Chimeric Antigen Receptor T cell (CAR-T) therapy has transformed cancer treatment. However, its application is hindered by complex manufacturing related to genetic engineering, high cost, toxicity, and resistance driven by antigen escape and tumor heterogeneity. To address these challenges, we developed a first-in-class pretargeted glycoengineered natural killer (PG-NK) cell platform that intentionally decouples the targeting moiety (ligand) from the effector cell (NK cell) using bioorthogonal click chemistry. This allows NK cells to be universally glycoengineered and paired with tumor-specific antibodies (Abs), enabling rapid, antigen-flexible targeting without genetic modification. For this, NK cells were modified with azide (NK-AZ), cyclopropene (NK-CP), or tetrazine (NK-TZ)-labeled glycans, while tumors were pretargeted with Abs functionalized with complementary click partners, dibenzocyclooctyne (Ab-DBCO) or trans-cyclooctene (Ab-TCO). The NK-TZ/Ab-TCO pair was selected as the lead configuration based on its superior affinity and efficacy. Initial glycoengineering experiments used CD16(-) NK92 cells. After labeling with azido-mannose, NK cells were modified with DBCO-TZ to display TZ on their surface. The combination of anti-CD20 (αCD20) Ab-TCO and NK-TZ resulted in a 15-fold increase in NK cell binding to CD20+ tumor cells (31.6% PG-NK vs 2.1% unmodified NK, P<0.0001) within 2 hours, promoting rapid and robust effector cell engagement. Cytotoxicity assays across multiple CD20+ lymphoma cell lines (Raji, DOHH2, DAUDI, and MINO) showed that the PG-NK cell platform (αCD20Ab-TCO + NK-TZ) consistently induced >3-fold higher cytotoxicity in a dose-dependent manner with increasing effector-to-target (E:T) ratios (e.g. 45.8% NK-TZ vs 13.1% control in MINO cells at E:T 2:1, p<0.01). Functional assays, including mass cytometry, revealed enhanced granzyme B and perforin expression associated with NK-TZ cells when co-cultured with target tumor cells, despite the absence of CD16 Fcɣ receptor. Glycoengineering of CD16(+) NK92 cells further enhanced therapeutic efficacy in the pretargeted setting, suggesting synergy between PG-NK and antibody-dependent cellular cytotoxicity (ADCC). To validate the off-the-shelf potential of the PG-NK platform, we treated Raji (CD20+/HER2-) cells with either αCD20 or αHER2 Ab-TCO. NK-TZ cells selectively eliminated Raji cells when pretargeted with αCD20Ab-TCO (33.5% αCD20 vs 16.3% αHER2, p<0.0001). Using the same batch of NK-TZ cells, BT474 (CD20-/HER2+) breast cancer cells were eliminated when pretargeted with αHER2Ab-TCO (30.3% αHER2 vs 14.0% αCD20, p<0.0001), demonstrating that NK-TZ cells are agnostic to tumor type or antigen profile, provided tumor cells are primed with the appropriate Ab-TCO. We next evaluated PG-NK therapy in vivo. In a subcutaneous Raji xenograft model, PG-NK treatment (αCD20Ab-TCO followed by NK-TZ 24 hours later) significantly reduced tumor volume (158 mm3 PG-NK vs 1639 mm3 unmodified NK, p<0.001) and improved survival after 3 weeks of therapy (80% PG-NK vs 0% all control groups at Day 50 of follow-up). A patient-derived mantle cell lymphoma model also showed superior efficacy of PG-NK therapy, with reduced bone marrow tumor burden (1.0% vs 11.6% CD20+ cells for PG-NK vs control, p<0.01) and extended survival (100% PG-NK vs 0% all control groups at Day 50; p< 0.001). Lastly, in an aggressive DOHH2 disseminated model, PG-NK treatment reduced minimal residual disease in peripheral blood (0.03% vs 0.3% CD20+ cells for PG-NK vs control, p<0.05) and significantly improved survival (67% PG-NK vs 0% all control groups at Day 60; p<0.001). Biodistribution studies confirmed selective tumor targeting and infiltration by PG-NK cells. ELISA assays showed elevated plasma levels of granzyme B, perforin, IFN-γ, and TNF-α in PG-NK-treated animals. Notably, these potent antitumor responses were achieved without discernible toxicity. Our study establishes a scalable, off-the-shelf, tumor-specific cell therapy platform that does not require genetic modification. Preclinical findings highlight Ab-TCO and NK-TZ as the optimal pairing, demonstrating rapid high-affinity binding and enhanced cytotoxicity. In vivo validation across three independent lymphoma models confirmed robust tumor killing and significant survival benefit. This PG-NK platform offers a novel framework for future cell therapy development, providing a versatile, antigen-adaptable strategy for targeted cell therapy.

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2025
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