The body weight of the mice remained normal until the end of this study (Fig

The body weight of the mice remained normal until the end of this study (Fig.5B), suggesting the acceptable safety of ROR1/CD3 TCE in this model. ROR1/CD3 antibody on TNBC cells was determined by the in vitro cytotoxicity assay and in vivo PBMC reconstituted mouse model. The activation of ROR1/CD3 on T cells was analyzed by the flow cytometry and ELISA assay. Pharmacokinetics study of ROR1/CD3 was performed in mouse. == Results == The ROR1/CD3 TCE triggered T cell activation and proliferation, which showed potent and specific killing to TNBC cells in ROR1-depedent manner. L-779450 In vivo mouse model indicated that ROR1/CD3 TCE redirected the cytotoxic activity of T cells to lyse TNBC cells and induced significant tumor regression. Additionally, the ROR1/CD3 bispecific antibody exhibited an extended half-life in mouse, which may enable intermittent administration in clinic. == Conclusions == Collectively, these results demonstrated that ROR1/CD3 TCE has a promising efficacy profile in preclinical studies, which suggested it as a possible option for the treatment of ROR1-expressing TNBC. == Supplementary Information == The online version contains supplementary material available at 10.1186/s13058-025-02005-w. Keywords:TNBC, ROR1, T cell engager, Immunotherapy == Background == Triple negative breast cancer (TNBC) is one of the mostly frequent malignancies and the leading cause of cancer-related death among women worldwide [1,2]. High invasiveness, proneness to relapse, and poor prognosis have been the typical hallmarkers of TNBC [35]. Due to lacking of human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), and progesterone receptor (PR), TNBC patients are not sensitive to endocrine therapy or therapies targeting HER2. Chemotherapy and radiotherapy are generally employed for the treatment of TNBC, however, the 5-year survival rate, especially for those diagnosed in advanced stage, is still poor [6,7]. Because the stage of cancer determines the treatment strategy and outcome, biomarkers that specifically or aberrantly expressed in TNBC that can be targeted for drug design need to be investigated. Over the past decade, immunotherapies are emerging outstanding clinical benefits beyond the traditional treatments in TNBC [812]. In particular, anti-programmed death-ligand 1 (PD-L1) Rabbit Polyclonal to ANXA2 (phospho-Ser26) monoclonal antibody atezolizumab and Sacituzumab Govitecan targeting Trop 2 show impressive response and are changing the paradigm of TNBC treatment, even for patients in advanced stage [13]. Nevertheless, the dark side is that only very limited patients can benefit from the current immunotherapy. Bispecific antibody that recognizes two targets or one target but different epitopes has drawn wide attention and shown exciting perspective in clinic [14,15]. The T cell-redirecting bispecific antibody specifically engages CD3 on T cells and antigens on tumor cells, leading to the activation of T cells and killing of cancer cells. These T cell engagers are considered more potent because T cells can be redirected to the tumor microenvironment regardless of the antigen specific T cell receptors [16]. Blinatumomab, a TCE L-779450 targeting CD19 and CD3, has been approved by the U.S. Food and Drug Administration for the treatment of hematological malignancies [17]. However, the application of TCEs in solid tumors calls for more attention and are still in clinical development to overcome the on-target off-tumor toxicity, which is caused by the low expression of antigens in normal tissues [18,19]. To avoid the insufficient tumor selectivity, antigens that have high tumor specificity are required for the safety and efficacy of TCE in clinical trials. ROR1 is a transmembrane glycoprotein member of the Receptor Tyrosine Kinase (RTK) superfamily, which consists of extracellular region, transmembrane domain and intercellular parts [20]. Besides the physiological roles of ROR1 in embryonic development, aberrant expression of ROR1 has been found from hematological malignancies to solid tumors [21]. Overexpressed ROR1 promoted cancer cell proliferation, migration and drug resistance [22,23]. Interestingly, ROR1 was highly expressed in breast cancer and associated with the advanced aggressive phenotypes, including TNBC [24]. Currently, ROR1 has emerged as a potential target for the development of anticancer drugs [23,2527]. So far, monoclonal antibodies, chimeric antigen receptor T cells (CAR-T cells), antibody-drug conjugates, and T cell engagers by targeting ROR1 have been investigated in preclinical studies in multiple settings [23,2527]. ROR1 targeted antibody-drug conjugates induced the growth inhibition of mantle cell lymphoma and diffuse large B-cell lymphoma [28]. Recent study reported that a ROR1/CD3 bispecific antibody in scFv-Fc format induced T-cell derived cytokine release, and recruited the infiltration of CD4+and CD8+T cells in the tumor tissues of NSCLC [29]. The anti-cancer efficacy of the ROR1/CD3 biAb was also validated in the xenograft mouse models. L-779450 These evidences suggested that it would be necessary to expand the role of ROR1 in TNBC by determining the function and exploring the possibility to target ROR1 for TCE as a strategy in TNBC treatment. In this study, we generated a ROR1/CD3 T cell engager in an IgG-based format L-779450 containing two chains that were covalently linked via disulfide bonds to the Fc hinge region. The biophysical properties of ROR1/CD3 antibody and killing potency of TNBC cells were investigated by in vitro assays and in vivo xenograft mouse.