Characterization and ex vivo Expansion of Human Placenta-Derived Natural Killer Cells for Cancer Immunotherapy

Abstract

Recent clinical studies suggest that adoptive transfer of donor-derived natural killer (NK) cells may improve clinical outcome in hematological malignancies and some solid tumors by direct anti-tumor effects as well as by reduction of graft versus host disease (GVHD). NK cells have also been shown to enhance transplant engraftment during allogeneic hematopoietic stem cell transplantation (HSCT) for hematological malignancies. The limited ex vivo expansion potential of NK cells from peripheral blood (PB) or umbilical cord blood (UCB) has however restricted their therapeutic potential. Here we define methods to efficiently generate NK cells from donor-matched, full-term human placenta perfusate (termed Human Placenta-Derived Stem Cell, HPDSC) and UCB. Following isolation from cryopreserved donor-matched HPDSC and UCB units, CD56+CD3- placenta-derived NK cells, termed pNK cells, were expanded in culture for up to 3 weeks to yield an average of 1.2 billion cells per donor that were >80% CD56+CD3-, comparable to doses previously utilized in clinical applications. Ex vivo-expanded pNK cells exhibited a marked increase in anti-tumor cytolytic activity coinciding with the significantly increased expression of NKG2D, NKp46, and NKp44 (p < 0.001, p < 0.001, and p < 0.05, respectively). Strong cytolytic activity was observed against a wide range of tumor cell lines in vitro. pNK cells display a distinct microRNA (miRNA) expression profile, immunophenotype, and greater anti-tumor capacity in vitro compared to PB NK cells used in recent clinical trials. With further development, pNK may represent a novel and effective cellular immunotherapy for patients with high clinical needs and few other therapeutic options.

Keywords: anti-tumor cytolytic activity; cellular immunotherapy; ex vivo expansion; miRNA; placental-derived natural killer cells.

Figure 1

Comparison of NK cells from donor-matched HPDSC and UCB units. (A) Immunophenotypic characterization of NK cells from donor-matched HPDSC and UCB units, the two-sample t-test was used to determine if population means are equal in HPDSC and UCB. (B) Cytotoxicity of expanded NK cells from donor-matched HPDSC and UCB units against K562 cells.

Figure 2

Phenotypic characterization of pNK cells in comparison with PB NK cells. (A) Flow cytometric identification of NK cells from Combo unit and PB. CD56+CD3− gated NK cells expressed a repertoire of receptors important for regulating NK-cell activity, including CD16, KIR3DL1, NKG2D, KIR2DL2/L3, NKp46, CD94, CD226, NKp44, NKp30, and 2B4. (B) Percentage of CD56+CD3− pNK cells from Combo unit prior to NK cell isolation. (C) About 90% CD56+CD3− NK cells from Combo unit was achieved after NK cell isolation.

Figure 3

Cell cycle analysis of expanded pNK cells. (A) Representative APC-BrdU/7-AAD cell cycle analysis of ex vivo-expanded pNK at different time points as indicated. (B) Cell cycle analysis at different phases from ex vivo-expanded pNK at different time points as indicated (n = 5).

Figure 4

Expansion, phenotype and functional characterization of ex vivo-expanded pNK cells. (A) Cell yield and cell viability of Day 21 expanded pNK cells. (B) Phenotype characterization of Day 21 expanded pNK cells. (C) Cytotoxicity of expanded pNK cells against K562 at different time points as indicated in comparison with Day 21 expanded PB NK cells.

Figure 5

Cytotoxicity of ex vivo-expanded Day 21 pNK cells against a wide range of tumor cell lines. Cytotoxicity of ex vivo-expanded pNK cells (n = 6) against a wide range of tumor cell lines at E:T ratio of 10:1, 5:1, 2:1, and 1:1 as indicated.