In Situ Generation of Claudin18.2-Specific CAR-T Cells via Lipid Nanoparticle–Mediated mRNA Delivery

Summary

CAR T-cell application to solid tumors is limited by complex ex vivo manufacturing and safety concerns. This protocol describes a method for the in situ generation of transient CAR-T cells via LNP–mediated delivery of CAR-encoding mRNA, providing a reproducible platform adaptable to other mRNA-based immune cell engineering applications.

Abstract

Chimeric antigen receptor (CAR) T-cell therapy has achieved remarkable clinical success in hematological malignancies; however, its broader application is limited by high manufacturing costs and insufficient efficacy against solid tumors. To overcome these challenges, an in situ CAR-T cell generation strategy was developed based on lipid nanoparticle (LNP)-mediated delivery of CAR mRNA targeting Claudin18.2, enabling the transient production of CAR-T cells directly within tumor tissue. Following LNP transfection, activated T cells exhibited efficient surface expression of CARs. These transient CAR-T cells demonstrated potent cytotoxic activity against Claudin18.2-positive gastric carcinoma cell lines, including NUGC4 and NCI-N87, and produced significant antitumor efficacy in tumor-bearing mouse models. This protocol describes detailed procedures for in vitro transcription of CAR mRNA, LNP formulation, T-cell activation and transfection, CAR expression analysis, and in vitro and in vivo functional evaluation of CAR-T-mediated cytotoxicity. Taken together, this strategy provides a cost-effective and versatile platform for generating transient CAR-T cells and offers a promising approach for treating solid tumors.

Introduction

The incidence and mortality of cancer are rising rapidly worldwide. This poses a threat to human survival and societal development. Gastric cancer is the third leading cause of cancer-related deaths globally and is highly prevalent¹. Due to low rates of early diagnosis, a significant proportion of patients are diagnosed at advanced stages. Traditional treatments include surgery, radiotherapy, and chemotherapy. Surgical resection for patients with mid-to-late-stage or complex gastric cancer may be incomplete. Furthermore, the significant surgical trauma and prolonged postoperative recovery can adversely affect patients' quality of life and p....

Protocol

All animal experiments were approved by the Ethics Committee for Laboratory Animals at Shanghai Tenth People’s Hospital (Approval No.: SHDSYY-2025-P0085; Approval Date: January 1, 2025). This study complies with local laws, regulations, and institutional requirements. NSG female mice aged 6–8 weeks were used. The Peripheral Blood Mononuclear Cells (PBMCs) used in this study were obtained from a commercial source. The reagents and equipment used in this study are listed in the Table of Materials.

1. Preparation of CAR mRNA

1. Linearized DNA templates preparation
   ​NOTE: The plasmid containing th....

Results

A CAR sequence targeting Claudin18.2 was employed, composed of the single-chain variable fragment (scFv) from a humanized anti-Claudin18.2 monoclonal antibody, the CD8 hinge region, the CD28 transmembrane region, the co-stimulatory domain, and the CD3ζ activation domain. A DNA template containing the T7 promoter and poly A tail was constructed based on this sequence for in vitro CAR mRNA transcription (Figure 1A). The CAR mRNA was approximately 1800 bp in length, as verified by capi.......

Discussion

This protocol provides a non-viral mRNA delivery system that avoids integration into the host genome, thereby circumventing numerous risks, including immune rejection. Its transient functionality also prevents off-target toxicity, enabling short-term, controllable expression in CAR-T cells to reduce side effects and safety risks.

Optimization of the CAR mRNA sequence design was performed to regulate its translation efficiency and expression duration. The 5'UTR, 3'UTR, 5'-Cap, and p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Absolute Ethanol	Sigma-Aldrich	459836	Organic phase
Agarose	Beyotime	ST004L	Gel electrophoresis
anti-G4S linker (B02H1) mAb PE	Hycells	GS-ARPE100	CAR detection
BspQI Restriction Enzyme	NEB	R0712	Plasmid linearization
BV421 Mouse Anti-Human CD25	BD Biosciences	564033	Activation marker
CD3/CD28 Activation Beads	Thermo Fisher Scientific	11161D	T cell activation
Cholesterol	Sigma-Aldrich	C8667	Structural lipid
CO2 Cell Culture Incubator	Thermo Fisher / equivalent	250i	For T cell and tumor cell culture
DMG-PEG2000	Avanti Polar Lipids	880151	PEGylated lipid
DNA Isolation Magnetic Beads	SYNTHGENE	10501-005	DNA purification
DNase I (RNase-free)	NEB	M0303L	Removal of DNA template
DSPC	Avanti Polar Lipids	850365	Phospholipid
Dynamic Light Scattering Analyzer	Malvern Panalytical	Zetasizer	For LNP size, PDI, zeta potential
EasySep Buffer	STEMCELL Technologies	20144	Magnetic separation
Fetal Bovine Serum (FBS)	Gibco	10099-141	Culture supplement
FITC Mouse Anti-Human CD3	BD Biosciences	555332	T cell identification
Flow Cytometer	BD Biosciences / equivalent	FACSCelesta / LSRFortessa	For CAR and activation marker
GelGreen Nucleic Acid Stain	Biotium	41005	DNA visualization
LIVE/DEAD Fixable Dye	Invitrogen	L34955	Viability staining
Luciferase Assay Kit	Promega	E1500	Cytotoxicity readout
Magnetic Separation Stand	STEMCELL Technologies / equivalent	18000	For PBMC and T cell isolation
Nanodrop Spectrophotometer	Thermo Fisher Scientific	NanoDrop 2000	For DNA/RNA quantification
NSG Female Mice (6–8 weeks)	Jackson Laboratory	5557	In vivo model
PBMCs	Shanghai Saili Biotechnology Company
PCR thermal cycler	Bio-Rad / equivalent	T100	For DNA amplification
PE Mouse Anti-Human CD69	BD Biosciences	557050	Early activation
Penicillin-Streptomycin	Gibco	15140-122	Antibiotics
Qsep Nucleic Acid Analyzer	BiOptic Inc.	Qsep100	For mRNA size/integrity analysis
Quant-iT RiboGreen RNA Kit	Invitrogen	R11490	Encapsulation efficiency
RapidSpheres	STEMCELL Technologies	15061	Magnetic labeling
Recombinant Human IL-15	PeproTech	200-15	Memory phenotype support
Recombinant Human IL-2	PeproTech	200-02	T cell expansion
Recombinant Human IL-7	PeproTech	200-07	T cell survival
Refrigerated Centrifuge	Eppendorf / equivalent	5425R	For LNP concentration and cell processing
RNA 6000 Ladder	Thermo Fisher Scientific	AM7152	RNA characterization
RNA Cartridge Kit(1pc)	Bioptic	C105110	RNA quality control
RNA Isolation Magnetic Beads	SYNTHGENE	10511-005	mRNA purification
RNase-free Water	Thermo Fisher Scientific	AM9937	IVT and RNA handling
RPMI 1640 GlutaMAX	Gibco	61870-036	T cell culture
SM-102	MedChemExpress / equivalent	HY-112563	Ionizable lipid
T Cell Isolation Cocktail	STEMCELL Technologies	15061	CD3+ T cell isolation
T7 Co-Transcription and Cap-Addition Kit	SYNTHGENE	10111-3011	IVT mRNA synthesis
TBS Buffer	Gibco	3005	LNP storage
Trypsin-EDTA	Gibco	25200-056	Cell detachment
UV Gel Imaging System	Syngene / equivalent	NuGenius/NuGenius+	For DNA digestion verification