 diabetes and / or is a first- or second-degree relative of a type 1 diabetes patient. In certain 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 immunospecific detection of antibodies described herein or known to one of skill in the art.

[0063] β-cell function before, during, and after treatment 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 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 requirement, C-peptide level / response, low blood glucose episodes, and / or FPIR can be used as markers of β-cell function or to establish a therapeutic index (Keymeulen et al., 2005, N. Engl. J. Med. 352: 2598-2608; Herold et al., 2006, N. Engl. J. Med. 352: 2598-2608, respectively). (see, e.g., Bingley et al., 1996, Diabetes 54:1763-1769; U.S. Patent Application Publication No. 2004 / 0038867 A1; and Greenbaum et al., 2001, Diabetes 50:470-476). For example, FPIR is calculated as the sum of the insulin values ​​at 1 and 3 minutes after an IGTT performed according to the Islet Cell Antibody Register User's Study protocol (see, e.g., Bingley et al., 1996, Diabetes 54:1763-1769; U.S. Patent Application Publication No. 2004 / 0038867 A1; and Greenbaum et al., 2001, Diabetes 50:470-476). s 45:1720-1728 and McCulloch et al., 1993, Diabetes Care 16:911-915).

[0064] In some embodiments, an individual predisposed to developing T1D may be a non-diabetic subject who is a relative of a T1D patient. In certain 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.

[0065] In various embodiments, the non-diabetic subject has impaired glucose tolerance on an oral glucose tolerance test (OGTT), defined as a fasting glucose level between 110-125 mg / dL, or a 2-hour plasma value between 140 mg / dL and <200 mg / dL, or an intermediate glucose value at 30, 60, or 90 minutes during the OGTT of >200 mg / dL.

[0066] In some embodiments, the non-diabetic subject responding to anti-CD3 antibody, such as teplizumab, does not have antibodies against ZnT8. In certain embodiments, such non-diabetic subject is HLA-DR4+ and not HLA-DR3+. In some embodiments, such non-diabetic subject responding to anti-CD3 antibody, such as teplizumab, demonstrates (e.g., by flow cytometry) an increase in the frequency (or relative amount) of TIGIT+KLRG1+CD8+T cells in peripheral blood mononuclear cells after administration (e.g., 1 month, 2 months, 3 months, or more or less).

[0067] In various embodiments, a prophylactically effective amount is an anti-CD3 antibody, such as teplizumab, in the range of 10 to 1000 micrograms per square meter (μg / m 2 ) by subcutaneous (SC) injection or intravenous (IV) infusion over a 10-14 day course. In one embodiment, the prophylactically effective amount includes an anti-CD3 antibody, such as 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 days 4–13 2 In certain 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, or more.

[0068] In certain 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 certain embodiments, the efficacy of treatment with an anti-CD3 antibody, e.g., teplizumab, is determined as described herein or 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.

[0069] In another embodiment, the 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. ... approximately 200μg / kg, 178μg / kg, 180μg / kg, 128μg / kg, 100μg / kg, 95μg / kg, 90μg / kg, 85μg / kg, 80μg / kg, 75μg / kg , 70μg / kg, 65μg / kg, 60μg / kg, 55μg / kg, 50μg / kg, 45μg / kg, 40μg / kg, 35μg / kg, 30μg / kg, 26μg / kg, 25μg / kg, 20 In one or more unit doses of an anti-CD3 antibody, e.g., teplizumab, is administered.

[0070] In certain embodiments, the subject is administered about 5 to 1200 μg / m 2 , preferably 51 to 826 μg / m 2 In another embodiment, the subject is administered one or more doses of 1200 μg / m2 of an anti-CD3 antibody, such as 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 , 1100μg / m 2 , 1050μg / m 2 , 1000μg / m 2 , 950 μg / m 2 , 900μg / 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 , 100μg / m 2 , 50 μg / m 2 , 40 μg / m 2 , 30 μg / m 2 , 20 μg / m 2 , 15 μg / m 2 , 10μg / m 2 , or 5 μg / m 2 of anti-CD3 antibody, e.g., teplizumab, is administered.

[0071] In another embodiment, the subject is administered a treatment regimen comprising one or more doses of a prophylactically effective amount of an anti-CD3 antibody, such as teplizumab, where the course of treatment is administered for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In one embodiment, the treatment regimen comprises administering a prophylactically effective amount of a dose every day, every other day, every second day, every third day, or every fourth day. In certain embodiments, the treatment regimen comprises administering a prophylactically effective amount of a dose on Monday, Tuesday, Wednesday, and Thursday of a given week, and not administering a prophylactically effective amount of a dose on Friday, Saturday, and Sunday of the same week, until 14, 13, 13, 12, 11, 10, 9, or 8 doses have been administered. In certain embodiments, the dose administered is the same for each day of the regimen.

[0072] In certain 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 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 , 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, 700 μg / m 2 / day, 650 μg / m 2 / day, 600μg / m 2 / day, 550 μg / 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, 30 μg / m 2 / day, 20 μg / m 2 / day, 15μg / m 2 / day, 10μg / m 2 / day or 5μg / m 2 / day.

[0073] In another embodiment, 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 2Below, 550μg / m 2 Below, 500μg / m 2 Below, 450μg / m 2 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 administered 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 certain embodiments, the total dose administered in the regimen may be less than 100 μg / m 2 ~200μg / m 2 , 100μg / m 2 ~500μg / m 2 , 100μg / m 2 ~1000μg / m 2 , or 500 μg / m 2 ~1000μg / m2 It is.

[0074] In a preferred embodiment, the dose is escalated over the first quarter, half or two-thirds of the dose of the 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 certain embodiments, a subject is administered a treatment regimen that includes one or more doses of a prophylactically effective amount of an anti-CD3 antibody, e.g., teplizumab, where the prophylactically effective amount is, 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, 4 μg / kg, 5 μg / kg, 6 μg / kg, 7 μg / kg, 8 μg / kg, 9 μg / kg, 10 μg / kg, 11 μg / kg, 12 μg / kg, 13 μg / kg, 14 μg / kg, 15 μg / kg, 16 μg / kg, 17 μg / kg, 18 μg / kg, 19 μg / kg, 20 μg / kg, 21 μg / kg, 22 μg / kg, 23 μg / kg, 24 μg / kg, 25 μg / kg, 26 μg / kg, 27 μg / kg, 28 μg / kg, 29 μg / kg, 30 μg / kg, 31 μg / kg, 32 μg / kg, 33 μg / kg, 34 μg / kg, 35 μg / kg, 36 μg / kg, 37 μg / kg, 38 μg / kg, 39 μg / kg, 40 μg / kg, 41 μg / kg, 42 μg / kg, 43 μg / kg, 44 μg / kg g, 4 μg / kg, 5 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 35 μg / kg, 40 μg / kg, 45 μg / kg, 50 μg / kg, 55 μg / kg, 60 μg / kg, 65 μg / kg, 70 μg / kg, 75 μg / kg, 80 μg / kg, 85 μg / kg, 90 μg / kg, 95 μg / kg, 100 μg / kg, or 125 μg / kg; or, for example, 1 μg / m daily 2 Each, 5μg / m 2 Each, 10μg / m 2 Each, 15μg / m 2 Each, 20μg / m 2 Each, 30μg / m 2 Each, 40μg / m 2 Each, 50μg / m 2 Each, 60μg / m 2 Each, 70μg / m 2 Each, 80μg / m 2 Each, 90μg / m 2 Each, 100μg / m 2Each, 150μg / m 2 Each, 200μg / m 2 Each, 250μg / m 2 Each, 300μg / m 2 Each, 350μg / m 2 Each, 400μg / m 2 Each, 450μg / m 2 Each, 500μg / m 2 Each , 550 μg / m 2 Each, 600μg / m 2 or 650μg / m 2 In certain 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 amount of the anti-CD3 antibody, e.g., teplizumab, is achieved.

[0075] In certain 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 0.5 μg / kg or less of an anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralaumab, is administered intramuscularly.

[0076] In another embodiment, 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 0.5 μg / kg or less of an anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralaumab, is administered subcutaneously.

[0077] In another embodiment, 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, 35 μg / kg or less, 40 μg / kg or less, 4 ... One or more doses of ≤ μg / kg, ≤ 30 μg / kg, ≤ 25 μg / kg, ≤ 20 μg / kg, ≤ 15 μg / kg, ≤ 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 ≤ 0.5 μg / kg of an anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralumab, are administered intravenously. In another embodiment, the 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, An intravenous dose of an anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralumab, is administered for 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, about 40 seconds, about 50 seconds, about 60 seconds, about 80 seconds, about 90 seconds, about 100 seconds, about 150 seconds, about 100 seconds, about 50 seconds, about 2 minutes, about 1 minute, about 30 seconds, about 5 ... The dose is administered over a period of 10 minutes or about 10 seconds.

[0078] In another embodiment, 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, 30 μg / kg or less, 50 μg / kg or less, 55 μg / kg or less, 50 μg / kg or less, 45 μg / kg or less, 40 μg / kg or less, 5 ... 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 0.5 μg / kg or less of an anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralaumab, are administered orally. In another embodiment, the dose is 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 20 μg / kg or less 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 0.5 μg / kg or less of an anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralumab, 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.

[0079] In certain embodiments in which escalating doses are administered over the first few days of a dosing regimen, the dose on day 1 of the regimen is 5 to 100 μg / m 2 / day, preferably 51 μg / m 2 / day and titrated up to the daily dose listed immediately above by day 3, 4, 5, 6, or 7. For example, subjects may receive approximately 51 μg / m 2 / day and approximately 103 μg / m on day 2 2 / day, approximately 207 μg / m on day 3 2 / day, approximately 413 μg / m on day 4 2 / day and 826 μg / m on subsequent days of the regimen (e.g., days 5–14). 2 In another embodiment, the subject is administered approximately 227 μg / m on day 1. 2 / day and approximately 459 μg / m on day 2 2 / day, approximately 919 μg / m on the 3rd day and for several days thereafter. 2 In another embodiment, 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 the 3rd day and several days thereafter. 2 Administered at 100 mg / day.

[0080] In other embodiments, the initial dose is ¼, ½, or the same amount as the last daily dose of the regimen, but in divided doses spaced 6, 8, 10, or 12 hours apart. For example, a dose of 13 μg / kg / day is administered in four doses of 3-4 μg / kg spaced 6 hours apart to reduce the level of cytokine release caused by administration of the antibody. In certain embodiments, the first one, two, three, or four or all doses of the regimen are administered more slowly by intravenous administration to reduce the possibility of cytokine release and other adverse effects. For example, a dose of 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 certain embodiments, the dose is administered by slow infusion, for example, over a period of 20 to 24 hours. In certain embodiments, the dose is infused by pump, preferably with increasing concentrations of antibody administered as the infusion progresses.

[0081] In other 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 certain 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, 5.2 μg / m on day 2, 5.3 μg / m on day 3, 5.4 μg / m on day 4, 5.5 μg / m on day 5, 5.6 μg / m on day 6, 5.7 μg / m on day 7, 5.8 μg / m on day 8, 5.9 μg / m on day 9, 5.8 μg / m on day 10, 5.9 μg / m on day 11, 5.8 μg / m on day 12, 5.9 μg / m on day 2 , and 10.3 μg / m on the second day. 2 , and 20.7 μg / m on the third day. 2 , and 41.3 μg / m on the fourth day. 2 , and 82.6 μg / m on days 5 to 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 to 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 to 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 to 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 to 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 to 14. 2 In other embodiments, the regimen is the same as one of the above regimens, but only for days 1-4, days 1-5, or days 1-6. For example, in certain embodiments, the dose is 17 μg / m on day 1, 17 μg / m on day 2, 17 μg / m on day 3, 17 μg / m on day 4, 17 μg / m on day 5, or 17 μg / m on day 6. 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 become.

[0082] In certain embodiments, the anti-CD3 antibody, e.g., teplizumab, otelixizumab, or foralumab, is administered by infusion in a continuous manner over 4, 6, 8, 10, 12, 15, 18, 20, 24, 30, or 36 hours, rather than administered in a daily dose over many days. The infusion may be constant, e.g., starting at 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 , 1000μg / m 2 , 1500μg / m2 , 2000μg / m 2 , 3000μg / m 2 , 4000μg / m 2 , 5000μg / m 2 , 6000μg / m 2 , 7000μg / m 2 , 8000μg / m 2 , or 9000 μg / m 2 In particular, the speed and duration of the infusion is designed to minimize the level of free anti-CD3 antibody, such as teplizumab, otelixizumab or foralumab, in the subject after administration. In certain embodiments, the level of free anti-CD3 antibody, such as 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.

[0083] In other embodiments, an anti-CD3 antibody, such as teplizumab, otelixizumab, or foralumab, is administered chronically to treat, prevent, or slow or delay the onset or progression of, or reverse, one or more symptoms of type 1 diabetes. For example, in certain embodiments, an anti-CD3 antibody, such as teplizumab, is administered at a lower dose once a month, twice a month, three times a month, once a week, or even 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 lower dose may be as low as 1 μg / m 2 to 100 μg / m 2 Any dose up to about 5 μg / m 2 , 10μg / 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 / m2 And so on.

[0084] In other embodiments, the subject may be re-dosed, or may be re-administered, at some time after administration of a dosing regimen of an anti-CD3 antibody, such as teplizumab, otelixizumab, or foralumab, for example, based on one or more physiological parameters. Such re-dosing 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 administering a course of treatment every 6 months, 9 months, 1 year, 15 months, 18 months, 2 years, 30 months, or 3 years indefinitely. EXAMPLES

[0085] [summary] Background: Type 1 diabetes (T1D) is a chronic autoimmune disease that leads to the destruction of insulin-producing β-cells and dependence on exogenous insulin for survival. Although several interventions have shown success in attenuating the loss of insulin production in patients with clinical disease, no intervention to date has been able to affect disease progression in individuals at high risk of developing the disease.

[0086] Methods: We conducted a randomized, placebo-controlled, double-blind trial of teplizumab (a non-FcR binding anti-CD3 mAb) in nondiabetic relatives of T1D patients at high risk for clinical disease. Patients were randomized to a single 14-day course of drug or placebo and followed up for disease progression at approximately 6-month intervals using oral glucose tolerance testing.

[0087] Results: A total of 76 subjects were randomly assigned, 44 to the teplizumab arm and 32 to the placebo arm. Seventy-two percent of participants were children. Treatment with teplizumab delayed the median time to clinical diagnosis of T1D from 24.4 to 48.4 months (Cox proportional hazards, p=0.006) and reduced the annual incidence of diabetes from 9.8% to 4.1%. Drug treatment increased the percentage of TIGIT+KLRG1+CD8+ T cells. Participants without antibodies to ZnT8 (p=0.01), those who were HLA-DR4+ (p=0.006), and those who were not HLA-DR3+ (p=0.05) were most likely to respond to teplizumab.

[0088] Conclusions: Teplizumab can delay the diagnosis of T1D in high-risk individuals. A subgroup of individuals may be more likely to respond to treatment and benefit.

[0089] [method] <Test participants> Participants in the TrialNet Natural History Study 14 Patients were identified through a randomized controlled trial (NCT0223466112) and randomized controlled trial (NCT0223466112). The study was conducted at sites in the United States, Canada, and Germany between July 2011 and November 2018. Institutional Review Board (IRB) approval was obtained at each participating site. Written informed consent was obtained from patients, their parents, or both prior to study entry.

[0090] Eligible participants were nondiabetic relatives of patients with type 1 diabetes, were over 8 years old at the time of randomization, and were at high risk for developing clinical diabetes. Eligible participants had two or more diabetes-associated autoantibodies on two specimen collections within 6 months prior to randomization. In addition, eligible participants had a fasting glucose level of 110-125 mg / dL or 140 mg / dL or 150 mg / dL or 160 mg / dL or 170 mg / dL or 180 mg / dL or 190 mg / dL or 200 mg / dL or 210 mg / dL or 220 mg / dL or 230 mg / dL or 240 mg / dL or 250 mg / dL or 260 mg / dL or 270 mg / dL or 280 mg / dL or 290 mg / dL or 300 mg / dL or 310 mg / dL or 320 mg / dL or 330 mg / dL or 340 mg / dL or 350 mg / dL or 360 mg / dL or 370 mg / dL or 380 mg / dL or 39 ... They had impaired glucose tolerance, defined as a 2-hour plasma value >200 mg / dL or interim glucose values ​​at 30, 60, or 90 minutes during the OGTT >200 mg / dL. In 2014, the protocol was amended to allow enrollment of participants under 18 years of age with a single abnormal OGTT, because the rate of T1D progression with and without a confirmatory OGTT was similar in this age group. Eight of these subjects (five in the teplizumab arm and three in the placebo arm) had a second pretreatment OGTT on day 1 of study drug administration. Individuals with other significant medical history, clinical chemistry, or blood count abnormalities were excluded.

[0091] Patient Identification Subjects were identified in the TrialNet Pathway to Prevention (PTP) study. See Figure 5. The PTP study enrolled first-degree relatives of patients with T1D aged 1-45 years, and second- or third-degree relatives up to 20 years of age, and assessed for diabetes autoantibodies to mini-insulin (mIAA), glutamic acid decarboxylase-65 (GAD), and insulinoma-associated antigen-2 (IA-2, or ICA512). Islet cell (ICA) and zinc transporter 8 (ZnT8) autoantibodies were measured if at least one of the other antibodies tested was positive.

[0092] Study design and interventions Participants were randomized to receive either teplizumab or placebo, with equal allocation to each group. Randomization was stratified by age (>18 or <18 years) and glucose during pre-randomization OGTT status at each TrialNet study site. Treatment allocation was double masked.

[0093] Participants were treated as outpatients at a clinical research center with a previously described drug dosing regimen. 9,10Specifically, participants assigned to receive the active study drug received a 14-day course of teplizumab or placebo at 51 micrograms per square meter (μg / m ) on study days 0–3, respectively. 2 ), 103 μg / m 2 , 207 μg / m 2 , and 413 μg / m 2 , and 826 μg / m on each of study days 4 through 13. 2 Participants randomized to the placebo arm received a 14-day course of the corresponding IV saline solution. Participants received ibuprofen and diphenhydramine before the infusion for the first 5 days, then additional doses of ibuprofen, diphenhydramine, and / or acetaminophen as needed to relieve symptoms. Protocol-defined discontinuation criteria for study drug infusion were followed.

[0094] During the entire study, all subjects had brief contact with study personnel for a formal interview regarding adverse events and diabetic symptoms.

[0095] <Endpoints and Evaluation> The primary outcome was a baseline score as defined by the American Diabetes Association. 15 The time elapsed from randomization to diagnosis of diabetes was used.

[0096] Scheduled OGTT studies were performed 3 and 6 months after infusion and every 6 months thereafter. Random glucose screening was performed every 3 months, and OGTT studies were performed if random glucose was >200 mg / dl and accompanied by diabetic symptoms. Sequential confirmation of diabetic OGTT studies was required, and the date of diagnosis was identified as the time of the second diagnostic test. Outcomes were examined without knowledge of treatment allocation.

[0097] Blood samples were collected from TrialNet core laboratories using the methods described in the experimental method. Flow cytometry was used to analyze CD8+ T cell subsets in peripheral blood (Figure 4).

[0098] <Experimental Method> C-peptide was measured from frozen plasma by enzyme-linked immunosorbent assay (Tosoh Bioscience, South San Francisco, CA) at two sites. HbA 1c were measured using ion-exchange high performance liquid chromatography (Variant II, Bio-Rad Diagnostics, Hercules, CA). For each assay, the reliability coefficient from duplicate samples was greater than 0.99. mIAA, GAD-65Ab, ICA-512Ab, ZnT8A were measured using radioimmunobinding assays at the Barbara Davis Diabetes Center, Anschultz CO, and ICA was measured using indirect immunofluorescence at the University of Florida at Gainesville. C-peptide, glucose, and HbA 1c C-peptide was measured from frozen plasma by enzyme-linked immunosorbent assay (Tosoh Bioscience, South San Francisco, CA) at two sites, and HbA 1c EBV and CMV viral loads in whole blood were measured using ion-exchange high-performance liquid chromatography (Variant II, Bio-Rad Diagnostics, Hercules, CA). For each assay, the reliability coefficient from duplicate samples was greater than 0.99. EBV and CMV viral loads in whole blood were measured using a method previously described at the University of Colorado. 1 The measurements were performed using the following method.

[0099] <Flow cytometry> Peripheral blood mononuclear cells (PBMCs) were processed and stored at the NIDDK repository. Frozen vials of PBMCs were sent to Benaroya Research Institute for analysis by flow cytometry using the antibody panel shown in Figure 4. T cell phenotyping was performed on PBMCs using FACS Diva software on an LSR-Fortessa (BD Biosciences) as previously described and analyzed using FlowJo software version 9.5 (Tree Star, Ashland, OR). TIGIT+KLRG+CD57-, TIGIT-KLRG1-CD57-, or CD4+CD127 lo The frequency of CD8+ T cells that are Foxp3+ (CD4+ Treg) was determined as previously described. 2 Quadrants were established based on staining controls.

[0100] <Exam Supervisor> The study was developed and conducted by Type 1 Diabetes TrialNet with funding from the National Institutes of Health and the Juvenile Diabetes Research Foundation.

[0101] Study coordination, laboratory testing, and data management were performed centrally, except that CBC with differential and routine chemistries were analyzed at the infusion site. An independent medical monitor (masked to treatment assignment) reviewed all available safety data.

[0102] <Statistical analysis> The cumulative incidence of incident diabetes over time from randomization within each group was estimated from Kaplan-Meier estimates of the "diabetes-free" survival function. 16 Differences between treatment groups in cumulative incidence functions over 6-month intervals were evaluated using hazard ratio (HR) and likelihood ratio tests. Both were based on the Cox proportional hazards (PH) model. 17 The critical value of the test statistic for the primary hypothesis was determined by a group sequential procedure.

[0103] Because of the slower than expected enrollment rate, the original protocol (n=144 subjects) was revised (one-sided) to detect a 60% (previously 50%) reduction in the hazard ratio (HR=0.4) with 80% power at an alpha level of 0.025. This protocol set the study goal to enroll at least 71 subjects and follow them until 40 subjects were diagnosed with T1D. 18 .

[0104] Safety and efficacy data were evaluated twice annually by an independent Data and Safety Monitoring Board (DSMB). An interim analysis was performed when 50% of the expected number of T1D cases were observed, at which point a formal comparison was submitted to the DSMB and the Lan DeMets stopping rules were used. 19 Data were analyzed according to the intention-to-treat principle. Tests of significance reported herein were one-sided using a significance threshold of 0.025 according to the design, except for two-sided tests for treatment interaction tests. 95% confidence intervals are reported unless indicated. Flow cytometry data were analyzed by repeated measures ANOVA. Statistical analyses were performed using either TIBCO Spotfire S+8.2 Workbech or SAS 9.4.

[0105] [result] Patients: Of 112 subjects screened for eligibility, 76 were enrolled: 44 were randomly assigned to teplizumab and 32 to placebo (Figure 1). The randomization process resulted in unequal proportions in the study arms, likely explained by randomization at study sites with small numbers of enrolled subjects (<3), resulting in unequal distribution between arms. All participants had at least two autoantibodies, with 71% of participants having three or more. Treatment arms were generally well balanced (Table 1). The majority of subjects (55, 72%) were children, and about half were siblings of T1D patients. Of subjects under 18 years of age, 47 had a confirmed dysglycemic OGTT prior to randomization. Of subjects randomized after a single dysglycemic OGTT, two had a "diabetic" OGTT and six had a normal pretreatment OGTT: these eight individuals were enrolled based on a dysglycemic OGTT prior to enrollment.

[0106] Ninety-three percent (41 / 44) and 87.5% (28 / 32) of subjects randomized to the teplizumab and placebo groups, respectively, completed the 14-day drug treatment regimen. The total dose of teplizumab administered was 9 (IQR: 9.01-9.37) μg / m 2 Was 14 Three drug-treated and four placebo-treated subjects did not complete treatment due to laboratory abnormalities (n = 4), inability to establish intravenous access (n = 2), or skin rash (n = 1). Median follow-up was 745 days (range 74-2683 days). Duration of follow-up exceeded 3 years in 75% of subjects. T1D was diagnosed in 42 (55%) of participants.

[0107] Efficacy: Treatment with a single course of teplizumab delayed the time to T1D (Figure 2A, p=0.006): the median time to T1D was 24.4 mos in the placebo group and 48.4 mos in the teplizumab group (hazard ratio=0.437 (IQR:0.229, 0.832). The annualized rates of T1D incidence were 9.8% and 4.1% for the placebo and teplizumab groups, respectively. Twenty-five subjects (57%) in the teplizumab group and 9 subjects (28%) in the placebo group were free of T1D at the end of the study (chi-square distribution, p=0.012). Hazard ratios remained statistically different when adjusted for the prespecified covariates of age, glucose during OGTT before randomization, or anti-GAD65 antibodies.

[0108] The overall rate of progression to T1D was greatest during the first year of study entry (n=17, 41%) compared with years 2 (n=10, 24%), 3 (n=6, 14%), or 4 (n=5, 12%). The benefit of treatment with teplizumab was also greatest during the first year of study entry (Figure 1B). Hazard ratios were lowest during the first 36 months after study entry and remained relatively constant and statistically significant thereafter (p<0.01).

[0109] Treatment Administration and Safety: In general, treatment with teplizumab was well tolerated. Adverse events designated as possibly, probably, or definitely related to study drug are shown in Table 2. Lymphocyte counts declined to nadir on day 5 in 72.3% (IQR 82.1, 68.4%) (p<0.0001), but recovered rapidly thereafter. Fifteen (34.1%) grade 3 events in the teplizumab group were associated with lymphopenia during the first 30 days after study drug administration. There were no cases of lymphopenia after day 30 in either treatment arm (Figure 1C). As noted above, a spontaneously resolving skin rash occurred in 36% of subjects treated with the drug. 11 Infection rates were similar in the two treatment arms.

[0110] At enrollment, 30 subjects (39%) (16 teplizumab-treated and 14 placebo-treated) had antibodies to the EBV virus. After treatment with study drug, quantifiable EBV viral load was present in seven participants, all in the teplizumab group, at weeks 3-6. Of the participants with detectable viral load, one had symptoms of pharyngitis, rhinorrhea, and cough on day 38. The EBV viral load decreased to below the level of quantification between days 43 and 134 (mean 74 days). At enrollment, 17 participants (10 teplizumab and 7 placebo) had antibodies to the CMV virus. One teplizumab subject who was CMV seropositive had detectable levels of CMV virus on day 20 that became undetectable by day 42.

[0111] Response biomarkers: We have previously described changes in CD8+ T cells, such as expression of markers TIGIT, KLRG1, and others, that are associated with decreased responsiveness following treatment with teplizumab. 12,13 To determine whether clinical outcomes were associated with changes in these CD8+ T cells, the frequency of CD8+KLRG1+TIGIT+CD57- T cells in the two treatment arms was compared. Treatment with teplizumab increased the frequency of these T cells compared to baseline at 3 and 6 months (p=0.009, 0.007, respectively), with levels at 3 and 6 mos being higher in teplizumab-treated participants than in placebo-treated participants (p=0.02, 0.04, respectively) (Figure 3A, Figure 6). No changes in these cells were identified in placebo-treated subjects. Not all T cell subsets were affected by teplizumab: there were no significant changes in CD4+Treg or CD8+KLRG1-TIGIT-CD57- cells in either group. 8,20 (Figures 7A and 7B).

[0112] To determine whether participant demographic characteristics were associated with clinical response, the effect of teplizumab in subgroups of participants based on age, HLA type, pretreatment C-peptide and glucose during OGTT, and autoantibodies was analyzed in prespecified analyses (Figure 3B). Participants without anti-ZnT8 antibodies showed a greater response to teplizumab compared to those with anti-ZnT8 antibodies (p=0.004) (Figure 3C). The presence or absence of other autoantibodies was not associated with clinical response. 49% and 65% of teplizumab subjects were HLA-DR3 and HLA-DR4, respectively. The presence of HLA-DR4 and the absence of HLA-DR3 were associated with a more robust response to teplizumab (p=0.004 and 0.01, respectively, two-tailed test) (Figure 3D, E).

[0113] [Consideration] In this phase II study, a single course of teplizumab was found to significantly slow the progression to T1D in non-diabetic relatives with impaired glucose tolerance during OGTT at study entry. The median delay in diabetes diagnosis was 2 years. Also, at the end of the study, diabetic individuals were twice as frequent in subjects treated with the drug (57%) as in those treated with placebo (28%). Safety findings in children and adults were favorable, with expected adverse events being skin rash and transient lymphopenia. According to our findings, this is the first treatment to delay or prevent the onset of T1D. Delaying the onset of clinical T1D with its challenges of daily management is clinically important. Furthermore, the younger the age at diagnosis, the worse the outcome. 2,4 Previous large, well-designed trials that failed to prevent disease did not utilize immune cell-directed immunotherapies, and our findings support the notion that T1D is a chronic T cell-mediated disease. 21,22 Furthermore, the finding that this treatment has an effect on disease progression prior to diagnosis and on the loss of β-cell function after diagnosis suggests the existence of a continuum of autoimmune processes and validates attempts to use immunomodulation prior to the onset of clinical disease. 9~11,23~25 .

[0114] The effect of the drug was greatest during the first 3 years of treatment. Forty-one percent of individuals who developed diabetes did so during the first year after randomization, when HRs were lowest in individuals exposed to teplizumab. The relatively rapid rate of progression to diabetes in the placebo group reflects the significantly higher risk for these individuals. 5 Indeed, our decision to enroll these subjects without clinical disease reflects the inevitability of progression when more than two autoantibodies and glycemic abnormalities are found, which is consistent with our report of high rates of β-cell death in these individuals. 26 Furthermore, the rapid onset of clinical T1D may reflect the prevalence of pediatric participants (72.4%) among those with rapid progression. 27,28 .

[0115] Differences in response to teplizumab were observed based on subject characteristics at study enrollment. The absence of one T1D-associated MHC allele, HLA-DR3, but the presence of another T1D-associated MHC allele, HLA-DR4, and the absence of anti-ZnT8 antibodies identify individuals most likely to respond. MHC may modulate responsiveness to teplizumab through its influence on the T-cell repertoire, possibly through altering the state of T-cell activation and susceptibility to the drug. It is also possible that anti-ZnT8 antibodies are a marker for individuals with a more virulent immune response or other features that make T cells susceptible to teplizumab. It is uncertain whether the treatment is effective in individuals with earlier stages of disease. Further immunological and metabolic testing may identify features that define individuals most likely to benefit from this treatment.

[0116] The transient effects of drug treatment on lymphocyte counts most likely reflect release of cells from the peripheral blood rather than cell depletion. 29,30Our flow cytometry studies suggest that changes in CD8+ T cell phenotype are markers of clinical response. These effects are associated with a non-responsive or "exhausted" phenotype, but not inactivation of CD8+ T cells, as vigorous responses to EBV and CMV were observed in individuals with elevated viral loads. 31,32 The functional effect of teplizumab on T cells may be influenced by the avidity of the T cells to the antigen, such that T cells with high avidity, e.g., viral antigen-reactive cells, may be unaffected, whereas T cells with low avidity, e.g., autoreactive T cells, may be inactivated. Further studies with antigen-reactive T cells will be required to address this hypothesis.

[0117] There are some limitations to consider in this clinical trial: the cohort was relatively small, and the rate of progression to T1D was rapid in the placebo group. Therefore, it is unclear whether these findings are generally applicable to unrelated individuals found to be at risk for T1D. Recent reports suggest that in genetically at-risk individuals, diabetes rates are similar in unrelated and related individuals. 33 Moreover, the population was predominantly non-Hispanic Caucasian, reflecting known disease incidence. Ideally, the median delay in disease onset would be increased. Only one course of drug was given in this study, and our analysis of HRs suggests that repeated dosing may be necessary to capture more individuals with active disease and to extend the therapeutic effect. 9,25 Identification of the target population for treatment and number of drug courses needs to be explored.

[0118] In summary, this is the first study to show delay or prevention of T1D. Since age at onset and duration of diabetes are important determinants of metabolic control and complications, and because of the burden of daily management, this has clinical implications whenever non-diabetic. Selection of individuals most likely to respond, repeated dosing, or combination of teplizumab with other agents with complementary mechanisms of action may allow for long-term prevention of clinical disease.

[0119] JPEG2025072504000003.jpg237170JPEG2025072504000004.jpg255170JPEG2025072504000005.jpg60170

[0120] JPEG2025072504000006.jpg148170

[0121] Modifications and variations of the methods and compositions described in the present disclosure 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 fall within the scope of the present disclosure as represented by the following claims, and will be understood by those skilled in the relevant fields to which the present disclosure pertains.

[0122] [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 and publication was specifically and individually indicated to be incorporated by reference.

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Claims

1. 1. A pharmaceutical composition comprising a prophylactically effective amount of teplizumab for preventing or delaying the onset of clinical type 1 diabetes (T1D) in a non-diabetic subject, The non-diabetic subject (1) has two or more diabetes-associated autoantibodies selected from islet cell antibodies (ICA), insulin autoantibodies (IAA), glutamic acid decarboxylase (GAD) antibodies, and tyrosine phosphatase (IA-2 / ICA512) antibodies, and / or (2) has impaired glucose tolerance in an oral glucose tolerance test (OGTT); and The non-diabetic subject (1) has substantially no antibodies to zinc transporter 8 (ZnT8), (2) is HLA-DR4+, and (3) is not HLA-DR3+. The pharmaceutical composition.

2. 10. The pharmaceutical composition of claim 1, wherein administration of teplizumab delays the onset of clinical T1D.

3. 3. The pharmaceutical composition according to claim 1, wherein the non-diabetic subject has (1) two or more diabetes-associated autoantibodies selected from islet cell antibodies (ICA), insulin autoantibodies (IAA), glutamic acid decarboxylase (GAD) antibodies, and tyrosine phosphatase (IA-2 / ICA512) antibodies, and (2) impaired glucose tolerance in an oral glucose tolerance test (OGTT).

4. 4. The pharmaceutical composition according to claim 1, wherein the frequency of TIGIT+KLRG1+CD8+ T cells in peripheral blood mononuclear cells of the non-diabetic subject is determined by flow cytometry, and an increase in the frequency after administration of teplizumab indicates responsiveness to teplizumab.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the non-diabetic subject is a relative of a T1D patient.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the composition is effective in improving glucose tolerance in the OGTT. The pharmaceutical composition, wherein the functional abnormality is a fasting glucose level of 110-125 mg / dL, a 2-hour plasma glucose level of 140 mg / dL or greater but less than 200 mg / dL, or a median glucose level of greater than 200 mg / dL at 30, 60, or 90 minutes in an OGTT.

7. 7. The pharmaceutical composition of claim 1, wherein the prophylactically effective amount is 10 to 1000 micrograms per square meter (μg / m 2 ) a 10-14 day course of subcutaneous (SC) injection or intravenous (IV) infusion of teplizumab.

8. 8. The pharmaceutical composition of claim 7, wherein the prophylactically effective amount is 51 μg / m 2 of teplizumab on days 0 to 3. 2 , 103 μg / m 2 , 207 μg / m 2 , and 413 μg / m 2 and 826 μg / m on each of days 4-13. 2 said pharmaceutical composition comprising a 14-day course of IV infusion of a single dose of

9. 1. A method for predicting responsiveness to teplizumab in preventing or delaying the onset of clinical type 1 diabetes (T1D), comprising: The method comprises determining by flow cytometry the frequency of TIGIT+KLRG1+CD8+ T cells in peripheral blood mononuclear cells obtained from a non-diabetic subject who has (1) two or more diabetes-associated autoantibodies selected from islet cell antibodies (ICA), insulin autoantibodies (IAA), glutamic acid decarboxylase (GAD) antibodies, and tyrosine phosphatase (IA-2 / ICA512) antibodies, and / or (2) impaired glucose tolerance in an oral glucose tolerance test (OGTT), and who has been administered a prophylactically effective amount of teplizumab, wherein an increase in the frequency indicates responsiveness to teplizumab.

10. 10. The method of claim 9, wherein the non-diabetic subject is a relative of a T1D patient.

11. 11. The method of claim 9 or 10, wherein the impaired glucose tolerance in the OGTT is a fasting glucose level of 110-125 mg / dL, a 2-hour plasma glucose level of 140 mg / dL or greater and less than 200 mg / dL, or a median glucose level of greater than 200 mg / dL at 30, 60, or 90 minutes in the OGTT.

12. The method of any one of claims 9 to 11, 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+.

13. 13. The method of any one of claims 9 to 12, wherein the prophylactically effective amount is 10 to 1000 micrograms per square meter (μg / m 2 ) for a 10-14 day course of subcutaneous (SC) injection or intravenous (IV) infusion of teplizumab.

14. 14. The method of claim 13, wherein the prophylactically effective amount is 51 μg / m teplizumab on days 0-3. 2 , 103 μg / m 2 , 207 μg / m 2 , and 413 μg / m 2 and 826 μg / m on each of days 4-13. 2 said pharmaceutical composition comprising a 14-day course of IV infusion of a single dose of

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