HK1203549B - Dimeric bacteriophage lysins - Google Patents

Description

Dimeric phage lysin

[ technical field ] A method for producing a semiconductor device

The present invention relates to the production and use of dimeric lysins, in particular mutant lysins, which dimerise to dimeric lysins with enhanced activity and/or stability, for detecting and killing bacteria of the genus Streptococcus (Streptococcus). The present invention relates to methods for the prophylactic and therapeutic amelioration, decolonization and treatment of bacteria, particularly strains of the genus Streptococcus (Streptococcus), and related conditions. The methods of the invention utilize dimeric phage lysins, particularly dimeric pneumococcal phage lysins, including Cpl-1 lytic enzymes and variants thereof.

[ background of the invention ]

The emergence of drug resistant bacteria has been a major problem in medicine as more antibiotics are used in a wide range of diseases and other conditions. Nosocomial infections are the leading cause of death number 8 in the united states, mostly due to drug-resistant and emerging pathogens. More antibiotic usage and number of bacteria showing resistance suggest a longer treatment period. In addition, broad-spectrum non-specific antibiotics are now used more frequently, some of which have deleterious effects on patients. A related problem with this increased use is that many antibiotics do not readily penetrate the mucous lining. In addition, the number of people allergic to antibiotics appears to increase. Thus, there is a commercial need for new antibacterial methods, especially those that operate in new modes or provide new means of killing pathogenic bacteria.

Gram-positive bacteria are surrounded by a cell wall containing polypeptides and polysaccharides. The gram-positive cell wall appears as a broad dense wall, is 20-80 nm thick and consists of multiple peptidoglycan interconnected layers. Between 60% and 90% of the gram-positive cell walls are peptidoglycans, providing cell shape, rigid structure, and resistance to osmotic shock. The cell wall does not exclude gram-stained crystal violet, allowing the cell to stain purple, hence the term "gram-positive". Gram-positive bacteria include, but are not limited to, actinomycetes (Actinomyces), Bacillus (Bacillus), Listeria (Listeria), Lactococcus (Lactococcus), Staphylococcus (Staphylococcus), Streptococcus (Streptococcus), Enterococcus (Enterococcus), Mycobacterium (Mycobacterium), Corynebacterium (Corynebacterium), and Clostridium (Clostridium). Medically relevant species include Streptococcus pyogenes (Streptococcus pyogenenes), Streptococcus pneumoniae (Streptococcus pneumoniae), Staphylococcus aureus (Staphylococcus aureus) and Enterococcus faecalis (Enterococcus faecalis).

Antibacterial agents that inhibit cell wall synthesis, such as penicillins and cephalosporins, interfere with the peptide interpolypeptide linkage and attenuate the cell wall of gram-positive and gram-negative bacteria. Gram-positive bacteria are more sensitive to these antibiotics because their peptidoglycan is exposed. Advantageously, eukaryotic cells lacking a cell wall are not sensitive to these drugs or other cell wall agents.

Attempts have been made to treat bacterial diseases by using bacteriophages. However, the direct introduction of bacteriophages into animals to prevent or fight diseases has certain disadvantages. In particular, the bacteria and phage need to be in the correct and synchronized growth cycle for phage attachment. In addition, the correct number of phage is necessary to attach to the bacteria; if there are too many or too few phages, no attachment or no lyase will be produced. The phage must also be sufficiently active. Bacteriophages are also inhibited by a number of substances, including bacterial debris from the organism they are intended to attack. Further complicating the direct use of bacteriophages for the treatment of bacterial infections is the possibility of immunological reactions that render the bacteriophages non-functional.

New antimicrobial treatment methods include enzyme-based antibiotics ("enzyme antibiotics") such as phage lysin. Bacteriophages use these lysins to digest the cell wall of their bacterial host, releasing viral progeny by hypotonic lysis. Similar results are caused when purified, recombinant lysin is added externally to gram-positive bacteria. The high lethal activity of lysins against gram-positive pathogens makes them attractive candidates for development as therapeutics. Phage lysins were originally proposed for eradication of nasopharyngeal colonies of pathogenic streptococci (Loeffler, J.M.et al (2001) Science 294: 2170-. Lysins are part of the lytic mechanism used by double-stranded DNA (dsDNA) phages to lyse hosts in coordination with completing viral assembly (Wang, I.N. et al (2000) Annu Rev Microbiol 54: 799-825). Bacteriophages encode perforin, which opens pores in the bacterial membrane, and peptidoglycan hydrolases, known as lysins that break bonds in the bacterial wall. During the late stages of infection, lysin translocates into the cell wall matrix where it rapidly hydrolyzes the covalent bonds necessary for peptidoglycan integrity, resulting in bacterial lysis and concomitant release of progeny phage.

The lysin family members exhibit a modular design in which the catalytic domain is fused to a specific or binding domain (Lopez, R. et al (1997) Microb Drug Resist 3: 199-211). Lysins can be cloned from viral prophage sequences within the bacterial genome and used in therapy (Beres, s.b. et al (2007) PLoS ONE 2(8): 1-14). When externally added, lysin can access the bonds of gram-positive cell walls (FIG. 1) (Fischetti, V.A. (2008) Curr OpinionMicrobiol 11: 393-. Lysins have been shown to exhibit high lethal activity against a variety of gram-positive pathogens, particularly bacteria from which they are cloned, raising their potential for development as therapeutics (Fischetti, V.A. (2008) Curreopinion Microbiol 11: 393-.

Phage lytic enzymes have been established to be useful in assessing and specifically treating various types of subject infections by various routes of administration. For example, U.S. Pat. No.5,604,109 (Fischetti et al) is directed to rapid detection of group A streptococci in clinical specimens by enzymatic digestion (by semi-purified group C streptococcal phage-associated lysin enzymes). This enzyme work has been the basis of additional research leading to methods of treating disease. Fischetti and Loomis Patents (U.S. Pat. Nos. 5,985,271,6,017,528 and 6,056,955) disclose the use of lysin enzymes produced by group C streptococci bacteria infected with the C1 bacteriophage. Us patent 6,248,324(Fischetti and Loomis) discloses compositions for dermatological infections using lytic enzymes in a carrier suitable for topical application to dermal tissue. U.S. patent 6,254,866(Fischetti and Loomis) discloses a method of treating bacterial infections of the digestive tract comprising administering a lytic enzyme specific for the infecting bacteria. The carrier for delivering at least one lytic enzyme to the digestive tract is selected from the group consisting of: suppository enema, syrup or enteric coated pill. U.S. patent 6,264,945(Fischetti and Loomis) discloses methods and compositions for treating bacterial infections by parenteral introduction (intramuscular, subcutaneous or intravenous) of at least one lytic enzyme produced by a bacterium infected with a bacteriophage specific for the bacterium and a suitable carrier for delivering the lytic enzyme into a patient.

Phage-associated lytic enzymes have been identified and cloned from various bacteriophages, each of which has been shown to be effective in killing a particular bacterial strain. U.S. Pat. nos. 7,402,309, 7,638,600 and published PCT application WO2008/018854 provide different phage-associated lytic enzymes useful as antibacterial agents for treating or reducing infection by Bacillus anthracis (Bacillus anthracaris). U.S. Pat. No. 7,569,223 describes the pneumococcal phage lytic enzyme Pal of Streptococcus pneumoniae (Streptococcus pneumoniae). Lysins useful for Enterococcus (Enterococcus) (e.faecalis) and Enterococcus faecium (e.faecium), including vancomycin-resistant strains, are described in us patent 7,582291. US 2008/0221035 describes a highly potent mutant Ply GBS lysin in killing group B streptococci. A chimeric lysin, denoted ClyS, which has activity against staphylococcus (staphyloccci) bacteria, including staphylococcus aureus (staphyloccocusareus), is described in detail in WO 2010/002959.

Streptococcus pneumoniae (Streptococcus pneumoniae) (s. pneumoconiae)), gram-positive encapsulated diplococcus, is a major causative agent of human diseases such as bacteremia, meningitis, pneumonia, otitis media, and sinusitis. This bacterium is responsible for >1 million deaths worldwide by the age of 5 years (English, M (2000) Paediator Respir Rev1:21-5) and community-acquired pneumonia is the 6 th most common cause of death in the USA (File, TM (2004) Am J Med117Suppl 3A: 39S-50S). Moreover, streptococcus pneumoniae (s. pneumoconiae) is the leading cause of acute otitis media worldwide, affecting more than 5 million children's disease In the USA annually (CDC (2009) Pneumococcal diseases, p.217-30.In w. atkinson, et al (ed.), epidemic and prevention of vaccine-preventive diseases (11 th), Public Health Foundation, Washington DC). Finally, secondary infections due to influenza pandemics account for > 90% of deaths, with streptococcus pneumoniae (s.pneumoniae) being the leading cause of these deaths (Brundage, JF Shanks GD (2008) emery fed Dis 14: 1193-9; Brundage, JFShanks GD (2007) J fed Dis 196: 1717-8; Morens, DM et al (2009) N Engl J Med 361: 225-9; Morens, DM et al (2009) Public Health Rep 124: 22-5). Pneumococcal infections are often treated with antibiotics, but are bacteriologically confirmed to have failed treatment due to the increased incidence of resistance reported for macrolides, fluoroquinolones and cephalosporins (Reinert, RR (2009) Clin Microbiol Infect 15Suppl 3:1-3) (Mandell, LAet al (2002) Clin Infect Dis 35: 721-7). Overuse and misuse of antibiotics to treat millions of otitis media cases contributes only to the emergence of resistant strains (Goossens, H (2009) Clin Microbiol infection 15Suppl 3: 12-5). Together, these observations have prompted the need for new drugs that act by entirely different mechanisms for the treatment and prevention of pneumococcal-associated diseases.

Before the discovery of antibiotics, bacteriophages, the natural primary predator of bacteria, were considered as possible methods of controlling pathogenic bacteria. Several reports of successful Phage therapy for infection were then published (Sulakveldze, A andBoard therapy In animals and aggregations, p.335-71.In E.Kutterand A.Sulakveld (ed.), Bacteriophohages: Biology and Applications, CRC Press, USA; Sulakveld, A and Kutter, E (2005) Bacteriophage therapy In humans, p.381-426 In E.Kutter and A.Sulakveld (ed.), bacteriophags: Biology and A.Sulakveld, CRC Press, USA), but the emergence of antibiotics of the 40 th generation led to a rapid reduction In western Phage therapy studies. New interest in phage therapy and phage-derived anti-bacterial compounds has been seen for the past decades (Borysowski, J et al (2006) Exp Biol Med 231: 366-77; Fischetti, VA (2008) Curr Opin Microbiol 11: 393-400).

One of these products, the phage endolysins or lysins, has been developed for their rapid killing of gram-positive bacteria (Borysowski, J et al (2006) Exp Biol Med 231: 366-77; Fischetti, VA (2008) Curr Opin Microbiol 11: 393-400.) these specific enzymes are produced when the phage progeny are to escape the bacterial host. pneumococcal phage Cp-1 produces lysin-1, 37kDa enzyme. this lysin, which is composed like all such endolysins, has 2 well-defined domains linked by a flexible linker catalytic activity restricted to the N-terminal domain, whereas the C-terminal part of the C-terminal tail containing 6 choline-binding repeats (ChBR) and 13 amino-acids, the substrate binding in pneumococcal cells requires that the N-acetylmuramic acid and the N-acetyl-D-glucose residue in the Cpl-1 targeting peptidoglycan (ChBR) and the N-acetyl-D-glucose residue, 851, glucosamine linkage between N-acetyl-D-glucose residue (JL-10) is highly expressed by the Liposomal phage Escherichia coli gene (1987, JL-10. Biotech) expressing the lysozyme-21. expressing the subunit gene (Biotech) III-21. 9-21. the Streptococcus pneumoniae gene expressed by the Streptococcus.

Purified Cpl-1 has been successfully tested in rodent models for the treatment of pneumococcal sepsis (Jado, I et al (2003) J Antimicrob Chemother 52: 967-73; Loeffler, JM and Fischetti, VA (2003) Antimicrob Agents Chemother 47:375-7), endocarditis (Entenza, JM et al (2005) Antimicrob Agents Chemother 49:4789-92), pneumococcal meningitis (Grandigrard, D et al (2008) J Infect 197:1519-22), and pneumonia (witzenith, M et al (2009) Crit Care Med37: 642-9). However, proteins are often cleared rapidly in vivo, and repeated injections or even continuous infusions of Cpl-1 are required in many studies conducted to date (Entenza, JM et al (2005) antibiotic Agents Chemother49: 4789-92.31; Witzenrath, M et al (2009) Crit Care Med37: 642-9).

These results may be a disadvantage of clinical development of Cpl-1 and similar lysins. There is a need in the art for improved lysins useful in the treatment of pneumococcal disease that have killing activity and enhanced clinically-relevant parameters, such as longer half-life or reduced clearance in vivo.

[ SUMMARY OF THE INVENTION ]

In a general aspect, the present invention provides mutant lysins mutated to have the ability to readily dimerize, thereby producing a dimeric lysin with enhanced activity or bactericidal activity, and greater stability, including longer stability in animals or mammals and longer bacterial killing in clinically relevant or biologically relevant environments.

In one aspect, the invention provides an isolated dimeric anti-bacteriophage lysin comprising 2 bacteriophage lysin monomers covalently linked to each other specific for a bacterium, wherein the dimer has bactericidal activity against one or more specific bacteria. In one aspect, the invention provides an isolated Streptococcus dimeric (Streptococcus) -specific phage lysin comprising 2 Streptococcus (Streptococcus) -specific phage lysin monomers covalently linked to each other, wherein the dimer has killing activity against one or more Streptococcus (Streptococcus) bacteria. In particular embodiments, the lysin monomer has at least 80%, at least 90%, at least 95% amino acid sequence identity to the unmutated Cpl-1 lysin shown in fig. 1 and 6 and SEQ ID NO: 1.In certain embodiments, the lysin monomer has at least 80%, at least 90%, at least 95% amino acid sequence identity to the unmutated Pal lysin shown in figure 7 and SEQ ID No. 5.

In particular embodiments, the lysin monomers are chemically cross-linked to each other. In one such example, the monomers can be chemically crosslinked via reactive groups or via amino acids in the monomer sequence, including modified, altered, or mutated amino acids. The lysin monomers may be covalently linked to each other by disulfide bonds. In a particular exemplary embodiment, each lysin monomer has a Cys residue immediately adjacent to the C-terminus, in particular between 14 and 20 amino acids from the C-terminus. In particular embodiments, the lysin monomer does not have a Cys residue in the first 45 residues. Exemplary mutant Cpl-1 lysins are provided herein, including in fig. 6 and table 1.In a particular embodiment, the lysin monomer has an amino acid sequence as shown in figure 6 or provided in table 1.In another embodiment, the lysin monomer has the amino acid sequence shown in figure 7. In particular embodiments, the lysin monomer comprises a catalytic domain of a Streptococcus sp (Streptococcus) -specific phage lysin 1 and a binding domain of a Streptococcus sp (Streptococcus) -specific phage lysin 2.

The present invention provides methods of treating a disease or condition in a mammal resulting from a streptococcal infection by administering a composition comprising a therapeutically effective amount of a dimeric lysin comprising 2 streptococcal (Streptococcus) -specific phage lysin monomers covalently linked to one another, wherein the dimer has bactericidal activity against one or more Streptococcus (Streptococcus) bacteria. In a particular embodiment, the infection is caused by Streptococcus pneumoniae (Streptococcus pneumoniae). The disease or condition caused by infection with Streptococcus pneumoniae (Streptococcus pneumoniae) may be at least one of bacteremia, meningitis, pneumonia, otitis media, or sinusitis.

The present invention provides methods of inhibiting or de-colonizing a streptococcal infection in a mammal by administering a composition comprising a therapeutically effective amount of a dimeric lysin comprising 2 streptococcal (Streptococcus) -specific phage lysin monomers covalently linked to one another, wherein the dimer has bactericidal activity against one or more Streptococcus (Streptococcus) bacteria. In a particular embodiment, the infection is caused by Streptococcus pneumoniae (Streptococcus pneumoniae). The disease or condition caused by infection with Streptococcus pneumoniae (Streptococcus pneumoniae) may be at least one of bacteremia, meningitis, pneumonia, otitis media, or sinusitis.

In one aspect of the invention, there is provided a method of killing gram positive bacteria comprising the step of contacting the bacteria with a composition comprising an amount of a mutant dimeric lysin polypeptide effective for killing gram positive bacteria, wherein the monomeric lysin is modified or altered to comprise 2 antibacterial bacteriophage lysin monomers covalently linked to each other and a dimeric lysin polypeptide effective for killing gram positive bacteria. In a particular aspect of this aspect, there is provided a method of killing a Streptococcus (Streptococcus) bacterium, comprising the step of contacting the bacterium with a composition comprising an amount of a mutant dimeric lysin polypeptide effective to kill the Streptococcus (Streptococcus) bacterium, wherein monomeric Streptococcus (Streptococcus) lysin is modified or altered to comprise 2 Streptococcus (Streptococcus) phage lysin monomers covalently linked to one another and a dimeric lysin polypeptide effective to kill the Streptococcus (Streptococcus) bacterium.

Thus, there is provided a method of killing a Streptococcus (Streptococcus) bacterium, comprising the step of contacting the bacterium with a composition comprising an amount of an isolated Streptococcus (Streptococcus) Cpl-1 lysin polypeptide (an isolated lysin polypeptide comprising a mutant Cpl-1 lysin comprising 2 Cpl-1 lysin monomers covalently linked to each other) effective to kill the Streptococcus (Streptococcus) bacterium. In a particular aspect, there is provided a method of killing a Streptococcus (Streptococcus) bacterium, comprising the step of contacting the bacterium with a composition comprising an amount of an isolated lysin polypeptide effective for killing Streptococcus (Streptococcus) bacteria (an isolated lysin polypeptide comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof having at least 80%, 85%, 90% or 95% homology to the polypeptide of SEQ ID NO:3) and effective for killing gram-positive bacteria.

Also provided is a method of killing Streptococcus (Streptococcus) bacteria, comprising the step of contacting the bacteria with a composition comprising an amount of an isolated Streptococcus (Streptococcus) Pal lysin polypeptide (an isolated lysin polypeptide containing a mutant Pal lysin comprising 2 Pal lysin monomers covalently linked to each other) effective to kill Streptococcus (Streptococcus) bacteria. In a particular aspect, there is provided a method of killing a Streptococcus (Streptococcus) bacterium, comprising the step of contacting the bacterium with a composition comprising an amount of an isolated lysin polypeptide effective for killing Streptococcus (Streptococcus) bacteria (an isolated lysin polypeptide comprising the amino acid sequence of SEQ ID NO:6 or a variant thereof having at least 80%, 85%, 90% or 95% homology to the polypeptide of SEQ ID NO: 6) and effective for killing gram-positive bacteria.

The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a dimeric lysin comprising 2 Streptococcus (Streptococcus) -specific phage lysin monomers covalently linked to each other, wherein the dimer has bactericidal activity against one or more Streptococcus (Streptococcus) bacteria, and a pharmaceutically acceptable carrier.

The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a dimeric lysin comprising 2 Streptococcus (Streptococcus) -specific phage lysin monomers covalently linked to each other, wherein said dimer has killing activity against one or more Streptococcus (Streptococcus) bacteria, and wherein said killing activity is greater than the killing activity of any of the Streptococcus (Streptococcus) -specific phage lysin monomers, and a pharmaceutically acceptable carrier.

In a further aspect, the present invention provides an anti-microbial composition for cleaning or decontaminating porous or non-porous surfaces, comprising a dimeric lysin comprising 2 Streptococcus (Streptococcus) -specific phage lysin monomers covalently linked to each other, wherein the dimer has bactericidal activity against one or more Streptococcus (Streptococcus) bacteria.

The compositions of the invention may exhibit or have killing activity specifically against one or more strains of bacteria of the genus Streptococcus (Streptococcus), in particular selected from: streptococcus suis (Streptococcus suis), Streptococcus equi (Streptococcus equi), Streptococcus agalactiae (Streptococcus agalactiae) (GBS), Streptococcus pyogenes (Streptococcus pyogenenes) (GAS), Streptococcus sanguis (Streptococcus sanguinis), Streptococcus grisea (Streptococcus gordonii), Streptococcus dysgalactiae (Streptococcus dysgalactiae), Streptococcus (Streptococcus) GES and Streptococcus pneumoniae (Streptococcus pneumaonia).

The present invention provides a method of decontaminating an inanimate surface suspected of containing infectious bacteria, comprising treating the surface with a bactericidally or bactericidally effective amount of an anti-microbial composition comprising a dimeric lysin comprising 2 Streptococcus (Streptococcus) -specific bacteriophage lysin monomers covalently linked to each other, wherein the dimer has bactericidal activity against one or more Streptococcus (Streptococcus) bacteria, for cleaning or decontaminating a porous or non-porous surface.

The present invention includes dimeric proteins comprising 2 Streptococcus (Streptococcus) -specific phage lysin binding domains covalently linked to each other. The present invention provides a dimeric protein comprising 2 Streptococcus (Streptococcus) -specific phage lysin binding domains covalently linked to each other, wherein at least one of the phage lysin binding domains is further conjugated to a label. The label may be any molecule that produces, or can be induced to produce, a detectable signal. Non-limiting examples of labels include radioisotopes, enzymes, enzyme fragments, enzyme substrates, enzyme inhibitors, coenzymes, catalysts, fluorophores, dyes, chemiluminescent agents, luminescent agents or sensitizers; non-magnetic or magnetic particles, solid supports, liposomes, ligands, or receptors.

The diagnostic utility of the present invention extends to the use of the lysin polypeptides in screening for the presence of gram positive bacteria, in screening for the presence of susceptible gram positive bacteria, or in determining the susceptibility of a bacterium to killing or lysis by one or more lysin polypeptides of the present invention.

Modified and chimeric or fusion proteins, or labeled lysin polypeptides are contemplated and provided herein. In chimeric or fusion proteins, the lysin polypeptides of the invention may be covalently attached to an entity that may provide additional functionality or enhance the use or application of the lysin polypeptide, including, for example, tags, labels, targeting moieties or ligands, cell binding or cell recognition motifs or agents, antibacterial agents, antibodies, antibiotics. Exemplary labels include radioactive labels, such as isotopes³H，¹⁴C，³²P，³⁵S，³⁶Cl，⁵¹Cr，⁵⁷Co，⁵⁸Co，⁵⁹Fe，⁹⁰Y，¹²⁵I，¹³¹I and¹⁸⁶re. The label may be an enzyme and detection of the labeled lysin polypeptide may be accomplished by any currently utilized or accepted colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gas quantitation technique known in the art.

Other objects and advantages will become apparent to those skilled in the art from the following description, with reference to the following illustrative drawings.

[ description of the drawings ]

FIG. 1 ClustalW alignment of amino acid sequences of Cpl-1(SEQ ID NO:1) and Streptococcus pneumoniae (S.pneumoconiae) LytA (SEQ ID NO: 2). 2 catalytic residues (D10 and E94) are shown in red. The 13 amino acids involved in the natural dimerization of lytA and the corresponding amino acids in Cpl-1 are shown underlined. Residue D324 is shown in blue.

FIGS. 2A-2C depict a Coomassie stained non-reducing SDS-PAGE gel of (A) purified Cpl-1. Lane 1, molecular weight marker, and Lane 2, purified Cpl-1 obtained by affinity purification on DEAE-agarose. (B) Purified Cpl-1^C45S,D324CCoomassie stained non-reducing SDS-PAGE gels of the mutants. Lane 1, molecular weight marker. Lane 2, purified Cpl-1 from DEAE-agarose affinity purification^C45S,D324CLane 3, Cpl-1 reduced with 10mM DTT^C45S,D324C. (C) Cpl-1 purified after gel filtration on Sephadex G100^C45S,D324CCoomassie stained non-reducing SDS-PAGE gels of mutant dimer enrichments. Lane 1, molecular weight marker. Lane 2, purified Cpl-1 from gel filtration on Sephadex G100^C45S,D324CDimer (purification 1), and lane 3, purified Cpl-1 from gel filtration on Sephadex G100^C45S,D324CDimer (2 nd purification).

FIG. 3 shows 5.10 of Cpl-1 vs. Streptococcus pneumoniae (S.pneumoconiae) DCC1490 after incubation at 37 ℃ for 15 minutes⁸In vitro anti-bacterial Activity of CFU/ml suspension. Cpl-1wt (filled circles), Cpl-1 in 1mM DTT^C45S,D324CMonomeric form (open rectangle), and Cpl-1^C45S,D324CDimer (ring opening). N-4 for each enzyme and condition.

Figure 4 plasma clearance of enzyme antibiotics in mice. Balb/c mice were injected with 100. mu.l PB50mM, 12.16nmol Cpl-1 (filled circles) or Cpl1 in pH7.4^C45S,D324CDimer (ring opening), n being 3 for each time point.

FIG. 5. 5.10 of several purified Cpl-1 mutant dimers against Streptococcus pneumoniae (S. pneumoconiae) DCC1490 at 37 ℃⁸In vitro anti-microbial Activity of CFU/ml suspension. Cpl-1wt (solid circle), Cpl-1^C45S,Q85CDimer (solid diamond), Cpl-1^C45S,D194CDimer (filled triangle), Cpl-1^C45S,N214CDimer (filled rectangle), Cpl-1^{C45S ,G216C}Dimer (hollow triangle), Cpl-^1C45S,D256CDimer (rhomboid), Cpl-1^C45S,S269CDimer (Ring opening), Cpl-1^C45S,D324CDimer (open rectangle). All enzymes were tested at a concentration of 0.5 mg/ml. Each point represents the average of 3 experiments.

FIG. 6 depicts the aligned amino acid sequences of (unmutated) Cpl-1(SEQ ID NO:1), and the mutants lysin C45S (SEQ ID NO:4) and C45S, D324C (SEQ ID NO: 3). Amino acid changes in the mutants are underlined.

FIG. 7 depicts aligned amino acid sequences of Cpl-1(SEQ ID NO:1), Pal (SEQ ID NO:5) and Streptococcus pneumoniae (S. pneumoconiae) LytA (SEQ ID NO:2) amino acid sequences. The same amino acids are indicated by asterisks throughout all 3 sequences. The 13 amino acids involved in the natural dimerization of LytA and similar amino acids in Cpl-1 and Pal are indicated by underlining and bolding. The corresponding amino acids mutated in exemplary dimeric mutants of Cpl-1 (residue D324) and Pal (residue D280) are boxed.

[ detailed description of the invention ]

Streptococcus dimerization (Streptococcus) -specific phage lysins having killing activity against Streptococcus pneumoniae (s. pneumoniae) are described herein. In general, dimeric phage lysins contain 2 Streptococcus (Streptococcus) -specific phage lysin monomers covalently linked to each other, wherein the dimer is active against one or more Streptococcus (Streptococcus) bacteria. The lysin monomers of the lysin dimer may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or even at least 99.5% amino acid sequence identity to unmutated Cpl-1 (FIG. 1, SEQ ID NO: 1). The lysin monomers of the lysin dimer may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or even at least 99.5% amino acid sequence identity to the unmutated Pal (fig. 7, SEQ ID NO: 5). The stabilized dimeric lysins provided herein exhibit increased bactericidal activity, approximately twice as much in vitro anti-bacterial activity as the original monomeric molecules. Dimeric lysins also show nearly 10-fold reduced plasma clearance between 5 minutes and 5 hours post-infection, either with reduced plasma clearance or increased stability in plasma or animals.

FIG. 1 shows the amino acid sequence of the C-terminal region of LytA and the homology of streptococcal (Streptococcus) lysin Cpl-1 (73/142 identical residues, and 55/69 residues are conservative substitutions). The C-terminal 13 amino acids are responsible for dimerization of Streptococcus pneumoniae (Streptococcus pneumoniae) autolysin LytA. Interestingly, the 10/13 amino acids were identical between Cpl-1 and LytA in this region. Fully active LytA is a choline-binding homodimer consisting of a tail association of 2 LytA monomers initiated by choline interactions. The main driving force for dimerization is provided by the hydrophobic core resulting from intermolecular hydrophobic interactions between several residues in the C-terminal choline binding regions 6 and 7 (8). The 13C-terminal residues of LytA are responsible for the formation of active homodimers whose activity is significantly greater than the native monomer. Indeed, the recombinant monomeric form of LytA lacking this 13 amino acid extension (27) retained less than 10% of the catalytic efficiency of the enzyme (24). Sequence alignment of Cpl-1 and LytA revealed similarity of extension, especially within the C-terminal tail of the enzymes involved in LytA dimerization (FIG. 1). The presence of a glomus filtration threshold estimated at approximately 60-65 kDa in humans (17) suggests that dimeric forms of Cpl-1 (MW of 74 kDa) may show a significant reduction compared to monomeric systemic clearance. Cpl-1 dimer, consisting of 2 monomers linked by covalent bonds and stabilized by disulfide bonds, has thus been processed and examined herein for its in vitro activity and in vivo plasma clearance compared to the monomeric form of Cpl-1. Moreover, and because several other phage lysins have been shown or suspected to dimerize (25,26), this represents a general way to increase the activity and/or pharmacokinetics of a particular phage lysin.

Conventional molecular biology, microbiology and recombinant DNA techniques within the skill of those in the art may be employed in accordance with the present invention. This technique is fully explained in the literature. See, e.g., Sambrook et al, "Molecular Cloning: laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-III [ Ausubel, R.M., ed. (1994) ]; "Cell Biology: A laboratory handbook" Volumes I-III [ J.E.Celis, ed. (1994)) ]; "Current Protocols in Immunology" Volumes I-III [ Coligan, J.E., ed. (1994) ]; "Oligonucleotide Synthesis" (m.j.gate ed.1984); "Nucleic Acid Hybridization" [ B.D.Hames & S.J.Higgins eds. (1985) ]; "TranscriptionnND transformation" [ B.D.Hames & S.J.Higgins, eds. (1984) ]; "Animal Cell Culture" [ r.i. freshney, ed. (1986) ]; "Immobilized Cells And Enzymes" [ IRL Press, (1986) ]; B.Perbal, "A Practical Guide To Molecular Cloning" (1984).

Thus, if appearing herein, the following terms shall have the definitions as provided and set forth below and in this section.

The terms "Streptococcus (Streptococcus) dimeric lysin", "Cpl-1 dimer", "dimeric Cpl-1", and any variants not specifically listed, may be used interchangeably herein, and as used throughout this application and the claims refer to proteinaceous material comprising single or multiple proteins, and specifically dimeric proteins, and extends to those proteins having the amino acid sequence data shown in fig. 6 and table 1, and SEQ ID NO:3, described herein, and the activity characteristics described herein and in the claims. Thus, proteins exhibiting substantially equivalent or altered activity are also contemplated. These modifications may be deliberate, such as, for example, those obtained by site-directed mutagenesis, or may be accidental, such as those obtained by mutation in the host which is the producer of the complex or its named subunit. Furthermore, the terms "Streptococcus (Streptococcus) dimeric lysin", "Cpl-1 dimer", "dimeric Cpl-1" are intended to include within their scope the proteins specifically referenced herein as well as all substantially homologous analogs, fragments or truncations, and allelic variations.

The terms "Streptococcus (Streptococcus) dimeric lysin", "Pal dimer", "dimeric Pal", and any variants not specifically listed, may be used interchangeably herein, and as used throughout this application and claims refer to proteinaceous material comprising single or multiple proteins, and in particular dimeric proteins, and extends to those proteins having the amino acid sequence data shown in fig. 7, and SEQ ID NO:5, as described herein and in the claims, and the activity characteristics described herein. Thus, proteins exhibiting substantially equivalent or altered activity are also contemplated. These modifications may be deliberate, such as, for example, those obtained by site-directed mutagenesis, or may be accidental, such as those obtained by mutation in the host which is the producer of the complex or its named subunit. Moreover, the terms "Streptococcus (Streptococcus) dimeric lysin", "Pal dimer", "dimeric Pal" are intended to include within the scope of the proteins specifically referenced herein as well as all substantially homologous analogs, fragments or truncations, and allelic variations.

[ Polypeptides and lytic enzymes ]

"lytic enzyme" includes any bacterial cell wall lytic enzyme that kills one or more bacteria under suitable conditions and during an associated period. Examples of lytic enzymes include, without limitation, various amidase cell wall lytic enzymes.

"Streptococcus (Streptococcus) lytic enzymes" include lytic enzymes that are capable of killing at least one or more Streptococcus (Streptococcus) bacteria under suitable conditions and during a relevant time period.

"phage lytic enzyme" refers to a lytic enzyme extracted or isolated from a bacteriophage or a synthetic lytic enzyme having a similar protein structure that maintains the functionality of the lytic enzyme.

Lytic enzymes are capable of specifically cleaving peptidoglycan linkages present in bacterial cells to disrupt the bacterial cell wall. It is also currently assumed that bacterial cell wall peptidoglycans are highly conserved among most bacteria, and that only a few bonded cleavages can disrupt the bacterial cell wall. The phage lytic enzyme may be an amidase, although other types of enzymes are possible. Examples of cleaving enzymes that cleave these bonds are various amidases such as muramidase, glucosaminidase, endopeptidase or N-acetyl-muramyl-L-alanine amidase. Fischetti et al (2008) reported that the C1 streptococcal phage lysin enzyme is an amidase. Garcia et al (1987,1990) reported that Cpl lysin from streptococcus pneumoniae (s. pneumoconiae) from Cp-1 phage is lysozyme. Caldentey and BamHord (1992) reported that the lytic enzyme from phi 6 Pseudomonas (Pseudomonas) phage is an endopeptidase that cleaves the peptide bridge formed by m-diaminopimelic acid and D-alanine. Coli (e.coli) T1 and T6 phage lytic enzymes are amidases such as lytic enzymes from Listeria (Listeria) phage (ply) (Loessner et al, 1996). Other lytic enzymes known in the art are also known which are capable of cleaving bacterial cell walls.

"lytic enzymes genetically encoded by a bacteriophage" include polypeptides that are capable of killing a host bacterium, for example, by having at least some cell wall lytic activity against the host bacterium. The polypeptide may have a sequence that includes a native sequence cleaving enzyme and variants thereof. The polypeptides can be isolated from various sources, such as from phage ("phage"), or prepared by recombinant or synthetic methods, such as those described by Garcia et al and also as provided herein. The polypeptide may comprise a choline binding moiety on the carboxy-terminal side and may be characterized by an enzymatic activity capable of cleaving a cell wall peptidoglycan (such as an amidase activity acting on amide bonds in the peptidoglycan) on the amino-terminal side. Lytic enzymes comprising multiple enzymatic activities, e.g. 2 enzymatic domains, such as PlyGBS lysin, have been described. In general, the molecular weight of the lytic enzyme may be between 25,000 and 35,000Da and comprise a single polypeptide chain; however, this may depend on the enzyme chain change. Molecular weight can be most conveniently determined by measurement on denaturing sodium dodecyl sulfate gel electrophoresis and comparison with molecular weight markers.

"native sequence phage-associated lytic enzymes" include polypeptides having the same amino acid sequence as the enzyme derived from the bacterium. The native sequence enzyme may be isolated or may be produced by recombinant or synthetic means.

The term "native sequence enzyme" includes naturally occurring forms (e.g., alternatively spliced or altered forms) and naturally occurring variants of the enzyme. In one embodiment of the invention, the native sequence enzyme is genetically encoded by a gene from a bacteriophage specific for Streptococcus (Streptococcus), and in particular a mature or full-length polypeptide of native Cpl-1 lysin or native Pal lysin. Of course, many variations are possible and known, as in publications such as Lopez et al, Microbial Drug Resistance 3: 199-; garcia et al, Gene 86:81-88 (1990); garcia et al, Proc.Natl.Acad.Sci.USA 85: 914-; garcia et al, Proc.Natl.Acad.Sci.USA 85: 914-; garcia et al, streptococcus Genetics (j.j.ferretti and Curtis eds., 1987); lopez et al, FEMS Microbiol. Lett.100: 439-; romero et al, J.Bacteriol.172: 5064-; ronda et al, Eur.J.biochem.164:621-624(1987) and Sanchez et al, Gene 61:13-19 (1987). The contents of each of these references, particularly the sequence listing and associated text comparing the sequences, including statements regarding sequence homology, are specifically incorporated herein by reference in their entirety.

"variant sequence lytic enzymes" include lytic enzymes characterized by a polypeptide sequence that is different from the lytic enzyme, but retains functional activity. The lytic enzyme may, in some embodiments, be genetically encoded by a bacteriophage specific to Streptococcus (Streptococcus), having a specific amino acid sequence identity to the present lytic enzyme sequence, such as the variant lytic enzymes provided in fig. 1,6 and 7, including SEQ ID NOs 3, 4 and 5. For example, in some embodiments, functionally active lytic enzymes can kill Streptococcus (Streptococcus) bacteria, and other susceptible bacteria, by disrupting the cell wall of the bacteria, as provided herein, or as previously described and known to those of skill in the art. An active lytic enzyme (e.g., a variant lytic enzyme provided in FIGS. 1,6, and 7, including SEQ ID NOS: 3, 4, and 5) can have 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 99.5% amino acid sequence identity to the instant lytic enzyme sequence. Such phage-associated lytic enzyme variants include, for example, lytic enzyme polypeptides comprising one or more amino acid residues altered or added or deleted at the N-or C-terminus of the sequence of the lytic enzyme sequence. In particular aspects, the bacteriophage-associated lytic enzyme will have at least about 80% or 85% amino acid sequence identity, particularly at least about 90% (e.g., 90%) amino acid sequence identity, to the native bacteriophage-associated lytic enzyme sequence. Most particular phage-associated dimeric lytic enzyme variants will have at least about 95% (e.g., 95%) amino acid sequence identity to the lytic enzyme sequences associated with the native phage (e.g., the present sequences provided in FIG. 1, FIG. 6 and FIG. 7 or Table 1 or including SEQ ID NO:1 or 5, or mutant sequences of SEQ ID NO:3, 4 or 6).

"percent amino acid sequence identity" with respect to a bacteriophage-associated lytic enzyme sequence identified herein is defined herein as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the bacteriophage-associated lytic enzyme sequence after aligning the sequences in the same reading frame and, if necessary, introducing a gap to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

"percent nucleic acid sequence identity" with respect to a bacteriophage associated lytic enzyme sequence identified herein is defined herein as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides in the bacteriophage associated lytic enzyme sequence after aligning the sequences and, if necessary, introducing a gap to achieve the maximum percent sequence identity.

To determine the percent identity of 2 nucleotide or amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of nucleotide sequence 1). The nucleotides or amino acids at corresponding nucleotide or amino acid positions are then compared. When a position in the 1 st sequence is occupied by the same nucleotide or amino acid as the corresponding position in the 2 nd sequence, the molecules are identical at that position. The percent identity between 2 sequences is a function of the number of identical positions shared by the sequences (i.e.,% identity ═ identical position #/total position # × 100).

Determination of percent identity between 2 sequences can be achieved by using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm for comparing 2 sequences is the algorithm of Karlin et al, Proc. Natl. Acad. Sci. USA,90: 5873-. The algorithm will incorporate the NBLAST program, which can be used to identify sequences having the desired identity to the nucleotide sequences of the present invention. For comparison purposes to obtain a gapped alignment, gapped BLAST can be used as described in Nucleic Acids Res,25: 3389-. When utilizing BLAST and gapped BLAST programs, the default parameters of the corresponding program (e.g., NBLAST) can be used. See the program supplied by National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health. In one embodiment, the parameter for sequence comparison may be set to W-12. The parameters may also be changed (e.g., W-5 or W-20). The value "W" determines how many consecutive nucleotides must be identical for the procedure to identify 2 sequences as containing a region of identity.

"polypeptide" includes polymer molecules consisting of a plurality of amino acids joined in a linear fashion. A polypeptide may, in some embodiments, correspond to a molecule encoded by a naturally occurring polynucleotide sequence. Polypeptides may include conservative substitutions, wherein a naturally occurring amino acid is substituted with an amino acid having similar properties, wherein the conservative substitution does not alter the function of the polypeptide (see, e.g., Lewis "Genes V" Oxford University Press Chapter 1, pp.9-131994).

The term "altered lytic enzyme" includes shuffled and/or chimeric lytic enzymes.

Phage lytic enzymes specific for bacteria infected with a particular bacteriophage have been found to efficiently and effectively disrupt the cell wall of the target bacteria. Lytic enzymes are believed to lack proteolytic enzymatic activity and are therefore non-destructive to mammalian proteins and tissues when present during digestion of the bacterial cell wall. Purified pneumococcal phage lytic enzymes, such as Pal, are shown to kill various pneumococci as demonstrated by loefler et al, "Rapid bagging of Streptococcus pneumoconiae with a bacteriophase Cell wall Hydrolase," Science,294: 2170-. Loeffler et al showed by these experiments that the lytic enzyme Pal was able to kill 15 clinical stains of streptococcus pneumoniae (s. pneumoconiae) in vitro, including the most commonly isolated serogroup and penicillin-resistant stains, within seconds after contact. Treatment of mice with Pal also eliminated or significantly reduced nasal colonies of serotype 14 in a dose-dependent manner. Furthermore, because it has been found that Pal acts (like other phage lytic enzymes, but unlike antibiotics) more specifically to target pathogens, it is possible that the normal flora will remain substantially intact (m.j.loessner, g.wendlinger, s.scherer, Mol Microbiol 16,1231-41 (1995) incorporated herein by reference). As demonstrated herein, for example, the mutant Cpl-1 lysin, particularly the dimeric lysin, dimeric Cpl-1 lysin, is effective in killing Streptococcus (Streptococcus) strains, including Streptococcus pneumoniae (Streptococcus pneumoniae).

The lytic enzymes or polypeptides of the invention may be produced by bacterial organisms following infection with a particular bacteriophage as a prophylactic treatment for preventing those who have been exposed to other symptoms of infection from becoming ill, or as a therapeutic treatment for those who have become ill from infection. As provided herein, the lysin polypeptide sequences and nucleic acids encoding lysin polypeptides, the lytic enzyme/polypeptide may preferably be produced via an isolated lytic enzyme gene from a phage genome, wherein the gene is placed into a transfer vector, and the transfer vector is cloned into an expression system (using standard methods in the art, including those exemplified herein). The lytic enzyme or polypeptide may be truncated, chimeric, shuffled or "native", and may be a combination of these. Associated U.S. Pat. No.5,604,109 is incorporated herein by reference in its entirety. An "altered" lytic enzyme can be produced in a number of ways. In a preferred embodiment, the altered lytic enzyme gene from the genome of the phage is introduced into a transfer or mobilizable vector, preferably a plasmid, and the plasmid is cloned into an expression vector or expression system. The expression vector used to produce the lysin polypeptides or enzymes of the present invention may be adapted for escherichia coli (e.coli), Bacillus (Bacillus) or many other suitable bacteria. The vector system may also be a cell-free expression system. All such methods of expressing a gene or combination of genes are known in the art. Lytic enzymes may also be produced by infecting Streptococcus (Streptococcus) with a bacteriophage specific for Streptococcus, wherein the at least one lytic enzyme exclusively lyses the cell wall of said Streptococcus having at most a minimal effect on other, e.g. natural or commensal, existing bacterial flora.

A "chimeric protein" or "fusion protein" comprises all or a (preferably biologically active portion or domain) portion of a polypeptide of the invention operably linked to a heterologous polypeptide. Chimeric proteins or peptides are produced, for example, by combining 2 or more proteins having 2 or more active sites. Chimeric proteins and peptides can act independently on the same or different molecules and thus have the potential to treat 2 or more different bacterial infections simultaneously. Chimeric proteins and peptides can also be used to treat bacterial infections by cleaving the cell wall at more than one position, thereby potentially providing a more rapid or effective (or synergistic) kill by a lysin molecule or chimeric peptide.

A "heterologous" region of a DNA construct or peptide construct is an identifiable segment of DNA within a larger DNA molecule or peptide within a larger peptide molecule that is not found naturally associated with the larger molecule. Thus, when a heterologous region encodes a mammalian gene, the gene will typically be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (e.g., a cDNA in which the genomic coding sequence contains introns, or a synthetic sequence having codons different from the native gene). Allelic variations or naturally occurring mutational events do not result in heterologous regions of DNA or peptides as defined herein.

The term "operably linked" means that the disclosed polypeptide and heterologous polypeptide are fused in frame. Heterologous polypeptides can be fused to the N-terminus or C-terminus of the disclosed polypeptides. Chimeric proteins are produced enzymatically by chemical synthetases or by recombinant DNA techniques. Many chimeric lytic enzymes have been generated and studied. The genes E-L constructed from phage phiX174 and MS2 cleavage proteins E and L, respectively, were subjected to chimeric cleavage by internal deletion to generate a series of new E-L clones with altered lytic or bactericidal properties. The lytic activity of the parental genes E, L, E-L, and the internally truncated forms of E-L were studied in this study to characterize different lytic mechanisms based on differences in the architecture of the different transmembrane domains. Electron microscopy and release of marker enzymes for the cytoplasmic and periplasmic spaces revealed that 2 different cleavage mechanisms can rely on the penetration of proteins of the inner or inner and outer membranes of Escherichia coli (E.coli) for differentiation (FEMS Microbiol. lett. (1998)164(1):159-67 (incorporated herein by reference.) an example of a useful fusion protein is a GST fusion protein in which the disclosed polypeptide is fused to the C-terminus of the GST sequence.

In another embodiment, the chimeric protein or peptide contains a heterologous signal sequence at its N-terminus. For example, the native signal sequence of the disclosed polypeptide can be removed and replaced with a signal sequence from another protein. For example, the gp67 secretory sequence of baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubelet al., eds., John Wiley & Sons,1992, incorporated herein by reference). Other examples of eukaryotic heterologous signal sequences include melittin and the secretory sequence of human placental alkaline phosphatase (Stratagene; La Jolla, Calif.). In yet another example, useful prokaryotic heterologous signal sequences include the phoA secretion signal (Sambrook et al, supra) and the protein A secretion signal (Pharmacia Biotech; Piscataway, N.J.).

Fusion proteins may combine lysin polypeptides with proteins or polypeptides having different abilities, or providing lysin polypeptides with additional abilities or added characteristics. The fusion protein can be an immunoglobulin fusion protein of all or part of the disclosed polypeptide fused to a sequence derived from a member of the immunoglobulin family. The immunoglobulin may be an antibody, for example, an antibody directed against a surface protein or epitope that is sensitive or targeted to the bacteria. Immunoglobulin fusion proteins can be incorporated into pharmaceutical compositions and administered to a subject to inhibit (soluble or membrane-bound) interaction between a ligand and a protein (receptor) on the surface of a cell, thereby inhibiting signal transduction in vivo. Immunoglobulin fusion proteins can alter the bioavailability of homologous ligands of the disclosed polypeptides. Inhibition of ligand/receptor interactions can be a useful treatment, both for the treatment of bacterial-associated diseases and disorders that modulate (i.e., promote or inhibit) cell survival. Furthermore, the disclosed immunoglobulin fusion proteins can be used as immunogens to generate antibodies against the disclosed polypeptides in a subject, to purify ligands, and to identify molecules that inhibit receptor-ligand interactions in screening assays. The disclosed chimeric and fusion proteins and peptides can be produced by standard recombinant DNA techniques.

The fusion gene can be synthesized by conventional techniques, including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that cause complementary overhang between 2 consecutive gene fragments that can then be annealed and reamplified to generate a chimeric gene sequence (see, i.e., Ausubel et al, supra). Moreover, many expression vectors are commercially available that already encode a fusion moiety (i.e., a GST polypeptide). Nucleic acids encoding the polypeptides of the invention can be cloned into the expression vector such that the fusion moiety is linked in-frame to the polypeptides of the invention.

As used herein, a shuffled protein or peptide, gene product, or peptide for more than one bacteriophage protein or protein peptide fragment of interest has been randomly cleaved and reassembled into a more active or specific protein. The shuffled oligonucleotide, peptide or peptide fragment molecules are selected or screened to identify molecules having the desired functional properties. This method is described, for example, in Stemmer, U.S. Pat. No.6,132,970 (method of shuffling polynucleotides); kauffman, U.S. Pat. No.5,976,862 (via codon-based synthetic evolution) and Huse, U.S. Pat. No.5,808,022 (direct codon synthesis). The contents of these patents are incorporated herein by reference. Shuffling can be used to produce more active proteins, for example proteins that have 10-100 fold activity as the template protein. The template protein was selected among different lysin proteins. Shuffled proteins or peptides, e.g., one or more binding domains and one or more catalytic domains. Each binding or catalytic domain is derived from the same or different bacteriophage or bacteriophage protein. Shuffled domains are oligonucleotide-based molecules, such as genes or gene products, alone or in combination with other genes or gene products translatable into peptide fragments, or they are peptide-based molecules. Gene fragments include any molecule of DNA, RNA, DNA-RNA hybrids, antisense RNA, ribozymes, ESTs, SNIPs, and other oligonucleotide-based molecules, alone or in combination with other molecules, that produce oligonucleotide molecules that may or may not be translated into peptides.

Dimeric forms of lysin proteins or peptides and peptide fragments as disclosed herein include proteins or peptides and peptide fragments that are chemically synthesized or prepared by recombinant DNA techniques, or both. These techniques include, for example, chimerization and shuffling. When the protein or peptide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in the synthesis of the protein. Thus, the protein preparation has less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.

The signal sequence of the polypeptide may assist in transmembrane transport of the disclosed proteins and peptides and peptide fragments to and from mucosa, and also in secretion and isolation of the protein or other protein secreted by the target. The signal sequence is generally characterized by a hydrophobic amino acid core that is normally cleaved from the mature protein during secretion in one or more cleavage events. The signal peptide contains processing sites that allow the signal sequence to be cleaved from the mature protein as they pass through the secretory pathway. Thus, the disclosure may relate to the described polypeptides having a signal sequence, as well as to the signal sequence itself and to polypeptides lacking the signal sequence (i.e., cleavage products). The disclosed nucleic acid sequences encoding signal sequences may be operably linked to a protein of interest, such as a protein that is not normally secreted or difficult to isolate, in an expression vector. The signal sequence directs secretion of the protein, such as from a eukaryotic host transformed into an expression vector, and the signal sequence is subsequently or simultaneously cleaved. The protein can then be readily purified from the extracellular medium by art-recognized methods. Alternatively, the signal sequence may be linked to the protein of interest using a sequence that aids purification, such as with a GST domain.

The invention also relates to other variants of the polypeptides of the invention. The variant may have an altered amino acid sequence which may function as an agonist (mimetic) or antagonist. Variants may result from mutagenesis, i.e., discrete point mutations or truncation. An agonist may retain substantially the same, or a subset, of the biological activity of the naturally occurring form of the protein. Antagonists of proteins may inhibit the activity of one or more naturally occurring forms of the protein by, for example, competitively binding to a downstream or upstream member of a cell signaling cascade that includes the protein of interest. Thus, a particular biological effect can be elicited by treatment with a variant having limited function. Treating a subject with a variant having a biological activity of a subset of the naturally-occurring form of the protein may have fewer side effects in the subject relative to treating with the naturally-occurring form of the protein. Disclosed protein variants that function as agonists (mimetics) or antagonists can be identified by screening a combinatorial library of mutants, i.e., truncated mutants, of the disclosed proteins for agonist or antagonist activity. In one embodiment, the variant heterolibrary is generated by combinatorial mutagenesis at the nucleic acid level and encoded by a heterogene library. Variant pools can be generated, for example, by enzymatically ligating a mixture of synthetic oligonucleotides into the gene sequences such that a degenerate set of potential protein sequences can be expressed as individual polypeptides, or alternatively, as a set of larger fusion proteins (i.e., for phage display). There are various methods that can be used to generate libraries of potential variants of the disclosed polypeptides from degenerate oligonucleotide sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39: 3; Itakura et al (1984) Annu. Rev. biochem.53: 323; Itakura et al (1984) Science198: 1056; Ike et al (1983) Nucleic Acid Res.11:477, all incorporated herein by reference).

In addition, a library of fragments of the coding sequence of the disclosed polypeptides can be used to generate polypeptides for screening and subsequent selection of variants, active fragments or truncated heteropopulations. For example, a library of coding sequence fragments can be generated by treating a double-stranded PCR fragment of the coding sequence of interest with a nuclease under conditions in which cleavage occurs only about once per molecule, denaturing the double-stranded DNA, renaturing the DNA from the products of the different cleavages to form double-stranded DNA that can include sense/antisense pairs, removing single-stranded portions from the modified duplex by treatment with S1 nuclease, and ligating the resulting library of fragments into an expression vector. By this method, expression libraries encoding N-terminal and internal fragments of proteins of interest of various sizes can be derived. Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncations, and for screening cDNA libraries for gene products having selected properties. The most widely used techniques for screening large gene banks, amenable to high throughput analysis, generally involve cloning the gene bank into replicable expression vectors, transforming appropriate cells with the resulting vector bank, and expressing the combined genes under conditions in which detection of the desired activity aids in isolation of the vector encoding the gene whose product is to be detected. Recursive Ensemble Mutagenesis (REM), a technique that enhances the frequency of functional mutants in the library, can be used in combination with screening assays to identify variants of the disclosed proteins (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89: 7811-valent 7815; Delgrave et al (1993) Protein Engineering6(3): 327-valent 331) the immunologically active portions of the proteins or peptide fragments include regions that bind to antibodies that recognize phage enzymes. In this context, the smallest part of the protein (or of the nucleic acid encoding the protein) is, according to an embodiment, an epitope that can be recognized as being specific for the phage that makes the lysin protein. Thus, the smallest polypeptide (and associated nucleic acid encoding the polypeptide) that can be expected to bind an antibody and be useful for some embodiments can be 8, 9, 10, 11, 12, 13, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 85, or 100 amino acids in length. While small sequences as short as 8, 9, 10, 11, 12 or 15 amino acids in length reliably contain enough structure to function as an epitope, shorter sequences of 5,6 or 7 amino acids in length may, under some conditions, exhibit epitope structure and may be of value in an embodiment.

Biologically active portions of the proteins or peptide fragments of the embodiments, as described herein, include polypeptides comprising an amino acid sequence sufficiently identical to or derived from the amino acid sequence of a disclosed bacteriophage protein that includes fewer amino acids than the full-length protein of the bacteriophage protein and exhibits at least one activity corresponding to the full-length protein. Generally, the biologically active portion comprises a domain or motif having at least one activity of the corresponding protein. Biologically active portions of the disclosed proteins or protein fragments can be, for example, polypeptides of less than or more than 10, 25, 50, 100 amino acids in length. Moreover, other biologically active portions of the deletion, or addition, of other protein regions may be prepared by recombinant techniques and evaluated for the functional activity of the native form of the polypeptide of one or more embodiments.

One skilled in the art will agree that homologous proteins and nucleic acids can be prepared that share functionality with the small protein and/or nucleic acid (or larger protein and/or nucleic acid regions). Such small molecules and short regions of larger molecules that may be homologous are specifically intended to be included in the embodiments. Preferably, the region of interest has at least 50%, 65%, 75%, 80%, 85% and preferably at least 90%, 95%, 97%, 98% or at least 99% homology to lysin polypeptides provided herein, including the polypeptides shown in figures 1 and 6. These percent homology values do not include changes due to conservative amino acid substitutions.

2 amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, at least about 85%, and preferably at least about 90 or 95%) are identical, or represent conservative substitutions. When one or more, or several, or up to 10%, or up to 15%, or up to 20% of the amino acids of a lysin polypeptide are substituted with similar or conservative amino acid substitutions, the sequence of a comparable lysin, such as a comparable Cpl-1 lysin, or a comparable Streptococcus (Streptococcus) lysin, is substantially homologous, and wherein the comparable lysin has the activity characteristics, anti-bacterial effects and/or bacterial specificity of a lysin disclosed herein, such as a Cpl-1 lysin.

The amino acid residues described herein are preferably in the form of the "L" isomer. However, any L-amino acid residue can be replaced with a residue in the form of a "D" isomer, so long as the polypeptide retains the desired functional properties of immunoglobulin-binding. NH (NH)₂Meaning that the free amino group is present at the amino terminus of the polypeptide. COOH means that the free carboxyl group is present at the carboxyl terminus of the polypeptide. Consistent with standard polypeptide nomenclature, J.biol.chem.,243:3552-59(1969), amino groupsAbbreviations for acid residues are shown in the following correspondence table:

[ correspondence table ]

[ amino acid symbols ]

1-letter	3-letter
			Y	Tyr	Tyrosine
G	Gly	Glycine
			F	Phe	Phenylalanine
M	Met	Methionine
			A	Ala	Alanine
S	Ser	Serine
			I	Ile	Isoleucine
L	Leu	Leucine
			T	Thr	Threonine
V	Val	Valine
			P	Pro	Proline
K	Lys	Lysine
			H	His	Histidine
Q	Gln	Glutamine
			E	Glu	Glutamic acid
W	Trp	Tryptophan
			R	Arg	Arginine
D	Asp	Aspartic acid
			N	Asn	Asparagine
C	Cys	Cysteine

It is to be understood that the entire amino-acid residue sequence is represented herein by a formula whose left and right orientations are in the conventional direction from amino-terminus to carboxy-terminus. Furthermore, it is to be understood that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues. The table above is provided to associate 3-letter and 1-letter designations that may appear interchangeably herein.

Mutations can be made in the amino acid sequences, or nucleic acid sequences encoding polypeptides and lysins herein, including the lysin sequences shown in fig. 1, fig. 6, or fig. 7, seq id NOs 1 or 5, or active fragments or truncations thereof, such that a particular codon is changed to a codon encoding a different amino acid, an amino acid is replaced with another amino acid, or one or more amino acids are deleted. The mutations are typically made by making the fewest amino acid or nucleotide changes possible. Such selected substitution mutations can be made to change amino acids in the resulting protein in a non-conservative manner (e.g., by changing a codon from an amino acid belonging to a group of amino acids having a particular size or characteristic to an amino acid belonging to another group) or in a conservative manner (e.g., by changing a codon from an amino acid belonging to a group of amino acids having a particular size or characteristic to an amino acid belonging to the same group). Such conservative changes typically result in fewer changes in the structure and function of the resulting protein. Non-conservative changes are more likely to alter the structure, activity or function of the resulting protein. The invention is to be considered to include sequences containing conservative changes that do not significantly alter the activity or binding characteristics of the resulting protein.

The following are examples of various groupings of amino acids:

amino acids with nonpolar R groups

Alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine

Amino acids with uncharged polar R groups

Glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine

Amino acids with charged polar R groups (negatively charged at pH6.0)

Aspartic acid, glutamic acid

Basic amino acids (positively charged at pH6.0)

Lysine, arginine, histidine (at pH6.0)

Another group may be those with phenyl groups:

phenylalanine, tryptophan, tyrosine

Another grouping may be based on molecular weight (i.e., R group size):

glycine	75	Alanine	89
				Serine	105	Proline	115
Valine	117	Threonine	119
				Cysteine	121	Leucine	131
Isoleucine	131	Asparagine	132
				Aspartic acid	133	Glutamine	146
Lysine	146	Glutamic acid	147
				Methionine	149	Histidine (at pH6.0)	155
Phenylalanine	165	Arginine	174
				Tyrosine	181	Tryptophan	204

Particularly preferred substitutions are:

● Lys for Arg and vice versa so that a positive charge can be maintained;

● Glu for Asp and vice versa, so that a negative charge can be maintained;

● Ser for Thr so that free-OH can be maintained; and

● Gln for Asn so that free NH can be maintained₂。

Exemplary and preferred conservative amino acid substitutions include any of:

glutamine (Q) for glutamic acid (E) and vice versa; leucine (L) for valine (V) and vice versa; serine (S) for threonine (T) and vice versa; isoleucine (I) for valine (V) and vice versa; lysine (K) in place of glutamine (Q) and vice versa; isoleucine (I) for methionine (M) and vice versa; serine (S) for asparagine (N) and vice versa; leucine (L) for methionine (M) and vice versa; lysine (L) for glutamic acid (E) and vice versa; alanine (a) for serine (S) and vice versa; tyrosine (Y) for phenylalanine (F) and vice versa; glutamic acid (E) for aspartic acid (D) and vice versa; leucine (L) for isoleucine (I) and vice versa; lysine (K) replaces arginine (R) and vice versa.

His can be introduced as a particularly "catalytic" site (i.e., His can function as an acid or base and is the most common amino acid in biochemical catalysis). Pro can be introduced because of its particularly planar structure, which induces a β -turn in the structure of the protein.

The polypeptides or epitopes described herein can be used to generate antibodies and also to detect binding to lysin or molecules that recognize lysin proteins. Another embodiment is a molecule, such as an antibody or other specific binding agent that can be generated, such as by conventional immunization or by phase display methods, through the use of an epitope, wherein the epitope can be used to screen the library for potential binding agents. The molecule recognizes one or more epitopes of a lysin protein or a nucleic acid encoding a lysin protein. The antibody recognizing the epitope may be a monoclonal antibody, a humanized antibody, or a partial antibody protein. Desirably, the epitope-recognizing molecule binds to an epitope with a specificity that is at least 10 times the specificity with which the molecule binds to serum albumin. Specific binding may be measured as affinity (Km). More desirably, specific binding is at least 10 times greater than binding to serum albumin under the same conditions²，10³，10⁴，10⁵，10⁶，10⁷，10⁸Or even higher.

In a desired embodiment, the antibody or antibody fragment is in a form useful for detecting the presence of a lysin protein, or alternatively detecting the presence of a bacterium that is sensitive to a lysin protein. In further embodiments, the antibodies may be attached or otherwise linked to a lysin polypeptide of the invention, e.g., in a chimeric or fusion protein, and may be used to direct lysin to a bacterial cell or strain or target of interest. Alternatively, the lysin polypeptide may be used to direct the antibody or act in combination with the antibody, for example in fully or partially lysing the bacterial cell wall, so that the antibody can specifically bind to a surface on the bacteria or to an epitope thereof under the surface or in the bacteria. For example, lysins of the invention may be attached to anti-streptococcal antibodies and the antibodies directed to epitopes thereof.

As will be appreciated by those skilled in the art, various formats and methods for antibody synthesis are known. The antibody may be conjugated to a (covalently complexed) reporter molecule or atom such as a fluorescent (fluor), an enzyme that generates an optical signal, a chemiluminescent group, a microparticle, or a radioactive atom. The antibody or antibody fragment can be synthesized in vivo following immunization of an animal, e.g., the antibody or antibody fragment can be synthesized by cell culture following genetic recombination. Antibodies or antibody fragments can be prepared by combining cellular synthesis and chemical modification.

An "antibody" is any immunoglobulin that binds a particular epitope, including antibodies and fragments thereof. The term includes polyclonal, monoclonal and chimeric antibodies, the last mentioned also being described in detail in U.S. Pat. Nos. 4,816,397 and 4,816,567. The term "antibody" describes an immunoglobulin, whether natural or produced in part or synthetically in whole. The term also encompasses any polypeptide or protein having a binding domain that is or is homologous to an antibody binding domain. CDR grafted antibodies are also encompassed by this term. An "antibody" is any immunoglobulin that binds a particular epitope, including antibodies and fragments thereof. The term includes polyclonal, monoclonal and chimeric antibodies, the last mentioned also being described in detail in U.S. Pat. Nos. 4,816,397 and 4,816,567. The term "antibody" includes wild-type immunoglobulin (Ig) molecules, typically comprising 4 full-length polypeptide chains, 2 heavy (H) chains and 2 light (L) chains, or equivalent Ig homologues thereof (e.g., camelid nanobodies, which comprise only heavy chains); full-length functional mutants, variants or derivatives thereof, including those that retain the essential epitope-binding characteristics of an Ig molecule, and including bispecific, multispecific and dual variable domain antibodies; immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2). Also included within the meaning of the term "antibody" are any "antibody fragments".

"antibody fragment" refers to a molecule comprising at least one polypeptide chain that is not full length, including (i) a Fab fragment, which is a monovalent fragment consisting of the Variable Light (VL), Variable Heavy (VH), Constant Light (CL) and constant heavy 1(CH1) domains; (ii) a F (ab')2 fragment which is a bivalent fragment comprising 2 Fab fragments linked by a disulfide bridge at the hinge region; (iii) the heavy chain portion of the fab (fd) fragment, consisting of the VH and CH1 domains; (iv) variable fragment (Fv) fragments consisting of VL and VH domains of a single arm of an antibody, (v) domain antibody (dAb) fragments comprising a single variable domain (Ward, e.s. et al, nature341, 544-546 (1989)); (vi) camelid antibodies; (vii) an isolated Complementarity Determining Region (CDR); (viii) single-chain Fv fragments in which the VH domain and the VL domain are connected by a peptide linker that allows the 2 domains to be joined to form an antigen-binding site (Bird et al, Science,242, 423-426, 1988; Huston et al, PNAS USA,85, 5879-; (ix) diabodies, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to pair between the 2 domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and generating 2 antigen binding sites (WO 94/13804; P.Holliger et al Proc. Natl.Acad. Sci. USA 649044-6448, (1993)); and (x) a linear antibody comprising a pair of tandem Fv fragments (VH-CH1-VH-CH1) that form a pair of antigen binding regions with a complementary light chain polypeptide; (xi) Multivalent antibody fragments (scFv dimers, trimers and/or tetramers (Power and Hudson, J Immunol. methods 242:193- & 2049 (2000)); and (xii) non-full length portions of other heavy and/or light chains, or mutants, variants or derivatives thereof, alone or in any combination.

Antibodies can be modified in many ways and the term "antibody" should be construed to cover any specific binding member or substance having a binding domain with the desired specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Thus included are chimeric molecules comprising an immunoglobulin binding domain fused to another polypeptide, or equivalents. The cloning and expression of chimeric antibodies is described in EP-A-0120694 and EP-A-0125023 and U.S. Pat. Nos. 4,816,397 and 4,816,567.

An "antibody combining site" is a structural portion of an antibody molecule that comprises a light chain or heavy and light chain variable and hypervariable regions that specifically bind an antigen.

The phrase "antibody molecule" in its various grammatical forms as used herein encompasses intact immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of immunoglobulin molecules that contain paratopes, including those portions known in the art, such as Fab, Fab ', F (ab')₂And f (v), which is preferably used in the therapeutic methods described herein.

The phrase "monoclonal antibody" in its various grammatical forms refers to an antibody having only one combined site of the antibody that is immunoreactive with a particular antigen. Monoclonal antibodies thus generally exhibit a single binding affinity for any antigen with which they are immunoreactive. Monoclonal antibodies may thus contain antibody molecules with multiple antibody combining sites, each specific for a different antigen; for example, bispecific (chimeric) monoclonal antibodies.

The term "specific" may be used to refer to a situation in which one member of a specific binding pair does not exhibit significant binding to a molecule to which it is not a specific binding partner. The term is also applicable, for example, where an antigen-binding domain is specific for a particular epitope carried by a number of antigens, in which case a specific binding member carrying the antigen-binding domain is capable of binding to a variety of epitope-carrying antigens.

The term "comprising" is generally used in an inclusive sense, that is to say allowing the presence of one or more features or components.

The term "consisting essentially of" refers to a product, particularly a peptide sequence that is not covalently attached to a defined number of residues of a larger product. In the case of the peptides of the invention herein, those skilled in the art will recognize that a few modifications to the N-or C-terminus of the peptide may be contemplated, such as chemical modification of the terminus to add a protecting group, etc., e.g., amidation of the C-terminus.

The term "isolated" refers to a lysin polypeptide of the invention, or a nucleic acid encoding such a polypeptide, in which state it would be according to the invention. The polypeptides and nucleic acids will be free or substantially free of materials with which they are naturally associated, such as other polypeptides or nucleic acids with which they are found in their natural environment or in the environment in which they are prepared (when such preparation is practiced in vitro or in vivo by recombinant DNA techniques, e.g., cell culture). The polypeptides and nucleic acids may be formulated with diluents or adjuvants and, for practical purposes, are isolated-for example the polypeptides will normally be mixed with polymers or mucoadhesives or other carriers, or with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy.

Dimeric lysin monomers of the present invention may be chemically cross-linked together by covalent bonds, including, inter alia, covalent bonds or other bonding chemistries, such as between amino acids located between about 14 and 20 amino acids from the C-terminus of a Cpl-1 lysin polypeptide (FIGS. 1 and SEQ ID NO:1) or Pal lysin (FIGS. 7 and SEQ ID NO: 5). The monomers can be crosslinked by using a chemical crosslinking agent. For example, the 1 st monomer may be crosslinked to the 2 nd monomer between 2 cysteine residues. Examples of cysteine-reactive crosslinking agents include agents such as 1, 6-bismaleimide hexane (BMH), 1, 3-dibromo-2-propanol (DBP), and mustard gas (bis (2-chloroethyl) sulfide; mustard). Alternative or other crosslinking reagents are known and may include amine to amine, maleimide linked N-hydroxysuccinic acid amide (NHS) and pyridyl dithiols linking sulfhydryl to sulfhydryl groups. Each may incorporate spacing to increase space and flexibility. Lysin monomers may be covalently bound or cross-linked via a linker peptide fused to their C-terminus or region. Any means in the art for dimerization can be utilized, as long as the C-terminal choline binding function and N-terminal enzymatic function of lysin are maintained.

The lysin monomers may be linked together by disulfide bonds between 2 cysteine residues. In a particular embodiment, the monomer is disulfide-bonded between 2 Cys residues located between 14 and 20 amino acids from the C-terminus of Cpl-1 or PalThe bonds are cross-linked together. The cross-linked cysteine residues may be at the same position on the lysin monomer polypeptide chain. In particular instances, a cysteine residue may not be present in the first 45 residues of lysin. An example of a lysin monomer cross-linked by a Cys residue at position 324 is Cpl-1^C45S,D324C(SEQ ID NO: 3). An example of a lysin monomer cross-linked by a Cys residue at position 280 is Pal^D280C(SEQ IDNO:6)。

In general, lysin has 2 different functional domains consisting of a catalytic domain for peptidoglycan hydrolysis and a binding domain for recognition of surface parts on bacterial cell walls. The catalytic domain is relatively conserved among lysins. Thus, the dimeric lysin may be naturally chimeric and include a dimer of lysin monomers comprising the catalytic domain of a Streptococcus pneumoniae (Streptococcus pneumoniae) -specific phage lysin and the binding domain of a Streptococcus sp. In particular embodiments the catalytic domain of the Streptococcus (Streptococcus) -specific phage lysin of 1 st is derived from Cpl-1 or other Streptococcus pneumoniae (Streptococcus pneumoniae) phage lysin catalytic domains. In other embodiments, the binding domain of the Streptococcus pneumoniae (Streptococcus pneumoniae) -specific phage lysin 2 is from Cpl-1 or other Streptococcus pneumoniae (Streptococcus pneumoniae) phage lysin binding domains. Examples of catalytic domains include amino acids 1-190 of Cpl-1. In other specific embodiments, the catalytic domain of the Streptococcus genus 1 (Streptococcus) -specific phage lysin is from Pal or other Streptococcus pneumoniae (Streptococcus pneumoniae) phage lysin catalytic domains. In other embodiments, the binding domain of the Streptococcus pneumoniae (Streptococcus pneumoniae) -specific phage lysin 2 is from Pal or other Streptococcus pneumoniae (Streptococcus pneumoniae) phage lysin binding domains. Other examples of catalytic domains are, inter alia, the N-terminal half of ClyS lysin and PlyG lysin. Examples of binding domains include amino acids 191-326 of Cpl-1. Examples of binding domains include amino acids 155-296 of Pal. Other examples of binding domains are, inter alia, the C-terminal half of ClyS and PlyG lysin. The exact amino acids comprising these domains can be readily determined by one skilled in the art by sequence analysis and sequence alignment.

The dimeric lysin exhibits killing activity against one or more streptococcal bacteria such as streptococcus pneumoniae (streptococcus pneumoniae). Killing activity can be determined by using a killing assay, such as the killing assay described in the examples section below.

[ nucleic acids ]

Nucleic acids encoding a dimeric lysin polypeptide of the invention form an aspect of the invention. Representative nucleic acid sequences in this context are polynucleotide sequences encoding dimeric polypeptide monomers of any of figures 1,6 and 7 and table 1, SEQ ID NO 3 or 6, and sequences that hybridize under stringent conditions to the complement of the nucleic acid sequences. Further variants of these sequences and nucleic acid sequences hybridizing to the sequences shown in these figures are also contemplated for use in generating the lytic enzymes according to the disclosure, including naturally occurring variants that are available. A wide variety of isolated nucleic acid sequences or cDNA sequences encoding phage-associated lytic enzymes and partial sequences that hybridize to the gene sequences are useful for recombinant production of the lysin enzymes or polypeptides of the invention.

A "replicon" is a function of an autonomous unit that functions in vivo in DNA replication; i.e., any genetic element (e.g., plasmid, chromosome, virus) capable of replicating under its own control.

A "vector" is a replicon, such as a plasmid, phage or cosmid, to which another DNA segment may be attached to replicate the attached segment.

"DNA molecule" refers to a polymeric form of deoxyribonucleotides (adenine, guanine, thymine or cytosine) in its single-stranded form, or in a double-stranded helix. This term refers only to the primary and secondary structure of the molecule and does not limit it to any particular tertiary form. Thus, the term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of a particular double-stranded DNA molecule, the sequence may be described herein according to the normal convention of giving only the sequence in the 5 'to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

"origin of replication" refers to those DNA sequences involved in DNA synthesis.

A DNA "coding sequence" is a double-stranded DNA sequence that is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Polyadenylation signal and transcription termination sequences will usually be located 3' to the coding sequence.

Transcriptional and translational control sequences are DNA regulatory sequences that ensure expression of a coding sequence in a host cell, such as promoters, enhancers, polyadenylation signals, terminators, and the like.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, a promoter sequence binds to the transcription initiation site at its 3 'end and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at detectable levels above background. Within the promoter sequence, a transcription initiation site (conveniently defined by the position marked with nuclease S1) and a protein binding domain (consensus sequence) responsible for RNA polymerase binding will be found. Eukaryotic promoters will often, but not always, contain "TATA" and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the-10 and-35 consensus sequences.

An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under the control" of transcriptional and translational control sequences in a cell, and when RNA polymerase transcribes the coding sequence into mRNA, the mRNA is then translated into the protein encoded by the coding sequence.

A "signal sequence" may be included before a coding sequence. This sequence encodes a signal peptide at the N-terminus of the polypeptide that is in communication with the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the culture medium, and this signal peptide is cleaved off by the host cell before the protein leaves the cell. The signal sequence may be found in association with a variety of proteins that are native in prokaryotes and eukaryotes.

The term "oligonucleotide" as used herein with respect to the probes of the invention is defined as a molecule comprising 2 or more ribonucleotides, preferably more than 3 ribonucleotides. The exact size will depend on many factors, which in turn depend on the ultimate function and use of the oligonucleotide.

The term "primer" as used herein refers to an oligonucleotide, whether naturally occurring or synthetically produced in a restriction digest, whether purified, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced in the presence of nucleotides and an inducing agent, such as a DNA polymerase, and at a suitable temperature and pH. The primer may be single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend on many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, oligonucleotide primers typically contain 15-25 or more nucleotides, although they may contain fewer nucleotides, depending on the complexity of the target sequence.

Primers are herein selected to be "substantially" complementary to different strands of a particular targeted DNA sequence. This means that the primers must be sufficiently complementary to hybridize to their respective strands. Thus, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment can be attached to the 5' end of a primer, while the remainder of the primer sequence is complementary to the strand. Alternatively, non-complementary bases or longer sequences may be interspersed within the primer, provided that the primer sequence has sufficient complementarity with the strand sequence to which it hybridizes, and thereby form a template for synthesis of the extension product.

As used herein, the terms "restriction enzyme" and "restriction enzyme" refer to bacterial enzymes, each cleaving double-stranded DNA at or near a particular nucleotide sequence.

When the DNA has been introduced into the cell, the cell has been "transformed" with exogenous or heterologous DNA. The transforming DNA may or may not be integrated (covalently linked) into the chromosomal DNA that makes up the genome of the cell. In prokaryotes, yeast and mammalian cells, for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has been integrated into a chromosome so that it is inherited by an offspring cell through chromosome replication. This stability is demonstrated by the ability of eukaryotic cells to establish cell lines or clones comprising a population of progeny cells containing the transformed DNA. "clones" are a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of primary cells that is capable of stable growth in vitro for many generations.

2 DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over a defined length of the DNA sequence. Substantially homologous sequences can be identified by comparing the sequences using standard software available in sequence data banks, or, for example, in a Southern hybridization experiment under stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of one in the art. See, e.g., Maniatis et al, supra; DNA Cloning, Vols.I & II, supra; nucleic Acid Hybridization, supra.

Many of the variant DNA molecules contemplated herein include those produced by standard DNA mutagenesis techniques, such as M13 primer mutagenesis. Details of these techniques are provided in Sambrook et al (1989) molecular cloning: a laboratory manual, cold spring harbor, n.y (incorporated herein by reference). By using this technique, variations slightly different from those disclosed can be produced. The present disclosure encompasses DNA molecules and nucleotide sequences that are derivatives of the DNA molecules and nucleotide sequences specifically disclosed herein and that differ from the disclosed DNA molecules and nucleotide sequences by deletion, addition or substitution of nucleotides, but still encode proteins having the functional characteristics of lysin polypeptides. Also included are small DNA molecules derived from the disclosed DNA molecules. The small DNA molecules include oligonucleotides suitable for use as hybridization probes or Polymerase Chain Reaction (PCR) primers. As such, these small DNA molecules will comprise at least one segment of a lytic enzyme genetically encoded by a bacteriophage of the genus Streptococcus (Streptococcus) and, for PCR purposes, will comprise at least 10-15 nucleotide sequences and, more preferably, 15-30 nucleotide sequences of a gene. DNA molecules and nucleotide sequences derived from the above disclosed DNA molecules can also be defined as DNA sequences that hybridize to the disclosed DNA sequences under stringent conditions, or fragments thereof.

Hybridization conditions corresponding to a particular degree of stringency will vary depending on the nature of the hybridization method selected and the composition and length of the hybridizing DNA used. In general, the hybridization temperature and the ionic strength of the hybridization buffer (especially the sodium ion concentration) will determine the stringency of hybridization. The calculation of hybridization conditions required to achieve a particular degree of stringency was performed by Sambrook et al. (1989) And (3) molecular cloning: a laboratory manual, cold spring harbor, n.y, chapters 9 and 11 (incorporated herein by reference).

An example of this calculation is as follows. Hybridization experiments can be performed by hybridization of a DNA molecule (e.g., a natural variation of a lytic enzyme genetically encoded by a bacteriophage specific for Bacillus anthracis) to a targeted DNA molecule. The targeted DNA may be, for example, the corresponding cDNA which has been electrophoresed on an agarose gel and transferred to nitrocellulose membrane by Southern blotting (Southern (1975). J.mol.biol.98:503), a technique well known in the art and described in Sambrook et al (1989) (molecular cloning: A laboratory Manual, Cold spring harbor, N.Y. incorporated herein by reference). With the isotope P³²Hybridization of labeled-dCTP labeled targeting probes is performed in a high ionic strength solution such as 6 XSSC (described below) at a temperature of 20 to 25 ℃ below the melting temperature, Tm. For the Southern hybridization experiment, the target DNA molecule on the Southern blot contains 10ng of DNA or more, and 1-2 ng/ml of radiolabeled probe is used for hybridizationFor 6 to 8 hours (specific activity: 10)⁹CPM/. mu.g or greater). After hybridization, the nitrocellulose filter was washed to remove background hybridization. Washing conditions are as stringent as possible to remove background hybridization while retaining specific hybridization signals. The term "Tm" refers to a temperature above which a radiolabeled probe molecule does not hybridize to its target DNA molecule under ion-rich conditions. The Tm of the hybrid molecule can be estimated according to the following equation: t is_m81.5 ℃ -16.6 (log 10 of sodium ion concentration) +0.41 (% G + C) -0.63 (% formamide) - (600/l) where l is the length of the hybrid in the base pair. This equation is valid for sodium ion concentrations in the range of 0.01M to 0.4M, and its calculation of Tm is less accurate for solutions with higher sodium ion concentrations (Bolton and McCarthy (1962) Proc. Natl. Acad. Sci. USA 48:1390) (incorporated herein by reference). This equation is also valid for DNA with G + C content within 30% to 75% and also applies to hybrids greater than 100 nucleotides in length. The behavior of oligonucleotide probes is described in detail in Sambrook et al (1989), molecular cloning: a laboratory manual, cold spring harbor, chapter 11 of n.y (incorporated herein by reference). Preferred exemplary conditions described herein are specifically contemplated for use in selecting variants of a lytic gene.

In a preferred embodiment of the present disclosure, stringent conditions may be defined as conditions under which DNA molecules with more than 25% sequence variation (also referred to as "mismatches") do not hybridize. In a more preferred embodiment, stringent conditions are conditions under which DNA molecules with more than 15% mismatches do not hybridize, and more preferably still, stringent conditions are conditions under which DNA sequences with more than 10% mismatches do not hybridize. Preferably, stringent conditions are conditions under which DNA sequences with more than 6% mismatches do not hybridize.

The degeneracy of the genetic code also widens the scope of the embodiments, as it results in variations in the nucleotide sequence of the main DNA molecule, while maintaining the amino acid sequence of the encoded protein. For example, a representative amino acid residue is alanine. This can be encoded in cDNA by the nucleotide codon triplet GCT. Because of the degeneracy of the genetic code, 3 other nucleotide codon triplets- -GCT, GCC and GCA- -also encode alanine. Thus, the nucleotide sequence of the gene can be changed to any such 3 codons at this position without affecting the amino acid composition or protein characteristics of the encoded protein. The genetic code for a particular amino acid and variations in nucleotide codons are well known to those skilled in the art. Based on the degeneracy of the genetic code, variant DNA molecules can be derived from the cDNA molecules disclosed herein using standard DNA mutagenesis techniques described above, or by synthesis of DNA sequences. DNA sequences that do not hybridize under stringent conditions to the disclosed cDNA sequences, depending on sequence variation, based on the degeneracy of the genetic code are included in the disclosure.

Thus, it will be appreciated that also within the scope of the present invention is a DNA sequence encoding a lysin of the present invention, including dimeric Cpl-1 and/or Pal lysins, which encodes a polypeptide having the same amino acid sequence as provided herein and in the figures and Table 1, but which is degenerate thereof. By "concatemer" is meant that different 3-letter codons are used to identify a particular amino acid.

Although the site of introducing an amino acid sequence variation is predetermined, the mutation itself need not be predetermined. For example, to optimize the performance of a mutation at a given site, random mutagenesis can be performed at the target codon or region and the expressed protein variants screened for the optimal combination of desired activities. Techniques for making substitution mutations at predetermined positions in DNA having the above-mentioned known sequences are well known.

Amino acid substitutions are typically single residue substitutions; insertions will typically be on the order of about 1-10 amino acid residues; and deletions will be in the range of about 1-30 residues. Deletions or insertions may be in single form, but are preferably made in adjacent pairs, i.e., a deletion of 2 residues or an insertion of 2 residues. Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at the final construct. It is clear that mutations made in the DNA encoding the protein do not allow the sequence to be out of reading frame and preferably do not produce complementary regions that can give rise to secondary mRNA structure (EP 75,444A).

Substitutional variants are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted at that position. The substitutions can be made so as not to have a significant effect on the protein characteristics, or when fine-tuning of the protein characteristics is desired. Amino acids that can be used to replace the original amino acids in a protein, and which are considered conservative substitutions, are described above and will be recognized by those skilled in the art.

Substantial changes in functional or immunological identity can be made by selecting substitutions that are less conservative, for example by selecting residues that differ more significantly in their effect of maintaining the following characteristics: (a) the structure of the polypeptide backbone in the substitution region, for example, in a sheet or helical conformation; (b) charge or hydrophobicity of the molecule at the target site; or (c) a pendant entity. Substitutions that are generally expected to produce the greatest change in protein properties will be those that are: (a) hydrophilic residues, e.g., seryl or threonyl, substituted with (or by) hydrophobic residues, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) cysteine or proline is replaced by (or by) any other residue; (c) residues having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, are replaced with (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue with a bulky side chain, e.g., phenylalanine, is replaced with (or by) an amino acid without a side chain, e.g., glycine.

The effect of these amino acid substitutions or deletions or additions can be assessed with respect to derivatives or variants of the lytic polypeptide, by analyzing the ability of the derivative or variant protein to lyse or kill sensitive bacteria, or to complement the sensitivity exhibited by the phage to DNA cross-linking agents in an infected bacterial host. These assays can be performed by transfecting DNA molecules encoding derivative or variant proteins into the bacteria described above or by incubating the bacteria with proteins expressed from hosts transfected with DNA molecules encoding derivative or variant proteins.

Although the site of introduction of the amino acid sequence variation can be predetermined, the mutation itself need not be predetermined. For example, to optimize performance of a mutation at a given site, random mutagenesis can be performed at the target codon or region and the expressed protein variants screened for the optimal combination of desired activities. Techniques for making substitution mutations at predetermined positions in DNA having the above-mentioned known sequences are well known.

Another feature of the invention is the expression of a DNA sequence encoding a dimeric lysin. The DNA sequences may be expressed by operably linking them to expression control sequences in a suitable expression vector and transforming a suitable unicellular host with the expression vector, as is well known in the art. The DNA sequence of the invention is operably linked to an expression control sequence, which, of course, includes, if not already part of the DNA sequence, providing an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence. A wide variety of host/expression vector combinations may be employed in the expression of the DNA sequences of the present invention. Useful expression vectors may, for example, consist of chromosomal segments, non-chromosomal and synthetic DNA sequences. Suitable vectors include SV40 and derivatives of known bacterial plasmids, for example, the escherichia coli (e.coli) plasmids colEl, pCR1, pBR322, pMB9 and derivatives thereof, plasmids such as RP 4; phage DNAS, e.g., various derivatives of phage λ, e.g., NM989 and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 □ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from a combination of plasmids and phage DNA, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

Any of a wide variety of expression control sequences- -sequences that control the expression of DNA sequences to which they are operably linked- -can be used in these vectors to express the DNA sequences of the invention. Such useful expression control sequences include, for example, 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 promoter regions of the major operators and phage lambda, the control regions of fd coat proteins, the promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatases (e.g., Pho5), the promoters of yeast-mating factors, and other sequences known to control expression of prokaryotic or eukaryotic cells or their viral genes, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressing the DNA sequences of the invention. These hosts may include well-known eukaryotic and prokaryotic hosts such as Escherichia coli (E.coli), Pseudomonas (Pseudomonas), Bacillus (Bacillus), Streptomyces (Streptomyces), fungi such as yeast strains, and animal cells in tissue culture such as CHO, Rl.l, B-W and L-M cells, African green monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40 and BMT10), insect cells (e.g., Sf9) and human and plant cells.

It is understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of the present invention. Not all hosts function equally well with the same expression system. However, one skilled in the art can select the appropriate vector, expression control sequence, and host without undue experimentation to achieve the desired expression without departing from the scope of the invention.

Libraries of fragments of the coding sequence of a polypeptide can be used to generate polypeptide hybrids for screening and subsequent selection of variants. For example, a library of coding sequence fragments can be generated by treating a double-stranded PCR fragment of the coding sequence of interest with a nuclease under conditions in which cleavage occurs only about once per molecule, denaturing the double-stranded DNA, renaturing the DNA from the products of the different cleavages to form double-stranded DNA that can include sense/antisense pairs, removing single-stranded portions from the modified duplex by treatment with S1 nuclease, and ligating the resulting library of fragments into an expression vector. By this method, expression libraries encoding N-terminal and internal fragments of proteins of interest of various sizes can be derived.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncations, and for screening cDNA libraries for gene products having selected properties. The most widely used techniques for screening large gene banks, amenable to high throughput analysis, generally involve cloning the gene bank into replicable expression vectors, transforming appropriate cells with the resulting vector bank, and expressing the combined genes under conditions in which detection of the desired activity aids in isolation of the vector encoding the gene whose product is to be detected. Recursive Ensemble Mutagenesis (REM), a technique that enhances the frequency of functional mutants in the library, can be used in combination with screening assays to identify variants of the disclosed proteins (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89: 7811. quadrature. 7815; Delgrave et al (1993) Protein Engineering6 (3: 327. quadrature. 331).

[ COMPOSITION ]

According to the present invention there are provided therapeutic or pharmaceutical compositions comprising the dimeric lytic enzymes/polypeptides of the invention, and related methods of use and methods of preparation. The therapeutic or pharmaceutical composition may comprise one or more dimeric lytic polypeptides, and optionally comprise a truncated, chimeric or shuffled lytic enzyme, optionally in combination with other components such as a carrier, vehicle, polypeptide, polynucleotide, perforin protein, one or more antibiotics or suitable excipients, carriers or vehicles. The present invention provides therapeutic or pharmaceutical compositions of lysins of the present invention, including dimeric Cpl-1, and embodiments Cpl-1^C45S,D324CFor killing, alleviating, decolonizing, preventing or treating gram-positive bacteria, including bacterial infections or associated conditions. The invention provides therapeutic or pharmaceutical compositions of lysins of the invention, including dimeric Pal, and embodiment Pal^D280CFor killing, alleviating, decolonizing, preventing or treating gram-positive bacteria, including bacterial infections or associated conditions. Provided herein are compositions comprising dimeric lysins, particularly dimeric Cpl-1, dimeric Pal, or combinations thereof, including truncations or variants thereof, for use in killing, palliating, decoloning, preventing or treating gram-positive bacteria, including bacterial infections or associated conditions, particularly infections of the Streptococcus genus (Streptococcus) or associated conditions.

The enzyme or polypeptide included in the therapeutic composition may be one or more or any combination of phage-associated dimeric lytic enzymes, truncated dimeric lytic polypeptides, variant dimeric lytic polypeptides, and chimeric and/or shuffled dimeric lytic enzymes. In addition, different lytic polypeptides genetically encoded by different phages used to treat the same bacterium can be used. These lytic enzymes may also be any combination of "unaltered" dimeric lytic enzymes or polypeptides, truncated dimeric lytic polypeptides, variant dimeric lytic polypeptides, and chimeric and shuffled dimeric lytic enzymes. The dimeric lytic enzymes/polypeptides used in the therapeutic or pharmaceutical compositions of gram-positive bacteria, including Streptococcus (Streptococcus), can be used alone or in combination with antibiotics or bactericidal or bacteriostatic agents or, if there are other invasive bacterial organisms to be treated, with lytic enzymes associated with other bacteriophages specific for the other bacteria targeted. Lytic enzymes, truncated enzymes, variant enzymes, chimeric enzymes and/or shuffled lytic enzymes may be used in conjunction with the perforin protein. The amount of perforin protein may also vary. Various antibiotics may optionally be included in the therapeutic compositions with the enzyme or polypeptide and in the presence or absence of lysostaphin. More than one lytic enzyme or polypeptide may be included in the therapeutic composition.

The pharmaceutical composition may also include one or more dimerization lyase enzymes produced by chemical synthesis or recombinant DNA techniques, including isozymes, analogs, or variants thereof. In particular, altered lytic proteins may be produced by amino acid substitutions, deletions, truncations, chimerism, shuffling or a combination thereof. The pharmaceutical composition may contain a combination of one or more dimeric lytic proteins and one or more truncated, variant, chimeric or shuffled lytic proteins. The pharmaceutical composition may also contain at least one peptide or peptide fragment of a dimeric lytic protein derived from the same or a different bacterial species, optionally together with one or more supplements, and a pharmaceutically acceptable carrier or diluent.

The present invention provides bacterial dimeric lysins, in particular recombinantly-produced dimeric lysins from natural monomeric lysins, including polypeptides comprising dimeric Cpl-1 lysin variants having bacteriocidal activity. Dimeric Cpl-1 lysin mutants containing a mutant cysteine for dimerization and retaining gram-positive antibacterial activity are described. Provided herein are compositions comprising dimeric Streptococcus (Streptococcus) lysin, including dimeric Cpl-1 mutant lysins, that are free of monomers compared to the monomeric formMutated Cpl-1 lysin proteins having equal or greater killing activity against Streptococcus (Streptococcus) cells include dimeric CPl-1 lysin Cpl-1^C45S,D342C。

The present invention provides bacterial dimeric lysins, particularly recombinantly-produced dimeric lysins from natural monomeric lysins, including polypeptides comprising dimeric Pal lysin variants having bacteriocidal activity. The present invention describes dimeric Pal lysin mutants containing a mutant cysteine for dimerization and retaining gram-positive antibacterial activity. Provided herein are compositions comprising dimeric Streptococcus (Streptococcus) lysin, including dimeric Pal mutant lysin, having equal or greater killing activity against Streptococcus (Streptococcus) cells than monomeric unmutated Pal lysin protein, including dimeric Pal lysin Pal^D280C。

The therapeutic composition may also comprise a perforin protein. Perforin proteins (or "perforins") are proteins that create pores in the cell membrane. Perforin proteins can form lethal membrane lesions that end up in bacteria in cell respiration. Such as lytic proteins, perforin proteins are encoded and carried by bacteriophages. Indeed, it is quite common for the genetic code of the perforin protein to be adjacent to or even within the coding of the bacteriophage lytic protein. Most perforin proteins are short in sequence and are generally hydrophobic with a highly hydrophilic carboxy-terminal domain. In many cases, the putative perforin protein is encoded in a different reading frame within the enzymatically active domain of the phage. In other cases, the perforin protein is encoded on DNA adjacent to or in close proximity to DNA encoding a cell wall lytic protein. Perforin proteins are often synthesized during the late stages of phage infection and are found in the plasma membrane of cells where they cause membrane damage. Perforins can be classified into 2 major classes based on primary structure analysis. Class I perforins are typically 95 residues or longer and may have 3 potential transmembrane domains. Class II perforins are generally smaller, roughly 65-95 residues, with charged and hydrophobic residues distributed indicating 2 TM domains (Young, et al. trends in Microbiology v.8, No.4, March 2000). However, at least for gram-positive host phages, two-component lysis systems may not be common. Although several phages have shown or suggested the presence of perforin, no putative perforin has been found whose gene encodes all phages. Perforin has been shown to be present in several bacteria, including, for example, lactococcus phage Tuc2009, lactococcus NLC3, pneumococcal phage EJ-1, Lactobacillus gasseri phage Nadh, Staphylococcus aureus (Staphylococcus aureus) phage Twort, listeria monocytogenes (listerinogenes) phage, pneumococcal phage Cp-1, Bacillus subtilis (Bacillus subtilis) phage M29, Lactobacillus delbruecki (Lactobacillus delbruecki) phage LL-H lysin, and Staphylococcus aureus (Staphylococcus aureus) phage N11. (Loessner, et al, Journal of Bacteriology, August 1999, p.4452-4460).

For example, perforin proteins can be used in conjunction with lytic enzymes to accelerate the speed and efficiency of bacterial killing. Perforin proteins may also be in the form of chimeric and/or shuffled enzymes. According to some embodiments, the perforin protein may also be used alone in the treatment of bacterial infections.

The pharmaceutical composition may contain a supplement comprising one or more antimicrobial agents and/or one or more conventional antibiotics. To accelerate the treatment of infections, the therapeutic agent may further include at least one supplement that may also enhance the bactericidal activity of the lytic enzyme. Antimicrobial agents function largely by interfering with the structure or function of bacterial cells by inhibiting cell wall synthesis, inhibiting cell-membrane function and/or inhibiting metabolic functions, including protein and DNA synthesis. Antibiotics can be broadly sub-grouped into those that affect cell wall peptidoglycan biosynthesis and those that affect DNA or protein synthesis in gram-positive bacteria. Cell wall synthesis inhibitors, including penicillins and antibiotics, etc., disrupt the rigid outer cell wall, thereby swelling and eventually rupturing the relatively unsupported cells. Antibiotics that affect cell wall peptidoglycan biosynthesis include: a glycopeptide which inhibits peptidoglycan synthesis by preventing incorporation of peptide subunits of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) into the peptidoglycan matrix. Glycopeptides that may be used include vancomycinPenicillin, which functions by inhibiting the formation of peptidoglycan cross-links, the functional group of penicillin, the β -lactam moiety, binds and inhibits DD-transpeptidase linked to peptidoglycan molecules in bacteria hydrolytic enzymes continue to break the cell wall, leading to cell lysis or death due to osmotic pressure₅₅Dephosphorylation of prenyl pyrophosphate (a molecule carrying the peptidoglycan building block outside the plasma membrane). The polypeptide affecting the cell wall is bacitracin.

The supplement can be an antibiotic such as erythromycin, clarithromycin, azithromycin, roxithromycin, vancomycin, oxacillin, doxycycline, other member of the macrolide family, penicillin, cephalosporin, and any combination thereof in an amount effective to synergistically enhance the therapeutic effect of the lytic enzyme. The antibiotic or bactericidal or bacteriostatic agent may be present in a clinically effective concentration or amount for bacterial growth or viability or may be sub-MIC or below or in a minimally inhibitory concentration. Indeed, any other antibiotic may be used with the lytic enzyme, altered and/or unaltered. Similarly, other lytic enzymes may be included in the vector to treat other bacterial infections. When treating different diseases, antibiotic supplements can be used in virtually all uses of the enzyme. The pharmaceutical composition may also contain at least one lytic protein, one perforin protein, or at least one perforin and one peptide or peptide fragment of a lytic protein, each of the lytic and perforin proteins being derived from the same or different bacterial species, optionally with the addition of a supplement, and a suitable carrier or diluent.

Also provided are compositions comprising nucleic acid molecules, alone or in combination with other nucleic acid molecules, capable of expressing an effective amount of a dimeric cleavable polypeptide or a peptide fragment of a dimeric cleavable polypeptide in vivo. Cell cultures containing these nucleic acid molecules, polynucleotides and vectors carrying and expressing these molecules in vitro or in vivo are also provided.

The therapeutic or pharmaceutical composition may comprise dimeric lytic polypeptides, including polypeptides directed against the same, differentOr one or more dimeric lysin polypeptides of a susceptible bacterium, in combination with various vectors, for treating a disease caused by a susceptible gram-positive bacterium. The carrier suitably contains minor amounts of additives such as substances which enhance isotonicity and chemical stability. The substance is non-toxic to the recipient at the dosages and concentrations employed, and includes buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or salts thereof; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; glycine; amino acids such as glutamic acid, aspartic acid, histidine or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, trehalose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counter ions such as sodium; non-ionic surfactants such as polysorbates, poloxamers or polyethylene glycols (PEG); and/or neutral salts, e.g. NaCl, KCl, MgCl₂，CaCl₂And others. Glycerol or glycerin (1,2, 3-propanetriol) for pharmaceutical use is commercially available. It can be diluted in sterile water for injection, or sodium chloride injection, or other pharmaceutically acceptable aqueous injection fluid, and used at a concentration of 0.1-100% (v/v), preferably 1.0-50%, more preferably about 20%. DMSO is an aprotic solvent with a significant ability to enhance penetration of many topically applied drugs. DMSO may be diluted in sterile water for injection, or sodium chloride injection, or other pharmaceutically acceptable aqueous injection fluid, and used at a concentration of 0.1-100% (v/v). Carrier media may also include Ringer's solution, buffer solutions and dextrose solutions, particularly when preparing intravenous solutions.

Any vector for dimerizing a lytic polypeptide can be produced by conventional means. However, it is preferred that any mouthwash or similar type product does not contain alcohol to prevent denaturation of the polypeptide/enzyme. Similarly, when the lytic polypeptide is placed into a cough drop, gum, candy or lozenge during the manufacturing process, the placement should be performed before the lozenge or candy hardens, but after the cough drop or candy has cooled somewhat, to avoid heat denaturation of the enzyme.

The dimeric lytic polypeptide may be added to these substances in liquid form or in a freeze-dried state, after which it will dissolve when it is encountered by body fluids such as saliva. The polypeptide/enzyme may also be in micelles or liposomes.

The effective dosage rate or amount of the dimeric lytic enzyme/polypeptide for treating an infection will depend, in part, on whether the dimeric lytic enzyme/polypeptide is to be used therapeutically or prophylactically, the duration of exposure of the recipient to the infectious bacteria, the size and weight of the individual, and the like. The duration of use of the enzyme/polypeptide containing composition will also depend on whether the use is for prophylactic purposes, where use may be for a short period of time in hours, days or weeks, or whether use would be for therapeutic purposes that may require a more intensive regimen of use of the composition, such that use may last for hours, days or weeks, and/or daily, or at timed intervals during the day. Any dosage form employed should ensure that a minimum number of units are present for a minimum amount of time. The concentration of active units of enzyme believed to ensure an effective amount or dose of enzyme may be in the range of about 100U/ml to about 500,000U/ml of fluid in the wet or moist environment of the nasal and oral tract, and may be in the range of about 100U/ml to about 50,000U/ml. More particularly, the time of exposure to the active enzyme/polypeptide unit may affect the desired concentration of active enzyme units/ml. A carrier classified as a "long" or "slow" release carrier (such as, for example, a particular nasal spray or lozenge) may have or provide a lower concentration of active (enzyme) units per ml, but for a longer period of time, whereas a "short" or "fast" release carrier (such as, for example, a mouthrinse) may have or provide a higher concentration of active (enzyme) units per ml, but for a shorter period of time. The amount of active units/ml and the duration of exposure depend on the nature of the infection, whether the treatment is prophylactic or therapeutic, and other variables. There are situations where it may be necessary to have a much higher unit/ml dose or a lower unit/ml dose.

The dimeric lyase/polypeptide should be in an environment having a pH that allows the lyase/polypeptide to be active. For example, if a human individual has been exposed to another human suffering from a bacterial upper respiratory disorder, the dimeric lytic enzyme/polypeptide will be located on the mucosal lining and prevent colonization of any infectious bacteria. Before or when the dimeric lytic enzyme is placed in the carrier system or oral delivery mode, it is preferred that the enzyme is in a stabilizing buffer environment that maintains a pH range of between about 4.0 and about 9.0, more preferably between about 5.5 and about 7.5.

The stabilizing buffer may allow for optimal activity of the dimeric lysin enzyme/polypeptide. The buffer may contain a reducing agent, such as dithiothreitol. The stabilizing buffer may also be or include a metal chelating agent, such as ethylenediaminetetraacetic acid disodium salt, or it may also contain a phosphate or citrate-phosphate buffer, or any other buffer. The DNA encoding of these and other phages may be altered to allow the recombinase to attack one cell wall at more than 2 positions, to allow the recombinase to cleave the cell wall of more than one species of bacteria, to allow the recombinase to attack other bacteria, or any combination thereof. The type and number of changes to the recombinant phage producing the enzyme cannot be calculated.

Suitable weak surfactants include, inter alia, esters of polyoxyethylene sorbitan and fatty acids (Tween series), octylphenoxypolyethoxyethanol (Triton-X series), n-octyl- β -D-glucopyranoside, n-octyl- β -D-thioglucopyranoside, n-decyl- β -D-glucopyranoside, n-dodecyl- β -D-glucopyranoside and biologically occurring surfactants, such as esters of fatty acids, glycerides, monoglycerides, deoxycholates and deoxycholates.

Preservatives may also be used in the present invention and are preferably included in an amount of from about 0.05% to about 0.5% by weight of the total composition. The use of a preservative ensures that if the product microorganisms become mixed, the formulation will prevent or reduce microbial growth. Some preservatives useful in the present invention include methylparaben, propylparaben, butylparaben, chloroxylenol, sodium benzoate, DMDM hydantoin, 3-iodo-2-propylbutylcarbamate, potassium sorbate, chlorhexidine digluconate, or combinations thereof.

Drugs or agents for use in all embodiments of the invention include antimicrobial agents, anti-inflammatory agents, antiviral agents, local anesthetics, corticosteroids, destructive therapeutic agents, antifungal agents, and antiandrogens. In the treatment of acne, active drugs that may be used include antimicrobial agents, particularly those with anti-inflammatory properties such as dapsone, erythromycin, minocycline, tetracycline, clindamycin and other antimicrobial agents. The preferred weight percentage of the antimicrobial agent is 0.5% to 10%.

Local anesthetics include tetracaine, tetracaine hydrochloride, lidocaine hydrochloride, dyclonine hydrochloride, quinicaine hydrochloride, cinchocaine hydrochloride, butamben picrate and pramoxine hydrochloride. A preferred concentration of local anesthetic is about 0.025% to 5% by weight of the total composition. Anesthetics such as benzocaine can also be used at preferred concentrations of about 2% to 25% by weight.

Corticosteroids that may be used include betamethasone dipropionate, fluocinolone actinide, betamethasone valerate, triamcinolone actinide, clobetasol propionate, desoximetasone, diflorasone diacetate, amcinonide, fludroxolone acetonide, hydrocortisone valerate, hydrocortisone butyrate, and desonide, suggested to be used at concentrations of about 0.01% to 1.0% by weight. Preferred concentrations of corticosteroids, such as hydrocortisone or methylprednisolone acetate, are from about 0.2% to about 5.0% by weight.

In addition, the therapeutic composition may further comprise other enzymes, such as the enzyme lysostaphin used to treat any Staphylococcus aureus (Staphylococcus aureus) bacteria present with susceptible gram-positive bacteria. Mucolytic peptides, such as lysostaphin, have been proposed to be effective in treating staphylococcus aureus (s. aureus) infection in humans (Schaffner et al, Yale j. biol. & Med, 39:230 (1967)). Lysostaphin, a gene product of Staphylococcus haemolyticus (s.aureus), exerts bacteriostatic and bactericidal effects on s.aureus (s.aureus) by enzymatically degrading polyglycine cross-links of the cell wall (Browder et al, res.comm., 19: 393-400 (1965)). U.S. Pat. No.3,278,378 describes a fermentation process for producing lysostaphin from the culture medium of Staphylococcus aureus (S.staphylolyticus) which was subsequently renamed to Staphylococcus hemolyticus (S.simulans). Other methods of producing lysostaphin are also described in U.S. Pat. Nos. 3,398,056 and 3,594,284. The gene for lysostaphin has subsequently been cloned and sequenced (Recsei et al, Proc. Natl. Acad. Sci. USA,84:1127-1131 (1987)). Recombinant mucolytic bactericidal proteins, such as r-lysostaphin, can potentially prevent problems associated with current antibiotic therapy because of their targeted specificity, low toxicity and possible reduction in biologically active residues. In addition, lysostaphin is active against non-dividing cells, whereas most antibiotics require actively dividing cells to mediate their effects (Dixon et al, Yale J.biology and Medicine, 41: 62-68 (1968)). Lysostaphin in combination with an altered lytic enzyme may be used in the presence or absence of an antibiotic. There is an additional degree of importance in using lysostaphin and lysin enzymes in the same therapeutic agent. Often, when a person is infected with bacteria, infection with one genus of bacteria weakens the person or alters the body's bacterial flora, allowing other potentially pathogenic bacteria to infect the body. One of the bacteria that sometimes co-infect the body is Staphylococcus aureus (Staphylococcus aureus). Many strains of Staphylococcus aureus (Staphylococcus aureus) produce penicillinase, which renders Staphylococcus (Staphylococcus), Streptococcus (Streptococcus) and other gram-positive bacterial strains non-killed by standard antibiotics. Thus, the use of lysin and lysostaphin, possibly in combination with antibiotics, may serve as the fastest and effective treatment for bacterial infections. The therapeutic composition may also include mutanolysin, and lysozyme. Therapeutic or antibacterial compositions may comprise a combination of dimeric lytic peptides, such as a combination of Cpl-1 dimer and Pal dimer.

Means of application of the therapeutic compositions comprising the dimeric lytic enzymes/polypeptides include, but are not limited to, direct, indirect, vectors and specific means or any combination of means. Direct application of the dimeric lytic enzyme/polypeptide may be by any suitable means to directly contact the polypeptide with the site of infection or bacterial colonization, such as to the nasal region (e.g., nasal spray), dermal or dermal application (e.g., transdermal formulation, topical ointment or formulation), suppository, plug application, and the like. Nasal applications include, for example, nasal sprays, nasal drops, nasal ointments, nasal washes, nasal injections, nasal tamponades, bronchial sprays and inhalers, or indirectly by using throat lozenges, mouthwashes or gargles, or by using ointments applied to the nostrils or the face or any combination of these and similar methods of application. The dimeric lytic enzymes may be administered in forms including, but not limited to, lozenges, troches, candies, injectables, chewing gums, tablets, powders, sprays, liquids, ointments and aerosols.

When the dimeric lytic enzyme/polypeptide is introduced directly by use of sprays, droplets, ointments, lotions, injections, tampons and inhalers, the enzyme is preferably in a liquid or gel environment, the liquid acting as a carrier. The altered enzyme may be administered in a dry, anhydrous form by inhaler and bronchial spray, although the liquid form of delivery is preferred.

The compositions comprising the dimeric lytic enzyme/polypeptide may be administered in the form of a candy, chewing gum, lozenge, troche, tablet, powder, aerosol, liquid spray, or toothpaste for preventing or treating bacterial infections associated with upper respiratory tract diseases. The lozenge, tablet or gum to which the dimerizing lyase/polypeptide is added may contain sugar, corn syrup, various dyes, non-sugar sweeteners, flavorings, any binders, or combinations thereof. Similarly, any gum-based product may contain acacia, carnauba wax, citric acid, corn starch, food coloring, flavoring, non-sugar sweeteners, gelatin, glucose, glycerin, mucilage, shellac, sodium saccharin, sugar, water, white wax, cellulose, other binders, and combinations thereof. Lozenges may also contain sucrose, corn starch, acacia, tragacanth, anethole, linseed, oleoresin, mineral oil, and cellulose, other binders, and combinations thereof. Sugar substitutes may also be used in place of dextrose, sucrose or other sugars.

Compositions comprising dimeric lytic enzymes, or dimeric peptide fragments thereof, may be introduced into the mucosal lining where they kill resident disease bacteria during the residence process. Mucosal linings, and are disclosed and described herein, including, for example, the upper and lower respiratory tract, eye, buccal cavity, nose, rectum, vagina, periodontal pocket, intestine, and colon. Conventional dosage forms do not remain at the site of application for any significant length of time due to the natural elimination or purification mechanism of the mucosal tissue.

It may be advantageous to have a substance that exhibits adhesion to mucosal tissue, which is administered over a period of time with one or more bacteriophage enzymes and other supplements. Substances with controlled release capability are particularly desirable, and the use of sustained release mucoadhesives has received a significant degree of attention. Robinson (U.S. patent No.4,615,697, incorporated herein by reference) provides a good overview of various controlled release polymer compositions used in mucosal drug delivery. This patent describes a controlled release treatment composition comprising a bioadhesive agent and an effective amount of a treatment agent. Bioadhesives are water swellable, but water insoluble fibrous, cross-linked, carboxyl functional polymers containing: (a) a plurality of repeating units containing at least one carboxyl functionality in at least about 80%, and (b) from about 0.05 to about 1.5% of a crosslinking agent substantially free of a polyalkenyl polyether. Whereas Robinson polymers are water swellable, but insoluble, they are crosslinked, are not thermoplastic, and are not as easily formulated with active agents and in various dosage forms as the copolymer systems of the present application. Micelles and multilamellar micelles can also be used to control the release of enzymes.

Other methods involving mucoadhesives that are a combination of hydrophilic and hydrophobic substances are known. From E.R. Rtm. orihesive of squibb & Co is a binder of a combination of pectin, gelatin and sodium carboxymethylcellulose in a viscous hydrocarbon polymer for adhesion to oral mucosa. However, the physical mixture of hydrophilic and hydrophobic components eventually separates. In contrast, the hydrophilic and hydrophobic domains in the present application result in insoluble copolymers. U.S. patent No.4,948,580 (also incorporated herein by reference) describes a bioadhesive oral drug delivery system. The compositions comprise a freeze-dried polymer blend formed from the copolymer poly (methyl vinyl ether/maleic anhydride) and gelatin dispersed in an ointment base, such as a mineral oil containing dispersed polyethylene. U.S. patent No.5,413,792 (incorporated herein by reference) discloses a paste-like preparation comprising: (A) a paste-like base comprising a polyorganosiloxane and a water-soluble polymeric material, preferably present in a ratio of 3:6 to 6:3 by weight, and (B) an active ingredient. U.S. patent No.5,554,380 claims a solid or semi-solid bioadhesive orally ingestible drug delivery system containing a water-in-oil system having at least 2 phases. One phase comprises from about 25% to about 75% by volume of an inner hydrophilic phase and the other phase comprises from about 23% to about 75% by volume of an outer hydrophobic phase, wherein the outer hydrophobic phase comprises 3 components: (a) an emulsifier, (b) a glyceride, and (c) a wax material. U.S. patent No.5,942,243, which is incorporated by reference, describes some representative release materials useful for administering antibacterial agents.

The therapeutic or pharmaceutical composition may also contain a polymeric mucoadhesive comprising a graft copolymer comprising a hydrophilic backbone and hydrophobic graft chains for controlled release of the biologically active agent. The graft copolymer is the reaction product of (1) a polystyrene macromer having a functional group that is ethylenically unsaturated, and (2) at least one hydrophilic acidic monomer having a functional group that is ethylenically unsaturated. The graft chain essentially consists of polystyrene, and a main polymer chain of hydrophilic monomer moieties, some of which have acidic functionality. The weight percent of polystyrene macromer in the graft copolymer is between about 1 and about 20%, and the weight percent of total hydrophilic monomer in the graft copolymer is between 80 and 99%, and wherein at least 10% of the total hydrophilic monomer is acidic, the graft copolymer having an equilibrium water content of at least 90% when fully hydrated. The copolymer-containing composition is gradually hydrated at the site of application by the absorption of tissue fluids to produce a very soft jelly-like mass that exhibits adhesion to mucosal surfaces. During this period, the composition adheres to the mucosal surface, which provides for sustained release of the pharmacologically active agent, which is absorbed by the mucosal tissue.

The compositions of the present application may optionally contain other polymeric materials such as poly (acrylic acid), poly, - (vinylpyrrolidone) and sodium carboxymethylcellulose plasticizers, and other pharmaceutically acceptable excipients, in amounts that do not cause deleterious effects on the mucoadhesiveness of the composition.

The dosage forms of the compositions of the present invention can be prepared by conventional methods. In case intramuscular injection is the mode of administration of choice, it is preferred to use an isotonic formulation. In general, additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. Vasoconstrictors may be added to the formulation. The pharmaceutical preparations of the present application are provided sterile and pyrogen-free.

The dimeric lytic enzymes/polypeptides of the invention may also be administered parenterally. For example, the dimeric lytic enzyme/polypeptide may be administered intramuscularly, intrathecally, subcutaneously or intravenously to treat infection by gram positive bacteria. Where parenteral injection is the mode of administration of choice, it is preferred to use an isotonic formulation. Generally, for the isotonic additives may include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. Vasoconstrictors may be added to the formulation. The pharmaceutical preparations according to the present application are provided sterile and pyrogen-free.

For any compound, a therapeutically effective dose can be estimated initially in cell culture assays or in animal models, typically mice, rabbits, dogs, or pigs. Animal models are also used to achieve the desired concentration range and route of administration. This information can then be used to determine useful doses and routes for administration in humans. The exact dosage is selected by the individual physician taking into account the patient to be treated. The dosage and administration are adjusted to provide a sufficient level of active moiety or to maintain the desired effect. Additional factors that may be considered include the severity, age, weight and sex of the patient's disease state; diet, desired duration of treatment, method of administration, time and frequency of administration, drug combination, sensitivity and tolerance/response to treatment. Long-acting pharmaceutical compositions may be administered every 3-4 days, weekly or every 2 weeks, depending on the half-life and clearance rate of the particular formulation.

The effective dosage rate or amount of the dimeric lytic enzyme/polypeptide to be administered parenterally, and the duration of treatment, will depend in part on the severity of the infection, the weight of the particular person of the patient, the duration of exposure of the recipient to the infectious bacteria, the number of square centimeters of the skin or tissue infected, the depth of infection, the severity of the infection, and a variety of other variables. The composition may be applied anywhere from once to several times per day, and may be applied for short or long periods of time. Utilization may last days or weeks. Any dosage form employed should ensure that a minimum number of units are present for a minimum amount of time. The concentration of the enzyme activity units is believed to provide an effective amount or dose of enzyme that can be suitably selected. The amount of active units/ml and the duration of exposure depend on the nature of the infection and the amount of contact the carrier allows for the lytic enzyme/polypeptide.

[ methods and assays ]

The bacterial killing capacity exhibited by the lysin polypeptides of the invention, and indeed significantly improved stability and reduced plasma clearance, provide various approaches based on the antibacterial effectiveness of the polypeptides of the invention. Thus, the present invention encompasses antibacterial methods, including methods of killing gram positive bacteria, reducing a gram positive bacterial population, treating or mitigating bacterial infection, treating a human subject exposed to pathogenic bacteria, and treating a human subject at risk of such exposure. Sensitive bacteria can be readily determined or identified by one of skill in the art, and are also identified herein to include bacteria from which the bacteriophage enzymes of the present invention are originally derived, Streptococcus pneumoniae (Streptococcus pneumoniae), and various other Streptococcus bacterial strains. Also provided are methods of treating various conditions, including prophylactic treatment of streptococcal infections, reduction of streptococcal flora or stroma, treatment of lower respiratory tract infections, treatment of otic infections, treatment of otitis media, treatment of endocarditis, and treatment or prevention of other local or systemic infections or conditions.

The invention is also useful for the treatment of sepsis, particularly in humans. To treat a sepsis infection, such as pneumonia, or bacterial meningitis, there should be a continuous intravenous inflow of the therapeutic agent into the bloodstream. The concentration of enzymes used to treat sepsis depends on the bacterial count in the blood and the amount of blood.

Also provided are methods for treating streptococcal infections, loci or populations comprising treating the infection with a therapeutic agent comprising an effective amount of at least one of the dimeric lytic enzymes/polypeptides of the invention, particularly dimeric Cpl-1 lysin, including lysins as particularly described herein. More particularly, the dimeric lytic enzyme/polypeptide capable of lysing the cell wall of a Streptococcus bacterial strain is produced from genetic material from a bacteriophage specific for Streptococcus (Streptococcus) or from one or more plasmids, vectors or other recombinant means. In the methods of the invention, dimeric lysin polypeptides of the invention, including Cpl-1 dimeric lysin, can be used in methods for the prevention and treatment of gram-positive bacteria, particularly streptococcal infections or bacterial colonization. The sensitive and related bacterial strains that are targets in the methods of the invention include and may be selected from: streptococcus suis (Streptococcus suis), Streptococcus equi (Streptococcus equi), Streptococcus agalactiae (Streptococcus agalactiae) (GBS), Streptococcus pyogenes (Streptococcus pyogenenes) (GAS), Streptococcus sanguis (Streptococcus sanguinis), Streptococcus grisea (Streptococcus gordonii), Streptococcus dysgalactiae (Streptococcus dysgalactiae), Streptococcus (Streptococcus) GES and Streptococcus pneumoniae (Streptococcus pneumoniae).

The present invention includes methods of decolonizing, treating, or ameliorating a streptococcal-related infection or condition, comprising an antibiotic-resistant bacterium, wherein the bacterium is either infected with or exposed to a particular bacterium, or a human subject suspected of being exposed or at risk for exposure is contacted with or administered an amount of an isolated dimeric lysin polypeptide of the present invention effective to kill the particular bacterium. Thus, contacting or administering one or more dimeric Cpl-1, including the polypeptide as provided herein and in fig. 6 and table 1 and SEQ ID NO:3, makes it effective to kill associated bacteria, to colonize associated bacteria, or otherwise mitigate or treat bacterial infection. In a further or additional aspect, contacting or administering one or more dimeric pals, including the polypeptide as provided herein and in fig. 7 and SEQ ID no 6, makes effective for killing the associated bacteria or otherwise mitigating or treating a bacterial infection.

The term 'agent' refers to any molecule, including polypeptides, antibodies, polynucleotides, compounds and small molecules. In particular, the term agent includes compounds such as test compounds, additional compounds added, or lysin enzyme compounds.

The term 'agonist' refers in the broadest sense to a ligand that stimulates the receptor to which the ligand binds.

The term 'determining' refers to any process by which a particular property of a compound is measured. By 'screening assay' is meant a process for characterizing or selecting compounds from a batch of compounds based on their activity.

The terms 'prevent' or 'prevent' refer to reducing the risk that a subject who may be exposed to a disease-causing agent, or who is predisposed to a disease prior to the onset of the disease, acquires or develops the disease or disorder (i.e., causes at least one clinical symptom of the disease not to develop).

The term 'prevention' is associated with and included in the term 'prevent' and refers to a measure or procedure that is intended to prevent, rather than treat or cure, a disease. Non-limiting examples of prophylactic measures can include administration of a vaccine; administering low molecular weight heparin to a hospital patient at risk for thrombosis due to, for example, immobility; and administering an anti-malarial agent such as chloroquine prior to visiting an area where malaria is endemic or at high risk of malaria.

By 'therapeutically effective amount' is meant an amount of a drug, compound, antimicrobial, antibody, polypeptide, or pharmaceutical agent that will elicit the biological or medical response of a subject that is being sought by a physician or other clinician. In particular, with respect to gram-positive bacterial infections and gram-positive bacterial growth, the term "effective amount" is intended to include a biologically significant reduction in the amount or extent of infection by gram-positive bacteria, including an effective amount of a compound or agent having a bactericidal and/or bacteriostatic effect. The phrase "therapeutically effective amount" is used herein to refer to an amount sufficient to prevent, and preferably reduce, the growth or amount of infectious bacteria, or other pathological features such as, for example, elevated fever or white blood cell count as its presence and activity is known, wherein a reduction refers to a reduction of at least about 30%, more preferably at least 50%, and most preferably at least 90% (a clinically significant change).

In one embodiment, the term 'treatment' or 'treatment' of any disease or infection refers to ameliorating the disease or infection (i.e., arresting the disease or growth of an infectious pathogen or bacterium or reducing the manifestation, extent or severity of at least one of its clinical symptoms). In another embodiment, 'treating' or 'treatment' refers to improving at least one physical parameter that is not discernible by the subject. In yet another embodiment, 'treating' or 'treatment' refers to modulating the disease or infection physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In a further embodiment, 'treating' or 'treatment' relates to slowing the progression of the disease or reducing the infection.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce allergic or similar adverse reactions, such as noisiness, dizziness and the like, when administered to a human.

It is to be understood that in the context of in vivo implemented therapeutic methods or medical and clinical therapeutic methods according to the present application and claims, the term subject, patient or individual is intended to refer to a human.

The terms "gram-positive bacterium", "gram-positive" and any variant not specifically listed, are used interchangeably herein, and as used throughout the application and claims refer to gram-positive bacteria that are known and/or can be identified by the presence of specific cell wall and/or cell membrane characteristics and/or by staining with a gram stain. Gram-positive bacteria are known and can be readily identified and can be selected from, but are not limited to, Listeria (Listeria), Staphylococcus (Staphylococcus), Streptococcus (Streptococcus), Enterococcus (Enterococcus), Mycobacterium (Mycobacterium), Corynebacterium (Corynebacterium) and Clostridium (Clostridium), and include any and all recognized or unrecognized species or strains thereof.

The term "bactericidal" refers to the ability to kill bacterial cells.

The term "bacteriostatic" refers to the ability to inhibit bacterial growth, including the inhibition of growing bacterial cells.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce allergic or similar adverse reactions, such as noisiness, dizziness and the like, when administered to a human.

The phrase "therapeutically effective amount" is used herein to refer to an amount sufficient to prevent, and preferably reduce, S-phase activity of a target cellular material, or other pathological feature such as, for example, elevated blood pressure, fever, or white blood cell count as its presence and activity is known, wherein the reduction refers to a reduction of at least about 30%, more preferably at least 50%, most preferably at least 90% (a clinically significant change).

Diseases or conditions caused by streptococcal infection, such as those caused by Streptococcus pneumoniae (Streptococcus pneumoniae), may be treated by administering to a mammal suffering from the disease or condition a composition comprising a therapeutically effective amount of a dimeric lysin.

One method of treating systemic or tissue bacterial infections caused by Streptococcus (Streptococcus) bacteria comprises parenterally treating the infection with a therapeutic agent comprising an effective amount of one or more dimeric lysin polypeptides of the invention, particularly dimeric Cpl-1, including the polypeptides provided herein, in fig. 6 and table 1, and/or dimeric Pal including the polypeptides provided herein in fig. 7 and SEQ ID NO:6, and a suitable carrier. Many other different methods may be used to introduce the dimeric lytic enzyme/polypeptide. These methods include intravenous, intramuscular, subcutaneous, intrathecal and subcutaneous introduction of the dimeric lytic enzyme/polypeptide. One skilled in the art, including medical personnel, will be able to evaluate and identify the most appropriate mode or means of administration, given the nature and extent of the bacterial conditions and strains or bacterial types involved or suspected. For example, intrathecal use and administration of one or more dimeric lytic polypeptides may be most beneficial for the treatment of bacterial meningitis.

Infections can also be treated by injecting infected tissue of a human patient with a therapeutic agent comprising an appropriate lytic enzyme/polypeptide and a carrier for the enzyme. The carrier may comprise distilled water, a salt solution, albumin, serum, or any combination thereof. More particularly, solutions for infusion or injection may be prepared in a conventional manner, for example with the addition of preservatives such as p-hydroxybenzoates or stabilizers such as alkali metal salts of ethylene-diamine tetraacetic acid, which may then be transferred into a fusion container, syringe vial or ampoule. Alternatively, the injectable compound may be lyophilized with or without other ingredients and suitably dissolved in a buffer solution or distilled water at the time of use. Non-aqueous vehicles such as fixed oils, liposomes, and ethyl oleate are also useful herein. Other phage-associated lytic enzymes, along with perforin proteins, may be included in the composition.

Various therapeutic approaches are provided to use dimeric lytic enzymes/polypeptides, such as dimeric Cpl-1 lysin or dimeric Pal lysin, or a combination thereof or in combination therewith, as exemplified herein, as a prophylactic treatment to eliminate or reduce susceptible bacterial loci, to prevent those people who have been exposed to other people with symptoms of infection from developing disease, or as a therapeutic treatment to those who have developed disease from infection. Similarly, the dimeric lytic enzymes/polypeptides may be used for the treatment or alleviation of, for example, lower respiratory tract diseases, particularly by bronchial nebulization or intravenous administration using the enzymes. For example, lytic enzymes may be used for prophylactic and therapeutic treatment of ocular infections, such as conjunctivitis. The method of treatment comprises administering eye drops or eye lotions comprising an effective amount of at least one dimeric lytic polypeptide of the invention and a carrier, which can be safely applied to the eye, with a carrier comprising a lytic enzyme. The eye drops or eye lotion are preferably in the form of an isotonic solution. The pH of the solution should be adjusted so that there is no irritation of the eye, which in turn may lead to possible infection by other organisms and possible damage to the eye. Although the pH range should be in the same range as other lytic enzymes, the optimal pH will be within the ranges shown and provided herein. Similarly, buffers of the above kind for other lytic enzymes should also be used. Other antibiotics suitable for use in eye drops may be added to the enzyme-containing composition. Bactericides and bacteriostatic compounds may also be added. The concentration of enzyme in the solution may range from about 100U/ml to about 500,000U/ml, a more preferred range from about 100 to about 5,000U/ml, and from about 100 to about 50,000U/ml. The concentration may be higher or lower than the ranges provided.

The dimeric lytic polypeptides of the invention may also be used in contact lens solutions for immersion and cleaning of contact lenses. This solution, which is a normally isotonic solution, may contain, in addition to the enzymes, sodium chloride, mannitol and other sugar alcohols, borates, preservatives, and the like. The lytic enzymes/polypeptides of the invention may also be administered to the ear of a patient. Thus, for example, the dimeric lytic polypeptides of the invention may be used to treat ear infections caused, for example, by Streptococcus pneumoniae (Streptococcus pneumoniae). Otitis media is inflammation of the middle ear characterized by symptoms such as ear pain, hearing loss and fever. One of the major causes of these symptoms is fluid formation in the middle ear (effusion). Complications include permanent hearing loss, tympanic membrane perforation, acquired cholesteatoma, mastoiditis and adhesive otitis media. Children with otitis media in the 1 st year after birth are at risk of recurrent acute or chronic disease. One of the major causes of otitis media is Streptococcus pneumoniae (Streptococcus pneumoniae). The lytic enzyme/polypeptide may be applied to the infected ear by delivering the enzyme to the ear canal in a suitable carrier. The carrier may comprise a sterile aqueous or oily solution or suspension. The lytic enzyme may be added to a carrier, which may also contain a suitable preservative, and preferably a surfactant. Preferred bactericides and fungicides for inclusion in the drops are nitric acid or phenylmercuric acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for preparing an oily solution include glycerol, diluted alcohols and propylene glycol. In addition, any number of other ear drop carriers can be used. Concentrations and preservatives for treatment of otitis media and other similar ear infections are the same as discussed for eye infections, and the enzyme-carrying vehicle is similar or identical to the vehicle used for treatment of eye infections. In addition, the carrier can generally include vitamins, minerals, carbohydrates, sugars, amino acids, proteinaceous substances, fatty acids, phospholipids, antioxidants, phenolic compounds, isotonic solutions, oil-based suspensions, and combinations thereof.

The diagnostic, prophylactic and therapeutic possibilities and the use thereof, which are raised by the recognition and the generation of dimeric lysin polypeptides of the invention, derive from the fact that the polypeptides of the invention cause direct and specific effects (e.g. killing) in susceptible bacteria. Thus, the polypeptides of the invention can be used to eliminate, characterize or identify related and sensitive bacteria.

Thus, the diagnostic methods of the invention may comprise examining a cell sample or culture medium for the purpose of determining whether it contains sensitive bacteria, or whether bacteria in the sample or culture medium are sensitive, using an assay comprising an effective amount of one or more dimeric lysin polypeptides and a means for characterizing one or more cells in the sample, or determining whether cell lysis has occurred or is occurring. Patients who can benefit from this method include those suffering from an undefined infection, an identified bacterial infection, or suspected of being exposed to or carrying a particular bacterium. Fluids, foods, medical devices, compositions or other such samples that may come into contact with a subject or patient may be examined for sensitive bacteria or associated bacteria may be eliminated. In one such aspect, the fluid, food, medical device, composition, or other such sample may be sterilized or otherwise treated to eliminate or remove any potentially associated bacteria by incubation with or exposure to one or more lytic polypeptides of the invention.

The procedures and their applications are all well known to those skilled in the art and can therefore be utilized within the scope of the present invention. In one example, a lytic polypeptide of the invention is complexed with or otherwise binds or associates with an associated or sensitive bacterium in a sample, and one member of the complex is labeled with a detectable label. The fact that a complex is formed and, if desired, the amount thereof can be determined by known methods applicable to the detection of a label. The labels most commonly used for these studies are radioactive elements, enzymes, chemicals that fluoresce when exposed to ultraviolet light, and others. Many fluorescent substances are known and can be used as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer yellow. The radioactive label can be detected by any currently available counting method. Preferred isotopes may be selected from³H，¹⁴C，³²P，³⁵S，³⁶Cl，⁵¹Cr，⁵⁷Co，⁵⁸Co，⁵⁹Fe，⁹⁰Y，¹²⁵I，¹³¹I and¹⁸⁶re. enzyme labels are equally useful and can be detected by any presently available colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gas-quantitative technique the enzyme is conjugated to selected particles by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like a number of enzymes are known and can be utilized which are useful in these processes, preferred are peroxidases, β -glucuronidase, β -D-glucosidase, β -D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase, and the disclosures of their alternative labeling materials and methods are cited by way of example in U.S. Pat. Nos. 3,654,090, 3,850,752, and 4,016,043.

The invention will be better understood by reference to the following non-limiting examples, which are provided as illustrations of the invention. However, the following examples are intended to more fully illustrate preferred embodiments of the invention and should not be construed as limiting the broad scope of the invention.

[ examples ] A method for producing a compound

Example 1: stable phage lysin (Cpl-1) dimers with increased anti-pneumococcal activity

Bacteriophages (phages) produce endolysins (lysins) as part of their lytic cycle to degrade the peptidoglycan layer of the infected bacteria for release of the phage progeny. Because these enzymes retain their lytic and lethal activity against gram-positive bacteria when not inherently added to cells, they have been actively developed as new anti-infective agents, sometimes referred to as enzyme antibiotics. Like other relatively small peptides, one problem in their clinical development is rapid inactivation by proteolytic degradation, immunological blockade or renal clearance. Anti-pneumococcal lysin Cpl-1 was shown to escape proteolysis and immunological blockade. However, its short plasma half-life (20.5 min in mice) may present a disadvantage of clinical usefulness. Here we report the construction of Cpl-1 dimers taking into account the increase in Cpl-1 anti-pneumococcal specific activity and plasma half-life. Dimerization is achieved by introducing a specific cysteine residue at the C-terminus of the enzyme, thereby facilitating disulfide bonding. The constructed dimer showed a 2-fold increase in specific anti-pneumococcal activity and almost a 10-fold increase in plasma half-life (i.e.. 0.028 vs. 0.27ml. min.) compared to the native monomer^-1). Since several lysins are suspected of dimerizing and thus fully activating upon contact with their cell wall substrates, stable pre-dimerized enzymes may represent a more efficient alternative to the natural monomers.

Streptococcus pneumoniae (Streptococcus pneumoniae) (s. pneumoconiae) is a gram-positive diplococcus responsible for the packaging of a wide range of human infections. It is the 1 st cause of otitis media affecting >5 million children annually in the USA (4), and a common cause of sinusitis, community-acquired pneumonia, bacteremia and meningitis (13). It is responsible for >1 million deaths per year in children under 5 years of age worldwide (5), and pneumococcal pneumonia remains the 6 th most common cause of death in the USA of all age groups (8). Streptococcus pneumoniae (s. pneumoniae) is also the major bacterial pathogen responsible for hyper-infection following influenza a respiratory tract infection (a complication of > 90% death during influenza pandemics) (2, 3, 20, 21).

However, their overuse in the treatment of millions of mild cases of otitis and sinusitis, often of viral origin, has given great pressure on resistance selection (11). therefore, there is now a widespread report of bacteriologically-confirmed therapeutic failure due to increased resistance to a variety of drugs, including the commonly used β -lactams, macrolides and fluoroquinolones (19, 23). thus, new drugs acting by entirely different mechanisms are highly desirable.

Bacteriophages, which are the natural major predators of bacteria, were considered as potential anti-bacterial agents (28, 29) decades before the clinical development of antibiotics, however, the complexity of developing natural bacteriophages to comply with industrial products led western countries to abandon their development since the 1940 s. Today, however, the problem of antibiotic resistance has expanded the interest in contributing phage-derived molecules as potentially clinically useful anti-bacterial compounds (9, 27).

One of these classes of compounds has been developed for its rapid killing of gram-positive bacteria, namely phage lysin (1, 9), which is produced in time when phage progeny are to escape the bacterial host, pneumococcal phage Cp-1 produces lysin Cpl-1, a 37kDa enzyme that specifically hydrolyses pneumococcal peptidoglycan, this lysin is constructed as a whole of this endolysin with 2 well-defined domains linked by a flexible linker catalytic activity is restricted to the N-terminal domain, while the C-terminal part containing 6 choline-binding repeats (ChBR) and 13 amino-acids C-terminal tails is used for specific substrate binding in the pneumococcal cell wall (10), Cpl-1 belongs to β 1,4 linked lysozyme family (22) between the N-acetylmuramic acid and N-acetyl-D-glucosamine residues targeting peptidoglycan, our laboratory clones the Cpl-1 gene into the high expression vector pina and achieves its overexpression and purification (15), the development of a model of the successful self-purifying lysozyme in vivo therapy for stroke (31), and the clinical clearance of pneumococcal infections with Cpl-1, similar model of the need for the rapid clearance of meningococcal infection (31, 31) and similar continuous clearance of the pneumococcal activity.

To construct more efficient Cpl-1, we utilized recent knowledge of lysin dimerization. To be fully active, the major pneumococcal autolysin (LytA) needs to dimerize first, which is initiated by the interaction of its C-terminal choline-binding domain with choline, leading to the formation of homodimers that are significantly more active than the natural monomers (7, 26, 30). Sequence alignment of Cpl-1 and LytA revealed extended similarity, especially within the C-terminal tail of the enzymes involved in dimerization. Thus, we hypothesized that Cpl-1 may have the same dimerization requirement as LytA for full activity. In addition, dimeric forms of Cpl-1 may have a significant reduction in renal clearance, as their molecular weight (74kDa vs 37kDa for monomer) is greater than 60-65 kDa, the threshold for glomerular filtration in humans (18).

In this work, we constructed stable Cpl-1 homodimers linked by disulfide bonds at the C-terminus and examined their in vitro activity and in vivo plasma clearance compared to the parent monomer. The results provide a basis for further studies of the biological activity of this type of dimer in infection models.

Materials and methods

[ reagent ]

Plasmid miniprep kit from Qiagen (Valencia, USA) and QuickChange II Site-Directed mutagenesis kit from Stratagene (Cedar Creek, USA). DEAE-Sepharose and gel filtration HiLoad 16/60Superdex^TMA200 prep grade column was obtained from GE Healthcare Bio-Sciences, Inc. (Piscataway, USA). Mutagenesis primers were synthesized by Fisher Scientific (Pittsburgh, USA). All other chemicals were purchased from Sigma-Aldrich (Saint Louis, USA). The rapid Start Bradford dye reagent and Bovine Gamma Globulin (BGG) standards used for protein concentration measurements were from Biorad (Hercules, USA). Amicon ultra centrifuge device from MillIcore (Billerica, USA) and 0.45 μm Acrodisc syringe filters from Pall (Ann Arbor, USA).

[ construction of Cpl-1 mutants ]

All mutants were constructed by using the QuickChange II Site-Directed mutagenesis kit, with appropriate primers designed to introduce the desired mutation, and according to the manufacturer's instructions. The presence of the mutation was confirmed by DNA sequencing performed in Genewiz (SouthClainfield, NJ USA).

[ Generation and purification of Cpl-1 and Cpl-1 mutant lysins ]

Plasmid DNA from selected clones carrying the wild-type and mutant Cpl-1 gene was isolated and further transformed into E.coli (E.coli) DH5 α to overexpress the corresponding protein wild-type Cpl-1 protein was obtained from the expression of pJML6 in DH5 α E.coli (E.coli) cells, lysin was generated and purified according to procedures already described elsewhere (15). briefly, the strain was grown in Luria-broth overnight under stirring at 225 rpm. protein expression was induced overnight with 20g/L lactose after harvesting and resuspending in enzyme buffer (50mM phosphate buffer, pH 7.4). cells were sonicated on ice (Sonopuls, Bandelin electronics, Berlin, Germany). cell debris was precipitated by centrifugation (15,000rpm,1h on 37 h supernatant treated with 20U (20U) enzyme buffer, filtered using a column containing 0.M buffer, filtered using a column containing 20 U.E) and the supernatant was stored in a column containing 20. mu.M buffer, filtered using a column containing 20. mu.M buffer, filtered using a column containing 20. mu.M buffer, 14. agarose, filtered, and the column containing the enzymes was stored at room temperature after harvesting and resuspended in a column buffer (10. mu.M).

[ isolation of Cpl-1 mutant dimers ]

All dimers were also separated on a Hiload 16/60Superdex column attached to AKTA Prime equipment (GE Healthcare Bio-sciences corporation, Piscataway, USA). Briefly, for each mutant, a mixture of monomers and dimers (see above) obtained spontaneously after induction of overexpression by lactose was applied at 1ml/min to a column previously equilibrated with enzyme buffer. Fractions containing dimer were combined and concentrated using an Amicon Ultra centrifugal filtration unit (MWCO 30,000, Millipore, carrigowhill, ireland) according to the manufacturer's recommendations. The concentrated sample was subjected to the 2 nd purification step on Hiload 16/60 Superdex. The final fractions containing dimer were combined, concentrated to 1mg/ml, and stored at-20 ℃ until further use.

[ in vitro killing assay ]

The kill assay was performed by using the streptococcus pneumoniae (s. pneumoconiae) strain DCC1490 and has been described elsewhere (17). Briefly, DCC1490 was grown to log phase (OD of 0.3) in brain cardiac infusion (BHI)_595nm). After centrifugation of the cells and resuspension to approximately 10 in enzyme buffer⁹After a concentration of CFU/ml, serial dilutions of lysin were added directly to the bacterial suspension in 96-well plates (approximately 5.10)⁸Final concentration of CFU/ml). OD was measured by measuring OD at 37 ℃ in an EL 80896-well plate reader (Biotek Instruments, Luzern, Switzerland)_595nmThe reaction kinetics were obtained over a 15 minute period. 1U of the enzyme was defined as 5.10 reaching Streptococcus pneumoniae (S.pneumoconiae) DCC1490 after 15 minutes at 37 ℃⁸OD of CFU/ml solution_595nmThe amount of enzyme required to reduce by half (corresponding to a1 log reduction in CFU/ml).

[ measurement of the clearance of lysin from the plasma of mice ]

All animal experiments were conducted according to federal and regulatory guidelines. Male Balb/c mice, obtained from Charles River Laboratories (Wilmington, USA) with an average weight of 22g, were injected intravenously (iv) in the lateral tail vein with the same molar amounts of wild-type and dimeric Cpl-1 enzyme. Thus, 2 different groups of 15 mice were given Cpl-1(4.5mg/ml) or Cpl-1^C45S,D324CA single bolus (100. mu.l) of dimer (9 mg/ml). After anesthesia, blood samples were collected by cardiac puncture 5, 30, 60, 180 and 300min after administration and placed directly on ice.

Prepared from ice-cold heparinized blood samples by centrifugation at 3000 Xg for 10min at 4 ℃And preparing blood plasma. Plasma samples were stored at-20 ℃ until use. Cpl-1 and Cpl-1^C45S,D324CPlasma concentrations of dimers were determined by using an indirect sandwich ELISA assay. 96-well plates were coated with 1 × (PBS 1 ×), 5 μ g/ml solutions of monoclonal anti-Cpl 1 antibody in pH7.4 (3 h at 37 ℃ C. and then overnight at 4 ℃). After 5 washes with 200. mu.l of wash buffer (PBS 1X, NaCl150mM, Brij-350.05%, sodium azide 0.02%, pH7.4), 100. mu.l of plasma sample and standard diluted in dilution buffer (PBS 1X, 0.5M NaCl, 0.25% Brij-35, 0.02% sodium azide, pH7.4) were added to the wells and the plates were incubated at 37 ℃ for 3 h. The plate was washed 5 more times with 200. mu.l wash buffer and 100. mu.l/well of primary antibody (rabbit polyclonal anti-Cpl-1 antibody) diluted to 2. mu.g/ml in dilution buffer was added to the wells and the plate was incubated at 37 ℃ for a further 3 h. After 5 additional washes with 200 μ l wash buffer, the plates were incubated overnight at room temperature in the presence of 100 μ l/well of secondary antibody (alkaline phosphatase conjugated goat anti-rabbit-IgG) diluted to 1/1000 in dilution buffer. The following morning, the wells were washed 5 times with 200. mu.l wash buffer and 200. mu.l alkaline phosphatase substrate (1 mg/ml in 10% diethanolamine, 1mM MgCl) at room temperature₂) After incubation the enzymatic activity at 405nm was measured by colorimetric detection. Using Spectra Max 5^ePlate reader (molecular Devices, Sunnyvale, USA), the results were analyzed with SoftMax Pro software.

Since monomeric and dimeric forms of Cpl-1 react differently with Cpl-1-specific antibodies, 2 different standards must be used in the ELISA assay. Using the known concentration of purified Cpl-1 monomer in experiments with native enzymes, the known concentration of purified Cpl-1 was used^C45S,D324CDimers for use with Cpl-1^C45S,D324CDimer experiments.

[ results and discussion ]

It is known that the amino acid sequences of the C-terminal regions of LytA and Cpl-1 are homologous (73/142 identical residues and 55/69 residues are conservative substitutions) and that the C-terminal 13 amino acids are responsible for the dimerization of LytA (FIG. 1). Interestingly, within this region, 10/13 amino acids were identical between Cpl-1 and LytA. We speculate from this that Cpl-1 may also dimerize and become fully active.

【Cpl-1^C45S,D324CConstruction and purification of dimers ]

Our strategy to generate pre-dimerized enzymes focuses on the formation of disulfide bridges between 2 monomers. The wild-type Cpl-1 enzyme contains 3 cysteine residues at positions 45, 108 and 239. Using the Accpro server on the SCRATCH protein predictor website (SCRATCH. proteomics. ics. uci. edu/index), only cysteine 45(C45) was predicted to be accessible to the solvent. Thus, to avoid possible interactions with this cysteine residue, we engineered the mutation Cpl-1^C45S. This mutant was soluble and fully active compared to native Cpl-1 (data not shown).

To construct a pre-dimerized form of Cpl-1, we will also refer to Cpl-1^C45SMutation to Cpl-1^C45S,D324C. We specifically introduced a new cysteine residue before the 13 amino-acid extension in the C-terminal tail of Cpl-1 to prevent interference with critical structures in this region. Mutations were confirmed by DNA sequencing. Cpl-1 and Cpl-1^C45S,D324CColi (E.coli) DH5 α cells were successfully over-expressed and purified to homogeneity on a DEAE-agarose column (FIG. 2A, lane 2 and FIG. 2B, lane 2)^C45S,D324CIt was predicted that band 2 was spontaneously represented at 74kDa on a Coomassie stained non-reducing SDS-PAGE gel (FIG. 2B, lane 2). Reduction of this sample with 10mM Dithiothreitol (DTT) resulted in complete disappearance of the 74kDa band (FIG. 2B, lane 3), confirming Cpl-1^C45S,D324CThe disulfide-related dimeric form of (a) is reducible. Cpl-1 was achieved by 2-step purification on Sephadex G-100^C45S,D324CThe dimer was further purified to homogeneity (-94%) (FIG. 2C, lanes 2, and 3).

Cpl-1^C45S,D324CIn vitro anti-microbial Activity of monomers

We found that 1mM DTT was sufficient to achieve Cpl-1^C45S,D324CThe dimer was completely reduced to monomer (data not shown). In this experiment, therefore, at 1mM DTTMeasuring purified Cpl-1 in the Presence of^C45S,D324CAnti-microbial activity in monomeric form. Under this condition, the mutant enzyme retained 100% of in vitro anti-bacterial activity compared to native Cpl-1. As seen in FIG. 3, using the in vitro kill assay, Cpl-1 and Cpl-1 were used at 0.5nmol/ml^C45S；D324CThe monomer reached the OD of Streptococcus pneumoniae (S.pneumoconiae) after 15 minutes_595nmA 50% reduction. The calculated specific activity was 6.7U/nmol of enzyme in both cases. It was concluded that the substitution of aspartic acid for Cpl-1 with cysteine within the C-terminal region^C45S,D324CHas no significant effect on the activity of (2). The substitution is located just before the 13 amino acid tail required for native dimerization of LytA (and possibly Cpl-1), which can therefore conclude that the new cysteine residue does not interfere with the suspected Cpl-1 native dimerization process required for full activity. Finally, we speculate that Cpl-1^C45S,D324CWhen converted to monomers by DTT treatment, they associate again with the same efficiency as dimers when interacting with choline in the cell wall.

【Cpl-1^C45S,D324CIn vitro anti-microbial Activity of dimers ]

Purified Cpl-1 in our in vitro assay^C45S,D324CThe dimer (FIG. 2C, lane 3) shows 2-fold increase in anti-bacterial activity compared to Cpl-1 (for Cpl-1)^C45S,D324CDimer and Cpl-1, respectively, at 50% OD from 0.25 vs. 0.5nmol/ml_595nmDecreased) (fig. 3). For Cpl-1^C45S,D324CDimer and Cpl-1, the calculated specific activities were 13.33 and 6.67U/nmol, respectively. Since this observation is not surprising based on mol-to-mol, twice as many molecules of monomer are required to produce a fixed number of dimers. Moreover, the dimer contains 2 active sites (1 in the comparative monomer) and is therefore expected to have twice the activity of the monomer. Finally, this result provides evidence that C-terminal dimerization does not impair Cpl-1 activity, i.e., that if Cpl-1 were to undergo substantial LytA-like dimerization, it would not impair enzyme activity.

[ Cpl-1 only ]^C45S,D324CDimers being fully active ]

13 Cpl-1C4 were mixedThe 5S mutants were each processed to have an additional cysteine at 13 different positions within the protein sequence. Among those, the 6/13 mutant showed the same anti-microbial activity as native Cpl-1 when tested for their monomeric state in the presence of 1mM DTT in an in vitro killing assay for streptococcus pneumoniae (s. pneumoconiae) DCC1490 (table 1). For all Cpl-1 mutants containing exposed cysteines, spontaneous dimerization occurred after lactose induction of the corresponding 6 mutant lysins. We purified the corresponding dimers and tested them for anti-microbial activity in our in vitro killing assay (figure 5). At 0.5mg/ml, all dimers (except Cpl-1)^C45S,D324COuter) showed a reduction of 87.5% or more in their anti-microbial activity. Thus, dimerization at other positions significantly impaired Cpl-1 enzymatic activity compared to the extreme end of the C-terminal moiety. These observations reveal the importance of a good positioning of the neocysteine residues involved in the formation of disulfide bridges between the 2 monomers.

Table 1: 0.5mg/ml anti-microbial Activity of Cpl-1 mutants

Name of mutation	The activity of the mutant monomer%	Purified mutant dimer Activity%
			Cpl-1C45S；Q85C	100	0
Cpl-1C45S；D194C	100	<1
			Cpl-1C45S；S206C	25	n.d
Cpl-1C45S；N214C	100	5
			Cpl-1C45S；G216C	100	3
Cpl-1C45S；F217C	25	n.d
			Cpl-1C45S；E249C	75	n.d
Cpl-1C45S；D256C	100	12.5
			Cpl-1C45S；S269C	100	3
Cpl-1C45S；M301C	18.75	n.d
			Cpl-1C45S；G310C	75	n.d
Cpl-1C45S；N319C	50	n.d
			Cpl-1C45S；D324C	100	100

wt, wild type, n.d, not determined

[ Cpl-1 and Cpl-1 in mice^C45S,D324CPlasmapheresis of dimers ]

Balb/c mice were injected in the lateral tail vein with 100. mu.l of either 4.5mg/ml (12.16 nmole/100. mu.l) native Cpl-1 or 9mg/ml (12.16 nmole/100. mu.l) Cpl-1^C45S,D324CA dimer bolus. Blood samples were taken at 5, 30, 60, 180 and 300 minutes post injection. Using a sandwich ELISA assay, Cpl-1 was found at each time point^C45S,D324CDimer concentrations were significantly higher compared to control monomers (figure 4). For example, 20.32 fold more dimeric molecules were detected in plasma 30 minutes post injection compared to Cpl-1 monomer (for Cpl-1^C45S,D324CDimer and Cpl-1, 4,764.32. + -. 788.55 vs 234.5. + -. 48.93pmol/ml, respectively). After 5 hours, the difference was 7.76 (for Cpl-1)^C45S,D324CDimer and Cpl-1, 116.8. + -. 22.85 vs 15.05. + -. 1.82pmol/ml, respectively). The calculated area under the curve (AUC) representing the residual lysin in plasma during the experiment is: the monomer is 44.417nmol/min^-1/ml^-1Comparative dimer was 435.026nmol/min^-1/ml^-1. Thus, for an injected dose of 12.16nmol, compare Cpl-1^C45S,D324CDimer 0.028ml/min^-10.274ml/min of Cpl-1 monomer was found^-1The clearance of stabilized dimer was reduced by a factor of-9.8 compared to monomer.

Multiplication by dimerizationThe size of Cpl-1 was found to have a significant effect on the clearance of the enzyme from the mouse plasma, suggesting that renal filtration may play a substantial role in its elimination. Our findings indicate that Cpl-1 is a natural Cpl-1 monomer that compares to that previously tested in a mouse model of pneumococcal septicaemia and shown to have a 20.5 minute half-life (12, 15)^C45S,D324CDimers are much more potent intravenous molecules. Furthermore, larger amounts of dimer are available in the blood for systemic distribution, thus Cpl-1^C45S,D324CDimers are expected to show improved therapeutic efficacy in other streptococcus pneumoniae (s. pneumoconiae) associated diseases. For example, in a recent publication, Cpl-1 in monomeric form was used to treat pneumonia mice by intraperitoneal delivery of lysin every 12 hours (31). Despite the short half-life of lysin, the effectiveness of this treatment suggests that the dimeric form may prove significantly more effective in treating similar infections. Of course, the bioavailability of the dimer at the site of infection is evaluated in an in vivo setting.

In summary, we report here that the constructed stabilized dimeric lysin is twice as active (at 1/1 molar ratio) as the original monomer in vitro, and has approximately 10-fold bioavailability in blood. We have also shown that the choice of the position of the newly introduced cysteine to be introduced, which is involved in dimerization, is critical. Persistent Cpl-1 lysin may represent a new alternative to antibiotic-sensitive and-resistant pneumococci. Moreover, since several other phage lysins have shown or are suspected of dimerizing (7, 24, 25), this strategy may represent a general way to increase the activity and/or pharmacokinetics of a particular phage lysin.

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Example 2: in vivo animal research ]

Intravenous killing effect. 4-to 6-week-old female C3H/HeJ mice were treated with 3X 10 through the tail vein⁷Log phase streptococcus pneumoniae (s.pneumoniae) serotype 14 (strain DCC1490, penicillin sensitive) infection of CFU and intravenous treatment with 100 μ Ι of different concentrations (100-2000 μ g) Cpl-1 or the same volume (100 μ Ι) of buffer 10h after infection blood samples were obtained before treatment and after 15 and 120min and the bacterial titer was determined by serial dilution and plated on blood agar the presence of streptococcus pneumoniae (s.pneumoniae) in α -haemolytic colonies was confirmed by using the opphoxing disc.

Example 3: dimerized PAL enzymes

Pal is a streptolysin isolated from pneumococcal phages that specifically digest pneumococcal cell walls within seconds (Loeffler, JM et al (2001) Science 294: 2170-2172; U.S. Pat. No. 7,569,223). Pal is a 296-amino acid-cleaving protein with a molecular mass of 34kDa, and is an amidase that cleaves peptidoglycan between N-acetylmuramic acid and L-alanine. Cpl-1, described above, is a 339 amino acid protein and is lysozyme, cleaving the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine (Garcia, P et al (1997) Microb Drug Resist 3: 165-176). Although the two enzymes have very different N-terminal catalytic sites, they share similar C-terminal cell wall attachment sites, which bind to choline. Enhanced killing efficacy of the combination of Pal and Cpl-1 against Streptococcus pneumoniae (S. pneumoconiae), including penicillin-resistant strains, has been described (Loeffler, JM and Fischetti, VA (2003) antimicrobial Agents and Chemoth47(1): 375-.

As with Cpl-1, the C-terminal region of the Pal enzyme includes the choline binding repeat corresponding to LytA and the C-terminal amino acid, and in fact, 11 of the C-terminal 14 amino acids of Pal are identical to those of the LytA sequence. When comparing Pal and Cpl-1, 9 of the C-terminal 14 amino acids of Pal are identical to those of Cpl-1. Pal shows overall similarity to Cpl-1 and LytA in its C-terminal region, particularly amino acids 155 to 296, with 60 of the 142 amino acids being identical among Pal, Cpl-1 and LytA. FIG. 7 depicts the amino acid sequences of Cpl-1, Pal and LytA and the homologous C-terminal regions displaying these 3 enzymes.

A Pal dimer consisting of 2 monomers covalently linked by disulfide bonds and stabilized was processed and constructed using an equivalent method described above for Cpl-1. Formation of disulfide bridges between 2 monomers using the Pal enzyme, as with Cpl-1, resulted in an exemplary pre-dimerized Pal enzyme.

In order to construct a pre-dimerized form of Pal, the C-terminal region amino acids were mutated to cys, new cysteine residues were specifically introduced into the C-terminal tail of Pal before interference of any key structures in this homologous region, mutation was confirmed by DNA sequencing and wild-type and mutant Pal was overexpressed in escherichia coli (e.coli) DH5 α cells and purified to homogeneity on a DEAE-agarose column, the pre-dimerized Pal was visualized by a non-reducing SDS-stained strip at about 2-70 th band on a PAGE-70 th band on a sars-reducing SDS-gel. Reduction with 10mM Dithiothreitol (DTT), which resulted in the disappearance of the higher kDa band, confirmed that the disulfide-related dimeric form of Pal was reducible. Construction of Pal with amino acid 280 mutated to cysteine^D280CConfirmed and tested to establish production of the pre-dimerized Pal enzyme. Pal^D280CThe mutant pre-dimerized with Cpl-1 at a comparable position adjacent to the C-terminal 14 amino acids analogous to LytA of Pal^C45S,D324CSimilarly. In vitro and in vivo tests for killing activity and efficacy of mutant dimerized Pal were performed as described above for Cpl-1 to demonstrate the anti-pneumococcal activity of mutant dimerized Pal.

Although the invention has been described and illustrated herein by reference to various specific materials, processes and examples, it is to be understood that the invention is not limited to the specific materials, combinations of materials and processes selected for this purpose. Various variations of the details may be suggested and will be within the purview of one skilled in the art.

Claims (13)

1. An isolated Streptococcus dimeric (Streptococcus) -specific phage lysin comprising 2 Streptococcus-specific phage lysin monomers covalently linked to each other, wherein the killing activity of the dimer against one or more Streptococcus bacteria is higher than the killing activity of any of the Streptococcus-specific phage lysin monomers, wherein the lysin monomers are selected from the group consisting of: a mutant Cpl 1-1 lysin having an amino acid sequence as shown in SEQ ID NO 1 and being mutated to a mutant Cpl 1-1 lysin which does not have a Cys residue within the first 45 residues of said lysin and comprises a Cys residue between the 14 and 20 amino acids from the C-terminus, a mutant Cpl-1 lysin consisting of the amino acid sequence shown in SEQ ID NO 3, a mutant Pal lysin having an amino acid sequence as shown in SEQ ID NO 5 and being mutated to a mutant Pal lysin comprising a Cys residue between the 14 and 20 amino acids from the C-terminus, and a mutant Pal lysin consisting of the amino acid sequence shown in SEQ ID NO 6.

2. The lysin of claim 1, wherein said lysin monomers are chemically cross-linked to each other.

3. The lysin of claim 2, wherein said lysin monomers are covalently linked to each other by disulfide bonds.

4. A lysin according to any one of claims 1-3, having killing activity against Streptococcus pneumoniae (Streptococcus pneumoniae).

5. The lysin of claim 1, wherein one or more lysin monomers is Cpl-1^C45S,D324COr Pal^D280C。

6. Use of a composition comprising a therapeutically effective amount of a lysin of any one of claims 1-5 in the manufacture of a medicament for treating a mammal having a disease or condition caused by streptococcal infection.

7. The use of claim 6, wherein the infection is caused by Streptococcus pneumoniae (Streptococcus pneumoniae).

8. The use of claim 6, wherein the disease or condition is one or more selected from the group consisting of: bacteremia, meningitis, pneumonia, otitis media and sinusitis.

9. Use of a composition comprising a therapeutically effective amount of a lysin of any of claims 1-5 in the preparation of a medicament for decolonizing streptococcus in a mammal having or at risk of a disease or condition caused by a streptococcal infection.

10. The use of claim 9, wherein the infection is caused by Streptococcus pneumoniae (Streptococcus pneumoniae).

11. A pharmaceutical composition comprising a therapeutically effective amount of a dimeric lysin of any of claims 1-5, and a pharmaceutically acceptable carrier.

12. An anti-microbial composition for cleaning or decontaminating porous or non-porous surfaces, comprising a dimeric lysin according to any one of claims 1-5.

13. A method of decontaminating an inanimate surface suspected of containing infectious bacteria, comprising treating the surface with a bactericidally or fungistatically effective amount of the composition of claim 12.