Journal: Frontiers in Immunology

Article Title: CLEC12A-directed immunocytokine with target cell–restricted IL-15 activity for treatment of acute myeloid leukemia

doi: 10.3389/fimmu.2025.1561823

Figure Lengend Snippet: Production of anti-CLEC12A IL-15 immunocytokines and characterization of on-target and off-target activity using leukemia cell lines. (A) Schematic illustrations of the MIC12 immunocytokine consisting of the humanized VH and VL domains of the 33C2, human IgG1 constant domains containing the SDIE modification (S239D; I332E), (G 4 S) 4 linker and a C-terminal IL15 E46K mutein (right) and a control Fc-optimized mAb without IL-15 moiety (33C2 SDIE ) (left). (B) SDS-PAGE electrophoresis of the humanized Fc-optimized 33C2 SDIE antibody and MIC12 in reducing (R) and non-reducing (N) conditions. Expected molecular weights: 23 kDa for the light chain, 63 kDa for the heavy chain of the MIC construct and 49 kDa for the heavy chain of the antibody. (C) Exemplary results of a size exclusion chromatography of the MIC12. Expected size of the full construct 172 kDa. (D) Binding of MIC12 and 33C2 SDIE to U937 cells at equimolar concentrations detected with donkey anti-human IgG-PE conjugate and flow cytometric analysis (grey peaks, anti-CLEC12A; outline peak, donkey anti-human IgG-PE conjugate). (E) Dose titration of MIC12 on U937 and EOL-1 cells detected with donkey anti-human IgG-PE conjugate and flow cytometric analysis. (F) Binding of MIC12 to IL-15Rα-expressing TF-1 (left) and IL15Rβ/γ-expressing M-07e cells (right). Binding was detected as described in (D) . A MIC construct with irrelevant specificity containing wild-type IL-15 was used as a positive control, 33C2 SDIE was used as a negative control. (G, H) PBMC from healthy donors (n=3) were cocultured with U937 (G) or EOL-1 (H) cells (E:T 2.5:1) with the indicated constructs at equimolar concentrations (1 µg/ml for the antibody and 1.2 µg/ml for the MIC). IFN-γ secretion in culture supernatants was determined after 24 h using ELISA. MIC-iso, – MIC construct with irrelevant specificity. P values were determined with Kruskal-Wallis test, followed by Dunn’s multiple comparison test. (I, J) PBMC from healthy donors were incubated with U937 and EOL-1 cells at an E:T ratio of 2.5:1 in the presence of constructs for 72h (1 µg/ml for the antibody and 1.2 µg/ml for the MIC). CD69 expression on CD3-CD56+ NK cells was determined by flow cytometry. (I) Exemplary flow cytometry plots derived with U937 target cells; the percentage of CD69 + NK cells is indicated. (J) Combined results obtained with 5 PBMC donors using U937 (left) and EOL-1 cells (right). Data are shown as means of triplicate measurements. P values were determined with Kruskal-Wallis test, followed by Dunn’s multiple comparison test. (K, L) PBMC from healthy donors were incubated for 72 h with CellTrace Violet (CTV)-labelled U937 or EOL-1 cells at an E:T ratio of 2.5:1 in the presence of the indicated constructs at equimolar concentration (1 µg/ml for the antibody and 1.2 µg/ml for the MIC). Target cell lysis was determined by flow cytometry. (K) Exemplary flow cytometry plots obtained with U937 target cells; the percentage of dead target cells (CTV + 7AAD + ) is indicated. (L) Combined results obtained with U937 (left) and EOL-1 (right) cells using 11 and 7 independent PBMC donors, respectively. Cell viability was normalized to the untreated control (mean of duplicate wells). P values were determined with Friedmans test, followed by Dunn’s multiple comparison test. (M, N) PBMC (M) or purified NK cells (N) from healthy donors were incubated for 72 h with CellTrace Violet (CTV)-labelled U937 cells at an E:T ratio of 2.5:1 (PBMC) or 1:1 (NK cells) in the presence of the indicated constructs titrated in a broad concentration range. Target cell lysis was determined by flow cytometry. Representative results from a total of three donors with similar outcomes are presented.

Article Snippet: Subsequently, analytical size exclusion chromatography (Superdex 200R PC3.2/30 column; Cytiva) and SDS-PAGE in 4-12% gradient gels (Invitrogen) were performed to assess antibody size, aggregation and integrity.

Techniques: Activity Assay, Modification, Control, SDS Page, Electrophoresis, Construct, Size-exclusion Chromatography, Binding Assay, Titration, Expressing, Positive Control, Negative Control, Enzyme-linked Immunosorbent Assay, Comparison, Incubation, Flow Cytometry, Derivative Assay, Concentration Assay, Lysis, Purification