key: cord-0872939-j1ifaouo
authors: Fu, Yanwen; Maruyama, Junki; Singh, Alok; Lim, Reyna; Ledesma, Arthur; Lee, Daniel; Rivero-Nava, Laura; Ko, Jamie; Rivera, Ianne; Sattler, Rachel A.; Manning, John T.; Kerwin, Lisa; Zhou, Heyue; Brunswick, Mark; Bresson, Damien; Ji, Henry; Paessler, Slobodan; Allen, Robert D.
title: Protective Effects of STI-2020 Antibody Delivered Post-Infection by the Intranasal or Intravenous Route in a Syrian Golden Hamster COVID-19 Model
date: 2020-10-29
journal: bioRxiv
DOI: 10.1101/2020.10.28.359836
sha: 6202eff5b0a9f9800be6ef176c287c7614a3787e
doc_id: 872939
cord_uid: j1ifaouo

We have previously reported that the SARS-CoV-2 neutralizing antibody, STI-2020, potently inhibits cytopathic effects of infection by genetically diverse clinical SARS-CoV-2 pandemic isolates in vitro, and has demonstrated efficacy in a hamster model of COVID-19 when administered by the intravenous route immediately following infection. We now have extended our in vivo studies of STI-2020 to include disease treatment efficacy, profiling of biodistribution of STI-2020 in mice when antibody is delivered intranasally (IN) or intravenously (IV), as well as pharmacokinetics in mice following IN antibody administration. Importantly, SARS-CoV-2-infected hamsters were treated with STI-2020 using these routes, and treatment effects on severity and duration of COVID-19-like disease in this model were evaluated. In SARS-CoV-2 infected hamsters, treatment with STI-2020 12 hours post-infection using the IN route led to a decrease in severity of clinical disease signs and a more robust recovery during 9 days of infection as compared to animals treated with an isotype control antibody. Treatment via the IV route using the same dose and timing regimen resulted in a decrease in the average number of consecutive days that infected animals experienced weight loss, shortening the duration of disease and allowing recovery to begin more rapidly in STI-2020 treated animals. Following IN administration in mice, STI-2020 was detected within 10 minutes in both lung tissue and lung lavage. The half-life of STI-2020 in lung tissue is approximately 25 hours. We are currently investigating the minimum effective dose of IN-delivered STI-2020 in the hamster model as well as establishing the relative benefit of delivering neutralizing antibodies by both IV and IN routes.

The SARS-CoV-2 neutralizing monoclonal antibody (mAb) STI-2020 is an affinity-matured variant of the parental antibody STI-1499, first isolated from the fully human G-MAB phage display antibody library and paired with a human IgG1 Fc domain bearing the double mutation in the hinge region, L234A, L235A (LALA). This modification is aimed at reducing the potential for antibody-dependent enhancement (ADE) of infection in the context of COVID-19 treatment 1, 2, 3, 4, 5 . STI-2020 administered IV immediately following infection has been previously shown to provide protection against pathogenesis in the hamster COVID-19 disease model 5 . The hamster model provides a ready means of evaluating the effects of candidate therapeutics on SARS-CoV-2-mediated respiratory disease, which has contributed greatly to the overall morbidity and mortality associated with the current pandemic 6, 7, 8 . Human SARS-CoV-2 infection along the respiratory tract has been detected in the ciliated epithelial cells of the trachea, alveolar cells, and upper airway epithelia in tissues from COVID-19 autopsies 9, 10 . Expression of angiotensin converting enzyme-2 (ACE2) and neuropilin-1 (NRP1), both of which have been identified as entry factors mediating uptake of SARS-CoV-2 into host cells, has been detected at varying levels in the epithelia of the upper and lower respiratory tract 9, 10, 11, 12 . Establishment and maintenance of a neutralizing antibody (nAb) blockade in regions of the respiratory tract that are most closely linked to primary virus infection, virus receptor expression, and progressive virus pathogenesis could provide a means of decreasing the severity of COVID-19 symptoms, preventing the dissemination of disease within the respiratory tract, and decreasing pharyngeal shedding of virus into the environment. Previously, it was demonstrated that mAbs directed against the influenza hemagglutinin (HA) stalk provide protection and therapeutic benefit to infected mice when administered as an aerosol or as a nasal droplet 13 . Recent studies have demonstrated the beneficial effects of nebulizer-based delivery of SARS-CoV-2 nAbs on severity of lung pathology and degree of virus replication in the lungs of infected hamsters 14 . To investigate alternative routes and timings of antibody administration, an intranasal STI-2020 formulation was developed for use in biodistribution, pharmacokinetic, and virus neutralization efficacy profiling experiments. Intranasally-delivered STI-2020 was detectable in mouse lung lavage and lung tissue within 10 minutes of administration. Following IN administration of a single 500 g dose of STI-2020 twelve hours post-infection in the Syrian golden hamster COVID-19 model, animals exhibited decreased weight loss as compared to isotype controltreated animals and underwent a more rapid and robust recovery from disease than animals in the control treatment group. Therapeutic effects including decreased duration of progressive disease following treatment with STI-2020 were also observed in animals administered an equivalent dose IV at 12 hours post-infection.

STI-2020 was produced, purified, and formulated as previously described 5 

Multi-Array 96-well plates (cat# L15XA-3, Meso Scale Discovery (MSD)) were coated with mouse antihuman IgG antibody (CH2 domain, cat# MA5-16929, ThermoFisher Scientific) at 2 µg/mL in 1X PBS (50 µL/well), sealed, and incubated overnight at 4°C. The following day, plates were washed 3X with 1X washing solution (KPL wash solution, cat no# 5150-0009, lot no# 10388555, Sera Care). Plates were then blocked using 50 µL/well of Blocker™ Casein in PBS (cat no# 37528, lot# QE220946, ThermoFisher) for 1 hour at room temperature on an orbital shaker. Plates were washed 3X with 1X washing solution. Samples from biodistribution or pharmacokinetic experiments were added in a volume of 50 µL to each well. STI-in PBS to generate the standard curve for the assay. Following addition of experimental samples or control samples, plates were incubated for 2 hours at room temperature on an orbital shaker. Plates were then washed 3X with 1X washing solution and 50 µL of Sulfo-Tag anti-human/NHP IgG antibody (cat no# D20JL-6, lot no# W0019029S, MSD), at 1/1,000 dilution in Blocker™ Casein in PBS was added to each well and plates were then incubated for 1-1.5 hours at room temperature on an orbital shaker. Plates were washed 3X with 1X washing solution and 150 µL of 2X read Buffer (cat# R92TC-3, MSD) was added to each well.

Plates were read immediately on an MSD instrument and the STI-2020 standard curve was used to calculate the concentration of antibody present in serum, lung lavage, and organ lysate materials.

Female CD-1-IGS (strain code #022) were obtained from Charles River at 6-8 weeks of age. For intravenous injection of STI-2020, 100 µL of antibody diluted in 1X HBSS was administered retro-orbitally to anesthetized animals. For intranasal injections, antibody was diluted in 1X HBSS and administered by inhalation into the nose of anesthetized animal in a total volume of 20 µL using a pipette tip. Organs, blood, and lung lavage samples were collected 24 hours post-antibody administration. Blood was collected by retro-orbital bleeding and then transferred to Microvette 200 Z-Gel tubes (Cat no# 20.1291, lot# 8071211, SARSTEDT). Tubes were then centrifuged at 10,000 x g for 5 minutes at room temperature.

Serum was transferred into 1.5 mL tubes and stored at −80°C. Lung lavage samples were collected following insertion of a 20G x 1-inch catheter (Angiocath Autoguard, Ref# 381702, lot# 6063946, Becton Dickinson) into the trachea. A volume of 0.8 mL of PBS was drawn into a syringe, placed into the open end of the catheter, and slowly injected and aspirated 4 times. The syringe was removed from the catheter, and the recovered lavage fluid was transferred into 1.5 mL tubes and kept on ice. Lavage samples were centrifuged at 800 × g for 10 min at 4°C. Supernatants were collected, transferred to fresh 1.5 mL tubes, and stored at −80°C. Total spleen, total large intestine, and 150 to 400 mg of lungs and small intestine were suspended in 300 µL of PBS in pre-filled 2.0 mL tubes containing zirconium beads (cat no# 155-40945, Spectrum). Tubes were processed in a BeadBug-6 homogenizer at a speed setting of 3000 and a 30 second cycle time for four cycles with a 30-second break after each cycle. Tissue homogenates were centrifuged at 15,000 rpm for 15 minutes at 4°C. Homogenate supernatants were then transferred into 1.5 mL tubes and stored at −80°C. STI-2020 antibody levels in each sample were quantified using the antibody detection ELISA method. Statistical significance was determined using the Welch's t-test. This study was reviewed and accepted by the animal study review committee (SRC) and conducted in accordance with IACUC guidelines.

Female CD-1-IGS (strain code #022) were obtained from Charles River Laboratories at 6-8 weeks of age.

STI-2020 dissolved in intranasal formulation buffer was administered as described for the IN biodistribution study. Lungs and blood were collected from 3 mice at each of the following timepoints: 10 min, 1.5 h, 6 h, 24 h, 72 h, 96 h, 168 h, 240 h, and 336 h. Serum and lung tissue samples were collected as described for the biodistribution study. STI-2020 antibody levels in each sample were quantified using the antibody detection ELISA method. Pharmacokinetic analysis of the collected ELISA data was performed with the Phoenix WiNnonlin suite of software (version 6.4, Certara) using a non-compartmental approach consistent with an IN bolus route of administration. Statistical significance was determined using the Welch's t-test. This study was reviewed and accepted by the animal study review committee (SRC) and conducted in accordance with IACUC guidelines.

Female Syrian golden hamsters were obtained from Charles River Laboratories at 6 weeks of age.

Hamsters were inoculated IN with 5x10 4 TCID50 of SARS-CoV-2 in 100 µL of sterile PBS on day 0. Antibody treatments were administered IV with monoclonal antibodies (mAbs) against SARS-CoV-2 Spike, or isotype control mAb in up to 350 µL of formulation buffer to anesthetized animals at 12 hours-post inoculation. For intranasal delivery of these antibodies, 100 L of formulated material was introduced directl into the nares and inhaled by anesthetized animals. Animals were monitored for illness and mortality for 9 days post-inoculation and clinical observations were recorded daily. Body weights and temperatures were recorded at least once daily throughout the experiment. Average % weight change on each experimental day was compared with the isotype control mAb-treated group using 2-way ANOVA followed by Fisher's LSD test. All animals were housed in animal biosafety level-2 (ABSL-2) and ABSL-3 facilities in Galveston National Laboratory at the University of Texas Medical Branch. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch and were conducted according to the National Institutes of Health guidelines.

Biodistribution studies of STI-2020 delivered by either the intravenous or intranasal route were carried out in CD-1 mice. Twenty-four hours following administration of a single antibody dose, samples of serum, lung lavage, and tissues including spleen, lung, small intestine, and large intestine were obtained from each of 5 treated mice at each dose level. Samples were processed and mAb levels were quantified using a human antibody detection ELISA readout. Following IV treatment at a dose level of 0.5 mg/kg, STI-2020 was detected in the serum, spleen, lungs, small intestine, and large intestine. Detected levels in the serum at 0.5 mg/kg dose averaged 4.5 g/mL, while STI-2020 was present at average concentrations less than 

To characterize STI-2020 pharmacokinetics following intranasal dosing at 5 mg/kg, antibody levels in CD-1 mouse lung lysates and serum were quantified at designated timepoints spanning a total of 336 h using a human antibody detection ELISA. The concentration of STI-2020 in lung lysates and serum for each individual mouse are shown in Figures 2A and 2B , respectively. There were no quantifiable concentrations of STI-2020 antibody in the pre-dose samples. Following IN administration of STI-2020, the antibody concentration was quantifiable up to 240 and 336 h in the lungs and serum, respectively. We observed a mouse-to-mouse variability at each time point that could be inherent to the delivery method 15 . When using intranasal instillation, the relative distribution between the upper and lower respiratory tract and the gastrointestinal tract is influenced by delivery volume and level of anesthesia. Average antibody concentration in the lung measured 10 minutes after dosing was nearly 70 percent of the maximum antibody concentration (Cmax) measured during the experiment. The Cmax value of STI-2020 in the lungs was measured at 1.5 hours post-administration at a value of 43 g/mL. In the lungs, an apparent terminal half-life (T1/2) of 32.21 hours was measured when analyzed between 0.15 and 240 h ( Table 1) . Under these conditions the R 2 value equaled 0.932, however when the data were analyzed between 0.15 and 168 hours the R 2 value increased to 0.987 but the T1/2 dropped to 25.07 hours for the lung samples. Kinetics of STI-2020 exposure in the lungs following intranasal administration was accompanied by a slower kinetic of detectable antibody in the serum of treated mice ( Figure 2C ). Antibody was first detected in the serum at 6 hours post-administration and the Cmax of 871 ng/mL was detected at the 240-h timepoint (Tmax).

Serum antibody concentrations were within 90% of the recorded Cmax by the 24-h timepoint. Antibody levels remained relatively constant in serum over the period spanning 24-240 h, which is in keeping with the calculated STI-2020 serum half-life observed following IV administration of 240 hours in mice (data not shown). The total systemic exposure (AUClast) was significantly higher in the lungs than in the serum of treated mice (AUClast were 1,861,645.8 and 248,675.5 h*ng/mL respectively, Table 1 ). Although some mice may have generated anti-drug antibody (ADA) during the course of the study the data (measured antibody concentrations and PK profiles) do not suggest that PK parameters were significantly influenced by immunogenicity.

Based on the observed kinetics of STI-2020 exposure in the lungs following IN dosing at 5 mg/kg and considering the protective efficacy of the 5 mg/kg IV dose in the Syrian golden hamster model of COVID-19, we chose 5 mg/kg as our IN and IV dose to be administered 12h post-infection in the hamster SARS-CoV-2 disease model. In this manner, we were able to directly compare the degree of disease severity and duration of disease in animals receiving a therapeutic 5 mg/kg dose of STI-2020 or a control IgG1 antibody (IsoCtl) by either the IV or the IN route. Animals were infected with 5x10 4 TCID50 of SARS-CoV-2

intranasally and subsequently treated with STI-2020 administered intravenously or intranasally at 12 h post-infection. Weight change as a percentage of starting weight was recorded and graphed for each animal.

Animals administered a 5 mg/kg dose of STI-2020 intravenously experienced a progression of disease similar to that of IsoCtl-treated animals for the first three days of infection. Based on the day over day average rate of weight change between the two treatment groups, the STI-2020-treated animals showed a slight decrease in the rate of weight loss between day 2 and day 3. By day 4 of infection, the weight loss rates had further separated along this same trend, and animals in the STI-2020 treatment group had begun to gain weight, on average. On day 5 of infection, the day on which the maximum average percentage weight loss in the IsoCtl group was observed (9.2%), the STI-2020-treated animals had already experienced two consecutive days of weight gain (average 2.2 grams/day). Average weight gain between day 3 and day 8 of the experiment between the STI-2020 and the IsoCtl IV-treated groups was 2.8 grams/day and 1.3 grams/day, respectively. Treatment with STI-2020 12h post-infection decreased duration clinical signs of disease by at least 24 h and led to an overall reduction of disease severity, as manifested by an average rate of weight gain double that of IsoCtl-treated animals between day 3 and day 8 of infection among STI-2020 treated animals. Of note, one of the animals in the STI-2020 treatment group exhibited a more profound course of disease than the other four animals in the same treatment group. Larger experimental groups as well as a dose-response study design will be required to better appreciate factors such as variable disease progression prior to antibody treatment that might contribute to this class of outcomes in the therapeutic treatment setting. Treatment of animals IV at 12 h post infection resulted in a maximum average weight loss of nearly 6%, which occurred on day 3 of infection. As such, IV-treated animals experienced an early course of disease that was indistinguishable from IsoCtl-treated animals and marginally more severe than the disease seen in IN-treated animals across that timespan. Once IV-administered antibody began to demonstrate antiviral efficacy, animals gained weight at a rate similar to that seen after day 5 of infection in IsoCtltreated animals. By day 5 in the IV STI-2020-treated group, animals had, on average, returned to their day 0 weight and continued to steadily gain weight until the experiment was ended on day 9. The improvements in early disease mitigation following IN dosing of hamsters may reflect the corresponding increases in the measured concentration of STI-2020 in lung lavage samples from mice in our biodistribution studies. Extravascular antibody in the lung may provide more acute protection against disease progression in the lung parenchyma than antibody present in the lung vasculature at early stages of disease. Combining the mitigating effects of IN-administered antibody on early disease severity with the effects of IV antibody dosing on disease duration may prove to be a regimen that maximizes the therapeutic effects of neutralizing antibodies on respiratory symptoms associated with COVID-19. Effects of varying the virus challenge dose and the single or combined IN and IV STI-2020 dose levels on disease duration and severity will be measured in future experiments using the hamster model. 

Protective Efficacy of STI-2020 Administered Intravenously or Intranasally in the Syrian Golden Hamster Model of COVID-19. (A) Female hamsters were inoculated with SARS-CoV-2 WA-1 isolate on day 0. Twelve hours post-infection, animals (n=5 per group) were administered a single intravenous dose of Control IgG (500 g

Twelve hours post-infection, a single dose of 500 g STI-2020 or Control IgG was administered intranasally (IN) and daily weight changes were recorded and (E) plotted for each individual animal

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Competing interests: Sorrento authors own options and/or stock of the company. This work has been described in one or more provisional patent applications. HJ is an officer at Sorrento Therapeutics, Inc..