Patient derived cancer organoids model the response to HER2-CD3 bispecific antibody (BsAbHER2) generated from hydroxyapatite gene delivery system.

HER2-positive cancer is a prevalent subtype of malignancy with poor prognosis, yet current targeted therapies, like Trastuzumab and pyrotinib, have resulted in remission in patients with HER2-positive cancer. This study provides a novel approach for immunotherapy based on a hydroxyapatite (HA) gene delivery system producing a bispecific antibody for HER2-positive cancer treatment. An HA nanocarrier has been synthesized by the classical hydrothermal method. Particularly, the HA-nanoneedle system was able to mediate stable gene expression of minicircle DNA (MC) encoding a humanized anti-CD3/anti-HER2 bispecific antibody (BsAbHER2) in vivo. The produced BsAbs exhibited a potent killing effect not only in HER2-positive cancer cells but also in patient-derived organoids in vitro. This HA-nanoneedle gene delivery system features simple large-scale preparation and clinical applicability. Hence, the HA-nanoneedle gene delivery system combined with minicircle DNA vector encoding BsAbHER2 reported here provides a potential immunotherapy strategy for HER2-positive tumors.

Engineering Bifunctional Calcium Alendronate Gene-Delivery Nanoneedle for Synergistic Chemo/Immuno-Therapy Against HER2 Positive Ovarian Cancer.

Ovarian cancer is the most lethal gynecological malignancy. Most patients are diagnosed at an advanced stage with widespread peritoneal dissemination and ascites. Bispecific T-cell engagers (BiTEs) have demonstrated impressive antitumor efficacy in hematological malignancies, but the clinical potency is limited by their short half-life, inconvenient continuous intravenous infusion, and severe toxicity at relevant therapeutic levels in solid tumors. To address these critical issues, the design and engineering of alendronate calcium (CaALN) based gene-delivery system is reported to express therapeutic level of BiTE (HER2×CD3) for efficient ovarian cancer immunotherapy. Controllable construction of CaALN nanosphere and nanoneedle is achieved by the simple and green coordination reactions that the distinct nanoneedle-like alendronate calcium (CaALN-N) with a high aspect ratio enabled efficient gene delivery to the peritoneum without system in vivo toxicity. Especially, CaALN-N induced apoptosis of SKOV3-luc cell via down-regulation of HER2 signaling pathway and synergized with HER2×CD3 to generate high antitumor response. In vivo administration of CaALN-N/minicircle DNA encoding HER2×CD3 (MC-HER2×CD3) produces sustained therapeutic levels of BiTE and suppresses tumor growth in a human ovarian cancer xenograft model. Collectively, the engineered alendronate calcium nanoneedle represents a bifunctional gene delivery platform for the efficient and synergistic treatment of ovarian cancer.

Anticancer effects of a single intramuscular dose of a minicircle DNA vector expressing anti-CD3/CD20 in a xenograft mouse model.

Bispecific antibodies (BsAbs) are a class of promising anticancer immunotherapies. Among them, the US Food and Drug Administration (FDA)-approved blinatumomab (BLI) is very effective in eliminating the minimum residual disease (MRD) of acute lymphoblastic leukemia (ALL), resulting in long-term remission in many individuals. However, the need for months-long intravenous delivery and high cost limit its clinical acceptance. Here we demonstrate that these problems can be solved by a BsAb expressed by one intramuscular (i.m.) dose of a minicircle DNA vector (MC). In a human B lymphoma xenograft mouse model, when microcancers became detectable in bone marrow, the mice received an i.m. dose of the MC encoding the BsAb anti-CD3/CD20 (BsAb.CD20), followed by 8 subsequent intravenous (i.v.) doses, one every other day (q2d), of human T cells to serve as effectors. The treatment resulted in persistent expression of a therapeutic level of serum BsAb.CD20 and complete regression or growth retardation of the cancers in the mice. These results suggest that the i.m. MC technology can eliminate the physical and financial burdens of i.v. delivered BLI without compromising anticancer efficacy and that cancer can be treated as easily as injecting a vaccine. This, together with other superior MC features, such as safety and affordability, suggests that the i.m. MC BsAb technology has great clinical application potential.

Calcium phosphate nanoneedle based gene delivery system for cancer genetic immunotherapy.

Ovarian cancer has become one of the most common gynecological cancers with a high mortality. However, conventional surgery together with combination chemotherapy is difficult to achieve ideal therapeutic effect. Although genetic immunotherapy is applied to active immune responses against cancer, the absence of efficient in vivo gene delivery technique is still an obstacle in clinical application. To overcome these problems, a minicircle DNA vector encoding humanized anti-EpCAM/CD3 bispecific antibody (BsAbEPH) has been constructed. Moreover, different shapes of calcium phosphate (CaPO) biomaterials were prepared. Specifically, the CaPO-nanoneedle-mediated "cell perforation" transfection technology achieves high levels of gene expression in peritoneal cavity. In an intraperitoneal xenograft model with human ovarian cancer cell line SKOV3, the CaPO-nanoneedle/minicircle DNA system expressed BsAbEPH resulted in significant retardation of cancer growth and extension of mouse life-span with limited toxicity. And this system can be made as off-the-shelf and easy-to-use products. Therefore, CaPO-nanoneedle based non-viral gene delivery technology will have great potential in clinical application.

Rhizopus nigricans polysaccharide activated macrophages and suppressed tumor growth in CT26 tumor-bearing mice.

In this study, a homogeneous polysaccharide (RPS-1) was extracted from liquid-cultured mycelia of Rhizopus nigricans. The weight-average molecular weight of RPS-1 was 1.617 × 107 g/mol and structural characterization indicated that RPS-1 was a non-starch glucan which consisted of a backbone structure of (1→4)-linked α-d-glucopyranosyl residues substituted at the O-6 position with α-d-glucopyranosyl branches. RPS-1 stimulated the production of nitric oxide and tumor necrosis factor-α by triggering phosphorylation of mitogen-activated protein kinases and nuclear translocation of nuclear factor kappa B p65 in RAW 264.7 macrophage cells. Moreover, intragastric administration of RPS-1 improved the immune function of CT26 tumor-bearing mice and significantly inhibited the growth of transplanted tumor. In combination with 5-FU, RPS-1 enhanced antitumor activity of 5-FU and alleviated its toxicity on immune system. These findings suggested that RPS-1 has the potential for the development of functional foods and dietary supplements.

Exopolysaccharide from Trichoderma pseudokoningii promotes maturation of murine dendritic cells.

Dendritic cells (DCs) are the key regulators of immune responses. In this study, the effect of an exopolysaccharide (EPS) from the culture broth of Trichoderma pseudokoningii on the phenotypic and functional maturation of murine DCs and its underlying molecular mechanisms were investigated. It showed that EPS induced the morphological changes of DCs and the enhanced expression of DCs featured surface molecules CD11c, CD86, CD80 and major histocompatibility complex II (MHC-II). Flow cytometry analysis showed that the treatment with EPS could reduce FITC-dextran uptake by DCs. Sequentially, the results of ELISA indicated that EPS could increase the production of interleukin-12p70 (IL-12p70) in culture supernatant of DCs. Immunofluorescence staining and western blot analysis further revealed that EPS significantly prompted nuclear factor (NF)-κB subunit p65 translocation, IκB-α protein degradation, and p38 mitogen-activated protein kinase (MAPK) phosphorylation. And the production of IL-12p70 was significantly decreased in condition of the inhibition of p38 or NF-κB signaling pathway. These findings suggested that EPS could induce DCs maturation through both p38 MAPK and NF-κB signaling pathways.

Anti-tumor and immunomodulatory activities of an exopolysaccharide from Rhizopus nigricans on CT26 tumor-bearing mice.

This study was aimed to investigate the anti-tumor and immunomodulatory activities of an exopolysaccharide (EPS) from Rhizopus nigricans. Our results showed EPS could significantly inhibit the tumor growth and increase the immune organs index of CT26 tumor-bearing mice. EPS treatment increased the productions of interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α) levels in serum. The increase of percentage of CD8(+) cytotoxic T cells among total spleen T lymphocyte was also observed. Furthermore, EPS remarkably stimulate spleen lymphocytes proliferation in the absence or presence of mitogens. In addition, we found that EPS had synergistic effect with chemotherapy and improved immunosuppressive effect induced by 5-Fu. In summary, these findings indicated that the antitumor effects of EPS might be partly due to immune function activation and it might have potential to be used in the treatment for colorectal cancer.

Purification, partial characterization and antitumor effect of an exopolysaccharide from Rhizopus nigricans.

In this study, a homogeneous exopolysaccharide (EPS1-1) was purified from the fermentation broth of Rhizopus nigricans. EPS1-1 was composed of glucose, mannose, galactose and fructose in the molar ratio of 5.89:3.64:3.20:1.00 with weight average molecular weight of 9.7×10(3)g/mol. EPS1-1 could significantly inhibit proliferation of human colorectal carcinoma HCT-116 cells in vitro. EPS1-1 also induced S phase cell cycle arrest and increased sub-G0/G1 population, a hallmark of apoptosis. The results of morphological characterization and flow cytometry showed that EPS1-1 induced apoptotic cell death in HCT-116 cells. EPS1-1 caused dissipation of mitochondrial membrane potential, accumulation of reactive oxygen species, up-regulation of Bax and p53 mRNA expression and down-regulation of Bcl-2 mRNA expression, which suggested that mitochondrial pathway was involved in the EPS1-1-induced apoptosis. These findings bring new insights into the potential use of EPS1-1 as antitumor drug against human colorectal carcinoma.

Activation of RAW 264.7 cells by a polysaccharide isolated from Antarctic bacterium Pseudoaltermonas sp. S-5.

The aim of this study was to examine the effect of extracellular polysaccharide (PEP) from Antarctic bacterium Pseudoaltermonas sp. S-5 on RAW 264.7 cells together with the underlying signaling pathways. Our results illustrated that PEP induced dendritic-like morphological change in RAW 264.7 cells, and increased the productions of nitric oxide (NO) and tumor necrosis factor-α (TNF-α). PEP could also enhance phagocytic activity of RAW 264.7 cells. Results of immunofluorescence staining and immunoblotting indicated that PEP caused the nuclear translocation of nuclear factor (NF)-κB subunit p65, the degradation of IκB-α and up-expression of phosphorylated p38 mitogen-activated protein kinase (MAPK) in RAW 264.7 cells. According to pharmacological evaluation with specific enzyme inhibitors, both NF-κB and p38 MAPK signaling pathways were involved in the generation of NO and TNF-α induced by PEP. All these results indicated that PEP from Antarctic bacterium Pseudoaltermonas sp. S-5 activated RAW 264.7 cells through NF-κB and p38 MAPK signaling pathways.

Exopolysaccharide of Antarctic bacterium Pseudoaltermonas sp. S-5 induces apoptosis in K562 cells.

The aim of this study was to investigate the anticancer activity of exopolysaccharide (PEP) of Antarctic bacterium Pseudoaltermonas sp. S-5 and elucidate the underlying molecular mechanisms. PEP significantly inhibited the growth of human leukemia K562 cells. Results of morphological characterization showed that PEP-treated cells displayed typical morphological characteristics of apoptosis such as condensation of chromatin and formation of apoptotic bodies. Flow cytometry analyses and colorimetric assay demonstrated that PEP induced collapse of mitochondrial membrane potential and activation of caspase-9, which indicated that intrinsic apoptotic signaling pathway was involved in apoptosis induced by PEP in K562 cells. Western blot analysis showed that PEP increased the ratio of Bax/Bcl-2. In addition, calcium signal might contribute to the cytotoxicity of PEP against K562 cells. These findings suggest that PEP may be potentially effective drug against human leukemia.

Polysaccharides from Rhizopus nigricans mycelia induced apoptosis and G2/M arrest in BGC-823 cells.

In this study, we reported that polysaccharides (RPS) from the mycelia of liquid-cultured Rhizopus nigricans inhibited the proliferation and clonogenic potential of human gastric cancer BGC-823 cells. Results of acridine orange/ethidium bromide and Hoechst 33258 staining showed that treated cells displayed typical morphological characteristics of apoptosis such as condensation of chromatin, nuclear pyknosis and formation of apoptotic bodies. Flow cytometry analyses and colorimetric assay indicated that RPS induced mitochondria-mediated apoptosis which was associated with collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, generation of intracellular reactive oxygen species and elevation of intracellular calcium in BGC-823 cells. Moreover, cell-cycle analysis revealed that RPS arrested cells in the G2/M phase of the cell cycle. These results provided further insights into the potential use of RPS as an anti-cancer agent against human gastric cancer.

Macrophage immunomodulatory activity of extracellular polysaccharide (PEP) of Antarctic bacterium Pseudoaltermonas sp.S-5.

Antarctic bacteria are a novel source of polysaccharides which might have potential applications as biological response modifiers (BRM). A heteropolysaccharide (PEP) was isolated from the liquid culture broth of the Antarctic bacterium Pseudoaltermonas sp.S-5. PEP contained Mannose, Glucose, and Galactose in a ratio of 4.8:50.9:44.3. High performance gel permeation chromatography of this polysaccharide showed a unimodal profile, and the molecular weight was 397 kDa. PEP was studied for its immunological effects on peritoneal macrophage cells. Morphological alterations were observed in macrophages treated with PEP. In vitro exposure to PEP increased the occurrence of activated macrophages and endocytic index in a dose-dependent pattern (2.5-50 µg/ml) after 24h of incubation, since increase of 136% and 133% was detected in the activated macrophage percentage and endocytic index respectively compared to untreated cells. At 200 µg/ml PEP caused a greatest increase (44.5%) in NO production when compared to the control group; however, this polysaccharide did not affect respiratory burst in the absence of PMA. Furthermore, it was demonstrated that PEP induces macrophages to secrete tumor necrosis factor (TNF)-α and interleukin (IL)-1ß. These results suggested that PEP from Pseudoaltermonas sp.S-5 can be classified as a BRM.

An exopolysaccharide from Trichoderma pseudokoningii and its apoptotic activity on human leukemia K562 cells.

In this study, a novel exopolysaccharide (EPS) was isolated from the fermentation broth of Trichoderma pseudokoningii and its anticancer activities on human leukemia K562 cells were studied. EPS could significantly inhibited K562 cells proliferation in a time- and concentration-dependent manner. Meanwhile, characteristic of apoptosis, including apoptotic morphological features and the apoptosis rate were obtained. Sequentially, the dissipation of mitochondrial membrane potential, increase production of Reactive oxygen species (ROS), enhancement of the concentration of intracellular, up-regulation of Bax and p53 mRNA, down-regulation of Bcl-2 mRNA were also detected. The results indicate that the EPS could induce of K562 cells apoptosis, primarily in involved the mitochondrial pathways. The present studies suggest that EPS could be a new potential adjuvant chemotherapeutic and chemo preventive agent against human leukemia.