2B4 (CD244) Signaling by Recombinant Antigen-specific Chimeric Receptors Costimulates Natural Killer Cell Activation to Leukemia and Neuroblastoma Cells

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 ζ 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 ζ 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-γ and tumor necrosis factor-α, 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 2B4ζ-expressing NK cells may be a powerful new tool for adoptive immunotherapy of leukemia and other malignancies.

NK cells and cancer: you can teach innate cells new tricks

“…160) and G D2 (REF. 161) showed increased responses to tumour cells in vitro and suppressed tumour growth when tested in vivo in xenograft models 162,163 . Although initially considered as receptors providing 'built-in' ADCC-like NCRL activity against specific tumour antigens, CARs actually elicit a significantly stronger NK cell cytotoxic response than ADCC mediated by anti bodies against the same targets 164 .…”

Bringing natural killer cells to the clinic: ex vivo manipulation

“…These data, as well as recent data showing the efficacy of T-cell-based CAR therapy targeting CD19 in B-cell malignancies, suggest that NK cells modified to express CARs after mRNA or viral transduction represent a therapeutic tool worthy of exploration in the clinical setting. 43,44,57,73,[76][77][78][79] The ability of CAR-expressing NK cells to home to the tumor may be a critical determinant of their efficacy in humans. As discussed previously, expanded NK cells may lack or down-regulate molecules that are critical for NK cell homing from the circulation.…”

NK cells are dysfunctional in human chronic myelogenous leukemia before and on imatinib treatment and in BCR–ABL-positive mice

“…Stimulation of PBMCs with irradiated K562 cells gene-modified to express membrane-bound IL-15 and 41BB ligand in the presence of low-dose (40 IU/ml) recombinant human IL-2 has been found to induce efficient and selective NK-cell expansion from the peripheral blood of both healthy donors 40 and patients in remission of acute leukemia. 42 In vitro stimulation with irradiated K562-mb15-4 1BBL at a 0.75:1 ratio, followed by 10-day in vitro expansion in the presence of recombinant human IL-2 (40 IU/ml) resulted in a median 37-fold (8-64-fold) increase in CD3ÀCD56 þ NK cells in healthy donors and a median purity of 94% (92-96%) CD3ÀCD56 þ NK cells within the stimulated bulk populations (Figure 3a). Absolute median numbers of 4.1 Â 10 6 (0.8-6.4 Â 10 6 ) NK cells were generated from 1 Â 10 6 PBMCs on day 10.…”