RT Journal Article
SR Electronic
T1 Functionalized microcarriers improve T cell manufacturing by facilitating migratory memory T cell production and increasing CD4/CD8 ratio
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 646760
DO 10.1101/646760
A1 Nathan J. Dwarshuis
A1 Hannah W. Song
A1 Anokhi Patel
A1 Theresa Kotanchek
A1 Krishnendu Roy
YR 2019
UL http://biorxiv.org/content/early/2019/05/23/646760.abstract
AB Adoptive cell therapies (ACT) using chimeric antigen receptor (CAR) T cells have shown promise in treating cancer, but manufacturing large numbers of high quality cells remains challenging. Critically, current T cell expansion technologies only partially recapitulate the in vivo microenvironment found in the human lymph nodes. In these organs, T cells expand at high cell density with autocrine/paracrine signaling, as well as signals from the extracellular matrix (ECM). Here we describe a T cell expansion system using degradable gelatin microcarriers functionalized with anti-CD3 and anti-CD28 monoclonal antibodies (mAbs), which address several of these shortcomings. We show that using this system, we can achieve approximately 2-fold greater expansion compared to functionalized magnetic beads, the current industry standard. Furthermore, carriers generated higher numbers of CCR7+CD62L+ migratory, central memory T cells and CD4+ T cells across multiple donors. Both these phenotypes have emerged as important for establishing durable and effective responses in patients receiving T cell immunotherapies. We further demonstrate that carriers can achieve greater memory cell yield compared to beads across a range of IL2 concentrations from 20 U/mL to 100 U/mL. These differences were greater at lower IL2 concentrations, indicating that the carriers are more efficient. We optimized this system using a design of experiments (DOE) approach and found that the carrier concentration affects the memory cell yield in a quadratic manner, where high or low concentrations are detrimental to memory formation. Finally, we show that carriers do not hinder CAR transduction and can maintain the CD4 and memory phenotype advantages in CAR-transduced T cells.ACTadoptive cell therapies. 1AICAkaike information criteria. 10AO/PIacridine orange/propidium iodide. 10, 17APCantigen presenting cell. 2BCAbicinchoninic acid assay. 17BSAbovine serum albumin. 17, 18CARchimeric antigen receptor. 1, 3, 5, 12–14, 16, 18CHOChinese hamster ovary. 2CuGCultispher-G. 3, 4, 16CuSCultispher-S. 3, 4, 16DCdendritic cell. 2DOEdesign of experiments. 1, 7, 8, 14–16, 19ECMextracellular matrix. 1, 2GMPGood Manufacturing Practices. 2IgGimmunoglobulin G. 3, 4, 17mAbmonoclonal antibody. 1–4, 7, 10–19MACSmagnetic activated cell sorting. 5, 17MHCmajor histocompatibility complex. 2MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. 3PBMCperipheral blood mononuclear cell. 5, 17PBSphosphate buffered saline. 16PDMSpolydimethylsiloxane. 2SNBsulfo-NHS-biotin. 3, 4, 16STPstreptavidin. 3, 4, 16–18TCRT Cell Receptor. 14, 16TEGT cell engineered to express γdTCR. 16TILtumor infiltrating lymphocyte. 16VSTvirus-specific T cell. 16