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. 2020 Jan;4(1):e1900224.
doi: 10.1002/adbi.201900224. Epub 2019 Nov 29.

Engineered Ovarian Cancer Cell Lines for Validation of CAR T Cell Function

Claire E Repellin  1 , Priya Ganesan  1 , Javier F Alcudia  2 , Haritha K Duggireddy Lakshmireddy  2 , Puja Patel  1 , Lucia Beviglia  1 , Harold S Javitz  3 , Lidia Sambucetti  1 , Parijat Bhatnagar  1
Affiliations

Engineered Ovarian Cancer Cell Lines for Validation of CAR T Cell Function

Claire E Repellin et al. Adv Biosyst. 2020 Jan.

Abstract

A set of genetically engineered isogenic cell lines is developed to express either folate receptor alpha or mesothelin, and a control cell line negative for both antigens. These cell lines also express fluorescent and bioluminescent reporter transgenes. The cell lines are used to authenticate specificity and function of a T-cell biofactory, a living vector that is developed to express proportionate amounts of engineered proteins upon engaging with disease cells through their specific antigenic biomarkers. The engineered cell lines are also used to assess the cytolytic function and specificity of primary T cells engineered with chimeric antigen receptors; and the specificity of monoclonal antibodies. The strategy described can be used to generate other cell lines to present different disease-specific biomarkers for use as quality control tools.

Keywords: CAR T cells; artificial antigen presenting cell; authentication of biologics; cell engineering; quality control.

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Conflict of interest statement

Conflict of Interest

The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.
Schematic of the plasmids and isogenic cell lines. A) PiggyBac vector plasmid modified with insert for engineering A2780cis cell line for expression of i) Luc2 and E2Crimson; ii) FRa, Luc2, and E2Crimson; iii) MSLN, Luc2, and E2Crimson. A common PiggyBac transposon vector plasmid backbone was prepared with a) puromycin N-acetyltransferase selection marker, b) Luc2 bioluminescent, and c) E2Crimson fluorescent reporter transgenes. An XbaI restriction endonuclease site was introduced at the 5’ end of the Ig kappa signal peptide nucleotide sequence and NheI site at its 3’ end. The rationale for the placement of these restriction endonuclease sites was that while Ig kappa signal peptide in the empty vector (control) could be used to display c-Myc on the cell surface, it could be exchanged with the nucleotide sequences for the antigens (i.e., FRa or MSLN). B) A2780cis cell engineered to express i) Luc2 and E2Crimson; ii) FRa, Luc2, and E2Crimson; iii) MSLN, Luc2, and E2Crimson.
Figure 2.
Figure 2.
Characterization of engineered A2780cis cell lines. The engineered A2780cis cell lines were generated using plasmids encoding desired transgenes sub-cloned in a PiggyBac transposon vector and PiggyBac transposase. The transformed cells were selected using puromycin treatment (negative selection) and selected by FACS-based enrichment (positive selection). The selected cells were expanded and assessed for A) E2Crimson fluorescent protein expression using flow cytometry, B,C) Luc2 (firefly luciferase) activity using bioluminescence analysis. B) The vertical histogram bars show the average Luc2 activity from a total of 5000 differently engineered A2780cis variants and non-engineered parental A2780cis. C) The Luc2 activity in the engineered A2780cis cell lines was fit using a linear regression model, Luc2 = m* (no. of engineered A2780cis cells) + c, where m is the slope (indicating the relationship between Luc2 activity and the number of engineered A2780cis cells) and c is the intercept (indicating background activity) (B,C) The error bars extend 1 standard deviation (SD) above the mean (n = 4) and can also be considered as one half-width of an 68% confidence interval for that mean. D) FRa and MSLN expression on the cell surface of respective A2780cis variants was analyzed using flow cytometry. The expression was compared to i) the A2780cis variant that is negative for both antigens (FRanegMSLNnegLuc2-2A-E2Crimson+A2780cis), ii) parental A2780cis (endogenous FRanegMSLNneg), and iii) parental OVCAR3 (endogenous FRa+MSLN+).
Figure 3.
Figure 3.
Application of engineered A2780cis cell lines as a quality control tool-set. T-cell biofactories (panel A) and primary CAR+ T cells (panel B) were assessed for their specificity against cell surface FRa and MSLN. A) The T-cell biofactory (see our recent work, ref. [8] for more details) were assessed for their engineered function to synthesize desired proteins/reporter) upon engaging their target antigen, that is, when incubated with the A2780cis variants that are i) negative for FRa and MSLN (FRanegMSLNnegLuc2-2A-E2Crimson+A2780cis), ii) express FRa but not MSLN (FRa+MSLNnegLuc2-2A-E2Crimson+A2780cis); and iii) express MSLN but not FRa (FRanegMSLN+Luc2-2A-E2Crimson+A2780cis). Nluc reporter activity from the T-cell biofactories is shown in the graphs. B) Primary CAR+ T cells—i) FRa-specific or ii) MSLN-specific—were assessed for their target-specific cytolytic function by assessing the Luc2 activity in the target/nontarget engineered A2780cis cell lines. The T-cell biofactory Nluc reporter activity (panel A) or Luc2 activity in (panel B) target/nontarget engineered A2780cis cell lines were fit using a four-parameter logistic model, Activity = Activitymin + (Activitymax - Activitymin)/(1 + 10(b × (X - log10EC50))) where X is the log10 of the number of Effector T 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 EC50 is an estimated parameter representing the X value corresponding to (Activitymax - Activitymin)/2, the error bars are 1 SD above and below the mean (n = 4) and can also be considered as one half-width of an 68% confidence interval for that mean.
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