Correction: Treatment with native heterodimeric IL-15 increases cytotoxic lymphocytes and reduces SHIV RNA in lymph nodes.

[This corrects the article DOI: 10.1371/journal.ppat.1006902.].

Treatment with native heterodimeric IL-15 increases cytotoxic lymphocytes and reduces SHIV RNA in lymph nodes.

B cell follicles in secondary lymphoid tissues represent an immune privileged sanctuary for AIDS viruses, in part because cytotoxic CD8+ T cells are mostly excluded from entering the follicles that harbor infected T follicular helper (TFH) cells. We studied the effects of native heterodimeric IL-15 (hetIL-15) treatment on uninfected rhesus macaques and on macaques that had spontaneously controlled SHIV infection to low levels of chronic viremia. hetIL-15 increased effector CD8+ T lymphocytes with high granzyme B content in blood, mucosal sites and lymph nodes, including virus-specific MHC-peptide tetramer+ CD8+ cells in LN. Following hetIL-15 treatment, multiplexed quantitative image analysis (histo-cytometry) of LN revealed increased numbers of granzyme B+ T cells in B cell follicles and SHIV RNA was decreased in plasma and in LN. Based on these properties, hetIL-15 shows promise as a potential component in combination immunotherapy regimens to target AIDS virus sanctuaries and reduce long-term viral reservoirs in HIV-1 infected individuals. TRIAL REGISTRATION: ClinicalTrials.gov NCT02452268.

Optimized administration of hetIL-15 expands lymphocytes and minimizes toxicity in rhesus macaques.

The common γ-chain cytokine interleukin-15 (IL-15) plays a significant role in regulating innate and adaptive lymphocyte homeostasis and can stimulate anti-tumor activity of leukocytes. We have previously shown that the circulating IL-15 in the plasma is the heterodimeric form (hetIL-15), produced upon co-expression of IL-15 and IL-15 Receptor alpha (IL-15Rα) polypeptides in the same cell, heterodimerization of the two chains and secretion. We investigated the pharmacokinetic and pharmacodynamic profile and toxicity of purified human hetIL-15 cytokine upon injection in rhesus macaques. We compared the effects of repeated hetIL-15 administration during a two-week dosing cycle, using different subcutaneous dosing schemata, i.e. fixed doses of 0.5, 5 and 50 µg/kg or a doubling step-dose scheme ranging from 2 to 64 µg/kg. Following a fixed-dose regimen, dose-dependent peak plasma IL-15 levels decreased significantly between the first and last injection. The trough plasma IL-15 levels measured at 48 h after injections were significantly higher after the first dose, compared to subsequent doses. In contrast, following the step-dose regimen, the systemic exposure increased by more than 1 log between the first injection given at 2 µg/kg and the last injection given at 64 µg/kg, and the trough levels were comparable after each injection. Blood lymphocyte cell count, proliferation, and plasma IL-18 levels peaked at day 8 when hetIL-15 was provided at fixed doses, and at the end of the cycle following a step-dose regimen, suggesting that sustained expansion of target cells requires increasing doses of cytokine. Macaques treated with a 50 µg/kg dose showed moderate and transient toxicity, including fever, signs of capillary leak syndrome and renal dysfunction. In contrast, these effects were mild or absent using the step-dose regimen. The results provide a new method of optimal administration of this homeostatic cytokine and may have applications for the delivery of other cytokines.

Dose Determination and Administration of Bacterial Extracellular Vesicles for In Vivo Preclinical Studies.

Essentially all bacteria secrete nano-sized (~20-200 nm) bacterial extracellular vesicles (bEVs) loaded with proteins, lipids, glycans, and nucleic acids. bEVs facilitate interactions among cells of the same species, different microbial species, and even with cells of multicellular organisms in the context of colonization or infection. Their interactions with host organism immune cell receptors vary depending on the producing bacterial species and are now being harnessed for the development of bEVs as a potential immunotherapeutic platform. Both basic/mechanistic and preclinical therapeutic development studies are thus increasing in number and require implementation of methods for multiparametric analytical characterization as well as in vivo administration in preclinical animal models of disease. We summarize a variety of analytical methods that can be used to calculate bEV dose for preparations made from diverse bacterial sources (including sterility testing, total protein concentration, particle concentration, and lipopolysaccharide concentration). We also describe basic methodology for intravenous administration of bEV preparations via tail vein injection in laboratory mice. Throughout the description of methodology, we highlight potential pitfalls and alternatives to further equip the reader for troubleshooting should challenges arise. Robust and reproducible characterization is a prerequisite of bEV preparation quality control and consistent dosing during preclinical development. This will allow for more streamlined testing of candidate therapeutic bEVs within a given research laboratory, and furthermore facilitate reproducibility of findings across laboratories.

Harnessing Bacterial Extracellular Vesicle Immune Effects for Cancer Therapy.

There are a growing number of studies linking the composition of the human microbiome to disease states and treatment responses, especially in the context of cancer. This has raised significant interest in developing microbes and microbial products as cancer immunotherapeutics that mimic or recapitulate the beneficial effects of host-microbe interactions. Bacterial extracellular vesicles (bEVs) are nano-sized, membrane-bound particles secreted by essentially all bacteria species and contain a diverse bioactive cargo of the producing cell. They have a fundamental role in facilitating interactions among cells of the same species, different microbial species, and even with multicellular host organisms in the context of colonization (microbiome) and infection. The interaction of bEVs with the immune system has been studied extensively in the context of infection and suggests that bEV effects depend largely on the producing species. They thus provide functional diversity, while also being nonreplicative, having inherent cell-targeting qualities, and potentially overcoming natural barriers. These characteristics make them highly appealing for development as cancer immunotherapeutics. Both natively secreted and engineered bEVs are now being investigated for their application as immunotherapeutics, vaccines, drug delivery vehicles, and combinations of the above, with promising early results. This suggests that both the intrinsic immunomodulatory properties of bEVs and their ability to be modified could be harnessed for the development of next-generation microbe-inspired therapies. Nonetheless, there remain major outstanding questions regarding how the observed preclinical effectiveness will translate from murine models to primates, and humans in particular. Moreover, research into the pharmacology, toxicology, and mass manufacturing of this potential novel therapeutic platform is still at early stages. In this review, we highlight the breadth of bEV interactions with host cells, focusing on immunologic effects as the main mechanism of action of bEVs currently in preclinical development. We review the literature on ongoing efforts to develop natively secreted and engineered bEVs from a variety of bacterial species for cancer therapy and finally discuss efforts to overcome outstanding challenges that remain for clinical translation.

'Slicing' glioblastoma drivers with the Swiss cheese model.

The Swiss cheese model is used to assess risks and explain accidents in a variety of industries. This model can be applied to dissect the homeostatic mechanisms whose cumulative dysregulation contributes to disease states, including cancer. Using glioblastoma (GBM) as an exemplar, we discuss how specific protumorigenic mechanisms collectively drive disease by affecting genomic integrity, epigenetic regulation, metabolic homeostasis, and antitumor immunity. We further highlight how host factors, such as hormonal differences and aging, impact this process, and the interplay between these 'system failures' that enable tumor progression and foster therapeutic resistance. Finally, we examine therapies that consider the interactions between these elements, which may comprise more effective approaches given the multifaceted protumorigenic mechanisms that drive GBM.

The Genomic, Transcriptomic, and Immunologic Landscape of HRAS Mutations in Solid Tumors.

Tipifarnib is the only targeted therapy breakthrough for HRAS-mutant (HRASmt) recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). The molecular profiles of HRASmt cancers are difficult to explore given the low frequency of HRASmt. This study aims to understand the molecular co-alterations, immune profiles, and clinical outcomes of 524 HRASmt solid tumors including urothelial carcinoma (UC), breast cancer (BC), non-small-cell lung cancer (NSCLC), melanoma, and HNSCC. HRASmt was most common in UC (3.0%), followed by HNSCC (2.82%), melanoma (1.05%), BC (0.45%), and NSCLC (0.44%). HRASmt was absent in Her2+ BC regardless of hormone receptor status. HRASmt was more frequently associated with squamous compared to non-squamous NSCLC (60% vs. 40% in HRASwt, p = 0.002). The tumor microenvironment (TME) of HRASmt demonstrated increased M1 macrophages in triple-negative BC (TNBC), HNSCC, squamous NSCLC, and UC; increased M2 macrophages in TNBC; and increased CD8+ T-cells in HNSCC (all p < 0.05). Finally, HRASmt was associated with shorter overall survival in HNSCC (HR: 1.564, CI: 1.16-2.11, p = 0.003) but not in the other cancer types examined. In conclusion, this study provides new insights into the unique molecular profiles of HRASmt tumors that may help to identify new targets and guide future clinical trial design.

γ-aminobutyric acid receptor B signaling drives glioblastoma in females in an immune-dependent manner.

Sex differences in immune responses impact cancer outcomes and treatment response, including in glioblastoma (GBM). However, host factors underlying sex specific immune-cancer interactions are poorly understood. Here, we identify the neurotransmitter γ-aminobutyric acid (GABA) as a driver of GBM-promoting immune response in females. We demonstrated that GABA receptor B (GABBR) signaling enhances L-Arginine metabolism and nitric oxide synthase 2 (NOS2) expression in female granulocytic myeloid-derived suppressor cells (gMDSCs). GABBR agonist and GABA analog promoted GBM growth in females in an immune-dependent manner, while GABBR inhibition reduces gMDSC NOS2 production and extends survival only in females. Furthermore, female GBM patients have enriched GABA transcriptional signatures compared to males, and the use of GABA analogs in GBM patients is associated with worse short-term outcomes only in females. Collectively, these results highlight that GABA modulates anti-tumor immune response in a sex-specific manner, supporting future assessment of GABA pathway inhibitors as part of immunotherapy approaches.

Spatial immunosampling of MRI-defined glioblastoma regions reveals immunologic fingerprint of non-contrast enhancing, infiltrative tumor margins.

Glioblastoma (GBM) treatment includes maximal safe resection of the core and MRI contrast-enhancing (CE) tumor. Complete resection of the infiltrative non-contrast-enhancing (NCE) tumor rim is rarely achieved. We established a safe, semi-automated workflow for spatially-registered sampling of MRI-defined GBM regions in 19 patients with downstream analysis and biobanking, enabling studies of NCE, wherefrom recurrence/progression typically occurs. Immunophenotyping revealed underrepresentation of myeloid cell subsets and CD8+ T cells in the NCE. While NCE T cells phenotypically and functionally resembled those in matching CE tumor, subsets of activated (CD69hi) effector memory CD8+ T cells were overrepresented. Contrarily, CD25hi Tregs and other subsets were underrepresented. Overall, our study demonstrated that MRI-guided, spatially-registered, intraoperative immunosampling is feasible as part of routine GBM surgery. Further elucidation of the shared and spatially distinct microenvironmental biology of GBM will enable development of therapeutic approaches targeting the NCE infiltrative tumor to decrease GBM recurrence.

Transferred mitochondria accumulate reactive oxygen species, promoting proliferation.

Recent studies reveal that lateral mitochondrial transfer, the movement of mitochondria from one cell to another, can affect cellular and tissue homeostasis. Most of what we know about mitochondrial transfer stems from bulk cell studies and have led to the paradigm that functional transferred mitochondria restore bioenergetics and revitalize cellular functions to recipient cells with damaged or non-functional mitochondrial networks. However, we show that mitochondrial transfer also occurs between cells with functioning endogenous mitochondrial networks, but the mechanisms underlying how transferred mitochondria can promote such sustained behavioral reprogramming remain unclear. We report that unexpectedly, transferred macrophage mitochondria are dysfunctional and accumulate reactive oxygen species in recipient cancer cells. We further discovered that reactive oxygen species accumulation activates ERK signaling, promoting cancer cell proliferation. Pro-tumorigenic macrophages exhibit fragmented mitochondrial networks, leading to higher rates of mitochondrial transfer to cancer cells. Finally, we observe that macrophage mitochondrial transfer promotes tumor cell proliferation in vivo. Collectively these results indicate that transferred macrophage mitochondria activate downstream signaling pathways in a ROS-dependent manner in cancer cells, and provide a model of how sustained behavioral reprogramming can be mediated by a relatively small amount of transferred mitochondria in vitro and in vivo.

Sex-Biased T-cell Exhaustion Drives Differential Immune Responses in Glioblastoma.

Sex differences in glioblastoma (GBM) incidence and outcome are well recognized, and emerging evidence suggests that these extend to genetic/epigenetic and cellular differences, including immune responses. However, the mechanisms driving immunologic sex differences are not fully understood. Here, we demonstrate that T cells play a critical role in driving GBM sex differences. Male mice exhibited accelerated tumor growth, with decreased frequency and increased exhaustion of CD8+ T cells in the tumor. Furthermore, a higher frequency of progenitor exhausted T cells was found in males, with improved responsiveness to anti-PD-1 treatment. Moreover, increased T-cell exhaustion was observed in male GBM patients. Bone marrow chimera and adoptive transfer models indicated that T cell-mediated tumor control was predominantly regulated in a cell-intrinsic manner, partially mediated by the X chromosome inactivation escape gene Kdm6a. These findings demonstrate that sex-biased predetermined behavior of T cells is critical for inducing sex differences in GBM progression and immunotherapy response. SIGNIFICANCE: Immunotherapies in patients with GBM have been unsuccessful due to a variety of factors, including the highly immunosuppressive tumor microenvironment in GBM. This study demonstrates that sex-biased T-cell behaviors are predominantly intrinsically regulated, further suggesting sex-specific approaches can be leveraged to potentially improve the therapeutic efficacy of immunotherapy in GBM. See related commentary by Alspach, p. 1966. This article is featured in Selected Articles from This Issue, p. 1949.

GAP43-dependent mitochondria transfer from astrocytes enhances glioblastoma tumorigenicity.

The transfer of intact mitochondria between heterogeneous cell types has been confirmed in various settings, including cancer. However, the functional implications of mitochondria transfer on tumor biology are poorly understood. Here we show that mitochondria transfer is a prevalent phenomenon in glioblastoma (GBM), the most frequent and malignant primary brain tumor. We identified horizontal mitochondria transfer from astrocytes as a mechanism that enhances tumorigenesis in GBM. This transfer is dependent on network-forming intercellular connections between GBM cells and astrocytes, which are facilitated by growth-associated protein 43 (GAP43), a protein involved in neuron axon regeneration and astrocyte reactivity. The acquisition of astrocyte mitochondria drives an increase in mitochondrial respiration and upregulation of metabolic pathways linked to proliferation and tumorigenicity. Functionally, uptake of astrocyte mitochondria promotes cell cycle progression to proliferative G2/M phases and enhances self-renewal and tumorigenicity of GBM. Collectively, our findings reveal a host-tumor interaction that drives proliferation and self-renewal of cancer cells, providing opportunities for therapeutic development.

Red man syndrome adverse reaction following intravenous infusion of cefepime.

We report the first case of cefepime-induced "red-man syndrome," which appeared 30 min following drug infusion and was confirmed with a rechallenge test. This syndrome is classically associated with vancomycin infusion and is the result of non-IgE mediated mast cell degranulation. While this adverse effect can be easily managed with drug withdrawal and antihistamine administration, it is unknown whether it can be prevented with slower cefepime infusion and preinfusion antihistamines, as is the case with vancomycin.

SerpinB3 drives cancer stem cell survival in glioblastoma.

Despite therapeutic interventions for glioblastoma (GBM), cancer stem cells (CSCs) drive recurrence. The precise mechanisms underlying CSC resistance, namely inhibition of cell death, are unclear. We built on previous observations that the high cell surface expression of junctional adhesion molecule-A drives CSC maintenance and identified downstream signaling networks, including the cysteine protease inhibitor SerpinB3. Using genetic depletion approaches, we found that SerpinB3 is necessary for CSC maintenance, survival, and tumor growth, as well as CSC pathway activation. Knockdown of SerpinB3 also increased apoptosis and susceptibility to radiation therapy. SerpinB3 was essential to buffer cathepsin L-mediated cell death, which was enhanced with radiation. Finally, we found that SerpinB3 knockdown increased the efficacy of radiation in pre-clinical models. Taken together, our findings identify a GBM CSC-specific survival mechanism involving a cysteine protease inhibitor, SerpinB3, and provide a potential target to improve the efficacy of GBM therapies against therapeutically resistant CSCs.

Distinct Cell Adhesion Signature Defines Glioblastoma Myeloid-Derived Suppressor Cell Subsets.

In multiple types of cancer, an increased frequency in myeloid-derived suppressor cells (MDSC) is associated with worse outcomes and poor therapeutic response. In the glioblastoma (GBM) microenvironment, monocytic (m) MDSCs represent the predominant subset. However, the molecular basis of mMDSC enrichment in the tumor microenvironment compared with granulocytic (g) MDSCs has yet to be determined. Here we performed the first broad epigenetic profiling of MDSC subsets to define underlying cell-intrinsic differences in behavior and found that enhanced gene accessibility of cell adhesion programs in mMDSCs is linked to their tumor-accelerating ability in GBM models upon adoptive transfer. Mouse and human mMDSCs expressed higher levels of integrin ß1 and dipeptidyl peptidase-4 (DPP-4) compared with gMDSCs as part of an enhanced cell adhesion signature. Integrin ß1 blockade abrogated the tumor-promoting phenotype of mMDSCs and altered the immune profile in the tumor microenvironment, whereas treatment with a DPP-4 inhibitor extended survival in preclinical GBM models. Targeting DPP-4 in mMDSCs reduced pERK signaling and their migration towards tumor cells. These findings uncover a fundamental difference in the molecular basis of MDSC subsets and suggest that integrin ß1 and DPP-4 represent putative immunotherapy targets to attenuate myeloid cell-driven immune suppression in GBM. SIGNIFICANCE: Epigenetic profiling uncovers cell adhesion programming as a regulator of the tumor-promoting functions of monocytic myeloid-derived suppressor cells in glioblastoma, identifying therapeutic targets that modulate the immune response and suppress tumor growth.

Large-scale production of extracellular vesicles: Report on the "massivEVs" ISEV workshop.

Extracellular vesicles (EVs) large-scale production is a crucial point for the translation of EVs from discovery to application of EV-based products. In October 2021, the International Society for Extracellular Vesicles (ISEV), along with support by the FET-OPEN projects, "The Extracellular Vesicle Foundry" (evFOUNDRY) and "Extracellular vesicles from a natural source for tailor-made nanomaterials" (VES4US), organized a workshop entitled "massivEVs" to discuss the potential challenges for translation of EV-based products. This report gives an overview of the topics discussed during "massivEVs", the most important points raised, and the points of consensus reached after discussion among academia and industry representatives. Overall, the review of the existing EV manufacturing, upscaling challenges and directions for their resolution highlighted in the workshop painted an optimistic future for the expanding EV field.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria.

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Phase I study of single agent NIZ985, a recombinant heterodimeric IL-15 agonist, in adult patients with metastatic or unresectable solid tumors.

BACKGROUND: NIZ985 is a recombinant heterodimer of physiologically active interleukin (IL-)15 and IL-15 receptor alpha. In preclinical models, NIZ985 promotes cytotoxic lymphocyte proliferation, killing function, and organ/tumor infiltration, with resultant anticancer effects. In this first-in-human study, we assessed the safety, pharmacokinetics, and immune effects of NIZ985 in patients with metastatic or unresectable solid tumors. METHODS: Single agent NIZ985 dose escalation data are reported from a phase I dose escalation/expansion study of NIZ985 as monotherapy. Adult patients (N=14) received 0.25, 0.5, 1, 2 or 4 µg/kg subcutaneous NIZ985 three times weekly (TIW) for the first 2 weeks of each 28-day cycle, in an accelerated 3+3 dose escalation trial design. IL-15 and endogenous cytokines were monitored by ELISA and multiplexed electrochemiluminescent assays. Multiparameter flow cytometry assessed the frequency, phenotype and proliferation of peripheral blood mononuclear cells. Preliminary antitumor activity was assessed by overall response rate (Response Evaluation Criteria in Solid Tumors V.1.1). RESULTS: As of March 2, 2020, median treatment duration was 7.5 weeks (range 1.1-77.1). Thirteen patients had discontinued and one (uveal melanoma) remains on treatment with stable disease. Best clinical response was stable disease (3 of 14 patients; 21%). The most frequent adverse events (AEs) were circular erythematous injection site reactions (100%), chills (71%), fatigue (57%), and fever (50%). Treatment-related grade 3/4 AEs occurred in six participants (43%); treatment-related serious AEs (SAEs) in three (21%). The per-protocol maximum tolerated dose was not reached. Pharmacokinetic accumulation of serum IL-15 in the first week was followed by significantly lower levels in week 2, likely due to more rapid cytokine consumption by an expanding lymphocyte pool. NIZ985 treatment was associated with increases in several cytokines, including interferon (IFN)-γ, IL-18, C-X-C motif chemokine ligand 10, and tumor necrosis factor-ß, plus significant induction of cytotoxic lymphocyte proliferation (including natural killer and CD8+ T cells), increased CD16+ monocytes, and increased CD163+ macrophages at injection sites. CONCLUSIONS: Subcutaneous NIZ985 TIW was generally well tolerated in patients with advanced cancer and produced immune activation paralleling preclinical observations, with induction of IFN-γ and proliferation of cytotoxic lymphocytes. Due to delayed SAEs at the two highest dose levels, administration is being changed to once-weekly in a revised protocol, as monotherapy and combined with checkpoint inhibitor spartalizumab. These alterations are expected to maximize the potential of NIZ985 as a novel immunotherapy. TRIAL REGISTRATION NUMBER: NCT02452268.

Myeloid-Derived Suppressor Cell Subsets Drive Glioblastoma Growth in a Sex-Specific Manner.

Myeloid-derived suppressor cells (MDSC) that block antitumor immunity are elevated in glioblastoma (GBM) patient blood and tumors. However, the distinct contributions of monocytic (mMDSC) versus granulocytic (gMDSC) subsets have yet to be determined. In mouse models of GBM, we observed that mMDSCs were enriched in the male tumors, whereas gMDSCs were elevated in the blood of females. Depletion of gMDSCs extended survival only in female mice. Using gene-expression signatures coupled with network medicine analysis, we demonstrated in preclinical models that mMDSCs could be targeted with antiproliferative agents in males, whereas gMDSC function could be inhibited by IL1ß blockade in females. Analysis of patient data confirmed that proliferating mMDSCs were predominant in male tumors and that a high gMDSC/IL1ß gene signature correlated with poor prognosis in female patients. These findings demonstrate that MDSC subsets differentially drive immune suppression in a sex-specific manner and can be leveraged for therapeutic intervention in GBM. SIGNIFICANCE: Sexual dimorphism at the level of MDSC subset prevalence, localization, and gene-expression profile constitutes a therapeutic opportunity. Our results indicate that chemotherapy can be used to target mMDSCs in males, whereas IL1 pathway inhibitors can provide benefit to females via inhibition of gMDSCs.See related commentary by Gabrilovich et al., p. 1100.This article is highlighted in the In This Issue feature, p. 1079.

Scalable, cGMP-compatible purification of extracellular vesicles carrying bioactive human heterodimeric IL-15/lactadherin complexes.

The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC. We applied this purification method for the isolation of engineered EV carrying multiple complexes of a novel human immunostimulatory cytokine-fusion protein, heterodimeric IL-15 (hetIL-15)/lactadherin. HEK293 cells stably expressing the fusion cytokine were cultured in a hollow-fibre bioreactor. Conditioned medium was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF + SEC. SEC demonstrated comparable particle recovery, size distribution, and hetIL-15 density as U/C purification. Relative to U/C, SEC preparations achieved a 100-fold reduction in ferritin concentration, a major protein-complex contaminant. Comparative proteomics suggested that SEC additionally decreased the abundance of cytoplasmic proteins not associated with EV. Combination of TFF and SEC allowed for bulk processing of large starting volumes, and resulted in bioactive EV, without significant loss in particle yield or changes in size, morphology, and hetIL-15/lactadherin density. Taken together, the combination of bioreactor culture with TFF + SEC comprises a scalable, efficient method for the production of highly purified, bioactive EV carrying hetIL-15/lactadherin, which may be useful in targeted cancer immunotherapy approaches.