Autologous antigen-presenting cells efficiently expand piggyBac transposon CAR-T cells with predominant memory phenotype

Abstract

The quality of chimeric antigen receptor (CAR)-T cell products, including the expression of memory and exhaustion markers, has been shown to influence their long-term functionality. The manufacturing process of CAR-T cells should be optimized to prevent early T cell exhaustion during expansion. Activation of T cells by monoclonal antibodies is a critical step for T cell expansion, which may sometimes induce excess stimulation and exhaustion of T cells. Given that piggyBac transposon (PB)-based gene transfer could circumvent the conventional pre-activation of T cells, we established a manufacturing method of PB-mediated HER2-specific CAR-T cells (PB-HER2-CAR-T cells) that maintains their memory phenotype without early T cell exhaustion. Through stimulation of CAR-transduced T cells with autologous peripheral blood mononuclear cell-derived feeder cells expressing both truncated HER2, CD80, and 4-1BBL proteins, we could effectively propagate memory-rich, PD-1-negative PB-HER2-CAR-T cells. PB-HER2-CAR-T cells demonstrated sustained antitumor efficacy in vitro and debulked the HER2-positive tumors in vivo . Mice treated with PB-HER2-CAR-T cells rejected the second tumor establishment owing to the in vivo expansion of PB-HER2-CAR-T cells. Our simple and effective manufacturing process using PB system and genetically modified donor-derived feeder cells is a promising strategy for the use of PB-CAR-T cell therapy.

PiggyBac Transposon-Mediated CD19 Chimeric Antigen Receptor-T Cells Derived From CD45RA-Positive Peripheral Blood Mononuclear Cells Possess Potent and Sustained Antileukemic Function

“…The human lymphoblastic leukemia cell line (REH) was purchased from the American Type Culture Collection (Manassas, VA). REH-expressing firefly luciferase (FFLuc) and green fluorescent protein (GFP) (REH-FFLuc-GFP) were obtained by introducing PB-based pIRII-FFLuc-puroR-GFP (18) in REH cells and subsequent fluorescent-activated cell sorting. REH and REH-FFLuc-GFP cells were cultured in Roswell Park Memorial Institute-1640 medium (Nacalai Tesque) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc. Waltham, MA) and maintained in a humidified incubator at 37°C in a 5% CO 2 atmosphere.…”

“…For the generation of antigen-presenting feeder cells for the stimulation of CD19-CAR-transduced T cells, we used a plasmid containing sequences encoding the extracellular, transmembrane, and 20 amino-acid-long intracellular portion of CD19 protein (tCD19) driven by CMV promoter, followed by CD80 and 4-1BBL (CD137L) with TA2 and P2A self-cleaving sites that enabled independent gene expression. The tCD19-CD80-4-1BBL sequence was artificially synthesized (Fasmac Inc., Kanagawa, Japan) and cloned into a pIRII PB transposon vector (pIRII-tCD19-CD80-4-1BBL) (Supplementary Figure S1) (17,18).…”

“…Non-viral gene transfer using piggyBac (PB) transposonbased genetic modifications is a potentially effective strategy for CAR-T cell manufacturing (11)(12)(13)(14)(15)(16). Our previous study and also other studies have reported that PB-CAR-T cells exhibit an enriched memory fraction and less exhaustionrelated markers, regardless of the type of CAR constructs, electroporation conditions, or expansion protocols (17,18). PB-CAR-T cells that redirected CD19, HER2, or ephrin type-B receptor 4 precursor (EPHB4) molecules were dominant in the naïve/stem cell memory-like T-cell fraction, which was characterized as CD45RA/C-C chemokine receptor type 7 (CCR7) double positive and is related to long-term functionality.…”

Homology mediated end joining enables efficient non-viral targeted integration of large DNA templates in primary human T cells

“…Current clinical studies using a non-viral approach to engineer CAR-T cells largely utilize DNA transposon systems to introduce the CAR construct into T cells (Clinical trial numbers NCT03389035, NCT04289220). While the use of transposons can allow for high level integration rates, on par with lentivirus, their use is limited by their mechanism of integrating at random locations within the genome [73][74][75][76][77] . This causes significant drawbacks, including the potential for integration into tumor suppressor genes or oncogenes, imprecise control of the number of copies integrated per cell, and an inability to use endogenous promoters and their regulatory elements to control expression of the integrated construct.…”

A lymphodepleted non‐human primate model for the assessment of acute on‐target and off‐tumor toxicity of human chimeric antigen receptor‐T cells

“…The CAR transgene was transduced into fresh, unstimulated PBMCs using the PB transposon system, as per methods described previously 7,26 (Supplementary figure 4). Briefly, the pCMV‐PB (7.5 μg) and pIRII‐EPHB4‐CAR‐28z (7.5 μg) were introduced into 4 × 10 7 PBMCs using the MaxCyte ATX electroporator (MaxCyte) with the optimised protocol for the introduction of DNA plasmid into resting T cells (Protocol; RTC 14‐3).…”