Sleeping beauty system to redirect T-cell specificity for human applications

Abstract

The Sleeping Beauty (SB) transposon/transposase DNA plasmid system is used to genetically modify cells for long-term transgene expression. We adapted the SB system for human application and generated T cells expressing a chimeric antigen receptor (CAR) specific for CD19. Electrotransfer of CD19-specific SB DNA plasmids in peripheral blood mononuclear cells and propagation on CD19 artificial antigen presenting cells was used to numerically expand CD3 T cells expressing CAR. By day 28 of coculture, >90% of expanded CD3 T cells expressed CAR. CAR T cells specifically killed CD19 target cells and consisted of subsets expressing biomarkers consistent with central memory, effector memory, and effector phenotypes. CAR T cells contracted numerically in the absence of the CD19 antigen, did not express SB11 transposase, and maintained a polyclonal TCR Vα and TCR Vβ repertoire. Quantitative fluorescence in situ hybridization revealed that CAR T cells preserved the telomere length. Quantitative polymerase chain reaction and fluorescence in situ hybridization showed CAR transposon integrated on average once per T-cell genome. CAR T cells in peripheral blood can be detected by quantitative polymerase chain reaction at a sensitivity of 0.01%. These findings lay the groundwork as the basis of our first-in-human clinical trials of the nonviral SB system for the investigational treatment of CD19 B-cell malignancies (currently under 3 INDs: 14193, 14577, and 14739).

Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1. K562-derived aAPC (clone #4) sustains proliferation of CAR+ T cells

(a) Phenotype of aAPC. Flow cytometry analyses revealed the expression of proteins derived from (i) the introduced transgenes CD19, CD64, CD86, and CD137L and (ii) membrane-bound IL-15 (mIL-15) co-expressed via IRES with EGFP. (b) Stability of the expression of introduced transgenes aAPC by flow cytometry. (c) CD19-specific CAR and CD3 expression and lack of CD32 expression on genetically modified and propagated T cells. Shown here are CD19-specific CAR and CD3 expression on Day 28 after electroporation. Although on Day 1, CAR expression was from episomal and integrated plasmids, at Day 28, the expression of this CAR transgene was from the integrated plasmid only. On Day 28, there was negligible (1%) expression of CD32, which was consistent with the loss of the aAPC which homogenously express endogenous CD32. (d) Numeric expansion of CAR+ and CD3+ T cells upon recursive additions of γ-irradiated aAPC. Upward arrows indicate additions of aAPC to culture. T cells were enumerated every 7 days, and viable cells were counted based on Trypan blue exclusion.

Figure 2. Redirected specificity of CAR+ T cells

(a) The percentage of specific lysis by red-fluorescent-labeled CD19-specific CAR+ T cells for EGFP+CD19neg U251T parental glioma cells and EGFP+CD19+ U251T target cells. Upper panel: U251T parental targets; lower panel, U251T genetically modified targets. (b) Specific lysis of the target cells by CAR+ T cells. (c) Targets used for CRA. Expression of truncated CD19 in mouse NSO cells and primary human CD3+ T cells (autologous targets). Also shown is the expression of CAR on genetically modified T cells. (d) CAR+ T cells lyse both CD19+ NS0 cells and autologous CD19+ T cells, while sparing CD19neg NS0 and CD19negCAR+ autologous T cells, as measured by 4 hour CRA.

Figure 3. In-process testing of genetically modified T cells harvested at Day 28 of culture on aAPC

(a) The assay to screen for lack of autonomous cell growth showed loss of T cells in the absence of antigen (aAPC clone #4) and IL-2. (b) Integrated SB11 transposase sequence was not detected in T-cell genome. Jurkat cell stably expressing SB11 transposase served as a positive control. (c) Messenger RNA coding for SB11 was not detected in T cells. Total cellular RNA from propagated CAR+ T cells was subjected to cDNA synthesis followed by PCR for the presence of SB11 transposase. Jurkat cell stably expressing SB11 transposase served as a positive control.

Figure 4. Measurement of TCR Vα and Vβ diversity in electroporated/propagated CAR+ T cells

The cellular lysate equivalent of 30,000 cells from one representative sample of CAR+ T cells harvested on Day 28 (filled bar) was used in digital multiplexed assay to determine the amount of mRNA coding for (a) 45 TCR Vα and (b) 46 TCR Vβ chains. Autologous CD3+ T cells harvested on Day 0 (unmodified T cells, open bar) were used as the pre-electroporation control. The mRNA counts were normalized against the house-keeping genes.

Figure 5. Comparison of telomere length in CD3+CARneg and CAR+ T cells

Two (a and b) independent experiments were performed using CD3+ T cells isolated from PBMC and autologous CARneg and CAR+ T cells propagated on γ-irradiated aAPC clone #4 with and without OKT3, respectively. Error bars define standard errors.

Figure 6. Determination of integrated CAR-transgene copy number in T-cell genome and detection of CAR+ T cells in the PBMC by Q-PCR

(a) CAR (CD19RCD28) transgene integration in Jurkat clone #12 by Southern blot analysis. Southern blot of control CAR transposon plasmid DNA (lanes 1–4) and Jurkat clone 12 genomic DNA (lanes 5–8). Plasmid and genomic DNA were digested with NheI and NcoI (lanes 1 and 5); ClaI and SphI (lanes 2 and 6); SacI and NcoI (lanes 3 and 7); and NheI and XbaI (lanes 4 and 8) and hybridized with a 770-bp (NheI-NcoI) CAR-specific probe; expected bands were revealed with plasmid DNA. (b) Integrated CD19-specific CAR transgene detection by FISH in Jurkat cells. Single Jurkat-cell clones expressing CAR transgene were sorted with CAR-specific antibody and FISH for CAR transgene was performed on several clones. Hybridized images of a single clone (clone 12) are shown and arrows denote the site of CAR integration. (c) Integrated CAR detected by FISH in primary T cells. Primary T cells from peripheral blood expressing the CD19-specific CAR were propagated for 28 days in the presence of irradiated aAPC (clone #4), harvested, and chromosomes were fixed. FISH analysis was then performed. Arrows denote the site of CAR integration (d) Genomic Q-PCR for determining the copy number of integrated CAR transgene in Jurkat clone #12. Q-PCR using CAR-specific primers revealed a single copy of introduced CAR transgene in electroporated and propagated Jurkat-cell clones. The Q-PCR primers could detect 0.1 fg of CAR+ plasmid. (10 pg is equivalent to DNA from the genome of ~1.6 cells) The amplification plot (Rn vs. cycle) displays baseline-corrected normalized reporter (RNase P) plotted against cycle number. The CT value of CAR and RNase P in relative quantity analyses of the CAR target copy number indicated that a single CAR transgene copy number per genome was present in Jurkat clone #12. Parental Jurkat cells were used as a reference and endogenous RNaseP was used as a normalizer. (e) Genomic Q-PCR was used to determine the copy number of integrated CAR in primary T cells. The amplification plot (Rn vs. cycle) displays baseline-corrected normalized reporter (RNase P) plotted against cycle number. Target copy number was determined using endogenous RNaseP as normalizer and Jurkat clone #12 containing single integrated transgene as a reference. (f) Q-PCR to undertake measurement of genetically modified T cells in peripheral blood. CD19RCD28mz(CoOp)/pSBSO plasmid DNA standard curve from seven 10-fold serial dilutions of CD19RCD28mz(CoOp)/pSBSO plasmid DNA expressing CD19RCD28 CAR transposon. Plasmid DNA concentration ranges from 100 pg to 0.1 fg. 0.1 fg of CAR plasmid DNA is equivalent to 14 copies of the CAR transgene. X-axis describes PCR cycles. The number of copies of the transgene was calculated by the following formula; number of copies = (amount of DNA*6.022×1023)/(length*109*650), where 6.022×1023 is the Avogadro’s number and that the one mole of a bp weighs 650 Daltons. (g) PBMC spiked at 1%, 0.1%, 0.01% and 0.001% and 0% with CAR+ Jurkat (clone #12) cells containing one copy of CD19RCD28 transgene per cell. Input genomic DNA was 10 ng per reaction. The experiment was performed twice with duplicate measurements at each time. The CT values of all measurements were within one cycle of each other and the measurements were averaged. The non-spiked control PBMC donor DNA was negative for CAR transgene.