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To circumvent the devastating pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection, a humanized decoy antibody (ACE2‐Fc fusion protein) was designed to target the interaction between viral spike protein and its cellular receptor, angiotensin‐converting enzyme 2 (ACE2). First, we demonstrated that ACE2‐Fc could specifically abrogate virus replication by blocking the entry of SARS‐CoV‐2 spike‐expressing pseudotyped virus into both ACE2‐expressing lung cells and lung organoids. The impairment of viral entry was not affected by virus variants, since efficient inhibition was also observed in six SARS‐CoV‐2 clinical strains, including the D614G variants which have been shown to exhibit increased infectivity. The preservation of peptidase activity also enables ACE2‐Fc to reduce the angiotensin II‐mediated cytokine cascade. Furthermore, this Fc domain of ACE2‐Fc was shown to activate NK cell degranulation after co‐incubation with Spike‐expressing H1975 cells. These promising characteristics potentiate the therapeutic prospects of ACE2‐Fc as an effective treatment for COVID‐19. Synopsis Currently, there is no effective strategy to fight against the COVID‐19 pandemic. We aim to design and develop a humanized decoy antibody to block SARS‐CoV‐2 infection. The ACE2‐Fc fusion protein can form a dimer that mimics a humanized antibody and specifically binds to the SARS‐CoV‐2 Spike protein. The ACE2‐Fc fusion protein abrogates virus replication by blocking SARS‐CoV‐2 entry in clinical isolates. The peptidase activity of ACE2‐Fc enables the decoy antibody to reduce angiotensin II‐mediated cytokine cascade. After binding to Spike‐expressing target cells, ACE2‐Fc activates degranulation of NK cells. Graphical Abstract Currently, there is no effective strategy to fight against the COVID‐19 pandemic. We aim to design and develop a humanized decoy antibody to block SARS‐CoV‐2 infection.

Natural killer (NK) cells are an attractive cell-type for adoptive immunotherapy, but challenges in preparation of therapeutic primary NK cells restrict patient accessibility to NK cell immunotherapy. NK-92 is a well-characterized human NK cell line that has demonstrated promising anti-cancer activities in clinical trials. Unlimited proliferation of NK-92 cells provides a consistent supply of cells for the administration and development of NK cell immunotherapy. However, the clinical efficacy of NK-92 cells has not reached its full potential due to reduced immune functions as compared to primary NK cells. Improvements of NK-92 functions currently rely on conventional transgene delivery by mRNA, plasmid and viral vector with limited efficiencies. To enable precise genetic modifications, we have established a robust CRISPR genome engineering platform for NK-92 based on the nucleofection of Cas9 ribonucleoprotein. To demonstrate the versatility of the platform, we have performed cell-based screening of Cas9 guide RNA, multiplex gene knockout of activating and inhibitory receptors, knock-in of a fluorescent gene, and promoter insertion to reactivate endogenous CD16 and DNAM-1. The CRISPR-engineered NK-92 demonstrated markedly enhanced cytotoxicity and could mediate antibody-dependent cellular cytotoxicity against hard to kill cancer cell lines. Our genome editing platform is straightforward and robust for both functional studies and therapeutic engineering of NK-92 cells.

BackgroundNatural 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.MethodsThird-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.ConclusionsWe 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)[Image Omitted. See PDF.]