Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Summary

This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells.

Abstract

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

Introduction

Adoptive T cell therapies expressing chimeric antigen receptors (CARs) have revolutionized the field of cancer immunotherapy by harnessing the power of the adaptive immune system to specifically target and eliminate antigen-positive cancer cells¹. While the success of CAR-T cell therapies targeting B cell malignancies has been clinically validated, preclinical studies performed in animal models remain vital for the development of new CARs targeting solid tumors. However, limited clinical efficacy has been demonstrated in solid tumor indications thus far, and it is becoming increasingly apparent that individual preclinical ....

Protocol

All animal procedures were performed with approval from the Institutional Animal Care and Use Committee (Columbia University, protocols AC-AABQ5551 and AC-AAAZ4470) using 6-8-week-old female BALB/c or CF57BL/6 mice weighing between 20-25 g. The animals were obtained from a commercial source (see Table of Materials). This protocol is structured around the 'days post-activation' of murine T cells, and viral production begins on Day -2. Retrovirus can be stored at -80 °C following initial produ.......

Discussion

This protocol describes the steps and reagents necessary for the retroviral transduction of murine T cells to generate CAR-T cells for in vivo studies. Optimizing retroviral transduction conditions achieves robust CAR expression without the need for viral concentration through ultracentrifugation or additional reagents. However, there are multiple modifications that can be applied to this methodology.

While this protocol describes the example generation of a GFP-specific CAR, these me.......

Materials

Name	Company	Catalog Number	Comments
0.45 μm filters	MilliporeSigma	SLHVR33RS
1 mL syringe	Fisher Scientific	14-955-450
1.5 mL microcentrifuge tubes	Fisher Scientific	05-408-135
10 mL syringe	BD	14-823-16E
100 μm strainer	Corning	07-201-432
15 cm TC treated cell culture dishes	ThermoFisher Scientific	130183
15 mL conical tubes	Falcon	14-959-70C
40 μm strainer	Corning	07-201-430
50 mL conical tubes	Falcon	14-959-49A
70 μm strainer	Corning	07-201-431
Attune NxT Flow Cytometer	ThermoFisher Scientific
BALB/C, 6-8 week old	Jackson Laboratory	651
B-Mercaptoethanol	Gibco	21985023
Bovine Serum Albumin	GOLDBIO	A-420-500
DMEM Medium	Gibco	11965092
Dulbecco's Phosphate Buffered Saline (PBS), without Calcium and Magnesium	Gibco	14-190-250
DynaMag-2 Magnet	Invitrogen	12-321-D
EasySep Magnet	Stemcell Technologies	18000
EasySep Mouse T cell Isolation Kit	Stemcell Technologies	19851
FACS buffer	BD	BDB554657
Fetal bovine serum (FBS)	Corning	MT35011CV
GlutaMAX	Gibco	35-050-061
G-Rex6	Wilson Wolf	80240M
HEPES Buffer Solution	Gibco	15-630-080
Human recombinant IL-15	Miltenyi Biotec	130-095-765
Human recombinant IL-2	Miltenyi Biotec	130-097-748
Human recombinant IL-7	Miltenyi Biotec	130-095-363
Lipofectamine 3000	Invitrogen	L3000008
MEM Non-Essential Amino Acids Solution	Gibco	11140-050
Mouse Anti-CD3 BV421	Biolegend	100228
Mouse Anti-CD3/CD28 Dynabeads	Gibco	11-453-D
Mouse Anti-CD4 BV605	BD	563151
Mouse Anti-CD44 APC	Biolegend	103011
Mouse Anti-CD62L PE-Cy7	Tonbo	SKU 60-0621-U025
Mouse Anti-CD8 APC-Cy7	Tonbo	SKU 25-0081-U025
Nikon Ti2 with Prime 95B camera	Nikon
Non-treated 24 well plates	CytoOne	CC7672-7524
Opti-MEM	Gibco	31-985-062
pCL-Eco	Addgene	#12371
Penicillin/Streptomycin Solution	Gibco	15-070-063
Phoenix Eco cells	ATCC	CRL-3214
pMDG.2	Addgene	#12259
pMSCV_PGK_GFP28z	N/A	Produced by R.LV.
Purified sfGFP	N/A	Produced by R.LV.
RetroNectin ('transduction reagent')	Takara Bio	T100B
RPMI 1640	Gibco	21875
Serological pipette 10 mL	Fisher Scientific	13-678-11E
Serological pipette 25 mL	Fisher Scientific	13-678-11
Serological pipette 5 mL	Fisher Scientific	13-678-11D
Sodium Pyruvate	Gibco	11-360-070
TC-treated 24 well plates	Corning	08-772-1
Trypan blue	Gibco	15-250-061