A non-genetic engineering platform for rapidly generating and expanding cancer-specific armed T cells.

BACKGROUND: Cancer-specific adoptive T cell therapy has achieved successful milestones in multiple clinical treatments. However, the commercial production of cancer-specific T cells is often hampered by laborious cell culture procedures, the concern of retrovirus-based gene transfection, or insufficient T cell purity. METHODS: In this study, we developed a non-genetic engineering technology for rapidly manufacturing a large amount of cancer-specific T cells by utilizing a unique anti-cancer/anti-CD3 bispecific antibody (BsAb) to directly culture human peripheral blood mononuclear cells (PBMCs). The anti-CD3 moiety of the BsAb bound to the T cell surface and stimulated the differentiation and proliferation of T cells in PBMCs. The anti-cancer moiety of the BsAb provided these BsAb-armed T cells with the cancer-targeting ability, which transformed the naïve T cells into cancer-specific BsAb-armed T cells. RESULTS: With this technology, a large amount of cancer-specific BsAb-armed T cells can be rapidly generated with a purity of over 90% in 7 days. These BsAb-armed T cells efficiently accumulated at the tumor site both in vitro and in vivo. Cytotoxins (perforin and granzyme) and cytokines (TNF-α and IFN-γ) were dramatically released from the BsAb-armed T cells after engaging cancer cells, resulting in a remarkable anti-cancer efficacy. Notably, the BsAb-armed T cells did not cause obvious cytokine release syndrome or tissue toxicity in SCID mice bearing human tumors. CONCLUSIONS: Collectively, the BsAb-armed T cell technology represents a simple, time-saving, and highly safe method to generate highly pure cancer-specific effector T cells, thereby providing an affordable T cell immunotherapy to patients.

Comparing the Therapeutic Mechanism and Immune Response of Human and Mouse Mesenchymal Stem Cells in Immunocompetent Mice With Acute Liver Failure.

Current mesenchymal stem cell (MSC) research is based on xenotransplantation of human MSCs (hMSCs) in immunodeficient mice and cannot comprehensively predict MSC repair mechanisms and immunomodulatory effects in damaged tissue. This study compared the therapeutic efficacy, mechanisms, and immune response of hMSCs and mouse MSCs (mMSCs) in immunocompetent mice with CCl4-induced acute liver failure. mMSCs maintained F4/80+ hepatic macrophage recruitment into the damaged liver region, increased IL-6-dependent hepatocyte proliferation, and reduced inflammatory TNF-α cytokine secretion. Moreover, mMSCs reduced α-SMA+ myofibroblast activation by lowering TGF-ß1 accumulation in damaged liver tissue. In contrast, hMSCs lowered TNF-α and TGF-ß1 by reducing the recruitment of F4/80+ hepatic macrophages, which lost the ability to remove debris and induce IL-6 liver regeneration. Finally, hMSCs, but not mMSCs, caused a significant antibody response in immunocompetent mice; therefore, hMSCs are unsuitable for long-term MSC studies. This comparative study provides reference information for further MSC studies of immunocompetent mice.

New ent-kaurene and germacrene derivatives from Mesona procumbens Hemseley and their biological activity.

Mesona procumbens Hemseley is a well-known traditional herbal medicine used for heat-related ailments. In Taiwan, boiled extracts of M. procumbens are also used as desserts called grass jelly. In this study, the hexane extract from 75% EtOH of M. procumbens showed potent activities on inhibition of E. coli ß-glucuronidase (eßG) and NO production and cytotoxicity against MCF-7 and HepG2 cancer cell lines. Furthermore, using various flash columns and HPLC chromatography on the bioactive layer led to the isolation of twelve compounds (1-12), including a new ent-kaurene, mesokaurol A (1), and a new germacrene derivative, mesogermapene A (2). Their structures were elucidated by extensive spectroscopic analyses, especially 2 D NMR and mass data. Biological assays showed that compound 9 (linolenic acid) had specific activity on inhibition of eßG (68.27%) at 100 µg/mL but was non-inhibitory to human ß-glucuronidase. Compound 1 possessed significant cytotoxicity against MCF-7 (EC50 = 9.76 µM) and HepG2 (EC50 = 8.64 µM) cancer cell lines.

Rational design of synthetically tractable HDAC6/HSP90 dual inhibitors to destroy immune-suppressive tumor microenvironment.

INTRODUCTION: The tumor microenvironment is mainly flooded with immunosuppressive cells and inhibitory cytokines, resulting in the inability of effective immune cells to infiltrate and recognize tumors and even the loss of anti-cancer ability. OBJECTIVES: We propose a novel HDAC6/HSP90 dual inhibitory strategy as well as a chemoimmunotherapeutic agent that does not only kill tumor cells but also destroys the tumor microenvironment and enhances anti-cancer immunity. METHODS: A hybrid scaffold construction approach was leveraged to furnish a series of rationally designed resorcinol-based hydroxamates as dual selective HDAC6/HSP90 inhibitors. The drug design campaign commenced with a fragment recruitment process to pinpoint validated structural units to inhibit HDAC6 and HSP90, followed by their installation in flexible HDAC inhibitory templates via an efficient and facile multistep synthetic route. Subsequent evaluations identified a strikingly potent selective HDAC6/HSP90 dual inhibitor (compound 17) via molecular and biological analysis in vitro and in vivo. RESULTS: Compound 17 exhibited not only direct cytotoxicity to cancer cells but also downregulated immune checkpoints (PD-L1 and IDO) expression in tumors via the inhibition of STAT1 pathway and degradation of oncogene proteins (Src, AKT, Rb, and FAK), leading to in vivo tumor growth inhibition. These multiple effects enabled the effector T cells to largely infiltrate into the tumor region and release granzyme B to kill cancer cells. In addition, compound 17 also decreased TGF-ß secretion from normal cells, resulting in the systemic reduction of immunosuppressive regulatory T cells. Delightfully, a cocktail treatment of compound 17 and anti-PD-1 antibodies demonstrated synergistic efficacy to eliminate solid tumors with 83.9% of tumor growth inhibition. CONCLUSION: In summary, the impressive activity profile of compound 17, as an effective anticancer agent and a potential immunosensitizer, forecasts the application of HDAC6/HSP90 dual inhibitory strategy to overcome the immunosuppressive tumor microenvironment.

Simply Mixing Poly Protein G with Detection Antibodies Enhances the Detection Limit and Sensitivity of Immunoassays.

An insufficient amount of detection antibodies bound to their antigens usually limits the sensitivity of immunoassays. Here, we describe a simple method to improve the detection limit and sensitivity of various immunoassays by mixing detection antibodies with a soluble poly protein G (named 8pG). 8pG was developed by fusing eight repeated fragment crystallizable (Fc) binding domains of streptococcal protein G to a linear polymer. Simply mixing detection antibodies with 8pG to form an antibody/8pG complex largely increased the accumulation of detection antibody to target molecules, which dramatically enhanced the sensitivity in direct ELISA, sandwich ELISA, Western blot, and flow cytometry systems, separately. The detection limit of Western blot for low-abundance PEGylated interferon (Pegasys) and recombinant human CTLA4 (rhCTLA4) improved by at least 13-fold and 31-fold, respectively, upon mixing detection antibodies with 8pG. Moreover, the nanoscale size of the antibody/8pG complex did not influence the granularity and dimension of target cells in the flow cytometry system. Collectively, we provide a quick and easy-to-operate method to make various immunoassays to sensitively detect low-abundance target molecules by just mixing their detection antibodies with 8pG.

Development of a highly sensitive enzyme-linked immunosorbent assay (ELISA) through use of poly-protein G-expressing cell-based microplates.

The sensitivity of traditional enzyme-linked immunosorbent assays (ELISAs) is limited by the low binding avidity and heterogeneous orientation of capture antibodies coated on polystyrene-based microplates. Here, we developed a highly sensitive ELISA strategy by fixing poly-protein G-expressing cells on microplates to improve the coating amount and displayed orientation of capture antibodies. One or eight repeated fragment crystallisable (Fc) binding domains of protein G are stably expressed on the surface of BALB/c 3T3 cells (termed 1pG cells or 8pG cells), which then act as highly antibody-trapping microparticles. The 8pG cells showed higher antibody-trapping ability than the 1pG cells did. The antibody-coating amount of the 8pG cell-based microplates was 1.5-23 times and 1.2-6.8 times higher than that of traditional polystyrene-based and commercial protein G-based microplates, respectively. The 8pG cell-based microplates were then applied to an anti-IFN-α sandwich ELISA and an anti-CTLA4 competitive ELISA, respectively, and dramatically enhanced their detection sensitivity. Importantly, direct coating unpurified capture antibody produced by mammalian cells did not impair the antigen-capturing function of 8pG cell-based microplates. The 8pG cell-based microplates exhibited a significant improvement in antibody-coating amount and preserved the homogeneous orientation of capture antibodies, making them a potential replacement for traditional microplates in various formats of ELISAs.

Real-time imaging of intestinal bacterial ß-glucuronidase activity by hydrolysis of a fluorescent probe.

Intestinal bacterial ß-glucuronidase (ßG) hydrolyzes glucuronidated metabolites to their toxic form in intestines, resulting in intestinal damage. The development of a method to inhibit ßG is thus important but has been limited by the difficulty of directly assessing enzyme activity in live animals. Here, we utilized a fluorescent probe, fluorescein di-ß-D-glucuronide (FDGlcU), to non-invasively image the intestinal bacterial ßG activity in nude mice. In vitro cell-based assays showed that the detection limit is 104 colony-forming units/well of ßG-expressing bacteria, and that 7.81 ng/mL of FDGlcU is enough to generate significant fluorescent signal. In whole-body optical images of nude mice, the maximum fluorescence signal for ßG activity in intestines was detected 3 hours after gavage with FDGlcU. Following pretreatment with a bacterial ßG inhibitor, the fluorescence signal was significantly reduced in abdomens and excised intestines images. For a 4-day antibiotic treatment to deplete intestinal bacteria, the FDGlcU-based images showed that the ßG activity was decreased by 8.5-fold on day 4 and then gradually increased after treatment stopped. The results suggested that FDGlcU-based imaging revealed the in vitro and in vivo activity of intestinal bacterial ßG, which would facilitate pharmacodynamic studies of specific bacterial ßG inhibitors in animal studies.