Recombinant antibody binding to penicillin binding protein 2a (PBP2a)

Abstract

The present invention relates to recombinant antibodies, isolated from mouse or rabbit phage library, that bind to penicillin binding protein 2A (PBP2a) of methicillin-resistant Staphylococcus aureus (MRSA) used for the detection of PBP2a.

Description

[0001] RECOMBINANT ANTIBODY BINDING TO PENICILLIN BINDING PROTEIN 2A (PBP2A)

[0002] REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT XML 1.0 FORMAT FILE (.xml)

[0003] Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (see MPEP § 2442.03(a)), Rule 30 EPC, and § 11 PatV, an electronic sequence listing compliant with WIPO standard ST.26 in the form of an XML 1.0 format file is submitted concurrently with the instant application, and the entire contents of the sequence listing are incorporated herein by reference. For the avoidance of doubt, if discrepancies exist between the sequences mentioned in the specification and the electronic sequence listing, the sequences in the specification shall be deemed to be the correct ones.

[0004] FIELD OF THE INVENTION

[0005] The present application relates to antibodies binding to PBP2a

[0006] BACKGROUND

[0007] Staphylococcus aureus is a Gram-positive coccoid bacterium that is both a human commensal organism part of the normal flora and a pathogen associated with significant morbidity and mortality. S. aureus may be found colonizing the nasal passages, vagina, urethra, perineum, pharynx, the entire gastrointestinal tract, skin, conjunctiva, and external ear. Although S. aureus may cause infections in healthy individuals who have no apparent risk factors, immunocompromised individuals have increased susceptibility. Infections typically manifest as skin and soft tissue infections (SSTIs), pneumonia, bone and joint infections, bacteremia, and endocarditis.

[0008] Staphylococcus aureus is one of the leading pathogens for deaths associated with antimicrobial resistance and the emergence of antibiotic-resistant strains, such as methicillin-resistant S. aureus (MRSA), is a worldwide problem in clinical medicine. Despite much research and development, no vaccine for S. aureus has been approved. Strains unable to resist these antibiotics are classified as methicillin-susceptible S. aureus, or MS SA.

[0009] MRSA infections result in approximately the same number of deaths annually in the U. S. as HIV-1, tuberculosis, and viral hepatitis combined. The prevalence of MRSA-colonized patients was reported to be 8.5% in health care and senior management facilities, and up to 29% of MRSA-colonized patients may subsequently develop a MRSA infection within 18 months. Greater than 50% of all hospital S. aureus infections are caused by methicillin- resistant S. aureus (MRSA). Compared with patients with bacteremia caused by methicillin-susceptible S. aureus, those with MRSA bacteremia have nearly twice the mortality rate, significantly longer hospital stays, and significantly higher median hospital costs.

[0010] It is hence one object of the present invention to provide diagnostic and therapeutic options to address MRSA infections. It is another object of the present invention to provide alternative diagnostic and therapeutic options to address MRSA infections. It is another object of the present invention to provide improved diagnostic and therapeutic options to address MRSA infections.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention provides, among others, antibodies that bind to PBP2a.

[0013] Brief description of the Figures:

[0014] Fig.1. Representative photo image for pre-selected clinically isolated MRSA and MSSA strains Fig.2. SDS-PAGE (2A) and Western Blotting analysis (2B) of expressed and purified MRSA PBP2a protein.

[0015] Fig.3 A. Selection of positive clones to PBP2a from Mouse antibody library by ELISA Fig.3B. Specific binding of two selected mouse antibodies to a coated PBP2a ELISA plate Fig.4A. Selection of positive clones to PBP2a from rabbit antibody library by ELISA Fig.4B. Specific binding of four selected rabbit antibodies to a coated PBP2a ELISA plate Fig.4C. Expression and purity of four selected rabbit antibodies, rAb-104=Ab4, rAb-105=Ab5, rAb-106=Ab6 and rAb-107=Ab7.

[0016] Fig.5. Western Blotting analysis of expressed and purified MRSA PBP2a antibodies, including rAb-101 and rAb-102.

[0017] Fig.6. Western Blotting analysis of expressed and purified MRSA PBP2a antibodies, including from rAb-104 to rAb-107.

[0018] Fig.6 A. Specifically binding to expressed PBP2a.

[0019] Fig.6B. Only specifically binding to PBP2a derived from MRSA not MS SA.

[0020] Fig.7A. Comparison binding of four antibodies against MRSA PBP2a by Indirect ELISA Fig.7B. Binding specificity of three biotinylating MRSA antibodies

[0021] Fig.7C. Pair combination of Mouse antibody with Rabbit antibody for detection of MRSA PBP2a.

[0022] Fig.7C-l. Mouse and rabbit antibodies were used for capture and detection reagents, respectively.

[0023] Fig.7C-2. Rabbit and Mouse antibodies were used for capture and detection reagents, respectively.

[0024] Fig.7D. Measurement of MRSA PBP2a using a antibody-based sandwich ELISA, Matched antibody pair, one mouse antibody and one rabbit antibody

[0025] Fig.8 A. Selected photo images for tested standard MRSA and MS SA stains by MRSA LFIA card.

[0026] Fig.8B. Selected photo images for tested clinically isolated MRSA and MSSA bacteria Fig.9A. Affinity measurement of mouse Antibody (rAb-101) against PBP2a of MRSA by SPR Fig.9B. Affinity measurement of mouse Antibody (rAb-102) against PBP2a of MRSA by SPR Fig.9C. Affinity measurement of rabbit Antibody (rAb-104) against PBP2a of MRSA by SPR Fig.9D. Affinity measurement of rabbit Antibody (rAb-106) against PBP2a of MRSA by SPR

[0027] DETAILED DESCRIPTION OF EMBODIMENTS Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts of the devices described or process steps of the methods described, as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include singular and / or plural referents unless the context clearly dictates otherwise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.

[0028] It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done.

[0029] Furthermore, the content of the prior art documents referred to herein is incorporated by reference. This refers, particularly, for prior art documents that disclose standard or routine methods. In that case, the incorporation by reference has mainly the purpose to provide sufficient enabling disclosure, and avoid lengthy repetitions.

[0030] According to one aspect of the invention, an antibody that binds to PBP2a, or a target-binding fragment or derivative of such antibody, is provided, which

[0031] a) comprises at least one set of three heavy chain and three light chain complementarity determining regions (CDR) comprised in the heavy chain / light variable domain sequence pairs selected from the list consisting of

[0032] SEQ ID NOs 7 and 8,

[0033] SEQ ID NOs 16 and 17,

[0034] SEQ ID NOs 25 and 26, SEQ ID NOs 34 and 35,

[0035] SEQ ID NOs 43 and 44, and / or

[0036] SEQ ID NOs 52 and 53,

[0037] b) comprises at least one set of three heavy chain and three light chain complementarity determining regions (CDR) selected from the list consisting of

[0038] • SEQ ID NOs 1 - 6,

[0039] • SEQ ID NOs 10 - 15,

[0040] • SEQ ID NOs 19 - 24,

[0041] • SEQ ID NOs 28 - 33,

[0042] • SEQ ID NOs 37 - 42, and / or

[0043] • SEQ ID NOs 46 - 51,

[0044] c) comprises the set of heavy chain / light chain complementarity determining regions (CDR) of b), with the proviso that at least one of the CDRs has up to 3 amino acid substitutions relative to the respective SEQ ID NOs, and / or

[0045] d) comprises the set of heavy chain / light chain complementarity determining regions (CDR) of b) or c), with the proviso that at least one of the CDRs has a sequence identity of > 66 % to the respective CDRs comprised in the SEQ ID NOs,

[0046] wherein the CDRs are embedded in a suitable protein framework, preferably a variable domain framework, so as to be capable to bind to PBP2a.

[0047] These sequences define the CDR regions of the antibodies according to the invention, or derivatives thereof.

[0048] Penicillin Binding Protein 2A (PBP2A) is a multi-modular class B penicillin-binding protein (PBP), which is located external to the membrane of all MRSA strains (von Eifif et al., 2002). The presence of PBP2a could be one of the key determinant of MRSA resistance. PBP2a allows cell wall biosynthesis in presence of most P-lactams antibiotics, like penicillin. The inventors have developed a set of anti-PBP2a antibodies which can protect subjects against MRSA infections or be used to identify the presence, or absence, of PBP2a in a sample, underscofing the presence of absence of MRS A strains.

[0049] According to one embodiment of the invention, the CDRs are determined according to the definition of Kabat, Chothia or MacCallum, preferably wherein the CDRs are determined according to the numbering set forth in Table 1.

[0050] As used herein, the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al. (1977), Chothia et al. (1987) and MacCallum et al., (1996) where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison.

[0051] Table 1: CDR definitions

[0052]

[0053] As used herein, the term “framework” when used in reference to an antibody variable domain is entered to mean all amino acid residues outside the CDR regions within the variable domain of an antibody. Therefore, a variable domain framework is between about 100-120 amino acids in length but is intended to reference only those amino acids outside of the CDRs.

[0054] As used herein, the term “capable to bind to target X” has to be understood as meaning that respective binding domain binds the target with a KD of 10'4or smaller. KD is the equilibrium dissociation constant, a ratio of koff / kon, between the antibody or fragment and its antigen. KD and affinity are inversely related. The KD value relates to the concentration of antibody or fragment (the amount of antibody or fragment needed for a particular experiment) and so the lower the KD value (lower concentration) and thus the higher the affinity of the binding domain. The following table shows typical KD ranges of monoclonal antibodies

[0055] Table 2: KDand Molar Values

[0056]

[0057] Preferably, the antibody or fragment has up to 2 amino acid substitutions, and more preferably up to 1 amino acid substitution.

[0058] Preferably, at least one of the CDRs of the antibody or fragment has a sequence identity of > 67 %; > 68 %; > 69 %; > 70 %; > 71 %; > 72 %; > 73 %; > 74 %; > 75 %; > 76 %; > 77 %; > 78 %; > 79 %; > 80 %; > 81 %; > 82 %; > 83 %; > 84 %; > 85 %; > 86 %; > 87 %; > 88 %; > 89 %; > 90 %; > 91 %; > 92 %; > 93 %; > 94 %; > 95 %; > 96 %; > 97 %; > 98 %; > 99 %, and most preferably 100 % to the respective SEQ ID NO.

[0059] “Percentage of sequence identity” as used herein, is determined by comparing two optimally aligned biosequences (amino acid sequences or polynucleotide sequences) over a comparison window, wherein the portion of the corresponding sequence in the comparison window may comprise additions or deletions (z.e., gaps) as compared to the reference sequence, which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0060] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (z.e., at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. The disclosure provides polypeptides that are substantially identical to the polypeptides exemplified herein. With respect to amino acid sequences, identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or over the full length of the reference sequence. With respect to shorter amino acid sequences, e.g., amino acid sequences of 20 or fewer amino acids, substantial identity exists when one or two amino acid residues are conservatively substituted, according to the conservative substitutions defined herein.

[0061] Preferably, at least one of the CDRs has been subject to CDR sequence modification, including

[0062] • affinity maturation, and / or

[0063] • reduction of immunogenicity

[0064] Affinity maturation in the process by which the affinity of a given antibody is increased in vitro. Like the natural counterpart, in vitro affinity maturation is based on the principles of mutation and selection. It has successfully been used to optimize antibodies, antibody fragments or other peptide molecules like antibody mimetics. Random mutations inside the CDRs are introduced using radiation, chemical mutagens or error-prone PCR. In addition, the genetic diversity can be increased by chain shuffling. Two or three rounds of mutation and selection using display methods like phage display usually results in antibody fragments with affinities in the low nanomolar range. For principles see Eylenstein et al. (2016) or US20050169925A1, the content of which is incorporated herein by reference for enablement purposes.

[0065] According to an embodiment of the invention, the CDRs are determined according to the definition of Kabat, Chothia or MacCallum. Preferably the CDRs are determined according to the numbering set forth in Table 1.

[0066] According to embodiments of the invention, the antibody, fragment or derivative comprises a) at least one the heavy chain / light chain variable domain (HCVD / LCVD) pair selected from the list consisting of:

[0067] • SEQ ID NOs 7 and 8,

[0068] • SEQ ID NOs 16 and 17,

[0069] • SEQ ID NOs 25 and 26,

[0070] • SEQ ID NOs 34 and 35,

[0071] • SEQ ID NOs 43 and 44, and / or

[0072] • SEQ ID NOs 52 and 53,

[0073] b) the heavy chain / light chain variable domains (HCVD / LCVD) pair of a), with the proviso that

[0074] • the HCVD has a sequence identity of > 80 % to the respective SEQ ID NO, and / or

[0075] • the LCVD has a sequence identity of > 80 % to the respective SEQ ID NO,

[0076] c) the heavy chain / light chain variable domains (VD) pairs of a) or b), with the proviso that at least one of the HCVD or LCVD has up to 10 amino acid substitutions relative to the respective SEQ ID NO,

[0077] said antibody or fragment still being capable to bind to PBP2a.

[0078] These sequences define the VH and VL domains of the anti-PBP2a antibodies, or derivatives thereof.

[0079] A “variable domain” when used in reference to an antibody or a heavy or light chain thereof is intended to mean the portion of an antibody which confers antigen binding onto the molecule and which is not the constant region. The term is intended to include functional fragments thereof which maintain some of all of the binding function of the whole variable region. Variable region binding fragments include, for example, functional fragments such as Fab, F(ab)2, Fv, single chain Fv (scfv) and the like. Such functional fragments are well known to those skilled in the art. Accordingly, the use of these terms in describing functional fragments of a heteromeric variable region is intended to correspond to the definitions well known to those skilled in the art. Such terms are described in, for example, Huston et al., (1993) or Plückthun and Skerra (1990).

[0080] Preferably, the HCVD and / or LCVD has a sequence identity of > 81 %; > 82 %; > 83 %; > 84 %; > 85 %; > 86 %; > 87 %; > 88 %; > 89 %; > 90 %; > 91 %; > 92 %; > 93 %; > 94 %; > 95 %; > 96 %; > 97 %; > 98 %; > 99 %; or most preferably 100 % to the respective SEQ ID NO.

[0081] According to embodiments of the invention, at least one amino acid substitution is a conservative amino acid substitution.

[0082] According to another aspect of the invention, an antibody that binds to PBP2a, or a targetbinding fragment or derivative of such antibody, is provided, which has a binding affinity of > 50 % to PBP2a compared to that of the antibody, derivative or fragment according to the above description.

[0083] As used herein the term “binding affinity” is intended to mean the strength of a binding interaction and therefore includes both the actual binding affinity as well as the apparent binding affinity. The actual binding affinity is a ratio of the association rate over the disassociation rate. Therefore, conferring or optimizing binding affinity includes altering either or both of these components to achieve the desired level of binding affinity. The apparent affinity can include, for example, the avidity of the interaction. For example, a bivalent heteromeric variable region binding fragment can exhibit altered or optimized binding affinity due to its valency.

[0084] A suitable method for measuring the affinity of a binding agent is through surface plasmon resonance (SPR). This method is based on the phenomenon which occurs when surface plasmon waves are excited at a metal / liquid interface. Light is directed at, and reflected from, the side of the surface not in contact with sample, and SPR causes a reduction in the reflected light intensity at a specific combination of angle and wavelength. Biomolecular binding events cause changes in the refractive index at the surface layer, which are detected as changes in the SPR signal. The binding event can be either binding association or disassociation between a receptorligand pair. The changes in refractive index can be measured essentially instantaneously and therefore allows for determination of the individual components of an affinity constant. More specifically, the method enables accurate measurements of association rates (kon) and disassociation rates (koff). Measurements of konand kOff values can be advantageous because they can identify altered variable regions or optimized variable regions that are therapeutically more efficacious. For example, an altered variable region, or heteromeric binding fragment thereof, can be more efficacious because it has, for example, a higher konvalued compared to variable regions and heteromeric binding fragments that exhibit similar binding affinity. Increased efficacy is conferred because molecules with higher konvalues can specifically bind and inhibit their target at a faster rate. Similarly, a molecule of the invention can be more efficacious because it exhibits a lower kOff value compared to molecules having similar binding affinity. Increased efficacy observed with molecules having lower kOff rates can be observed because, once bound, the molecules are slower to dissociate from their target. Although described with reference to the altered variable regions and optimized variable regions of the invention including, heteromeric variable region binding fragments thereof, the methods described above for measuring associating and disassociation rates are applicable to essentially any antibody or fragment or fragment thereof for identifying more effective binders for therapeutic or diagnostic purposes.

[0085] Another suitable method for measuring the affinity of a binding agent is through surface is by FACS / scatchard analysis. See inter alia example 1 for a respective description.

[0086] Methods for measuring the affinity, including association and disassociation rates using surface plasmon resonance are well known in the arts and can be found described in, for example, Jonsson and Malmquist, (1992) and Wu et al. (1998). Moreover, one apparatus well known in the art for measuring binding interactions is a BIAcore 2000 instrument which is commercially available through Pharmacia Biosensor, (Uppsala, Sweden).

[0087] Preferably said target binding affinity is > 51%, > 52%, > 53%, > 54%, > 55%, > 56%, > 57%, > 58%, > 59%, > 60%, > 61%, > 62%, > 63%, > 64%, > 65%, > 66%, > 67%, > 68%, > 69%, > 70%, > 71%, > 72%, > 73%, > 74%, > 75%, > 76%, > 77%, > 78%, > 79%, > 80%, > 81%, > 82%, > 83%, > 84%, > 85%, > 86%, > 87%, > 88%, > 89%, > 90%, > 91%, > 92%, > 93%, > 94%, > 95%, > 96%, > 97%, > 98%, and most preferably > 99 % compared to that of the reference binding agent. According to another aspect of the invention, an antibody that binds to PBP2a, or a targetbinding fragment or derivative of such antibody is provided, which competes for binding to PBP2a with the antibody, derivative or fragment according to the above description.

[0088] According to another aspect of the invention, an antibody or a target-binding fragment or derivative of such antibody is provided that binds to essentially the same, or the same, region on PBP2a as the antibody, derivative or fragment according to the above description.

[0089] As used herein, the term “region” shall be understood to mean an extracellular region, a domain, a subdomain, or a secondary structure (e.g. loop), or preferably an epitope.

[0090] As regards the format or structure of such antibody or fragment, the same preferred embodiments as set forth above apply. In one embodiment, said antibody or fragment is a monoclonal antibody, or a target-binding fragment or derivative thereof retaining target binding capacities, or an antibody mimetic.

[0091] As used herein, the term "competes for binding" is used in reference to one of the antibodies defined by the sequences as above, meaning that the actual antibody or fragment as an activity which binds to the same target, or target epitope or domain or subdomain, as does said sequence defined antibody or fragment, and is a variant of the latter. The efficiency (e.g., kinetics or thermodynamics) of binding may be the same as or greater than or less than the efficiency of the latter. For example, the equilibrium binding constant for binding to the substrate may be different for the two antibodies.

[0092] Such competition for binding can be suitably measured with a competitive binding assay. Such assays are disclosed in Finco et al. 2011, the content of which is incorporated herein by reference for enablement purposes, and their meaning for interpretation of a patent claim is disclosed in Deng et al 2018, the content of which is incorporated herein by reference for enablement purposes.

[0093] In order to test for this characteristic, suitable epitope mapping technologies are available, including, inter alia, • X-ray co-crystallography and cryogenic electron microscopy (cryo-EM) • Array-based oligo-peptide scanning

[0094] • Site-directed mutagenesis mapping

[0095] • High-throughput shotgun mutagenesis epitope mapping

[0096] • Hydrogen-deuterium exchange

[0097] • Cross-linking-coupled mass spectrometry

[0098] These methods are, inter alia, disclosed and discussed in Banik et al (2010), and DeLisser (1999), the content of which is herein incorporated by reference for enablement purposes.

[0099] According to an embodiment of the invention, the antibody or fragment or derivative is a monoclonal antibody, or a target-binding fragment or derivative thereof retaining target binding capacities.

[0100] According to an embodiment of the invention, the antibody or fragment or derivative is in at least one of the formats selected from the group consisting of: IgG, scFv, scFv-Fc, scFv-diabody, Fab, or (Fab)2.

[0101] As used herein, the term “monoclonal antibody (mAb)” shall refer to an antibody composition having a homogenous antibody population, i.e., a homogeneous population consisting of a whole immunoglobulin, or a fragment or derivative thereof retaining target binding capacities.

[0102] Particularly preferred, such antibody is an IgG antibody, or a fragment or derivative thereof retaining target binding capacities. Immunoglobulin G (IgG) is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. IgG molecules are created and released by plasma B cells. Each IgG has two antigen binding sites.

[0103] IgG antibodies are large molecules with a molecular weight of about 150 kDa made of four peptide chains. It contains two identical class y heavy chains of about 50 kDa and two identical light chains of about 25 kDa, thus a tetrameric quaternary structure. The two heavy chains are linked to each other and to a light chain each by disulfide bonds. The resulting tetramer has two identical halves, which together form the Y-like shape. Each end of the fork contains an identical antigen binding site. The Fc regions of IgGs bear a highly conserved N-glycosylation site. The N-glycans attached to this site are predominantly core-fucosylated diantennary structures of the complex type. In addition, small amounts of these N-glycans also bear bisecting GlcNAc and a-2,6-linked sialic acid residues.

[0104] There are four IgG subclasses (IgGl, 2, 3, and 4) in humans, named in order of their abundance in serum (IgGl being the most abundant).

[0105] As used herein, the term “fragment” shall refer to fragments of such antibody retaining target binding capacities, e.g.

[0106] • a CDR (complementarity determining region)

[0107] • a hypervariable region,

[0108] • a variable domain (Fv)

[0109] • an IgG or IgM heavy chain (consisting of VH, CHI, hinge, CH2 and CH3 regions)

[0110] • an IgG or IgM light chain (consisting of VL and CL regions), and / or

[0111] • a Fab and / or F(ab)2.

[0112] As used herein, the term “derivative” shall refer to protein constructs being structurally different from, but still having some structural relationship to, the common antibody concept, e.g., scFv, Fab and / or F(ab)2, as well as bi-, tri- or higher specific antibody constructs, and further retaining target binding capacities. All these items are explained below.

[0113] Other antibody derivatives known to the skilled person are Diabodies, Camelid Antibodies, Nanobodies, Domain Antibodies, bivalent homodimers with two chains consisting of scFvs, IgAs (two IgG structures joined by a J chain and a secretory component), shark antibodies, antibodies consisting of new world primate framework plus non-new world primate CDR, dimerized constructs comprising CH3+VL+VH, and antibody conjugates (e.g. antibody or fragments or derivatives linked to a toxin, a cytokine, a radioisotope or a label). These types are well described in the literature and can be used by the skilled person on the basis of the present disclosure, without adding further inventive activity.

[0114] Methods for the production of a hybridoma cell are disclosed in Kohler & Milstein (1975). Methods for the production and / or selection of fully human mAbs are known in the art. These can involve the use of a transgenic animal which is immunized with the respective protein or peptide, or the use of a suitable display technique, like yeast display, phage display, B-cell display or ribosome display, where antibodies from a library are screened against FGFR1 in a stationary phase.

[0115] In vitro antibody libraries are, among others, disclosed in US6300064 by MorphoSys and US6248516 by MRC / Scripps / Stratagene. Phage Display techniques are for example disclosed in US5223409 by Dyax. Transgenic mammal platforms are for example described in EP1480515A2 by Taconic Artemis.

[0116] IgG, IgM, scFv, scFv-Fc, scFv-diabody, Fab and / or F(ab)2 are antibody formats well known to the skilled person. Related enabling techniques are available from the respective textbooks.

[0117] As used herein, the term “Fab” relates to an IgG / IgM fragment comprising the antigen binding region, said fragment being composed of one constant and one variable domain from each heavy and light chain of the antibody.

[0118] As used herein, the term “F(ab)2” relates to an IgG / IgM fragment consisting of two Fab fragments connected to one another by disulfide bonds.

[0119] As used herein, the term “scFv” relates to a single-chain variable fragment being a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short linker, usually serine (S) or glycine (G). This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. scFv-Fc and scFv-diabody are modified formats of scFv. scFv-Fc consists of two scFv domains each of which is fused to an Fc domain. Both resulting chains are typically held together by two disulfide bonds arranged in the hinge region. A diabody is composed of two scFv. Depending on the specificities of these two parts a diabody can be monospecific, i.e. both scFv are directed against the same antigen, or bispecific, i.e. the two scFv are directed against two different antigens.

[0120] Modified antibody formats are for example bi- or trispecific antibody constructs, antibodybased fusion proteins, immunoconjugates and the like. These types are well described in the literature and can be used by the skilled person on the basis of the present disclosure, without adding further inventive activity.

[0121] According to yet another aspect of the invention, the antibody, conjugate or fusion protein according to the above description is provided for (the manufacture of a medicament for) use in the treatment of a human or animal subject

[0122] • being diagnosed for,

[0123] • suffering from or

[0124] • being at risk of developing

[0125] an infection by MRSA.

[0126] This language is deemed to encompass both the swiss type claim language accepted in some countries (in this case, brackets are deemed absent) and EPC2000 language (in this case, brackets and content within the brackets is deemed absent).

[0127] According to yet another aspect of the invention, a method for treating or preventing an infection by MRSA in a human or animal subject is provided, which method comprises administration of the antibody, conjugate or fusion protein according to the above description to a patient, in one or more therapeutically sufficient doses.

[0128] According to yet another aspect of the invention, the antibody or fragment or derivative according to the above description is provided for (the manufacture of a medicament for) use in the diagnosis of a human or animal subject for an infection by MRSA.

[0129] This language is deemed to encompass both the swiss type claim language accepted in some countries (in this case, brackets are deemed absent) and EPC2000 language (in this case, brackets and content within the brackets is deemed absent).

[0130] According to embodiments, the antibody or fragment according to the above description is labelled with a detectable marker.

[0131] According to embodiments, the detectable marker is selected from the group consisting of enzymes, paramagnetic materials, members of the avidin-biotin specific binding pair, fluorophores, chromophores, chemiluminophores, heavy metals, and radioisotopes. According to one embodiment, the antibody is labelled with a peroxidase enzyme.

[0132] According to yet another aspect of the invention, an ELISA method, a WB method or a LFIA method is provided, in which method an antibody or fragment according to the above description is used. The method serves to identify MRSA by detecting the presence of absence of PBP2a in a sample.

[0133] Such method is sometimes called the “MRSA assay according to the invention” herein.

[0134] According to yet another aspect of the invention, An ELISA kit, a WB kit or a LFIA kit, which kit comprises an antibody or fragment according to the above description.

[0135] As used herein, the term ELISA stands for enzyme-linked immunosorbent assay, which is a commonly used analytical biochemistry assay. The assay is a solid-phase type of enzyme immunoassay (EIA) to detect the presence of a ligand (commonly an amino acid) in a liquid sample using antibodies directed against the ligand to be measured. ELISA has been used as a diagnostic tool in medicine, plant pathology, and biotechnology, as well as a quality control check in various industries.

[0136] Typically, antigens from the sample to be tested are attached to a surface. Then, a matching antibody is applied over the surface so it can bind the antigen. This antibody is linked to an enzyme, and then any unbound antibodies are removed. In the final step, a substance containing the enzyme's substrate is added. If there was binding, the subsequent reaction produces a detectable signal, most commonly a color change.

[0137] Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation. Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are non-specifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.

[0138] As used herein, the term WB stands for western blot. This method is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract, and to visualize, distinguish, and quantify the different proteins in a complicated protein combination. Western blot technique uses three elements to achieve its task of separating a specific protein from a complex: separation by size, transfer of protein to a solid support, and marking target protein using a primary and secondary antibody to visualize. An antibody (known as the primary antibody) is created that recognizes and binds to a specific target protein. The electrophoresis membrane is washed in a solution containing the primary antibody, before excess antibody is washed off. A secondary antibody is added which recognizes and binds to the primary antibody. The secondary antibody is visualized through various methods such as staining, immunofluorescence, and radioactivity, allowing indirect detection of the specific target protein.

[0139] As used herein, the term LFIA stands for lateral flow immunoassay, which operates on the same principles of affinity chromatography as the enzyme-linked immunosorbent assays (ELISA). In essence, these tests run the liquid sample along the surface of a pad with reactive molecules that show a visual positive or negative result. The pads are based on a series of capillary beds, such as pieces of porous paper, microstructured polymer, or sintered polymer. Each of these pads has the capacity to transport fluid (e.g., urine, blood, saliva) spontaneously.

[0140] The sample pad acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid flows to the second conjugate pad in which the manufacturer has stored freeze dried bio-active particles called conjugates (see below) in a salt-sugar matrix. The conjugate pad contains all the reagents required for an optimized chemical reaction between the target molecule (e.g., an antigen) and its chemical partner (e.g., antibody) that has been immobilized on the particle's surface. This marks target particles as they pass through the pad and continue across to the test and control lines. The test line shows a signal, often a color as in pregnancy tests. The control line contains affinity ligands which show whether the sample has flowed through and the biomolecules in the conjugate pad are active. After passing these reaction zones, the fluid enters the final porous material, the wick, that simply acts as a waste container. LFIAs can operate as either competitive or sandwich assays.

[0141] EXAMPLES

[0142] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0143] All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'.

[0144] 1. Materials

[0145] 1.1. Antigen and Antibody

[0146] Both MRSA PBP2a antigen and the two mouse and four rabbit antibodies against PBP2a listed in the Table 3 below were made and prepared in-house.

[0147] Table 3. MRRSA protein and antibodies disclosed herein

[0148]

[0149]

[0150] In brief, the DNA sequence encoding PBP2a protein was codon-optimized for E. Coli and synthesized (General Bio). A hexa-histidine tag was fused to the C terminus of the expressed PBP2a for protein detection and purification purposes. The expression and purity of PBP2a protein were assessed by standard SDS-PAGE and Western Blotting methods (See Figs.2A and 2B below7, also see Refs 1-3).

[0151] DNA fragments of the heavy and light chains were cloned into the pATXI expression vector and transfected into CHO-S cells via electroporation. The transfected cells were screened in medium containing 10 or 20 gg / mL puromycin (Gibco) for 7 days. The medium was changed every 3 days during screening. Positive cells were inoculated into medium at a concentration of 5 x 105cells / mL and incubated for 10 days. The glucose concentration was maintained at 5 g / L. Then, the culture supernatant was collected, and the antibody was purified using the AKTA Prime Plus System (GE Healthcare) and a HiTrap Protein G HP antibody purification column (Cytiva).

[0152] 1.2. MRSA and MSSA bacteria and bacterial preparation

[0153] 1.2.1. Standard strains

[0154] Two standard S. aureus strains, positive (43300 KS) and negative (29213 KS) listed in the Table 2 were chosen for binding characterization of selected six MRSA antibodies by ELISA, Western Blotting, Lateral Flow7ImmunoAssay (LFIA) as described in Sections 2.1, 2.2 and 2.3 below. Table 2. Selected MRSA and MSSA strains

[0155]

[0156] 1.2.2. Clinical Isolates

[0157] Identification of S. aureus, confirmation of methicillin resistance (MRSA) and antibiotic susceptibility (MSSA) testing were carried using the VITEK 2 automated platform (bioMe rieux, Marcy-l’E toile, France) in accordance to manufacturer’s instructions and Clinical and Laboratory Standards Institute guidelines (Ref.4). Isolates meeting the inclusion criteria were studied for lateral flow immunoassay and molecular characterization (See Staph Colony Image as shown in Fig.l). A total of 309 isolates (MRSA: n=186; MSSA: n=123) were tested by LFIA.

[0158] 1.2.3. Preparation of MRSA and MSSA bacterial sample

[0159] Collect 10 L grown bacteria pellet from broth tube after centrifuging it at 2500 g RCF and transfer it to the 1.5 ml clean Eppendorf tube containing 100 l MRSA / MSSA assay buffer (80 mM Tris, 1% NaCl, 1% S9 Surfactant, 1% Triton X- 100, 1% PVP K30, 0.5% Casein, pH 9.0 to 9.1). Alternatively, pick up single colony from agar plate by using inoculating loop, then put the loop into the pre-loaded buffer tube, mix it well and keep it at RM for 2 mins. Centrifuge the tube above at 2500 g RCF for 2 mins. Add 15 pl of the treated MRSA or MSSA supernatant from previous steps into new Eppendorf tube containing 55 pl of MRSA assay buffer, gently stir it and keep it at RT for 2 minutes. The sample is ready for LFIA testing.

[0160] 1.3. Assembled LFIA strip

[0161] The sample pad (glass fiber, grade 8951, Ahlstrom-Munksjb, Helsinki, Finland) was treated with PBS (pH 7.4) containing 1% BSA and 0.05% Tween 20 and the conjugate pad (glass fiber, grade 8950, Ahlstrom) was treated with sodium phosphate dibasic solution (5 mM, pH 7.4) containing 0.5% BSA and 1% Tween 20. After pre-treatment of the sample and conjugate pad, these pads were dried at 37 °C for 24 h. The dried conjugate pad and nitrocellulose (NC) membrane (CN 110, Sartorius, Gottingen, Germany) were then attached to a plastic backing card (PJEAGO, Seoul, South Korea), and an absorbent pad (cellulose fiber, C083, Merck Millipore, Darmstadt, Germany) was stacked onto the NC membrane. The laminated lateral flow assay strip was cut into 3.8 mm-wide strips using a guillotine cutter (Zeta Corporation, Gunpo, South Korea). In each cut, the conjugate pad was prepared by adding 1 pL of different concentration of AuNP probes and then incubated at 37°C for 1.5 h. For the test and control line zone, 0.4 pL of anti-PBP2a capture antibody and anti-mouse IgG was added on the NC membrane and dried at 37°C for 1.5 h. Then, the LFA test strips were prepared and stored in a 4°C refrigerator for testing.

[0162] 2. Methods

[0163] 2.1. Enzyme-Linked Immunosorbent Assay (ELISA) Assay

[0164] Purified PBP2a protein was suspended in carbonate / bicarbonate buffer (Sigma) and 100 pl / well was coated onto the surface of Immulon 2HB plates at a concentration of 100 pg / mL. Plates were allowed to incubate overnight at 4° C. and the coating solution was removed prior to blocking plates. Plates were blocked with 300 pL / well of Neptune block (Immunochemistry Technologies LLC) and allowed to incubate for two hours at 37° C. Plates were then washed one time in PBS-T using a plate washer set to aspirate blocking buffer, add 300 pL / well of wash buffer to the plate, and then aspirate the wash buffer. Next, own-made recombinant antibodies, serially diluted at from 0.015625 pg to Ipg, were added to the plate in duplicate in 100 pL volumes and allowed to incubate for one hour at 37°C. Plates were then washed three times in PBS-T using the same volume setting as the first wash. Conjugate (peroxidase labeled antimouse or anti-rabbit secondary antibody) was added to the plate at a 1 / 2000 dilution and allowed to incubate for one hour at 37° C. After the conjugate incubation, plates were washed four times in PBS-T and developed using TMB chromogen. The chromogen was added at 100 pL / well and allowed to develop for ten minutes at room temperature. The reaction was stopped by the addition of IN sulfuric acid added to the plate at 100 pL / well and then absorbance was detected by Biotek Synergy Hl plate reader at 450 nm (See refs.9-10).

[0165] 2.2. SDS-PAGE and Western Blot Assay

[0166] PBP2a protein, extracted protein from cultured MRSA and MSSA bacteria and unrelated control protein were separately incubated with SDS-PAGE sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% beta-mercaptoethanol, and 0.004% bromophenol blue) at 100 °C for 5 min. Tris-glycine SDS running buffer (25 mM Tris, 250 mM glycine, 0.1% SDS) was used for electrophoresis. After electrophoresis, the proteins were transferred onto the nitrocellulose membranes (Millipore) at 80 mA for 1 h by using Tris-glycine SDS (48 mM Tris, 39 mM glycine, 0.037% SDS) transfer buffer with 20% methanol and then stained with Coomassie Brilliant Blue (CBB) R-250 dye for the rapid reversible detection of protein bands for Western Blotting.

[0167] For the conventional Western Blotting, the blotted membranes were directly blocked with a blocking buffer (5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBST) for 1 h and then probed with primary six antibodies we generated and characterized, including rAb-101, rAb-102. rAb-104. rAb-105, rAb-106 and rAb-107, followed by HRP-conjugated anti-mouse and anti-rabbit secondary antibody, respectively. After washing with TBST 3 times, the immunoreactive bands were detected by ChemiDoc XRS Image System (Bio-Rad Laboratories, Hercules, CA, USA).

[0168] 2.3, Colloidal gold-based Lateral Flow ImmunoAssay (LFIA)

[0169] The Lateral Flow Immunoassay is performed with sample being applied to sample pad which when goes forward binds to the gold nanoparticles biosensor conjugate and flows in order for the specific analyte to be captured by capture antibody and in doing so the color develops to red and then changes to purple. Next the gold nanoparticle biosensor binds to control protein to give color development (See refs.11-12).

[0170] Conjugation of gold nanoparticles with an antibody: 300 pL of 15 nm the conjugation of gold nanoparticles (AuNPs) was pipetted into an Eppendorf tube, and 30 u L of 6 pg / ml of MRS A PBP2a detection antibody was added and vortexed to mix the solution. The solution was allowed to sit for 30 minutes, then 100 pL of 10% BSA was added. The mixture was centrifuged at 3220 x G (6000 RPM) for 1 hour, and the supernatant was removed. The pellets were resuspended in 100 pL of 1% BSA. The mixture was centrifuged at 3220 x G for 1 hour, and the supernatant was removed again. The pellets were resuspended in 1000 pL of 1% BSA. The mixture was centrifuged at 3220 x G for 1 hour, the supernatant was removed, and the pellets were resuspended in 200 pL of storage buffer and stored at 4° C until use. The conjugation process for each solution was measured by DLS and UV-visible spectroscopy and the changes in absorption peak after conjugation of antibody to the AuNPs were detected.

[0171] Creating Test and Control lines: The Auto-Laminator applicator was employed to construct the Test line (T) and the Control line (C) by loading MRS A PBP2a antibody (Pre-selected capture antibody, i.e. rAb-106 and so on) and goat anti-mouse antibody on the membrane, respectively. The C and T lines were constructed at distances of 15 and 21 mm from the edge of the membrane, respectively.

[0172] In details, for the T line, a solution consisting of 25 pg / mL purified PBP2a antibody, 50 mL / L methanol, and 20 g / L sucrose in freshly prepared 100 mM NaHCO3 buffer (pH 8.5) was loaded at a density of 500 ng per strip, with dispensing velocity 60 nL / s.

[0173] For C line, a solution containing 25 pg / mL goat anti-mouse antibody, 50 mL / L methanol, and 20 g / L sucrose in 100 mM NaHCO3 buffer (pH 8.5) was loaded at a density of 500 ng per strip, with dispensing velocity 60 nL / s. The membrane was dried in an oven for at 37°C overnight and stored at room temperature. The final strips were assembled as described below. Strips with a 5-mm width were cut using a Guillotine cutter and stored dry, at ambient temperature.

[0174] Assembly of the strip: The pretreated nitrocellulose membranes with laminated backing were used with dimensions of 6 cm (length) x 0.5 cm per strip. A pretreated conjugate pad containing the immobilized gold-nanoparticle conjugated antibody was attached to the backing next to the nitrocellulose membrane on the Test line side. Next, the pretreated sample pad was attached to the backing next to the conjugate pad. Then, the absorbent pad was attached to the backing next to the nitrocellulose membrane on the control line side as shown in Schematic diagram of immunochromatographic test to detect antibodies against MRSA PBP2a below (see Strip in Fig. 10A.). All the pads were overlapping by 0.2 cm. Lastly, each fully assembled strip was cut with a width of 0.5 cm. The strips were stored at room temperature until used.

[0175] Running the strips: See Section 4.6 below.

[0176] 2.4. Surface Plasmon Resonance (SPR) Assay — Antibody Binding Affinity Measurement

[0177] The Biacore® technology monitors the binding between biomolecules in real time and without the requirement for labeling. One of the interactants, termed the ligand, is either immobilized directly or captured on the immobilized surface while the other, termed the analyte, flows in solution over the captured surface. The sensor detects the change in mass on the sensor surface as the analyte binds to the ligand to form a complex on the surface. This corresponds to the association process. The dissociation process is monitored when the analyte is replaced by buffer (See refs.13-14). In the affinity Biacore® assay, the analyte is six anti~PBP2a antibodies, such as rAb-106 described above, and the ligand is MRSA PBP2a protein.

[0178] Details of the method are as follows:

[0179] Instrument: Biacore® 3000, Biacore AB, Uppsala, Sweden.

[0180] Sensor chip: CM5 (research grade) Catalogue Number: BR-1001-14, Biacore AB, Uppsala, Sweden. Chips were stored at 4° C.

[0181] Buffers: Running buffer is HBS-EP (being 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM ED A, 0.005% Surfactant P20). Catalogue Number: BR-1001-88, Biacore AB, Uppsala, Sweden. Buffer stored at 4° C. Immobilization buffer is Acetate 5.0 (being 10 mM sodium acetate pH 5.0). Catalogue number: BR-1003-51, Biacore AB, Uppsala, Sweden. Buffer stored at 4° C. Ligand: Recombinant MRSA PBP2a protein.

[0182] Analyte: six anti-PBP2a antibodies (See Table 1 and antibody list in provisional application). The materials were prepared at 2 mg / mL (40 pM) in phosphate-buffered saline, stored at 4°C, and diluted in HBE-EP running buffer for the assays. Typically anti-PBP2a antibodies were diluted from ~0.5 nM to ~10 pM by doubling dilutions for the affinity assay.

[0183] Regeneration Solution: 40 mM HC1 prepared by dilution with distilled water from an 11.6 M stock solution (BDH, Poole, England. Catalogue number: 101254H). 5 mM NaOH prepared by dilution with distilled water from a 50 mM stock solution. Catalogue number: BR- 1003-58, Biacore AB, Uppsala, Sweden.

[0184] Assay Method: BIA (Biamolecular interaction Analysis) was performed using a Biacore® 3000 (Biacore AB). MRSA PBP2a protein was immobilized on a CMS Sensor Chip. HBS-EP buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore AB) was used as the running buffer with a flow rate of 10 pl / min. Anti-PBP2a antibodies were used at concentrations such that, once captured by the immobilized antigen surface, gave a signal. MRSA PBP2a antibodies were titrated over the captured PBP2a antigen, at various concentrations. 90 uL of MRSA PBP2a antibody was injected over the surface (association phase), followed by a 240 second dissociation phase, all at a flow rate of 30 pL / min. The surface was regenerated by two 10 pL injections of 40 mM HC1, followed by a 5 pL injection of 5 M NaOH at a flow rate of 10 gL / min. Background subtraction binding curves were analyzed using the BIA evaluation software (version 3.2) following standard procedures. Kinetic parameters were determined from the fitting algorithm.

[0185] 3. Examples

[0186] 3.1. Generation of MRSA PBP2a protein antigen

[0187] To test the utility and specificity of immunization antigen and detection reagent for PBP2a protein containing HisTag, we used mouse anti-HisTag monoclonal antibody as a probe for Western Blot analysis. We found that the HisTag antibody was able to react with denatured protein adhered to a nitrocellulose membrane, and that this reaction was highly specific - as evidenced by the presence of only a single band in the lane corresponding to A. Coli lysate wherein PBP2a was expressed, and no visible bands in the lane corresponding to cells expressing an empty vector control (Fig.2A and 2B),

[0188] This figure shows how the PBP2a protein partitions during purification and demonstrates the purity of the final antigen preparation.

[0189] 3.2. Generation of Recombinant Monoclonal Antibodies Using Rabbit B Cells

[0190] As described 4.1. Section above, the PBP2a antigen of MRS A was produced. The successful procedure is outlined in Fig. 10B and is based around standard animal immunization and single" B cell technology, which enabled the selection of antibodies of a given specificity and ensured that they were affinity matured. Throughput was increased by immunizing mice and rabbit with the antigen from our two constructed antibody libraries, mouse and rabbit libraries, so that, antibodies to the PBP2a protein could be selected simultaneously from a single animal library (See Refs.5-8).

[0191] 3.2.1. Immunization

[0192] Purified, E. Coli -derived PBP2a was dissolved in NaCl-Histidin buffer pH 6.1 and mixed with an equal volume of complete Freund's adjuvant (CFA) till generation of a stable emulsion. We immunized 6 mice and 3 rabbits following an immunization protocol which has been established. The immunization protocol includes the repeated injection of immunogen emulsified with CFA into the same animal rotating different application routes. Titers were monitored regularly during the immunization period. From each animal samples of peripheral whole blood were collected 4-6 days after the third and fourth injection by bleed, respectively, and used for B-cell isolation.

[0193] Two single chain variable fragment (scFv) libraries were constructed with the resultant antibody repertoire. Mouse library contained 1.2 x 10 unique clones and rabbit libraiy contained 3.6 x 109clones. Biopanning strategies were designed to drive the enrichment of high-affinity binders to the PBP2a. To illustrate the success of the selection strategies, 20 unique scFv sequences in total were isolated and expressed as soluble scFv in a bacterial expression system for further characterization.

[0194] A series of ranking protocols were employed to narrow down the number of scFvs to a panel of lead antibodies.

[0195] After the initial B-Cell Cloning using peripheral B cells of immunized mouse and rabbits, the supernatants of the individual B-cell clones (ASCs) are analyzed by several appropriate ELISA screening assays (primary screening), while the mRNA containing lysates of all B-cell clones are preserved at -20°C. After identification of the supernatants containing the recombinant monoclonal antibodies with the desired properties, the corresponding preserved lysates of the B-cell clones are hit-picked and fed into the B-Cell PCR process. After recombinant expression in mammalian cells, the rabbit recombinant antibodies are purified and thereby concentrated using a chemically defined buffer / medium. The secondary screening round confirms the primary screening and allows additional functional and epitope characterizing assays. After the aforementioned criteria have been satisfied, the expression plasmid of the antibody with the desired properties can be used for a larger scale production.

[0196] 3.3, Selection and screening of two mouse and four rabbit recombinant antibodies to PBP2a

[0197] Two phage libraries expressing mouse and rabbit scFvs were separately incubated with purified PBP2a protein and then the phage that had bound to specific antigen were captured using immunotubes precoated with decreasing concentration of PBP2a protein. Panning of the library on immunotubes with coated PBP2a showed enrichment with regards to phage titer (See Fig.

[0198] 3A / 4A). This procedure gave a better enrichment and resulted in the isolation of multiple new antibodies against PBP2a antigen.

[0199] With each round of panning, we detected the anti-PBP2a antibody by ELISA. After each round of panning, we obtained multiple positive clones with PBP2a binding activities, respectively. After 3 rounds of selection, 57 of the 80 clones were found to bind PBP2a protein and gave various ELISA readings in the presence of PBP2a antigen using indirect ELISA as shown in Fig. 3B / 4B Of the clones thus screened, these 20 clones were selectively sequenced. For six phage clones, the binding activities were detected using soluble scFv fragments, instead of phage scFv. Six different antibodies were identified in round 3 (rAb-101, rAb-102, rAb-104, rAb-105, rAb-106 and rAb-107). These clones strongly bound PBP2a by ELISA. One antibody (rAb-102) had a low absorbance, whereas rest five antibodies reacted strongly, with signals above A4502.0.

[0200] Two mouse and four rabbit rAbs were produced and the binding with PBP2a was confirmed by indirect ELIS A (Fig.3A-3B / 4A-4B). The five rAbs, rAb-101, rAb-104, rAb-105, rAb-106 and rAb-107 showed higher binding affinity against a E. Coli-expressed PBP2a recombinant protein. Predicted molecular weight of 76 kDa protein was detected by SDS-PAGE analysis with potential post translational modifications (Figs. 3C and 4C). All five purified rAbs specifically recognized PBP2a recombinant protein and showed similar pattern of bands and sizes on Western blot.

[0201] This figure shows how the screened PBP2a antibody partitions (from rAb-104 throughout rAb-107,) during purification and demonstrates the purity of the final antibody preparation.

[0202] 3.4. Specificity, expression and purification of mouse and rabbit recombinant antibodies

[0203] In order to achieve overexpression and purification of full length of IgG antibodies in CHO cell, the gene encoding the single-chain variable fragment (scFv) was inserted into vector pTAXl, which results in fusion of scFv and Fc fragments. All full IgG antibodies (rAb) were purified on a small-scale using Protein A / G affinity column chromatography. As shown in Fig.3C and 4C, all six rAbs were detected in all. A single band of approximately 150 Kda was detectable. SDS-PAGE analysis revealed that non-denatured and denatured rAb of over 95% purity were obtained. The soluble fractions obtained from 1 L of culture yielded about 1-3 mg of rAbs.

[0204] To determine whether the antibodies were specific for PBP2a protein of MRSA, we performed an ELISA with the PBP2a. The selected antibodies gave different ELISA results. The binding characteristics of the converted full mouse and rabbit IgG antibodies were examined in further detail by immunoblot analysis. As shown in Figs.5A and 6A, two mouse antibodies (rAb-101 and rAb-102) and four rabbit antibodies (rAb-104, rAb-105, rAb-106 and rAb-107) bound specifically to purified PBP2a (76 kDa) when compared to commercial anti-PBP2a monoclonal antibody (Fig.5B).

[0205] On the Western Blot, six selected antibodies also detected a strong band from real preparation samples MRSA but not from MSSA bacteria in indicated lane, a size consistent with that of the PBP2a capsid protein (See Fig.6B). From these results, the single chain antibodies and full antibodies with Fc fragment that we obtained are able to recognize and bind to expressed and bacterial PBP2a protein.

[0206] The scFv was purified to homogeneity from soluble protein obtained from the supernatant and the periplasmic fractions by affinity chromatography. This figure shows how the screened six PBP2a antibodies partition during purification and demonstrates the purity of the final antibody preparation.

[0207] 3.5. Determination of pairing PBP2a antibody

[0208] At least four antibodies sandwich ELISA detecting PBP2a were developed (Fig. 7A). The three ELISA-positive mouse and rabbit antibodies were biotinylated and used to determine the best antibody pairing showing the strongest signal in a sandwich ELISA platform (Fig.7B).

[0209] Interestingly, the three defined mouse and rabbit recombinant antibody combinations showing strong affinity were found in the pairing assay with one mouse rAb (rAb-101) as a capture antibody (cAb) and two rabbit rAbs (rAb- 104 or rAb- 106 ) as a detection antibody (dAb). Similarly, in the reverse combination using two rabbit rAbs (rAb-104 or rAb-106 ) as a capture Ab, rAb-101 and rAb-106 were the best detection antibody pair with high sensitivity. Two different pair combinations (rAb-101 / rAb-104) and (rAb-101 / rAb-106) were selected for sandwich ELISA and standard curve was established using PBP2a recombinant protein (Figs.

[0210] 7C to 7D).

[0211] 3.6. Detection of MRSA by sandwich antibody-based Lateral Flow Immunoassay

[0212] In brief, 70 pL of MRSA or MSSA samples (See Method Section: 1.2.3 above) were added onto the sample well. After a short time (no longer than 15 min) of incubation, MRSA positive sample containing PBP2a (Fig. 8A-B above) could show up both clear color T line (Test) and C line (Control); the MSSA negative sample with containing no PBP2a only color C line were regarded as negative (Fig. 8A-B above); the strips with no C line showed up should be considered as the invalid test.

[0213] Furthermore, a total of 309 isolates (MRSA: n=186; MSSA: n=123) were tested by Lateral Flow Immunoassay (See Table 4). Eight different kinds of clinical samples have been examined using the MRSA LFIA according to the invention assay thus far. Of the 309 samples, patient saliva has the biggest sample size, followed by abscess samples.

[0214] Table 4. Collected and Prepared clinical specimen

[0215]

[0216] Of the 186 MRSA isolates identified by Oxacillin antibiotics test, 186 isolates were detected positively by LFIA and as resistant isolates. However, there were four isolates which yielded weak positive signals on the lateral flow immunoassay.

[0217] Of the 123 MSSA isolates identified by Oxacillin antibiotics test, 123 isolates were detected negatively by LFIA and as susceptible isolates.

[0218] The sensitivity and specificity of the LFIA in the detection of MRSA isolates compared to by Oxacillin antibiotics test is almost 100.00% and 100.00%, respectively.

[0219] The results of our study have acceptable sensitivity and specificity compared to the Abbott CLEARVIEW™ and Oxacillin antibiotics test methods (see Table 5). Our new MRSA assay has shown a good performance compared to existed MRSA assay from Abbott.

[0220] Table 5. Comparison of LFIA assay according to the invention with Abbott CLEARVIEW™

[0221]

[0222]

[0223] A comparative study was conducted on the MRSA assay according to the invention and the Abbott CLEARVIEW™ assay by evaluating six clinically isolated strains, as detailed in Table 6. The MRSA assay according to the invention is capable of detecting all six isolates as MRSA, whereas the Abbott CLEARVIEW™ assay can only detect 2 of the six isolates.

[0224] Table 6. Comparison of the MRSA assay according to the invention with ABBOTT'S Kit

[0225]

[0226] 3.7. High-affinity mouse and rabbit anti-PBP2a antibody panel

[0227] For full kinetics and affinity measurements, Fig.9A to Fig.9D and Table 7 summarize the affinity data obtained for six recombinant antibodies (rAbs) binding their purified recombinant as well as bacterial PBP2a antigen. Biacore® analysis of two mouse (rAb-101 and rAb-102) and four rabbit (rAb-104, rAb-105, rAb-106 and rAb-107) antibodies binding to MRSA PBP2a protein indicated that rAb-101, rAb-104 and rAb-106 displayed strong binding to PBP2a when added to the PBP2a (Figs. 9A, 9C and 9D). rAb-102 was not able to binding to PBP2a when added to the PBP2a (Fig. 9B). Table 7. Binding activity and application of six MRSA multiple Antibodies

[0228]

[0229] Antibodies rAb-105 and rAb-107 exhibited comparable binding results to PBP2a in the ELISA competition experiment, although they were not evaluated using SPR.

[0230] Affinity ranking was undertaken using ELISA (Figs. 3B and 4B) and subsequently, Surface Plasmon Resonance (SPR), using immobilized PBP2a ligand as antigen. The binding affinity of rAbs ranged from high nanomolar (KD 5.65 nM) to sub-picomolar (KD 38.6 pM) and their affinities largely correlated with their ranking using ELISA, WB and LFIA data (see Supplementary Table 7). References

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[0267] SEQUENCES

[0268] The following sequences form part of the disclosure of the present application. A WIPO ST 26 compatible electronic sequence listing is provided with this application, too. For the avoidance of doubt, if discrepancies exist between the sequences in the following table and the electronic sequence listing, the sequences in this table shall be deemed to be the correct ones.

[0269] In some cases, signal peptides may be encompassed in the reproduced sequences. In such case, the sequences shall be deemed disclosed with and without signal peptides (like e.g a peptide with AA sequence MKHLWFFLLLVAAPRWVLS). A readily available tool to identify signal peptides in a given protein sequence is SignalP - 6.0 provided by Dansk Technical University under https: / / services.healthtech.dtu.dk / service.php7Si nalP. The same applies to His tags, C-Myc tags, or S-tags (like e.g. an S-tag with the AA sequence GSWSHPQFEK). Table 8: Sequence listing

[0270]

[0271]

[0272]

Claims

What is claimed:

1. An antibody that binds to PBP2a, or a target-binding fragment or derivative of such antibody, whicha) comprises at least one set of three heavy chain and three light chain complementarity determining regions (CDR) comprised in the heavy chain / light variable domain sequence pairs selected from the list consisting of• SEQ ID NOs 7 and 8,• SEQ ID NOs 16 and 17,• SEQ ID NOs 25 and 26,• SEQ ID NOs 34 and 35,• SEQ ID NOs 43 and 44, and / or• SEQ ID NOs 52 and 53,b) comprises at least one set of three heavy chain and three light chain complementarity determining regions (CDR) selected from the list consisting of• SEQ ID NOs 1 - 6,• SEQ ID NOs 10 - 15,• SEQ ID NOs 19 - 24,• SEQ ID NOs 28 - 33,• SEQ ID NOs 37 - 42, and / or• SEQ ID NOs 46 - 51,c) comprises the set of heavy chain / light chain complementarity determining regions (CDR) of b), with the proviso that at least one of the CDRs has up to 3 amino acid substitutions relative to the respective SEQ ID NOs, and / ord) comprises the set of heavy chain / light chain complementarity determining regions (CDR) of b) or c), with the proviso that at least one of the CDRs has a sequence identity of > 66 % to the respective CDRs comprised in the SEQ ID NOs,wherein the CDRs are embedded in a suitable protein framework, preferably a variable domain framework, so as to be capable to bind to PBP2a.

2. The antibody or fragment or derivative according to claim 1, wherein the CDRs are determined according to the definition of Kabat, Chothia or MacCallum, preferably wherein the CDRs are determined according to the numbering set forth in Table 1.

3. The antibody or fragment or derivative according to any one of claims 1 - 2, which comprisesa) at least one the heavy chain / light chain variable domain (HCVD / LCVD) pair selected from the list consisting of:• SEQ ID NOs 7 and 8,• SEQ ID NOs 16 and 17,• SEQ ID NOs 25 and 26,• SEQ ID NOs 34 and 35,• SEQ ID NOs 43 and 44, and / or• SEQ ID NOs 52 and 53,b) the heavy chain / light chain variable domains (HCVD / LCVD) pair of a), with the proviso that• the HCVD has a sequence identity of > 80 % to the respective SEQ ID NO, and / or• the LCVD has a sequence identity of > 80 % to the respective SEQ ID NO,c) the heavy chain / light chain variable domains (VD) pairs of a) or b), with the proviso that at least one of the HCVD or LCVD has up to 10 amino acid substitutions relative to the respective SEQ ID NO,said antibody or fragment still being capable to bind to PBP2a.

4. The antibody or fragment according to any one of claims 1 -3, wherein at least one amino acid substitution is a conservative amino acid substitution.

5. An antibody that binds to PBP2a, or a target-binding fragment or derivative of such antibody, which• has a binding affinity of > 50 % to PBP2a compared to that of the antibody or fragment or derivative according to any one of the aforementioned claims 1 - 4, and / or• competes for binding to PBP2a with an antibody according to any one of claims 1 - 4.

6. An antibody or a target-binding fragment or derivative of such antibody that binds to essentially the same, or the same, region on PBP2a as an antibody according to any one of claims 1 - 5.

7. The antibody or fragment or derivative according to any one of the aforementioned claims, which is a monoclonal antibody, or a target-binding fragment or derivative thereof retaining target binding capacities.

8. The antibody or fragment or derivative according to any one of the aforementioned claims, which is in at least one of the formats selected from the group consisting of: IgG, scFv, Fab, or (Fab)2.

9. The antibody or fragment or derivative according to any one of the aforementioned claims for (the manufacture of a medicament for) use in the treatment of a human or animal subject• being diagnosed for,• suffering from or• being at risk of developingan infection by MRSA.

10. The antibody or fragment or derivative according to any one of the aforementioned claims for (the manufacture of a medicament for) use in the diagnosis of a human or animal subject for an infection by MRSA.

11. The antibody or fragment according to any one of the aforementioned claims, which is labelled with a detectable marker.

12. The antibody or fragment according to claim 11, wherein the detectable marker is selected from the group consisting of enzymes, paramagnetic materials, members of the avidin-biotin specific binding pair, fluorophores, chromophores, chemiluminophores, heavy metals, and radioisotopes.

13. An ELISA method, a WB method or a LFIA method, in which method an antibody or fragment according to any one of the aforementioned claims is used.

14. An ELISA kit, a WB kit or a LFIA kit, which kit comprises an antibody or fragment according to any one of the aforementioned claims.

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