Volume 2 Supplement 3
A clinical grade cell-based artificial APT, aAPC/mOKT3, for unbiased expansion of CD3+ T lymphocytes
© Sotov et al.; licensee BioMed Central Ltd. 2014
Published: 6 November 2014
Recent clinical results confirm that adoptive cell therapy, whereby expanded antitumor lymphocytes are infused into cancer patients, is a promising new therapy. Standardized methods to reproducibly and efficiently expand high quality antitumor lymphocytes in vitro are needed. We and others have successfully used cell-based artificial APCs (aAPCs) in the clinic as an off-the-shelf, standardized, and renewable reagent to reliably expand antitumor T cells in vitro for adoptive therapy. Recently, we have reported a genetically engineered novel human cell-based aAPC, aAPC/mOKT3, which expresses a membranous form of anti-CD3 mAb (mOKT3) in conjunction with immunostimulatory molecules, CD80 and CD83. Without requiring allogeneic feeder cells, aAPC/mOKT3 induces the robust expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction, but not Foxp3+ regulatory T cells or NK cells. Expanded T cells predominantly secreted Th1-type cytokines such as interferon-γ and IL-2. Unlike anti-CD3/CD28 mAb-coated beads, aAPC/mOKT3 enabled significantly improved CD8+ T cell expansion in part through IL-21 secreted by cocultured CD4+ T cells. To generate a clinical grade version of this aAPC, the parental cell line K562 was cotransfected with five linearized DNA plasmids encoding the light and heavy chains of mOKT3, CD80, CD83, and a puromycin N-acetyl-transferase gene. Simultaneous transfection of the 5 plasmids and subsequent drug selection resulted in aAPC/mOKT3 lines with >20% triple positivity (mOKT3, CD80, and CD83). Using a limiting dilution method, >100 candidate clones were established. Based on high expression of all 3 surface molecules, 41 clones were selected for long-term culture and monitoring for stable, high triple expression. After 3 months of continuous culture, 9 clones demonstrated stable expression and had suitable doubling time (24-40 hours). As seen with research grade aAPC/mOKT3, all clones induced the preferential expansion of CD8+ T cells in the presence of CD4+ T cells. Two clones, which consistently induced superior CD8+ T cell proliferation (>1,000 fold within 4 weeks), are lead candidates for selection to generate a master cell bank of clinical grade aAPC/mOKT3. Further criteria for selection include the ability of clones to expand T cells with a non-exhausted, young phenotype without contaminating immunosuppressive cell populations. Ability of expanded T cells to secrete Th1 cytokines and not immunosuppressive cytokines such as IL-10 or TGF-β will be confirmed. Once these data are obtained, a single clone will be chosen to generate a master cell bank and clinical lots for use in future clinical trials.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.