No abstract

Targeting and modification of important intracellular proteins using efficient vehicles are invaluable in diagnostic and therapeutic fields. Cell-penetrating antibodies and their fragments can be utilized as vehicles for the delivery of modifiers into cells. In this study, we explored the applicability of variable heavy chain (VH) domain as delivery vehicles for mammalian cells. The characteristics of the recombinant VH domain produced from a cell-penetrating monoclonal anti-double stranded DNA antibody 2C10 were analyzed using flow cytometry, confocal microscopy, cell proliferation assay, and cell cycle analysis in various mammalian cell lines. The VH domain penetrated into various cell lines in a time- and dose-dependent manner, although the internalization efficiency varied. The domain was localized in the nuclei as well as the cytoplasm of living cells. It was also internalized into cells mainly through the clathrin-mediated endocytosis pathway. We tested further its efficiency in delivering specific biomolecule(s) using the conjugates of the single domain molecule and small interfering RNA (siRNA) for the testicular nuclear auto-antigenic sperm protein (tNASP). It was found that the siRNA was successfully delivered by the VH domain into cancer cells, and knockdown effects from the delivered tNASP-siRNA were observed. The levels of the RNA transcript and protein of tNASP were decreased and the down-regulated tNASP inhibited cell proliferation and caused G0G1 phase arrest of the cell cycle. These results indicate that the recombinant 2C10 VH domain could be applied as an efficient vehicle capable of delivering valuable biomolecule into the cytoplasm or cell nuclei for clinical uses.

Since the Spike protein on the surface of SARS coronavirus 2 (SARS-CoV-2) binds to the ACE2 receptor in human cells, the development of neutralizing proteins or antibodies targeting the receptor binding domain (RBD) of the spike protein is an important strategy for SARS-COV-2 therapy. We chose to develop molecularly-evolved soluble ACE2 protein on three grounds; 1) it can trap and neutralize the SARS-CoV-2 as neutralizing antibodies do, 2) it can supplement angiotensin II-converting enzyme activities that protect lung, heart, and kidneys of severe cases of infections and patients with underlying diseases, and 3) it may trap effectively SARS-CoV-2 mutants even though the mutations compromise the protection by neutralizing antibodies or vaccine. For the enhancement of ACE2 binding affinity to RBD, we used the 3D complex structure between ACE2 and RBD to select the major contributing ACE2 amino acids, a library targeting selected amino acids and random mutations were constructed and screened using yeast surface display. The engineered ACE2, EU129, was fused with the human IgG1 Fc for long half-life and viral clearance. The binding affinity of EU129 to RBD was increased by about 500-folds compared to ACE2 wild-type in SPR analysis, and the neutralizing activity was also increased by about 130-folds compared to ACE2 wild-type in surrogate virus neutralization test (sVNT). In addition, it was confirmed that the enzymatic activity of ACE2, which prevents organ damage due to SARS-CoV-2 infection in the human, is maintained at a level similar to that of ACE2 wild-type. In vitro assays using live SARS-CoV-2 virus and Vero E6 cells, EU129 was shown to be more effective in inhibiting viral infection and amplification than ACE2 wild-type, which was confirmed through protein and RNA level and cell morphology change of the live virus. In vivo stability assays using BALB/c mice, EU129 showed enhanced binding to the RBD and maintained enzymatic activity similar to ACE2 wild-type. We generated EU129 with the improved binding affinity and neutralizing activity through ACE2 receptor engineering. It has angiotensin II-converting enzymatic activity for organ protection, thus EU129 is a better therapeutic candidate for severe cases of SARS-CoV-2 infection and patients with underlying diseases such as cancers. Citation Format: Byoung S. Kwon, Seunghyun Lee, Jin-Kyung Choi, Bora Hwang, Sun-Woo Im, Yun-Sook Lim, Bumseok Kim, Soon B. Hwang, HoonSung Jeh. Development of SARS-CoV-2 neutralizing protein by ACE2 receptor engineering for severe infection and patients with underlying diseases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB075.