Abstract 3498: Generation of an NG2 targeting bispecific antibody for induction of T cell immunity against melanoma

Abstract Analysis of mRNA levels using The Cancer Genome Atlas (TCGA) identifies high expression of neuron-glial antigen 2 (NG2) in several tumor tissues, particularly in melanoma, where it is selectively overexpressed in 90% of cases. NG2, also known as Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP) was originally identified on the surface of cutaneous melanoma. Due to its tumor-restricted expression, NG2 is an attractive target for immunotherapeutic approaches. Strategies that aim at the direct and specific lysis of malignant cells are needed in melanoma treatment despite the advent of immune checkpoint inhibiting antibodies. The latter lack tumor-specific activity and the capacity to directly recruit T cells to the tumor site. As a consequence, a considerable proportion of patients do not respond to immune checkpoint inhibitors, or for a limited time only. Here we report on the generation and characterization of two NG2 bispecific antibodies (bsAbs) with differing T cell binding properties. NG2xUCHT-1 contains a CD3 binder with high affinity, whereas NG2xM18 was generated using a modified UCHT-1 with approximately 100-fold lower CD3 affinity. The purity and integrity of the generated bsAbs were biochemically confirmed using SDS-PAGE and size exclusion chromatography. Dose titration of the two bsAbs on NG2 expressing cell lines documented dose-dependent binding with similar EC50 values of ∼0.5nM. Functional analysis in-vitro revealed that both bsAbs potently induce T cell activation and proliferation, resulting in strictly target-dependent tumor cell lysis. Of note, despite the significantly reduced binding affinity to T cells, NG2xM18 mediated comparable antitumor efficacy as NG2xUCHT-1, but induced substantially lower secretion of cytokines like IL-2, IFN-γ, and TNF, pointing to an optimized ratio of efficacy and potentially dose limiting side effects caused by cytokine release syndrome (CRS). Our findings indicate that NG2xM18 is a promising therapeutic compound that may allow for effective clinical dosing with reduced side effects. Citation Format: Nisha Prakash, Sebastian Hörner, Ilona Hagelstein, Gundram Jung, Helmut R. Salih. Generation of an NG2 targeting bispecific antibody for induction of T cell immunity against melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3498.