In vitro potency, in vitro and in vivo efficacy of liposomal alendronate in combination with γδ T cell immunotherapy in mice

Abstract

Nitrogen-containing bisphosphonates (N-BP), including zoledronic acid (ZOL) and alendronate (ALD), have been proposed as sensitisers in γδ T cell immunotherapy in pre-clinical and clinical studies. Therapeutic efficacy of N-BPs is hampered by their rapid renal excretion and high affinity for bone. Liposomal formulations of N-BP have been proposed to improve accumulation in solid tumours. Liposomal ALD (L-ALD) has been suggested as a suitable alternative to liposomal ZOL (L-ZOL), due to unexpected mice death experienced in pre-clinical studies with the latter. Only one study so far has proven the therapeutic efficacy of L-ALD, in combination with γδ T cell immunotherapy, after intraperitoneal administration of γδ T cell resulting in delayed growth of ovarian cancer in mice. This study aims to assess the in vitro efficacy of L-ALD, in combination with γδ T cell immunotherapy, in a range of cancerous cell lines, using L-ZOL as a comparator. The therapeutic efficacy was tested in a pseudo-metastatic lung mouse model, following intravenous injection of γδ T cell, L-ALD or the combination. In vivo biocompatibility and organ biodistribution studies of L-N-BPs were undertaken simultaneously. Higher concentrations of L-ALD (40–60 μM) than L-ZOL (3–10 μM) were required to produce a comparative reduction in cell viability in vitro, when used in combination with γδ T cells. Significant inhibition of tumour growth was observed after treatment with both L-ALD and γδ T cells in pseudo-metastatic lung melanoma tumour-bearing mice after tail vein injection of both treatments, suggesting that therapeutically relevant concentrations of L-ALD and γδ T cell could be achieved in the tumour sites, resulting in significant delay in tumour growth.

Organ Biodistribution of Radiolabelled γδ T Cells Following Liposomal Alendronate Administration in Different Mouse Tumour Models

“…Using an ovarian tumour model established by intraperitoneal (IP) inoculation, liposomal alendronate (L-ALD) has been shown to be more effective at slowing tumour growth than ALD when administered intravenously in combination with Vγ9Vδ2 T cells that were injected into the peritoneal cavity of mice [9]. Additionally, we have recently reported that only the combinatory treatment of L-ALD and γδ T cells led to a significant reduction in tumour growth in the experimental metastatic lung melanoma model, after 3 successive intravenous injections [20,21].…”

“…Lipid stock solutions were prepared in chloroform/methanol (4:1 v/v) at concentrations of 20-40 mg/ml and were stored under nitrogen at -20°C to avoid degradation. Alendronic acid liposomes (L-ALD) were prepared by the thin film hydration (TFH) method as previously described [20,21,25]. DSPC, cholesterol and DSPE-PEG2000 (55:40:5 molar volume) were added to a 25 ml round-bottom flask and 2 ml chloroform/ methanol (4:1 v/v) was added.…”

“…The γδ T cells were isolated and expanded as previously described [20,21]. Blood samples of 20-30 ml were obtained from healthy volunteers in an ethically approved protocol ("Use of Donor Blood Samples for Pre-Clinical Development of Active and Passive Immunotherapy for Cancer"; Ref.09/H0804/ 92).…”

“…Liposomes containing alendronate (L-ALD) were prepared and characterised for their size, charge and drug loading as previously described [20,21]. Compositions are DSPC, cholesterol, DSPE-PEG2000 and DSPE-DTPA (54:40:5:1 molar volume).…”

“…Compositions are DSPC, cholesterol, DSPE-PEG2000 and DSPE-DTPA (54:40:5:1 molar volume). Liposomes were then radiolabelled with 111 In using protocols as described previously [20,21]. In brief, the required volume of 111 In, containing 1 MBq per mouse for gamma counting studies, was added to 2 M ammonium acetate buffer (one-ninth of the reaction volume, pH 5.5).…”