Current treatments for cholangiocarcinoma (CCA) are largely unsuccessful due to late diagnosis at advanced stage, leading to high mortality rate. Consequently, improved therapeutic approaches are urgently needed. Chimeric antigen receptor (CAR) T cell therapy is a newly potential therapy that can recognize specific surface antigen without major histocompatibility complex (MHC) restriction. Mucin 1 (MUC1) is an attractive candidate antigen as it is highly expressed and associated with poor prognosis and survival in CCA. We, therefore, set forth to create the fourth-generation CAR (CAR4) construct containing anti-MUC1-single-chain variable fragment (scFv) and three co-stimulatory domains (CD28, CD137, and CD27) linked to CD3ζ and evaluate anti-MUC1-CAR4 T cells in CCA models. Compared to untransduced T cells, anti-MUC1-CAR4 T cells produced increased levels of TNF-α, IFN-γ and granzyme B when exposed to MUC1-expressing KKU-100 and KKU-213A CCA cells (all p < 0.05). Anti-MUC1-CAR4 T cells demonstrated specific killing activity against KKU-100 (45.88 ± 7.45%, p < 0.05) and KKU-213A cells (66.03 ± 3.14%, p < 0.001) at an effector to target ratio of 5:1, but demonstrated negligible cytolytic activity against immortal cholangiocytes. Furthermore, the anti-MUC1-CAR4 T cells could effectively disrupt KKU-213A spheroids. These activities of anti-MUC1-CAR4 T cells supports the development of this approach as an adoptive T cell therapeutic strategy for CCA.
Cholangiocarcinoma (CCA) is a lethal cancer of bile duct epithelial cells with a high mortality rate and limited therapeutic options. An effective treatment is, therefore, urgently needed to improve treatment outcomes for these patients. To develop a new therapeutic option, we engineered T cells secreting αCD133-αCD3 bispecific T-cell engager and evaluated their antitumor effects against CD133-expressing CCA cells. The cDNA encoding αCD133-αCD3 bispecific T-cell engager (αCD133-αCD3-ENG) was cloned into pCDH lentiviral construct and its expression was tested in Lenti-X 293T cells. T cells from healthy donors were then transduced with engineered lentiviruses to create T cells secreting αCD133-αCD3 engager to evaluate their antitumor activities. The average transduction efficiency into T cells was approximately 60.03±21.65%. In the co-culture system containing T cells secreting αCD133-αCD3 engager (as effector cells) and mWasabi-luciferase-expressing CCA cells (KKU-100 and KKU-213A; as target cells), the effector T cells exhibited significantly higher cytolytic activities against the target CCA cells (49.0±9.76% and 64.10±13.18%, respectively) than those observed against the untransduced T cells (10.97±10.65%; p = 0.0103 and 9.80±11.05%; p = 0.0054) at an effector-to-target ratio of 5:1. In addition, the secreted αCD133-αCD3 engager significantly redirected both transduced T cells and bystander T cells to kill the target CCA cells (up to 73.20±1.68%; p<0.05). Moreover, the transduced and bystander T cells could kill the target CCA spheroids at a rate approximately 5-fold higher than that of the no treatment control condition (p = 0.0011). Our findings demonstrate proof-of-principle that T cells secreting αCD133-αCD3 engager can be an alternative approach to treating CD133-positive CCA, and they pave the way for future in vivo study and clinical trials.
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