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.

Bispecific T cell engager-armed T cells targeting integrin ανß6 exhibit enhanced T cell redirection and antitumor activity in cholangiocarcinoma.

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 αvß6, an antigen elevated in various epithelial malignancies, on cancer cells. The novel BTE was generated by fusing an integrin αvß6-binding peptide (A20) to an anti-CD3 (OKT3) single-chain variable fragment (scFv) through a G4S 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 αvß6 on target cells, effectively redirecting T cells towards these targets. After co-culture, A20/αCD3-armed T cells exhibited significantly heightened cytotoxicity against integrin αvß6-expressing target cells compared to unarmed T cells in both KKU-213A cells and A375.ß6 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 αvß6-dependent manner. These findings support the potential of A20/αCD3-armed T cells as a novel therapeutic approach for integrin αvß6-expressing cancers.

Therapeutic potential of third-generation chimeric antigen receptor T cells targeting B cell maturation antigen for treating multiple myeloma.

Multiple myeloma (MM) is an incurable hematologic malignancy characterized by the rapid proliferation of malignant plasma cells within the bone marrow. Standard therapies often fail due to patient resistance. The US FDA has approved second-generation chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (anti-BCMA-CAR2 T cells) for MM treatment. However, achieving enduring clinical responses remains a challenge in CAR T cell therapy. This study developed third-generation T cells with an anti-BCMA CAR (anti-BCMA-CAR3). The CAR incorporated a fully human scFv specific to BCMA, linked to the CD8 hinge region. The design included the CD28 transmembrane domain, two co-stimulatory domains (CD28 and 4-1BB), and the CD3ζ signaling domain (28BBζ). Lentiviral technology generated these modified T cells, which were compared against anti-BCMA-CAR2 T cells for efficacy against cancer. Anti-BCMA-CAR3 T cells exhibited significantly higher cytotoxic activity against BCMA-expressing cells (KMS-12-PE and NCI-H929) compared to anti-BCMA-CAR2 T cells. At an effector-to-target ratio of 10:1, anti-BCMA-CAR3 T cells induced lysis in 75.5 ± 3.8% of NCI-H929 cells, whereas anti-BCMA-CAR2 T cells achieved 56.7 ± 3.4% (p = 0.0023). Notably, after twelve days of cultivation, anti-BCMA-CAR3 T cells nearly eradicated BCMA-positive cells (4.1 ± 2.1%), while anti-BCMA-CAR2 T cells allowed 36.8 ± 20.1% to survive. This study highlights the superior efficacy of anti-BCMA-CAR3 T cells against both low and high BCMA-expressing MM cells, surpassing anti-BCMA-CAR2 T cells. These findings suggest potential for advancing anti-BCMA-CAR3 T cells in chimeric antigen receptor T (CAR-T) therapy for relapsed/refractory MM.

Cytotoxicity of fourth-generation anti-Trop2 CAR-T cells against breast cancer.

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.

Anti-PD-L1 × anti-CD3 bispecific T-cell engager-armed T cells can overcome immunosuppression and redirect T cells to kill breast cancer cells expressing PD-L1.

T cell-based immunotherapy has transformed cancer treatment. Nonetheless, T cell antitumor activity can be inhibited by an immune checkpoint molecule expressed on cancer cells, program death ligand 1 (PD-L1), which interacts with the PD-1 on T cells. We generated αPD-L1 × αCD3 bispecific T-cell engager-armed T cells (BATs) to prevent PD-L1/PD-1 interaction and hence to redirect T cells to kill cancer cells. αPD-L1 × αCD3 bispecific T-cell engagers (BTEs) were produced from Chinese hamster ovary (CHO) cells to arm human primary T cells. Flow cytometry was used to investigate BTE binding to BATs. The cytotoxicity of BATs against PD-L1-expressing breast cancer (BC) cell lines was assessed in 2-dimensional (2D) and 3-dimensional (3D) culture models. The binding stability of BTE on BATs and their efficacy after cryopreservation were also examined. The CHO cell BTE expression yield was 3.34 mg/ml. The binding ability on T cells reached 91.02 ± 4.2 %. BATs specifically lysed PD-L1-expressing BC cells, with 56.4 ± 15.3 % HCC70 cells and 70.67 ± 15.6 % MDA-MB-231 cells lysed at a 10:1 effector-to-target ratio. BATs showed slight, nonsignificant lysis of PD-L1-negative BC cells, MCF-7, and T47D. Moreover, BATs significantly disrupted MDA-MB-231 3D spheroids expressing PD-L1 after 48 and 72 h of coculture. Cryopreserved BATs maintained BTE binding stability, cell viability, and anticancer activity, comparable to fresh BATs. αPD-L1 × αCD3 BATs induced the cytolysis of PD-L1-expressing BC cells in 2D and 3D coculture assays. BATs can be prepared and preserved, facilitating their use and transportation. This study demonstrates the potential of αPD-L1 × αCD3 BATs in treating cancers with positive PD-L1 expression.

Enhancement of PD-L1-attenuated CAR-T cell function through breast cancer-associated fibroblasts-derived IL-6 signaling via STAT3/AKT pathways.

BACKGROUND: Carcinoma-associated fibroblasts (CAFs) play a critical role in cancer progression and immune cell modulation. In this study, it was aimed to evaluate the roles of CAFs-derived IL-6 in doxorubicin (Dox) resistance and PD-L1-mediated chimeric antigenic receptor (CAR)-T cell resistance in breast cancer (BCA). METHODS: CAF conditioned-media (CM) were collected, and the IL-6 level was measured by ELISA. CAF-CM were treated in MDA-MB-231 and HCC70 TNBC cell lines and siIL-6 receptor (IL-6R) knocked down (KD) cells to determine the effect of CAF-derived IL-6 on Dox resistance by flow cytometry and on increased PD-L1 through STAT3, AKT and ERK1/2 pathways by Western blot analysis. After pre-treating with CM, the folate receptor alpha (FRα)-CAR T cell cytotoxicity was evaluated in 2D and 3D spheroid culture assays. RESULTS: The results showed a significant level of IL-6 in CAF-CM compared to that of normal fibroblasts (NFs). The CM with high IL-6 level significantly induced Dox resistance; and PD-L1 expression through STAT3 and AKT pathways in MDA-MB-231 and HCC70 cells. These induction effects were attenuated in siIL-6R KD cells. Moreover, the TNBC cell lines that were CM-treated with STAT3 and an AKT inhibitor had a reduced effect of IL-6 on PD-L1 expression. BCA cells with high IL-6 containing-CM treatment had resistance to cancer cell killing by FRα CAR-T cells compared to untreated cells. CONCLUSION: These results highlight CAF-derived IL-6 in the resistance of chemotherapy and T cell therapy. Using inhibitors of IL6-STAT3/AKT-PD-L1 axis may provide a potential benefit of Dox and CAR-T cell therapies in BCA patients.

High prevalence of Wilms tumor 1 expression in multiple myeloma and plasmacytoma: A cohort of 142 Asian patients' samples.

Wilms tumor 1 (WT1) is a promising target antigen for cancer immunotherapy. However, WT1 protein expression and its clinical correlation in multiple myeloma (MM) patients are still limited. We, therefore, investigated WT1 expression in 142 bone marrow and plasmacytoma samples of MM patients at different stages of the disease by immunohistochemistry. The correlations between WT1 expression and clinical parameters or treatment outcomes were evaluated. The overall positive rate of WT1 expression was 91.5%; this high prevalence was found in both bone marrow and plasmacytoma samples, regardless of the disease status. Cytoplasmic WT1 expression was correlated with high serum free light chain ratio at presentation. However, no significant association between WT1 expression and treatment outcome was observed. This study confirms the high prevalence of WT1 expression in an Asian cohort of MM, encouraging the development of immunotherapy targeting WT1 in MM patients, particularly in those with extramedullary plasmacytoma or relapsed disease.

Cytotoxic activity of anti-mucin 1 chimeric antigen receptor T cells expressing PD-1-CD28 switch receptor against cholangiocarcinoma cells.

BACKGROUND AIMS: Cholangiocarcinoma (CCA) is a lethal bile-duct cancer that is difficult to treat by current standard procedures. This drawback has prompted us to develop adoptive T-cell therapy for CCA, which requires an appropriate target antigen for binding of chimeric antigen receptor (CAR) T cells. Mucin 1 (MUC1), an overexpressed protein in CCA cells, is a potential target antigen for the CAR T-cell development. However, MUC1 overexpression also is associated with the upregulation of programmed death-ligand 1 (PD-L1), an immune checkpoint protein that prohibits anti-tumor functions of T cells, probably causing poor overall survival of patients with CCA. METHODS: To overcome this problem, we developed anti-MUC1-CAR T cells containing PD-1-CD28 switch receptor (SR), namely αM.CAR/SR T cells, to target MUC1 and switch on the inhibitory signal of PD-1/PD-L1 interaction to activate CD28 signaling. Our lentiviral construct contains the sequences that encode anti-MUC1-single chain variable fragment, CD137 and CD3ζ, linked with P2A, PD-1 and CD28. RESULTS: Initially, the upregulations of MUC1 and PD-L1 proteins were confirmed in CCA cell lines. αM.CAR and SR were co-expressed in 53.53 ± 13.89% of transduced T cells, mainly CD8+ T cells (85.7 ± 0.75%, P<0.0001) with the effector memory phenotype (59.22 ± 16.31%, P < 0.01). αM.CAR/SR T cells produced high levels of intracellular tumor necrosis factor-α and interferon-γ in response to the activation by CCA cells expressing MUC1, including KKU-055 (27.18 ± 4.38% and 27.33 ± 5.55%, respectively, P < 0.05) and KKU-213A (47.37 ± 12.67% and 54.55 ± 8.66%, respectively, P < 0.01). Remarkably, the cytotoxic function of αM.CAR/SR T cells against KKU-213A cells expressing PD-L1 was significantly enhanced compared with the αM.CAR T cells (70.69 ± 14.38% versus 47.15 ± 8.413%, respectively; P = 0.0301), correlated with increased granzyme B production (60.6 ± 9.89% versus 43.2 ± 8.95%, respectively; P = 0.0402). Moreover, the significantly enhanced disruption of KKU-213A spheroids by αM.CAR/SR T cells (P = 0.0027), compared with αM.CAR T cells, was also observed. CONCLUSION: Taken together, the cytotoxic function of αM.CAR/SR T cells was enhanced over the αM.CAR T cells, which are potential to be further tested for CCA treatment.

Anti-CD19 chimeric antigen receptor T cells secreting anti-PD-L1 single-chain variable fragment attenuate PD-L1 mediated T cell inhibition.

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.

Activation of cytotoxic T lymphocytes by self-differentiated myeloid-derived dendritic cells for killing breast cancer cells expressing folate receptor alpha protein.

Adoptive cell transfer (ACT) is a promising approach for cancer treatment. Activation of T lymphocytes by self-differentiated myeloid-derived antigen-presenting-cells reactive against tumor (SmartDC) resulted in specific anti-cancer function. Folate receptor alpha (FRα) is highly expressed in breast cancer (BC) cells and thus potential to be a target antigen for ACT. To explore the SmartDC technology for treatment of BC, we create SmartDC expressing FRα antigen (SmartDC-FRα) for activation of FRα-specific T lymphocytes. Human primary monocytes were transduced with lentiviruses containing tri-cistronic complementary DNA sequences encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and FRα to generate SmartDC-FRα. Autologous T lymphocytes were activated by SmartDC-FRα by coculture. The activated T lymphocytes exhibited enhanced cytotoxicity against FRα-expressing BC cell cultures. Up to 84.9 ± 6.2% of MDA-MB-231 and 89.7 ± 1.9% of MCF-7 BC cell lines were specifically lysed at an effector-to-target ratio of 20:1. The cytotoxicity of T lymphocytes activated by SmartDC-FRα was also demonstrated in three-dimensional (3D) spheroid culture of FRα-expressing BC cells marked by size reduction and spheroid disruption. This study thus portray the potential development of T lymphocytes activated by SmartDC-FRα as ACT in FRα-expressing 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.

Cytotoxic T Cells Activated by Self-differentiated Monocyte-derived Dendritic Cells Against Multiple Myeloma Cells.

BACKGROUND/AIM: B cell maturation antigen (BCMA) is an ideal target for adoptive T cell therapy of multiple myeloma (MM). In this study, we evaluated self-differentiated monocyte-derived dendritic cells expressing BCMA (SD-DC-BCMA) to activate T cells for killing MM cells. MATERIALS AND METHODS: Lentivirus-modified SD-DC-BCMA harboring tri-cistronic cDNAs encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and BCMA was generated. Cytotoxicity of T cells activated by SD-DC-BCMA against MM cells was evaluated. RESULTS: T cells activated by SD-DC-BCMA exhibited a dose-dependent cytotoxicity against BCMA-expressing MM cells and produced high IFN-γ levels, compared to inactivated T cells or control T cells. A significantly higher killing ability of T cells activated by SD-DC-BCMA was further demonstrated in BCMA-overexpressing cells when compared with BCMA-negative cells. CONCLUSION: The potency of SD-DC-BCMA to activate T cells for antigen-specific cancer killing provides a framework for therapeutic application of adoptive T cell therapy in MM.

Antitumor activity of T cells secreting αCD133-αCD3 bispecific T-cell engager against cholangiocarcinoma.

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.

Dexamethasone induces pancreatic ß-cell apoptosis through upregulation of TRAIL death receptor.

Long-term medication with dexamethasone - a synthetic glucocorticoid (GC) drug - results in hyperglycemia, or steroid-induced diabetes. Although recent studies revealed that dexamethasone directly induces pancreatic ß-cell apoptosis, its molecular mechanisms remain unclear. In our initial analysis of mRNA transcripts, we discovered the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway may be involved in dexamethasone-induced pancreatic ß-cell apoptosis. In the present study, a mechanism of dexamethasone-induced pancreatic ß-cell apoptosis through the TRAIL pathway was investigated in cultured cells and isolated mouse islets. INS-1 cells were cultured with and without dexamethasone in the presence or absence of a glucocorticoid receptor (GR) inhibitor, RU486. We found that dexamethasone induced pancreatic ß-cell apoptosis in association with the upregulation of TNSF10 (TRAIL) mRNA and protein expression. Moreover, dexamethasone upregulated the TRAIL death receptor (DR5) protein but suppressed the decoy receptor (DcR1) protein. Similar findings were observed in mouse isolated islets: dexamethasone increased TRAIL and DR5 compared to that of control mice. Furthermore, dexamethasone stimulated pro-apoptotic signaling including superoxide production, caspase-8, -9, and -3 activities, NF-κB, and Bax but repressed the anti-apoptotic protein, Bcl-2. All these effects were inhibited by the GR-inhibitor, RU486. Furthermore, knock-down DR5 decreased dexamethasone-induced caspase 3 activity. Caspase-8 and caspase-9 inhibitors protected pancreatic ß-cells from dexamethasone-induced apoptosis. Taken together, dexamethasone induced pancreatic ß-cell apoptosis by binding to the GR and inducing DR5 and TRAIL pathway.

Targeting UCHL1 Induces Cell Cycle Arrest in High-Risk Multiple Myeloma with t(4;14).

Multiple myeloma (MM) patients considered to be at high cytogenetic risk commonly fail to respond to standard treatment. A thorough understanding of the molecular mechanism of MM development is, therefore, needed. We endeavored to explore the transcriptional signature among different subgroups of newly diagnosed MM using gene chip-based expression microarray. Bone marrow samples of 15 newly diagnosed Thai MM patients were included. The chromosomal translocation t(4;14) was the most frequently identified genetic alteration in the high-risk subgroup. Cluster analysis from expression profiling demonstrated that high-risk MM have a distinctly different expression pattern compared to standard-risk patients. The most significant differentially expressed gene was UCHL1. Functional enrichment analysis by Gene Set Enrichment Analysis, FUNRICH, and Gene Ontology Panther pathway revealed the gene sets involved in cell cycle control to be enriched in the t(4;14) high-risk group. Interestingly, among the well-established downstream targets of UCHL1, only CCND2 was significantly expressed in the t(4;14) high-risk group. Suppression of UCHL1 protein level by LDN-5744 inhibitor could arrest the cell cycle in G1 phase in cell lines. These findings shed light on the molecular mechanism of UCHL1 in t(4;14) high-risk MM and support the evidence that alteration of the UCHL1 pathway may play a role in the pathogenesis of high-risk MM.

Chimeric Antigen Receptor T Cells Targeting Integrin αvß6 Expressed on Cholangiocarcinoma Cells.

Cholangiocarcinoma (CCA) is a lethal bile duct cancer that responds poorly to current standard treatments. A new therapeutic approach is, therefore, urgently needed. Adoptive T cell transfer using chimeric antigen receptor (CAR) T cells is a new therapeutic modality with demonstrated efficacy in hematologic malignancies. However, its efficacy against solid tumors is modest, and further intensive investigation continues. An important factor that influences the success of CAR T cell therapy is the selection of a target antigen that is highly expressed on cancer cells, but markedly less so in normal cells. Integrin αvß6 is upregulated in several solid tumors, but is minimally expressed in normal epithelial cells, which suggests integrin αvß6 as an attractive target antigen for CAR T cell immunotherapy in CCA. We investigated integrin αvß6 expression in pathological tissue samples from patients with liver fluke-associated CCA. We then created CAR T cells targeting integrin αvß6 and evaluated their anti-tumor activities against CCA cells. We found overexpression of the integrin αvß6 protein in 23 of 30 (73.3%) CCA patient tissue samples. Significant association between high integrin αvß6 expression and short survival time (p = 0.043) was also observed. Lentiviral constructs were engineered to encode CARs containing an integrin αvß6-binding peptide (A20) derived from foot-and-mouth disease virus fused with a second-generation CD28/CD3ζ signaling domain (A20-2G CAR) or with a fourth-generation CD28/4-1BB/CD27/CD3ζ signaling domain (A20-4G CAR). The A20-2G and A20-4G CARs were highly expressed in primary human T cells transduced with the engineered lentiviruses, and they exhibited high levels of cytotoxicity against integrin αvß6-positive CCA cells (p < 0.05). Interestingly, the A20-2G and A20-4G CAR T cells displayed anti-tumor function against integrin αvß6-positive CCA tumor spheroids (p < 0.05). Upon specific antigen recognition, A20-4G CAR T cells produced a slightly lower level of IFN-γ, but exhibited higher proliferation than A20-2G CAR T cells. Thus, the A20-4G CAR T cells with lower level of cytokine production, but with higher proliferation represents a promising potential adoptive T cell therapy for integrin αvß6-positive CCA.

Anti-mucin 1 chimeric antigen receptor T cells for adoptive T cell therapy of cholangiocarcinoma.

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.

Fourth-generation chimeric antigen receptor T cells targeting folate receptor alpha antigen expressed on breast cancer cells for adoptive T cell therapy.

PURPOSE: Treatment of breast cancer (BC) by standard methods is effective in the early stage, but ineffective in the advanced stage of disease. To develop an adoptive T cell therapy for advanced and severe BC, we generated fourth-generation chimeric antigen receptor (CAR) T cells targeting folate receptor alpha antigen (FRα) expressed on BC cells, and preclinically evaluated their anti-BC activities. METHODS: The fourth-generation FRα-CAR T cells containing extracellular FRα-specific single-chain variable fragment (scFv) and three intracellular costimulatory domains (CD28, 4-1BB, and CD27) linked to CD3ζ were generated using a lentiviral system, and then were evaluated for their anti-BC activities in two-dimensional and three-dimensional (spheroid) cultures. RESULTS: When our fourth-generation FRα-CAR T cells were cocultured with FRα-expressing MDA-MB-231 BC cell line at an effector to target ratio of 20:1, these CAR T cells specifically lysed 88.7 ± 10.6% of the target cells. Interestingly, the cytotoxic lysis of FRα-CAR T cells was more pronounced in target cells with higher surface FRα expression. This specific cytotoxicity of the CAR T cells was not observed when cocultured with FRα-negative MCF10A normal breast-like cell line at the same ratio (34.3 ± 4.7%). When they were cocultured with MDA-MD-231 spheroid, the FRα-CAR T cells exhibited antitumor activity marked with spheroid size reduction and breakage. CONCLUSION: This proof-of-concept study thus shows the feasibility of using these fourth-generation FRα-CAR T cells for adoptive T cell therapy in BC.

Development of a Novel Anti-CD19 CAR Containing a Fully Human scFv and Three Costimulatory Domains.

Second-generation anti-CD19-chimeric antigen receptor T cells (anti-CD19-CAR2 T cells) are effective for treating B-cell malignancies; however, anti-CD19-CAR2 T cells can induce human anti-mouse immune responses because anti-CD19 single-chain variable fragment (scFv) in the CAR molecules is derived from a murine FMC63 (mFMC63) monoclonal antibody. Consequently, the persistence of mFMC63-CAR2 T cells and their therapeutic efficiency in patients are decreased, which results in tumor relapse. In an attempt to remedy this shortcoming, we generated a new anti-CD19-CAR T cells containing fully human anti-CD19 scFv (Hu1E7-CAR4 T cells) to pre-clinically evaluate and compare with mFMC63-CAR4 T cells. The human anti-CD19 scFv (Hu1E7) was isolated from a human scFv phage display library and fused to the hinge region of CD8α, the transmembrane domain of CD28, three intracellular costimulatory domains (CD28, 4-1BB, and CD27), and a CD3ζ signaling domain (28BB27ζ). Compared to mFMC63-CAR2 T cells (BBζ) and mFMC63-CAR3 (BB27ζ), the mFMC63-CAR4 T cells (28BB27ζ) exerted superior anti-tumor activity against Raji (CD19+) target cell. The Hu1E7-CAR4 and mFMC63-CAR4 T cells demonstrated comparable cytotoxicity and proliferation. Interestingly, compared to mFMC63-CAR4 T cells, the Hu1E7-CAR4 T cells secreted lower levels of cytokines (IFN-γ and TNF-α), which may be due to the lower binding affinity of Hu1E7-CAR4 T cells. These findings demonstrated the successfulness in creation of a new CAR T cells containing a novel fully human-derived scFv specific to CD19+ cancer cells. In vivo studies are needed to further compare the anti-tumor efficacy and safety of Hu1E7-CAR4 T cells and mFMC63-CAR4 T cells.

Anti-tumour effect of the fourth-generation chimeric antigen receptor T cells targeting CD133 against cholangiocarcinoma cells.

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.