WO2017070567A1 - Klebsiella pneumoniae antibodies and methods to treat klebsiella pneumoniae infections - Google Patents

Abstract

The instant invention relates to antibodies (which includes whole antibodies and functional parts thereof) as therapeutic and diagnostics tools to combat K. pneumoniae (Kp) infections, and diseases and disorders which are caused by or associated with Kp infections.

Description

KLEBSIELLA PNEUMONIAE ANTIBODIES AND

METHODS TO TREAT KLEBSIELLA PNEUMONIAE INFECTIONS BACKGROUND OF THE INVENTION

Klebsiella pneumoniae is a gram-negative pathogen of the Enterobacteriaceae family that can cause pulmonary, urinary tract, wound and soft tissue infections in hospitalized patients. Additionally, Klebsiella pneumoniae can be carried

asymptomatically in healthy people, mainly in the intestinal tract but also in the nose, throat and skin [1]. The mechanisms as to how Klebsiella pneumoniae can cause invasive disease are not completely understood [2]. Due to acquisition of multidrug resistance genes, in the recent years, Klebsiella pneumoniae infections have become a worldwide threat [3]. For example, carbapenem-resistant K. pneumoniae has been spreading worldwide; the most successful clonal type is ST258.

A variant strain of K. pneumoniae has been emerging, the hypervirulent K. pneumoniae

This variant strain can cause invasive infections including pyogenic liver abscesses, pneumonia, ophthalmitis and meningitis [2]. It has been reported worldwide [4-6] but the majority of this strain comes from Asia, where it is the leading cause of liver abscesses in Taiwan [7], Singapore [8], Hong Kong [9] and South Korea [10]. In fact in Taiwan, the annual incidence of pyogenic liver abscess increased between 1996 to 2004 from 11 to 17 cases per 100,000 persons [11PMCPMC2609891] As high as 13% of

infections can have a metastatic spread to the eye or meninges [11]. A significant percentage of

infections disseminate to the eye or meninges [12, Fang CT, PMCPMC4076766] commonly resulting in irreversible damage like eye vision loss, neurologic deficits or loss of a limb. Mortality rates range from 3% to 42% [2]. Recent epidemiological studies demonstrated that healthy adults carry virulent strains in their gastrointestinal tracts and based on MLST typing it was concluded that colonizing strains cause invasive infections in patients [13].

Fortunately, and in contrast to ST258 strains [12],

strains exhibit less diversity of their capsular polysaccharide (CPS). The Kl serotype is the prevailing CPS and reported to be present on up to 81% of

strains, followed by K2 serotype and non-Kl/K2 serotypes, respectively [10, 11, 13, 14]. Historically hvKp were observed to be pan-sensitive to standard antibiotics so if recognized early these infections could be commonly successfully treated despite their invasive nature. With the emergence of resistant

strains one of the main concerns is that these variants could acquire multidrug resistance (MDR) conferring plasmids like the ones earring bla ^" and bla ^" . Accordingly, on a recent report from China, strains were found to be still sensitive to carbapenems and amikacin but resistant to 14 of 19 tested antimicrobials [15]. Furthermore the report indicated that antibiotic resistance was consistently rising from December 2010 to June 2012 [15]. In vitro the acquisition by a hipervirulent strain of a KPC-bearing plasmid has been demonstrated [16]. Moreover most recently

strains that exhibit carbapenem resistance have been isolated in China [17].

Accordingly, given the looming threat of emerging multi-drug resistant

strains, clearly there is a critical need to find diagnostic and therapeutic tools to combat K. pneumoniae infections, in particular, to combat

serotype Kl infections.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1: Chromatogram of the S200HR gel filtration chromatography recorded at A280nm. Left panel shows the chromatogram of the Kl-CPS after conjugation with PA protein; right panel shows the chromatogram of before the conjugation. Lower line point out the fractions that contained Kl-CPS detected by the phenol-sulfuric acid method [26].

Figure 2: In vitro characterization of mAbs effects. A, B) Viability of Kl

in the presence of 4C5 or 19A10, respectively. Growth was recorded by CFU/ml at different time points. Three independent were monitored. C) Agglutination assays of Kl

in the presence or absence of mAbs. 10 μg of mAb were added to cells and incubated for lh. The lower panel shows the agglutination of the transformed with the GFP- expressing plasmid pPROBEKT-Km strain. D) Human serum resistance assays.

Growth of CR-Kp strains in the presence of normal human serum. Levels of viable CFU of the Kl

after incubation for 2 hours with NHS were assessed. Levels of viable cells relative to the number of CFU at time 0' are shown. E) Complement deposition assay. Deposition was measured by fluorescence signal of anti -human C3c FITC. Three independent experiments were obtained and the % increment of the signal of mAb treatment with respect to PBS incubation is shown. F) Shows preincubation of Kl-Kp with 25 μg of 4C5 or 19A10 enhances survival of infected G. mellonella.

Significance was determined by Log-rank (Mantel-Cox) test (n= 20 worms per group) corrected with Bonferroni's multiple comparisons test. G) MAC deposition was enhanced by 19A10Ab treatment. In 3 independent experiments signal of anti-human C5b-9 was measured by ELISA in Kl-Kp bacteria incubated with NHS or HI- NHS and mABs, aggregated human IgG or PBS. Mean percent increase of AP signal relative to each PBS control is shown with standard deviation, p-values are calculated by ANOVA with post hoc Tukey's multiple comparisons test. H) Growth of Kl-Kp in the presence of IgG Control, 4C5 or 19A10 in LB media. Experiments were done in quadruplicates. Bars represent the calculated means + standard deviation, p-values were determined by 2- way ANOVA with post hoc Tukey's multiple comparisons test.

Figure 3: Phagocytosis assays. A) Murine macrophages J744 phagocytosis of Kl hv- Kp in the presence of PBS (blue), an IgG control (red), 4C5 (green) or 19A10 (purple). B) THP-1 -differentiated human macrophages phagocytosis of Kl hv-Kp in the presence of PBS (blue), an IgG control (red), 4C5 (green) or 19A10 (purple). C) Bone marrow- derived macrophages of wild-type (solid) or FcR-KO (squared) phagocytosis of Kl hv- Kp in the presence of PBS (blue), an IgG control (red), 4C5 (green) or 19A10 (purple). In all experiments the % of macrophages with pH-rodo labeled bacteria inside it is shown 67 min and 150 min after starting the coincubation. Three independent experiments were conducted. Figure 3 A) Number of bacteria passing a fixed point in the sinusoid blood vessel (diameter between 5μιη and 8μιη) at various times points after injection. B) The number of stationary bacteria captured in a field of view at various time points after injection. For each group, mouse with/without treatment of mAb received i.v. injection of lxlO⁸ GFP-expressing Kp, an one-hour IVM video was taken based on the protocol described above. * denotes p-value<0.05, ** p-value<0.01 and *** p-value<0.001. (D) Shows murine macrophage J744.16 killing of Kl-Kp. p-values were determined by one-way ANOVA with Sidak's multiple comparisons test. (E)

Quantitation of NET release (shown in F) by human neutrophils after stimulation with C. albicans, Kl-Kp or Kl-Kp incubated with mAbs 4C5, 19A10 and IgG control. Bars reflect mean percentage of neutrophils' nuclei with an area exceeding 1,000 μηι2 over the total neutrophils in field with standard deviation (3 independent replicates). The p- value was determined by one-way ANOVA with Sidak's multiple comparisons test. (E) Enhanced NET release by human neutrophils is induced by 4C5 and 19A10 coincubated Kl-Kp bacteria but not by isotype matched control Ab.

Figure 4. A) and B) mice survival and lung, liver and spleen bacterial counts after i.p. injection of 5xl0⁴ Kl-Kp CFUs, respectively. C) and D) survival and lung, liver and spleen bacterial counts after i.t. injection of lxlO⁴ Kl-Kp CFUs, respectively. Mice were treated with PBS, IgG control, 4C5, 19A10 or 4C5 and 19A10. Oran bacterial loads are expresses by CFU/ml. * denotes /?-value<0.05, ** /?-value<0.01 and *** p- value<0.001 and *** / value<0.0001. Comparison of mean cytokine levels (24h post i.p. infection with 5x104 Kl-Kp) in liver (E) and spleen (F) are shown with standard deviation (n=4 mice per group), black * denotes comparison of PBS with 4C5 and blue * with 19A10. 2-way ANOVA with Dunnett's multiple comparisons test was performed for p- value determination.

Figure 5. Intravital microscopy. A) Number of bacteria passing a fixed point in the sinusoid blood vessel (diameter between 5μηι and 8μιη) at various times points after injection. B) The number of stationary bacteria captured in a field of view at various time points after injection. For each group in A) and B), mouse with/without treatment with mAb received i.v. injection of lxlO⁸ GFP-expressing Kl hv-Kp, an one-hour IVM video was taken based on the protocol described above. C) Number of bacteria passing a fixed point in the sinusoid blood vessel (diameter between 5μιη and 8μιη) 24 hours post injection. D) The number of stationary bacteria captured in a field of view 24 hours after injection. For each group in C) and D), mouse with/without treatment with mAb received i.p. injection of 5xl0⁴ GFP-expressing hv-Kp, an one -hour IVM video was taken based on the protocol described above. E) Microscopy image 200x of the intra-abdominal cavity of untreated muse after i.p. injection 5xl0⁴ GFP-expressing hv- Kp. F) Number of bacteria passing a fixed point in the sinusoid blood vessel (diameter between 5μιη and 8μιη) 24 hours post i.v. injection lxlO⁴ GFP-expressing hv-Kp. Mice with or without treatment with mAbs received i.p lxlO⁴ CFUs GFP-expressing hv-Kp. ** denotes / value<0.01.

Figure 6. Kl-CPS detection method in infected mice. A) Schema of the designed sandwich ELISA. ELISA plates are coated with 4C5 mAb and then incubated with the sample that contains or not Kl-CPS. Then 19A10 mAb is bound and detected via anti- mouse IgG3 conjugated mAb. B) Kl-CPS detection in a mouse i.v. injected with lxlO⁴ CFUs Kl hv-Kp in serum obtained at different time points. C) Kl-CPS detection in mice i.p. injected with 5xl0⁴ CFUs Kl hv-Kp in urine obtained at different time points. D) Kl-CPS detection in mice i.t. injected with 1x104 CFUs Kl-Kp in urine obtained at different time points.

Figure 7. In vivo protection studies. (A) Survival analysis of mice (n=6, per group) infected i.p. with 5x104 Kl-Kp and treated with PBS, 250 μg 4C5, 19A10 or 125 μg 4C5 and 19A10, respectively, p- value was determined with log-rank (Mantel-Cox) corrected with Bonferroni's multiple comparisons test. (B) 24h post infection mean bacterial organ loads. Mean log CFU/ml + standard deviation (n=4 mice per group) are shown. Black * denotes comparison of PBS and both mAbs. 2-way ANOVA with Tukey's multiple comparisons test was performed in (B) for p- value determination. (C) Comparison of mean cytokine levels (24h post i.p. infection with 5x104 Kl-Kp in lung is shown with standard deviation (n=4 mice per group), black * denotes comparison of PBS with 4C5 and blue * with 19A10. 2-way ANOVA with Dunnett's multiple comparisons test was performed for p- value determination.

Figure 8. Bacterial colonization and dissemination protection studies. Mean bacterial loads in organs post-dissemination and in feces on day 8 post-colonization with 1x108 Kl-Kp. Mean log CFU/ml + standard deviation (n=3 mice per group) are shown. 2-way ANOVA with Dunnet's multiple comparisons test was performed for p-value determination. Black * denotes comparison of PBS.

Figure 9. (A) Bacterial CFU counts feces of colonized mice increase more than 5 logs after ampicillin treatment. (B) Time of ampicillin and mAb treatment in colonized mice. Supplementary videos 1-6. Three video clips for i.v injection are all 30 min post infection played at 5 fps, showing treatment with PBS, 4C5 or 19A10. Video clips for i.p infection are 24 h post infection played at 5 fps showing treatment with PBS, 4C5 or 19A10.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention relates to antibodies (which includes whole antibodies and functional parts thereof) as therapeutic and diagnostic tools to combat K. pneumoniae (Kp) infections, and diseases and disorders which are caused by or associated with Kp infections.

The terms "antibody" or "antibodies" as used herein are art recognized and are understood to refer to polypeptide molecule(s) or active fragments of polypeptide molecule(s) that bind to known antigens.

In one embodiment of the invention, monoclonal antibodies (mAbs) produced by hybridoma 4C5 (IgGl isotype) are provided. In another embodiment, monoclonal antibodies produced by hybridoma 19A10 (IgG3 isotype) are provided. These IgG mAbs have high affinity against the capsular polysaccharide (CPS) of the hypervirulent Kp Kl serotype. The isolated nucleotide and amino acid sequences of the heavy chain of mAb 4C5 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The amino acid sequences of CDRs 1-3 of the heavy chain are shown in SEQ ID NOs: 12-14, respectively. The nucleotide and amino acid sequences of the light chain of mAb 4C5 are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The amino acid sequences of CDRs 1-3 of the light chain are shown in SEQ ID NOs: 18-20, respectively.

The isolated nucleotide and amino acid sequences of the heavy chain of mAb 19A10 are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The amino acid sequences of CDRs 1-3 of the heavy chain are shown in SEQ ID NOs: 24-26, respectively. The nucleotide and amino acid sequences of the light chain of mAb 19A10 are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The amino acid sequences of CDRs 1-3 of the light chain are shown in SEQ ID NOs: 30-32, respectively.

By "isolated" is meant a biological molecule free from at least some of the components with which it naturally occurs. The term "CDR" refers to the hypervariable region of an antibody. The mAbs comprise six hypervariable regions; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3).

In another embodiment, any functionally equivalent antibodies, or functional parts of, mAb 4C5 and mAb 19A10 are provided. "Functionally equivalent" antibodies/parts substantially share at least one major functional property with the mAbs, as described herein, including, passively immunizing against Kp infections.

For example, mAb 4C5 and mAb 19A10 can have amino acid sequences which are altered by at least one, particularly at least two, more particularly at least 3 or more conservative substitutions in their sequences, such that the mAbs essentially maintain their functionalities. A conservative amino acid substitution does not render the antibody incapable of binding to the subject receptor. A skilled artisan would be able to predict which amino acid substitutions can be made while maintaining a high probability of being conformationally and antigenically neutral. Factors to be considered that affect the probability of maintaining conformational and antigenic neutrality include, but are not limited to: (a) substitution of hydrophobic amino acids is less likely to affect antigenicity because hydrophobic residues are more likely to be located in a protein's interior; (b) substitution of physiochemically similar, amino acids is less likely to affect conformation because the substituted amino acid structurally mimics the native amino acid; and (c) alteration of evolutionarily conserved sequences is likely to adversely affect conformation as such conservation suggests that the amino acid sequences may have functional importance.

Also, the invention provides functional peptide fragments which comprise at least one of the CDRs described herein. Examples of functional fragments include Fab, F(ab')2, scFv and Fv fragments.

In one embodiment, the invention relates to a heavy chain variable region exhibiting an isolated amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences given in SEQ ID NO: 2 or 6, or functional parts thereof, comprising at least one, typically at least two, more typically at least 3, of the heavy chain CDRs having the polypeptide sequences SEQ ID NOs: 12-14 or 24-26, respectively, but especially all CDRs embedded in their natural framework regions.

In one embodiment, the invention relates to a light chain variable region exhibiting an isolated amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%,

91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences given in SEQ ID NO: 4 or 8, or a functional part thereof, comprising at least one, typically at least two, more typically at least 3 of the light chain CDRs, having the polypeptide sequences SEQ ID NOs: 18-20 or 30-32, respectively, but especially all CDRs embedded in their natural framework regions.

Sequence identity can be determined conventionally with the use of computer programs such as, for example, the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, Wis. 53711).

In one embodiment, the invention provides polynucleotides comprising isolated nucleotide sequences which encode the amino acid sequences described herein.

Therapeutic Methods

In one embodiment of the instant invention, passive immunotherapy is provided for K. pneumoniae infections, particularly infections by hypervirulent strains of the Kl serotype, by administering therapeutic compositions to subjects in need thereof. The therapeutic compositions can prevent, inhibit, treat and/or alleviate the effects of diseases and disorders in a subject, which are caused by or associated with Kp infections including, but not limited to, for example, pyogenic liver abscesses, pneumonia, ophthalmitis, meningitis and neurologic disorders. Subjects include mammals, in particular humans, who are at risk of acquiring a Kp infection, or have a Kp infection and/or a disorder/disease associated with a Kp infection.

Therapeutic compositions comprise at least one of the mAbs as described herein, including any functionally equivalent antibody or functional parts thereof, in a therapeutically effective amount. In some embodiments, the compositions comprise only one of mAb 4C5 or mAb 19A10, and/or functional equivalents/fragments thereof. In other embodiments, compositions comprise both mAb 4C5 and mAb 19A10, and/or functional equivalents/fragments thereof.

The antibodies can be prepared in a physiologically acceptable formulation and can comprise a pharmaceutically acceptable carrier, diluent and/or excipient using known techniques. For example, the antibodies can be combined with a

pharmaceutically acceptable carrier, diluent and/or excipient to form a therapeutic composition. Suitable pharmaceutical carriers, diluents and/or excipients are well known in the art and include, for example, phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, sterile solutions, etc. The pharmaceutical composition can further comprise proteinaceous carriers such as, for example, serum albumin or immunoglobulin, particularly of human origin.

Formulation of the pharmaceutical composition can be accomplished according to standard methodology know to those of ordinary skill in the art.

The compositions can be administered to a subject in the form of a solid, liquid or aerosol at a suitable, pharmaceutically effective dose. Examples of solid

compositions include pills, creams, and implantable dosage units. Pills may be administered orally. Therapeutic creams may be administered topically. Implantable dosage units may be administered locally, for example to an infected site (e.g., the liver) or may be implanted for systematic release of the therapeutic composition, for example, subcutaneously. Examples of liquid compositions include formulations adapted for injection intramuscularly, subcutaneously, intravenously, intra-arterially, and formulations for topical and intraocular administration. Examples of aerosol formulations include inhaler formulations for administration to the lungs.

The compositions can be administered by standard routes of administration. In general, the composition may be administered by topical, oral, rectal, nasal, interdermal, intraperitoneal, or parenteral (for example, intravenous, subcutaneous, or intramuscular) routes. In addition, the composition may be incorporated into sustained release matrices such as biodegradable polymers, the polymers being implanted in the vicinity of where delivery is desired. The method includes administration of a single dose, administration of repeated doses at predetermined time intervals, and sustained administration for a predetermined period of time.

It is well known to those of ordinary skill in the pertinent art that the dosage of the composition will depend on various factors such as, for example, the condition of being treated, the particular composition used, and other clinical factors such as weight, size, sex and general health condition of the patient, body surface area, the particular compound or composition to be administered, other drugs being administered concurrently, and the route of administration.

The composition can be administered in combination with other compositions comprising a biologically active substance or compound, for example, with antibiotics (e.g., carbapenems and amikacin) and/or analgesics and/or anti-inflammatory compounds. The other biologically active substances can be part of the same composition already comprising an antibody, in the form of a mixture, wherein the antibody and the other biologically active substance are intermixed in or with the same pharmaceutically acceptable solvent and/or carrier or the antibody; or the other biologically active substance can be provided separately as part of a separate composition. The antibodies can be administered to the subject at the same time with the other biologically active substance or substances, intermittently or sequentially.

Proteinaceous pharmaceutically active matter can be present in amounts between

1 ng and 10 mg per dose. Generally, the regime of administration should be in the range of between 0.1 μg and 10 mg of the antibody according to the invention, particularly in a range 1.0 μg to 1.0 mg, and more particularly in a range of between 1.0 μ-g and 100 μ-g. If the administration occurs through continuous infusion a more proper dosage may be in the range of between 0.01 μg and 10 mg units per kilogram of body weight per hour.

Administration will generally be parenterally, e.g. intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Non-aqueous solvents include without being limited to it, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous solvents may be chosen from the group consisting of water, alcohol/aqueous solutions, emulsions or suspensions including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose) and others. Preservatives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases, etc.

Diagnostic Methods

In one embodiment, the invention relates to a method of diagnosis of a Kp infection. Diagnosis of Kp infection in a subject can be achieved by detecting the immunospecific binding of an antibody of the invention, particularly a monoclonal antibody or an active fragment thereof to an epitope of a Kp polysaccharide in a sample or in situ, which includes bringing the sample or a specific body part or body area suspected to contain the Kp polysaccharide into contact with an antibody which binds an epitope of the Kp polysaccharide, allowing the antibody to bind to the polysaccharide to form an immunological complex, detecting the formation of the immunological complex and correlating the presence or absence of the immunological complex with the presence or absence of Kp polysaccharide in the sample or specific body part or area, optionally comparing the amount of said immunological complex to a normal control value, wherein an increase in the amount of said immunologic complex compared to a normal control value indicates that said subject is suffering from or is at risk of developing a Kp infection or associated disease or condition.

Biological samples that can be used in the diagnosis of a Kp infection or associated disease or condition, or for monitoring minimal residual disease in a subject are, for example, fluids such as urine, serum, plasma, saliva, gastric secretions, mucus, cerebrospinal fluid, lymphatic fluid and the like or tissue or cell samples obtained from the subject. For determining the presence or absence of the Kp polysaccharide in a sample, any immunoassay known to those of ordinary skill in the art can be used such as, for example, assays which utilize indirect detection methods using secondary reagents for detection, ELISA's and immunoprecipitation and agglutination assays. A detailed description of these assays is, for example, given in Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New York 1988 555-612, WO96/13590 to Maertens and Stuyver, Zrein et al. (1998) and WO96/29605. Method of Obtaining mAb 4C5 and mAb 19A10, and Characteristic Thereof

The highly affine IgG mAbs against the capsular polysaccharide (CPS) of the hypervirulent Kl serotype of the instant invention do not only target the strain used in the immunization but also other unrelated Kl-Kp strains. The CPS covalent conjugation to an immunogenic protein, Bacillus anthracis Protective Antigen (PA), was employed to enhance the isolation of IgG mAbs, as has been previously described for other polysaccharides [33-36]. This conjugation drives the immunogenic response towards a Thymus-Dependent pathway that allows the isolation of memory IgGs mAbs [37], as polysaccharides antigens cannot be processed and presented via major histocompatibility complex.

Mice were immunized with capsular polysaccharide (CPS) obtained from the capsule of K. pneumoniae Kl serotype conjugated to the PA protein to enhance the immunological response. Every two weeks, antibody titers were measured specific to the Kl polysaccharide. When antibody titers were adequately high, fusion with myeloma cells was performed to obtain specific hybridomas. Hybridomas with high specific signal against Kl serotype were selected and monoclonal hybridomas were purified. Two highly specific hybridomas producing mAbs 4C5 and 19A10, IgGl and IgG3 isotypes, respectively, were obtained.

Both mAbs recognized Kl serotypes specifically. Both mAbs have high binding affinity to the Kl polysaccharide in the subnanomolar range. Both mAbs can bind simultaneously to the Kl capsular polysaccharide and a sandwich ELISA was developed to detect Kl CPS in urine and serum of mice infected with Kl serotype intra- peritoneally, intra- venously and intra-tracheally. Both mAbs decrease the human serum resistance of these bacteria. Both mAbs promote complement deposition and promote phagocytosis of the bacteria by murine and human macrophages. In animal infection models, both mAbs decrease the number of bacteria found in spleen, liver and lungs after intra-tracheal and intra-peritoneal infections. In intra-tracheal infections, the survival of the mice was improved when both mAbs were used simultaneously. In intra- peritoneal infections, the survival of mice was improved with both mAbs independently and is further improved when both are used simultaneously.

Common protective characteristics were observed with both mAbs, although they differed in their isotype and also bind different non-overlapping epitopes. Both of them were able to agglutinate and promote capsule swelling as described with other mAbs targeting Kp CPS [12, 25]. Furthermore, both mAbs were able to prevent equally the survival of Kl-Kp in the presence human serum. [26, 27] Serum resistance is an important virulence trait [26, 27]. The capsule evades the human immune response by inhibiting binding of complement which can kill on its own or promote phagocytosis [28]. Consistent with their protective efficacy, the instant data demonstrates that both mAbs promote complement deposition. The IgGl 4C5 mAb exhibited both enhanced complement deposition as well as more effective opsono-phagocytosis than the mAb 19A10. Consistent with this finding, enhanced protection of mAb 4C5 compared to 19A10 in two murine infection models was also demonstrated herein.

Multiphoton microscopy is a powerful technique to study in real-time pathogen host interactions, especially in cases where the hepatic intravascular immune response is deemed relevant for the pathogen's ability to disseminate [29-31]. Old studies with labeled Escherichia coli bacteria and more recent studies with Mycobacterium bovis and Borrelia burghdorferi document rapid removal from circulation of bacteria by the liver and Kupfer cells, respectively (KC) [32, 33]. Since Kl-Kp causes predominantly invasive liver abscesses, intravital microscopy was employed herein to investigate trafficking of

through the liver in the presence and absence of Kl-specific mAbs [2]. Liver sinusoids form an extensive network of narrow capillaries lined by resident macrophages called Kupffer cells. They capture and ingest pathogens present antigen via CD Id, creating a highly efficient surveillance and filtering system. Intravenous high doses injection of

bacteria demonstrated that co-treatment with mAbs captured bacteria in the liver and progressively enhanced removal of bacteria from the circulation over time. In contrast, sham infected mice could not clear

from blood and CFU counts remained high in blood. When fewer bacteria were injected, bacteria were rapidly cleared from liver and blood in mAb treated mice. Again, and consistent with enhanced protection in murine models, more rapid clearance was observed with mAb4C5 compared to mAb 19A10. For pathogens, phagocytosis by Kupfer cells may also alter host response by inducing chemokine and cytokine production and enhancing recruitment of neutrophils and natural killer cells [34-36]. Noteworthy, intravital microscopy also revealed the role of peritoneal macrophages on initial clearance of Kl- bacteria similar to the initial containment of intra-peritoneal infections with Enterococcus faecium or E. coli [37, 38].

Bone marrow-derived macrophages from FcyR deficient mice showed impaired Ab mediated phagocytosis of opsonized Kl-hvKp. Interestingly, Kupffer cells have also been shown to eliminate circulating tumor cells through rapid phagocytosis, which was dependent on binding to high (FcyRI) as well as low- affinity Fc receptors (FcyRIV) [39]. Some residual phagocytosis in macrophages deficient of FcyR was documented herein, which is probably complement mediated and also enhanced by Ab treatment. Both, murine IgG3 and IgGl bind to low affinity receptors FcyRIII and FcyR I [40] as well as to the high affinity neonatal FcyR (FcRn) [41, 42]. The latter receptor binds to opsonized bacteria in the acidic environment of endocytic vacuoles [42]. The instant microscopic data demonstrated that

do not escape the phagolysosome compartment opposed to other gram-negative bacteria. It was concluded therefore that mAbs promotes uptake into the phagolysosome and killing of bacteria occurs there. Intracellular TRIM21 signaling as a result of escaped Ab-pathogen complexes as described in Samonella is therefore not expected [43].

The instant work does not clarify which isotype would constitute the most favorable but does indicate that genetically engineered mAbs where FcyR receptor engagement is blocked would not be effective. Bacterial CPS induce predominantly IgM and IgG3 antibodies in mouse [23] and IgG2 in humans [44]. Accordingly, experimental data suggests that IgG3 antibodies are important for bacterial immunity [45-47] and anti-CPS IgG3 exhibits increased affinity for binding multivalent CPS epitopes via an Fc-mediated cooperative binding [48]. The conjugation of the PS to a protein shifts the response towards generation of IgGl antibodies that could be equally protective [49]. In the instant case, it was found that both, IgGl and IgG3 exert protection but 4C5, an IgGl higher protective efficacy.

Different classes of IgG antibodies differ in their effector responses. Classically IgG2a and 2b play a key role for antiviral immunity and they are also potent Abs mediating autoimmune diseases [50]. Mouse IgGl and IgG3 exhibit FcyR activation. Binding affinities to activating receptor inhibitory FcyR receptors, measured as activation-to-inhibition ratio, influences their overall activities [50]. IgGl Abs have a superior activation signaling compared to IgG3 Abs [50]. This could explain the observed lower effectors processes exerted by IgG3 antibodies in the context of comparable binding affinities. Past studies have shown antibody-mediated protection can depend on the IgG subclass but the results also depends on the specific Ab. For example, two IgG3 mAbs against meningococcal PorA protein and the Bacillus anthracis capsule were found to be more protective than other subclasses [47, 51]. Those differences were explained by differential interactions of CH2-CH3 domains in each of the subclasses that drive their binding affinity. On the other hand, an IgG3 mAb against Cryptococcus neoformans capsule was found not to be protective unless it was switched to an IgGl isotype switch variant [52, 53]. In this latter case, the lack of protection was explained by the contribution of CH domains to the binding affinity of the antibodies to their target polysaccharide. Other investigators compared the relative opsonic activity of antibodies to the cryptococcal polysaccharide capsule and concluded that for that anti-capsular activity was greatest for IgG2a followed by IgGl then IgG2b [54]. All of the above highlights the complexity of predicting the contribution of the FcR interaction to efficacy and indicates this quality is important and has to be experimentally assessed for every anti-infective Ab.

Most importantly, clinical data derived from patients with recurrent

mediated liver abcesses indicate that opsonizing and killing efficacy of serum increases over time consistent with an evolving protective immune response [55]. Passive immunotherapy mimics effective immune response and high titer preparations of protective Abs could be used alone or in combination with antibiotics to control the invasive infection caused by Kl-Kp. Importantly, prior to treatment, rapid confirmation of the appropriate infection

strain (Kl-CPS) would be required. It was demonstrated herein that CPS Ag secreted in the urine can be diagnosed by ELISA with Kl-CPS specific Abs. Such a sandwich-based ELISA could then be easily further developed into a lateral flow based assay similar to other commercially available diagnostic Ag tests. It was demonstrated herein that CPS Ag is secreted both in urine as well as in blood.

In times of re-emergence of well-known pathogenic microbes, in drug-resistant form, as exemplified by MRSA, multidrug-resistant (MDR) Mycobacterium

tuberculosis and now MDR

it seems prudent to include upfront development of anti-infective Abs in the fight of combating emerging resistant pathogens. The restraint of companies and research foundations to more proactively develop mAb therapies against infectious diseases is short sighted given that even in the best scenario it takes years to develop Ab therapies. Treatment with anti-effective Abs against a gut colonizing bacteria like

is a targeted intervention that should not disturb the microbiome. As evidenced by the efficacy of palivizumab in preterm infants, reducing hospitalizations for RSV-associated disease mAbs have been shown to be effective in immunocompromised hosts [56]. With continuous fast moving globalization, and unrestrained antibiotic usage in most parts of the world, the far away residing

"supersensitive" pathogen becomes an unwanted highly resistant pathogen, which should be anticipated to arrive.

In the instant invention, it has been shown that passive immunotherapy is also a successful treatment to control the invasive infections caused by Kl-Kp. The protective characteristics of both mAbs 4C5 and 19A10 have been demonstrated. EXAMPLES

Materials and Methods

Kl-Kp strains

Kl-Kp strains (n= 4) were collected from inpatients at Montefiore Medical Center (MMC) in Bronx, and Stony Brook University Hospital in Stony Brook. Kp strains were cultured in Luria-Bertani (LB) broth or agar plates at 37 °C. Kl-Kp strain #20 of MMC was used for CPS purification and for in vitro in vivo experiments. To obtain a stable GFP-labeled strain, the strain was transformed with pPROBEKT plasmid kindly provided by Dr. Triplett [22] .

CPS purification

CPS was isolated as previously described with little modifications [23, 24]. Briefly, Kp strain #20 was inoculated in 1L of LB broth and grown o/n at 37°C. Cells were pelleted by centrifugation and washed with PBS. Cells were resuspended to a 5% (wt/vol) in distilled water and CPS was extracted by phenol-water. CPS, present in the aqueous phase, was precipitated by adding 5 volumes of methanol plus 1 % (v/v) of a saturated solution of sodium acetate in methanol and incubated for lh at 24°C. After dissolving the pellet in water, it was dialyzed against water for 6 hours prior to freeze-drying. The lyophilized polysaccharide was dissolved in in 0.8% NaCl, 0.05% NaN3, 0.1M Tris- HC1 (pH 7) to a 10 mg/ml concentration and digested with nucleases (50 mg/ml of

DNase I and RNase A) twice for 24h at 37°C. Then proteinase K was added (50 mg/ml) and incubated for 1 h at 55 °C and for 24 h at room temperature twice. Polysaccharides were precipitated as stated above and dissolved in water. LPS was removed by ultracentrifugation (105000 x g, 16 h, 4°C) and samples were freeze-dried. CPS was further extracted with phenol following previously published protocol [25]. CPS was further purified by a size-exclusion chromatography on a column S200HR (GE

Healthcare Life Sciences) with PBS. Fractions were collected and the presence of polysaccharide was analyzed by the phenol- sulfuric acid method [26].

CPS-PA conjugation

For conjugation isolated Kp CPS was activated with l-cyano-4-dimethylamino- pyridinium tetrafluoroborate (CDAP) as described, with modifications [27]. PA (Protective Antigen) from Bacillus anthracis was obtained from the Wadsworth Center, NYS Department of Health and it was previously used for a PS-protein conjugation [28]. Briefly, CPS was dissolved in NaCl 0.15 M to 10 mg/ml (1ml). At t= 0 s, 100 μΐ of CDAP (100 mg/ml in acetonitrile) (1 mg CDAP/mg PS) was slowly added while stirring. 30 s later 0.2 M TEA (10 μΐ/mg PS) was added to raise the pH. At t = 2' 30 s, 0.1M sodium borate pH 9.3 was added to adjust pH to 9 and 10 mg of PA in 0.15 M NaCl were added. The reaction was incubated o/n at 4°C and the dialyzed against water. PS-protein conjugate product was purified using an S200HR gel filtration column (GE Healthcare Life Sciences). The A280nm in the chromatogram monitored the presence of protein while the PS content was monitored by phenol- sulfuric acid method [26].

mAb generation

MAbs to Kl CPS were generated by immunization with 100 μg of PA-conjugated Kl- CPS in complete Freund's adjuvant (CFA) followed by boosters of PA-conjugated Kl- CPS in incomplete Freund's adjuvant (IF A) of BALB/c mice. Fusion and cloning was performed as described [29].

Agglutination assays

Agglutination was carried out as previously described on glass slides [21].

Human serum resistance assays

Resistance assays were performed as previously described with minor modifications [18]. 1.4xl0⁶ cfu of Kl-Kp were incubated with 20μg of 4C5 or 19A10 mAbs at room temperature for lh. Then they were mixed with 3: 1 (vol: vol) of normal human serum (time 0) and incubated for 2h at 37°C. Aliquots were taken on time 0, 1 and 2, and colony forming units (CFUs) were calculated. Survival ratio was plotted as the number of Kl-Kp found compared to the time 0. Three independent experiments were performed.

C3b-deposition assays

Deposition of complement was measured by flow cytometry with modifications to a protocol previously described [30]. Briefly, an LB o/n culture was diluted and it grew to reach mid-logarithmic phase. 5xl0⁷ cells were resuspended in 1ml of PBS + 20% Normal Human Serum + 1% BSA. PBS, 20μg of 4C5 or 1910 mAbs were added and incubated for 15'. Then, cells were washed 2x with PBS + 1% BSA and divided in half. One half was incubated with 1 :500 sheep anti-human C3c FITC (Bio-Rad) in PBS + 1% BSA. The other was incubated in the absence of antibody. Samples were incubated for 25 min at 4°C. A sample with bacteria incubated without serum was used as a negative control to set the threshold and fluorescence intensity. Intensity higher than 10 was considered positive for C3 binding. Quantification was performed multiplying the percentage of bacteria moving into the gate by the average fluorescence of a defined population (X-mean), to give a Fluorescence Index (FI). Results are expressed by the increment of the signal of mAb treatment with respect to PBS incubation.

Phagocytosis experiments

Bacteria were pH-rodo^® (Life Technologies) labeled following the manufacturer instructions. J744.16 murine macrophage cell line or THP-1 human monocyte cell line were used for phagocytosis assays. THP-1 cells were differentiated with phorbol 12- myristate 12-acetate (PMA), 2xl0⁵ c/ml were treated with 100 nM PMA for 3 days. One day before the phagocytosis experiment both J744.16 and THP-1 cells were seeded at a concentration 5xl0⁵ c/ml in 35mm glass bottom microwell dishes (MatTek

Corporation). On the day of the experiment, 5xl0⁶ pHrodo^®-labeled Kl-Kp cells were incubated for 1 hour with PBS, 20μg of isotype control, 4C5 or 19A10 mAbs.

Macrophages were infected with pHrodo^®-labeled Kl-Kp with a MOI 10: 1.

Phagocytosis was immediately assessed using a Zeiss Axiovert 200 M inverted microscope with a lOx objective with the dish housed in an enclosed chamber under conditions of 5% CO₂ and 37°C, images were taken every 4 min for the 2h of duration of the experiment. Phagocytosis was measured by counting the percentage of macrophages that display labeled bacteria at 67 and 150 min.

For experiments with primary macrophages, bone marrow macrophages were isolated as described [31] from Wild-type or FcR-Knock out mice (Fcerlg^mlRav, The Jackson Laboratory). After 7 days of macrophage differentiation the phagocytosis experiments, as outlined above, were carried out.

In vivo animal experiments

Six- to eight-week-old female BALB/c or Swiss Webster mice were purchased from Taconic Biosciences, Inc. Mice (n = 10) were treated intraperitoneally (i.p.) with 500 μg of PBS, control isotype, 4C5, 19A10 or 4C5 + 19A10 mAbs 120 min prior to challenge with 5xl0⁴ CFUs of Kl-Kp i.p. or 24h prior to challenge with lxlO⁴ CFUs

intratracheally (i.t.). Survival of 6 mice was monitored for 15 days. Liver, spleen and lungs of 4 mice were processed to enumerate bacteria in homogenized tissue.

All animal experiments were carried out with the approval of the Animal Institute

Committee (AIC), in accordance with the rules and regulations set forth by the Albert Einstein College of Medicine and Stony Brook University. Intravital microscopy (IVM)

Liver intravital protocol was performed as previously described [32]. Briefly, mice were anesthetized by i.p. injection of a mixture of ketamine (120 mg/kg) and xylazine (10 mg/Kg). Tail vein was cannulated for administration of additional anesthetic and experimental reagents as required. A midline laparotomy was performed followed by removal of the skin and abdominal muscle along the costal margin to the mid-axillary line to expose the liver. Mice were placed in the right lateral position and a single liver lobe was exteriorized on a custom-made stage. Exposed tissues were moistened with saline-soaked gauze to prevent dehydration during imaging. Body temperature was maintained with an infrared heat lamp, and the liver was continuously superfused with physiological saline buffer. Intravital microscopy was performed by Examiner Zl system (Zeiss), time-lapse video was taken at a speed of 1 frame/s. Videos were further analyzed by Zen2012 software (Zeiss).

Statistical analysis

Data are presented in mean + standard deviation or median range. Survival data were analyzed with a log-rank test. Statistical tests were performed with GraphPad Prism 6 for Mac.

Results

CPS-PA conjugation and mAbs production

CPS of Kp strain #20, Kl serotype was purified as previously reported [12]. Given that CPSs are T-cell independent antigens with limited immunogenicity, the probability of recovering IgG producing mAbs is low. Therefore, CPS was conjugated to Protective Antigen (PA), a Bacillus anthracis protein that has been used successfully in other polysaccharide conjugations [62]. The conjugation was carried out by CDAP-mediated activation of the polysaccharide [61]. The chromatograms of a non-conjugated CPS +PA and a conjugated CPS-PA are shown in Figure 1. As expected, conjugation increased the protein content of the CPS fractions that were subsequently pooled.

Immunization with conjugated Kl-PA CPS yielded 6 different hybridomas each producing IgGs that were specific for Kl-CPS (Table 1). Two of them were selected for further development, as they were higher mAb producers and exhibited different isotype classes (4C5 -IgGl- and 19A10 -IgG3-).

Binding properties to purified Kl-CPS by ELISA were found to be in the sub- nanomolar range. Kd of 4C5 was calculated 0.3642+/- 0.07244 nM, whereas 19A10 has a Kd of 0.2419+/-0.3210 nM confirming a high binding capacity of both mAbs to Kl-CPS. ELISA assays employing isotype specific secondary Abs indicated that both Abs can bind simultaneously to purified CPS. Some inhibition was only detected at higher concentration (data not shown).

In vitro antibody-mediated activities

First, the effect of the mAb on bacterial growth was examined. For this purpose, the viability of the Kl-Kp growing in the presence of either 4C5 or 19A10 mAbs was assayed. Both mAbs induced a significant decrease of bacterial viability after 2 h of co- incubation (Figure 2 A and 2B).

In addition, both mAbs induced agglutination and led to capsular swelling when co- incubated with Kl-Kp (Figure 2C). Agglutination also occurred with the GFP- expressing strain, Kl-pPROBEKT, and three other clinical Kl-Kp strains that were not used for the immunization (data not shown). No agglutination was observed with non Kl-hvKp strains that did not express a Kl-CPS.

Co-incubation with either mAbs decreased Kl-Kp virulence in the invertebrate animal model Galleria mellonella (p-value 0.0172 and 0.032, respectively) (Fig 2F).

In vitro growth experiments demonstrated that Kl-hvZ/?'s generic ability to readily replicate in serum was 30-fold reduced by both mAbs (2h / value=0.003) (Figure 2D).

In addition, C3-complement deposition on the capsule in the presence of mAbs was quantified and compared to that of Kp bacteria in the presence of PBS and Normal Human Serum (NHS) as described in the Material and Methods. These experiments found that 4C5 incubation increased by 1.44x and 19A10 1.17x the C3b deposition

(Figure 2E) (/ value<0.05).

When evaluating the deposition of the Membrane Attack complex (MAC) in Kl-Kp bacteria, a significant deposition after the binding of 19A10 compared to the PBS or IgG controls was observed (Fig 2G) (2-way ANOVA /?-value<0.0001, PBS vs. 4C5 n.s, PBS vs 19A10 /?-value<0.0001 and PBS vs. positive control Aggregated human IgG p- value<0.0001)

4C5 mAb promotes phagocytosis of Kl-fip in professional phagocytic cells

Next, the ability of 4C5 to promote uptake of Kl-Kp cells by a murine macrophage cell line, J744.16, was investigated labeling Kp bacteria with pHrodo^® (LifeTechnologies) which allowed proper detection of real phagocytosis events opposed to only attached Kp bacteria, as rodamine-fluorescence depends on acidic pH acid of the phagolysosomes. The phagocytosis index was determined by the number of macrophages that were phagocytosing bacteria at a certain time point No phagocytosis was recorded when bacteria were incubated with PBS (0%

phagocytizing macrophages) (Figure 3A-B and supplementary video 1). In contrast, when Kl-Kp was co-incubated with 4C5 mAb, 13.85 and 27.65% of macrophages exhibited phagocytized bacteria 67 and 150 min after infection (/?-value= 0.0004 and 0.005, respectively). For 19A10 mAb, similar results were found (27.45% and 38.05%, 66.7 and 150 min after infection, respectively) (/ value= 0.0002 and 0.003, respectively).

Opsonophagocytic properties of the mAbs were also documented within the human macrophage cell line. Both mAb promoted phagocytosis, 4C5 (Figure 3D-C and supplementary video 2) (67min 22% vs. 0% /?-value=0.008; 150min 30% vs. 0% p- value=0.003), as well as 19A10 mAb, (6.83% and 20.04% of macrophages were phagocytizing at 67min and 150min, /?-value= 0.0005 and 0.042, respectively). When phagocytosis for 8h after the initial phagocytosis was observed, no bacteria was seen escaping from the phagolysosome (data not shown).

It was furthermore observed that bone marrow-derived macrophages from FcyR- deficient mice exhibited significantly reduced phagocytosis compared to wild type derived bone marrow macrophages (p-value<0.05 for 4C5 and p-value<0.01 for 19A10) demonstrating that opsonophagocytic effect was dependent on Fc receptor engagement. With respect to killing, it was found that J744.16 murine macrophages killed Kl-Kp when opsonized with 4C5 or 19A10 (Fig 3D) (ANOVA p-value<0.0001, /?-value<0.01 for 4C5 and 19A10 treatment). No phagocytosis events were observed when bacteria were incubated with PBS or IgG control.

Neutrophils do not only kill pathogens by phagocytosis, but can also release of web-like structures called neutrophil extracellular traps (NETs) that trap and kill a variety of microbes. This process is called NETosis and is implicated in clearance of many infectious pathogens including fungi, parasites and bacteria. Kl-Kp alone did not induce release of NETs whereas (Fig. 3E-F) hyphae of Candida albicans did, as previously reported. However Kl-Kp co-incubated with either 4C5 or 19A10 led to release of NETs (ANOVA p-value<0.0001, / value<0.0001 for 4C5 and 19A10 treatment). NETosis was only seen with Kl-Kp specific mAbs and absent in control experiments that used an IgG control Ab. 4C5 and 19A10 elicit a protective response in murine Kl-Kp infections being enhanced when both mAbs were combined.

Next it was tested if opsonophagocytic in vitro activities correlated with protective efficacy in vivo in two different murine infection models using Kl-Kp strain#20. For these experiments, Swiss-Webster mice were pre-treated either with PBS, 500 μg of an IgG isotype control or 500 mg of 4C5 mAb or 500 mg of 19A10 mAb 2h prior to the introperitoneal inoculation with 5xl0⁴ Kl-Kp CFUs. Treatment with 4C5 enhanced survival when compared to PBS- or Control isotype-treated mice (/?-value=0.0027) (Figure 4A). Treatment with 4C5 significantly reduced the bacterial load (CFU) in liver, as well as well as lung and spleen, when compared to PBS or isotype control treatment (p-value<0.001 in all cases) (Figure 4B). For mice treated with 19A10 mAb, a similar protective trend (/ value=0.0170) was observed and treatment with 19A10 also significantly reduced bacterial organ burden (p-value<0.001 in all cases).

Treatment with combination of mAbs enhanced protective efficacy (/ value=0.014) and also reduced bacterial burden significantly (/ value<0.001).

Next protection was tested in an intratracheal infection model. Mice were treated with PBS, isotype control and mAbs, 24h before intratracheal inoculation with lxlO⁴ Kl-Kp CFUs. Again treatment with 4C5 significantly enhanced surivival in mice in comparison to PBS- or isotype control-treated (Figure 4C and 4D) (/ value=0.0027). Protective efficacy as well as lower bacterial organ burden was also observed after treatment with mAbl9A10 (/?-value=0.0051). Survival as well as bacterial clearance was further enhanced by combination therapy in this model as well (p-value=0.0009) (Figure 4C and 4D).

Finally cytokine and chemokine induction in tissue of mAbs- and PBS-treated mice that were i.p. infected with Kl-Kp strain was compared. Pro-inflammatory cytokines levels in liver and spleen homogenates of mAb treated mice were significantly lower compared to PBS treated mice, such as IFN-γ, ILl-β, IL-2, IL-6 and TNF-a (2-way ANOVA treatment /?-value<0.0001 for both liver and spleen, Fig. 4E-F). Similar trends were noted in lungs (Fig. 7A-C).

Antibodies promote capture of Kl-Kp by Kupffer cells in the liver

In order to decipher the mechanisms that promoted the clearance of Kl-Kp from the organs, intravital microscopy assays were carried out. When 10⁸ CFUs were i.v.

injected in the absence of treatment, the number of bacteria flowing through blood vessels was significantly higher and lasted longer than when either of the mAbs was used (p<0.05) (Figure 6A). Concomitantly the number of bacteria being capture in the liver tissue was significantly higher when both antibodies were used (Figure 6B). When 5xl0⁴ CFU were i.p. injected, when either of the mAbs were used, no bacteria was detected in the blood vessels or captured in the tissue (Figure 6C and D). Furthermore, in the absence of treatment,

was found abundantly in the abdominal cavity, whereas hv-Kp was cleared when mAbs were injected (data not shown). Reducing inoculum size of bacteria, injected i.v., to 5x104, resulted in complete clearance of the bacteria (Figure 5F).

Treatment with mAbs decreased dissemination of bacteria in Kl-Kp colonized mice that were treated with antibiotics.

Protective efficacy of mAbs was examined in a murine model of gastrointestinal colonization, where bacterial dissemination to liver, spleen and mesenteric lymph nodes (MLN) is promoted by treatment with antibiotics. Mice colonized with 10⁸ GFP- labeled kvKp via oral ingestion were given ampicillin, leading to a significant increase in bacterial burden in feces and subsequent translocation and dissemination to mesenteric lymphnodes (MLN), spleen and liver. Whether mAbs treatment protected against dissemination and decreased bacterial burden in MLN, spleen and liver was investigated. The data indicated that bacterial dissemination to the MLN, liver, and spleen was significantly decreased and bacterial burden was up to 6 logs lower in those organs compared to the PBS-treated mice (Fig 8) (2-way ANOVA /?-value<0.0001). In contrast bacterial colonization seen in feces showed no significant differences with mAb treatment indicating that colonization per se is not affected by systemic mAb treatment. Both mAbs provide a detection tool of Kl-infections both in urine and serum samples.

Lastly, mAbs were used to design a sandwich ELISA to enable rapid non- invasive detection of Kl-Kp CPS in murine body fluids (Figure 6A). ELISA tests employing both mAbs exhibited detection sensitivity of CPS as low as 0.20 μg/ml in serum and 0.11 μg/ml in urine (Figure 6B and 6D). CPS was detected in mice infected i.v. in serum samples, and i.p.- and i.t-infected mice in urine samples. Quantities as low as 0.11 μg/ml of CPS were found in different mice in urine over time and corresponded with the severity of the disease (Figure 6D). Importantly, the ELISA was specific and did not detect CPS from other non Kl-CPS expressing Kp strains. Treatment mAbs reduces Ampicillin induced dissemination from gut in Kl-Kp colonized mice

Protective efficacy of mAbs was examined in a murine model of gastrointestinal colonization, where bacterial dissemination to liver, spleen and mesenteric lymph nodes (MLN) is promoted by treatment with antibiotics. Mice colonized with 10⁸ GFP- labeled Kl-Kp via oral ingestion were given ampicillin, leading to a significant increase in bacterial burden in feces and subsequent translocation and dissemination to mesenteric lymphnodes (MLN), spleen and liver (Figures 9A-B and 8). Whether mAbs treatment protected against dissemination and decreased bacterial burden in MLN, spleen and liver was investigated (Figure 9B). The data indicate that bacterial dissemination to the MLN, liver, and spleen was significantly decreased and bacterial burden was up to 6 logs lower in those organs compared to the PBS-treated mice (Figure 8) (2- way ANOVA / value<0.0001). In contrast bacterial colonization seen in feces showed no significant differences with mAb treatment indicating that colonization per se is not affected by systemic mAb treatment.

Sequencing of the Monoclonal Antibodies

Total RNA was extracted from frozen hybridoma cells and cDNA was synthesized from the RNA. PCR was then performed to amplify the variable regions (heavy and light chains) of the antibody, which were then cloned into a standard cloning vector separately and sequenced.

Materials

Hybridoma cells; TRIzol® Reagent (Ambion, Cat. No. : 15596-026); PrimeScriptTM 1st Strand cDNA Synthesis Kit (Takara, Cat. No. : 6110A).

Methods

Total RNA extraction

Total RNA was isolated from the hybridoma cells following the technical manual of TRIzol® Reagent. The total RNA was analyzed by agarose gel electrophoresis.

RT-PCR

Total RNA was reverse transcribed into cDNA using isotype-specific anti-sense primers or universal primers following the technical manual of PrimeScriptTM 1st Strand cDNA Synthesis Kit. The antibody fragments of VH and VL were amplified according to the standard operating procedure of RACE of GenScript. Cloning of antibody genes

Amplified antibody fragments were separately cloned into a standard cloning vector using standard molecular cloning procedures.

Screening and sequencing

Colony PCR screening was performed to identify clones with inserts of correct sizes. No less than five single colonies with inserts of correct sizes were sequenced for each antibody fragment.

Total RNA extraction

The isolated total RNA of the sample was run alongside a DNA marker Marker III (TIANGEN, Cat. No. : MD 103) on a 1.5% agarose/GelRed™ gel.

PCR product of antibody genes

Four microliters of PCR products of each sample were run alongside the DNA marker

Marker III on a 1.5% agarose/GelRedTM gel. The PCR products were purified and stored at -20 C until further use.

Sequencing results and analysis

Five single colonies with correct VH and VL insert sizes were sent for sequencing. The

VH and VL genes of five different clones were found nearly identical.

The consensus sequence, listed below, is the sequence of the antibody produced by the hybridoma 4C5

Heavy chain: DNA sequence (405 bp)

Leader sequence-FRl-CDRl-FR2-CDR2-FR3-CDR3-FR4

ATGGACTGGAGTTGGGTCTTTCTCTTCCTCCTGTCAGTAAATGAAGGTGTCT ACTGTCAGGTCCAGCTGCAGCAGTCTGGAGATGATCTGGTAACGCCTGGGG CCTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACT GGATTAACTGGATAAAACAGAGGCCTGGACAGGGCCTTGAGTGGGTAGGA CGTATTACTCCTGGACGTGGTAATACTTACTACAATGAAATGTTCAAGG ACAAGGCAACACTGACTGTAGACACATCCTCCAGAACAGCCTACATTCAGC TCAGCAGCCTGTCATCTGAGGACTCTGCTGTCTATTTCTGTGCAAGGGGGG GTGTCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

(SEQ ID NO: 1)

The nucleotide sequences of CDR1, CDR2 and CDR3 of the mAb 4C5 heavy chain are SEQ ID NO: 9, 10 and 11, respectively, as follows:

AGCTACTGGATTAAC (SEQ ID NO: 9) CGTATTACTCCTGGACGTGGTAATACTTACTACAATGAAATGTTCAAGGAC (SEQ ID NO: 10)

GGGGGTGTCTGGTTTGCTTAC (SEQ ID NO: 11)

Heavy chain: Amino acids sequence (135 AA)

Leader sequence-FRl-CDRl-FR2-CDR2-FR3-CDR3-FR4

MDWSWVFLFLLSVNEGVYCOVOLQOSGDDLVTPGASVKLSCKASGYTFTSYW INWIKQRPGQGLEWVGRITPGRGNTYYNEMFKDKATLTVDTSSRTAYIQLSSL SSEDSAVYFCARGGVWFAYWGQGTLVTVSA (SEQ ID NO: 2)

The amino acid sequences of CDR1, CDR2 and CDR3 of the mAb 4C5 heavy chain are SEQ ID NO: 12, 13 and 14, respectively, as follows:

SYWIN (SEQ ID NO: 12)

RITPGRGNTYYNEMFKD (SEQ ID NO: 13)

GGVWFAY (SEQ ID NO: 14)

Light chain: DNA sequence (393 bp)

Leader sequence-FRl-CDRl-FR2-CDR2-FR3-CDR3-FR4

ATGGGCATCAAGATGGAGTCACAGATTCAGGCATTTGTATTCGTGTTTCTCT GGTTGTCTGGTGTTGACGGAGACATTGTGATGACCCAGTCTCACAAATTCAT GTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGG ATGTGAGTACTGCTGTAGCCTGGTATCAGCAAAAACCAGGGCAATCTCCT AAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCG CTTCACAGGCAGTGGATCTGGGACAGATTATACTCTCACCATCAGCAGTGTG CAGGCTGAAGACCTGGCACTTTATTACTGTCAGCAACATTATAGCACTCCG TACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 3)

The nucleotide sequences of CDR1, CDR2 and CDR3 of the mAb 4C5 light chain are SEQ ID NO: 15, 16 and 17, respectively, as follows:

AAGGCCAGTCAGGATGTGAGTACTGCTGTAGCC (SEQ ID NO: 15)

TGGGCATCCACCCGGCACACT (SEQ ID NO: 16)

CAGCAACATTATAGCACTCCGTACACG (SEQ ID NO: 17)

Light chain: Amino acids sequence (131 AA)

Leader sequence-FRl-CDRl-FR2-CDR2-FR3-CDR3-FR4

MGIKMESOIOAFVFVFLWLSGVDGDIVMTOSHKFMSTSVGDRVSITCKASODV STAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAED LALYYCOOHYSTPYTFGGGTKLEIK (SEQ ID NO: 4) The amino acid sequences of CDR1, CDR2 and CDR3 of the mAb 4C5 light chain are SEQ ID NO: 18, 19 and 20, respectively, as follows:

KASQDVSTAVA (SEQ ID NO: 18)

WASTRHT (SEQ ID NO: 19)

QQHYSTPYT (SEQ ID NO: 20)

The consensus sequence, listed below, is the sequence of the antibody produced by the hybridoma 19A10

Heavy chain: DNA sequence (399 bp)

Leader sequence-FRl-CDRl-FR2-CDR2-FR3-CDR3-FR4

ATGGCTGTCCTGGTGCTGTTCCTCTGCCTGGTTGCATTTCCAAGCTGTGTCCT GTCCCAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACA GAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTAACCAGCTATGGT GTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGT AATATGGGCTGGTGGAAGCACAAATTATAATTCGGCTCTCATGTCCAGA CTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAAC AGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCTTACTATGGCTA AGAGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 5)

The nucleotide sequences of CDR1, CDR2 and CDR3 of the mAb 19A10 heavy chain are SEQ ID NO: 21, 22 and 23, respectively, as follows:

AGCTATGGTGTACAC (SEQ ID NO: 21)

GTAATATGGGCTGGTGGAAGCACAAATTATAATTCGGCTCTCATGTCC (SEQ ID NO: 22)

CTATGGCTAAGAGCTTAC (SEQ ID NO: 23)

Heavy chain: Amino acids sequence (133 AA)

Leader sequence-FRl-CDRl-FR2-CDR2-FR3-CDR3-FR4

MAVLVLFLCLVAFPSCVLSQVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVH WVROPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSOVFLKMNSLOT DDTAMYYCALLWLRAYWGQGTLVTVSA (SEQ ID NO: 6)

The amino acid sequences of CDR1, CDR2 and CDR3 of the mAb 19A10 heavy chain are SEQ ID NO: 24, 25 and 26, respectively, as follows:

SYGVH (SEQ ID NO: 24)

VIWAGGSTNYNSALMS (SEQ ID NO: 25)

LWLRAY (SEQ ID NO: 26) Light chain: DNA sequence (393 bp)

Leader sequence-FRl-CDRl-FR2-CDR2-FR3-CDR3-FR4

ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCA GCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGG AGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTA ATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCA AAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAG GTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGT GGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCC GTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 7)

The nucleotide sequences of CDR1, CDR2 and CDR3 of the mAb 19A10 light chain are SEQ ID NO: 27, 28 and 29, respectively, as follows:

AGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAA (SEQ ID NO: 27)

AAAGTTTCCAACCGATTTTCT (SEQ ID NO: 28)

TTTCAAGGTTCACATGTTCCGTGGACG (SEQ ID NO: 29)

Light chain: Amino acids sequence (131 AA)

Leader sequence-FRl-CDRl-FR2-CDR2-FR3-CDR3-FR4

MKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCRSSOSIVHSNGN TYLEWYLQKPGOSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLG

VYYCFQGSHVPWTFGGGTKLEIK (SEQ ID NO: 8)

The amino acid sequences of CDR1, CDR2 and CDR3 of the mAb 19A10 light chain are SEQ ID NO: 30, 31 and 32, respectively, as follows:

RSSQSIVHSNGNTYLE (SEQ ID NO: 30)

KVSNRFS (SEQ ID NO: 31)

FQGSHVPWT (SEQ ID NO: 32)

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Claims

CLAIMS:

1. An isolated polynucleotide encoding a light chain variable region (LCVR) of an antibody, or active fragment thereof, said LCVR comprising an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 4, wherein the sequence comprises light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 18-20.

2. An isolated polynucleotide encoding a heavy chain variable region (HCVR) of an antibody, or active fragment thereof, said HCVR comprising an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 2, wherein the sequence comprises heavy chain CDRs comprising the amino acid sequences of SEQ ID NOs: 12-14.

3. The isolated polynucleotide of claim 1, wherein the amino acid sequence is at least 99% identical to the sequence set forth in SEQ ID NO: 4.

4. The isolated polynucleotide of claim 2, wherein the amino acid sequence is at least 99% identical to the sequence set forth in SEQ ID NO: 2.

5. An isolated polynucleotide encoding a light chain variable region (LCVR) of an antibody, or active fragment thereof, said LCVR comprising an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 8, wherein the sequence comprises light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 30-32.

6. An isolated polynucleotide encoding a heavy chain variable region (HCVR) of an antibody, or active fragment thereof, said HCVR comprising an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 6, wherein the sequence comprises heavy chain CDRs comprising the amino acid sequences of SEQ ID NOs: 24-26.

7. The isolated polynucleotide of claim 5, wherein the amino acid sequence is at least 99% identical to the sequence set forth in SEQ ID NO: 8.

8. The isolated polynucleotide of claim 6, wherein the amino acid sequence is at least 99% identical to the sequence set forth in SEQ ID NO: 6.

9. An isolated polypeptide comprising a light chain variable region (LCVR) of an antibody, or active fragment thereof, said LCVR comprising an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 4, wherein the sequence comprises light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 18-20.

10. An isolated polypeptide comprising a heavy chain variable region (HCVR) of an antibody, or active fragment thereof, said HCVR comprising an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 2, wherein the sequence comprises heavy chain CDRs comprising the amino acid sequences of SEQ ID NOs: 12-14.

11. The isolated polypeptide of claim 9, wherein the amino acid sequence is at least 99% identical to the sequence set forth in SEQ ID NO: 4.

12. The isolated polypeptide of claim 10, wherein the amino acid sequence is at least 99% identical to the sequence set forth in SEQ ID NO: 2.

13. An isolated polypeptide comprising a light chain variable region (LCVR) of an antibody, or active fragment thereof, said LCVR comprising an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 8, wherein the sequence comprises light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 30-32.

14. An isolated polypeptide comprising a heavy chain variable region (HCVR) of an antibody, or active fragment thereof, said HCVR comprising an amino acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 6, wherein the sequence comprises heavy chain CDRs comprising the amino acid sequences of SEQ ID NOs: 24-26.

15. The isolated polypeptide of claim 13, wherein the amino acid sequence is at least 99% identical to the sequence set forth in SEQ ID NO: 8.

16. The isolated polypeptide of claim 14, wherein the amino acid sequence is at least 99% identical to the sequence set forth in SEQ ID NO: 6.

17. A method of inhibiting a K. pneumoniae infection in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising at least one polypeptide of claims 9-16.

18. The method of claim 17, wherein the K. pneumoniae infection is a hypervirulent K. pneumoniae infection of serotype Kl .

19. A method of diagnosing a K. pneumoniae infection in a subject, the method comprising contacting a biological sample of the subject with at least one polypeptide of claims 9-16; determining whether an immunological complex is formed; and comparing amount of the complex formed to a normal control value.