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. 2009 Aug 1;15(15):4857-66.
doi: 10.1158/1078-0432.CCR-08-2810. Epub 2009 Jul 28.

2B4 (CD244) signaling by recombinant antigen-specific chimeric receptors costimulates natural killer cell activation to leukemia and neuroblastoma cells

Bianca Altvater  1 , Silke Landmeier, Sibylle Pscherer, Jaane Temme, Katharina Schweer, Sareetha Kailayangiri, Dario Campana, Heribert Juergens, Martin Pule, Claudia Rossig
Affiliations

2B4 (CD244) signaling by recombinant antigen-specific chimeric receptors costimulates natural killer cell activation to leukemia and neuroblastoma cells

Bianca Altvater et al. Clin Cancer Res. .

Abstract

Purpose: Novel natural killer (NK) cell-directed strategies in cancer immunotherapy aim at specifically modulating the balance between NK cell receptor signals toward tumor-specific activation. The signaling lymphocyte activation molecule-related receptor 2B4 (CD244) is an important regulator of NK cell activation. We investigated whether 2B4-enhanced activation signals can redirect the cytolytic function of human NK cells to NK cell-resistant and autologous leukemia and tumor targets.

Experimental design: In vitro-stimulated NK cells from healthy donors and pediatric leukemia patients were gene modified with CD19 or G(D2)-specific chimeric receptors containing either the T-cell receptor zeta or 2B4 endodomain alone or combined.

Results: Chimeric 2B4 signaling alone failed to induce interleukin-2 receptor up-regulation and cytokine secretion but triggered a specific degranulation response. Integration of the 2B4 endodomain into T-cell receptor zeta chimeric receptors significantly enhanced all aspects of the NK cell activation response to antigen-expressing leukemia or neuroblastoma cells, including CD25 up-regulation, secretion of IFN-gamma and tumor necrosis factor-alpha, release of cytolytic granules, and growth inhibition, and overcame NK cell resistance of autologous leukemia cells while maintaining antigen specificity.

Conclusion: These data indicate that the 2B4 receptor has a potent costimulatory effect in NK cells. Antigen-specific 2B4zeta-expressing NK cells may be a powerful new tool for adoptive immunotherapy of leukemia and other malignancies.

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Figures

Figure 1
Figure 1. Expansion and immunophenotypes of NK cells cocultured with K562-mb15-41BBL cells
(A) Peripheral blood mononuclear cells from five healthy donors and from four pediatric patients in first hematological remission of B-cell precursor ALL were cocultured with irradiated K562mb15-4-1BBL at a 0.75:1 ratio in the presence of rhIL-2 (40 IU/ml), and absolute numbers of NK cells were calculated every 5 days after staining of viable cells with CD3- and CD56-specific antibodies. (B) Cell surface expression of CD3 and CD56 prior to and on days 5 and 10 after stimulation by flow cytometry, and expression of CD56, CD16, CD244 (2B4) and CD48 on day 10 of coculture with K562-mb15-41BBL cells. Shown are representative histograms for one of five cell cultures obtained from four donors.
Figure 2
Figure 2. NK cells expanded from peripheral blood mononuclear cells are efficiently transduced with the CAR genes
The various CAR constructs are depicted schematically. Following retroviral transduction of NK cells, gene expression was determined by flow cytometric quantification of GFP-expressing cells. Shown is one representative example of four.
Figure 3
Figure 3. CAR transduced NK cells functionally interact with antigen-expressing tumor targets
(A) Expression of the 2B4 ligand CD48 on the leukemia target cell lines (dashed line, isotype control; solid line, antibody). (B) CD25 upregulation and (C) intracellular interferon (IFN)-γ and tumor necrosis factor (TNF)-α expression by non-transduced (NT), CD19-ζ, CD19-2B4, CD19-t2B4, CD19-2B4ζ, CD19-t2B4ζ, and (B) CD19-41BBζ transduced NK cells were quantified by flow cytometry after 24-hour (B) or 6-hour (C) coincubation with CD19+ REH or SupB15 leukemia cells, CD19-negative ML-2 leukemia cells, K562, or medium alone, as indicated. To exclude non-NK lymphocytes and nontransduced NK cells within the cultures from analysis, the gate was set on GFP/CD56-coexpressing cells. Shown is one representative experiment of three. *p<0.01
Figure 4
Figure 4. CAR transduced NK cells show powerful cytolytic responses against antigen-expressing tumor targets
(A) The percentages of CD107a-expressing, degranulating NK cells were determined by flow cytometry after 4-hour coincubation of nontransduced (NT), CD19-ζ, CD19-2B4, CD19-t2B4, CD19-2B4ζ, and CD19-t2B4ζ transduced NK cells with CD19-negative ML-2 leukemia cells, CD19+ REH leukemia cells, or K562 cells at an effector-to-target (E:T) ratio of 1:1, or in the presence of medium alone, as indicated. To exclude non-NK lymphocytes and nontransduced NK cells within the cultures from analysis, the gate was set on GFP/CD56-coexpressing cells. Shown is one representative experiment of two. (B) To directly assess the cytotoxic effects of transduced NK cells on CD19+ leukemia cells, FACS-purified (GFP+) NK cells expressing the various CARs were coincubated for 16 hours with the CD19+ leukemia cell line REH at a 1:3 E:T ratio. Each data point represents the mean percentage of target cytolysis compared to parallel cultures of REH cells in the absence of NK cells. Non-transduced NK cells were used as negative controls. Shown is one representative experiment of two, each performed in duplicate wells.
Figure 5
Figure 5. NK cells obtained from pediatric leukemia patients and gene-modified with CARs are highly cytolytic against autologous leukemia cell lines
(A) The percentages of CD107a-expressing NK cells were determined by flow cytometry after 4-hour coincubation of non-transduced (NT), CD19-ζ, CD19-2B4ζ, and CD19-t2B4 transduced NK cells at a 1:1 effector-to-target (E:T) ratio. To restrict the analysis to transduced NK cells, the gate was set on GFP/CD56-coexpressing cells. Shown is one representative experiment of three, performed with three individual donors. (B) FACS-purified (GFP+) NK cells expressing the various CARs were incubated for 16 hours with autologous leukemic cells at an E:T ratio of 1:8. Each data point represents the mean percentage of lysis of autologous leukemia cells compared to parallel cultures in the absence of NK cells. Non-transduced NK cells were used as negative controls.
Figure 6
Figure 6. 14.G2a-2B4ζ expression in NK cells enhances the activation response to GD2+ neuroblastoma cells and can overome their resistance to NK cell lysis
(A) 14.G2a-ζ and −2B4ζ CAR surface expression was determined by staining with F(ab)-specific antibody. One of four representative experiments. (B) CD25 upregulation by nontransduced (NT), 14.G2a-ζ and 14.G2a-2B4ζ transduced NK cells was quantified by flow cytometry after 24-hour coincubation with GD2+ JF neuroblastoma cells at an effector-to-target ratio of 1:1. To exclude nontransduced NK cells within the cultures from analysis, the cells were FACS-purified based on F(ab) staining prior to analysis. Shown is one representative experiment of two. (C) FACS-purified transduced NK cells from three individual healthy donors were incubated for 16 hours with autologous leukemic cells at an E:T ratio of 2:1. Each data point represents the mean percentage of lysis of JF cells compared to parallel cultures in the absence of NK cells. Non-transduced NK cells were used as negative controls.
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