Teplizumab preserves C-peptide in recent-onset type 1 ... · 17/06/2013 · Teplizumab is a...

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Teplizumab preserves C-peptide in recent-onset type 1 diabetes: 2-year results from the

randomized, placebo-controlled Protégé trial

Running title: 2-year results of the Protégé trial

William Hagopian, MD, PhD,1 Robert J. Ferry Jr, MD,2 Nicole Sherry, MD,3 David Carlin,

PhD,4 Ezio Bonvini, MD,4 Syd Johnson, PhD,4 Kathryn E. Stein, PhD,4 Scott Koenig, MD, PhD4

Anastasia G. Daifotis, MD,4 Kevan C. Herold, MD,5 Johnny Ludvigsson, MD, PhD6 for the

Protégé Trial Investigators*

1Pacific Northwest Diabetes Research Institute, Seattle, WA; the 2Division of Pediatric

Endocrinology and Metabolism, Le Bonheur Children’s Hospital and University of Tennessee

Health Science Center, Memphis, TN; the 3Massachusetts General Hospital, Boston, MA;

4MacroGenics, Rockville, MD; 5Departments of Immunobiology and Internal Medicine, 5Yale

University, New Haven, CT; and the 6Division of Pediatrics, Department of Clinical and

Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden

*A list of all investigators appears online in the Appendix

Corresponding author: William Hagopian, Pacific Northwest Diabetes Research Institute, 720

Broadway, Seattle, WA 98122 phone: (206) 860-6759; fax: (206) 320-1448; [email protected]

Word count: 3977; 4 Tables, 3 Figures

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Diabetes Publish Ahead of Print, published online June 25, 2013

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Abstract [176 words (limit 200)]

Protégé was a Phase 3, randomized, double-blind, parallel, placebo-controlled 2-year study of

three intravenous teplizumab dosing regimens, administered daily for 14 days at baseline and

again after 26 weeks, in new-onset type 1 diabetes. We sought to determine efficacy and safety

of teplizumab immunotherapy at 2 years, and to identify characteristics associated with

therapeutic response. Of 516 randomized patients, 513 were treated, and 462 completed 2 years

follow-up. Teplizumab (14-day full-dose) reduced the loss of C-peptide mean AUC (a pre-

specified secondary endpoint) at 2 years versus placebo. In analyses of pre-specified and post-

hoc subsets at entry, US residents, patients with C-peptide mean AUC >0.2nmol/L, those

randomized <6 weeks after diagnosis, HbA1c <7.5% (58 mmol/mol), insulin use <0.4U/kg/day,

and ages 8-17 each had greater teplizumab-associated C-peptide preservation than their

counterparts. Exogenous insulin needs tended to be reduced versus placebo. Anti-drug antibodies

developed in some patients without apparent change in drug efficacy. No new safety or

tolerability issues were observed during Year 2. In summary, anti-CD3 therapy reduced C-

peptide loss 2 years after diagnosis using a tolerable dose.

ClinicalTrials.gov identifier: NCT00385697

Introduction

Immunotherapy that directly inhibits ß-cell destruction is an unfulfilled need for treatment of

autoimmune type 1 diabetes. While it may eventually be useful in prediabetes, treatment at

clinical onset is an excellent opportunity, when patients are easily identified and functional ß-cell

mass remains (1). Preservation of residual ß-cell function (represented by higher levels of C-

peptide) facilitates better glycemic control to lessen retinopathy, nephropathy, hypoglycemia,

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and ketoacidosis (2-4). Immunotherapy given at diagnosis aims to prolong and augment this

effect both by preventing further ß-cell death and possibly also by enabling living ß-cells to

recover function after resolution of inflammation (5). Clinical trials of different agents have had

modest success in this regard, but treatment responses have often waned within 2 years (6-8).

Teplizumab is a non-activating Fc-modified, anti-CD3 monoclonal antibody thought to

attenuate activated autoreactive T-cells mediating ß-cell death. These T-cells disappear from the

peripheral circulation during immunotherapy, but return within weeks after stopping treatment

(9). Preclinical and clinical studies suggest that the drug may induce regulatory T-cell activity,

suggesting augmented immune tolerance (10).

Protégé was a large, randomized, placebo-controlled, double-blinded trial of immunotherapy

in type 1 diabetes (11). Recently diagnosed patients (ages 8–35 yr) were randomized to receive

daily infusions of placebo or one of three teplizumab regimens at baseline and at 6 months. The

primary outcome (a composite of insulin <0.5 U/kg/day and HbA1C <6.5% (48 mmol/mol) at

Year 1) had not been validated previously and did not achieve statistical significance. In

exploratory analyses, a significant improvement in AUC mean C-peptide (4-hour mixed meal

tolerance test) was observed in the group treated with a full-dose 14-day course. In certain

prespecified subgroups, the AUC mean C-peptide differences versus placebo appeared to be

most pronounced in recently diagnosed patients, patients in the United States, and younger

patients. The drug was generally well tolerated.

A recent study reported that teplizumab treatment reduced ß-cell death at 1 year, but the

differences versus placebo were not significant earlier, at 6 months (12). The acute (i.e. within 1

year) effects of immunotherapy on ß-cell function may not occur through the same mechanisms

as longer-term effects that have greater clinical importance. Improvement in C-peptide responses

may be seen in type 1 diabetes trials, even with therapies that do not affect immune responses,

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through mechanisms that may involve recovery of dysfunctional ß-cells when inflammation is

acutely resolved (5,13). To be of value, a lasting effect on ß-cell function and survival is needed.

The objective of this report is to characterize the efficacy and safety of teplizumab over 2

years and identify characteristics associated with response to therapy. Regarding efficacy, we

focus on the 14-day full-dose regimen that was administered versus placebo, because at 1 year

efficacy was seen in the 14-day full-dose arm but not the reduced dose or curtailed dose arms

(11). Emphasis is given to AUC mean C-peptide because this has become the preferred measure

of efficacy in type 1 diabetes immunotherapy (14). To explore the potential implications for

dosing in future studies, we also describe the pharmacokinetics and pharmacodynamics of

teplizumab, the effect of antidrug antibodies, and the safety profiles of all three dosing regimens.

Methods

Details of the trial methodology were published previously (11) and are summarized briefly

here and in the Appendix. Participation was restricted to patients with type 1 diabetes mellitus

diagnosed according to American Diabetes Association criteria (15) within the prior 12 weeks,

and who required injected insulin therapy. Inclusion also required detectable levels of fasting or

stimulated C-peptide, and autoantibodies to one or more standard islet autoantigens. Exclusion

criteria focused on medical disorders that might confound results or interfere with safe trial

completion, such as active infections. The research protocol was approved by institutional review

boards, and all participants or guardians gave written informed consent.

Patients were randomly assigned (2:1:1:1) to one of four parallel treatment groups, with an

escalating dose, 14-day course of daily intravenous treatment starting at baseline, and another

14-day course at Week 26. For each treatment course, the 14-day full-dose group (n=209)

received a total cumulative teplizumab dose of ~9034 µg/m²; the 14-day low-dose group (n=102)

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received a total of ~2985 µg/m²; the 6-day full-dose group received a total of ~2426 µg/m² over 6

days followed by 8 days of placebo; the placebo group (n=99) received 14-days of placebo

infusions. Randomization was stratified by age group (8–11, 12–17, and 18–35 years) and by

country. Dosing was double blind (patients and study personnel) to conceal allocation. Patients

received a non-steroidal, anti-inflammatory drug (e.g. ibuprofen) and/or antihistamine (e.g.

diphenhydramine) to minimize adverse events during each treatment cycle.

Intensive diabetes care was provided for all patients. Investigators were instructed to

aggressively treat diabetes to a target HbA1C of <6.5%, and to maintain an insulin dose of >0.25

U/kg per day, but insulin adjustment algorithms were not pre-specified. Patients used diary cards

to record insulin use at screening and for 3 days before each visit at Days 91, 140, 273, 364, 448,

546, 616, and 728. Use of agents that might affect islet growth, endogenous insulin secretion,

insulin sensitivity, or immune function were not permitted during the study.

HbA1C was measured and a 4-hour mixed-meal tolerance test (MMTT) was performed at a

screening visit and on Days 140, 364, 546, and 728 (HbA1C was also measured on Days 273,

448, and 616); total AUC mean C-peptide during the MMTT was then calculated (1). After

interim analyses determined that the primary endpoint at 1 year was not met, patients not yet at

Day 728 continued follow-up, but AUC mean C-peptide, flow cytometry, and anti-drug

antibodies were no longer measured, to reduce the burden on participants and cost. Anti-CMV

IgG, anti-EBV IgG, and IgM titers were measured at screening and Days 28, 91, 140, 210, 273,

364, and 728 to evaluate seropositivity for Epstein-Barr virus (EBV) and cytomegalovirus

(CMV); semi-quantitative PCR was used to measure viral load for seropositive patients.

Adverse events (including clinically significant hypoglycemia) and abnormal laboratory

values were reported by investigators, coded using the Medical Dictionary for Regulatory

Activities and graded using the Common Terminology Criteria for Adverse Events (version 3.0).

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Statistical Analysis

Changes from baseline for AUC mean C-peptide (a pre-specified secondary endpoint),

HbA1C, and other measures in teplizumab groups were compared to the placebo group using

mixed-model repeated measures analysis (MMRM) models, adjusted for age-group and baseline

values. A Mantel-Haenszel test stratified by age group (8-11, 12-17, and 18-35 years) or Fisher

Exact test was used for exploratory efficacy analyses of dichotomous outcomes. Two-sided

testing was done at an α level of 0.05. Subset analyses compared the 14-day full-dose regimen

with placebo; age groups, regions, and time from diagnosis to randomization were pre-specified

subsets (11). These analyses were done for hypothesis generation (because the primary outcome

was not significant at 1 year); therefore, we did not adjust for multiple comparisons. Similar

analyses were conducted for the other treatment groups but no meaningful findings were

observed, so the results are not presented.

The 1 year analysis employed a non-parametric analysis and reported median values for

AUC mean C-peptide because the distribution was not normal (11); a last observation carried

forward (LOCF) analysis was used at the request of regulators, as too few time points existed for

a longitudinal analysis at 1 year. For the current report at 2 years, longitudinal analysis

(MMRM) was employed instead of LOCF. AUC mean C-peptide change from baseline was

calculated using [ln(AUC mean C-peptideDay x + 1) - ln(AUC mean C-peptidebaseline + 1)]. The

adjusted mean values reported here reflect the logarithm values after adjustment for covariates

(listed above). Consequently, the adjusted means and statistical significance reported here differ

from the unadjusted medians and p-value reported earlier at 1 year (11). The overall mean of

insulin use was calculated for each group using all values after baseline.

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Safety and tolerability through Year 2 were assessed primarily by summarizing adverse

experiences, serious adverse experiences (life-threatening, death, persistent disability or

hospitalization) and adverse experiences of special interest (acute mononucleosis-like illness,

infection requiring intravenous antibiotic treatment, demyelinating disease, lymphoma or other

malignancy, clinically significant hypoglycemia requiring assistance, Grade 3 liver function

abnormalities, Grade 3 thrombocytopenia, Grade 3 neutropenia; through Year 1: rash, Grade 4

allergic/hypersensitivity, Grade 4 cytokine-release syndrome).

Results

A high proportion (90% overall) of randomized patients completed 2 years of follow-up

(Supplementary Table A). In the 14-day full-dose group at 2 years, 89% had HbA1C measured

and insulin therapy recorded, but only 64% had Year 2 AUC mean C-peptide measurements

because these were discontinued after final analysis of Year 1 data (see Methods) (Figure 1A).

Baseline characteristics, including diabetes measures (autoantibodies, C-peptide, insulin dose,

and HbA1C) were balanced across treatment groups, but not geographic regions. In particular,

patients in India had less frequent ICA512 autoantibodies, higher HbA1C, higher insulin use, and

lower AUC mean C-peptide, the latter suggesting more advanced disease on average than other

regions (11).

HbA1C. Intensive diabetes care with insulin was provided for all patients. There was no

significant difference in HbA1C change from baseline comparing the teplizumab and placebo

groups over the 2-year study at any time point (See Table 1), suggesting that glycemic control

was maintained to a comparable extent across treatment groups.

Efficacy measures during 2 years of follow-up

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AUC mean C-peptide. Teplizumab treatment (14-day full-dose) reduced the loss of AUC

mean C-peptide at 2 years versus placebo (p = 0.027) (Figure 1A, Table 1). The adjusted mean

differences in AUC mean C-peptide change from baseline at 2 years favored the 14-day full-dose

regimen versus placebo in analyses of all patients, patients in the US, and patients randomized

<6 weeks after diagnosis (Figures 1B, 1C, and 2, and Table 2). The results in these pre-specified

subsets suggested larger treatment effects in patients with characteristics consistent with less

advanced disease. Therefore, additional analyses were conducted to explore the treatment effects

in other patient subsets at entry defined by a) HbA1c<7.5%, the ADA recommendation for type 1

diabetes control in children aged 13-19 (16); b) insulin use <0.4 U/kg/d, the lower limit of typical

type 1 diabetes insulin needs (17); c) AUC mean C-peptide>0.65nmol/L, the mean at baseline

(11), and d) AUC mean C-peptide>0.2 nmol/L, a value including ≥90% of newly diagnosed

patients and comparable (18) to an amount of insulin reserve thought to be clinically beneficial

(19).

Importantly, patients randomized <6 weeks after diagnosis had the largest treatment

difference versus placebo, among the baseline subsets examined (Table 2, Figure 2). Subsets

with US residence, HbA1c <7.5%, insulin use <0.4U/kg/day, AUC mean C-peptide >0.65 nmol/L

at entry, or AUC mean C-peptide >0.2 nmol/L also had much larger differences versus placebo

compared to subsets of patients from India, higher HbA1c, higher insulin use, and lower C-

peptide, respectively. Of note, for patients in the US and India, mean baseline HbA1c was 7.6%

(60 mmol/mol) and 9.7% (83 mmol/mol), insulin use was 0.47 and 0.98 U/kg/day, and AUC

mean C-peptide was 0.77 and 0.53 nmol/L, respectively (11).

Age groups were also pre-specified for analyses, served as an enrollment stratification

criterion, and served as an adjustment covariate in analyses. While the difference versus placebo

was small and not statistically significant in 18-35 year olds, treatment effects were larger in the

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8-11 and 12-17 year-olds and these groups were combined (Table 2). In the combined 8-17 year-

old subset, differences in AUC mean C-peptide change from baseline favored the 14-day full-

dose versus placebo group (p<0.05 at 1 year and all subsequent time points; Figure 1D, Figure 2,

and Table 2). Among age subsets, a large difference versus placebo was seen in ages 8-11, but

the p-value was not significant until combined with ages 12-17, perhaps due to the smaller

number of patients in the youngest group.

Insulin Use. After an initial decline from baseline, adjusted mean insulin use increased

progressively over time (Supplementary Figure A, Table 1). It was pre-specified to look at the

largest countries in the trial (US and India), and there were important regional differences in

insulin use, HbA1c and C-peptide at study entry, as described above. The overall adjusted mean

insulin use (U/kg/day) at all times after baseline in the 14-day full-dose versus placebo groups

was 0.59 versus 0.62 for all patients (not significant), and 0.44 versus 0.50 (p=0.02) for US

patients (data not shown). For individual time points, the difference versus placebo was

statistically significant at Day 448 in US patients (Supplementary Figure A). Compared to

placebo, a greater proportion of patients in the 14-day full-dose group met the modified

composite endpoint (HbA1c <7% (53 mmol/mol) and insulin use <0.25 U/kg/day), and the

differences were statistically significant at days 91, 273, 364, and 616 (Supplementary Figure B).

Despite being blind to treatment, at 1 year, 5.3% (11/207) of patients in the 14-day full-dose

group were not taking insulin, compared with no patients (0/98) in the placebo group (p=0.02).

At Year 2, three of these 11 patients remained off insulin, whereas all placebo patients were still

taking insulin (p>0.05).

Teplizumab pharmacokinetics, immunogenicity, and effects on T-cells

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Higher levels of anti-teplizumab (antidrug) antibodies were seen in cycle 2 than cycle 1 for

all three teplizumab regimens (Supplementary Table B). For typical patients in the 14-day full-

dose group who did not make antidrug antibodies, teplizumab levels peaked on Day 14 with a

Cmin and Cmax (mean±SD) of 418±225 ng/mL and 826±391 ng/mL, respectively. However,

teplizumab clearance increased with maximum observed antidrug antibody concentrations, and

some patients demonstrated a strong antidrug antibody response after about 10 days of cycle 2

dosing, with an abrupt reduction of bioavailability and increase in drug clearance

(Supplementary Figure C). There did not appear to be a meaningful correlation between antidrug

antibody levels and AUC mean C-peptide changes from baseline (Supplementary Table C), nor

with response using the modified composite HbA1C + insulin usage endpoint (not shown).

Additional details are in Supplementary Materials.

Circulating levels of CD4+ and CD8+ T-cells were transiently reduced during each cycle of

treatment, but not in the placebo group (Figure 3A). The effects of teplizumab on CD4+ T-cell

appeared to diminish at antidrug antibody levels >5000 ng/mL (Supplementary Figure D). This

level was observed in 19% of patients in the 14-day full-dose group after the second course of

drug (Supplementary Table B). During treatment, teplizumab was transiently bound to CD3

molecules on surfaces of both CD4+ and CD8+ T-cells (Figure 3B). There was some evidence

of down-modulation (Figure 3C) and an increase in the percentage of circulating Foxp3-positive

CD8+ (but not CD4+) T-cells during teplizumab dosing (Figure 3D).

Safety and Tolerability

There were no differences in adverse events or serious adverse events between groups at year

2 (Table 3). While grade 3 adverse events were increased in teplizumab groups, this difference

versus placebo was primarily due to lymphopenia, an expected consequence of the mechanism of

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action. In particular, there were no apparent differences between groups in the incidence of

infections overall, or by specific types, with the possible exception of herpes zoster (ten

teplizumab patients versus no placebo patients, all non-serious) (Table 4). Information on prior

history was incomplete, but there was no convincing evidence of prior history of varicella or

varicella vaccination in any of the patients who reported herpes zoster (age range, 12-34 years).

Three events occurred within 28 days of starting cycle 1 of treatment, while 5 occurred after 270

days when drug is no longer detectable in the circulation. Other herpes virus infections including

CMV and EBV did not appear to increase in frequency during the 2 years of the trial. The most

common infection was upper respiratory infection (16.3% of placebo versus 15.5% of 14-day

full-dose patients) which did not differ appreciably between teplizumab groups and placebo

(Table 4). As expected, there were no rashes or cytokine release events during the second year,

because drug was not administered during this period.

Discussion

In this report of data from the complete Protégé trial, patients with new-onset type 1 diabetes

who received a full course of teplizumab (14-day full-dose) had significant improvement in

stimulated C-peptide responses compared to placebo-treated subjects (p=0.027). This effect was

strongest in particular subsets including children, those randomized <6 weeks following

diagnosis, and US participants. As reported earlier, no significant differences were observed

between the teplizumab and placebo treatment groups using a previously unvalidated primary

composite endpoint (insulin <0.5 U/kg/day and HbA1C <6.5% at Year 1). Although investigators

were instructed to treat aggressively to HbA1c below 6.5% and to maintain insulin >0.25

U/kg/day, this may have been unrealistic given that, after a nadir of ~6.9% (52 mmol/mol) at 90

days, mean HbA1c increased to ~7.9% (63 mmol/mol) at Year 1 (11) and 8.4% (68 mmol/mol) at

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Year 2. Further, C-peptide may be a more objective and reliable outcome than insulin use and

HbA1C, because it is a more direct indicator of endogenous insulin secretion and cannot be easily

measured or manipulated by patients or their physicians. AUC mean C-peptide is now the most

widely used endpoint for type 1 diabetes interventions, and is accepted by the FDA as a primary

endpoint for these trials (6,8, 20-22).

The AUC mean C-peptide treatment difference versus placebo did not appear to change

markedly during the second year of follow-up (Figure 1A), although the study was not designed

to test hypotheses regarding time-trends. A particularly strong treatment effect was found in

patient subsets who share characteristics of early type 1 diabetes (treatment sooner after

diagnosis, lower baseline insulin use, greater C-peptide, and lower HbA1c at baseline). The

higher baseline HbA1c and lower C-peptide levels suggest the patients in India had more

advanced disease. Larger treatment effects on AUC mean C-peptide were also observed in ages

8-17 years who have a more rapid C-peptide decline, on average, compared to adults.

The modified composite endpoint (insulin <0.25 U/kg/day and HbA1C <7.0%) and insulin

use also suggested a treatment benefit on insulin use. In the US, the overall insulin use was less

in the teplizumab-treated subjects compared to those receiving placebo. This trend was not seen

when subjects outside the US were included, perhaps reflecting different patterns of insulin use

in other countries. Together, the results suggest teplizumab treatment preserves endogenous

insulin, thereby reducing needs for exogenous insulin to maintain glycemic control.

Immunotherapy must meet a high safety standard because clinical type 1 diabetes can be

managed using insulin. However, good metabolic control is often difficult to achieve safely with

insulin; a recent study of 25,833 type 1 diabetes patients revealed that 7% of patients reported

severe hypoglycemic events (seizure or coma) and 8% reported diabetic ketoacidosis in the prior

12 months (23). High doses of anti-CD3 immunotherapy are associated with tolerability/toxicity

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issues (9,24), whereas low doses appear to be ineffective (11,25). One phase 2 trial of

teplizumab (9) used a high dose (37 mg total per course per 1.9m2 subject) and observed a high

(28%) incidence of grade 2 or greater adverse events associated with infusion (primarily fever,

nausea, vomiting and rigors), whereas the incidence was only 6% in an earlier trial (6). The

Protégé trial used a dose of 17 mg total per course (for a 1.9m2 patient), comparable with that

used in the earlier trial (6), and experienced similar excellent tolerability. Conversely, very low

dose otelixizumab (another non-activating Fc-modified anti-CD3 monoclonal antibody) dosed at

3.1 mg over 8 days did not preserve ß-cell function in a double-blinded phase 3 study (25), and

the Protégé treatment arms with lower cumulative dose were also ineffective (11). Overall, the

14-day full-dose regimen of Protégé appears to provide sufficient drug to influence efficacy

measures, with acceptable tolerability and safety.

Treatment-related adverse experiences were mostly limited to the dosing period and

generally resolved within 14 days (11). Most (transient cytopenias, transient mild laboratory or

clinical manifestations of cytokine release such as rash, headache, nausea, and vomiting) were

moderate, manageable, and expected as a manifestation of the intended mechanism of action.

Along with transient, small increases in aminotransferases, these also represented the main

differences versus placebo in Year 1 safety analyses (11). Use of effective stopping rules (based

on liver function tests to delimit cytokine release syndrome) served to lessen adverse events

compared to earlier studies, allowing 90.6% of treated patients to complete a full course of drug

(11).

The observed reduction in circulating CD4+ and CD8+ cells likely reflects both transient

margination of the T-cell compartment and apoptosis of some activated T-cell subsets. Both may

be relevant mechanisms of action of teplizumab, wherein the T-effector cells that are maintaining

an inflammatory environment in the pancreas are preferentially depleted while regulatory T-cells

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are favored. Flow cytometry analysis of peripheral blood in the treated Protégé patients

suggested that there may be an increase in Foxp3 expression in CD8+ but not CD4+ T-cells

during periods of maximum drug binding to T-cells. Previous studies have shown that

teplizumab induces activation of CD8+ T-cells with regulatory function (26,27). In addition,

CD4+ and CD8+ cells are directed to the lamina propria where they appear to acquire regulatory

function, although cell deletion may also be involved in the drug action.

Longer-term changes in patient immune function, such as persistent low CD4 counts (9) and

reactivation of EBV infection, were reported previously from studies that used much higher anti-

CD3 doses (28). At the lower doses used in Protégé, EBV reactivation was rare and acute

mononucleosis syndrome was not increased versus placebo. A possible dose-related increase in

herpes zoster was seen, with ten cases (that could not be subsequently confirmed) reported

among teplizumab patients; no cases occurred in the placebo group. Of note, in a subsequent

phase 3 double-blind randomized study (n=254) with identical teplizumab dosing

(NCT00920582), after 2 years of follow-up the only patient with herpes zoster was a placebo

patient (data on file).

To be meaningful, treatment effects must be maintained for multiple years. Repeated dosing

might be advantageous if it increases durability without causing new or cumulative side effects.

Protégé did not include an arm with a single drug cycle and cannot answer whether two drug

cycles confer greater benefit or duration than a single cycle. Nonetheless, Protégé did not

identify any cumulative, persistent, or unexpected safety or tolerability issues. Although high

levels of antidrug antibodies occurred late in the second cycle in about one-sixth of all patients

and appeared to accelerate drug clearance, this did not appear to affect efficacy endpoints.

The large number and diverse characteristics of Protégé patients enables more precise

estimates of treatment effects than smaller trials, increases generalizability, and allows for

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meaningful subset analyses. The 2-year follow-up provides evaluation of efficacy and safety

during placebo-controlled double-blind conditions for a longer period than previous trials. The

double-blind design reduces the potential for bias. Limitations of the study include the

heterogeneous baseline patient disease status, post-hoc analyses without adjustment for multiple

comparisons, and elimination of AUC mean C-peptide measurements in some patients after the

primary analysis at Year 1, which may have reduced statistical power. Another limitation is the

lack of information on HLA-DQ/DR or other genotypes that might identify patient subsets with

greater response.

Rodent studies reported full reversal of diabetes using anti-CD3 immunotherapy, but only

when given immediately at disease onset (29,30). In clinical trials, delays due to required

screening and enrollment procedures may lead to lower drug efficacy. In actual clinical settings,

immunotherapy could be initiated promptly at the time of diagnosis. Further, the peak incidence

of diabetes occurs in 8-11 year olds, and ages 8-17 appeared to have a greater drug response than

older patients.

In summary, continued follow-up for a second year demonstrated a benefit of teplizumab

treatment on AUC mean C-peptide and a possible benefit on insulin needs. Most importantly,

these analyses identified baseline characteristics associated with greater treatment efficacy. No

new safety or tolerability issues emerged. These post-hoc findings are hypothesis-generating and

confirmation is needed; nevertheless, they suggest that future studies of CD3 immunotherapy

should consider recruiting young patients with better glucose control and greater remaining

endogenous insulin secretion, and initiating treatment immediately upon diagnosis.

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Acknowledgments

This research was supported by Macrogenics, Inc., Rockville, Maryland. D.C., E.B., S.J., S.K.,

and A.G.D. are full-time employees and K.E.S. is a part-time employee of Macrogenics, Inc. and

have stock options as part of an employee offering program. W.H., R.J.F., N.S., and J.L. received

research support for this study from MacroGenics. J.L. has received advisory board support from

Johnson & Johnson. R.J.F. has received research support within the past 3 years from Tolerx (to

study otelixizumab for recent-onset type 1 diabetes); unrelated research support from the US

National Institutes of Health (grants R21 HD059292, T35 DK007405, and U01 DK085465),

Juvenile Diabetes Research Foundation (grant 2011-597), Gabrielle’s Angel Foundation, Eli

Lilly, Diamyd, Pfizer, Bristol-Myers Squibb, Takeda, and Novo Nordisk; and unrestricted

research support from Le Bonheur Foundation (Memphis TN, USA). W.H. chaired the data

safety and monitoring board for the BHT-3021-01 insulin plasmid trial (Bayhill

Pharmaceuticals). K.C.H. has received research support from MacroGenics, and support from the

Juvenile Diabetes Research Foundation (grant 2008-502) for laboratory studies on patients’

samples from Protégé.

E.B., S.J., K.E.S., K.C.H., S.K., and J.L. contributed to the study design. All authors

contributed to study implementation and supervision of data collection at the sites. A.G.D., D.C.,

W.H., K.C.H., and N.S. contributed to planning of protocol-stated analyses and post-hoc

analyses. D.C. designed and did the statistical analysis and verified its accuracy. D.C. and

A.G.D. contributed to compiling the official clinical study report. All authors had full access to

the data, helped draft the report or critically revise the draft, contributed to data interpretation,

and reviewed and approved the final version of the report. W.H. and J.L. are guarantors of this

work. As such, they had full access to all the data, and they take full responsibility for the

integrity of the data and its analysis.

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Parts of this study were presented in abstract form at the 71st Scientific Sessions of the

American Diabetes Association (San Diego, CA, June 24-28, 2011), the 47th European

Association for the Study of Diabetes (Lisbon, Portugal, September 12-16, 2011), and the 48th

European Association for the Study of Diabetes (Berlin, Germany, October 1-5, 2012). The

authors thank Philip Ross, MedStrat Communications, for editorial assistance in preparing this

manuscript with support from Macrogenics, Inc.

References

1. Steele C, Hagopian WA, Gitelman S, Masharani U, Cavaghan M, Rother KI, Donaldson

D, Harlan DM, Bluestone J, Herold KC. Insulin secretion in type 1 diabetes. Diabetes

2004;53:426-433.

2. Binder C, Faber OK. Residual beta-cell function and its metabolic consequences.

Diabetes 1978;27 (Suppl 1):226-229

3. Steffes MW, Sibley S, Jackson M, Thomas W. Beta-cell function and the development of

diabetes-related complications in the diabetes control and complications trial. Diabetes

Care 2003;26:832-836

4. Madsbad S, Alberti KG, Binder C, Burrin JM, Faber OK, Krarup T, Regeur L. Role of

residual insulin secretion in protecting against ketoacidosis in insulin-dependent diabetes.

Br Med J 1979;2:1257-1259

5. Sherry NA, Kushner JA, Glandt M, Kitamura T, Brillantes AM, Herold KC. Effects of

autoimmunity and immune therapy on beta-cell turnover in type 1 diabetes. Diabetes

2006;55:3238-3245.

6. Herold KC, Hagopian WA, Auger JA, Poumian-Ruiz E, Taylor L, Donaldson D,

Gitelman SE, Harlan DM, Xu D, Zivin RA, Bluestone JA. Anti-CD3 monoclonal

antibody in new-onset type 1 diabetes mellitus. N Engl J Med 2002;346:1692-1698.

7. Pescovitz MD, Greenbaum CJ, Krause-Steinrauf H, Becker DJ, Gitelman SE, Goland R,

Gottlieb PA, Marks JB, McGee PF, Moran AM, Raskin P, Rodriguez H, Schatz DA,

Wherrett D, Wilson DM, Lachin JM, Skyler JS; Type 1 Diabetes TrialNet Anti-CD20

Study Group. Rituximab, B-lymphocyte depletion, and preservation of beta-cell function.

N Engl J Med 2009;361:2143-2152

8. Buzzetti R, Cernea S, Petrone A, Capizzi M, Spoletini M, Zampetti S, Guglielmi C,

Venditti C, Pozzilli P; DiaPep Trialists Group. C-peptide response and HLA genotypes in

subjects with recent-onset type 1 diabetes after immunotherapy with DiaPep277: an

exploratory study. Diabetes 2011;60:3067-3072

of 38 Diabetes

18

9. Herold KC, Gitelman S, Greenbaum C, Puck J, Hagopian W, Gottlieb P, Sayre P,

Bianchine P, Wong E, Seyfert-Margolis V, Bourcier K, Bluestone JA; Immune Tolerance

Network ITN007AI Study Group. Treatment of patients with new onset Type 1 diabetes

with a single course of anti-CD3 mAb Teplizumab preserves insulin production for up to

5 years. Clin Immunol 2009;132:166-173

10. Herold KC, Gitelman SE, Masharani U, Hagopian W, Bisikirska B, Donaldson D, Rother

K, Diamond B, Harlan DM, Bluestone JA. A single course of anti-CD3 monoclonal

antibody hOKT3gamma1(Ala-Ala) results in improvement in C-peptide responses and

clinical parameters for at least 2 years after onset of type 1 diabetes. Diabetes

2005;54:1763-1769

11. Sherry N, Hagopian W, Ludvigsson J, Jain SM, Wahlen J, Ferry RJ Jr, Bode B, Aronoff

S, Holland C, Carlin D, King KL, Wilder RL, Pillemer S, Bonvini E, Johnson S, Stein

KE, Koenig S, Herold KC, Daifotis AG; Protégé Trial Investigators. Teplizumab for

treatment of type 1 diabetes (Protégé study): 1-year results from a randomised, placebo-

controlled trial. Lancet 2011;378:487-497

12. Lebastchi J, Deng S, Lebastchi AM, Beshar I, Gitelman S, Willi S, Gottlieb P, Akirav E,

Bluestone JA, Herold KC. Immune therapy and beta cell death in type 1 diabetes.

Diabetes (in press)

13. Ablamunits V, Sherry NA, Kushner JA, Herold KC. Autoimmunity and beta cell

regeneration in mouse and human type 1 diabetes: the peace is not enough. Ann N Y

Acad Sci 2007;1103:19-32

14. Greenbaum CJ, Beam CA, Boulware D, Gitelman SE, Gottlieb PA, Herold KC, Lachin

JM, McGee P, Palmer JP, Pescovitz MD, Krause-Steinrauf H, Skyler JS, Sosenko JM;

Type 1 Diabetes TrialNet Study Group. Fall in C-peptide during first 2 years from

diagnosis: evidence of at least two distinct phases from composite Type 1 Diabetes

TrialNet data. Diabetes 2012;61:2066-2073

15. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diab

Care 2006; 29 (Suppl 1):S43-S48

16. ADA. Standards of Medical Care in Diabetes—2011. Diabetes Care 2011;34(Suppl

1):S11-S61.

17. Hirsch IB, Skyler JS. Chapter 17 – The Management of Type 1 Diabetes. Updated 2009.

At: http://www.endotext.org/diabetes/diabetes17/diabetesframe17.htm Accessed on

November 5, 2012.

18. Besser R, Shields B, Casas R, Hattersley A, Ludvigsson J. Lessons from the Mixed Meal

Tolerance Test. Diabetes Care 2013;36:195-201

19. Diabetes Control and Complication Research Group. Effect of intensive therapy on

residual beta cell function in patients with Type 1 diabetes in the Diabetes Control and

Complications Trial. Ann Int Med 1998;128 517-523

20. Ludvigsson J, Faresjö M, Hjorth M, Axelsson S, Chéramy M, Pihl M, Vaarala O,

Forsander G, Ivarsson S, Johansson C, Lindh A, Nilsson NO, Aman J, Ortqvist E,

of 38Diabetes

19

Zerhouni P, Casas R. GAD treatment and insulin secretion in recent-onset type 1

diabetes. N Engl J Med 2008;359:1909-1920

21. Mandrup-Poulsen T. IAPP boosts islet macrophage IL-1 in type 2 diabetes. Nat Immunol

2010;11:881-883

22. Ludvigsson J, Krisky D, Casas R, Battelino T, Castaño L, Greening J, Kordonouri O,

Otonkoski T, Pozzilli P, Robert JJ, Veeze HJ, Palmer J, Samuelsson U, Elding Larsson

H, Åman J, Kärdell G, Neiderud Helsingborg J, Lundström G, Albinsson E, Carlsson A,

Nordvall M, Fors H, Arvidsson CG, Edvardson S, Hanås R, Larsson K, Rathsman B,

Forsgren H, Desaix H, Forsander G, Nilsson NÖ, Åkesson CG, Keskinen P, Veijola R,

Talvitie T, Raile K, Kapellen T, Burger W, Neu A, Engelsberger I, Heidtmann B,

Bechtold S, Leslie D, Chiarelli F, Cicognani A, Chiumello G, Cerutti F, Zuccotti GV,

Gomez Gila A, Rica I, Barrio R, Clemente M, López Garcia MJ, Rodriguez M, Gonzalez

I, Lopez JP, Oyarzabal M, Reeser HM, Nuboer R, Stouthart P, Bratina N, Bratanic N, de

Kerdanet M, Weill J, Ser N, Barat P, Bertrand AM, Carel JC, Reynaud R, Coutant R,

Baron S. GAD65 antigen therapy in recently diagnosed type 1 diabetes mellitus. N Engl J

Med 2012;366:433-442

23. Beck RW, Tamborlane WV, Bergenstal RM, Miller KM, Dubose SN, Hall CA; for the

T1D Exchange Clinic Network. The T1D Exchange Clinic Registry. J Clin Endocrinol

Metab 2012;97:4383-4389

24. Keymeulen B, Vandemeulebroucke E, Ziegler AG, Mathieu C, Kaufman L, Hale G,

Gorus F, Goldman M, Walter M, Candon S, Schandene L, Crenier L, De Block C,

Seigneurin JM, De Pauw P, Pierard D, Weets I, Rebello P, Bird P, Berrie E, Frewin M,

Waldmann H, Bach JF, Pipeleers D, Chatenoud L. Insulin needs after CD3-antibody

therapy in new-onset type 1 diabetes. N Engl J Med 2005;352:2598-2608

25. Sprangers B, Van der Schueren B, Gillard P, Mathieu C. Otelixizumab in the treatment of

type 1 diabetes mellitus. Immunotherapy 2011;3:1303-1316

26. Bisikirska B, Colgan J, Luban J, Bluestone JA, Herold KC. TCR stimulation with

modified anti-CD3 mAb expands CD8+ T cell population and induces CD8+CD25+

Tregs. J Clin Invest 2005;115:2904-2913

27. Ablamunits V, Bisikirska B, Herold KC. Acquisition of regulatory function by human

CD8(+) T cells treated with anti-CD3 antibody requires TNF. Eur J Immunol

2010;40:2891-2901

28. Keymeulen B, Candon S, Fafi-Kremer S, Ziegler A, Leruez-Ville M, Mathieu C,

Vandemeulebroucke E, Walter M, Crenier L, Thervet E, Legendre C, Pierard D, Hale G,

Waldmann H, Bach JF, Seigneurin JM, Pipeleers D, Chatenoud L. Transient Epstein-Barr

virus reactivation in CD3 monoclonal antibody-treated patients. Blood 2010;115:1145-

1155

29. Chatenoud L, Thervet E, Primo J, Bach JF. Anti-CD3 antibody induces long-term

remission of overt autoimmunity in nonobese diabetic mice. Proc Natl Acad Sci USA

1994;91:123-127

of 38 Diabetes

20

30. Chatenoud L, Primo J, Bach J-F. CD3 Antibody-Induced Dominant Self-Tolerance in

Overtly Diabetic NOD Mice. J Immunol 1997;158:2947-2954

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Table 1. Outcomes at Year 2.

Outcome

14-day full-

dose (n=207)

14-day low-

dose

(n=102)

6-day full-

dose (n=106)

Placebo

(n=98)

*Adjusted mean change in AUC of C-peptide from baselinea

-0.136 p=0.027

-0.198 p=0.968

-0.174 p=0.312

-0.191

*Composite of insulin dose <0.5 U/kg/day and HbA1C <6.5%b (N (%))

17 (8.2%) p=0.775

6 (5.9%) p=0.402

10 (9.4%) p= 0.859

9 (9.2%)

Composite of insulin dose <0.25 U/kg/ day and HbA1C <7.0%b (N (%))

11 (5.3%) p=0.070

4 (3.9%) p= 0.183

3 (2.8%) p= 0.339

1 (1.0%)

*Adjusted mean change in HbA1C from baselinea (%)

0.233 p= 0.706

0.220 p= 0.868

0.149 p= 0.606

0.135

Adjusted mean change in insulin use from baselinea (U/kg/day)

0.067 p= 0.963

0.010 p= 0.142

0.105 p= 0.861

0.070

Sample sizes shown are at baseline – numbers of patients at each time point are shown in Figure 1A for AUC mean C-peptide and Supplementary Figure A for insulin use. *Pre-specified endpoints at Year 1. aAdjusted mean changes from baseline were calculated using MMRM models adjusted for age-group and baseline values; adjusted means for placebo group were calculated using the placebo vs. 14-day full-dose models. AUC mean C-peptide change from baseline was calculated by: [ln(AUC mean C-peptideDay x + 1) - ln(AUC mean C-peptidebaseline + 1)]. bp-values were calculated using a Mantel-Haenszel test stratified by age group (8-11, 12-17, and 18-35).

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Table 2. Adjusted mean change from baseline at Year 2 for AUC mean C-peptide in the 14-day full-dose and placebo groups, by characteristics at study entry.

Baseline Subset Totala

patients at

baseline N

14-day full-

dose

Placebo Adjusted

mean

difference

p-value

USA 95 -0.169 -0.289 -0.120 0.01

India 85 -0.102 -0.147 -0.045 0.28

Diagnosed <6 weeks 67 -0.068 -0.211 -0.142 0.006

Diagnosed >6 Weeks 238 -0.155 -0.189 -0.034 0.23

HbA1c <7.5% 124 -0.153 -0.248 -0.095 0.024

HbA1c >7.5% 181 -0.123 -0.157 -0.034 0.28

Insulin <0.4U/kg/day 74 -0.161 -0.282 -0.121 0.034

Insulin >0.4U/kg/day 231 -0.122 -0.169 -0.047 0.1

AUC mean C-peptide

<0.65nmol/L

185 -0.104 -0.113 -0.009 0.73

AUC mean C-peptide

>0.65nmol/L

120 -0.176 -0.286 -0.111 0.02

AUC mean C-peptide

<0.2nmol/L

31 -0.038 -0.064 -0.025 0.52

AUC mean C-peptide

>0.2nmol/L

274 -0.149 -0.207 -0.058 0.036

Ages 8-11 years 46 -0.155 -0.264 -0.109 0.15

Ages 12-17 years 119 -0.157 -0.217 -0.060 0.12

Ages 8-17 years 165 -0.156 -0.230 -0.075 0.031

Ages 18-35 years 140 -0.103 -0.142 -0.039 0.28 aTotal in the 14-day full-dose and placebo groups combined; overall N=305. All analyses were MMRM; p-values are for treatment effect from ANCOVA models. Sample sizes are from baseline measurements; see Figure 1 for sample sizes at each time point. ~66% of all patients (~48% of US patients) had AUC mean C-peptide at Year 2, because measurements were discontinued after analysis of Year 1 data. C-peptide change of AUC from study entry was calculated using [ln(AUC mean C-peptideDay x + 1) - ln(AUC mean C-peptideBaseline + 1)].

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Table 3. Adverse events in the safety population in the complete 2-year study. Adverse Event 14-day full-

dose (n=207)

14-day low-

dose

(n=102)

6-day full-

dose (n=106)

Placebo

(n=98)

Any Adverse event 207 (100%) 101 (99.0%) 105 (99.1%) 98 (100%) Adverse event leading to drug withdrawal

35 (16.9%) 12 (11.8%) 17 (16.0%) 5 (5.1%)

Adverse event leading to study discontinuation

3 (1.4%) 1 (1.0%) 0 0

Grade 3 or higher adverse event

135 (65.2%) 55 (53.9%) 71 (67.0%) 28 (28.6%)

Serious adverse event 23 (11.1%) 14 (13.7%) 12 (11.3%) 12 (12.2%) Deaths 1 (0.5%) 1 (1.0%) 0 0

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Table 4. Incidence of infections in the complete 2-year study.

14-day

full-dose

(n=207)

14-day

low-dose

(n=102)

6-day

full-dose

(n=106)

Placebo

(n=98)

All infections 48.3% 48.0% 50.9% 58.2%

Respiratory infection 15.5% 20.6% 22.6% 16.3%

Acute mono-like illness

7.7% 4.9% 3.8% 8.2%

Herpes (all) 8.7% 8.8% 6.6% 8.2%

Herpes zoster 3.4% 1.0% 1.9% 0%

Mononucleosis 1.4% 0% 0.9% 1.0%

Tuberculosis 0% 1.0% 0% 1.0%

Herpes zoster cases were presumed but not confirmed; subjects with herpes zoster were asked retrospectively, after study completion, to provide data on their history of chicken pox, herpes zoster, or prior varicella vaccination. The information provided was incomplete (eg, not all subjects responded to the request and data for other subjects were provided by family members and not confirmed by a health care professional). The data received supported no prior history of varicella or vaccination in any of the subjects who were reported to have herpes zoster.

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Figure Legends

Figure 1. Adjusted mean changes in AUC mean C-peptide from baseline over time in the 14-day full-dose and placebo groups. Bars indicate standard errors; number of patients are above (teplizumab) or below (placebo). P-values are indicated where significant. Changes in AUC mean C-peptide from baseline were calculated using [ln(AUC mean C-peptideDay x + 1) - ln(AUC mean C-peptideBaseline + 1)]. A) All patients. B) Patients in the US. C) Patients randomized <6 weeks after diagnosis. D) Ages 8-17 years. Figure 2. Adjusted mean difference in AUC mean C-peptide change from baseline at 2 years among subsets at study entry; 14-day full-dose group versus placebo. Diamond symbols indicate least squares means; bars indicate 95% confidence intervals. AUC mean C-peptide change from baseline was calculated using [ln(AUC mean C-peptideDay x + 1) - ln(AUC mean C-peptideBaseline + 1)]. Figure 3. Flow cytometry for CD4+ (LEFT) and CD8+ T-cells (RIGHT). A) Cell counts. B) CD3 occupancy/cell. C) CD3/TCR (T-cell receptor) modulation on cells. D) Percent of cells positive for Foxp3 marker. Symbols indicate means, and bars indicate standard errors; open circles = teplizumab 14-day full-dose, closed squares = placebo. A & C; number of patients are above (placebo) or below (teplizumab). B & D; number of patients are above (teplizumab) or below (placebo).

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Figure 1

64 62

53

48

30

30

30

26

25

14

-0.35

-0.3

-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05

0 100 200 300 400 500 600 700 800

Ad

just

ed

me

an c

han

ge in

AU

C m

ean

C

-pe

pti

de

fro

m b

ase

line

Study Day

B. U.S. Patients

14-day full-dose

Placebo

p=0.050

p=0.010

p=0.010

207

200

188

173

131

98 95

91

84

64

-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05

0.1

0 100 200 300 400 500 600 700 800

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just

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AU

C m

ean

C

-pe

pti

de

fro

m b

ase

line

Study Day

A. All Patients

14-day full-dose

Placebo

p=0.036

p=0.027

46

44

44

39

34 21 20

20

17

14

-0.3

-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05

0.1

0.15

0.2

0 100 200 300 400 500 600 700 800

Ad

just

ed

me

an c

han

ge in

AU

C m

ean

C

-pe

pti

de

fro

m b

ase

line

Study Day

C. Randomized <6 weeks

14-day full-dose

Placebo

p=0.045

p=0.005

p=0.006

p=0.034 112

107

100

91

69

53 52

48

46

32

-0.3

-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05

0.1

0 100 200 300 400 500 600 700 800

Ad

just

ed

me

an c

han

ge in

AU

C m

ean

C

-pe

pti

de

fro

m b

ase

line

Study Day

D. Ages 8-17 years

14-day full-dose

Placebo

p=0.014

p=0.024

p=0.031

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Figure 2

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Figure 3

200

192

197

192 196

186

166

190

186 183 193 178 123

94 90 95 98 95 90

84 89

89

91 93

83 59

0

200

400

600

800

1000

1200

0 100 200 300 400 500 600 700 800

CD

4+

T-ce

ll co

un

ts (

cells

/mm

3)

Study Day

A. CD4+

14-day full-dose

Placebo

200

192

197

193 196

186

166

190

187 183 193 177 123

94

90 95 98

95 90

84 89

89

91 93

82 59

0

100

200

300

400

500

600

700

800

0 100 200 300 400 500 600 700 800

CD

8+

T-ce

ll co

un

ts (

cells

/mm

3)

Study Day

A. CD8+

14-day full-dose

Placebo

34

37

37 35 34

32

31

28

29 31 27 18

16 17 18 17 12 13 15 15 13 16 14 9

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0 100 200 300 400 500 600 700 800

CD

3 O

ccu

pan

cy/C

D4

+ T

Ce

lls

Study Day

B. CD4+

14-day full-dose

Placebo

34

37

37 35 34

32

31

28

29 31 27 18

16 17 18 17 12 13 15 15 13 16 14 9 -0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 100 200 300 400 500 600 700 800

CD

3 O

ccu

pan

cy/C

D8

+ T

Ce

lls

Study Day

B. CD8+

14-day full-dose

Placebo

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Figure 3

34 38

37 35

34

32

31

28 29

31 27 18

16 17

18 17

12

13 15 15 13

16 14 9

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 100 200 300 400 500 600 700 800

CD

3/T

CR

Mo

du

lati

on

on

CD

4+

T C

ells

Study Day

C. CD4+

14-day full-dose

Placebo

42

36

39 40 39

38

36

37

33 33 33

32

31 22

17

20 20

20

14

16 17

17

15

17 15

13

0

1

2

3

4

5

6

7

8

0 100 200 300 400 500 600 700 800

CD

4+F

oxP

3+

Ce

lls (

%)

Study Day

D. CD4+

14-day full-dose

Placebo

43

37

40

40

39 38

36

37 33 33

33 32 31

21 17 19 20

20 14 16 17 17 14 17 15 13 0

0.5

1

1.5

2

2.5

3

3.5

4

0 100 200 300 400 500 600 700 800

CD

8+F

oxP

3+

Ce

lls (

%)

Study Day

D. CD8+

14-day full-dose

Placebo

37 39

39 37

35

33 32

30

33 30

20

16 17

18 17

12

13 15

15

16 14

9

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 100 200 300 400 500 600 700 800

CD

3/T

CR

Mo

du

lati

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on

CD

8+

T C

ells

Study Day

C. CD8+

14-day full-dose

Placebo

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1

Supplementary Materials (online only)

Laboratory measurements

Mixed-meal tolerance tests (MMTT) were performed by measuring C-peptide and glucose

levels in blood samples using a validated chemiluminescent assay on the Immulite® 2000

(Siemens Healthcare Diagnostics Inc., Tarrytown, NY) at pre-specified time points before and

after the meal. The C-peptide lower limit of quantitation was 0.03 nmol/L; the lower limit was

0.17 nmol/L for North America sites before July 2007 and other sites before January 2008.

HbA1C was measured by immunologic method on Roche Modular or Cobas® Integra 400, both

of which were highly correlated (r = 0.9922). Bio-Rad Liquichek™ Diabetes Control Level 1

and 3 were used to monitor assay quality. All glucose, C-peptide, and autoantibody assays were

done in a licensed central laboratory (Esoterix Clinical Trials Services). Validated quantitative

detection of Epstein Barr virus (EBV) DNA and Cytomegalovirus (CMV) DNA in plasma were

performed at a licensed central laboratory (ViroMed). EBV IgM and IgG and CMV IgG were

measured by Esoterix Clinical Trials Services.

Multicolor flow cytometry was performed on peripheral blood samples by LabCorp

(Brentwood, TN) from a subset of subjects (all 38 patients in a related open-label study (11), and

a subset (n=100) from the US sites in the double-blind portion; total n=138) before and after

(Days 6, 14, 28, 91,140, 546, and 728) study drug administration. Peripheral blood mononuclear

cells were prepared and frozen at -80°C until use. Thawed cells were stained appropriately, and a

Becton Dickinson FACSCalibur™ flow cytometer was used to acquire 75,000 total events. After

acquisition, the listmode files were analyzed offline using WinList 3.0™ to generate the

reportable data. Flow cytometry was also performed in the first 100 subjects randomized in

North America to determine if the CD3 epitope recognized by teplizumab had been bound (or

coated) off the surface of the T lymphocytes.

Pharmacokinetics

The pharmacokinetics of teplizumab following intravenous administration were best

described using a two-compartment model with saturable binding in central and peripheral

compartments. Clearance of teplizumab was estimated to be 2.3 L/day (33.1% coefficient of

variation [CV]). Central volume of distribution was 3.4 L, and the peripheral volume of

distribution was 6.9 L, resulting in a volume of distribution at steady state of 10.4 L. At

concentrations much higher than the binding capacity of the target (129 ng/mL), teplizumab

pharmacokinetics were characterized by bi-exponential decay with the distribution and terminal

half-lives of t1/2α=0.19 day (SD=0.22 day) and t1/2β = 4.01 days (SD = 1.75 days), respectively.

Clearance following 14-day low-dose and 6-day full-dose regimens was higher than following

the 14-day full-dose regimen.

For typical subjects, without anti-drug antibodies (ADA), administered the 14-day full-dose

regimen, the predicted mean (± SD) total AUC was 6421 ± 1940 ng•day/mL. The accumulation

ratio for AUC between Day 5 and Day 14 (the first and the last day with the full dose

administration) was 3.43 ± 1.16. With a BSA-proportional dose, teplizumab AUC, Cmin and Cmax

were independent of body weight. There was no evidence of clinically relevant dependencies of

pharmacokinetics on age, gender, race, region, baseline CD4+ and CD8+ cell counts, disease

state or disease onset time. The predicted concentration-time courses following the 14-day full-

dose regimen are illustrated in Supplementary Figure B.

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2

Teplizumab pharmacokinetics were influenced by the development of ADA in many

subjects. Strong immunogenicity (defined as an abrupt reduction of bioavailability and increase

in clearance after about 10 days of cycle 2 dosing) was estimated to occur in 47.5% (10%

relative standard error [RSE]) and 65.0% (13% RSE) of subjects administered the 14-day full-

dose and 14-day low-dose regimens, respectively. The immunogenic reaction was assumed to be

developed after 10 days of dosing and resulted in a decrease of bioavailability to zero and up to

438% increase in clearance (proportional to the log of maximum antidrug antibody

concentrations observed after the end of cycle 2 dosing). Clearance in cycle 1 was increased by

up to 67% in subjects with high antidrug antibody concentrations (22 mcg/mL) observed after

cycle 1 dosing. The effect of immunogenicity on clearance in cycle 2 was stronger, with

clearance increasing by up to 105% in subjects with high antidrug antibody concentrations

observed after cycle 1 dosing. In addition, clearance in cycle 2 increased by up to 194% in

subjects with high antidrug antibody concentration (3 mcg/mL) observed during cycle 2 dosing.

Serum antidrug antibody samples were taken on days 0, 28, 56, 91, 182, 201, 224, 273, 364,

546, and 728. However, after the DMC determined that the primary endpoint was not met,

antidrug antibody samples were no longer collected to decrease the burden on the subjects.

Levels of antidrug antibody were assayed using a ‘bridging’ (or sandwich) ELISA format where

teplizumab was used as both the coating and detecting reagent. Supplementary Table B shows

the incidence and extent of antidrug antibody levels in subjects that received teplizumab.

There did not, however, appear to be a significant correlation between antidrug antibody

levels and either the changes in AUC mean C-peptide or with response using the modified

composite HbA1c + insulin usage endpoint (Supplementary Table C). These results suggest that

even in spite of high antidrug antibody levels some subjects achieved a change in C-peptide

AUC level that placed them in the highest quartile of change from baseline. For example, at Day

364, 40 of the 55 subjects in this highest quartile experienced some level of antidrug antibody

thought to be neutralizing (antidrug antibody>100 ng/mL).

Appendix. The investigators and institutions participating in the Protégé trial.

James Lenhard, Christiana Care Research Institute; Stephen Aronoff, Research Institute of Dallas; Bruce

Bode, Atlanta Diabetes Associates; Jolene Berg, Diabetes and Glandular Disease Research Associates,

PA; Dennis Brenner, Saint Barnabas Medical Center; Holley Allen, Baystate Medical Center; Nicole

Sherry, Massachusetts General Hospital; Eda Cengiz, Yale Diabetes Research Program; Marc Rendell,

Creighton Diabetes Center; Fernando Ovalle, University of Alabama at Birmingham School of Medicine;

Celeste Hart, North Florida Thyroid Center; Richard Guthrie, Mid-America Diabetes Associates, PA;

David Liljenquist, Rocky Mountain Diabetes and Osteoporosis Center; Richard Hays; Kevin Ganong,

NEA Baptist Clinic Clinical Research Center; Jack Wahlen, Endocrine Reseach Specialists; Mary

Luidens, Division of Endocrinology, Albany Medical College; Roberto Izquierdo, Joslin Diabetes Center;

Wayne Moore, The Children's Mercy Hospital; Eva Tsalikian, University of Iowa Children's Hospital;

David M. Huffman, University Diabetes & Endocrine Consultants; Barry Reiner,; Peter Gottlieb,

University of Colorado Health Sciences Center; William Hagopian, University of Washington; Todd

Nebesio, Indiana University School of Medicine; Richard Christensen, Humphreys Diabetes Center;

Robert J. Ferry Jr., Le Bonheur Children's Medical Center Hospital and The University of Tennessee

Health Science Center at Memphis; Danièle Pacaud, Alberta Children's Hospital; Shayne Taback,

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University of Manitoba; Miguel Escalante, Hospital Mexico-Americano; Jaime Rodriguez Rivera,

Hospital Central; Ignacio Morones Prieto, Endocrinology Department; Hector E. Tamez Perez, Hospital

CIMA Santa Engracia; Zdenek Sumnik, Pediatricka klinika UK 2.LF, Fakultni nemocnice v Motole;

Jaroslav Skvor, Detska klinika, Masarykova nemocnice v Usti nad Labem; David Neumann, Detska

klinika, Fakultni nemocnice Hradec Kralove; Jaroslav Michalek, Pediatricka klinika, Fakultni nemocnico

Brno; Jan Vavrinec, Klinika deti a dorostu, Fakultni nemocnice Kralovske Vinohrady; Boris Mankovsky,

State Institution "Institute of Endocrinology and Metabolism named after V. Komisarenko of AMS

Ukraine"; Yuriy Karachentsev, V. Danilevsky Institute of Endocrine Pathology Problems; Nataliya

Zelinskaya, Ukrainian Children Specialised Clinical Hospital Ohmatdet; Gennadiy Lezhenko,

Zaporizhzhya Regional Pediatric Hospital; Tatyana Mykhaylychenko, Donetsk Regional Children

Clinical Hospital; Oksana Khyzhnyak, Kharkiv Regional Clinical Children Hospital; Maryna Vlasenko,

Regional Clinical Endocrinological Dispensary; Gottfried Rudofsky, Innere Medizin I, Endokrinologie

und Stoffwechsel, Universitaetsklinikum Heidelberg; Prasanna Kumar, Bangalore Diabetes centre; Richa

Chaturvedi, Pushpawati Singhania Research Institute; Uday Phadke, Ruby Hall Clinic; Sailesh Lodha,

Fortis Escorts Hospital; Rakesh Sahay, Mediciti Hospitals; K.D. Modi, Medwin Hospitals; Sanjiv Shah,

Diabetes Action Center; P.V. Rao, DiabetOmics India Pvt. Ltd; Pramod Gandhi, Gandhi Endocrinology

and Diabetes Centre; Parag Shah, Gujarat Endocrine Centre; K.A.V. Subrahmanyam, King George

Hospital; Sanjeev Pathak, DHL Research Center; Sanjay Kalra, Bharti Research Institute of Diabetes and

Endocrinology; Sunil Jain, TOTALL Diabetes Hormone Research Institute Pvt Ltd; Manojit

Mukhopadyay, B.P. Poddar Hospital and Medical Research Ltd; Sujeet Chandratreya, Endocare Clinic;

Aaron Hanukoglu, Pediatric Endocrinology, Wolfson Medical Center; Anat Jaffe, Hillel Yaffe Medical

Center; Moshe Phillip, Schneider Children's Medical Center of Israel; Orit Pinchas-Hamiel, Pediatric

Endocrine and Diabetic Unit, The Chaim Sheba Medical Center; Naim Shehadeh, Rambam Health Care

Campus; Maria Gorska, , Klinika Endokrynologii, Diabetologii i Chorób Wewnętrznych, Uniwersytecki

Szpital Kliniczny w Białymstoku; Wojciech Mlynarski, Klinika Pediatrii, Onkologii, Hematologii I

Diabetologii, Samodzielny Publiczny Zakład Opieki Zdrowotnej Uniwersytecki Szpital Kliniczny Nr 4,

im. Marii Konopnickiej Uniwersytetu Medycznego; Anna Noczynska, Klinika Endokrynologii i

Diabetologii Wieku Rozwojowego, Samodzielny Publiczny Szpital Kliniczny Nr 1; Maciej Pregiel,

Odzial Chorob Wewnetrznych, Powiatowy Zespol Szpitali w Olesnicy; Mieczyslaw Szalecki, III Oddział

Chorób Dziecięcych (Endokrynologiczno-Diabetologiczny), Wojewodzki Specjalistyczny Szpital

Dzieciecy im. Wladyslawa Buszkowskiego w Kielcach; Malgorzata Mysliwiec, Oddział Diabetologiczny

Klinika Pediatrii, Hematologii, Onkologii i Endokrynologii; Adriana Dumitrescu, Centrul Medical

Sanatatea ta; Nicolae Dragos Hancu, Centrul de Diabet, Nutritie si Boli Metabolice, Spital Clinic

Judetean de urgenta Cluj; Valerica Nafornita, S.C. MINIMED S.R.L.; Iosif Szilagyi, Sectia de

Diabetologie si Boli de Nutritie, Spitalul Judetean Satu Mare; Eugenia Farcasiu, Institutul de Diabet,

Nutritie si Boli Metabolice N.C. Paulescu; Ulle Jakovlev, East Tallinn Central Hospital; Tarvo Rajasalu,

Tartu University Hospital; Valdis Pirags, P. Stradins Clinical University Hospital; Wilfredo Ricart Engel,

Servicio de Endocrinología, Hospital Dr. Josep Trueta de Girona; Marta Botella Serrano, Servicio de

Endocrinología, Hospital Príncipe de Asturias; Johnny Ludvigsson, Barnkliniken Universitetssjukhuset.

See ClinicalTrials.gov identifier: NCT00385697 for additional information.

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Supplementary Table A. Summary of patient disposition.

14-day

full-dose

14-day

low-dose

6-day full-

dose

Placebo Total

Enrolled/Randomized 209 (100) 102 (100) 106 (100) 99 (100) 516 (100)

ITT/Safety Population 207 (99.0)a 102 (100) 106 (100) 98 (99.0)

b 513 (99.4)

Early withdrawal from

dosing:

47 (22.5) 23 (22.5) 24 (22.6) 12 (12.1) 106 (20.5)

Adverse event 35 (16.7) 12 (11.8) 17 (16.0) 5 (5.1) 69 (13.4)

Lost to follow-up 1 (0.5) 0 0 0 1 (0.2)

Withdrawal of consent 6 (2.9) 5 (4.9) 3 (2.8) 2 (2.0) 16 (3.1)

Other 5 (2.4) 6 (5.9) 4 (3.8) 5 (5.1) 20 (3.9)

Early withdrawal from

study:

26 (12.4) 12 (11.8) 9 (8.5) 7 (7.1) 54 (10.5)

Adverse event 3 (1.4) 1 (1.0) 0 0 4 (0.8)

Lost to follow-up 11 (5.3) 5 (4.9) 2 (1.9) 5 (5.1) 23 (4.5)

Withdrawal of consent 10 (4.8) 6 (5.9) 7 (6.6) 2 (2.0) 25 (4.8)

Other 2 (1.0) 0 0 0 2 (0.4)

Completed visits

Year 1 198 (94.7) 99 (97.1) 101 (95.3) 96 (97.0) 494 (95.7)

Year 2 183 (87.6) 90 (88.2) 97 (91.5) 92 (92.9) 462 (89.5)

Values in table are number of patients (percent of randomized patients)

ITT = intent-to-treat a Two subjects did not receive any study drug: one withdrew consent and one met the

discontinuation criteria before dosing. b One subject met the discontinuation criteria before dosing and did not receive any study drug.

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Supplementary Table B. Incidence (percent) of subjects who developed antidrug antibody and

the extent of antidrug antibody response.

Regimen Course Incidence

Antidrug antibody rangea

<103 ng/mL 1-5x10

3 ng/mL >5×10

3 ng/mL

14-day full-

dose

First 56% 93% 3% 4%

Second 60% 45% 36% 19%

14-day low-

dose

First 56% 95% 5% 0%

Second 64% 30% 34% 36%

6-day full-

dose

First 68% 82% 10% 7%

Second 77% 34% 27% 39% aThe subjects with positive antidrug antibody were classified into three classes based on the

extent of antidrug antibody response; ng/mL refers to a rabbit polyclonal anti-idiotypic standard,

not actual human antidrug antibody.

Supplementary Table C. Number of patients with maximum antidrug antibody by quartile of

AUC mean C-peptide change from baseline in the 14-day full-dose group.

Level of Antidrug Antibody (ng/mL)

Quartile <100 100-1000 1000-1500 1500-5000 >5000

Year 1

Lowest 9 11 2 3 10

Second 7 16 5 5 16

Third 17 14 5 2 11

Highest 15 19 6 4 11

Year 2

Lowest 18 4 0 1 0

Second 25 8 1 0 0

Third 28 5 2 1 0

Highest 26 10 0 1 1

AUC = area-under-the-curve. Antidrug antibodies were measured using a rabbit anti-teplizumab

reference standard. Quartiles of C-peptide change of AUC from baseline were calculated using

values of [ln(C-peptide AUCDay x + 1) - ln(C-peptide AUCBaseline + 1)].

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Supplementary Figure A. Adjusted mean insulin use over time in the 14-day full-dose and

placebo groups. All patients (TOP) and patients in the US (BOTTOM). Bars indicate standard

errors. *p=0.03

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Supplementary Figure B. Percent of patients with HbA1c <7% and insulin use <0.25 U/kg/day

over 2 years.

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Supplementary Figure C. Predicted teplizumab concentration-time course in Cycle 2 for the 14-

Day Full-dose group.

The red line illustrates the predicted teplizumab levels for a typical 60 kg male subject with no

observed antidrug antibody. The blue line illustrates the predicted levels for a subject with no

observed antidrug antibody initially, but strong immune response on the tenth day of dosing.

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Supplementary Figure D. Levels of CD4+ T-cells on sixth day of dosing of the 14-day full-dose

regimen in relation to antidrug antibody (ADA) levels on either Day 28 (first cycle) or Day 210

(second cycle). Similar results were observed for the other dosing regimens (data not shown).

of 38Diabetes

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