In vitro characterization of a novel anti-GD2/ROR1 bispecific antibody exhibiting tumor cell killing efficacy in neuroblastoma cross-cancer activity in ROR1-positive triple-negative breast cancer.

BACKGROUND

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Although anti-disialoganglioside 2 (GD2) monoclonal antibodies have become the standard immounotherapy for high-risk petients, antigen escape and drug resistance still lead to relapse or suboptimal responses in some cases. This study aims to develop a novel bispecific antibody (BsAb) G/R-001, which specifically binds to both GD2 and receptor tyrosine kinase-like orphan receptor 1 (ROR1), for the treatment of NB and other tumors.

METHODS

G/R-001 BsAb (IgG1 subtype) was constructed by fusing anti-ROR1 scFv to the C-terminus of anti-GD2 heavy chain. G/R-001 was transiently expressed in HEK293 cells, purified by affinity chromatography, and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for structure and purity. Enzyme-linked immunosorbent assay (ELISA) and flow cytometry were used to evaluate the binding capacity of G/R-001 to GD2 and ROR1. The in vitro cytotoxic activity of G/R-001 against GD2/ROR1-positive NB cells and other cancer cell lines was assessed using the lactate dehydrogenase (LDH) assay.

RESULTS

The theoretical molecular weight of G/R-001 was 198.82 kDa, with SDS-PAGE results showing high purity (the molecular weight of the intact antibody with glycosylation modification corresponded to approximately 248.68 kDa on non-reducing electrophoresis). Under reducing and non-reducing conditions, bands corresponding to heavy chain, light chain, and intact antibody were observed respectively. ELISA results showed that G/R-001 had a binding capacity of 12.1 nM for GD2 and 3.5 nM for hROR1. Flow cytometry further confirmed the specific binding of G/R-001 to GD2 and ROR1. LDH assay results showed that, compared with Naxitamab or Zilovertamab alone, G/R-001 exhibited significant cytotoxic activity against GD2+/ROR1- LA-1, GD2-/ROR1+ MDA-MB-468, and GD2+/ROR1+ MHH-NB-11 cells in a concentration-dependent manner, with cell-killing rates of 59.03%, 67.58%, and 84.50% at 100 nM, respectively. Among these, G/R-001 demonstrated the strongest cytotoxic effect against GD2+/ROR1+ MHH-NB-11 cells. G/R-001 may synergistically activates natural killer (NK) cells to potently kill NB and minimize antigen-escape risk.

CONCLUSIONS

The G/R-001 BsAb can simultaneously bind to GD2 and ROR1, and shows significant anti-tumor activity in vitro. These results provide strong experimental evidence for the further development of G/R-001 as a novel anti-tumor therapeutic agent.