Immunotherapy using peptides has been successful for some patients with allergies, but has not yet been deployed in autoimmune diseases, which may involve greater safety risks. Alhadj Ali et al. designed a placebo-controlled trial to determine whether a proinsulin peptide could safely elicit immune and metabolic responses in people recently diagnosed with type 1 diabetes without accelerating disease. This small trial showed that treatment seemed to modify T cell responses and did not interfere with residual β cell function. In contrast to subjects in the placebo arm, treated subjects did not need to increase their insulin use. These encouraging results support a larger trial to investigate efficacy of the peptide therapy for treating disease.

Immunotherapy using short immunogenic peptides of disease-related autoantigens restores immune tolerance in preclinical disease models. We studied safety and mechanistic effects of injecting human leukocyte antigen–DR4(DRB1*0401)–restricted immunodominant proinsulin peptide intradermally every 2 or 4 weeks for 6 months in newly diagnosed type 1 diabetes patients. Treatment was well tolerated with no systemic or local hypersensitivity. Placebo subjects showed a significant decline in stimulated C-peptide (measuring insulin reserve) at 3, 6, 9, and 12 months versus baseline, whereas no significant change was seen in the 4-weekly peptide group at these time points or the 2-weekly group at 3, 6, and 9 months. The placebo group’s daily insulin use increased by 50% over 12 months but remained unchanged in the intervention groups. C-peptide retention in treated subjects was associated with proinsulin-stimulated interleukin-10 production, increased FoxP3 expression by regulatory T cells, low baseline levels of activated β cell–specific CD8 T cells, and favorable β cell stress markers (proinsulin/C-peptide ratio). Thus, proinsulin peptide immunotherapy is safe, does not accelerate decline in β cell function, and is associated with antigen-specific and nonspecific immune modulation.

This knowledge promotes consideration of antigen-specific immunotherapy (ASI) as an approach for type 1 diabetes, because it has been shown in preclinical models of inflammation and autoimmunity to limit disease by deletional effects on effector T cells and by promoting cohorts of CD4 T cells with regulatory properties, including those that secrete IL-10 ( 15 ). One ASI approach involves administration of short peptides, representing epitopes of disease-related autoantigens. This strategy, termed peptide immunotherapy (PIT), has gained considerable traction in clinical allergy, where it avoids the problem of using whole antigens that might trigger immunoglobulin E–mediated hypersensitivity; it is also under development in autoimmune inflammatory conditions, such as celiac disease and multiple sclerosis ( 15 ). We have previously described how administration of a peptide representing an immunodominant region of proinsulin presented by the human leukocyte antigen (HLA) class II diabetes risk molecule HLA-DR4 (DRB1*0401) can modulate autoreactive CD4 T cells in patients with long-standing type 1 diabetes, but in that study, circulating C-peptide was absent, and therefore, safety and disease-modifying effects in a clinically relevant target population could not be evaluated ( 16 ). Therefore, here, we examined the proinsulin mono-PIT approach in adults ascertained within 100 days of type 1 diabetes diagnosis and with residual C-peptide to examine safety and tolerability in a relevant therapeutic setting and study early indications of mechanistic and metabolic effects.

In the same timeframe, an understanding of the numerous immunological pathways that contribute to β cell loss has emerged. These include delineation of effector pathways, such as autoreactive CD4 T cells secreting proinflammatory cytokines and CD8 T cells with cytotoxic activity upon recognition of β cell targets ( 8 – 10 ). There is also evidence that immune regulatory pathways may be compromised or unable to adequately control effector responses ( 11 , 12 ). These findings not only relate to conventional FoxP3 + CD25 hi regulatory T cells (T regs ) but also to regulatory autoreactive CD4 T cells that secrete the immune suppressive cytokine interleukin-10 (IL-10) ( 8 , 13 ), which have been shown at the clonal level to mediate linked suppression of inflammatory T cells ( 14 ).

Despite over 25 years of efforts to develop immunomodulatory therapies to divert the course of type 1 diabetes, no therapeutic has yet emerged that balances robust efficacy with acceptable safety and tolerability for patients. This pharmacopoeial poverty comes at a time when there is increasingly clear evidence that retained C-peptide secretion, even down to the limits of conventional detection, is associated with significantly improved metabolic control and reduced risk of the serious diabetic complications that impact upon quality and duration of life ( 5 – 7 ).

Type 1 diabetes is a chronic autoimmune disease characterized by progressive, immune-mediated loss of β cell mass and function. After clinical presentation, most patients undergo continued attrition of remaining functional β cell mass and progress to the point at which residual C-peptide, a surrogate marker for insulin secretion, is absent or present at very low levels in the circulation ( 1 , 2 ). Two factors compound the clinical burden of type 1 diabetes. First, despite optimized insulin administration regimes, chronic hyperglycemia and hyperglycemic excursions are unavoidable in most patients and result in complications including retinopathy, nephropathy, and neuropathy, which reduce life expectancy by an average of over 10 years ( 3 ). Second, it is apparent that the incidence of the disease has been increasing by about 4% per year in recent decades, most notably in children and adolescents ( 4 ).

To examine whether peptide-treated C-peptide responders/nonresponders differ by additional metabolic markers, we also measured the proinsulin/C-peptide ratio during the MMTT, high levels of which are an indicator of β cell stress. We observed that both fasting and 90-min proinsulin/C-peptide ratio are significantly higher compared to baseline at multiple time points in peptide-treated C-peptide nonresponders (for fasting, P = 0.03, P = 0.004, and P = 0.02 at 3, 6, and 12 months, respectively; for 90-min, P = 0.004, P = 0.008, P = 0.03, and P = 0.047 at 3, 6, 9, and 12 months, respectively) ( Fig. 5 , A and B, and table S13). No change over time was observed in peptide-treated C-peptide responders, consistent with there being less β cell stress in this group ( Fig. 5 , C and D).

Extending these studies to the high-dimensional analysis of T regs , we noted an increase in levels of T reg expression of FoxP3, the master transcriptional regulator of these cells, during the treatment period (between baseline and 3 months) in peptide-treated C-peptide responders ( Fig. 4D , fig. S2, and table S8). Levels returned to baseline at 12 months and were unchanged throughout the study in peptide-treated C-peptide nonresponders. The greatest fold change in FoxP3 expression was seen in CD45RA − (memory) T reg subpopulations that lacked Helios expression, especially those coexpressing CD39 ( Fig. 4 , E and F). In contrast, Helios expression levels on T regs did not change in either study group ( Fig. 4G ). The proportion of CD8 T cells specific for β cells that expressed the marker of antigen experience (CD57 + ) was significantly lower in treated C-peptide responders compared to placebo and nonresponders at baseline (P = 0.01 and P = 0.04, respectively) and remained lower than placebo at 6 months ( Fig. 4H , fig. S3, and table S9). As observed in our previous studies in new-onset type 1 diabetes patients ( 8 ), detectable T cell responses to C19-A3 at baseline were present in a small minority of patients; significant treatment- and responder-related changes were not observed (table S10). There were no treatment- or response-related changes in autoantibodies or enzyme-linked immunospot (ELISPOT) responses to the control recall antigen (tables S11 and S12).

We next measured immune changes in peptide-treated C-peptide responders/nonresponders. We found a statistically significant difference between these groups over the duration of the treatment period (P = 0.0029). Higher levels of IL-10 responses appear to have been maintained in the responder group, and subsequent Bonferroni-adjusted, month-wise testing indicates that at 2, 5, and 6 months, IL-10 responses against proinsulin were specifically, significantly higher in peptide-treated C-peptide responders (P = 0.007, P = 0.047, and P = 0.03, respectively) ( Fig. 4B and table S7). Peptide-treated C-peptide responders (but not nonresponders) showed a trend for IFN-γ response levels against proinsulin to decline between starting therapy and the first assay performed at 1 month (P = 0.08) ( Fig. 4C ).

To provide further mechanistic insight, we also performed analyses on subjects divided according to the approach validated by Beam et al. ( 18 ) in which response to treatment is defined as a post-baseline value that is 100% or more of the baseline value of C-peptide AUC. There were 10 such “C-peptide responder” subjects identifiable during the treatment period (6 months)—1 in the placebo, 6 in the low-frequency, and 3 in the high-frequency groups. C-peptide responders were significantly more frequent in the low-frequency group than in the placebo group at 3 months (P = 0.03).

( A ) Cumulative mean CD4 T cell IL-10 and IFN-γ responses to proinsulin stimulation measured over the duration of the 6-month treatment period shown according to treatment group. ( B ) Mean CD4 T cell IL-10 and ( C ) IFN-γ responses to proinsulin stimulation measured at each month during therapy in peptide-treated subjects divided according to C-peptide responder status. Bars and symbols represent mean stimulation index (SI) at each time point, and error bars are the 95% confidence intervals (CI). For analysis over the treatment period, longitudinal measurements of the SI were transformed using the natural logarithm (“Ln”) and were analyzed with linear models having visit and treatment as main factors and a repeated-measures error structure. Estimates of the mean SI across visits were computed using model-based estimates (least-squares means). ( D ) Change in FOXP3 expression levels [mean fluorescence intensity (MFI)] on all T reg subsets (CD4 + CD25 hi FOXP3 + ), ( E ) on memory (CD45RA − ) adaptive T regs , and ( F ) on memory CD39 + T regs in peptide-treated subjects divided according to C-peptide responder status. ( G ) Change in Helios expression by T regs in the same period and same groups. ( H ) Mean percentage levels of antigen-experienced (CD57 + ) CD8 T cells stained with peptide-HLA tetramers loaded with β cell peptides at baseline and at 6 months in peptide-treated C-peptide responders, compared with placebo and nonresponder subjects. Error bars show means and SEM. (B to H) C-peptide responders/nonresponders defined as having a post-baseline value that is 100% or more of the baseline value of C-peptide AUC during the treatment period. There were 9 peptide-treated C-peptide responders (6 of 9 subjects in the low-frequency and 3 of 7 in the high-frequency groups) and 10 nonresponders.

We examined additional markers representing β cell stress and effector, regulatory, functional, and phenotypic features of global and antigen-specific adaptive immune responses. Over the duration of the treatment period, cumulative CD4 T cell IL-10 responses to proinsulin stimulation were significantly higher in the blood of the high-frequency compared with placebo (P = 0.015) and low-frequency groups (P = 0.003; Fig. 4A ). There were no significant differences in CD4 T cell interferon-γ (IFN-γ) responses to proinsulin, circulating subsets of T regs or activated CD8 T cells specific for β cell target peptides between these groups.

The study was designed to manage glycemic control intensively with a target HbA1c of less than 48 mmol/mol (6.5%). Therefore, differences in HbA1c between study groups would not be expected, and significant changes were not seen; however, there was a trend for HbA1c levels to increase over time in the placebo group, whereas in the treatment groups, there was a trend for values to decline and then stabilize after 6 months ( Fig. 3B and tables S5 and S6). To examine the combined impact of changes in HbA1c and insulin usage on metabolic control, we examined the IDAA1c according to the formula of Mortensen et al. ( 17 ). IDAA1c increased significantly over 12 months in the placebo group compared with baseline (P = 0.04; Fig. 3C ), consistent with a decline in endogenous insulin production, but was maintained at baseline levels in the intervention groups, consistent with C-peptide preservation. IDAA1c values were significantly lower in the high-frequency arm at baseline and 3, 6, 9, and 12 months (P = 0.02, P = 0.001, P = 0.003, P = 0.01, and P = 0.002, respectively) and significantly lower in the low-frequency arm at 6 and 12 months (P = 0.047 and P = 0.01, respectively) compared with placebo.

Other potential effects of proinsulin PIT on metabolic responses were assessed by changes in insulin use during the study. Mean change in average insulin dose (unit kg −1 day −1 ) showed a progressive rise in subjects in the placebo arm ( Fig. 3A and tables S3 and S4). In contrast, there was no significant change in average insulin dose in the high- and low-frequency arms of the study. As a result, mean changes in insulin use were significantly lower in the high-frequency arm at 6, 9, and 12 months (P = 0.03, P = 0.04, and P = 0.01, respectively) and significantly lower in the low-frequency arm at 12 months (P = 0.009) compared with placebo, with an overall difference between the treatment and placebo groups across all time points in MMRM analysis (P = 0.01).

However, we noted differences in C-peptide changes during the study that are worthy of discussion. The decline in stimulated C-peptide was different between study groups, and at the 3-month time point, mean loss of C-peptide in the placebo group exceeded that of the high-frequency (P = 0.03) and low-frequency groups ( Fig. 2A ). This difference in C-peptide decline was evident in individual data plots ( Fig. 2B and table S2): Compared with baseline, C-peptide levels in subjects receiving placebo showed a decline at every time point in every subject (apart from one subject, at 6, 9, and 12 months), and means declined significantly (P = 0.003, P = 0.03, P = 0.03, and P = 0.03 at 3, 6, 9, and 12 months, respectively) in paired analyses compared to baseline. This contrasted with findings in the treatment groups, in which the mean percent change was more modest, fewer individual subjects showed actual loss of C-peptide, and significant changes in means were only seen when comparing baseline with 12-month levels in the high-frequency group (P = 0.03). Thus, in this study, patients on placebo manifest an early decline of measurable C-peptide production, although this is not seen during administration of proinsulin C19-A3 peptide injections.

As specified in the predetermined analysis plan, C-peptide area under the curve (AUC) was compared between the treatment groups over time (3, 6, 9, and 12 months) by multilevel model repeated measures (MMRM) analysis adjusted for the baseline value of AUC, and no significant treatment-related effects were observed. There was no evidence of accelerated C-peptide loss in the treated groups compared to placebo.

Subjects enrolled were treated according to the regimen in Fig. 1 , and the participant flow is summarized in fig. S1. Peptide injection was very well tolerated with no serious adverse events considered to be treatment-emergent, and there was no evidence of hypersensitivity reactions at any time during the treatment course. Local erythematous skin reactions without local wheal or swelling have been observed previously with this peptide ( 16 ) and were seen in 8 of 9, 10 of 10, and 4 of 8 subjects in the high-frequency, low-frequency, and placebo groups, respectively, but did not change in quality or size over time.

Of the 233 patients referred to the study sites, 84 were assessed for eligibility and attended screening visits. Of these, 56 subjects did not have either the HLA-DRB1*0401 genotype or autoantibodies, and 1 subject had stimulated C-peptide <0.2 nM; all were excluded (fig. S1). After 24 subjects had been randomized, subjects who did not complete a minimum of 11 of 12 treatments (n = 1 in the low-frequency and n = 2 in the high-frequency groups) were replaced with additional study subjects (n = 2 in the low-frequency and n = 1 in the high-frequency groups by randomization) to maximize information on treatment exposure, but all subjects (n = 27) were retained in the analysis. Four subjects missed follow-up assessments (n = 3 in the low-frequency and n = 1 in the high-frequency groups; two subjects declined these visits, and two subjects were lost to follow-up). Baseline characteristics are shown in Table 1 and did not differ between groups, except for HbA1c, which was significantly higher in the placebo group compared with high-frequency group (P = 0.02). Planned primary and secondary end points are shown in table S1.

DISCUSSION

The principle that simple administration of antigens that are targeted in inflammatory diseases, such as autoimmunity and allergy, can have a therapeutic benefit has been borne out by many robust studies in preclinical models, as well as by more recent indications of success in the clinic (15, 19–21). Our group has developed a distinctive approach to this in type 1 diabetes, through HLA-guided identification of naturally processed and presented epitopes of major autoantigens, such as proinsulin, that can be developed for PIT (8, 22). The current phase 1b study was designed to explore safety (notably the risks of hypersensitivity and acceleration of loss of β cell function) and examine immunological effects of repeated dosing with such a native peptide sequence at the point of diagnosis of type 1 diabetes. We find that this approach is very well tolerated by patients even with dosing every 2 weeks for 6 months with no evidence of development of hypersensitivity.

We also find no evidence for accelerated loss of C-peptide secretion as an indicator of augmented β cell damage. Early C-peptide loss after diagnosis was apparent and significant in the placebo group but much less so in either of the treated groups, and C-peptide loss was significantly lower in the high-frequency group at 3 months. These results should be viewed with caution because C-peptide measurements can be variable, there were small numbers of subjects in each group with some imbalance between groups in baseline metabolic data (Table 1), and the study was not powered to examine efficacy, which would require many more subjects. However, patients receiving proinsulin PIT showed stable daily insulin use, compared with rising use in the placebo group. Stable insulin use in the treatment groups was not associated with poorer glycemic control; IDAA1c levels fell or stabilized, compared with an overall increase in the placebo group. Both treatment groups (high and low frequency) showed similar behavior in relation to C-peptide, insulin use, and HbA1c stabilization, consistent with a treatment effect. Although more frequent dosing was also safe, it did not appear to confer additional effects.

In exploratory analyses, we used validated criteria (18) to define a group of clinical responders by their retention of stimulated C-peptide secretion during the treatment period and found such subjects to be enriched in the peptide-treated groups. Note that these peptide-treated C-peptide responders/nonresponders also differed according to changes in proinsulin/C-peptide ratio during the study. Under normal conditions, very small amounts of proinsulin are secreted, but stressed β cells release more relative to mature insulin/C-peptide, due to endoplasmic reticulum dysfunction (23). Thus, the circulating proinsulin/C-peptide ratio is a measure of β cell stress, typically showing a rise shortly after diagnosis (24, 25) followed by reduction later in the disease (26). Our data can be interpreted as indicating that peptide-treated C-peptide responders have less β cell stress compared to nonresponders. Heterogeneity of response to treatment has been recognized in other intervention studies in type 1 diabetes, and understanding its underlying basis is important for maximizing therapeutic effects. Differences in the T cell response to proinsulin according to treatment group were also observed over the course of the current study, and there was a trend for several important differences in immunological responsiveness to emerge between responder/nonresponder groups. First, in relation to immune regulation, we observed a higher level of IL-10 responses to proinsulin in association with high-frequency treatment and trends for higher IL-10 responses in peptide-treated responders. There have been numerous reports that ASI and PIT induce IL-10 responses and that this is a key component of the therapeutic mechanism, although other mechanisms, including effects on conventional FoxP3+CD25high T regs , have also been observed in preclinical models (21). Linked to this, our finding of a higher fold change in T reg expression of FoxP3 in peptide-treated responders is of considerable interest, because it was most marked in a population of memory T regs coexpressing CD39, which is associated with controlling inflammation via IL-10 secretion (27). Moreover, the memory subsets markedly up-regulating FoxP3 expression were Helios-negative, suggesting that they are peripherally generated, adaptive T regs arising after treatment (28). It is proposed that autoantigen-specific CD4 T cells with immunoregulatory properties are induced and suppress bystander inflammatory responses to the same epitope, autoantigen, or related autoantigens being presented in cis by the same antigen-presenting cells (APCs) (14). In an extension of this effect, there is also evidence that under these conditions APCs are licensed to induce new cohorts of T regs (“infectious tolerance”) (29). It is tempting to speculate that administration of C19-A3 has resulted in the generation of IL-10+ proinsulin-specific CD4 T cells and/or adaptive T regs through infectious tolerance and that this response is causally related to the C-peptide retention observed in selected subjects, but this remains to be formally shown. It is also intriguing to note that a single subject in the placebo group is a C-peptide responder by the same criteria. Adequately powered, observational studies on similar subjects, using similar immune and metabolic biomarker assays, will be required to explore the underlying basis for this nonprogressor phenotype.

Why some peptide-treated subjects should respond whereas others do not is a common conundrum of the immunotherapy field. We have previously shown that a distinguishing feature of type 1 diabetes is the presence of circulating β cell–specific effector memory CD8 T cells that show evidence (CD57 expression) of recent antigen exposure (30). We found that baseline levels of this subset were low in treated C-peptide responders, raising the intriguing possibility that patients with restricted activation of autoreactive cytotoxic T lymphocytes represent a disease stratum that is more permissive to the immune regulatory effects inducible by PIT.

Our study extends experience with ASI and PIT in type 1 diabetes and provides further evidence that it has a very favorable safety profile, especially by comparison with biologic agents that carry the risk of acute toxicities, such as cytokine storm and circulatory compromise, as well as chronic effects, such as increased infection risk. The safety signal in PIT is coupled with strong evidence against any deleterious effect on β cell function. In combination, these two features make this an appealing strategy for prevention, both in stage 1 disease (defined as the presymptomatic presence of β cell autoimmunity evidenced by two or more islet autoantibodies with normoglycemia) and in those identified early in life as being at high genetic risk (31).

In summary, our study demonstrates that PIT using proinsulin peptide appears safe and well tolerated, even when administered over several months and during the autoinflammatory process that is associated with the immediate period after diagnosis of type 1 diabetes. Two-weekly dosing does not appear to confer any benefit over 4-weekly dosing. However, the major restriction of our study is the small number of subjects enrolled. Combined with disease heterogeneity, this limits opportunities to better understand dosing and identify robust immunological effects and biomarkers. Future studies will need to be powered for these and for efficacy, should examine whether children are similarly responsive, and begin to explore opportunities for prevention.