Lentivirus Manufacturing Process for Primary T‐Cell Biofactory Production

Abstract

A process for maximizing the titer of lentivirus particles, deemed to be a necessity for transducing primary cells, is developed. Lentivirus particles, with a set of transgenes encoding an artificial cell‐signaling pathway, are used to transform primary T cells as vectors for calibrated synthesis of desired proteins in situ, that is, T‐cell biofactory cells. The process is also used to generate primary T cells expressing antigen‐specific chimeric antigen receptors, that is, CAR T cells. The two differently engineered primary T cells are expanded and validated for their respective functions, that is, calibrated synthesis of desired proteins upon engaging the target cells, which is specific for the T‐cell biofactory cells, and cytolysis of the target cells common to both types of cells. The process is compliant with current Good Manufacturing Practices and can be used to support the scale‐up for clinical translation.

NK‐Cell Biofactory as an Off‐the‐Shelf Cell‐based Vector for Targeted In Situ Synthesis of Engineered Proteins

“…Successful implementation of the artificial cell‐signaling pathway in an NK‐cell line presented in this work, in addition to T‐cell line [ 6 ] and primary T cells, [ 7 ] extend the options for selecting a cellular chassis based on the advantages they offer. This work additionally raises curiosity for investigating other cell types, for example, primary NK cells, iPS cells, mesenchymal stem cells, TALL‐104, K562, as cellular chassis, which may offer alternative therapeutic benefits.…”

“…To address this challenge, we recently reported on an antigen‐specific T‐cell Biofactory , which is a T‐cell‐based living vector that, upon interacting with the antigen‐presenting target cell, synthesizes proportionate amounts of desired proteins in situ. [ 6,7 ] However, these and other T‐cell therapies, such as T cells engineered with chimeric antigen receptors (CAR T cells), are primarily autologous in nature and present a pragmatic challenge in terms of manufacturing cost. Lymphodepleted patients have a low primary T cell count to draw from, and this number is further reduced when the cells are genetically modified.…”

“…To address this challenge, we recently reported on an antigen-specific T-cell Biofactory, which is a T-cell-based living vector that, upon interacting with the antigen-presenting target cell, synthesizes proportionate amounts of desired proteins in situ. [6,7] However, these and the Effector with a therapeutic transgene to neutralize the pathology that triggered the NK-cell Biofactory, essentially creating an off-the-shelf living vector, further enhanced by the innate cytolytic activity of the NK-92MI cell line.…”

“…Primarily due to the autologous nature of adoptive cell therapies, which contributes to high manufacturing cost, product variability, and potential for adverse events; we recognize the possibility of a clinical obstacle that may emerge during the translation of our previously reported primary T-cell Biofactory. [6,7] Therefore, we have utilized the NK-92MI cell line as a cellular chassis, which allowed us to generate and test NK-cell Biofactory in parallel to the T-cell Biofactory. [6,7,14] With a track record for exerting anti-tumor effect in the clinic [17,[19][20][21] and doubling time of ≈24 h, [19,21] NK-92MI is a strong candidate offering the potential for off-the-shelf allogeneic option.…”

Optimized protocol for high‐titer lentivirus production and transduction of primary fibroblasts

“…The density of 293T cells should also be an important concern for higher transfection efficiency and lentiviral titer [24]. At the transfection step, 80% confluency was optimal to lower the cytotoxicity of the transfection reagent [25].…”