Ex-vivo expanded human NK cells express activating receptors that mediate cytotoxicity of allogeneic and autologous cancer cell lines by direct recognition and antibody directed cellular cytotoxicity

Caroline J Voskens, Ryuko Watanabe, Sandra Rollins, Dario Campana, Kenichiro Hasumi, Dean L Mann, Caroline J Voskens, Ryuko Watanabe, Sandra Rollins, Dario Campana, Kenichiro Hasumi, Dean L Mann

Abstract

Background: The possibility that autologous NK cells could serve as an effective treatment modality for solid tumors has long been considered. However, implementation is hampered by (i) the small number of NK cells in peripheral blood, (ii) the difficulties associated with large-scale production of GMP compliant cytolytic NK cells, (iii) the need to activate the NK cells in order to induce NK cell mediated killing and (iv) the constraints imposed by autologous inhibitory receptor-ligand interactions. To address these issues, we determined (i) if large numbers of NK cells could be expanded from PBMC and GMP compliant cell fractions derived by elutriation, (ii) their ability to kill allogeneic and autologous tumor targets by direct cytotoxicity and by antibody-mediated cellular cytotoxicity and (iii) defined NK cell specific receptor-ligand interactions that mediate tumor target cell killing.

Methods: Human NK cells were expanded during 14 days. Expansion efficiency, NK receptor repertoire before and after expansion, expression of NK specific ligands, cytolytic activity against allogeneic and autologous tumor targets, with and without the addition of chimeric EGFR monoclonal antibody, were investigated.

Results: Cell expansion shifted the NK cell receptor repertoire towards activation and resulted in cytotoxicity against various allogeneic tumor cell lines and autologous gastric cancer cells, while sparing normal PBMC. Blocking studies confirmed that autologous cytotoxicity is established through multiple activating receptor-ligand interactions. Importantly, expanded NK cells also mediated ADCC in an autologous and allogeneic setting by antibodies that are currently being used to treat patients with select solid tumors.

Conclusion: These data demonstrate that large numbers of cytolytic NK cells can be generated from PBMC and lymphocyte-enriched fractions obtained by GMP compliant counter current elutriation from PBMC, establishing the preclinical evidence necessary to support clinical trials utilizing autologous expanded NK cells, both directly and in combination with monoclonal antibodies in future cell-based immunotherapy in select solid tumors.

Figures

Figure 1
Figure 1
Cytolytic NK cells are efficiently expanded from PBMC. In the presence of K562-IL15-41BBL (A) expanded cells become significantly enriched (P = 0.0307) in NK cells (defined by CD56+CD3- cells) after 14 days of culture. Expanded cells were evaluated for cytolytic activity using 4 hour 51Cr release assays. Ex-vivo expanded cells from PBMC (■ donor 1 and △ donor 2), but not freshly purified non-expanded NK cells (◇), efficiently lysed allogeneic tumor cell lines derived from breast (MCF-7) and prostate (LNCaP) cancers but not allogeneic or autologous PBMC derived from donor 1 (B). The mean percentage cytotoxicity is shown from triplicate wells. Error bars represent the SD. Lytic activity is likely mediated by NK cells in the expanded cell population (○) since separation in individual populations of NK cells (◇) and NKT/T cells (△) resulted in allogeneic cytolytic activity of the expanded cell population and the purified NK cell population. Little lytic activity was observed in the presence of NKT/T cells alone (C). The mean percentage cytotoxicity is shown from triplicate wells from one representative experiment. Error bars represent the SD. Experiment shown represents one of three individual experiments with three different donors.
Figure 2
Figure 2
Ex-vivo expanded NK cells recognize autologous gastric tumor cells through different activating receptor-ligand interactions. PBMC from two gastric cancer patients were ex-vivo expanded for 14 days and then tested for cytolytic activity against autologous gastric tumor cells in 4 hour 51Cr release assays. (A) Ex-vivo expanded cells from both patients (patient 1 and patient 2), but not freshly purified non-expanded NK cells from patient 2, efficiently lysed autologous gastric tumor cells (Effector to Target Ratio 20:1). To evaluate the impact of activating receptor-ligand interactions on autologous tumor cell lysis indicated blocking antibodies (10 μg/ml) were added during 4 hours of incubation. (B) Cytotoxicity was reduced in the presence of DNAM-1 (P = 0.0309) and NKp30 (P = 0.0056) for patient 1 and in the presence of NKp46 (P = 0.0003) for patient 2. In both patients autologous cytolytic activity was abrogated in the presence of all four blocking antibodies with P = 0.0111 and P = 0.0001, respectively. Statistical analysis is based on triplicate wells of four (patient 1) and two (patient 2) experiments performed, respectively. Error bars represent the SD. * P < 0.05. MoIgG1 indicates mouse IgG1.
Figure 3
Figure 3
Cetuximab significantly enhances cytolytic activity and ADCC is negatively affected by inhibition of activating receptor-ligand interactions. Ex-vivo expanded cells from cancer patient 1 were evaluated for their ability to mediate ADCC against autologous (patient 1) and allogeneic (TU-167 and H-358) EGFR expressing lung cancer cells. (A) Cytolytic activity of ex-vivo expanded cells was enhanced in the presence of Cetuximab (10 μg/ml, black bar) but not in the presence of control human IgG1 (10 μg/ml; dotted bar) or media alone (white bar). The mean percentage cytotoxicity is shown from triplicate wells from one representative experiment. Error bars represent the SD. Experiment shown represents one of two individual experiments. (B) The addition of blocking antibodies (10 μg/ml) against DNAM-1, NKp46, NKp44 and NKp30 (= all) significantly reduced (P = 0.0176) Cetuximab-mediated ADCC. Statistical analysis is based on three experiments performed. Error bars represent the SD. * P < 0.05. HuIgG1 indicates human IgG1, Ctx; Cetuximab and moIgG1; mouse IgG1.
Figure 4
Figure 4
Distribution of lineage-specific phenotypic markers on PBMC and separate cell fractions obtained after counter current elutriation. PBMC and elutriated cell fractions were stained with various lineage-specific directly-conjugated antibodies and analyzed by flow cytometry (A). Average number of cells and phenotypic distribution (%) expressing lineage-markers in elutriated cell fractions (n = 11) (B).
Figure 5
Figure 5
Ex-vivo expanded cells from elutriated cell fractions efficiently lyse allogeneic prostate cancer cells. PBMC and elutriated fractions 2, 3 and 4 from the same healthy individual were expanded ex-vivo in the presence of K562-mbIL15-41BBL and IL-2 for 14 days and then tested for in vitro cytolytic activity. Cytolytic activity was evaluated in 4 hour 51Cr release assays against (A) prostate cancer (DU-145, PC-3 and LNCaP) cells. Ex-vivo expanded cells from elutriated cell fractions 2 (◇), 3 (△) and 4 (□) lysed prostate cancer cells in a similar fashion as ex-vivo expanded cells from PBMC (○). (B) Elutriated cell fractions become enriched in NK cells (defined by CD56+CD3- cells) after 14 days of culture regardless the cellular content of these fractions. The mean percentage cytotoxicity is shown from triplicate wells from one representative experiment. Bars represent the SD. Experiment shown represents one of four individual experiments.

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