A 1-year pilot study of intralymphatic injections of GAD-alum in individuals with latent autoimmune diabetes in adults (LADA) with signs of high immunity: No safety concerns and resemblance to juvenile type 1 diabetes.

AIMS: To test, for the first time in latent autoimmune diabetes in adults (LADA), the effects of autoantigen-specific immunotherapy by intralymphatic administration of aluminium-formulated recombinant human glutamic acid decarboxylase 65 (GAD-alum); specifically, to test if this treatment is safe, to test whether it induces a strong immunological response akin to a similar protocol in type 1 diabetes and to look for associations with preserved beta-cell function. MATERIALS AND METHODS: Three GAD-alum injections, 4 µg each, were administered 1 month apart into an inguinal lymph node in 14 people with newly diagnosed LADA (age 30-62 years) presenting with high levels of antibodies against glutamic acid decarboxylase (GADA). Adverse effects, immunological variables and beta-cell function were monitored, with detailed measurements at 5 and 12 months from baseline. RESULTS: Clinical adverse effects were minor and transient and measured laboratory variables were unaffected. All participants completed the study. Treatment raised levels of GADA, elicited strong effects on reactivity of peripheral blood mononuclear cells to GAD and raised cytokine/chemokine levels. Beta-cell function appeared stable preferentially in the seven participants carrying human leukocyte antigen (HLA) haplotypes DR3DQ2, as assessed by C-peptide glucagon tests (P < 0.05 vs. seven non-carriers). CONCLUSION: Intralymphatic treatment with GAD-alum in LADA is without clinical or other safety concerns over a 12-month period. As in a similar protocol used in type 1 diabetes, treatment exerts a strong immunological impact and is compatible with protection of beta-cell function preferentially in HLA-DR3DQ2 LADA patients. These findings pave the way for a randomized controlled trial in this important subgroup of LADA patients.

Nivolumab-induced autoimmune diabetes mellitus presenting as diabetic ketoacidosis in a patient with metastatic mucosal melanoma.

INTRODUCTION: Nivolumab is an immune checkpoint inhibitor used in the treatment of several malignancies. A number of immune-related endocrinopathies have been linked to its use. CASE REPORT: We report a unique case of a 74-year-old man with well-controlled diabetes mellitus type 2 and metastatic mucosal anorectal melanoma who presented with diabetic ketoacidosis after receiving his third cycle of nivolumab 240 mg intravenous (IV) every 2 weeks. He was found to have autoantibodies against glutamic acid decarboxylase 65. Genotyping for human leukocyte antigens showed the presence of DQB1*02:01 and DRB1*03:01. MANAGEMENT AND OUTCOME: His presentation was complicated by acute renal failure. He required aggressive fluid resuscitation and insulin supplementation to reverse severe acid-base disturbance and multiple electrolyte abnormalities. After an 8-week interruption, the patient restarted nivolumab without any further evidence of adverse events over the next 12 weeks. He continues to require insulin replacement therapy. DISCUSSION AND CONCLUSION: Development of type 1 diabetes with the use of immune checkpoint inhibitors has been increasingly reported in the literature. The exact mechanism for autoimmune diabetes precipitated by nivolumab is yet to be elucidated. Patient education about the symptoms of diabetes and regular glucose monitoring cannot be overemphasized. Testing for antibodies against glutamic acid decarboxylase 65, insulin receptors, and islet cells may also prove useful. Human leukocyte antigen DQ and DR haplotyping prior to immune checkpoint inhibitor treatment might help determine susceptibility toward developing type 1 diabetes, and provide opportunities for earlier recognition, intervention, and possibly prevention.

Assessment of CD4+ T cell responses to glutamic acid decarboxylase 65 using DQ8 tetramers reveals a pathogenic role of GAD65 121-140 and GAD65 250-266 in T1D development.

Susceptibility to type 1 diabetes (T1D) is strongly associated with MHC class II molecules, particularly HLA-DQ8 (DQ8: DQA1*03:01/DQB1*03:02). Monitoring T1D-specific T cell responses to DQ8-restricted epitopes may be key to understanding the immunopathology of the disease. In this study, we examined DQ8-restricted T cell responses to glutamic acid decarboxylase 65 (GAD65) using DQ8 tetramers. We demonstrated that GAD65 121-140 and GAD65 250-266 elicited responses from DQ8+ subjects. Circulating CD4+ T cells specific for these epitopes were detected significantly more often in T1D patients than in healthy individuals after in vitro expansion. T cell clones specific for GAD65 121-140 and GAD65 250-266 carried a Th1-dominant phenotype, with some of the GAD65 121-140-specific T cell clones producing IL-17. GAD65 250-266-specific CD4+ T cells could also be detected by direct ex vivo staining. Analysis of unmanipulated peripheral blood mononuclear cells (PBMCs) revealed that GAD65 250-266-specific T cells could be found in both healthy and diabetic individuals but the frequencies of specific T cells were higher in subjects with type 1 diabetes. Taken together, our results suggest a proinflammatory role for T cells specific for DQ8-restricted GAD65 121-140 and GAD65 250-266 epitopes and implicate their possible contribution to the progression of T1D.

Combinational spinal GAD65 gene delivery and systemic GABA-mimetic treatment for modulation of spasticity.

BACKGROUND: Loss of GABA-mediated pre-synaptic inhibition after spinal injury plays a key role in the progressive increase in spinal reflexes and the appearance of spasticity. Clinical studies show that the use of baclofen (GABA(B) receptor agonist), while effective in modulating spasticity is associated with major side effects such as general sedation and progressive tolerance development. The goal of the present study was to assess if a combined therapy composed of spinal segment-specific upregulation of GAD65 (glutamate decarboxylase) gene once combined with systemic treatment with tiagabine (GABA uptake inhibitor) will lead to an antispasticity effect and whether such an effect will only be present in GAD65 gene over-expressing spinal segments. METHODS/PRINCIPAL FINDINGS: Adult Sprague-Dawley (SD) rats were exposed to transient spinal ischemia (10 min) to induce muscle spasticity. Animals then received lumbar injection of HIV1-CMV-GAD65 lentivirus (LVs) targeting ventral α-motoneuronal pools. At 2-3 weeks after lentivirus delivery animals were treated systemically with tiagabine (4, 10, 20 or 40 mg/kg or vehicle) and the degree of spasticity response measured. In a separate experiment the expression of GAD65 gene after spinal parenchymal delivery of GAD65-lentivirus in naive minipigs was studied. Spastic SD rats receiving spinal injections of the GAD65 gene and treated with systemic tiagabine showed potent and tiagabine-dose-dependent alleviation of spasticity. Neither treatment alone (i.e., GAD65-LVs injection only or tiagabine treatment only) had any significant antispasticity effect nor had any detectable side effect. Measured antispasticity effect correlated with increase in spinal parenchymal GABA synthesis and was restricted to spinal segments overexpressing GAD65 gene. CONCLUSIONS/SIGNIFICANCE: These data show that treatment with orally bioavailable GABA-mimetic drugs if combined with spinal-segment-specific GAD65 gene overexpression can represent a novel and highly effective anti-spasticity treatment which is associated with minimal side effects and is restricted to GAD65-gene over-expressing spinal segments.

GAD65 antigen therapy in recently diagnosed type 1 diabetes mellitus.

BACKGROUND: The 65-kD isoform of glutamic acid decarboxylase (GAD65) is a major autoantigen in type 1 diabetes. We hypothesized that alum-formulated GAD65 (GAD-alum) can preserve beta-cell function in patients with recent-onset type 1 diabetes. METHODS: We studied 334 patients, 10 to 20 years of age, with type 1 diabetes, fasting C-peptide levels of more than 0.3 ng per milliliter (0.1 nmol per liter), and detectable serum GAD65 autoantibodies. Within 3 months after diagnosis, patients were randomly assigned to receive one of three study treatments: four doses of GAD-alum, two doses of GAD-alum followed by two doses of placebo, or four doses of placebo. The primary outcome was the change in the stimulated serum C-peptide level (after a mixed-meal tolerance test) between the baseline visit and the 15-month visit. Secondary outcomes included the glycated hemoglobin level, mean daily insulin dose, rate of hypoglycemia, and fasting and maximum stimulated C-peptide levels. RESULTS: The stimulated C-peptide level declined to a similar degree in all study groups, and the primary outcome at 15 months did not differ significantly between the combined active-drug groups and the placebo group (P=0.10). The use of GAD-alum as compared with placebo did not affect the insulin dose, glycated hemoglobin level, or hypoglycemia rate. Adverse events were infrequent and mild in the three groups, with no significant differences. CONCLUSIONS: Treatment with GAD-alum did not significantly reduce the loss of stimulated C peptide or improve clinical outcomes over a 15-month period. (Funded by Diamyd Medical and the Swedish Child Diabetes Foundation; ClinicalTrials.gov number, NCT00723411.).

GAD-alum immunotherapy in Type 1 diabetes mellitus.

Glutamic acid decarboxylase (GAD)-alum (Diamyd(®), Diamyd Medical, Stockholm, Sweden) is an adjuvant-formulated vaccine incorporating recombinant human GAD65, the specific isoform of GAD expressed in human pancreatic ß-cells and a major antigen targeted by autoreactive T lymphocytes in Type 1 diabetes mellitus. Intermittent vaccination with this protein is theorized to induce immune tolerance to GAD65, thereby potentially interrupting further ß-cell destruction. Hence, clinical trials are ongoing to examine the efficacy and safety of GAD-alum immunotherapy in patients with autoimmune-mediated forms of diabetes, including Type 1 diabetes and latent autoimmune diabetes in adults.

Extended evaluation of the safety and efficacy of GAD treatment of children and adolescents with recent-onset type 1 diabetes: a randomised controlled trial.

AIMS/HYPOTHESIS: The aim of this study was to investigate the safety and efficacy of alum formulated glutamic acid decarboxylase GAD(65) (GAD-alum) treatment of children and adolescents with type 1 diabetes after 4 years of follow-up. METHODS: Seventy children and adolescents aged 10-18 years with recent onset type 1 diabetes participated in a phase II, double-blind, randomised placebo-controlled clinical trial. Patients identified as possible participants attended one of eight clinics in Sweden to receive information about the study and for an eligibility check, including a medical history. Participants were randomised to one of the two treatment groups and received either a subcutaneous injection of 20 µg of GAD-alum or placebo at baseline and 1 month later. The study was blinded to participants and investigators until month 30. The study was unblinded at 15 months to the sponsor and statistician in order to evaluate the data. At follow-up after 30 months there was a significant preservation of residual insulin secretion, as measured by C-peptide, in the group receiving GAD-alum compared with placebo. This was particularly evident in patients with <6 months disease duration at baseline. There were no treatment-related serious adverse events. We have now followed these patients for 4 years. Overall, 59 patients, 29 who had been treated with GAD-alum and 30 who had received placebo, gave their informed consent. RESULTS: One patient in each treatment group experienced an episode of keto-acidosis between months 30 and 48. There were no treatment-related adverse events. The primary efficacy endpoint was the change in fasting C-peptide concentration from baseline to 15 months after the prime injection for all participants per protocol set. In the GAD-alum group fasting C-peptide was 0.332 ± 0.032 nmol/l at day 1 and 0.215 ± 0.031 nmol/l at month 15. The corresponding figures for the placebo group were 0.354 ± 0.039 and 0.184 ± 0.033 nmol/l, respectively. The decline in fasting C-peptide levels between day 1 and month 1, was smaller in the GAD-alum group than the placebo group. The difference between the treatment groups was not statistically significant. In those patients who were treated within 6 months of diabetes diagnosis, fasting C-peptide had decreased significantly less in the GAD-alum group than in the placebo-treated group after 4 years. CONCLUSION/INTERPRETATION: Four years after treatment with GAD-alum, children and adolescents with recent-onset type 1 diabetes continue to show no adverse events and possibly to show clinically relevant preservation of C-peptide. TRIAL REGISTRATION: ClinicalTrials.gov NCT00435981 FUNDING: The study was funded by The Swedish Research Council K2008-55X-20652-01-3, Barndiabetesfonden (The Swedish Child Diabetes Foundation), the Research Council of Southeast Sweden, and an unrestricted grant from Diamyd Medical AB.

GAD-alum (Diamyd)--a new concept for preservation of residual insulin secretion.

IMPORTANCE OF THE FIELD: Type 1 diabetes is a common and very serious disease. There has been very active research going on for a long time aiming at preservation of the residual insulin secretion by some kind of intervention to stop the destructive autoimmune process. This review covers a new type of immune intervention using auto-antigen treatment. AREAS COVERED IN THIS REVIEW: Immune interventions in type 1 diabetes have been tried during the last 30 years, this review mentions some of them, but the main topic is the use of the auto-antigen glutamic acid decarboxylase (GAD) to create tolerance to stop the autoimmune process. The clinical trials have been performed during the last 15 years and are all covered. WHAT THE READER WILL GAIN: This review will give the reader a picture of the research behind treatment with GAD as an immune intervention in type 1 diabetes. TAKE HOME MESSAGE: The key finding so far is that treatment with Diamyd has not only been shown to preserve residual beta cell function in type 1 diabetes, but this treatment may be the proof in humans of a new concept of treating and perhaps even preventing autoimmune diseases.

GAD65 vaccination: 5 years of follow-up in a randomised dose-escalating study in adult-onset autoimmune diabetes.

AIMS/HYPOTHESIS: The aim of this study was to ascertain whether treatment of GAD65 autoantibody (GADA)-positive diabetic patients with alum-formulated recombinant GAD65 (GAD-alum) is safe and does not compromise beta cell function. METHODS: This Phase 2, placebo-controlled, dose-escalation clinical trial, which was randomized through a central office, was performed in 47 GADA-positive type 2 diabetic patients, who received subcutaneous injections of GAD-alum (4 [n = 9], 20 [n = 8], 100 [n = 9] or 500 [n = 8] microg) or placebo (n = 13) at weeks 1 and 4 of the trial. Participants and caregivers were blinded to group assignments. The primary outcome was safety as assessed by neurological tests, medications and beta cell function evaluated over 5 years, representing the end of the trial. RESULTS: No severe study-related adverse events occurred during the 5 year follow-up. None of the dose groups was associated with an increased risk of starting insulin treatment compared with the placebo group. The use of oral hypoglycaemic agents did not differ between the dose groups. After 5 years, fasting C-peptide levels declined in the placebo group (-0.24; 95% CI -0.41 to -0.07 log(10) nmol/l; p = 0.01) and the 500 microg dose group (-0.37; 95% CI -0.57 to -0.17 log(10) nmol/l; p = 0.003), but not in the 4 microg (-0.10; 95% CI -0.28 to 0.07 log(10) nmol/l; p = 0.20), 20 microg (0.04; 95% CI -0.12 to 0.19 log(10) nmol/l; p = 0.58) and 100 microg (0.00; 95% CI -0.20 to -0.20 log(10) nmol/l; p = 0.98) dose groups. CONCLUSIONS/INTERPRETATION: The primary outcome of safety was achieved, since no severe study-related adverse events occurred. TRIAL REGISTRATION: Because the study was initiated before 1 July 2005, the protocol was not registered in a registry.

GAD treatment and insulin secretion in recent-onset type 1 diabetes.

BACKGROUND: The 65-kD isoform of glutamic acid decarboxylase (GAD) is a major autoantigen in patients with type 1 diabetes mellitus. This trial assessed the ability of alum-formulated GAD (GAD-alum) to reverse recent-onset type 1 diabetes in patients 10 to 18 years of age. METHODS: We randomly assigned 70 patients with type 1 diabetes who had fasting C-peptide levels above 0.1 nmol per liter (0.3 ng per milliliter) and GAD autoantibodies, recruited within 18 months after receiving the diagnosis of diabetes, to receive subcutaneous injections of 20 microg of GAD-alum (35 patients) or placebo (alum alone, 35 patients) on study days 1 and 30. At day 1 and months 3, 9, 15, 21, and 30, patients underwent a mixed-meal tolerance test to stimulate residual insulin secretion (measured as the C-peptide level). The effect of GAD-alum on the immune system was also studied. RESULTS: Insulin secretion gradually decreased in both study groups. The study treatment had no significant effect on change in fasting C-peptide level after 15 months (the primary end point). Fasting C-peptide levels declined from baseline levels significantly less over 30 months in the GAD-alum group than in the placebo group (-0.21 vs. -0.27 nmol per liter [-0.62 vs. -0.81 ng per milliliter], P=0.045), as did stimulated secretion measured as the area under the curve (-0.72 vs. -1.02 nmol per liter per 2 hours [-2.20 vs. -3.08 ng per milliliter per 2 hours], P=0.04). No protective effect was seen in patients treated 6 months or more after receiving the diagnosis. Adverse events appeared to be mild and similar in frequency between the two groups. The GAD-alum treatment induced a GAD-specific immune response. CONCLUSIONS: GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent-onset type 1 diabetes, although it did not change the insulin requirement. (ClinicalTrials.gov number, NCT00435981.)

Clinical evidence for the safety of GAD65 immunomodulation in adult-onset autoimmune diabetes.

The purpose of this Phase II study was to evaluate if alum-formulated human recombinant GAD65 is safe and does not compromise beta cell function. The study was conducted as a randomized, double blind, placebo-controlled, dose-escalation clinical trial in a total of 47 Latent Autoimmune Diabetes in Adults (LADA) patients who received either placebo or 4, 20, 100, or 500 microg Diamyd subcutaneously at Weeks 1 and 4. Safety evaluations, including neurology, beta cell function tests, diabetes status assessment, hematology, biochemistry, and cellular and humoral immunological markers, were repeatedly assessed over 24 weeks. None of the patients had significant study-related adverse events (AE). Fasting c-peptide levels at 24 weeks were increased compared with placebo (P=.0015) in the 20 microg but not in the other dose groups. In addition, both fasting (P=.0081) and stimulated (P=.0236) c-peptide levels increased from baseline to 24 weeks in the 20 microg dose group. GADA log levels clearly increased (P=.0002) in response to 500 microg Diamyd. The (CD4+)(CD25+)/(CD4+)(CD25-) cell ratio increased (P=.0128) at 24 weeks in the 20 microg group. No sudden increase in HbA1c or plasma glucose or decrease in beta cell function was observed in any of the dose groups. These positive findings for clinical safety further support the clinical development of Diamyd as a therapeutic to prevent autoimmune diabetes.