Abstract
Dasiglucagon is a next-generation glucagon analogue that is stable in aqueous formulation. This dedicated immunogenicity trial to support use as rescue treatment for severe hypoglycemia was conducted to evaluate the immunogenicity of repeated subcutaneous doses of dasiglucagon in subjects with type 1 diabetes. A total of 112 subjects were randomized 1:1 to receive three subcutaneous weekly doses of either 0.6 mg dasiglucagon or 1.0 mg recombinant glucagon (GlucaGen®) according to a double-blind parallel-group trial design. Subjects were followed for 15 weeks, with a multitiered testing approach planned for assessment of antidrug antibody (ADA) formation. For the primary immunogenicity endpoint, the overall ADA incidence was zero, as no subject demonstrated any treatment-induced or treatment-boosted ADA response at any time point in this trial involving three consecutive weekly doses of trial drug. No injection site reactions were reported for subjects receiving dasiglucagon. There were no unexpected safety findings for the trial.
Introduction
Dasiglucagon is a next-generation glucagon analogue that is stable in aqueous formulation. In addition to enabling continuous infusion through pump for glycemic control in diabetes and for other clinical settings, the stability in aqueous solution has enabled dasiglucagon development and approval (U.S. trade name Zegalogue) as a ready-to-use treatment for severe hypoglycemia through subcutaneous injection in patients with diabetes. 1 For treatment of severe hypoglycemia, dasiglucagon provides an alternative to glucagon products that require reconstitution before use (glucagon for injection, Eli Lilly; and GlucaGen® HypoKit®, Novo Nordisk) as well as to the newer ready-to-use glucagon products for subcutaneous injection (Gvoke®; formulation in dimethyl sulfoxide) 2 or intranasal administration (Baqsimi®; powder formulation). 3
While retaining specificity for the glucagon receptor and potency similar to native glucagon, the improved physical and chemical stability of dasiglucagon relative to native glucagon is achieved by substituting 7 of the 29 amino acids of which native glucagon is comprised. Aqueous formulation is thereby enabled, partly due to the fact that the propensity of glucagon to form fibrils and aggregate is significantly reduced by increasing the electrostatic repulsions between peptide molecules and removing amino acids involved in fibril formation. 4
The reduced aggregation potential of dasiglucagon would also be expected to reduce the risk of generating an immune response, given that aggregation and adduct formation of proteins may reveal new epitopes or lead to the formation of multivalent epitopes, which may stimulate the immune system. 5 However, as with any therapeutic administration of peptides, there is a potential risk of antibody formation and/or hypersensitivity reactions toward dasiglucagon. Clinical data from the comprehensive development program to support treatment of severe hypoglycemia indicate a relatively low immunogenicity-related risk for dasiglucagon, with no safety or efficacy concerns noted for the few subjects who had an antidasiglucagon antibody response. 6
However, in previous studies, the majority of subjects were exposed only to a single dose of dasiglucagon, which does not take into account that—although severe hypoglycemia occurs infrequently in most people with diabetes—any given person with type 1 diabetes is likely to experience several treatment-requiring events of severe hypoglycemia over time. We, therefore, conducted a dedicated immunogenicity trial to evaluate the immunogenicity of repeated subcutaneous doses of dasiglucagon and recombinant glucagon (GlucaGen) in subjects with type 1 diabetes. Secondary trial objectives comprised evaluation of safety, tolerability, and pharmacodynamic response.
Methods
The trial (
On dosing days, subjects were allowed to consume a small breakfast with corresponding administration of rapid-acting insulin, in accordance with their routine diabetes management. The use of long-acting insulin and the rate of basal insulin infusion were continued according to the subjects' individual blood glucose management plan. Subjects were to be at a pretreatment plasma glucose target of 70–150 mg/dL when dosed, which was ensured by subcutaneous administration of a fast-acting insulin analogue and/or consumption of oral glucose as needed.
To enable glucose response assessment at the 1st and 3rd dosing visits, subjects were asked to have similarly sized breakfasts (and a corresponding rapid-acting insulin dose) and were instructed to fast for 90 min after dosing of trial product. Subjects were monitored for at least 5 h after dosing at the trial center for safety, including blood glucose monitoring, and subjects were not discharged until they were considered stable with a blood glucose level in the range of 70–180 mg/dL.
Subjects were followed for 15 weeks from the day of the first dose, with follow-up visits on days 35, 60, and 104 after the first dose. It was planned that subjects be monitored until the antidrug antibody (ADA) levels had returned to baseline, and samples from ADA-positive subjects were planned to be tested for neutralizing potential in a neutralizing antibody assay. Furthermore, any subjects with treatment-induced or treatment-boosted ADA responses were planned to be called in for an additional (unscheduled) visit to evaluate the clinical effect of immunogenicity on pharmacodynamic and pharmacokinetic responses.
The trial was performed in accordance with the Declaration of Helsinki, International Conference on Harmonization guidelines, and Good Clinical Practice. An institutional review board or independent ethics committee approved the trial at each center, and all subjects gave written informed consent before undergoing any trial procedures or assessments.
ADA assay methods
The ADA assessment was planned as a multitiered testing approach. Tier 1 screening for antidasiglucagon antibodies in dasiglucagon-treated subjects was done using an enzyme-linked immunosorbent assay (ELISA). The same assay with inclusion of excess dasiglucagon was used as confirmatory analysis in Tier 2. Any confirmed positive samples were planned to be analyzed for potentially neutralizing antibodies including titer in a cell-based assay (Tier 3). A similar set of assays were used to analyze for antiglucagon antibodies in glucagon-treated subjects. Furthermore, antidasiglucagon positive samples were planned to be analyzed in the antiglucagon antibody analyses to assess for cross-reactivity to glucagon.
Comparable ADA assays for dasiglucagon and glucagon antibodies were developed. To overcome the tendency of glucagon to fibrillate and aggregate during handling, use of a direct ELISA format for screening, confirmatory, and titer assays was necessitated.
In the antidasiglucagon antibody screening assay, dasiglucagon was coated on microtiter plates. Thereafter, serum (minimum required dilution of 10) was incubated without dasiglucagon (for screening) and with excess dasiglucagon (for confirmation) on the plates. Captured antidasiglucagon antibodies were detected by an antimouse immunoglobulin (Ig) capable of detecting mouse IgG and IgM and cross-reacting with the human isoforms (IgG1–4κ,λ and IgM). The assay was validated using a dasiglucagon-specific monoclonal mouse IgG antibody. The assay sensitivity using the monoclonal mouse antibody was determined to be 13.6 ng/mL.
Based on the affinity difference of the detection antibodies for mouse and human Ig isoforms, the assay was expected to be five- to ninefold less sensitive to the human isoforms, resulting in an acceptable sensitivity of ∼100 ng/mL antidasiglucagon antibodies. To document detection of human Ig and to ensure assay control, quality control samples with human IgG and IgM were included during sample analysis by coating defined concentrations of human IgG and IgM directly to selected wells of the ELISA plate. Partial validations for the detection of human IgG and IgM were performed, which demonstrated detection of IgG down to 60 ng/mL and IgM down to 300 ng/mL. The assay drug tolerance was tested up to 4000 ng/mL dasiglucagon (1180 nM), which indicated no interference of remaining drug at scheduled ADA sampling time points.
A similar set of ADA assays was developed and validated for evaluation of the immunogenicity of recombinant glucagon. The validation results were comparable with those for the antidasiglucagon ADA assays. For further details of validation parameters, see Supplementary Tables S1 and S2 available online.
Primary immunogenicity endpoint methodology
Determination of ADA incidence comprised counting subjects who were ADA negative at baseline and ADA positive after trial drug administration (treatment-induced ADA) and subjects who were ADA positive at baseline with significant increases (greater than or equal to fivefold) in ADA titer after trial drug administration (treatment-boosted ADA). An informal analysis of incidence difference between treatment groups was planned but was not done, as no ADA-positive subjects were identified.
Results
Of 131 subjects screened for the trial, 112 subjects were randomly assigned to treatment groups, and 111 subjects received at least 1 dose of either dasiglucagon or GlucaGen (safety analysis set). A total of 102 (91%) subjects received all three planned doses. Overall, the subject characteristics at baseline were well matched between treatment groups, although the mean age of subjects was slightly higher in the dasiglucagon group than in the GlucaGen group (Table 1).
Baseline Characteristics of Subjects
Data are mean (standard deviation) unless otherwise stated.
The overall ADA incidence was zero. No treatment-induced or treatment-boosted ADA response was identified for any patient at any time point after dosing.
No major differences between treatment groups were noted for adverse events (AEs). Consistent with nausea and vomiting being common side effects after administration of glucagon, nausea was reported for 45.6% of subjects receiving dasiglucagon and 42.6% of subjects receiving GlucaGen, with corresponding proportions of 21.1% and 14.8% for vomiting, respectively. No injection site reactions were reported for subjects receiving dasiglucagon, whereas eight events in six subjects receiving GlucaGen were recorded. None of the reported hypoglycemic events were assessed as related to trial drug.
A single serious AE was recorded. This concerned an event of hypoglycemia with onset at ∼26 h after dosing with dasiglucagon. The event was evaluated as unlikely related to trial drug by the investigator and was concluded to be due to a temporary mismatch between administered and required insulin.
As there were no subjects with a treatment-induced or treatment-boosted ADA response, the planned assessment of the effect of immunogenicity on pharmacodynamic and pharmacokinetic response was not performed.
Discussion
This trial was a dedicated immunogenicity trial designed to evaluate the immunogenicity of the novel glucagon analogue dasiglucagon. Aiming to provide data supporting use as rescue treatment for severe hypoglycemia, the occurrence of ADAs, neutralizing ADAs, and cross-reactivity toward native glucagon was assessed after three subcutaneous weekly doses of dasiglucagon or recombinant glucagon (GlucaGen) in individuals with type 1 diabetes. Trial participants were followed for 15 weeks from the day of the first dose.
No incidences of treatment-induced or treatment-boosted ADA were detected in any subject in the dasiglucagon group (n = 57) or GlucaGen group (n = 54). As no ADA development was observed, the impact of potential ADAs on pharamacokinetics, pharmacodynamics, and other safety parameters could not be evaluated. The absence of ADA is reassuring given that this was a dedicated immunogenicity trial involving consecutive exposures to the test products. The results are consistent with the immunogenicity profile observed in the clinical trials supporting dasiglucagon treatment of severe hypoglycemia, in which a treatment-induced antibody response was observed in ∼1% of subjects exposed to dasiglucagon, with no clinical consequences identified. 6 This trial does not provide conclusive evidence of the potential immunogenicity of dasiglucagon during continuous or long-term treatment; this is being investigated as part of other ongoing dasiglucagon clinical development programs.
Dasiglucagon appeared well tolerated, and overall, the safety data did not indicate clinically relevant differences between groups. In addition to episodes of hypoglycemia, which are expected in a population with type 1 diabetes, the most frequent AEs were nausea and vomiting. These are well established common side effects of glucagon treatment. 2,3,7 No injection site reactions were reported for subjects receiving dasiglucagon.
Conclusion
In this dedicated immunogenicity trial, no ADA development after three consecutive weekly exposures to dasiglucagon or recombinant glucagon was identified, and there were no unexpected safety findings. These data support a low immunogenicity risk for dasiglucagon for the treatment of severe hypoglycemia.
Footnotes
Authors' Contributions
T.R.P. and R.T. wrote the article. T.R.P., O.A., O.S., D.D., L.E.H., and R.T. reviewed, edited, and approved the article for submission.
Acknowledgments
The authors thank the subjects who participated in the trial. Zealand Pharma sponsored this trial and was involved in the design and conduct of the trial as well as analysis and interpretation of the data. T.R.P., O.A., O.S., and D.D. were involved in conducting the trial. Karsten Soendergaard of Zealand Pharma provided medical writing support.
Author Disclosure Statement
T.R.P. has received research funding (paid to the university) from Zealand Pharma, AstraZeneca, Novo Nordisk, and Sanofi and consulting fees from Adocia, Arecor, AstraZeneca, Eli Lilly, Novo Nordisk, Sanofi, and The Longevity Labs. O.A. has received research support from Zealand Pharma. O.S. has received research funding from AstraZeneca, Boehringer Ingelheim, Eli Lilly, Gilead, Kowa, Novo Nordisk, Pfizer, Sanofi, and Zucare, is on the speakers bureau for AstraZeneca, Boehringer-Inhelheim, Eli Lilly, Novo Nordisk, and Sanofi, and has received consulting fees from Acerus, Boehringer-Ingelheim, Eli Lilly, Merck, Novo Nordisk, and Sanofi. D.D. has received research support from Zealand Pharma, Novo Nordisk, Eli Lilly, and Novartis. L.E.H. and R.T. are employed at Zealand Pharma.
Funding Information
The trial was funded by Zealand Pharma.
Supplementary Material
Supplementary Table S1
Supplementary Table S2
References
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
