Journal: mAbs

Article Title: Artificial intelligence-assisted design of MAGE-A4 ×CD16 T-cell receptor-like natural killer cell engagers

doi: 10.1080/19420862.2026.2717991

Figure Lengend Snippet: Characterization and in vitro functional assessment of the MAGE-A4-targeting NKCE. (A) Schematic diagram of the NKCE molecular structure. NKCE is composed of a humanized anti-MAGE-A4 IgG framework fused with an anti-CD16 VHH, with an Fc N297A mutation to block Fc mediated ADCC activity. (B) SDS-PAGE analysis under reducing conditions. Lane 1, NKCE; lane 2, 5C4Hu IgG; lane 3, 5C4HuN297A. (C) Thermal stability analysis. The thermal denaturation temperature of the NKCE molecule was determined by circular dichroism (CD). (D) Tumor cell binding affinity. Binding ability of NKCE to HLA-A*02:01 + /MAGE-A4 + tumor cells (NCI-H1703, NCI-H1755, A375, and A375 HLA-A2 overexpress) was measured, CON: blank control with only secondary antibody staining. (E-F) Primary NK cell binding analysis. Flow cytometry demonstrated that NKCE effectively targets the CD3 − CD56 + population in human PBMCs via its anti-CD16 VHH domain. (E) Identification of NK cells in human PBMCs. NK cells were defined as the CD3 − CD56 + population by sequential flow-cytometric gating. (F) NKCE binding was subsequently evaluated within the pre-gated CD3 − CD56 + NK-cell population using a FITC-conjugated anti-human Fc secondary antibody. (G) ELISA comparison of 5C4Hu IgG, 5C4HuN297A, and NKCE binding to the GVY-HLA-A *02:01 complex. (H) Concentration-dependent ELISA binding of NKCE to recombinant CD16a and determination of the apparent EC50. (I) Jurkat-Lucia™ NFAT-CD16a reporter assay evaluating Fc silencing, target-enhanced CD16a reporter activation, and CD16a blockade. The indicated conditions included target cells alone, NKCE without target cells, 5C4Hu IgG with target cells, 5C4HuN297A with target cells, NKCE with target cells, and NKCE with target cells in the presence of a CD16a-blocking antibody. Antibodies were used at 100 nM, and reporter activation was measured after 6 h. Data are presented as mean ± SD of three technical replicate wells from one representative experiment. (J–L) Concentration-dependent cytotoxicity against (J) A375, (K) A375-OVER, and (L) NCI-H1755 cells. Data are presented as mean ± standard error of the mean, n = 3. Statistical significance was assessed using two-way ANOVA followed by multiple-comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: To evaluate the silencing of ADCC activity by the N297A mutation, an ADCC reporter assay was performed using Jurkat-LuciaTM NFAT-CD16 effector cells (InvivoGen, Cat. Code: jktl-nfat-cd16).

Techniques: In Vitro, Functional Assay, Mutagenesis, Blocking Assay, Activity Assay, SDS Page, Circular Dichroism, Binding Assay, Control, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Comparison, Concentration Assay, Recombinant, Reporter Assay, Activation Assay