CN112839707A

CN112839707A - Methods and compositions for preventing type I diabetes

Info

Publication number: CN112839707A
Application number: CN202080005325.2A
Authority: CN
Inventors: 弗朗西斯科·莱昂; 凯万·C·赫罗尔德; 杰伊·S·斯凯勒
Original assignee: Previnson Biology Ltd
Current assignee: Previnson Biology Ltd
Priority date: 2019-05-14
Filing date: 2020-05-14
Publication date: 2021-05-25
Also published as: EP3873601A1; EP3873601A4

Abstract

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) has substantially no 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-CD 3 antibody to the non-diabetic subject.

Description

Methods and compositions for preventing type I diabetes

Cross Reference to Related Applications

Priority and benefit of the present application claims priority and benefit of U.S. provisional application No.62/847,466 filed on 5/14/2019 and U.S. patent application No.15/931,685 filed on 5/14/2020, the entire disclosures of which are incorporated herein by reference.

Sequence listing

An ASCII text file named "010701 seq. txt", filed on 14/5/2020 by EFS-Web, which was created on 14/5/2020 and is 6,083 bytes in size, is incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates generally to compositions and methods for preventing or delaying the onset of clinical type 1 diabetes (T1D) in a subject at risk, and more particularly to the use of anti-CD 3 antibodies.

Background

Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing beta cells in the islets of langerhans, which results in reliance on exogenous insulin injections for survival. About 160 million americans have type 1 diabetes and, following asthma, it remains one of the most common diseases in childhood¹. Despite the improved care, most affected individuals with T1D still fail to consistently achieve the desired glycemic target². For individuals with type 1 diabetes, there is a constant concern about increased risk of both morbidity and mortality. Two recent studies have indicated that children diagnosed before the age of 10 lost 17.7 years of life, while scotland men and women diagnosed annually lost 11 and 13 years of life, respectively^3,4。

In genetically susceptible individuals, T1D undergoes an asymptomatic phase prior to overt (overt) hyperglycemia, characterized by the appearance of autoantibodies first (phase 1) and then blood glucose abnormalities (phase 2). In phase 2, the metabolic response to glucose load is impaired, but other metabolic indicators (e.g., glycosyl groups)Hemoglobin) normal, and does not require insulin therapy⁵. These immunological and metabolic features identify individuals at high risk of developing clinical disease (stage 3) with overt hyperglycemia and in need of insulin therapy. In a recently initiated clinical T1D study, several immune interventions have been shown to delay the decline of beta cell function⁶. One promising therapy is the FcR non-binding anti-CD 3 monoclonal antibody telithromumab (teplizumab), since several studies have shown that short-term therapy persistently reduces the loss of beta-cell function, with observable effects seen 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。

To date, interventions initiated prior to clinical diagnosis (i.e., at stage 1 or 2) have failed to alter progression to clinical stage 3T 1D. Thus, there is a need for treatments that prevent or delay the onset of clinical T1D in high risk individuals.

Disclosure of Invention

In one aspect, 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 of T1D;

determining that the non-diabetic subject is (1) substantially free of antibodies to 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-CD 3 antibody.

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 glutamate decarboxylase (GAD), tyrosine phosphatase (IA-2/ICA512), or ZnT 8.

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

In some embodiments, the non-diabetic subject has an infection with coxsackie B virus (CVB) and/or other beta cell tropic virus. In some embodiments, the subject infected with the beta cell tropic virus has HLA-DR4 and is more responsive to telithromumab.

In various embodiments, the non-diabetic subject has abnormal glucose tolerance in an Oral Glucose Tolerance Test (OGTT). Abnormal glucose tolerance in the OGTT is defined as fasting glucose levels of 110 to 125mg/dL, or 2 hour plasma of ≥ 140 and <200mg/dL, or a median glucose value at 30, 60 or 90 minutes in the OGTT of >200 mg/dL.

In some embodiments, the non-diabetic subject does not have antibodies to ZnT 8. In certain embodiments, the non-diabetic subject is HLA-DR4+ and is not HLA-DR3 +.

In one embodiment, the anti-CD 3 antibody is telithromumab.

In various embodiments, a prophylactically effective amount includes from 10 to 1000 micrograms per square meter per day (μ g/m)²) Subcutaneous (SC) injection or Intravenous (IV) infusion of anti-CD 3 antibody (e.g., telithromumab) for a course of 10 to 14 days at a total dose of 6 to 15mg of anti-CD 3/telithromumab; preferably 51. mu.g/m on days 0 to 3, respectively²、103μg/m²、207μg/m²And 413. mu.g/m²And 826 μ g/m daily on days 4 to 13²One dose IV of telithromumab was infused over the course of 14 days. In certain embodiments, a prophylactically effective amount of an anti-CD 3 antibody (e.g., telithromumab) 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.

In some embodiments, the anti-CD 3 antibody (e.g., telithromab, otelixizumab, or forramumab) is administered in an SC pump or embedded in a slow release biomaterial or administered orally. In other embodiments, the anti-CD 3 antibody (e.g., telithromumab, oxizumab, or fralizumab) is embedded in a biomaterial that encapsulates, or is provided parenterally in combination with, a beta cell precursor or beta cell.

In some embodiments, the anti-CD 3 antibody (e.g., telithromumab, oxizumab, or forrauzumab) is administered in combination with other pharmacological agents (e.g., a metabolic agent, a B-cell inhibitor, or other immunomodulator).

In some embodiments, the anti-CD 3 antibody (e.g., telithromumab, oxizumab, or forrauzumab) is administered with an antigen-specific immunotherapy and/or vaccine.

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, wherein the increase in frequency after administration of the anti-CD 3 antibody (e.g., telithrombin, oxlizumab, or forranibizumab) is indicative of responsiveness to the anti-CD 3 antibody (e.g., telithrombin).

Another aspect relates to a method of prognosing the responsiveness of an anti-CD 3 antibody (e.g., telithromumab, oxzezumab, or forrauzumab) in preventing or delaying the onset of type 1 diabetes (T1D), comprising:

providing a non-diabetic subject at risk of T1D;

administering to the non-diabetic subject a prophylactically effective amount of telithromumab, oxizumab, or framumab; and

determining the frequency of TIGIT + KLRG1+ CD8+ T cells in peripheral blood mononuclear cells of the non-diabetic subject by flow cytometry, wherein the increase in frequency is indicative of responsiveness to an anti-CD 3 antibody (e.g., telithromumab, oxzezumab, or forranibizumab).

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

It will be apparent to those skilled in the art that the aspects and/or embodiments described herein may be combined.

Drawings

FIG. 1: screening, enrollment and follow-up of participants. A total of 112 subjects were screened for eligibility at the TrialNet site (list of relevant study sites, see appendix). 76 subjects were randomized to either the drug or placebo group. Study medication was infused to one of the 14 triallet sites and followed according to the study protocol at one of the 33 sites. All randomized objects were included in the analysis.

Fig. 2A to 2C: effects of telithromumab treatment on T1D. FIG. 2A. Kaplan-Meier estimation of the proportion of participants without diabetes. The overall risk ratio was 0.437 (95% CI:0.229,0.832) (p ═ 0.006, unilateral, Cox model). Median T1D time was 48.4 months for the telithromumab group and 24.4 months for the placebo group. The insert shows the total number of subjects with T1D and no diabetes at the end of the study. Figure 2b. frequency of type I diabetes by treatment group and cumulative and interval risk ratio (95% confidence interval) by study year (the number of participants who experienced T1D in each treatment group within the year interval is shown.

Mantel-Haenszel method applied to time-event data: chi-square test and risk ratio estimation³⁴. The check for likelihood and risk ratio estimates for the color and 95% CI from the Cox model). Fig. 2c Absolute Lymphocyte Counts (ALC) in the study group.

Fig. 3A to 3E: immunological effects and subgroup analysis of responses to telithrombin. FIG. 3A. frequency of CD8+ KLRG1+ TIGIT + CD57-T cells in subjects treated with telithromycin and placebo. (p <0.05, telithromumab treated participants versus placebo treated participants). Mean ± 95% CI are shown. Each time point was compared using ANCOVA and corrected for baseline values. Furthermore, the frequency of these cells increased significantly at 3 months (p ═ 0.009) and 6 months (p ═ 0.007) in the telithromumab treated participants, but not in the placebo treated participants (paired t-test). Fig. 3b. ladder diagram shows the risk ratio for each of the indicative features of the participant at baseline. The absence of anti-ZnT 8 antibody (p 0.004, HR: negative 0.031, positive 0.657), the presence of HLA-DR4(p 0.004, HR: negative 1.47, positive 0.201), and the absence of HLA-DR3(p 0.01, HR: negative 0.181, positive 0.907) had a significant impact on risk ratio. The incidence of diabetes in patients positive or negative at baseline with or without anti-ZnT 8 antibody (fig. 3C), or HLA-DR3 (fig. 3D) or HLA-DR4 (fig. 3E) is shown.

FIG. 4: monoclonal antibodies for use in flow cytometry.

FIG. 5: and (6) test registration.

FIG. 6: FACS profiles of staining for TIGIT (Y-axis) compared to KLRG1 (X-axis) are shown. Electronic gating was set on live CD8+ CD57-T cells and shows the expression of KLRG1 and TIGIT in peripheral blood cells from 3 subjects treated with telithromumab (upper row) and 3 subjects treated with placebo. The numbers refer to the proportion of total gated cells in each quadrant. Quadrants were set based on staining controls.

Fig. 7A to 7B: frequency of T cell subpopulations in treatment group. CD4+ Treg (FIG. 7A) CD4+ CD127 at study visit are shown_loFrequency of Foxp3+ and (FIG. 7B) CD8+ TIGIT-KLRG1-CD57-T cells. When each time point was compared by ANCOVA and corrected for baseline values, the differences between telithromumab and placebo and in the two cell subsets from baseline to post-treatment for each treatment group were not statistically significant.

Detailed Description

In some embodiments, the present disclosure provides an unexpected discovery that non-diabetic subjects that would respond to anti-CD 3 antibody (e.g., telithromumab) treatment do not have antibodies to ZnT 8. In certain embodiments, such non-diabetic subjects are HLA-DR4+ and are not HLA-DR3 +. Unexpectedly, such non-diabetic subjects who will respond to anti-CD 3 antibody therapy exhibit an increase in the frequency (or relative amount) of TIGIT + KLRG1+ CD8+ T cells in peripheral blood mononuclear cells (e.g., by flow cytometry) following telithromumab administration (e.g., after 1 month, after 2 months, after 3 months, or longer or shorter).

In some embodiments, provided herein are methods 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 is (1) 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-CD 3 antibody, e.g., telithromumab, to the non-diabetic subject.

In certain embodiments, methods of prognosing the responsiveness of an anti-CD 3 antibody (e.g., telithromumab) in preventing or delaying the onset of T1D are provided. The method may comprise: providing a non-diabetic subject at risk of T1D; administering to the non-diabetic subject a prophylactically effective amount of an anti-CD 3 antibody, e.g., telithromumab; and determining the frequency of TIGIT + KLRG1+ CD8+ T cells in peripheral blood mononuclear cells of the non-diabetic subject by flow cytometry, wherein the increase in frequency is indicative of responsiveness to an anti-CD 3 antibody (e.g., telithromumab).

Definition of

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

As used herein, an unqualified noun refers to one or more than one, for example, to at least one. The use of an indefinite article "a" or "an" when used herein in conjunction with the term "comprising" may mean "one", but it is also intended to cover "one or more", "at least one", and "one or more than one".

As used herein, "about" and "approximately" generally refer to an acceptable degree of error in the measured quantity, taking into account the nature or accuracy of the measurement. Exemplary degrees of error are within 20 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%.

As used herein, the terms "comprises" or "comprising" are used to indicate that the compositions, methods, and their respective components present in a given embodiment are open-ended, containing unspecified elements.

As used herein, the term "consisting essentially of … …" refers to those elements required for a given embodiment. The terms allow for the presence of additional elements that do not materially affect the basic and novel or functional characteristics of this embodiment of the disclosure.

The term "consisting of … …" refers to the compositions, methods, and their corresponding components described herein, which do not contain any elements not listed in this description of the embodiments.

The term "antibody" herein is used 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.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the 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 '-SH, F (ab')₂(ii) a Doublets (diabodies); a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

As used herein, the term "prophylactic agent" refers to a CD3 binding molecule, e.g., telithrombin, that can be used to prevent, treat, manage, or ameliorate one or more symptoms of T1D.

As used herein, with respect to type 1 Diabetes, the term "onset" of disease means that the patient meets the criteria established by the American Diabetes Association for diagnosing type 1 Diabetes (see Mayfield et al, 2006, am. fam. physics 58: 1355-.

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

As used herein, "regimen" includes schedule of administration and regimen of administration. The regimens herein are methods of use and include prophylactic and therapeutic regimens. A "dosing regimen" or "course of treatment" may include administration of several doses of a therapeutic or prophylactic agent over a period of 1 to 20 days.

As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the term "subject" refers to an animal, preferably a mammal, including non-primates (e.g., cows, pigs, horses, cats, dogs, rats, and mice) and primates (e.g., monkeys or humans), more preferably a human.

As used herein, the term "prophylactically effective amount" refers to an amount of telithromumab sufficient to result in the delay or prevention of the onset, recurrence, or onset of one or more symptoms of T1D. In some embodiments, a prophylactically effective amount is preferably an amount of telithromumab 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%.

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

anti-CD 3 antibodies and pharmaceutical compositions

The terms "anti-CD 3 antibody" and "antibody that binds to CD 3" refer to an antibody or antibody fragment that is capable of binding cluster of differentiation 3(CD3) with sufficient affinity such that the antibody is useful as a prophylactic, diagnostic and/or therapeutic agent targeting CD 3. In one embodiment, the anti-CD 3 antibody binds to an unrelated, non-CD 3 protein to less than about 10% of the binding of the antibody to CD3, as measured, for example, by Radioimmunoassay (RIA). In certain embodiments, an antibody that binds CD3 has a dissociation constant (Kd) of<1μΜ、<100nM、<10nM、<1nM、<0.1nM、<0.01nM, or<0.001nM (e.g., 10)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M). In certain embodiments, the anti-CD 3 antibody binds to a CD3 epitope that is conserved between CD3 from different species.

In one embodiment, the anti-CD 3 antibody may be chagalyc 3 (oxvacizumab). Oxzezumab is a humanized Fc non-binding anti-CD 3 originally evaluated by Belgian Diabetes Registry (BDR) in phase 2 studies and then developed by Tolerx, which then partnered with GSK in phase 3 DEFEND new episode T1D trials (NCT00678886, NCT01123083, NCT 00763451). Oxzezumab was administered by infusion IV for 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-; 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, Diabet med.2014; 31: 399-; bolt et al, Eur.J.Immunol.lY3.23: 403-411; vlasakakis et 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-; keymeulen et al, BLOOD 2010, VOL 115, No. 6; spransers et al, Immunotherapy (2011)3(11), 1303-; daifotis et al, Clinical Immunology (2013)149, 268-278; which is incorporated herein by reference in its entirety.

In another embodiment, the anti-CD 3 antibody can be vislizumab (visilizumab) (also known as HuM 291; Nuvion). Vislizumab is a humanized anti-CD 3 monoclonal antibody characterized by a mutated IgG2 isotype, lacking binding to Fc γ receptors and capable of selectively inducing apoptosis in activated T cells. They have been evaluated in patients with graft versus host Disease (NCT 00720629; NCT00032279) as well as ulcerative colitis (NCT00267306) and Crohn's Disease (NCT 00267709). See, e.g., Sandborn et al, Gut 59(11) (Nov 2010) 1485-.

In another embodiment, the anti-CD 3 antibody may be forrestitumumab, a fully human anti-CD 3 monoclonal antibody developed by Tiziana Life Sciences, PLC in NASH and T2D (NCT 03291249). 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; which is incorporated herein by reference in its entirety.

In another embodiment, the anti-CD 3 antibody can be telithromumab. Telithromumab, also known as hcokt 3yl (Ala-Ala) (containing alanines at positions 234 and 235), is an anti-CD 3 antibody that has been engineered to alter the function of T lymphocytes to mediate destruction of the insulin-producing beta cells of the islets of langerhans. Telithromumab binds to an epitope of the CD3s chain expressed on mature T cells and thereby alters its function. The sequence and composition of telithromumab is 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 portions are complementarity determining regions.

Telithromycin light chain (SEQ ID NO:1)

Tigli group monoclonal antibody heavy chain (SEQ ID NO:2)

In some embodiments, provided herein are pharmaceutical compositions. Such compositions comprise a prophylactically effective amount of an anti-CD 3 antibody and a pharmaceutically acceptable carrier. In a particular embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., freund's adjuvant (complete and incomplete)), excipient, or carrier with which the therapeutic agent 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 as well as aqueous dextrose and glycerol solutions may also be employed 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, skim milk, glycerol, propylene, glycols, 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, d.c.

The composition may also contain minor amounts of wetting or emulsifying agents or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Some examples of suitable Pharmaceutical carriers are described in e.w. martin, "Remington's Pharmaceutical Sciences". Such compositions comprise a prophylactically or therapeutically effective amount of the prophylactic or therapeutic agent, preferably in purified form, and an appropriate amount of carrier to provide a form for appropriate administration to a patient. The formulation should be suitable for the mode of administration. In a preferred embodiment, the pharmaceutical composition is sterile and in a suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject.

In a particular embodiment, it may be desirable to administer the pharmaceutical composition topically to the area in need of treatment; this may be achieved by, for example but not limited to, local infusion, by injection or by means of an implant which is a porous, non-porous or gel-like material, including membranes, such as elastic membranes, or fibres. Preferably, when administering anti-CD 3 antibodies, care must be taken to use materials that do not absorb anti-CD 3 antibodies.

In another embodiment, the composition may be delivered in vesicles, particularly Liposomes (see Langer, Science 249: 1527-.

In another embodiment, the composition may be delivered in a controlled release or sustained release system. In one embodiment, controlled or sustained release can be achieved using a pump (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 may be used to achieve Controlled or sustained Release of the antibodies or fragments thereof of the present 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); Range 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; Hot et al, 1989, J. Neurol.105: 71); U.S. patent nos. 5,679,377; U.S. patent nos. 5,916,597; U.S. patent nos. 5,912,015; U.S. patent nos. 5,989,463; U.S. patent nos. 5,128,326; PCT publication nos. 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 upon storage, sterile and biodegradable. In yet another embodiment, a Controlled or sustained Release system can be placed in proximity to the therapeutic target (i.e., the lung), 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)).

Controlled release systems are discussed in the review by Langer (1990, Science 249: 1527) -1533). Any technique known to those skilled in the art can be used to produce sustained release formulations comprising one or more antibodies or fragments thereof of the invention. See, for example, U.S. Pat. Nos. 4,526,938; PCT publications WO 91/05548; PCT publications WO 96/20698; ning et al, 1996, radiothergy & 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.

The pharmaceutical composition may be formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. In a specific embodiment, the composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to humans. In a preferred embodiment, the pharmaceutical composition is formulated for subcutaneous administration to a human according to conventional procedures. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may further comprise a solubilizing agent and a local anesthetic such as lidocaine (lignocamine) to reduce pain at the injection site.

The compositions may be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

In some embodiments, the present disclosure provides for allowing a time of hours or days (e.g., associated with a pump or other device for such delivery)For example, the dosage form of the anti-CD 3 antibody is administered continuously 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 invention provides dosage forms that allow for administration of consecutive ascending doses, e.g., from 51ug/m over a period of 24 hours, 30 hours, 36 hours, 4 days, 5 days, 7 days, 10 days, or 14 days²The number of the water increases to 826ug/m²The day is.

The compositions may be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and salts with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

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 pouch (sachette)) that indicates the amount of 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 may be provided so that the ingredients may be mixed prior to administration.

In particular, the present disclosure provides that the anti-CD 3 antibody or pharmaceutical composition thereof may be packaged in a hermetically sealed container, such as an ampoule or a sachet, indicating the amount of medicament. In one embodiment, the anti-CD 3 antibody or pharmaceutical composition thereof is provided as a dried sterilized lyophilized powder or anhydrous concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline, to an appropriate concentration for administration to a subject. Preferably, the anti-CD 3 antibody or pharmaceutical composition thereof is provided in a unit dose of at least 5mg, more preferably at least 10mg, at least 15mg, at least 25mg, at least 35mg, at least 45mg, at least 50mg, at least 75mg, or at least 100mg in a dry sterile lyophilized powder in a hermetically sealed container. The lyophilized prophylactic or pharmaceutical composition herein should be stored in its original container at 2 ℃ to 8 ℃, and the prophylactic or therapeutic agent or pharmaceutical composition of the invention should be administered within 1 week, 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 after reconstitution. In an alternative embodiment, the pharmaceutical composition is provided in liquid form in a hermetically sealed container that indicates the amount and concentration of the medicament. Preferably, the administered composition in liquid form is provided in a hermetically sealed container at least 0.25mg/ml, more preferably at least 0.5mg/ml, at least 1mg/ml, at least 2.5mg/ml, at least 5mg/ml, at least 8mg/ml, at least 10mg/ml, at least 15mg/kg, at least 25mg/ml, at least 50mg/ml, at least 75mg/ml or at least 100 mg/ml. The liquid form should be stored in its original container at 2 ℃ to 8 ℃.

In a specific embodiment, the present disclosure provides for packaging the compositions of the present invention in a hermetically sealed container (e.g., ampoule or sachet) that indicates the amount of anti-CD 3 antibody.

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

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 to be employed in the formulation will also depend on the route of administration and the severity of the condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Method and use

In certain embodiments, the disclosure includes administering an anti-human CD3 antibody (e.g., telithrombin) to an individual susceptible to developing type 1 Diabetes or having a preclinical stage of type 1 Diabetes but not meeting the diagnostic criteria established by the american Diabetes association or the Diabetes Immunology Society to prevent or delay the onset of type 1 Diabetes and/or to prevent or delay the need to administer insulin to such a patient. In certain embodiments, high risk factors for identifying a susceptible subject include: having primary or secondary relatives diagnosed with type 1 diabetes, a reduced fasting glucose level (e.g., 100 to 125mg/dl as measured at least once after fasting (8 hours of non-eating)), a reduced glucose tolerance in response to 75g OGTT (e.g., 140 to 199mg/dl as measured at least once as measured at 2-hour glucose level in response to 75g OGTT), HLA type DR3, DR4 or DR7 in Caucasian, HLA type DR3 or DR4 in African, HLA type DR3, DR4 or DR9 in Japanese, exposure to a virus (e.g., Coxsackie B virus, enterovirus, adenovirus, rubella, cytomegalovirus, EB virus (steEpin-Barr virus)), a positive diagnosis of at least one other autoimmune disorder (e.g., thyroid disease, celiac disease) according to art-recognized criteria, and/or the detection of autoantibodies, particularly ICA and type 1 diabetes related autoantibodies, in serum or other tissues. In certain embodiments, a subject identified as susceptible to developing type 1 diabetes has at least one of the risk factors described herein and/or known in the art. The present disclosure also includes identifying a subject susceptible 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.

Serum autoantibodies associated with type 1 diabetes or associated with susceptibility to type 1 diabetes are islet cell autoantibodies (e.g., anti-ICA 512 autoantibodies), glutamate decarboxylase autoantibodies (e.g., anti-GAD 65 autoantibodies), IA2 antibody, ZnT8 antibody and/or anti-insulin autoantibodies. Thus, in a specific example according to this embodiment, the invention encompasses treating an individual having detectable autoantibodies (e.g., anti-IA 2, anti-ICA 512, anti-GAD, or anti-insulin autoantibodies) associated with susceptibility to developing type 1 diabetes or associated with early stage type 1 diabetes, wherein the individual has not been diagnosed with type 1 diabetes and/or is a primary or secondary relative of a type 1 diabetes patient. In certain embodiments, the presence of autoantibodies is detected by ELISA, Electrochemoluminescence (ECL), by radiometry (see, e.g., Yu et al, 1996, J.Clin.Endocrinol.Metab.81: 4264-.

Beta cell function before, during, and after treatment can be assessed by the methods described herein or by any method known to one of ordinary skill in the art. For example, the Diabetes Control and Complications Trial (DCCT) research group HAs established the percentage monitoring of glycosylated hemoglobin (HA1 and HA1c) as a criterion for assessing glycemic Control (DCCT,1993, N.Engl. J.Med.329: 977-. Alternatively, characterization of daily insulin requirements, C-peptide levels/responses, hypoglycemic episodes and/or FPIR may be used as markers of beta cell function or to establish therapeutic indices (see Keymeulen et al, 2005, N.Engl. J.Med.352: 2598-. For example, FPIR is calculated as the sum of insulin values at 1 minute and 3 minutes after the IGTT, according to the Islet Cell Antibody registry User's Study protocol (see, e.g., Bingley et al, 1996, Diabetes 45: 1720-.

In some embodiments, an individual susceptible to T1D may be a non-diabetic subject who is a relative of the 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 against glutamate decarboxylase (GAD), tyrosine phosphatase (IA-2/ICA512) or ZnT 8.

In various embodiments, the non-diabetic subject has abnormal glucose tolerance in an Oral Glucose Tolerance Test (OGTT). Abnormal glucose tolerance in the OGTT is defined as fasting glucose levels of 110 to 125mg/dL, or 2 hour plasma of ≧ 140 and <200mg/dL, or a median glucose value at 30, 60, or 90 minutes in the OGTT of >200 mg/dL.

In some embodiments, a non-diabetic subject that will respond to an anti-CD 3 antibody (e.g., telithromumab) does not have an antibody to ZnT 8. In certain embodiments, such non-diabetic subjects are HLA-DR4+ and are not HLA-DR3 +. In some embodiments, such non-diabetic subjects that will respond to an anti-CD 3 antibody (e.g., telithromumab) exhibit 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).

In various embodiments, a prophylactically effective amount includes from 10 to 1000 micrograms per square meter (μ g/m)²) Subcutaneous (SC) injection or Intravenous (IV) infusion of an anti-CD 3 antibody (e.g., telithromumab) for a course of 10 to 14 days. In one example, a prophylactically effective amount comprises 51 μ g/m on days 0 to 3, respectively²、103μg/m²、207μg/m²And 413. mu.g/m²And 826 μ g/m daily on days 4 to 13²One dose IV 14 day course of infusion of anti-CD 3 antibody (e.g., telithromumab). In certain embodiments, a prophylactically effective amount delays the median time to clinical diagnosis of T1D by at least 50%, at least 80%, or at least 90%, or 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.

In certain embodiments, the course of administration of the anti-CD 3 antibody (e.g., telithromumab) may be repeated at 2 month, 4 month, 6 month, 8 month, 9 month, 10 month, 12 month, 15 month, 18 month, 24 month, 30 month, or 36 month intervals. In some embodiments, the efficacy of treatment with an anti-CD 3 antibody (e.g., telithromumab) is determined as described herein or at 2 months, 4 months, 6 months, 9 months, 12 months, 15 months, 18 months, 24 months, 30 months, or 36 months after the prior treatment as known in the art.

In another embodiment, the subject is administered one or more unit doses of about 0.5 to 50 μ g/kg, about 0.5 to 40 μ g/kg, about 0.5 to 30 μ g/kg, about 0.5 to 20 μ g/kg, about 0.5 to 15 μ g/kg, about 0.5 to 10 μ g/kg, about 0.5 to 5 μ g/kg, about 1 to 10 μ g/kg, about 20 to 40 μ g/kg, about 20 to 30 μ g/kg, about 22 to 28 μ g/kg, or about 25 to 26 μ g/kg of an anti-CD 3 antibody (e.g., telithromumab) to prevent, treat, or ameliorate one or more symptoms of T1D. In another embodiment, the subject administers about 200 μ g/kg, 178 μ g/kg, 180 μ g/kg, 128 μ g/kg, 100 μ g/kg, 95 μ g/kg, 90 μ g/kg, 85 μ g/kg, 80 μ g/kg, 75 μ g/kg, 70 μ g/kg, 65 μ g/kg, 60 μ g/kg, 55 μ g/kg, 50 μ g/kg, 45 μ g/kg, 40 μ g/kg, 35 μ g/kg, 30 μ g/kg, 26 μ g/kg, 25 μ g/kg, 20 μ g/kg, 15 μ g/kg, 13 μ g/kg, 10 μ g/kg, 6.5 μ g/kg, 5 μ g/kg, 3.2 μ g/kg, 3 μ g/kg, an anti-CD 3 antibody (e.g., telithrombin) at 2.5 μ g/kg, 2 μ g/kg, 1.6 μ g/kg, 1.5 μ g/kg, 1 μ g/kg, 0.5 μ g/kg, 0.25 μ g/kg, 0.1 μ g/kg, or 0.05 μ g/kg to prevent, treat, or ameliorate one or more symptoms of T1D.

In some embodiments, the subject is administered one or more doses of an anti-CD 3 antibody (e.g., telithromumab) at about 5 to 1200 μ g/m2, preferably 51 to 826 μ g/m 2. In another embodiment, a subject is administered one or more unit doses of 1200 μ g/m2, 1150 μ g/m2, 1100 μ g/m2, 1050 μ g/m2, 1000 μ g/m2, 950 μ g/m2, 900 μ g/m2, 850 μ g/m2, 800 μ g/m2, 750 μ g/m2, 700 μ g/m2, 650 μ g/m2, 600 μ g/m2, 550 μ g/m2, 500 μ g/m2, 450 μ g/m2, 400 μ g/m2, 350 μ g/m2, 300 μ g/m2, 250 μ g/m2, 200 μ g/m2, 150 μ g/m2, 100 μ g/m2, 50 μ g/m2, 40 μ g/m2, 30 μ g/m2, 20 μ g/m 3615 μ g/m2, 100 μ g/m2, 3615 μ g/m2, 2 μm, Or 5 μ g/m2 of an anti-CD 3 antibody (e.g., telithromumab) to prevent, treat, slow progression of, delay onset of, or ameliorate one or more symptoms of T1D, T1D.

In another embodiment, the subject is administered a treatment regimen comprising one or more doses of a prophylactically effective amount of an anti-CD 3 antibody (e.g., telithromumab), wherein the course of treatment 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 comprises administering a prophylactically effective amount of the dose daily, every 2 days, every 3 days, or every 4 days. In certain embodiments, the treatment regimen comprises administering a prophylactically effective amount of a dose on monday, tuesday, wednesday, 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, 12 doses, 11 doses, 10 doses, 9 doses, or 8 doses are administered. In certain embodiments, the daily dosages administered for a regimen are the same.

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

In another embodiment, the intravenous dose is 1200 μ g/m2 or less, 1150 μ g/m2 or less, 1100 μ g/m2 or less, 1050 μ g/m2 or less, 1000 μ g/m2 or less, 950 μ g/m2 or less, 900 μ g/m2 or less, 850 μ g/m2 or less, 800 μ g/m2 or less, 750 μ g/m2 or less, 700 μ g/m2 or less, 650 μ g/m2 or less, 600 μ g/m2 or less, 550 μ g/m2 or less, 500 μ g/m2 or less, 450 μ g/m2 or less, 400 μ g/m2 or less, 350 μ g/m2 or less, 300 μ g/m2 or less, 250 μ g/m 5 or less, 200 μ g/m2 or less, 150 μ g/m2 or less, or 57324 μ g/m 5738 or less, or 200 μ g/m, 100 μ g/m2 or less, 50 μ g/m2 or less, 40 μ g/m2 or less, 30 μ g/m2 or less, 20 μ g/m2 or less, 15 μ g/m2 or less, 10 μ g/m2 or less, or 5 μ g/m2 or less of an anti-CD 3 antibody (e.g., telithromumab) 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. mu.g/m 2, 8000. mu.g/m 2, 7000. mu.g/m 2, 6000. mu.g/m 2, and may be less than 5000. mu.g/m 2, 4000. mu.g/m 2, 3000. mu.g/m 2, 2000. mu.g/m 2 or 1000. mu.g/m 2. In some embodiments, the total dose administered in the regimen is from 100 μ g/m2 to 200 μ g/m2, 100 μ g/m2 to 500 μ g/m2, 100 μ g/m2 to 1000 μ g/m2, or 500 μ g/m2 to 1000 μ g/m 2.

In some preferred embodiments, the dose is escalated in the first quarter, half, or 2/3 of the dose of the treatment regimen (e.g., within the first 2, 3, 4,5, or 6 days of the 10, 12, 14, 16, 18, or 20 day regimen of a dose per day) until a daily prophylactically effective amount of an anti-CD 3 antibody (e.g., telithromumab) is achieved. In certain embodiments, the subject is administered a therapeutic regimen comprising one or more doses of a prophylactically effective amount of an anti-CD 3 antibody (e.g., telithromumab), wherein the prophylactically effective amount increases daily, e.g., by 0.01 μ g/kg, 0.02 μ g/kg, 0.04 μ g/kg, 0.05 μ g/kg, 0.06 μ g/kg, 0.08 μ g/kg, 0.1 μ g/kg, 0.2 μ g/kg, 0.25 μ g/kg, 0.5 μ g/kg, 0.75 μ g/kg, 1 μ g/kg, 1.5 μ g/kg, 2 μ g/kg, 4 μ g/kg, 5 μ g/kg, 10 μ g/kg, 15 μ g/kg, 20.xi.g/kg, 25 μ g/kg, 30 μ g/kg, 35 μ g/kg, 40 μ g/kg, 45 μ g/kg, 50 μ g/kg, 55 μ g/kg, 60 μ g/kg, 65 μ g/kg, 70 μ g/kg, 75 μ g/kg, 80 μ g/kg, 85 μ g/kg, 90 μ g/kg, 95 μ g/kg, 100 μ g/kg, or 125 μ g/kg; or increased daily, for example, by 1. mu.g/

m

2, 5. mu.g/

m

2, 10. mu.g/

m

2, 15. mu.g/

m

2, 20. mu.g/

m

2, 30. mu.g/

m

2, 40. mu.g/

m

2, 50. mu.g/

m

2, 60. mu.g/

m

2, 70. mu.g/

m

2, 80. mu.g/m 2, 90. mu.g/m 2, 100. mu.g/m 2, 150. mu.g/m 2, 200. mu.g/m 2, 250. mu.g/m 2, 300. mu.g/m 2, 350. mu.g/m 2, 400. mu.g/m 2, 450. mu.g/m 2, 500. mu.g/m 2, 550. mu.g/m 2, 600. mu.g/m 2, or 650. mu.g/m 2. In certain embodiments, the subject is administered a treatment regimen comprising one or more doses of a prophylactically effective amount of an anti-CD 3 antibody (e.g., telithromumab), wherein the prophylactically effective amount is increased 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-CD 3 antibody (e.g., telithromumab) is achieved.

In a specific embodiment, the subject is administered one or more doses of 200 μ g/kg or less intramuscularly, preferably 175 μ g/kg or less, 150 μ g/kg or less, 125 μ g/kg or less, 100 μ g/kg or less, 95 μ g/kg or less, 90 μ g/kg or less, 85 μ g/kg or less, 80 μ g/kg or less, 75 μ g/kg or less, 70 μ g/kg or less, 65 μ g/kg or less, 60 μ g/kg or less, 55 μ g/kg or less, 50 μ g/kg or less, 45 μ g/kg or less, 40 μ g/kg or less, 35 μ g/kg or less, 30 μ g/kg or less, 25 μ g/kg or less, 20 μ g/kg or less, An anti-CD 3 antibody (e.g., telithrombin, oxizumab, or fomitumumab) at 15 μ g/kg or less, 10 μ g/kg or less, 5 μ g/kg or less, 2.5 μ g/kg or less, 2 μ g/kg or less, 1.5 μ g/kg or less, 1 μ g/kg or less, 0.5 μ g/kg or less, or 0.5 μ g/kg or less to prevent, treat, or ameliorate one or more symptoms of T1D.

In another embodiment, the subject is administered one or more doses of 200 μ g/kg or less, preferably 175 μ g/kg or less, 150 μ g/kg or less, 125 μ g/kg or less, 100 μ g/kg or less, 95 μ g/kg or less, 90 μ g/kg or less, 85 μ g/kg or less, 80 μ g/kg or less, 75 μ g/kg or less, 70 μ g/kg or less, 65 μ g/kg or less, 60 μ g/kg or less, 55 μ g/kg or less, 50 μ g/kg or less, 45 μ g/kg or less, 40 μ g/kg or less, 35 μ g/kg or less, 30 μ g/kg or less, 25 μ g/kg or less, 20 μ g/kg or less, or, An anti-CD 3 antibody (e.g., telithrombin, oxizumab, or fomitumumab) at 15 μ g/kg or less, 10 μ g/kg or less, 5 μ g/kg or less, 2.5 μ g/kg or less, 2 μ g/kg or less, 1.5 μ g/kg or less, 1 μ g/kg or less, 0.5 μ g/kg or less, or 0.5 μ g/kg or less to prevent, treat, or ameliorate one or more symptoms of T1D.

In another embodiment, the subject intravenously administers one or more doses of 100 μ g/kg or less, preferably 95 μ g/kg or less, 90 μ g/kg or less, 85 μ g/kg or less, 80 μ g/kg or less, 75 μ g/kg or less, 70 μ g/kg or less, 65 μ g/kg or less, 60 μ g/kg or less, 55 μ g/kg or less, 50 μ g/kg or less, 45 μ g/kg or less, 40 μ g/kg or less, 35 μ g/kg or less, 30 μ g/kg or less, 25 μ g/kg or less, 20 μ g/kg or less, 15 μ g/kg or less, 10 μ g/kg or less, 5 μ g/kg or less, 2.5 μ g/kg or less, An anti-CD 3 antibody (e.g., telithrombin, oxizumab, or forrauzumab) at 2 μ g/kg or less, 1.5 μ g/kg or less, 1 μ g/kg or less, 0.5 μ g/kg or less, or 0.5 μ g/kg or less to prevent, treat, or ameliorate one or more symptoms of T1D. In another embodiment, the intravenous dose is 100 μ g/kg or less, 95 μ g/kg or less, 90 μ g/kg or less, 85 μ g/kg or less, 80 μ g/kg or less, 75 μ g/kg or less, 70 μ g/kg or less, 65 μ g/kg or less, 60 μ g/kg or less, 55 μ g/kg or less, 50 μ g/kg or less, 45 μ g/kg or less, 40 μ g/kg or less, 35 μ g/kg or less, 30 μ g/kg or less, 25 μ g/kg or less, 20 μ g/kg or less, 15 μ g/kg or less, 10 μ g/kg or less, 5 μ g/kg or less, 2.5 μ g/kg or less, 2 μ g/kg or less, or, 1.5 μ g/kg or less, 1 μ g/kg or less, 0.5 μ g/kg or less, or 0.5 μ g/kg or less of an anti-CD 3 antibody (e.g., telithromumab, oxizumab, or forralumab) 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.

In another embodiment, the subject orally administers one or more doses of 100 μ g/kg or less, preferably 95 μ g/kg or less, 90 μ g/kg or less, 85 μ g/kg or less, 80 μ g/kg or less, 75 μ g/kg or less, 70 μ g/kg or less, 65 μ g/kg or less, 60 μ g/kg or less, 55 μ g/kg or less, 50 μ g/kg or less, 45 μ g/kg or less, 40 μ g/kg or less, 35 μ g/kg or less, 30 μ g/kg or less, 25 μ g/kg or less, 20 μ g/kg or less, 15 μ g/kg or less, 10 μ g/kg or less, 5 μ g/kg or less, 2.5 μ g/kg or less, An anti-CD 3 antibody (e.g., telithrombin, oxizumab, or forrauzumab) at 2 μ g/kg or less, 1.5 μ g/kg or less, 1 μ g/kg or less, 0.5 μ g/kg or less, or 0.5 μ g/kg or less to prevent, treat, or ameliorate one or more symptoms of T1D. In another embodiment, the oral dose is 100 μ g/kg or less, 95 μ g/kg or less, 90 μ g/kg or less, 85 μ g/kg or less, 80 μ g/kg or less, 75 μ g/kg or less, 70 μ g/kg or less, 65 μ g/kg or less, 60 μ g/kg or less, 55 μ g/kg or less, 50 μ g/kg or less, 45 μ g/kg or less, 40 μ g/kg or less, 35 μ g/kg or less, 30 μ g/kg or less, 25 μ g/kg or less, 20 μ g/kg or less, 15 μ g/kg or less, 10 μ g/kg or less, 5 μ g/kg or less, 2.5 μ g/kg or less, 2 μ g/kg or less, or, 1.5 μ g/kg or less, 1 μ g/kg or less, 0.5 μ g/kg or less, or 0.5 μ g/kg or less of an anti-CD 3 antibody (e.g., telithromumab, oxizumab, or forralumab) 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.

In some embodiments where escalating doses are administered several days prior to the dosing regimen, the dose on day 1 of the regimen is 5 to 100 μ g/m²Per day, preferably 51. mu.g/m²Day by day, and gradually increased by

day

3, 4,5, 6 or 7 as noted immediately aboveDaily dose of vehicle. For example, the subject is administered a dose of about 51 μ g/m on day 1² Day 2 is about 103. mu.g/m²Day 3 was about 207. mu.g/m²Day 4 was about 413. mu.g/m²Day and 826 μ g/m on subsequent days of the regimen (e.g., days 5 to 14)²The day is. In another embodiment, the subject is administered a dose of about 227 μ g/m on day 1² Day 2 was about 459. mu.g/m²Day 3 and subsequent days was about 919 μ g/m²The day is. In another embodiment, the subject is administered a dose of about 284 μ g/m on day 1² Day 2 was about 574. mu.g/m²Day 3 and subsequent days was about 1148. mu.g/m²The day is.

In other embodiments, the initial dose is 1/4, to 1/2, to equal the daily dose at the end of the regimen, but is administered in portions at 6,8, 10, or 12 hour intervals. For example, a 13 μ g/kg/day dose is administered at four doses of 3 to 4 μ g/kg at 6 hour intervals to reduce the level of cytokine release resulting from administration of the antibody. In some embodiments, to reduce the likelihood of cytokine release and other adverse effects, the first 1, 2, 3, or 4 doses or all doses in a regimen are administered more slowly by intravenous administration. For example, 51. mu.g/m²A dose per day may be administered within 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 embodiments, the dose is infused in a pump, preferably increasing the concentration of antibody administered as the infusion progresses.

In other embodiments, the foregoing 51 μ g/m²Day to 826 mug/m²The collection of fractions of doses per day regimen was 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 regimen described above. Thus, when the score is 1/10, the daily dose on day 1 will be 5.1 μ g/m²Of 1 at10.3. mu.g/m for 2 days²20.7g/m on day 3²41.3. mu.g/m on day 4²And 82.6. mu.g/m on days 5 to 14². When the score is 1/4, the dose on day 1 will be 12.75 μ g/m²25.5. mu.g/m on day 2²51. mu.g/m on day 3²103. mu.g/m on day 4²And 207. mu.g/m on days 5 to 14². When the score is 1/3, the dose on day 1 will be 17 μ g/m²34.3. mu.g/m on day 2²69 μ g/m on day 3²137.6. mu.g/m on day 4²And 275.3 μ g/m on days 5 to 14². When the score is 1/2, the dose on day 1 will be 25.5. mu.g/m²51. mu.g/m on day 2²103. mu.g/m on day 3²207. mu.g/m on day 4²And 413. mu.g/m on days 5 to 14². When the score is 2/3, the dose on day 1 will be 34 μ g/m²69 μ g/m on day 2²137.6. mu.g/m on day 3²275.3 μ g/m on day 4²And 550.1. mu.g/m on days 5 to 14². When the score is 3/4, the dose on day 1 will be 38.3. mu.g/m²77.3. mu.g/m on day 2²155.3. mu.g/m on day 3²309.8. mu.g/m on day 4²And 620. mu.g/m on days 5 to 14². In other embodiments, the regimen is the same as one of the regimens described above, except only on days 1 to 4, 1 to 5, or 1 to 6. For example, in one embodiment, the dose at day 1 will be 17 μ g/m²34.3. mu.g/m on day 2²69 μ g/m on day 3²137.6. mu.g/m on day 4²And 275.3 μ g/m on

days

5 and 6²。

In some embodiments, the anti-CD 3 antibody (e.g., telithromumab, oxizumab, or forranibizumab) is not administered by daily dose over a few days, but 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. Infusion may be constant, or may be initiated at a lower dose, e.g., 1, 2, 3, 5,6, or 8 hours prior to infusion, and then escalated to a subsequent higher doseAmount of the compound (A). During the course of infusion, the patient received a dose equal to the amount administered in the 5 to 20 day regimen described above. For example, the dosage is about 150. mu.g/m²、200μg/m²、250μg/m²、500μg/m²、750μg/m²、1000μg/m²、1500μg/m²、2000μg/m²、3000μg/m²、4000μg/m²、5000μg/m²、6000μg/m²、7000μg/m²、8000μg/m²Or 9000. mu.g/m². In particular, the rate and duration of infusion is designed to minimize the level of free anti-CD 3 antibody (e.g., telithromumab, oxvacizumab, or forrafluzumab) in the subject after administration. In certain embodiments, the level of free anti-CD 3 antibody (e.g., telithromumab) should not exceed 200ng/ml free antibody. In addition, the infusions are designed to achieve combined T cell receptor coating and at least 50%, 60%, 70%, 80%, 90%, 95% or 100% modulation.

In other embodiments, the anti-CD 3 antibody (e.g., telithromumab, oxizumab, or forranibizumab) is administered chronically to treat, prevent, or slow or delay the onset or progression of type 1 diabetes, or to ameliorate one or more symptoms thereof. For example, in certain embodiments, a low dose of an anti-CD 3 antibody (e.g., telithrombin) is administered monthly, twice monthly, three times monthly, weekly, or even more frequently following administration of such a regimen to potentiate or maintain its effect as an alternative to the 6-to 14-day dosage regimen described above. Such low doses may be 1 μ g/m²To 100. mu.g/m²Any value of (a), e.g. about 5. mu.g/m²、10μg/m²、15μg/m²、20μg/m²、25μg/m²、30μg/m²、35μg/m²、40μg/m²、45μg/m²Or 50. mu.g/m²。

In other embodiments, the subject may be re-dosed at a time after administration of the anti-CD 3 antibody (e.g., telithromumab, oxizumab, or forranibizumab) dosing regimen, e.g., based on one or more physiological parameters, or as usual. Such re-dosing and/or the need for such re-dosing can 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 can include administering the course of treatment sporadically every 6 months, 9 months, 1 year, 15 months, 18 months, 2 years, 30 months, or 3 years.

Examples

SUMMARY

Background: type 1 diabetes (T1D) is a chronic autoimmune disease that results in the destruction of insulin-producing beta cells and relies on exogenous insulin for survival. Some interventions have shown success in attenuating the loss of insulin production in patients presenting with clinical disease, but to date, no intervention has been able to affect the progression of the disease in individuals at high risk of developing the disease.

Method: we performed a randomized placebo-controlled double-blind study of telithrombin (FcR non-binding anti-CD 3mAb) in non-diabetic relatives at high risk of clinical disease in patients with T1D. 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.

Results: in total 76 subjects were randomized, 44 in the temizumab group and 32 in the placebo group. Seventy-two percent of participants are children. Telithromumab treatment delayed the median time to clinical diagnosis for T1D from 24.4 months to 48.4 months (Cox proportional risk, p ═ 0.006), and reduced the incidence of diabetes from 9.8% per year to 4.1%. Drug treatment increased the proportion of TIGIT + KLRG1+ CD8+ T cells. Participants who did not have an antibody against ZnT8 (p ═ 0.01), were HLA-DR4+ (p ═ 0.006) and not HLA-DR3+ (p ═ 0.05) were most likely to respond to telithromumab.

Conclusion: telithromumab could delay the diagnosis of T1D in high risk individuals. Some subgroups of individuals may be more likely to respond to treatment and action.

Method

Test participants

Participants were identified by TrialNet Natural History Study (TrialNet Natural History Study)¹⁴. The test was conducted at sites in the united states, canada and germany from 7 months 2011 to 11 months 2018. Approval by the Institutional Review Board (IRB) was obtained at each participating site. The patient, their parents, or both provided written informed consent prior to entry into the trial.

Eligible participants were non-diabetic relatives of type 1 diabetic patients, age at randomization ≥ 8 years, at high risk for developing clinical diabetes. Within 6 months prior to randomization, they had 2 or more diabetes-associated autoantibodies at 2 sample collections. In addition, they have abnormal glucose tolerance in the Oral Glucose Tolerance Test (OGTT), which is defined as: fasting glucose levels were 110 to 125mg/dL, or 2 hour plasma >140 and <200mg/dL, or intermediate glucose values in OGTT at 30, 60 or 90 minutes >200mg/d, occurring twice within 52 days after enrollment. The protocol was revised in 2014 to allow enrolling participants with age <18 with a single abnormal OGTT, since the rate of T1D progression in this age group is similar with or without a definitive OGTT. In these 8 subjects (5 in the telithromab group and 3 in the placebo group), OGTT was performed before the second treatment on the first day of study drug administration. Individuals with other important medical histories, abnormal laboratory chemistry or blood cell counts were excluded.

Patient identification

Subjects were identified in TrialNet prevention pathway (PTP) studies. See fig. 5. The PTP study enrolled a first-degree relative of T1D patients, aged 1 to 45 years, and a second-or third-degree relative aged up to 20 years, evaluated diabetic autoantibodies to micro-insulin (mIAA), glutamic acid decarboxylase-65 (GAD), and insulinoma-associated antigen 2(IA-2 or ICA 512). Islet Cells (ICA) and zinc transporter 8(ZnT8) autoantibodies were measured if at least one other antibody tested positive.

Design and intervention of experiments

Participants were randomized to receive either telithrombin or placebo, each group was equally divided. At each TrialNet study site, randomization was stratified by age (< or >18 years) and glucose during the OGTT state prior to randomization. The treatment allocation is double masked.

Participants received a 14 day course of telithrombin or placebo administered as outpatient in a clinical study center using the previously described drug dosing regimen^9,10. In particular, those assigned to the active study drugs received telithromumab via daily IV infusion of telithromumab according to the following daily schedule: on study days 0 to 3, 51 micrograms per square meter (. mu.g/m), respectively²)、103μg/m²、207μg/m²And 413. mu.g/m²And a daily dose of 826. mu.g/m on study days 4 to 13². Participants randomized to the placebo group received a 14 day course of matched IV saline. Participants received ibuprofen (ibuprofen) and diphenhydramine (diphenhydramine) for the first 5 days prior to infusion and then further administered ibuprofen, diphenhydramine and/or acetaminophen as needed for symptom relief. The protocol defining the stopping criteria for study drug infusion was followed.

All subjects were in temporary contact with the investigator for formal interrogation of adverse events and diabetic symptoms throughout the study.

Endpoint and evaluation

Using the standards defined by the American diabetes Association, the primary outcome is the time elapsed from randomization to diagnosis of diabetes¹⁵。

The scheduled OGTT test was performed at 3 and 6 months after infusion and every 6 months thereafter. Random glucose screens were performed at 3 month intervals and the OGTT test was performed if random glucose was >200mg/dl with symptoms of diabetes. The diabetic OGTT test requires sequential confirmation and the date of diagnosis is determined as the time of the second diagnostic test. Outcome review is performed without knowledge of treatment allocation.

Blood samples were analyzed centrally in the triallet core laboratory using the method described in the laboratory method. Flow cytometry was used to analyze CD8+ T cell subsets in peripheral blood (fig. 4).

Laboratory method

C-peptide was measured from frozen plasma by a two-site immunoenzyme assay (Tosoh Bioscience, South San Francisco, Calif.). HbA was measured using ion exchange high performance liquid chromatography (II th edition, Bio-Rad Diagnostics, Hercules, Calif.)_1c. The reliability factor for each assay in the isolated replicate samples was greater than 0.99. mIAA, GAD-65Ab, ICA-512Ab, ZnT8A were measured using radioimmunoconjugation assay at the Barbara Davis diabetes center of Anschultz CO, and ICA was measured using indirect immunofluorescence at the university of Florida, Gernsvell. C-peptide, glucose and HbA1C were measured at the northwest research laboratory in Seattle, Washington (WA). HbA was measured using ion exchange high performance liquid chromatography (II th edition, Bio-Rad Diagnostics, Hercules, Calif.)_1cIn time, C peptide was determined from frozen plasma by a two-site immunoenzyme assay (Tosoh Bioscience, South San Francisco, CA). The reliability factor for each assay in the isolated replicate samples was greater than 0.99. EBV and CMV viral loads were measured in whole blood at the University of Colorado (University of Colorado) using the previously described methods¹。

Flow cytometry

Peripheral Blood Mononuclear Cells (PBMCs) were treated and stored in NIDDK reservoirs. Frozen PBMC vials were sent to the Benaroya institute for analysis by flow cytometry using the antibody panel shown in figure 4. T cell phenotype sorting (phenotyping) was performed on PBMC as previously described on LSR-fortessa (bd biosciences) with FACS Diva software and analyzed with FlowJo software version 9.5 (Tree Star, Ashland, OR). Determined as TIGIT + KLRG + CD57-, TIGIT-KLRG1-CD 57-or CD4+ CD127 as described previously^loFrequency of CD8+ T cells of Foxp3+ (CD4+ Treg)². Quadrants were placed based on staining controls.

Test supervision

The study was developed and conducted by type 1 Diabetes mellitus TrialNet sponsored by the national institutes of health and the Juvenile Diabetes Research Foundation.

In addition to analysis of CBC at infusion sites, as well as differential and routine chemistry, study coordination, laboratory testing, and data management were focused. An independent medical monitor (masked from treatment distribution) reviews all accumulated safety data.

Statistical analysis

Estimating cumulative incidence of diabetes onset over time within each group since randomization from Kaplan-Meier estimation of "diabetes free" survival function¹⁶. The differences between treatment groups in the cumulative incidence function at six month intervals were estimated by the risk ratio (HR) and the hypothesis test employed the likelihood ratio test; both based on the Cox proportional Risk (PH) model¹⁷. The cut-off values of the test statistics of the main hypotheses were determined by a group-sequential procedure.

The original protocol (censored 144 subjects with 60% (previous 50%) risk rate (HR 0.4) decreased with an alpha level of 0.025 and a statistical efficacy of 80% (one-sided) due to lower than expected accreditation rate, setting the study objective to enroll at least 71 subjects and follow them until 40 subjects were diagnosed with T1D¹⁸。

Data on Safety and efficacy were evaluated twice a year by the independent Data Safety Monitoring Board (DSMB). Metaphase analysis was performed when 50% of the expected number of T1D cases were observed, at which time formal comparisons were presented to the DSMB and the Lan DeMets stopping rule was applied¹⁹. The data was analyzed according to the principle of intention treatment. The significance test reported herein is unilateral, using a significance threshold of 0.025 according to the present design, but the therapeutic interaction test is bilateral. Unless otherwise stated, 95% confidence intervals are reported. Flow cytometry data were analyzed by repeated measures ANOVA. Statistical analysis was performed using TIBCO Spotfire S +8.2 Workbench or SAS 9.4.

Results

Patient's health: among 112 objects for eligibility screening, 76: 44 were randomized to telithromumab and 32 were randomized to placebo (figure 1). The randomization process results in unequal proportions in the study groups, which may beWith a few enrolled subjects through some study sites (<3) in which randomization may result in unequal assignments 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 the T1D patient. At age<Of the 18 subjects, 47 confirmed blood glucose abnormalities OGTT before randomization. Of those randomized after a single dysglycaemic OGTT, 2 had "diabetes" and 6 had normal pre-treatment OGTT: these 8 individuals were enrolled based on blood glucose abnormalities OGTT prior to enrollment.

Ninety-three percent (41/44) and 87.5 percent (28/32) of subjects randomized to the telithromumab and placebo groups, respectively, completed 14 days of drug treatment. The total dose of telithromumab administered was 9.¹⁴(IQR: 9.01 to 9.37) μ g/m². Treatment was incomplete in 3 drug-treated subjects and 4 placebo-treated subjects due to laboratory abnormalities (n-4), inability to establish venous access (n-2), or rash (n-1). Median follow-up was 745 days (range 74 to 2683 days). The duration of follow-up in 75% of subjects was more than 3 years. T1D was diagnosed in 42 (55%) participants.

Efficacy: treatment with the monoterpenizumab procedure delayed T1D time (fig. 2A, p ═ 0.006): median T1D time was 24.4 months in the placebo group and 48.4 months in the teli group mab group (risk ratio 0.437 (IQR: 0.229, 0.832). the annual incidence of T1D for the placebo and teli group mab groups was 9.8% and 4.1%, respectively.) 25 subjects from the teli group mab group (57%) and 9 subjects from the placebo group (28%) did not have T1D (chi fang, p ═ 0.012) at the end of the trial.

The overall rate of progression of T1D was highest in the first year after entry into the test (n 17, 41%) compared to year 2 (

n

10, 24%), year 3 (

n

6, 14%) or year 4 (

n

5, 12%). The effect of telithromumab treatment was also maximal during this period (fig. 1B). The risk ratio was lowest in the first 36 months after study enrollment and remained relatively constant and statistically significant after this (p < 0.01).

Therapeutic administration and safety profile: overall, telithromumab treatment was well tolerated-adverse events designated as likely, likely or absolutely relevant to the study drug are shown in table 2. Lymphocyte counts were lowest at day 5, a 72.3% drop (IQR 82.1, 68.4%) (p)<0.0001), but then quickly recovers. 15 of the grade 3 events in the telithromumab group (34.1%) were involved in lymphopenia during the first 30 days after study drug administration. After day 30, there were no lymphopenia cases in any of the treatment groups (fig. 1C). Spontaneous regression of skin rash as described previously occurred in 36% of drug-treated subjects¹¹. The infection rates were similar in both treatment groups.

At entry, 30 subjects (39%) (16 treated with telithromumab and 14 treated with placebo) had antibodies against the EBV virus. Quantifiable EBV viral loads were present in 7 participants (all in the telithromumab group) at weeks 3 to 6 after study drug treatment. Of these with detectable viral loads, 1 had symptoms of pharyngitis, runny nose and cough on day 38. EBV viral load dropped below the quantitative level on days 43 to 134 (average 74 days). At entry, 17 participants (10 temepratumab and 7 placebo) had antibodies against CMV virus. One of the subjects with telithromumab (which was CMV seropositive) had detectable CMV virus levels at day 20 and was undetectable by day 42.

Response biomarkers: we previously described changes in CD8+ T cells following telithromumab treatment, e.g. expression of markers TIGIT, KLRG1, etc. associated with decreased responsiveness^12,13. To determine whether clinical outcome correlates with these changes in CD8+ T cells, we compared the frequency of CD8+ KLRG1+ TIGIT + CD57-T cells in the two treatment groups. Telithromumab treatment increased the frequency of these T cells (p ═ 0.009, 0.007, respectively) at month 3 and month 6, and at

month

3 and 6, compared to baselineAt month time, levels were higher in the telithromumab-treated participants relative to placebo-treated participants (p ═ 0.02, 0.04, respectively). (FIG. 3A, FIG. 6). We did not identify changes in these cells in placebo treated subjects. Not all T cell subsets were affected by telithrombin: in either group, there was no significant change in CD4+ Treg or CD8+ KLRG1-TIGIT-CD 57-cells^8,20(FIGS. 7A, 7B).

To determine whether demographic characteristics of the participants correlated with clinical response, in a pre-assigned analysis, we analyzed the role of telithromumab in a subgroup of participants based on age, HLA type, pre-treatment C-peptide and glucose during OGTT, as well as autoantibodies (fig. 3B). Participants without the anti-ZnT 8 antibody showed a greater response to telithrombin (p ═ 0.004) compared to those with anti-ZnT 8 antibody (fig. 3C). The presence or absence of other autoantibodies is not relevant to clinical response. The subjects with 49% and 65% of telithromumab were HLA-DR3 and HLA-DR4, respectively. The presence and absence of HLA-DR4 and 3 correlated with a more robust response to telithrombin (p ═ 0.004 and 0.01, respectively, two-sided assay) (fig. 3D, E).

Discussion of the related Art

In this phase II study, we found that the mono-teicoplasma process significantly slowed progression to T1D in non-diabetic relatives with abnormal glucose tolerance during the OGTT at the beginning of the study. The median delay in diabetes diagnosis was 2 years and at the end of the trial, the frequency of non-diabetic individuals doubled (57%) in drug-treated subjects relative to placebo-treated subjects (28%). The safety experience is good for children and adults in the case of expected adverse events of rash and transient lymphopenia. To our knowledge, this is the first treatment to delay or prevent the onset of T1D. The delay in onset of clinical T1D and the challenges of daily management are of clinical importance. In addition, the smaller the age of diagnosis is associated with worse outcome^2,4. Previous large well-designed but unsuccessful preventive trials have not utilized immunotherapy against immune cells, and our findings support the concept that T1D is a chronic T cell-mediated disease^21,22. In addition, the appearanceThe observation that this treatment affects disease progression prior to diagnosis and loss of beta cell function after diagnosis indicates continuity in the autoimmune process and confirms attempts to use immunomodulation prior to onset of clinical disease^9-11,23-25。

The effect of the drug was greatest in the first three years after administration. This was indeed true in 41% of patients with diabetes in the first year after randomization, and HR was lowest for patients exposed to telithromumab at that time. The relatively rapid progression to diabetes in the placebo group reflects a very high risk for these individuals⁵. In fact, our decision to enroll these subjects without clinical disease reflects when finding>2 autoantibodies and the unavailability of progression at abnormal glycemia, consistent with our reports of high beta cell killing rates in these individuals²⁶. In addition, the rapid onset of clinical T1D may reflect an enrichment (72.4%) of pediatric participants in which the rate of progression is rapid^27,28。

There were differences in the response to telithrombin based on the characteristics of the subjects at study enrollment. The absence of one T1D-related MHC allele (HLA-DR3) but the presence of the other (HLA-DR4) and the absence of anti-ZnT 8 antibody determined the individuals most likely to respond. MHC can modulate responsiveness to telithromumab through its action on the T cell reservoir, possibly altering the T cell activation state and sensitivity to drug action. The anti-ZnT 8 antibody may also be a marker for individuals with a more explosive immune response, or other characteristic that sensitizes T cells of an individual to telithromumab. We cannot determine whether the treatment will be effective in those at an earlier stage of the disease. Further immunological and metabolic studies may determine features that define individuals most likely to benefit from the treatment.

The transient effect of drug treatment on lymphocyte counts most likely reflects export from peripheral blood rather than cell depletion^29,30. Our flow cytometry studies showed that changes in the CD8+ T cell phenotype were one indicator of clinical response. However, these effects associated with the non-responsive or "depleted" phenotype did not inactivate CD8+ T cells asTo present an active response to EBV and CMV in those in which viral load is increased^31,32. The functional effect of telithromumab on T cells may be influenced by its affinity for antigen: t cells with high avidity (e.g., viral antigen reactive cells) may be unaffected, while those with low avidity (e.g., autoreactive T cells) may become inactive. Further studies on antigen-reactive T cells are needed to address this hypothesis.

Consider some limitations that exist with this clinical study. The cohort was relatively small and the rate of progression to T1D was more rapid in the placebo group. The study subjects were relatives of patients with T1D, and therefore we did not know whether these findings would be generally applicable to the discovery of non-relatives at risk for T1D. Recent reports indicate that the rate of diabetes in non-relatives versus relatives is similar in genetically high risk individuals³³. Furthermore, despite reflecting the known incidence of disease, the majority of this population is non-hispanic caucasians. It is desirable to increase the median delay in onset of disease. In this study, the drug was given for only one course, and our analysis of HR suggested that repeated dosing may be required to obtain more individuals with active disease and prolong the therapeutic effect^9,25. The following needs to be explored: the target population and number of drug processes for treatment are identified.

Overall, this was the first trial to show delay or prevention of T1D. Since the age and duration of onset of diabetes are important determinants of metabolic management and complications, as well as the burden of daily management, any time without diabetes is clinically significant. Selecting those that are most likely to respond, repeated administration, or combining telithromumab with other agents with complementary mechanisms of action may enable prevention of clinical disease for an extended period of time.

Table 1: subject characteristics at baseline for treatment group

Values in parentheses: the quartile range is shown in median and the object percentage is shown in number of objects.

At randomization. All subjects had at least 2+ autoantibodies prior to randomization.

Deletion (c): loss of HLA allelic State in 1 subject

Table 2: adverse events assigned as likely, likely or absolutely relevant to study drug during active follow-up

P <0.001, telithromumab to placebo

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

Is incorporated 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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Sequence listing

<110> Provention Bio, Inc.

<120> methods and compositions for preventing type 1 diabetes

<130> 178833-010701/US

<150> US 62/847,466

<151> 2019-05-14

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<170> PatentIn version 3.5

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Lys

Claims

1. 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) has substantially no antibodies to zinc transporter 8(ZnT8), (2) is HLA-DR4+, and/or (3) is not HLA-DR3 +; and

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

3. The method of claim 1, wherein 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 glutamate decarboxylase (GAD), tyrosine phosphatase (IA-2/ICA512), or ZnT 8.

4. The method of claim 1, wherein the non-diabetic subject has abnormal glucose tolerance in the Oral Glucose Tolerance Test (OGTT).

5. The method of claim 4, wherein the abnormal glucose tolerance in the OGTT is a fasting glucose level of 110 to 125mg/dL, or a2 hour plasma of ≥ 140 and <200mg/dL, or a median glucose value at 30, 60, or 90 minutes in the OGTT of >200 mg/dL.

6. The method of claim 1, wherein the non-diabetic subject does not have antibodies to ZnT 8.

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

8. The method of claim 1, wherein the anti-CD 3 antibody is selected from the group consisting of telithromumab, oxizumab, or forralumab.

9. The method of claim 8, wherein the prophylactically effective amount comprises from 10 to 1000 micrograms per square meter (μ g/m)²) Subcutaneous (SC) injection or Intravenous (IV) infusionOr orally administering the anti-CD 3 antibody for a period of 10 to 14 days, preferably 51 μ g/m on days 0 to 3, respectively²、103μg/m²、207μg/m²And 413. mu.g/m²And 826 μ g/m daily on days 4 to 13²One dose of (a) was given for 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 the increase in frequency after administration of telithromumab is indicative of responsiveness to the anti-CD 3 antibody.

12. A method of prognosing responsiveness of an anti-CD 3 antibody in preventing or delaying the onset of type 1 diabetes (T1D), comprising:

providing a non-diabetic subject at risk of T1D;

administering to the non-diabetic subject a prophylactically effective amount of an anti-CD 3 antibody; and

determining the frequency of TIGIT + KLRG1+ CD8+ T cells in peripheral blood mononuclear cells of the non-diabetic subject by flow cytometry, wherein the increase in frequency is indicative of responsiveness to the anti-CD 3 antibody.

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

14. The method of claim 12, wherein 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 glutamate decarboxylase (GAD), tyrosine phosphatase (IA-2/ICA512), or ZnT 8.

15. The method of claim 12, wherein the non-diabetic subject has abnormal glucose tolerance in the Oral Glucose Tolerance Test (OGTT).

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

17. The method of claim 12, further comprising determining that the non-diabetic subject is (1) 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 to ZnT 8.

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

20. The method of claim 12, wherein the anti-CD 3 antibody is selected from the group consisting of telithromumab, oxizumab, or forralumab.

21. The method of claim 20, wherein the prophylactically effective amount comprises from 10 to 1000 micrograms per square meter (μ g/m)²) Subcutaneous (SC) injection or Intravenous (IV) infusion or oral administration of the anti-CD 3 antibody for a period of 10 to 14 days, preferably 51 μ g/m on days 0 to 3, respectively²、103μg/m²、207μg/m²And 413. mu.g/m²And 826 μ g/m daily on days 4 to 13²One dose IV of telithromumab was infused over the course of 14 days.

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.