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Background Natural killer (NK) cells are lymphocytes of the innate immune system that provide key anti-tumor surveillance. NK cells can be given as allogeneic products and, unlike T cells, do not induce cytokine- release syndrome or neurotoxicity. Due to their anti-viral effects, NK cells have proven difficult to stably transduce with viral constructs. We have developed a novel dual camelid (cam) Tri-specific Killer Engager (TriKE) molecule (containing WT IL-15 as a linker) and a Bi-Specific Killer Engager (BiKE without IL-15) and two cam engagers targeting CD16 on NK cells and B7H3 (CD276) a tumor-associated antigen. In order to deliver BiKE or TriKE (engager) directly to the tumor microenvironment, we sought to develop lentiviral techniques to transduce NK cells to secrete these immune engagers.Methods Third-generation lentivirus was generated containing camB7-H3 BiKE, camB7-H3 TriKE, or empty vector, all with mNeon green reporter. Transduction conditions were optimized and healthy donor NK cells were expanded and stimulated for 7 days, then transduced with lentiviral constructs. Cells were sorted and expanded for an additional 21 days under optimized cytokine conditions. Expanded, transduced NK cells were analyzed by flow cytometry pre- and post-expansion for retention of mNeon green reporter expression. The presence of BiKE or TriKE was measured targeting the 10x Histidine tag on BiKE and TriKE using an anti- His PE antibody and evaluated by flow cytometry. NK cell degranulation (CD107a), inflammatory cytokine (interferon-gamma or tumor necrosis factor-alpha), and proliferation induction by secreted BiKE or TriKE are pending with n=4 donors.Results camB7-H3 TriKE and camB7-H3 were successfully cloned into vector and lentivirus was produced. Transduced NK cells demonstrated persistent mNeon green expression persisted through the 21-day expansion ( figure 1A). Secreted engager was detected on the surface of transduced, expanded NK cells and B7-H3+ tumor targets (figure 1B) and secreted camB7-H3 TriKE maintained NK viability for 7 days (figure 1C). In vitro and in vivo functional and killing assays are pending.Conclusions We have successfully optimized and validated a method to efficiently transduce and subsequently expand normal donor NK cells to secrete engager molecules targeting the tumor antigen B7-H3. We have shown that transduced NK cells secrete functional engager that binds to the surface of both NK cells and tumor targets. This study demonstrates the feasibility of efficient lentiviral transduction of peripheral blood NK cells as a source of local and continuous immune engager production for the treatment of prostate cancer.Abstract 1032 Figure 1Transduced NK cells continue to express GFP after 32 days (A). Secreted B7-H3 TriKE binds ovarian cancer (SK-OV3) and normal donor NK cells (B). B7-H3 TriKE supports NK cell survival (GFP+) compared to empty vector (C)