Engineering Chimeric Antigen Receptor-Natural Killer Cells Targeting Fungal Infections Using the Non-viral Sleeping Beauty Transposon System

Summary

We outline a transfection protocol for producing chimeric antigen receptor-natural killer (CAR-NK) cells targeting fungal pathogens using the non-viral Sleeping Beauty transposon system. To assess antigen-specific activation, we co-cultured the engineered cells with Aspergillus fumigatus germ tubes and measured IFN-γ secretion.

Abstract

Chimeric antigen receptor (CAR)-based cell therapies have shown impressive efficacy in the treatment of hematological malignancies. Recently, these therapies are being developed for infectious diseases, yet studies targeting fungal infections remain scarce. To identify optimal targets and optimize cellular products, we developed a method to engineer chimeric antigen receptor-natural killer (CAR-NK) cells and evaluated their response to stimulation by fungi. This paper describes a straightforward and robust method for generating CAR-NK cells tailored to fungal targets using the non-viral Sleeping Beauty transposon system. NK-92 cells are transfected with the hyperactive transposase SB100X vectorized as minicircle DNA (MC) along with a plasmid-encoded CAR transposon. Transfection efficiency is assessed 1 week later using flow cytometric analysis. Prior to functional testing, the cells expressing the transgene are enriched using magnetic-activated cell sorting and cultured for 1 more week. To evaluate antigen-specific activation, the engineered cells are co-cultured with Aspergillus fumigatus germ tubes for at least 6 h. Subsequently, the concentration of the secreted interferon-gamma (IFN-γ) is measured using an enzyme-linked immunosorbent assay.

Introduction

Aspergillus fumigatus (A. fumigatus) is a ubiquitous fungus, with the average person inhaling between 100 and 1,000 conidia on a daily basis¹. In patients with acquired or congenital immunodeficiencies, such as those undergoing chemotherapy or who are suffering from leukemia, A. fumigatus can result in severe invasive apsergillosis. Each year, more than 200,000 people worldwide contract aspergillosis. Despite the use of antimycotics for prevention and therapy with azole derivatives, the mortality rate remains between 30% and 95%^2,3. Additionally, the treatment....

Protocol

All experiments described here must be conducted under sterile conditions. In this protocol, we show two conditions: (I) Mock transfection without DNA that will serve as control and (II) CAR transfection using the previously described Af-CAR⁶. The Af-CAR plasmid contains a truncated epidermal growth factor receptor (EGFRt) sequence downstream of the CAR, which will be used as a transfection marker (Supplemental Figure S1). For each condition, 4 × 10⁶ NK-92 cells ar.......

Discussion

The provided protocol offers a simple and reliable method for creating CAR-NK cells that target fungal pathogens using the non-viral Sleeping Beauty transposon system. This method allows for the stable and permanent insertion of large transgenes, which enables the expansion of cells to obtain the necessary quantities for further analysis^14,16.

Several key factors can impact the success of the transfection, including cell number and via.......

Materials

Name	Company	Catalog Number	Comments
15 ML conical bottom tubes	Greiner Bio-One	188271	PP, 17/120 MM, CELLSTAR, STERILE
50 ML conical bottom tubes	Greiner Bio-One	227270	PP, 30/115 MM, CELLSTAR, STERILE
6-and 96-Well- Cell culture plate	Corning Inc. LifeSciences	83-3736/ 83-3474	TC-treated Multiple Well Plates, Flat bottoms, Treated for optimal cell attachment, Sterilized by gamma irradiation, Nonpyrogenic
7-AAD (7-Aminoactinomycin D)	BD Biosciences	559925	eady-to-use nucleic acid dye for the exclusion of nonviable cells in flow cytometric assays
Alexa Fluor 647 anti-human EGFR Antibody	Biolegend	352918	Clone AY13
Anti-Biotin MicroBeads UltraPure	Miltenyi Biotec	130-105-637	UltraPure MicroBeads conjugated to monoclonal mouse anti-biotin antibodies
Aspergillus fumigatus	ATCC	46645
Biosphere Filter Tips (20 and 100 μL)	Sarstedt	70.3030.365 / 70.3030.275
Dulbecco′s Phosphate Buffered Saline (PBS)	Sigma-Aldrich	D8537	Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture, endotoxin, tested
ELISA MAX Deluxe Set Human IFN-γ	Biolegend	430104
Ethylenediaminetetraacetic acid disodium salt solution (EDTA)	Sigma-Aldrich	E7889	for molecular biology, 0.5 M in H2O, DNase, RNase, NICKase and protease, none detected, 0.2 μm filtered.
FACS clean	BD Biosciences	340345
FACS Flow Sheath Fluid	BD Biosciences	342003
FACS Rinse Solution	BD Bioscience	340346
Fetal Bovine Serum (FBS)	Sigma-Aldrich	F7524	sterile-filtered, suitable for cell culture, heat inactivated before use
Gibco, MEM α, nucleosides	Thermofisher Scientific	22571020	Phenol Red, Ribonucleosides, Deoxyribonucleosides, Sodium Bicarbonate
Horse serum	Thermo Fisher Scientific	16050122	Sterile-filtered, heat inactivated before use
Human IL-2 IS, research grade	Miltenyi Biotec	130-097-742	Research grade. The ED50 is ≤0.3 ng/mL, corresponding to an activity of ≥3 × 106 IU/mg.
Ionomycin	Sigma-Aldrich	I0634-1MG	from Streptomyces conglobatus, used as positive control for cytokine secretion
L-Glutamine	Sigma-Aldrich	W368401	For cell culture
MACS LS Column	Miltenyi Biotec	130-042-401	Columns and plungers, sterile packed
NK-92	DSMZ	ACC 488
Neon Transfektionssystem 100 μL-Kit	Thermo Fisher Scientific	MPK10096	Resuspension Buffer R, Electrolytic Buffer E2, 100 μL Neon Tips, Neon Electroporation Tubes
Phorbol-12-myristat-13-acetat (PMA)	Sigma-Aldrich	P1585-1MG	used as positive control for cytokine secretion
Polystyrene round-bottom tube, 5 mL	BD Bioscience	S-452400	used for flow cytometry
Reaction tube, 1.5 ML	Greiner Bio-One	7,26,90,001	PP, transparent, cap attached, with injected graduation. Sterilized before use.
RPMI 1640 Medium, GlutaMAX	Thermo Fisher Scientific	72400021	GlutaMax I,  Phenol Red, HEPES, Sterile-filtered
TipOne 1000 μL XL Graduated Filter Tip	StarLab	S1122-1730
Equipment
BD FACSCalibur	BD Bioscience
CO2 Incubator C60	Labotect
Heraeus Multifuge 3SR	Thermo Scientific
HydroFLEX microplate washer	Tecan
NanoQuant Infinite M200 Pro	Tecan
Neon Transfection System	Invitrogen
Pipettes	Eppendorf
Quadro MACS Separator	Miltenyi Biotec