Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells

Abstract

Natural killer (NK) cells hold promise for improving the therapeutic potential of allogeneic hematopoietic transplantation, but their effectiveness is limited by inhibitory HLA types. We sought to overcome this intrinsic resistance by transducing CD56+CD3- NK cells with chimeric receptors directed against CD19, a molecule widely expressed by malignant B cells. An abundance of NK cells for transduction was secured by culturing peripheral blood mononuclear cells with K562 cells expressing the NK-stimulatory molecules 4-1BB ligand and interleukin 15, which yielded a median greater than 1000-fold expansion of CD56+CD3- cells at 3 weeks of culture, without T-lymphocyte expansion. Expression of anti-CD19 receptors linked to CD3zeta overcame NK resistance and markedly enhanced NK-cell-mediated killing of leukemic cells. This result was significantly improved by adding the 4-1BB costimulatory molecule to the chimeric anti-CD19-CD3zeta receptor; the cytotoxicity produced by NK cells expressing this construct uniformly exceeded that of NK cells whose signaling receptors lacked 4-1BB, even when natural cytotoxicity was apparent. Addition of 4-1BB was also associated with increased cell activation and production of interferon gamma and granulocyte-macrophage colony-stimulating factor. Our findings indicate that enforced expression of signaling receptors by NK cells might circumvent inhibitory signals, providing a novel means to enhance the effectiveness of allogeneic stem cell transplantation.

Figure 1.

Expansion of NK cells after 1 week of culture with genetically modified K562 cells. Peripheral blood mononuclear cells from 7 healthy individuals (represented by different symbols) were cultured with various preparations of K562 at a 1:1 ratio in the presence of low-dose (10 U/mL) IL-2. Percentages of CD56⁺CD3^- NK cells and CD3⁺ T lymphocytes after 7 days of culture relative to the number of input cells are shown. Each data point represents the average of 2 measurements; bars correspond to the median expansion in each group. K562 cells expressing both membrane-bound IL-15 and 4-1BBL (K562-mb15-41BBL) induced a markedly superior expansion of NK cells (P < .001 by the Tukey honest significant difference test) without inducing T-cell proliferation; there were no significant differences among other pairwise comparisons of NK expansions obtained with K562, K562-41BBL, and K562-mb15.

Figure 2.

Immunophenotypic features of NK cells before and after expansion with K562-mb15-41BBL cells. Expression of CD3 and CD56, as well as expression of the KIRs CD158a (2DL1), CD158b (2DL2), NKB1 (3DL1), and NKAT2 (2DL3) on CD56⁺CD3^- cells were examined in peripheral blood mononuclear cells from a healthy donor before (top row) and after (bottom row) 3 weeks of coculture with K562-mb15-41BBL cells and low-dose (10 U/mL) IL-2.

Figure 3.

Schematic representation of the chimeric receptors used in this study. LTR indicates long terminal repeat; AMP, ampicillin resistance; and bp, base pair.

Figure 4.

Expression of chimeric receptors by NK cells expanded from peripheral blood mononuclear cells. (A) Surface receptor expression was visualized by flow cytometry after staining with a goat anti-mouse (Fab)₂ polyclonal antibody conjugated with biotin followed by streptavidin PerCP (y-axes); expression of GFP is also shown (x-axes). (B) Western blot analysis of chimeric receptor expression in NK cells, under reducing or nonreducing conditions. Filter membranes were labeled with an antihuman CD3ζ antibody and a goat anti-mouse IgG horseradish peroxidase-conjugated second antibody.

Figure 5.

Chimeric receptors bearing CD3ζ overcome the NK resistance of leukemic cells. The results shown for 3 cell lines, 380, RS4;11, and 697, are expressed as the mean (± SD; n = 4) percentage of leukemic cell recovery after 24 hours of culture at a 1:1 E/T ratio relative to cultures without NK cells, as measured by flow cytometry. NK cells expressing anti-CD19-ζ receptors were more cytotoxic than NK cells expressing anti-CD19-DAP10 receptors, anti-CD19 receptors without signaling capacity (anti-CD19-trunc), or NK cells transduced with GFP control vector (P < .001).

Figure 6.

Addition of the 4-1BB costimulatory molecule to the chimeric receptors augments their capacity to induce NK cytotoxicity against NK-resistant leukemic cells. (A) Expanded primary NK cells expressing chimeric receptors were incubated for 4 hours with the B-lineage ALL cell lines 380 and RS4;11 at the indicated E/T ratios. Each data point represents the mean (± SD; n = 4) percentage of ALL cell killing after culture as compared to that of parallel cultures without NK cells. At all E/T ratios, cytotoxicity of NK cells expressing chimeric receptors containing 4-1BB was significantly higher than that induced by receptors without 4-1BB (P < .001). (B) Flow cytometric dot plots show staining with anti-CD56 and anti-CD22 after a 4-hour coculture of NK cells (CD56⁺) and ALL cells (380; CD22⁺) at a 2:1 ratio. The percentage of cell killing obtained with NK cells expressing different chimeric receptors (% kill) was calculated by comparing the number of viable CD22⁺ ALL cells recovered after the test culture to that of parallel cultures without NK cells.

Figure 7.

NK cells expressing 4-1BB-augmented chimeric receptors show powerful cytotoxicity against leukemic cells from patients. Expanded primary NK cells expressing chimeric receptors were incubated for 4 hours with leukemic cells from children with different subtypes of B-lineage ALL (patient [Pt] 1, hyperdiploid 47-50; Pt 2 and Pt 5, t(4;11)(q21;q23); Pt 3, t(14;?)(q32;?); Pt 4, der8, t(8;?)) at the indicated E/T ratios. Each data point represents the mean (± SD; n = 4) percentage of ALL cell killing after culture as compared to that of parallel cultures without NK cells. With the exception of the results obtained in patient 2 at a 1:1 ratio, the cytotoxicity of NK cells expressing chimeric receptors containing 4-1BB was significantly higher than that induced by receptors without 4-1BB (P < .005).

Figure 8.

Chimeric receptors bearing 4-1BB induce a superior NK-cell activation. (A) Expanded primary NK cells expressing chimeric receptors were incubated for 24 hours with the ALL cell line RS4;11 at a 1:1 ratio. Flow cytometric dot plots illustrated CD25 expression (in the y-axes) and side scatter (SSC; in the x-axes) of GFP⁺ cells before and after culture. The percentages of CD25⁺ NK cells are indicated. (B) Production of IFN-γ and GM-CSF by NK cells expressing different chimeric receptors after 24 hours of culture with 697 cells at a 1:1 ratio (mean ± SD of 3 measurements). The 4-1BB receptors elicited a significantly higher production of both cytokines (P < .005).

Figure 9.

NK cells expressing anti-CD19 signaling receptors become highly cytotoxic against autologous leukemic cells. Peripheral blood NK cells were obtained from patients with B-lineage ALL in clinical remission. After expansion and transduction, cytotoxicity was tested against autologous leukemic lymphoblasts from diagnostic bone marrow samples. Each data point represents the mean (± SD; n = 4) percentage of ALL cell killing after culture as compared to that of parallel cultures without NK cells. The cytotoxicity of NK cells expressing anti-CD19-BB-ζ receptors (□ and solid lines) was markedly higher than that exerted by NK cells transduced with anti-CD19 truncated nonsignaling receptor (▵ and dotted line; patients 6-8) or empty vector (▵ and dotted line; patient 9).