Precision immunotherapy for cholangiocarcinoma: Pioneering the use of human-derived anti-cMET single chain variable fragment in anti-cMET chimeric antigen receptor (CAR) NK cells.

Cholangiocarcinoma (CCA) presents a significant clinical challenge which is often identified in advanced stages, therby restricting the effectiveness of surgical interventions for most patients. The high incidence of cancer recurrence and resistance to chemotherapy further contribute to a bleak prognosis and low survival rates. To address this pressing need for effective therapeutic strategies, our study focuses on the development of an innovative cellular immunotherapy, specifically utilizing chimeric antigen receptor (CAR)-engineered natural killer (NK) cells designed to target the cMET receptor tyrosine kinase. In this investigation, we initiated the screening of a phage library displaying human single-chain variable fragment (ScFv) to identify novel ScFv molecules with specificity for cMET. Remarkably, ScFv11, ScFv72, and ScFv114 demonstrated exceptional binding affinity, confirmed by molecular docking analysis. These selected ScFvs, in addition to the well-established anti-cMET ScFvA, were integrated into a CAR cassette harboring CD28 transmembrane region-41BB-CD3ζ domains. The resulting anti-cMET CAR constructs were transduced into NK-92 cells, generating potent anti-cMET CAR-NK-92 cells. To assess the specificity and efficacy of these engineered cells, we employed KKU213A cells with high cMET expression and KKU055 cells with low cMET levels. Notably, co-culture of anti-cMET CAR-NK-92 cells with KKU213A cells resulted in significantly increased cell death, whereas no such effect was observed with KKU055 cells. In summary, our study identified cMET as a promising therapeutic target for CCA. The NK-92 cells, armed with the anti-cMET CAR molecule, have shown strong ability to kill cancer cells specifically, indicating their potential as a promising treatment for CCA in the future.

Genistein Sensitizes Human Cholangiocarcinoma Cell Lines to Be Susceptible to Natural Killer Cells.

Cholangiocarcinoma (CCA) is a lethal bile duct cancer, which has poor treatment outcomes due to its high resistance to chemotherapy and cancer recurrence. Activation of aberrant anti-apoptotic signaling pathway has been reported to be a mechanism of chemoresistance and immune escape of CCA. Therefore, reversal of anti-apoptotic signaling pathway represents a feasible approach to potentiate effective treatments, especially for CCA with high chemoresistance. In this study, we demonstrated the effects of genistein on reactivation of apoptosis cascade and increase the susceptibility of CCA cells to natural killer (NK-92) cells. Genistein at 50 and 100 µM significantly activated extrinsic apoptotic pathway in CCA cells (KKU055, KKU100, and KKU213A), which was evident by reduction of procaspase-8 and -3 expression. Pretreatment of CCA cells with genistein at 50 µM, but not NK-92 cells, significantly increased NK-92 cell killing ability over the untreated control, suggesting the ability of genistein to sensitize CCA cells. Interestingly, genistein treatment could greatly lower the expression of cFLIP, an anti-apoptotic protein involved in the immune escape pathway, in addition to upregulation of death receptors, Fas- and TRAIL-receptors, in CCA cells, which might be the underlying molecular mechanism of genistein to sensitize CCA to be susceptible to NK-92 cells. Taken together, this finding revealed the benefit of genistein as a sensitizer to enhance the efficiency of NK cell immunotherapy for CCA.

Doxorubicin sensitizes breast cancer cells to natural killer cells in connection with increased Fas receptors.

Breast cancer (BC) is the most common cancer in women. Although standard treatments are successful in patients with BC diagnosed at an early stage, an alternative treatment is required for patients with advanced­stage disease who do not respond to these treatments. The concept of using chemotherapy to sensitize cancer cells to become susceptible to immunotherapy was recently introduced and may be used as an alternative treatment for BC. The chemotherapeutic drug doxorubicin has been reported to sensitize cancer cells; however, the efficacy to sensitize the solid spheroids, in addition to its underlying mechanism regarding how doxorubicin sensitizes BC, has not previously been explored. In the present study, the effectiveness of a combined treatment of doxorubicin and natural killer­92 (NK­92) cells against BC in either 2D or 3D spheroid models, and its association with Fas receptor (FasR) expression, was demonstrated. The BC (MCF7) cell line expressing a higher level of FasR was more sensitive to NK­92 cell killing than the MDA­MB­231 cell line, which expressed a lower level of FasR. A sublethal dose of doxorubicin caused a significant improvement in NK cytotoxicity. Concordantly, a significant reduction in cell viability was observed in the doxorubicin­treated MCF7 spheroids. Notably, flow cytometric analysis revealed significantly increased FasR expression in the MCF7 cells, suggesting the underlying sensitization mechanism of doxorubicin in BC was related to the FasR upregulation. The present findings supported the use of combined doxorubicin and NK immunotherapy in BC treatment.

Combination gemcitabine and PD-L1xCD3 bispecific T cell engager (BiTE) enhances T lymphocyte cytotoxicity against cholangiocarcinoma cells.

Cholangiocarcinoma (CCA) is a lethal cancer with rapid progression and poor survival. Novel and more effective therapies than those currently available are, therefore, urgently needed. Our research group previously reported the combination of gemcitabine and cytotoxic T lymphocytes to be more effective than single-agent treatment for the elimination of CCA cells. However, gemcitabine treatment of CCA cells upregulates the expression of an immune checkpoint protein (programmed death-ligand 1 [PD-L1]) that consequently inhibits the cytotoxicity of T lymphocytes. To overcome this challenge and take advantage of PD-L1 upregulation upon gemcitabine treatment, we generated recombinant PD-L1xCD3 bispecific T cell engagers (BiTEs) to simultaneously block PD-1/PD-L1 signaling and recruit T lymphocytes to eliminate CCA cells. Two recombinant PD-L1xCD3 BiTEs (mBiTE and sBiTE contain anti-PD-L1 scFv region from atezolizumab and from a published sequence, respectively) were able to specifically bind to both CD3 on T lymphocytes, and to PD-L1 overexpressed after gemcitabine treatment on CCA (KKU213A, KKU055, and KKU100) cells. mBiTE and sBiTE significantly enhanced T lymphocyte cytotoxicity against CCA cells, especially after gemcitabine treatment, and their magnitudes of cytotoxicity were positively associated with the levels of PD-L1 expression. Our findings suggest combination gemcitabine and PD-L1xCD3 BiTE as a potential alternative therapy for CCA.