(366m) Phage-Drug Conjugates for Antimicrobial Drug Delivery

The rate of drug development is insufficient to meet the current and future clinical needs to combat antimicrobial resistance, especially in gram-negative bacteria. Polymyxins are a family of cyclic lipopeptides that display exceptional antibiotic activities toward gram-negative pathogens such as E. coli, Pseudomonas aeruginosa, and Acinetobacter baumannii4. Polymyxin B (PMB) is composed of a positively charged peptide ring that interacts non-specifically with the negatively charged bacterial surface, and a fatty acyl chain that embeds into and disrupts the cell membrane, leading to cell death. PMB and polymyxin E (colistin) are the only polymyxins in clinical use. Between them, PMB is usually preferred for treating systemic infections intravenously due to better pharmacokinetic properties compared to colistin (a prodrug). Polymyxins are positively charged and have moderate nonspecific affinity for both bacterial and mammalian cells, leading to serious side effects. PMB was introduced clinically in the late 1950s, but due to its high (up to 60%) rate of nephrotoxicity, it was sidelined in the 1970s in favor of safer antibiotics. The PMB label includes an FDA black box warning for serious nephrotoxicity and neurotoxicity. However, today, with the growing emergence of “superbugs” and a dry antibiotic discovery pipeline, PMB is increasingly used as a last-line therapy to treat systemic infections, including from carbapenem-resistant or multidrug-resistant E. coli, when the clinical pathogens are resistant to frontline antibiotics such as -lactams, aminoglycosides and fluoroquinolones. Nevertheless, the use of PMB is still extremely conserved, with nephrotoxicity leading to renal failure from acute tubular necrosis being the major dose-limiting factor for PMB administration. Clinical use is currently limited to hospitalized patients under physician supervision due to the need to closely monitor renal function. Thus, although PMB is a potent antibacterial agent, its toxicity currently prevents its use as an earlier or more widespread treatment.

A general strategy to overcome toxic side effects is to deliver drugs specifically to the targeted cells, avoiding harm to other tissues. In this case, PMB should be delivered specifically to bacterial cells and not to the general circulation or mammalian cells (especially kidney cells). Bacteriophages (phages) are viruses that are specific to bacteria and appear to be safe when used in therapy for humans. Due to their biocompatibility and therapeutic potential, phages are increasingly studied and engineered for various applications such as drug delivery, biosensing, and cancer therapy. A well-characterized phage is M13, a filamentous phage that is about 1 in length and 6 in diameter, that normally infects E. coli (F+ strains) through attachment to the conjugative F pilus. M13 has a 6.4 kb single-stranded, positive-sense, circular DNA genome packaged inside its capsid, which is normally composed of ~2700 copies of the major capsid protein pVIII (also called g8p), forming the bulk of the filamentous structure, and three to five copies of four minor capsid proteins, including the receptor-binding protein pIII (also called g3p). M13 phage is used prominently for phage display technology, in which protein variants can be rapidly selected for binding activity (‘biopanning’). Phage display has generated recombinant M13 phages that bind to a wide variety of targets through display of antibody fragments fused to pIII. At the same time, since pVIII carries solvent-exposed carboxylates as well as primary amine groups, M13 phages have the potential for chemical surface modifications to achieve various functionalities and deliver payloads such as drugs. While antibodies can also bind specifically to targeted cells, due to its much greater surface area, M13 phages could potentially deliver hundreds of times more cargo compared to antibodies. Thus, in our project, we aim to prove the hypothesis that M13 phages can be engineered to bind to specific targets, such as bacterial pathogens, and can deliver thousands of drug molecules, specifically polymyxin B, at once to the targeted cell surface. Targeting PMB to bacteria by loading the drug onto phages could simultaneously deliver large concentrations of PMB to the bacterial cell surface and avoid uptake of PMB by mammalian cells, improving both safety and efficacy.

Wild-type phages are usually extremely specific for their hosts, which can limit clinical utility of phages in bacteremia. For example, wild-type M13 only infects E. coli strains that express the F pilus. Defining the surface antigens of a clinical bacterial isolate from a given patient would be a time-consuming step. Bacteremia can become critical rapidly, and a delay of a few hours can significantly worsen patient outcomes. Therefore, we sought to create a phage that would bind to a wide range of gram-negative bacterial organisms. In preliminary work, we engineered M13 to bind the core antigen of lipopolysaccharide (LPS), a structure common to gram-negative bacteria and a critical component of their outer membranes. LPS is composed of three domain sections, the lipid A, core oligosaccharide and O-antigen. This recombinant anti-LPS phage was by design to bind to the core-oligosaccharide domain of LPS and was verified to bind to a variety of top-priority pathogenic gram-negative bacterial species recognized by the CDC and WHO, including E. coli, P. aeruginosa, A. baumannii, Klebsiella pneumoniae, and Burkholderia cepacia. Since the anti-LPS phages are able to bind but do not infect (nonviable), they are not cytotoxic by themselves. However, delivery of PMB by the anti-LPS phage confers antibiotic activity, since the mechanism of action for PMB, disruption of the cell membrane, occurs at the cell surface exterior without drug internalization. Thus, in our strategy, PMB molecules are conjugated to the anti-LPS phage capsid, and delivered specifically to gram-negative bacterial cells through the specificity and high affinity of the anti-LPS binding domain. We have shown that the PMB – anti-LPS phage conjugate (termed “PMB–Phage” hereafter) effectively lowers the minimal inhibitory concentration of PMB by multiple orders of magnitude in vitro compared to PMB in the standard free drug formulation (PMB sulfate). In addition, by essentially achieving a higher local concentration, bacterial strains that are resistant to PMB sulfate are actually sensitive to PMB-Phage. Thus, we hypothesize that our new formulation, PMB–Phage, will solve the problematic toxicity of PMB and increase its therapeutic index, allowing clinicians to take full advantage of this highly potent bactericidal drug to treat patients suffering from both system infections such as bloodstream infection, pneumonia and local infections such as corneal keratitis.

Through our engineering, we have obtain the following results

We have further demonstrated that the phage-drug conjugate can be expanded in general as a platform technology for other drugs and other therapeutic purposes. The phages can be accessibly engineered to bind specifically to other cell surface antigens. Other drug compound current be developed in our lab can also be delivered in this mode, achieving enhanced therapeutic results. This technology has been submitted and filed in US Provisional Patent 63/597,619 owned by UCLA.