Expansion and Enrichment of Gamma-Delta (γδ) T Cells from Apheresed Human Product

Abstract

Although Vγ9Vδ2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (γδ) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure γδ T cells drug product for first-in-human use of allogeneic γδ T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density.The lymphocyte-rich fraction is utilized, and the γδ T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of γδ T cells, a clinical-grade magnetic cell-separation device and TCRαβ beads are used to deplete contaminating T-cell receptor (TCR)αβ T cells. The highly enriched γδ T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cellsgenetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched γδ T cells in co-culture with the aAPCs facilitates the manufacture of highly pure γδ T cells with an average fold expansion of >229,000-fold from healthy donor blood.

γδ-Enriched CAR-T cell therapy for bone metastatic castrate-resistant prostate cancer

“…Because the Vγ9Vδ2 TCR is activated by accumulation of phosphoantigens induced by bisphosphonates such as ZOL ( 19 ), we tested whether γδ CAR-T cells maintained the functionality of their endogenous TCR. To this end, we monitored tumor cell viability over time following exposure to γδ CAR-T cells or control UT γδ T cells in the presence or absence of 4 μM ZOL, the concentration used for γδ T cell expansion ex vivo ( 18 , 29 ). C4-2B PSCA + and 22Rv1 PSCA + cells were used as target cells, as they both express CD277 (BTN3A1/2/3) (fig.…”

“…Expansion from PBMCs was performed as previously described ( 18 ). Briefly, a total of 48 × 10 6 cells were plated in 24-well plates at a concentration of 1 × 10 6 cells/ml with 4 μM ZOL ( 18 , 29 ). After 3 days, the medium was replaced with new complete RPMI 1640 medium with 4 μM ZOL.…”

Ex vivo assays show human gamma-delta T cells specific for common allergens are Th1-polarized in allergic donors

“…This is of importance as current methodologies employed in functional studies of γδ T cells rely exclusively on long-term in vitro expansion and physiological manipulation. 53 , 54 , 55 …”

Vγ9Vδ2 T-cell immunotherapy in blood cancers: ready for prime time?

“…The use of feeder cells is another workable tool to improve the efficacy of in vitro Vg9Vd2 T-cell expansion protocols (117)(118)(119)(120). Side-by-side comparison of ZA + IL-2 versus K562-based artificial antigen-presenting cells (aAPCs) has shown in mouse models that the latter induces Vg9Vd2 T cells with stronger antitumor activity and enhanced capacity to survive in vivo (118).…”