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. 2020 Jun;4(6):e1900288.
doi: 10.1002/adbi.201900288. Epub 2020 May 10.

Lentivirus Manufacturing Process for Primary T-Cell Biofactory Production

Harikrishnan Radhakrishnan  1 , Harold S Javitz  2 , Parijat Bhatnagar  1
Affiliations

Lentivirus Manufacturing Process for Primary T-Cell Biofactory Production

Harikrishnan Radhakrishnan et al. Adv Biosyst. 2020 Jun.

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.

Keywords: CAR T cells; cell engineering; cell manufacturing; drug delivery; high-titer lentivirus production.

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Conflict of interest statement

Competing financial interests. The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.. Schematic of the process.
Process flow for lentivirus production scaled-up to obtain functional lentivirus titer of ~6 × 106 Infectious Units (IFU)/cm2 of cell growth surface (i.e. ~109 IFU for a single T175 flask).
Figure 2.
Figure 2.. Factors affecting the lentivirus process yield.
Various factors associated with the lentivirus production were compared and quantified using Lenti-X™ p24 Rapid titer ELISA. As per a previous report (see Ref.[11]) and manufacturer’s instructions, total lentivirus particles were normalized to Infectious Units (IFU) using the ratio of 1 IFU for every 100 lentivirus particles. Effect of (A) producer cell seeding densities; (B) types of producer cell lines; (C) DNA quantities used for the transfection; (D) types of transfection reagents; (E) types of cell growth surfaces; (F) types of supernatant clarification processes; (G) supernatant harvesting times; (H) Sodium Butyrate (HDAC inhibitor); (I) 2AP (Protein Kinase R inhibitor); (J) pAdVAntange™ and its combination with 2AP. A unified protocol, based on the favorable values for the above factors, was generated and compared (K) with the generally reported protocol, that has also been used by us (see Ref.[6]). The process was scaled-up (L) from 100-mm plate (55cm2) to 175cm2 flask Note: HEK293T cell line was used as a producer cell for (A, C – E, G, H); HEK293T/17 was used as a producer cell for (F, I – L). GraphPad Prism 8.1.1 (GraphPad Software, Inc) was utilized. All data were represented on a Tukey box-and-whisker plot (n ≥ 3). Statistical analysis was performed using parametric two-tailed t-test for all comparisons involving two groups and one-way ANOVA for those comparisons involving more than two groups. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Legend: (A-L) TU: Transduction units (functional titer), (D) Lipo 2000: Lipofectamine® 2000, T5: Transporter 5™; (E) PCA: PureCoat™ Amine, TCT: Tissue culture treated; (F) (i) filtered daily (0.45 μm), (ii) filtered (0.45 μm) total harvest on the 3rd day, or (iii) centrifuged daily (500g, 10 min at 25°C) and filtered (0.45 μm) the total harvest on the 3rd day; (G) 24-48-72: supernatant harvested at 24, 48, and 72 hr, 48-72-96: supernatant harvested at 48, 72, and 96 hr; (I, J) 2AP: 2-amino purine, PKRi: Protein Kinase R inhibitor, pAdV: pAdVAntage. (A-L)
Figure 3.
Figure 3.. Functional evaluation and applications of lentivirus vectors.
(A) Schematics of the plasmid constructs used to generate the two lentivirus vectors for (i) FRα-CAR T cells, and (ii) FRα-specific T-cell Biofactory cells; both of which can also be redirected to other cell-surface antigens. (B) Transduction efficiency of the lentivirus particles was calculated by assessing the GFP expression in HEK293T and acute T leukemia cell line (Jurkat clone E6–1) transduced at various Multiplicity of Infection (MOI). Target-specific cytolytic activities of (C) primary FRα-CAR+ T cells and (D) primary FRα-specific T-cell Biofactory were calculated by assessing the reduction in Luc2 activity in a co-culture with Luc2 expressing OVCAR3 (FRα+) or A2780cis (FRαneg) cell lines. (C, D) Luc2 activity were fit using a four-parameter logistic model, Activity = Activitymin + {Activitymax − Activitymin}/{1+10^ [b* (log10[η(E:T)50] − X)]}. (E) Target-specific engineered function of synthesizing the desired protein from the primary FRα-specific T-cell Biofactory was calculated by assessing the Nluc activity from the T-cell Biofactory cells in a co-culture with OVCAR3 (FRα+) or A2780cis (FRαneg) cell lines. The Nluc activity were fit using a four-parameter logistic model, Activity = Activitymin + {Activitymax – Activitymin}/ {1+10^ [b* (log10[η(E:T)50] – X)]}. (C-E) X is the log10 of the number of Effector T cells (primary FRα-CAR+ T cells or primary FRα-specific T-cell Biofactory cells), Activitymax is an estimated parameter defining an upper asymptote for the reporter activity, Activitymin is an estimated parameter defining a lower asymptote for the reporter activity, b is a “Hill” parameter defining the slope at the inflection point of the fitted curve, and η(E:T)50 is an estimated E:T value corresponding to (Activitymax – Activitymin)/2. The error bars are 1 standard deviation above and below the mean and can also be considered as one half-width of an 68% confidence interval for that mean. The specificity of T-cell Biofactory was plotted as the signal-to-noise ratio (S/N) and was calculated as the ratio of the mean Nluc activity in primary T-cell Biofactory cells when stimulated by the FRα+ OVCAR3 cells and the mean Nluc activity when stimulated by the FRαneg A2780cis cells. The specificity (S/N) data were fit using second order polynomial equation, Y = a + b*X + C*X^2, where X is the log10 of the number of T cell Biofactory cells. Statistical analysis to determine p-values was performed using Two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli at each E:T with a false discovery rate of less than 1%. GraphPad Prism 8.1.1 (GraphPad Software, Inc) was utilized.
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