Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells

Summary

Here, we present a method to differentiate and expand human iPSC-derived chimeric antigen receptor (CAR)-expressing natural killer cells with improved killing against various malignancies. This protocol demonstrates the differentiation and expansion of natural killer (NK) optimized iPSC-derived CAR-NK cells and the measurement of antitumor activity against various tumor cell lines.

Abstract

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of human induced pluripotent stem cell (iPSC)-derived chimeric antigen receptor (CAR) expressing NK cells has emerged as a promising avenue for "off the shelf" cancer immunotherapy. Here, we utilized an NK cell-optimized CAR construct that includes the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3ζ signaling domain, which has been demonstrated to stimulate robust antigen-specific NK cell-mediated antitumor activity. The use of iPSCs for CAR NK cell generation offers several advantages, including homogenous CAR expression, scalability, reproducibility, and the potential for clinical application. This detailed step-by-step protocol from cell engineering to differentiation enables the generation of NK cell-optimized iPSC-derived CAR-expressing NK cells, providing a standardized and targeted cancer immunotherapy with improved antitumor activity and highlighting their potential as a promising treatment option for various malignancies.

Introduction

NK cells, a type of lymphocyte within the innate immune system, are pivotal in the early defense against tumors and virally infected cells^1,2,3. Unlike T cells, NK cells do not require antigen presentation via major histocompatibility complex (MHC) molecules. Instead, NK cells have a repertoire of activating and inhibitory receptors that regulate their activity³. NK cell-mediated cytotoxic activity utilizes various mechanisms, including the release of perforin and granzymes, engagement of death receptors, and the production of pro-inflammatory cytokine....

Protocol

1. Feeder-free culture method of human iPSCs

NOTE: Thaw the frozen undifferentiated human iPSCs and culture them with the use of mTeSR 1-plus on Matrigel (henceforth referred to as basement membrane matrix [BMM]) pre-coated plates, as previously described^23,34. It is very important to make sure the iPSCs are not differentiated before and after engineering. Freshly thawed iPSCs should be cultured for about 2-3 passages to exhibit pluripotency morphology consistent with the human pluripotent stem cells. The following instructions are used for passaging cells from one we....

Discussion

This protocol outlines a standardized and reproducible approach for generating CAR-expressing, iPSC-derived NK cells from a consistent cell source aimed at facilitating targeted "off-the-shelf" cancer immunotherapy. Multiple preclinical and clinical studies have shown the efficacy of adoptive NK cell-based immunotherapy in treating cancers while minimizing toxicities, like graft versus host diseases (GvHD) or cytokine-release syndrome (CRS)^23,42,

Materials

Name	Company	Catalog Number	Comments
aAPC	Dean A. Lee lab	N/A
a-MEM culture medium	Fisher Scientific	Cat#12634
APC anti-DYKDDDDK (FLAG Tag)	BD Biosciences	Cat#637308
APC-anti-human TRA-1-81	ThermoFisher	17-8883-42
APC-CD16	BD Biosciences	Cat#302015
APC-CD43	BD Biosciences	Cat# 560198
APC-CD45	BD Biosciences	Cat# 555485
APC-GPC3	BD Biosciences	Cat#DB100B
APC-NKG2D	BD Biosciences	Cat# 558071
CellEvent Caspase-3/7 Green Detection Reagent	Thermo fisher	Cat#C10423
CellTrace Violet Cell Proliferation Kit	Thermo fisher	Cat#C34571	Cell tracing fluorescent dye
CryoStor solution	Stem Cell Technologies	https://www.stemcell.com/products/cryostor-cs10.html	Cryopreservation medium
CTS NK Xpander	Gibco	A5019001
CTS NK-Xpander Medium	Life Technologies	Cat#A5019001
DMEM	Gibco	11965084
DMEM, high glucose, GlutaMAX Supplement, pyruvate	Gibco	10569010
EasySep Human NK Cell Enrichment Kit	StemCell Technologies, Inc.	Cat#19055
Ethanolamine	Sigma Aldrich	E9508
Fetal bovine serum	Fisher Scientific	Cat# 10437010
FITC-CD94	BD Biosciences	Cat#555888
GlutaMAX Supplement	Gibco	35050061
GolgiPlug	BD Biosciences	Cat#555029
GolgiStop	BD Biosciences	Cat#554724
Ham's F-12 Nutrient Mix, GlutaMAX Supplement	Gibco	31765035
Horse serum	Fisher Scientific	Cat#16050130
Human Serum	AB Sigma-Aldrich	Cat#BP2525100
Human Stem Cell NucleofectorTM Kit	Lonza	Cat# VPH-5012
Human: HePG2 cells	ATCC	Cat#HB-8065
Human: HePG2-td-tomato-luc cells	Dan S. Kaufman lab	N/A
Human: iPS cells	Dan S. Kaufman lab	N/A
Human: SNU-449 cells	ATCC	Cat#CRL-2234
Human: SNU-449-td-tomato-luc cells	Dan S. Kaufman lab	N/A
IncuCyte Caspase-3/7 Green Apoptosis Assay	Essenbioscience	Cat#4440
L-Ascorbic acid	Sigma Aldrich	A5960
MP Biomedicals Human Serum, Type AB	MP Biomedicals	ICN2938249
mTeSR plus	StemCell Technologies, Inc.	100-0276
NovoExpress software	ACEA Biosciences	https://www.agilent.com/en/product/research-flow-cytometry/flow-cytometry-software/novocyte-novoexpress-software-1320805
PE/Cy7 anti-human SSEA-4 Antibody	Biolegend	330420
PE-CD16	BD Biosciences	Cat#560995
PE-CD226	BD Biosciences	Cat#559789
PE-CD34	BD Biosciences	Cat# 555822
PE-CD45	BD Biosciences	Cat# 555483
PE-CD94	BD Biosciences	Cat#555888
PE-cy7-CD56	BioLegend	Cat# 318318
PE-FAS Ligand	BD Biosciences	Cat#564261
PE-NKp30	BD Biosciences	Cat# 558407
PE-NKp44	BD Biosciences	Cat#558563
PE-NKp46	BD Biosciences	Cat#331908
PE-NKp46	BD Biosciences	Cat#557991
Peripheral blood buffy coat	San Diego Blood Bank (https://www. sandiegobloodbank.org/)	N/A
PE-TRAIL	BD Biosciences	Cat#565499
pKT2-mCAG-IRES-GFP-ZEO	Branden Moriarity lab	N/A
pMAX-GFP plasmid	Lonza	N/A	GFP positive control
Prism 9	Graphpad	Version 9
pSpCas9	GenScript	PX165
RBC Lysis Buffer (10x)	Biolegend	Cat#420301
Recombinant human bFGF basic	R&D Systems	Cat#4114-TC
Recombinant human BMP-4	PeproTech	Cat#120-05
Recombinant human FLT-3 Ligand	PeproTech	Cat# 300-19
Recombinant human IL-15	PeproTech	Cat# 200-15
Recombinant human IL-2	PeproTech	Cat# 200-02
Recombinant human IL-3	PeproTech	Cat#200-03
Recombinant human IL-7	PeproTech	Cat# 200-07
Recombinant Human Nodal Protein	R&D Systems	Cat#3218-ND-025
Recombinant human SCF	PeproTech	Cat# 300-07
Recombinant Human TGF-β1	PeproTech	Cat#100-21
Recombinant human VEGF	PeproTech	Cat# 100-20
RPMI1640	Gibco	11875093
Sodium selenite	Sigma Aldrich	214485
STEMdiff APEL 2 Medium	StemCell Technologies, Inc.	5270	EB formation medium
STEMdiff APEL2 Medium	StemCell Technologies, Inc.	Cat#05270
Super piggyBac Transposase expression vector	SBI	Cat#PB210PA-1
SYTOX AADvanced Dead Cell Stain Kit	ThermoFisher Scientific	S10274, S10349	Dead cell staining solution kit
β-mercaptoethanol	Gibco	21985023