Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Summary

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

Abstract

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Introduction

Chimeric antigen receptors (CARs) provide a promising approach to redirect T cells against distinct tumor antigens. CARs are synthetic receptors that bind an antigen target. While their precise composition is variable, CARs generally contain 3 distinct domains. The extracellular domain directs binding to a target antigen and is typically comprised of a single chain antibody fragment linked to the CAR via an extracellular hinge. The second domain, commonly derived from the CD3ζ chain of the T cell receptor (TCR) complex, promotes T cell activation following CAR engagement. A third costimulatory domain is included to enhance T cell function, engraftment, metabolism....

Protocol

All animal studies are approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

1. T Cell Activation, Transduction, and Expansion

1. Activate fresh or cryopreserved primary human T cells by mixing with anti CD3/CD28 magnetic beads (e.g., dynabeads) at a ratio of 3 beads per T cell in 6-well cell culture dishes. Culture T cells in X-VIVO 15 medium supplemented with 5% normal human AB serum, 2 mM L-glutamine, 20 mM HEPES, and IL2 (100 units/mL). Maintain T cells at a concentration of 10⁶ T cells/mL during expansion. Culture T cells at 37 °C, 20% O₂, and 95% humidity wit....

Results

Using the methods described above, we stimulated and expanded T cells for either 3 or 9 days (Figure 1A,B). We also analyzed their differentiation profile, as indicated by the gating strategy outlined in Figure 1C, by measuring the abundance of distinct glycoproteins expressed on the cell surface. We show a progressive shift towards effector differentiation over time during ex vivo culture (Figure 1.......

Discussion

Here we describe approaches to measure the function and efficacy of CAR T cells harvested at varying intervals throughout ex vivo culture. Our methods provide comprehensive insight into assays designed to assess proliferative capacity as well as effector function in vitro. We describe how to measure CAR T cell activity following stimulation through the CAR and detail xenograft models of leukemia using CAR T cells harvested at day 3 vs day 9 of their logarithmic expansion phase.

There are inher.......

Materials

Name	Company	Catalog Number	Comments
Anti CD3/CD28 dynabeads	Thermo Fisher	40203D
APC Mouse Anti-Human CD8	BD Biosciences	555369	RRID:AB_398595
APC-H7 Mouse anti-Human CD8 Antibody	BD Biosciences	560179	RRID:AB_1645481
BD FACS Lysing Solution 10X Concentrate	BD Biosciences	349202
BD Trucount Absolute Counting Tubes	BD Biosciences	340334
Brilliant Violet 510 anti-human CD4 Antibody	BioLegend	317444	RRID:AB_2561866
Brilliant Violet 605 anti-human CD3 Antibody	BioLegend	317322	RRID:AB_2561911
CellTrace CFSE Cell Proliferation Kit	Life Technolohgies	C34554
CountBright Absolute Counting Beads,	Invitrogen	C36950
FITC anti-Human CD197 (CCR7) Antibody	BD Pharmingen	561271	RRID:AB_10561679
FITC Mouse Anti-Human CD4	BD Biosciences	555346	RRID:AB_395751
HEPES	Gibco	15630-080
Human AB serum	Valley Biomedical	HP1022
Human IL-2 IS, premium grade	Miltenyi	130-097-744
L-glutamine	Gibco	28030-081
Liquid scintillation counter, MicroBeta trilux	Perkin Elmer
LIVE/DEAD Fixable Violet	Molecular Probes	L34964
Multisizer Coulter Counter	Beckman Coulter
Na251CrO4	Perkin Elmer	NEZ030S001MC
Pacific Blue anti-human CD14 Antibody	BioLegend	325616	RRID:AB_830689
Pacific Blue anti-human CD19 Antibody	BioLegend	302223
PE anti-human CD45RO Antibody	BD Biosciences	555493	RRID:AB_395884
PE/Cy5 anti-human CD95 (Fas) Antibody	BioLegend	305610	RRID:AB_493652
PE/Cy7 anti-human CD27 Antibody	Beckman Coulter	A54823
Phenol red-free medium	Gibco	10373-017
UltraPure SDS Solution, 10%	Invitrogen	15553027
Via-Probe	BD Biosciences	555815
X-VIVO 15	Gibco	04-418Q
XenoLight D-Luciferin - K+ Salt	Perkin Elmer	122799