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

Human blood samples were purchased (BRT Laboratories, Baltimore, MD) and whole peripheral blood mononuclear cells (PBMC) were isolated using density-gradient centrifugation. Using leukapheresis products purchased from the same source, the constitutive cell populations were fractionated by continuous-counterflow elutriation following protocols established by the manufacturer of cell separator (Elutra, Gambro BCT). This instrument uses continuous counter-flow elutriation technology to separate cells fractions based primarily by size and secondarily by specific gravity. In brief, the leukapheresis product was loaded via an inlet pump into a constantly rotating (2400 rpm) elutriation chamber. Based on centrifuge speed and cell density, five elutriated cell fractions were collected. PBMC and various elutriated cell fractions were viably frozen in RPMI-1640 (Invitrogen Corp., Grand Island, NY) supplemented with 20% human AB serum (Gemini Bio-Products, Woodland, CA) and 10% Dimethylsulfoxide (Sigma, St. Louis, MO) using an automated cell freezer (Gordinier Electronics, Roseville, MI) and stored in the vapor phase of liquid nitrogen until used.

The myeloid cell line K562, prostate cancer cell lines LNCaP, PC-3 and DU-145 and breast cancer cell line MCF-7 were available in our laboratory. The lung cancer cell line H358 was kindly provided by Dr. S. Ostrand-Rosenberg (Department of Biological Sciences, University of Maryland Baltimore County, Catonsville, MD) and the Head and Neck cancer cell line TU-167 was kindly provided by Dr. S. Strome (Department of Otorhinolaryngology-Head and Neck Surgery, University of Maryland, Baltimore, MD).

With patient consent and under approval of the Institutional Review Board, peripheral blood mononuclear cells were obtained from 2 patients with gastric cancer undergoing treatment at the Tokyo Clinic and Research Institute. Cell lines (tumor 1 and tumor 2) were established from biopsies of metastatic gastric tumor lesions from the respective patients. All tumor cell lines were cultured in RPMI 1640 supplemented with 10% Fetal Bovine Serum, 1% P/S and 1% Glutamax-1 (cRPMI).

NK cells were expanded from PBMC as previously described with some minor modifications [12]. In brief, PBMC (1.5 × 10⁶) were incubated with irradiated (14,000 rad) K562-mbIL15-41BBL cells (10⁶) in a 24-well tissue culture plate in the presence of 200 IU/ml human IL-2 (R&D Systems Inc) in cRPMI. Half of the culture medium was replaced every 2-3 days with fresh culture medium for the first 6 days. After 6 days of expansion, cells were harvested, washed, counted and re-cultured at a starting cell density of 1 × 10⁵-3 × 10⁵/ml in T-25 or T-75 culture flasks in cRPMI supplemented with IL-2. Cells were expanded for and additional 8 days. Additional cRPMI was added to the flasks if necessary based on cell density.

Cell surface expression was determined before and after 14 days of cell expansion by staining with directly conjugated mouse anti-human mAb's against CD3, CD56, αβTCR, γδTCR, HLA class I, HLA-DR, Fas, Fas-ligand, KLRD1, NKG2a, KIR3DL1, ILT2, CD62L, KIR3DL2/3, NKG2d, DNAM-1, NKp46, NKp44 and NKp30 (BD Biosciences). Gates were set around NK cells which were defined as CD3^-CD56⁺ cells. Surface expression of NK cell ligands was determined on both autologous gastric tumor cell lines and included directly conjugated mouse anti-human nectin-2, PVR, MIC A/B, Fas, HLA class I, HLA class II, HLA-G and purified mouse anti-human HLA-E, ULPB-1, ULBP-2 and ULBP-3. For EGFR-mediated ADCC, gastric tumors were stained with mouse anti-human EGFR mAb. Mouse IgGs were used as isotype controls and purified mAbs were secondarily stained with FITC labelled goat anti-mouse mAb. A minimum of 10000 events were acquired using a BD™ LSR II flow cytometer. Data was analyzed with BD™ FACS DIVA Software.

Cytolytic NK cell activity was measured by 4 hour chromium 51 (⁵¹Cr)-release assays as previously described [19]. K562 cells were included as target cells in all cytotoxicity assays to assess overall cytotoxicity performance (data not shown). Expanded day 14 cells were purified into separate populations of NK cells (CD3^-CD56⁺) and NKT/T (CD3⁺CD56⁺/CD3⁺CD56^-) cells using MACS human CD3 microbeads and non-expanded NK cells were purified from PBMC using a MACS human NK cell isolation kit. (Miltenyi Biotec Inc). Cell purity was determined to be >92% and 95% respectively. To determine ADCC, 10 μg/ml human IgG1 (huIgG1, Sigma-Aldrich Corp, St. Louis, MO, USA) or Cetuximab (University of Maryland Marlene and Stewart Greenebaum Cancer Center Pharmacy) was added to defined wells. Purified mouse IgG1, mouse anti-DNAM-1, NKp46, NKp44, NKp30 or all four together (all at 10 μg/ml) were added to defined wells during 4 hours of cytotoxicity in order to assess specific activating NK cell receptor-tumor ligand interactions. Reduction in cytotoxicity was calculated based on percentage cytotoxicity in the presence of indicate blocking mAb(s) versus percentage cytotoxicity in the presence of mouse control mAb. The % reduction in ADCC was calculated with percentage cytotoxicity in the presence of human IgG1 set at 100%. To minimize changes that may occur when cell lines are established from primary tumors, the gastric cell lines used in these studies were cultured for less than 10 passages after isolation from the primary tumor tissue.

Paired two-tailed Student's t tests were used to calculate p values. P < 0.05 was considered to be significant.

To achieve large-scale expansion of human NK cells, PBMC were co-cultured in a 1 to 1.5 ratio with lethally irradiated K562 cells expressing membrane-bound IL-15 and 4-1BBLigand (K562-mbIL15-4-1BBL) in culture media containing 200 units IL2/ml. After 14 days of culture, NK cells (CD56⁺CD3^- as defined by flow cytometry) expanded greater than 2 orders of magnitude from PBMC (mean 165 fold; range 4-567 fold, n = 6) and cell products became significantly enriched in NK cells (day 0 with mean 7%, range 3.2%-12.6% versus day 14 with mean 45.6%, range 7.4%-76.4%; P = 0.0140). At the same time, NKT cells (CD56⁺CD3⁺ as defined by flow cytometry) expanded at an average of 57 fold (range 7-234), although no significant enrichment (day 0 with mean 3.8%, range 0.8%-8.1% versus day 14 with mean 11.4%, range 2.3%-17.9%; P = 0.1907) was observed. In contrast, a significant decrease in T cells (CD3⁺ as defined by flow cytometry) was noted after 14 days of expansion (day 0 with mean 54.5%, range 39.9%-71.2% versus day 14 with mean 30.0%, range 4.2%-58.4%; P = 0.0436) with an absolute expansion of 7 fold (range 2-19). The distribution of NK cells and NKT cells in PBMC after expansion is shown in Figure 1A.

Importantly, ex-vivo expanded NK cells from healthy donor PBMC efficiently lysed allogeneic breast-and prostate-derived tumor targets but not allogeneic or autologous PBMC (Figure 1B). We did observe that cytotoxicity was associated with overall expansion efficiency. Specifically, the one donor whose cells expanded 4 fold after 14 days of culture demonstrated an average of 11.7% cytotoxicity (effector to target ratio 1:10) against K562 cells whereas donors who expanded an average of 202 fold (range 34-576; n = 4) possessed an average of 59.8% cytotoxicity (range 56.0%-65.9%; n = 4) against K562 cells (data not shown).

Based on CD3 and/or CD56 phenotype, the majority of cells in the expanded cell products represented NK cells while a much smaller proportion represented NKT and T cells (Table 1). To determine if both the NK cells and NKT/T cells mediated cytolytic activity, the two populations were isolated by immunomagnetic bead selection and killing assays against prostate-derived tumor cell targets were performed. Cytolytic activity was mediated by NK cells and not NKT cells (Figure 1C). Interestingly, little to no killing was observed with the NKT/T cell population even though a subpopulation of the T cells was confirmed to be γδ-TCR⁺ by flow cytometry (data not shown). Although γδ-TCR⁺ T cells are reported to have lytic activity against allogeneic tumor cells, they first require in vitro activation with isopentenyl pyrophosphate (IPP) and IL-2 [20]. Studies are underway to determine if addition of IPP will expand a cytolytic γδ-TCR⁺ population.

The capacity of K562-mb15-41BBL to stimulate expansion of NK cells from peripheral blood of healthy individuals and children with leukemia in remission was previously demonstrated [12, 17]. However, there is little information in reference to expand NK cells from PBMC derived from patients with solid tumors. With informed consent, NK cells were expanded from PBMC derived from patients with various solid tumors, including gastric cancer, lung cancer, colon cancer and hepatocellular cancer. After 14 days of culture, cell products became significantly enriched in NK cells (day 0 with mean 23.5%; range 5%-46% versus day 14 with mean 80%; range 60%-95%, n = 6, P = 0.0001 data not shown). Expansion efficiency was comparable between PBMC derived from solid tumor patients versus healthy donor PBMC (mean 316 fold; range 1-1795 with n = 6 versus mean 165 fold; range 4-567 with n = 6, P = 0.6685).

These data suggest that NK cells are efficiently expanded from PBMC from normal individuals and more importantly, from patients with various solid tumors without the need of primary enrichment protocols.

Human NK cells maintain self tolerance by the expression of at least one inhibitory receptor specific for autologous HLA class I which prevents cytotoxicity against autologous cells [21]. To establish cytotoxicity against autologous target cells, inhibitory signals must be overcome, either by (i) down-regulation of inhibitory ligands on the tumor cell, (ii) enhanced expression of activating receptors on NK cells, (iii) expression of ligands on the tumor target that activate the NK cell or (iv) a combination of thereof. Since NK cell activation is affected by cytokines such as IL-2 and IL-15 [22], we sought to determine if NK cells expanded from PBMC were phenotypically different from non-expanded NK cells (Table 2).

In expanded NK cells from normal individuals, no significant change was observed in inhibitory receptors KIR3DL1 (P = 0.1526), KIR3DL2/3 (P = 0.7858) and the activating receptor NKG2D (P = 0.1074). In contrast, activating receptors DNAM-1 (P = 0.0061), NKp46 (P = 0.0161), NKp44 (P = 0.0039) and NKp30 (P = 0.0131) were significantly increased in expression after 14 days of expansion. Interestingly, KLRD1 (P = 0.0012) and NKG2A (P = 0.0118), which both form a complex with the inhibitory non-classical HLA class I ligand HLA-E [23, 24] were also significantly increased after expansion. Importantly, expression of the inhibitory receptor ILT2 (P = 0.0142) which recognizes multiple HLA class I alleles, including non-classical HLA class I, HLA-G [25], was significantly decreased after expansion.

In order to define the ability of expanded NK cells derived from patients with solid tumors to kill their autologous tumors, tumor cell lines were established from tumor biopsies from two metastatic gastric cancer patients undergoing immunotherapy at the Tokyo Clinic and Research Institute. Of note, the expression of inhibitory and activating receptors on expanded NK cells from the gastric cancer donors were generally not different from expression on expanded NK cells from normal donors (Table 2).

Since autologous NK cell cytotoxicity is the net result of engagement of activating NK cell receptors with activating target cell ligands, the two gastric tumor cell lines were first phenotypically characterized for expression of ligands (Table 3) that are known to engage the NK cell receptors identified in table 2. While the ligands for human NKp46, NKp44 and NKp30 are to be defined, both patient cell lines expressed high levels of the inhibitory ligands HLA class I (75% and 67%, respectively) and HLA-G (42% and 57%, respectively) and relatively small amounts of the activation ligands MHC class I chain-related (MIC) A/B (2% and 1%, respectively), UL16 binding protein (ULBP)-1 (both 3%), ULBP-3 (both 3%) and polio-virus receptor (PVR; 8% and 9%, respectively). Importantly, both cell lines expressed the activating ligand nectin-2 (both 92%; specific for DNAM-1) which prompted us to evaluate both cell lines for their sensitivity against autologous NK cells.

Subsequent 4 hour chromium-release (⁵¹Cr-release) assays confirmed that gastric tumor cells derived from both patients were killed by autologous expanded NK cells (Figure 2A) and not by resting (non-expanded) NK cells from patient 2. Unfortunately, insufficient numbers of PBMC from patient 1 were available to isolate and test resting NK cells.

Since expanded NK cells significantly up-regulated DNAM-1, NKp46, NKp44 and NKp30, we performed blocking studies in order to evaluate the importance of these activating receptor-ligand interactions in autologous tumor cell recognition (Figure 2B). As expected, autologous lytic activity was significantly reduced (P = 0.0111 for patient 1 and P = 0.0001 for patient 2) when activating receptor-ligand interactions were interrupted by all four blocking antibodies (mAbs). Specifically, lytic activity of autologous NK cells from patient 1 was significantly reduced in the presence of mAb against DNAM-1 (P = 0.0309) or NKp30 (P = 0.0056) while lytic activity of autologous NK cells from patient 2 was only affected in the presence of mAb against NKp46 (P = 0.003).

Over many years, it has been postulated that eradication of human tumors may best be accomplished by combining cancer treatments modalities [26, 27]. Monoclonal antibodies that react with cell surface structures expressed on cancer cells represent the most successful cancer immunotherapy to date. It is quite clear that their mechanism of action is, at least partially, due to NK cell-mediated ADCC [28]. Since expanded NK cells expressed high levels of CD16 (data not shown), an Fc receptor that mediates ADCC, we sought to determine if lytic activity against the gastric tumor cells could be enhanced in the presence of Cetuximab (Erbitux^®), a chimeric monoclonal antibody that reacts with the EGFR receptor and is used to treat patients with a variety of solid tumors [29].

Both gastric tumor cell lines were screened for EGFR and only one of the two patient tumor cell lines (patient 1) expressed EGFR (Table 2). Subsequent ⁵¹Cr-release assays confirmed that allogeneic and autologous cytolytic activity is greatly enhanced in the presence of chimeric anti-EGFR mAb but not in the presence of human IgG1 control antibody (Figure 3A). As expected, the enhancement in cytotoxicity was far more dramatic in the autologous setting if compared to the allogeneic setting since allogeneic tumor cells do not maintain self tolerance through specific inhibitory receptor-ligand interactions.

Importantly, the expression of activating receptors on the ex-vivo expanded NK cells positively affected overall cytotoxic activity (Figure 3B) since blocking all four activating receptors on the NK cell surface decreased autologous cytotoxicity if compared with control mAb (P = 0.0176 and P = 0.1019, respectively). These data suggest that the combined strategy of adoptively transferred ex-vivo expanded autologous NK cells with infusion of an mAb that is used for cancer immunotherapy may provide clinical benefit for the treatment of select human solid tumors. To extend these observations, we are attempting to establish cell lines from other solid tumors where PBMC would be available to test NK expansion and direct cytotoxicity and ADCC capability.

A GMP compliant system has successfully been established for the enrichment of monocytes from PBMC using an Elutra cell separator. In this closed system, PBMC are fractionated by centrifugal elutriation and five cell fractions are obtained. In general, these fractions consist of platelets (fraction 1), erythrocytes mixed with lymphocytes (fraction 2), lymphocytes (fraction 3), lymphocytes mixed with monocytes (fraction 4) and mainly monocytes (fraction 5) as demonstrated in Figure 4 (n = 11). Current clinical cellular therapy protocols use monocytes obtained from elutriated fraction 5 to generate dendritic cells for cancer immunotherapy while the cells from fractions 2, 3 and 4 are usually "archived" in liquid nitrogen. As a means to facilitate clinical translation, we explored the possibility of these GMP compliant cell fractions to serve in future NK cell-based immunotherapy studies. PBMC and separate elutriated cell fractions were expanded with the aforementioned expansion strategy. After 14 days of culture in the presence of K562-mbIL15-41BBL cells and exogenous IL-2, NK cells expanded greater than two orders of magnitude from PBMC (mean 165 fold; range 4-567 fold with n = 6, data not shown), elutriated cell fraction 2 (mean 209 fold; range 3-615 fold with n = 3, data not shown), elutriated cell fraction 3 (mean 131 fold; range 4-339 fold with n = 3, data not shown) and elutriated cell fraction 4 (mean 91 fold; range no expansion-358 fold with n = 4, data not shown). Importantly, expanded cells from PBMC and separate elutriated cell fractions became significantly enriched in NK cells and lysed allogeneic prostate-derived tumor cell lines in a similar fashion (Figure 5A-B). Thus, these data show that large quantities of cytolytic NK cells can be expanded from various elutriated cell fractions collected with the GMP compliant Elutra system.