ABSTRACT
In the original publication [...].
ABSTRACT
Adeno-associated viruses (AAVs) have been well characterized and used to deliver therapeutic genes for diseases treatment in clinics and basic research. This study used the triple transient transfection of AAV-DJ/8 as a model expression system to develop and optimize the laboratory production of AAV for research and pre-clinical applications. Specifically, various production parameters, including host cell, transfection reagent, cell density, ratio of plasmid DNA and cells, gene size, and production mode, were tested to determine the optimal process. Our results showed that the adherent production using HEK 293AAV with calcium transfection generated the highest volumetric productivity of 7.86x109 gc/mL. The optimal suspensive production using HEK 293F had best AAV productivity of 5.78x109 gc/mL in serum-free medium under transfection conditions of transfection density of 0.4x106 cells/mL, plasmid DNA:cells ratio of 1.6 µg:106 cells and synthesized cationic liposomes as transfection reagent. The similar AAV productivity was confirmed at scales of 30 mL - 450 mL in shaker and/or spinner flasks. The in vitro transfection and in vivo infection efficiency of the harvested AAV-DJ/8 carrying luciferase reporter gene was confirmed using cell line and xenograft mouse model, respectively. The minimal or low purification recovery rate of AAV-DJ/8 in ion-exchange chromatography column and affinity column was observed in this study. In summary, we developed and optimized a scalable suspensive production of AAV to support the large-scale preclinical animal studies in research laboratories.
ABSTRACT
Triple-negative breast cancers (TNBCs) are heterogeneous and metastatic, and targeted therapy is highly needed for TNBC treatment. Recent studies showed that extracellular vesicles (EV) have great potential to deliver therapies to treat cancers. This study aimed to develop and evaluate a natural compound, verrucarin A (Ver-A), delivered by targeted EV, to treat TNBC. First, the surface expression of epidermal growth factor receptor (EGFR) and CD47 were confirmed with immunohistochemistry (IHC) staining of patient tissue microarray, flow cytometry and Western blotting. EVs were isolated from HEK 293F culture and surface tagged with anti-EGFR/CD47 mAbs to construct mAb-EV. The flow cytometry, confocal imaging and live-animal In Vivo Imaging System (IVIS) demonstrated that mAb-EV could effectively target TNBC and deliver the drug. The drug Ver-A, with dosage-dependent high cytotoxicity to TNBC cells, was packed in mAb-EV. The anti-TNBC efficacy study showed that Ver-A blocked tumor growth in both 4T1 xenografted immunocompetent mouse models and TNBC patient-derived xenograft models with minimal side effects. This study demonstrated that the targeted mAb-EV-Ver-A had great potential to treat TNBCs.
ABSTRACT
Glioblastomas, accounting for approximately 50% of gliomas, comprise the most aggressive, highly heterogeneous, and malignant brain tumors. The objective of this study was to develop and evaluate a new targeted therapy, i.e., highly potent natural compound verrucarin A (Ver-A), delivered with monoclonal antibody-directed extracellular vesicle (mAb-EV). First, the high surface expression of epidermal growth factor receptor (EGFR) in glioblastoma patient tissue and cell lines was confirmed using immunohistochemistry staining, flow cytometry, and Western blotting. mAb-EV-Ver-A was constructed by packing Ver-A and tagging anti-EGFR mAb to EV generated from HEK293F culture. Confocal microscopy and the In Vivo Imaging System demonstrated that mAb-EV could penetrate the blood-brain barrier, target intracranial glioblastoma xenografts, and deliver drug intracellularly. The in vitro cytotoxicity study showed IC50 values of 2-12 nM of Ver-A. The hematoxylin and eosin staining of major organs in the tolerated dose study indicated minimal systemic toxicity of mAb-EV-Ver-A. Finally, the in vivo anti-tumor efficacy study in intracranial xenograft models demonstrated that EGFR mAb-EV-Ver-A effectively inhibited glioblastoma growth, but the combination with VEGF mAb did not improve the therapeutic efficacy. This study suggested that mAb-EV is an effective drug delivery vehicle and natural Ver-A has great potential to treat glioblastoma.
ABSTRACT
Triple-negative breast cancers (TNBCs) are highly aggressive and recurrent. Standard cytotoxic chemotherapies are currently the main treatment options, but their clinical efficacies are limited and patients usually suffer from severe side effects. The goal of this study was to develop and evaluate targeted liposomes-delivered combined chemotherapies to treat TNBCs. Specifically, the IC50 values of the microtubule polymerization inhibitor mertansine (DM1), mitotic spindle assembly defecting taxane (paclitaxel, PTX), DNA synthesis inhibitor gemcitabine (GC), and DNA damage inducer doxorubicin (AC) were tested in both TNBC MDA-MB-231 and MDA-MB-468 cells. Then we constructed the anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) tagged liposomes and confirmed its TNBC cell surface binding using flow cytometry, internalization with confocal laser scanning microscopy, and TNBC xenograft targeting in NSG female mice using In Vivo Imaging System. The safe dosage of anti-EGFR liposomal chemotherapies, i.e., <20% body weight change, was identified. Finally, the in vivo anti-tumor efficacy studies in TNBC cell line-derived xenograft and patient-derived xenograft models revealed that the targeted delivery of chemotherapies (mertansine and gemcitabine) can effectively inhibit tumor growth. This study demonstrated that the targeted liposomes enable the new formulations of combined therapies that improve anti-TNBC efficacy.
ABSTRACT
Triple-negative breast cancers (TNBCs) are frequently recurrent due to the development of drug resistance post chemotherapy. Both the existing literature and our study found that surface receptor CD47 (cluster of differentiation 47) was upregulated in chemotherapy-treated TNBC cells. The goal of this study was to develop a monoclonal antibody (mAb)-based targeting strategy to treat TNBC after standard treatment. Specifically, a new mAb that targets the extracellular domain of receptor CD47 was developed using hybridoma technology and produced in fed-batch culture. Flow cytometry, confocal microscopy, and in vivo imaging system (IVIS) showed that the anti-CD47 mAb effectively targeted human and mouse TNBC cells and xenograft models with high specificity. The antibody-drug conjugate (ADC) carrying mertansine was constructed and demonstrated higher potency with reduced IC50 in TNBC cells than did the free drug and significantly inhibited tumor growth post gemcitabine treatment in MDA-MB-231 xenograft NSG model. Finally, whole blood analysis indicated that the anti-CD47 mAb had no general immune toxicity, flow cytometry analysis of lymph nodes revealed an increase of CD69+ NK, CD11c+ DC, and CD4+ T cells, and IHC staining showed tumoral infiltration of macrophage in the 4T1 xenograft BALB/cJ model. This study demonstrated that targeting CD47 with ADC has great potential to treat TNBCs as a targeted therapy.
ABSTRACT
Meningiomas are primary tumors of the central nervous system with high recurrence. It has been reported that somatostatin receptor 2 (SSTR2) is highly expressed in most meningiomas, but there is no effective targeted therapy approved to control meningiomas. This study aimed to develop and evaluate an anti-SSTR2 antibody-drug conjugate (ADC) to target and treat meningiomas. The meningioma targeting, circulation stability, toxicity, and anti-tumor efficacy of SSTR2 ADC were evaluated using cell lines and/or an intracranial xenograft mouse model. The flow cytometry analysis showed that the anti-SSTR2 mAb had a high binding rate of >98% to meningioma CH157-MN cells but a low binding rate of <5% to the normal arachnoidal AC07 cells. The In Vivo Imaging System (IVIS) imaging demonstrated that the Cy5.5-labeled ADC targeted and accumulated in meningioma xenograft but not in normal organs. The pharmacokinetics study and histological analysis confirmed the stability and minimal toxicity. In vitro anti-cancer cytotoxicity indicated a high potency of ADC with an IC50 value of <10 nM. In vivo anti-tumor efficacy showed that the anti-SSTR2 ADC with doses of 8 and 16 mg/kg body weight effectively inhibited tumor growth. This study demonstrated that the anti-SSTR2 ADC can target meningioma and reduce the tumor growth.
