Phase I clinical trial of autologous NK cell therapy using novel expansion method in patients with advanced digestive cancer

Abstract

Background: NK cells can destroy tumor cells without prior sensitization or immunization. Tumors often lose expression of MHC molecules and/or antigens. However, NK cells can lyse tumor cells in a non-MHC-restricted manner and independent of the expression of tumor-associated antigens. NK cells are therefore considered ideal for adoptive cancer immunotherapy; however the difficulty of obtaining large numbers of fully functional NK cells that are safe to administer deters its clinical use. This phase I clinical trial seeks to address this obstacle by first developing a novel system that expands large numbers of highly activated clinical grade NK cells, and second, determining if these cells are safe in a mono-treatment so they can be combined with other reagents in the next round of clinical trials.

Methods: Patients with unresectable, locally advanced and/or metastatic digestive cancer who did not succeed with standard therapy were enrolled. NK cells were expanded ex vivo by stimulating PBMCs with OK432, IL-2, and modified FN-CH296 induced T cells. Patients were administered autologous natural killer cell three times weekly via intravenous infusions in a dose-escalating manner (dose 0.5 × 10(9), 1.0 × 10(9), 2.0 × 10(9) cells/injection, three patients/one cohort).

Results: Total cell population had a median expansion of 586-fold (range 95-1102), with a significantly pure (90.96 %) NK cell population. Consequently, NK cells were expanded to approximately 4720-fold (range 1372-14,116) with cells being highly lytic in vitro and strongly expressing functional markers such as NKG2D and CD16. This NK cell therapy was very well tolerated with no severe adverse events. Although no clinical responses were observed, cytotoxicity of peripheral blood was elevated approximately twofolds up to 4 weeks post the last transfer.

Conclusion: We successfully generated large numbers of activated NK cells from small quantities of blood without prior purification of the cells. We also determined that the expanded cells were safe to administer in a monotherapy and are suitable for the next round of clinical trials where their efficacy will be tested combined with other reagents.

Trial registration: UMIN UMIN000007527.

Fig. 1

Treatment protocol. PBMCs were separated to prepare RN-T as stimulator cells. One week later, PBMCs were again separated similarly, and re-suspended in culture medium supplemented with heat-inactivated autologous plasma, IL-2 and OK-432. RN-T cells were added to the same flask or culture bag on day 0 and day 7. On days 21–22, the cultured cells were harvested and administered to the patients immediately. Expanded NK cells were intravenously injected for 60 min on days 0, 7, 14 in a dose-escalating manner (dose 0.5 × 10⁹, 1 × 10⁹, 2 × 10⁹ cells/injection, three patients/one cohort). We investigated the dose-limiting toxicity (DLT) occurring over a 28-day period after the last administration of cultured cells. Blood samplings for immune monitoring were done just before the 1st and 3rd administration and 4 weeks after the 3rd administration. PBMCs peripheral blood mononuclear cells, RN-T cells RetroNectin^®-induced T cells

Fig. 2

Expansion and NK purity dynamics of PBMCs obtained from 14 patients. a Fold expansion of total cell and NK cell population (CD3⁻CD56⁺ cell) during the culture period. Dots represent mean values of each patient in triplicate cultures. Horizontal bars indicate median values. b Relation of NK cell expansion fold and cytotoxic activity of PBMCs. Expansion fold of NK cells in the first culture significantly correlated with cytotoxic activity of PBMCs at baseline (ρ = 0.661, p = 0.044). PBMCs peripheral blood mononuclear cells. c The purity of NK cell population (CD3⁻CD56⁺ cell) at baseline and 21 or 22 days after the initiation of the culture

Fig. 3

Cytotoxic activity of expanded NK cells against K-562 cells. 4 h cytotoxicity from 14 patients against K-562 target cells. Mean cell death in each patient at the indicated E:T ratios in triplicate cultures

Fig. 4

Longitudinal plots of NK cell population in PBLs plotted according to their deviation from the baseline. Mean levels in all patients (a) and levels in each cohort (b) are shown. PBLs peripheral blood lymphocytes

Fig. 5

Longitudinal plots of cytotoxic activity of PBMCs against K-562 cells plotted according to the deviation from the baseline. Mean levels in all patients at indicated E:T ratios (a blue 20:1, red 40:1) and levels for the tumor responses at 40:1 E/T ratio (b) are shown. PBMCs peripheral blood mononuclear cells, SD stable disease, PD progressive disease