FIELD OF THE INVENTION

The invention refers to a monoclonal antibody that specifically recognizes a unique galactan-based O-antigen structure, which is associated with the majority of epidemic multi-drug resistantKlebsiella pneumoniaeST258.

BACKGROUND OF THE INVENTION

Klebsiella pneumoniaeis an important enterobacterial pathogen responsible for urinary tract infections, pneumonia, and septicaemia that cause significant morbidity and mortality. Multi-drug resistant (MDR) strains have recently emerged and spread globally, against which therapeutic options are limited.

Lipopolysaccharide (LPS) is the major constituent of the outer leaflet of the outer membrane of Gram-negative bacteria, such asKlebsiella pneumoniae. LPS has three major structurally and functionally diverse parts: i) lipid A, which is also known as endotoxin, ii) core-oligosaccharide, and iii) O-antigen, which is built up of repeating units of oligosaccharide blocks.

K. pneumoniaeO-antigens are surface antigens of diverse structure, defining different O-types. The most common serotypes among the currently recognized 7 O-types appear to be O1 and O2, which together were reported to be expressed by the majority (i.e. >50%) of all isolates(1;2). Both O1 and O2 antigens are composed of galactose polymers, i.e. galactans. The O2 antigen (also known as O2a or O2ab in order to differentiate from O2ac described below) is made up of repeats of the so-called galactan-I disaccharide (seeFIG. 3). In contrast, the O1 and O2ac antigens do contain additional distinct structures besides galactan-I as follows: the LPS core-proximal portion is constituted of repeats of galactan-I, which is capped by either galactan-II (a different homopolymer of galactose in case of O1) or a non-galactan repeating unit (in case of O2ac).

The serotypes sharing the galactan-I O-subunit carry a highly similar rfb locus encoding synthesis and export of this structure. The nucleotide sequence of the galactan-I encoding operon has been partially determined (3). The locus was described to be 7.3 kb long comprising 6 genes. Complementation of rough mutants ofK. pneumoniaeO1,E. coliK-12 orSalmonella entericaserovar Typhimurium by the cloned rfb locus restored production of smooth LPS consisting of galactan-I O-antigen repeats. This suggests that these 6 genes are essential and sufficient for the production of galactan-I O-antigen side chains (3). Structural modification of D-galactan-I in different O2Klebsiellastrains was published by Kelly et al. (5). Nevertheless, the genetic background for these modifications remains to be elucidated. The genetic determinants encoding galactan-II repeating units (i.e. those capping either galactan-I or galactan-III presented herewith) were recently described (4). Importantly, these genes are unrelated to the genetic determinants responsible for the conversion of galactan-I units to galactan-III. Consequently, O1 serotype strains, besides expressing the serotype determining surface located galactan-II repeating units can express either galactan-I or galactan-III repeating units bridging the Lipid A-core and galactan-II repeats.

Multi-drug resistant (MDR) strains ofK. pneumoniaethat have emerged recently cause a significant proportion ofK. pneumoniaeinfections. Treatment options against MDR strains are getting very limited as they have evolved resistance to most classes of clinically relevant antibiotics. Therefore, alternative treatment options, e.g. passive immunization with monoclonal antibodies (mAbs) hold a great promise for the future.

There is a need for new targets ofKlebsiella pneumoniae. In particular, targets need to be identified which are immunorelevant and may be used for developing therapies and diagnostics.

SUMMARY OF THE INVENTION

It is the objective of the present invention to provide for an antibody directed againstK. pneumoniae, in particular MDR strains, with improved relevance to target the pathogen, to be used for the prevention or therapy ofK. pneumoniaeinfections. It is further the objective to provide means and methods that are capable of diagnosingK. pneumoniaebacteria, such as MDR strains, in a rapid and reliable manner.

The object is solved by the subject of the present invention.

According to the invention there is provided an isolated antibody that specifically recognizes a galactan-III (gal-III) epitope of the lipopolysaccharide (LPS) O-antigen structure ofKlebsiella pneumoniae, which epitope is incorporated in galactan-III repeating units, wherein the galactan-III repeating unit is a branched galactose homopolymer of Formula (I)



Specifically the galactan-III epitope is incorporated in an O-antigen structure comprising at least 2 gal-III repeating units, or at least 3, 4, or 5.

According to a specific aspect, the antibody preferentially binds to the galactan-III epitope relative to the galactan-I epitope, or which does not cross-react with the galactan-I epitope, wherein the galactan-I (gal-I) epitope is incorporated in galactan-I repeating units of the LPS O2a-antigen structure ofKlebsiella pneumoniae, and wherein the galactan-I repeating unit is a linear galactose homopolymer of Formula (II)
[→3)-β-D-Galf-(1→3)-α-D-Galp-(1→]  Formula (II)

For example, the antibody of the invention is a gal-III specific antibody which is specifically recognizing or binding the O-antigen structure comprising the gal-III antigen. Exemplary antibodies are listed inFIGS. 1 and 2, or variants of such antibodies. For the purpose of providing variants, the antibodies are herein referred to as parent antibodies, and CDR or framework sequences are herein referred to as parent CDR or parent framework sequences.

According to a specific aspect, the antibody comprises recombinant CDR and framework sequences, e.g. of different origin, wherein at least one of the CDR and framework sequences includes human, humanized, chimeric, murine or affinity matured sequences, preferably wherein the framework sequences are of any immunoglobulin isotype, and in particular of an IgG antibody.

Specifically, the antibody of the invention is cross-specific to bind the gal-III and gal-I epitopes, and preferentially binds to the gal-III antigenic structure relative to a gal-I antigenic structure of an O2 antigen ofK. pneumoniae, e.g. with an affinity which is higher to bind the gal-III as compared to the gal-I antigen. According to a specific embodiment, the antibody has at least two-fold greater affinity for binding the gal-III antigen as compared to the gal-I antigen, specifically with at least two-fold difference, or at least three-fold, at least four-fold, at least 5-fold, or even at least 10-fold difference, e.g. difference in affinity and/or avidity. For example, the Kd difference to preferentially bind the gal-III antigen over the gal-I antigen is at least 0.5 or 1 log, or even at least 2 logs, or at least 3 logs different, as determined by an immunoassay, preferably immunoblotting, ELISA or other immunological methods.

The antibody of the invention is specifically further characterized that it does not cross-react with any otherK. pneumoniaeantigen, and/or the antibody binds to any otherK. pneumoniaeantigen with a lower affinity, e.g. where the Kd difference to preferentially bind the gal-III antigen over otherK. pneumoniaeantigens (other than the gal-III or gal-I antigens) is at least 2 logs, preferably at least 3 logs.

Specifically, the functionally active variant is a functionally active CDR variant which comprises at least one point mutation in the parent CDR sequence, and comprises or consists of the amino acid sequence that has at least 60% sequence identity with the parent CDR sequence, preferably at least 70%, at least 80%, at least 90% sequence identity.

A specific variant is e.g., a humanized variant of the parent antibody, wherein the parent CDR sequences are incorporated into human or humanized framework sequences, wherein optionally 1, 2, 3, or 4 amino acid residues of each of the parent CDR sequences may be further mutated by introducing point mutations to improve the stability, specificity and affinity of the parent or humanized antibody.

Specifically, the VH or heavy chain (HC) sequences of such variants may be substituted by VH and HC sequences of another variant, respectively, in particular where the other variant is any other variant of the same parent antibody.

Specifically, the VL or light chain (LC) sequences of such variants may be substituted by VL and LC sequences of another variant, respectively, in particular where the other variant is any other variant of the same parent antibody.

Specifically, the galactan-III epitope is expressed by multi-drug resistant (MDR)Klebsiella pneumoniae, more specifically the MDR clone ST258. Specifically, the galactan-III epitope is of the multi-drug resistant (MDR)Klebsiella pneumoniae.

According to a specific embodiment, the antibody has an affinity to bind the galactan-III epitope with a Kd of less than 10−7M, preferably less than 10−8M, even more preferably less than 10−9M.

Variants of parent antibodies which are produced by affinity maturation, herein referred to as affinity-maturated variants, may have an increased binding affinity, with a Kd difference of at least 1 log, or 2 logs, or 3 logs, as compared to the parent antibody. Affinity maturated variants typically have an affinity to bind the gal-III antigen with a Kd of less than 10−8M, or less than 10−9M. If the parent antibody has an affinity with a Kd of less than 10−8M, or less than 10−9M, and the parent antibody is undergoing affinity maturation, the affinity matured variant may have an even higher affinity with a Kd of less than 10−9M and less than 10−10M, respectively.

According to a specific aspect, the antibody is a neutralizing antibody. Specifically the antibody is neutralizing endotoxin (i.e. LPS) ofKlebsiella pneumoniaestrains expressing the galactan-III epitope, as determined by an in vitro or in vivo detection method. Specifically, the antibody neutralizes endotoxic effect of specific LPS molecules in vitro.

Specifically, the antibody is neutralizing endotoxin ofKlebsiella pneumoniaestrains expressing the galactan-III epitope, wherein the neutralization potency is at least the potency of a reference antibody (e.g. the reference antibody 2D8-A10), which comprisesa) a CDR1 consisting of the amino acid sequence of SEQ ID 10; andb) a CDR2 consisting of the amino acid sequence of SEQ ID 11; andc) a CDR3 consisting of the amino acid sequence of SEQ ID 12; andd) a CDR4 consisting of the amino acid sequence of SEQ ID 19; ande) a CDR5 consisting of the amino acid sequence of SEQ ID 17; andf) a CDR6 consisting of the amino acid sequence of SEQ ID 18,

according to the nomenclature of Kabat. Such CDR sequences are designated according to the numbering system of Kabat.

In the following, unless indicated otherwise, reference is made to the CDR sequences as numbered according to Kabat, i.e. as determined according to Kabat nomenclature (see Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, U.S. Department of Health and Human Services. (1991)), and in particular those CDR sequences as listed in Table 1. It is well understood that the invention and the scope of the claims shall also encompass the same antibodies and CDR, yet with a different numbering and designated CDR region, where CDR regions are defined according to the IMGT system (The international ImMunoGeneTics, Lefranc et al., 1999, Nucleic Acids Res. 27: 209-212).

Specifically, theKlebsiella pneumoniaestrain targeted by the antibody of the invention is characterized by a rfbgal-Ilocus incorporating additional glycosyl transferase (gtr) genes.

According to a specific aspect, the antibody recognizes the MDRKlebsiella pneumoniaeclone ST258, in particular, a strain expressing the galactan-III epitope.

A specific embodiment refers to an antibody which is any of a full-length monoclonal antibody, an antibody fragment thereof comprising at least one antibody domain incorporating the binding site, or a fusion protein comprising at least one antibody domain incorporating the binding site, specifically wherein the antibody is a non-naturally occurring antibody which comprises a randomized or artificial amino acid sequence.

Specifically, the antibody is an antibody selected from the group consisting of murine, lama, rabbit, goat, cow, chimeric, humanized or human antibodies, heavy-chain antibodies, Fab, Fd, scFv and single-domain antibodies like VH, VHH or VL, preferably a human IgG antibody or a murine IgG antibody.

Specifically, the antibody is a monoclonal antibody.

According to a specific embodiment, the antibody comprises at least an antibody heavy chain variable region or domain (VH), which is characterized by any of the CDR1 to CDR3 sequences as listed in Table 1, which are designated according to the numbering system of Kabat, or functionally active CDR variants thereof.

According to a specific aspect, the invention provides for exemplary (parent) antibodies as detailed in the figures provided herein, and further antibody variants of such parent antibodies, in particular including variants binding to essentially the same epitope, as the parent antibody which is characterized by the specific binding site formed by the VH and the VL amino acid sequences ofFIG. 2, or else by the respective CDR sequences of Table 1. Such antibodies may e.g. be functionally active variant antibodies obtained by modifying the respective CDR or antibody sequence of the parent antibody. It is well understood that any antibody sequence as described herein is considered a “parent” sequence which is subject to variation, e.g. by point mutations.

The antibodies described in the examples are of murine origin or humanized forms thereof. Variants which are obtained by humanization and optionally affinity maturation may be engineered using well-known techniques. These variant antibodies bind to the target antigen, thus, are considered functionally active. It is feasible that also variant VH or VL domains, e.g. with modifications in the respective FR or CDR sequences may be used, which are functionally active, e.g. binding to the same epitope or comprising the same binding site or having the same binding characteristics as the parent antibody. It is also feasible that some of the FR or CDR sequences of the antibodies described herein may be exchanged by those of other antibodies, e.g. of antibodies as listed in Table 1.

Specifically, the antibody of the invention comprises any of the CDR sequences of the antibody heavy chain variable region as depicted inFIG. 1(Table 1) or functionally active CDR variants thereof and/or a VH amino acid sequence selected from any of the VH sequences as depicted inFIG. 2or functionally active variants thereof, e.g. an antibody heavy chain (HC) or VH amino acid sequence which is comprising CDR1, 2, and 3, wherein any of the CDR1 sequences 1, 4, 7, or 10; and/or any of the CDR2 sequences 2, 5, 8, or 11; and/or any of the CDR3 sequences 3, 6, 9, or 12; or comprising any of the VH sequences 19, 21, 23, or 25.

Specifically, the antibody is

A)

selected from the group consisting of group members i) to iv), wherein

i)

is an antibody which comprisesa) a CDR1 consisting of the amino acid sequence of SEQ ID 1; andb) a CDR2 consisting of the amino acid sequence of SEQ ID 2; andc) a CDR3 consisting of the amino acid sequence of SEQ ID 3;

ii)

is an antibody which comprisesa) a CDR1 consisting of the amino acid sequence of SEQ ID 4; andb) a CDR2 consisting of the amino acid sequence of SEQ ID 5; andc) a CDR3 consisting of the amino acid sequence of SEQ ID 6;

iii)

is an antibody which comprisesa) a CDR1 consisting of the amino acid sequence of SEQ ID 7; andb) a CDR2 consisting of the amino acid sequence of SEQ ID 8; andc) a CDR3 consisting of the amino acid sequence of SEQ ID 9;

iv)

is an antibody which comprisesa) a CDR1 consisting of the amino acid sequence of SEQ ID 10; andb) a CDR2 consisting of the amino acid sequence of SEQ ID 11; andc) a CDR3 consisting of the amino acid sequence of SEQ ID 12;

or

B) an antibody which is a functionally active variant of a parent antibody that is any of the group members of A, which comprises at least one functionally active CDR variant of any of the CDR1, CDR2 or CDR3 of the parent antibody.

Specifically, the functionally active variant is a functionally active CDR variant which comprises at least one point mutation in the parent CDR sequence, and comprises or consists of the amino acid sequence that has at least 60% sequence identity with the parent CDR sequence, preferably at least 70%, at least 80%, at least 90% sequence identity.

Specifically, the antibody comprises a VH amino acid sequence selected from any of the VH sequences as depicted inFIG. 2.

According to a specific embodiment, the antibody further comprises an antibody light chain variable region or domain (VL), which comprises any of the CDR4 to CDR6 sequences as listed in Table 1, which are designated according to the numbering system of Kabat, or functionally active CDR variants thereof.

Specifically, the antibody of the invention comprises any of the CDR sequences of the antibody light chain variable region as depicted inFIG. 1(Table 1) or functionally active CDR variants thereof and/or a VL amino acid sequence selected from any of the VL sequences as depicted inFIG. 2or functionally active variants thereof, e.g. an antibody light chain (LC) or VL amino acid sequence which is comprising CDR4, 5, and 6, wherein any of the CDR4 sequences 13, 16, or 19; and/or any of the CDR5 sequences 14, 17, or 20; and/or any of the CDR6 sequences 3, 6, 9, or 12; or comprising any of the VH sequences 15 or 18, or comprising any of the VL sequences 20, 22, 24, or 26.

Specifically, the antibody is

A)

selected from the group consisting of group members i) to iv), wherein

i)

is an antibody which comprisesa) a CDR4 consisting of the amino acid sequence of SEQ ID 13; andb) a CDR5 consisting of the amino acid sequence of SEQ ID 14; andc) a CDR6 consisting of the amino acid sequence of SEQ ID 15;

ii)

is an antibody which comprisesa) a CDR4 consisting of the amino acid sequence of SEQ ID 16; andb) a CDR5 consisting of the amino acid sequence of SEQ ID 17; andc) a CDR6 consisting of the amino acid sequence of SEQ ID 18;

iii)

is an antibody which comprisesa) a CDR4 consisting of the amino acid sequence of SEQ ID 19; andb) a CDR5 consisting of the amino acid sequence of SEQ ID 20; andc) a CDR6 consisting of the amino acid sequence of SEQ ID 18;

iv)

is an antibody which comprisesa) a CDR4 consisting of the amino acid sequence of SEQ ID 19; andb) a CDR5 consisting of the amino acid sequence of SEQ ID 17; andc) a CDR6 consisting of the amino acid sequence of SEQ ID 18;

or

B) an antibody which is a functionally active variant of a parent antibody that is any of the group members of A, which comprises at least one functionally active CDR variant of any of the CDR4, CDR5 or CDR6 of the parent antibody.

Specifically, the functionally active variant is a functionally active CDR variant which comprises at least one point mutation in the parent CDR sequence, and comprises or consists of the amino acid sequence that has at least 60% sequence identity with the parent CDR sequence, preferably at least 70%, at least 80%, at least 90% sequence identity.

Specifically, the antibody comprises a VL domain characterized bya) a CDR4 consisting of the amino acid sequence of SEQ ID 19 or a functionally active CDR variant of the CDR4; andb) a CDR5 consisting of the amino acid sequence of SEQ ID 20 or a functionally active CDR variant of the CDR5; andc) a CDR6 consisting of the amino acid sequence of SEQ ID 18 or a functionally active CDR variant of the CDR6;preferably in combination with any of the VH sequences as described herein.

Specifically, the antibody comprises a VL amino acid sequence selected from any of the VL sequences as depicted inFIG. 2.

Specifically, the antibody comprises both, VH and VL amino acid sequences, and optionally further framework sequences of a full-length antibody or an antibody fragment, in particular any of a full-length antibody or Fab fragment.

Specifically, the antibody comprisesa) the CDR1-CDR6 sequences of any of the antibodies as listed in Table 1; orb) the VH and VL sequences of any of the antibodies as depicted inFIG. 2; orc) which is a functionally active variant of a parent antibody that is characterized by the sequences of a)-c),preferably whereini. the functionally active variant comprises at least one functionally active CDR variant of any of the CDR1-CDR6 of the parent antibody; and/orii. the functionally active variant comprises at least one point mutation in the framework region of any of the VH and VL sequences;and further whereiniii. the functionally active variant has a specificity to bind the same epitope as the parent antibody; and/oriv. the functionally active variant is a human, humanized, chimeric or murine and/or affinity matured variant of the parent antibody.

Specifically, the antibody is selected from the group consisting ofa) an antibody comprisinga. the CDR1 sequence of SEQ ID 1; andb. the CDR2 sequence of SEQ ID 2; andc. the CDR3 sequence of SEQ ID 3; andd. the CDR4 sequence of SEQ ID 13; ande. the CDR5 sequence of SEQ ID 14; andf. the CDR6 sequence of SEQ ID 15;b) an antibody comprisinga. the CDR1 sequence of SEQ ID 4; andb. the CDR2 sequence of SEQ ID 5; andc. the CDR3 sequence of SEQ ID 6; andd. the CDR4 sequence of SEQ ID 16; ande. the CDR5 sequence of SEQ ID 17; andf. the CDR6 sequence of SEQ ID 18;c) an antibody comprisinga. the CDR1 sequence of SEQ ID 7; andb. the CDR2 sequence of SEQ ID 8; andc. the CDR3 sequence of SEQ ID 9; andd. the CDR4 sequence of SEQ ID 19; ande. the CDR5 sequence of SEQ ID 20; andf. the CDR6 sequence of SEQ ID 18;d) an antibody comprisinga. the CDR1 sequence of SEQ ID 10; andb. the CDR2 sequence of SEQ ID 11; andc. the CDR3 sequence of SEQ ID 12; andd. the CDR4 sequence of SEQ ID 19; ande. the CDR5 sequence of SEQ ID 17; andf. the CDR6 sequence of SEQ ID 18;e) an antibody comprisinga. the CDR1 sequence of SEQ ID 4; andb. the CDR2 sequence of SEQ ID 5; andc. the CDR3 sequence of SEQ ID 6; andd. the CDR4 sequence of SEQ ID 19; ande. the CDR5 sequence of SEQ ID 20; andf. the CDR6 sequence of SEQ ID 18;

andf) an antibody comprisinga. the CDR1 sequence of SEQ ID 10; andb. the CDR2 sequence of SEQ ID 11; andc. the CDR3 sequence of SEQ ID 12; andd. the CDR4 sequence of SEQ ID 19; ande. the CDR5 sequence of SEQ ID 20; andf. the CDR6 sequence of SEQ ID 18;

or a functionally active CDR variant of any of the foregoing, which has an affinity to bind the gal-III antigen with a Kd of less than 10−8M, preferably less than 10−9M, preferably less than 10−10M, preferably less than 10−11M, e.g. with an affinity in the picomolar range.

Specifically the antibody comprises a functionally active CDR variant of any of the CDR sequences as listed in Table 1, wherein the functionally active CDR variant comprises at least one of

a) 1, 2, or 3 point mutations in the parent CDR sequence; and/or

b) 1 or 2 point mutations in any of the four C-terminal or four N-terminal, or four centric amino acid positions of the parent CDR sequence; and/or

c) at least 60% sequence identity with the parent CDR sequence;

preferably wherein the functionally active CDR variant comprises 1 or 2 point mutations in any CDR sequence consisting of less than 4 or 5 amino acids.

Specifically, the functionally active variant antibody comprises at least one of the functionally active CDR variants of the invention. Specifically, the functionally active variant antibody comprising one or more of the functionally active CDR variants has a specificity to bind the same epitope as the parent antibody.

Specifically, the functionally active variant is a CDR variant, e.g. which comprises a CDR, more specifically a CDR loop sequence, with an amino acid sequence having at least 60% sequence identity, preferably at least 70%, 80% or 90% sequence identity.

According to a specific aspect, the at least one point mutation is any of an amino acid substitution, deletion and/or insertion of one or more amino acids.

Specifically, the functionally active variant differs from the parent antibody in at least one point mutation in the amino acid sequence, preferably in the CDR, wherein the number of point mutations in each of the CDR amino acid sequences is either 0, 1, 2 or 3.

Specifically, the antibody is derived from such antibodies, employing the respective CDR sequences, or CDR mutants, including functionally active CDR variants, e.g. with 1, 2 or 3 point mutations within one CDR loop, e.g. within a CDR length of 5-18 amino acids, e.g. within a CDR region of 5-15 amino acids or 5-10 amino acids. Alternatively, there may be 1 to 2 point mutations within one CDR loop, e.g. within a CDR length of less than 5 amino acids, to provide for an antibody comprising a functionally active CDR variant. Specific CDR sequences might be short, e.g. the CDR2 or CDR5 sequences. According to a specific embodiment, the functionally active CDR variant comprises 1 or 2 point mutations in any CDR sequence consisting of less than 4 or 5 amino acids.

According to a specific aspect, the antibody of the invention comprises CDR and framework sequences, wherein at least one of the CDR and framework sequences includes human, humanized, chimeric, murine or affinity matured sequences, preferably wherein the framework sequences are of an IgG antibody, e.g. of an IgG1, IgG2, IgG3, or IgG4 subtype, or of an IgA1, IgA2, IgD, IgE, or IgM antibody.

Specific antibodies are provided as framework mutated antibodies, e.g. to improve manufacturability or tolerability of a parent antibody, e.g. to provide an improved (mutated) antibody which has a low immunogenic potential, such as humanized antibodies with mutations in any of the CDR sequences and/or framework sequences as compared to a parent antibody.

Further specific antibodies are provided as CDR mutated antibodies, e.g. to improve the affinity of an antibody and/or to target the same epitope or epitopes near the epitope that is targeted by a parent antibody (epitope shift).

Accordingly, any of the antibodies as listed in Table 1 orFIG. 2may be used as parent antibodies to engineer improved versions.

According to a specific aspect, the antibody of the invention comprises CDR combinations as listed inFIG. 1(Table 1), provided, that the antibody is still functionally active.

Specifically, the antibody of the invention comprises the CDR1-6 of any of the antibodies as listed in Table 1. However, according to an alternative embodiment, the antibody may comprise different CDR combinations, e.g. wherein an antibody as listed in Table 1 comprises at least one CDR sequence, such as 1, 2, 3, 4, 5, or 6 CDR sequences of one antibody and at least one further CDR sequence of a different antibody of any of the antibodies as listed in Table 1. According to a specific example, the antibody comprises 1, 2, 3, 4, 5, or 6 CDR sequences, wherein the CDR sequences are CDR combinations of more than 1 antibody, e.g. 2, 3, 4, 5, or 6 different antibodies. For example, the CDR sequences may be combined to preferably comprise 1, 2, or all 3 of CDR1-3 of any of the antibodies as listed in Table 1, and 1, 2, or all 3 of CDR4-6 of the same or any other antibody listed in Table 1.

For example, the CDR sequences may be combined to preferably comprise at least CDR1-3 of any of the antibodies as listed in Table 1, e.g. any of the antibodies designated 8E3-E5, 9H9-H7, 5A4-A7, or 2D8-A10 and/or at least CDR4-6 of any (other) of the antibodies as listed in Table 1, e.g. of the antibody designated 5A4-A7, or at least its CDR4 and CDR6 sequences in combination with a functionally active CDR variant of its CDR5. According to a specific embodiment, the antibody of the invention comprises the CDR1-6 of any of the antibodies as listed in Table 1, e.g. any of the antibodies designated 8E3-E5, 9H9-H7, 5A4-A7, or 2D8-A10. However, according to a further specific aspect, the antibody may comprise different CDR combinations, e.g. wherein an antibody as listed in Table 1, e.g. any of the antibodies designated 8E3-E5, 9H9-H7, 5A4-A7, or 2D8-A10 comprises at least one CDR sequence, such as 1, 2, 3, 4, 5, or 6 CDR sequences, of a different antibody, e.g. a CDR sequence of any different antibody of any of the antibodies as listed in Table 1. For example, the antibody comprises 1, 2, 3, 4, 5, or 6 CDR sequences, wherein the CDR sequences are CDR combinations of more than 1 antibody, e.g. 2, 3, 4, 5, or 6 different antibodies. Exemplary antibodies comprising CDR sequences of different antibodies are provided inFIG. 2.

In particular,i. antibody G3-77 comprises VH-CDR sequences (CDR1, 2, and 3) of antibody 9H9-H7; and VL-CDR sequences (CDR4, 5, and 6) of antibody 5A4-A7;ii. antibody G3-78 comprises VH-CDR sequences (CDR1, 2, and 3) of antibody 9H9-H7; and VL-CDR sequences (CDR4, 5, and 6) of antibody 5A4-A7;iii. antibody G3-97 comprises VH-CDR sequences (CDR1, 2, and 3) of antibody 2D8-A10; and VL-CDR sequences (CDR4, 5, and 6) of antibody 5A4-A7.

According to a specific embodiment, the antibody only comprises a VH domain as antigen binding moiety, thus, comprises CDR1-3, without a respective VL domain.

It is herein specifically understood that the CDRs numbered CDR1, 2, and 3 represent the binding region of the VH domain, and CDR4, 5, and 6 represent the binding region of the VL domain.

According to a specific aspect, the antibody of the invention comprises any of the VH and VL amino acid sequence combinations as depicted inFIG. 2, or the binding site formed by such combination of VH and VL amino acid sequences. Alternatively, combinations of the immunoglobulin domains of two different antibodies may be used, provided, that the antibody is still functionally active. For example, the VH sequence of one antibody may be combined with a VL sequence of another antibody. According to further specific embodiments, any of the framework regions as provided inFIG. 2may be employed as a framework to any of the CDR sequences and/or VH/VL combinations as described herein.

According to a specific aspect, the antibody of the invention comprises any of the VH and VL amino acid sequence combinations as depicted inFIG. 2, or the binding site formed by such combination of VH and VL amino acid sequences.

It is understood that the antibody of the invention optionally comprises such amino acid sequences ofFIG. 2with or without a suitable signal or leader sequence.

According to a specific aspect, each of the sequences ofFIG. 2may be terminally extended or deleted in the constant region, e.g. a deletion of one or more of the C-terminal amino acids.

FIG. 2shows different VH sequences and different VL sequences of parent antibodies referred to as 8E3-E5, 9H9-H7, 5A4-A7, 2D8-A10, G3-43, G3-46, G3-77, G3-78, and G3-97, and supports any VH/VL combination, thus a series of different VH/VL combinations for each of the parent antibodies, e.g. such as depicted inFIG. 9. Therefore, specific variants of a parent antibody may include a VH sequence of one parent antibody and a VL sequence of another parent antibody, or a combination of functionally active variants of such VH and VL sequences, e.g. functionally active variants that derive from the same parent antibody.

In particular,FIG. 2shows different VH sequences and different VL sequences of the parent antibodies referred to as 8E3-E5, 9H9-H7, 5A4-A7, 2D8-A10, G3-43, G3-46, G3-77, G3-78, and G3-97. For example, 81 different VH/VL combinations are feasible combining a VH sequence of one parent antibody and a VL sequence of another parent antibody, and many more variants are possible, if any of the VH or VL sequences is a functionally active variant of the parent sequence, e.g. a variant which includes any of a CDR mutation and/or a framework mutation.

The CDR sequences included in the VH and VL sequences ofFIG. 2are identical to the respective CDR sequences as listed inFIG. 1.

The invention further provides for a method of producing functionally active antibody variants of a parent antibody which is any of the antibodies of the invention, e.g. an antibody as listed in Table 1, or comprising any of the VH or VL amino acid sequence combinations as depicted inFIG. 2, or comprising the binding site formed by such combination of VH and VL amino acid sequences, which method comprises engineering at least one point mutation in any of the framework regions (FR) or constant domains, or complementarity determining regions (CDR1 to CDR6) to obtain a variant antibody, and determining the functional activity of the variant antibody, specifically by the affinity to bind the gal-III epitope with a Kd of less than 10−6M, preferably less than 10−7M, or less than 10−8M, or less than 10−9M, even less than 10−10M, or less than 10−11M, e.g. with an affinity in the picomolar range. Upon determining the functional activity, the functionally active variants are selected for further use and optionally for production by a recombinant production method.

According to a specific aspect, the variant antibody binds the same epitope as the parent antibody.

According to a further specific aspect, the variant antibody comprises the same binding site as the parent antibody.

Functionally active variant antibodies may differ in any of the VH or VL sequences, or share the common VH and VL sequences, and comprise modifications in the respective FR. The variant antibody derived from the parent antibody by mutagenesis may be produced a methods well-known in the art.

Exemplary parent antibodies are described in the examples section below and inFIG. 1(Table 1) andFIG. 2. Specifically, the antibody is a functionally active derivative of a parent antibody that is characterized by the sequences as listed in Table 1 orFIG. 2. Variants with one or more modified CDR sequences, and/or with one or more modified FR sequences, such as sequences of FR1, FR2, FR3 or FR4, or a modified constant domain sequence may be engineered.

For example, functionally active variant antibodies may be obtained by mutagenesis, specifically by affinity maturation and/or humanization. Though the variant antibodies may still share common CDR sequences CDR1-6 or common VH and VL sequences of a parent antibody, it is feasible that also variant antibodies or antibody domains are produced, e.g. with modifications in the respective FR or CDR sequences, which are functionally active.

Exemplary variant antibodies of a parent antibody comprise at least one point mutation in any of the CDR1-CDR6, and/or at least one point mutation in any of the FR sequences, preferably wherein the antibody has a specificity to bind the same epitope as the parent antibody.

In certain aspects, the invention provides for such functionally active variant antibodies, preferably monoclonal antibodies, most preferably humanized or human antibodies, comprising a heavy chain and a light chain, wherein any of the light chain or VL variable region or the respective CDRs comprises an amino acid sequence as derived from a parent antibody, which is the antibody designated 8D5-1G10 or 4D5-D4 or any other antibody as listed in Table 1 orFIG. 2, by modification of at least one FR or CDR sequences.

The invention further provides for an antibody of the invention, for use in treating a subject at risk of or suffering fromKlebsiella pneumoniaeinfection or colonization comprising administering to the subject an effective amount of the antibody to limit the infection in the subject or to ameliorate a disease condition resulting from said infection, preferably for treatment or prophylaxis of any of primary and secondary bacteremia, pneumonia, urinary tract infection, liver abscess, peritonitis, or meningitis.

Accordingly, the invention further refer to a method of treating a subject at risk of or suffering fromKlebsiella pneumoniaeinfection or colonization comprising administering to the subject an effective amount of the antibody to limit the infection in the subject or to ameliorate a disease condition resulting from said infection, preferably for treatment or prophylaxis of any of primary and secondary bacteremia, pneumonia, urinary tract infection, liver abscess, peritonitis, or meningitis.

The antibody is specifically able to neutralize lethal endotoxemia. Such functional activity may be determined in an appropriate in vivo model (challenge with purified LPS).

Specifically, the antibody may provide bactericidal activity againstKlebsiella pneumoniaeof the gal-III O-type, in particular MDRKlebsiella pneumoniae, preferably MDRKlebsiella pneumoniaeST258.

According to a specific aspect, immunotherapy using the antibody of the invention may effectively protect against live bacterial challenge, e.g. as determined in various animal models.

The antibody can be specifically effective againstKlebsiella pneumoniaeof the gal-III O-type by complement-mediated killing, e.g. as determined by an in vitro serum bactericidal assay (SBA), e.g. with at least 20% killing of bacteria above the control samples (no antibody or irrelevant control mAb added).

The antibody can be specifically effective againstKlebsiella pneumoniaeof the gal-III O-type by antibody mediated phagocytosis, e.g. as determined by an in vitro opsonophagocytotic killing assay (OPK), e.g. with at least 20% uptake of input bacteria or 20% lower end CFU count above the control samples (no antibody or irrelevant control mAb added).

The antibody is specifically effective againstKlebsiella pneumoniaeof the gal-III O-type by neutralizing endotoxin functions, e.g. as determined by an in vitro LAL assay, or toll-like receptor 4 (TLR4) reporter assay e.g. with at least 20% reduction in endotoxin activities in comparison to control samples (no antibody or irrelevant control mAb added).

According to a further specific aspect, the antibody neutralizes the targeted pathogen in animals, including both, human and non-human animals, and inhibits pathogenesis in vivo, preferably any models of primary and secondary bacteremia, pneumonia, urinary tract infection, liver abscess, peritonitis, or meningitis.

As a reference (positive control) for determining the neutralization potency, any of the antibodies described in the examples section may be use. Preferably, the neutralization potency of an antibody of the invention is equal or higher than the antibody characterized by the CDR1-6 sequences of the antibody herein referred to as 2D8-A10, in particular the chimeric IgG1 antibody as described in the Examples.

The invention further provides for a pharmaceutical preparation comprising the antibody of the invention, preferably comprising a parenteral or mucosal formulation, optionally containing a pharmaceutically acceptable carrier or excipient.

Such pharmaceutical composition may contain the antibody as the sole active substance, or in combination with other active substances, or a cocktail of active substances, such as a combination or cocktail of at least two or three different antibodies.

According to the invention, the antibody of the invention is specifically provided for medical, diagnostic or analytical use.

The invention further provides for the use of the antibody of the invention for diagnostic purposes, specifically for the diagnosis ofKlebsiella pneumoniae(especially ST258) infection or colonization, or an associated disease such as primary and secondary bacteremia, pneumonia, urinary tract infection, liver abscess, peritonitis, or meningitis in a subject.

Specifically, the subject is a human being, in particular an immunocompromised or immunosuppressed patient, or a contact thereof.

Specifically, the antibody is provided for use according to the invention, wherein a systemic infection or colonization withKlebsiella pneumoniaeof the gal-III O-type in a subject is determined ex vivo by contacting a biological sample of said subject with the antibody, wherein a specific immune reaction of the antibody determines the infection or colonization.

Specifically, the biological samples is a body fluid or tissue sample, preferably a sample selected from the group consisting of a blood sample, stool sample, skin sample, urine sample, cerebrospinal fluid, and a respiratory tract specimen such as endotracheal aspirates, pleural fluid, lung tap, nasal swab or sputum, or aKlebsiella pneumoniaeisolate originating from any of the foregoing. Specifically, a sample of body fluid is tested for the specific immune reaction, which sample is selected from the group consisting of urine, blood, blood isolates or blood culture, aspirate, sputum, lavage fluid of intubated subjects and stool.

Specifically, the biological sample is treated to produce aKlebsiella pneumoniaeisolate originating from the biological sample, which isolate may be further characterized for its gal-III genotype or phenotype, and/or the level of gal-III antigen expression. Preferable sample preparation methods for producing bacterial isolates are employing bacterial enrichment and cultivation steps.

Specifically, the biological sample is treated to determine the gal-III level directly in the sample, optionally following preparatory steps of enrichment or purification to reduce matrix effects and to increase the specificity and sensitivity of the test. Preparatory steps include culturing of the biological specimen according to standard culture procedures such as but not exclusively being hemocultures in standard growth media as well as the culturing of specimens on solid agar (including phenotyping—i.e. antibiogram) as performed in routine microbiology laboratories. Bacteria may be sub-cultured for expansion of CFU in different growth media (standard media and/or chemically defined media; high nutrient, low nutrient, limited growth media composition) to enhance expression of virulence factors Bacterial suspensions may be prepared and washed in standard buffer solutions to remove potential matrix effects.

Specifically, the gal-III antigen is determined by at least one of an immunoassay, preferably any of ELISA, CIA, RIA, IRMA, agglutination assay, immunochromatography, dipstick assay and Western-blot, or mass-spectrometry, nuclear magnetic resonance (NMR), or a method of determining corresponding DNA or RNA indicative of gal-III expression, in particular determining a nucleic acid sequence specific to the gtr genes, preferably employing a nucleic acid hybridization assay or a nucleic acid amplification assay.

Specifically, the diagnostic use according to the invention refers to determining the serotype ofKlebsiella pneumoniaein vitro from a pureKlebsiella pneumoniaeculture recovered from a clinical specimen, to determine whether the bacterium is of the gal-III O-type, or not.

The invention further provides for a diagnostic preparation of the antibody of the invention, comprising the antibody and a further diagnostic reagent in a composition or a kit of parts, comprising the componentsa) the antibody; andb) the further diagnostic reagent;c) and optionally a solid phase to immobilize at least one of the antibody and the diagnostic reagent.

The diagnostic preparation optionally comprises the antibody of the invention and the further diagnostic reagent in a composition or a kit of parts.

The diagnostic kit preferably comprises all essential components to determine the gal-III expression in the biological sample, optionally without common or unspecific substances or components, such as water, buffer or excipients. The storage stable kit can be stored preferably at least 6 months, more preferably at least 1 or 2 years. It may be composed of dry (e.g. lyophilized) components, and/or include preservatives.

The preferred diagnostic kit is provided as a packaged or prepackaged unit, e.g. wherein the components are contained in only one package, which facilitates routine experiments. Such package may include the reagents necessary for one or more tests, e.g. suitable to perform the tests of a series of biological samples. The kit may further suitably contain a gal-III antigen preparation as a standard or reference control.

The diagnostic composition may be a reagent ready-to-use in a reaction mixture with the biological sample, or a conserved form of such reagent, e.g. a storage-stable form such as lyophilized; snap-frozen (e.g. in liquid nitrogen), ultra low-temperature storage (−70° C. and −80° C.), cold-storage (−20° C. and 5° C.) and controlled room temperature (15° C.-27° C.); standard sample storage as e.g. glycerol-stocks, tissue paraffin-blocks, (buccal) swabs and other standard biological sample storage methods, which conserved form of a reagent can be reconstituted or prepared to obtain a ready-to-use reagent. Such ready-to-use reagent is typically in the form of an aqueous solution, specifically (physiological) buffer conditions (e.g. EDTA buffered, phosphate buffer, HBSS, citrate buffer etc.).

Specifically, the further diagnostic reagent is a reagent specifically reacting with the antibody and/or the reaction product of the antibody binding to its antigen. An appropriate diagnostic reagent is suitably used for performing an immunoassay for diagnosing or monitoring, in a subject, theKlebsiella pneumoniaeinfection or colonization. The appropriate diagnostic reagent can be a solvent, a buffer, a dye, an anticoagulant, a ligand that specifically binds to the antibody of the invention and/or the antibody-antigen immune complex.

Specifically, the invention provides for a diagnostic preparation of an antibody of the invention, optionally containing the antibody with a label and/or a further diagnostic reagent with a label, such as a reagent specifically recognizing the antibody or an immune complex of the antibody with the respective target antigen, and/or a solid phase to immobilize at least one of the antibody and the diagnostic reagent.

The antibody or the diagnostic reagent can be directly labeled or indirectly labeled. The indirect label may comprise a labeled binding agent that forms a complex with the antibody or diagnostic reagent to the gal-III antigen.

The label is typically a molecule or part of a molecule that can be detected in an assay. Examplary labels are chromophores, fluorochromes, or radioactive molecules. In some embodiments the antibody or diagnostic reagent is conjugated to a detectable label which may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, metal chelates, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or by a specific binding molecule which itself may be detectable (e.g., biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

Preferred diagnostic preparations or assays comprise the antibody of the invention immobilized on a solid phase, e.g. latex beads, gold particles, etc., e.g. to test agglutination by the antibody of bacteria of the gal-III type obtained from a sample to be tested.

The invention further provides for a method of diagnosingKlebsiella pneumoniaeinfection or colonization in a subject caused by aKlebsiella pneumoniaestrain, comprisinga) providing an antibody according to the invention, andb) detecting if the antibody specifically immunoreacts with the galactan-III epitope in a biological sample of the subject to be tested, thereby diagnosingKlebsiella pneumoniaeinfection or colonization.

Such diagnosis is specifically indicated in case of a MDRKlebsiella pneumoniaeinfection of colonization, in particular addressing MDRKlebsiella pneumoniaeof the gal-III type. Optionally, a diagnostic assay may involve two different antibodies with different specificity and/or affinity to bind gal-III and/or gal-I, so to possibly differentiate between the gal-III and gal-I antigens.

According to a specific aspect, the invention provides for companion diagnostics to determine the infection of a subject withKlebsiella pneumoniae, in particular with MDRKlebsiella pneumoniae, by the diagnostics of the invention or the diagnostic method of the invention, to provide for the basis of treatment with a therapeutic against such infection, e.g. employing immunotherapy, such as treating with an antibody of the invention.

According to a specific aspect, the invention provides for a sensitive bedside diagnostics to diagnose infection of a subject withKlebsiella pneumoniae, in particular with MDRKlebsiella pneumoniae, by determining free LPS, e.g. from clinical specimen where the amount of live bacteria is limited. The sensitivity of such assay is specifically less than 100 ng preferably less than 10 ng of LPS.

The invention further provides for an isolated nucleic acid encoding an antibody of any of the invention.

The invention further provides for an expression cassette or a plasmid comprising a coding sequence to express a protein comprising a VH and/or VL of an antibody of the invention.

The invention further provides for a host cell comprising an expression cassette or a plasmid of the invention.

The invention further provides for a method of producing an antibody of the invention, wherein a host cell of the invention is cultivated or maintained under conditions to produce said antibody.

Specifically preferred is a host cell and a production method employing such host cell, which host cell comprisesthe plasmid or expression cassette of the invention, which incorporates a coding sequence to express the antibody light chain; andthe plasmid or expression cassette of the invention, which incorporates a coding sequence to express the antibody heavy chain.

According to a further aspect, the invention provides for a method of producing an antibody of the invention, comprisinga) immunizing a non-human animal with the gal-III antigen ofKlebsiella pneumoniaeand isolating B-cells producing antibodies;b) forming immortalized cell lines from the isolated B-cells;c) screening the cell lines to identify a cell line producing a monoclonal antibody that specifically binds to the gal-III antigen and optionally the gal-I antigen, e.g. wherein preferential binding to gal-III as compared to gal-I is determined; andd) producing the monoclonal antibody, or a humanized or human form of the antibody, or a derivative thereof with the same epitope binding specificity as the monoclonal antibody.

The invention further provides for a method of identifying a candidate antibody comprising:a) providing a sample containing an antibody or antibody-producing cell; andb) assessing for binding of an antibody in or produced by the sample with a galactan-III epitope, wherein a positive reaction between the antibody and the epitope identifies the antibody as candidate antibody.

The invention further provides for a method of identifying a candidate antibody comprising:a) providing a sample containing an antibody or antibody-producing cell; andb) assessing for binding of an antibody in or produced by the sample with the galactan-III epitope, wherein a specific positive reaction between the antibody and the galactan-III epitope relative to the galactan-I epitope identifies the antibody as candidate antibody.

The invention further provides for a method of producing an antibody of the invention, comprising

a) providing a candidate antibody identified according to the invention; and

b) producing a monoclonal antibody, or a humanized or human form of the candidate antibody, or a derivative thereof with the same epitope binding specificity as the candidate antibody.

