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Cooperative function of superoxide dismutase (SOD) and catalase (CAT), in protection against oxidative stress, is known to be more effective than the action of either single enzyme. Chemical conjugation of the two enzymes resulted in molecules with higher antioxidant activity and therapeutic efficacy. However, chemical methods holds several drawbacks; e.g., loss of enzymatic activity, low homogeneity, time-consuming, and the need of chemical residues removal. Yet, the conjugated enzymes have never been proven to internalize into target cells. In this study, by employing genetic and protein engineering technologies, we reported designing and production of a bi-functional protein with SOD and CAT activities for the first time. To enable cellular internalization, cell penetrating peptide from HIV-1 Tat (TAT) was incorporated. Co-expression of CAT-MnSOD and MnSOD-TAT fusion genes allowed simultaneous self-assembly of the protein sequences into a large protein complex, which is expected to contained one tetrameric structure of CAT, four tetrameric structures of MnSOD and twelve units of TAT. The protein showed cellular internalization and superior protection against paraquat-induced cell death as compared to either complex bi-functional protein without TAT or to native enzymes fused with TAT. This study not only provided an alternative strategy to produce multifunctional protein complex, but also gained an insight into the development of therapeutic agent against oxidative stress-related conditions. [ABSTRACT FROM AUTHOR]

Adoptive cell transfer (ACT) is a promising approach for cancer treatment. Activation of T lymphocytes by s elf-differentiated m yeloid-derived a ntigen-presenting-cells r eactive against t umor (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.

Dendritic cell (DC)-based T-cell activation is an alternative immunotherapy in breast cancer. The anti-programmed death ligand 1 (PD-L1) can enhance T-cell function. Nucleolin (NCL) is overexpressed in triple-negative breast cancer (TNBC). The regulation of PD-L1 expression through autophagy and the anti-PD-L1 peptide to help sensitize T cells for NCL-positive TNBC cell killing has not been evaluated. Results showed the worst clinical outcome in patients with high NCL and PD-L1. Self-differentiated myeloid-derived antigen-presenting cells reactive against tumors presenting NCL or SmartDCs-NCL producing GM-CSF and IL-4, could activate NCL-specific T cells. SmartDCs-NCL plus recombinant human ribosomal protein substrate 3 (RPS3) successfully induced maturation and activation of DCs characterized by the reduction of CD14 and the induction of CD11c, CD40, CD80, CD83, CD86, and HLA-DR. Interestingly, SmartDCs-NCL plus RPS3 in combination with anti-PD-L1 peptide revealed significant killing activity of the effector NCL-specific T cells against NCLHigh/PD-L1High MDA-MB-231 and NCLHigh/PD-L1High HCC70 TNBC cells at the effector: a target ratio of 5:1 in 2-D and 10:1 in the 3-D culture system; and increments of IFNγ by the ELISpot assay. No killing effect was revealed in MCF-10A normal mammary cells. Mechanistically, NCL-specific T-cell-mediated TNBC cell killing was through both apoptotic and autophagic pathways. Induction of autophagy by curcumin, an autophagic stimulator, inhibited the expression of PD-L1 and enhanced cytolytic activity of NCL-specific T cells. These findings provide the potential clinical approaches targeting NCLHigh/PD-L1High TNBC cells with NCL-specific T cells in combination with a PD-L1 inhibitor or autophagic stimulator., (©2022 The Authors; Published by the American Association for Cancer Research.)

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., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)