WO2024261344A1 - Novel binders targeting the multi-drug resistant pathogen acinetobacter baumannii - Google Patents

Abstract

The invention relates to antigen-binding proteins specifically binding Acinetobacter cells, more specifically, wherein said binding occurs through targeting a binding site of Outer membrane protein 25 (Omp25) exposed on the surface of said pathogenic Acinetobacter cells in capsulated and non- capsulated form. More specifically, the invention relates to a family of immunoglobulin single variable domains (ISVDs) capable of specifically binding the Acinetobacter baumannii Outer membrane protein 25 (Omp25) at an epitope located in the surface-exposed regions. The invention further relates to fusions of said antigen-binding proteins as multivalent, multispecific or functional fusions with further moieties, and their use in functionalized targeting of A. baumannii, as a scavenger or to induce killing activity. More particular said antigen-binding proteins or fusions are provided as a tool for detection of Acinetobacter, or for depletion of bacterial cells from a sample upon binding. Finally, the invention relates to said antigen-binding proteins for use as a diagnostic or as a medicine, preferably in prevention or treatment of Acinetobacter baumannii infection.

Description

NOVEL BINDERS TARGETING THE MULTI-DRUG RESISTANT PATHOGEN ACINETOBACTER BAUMANNII

FIELD

The invention relates to antigen-binding proteins specifically binding Acinetobacter cells, more specifically, wherein said binding occurs through targeting a binding site of Outer membrane protein 25 (Omp25) exposed on the surface of said pathogenic Acinetobacter cells in capsulated and noncapsulated form. More specifically, the invention relates to a family of immunoglobulin single variable domains (ISVDs) capable of specifically binding the Acinetobacter baumannii Outer membrane protein 25 (Omp25) at an epitope located in the surface-exposed regions. The invention further relates to fusions of said antigen-binding proteins as multivalent, multispecific or functional fusions with further moieties, and their use in functionalized targeting of A. baumannii, as a scavenger or to induce killing activity. More particular said antigen-binding proteins or fusions are provided as a tool for detection of Acinetobacter, or for depletion of bacterial cells from a sample upon binding. Finally, the invention relates to said antigen-binding proteins for use as a diagnostic or as a medicine, preferably in prevention or treatment of Acinetobacter baumannii infection.

BACKGROUND

The multi-drug-resistant Acinetobacter baumannii is a nosocomial pathogen thriving in hospital environment and endangering patients, and is thus considered as a global health threat requiring urgent and effective approaches for improving early diagnosis and curative options. This Gramnegative bacterium has a peculiar virulence based on a 'persist and resist' strategy and is part of the most problematic ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa and Enterobacter species) group of human pathogens, against which development of new antibiotics is critically needed as stated by the World Health Organization. An obvious hallmark of A. baumannii bacteria is the high heterogeneity observed among the isolates, partially by their dynamic genome, with an estimated conserved core genome of only 16.5%, while 25% of the genome is unique to each strain, as well as observed on the phenotypic level ⁽⁵⁾. For instance, the phenotypic heterogeneity, observable using electron microscopy where the thickness and rigidity of the capsule differ, along with different cellular densities, is at least in part a translation of the genetic variability wherein capsular polysaccharide - impacting antibiotic and environmental resistances, host response and virulence- and the outer core of lipooligosaccharide, are both encoded by a plurality of locus types. The established strains such as ATCC19606, ATCC17978, DSM30011 or AB5075 are widely used (reference) type strains from which significant and validated observations were and are still generated. Altogether, these established strains greatly contributed to the state of the art of the current and growing A. baumannii field. The established strains AB5075, ATCC17978 and ATCC19606 are in fact multidrug-resistant (MDR) strains of clinical origin, whereas DSM30011 is a non-MDR environmental strain obtained from plant microbiota. However in view of developing clinically relevant tools, the recent release of the Acinetobase database is now, by providing the genotypic and phenotypic data on a large collection of clinical isolated ⁽⁵⁾, a huge step forward to form a representative platform highly valuable for developing and testing novel therapeutics and diagnostics.

The Acinetobacter baumannii 25 kDa outer membrane protein (Omp25) is predicted to have a role as a porin in the outer membrane, and suggested to be involved in bacterial multidrug resistance, more specifically, it was already anticipated that together with CarO, Omp25 may form an important target of regulation by BfmRS related to antibiotic resistance ^{(9 10)}. The protein seems highly conserved within A. baumanni clinical isolates, but does not show closely related proteins in distant bacterial strains, positioning this as a novel Acinetobacter target for development of specific and selective antibacterials.

Since A. baumannii infection routes and factors known to favoring infections are colonization of mechanical devices such as catheters and ventilation equipment, open wounds, major trauma or burns, prolonged hospital stays, and immunocompromisation, often leading to a therapeutic dead end because of last-resort antibiotic resistance, there is a need to find selective molecules for use in diagnosis of A.baumannii infection, but also for therapeutic approaches, preferably for targets not yet considered in the past or in other bacterial pathogens.

SUMMARY OF THE INVENTION

The invention relates to antigen-binding agents that specifically target Acinetobacter baumannii Outermembrane protein 25, a 25 kDa protein, putatively with a porin function, which is highly conserved among (clinical) A. baumannii isolates, though not known to be conserved in other bacteria, such as E.coli. Moreover, the antigen-binding proteins were surprisingly found to be capable of binding the Acinetobacter cells in non-capsulated as well as in capsulated strains, which is favorable for development of novel antibacterials with high clinical value as to attack highly virulent pathogenic strains. Although the role of Omp25 (UniprotQ4A208, SEQ. ID NO: 43) in Acinetobacter baumannii virulence or functioning is not entirely clear, it was established herein that antigen-binding proteins are capable to specifically and selectively target said protein on capsulated Acinetobacter cells by binding a surface-exposed binding site. Thus, a novel therapeutically relevant binding agent is disclosed herein providing for first-on-target means for selective targeting of this human pathogen.

The invention specifically relates to antigen-binding proteins in the minimal format of active antibody fragments presented herein as a VHH or Nb, known to have a low molecular size (ca. 15 kDa), which can be recombinantly produced via single gene expression and for which the antigen-binding protein can reach the favorable high affinities and specificities for antigens of classical heterotetrameric monoclonal antibodies. The size reduction of Nbs provides favorable properties over monoclonal antibodies, including lower production costs, enhanced thermal and chemical stabilities, improved tissue penetration, lowered immunogenic responses and the ability to access cavities on the target surface.

So the invention discloses for the first time highly selective and specific binding agents against an Acinetobacter baumannii cell-surface membrane target protein, Omp25, unique in its binding to the surface of Acinetobacter cells containing a virulent capsule. Finally, said novel antibodies, herein in the format of Nbs, bring forward a novel innovative asset for tackling Acinetobacter baumannii in future biotechnological and medical advances for clinical applications.

A first aspect of the invention discloses means, in particular antigen-binding proteins, specifically binding the Acinetobacter Omp25 protein, more particularly, Omp25 as present in living bacterial cells, with said membrane protein exposed at the surface of the capsulate. So in a specific embodiment, said antigen-binding protein specifically bind an Omp25 epitope exposed or present at the surface of the Acinetobacter outer membrane, thereby reachable by extracellularly presented antigen-binding agents.

In a further specific embodiment, said antigen-binding proteins described herein specifically bind A. baumannii Omp25, as provided herein as SEQ ID NO:4, 5, or 43, in particular wherein said antigenbinding proteins bind the surface exposed regions of said Omp25, more specifically said surface- exposed regions present on the surface of A.baumannii cells, even more specifically, said surface exposed regions 2-6, as defined herein, and as provided in SEQ ID NOs: 19-25, more specifically SEQ ID NO: 20-24, or said surface-exposed regions of Acinetobacter species Omp25 protein wherein the sequences of said surface exposed regions defined herein for A.baumannii are conserved in sequence. In a specific embodiment, said antigen-binding protein as described herein specifically bind the epitope of Omp25 as present in A.baumannii Omp25 (SEQ ID NO:4) at amino acids at position 60, 93-106, 131, 165, 166, 167, and 190-193, or alternatively bind the epitope of Omp25 as present in Acinetobacter species at the corresponding positions of the surface-exposed regions of said Omp25 based on alignment of said Omp25, and wherein the corresponding Omp25 surface-exposed regions 2-6 are at least 90 % identical to the surface exposed regions 2-6 of A.baumannii Omp25.

The invention further relates to specific antigen-binding proteins comprising Immunoglobulin single variable domains binding Omp25 as described herein, wherein the CDR1, 2 and 3 loops are providing the paratope, more specifically the paratope being defined by SEQ ID NO:s 11-13 representing the CDR1, 2 and 3 loops, respectively. Said ISVDs provide for antigen-binding proteins wherein the antigen- binding paratope is defined by as little as 3 CDR loops, thereby providing a minimal antibody structural unit for these novel antibodies against A.baumannii Omp25.

Another aspect of the invention relates to moieties, agents or compositions comprising said antigenbinding proteins of the present invention, which may be multivalent or multispecific antigen-binding proteins, and/or fusions to further molecules or moieties provided by translational fusions or conjugates.

Further aspects of the invention relates to nucleic acid molecules including DNA and RNA encoding said antigen-binding agents described herein; to pharmaceutical compositions comprising any of the above; and applications of said antigen-binding proteins, moieties, nucleic acids or pharmaceutical compositions as medicine, as vaccine, or in treatment of Acinetobacter infections, specifically Acinetobacter baumannii infection; for use in diagnosis of Acinetobacter infection; or to use as a tool in scavenging, concentration, or depletions of bacterial cells.

DESCRIPTION OF THE FIGURES

Figure 1. NbH7 binds the cell surface of AB5075-VUB-/t/'A.^,. SAbal3 (AB5075C-). A Fluorescence micrographs of NbH7 and a control Nb (CtrINb) tested on AB5075-VUBC- in exponential and stationary phase are shown. Per condition, from left to right, the phase contrast, GFP, DL650 and an overlay image are shown. B Enlargement of NbH7-bound bacteria in stationary phase. One representative cell was picked to illustrate the membrane labeling of NbH7 by showing its intensity profile of GFP (expressed in cytoplasm) and DL650 (labelled NbH7). A black bar is shown on the cell to represent where the intensity profile was measured.

Figure 2. NbH7 binds AB5075-VUB. Fluorescence micrographs of NbH7 and a control Nb (CtrINb) tested on AB5075-VUB in exponential and stationary phase are shown. Per condition, from left to right, the phase contrast, GFP, DL650 and an overlay image are shown.

Figure 3. NbH7 binds clinical isolate AB3-VUB. Fluorescence micrographs of NbH7 and a control Nb (CtrINb) tested on AB3-VUB in exponential and stationary phase are shown. Per condition, from left to right, the phase contrast, DL650 and an overlay image are shown.

Figure 4. NbH7 binds clinical isolate AB180-VUB. Fluorescence micrographs of NbH7 and a control Nb (CtrINb) tested on AB180-VUB in exponential and stationary phase are shown. Per condition, from left to right, the phase contrast, DL650 and an overlay image are shown. Figure 5. NbH7 binds clinical isolate AB220-VUB. Fluorescence micrographs of NbH7 and a control Nb (CtrINb) tested on AB220-VUB in exponential and stationary phase are shown. Per condition, from left to right, the phase contrast, DL650 and an overlay image are shown.

Figure 6. NbH7 binds classically used strain ATCC17978-VUB-VUB. Fluorescence micrographs of NbH7 and a control Nb (CtrINb) tested on ATCC17978-VUB in exponential and stationary phase are shown. Per condition, from left to right, the phase contrast, DL650 and an overlay image are shown.

Figure 7. NbH7 does not bind E. coli. Fluorescence micrographs of NbH7 and a control Nb (CtrINb) tested on E. coli S17 in exponential and stationary phase are shown. Per condition, from left to right, the phase contrast, DL650 and an overlay image are shown.

Figure 8. Omp25 is the target of NbH7. A Coomassie stained SDS-PAGE of the pull down eluate. A black frame shows the band visible for the pull down sample with NbH7, but not with other tested nanobodies (E3 and B9). Mass spectrometry analysis indicated that this band is Omp25. B Fluorescence micrographs of the binding ability of NbH7 on the AB5075C- strain and the derivative where the omp25 gene was deleted, namely AB5075C-Aomp25.

Figure 9. NbH7 retains its binding ability on AB5075C- when fused to sfGFP. A Fluorescence micrographs of the NbH7-sfGFP construct tested on AB5075C- and E. coli S17. E. coli S17 is used as negative control because NbH7 does not bind this strain. B The max gray values of the fluorescence intensity signal measured for 15 randomly picked cells of the Nb-construct (Nb_GFP) and cells only (Ctrl) tested on both AB5075C- and E. coli S17 are shown. Gray value measurements are done with ImageJ software on the raw data. The statistical significance was assessed by an unpaired t test using Graphpad Prism software.

Figure 10. NbH7 captures AB5075C- from liquid samples. A Capture efficiency of NbH7 in PBS for 5, 30 and 120 minutes incubation. B Capture efficiency of NbH7 for consecutive incubation with new coupled beads. The capture efficiency is the inverse of the ratio of CFU/ml remaining in the supernatant before and after incubation with the coupled beads.

Figure 11. NbA8 and NbE12 bind the non-capsulated strain AB5075-VUB-/trA;.7SAbol3 (AB5075C-).

Per Nb, from left to right: Phase contrast images of the bacteria, fluorescent micrographs of GFP expressed in the cytoplasm, the DL650-labelled Nb and an overlay image. NbA8 and NbE12 belong to the same Nb family. A nanobody know not to bind A. baumannii (CtrINb) was also included.

Figure 12. NbA8 and NbE12 bind a highly capsulated, clinically isolated A. baumannii strain (AB3- VUB). Per Nb, from left to right: Phase contrast images of the bacteria, the DL650-labelled Nb and an overlay image. NbA8 and NbE12 belong to the same Nb family. A nanobody known not to bind A. baumannii (CtrINb) was also included.

Figure 13. CDR annotations as provided here for the Nb H7 sequence. The MacCallum, AbM, Chothia, Kabat and IMGT annotations as commonly known are shown in grey labeled boxes.

Figure 14. BLI measurements to determine the affinity of NbH7 and NbA8 for Omp25 including the negative control. In each measurement, a control Nb (CtrINb) was measured against the highest concentration of Omp25 to monitor aspecific binding. This aspecific binding signal was subtracted from the binding signals of the other Nb before analysis of the measurement.

Figure 15. Affinity determination of the Nb family representatives H7 and A8 by BLI. The affinity of two nanobodies grouped into the same nanobody family was determined by means of BLI using the Octet® R8 apparatus. A Schematic overview of an Octet® Ni-NTA sensor in the experiment: the Nbs are loaded on the Ni-NTA sensor through binding the hexahistidine tag and consequently dipped into a well with different Omp25 concentrations. B Association and dissociation curves for the Nbs with a two-fold dilution series of Omp25 concentrations. In each test, a control Nb was tested with the highest Omp25 concentration (80 nM) to monitor aspecific binding. This aspecific binding signal was subtracted from the binding signals plotted here. C Overview of the K_D values obtained for each Omp25 concentration per Nb. The binding curve of the lowest Omp25 concentration could not be fitted and calculated. The K_D values were calculated using Octet® Analysis Studio software and the graphs were plotted using Graphpad Prism.

Figure 16. The humanized NbH7 variants can bind the capsulated AB5075-VUB bacteria. A Alignment of the humanized NbH7 variants. The amino acids that differ from the NbH7 sequence are highlighted in bold. B Binding assessment of each Nb with fluorescence microscopy. For every condition, the top image is a phase contrast image of the bacteria. In the bottom, fluorescence micrographs of DL650- labelled NbH7, the humanized variants (NbH7_hl-h5) and a negative control nanobody (CtrINb) are shown. All tests are done on a 1:1 ratio of stationary and exponential phase, living bacteria.

Figure 17. Affinity determination of the humanized variants of NbH7. The affinity of 5 humanized Nb variants of NbH7 (NbH7_hl-5; SEQ ID NOs: 14-18, resp.) was determined by means of BLI using the Octet® R8 apparatus. The Nbs were loaded on the Ni-NTA sensor through binding of the hexahistidine tag and consequently dipped into a well with different Omp25 concentrations. In each test, a negative control Nb was tested with the highest Omp25 concentration (80 nM) to monitor aspecific binding. Table: Overview of the K_D values obtained for each Omp25 concentration per Nb. Graph: the obtained K_D values. The data was statistically analyzed by a one-way ANOVA, followed by a multiple comparisons test. The K_D values were calculated using Octet® Analysis Studio software, and the graph was made and statistics were done using Graphpad Prism.

Figure 18. Alphafold prediction of A. baumannii OMP25 protein. The predicted surface-exposed regions are colored in green. The first amino acid per region is indicated on the figure with its one-letter code and position in the Omp25 sequence (SEQ ID NO:5).

Figure 19. Multiple sequence alignment of the surface exposed regions of Omp25 to TtAac, the antigen target of the negative control Nb (CtrINb). The surface-exposed regions of the Omp25 protein (SEQ ID NO:5) are indicated in the alignment as regions 1-7 (SEQ ID NOs:19-25). The alignment was generated using Clustal Omega and Jalview software.

Figure 20. NbH7 does not bind Brucella spp. cell surfaces. For every condition, the top image is a phase contrast image of the bacteria. In the bottom, a fluorescence micrograph of DL650-labelled NbH7 is shown. Fixed, stationary phase bacteria of three Brucella strains, belonging to two different species, were tested: B. melitensis 16M, B. abortus 544 and B. abortus 2308. The same batch of DL650-labelled NbH7 was in parallel tested on AB5075-VUB as positive control, in the same conditions as described in previous tests on Acinetobacter spp.

Figure 21. NbH7 can bind other Acinetobacter spp., but not all Gram-negative bacteria. For every condition, the top image is a phase contrast image of the bacteria. In the bottom, a fluorescence micrograph of DL650-labelled NbH7 and a control nanobody (CtrINb) is shown. A Binding assessment of NbH7 on Acinetobacter calcoaceticus, Acinetobacter pittii, and Acinetobacter junii. B Binding assessment of NbH7 on Escherichia coli S17 and Klebsiella pneumoniae. All tests are done on a 1:1 ratio of stationary and exponential phase, living bacteria.

Figure 22. Multiple sequence alignment of Omp25 proteins of A.baumannii AB5075-VUB Omp25 and Acinetobacter spp. strains A. pittii, A. calcoaceticus and A. junii Omp25 amino acid sequences. The less conserved amino acids are lighter or not colored. More information on the strains is provided in Table 1. The alignment was generated using ClustalWS and Jalview software.

Figure 23. Recombinant expression of AB5075-VUB Omp25 in the outer membrane of E. coli. On the left, a scheme is given of the expression construct used for recombinant expression of Omp25 in E. coli: the E. coli OmpA signal peptide (Ec OmpA SP), a streptavidin tag (Strep) and the AB5075 omp25 (Ab omp25) sequence without its endogenous signal peptide coding sequence. On the right, the fluorescence micrographs to assess binding of labelled NbH7 on the E. coli cells before (non-induced) and after (induced) expression of AbOmp25 are shown. Figure 24. X-ray structure of Omp25 in complex with NbH7. The crystal structure of the complex was solved at 2.91A. A Ribbon presentation of the complex. Omp25 is the top structure, NbH7 is the bottom structure. The boxes show a close-up view of the interface contacts of CDR1 (B), CDR2 (C) and CDR3 (D) of NbH7 with Omp25. The side chains are shown in stick representation. Water atoms are shown as dots and Hydrogen bonds are represented by dotted lines. Amino acid numbering is indicated by sequential numbering.

Figure 25. Omp25 and NbH7 sequence with the epitope and paratope highlighted, respectively. The amino acid sequences of NbH7 and A.baumannii Omp25 (Uniprot Q4A208; SEQ. ID NO:43) are shown. Based on analysis of the crystal structure of the complex, the epitope and paratope were determined and are highlighted by the darker label. Amino acids involved in hydrogen bonds between both proteins are indicated by a dot. Amino acid numbering is indicated by sequential numbering.

Figure 26. NbH7 conjugated with GFP label by the SpyTag-SpyCatcher method retains its Acinetobacter binding ability. Membrane labelling of AB5075-VUB-A/trA cells was determined by fluorescence microscopy on the bacteria incubated with A GFP_SpyTag B NbH7_SpyCatcher C a NbH7- GFP conjugate. D shows a quantitative analysis on randomly picked bacterial cells of the calculated maximal gray values, subtracted with their respective background signal. The maximal gray values, as well as the background signals, were determined along lines drawn through nine cells for the controls or eighteen cells for the NbH7-GFP conjugate labelling. Additionally, the median and interquartile range were indicated with horizontal bars. Statistical analysis was done by a Mann-Whitney test using GraphPad Prism software and revealed a highly significant difference in distribution of the data sets from each control compared to the NbH7-GFP labelled group (****: extremely significant, p < 0.0001, U = 0).

DETAILED DESCRIPTION

Definitions

Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments, of the invention described herein are capable of operation in other sequences than described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4^th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in molecular biology, biochemistry, structural biology, and/or computational biology).

"Nucleotide sequence", "DNA sequence" or "nucleic acid molecule(s)" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and singlestranded DNA, and RNA. It also includes known types of modifications, for example, methylation, "caps" substitution of one or more of the naturally occurring nucleotides with an analog. By "nucleic acid construct" or "construct sequence(s)" it is meant a nucleic acid sequence that has been constructed to comprise one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising non-native nucleic acid sequences, and the like. "Coding sequence" or a "nucleic acid molecule encoding" is a nucleotide sequence, which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances. An "expression vector" comprises an expression cassette which in turn comprises any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest, which is operably linked to a promoter of the expression cassette. Expression cassettes are generally DNA constructs preferably including (5' to 3' in the direction of transcription): a promoter region, a polynucleotide sequence, homologue, variant or fragment thereof operably linked with the transcription initiation region, and a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal. It is understood that all of these regions should be capable of operating in biological cells, such as prokaryotic or eukaryotic cells, to be transformed. The promoter region comprising the transcription initiation region, which preferably includes the RNA polymerase binding site, and the polyadenylation signal may be native to the biological cell to be transformed or may be derived from an alternative source, where the region is functional in the biological cell. The terms "protein", "polypeptide", and "peptide" are interchangeably used further herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. A "peptide" may also be referred to as a partial amino acid sequence derived from its original protein, for instance after tryptic digestion. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. This term also includes posttranslational modifications of the polypeptide, such as glycosylation, phosphorylation and acetylation, and also myristoylation. Based on the amino acid sequence and the modifications, the atomic or molecular mass or weight of a polypeptide is expressed in (kilo)dalton (kDa). A "protein domain" is a distinct functional and/or structural unit in a protein. Usually a protein domain is responsible for a particular function or interaction, contributing to the overall role of a protein. Domains may exist in a variety of biological contexts, where similar domains can be found in proteins with different functions.

By "isolated" or "purified" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated polypeptide" or "purified polypeptide" refers to a polypeptide which has been purified from the molecules which flank it in a naturally-occurring state, e.g., an antibody or VHH as identified and disclosed herein which has been removed from the molecules present in the sample or mixture, such as a production host, that are adjacent to said polypeptide. An isolated protein or peptide can be generated by amino acid chemical synthesis or can be generated by recombinant production or by purification from a complex sample.

The term "linked to", or "fused to", as used herein, and interchangeably used herein as "connected to", "conjugated to", "ligated to" refers, in particular, to "genetic fusion", e.g., by recombinant DNA technology, as well as to "chemical and/or enzymatic conjugation" resulting in a stable covalent link.

"Homologue", "Homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. The term "amino acid identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met, also indicated in one-letter code herein) 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 (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Preferably, the percentage of identity is calculated over a window of the full-length sequence referred to, or a particular length of a part of a sequence referred to, such as the homology to a surface exposed sequence region of a protein, as used herein. A "substitution", or "mutation", or "variant" as used herein, results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental protein or a fragment thereof. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity, which is hereby defined as a 'functional variant'. A functional variant thus also refers to variants comprising one or more substitutions or mutations, resulting in a homologue, preferably of at least 70 %, at least 80%, or at least 90 % amino acid identity, wherein the functionality is retained or at least similar as compared to the wild type protein or reference protein. In view of the present invention, antigen-binding proteins are functional in specifically binding to Acinetobacter Omp25 protein, so functional variants are defined as agents functional in binding Omp25 protein. Conserved amino acid substitutions are hence defined herein as those substitutions as compared to the sequence of A.baumannii Omp25, preferably in the surface-exposed regions as defined herein, which do not significantly impact the structure of the protein surface and thereby allow to retain said binding. For instance, conserved substitutions involve amino acid replacements of the same type of amino acid (so aliphatic amino acids may be replaced with another aliphatic amino acid, such as Vai with Leu or He, or alternatively small hydrophobic with another small hydrophobic amino acid such as Gly and Ala, or polar with another polar such as Ser with Thr, charged amino acids such as Lys and Arg both positive in charge, or Asp and Glu both negative in charge, etc.).

The term "wild type" refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene. In contrast, the term "modified", "mutant", "engineered" or "variant" refers to a gene or gene product that displays modifications in sequence, post-translational modifications and/or functional properties (i.e., altered characteristics) when compared to the wildtype gene or gene product. It is noted that naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product. A functional variant thus also refers to 'variants' as comparted to the wild type, though with the limitation that such a functional variant has retained function and properties relevant for its function. With 'function' in view of the present disclosure is referred for instance to the function of the VHH or VHH-based product, which preferably is the specificity for surface-binding of Acinetobacter cells, preferably A. baumannii cells, preferably capsulated Acinetobacter cells, and more preferably wherein Acinetobacter Omp25 protein is bound on the surface of said cells with the capacity to specifically target or label or detect A. baumannii bacteria.

"Binding" means any interaction, be it direct or indirect. A direct interaction implies a contact between the binding partners. An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two molecules. The interaction can be completely indirect, with the help of one or more bridging molecules, or partly indirect, where there is still a direct contact between the partners, which is stabilized by the additional interaction of one or more molecules. By the term "specifically binds," as used herein is meant a binding domain which recognizes a specific target, but does not substantially recognize or bind other molecules in a sample. Specific binding does not mean exclusive binding. However, specific binding does mean that proteins have a certain increased affinity or preference for one or a few of their binders. The term "affinity", as used herein, generally refers to the degree to which a ligand, chemical, protein or peptide binds to another (target) protein or peptide so as to shift the equilibrium of single protein monomers toward the presence of a complex formed by their binding.

A "binding agent", or "agent" as used interchangeably herein, relates to a molecule that is capable of binding to another molecule, via a binding region or binding domain located on the binding agent, wherein said binding is preferably a specific binding, recognizing a defined binding site, pocket or epitope. The binding agent may be of any nature or type and is not dependent on its origin. The binding agent may be chemically synthesized, naturally occurring, recombinantly produced (and purified), as well as designed and synthetically produced. Said binding agent may hence be a small molecule, a chemical, a peptide, a polypeptide, an antibody, or any derivatives thereof, such as a peptidomimetic, an antibody mimetic, an active fragment, a chemical derivative, among others. A "protein binding agent" is a binding agent of protein nature. An "antigen-binding protein" is a binding agent of protein nature specifically binding an antigen.

The term "binding pocket" or "binding site" refers to a region of a molecule or molecular complex, that, as a result of its shape and charge, favourably associates with another chemical entity or binding domain, such as a compound, proteins, peptide, antibody or Nb, among others. For antibody-related molecules, the term "epitope" or "conformational epitope" is also used interchangeably herein. The Acinetobacter Omp25 protein herein described comprises a binding pocket or binding site which includes, but is not limited to a Nanobody binding site, herein provided as a conformational epitope or binding site on the surface exposed or extracellular side of the OMP25 protein when present in the outer membrane. The term "part of a binding pocket/site" refers to less than all of the amino acid residues that define the binding pocket, binding site or epitope. For example, the atomic coordinates of residues that constitute part of a binding pocket may be specific for defining the chemical environment of the binding pocket, or useful in designing fragments of an inhibitor that may interact with those residues. For example, the portion of residues may be key residues that play a role in ligand binding, or may be residues that are spatially related and define a three-dimensional compartment of the binding pocket. The residues may be contiguous or non-contiguous in primary sequence.

The term "epitope" refers to a region of a molecule or molecular complex, that, as a result of its shape and charge, favourably associates with another chemical entity or binding domain, such as a compound, proteins, peptide, antibody or Nb, among others. An "epitope", as used herein, thus refers to an antigenic determinant of a polypeptide, constituting a binding site or binding pocket on a target molecule, such as Acinetobacter Omp25. Said epitope may comprise at least one amino acid that is essential for binding the binding agent, though preferably comprise at least 3 amino acids in a spatial conformation, which is unique to the epitope. Generally, an epitope consists of at least 4, 5, 6, 7 such amino acids, and more usually, consists of at least 8, 9, 10 such amino acids.

A "conformational epitope", as used herein, refers to an epitope comprising amino acids in a spatial conformation that is unique to a folded 3-dimensional conformation of a polypeptide. Generally, a conformational epitope consists of amino acids that are discontinuous in the linear sequence but that come together in the folded structure of the protein. However, a conformational epitope may also consist of a linear sequence of amino acids that adopts a conformation that is unique to a folded 3- dimensional conformation of the polypeptide (and not present in a denatured state). In protein complexes, conformational epitopes consist of amino acids that are discontinuous in the linear sequences of one or more polypeptides that come together upon folding of the different folded polypeptides and their association in a unique quaternary structure. The term "conformation" or "conformational state" of a protein refers generally to the range of structures that a protein may adopt at any instant in time. A conformational epitope may thus comprise amino acid interactions from different protein domains of the Acinetobacter Omp25 protein, in particular, the conformation epitope may comprise amino acids from different sequence regions of the protein, preferably wherein the entire conformational epitope is present or exposed at the membrane surface or exterior when the Omp25 protein is folded and present in a membrane.

Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, X-ray crystallography and multi-dimensional nuclear magnetic resonance, cryo-EM, or other structural analyses.

One of skill in the art will recognize that determinants of conformation or conformational state include a protein's primary structure as reflected in a protein's amino acid sequence (including modified amino acids) and the environment surrounding the protein. The conformation or conformational state of a protein also relates to structural features such as protein secondary structures (e.g., a-helix, p-sheet, among others), tertiary structure (e.g., the three dimensional folding of a polypeptide chain), and quaternary structure (e.g., interactions of a polypeptide chain with other protein subunits). Posttranslational and other modifications to a polypeptide chain such as ligand binding, phosphorylation, sulfation, glycosylation, or attachments of hydrophobic groups, among others, can influence the conformation of a protein. Furthermore, environmental factors, such as pH, salt concentration, ionic strength, and osmolality of the surrounding solution, and interaction with other proteins and co-factors, among others, can affect protein conformation. The conformational state of a protein may be determined by either functional assay for activity or binding to another molecule or by means of physical methods such as X-ray crystallography, NMR, or spin labeling, among other methods. For a general discussion of protein conformation and conformational states, one is referred to Cantor and Schimmel, Biophysical Chemistry, Part I: The Conformation of Biological. Macromolecules, W.H. Freeman and Company, 1980, and Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman and Company, 1993.

The term "antibody" refers to an immunoglobulin (Ig) molecule or a molecule comprising an immunoglobulin (Ig) domain, which specifically binds with an antigen. "Antibodies" can further be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The term "active antibody fragment" refers to a portion of any antibody or antibody-like structure that by itself has high affinity for an antigenic determinant, or epitope, and contains one or more complementarity determining regions (CDRs) accounting for such specificity, typically at least 3 CDRs, or in conventional antibodies, defined by 6 CDRs. Non-limiting examples of active antibody fragments include immunoglobulin domains, Fab, F(ab)'2, scFv, heavy-light chain dimers, immunoglobulin single variable domains (ISVDs), Nanobodies (or VHH antibodies), domain antibodies, and single chain structures, such as a complete light chain or complete heavy chain. The term "antibody fragment" and "active antibody fragment" or "functional variant" as used herein refers to a protein comprising an immunoglobulin domain or an antigen-binding domain capable of specifically binding A. baumannii Omp25. Antibodies are typically tetramers of immunoglobulin molecules. The term "immunoglobulin (Ig) domain", or more specifically "immunoglobulin variable domain" (abbreviated as "IVD") means an immunoglobulin domain essentially consisting of four "framework regions" which are referred to in the art and herein below as "framework region 1" or "FR1"; as "framework region 2" or "FR2"; as "framework region 3" or "FR3"; and as "framework region 4" or "FR4", respectively; which framework regions are interrupted by three "complementarity determining regions" or "CDRs", which are referred to in the art and herein below as "complementarity determining region 1" or "CDR1"; as "complementarity determining region 2" or "CDR2"; and as "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. It is the immunoglobulin variable domain(s) (IVDs) that confer specificity to an antibody for the antigen by carrying the antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VLwill contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation. In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, binds to the respective epitope of an antigen by a pair of (associated) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen. An "immunoglobulin single variable domain (ISVD)" as used herein, refers to a protein with an amino acid sequence comprising 4 Framework regions (FR) and 3 complementary determining regions (CDR) according to the format of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An "immunoglobulin domain" of this invention refers to "immunoglobulin single variable domains" (abbreviated as "ISVD"), equivalent to the term "single variable domains", and defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from "conventional" immunoglobulins or their fragments, wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. The binding site of an immunoglobulin single variable domain is formed by a single VH/VHH or VL domain. Hence, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDR's. As such, the single variable domain may be a light chain variable domain sequence (e.g., a VL- sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH- sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit). In one embodiment of the invention, the immunoglobulin single variable domains are heavy chain variable domain sequences (e.g., a VH- sequence); more specifically, the immunoglobulin single variable domains can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody. For example, the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence that is suitable for use as a (single) domain antibody), a "dAb" or dAb (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody (as defined herein, and including but not limited to a VHH); other single variable domains, or any suitable fragment of any one thereof. In particular, the immunoglobulin single variable domain may be a Nanobody (as defined herein) or a suitable fragment thereof. Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N.V. (a Sanofi Company). For a general description of Nanobodies, reference is made to the further description below, as well as to the prior art cited herein, such as e.g. described in W02008/020079. "VHH domains", also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin (Ig) (variable) domain of "heavy chain antibodies" (i.e., of "antibodies devoid of light chains"; Hamers-Casterman et al (1993) Nature 363: 446-448). The term "VHH domain" has been chosen to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VH domains") and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VL domains"). For a further description of VHHs and Nanobody , reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx N.V.; WO 01/90190 by the National Research Council of Canada; WO 03/025020 (= EP 1433793) by the Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V. As described in these references, Nanobody (in particular VHH sequences and partially humanized Nanobody) can in particular be characterized by the presence of one or more "Hallmark residues" in one or more of the framework sequences.

VHHs or Nbs are often classified in different families according to amino acid sequences, or even in superfamilies, as to cluster the clonally related sequences derived from the same progenitor during B cell maturation (Deschaght et al. 2017, Front Immunol 8:420). This classification is often based on the CDR sequence of the VHHs or Nbs, and wherein for instance each VHH (or Nb) family is defined as a cluster of clonally) related sequences with a sequence identity threshold of the CDR3 region. Within a single VHH family (also called CDR3-based VHH family or CDR3-family herein) defined herein, the CDR3 sequence is thus identical or very similar in amino acid composition, preferably with at least 80 % identity, or at least 85 % identity, or at least 90 % identity in the CDR3 sequence, resulting in Nbs of the same family binding to the same binding site, and having the same effect such as functional effect.

For numbering of the amino acid residues of an IVD different numbering schemes can be applied. For example, numbering can be performed according to the AHo numbering scheme for all heavy (VH) and light chain variable domains (VL) given by Honegger, A. and Pluckthun, A. (J. Mol. Biol. 309, 2001), as applied to VHH domains from camelids. Alternative methods for numbering the amino acid residues of VH domains, which can also be applied in an analogous manner to VHH domains, are known in the art. For example, the delineation of the FR and CDR sequences can be done by using the Kabat numbering system as applied to VHH domains from camelids in the article of Riechmann, L. and Muyldermans, S., 231(1-2), J Immunol Methods. 1999. It should be noted that - as is well known in the art for VH domains and for VHH domains - the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein. Determination of CDR regions may also be done according to different methods, such as the designation based on contact analysis and binding site topography as described in MacCallum et al. (J. Mol. Biol. (1996) 262, 732-745). Or alternatively the annotation of CDRs may be done according to AbM (AbM is Oxford Molecular Ltd.'s antibody modelling package as described on http://www.bioinf.org.uk/abs/index.html), Chothia (Chothia and Lesk, 1987; Mol Biol. 196:901-17), Kabat (Kabat et al., 1991; 5th edition, NIH publication 91-3242), IMGT (LeFranc, 2014; Frontiers in Immunology. 5 (22): 1-22) see, e.g., Dondelinger et al. 2018, Front Immunol 9:2278 for a review). Those annotations exist for numbering amino acids in immunoglobulin protein sequences, though in the present application solely the Kabat numbering is used, or the specific SEQ. ID numbering, as indicated. Said annotations further include delineation of CDRs and framework regions (FRs) in immunoglobulin-domain-containing proteins, and are known methods and systems to a skilled artisan who thus can apply these annotations onto any immunoglobulin protein sequences without undue burden. These annotations differ slightly, but each intend to comprise the regions of the loops involved in binding the target. Immunoglobulin single variable domains such as Domain antibodies and Nanobody® (including VHH domains) can be subjected to humanization, i.e. increase the degree of sequence identity with the closest human germline sequence. In particular, humanized immunoglobulin single variable domains, such as Nanobody® (including VHH domains) may be immunoglobulin single variable domains in which at least one amino acid residue is present (and in particular, at least one framework residue) that is and/or that corresponds to a humanizing substitution. Potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring VHH sequence with the corresponding framework sequence of one or more closely related human VH sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said VHH sequence (in any manner known per se, as further described herein) and the resulting humanized VHH sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person. Also, based on what is described before, (the framework regions of) an immunoglobulin single variable domain, such as a Nanobody® (including VHH domains) may be partially humanized or fully humanized.

Humanized immunoglobulin single variable domains, in particular Nanobody®, may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring VHH domains. By humanized is meant mutated so that immunogenicity upon administration in human patients is minor or non-existent. The humanizing substitutions should be chosen such that the resulting humanized amino acid sequence and/or VHH still retains the favourable properties of the VHH, such as the antigen-binding capacity. Based on the description provided herein, the skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favourable properties provided by the humanizing substitutions on the one hand and the favourable properties of naturally occurring VHH domains on the other hand. Such methods are known by the skilled addressee. A human consensus sequence can be used as target sequence for humanization, but also other means are known in the art. One alternative includes a method wherein the skilled person aligns a number of human germline alleles, such as for instance but not limited to the alignment of IGHV3 alleles, to use said alignment for identification of residues suitable for humanization in the target sequence. Also a subset of human germline alleles most homologous to the target sequence may be aligned as starting point to identify suitable humanisation residues. Alternatively, the VHH is analyzed to identify its closest homologue in the human alleles and used for humanisation construct design. A humanisation technique applied to Camelidae VHHs may also be performed by a method comprising the replacement of specific amino acids, either alone or in combination. Said replacements may be selected based on what is known from literature, are from known humanization efforts, as well as from human consensus sequences compared to the natural VHH sequences, or the human alleles most similar to the VHH sequence of interest. As can be seen from the data on the VHH entropy and VHH variability given in Tables A-5-A-8 of WO 08/020079, some amino acid residues in the framework regions are more conserved between human and Camelidae than others. Generally, although the invention in its broadest sense is not limited thereto, any substitutions, deletions or insertions are preferably made at positions that are less conserved. Also, generally, amino acid substitutions are preferred over amino acid deletions or insertions. For instance, a human-like class of Camelidae single domain antibodies contain the hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species, but compensating this loss in hydrophilicity by other substitutions, such as at position 103 that substitutes the conserved tryptophan residue present in VH from double-chain antibodies. As such, peptides belonging to these two classes show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanisation. Indeed, some Camelidae VHH sequences display a high sequence homology to human VH framework regions and therefore said VHH might be administered to patients directly without expectation of an immune response therefrom, and without the additional burden of humanization.

As used herein, a "therapeutically active agent" or "therapeutically active composition" means any molecule or composition of molecules that has or may have a therapeutic effect (i.e. curative or prophylactic effect) in the context of treatment of a disease (as described further herein). Preferably, a therapeutically active agent is a disease-modifying agent, which can be a cytotoxic agent, such as a toxin, or a cytotoxic drug, or an enzyme capable of converting a prodrug into a cytotoxic drug, or a radionuclide, or a cytotoxic cell, or which can be a non-cytotoxic agent. Even more preferably, a therapeutically active agent has a curative effect on the disease. The binding agent or the composition, or pharmaceutical composition (described below), of the invention may act as a therapeutically active agent, when beneficial in treating patients infected with or at risk of infection with Acinetobacter. The therapeutically active agent/binding agent or therapeutically active composition may include an agent comprising an ISVD specifically binding the A. baumannii Omp25 protein target and/or may contain or be coupled to additional "functional groups", interchangeably called "functional moieties" herein, which are advantageous when administered to a subject. Examples of such functional groups and of techniques for introducing them will be clear to the skilled person, and can generally comprise all functional groups and techniques mentioned in the art as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments, for which reference is for example made to Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA (1980). Such functional groups may for example be linked directly (for example covalently) to the ISVD, or optionally via a suitable linker or spacer, as will again be clear to the skilled person. One of the most widely used techniques for increasing the half-life and/or reducing immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). Another technique for increasing the half-life of a binding domain may comprise the engineering into bifunctional or bispecific domains (for example, one ISVD or active antibody fragment against A. baumannii and one against a serum protein such as albumin aiding in prolonging half-life) or into fusions of antibody fragments, in particular immunoglobulin single variable domains, with peptides (for example, a peptide against a serum protein such as albumin).

As used herein, the terms "determining," "measuring," "assessing,", "identifying", "screening", "addressing", "testing", and "assaying" are used interchangeably and include both quantitative and qualitative determinations. "Similar" as used herein, is interchangeable for alike, analogous, comparable, corresponding, and -like or alike, and is meant to have the same or common characteristics, and/or in a quantifiable manner to show comparable results i.e. with a variation of maximum 20 %, 10 %, more preferably 5 %, or even more preferably 1 %, or less.

The term "subject", "individual" or "patient", used interchangeably herein, relates to any organism such as a vertebrate, particularly any mammal, including both a human and another mammal, for whom diagnosis, therapy or prophylaxis is desired, e.g., an animal such as a rodent, a rabbit, a cow, a sheep, a horse, a dog, a cat, a lama, a pig, or a non-human primate (e.g., a monkey). The rodent may be a mouse, rat, hamster, guinea pig, or chinchilla. In one embodiment, the subject is a human, a rat or a non-human primate. Preferably, the subject is a human. In one embodiment, a subject is a subject with or suspected of having a disease or disorder, or is expected to be at high risk of developing a disease or disorder, in particular a disease or disorder as disclosed herein, also designated "patient" herein. However, it will be understood that the aforementioned terms do not imply that symptoms are present.

The term "medicament", as used herein, refers to a substance/composition used in therapy, i.e., in the prevention or treatment of a disease or disorder. According to the invention, the terms "disease" or "disorder" refer to any pathological state, in particular to the diseases or disorders as defined herein.

The term "treatment" or "treating" or "treat" can be used interchangeably and are defined by a therapeutic intervention that slows, interrupts, arrests, controls, stops, reduces, or reverts the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders. Therapeutic treatment is thus designed to treat an illness or to improve a person's health, rather than to prevent an illness. Treatment may also refer to a prophylactic treatment which relates to a medication or a treatment designed and used to prevent a disease from occurring, herein referred to as "prevention".

A "composition" relates to a combination of one or more active molecules, and may further include buffered solutions and/or solutes such as pH buffering substances, water, saline, physiological salt solutions, glycerol, preservatives, etc. for which a person skilled in the art is aware of the suitability to obtain optimal performance. Suitable conditions as used herein could also refer to suitable binding conditions, for instance when Nbs or test compounds are aimed to bind Acinetobacter cells, preferably A.baumannii cells expressing Omp25 at the outer surface of the membrane.

A "pharmaceutical composition" is a therapeutically active composition comprising the one or more antigen-binding agents or therapeutically active agents or therapeutically active compositions and optionally comprising a carrier, diluent or excipient. A "carrier", or "adjuvant", in particular a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable adjuvant" is any suitable excipient, diluent, carrier and/or adjuvant which, by themselves, do not induce the production of antibodies harmful to the individual receiving the composition nor do they elicit protection. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. A pharmaceutically acceptable carrier is preferably a carrier that is relatively non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient. Preferably, a pharmaceutically acceptable carrier or adjuvant enhances the immune response elicited by an antigen. Suitable carriers or adjuvantia typically comprise one or more of the compounds included in the following non-exhaustive list: large slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. The term "excipient", as used herein, is intended to include all substances which may be present in a pharmaceutical composition and which are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffer substances, stabilizing agents, flavouring agents or colorants. A "diluent" includes vehicles such as water, saline, physiological salt solutions, glycerol, ethanol, etc. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, or preservatives may be included in such vehicles. A pharmaceutically effective amount of polypeptides, or conjugates of the invention and a pharmaceutically acceptable carrier is preferably that amount which produces a result or exerts an influence on the particular condition being treated. For therapy, the pharmaceutical composition of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including orally, parenterally, topically, nasally, ophthalmically, intrathecally, intracerebroventricularly, sublingually, rectally, vaginally, and the like. Still other techniques of formulation as nanotechnology and aerosol and inhalant are also within the scope of this invention. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counter-indications and other parameters to be taken into account by the clinician. The pharmaceutical composition of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. When prepared as lyophilization or liquid, physiologically acceptable carrier, excipient, stabilizer need to be added into the pharmaceutical composition of the invention (Remington's Pharmaceutical Sciences 22nd edition, Ed. Allen, Loyd V, Jr. (2012). The dosage and concentration of the carrier, excipient and stabilizer should be safe to the subject (human, mice and other mammals), including buffers such as phosphate, citrate, and other organic acid; antioxidant such as vitamin C, small polypeptide, protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as PVP, amino acid such as amino acetate, glutamate, asparagine, arginine, lysine; glycose, disaccharide, and other carbohydrate such as glucose, mannose or dextrin, chelate agent such as EDTA, sugar alcohols such as mannitol, sorbitol; counterions such as Na+, and /or surfactant such as TWEEN™, PLURONICS™ or PEG and the like.

Detailed description

The protein binding agents, specifically the antigen-binding domains of said protein binding agents, as provided herein, specifically bind the Acinetobacter baumannii cell surface, more specifically via binding to the Outer membrane protein 25 (Omp25) target, which is present on the outer membrane, at least with its surface-exposed structural parts, as clear from the structure described for the first time herein. The importance of the capsule on Acinetobacter in relation to its virulence is clear from the fact that it protects the bacteria against several host processes, and aids in resistance to desiccation, disinfectants, antimicrobials, and antibiotics. Hence targeting an outer membrane protein present on the surface of pathogenic Acinetobacter cells ultimately requires that the target is also bound in the presence of a bacterial capsule. Surprisingly, this has been observed for the antibodies disclosed in the present invention by targeting a conformational epitope constituted by surface-exposed regions of the protein. Moreover, even though it is known that genetic variability is high between Acinetobacter baumannii strains, looking at reference classical and clinically isolated ones, the high conservation degree of the Omp25 protein within the species has been shown herein to provide for a selective antigen-binding protein tool that is usable in detection and targeting of many A. baumannii isolates. Among Acinetobacter baumannii strains, the Omp25 sequence is thus highly conserved. As deducible from the Acinetobase ⁽⁵⁾, and as exemplified for instance herein, the sequence of a number of the tested clinical isolates or classical strain (SEQ ID NO: 5 and 10) is identical or very close to the one of the reference strains (SEQ ID NO: 4-5, 43), with minor amino acid differences in the signal peptide N- terminal region.

Moreover, the surface exposed regions of the predicted 3D structure of A.baumannii Omp25 protein are thus highly conserved within the different isolates of Acinetobacter baumannii species. As also confirmed herein by resolving the structure of A. baumanii Omp25 in complex with the antigen-binding protein, the surface-exposed protein regions, i.e. the regions present on the extracellular side of the putative porin beta barrel, composed of beta strands and loops, confirmed to be positioned as such to be accessible to the Nb CDR loops. Finally, the high resolution structure obtained for A.baumannii Omp25 in complex with NbH7 revealed the epitope and paratope residues of the binding interaction. In view of that information, the selectivity of said antigen-binding proteins described herein may perhaps be defined as a binding site provided in said gram-negative bacteria of the Acinetobacter genus with a homologous Omp25 protein sequence of at least 90 % identity, and/or wherein at least the surface-exposed protein sequence or epitope as described therein are conserved.

Moreover, as the Omp25 protein is very divergent in structure among Gram-negative bacteria, and functional equivalence is not known so far, the selectivity of the Acinetobacter Omp25 antigen-binding proteins has been confirmed not to cross-bind to other cell surfaces such as E.coli or Klebsiella pneumoniae, and even more importantly to Brucella spp., for which the Omp25 has been more extensively described, but with a low sequence conservation to Acinetobacter Omp25. Hence the antigen-binding proteins of the present invention were shown not to bind Brucella spp. Omp25 and are thus selective in targeting a cell surface-exposed epitope on Acinetobacter species with an Omp25 protein homologue of at least 90 % identity to SEQ ID NO:43.

In another embodiment, the antigen-binding protein specifically binding the Acinetobacter cell surface of Omp25 more specifically binds the epitope comprising the A. baumannii Omp25 surface-exposed (region) amino acids at position 60, 93-106, 131, 165, 166, 167, and 190-193 as present in SEQ ID NO:43, or an epitope comprising the Acinetobacter Omp25 surface-exposed (region) amino acids in an Omp25 homologous protein corresponding to said residues at position 60, 93-106, 131, 165, 166, 167, and 190- 193 as present in SEQ ID NO:43, wherein said Acinetobacter Omp25 homologue is at least 90% identical to A.baumannii Omp25 of SEQ ID NO:43, and/or preferably wherein said Omp25 homologue is conserved in the nature of the amino acids present in said surface exposed regions constituting said epitope. With "corresponding" as used herein we refer to the observed correspondence in the position of these amino acids when the protein sequences are aligned under a pairwise or multiple alignment algorithm of protein sequences, as for instance done by using ClustalW.

In another embodiment, the antigen-binding protein specifically binding the Acinetobacter cell surface of Omp25 more specifically binds the epitope comprising the Omp25 surface-exposed regions 2 and 3, and optionally regions 4, 5, and 6 as present in SEQ ID NO:43, or an epitope comprising the Acinetobacter Omp25 surface-exposed regions 2 and 3, and optionally regions 4, 5, and 6 in an Omp25 homologous protein wherein said regions as defined for A.baumanni to be presented in SEQ ID NOs: 20, 21, and 22, 23, and 25, respectively, correspond to said regions in said Acinetobacter Omp25 homologue and wherein said Acinetobacter Omp25 homologue is at least 90 % identical toA.baumannii Omp25 of SEQ ID NO:43, and/or preferably wherein said Omp25 homologue is conserved in the nature of the amino acids present in said surface exposed regions constituting said epitope.

In a specific embodiment, the antigen-binding protein as presented here comprises an ISVD, wherein said ISVD comprises the CDR1, 2, and 3 sequences as part of one of the ISVD sequences of Nb H7, A8 and E12 (SEQ ID NO:l-3). The annotations used for defining such CDRs is known for the skilled person, and/or can be derived from the example of annotated CDRs for SEQ ID NO:1 (NbH7) as shown in Figure 13.

The CDR region annotation for each VHH sequence described herein according to Chothia (Chothia and Lesk, 1987; J Mol Biol. 196:901-17) is shown in the Figure 13. Alternatively, slightly different CDR annotations known in the art may be applied here and relate to the AbM (AbM is Oxford Molecular Ltd.'s antibody modelling package as described on http://www.bioinf.org.uk/abs/index.html), IMGT (LeFranc, 2014; Frontiers in Immunology. 5 (22): 1-22), Kabat (Kabat et al., 1991; Sequences of Proteins of Immunological Interest. 5th edition, NIH publication 91-3242), or MacCallum et al. (J. Mol. Biol. (1996) 262, 732-745) annotation, which are all applicable to identify the CDR regions of the ISVDs as disclosed herein for SEQ ID NO: 1-3. It should be noted that - as is well known in the art for VH domains and for VHH domains - the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering, see for instance Figure 13, where Kabat numbering is indicated for NbH7). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.

VHHs or Nbs are often classified in different sequences families or even superfamilies, as to cluster the clonally related sequences derived from the same progenitor during B cell maturation (Deschaght et al. 2017. Front Immunol. 10; 8 :420). This classification is often based on the CDR sequence of the Nbs, and wherein for instance each Nb family is defined as a cluster of (clonally) related sequences with a sequence identity threshold of the CDR3 region. Within a single VHH family defined herein, the CDR3 sequence is thus identical or very similar in amino acid composition, preferably with at least 80 % identity, or at least 85 % identity, or at least 90 % identity in the CDR3 sequence, resulting in Nbs of the same family binding to the same binding site, having the same effect or functional impact. Within the VHH family exemplified herein, CDR3 is identical for all residues among said 3 Nbs, H7, A8 and E12, except for one residue at the end of CDR3 (or in FR4), depending on the applied annotation), so these fall within the same family, and are expected to bind the same epitope. Indeed, the 3 family members were shown to all bind to the conformational epitope present on the surface exposed region of Omp25 as present on living A.baumannii cells.

A further embodiment relates to the antigen-binding protein as described herein, specifically binding Omp25, which comprises an ISVD comprising a sequence selected from the group of sequences of SEQ ID NOs: 1-3, or a functional variant of any one thereof with at least 90 %, or 95 %, or 99 % amino acid sequence identity over the full length of the ISVD sequence wherein the non-identical amino acids are located in one or more Framework residues. In a specific embodiment, the antigen-binding protein specific for A. baumannii Omp S as described herein comprises an ISVD comprising a humanized variant of a sequence selected from the group of sequences of SEQ ID NOs: 1-3, such as for instance but not limited to SEQ ID NOs:14-18 or of any functional variant thereof as described herein.

In a further specific embodiment, said humanized variant of the antigen-binding protein comprising SEQ ID NO:l-3 are disclosed wherein any combination of the single humanization substitutions made in SEQ ID NO:14-18 are used to provide a further humanized variant. Indeed, as exemplified herein, said humanization variants disclosed herein have shown to retain the binding affinity to Omp25 and the specific Acinetobacter cell-surface binding ability, even on capsulated cells, thereby proving that such substitutions or a combination of several of these do not affect the binding and functionality of the antigen-binding proteins. Moreover, as the first structure of this target was solved in complex with one of the antibodies disclosed herein, it is clear that the paratope is located solely in the CDRs and not in the framework, which does allow for conventional substitutions for which conservation of the antigen-binding capacity is known in the art. Humanized and/or functional variants are obtained as described herein, and are based on primary sequence alignment with the human IGHv3 coding sequence, to substitute one or more key residues of the alpaca-derived framework regions of the VHHs, followed by biophysical analysis of the resulting VHHs after recombinant production. Specifically, the stability and neutralizing properties of the resulting VHHs are analysed. Moreover, said original and/or humanized variant sequence can be fused directly or via a linker, as to provide for (humanized) bivalent VHH variants, as tandem repeats, or head- to-tail fusion, as interchangeably used herein. Alternatively, they may be additionally fused to an Fc tail, more specifically a human IgGl Fc.

So, in another embodiment the A.baumannii Omp25-binding protein as described herein is a multivalent or multispecific binding agent. The binding moieties within said multivalent or multispecific agent may be directly linked, or fused by a linker or spacer. The composition or binding agent(s) as described herein may appear in a "multivalent" or "multispecific" form and thus be formed by bonding, chemically or by recombinant DNA techniques, together two or more identical or different binding agents. Said multivalent forms may be formed by connecting the building blocks directly or via a linker, or through fusing the building block(s) with an Fc domain encoding sequence. Non-limiting examples of multivalent constructs include "bivalent" constructs, "trivalent" constructs, "tetravalent" constructs, and so on. The immunoglobulin single variable domains comprised within a multivalent construct may be identical or different, preferably binding to the same or overlapping binding site. In another particular embodiment, the binding agent(s) of the invention are in a "multispecific" form and are formed by bonding together two or more building blocks or agents, of which at least one binds to Omp25, as shown herein, and at least one binds to a further target or alternative molecule, so when present in multispecific fusion, presenting a binding agent or composition that is capable of specifically binding both epitopes or targets, thus comprising binders with a different specificity. Non-limiting examples of multi-specific constructs include "bispecific" constructs, "trispecific" constructs, "tetraspecific" constructs, and so on. To illustrate this further, any multivalent or multispecific (as defined herein) form of the invention may be suitably directed against one or more different epitopes on the same Omp25 antigen, or on epitopes of Omp25 proteins from different species or pathogens, or may be directed against two or more different antigens, for example one building block against Omp25 and one building block as a half-life extension against Serum Albumin, or another target. Multivalent or multi-specific ISVDs of the invention may also have (or be engineered and/or selected for) increased avidity and/or improved selectivity for the desired A. baumannii Omp25 interaction, and/or for any other desired property or combination of desired properties that may be obtained by the use of such multivalent or multispecific immunoglobulin single variable domains. Upon binding Omp25-expressing cells, said multi-specific or multivalent binding agent may have an additive or synergistic impact on the binding and/or therapeutic effect on Omp2-expressing cells, such as a killing potency or other antibacterial effect, preferably inherent to the binder, or alternatively by using a fusion or conjugated product containing the binder. In another embodiment, the invention provides a polypeptide comprising any of the immunoglobulin single variable domains according to the invention, either in a monovalent, multivalent or multispecific form. Thus, polypeptides comprising monovalent, multivalent or multispecific nanobodies are included here as non-limiting examples. The multivalent or multispecific binders or building blocks may be fused directly or fused by a suitable linker, as to allow that the at least two binding sites can be reached or bound simultaneously by the multivalent or multispecific agent.

Alternatively, at least one ISVD as described herein may be fused at its C-terminus to an Fc domain, for instance an Fc-tail of an Ig, resulting in an antigen-binding protein of bivalent format wherein two of said VHH-lg Fes, or humanized forms thereof, form a heavy chain only-antibody-type molecule through disulfide bridges in the hinge region of the Fc part, called "Fc fusion" herein. In a specific embodiment, the multivalent or multispecific agent as described herein is an Fc fusion or an antibody. Another embodiment comprises a humanized ISVD specifically binding Omp25 as described herein, comprised in a multivalent or multispecific agent, which may be provided as a humanized ISVD-IgG fusion, and which may further include but is not limited to the use of IgG humanization variants known in the art.

In an alternative setup, the "Fc fusion" is designed by linking the C-terminus of such a bivalent or bispecific binder fused by a linker to an Fc domain, which then upon expression in a host forms a multivalent or multispecific-antibody-type molecule through disulfide bridges in the hinge region of the Fc part.

In a specific embodiment, the Omp25-binding multivalent or multispecific agent is a bivalent or bispecific binder. In a further specific embodiment, the multivalent or multispecific agent that specifically interacts with the A.baumannii Omp25 protein comprises at least one sequence selected from the group of sequences of SEQ. ID NOs: 1-3, or a functional variant of any one thereof with at least 90 %, or 95 %, or 99 % sequence identity over the full length of the ISVD sequence wherein the nonidentical amino acids are located in one or more Framework residues, or a humanized variant of any one thereof. In a further specific embodiment, said multivalent or multispecific agent comprises a bivalent Omp25-specific ISVD or a homologues functional variant of any one thereof with at least 90 %, or 95 %, or 99 % sequence identity over the full length of the sequence wherein the non-identical amino acids are located in one or more framework residues, or a humanized variant of any one thereof or a homologue with at least 90 %, or 95 %, or 99 % sequence identity over the full length of the sequence wherein the non-identical amino acids are located in one or more framework residues, thereof. In a further specific embodiment said multivalent or multispecific binding agent described herein comprises an Fc fusion of any one of the Omp25-specific ISVDs as described herein, or of a humanized variant thereof.

In further aspects of the invention, the antigen-binding protein as described herein may be labelled, tagged or conjugated, or fused to a labelling or detectable protein. More specifically, a detection label may be useful for localizing, visualizing, and quantitating a binding or recognition event, hence also for in vivo imaging or for diagnostic purposes. The labelled binding agents as described herein can detect Acinetobacter, more specifically A.baumannii cells in vitro and in vivo. Another use for detectably labelled binding agents is a method of bead-based immunocapture comprising conjugating a bead with a fluorescent labelled binding agent and detecting a fluorescence signal upon binding of a ligand. Similar binding detection methodologies utilize the surface plasmon resonance (SPR) effect to measure and detect antigen-binding protein/antigen interactions.

The term detectable label or tag, as used herein, refers to detectable labels or tags allowing the detection and/or quantification of the Omp25-specific binding agent as described herein, and/or to detect Acinetobacter cells bound to said antigen-binding agent, and is meant to include any labels/tags known in the art for these purposes. Particularly preferred, but not limiting, are affinity tags, such as chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His) (e.g., 6x His or His6), biotin or streptavidin, such as Strep-tag®, Strep-tag II® and Twin-Strep-tag®; solubilizing tags, such as thioredoxin (TRX), poly(NANP) and SUMO; chromatography tags, such as a FLAG-tag; epitope tags, such as V5-tag, myc-tag and HA-tag; fluorescent labels or tags (i.e., fluorochromes/-phores), such as fluorescent proteins (e.g., GFP, YFP, RFP etc.) and fluorescent dyes (e.g., FITC, TRITC, coumarin and cyanine); luminescent labels or tags, such as luciferase, bioluminescent or chemiluminescent compounds (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs); phosphorescent labels; a metal chelator; and (other) enzymatic labels (e.g., peroxidase, alkaline phosphatase, beta-galactosidase, urease or glucose oxidase); radioisotopes. Also included are combinations of any of the foregoing labels or tags. Technologies for generating labelled polypeptides and proteins are well known in the art. An antigen-binding protein comprising an Omp25-specific ISVD of the invention, coupled to, or further comprising a label or tag allows for instance immune-based detection. Immune-based detection is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as described above. See, for example, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. In the case where multiple antibodies are reacted with a single array, each antibody can be labelled with a distinct label or tag for simultaneous detection. Yet another embodiment may comprise the introduction of one or more detectable labels or other signal-generating groups or moieties, or tags, depending on the intended use of the labelled or tagged Omp25-specific or A.baumannii-spec ic binding agent of the present invention. Other suitable labels will be clear to the skilled person, and for example include moieties that can be detected using NMR or ESR spectroscopy. Such labelled binding agents, such as Omp25-specific ISVDs or Nanobodies as described herein may for example be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other "sandwich assays", etc.) as well as in vivo imaging purposes, depending on the choice of the specific label.

The labelled or tagged binding agents as described herein may also be used as an affinity purification agent. In this process, the labelled agent or antigen-binding protein is immobilized on a solid surface, such as a Sephadex, Sepharose or other polymeric resin, or filter paper, or a cartridge, using methods well known in the art. The immobilized binding agent is subsequently contacted with a sample containing the antigen to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the antigen to be purified, which is bound to the immobilized binding agent. Finally, the support is washed with another suitable solvent, which is capable to outcompete the binding.

In a further aspect of the invention, the antigen-binding protein as described herein may be conjugated to a further functional moiety, such as a therapeutic or half-life extension moiety, or to a cell-penetrant carrier, or an effector molecules such as a toxin or antibacterial killing agent. More specifically, the binders as described herein may as fusion be further coupled or operably linked to further binding moieties, which may be additional ISVDs, or antigen-binding domains specific for a target protein, preferably a target present on the cell surface or extracellularly, or to extend the half-life (e.g. serum albumin specific binders), or alternative compounds that are providing a function. One of the most widely used techniques for increasing the half-life and/or reducing immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). Another technique for increasing the half-life of a binding agent may comprise the engineering into bifunctional or bispecific domains (for example, one or more ISVDs or active antibody fragments against Omp25 coupled to one ISVD or active antibody fragment against serum albumin aiding in prolonging half-life)) or into fusions of antibody fragments, in particular immunoglobulin single variable domains, with peptides (for example, a peptide against a serum protein such as albumin). The coupling to additional moieties will result in multispecific binding agent, as further disclosed herein. In a final aspect of the invention, the antigen-binding protein as described herein which specifically binds A.baumannii Omp25, as expressed on A.baumannii cells, with selective binding properties to a capsulated as well as non-capsulated bacterial cell, is used as a medicament, or for prevention or treatment of bacterial infection, specifically A.baumannii infection.

It is to be understood that although particular embodiments, specific configurations as well as materials and/or molecules, have been discussed herein for methods, samples and products according to the disclosure, various changes or modifications in form and detail may be made without departing from the scope of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.

Aspects of the disclosure

In one aspect the invention relates to an antigen-binding protein specifically binding Acinetobacter Outer membrane protein 25 (Omp25).

A further embodiment relates to said antigen-binding protein, which binds the Acinetobacter Omp25 protein as present on the surface of Acinetobacter capsulated and non-capsulated cells.

A further embodiment relates to said antigen-binding protein, wherein said Acinetobacter Omp25 protein comprises Acinetobacter baumannii OmplS as present in SEQ ID NO:4, or a homologue with at least 90 % identity thereof.

A further embodiment relates to said antigen-binding protein, wherein said antigen-binding protein comprises an antibody, an antibody mimetic, a single chain variable fragment (ScFv), an immunoglobulin single variable domain (ISVD), a VHH, or a nanobody, or an active antibody fragment.

A further embodiment relates to said antigen-binding protein, comprising an ISVD comprising the complementarity determining regions (CDRs) as present in any of SEQ ID NOs: 1 to 3, wherein the CDRs are annotated according to Kabat, MacCallum, IMGT, AbM, or Chothia.

A further embodiment relates to said antigen-binding protein, wherein the ISVD comprises a sequence selected from the group of sequences of SEQ ID NOs: 1- 3, or a functional variant of any one thereof with at least 90 % amino acid identity over the full length of the ISVD sequence wherein the nonidentical amino acids are located in one or more Framework residues, or a humanized variant of any one thereof.

A further embodiment relates to said antigen-binding protein, which is a multivalent or multispecific antigen-binding agent, preferably a bivalent or bispecific antigen-binding agent, which may be in an Fc fusion or an antibody format. A further embodiment relates to said antigen-binding protein, wherein said antigen-binding protein is linked to a further moiety, wherein said moiety is linked via conjugation or via genetic fusion.

A further embodiment relates to said antigen-binding protein, wherein said further moiety comprises a detectable label, such as a dye or fluorophore, an effector molecule, such as a toxin, or a functional moiety, such as a therapeutic moiety or a half-life extension.

Another aspect of the disclosure relates to a nucleic acid molecule encoding any of said preceding antigen-binding proteins.

Another aspect of the disclosure relates to a pharmaceutical composition comprising said antigenbinding protein, and optionally a further therapeutically active agent, a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

Another aspect of the disclosure relates to said antigen-binding protein or said pharmaceutical composition, for use as a medicament, in particular for use in treatment of Acinetobacter infection, preferably Acinetobacter baumannii infection.

Another aspect of the disclosure relates to said antigen-binding protein or said pharmaceutical composition, for use in in vivo imaging, or for use as a diagnostic.

Another aspect of the disclosure relates to the use of said antigen-binding protein, or said pharmaceutical composition, for depletion of Acinetobacter cells from a sample in vitro.

EXAMPLES

Example 1. Immunization and generation of a Nb library for selection of A. baumannii-b'md'mg Nbs.

A llama (lama glama) was immunized weekly with a mix of fixed A. baumannii cells (AB5075-VUB; AB5075-VUB-/t :. SAbol3; approximately 1.6 10⁸ CFU). After the final injection, total RNA was extracted from peripheral blood mononuclear cells from which cDNA was synthesized, as described earlier.¹- ² The cDNA was cloned into the phagemid vector pMESy4, enabling expression of Nbs with a N-terminal periplasmic leader sequence and a C-terminal 6-His-EPEA tag. The resulting phage library, consisting of approximately 4.10⁸ clones, was enriched by two phage display selection rounds using fixed A. baumannii cells (approximately 2.56 10⁹ CFU). Out of the enriched library, clones were transformed to E. coli WK6. Cells were grown in Terrific Broth (Duchefa Biochemie) and then induced by IPTG for periplasmic expression and purification of the Nbs as described, with each Nb fused at its C -terminus to a His-EPEA affinity tag (SEQ ID NO:9) for detection purposes.³ Example 2. NbH7 specifically binds the membrane of A. baumannii cells.

A collection of 3 classically used reference strains (AB5075-VUB - CP070362; AB5075-VUB-/t :.7SAbol3 - CP070358, and ATCC17978-VUB) and 3 clinically isolated A. baumannii strains (AB3-VUB; AB180-VUB; AB220-VUB) and 1 E. coli strain (E. coli S17) were used to further test binding of the primary selected Nb, NbH7 (SEQ ID NO:1). We refer to (5) and https://acinetobase.vib.be/ for more detailed information on the particular strains used herein. In particular, in view of the phenotypes and description of the capsule presence for the strains comprised in Acinetobase, we refer to (6) for further details.

The bacterial cultures were started from a single clone and were grown for 17 h at 37°C under agitation (165 rpm) in low salt broth (Luria-Bertani formulation, Duchefa Biochemie). Cells were collected by centrifugation (8000g) and normalized to ODgoo=3 (10⁹ CFU/ml) in phosphate buffered saline (PBS) for further steps.

Labelling of the Nbs was done by incubation with molar 3-fold of DyLight 650 NHS ester (ex:652/em:672, Thermo Scientific) for 1 h, quenching of the reaction with 1 mM Tris buffer, followed by overnight dialysis in PBS. To allow binding, 100 pl of the labelled NbH7 (10 pM) was incubated with 100 pl of fixed or living bacterial cells (approximately 10⁵ CFU) for 30 min at 37°C, under agitation (165 rpm). To remove unbound Nb, the cells were centrifugated (8000g). Finally, the bacteria were spotted on an 1.5% agarose pad (Thermo Scientific Gene Frame). Microscopy images were acquired using a Leica DMi8 fluorescence microscope with a DFC7000 GT camera (Leica Microsystems CMS GmbH). The GFP expressed in the cytoplasm of the AB5075-VUB strain was used to determine the right focal plane, after which phase contrast and fluorescent images were acquired. The fluorescent images were acquired with a Leica FRAP450 and Y5 filter set. The raw data was processed by using ImageJ software where brightness was adjusted equally for all fluorescence micrographs.

We observed that NbH7 bound the non-capsulated AB5075-VUB-itrA::ISAbal3 (also referred to as AB5075C-) strain both in exponential and stationary phase (Figure 1A), and moreover the labelled NbH7 showed membrane labelling on the bacterial cell, as seen in the intensity profile in Figure IB.

Furthermore, we showed in Figures 2-6 that NbH7 binds a variety of A. baumannii strains, including a multidrug-resistant (AB180-VUB), an extensively drug-resistant (AB220-VUB) and a pandrug-resistant (AB3-VUB) strain and another classically used strain (ATCC17978-VUB; also see ), while the same experiment using E. coli cells did not result in binding of NbH7 (Figure 7), indicating specificity of the Nb binding site to a certain degree. Example 3. NbH7 specifically targets the Acinetobacter baumannii Outer-membrane-protein 25 (Omp25).

To explore the binding site of the Nb on the cells, we performed a pull down experiment, for which we used approximately IO¹⁰ CFU of AB5075-VUB and AB5075-VUB-/t :.7SAbol3, referred to as AB5075C+ and AB5075C-, respectively, wherein 'C+' refers to the presence of capsule on the bacterial cells, and 'C-' to the absence of a capsule on the bacterial cells. The cells were lysed by PBS supplemented with 1 % DDM, 300 mM NaCI, 10 mM imidazole, 50 pg/ml DNase and 0.1 mg/ml lysozyme for 2 h. The lysed cultures were then incubated with 200 pM NbH7, and two control Nbs for 1 h at 37°C under agitation. The controls used herein are NbE3 (SEQ ID NO: 7), which binds Acinetobacter baumannii but at a different binding site, and NbB9 (SEQ ID NO: 8) which does not bind to Acinetobacter baumannii cells ⁽⁷⁾. To pull out the Nb-target complexes based on the hexahistidin tag of the Nb, the cell lysates were loaded over His SpinTrap columns (Cytiva) according to the manufacturer's instructions. The columns were washed with PBS supplemented with 0.03% DDM, 20 mM imidazole and 300 mM NaCI, and eluted with PBS supplemented with 0.03% DDM, 500 mM imidazole and 300 mM NaCI. The eluted samples were consequently analyzed by SDS-PAGE (Figure 8A). The band visible around 25 kDa in the NbH7 sample, but not in the E3 or B9 pull down samples, was excised and analyzed by Mass spectrometry analysis.

The mass spectrometry analysis revealed that Acinetobacter Omp25 (SEQ ID NO:5) is potentially the antigen target of NbH7, the AB5075C+Aomp25 and AB5075C-Aomp25 strains were generated. The omp25 gene deletion was done by generating a construct of the sacB-aaC selection/counterselection cassette flanked by 2 kb up and downstream homologous regions of the omp25 gene, and transforming it to as described in Whiteway et al *

In Figure 8B, we show that the strain lacking the omp25 gene did not result in binding of the NbH7 to the cells, providing the confirmation to conclude that Acinetobacter baumannii Omp25 is the antigen target of NbH7.

Example 4. NbH7 translationally fused to super folder Green fluorescent protein (sfGFP) retains its binding to Acinetobacter baumannii cells.

The NbH7 sequence was cloned into a customized pET21b vector to obtain a fusion protein upon expression of the construct wherein NbH7 has a hexahistidin tag, a glycine-serine linker and super folder GFP (sfGFP) fused at its C-terminal end (SEQ ID NO: 39). The plasmid was transformed into the E. coli T7 SHuffle strain for optimal expression and formation of disulfide bonds in the cytoplasm. An overnight culture (lysogeny broth medium, 30°C, 100 pg/ml ampicillin) of the transformed strain was used to inoculate Terrific broth medium, supplemented with 0.1% glucose, 100 pg/ml ampicillin and 2 mM MgCL. Expression of the construct was induced by 0.1 mM IPTG at ODsoo=0.6 for 4 h at 30°C. The cultures were then centrifuged (15 min, 4788 g) and the pellet was resuspended in sonication buffer (50 M Tris base pH=8, 300 mM NaCI, 10 % Glycerol, 10 mM Imidazole, 50 pg/mL DNase I, 0.2 % AEBSF Hydrochlorid, 0.1 Leupeptin Hemisulfat) for sonication (Amplitude=60%, pulse=10s) for 3 min. The cell lysate was centrifuged (40 min, 10976 g) to obtain the supernatant and thus the soluble fraction.

Purification was done using Nickel-based Immobilized Metal Affinity Chromatography (Ni²⁺-IMAC). The cell lysate was loaded on a 1ml HisTrap HP His tag purification column (Cytiva) after equilibration of the column with 5 column volumes (CV) wash buffer (20 mM Tris base pH=8, 500 mM NaCI, 20 mM Imidazole). After the column was loaded, the column was washed with 5 CV wash buffer and eluted by elution buffer (20 mM Tris base pH=8, 500 mM NaCI, 500 mM Imidazole). The eluted fractions were then analyzed by SDS-PAGE and the fractions corresponding to the expected molecular weight were pooled, concentrated a smaller volume and separated according to size by Size Exclusion Chromatography (SEC). The Enrich SEC 70 10x300 column was equilibrated with PBS and fractions were collected. By SDS-PAGE, the fractions containing the correct construct based on the expected molecular weight were pooled and used for microscopy analysis.

To test the binding ability of the NbH7_sfGFP construct, the pooled fractions of the size exclusion chromatography were added to approximately to 100 pl of ODgoo=3 of AB5075C- and E. coli S17 of which the latter serves as a negative control because NbH7 does not bind this strain. The cells and construct were incubated for 30 min at 37°C, washed three times with PBS and then put on an 1.5 % agarose pad (Gene Frame, Thermo Scientific) for fluorescence microscopy acquisition. The fluorescent images were acquired with a Leica FRAP450 filter set. The raw data was processed by using ImageJ software where brightness was adjusted equally for all fluorescence micrographs. To quantify the fluorescence in the micrographs, 15 cells were picked randomly for both strains and the max gray value was measured by ImageJ software. The graphs and statistical analysis were done using Graphpad Prism.

As shown in Figure 9, NbH7 retains its binding ability and specificity on AB5075C- when translationally fused to sfGFP, indicating that the Nb can be used in further applications using Nb fusions with additional functional moieties.

Example 5. NbH7 can scavenge bacterial cells from liquid samples.

Nb proteins were coupled to magnetic beads (Invitrogen™ Dynabeads™ M-280 Tosylactivated, Fisher Scientific) by adding 100 pg NbH7 or NbB9 (Ctrl Nb) per 5 mg of beads. Preparation of the beads, labelling and washing steps after labelling were done as instructed by the manufacturer. For the control with uncoupled beads, beads were treated with all buffers, but no proteins were added. In the experiments, 1 mg of coupled beads was added to 1 ml of approximately 3 10⁵ CFU/ml of stationary phase bacteria in PBS. For accurate estimation of the number of cells, the CFU were determined before and after incubation with the coupled beads. CFU determination was done by plating serial dilutions on LB agar plates and overnight incubation at 37°C, after which the CFU were calculated.

As shown in Figure 10A, NbH7 coupled beads allowed to remove up to 97 % of bacteria in PBS, when incubated for 30 mins, and an increase in incubation time up to 120 minutes further increased capture efficiency, though also non-specific interactions with the beads were increased as shown in the 'Beads' and 'ctrINb' sample (Figure 10A).

Furthermore, several short passages (Pl, P2, P3 in Figure 10B; of 5 min) with new NbH7-coupled beads increases efficiency with less non-specific binding.

Example 6. VHH family members confirm specific recognition of Acinetobacter baumannii cells.

Two additional nanobodies classified to belong to the same family of NbH7, NbA8 (SEQ ID NO: 2) and NbE12 (SEQ ID NO:3), were tested for binding on AB5075C- by fluorescence microscopy. The strain was grown and handled, and the nanobodies labelled, as described in Example 2.

As shown in Figure 11, both NbA8 and NbE12 bind the non-capsulated AB5075C- strain.

Next, NbA8 and NbE12 were tested for binding on stationary phase bacteria of AB3-VUB by fluorescence microscopy. AB3-VUB is a clinically isolated strain which is classified as pandrug-resistant and is highly capsulated consequently making it a highly relevant, but potentially difficult target^5-6. The strain was grown and handled, and the nanobodies labelled, as described in Example 2.

As shown in Figure 12, these 2 Nb belonging to the same VHH family as NbH7 were shown to bind the AB3-VUB strain, so this confirms that capsulated as well as non-capsulated bacterial cells are recognized by the Nbs, while the control Nb does not show binding.

Example 7. Binding kinetics and resolving the binding site of the Nbs using recombinantly expressed Acinetobacter baumannii Omp25 protein.

Next, the Acinetobacter baumannii Omp25 protein (SEQ ID NO:6) was recombinantly produced in E.coli, and purified for determination of the affinity kinetics upon binding of the Nbs /n vitro (also see methods and Figure 15A). NbH7 and NbA8 were tested for their affinity to the recombinant Acinetobacter baumannii Omp25 protein, and the specific binding to Omp25 was analyzed using Biolayer interferometry using the Octet® R8 apparatus (also see methods and Figure 15B). As shown in Figure 14, all measurements included a negative control Nb (CtrINb) tested for the highest concentration of Omp25 to monitor aspecific binding or background signal, which was used to normalize the data. Overview of the K_D values obtained for each Omp25 concentration per Nb, as well as the average K_D are in the low nM range, confirming that Omp25 specific binding is also obtained in vitro for both Nbs of this VHH family.

Example 8. Humanized NbH7 variants bind capsulated AB5075-VUB (AB5075C+).

To improve the homology with human VH domains the NbH7 sequence was aligned with the closest human homologue sequence human VH3-23 (GenBank: P01764.2)/J5). Typical residues common in most VHH sequences are often left unchanged as these are considered critical for the VHH properties. However, mutations in framework regions which appear less typical are mutated towards the human amino acid at their respective positions. As non-limiting examples of humanized variants of SEQ ID NO:1, five different humanized sequences were generated for the NbH7 denoted as H7hl-H7h5, and as provided by SEQ ID NOs: 14-18, resp.

The substitutions made in the framework regions are shown in Figure 16A as compared to the NbH7 original camelid sequence. All 5 variants were produced and purified according to the method used for the NbH7.

First, their binding capability to capsulated A. baumannii AB5075-VUB was analyzed (Figure 16B) confirming that surface-binding to A.baumannii is retained in the framework region-substituted variants, Furthermore, binding affinity to recombinantly produced and purified A.baumannii Omp25 protein was analyzed by BLI (see methods as applied also above for the original Nbs). As shown in the table in Figure 17, the binding affinity of said humanized variants is also in the low nM range, and thus not affected by the substitutions as compared to the original H7 Nb.

Example 9. The binding site of the Nb family is located in the Omp25 surface exposed regions.

In view of resolving the structure of the Nb binders in complex with the Acinetobacter Omp25 target, we applied AlphaFold v2.0¹⁴ to define the surface-exposed regions of the protein. As shown in Figure 18, seven different stretches of the polypeptide are facing the extracellular surface, with for each surface-exposed region the position of the first amino acid per region indicated on the figure (numbering according to the position in SEQ ID NO:5, which provides for the A.baumanii Omp25 including a signal peptide.

These multiple sequence regions (as provided in SEQ ID NOs: 19-25) that are exposed on the surface of the bacterial cell, i.e. which are present on the extracellular side of the membrane, may thus be of importance for binding the Nb. In Figure 19, a sequence alignment is provided showing the Acinetobacter baumannii Omp25 sequence (SEQ ID NO:5) followed by the aligned surface-exposed stretches determined based on the Alphafold prediction. In addition, the Thermothelomyces thermophila ADP/ATP carrier (TtAac) amino acid sequence was aligned to it as well, which is the antigen target of the negative control Nb used herein⁷ (NbB9 of SEQ. ID NO:8), confirming that there are indeed no similar sequence stretches present in this negative control target of NbB9 which does not bind to the Acinetobacter cells.

Example 10. The Nb binding is specific for Acinetobacter spp. Omp25 protein.

The Omp25 protein studied in Brucella species has been the antigen for clinical antibody development ⁿ, though this protein is quite diverse in sequence as compared to the A. baumannii Omp25 , hence the Brucella Omp25 is not considered as a true orthologue of the Acinetobacter Omp25 protein. However, to check whether the Acinetobacter Omp25 Nb binders described herein specifically bind the surface of Brucella spp., we analyzed the binding in a similar manner as previously described for Acinetobacter cells herein. As shown in Figure 20, Nb H7 was not capable of binding three Brucella spp. belonging to two different species: B. melitensis 16M, B. abortus 544 and B. abortus 2308, as expected since the surface-exposed Omp25 regions of Brucella spp. are different in sequence as compared to the Acinetobacter baumannii Omp25 regions.

Furthermore, other Gram-negative bacteria such as E.coli S17 and Klebsiella pneumoniae also lack this cell-surface epitope (Figure 21), strengthening the selectivity of the Nb binders towards Acinetobacter species Outer membrane protein binding.

Finally, we analyzed whether the surface-exposed epitope of Omp25 would be sufficiently conserved among further Acinetobacter species as to allow specific binding of the Nbs described herein across the Acinetobacter genus.

Binding assessment of NbH7 on Acinetobacter calcoaceticus, Acinetobacter pittii and Acinetobacter junii revealed cell-surface binding to A. pitii and A. calcoaceticus, which both have an Omp25 sequence identical over more than 90 % to A.baumannii Omp25 (Table 1), but not to A. junii, wherein the sequence identity is found to be less than 82 %. Looking into the conservation of the surface-exposed regions, indeed, this latter species shows significant sequence diversity within said regions (Figure 22), which may explain the lack of or weak binding to these Acinetobacter cells.

Table 1. Homologous Omp25 sequences in A. pittii, A. calcoaceticus and A. junii.

% ID with AB5075 Omp25 Accession number Length (#aa)

Acinetobacter 95,71 WP_004639960.1 255 calcoaceticus

95,28 WP_199966399.1 255

94,85 VAX43036.1 255

93,13 CAI3136485.1 255

Acinetobacter junii 81,97 WP_004951331.1 255

81,55 RXS99878.1 255

81,12 WP_262579446.1 255

A Protein BLAST was done using AB5075-VUB Omp25 against a non-redundant protein sequences database in Acinetobacter (taxid: 469), excluding Acinetobacter baumannii (taxid: 470). The results were filtered for each species separately and one representative of differing sequence identity (% ID) was selected for each hit with the same amino acid sequence length (#aa): 255. For each hit, the accession number in Genbank is given.

Example 11. Crystal structure of NbH7 in complex with Acinetobacter baumannii Omp25 protein

Furthermore, the binding site has been determined by X-ray crystal structure analysis ) to a resolution of 2.91A providing the NbH7 in complex with the recombinantly produced Acinetobacter baumannii Omp25 protein (see methods (Figure 24)). The structure confirmed our previously predicted surface- exposed regions to be positioned at the extracellular side and further allowed to distinguish the contact points or binding site of the Nb to said exposed surface of the Omp25 protein. As shown in the details of Figure 24 B to D, a number of amino acids present in the CDR1, 2, and 3 loops respectively, are responsible for the specific binding site as indicated in the sequence presented in Figure 25. In addition, the epitope or binding residues of Omp25 in contact with the Nb CDR loops were confirmed to mainly be located in exposed region 2, 3, 4, 5 and 6, as also indicated in Figure 25.

From these data we can conclude that the binding site of the antibody protein binding agents disclosed in this application specifically bind an epitope present on the cell-surface exposed protein regions of Acinetobacter baumanni Omp25, and said epitope specifically being composed of the Omp25 residues with position G60, E93-T106, E131, D165, L166, D167, and N190-T193 as present in SEQ ID NO:43. Conversely, the paratope of the Nb H7 (SEQ ID NO:1) for binding via its CDR loops to Omp25 comprises or consists of the amino acids with positions R29, 30S, and N31 in CDR1, K52, H58, Y59, A60, and D61 in CDR2, and Y96, Y97, S98, G99, FIDO, YlOOa, LlOOb, PlOOc, AlOOd, AlOOe, LlOOf, and ElOOi in CDR3, according to Kabat numbering.

The epitope region of Omp25 as observed herein for NbH7 is conserved in a number of Acinetobacter species, as shown herein for at least 2 out of 3 tested Acinetobacter species, and confirming that the loss of binding of the Nbs for A.junii is explained through its divergence in the exposed region 2 and 3 residues of the epitope, since these 2 regions do not show a highly similar amino acid sequence, hence low conservation, in A.junii Omp25. So, at least the surface-exposed regions 2 and 3 provide for a number of essential residues of the epitope, more specifically the residues corresponding to G60, E93- T106 of SEQ ID NO:43 are key for binding to bind Acinetobacter Omp25, and hence the Acinetobacter surface.

Example 12. NbH7 can bind recombinantly expressed A.baumannii Omp25 in E. coli.

Recombinant expression of AB5075-VUB Omp25 in the outer membrane of f. coli was performed using a construct for Omp25 expression containing the E. coli OmpA signal peptide (Ec OmpA SP), a streptavidin tag (Strep) and the AB5075 omp25 (Ab omp25) sequence without its endogenous signal peptide coding sequence (SEQ ID NO:42), in an inducible expression cassette (see methods). Upon culturing the transformed E.coli cells, the cultured cells were analyzed by fluorescence microscopy to assess binding of labelled NbH7 on the surface of the E. coli cells comparing before (non-induced) and after (induced) expression of AbOmp25 (Figure 22).

As observed, the recombinant expression of this Acinetobacter Outer membrane 25 protein allows for detection and/or scavenging of those vehicles or cells wherein said recombinant protein is expressed and present on its membrane surface.

Example 13. NbH7 fused to GFP by conjugation retains Acinetobacter binding ability.

As described in the methods below, it was shown herein that the Acinetobacter Omp25-specific Nbs may also be applied in cell or membrane labelling wherein the Omp25 protein is present with its surface exposed regions on the outside surface by assembling a fluorescently labeled Nb construct which is composed of the Omp25-Nb binder fused to a SpyCatcher followed by conjugation to a Spytag-GFP protein, resulting in a labelled Nb conjugate. Indeed, as shown in Figure 26, the AB5075-VUB-A/trA cells were subjected to fluorescence microscopy after incubation with the NbH7-GFP conjugate, or alternatively, as a control with Spytag-GFP or NbH7-Spycatcher (unconjugated) proteins only. From the quantitative analysis we can conclude that a highly significant difference was present in distribution of the data sets from each control compared to the NbH7-GFP labelled conjugate group, which confirms that in addition to translational fusions, the Omp25-specific binders may also be applied as labelled conjugates in order to specifically bind and scavenge Omp25-expressing cells or organisms. Methods

Microscopy

The classically used AB5075-VUB (CP070362), 3 Acinetobacter spp. isolated from environmental sources (A. calcoaceticus, A. junii and A. pittii), Klebsiella pneumoniae, and an E. coli S17 strain were used in the microscopy assays on living bacteria. The bacterial cultures were started from a single clone and were grown for 17 h at 37°C under agitation (165 rpm) in low salt broth (Luria-Bertani formulation, Duchefa Biochemie). The Brucella strains (B. melitensis 16M, B. abortus 544 and B. abortus 2308) were grown overnight and then fixed with 4% paraformaldehyde for l,5h before being used in the microscopy assays.

Cells were collected by centrifugation (8000g) and normalized to ODgoo=3 (10⁹ CFU/ml) in phosphate buffered saline (PBS) for further steps. Labelling of the nanobody was done by incubation with molar 3-fold of DyLight 650 NHS ester (ex:652/em:672, Thermo Scientific) for 1 h, quenching of the reaction with 50 mM Tris buffer, followed by overnight dialysis in PBS. To allow binding, 100 pl of the labelled Nb (10 pM) was incubated with 100 pl of fixed or living bacterial cells (approximately 10⁵ CFU) for 30 min at 37°C, under agitation (165 rpm). To remove unbound Nb, the cells were centrifugated (8000g). Finally, the bacteria were spotted on an 1.5% agarose pad (Thermo Scientific Gene Frame). Microscopy images were acquired using a Leica DMi8 fluorescence microscope with a DFC7000 GT camera (Leica Microsystems CMS GmbH). The fluorescent images were acquired with a Leica FRAP450 and Y5 filter set. The raw data was processed by using ImageJ software where brightness was adjusted equally for all fluorescence micrographs within one experiment.

Biolayer interferometry assays

To determine the affinity of NbH7 and its target Omp25, biolayer interferometry (BLI) was done on an Octet® R8 instrument (Sartorius). All measurements were done at 30°C in 50 mM Tris (pH 8), 150 mM NaCI, 1% n-octylpolyoxyethylene (nOPOE) and 0.1% BSA. The nanobodies were loaded on Ni-NTA sensors at a concentration of 10 pg/ml for 120 s to obtain approximately 1 nm signal increase. After a baseline of 480 s, the association was measured for 100 s by dipping the sensors in a two-fold dilution series starting at 80 pM Omp25. The dissociation was measured for 600 s. To monitor aspecific binding, a control Nb (CtrINb) was tested in parallel in each measurement against the highest concentration of Omp25 (80 pM). This aspecific binding signal was subtracted from each binding signal before calculation of the dissociation constant (K_D). The parameters (k_a, kdis and K_D) were calculated using the Octet® Analysis Studio software (version 13.0.2.46). The graphs were plotted using Graphpad Prism (version 9.0.1). Recombinant expression ofAB5075-VUB Omp25 in the outer membrane of E. coli

For recombinant expression of AB5075-VUB Omp25 in the outer membrane of f. coli, the signal peptide (SP) coding sequence of E. coli OmpA was used. By means of PCR, the open reading frame of AB5075- VUB Omp25, excluding its endogenous SP coding sequence, was cloned into a pASK vector downstream from a N-terminal streptavidin tag, a short glycine-serine linker, and a TEV cleavage site to construct pASK-Omp25 (wherein the Omp25 protein is encoded as SEQ ID NO:42). The pASK-Omp25 plasmid was transformed into E. coli BL21 (DEB) pLysS AtonA cells.

For fluorescence microscopy, an overnight culture started from a transformant was used to inoculate LB supplemented with ampicillin and magnesium chloride. The culture was grown to an ODgoo of 0.5- 0.7, after which Omp25 expression was induced with 0.2 pg/ml anhydrotetracycline hydrochloride. After 3h, the cells were tested for binding of NbH7 with fluorescence microscopy.

Omp25 recombinant protein production and purification for BLI and crystallization experiments was performed as follows. The E. coli BL21 (DE3) pLysS AtonA cells expressing the Acinetobacter baumannii Omp25 protein (SEQ ID NO:42) were harvested by centrifugation (10 min, 6000 g) and resuspended in lysis buffer containing 50 mM Tris-HCI pH 8.0, 300 mM NaCI, 10% glycerol, 1 mM p-mercaptoethanol, DNase and an antiprotease cocktail. The cell suspension was run through a LM10 Microfluidizer at 15000 psi to lyse the cells. The cell lysate was centrifuged (45 min, 48000 g) to obtain the membrane fraction which was resuspended in buffer containing 50 mM Tris-HCI pH 8.0, 300 mM NaCI, 3% OPOE and an antiprotease cocktail. After lh incubation at 4°C the membrane extract was centrifuged (45 min, 48000 g) to obtain the solubilized membrane fraction containing the strep-tagged protein. This membrane fraction was loaded onto a Strep-Tactin®XT 4Flow® cartridge (IBA) and washed with 100 mM Tris-HCI pH 8.0, 150 mM NaCI, 1% OPOE and 1 mM EDTA.

Protein used for BLI analysis was eluted with 100 mM Tris-HCI pH 8.0, 150 mM NaCI, 1% OPOE, 1 mM EDTA and 50 mM biotin. The eluted protein was analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to assess the purity, then concentrated by using an AMICON 3 kDa MWCO and flash frozen in liquid nitrogen to be stored at -80°C.

Protein used for crystallization was first detergent-exchanged on column by washing with 100 mM Tris- HCI pH 8.0, 150 mM NaCI, 5 mM LDAO, 1 mM EDTA and then eluted by 100 mM Tris-HCI pH 8.0, 150 mM NaCI, 5 mM LDAO, 1 mM EDTA, 50 mM biotin. Eluted protein was loaded onto a Superdex 100 16/60 size-exclusion column (GE life Sciences) that was equilibrated with SEC buffer containing 50 mM Tris-HCI pH 8.0, 150 mM NaCI and 2 mM LDAO. The fractions corresponding to Omp25 were pooled and consequently used for crystallization. Crystallization, structure determination and analysis

The crystallization screens were setup with a mix of freshly purified Omp25 with 1.2-fold molar excess of NbH7. The mixture was concentrated to 35 mg/ml by using an AMICON 3 kDa MWCO. Crystals were obtained by using the sitting drop vapor diffusion method and a Mosquito nanoliter-dispensing robot at room temperature (TTP Labtech, Melbourn, UK). After 3 months, hexagonal-shaped crystals appeared in a crystallization buffer containing 0.2 M calcium chloride dihydrate, 0.1 M Tris-HCI pH 8.0 and 44 % v/v PEG 400. After supplementing the buffer with 10% glycerol, the crystals were mounted in nylon loops and flash-cooled in liquid nitrogen. The X-ray diffraction data was collected at 100 K using the Beamline Proxima 2A (wavelength = 0.9801 A) at the Soleil Synchrotron (Gif-sur-Yvette, France). The diffraction data was processed using XDS software¹² at 2.91 A in P2 (1) 3 with unit-cell dimensions of a = 133.7, b = 134.4, c = 133.5, a = 89.2, = 90.4, y = 89.7. To determine the crystal structure, molecular replacement was done using phaser from the phenix suite¹³ with as search models the NbH7 and Omp25 structures predicted by AlphaFold2¹⁴. Refinement of the structure was done by iterative cycles of manual model building with COOT¹⁵ and refinement with phenix.refine¹⁶ and Buster¹⁷ until R values of Rwork/Rfree of 0.24/0.28 were obtained. More crystallographic parameters can be found in table 2. Figures of the structures were generated with ChimeraX¹⁸.

Table 2. Crystallographic parameters of the data collection.

The values given in parenthesis refer to the highest recorded resolution shell. NbH7- Spycatcher- Spytag-GFP labeling

The NbH7_SpyCatcher fusion (SEQ ID NO:40) was expressed in the periplasm of E. coll WK6 by a N terminal pelB signal peptide. The bacteria were grown by inoculating Terrific Broth (TB) medium supplemented with 0.1% (w/v) D-(+)-Glucose, 2 mM MgCL, and 100 pg/ml Ampicillin until an ODgoo of 0.7 was obtained. Protein expression was induced by 1 mM Isopropyl p-D-l-thiogalactopyranoside (IPTG). After induction, the cells were further incubated overnight at 28°C. The following day, the culture was centrifuged (15 minutes, 6227 g) to harvest the cells. The pellet was resuspended in periplasm extraction buffer containing 20 mM Tris pH 8.0, 500 mM NaCI, 10 mM imidazole, 20% (w/v) glucose, 2 mM EDTA, 0.1 mg/ml lysozyme. The suspension was incubated for 1 h at 4°C and centrifuged (40 min, 17664 g) to obtain the periplasm extract. The extract was incubated with Ni-NTA beads (Workbeads 40 IDA, Bio-Works) for 1 h. The beads were washed with buffer containing 20 mM Tris pH 8.0, 500 mM NaCI and 20 mM imidazole. The protein was eluted with buffer containing 20 mM Tris pH 8.0, 500 mM NaCI and 500 mM imidazole.

The GFP_SpyTag fusion (SEQ ID NO: 41) was expressed in the cytoplasm of E. coll BL21 (DE3) pLysS AtonA cells. The bacteria were grown by inoculating Terrific Broth (TB) medium supplemented 0.1% (w/v) D-(+)-Glucose, 2 mM MgCL, and 100 pg/ml Ampicillin until an ODgoo of 0.5-0.7 was obtained. Protein expression was induced by 2 pg/ml anhydrotetracycline hydrochloride. After induction, the cells were further incubated overnight at 23°C and the following day, the culture was centrifuged (15 minutes, 6227 g) to harvest the cells. The pellet was resuspended in 50 mM Tris-HCI pH=8, 300 mM NaCI, 10% glycerol, 1 mM mercaptoethanol, antiprotease cocktail, DNase and MgCL and lysed by a LM10 Microfluidizer at 15000 psi. The cell lysate was loaded onto a Strep-Tactin®XT 4Flow® cartridge (IBA) and washed with 100 mM Tris-HCI pH 8.0, 150 mM NaCI and 1 mM EDTA. The protein was eluted with 100 mM Tris-HCI pH 8.0, 150 mM NaCI, 1 mM EDTA and 50 mM biotin.

The eluted proteins were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) to assess the purity and dialysed to phosphate buffered saline (PBS) for further use.

For the microscopy analysis, equimolar amounts of NbH7_SpyCatcher and GFP_SpyTag, both at a concentration of 100 pM were combined and incubated for 45 minutes at room temperature to allow coupling. The incubation of the construct with the cells and the microscopy analysis was done as described for the other microscopy assays.

Sequence listing

>SEQ ID NO:1: Nb H7 amino acid sequence

>SEQ ID NO:2: Nb A8 amino acid sequence >SEQ ID N0:3: Nb E12 amino acid sequence

>SEQ ID NO:4: Acinetobacter baumannii Omp25 amino acid sequence of AB5075-VUB (mature protein, without signal peptide)

>SEQ ID NO:5: Acinetobacter baumannii Omp25 amino acid sequence of AB5075-VUB and ATCC17978- VUB clinical isolates (signal peptide underlined)

MKKLAIASALLSALAVSGTANAYQAEVGGSYNYLDPDNGSSVSKFGVDGTYYFNPVQTRNAPLAEAAFLNRASNVN

AHVNYGDNSGTKDTQYGVGVEYFVPNSDFYLSGDVGRNEREIDNTNIDSKVTTYAAEVGYLPAPGLLLALGVKGYDE

KDGKDGADPTVRAKYVTQVGQHDVNLEAYGAFGDLDEYKVRGDYYIDKTLSLGVDYYNNDLTDKDEFGINAKKFLN QQVSVEGRVGFGDNDNTYGVRAAYRF

>SEQ ID NO:6: recombinant Omp25 amino acid sequence (including an N terminal E. coli OmpA signal peptide (underlined), a 6xHis tag and TEV cleavage site (bold))

MKKTAIAIAVALAGFATVAQAGGHHHHHHENLYFQGAYQAEVGGSYNYLDPDNGSSVSKFGVDGTYYFNPVQTR

NAPLAEAAFLNRASNVNAHVNYGDNSGTKDTQYGVGVEYFVPNSDFYLSGDVGRNEREIDNTNIDSKVTTYAAEVG

YLPAPGLLLALGVKGYDEKDGKDGADPTVRAKYVTQVGQHDVNLEAYGAFGDLDEYKVRGDYYIDKTLSLGVDYYN

NDLTDKDEFGINAKKFLNQQVSVEGRVGFGDNDNTYGVRAAYRFYPYDV

>SEQ ID NO:7: control Nb E3 amino acid sequence

>SEQ ID NO:8: control Nb B9 amino acid sequence

>SEQ ID NO:9: His6-EPEA tag amino acid sequence

>SEQ ID NO:10: Acinetobacter baumannii Omp25 amino acid sequence of AB3-VUB, AB180-VUB, and AB220-VUB clinical isolates

>SEQ ID NO:11: CDR1 of NbH7 family

>SEQ ID NO:12: CDR2 of NbH7 family

>SEQ ID NO:13: CDR3 of NbH7 family

>SEQ ID NO:14:_humanized Nb H7 variant hl amino acid sequence (H7hl)

>SEQ ID NO:15: humanized Nb H7 variant h2 amino acid sequence (H7h2)

>SEQ ID NO:16: humanized Nb H7 variant h3 amino acid sequence (H7h3)

>SEQ ID NO:17: humanized Nb H7 variant h4 amino acid sequence (H7h4)

>SEQ ID NO:18:humanized Nb H7 variant h5 amino acid sequence (H7h5)

>SEQ ID NO:19-25: OM P25 Surface-exposed region 1-7, respectively

>SEQ ID NO:26: WP_337077925.1 PUTATIVE PORIN [ACINETOBACTER PITTII] >SEQ ID NO:27: RQL72781.1 PUTATIVE PORIN [ACINETOBACTER PITTII]

>SEQ ID NO:28: WP_063097693.1 PUTATIVE PORIN [ACINETOBACTER PITTII]

>SEQ ID NO:29: WP_216946930.1 PUTATIVE PORIN [ACINETOBACTER PITTII]

>SEQ ID NO:30: WP_016139874.1 PUTATIVE PORIN [ACINETOBACTER CALCOACETICUS]

>SEQ ID NO:31: WP_004639960.1 PUTATIVE PORIN [ACINETOBACTER CALCOACETICUS]

>SEQ ID NO:32: WP_199966399.1 PUTATIVE PORIN [ACINETOBACTER CALCOACETICUS]

>SEQ ID NO:33: VAX43036.1 UNCHARACTERISED PROTEIN [ACINETOBACTER CALCOACETICUS]

>SEQ ID NO:34: CAI3136485.1 HYPOTHETICAL PROTEIN MWMV8_IVIWIVIV8_01065 [ACINETOBACTER CALCOACETICUS]

>SEQ ID NO:35: WP_004951331.1 PUTATIVE PORIN [ACINETOBACTER JUNII]

>SEQ ID NO:36: RXS99878.1 PUTATIVE PORIN [ACINETOBACTER JUNII]

>SEQ ID NO:37: WP_262579446.1 PUTATIVE PORIN [ACINETOBACTER JUNII]

>SEQ ID NO:38: Thermothelomyces thermophila ADP/ATP carrier (TtAac) amino acid sequence

>SEQ ID NO:39: NbH7_sfGFP translational fusion protein

>SEQ ID NO:40: NbH7_SpyCatcher fusion protein

>SEQ ID NO:41: SpyTag_GFP fusion protein

>SEQ ID NO:42: recombinant Omp25 amino acid sequence (including an N terminal E. coll OmpA signal peptide, a Strep tag and TEV cleavage site)

>SEQ ID NO:43: Acinetobacter baumannii Omp25 amino acid sequence (mature protein; corresponding to UniProt Q4A208); as shown in Fig25

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Claims

1. An antigen-binding protein specifically binding Acinetobacter Outer membrane protein 25 (Omp25).

2. The antigen-binding protein of claim 1, which binds the Acinetobacter Omp25 protein as present on the surface of Acinetobacter capsulated and non-capsulated cells.

3. The antigen-binding protein of any one of claims 1 or 1, which specifically binds the surface- exposed regions of Acinetobacter Omp25.

4. The antigen-binding protein of any one of claims 1 to 3, wherein said Acinetobacter Omp25 protein comprises Acinetobacter baumannii Omp25 as present in SEQ ID NO:43, or a homologue with at least 90 % identity thereof.

5. The antigen-binding protein of any one of claims 1 to A, which specifically binds the epitope comprising the Omp25 surface-exposed region amino acids at position 60, 93-106, 131, 165, 166, 167, and 190-193 as present in SEQ ID NO:43, or alternatively the epitope comprising the Omp25 surface-exposed regions 2 and 3, and optionally regions 4, 5, and 6 as present in SEQ ID NO:43, or the Omp25 surface-exposed regions or amino acids corresponding to said regions or positions in an Acinetobacter Omp25 homologue.

6. The antigen-binding protein of any one of claims 1 to 5, wherein said antigen-binding protein comprises an antibody, an antibody mimetic, a single chain variable fragment (ScFv), an immunoglobulin single variable domain (ISVD), a VHH, or a nanobody, or an active antibody fragment.

7. The antigen-binding protein of any one of claims 1 to 6, comprising an ISVD comprising the complementarity determining regions (CDRs) as present in any of SEQ ID NOs: 1 to 3, wherein the CDRs are annotated according to Kabat, MacCallum, IMGT, AbM, or Chothia, preferably comprising the CDR1 of SEQ ID NO:11, CDR2 of SEQ ID NO:12, and CDR3 of SEQ ID NO:13.

8. The antigen-binding protein of claim 7, wherein the ISVD comprises a sequence selected from the group of sequences of SEQ ID NOs: 1- 3, or a functional variant of any one thereof with at least 90 % amino acid identity over the full length of the ISVD sequence wherein the non-identical amino acids are located in one or more Framework residues, or a humanized variant of any one thereof, preferably a humanized variant as present in any one of SEQ ID NOs: 14-18.

9. The antigen-binding protein of any one of claims 1 to 8, which is a multivalent or multispecific antigen-binding agent, preferably a bivalent or bispecific antigen-binding agent, which may be in an Fc fusion or an antibody format.

10. The antigen-binding protein of any one of claims 1 to 9, wherein said antigen-binding protein is linked to a further moiety, wherein said moiety is linked via conjugation or via genetic fusion.

11. The antigen-binding protein of claim 10, wherein said further moiety comprises a detectable label, such as a dye or fluorophore, an effector molecule, such as a toxin, or a functional moiety, such as a therapeutic moiety or a half-life extension.

12. A nucleic acid molecule encoding the antigen-binding protein of any one of claims 1 to 11.

13. A pharmaceutical composition comprising an antigen-binding protein of any one of claims 1 to 11, or the nucleic acid molecule of claim 12, and optionally a further therapeutically active agent, a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

14. The antigen-binding protein of any one of claims 1 to 11, the nucleic acid molecule of claim 12, or the pharmaceutical composition of claim 13, for use as a medicament.

15. The antigen-binding protein of any one of claims 1 to 11, the nucleic acid molecule of claim 12, or the pharmaceutical composition of claim 13, for use in treatment of Acinetobacter infection, preferably Acinetobacter baumannii infection.

16. The antigen-binding protein of any one of claims 1 to 11, the nucleic acid molecule of claim 12, or the pharmaceutical composition of claim 13, for use in in vivo imaging.

17. The antigen-binding protein of any one of claims 1 to 11, or the pharmaceutical composition of claim 13, for use as a diagnostic.

18. Use of the antigen-binding protein of any one of claims 1 to 11, or the pharmaceutical composition of claim 13, for depletion of Acinetobacter cells from a sample in vitro.

19. Use of the antigen-binding protein of any one of claims 1 to 11, or the pharmaceutical composition of claim 13, for scavenging Acinetobacter cells.