Bisphosphonate-Based γδ T Cell Expansion

Owing to their repertoire of activating receptors, γδ-T cells have been reported to kill a wide variety of cancer cell lines in vitro such as leukemia, melanoma, lymphomas and other carcinomas. The growing interest in the use of γδ-T cells as an alternative form of immunotherapy has been fueled. Future clinical trials involving Vγ9Vδ2 T cells would explore the combinatorial use with other therapeutic agents, such as CAR and immune checkpoint inhibitors. Creative Biolabs provides comprehensive bisphosphonate-based γδ T cell expansion services to boost our clients’ projects to clinical research.

Introduction to Bisphosphonate

Bisphosphonates (BPs) are a class of drugs successfully used to treat a wide variety of diseases characterized by excessive osteoclast-mediated bone resorption, such as tumor-associated osteolysis, Paget’s disease of bone, and post-menopausal osteoporosis. BPs consist of a common geminal carbon atom linked to two phosphonate groups, to yield a P-C-P structure and, as such, BPs are considered to be analogs of naturally occurring pyrophosphate (P-O-P). The two phosphonate groups present in BPs also endow these compounds with a high affinity for divalent ions, such as calcium. Due to the abundance of calcium in bone mineral, one of the key pharmacological features of these drugs is rapid sequestration by the skeleton. Recently, N-BPs were thought to activate Vγ9Vδ2 T cells by acting as agonists for the Vγ9Vδ2-TCR.

Bisphosphonate and γδ T Cell

The intrinsic anti-tumor activity of γδ cells, coupled with their MHC-independent modus operandi, makes them ideal allogenic off-the-shelf effector cells for cancer immunotherapy. The bottleneck had been the large-scale expansion of these scarce cells and the BPs-based approach is one of the economical methods to achieve this.

Fig.1 Activation of γδ T cells by bisphosphonates. (Thompson, 2010)

• Bisphosphonate-based γδ T cell activation

It is now apparent that nitrogen-BPs (N-BPs) indirectly activate Vγ9Vδ2 T cells through the intracellular inhibition of the molecular target of the N-BPs, FPP synthase. Farnesyl diphosphate (FPP) synthase inhibition, as well as causing a depletion of the downstream metabolites FPP and geranylgeranyl diphosphate (GGPP) (and the consequent inhibition of protein prenylation), also induces the accumulation of the substrates of this enzyme, isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are both agonists for the Vγ9Vδ2-TCR. Recent reports demonstrated that the stimulatory effect of Zoledronate (ZOL) on Vγ9Vδ2 T cell activation by tumor cells was caused by the accumulation of these upstream mevalonate pathway intermediates in the tumor cells. Another parallel study using peripheral blood mononuclear cell (PBMC) cultures, revealed that the stimulatory capacity of N-BPs for inducingVγ9Vδ2 T cell activation was determined by the potency of the N-BP for inhibiting FPP synthase. Furthermore, N-BP-induced Vγ9Vδ2 T cell activation could be blocked by a statin, which inhibits the upstream enzyme HMG-CoA reductase, through a mechanism most likely involving inhibition of IPP/DMAPP synthesis.

• Bisphosphonate-based γδ T cell Expansion

The infrequent nature of Vγ9Vδ2 T cells necessitates a large starting number of PBMC to generate adequate effector Vγ9Vδ2 T cells for ACT. In order to develop off-the-shelf cancer immunotherapy, a larger expansion rate of the Vγ9Vδ2 T cell is needed to streamline the process and lower the cost of production. Cytokine-based approaches for expansion of γδ-T cells have thus far revolved around the combinatorial use of both IL-2 and ZOL or the synthetic phosphoantigen bromohydrin pyrophosphate (BrHPP). Nitrogen-containing bisphosphonates (N-BPs) such as ZOL inhibit the FPP synthase enzyme through competitive inhibition. The addition of ZOL or BrHPP to PBMC culture blocks FPP synthase activity and causes an accumulation of IPP in monocytes12 and MHC Class II⁺ cells, which collectively activates Vγ9Vδ2 T cells through cell-to-cell interactions. In these cytokine-based approaches, the length of culture was often kept between 10-14 days. Vγ9Vδ2 T cells harvested from day 12 to day 14 might be most optimal for therapy as cytotoxic activity peaked on day 12 and declined by day 14. Prolonged exposure of Vγ9Vδ2 T cells to phosphoantigens beyond 14 days caused activation-induced cell death (AICD). Therefore, lengthening the culture period in pursuit of increased Vγ9Vδ2 T cell expansion runs the risk of producing exhausted Vγ9Vδ2 T cells with lower cytotoxic activity.

Services of Bisphosphonate-based γδ T cell Expansion at Creative Biolabs

Focus on cellular immunotherapies for years, Creative Biolabs has extensive experience in bisphosphonate-based γδ T cell expansion services. In order to provide global customers with perfect services, we have established an experienced team with a number of Ph.D. level scientists specialized in cellular immunotherapies. We will tailor suitable experimental programs for our customers.

Please feel free to contact us for detailed information.

Reference

1. Thompson, K.; et al. Activation of γδ t cells by bisphosphonates. Advances in experimental medicine and biology. 2010, 658: 11-20.