A high throughput microelectroporation device to introduce a chimeric antigen receptor to redirect the specificity of human T cells

Abstract

It has been demonstrated that a chimeric antigen receptor (CAR) can directly recognize the CD19 molecule expressed on the cell surface of B-cell malignancies independent of major histocompatibility complex (MHC). Although T-cell therapy of tumors using CD19-specific CAR is promising, this approach relies on using expression vectors that stably integrate the CAR into T-cell chromosomes. To circumvent the potential genotoxicity that may occur from expressing integrating transgenes, we have expressed the CD19-specific CAR transgene from mRNA using a high throughput microelectroporation device. This research was accomplished using a microelectroporator to achieve efficient and high throughput non-viral gene transfer of in vitro transcribed CAR mRNA into human T cells that had been numerically expanded ex vivo. Electro-transfer of mRNA avoids the potential genotoxicity associated with vector and transgene integration and the high throughput capacity overcomes the expected transient CAR expression, as repeated rounds of electroporation can replace T cells that have lost transgene expression. We fabricated and tested a high throughput microelectroporator that can electroporate a stream of 2 x 10(8) primary T cells within 10 min. After electroporation, up to 80% of the passaged T cells expressed the CD19-specific CAR. Video time-lapse microscopy (VTLM) demonstrated the redirected effector function of the genetically manipulated T cells to specifically lyse CD19+ tumor cells. Our biomedical microdevice, in which T cells are transiently and safely modified to be tumor-specific and then can be re-infused, offers a method for redirecting T-cell specificity, that has implications for the development of adoptive immunotherapy.

Fig. 1

Outline of the experimental platform. There are three major parts for this research, ex vivo T-cell proliferation, a microelectroporator, and electro-transfer of mRNA to redirect T-cell specificity and function

Fig. 2

DNA plasmids for in vitro transcription. (a) The second generation CAR (CD19RCD28) cDNA under control of T7 promoter. (b) EGFP cDNA under control of T7 promoter. Amp(R): Ampicillin resistance, Kan(R): Kanamycin resistance, Hygro(R): Hygromycin resistance, BGH: bovine growth hormone

Fig. 3

Description of HiTMeD1 to electroporate T cells in continuous flow. (a) HiTMeD1 is connected to multi-syringe pump. Multiple pulsing mode (square wave) of BTX ECM830 has been used for electroporation (1 kV/cm, 6 ms). (b) Schematic view of HiTMeD1. Multiple inlet ports and polymer films are used to make electrodes and 400 μm gap

Fig. 4

Phenotypic characteristics of aAPC and propagation of human T cells on aAPC. (a) Surface expression of co-stimulatory molecules and adsorbed OKT3 on K562-aAPC. Open histograms show the expression on K562 parental cells and filled histograms show the expression on genetically modified K562 which function as aAPC. Expression of CD64, CD86, CD137L, membrane-bound IL-15, and OKT3 were detected with antibodies. (b) Propagation of primary T cells on aAPC. Irradiated OKT3-loaded K562-aAPC were added to the T-cell culture for every 7 days at two different ratios. (c) T-cell subsets after 7 and 14 days of numeric expansion on OKT3-loaded aAPC at 1 : 1 (T cells : aAPC) ratio

Fig. 5

Electro-transfer of mRNA into T cells. (a) Flow cytometry analysis of CD19-specific CAR and EGFP transgenes in gated CD3⁺ T cells 12 h after electroporation. (b) CD19-specific CAR expression on CD3⁺ T cells by flow cytometry 12 and 48 h after electro-transfer of mRNA. (c) Four experiments with HiTMeD1 in which T cells were electroporated without mRNA, with CAR mRNA, with EGFP mRNA, and with both CAR and EGFP mRNA species

Fig. 6

Redirected specificity of electroporated T cells. Killing of CD19⁺GFP⁺U251T target cells within 7 h of co-culture with CD19-specific T cells (12 h after processing through HiTMeD1). Orange⁺ adherent CD19⁺GFP⁺U251T cells can be differentiated from CD19^negGFP⁺U251T cells. T cells (approximately 10 μm diameter) can be seen as black (mobile) structures. See supplementary material 2 (Movie 2) for the original data used to perform this analysis

Fig. 7

CD19-specific time-dependent killing of tumor cells by CAR⁺ T cells. The killing of U251T cells over time was analyzed for 14 h by Nikon imaging software (NIS-Elements AR v3.00). 50% killing of CD19⁺U251T target cells was observed at approximately 4 h after the beginning of the assay. CD19^negU251T cells were not targeted by CD19-specific T cells and cell division was observed which resulted in cell viability over 110%