TWI773670B - 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 (KLEBSIELLA PNEUMONIAE) antibody and method for treating Klebsiella pneumoniae infection

Klebsiella pneumoniae is a gram-negative pathogen of the Enterobacteriaceae family that causes lung, urinary tract, wound and soft tissue infections in hospitalized patients. In addition, Klebsiella pneumoniae can be asymptomatically carried in healthy individuals, mainly in the gut, but also in the nose, throat, and skin [1]. The mechanisms by which Klebsiella pneumoniae can cause invasive disease are not fully understood [2]. Due to the acquisition of multi-drug resistance genes, Klebsiella pneumoniae infection has become a worldwide threat in recent years [3]. For example, carbapenem-resistant Klebsiella pneumoniae spreads worldwide; the most successful pure phylotype is ST258. A variant strain of Klebsiella pneumoniae, the hypervirulent Klebsiella pneumoniae (hv Kp ), has emerged. This variant strain can cause invasive infections, including purulent liver abscesses, pneumonia, ophthalmia, and meningitis [2]. It has been reported worldwide [4-6], but most of this strain is from Asia, where it is the main cause of liver abscesses in Taiwan [7], Singapore [8], Hong Kong [9] and Korea [10]. In fact, in Taiwan, the annual incidence of purulent liver abscess increased by 11 to 17 cases per 100,000 population between 1996 and 2004 [11PMCPMC2609891]. Up to 13% of hv Kp infections have metastatic spread to the eye or meninges [11]. A large percentage of hv Kp infections spread to the eye or meninges [12, Fang CT, PMCPMC4076766], which often results in irreversible damage such as vision loss, neurological deficits, or loss of amine bodies. Mortality rates range from 3 to 42 percent [2]. Recent epidemiological studies have shown that healthy adults carry virulent strains in their gastrointestinal tract, and based on MLST typing, colonizing strains are inferred to cause invasive infections in patients [13].

Fortunately, and in contrast to the ST258 strain [12], the hv Kp strain exhibited less diversity in its capsular polysaccharide (CPS). The K1 serotype is the prevalent CPS and has been reported to be present in up to 81% of hv Kp strains, followed by the K2 serotype and the non-K1/K2 serotype, respectively [10, 11, 13, 14].

Historically, hv Kp has been observed to be pan-susceptible to standard antibiotics and thus, if identified early, these infections can often be successfully treated, despite their invasive nature. With the emergence of resistant hv Kp strains, one of the major concerns is that these variants can acquire multidrug resistance (MDR) conferring plastids, such as those carrying bla ^{KPC -2} and bla ^{KPC -3} . Therefore, according to a recent report from China, the hv Kp strain was found to be still sensitive to carbapenems and amikacin, but resistant to 14 out of 19 tested antimicrobials [15]. In addition, the report noted that antibiotic resistance continued to rise from December 2010 to June 2012 [15]. In vitro acquisition of highly virulent strains with KPC plastids has been demonstrated [16]. In addition, hv Kp strains exhibiting carbapenem resistance have recently been isolated in China [17].

Therefore, given the imminent threat of emerging multi-drug resistant hv Kp strains, it is clear that there is an urgent need to discover diagnostic and therapeutic tools to combat P. lebsiella infection, in particular hv Kp serotype K1 infection.

The present invention relates to antibodies (which include whole antibodies and functional parts thereof) as therapeutic and diagnostic tools for combating Klebsiella pneumoniae ( Kp ) infection and diseases and disorders caused by or associated with Kp infection. The term "antibody" or "antibodies" as used herein is art recognized and should be understood to refer to a polypeptide molecule or an active fragment of a polypeptide molecule that binds to a known antigen. In one embodiment of the present 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 for the capsular polysaccharide (CPS) of the highly virulent Kp K1 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. "Isolated" means that the biomolecule is free of at least some of the components with which it naturally occurs. The term "CDR" refers to the hypervariable regions of an antibody. The mAb contains 6 hypervariable regions: 3 are located in the VH (H1, H2, H3) and 3 are located in the VL (L1, L2, L3). In another embodiment, any functionally equivalent antibody or functional portion of mAb 4C5 and mAb 19A10 is provided. "Functionally equivalent" antibodies/portions substantially share at least one major functional property with mAbs as described herein, including passive immunity against Kp infection. For example, mAb 4C5 and mAb 19A10 can have amino acid sequences altered by at least one, specifically at least 2, more specifically at least 3 or more conservative substitutions in their sequences such that the mAbs substantially maintain their Function. Conservative amino acid substitutions do not render the antibody incapable of binding to the individual receptor. One skilled in the art should be able to predict what amino acid substitutions can be made while maintaining a high probability of conformation and antigenic neutrality. Considerations that affect the probability of maintaining conformation and antigenic neutrality include (but are not limited to): (a) substitution of hydrophobic amino acids is unlikely to affect antigenicity since hydrophobic residues are more likely to be internal to the protein; ( b) substitution of physiochemically similar amino acids is unlikely to affect conformation, since substituted amino acids structurally mimic natural amino acids; and (c) changes to evolutionarily conserved sequences may adversely affect conformation impact, as these conserved properties suggest that the amino acid sequence may be of functional importance. The present invention also provides functional peptide fragments comprising 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 exhibiting 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% of the sequence given in SEQ ID NO: 2 or 6 , 94%, 95%, 96%, 97%, 98%, or 99% identical heavy chain variable regions or functional portions thereof of isolated amino acid sequences comprising at least one, usually at least 2, more usually at least 3 heavy chain CDRs with polypeptide sequences SEQ ID NOs: 12-14 or 24-26, respectively, but in particular all CDRs are embedded in their native framework regions. In one embodiment, the invention relates to exhibiting 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% of the sequence given in SEQ ID NO: 4 or 8 , 94%, 95%, 96%, 97%, 98% or 99% identical light chain variable regions or functional portions thereof of isolated amino acid sequences comprising at least one, usually at least 2, more usually at least 3 light chain CDRs with polypeptide sequences SEQ ID NOs: 18-20 or 30-32, respectively, but in particular all CDRs are embedded in their native framework regions. Conventionally, sequence identity can be determined using a computer program such as the Bestfit program (Wisconsin Sequence Analysis Package, Unix version 8, Genetics Computer Group, University Research Park, 575 Science Drive Madison, Wis. 53711). In one embodiment, the present invention provides polynucleotides comprising isolated nucleotide sequences encoding amino acid sequences described herein. Methods of Treatment In one embodiment of the present invention, passive immunotherapy is provided for Klebsiella pneumoniae infection, in particular infection by a highly virulent strain of serotype K1, by administering to an individual in need thereof This is accomplished by administering a therapeutic composition. Therapeutic compositions can prevent, inhibit, treat and/or ameliorate the effects of diseases and disorders in individuals caused by or associated with Kp infection, including but not limited to, for example, purulent liver abscesses, pneumonia, ophthalmia, meninges inflammation and neurological disorders. Individuals include mammals, in particular humans, who are at risk of acquiring a Kp infection, or have a Kp infection and/or a condition/disease associated with a Kp infection. Therapeutic compositions comprise a therapeutically effective amount of at least one of the mAbs described herein, including any functionally equivalent antibody or functional portion thereof. In some embodiments, the composition comprises only one of mAb 4C5 or mAb 19A10, and/or functional equivalents/fragments thereof. In other embodiments, the composition comprises mAb 4C5 and mAb 19A10, and/or functional equivalents/fragments thereof. Antibodies can be prepared in physiologically acceptable formulations and can include pharmaceutically acceptable carriers, diluents and/or excipients using known techniques. For example, antibodies can be combined with pharmaceutically acceptable carriers, diluents and/or excipients to form therapeutic compositions. Suitable pharmaceutical carriers, diluents and/or excipients are well known in the art and include, for example, phosphate buffered saline solutions, water, emulsions (eg, oil/water emulsions), various types of wetting agents, sterile solutions, and the like. The pharmaceutical composition may further comprise a proteinaceous carrier, such as serum albumin or immunoglobulins of human origin in particular. Formulation of pharmaceutical compositions can be accomplished according to standard methods known to those skilled in the art. The compositions can be administered to a subject in solid, liquid or aerosol form in a suitable pharmaceutically effective dose. Examples of solid compositions include pills, creams, and implantable dosage units. Pills can be administered orally. Therapeutic creams can be administered topically. Implantable dosage units can be administered locally, eg, at a site of infection (eg, the liver), or can be implanted, eg, subcutaneously, for systemic delivery of the therapeutic composition. Examples of liquid compositions include formulations suitable for intramuscular, subcutaneous, intravenous, intraarterial injection, 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, compositions can be administered by topical, oral, rectal, nasal, interdermal, intraperitoneal, or parenteral (eg, intravenous, subcutaneous, or intramuscular) routes. Additionally, the composition can be incorporated into a sustained release matrix (eg, a biodegradable polymer) that is implanted near where delivery is desired. The method includes administering a single dose, administering repeated doses at predetermined time intervals, and administering continuously over a predetermined period of time. It is well known to those skilled in the relevant art that the dosage of the composition will depend on various factors, such as the condition being treated, the particular composition being used, and other clinical factors such as the patient's weight, size, gender and general health, body surface area, desire for The particular compound or composition is administered along with other drugs and routes of administration. The compositions can be administered in combination with other compositions comprising biologically active substances or compounds, such as with antibiotics (eg, carbapenems and amikacin) and/or pain relievers and/or anti-inflammatory compounds. The other biologically active substance may be part of the same composition already comprising the antibody in a mixture in which the antibody and the other biologically active substance are intermixed in or with the same pharmaceutically acceptable solvent and/or carrier or antibody; or other biologically active substances may be provided separately as part of a separate composition. Antibodies can be administered to an individual simultaneously, intermittently, or sequentially with one or more other biologically active substances. The proteinaceous pharmaceutically active substance may be present in amounts between 1 ng/dose and 10 mg/dose. Generally, the dosing regimen should be in the range between 0.1 μg and 10 mg of the antibody of the invention, specifically in the range of 1.0 μg to 1.0 mg, and more specifically between 1.0 μg and 100 μg within the range. If administration occurs via continuous infusion, more appropriate doses may range between 0.01 μg and 10 mg units/kg body weight/hour. Administration will typically be parenteral, eg, intravenous. Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Non-aqueous solvents include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils (eg, olive oil), and injectable organic esters (eg, ethyl oleate). Aqueous solvents can be selected from the group consisting of water, alcoholic/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 (eg, those based on Ringer's dextrose), and others. Preservatives, such as antimicrobials, antioxidants, chelating agents, inert gases, and the like, may also be present. Diagnostic Methods In one embodiment, the present invention pertains to methods of diagnosing Kp infection. Diagnosis of Kp infection in an individual can be achieved by detecting, in a sample or in situ, the immunospecific binding of an antibody of the invention, in particular a monoclonal antibody or an active fragment thereof, to an epitope of a Kp polysaccharide, which includes using Contacting a sample or specific body part or body region suspected of containing Kp polysaccharide with an antibody that binds an epitope of Kp polysaccharide, allowing the antibody to bind to the polysaccharide to form an immune complex, detecting the formation of an immune complex and allowing the presence or absence of an immune complex Absence correlates with the presence or absence of Kp polysaccharide in the sample or specific body part or region, as appropriate comparing the amount of the immune complex to a normal control value, wherein an increase in the amount of the immune complex compared to the normal control value is indicative The individual has or is at risk of developing a Kp infection or related disease or condition. Biological samples that can be used in individuals to diagnose Kp infection or related diseases or conditions, or to monitor minimal residual disease, are, for example, fluids such as urine, serum, plasma, saliva, gastric secretions, mucus, cerebrospinal Fluid, lymph, and the like or tissue or cell samples obtained from an individual. To determine the presence or absence of Kp polysaccharides in a sample, any immunoassay known to those skilled in the art can be used, such as assays using secondary reagents for detection by indirect detection methods, ELISA assays, and immunoprecipitation and agglutination assays. A detailed description of these assays is given, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New York 1988 555-612, WO 96/13590 to Maertens and Stuyver, Zrein et al. (1998) and In WO 96/29605. Methods of obtaining mAb 4C5 and mAb 19A10 and their characteristics High affinity IgG mAbs directed against the capsular polysaccharide (CPS) of the highly virulent K1 serotype of the present invention target not only the strain used for immunization, but also target To other unrelated strains K1- Kp . Covalent coupling of CPS to the immunogenic protein, Bacillus anthracis protective antigen (PA), was used to enhance the isolation of IgG mAbs as previously described for other polysaccharides [33-36]. This coupling drives an immunogenic response against the thymus-dependent pathway, which allows the isolation of memory IgG mAbs [37], as polysaccharide antigens cannot be processed and presented by the major histocompatibility complex. Capsular polysaccharide (CPS) obtained from the capsule of Klebsiella pneumoniae K1 serotype was coupled to PA protein to immunize mice to enhance immune response.The titers of antibodies specific to K1 polysaccharide were measured every two weeks. When the antibody titer is high enough, fusion with myeloma cells is performed to obtain specific hybridomas. Hybridomas with high specific signal for K1 serotype are selected and individual hybridomas are purified. Two mAbs, 4C5 and 4C5, respectively, are obtained. Highly specific hybridoma of 19A10, IgG1 and IgG3 isotypes. Both mAbs specifically recognize the K1 serotype. Both mAbs have high binding affinity to K1 polysaccharide in the subnanomolar range. Both mAbs can Simultaneously binds to K1 capsular polysaccharide and a sandwich ELISA was developed to detect K1 CPS in urine and serum of mice infected with K1 serotype intraperitoneally, intravenously and intratracheally. Both mAbs reduced the human levels of these bacteria Serum resistance. Both mAbs promote complement deposition and promote bacterial phagocytosis by murine and human macrophages. In animal infection models, both mAbs reduce spleen, liver and lung after intratracheal and intraperitoneal infection The number of bacteria found in the tracheal infection. In the intratracheal infection, the survival of the mice was improved when the two mAbs were used simultaneously. In the intraperitoneal infection, the survival of the mice was improved by the two mAbs independently and when the two mAbs were used simultaneously. A further improvement was observed with the two mAbs. A common protective profile was observed for both mAbs, although they were of different isotypes and also bound different non-overlapping epitopes. Both were able to agglutinate and promote capsular swelling, as described for other mAbs targeting Kp CPS [12] , 25]. Furthermore, both mAbs were able to equally prevent K1- Kp survival in the presence of human serum.[26, 2 7] Serum resistance is an important virulence trait [26, 27]. Capsules evade human immune responses by inhibiting suicidal or phagocytosis-promoting complement binding [28]. Consistent with their protective efficacy, the present data demonstrate that both mAbs promote complement deposition. IgG1 4C5 mAb exhibited enhanced complement deposition and more efficient opsonophagocytosis than mAb 19A10. Consistent with this finding, enhanced protection by mAb 4C5 compared to 19A10 in two murine infection models is also demonstrated herein. Multiphoton microscopy is a powerful technique for studying immediate pathogen-host interactions, especially where intravascular immune responses in the liver are considered to correlate with the ability of the pathogen to disseminate [29-31]. Old studies using labeled Escherichia coli bacteria and recent studies using Mycobacterium bovis and Borrelia burghdorferi document cycling from bacteria from liver and Kupfer cells (KC), respectively Fast removal [32, 33]. Since K1- Kp primarily causes invasive liver abscesses, in vivo microscopy was employed here to study the transport of hv Kp through the liver in the presence and absence of K1-specific mAbs [2]. The hepatic sinusoids form an extensive network of narrow capillaries lined with resident macrophages, called Kupffer cells. It captures and ingests antigens present in pathogens via CDld, resulting in an efficient surveillance and filtering system. Intravenous high-dose injection of hv Kp bacteria demonstrated that co-treatment with mAbs captured bacteria in the liver and progressively enhanced bacterial removal from the circulation over time. In contrast, mock-infected mice were unable to clear hv Kp from the blood, and CFU counts remained high in the blood. When less bacteria were injected, bacteria were rapidly cleared from the liver and blood in mAb-treated mice. Again, and consistent with the enhanced protection in the murine model, faster clearance was observed with mAb4C5 compared to mAb 19A10. For pathogens, phagocytosis by Kupffer cells can also alter the host response by inducing chemokine and cytokinin production and enhancing the recruitment of neutrophils and natural killer cells [34-36]. Notably, intravital microscopy also revealed a role for the initial clearance of K1-hv Kp bacteria by peritoneal macrophages, similar to the initial suppression of Enterococcus faecium or E. coli intraperitoneal infection [37,38] . Bone marrow-derived macrophages from FcγR-deficient mice displayed impaired Ab-mediated phagocytosis of opsonized K1-hv Kp . Interestingly, Kupffer cells have also been shown to eliminate circulating tumor cells by rapid phagocytosis, which is dependent on binding to both high (FcyRI) and low affinity Fc receptors (FcyRIV) [39]. Described herein is some residual phagocytosis in FcyR-deficient macrophages, possibly mediated by complement and also enhanced by Ab treatment. Both murine IgG3 and IgG1 bind to the low affinity receptors FcyRIII and FcyR I [40] and the high affinity nascent FcyR (FcRn) [41, 42]. The latter receptor binds to opsonized bacteria in the acidic environment of endocytic vacuoles [42]. The microscopic data of the present invention demonstrate that hv Kp cannot escape the phagolysosomal compartment in contrast to other Gram-negative bacteria. It is therefore inferred that mAbs facilitate uptake into phagolysosomes, where they kill bacteria. Intracellular TRIM21 signaling is undesirable due to the escape of Ab-pathogen complexes as described in Salmonella [43]. The work of the present invention did not define which isotype would constitute the most favorable, but indicated that genetically engineered mAbs in which FcyR receptor engagement was blocked would not be effective. Bacterial CPS mainly induces IgM and IgG3 antibodies in mice [23] and IgG2 antibodies in humans [44]. Thus, experimental data suggest that IgG3 antibodies are important for bacterial immunity [45-47], and anti-CPS IgG3 exhibits increased affinity for binding to multivalent CPS epitopes via Fc-mediated cooperative binding [48]. Conjugation of PS to protein shifts the reaction to the production of IgG1 antibodies that can also protect [49]. In the context of the present invention, both IgG1 and IgG3 were found to exert protective effects, while 4C5 had higher IgG1 protective efficacy. Different classes of IgG antibodies have different effector responses. Traditionally, IgG2a and 2b play a key role in antiviral immunity and are also potent Ab-mediated autoimmune diseases [50]. Mouse IgG1 and IgG3 exhibit FcγR activation. Binding affinity to an activating receptor-inhibiting FcγR receptor, measured as an activation-to-inhibition ratio, affects its overall activity [50]. IgG1 Abs have superior activation signaling compared to IgG3 Abs [50]. This may explain the observed lower effector process applied by the IgG3 antibody in the context of comparable binding affinity. Past studies have shown that antibody-mediated protection can depend on the IgG subclass, but the results also depend on the specific Ab. For example, two IgG3 mAbs against the meningococcal PorA protein and the B. anthracis capsule were found to be more protective than the other subclasses [47, 51]. These differences are explained by the differential interactions of the CH2-CH3 domains in each subclass that drive their binding affinity. On the other hand, an IgG3 mAb against the capsule of Cryptococcus neoformans was found not to be protective unless it was switched to an IgGl isotype-switched variant [52, 53]. In the latter case, the lack of protection is explained by the contribution of the CH domain to the binding affinity of the antibody to its target polysaccharide. Other investigators compared the relative opsonization activity of antibodies against the cryptococcal polysaccharide capsule and concluded that the anti-capsule activity was greatest for IgG2a, followed by IgG1, and then IgG2b [54]. All of the above underscore the complexity of predicting the contribution of FcR interactions to potency and indicate that this quality is important and must be experimentally evaluated for each anti-infective Ab. Most importantly, clinical data from patients with recurrent hv Kp -mediated liver cysts indicate that the opsonizing and killing potency of serum increases over time, consistent with an evolutionary protective immune response [55]. Passive immunotherapy mimics an effective immune response and high titer formulations of protective Ab can be used alone or in combination with antibiotics to control invasive infections caused by K1- Kp . Importantly, the appropriate infectious hv Kp strain (K1-CPS) needs to be rapidly confirmed prior to treatment. It is shown here that CPS Ag secreted in urine can be diagnosed by ELISA using K1-CPS specific Ab. This sandwich-based ELISA can then be easily further developed into a lateral flow-based assay similar to other commercially available diagnostic Ag tests. It is demonstrated herein that CPS Ag is secreted in both urine and blood. In the re-emergence of well-known pathogenic microorganisms in drug-resistant forms as exemplified by MRSA, multidrug-resistant (MDR) M. tuberculosis and now MDR hv Kp , it seems prudent to include anti-infective Ab pre-development against emerging resistant pathogens. Because even in the best-case scenario, developing Ab therapeutics takes years, it is short-sighted for companies and research foundations to limit more proactive development of mAb therapeutics for infectious diseases. Treatment with anti-effective Abs against gut-colonizing bacteria such as hv Kp should not interfere with targeted interventions in the microbiota. mAbs have been shown to be effective in immunocompromised hosts, as demonstrated by the efficacy of palivizumab in reducing hospitalizations for RSV-related disease in preterm infants [56]. With the continuous rapid advance of globalization, and the unrestricted use of antibiotics in most parts of the world, "hypersensitive" pathogens that are far from inhabiting become undesirably highly resistant pathogens that should be expected to arrive. In the present invention, passive immunotherapy has also been shown to be a successful treatment to control invasive infections caused by K1- Kp . The protective characteristics of both mAbs 4C5 and 19A10 have been demonstrated. EXAMPLES Materials and Methods K1- Kp strains K1- Kp strains (n=4) were collected from hospitalized patients at Montefiore Medical Center (MMC) in Bronx and Stony Brook University Hospital in Stony Brook. Kp strains were grown in Luria-Bertani (LB) broth or agar plates at 37°C. CPS purification and in vitro and in vivo experiments were performed using the K1- Kp strain of MMC No. 20. To obtain stable GFP-tagged strains, the strains were transformed with pPROBEKT plastids kindly provided by Dr. Triplett [22]. CPS purification CPS was isolated as previously described with minimal modifications [23, 24]. Briefly, Kp strain #20 was inoculated in 1 L of LB broth and grown o/n at 37°C. Cells were pelleted by centrifugation and washed with PBS. Cells were resuspended in distilled water to 5% (wt/vol) and CPS was extracted by phenol-water. The CPS present in the aqueous phase was precipitated by adding 5 volumes of methanol plus 1% (v/v) saturated solution of sodium acetate in methanol and incubated at 24°C for 1 h. After dissolving the pellets in water, they were dialyzed against water for 6 hours and then freeze-dried. The lyophilized polysaccharide was dissolved in 0.8% NaCl, 0.05% NaN3, 0.1M Tris-HCl (pH 7) to a concentration of 10 mg/ml and treated with nuclease (50 mg/ml DNase I and RNase A) at 37°C Digest twice and keep for 24h. Subsequently, proteinase K (50 mg/ml) was added and incubated twice at 55°C for 1 h and at room temperature for 24 h. The polysaccharide was precipitated and dissolved in water as described above. The LPS was removed by ultracentrifugation (105000 x g, 16 h, 4°C) and the samples were freeze-dried. CPS was further extracted with phenol following a previously published protocol [25]. CPS was further purified by size exclusion chromatography on a column S200HR (GE Healthcare Life Sciences) using PBS. Fractions were collected and analyzed for the presence of polysaccharides by the phenol-sulfuric acid method [26]. CPS-PA coupling For coupling, the isolated Kp CPS was activated using 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) as described with modifications [27]. PA (protective antigen) of B. anthracis was obtained from Wadsworth Center, NYS Department of Health and was previously used for PS-protein conjugation [28]. Briefly, CPS was dissolved in NaCl 0.15 M to 10 mg/ml (1 ml). At t=0 s, 100 μl CDAP (100 mg/ml in acetonitrile) (1 mg CDAP/mg PS) was added slowly while stirring. After 30 s, 0.2 M TEA (10 µl/mg PS) was added to raise the pH. At t = 2' 30 s, 0.1 M sodium borate pH 9.3 was added to adjust the pH to 9 and 10 mg of PA in 0.15 M NaCl was added. The reaction was incubated o/n at 4°C and dialyzed against water. The PS-protein conjugated product was purified using S200HR gel filtration columns (GE Healthcare Life Sciences). The presence of protein was monitored at A _{280 nm} in the chromatogram, while PS content was monitored by the phenol-sulfuric acid method [26]. mAbs were generated by K1-CPS conjugated with 100 µg of PA in complete Freund's adjuvant (CFA) followed by K1-conjugated with PA in incomplete Freund's adjuvant (IFA). Booster injection of CPS BALB/c mice were immunized to generate MAbs of K1 CPS. Fusion and colonization were performed as described [29]. Agglutination assay Agglutination was performed on glass slides as previously described [21]. Human Serum Resistance Assays Resistance assays were performed as previously described with minimal modifications [18]. 1.4×10 ⁶ cfu of K1- Kp was incubated with 20 μg of 4C5 or 19A10 mAb for 1 h at room temperature. It was then mixed with 3:1 (vol:vol) normal human serum (time 0) and incubated at 37°C for 2h. Aliquots were taken at times 0, 1 and 2 and colony forming units (CFU) were calculated. Survival ratios are plotted as a graph of the number of K1- Kp found compared to time 0. Three independent experiments were performed. C3b- Deposition Assay Complement deposition was measured by flow cytometry with a modification of the previously described protocol [30]. Briefly, LB o/n cultures were diluted and grown to mid-log phase. ⁵ x 107 cells were resuspended in 1 ml PBS + 20% normal human serum + 1% BSA. Add PBS, 20 µg 4C5 or 1910 mAb and incubate for 15'. Subsequently, cells were washed 2 times with PBS + 1% BSA and divided in half. Half were incubated with 1:500 sheep anti-human C3c FITC (Bio-Rad) in PBS + 1% BSA. The other half was incubated in the absence of antibody. The samples were incubated at 4°C for 25 min. Serum-free incubated bacterial samples were used as negative controls to set threshold values and fluorescence intensity. Intensities above 10 were considered positive for C3 binding. Quantification was performed by multiplying the percentage of bacteria that moved into the gate by the mean fluorescence (X-mean) of the defined population to obtain the fluorescence index (FI). Results are represented by the increase in signal treated with mAb relative to PBS incubation. Phagocytosis Assay Bacteria were pH-rodo ^® (Life Technologies) labeled following the manufacturer's instructions. Phagocytosis assays were performed using the J744.16 murine macrophage cell line or the THP-1 human monocyte cell line. THP-1 cells were differentiated with phorbol 12-myristate 12-acetate (PMA) and treated with 100 nM PMA at 2 x 105 ^c /ml for 3 days. One day prior to phagocytosis experiments, both J744.16 and THP-1 cells were seeded at a concentration of ^5x105 c/ml in 35 mm glass bottom microwell dishes (MatTek Corporation). On the day of the experiment, 5×10 ⁶ pHrodo ^® labeled K1- Kp cells were incubated with PBS, 20 μg isotype control, 4C5 or 19A10 mAb for 1 hour. Macrophages were infected with ^pHrodo® -labeled K1- Kp at MOI 10:1. Phagocytosis was assessed immediately using a Zeiss Axiovert 200M inverted microscope with a 10× objective at 5% CO ₂ and 37° C. using petri dishes housed in a closed chamber, with images acquired every 4 min for the duration of the 2 h experiment. Phagocytosis was measured by counting the percentage of macrophages displaying labeled bacteria at 67 and 150 min. For experiments with primary macrophages, bone marrow macrophages were isolated from wild-type or FcR knockout mice ( Fcerlg ^tm1Rav , The Jackson Laboratory) as described [31]. After 7 days of macrophage differentiation, phagocytosis experiments as outlined above were performed. In vivo animal experiments 6-8 week old female BALB/c or Swiss Webster mice were purchased from Taconic Biosciences, Inc. 500 μg PBS, control isotype, 4C5, 19A10 or 4C5 + 19A10 mAb 120 min prior to challenge with 5×10 ⁴ CFU of K1- Kp ip or 24 h prior to challenge with 1×10 ⁴ CFU intratracheal (it) Mice (n = 10) were treated intraperitoneally (ip). The survival of 6 mice was monitored for 15 days. The liver, spleen and lung of 4 mice were processed to enumerate bacteria in the homogenized tissue. All animal experiments were performed with the approval of the Animal Institute Committee (AIC) in accordance with the rules and regulations described by the Albert Einstein College of Medicine and Stony Brook University. Intravital microscopy (IVM) Liver in vivo protocols were performed as previously described [32]. Briefly, mice were anesthetized by ip injection of a mixture of ketamine (120 mg/kg) and xylazine (10 mg/Kg). The tail vein is cannulated for administration of additional anesthetics and experimental reagents (if needed). A midline laparotomy was performed, followed by removal of the skin and abdominal muscles along the costal margin to the midaxillary line to expose the liver. The mouse was placed in the right position and a single liver lobe was removed from the abdomen on a custom made table. Wet exposed tissue with saline soaked gauze to prevent dehydration during imaging. Body temperature was maintained with an infrared heat lamp, and the liver was continuously perfused with saline buffer. Intravital microscopy was performed by the Inspector Z1 system (Zeiss), with temporal video taken at 1 frame/sec. The video was further analyzed by Zen2012 software (Zeiss). Statistical Analysis Data are presented as mean ± standard deviation or median range. Survival data were analyzed using the log-rank test. Statistical tests were performed using GraphPad Prism 6 for Mac. Results CPS-PA conjugation and mAb production CPS of strain No. 20, K1 serotype Kp was purified as previously reported [12]. Considering that CPS is a T cell-independent antigen with limited immunogenicity, the probability of restoring IgG production is low. Thus, CPS is conjugated to the protective antigen (PA), a B. anthracis protein that has been used successfully in other polysaccharide conjugates [62]. Coupling was carried out by CDAP-mediated activation of the polysaccharide [61]. Chromatograms of unconjugated CPS+PA and conjugated CPS-PA are shown in FIG. 1 . As expected, conjugation increased the protein content of the subsequently pooled CPS fractions. Immunization with conjugated K1-PA CPS produced 6 different hybridomas, each producing IgG specific for K1-CPS (Table 1). Two of them were selected for further development because they are higher mAb producers and display different isotype classes (4C5-IgG1- and 19A10-IgG3-). The binding properties by ELISA to purified K1-CPS were found to be in the subnaimole concentration range. The Kd for 4C5 was calculated to be 0.3642 +/- 0.07244 nM, while the Kd for 19A10 was 0.2419 +/- 0.3210 nM, confirming the high binding capacity of both mAbs to K1-CPS. ELISA analysis using isotype-specific secondary Abs indicated that both Abs could bind to purified CPS simultaneously. Some inhibition was detected only at higher concentrations (data not shown). In Vitro Antibody-Mediated Activity First, the effect of mAbs on bacterial growth was examined. For this purpose, the survival of K1- Kp grown in the presence of 4C5 or 19A10 mAbs was analyzed. Both mAbs induced a significant reduction in bacterial viability after 2 h of co-incubation (Figures 2A and 2B). Furthermore, both mAbs induced agglutination and resulted in capsule swelling when co-incubated with K1- Kp (Fig. 2C). Agglutination also occurred with the GFP-expressing strain K1-pPROBEKT and three other clinical K1- Kp strains not used for immunization (data not shown). Agglutination of non-K1-hv Kp strains not expressing K1-CPS was not observed. Co-incubation with mAbs reduced K1- Kp virulence in the invertebrate animal model G. mellonella (p values 0.0172 and 0.032, respectively) (Figure 2F). In vitro growth experiments showed that the heritability of K1-hv Kp to be easily replicated in serum was reduced 30-fold by both mAbs (2h p-value = 0.003) (Fig. 2D). Additionally, C3-complement deposition on the capsule in the presence of mAb was quantified and compared to Kp bacteria in the presence of PBS and normal human serum (NHS), as described in Materials and Methods. These experiments found that 4C5 incubation increased C3b deposition by 1.44x and 19A10 by 1.17x (Fig. 2E) (p value < 0.05). When evaluating membrane attack complex (MAC) deposition in K1- Kp bacteria, significant deposition was observed following 19A10 binding compared to PBS or IgG controls (Fig. 2G) (2-factor ANOVA p-value < 0.0001, PBS vs 4C5 ns, PBS vs. 19A10 p-value < 0.0001 and PBS vs. positive control aggregated human IgG p-value < 0.0001). The 4C5 mAb promotes phagocytosis of K1- Kp in specialized phagocytes Next, the ability of 4C5 to promote the uptake of K1- Kp cells by the murine macrophage cell line J744.16 was investigated, labeling Kp bacteria with pHrodo ^® (LifeTechnologies), which allows appropriate True phagocytosis events were detected as opposed to only attached Kp bacteria, since rhodamine-fluorescence is dependent on the acidic pH of phagolysosomes. The phagocytosis index is determined by the number of macrophages phagocytosing bacteria at a specific time point. Phagocytosis (0% phagocytosed macrophages) was not recorded when bacteria were incubated with PBS (Fig. 3A-B and Supplementary Video 1). In contrast, when K1- Kp was co-incubated with 4C5 mAb, 13.85 and 27.65% of macrophages exhibited phagocytosis of bacteria at 67 and 150 min after infection (p-value = 0.0004 and 0.005, respectively). Similar results were found for the 19A10 mAb (66.7 and 150 min post-infection, 27.45% and 38.05%, respectively) (p-value = 0.0002 and 0.003, respectively). The opsonophagocytic properties of the mAbs were also documented in human macrophage cell lines. Both mAbs promoted phagocytosis, 4C5 (Fig. 3D-C and Supplementary Video 2) (67min 22% vs. 0% p-value=0.008; 150min 30% vs. 0% p-value=0.003), and 19A10 mAb (6.83% and 20.04% of macrophages were phagocytosed at 67 min and 150 min, p-value = 0.0005 and 0.042, respectively). At 8 h after initial phagocytosis was observed, no bacterial escape from the phagolysosome was observed (data not shown). It was also observed that bone marrow-derived macrophages from FcγR-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), thus Exhibiting opsonophagocytic effects depends on Fc receptor engagement. Regarding killing, J744.16 murine macrophages were found to kill K1- Kp when opsonized with 4C5 or 19A10 (Fig. 3D) (ANOVA p-value < 0.0001, p-value < 0.01 for 4C5 and 19A10 treatments). Phagocytosis was not observed when bacteria were incubated with PBS or IgG controls. Neutrophils not only kill pathogens by phagocytosis, but also release a network that captures and kills various microorganisms, called neutrophil extracellular traps (NETs). This process is called NETosis and is involved in the removal of many infectious pathogens, including fungi, parasites and bacteria. K1- Kp alone did not induce release of NETs, whereas (Fig. 3E-F) hyphae of C. albicans did, as previously reported. However, K1- Kp co-incubated with 4C5 or 19A10 resulted in the release of NETs (ANOVA p-value < 0.0001, p-value < 0.0001 for 4C5 and 19A10 treatments). NETosis was only observed with the K1- Kp specific mAb and was absent in the control experiments with the IgG control Ab. 4C5 and 19A10 elicited a protective response in murine K1- Kp infection, which was enhanced when the two mAbs were combined. Next, it was tested whether opsonophagocytic in vitro activity correlates with in vivo protective efficacy in two different murine infection models using K1- Kp strain number 20. For these experiments, Swiss-Webster mice were pretreated with PBS, 500 μg IgG isotype control or 500 mg 4C5 mAb or 500 mg 19A10 mAb 2 h prior to intraperitoneal inoculation with 5×10 ⁴ K1- Kp CFU. Treatment with 4C5 enhanced survival when compared to PBS- or control isotype-treated mice (p-value=0.0027) (Figure 4A). Treatment with 4C5 significantly reduced bacterial load (CFU) in liver as well as lung and spleen (p-value < 0.001 in all cases) when compared to PBS or isotype control treatment (Figure 4B). Similar protective trends were observed for mice treated with 19A10 mAb (p value = 0.0170) and treatment with 19A10 also significantly reduced bacterial organ burden (p value < 0.001 in all cases). Treatment with the combination of mAbs enhanced protective efficacy (p-value=0.014) and also significantly reduced bacterial load (p-value<0.001). Next, protection was tested in an endotracheal infection model. Mice were treated with PBS, isotype controls and mAbs 24 h prior to intratracheal inoculation of 1×10 ⁴ K1- Kp CFU. Retreatment with 4C5 significantly enhanced the survival of mice compared to PBS- or isotype control treatment (Figures 4C and 4D) (p-value=0.0027). Protective efficacy and lower bacterial organ burden were also observed after treatment with mAb19A10 (p-value=0.0051). Combination therapy also further enhanced survival and bacterial clearance in this model (p-value=0.0009) (Figures 4C and 4D). Finally, the induction of cytokines and chemokines in tissues of mAb- and PBS-treated mice ip infected with K1- Kp strain was compared. The liver and spleen homogenates of mAb-treated mice had significantly lower levels of pro-inflammatory cytokines such as IFN-γ, IL1-β, IL-2, IL-6 and TNF compared to PBS-treated mice -α (2-factor ANOVA treatment p-value < 0.0001 for both liver and spleen, Figures 4E-F). Similar trends were observed in the lungs (Figure 7A-C). Antibodies promote capture of K1- Kp from K1-Kp cells in the liver To decipher the mechanisms that promote the clearance of K1- Kp from organs, in vivo microscopy analysis was performed. When ¹⁰⁸ CFU was injected iv without treatment, the number of bacteria flowing through the blood vessels was significantly higher and longer (p < 0.05) than with either mAb (Fig. 6A). At the same time, the number of bacteria captured in liver tissue was significantly higher when both antibodies were used (Figure 6B). At ⁵ x 104 CFU injected ip, no bacteria were detected in blood vessels or captured in tissues with either mAb (Figure 6C and D). Furthermore, high amounts of hv- Kp were found in the peritoneal cavity without treatment, whereas hv- Kp was cleared upon mAb injection (data not shown). Reducing the size of the iv injected bacterial inoculum to ⁵ x 104 resulted in complete bacterial clearance (Fig. 5F). Treatment with mAb reduced bacterial dispersal in antibiotic-treated K1- Kp colonized mice. The protective efficacy of mAbs was examined in a murine model of gastrointestinal colonization in which bacterial dissemination to the liver, spleen and mesenteric lymph nodes (MLN) was promoted by antibiotic treatment. Oral administration of ampicillin to mice colonized with ¹⁰⁸ GFP-tagged kv Kp resulted in a marked increase in bacterial load in the feces and subsequent translocation and dissemination to mesenteric lymph nodes (MLN), spleen and liver. It was investigated whether mAb treatment protects against disseminated and reduced bacterial load in MLN, spleen and liver. The data indicated that bacterial dispersal to MLN, liver and spleen was significantly reduced compared to PBS-treated mice, and bacterial loads in these organs were below 6 log (Figure 8) (2-factor ANOVA p-value<0.0001) . In contrast, bacterial colonization observed in feces showed no significant difference from mAb treatment, indicating that colonization itself was not affected by systemic mAb treatment. Both mAbs provided tools for the detection of K1 -infection in both urine and serum samples . Recently, a sandwich ELISA was designed using the mAbs to enable rapid non-invasive detection of K1- Kp CPS in murine body fluids (Figure 6A). ELISA testing with both mAbs demonstrated detection sensitivity of CPS as low as 0.20 μg/ml in serum and as low as 0.11 μg/ml in urine (Figures 6B and 6D). CPS was detected in serum samples from iv-infected mice, and in urine samples from ip- and it-infected mice. Amounts of CPS as low as 0.11 μg/ml were found in the urine of different mice over time and corresponded to the severity of the disease ( FIG. 6D ). Importantly, the ELISA was specific and did not detect CPS of other non-K1-CPS expressing Kp strains. Treatment of the mAb reduced ampicillin-induced dispersal from the gut in K1- Kp colonized mice. The protective efficacy of mAbs was examined in a murine model of gastrointestinal colonization in which bacterial dissemination to the liver, spleen and mesenteric lymph nodes (MLN) was promoted by treatment with antibiotics. Administration of ampicillin to mice colonized with ¹⁰ GFP-tagged K1- Kp via oral ingestion resulted in increased bacterial load in feces and subsequent translocation and dissemination to mesenteric lymph nodes (MLN), spleen and liver ( 9A-B and 8). It was investigated whether mAb treatment protected against MLN, dissemination in spleen and liver and reduced bacterial load (Figure 9B). The data indicated that bacterial dispersal to MLN, liver and spleen was significantly reduced and bacterial burden was up to 6 log lower in these organs compared to PBS-treated mice (Figure 8) (2-factor ANOVA p-value<0.0001). In contrast, bacterial colonization observed in feces showed no significant difference from mAb treatment, indicating that colonization itself was not affected by systemic mAb treatment. Sequencing of Monoclonal Antibodies Total RNA was extracted from frozen hybridoma cells and cDNA was synthesized from RNA. PCR was then performed to amplify the variable regions (heavy and light chains) of the antibody, which were then individually cloned into standard clone vectors and sequenced. Materials Hybridoma cells; TRIzol® reagent (Ambion, catalog number: 15596-026); PrimeScript™ 1st strand cDNA synthesis kit (Takara, catalog number: 6110A). Methods Total RNA extraction Total RNA was isolated from hybridoma cells following the technical manual for TRIzol® reagents. Total RNA was analyzed by agarose gel electrophoresis. RT-PCR followed the technical manual of the PrimeScript™ 1st Strand cDNA Synthesis Kit using isotype-specific antisense primers or universal primers to reverse-transcribe total RNA into cDNA. Antibody fragments of VH and VL were amplified according to the standard operating procedure of GenScript's RACE. Cloning of Antibody Genes Amplified antibody fragments are individually cloned into standard cloning vectors using standard molecular cloning procedures. Screening and Sequencing Colony PCR screening was performed to identify clones with inserts of appropriate size. A single population of no less than 5 inserts of the appropriate size was sequenced for each antibody fragment. Total RNA extraction The isolated total RNA of the samples was run on a 1.5% agarose/GelRed ^™ gel with DNA marker Marker III (TIANGEN, catalog number: MD 103). PCR product of antibody gene 4 microliters of PCR product from each sample was run on a 1.5% agarose/GelRed™ gel with DNA marker Marker III. PCR products were purified and stored at -20 ^° C until further use. Sequencing Results and Analysis Five single colonies with appropriate VH and VL insert sizes were sent for sequencing. The VH and VL genes of the two different clones were found to be almost identical.下文列舉之共有序列係由雜交瘤4C5產生之抗體之序列重鏈 ： DNA 序列 (405 bp)前導序列- FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 ATGGACTGGAGTTGGGTCTTTCTCTTCCTCCTGTCAGTAAATGAAGGTGTCTACTGT CAGGTCCAGCTGCAGCAGTCTGGAGATGATCTGGTAACGCCTGGGGCCTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACC AGCTACTGGATTAAC TGGATAAAACAGAGGCCTGGACAGGGCCTTGAGTGGGTAGGA CGTATTACTCCTGGACGTGGTAATACTTACTACAATGAAATGTTCAAGGAC AAGGCAACACTGACTGTAGACACATCCTCCAGAACAGCCTACATTCAGCTCAGCAGCCTGTCATCTGAGGACTCTGCTGTCTATTTCTGTGCAAGG GGGGGTGTCTGGTTTGCTTAC TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 1) The nucleotide sequences of CDR1, CDR2 and CDR3 of the heavy chain of mAb 4C5 are the following SEQ ID NOs: 9, 10 and 11, respectively: AGCTACTGGATTAAC (SEQ ID NO: 9) CGTATTACTCCTGGACGTGGTAATACTTACTACAATGAAATGTTCAAGGAC (SEQ ID NO: 10) GGGGGTGTCTGGTTTGCTTAC (SEQ ID NO: 11) 重鏈：胺基酸序列(135 AA) 前導序列- FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 MDWSWVFLFLLSVNEGVYC QVQLQQSGDDLVTPGASVKLSCKASGYTFT SYWIN WIKQRPGQGLEWVG RITPGRGNTYYNEMFKD KATLTVDTSSRTAYIQLSSLSSEDSAVYFCAR GGVWFAY WGQGTLVTVSA ( SEQ ID NO: 2) mAb The amino acid sequences of CDR1, CDR2 and CDR3 of the 4C5 heavy chain are the following SEQ ID NOs: 12, 13 and 14, respectively: SYWIN (SE Q ID NO: 12) RITPGRGNTYYNEMFKD (SEQ ID NO: 13) GGVWFAY (SEQ ID NO: 14)輕鏈 ： DNA 序列 (393 bp)前導序列- FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 ATGGGCATCAAGATGGAGTCACAGATTCAGGCATTTGTATTCGTGTTTCTCTGGTTGTCTGGTGTTGACGGA GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGC AAGGCCAGTCAGGATGTGAGTACTGCTGTAGCC TGGTATCAGCAAAAACCAGGGCAATCTCCTAAACTACTGATTTAC TGGGCATCCACCCGGCACACT GGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTATACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCACTTTATTACTGT CAGCAACATTATAGCACTCCGTACACG TTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 3) mAb 4C5輕鏈之CDR1、CDR2及CDR3之核苷酸序列分別係如下SEQ ID NO: 15、16及17： AAGGCCAGTCAGGATGTGAGTACTGCTGTAGCC (SEQ ID NO: 15) TGGGCATCCACCCGGCACACT ( SEQ ID NO: 16) CAGCAACATTATAGCACTCCGTACACG (SEQ ID NO: 17)輕鏈 ：胺基酸序列 (131 AA)前導序列- FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 MGIKMESQIQAFVFVFLWLSGVDG DIVMTQSHKFMSTSVGDRVSITC KASQDVSTAVA WYQQKPGQSPKLLIY WASTRHT GVPDRFTGSGSGTDYTLTISSVQAEDLALYYC QQHYSTPYT FGGGTKLEIK ( SEQ ID NO: 4) The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain of mAb 4C5 are respectively the following SEQ IDs NO: 18, 19 and 20: KASQDVSTAVA (SEQ ID NO: 18) WASTRHT (SEQ ID NO: 19) QQHYSTPYT (SEQ ID NO: 20) The consensus sequence listed below is the sequence heavy chain of the antibody produced by hybridoma 19A10 : DNA 序列 (399 bp)前導序列- FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 ATGGCTGTCCTGGTGCTGTTCCTCTGCCTGGTTGCATTTCCAAGCTGTGTCCTGTCC CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTAACC AGCTATGGTGTACAC TGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGA GTAATATGGGCTGGTGGAAGCACAAATTATAATTCGGCTCTCATGTCC AGACTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCTTA CTATGGCTAAGAGCTTAC TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 5) mAb 19A10重鏈之CDR1、CDR2及CDR3之核苷The acid sequences are the following SEQ ID NOs: 21, 22 and 23, respectively: AGCTATGGTGTACAC (SEQ ID NO: 21) GTAATATGGGCTGGTGGAAGCACAAATTATAATTCGGCTCTCATGTCC (SEQ ID NO: 22) CTATGGCTAAGAGCTTAC (SEQ ID NO: 23) Heavy chain : amino acid sequence (133 AA) Preamble - FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 MAVLVLFLCLVAFPSCVLS QVQLKESGPGLVAPSQSLSITCTVSGFSLT SYGVH WVRQPPGKGLEWLG VIWAGGSTNYNSALMS RLSISKDNSKSQVFLKMNSLQTDDTAMYYCAL LWLRAY WGQGTLVTVS A (SEQ ID NO: 6) The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of mAb 19A10 are the following SEQ ID NOs: 24, 25 and 26, respectively: SYGVH (SEQ ID NO: 24) VIWAGGSTNYNSALMS (SEQ ID NO: 25) LWLRAY (SEQ ID NO: 26)輕鏈 ： DNA 序列 (393 bp)前導序列- FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGT GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGC AGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAA TGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTAC AAAGTTTCCAACCGATTTTCT GGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGC TTTCAAGGTTCACATGTTCCGTGGACG TTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 7) The nucleotide sequences of CDR1, CDR2 and CDR3 of the light chain of mAb 19A10 are the following SEQ ID NOs: 27, 28 and 29, respectively: AGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAA (SEQ ID NO: 27) AAAGTTTCCAACCGATTTTCT (SEQ ID NO: 28) TTTCAAGGTTCACATGTTCCGTGGACG (SEQ ID NO: 29) Light chain : amino acid sequence (131 AA) leader sequence - FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 MKLPVRLLVLMFWIPASSS DVLMTQTPLSLPVSLGDQASISC RSSQSIVHSNGNTYLE WYLQKPGQSPKLLIY KVSNRFS GVPDRFSGSGSG The amino acid sequences of CDR1, CDR2 and CDR3 of TDFTLKISRVEAEDLGVYYC FQGSHVPWT FGGGTKLEIK (SEQ ID NO: 8) mAb 19A10 light chain are the following SEQ ID NOs: 30, 31 and 32, respectively: RSSQSIVHSNGNTYLE (SEQ ID NO: 30) KVSNRFS (SEQ ID NO: 30) NO: 31) FQGSHVPWT (SEQ ID NO: 32) Bibliography 1. 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Figure 1: Chromatogram of S200HR gel filtration chromatography recorded at A280nm. 1A shows the chromatogram of K1-CPS after coupling with PA protein; 1B shows the chromatogram before coupling. The lower line indicates the fraction containing K1-CPS detected by the phenol-sulfuric acid method [26].

Figure 2: In vitro characterization of mAb effects. A, B) Survival of K1 hv- Kp in the presence of 4C5 or 19A10, respectively. Growth was recorded by CFU/ml at different time points. Three independent experiments were monitored. C) Agglutination analysis of K1 hv- Kp in the presence or absence of mAb. 10 μg mAb was added to cells and incubated for 1 h. The lower panel shows agglutination transformed by the GFP expressing plastid pPROBEKT-Km strain. D) Human serum resistance assay. Growth of the CR- Kp strain in the presence of normal human serum. The content of live CFU of K1 hv- Kp was evaluated after incubation with NHS for 2 hours. The content of viable cells at time 0' is shown relative to the number of CFUs. E) Complement deposition analysis. Deposition was measured by the fluorescent signal of anti-human C3c FITC. Three independent experiments were obtained and show % signal gain relative to PBS-incubated mAb treatment. F) Shows that preincubation of K1- Kp with 25 μg 4C5 or 19A10 prolongs the survival of infected G. mellonella. Significance was determined by the log-rank (Mantel-Cox) test adjusted for Bonferroni's multiple comparisons test (n=20 worms/group). G) MAC deposition was enhanced by 19A10Ab treatment. In 3 independent experiments, the anti-human C5b-9 signal was measured by ELISA in K1- Kp bacteria incubated with NHS or HI-NHS and mAB, aggregated human IgG or PBS. The mean % increase in AP signal relative to each PBS control is shown as standard deviation. p-values were calculated by ANOVA with post hoc Tukey's multiple comparison test. H) Growth of K1- Kp in LB medium in the presence of IgG control, 4C5 or 19A10. Experiments were performed in quadruplicate. Bars represent calculated mean ± standard deviation. p-values were determined by 2-way ANOVA with post hoc Tukey's multiple comparison test.

Figure 3: Phagocytosis assay. A) Phagocytosis of K1 hv- Kp murine macrophages J744 in the presence of PBS (blue), IgG control (red), 4C5 (green) or 19A10 (purple). B) Phagocytosis of THP-1 differentiated human macrophages of K1 hv- Kp in the presence of PBS (blue), IgG control (red), 4C5 (green) or 19A10 (purple). C) Bone marrow-derived macrophages by wild-type (solid) or FcR-KO (tetragonal) phagocytosis of K1 hv- Kp in the presence of PBS (blue), IgG control (red), 4C5 (green) or 19A10 (purple). phagocytes. In all experiments, the % of macrophages showing pH-rodo labeled bacteria inside after 67 min and 150 min after initial co-incubation. Three independent experiments were performed. (D) Murine macrophage J744.16 killing of K1- Kp is shown. p-values were determined by one-way ANOVA with Sidak's multiple comparison test. (E) Quantification of NET release from human neutrophils following stimulation with C. albicans, K1- Kp or K1- Kp incubated with mAbs 4C5, 19A10 and IgG controls (shown in F ). The bars reflect the average percentage of neutrophil nuclei with an area greater than 1,000 μm2 of the total neutrophils in the field (3 independent replicates) with standard deviation. p-values were determined by one-way ANOVA with Sidak's multiple comparison test. (F) Enhanced NET release from human neutrophils was induced by K1- Kp bacteria co-cultured with 4C5 and 19A10 but not by the isotype-matched control Ab.

Figure 4. A) and B) mouse survival and lung, liver and spleen bacterial counts after ip injection of 5 x 104 K1 ^- Kp CFU, respectively. C) and D) Survival and lung, liver and spleen bacterial counts after it injection of 1×10 ⁴ K1- Kp CFU, respectively. Mice were treated with PBS, IgG control, 4C5, 19A10 or 4C5 and 19A10. Organ bacterial load is expressed as CFU/ml. * indicates p-value < 0.05, **p-value < 0.01 and ***p-value < 0.001 and ***p-value < 0.0001. A comparison of mean interleukin content in liver (E) and spleen (F) (24 h after ip infection with 5 x 104 K1 ^- Kp ) with standard deviation is shown (n=4 mice/group). Black* indicates comparison of PBS with 4C5 and blue* indicates comparison with 19A10. A 2-way ANOVA with Dunnett's multiple comparisons test was performed for p-value determination.

Figure 5. Intravital microscopy. A) Number of bacteria passing through immobilization points in sinusoids (between 5 and 8 μm in diameter) at different time points after injection. B) Number of immobilized bacteria captured in the field of view at different time points after injection. For each group in A) and B), mice with/without mAb treatment received iv injections of ¹ x 108 GFP expressing K1 hv- Kp with 1 hour IVM video based on the above protocol. C) Number of bacteria passing through the fixed point in sinusoids (between 5 and 8 μm in diameter) 24 hours after injection. D) Number of immobilized bacteria captured in the field of view 24 hours after injection. For each group in C) and D), mice with/without mAb treatment received ⁵ x 104 ip injections of GFP-expressing hv- Kp for 1 hour IVM video based on the protocol described above. E) 200x microscopic images of the intraperitoneal cavity of untreated mice following ip injection of ^5x104 hv- Kp expressing GFP. F) Number of bacteria passing through the fixed point in sinusoids (between 5 and 8 μm in diameter) 24 hours after iv injection of 1×10 ⁴ hv- Kp expressing GFP. Mice with or without mAb treatment received ip ¹ x 104 CFU of hv- Kp expressing GFP. ** denotes p-value &lt; 0.01. Figure 6. K1-CPS detection method in infected mice. A) Protocol for the designed sandwich ELISA. ELISA plates were coated with 4C5 mAb and subsequently incubated with samples with or without K1-CPS. Subsequently, the 19A10 mAb was bound and detected via an anti-mouse IgG3-conjugated mAb. B) K1-CPS detection in mice injected iv with 1×10 ⁴ CFU K1 hv- Kp in serum obtained at different time points. C) K1-CPS detection in mice injected ip with 5×10 ⁴ CFU K1 hv- Kp in urine obtained at different time points. D) K1-CPS detection in mice it injected with 1×10 ⁴ CFU K1- Kp in urine obtained at different time points. Figure 7. In vivo protection study . (A) Survival analysis of mice (n=6/group) injected ip with 5×10 ⁴ K1- Kp and treated with PBS, 250 μg 4C5, 19A10 or 125 μg 4C5 and 19A10, respectively . p-values were determined using log-rank (Mantel-Cox) corrected for Bonferroni's multiple comparisons test. (B) Mean bacterial organ load 24 h after infection. Mean log CFU/ml + standard deviation is shown (n=4 mice/group). Black * indicates the comparison of PBS and two mAbs. A 2-factor ANOVA with Tukey's multiple comparison test was performed in (B) for p-value determination. (C) Shows a comparison of mean interleukin content in lungs (24 h after ip infection with 5 x 104 K1 ^- Kp ) with standard deviation (n=4 mice/group). Black* indicates comparison of PBS with 4C5 and blue* indicates comparison with 19A10. A 2-way ANOVA with Dunnett's multiple comparisons test was performed for p-value determination. Figure 8. Bacterial colonization and dispersal protection studies . Mean bacterial loads in organs and feces after dissemination on day ⁸ after colonization with 1 x 108 K1- Kp . Mean log CFU/ml + standard deviation is shown (n=3 mice/group). A 2-factor ANOVA with Dunnet's multiple comparison test was performed for p-value determination. Black* indicates PBS comparison. Figure 9. (A) Bacterial CFU counts in feces of colonized mice increased by more than 5 log following ampicillin treatment. (B) Time of ampicillin and mAb treatment in colonized mice. Supplementary Videos 1-6 . The three video clips of iv injections were all played at 5 fps at 30 min post infection, showing treatment with PBS, 4C5 or 19A10. Video clips on ip infection were shown at 5 fps 24 h after infection showing treatment with PBS, 4C5 or 19A10.

<110> THE RESEARCH FOUNDATION FOR THE STATE UNIVERSITY OF NEW YORK

<120> Klebsiella pneumoniae (KLEBSIELLA PNEUMONIAE) antibody and method for treating Klebsiella pneumoniae infection

<130> 178-437P

<140>

<141> 2015-10-21

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 405

<212> DNA

<213> Artificial sequences

<220>

<223> Synthetic sequences

<400> 1

<210> 2

<211> 135

<212> PRT

<213> Artificial sequences

<220>

<223> Synthetic sequences

<400> 2

Claims (19)

An isolated polynucleotide encoding the light chain variable region (LCVR) of an antibody or active fragment thereof, the LCVR comprising at least 95% identity to the sequence set forth in SEQ ID NO:4 and identical to SEQ ID NO:4 An amino acid sequence that shares at least one major functional property, wherein the sequence includes light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 18-20. An isolated polynucleotide encoding the heavy chain variable region (HCVR) of an antibody or active fragment thereof, the HCVR comprising at least 95% identity to the sequence set forth in SEQ ID NO:2 and identical to SEQ ID NO:2 An amino acid sequence that shares at least one major functional property, wherein the sequence includes heavy chain CDRs comprising the amino acid sequences of SEQ ID NOs: 12-14. 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. 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. An isolated polynucleotide encoding the light chain variable region (LCVR) of an antibody or an active fragment thereof, the LCVR comprising at least 95% identity to the sequence set forth in SEQ ID NO:8 and identical to SEQ ID NO:8 An amino acid sequence that shares at least one major functional property, wherein the sequence includes light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 30-32. An isolated polynucleotide encoding the heavy chain variable region (HCVR) of an antibody or active fragment thereof, the HCVR comprising at least 95% identity to the sequence set forth in SEQ ID NO:6 and identical to SEQ ID NO:6 An amino acid sequence that shares at least one major functional property, wherein the sequence includes heavy chain CDRs comprising the amino acid sequences of SEQ ID NOs: 24-26. 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. 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. An isolated polypeptide comprising a light chain variable region (LCVR) of an antibody or an active fragment thereof, the LCVR comprising at least 95% identical to the sequence set forth in SEQ ID NO:4 and sharing at least 4 with SEQ ID NO:4 An amino acid sequence of primary functional properties, wherein the sequence includes light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 18-20. An isolated polypeptide comprising a heavy chain variable region (HCVR) of an antibody or an active fragment thereof, the HCVR comprising at least 95% identical to the sequence set forth in SEQ ID NO:2 and sharing at least 2 with SEQ ID NO:2 An amino acid sequence of primary functional properties, wherein the sequence includes heavy chain CDRs comprising the amino acid sequences of SEQ ID NOs: 12-14. 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. 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. An isolated polypeptide comprising a light chain variable region (LCVR) of an antibody or an active fragment thereof, the LCVR comprising at least 95% identical to the sequence set forth in SEQ ID NO:8 and sharing at least 8 with SEQ ID NO:8 An amino acid sequence of primary functional properties, wherein the sequence includes light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 30-32. An isolated polypeptide comprising a heavy chain variable region (HCVR) of an antibody or an active fragment thereof, the HCVR comprising at least 95% identical to the sequence set forth in SEQ ID NO:6 and sharing at least 6 with SEQ ID NO:6 An amino acid sequence of primary functional properties, wherein the sequence includes heavy chain CDRs comprising the amino acid sequences of SEQ ID NOs: 24-26. 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. 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. Use of at least one polypeptide according to any one of claims 9, 11, 13 and 15 and at least one polypeptide according to any one of claims 10, 12, 14 and 16 for the preparation of inhibiting an individual in need thereof Drugs for Klebsiella pneumoniae ( K.pneumoniae ) infection. The use of claim 17, wherein the Klebsiella pneumoniae infection is a hypervirulent Klebsiella pneumoniae infection of serotype K1. A method of diagnosing Klebsiella pneumoniae infection in an individual, the method comprising combining a biological sample of the individual with at least one polypeptide as claimed in any one of claims 9, 11, 13 and 15 and at least one as claimed in claim 10 contacting the polypeptide of any one of , 12, 14, and 16; determining whether an immune complex is formed; and comparing the amount of the complex formed to a normal control value.

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