Investigation into the difference in mitochondrial-cytosolic calcium coupling between adult cardiomyocyte and hiPSC-CM using a novel multifunctional genetic probe.

Ca2+ cycling plays a critical role in regulating cardiomyocyte (CM) function under both physiological and pathological conditions. Mitochondria have been implicated in Ca2+ handling in adult cardiomyocytes (ACMs). However, little is known about their role in the regulation of Ca2+ dynamics in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). In the present study, we developed a multifunctional genetically encoded Ca2+ probe capable of simultaneously measuring cytosolic and mitochondrial Ca2+ in real time. Using this novel probe, we determined and compared mitochondrial Ca2+ activity and the coupling with cytosolic Ca2+ dynamics in hiPSC-CMs and ACMs. Our data showed that while ACMs displayed a highly coordinated beat-by-beat response in mitochondrial Ca2+ in sync with cytosolic Ca2+, hiPSC-CMs showed high cell-wide variability in mitochondrial Ca2+ activity that is poorly coordinated with cytosolic Ca2+. We then revealed that mitochondrial-sarcoplasmic reticulum (SR) tethering, as well as the inter-mitochondrial network connection, is underdeveloped in hiPSC-CM compared to ACM, which may underlie the observed spatiotemporal decoupling between cytosolic and mitochondrial Ca2+ dynamics. Finally, we showed that knockdown of mitofusin-2 (Mfn2), a protein tethering mitochondria and SR, led to reduced cytosolic-mitochondrial Ca2+ coupling in ACMs, albeit to a lesser degree compared to hiPSC-CMs, suggesting that Mfn2 is a potential engineering target for improving mitochondrial-cytosolic Ca2+ coupling in hiPSC-CMs. Physiological relevance: The present study will advance our understanding of the role of mitochondria in Ca2+ handling and cycling in CMs, and guide the development of hiPSC-CMs for healing injured hearts.

MitoQ regulates redox-related noncoding RNAs to preserve mitochondrial network integrity in pressure-overload heart failure.

Evidence suggests that mitochondrial network integrity is impaired in cardiomyocytes from failing hearts. While oxidative stress has been implicated in heart failure (HF)-associated mitochondrial remodeling, the effect of mitochondrial-targeted antioxidants, such as mitoquinone (MitoQ), on the mitochondrial network in a model of HF (e.g., pressure overload) has not been demonstrated. Furthermore, the mechanism of this regulation is not completely understood with an emerging role for posttranscriptional regulation via long noncoding RNAs (lncRNAs). We hypothesized that MitoQ preserves mitochondrial fusion proteins (i.e., mitofusin), likely through redox-sensitive lncRNAs, leading to improved mitochondrial network integrity in failing hearts. To test this hypothesis, 8-wk-old C57BL/6J mice were subjected to ascending aortic constriction (AAC), which caused substantial left ventricular (LV) chamber remodeling and remarkable contractile dysfunction in 1 wk. Transmission electron microscopy and immunostaining revealed defective intermitochondrial and mitochondrial-sarcoplasmic reticulum ultrastructure in AAC mice compared with sham-operated animals, which was accompanied by elevated oxidative stress and suppressed mitofusin (i.e., Mfn1 and Mfn2) expression. MitoQ (1.36 mg·day-1·mouse-1, 7 consecutive days) significantly ameliorated LV dysfunction, attenuated Mfn2 downregulation, improved interorganellar contact, and increased metabolism-related gene expression. Moreover, our data revealed that MitoQ alleviated the dysregulation of an Mfn2-associated lncRNA (i.e., Plscr4). In summary, the present study supports a unique mechanism by which MitoQ improves myocardial intermitochondrial and mitochondrial-sarcoplasmic reticulum (SR) ultrastructural remodeling in HF by maintaining Mfn2 expression via regulation by an lncRNA. These findings underscore the important role of lncRNAs in the pathogenesis of HF and the potential of targeting them for effective HF treatment.NEW & NOTEWORTHY We have shown that MitoQ improves cardiac mitochondrial network integrity and mitochondrial-SR alignment in a pressure-overload mouse heart-failure model. This may be occurring partly through preventing the dysregulation of a redox-sensitive lncRNA-microRNA pair (i.e., Plscr4-miR-214) that results in an increase in mitofusin-2 expression.

A Dual-Payload Antibody-Drug Conjugate Targeting CD276/B7-H3 Elicits Cytotoxicity and Immune Activation in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous disease that often relapses following treatment with standard radiotherapies and cytotoxic chemotherapies. Combination therapies have potential for treating refractory metastatic TNBC. Here, we aimed to develop an antibody-drug conjugate with dual payloads (DualADC) as a chemo-immunotherapy for TNBC. The overexpression of an immune checkpoint transmembrane CD276 (also known as B7-H3) was associated with angiogenesis, metastasis, and immune tolerance, in over 60% of TNBC patients. Development of a monoclonal antibody (mAb) capable of targeting the extracellular domain of surface CD276 enabled delivery of payloads to tumors, and a platform was established for concurrent conjugation of a traditional cytotoxic payload and an immunoregulating toll-like receptor 7/8 agonist to the CD276 mAb. The DualADC effectively killed multiple TNBC subtypes, significantly enhanced immune functions in the tumor microenvironment, and reduced tumor burden by up to 90-100% in animal studies. Single-cell RNA-sequencing, multiplex cytokine analysis, and histology elucidated the impact of treatment on tumor cells and the immune landscape. This study suggests that the developed DualADC could represent a promising targeted chemo-immunotherapy for TNBC.

mLumiOpto is a Mitochondrial-Targeted Gene Therapy for Treating Cancer.

Mitochondria are important in various aspects of cancer development and progression. Targeting mitochondria in cancer cells holds great therapeutic promise, yet current strategies to specifically and effectively destroy cancer mitochondria in vivo are limited. Here, we developed mLumiOpto, an innovative mitochondrial-targeted luminoptogenetics gene therapy designed to directly disrupt the inner mitochondrial membrane (IMM) potential and induce cancer cell death. The therapeutic approach included synthesis of a blue light-gated cationic channelrhodopsin (CoChR) in the IMM and co-expression of a blue bioluminescence-emitting nanoluciferase (NLuc) in the cytosol of the same cells. The mLumiOpto genes were selectively delivered to cancer cells in vivo by an adeno-associated virus (AAV) carrying a cancer-specific promoter or cancer-targeted monoclonal antibody-tagged exosome-associated AAV (mAb-Exo-AAV). Induction with NLuc luciferin elicited robust endogenous bioluminescence, which activated CoChR, triggering cancer cell mitochondrial depolarization and subsequent cell death. Importantly, mLumiOpto demonstrated remarkable efficacy in reducing tumor burden and killing tumor cells in glioblastoma and triple-negative breast cancer xenograft mouse models. Furthermore, the approach induced an anti-tumor immune response, increasing infiltration of dendritic cells and CD8+ T cells in the tumor microenvironment. These findings establish mLumiOpto as a promising therapeutic strategy by targeting cancer cell mitochondria in vivo.

An Innovative Mitochondrial-targeted Gene Therapy for Cancer Treatment.

Targeting cancer cell mitochondria holds great therapeutic promise, yet current strategies to specifically and effectively destroy cancer mitochondria in vivo are limited. Here, we introduce mLumiOpto, an innovative mitochondrial-targeted luminoptogenetics gene therapy designed to directly disrupt the inner mitochondrial membrane (IMM) potential and induce cancer cell death. We synthesize a blue light-gated channelrhodopsin (CoChR) in the IMM and co-express a blue bioluminescence-emitting Nanoluciferase (NLuc) in the cytosol of the same cells. The mLumiOpto genes are selectively delivered to cancer cells in vivo by using adeno-associated virus (AAV) carrying a cancer-specific promoter or cancer-targeted monoclonal antibody-tagged exosome-associated AAV. Induction with NLuc luciferin elicits robust endogenous bioluminescence, which activates mitochondrial CoChR, triggering cancer cell IMM permeability disruption, mitochondrial damage, and subsequent cell death. Importantly, mLumiOpto demonstrates remarkable efficacy in reducing tumor burden and killing tumor cells in glioblastoma or triple-negative breast cancer xenografted mouse models. These findings establish mLumiOpto as a novel and promising therapeutic strategy by targeting cancer cell mitochondria in vivo.

Advanced biomanufacturing and evaluation of adeno-associated virus.

Recombinant adeno-associated virus (rAAV) has been developed as a safe and effective gene delivery vehicle to treat rare genetic diseases. This study aimed to establish a novel biomanufacturing process to achieve high production and purification of various AAV serotypes (AAV2, 5, DJ, DJ8). First, a robust suspensive production process was developed and optimized using Gibco Viral Production Cell 2.0 in 30-60 mL shaker flask cultures by evaluating host cells, cell density at the time of transfection and plasmid amount, adapted to 60-100 mL spinner flask production, and scaled up to 1.2-2.0-L stirred-tank bioreactor production at 37 °C, pH 7.0, 210 rpm and DO 40%. The optimal process generated AAV titer of 7.52-8.14 × 1010 vg/mL. Second, a new AAV purification using liquid chromatography was developed and optimized to reach recovery rate of 85-95% of all four serotypes. Post-purification desalting and concentration procedures were also investigated. Then the generated AAVs were evaluated in vitro using Western blotting, transmission electron microscope, confocal microscope and bioluminescence detection. Finally, the in vivo infection and functional gene expression of AAV were confirmed in tumor xenografted mouse model. In conclusion, this study reported a robust, scalable, and universal biomanufacturing platform of AAV production, clarification and purification.

Characterization of the far-red fluorescent probe MitoView 633 for dynamic mitochondrial membrane potential measurement.

Introduction: MitoView 633, a far-red fluorescent dye, exhibits the ability to accumulate within mitochondria in a membrane potential-dependent manner, as described by the Nernst equation. This characteristic renders it a promising candidate for bioenergetics studies, particularly as a robust indicator of mitochondrial membrane potential (DYm). Despite its great potential, its utility in live cell imaging has not been well characterized. Methods: This study seeks to characterize the spectral properties of MitoView 633 in live cells and evaluate its mitochondrial staining, resistance to photobleaching, and dynamics during DYm depolarization. The co-staining and imaging of MitoView 633 with other fluorophores such as MitoSOX Red and Fluo-4 AM were also examined in cardiomyocytes using confocal microscopy. Results and Discussion: Spectrum analysis showed that MitoView 633 emission could be detected at 660 ± 50 nm, and exhibited superior thermal stability compared to tetramethylrhodamine methyl ester (TMRM), a commonly used DYm indicator, which emits at 605 ± 25 nm. Confocal imaging unequivocally illustrated MitoView 633's specific localization within the mitochondrial matrix, corroborated by its colocalization with MitoTracker Green, a well-established mitochondrial marker. Furthermore, our investigation revealed that MitoView 633 exhibited minimal photobleaching at the recommended in vitro concentrations. Additionally, the dynamics of MitoView 633 fluoresce during carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, a mitochondrial uncoupler)-induced DYm depolarization mirrored that of TMRM. Importantly, MitoView 633 demonstrated compatibility with co-staining alongside MitoSOX Red and Fluo-4 AM, enabling concurrent monitoring of DYm, mitochondrial ROS, and cytosolic Ca2+ in intact cells. Conclusion: These findings collectively underscore MitoView 633 as a superb molecular probe for the singular or combined assessment of DYm and other indicators in live cell imaging applications.

A CD276-Targeted Antibody-Drug Conjugate to Treat Non-Small Lung Cancer (NSCLC).

Non-small cell lung cancer (NSCLC) patients, accounting for approximately 85% of lung cancer cases, are usually diagnosed in advanced stages. Traditional surgical resection and radiotherapy have very limited clinical benefits. The objective of this study was to develop and evaluate a targeted therapy, antibody-drug conjugate (ADC), for NSCLC treatment. Specifically, the CD276 receptor was evaluated and confirmed as an ideal surface target of NSCLC in the immunohistochemistry (IHC) staining of seventy-three patient tumor microarrays and western blotting analysis of eight cell lines. Our anti-CD276 monoclonal antibody (mAb) with cross-activity to both human and mouse receptors showed high surface binding, effective drug delivery and tumor-specific targeting in flow cytometry, confocal microscopy, and in vivo imaging system analysis. The ADC constructed with our CD276 mAb and payload monomethyl auristatin F (MMAF) showed high anti-NSCLC cytotoxicity to multiple lines and effective anti-tumor efficacy in both immunocompromised and immunocompetent NSCLC xenograft mouse models. The brief mechanism study revealed the integration of cell proliferation inhibition and immune cell reactivation in tumor microenvironments. The toxicity study did not detect off-target immune toxicity or peripheral toxicity. Altogether, this study suggested that anti-CD276 ADC could be a promising candidate for NSCLC treatment.

Correction: Chen et al. Targeted Extracellular Vesicles Delivered Verrucarin A to Treat Glioblastoma. Biomedicines 2022, 10, 130.

In the original publication [...].

Multiscale Modeling of the Mitochondrial Origin of Cardiac Reentrant and Fibrillatory Arrhythmias.

While mitochondrial dysfunction has been implicated in the pathogenesis of cardiac arrhythmias, how the abnormality occurring at the organelle level escalates to influence the rhythm of the heart remains incompletely understood. This is due, in part, to the complexity of the interactions formed by cardiac electrical, mechanical, and metabolic subsystems at various spatiotemporal scales that is difficult to fully comprehend solely with experiments. Computational models have emerged as a powerful tool to explore complicated and highly dynamic biological systems such as the heart, alone or in combination with experimental measurements. Here, we describe a strategy of integrating computer simulations with optical mapping of cardiomyocyte monolayers to examine how regional mitochondrial dysfunction elicits abnormal electrical activity, such as rebound and spiral waves, leading to reentry and fibrillation in cardiac tissue. We anticipate that this advanced modeling technology will enable new insights into the mechanisms by which changes in subcellular organelles can impact organ function.

Targeted EV to Deliver Chemotherapy to Treat Triple-Negative Breast Cancers.

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.

Targeted Extracellular Vesicles Delivered Verrucarin A to Treat Glioblastoma.

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.

Process improvement of adeno-associated virus (AAV) production.

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.

Antibody-Drug Conjugate to Treat Meningiomas.

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.

Anti-EGFR antibody-drug conjugate for triple-negative breast cancer therapy.

Triple-negative breast cancers (TNBCs) are highly aggressive, metastatic and recurrent. Cytotoxic chemotherapies with limited clinical benefits and severe side effects are the standard therapeutic strategies, but, to date, there is no efficacious targeted therapy. Literature and our data showed that epidermal growth factor receptor (EGFR) is overexpressed on TNBC cell surface and is a promising oncological target. The objective of this study was to develop an antibody-drug conjugate (ADC) to target EGFR+ TNBC and deliver high-potency drug. First, we constructed an ADC by conjugating anti-EGFR monoclonal antibody with mertansine which inhibits microtubule assembly via linker Sulfo-SMCC. Second, we confirmed the TNBC-targeting specificity of anti-EGFR ADC by evaluating its surface binding and internalization in MDA-MB-468 cells and targeting to TNBC xenograft in subcutaneous mouse mode. The live-cell and live-animal imaging with confocal laser scanning microscopy and In Vivo Imaging System (IVIS) confirmed the TNBC-targeting. Finally, both in vitro toxicity assay and in vivo anti-cancer efficacy study in TNBC xenograft models showed that the constructed ADC significantly inhibited TNBC growth, and the pharmacokinetics study indicated its high circulation stability. This study indicated that the anti-EGFR ADC has a great potential to against TNBC.

Anti-SSTR2 antibody-drug conjugate for neuroendocrine tumor therapy.

Neuroendocrine (NE) tumors include a diverse spectrum of hormone-secreting neoplasms that arise from the endocrine and nervous systems. Current chemo- and radio-therapies have marginal curative benefits. The goal of this study was to develop an innovative antibody-drug conjugate (ADC) to effectively treat NE tumors (NETs). First, we confirmed that somatostatin receptor 2 (SSTR2) is an ideal cancer cell surface target by analyzing 38 patient-derived NET tissues, 33 normal organs, and three NET cell lines. Then, we developed a new monoclonal antibody (mAb, IgG1, and kappa) to target two extracellular domains of SSTR2, which showed strong and specific surface binding to NETs. The ADC was constructed by conjugating the anti-SSTR2 mAb and antimitotic monomethyl auristatin E. In vitro evaluations indicated that the ADC can effectively bind, internalize, release payload, and kill NET cells. Finally, the ADC was evaluated in vivo using a NET xenograft mouse model to assess cancer-specific targeting, tolerated dosage, pharmacokinetics, and antitumor efficacy. The anti-SSTR2 ADC exclusively targeted and killed NET cells with minimal toxicity and high stability in vivo. This study demonstrates that the anti-SSTR2 ADC has a high-therapeutic potential for NET therapy.

Targeted Exosomes for Drug Delivery: Biomanufacturing, Surface Tagging, and Validation.

Exosomes hold great potential to deliver therapeutic reagents for cancer treatment due to its inherent low antigenicity. However, several technical barriers, such as low productivity and ineffective cancer targeting, need to be overcome before wide clinical applications. The present study aims at creating a new biomanufacturing platform of cancer-targeted exosomes for drug delivery. Specifically, a scalable, robust, high-yield, cell line based exosome production process is created in a stirred-tank bioreactor, and an efficient surface tagging technique is developed to generate monoclonal antibody (mAb)-exosomes. The in vitro characterization using transmission electron microscopy, NanoSight, and western blotting confirm the high quality of exosomes. Flow cytometry and confocal laser scanning microscopy demonstrate that mAb-exosomes have strong surface binding to cancer cells. Furthermore, to validate the targeted drug delivery efficiency, romidepsin, a histone deacetylase inhibitor, is loaded into mAb-exosomes. The in vitro anti-cancer toxicity study shows high cytotoxicity of mAb-exosome-romidepsin to cancer cells. Finally, the in vivo study using tumor xenograft animal model validates the cancer targeting specificity, anti-cancer efficacy, and drug delivery capability of the targeted exosomes. In summary, new techniques enabling targeted exosomes for drug delivery are developed to support large-scale animal studies and to facilitate the translation from research to clinics.

Dual-Targeted Extracellular Vesicles to Facilitate Combined Therapies for Neuroendocrine Cancer Treatment.

Neuroendocrine (NE) cancers arise from cells within the neuroendocrine system. Chemotherapies and endoradiotherapy have been developed, but their clinical efficacy is limited. The objective of this study was to develop a dual-targeted extracellular vesicles (EV)-delivered combined therapies to treat NE cancer. Specifically, we produced EV in stirred-tank bioreactors and surface tagged both anti-somatostatin receptor 2 (SSTR 2) monoclonal antibody (mAb) and anti-C-X-C motif chemokine receptor 4 (CXCR4) mAb to generate mAbs-EV. Both live-cell confocal microscopy imaging and In Vivo Imaging System (IVIS) imaging confirmed that mAbs-EV specifically targeted and accumulated in NE cancer cells and NE tumor xenografts. Then the highly potent natural cytotoxic marine compound verrucarin A (Ver-A) with IC50 of 2.2-2.8 nM and microtubule polymerization inhibitor mertansine (DM1) with IC50 of 3.1-4.2 nM were packed into mAbs-EV. The in vivo maximum tolerated dose study performed in non-tumor-bearing mice indicated minimal systemic toxicity of mAbs-EV-Ver-A/DM1. Finally, the in vivo anticancer efficacy study demonstrated that the SSTR2/CXCR4 dual-targeted EV-Ver-A/DM1 is more effective to inhibit NE tumor growth than the single targeting and single drug. The results from this study could expand the application of EV to targeting deliver the combined potent chemotherapies for cancer treatment.

Novel biomanufacturing platform for large-scale and high-quality human T cells production.

The adoptive transfer of human T cells or genetically-engineered T cells with cancer-targeting receptors has shown tremendous promise for eradicating tumors in clinical trials. The objective of this study was to develop a novel T cell biomanufacturing platform using stirred-tank bioreactor for large-scale and high-quality cellular production. First, various factors, such as bioreactor parameters, media, supplements, stimulation, seed age, and donors, were investigated. A serum-free fed-batch bioproduction process was developed to achieve 1000-fold expansion within 8 days after first stimulation and another 500-fold expansion with second stimulation. Second, this biomanufacturing process was successfully scaled up in bioreactor with dilution factor of 10, and the robustness and reproducibility of the process was confirmed by the inclusion of different donors' T cells of various qualities. Finally, T cell quality was monitored using 12 surface markers and 3 intracellular cytokines as the critical quality assessment criteria in early, middle and late stages of cell production. In this study, a new biomanufacturing platform was created to produce reliable, reproducible, high-quality, and large-quantity (i.e. > 5 billion) human T cells in stirred-tank bioreactor. This platform is compatible with the production systems of monoclonal antibodies, vaccines, and other therapeutic cells, which provides not only the proof-of-concept but also the ready-to-use new approach of T cell expansion for clinical immune therapy.