# Patent

## Title
Anti-staphylococcal antibodies

## Abstract

This disclosure generally relates to antibodies or fragments thereof which interact with the bacterial protein MprF. The disclosure further discloses antibodies, which bind to specific extracellular motifs of MprF. The disclosure further relates to therapeutics comprising MprF-specific antibodies and methods of treatment using MprF-specific antibodies or fragments thereof.

## Background
CROSS REFERENCE
This application is the U.S. National Phase of PCT/EP2013/077633, filed Dec. 20, 2013, which claims benefit of the U.S. provisional application Ser. No. 61/739,759 filed Dec. 20, 2012 and of the U.S. provisional application Ser. No. 61/775,716 filed Mar. 11, 2013, which both are incorporated by reference in its entirety.


BACKGROUND OF THE INVENTION
This disclosure generally relates to antibodies or fragments thereof which interact with the bacterial protein MprF. In particular antibodies or fragments are disclosed, which bind to specific extracellular motifs of MprF. The disclosure further relates to therapeutics comprising MprF-specific antibodies and methods of treatment using MprF-specific antibodies or fragments thereof.
BACKGROUND OF THE INVENTION
Staphylococcus aureus (S. aureus) is a facultative anaerobic, gram positive, spherical bacterium considered to be an opportunistic pathogen. S. aureus commonly colonizes the nose, skin and mucosal surfaces of healthy humans. Approximately 20-30% of the population is colonized with S. aureus at any given time. These bacteria often cause minor infections, such as pimples and boils in healthy individuals. Normally, mucosal and epidermal barriers (skin) protect against S. aureus infections. Interruption of these natural barriers as a result of injuries—such as burns, trauma or surgical procedures—dramatically increases the risk of infection and could cause severe and/or systemic infections. Furthermore also diseases that compromise the immune system (e.g., diabetes, end-stage renal disease, cancer, AIDS and other viral infections), but also immunosuppressive therapies—e.g. as radiation, chemotherapeutic and transplantation therapies—increase the risk of infection. Opportunistic S. aureus infections can become quite serious, causing endocarditis, bacteremia, osteomyelitis and abscess formation, which might result in severe morbidity or mortality.
S. aureus is a leading cause of bloodstream, skin, soft tissue, and lower respiratory tract infections worldwide. The frequencies of both nosocomial and community-acquired infections have increased steadily over the years. During a localized infection such as pneumonia in humans, approx. 40% of patients with S. aureus pneumonia develop blood stream infections and disseminated disease. The dissemination of the bacterial infection can lead to blood stream infection and distant organ seeding. The blood stream infection can lead to septicemia, a rapidly progressing and frequently fatal complication of S. aureus infections.
In addition, treatment of these infections has become more challenging due to the emergence of multi-drug resistant strains. In almost all developed countries, resistance to antibiotics in methicillin-resistant S. aureus strains (MRSA) is a major problem in hospitals and other healthcare settings and although data on structure and function of S. aureus proteins became more comprehensive the development of an effective vaccine remains a challenge. Notably, the incidence rate of all invasive MRSA infections, including those outside of hospitals, in comparison to other bacterial pathogens is quite high and 20% of these infections result in death. In addition the occurrence of acquired resistance to vancomycin further limited the options for treatment of severe S. aureus infections.
MprF was identified as the enzyme catalyzing Lysyl-phosphatidylglycerol (Lys-PG) biosynthesis in Staphylococcus aureus which is a major component of the bacterial membrane (Peschel et al. 2001). MprF does not only synthesize Lys-PG but also accomplishes the translocation of Lys-PG from the inner to the outer leaflet of the membrane or lipid bilayer resulting in a reduced negative charge of the membrane surface. The mortality of mice infected with a S. aureus mutant strain with an inactivated MprF gene was found to be significantly lower than that of mice infected with the wildtype strain (Peschel et al, 2001). In addition the mutant strain was cleared more efficiently from the bloodstream and showed impaired capacity to proliferate within cardiac vegetations compared to the wildtype in a model of endovascular infection of rabbits (Weidenmaier et al., 2005). Together these findings strongly support the hypothesis of a role of MprF in Staphylococcal virulence (Peschel et al., 2001).
Furthermore, in S. aureus Lys-PG and MprF also affect the susceptibility to cationic antibiotics, e.g. gentamycin (Nishi et al., 2004), and daptomycin (Ernst et al., 2009). The lipopeptide daptomycin is an approved last-resort antibiotic for the treatment of methicillin- and vancomycin resistant S. aureus. An analysis of daptomycin non-susceptible clinical strains revealed that many of them harboured point mutations in the mprF gene, which were interpreted as gain-of-function mutations (Jones et al., 2008, Peleg et al., 2012).
MprF is highly conserved within laboratory and clinically relevant strains of the species Staphylococcus aureus (FIGS. 12A and 12B). The importance of Lys-PG in resistance to antimicrobial peptides has also been demonstrated in other bacterial species. A mutant of Mycobacterium tuberculosis defective in Lys-PG production showed increased sensitivity to vancomycin and defective growth in mouse and guinea pig lungs and reduced pathology relative to wild type (Maloney et al., 2009). Deletion of an mprF-homologue in Listeria monocytogenes resulted in a strain less resistant to specific antimicrobial peptides, with reduced ability to infect macrophages and epithelial cells and the deletion mutant was attenuated in a mouse infection model (Thedieck et al., 2006). Recently a publication described that the expression of an MprF-homologue from Clostridium perfringens in a S. aureus mprF-deletion mutant restores the resistance towards daptomycin indicating a role of C. perfringens MprF in resistance to antimicrobial peptides (Slavetinsky et al., 2012).
Thus, an object of the invention is the provision of products and methods for prophylaxis and therapy of clinically complex S. aureus infection. In particular the present disclosure provides antibodies or fragments specific to MprF from S. aureus, wherein the antibody has protective capacity in vivo, against clinically complex S. aureus infection. Furthermore, the present disclosure provides antibodies or fragments specific for MprF which enhance the susceptibility of S. aureus to antimicrobial peptides and antibiotics which interfere with the bacterial membrane. Furthermore, the present disclosure provides a combinatorial therapy comprising MprF antibodies or fragments together with cationic antimicrobial peptides (CAMP) or CAMP-like antibiotics, e.g. daptomycin, for the treatment of bacterial infections.
SUMMARY OF THE INVENTION
The applicant for the first time discloses antibodies or antibody fragments which specifically bind to MprF. MprF antibodies or antibody fragments interfering with the translocation of lysyl-phosphatidyglycerol provide a promising approach to enhance susceptibility of multi-resistant S. aureus to antimicrobial peptides and antibiotics, like e.g. daptomycin. Additionally, since the structure of specific extracellular loops of MprF are highly conserved not only within S. aureus strains but also within MprF orthologues from other gram-positive or gram-negative bacteria the present disclosure provides a therapeutic approach bearing high potential in broad infectious disease treatments.
The antibodies were identified upon selection strategies using recombinant cyclic and linear peptides mimicking the extracellular loops of MprF. Based on ELISA screening binding of specific antibodies to each of the peptides were detected. Identified clones were converted into IgG format and expressed in eukaryotic cells. After purification further characterization of the selected antibodies confirmed binding to S. aureus in a whole cell ELISA approach. Additionally, further functional analyses were performed to demonstrate functional activity of the selected antibodies. Consequently, an activity and efficacy of MprF-specific antibodies or antibody fragments is predicted in human in treatment of S. aureus infection, specifically of infection caused by antibiotic resistant S. aureus strains.



FIGURE LEGENDS
FIG. 1: Membrane topology of MprF of Staphylococcus aureus showing Peptide mprf1 (SEQ ID NO:2), Peptide mprf2 (SEQ ID NO:3), Peptide mprf3 (SEQ ID NO:4), Peptide mprf4 (SEQ ID NO:5), Peptide mprf5 (SEQ ID NO:6), Peptide mprf6 (SEQ ID NO:7).
FIG. 2: Specific binding of selected IgGs on linear or cyclic peptide was analyzed by ELISA according to Example 3. Respective biotinylated linear peptide representing loop 1 and cyclic peptide representing loop 1 were incubated with IgGs (M-L1, M-C1) in PBS. M-L1 was shown to specifically bind to linear loop 1 peptide in a dose-dependent manner. M-C1 was shown to specifically bind to cyclic loop 1 peptide in a dose-dependent manner.
FIG. 3: Specific binding of selected IgGs on linear or cyclic peptide was analyzed by ELISA according to Example 3. Respective biotinylated linear peptide representing loop 4 and cyclic peptide representing loop 4 were incubated with IgGs (M-L4, M-C4.1, M-C4.2 and M-C4.3) in PBS. M-L4 was shown to specifically bind to linear loop 4 peptide in a dose-dependent manner. M-C4.1, M-C4.2 and M-C4.3 were shown to specifically bind to cyclic loop 4 peptide in a dose-dependent manner.
FIG. 4: Specific binding of selected IgGs on linear or cyclic peptide was analyzed by ELISA according to Example 3. Respective biotinylated cyclic peptides representing loop 5 or loop 6 were incubated with IgGs (M-C5.1, M-C5.2, M-C6.1 and M-C6.2) in PBS. M-C5.1 and M-C5.2, were shown to specifically bind to cyclic loop 5 peptide in a dose-dependent manner. M-C6.1 and M-C6.2 were shown to specifically bind to cyclic loop 6 peptide in a dose-dependent manner.
FIG. 5: Specific binding of selected IgGs on S. aureus was analyzed by ELISA according to Example 4. Representative antibodies M-L1, M-C4.1 and M-C5.1 proved to detect MprF on S. aureus and showed significantly weaker binding to S. aureus mutants lacking MprF expression (delta spa delta mprF).
FIG. 6: Killing of Staphylococcus aureus by nisin was analyzed in the presence and absence of MprF antibodies. An anti-lysozyme antibody was used as a negative control. Antibodies of the present invention increased the sensitivity of Staphylococcus aureus towards nisin.
FIG. 7: Growth of Staphylococcus aureus by nisin was analyzed in the presence and absence of MprF antibodies. An anti-lysozyme antibody was used as a negative control. Antibodies of the present invention increased the sensitivity of Staphylococcus aureus towards nisin and reduced growths of bacteria.
FIG. 8: Killing of Staphylococcus aureus by daptomycin was analyzed in the presence and absence of the MprF antibody M-C4.1. An anti-lysozyme antibody was used as a negative control. M-C4.1 increased the sensitivity of Staphylococcus aureus towards daptomycin.
FIG. 9: Killing of Staphylococcus aureus by LL-37 was analyzed in the presence and absence of the MprF antibody M-C4.1. An anti-lysozyme antibody was used as a negative control. M-C4.1 increased the sensitivity of Staphylococcus aureus towards LL-37.
FIG. 10: The repulsion of positively charged cytochrome C was analyzed in the presence and absence of the MprF antibody M-C4.1. Pre-incubation of M-C4.1 with S. aureus SA113 Δspa led to 40% decreased repulsion of cytochrome C, compared to pre-incubation with anti-lysozyme antibodies, indicating that M-C4.1 impairs the flippase reaction of MprF. The S. aureus SA113 spa-mprF double deletion mutant treated with either M-C4.1 or anti-lysozyme served as negative controls and were significantly impaired in their capacity to repulse cytochrome C (55-75% reduced repulsion).
FIG. 11: Positively charged Annexin V binding to S. aureus was analyzed in the presence and absence of the MprF antibody M-C4.1. Cells were grown in the presence of M-C4.1 or anti-lysozyme antibody and then treated with annexin V. Cells pre-treated with M-C4.1 bound significantly more annexin V (70% increased binding) indicating that the pre-treatment with M-C4.1 had led to impaired flipping of lysyl-phosphatidylglycerol to the outer leaflet of the membrane.
FIGS. 12A and 12B: Sequence alignment of polypeptide sequences of MprF variants from specific S. aureus strains (MRSA252, SEQ ID NO.:114; MSSA479, SEQ ID NO.:115; MW2, SEQ ID NO.:116; N315, SEQ ID NO.:117, NCTC8325, SEQ ID NO.:1 and USA300, SEQ ID NO.:118). Extracellular loops 1-6 are underlined and bold.
FIG. 13: Killing of Staphylococcus aureus clinical isolate 703 by daptomycin was analyzed in the presence and absence of the MprF antibody M-C4.1. An anti-lysozyme antibody was used as a negative control. While in the presence of the anti-lysozyme antibody, daptomycin had no effect on S. aureus 703, the application of the MprF specific antibody M-C4.1 restored susceptibility of S. aureus 703 bacteria to daptomycin and bacterial killing was induced.
FIG. 14: Killing of Staphylococcus aureus by nisin was analyzed in the presence and absence of MprF antibodies. An anti-lysozyme antibody was used as a negative control. Antibodies of the present invention increased the sensitivity of the clinical isolate Staphylococcus aureus strain USA300 towards nisin.
FIG. 15: Specific binding of selected IgGs on linear or cyclic peptide was analyzed by ELISA according to Example 3. Respective biotinylated cyclic peptide representing loop 4 was incubated with IgGs (M-C4.4, M-C4.5) in PBS. Both antibodies were shown to specifically bind to cyclic loop 4 peptide in a dose-dependent manner.
FIG. 16: Killing of Staphylococcus aureus by daptomycin was analyzed in the presence and absence of MprF antibodies. An anti-lysozyme antibody was used as a negative control antibody. Antibodies of the present invention increased the sensitivity of the clinical isolate Staphylococcus aureus strain USA300 towards daptomycin.



DETAILED DESCRIPTION
The MprF protein of Staphylococcus aureus has a length of 840 amino acids. The amino acid sequence is shown in SEQ ID No.: 1.
SEQ ID NO.:1 (MprF of Staphylococcus aureus):







MNQEVKNKIFSILKITFATALFIFVAITLYRELSGINFKDTLVEFSKINR


 


MSLVLLFIGGGASLVILSMYDVILSRALKMDISLGKVLRVSYIINALNAI


 


VGFGGFIGAGVRAMVYKNYTHDKKKLVHFISLILISMLTGLSLLSLLIVF


 


HVFDASLILDKITWVRWVLYVVSFFLPLFIlYSMVRPPDKNNRFVGLYCT


 


LVSCVEWLAAAVVLYFCGVIVDAHVSFMSFIAIFIIAALSGLVSFIPGGF


 


GAFDLVVLLGFKTLGVPEEKVLLMLLLYRFAYYFVPVIIALILSSFEFGT


 


SAKKYIEGSKYFIPAKDVTSFLMSYQKDIIAKIPSLSLAILVFFTSMIFF


 


VNNLTIVYDALYDGNHLTYYILLAIHTSACLLLLLNVVGIYKQSRRAIIF


 


AMISILLITVATFFTYASYILITWLAIIFVLLIVAFRRARRLKRPVRMRN


 


IVAMLLFSLFILYVNHIFIAGTLYALDIYTIEMHTSVLRYYFWLTILIIA


 


IIIGMIAWLFDYQFSKVRISSKIEDCEEIINQYGGNYLSHLIYSGDKQFF


 


TNENKTAFLMYRYKASSLVVLGDPLGDENAFDELLEAFYNYAEYLGYDVI


 


FYQVTDQHMPLYHNFGNQFFKLGEEAIIDLTQFSTSGKKRRGFRATLNKF


 


DELNISFEIIEPPFSTEFINELQHVSDLWLDNRQEMHFSVGEFNEEYLSK


 


APIGVMRNEENEVIAFCSLMPTYFNDAISVDLIRWLPELDLPLMDGLYLH


 


MLLWSKEQGYTKFNMGMATLSNVGQLHYSYLRERLAGRVFEHFNGLYRFQ


 


GLRRYKSKYNPNWEPRFLVYRKDNSLWESLSKVMRVIRHK






The membrane topology of MprF protein of Staphylococcus aureus is shown in FIG. 1. The extracellular loops of MprF are characterized by the following sequences:









TABLE 1







Loop 1
ELSGINFKDTLVEFSKINR
(SEQ ID NO.: 2)


 


Loop 2
YKNYTHDKKKLVHF
(SEQ ID NO.: 3)


 


Loop 3
SMVRPPDKNNRFVG
(SEQ ID NO.: 4)


 


Loop 4
LGFKTLGVPEEKV
(SEQ ID NO.: 5)


 


Loop 5
DALYDGNHLT
(SEQ ID NO.: 6)


 


Loop 6
DIYTIEMHTSVLR
(SEQ ID NO.: 7)









Accordingly, in one aspect the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1, or an orthologue thereof. In another embodiment said antibody or antibody fragment is specific for MprF, or an orthologue thereof. In one embodiment the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1, or an orthologue thereof wherein said antibody or antibody fragment increases the susceptibility of a pathogen to a cationic antimicrobial peptide. In one embodiment the antibody or antibody fragment is specific for a polypeptide comprising SEQ ID NO.: 1 or an orthologue thereof, induces the susceptibility of a pathogen to a cationic antimicrobial peptide.
In another aspect the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1, or an orthologue thereof, wherein said antibody or antibody fragment inhibits the flippase activity of a polypeptide comprising SEQ ID NO.: 1, or an orthologue thereof. In one embodiment the disclosure pertains to an antibody or antibody fragment specific for MprF, or an orthologue thereof, wherein said antibody or antibody fragment inhibits the flippase activity of MprF. In another embodiment said antibody or antibody fragment inhibits the flippase activity of MprF and decreases the repulsion of cytochrome C from the membrane of bacteria. In another embodiment the repulsion of cytochrome C from the membrane of bacteria is decreased by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90%. In another embodiment the repulsion of cytochrome C from the membrane of bacteria is decreased by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% in comparison to a control antibody. In another embodiment the repulsion of cytochrome C from the membrane of bacteria is decreased by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% in an cytochrome C repulsion assay as described in Example 8. In a further embodiment the bacteria is S. aureus. In a further embodiment the bacteria is S. aureus SA113 Δspa.
In another embodiment said antibody or antibody fragment inhibits the flippase activity of MprF and increases the binding of annexin V to the membrane of bacteria. In another embodiment the binding of annexin V to the membrane of bacteria is increased by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90%. In another embodiment the binding of annexin V to the membrane of bacteria is increased by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% in comparison to a control antibody. In another embodiment the binding of annexin V to the membrane of bacteria is increased by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% in an annexin V binding assay as described in Example 8. In a further embodiment the bacteria is S. aureus. In a further embodiment the bacteria is S. aureus SA113 Δspa.
In another aspect, the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1, or an orthologue thereof, wherein said antibody or antibody fragment binds to a polypeptide comprising SEQ ID NO.: 1, or an orthologue thereof, with a dissociation constant (KD) of less than 1×107 M−1, 108 M−1, 109 M−1, 1010 M−1, 1011 M−1, 1012 M−1 or 1013 M−1.
In one aspect, the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1. In another aspect, the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1, or an orthologue thereof, wherein said antibody or antibody fragment is an isolated antibody or antibody fragment. In one embodiment said antibody or antibody fragment is a monoclonal or polyclonal. In one embodiment said antibody or antibody fragment is human or humanized. In one embodiment said antibody or an antibody fragment is a chimeric antibody or antibody fragment. In one embodiment said antibody or antibody fragment comprises a human heavy chain constant region and a human light chain constant region. In one embodiment said antibody or antibody fragment is an IgG isotype. In another embodiment the antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or derivative thereof (e.g. IgG1 LALA). In one embodiment the antibodies are of IgG1 LALA isotype. In one embodiment said antibody fragment is an antigen binding fragment. In another embodiment said antibody or antibody fragment or antigen binding fragment is selected from the group consisting of a Fab, F(ab2)′, F(ab)2′ and scFV. In one embodiment the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, and a synthetic antibody. In one embodiment, the antibody or antibody fragment is a human or humanized antibody. In another embodiment, the antibody or antibody fragment is a human or humanized monoclonal antibody.
In one embodiment, the antibody or antibody fragment is a single chain antibody. In one embodiment, the antibody or antibody fragment is bispecific. In one embodiment the antibody or antibody fragment is a bispecific antibody-derived scaffold wherein said bispecific antibody-derived scaffold is selected from the group consisting of a bispecific-scFv, a tetravalent bispecific antibody, a cross-linked Fab or a bispecific IgG.
In one aspect, the disclosure pertains to an antibody or antibody fragment, wherein the antibody or antibody fragment is selected from the group consisting of single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, camelid antibodies, ankyrins, domain antibodies, lipocalins, small modular immuno-pharmaceuticals, maxybodies, Protein A and affilins.
In another aspect the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1 or an orthologue thereof, wherein said antibody or antibody fragment increases the susceptibility of a gram-positive bacterium to cationic antimicrobial peptides. In one embodiment the susceptibility of a gram-positive bacterium to cationic antimicrobial peptides is induced. In another embodiment the susceptibility of a gram-positive bacterium to cationic antimicrobial peptides is enhanced. In a preferred embodiment the gram-positive bacterium is Staphylococcus aureus. 
In another aspect the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1 or an orthologue thereof, wherein said antibody or antibody fragment increases the susceptibility of a gram-positive bacterium to a cationic antimicrobial peptide and wherein said cationic antimicrobial peptide is a lipopeptide. In a preferred embodiment, the lipopeptide interacts with membranes via their lipid tails. In a more preferred embodiment, the lipopeptide has an antimicrobial activity. In a preferred embodiment said lipopeptide is a cationic amphiphilic peptide with an acetylated N-terminus. In another embodiment said acylated N-terminus comprises a chain of at least 8 fatty acids, at least 9 fatty acids, at least 10 fatty acids, at least 11 fatty acids, at least 12 fatty acids, at least 13 fatty acids, at least 14 fatty acids, at least 15 fatty acids, at least 16 fatty acids, at least 17 fatty acids, at least 18 fatty acids.
In another aspect the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1 or an orthologue thereof, wherein said antibody or antibody fragment increases the susceptibility of a gram-positive bacterium to a cationic antimicrobial peptide and wherein said cationic antimicrobial peptide is a lipopeptide which is a member of the polymyxin family.
In another aspect the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1 or an orthologue thereof, wherein said antibody or antibody fragment increases the susceptibility of a gram-positive bacterium to a cationic antimicrobial peptide and wherein said cationic antimicrobial peptide is an amphipathic antimicrobial peptide. In another embodiment the cationic antimicrobial peptide is an alpha-helical or beta-helical peptide. In a further embodiment the cationic antimicrobial peptide is a cathelicidin. In one embodiment the cathelicidin is LL-37.
In another aspect the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1 or an orthologue thereof, wherein said antibody or antibody fragment increases the susceptibility of a gram-positive bacterium to an antimicrobial peptide and wherein said antimicrobial peptide is a lipopeptide and wherein said lipopeptide is a cyclic lipopeptide. In a preferred embodiment said cyclic lipopeptide is a daptomycin-related lipopeptide. In a more preferred embodiment said daptomycin-related lipopeptide is A54145 or an A-21978C cyclic peptide in which the n-decanoyl fatty acid side chain of daptomycin is replaced by an n-heptanoyl, n-octanoyl, n-nonanoyl, n-undecanoyl, n-lauroyl, n-dodecanoyl, n-tridecanoyl, n-myristoyl, n-pentadecanoyl, 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl or n-tetradecanoyl fatty. In a more preferred embodiment, the cyclic lipopeptide is daptomycin (LY 146032).
In another aspect the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1 or an orthologue thereof, wherein said antibody or antibody fragment increases the susceptibility of a gram-positive bacterium to an antimicrobial peptide and wherein said antimicrobial peptide is a lantiobiotic. In another embodiment said lantibiotic is nisin.
In another aspect the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1 or an orthologue thereof, wherein said antibody or antibody fragment binds to an extracellular loop of MprF. In one embodiment said antibody or antibody fragment binds to an extracellular loop of MprF wherein said loop comprises an amino acid sequence of ELSGINFKDTLVEFSKINR (SEQ ID NO.: 2), YKNYTHDKKKLVHF (SEQ ID NO.: 3), SMVRPPDKNNRFVG (SEQ ID NO.: 4), LGFKTLGVPEEKV (SEQ ID NO.: 5), DALYDGNHLT (SEQ ID NO.: 6) or DIYTIEMHTSVLR (SEQ ID NO.: 7). In one embodiment said antibody or antibody fragment binds to an extracellular loop of MprF wherein said loop comprises the amino acid sequence ELSGINFKDTLVEFSKINR (SEQ ID NO.: 2). In one embodiment said antibody or antibody fragment binds to an extracellular loop of MprF wherein said loop comprises the amino acid sequence DALYDGNHLT (SEQ ID NO.: 6). In one embodiment said antibody or antibody fragment binds to an extracellular loop of MprF wherein said loop comprises the amino acid sequence DIYTIEMHTSVLR (SEQ ID NO.: 7). In a more preferred embodiment said antibody or antibody fragment binds to an extracellular loop of MprF wherein said loop comprises the amino acid sequence LGFKTLGVPEEKV (SEQ ID NO.: 5).
In another aspect the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising ELSGINFKDTLVEFSKINR (SEQ ID NO.: 2), YKNYTHDKKKLVHF (SEQ ID NO.: 3), SMVRPPDKNNRFVG (SEQ ID NO.: 4), LGFKTLGVPEEKV (SEQ ID NO.: 5), DALYDGNHLT (SEQ ID NO.: 6) or DIYTIEMHTSVLR (SEQ ID NO.: 7). In one embodiment the peptides are isolated polypeptides peptides. In another aspect the disclosure pertains to an antibody or antibody fragment specific for one of the peptides of ELSGINFKDTLVEFSKINR (SEQ ID NO.: 2), YKNYTHDKKKLVHF (SEQ ID NO.: 3), SMVRPPDKNNRFVG (SEQ ID NO.: 4), LGFKTLGVPEEKV (SEQ ID NO.: 5), DALYDGNHLT (SEQ ID NO.: 6) or DIYTIEMHTSVLR (SEQ ID NO.: 7). In one embodiment the peptides are isolated peptides.
In another aspect the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising CELSGINFKDTLVEFSKINR (SEQ ID NO.: 108), CYKNYTHDKKKLVHF (SEQ ID NO.: 109), CSMVRPPDKNNRFVG (SEQ ID NO.: 110), CGLGFKTLGVPEEKV (SEQ ID NO.: 111), CGGDALYDGNHLT (SEQ ID NO.: 112) or CDIYTIEMHTSVLR (SEQ ID NO.: 113). In one embodiment the peptides are isolated polypeptides peptides. In another aspect the disclosure pertains to an antibody or antibody fragment specific for one of the peptides of CELSGINFKDTLVEFSKINR (SEQ ID NO.: 108), CYKNYTHDKKKLVHF (SEQ ID NO.: 109), CSMVRPPDKNNRFVG (SEQ ID NO.: 110), CGLGFKTLGVPEEKV (SEQ ID NO.: 111), CGGDALYDGNHLT (SEQ ID NO.: 112) or CDIYTIEMHTSVLR (SEQ ID NO.: 113). In one embodiment the peptides are isolated peptides. In another embodiment the peptides are linear peptides. In a further embodiment the peptides are cyclic peptides.
In another aspect the disclosure pertains to a kit comprising an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1, or an orthologue thereof, and a cationic antimicrobial peptide. In another aspect the disclosure pertains to a kit comprising an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1, or an orthologue thereof, wherein said antibody or antibody fragment increases the susceptibility of a pathogen to a cationic antimicrobial peptide and a cationic antimicrobial peptide. In one embodiment the disclosure pertains to a kit comprising an antibody or antibody fragment specific for a polypeptide comprising ELSGINFKDTLVEFSKINR (SEQ ID NO.: 2), YKNYTHDKKKLVHF (SEQ ID NO.: 3), SMVRPPDKNNRFVG (SEQ ID NO.: 4), LGFKTLGVPEEKV (SEQ ID NO.: 5), DALYDGNHLT (SEQ ID NO.: 6) or DIYTIEMHTSVLR (SEQ ID NO.: 7) and a cationic antimicrobial peptide. In one embodiment the disclosure pertains to a kit comprising an antibody or antibody fragment specific for one of peptides of ELSGINFKDTLVEFSKINR (SEQ ID NO.: 2), YKNYTHDKKKLVHF (SEQ ID NO.: 3), SMVRPPDKNNRFVG (SEQ ID NO.: 4), LGFKTLGVPEEKV (SEQ ID NO.: 5), DALYDGNHLT (SEQ ID NO.: 6) or DIYTIEMHTSVLR (SEQ ID NO.: 7) and a cationic antimicrobial peptide. In one embodiment the disclosure pertains to a kit comprising an antibody or antibody fragment specific for one of peptides of CELSGINFKDTLVEFSKINR (SEQ ID NO.: 108), CYKNYTHDKKKLVHF (SEQ ID NO.: 109), CSMVRPPDKNNRFVG (SEQ ID NO.: 110), CGLGFKTLGVPEEKV (SEQ ID NO.: 111), CGGDALYDGNHLT (SEQ ID NO.: 112) or CDIYTIEMHTSVLR (SEQ ID NO.: 113).
In another aspect the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1, or an orthologue thereof, for use in medicine wherein said antibody or antibody fragment increases the susceptibility of a pathogen to a cationic antimicrobial peptide. In one embodiment the disclosure pertains to the use of an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1, or an orthologue thereof, in the treatment of an infectious disease, wherein said antibody or antibody fragment increases the susceptibility of a pathogen to a cationic antimicrobial peptide. In another embodiment the infectious diseases is an infection with gram-positive or gram-negative bacteria. In a preferred embodiment said infectious disease is an infection with Staphylococcus aureus. In another aspect the disclosure pertains to a pharmaceutical composition comprising an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1, or an orthologue thereof, for use in medicine wherein said antibody or antibody fragment increases the susceptibility of a pathogen to a cationic antimicrobial peptide.
In another aspect the disclosure pertains to a combination comprising an antibody or antibody fragment specific for a polypeptides comprising SEQ ID NO.: 1, or an orthologue thereof, and a cationic antimicrobial peptide for use in medicine. In another aspect the disclosure pertains to a synergistic combination comprising an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1, or an orthologue thereof, and a cationic antimicrobial peptide for use in medicine. In one embodiment the combination is used in the treatment of an infectious disease. In one embodiment the combination is used in the treatment of an infectious disease with gram-positive or gram-negative bacteria. In a preferred embodiment said infectious disease is an infection with Staphylococcus aureus. In another embodiment the combination is used in the treatment of an infectious disease with antibiotic resistant bacteria. In another embodiment said antibiotic resistant bacteria is resistant to beta-lactam antibiotics. In another embodiment said antibiotic resistant bacteria is a Methicillin-resistant Staphylococcus aureus. 
In another aspect the disclosure pertains to a pharmaceutical composition comprising an antibody or antibody fragment specific for a polypeptides comprising SEQ ID NO.: 1, or an orthologue thereof, and a cationic antimicrobial peptide for use in medicine.
In another aspect the disclosure pertains to a method of treating a bacterial infection in an individual in need thereof, which method comprises administration of an antibody or antibody fragment specific for a polypeptides comprising SEQ ID NO.: 1, or an orthologue thereof. In another embodiment the disclosure pertains to a method of treating a bacterial infection in an individual in need thereof, which method comprises administration of an antibody or antibody fragment specific for a polypeptides comprising SEQ ID NO.: 1, or an orthologue thereof and a cationic antimicrobial peptide. In another embodiment the disclosure pertains to a method of treating a bacterial infection in an individual in need thereof, which method comprises administration of a pharmaceutical composition comprising an antibody or antibody fragment specific for a polypeptides comprising SEQ ID NO.: 1, or an orthologue thereof and a cationic antimicrobial peptide.
In certain aspects of the present invention the antibody of the synergistic combination of the present invention is an antibody specific for a polypeptides comprising SEQ ID NO.: 1. In other aspects the antibody of the synergistic combination of the present invention is an antibody specific for a peptide of SEQ ID NO.: 2, an antibody specific for a peptide of SEQ ID NO.: 3, an antibody specific for a peptide of SEQ ID NO.: 4, an antibody specific for a peptide of SEQ ID NO.: 5, an antibody specific for a peptide of SEQ ID NO.: 6, or an antibody specific for a peptide of SEQ ID NO.: 7. In most preferred aspects, the antibody of the synergistic combination of the present invention is an antibody specific for a peptide consisting of any one of SEQ ID NO's: 5, 6 or 7.
In another aspect the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1 or an orthologue thereof, wherein said antibody or antibody fragment increases bacterial killing of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% in the presence of a cationic antimicrobial peptide.
In another aspect the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1 or an orthologue thereof, wherein said antibody or antibody fragment increases bacterial killing of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% in the presence of a cationic antimicrobial peptide in an experimental setting as described in Example 5, Example 6 or Example 7 of the present disclosure.
In other aspects of the present invention the antibodies of the present invention are specific for an orthologue of the polypeptides of SEQ ID NO.:1. MprF homologues can be found in most bacterial kingdoms and antibodies against these orthologues are contemplated in the present invention. In certain embodiments the orthologues have a sequence identity of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% to the polypeptides of SEQ ID NO.:1.
In another aspect the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1 or an orthologue thereof, comprising 6 CDRs defined by Kabat of any of the antibodies in Table 3.
In another aspect the disclosure pertains to an antibody or antibody fragment specific for a polypeptide comprising SEQ ID NO.: 1 or an orthologue thereof, that cross-competes with an antibody described in Table 3.
In a certain embodiment, the antibody that cross-competes with an antibody described in Table 3 reduces the binding of one of the antibodies described in Table 3 to a polypeptide comprising SEQ ID NO.: 1 or an orthologue thereof, by at least 50%, 60%, 70%, 80% or 90% in an ELISA-based cross-competition assay.
In a certain embodiment, the antibody that cross-competes with an antibody described in Table 3 reduces the binding of one of the antibodies described in Table 3 to a polypeptide comprising SEQ ID NO.: 1 or an orthologue thereof, by at least 50%, 60%, 70%, 80% or 90% in an ELISA-based cross-competition assay according to Example 9 in comparison to the positive control.
In a certain embodiment, the antibody that cross-competes with an antibody described in Table 3 reduces the binding of one of the antibodies described in Table 3 to one of the peptides of ELSGINFKDTLVEFSKINR (SEQ ID NO.: 2), YKNYTHDKKKLVHF (SEQ ID NO.: 3), SMVRPPDKNNRFVG (SEQ ID NO.: 4), LGFKTLGVPEEKV (SEQ ID NO.: 5), DALYDGNHLT (SEQ ID NO.: 6) or DIYTIEMHTSVLR (SEQ ID NO.: 7) by at least 50%, 60%, 70%, 80% or 90% in an ELISA-based cross-competition assay.
In a certain embodiment, the antibody that cross-competes with an antibody described in Table 3 reduces the binding of one of the antibodies described in Table 3 to one of peptides of ELSGINFKDTLVEFSKINR (SEQ ID NO.: 2), YKNYTHDKKKLVHF (SEQ ID NO.: 3), SMVRPPDKNNRFVG (SEQ ID NO.: 4), LGFKTLGVPEEKV (SEQ ID NO.: 5), DALYDGNHLT (SEQ ID NO.: 6) or DIYTIEMHTSVLR (SEQ ID NO.: 7) by at least 50%, 60%, 70%, 80% or 90% in an ELISA-based cross-competition assay according to Example 9 in comparison to the positive control.
In another aspect, the disclosure pertains to an antibody or fragment thereof specific for a polypeptide comprising SEQ ID NO.: 1 or an orthologue thereof, and interacts with (e.g., by binding, stabilizing, spatial distribution) the same epitope as an antibody described in Table 3.
In a certain embodiment, the antibody or fragment thereof specific for a polypeptide comprising SEQ ID NO.: 1 or an orthologue thereof, binds to the same epitope as an antibody described in Table 3, wherein said epitope is an extracellular loop of MprF. In a certain embodiment, the antibody or fragment thereof specific for a polypeptide comprising SEQ ID NO.: 1 or an orthologue thereof, binds to the same epitope as an antibody described in Table 3, wherein said epitope is an extracellular loop of MprF and wherein said extracellular loop comprises an amino acid sequence of ELSGINFKDTLVEFSKINR (SEQ ID NO.: 2), YKNYTHDKKKLVHF (SEQ ID NO.: 3), SMVRPPDKNNRFVG (SEQ ID NO.: 4), LGFKTLGVPEEKV (SEQ ID NO.: 5), DALYDGNHLT (SEQ ID NO.: 6) or DIYTIEMHTSVLR (SEQ ID NO.: 7). In another embodiment said antibody or fragment thereof is a human monoclonal antibody. Such human monoclonal antibodies can be prepared and isolated as described herein.
Definitions
The term “antibody” as used herein includes whole antibodies. A naturally occurring “antibody” is a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised specific CH domains (e.g. CH1, CH2 and CH3). Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementary determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), subclass or modified version thereof (e.g. IgG1 LALA). The antibodies can be of any species, chimeric, humanized or human.
The terms “heavy chain variable region CDR1” and “H-CDR1” are used interchangeably, as are the terms “heavy chain variable region CDR2” and “H-CDR2”, the terms “heavy chain variable region CDR3” and “H-CDR3”, the terms “light chain variable region CDR1” and “L-CDR1”; the terms “light chain variable region CDR2” and “L-CDR2” and the terms “light chain variable region CDR3” and “L-CDR3” antibody fragment
Antigen binding can be performed by “fragments” or “antigen binding fragments” of an intact antibody. Herein, both terms are used interchangeably. Examples of binding fragments encompassed within the term “antibody fragment” of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and an isolated complementary determining region (CDR).
A “single chain Fragment (scFv)” is a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Although the two domains VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by an artificial peptide linker that enables them to be made as a single protein chain. Such single chain antibodies include one or more antigen binding moieties. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
The term “epitope” includes any proteinaceous region which is specifically recognized by an immunoglobulin or T-cell receptor or otherwise interacts with a molecule. Generally epitopes are of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally may have specific three-dimensional structural characteristics, as well as specific charge characteristics. As will be appreciated by one of skill in the art, practically anything to which an antibody can specifically bind could be an epitope.
The term “cross-competes” refers to antigen binding moieties (such as antibodies) which share the ability to bind to a specific region of an antigen. In the present disclosure an antigen binding moiety that is “cross-competitive” has the ability to interfere with the binding of another antigen binding moiety for MprF in a standard competitive binding assay. Such an antibody may, according to non-limiting theory, bind to the same or a related or nearby (e.g., a structurally similar or spatially proximal) epitope on MprF or an extracellular loop of MprF as the antibody with which it competes. Cross-competition studies to find antibodies that competitively bind with one another, e.g., the antibodies compete for binding to the antigen can be performed. For example the present disclosure provides antibodies that cross-compete with (e.g., by binding, stabilizing, spatial distribution) the antibodies described in Table 3. The ability or extent to which an antibody or other binding agent is able to interfere with the binding of another antibody or binding molecule to MprF or an extracellular loop of MprF and therefore whether it can be said to cross-compete according to the invention, can be determined using standard competition binding assays. Cross-competition is present if antibody A reduces binding of antibody B at least by 50%, at least by 60%, specifically at least by 70% and more specifically at least by 80% and vice versa in comparison to the positive control which lacks one of said antibodies. As the skilled artisan appreciates competition may be assessed in different assay set-ups. One suitable assay involves the use of the Biacore technology (e.g. by using the BIAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the extent of interactions using surface plasmon resonance technology. Another assay for measuring cross-competition uses an ELISA-based approach (e.g. Example 9). Furthermore, a high throughput process for “binning” antibodies based upon their cross-competition is described in International Patent Application No. WO2003/48731. Cross-competition is present if the antibody under investigation reduces the binding of one of the antibodies described in Table 3 by 60% or more, specifically by 70% or more and more specifically by 80% or more and if one of the antibodies described in Table 3 reduces the binding of said antibody to MprF or an extracellular loop of MprF by 60% or more, specifically by 70% or more and more specifically by 80% or more.
The term “human antibody”, as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. As used herein, a human antibody comprises heavy or light chain variable regions or full length heavy or light chains. In certain cases, a human antibody may be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Thereby said human antibody can be obtained from technology platforms which comprise antibodies derived from human germline genes either generated by PCR-amplification of VH/VL repertoire isolated from B-cells or are generated synthetically. Technology platforms include library based approaches comprising human immunoglobulin genes displayed on phage, ribosome or yeast. Respective display technologies are standard in the scientific community. Furthermore immunization of a transgenic mouse carrying human immunoglobulin repertoire is another approach to generate human antibodies against an antigen of interest. Antibodies or fragments thereof selected from an antibody library based on the MorphoSys HuCAL® concept (Knappik et al., (2000) J Mol Biol 296:57-86) are considered as fully human.
The terms “monoclonal antibody” as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a unique binding site having a unique binding specificity and affinity for particular epitopes.
A “humanized” antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts (i.e., the constant region as well as the framework portions of the variable region). See, e.g., Morrison et al (1994) Proc. Natl. Acad. Sci. USA, 81:6851-6855; Morrison and Oi (1988) Adv. Immunol., 44:65-92; Verhoeyen et al. (1988) Science, 239:1534-1536; Padlan, Molec (1991) Immun., 28:489-498; and Padlan, Molec (1994) Immun., 31:169-217. Other examples of human engineering technology include, but are not limited to Xoma technology disclosed in U.S. Pat. No. 5,766,886.
The term “chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity. For example, a mouse antibody can be modified by replacing its constant region with the constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced antigenicity in human as compared to the original mouse antibody.
The term “isolated” refers to a compound which can be e.g. an antibody or an antigen binding moiety that is substantially free of other antibodies or antigen binding moieties having different antigenic specificities. Moreover, an isolated antibody antigen binding moiety may be substantially free of other cellular material and/or chemicals.
The term “isotype” refers to the antibody class (e.g., IgM, IgE, IgG such as IgG1 or IgG4) that is provided by the heavy chain constant region genes. Isotype also includes modified versions of one of these classes, where modifications have been made to alter the Fc function, for example, to enhance or reduce effector functions or binding to Fc receptors. For example IgG1 LALA is a modified version of the IgG isotype having significantly reduced effector functions. Specific substitutions of amino acids reduced the binding affinity for Fc gamma RI receptor as compared with unmodified antibody. IgG1 LALA is described in U.S. Ser. No. 08/479,752 (SCOTGEN BIOPHARMACEUTICALS INC.) which is incorporated by reference in its entirety. In certain embodiments of the present disclosure the antigen-binding moieties of are antibodies and are of the type IgG, IgM, IgA, IGE or IgD. In specific embodiments the antibodies are of the type IgG. In certain embodiments of the present disclosure the antibodies are of the subtype IgG1, IgG2, IgG3 or IgG4. In specific embodiments the antibodies are of the subtype IgG1 or IgG4. In other specific embodiments the antibodies are of the subtype IgG1 or IgG1 LALA.
The term “affinity” as used herein refers to the strength of interaction between an antigen binding moiety, like e.g. a monoclonal antibody and an antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody “arm” interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity.
The term “KD”, as used herein, refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M). KD values for antigen binding moieties like e.g. monoclonal antibodies can be determined using methods well established in the art. Methods for determining the KD of an antigen binding moiety like e.g. a monoclonal antibody are SET (soluble equilibrium titration) or surface plasmon resonance using a biosensor system such as a Biacore® system. Antibodies of the present disclosure typically have a dissociation rate constant (KD) (koff/kon) of less than 5×10−2M, less than 10−2M, less than 5×10−3M, less than 10−3M, less than 5×10−4M, less than 10−4M, less than 5×10−5M, less than 10−5M, less than 5×10−6M, less than 10−6M, less than 5×10−7M, less than 10−7M, less than 5×10−8M, less than 10−8M, less than 5×10−9M, less than 10−9M, less than 5×10−10M, less than 10-10M, less than 5×10−11M, less than 10−11M, less than 5×10−12M, less than 10−12M, less than 5×10−13M, less than 10−13M, less than 5×10−14M, less than 10−14M, less than 5×10−15M, or less than 10−15M or lower.
The terms “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
The term “nucleic acid” is used herein interchangeably with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, as detailed below, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608; and Rossolini et al. (1994) Mol. Cell. Probes 8:91-98).
The term “recombinant host cell” (or simply “host cell”) refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
The term “vector” is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
“Synergy”, “synergism” or “synergistic activity” mean more than the expected additive effect of a combination. The “synergy”, “synergism” or “synergistic activity” of a combination can be determined by different methods of like e.g. Chou et al., Clarke et al., and/or Webb et al. See Ting-Chao Chou, Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in Drug Combination Studies, Pharmacol Rev 58:621-681 (2006), which is incorporated by reference in its entirety. See also Clarke et al., Issues in experimental design and endpoint analysis in the study of experimental cytotoxic agents in vivo in breast cancer and other models, Breast Cancer Research and Treatment 46:255-278 (1997), which is incorporated by reference in its entirety. See also Webb, J. L. (1963) Enzyme and Metabolic Inhibitors, Academic Press, New York, which is incorporated by reference in its entirety.
The term “orthologue” denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologues are the result of speciation. “Orthologues” as used herein encompass polypeptides derived from another origin than the shown origin (SEQ ID NO: 1) which have substantially the same amino acid sequence and substantially the same biological activity as MprF set forth in SEQ ID NO: 1. “Orthologues” as used herein encompass MprF-orthologues derived from gram-negative or gram-positive bacteria. Representative MprF-orthologues are for example polypeptides (FIG. 12) encoded by SEQ ID NO.:114, SEQ ID NO.:115, SEQ ID NO.:116, SEQ ID NO.:117, SEQ ID NO.:118. Respective polypeptides are derived from the strains MSSA479 (a methicillin-sensitive strain, isolated from a patient in the community who developed severe invasive disease), N315 (isolated as an methicillin-resistant S. aureus that was a major pathogen causing hospital-acquired infections in 1982), MRSA252 (representative of the epidemic EMRSA-16 lineage endemic in UK hospitals), MW2 (methicillin resistant, community acquired) and USA300 (methicillin resistant, community acquired (CA-MRSA). USA300 was first identified in 1998, and is thought to be the primary causal strain of community-acquired Staph infections throughout the United States, Canada and Europe. In 2006 the CDC reported that 64% of MRSA isolated from infected patients were of the USA300 strain. USA300 causes an estimated 20 thousands deaths annually in the United States only. Further representative MprF-orthologues can be for example derived from the clinical isolate Staphylococcus aureus 703 (described in Jones et al., AAC 2008).
As used herein, the phrase “pathogen” means an agent which causes a disease state or affliction in an animal. Included within this definition, for examples, are bacteria (gram-negative or gram-positive bacteria), protozoans, fungi, viruses and metazoan parasites which either produce a disease state or render an animal infected with such an organism susceptible to a disease state (e.g., a secondary infection). Further included are species and strains of the genus Staphylococcus which produce disease states in animals. As used herein, the term “organism” means any living biological system, including viruses, regardless of whether it is a pathogenic agent.
The term “Gram-negative bacteria” as used herein refer to bacteria characterized by the presence of a double membrane surrounding each bacterial cell. Representative Gram-negative bacteria include Acinetobacter calcoaceticus, Bacteroides, Bacteroides fragilis, Bartonella bacilliformis, Bordetella spp., Borrelia burgdorferi, Branhamella catarrhalis, Brucella spp., Campylobacter spp., Chalmydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Enterobacter aerogenes, Escherichia coli, Flavobacterium meningosepticum, Fusobacterium spp., Haemophilus influenzae, Haemophilus spp., Helicobacter pylori, Klebsiella spp., Legionella spp., Leptospira spp., Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Plesiomonas shigelloides, Prevotella spp., Proteus spp., Providencia rettgeri, Pseudomonas aeruginosa, Pseudomonas spp., Rickettsia prowazekii, Rickettsia rickettsii, Rochalimaea spp., Salmonella spp., Salmonella typhi, Serratia marcescens, Shigella spp., Treponema carateum, Treponema pallidum, Treponema pallidum endemicum, Treponema pertenue, Veillonella spp., Vibrio cholerae, Vibrio vulnificus, Yersinia enterocolitica and Yersinia pestis. 
The term “Gram-positive bacteria” as used herein refers to bacteria characterized by having a single membrane and teichoic acids or teichoic-like acids as part of their cell wall and are characterized by their blue-violet color reaction in the Gram-staining procedure. Representative Gram-positive bacteria include: Actinomyces spp., Bacillus anthracis, Bifidobacterium spp., Clostridium botulinum, Clostridium perfringens, Clostridium spp., Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium jeikeium, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Eubacterium spp., Gardnerella vaginalis, Gemella morbillorum, Leuconostoc spp., Mycobacterium abcessus, Mycobacterium avium complex, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium haemophilium, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium smegmatis, Mycobacterium terrae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Nocardia spp., Peptococcus niger, Peptostreptococcus spp., Proprionibacterium spp., Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdanensis, Staphylococcus saccharolyticus, Staphylococcus saprophyticus, Staphylococcus schleiferi, Staphylococcus similans, Staphylococcus warneri, Staphylococcus xylosus, Streptococcus agalactiae (group B streptococcus), Streptococcus anginosus, Streptococcus bovis, Streptococcus canis, Streptococcus equi, Streptococcus milleri, Streptococcus mitior, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes (group A streptococcus), Streptococcus salivarius, Streptococcus sanguis. 
As used herein, the term “Staphylococcus” means any species or strain of bacteria which is members of the genus Staphylococcus regardless of whether they are known pathogenic agents.
As used herein, “bacteremia” means the presence of viable bacteria in the blood or organs of an individual (human or other animal). “Bacteremia caused by S. aureus” or “S. aureus bacteremia” refers to bacteremia in which at least some of the bacteria in the blood or organs are S. aureus. Other species of bacteria also may be present.
“Infection” as used herein is an invasion and multiplication of microorganisms in tissues of a subject's body. The infection or “infectious disease” may be clinically inapparent or result in local cellular injury due to competitive metabolism, toxins, intracellular replication, or antigen-antibody response. The infection may remain localized, subclinical and temporary if the body's defensive mechanisms are effective. A local invention may persist and spread by extension to become an acute, subacute, or chronic clinical infection or disease state. A local infection may also become systemic when the microorganisms gain access to the lymphatic or vascular system. Infectious diseases include bacterial, viral, parasitic, opportunistic, or fungal infections.
As used herein “antibiotic” refers to an aminoglycoside such as gentamycin or a beta-lactam such as penicillin, cephalosporin and the like. Also included are known antifungals and anti-virals.
The term “antimicrobial agent” as used herein refers to any entity with antimicrobial activity, i.e. the ability to inhibit the growth and/or kill bacterium, for example gram positive- and gram negative bacteria. An antimicrobial agent is any agent which results in inhibition of growth or reduction of viability of a bacteria by at least about 30% or at least about 40%, or at least about 50% or at least about 60% or at least about 70% or more than 70%, or any integer between 30% and 70% or more, as compared to in the absence of the antimicrobial agent. In one embodiment, an antimicrobial agent is an agent which specifically targets a bacteria cell. In another embodiment, an antimicrobial agent modifies (i.e. inhibits or activates or increases) a pathway which is specifically expressed in bacterial cells. An antimicrobial agent can include any chemical, peptide (i.e. an antimicrobial peptide), peptidomimetic, entity or moiety, or analogues of hybrids thereof, including without limitation synthetic and naturally occurring non-proteinaceous entities. In one embodiment an antimicrobial agent includes cationic antimicrobial peptides further including lipopeptides, cyclic lipopeptides and daptomycin-related lipopeptides and daptomycin.
The term “cationic antimicrobial peptide” or “CAMP” as used herein refers to polypeptides or peptides having lengths of 10-50 amino acids and affect the cytoplasmic membrane of bacteria by typically forming channels. According to the present disclosure and also according to the scientific community (see e.g. Mishra et al. 2001, Antimicrobial agents and chemotherapy) the term “cationic antimicrobial peptide” also includes “CAMP-like antibiotics” like e.g. daptomycin and daptomycin-related lipopeptides. Most cationic antimicrobial peptides have a net positive charge due an excess of basic lysine and arginine residues over acidic residues. Typically, their mode of action is to specifically bind to cell wall precursor lipid II or bind to bacterial membrane and other bacterial surfaces, and disrupt specific cellular processes such as cell wall and membrane biosynthesis, as well as DNA replication upon translocation. In one embodiment the cationic antimicrobial peptide is an amphipathic antimicrobial peptide. In another embodiment the cationic antimicrobial peptide is an alpha-helical or beta-helical peptide. In a further embodiment the cationic antimicrobial peptide is a cathelicidin. In one embodiment the cathelicidin is LL-37. In one embodiment the cationic antimicrobial peptide is a lantibiotic, like e.g. nisin. In one embodiment, the cationic antimicrobial peptide is a lipopeptide, and in some embodiments, the lipopeptide is a cyclic lipopeptide. In another embodiment, the cyclic lipopeptide is daptomycin or a daptomycin-related lipopeptide.
The term “CAMP-like antibiotics” as used herein refers to polypeptides acting as a CAMP, like e.g. daptomycin and daptomycin-related lipopeptides, such as e.g. friulimicin B.
The term “lipopeptide” refers to a molecule that comprises a lipid-like moiety covalently linked to a peptide moiety, as well as salts, esters, amides and ethers thereof. The term “lipopeptide” also encompasses protected forms of lipopeptide in which one or more amino, carboxylate or hydroxyl groups are protected (see, e.g., “Protective Groups in Organic Synthesis” by Theodora W. Greene, John Wiley and Sons, New York, 1981 for examples of protecting groups) and lipopeptides consisting of cationic amphiphilic peptides with an acetylated N-terminus (C8-C18 fatty acid chain length). Furthermore, members of the polymyxin family are included. In a preferred embodiment, the lipopeptide is a cyclic lipopeptide. In a preferred embodiment, the lipopeptide interacts with membranes via their lipid tails. In a more preferred embodiment, the lipopeptide has an antimicrobial activity.
The term “cyclic lipopeptide” as used in this specification means a lipopeptide having a polypeptide ring and, on said ring, a side-chain acyl-amino group, optionally with or without one or more other side chains. In a preferred embodiment, the cyclic lipopeptide is a daptomycin-related molecule.
The term “daptomycin-related lipopeptide” disclosed in the above-identified application relates to synthetic and semisynthetic lipopeptides in which the ornithine and/or kynurine residues, and/or the fatty acid side chain of daptomycin, are modified. A “daptomycin-related lipopeptide” includes, inter alia, daptomycin, A54145, a daptomycin-related lipopeptide disclosed in U.S. Pat. Nos. 4,537,717, 4,482,487, Re. 32,311, Re. 32,310, U.S. Pat. No. 5,912,226, currently in reissue as U.S. Ser. No. 09/547,357, U.S. Provisional Applications No. 60/170,943, 60/170,946 or 60/170,945, filed Dec. 15, 1999, U.S. Provisional Application No. 60/208,222, filed May 30, 2000, all of which are specifically incorporated herein by reference, or an A-21978C cyclic peptide in which the n-decanoyl fatty acid side chain of daptomycin is replaced by an n-heptanoyl, n-octanoyl, n-nonanoyl, n-undecanoyl, n-lauroyl, n-dodecanoyl, n-tridecanoyl, n-myristoyl, n-pentadecanoyl, 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl or n-tetradecanoyl fatty. In a more preferred embodiment, the cyclic lipopeptide is daptomycin (LY 146032). The term daptomycin-related lipopeptide refers to compounds described above, and salts thereof.
The term “lantibiotic” is derived from “lanthionine-containing peptide antibiotic”. Bacteria are known to produce a family of lantibiotics, which are ribosomally synthesized and usually activated by post-translational modification involving dehydration (by a dehydratase) and then cyclisation (by a cyclase) to create the active bacteriocin. In one embodiment the lantibiotic is nisin.
The term “antibiotic resistance” refers to a type of drug resistance where a microorganism (e.g., S. aureus bacterium) has developed the ability to survive exposure to an antibiotic or antimicrobial peptide. Evolutionary stress such as exposure to antibiotics selects for the antibiotic resistant trait. A bacterium may carry several resistance genes. “Antibiotic resistance” has an opposite meaning as compared to “antibiotic susceptibility”, that is, a high antibiotic resistance means a low antibiotic susceptibility and vice versa.
“Administration” and “treatment,” as it applies to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. “Administration” and “treatment” can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. “Administration” and “treatment” also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition, or by another cell. “Treatment,” as it applies to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, to research and diagnostic applications. “Treatment” as it applies to a human, veterinary, or research subject, or cell, tissue, or organ, encompasses contact of an agent with animal subject, a cell, tissue, physiological compartment, or physiological fluid. “Treatment of a cell” also encompasses situations where the agent contacts PILR, e.g., in the fluid phase or colloidal phase, but also situations where the agonist or antagonist does not contact the cell or the receptor.
EXAMPLES
Generation of Fab Fragments and Antibodies that are Specific for MprF
For the selection of antibodies specifically recognizing MprF a commercially available phage display library, the MorphoSys HuCAL PLATINUM® library was used. Said antibody library is based on the HuCAL® concept (Knappik et al., (2000) J Mol Biol 296:57-86) and employs the CysDisplay® technology for displaying the Fab on the phage surface (WO2001/05950 to Lohning). M-C4.4 and M-C4.5 were selected form the commercially available MorphoSys Ylanthia® library. However, any other available antibody library would be suitable to identify MprF antibodies.
To identify MprF antibodies specific panning strategies had been developed to target MprF. Thereby specific peptides mimicking the extracellular loops of MprF were generated and used as antigen for respective pannings. All described panning strategies and antigens were used for the antibody selection process. Each panning strategy comprised at least 3 individual rounds of panning and contained unique antigens, antigen concentrations and washing stringency.
Example 1: Selection and Synthesis of the Peptides Used for Panning
The in silico analysis of the MprF sequence led to the prediction of two possible membrane topologies of MprF. The correct topology was determined using lacZ- and phoA-fusions. Results are shown in FIG. 1.
The extracellular peptides of MprF, i.e. the peptides of SEQ ID NOs.: 2-7 were custom-synthesized as linear and cyclic peptides by JPT Peptide Technologies GmbH (Berlin).
In case of cyclic peptides an N-terminal and a C-terminal Cysteine was added to enable cyclization. Biotin was coupled to the peptide via a Ttds-linker. Cyclization and coupling of linker and Biotin was performed by JPT Peptide Technologies.
In case of linear peptides an N-terminal Cysteine was introduced (SEQ ID NOs.: 108-113) to enable coupling to carrier proteins. For some peptides (SEQ ID No.: 111 and SEQ ID NO.: 112) additional Glycins had to be introduced to improve solubility. Amino acids within the peptides not representing S. aureus amino acids are indicated in bold underlined.










CELSGINFKDTLVEFSKINR,
(SEQ ID NO.: 108)



 



CYKNYTHDKKKLVHF,
(SEQ ID NO.: 109)



 



CSMVRPPDKNNRFVG,
(SEQ ID NO.: 110)



 



CGLGFKTLGVPEEKV,
(SEQ ID NO.: 111)



 



CGGDALYDGNHLT,
(SEQ ID NO.: 112)



 



CDIYTIEMHTSVLR.
(SEQ ID NO.: 113)






Prior to their use in pannings the linear peptides were coupled using NHS/EDC chemistry and two different linkers to the carrier proteins bovine serum albumin (BSA) and human transferrin (Trf).
Example 2: Generation and Characterization of Fab Fragments and Antibodies Specific for MprF
All described panning strategies and antigens were used for the antibody selection process. Each panning strategy comprised of at least 3 individual rounds of panning and contained unique antigens, antigen concentrations and washing stringency. Furthermore all described panning strategies and antigens can be combined and mixed and used as various differential panning strategies.
a) Panning with Linear Peptide-Conjugates as Antigens
Recombinant antibodies were generated from the HuCAL PLATINUM® library by three iterative rounds of panning on the peptide-carrier protein conjugates coupled to magnetic Dynal M-450 Epoxy beads (Invitrogen 140-11).
The antigen coupled beads were incubated over night at room temperature, blocked by addition of Tris, pH7.4, and subsequently re-suspended in PBS.
The antigen used for panning was alternated from Trf conjugate to BSA conjugate in each round to deplete carrier- or linker-specific antibodies. In addition, the phage library was blocked with BSA and Trf prior to every panning round with a blocking solution containing 5% BSA and 0.5% Trf for 2 h at room temperature.
b) Panning with Cyclic Biotinylated Peptides as Antigen
Recombinant antibodies were generated from the HuCAL PLATINUM® library by three iterative rounds of panning; wherein the phage antibody library was incubated with the antigen in solution and antigen-antibody-phage complexes were captured with streptavidin coated beads. (Dynal M-280)
c) Washing and Elution for all Pannings
Unspecific phages were washed away from the bead-antigen-antibody-phage complexes extensively by using PBST and PBS. Remaining phages were eluted, and eluted phages were used immediately for infection of E. coli TG1 bacteria. After rescue of the phages by using helper phage the polyclonal amplified phage output was used in consecutive selection steps.
Subsequent panning round 2 and 3 were performed in a similar fashion with prolonged washing steps and reduced antigen concentration to increase stringency and discard antibodies having low specificity and affinity. The peptide antigens were used either consistently (cyclic peptides) throughout the 3 rounds of panning or in an alternating manner (carrier protein was alternated).
d) Cloning of Fab-Encoding DNA into Expression Vector and Expression/Purification
After the 3rd round of panning the DNA of the eluted antigen-specific phages was isolated from the infected bacteria and the Fab-encoding DNA was subcloned via PCR into specific Fab expression vectors.
After transformation of TG1-F bacteria, using the Fab-encoding vectors, 368 individual colonies were randomly picked for each panning and expression and preparation of cell lysates containing HuCAL-Fab fragments were performed. Fab-containing crude extracts were used for the initial screening and characterization.
For further characterization purified Fabs had been used. E. coli TG1F− cultures (250 mL) containing the chosen antibody genes were grown at 30° C. until OD600 nm reached 0.5, and the antibody expression was induced by adding IPTG to a final concentration of 1 mM. After further incubation for at least 14 hours at 30° C., the cells were harvested, chemically lysed, and the soluble crude extract was subjected to one-step affinity chromatography (Ni-NTA agarose, Qiagen). After elution of the purified antibodies from the column, the buffer was changed from elution buffer to 3×PBS, pH 7.4, and the concentration was determined by UV280 nm measurement. Purity and activity was tested subsequently by Coomassie-stained SDS-PAGE under reducing conditions and ELISA, respectively.
Altogether Fabs against all six peptides could be isolated from the pannings and showed specific binding to the respective peptides (signal at least 5-fold over background). A representative selection of 36 unique Fabs against all six peptides was selected for conversion into IgG1 format. Altogether 22 out of 36 clones were successfully converted into a full length human IgG1 format, expressed in a human cell line and purified via protein A chromatography for further analysis.
Example 3: Characterization of Selected MprF Antibodies in ELISA
The IgGs were tested for binding to the antigen, which was used in their selection. Also binding to other MprF-peptides was tested to check for selectivity.
For ELISAs with linear antigen, the peptides coupled to BSA or transferrin were coated at a concentration of 5 μg/ml to Maxisorp plates (Nunc). The plates were blocked using 5% milk powered in PBS. After blocking of the plates the IgGs were added at various concentrations. MprF-specific IgGs were detected using goat-anti-human IgG (Sigma).
For ELISAs with cyclic antigen, the biotinylated cyclic peptides (final conc. 5 μg/ml) were incubated with IgGs in PBS. Then the mix was transferred to a pre-blocked Neutravidin plate (Thermo Scientific), incubated and then the plate was washed with PBS containing 0.5% of TWEEN20. MprF-specific IgGs were detected using goat-anti-human IgG (Sigma).
In both cases washing was performed using PBST (TBS with 0.05% TWEEN20. Detection was performed using alkaline phosphatase coupled Goat anti-human IgG (Sigma Cat. # A-8542 and AttophosTM substrate Roche, Cat. #11681982001). Fluorescence was measured at an excitation of 440±25 nm and an emission of 550±35 nm. Full signal intensity is typically reached after 5-15 min at room temperature.
The following twelve antibodies were further investigated:











TABLE 2







ID number
Antigen
Framework









M-L1
Linear peptide, loop 1
VH1A kappa 3



M-L4
Linear peptide, loop 4
VH3-23 kappa 1



M-C1
Cyclic peptide, loop 1
VH3-23 kappa 3



M-C4.1
Cyclic peptide, loop 4
VH1A kappa 1



M-C4.2
Cyclic peptide, loop 4
VH3-23 kappa 1



M-C4.3
Cyclic peptide, loop 4
VH1A kappa 1



M-C4.4
Cyclic peptide, loop 4
VH3-23



M-C4.5
Cyclic peptide, loop 4
VH6-1



M-C5.1
Cyclic peptide, loop 5
VH1A kappa 1



M-C5.2
Cyclic peptide, loop 5
VH3-23 kappa 1



M-C6.1
Cyclic peptide, loop 6
VH1A kappa 1



M-C6.2
Cyclic peptide, loop 6
VH1A kappa 1










Results and specific binding of each of the disclosed antibodies to its respective antigen are shown in FIGS. 2, 3, 4, and 15.

Example 4: MprF Antibodies Specifically Bind to S. aureus in ELISA
The specificity of the purified antibodies was tested for binding to whole cell Staphylococcus aureus in an ELISA assay. Escherichia coli bacteria were used as a negative control. Antibodies in Table 2 showed specific binding to whole Staphylococcus aureus. 
Representative examples are shown in FIG. 5.
For ELISA on S. aureus the Protein A deletion mutant (delta spa) or the mutant with an additional deletion in MprF (delta spa delta mprf) were grown to mid log phase, than washed in PBS and resuspended in saline to a final OD600 of 0.6. This bacterial culture was coated on Maxisorp plates (Nunc). Plates were blocked with 2% BSA/PBS and washed with TSB containing 0.5% TWEEN20. MprF-specific IgGs were detected using goat-anti-human IgG (Sigma).
Example 5: Functional Characterization: Studies with Nisin
In this experiment killing of Staphylococcus aureus was tested. Nisin was used as an exemplary cationic antimicrobial compound. Nisin was tested in combination with selected antibodies of the present invention. An anti-lysozyme antibody was used as a negative control.
Overnight cultures of S. aureus SA113 Δspa were diluted in fresh MHB medium and adjusted to OD600 0.25 ( ̃1.5×107 cells). Antibodies were adjusted to a concentration of 1 mg/ml and 10 μl were applied per well of a 96 well plate and diluted in 90 μl of the adjusted cell suspension (final antibody concentration: 100 μg/ml). Cells were grown in the presence of anti-lysozyme antibody (control) or with the anti-MprF antibodies. Duplicates were employed for each assay. After 3 hours of incubation under shaking conditions at 37° C., optical density was determined, adjusted to OD600 0.025 in 500 μl ice cold PBS buffer. 80 μl of the adjusted cell suspension were diluted with 20 μl of antimicrobial substances to final concentrations of 22.5 μg/ml nisin. After incubation for 2 hours under shaking conditions at 37° C., the cell suspensions were diluted 1:2000 and 100 μl of each duplicate was plated in triplicates on TSB medium containing agar plates to obtain a representative value of bacterial survival. Cells treated without antimicrobial substance were set at 100% and survival values were calculated with cells treated with antimicrobial substances. At least three independent assays were employed.
Results are depicted in FIG. 6. The antibodies of the present invention strongly increased the sensitivity of Staphylococcus aureus towards nisin, indicated by a strongly increased killing of staphylococci. The most pronounced effects were observed with antibodies M-C4.1, M-C4.2, M-C5.1 and M-C6.2.
Comparable effects were observed in a growth inhibition assay using S. aureus SA113. Therefore S. aureus (S. aureus SA113) cells grown to mid-log phase were used to inoculate fresh TSB medium. These cultures were mixed with anti-MprF (1 μM) antibody or control IgG respectively or with anti-MprF (1 μM) antibody or control IgG in combination with nisin (6 μg/ml). The growth of the S. aureus in presence of anti-MprF antibody was monitored in culture plates shaking at 37° C. by measuring OD600. While control antibody or anti-MprF antibody alone did not significantly impair growths of bacteria, an effect of nisin alone in terms of reducing growths of bacteria was observable. However, the antimicrobial effect of nisin was significantly increased in the presence of anti-MprF antibody. (FIG. 7)
Example 6: Functional Characterization: Studies with Daptomycin
The previous experiment was repeated, but instead of nisin, daptomycin is used. Again, a strong increase of sensitivity of Staphylococcus aureus towards daptomycin is observed when combined with the antibodies of the present invention (representative example in FIG. 8).
The experiment was performed as described in Example 5. Instead of nisin, 12.5 μg/ml Daptomycin was used.
Example 7: Functional Characterization: Studies with LL-37
The previous experiment was repeated, but instead of nisin LL-37 is used. Again, a strong increase of sensitivity of Staphylococcus aureus towards LL-37 is observed when combined with the antibodies of the present invention.
The experiment was performed as described in Example 5. Instead of nisin, 45.0 μg/ml of LL-37 was used (representative example in FIG. 9).
Example 8: Functional Characterization: Studies to Analyze Effect of MprF Antibodies on Charge of S. aureus Membrane and Flippase Activity of MprF
The flippase domain of MprF translocates the positively charged lipid lysyl-phosphatidylglycerol into the outer leaflet of the membrane and thereby prevents the interaction with antimicrobial peptides and daptomycin with the membrane. M-C4.1 targets an extracellular loop of the flippase and is able to increase the killing activity of nisin, daptomycin and LL-37, suggesting that M-C4.1 interferes with the flippase reaction of MprF.
Cytochrome C Repulsion Assay
The repulsion of positively charged cytochrome C has been repeatedly employed to investigate charge dependent cell surface modifications in bacteria, including the flippase reaction of MprF, which leads to increased positively charged lipids in the outer leaflet of the membrane and to increased repulsion of cytochrome C (Ernst et al., PLoS Pathogens 2009). Briefly, cells were grown in the presence of M-C4.1 or anti-lysozyme for 3 hours, followed by incubation with cytochrome C for 15 minutes and measurement of optical density of the supernatant in order to determine repulsion of positively charged cytochrome C. The established cytochrome C repulsion assay was scaled down to accommodate for the reduced amount of bacteria employed in the killing-assays.
Pre-incubation of M-C4.1 with S. aureus SA113 Δspa led to 40% decreased repulsion of cytochrome C, compared to pre-incubation with anti-lysozyme antibodies, indicating that M-C4.1 inhibits the flippase reaction of MprF. The S. aureus SA113 spa-mprF double deletion mutant treated with either M-C4.1 or anti-lysozyme served as negative controls and were significantly impaired in their capacity to repulse cytochrome C (55-75% reduced repulsion). (FIG. 10)
Annexin V Assay
Annexin V binds preferentially to negatively charged phospholipids and is therefore a useful tool for measuring the relative content of MprF-mediated flipping of lysyl-phosphatidylglycerol, as translocation leads to reduced accessibility of negatively charged phospholipids in the outer leaflet of the membrane and therefore to reduced binding of annexin V. Cells (S. aureus SA113 Δspa) were grown in the presence of M-C4.1 or anti-lysozyme antibody and then treated with annexin V for 15 minutes before being analyzed by flow cytometry for surface-bound fluorophore (FL-4 channel). Data are expressed in relative fluorescence units and compared to cells pre-treated with anti-lysoyzme set at 100% fluorescence.
Cells pre-treated with M-C4.1 bound significantly more annexin V (70% increased binding) indicating that the pre-treatment with M-C4.1 had led to impaired flipping of lysyl-phosphatidylglycerol to the outer leaflet of the membrane. (FIG. 11)
Example 9: ELISA-Based Cross-Competition Assay
Cross-competition of an anti-MprF antibody or another MprF binding agent may be detected by using an ELISA assay according to the following standard procedure. Likewise, cross-competition of an anti-MprF antibody or another MprF binding agent may be detected.
The general principle of the ELISA-assay involves coating of an anti-MprF antibody onto the wells of an ELISA plate. An excess amount of a second, potentially cross-competitive, anti-MprF antibody is then added in solution (i.e. not bound to the ELISA plate). Subsequently a limited amount of antigen (representing MprF specific structures) is then added to the wells.
The antibody which is coated onto the wells and the antibody in solution will compete for binding of the limited number of antigen molecules. The plate is then washed to remove antigen molecules that has not bound to the coated antibody and to also remove the second, solution phase antibody as well as any complexes formed between the second, solution phase antibody and the antigens. The amount of bound antigen is then measured using an appropriate antigen detection reagent. Therefore, the antigen may be fused with a tag, e.g. Fc, Flag, etc. which can be detected via an appropriate tag-specific antibody.
An antibody in solution that is cross-competitive to the coated antibody will be able to cause a decrease in the number of antigen molecules that the coated antibody can bind relative to the number of antigen molecules that the coated antibody can bind in the absence of the second, solution phase antibody.
This assay is described in more detail further below for two antibodies termed Ab-X and Ab-Y. In the instance where Ab-X is chosen to be the immobilized antibody, it is coated onto the wells of the ELISA plate, after which the plates are blocked with a suitable blocking solution to minimize non-specific binding of reagents that are subsequently added. An excess amount of Ab-Y is then added to the ELISA plate such that the moles of Ab-Y MprF binding sites per well are at least 10 fold higher than the moles of Ab-X MprF specific structures binding sites that are used, per well, during the coating of the ELISA plate. Antigen (representing MprF specific structures, e.g. linear or cyclic extracellular loop) is then added such that the moles of antigen added per well were at least 25-fold lower than the moles of Ab-X MprF binding sites that are used for coating each well. Following a suitable incubation period, the ELISA plate is washed and an antigen detection reagent is added to measure the amount of antigen molecules specifically bound by the coated anti-MprF antibody (in this case Ab-X). The background signal for the assay is defined as the signal obtained in wells with the coated antibody (in this case Ab-X), second solution phase antibody (in this case Ab-Y), buffer only and antigen detection reagents. The positive control signal for the assay is defined as the signal obtained in wells with the coated antibody (in this case Ab-X), second solution phase antibody buffer only (i.e. no second solution phase antibody), antigen detection reagents. The ELISA assay needs to be run in such a manner so as to have the positive control signal be at least 6 times the background signal.
To avoid any artifacts (e.g. significantly different affinities between Ab-X and Ab-Y for MprF or MprF specific structures) resulting from the choice of which antibody to use as the coating antibody and which to use as the second (competitor) antibody, the cross-blocking assay needs to be run in two formats: 1) format 1 is where Ab-X is the antibody that is coated onto the ELISA plate and Ab-Y is the competitor antibody that is in solution and 2) format 2 is where Ab-Y is the antibody that is coated onto the ELISA plate and Ab-X is the competitor antibody that is in solution.
Example 10: Functional Characterization: Studies with Clinical S. aureus Strains and Daptomycin
In this experiment killing of Staphylococcus aureus clinical isolate 703 (described in Jones et al., AAC 2008) by daptomycin in the presence of an MprF specific antibody was analyzed. The experiment was performed as described in Example 5. Instead of nisin, 11 μg/ml daptomycin was used and instead of S. aureus SA113 Δspa the Staphylococcus aureus clinical isolate 703 was used.
Staphylococcus aureus clinical isolate 703 was described in Jones et al., AAC 2008 to be non-responsive to daptomycin. However, in the presence of an anti-MprF antibody survival of S. aureus clinical 703 bacteria is significantly reduced. Therefor interference with MprF using a MprF-specific antibody induced susceptibility of S. aureus clinical 703 bacteria to daptomycin (FIG. 13).
Example 11: Growth Inhibition of Clinical Isolate S. aureus USA300 (MRSA)
In this experiment growth inhibition of Staphylococcus aureus clinical isolate USA300 by nisin was tested in the presence of an MprF specific antibody. While control antibody or anti-MprF antibody alone did not impair growths of bacteria, an effect of nisin alone in terms of reducing bacterial growths was observed. However, in the presence of anti-MprF antibody the antimicrobial effect of nisin was significantly increased, and growth of bacteria was abolished over a period of 24 h (FIG. 14).
The experiment was performed as described in Example 5. Instead of S. aureus SA113 the clinical isolate S. aureus USA300 was used. The concentration of nisin was 4.5 μg/ml instead of 6 μg/ml nisin.










TABLE 3





ID#

Seq. ID:
[aa]/DNA







M-L1
HCDR1
Seq. ID: 8
GGTFNSYAIH


 



HCDR2
Seq. ID: 9
GIIPIFGIANYAQKFQG


 



HCDR3
Seq. ID: 10
DQYTYDYYRAFDY


 



LCDR1
Seq. ID: 11
RASQSVRDNLA


 



LCDR2
Seq. ID: 12
GASNRAT


 



LCDR3
Seq. ID: 13
QQYDHSPRT


 



VL
Seq. ID: 14
DIVLTQSPATLSLSPGERATLSCRASQSVRDNLAWYQ





QKPGQAPRLLIYGASNRATGIPARFSGSGSGTDFTLTI





SSLEPEDFAVYYCQQYDHSPRTFGQGTKVEIKRT


 



VH
Seq. ID: 15
QVQLVQSGAEVKKPGSSVKVSCKASGGTFNSYAIHW





VRQAPGQGLEWMGGIIPIFGIANYAQKFQGRVTITADE





STSTAYMELSSLRSEDTAVYYCARDQYTYDYYRAFDY





WGQGTLVTVSS


 



VL (DNA)
Seq. ID: 16
GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAG





CCTGAGCCCGGGTGAACGTGCCACCCTGAGCTGCA





GAGCGAGCCAGTCTGTTCGTGACAACCTGGCTTGG





TACCAGCAGAAACCGGGCCAGGCCCCGCGTCTATT





AATCTACGGTGCTTCTAACCGTGCGACCGGCATTCC





GGCGCGTTTTAGCGGCAGCGGATCCGGCACCGATT





TCACCCTGACCATTAGCAGCCTGGAACCGGAAGAC





TTTGCGGTGTATTATTGCCAGCAGTACGACCATTCT





CCGCGTACCTTTGGCCAGGGCACGAAAGTTGAAAT





TAAACGTACG


 



VH (DNA)
Seq. ID: 17
CAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAA





AAAACCGGGCAGCAGCGTGAAAGTTAGCTGCAAAG





CATCCGGAGGGACGTTTAACTCTTACGCTATCCATT





GGGTGCGCCAGGCCCCGGGCCAGGGCCTCGAGTG





GATGGGCGGTATCATCCCGATCTTCGGCATCGCGA





ACTACGCCCAGAAATTTCAGGGCCGGGTGACCATT





ACCGCCGATGAAAGCACCAGCACCGCCTATATGGA





ACTGAGCAGCCTGCGCAGCGAAGATACGGCCGTGT





ATTATTGCGCGCGTGACCAGTACACTTACGACTACT





ACCGTGCTTTCGATTACTGGGGCCAAGGCACCCTG





GTGACTGTTAGCTCA


 


M-L4
HCDR1
Seq. ID: 18
SYAMS


 



HCDR2
Seq. ID: 19
IISYDGSSTYYADSVKG


 



HCDR3
Seq. ID: 20
TATSGSRGYFDY


 



LCDR1
Seq. ID: 21
RASQDISSWLN


 



LCDR2
Seq. ID: 22
GASTLQS


 



LCDR3
Seq. ID: 23
QQHYTSPVT


 



VL
Seq. ID: 24
DIQMTQSPSSLSASVGDRVTITCRASQDISSWLNWYQ





QKPGKAPKLLIFGASTLQSGVPSRFSGSGSGTDFTLTI





SSLQPEDFATYYCQQHYTSPVTFGQGTKVEIKRT


 



VH
Seq. ID: 25
EVQLLESGGGLVQPGGSLRLSCAASGFTFRSYAMSW





VRQAPGKGLEWVSIISYDGSSTYYADSVKGRFTISRDN





SKNTLYLQMNSLRAEDTAVYYCARTATSGSRGYFDY





WGQGTLVTVSS


 



VL (DNA)
Seq. ID: 26
GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAG





CGCCAGCGTGGGCGATCGCGTGACCATTACCTGCA





GAGCCAGCCAGGACATTTCTTCTTGGCTGAACTGGT





ACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTA





ATCTTCGGTGCTTCTACTCTGCAAAGCGGCGTGCC





GAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATT





TCACCCTGACCATTAGCTCTCTGCAACCGGAAGACT





TTGCGACCTATTATTGCCAGCAGCATTACACTTCTC





CGGTTACCTTTGGCCAGGGCACGAAAGTTGAAATTA





AACGTACG


 



VH (DNA)
Seq. ID: 27
GAAGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGT





GCAGCCGGGTGGCAGCCTGCGTCTGAGCTGCGCG





GCGTCCGGATTCACCTTTCGTTCTTACGCTATGTCT





TGGGTGCGCCAGGCCCCGGGCAAAGGTCTCGAGT





GGGTTTCCATTATCTCTTACGACGGTTCTTCTACCTA





CTATGCGGATAGCGTGAAAGGCCGCTTTACCATCA





GCCGCGATAATTCGAAAAACACCCTGTATCTGCAAA





TGAACAGCCTGCGTGCGGAAGATACGGCCGTGTAT





TATTGCGCGCGTACTGCTACTTCTGGTTCTCGTGGT





TACTTCGATTACTGGGGCCAAGGCACCCTGGTGAC





TGTTAGCTCA


 


M-C1
HCDR1
Seq. ID: 28
SYAMN


 



HCDR2
Seq. ID: 29
VISSDGSDTYYADSVKG


 



HCDR3
Seq. ID: 30
ESYYIGWDY


 



LCDR1
Seq. ID: 31
RASQRVSSSFLA


 



LCDR2
Seq. ID: 32
LASNRAT


 



LCDR3
Seq. ID: 33
LQYGHYPPT


 



VL
Seq. ID: 34
DIVLTQSPATLSLSPGERATLSCRASQRVSSSFLAWY





QQKPGQAPRLLIYLASNRATGIPARFSGSGSGTDFTLT





ISSLEPEDFAVYYCLQYGHYPPTFGQGTKVEIKRT


 



VH
Seq. ID: 35
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNW





VRQAPGKGLEWVSVISSDGSDTYYADSVKGRFTISRD





NSKNTLYLQMNSLRAEDTAVYYCARESYYIGWDYWG





QGTLVTVSS


 



VL (DNA)
Seq. ID: 36
GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAG





CCTGAGCCCGGGTGAACGTGCCACCCTGAGCTGCA





GAGCGAGCCAGCGTGTTTCTTCTTCTTTCCTGGCTT





GGTACCAGCAGAAACCGGGCCAGGCCCCGCGTCTA





TTAATCTACCTGGCTTCTAACCGTGCGACCGGCATT





CCGGCGCGTTTTAGCGGCAGCGGATCCGGCACCG





ATTTCACCCTGACCATTAGCAGCCTGGAACCGGAAG





ACTTTGCGGTGTATTATTGCCTGCAGTACGGTCATT





ACCCGCCGACCTTTGGCCAGGGCACGAAAGTTGAA





ATTAAACGTACG


 



VH (DNA)
Seq. ID: 37
CAGGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGT





GCAGCCGGGTGGCAGCCTGCGTCTGAGCTGCGCG





GCGTCCGGATTCACCTTTTCTTCTTACGCTATGAACT





GGGTGCGCCAGGCCCCGGGCAAAGGTCTCGAGTG





GGTTTCCGTTATCTCTTCTGACGGTTCTGACACCTA





CTATGCGGATAGCGTGAAAGGCCGCTTTACCATCA





GCCGCGATAATTCGAAAAACACCCTGTATCTGCAAA





TGAACAGCCTGCGTGCGGAAGATACGGCCGTGTAT





TATTGCGCGCGTGAATCTTACTACATCGGTTGGGAT





TACTGGGGCCAAGGCACCCTGGTGACTGTTAGCTC





A


 


M-C4.1
HCDR1
Seq. ID: 38
SYAIS


 



HCDR2
Seq. ID: 39
GIIPIFGMATYAQKFQG


 



HCDR3
Seq. ID: 40
SLFGRAY


 



LCDR1
Seq. ID: 41
RASQSISSFLA


 



LCDR2
Seq. ID: 42
DASSLQS


 



LCDR3
Seq. ID: 43
LQADSTSYT


 



VL
Seq. ID: 44
DIQMTQSPSSLSASVGDRVTITCRASQSISSFLAWYQQ





KPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTIS





SLQPEDFATYYCLQADSTSYTFGQGTKVEIKRT


 



VH
Seq. ID: 45
QVQLVQSGAEVKKPGSSVKVSCKASGGTFNSYAISW





VRQAPGQGLEWMGGIIPIFGMATYAQKFQGRVTITAD





ESTSTAYMELSSLRSEDTAVYYCARSLFGRAYWGQG





TLVTVSS


 



VL (DNA)
Seq. ID: 46
GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAG





CGCCAGCGTGGGCGATCGCGTGACCATTACCTGCA





GAGCCAGCCAGTCTATTTCTTCTTTCCTGGCTTGGT





ACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTA





ATCTACGACGCTTCTTCTCTGCAAAGCGGCGTGCC





GAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATT





TCACCCTGACCATTAGCTCTCTGCAACCGGAAGACT





TTGCGACCTATTATTGCCTGCAGGCTGACTCTACTT





CTTACACCTTTGGCCAGGGCACGAAAGTTGAAATTA





AACGTACG


 



VH (DNA)
Seq. ID: 47
CAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAA





AAAACCGGGCAGCAGCGTGAAAGTTAGCTGCAAAG





CATCCGGAGGGACGTTTAACTCTTACGCTATCTCTT





GGGTGCGCCAGGCCCCGGGCCAGGGCCTCGAGTG





GATGGGCGGTATCATCCCGATCTTCGGCATGGCGA





CTTACGCCCAGAAATTTCAGGGCCGGGTGACCATTA





CCGCCGATGAAAGCACCAGCACCGCCTATATGGAA





CTGAGCAGCCTGCGCAGCGAAGATACGGCCGTGTA





TTATTGCGCGCGTTCTCTGTTCGGTCGTGCTTACTG





GGGCCAAGGCACCCTGGTGACTGTTAGCTCA


 


M-C4.2
HCDR1
Seq. ID: 48
SYAMH


 



HCDR2
Seq. ID: 49
VISSVGSSTYYADSVKG


 



HCDR3
Seq. ID: 50
GGLDV


 



LCDR1
Seq. ID: 51
RASQDISKYLN


 



LCDR2
Seq. ID: 52
AASRLQS


 



LCDR3
Seq. ID: 53
QQGDSTPFT


 



VL
Seq. ID: 54
DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQ





QKPGKAPKLLIYAASRLQSGVPSRFSGSGSGTDFTLTI





SSLQPEDFATYYCQQGDSTPFTFGQGTKVEIKRT


 



VH
Seq. ID: 55
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMHW





VRQAPGKGLEWVSVISSVGSSTYYADSVKGRFTISRD





NSKNTLYLQMNSLRAEDTAVYYCARGGLDVWGQGTL





VTVSS


 



VL (DNA)
Seq. ID: 56
GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAG





CGCCAGCGTGGGCGATCGCGTGACCATTACCTGCA





GAGCCAGCCAGGACATTTCTAAATACCTGAACTGGT





ACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTA





ATCTACGCTGCTTCTCGTCTGCAAAGCGGCGTGCC





GAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATT





TCACCCTGACCATTAGCTCTCTGCAACCGGAAGACT





TTGCGACCTATTATTGCCAGCAGGGTGACTCTACTC





CGTTCACCTTTGGCCAGGGCACGAAAGTTGAAATTA





AACGTACG


 



VH (DNA)
Seq. ID: 57
GAAGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGT





GCAGCCGGGTGGCAGCCTGCGTCTGAGCTGCGCG





GCGTCCGGATTCACCTTTTCTTCTTACGCTATGCATT





GGGTGCGCCAGGCCCCGGGCAAAGGTCTCGAGTG





GGTTTCCGTTATCTCTTCTGTTGGTTCTTCTACCTAC





TATGCGGATAGCGTGAAAGGCCGCTTTACCATCAG





CCGCGATAATTCGAAAAACACCCTGTATCTGCAAAT





GAACAGCCTGCGTGCGGAAGATACGGCCGTGTATT





ATTGCGCGCGTGGTGGTCTGGACGTTTGGGGCCAA





GGCACCCTGGTGACTGTTAGCTCA


 


M-C4.3
HCDR1
Seq. ID: 58
DHTIS


 



HCDR2
Seq. ID: 59
GIIPIVGIANYAQKFQG


 



HCDR3
Seq. ID: 60
EYYVPDSGWFDY


 



LCDR1
Seq. ID: 61
RASQDISDYLV


 



LCDR2
Seq. ID: 62
DASNLQS


 



LCDR3
Seq. ID: 63
QQYYHYRT


 



VL
Seq. ID: 64
DIQMTQSPSSLSASVGDRVTITCRASQDISDYLVWYQ





QKPGKAPKLLIYDASNLQSGVPSRFSGSGSGTDFTLTI





SSLQPEDFATYYCQQYYHYRTFGQGTKVEIKRT


 



VH
Seq. ID: 65 
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDHTISW





VRQAPGQGLEWMGGIIPIVGIANYAQKFQGRVTITADE





STSTAYMELSSLRSEDTAVYYCAREYYVPDSGWFDY





WGQGTLVTVSS


 



VL (DNA)
Seq. ID: 66
GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAG





CGCCAGCGTGGGCGATCGCGTGACCATTACCTGCA





GAGCCAGCCAGGACATTTCTGACTACCTGGTTTGGT





ACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTA





ATCTACGACGCTTCTAACCTGCAAAGCGGCGTGCC





GAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATT





TCACCCTGACCATTAGCTCTCTGCAACCGGAAGACT





TTGCGACCTATTATTGCCAGCAGTACTACCATTACC





GTACCTTTGGCCAGGGCACGAAAGTTGAAATTAAAC





GTACG


 



VH (DNA)
Seq. ID: 67
CAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAA





AAAACCGGGCAGCAGCGTGAAAGTTAGCTGCAAAG





CATCCGGAGGGACGTTTTCTGACCACACTATCTCTT





GGGTGCGCCAGGCCCCGGGCCAGGGCCTCGAGTG





GATGGGCGGTATCATCCCGATCGTTGGCATCGCGA





ACTACGCCCAGAAATTTCAGGGCCGGGTGACCATT





ACCGCCGATGAAAGCACCAGCACCGCCTATATGGA





ACTGAGCAGCCTGCGCAGCGAAGATACGGCCGTGT





ATTATTGCGCGCGTGAATACTACGTTCCGGACTCTG





GTTGGTTCGATTACTGGGGCCAAGGCACCCTGGTG





ACTGTTAGCTCA


 


M-05.1
HCDR1
Seq. ID: 68
SYAIS


 



HCDR2
Seq. ID: 69
GIVPIFGTANYAQKFQG


 



HCDR3
Seq. ID: 70
VRYGYWDV


 



LCDR1
Seq. ID: 71
RASQDIANWLN


 



LCDR2
Seq. ID: 72
AASSLQS


 



LCDR3
Seq. ID: 73
QQYISLPIT


 



VL
Seq. ID: 74
DIQMTQSPSSLSASVGDRVTITCRASQDIANWLNWYQ





QKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTI





SSLQPEDFATYYCQQYISLPITFGQGTKVEIKRT


 



VH
Seq. ID: 75
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISW





VRQAPGQGLEWMGGIVPIFGTANYAQKFQGRVTITAD





ESTSTAYMELSSLRSEDTAVYYCARVRYGYWDVWGQ





GTLVTVSS


 



VL (DNA)
Seq. ID: 76
GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAG





CGCCAGCGTGGGCGATCGCGTGACCATTACCTGCA





GAGCCAGCCAGGACATTGCTAACTGGCTGAACTGG





TACCAGCAGAAACCGGGCAAAGCGCCGAAACTATT





AATCTACGCTGCTTCTTCTCTGCAAAGCGGCGTGCC





GAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATT





TCACCCTGACCATTAGCTCTCTGCAACCGGAAGACT





TTGCGACCTATTATTGCCAGCAGTACATCTCTCTGC





CGATCACCTTTGGCCAGGGCACGAAAGTTGAAATTA





AACGTACG


 



VH (DNA)
Seq. ID: 77
CAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAA





AAAACCGGGCAGCAGCGTGAAAGTTAGCTGCAAAG





CATCCGGAGGGACGTTTTCTTCTTACGCTATCTCTT





GGGTGCGCCAGGCCCCGGGCCAGGGCCTCGAGTG





GATGGGCGGTATCGTTCCGATCTTCGGCACTGCGA





ACTACGCCCAGAAATTTCAGGGCCGGGTGACCATT





ACCGCCGATGAAAGCACCAGCACCGCCTATATGGA





ACTGAGCAGCCTGCGCAGCGAAGATACGGCCGTGT





ATTATTGCGCGCGTGTTCGTTACGGCTACTGGGATG





TTTGGGGCCAAGGCACCCTGGTGACTGTTAGCTCA


 


M-05.2
HCDR1
Seq. ID: 78
SYAVH


 



HCDR2
Seq. ID: 79
VISGRGGSTYYADSVKG


 



HCDR3
Seq. ID: 80
DHGYFDY


 



LCDR1
Seq. ID: 81
RASQTISNHLG


 



LCDR2
Seq. ID: 82
TASNLQS


 



LCDR3
Seq. ID: 83
QQYSHSSYT


 



VL
Seq. ID: 84
DIQMTQSPSSLSASVGDRVTITCRASQTISNHLGWYQ





QKPGKAPKLLIYTASNLQSGVPSRFSGSGSGTDFTLTI





SSLQPEDFATYYCQQYSHSSYTFGQGTKVEIKRT


 



VH
Seq. ID: 85
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAVHW





VRQAPGKGLEWVSVISGRGGSTYYADSVKGRFTISRD





NSKNTLYLQMNSLRAEDTAVYYCARDHGYFDYWGQG





TLVTVSS


 



VL (DNA)
Seq. ID: 86
GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAG





CGCCAGCGTGGGCGATCGCGTGACCATTACCTGCA





GAGCCAGCCAGACTATTTCTAACCATCTGGGTTGGT





ACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTA





ATCTACACTGCTTCTAACCTGCAAAGCGGCGTGCCG





AGCCGCTTTAGCGGCAGCGGATCCGGCACCGATTT





CACCCTGACCATTAGCTCTCTGCAACCGGAAGACTT





TGCGACCTATTATTGCCAGCAGTACTCTCATTCTTCT





TACACCTTTGGCCAGGGCACGAAAGTTGAAATTAAA





CGTACG


 



VH (DNA)
Seq. ID: 87
GAAGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGT





GCAGCCGGGTGGCAGCCTGCGTCTGAGCTGCGCG





GCGTCCGGATTCACCTTTTCTTCTTACGCTGTTCATT





GGGTGCGCCAGGCCCCGGGCAAAGGTCTCGAGTG





GGTTTCCGTTATCTCTGGTCGTGGTGGTTCTACCTA





CTATGCGGATAGCGTGAAAGGCCGCTTTACCATCA





GCCGCGATAATTCGAAAAACACCCTGTATCTGCAAA





TGAACAGCCTGCGTGCGGAAGATACGGCCGTGTAT





TATTGCGCGCGTGACCATGGTTACTTCGACTACTGG





GGCCAAGGCACCCTGGTGACTGTTAGCTCA


 


M-C6.1
HCDR1
Seq. ID: 88
IYAIS


 



HCDR2
Seq. ID: 89
GIIPEFGTANYAQKFQG


 



HCDR3
Seq. ID: 90
SQIYTLSYPKWFDF


 



LCDR1
Seq. ID: 91
RASQDISNYLN


 



LCDR2
Seq. ID: 92
DASNLQS


 



LCDR3
Seq. ID: 93
LQYLQSPKT


 



VL
Seq. ID: 94
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQ





QKPGKAPKLLIYDASNLQSGVPSRFSGSGSGTDFTLTI





SSLQPEDFATYYCLQYLQSPKTFGQGTKVEIKRT


 



VH
Seq. ID: 95
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSIYAISWV





RQAPGQGLEWMGGIIPEFGTANYAQKFQGRVTITADE





STSTAYMELSSLRSEDTAVYYCARSQIYTLSYPKWFDF





WGQGTLVTVSS


 



VL (DNA)
Seq. ID: 96
GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAG





CGCCAGCGTGGGCGATCGCGTGACCATTACCTGCA





GAGCCAGCCAGGACATTTCTAACTACCTGAACTGGT





ACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTA





ATCTACGACGCTTCTAACCTGCAAAGCGGCGTGCC





GAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATT





TCACCCTGACCATTAGCTCTCTGCAACCGGAAGACT





TTGCGACCTATTATTGCCTGCAGTACCTGCAGTCTC





CGAAAACCTTTGGCCAGGGCACGAAAGTTGAAATTA





AACGTACG


 



VH (DNA)
Seq. ID: 97
CAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAA





AAAACCGGGCAGCAGCGTGAAAGTTAGCTGCAAAG





CATCCGGAGGGACGTTTTCTATCTACGCTATCTCTT





GGGTGCGCCAGGCCCCGGGCCAGGGCCTCGAGTG





GATGGGCGGTATCATCCCGGAATTCGGCACTGCGA





ACTACGCCCAGAAATTTCAGGGCCGGGTGACCATT





ACCGCCGATGAAAGCACCAGCACCGCCTATATGGA





ACTGAGCAGCCTGCGCAGCGAAGATACGGCCGTGT





ATTATTGCGCGCGTTCTCAGATCTACACTCTGTCTTA





CCCGAAATGGTTCGACTTCTGGGGCCAAGGCACCC





TGGTGACTGTTAGCTCA


 


M-C6.2
HCDR1
Seq. ID: 98
SYAIS


 



HCDR2
Seq. ID: 99
GIIPILGIANYAQKFQG


 



HCDR3
Seq. ID: 100
SLPYRSDLYGFSRWSYHRVGMDV


 



LCDR1
Seq. ID: 101
RASQDISNTLN


 



LCDR2
Seq. ID: 102
AASTLQS


 



LCDR3
Seq. ID: 103
QQVGSFPYT


 



VL
Seq. ID: 104
DIQMTQSPSSLSASVGDRVTITCRASQDISNTLNWYQ





QKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTI





SSLQPEDFATYYCQQVGSFPYTFGQGTKVEIKRT


 



VH
Seq. ID: 105
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISW





VRQAPGQGLEWMGGIIPILGIANYAQKFQGRVTITADE





STSTAYMELSSLRSEDTAVYYCARSLPYRSDLYGFSR





WSYHRVGMDVWGQGTLVTVSS


 



VL (DNA)
Seq. ID: 106
GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAG





CGCCAGCGTGGGCGATCGCGTGACCATTACCTGCA





GAGCCAGCCAGGACATTTCTAACACTCTGAACTGGT





ACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTA





ATCTACGCTGCTTCTACTCTGCAAAGCGGCGTGCC





GAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATT





TCACCCTGACCATTAGCTCTCTGCAACCGGAAGACT





TTGCGACCTATTATTGCCAGCAGGTTGGTTCTTTCC





CGTACACCTTTGGCCAGGGCACGAAAGTTGAAATTA





AACGTACG


 



VH (DNA)
Seq. ID: 107
CAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAA





AAAACCGGGCAGCAGCGTGAAAGTTAGCTGCAAAG





CATCCGGAGGGACGTTTTCTTCTTACGCTATCTCTT





GGGTGCGCCAGGCCCCGGGCCAGGGCCTCGAGTG





GATGGGCGGTATCATCCCGATCCTGGGCATCGCGA





ACTACGCCCAGAAATTTCAGGGCCGGGTGACCATT





ACCGCCGATGAAAGCACCAGCACCGCCTATATGGA





ACTGAGCAGCCTGCGCAGCGAAGATACGGCCGTGT





ATTATTGCGCGCGTTCTCTGCCGTACCGTTCTGACC





TGTACGGTTTCTCTCGTTGGTCTTACCATCGTGTTG





GTATGGATGTTTGGGGCCAAGGCACCCTGGTGACT





GTTAGCTCA


 


M-C4.4
HCDR1
Seq. ID: 119
FTFSSYAIS


 



HCDR2
Seq. ID: 120
VSAISGSGGSTYYAESVKG


 



HCDR3
Seq. ID: 121
YPYPGYFDL


 



LCDR1
Seq. ID: 122
SGDKLGDKYAY


 



LCDR2
Seq. ID: 123
LVIYQDSKRPS


 



LCDR3
Seq. ID: 124
QTWVSSISAF


 



VL
Seq. ID: 125
SYELTQPPSVSVSPGQTASITCSGDKLGDKYAYWYQQ





KPGQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLTIS





GTQAEDEADYYCQTWVSSISAFVFGGGTKLTVLGQ


 



VH
Seq. ID: 126
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAISWV





RQAPGKGLEWVSAISGSGGSTYYAESVKGRFTISRDN





SKNTLYLGMNSLRAEDTAVYYCARYPYPGYFDLWGQ





GTLVTVSS


 



VL (DNA)
Seq. ID: 127
AGCTATGAACTGACCCAGCCGCCGAGCGTTAGCGT





TAGCCCAGGCCAGACCGCCAGCATTACCTGTAGCG





GCGACAAACTGGGCGACAAATACGCCTACTGGTAT





CAGCAGAAACCGGGCCAGAGCCCGGTGCTGGTTAT





CTATCAGGATAGCAAACGCCCGAGCGGCATTCCAG





AACGCTTTAGCGGCAGCAACAGCGGCAACACCGCC





ACCCTGACCATTAGCGGCACCCAGGCCGAAGACGA





AGCCGATTATTACTGCCAGACTTGGGTTTCTTCTAT





CTCTGCTTTCGTGTTTGGCGGCGGTACCAAGCTGA





CCGTGCTGGGCCAG


 



VH (DNA)
Seq. ID: 128
GAAGTGCAGCTGCTGGAAAGCGGTGGCGGTCTGGT





GCAGCCAGGTGGTAGCCTGCGCCTGAGCTGTGCC





GCAAGCGGCTTTACCTTTAGCAGCTATGCCATTAGC





TGGGTGCGCCAAGCACCAGGCAAAGGCCTGGAATG





GGTGAGCGCCATTAGCGGCAGCGGTGGCAGCACC





TATTATGCCGAGAGCGTGAAAGGTCGCTTTACCATT





AGTCGCGATAACAGCAAAAACACCCTGTATCTGCAA





ATGAACAGCCTGCGGGCAGAAGATACCGCAGTTTA





TTATTGCGCGCGTTACCCTTACCCTGGTTATTTCGA





CCTGTGGGGCCAGGGCACCCTGGTTACTGTCTCGA





GC


 


M-C4.5
HCDR1
Seq. ID: 129
DSVSSNSAAWN


 



HCDR2
Seq. ID: 130
LGRTYYRSKWYNDYAVSVKS


 



HCDR3
Seq. ID: 131
SAEPSYAYYHGFDY


 



LCDR1
Seq. ID: 132
RASQGISSYLA


 



LCDR2
Seq. ID: 133
LLIYAASTLQS


 



LCDR3
Seq. ID: 134
QQRIIFPQ


 



VL
Seq. ID: 135
DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQ





KPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTIS





SLQPEDFATYYCQQRIIFPQTFGQGTKVEIKRT


 



VH
Seq. ID: 136
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWN





WIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITIN





PDTSKNQFSLQLNSVTPEDTAVYYCARSAEPSYAYYH





GFDYWGQGTLVTVSS


 



VL (DNA)
Seq. ID: 137
GATATTCAGCTGACCCAGAGCCCGAGCTTTCTGAG





CGCCAGCGTGGGCGATCGCGTGACCATTACCTGCC





GCGCCAGCCAGGGCATTAGCAGCTATCTGGCCTGG





TATCAGCAGAAACCGGGCAAAGCCCCGAAACTGCT





GATCTATGCCGCCAGCACCCTGCAAAGCGGCGTGC





CAAGCCGCTTTAGCGGCAGCGGTAGCGGCACCGA





GTTCACCCTGACCATTAGCAGCCTGCAACCGGAAG





ACTTTGCCACCTATTATTGCCAGCAGCGTATCATCTT





CCCGCAGACCTTCGGCCAGGGTACCAAAGTGGAAA





TCAAGCGGACC


 



VH (DNA)
Seq. ID: 138
CAGGTGCAGCTGCAACAGAGCGGCCCAGGCCTGG





TTAAACCGAGCCAGACCCTGAGCCTGACCTGCGCC





ATTAGCGGCGATAGCGTTAGCAGCAACAGCGCCGC





CTGGAACTGGATTCGCCAGAGCCCGAGCCGCGGTC





TGGAATGGCTGGGCCGCACCTATTATCGCAGCAAA





TGGTACAACGATTACGCCGTTAGCGTGAAAAGCCG





CATTACCATTAACCCGGATACCAGCAAAAACCAGTT





CAGCCTGCAACTGAACAGCGTGACCCCGGAAGATA





CCGCCGTGTATTACTGCGCGCGTAGCGCAGAGCCT





AGCTACGCATACTATCACGGTTTTGACTATTGGGGC





CAGGGCACCCTGGTTACTGTCTCGAGC

## Claims

1. An antibody or antibody fragment that is specific to an extracellular loop of the multiple peptide resistance factor (MprF) of Staphylococcus aureus, wherein said MprF is of SEQ ID NO:1 or an orthologue of SEQ ID NO: 1, wherein said antibody binds specifically to an extracellular loop of the MprF or the orthologue, wherein said antibody is a monoclonal antibody, chimeric antibody, humanized antibody, synthetic antibody or human monoclonal antibody, or a fragment of said antibody and wherein said antibody or antibody fragment increases susceptibility of a Staphylococcus aureus to a cationic antimicrobial peptide.

2. The antibody or antibody fragment of claim 1, wherein the cationic antimicrobial peptide is nisin, daptomycin, or cathelicidin.

3. The antibody or antibody fragment according to claim 1, wherein said antibody binds to the extracellular loop of the MprF, wherein the loop is of the amino acid sequence of ELSGINFKDTLVEFSKINR (SEQ ID NO: 2), YKNYTHDKKKLVHF (SEQ ID NO: 3), SMVRPPDKNNRFVG (SEQ ID NO: 4), LGFKTLGVPEEKV (SEQ ID NO: 5), DALYDGNHLT (SEQ ID NO: 6) or DIYTIEMHTSVLR (SEQ ID NO: 7).

4. The antibody or antibody fragment according to claim 1, wherein said antibody binds to an isolated peptide of ELSGINFKDTLVEFSKINR (SEQ ID NO: 2), YKNYTHDKKKLVHF (SEQ ID NO: 3), SMVRPPDKNNRFVG (SEQ ID NO: 4), LGFKTLGVPEEKV (SEQ ID NO: 5), DALYDGNHLT (SEQ ID NO: 6) or DIYTIEMHTSVLR (SEQ ID NO: 7).

5. A combination comprising the antibody or antibody fragment according to claim 1, and a cationic antimicrobial peptide.

6. The combination according to claim 5, wherein said antimicrobial peptide is a cyclic lipopeptide.

7. The combination according to claim 6, wherein said cyclic lipopeptide is daptomycin or a daptomycin-related lipopeptide.

8. The combination according to claim 5, wherein said combination is synergistic.

9. A pharmaceutical composition comprising the combination according to claim 5.

10. The antibody or antibody fragment according to claim 1 which is an IgG isotype.