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CFRX: Developing Gram-Negative Lysin Targeting P. aeruginosa

06/18/2018
By David Bautz, PhD

NASDAQ:CFRX

READ THE FULL RESEARCH REPORT

Business Update

Presentations at ASM 2018 Highlight New Gram-Negative Lysin Program and CF-301

In June 2018, ContraFect Corp. (NASDAQ:CFRX) had multiple presentations at the American Society for Microbiology (ASM) Microbe 2018 on the company’s lead drug candidate CF-301 and the Gram-negative lysin discovery program. A summary of each of the poster’s is presented below.

Bacteriophage-Derived Lysins can be Engineered to Exert a Rapid and Potent Bactericidal Effect Against Pseudomonas aeruginosa in Serum

This study was undertaken as part of ContraFect’s Gram-negative lysin discovery program. Gram-negative (GN) bacteria are responsible for a number of different bacterial infections and the emergence of antibiotic resistant strains is leading to a potential public health crisis. Lysins represent a novel means to treat these infections through their ability to cleave peptidoglycan bonds (peptidoglycan is the main structural component of bacterial cell walls), however their use against GN pathogens has been hindered by their inability to penetrate the outer membrane. The following figure shows how lysins are effective against Gram-positive bacteria due to their ability to easily interact with the peptidoglycan layer. However, Gram-negative bacteria have an outer membrane that acts as a barrier against most lysins, thus preventing them from reaching the peptidoglycan layer. While the majority of purified GN lysins have no antimicrobial activity, there are a select few that have some activity in low ionic strength buffers (indicated by the asterisk in the following figure on the right). It is these lysins that ContraFect used as lead compounds to modify in order to increase their anti-microbial activity in human serum.



View Image I

Following screening of naturally occurring GN lysins for anti-microbial activity and targeted sequence modifications, lead lysins were tested in a variety of assays including minimum inhibitory concentration (MIC) determination, anti-biofilm activity, synergistic assays with antibiotics, and hemolysis assays. Lead lysins exhibited MIC values between 0.06 and 16 μg/mL, anti-biofilm activity that was below the MIC value, were synergistic with a broad range of antibiotics, and did not demonstrate any hemolytic activity. An in vitro screening assay was then performed to determine which of those lead lysins had rapid bactericidal activity. The following show examples of lead lysins in a bactericidal activity assay after incubation with P. aeruginosa for 15 minutes in 100% human sera.



View Image II

The most important takeaway from these studies is that it is possible to engineer lysins with rapid bactericidal activity against GN pathogens, which greatly broadens the use of lysins and could greatly expand ContraFect’s pipeline. We look forward to learning more about the company’s GN pipeline and selection of lead development lysins for use against P. aeruginosa as well as what specific indications the company will be pursuing.

Lysin CF-301 (exebacase) Administered in Addition to Vancomycin (VAN) Suppresses the Emergence of Reduced Susceptibilities to VAN Within Cardiac Vegetations in a Rabbit Model of MRSA Infective Endocarditis (IE)

This study was performed to test whether treating animals with IE with CF-301 had any effect on the development of resistance to VAN. Rabbits with IE were treated with 1) VAN alone; 2) CF-301 alone; 3) VAN + CF-301; or 4) vehicle control. Vegetations (microbial growth) were isolated from rabbits with IE and tested for resistance to VAN, CF-301, and oxacillin. MIC values against oxacillin were determined based on previous data showing that treatment with CF-301 resulted in decreased oxacillin MICs.

The following two tables show analysis of MIC from isolates recovered from non-selective growth plates and selective growth plates with CF-301 added. The selective growth plates were utilized as a means to enrich for isolates with increased CF-301 MICs. For isolates from the non-selective plates, only two showed a 2-fold increase in MIC, one of which was from the control group of rabbits while the other was from a group of 32 isolates tested from rabbits treated with 1.4 μg/kg CF-301. In addition, CF-301 suppressed any increase in VAN MIC (red box) and caused an increase in susceptibility to oxacillin treatment in certain isolates (gray boxes).



View Image III

Similar results were seen for isolates plated on the selective growth plates, although there were a few more isolates with a modest increase in MIC (≤ 2-fold increase). Some isolates showed increased susceptibility to oxacillin (gray boxes), which was similar to what was seen in the same assay performed with CF-301 and daptomycin.



View Image IV

The most important conclusion from this study is that while a modest increase in CF-301 MIC was observed, based on PK modeling this likely will not change the susceptibility of those variants to the clinical dose of 0.25 mg/kg being utilized in the ongoing Phase 2 study.

Lysin CF-301 (exebacase) Activates Latent Host Factors in Human Blood to Potentiate Bacteriolysis

The purpose of this study was to test the activity of CF-301 in various mammalian and rodent blood matrices compared to cation-supplemented Mueller Hinton Broth (caMHB). A wide variance in activity was discovered between the different blood matrices and caMHB. CF-301 MICs observed in rabbit, dog, horse, and human blood matrices were up to 64-fold lower compared to MICs obtained in rat and mouse blood. The following graphs show that minimum sterilizing concentrations for CF-301 in human blood was 3.2 μg/mL, 32 μg/mL for rat blood, and ≥ 320 μg/mL in mouse blood. These results were seen when testing 10 different staphylococcal strains and in blood from multiple pooled individuals and polled sources.



View Image V

These results led to the hypothesis that CF-301 synergizes with some component(s) or activates latent anti-staphylococcal activity in the blood. To test this, CF-301 was tested with a wide range of serum proteins in caMHB. The following chart shows that human lysozyme (HuLYZ) and albumin (HSA) were identified in checkerboards (A), lytic assay (B), and time-kill assays (C).



View Image VI

The mechanism by which this enhancement occurred was investigated and found to be due to the accumulation of CF-301 at the surface of S. aureus cells based on its interactions with HSA and facilitation of HuLYZ activity. The following image shows much greater binding of CF-301 to S. aureus cells (depicted by red color) in human and rabbit blood compared to rat or mouse blood.



View Image VII

These in vitro results were confirmed by an in vivo test performed in rabbit and rat IE models. The following graphs show that a >50-fold higher dose of CF-301 was required for similar efficacy in the rat model (10 mg/kg) compared to the rabbit model (0.09 mg/kg).



View Image VIII

The results of these studies show that CF-301 has differentiated characteristics compared to small molecule antibiotics in that binding to albumin increases activity of the enzyme, while binding of small molecule antibiotics to albumin typically decreases their activity. In addition, it reaffirms the company’s use of 0.25 mg/kg dose in the Phase 2 study.

Lysin CF-301 (exebacase) Demonstrates Potent in vitro Activity Against a Range of Staphylococcus and Streptococcus Species Associated with Complicated Bacteremia and Infective Endocarditis (IE) in Humans

This experiment was performed to determine which species of staphylococcal and streptococcal bacteria known to cause IE are susceptible to CF-301. MIC values for CF-301, vancomycin (VAN), and daptomycin (DAP) were determined against a total of six staphylococcal and 13 streptococcal species. The following two tables show the MIC values for CF-301 against the tested strains. Importantly, all of the staphylococcal strains tested were susceptible with MIC values for CF-301 of 1-2 μg/mL.



View Image IX

MIC values for CF-301 were higher for most of the streptococcus species, although S. intermedius, S. dysgalactiae, S. pyogenes and S. agalactiae were all susceptible (MIC = 0.5, 2, 2, and 2 μg/mL, respectively).



View Image X

The important take away from this study was that CF-301 is active against a wide range of staphylococcal species, and some streptococcal species, and may have the potential to treat IE caused by these species.

CF-301 (Exebacase) Activity versus Contemporary Staphylococcus aureus Clinical Isolates from Six US Hospitals

This study was undertaken to determine the effectiveness of CF-301 against S. aureus clinical isolates collected in the U.S. A total of 300 samples were tested that included both methicillin-sensitive (MSSA, n = 150) and methicillin-resistant (MRSA, n=150) S. aureus, thus they represent a “real-world” test of CF-301 activity. The following table shows the number of isolates that were inhibited by various concentrations of CF-301. This data is in agreement with a clinical isolate study from 2011 performed in the U.S. and a recently reported clinical isolate study from European hospitals. Importantly, none of the isolates had a MIC value higher than 1 μg/mL, which is expected to correspond to susceptibility to the clinical dose of 0.25 mg/kg being used in the Phase 2 study.



View Image XI

No Safety Issues Thus Far in Phase 2 Trial of CF-301

ContraFect is currently conducting a Phase 2 clinical trial of CF-301 in patients with bacteremia, including those with endocarditis, which is caused by both methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) strains of S. aureus. The trial is an international, multicenter, randomized, double blind, placebo controlled study with a superiority comparison between CF-301 or placebo combined with the standard of care antibiotics. The study will include 115 patients randomized 3:2 to receive a single dose of 0.25 mg/kg CF-301 administered via a two-hour infusion or placebo along with standard of care antibiotics. The primary endpoint of the study will be early clinical response. Safety, tolerability, and pharmacokinetics will also be examined along with additional exploratory clinical and health economic endpoints. We anticipate topline results in the fourth quarter of 2018. The company is not planning to perform an interim analysis.

Based on a review of safety data examined after approximately one-half of target enrollment was reached in the Phase 2 trial, there have been no serious adverse events related to study drug, no reports of adverse events due to hypersensitivity reactions related to study drug, and no discontinuations of study drug due to adverse events. Since CF-301 is the first lysin to be tested in humans it is important to show that it can be safety administered, thus we are encouraged by the fact that no safety issues have been reported thus far. ContraFect previously conducted a Phase 1 clinical trial of CF-301 in healthy volunteers, with no adverse safety signals reported, and the encouraging safety data from the Phase 2 trial adds to our confidence in the ability of CF-301 to be used safely to treat patients.

The Data Safety Monitoring Board (DSMB) has recommended that patients with moderate to severe renal insufficiency be administered 0.12 mg/kg CF-301 instead of 0.25 mg/kg such that those patients attain the target pharmacokinetic exposure. This recommendation was not made due to any observed safety concerns.

Conclusion

The Gram-negative lysin program is an exciting opportunity for the company and the data that has been compiled thus far for the development candidates is very encouraging. We look forward to getting updates on this program as it progresses.

The data presented at ASM for CF-301 gives us further confidence in its ability to treat S. aureus infections (including those caused by MRSA) and we are looking forward to seeing the data from the Phase 2 study in the fourth quarter of 2018. We believe interest in ContraFect and the lysin story will begin to increase as we get closer to the data readout. For example, Reuters recently had a report on lysins that featured ContraFect CMO Dr. Cara Cassino, which can be viewed here. Our valuation currently stands at $7 and given the wide disparity between our valuation and the current stock price we believe investors would be well served to take a closer look at the company in the lead up to the Phase 2 data.

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