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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. [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]

• Anti-PD-L1 × anti-CD3 bispecific T-cell engagers (BTEs) were produced from CHO cells. • The BTEs were used to arm T cells to generate BTE-armed T cells (BATs). • BATs allow blockage of PD-L1/PD-1 and redirect T cells to kill PD-L1 + cancer cells. • Cryopreserved BATs retained stability, supporting their use and transportation. 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. [ABSTRACT FROM AUTHOR]

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]

Precision immunotherapy, driven by genomic and bioinformatic advancements, has emerged as a promising and viable approach to combat cancer. Targeting neoantigens offers the advantage of specific immune responses with minimal off-tumor toxicity. In this study, we investigated the potential of adoptive T cells activated by HLA-restricted neoantigen peptides from driver gene mutations for treating cholangiocarcinoma (CCA), a highly aggressive cancer with poor prognosis and high mortality rates. Through whole exome sequencing of CCA cell lines, KKU-213A and KKU-100, we identified mutations in common driver genes and predicted corresponding HLA-restricted peptides. Peptides from KRAS , RNF43 , and TP53 mutations exhibited strong binding affinity to HLA-A11, as validated through molecular docking and T2-cell binding assays. Dendritic cells (DCs) from healthy donors expressing HLA-A* 11:01, pulsed with individual or pooled peptides, showed comparable levels of costimulatory molecules (CD11c, CD40, CD86, and HLA-DR) to conventional DCs but higher expression of maturation markers, CD80 and CD86. Autologous HLA-A* 11:01-restricted T cells, activated by peptide-pulsed DCs, effectively lysed KKU-213A (HLA-A*11:01) cells, outperforming conventional tumor lysate-pulsed DCs. This effect was specific to HLA-A* 11:01-restricted T cells and not observed in KKU-100 (HLA-A*33:03) cells. Moreover, HLA-A* 11:01-restricted T cells exhibited elevated levels of IFN-gamma, granulysin, and granzyme B, indicating their potent anti-tumor capabilities. These findings underscore the specificity and efficiency of HLA-A* 11:01-restricted T cells targeting KRAS , RNF43 , TP53 mutated CCA cells, and offer valuable insights for developing immunotherapeutic strategies and therapeutic peptide-vaccines for CCA treatment. • KRAS , RNF43 , and TP53 neoantigen peptides bind to HLA-A* 11. • Neoantigen peptide-pulsed dendritic cells show high CD80 and CD83 expression. • Neoantigen-pulsed dendritic cells-activated T cells effectively killed mutated gene-harboring CCA. • T cell killing was HLA-A* 11-specific. [ABSTRACT FROM AUTHOR]