Fig. 5. In vitro virus neutralization by CTC-445.2d.

(A) Top left: In vitro neutralization of NanoLuc SARS-CoV-2 by CTC-445.2d in Calu-3 cells after 72 hours of incubation at a MOI of 1.0. Top right: A cell viability assay (48 hours) confirmed that the decoys are not cytotoxic to Calu-3. Bottom left: In vitro neutralization of live BetaCoV/Hong Kong/VM20001061/2020 SARS-CoV-2 virus in Vero E6 cells at a MOI of 1.0. The cells were incubated with CTC-445.2d throughout infection and the colors indicate the following: orange, before infection, during infection, and after infection; black, after infection only; and gray, before infection only. SARS-CoV-2 RNA copy numbers were determined by quantitative real-time reverse transcription polymerase chain reaction. All assays were performed in triplicate unless otherwise noted, and all data points are shown. Bottom right: Cell viability in Vero E6 cells was independently performed (CCK8 assay) and it was confirmed that the de novo decoys are not cytotoxic. (B) In vivo mouse pharmacokinetics and tolerability of intranasally administered CTC-445.2d. Left: Plot showing the concentration of fully functional CTC-445.2d (i.e., capable of binding to the SARS-CoV-2 RBD; see the materials and methods) found in homogenized lungs of Balb/c mice after a single 100 μg dose, measured at various times after dosing (n = 5 mice). Right: Body weight of mice after repeat daily intranasal doses of CTC-445.2d (100 μg; n = 18 at day 0) compared with control [phosphate-buffered saline (PBS)–treated] mice (n = 5). At each time point, three CTC-445.2d–treated mice were sacrificed for lung examination. Weight data shown are for the remaining mice (n = 18, 15, 12, 9, 6, and 3 at days 1, 2, 4, 8, 11, and 14, respectively). No significant weight loss or lung abnormalities were observed. Error bars indicate the standard deviation. (C) In vivo Syrian hamster SARS-CoV-2 challenge. Left: Body weight measurements through day 10 for unchallenged hamsters (n = 5, red) compared with SARS-CoV-2–challenged hamsters treated either with a single dose of CTC-445.2d (day 0 at –12 hours; n = 8, orange) or PBS (day –1, day 0 at –12 hours, day 1, and day 2; n = 7, gray). Right: Survival plot. Hamsters were euthanized when they displayed clinical signs of distress according to protocol clinical scoring criteria (see the materials and methods). At the end of the experiment, all hamsters treated with the de novo decoy CTC-445.2d survived, exhibiting moderate weight loss, whereas hamsters treated with vehicle did not survive past day 7 because of severe weight loss and other complications from the viral infection (see table S5).