Targeting Intracellular Mutant NPM1 in Acute Myeloid Leukemia with a Novel Peptide-MHC-Specific T-Cell Bispecific Antibody

• Published in: Blood 2024-11, Vol.144, p.2210-2210
• Main Authors: Hong, Yunda, Chen, Dabing, Xu, Jingjing, Chen, Yanxin, Wu, Yangtao, Shi, Yang, Zhang, Tianying, Yuan, Quan, Xia, Ningshao, Yang, Ting, Hu, Jianda
• Format: Article
• Language: English
• ISSN: 0006-4971
• DOI: 10.1182/blood-2024-209636

Background High-frequency NPM1 mutations are essential for initiating malignant transformation at the early stages of leukemogenesis of acute myeloid leukemia (AML). Mutated NPM1 protein is retained in AML cells to maintain its malignant phenotype, with a relatively conserved alternative reading frame, positioning it as an ideal target for AML immunotherapy. However, the current therapeutic antibodies are capable of recognizing surface antigens, but failing to penetrate intracellular domains, thereby limiting the development of therapeutics aimed at NPM1 mutant proteins. To mimic the feature of T-cell receptor (TCR) that recognizes the complex of major histocompatibility class I and peptide on the cell surface derived from the processed intracellular antigen, we used NPM1mutA283-291/HLA0201 complex, as neoantigen, to develop a TCR-like fully human IgG1 antibody, for AML immunotherapy. Methods In this study, the NPM1mutA283-291/HLA0201 complex was synthesized and mouse hybridoma technology was used to prepare monoclonal antibody cells. Based on the human IgG1 antibody, we modified the positive clones to construct a variety of chimeric antibodies and humanized antibodies. The physicochemical properties and anti-tumor activity of these antibodies were evaluated in vitro and in vivo. Result Utilizing the CHO expression system, we successfully produced the recombinant NPM1mutA283-291/HLA0201 complex. To generate antibodies specific to this complex, hybridoma technology was employed, and protein-specific monoclonal antibodies were screened using flow cytometry. Notably, among these antibodies, mouse anti-2E2 demonstrated the most significant disparity in binding affinity to NPM1 compared to the control peptide. To explore the kinetics of antibody binding, surface plasmon resonance analyses were conducted. The Biacore sensorgrams depicting the interaction of mouse 2E2 antibody at different concentrations with NPM1mutA283-291/HLA0201 complex are presented, revealing an affinity constant (KD) of 158nM. The in vitro binding experiment between different concentrations of monoclonal antibody 2E2 and T2-peptide is depicted using flow cytometry. At varying concentrations, the binding strength of monoclonal antibody 2E2 to T2-NPM1 surpassed that to the T2 control peptide, reaching a maximum of 3.75 times. The OCI-AML3 cell line was utilized with the presence of NPM1 A-type mutation and HLA-A2 positivity. Monoclonal antibody 2E2 demonstrated consistently higher binding in