Search

Advanced cholangiocarcinoma (CCA) presents a clinical challenge due to limited treatment options, necessitating exploration of innovative therapeutic approaches. Bispecific T cell engager (BTE)-armed T cell therapy shows promise in hematological and solid malignancies, offering potential advantages in safety over continuous BTE infusion. In this context, we developed a novel BTE, targeting CD3 on T cells and integrin αvβ6, an antigen elevated in various epithelial malignancies, on cancer cells. The novel BTE was generated by fusing an integrin αvβ6-binding peptide (A20) to an anti-CD3 (OKT3) single-chain variable fragment (scFv) through a G 4 S peptide linker (A20/αCD3 BTE). T cells were then armed with A20/αCD3 BTE (A20/αCD3-armed T cells) and assessed for antitumor activity. Our results highlight the specific binding of A20/αCD3 BTE to CD3 on T cells and integrin αvβ6 on target cells, effectively redirecting T cells towards these targets. After co-culture, A20/αCD3-armed T cells exhibited significantly heightened cytotoxicity against integrin αvβ6-expressing target cells compared to unarmed T cells in both KKU-213A cells and A375.β6 cells. Moreover, in a five-day co-culture, A20/αCD3-armed T cells demonstrated superior cytotoxicity against KKU-213A spheroids compared to unarmed T cells. Importantly, A20/αCD3-armed T cells exhibited an increased proportion of the effector memory T cell (Tem) subset, upregulation of T cell activation markers, enhanced T cell proliferation, and increased cytolytic molecule/cytokine production, when compared to unarmed T cells in an integrin αvβ6-dependent manner. These findings support the potential of A20/αCD3-armed T cells as a novel therapeutic approach for integrin αvβ6-expressing cancers. [Display omitted] • A20/αCD3 BTE targets both CD3 on T cells and integrin αvβ6 on cancer cells. • A20/αCD3-armed T cells exhibit higher antitumor activity compared to unarmed T cells. • A20/αCD3 BTE enhances T cell activation, proliferation, and cytokine secretion. [ABSTRACT FROM AUTHOR]

• The superior antitumor function of fourth-generation CAR T compared to second-generation. • We developed a fourth-generation CAR targeting Trop2 (referred to as anti-Trop2 CAR4), which includes three co-stimulatory domains: CD28, 4-1BB, and CD27. • Anti-Trop2 CAR4-T cells exhibited potent cytotoxicity against breast cancer cells in both 2D and 3D cell cultures. • Anti-Trop2 CAR4-T cell proliferation and cytokine secretion were enhanced when exposed to Trop2-expressing cells. • Anti-Trop2 CAR4-T cells hold promise for improving the effectiveness of breast cancer treatment. The treatment of breast cancer (BC) remains a formidable challenge due to the emergence of drug resistance, necessitating the exploration of innovative strategies. Chimeric antigen receptor (CAR)-T cell therapy, a groundbreaking approach in hematologic malignancies, is actively under investigation for its potential application in solid tumors, including BC. Trophoblast cell surface antigen 2 (Trop2) has emerged as a promising immunotherapeutic target in various cancers and is notably overexpressed in BC. To enhance therapeutic efficacy in BC, a fourth-generation CAR (CAR4) construct was developed. This CAR4 design incorporates an anti-Trop2 single-chain variable fragment (scFv) fused with three costimulatory domains –CD28/4-1BB/CD27, and CD3ζ. Comparative analysis with the conventional second-generation CAR (CAR2; 28ζ) revealed that anti-Trop2 CAR4 T cells exhibited heightened cytotoxicity and interferon-gamma (IFN-γ) production against Trop2-expressing MCF-7 cells. Notably, anti-Trop2 CAR4-T cells demonstrated superior long-term cytotoxic functionality and proliferative capacity. Crucially, anti-Trop2 CAR4-T cells displayed specific cytotoxicity against Trop2-positive BC cells (MDA-MB-231, HCC70, and MCF-7) in both two-dimensional (2D) and three-dimensional (3D) culture systems. Following antigen-specific killing, these cells markedly secreted interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), IFN-γ, and Granzyme B compared to non-transduced T cells. This study highlights the therapeutic potential of anti-Trop2 CAR4-T cells in adoptive T cell therapy for BC, offering significant promise for the advancement of BC treatment strategies. [ABSTRACT FROM AUTHOR]

In order to investigate the role of translationally- controlled tumor protein (TCTP) in cholangiocarcinoma (CCA) progression and metastasis, TCTP protein staining in paraffin-embedded sections of human CCA tissue samples was examined using immunohistochemistry, and its expression was subsequently compared with clinicopathological parameters. Small interfering RNA (siRNA) targeting TCTP (siTCTP) were transfected into CCA cell lines to evaluate its effects on cellular functions. The proliferation, tumorigenicity and migration abilities of the transfected cells were measured using sulforhodamine B, clonogenic and would healing assays, respectively. The protein levels of TCTP and its associated molecules were evaluated by western blot analysis. Of the 119 individual cases of CCA tissues analyzed, high TCTP scores were significantly correlated with overall metastasis (P=0.044) and a shorter survival time (P<0.001). Multivariate proportional hazards analysis revealed that TCTP is an independent indicator of poor prognosis in CCA (hazard ratio =2.864; P<0.001). siTCTP transfection suppressed CCA cell growth and migration abilities, compared with the control cells (P<0.01). The siTCTP reduced the protein levels of focal adhesion kinase (FAK), phospho-FAK, nuclear factor kappa-light-chain-enhancer of activated B cells and matrix metalloproteinase 9, suggesting potential roles of TCTP in regulating CCA progression and metastasis. In conclusion, the upregulation of TCTP is clinically significant in patients with CCA, serving roles in CCA progression, particularly in cell survival and metastasis. Suppression of TCTP may serve as a potential target in CCA prevention and treatment. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) is considered the third leading cause of cancer mortality in the western world, offering advanced stage patients with few viable treatment options. Consequently, there remains an urgent unmet need to develop novel therapeutic strategies that can effectively inhibit pro-oncogenic molecular targets underpinning PDACs pathogenesis and progression. One such target is c-RAF, a downstream effector of RAS that is considered essential for the oncogenic growth and survival of mutant RAS-driven cancers (including KRASMT PDAC). Herein, we demonstrate how a novel cell-penetrating peptide disruptor (DRx-170) of the c-RAF–PDE8A protein–protein interaction (PPI) represents a differentiated approach to exploiting the c-RAF–cAMP/PKA signaling axes and treating KRAS–c-RAF dependent PDAC. Through disrupting the c-RAF–PDE8A protein complex, DRx-170 promotes the inactivation of c-RAF through an allosteric mechanism, dependent upon inactivating PKA phosphorylation. DRx-170 inhibits cell proliferation, adhesion and migration of a KRASMT PDAC cell line (PANC1), independent of ERK1/2 activity. Moreover, combining DRx-170 with afatinib significantly enhances PANC1 growth inhibition in both 2D and 3D cellular models. DRx-170 sensitivity appears to correlate with c-RAF dependency. This proof-of-concept study supports the development of DRx-170 as a novel and differentiated strategy for targeting c-RAF activity in KRAS–c-RAF dependent PDAC. [ABSTRACT FROM AUTHOR]

[Display omitted] • The expression of cMET was predominantly observed in cholangiocarcinoma (CCA) cell lines which supported the cMET as a target for CCA. • In this study, we screened and characterized novel single chain variable fragment (ScFv) targeted cMET and generated an anti-cMET CAR-NK cells to treat CCA. • The anti-cMET CAR-NK cells exhibited specificity and favorable cytotoxicity against high-cMET CCA cell line in in vitro model. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cholangiocarcinoma (CCA) is a cancer of the bile ducts that is associated with poor prognosis and poor treatment outcome. Approximately one-third of CCA patients can undergo surgery, but the recurrence rate is high and chemotherapy often cannot satisfactorily prolong survival. Cellular immunotherapy based on adoptive T-cell transfer is a potential treatment for CCA; however, the development of this technology and the search for an appropriate tumor-associated antigen are still ongoing. To enhance the cytotoxic activity of effector T cells against CCA, we developed self-differentiated monocyte-derived dendritic cells (SD-DC) presenting cAMP-dependent protein kinase type I-alpha regulatory subunit (PRKAR1A), which is an overexpressed protein that plays a role in the regulation of tumor growth to activate T cells for CCA cell killing. Dendritic cells (DCs) transduced with lentivirus harboring tri-cistronic cDNA sequences (SD-DC-PR) could produce granulocyte-macrophage colony-stimulating factor, interleukin-4, and PRKAR1A. SD-DC showed similar phenotypes to those of DCs derived by conventional method. Autologous effector T cells (CD3+, CD8+) activated by SD-DC-PR exhibited greater cytotoxic activity against CCA than those activated by conventionally-derived DCs. Effector T cells activated by SD-DC-PR killed 60% of CCA cells at an effector-to-target ratio of 15:1, which is approximately twofold greater than the cell killing performance of those stimulated with control DC. The cytotoxic activities of effector T cells activated by SD-DC-PR against CCA cells were significantly associated with the expression levels of PRKR1A in CCA cells. This finding that SD-DC-PR effectively stimulated autologous effector T cells to kill CCA cells may help to accelerate the development of novel therapies for treating CCA. [ABSTRACT FROM AUTHOR]

[Display omitted] • Anti-CD19-CAR5-T cells exerted more effective cytotoxicity than anti-CD19-CAR4-T cells at a low effector-to-target ratio. • Cytotoxicity of anti-CD19-CAR4-T cells was inhibited by upregulations of PD-1 on CAR-T cells and PD-L1 on the target cancer cells. • The secreted anti-PD-L1 scFv from anti-CD19-CAR5-T cells could restore the cytotoxic effect of anti-CD19-CAR4-T cells. • The secreted anti-PD-L1 scFv promoted self-proliferation of anti-CD19-CAR5-T cells. • PD-L1 blockade by anti-PD-L1 scFv secreted from CAR5-T cells is a feasible strategy to improve CAR-T antitumor efficiency. Adoptive T cell therapy using second-generation anti-CD19 chimeric antigen receptor T cells (anti-CD19-CAR2-T) induced complete remission in many heavily pretreated patients with B cell acute lymphoblastic leukemia (B-ALL) or diffuse large B cell lymphoma (DLBCL). However, poor clinical efficacy was observed in treating aggressive B cell lymphomas (BCL). The limited T cell function was reported by programmed cell death protein 1 ligand (PD-L1) expressed on BCL cells bound to the PD-1 receptor on T cells. To overcome this problem, we generated anti-CD19-CAR4-T cells secreting anti-PD-L1 single-chain variable fragment (scFv), namely anti-CD19-CAR5-T cells, and evaluated their functions in vitro. Both anti-CD19-CAR-T cells contain an anti-CD19 scFv derived from a monoclonal antibody, FMC63, linked to CD28/4-1BB/CD27/CD3ζ. The secreting anti-PD-L1 scFv is derived from atezolizumab. Our results showed that secreted anti-PD-L1 scFv could bind to PD-L1 and block the binding of anti-PD-L1 monoclonal antibodies on PD-L1high tumor cells. Anti-CD19-CAR4-T and anti-CD19-CAR5-T cells efficiently killed CD19+ target tumor cells in two-dimensional (2D) and three-dimensional (3D) co-culture systems. However, anti-CD19-CAR5-T cells demonstrated superior proliferative ability. Interestingly, at a low effector (E) to target (T) ratio of 0.5:1, anti-CD19-CAR5-T cells showed higher cytotoxicity against CD19+/PD-L1high cells compared to that of anti-CD19-CAR4-T cells. The cytotoxicity of anti-CD19-CAR4-T cells against CD19+/PD-L1high could be restored by adding anti-PD-L1 scFv. Our findings demonstrate the combination antitumor efficiency of anti-CD19-CAR4-T cells and anti-PD-L1 scFv against CD19+/PD-L1high tumors. As such, anti-CD19-CAR5-T cells should be further investigated in vivo antitumor efficiency and clinical trials as a treatment for aggressive B cell lymphoma. [ABSTRACT FROM AUTHOR]

[Display omitted] • Anti-CD19-CAR5-T cells exerted more effective cytotoxicity than anti-CD19-CAR4-T cells at a low effector-to-target ratio. • Cytotoxicity of anti-CD19-CAR4-T cells was inhibited by upregulations of PD-1 on CAR-T cells and PD-L1 on the target cancer cells. • The secreted anti-PD-L1 scFv from anti-CD19-CAR5-T cells could restore the cytotoxic effect of anti-CD19-CAR4-T cells. • The secreted anti-PD-L1 scFv promoted self-proliferation of anti-CD19-CAR5-T cells. • PD-L1 blockade by anti-PD-L1 scFv secreted from CAR5-T cells is a feasible strategy to improve CAR-T antitumor efficiency. Adoptive T cell therapy using second-generation anti-CD19 chimeric antigen receptor T cells (anti-CD19-CAR2-T) induced complete remission in many heavily pretreated patients with B cell acute lymphoblastic leukemia (B-ALL) or diffuse large B cell lymphoma (DLBCL). However, poor clinical efficacy was observed in treating aggressive B cell lymphomas (BCL). The limited T cell function was reported by programmed cell death protein 1 ligand (PD-L1) expressed on BCL cells bound to the PD-1 receptor on T cells. To overcome this problem, we generated anti-CD19-CAR4-T cells secreting anti-PD-L1 single-chain variable fragment (scFv), namely anti-CD19-CAR5-T cells, and evaluated their functions in vitro. Both anti-CD19-CAR-T cells contain an anti-CD19 scFv derived from a monoclonal antibody, FMC63, linked to CD28/4-1BB/CD27/CD3ζ. The secreting anti-PD-L1 scFv is derived from atezolizumab. Our results showed that secreted anti-PD-L1 scFv could bind to PD-L1 and block the binding of anti-PD-L1 monoclonal antibodies on PD-L1high tumor cells. Anti-CD19-CAR4-T and anti-CD19-CAR5-T cells efficiently killed CD19+ target tumor cells in two-dimensional (2D) and three-dimensional (3D) co-culture systems. However, anti-CD19-CAR5-T cells demonstrated superior proliferative ability. Interestingly, at a low effector (E) to target (T) ratio of 0.5:1, anti-CD19-CAR5-T cells showed higher cytotoxicity against CD19+/PD-L1high cells compared to that of anti-CD19-CAR4-T cells. The cytotoxicity of anti-CD19-CAR4-T cells against CD19+/PD-L1high could be restored by adding anti-PD-L1 scFv. Our findings demonstrate the combination antitumor efficiency of anti-CD19-CAR4-T cells and anti-PD-L1 scFv against CD19+/PD-L1high tumors. As such, anti-CD19-CAR5-T cells should be further investigated in vivo antitumor efficiency and clinical trials as a treatment for aggressive B cell lymphoma. [ABSTRACT FROM AUTHOR]

• CD133 expression in patient tissues and cholangiocarcinoma (CCA) cells were shown. • Anti-CD133 CAR4 T cells containing CD28/CD137/CD27/CD3ς was successfully created. • The concept of using anti-CD133 CAR4 T cells against CD133+ CCA cells is proved. • Anti-CD133 CAR4 T cells is a potential cancer immunotherapy for CCA treatment. Current treatment of cholangiocarcinoma (CCA) – a lethal bile duct cancer – is ineffective because the disease is usually diagnosed at late and advanced stage. Thus, a novel therapeutic modality is urgently required. Fourth-generation chimeric antigen receptor (CAR4) T cells was created to target CD133, a well-known cancer stem cell marker, that is highly expressed and associates with cancer progression. The anti-CD133-CAR4 T cells showed high efficacy against CD133-expressing CCA cells. Tumour cell lysis occurred in a dose- and CD133 antigen-dependent manner, and significantly higher, up to 57.59% ± 9.62 at effector to target ratio of 5:1 in a CCA cell line – KKU-213A cells, compared to mock control (p = 0.008). Similarly, significant IFN-γ (p = 0.011) and TNF-α (p = 0.002) upregulation was observed upon tumour treatment. The effectiveness of our anti-CD133-CAR4 T cells will be beneficial not only for CD133-expressing CCA, but also for other CD133-expressing tumours. This study may guide future in vivo study and clinical trials. [ABSTRACT FROM AUTHOR]