Patent Application
20230277632
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 18, 2021 is named 0341_0027-PRO_ST25.txt and is 36,229 bytes in size.
The present disclosure relates generally to antibacterial agents and more specifically to modified, non-naturally occurring lysin polypeptides, notably, modified PlySs2 lytic enzymes, and the use of these peptides in combination with antibiotics in killing Gram-positive bacteria and combatting bacterial infection and contamination.
Antibiotic resistance is on the increase worldwide, influenced, inter alia, by (a) increased and prolonged use of antibiotics administered to treat a variety of illnesses and other conditions; (b) poor patient compliance; and (c) a paucity of new antimicrobial agents that can be deployed against pathogens that have developed resistance to existing antibiotics.
Bacteriophage endolysins (lysins) represent a promising alternative or complementary approach to combating bacterial infections and to overcoming bacterial resistance. Lysins are peptidoglycan hydrolases that can be produced naturally by bacteriophages. When contacting the bacteria from the outside, recombinantly-produced lysin polypeptides directly lyse and kill the bacteria [1], [2]. Lysins may also overcome antibiotic resistance by facilitating access of the antibiotic agents to pathogens. Several studies have recently demonstrated the strong potential of these enzymes in human and veterinary medicine to control pathogens on mucosal surfaces, in organ-confined infections, and in systemic infections.
Gram-positive bacteria are surrounded by a cell wall containing polypeptides and polysaccharides. The gram-positive cell wall appears as a broad, dense wall that may be about 20-80 nm thick and contains numerous interconnecting layers of peptidoglycan. Between 60% and 90% of the gram-positive cell wall is peptidoglycan, providing cell shape, a rigid structure, and resistance to osmotic shock. The cell wall does not exclude the Gram stain crystal violet, allowing cells to be stained purple, and therefore classified as “Gram-positive.”
Bacteriophage lytic enzymes have been established as useful in the specific treatment of various types of infection in subjects through various routes of administration. See e.g., U.S. Pat. Nos. 5,985,271; 6,017,528; 6,056,955; 6,248,324; 6,254,866; and 6,264,945. U.S. Pat. No. 9,034,322 to Fischetti et al., which is hereby incorporated by reference in its entirety, is directed to bacteriophage lysins derived from Streptococcus suis bacteria, including the lysin PlySs2. These lysin polypeptides demonstrate broad killing activity against multiple bacteria, including Gram-positive bacteria such as Staphylococcus, Streptococcus Group B, Enterococcus, and Listeria bacterial strains.
The PlySs2 lysin is capable of killing Staphylococcus aureus bacteria in animal models, synergizing with antibiotics, and overcoming (or preventing) antibiotic resistance. PlySs2 was shown to be effective against antibiotic-resistant Staphylococcus aureus, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA).
The elicitation of an inflammatory immune response by a therapeutic protein is undesirable [6], [8]. Protein immunogenicity may be of concern for exogenous proteins and peptides, whether they are derived from synthetic or biological (such as recombinant) sources. In some instances, therapeutic proteins have caused severe adverse events (for example, anemia, thrombocytopenia, anaphylaxis, and innate immune responses) that can even be fatal in certain instances. Well-known examples include thrombocytopenia in patients treated with thrombopoietin and pure red cell aplasia in chronic renal disease patients treated with EPREX®, an approved erythropoietin (EPO) product, following induction of neutralizing antibodies that cross-reacted with the functionally non-redundant endogenous EPO. Since immunogenicity has been reported with various products, including monoclonal antibodies, thrombokinase, and PULMOZYME® (dornase alfa), immunogenicity continues to receive significant attention from regulatory bodies, industry, and clinicians. Such immune responses may be potentially exacerbated in a patient who is already experiencing inflammatory response because of the underlying bacterial infection.
As lysins, such as PlySs2, are proteins, they have the potential of eliciting an immune response when administered to a host. In addition to causing severe adverse effects, as discussed above, such immune responses can also decrease lytic activity of a lysin. Indeed, some immune responses have been observed in animals with other types of lysins, such as Cpl-1, which caused a slowdown in the lytic activity but did not substantially inhibit it. Other researchers, however, observed that administration of Cpl-1 was accompanied by a substantial increase in inflammatory cytokine secretion [9].
In silico, computationally-guided tools have been developed to facilitate the identification of epitopic regions of proteins and to design variants that are less predisposed to being immunogenic [11]-[18]. Although such techniques may be helpful, they have some limitations. These limitations may include, for example: antigen processing, which can eliminate some putative T-cell epitopes; inability to predict T-cell receptor affinity using in silico methods due to the pleiomorphism and three-dimensional complexity of the T-cell receptor; inability to predict T-cell epitope phenotypes; limitations in the applicability of statistical techniques devised with patient populations in mind to an individual patient and influence of post-translational factors; and inherent limitations of in silico techniques and the assumptions underlying them [8]. Accordingly, there remains trial and error and uncertainty in efforts to reduce the immunogenicity of peptides.
Thus, the discovery of modified lysins, such as modified PlySs2 lysins, that retain desired antibacterial activity, including enhanced antibacterial activity when used in combination with standard-of-care antibiotics, yet have reduced immunogenicity, would be beneficial.
In one aspect, a method is provided for preventing or treating a bacterial infection caused by at least one species of Gram-positive bacteria, the method comprising co-administering to a subject diagnosed with, at risk for, or exhibiting symptoms of the bacterial infection (1) a first amount of a modified lysin polypeptide having at least one amino acid substitution relative to a counterpart wild-type PlySs2 lysin; and (2) a second amount of an antibiotic suitable for the treatment of a Gram-positive bacterial infection.
In another aspect, a method is provided for augmenting the efficacy of an antibiotic suitable for the treatment of a Gram-positive bacterial infection, comprising co-administering the antibiotic in combination with a modified lysin polypeptide having at least one amino acid substitution relative to a counterpart wild-type PlySs2 lysin, wherein co-administration is more effective in inhibiting the growth, or reducing the population, or killing the Gram-positive bacteria than administration of either the antibiotic or the modified lysin polypeptide individually.
In another aspect, a combination is provided, wherein the combination comprises a modified lysin polypeptide and an antibiotic. In certain embodiments, the minimum amount of the modified lysin polypeptide, the antibiotic, or both that is effective in the combination is below the respective MIC amount of the modified lysin polypeptide and/or the antibiotic. In some embodiments, the modified lysin polypeptide and the antibiotic are provided in the same composition, and in certain embodiments, the modified lysin polypeptide and the antibiotic are provided in different compositions.
In some embodiments of all aspects of the disclosure, the antibiotic is one or more of a penicillin such as methicillin or oxacillin, glycopeptides such as vancomycin, cyclic lipopeptides such as daptomycin, mupirocin, and lysostaphin. In certain embodiments, the antibiotic is one or more of vancomycin and daptomycin. In other embodiments, the antibiotic is oxacillin.
In some embodiments of all aspects of the disclosure, the amount of the modified lysin polypeptide used in the foregoing methods may be below that which would result in a concentration equal to the MIC of the modified lysin polypeptide when used in the absence of antibiotic (i.e., a “sub-MIC lysin amount”); alternatively or additionally, the amount of antibiotic used in the foregoing methods may be below that which corresponds, i.e., which would result in, a concentration equal to the MIC for the antibiotic when used in the absence of the modified lysin polypeptide (i.e., a “sub-MIC antibiotic amount”).
Typically, the modified lysin polypeptide has reduced immunogenicity as compared to the counterpart wild-type PlySs2 lysin. The wild-type PlySs2 lysin has a cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) endopeptidase domain, which is the enzymatically active domain (EAD) of the PlySs2 polypeptide, and a C-terminal SH3b_5 (SH3b) cell wall-binding domain (CBD). In certain embodiments of all aspects of the disclosure, the modified lysin polypeptides comprise at least one amino acid substitutions in the CHAP and/or one or more amino acid substitutions in the SH3b.
In certain embodiments of all aspects of the disclosure, the modified lysin polypeptide comprises at least one amino acid substitution as compared to a wild-type PlySs2 lysin polypeptide, wherein the wild-type PlySs2 lysin polypeptide has an amino acid sequence of SEQ ID NO: 1, a cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain, and a cell wall binding (SH3b) domain, and wherein the at least one amino acid substitution is in the CHAP domain and/or the SH3b domain, wherein the modified lysin polypeptide inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria.
In certain embodiments of all aspects of the disclosure, the modified lysin polypeptide comprises the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1: L92W, V104S, V128T, Y137S, Y164K, N184D, and S198Q (pp296).
Also disclosed are active fragments of the modified lysin polypeptides disclosed herein, where the active fragments include one or more of the amino acid substitutions in the CHAP domain and/or the SH3b domain.
As used herein, the following terms and cognates thereof shall have the following meanings unless the context clearly indicates otherwise:
“Carrier,” refers to a solvent, additive, excipient, dispersion medium, solubilizing agent, coating, preservative, isotonic and absorption delaying agent, surfactant, propellant, diluent, vehicle and the like with which an active compound is administered. Such carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
“Pharmaceutically acceptable carrier” refers to any and all solvents, additives, excipients, dispersion media, solubilizing agents, coatings, preservatives, isotonic and absorption delaying agents, surfactants, propellants, diluents, vehicles and the like that are physiologically compatible. The carrier(s) must be “acceptable” in the sense of not being deleterious to the subject to be treated in amounts typically used in medicaments. Pharmaceutically acceptable carriers are compatible with the other ingredients of the composition without rendering the composition unsuitable for its intended purpose.
Furthermore, pharmaceutically acceptable carriers are suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are “undue” when their risk outweighs the benefit provided by the composition. Non-limiting examples of pharmaceutically acceptable carriers or excipients include any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, and emulsions such as oil/water emulsions and microemulsions. Suitable pharmaceutical carriers are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin, 18th Edition.
“Bactericidal” refers to the property of causing the death of bacteria or capable of killing bacteria to an extent of at least a 3-log 10 (99.9%) or better reduction among an initial population of bacteria over an 18-24 hour period.
“Bacteriostatic” refer to the property of inhibiting bacterial growth, including inhibiting growing bacterial cells, thus causing a 2-log 10 (99%) or better and up to just under a 3-log reduction among an initial population of bacteria over an 18-24 hour period.
“Antibacterial” refers to both bacteriostatic and bactericidal agents.
“Antibiotic” refers to a compound having properties that have a negative effect on bacteria, such as lethality or reduction of growth. An antibiotic can have a negative effect on Gram-positive bacteria, Gram-negative bacteria, or both. By way of example, an antibiotic can affect cell wall peptidoglycan biosynthesis, cell membrane integrity, or DNA or protein synthesis in bacteria. Nonlimiting examples of antibiotics active against Gram-positive bacteria include methicillin, oxacillin, vancomycin, daptomycin, mupirocin, lysostaphin, penicillins, cloxacillin, erythromycin, carbapenems, cephalosporins, glycopeptides, lincosamides, azithromycin, clarithromycin, roxithromycin, telithromycin, spiramycin, and fidaxomicin.
“Drug resistant” generally refers to a bacterium that is resistant to the antibacterial activity of a drug. When used in certain ways, drug resistance may specifically refer to antibiotic resistance. In some cases, a bacterium that is generally susceptible to a particular antibiotic can develop resistance to the antibiotic, thereby becoming a drug resistant microbe or strain. A “multi-drug resistant” (“MDR”) pathogen is one that has developed resistance to at least two classes of antimicrobial drugs, each used as monotherapy. For example, certain strains of S. aureus have been found to be resistant to several antibiotics including methicillin and/or vancomycin (Antibiotic Resistant Threats in the United States, 2013, U.S. Department of Health and Services, Centers for Disease Control and Prevention). One skilled in the art can readily determine if a bacterium is drug resistant using routine laboratory techniques that determine the susceptibility or resistance of a bacterium to a drug or antibiotic.
“Effective amount” refers to an amount which, when applied or administered in an appropriate frequency or dosing regimen, is sufficient to prevent, reduce, inhibit, or eliminate bacterial growth or bacterial burden or to prevent, reduce, or ameliorate the onset, severity, duration, or progression of the disorder being treated (for example, bacterial pathogen growth or infection), prevent the advancement of the disorder being treated, cause the regression of the disorder being treated, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy, such as antibiotic or bacteriostatic therapy.
“Co-administer” is intended to embrace separate administration of two agents, such as a lysin peptide and an antibiotic or any other antibacterial agent in a sequential manner as well as administration of these agents in a substantially simultaneous manner, such as in a single mixture/composition or in doses given separately, but nonetheless administered substantially simultaneously to the subject, for example at different times in the same day or 24-hour period. Such co-administration of lysin peptides with one or more additional antibacterial agents can be provided as a continuous treatment lasting up to days, weeks, or months. Additionally, depending on the use, the co-administration need not be continuous or coextensive. For example, if the use were as a topical antibacterial agent to treat, e.g., a bacterial ulcer or an infected diabetic ulcer, the lysin polypeptide could be administered only initially within 24 hours of the first antibiotic use, and then the antibiotic use may continue without further administration of the lysin polypeptide.
“Subject” refers to a mammal, a plant, a lower animal, a single cell organism, or a cell culture. For example, the term “subject” is intended to include organisms, e.g., prokaryotes and eukaryotes, which are susceptible to or afflicted with bacterial infections, for example Gram-positive or Gram-negative bacterial infections. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, e.g., a human suffering from, at risk of suffering from, or susceptible to infection by Gram-positive bacteria, whether such infection be systemic, topical or otherwise concentrated or confined to a particular organ or tissue.
“Polypeptide” is used interchangeably with the term “protein,” “peptide,” and refers to a polymer made from amino acid residues. In certain embodiments, the polypeptide has at least about 30 amino acid residues. The term may include not only polypeptides in isolated form, but also active fragments and derivatives thereof. The term “polypeptide” also encompasses fusion proteins or fusion polypeptides comprising a modified lysin polypeptide as described herein and maintaining the lysin function. Depending on context, a polypeptide can be a naturally-occurring polypeptide or a recombinant, engineered, or synthetically-produced polypeptide. A particular lysin polypeptide can be, for example, derived or removed from a native protein by enzymatic or chemical cleavage, or can be prepared using conventional peptide synthesis techniques (e.g., solid phase synthesis) or molecular biology techniques (such as those disclosed in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)) or can be strategically truncated or segmented yielding active fragments, maintaining lytic activity against the same or at least one common target bacterium.
“Fusion polypeptide” refers to an expression product resulting from the fusion of two or more nucleic acid segments, resulting in a fused expression product typically having two or more domains or segments with different properties or functionality. In certain embodiments, the term “fusion polypeptide” also refers to a polypeptide or peptide comprising two or more heterologous polypeptides or peptides covalently linked, either directly or via an amino acid or peptide linker. The polypeptides forming the fusion polypeptide are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The term “fusion polypeptide” can be used interchangeably with the term “fusion protein.” Thus, the open-ended expression “a polypeptide comprising” a certain structure includes larger molecules than the recited structure such as fusion polypeptides or constructs. The constructs referred to herein can be made as fusion polypeptides or as conjugates (by linking two or more moieties).
“Heterologous” refers to nucleotide, peptide, or polypeptide sequences that are not naturally contiguous. For example, in the context of the present disclosure, the term “heterologous” can be used to describe a combination or fusion of two or more peptides and/or polypeptides wherein the fusion peptide or polypeptide is not normally found in nature, such as for example a modified lysin polypeptide and a cationic and/or a polycationic peptide, an amphipathic peptide, a sushi peptide (Ding et al. Cell Mol Life Sci., 65(7-8):1202-19 (2008)), a defensin peptide (Ganz, T. Nature Reviews Immunology 3, 710-720 (2003)), a hydrophobic peptide, and/or an antimicrobial peptide which may have enhanced lytic activity. Included in this definition are two or more lysin polypeptides or active fragments thereof. These can be used to make a fusion polypeptide with lytic activity.
“Active fragment” refers to a portion of a polypeptide that retains one or more functions or biological activities of the isolated polypeptide from which the fragment was taken. As used herein, an active fragment of a modified lysin polypeptides inhibits the growth, or reduces the population, or kills at least one Gram-positive bacterial species, such as S. aureus.
“Amphipathic peptide” refers to a peptide having both hydrophilic and hydrophobic functional groups. In certain embodiments, secondary structure places hydrophobic and hydrophilic amino acid residues at opposite sides (e.g., inner side vs outer side when the peptide is in a solvent, such as water) of an amphipathic peptide. These peptides may in certain embodiments adopt a helical secondary structure, such as an alpha-helical secondary structure.
“Cationic peptide” refers to a peptide having a high percentage of positively charged amino acid residues. In certain embodiments, a cationic peptide has a pKa-value of 8.0 or greater. The term “cationic peptide” in the context of the present disclosure also encompasses polycationic peptides which are synthetically produced peptides composed of mostly positively charged amino acid residues, such as lysine and/or arginine residues. The amino acid residues that are not positively charged can be neutrally charged amino acid residues, negatively charged amino acid residues, and/or hydrophobic amino acid residues.
“Hydrophobic group” refers to a chemical group such as an amino acid side chain which has low or no affinity for water molecules but higher affinity for oil molecules. Hydrophobic substances tend to have low or no solubility in water or aqueous phases and are typically apolar but tend to have higher solubility in oil phases. Examples of hydrophobic amino acids include glycine (Gly), alanine (Ala), valine (Val), Leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp).
“Augmenting” as used herein refers to a degree of activity of an agent, such as antimicrobial activity, that is higher than it would be otherwise. “Augmenting” encompasses additive as well as synergistic (superadditive) effects.
“Synergistic” or “superadditive” refers to a beneficial effect brought about by two substances in combination that exceeds the sum of the effects of the two agents working independently. In certain embodiments the synergistic or superadditive effect significantly, i.e., statistically significantly, exceeds the sum of the effects of the two agents working independently. One or both active ingredients may be employed at a subthreshold level, i.e., a level at which if the active substance is employed individually produces no or a very limited effect. The effect can be measured by assays such as a checkerboard assay, described here.
“Treatment” refers to any process, action, application, therapy, or the like, wherein a subject, including a human being, is subjected to medical aid with the object of curing a disorder, eradicating a pathogen, or improving the subject's condition, directly or indirectly. Treatment also refers to reducing incidence, alleviating symptoms, eliminating recurrence, preventing recurrence, preventing incidence, reducing the risk of incidence, improving symptoms, improving prognosis, or combinations thereof. “Treatment” may further encompass reducing the population, growth rate, or virulence of the bacteria in the subject and thereby controlling or reducing a bacterial infection in a subject or bacterial contamination of an organ, tissue, or environment. Thus “treatment” that reduces incidence is effective to inhibit growth of at least one Gram-positive bacterium in a particular milieu, whether it be a subject or an environment. On the other hand, “treatment” of an already established infection refers to reducing the population, killing, inhibiting the growth, and/or eradicating the Gram-positive bacteria responsible for an infection or contamination.
The term “preventing” and includes the prevention of the incidence, recurrence, spread, onset, or establishment of a disorder such as a bacterial infection. It is not intended that the present disclosure be limited to complete prevention or to prevention of establishment of an infection. In some embodiments, the onset is delayed, or the severity of a subsequently contracted disease or the chance of contracting it is reduced, and such constitute examples of prevention. With specific reference to biofilm prevention, the term includes prevention of the formation of biofilm, for example by interfering with the adherence of bacteria on a surface of interest, such as the surface of a medical device (e.g., inhaler, catheter, intubation, valve, or other prosthesis).
“Contracted disease” refers to a disease manifesting with clinical or subclinical symptoms, such as the detection of fever, sepsis, or bacteremia, as well as disease that may be detected by growth of a bacterial pathogen (e.g., in culture) when symptoms associated with such pathology are not yet manifest. With respect to medical devices, in particular, a contracted disease shall include a biofilm containing bacteria, such as Staphylococcus or Streptococcus bacteria, and forming when such a device is in use.
“Percent amino acid sequence identity” refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, such as a lysin polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as a part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available software such as BLAST or software available commercially for example from DNASTAR. Two or more polypeptide sequences can be anywhere from 0-100% identical, or any integer value there between. In the context of the present disclosure, two polypeptides are “substantially identical” when at least 80% of the amino acid residues (preferably at least about 85%, at least about 90%, and preferably at least about 95%, at least about 98%, or at least 99%) are identical. The term “percent (%) amino acid sequence identity” as described herein applies to peptides as well. Thus, the term “Substantially identical” will encompass mutated, truncated, fused, or otherwise sequence-modified variants of isolated polypeptides and peptides, such as those described herein, and active fragments thereof, as well as polypeptides with substantial sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95% identity, at least 98% identity, or at least 99% identity as measured for example by one or more methods referenced above) as compared to the reference (wild type or other intact) polypeptide. Two amino acid sequences are “substantially homologous” when at least about 80% of the amino acid residues (preferably at least about 85%, at least about 90%, at least about 95%, at least about 98% identity, or at least about 99% identity) are identical, or represent conservative substitutions. The sequences of polypeptides of the present disclosure, are substantially homologous when one or more, or several, or up to 10%, or up to 15%, or up to 20% of the amino acids of the polypeptide, such as the lysin and/or fusion polypeptides described herein, are substituted with a similar or conservative amino acid substitution, and wherein the resulting polypeptide, such as the lysin and/or fusion polypeptides described herein, have at least one activity, antibacterial effects, and/or bacterial specificities of the reference polypeptide, such as the lysin and/or fusion polypeptides described herein.
As used herein, a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
“Inhalable composition” refers to pharmaceutical compositions of the present disclosure that are formulated for direct delivery to the respiratory tract during or in conjunction with routine or assisted respiration (e.g., by intratracheobronchial, pulmonary, and/or nasal administration), including, but not limited to, atomized, nebulized, dry powder, and/or aerosolized formulations.
“Biofilm” refers to bacteria that attach to surfaces and aggregate in a hydrated polymeric matrix that may be comprised of bacterial- and/or host-derived components. A biofilm is an aggregate of microorganisms in which cells adhere to each other on a biotic or abiotic surface. These adherent cells are frequently embedded within a matrix comprised of, but not limited to, extracellular polymeric substance (EPS). Biofilm EPS, which is also referred to as slime (although not everything described as slime is a biofilm) or plaque, is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides. In certain embodiments, the biofilm may contain Staphylococcus and/or Streptococcus bacteria.
“Suitable” in the context of an antibiotic being suitable for use against certain bacteria refers to an antibiotic that was found to be effective against those bacteria even if resistance subsequently developed.
“Wild-type PlySs2 lysin” and “PlySs2 lysin,” refer to a polypeptide having the amino acid sequence:
“Modified lysin polypeptide” as used herein refers to a non-naturally occurring variant (or active fragment thereof) of the wild-type PlySs2 lysin. The modified lysin polypeptide has at least one amino acid substitution in the CHAP domain and/or the SH3b domain, and inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria, such as S. aureus.
“Immunogenic” or “immunogenicity” means predicted to be immunogenic or to have immunogenicity by establishing (for example, through computationally guided in silico methods) the existence of one or more T-cell epitopes Immunogenicity of a modified lysin polypeptide as disclosed herein can be measured by TCE score, using any available in silico computationally guided method for obtaining such score and compared to the similarly derived TCE score of a wild-type PlySs2 lysin having the amino acid sequence of SEQ ID NO: 1. Alternatively, immunogenicity of a modified lysin polypeptide as disclosed herein can be measured by an in vitro T cell response. By extension, “less immunogenic,” “reduced immunogenicity,” or the like means predicted to be less immunogenic or to have reduced immunogenicity by depletion (which includes elimination or attenuation by amino acid replacement) of one or more T-cell epitopes (i.e., have a lower TCE score as compared to a reference polypeptide) or that the modified lysin polypeptide as disclosed herein elicits a reduced T cell response. Therefore, as used herein, a modified lysin polypeptide is “less immunogenic,” or has “reduced immunogenicity,” or the like if the modified lysin polypeptide has either 1) a lower TCE score than a wild-type PlySs2 lysin having the amino acid sequence of SEQ ID NO: 1 or 2) a reduced T cell response.
“Reduced T cell response” means that the modified lysin polypeptide induces less T cell activation than a wild-type PlySs2 lysin having the amino acid sequence of SEQ ID NO: 1, as measured by an in vitro T cell proliferation (3{H}-thymidine incorporation) assay using CD8+ depleted, human peripheral blood mononuclear cells in which the human peripheral blood mononuclear cells are exposed to fluorescein isothiocyanate-labeled anti-cytokine antibodies and the response measured.
“Substantially” used in the context of lytic activity (antimicrobial activity) of a modified lysin polypeptide of the present disclosure means at least a considerable portion of the antibacterial activity of the wild-type PlySs2 lysin, such that, on the basis of such activity, the modified lysin polypeptide would be useful alone or together with other antimicrobial agents, such as one or more antibiotics and/or lysostaphin, to inhibit, combat, or eliminate Staphylococcal or Streptococcal bacterial infection by killing these bacteria. Nonlimiting examples of such substantial activity compared to the wild-type PlySs2 lysin include no more than about 5, such as no more than about 4, no more than about 3, or no more than about 2, times the MIC of the wild-type lysin. Other measures of activity can be, for example, minimum biofilm eliminating concentration (MBEC) or in vivo efficacy using, for example, an animal model, such as the mouse neutropenic thigh infection model (MNTI). Still other measures can be the ability to synergize with antibiotics, such as vancomycin, daptomycin or oxacillin, or the ability to ameliorate, prevent, or delay development of, bacterial resistance of antibiotics, such as vancomycin, daptomycin or oxacillin, similar to the wild-type PlySs2 lysin. The same term “substantially” used in the context of reduced immunogenicity means having at most 65%, such as at most 50%, at most 40%, at most 30%, or at most 25% of the immunogenicity of the wild-type PlySs2 lysin, as measured for example by a TCE score [19].
Modified Lysin Polypeptides
The present disclosure is directed to methods of using a modified lysin polypeptide having lytic activity and reduced immunogenicity as compared to a wild-type PlySs2 lysin. As used herein “lytic activity” encompasses the ability of a lysin to kill bacteria, reduce the population of bacteria or inhibit bacterial growth. Lytic activity also encompasses the ability to remove or reduce a biofilm and/or the ability to reduce the minimum inhibitory concentration (MIC) of an antibiotic.
Typically, the present modified lysin polypeptides are capable of degrading peptidoglycan, a major structural component of the bacterial cell wall, resulting in cell lysis. The modified lysin polypeptides are further capable of reducing immunogenicity and/or reducing inflammatory response-related toxicity compared to a wild-type PlySs2 lysin.
Suitable methods for assessing the activity of a modified lysin polypeptide as disclosed herein are well known in the art and described in the examples. Briefly, a MIC value (i.e., the minimum concentration of peptide sufficient to suppress at least 80% of the bacterial growth compared to control) may be determined for a modified lysin polypeptide and compared to, e.g., a wild-type PlySs2 lysin or inactive compound. For example, MIC values for a modified lysin polypeptide may be determined against e.g., laboratory Staphylococcus aureus strains, in e.g., Mueller-Hinton broth or Mueller-Hinton broth supplemented with serum, such as horse serum.
In some embodiments, the present modified lysin polypeptides are capable of reducing a biofilm. Methods for assessing the Minimal Biofilm Eradicating Concentration (MBEC) of a modified lysin polypeptide may be determined using a variation of the broth microdilution MIC method with modifications (See Ceri et al. 1999. J. Clin Microbial. 37:1771-1776, which is herein incorporated by reference in its entirety and Schuch et al., 2017, Antimicrob. Agents Chemother. 61, pages 1-18, which is herein incorporated by reference in its entirety.) In this method, colonies of bacteria, e.g., Staphylococcus aureus such as methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA), are suspended in medium, e.g., phosphate buffer solution (PBS) diluted e.g., 1:100 in TSBg (tryptic soy broth supplemented with 0.2% glucose), added as e.g., 0.15 ml aliquots, to a Calgary Biofilm Device (96-well plate with a lid bearing 96 polycarbonate pegs; Innovotech Inc.) and incubated e.g., 24 hours at 37° C. Biofilms are then washed and treated with e.g., a 2-fold dilution series of the lysin in TSBg at e.g., 37° C. for 24 hours. After treatment, wells are washed, air-dried at e.g., 37° C. and stained with e.g., 0.05% crystal violet for 10 minutes. After staining, the biofilms are destained in e.g., 33% acetic acid and the OD600 of e.g., extracted crystal violet is determined. The MBEC of each sample is the minimum lysin concentration required to remove >95% of the biofilm biomass assessed by crystal violet quantitation.
In some embodiments, the present modified lysin polypeptides reduce the minimum inhibitory concentration (MIC) of an antibiotic. Any known method to assess MIC may be used. In some embodiments, a checkerboard assay is used to determine the effect of a lysin on antibiotic concentration. The checkerboard assay is based on a modification of the CLSI method for MIC determination by broth microdilution (See Clinical and Laboratory Standards Institute (CLSI), CLSI. 2015. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-10th Edition. Clinical and Laboratory Standards Institute, Wayne, Pa., which is herein incorporated by reference in its entirety and Ceri et al. 1999. J. Clin. Microbiol. 37: 1771-1776, which is also herein incorporated by reference in its entirety).
Checkerboards are constructed by first preparing columns of e.g., a 96-well polypropylene microtiter plate, wherein each well has the same amount of antibiotic diluted 2-fold along the horizontal axis. In a separate plate, comparable rows are prepared in which each well has the same amount of lysin diluted e.g., 2-fold along the vertical axis. The lysin and antibiotic dilutions are then combined, so that each column has a constant amount of antibiotic and doubling dilutions of lysin, while each row has a constant amount of lysin and doubling dilutions of antibiotic. Each well thus has a unique combination of lysin and antibiotic. Bacteria are added to the drug combinations at a given concentration. The MIC of each drug, alone and in combination, is then recorded after e.g., 16 hours at 37° C. in ambient air. Summation fractional inhibitory concentrations (ΣFICs) are calculated for each drug and the minimum ΣFIC value (ΣFICmin) is used to determine the effect of the lysin/antibiotic combination.
The lysin polypeptides disclosed herein have been modified from a wild-type PlySs2 lysin. PlySs2 is a bacteriophage lysin that may be derived from Streptococcus suis bacteria. PlySs2 demonstrates broad killing activity against multiple bacteria, including Gram-positive bacteria, including Staphylococcus, Streptococcus, Enterococcus, and Listeria bacterial strains, including antibiotic-resistant Staphylococcus aureus, such as MRSA and vancomycin-resistant Staphylococcus aureus (VRSA). Wild-type PlySs2 has the following amino acid sequence: PPGTVAQSAP
(SEQ ID NO: 1). SEQ ID NO: 1 has 245 amino acid residues, including the initial methionine residue which is removed during post-translational processing, leaving a 244-amino acid polypeptide. The italicized amino acids indicates the CHAP domain (amino acids 1 to 146) and the dotted underline indicates the SH3b domain (amino acids 157 to 245). The naturally occurring linker between the two domains is PPGTVAQSAP (SEQ ID NO: 2).
As disclosed herein, wild-type PlySs2 comprises both a CHAP domain and a SH3b domain, each of which in turn comprises multiple T-cell epitopes (TCE). TCE 1, TCE 2, TCE 3, and TCE 4 are located in the CHAP domain, while TCE 5, TCE 6, TCE 7, and TCE 8 are located in the SH3b domain. TCE 1 corresponds to amino acid residues 32-45 of SEQ ID NO: 1. TCE 2 corresponds to amino acid residues 84-98 of SEQ ID NO: 1. TCE 3 corresponds to amino acid residues 100-112 of SEQ ID NO: 1. TCE 4 corresponds to amino acid residues 128-145 of SEQ ID NO: 1. TCE 5 corresponds to amino acid residues 164-170 of SEQ ID NO: 1. TCE 6 corresponds to amino acid residues 172-187 of SEQ ID NO: 1. TCE 7 corresponds to amino acid residues 189-201 of SEQ ID NO: 1, and TCE 8 corresponds to amino acid residues 204-221 of SEQ ID NO: 1.
In certain embodiments, the modified lysin polypeptide comprises at least one substitution as compared to the wild-type PlySs2 polypeptide (SEQ ID NO: 1), wherein the at least one substitution is in one or more of TCE 1, TCE 2, TCE 3, or TCE 4, wherein the modified lysin polypeptide or fragment thereof inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria. In certain embodiments, the modified lysin polypeptide comprises at least one substitution as compared to the wild-type PlySs2 polypeptide (SEQ ID NO: 1), wherein the at least one substitution is in one or more of TCE 5, TCE 6, TCE 7, or TCE 8, wherein the modified lysin polypeptide or fragment thereof inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria. In certain embodiments, the modified lysin polypeptide comprises at least a first substitution and at least a second substitution as compared to the wild-type PlySs2 polypeptide (SEQ ID NO: 1), wherein the at least the first substitution is in one or more of TCE 1, TCE 2, TCE 3, or TCE 4 and at least the second substitution is in one or more of TCE 5, TCE 6, TCE 7, or TCE 8, wherein the modified lysin polypeptide or fragment thereof inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria. Typically, the modified lysin polypeptide has reduced immunogenicity as compared to a wild-type PlySs2 having the amino acid sequence of SEQ ID NO: 1.
In certain embodiments, the modified lysin polypeptide comprises at least two substitutions as compared to the wild-type PlySs2 polypeptide (SEQ ID NO: 1), wherein the at least two substitutions are in TCE 4. In certain embodiments, the modified lysin polypeptide comprises at least four substitutions as compared to the wild-type PlySs2 polypeptide (SEQ ID NO: 1), wherein at least one substitution is in TCE 2, at least one substitution is in TCE 3, and at least two substitutions are in TCE 4.
In certain embodiments, a modified lysin polypeptide as disclosed herein may result from modifying the amino acid sequence of SEQ ID NO: 1 by an amino acid substitution in the CHAP domain in at least one position selected from amino acid residue 35, 92, 104, 128, and 137 and/or an amino acid substitution in the SH3b domain in at least one position selected from amino acid residue 164, 184, 195, 198, 204, 206, 212, and 214. Accordingly, in certain embodiments, disclosed herein is a modified lysin polypeptide having at least one amino acid substitution as compared to the wild-type PlySs2 polypeptide (SEQ ID NO: 1), wherein the modified lysin polypeptide comprises at least one amino acid substitution in the CHAP domain in at least one position selected from amino acid residue 35, 92, 104, 128, and 137 of SEQ ID NO: 1 and/or at least one amino acid substitution in SH3b domain in at least one position selected from amino acid residue 164, 184, 195, 198, 204, 206, 212, and 214 of SEQ ID NO: 1, wherein the modified lysin polypeptide or fragment thereof inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria. In certain embodiments, the modified lysin polypeptide comprises an amino acid substitution in amino acid residues of 92, 104, 128, and 137 of SEQ ID NO: 1. In certain embodiments, the modified lysin polypeptide comprises an amino acid substitution in amino acid residues 92, 104, 128, 137, 164, 184, and 198 of SEQ ID NO: 1. Typically, the modified lysin polypeptide has reduced immunogenicity as compared to a wild-type PlySs2 having the amino acid sequence of SEQ ID NO: 1.
In certain embodiments, the modified lysin polypeptide may contain at least 3 amino acid substitutions, such as at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 amino acid substitutions. In certain embodiments, the modified lysin polypeptide may contain 3-9 amino acid substitutions, such as 4-9, 5-9, 6-9, 7-9, 8-9, or 9 amino acid substitutions relative to SEQ ID NO: 1. In certain embodiments, the modified lysin polypeptide may comprise at least two, such as at least three or at least four, amino acid substitutions relative to SEQ ID NO: 1 in the CHAP domain, and in certain embodiments, the modified lysin polypeptide may comprise at least two, such as at least three or at least four, amino acid substitutions relative to SEQ ID NO: 1 in the SH3b domain. In certain embodiments, the modified lysin polypeptide may consist of two, three or four amino acid substitutions relative to SEQ ID NO: 1 in the CHAP domain, and in certain embodiments, the modified lysin polypeptide may consist of two, three, or four amino acid substitutions relative to SEQ ID NO: 1 in the SH3b domain.
In certain embodiments, the modified lysin polypeptide comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 1: R35E, L92W, V104S, V128T, Y137S, Y164N, Y164K, N184D, R195E, S198H, S198Q, V204K, V204A, 1206E, V212E, V212A, and V214G. In certain embodiments, the modified lysin polypeptide comprises one or more of the following amino acid substitutions located in the CHAP domain: R35E, L92W, V104S, V128T and Y137S, and/or one or more of the following amino acid substitutions located in the SH3b domain: Y164N, Y164K, N184D, R195E, S198H, S198Q, V204K, V204A, I206E, V212A, V212E, and V214G, wherein the modified lysin polypeptide or fragment thereof inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria. Typically, the modified lysin polypeptide has reduced immunogenicity as compared to a wild-type PlySs2 having the amino acid sequence of SEQ ID NO: 1.
The substitutions herein are designated using the one-letter amino acid code of the original amino acid in SEQ ID NO: 1 that is replaced, followed by the amino acid position in SEQ ID NO: 1, followed by the amino acid that is substituted into the sequence to result in the modified lysin polypeptide. Accordingly, by way of example, R35E indicates a substitution wherein the arginine at amino acid number 35 of SEQ ID NO: 1 is replaced with glutamic acid.
Exemplary modified lysin polypeptides are disclosed herein as pp55, pp61, pp65, pp296, pp324, pp325, pp341, pp338, pp388, pp400, pp616, pp619, pp628, pp632, and pp642.
The exemplary modified lysin polypeptides comprise the amino acid substitutions relative to the amino acid sequence of SEQ ID NO:1 as shown below in Table 1.
In certain embodiments disclosed herein, the modified lysin polypeptide is pp55 and comprises the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1: L92W, V104S, V128T, and Y137S. In certain embodiments, the modified lysin polypeptide comprises the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the modified lysin polypeptide has at least 80% sequence identity with SEQ ID NO: 3, wherein the modified lysin polypeptide inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria and optionally wherein the modified lysin polypeptide has reduced immunogenicity as compared to the wild-type PlySs2 (SEQ ID NO: 1). In certain embodiments, the modified lysin polypeptide has at least 85% sequence identity with SEQ ID NO: 3. In certain embodiments, the modified lysin polypeptide has at least 90% sequence identity with SEQ ID NO: 3. In certain embodiments, the modified lysin polypeptide has at least 95% sequence identity with SEQ ID NO: 3. In certain embodiments, the modified lysin polypeptide has at least 98% sequence identity with SEQ ID NO: 3. In certain embodiments, the modified lysin polypeptide has at least 99% sequence identity with SEQ ID NO: 3.
In certain embodiments disclosed herein, the modified lysin polypeptide is pp61 and comprises the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1: L92W, V104S, V128T, Y137S, S198H, and 1206E. In certain embodiments, the modified lysin polypeptide comprises the amino acid sequence of SEQ ID NO: 4, wherein the modified lysin polypeptide inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria and optionally wherein the modified lysin polypeptide has reduced immunogenicity as compared to the wild-type PlySs2 (SEQ ID NO: 1). In certain embodiments, the modified lysin polypeptide has at least 80% sequence identity with SEQ ID NO: 4. In certain embodiments, the modified lysin polypeptide has at least 85% sequence identity with SEQ ID NO: 4. In certain embodiments, the modified lysin polypeptide has at least 90% sequence identity with SEQ ID NO: 4. In certain embodiments, the modified lysin polypeptide has at least 95% sequence identity with SEQ ID NO: 4. In certain embodiments, the modified lysin polypeptide has at least 98% sequence identity with SEQ ID NO: 4. In certain embodiments, the modified lysin polypeptide has at least 99% sequence identity with SEQ ID NO: 4.
In certain embodiments disclosed herein, the modified lysin polypeptide thereof is pp65 and comprises the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1: L92W, V104S, V128T, Y137S, S198Q, V204A, and V212A. In certain embodiments, the modified lysin polypeptide comprises the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the modified lysin polypeptide has at least 80% sequence identity with SEQ ID NO: 5, wherein the modified lysin polypeptide inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria and optionally wherein the modified lysin polypeptide has reduced immunogenicity as compared to the wild-type PlySs2 (SEQ ID NO: 1). In certain embodiments, the modified lysin polypeptide has at least 85% sequence identity with SEQ ID NO: 5. In certain embodiments, the modified lysin polypeptide has at least 90% sequence identity with SEQ ID NO: 5. In certain embodiments, the modified lysin polypeptide has at least 95% sequence identity with SEQ ID NO: 5. In certain embodiments, the modified lysin polypeptide has at least 98% sequence identity with SEQ ID NO: 5. In certain embodiments, the modified lysin polypeptide has at least 99% sequence identity with SEQ ID NO: 5.
In certain embodiments disclosed herein, the modified lysin polypeptide is pp296 and comprises the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1: L92W, V104S, V128T, Y137S, Y164K, N184D, and S198Q, such that the amino acid sequence is MTTVNEALNNVRAQVGSGVSVGNGECYALASWYERMISPDATVGLGAGVGWVSG AIGDTISAKNIGSSYNWQANGWTVSTSGPFKAGQIVTWGATPGNPYGHVSIVEAVDG DRLTILEQNYGGKRYPTRNYYSAASSRQQVVHYITPPGTVAQSAPNLAGSRSKRETG TMTVTVDALNVRRAPDTSGEIVAVYKRGEQFDYDTVIIDVNGYVWVSYIGGSGKRN YVATGATKDGKRFGNAWGTFK (SEQ ID NO: 6). In certain embodiments, the modified lysin polypeptide comprises the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the modified lysin polypeptide has at least 80% sequence identity with SEQ ID NO: 6, wherein the modified lysin polypeptide inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria and optionally wherein the modified lysin polypeptide has reduced immunogenicity as compared to the wild-type PlySs2 (SEQ ID NO: 1). In certain embodiments, the modified lysin polypeptide has at least 85% sequence identity with SEQ ID NO: 6. In certain embodiments, the modified lysin polypeptide has at least 90% sequence identity with SEQ ID NO: 6. In certain embodiments, the modified lysin polypeptide has at least 95% sequence identity with SEQ ID NO: 6. In certain embodiments, the modified lysin polypeptide has at least 98% sequence identity with SEQ ID NO: 6. In certain embodiments, the modified lysin polypeptide has at least 99% sequence identity with SEQ ID NO: 6.
In certain embodiments disclosed herein, the modified lysin polypeptide is pp324 and comprises the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1: L92W, V104S, V128T, Y137S, Y164K, and N184D. In certain embodiments, the modified lysin polypeptide comprises the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the modified lysin polypeptide has at least 80% sequence identity with SEQ ID NO: 7, wherein the modified lysin polypeptide inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria and optionally wherein the modified lysin polypeptide has reduced immunogenicity as compared to the wild-type PlySs2 (SEQ ID NO: 1). In certain embodiments, the modified lysin polypeptide has at least 85% sequence identity with SEQ ID NO: 7. In certain embodiments, the modified lysin polypeptide has at least 90% sequence identity with SEQ ID NO: 7. In certain embodiments, the modified lysin polypeptide has at least 95% sequence identity with SEQ ID NO: 7. In certain embodiments, the modified lysin polypeptide has at least 98% sequence identity with SEQ ID NO: 7. In certain embodiments, the modified lysin polypeptide has at least 99% sequence identity with SEQ ID NO: 7.
In certain embodiments disclosed herein, the modified lysin polypeptide is pp325 and comprises the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1: L92W, V104S, V128T, Y137S, Y164N, and R195E. In certain embodiments, the modified lysin polypeptide comprises the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the modified lysin polypeptide has at least 80% sequence identity with SEQ ID NO: 8, wherein the modified lysin polypeptide inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria and optionally wherein the modified lysin polypeptide has reduced immunogenicity as compared to the wild-type PlySs2 (SEQ ID NO: 1). In certain embodiments, the modified lysin polypeptide has at least 85% sequence identity with SEQ ID NO: 8. In certain embodiments, the modified lysin polypeptide has at least 90% sequence identity with SEQ ID NO: 8. In certain embodiments, the modified lysin polypeptide has at least 95% sequence identity with SEQ ID NO: 8. In certain embodiments, the modified lysin polypeptide has at least 98% sequence identity with SEQ ID NO: 8. In certain embodiments, the modified lysin polypeptide has at least 99% sequence identity with SEQ ID NO: 8.
In certain embodiments disclosed herein, the modified lysin polypeptide is pp381 and comprises the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1: L92W, V104S, V128T, Y137S, N184D, and S198H. In certain embodiments, the modified lysin polypeptide comprises the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the modified lysin polypeptide has at least 80% sequence identity with SEQ ID NO: 9, wherein the modified lysin polypeptide inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria and optionally wherein the modified lysin polypeptide has reduced immunogenicity as compared to the wild-type PlySs2 (SEQ ID NO: 1). In certain embodiments, the modified lysin polypeptide has at least 85% sequence identity with SEQ ID NO: 9. In certain embodiments, the modified lysin polypeptide has at least 90% sequence identity with SEQ ID NO: 9. In certain embodiments, the modified lysin polypeptide has at least 95% sequence identity with SEQ ID NO: 9. In certain embodiments, the modified lysin polypeptide has at least 98% sequence identity with SEQ ID NO: 9. In certain embodiments, the modified lysin polypeptide has at least 99% sequence identity with SEQ ID NO: 9.
In certain embodiments disclosed herein, the modified lysin polypeptide is pp341 and comprises the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1: L92W, V104S, V128T, Y137S, N184D, V204A, and V212A. In certain embodiments, the modified lysin polypeptide comprises the amino acid sequence of SEQ ID NO: 10, wherein the modified lysin polypeptide inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria and optionally wherein the modified lysin polypeptide has reduced immunogenicity as compared to the wild-type PlySs2 (SEQ ID NO: 1). In certain embodiments, the modified lysin polypeptide has at least 80% sequence identity with SEQ ID NO: 10. In certain embodiments, the modified lysin polypeptide has at least 85% sequence identity with SEQ ID NO: 10. In certain embodiments, the modified lysin polypeptide has at least 90% sequence identity with SEQ ID NO: 10. In certain embodiments, the modified lysin polypeptide has at least 95% sequence identity with SEQ ID NO: 10. In certain embodiments, the modified lysin polypeptide has at least 98% sequence identity with SEQ ID NO: 10. In certain embodiments, the modified lysin polypeptide has at least 99% sequence identity with SEQ ID NO: 10.
In certain embodiments disclosed herein, the modified lysin polypeptide is pp388 and comprises the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1: Y164N, N184D, R195E, V204K, and V212E. In certain embodiments, the modified lysin polypeptide comprises the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the modified lysin polypeptide has at least 80% sequence identity with SEQ ID NO: 11, wherein the modified lysin polypeptide inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria and optionally wherein the modified lysin polypeptide has reduced immunogenicity as compared to the wild-type PlySs2 (SEQ ID NO: 1). In certain embodiments, the modified lysin polypeptide has at least 85% sequence identity with SEQ ID NO: 11. In certain embodiments, the modified lysin polypeptide has at least 90% sequence identity with SEQ ID NO: 11. In certain embodiments, the modified lysin polypeptide has at least 95% sequence identity with SEQ ID NO: 11. In certain embodiments, the modified lysin polypeptide has at least 98% sequence identity with SEQ ID NO: 11. In certain embodiments, the modified lysin polypeptide has at least 99% sequence identity with SEQ ID NO: 11.
In certain embodiments disclosed herein, the modified lysin polypeptide is pp400 and comprises the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1: R35E, L92W, V104S, V128T, and Y137S. In certain embodiments, the modified lysin polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the modified lysin polypeptide has at least 80% sequence identity with SEQ ID NO: 12, wherein the modified lysin polypeptide inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria and optionally wherein the modified lysin polypeptide has reduced immunogenicity as compared to the wild-type PlySs2 (SEQ ID NO: 1). In certain embodiments, the modified lysin polypeptide has at least 85% sequence identity with SEQ ID NO: 12. In certain embodiments, the modified lysin polypeptide has at least 90% sequence identity with SEQ ID NO: 12. In certain embodiments, the modified lysin polypeptide has at least 95% sequence identity with SEQ ID NO: 12. In certain embodiments, the modified lysin polypeptide has at least 98% sequence identity with SEQ ID NO: 12. In certain embodiments, the modified lysin polypeptide has at least 99% sequence identity with SEQ ID NO: 12.
In certain embodiments disclosed herein, the modified lysin polypeptide is pp616 and comprises the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1: V128T, Y137S, and Y164K. In certain embodiments, the modified lysin polypeptide comprises the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the modified lysin polypeptide has at least 80% sequence identity with SEQ ID NO: 13, wherein the modified lysin polypeptide inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria and optionally wherein the modified lysin polypeptide has reduced immunogenicity as compared to the wild-type PlySs2 (SEQ ID NO: 1). In certain embodiments, the modified lysin polypeptide has at least 85% sequence identity with SEQ ID NO: 13. In certain embodiments, the modified lysin polypeptide has at least 90% sequence identity with SEQ ID NO: 13. In certain embodiments, the modified lysin polypeptide has at least 95% sequence identity with SEQ ID NO: 13. In certain embodiments, the modified lysin polypeptide has at least 98% sequence identity with SEQ ID NO: 13. In certain embodiments, the modified lysin polypeptide has at least 99% sequence identity with SEQ ID NO: 13.
In certain embodiments disclosed herein, the modified lysin polypeptide is pp619 and comprises the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1: L92W, V104S, V128T, Y137S, and Y164K. In certain embodiments, the modified lysin polypeptide comprises the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the modified lysin polypeptide has at least 80% sequence identity with SEQ ID NO: 14, wherein the modified lysin polypeptide inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria and optionally wherein the modified lysin polypeptide has reduced immunogenicity as compared to the wild-type PlySs2 (SEQ ID NO: 1). In certain embodiments, the modified lysin polypeptide has at least 85% sequence identity with SEQ ID NO: 14. In certain embodiments, the modified lysin polypeptide has at least 90% sequence identity with SEQ ID NO: 14. In certain embodiments, the modified lysin polypeptide has at least 95% sequence identity with SEQ ID NO: 14. In certain embodiments, the modified lysin polypeptide has at least 98% sequence identity with SEQ ID NO: 14. In certain embodiments, the modified lysin polypeptide has at least 99% sequence identity with SEQ ID NO: 14.
In certain embodiments disclosed herein, the modified lysin polypeptide is pp628 and comprises the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1: L92W, V104S, V128T, Y137S, Y164K, V204K, and V212E. In certain embodiments, the modified lysin polypeptide comprises the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the modified lysin polypeptide has at least 80% sequence identity with SEQ ID NO: 15, wherein the modified lysin polypeptide inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria and optionally wherein the modified lysin polypeptide has reduced immunogenicity as compared to the wild-type PlySs2 (SEQ ID NO: 1). In certain embodiments, the modified lysin polypeptide has at least 85% sequence identity with SEQ ID NO: 15. In certain embodiments, the modified lysin polypeptide has at least 90% sequence identity with SEQ ID NO: 15. In certain embodiments, the modified lysin polypeptide has at least 95% sequence identity with SEQ ID NO: 15. In certain embodiments, the modified lysin polypeptide has at least 98% sequence identity with SEQ ID NO: 15. In certain embodiments, the modified lysin polypeptide has at least 99% sequence identity with SEQ ID NO: 15.
In certain embodiments disclosed herein, the modified lysin polypeptide is pp632 and comprises the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1: L92W, V104S, V128T, Y137S, Y164K, N184D, S198Q, V204K, and V212E. In certain embodiments, the modified lysin polypeptide comprises the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the modified lysin polypeptide has at least 80% sequence identity with SEQ ID NO: 16, wherein the modified lysin polypeptide inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria and optionally wherein the modified lysin polypeptide has reduced immunogenicity as compared to the wild-type PlySs2 (SEQ ID NO: 1). In certain embodiments, the modified lysin polypeptide has at least 85% sequence identity with SEQ ID NO: 16. In certain embodiments, the modified lysin polypeptide has at least 90% sequence identity with SEQ ID NO: 16. In certain embodiments, the modified lysin polypeptide has at least 95% sequence identity with SEQ ID NO: 16. In certain embodiments, the modified lysin polypeptide has at least 98% sequence identity with SEQ ID NO: 16. In certain embodiments, the modified lysin polypeptide has at least 99% sequence identity with SEQ ID NO: 16.
In certain embodiments disclosed herein, the modified lysin polypeptide is pp642 and comprises the following amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1: L92W, V104S, V128T, Y137S, Y164K, 1206E, and V214G. In certain embodiments, the modified lysin polypeptide comprises the amino acid sequence of SEQ ID NO: 17. In certain embodiments, the modified lysin polypeptide has at least 80% sequence identity with SEQ ID NO: 17, wherein the modified lysin polypeptide inhibits the growth, reduces the population, or kills at least one species of Gram-positive bacteria and optionally wherein the modified lysin polypeptide has reduced immunogenicity as compared to the wild-type PlySs2 (SEQ ID NO: 1). In certain embodiments, the modified lysin polypeptide has at least 85% sequence identity with SEQ ID NO: 17. In certain embodiments, the modified lysin polypeptide has at least 90% sequence identity with SEQ ID NO: 17. In certain embodiments, the modified lysin polypeptide has at least 95% sequence identity with SEQ ID NO: 17. In certain embodiments, the modified lysin polypeptide has at least 98% sequence identity with SEQ ID NO: 17. In certain embodiments, the modified lysin polypeptide has at least 99% sequence identity with SEQ ID NO: 17.
In addition to the at least one substitution in the CHAP and/or Ch3b domains, the modified lysin polypeptides can also include one or more amino acid insertions and/or deletions, provided those modifications do not interfere with the lytic activity and/or reduced immunogenicity of the modified lysin polypeptide.
Also disclosed are chimeric lysin polypeptides. Chimeric lysin polypeptides are known in the art. For example, ClyF is a chimeric lysin that combines the catalytic domain of Ply187 lysin (the N-terminal 157 amino acid residues) with the binding domain of PlySs2 (the C-terminal 99 residues) [10]. In certain embodiments, the chimeric lysin polypeptide comprises a modified PlySs2 CHAP domain, as disclosed herein, and the binding domain of another lysin. In certain embodiments, the chimeric lysin polypeptide comprises the catalytic domain of another lysin and a modified PlySs2 SH3b domain, as disclosed herein.
In some embodiments, an active fragment of the modified lysin polypeptide is obtained. The term “active fragment” refers to a portion of a full-length lysin, which retains one or more biological activities of the reference lysin. Thus, as used herein, an active fragment of a modified lysin polypeptides inhibits the growth, or reduces the population, or kills at least one Gram-positive bacterial species.
Nucleic acids encoding the modified lysin polypeptides disclosed herein can be introduced into an appropriate vector for expressing the modified lysin polypeptides. Generally, any system or vector suitable to maintain, propagate or express a polypeptide in a host may be used for expression of the modified lysin polypeptides disclosed herein or fragments thereof. For example, “recombinant expression vectors” or “expression vectors,” can direct the expression of genes to which they are operatively linked. The appropriate DNA/polynucleotide sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory (2001). Additionally, tags can also be added to the modified lysin polypeptides of the present disclosure to provide convenient methods of isolation, e.g., c-myc, biotin, poly-His, etc. Kits for such expression systems are commercially available.
A wide variety of host/expression vector combinations may be employed in expressing the polynucleotide sequences encoding the present modified lysin polypeptides. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available. Examples of suitable vectors are provided, e.g., in Sambrook et al, eds., Molecular Cloning: A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory (2001). Such vectors include, among others, chromosomal, episomal and virus derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
Furthermore, the vectors may provide for the constitutive or inducible expression of the modified lysin polypeptides of the present disclosure. Suitable vectors include but are not limited to derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids colEl, pCRl, pBR322, pMB9 and their derivatives, plasmids such as RP4, pBAD24 and pBAD-TOPO; phage DNAS, e.g., the numerous derivatives of phage A, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 D plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like. Many of the vectors mentioned above are commercially available from vendors such as New England Biolabs Inc., Addgene, Takara Bio Inc., ThermoFisher Scientific Inc., etc.
Additionally, vectors may comprise various regulatory elements (including promoter, ribosome binding site, terminator, enhancer, various cis-elements for controlling the expression level) wherein the vector is constructed in accordance with the host cell. Any of a wide variety of expression control sequences (sequences that control the expression of a polynucleotide sequence operatively linked to it) may be used in these vectors to express the polynucleotide sequences encoding the modified lysin polypeptides of the present disclosure. Useful control sequences include, but are not limited to: the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast-mating factors, E. coli promoter for expression in bacteria, and other promoter sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. Typically, the polynucleotide sequences encoding the modified lysin polypeptides are operatively linked to a heterologous promoter or regulatory element. A polynucleotide sequence is “operatively linked” when it is placed into a functional relationship with another nucleotide sequence. For example, a promoter or regulatory DNA sequence is said to be “operatively linked” to a DNA sequence that codes for an RNA and/or a protein if the two sequences are operatively linked, or situated such that the promoter or regulatory DNA sequence affects the expression level of the coding or structural DNA sequence. Operatively linked DNA sequences are typically, but not necessarily, contiguous.
A wide variety of host cells are useful in expressing the present polypeptides. Non-limiting examples of host cells suitable for expression of the present polypeptides include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, R1.1, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSCl, BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture.
While the expression host may be any known expression host cell, in a typical embodiment the expression host is one of the strains of E. coli. These include, but are not limited to commercially available E. coli strains such as Top10 (ThermoFisher Scientific, Inc.), DH5a (Thermo Fisher Scientific, Inc.), XLI-Blue (Agilent Technologies, Inc.), SCSllO (Agilent Technologies, Inc.), JM109 (Promega, Inc.), LMG194 (ATCC), and BL21 (Thermo Fisher Scientific, Inc.). There are several advantages of using E. coli as a host system including: fast growth kinetics, where under the optimal environmental conditions, its doubling time is about 20 min (Sezonov et al., J. Bacterial. 189 8746-8749 (2007)), easily achieved high density cultures, easy and fast transformation with exogenous DNA, etc. Details regarding protein expression in E. coli, including plasmid selection as well as strain selection are discussed in details by Rosano, G. and Ceccarelli, E., Front Microbial., 5: 172 (2014).
Efficient expression of the present modified lysin polypeptides depends on a variety of factors such as optimal expression signals (both at the level of transcription and translation), correct protein folding, and cell growth characteristics. Regarding methods for constructing the vector and methods for transducing the constructed recombinant vector into the host cell, conventional methods known in the art can be utilized. While it is understood that not all vectors, expression control sequences, and hosts will function equally well to express the polynucleotide sequences encoding the modified lysin polypeptides of the present disclosure, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this disclosure.
The modified lysin polypeptides of the present disclosure can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography can also employed for lysin polypeptide purification.
Alternatively, the vector system used for the production of the modified lysin polypeptides of the present disclosure may be a cell-free expression system. Various cell-free expression systems are commercially available, including, but are not limited to those available from Promega, LifeTechnologies, Clonetech, etc.
The modified lysin polypeptides disclosed herein may be incorporated into antimicrobial and bactericidal compositions and unit dosage forms thereof alone or with one or more conventional antibiotics and other bactericidal agents.
Typically, the compositions contain the modified lysin polypeptide as disclosed herein in an amount effective for killing Gram-positive bacteria selected from the group consisting of Staphylococcus aureus; Listeria monocytogenes; a coagulase negative staphylococcus such as from the Staphylococcus epidermidis group, the Staphylococcus saprophyticus group, the Staphylococcus simulans group, the Staphylococcus intermedius group, the Staphylococcus sciuri group, and the Staphylococcus hyicus group; Streptococcus suis; Streptococcus pyogenes; Streptococcus agalactiae; Streptococcus dysgalactiae; Streptococcus pneumoniae; species included in the viridans streptococci group such as the Streptococcus anginosis group, Streptococcus mitis group, Streptococcus sanguinis group, Streptococcus bovis group, Streptococcus salivarius group, and Streptococcus mutans group; Enterococcus faecalis; and Enterococcus faecium.
The compositions disclosed herein can take the form of solutions, suspensions, emulsions, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, tampon applications, aerosols, sprays, lozenges, troches, candies, injectables, chewing gums, ointments, smears, time-release patches, liquid-absorbed wipes, and combinations thereof. Hence, the compositions can be employed as solids, such as tablets, lyophilized powders for reconstitution, liposomes or micelles, or the compositions can be employed as liquids, such as solutions, suspensions, gargles, emulsions, or capsules filled solids or liquids, such as for oral use. In certain embodiments, the compositions can be in the form of suppositories or capsules for rectal administration or in the form of sterile injectable or inhalable solutions or suspensions for parenteral (including, for example, intravenous or subcutaneous) or topical, such as dermal, nasal, pharyngeal or pulmonary, use. Such compositions include pharmaceutical compositions, and unit dosage forms thereof may comprise conventional or new ingredients in conventional or special proportions, with or without additional active compounds or principles. Such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.
Carriers and excipients can be selected from a great variety of substances acceptable for human or veterinary use. Non-limiting examples of pharmaceutically acceptable carriers or excipients include any of the standard pharmaceutical carriers, such as phosphate buffered saline solutions, water, polyols, disaccharides or polysaccharides, and emulsions such as oil/water emulsions and microemulsions. Other stabilizing excipients include proprietary blends of stabilizing and protecting solutions (SPS), cyclodextrins and recombinant human albumin (rHSA). Other excipients may include bulking agents, buffering agents, tonicity modifiers (e.g., salts and amino acids), surfactants, preservatives, antioxidants, and co-solvents. For solid oral compositions comprising a modified lysin polypeptide disclosed herein, suitable pharmaceutically acceptable excipients include, but are not limited to, starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like. For liquid oral compositions, suitable pharmaceutically acceptable excipients may include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and the like. For topical solid compositions such as creams, gels, foams, ointments, or sprays, suitable excipients may include, but are not limited to a cream, a cellulosic, or an oily base, emulsifying agents, stiffening agents, rheology modifiers or thickeners, surfactants, emollients, preservatives, humectants, alkalizing or buffering agents, and solvents.
For example, the modified lysin polypeptides disclosed herein can be combined with buffers that maintain the pH of a liquid suspension, solution, or emulsion within a range that does not substantially affect the activity of the modified lysin polypeptide. For example, a desirable pH range of the composition or of the environment wherein the active ingredient is found upon administration may be between about 4.0 and about 9.0, for example between about 4.5 and about 8.5.
A stabilizing buffer may be optionally included to permit the modified lysin polypeptide to exert its activity in an optimized fashion. The buffer may contain a reducing reagent, such as dithiothreitol. The stabilizing buffer may also be or include a metal chelating reagent, such as ethylenediaminetetracetic acid disodium salt, or it may contain a phosphate or citrate-phosphate buffer, or any other buffering agent, such as Tris or succinate.
A mild surfactant can be included in a pharmaceutical composition in an amount effective to potentiate the therapeutic effect of the modified lysin polypeptides used in the composition. Suitable mild surfactants may include, inter alia, esters of polyoxyethylene sorbitan and fatty acids (such as the Tween series), octylphenoxy polyethoxy ethanol (such as the Triton-X series), n-Octyl-β-D-glucopyranoside, n-Octyl-β-D-thioglucopyranoside, n-Decyl-β-D-glucopyranoside, n-Dodecyl-β-D-glucopyranoside, poloxamer, polysorbate 20, polysorbate 80, polyethylene glycol, and biologically occurring surfactants, e.g., fatty acids, glycerides, monoglycerides, deoxycholate, and esters of deoxycholate.
Preservatives may also be used in the compositions disclosed herein, and may, for example, comprise about 0.05% to about 0.5% by weight of the total composition. The use of preservatives may assure that if the product is microbially-contaminated, the formulation will prevent or diminish microorganism growth (or attenuate the potency of the formulation). Exemplary preservatives include methylparaben, propylparaben, butylparaben, chloroxylenol, sodium benzoate, DMDM Hydantoin, 3-Iodo-2-Propylbutyl carbamate, potassium sorbate, chlorhexidine digluconate, or a combination thereof.
For oral administration, the modified lysin polypeptides disclosed herein can be formulated into solid or liquid preparations, for example tablets, capsules, powders, solutions, suspensions, and dispersions. For oral administration in the form of a tablet or capsule, the active ingredient may be combined with one or more pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, sucrose, glucose, mannitol, sorbitol, other reducing and non-reducing sugars, microcrystalline cellulose, calcium sulfate, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica, steric acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate, and the like); disintegrants (e.g., potato starch or sodium starch glycolate); wetting agents (e.g., sodium lauryl sulphate), coloring and flavoring agents, gelatin, sweeteners, natural and synthetic gums (such as acacia, tragacanth or alginates), buffer salts, carboxymethylcellulose, polyethyleneglycol, waxes, and the like. For oral administration in liquid form, the drug components can be combined with non-toxic, pharmaceutically acceptable inert carriers (e.g., ethanol, glycerol, water), suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils), preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid), and the like. Stabilizing agents such as antioxidants (e.g., BHA, BHT, propyl gallate, sodium ascorbate, or citric acid) can also be added to stabilize the dosage forms.
In certain embodiments, the tablets can be coated by methods well-known in the art. The compositions disclosed herein can be also introduced in microspheres or microcapsules, e.g., fabricated from polyglycolic acid/lactic acid (PGLA). Liquid preparations for oral administration can take the form of, for example, solutions, syrups, emulsions, or suspensions, or they can be presented as a dry product for reconstitution with water or other suitable vehicle before use. Preparations for oral administration can be suitably formulated to give controlled or postponed release of the active compound.
The active agents can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines, as is well known.
For preparing solid compositions such as tablets and pills, a modified lysin polypeptide as disclosed herein may be mixed with a pharmaceutical excipient to form a solid preformulation composition. If desired, tablets may be sugar coated or enteric coated by standard techniques. The tablets or pills may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged or delayed action. For example, the tablet or pill can include an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer, which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be further delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate. Similarly, the orally-administered medicaments may be administered in the form of a time-controlled release vehicle, including diffusion-controlled systems, osmotic devices, dissolution-controlled matrices, and erodible/degradable matrices.
Topical compositions as disclosed herein may further comprise a pharmaceutically or physiologically acceptable carrier, such as a dermatologically or an otically acceptable carrier. Such carriers, in the case of dermatologically acceptable carriers, may be compatible with skin, nails, mucous membranes, tissues, and/or hair, and can include any conventionally used dermatological carrier meeting these requirements. In the case of otically acceptable carriers, the carrier may be compatible with all parts of the ear. Such carriers can be readily selected by one of ordinary skill in the art. Carriers for topical administration of the compounds disclosed herein include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene and/or polyoxypropylene compounds, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. In formulating skin ointments, the active components of the present disclosure may be formulated in an oleaginous hydrocarbon base, an anhydrous absorption base, a water-in-oil absorption base, an oil-in-water water-removable base, and/or a water-soluble base. In formulating otic compositions, the active components of the present disclosure may be formulated in an aqueous polymeric suspension including such carriers as dextrans, polyethylene glycols, polyvinylpyrrolidone, polysaccharide gels, Gelrite®, cellulosic polymers like hydroxypropyl methylcellulose, and carboxy-containing polymers such as polymers or copolymers of acrylic acid, as well as other polymeric demulcents. The topical compositions as disclosed herein may be in any form suitable for topical application, including aqueous, aqueous-alcoholic or oily solutions; lotion or serum dispersions; aqueous, anhydrous or oily gels; emulsions obtained by dispersion of a fatty phase in an aqueous phase (O/W or oil in water) or, conversely, dispersion of an aqueous phase in a fatty phase (W/O or water in oil), microemulsions or alternatively microcapsules, microparticles or lipid vesicle dispersions of ionic and/or nonionic type, creams, lotions, gels, foams (which may use a pressurized canister, a suitable applicator, an emulsifier, and an inert propellant), essences, milks, suspensions, or patches. Topical compositions disclosed herein may also contain adjuvants such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preserving agents, antioxidants, solvents, fragrances, fillers, sunscreens, odor-absorbers, and dyestuffs. In a further aspect, the topical compositions disclosed herein may be administered in conjunction with devices such as transdermal patches, dressings, pads, wraps, matrices and bandages capable of being adhered or otherwise associated with the skin or other tissue or organ of a subject, being capable of delivering a therapeutically-effective amount of one or more modified lysin polypeptides as disclosed herein.
In some embodiments, the topical compositions disclosed herein additionally comprise one or more components used to treat topical burns. Such components may include, but are not limited to, a propylene glycol hydrogel; a combination of a glycol, a cellulose derivative and a water-soluble aluminum salt; an antiseptic; an antibiotic; and a corticosteroid. Humectants (such as solid or liquid wax esters), absorption promoters (such as hydrophilic clays, or starches), viscosity building agents, and skin-protecting agents may also be added. Topical formulations may be in the form of rinses such as mouthwash. See, e.g., WO2004/004650.
The modified lysin polypeptides disclosed herein may also be administered by injection of a therapeutic agent comprising the appropriate amount of a modified lysin polypeptide and a carrier. For example, the modified lysin polypeptides can be administered intramuscularly, intracerebrovetricularly, intrathecally, subdermally, subcutaneously, intreaperitoneally, intravenously, or by direct injection or continuous infusion to treat infections by bacteria, such as gram-positive bacteria. The carrier may be comprised of distilled water, a saline solution, albumin, a serum, or any combinations thereof. Additionally, pharmaceutical compositions of parenteral injections can comprise pharmaceutically-acceptable aqueous or nonaqueous solutions of modified lysin polypeptides in addition to one or more of the following: pH buffered solutions, adjuvants (e.g., preservatives, wetting agents, emulsifying agents, stabilizing agents, and dispersing agents), liposomal formulations, nanoparticles, dispersions, suspensions, and emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
In certain embodiments, formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, and in certain embodiments may include an added preservative. The compositions can take such forms as excipients, suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, bulking, and/or dispersing agents. The active ingredient can be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Examples of buffering agents may include histidine, Tris, phosphate, succinate citrate, methionine, cystine, glycine, mild surfactants, calcium, and magnesium. A reducing agent such as dithiothreitol can also be included.
In cases where parenteral injection is the chosen mode of administration, an isotonic formulation may be used. Generally, additives for isotonicity can include sodium chloride, dextrose, sucrose, glucose, trehalose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate buffered saline may be used. Stabilizers can include histidine, methionine, glycine, arginine, gelatin, and albumin, such as human or bovine serum albumin. A person of ordinary skill will readily appreciate that many of the foregoing excipients can also be used in compositions for injection.
A vasoconstriction agent can be added to the compositions disclosed herein. In certain embodiments, the compositions may be provided sterile and pyrogen-free.
In another embodiment, the compositions disclosed herein may be dry inhalable powders or other inhalable compositions, such as aerosols or sprays. The inhalable compositions disclosed herein can further comprise a pharmaceutically acceptable carrier. For administration by inhalation, the modified lysin polypeptides may be conveniently delivered in the form of an aerosol spray presentation from such devices as inhalers, pressurized aerosol dispensers, or nebulizers, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the active ingredient and a suitable powder base such as lactose or starch.
In one embodiment, modified lysin polypeptides disclosed herein may be formulated as a dry, inhalable powder or as an aerosol or spray. In specific embodiments, modified lysin polypeptide inhalation solution may further be formulated with a propellant for aerosol delivery. In certain embodiments, solutions may be nebulized. Many dispensing devices are available in the art for delivery of pharmaceutical compositions, including polypeptides, by inhalation. These include nebulizers, pressurized aerosol dispensers, and inhalers.
A surfactant can be added to an inhalable pharmaceutical composition as disclosed herein in order to lower the surface and interfacial tension between the medicaments and the propellant. Where the medicaments, propellant, and excipient are to form a suspension, a surfactant may or may not be required. Where the medicaments, propellant, and excipient are to form a solution, a surfactant may or may not be necessary, depending in part on the solubility of the particular medicament and excipient. The surfactant may be any suitable, non-toxic compound that is non-reactive with the medicament and that reduces the surface tension between the medicament, the excipient, and the propellant and/or acts as a valve lubricant.
Examples of suitable surfactants include, but are not limited to: oleic acid; sorbitan trioleate; cetyl pyridinium chloride; soya lecithin; polyoxyethylene(20) sorbitan monolaurate; polyoxyethylene (10) stearyl ether; polyoxyethylene (2) oleyl ether; polyoxypropylene-polyoxyethylene ethylene diamine block copolymers; polyoxyethylene(20) sorbitan monostearate; polyoxyethylene(20) sorbitan monooleate; polyoxypropylene-polyoxyethylene block copolymers; castor oil ethoxylate; and combinations thereof.
Examples of suitable propellants include, but are not limited to: dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, and carbon dioxide.
Examples of suitable excipients for use in inhalable compositions include, but are not limited to: lactose, starch, propylene glycol diesters of medium chain fatty acids; triglyceride esters of medium chain fatty acids, short chains, or long chains, or any combination thereof; perfluorodimethylcyclobutane; perfluorocyclobutane; polyethylene glycol; menthol; lauroglycol; diethylene glycol monoethylether; polyglycolized glycerides of medium chain fatty acids; alcohols; eucalyptus oil; short chain fatty acids; and combinations thereof.
In some embodiments, the compositions disclosed herein comprise nasal applications. Nasal applications include, for instance, nasal sprays, nasal drops, nasal ointments, nasal washes, nasal injections, nasal packings, bronchial sprays and inhalers, or indirectly through use of throat lozenges, mouthwashes or gargles, or through the use of ointments applied to the nasal nares, or the face or any combination of these and similar methods of application.
Compositions disclosed herein can also be formulated for rectal administration, e.g., as suppositories or retention enemas (e.g., containing conventional suppository bases such as cocoa butter or other glycerides).
In certain embodiments, the compositions disclosed herein may further comprise at least one antibiotic, such as at least one antibiotic effective to inhibit the growth, reduce the population, or kill at least one species of Gram-positive bacteria. In certain embodiments, the at least one antibiotic is effective against one or more of Staphylococcus aureus; Listeria monocytogenes; a coagulase negative Staphylococcus such as from the Staphylococcus epidermidis group, the Staphylococcus saprophyticus group, the Staphylococcus simulans group, the Staphylococcus intermedius group, the Staphylococcus sciuri group, and the Staphylococcus hyicus group; Streptococcus suis; Streptococcus pyogenes; Streptococcus agalactiae; Streptococcus dysgalactiae; Streptococcus pneumoniae; species included in the viridans streptococci group such as the Streptococcus anginosis group, Streptococcus mitis group, Streptococcus sanguinis group, Streptococcus bovis group, Streptococcus salivarius group, and Streptococcus mutans group; Enterococcus faecalis; and Enterococcus faecium.
In certain embodiments of the compositions disclosed herein, the modified lysin polypeptide in combination with the at least one antibiotic may exhibit synergism, for example synergism in the modified lysin polypeptide's or the antibiotic's ability to inhibit the growth, reduce the population, or kill at least one species of Gram-positive bacteria. Synergy may refer to the inhibitory activity of a combination of two active agents, wherein the fractional inhibitory concentration (FIC) index for the combination is less than 1, and for strong synergy, less than or equal to 0.5. The FIC of an agent is the minimum concentration of that agent that kills bacteria when used in combination with another agent divided by the concentration of the first agent that has the same effect when the first agent is used alone. The FIC index for the combination of A and B is the sum of their individual FIC values.
Synergy may be evaluated in a checkerboard assay (and can be validated by time-kill curves). Each checkerboard assay generates many different combinations, and, by convention, the FIC values of the most effective combination are used in calculating the FIC index. The FIC index defines the nature of the interaction. Antimicrobial agents with additive interactions have a FIC index of 1; an FIC index of <1 defines synergistic interactions; combinations with an FIC index>1 are antagonistic. The lower the FIC index, the more synergistic a combination. See, e.g., Singh, P. K. et al, Am J Physiol Lung Cell Mol Physiol 279: L799-L805, 2000. Synergy has implications for an efficacious, new general anti-infective strategy based on the co-administration of modified lysin polypeptides and antibiotics. In particular each and both modified lysin polypeptides and antibiotics may be administered at reduced doses and amounts, with enhanced bactericidal and bacteriostatic activity and with reduced risk of resistance development. In other words, the benefits of synergy are not only realized when one or both agents are used at sub-MIC concentrations, although the existence of synergy may be revealed by testing with sub-MIC concentrations of each agent.
Due to their high degree of activity and their low toxicity, together presenting a favorable therapeutic index, the modified lysin polypeptides disclosed herein may be administered to a subject in need thereof, e.g., a living animal (including a human) for the treatment, alleviation, or amelioration, palliation, or elimination of an indication or condition which is susceptible thereto.
In one aspect, the present disclosure is directed to a method of preventing or treating a bacterial infection comprising co-administering to a subject diagnosed with, at risk for, or exhibiting symptoms of a bacterial infection, a combination of a first effective amount of the composition containing an effective amount of a modified lysin polypeptide as described herein, and a second effective amount of an antibiotic suitable for the treatment of Gram-positive bacterial infection.
The modified lysin polypeptides of the present disclosure can be co-administered with standard care antibiotics or with antibiotics of last resort, individually or in various combinations as within the skill of the art. Traditional antibiotics used against Gram-positive bacteria are described herein and may include, for example, antibiotics of different types and classes, such as beta-lactams including penicillins (e.g., methicillin, oxacillin, cloxacillin), cephalosporins (e.g., cefalexin and cefactor), carbapenems (e.g., imipenem and entapenem); macrolides (e.g., erythromycin, azithromycin, clarithromycin, roxithromycin, spiramycin, fidaxomicin), aminoglycosides (e.g., gentamicin, tobramycin, amikacin), glycopeptides (e.g., vancomycin, teicoplanin), oxazolidinones (e.g., linezolid and tedizolid), cyclic lipopeptides (e.g., daptomycin, mupirocin, lysostaphin), ketolides (e.g., telithromycin), and lincosamides (e.g., clindamycin). In certain embodiments of all aspects of the disclosure, the antibiotic is vancomycin. In certain embodiments of all aspects of the disclosure, the antibiotic is daptomycin. In certain embodiments of all aspects of the disclosure, the antibiotic is vancomycin and daptomycin. In certain embodiments of all aspects of the disclosure, the antibiotic is oxacillin.
Combining the modified lysin polypeptides of the present disclosure with antibiotics provides an efficacious antibacterial regimen. In some embodiments, co-administration of the modified lysin polypeptides of the present disclosure with one or more antibiotics may be carried out at reduced doses and amounts of either the modified lysin polypeptides or the antibiotic or both, and/or reduced frequency and/or duration of treatment with augmented bactericidal and bacteriostatic activity, reduced risk of antibiotic resistance and with reduced risk of deleterious neurological or renal side effects (such as those associated with colistin or polymyxin B use). As used herein the term “reduced dose” refers to the dose of one active ingredient in the combination compared to monotherapy with the same active ingredient. In some embodiments, the dose of the modified lysin polypeptide or the antibiotic in a combination may be suboptimal or even subthreshold compared to the respective monotherapy.
In some embodiments, the present disclosure provides a method of augmenting antibiotic activity of one or more antibiotics against Gram-positive bacteria compared to the activity of said antibiotics used alone by administering to a subject one or more modified lysin polypeptides disclosed herein together with an antibiotic of interest. The combination is effective against the bacteria and permits resistance against the antibiotic to be overcome and/or the antibiotic to be employed at lower doses, decreasing undesirable side effects.
The terms “infection” and “bacterial infection” are meant to include respiratory tract infections (RTIs), such as respiratory tract infections in patients having cystic fibrosis (CF), lower respiratory tract infections, such as acute exacerbation of chronic bronchitis (ACEB), acute sinusitis, community-acquired pneumonia (CAP), hospital-acquired pneumonia (HAP) and nosocomial respiratory tract infections; sexually transmitted diseases, such as gonococcal cervicitis and gonococcal urethritis; urinary tract infections; acute otitis media; sepsis including neonatal septisemia and catheter-related sepsis; and osteomyelitis. Infections caused by drug-resistant bacteria and multidrug-resistant bacteria are also contemplated.
Non-limiting examples of infections caused by Gram-positive bacterial may include: A) Nosocomial infections: 1. Respiratory tract infections especially in cystic fibrosis patients and mechanically-ventilated patients; 2. Bacteraemia and sepsis; 3. Wound infections, particularly those of burn victims; 4. Urinary tract infections; 5. Post-surgery infections on invasive devises; 6. Endocarditis by intravenous administration of contaminated drug solutions; 7. Infections in patients with acquired immunodeficiency syndrome, cancer chemotherapy, steroid therapy, hematological malignancies, organ transplantation, renal replacement therapy, and other conditions with severe neutropenia. B) Community-acquired infections: 1. Community-acquired respiratory tract infections; 2. Meningitis; 3. Folliculitis and infections of the ear canal caused by contaminated water; 4. Malignant otitis externa in the elderly and diabetics; 5. Osteomyelitis of the caleaneus in children; 6. Eye infections commonly associated with contaminated contact lens; 7. Skin infections such as nail infections in people whose hands are frequently exposed to water; 8. Gastrointestinal tract infections; and 9. Muscoskeletal system infections.
The one or more species of Gram-positive bacteria of the present methods may include any of the species of Gram-positive bacteria as described herein or known in the art. Typically, the species of Gram-positive bacteria may include Listeria monocytogenes, Staphylococcus aureus, coagulase negative staphylococci (including at least 40 recognized species including, but not limited to, the Staphylococcus epidermidis group, the Staphylococcus saprophyticus group, the Staphylococcus simulans group, the Staphylococcus intermedius group, the Staphylococcus sciuri group, the Staphylococcus hyicus group, and any isolates referred to as from the “unspecified species group”), Streptococcus suis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus pneumoniae, any additional species included in the viridans streptococci group (including, but not limited to, all species and strains included in the Streptococcus anginosis group, Streptococcus mitis group, Streptococcus sanguinis group, Streptococcus bovis (now gallolyticus) group, Streptococcus salivarius group, and Streptococcus mutans group), Enterococcus faecalis, and Enterococcus faecium. Other examples of Gram-positive bacteria include but are not limited to the genera Actinomyces, Bacillus, Lactococcus, Mycobacterium, Corynebacterium, and Clostridium.
In some embodiments of all aspects of the disclosure, coadministering the present modified lysin polypeptides and an antibiotic of interest may be used for the prevention, control, disruption, and treatment of bacterial biofilm formed by Gram-positive bacteria Biofilm formation occurs when microbial cells adhere to each other and are embedded in a matrix of extracellular polymeric substance (EPS) on a surface. The growth of microbes in such a protected environment that is enriched with biomacromolecules (e.g. polysaccharides, nucleic acids and proteins) and nutrients allow for enhanced microbial cross-talk and increased virulence. Biofilm may develop in any supporting environment including living and nonliving surfaces such as the mucus plugs of the CF lung, contaminated catheters, implants, contact lenses, etc (Sharma et al. Biologicals, 42(1):1-7 (2014), which is herein incorporated by reference in its entirety). Because biofilms protect the bacteria, they are often more resistant to traditional antimicrobial treatments, making them a serious health risk, which is evidenced by more than one million cases of catheter-associated urinary tract infections (CAUTI) reported each year, many of which can be attributed to biofilm-associated bacteria (Donlan, R M (2001) Emerg Infect Dis 7(2):277-281; Maki D and Tambyah P (2001) Emerg Infect Dis 7(2):342-347).
Thus, in one embodiment of all aspects of the disclosure, the modified lysin polypeptides of the present disclosure are coadministered with an antibiotic of interest and used for the prevention, control, disruption, and treatment of bacterial infections due to Gram-positive bacteria when the Gram-positive bacteria are protected by a bacterial biofilm.
In some embodiments of all aspects of the disclosure, inhibiting the growth, or reducing the population, or killing at least one species of Gram-positive bacteria comprises contacting bacteria with the modified lysin polypeptides as described herein and an antibiotic of interest, wherein the bacteria are present on a surface of e.g., medical devices, floors, stairs, walls and countertops in hospitals and other health related or public use buildings and surfaces of equipment in operating rooms, emergency rooms, hospital rooms, clinics, and bathrooms and the like.
Examples of medical devices that can be protected using the modified lysin polypeptides described herein include but are not limited to tubing and other surface medical devices, such as urinary catheters, mucous extraction catheters, suction catheters, umbilical cannulae, contact lenses, intrauterine devices, intravaginal and intraintestinal devices, endotracheal tubes, bronchoscopes, dental prostheses and orthodontic devices, surgical instruments, dental instruments, tubings, dental water lines, fabrics, paper, indicator strips (e.g., paper indicator strips or plastic indicator strips), adhesives (e.g., hydrogel adhesives, hot-melt adhesives, or solvent-based adhesives), bandages, tissue dressings or healing devices and occlusive patches, and any other surface devices used in the medical field. The devices may include electrodes, external prostheses, fixation tapes, compression bandages, and monitors of various types. Medical devices can also include any device which can be placed at the insertion or implantation site such as the skin near the insertion or implantation site, and which can include at least one surface which is susceptible to colonization by Gram-positive bacteria.
Dosages administered depend on a number of factors such as the activity of infection being treated; the age, health and general physical condition of the subject to be treated; the activity of a particular modified lysin polypeptide; the nature and activity of the antibiotic if any with which a modified lysin polypeptide according to the present disclosure is being paired; and the combined effect of such pairing. In certain embodiments, effective amounts of the modified lysin polypeptide to be administered may fall within the range of about 0.1-100 mg/kg (or 1 to 100 mcg/ml), such as from 0.5 mg/kg to 30 mg/kg. In certain embodiments, the modified lysin polypeptide may be administered 1-4 times daily for a period ranging from 1 to 14 days. The antibiotic if one is also used may be administered at standard dosing regimens or in lower amounts in view of any synergism. All such dosages and regimens, however, (whether of the modified lysin polypeptide or any antibiotic administered in conjunction therewith) are subject to optimization. Optimal dosages can be determined by performing in vitro and in vivo pilot efficacy experiments as is within the skill of the art but taking the present disclosure into account.
It is contemplated that the modified lysin polypeptides disclosed herein may provide a rapid bactericidal and, when used in sub-MIC amounts, may provide a bacteriostatic effect. It is further contemplated that the modified lysin polypeptides disclosed herein may be active against a range of antibiotic-resistant bacteria. Based on the present disclosure, in a clinical setting, the present modified lysin polypeptides may be a potent alternative (or additive) for treating infections arising from drug- and multidrug-resistant bacteria alone or together with antibiotics (including antibiotics to which resistance has developed).
In some embodiments, time exposure to the modified lysin polypeptides disclosed herein may influence the desired concentration of active polypeptide units per ml. Carriers that are classified as “long” or “slow” release carriers (such as, for example, certain nasal sprays or lozenges) may possess or provide a lower concentration of polypeptide units per ml but over a longer period of time, whereas a “short” or “fast” release carrier (such as, for example, a gargle) may possess or provide a high concentration of polypeptide units (mcg) per ml but over a shorter period of time. There are circumstances where it may be desirable to have a higher unit/ml dosage or a lower unit/ml dosage.
For the modified lysin polypeptides of the present disclosure, the therapeutically effective dose may be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model can also be used to achieve a desirable concentration range and route of administration. Obtained information can then be used to determine the effective doses, as well as routes of administration, in humans Dosage and administration can be further adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Additional factors that may be taken into account include the severity of the disease state; age, weight and gender of the patient; diet; desired duration of treatment; method of administration; time and frequency of administration; drug combinations; reaction sensitivities; tolerance/response to therapy; and the judgment of a treating physician.
A treatment regimen can entail daily administration (e.g., once, twice, thrice, etc. daily), every other day (e.g., once, twice, thrice, etc. every other day), semi-weekly, weekly, once every two weeks, once a month, etc. In one embodiment, treatment can be given as a continuous infusion. Unit doses can be administered on multiple occasions. Intervals can also be irregular as indicated by monitoring clinical symptoms. Alternatively, the unit dose can be administered as a sustained release formulation, in which case less frequent administration may be used. Dosage and frequency may vary depending on the patient. It will be understood by one of skill in the art that such guidelines will be adjusted for localized administration, e.g., intranasal, inhalation, rectal, etc., or for systemic administration, e.g., oral, rectal (e.g., via enema), intramuscular (i.m.), intraperitoneal (i.p.), intravenous (i.v.), subcutaneous (s.c.), transurethral, and the like.
The modified lysin polypeptides described herein and their preparation, characterization, and use will be better understood in connection with the following examples, which are intended as an illustration of and not a limitation upon the scope of the present disclosure.
The ability of CF-301 or a modified lysin polypeptide pp296 to synergize with oxacillin was assessed using a checkerboard analysis. For the checkerboard assay, a total of 50 μl of Mueller-Hinton broth is distributed into each well of microdilution plates. Then, a first antibiotic of a combination is serially diluted along the ordinate, while a second drug is diluted along the abscissa. An inoculum equal to a 0.5 McFarland turbidity standard is prepared from each bacterial isolate in Mueller-Hinton broth. Each microtiter well is then inoculated with 100 μl of a bacterial inoculum of 5×105 CFU/ml, and the plates are incubated at 37° C. for 18 h under aerobic conditions. The resulting checkerboard contains each combination of two antibiotics, with wells that contain the highest concentration of each antibiotic at opposite corners. According to the CLSI guidelines for broth microdilution, the MIC is defined as the lowest concentration of antibiotic that completely inhibits the growth of the organism as detected with the naked eye. To quantify the interactions between the antibiotics being tested, the FIC index (the combination of antibiotics that produced the greatest change from the individual antibiotic's MIC) value is calculated for each strain and antibiotic combination as follows:
(A/MICA)+(B/MICB)=FICA+FICB=FIC index,
where A and B are the MIC of each agent in combination in a single well, and MICA and MICB are the MIC of each drug when used individually. The combination is considered strongly synergistic when the FIC index is ≤0.5 and synergistic when the FIC index is >0.5-<1.
Checkerboards were generated using combinations of pp296 or CF-301 and oxacillin against 10 MSSA strains. As shown in Table A1, below, pp296 and CF-301 exhibited identical FIC index values against 6 of the 10 MSSA strains tested when combined with oxacillin. pp296 exhibited slightly superior FIC index values than those for CF-301 against MSSA strains JMI-40671 (0.375 and 0.3125, respectively) and JMI-46142 (0.3125 and 0.375, respectively). CF-301 in combination with oxacillin exhibited slightly superior FIC index values than those for pp296 against MSSA strains JMI-6625 (0.375 and 0.5, respectively) and JMI-6885 (0.375 versus 0.3125, respectively). As explained above, the combination is considered strongly synergistic when the FIC index is ≤0.5 and synergistic when the FIC index is >0.5-<1. These findings indicate that pp296 exhibits levels of synergy with oxacillin very similar to those of CF-301.
Staphylococcus
aureus type
Table A2, below, shows the individual strain data for the pp296 and oxacillin checkerboard assay. As is evident in Table A2, the MIC concentrations are reduced for pp296 and oxacillin when combined for each of the MSSA strains tested (4 to 16-fold reduction in MIC). As noted previously, the combination is considered strongly synergistic when the FIC index is ≤0.5 and synergistic when the FIC index is >0.5-<1.
To confirm the synergy results in the checkerboard assay using pp296 and oxacillin, a standard time-kill assay was used against 5 MSSA strains (JMI-6886, JMI-7054, JMI-7160, JMI-6885 and JMI-6625.) To perform the assay, the bacteria for each strain was grown overnight, diluted 1:100 in CAMHB-HSD and then incubated at 37° C. for 2.5 hours while shaking at 225 revolutions per minute (rpm). The culture was adjusted to a McFarland turbidity of 0.5 and then added to a conical tube with 0.25×MIC of pp296 and/or oxacillin. Samples were collected at 0, 0.15, 1, 3, 5 and 24 hours. The samples were then serially diluted 1:50. Each dilution was plated on a tryptic soy agar (TSA) blood plate and incubated overnight at 37° C. Colony forming unit (CFU) counts were then calculated via the Log10.
Synergy in the time kill assay format is defined as a ≥2-log10 decrease in CFU/ml between the combination and its most active constituent after 24 hours (at least one of the drugs must be present at a concentration that has a minimal effect on the growth curve of the test organism), and the number of surviving organisms in the presence of the combination must be ≥2 log10 CFU/ml below the starting inoculum. The resulting Δ log10 CFU/mL values for the combination of pp296 and oxacillin were 7.2 (JMI-6886), 4.0 (JMI-7054), 5.7 (JMI-7160), 3.9 (JMI-6885) and 4.8 (JMI-6625), thereby confirming the synergy of pp296 and oxacillin for all of the tested strains.
In vitro synergy studies with antibiotics suitable for treatment of Gram-positive bacteria, for example, daptomycin (DAP) and vancomycin (VAN), were previously performed using a checkerboard assay and the results reported in PCT publication WO 2019/165454. Fractional inhibitory concentration (FIC) index values were determined for modified lysin polypeptide pp296 and antibiotic combinations against 5 methicillin-sensitive Staphylococcus aureus (MSSA) and 5 methicillin resistant Staphylococcus aureus (MRSA) isolates. The FIC index values for modified lysin polypeptide pp296 were predominantly 0.5 with both daptomycin and vancomycin and were nearly identical to wild type, indicating that the in vitro synergistic activities of pp296 with both daptomycin and vancomycin are nearly identical to that of wild-type lysin. In vivo evidence of the synergy of pp296 with both vancomycin and daptomycin against S. aureus is presented herein.
Antimicrobial test agents: pp296 (10.5 mg/mL), daptomycin (500 mg, intravenous-use vials), and vancomycin (1000 mg, intravenous-use vials) were obtained for in vivo testing. Dilutions of pp296 were made in 0.9% sodium chloride (normal saline) to obtain final concentrations for delivery in the studies described herein. Daptomycin was reconstituted with 10 mL normal saline and vancomycin was constituted with 20 mL sterile water to obtain stock concentrations of 50 mg/mL of each antibiotic. Subsequent dilutions of the vancomycin and daptomycin stock solutions were made in normal saline to obtain the final concentrations needed to deliver the dosages reported herein.
Bacterial isolates: A total of 8 S. aureus isolates (1 MSSA and 7 MRSA) were selected for the assessment of the in vivo efficacy of pp296 alone and in combination with vancomycin or daptomycin. The pp296 MICs were determined based on (1) cation-adjusted Mueller-Hinton Broth with 25% horse serum and 0.5 mM DTT (caMHB-HSD); (2) human serum (100% serum); and (3) ICR mice (100% serum). Vancomycin and daptomycin MICs of the isolate strains were determined in accordance with the broth microdilution method as outlined by CLSI. Results of the MIC determination are shown below in Table 2. The bacterial isolates were frozen at −80° C. in skim milk.
Study animals: Specific pathogen-free female ICR mice (average body weight 23 grams) were provided food and water ad libitum and maintained and used in accordance with National Research Council recommendations. Animals were allowed a 48-hour post-delivery acclimation period before every experiment. Mice were rendered transiently neutropenic by injection of cyclophosphamide via intraperitoneal (IP) route at a dose of 150 mg/kg of body weight at four days before bacterial inoculation and 100 mg/kg of body weight at one day before bacterial inoculation. Three days prior to bacterial inoculation, mice also received a single IP injection of uranyl nitrate (5 mg/kg) to induce a predictable degree of renal impairment to assist with achieving appropriate vancomycin and daptomycin exposures.
Neutropenic Thigh Infection Model: Two transfers of the bacterial isolates were performed onto trypticase soy agar plates with 5% sheep blood and placed into an incubator at 37° C. for approximately 24 hours. After an 18-24 hour incubation of the second transfer, a suspension of approximately 107 CFU/mL was made for inoculation. Final bacterial inoculum concentrations were confirmed by serial dilution and plating techniques. Thigh infection with the test isolate was produced by intramuscular injection of 0.1 mL of the bacterial inoculum into each thigh of the mice. Antimicrobial therapy was initiated 2 hours after bacterial inoculation, as detailed below.
Antimicrobial therapy: Mice were divided into four study groups: pp296 alone, vancomycin alone, pp296 in combination with vancomycin, and pp296 in combination with daptomycin. For all studies, at time points ranging from 0.5 to 24 hours after dosing, groups of 6 mice were euthanized by CO2 exposure followed by blood collection in vials containing K2EDTA. The blood was centrifuged for 10 minutes at 4° C. at 10,000×g, and the plasma transferred into polypropylene tubes. The tubes were stored at −80° C. until analyzed for drug concentrations. Once pp296 and vancomycin concentration data are determined, the pharmacokinetic parameters for each of the administered doses will be estimated by nonlinear least-square techniques.
Vancomycin dose-ranging studies: The in vivo bactericidal activity of vancomycin alone against 8 S. aureus isolates was evaluated in order to identify the vancomycin exposition (i.e., a sub-therapeutic regimen) that would provide approximate mean bacterial stasis across all isolates at 24 hours relative to the 0 hour untreated controls. As discussed below, this sub-therapeutic vancomycin dosing regimen was then administered in addition to pp296 to assess the capability of pp296 to derive additional bacterial killing.
Dose-ranging studies of vancomycin were undertaken with the following subcutaneous administration (n=6): (1) 25 mg/kg; (2) 12.5 mg/kg; (3) 6.25 mg/kg; and (4) 3.125 mg/kg, all of which were administered every 12 hours (q12h) over 24 hours. Two hours post-bacterial inoculation, one group of mice (n=3) was sacrificed to serve as a 0 hour control, representing initial bacterial burden in the thigh, wherein bacterial burden is determined as the average CFU from 6 thighs, i.e., 2 thighs per mouse. The other inoculated groups of mice (n=3) were sacrificed at the end of the 24 hours, and both thighs from each mouse were individually homogenized in normal saline. Serial dilutions were plated on trypticase soy agar with 5% sheep blood and incubated for 18 hours at 37° C. to determine the average CFU.
The average bacterial burden was 5.68±0.28 log10 CFU/thigh at 0-hr (pre-treatment control). All of the tested isolates grew in the neutropenic thigh infection model, and the bacterial burden increased over 24 hours by an average magnitude of 2.76±0.45 log10 CFU/thigh in the untreated control mice. A dose-response in bacterial burden reduction (ranging from −0.05±1.16 log10 CFU/thigh to −1.15±0.40 log10 CFU/thigh) was observed with increasing vancomycin doses compared with the 0-hour controls for all isolates. On average, the vancomycin dose of 3.125 mg/kg q12h SC was associated with stasis (mean bacterial burden: −0.05±1.16 log10 CFU/thigh) across all isolates. Accordingly, for the purpose of the in vivo efficacy studies with vancomycin described below, a rounded vancomycin dose of 3 mg/kg q12h was utilized.
Efficacy in vivo of vancomycin in combination with pp296: The in vivo efficacy of vancomycin in addition to escalating exposures of pp296 against S. aureus was determined, using the sub-therapeutic vancomycin regimen of 3 mg/kg q12h as discussed above.
After inoculation of the mice as described above, the following treatment groups were evaluated: (1) vancomycin (3 mg/kg) administered SC alone q12h; (2) vancomycin (3 mg/kg) administered SC q12h in addition to a single IV administration of 0.5 mg/kg pp296; (3) vancomycin (3 mg/kg) administered SC q12h in addition to a single IV administration of 2.5 mg/kg pp296; (4) vancomycin (3 mg/kg) administered SC q12h in addition to a single IV administration of 5 mg/kg pp296; (5) vancomycin (3 mg/kg) administered SC q12h in addition to a single IV administration of 25 mg/kg pp296; (6) vancomycin (3 mg/kg) administered SC q12h in addition to a single IV administration of 50 mg/kg pp296; and (7) a single IV administration of 50 mg/kg pp296 alone. Treatment for each group was initiated two hours after inoculation for each of the 8 bacterial isolates.
Additionally, in order to determine the in vivo efficacy of pp296 while varying the exposure to vancomycin against S. aureus, the following treatment groups were also evaluated: (1) vancomycin (1.5 mg/kg) administered SC alone q12h; (2) vancomycin (5 mg/kg) administered SC alone q12h; (3) vancomycin (1.5 mg/kg) administered SC q12h in addition to a single IV administration of 0.5 mg/kg pp296; (4) vancomycin (1.5 mg/kg) administered SC q12h in addition to a single IV administration of 2.5 mg/kg pp296; (5) vancomycin (1.5 mg/kg) administered SC q12h in addition to a single IV administration of 5 mg/kg pp296; (6) vancomycin (1.5 mg/kg) administered SC q12h in addition to a single IV administration of 25 mg/kg pp296; (7) vancomycin (1.5 mg/kg) administered SC q12h in addition to a single IV administration of 50 mg/kg pp296; and (8) a single IV administration of 50 mg/kg pp296 alone. Treatment for each group was initiated two hours after inoculation for each of the 8 bacterial isolates.
Untreated control mice (n=3) were sacrificed just prior to antibiotic administration and 24 hours after bacterial inoculation. Animals sacrificed prior to antibiotic initiation served as 0-hour control animals, representing initial bacterial burden. The 24-hour control animals received 0.9% NaCl in the same volume, route, and schedule as the vancomycin regimen, as it was the most frequently administered drug regimen). Treatment mice (n=3/group) were sacrificed at the end of the 24 hour treatment period. After sacrifice, both thighs for each mouse were homogenized in normal saline. Serial dilutions were plated on Trypticase soy agar with 5% sheep blood and incubated for 18 to 20 hours at 37.0 to determine the CFU. Efficacy was calculated as the change in bacterial density obtained in treated mice after 24 hours compared with the numbers in the 0-hour control animals. Bacterial density represents the average CFU determined from 3 mice, each contributing 2 thighs.
For the study using vancomycin 3 mg/kg q12h SC against 8 S. aureus isolates, the average bacterial burden was 5.83±0.23 log10 CFU/thigh at 0-hr. All of the tested isolates grew in the neutropenic thigh infection model; on average, bacterial density in controls increased to 8.13±1.05 log10 CFU/thigh at 24 h after initiation of study. Across all isolates, the administration of vancomycin alone was associated with an average of 0.90±1.21 log10 CFU/thigh bacterial growth compared with starting inoculum, and the administration of pp296 alone (50 mg/kg q24h IV) resulted in an average growth of 0.37±1.27 log10 CFU/thigh compared with starting inoculum. When used in combination with vancomycin (3 mg/kg), escalating pp296 exposures (0.5-50 mg/kg IV) resulted in an augmented dose-response, ranging from a mean bacterial reduction of −0.26±1.10 log10 CFU/thigh (with addition of pp296 0.5 mg/kg) to −1.01±0.41 log10 CFU/thigh (with addition of pp296 50 mg/kg), compared with starting inoculum. The observed data for the individual animals are provided in Table 3, below. In vivo efficacy studies for 4 isolates (NRS217, NRS100, HPV107, and CAIRD426) were repeated due to CFU variability observed in the model. Repeat data (Run #2) was similar to Run #1, so isolate-specific data were compiled and averaged such that no data were excluded.
For the study using vancomycin (1.5 mg/kg) against 4 S. aureus isolates, the average bacterial burden was 5.46±0.24 log10 CFU/thigh at 0-hr (pre-treatment control). All the tested isolates grew in the neutropenic thigh infection model; on average, bacterial density in controls increased to 8.21±0.61 log10 CFU at 24 h after initiation of study. The administration of vancomycin 1.5 mg/kg Q12h SC alone was associated with an average bacterial growth of 2.75±0.52 log10 CFU/thigh compared with starting inoculum, and the administration of pp296 (50 mg/kg) alone resulted in an average growth of 0.75±1.24 log10 CFU/thigh compared with starting inoculum. The administration of escalating dosages of pp296 (0.5-50 mg/kg IV) in addition to vancomycin 1.5 mg/kg SC resulted in an augmented dose-response, ranging from a mean bacterial density of 0.92±1.24 log10 CFU/thigh (0.5 mg/kg) to −0.61±0.82 log10 CFU/thigh (50 mg/kg), compared with starting inoculum.
For the study using vancomycin (5 mg/kg) against 4 S. aureus isolates, the average bacterial burden was 5.57±0.22 log10 CFU/thigh at 0-hr (pre-treatment control). All of the tested isolates grew in the neutropenic thigh infection model; on average, bacterial density in controls increased to 7.97±1.47 log10 CFU at 24 h after initiation of study. The administration of vancomycin 5 mg/kg q12h SC alone was associated with an average bacterial reduction of −0.38±1.10 log10 CFU/thigh compared with starting inoculum, and the administration of pp296 (50 mg/kg) alone resulted in an average growth of 1.44±1.43 log10 CFU/thigh compared with starting inoculum. The administration of escalating dosages of pp296 (0.5-50 mg/kg IV) in addition to vancomycin 5 mg/kg SC resulted in an augmented dose-response, ranging from a mean bacterial reduction of −0.59±0.79 log10 CFU/thigh (0.5 mg/kg) to −0.85±0.54 log10 CFU/thigh (50 mg/kg) compared with starting inoculum. These results suggest that at a given dose of pp296, a greater magnitude of augmented bacterial killing is observed with higher exposures of the standard of care antibiotic. The results are shown below in Tables 4 and 5.
Efficacy in vivo of daptomycin in combination with pp296: The in vivo efficacy of daptomycin in combination with pp296 was evaluated using the same methodology described above for vancomycin, wherein a sub-therapeutic daptomycin dosage regimen was administered as follows for the 4 S. aureus isolates NRS217, NRS100, JMI4789, and ATCC2921: 0.475 mg/kg at 0 hrs, 0.25 mg/kg at 4 hrs, 0.15 mg/kg at 10 hrs, and 0.075 mg/kg at 17 hrs. For the 4 S. aureus isolates HPV107, CAIRD426, JMI227, and NRS123, the following sub-therapeutic daptomycin dosage regimen was adopted: 0.76 mg/kg at 0 hrs, 0.4 mg/kg at 4 hrs, 0.24 mg/kg at 10 hrs, and 0.12 mg/kg at 17 hrs.
The average bacterial burden was 5.73±0.23 log10 CFU/thigh at 0-hr (pre-treatment control). All of the tested isolates grew in the neutropenic thigh infection model; on average, bacterial density in controls increased to 8.43±0.63 log10 CFU at 24 h after initiation of the study. The administration of daptomycin alone was associated with an average of 1.47±0.80 log10 CFU/thigh bacterial growth compared with starting inoculum, and the administration of pp296 alone (50 mg/kg) resulted in an average growth of 1.36±1.35 log10 CFU/thigh compared with starting inoculum. In combination with daptomycin, increasing dosages of pp296 (0.5-50 mg/kg IV) resulted in an augmented dose-response, ranging from a mean bacterial density of 0.80±1.19 log10 CFU/thigh (0.5 mg/kg) to −0.72±0.59 log10 CFU/thigh (50 mg/kg). The results are shown below in Table 6.
pp296 dose fractionation studies: The PK/PD index (Cmax/MIC, AUC/MIC, and % T>MIC) that correlated most closely with efficacy of pp296 in additional to vancomycin and daptomycin was sought to be determined.
Mice were inoculated as described above and treatment with pp296, vancomycin, and daptomycin was administered as described above, wherein two total daily doses (TDD) of pp296 (5 mg/kg/day and 50 mg/kg/day) were evaluated. Each of the pp296 TDDs was fractionated into three regimens, i.e., one-third of the entire dose was administered via IV three times (q8h); one-half of the entire does was administered via IV two times (q12h), or the entire dose was administered via IV once daily over a 24-hour period, for a total of three groups per each TDD.
Untreated control mice (n=3/group) were sacrificed just prior to antibiotic administration, as well as 24 hours later. Animals sacrificed prior to antibiotic administration served as 0-hour control animals, representing initial bacterial burden. The 24-hour control animals received sterile normal saline in the same volume and schedule as the most frequent active drug regimen. One group (n=3) was administered either vancomycin (3 mg/kg, Q12h, SC) or the sub-therapeutic daptomycin regimen (SC) alone to serve as a comparator controls. After the 24-hour treatment period, all animals were euthanized and thighs were cultured quantitatively as described above. The change in bacterial density was calculated for each of the dosing regimen. Comparisons were made between the different regimens of the same total daily dose on bacterial burdens at 24 hours using one-way Analysis of Variance (ANOVA) test followed by Tukey's test where the P value was 0.05.
At 0 hours, the average bacterial burden in the thighs for the tested isolate was 6.03±0.13 log10 CFU/thigh. Adequate growth in the neutropenic thigh infection model was achieved; the bacterial burdens increased over 24 hours by an average magnitude of 2.77±0.53 log10 CFU/thigh in the untreated control mice. Compared with the 0-hour control mice, the bacterial burden observed in the thighs of the mice receiving sub-therapeutic daptomycin SC alone was 0.82±0.44 log10 CFU/thigh and 0.41±0.62 log10 CFU/thigh in mice receiving vancomycin 3 mg/kg Q12h SC alone. Further pharmacokinetic analyses may be performed to interpret the dose-fractionation results. However, pp296 dose-fractionation (q12h v. q8h) does not appear to provide enhanced efficacy compared with pp296 q24h, when administered in combination with either vancomycin or daptomycin. Thus, the driver of efficacy appears to be AUC related.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description.
All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference.
This application claims the benefit of, and relies on the filing date of, U.S. provisional patent application No. 63/026,954, filed 19 May 2020, the entire disclosure of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/032958 | 5/18/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63026954 | May 2020 | US |
