Diabetes Care Volume 38, June 2015 997 CROSSROADS A AT DIABETES 1 TYPE

Prevention and Reversal of Type 1 Jay S. Skyler DiabetesdPast Challenges and Future Opportunities Diabetes Care 2015;38:997–1007 | DOI: 10.2337/dc15-0349

Over the past three decades there have been a number of clinical trials directed at interdicting the (T1D) disease process in an attempt to prevent the development of the disease in those at increased risk or to stabilizedpotentially even reversedthe disease in people with T1D, usually of recent onset. Unfortu- nately, to date there has been no prevention trial that has resulted in delay or prevention of T1D. And, trials in people with T1D have had mixed results with some showing promise with at least transient improvement in b-cell function compared with randomized control groups, while others have failed to slow the decline in b-cellfunctionwhencomparedwith placebo. This Perspective will assess the past and present challenges in this effort and provide an outline for potential future opportunities.

THE BEGINNINGS The first randomized, double-masked, controlled trials with sufficient statistical power to give confidence for the outcome were conducted in the mid-1980s with cyclosporine (1,2). Two large studies were conducteddthe French cyclosporine study that included 122 patients with type 1 diabetes (T1D) aged 15–40 years who had been symptomatic for 6 months or less and were on insulin therapy for 2 months or less (1) and the Canadian- European cyclosporine study that included 188 subjects aged 9–35 years who had been symptomatic for 14 weeks or less and were on insulin therapy for 6 weeks or less (2). Both trials used as their primary outcome the achievement of remission defined two ways. First, “complete remission” was defined as good metabolic control (fasting glucose ,140 mg/dL [7.8 mmol/L], postprandial glucose ,200 mg/dL [11.1 mmol/L], HbA1c #7.5%) in the absence of insulin treatment. Second, “partial remission” was defined as good metabolic control with insulin dose ,0.26 units/kg per day. Both studies found a greater proportion of cyclosporine patients than placebo patients achieving and main- taining remissions. In both studies, cyclosporine could be given for 1 year, with stopping Diabetes Research Institute, University of Miami rules in place that led to blinded substitution of placebo for cyclosporine if remission was Miller School of Medicine, Miami, FL lost. Although both studies found that cyclosporine had superior efficacy than placebo, fi fi Corresponding author: Jay S. Skyler, jskyler@ the magnitude and duration of bene tdidnotappearsuf cient to justify cyclosporine miami.edu. treatment in clinical practice, given the potential of cyclosporine-induced nephrotoxicity. Received 16 February 2015 and accepted 17 The importance of the studies, however, was that they demonstrated the impact of March 2015. fi immune intervention on the evolution of T1D, in a sense ful lling Koch postulates, which © 2015 by the American Diabetes Association. were developed for infectious diseases, but the response to immune therapy can be Readers may use this article as long as the work considered as indicative that T1D is immune mediated. is properly cited, the use is educational and not for profit, and the work is not altered. CHALLENGES COMPLICATING PREVIOUS STUDIES See accompanying articles, pp. 968, Since the completion of the early trials, particularly during the past decade, a num- 971, 979, 989, 1008, 1016, 1030, ber of additional randomized, double-masked, adequately powered, controlled and 1036. 998 Prevention and Reversal of T1D Diabetes Care Volume 38, June 2015

clinical trials have been conducted using A number of other strategies have journals that take a bleak look at the field. many different immunological strate- been without benefit in preserving Rather than looking negatively on the re- gies. For the most part, these have been b-cell function (12–17). In addition, sults to date, an opportunity exists to ex- disappointing, with none showing unam- some studies had ambiguous effects, amine the details of previous studies to biguous benefit in preserving b-cell func- not meeting the primary outcome mea- identify what can be learned and what tion. Some studies have shown transient sure (i.e., preservation of b-cell function) can be applied to future studies. To that benefit with anti-CD3 monoclonal anti- but showing potential benefit either on end, several problems are notable, and bodies targeting T cells (Fig. 1A and B) secondary outcome measures or mech- these are detailed below. (3–6), an anti-CD20 monoclonal anistic measures or in only a subgroup of (i.e., ) targeting B cells (Fig. 1C) subjects (18–20). MISLEADING PILOT STUDIES (7,8), and with a costimulation blocking Thus, most immune intervention trials The Diabetes Prevention Trial–Type 1 agent (i.e., ) that prevents im- in T1D have either failed to achieve suc- Parenteral Insulin Trial was a fully pow- mune activation (Fig. 1D)(9,10).Onepilot cess in preserving b-cell function or ered, randomized, controlled clinical tri- study combining antithymocyte globulin have met that hurdle but have nonethe- al that enrolled 339 relatives of patients (ATG) and granulocyte colony-stimulating less shown only a transient effect. This with T1D who were estimated to have at factor (GCSF) suggested benefit(Fig.1E)but has resulted in a flurry of editorial and least a 50% risk of developing T1D in awaits confirmationinalargertrial(11). commentary articles in peer-reviewed the next 5 years (21). To enroll these

Figure 1—There has been a progressive decline in b-cell function, as measured by C-peptide, even in most studies that have been “successful,” thus showing only a transient benefit, as depicted for the anti-CD3 (A) (3,4), the anti-CD3 monoclonal antibody (B) (5,6), the anti-CD20 monoclonal antibody rituximab (C) (7,8), and the costimulation blocker abatacept (D) (9,10). E:Alsoshown is the preservation of b-cell function in a pilot study (only 25 subjects randomized) with the combination of low-dose ATG plus GCSF (11), in which there is preservation of b-cell function at 1 year, something needing confirmation in a larger study. care.diabetesjournals.org Skyler 999

subjects, more than 93,000 relatives treated group was younger (mean age fasting C-peptide at 15 months, were screened. The trial showed no dif- 10 years) than the untreated group showed no difference between groups. ference in the rate of development of (mean age 20.7 years), and one would However, mixed-meal tolerance test T1D (Fig. 2A) (21). This was a surprising have expected younger individuals to (MMTT)-stimulated C-peptide was outcome, as two pilot studies had sug- progress faster, which did not happen. higher in GAD subjects who were treated gested that the interventiondlow-dose In the other pilot trial, 14 subjects were within 6 months of diagnosis, but insulindcould delay the disease. One of randomized to insulin treatment or to this included only 11 subjects treated those apparently promising pilot studies the control group, with the insulin group with GAD and 14 treated with placebo was a nonrandomized study of 12 indi- having longer diabetes-free survival (Fig. 2C). On the basis of this secondary viduals offered the intervention, 7 of (P , 0.03) (23). subgroup outcome, three additional whom declined and were analyzed as a The first study in T1D using GAD in a studiesdall enrolling subjects within 3 comparison group, along with a group of vaccine formulation with alum (GAD- months of diagnosisdwere undertaken, historical control subjects of undefined alum) randomized 70 subjects, aged including two phase 3 trials. A TrialNet number (Fig. 2B) (22). Life-table analysis 10–18 years, within 18 months of diag- study enrolled 145 subjects aged 3–45 suggested a statistically significant (P = nosis, to receive either two doses of years (Fig. 2D), a European phase 3 trial 0.002) and dramatic difference in rate of GAD-alum or two doses of alum alone enrolled 334 subjects aged 10–20 years development of T1D. Interestingly, the (24). The primary outcome measure, (Fig. 2E), and a U.S. phase 3 trial enrolled

Figure 2—A: Life-table analysis showing lack of benefit in the fully powered DPT-1 Parenteral Insulin Trial (21), despite perceived benefit (by life-table analysis) in a small pilot study (B) (22). C: Putative benefit in a small subgroup (those treated within 6 months of diagnosis) in a GAD-alum vaccine trial (24) that was not confirmed in two larger trials (D and E) with GAD-vaccine (12,13). 1000 Prevention and Reversal of T1D Diabetes Care Volume 38, June 2015

Figure 3—A: Transient effect of the anti-CD3 monoclonal antibody teplizumab in all treated subjects in one study (62). B: In that same study, retention of b-cell function for 2 years in responders, whereas nonresponders were identical to the comparison control group.

328 subjects aged 10–20 years (12,13,25). report alleged that glucagon-stimulated Another pilot study involved the use of In all three of these trials, there was no test (GST) of C-peptide at 24 months was Bacillus Calmette-Guerin´ (BCG) vaccine in evidence of a treatment effect with improved in the DiaPep277 group versus six subjects with long-standing T1D who GAD-alum. Yet, it should be appreciated the placebo group, although there was no were randomized to receive either two that the success of GAD in mice was difference in MMTT-stimulated C-peptide. doses of BCG or placebo (32). The authors approached differently. For example, However, MMTT was the original primary claimed that the BCG subjects, and one of the GAD-alum studies perhaps used the outcome measure and therefore was mea- the three control subjects who devel- wrong formulation (with adjuvant). The sured at randomization (month 0) and after oped acute Epstein-Barr virus (EBV) in- studies could have used GAD-alum by 6, 12, 18, and 24 monthsda total of 5 fection, showed increases in dead the wrong route (subcutaneous) and at measurements. As GST originally was a sec- insulin-autoreactive T cells and induction the wrong time (after clinical diagnosis ondary outcome measure, it was per- of regulatory T cells. They also claimed of T1D). Although GAD vaccine still may formed at month 1 (defined as “baseline” that there was transient increase in be able to prevent or delay the develop- for the GST but 1 month after the first treat- C-peptidelevels notseenina nonrandom- ment of T1D if used as a vaccine prior to menthadbeengiven)andat12and24 ized cohort of subjects with T1D in their disease onset, the results of these trials monthsda total of 3 measurements. The institution. They have garnered a lot of have greatly diminished the enthusiasm authors intended, initially, to have MMTT publicity for this very tiny study. Although for GAD that once existed in our field. be the primary outcome measuredthey the authors reported an increase in Another approach considered was performed the first MMTT before initiating C-peptide levels using an ultrasensitive DiaPep277. DiaPep277 is a peptide de- treatment (a true baseline measurement) assay, there are no data that the trivial rived from positions 437–460 of the hu- and conducted the test at more frequent increases in C-peptide have any biological man heat shock protein 60 (Hsp60), intervals. However, the primary outcome importance. Moreover, two larger previous named peptide 277, that is alleged to in- measure was changed from the MMTT to randomized clinical trials of BCG, involving duce anti-inflammatory T cells. The first the GST. Specifically, it was stated “the 26 and 47 subjects, showed no effect of report of its use in T1D was a pilot study study protocol was amended and the Sta- BCG on preservation of b-cell function, in which 35 subjects, aged 16–55 years, tistical Analysis Plan was planned and final- but in both trials there was a trend to with recent onset T1D, received either ized before the study was unblinded, with greater decline of b-cell function in the BCG three doses of DiaPep277 or three doses the GST clearly defined as the primary end- group than in the control group (33,34). of placebo (26). Primary outcome point” (14). Had that really been the case, it What can one then conclude about was glucagon-stimulated C-peptide at raised questions of why differences in these the value of pilot studies in this area? 10 months, which was higher in the two outcome measures existed and led to Essentially, the bottom line is that pilot DiaPep277 group than in the placebo group, the question of whether both measures studies must be viewed with great cau- an effect that was sustained with follow- would be needed in future trials (31). How- tion and definitive answers can only be up to 18 months (27). Four additional ever, subsequently the article was retracted obtained by adequately powered ran- phase 2 studies showed results that with a statement that there had been evi- domized controlled trials. were ambiguous at best, with no clear dence uncovered that some employees of benefit (28–30). Nonetheless, the spon- the sponsor had engaged in serious miscon- FAILURE TO DISTINGUISH sor mounted two full-scale phase 3 trials. duct, including collusionwithathird-party TRANSIENT SYMPTOMS FROM The first enrolled 457 subjects, aged 16– biostatistics firm to improperly receive un- SERIOUS ADVERSE EVENTS 45 years, with recent-onset T1D, who re- blinded trial data and to use such data in A number of interventions used in ceived injections of DiaPep277 or placebo order to manipulate the analyses to recent-onset T1D have shown transient quarterly for 2 years (14,15). The initial obtain a favorable result. symptoms related to cytokine release care.diabetesjournals.org Skyler 1001

Table 1—Immune intervention trials in T1D Year Study name Intervention Outcome Result reported Reference Primary prevention studies Finnish TRIGR pilot Casein hydrolysate formula Autoantibodies Apparent 2010 66 benefit TRIGR Casein hydrolysate formula Autoantibodies No difference 2014 67 FINDIA Insulin-free whey-based formula Autoantibodies Apparent 2012 68 benefit BABYDIET Gluten-free diet Autoantibodies No difference 2011 69 TRIGR Casein hydrolysate formula Diagnosis of T1D * Ongoing 67 Secondary prevention studies DENIS Nicotinamide Diagnosis of T1D No difference 1998 70 ENDIT Nicotinamide Diagnosis of T1D No difference 2004 71 DPT-1 Parenteral Insulin Injected insulin Diagnosis of T1D No difference 2002 21 DPT-1 Oral Insulin Oral insulin Diagnosis of T1D No difference 2005 20 Belgian parenteral insulin Injected insulin Diagnosis of T1D No difference 2009 51 DIPP birth cohort Nasal insulin Diagnosis of T1D No difference 2008 52 DIPP sibling cohort Nasal insulin Diagnosis of T1D No difference 2008 52 INIT II Nasal insulin Diagnosis of T1D * Ongoing 60 DIAPREV-IT GAD Diagnosis of T1D * Ongoing 58 TrialNet oral insulin Oral insulin Diagnosis of T1D * Ongoing 59 TrialNet teplizumab Anti-CD3, teplizumab Diagnosis of T1D * Ongoing 72 TrialNet abatacept Abatacept Diagnosis of T1D * Ongoing 73 Studies in recent-onset T1D French cyclosporine Cyclosporine Remission Benefit19861 Canadian-European C-pep $0.6 nmol/L or noninsulin cyclosporine Cyclosporine treated Benefit19882 + Azathioprine glucocorticoids and prednisone Peak C-pep/glucose ratio Benefit198874 Azathioprine, adults Azathioprine Remission Benefit198575 Azathioprine, children Azathioprine Partial remission No difference 1989 76 Linomide French trial Linomide Glucagon-stimulated C-pep Benefit199877 BCG BCG vaccine Glucagon-stimulated C-pep No difference 1998 33 BCG BCG vaccine Primary, remission; secondary, No difference 1999 34 MMTT C-pep French oral insulin Oral insulin Glucagon-stimulated C-pep No difference 2000 53 Italian oral insulin Oral insulin Fasting C-pep No difference 2000 54 U.S. oral insulin Oral insulin Loss of C-pep # 2004 55 Herold anti-CD3 Teplizumab MMTT C-pep Benefit 2002, 2005 3,4 Keymeulen anti-CD3 Otelixizumab C-pep after clamp Benefit 2005, 2010 5,6

Proteg´ e´ Teplizumab Insulin ,0.5 unit/kg + HbA1c No difference 2011, 2013 35,36 ,6.5%

Proteg´ e´ Encore Teplizumab Insulin ,0.5 unit/kg + HbA1c *201178 ,6.5% DEFEND-1 Otelixizumab MMTT C-pep No difference 2014 37 DEFEND-2 Otelixizumab MMTT C-pep No difference 2014 38 AbATE (ITN study) Teplizumab MMTT C-pep Benefit201362 DELAY Teplizumab MMTT C-pep Benefit201379 GAD pilot GAD-alum vaccine Fasting C-pep Apparent 2008 24 benefit in secondary outcome in subgroup GAD TrialNet GAD-alum vaccine MMTT C-pep No difference 2011 12 GAD Europe GAD-alum vaccine MMTT C-pep No difference 2012 13 GAD U.S. (DiaPrevent) GAD-alum vaccine MMTT C-pep N/A 2011 25 DiaPep–Israeli adults DiaPep277 peptide Glucagon-stimulated C-pep Benefit 2001, 2007 26,27 DiaPep–Israeli pediatrics DiaPep277 peptide MMTT C-pep No difference 2007 28

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Table 1—Continued Year Study name Intervention Outcome Result reported Reference

DiaPep–Belgian adults DiaPep277 peptide Glucagon-stimulated C-pep Benefitatone 2007 29 dose DiaPep–Europe adults DiaPep277 peptide Glucagon-stimulated C-pep No difference 2007 30 DiaPep–Europe pediatrics DiaPep277 peptide Glucagon-stimulated C-pep No difference 2007 30 DiaPep–phase III DiaPep277 peptide Glucagon-stimulated C-pep ♓ 2014 14,15 MMF/DZB Mycophenolate mofetil MMTT C-pep No difference 2010 16 with/without Anti-CD20 TrialNet Anti-CD20 rituximab MMTT C-pep Benefit 2009, 2014 7,8 Abatacept TrialNet Abatacept MMTT C-pep Benefit 2011, 2014 9,10 TrialNet Anti-IL1b canakinumab MMTT C-pep No difference 2013 17 START thymoglobulin ITN Thymoglobulin MMTT C-pep No difference 2013 18 T1DAL– ITN Alefacept MMTT C-pep No difference 2013 19 IL-2 & rapamycin Transient safety ITN IL-2 and rapamycin MMTT C-pep worsening 2012 45 AIDA trial Anakinra MMTT C-pep No difference 2013 17 a1-Antitrypsin a1-Antitrypsin MMTT C-pep § 2014 80 Altered peptide ligand B9–23 altered peptide ligand MMTT C-pep No difference 2009 56 Plasmid-encoded Plasmid-encoded proinsulin proinsulin Safety MMTT C-pep ¶ 2013 57 Proinsulin peptide Proinsulin peptide Safety study No safety issues 2009 61 ATG–GCSF trial ATG and GCSF MMTT C-pep Benefit201511 DIATOR Atorvastatin MMTT C-pep No difference 2011 81 Etanercept MMTT C-pep Benefit200982 Low-dose IL-2 safety trial IL-2 (3 doses) T-reg number Increased 2013 46 REPAIR-T1D Sitagliptin and lansoprazole MMTT C-pep No difference 2014 48 AHSCT + profound Cyclophosphamide, 2007, 2009, GCSF, ATG, AHSCT MMTT C-pep Benefit 2009 83–85 AHSCT + profound Cyclophosphamide, immunosuppression GCSF, ATG, AHSCT MMTT C-pep Benefit201486 ATG–GCSF trial ATG and GCSF MMTT C-pep * Ongoing 87 EXTEND trial MMTT C-pep * Ongoing 88 Otelixizumab dose-ranging trial Otelixizumab MMTT C-pep * Ongoing 89 a1-Antitrypsin trial a1-Antitrypsin Basal C-pep * Ongoing 90 a1-Antitrypsin trial a1-Antitrypsin MMTT C-pep * Ongoing 91 pilot Ustekinumab Safety * Ongoing 92 Imatinib trial Imatinib MMTT C-pep * Ongoing 93 Tauroursodeoxycholic acid trial Tauroursodeoxycholic acid MMTT C-pep * Ongoing 94 DIABGAD GAD-alum & vitamin D with/without MMTT C-pep * Ongoing 95 ibuprofen Proinsulin peptide Proinsulin peptide Safety * Ongoing 96 Methyldopa Methyldopa Inhibition of DQ8 Ag * Ongoing 97 Low-dose IL-2 IL-2 T-reg number * Ongoing 98 AHSCT, autologous hematopoietic stem cell transplantation; C-pep, C-peptide; INT, Immune Tolerance Network; T-reg, regulatory . *Data not yet available. #Data ambiguousdauthors claim benefit but only seen in one dose in post hoc subgroup. N/Adactual data not available; press release announced negative result and study discontinuation. ♓Article retracted. §Data ambiguousdauthors claim benefit but single-arm trial and “benefit” unclear. ¶Data ambiguous (as discussed in text).

syndrome, including headache, fever, rituximab (7), and thymoglobulin benefit versus the discomfort. A few and hypotension, at the time of infusion (11,18). Yet, although the infusions daysofsymptomsearlyonneedtobe of the treatment. These symptoms are have been completed within the first balanced with a sustained beneficial not intolerable and are both transient days or within the first month after outcome over a protracted time frame. and fully reversible. Thus, they do not randomization, beneficial effects from Some of these trials have used anti- constitute major adverse effects that these interventions have been seen histamine and analgesic prophylaxis to would justify withdrawal of the sub- 12 months to 4 years after randomiza- obviate symptoms related to cytokine ject from the study. Cytokine release tion (3–7,11). Thus, subjects may have release. At least one trial also used syndrome has been seen with infusions some discomfort, but there needs to low doses of glucocorticoids (18) but of anti-CD3 monoclonal (3,5), be a reality check as to the potential only in the experimental group, not in care.diabetesjournals.org Skyler 1003

in the first trial with otelixizumab was transient EBV reactivation (39). Al- though the authors concluded that such EBV reactivation was of no appar- ent clinical concern over the long term, others have asserted that this must be avoided at all costs (40). I was Chair of the Data Safety Monitoring Committee for that study, and prior to the study the committee had concluded that tran- sient EBV reactivation was possible and would neither constitute a reason to halt the study nor was a side effect that needed to be avoided. So, in the phase 3 trials with use of the lower dose, side effects were obviated; however, benefi- cial effects were also completely obvi- ated. This unfortunate dose reduction — Figure 4 Potential scheme of combination therapy using agents with complementary effects. reminds us that all effective therapies This scheme includes anti-inflammatory therapy targeting innate immunity, immunomodulatory therapy targeting adaptive immunity, therapy driving regulatory immunity, -based ther- are likely to have some side effects and apy directing regulation to b-cells, and an agent promoting b-cell health. T-reg, regulatory T cell. that if one lowers the dose to eliminate all side effects, the drug may no longer have benefit. the placebo group. One might consider outcome that requires a subject to meet givingthesameprophylaxistobothex- two criteria, the outcome becomes a DOSING ISSUES perimental and placebo groups in order dichotomous measure that dilutes the Getting the dose right is important. In to minimize the risk of unblinding. effect of two continuous variablesdHbA1c addition to the otelixizumab dosing and insulin dose. More important, when issue, several other examples are worth ERRORS IN TRIAL DESIGN the conventional outcome measure of noting. -2 (IL-2) stimulates There have been extensive studies with C-peptide was assessed, there was evidence regulatory T cells at low doses but also two anti-CD3 monoclonal antibodiesd of efficacy both at 1 year (35) and at 2 years stimulates effector T cells at higher teplizumab and otelixizumab. The first (36) following two 14-day courses of tepli- doses (41). Thus, it has been noted study with teplizumab (given over 14 zumab (at entry and at 26 weeks into the that the use of IL-2 is a double-edged days) demonstrated a slower decline of study). This was especially evident in sub- sword (42). In other conditions (e.g., b-cell function (by MMTT) at 1 year (3), jects enrolled in the U.S., in younger sub- hepatitis-C virus–induced vasculitis with sustained improvement on b-cell jects (age 8–17 years), in subjects enrolled and graft-versus-host disease), low- function at 2 years (4). Meanwhile, the within 6 weeks of diagnosis, and in sub- dose IL-2 has shown beneficial effects first study with otelixizumab (given over jects with higher levels of C-peptide at en- (43,44). The first human study of IL-2 6 days) showed a slower decline of b-cell try (35). In addition, the phase 3 study also in T1D used a relatively high dose and function (measured using a hyperglyce- enrolled subjects in South Asia. Although combined its use with rapamycin (45). mic clamp followed by glucagon stimula- these subjects met the clinical criteria used An adverse effect on b-cell function was tion) at 18 months (5). After 4 years of for enrollment, it is important to note that observed. A subsequent study using follow-up, although b-cell function was typical immune-mediated T1D (also called low-dose IL-2 appeared to be relatively not measured, the otelixizumab group type 1A diabetes) is a disease principally of safe, without a decrease in b-cell had lower insulin requirements despite Europoid Caucasians. Enrollment of Asian function and with the expected in- similar glycemic control as measured by subjects may have confounded the results. crease in regulatory T cells (46). There- HbA1c (6). Thus, effects of a 6-day treat- Another issue was that the control group fore, studies are currently under way ment course appeared to be evident in the phase 3 study maintained residual to study low-dose IL-2 in T1D. 4 years later. C-peptide to a greater extent and longer Another example of a dosing issue is The results from these early studies than expected, which was especially true thymoglobulin (ATG). In a study using a with anti-CD3 led to the initiation of phase for adult subjects. This made it more diffi- relatively high dose of ATG, no beneficial 3 clinical trials with both agents. Unfor- cult to detect differences between groups. effect was seen, and it was observed that tunately, the phase 3 studies did not For otelixizumab, the phase 3 studies there was suppression of both effector meet their primary outcome criteria. For used a dose that was one-sixteenth (to- and regulatory T cells (18). In another teplizumab, the primary outcome was tal of 3.1 mg over 8 days) of that used in study in which a lower dose of ATG was the combination of HbA1c ,6.5% and in- the original phase 2 study (total of 48 used, regulatory T cells were not sup- sulin dose ,0.5 units/kg/day (35). This mg), in an effort to avoid any side effects pressed (11). Interpretation of that study outcome measure was arbitrarily selected (37,38). One has to question what the is confounded, however, because low- without sufficient data to justify its selec- investigators were trying to avoid. The dose ATG was used in combination with tion. Moreover, by using a composite one noninfusion-related side effect seen GCSF. 1004 Prevention and Reversal of T1D Diabetes Care Volume 38, June 2015

A related issue providing an addi- nasal insulin (60) prevention trials and criteria for future use of a given tional confounder is choosing surrogate of the development of an approach therapy. agents. A study in NOD mice found that that uses a combination of peptides combination therapy with glucagon-like derived from proinsulin (61). Yet, the FUTURE DIRECTIONS peptide 1 (GLP-1) and gastrin restores desirable concept of an antigen-based As discussed above, there have been normoglycemia (47). This combination therapy sustains the efforts to find many attempts at immune intervention resulted in increases in pancreatic insu- an effective one, perhaps as a compo- in T1D, with mixed results. Table 1 lin content, b-cell mass, b-cell prolifera- nent of a combination therapeutic provides a listing of all of the major stud- tion, and b-cell neogenesis; a reduction approach. ies to date, including randomized con- in b-cell apoptosis; and a beneficial ef- trolled trials and other studies mentioned fect on the immune response (47). It NOT ALL PEOPLE MAY RESPOND in this Perspective; not included are seemed like a natural combination to TO THERAPY small pilot studies not discussed in this test in human beings. Yet, rather than In one study using the anti-CD3 mono- article. testing the combination of GLP-1 and gas- clonal antibody teplizumab, a group of It should be appreciated that in the trin, a study evaluated the combination of “responders” to treatment was identi- evolution of T1D several immune path- the dipeptidyl peptidase-4 inhibitor sita- fied, who at 2 years maintained ways are involved. This complicates the gliptin and the proton-pump inhibitor lan- C-peptide better than the randomized design of an ideal therapeutic strategy soprazole, as these agents, respectively, but untreated comparison group (Fig. to control the and pre- increase circulating levels of GLP-1 and 3) (62). In that study, responders consti- vent the loss of b-cell function and b-cell gastrin (48). The primary end point was tuted 48% of subjects treated with tepli- mass. Indeed, if one pathway is con- not achieved, but not all participants zumab. Interestingly, the responders trolled, another pathway may become had the expected increases in GLP-1 not only did better than the comparison more active. Thus, success may require and gastrin levels. Thus, they effectively group but also, as a group, actually that a combination approach be used. did not get the doses that may have maintained b-cell function essentially Such a combination might include been needed to achieve the desired at the level seen at randomization, (Fig. 4) one or more anti-inflammatory effects. whereas the nonresponders had lost agents targeting innate immunity, b-cell function at a rate similar to the such as agents that target IL-1 (IL- ANTIGEN-BASED THERAPIES HAVE comparison group. Had the analysis 1b) or tumor necrosis factor (TNF-a); NOT WORKED YET been confinedtothetotaltreated one or more immunomodulatory agents The desirability of antigen-based groupdincluding both responders and targeting adaptive immunity, such as is grounded on the no- nonrespondersdthe full retention of anti-CD3, anti-CD20, or costimulation tion that such therapies are specificfor b-cell function in nearly half of the sub- blockade (three agents that already T1D and are unlikely to have adverse off- jects would have been missed. Some have shown some beneficial effect on target effects. Thus, they should have a type of responder analysis should be ap- preserving b-cell function) or ATG; per- high degree of safety. The two diabetes- plied to all intervention studies. Indeed, haps some agent that would drive regu- specific that have been used it has been suggested that a responder latory T cells, such as low-dose IL-2 or are insulin and GAD, both of which had analysis be included in the statistical GCSF or the infusion of regulatory T cells great success in animal models of T1D plan for all T1D intervention studies, themselves; any of these perhaps cou- (49,50). Yet, to date, there has been no and for that purpose, the definition pled with a diabetes-related antigen unambiguous success with antigen- of a responder should be the mainte- that might help target the regulatory T based therapies in human T1D. GAD- nance of 100% of baseline b-cell func- cells to b-cells; and agents that help pre- alum vaccine has failed in new-onset tion (63). serve b-cell health, such as GLP-1 (64). T1D (12,13). Injected insulin has failed A fundamental question is why In such a strategy, the combination of in prevention trials (21,51). Nasal insulin some subjects fail to respond. It could agents is selected for having potential has failed in prevention trials (52). Oral be that the immunotherapy used was complementary effects. It may also be insulin has failed in new-onset T1D (53– ineffective (at least at the dose necessary to tailor the selection of 55) and did not meet its primary tested), that the immunological agents for different individuals, i.e., we outcome in a prevention trial (20). An processdperhaps a relapsing and re- may need to move to a personalized altered peptide ligand of insulin failed in mitting onedwas in a latent period at medicine approach with different treat- new-onset T1D (56). A plasmid-encoding thetimeofdrugadministrationand ments for different subtypes, if these proinsulin was claimed to have a benefit thus not responsive to immunother- become better defined (65). on b-cell function, but actually the ef- apy, that b-cell mass or b-cell function Therefore as outlined, there are many fect was seen in but one of four doses had already deteriorated to a point of potential interventions that hold prom- tested and only at one time point (57). no return, that the immunological pro- ise, particularly if they are used as com- Moreover, the number of subjects stud- cesses damaging b-cells are different ponents of combination therapy. Several ied at each dose was small, and there did among individuals, or for some other new strategies are approaching clinical not appear to be statistical adjustment reason. It is important to assess poten- evaluation. To be successful, we must for multiple comparisons. The field is ea- tial biomarkers that might discrimi- be patient, yet proceed with diligence. gerly awaiting the conclusion of ongoing nate responders from nonresponders Moreover, it is important that trials be GAD-alum (58), oral insulin (59), and and thus might be used as enrollment carefully designed, well controlled, and care.diabetesjournals.org Skyler 1005

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