Later, we analyzed the binding specificity of the MUC1/CD3 BsAb via flow cytometry and ELISA assay

Later, we analyzed the binding specificity of the MUC1/CD3 BsAb via flow cytometry and ELISA assay. and CD3-positive cells and efficiently enhance T cell activation, cytokine release, and cytotoxicity. Furthermore, our study demonstrated that this BsAb could potently redirect T cells to eliminate MUC1-expressing tumor cells in vitro and significantly suppress MUC1-positive tumor growth in a xenograft mouse model. Thus, T cell-engaging MUC1/CD3 BsAb could be an effective therapeutic approach to combat MUC1-positive tumors and our MUC1/CD3 BsAb could be a promising candidate in clinical RV01 applications for the treatment of MUC1-positive cancer patients. Keywords: Mucin1, epithelial cancers, RV01 oncogenic molecule, immunotherapy, BsAb 1. Introduction Mucins are large and highly glycosylated proteins and are expressed in many epithelial cells. They play important functions in lubricating and hydrating epithelial cell surfaces and function as a physical barrier against invading pathogens in the epithelium [1]. Mucin1 (also known as MUC1, CD227, EMA, PEM, CA15-3, KL-6, MCD, or MAM6) is the first mucin to be identified and characterized [2]. MUC1 is normally expressed in the apical surface of glandular epithelial cells, including the stomach, kidneys, pancreas, prostate, lungs, and esophagus, and it provides protection to the underlying epithelia [3,4,5]. However, MUC1 is usually highly overexpressed in a wide range of cancers, including breast cancer, ovarian cancer, pancreatic cancer, colon cancer, and uterine corpus endometrial carcinoma [6]. MUC1 is usually expressed in more than 90% of breast cancer samples, 25C70% of colon cancer samples, and 10C90% of other forms of cancer tissues. The overexpression of MUC1 is usually associated with tumor progression and decreased overall survival in patients with various tumors [7,8,9]. In addition, MUC1 expression loses apicalCbasal polarity, which causes the redistribution of MUC1 over the tumor cell surface [10]. Tumor-associated MUC1 displays the hypo-glycosylation of core glycans, in which the long-branched glycan chains in normal tissues are truncated. The abnormal glycosylation of MUC1 will lead to the formation of tumor-related antigen epitopes (new protein or carbohydrate epitopes) [11,12,13]. Moreover, the aberrant tumor-associated MUC1 can contribute to the change in MUC1 downstream signals through its cytoplasmic domain name, which further regulates different aspects of tumor functions (cell growth, proliferation, developmental processes, metastasis, apoptosis, etc.) [14,15,16,17,18]. These features have made tumor-associated MUC1 an ideal target for immunotherapy. Antibody-based immunotherapy has been developed for cancer treatment over the past few decades and is considered one of the most effective methods to treat solid tumors RV01 as well as hematologic malignancies [19]. For the treatment of MUC1-positive tumors, mAbs that target different epitopes of MUC1 have been developed in preclinical and clinical studies. Gatipotuzumab (also known as PankoMab-GEX? or PMG) RV01 is a humanized and glyco-optimized mAb that recognizes tumor-associated MUC1 glycopeptide [20]. It has a high binding affinity and specificity to tumor-associated MUC1, decreased binding to circulating MUC1 from colon and pancreatic cancer patients, and no binding to RV01 peripheral blood mononuclear cells (PBMCs) [21]. Gatipotuzumab shows enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and can induce the apoptosis of MUC1-expressing tumor cells [20]. A Phase I clinical study revealed that gatipotuzumab was Aspn safe, well tolerated, and had clinical benefits in MUC1-positive patients with advanced solid tumors, while a Phase II clinical study showed that gatipotuzumab treatment had no obvious benefits over a placebo in tumor patients [22,23]. In another study, the combination therapy of gatipotuzumab with the EGFR-targeting antibody tomuzotuximab showed some anti-tumor activity in heavily pretreated EGFR-positive non-small cell lung cancer (NSCLC) patients and colorectal cancer (CRC) [24]. However, other MUC1-specific mAbs, such as huHMFG1, hPAM4, BrE-3, and CMB-401, usually showed limited anti-tumor activity as a monotherapy in clinical studies [25,26,27,28]. To further combat MUC1-positive tumors, one approach that can be tested is usually bispecific antibodies. BsAbs can redirect the immune cells to tumor cells by binding antigens on both tumor cells and immune cells, like T cells, natural.