Exebacase Is Active In Vitro in Pulmonary Surfactant and Is Efficacious Alone and Synergistic with Daptomycin in a Mouse Model of Lethal Staphylococcus aureus Lung Infection

ABSTRACT Exebacase (CF-301) is a novel antistaphylococcal lysin (cell wall hydrolase) in phase 3 of clinical development for the treatment of Staphylococcus aureus bacteremia, including right-sided endocarditis, used in addition to standard-of-care antibiotics. In the current study, the potential for exebacase to treat S. aureus pneumonia was explored in vitro using bovine pulmonary surfactant (Survanta) and in vivo using a lethal murine pneumonia model. Exebacase was active against a set of methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains, with an MIC90 of 2 μg/ml (n = 18 strains), in the presence of a surfactant concentration (7.5%) inhibitory to the antistaphylococcal antibiotic daptomycin, which is inactive in pulmonary environments due to specific inhibition by surfactant. In a rigorous test of the ability of exebacase to synergize with antistaphylococcal antibiotics, exebacase synergized with daptomycin in the presence of surfactant in vitro, resulting in daptomycin MIC reductions of up to 64-fold against 9 MRSA and 9 MSSA strains. Exebacase was also observed to facilitate the binding of daptomycin to S. aureus and the elimination of biofilm-like structures formed in the presence of surfactant. Exebacase (5 mg/kg of body weight 1 time every 24 h [q24h], administered intravenously for 3 days) was efficacious in a murine model of staphylococcal pneumonia, resulting in 50% survival, compared to 0% survival with the vehicle control; exebacase in addition to daptomycin (50 mg/kg q24h for 3 days) resulted in 70% survival, compared to 0% survival in the daptomycin-alone control group. Overall, exebacase is active in pulmonary environments and may be appropriate for development as a treatment for staphylococcal pneumonia.

S taphylococcus aureus colonizes the skin and mucosal surfaces of up to 60% of the adult human population on a permanent or intermittent basis (1, 2) and is associated with clinical manifestations ranging from mild skin and soft tissue infections to severe and life-threatening diseases such as bacteremia, endocarditis, osteomyelitis, and pneumonia (3). Notably, staphylococci are the most common cause of bacterial pneumonia, at 19.2 cases/ 100,000 population, and are associated with the highest case-fatality rate for bacterial pneumonia, at 15.6 deaths/100 cases, despite antibiotic intervention (4). In the setting of an influenza epidemic, secondary respiratory infections with S. aureus are associated with increased morbidity, particularly in at-risk groups such as the immunocompromised/immunosuppressed (5,6). During the severe acute respiratory syndrome coronavirus (SARS-CoV) outbreak in 2003, up to 30% of patients were diagnosed with secondary bacterial infections (including S. aureus), and coinfection was positively associated with disease severity (7). In a recent multicenter study that included 476 coronavirus disease 2019 (COVID-19) patients, secondary bacterial infections were also significantly associated with outcome severity (8).
For a growing number of infection types, including staphylococcal pneumonia, treatment is confounded by antibiotic resistance (9)(10)(11), resulting in longer hospital stays, higher medical costs, and increased mortality. This issue of rising and globally disseminated antibiotic resistance has created a public health crisis, which requires the development of novel antimicrobial agents, including those with mechanisms of action differentiated from those of traditional antibiotics (12).
Direct lytic agents (DLAs), including lysins, are a new antimicrobial modality to address the unmet need arising from antibiotic resistance (13,14). Lysins are recombinantly produced cell wall peptidoglycan hydrolytic enzymes that elicit rapid cell wall cleavage and concomitant osmotic lysis. Exebacase is an antistaphylococcal lysin with the following microbiological attributes: (i) rapid, targeted bactericidal activity; (ii) the ability to eradicate staphylococcal biofilms; (iii) synergy with antistaphylococcal antibiotics, including daptomycin (DAP) and vancomycin; (iv) a low propensity for the development of resistance; (v) no cross-resistance with antibiotics; (vi) the capacity to both suppress antibiotic resistance and "resensitize" antibiotic-resistant bacteria; and (vii) an extended in vitro and in vivo postantibiotic effect (15)(16)(17)(18)(19)(20). Exebacase recently became the first lysin with published results from a phase 2 proof-of-concept clinical trial, which demonstrated 42.8% higher clinical responder rates with a single dose of exebacase used in addition to standard-of-care antibiotics (SOCAs) than with SOCAs alone for the treatment of methicillin-resistant S. aureus (MRSA) bacteremia, including endocarditis (21). Breakthrough therapy designation has been granted by the U.S. Food and Drug Administration (FDA) for exebacase, which is now also in phase 3 of development (22).
In the present study, the antistaphylococcal activity of exebacase was tested in vitro in the presence of bovine pulmonary surfactant and in vivo in a murine pneumonia model to explore the use of exebacase as a treatment for staphylococcal lung infections, including pneumonia. In a rigorous test of the ability of exebacase to synergize with antistaphylococcal antibiotics, the potency of exebacase in pulmonary environments was examined in addition to daptomycin, a lipopeptide antibiotic approved for use in treating S. aureus skin and soft tissue infections and bacteremia but which is ineffective in treating bronchoalveolar pneumonia because of selective sequestration by and inactivation in pulmonary surfactant (23). The capacity of exebacase to synergize with daptomycin in this proof-of-principle study would highlight the potency of exebacase in pulmonary environments and its promise as a treatment for staphylococcal lung infections.

RESULTS
Exebacase is active in bovine pulmonary surfactant. The activity of exebacase was tested in medium supplemented with increasing amounts of bovine pulmonary surfactant (Survanta), a natural lung extract containing phospholipids, neutral lipids, fatty acids, and surfactant-associated proteins that is used to mimic the surface-tension-lowering properties of natural lung surfactant (24). As indicated in Fig. 1, exebacase was highly active against each of three S. aureus strains, over a range of surfactant concentrations from 1 to 15%. In contrast, daptomycin exhibited a 16-to 512-fold loss of activity for each S. aureus strain across the range of concentrations tested.
Exebacase synergizes with daptomycin in 7.5% bovine pulmonary surfactant. To inform concentration selection in checkerboard assays, single-agent MICs for exebacase and daptomycin were first determined by broth microdilution (BMD) against each of 9 methicillin-sensitive S. aureus (MSSA) and 9 MRSA strains in medium with 7.5% bovine pulmonary surfactant. Exebacase was highly active, with MICs ranging from 1 to 2 mg/ml for all strains tested (Table 1). Daptomycin MICs ranged from 16 to 128 mg/ml, consistent with inhibition of activity in the presence of surfactant. In contrast, MICs for exebacase and daptomycin in the absence of surfactant were each 0.5 to 1 mg/ml.
Based on the single-agent MICs determined as described above, exebacase was tested in addition to daptomycin against each of the 9 MSSA and 9 MRSA strains using a standard checkerboard assay format in medium with 7.5% pulmonary surfactant. Fractional inhibitory concentration index (FICI) values were assessed according to the following criteria: synergy at an FICI of #0.5, additivity at an FICI of .0.5 to #1, no interaction (indifference) at an FICI of .1 to #4, and antagonism at an FICI of .4. Exebacase synergized with daptomycin against each of the 18 strains tested (Table 1). Exebacase MICs were reduced 4-fold, while daptomycin MICs were reduced up to 64-fold. When used in addition to exebacase, the daptomycin MICs were at or near the susceptibility breakpoint of #1mg/ml established by the Clinical and Laboratory Standards Institute (CLSI) (25).
Exebacase promotes daptomycin binding to S. aureus in surfactant. The binding of BODIPY FL-labeled daptomycin (used at a sub-MIC) to MRSA strain MW2 was examined in 7.5% surfactant in the presence or absence sub-MIC exebacase, using both epifluorescence ( Fig. 2) and confocal (Fig. 3) microscopy. Fluorescently labeled boron-dipyrromethene (BODIPY-FL)-labeled daptomycin normally fluoresces green when inserted into the bacterial membrane target but can have a red shift in fluorescence emission when the signal is very intense from a high probe density (26). In the presence of CF-301, BODIPY FL-labeled daptomycin stained S. aureus green or red within 30 min, whereas without CF-301, no staining was observed.
Exebacase and daptomycin act synergistically against S. aureus biofilms in surfactant. The activity of exebacase and/or daptomycin (each at sub-MICs) against biofilms formed by MRSA strain MW2 in 7.5% surfactant was examined by scanning electron microscopy (SEM). Treatments with daptomycin alone for 20 min resulted in densely packed formations of cells similar to those observed in the vehicle control (Fig. 4). While the 20-min treatment with sub-MIC exebacase alone was not disruptive, the biofilms were nonetheless porous and less dense than those observed with either the vehicle or daptomycin treatments. In contrast, exebacase in addition to daptomycin removed the biofilm, leaving only debris and scattered groups of individual bacteria after the 20-min treatment.  (Fig. 5A). ATCC BAA-42 is a human respiratory isolate also known as strain HDE288 (36). At 14 days, mice treated with exebacase alone or in addition to daptomycin yielded 50% and 70% survival rates, respectively, whereas treatment with daptomycin alone or the vehicle yielded no survivors by 8 days. Exebacase alone and in addition to daptomycin was superior to either daptomycin alone or the vehicle control (P , 0.05 by a log rank test). 5% pulmonary surfactant and when tested in addition to each other in the presence of 7.5% pulmonary surfactant. The fold reduction in the MIC is based on the decrease observed for each agent in the synergistic combination compared to the value obtained as a single agent. b The bacterial strains are described in Table S1 in the supplemental material. While survival was the primary endpoint in this proof-of-concept study, bacterial loads were also determined in the lungs of infected animals at the 1-and 3-day time points after the start of treatment. Only the exebacase plus daptomycin treatment group exhibited significant 1-and 2-log 10 decreases compared to the starting bacterial inoculum at 1 and 3 days, respectively (Fig. 5B). Bacterial loads were not determined at the later time points up to 14 days.

DISCUSSION
Novel antimicrobial agents with differentiated mechanisms of action compared to those of current and long-standing antibiotic classes are needed to confront the urgent unmet medical need resulting from drug-and multidrug-resistant bacteria, including S. aureus, a ubiquitous and versatile human pathogen. Direct lytic agents, in particular lysins, represent novel therapeutic modalities distinguished by a notably potent enzymatic mechanism of action (peptidoglycan hydrolysis) and bacteriolytic effect. The therapeutic potential of lysins is strongly supported by positive proof-of-concept data from a completed phase 2 clinical trial of exebacase and the initiation of the phase 3 DISRUPT trial to assess the efficacy and safety of exebacase used in addition to standard antibiotics in patients with S. aureus bacteremia, including right-sided endocarditis (21,22).
In the current study, the potential for using exebacase as a treatment for another staphylococcal infection with a high unmet need (i.e., bronchopneumonia) was demonstrated. Exebacase exhibited potent activity and low MICs in growth media supplemented with bovine pulmonary surfactant, unlike daptomycin, which was inhibited by up 512-fold. The inactivity of daptomycin has been attributed to insertion into lipid aggregates within the surfactant, which then precludes insertion into the membrane of target Gram-positive bacteria (23); sequestration within and inhibition by the surfactant may explain the failure of daptomycin in clinical trials for community-acquired pneumonia. Conversely, the potent activity demonstrated for exebacase in bovine surfactant predicts a high level of activity for the enzyme in the presence of natural surfactant in pulmonary environments. Indeed, systemically administered exebacase, tested only at a 5-mg/kg dose, resulted in 50% survival among mice infected intranasally with an otherwise lethal dose of S. aureus. The efficacy of exebacase in the murine pneumonia model is consistent with the capacity to both penetrate the pulmonary environment and exert antistaphylococcal activity in the presence of a "natural" surfactant. Synergy between exebacase and daptomycin was previously demonstrated in checkerboard and time-kill assays performed using cation-adjusted Mueller-Hinton broth supplemented with 25% horse serum and 0.5 mM dithiothreitol (CAMHB-HSD) albeit in the absence of surfactant (16). The synergy now demonstrated in the presence of surfactant is particularly compelling considering that daptomycin is inactive in surfactant in the absence of exebacase. By reducing the daptomycin MICs by up to 64fold to nearly breakpoint values, synergy with exebacase "activated" the antibiotic. This activation was visualized and observed to be based on the enhanced binding of BODIPY-labeled daptomycin to the staphylococcal membrane target in the presence of exebacase. Synergy was furthermore observed to facilitate antibiofilm activity in vitro and enhanced efficacy (70% survival versus no survival in the vehicle control group) in the murine pneumonia model.
The mechanism by which exebacase promotes daptomycin binding and synergistic activity remains to be determined. Exebacase may diminish the sequestration of daptomycin within lipid aggregates in pulmonary surfactants, or the cell wall hydrolytic activity of exebacase may facilitate the access of nonaggregated antibiotics to the target membrane. While the pattern of BODIPY-DAP surface labeling observed in our study, which is defined by distinct foci of enhanced fluorescence, is similar to that previously reported (28,29), it does not show distinct septal binding as has also been observed for daptomycin (30). Exebacase may thus displace the antibiotic from the division plane and favor accumulation at other sensitive sites.
Overall, we have provided evidence of the capacities of exebacase to kill S. aureus under in vitro conditions mimicking the respiratory environment and to confer a survival benefit in vivo in the lungs of experimentally infected mice. These findings support consideration for exebacase development as a novel treatment, with a differentiated mechanism of action, for staphylococcal pneumonia and other difficult-to-treat respiratory infections, including MRSA-related pulmonary exacerbations of cystic fibrosis. While exebacase has been shown to synergize with a wide range of antistaphylococcal antibiotics in checkerboard and time-kill assay formats (15,16) and in rat and rabbit models of bacteremia and endocarditis (31), until now, the potential ability of exebacase to synergize with antibiotics in the setting of in vivo lung infections has not been demonstrated. Daptomycin, an antistaphylococcal antibiotic known to be inactive in the pulmonary environment, was specifically chosen as the "partner antibiotic" in this proof-of-principle study to provide the most rigorous test of the potential ability of exebacase to synergize with antistaphylococcal antibiotics. Based on the activity of exebacase observed in the current work, including the ability to synergize with daptomycin in the pulmonary environment, future work will include translational studies in higher-order species (e.g., the rabbit pulmonary infection model) testing the ability of exebacase to improve outcomes when used in addition to antistaphylococcal antibiotics commonly used clinically to treat pneumonia caused by S. aureus (e.g., vancomycin and linezolid, etc.) (32,33).

MATERIALS AND METHODS
Bacterial strains and reagents. Exebacase (CF-301) (.99% pure) was prepared by ContraFect Corporation (Yonkers, NY). Daptomycin was obtained from Sigma-Aldrich (St. Louis, MO). All S. aureus strains were obtained from the American Type Culture Collection (ATCC), BEI Resources (NRS), and JMI Laboratories, as indicated in Table S1 in the supplemental material. Frozen strains were revived on BBL Trypticase soy agar plates with 5% sheep blood (TSAB; Becton, Dickinson and Company [BD]) and incubated at 37°C overnight for single colonies. DAPI (49,6-diamidino-2-phenylindole dihydrochloride) was obtained from Thermo Fisher Scientific. Other growth media included tryptic soy broth (TSB) from Hardy Diagnostics (VWR International), TSB with 0.2% D-glucose (TSBg), and BBL cation-adjusted Mueller-Hinton II broth (CAMHB; Becton, Dickinson and Company). Horse serum (donor herd, sterile filtered, and not heat inactivated) was obtained from Sigma-Aldrich. D-Glucose and DL-dithiothreitol (DTT) were obtained from Sigma-Aldrich. Survanta (Beractant) was obtained from Myonex Incorporated and is a modified bovine pulmonary surfactant consisting of 25 mg/ml phospholipids, 0.5 to 1.75 mg/ml triglycerides, 1.4 to 3.5 mg/ml free fatty acids, and ,1.0 mg/ml total surfactant proteins.
MIC assays. Exebacase MICs were measured by broth microdilution (BMD) according to the CLSI M07-A11 methodology (34) and incorporating a CLSI-approved antimicrobial susceptibility testing (AST) medium comprised of CAMHB supplemented with horse serum (Sigma-Aldrich) and DL-dithiothreitol