Immunological Characterization and Therapeutic Activity of an Altered-Peptide Ligand, NBI-6024, Based on the Immunodominant Type 1 Diabetes Autoantigen Insulin B-Chain (9–23) Peptide

Female 3- to 4-week-old NOD mice were purchased from The Jackson Laboratories (Bar Harbor, ME) and housed in a vivarium under pathogen-free conditions at Neurocrine Biosciences (San Diego, CA). Animal experimentation was approved by Neurocrine Biosciences’ Institutional Animal Care and Use Committee.

Mouse cells were cultured in Dulbecco’s minimal essential medium with high glucose supplemented with 2 mmol/l l-glutamine, 10 mmol/l HEPES (Cellgro, Herndon, VA), nonessential amino acids (Sigma, St. Louis, MO), 1 mmol/l sodium pyruvate, 50 μg/ml gentamicin, 126 μg/ml l-arginine, 20 μg/ml l-aspartic acid (Life Technologies, Grand Island, NY), 50 μmol/l 2-mercaptoethanol (Sigma), 100 units/ml penicillin, and 5 μg/ml streptomycin (Life Technologies). Heat-inactivated 10% fetal bovine serum (HyClone, Logan, UT) was added to the medium before use. Human T-cell lines were cultured in RPMI-1640 medium (Cellgro) with the supplements described above. All peptides, including the native insulin B_(9–23) (SHLVEALYLVCGERG) and several alanine-substituted APLs of B_(9–23), sperm whale myoglobin (110–121) [SWM_(110–121)], and biotinylated herpes simplex virus peptide (HSV)-2 VP16 (430–444) (EEVDMTPADALDDFD), were synthesized in house by Merrifield’s solid-phase method (22) and purified by high-performance liquid chromatography (HPLC) (purity >95%).

B_(9–23)-specific pathogenic T-cell clones PD12-2.35, PD12-2.40, PD12-4.1, and PD12-4.9 were isolated from the pancreatic islets of NOD mice with insulitis (6,7) and were maintained by stimulation with B_(9–23) peptide (25 μg/ml; Sigma) and irradiated (3,000 rad) syngeneic spleen cells for 4 days, followed by a resting phase of 5 days with the addition of recombinant human IL-2 (10 units/ml; Boehringer Mannheim, Mannheim, Germany). To assess reactivity to peptide antigens, T-cells (10⁵/well) and irradiated syngeneic spleen cells (5 × 10⁵/well) were cultured in 96-well flat-bottom tissue-culture plates (Costar) in the presence of several concentrations of the APL, B_(9–23) native peptide, or control peptides in a 7.5% CO₂ incubator. In some cultures, conditioned medium was removed after 48 h of incubation and analyzed for cytokine levels by enzyme-linked immunosorbent assay (ELISA) (monoclonal antibodies from PharMingen, San Diego, CA; anti-IL-2: JES6-1A12/JES6-5H4; anti-IL-4: BVD4-1D11/BVD6-24G2; anti-IL-5: TRFK5/TRFK4; anti-IL-10: JES5-2A5/JES5-16E3; anti-IFN-γ: R4-6A2/XMG1.2). In other cultures, T-cell proliferation was assessed by pulsing cultures with 1 μCi [³H]thymidine specific activity 25 Ci/mmol; Amersham Life Sciences, Arlington Heights, IL) for the last 18 h of a 72-h incubation. Cells were harvested onto glass fiber-lined plates (Unifilter-96 GF/B; Packard, Meriden, CT), and the amount of radioactivity incorporated into de novo synthesized DNA was measured in a scintillation counter (Top Count NXT; Packard).

Different doses of peptide antigens were diluted in a soluble vehicle (i.e., 10 mmol/l acetate buffer with 30 mg/ml d-mannitol, pH 6.0) and injected subcutaneously at different intervals in prediabetic female NOD mice (i.e., 4-week-old, insulitis-free). Peptide doses and frequency of administration are indicated in the figure legends. Mice were usually killed 3 days after the last injection and tested for responsiveness to antigen by culturing single-cell suspensions of lymph node or spleen cells (6 × 10⁵/well) with antigen, and proliferation and cytokine production was assessed as described above. In some experiments, peptides were also administered subcutaneously with incomplete Freund’s adjuvant (IFA) or complete Freund’s adjuvant (CFA), which contained 1–5 mg/ml heat-killed Mycobacterium (Difco).

T-cell lines were generated by culturing spleen cells (5 × 10⁶/well) from APL-vaccinated mice in 24-well culture plates (Costar) in the presence of 10 μmol/l of the appropriate APL used for vaccination for 7 days. After washing, 5 × 10⁶ T-cells were restimulated in 25-cm² tissue culture flasks with 20 × 10⁶ irradiated (3,000 rad) NOD spleen cells as antigen-presenting cells in the presence of APL (10 μmol/l) and recombinant human IL-2 (5 units/ml; Boehringer Mannheim) for another 7 days. After an additional eight stimulation cycles, T-cells were used in proliferation and cytokine assays. T-cell clones were derived from these cell lines by a limiting dilution procedure, as previously described (6).

Blood glucose was monitored using a glucometer (Encore Glucometer; Bayer, Elkhart, IN) at weekly intervals, beginning at 10 weeks of age. Mice with blood glucose levels ≥200 mg/dl on two consecutive occasions were considered diabetic. The data are presented as the percentage of animals remaining diabetes-free over the course of the experiment. The differences between curves were tested using the log-rank test, which compared the distributions over the entire observation period.

The B_(9–23) epitope of insulin appears to be an immunodominant target antigen for pathogenic autoreactive T-cells in NOD mice (6–9). An APL derived from B_(9–23) that lacks the ability to activate B_(9–23)-specific pathogenic T-cells, but that can induce an antigen cross-reactive regulatory T-cell population, may be a useful therapeutic agent. Therefore, we first determined the critical amino acid residues of the B_(9–23) peptide involved in stimulating pathogenic T-cells by generating several APLs with single alanine residue substitutions at each position of B_(9–23) and tested whether these APLs were able to stimulate a panel of five B_(9–23)-specific pathogenic CD4⁺ T-cell clones derived from pancreata of NOD mice (6,7). Alanine was used as the test replacement amino acid because of its small neutral side-chain. Although several APLs lost their ability to stimulate at least one clone (i.e., stimulation index [SI] ≤2 for positive stimulation), those APLs with alanine substitutions at positions 13, 15, and 16 (i.e., A¹³, A¹⁵, and A¹⁶, respectively) lost stimulatory activity on all five clones tested (Table 1), suggesting that these alterations disrupted either the TCR contact site or the MHC class II binding site of B_(9–23). In an effort to minimize cysteine-cysteine interactions, A¹³, A¹⁵, and A¹⁶ were modified to increase their stability by replacing the free cysteine residue at position 19 with alanine (i.e., A^13,19, A^15,19, and A^16,19). The alanine substitution at position 19 had no effect on peptide activity because it did not cause APLs to stimulate pathogenic T-cell clones (Table 2) and, in most cases, did not substantially alter the stimulatory activity of the native B_(9–23) (i.e., A¹⁹) peptide (Tables 1 and 2). These stable double alanine-substituted APLs were further characterized.

Although APLs must bind the MHC class II molecule for stimulation of a protective regulatory T-cell population, they must not activate the TCR of B_(9–23)-specific pathogenic T-cells. As an indirect assessment of whether A^13,19, A^15,19, and A^16,19 could bind MHC class II molecules, APLs were tested for their ability to competitively inhibit B_(9–23) from activating a pathogenic T-cell clone. All three APLs competitively inhibited B_(9–23) from activating clone 12-2.35, and this inhibition was specific because negative control peptides, SWM_(110–121) and neurotensin (NT), did not interfere with B_(9–23) stimulatory activity (Fig. 1). In addition, competitive inhibitory activity by these APLs was specific for binding the TCR of the clone and not for binding MHC class II molecules on antigen-presenting cells because the control peptides [i.e., SWM_(110–121) and NT], which are known to bind the NOD MHC class II molecules by inducing immune responses in NOD mice (A.G., L.J., unpublished data), did not compete with B_(9–23) for binding MHC. Thus, these APLs were able to bind MHC molecules of the NOD mouse and engage in a nonproductive (i.e., nonstimulatory) binding event with TCRs that competed with TCR binding of the native B_(9–23) antigen.

The disease-protective activity of APLs is thought to exist because of their ability to induce regulatory T-cell populations in vivo that cross-react with their native antigen counterpart, as has been demonstrated by APLs of neuroantigenic epitopes involved in EAE (12–20). We, therefore, determined whether these B_(9–23)-derived APLs could induce a specific T-cell response in vivo after subcutaneous administration of A^13,19, A^15,19, and A^16,19 in a buffered aqueous vehicle (soluble vehicle) to young prediabetic female NOD mice. The soluble vehicle, rather than CFA, was used in these experiments to promote the skewing of APL-induced T-cell responses toward a disease-protective Th2-like cytokine profile, as has been previously described for administration of native insulin peptides (23). Vaccination with each APL induced strong (SI >10) antigen-specific proliferative T-cell responses in the spleen (Fig. 2A). However, only modest cross-reactive responses to the native antigen, B_(9–23), were apparent in the spleens of APL-vaccinated mice (Fig. 2B). The degree of these cross-reactive responses (i.e., SI ≤2) was similar to those responses observed in naive age-matched NOD mice (Fig. 2C; SI ∼2), which makes it difficult to determine whether the APL induced the cross-reactive response. Note that vaccination with B_(9–23) in either soluble vehicle or CFA induced antigen-specific responses that were stronger than those in naive mice (SIs were roughly 20 and 6, respectively; Fig. 2C), demonstrating the capacity of NOD mice to respond to the native antigen. These results show that B_(9–23)-derived APLs themselves, given in a soluble vehicle, were able to induce antigen-specific responses in young prediabetic NOD mice, suggesting that this mode of administration could lead to regulatory T-cell responses that may protect against disease.

To further evaluate APL-induced cross-reactivity to B_(9–23) and to determine the cytokine profile of APL-induced T-cell responses, APL-reactive T-cell lines were generated from spleens of vaccinated mice. After eight cycles of restimulation with the appropriate APLs (i.e., A^13,19, A^15,19, and A^16,19), all three APL-derived T-cell lines showed a strong proliferative response to their respective APL (Fig. 3A), while maintaining a small but significant cross-reactivity to B_(9–23) (Fig. 3B). Although this cross-reactivity was greater than that seen immediately after vaccination (Fig. 2C), the response was nevertheless modest (i.e., SI for A^13,19 = 7.6, for A^15,19 = 2.7, and for A^16,19 = 2.5), which demonstrated that vaccination with APLs predominantly induces an APL-specific response. By stimulation with the appropriate APL, these cell lines showed Th0- or Th2-like cytokine profiles (Fig. 4). Although each cell line produced similar levels of the Th2-derived cytokines IL-4 and IL-5, A^13,19 produced large quantities of the Th1-derived cytokine IFN-γ, whereas A^15,19 and A^16,19 produced 8- to 10-fold less IFN-γ (Fig. 4). Therefore, the A^13,19-specific cell line was characterized as having a Th0 phenotype, and those specific for A^15,19 and A^16,19 were characterized as having a Th2 phenotype.

To further investigate the cross-reactive and Th2-inducing nature of APLs, we used A^16,19 (NBI-6024) to generate T-cell clones from the A^16,19-derived T-cell line because this APL showed the ability to induce both cross-reactive and Th2 responses. After challenging these A^16,19-specific T-cell clones with B_(9–23) for proliferative responses, 6 of 35 clones tested (i.e., 17.1%) scored positively in their response to B_(9–23) using an SI >2 as a criterion (Table 3). In addition, 35 of 38 clones tested (i.e., 92.1%) showed a Th2 phenotype (stimulated with A^16,19) by producing substantial levels of IL-4 and IL-10, and 3 of 38 clones tested (i.e., 7.9%) showed a Th0 phenotype by producing substantial levels of IFN-γ in addition to IL-4 or IL-10 (Table 4). Criteria for identifying positive cytokine-producing clones were derived from the levels of cytokines produced by the B_(9–23)-specific NOD Th1 pathogenic clones PD12-2.35, PD12-4.1, and PD12-4.9 (Table 4). These results are consistent with our characterization of the parental A^16,19-specific T-cell line, in that the frequency of cross-reactive clones (17.1%) derived from this line was similar to the degree of cross-reactivity induced by A^16,19 vaccination (Fig. 2) or to the degree of cross-reactivity in the A^16,19 T-cell line (Fig. 3). Furthermore, the frequency of Th2 (i.e., IL-4 and IL-10) versus Th0 (i.e., Th2 plus IFN-γ) clones, which was 92.1 vs. 7.9%, respectively, was similar to the relative proportions of these cytokines produced by the A^16,19 cell line (Fig. 4).

Because A^16,19 induced a Th2 phenotype in addition to a T-cell population that cross-reacted with the native B_(9–23) antigen, we chose this APL for its ability to modulate disease in NOD mice. The optimal dose of this APL was first determined by injecting single doses ranging from 0.005 to 20 mg/kg into young prediabetic NOD female mice and determining APL-induced proliferative responses 7 days later (Fig. 5). Although 0.005, 0.05, and 0.5 mg/kg did not induce detectable splenic T-cell proliferative responses, a dose-dependent induction of responses was observed with 5, 10, and 20 mg/kg, in which maximum responsiveness was achieved with 20 mg/kg (Fig. 5). Lymph node T-cell responses were less pronounced than splenic responses, a feature that is consistently observed with APL vaccinations in the soluble vehicle, whereas stronger lymph node responses were observed using CFA as a vehicle (data not shown).

To determine whether A^16,19 could effectively modulate the onset of disease in NOD mice, 20 mg/kg of APL was administered to young (4-week-old) predisease NOD female mice at weekly intervals for 12 weeks; one injection was administered every 2 weeks thereafter until termination of the experiment (i.e., 35 weeks), and spontaneous disease was monitored (i.e., glucose levels >200 mg/dl on two consecutive weekly readings). Females were used because disease incidence is dramatically greater in female NOD mice than in male NOD mice (i.e., 80–90% in female cohorts vs. 10–20% in male cohorts) (2), in which the onset of frank diabetes usually occurs by 15–20 weeks. In this experiment, the onset of disease occurred at 15 weeks of age in the control peptide group, in which disease incidence reached ∼50% by 20 weeks and 88–100% by 30 weeks in both control groups (Fig. 6A). However, disease onset in A^16,19-treated mice was significantly delayed (20% incidence at 21–30 weeks) and the maximum incidence achieved was reduced to 47% (53% diabetes-free), even at 32–39 weeks (Fig. 6A). This therapeutic activity prompted us to determine whether APL treatment of mice with severe insulitis or new-onset diabetes could modulate disease progression, an approach that would mimic most clinical settings with recently diagnosed new-onset diabetic subjects. APL was administered to 12- to 20-week-old female diabetic mice with severe insulitis in a single injection emulsified in IFA to maximize the delivery of the APL without inducing a Th1 response, as would be induced with CFA. Strikingly, treatment with a single dose of the APL reduced disease incidence from 81 to 53% in the control peptide-treated group (tetanus toxoid) (Fig. 6B). These results demonstrate that APL therapy may be more versatile than expected in clinical settings, where it could be administered to patients either before or after diagnosis.

Here, we report the first demonstration of a therapeutic activity of an APL derived from an islet β-cell-specific antigen in type 1 diabetes. This APL, A^16,19 (i.e., NBI-6024), was based on the B_(9–23) autoantigenic immunodominant epitope in the NOD mouse and was identified by generating a series of peptides containing single alanine substitutions at each amino acid residue of B_(9–23) and testing their ability to stimulate or competitively inhibit proliferation of B_(9–23)-specific NOD pathogenic T-cell clones. These experiments allowed us to identify at least three critical TCR contact residues at positions 13, 15, and 16 of B_(9–23) that prevented the activation of T-cell clones while allowing these peptides to maintain their binding to MHC class II molecules (i.e., a requirement for competitive inhibition). To increase peptide stability and to avoid adverse reducing effects of the cysteine at position 19, this position was substituted with alanine in all three of the APLs and conveyed a neutral function. These APLs did not appear to compete with B_(9–23) in MHC class II binding because the control peptides SWM_(110–121) and NT, which are known to bind the NOD MHC, did not alter the ability of B_(9–23) to stimulate T-cell clones. Additional evidence that these APLs could bind NOD MHC class II molecules for interaction with T-cells was demonstrated by their ability to induce immune responses in vivo, which led to the generation of APL-specific T-cell lines and clones.

Others have recently reported the development of APLs of other type 1 diabetes antigens involved in the human disease, such as GAD65 (24) and Imogen 38 (25). These APLs have been characterized by their ability to bind MHC class II molecules and to activate T-cell clones. Nepom et al. (24) identified an APL of the native GAD65_(553–585) peptide with an alanine substitution at position 561 (methionine) that resulted in complete inhibition of proliferation of a T-cell clone specific for GAD_(553–585). This APL maintained binding to two HLA-DR4 haplotypes, *0404 and *0405, which are highly associated with type 1 diabetes. An APL based on the 55–70 region of the mitochondrial 38-kDa pancreatic islet autoantigen, Imogen 38, was shown to block proliferation of an Imogen 38-specific Th1 cell clone from a type 1 diabetic patient (25). However, without in vivo efficacy data on their ability to modulate disease, the therapeutic activity of these APLs remains to be determined.

The B_(9–23)-based APLs in this study did not activate pathogenic B_(9–23)-specific T-cell clones but did induce a regulatory Th2 cell population that was cross-reactive with the native B_(9–23) antigen, a requisite for APL therapeutic activity (12,16,18–21). This cross-reactive population was relatively small compared with the non-cross-reactive APL-specific population because 1) the cross-reactive response of spleen cells after vaccination was weak, 2) the cross-reactive proliferative response of APL-induced T-cell lines was modest, and 3) the frequency of cross-reactive APL-induced T-cell clones was only 17.9%. This small cross-reactive population induced by APL vaccinations is consistent with that induced by another APL, Q144, based on the neuroantigenic peptide, PLP_(139–151) (i.e., W144), used in EAE (20). The number of cross-reactive T-cell clones derived from vaccinations with the Q144 APL constituted a small fraction of the total number of Q144-derived T-cell clones, in which Anderson et al. describe this heterogeneity in TCR specificity as “unfocused” cross-reactivity rather than “focused” cross-reactivity (20). Focused cross-reactivity would entail that each T-cell in an immune response to an antigen demonstrate cross-reactivity with another related antigen, which could lead to autoimmunity. However, unfocused cross-reactivity in which only a small subset of the T-cell population shows cross-reactivity would ensure immune diversity without the threat of a hyper-expansion of potentially harmful autoreactive T-cells (20). In the case of therapeutic APLs, the large non-cross-reactive APL-specific T-cell population may provide bystander Th2 cytokines for the small cross-reactive population being activated because of the close proximity between these populations during antigen presentation. The subsequent expansion of this cross-reactive population would influence both the Th2 development and activity of B_(9–23)-specific pathogenic T-cells. Thus, the therapeutic activity of the APL may work in a two-stage fashion in which a small cross-reactive population is first activated and expanded into a Th2 population in the draining lymph nodes near the site of vaccination. This Th2 population could then migrate to draining lymph nodes of the pancreas and dampen the activation of or promote the Th2 development of endogenous B_(9–23)-specific pathogenic T-cells using its cross-reactive capabilities. In addition, these regulatory Th2 cells could migrate directly to the inflamed pancreas where they suppress the activity of pathogenic Th1 cells. Indeed, we have observed, in a phase II clinical trial with MS patients receiving APL of MBP_(83–99), an initial APL-specific Th2 response immediately after dosing, which was modestly cross-reactive and was followed by a strong Th2 response to the native MBP_(83–99) antigen several weeks after dosing (18).

Perhaps this APL-induced mechanism may shed light on how aerosol or soluble forms of administration of the native insulin B-chain or B-chain peptides induce protective Th2 immunity in NOD mice (8,26–28). That is, the induction of any Th2 population via a soluble vehicle that reacts with B_(9–23) should be able to provide protection via a bystander mechanism involving Th2 cytokine production that dampens pathogenic B_(9–23)-specific Th1 cell activity. This Th2 population could be either an unfocused cross-reactive population, such as that induced by APL, or a non-cross-reactive B_(9–23)-specific population induced with B_(9–23) in a Th2-promoting vehicle. To ensure that Th2 responses were induced by APLs in this study, the peptides were administered in a soluble vehicle instead of CFA because CFA is known to induce strong Th1 responses, in part because Mycobacterium constituents in the adjuvant stimulate antigen-presenting cell production of the Th1-promoting cytokine IL-12. Alternatively, the absence of adjuvants usually leads to induction of weak, if any, T-cell responses, especially to foreign antigens. It was striking that even a single injection of APLs in the absence of adjuvant (i.e., soluble vehicle only) could induce strong T-cell responses in the spleen of NOD mice. Although lymph node responses were not readily detectable using a soluble vehicle, we have shown that injection of APLs emulsified in CFA induces robust responses in both the spleen and lymph nodes (A.G., L.J., unpublished data), demonstrating that adjuvants enhance the immunogenicity of APLs. Yet these APLs appear to have greater immunogenicity than conventional foreign antigens because of their elevated potency in soluble vehicles.

Through the use of the ELISPOT assay and short-term T-cell lines, we demonstrated that only type 1 diabetic subjects in the study had endogenous Th1 responses to B_(9–23) (10), which supports that B_(9–23) may be one of the immunodominant autoantigenic epitopes in humans as well as in the NOD mouse. In addition, we demonstrated that A^16,19 APL could bind the disease-associated HLA haplotype HLA-DQB1*0302 and was effective in competitively inhibiting B_(9–23) from stimulating short-term T-cell lines from a type 1 diabetic patient (P.A.G., unpublished data), a cell line that we previously characterized as having a Th1 phenotype (10). Therefore, the activity of A^16,19 in NOD mice demonstrates a potential therapeutic value, and, thus, APL therapies directed at this autoantigenic response might be beneficial in controlling type 1 diabetes in humans. Indeed, such efforts are currently underway in clinical trials.

The authors thank Taisho Pharmaceutical for their critical review of the manuscript and Dr. Lawrence Steinman for his helpful discussions and scientific expertise.