Afuco™ Anti-Human IAPP ADCC Recombinant Antibody (NI-203), ADCC Enhanced (CAT#: AFC-360CL)

Figure 1 NI-203.26C11 antibody selectively and dose-dependently recognizes pathological hIAPP aggregates in the pancreas of patients diagnosed with diabetes mellitus type 2 (T2D).

Thio flavin S (ThioS, left panels) staining of islet amyloid in pancreatic islets of T2D patients but not in healthy controls. NI-203.26C11 antibody shows a staining in T2D pancreatic islets loaded with islet amyloids (Subjects 1 and 2) but not in healthy control pancreatic islets lacking islet amyloids (Subjects 3 and 4). Detection of hIAPP aggregates on amyloid positive T2D pancreatic islets with recombinant mouse chimeric antibody NI-203.26C11 (NI-203.26C11-ch) at 3, 10, 30 nM (strong staining), and 1 nM (weak staining). NI-203.26C11 antibody does not recognize physiological hIAPP on healthy control pancreases. Anti-IAPP antibody (1 :100; anti-IAPP) and secondary donkey anti-mouse antibody only (anti-mouse secondary) were used as positive and negative controls, respectively. Counterstaining was performed to visualize cell nuclei (faint blue i.o., faint staining here).

Figure 2 NI-203.26C11 antibody selectively recognizes pathological hIAPP aggregates in T2D patients.

NI-203.26C11 antibody shows a staining in T2D pancreatic islets but does not recognize physiological IAPP on healthy control pancreatic islets lacking pathological hIAPP aggregates. Detection of hIAPP aggregates on T2D pancreatic islets with recombinant mouse chimeric antibody NI-203.26C11 (NI-203.26C11-ch; blue; 100 nM; top panels). NI-203.26C11 staining of hIAPP aggregates is restricted to islet areas deprived of β-cells (no merge with insulin staining in red). Detection of insulin on islet β-cells with anti- insulin antibody (1:3; red). Anti-IAPP antibody (1:100; anti-IAPP; bottom panels) recognizes physiological hIAPP on T2D and healthy control pancreatic islet β-cells, as shown by co- localization with insulin staining (merge with insulin staining in red).

Figure 3 NI-203.26C11 antibody targets aggregated hIAPP after a single administration in hIAPP transgenic mice.

Recombinant human NI-203.26C11 or isotype control (control IgG) antibodies were administered to 16 week-old hIAPP transgenic and wild- type mice at 10 mg/kg (i.p.) and antibody binding was evaluated 2 days after administration using an anti-human secondary antibody. Recombinant human NI- 203.26C11 (brown staining here) targets transgenic islets showing ThioS-positive amyloids (green staining here) but not ThioS -negative wild-type islets. No staining was observed with the control IgG. Counterstaining was performed to visualize cell nuclei (faint blue i.o., faint staining here).

Figure 4 NI-203.26C11 antibody protects against β-cell loss in hIAPP transgenic mice.

(A) Representative image of a mouse pancreatic islet stained with anti- insulin antibody to visualize β-cells (red i.o., strong staining here). (B) Once-weekly treatment with recombinant mouse chimeric NI-203.26C11 antibody in hIAPP transgenic mice (tg NI-203.26Cl l-ch, n=23; 10 mg/kg i.p. for 12 weeks) increases pancreatic insulin (insulin-positive area in % pancreas area and % islet area; top left and top middle panels), islet area (mean islet area; top right panel) and insulin secretion (plasma insulin levels; bottom left panel) compared with hIAPP transgenic mice receiving PBS (tg PBS, n=28). Islet density and pancreatic mass were unchanged after NI- 203.26C11-ch treatment (bottom middle and bottom right panel, respectively).

Figure 5 NI-203.26C11 antibody decreases fasting blood glucose, improves glucose tolerance and normalizes body weight gain in hIAPP transgenic mice.

Recombinant mouse chimeric NI-203.26C11 antibody was administered once-weekly to hIAPP transgenic mice (tg NI-203.26Cl l-ch, n=24; 10 mg/kg i.p.). PBS was used as vehicle (tg PBS, n=27). (A) NI-203.26C11 antibody significantly decreases fasting blood glucose after 8 and 12 weeks of treatment in hIAPP transgenic mice compared with PBS group. Blood glucose levels were measured after overnight fasting. (B) NI-203.26C11 antibody significantly improves glucose tolerance in hIAPP transgenic mice compared with PBS group. Blood glucose levels during an oral glucose tolerance test (oGTT) performed in hIAPP transgenic mice after 10 weeks of treatment. (C) Incremental body weight (%) was normalized in NI-203.26C11 -treated hIAPP transgenic mice over 12 weeks of treatment, as compared with wild-type mice injected with PBS (wt PBS, n=31).

Figure 6 NI-203.26C11 recognizes predominantly early fibrillar hIAPP aggregate species.

(A) Thioflavin-T (Thio-T) aggregation assay with a classical sigmoidal aggregation curve for hIAPP (■) but no aggregation for rodent IAPP (□; rIAPP). (B) Dotblot analysis of samples (1:3 dilution serie) taken from the Thio-T experiment at the time points indicated revealed unselective binding of anti-IAPP antibody to all hIAPP samples collected as well as to rIAPP taken after 20 min. In contrast, NI-203.26C11 displayed a selective binding for hIAPP preparations taken at the growth phase of hIAPP aggregation (5'), with decreased binding to hIAPP samples taken at later time points (10' and 20'). Importantly, NI-203.26C11 remained immuno-negative for non- aggregated hIAPP fractions (0') and did not react to rIAPP. In filter retardation assay (FRA), the anti-IAPP antibody recognized SDS-resistant fibrillar hIAPP species at all time-points assessed, whereas no reactivity could be seen for NI-203.26C11 against these hIAPP species. (C) Transmission electron microscopy (TEM) analysis of samples taken from the Thio-T aggregation assay revealed a diverse morphological spectrum. No large fibrillar aggregates could be detected for hIAPP taken before aggregation (0') neither for rIAPP taken at late stage (20'). Intermediate hIAPP species showed both, amorphous non- fibrillar as well as fibrillar characteristics (5') while samples taken at the border to (10') or within the equilibrium phase (20') predominantly showed a fibrillar morphology.

Figure 7 NI-203.26C11 antibody normalizes glucose tolerance in a transgenic rat model of type-2 diabetes (hIAPP transgenic rats).

Recombinant rat chimeric NI-203.26C11 antibody was administered once-weekly to hIAPP transgenic rats (tg NI-203.26C11-r, n=9; 3 mg/kg i.p.) and wild-type rats (wt NI-203.26Cl 1-r, n=4; 3 mg/kg i.p.). PBS was used as vehicle in hIAPP transgenic rats (tg PBS, n=10) and wild-type rats (wt PBS, n=5). (A) Oral glucose tolerance test (oGTT) before treatment in 3 month-old hIAPP transgenic rats and wild-type rats showing equivalent blood glucose levels. (B) NI- 203.26C11-r antibody normalizes blood glucose levels in hIAPP transgenic rats during an oGTT performed after 8 weeks of treatment, as compared with tg PBS and wt PBS rats. NI-203.26Cl l-r antibody does not affect blood glucose levels in wild-type rats (wt NI-203.26C11-r). tg NI-203.26C11-r compared with tg PBS: ** p < 0.01.