Abstract
γ9δ2T cells play a critical role in daily cancer immune surveillance by sensing cancer-mediated metabolic changes. However, a major limitation of the therapeutic application of γ9δ2T cells is their diversity and regulation through innate co-receptors. In order to overcome natural obstacles of γ9δ2T cells, we have developed the concept of T cells engineered to express a defined γδT cell receptor (TEGs). This next generation of chimeric antigen receptor engineered T (CAR-T) cells not only allows for targeting of hematological but also of solid tumors and, therefore, overcomes major limitations of many CAR-T and γδT cell strategies. Here, we report on the development of a robust manufacturing procedure of T cells engineered to express the high affinity Vγ9Vδ2T cell receptor (TCR) clone 5 (TEG001). We determined the best concentration of anti-CD3/CD28 activation and expansion beads, optimal virus titer, and cell density for retroviral transduction, and validated a Good Manufacturing Practice (GMP)-grade purification procedure by utilizing the CliniMACS system to deplete non- and poorly-engineered T cells. To the best of our knowledge, we have developed the very first GMP manufacturing procedure in which αβTCR depletion is used as a purification method, thereby delivering untouched clinical grade engineered immune cells. This enrichment method is applicable to any engineered T cell product with a reduced expression of endogenous αβTCRs. We report on release criteria and the stability of TEG001 drug substance and TEG001 drug product. The GMP-grade production procedure is now approved by Dutch authorities and allows TEG001 to be generated in cell numbers sufficient to treat patients within the approved clinical trial NTR6541. NTR6541 will investigate the safety and tolerability of TEG001 in patients with relapsed/refractory acute myeloid leukemia, high-risk myelodysplastic syndrome, and relapsed/refractory multiple myeloma.
Highlights
Chimeric antigen receptor engineered T (CAR-T) cells are currently entering clinical practice with remarkable response rates resulting in multiple FDA approvals in 2017 [1]
In order to define the best anti-CD3/28 bead to CD3+ T cell number for engineering TEGs, Peripheral blood mononuclear cells (PBMCs) were first analyzed for CD3+ T cell content by flow cytometry, and incubated with various ratios of anti-CD3/CD28 beads in the presence of the cytokines interleukin (IL)-7 and IL-15
We have developed a robust Good Manufacturing Practice (GMP)-grade TEG production protocol, which includes a conventional transduction and expansion step and a very stringent CliniMACS enrichment procedure to guarantee high purity of the drug substance
Summary
Chimeric antigen receptor engineered T (CAR-T) cells are currently entering clinical practice with remarkable response rates resulting in multiple FDA approvals in 2017 [1]. Major limitations of current clinical strategies are, that CAR-T cells rarely offer solutions to solid tumors Another restriction of current CAR-T approaches is that target antigens are often present on healthy. For clinical testing of the TEG concept, we recently selected a highly tumor reactive Vγ9Vδ2TCR clone (clone 5) from the natural repertoire of a healthy individual [2]. This particular Vγ9Vδ2TCR showed a strong reactivity toward a broad range of tumor cells within the TEG format, including primary leukemic blasts [8] as well as primary multiple myeloma cells [9]. Given that this process requires connecting two completely different worlds, a flexible research environment with a rigid GMP environment, the reported developmental process can be of high interest to researchers who aim at translating research findings to the clinic
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