B19 New Potential Targets of Antibody-Drug Conjugates for Small-Cell Lung Carcinoma

Abstract

Small-cell lung cancer (SCLC) remains one of the high-grade malignancies, whereas non-small cell lung cancer benefits from molecular target drugs or immune checkpoint inhibitors as a result of investigating driver mutations and immune microenvironment. However, novel driver mutation was not identified through genome-wide sequence analyses, resulting in invariable therapeutic strategy for SCLC. Therefore, we have to shift from cytotoxic agent and molecular target drug in the care of SCLC. In recent years, different approaches to hematologic malignancies and solid tumors were established in clinical situation. Antibody-drug conjugates (ADCs) is the key technique. In this study, we aimed to search new therapeutic targets for ADCs toward a paradigm shift in treatment and research of SCLC. We sought to transmembrane proteins of SCLC as new targets for ADCs with computational-biologic approach. We demonstrated 565 genes were overexpressed on 51 small-cell lung cancer cell lines compared to 30 normal lung tissue samples by investigating gene expression profile available in open source of Cancer Cell Line Encyclopedia and National Center for Biotechnology Information (NCBI) with Human Genome U133 Plus 2.0 Array (ThermoFisher Scientific). Among the 565 genes, 31 genes manifested increased value of compensated fluorescence signal on average by 3 or more. Of the 31 genes, by investigating RNA sequence data for normal tissue in NCBI, we identified 7 genes expressed in limited organs. We adopted these 7 selected genes as candidates for new targets of ADCs. We examined these new target genes by evaluating in vitro cytotoxicity of corresponding monoclonal antibodies followed by secondary ADCs comprising PNU-159682, a derivative of nemorubicin, using SCLC cell lines with and without overexpression of these genes. Cytotoxicity assay targeting a certain transmembrane protein, one of the candidate molecules, showed distinct effect of secondary ADC, inducing a large amount of cell death in concentration-dependent manner while secondary ADC following murine IgG isotype control exhibited lack of cytotoxicity. Secondary ADC targeting the protein showed about fourfold greater potency than that using murine IgG isotype control as a primary antibody (EC50 3.3 nM versus 13.0 nM). Conversely, CRISPR-Cas9 mediated knockout of the gene showed explicit loss of the cytotoxic effect. The expression of the gene in normal organs were examined using human total RNA, which demonstrated lower expression of the gene in many organs than in brain. The distribution of the gene expression is preferable in the viewpoint of reducing side effects of the ADC, which cannot cross the blood-brain barrier. We successfully estimated new targets for ADCs by investigating membrane proteins and narrowing these proteins with computational-biologic approach. Through in vitro cytotoxicity assays, the protein -mediated ADC exhibited specific killing of SCLC cell lines overexpressing the gene, suggesting the gene can be a potential target of ADCs.