Methods and compositions for preventing type i diabetes

Abstract

The present invention relates to methods and compositions for preventing type I diabetes. In one aspect, 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 a prophylactically effective amount of an anti-CD3 antibody to the non-diabetic subject.

Description

[0001] This application is a divisional application of a Chinese patent application filed on May 14, 2020, with application number 202080005325.2 and invention name “Methods and compositions for preventing type 1 diabetes”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 847,466, filed on May 14, 2019, and U.S. Utility Patent Application No. 15 / 931,685, filed on May 14, 2020, the entire disclosures of which are incorporated herein by reference.

[0004] Sequence Listing

[0005] The ASCII text file named "010701seq.txt" filed via EFS-Web on May 14, 2020, created on May 14, 2020, and 6,083 bytes in size, is incorporated herein by reference in its entirety. Technical Field

[0006] The present disclosure relates generally to compositions and methods for preventing or delaying the onset of clinical type 1 diabetes (T1D) in at-risk subjects, and more particularly to the use of anti-CD3 antibodies. Background Art

[0007] Type 1 diabetes (T1D) is caused by autoimmune destruction of the insulin-producing beta cells in the pancreas, which results in dependence on exogenous insulin injections for survival. Approximately 1.6 million Americans have type 1 diabetes, and after asthma, it remains one of the most common childhood diseases. 1 Despite improvements in care, most individuals with T1D are unable to consistently achieve desired glycemic goals. 2 For individuals with type 1 diabetes, there is concern about the increased risk of both morbidity and mortality. Two recent studies indicate that children diagnosed before the age of 10 lose 17.7 life years, while Scottish men and women diagnosed as adults lose 11 and 13 life years, respectively. 3,4 .

[0008] In genetically susceptible individuals, T1D progresses through an asymptomatic phase before overt hyperglycemia, characterized by the first appearance of autoantibodies (phase 1) and then the development of dysglycemia (phase 2). In phase 2, the metabolic response to a glucose load is impaired, but other metabolic markers (e.g., glycosylated hemoglobin) are normal, and insulin therapy is not required. 5These immunological and metabolic features identify individuals at high risk for developing clinical disease (stage 3) with overt hyperglycemia requiring insulin therapy. In recently initiated clinical T1D studies, several immune interventions have been shown to delay the decline in beta cell function. 6 One promising treatment is the FcR-nonbinding anti-CD3 monoclonal antibody teplizumab, as several studies have shown that short-term treatment reduces the loss of beta-cell function in a durable manner, with effects observed up to 7 years after diagnosis and treatment. 7-11 The drug alters the function of CD8+ T lymphocytes, which are considered important effector cells that lead to beta cell killing. 12,13 .

[0009] To date, interventions initiated prior to clinical diagnosis (ie, at stage 1 or 2) have failed to alter progression to clinical stage 3 T1D. Therefore, there is a need for treatments that prevent or delay the onset of clinical T1D in high-risk individuals. Summary of the invention

[0010] In one aspect, a method of preventing or delaying the onset of clinical type 1 diabetes (T1D) is provided, comprising:

[0011] Provide non-diabetic subjects at risk for T1D;

[0012] 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+; and

[0013] A prophylactically effective amount of an anti-CD3 antibody is administered to the non-diabetic subject.

[0014] In some embodiments, the non-diabetic subject is a relative of a T1D patient. In certain embodiments, the non-diabetic subject has 2 or more diabetes-associated autoantibodies selected from the group consisting of islet cell antibodies (ICA), insulin autoantibodies (IAA), and antibodies to glutamic acid decarboxylase (GAD), tyrosine phosphatase (IA-2 / ICA512), or ZnT8.

[0015] In some embodiments, the detection of T1D-associated autoantibodies is performed by point-of-care (POC) screening methods in the general population or relatives of T1D patients. These POC methods can be qualitative rapid lateral flow tests.

[0016] In some embodiments, the non-diabetic subject has an infection with coxsackie B virus (CVB) and / or other β-cell tropic viruses. In some embodiments, the subject infected with a β-cell tropic virus has HLA-DR4 and is more responsive to teligrumab.

[0017] In various embodiments, the non-diabetic subject has abnormal glucose tolerance in an oral glucose tolerance test (OGTT). Abnormal glucose tolerance in an OGTT is defined as a fasting glucose level of 110 to 125 mg / dL, or a 2-hour plasma of ≥140 and <200 mg / dL, or an intermediate glucose value of >200 mg / dL at 30, 60, or 90 minutes in an OGTT.

[0018] In some embodiments, the non-diabetic subject does not have antibodies against ZnT8.In certain embodiments, the non-diabetic subject is HLA-DR4+ and not HLA-DR3+.

[0019] In one embodiment, the anti-CD3 antibody is teliguzumab.

[0020] In various embodiments, a prophylactically effective amount comprises 10 to 1000 μg / m2 (μg / m 2 ) subcutaneous (SC) injection or intravenous (IV) infusion of an anti-CD3 antibody (e.g., teligrum) over a course of 10 to 14 days, with a total dose of 6 to 15 mg of anti-CD3 / teligrum; preferably 51 μg / m on days 0 to 3, respectively 2 , 103 μg / m 2 207 μg / m 2 and 413 μg / m 2 and 826 μg / m2 per day from day 4 to day 13 2 In certain embodiments, a prophylactically effective amount of an anti-CD3 antibody (e.g., teligrumumab) delays the median time to clinical diagnosis of T1D by at least 50%, at least 80%, or at least 90%, or 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.

[0021] In some embodiments, the anti-CD3 antibody (e.g., otelixizumab, otelixizumab, or foralumab) is administered in a SC pump or embedded in a slow-release biomaterial or administered orally. In other embodiments, the anti-CD3 antibody (e.g., otelixizumab, otelixizumab, or foralumab) is embedded in a biomaterial encapsulating a β cell precursor or β cell, or is provided parenterally in combination with a β cell precursor or β cell.

[0022] In some embodiments, an anti-CD3 antibody (eg, teliguzumab, otelizumab, or foraminib) is administered in combination with other pharmacological agents (eg, metabolic agents, B cell inhibitors, or other immunomodulators).

[0023] In some embodiments, an anti-CD3 antibody (eg, teliguzumab, oxicizumab, or foraminib) is administered with an antigen-specific immunotherapy and / or vaccine.

[0024] In some embodiments, the method further comprises determining the frequency of TIGIT+KLRG1+CD8+T cells in peripheral blood mononuclear cells of non-diabetic subjects by flow cytometry, wherein an increase in the frequency after administration of an anti-CD3 antibody (e.g., teliguzumab, oteliguzumab, or foraminib) indicates responsiveness to the anti-CD3 antibody (e.g., teliguzumab).

[0025] Another aspect relates to a method of prognosing responsiveness to an anti-CD3 antibody (e.g., teligrum, oxicizumab, or foraminolamide) in preventing or delaying the onset of type 1 diabetes (T1D), comprising:

[0026] Provide non-diabetic subjects at risk for T1D;

[0027] administering to the non-diabetic subject a prophylactically effective amount of teligrumumab, otelizumab, or foraminumab; and

[0028] The frequency of TIGIT+KLRG1+CD8+ T cells in peripheral blood mononuclear cells of the non-diabetic subject is determined by flow cytometry, wherein an increase in the frequency indicates responsiveness to an anti-CD3 antibody (eg, teliguzumab, otelizumab, or foraminib).

[0029] In some embodiments, the method can 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+.

[0030] It will be apparent to those skilled in the art that aspects and / or embodiments described herein may be combined. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Screening, enrollment, and follow-up of participants. A total of 112 subjects were screened for eligibility at TrialNet sites (see Appendix for a list of study sites). 76 subjects were randomized to either the drug group or the placebo group. The study drug was infused to them at one of the 14 TrialNet sites and followed according to the study protocol at one of the 33 sites. All randomized subjects were included in the analysis.

[0032] Figures 2A to 2C : Effect of teliguzumab treatment on the development of T1D. Figure 2A .Kaplan-Meier estimate of the proportion of participants without diabetes. 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 months in the teligrumumab group and 24.4 months in the placebo group. The insert shows the total number of subjects with T1D and without diabetes at the end of the study. Figure 2B Frequency of type 1 diabetes by treatment group and cumulative and interval hazard ratios (95% confidence intervals) by study year (* shows the number of participants who developed T1D in each treatment group within the year interval. In addition, the cumulative HR and the HR for each year interval were calculated. Mantel-Haenszel methods applied to time-to-event data: both chi-square tests and hazard ratio estimates 34 €Likelihood ratio test and hazard ratio estimates with 95% CI from Cox models). Figure 2C .Absolute lymphocyte count (ALC) in the study groups.

[0033] Figures 3A to 3E : Immunological effects and subgroup analyses of response to teikyostatin. Figure 3A .Frequency of CD8+KLRG1+TIGIT+CD57-T cells in teliguzumab and placebo treated subjects. (*p<0.05, teliguzumab treated participants vs. placebo treated participants). Mean ± 95% CI is shown. ANCOVA was used to compare each time point and corrected for baseline values. In addition, the frequency of these cells was significantly increased at 3 months (p=0.009) and 6 months (p=0.007) in teliguzumab treated participants, but not in placebo treated participants (paired t-test). Figure 3BThe ladder diagram shows the hazard ratio for each indicated characteristic of the participants at baseline. The absence of anti-ZnT8 antibodies (p = 0.004, HR: 0.031 for negative and 0.657 for positive), the presence of HLA-DR4 (p = 0.004, HR: 1.47 for negative and 0.201 for positive), and the absence of HLA-DR3 (p = 0.01, HR: 0.181 for negative and 0.907 for positive) had a significant effect on the hazard ratio. The presence or absence of anti-ZnT8 antibodies ( Figure 3C ), or HLA-DR3( Figure 3D ) or HLA-DR4( Figure 3E The occurrence of diabetes in patients with positive or negative .

[0034] Figure 4 : Monoclonal antibodies used in flow cytometry.

[0035] Figure 5 : Trial registration.

[0036] Figure 6 : FACS profiles showing staining of TIGIT (Y-axis) compared to KLRG1 (X-axis). Electronic gates were set on live CD8+CD57- T cells and expression of KLRG1 and TIGIT in peripheral blood cells from 3 subjects treated with Tilizumab (upper row) and 3 subjects treated with placebo are shown. Numbers refer to the proportion of total gated cells in each quadrant. Quadrants were set based on staining controls.

[0037] Figures 7A to 7B : The frequency of T cell subsets in the treatment group. CD4+Treg ( Fig. 7A )CD4+CD127 lo Foxp3+ and ( Figure 7B ) The frequency of CD8+TIGIT-KLRG1-CD57-T cells. When each time point was compared by ANCOVA and corrected for baseline values, the differences in both cell subsets between tilimumab and placebo and in each treatment group from baseline to post-treatment were not statistically significant. DETAILED DESCRIPTION

[0038] In some embodiments, the present disclosure provides the unexpected discovery that non-diabetic subjects that will respond to treatment with anti-CD3 antibodies (e.g., tilizumab) do not have antibodies against ZnT8. In certain embodiments, such non-diabetic subjects are HLA-DR4+ and not HLA-DR3+. Unexpectedly, such non-diabetic subjects that will respond to treatment with anti-CD3 antibodies show 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 of tilizumab (e.g., 1 month later, 2 months later, 3 months later, or longer or shorter).

[0039] In some embodiments, provided herein is a method for preventing or delaying the onset of clinical type 1 diabetes (T1D), comprising: providing a non-diabetic subject at risk for T1D; 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+; and administering a prophylactically effective amount of an anti-CD3 antibody, e.g., teligrumumab, to the non-diabetic subject.

[0040] In certain embodiments, a method for prognosing the responsiveness of an anti-CD3 antibody (e.g., tilizumab) in preventing or delaying the onset of T1D is provided. The method may include: providing a non-diabetic subject at risk for T1D; administering a preventively effective amount of an anti-CD3 antibody, such as tilizumab, to the non-diabetic subject; and determining the frequency of TIGIT+KLRG1+CD8+T cells in peripheral blood mononuclear cells of the non-diabetic subject by flow cytometry, wherein an increase in the frequency indicates responsiveness to an anti-CD3 antibody (e.g., tilizumab).

[0041] definition

[0042] Certain terms are defined below. Additional definitions are provided throughout the application.

[0043] As used herein, a noun not qualified by a quantifier refers to one or more than one, for example, at least one. When used in conjunction with the term "comprising" herein, the use of a noun not qualified by a quantifier may mean "one", but it also complies with the meaning of "one or more", "at least one", and "one or more than one".

[0044] As used herein, "about" and "approximately" generally refer to an acceptable degree of error for a measured amount, taking into account the nature or precision of the measurement. Exemplary degrees of error are within twenty percent (20%), typically within 10%, and more typically within 5% of a given value range. The term "substantially" means greater than 50%, preferably greater than 80%, and most preferably greater than 90% or 95%.

[0045] As used herein, the terms "comprising" or "including" are used to indicate that compositions, methods, and their corresponding components are present in a given embodiment, but are also open ended and include unspecified elements.

[0046] As used herein, the term "consisting essentially of refers to those 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 characteristics of the embodiment of the disclosure.

[0047] The term "consisting of" refers to the compositions, methods, and corresponding components described herein, which do not include any elements not listed in this description of the embodiment.

[0048] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0049] "Antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to the antigen bound by the intact antibody. 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.

[0050] As used herein, the term "prophylactic agent" refers to a CD3 binding molecule that can be used to prevent, treat, manage or improve one or more symptoms of T1D, such as teligrumumab.

[0051] As used herein with respect to type 1 diabetes, the term "onset" of the disease refers to a patient meeting the criteria established by the American Diabetes Association for diagnosing type 1 diabetes (see, Mayfield et al., 2006, Am. Fam. Physician 58: 1355-1362).

[0052] As used herein, the term "prevention" refers to preventing the onset of one or more symptoms of T1D in a subject by administering a prophylactic or therapeutic agent.

[0053] As used herein, "regimen" includes dosing schedules and dosing regimens. Regimens herein are methods of use and include both prophylactic and therapeutic regimens. A "dosing regimen" or "course of treatment" may include administering several doses of a therapeutic or prophylactic agent over 1 to 20 days.

[0054] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the term "subject" refers 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.

[0055] As used herein, the term "prophylactically effective amount" refers to an amount of tilizumab sufficient to delay or prevent the onset, recurrence or onset of one or more symptoms of T1D. In some embodiments, the prophylactically effective amount preferably refers to an amount of tilizumab 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%.

[0056] Various aspects of the disclosure are described in further detail below.Additional definitions are listed throughout the specification.

[0057] Anti-CD3 antibodies and pharmaceutical compositions

[0058] The terms "anti-CD3 antibody" and "antibody that binds to CD3" refer to an antibody or antibody fragment that is capable of binding to cluster of differentiation 3 (CD3) with sufficient affinity such that the antibody can be used as a prophylactic, diagnostic, and / or therapeutic agent targeting CD3. In one embodiment, the extent of binding of the anti-CD3 antibody to unrelated, non-CD3 proteins is less than about 10% of the binding of the antibody to CD3, for example as measured by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of the 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 smaller, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M). In certain embodiments, the anti-CD3 antibody binds to a CD3 epitope that is conserved between CD3 from different species.

[0059] In one embodiment, the anti-CD3 antibody can be ChAglyCD3 (Oxetizumab). Oxetizumab is a humanized Fc non-binding anti-CD3 that was initially evaluated by the Belgian Diabetes Registry (BDR) in a Phase 2 study and then developed by Tolerx, which then partnered with GSK to conduct the Phase 3 DEFEND new-onset T1D trial (NCT00678886, NCT01123083, NCT00763451). Oxetizumab is administered by IV infusion for 8 days. See, e.g., Wiczling et al., J. Clin. Pharmacol. 50(5) (May 2010) 494–-506; Keymeulen et al., NEngl J Med. 2005; 352:2598-608; Keymeulen et al., Diabetologia. 2010; 53:614-23; Hagopian et al. al., Diabetes.2013;62:3901-8; Aronson et al.,Diabetes Care.2014;37:2746-54; Ambery et al.,Diabet Med.2014;31:399-402; Bolt et al.,Eur.J.Immunol.lYY3.23:403-411;Vlasakakis et al. al.,Br J Clin Pharmacol (2019) 85704-–714;Guglielmi et al, Expert Opinion on Biological Therapy, 16: 6, 841-846;Keymeulen et al., N Engl J Med 2005;352: 2598-608;Keymeulen et al., BLOOD 2010, VOL 115, No. 6;Sprangers et al., Immunotherapy (2011) 3(11), 1303-–1316;Daifotis et al., Clinical Immunology (2013) 149, 268-–278;all of which are incorporated herein by reference.

[0060] In another embodiment, the anti-CD3 antibody can be visilizumab (also known as HuM291; Nuvion). Visilizumab is a humanized anti-CD3 monoclonal antibody characterized by a mutated IgG2 isotype, lacks binding to Fcγ receptors and is able to selectively induce apoptosis in activated T cells. It has been evaluated in patients with graft-versus-host disease (NCT00720629; NCT00032279) and ulcerative colitis (NCT00267306) and Crohn's Disease (NCT00267709). See, e.g., Sandborn et al., Gut 59 (11) (Nov 2010) 1485--1492, incorporated herein by reference.

[0061] In another embodiment, the anti-CD3 antibody can be forracirumab, a fully human anti-CD3 monoclonal antibody developed by Tiziana Life Sciences, PLC in NASH and T2D (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 Immunol. 2010; 185 (6): 3401-7; all of which are incorporated herein by reference.

[0062] In another embodiment, the anti-CD3 antibody can be tilizumab. Tilizumab, also known as hOKT3yl(Ala-Ala) (containing alanine at positions 234 and 235), is an anti-CD3 antibody that has been modified to change the function of T lymphocytes to mediate the destruction of insulin-producing beta cells of pancreatic islets. Tilizumab binds to the epitope of the CD3s chain expressed on mature T cells and thereby changes its function. The sequence and composition of tilizumab 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 given below. The bold portion is the complementary determining region.

[0063] Tilimumab light chain (SEQ ID NO: 1)

[0064]

[0065] Tilimumab heavy chain (SEQ ID NO: 2)

[0066]

[0067] In some embodiments, pharmaceutical compositions are provided herein. Such compositions comprise a preventive effective amount of anti-CD3 antibodies and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more particularly for humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle administered with a therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous solutions of glucose and glycerol 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, glyceryl monostearate, talc, sodium chloride, skim milk, glycerol, propylene, glycol, water, ethanol, and the like (see, e.g., Handbook of Pharmaceutical Excipients, Arthur H. Kibbe (2000 edition, which is incorporated herein by reference in its entirety), Am. Pharmaceutical Association, Washington, DC.

[0068] If necessary, the composition may also include a small amount of wetting agent or emulsifier or pH buffer. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. Oral formulations may include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Some examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EWMartin. Such compositions include a preventive or therapeutically effective amount of a preventive or therapeutic agent, preferably a preventive or therapeutic agent in a purified form, and an appropriate amount of carrier to provide a form for appropriate administration to a patient. The preparation should be suitable for administration. In a preferred embodiment, the pharmaceutical composition is sterile and in a suitable form for administration to an object, preferably an animal object, more preferably a mammalian object, and most preferably a human object.

[0069] In a specific embodiment, it may be desirable to administer the pharmaceutical composition locally to the area in need of treatment; this may be achieved, for example, but not limited to, by local infusion, by injection, or by means of an implant, which is a porous, non-porous or gel-like material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering an anti-CD3 antibody, care must be taken to use a material that does not absorb the anti-CD3 antibody.

[0070] In another embodiment, the composition can be delivered in a vesicle, particularly a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al., Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, supra, pp. 317-327; see generally supra).

[0071] In another embodiment, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump can be used to achieve 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, 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; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1990). al., 1989, J. Neurosurg. 71:105); 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 for 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), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In a preferred embodiment, the polymer used in the sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.In yet another embodiment, a controlled or sustained release system can be placed near the therapeutic target (ie, the lungs), thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

[0072] Controlled release systems are discussed in the review by Langer (1990, Science 249: 1527-1533).Sustained release formulations comprising one or more antibodies of the invention or fragments thereof may be produced using any technique known to those skilled in the art. See, e.g., U.S. Pat. No. 4,526,938; PCT Publication WO 91 / 05548; PCT Publication 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 herein by reference in its entirety.

[0073] The pharmaceutical composition can be formulated to be compatible with its expected route of administration. The example of route of administration includes but is not limited to parenteral, such as intravenous, intradermal, subcutaneous, oral, intranasal (such as inhalation), transdermal (surface), transmucosal and rectal administration. In a specific embodiment, according to conventional procedures, the composition is formulated as a pharmaceutical composition suitable for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to people. In a preferred embodiment, according to conventional procedures, the pharmaceutical composition is formulated for subcutaneous administration to people. Usually, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic (such as lidocaine (lignocamne)) to alleviate the pain at the injection site.

[0074] The composition can be formulated for parenteral administration by injection (e.g., by push injection or continuous infusion). Injectable formulations can be present in unit dosage form, for example, in ampoules or multidose containers with added preservatives. The composition can take the form of a suspension, solution, or emulsion, such as in an oily or aqueous vehicle, and can include preparatants, such as suspending agents, stabilizers, and / or dispersants. Alternatively, the active ingredient can be in powder form for construction with a suitable vehicle (e.g., sterile pyrogen-free water) prior to use.

[0075] In some embodiments, the disclosure provides dosage forms that allow for continuous administration of anti-CD3 antibodies over a period of hours or days (e.g., in connection with a pump or other device for such delivery), e.g., over a period of 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 embodiments, the disclosure provides dosage forms that allow for administration of continuously increasing doses, e.g., from 51 ug / m to 121 ug / m over a period of 24 hours, 30 hours, 36 hours, 4 days, 5 days, 7 days, 10 days, or 14 days. 2 / day increased to 826ug / m 2 / sky.

[0076] The composition can be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions (e.g., anions derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.), and salts formed with cations (e.g., cations derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.).

[0077] Typically, the components of the compositions disclosed herein are provided separately or mixed together in a unit dosage form, for example as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container (e.g., an ampoule or sachette) indicating the amount of the active agent. When the composition is to be administered by infusion, it can be dispensed with 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.

[0078] In particular, the present disclosure provides that the anti-CD3 antibody or its pharmaceutical composition can be packaged in an airtight sealed container, such as an ampoule or a pouch, indicating the amount of the medicament. In one embodiment, the anti-CD3 antibody or its pharmaceutical composition is provided in a dry sterilized lyophilized powder or anhydrous concentrate in an airtight sealed container, and can be reconstituted to an appropriate concentration, for example, with water or saline, for administration to a subject. Preferably, the anti-CD3 antibody or its pharmaceutical composition is provided 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 in a dry sterile lyophilized powder in an airtight sealed container. The lyophilized prophylactic 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 1 week after reconstitution, preferably within 5 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. In an alternative embodiment, the pharmaceutical composition is provided in a gas-tight sealed container indicating the amount and concentration of the medicament in liquid form. Preferably, the applied composition in liquid form is provided in a gas-tight sealed container 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 2 ° C to 8 ° C.

[0079] In a specific embodiment, the present disclosure provides that a composition of the invention is packaged in a hermetically sealed container (eg, an ampoule or a sachet) indicating the amount of anti-CD3 antibody.

[0080] 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 may, for example, comprise metal or plastic foil, such as a blister pack.

[0081] The amount of the composition of the invention effective to prevent or ameliorate one or more symptoms associated with T1D can be determined by standard clinical techniques. The precise dose 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 the circumstances of each patient. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0082] Methods and uses

[0083] In certain embodiments, the present disclosure comprises administering an anti-human CD3 antibody (e.g., teligrumumab) to an individual who is susceptible to developing type 1 diabetes or has a preclinical stage of type 1 diabetes but does 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 prevent or delay the need to administer insulin to such a patient. In certain embodiments, high risk factors for identifying susceptible subjects include: having a first or second degree relative diagnosed with type 1 diabetes, reduced fasting glucose levels (e.g., at least one measurement of glucose levels after fasting (8 hours without food) of 100 to 125 mg / dl), impaired glucose tolerance in response to a 75 g OGTT (e.g., at least one measurement of 2-hour glucose levels in response to a 75 g OGTT of 140 to 199 mg / dl), HLA types of DR3, DR4, or DR7 in Caucasians, HLA types of DR3 or DR4 in people of African descent, HLA types of DR3, DR4, or DR9 in people of Japanese descent, exposure to viruses (e.g., Coxsackie B virus, enterovirus, adenovirus, rubella, cytomegalovirus, Epstein-Barr virus, virus), positive diagnosis of at least one other autoimmune disorder (e.g., thyroid disease, celiac disease) according to art-recognized 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 being susceptible to type 1 diabetes have at least one of the risk factors described herein and / or known in the art. The present disclosure also includes identifying subjects susceptible to 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.

[0084] Serum autoantibodies associated with type 1 diabetes or associated with susceptibility to type 1 diabetes are islet cell autoantibodies (e.g., anti-ICA512 autoantibodies), glutamic acid decarboxylase autoantibodies (e.g., anti-GAD65 autoantibodies), IA2 antibodies, ZnT8 antibodies and / or anti-insulin autoantibodies. Therefore, in a specific example according to this embodiment, the present invention includes treating an individual with detectable autoantibodies associated with susceptibility to type 1 diabetes or associated with early type 1 diabetes (e.g., anti-IA2, anti-ICA512, anti-GAD or anti-insulin autoantibodies), wherein the individual has not yet 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, electrochemoluminescence (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 by any other method described herein or known to those of ordinary skill in the art for immunospecific detection of antibodies.

[0085] Beta cell function before, during and after treatment can be assessed by the methods described herein or by any method known to those of ordinary skill in the art. For example, the Diabetes Control and Complications Trial (DCCT) research group has established glycosylated hemoglobin (HA1 and HA1c) percentage monitoring as a standard for evaluating glycemic control (DCCT, 1993, N. Engl. J. Med. 329: 977-986). Alternatively, characterization of daily insulin requirements, C-peptide levels / responses, hypoglycemic episodes, and / or FPIR can be used as markers of β-cell function or to establish treatment targets (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 / 0038867A1; and Greenbaum et al., 2001, Diabetes 50:470-476, respectively). For example, FPIR is calculated as the sum of insulin values ​​at 1 minute and 3 minutes after IGTT, which is 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).

[0086] In some embodiments, the individual susceptible 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 2 or more diabetes-associated autoantibodies selected from the group consisting of islet cell antibodies (ICA), insulin autoantibodies (IAA), and antibodies to glutamic acid decarboxylase (GAD), tyrosine phosphatase (IA-2 / ICA512), or ZnT8.

[0087] In various embodiments, the non-diabetic subject has abnormal glucose tolerance in an oral glucose tolerance test (OGTT). Abnormal glucose tolerance in an OGTT is defined as a fasting glucose level of 110 to 125 mg / dL, or a 2-hour plasma of ≥140 and <200 mg / dL, or an intermediate glucose value of >200 mg / dL at 30, 60, or 90 minutes in an OGTT.

[0088] In some embodiments, non-diabetic subjects that will respond to anti-CD3 antibodies (e.g., tilizumab) do not have antibodies against ZnT8. In certain embodiments, such non-diabetic subjects are HLA-DR4+ and not HLA-DR3+. In some embodiments, such non-diabetic subjects that will respond to anti-CD3 antibodies (e.g., tilizumab) show an increase in the frequency (or relative amount) of TIGIT+KLRG1+CD8+T cells (e.g., by flow cytometry) in peripheral blood mononuclear cells after administration (e.g., after 1 month, after 2 months, after 3 months, or longer or shorter).

[0089] In various embodiments, a prophylactically effective amount comprises 10 to 1000 micrograms per square meter (μg / m 2 ) subcutaneous (SC) injection or intravenous (IV) infusion of anti-CD3 antibody (e.g., teligrumumab) for 10 to 14 days. In one example, the prophylactically effective amount comprises 51 μg / m on days 0 to 3, respectively. 2 , 103 μg / m 2 207 μg / m 2 and 413 μg / m 2 and 826 μg / m2 per day from day 4 to day 13 2 A 14-day course of one dose IV infusion of an anti-CD3 antibody (e.g., teligrumumab). In certain embodiments, 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 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.

[0090] In certain embodiments, the course of administration of an anti-CD3 antibody (e.g., telizumab) 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 specific embodiments, the efficacy of treatment with an anti-CD3 antibody (e.g., telizumab) is determined as described herein or as known in the art at 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.

[0091] In another embodiment, the subject is administered one or more unit doses of about 0.5 to 50 μg / kg, about 0.5 to 40 μg / kg, about 0.5 to 30 μg / kg, about 0.5 to 20 μg / kg, about 0.5 to 15 μg / kg, about 0.5 to 10 μg / kg, about 0.5 to 5 μg / kg, about 1 to 5 μg / kg, about 1 to 10 μg / kg, about 20 to 40 μg / kg, about 20 to 30 μg / kg, about 22 to 28 μg / kg, or about 25 to 26 μg / kg of an anti-CD3 antibody (e.g., teligrumumab) to prevent, treat, or ameliorate one or more symptoms of T1D. In another embodiment, the subject is administered one or more unit doses of 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, 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., teligrum) to prevent, treat or ameliorate one or more symptoms of T1D.

[0092] In some specific embodiments, the subject is administered one or more doses of an anti-CD3 antibody (e.g., teligrumumab) at about 5 to 1200 μg / m2, preferably 51 to 826 μg / m2. In another embodiment, the subject is administered one or more unit doses of 1200 μg / m2, 1150 μg / m2, 1100 μg / m2, 1050 μg / m2, 1000 μg / m2, 950 μg / m2, 900 μg / m2, 850 μg / m2, 800 μg / m2, 750 μg / m2, 700 μg / m2, 650 μg / m2, 600 μg / m2, 550 μg / m2, 500 μg / m2, 450 μg / m2. 2. 400 μg / m2, 350 μg / m2, 300 μg / m2, 250 μg / m2, 200 μg / m2, 150 μg / m2, 100 μg / m2, 50 μg / m2, 40 μg / m2, 30 μg / m2, 20 μg / m2, 15 μg / m2, 10 μg / m2, or 5 μg / m2 of an anti-CD3 antibody (e.g., teligrum) to prevent, treat, slow the progression of, delay the onset of, or improve one or more symptoms of T1D.

[0093] 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 (e.g., tilizumab), wherein the treatment course is administered within 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In one embodiment, the treatment regimen includes administering a prophylactically effective amount of a dose every day, every 2 days, every 3 days, or every 4 days. In certain embodiments, the treatment regimen includes 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 doses, 13 doses, 13 doses, 12 doses, 11 doses, 10 doses, 9 doses, or 8 doses have been administered. In certain embodiments, the doses administered every day of the regimen are the same.

[0094] In certain 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., teligrum), 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, or 100 μg / kg / day. g / kg / day, 50μg / kg / day, 45μg / kg / day, 40μg / kg / day, 35μg / kg / day, 30μg / kg / day, 26μg / kg / day, 25μg / kg / day, 20μg / kg / day, 15μg / kg / day, 13μg / kg / day, 10μg / kg / day, 6.5μg / kg / day, 5μg / kg / day, 3.2μg / kg / day, 3μg / kg / day, 2.5μg / kg / day, 2μg / kg / day, 1.6μg / kg / day, 1.5μg / 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 wherein the prophylactic effective amount is 1200 μg / m2 / day, 1150 μg / m2 / day, 1100 μg / m2 / day, 1050 μg / m2 / day, 1000 μg / m2 / day, 950 μg / m2 / day, 900 μg / m2 / day, 850 μg / m2 / day, 800 μg / m2 / day, 750 μg / m2 / day, 700 μg / m2 / day, 650 μg / m2 / day g / m2 / day, 600μg / m2 / day, 550μg / m2 / day, 500μg / m2 / day, 450μg / m2 / day, 400μg / m2 / day, 350μg / m2 / day, 300μg / m2 / day, 250μg / m2 / day, 200μg / m2 / day, 150μg / m2 / day, 100μg / m2 / day, 50μg / m2 / day, 40μg / m2 / day, 30μg / m2 / day, 20μg / m2 / day, 15μg / m2 / day, 10μg / m2 / day, or 5μg / m2 / day.

[0095] In another embodiment, the intravenous dose is 1200 μg / m2 or less, 1150 μg / m2 or less, 1100 μg / m2 or less, 1050 μg / m2 or less, 1000 μg / m2 or less, 950 μg / m2 or less, 900 μg / m2 or less, 850 μg / m2 or less, 800 μg / m2 or less, 750 μg / m2 or less. or less, 700μg / m2 or less, 650μg / m2 or less, 600μg / m2 or less, 550μg / m2 or less, 500μg / m2 or less, 450μg / m2 or less, 400μg / m2 or less, 350μg / m2 or less, 300μg / m2 or less, 250μg / m2 or less, 200μg / m2 or less, 15 0 μg / m2 or less, 100 μg / m2 or less, 50 μg / m2 or less, 40 μg / m2 or less, 30 μg / m2 or less, 20 μg / m2 or less, 15 μg / m2 or less, 10 μg / m2 or less, or 5 μg / m2 or less of an anti-CD3 antibody (e.g., teligrum) is administered within 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 to prevent, treat, or ameliorate one or more symptoms of type 1 diabetes. The total dose over the duration of the regimen preferably amounts to less than 9000 μg / m2, 8000 μg / m2, 7000 μg / m2, 6000 μg / m2, and may be less than 5000 μg / m2, 4000 μg / m2, 3000 μg / m2, 2000 μg / m2, or 1000 μg / m2. In some specific embodiments, the total dose administered in the regimen is 100 μg / m2 to 200 μg / m2, 100 μg / m2 to 500 μg / m2, 100 μg / m2 to 1000 μg / m2, or 500 μg / m2 to 1000 μg / m2.

[0096] In some preferred embodiments, the dose is increased gradually over the first quarter, first half, or first two-thirds of the treatment regimen dose (e.g., within the first 2, 3, 4, 5, or 6 days of a 10, 12, 14, 16, 18, or 20-day regimen of once-daily dosing) until a daily prophylactically effective amount of the anti-CD3 antibody (e.g., teligrum) is achieved. In certain 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., teligrum), wherein the prophylactically effective amount is increased daily as the treatment progresses, e.g., 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, 10 μg / kg, 15 μg / kg, 20.X1.g / kg, 25 μg / kg, 30 μg / kg, 35 μg / kg, 40 μg / kg, 45 μg / kg, 50 μg / kg, 55 μg / kg g, 60μg / kg, 65μg / kg, 70μg / kg, 75μg / kg, 80μg / kg, 85μg / kg, 90μg / kg, 95μg / kg, 100μg / k g, or 125μg / kg; or increase daily such as 1μg / m2, 5μg / m2, 10μg / m2, 15μg / m2, 20μg / m2, 30μg / m2, 40μg / m2, 5 0 μg / m2, 60 μg / m2, 70 μg / m2, 80 μg / m2, 90 μg / m2, 100 μg / m2, 150 μg / m2, 200 μg / m2, 250 μg / m2, 300 μg / m2, 350 μg / m2, 400 μg / m2, 450 μg / m2, 500 μg / m2, 550 μg / m2, 600 μg / m2, or 650 μg / m2. In certain 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., teligrumumab), wherein 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 an anti-CD3 antibody (e.g., teligrumumab) is achieved.

[0097] In a specific embodiment, the subject is administered one or more doses of 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. 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., teligrum, otelizumab, or foraminumab) to prevent, treat, or ameliorate one or more symptoms of T1D.

[0098] In another embodiment, the subject is administered one or more doses of 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 more subcutaneously. 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., teligrum, otelizumab, or foraminumab) to prevent, treat, or ameliorate one or more symptoms of T1D.

[0099] In another embodiment, the subject is administered one or more doses of 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 ...0 μg / kg or less, 35 μg / kg or less, 40 μg / kg or less, 40 μg / kg or less, 40 μg / kg or less, 40 μg / kg or less, 40 μg / kg or less, 40 μg / kg or less, 40 μg 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., teliguzumab, otelizumab, or foraminib) to prevent, treat, or ameliorate one or more symptoms of T1D. In another embodiment, the intravenous 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, 10 μg / kg or less. / 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., teligrum, otelizumab, or foraminumab) is administered within 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 to prevent, treat, or ameliorate one or more symptoms of T1D.

[0100] In another embodiment, the subject is orally administered one or more doses of 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 ... 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., teliguzumab, otelizumab, or foraminib) to prevent, treat, or ameliorate one or more symptoms of T1D. In another embodiment, the oral 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, 10 μg / kg or less. / 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., teligrum, otelizumab, or foraminumab) is administered within 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 to prevent, treat, or ameliorate one or more symptoms of T1D.

[0101] In some embodiments where increasing doses are administered over the first few days of the 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 gradually increase to the daily dose as described immediately above on day 3, 4, 5, 6 or 7. For example, the dose administered to a subject on day 1 is about 51 μg / m 2 / day, about 103 μg / m on the second day2 / day, about 207 μg / m on the third day 2 / day, about 413 μg / m on the 4th day 2 / day and 826 μg / m on subsequent days of the regimen (e.g., Days 5 to 14) 2 In another embodiment, the dose administered to the subject on day 1 is about 227 μg / m 2 / day, and about 459 μg / m on the second day 2 / day, about 919 μg / m on the third and subsequent days 2 In another embodiment, the dose administered to a subject on day 1 is about 284 μg / m 2 / day, about 574 μg / m on the second day 2 / day, about 1148 μg / m on the third and subsequent days 2 / sky.

[0102] In other embodiments, the initial dose is 1 / 4, to 1 / 2, to equal to the daily dose at the end of the regimen, but is administered in batches at intervals of 6, 8, 10 or 12 hours. For example, a 13 μg / kg / day dose is administered in four 3 to 4 μg / kg doses at 6-hour intervals to reduce the level of cytokine release caused by the administration of the antibody. In some specific embodiments, in order to reduce the possibility of cytokine release and other adverse effects, the first 1, 2, 3 or 4 doses or all doses in the regimen are administered more slowly by intravenous administration. For example, 51 μg / m 2 The dose per day 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 over a period of, for example, 20 to 24 hours. In some specific embodiments, the dose is infused in a pump, preferably increasing the concentration of the administered antibody as the infusion progresses.

[0103] In other embodiments, the above 51 μg / m 2 / day to 826 μg / m 2 A set of fractions of the dose of the / day regimen is administered in increasing 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, when the fraction is 1 / 10, the daily dose on day 1 will be 5.1 μg / m 2 , 10.3 μg / m on the second day 2 , 20.7 g / m on the third day 2 , 41.3 μg / m on the 4th day 2and 82.6 μg / m on days 5 to 14 2 When the fraction is 1 / 4, the dose on day 1 will be 12.75 μg / m 2 , 25.5 μg / m on the second day 2 , 51 μg / m on the third day 2 , 103 μg / m on the 4th day 2 and 207 μg / m on days 5 to 14 2 When the fraction is 1 / 3, the dose on day 1 will be 17 μg / m 2 , 34.3 μg / m on the second day 2 , 69 μg / m on the third day 2 , 137.6 μg / m on the 4th day 2 and 275.3 μg / m on days 5 to 14 2 When the fraction is 1 / 2, the dose on day 1 will be 25.5 μg / m 2 , 51 μg / m on the second day 2 , 103 μg / m on the third day 2 , 207 μg / m on the 4th day 2 and 413 μg / m on days 5 to 14 2 When the fraction is 2 / 3, the dose on day 1 will be 34 μg / m 2 , 69 μg / m on the second day 2 , 137.6 μg / m on the third day 2 , 275.3 μg / m on the 4th day 2 and 550.1 μg / m on days 5 to 14 2 When the fraction is 3 / 4, the dose on day 1 will be 38.3 μg / m 2 , 77.3 μg / m on the second day 2 , 155.3 μg / m on the third day 2 , 309.8 μg / m on the 4th 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, except that it is only on days 1 to 4, days 1 to 5, or days 1 to 6. For example, in one specific embodiment, the dose on day 1 would be 17 μg / m 2 , 34.3 μg / m on the second day 2 , 69 μg / m on the third day 2 , 137.6 μg / m on the 4th day 2 And on the 5th and 6th days, it was 275.3 μg / m 2 .

[0104] In some embodiments, the anti-CD3 antibody (e.g., tilizumab, oxicizumab, or forerunner) is not administered in daily doses over several days, but rather is administered by infusion in an uninterrupted manner over 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 24 hours, 30 hours, or 36 hours. The infusion can be constant, or can be started at a lower dose, for example, 1, 2, 3, 5, 6, or 8 hours before the infusion, and then increased to subsequent higher doses. During the infusion process, the patient receives a dose equal to that administered in the above 5 to 20 day regimen. For example, a dose of about 150 μg / m 2 , 200 μg / m 2 , 250 μg / m 2 , 500 μg / m 2 , 750 μg / m 2 , 1000 μg / m 2 , 1500μg / m 2 , 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 rate and duration of the infusion are designed to minimize the level of free anti-CD3 antibodies (e.g., teliguzumab, oteliguzumab, or forerunnerumab) in the subject after administration. In certain embodiments, the level of free anti-CD3 antibodies (e.g., teliguzumab) should not exceed 200 ng / ml free antibody. In addition, the infusion is designed to achieve combined T cell receptor coating and at least 50%, 60%, 70%, 80%, 90%, 95%, or 100% modulation.

[0105] In other embodiments, anti-CD3 antibodies (e.g., tilizumab, oxicizumab, or forerunner) are administered chronically to treat, prevent, slow or delay the onset or progression of type 1 diabetes, or improve one or more symptoms thereof. For example, in certain embodiments, as an alternative to the above-mentioned 6 to 14 day dosage regimen or after the administration of such a regimen, a low dose of an anti-CD3 antibody (e.g., tilizumab) is administered once a month, twice a month, three times a month, once a week, or even more frequently to enhance or maintain its effect. Such a low dose may be 1 μg / m 2 Up to 100 μg / m 2 Any value, such as about 5 μg / m 2 , 10μg / m2 , 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 2 .

[0106] In other embodiments, the subject may be re-administered at a certain time after administration of the anti-CD3 antibody (e.g., tilizumab, oxicizumab or forerunner) dosing regimen, for example based on one or more physiological parameters, or may be performed routinely. Such re-administration and / or the need for such re-administration may be administered 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 re-administration may include administering the treatment course irregularly every 6 months, 9 months, 1 year, 15 months, 18 months, 2 years, 30 months or 3 years.

[0107] Example

[0108] Overview

[0109] 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. Some interventions have shown success in attenuating the loss of insulin production in patients showing clinical disease, but to date, no intervention has been able to influence the development of the disease in individuals at high risk for developing the disease.

[0110] method We conducted a randomized, placebo-controlled, double-blind study of teligrumumab, an FcR-nonbinding anti-CD3 mAb, in nondiabetic relatives of patients with T1D who were at high risk for clinical disease. Patients were randomized to a single 14-day course of drug or placebo, and disease progression was followed at approximately 6-month intervals using an oral glucose tolerance test.

[0111] result: A total of 76 subjects were randomized, 44 in the teligrum group and 32 in the placebo group. Seventy-two percent of the participants were children. Teligrum treatment delayed the median time to clinical diagnosis of T1D from 24.4 months to 48.4 months (Cox proportional hazards, p=0.006), and reduced the incidence of diabetes from 9.8% to 4.1% per year. Drug treatment increased the proportion of TIGIT+KLRG1+CD8+T cells. Participants who did not have antibodies to ZnT8 (p=0.01), were HLA-DR4+ (p=0.006), and were not HLA-DR3+ (p=0.05) were most likely to respond to teligrum.

[0112] in conclusion Conclusions: Tilimumab may delay the diagnosis of T1D in high-risk individuals. Some subgroups of individuals may be more likely to respond to treatment and action.

[0113] method

[0114] Trial Participants

[0115] Participants were identified through the TrialNet Natural History Study 14 The trial was conducted at sites in the United States, Canada, and Germany from July 2011 to November 2018. Institutional Review Board (IRB) approval was obtained at each participating site. Patients, their parents, or both provided written informed consent before entry into the trial.

[0116] Eligible participants were nondiabetic relatives of patients with type 1 diabetes aged 1-2 years at randomization. >8 years old, at high risk of clinical diabetes. Within 6 months before randomization, they had 2 or more diabetes-related autoantibodies at 2 sample collections. In addition, they had abnormal glucose tolerance in the oral glucose tolerance test (OGTT), which was defined as: fasting glucose level of 110 to 125 mg / dL, or 2-hour plasma>140 and <200 mg / dL, or the intermediate glucose value at 30 minutes, 60 minutes or 90 minutes in the OGTT>200 mg / d, which appeared twice within 52 days after registration. The program was revised in 2014 to allow participants with a single abnormal OGTT of age <18 to be registered, because the T1D progression rate in this age group was similar with or without a definitive OGTT. In these 8 subjects (5 in the tilizumab group and 3 in the placebo group), a second pre-treatment OGTT was performed on the first day of the study drug administration. Individuals with other important medical histories, abnormal laboratory chemistry or blood cell counts were excluded.

[0117] Patient identification

[0118] Identify subjects in the TrialNet Prevention Pathway (PTP) study. Figure 5 The PTP study enrolled first-degree relatives of patients with T1D, aged 1 to 45 years, and second- or third-degree relatives up to 20 years old, and evaluated diabetes autoantibodies against microinsulin (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 other antibody tested positive.

[0119] Trial Design and Interventions

[0120] Participants were randomized to receive either telizumab or placebo, with equal allocation in each group. Randomization was stratified by age (< or > 18 years) and glucose during OGTT status before randomization at each TrialNet study site. Treatment allocation was double-masked.

[0121] Participants received a 14-day course of teligrumumab or placebo administered as an outpatient in a clinical research center using the drug dosing regimen described previously. 9,10 Specifically, those assigned to active study drug received telucam via daily IV infusion according to the following daily regimen: 51 μg / m 2 ), 103 μg / m 2 207 μg / m 2 and 413 μg / m 2, and a daily dose of 826 μg / m 2 Participants randomized to the placebo group received a 14-day course of matching IV saline. Participants received ibuprofen and diphenhydramine for the first 5 days prior to infusion, and further dosing of ibuprofen, diphenhydramine, and / or acetaminophen was subsequently required as needed for symptom relief. Protocol-defined stopping criteria for study drug infusion were followed.

[0122] Throughout the study period, all subjects had ad hoc contact with the study staff for formal questioning regarding adverse events and diabetic symptoms.

[0123] End Points and Assessments

[0124] The primary outcome was the time from randomization to diagnosis of diabetes using criteria defined by the American Diabetes Association. 15 .

[0125] Scheduled OGTTs were performed at 3 and 6 months after infusion and every 6 months thereafter. Random glucose screening was performed at 3-month intervals, and an OGTT was performed if random glucose >200 mg / dl and symptoms of diabetes were present. Diabetes OGTTs required sequential confirmation, and the diagnosis date was determined as the time of the second diagnostic test. Outcome censoring was performed blinded to treatment assignment.

[0126] Blood samples were analyzed centrally at the TrialNet core laboratory using methods described in Laboratory Methods. Flow cytometry was used to analyze CD8+ T cell subsets in peripheral blood ( Figure 4 ).

[0127] Laboratory methods

[0128] C-peptide was measured from frozen plasma by a two-site immunoenzymatic assay (Tosoh Bioscience, South San Francisco, CA). HbA was measured using ion-exchange high-performance liquid chromatography (version II, Bio-Rad Diagnostics, Hercules, CA). 1c. The reliability coefficients for each assay in separate replicates were above 0.99. mIAA, GAD-65Ab, ICA-512Ab, ZnT8A were measured using radioimmunoassay at the Barbara Davis Diabetes Center at Anschultz CO, and ICA was measured using indirect immunofluorescence at the University of Florida in Gainesville. C-peptide, glucose, and HbA1c were measured at Northwest Research Laboratories in Seattle, WA. HbA1c was measured using ion exchange high performance liquid chromatography (version II, Bio-Rad Diagnostics, Hercules, CA). 1c C-peptide was measured from frozen plasma by a two-site immunoenzymatic assay (Tosoh Bioscience, South San Francisco, CA) at 4 °C. The reliability coefficients for each assay were higher than 0.99 in separate replicates. EBV and CMV viral loads were measured in whole blood using previously described methods at the University of Colorado. 1 .

[0129] Flow cytometry

[0130] Peripheral blood mononuclear cells (PBMCs) were processed and stored at the NIDDK repository. Frozen PBMC vials were sent to the Benaroya Institute for use Figure 4 The antibody panels shown in were analyzed by flow cytometry. T cell phenotyping was performed on PBMCs as previously described on an LSR-Fortessa (BD Biosciences) with FACSDiva software and analyzed using FlowJo software version 9.5 (Tree Star, Ashland, OR). TIGIT+KLRG+CD57-, TIGIT-KLRG1-CD57-, or CD4+CD127 were determined as previously described. lo The frequency of Foxp3+ (CD4+ Treg) CD8+ T cells 2 . Quadrants were placed based on staining controls.

[0131] Trial supervision

[0132] The study was developed and conducted by Type 1 Diabetes TrialNet, which was funded by the National Institutes of Health and the Juvenile Diabetes Research Foundation.

[0133] Study coordination, laboratory testing, and data management were performed centrally, except for analysis of CBC and differential and routine chemistries at the infusion site.An independent medical monitor (masked to treatment assignment) reviewed all accumulated safety data.

[0134] Statistical analysis

[0135] Cumulative incidence of diabetes onset over time within each group since randomization was estimated based on Kaplan-Meier estimates of the “diabetes-free” survival function 16 The difference between treatment groups in the cumulative incidence function at the six-month interval was estimated by hazard ratio (HR), and hypothesis testing was performed using the likelihood ratio test; both were based on the Cox proportional hazards (PH) model. 17 The critical values ​​of the test statistics for the main hypotheses were determined by a group-sequential procedure.

[0136] Due to the lower than expected accrual rate, the original protocol (n = 144 subjects) underwent censoring to detect a 60% (previously 50%) reduction in hazard rate (HR = 0.4) with an alpha level of 0.025 and a power of 80% (one-sided). This set the study goal to enroll at least 71 subjects and follow them until 40 subjects were diagnosed with T1D. 18 .

[0137] Data on safety and efficacy will be reviewed twice a year by an independent Data Safety Monitoring Board (DSMB). An interim analysis will be conducted when 50% of the expected number of T1D cases are observed, at which time a formal comparison will be presented to the DSMB and the Lan DeMets stopping rule will be used. 19 . Data were analyzed according to the intention-to-treat principle. Significance tests reported in this article were one-sided, using a significance threshold of 0.025 according to the present design, but treatment interaction tests were two-sided. Unless otherwise stated, 95% confidence intervals are reported. Flow cytometry data were analyzed by repeated measures ANOVA. Statistical analyses were performed using TIBCO Spotfire S+8.2 Workbench or SAS 9.4.

[0138] result

[0139] patient : Of 112 subjects screened for eligibility, 76 were enrolled: 44 were randomized to teliguzumab and 32 were randomized to placebo ( Figure 1). The randomization process resulted in unequal proportions in the study groups, which can be explained by the fact that some study sites had a small number of enrolled subjects (<3), in which randomization may have resulted in unequal allocation between groups. All participants had at least 2+ autoantibodies, and 71% of participants had 3 or more. The treatment groups were well balanced overall (Table 1). Most subjects (55, 72%) were children and about half were siblings of T1D patients. Of the subjects aged <18, 47 had confirmed dysglycemic OGTT prior to randomization. Of those randomized after a single dysglycemic OGTT, 2 had "diabetes" and 6 had a normal pre-treatment OGTT: these 8 individuals were enrolled based on the dysglycemic OGTT prior to enrollment.

[0140] Ninety-three percent (41 / 44) and 87.5% (28 / 32) of subjects randomized to telizumab and placebo, respectively, completed the 14-day treatment period. The total dose of telizumab administered was 9. 14 (IQR: 9.01 to 9.37) μg / m 2 . Three drug-treated subjects and four placebo-treated subjects did not complete treatment due to laboratory abnormalities (n=4), inability to establish intravenous access (n=2), or rash (n=1). Median follow-up was 745 days (range, 74 to 2683 days). Follow-up duration was greater than 3 years in 75% of subjects. T1D was diagnosed in 42 (55%) participants.

[0141] Effectiveness : Treatment with a single-course monoclonal antibody delayed the time to T1D ( Figure 2A , p=0.006): median time to T1D was 24.4 months in the placebo group and 48.4 months in the teligrum group (hazard ratio=0.437 (IQR: 0.229, 0.832). The annual incidence of T1D was 9.8% and 4.1% in the placebo and teligrum groups, respectively. Twenty-five subjects (57%) from the teligrum group and nine subjects (28%) from the placebo group were free of T1D at the end of the trial (chi-square, p=0.012). The hazard ratios remained statistically different when adjusted for the prespecified covariates: age, glucose during OGTT before randomization, or anti-GAD65 antibodies.

[0142] The overall rate of progression to T1D was highest in the first year after trial entry (n=17, 41%) compared with year 2 (n=10, 24%), year 3 (n=6, 14%), or year 4 (n=5, 12%). The effect of teligrumumab treatment was also greatest during this period ( Figure 2B ). The hazard ratio was lowest in the first 36 months after study enrollment and remained relatively constant and statistically significant thereafter (p<0.01).

[0143] Treatment administration and safety : Overall, treatment with teligrumumab was well tolerated - adverse events designated as possibly, probably, or definitely related to the study drug are shown in Table 2. Lymphocyte counts reached their nadir on day 5, with a decrease of 72.3% (IQR 82.1, 68.4%) (p<0.0001), but recovered rapidly thereafter. Fifteen (34.1%) of the grade 3 events in the teligrumumab group involved lymphopenia during the first 30 days after study drug administration. After day 30, there were no cases of lymphopenia in either treatment group ( Figure 2C Spontaneously resolving rashes, as described above, occurred in 36% of drug-treated subjects. 11 The infection rates were similar in both treatment groups.

[0144] At the time of entry, 30 subjects (39%) (16 treated with tilizumab and 14 treated with placebo) had antibodies against EBV virus. At the 3rd to 6th week after study drug treatment, 7 participants (all in the tilizumab group) had quantifiable EBV viral load. Among those with detectable viral load, 1 had symptoms of pharyngitis, runny nose and cough on the 38th day. The EBV viral load dropped below the quantitative level from the 43rd to 134th day (average 74 days). At the time of entry, 17 participants (10 tilizumab and 7 placebo) had antibodies against CMV virus. A tilizumab subject (who was CMV seropositive) had detectable CMV viral levels on the 20th day, which were undetectable on the 42nd day.

[0145] Response biomarkers : We previously described changes in CD8+ T cells after teligrumab treatment, such as expression of markers such as TIGIT and KLRG1, which are associated with reduced responsiveness12 ,13 . To determine whether clinical outcomes were associated with these changes in CD8+ T cells, we compared the frequencies of CD8+ KLRG1+ TIGIT+ CD57- T cells in the two treatment groups. Compared with baseline, teligrumumab treatment increased the frequencies of these T cells at months 3 and 6 (p=0.009, 0.007, respectively), and levels were higher in teligrumumab-treated participants relative to placebo-treated participants at months 3 and 6 (p=0.02, 0.04, respectively). ( Figure 3A , Figure 6 ). We did not identify changes in these cells in placebo-treated subjects. Not all T-cell subsets were affected by tilimab: there were no significant changes in CD4+Treg or CD8+KLRG1-TIGIT-CD57- cells in either group. 8,20 ( Fig. 7A , Figure 7B ).

[0146] To determine whether demographic characteristics of the participants were associated with clinical response, in prespecified analyses, we analyzed the effect of telizumab in subgroups of participants based on age, HLA type, pretreatment C-peptide and glucose during OGTT, and autoantibodies ( Figure 3B Participants without anti-ZnT8 antibodies showed a greater response to teligrumab 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 teliguzumab subjects had 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 teliguzumab (p=0.004 and 0.01, two-sided test, respectively) ( Figure 3D , Figure 3E ).

[0147] discuss

[0148] In this Phase II study, we found that a course of tadalafil significantly slowed progression to T1D in nondiabetic relatives who had abnormal glucose tolerance during an OGTT at the start of the study. The median delay in diabetes diagnosis was 2 years, and at the end of the trial, the frequency of individuals without diabetes doubled in drug-treated subjects (57%) relative to placebo-treated subjects (28%). The safety experience in children and adults was good, with the expected adverse events of rash and transient lymphocytopenia. To our knowledge, this is the first treatment that delays or prevents the onset of T1D. The delay in the onset of clinical T1D and the challenges of daily management are clinically important. In addition, a younger age at diagnosis is associated with worse outcomes. 2,4 Previous large, well-designed but unsuccessful prevention trials have not utilized immunotherapy targeting immune cells, and our findings support the concept that T1D is a chronic, T-cell-mediated disease. 21,22 Furthermore, the observation that this treatment affected disease progression prior to diagnosis as well as loss of beta-cell function after diagnosis suggests that there is a continuum in the autoimmune process and validates attempts to use immune modulation prior to the onset of clinical disease. 9-11,23-25 .

[0149] The effect of the drug was greatest in the first three years after administration. 41% of patients who developed diabetes did so in the first year after randomization, and the HR for patients exposed to teligrumumab was lowest at that time. The relatively rapid rate of progression to diabetes in the placebo group reflects the very high risk of these individuals. 5Indeed, our decision to enroll these subjects without clinical disease reflects the inevitability of progression when >2 autoantibodies and dysglycemia are found, consistent with our reports of high rates of β-cell killing in these individuals. 26 Additionally, the rapid onset of clinical T1D may reflect the enrichment of pediatric participants with rapid progression rates (72.4%). 27,28 .

[0150] There are differences in responses to tilizumab based on the characteristics of the subjects at the time of study enrollment. The absence of one T1D-associated MHC allele (HLA-DR3) but the presence of another (HLA-DR4) and the absence of anti-ZnT8 antibodies identify individuals who are most likely to respond. MHC can regulate responsiveness to tilizumab through its effects on T cell reservoirs, potentially changing T cell activation state and sensitivity to drug effects. Anti-ZnT8 antibodies may also be a marker for individuals with more explosive immune responses, or other features that make an individual's T cells sensitive to tilizumab. We cannot be sure whether the treatment will be effective in those in earlier stages of the disease. Further immunological and metabolic studies can determine the characteristics that define individuals who are most likely to benefit from the treatment.

[0151] Transient effects of drug therapy on lymphocyte counts most likely reflect egress from peripheral blood rather than cellular depletion 29,30 Our flow cytometric studies suggest that changes in CD8+ T cell phenotype are a hallmark of clinical response. However, these effects associated with an unresponsive or "exhausted" phenotype did not inactivate CD8+ T cells, as active responses to EBV and CMV were present in those with elevated viral loads. 31,32 The functional effects of teligrum on T cells may be influenced by their avidity for the antigen: T cells with high avidity (e.g., viral antigen-reactive cells) may be unaffected, whereas those with low avidity (e.g., autoreactive T cells) may become inactivated. Further studies on antigen-reactive T cells are needed to resolve this hypothesis.

[0152] Consider some limitations of this clinical study. The cohort was relatively small and the rate of progression to T1D was more rapid in the placebo group. The subjects were relatives of patients with T1D, and therefore, we do not know whether these findings will generalize to non-relatives at risk for T1D. Recent reports suggest that the rates of diabetes in non-relatives are similar to those in relatives of individuals at genetic high risk. 33. In addition, although reflecting known disease incidence, this group was overwhelmingly non-Hispanic Caucasian. Improving the median delay to disease onset would be ideal. In this study, the drug was given for only one course, and our analysis of the HR suggests that repeated dosing may be necessary to obtain more individuals with active disease and prolong the treatment effect. 9,25 Need to explore: Identify target groups and number of drug courses for treatment.

[0153] In conclusion, this is the first trial to show delay or prevention of T1D. Because age of onset and duration of diabetes are important determinants of metabolic management and complications, as well as the burden of daily management, any time free of diabetes is clinically significant. Selecting those most likely to respond, repeat dosing, or combining teligrumumab with other agents with complementary mechanisms of action may enable prevention of clinical disease to continue for extended periods of time.

[0154] Table 1: Subject Characteristics by Treatment Group at Baseline

[0155]

[0156]

[0157] *Values ​​in brackets: interquartile ranges are shown with medians and percentages of subjects are shown with numbers of subjects.

[0158] ^At randomization. All subjects had at least 2+ autoantibodies prior to randomization.

[0159] Missing: HLA allele status missing for 1 subject

[0160] Table 2: Adverse events designated as possibly, probably, or definitely related to study drug during active follow-up

[0161]

[0162] ***p<0.001, teliguzumab versus placebo

[0163] Without departing from the scope and spirit of the present disclosure, the modification and variation of the methods and compositions described in the present disclosure will be apparent to those skilled in the art. Although the present disclosure has been described in conjunction with some specific embodiments, it should be understood that the disclosure claimed for protection should not be unduly limited to such specific embodiments. In fact, multiple modifications to the described modes for implementing the present disclosure are intended to and understood by those skilled in the relevant art to which the present disclosure belongs to fall within the scope of the present disclosure represented by the appended claims.

[0164] Incorporated by Reference

[0165] 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. A method for preventing or delaying the onset of clinical type 1 diabetes (T1D), comprising: Provide non-diabetic subjects at risk for T1D; 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+; and A prophylactically effective amount of an anti-CD3 antibody is administered to the non-diabetic subject.

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

3. The method of claim 1, wherein the non-diabetic subject has two or more diabetes-associated autoantibodies selected from the group consisting of islet cell antibodies (ICA), insulin autoantibodies (IAA), and antibodies to glutamic acid decarboxylase (GAD), tyrosine phosphatase (IA-2 / ICA512), or ZnT8.

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

5. The method of claim 4, wherein the abnormal glucose tolerance in an OGTT is a fasting glucose level of 110 to 125 mg / dL, or a 2-hour plasma of ≥140 and <200 mg / dL, or an intermediate glucose value at 30, 60, or 90 minutes in an OGTT of >200 mg / dL.

6. The method of claim 1, wherein the non-diabetic subject does not have antibodies against ZnT8.

7. The method of claim 1, wherein the non-diabetic subject is HLA-DR4+ and not HLA-DR3+.

8. The method of claim 1, wherein the anti-CD3 antibody is selected from teligrumumab, otelizumab, or foraminib.

9. The method of claim 8, wherein the prophylactically effective amount comprises 10 to 1000 micrograms / square meter (μg / m 2 ) subcutaneous (SC) injection or intravenous (IV) infusion or oral administration of the anti-CD3 antibody for 10 to 14 days, preferably at 51 μg / m on days 0 to 3, respectively 2 , 103 μg / m 2 207 μg / m 2 and 413 μg / m 2 and 826 μg / m2 per day from day 4 to day 13 2 One dose is given as an IV infusion over the course of 14 days.

10. The method of claim 8, 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 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.

11. The method of any one of claims 1 to 10, further comprising determining the frequency of TIGIT+KLRG1+CD8+T cells in peripheral blood mononuclear cells of the non-diabetic subject by flow cytometry, wherein an increase in the frequency after administration of tilizumab indicates responsiveness to the anti-CD3 antibody.

12. A method for prognosing the responsiveness of an anti-CD3 antibody in preventing or delaying the onset of type 1 diabetes (T1D), comprising: Provide non-diabetic subjects at risk for T1D; administering to the non-diabetic subject a prophylactically effective amount of an anti-CD3 antibody; as well as The frequency of TIGIT+KLRG1+CD8+ T cells in peripheral blood mononuclear cells of the non-diabetic subject is determined by flow cytometry, wherein an increase in the frequency indicates responsiveness to the anti-CD3 antibody.

13. The method of claim 12, wherein the non-diabetic subject is a relative of a patient with T1D.

14. The method of claim 12, wherein the non-diabetic subject has two or more diabetes-associated autoantibodies selected from the group consisting of islet cell antibodies (ICA), insulin autoantibodies (IAA), and antibodies to glutamic acid decarboxylase (GAD), tyrosine phosphatase (IA-2 / ICA512), or ZnT8.

15. The method of claim 12, wherein the non-diabetic subject has abnormal glucose tolerance in an oral glucose tolerance test (OGTT).

16. The method of claim 15, wherein the abnormal glucose tolerance in an OGTT is a fasting glucose level of 110 to 125 mg / dL, or a 2-hour plasma of ≥140 and <200 mg / dL, or an intermediate glucose value at 30, 60, or 90 minutes in an OGTT of >200 mg / dL.

17. The method of claim 12, further comprising 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+.

18. The method of claim 17, wherein the non-diabetic subject does not have antibodies against ZnT8.

19. The method of claim 17, wherein the non-diabetic subject is HLA-DR4+ and not HLA-DR3+.

20. The method of claim 12, wherein the anti-CD3 antibody is selected from teligrumumab, otelizumab, or foraminib.

21. The method of claim 20, wherein the prophylactically effective amount comprises 10 to 1000 micrograms per square meter (μg / m 2 ) subcutaneous (SC) injection or intravenous (IV) infusion or oral administration of the anti-CD3 antibody for 10 to 14 days, preferably at 51 μg / m on days 0 to 3, respectively 2 , 103 μg / m 2 207 μg / m 2 and 413 μg / m 2 and 826 μg / m2 per day from day 4 to day 13 2 A 14-day course of one dose of IV infusion of telizumab.

22. The method of claim 20, 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 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.

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