Abstract
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.
Acronyms
- ACT
- adoptive cell therapies. 1
- AIC
- Akaike information criteria. 10
- AO/PI
- acridine orange/propidium iodide. 10, 17
- APC
- antigen presenting cell. 2
- BCA
- bicinchoninic acid assay. 17
- BSA
- bovine serum albumin. 17, 18
- CAR
- chimeric antigen receptor. 1, 3, 5, 12–14, 16, 18
- CHO
- Chinese hamster ovary. 2
- CuG
- Cultispher-G. 3, 4, 16
- CuS
- Cultispher-S. 3, 4, 16
- DC
- dendritic cell. 2
- DOE
- design of experiments. 1, 7, 8, 14–16, 19
- ECM
- extracellular matrix. 1, 2
- GMP
- Good Manufacturing Practices. 2
- IgG
- immunoglobulin G. 3, 4, 17
- mAb
- monoclonal antibody. 1–4, 7, 10–19
- MACS
- magnetic activated cell sorting. 5, 17
- MHC
- major histocompatibility complex. 2
- MTT
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. 3
- PBMC
- peripheral blood mononuclear cell. 5, 17
- PBS
- phosphate buffered saline. 16
- PDMS
- polydimethylsiloxane. 2
- SNB
- sulfo-NHS-biotin. 3, 4, 16
- STP
- streptavidin. 3, 4, 16–18
- TCR
- T Cell Receptor. 14, 16
- TEG
- T cell engineered to express γdTCR. 16
- TIL
- tumor infiltrating lymphocyte. 16
- VST
- virus-specific T cell. 16