Background
NK cells are the body’s first line of defense against cancer, able to recognize and kill tumor cells without having been exposed to the tumor cell previously. Unlike T cells, NK cells do not induce graft-versus-host disease. Therefore, using NK cells in adoptive cell therapy (NK-ACT) holds promise as a true ‘off-the-shelf’ cellular immunotherapy for cancer, with the potential to circumvent many of the hurdles associated with autologous cell therapies. NK-ACT has demonstrated potential against hematological cancers, but the activity of NK cells in ACT should be enhanced to improve clinical efficacy. Cbl-b, an E3 ubiquitin ligase, is an important gatekeeper, which limits NK cell activation. In NK cells, Cbl-b is activated and stabilized through inhibitory receptor signaling and has been shown to reduce NK cell degranulation and cytotoxicity. Strategies to reduce Cbl-b in NK-ACT products are under active exploration as means to improve the efficacy of NK-ACT. Incorporating RNAi treatment into ex vivo NK cell expansion protocols prior to ACT represents one such strategy. We have developed a new class of stable, self-delivering RNAi compounds (INTASYL™) that incorporate features of RNAi and antisense technology. INTASYL compounds demonstrate potent activity, stability, and are rapidly and efficiently taken up by cells. INTASYL 27457 targeting Cbl-b enhances the cytotoxic activity of expanded human NK cells in vitro.
Materials and Methods
Primary human CD56+ NK cells were expanded using the ImmunoCult™ NK Cell Expansion Kit. Following the 14-day expansion protocol, cells were seeded directly into 24-well plates containing Cbl-b compound and cultured for 3 days. After the 3-day exposure to INTASYL, compound was washed off and cells were cultured for an additional 3 days. At the end of the culture period, cell viability and expression levels of Cbl-b mRNA were determined. Flow cytometry was performed to explore the impact on various proliferation and activation makers. Cytotoxic capability against the K562 (Chronic Myelogenous Leukemia) cancer cell line was tested in a DELFIA cell cytotoxicity assay. Following co-culture with K562 cells, culture supernatants were analyzed by ELISA for IFN-.
Results
INTASYL treatment resulted in consistent Cbl-b mRNA silencing without negative impact on NK cell viability. Following silencing of Cbl-b markers of NK cell activation and proliferation as well as cytotoxic capability of NK cells against K562 cancer cells were increased.
Conclusions
We demonstrate the potential of INTASYL self-delivering siRNA targeting Cbl-b to improve NK cell activity for ACT. By treating NK cells with INTASYL targeting the inhibitory protein Cbl-b ex vivo, during NK cell expansion, the proliferation, activation, and anti-tumor response of these cells was enhanced. This may result in a more effective cell therapy for hematological malignancies.
M. Maxwell: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. D. Yan: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. B. Rivest: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. J. Cardia: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. E. Noessner: A. Employment (full or part-time); Significant; Helmholtz Zentrum Munich.