Ruifang Sun ¹ Qianqian Yu ¹ Chen Lin ² Long Zhang ² Yilu Zhou ^2* Runzhe Sun ^3* Yongsheng Meng ^1* Ningning Yao ⁴ Haizhen Wang ^1* Feilin Cao ^1* Meilin Liu ^1* Cheng Liao ^2* Jinfeng Ma ^4*

• ¹ Department of Tumor Biobank, Shanxi Province Cancer Hospital, Taiyuan, China
• ² Shanghai Shengdi Medicine Co. Ltd., Shanghai, China
• ³ School of Basic Medicine, Shanxi Medical University, Taiyuan, China
• ⁴ Department of Radiobiology, Shanxi Province Cancer Hospital, Taiyuan, China

Background: Epidermal growth factor receptor (EGFR) is a key protein in cellular signaling that is overexpressed in many human cancers, making it a compelling therapeutic target. On-target severe skin toxicity has limited its clinical application.Dual-targeting therapy represents a novel approach to overcome the challenges of EGFR-targeted therapies.: A single-cell tumor-normal RNA transcriptomic meta-atlas of lung adenocarcinoma (LUAD) and normal lung tissues was constructed from published data. Tumor associated antigens (TAAs) were screened from the genes which were expressed on cell surface and could distinguish cancer cells from normal cells. Expression of MUC1 and EGFR in tumors and normal tissues was detected by immunohistochemistry (IHC), bulk transcriptomic and single-cell transcriptomic analyses. RNA cut-off values were calculated using paired analysis of RNA sequencing and IHC in patient-derived tumor xenograft samples. They were used to estimate the abundance of EGFR-and MUC-positive subjects in The Cancer Genome Atlas Program (TCGA) database. Survival analysis of EGFR and MUC1 expression was carried out using the transcription and clinical data from TCGA. Results: A candidate TAA target, transmembrane glycoprotein mucin 1 (MUC1), showed strong expression in cancer cells and low expression in normal cells. Singlecell analysis suggested EGFR and MUC1 together had better tumor specificity than the combination of EGFR with other drug targets. IHC data confirmed that EGFR and MUC1 were highly expressed on LUAD and colorectal cancer (CRC) clinical samples but not on various normal tissues. Notably, co-expression of EGFR and MUC1 was observed in 98.4% (n=64) of patients with LUAD and in 91.6% (n=83) of patients with CRC. It was estimated that EGFR and MUC1 were expressed in 97.5% of LUAD samples in the TCGA dataset. Besides, high expression of EGFR and MUC1 was significantly associated with poor prognosis of LUAD and CRC patients.Conclusions: Single-cell RNA, bulk RNA and IHC data demonstrated the high expression levels and co-expression patterns of EGFR and MUC1 in tumors but not normal tissues. Therefore, it is a promising dual-TAA strategy combination for therapeutic targeting which could enhance on-tumor efficacy while reducing offtumor toxicity.