Therapeutic administration of Tregitope-Human Albumin Fusion with Insulin Peptides to promote Antigen-Specific Adaptive Tolerance Induction

Type 1 Diabetes (T1D) is an autoimmune disease that is associated with effector T cell (Teff) destruction of insulin-producing pancreatic beta-islet cells. Among the therapies being evaluated for T1D is the restoration of regulatory T cell (Treg) activity, specifically directed toward down-modulation of beta-islet antigen-specific T effector cells. This is also known as antigen-specific adaptive tolerance induction for T1D (T1D ASATI). Tregitopes (T regulatory cell epitopes) are natural T cell epitopes derived from immunoglobulin G (IgG) that were identified in 2008 and have been evaluated in several autoimmune disease models. In the T1D ASATI studies presented here, Tregitope peptides were administered to non-obese diabetic (NOD) mice at the onset of diabetes within two clinically-relevant delivery systems (liposomes and in human serum albumin [HSA]-fusion products) in combination with preproinsulin (PPI) target antigen peptides. The combination of Tregitope-albumin fusions and PPI peptides reduced the incidence of severe diabetes and reversed mild diabetes, over 49 days of treatment and observation. Combining HSA-Tregitope fusions with PPI peptides is a promising ASATI approach for therapy of T1D.

performed to confirm a BG levels between 200-300mg/dL for study entry. Prior to initiating the formal study, it was noted that many mice progressed rapidly beyond 300mg/dL at the second glycosuria test and were not able to be randomized. Therefore, the recruitment protocol was amended to perform two consecutive BG to confirm diabetes onset and to check that the BG level remained between 200-350 mg/dL at enrollment.
The initial number of mice in each group is indicated to the right of each colored bar. The goal was to include at least 10 mice per group. A total of 113 mice were initially enrolled, but many were excluded after the first BG due to rapid increase of the level at the second check (see recruitment details and protocol modification above). Since some mice that qualified over the target number of 60 became available, a total of five extra mice were split between two study groups (G4 and G5) and were also included in the final data analysis.
Each liposome study group G1-G6 received a total of six treatments (on Days 0,7,14,35,42 and 49) via subcutaneous (s.c.) injection with combinations of PBS (vehicle control with identical DMSO concentration as the peptides), PPI peptides and/or Tregitopes or scrambled Tregitope peptides (control). In all arms of the study, liposomes were formulated with one or combined (e.g. PPI and Tregitopes) test articles. For example, in the proposed T1D-ASATI "drug treatment" arm (starred), PPI peptides were formulated with the Tregitopes and co-administered in the same liposomes.

S1B. Pilot toxicity study
Legend for Supplemental Figure S1B: Due to well established concern about adverse reactions to human serum albumin in NOD mice, a pilot study was performed to determine the optimal dosing approach for the proposed HSA-Tregitope versus control group (HSA without Tregitope) studies. Four groups of mice (N=6 in each arm) were included in the human serum albumin pilot study. Group 1 mice received HSA as a full dose (800 µg), Group 2 received HSA as a split dose one day apart (400 µg); Group 3 received a split dose injection of the HSA in left and right flanks on the same day (2 x 400 µg), and Group 4 received HSA as a full dose (800 µg) but were pretreated with diphenhydramine, an antihistamine, 15 minutes prior to injection. Deaths were recorded in all groups. These deaths only occurred only on the second or later dose of therapy. Mice who died developed illness (lethargy, prostration) within 1-2 hours of dosing. Two thirds, or 67% of mice in the HSA (full dose) arm died soon after the second dose of HSA.
Antihistamine pre-treatment reduced mortality (from 67% to 17%), however, all mice developed antihistamine injection site reactions, characterized by hair loss and skin irritation. Split HSA dose by location (administered on the same day) did not reduce mortality (67%). Split dose by time (two doses of HSA, one day apart) led to the most favorable outcome for HSA administered without concurrent Tregitope, with good tolerance and reduced mortality (one death, third administration, 17%). Based on these results, the split dose by time was selected for both HSA (control treatment) and HSA-Tregitope fusion in the planned HSA-Tregitope fusion study.

S1C. HSA-Tregitope Fusion Study Design
Legend for Supplemental Figure S1C: Beginning at 16 weeks of age, a total of 246 NOD/ShiltJ (JAX# 1976) female mice were monitored for enrollment into the albumin-Tregitope fusion study. BG monitoring was performed twice weekly using a hand-held glucometer. Mice that had BG values between 200-350 mg/dl were retested as early as possible on the following day and those with a second confirmed BG level between 200-350mg/dl were enrolled into the study at the time of the second testing.
Mice were assigned to seven study groups on a rolling basis until each group contained 12 mice. Mice in groups 2-6 received split dosing of the treatments (Days 0/1, 13/14, 27/28, and 41/42) via subcutaneous injection; mice in group 1 received no treatment and mice in group 7 received single dose HSA-Tregitope treatment (without PPI co-treatment) on days 0, 13, 27 and 41. For those arms receiving peptides (PPI or  the Liposome and HSA-Fusion studies

S5B. HSA-Tregitope Fusion and PPI Study
Supplemental Figure S5: Lines representing mean blood glucose for each study group are provided for side-by-side comparisons. Liposome study data is shown in Figure S5A and Blood glucose (BG) trajectories are shown for mice who controlled their diabetes for three study arms of the HSA-Tregitope fusion study. Mice who did not control their diabetes (BG>200) are not included in these figures. More mice in the HSA-Fusion E arm were able to control BG even after having developed diabetes.
Supplemental Figure S7. Insulitis Score vs. Q-Twist for the HSA-Fusion Study Supplemental Figure S7. Pancreatic tissue was obtained from a subset of mice (n=22) from the HSA-Tregitope fusion study and submitted for pathological analysis. Three mice were examined from the G1 (No Treatment) and G2 (HSA) groups. Four mice were evaluated from the G3 HSA-Fusion 4 mice, five mice were included from the G4 group, HSA + PPI, and six mice were included from the G6 HSA-Fusion E + PPI Group. Mice were euthanized and pancreases were dissected free and fixed in 10% neutral-buffered formalin. Each pancreas was sectioned into 3 levels 100 µm apart. Granulated cells were stained with aldehyde fuchsin (AF) and leukocytes were stained with a hematoxylin and eosin counterstain.
Islets were individually assessed microscopically and score for insulitis. Previously described 35, 36 criteria were used to establish scoring criteria as follows: 0, no lesions; 1, peri-insular leukocytic aggregates; 2, <25% islet destruction; 3, >25% islet destruction; 4, complete islet destruction. An insulitis score for each mouse was obtained by dividing the total score for each pancreas by the total number of islets examined.
Microscopic examination was performed by expert technicians who were blinded to treatment group. Group data are presented as mean insulitis score. Slide images were obtained with an Olympus DP72 microscope digital camera. The average insulitis score for each group is plotted along with the Q-Twist scores calculated for each group .
The average insulitis scores per group were plotted against the Q-TWiST scores for quality of life (see Figure 7, higher Q-TWiST indicates higher cumulative duration of time in a lower blood glucose group).
Although this study was not powered to determine significant differences in insulitis scores, a trend is observed that mice treated with HSA-Fusion E + PPI (the 'drug treatment arm') had lower scores compared to the control groups. Lower insulitis scores were associated with higher Q-TWiST scores.