Generation of an NG2 targeting bispecific antibody for the induction of T-cell immunity against melanoma

Abstract Background Melanoma remains a highly aggressive malignancy, and many patients fail to achieve durable benefit despite immune checkpoint inhibitor (ICI) therapy. ICIs improve outcomes but lack tumor specificity and do not directly recruit T-cells to tumor cells resulting in substantial side effects, highlighting the need for targeted immunotherapies selectively toward melanoma. Neuron glial antigen-2 (NG2) represents an attractive antigen for T-cell redirecting strategies due to its largely tumor restricted expression. Methods Here we report the generation and characterization of T-cell engaging NG2‑targeting bispecific antibodies. (bsAbs). To finetune CD3 affinity, we generated two constructs with differing CD3-affinities. NG2xCD3 high contains a UCHT-1-based CD3-binder with high affinity, whereas NG2xCD3 low carries a UCHT‑1 variant with approximately 100‑fold reduced CD3 affinity. Both constructs were functionally assessed in NG2-high and NG2-low melanoma cell lines covering several key aspects of T-cell mediated immune response. Results In cultures with NG2-expressing melanoma cells, both constructs mediated immune-synapse formation, T-cell activation, proliferation, and strictly NG2-dependent tumor cell killing. Both NG2xCD3 high and NG2xCD3 low supported differentiation into memory T‑cell subsets, but NG2xCD3 low induced less cytokine secretion and diminished functional activity, particularly against NG2 lower expressing targets. NG2xCD3 high maintained robust cytokine production and sustained cytotoxicity across both NG2‑high and NG2‑low tumor models. Conclusion Our data identify NG2 as a promising target for T-cell-redirecting therapy in melanoma and demonstrate that, within the IgG-scFv format, high CD3 affinity is required to achieve potent effector function across differing NG2-expression levels. NG2xCD3 high was thus identified as lead candidate for further development in NG2-expressing malignancies.