Fig. 7: AFM28 induces basophil depletion and shows a lower cytokine release than a T cell-engager.

A Comparison of cytokine release from primary human leukocytes treated with AFM28, a CD123/CD3 T cell engager or without antibody (w/o) after 24 h incubation. E_max denotes maximal induction of the respective cytokine. Cytokines were measured using the Luminex 24-plex human cytokine panel (only the 16 induced cytokines are shown). Individual data from two donors are shown. B Dose-dependent depletion of CD123⁺ basophils and dose-dependent changes in CD137 expression on NK cells after 24 h incubation of primary human leukocytes in the presence of titrated AFM28 or RSV/CD16A control engager (for further details and gating strategy see Supplementary Fig. 5B). Data are represented as mean ± SD of six donors and were analyzed using two-way ANOVA and Šídák’s multiple comparisons test. C Circulating healthy donor whole blood loops in the presence of titrated AFM28. Dose-dependent depletion of CD123⁺ peripheral blood basophils and dose-dependent changes in CD69 and CD16A expression on NK cells. The anti-CD16 3G8 antibody does not compete with AFM28 for CD16A binding, as further illustrated in Supplementary Fig. 6 and as previously shown for the innate cell engager acimtamig (AFM13) targeting the same CD16A epitope⁶⁶. For gating strategy see Supplementary Fig. 8. The E:T cell ratio between NK cells and basophils ranged between 2.1-9.2 amongst donors and did not correlate with the extend of basophil depletion. Individual data from n = 5 donors are shown (% remaining basophils, normalized versus formulation buffer). The population was normally distributed tested by the Shapiro-Wilk test, and significance was tested by two-way ANOVA and Turkey’s multiple comparisons test. Data are represented as mean ± SD. D AFM28 induces basophil depletion in cynomolgus monkeys, indicating PD activity towards CD123⁺ cells (gated as CD3⁻/CD14⁻/CD20⁻/CD159a⁻/HLA⁻DR⁻/FcεR1a⁺). Basophil recovery after day 15 was concurrent with ADA presence and corresponding loss of exposure. Arrows indicate dosing occasions. Data are represented as mean ± SD (n = 36 animals; 10 animals per vehicle, 20 mg/kg and 100 mg/kg groups; 6 animals per 4 mg/kg group). E Increases in IL-6 levels upon treatment of cynomolgus monkeys (n = 36 animals; 10 animals per vehicle, 20 mg/kg and 100 mg/kg groups; 6 animals per 4 mg/kg group) with AFM28 were detected only at later dosing time points correlating with the onset of an ADA response, mostly at 4 and 20 mg/kg doses of AFM28. ADA anti-drug antibodies.