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

Sakamoto N, Ishikawa T, Kokura S, Okayama T, Oka K, Ideno M, Sakai F, Kato A, Tanabe M, Enoki T, Mineno J, Naito Y, Itoh Y, Yoshikawa T - J Transl Med (2015)

Bottom Line: Total cell population had a median expansion of 586-fold (range 95-1102), with a significantly pure (90.96 %) NK cell population.This NK cell therapy was very well tolerated with no severe adverse events.We successfully generated large numbers of activated NK cells from small quantities of blood without prior purification of the cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan. jrsaka@koto.kpu-m.ac.jp.

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.

No MeSH data available.


Related in: MedlinePlus

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
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Fig2: 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

Mentions: PBMCs from 14 enrolled patients were cultured. The median of NK cell (CD3−CD56+) in lymphocytes was 13.59 % (range 4.43–34.85). The median of total cell or NK cell expansion rate after 21 and 22 days of culture was 586-fold (range 95–1102) and 4720-fold (range 1372–14,116), respectively (Fig. 2a). The total cell expansion fold did not correlate with the percentage of NK cells in lymphocytes (ρ = 0.24, P = 0.40), but in the 1st culture it significantly correlated with the cytotoxicity activity of PBMCs on day 0 (ρ = 0.66, P = 0.04, Fig. 2b). As shown in Fig. 2c, the purity of expanded NK (CD3−CD56+) cells markedly increased after culture, with the exception of one patient (Pt no. 1). The median purity of NK cell (CD3−CD56+) was 90.96 % (range 65.94–99.45) in the intention-to treat (ITT) population and 96.14 % (range 65.94–99.45) in the per protocol (PP) population. As shown in Table 2 however, the percentage of CD3+CD56+, CD3+CD4+ and CD3+CD8+ cells was minimal (median 8.60, 3.50 and 0.24 %, respectively). Expanded NK cells highly expressed cell surface markers such as NKG2D and CD16, which are considered viable and functional markers of NK cells. In the ITT population, the median percentage of NKG2D+ and CD16+ cells in NK population was 98.36 % (range 95.20–99.59) and 61.75 % (range 20.84–73.50), respectively. We also found relatively high expression levels of chemokine receptors such as CXCR3, CXCR4 and CX3CR1 on expanded NK cells (median in ITT population, 45.47, 37.71, and 43.74 %, respectively). Additional file 2: Figure S1 shows representative flow cytometry dot-plots for each population of expanded cells in patient no. 14.Fig. 2


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

Sakamoto N, Ishikawa T, Kokura S, Okayama T, Oka K, Ideno M, Sakai F, Kato A, Tanabe M, Enoki T, Mineno J, Naito Y, Itoh Y, Yoshikawa T - J Transl Med (2015)

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
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4548900&req=5

Fig2: 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
Mentions: PBMCs from 14 enrolled patients were cultured. The median of NK cell (CD3−CD56+) in lymphocytes was 13.59 % (range 4.43–34.85). The median of total cell or NK cell expansion rate after 21 and 22 days of culture was 586-fold (range 95–1102) and 4720-fold (range 1372–14,116), respectively (Fig. 2a). The total cell expansion fold did not correlate with the percentage of NK cells in lymphocytes (ρ = 0.24, P = 0.40), but in the 1st culture it significantly correlated with the cytotoxicity activity of PBMCs on day 0 (ρ = 0.66, P = 0.04, Fig. 2b). As shown in Fig. 2c, the purity of expanded NK (CD3−CD56+) cells markedly increased after culture, with the exception of one patient (Pt no. 1). The median purity of NK cell (CD3−CD56+) was 90.96 % (range 65.94–99.45) in the intention-to treat (ITT) population and 96.14 % (range 65.94–99.45) in the per protocol (PP) population. As shown in Table 2 however, the percentage of CD3+CD56+, CD3+CD4+ and CD3+CD8+ cells was minimal (median 8.60, 3.50 and 0.24 %, respectively). Expanded NK cells highly expressed cell surface markers such as NKG2D and CD16, which are considered viable and functional markers of NK cells. In the ITT population, the median percentage of NKG2D+ and CD16+ cells in NK population was 98.36 % (range 95.20–99.59) and 61.75 % (range 20.84–73.50), respectively. We also found relatively high expression levels of chemokine receptors such as CXCR3, CXCR4 and CX3CR1 on expanded NK cells (median in ITT population, 45.47, 37.71, and 43.74 %, respectively). Additional file 2: Figure S1 shows representative flow cytometry dot-plots for each population of expanded cells in patient no. 14.Fig. 2

Bottom Line: Total cell population had a median expansion of 586-fold (range 95-1102), with a significantly pure (90.96 %) NK cell population.This NK cell therapy was very well tolerated with no severe adverse events.We successfully generated large numbers of activated NK cells from small quantities of blood without prior purification of the cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan. jrsaka@koto.kpu-m.ac.jp.

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.

No MeSH data available.


Related in: MedlinePlus