A composition for treating klebsiella infections and methods thereof

Abstract

The present disclosure provides compositions and formulations comprising a mixture of antibacterial proteins, such as bacteriocins for the treatment of Klebsiella infections. The composition and formulations, as disclosed, comprises a mixture of klebicins (proteins or variants thereof) with broader target cell ranges in order to effectively treat Klebsiella infections. The present disclosure provides kits and methods for treating Klebsiella infections.

Description

A COMPOSITION FOR TREATING KLEBSIELLA INFECTIONS AND METHODS THEREOFFIELD OF INVENTION

[0001] The present disclosure broadly relates to the field of bacterial infections and particularly discloses compositions and formulations comprising a mixture of antibacterial proteins, such as bacteriocins for the treatment of Klebsiella infections. The present disclosure also relates to a method of treating Klebsiella infections using said compositions.BACKGROUND OF THE INVENTION

[0002] Bacteriocins are protein antibacterials produced by bacteria to kill similar or closely related species, as a survival mechanism in competing ecological niches. Bacteriocins parasitize on components of bacterial machinery involved in transport of essential nutrients and minerals such as iron and vitamin B12 for cell entry. Bacteriocin-producing bacteria are themselves protected from lethal activity by the presence of immunity proteins that bind to the bacteriocin and sterically prevent its activity. Bacteriocins are highly genus specific and target the same genus as the producer organism. Large bacteriocins (>30kDa) are modular proteins and many of them are natural chimeric proteins, evolved from varied sources having receptor binding, translocation and killing domains, functioning as a single unit to kill susceptible bacteria. Using the receptor binding and translocation domains, bacteriocins enter bacterial cells by parasitizing on cell-surface receptors. Bacteriocins, after binding to their cognate receptors at the cell surface, are transported through the outer membrane of susceptible bacteria by using either the Tol system (Group A) or the Ton system (Group B). After entry into the cell, the killing domain can affect bactericidal action, through multiple modes- membrane damage, or by interfering in metabolic pathways, and DNase or RNase activities.

[0003] Susceptibility to bacteriocins is primarily driven by the presence of cell surface receptor and translocation proteins expressed by susceptible bacteria, and to a lesser extent by the absence of a cognate immunity protein. Hence most bacteriocins exhibit a narrow coverage. Although bacteria can remain sensitive to a given bacteriocin even when the immunity is present, bacteria lacking its cognate receptor will be completely insensitive to the bacteriocin. Bacteriocins utilize only a single cell surface receptor and a single translocation system for cell entry into susceptible bacteria. Any mutation / s in genes involved in either the receptor or translocation machinery will render the bacteria resistant to bacteriocins adding to the restricted coverage. Under laboratory conditions, many bacteriocins studied so far demonstrate an in vitro frequency of resistance (FoR) of 10’6to 10’7.

[0004] Klebsiella pneumoniae is a highly drug-resistant pathogenic bacteria and in humans, it causes serious infections including pneumonia, bacteremia, and urinary tract infections. Due to the high burden of antimicrobial resistance, new interventions are required for treatment of infections caused by these bacteria. Klebicins are large molecular weight bacteriocins produced by Klebsiella spp. and specifically kill other susceptible members of the Klebsiella genus including the pathogenic Klebsiella pneumoniae with possible utility as a therapeutic option. However, as is known for other bacteriocins, klebicins also have properties such as narrow coverage within the Klebsiella and high FoR. Since the clinical utility of klebicins is yet to be explored, pre-existing resistance may be very minimal, and in case of emergence of resistance, it will be only limited to the susceptible bacteria.SUMMARY OF INVENTION

[0005] In an aspect of the present disclosure, there is provided a composition, comprising a mixture of proteins or variants thereof, wherein the proteins have an amino acid sequence of at least 90% sequence identity to SEQ ID NO. 1, SEQ ID NO.2, and SEQ ID NO.3; and a buffer component; wherein the composition has a pH in the range of 4 to 7.

[0006] In another aspect of the present disclosure, there is provided a pharmaceutical formulation comprising the composition as disclosed herein, and a pharmaceutically acceptable carrier.

[0007] In an aspect of the present disclosure, there is provided a method of treating an infection caused due to Klebsiella in a subject, comprising administering the composition as disclosed herein or the pharmaceutical formulation as disclosed herein to the subject.

[0008] In an aspect of the present disclosure, there is provided a kit comprising the composition as disclosed herein or the pharmaceutical formulation as disclosed herein; and an instruction manual.

[0009] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the disclosed subject matter, nor is it intended to be used to limit the scope of the disclosed subject matter.BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[0010] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0011] Figure 1 depicts the domains architecture of proteins: P628 (SEQ ID NO.l), P764 (SEQ ID NO.2) and P801 (SEQ ID NO.3), in accordance with the embodiments herein.DETAILED DESCRIPTION OF THE INVENTION

[0012] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.Definitions

[0013] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0014] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0015] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0016] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0017] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[0018] The term “additive”, as used herein refers to a substance that is added to a composition to improve its stability properties but does not affect the action of the active substances or the therapeutic agent in the composition.

[0019] The term “administering”, as used herein refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Administering of the composition may be through intravenous, intranasal, inhalation, subcutaneous, or intraperitoneal.

[0020] The term “buffer component”, as used herein refers to a solution that can resist pH change upon the addition of an acidic or basic components. The buffer component aids in maintaining the pH of the disclosed composition at a desired range or value.

[0021] The term “drug resistant”, as used herein refers to microorganisms, such as bacteria that no longer respond to a drug that is usually able to kill or weaken them. Further, the terms “mono drug resistant” and “multi-drug-resistant”, as used herein refer to resistance of a microorganism to a single first-line drug, and two or more first- line drugs, respectively.

[0022] The term “frequency of resistance (FoR)”, as used herein refers to frequency at which detectable mutant cells (capable of providing drug-resistance to the bacteria) emerge in a bacterial population in the presence of an antibiotic. The FoR is calculated using the formulae:

[0023] The term ''Klebsiella" , as used herein refers to a bacterium of the family Enterobacteriaceae . The genus Klebsiella consists of six species that can cause infections, namely, K. pneumoniae, K. ozaenae, K. aerogenes, K. quasipneumoniae , K. rhinoscleromatis, and K. oxytoca. K. pneumoniae is one of a few gram-negative rods that can cause a primary pneumonia.

[0024] The term “minimum inhibitory concentration” or “MIC”, as used herein refers to the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation.

[0025] The term “mpk” or “mg per kg”, as used herein refers to a measurement used for dosing medications and substances based on a subject’s weight.

[0026] The term “pharmaceutically acceptable carrier” as used herein refers to a substance that can be used in preparing pharmaceutical formulations. The term pharmaceutically acceptable carrier includes buffer component, stabilizing agents, pH adjusters, surfactants, adjuvants, excipients, or combination thereof.

[0027] The term "sequence identity" as used herein refers to the relationship between two or more amino acid (peptide, polypeptide, or protein) sequences or two or more nucleic acid (nucleotide, polynucleotide) sequences, as determined by comparing the sequences. Various methods are known in the art to determine sequence identity between two sequences. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include for e.g. the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S„ et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S„ et al., J. Mol. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.

[0028] The term “subject”, as used herein, refers to any animal classified as a mammal, e.g., human, and non-human mammals. Examples of non-human animals include nonhuman primates, dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, mice, rats, hamsters, guinea pigs and etc. Unless otherwise noted, the terms “patient” or “subject” are used herein interchangeably. Preferably, the subject is human.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0030] Sequences used in the present disclosure

[0031] Klebsiella pneumoniae is a highly drug-resistant pathogenic bacteria in humans as it causes serious infections including pneumonia, bacteremia, and urinary tract infections. Due to the high burden of antimicrobial resistance, new interventions are required for treatment of infections caused by these bacteria. Klebicins are known to kill other susceptible members of the Klebsiella genus individually. Therefore, the present disclosure provides a composition comprising a mixture of klebicins (proteins or variants thereof) with broader target cell ranges in order to effectively treat Klebsiella infections.

[0032] In an embodiment of the present disclosure, there is provided a composition, comprising a mixture of proteins or variants thereof, wherein the proteins have an amino acid sequence of at least 90% sequence identity to SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO.3; and a buffer component; wherein the composition has a pH in the range of 4 to 7. In another embodiment of the present disclosure, there is provided a composition, comprising a mixture of proteins or variants thereof, wherein the mixture of proteins have an amino acid sequence of at least 90%, at least 92%, at least 94%, at least 98%, sequence identity to SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO.3; and a buffer component; wherein the composition has a pH in the range of 5 to 6. Figure 1 depicts the domains architecture of proteins: P628 (SEQ ID NO.l), P764 (SEQ ID NO.2) and P801 (SEQ ID NO.3), in accordance with the embodiments herein.

[0033] The variants of the proteins as described herein may comprise one or more modifications from the amino acid sequence of a reference sequence (SEQ ID NO.l or SEQ ID NO.2 or SEQ ID NO.3) by way of substitution, deletion and / or addition. For example, the modification may comprise, where appropriate, up to 50, or up to 40, 30,20, 15, 10, 8, 6, 5, 4, 3, 2 or 1 amino acid substitutions, additions and / or deletions from the amino acid sequence of the reference sequence. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue is inserted in its place.

[0034] In an embodiment of the present disclosure, the protein or variant thereof as set forth in SED ID NO. 1 comprises at least one substitution selected from a group consisting of V548I, I352L, V298A, V218M, V241L, K468Q, M97L, A416S, I407T, A21V, A130V, T496A, A126V, D469N, A452V, S441I, Q397H, S394P, F475C, K158N, P46S, R307H, H472Y, P553Q, R313C, P212S, A434E, A109D, K598N, K599R, P463T, V228G, P542L, G64R, G33D, D381Y, L299P, D184G, D319G, Y529D, N26K, T28R, D41Y, G64W, V165M, S180T, S189R, T191R, K192Q, A195V, I197V, T209A, E412A, T419A, E423A, K484N, K513G, V116F, A123S, V178A, S238A, I258V, S286T, L308Q, A346T, S350G, A353T, E426D, S427A, D451E, L427F, T172A, E330D, S436T, K461N, F475H, D477V, E491R, K493D, A194T, I143M, K312N, I345L, S71D, A94T, A249T, K460R, D469E, Y470H, and E435K.

[0035] In an embodiment of the present disclosure, the protein or variant thereof as set forth in SED ID NO. 2 comprises at least one amino acid mutation selected from a group consisting of A339S, I295V, A339T, N238S, E289D, V9I, N205S, K286R, V342I, L137Q, R267Q, A334T, T324A, A90P, G104S, D299N, E176A, S116Y, S349A, M242V, F305L, G114R, S180L, G106V, G91V, D143G, K209R, V319I, G374S, T185A, A274T, D52Y, G352S, G91R, F226L, G37D, T296E, G317S, A318G, M327F, T330A, V331A, S343T, S110N, V292F, A148G, V150I, N155S, H159D, I166V, VI 131, S116A, Y117S, S128N, L137M, D78N, I233V, T325S, S 1281, V252F, L271R, V347G, A318S, T330G, V331L, A341V, L356A, K216R, V333S, A334L, L335V, S336A, L337V, S354A, L373I, K101H, T102A, S103G, G104K, G109N, V112A, G114N, S116L, Y117N, S118D, S119R, V122K, D123Q, T124Y, Y125F, S126Q, L137I, N139D, R140S, P157K, H159Q, S160L, K162D, I166L, S177R, A179S, S180A, I182L, and K302N.

[0036] In an embodiment of the present disclosure, the protein or variant thereof as set forth in SED ID NO. 3 comprises at least one amino acid mutation selected from a group consisting of I87V, S16T, T121S, I196N, A115S, S155T, N221D, I160V, S271N, T10A, T4A, S16V, A27S, A33P, N34S, S37N, I57L, K111Q, F119Y, S123T, E145D, K147Q, K173N, I192V, T258K, L268I, D3E, T4N, M5Y, I6Y, V8I, T10P, S19T, S154T, A176G, N223D, L251M, S271K, and S273T.

[0037] In an embodiment of the present disclosure, the weight ratio of SEQ ID NO.l, SEQ ID NO.2, and SEQ ID NO.3 in the mixture of proteins is in a range of 1 : 0.40 :0.02 to 1:1: 1. In another embodiment of the present disclosure, the weight the ratio of SEQ ID NO.l, SEQ ID NO.2, and SEQ ID NO.3 in the mixture of proteins is in a range of 1: 0.45 :0.03 to 1: 1: 1.

[0038] In an embodiment of the present disclosure, the proteins or variants thereof have a nucleotide sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO.5, and SEQ ID NO.6. The protein represented by SEQ ID NO. 1 has a nucleotide sequence of SEQ ID NO. 4. The protein represented by SEQ ID NO. 2 has a nucleotide sequence of SEQ ID NO. 5. The protein represented by SEQ ID NO. 3 has a nucleotide sequence of SEQ ID NO. 6.

[0039] In an embodiment of the present disclosure, the mixture of proteins or variants thereof is in a weight range of 2.5 to 60% with respect to the composition.

[0040] In an embodiment of the present disclosure, the buffer component is selected from sodium phosphate buffer, sodium acetate buffer, sodium citrate buffer, phosphate buffered saline, saline, Ringer’s lactate solution, 5% dextrose solution, or mixtures thereof. In another embodiment of the present disclosure, the buffer component is selected from sodium phosphate buffer, sodium acetate buffer, or mixtures thereof.

[0041] In an embodiment of the present disclosure, the sodium phosphate buffer is in a concentration in the range of 20mM to 50mM and has a pH in the range of 6 to 7; and the sodium acetate buffer is in a concentration in the range of 10 mM to 50 mM and has a pH in the range of 4 to 6.

[0042] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the composition comprises water in quantity sufficient.

[0043] In an embodiment of the present disclosure, the composition further comprises an additive selected from polysorbate 80 (tween 80), n-Dodecyl-beta-D-maltoside (DDM), octylphenol ethylene oxide condensate (triton X-100), polysorbate 20(tween 20), 3- [ (3- cholamidopropyl)dimethyl ammonio]-!- propane sulfonate (CHAPS), 30% aqueous solution of lauryl betaine (EMPIGEN BB), or mixtures thereof. In another embodiment of the present disclosure, the composition further comprises an additive selected from polysorbate 80 (tween 80), n-Dodecyl-beta-D-maltoside (DDM), or mixtures thereof.

[0044] In an embodiment of the present disclosure, there is provided a pharmaceutical formulation comprising the composition as described herein and a pharmaceutically acceptable carrier. In an example, the pharmaceutically acceptable carrier is selected from buffer component, stabilizing agents, pH adjusters, tonicity / osmolality adjusting agents, surfactants, adjuvants, excipients, or combination thereof. Non-limiting examples of buffer component in the formulation may be selected from the buffer components described previously herein above, phosphate buffer, acetate buffer, histidine buffer, tromethamine buffer, or mixtures thereof. Non-limiting examples of buffer component in the formulation includes, but are not limited to, stabilizing agents such as sugars including sucrose and trehalose; and polyols including mannitol and sorbitol. Non-limiting examples of tonicity / osmolality adjusting agents include, but are not limited to, sodium chloride, sucrose, and dextrose. Non-limiting examples of surfactants include, but are not limited to, polysorbate 20 and polysorbate 80.

[0045] In an embodiment of the present disclosure, the pharmaceutically acceptable carrier is selected from buffer component, preferably selected from phosphate buffer, acetate buffer, histidine buffer, tromethamine buffer, or mixtures thereof; stabilizing agent preferably selected from sugars, preferably selected from sucrose, trehalose, or mixtures thereof, and / or polyols preferably selected from mannitol, sorbitol, or mixtures thereof; tonicity / osmolality adjusting agents preferably selected from sodiumchloride, sucrose, dextrose, or combination thereof; and / or surfactant preferably selected from polysorbate 20, polysorbate 80, or mixtures thereof.

[0046] In an embodiment of the present disclosure, the pharmaceutically acceptable carrier is selected from stabilizing agent preferably selected from sugars, preferably selected from sucrose, trehalose, or combination thereof, and / or polyols preferably selected from mannitol, sorbitol, or combination thereof; tonicity / osmolality adjusting agents preferably selected from sodium chloride, sucrose, dextrose, or combination thereof; and / or surfactant preferably selected from polysorbate 20, polysorbate 80, or mixtures thereof.

[0047] In an embodiment of the present disclosure, the pharmaceutically acceptable carrier is selected from phosphate buffer, acetate buffer, histidine buffer, tromethamine buffer, sugars, polyols sodium chloride, sucrose, dextrose, polysorbate 20, polysorbate 80, or mixtures thereof.

[0048] In an embodiment, there is provided a formulation, wherein the formulation comprises the proteins or variant thereof, or composition, as disclosed herein; and at least one pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is selected from buffer component, stabilizing agents, tonicity / osmolality adjusting agents, pH adjusters, surfactants, or combination thereof.

[0049] In another embodiment, there is provided a formulation, wherein the formulation comprises the proteins or variant thereof, or composition, as disclosed herein; and at least one pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is selected from buffer component, preferably selected from phosphate buffer, acetate buffer, histidine buffer, tromethamine buffer, or combination thereof; stabilizing agent preferably selected from sugars, preferably selected from sucrose, trehalose, or combination thereof, and / or polyols preferably selected from mannitol, sorbitol, or combination thereof; tonicity / osmolality adjusting agents preferably selected from sodium chloride, sucrose, dextrose, or combination thereof; and / or surfactant preferably selected from polysorbate 20, polysorbate 80, or mixtures thereof

[0050] In an embodiment, the pH of the composition having the proteins or variant thereof and buffering, is in the range of 4 to 6.

[0051] In an embodiment, there is provided a formulation comprising a mixture of proteins or variants thereof, wherein the proteins have an amino acid sequence of at least 90% sequence identity to SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO.3; and a pharmaceutically acceptable carrier selected from buffer component; stabilizing agent; tonicity / osmolality adjusting agents; surfactant; or mixtures thereof.

[0052] In various embodiments, the stabilizing agent may be present in the range of 2 wt% to 20 wt%, preferably 2 wt% to 12 wt%, or 2 wt% to 10 wt%, in respect of the formulation. In some embodiments, the surfactant may be present in the range of 0.01 wt% to 5 wt%, preferably 0.01 wt% to 2 wt%, or 0.02 wt% to 1 wt%, in respect of the formulation. In some embodiments, the concentration of the tonicity / osmolality adjusting agents is in the range of lOOmM to 250Mm, preferably 150mM.

[0053] In an embodiment of the present disclosure, the pharmaceutically acceptable carrier is selected from is an inert component which is pharmaceutically compatible or non-toxic compatible substances employed in pharmaceutical formulations, which is known in the art.

[0054] In an embodiment of the present disclosure, there is provided a method of treating an infection caused due to Klebsiella in a subject, comprising administering the composition as described herein or the pharmaceutical formulation as described herein to the subject. In a preferred embodiment, the subject is a human.

[0055] In an embodiment of the present disclosure, the Klebsiella is selected from a group consisting of Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella oxytoca, Klebsiella granulomatis, and Klebsiella aerogenes.

[0056] In an embodiment of the present disclosure, the Klebsiella may be common Klebsiella or hypervirulent Klebsiella-, and may be mono drug resistant or multi-drug resistant.

[0057] In an embodiment of the present disclosure, the method of administering is selected from intravenous, intranasal, inhalation, subcutaneous, or intraperitoneal.

[0058] In an embodiment of the present disclosure, there is provided a kit comprising the composition as described herein or the pharmaceutical formulation as described herein, and an instruction manual.

[0059] In an embodiment of the present disclosure, there is provided a use of the composition as described herein or the formulation as described herein for the treatment of Klebsiella infections.

[0060] In yet another embodiment, there is provided a use of the kit as described herein above for the treatment of Klebsiella infections.

[0061] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.EXAMPLES

[0062] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.Materials

[0063] The reagents and chemicals used in the present disclosure were procured from Sigma; and the growth media were procured from Himedia Labs and Thermofisher Scientific.Example 1: Preparation of Composition

[0064] The compositions Cl to C3 were prepared by mixing the three klebicins (SEQ ID NO.1-P628 + SEQ ID NO. 2-P764+ SEQ ID NO. 3-P801) in different weight ratios as described below. Individual klebicin gene was synthesized by gene synthesis and cloned into an expression plasmid (pET26b). The plasmid was transformed into competent E. coli expression strain ER2566AbcZ and the resulting expressed protein was purified using methods well known in the art. Compositions were prepared by mixing the three klebicins in respective ratio and were used for in vitro microbiological potency animal efficacy and FoR studies as described in subsequent examples of the present disclosure.

[0065] The composition C 1 was prepared by mixing 6 mg / ml of each of the klebicins (i.e. (SEQ ID NO.1-P628 + SEQ ID NO. 2-P764+ SEQ ID NO. 3-P801) in a weight ratio of 1 : 1 : 1) and buffered in lOmM sodium acetate buffer at pH 5.1.

[0066] The composition C2 was prepared by mixing 12 mg / ml of SEQ ID NO.1 -P628, 8 mg / ml of SEQ ID NO. 2-P764, and 1.5 mg / ml of SEQ ID NO. 3-P801 (i.e. 1:0.7:0.125) and buffered in lOmM sodium acetate buffer at pH 5.1.

[0067] The composition C3 was prepared by mixing 12 mg / ml of SEQ ID NO.1-P628, 8 mg / ml of SEQ ID NO. 2-P764, and 0.5 mg / ml of SEQ ID NO. 3-P801 (i.e. 1 :0.7:0.04) and buffered in lOmM sodium acetate buffer at pH 5.1.

[0068] The compositions Cl to C3 were found to be stable at different temperatures - 20 °C, and 5 °C, even after 1 to 12 months of storage.

[0069] Further, the prepared compositions were mixed with pharmaceutically acceptable carriers to prepare the corresponding pharmaceutical formulations.Example 2: Determination of biological activity of klebicin compositions on various clinical Klebsiella pneumoniae strains by minimum inhibitory concentration (MIC) assay

[0070] Klebsiella pneumoniae bacterial suspensions (0.5 McFarland standard) were diluted in CAA (Cas Amino Acid) iron deficient media to achieve 105CFU per mL. Cell suspension (50 pL) was added to wells containing the respective individual klebicins, and the klebicin mixture containing compositions Cl to C3 as prepared in Example 1 (diluted two-fold starting with 256 pg / mL) to a total assay volume of 100 pL. The plates were incubated under static conditions at 35 °C for 18 hours and the MIC was recorded as the lowest concentration of the drug that shows visible inhibition of bacterial growth.Results:

[0071] The MIC obtained for the compositions Cl to C3 was not a sum total of the MICs obtained for individual klebicins (Table 1).

[0072] Further, the MICs obtained for individual sensitive strains (S. No. 11-18 & 20) were much lower in the case of compositions Cl to C3 indicating a potentiation effect of the individual klebicins in the mixture.Table 1: MIC of individual klebicins and different compositions of the klebicin mixture on clinical Klebsiella pneumoniae strains.Example 3: Lung infection efficacy studies of the compositions a. Lung infection efficacy studies against Klebsiella pneumoniae strains:

[0073] Lung infection efficacy studies were performed in a 24-hour neutropenic lung infection model. Briefly, 6-8-week-old immunocompromised BalB / c mice were first intranasally challenged with Klebsiella pneumoniae at either 105or 106CFU per animal. Treatment with all individual klebicins and the klebicin mixture were administered intravenously (IV) after 2-hours of intranasal challenge with test Klebsiella pneumoniae strains. Individual klebicins were administered at either 30 or 60 mpk for P628, 30 or 60 mpk for P764 and 30 mpk for P801. The composition Cl ofP628+P764+P801 at 30 mpk of each klebicin was used for IV treatment. The dosing regimen was twice a day (b.i.d.) at every 12- hour interval. Ciprofloxacin was used as a positive control for the lung infection animal efficacy studies. End point of the study was enumeration of the residual lung CFUs after 24-hours of treatment. The control and treated animals were humanely euthanized and bacterial load in the lungs were enumerated after harvesting the animal lungs and plating the macerated lung tissue on LB plates and incubating them at 37°C for 18 hours.Results:

[0074] The composition Cl exhibited significant lung CFU reduction in infected animals for all K. pneumoniae strains that were tested in the study (Table 2).

[0075] No antagonism was observed when klebicins are in a mixture (composition Cl) in the animal efficacy study.

[0076] Table 2: In vivo efficacy of individual klebicins and the klebicin mixture containing equal amounts of all three individual klebicins (composition Cl).R: Indicates strain resistant to antibiotic ciprofloxacin b. Dose-response efficacy studies of the klebicin mixture on clinical Klebsiella pneumoniae strains:

[0077] Dose-response efficacy studies were performed in a 24-hour neutropenic lung infection model. Briefly, 6 to 8 weeks old immunocompromised BalB / c mice were first intranasally challenged with K. pneumoniae at either 105or 106CFU / animal. Treatment with all klebicins and the klebicin mixture were administered intravenously (IV) after 2 -hours of intranasal challenge with test Klebsiella pneumoniae strains. Individual klebicins were administered at either 60 mpk for P628, 40 mpk for P764 and 7.5, 5 or 2.5 mpk for P801. The klebicins mixture containing P628+P764+P801 at 60+40+7.5 (C2), and 60+40+2.5 (C3) mpk were used for IV treatment. The dosing regimen was thrice a day (t.i.d.) at every 8-hour interval. Ciprofloxacin was used as positive control for the lung infection animal efficacy studies. End point of the study was enumeration of the residual lung CFUs after 24-hours of treatment. The control and treated animals were humanely euthanized and bacterial load in the lungs were enumerated after harvesting the animal lungs and plating the macerated lung tissue on LB plates and incubating them at 37 °C for 18 hours.Results:

[0078] Klebicin mixture composition at 60+40+2.5mpk (P628+P764+P801, composition C3) when administered IV in TID dosing regimen afforded significant lung CFU reduction with all nine challenge K. pneumoniae strains tested here.

[0079] No antagonism was observed when klebicins are in a mixture in the animal efficacy studies.

[0080] Table 3: In vivo efficacy of individual klebicins and the klebicin mixture containing different composition of individual klebicins.c. Efficacy studies of the Composition C3 on clinical Klebsiella pneumoniae strains:

[0081] Lung infection efficacy studies of the Composition C3 was performed in a 24- hour neutropenic lung infection model with 18 different clinical Klebsiella pneumoniae strains including multi-drug resistant strains resistant to most standard of care antibiotics.

[0082] Briefly, 6-8 weeks old immunocompromised BalB / c mice were first intranasally challenged with K. pneumoniae at either 105or 106CFU / animal. Treatment with all klebicins and the Composition C3 were administered intravenously (IV) after 2-hours of intranasal challenge with test Klebsiella pneumoniae strains. Individual klebicins were administered at 60 mpk for P628, 40 mpk for P764 and 2.5 mpk for P801. The Composition C3 containing P628+P764+P801 at 60+40+2.5 mpk of each klebicin (i.e. weight ratio of 1:0.7:0.04) were used for IV treatment. The dosing regimen was thrice day (t.i.d.) at every 8- hour interval. Ciprofloxacin was used aspositive control for the lung infection animal efficacy studies. The end point of the study was enumeration of the residual lung CFUs after 24-hours of treatment. The control and treated animals were humanely euthanized and bacterial load in the lungs were enumerated after harvesting the animal lungs and plating the macerated lung tissue on LB plates and incubating them at 37°C for 18 hours. Log CFU was calculated as the CFU reduction in the treatment group compared to the lung CFUs at the time of initiation of the treatment.

[0083] Composition C3 dosage: 60+40+2.5mpk with total daily dosage of 180-120- 7.5mpk (P628 + P764 + P801); pH 5.1 Results:

[0084] All tested strains were sensitive to the Composition C3 in the lung infection efficacy model.

[0085] Composition C3 at 60+40+2.5mpk (P628+P764+P801) afforded >1 log lung CFU reduction with all Klebsiella pneumoniae challenge strains.

[0086] A potentiation effect was seen in the efficacy studies when the klebicins were administered as a mixture, especially in the optimized composition C3.

[0087] Table 4 : In vivo efficacy of individual klebicins and the optimized klebicin composition C3 containing 60-40-2.5 mpk equivalent of the three individual klebicins.s: Indicates strain sensitive only to klebicins P764 or P801 by in vitro MIC,R: Indicates strain resistant to antibiotic ciprofloxacinExample 4: Determination of Frequency of resistance (FoR) in clinical K. pneumoniae strains with the optimized composition C3 mixture (P628+P764+P801)

[0088] Frequency of resistance (FoR) studies were performed on 10 different clinical Klebsiella penumoniae strains. The Klebsiella pneumoniae strains, ATCC13883, B2192, B225, B2230, B2236, B2242, B2267, B2344, B2351 and B2597 were grown in CAA at 37°C until ODeoo reached 1.0. The cells were centrifuged and pelleted to get concentrated cells. CAA agar plates were prepared with 0.75% agar base and klebicin mixture containing 12 mg / ml of P628, 8 mg / ml of P764 and 0.5 mg / ml of P801 (Composition C3 equivalent to 60, 40 & 2.5 mpk of P628, P764 & P801; pH 5.1 respectively) were incorporated in CAA agar plates at 8X MIC of composition C3. Higher cell numbers were plated on drug containing plates. Appropriate dilutions were also plated on drug-free CAA agar plates for determining input cell numbers. All CAA agar plates were incubated at 37°C for 24 hours and at room temperature for an additional 18 hours. Frequency of resistance (FoR) was calculated based on the recovered cell numbers on drug-containing and drug-free CAA agar plates.Results:

[0089] Optimized composition C3 has very low risk for resistance development with the projected FoR of <10-9with multiple Klebsiella pneumoniae strains.

[0090] In addition, FoR was determined with other combinations of klebicin mixture, containing equal concentrations of all three klebicins (Composition Cl) and a mixture containing 12 mg / mL of P628, 8 mg / mL of P764 and 1.5mg / mL of P801 (Composition C2 equivalent to 60-40-7.5 mpk of P628, P764 and P801 respectively). These compositions also showed FoR of <10-9on multiple Klebsiella pneumoniae strains.

[0091] Table 5: FoR of the klebicin mixture against 10 Klebsiella pneumoniae strains.Example 5: Preparation and Evaluation of the Formulation

[0092] The compositions prepared using a method as described in Example 1 were mixed with pharmaceutically acceptable carriers to prepare pharmaceutical formulations. Various formulations were prepared by mixing the three klebicins (SEQID NO.1-P628 + SEQ ID NO. 2-P764+ SEQ ID NO. 3-P801) buffered in lOmM sodium acetate buffer at pH 5.1, which was further mixed with the pharmaceutically acceptable carriers. Table 6 depicts exemplary formulations Fl to F17 prepared using the composition C3.

[0093] For example, Fl was prepared by mixing the composition C3 (refer Example1) with 150mM NaCl (tonicity / osmolality adjusting agent), to obtain Fl formulation.

[0094] F2 was prepared by mixing composition C3 (refer Example 1) with 4 wt% Sorbitol (stabilizing agent), to obtain F2 formulation.

[0095] F9 was prepared by mixing composition C3 (refer Example 1) with 0.05 wt% of Polysorbate 80 (PS80) (surfactant), to obtain F9 formulation.

[0096] F10 was prepared by mixing composition C3 (refer Example 1) with 0.05 wt% of Polysorbate 80 (PS80) (surfactant) and 150mM NaCl (tonicity / osmolality adjusting agent), to obtain F9 formulation.

[0097] Similarly, the formulations F3-F8 and F11-F17, as depicted in Table 6, were prepared using various combinations of stabilizing agents, surfactants, and tonicity / osmolality adjusting agent.

[0098] The formulations prepared were evaluated by minimum inhibitory concentration (MIC) method for activity against a Control CT (composition C3, i.e. the klebicins (i.e. (SEQ ID NO.1-P628 + SEQ ID NO. 2-P764+ SEQ ID NO. 3-P801) in a weight ratio of 1:0.7:0.04, and buffered sodium acetate buffer at pH 5.1).Results:

[0099] The formulations depicted in Table 6 were active in the presence of the pharmaceutically acceptable carriers.Table 6: Depicts exemplary formulations Fl to F17.Advantages of the present disclosure

[0100] The present disclosure provides a composition comprising a mixture of klebicin proteins and variants thereof. Klebicins are known to kill other susceptible members of the Klebsiella genus individually. Therefore, this present disclosure provides a klebicin mixture with broader target cell ranges in order to effectively treat Klebsiella infections. Further, potentiation effect was observed in both in vitro and in vivo efficacy studies with the disclosed composition against several clinical and drug resistant strains of Klebsiella. Furthermore, the disclosed composition improved coverage and FoR of <10-9on multiple Klebsiella pneumoniae strains when compared with individual klebicins.

Claims

CLAIMSI / We claim:

1. A composition, comprising: a) a mixture of proteins or variants thereof, wherein the proteins have an amino acid sequence of at least 90% sequence identity to SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO.3; and b) a buffer component; wherein the composition has a pH in the range of 4 to 7.

2. The composition as claimed in claim 1, wherein the weight ratio of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 in the mixture is in a range of 1: 0.4 :0.02 to 1: 1: 1.

3. The composition as claimed in claim 1 , wherein the proteins or variants thereof have a nucleotide sequence selected from the group consisting of SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6.

4. The composition as claimed in claim 1, wherein the proteins or variants thereof is in a weight range of 2.5 to 60% with respect to the composition.

5. The composition has claimed in claim 1, wherein the buffer component is selected from sodium phosphate buffer, sodium acetate buffer, sodium citrate buffer, phosphate buffered saline, saline, Ringer’s lactate solution, 5% dextrose solution, or mixtures thereof.

6. The composition as claimed in claim 5, wherein the sodium phosphate buffer is in a concentration in the range of 20mM to 50mM, and has a pH in the range of 6 to 7; and wherein the sodium acetate buffer is in a concentration in the range of 10 mM to 50 mM and has a pH in the range of 4 to 6.

7. The composition as claimed in claim 1, wherein the composition comprises water in quantity sufficient.

8. The composition as claimed in anyone of claims 1 to 7, wherein the composition further comprises an additive selected from polysorbate 80 (tween 80), n- Dodecyl-beta-D-maltoside (DDM), octylphenol ethylene oxide condensate(triton X-100), polysorbate 20(tween 20), (3-((3-cholamidopropyl) dimethylammonio)- 1 -propanesulfonate) (CHAPS), 30% aqueous solution of lauryl betaine (EMPIGEN BB), or mixtures thereof.

9. A pharmaceutical formulation comprising the composition as claimed in any one of claims 1 to 8; and a pharmaceutically acceptable carrier.

10. A method of treating an infection caused due to Klebsiella in a subject, comprising administering the composition as claimed in claim 1; or the pharmaceutical formulation as claimed in claim 9 to the subject.

11. The method as claimed in claim 10, wherein the Klebsiella is selected from a group consisting of Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella oxytoca, Klebsiella granulomatis, and Klebsiella aerogenes.

12. The method as claimed in claim 11, wherein the Klebsiella may be common Klebsiella or hypervirulent Klebsiella; and may be mono drug resistant or multi-drug resistant.

13. The method as claimed in claim 10, wherein the method of administering is selected from intravenous, intranasal, inhalation, subcutaneous, or intraperitoneal.

14. A kit comprising the composition as claimed in claim 1; or the pharmaceutical formulation as claimed in claim 9; and an instruction manual.

15. Use of the composition as claimed in claim 1; or the formulation as claimed in claim 9 for the treatment of Klebsiella infections.

16. Use of the kit as claimed in claim 14 for the treatment of Klebsiella infections.

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