A clinically relevant large‐scale biomanufacturing workflow to produce natural killer cells and natural killer cell‐derived extracellular vesicles for cancer immunotherapy

Abstract

Natural killer cell‐derived extracellular vesicles (NK‐EVs) have shown promising potential as biotherapeutics for cancer due to their unique attributes as cytotoxic nanovesicles against cancer cells and immune‐modulatory activity towards immune cells. However, a biomanufacturing workflow is needed to produce clinical‐grade NK‐EVs for pre‐clinical and clinical applications. This study established a novel biomanufacturing workflow using a closed‐loop hollow‐fibre bioreactor to continuously produce NK‐EVs from the clinically relevant NK92‐MI cell line under serum‐free, Xeno‐free and feeder‐free conditions following GMP‐compliant conditions. The NK92 cells grown in the bioreactor for three continuous production lots resulted in large quantities of both NK cell and NK‐EV biotherapeutics at the end of each production lot (over 109 viable cells and 1013 EVs), while retaining their cytotoxic payload (granzyme B and perforin), pro‐inflammatory cytokine (interferon‐gamma) content and cytotoxicity against the human leukemic cell line K562 with limited off‐target toxicity against healthy human fibroblast cells. This scalable biomanufacturing workflow has the potential to facilitate the clinical translation of adoptive NK cell‐based and NK‐EV‐based immunotherapies for cancer with GMP considerations.

Evaluation of resazurin phenoxazine dye as a highly sensitive cell viability potency assay for natural killer cell‐derived extracellular vesicle‐based cancer biotherapeutics

“…Cells were maintained between 3–4 × 10 4 cells/cm 2 and passaged every 2–3 days. The FreeStyle™ 293F cell line (Thermo Fisher Scientific, R790‐07) was cultured in FreeStyle™ 293 Expression Medium (Gibco, cat#12338018) to produce non‐therapeutic control EVs (i.e., 293F‐EVs), as previously done (St‐Denis‐Bissonnette et al., 2023 ). Cells were maintained between 1–3 × 10 6 cells/mL on a heat‐resistant shaker (Thermo Fisher Scientific, cat#88881127) at 135 RPM.…”

“…As previously done, NK‐EVs were manufactured using a large‐scale biomanufacturing workflow that adheres to Good Manufacturing Practices and uses NK92‐MI cells (St‐Denis‐Bissonnette et al., 2023 ). Briefly, the biomanufacturing of NK92‐MI cells and NK‐EVs was performed using a closed‐loop hollow fibre bioreactor to generate large quantities of clinical‐grade NK‐EVs.…”

“…Research in extracellular vesicle (EV)‐based biotherapeutics in cancer treatment is soaring worldwide (Gandham et al., 2020 ; Lener et al., 2015 ; Nguyen et al., 2020 ; Nieuwland et al., 2020 ; Reiner et al., 2017 ). However, a considerable barrier to the advancement of natural killer cell‐derived EVs (NK‐EVs) as a therapy for cancer has been the lack of validated standardised potency assays to measure NK‐EVs’ cytotoxic activity (Dosil et al., 2022 ; EL Andaloussi et al., 2013 ; Elsharkasy et al., 2020 ; Farcas & Inngjerdingen, 2020 ; Federici et al., 2014 ; Geeurickx et al., 2019 ; Jiang et al., 2021 ; Lugini et al., 2012 ; Neviani et al., 2019 ; St‐Denis‐Bissonnette et al., 2022 ; St‐Denis‐Bissonnette et al., 2023 ; Sun et al., 2019 ; Yáñez‐Mó et al., 2015 ). Such standardised quantitative potency assays must meet key attributes aligned with regulatory guidelines, such as the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) (FDA, 2011 ; Gimona et al., 2017 ; ICH, 2022 ; Nguyen et al., 2020 ; Reiner et al., 2017 ).…”

“…Such standardised quantitative potency assays must meet key attributes aligned with regulatory guidelines, such as the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) (FDA, 2011 ; Gimona et al., 2017 ; ICH, 2022 ; Nguyen et al., 2020 ; Reiner et al., 2017 ). Other challenges include: i ) comparing novel EV‐based therapeutics across studies, EV lots/preparations, and individual experiments (St‐Denis‐Bissonnette et al., 2023 ); ii ) the need for more essential quality controls and consistent dosing methodologies (Gimona et al., 2017 ; Nguyen et al., 2020 ; Reiner et al., 2017 ; St‐Denis‐Bissonnette et al., 2023 ).…”

“…To thisend, we evaluated a high sensitivity (HS) cell viabilityassay, the PrestoBlue HS cell viability assay, which relies on reducing resazurin to resorufin (detectable by absorbance or fluorescence), as a measurement of the metabolic activity of living cells. Here, we perform a rigorous analysis of the performance of this highly sensitive resazurin‐based cell viability assay against other commonly reported assays in the field using the NK92‐MI cell line‐derived‐EVs, which is a clinically relevant model (Cochran & Kornbluth, 2021 ; Enomoto et al., 2022 ; Kaban et al., 2021 ; Kim et al., 2022 ; St‐Denis‐Bissonnette et al., 2023 ; Wu et al., 2019 ; Wu et al., 2021 ; Zhu et al., 2017 ; Zhu et al., 2019 ). This resazurin‐based reagent, marketed by Thermo Fisher Scientific, is promoted as an exceptional pure resazurin solution with better signal‐to‐background ratio compared to the standard version.…”