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.

Enhanced antitumor efficacy, proliferative capacity, and alleviation of T cell exhaustion by fifth-generation chimeric antigen receptor T cells targeting B cell maturation antigen in multiple myeloma.

Chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) has been approved for treating multiple myeloma (MM). Some clinical studies reported suboptimal outcomes, including reduced cytotoxicity of CAR-T cells and tumor evasion through increased expression of programmed death-ligand 1 (PD-L1). To enhance CAR-T cell efficiency and overcome PD-L1-mediated T cell suppression, we developed anti-BCMA-CAR5-T cells equipped with three costimulatory domains and the ability to secrete anti-PD-L1 single-chain variable fragment (scFv) blockade molecules. Anti-BCMA-CAR4-T cells contained a fully human anti-BCMA scFv and three intracellular domains (CD28, 4-1BB, and CD27) joined with CD3ζ. Anti-BCMA-CAR5-T cells were generated by fusing anti-BCMA-CAR4 with anti-PD-L1 scFv. Both anti-BCMA-CAR4-T and anti-BCMA-CAR5-T cells demonstrated comparable antitumor activity against parental MM cells. However, at an effector-to-target ratio of 1:2, only anti-BCMA-CAR5-T cells maintained cytolytic activity against PD-L1 high MM cells, unlike anti-BCMA-CAR4 T cells. Anti-BCMA-CAR5-T cells were specifically activated by BCMA-expressing target cells, resulting in increased CAR-T cell proliferation, release of cytolytic mediators, and pro-inflammatory cytokines. Anti-BCMA-CAR5-T cells demonstrated specific cytotoxicity against BCMA-expressing target cells, leading to decreased target cell numbers, increased CAR-T cell numbers, and preserved CAR expression during antigenic re-stimulation. Interestingly, only anti-BCMA-CAR5-T cells showed reduced PD-1 receptor levels, which correlated with decreased PD-L1 expression on target cells. We successfully generated anti-BCMA-CAR5-T cells capable of secreting anti-PD-L1 scFv. These cells exhibited superior antitumor efficiency, proliferative capacity, and alleviated T-cell exhaustion against MM cells. Further investigation into the antitumor efficacy of anti-BCMA-CAR5-T cells is warranted in ex vivo and clinical research settings.

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.

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.

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.

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.

Autosomal dominant diabetes associated with a novel ZYG11A mutation resulting in cell cycle arrest in beta-cells.

Diabetes is a genetically heterogeneous disease, for which we are aiming to identify causative genes. Here, we report a missense mutation (c.T1424C:p.L475P) in ZYG11A identified by exome sequencing as segregating with hyperglycemia in a Thai family with autosomal dominant diabetes. ZYG11A functions as a target recruitment subunit of an E3 ubiquitin ligase complex that plays an important role in the regulation of cell cycle. We demonstrate an increase in cells arrested at G2/mitotic phase among beta-cells deficient for ZYG11A or overexpressing L475P-ZYG11A, which is associated with a decreased growth rate. This is the first evidence linking a ZYG11A mutation to hyperglycemia, and suggesting ZYG11A as a cell cycle regulator required for beta-cell growth. Since most family members were either overweight or obese, but only mutation carriers developed hyperglycemia, our data also suggests the ZYG11A mutation as a genetic factor predisposing obese individuals to beta-cell failure in maintenance of glucose homeostasis.

GD2-specific chimeric antigen receptor-modified T cells targeting retinoblastoma - assessing tumor and T cell interaction.

A novel disialoganglioside 2 (GD2)-specific chimeric antigen receptor (CAR)-modified T cell therapy against retinoblastoma (RB) were generated. GD2-CAR consists of a single-chain variable fragment (scFv) derived from a monoclonal antibody, hu3F8, that is linked with the cytoplasmic signaling domains of CD28, 41BB, a CD3ζ, and an inducible caspase 9 death fusion partner. GD2 antigen is highly expressed in Y79RB cell line and in several surgical RB tumor specimens. In vitro co-culture experiments revealed the effective killing of Y79RB cells by GD2-CAR T cells, but not by control CD19-CAR T cells. The killing activities of GD2-CAR T cells were diminished when repeatedly exposed to the tumor, due to an attenuated expression of GD2 antigen on tumor cells and upregulation of inhibitory molecules of the PD1 and PD-L1 axis in the CAR T cells and RB tumor cells respectively. This is the first report to describe the potential of GD2-CAR T cells as a promising therapeutic strategy for RB with the indication of potential benefit of combination therapy with immune checkpoint inhibitors.

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.

Defective functions of HNF1A variants on BCL2L1 transactivation and beta-cell growth.

Maturity-onset diabetes of the young type 3 (MODY3) is caused by mutations in a gene encoding transcription factor hepatocyte nuclear factor 1-alpha (HNF1A). Although the roles of HNF1A in regulation of hepatic and pancreatic genes to maintain glucose homeostasis were investigated, the functions of HNF1A are not completely elucidated. To better understand the functions of HNF1A, we characterized mutations of HNF1A in Thai MODY3 patients and studied the functions of wild-type HNF1A and variant proteins. We demonstrate for the first time that HNF1A upregulates transactivation of an anti-apoptotic gene BCL2 Like 1 (BCL2L1) and that all the identified HNF1A variants including p.D80V, p.R203C, p.P475L, and p.G554fsX556, reduce this ability. The four HNF1A variants impair HNF1A function in promoting INS-1 cell transition from G1 to S phase of cell cycle, which thereby retard cell growth. This finding indicates the role of HNF1A in beta-cell viability by upregulation of anti-apoptotic gene expression and also reaffirms its role in beta-cell growth through cell cycle control.

Suppression of TGF-ß and IL-10 receptors on self-differentiated dendritic cells by short-hairpin RNAs enhanced activation of effector T-cells against cholangiocarcinoma cells.

Cholangiocarcinoma (CCA) is an aggressive tumor that is associated with high rates of recurrence and mortality. This is due, in part, to the fact that CCA cells and their microenvironment secrete immunosuppressive cytokines, transforming growth factor-ß (TGF-ß) and interleukin-10 (IL-10), that inhibit dendritic cell (DC) functions, which, in turn, results in the decreased anti-tumor activity of T-cells. We hypothesized that the TGF-ß receptor and IL-10 blockade on dendritic cells would improve DC function, thereby allowing improved activation of T cells against CCA cells. To test our hypothesis, we generated self-differentiated DCs (SD-DCs) via transduction of human peripheral blood monocytes with lentivirus expressing IL-4 and GM-CSF. SD-DCs were transduced with a second lentivirus containing short-hairpin RNAs (shRNAs) to knock-down TGF-ßRII and IL-10RA mRNAs. Immunoblot confirmed the reduced expression levels of TGF-ß and IL-10 receptors in both SD-DCs that were transduced with a single and/or combination of lentiviruses containing shRNAs. SD-DCs were thereafter pulsed with tumor antigens extracted from CCA cell lines in an effort to activate DC function. MHC class II (HLA-DR) and co-stimulatory molecules (CD40 and CD86) on SD-DCs were upregulated to levels comparable to those on DCs generated by the conventional method. Suppression of TGF-ß and IL-10 receptors on SD-DCs influenced the effector T-cells to produce IFN-γ, which enhanced their ability to kill CCA cells. The preparation of adoptive effector T-cells holds the potential of becoming a novel therapy for cellular immunotherapy in CCA.

Gemcitabine enhances cytotoxic activity of effector T-lymphocytes against chemo-resistant cholangiocarcinoma cells.

Cholangiocarcinoma (CCA) can resist chemotherapy resulting in treatment failure. Gemcitabine, a chemotherapeutic drug, can sensitize cancer cells to become susceptible to cytotoxic T-lymphocytes (CTLs). We, therefore, hypothesized that a combination of gemcitabine and CTLs would be more effective for CCA treatment than individual therapy. To test this hypothesis, we conducted an in vitro study using gemcitabine combined with CTLs to treat gemcitabine-resistant CCA (KKU-213) cells. KKU-213 cells were pretreated with gemcitabine and tested for killing by CTLs activated by dendritic cells that were prepared by three different methods, including: (i) monocyte-derived dendritic cells pulsed with cancer cell lysate (Mo-DC + Lys), (ii) self-differentiated dendritic cells pulsed with cancer-cell lysate (SD-DC + Lys), and (iii) SD-DC presenting tumor-associated antigen PRKAR1A (SD-DC-PR). KKU-213 cells pretreated with gemcitabine were killed by CTLs activated by either SD-DC + Lys or SD-DC-PR more efficiently than those activated by Mo-DC + Lys. Furthermore, KKU-213 cells pretreated with a low dose (2 µM) of gemcitabine significantly enhanced the cytotoxic activity of CTLs activated by either SD-DC + Lys or SD-DC-PR at all evaluated effector (E) to target cell (T) ratios. At an E:T ratio of 5:1, KKU-213 cells pretreated with gemcitabine enhanced the cytotoxic activity of CLTs by approximately 2.5-fold (greater than 50% cell death) compared to untreated condition. The upregulation of HLA class I upon pretreatment of KKU-213 cells with gemcitabine may suggest a mechanism that leads to alteration of the antigen presentation process to promote CTL functions. These findings support the concept of combination therapy for overcoming chemo-resistant CCA.

Estradiol Prevents High Glucose-Induced ß-cell Apoptosis by Decreased BTG2 Expression.

Hyperglycemia stimulates several pathways to induce pancreatic ß-cell apoptosis. In our previous study by mRNA analysis, we demonstrated that B-cell translocation gene 2 (BTG2) expression was up-regulated in INS-1 cells cultured under high glucose conditions, but this effect was reversed by estrogen. In the present study, we demonstrated that BTG2 mRNA and protein expressions in both INS-1 cells and mouse pancreatic islets increased under high glucose conditions compared to those cultured under basal glucose conditions, while in the presence of estrogen, the BTG2 mRNA and protein expressions decreased. SiRNA-BTG2 significantly reduced cell apoptosis, cleaved-caspase 3, and Bax, compared to the siRNA-control in INS-1 cultured under high glucose conditions. We further demonstrated that BTG2 promoter activity was activated under high glucose conditions whereas estrogen significantly reduced it. The effects of estrogen on BTG2 expression were inhibited by estrogen receptor inhibitors. Also, under high glucose conditions, p53 and Bax mRNA and protein expressions increased, but they decreased in the presence of estrogen. Again, the effect of estrogen on p53 and Bax expression was inhibited by estrogen receptor inhibitors. Taken together, this study demonstrates that estrogen reduces pancreatic ß-cell apoptosis under high glucose conditions via suppression of BTG2, p53, and Bax expressions.

Cytotoxic activity of effector T cells against cholangiocarcinoma is enhanced by self-differentiated monocyte-derived dendritic cells.

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.

DNAJC3 mutation in Thai familial type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2D) is a heterogeneous disease, with certain cases presenting an autosomal dominant type. The rare coding variants of disease­causing genes in T2D remain mostly unclear. The present study aimed to identify the disease­causing gene conducting whole exome sequencing in a Thai T2D family with an autosomal dominant transmission of T2D with no evidence of mutations in known maturity­onset diabetes of the young (MODY) genes. Candidate variants were selected according to certain criteria of mutation prediction programs, followed by segregation analysis with diabetes in the family. The results demonstrated that, of the 68,817 variants obtained, 122 were considered as candidate variants subsequent to the filtering processes. Genotyping of these variants revealed that DnaJ homolog subfamily C member 3 (DNAJC3) p.H238N segregated with diabetes in the family. This mutation was also identified in another proband from the autosomal dominant T2D family without mutation in known MODY genes and was segregated with diabetes. This variant was also identified in 14/1,000 older­onset T2D patients [minor allele frequency (MAF)=0.007], 2/500 non­diabetic controls (MAF=0.002) and 3 prediabetic individuals who were previously classified as non­diabetic controls. In silico mutagenesis and protein modeling of p.H238N revealed changes of the polar contacts across the tetratricopeptide repeat (TPR) motif and TPR subdomains, which may affect the protein tertiary structure. Furthermore, the expression of DNAJC3 H238N protein was 0.68±0.08 fold (P<0.05) lower when compared with that of the wild­type, possibly due to protein instability. Thus, DNAJC3 p.H238N is likely to be a variant causing diabetes.