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Flow Cytometry-based Assay for the Monitoring of NK Cell Functions
Published on: October 30, 2016
Dual-functioning Targeted ADAM17 Blocker CD16 (TAB16) mediates selective ADAM17 inhibition in NK cells and engages
Kate J Dixon1, Robert Hullsiek1, Sam Wang1
1Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, MN, United States.
Introduction:
Natural killer (NK) cells are innate lymphocytes that kill tumor cells by natural cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). Human NK cells mediate the latter process exclusively by the IgG Fc receptor CD16 (FcγRIIIA). Cell surface levels of this activating receptor are tightly regulated by the metalloprotease ADAM17, which cleaves CD16 upon NK cell activation or cellular stress. We have reported that Medi-1, a fully human IgG1 mAb, blocks ADAM17, and its Fc region is simultaneously engaged by CD16, inducing and prolonging its signaling, which synergizes with cytokine stimulation, such as IL-15. To exploit these distinctive features of Medi-1 while also addressing limitations of the mAb, such as the varied affinity by which CD16 binds to it due to receptor polymorphisms and the risk of broadly blocking ADAM17 activity, we engineered Targeted ADAM17 Blocker CD16 (TAB16).
Methods:
TAB16 was generated with a camelid heavy-chain variable domain specific to CD16 linked to a single-chain variable fragment derived from Medi-1. TAB16 was further modified by the linkage of an IL-15 moiety to generate TAB16/15. Primary human NK cells were treated with TAB16 or TAB16/15 and evaluated for proliferation by cell dilution dye, ADAM17 blocking, activation marker expression, and cytotoxicity against ovarian cancer cell lines in real-time by IncuCyte assays.
Results:
The TAB16 bispecific engager targeted NK cells and blocked ADAM17. A novel feature of TAB16 is its dual functionality, as it synergizes with IL-15 to enhance NK cell activation and proliferation and targets ADAM17 overexpressed on cancer cells to induce ADCC. TAB16 is a modifiable backbone to which additional functional components can be added, such as IL-15 (TAB16/15) for consolidated and multifaceted activity.
Discussion:
Our ADAM17-engaging platform offers a unique approach for targeted ADAM17 inhibition to augment the anti-tumor function of endogenous and therapeutic NK cells.
Insights
Engineered Targeted ADAM17 Blocker CD16 (TAB16) enhances natural killer (NK) cell anti-tumor activity by blocking ADAM17 and engaging CD16. This novel bispecific molecule synergizes with IL-15 for improved NK cell function.
Area of Science:
- Immunology
- Cell Biology
- Cancer Therapeutics
Background:
- Natural killer (NK) cells are crucial for tumor surveillance, utilizing cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC).
- Human NK cell ADCC relies on the CD16 (FcγRIIIA) receptor, whose surface levels are regulated by the metalloprotease ADAM17.
- Previous work showed a human IgG1 mAb, Medi-1, blocks ADAM17 and engages CD16, enhancing NK cell signaling.
Purpose of the Study:
- To engineer a novel bispecific molecule, Targeted ADAM17 Blocker CD16 (TAB16), to overcome limitations of Medi-1.
- To enhance NK cell anti-tumor functions through targeted ADAM17 inhibition and CD16 engagement.
- To develop a versatile platform for augmenting NK cell-mediated cancer therapy.
Main Methods:
- TAB16 was constructed by linking a CD16-specific camelid variable domain to a Medi-1 derived single-chain variable fragment.
- An IL-15 moiety was added to create TAB16/15 for enhanced functionality.
- Primary human NK cells were treated with TAB16/TAB16/15 and assessed for proliferation, ADAM17 blockade, activation markers, and cytotoxicity against ovarian cancer cells.
Main Results:
- TAB16 effectively targeted NK cells and inhibited ADAM17.
- The bispecific engager demonstrated dual functionality, synergizing with IL-15 to boost NK cell activation and proliferation.
- TAB16 targeted ADAM17 on cancer cells, inducing ADCC and showing potential for multifaceted anti-tumor activity.
Conclusions:
- The engineered TAB16 platform provides a novel strategy for targeted ADAM17 inhibition.
- This approach augments the anti-tumor functions of both endogenous and therapeutic NK cells.
- TAB16 serves as a modifiable backbone for developing advanced NK cell-based cancer therapies.
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