AN ANTIBODY FRAGMENT BASED ANTIFUNGAL CONJUGATE SELECTIVELY TARGETING CANDIDA

Abstract

The present invention provides a novel antibody fragment based antifungal conjugate selectively targeting Candida spp. comprising of at least one antimicrobial peptide at one end of the conjugate, more particularly, human Histatin-5; an antibody fragment at the other end of the conjugate, specific against Candida spp. enolase, a virulence factor protease and biofilm specific antigen of Candida spp.; at least one signal protease cleavage sequence susceptible to cleavage by virulent protease secreted by Candida spp., secreted aspartyl proteinase-1 (SAP1); and at least one flexible polypeptide linker. The signal protease cleavage sequence and the flexible polypeptide linker are in tandem with each other and placed in between the antimicrobial peptide and the antibody. The in vitro MIC-99 of the conjugate against Candida spp., is in the range of 0.2-0.3 ?M, more specifically, 0.25 ?M or 250 nM.

Claims

1. An antibody fragment based antifungal conjugate selectively targeting Candida spp., comprising an antimicrobial peptide at one end of the conjugate, an antibody fragment at the other end of the conjugate, preferably, a camelid heavy chain antibody variable region fragment (VHH) specific against the antigen of Candida spp. selected from the group consisting of surface antigen, and extracellular matrix antigen of Candida biofilm, at least one signal protease cleavage sequence susceptible to cleavage by proteases selected from the group consisting of membrane proteases, cell wall associated proteases, and secreted proteases of Candida spp., and proteases of host neutrophils; and at least one flexible polypeptide linker in tandem with the signal protease cleavage sequence, with the signal protease cleavage sequence and the polypeptide linker placed in between the antimicrobial peptide and antibody fragment; wherein, said antimicrobial peptide belongs to the group comprising of cationic histidine-rich antimicrobial peptides, more particularly, human Histatin-5 represented by amino acids selected from the group comprising of Seq. ID 1 and Seq. ID 2; VHH, targeting Candida spp. specific enolase which is a virulence factor protease and biofilm specific antigen of Candida spp., belongs to the group comprising of amino acid sequences represented by Seq. ID 6 and Seq. ID 9, preferably, Seq. ID 9; said VHH represented by Seq. ID 9 is humanized form of Seq. ID 6, wherein, Seq. ID 9 is generated by mutating Seq. ID 6 at position 49 from glutamic acid to glycine, and at position 50 from arginine to leucine; said protease specific cleavage sequence is represented by Seq. ID 10 which is susceptible to cleavage by virulent protease secreted by Candida spp., secreted as partyl proteinase-1 (SAP1); the flexible polypeptide linker is of formula {(Gly).sub.4S}.sub.n, where n is 1-9, preferably, selected from the group comprising of Seq. ID 14 and Seq. ID 15; the amino acid sequence of the conjugate is represented by Seq. ID 19; said conjugate is specific against Candida spp., preferably, C. albicans, C. tropicalis, C. krusei, C. parasilopsis, and C. glabrata. the in vitro MIC-99 of the conjugate represented by Seq. ID 19 against Candida albicans is 0.25 ?M; and the in vitro MIC-99 of the VHH represented by Seq. ID 9 against Candida albicans is 6.5 ?M.

2. The antibody fragment based antifungal conjugate as claimed in claim 1, wherein, the amino acid sequence of the conjugate is selected from the group consisting of Seq. ID 19, and Seq. ID 20.

3. The antibody fragment based antifungal conjugate as claimed in claim 1, wherein, said antimicrobial peptide belongs to the group comprising of cationic histidine-rich antimicrobial peptides represented by amino acid sequence Seq. ID 1, and Seq. ID 2; mucin family of proteins represented by amino acid sequence Seq. ID 3, and Seq. ID 4; and human beta defensins, preferably, amino acid sequence Seq. ID 5.

4. The antibody fragment based antifungal conjugate claimed in claim 1, wherein, the VHH fragment targeting Candida spp. is derived from immunized Camelus dromedarius having amino acid sequence represented by Seq. ID no. 6, Seq. ID7, Seq. ID 8, and Seq. ID 9.

5. The antibody fragment based antifungal conjugate as claimed in claim 1, wherein, the protease specific cleavage sequence is susceptible to cleavage by proteases selected from the group consisting of Candida spp. specific secreted aspartyl proteinases (SAP), a secreted virulent protease, preferably, Seq. ID 10 and Seq. ID 11 susceptible to cleavage by SAP1; membrane or cell wall associated proteases of Candida spp. preferably, Seq. ID 12 susceptible to cleavage by Candida spp. specific signal peptidase 3; host neutrophil protease, preferably Seq. ID 13 susceptible to cleavage by multiple host neutrophil proteases like Elastase, Proteinase 3, Matrix metalloproteinases 1 & 13, Thrombin and Activated protein C; or a combination thereof.

6. The antibody fragment based antifungal conjugate as claimed in claim 1, wherein, the flexible polypeptide linker is selected from the amino acid sequence with Glycine and Serine in tandem with the formula {(Gly).sub.4Ser}.sub.n, where n is 1-9, preferably Seq. ID 14, or Seq. ID 15; or from amino acid sequence represented by Seq. ID 16 where Glutamic acid can be substituted with Aspartate (D), or from Lysine rich sequences as represented by Seq. ID 17 or Seq. ID 18 or a combination thereof.

7. The antibody fragment based antifungal conjugate as claimed in claim 1, wherein, the antibody is derived from a library of heavy chain antibody variable region fragment (VHH) from camelids selected from the group comprising of dromedary camel, Bactrian camels, wild or feral camels, llamas, alpacas, vicu?as, or guanacos, preferably Camelus dromedarius.

8. The antibody fragment based antifungal conjugate as claimed in claim 1, wherein, the conjugate is a non-toxic prodrug and gets activated only upon interaction of the VHH of the conjugate with Candida spp., thereby initiating a cascade of reactions leading to cleavage of the protease cleavage site of the conjugate releasing the antimicrobial peptide from the conjugate to act against the Candida spp.

9. The antibody fragment based antifungal conjugate as claimed in claim 1, wherein, the conjugate can constitute pharmaceutical compositions for topical application, systemic delivery, or oral consumption.

10. The antibody fragment based antifungal conjugate as claimed in claim 1, wherein, the conjugate can constitute formulations for coating medical implants to reduce infections.

Description

BRIEF DESCRIPTION OF THE DRAWING(S)

[0027] The object of the invention may be understood in more details and more particularly description of the invention briefly summarized above by reference to certain embodiments thereof which are illustrated in the appended drawings, which drawings form a part of this specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective equivalent embodiments.

[0028] FIG. 1a is a schematic of the antibody fragment based antifungal conjugate (100) depicting the cleavage of the conjugate (100) at the protease cleavage site (103) separating antimicrobial peptide (101) and the antibody (102);

[0029] FIG. 1b is a schematic depicting the mode of action of the antibody fragment based antifungal conjugate (100) at the surface of the pathogen cell membrane (104) by membrane or cell wall associated proteases (105);

[0030] FIG. 1c is a schematic depicting the mode of action of the antibody fragment based antifungal conjugate (100) at the vicinity of the pathogen by proteases secreted by the pathogen (105);

[0031] FIG. 1d is a schematic depicting the mode of action of the antibody fragment based antifungal conjugate (100) on host neutrophil ingested pathogen by host neutrophil specific proteases (105);

[0032] FIG. 2a provides the amino acid sequence of antibody fragment based antifungal conjugate with amino acid sequence represented by Seq. ID 19;

[0033] FIG. 2b provides the amino acid sequence of antibody fragment based antifungal conjugate with amino acid sequence represented by Seq. ID 20;

[0034] FIG. 3a depicts achromatogram that shows affinity purification by Ni-NTA (1st round) of conjugate Seq. ID 19 from solubilized inclusion bodies and purified using AKTA-prime plus purification system;

[0035] FIG. 3b depicts SDS-PAGE image of conjugate Seq. ID 19 purified from solubilized inclusion bodies and purified using AKTA-prime plus purification system;

[0036] FIG. 3c depict Western blot using the His tagged Seq. ID 9 as the primary antibody and the anti His secondary antibody with cell lysates of E. coli strain E2B (Rosetta Gami2 DE3) expressing empty pET vector uninduced in lane 1, and pET vector containing the Candida Enolase gene (NCBI No GenBank: M93712.1) uninduced in lane 2 and induced in lane 3; cell lysates of E. coli strain C41(DE3) expressing empty pET vector uninduced in lane 4, and pET vector containing the Candida Enolase gene uninduced in lane Sand induced in lane 6; cell lysates of E. coli strain BL21(DE3) expressing empty pET vector uninduced in lane 7, and the pET vector containing the Candida Enolase gene uninduced in lane 8 and induced in lane 9; and marker peptides mix in lane 10;

[0037] FIG. 4 is a representative image of SDS PAGE of lysates from E. coli expressing the antibody fragment based antifungal conjugate represented by Seq. ID 19 (lane 1) and lysates of Candida albicans treated with purified Seq. ID 19 were compared by analysis for the VHH of Seq. ID 9 after 1 hour (lane 2) and 2 hour (lane 3); and

[0038] FIG. 5 is representative microbiological agar-plate assay to determine MIC-99 of purified Seq. ID19 and Seq. ID 9.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention now will be described hereinafter with reference to the detailed description, in which some, but not all embodiments of the invention are indicated. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. The present invention is described fully herein with non-limiting embodiments and exemplary experimentation.

Definitions

[0040] The term antibody fragment as used herein refers to polypeptides or proteins that bind to specific antigens. It also means immunoglobulins, not limited to polyclonal, monoclonal, chimeric, humanized antibodies, Fab fragments, F(ab)2 fragments and likewise.

[0041] The term antifungal conjugate as used herein refers to a molecule which shows antimicrobial properties against fungi including single cellular fungi, multicellular fungi, yeast, molds, filamentous fungi, non-filamentous fungi, and other types of fungi.

[0042] The term antimicrobial peptide as used herein refers to a polymer of amino acid residues typically ranging in length from 10 to about 50 which show antimicrobial properties by associating with membranes of microorganisms and causing membrane permeabilization, thereby killing the microorganisms.

[0043] The term MIC as used herein refers to minimal inhibitory concentration.

[0044] The term MIC-99 as used herein refers to minimal inhibitory concentration for killing 99% microorganisms.

[0045] The term next generation as used herein refers to product that has been developed using latest technology to replace existing less efficient form of the drug.

[0046] The term prodrug as used herein refers to a compound that, after administration, is metabolized (i.e., converted within the body) into a pharmacologically active drug.

[0047] The term in tandem as used herein refers to one behind another. A sequence in tandem with another is adjacent sequences in continuation.

[0048] The term VHH as used herein refers to an antigen binding fragment of antibody derived from camels which is composed only of fragments of heavy chains and does not comprise any light chains; it is also called as nanobody. Typically, an IgG antibody comprises two heavy chains and two light chains. Each heavy chain comprises a variable region (encoded by VHH, D and J elements) and a constant region.

[0049] The term virulent protease as used herein refers to proteases naturally produced by pathogens to attack their host cells and aids in pathogenicity.

[0050] The company AbGenics Lifesciences Pvt. Ltd. has developed new generation of antibody fragment based antimicrobial conjugates known by the trademark AbTids? for providing a solution to management of drug-resistant Candida spp.

[0051] The genus Candida represents a highly heterogeneous group of >50 known species. Candida species are found as normal flora in healthy individuals and are known to cause opportunistic infections with high rates of mortality, especially in immunocompromised individuals. Candida spp. cause systemic diseases which are the fourth leading cause of nosocomial bloodstream infections in modern hospitals. Nevertheless, >90% of the invasive Candida infections are caused by C. albicans, C. glabrata, C. parapsilopsis, C. tropicalis, or C. krusei in many parts of the world.

[0052] Therefore, the main embodiment of the present invention provides a novel antibody fragment based antifungal conjugate, selectively targeting Candida spp., comprising of: [0053] at least one antimicrobial peptide at one end of the conjugate belonging to the group comprising of cationic histidine-rich antimicrobial peptides, preferably Histatin family of peptides, more preferably, the active fragment of human Histatin-5 represented by amino acid sequence Seq. ID 1, and Seq. ID 2 as listed in Table 1; mucin family of proteins Mucin 1-22, preferably human Mucin 7 represented by amino acid sequence Seq. ID 3, and Seq. ID 4 as listed in Table 1; and human beta defensins, preferably, amino acid sequence Seq. ID 5 as listed in Table 1; [0054] at least one antibody fragment at the other end of the conjugate, preferably, a camelid heavy chain antibody variable region fragment (VHH) targeting antigen present on the cell walls and biofilms of Candida spp., more preferably, enolase extracellular matrix antigen, that is also a virulent protease wherein, the sequence of the VHH is selected from the group of amino acid sequences Seq. ID. 6, Seq. ID 7, Seq. ID. 8, and Seq. ID 9 as listed in Table 1, preferably, Seq. ID. 9; [0055] at least one signal protease cleavage sequence susceptible to proteases selected from the group consisting of membrane proteases, cell wall associated proteases, or secreted proteases of Candida spp., or proteases of host neutrophils; wherein, the protease specific cleavage sequence is susceptible to cleavage by proteases selected from the group consisting of Candida spp. specific secreted aspartyl proteinases (SAP), a secreted virulent protease, preferably, Seq. ID 10 or Seq. ID 1 las listed in Table 1 susceptible to cleavage by SAP1; membrane or cell wall associated proteases of Candida spp. preferably, Seq. ID 12 as listed in Table 1 susceptible to cleavage by Candida spp. specific signal peptidase 3; and by host neutrophil proteases, preferably Seq. ID 13 as listed in Table 1 susceptible to cleavage by Elastase, Proteinase 3, Matrix metalloproteinases 1 & 13, Thrombin and Activated protein C; or a combination thereof; and [0056] at least one flexible polypeptide linker tandem to the protease cleavage sequence, wherein, the linker is selected from the amino acid sequence with Glycine and Serine in tandem with the formula {(Gly).sub.4Ser}.sub.n, where n is 1-9, preferably Seq. ID 14, or Seq. ID 15; or from amino acid sequence represented by Seq. ID16 where Glutamic acid can be substituted with Aspartate (D), or from Lysine rich sequences as represented by Seq. ID 17 or Seq. ID 18 or a combination thereof.

TABLE-US-00001 TABLE1 Listofaminoacidsequences Sequence IDno. Aminoacidsequence Aminoacidsequencesofantimicrobialpeptide 1 MetXaaAlaLysArgHisHisGlyTyrLys ArgLysPheHisXaaXaa whereinXaaisanyaminoacid 2 AlaLysArgHisHisGlyTyrLysArgLys PheHis 3 XaaXaaLeuAlaHisGlnLysProPheIleArgLysSer TyrLysCysLeuHisLysArgCysArgXaaXaa whereinXaaisanyaminoacid 4 GlyCysLeuAlaHisGlnLysProPheIleArgLysSer TyrLysCysLeuHisLysArgCysArg 5 GlyIleGlyAspProValThrCysLeuLysSerGlyAla IleCysHisProValPheCysProArgArgTyrLysGln IleGlyThrCysGlyLeuProGlyThrLysCysCysLys LysPro AminoacidsequencesofCamelidheavychain antibodyvariableregionfragment (VHH)specifictoCandidaspp. 6 MetAlaAspValGlnLeuGlnGluSerGlyGlyGlySer ValGlnAlaGlyGlySerLeuArgLeuSerCysThrIle SerGlyTyrSerGlyTyrSerPheArgThrLeuCysVal TyrTrpPheArgGlnAlaProGlyLysGluArgGluGly ValAlaArgIleAsnIleGlySerThrSerThrTyrTyr GlyAspSerValLysGlyArgPheThrValSerSerAsp AsnAspLysAsnThrValTyrLeuGluMetAsnSerLeu GluProGluAspThrValThrTyrTyrCysAlaAlaGln ProAsnTyrCysTyrLeuPheSerTyrSerAsnLysLeu ThrTyrTrpGlyGlnGlyThrGlnValThrValSerSer 7 MetAspValGlnLeuGlnGluSerGlyGlyGlyLeuVal GlnProGlyGlySerLeuArgLeuSerCysAlaThrSer GlyPheThrPheAsnSerTyrTrpMetTyrTrpValArg GlnAlaProGlyLysGlyProGluTrpValAlaArgIle AsnThrArgThrProArgIleThrTyrThrAspSerVal LysGlyArgPheThrIleSerArgAspAsnAlaLysAsn ThrLeuTyrLeuGlnMetAsnSerLeuLysProGluAsp ThrAlaLeuTyrTyrCysThrThrAsnArgAsnArgVal ValGlyGlyGlyThrGlnValThrValSerSer 8 MetAspValGlnLeuGlnGluSerGlyGlyGlySerVal GlnAlaGlyGlySerLeuArgLeuSerCysValAlaSer GlyValThrTyrSerProTyrTyrCysMetGlyTrpPhe ArgGlyGlnGluArgGluAlaValAlaSerIleThrIle GlyArgIleGlyGlyGlyGlyThr PheValAlaAspSerValLysGlyArgPheThrIleSer GlnAspAlaAlaLysAsnSerAlaTyrLeuGlnLeuAsn SerLeuArgProGluAspSerAlaIleTyrTyrCysAla AlaGlyValGlyTyrCysTyrThrArgArgLeuAspTyr AspHisTrpGlyTyrGlyThrGlnValThrValSerSer 9 MetAspValGlnLeuGlnGluSerGlyGlyGlySerVal GlnAlaGlyGlySerLeuArgLeuSerCysThrIleSer GlyTyrSerGlyTyrSerPheArgThrLeuCysValTyr TrpPheArgGlnAlaProGlyLysGlyLeuGluGlyVal AlaArgIleAsnIleGlySerThrSerThrTyrTyrGly AspSerValLysGlyArgPheThrValSerSerAspAsn AspLysAsnThrValTyrLeuGluMetAsnSerLeuGlu ProGluAspThrValThrTyrTyrCysAlaAlaGlnPro AsnTyrCysTyrLeuPheSerTyrSerAsnLysLeuThr TyrTrpGlyGlnGlyThrGlnValThrValSerSer AminoacidsequencesofProteasecleavage sequence 10 GluLeuTyrLeuVal 11 LeuValGluLeuLeuTyrLeu 12 LysArgGluAla 13 AsnAlaThrLeuAspProArgSerPheLeu LeuArgAsn Aminoacidsequencesofflexiblelinker peptides 14 GlyGlyGlyGlySer 15 GlyGlyGlyGlySerGlyGlyGlyGlySer 16 GluGluGlyGluPheSerGluAlaArg WhereGluisGlu(E)orAsp(D) 17 GlySerAlaAspAspAlaLysLysAspAla AlaLysLysAspGlyLysSer 18 SerSerAlaAspAspAlaLysLysAspAla AlaLysLysAspAspAlaLysLysAspAla

[0057] The invention provides an antibody fragment based antifungal conjugate Seq. ID 19 having in vitro MIC-99 of the conjugate against Candida spp. in the range of 0.2 ?M-0.3 ?M, more specifically, 0.25 ?M or 250 nM.

[0058] The most challenging clinical problem is the rapidly growing resistance of Candida spp. Biofilm production is one of the most important factors in developing high level of antimicrobial resistance in Candid spp. Most of the diseases caused by Candida spp. are due to biofilm formation. Biofilms are the group of microorganisms that are embedded in an extracellular matrix, forming a complex three-dimensional architecture on biotic and abiotic surfaces. Biofilms are genetically resistant to antifungal agents including amphotericin B and fluconazole. Biofilms prevent the access of the drugs to the pathogens and consequently these fungi become drug resistant that increases the morbidity and mortality of the infected patients. As currently available antifungals have minimal activity against biofilms, new drugs to treat these recalcitrant infections are urgently needed. Peptide-based therapeutics to treat drug resistant pathogens might be an alternative to conventional antibiotics.

[0059] One of the most potent salivary peptides called Histatin 5 is a cationic histidine-rich peptide present in humans and higher primates and has both antibacterial and antifungal activity. Salivary innate immunity is the first line of defense against pathogens in the oral cavity. Histatin is normally present in the oral cavity. The sequence of Histatin-5 used is conserved in Humans, Chimpanzee and Gorilla. It has 83.8% homology to Homo sapiens, 80.4 to Chimpanzees and 70% with Gorilla. The mode of action has been demonstrated to be by internalization followed by intercalation with the DNA or by membrane disruption resulting in leakage from the cells and non-energy dependent lysis. However, such broad-spectrum peptide needs to be diligently inserted into an antimicrobial peptide conjugate to control its non-specific toxicity.

[0060] Similarly, mucins, the primary structural components of mucus that creates its viscoelastic properties, are critical components of the gel layer that protect against invading pathogens. Different types of mucins exist throughout the body in various locations. In the oral cavity, decreased salivary flow is linked to the increased incidence of candidiasis and dental caries, which could be caused by reduced levels of salivary mucins. Mucins identified so far are Mucin 1-22, of which Mucin 7 is found in the oral cavity and has anti-Candida properties.

[0061] Hence, an important aspect of the present invention is to design a novel antibody fragment based antifungal conjugate targeting Candida spp. capable to assert antifungal properties on Candida biofilms as well. Therefore, the present invention provides a novel antibody fragment based antifungal conjugate, comprising of at least one antimicrobial peptide at one end of the conjugate either on the C or the N terminal end, at least one antibody fragment at the other end of the fragment, at least one protease cleavage sequence, and at least one flexible polypeptide linker, wherein, said antimicrobial peptide belongs to group comprising of cationic histidine-rich antimicrobial peptides, Histatin family of peptides, more preferably, Histatin-5; mucin family of proteins Mucin1-22, preferably, Mucin 7; and human beta defensins. The antimicrobial peptides are derived from Human histatin and mucin amino acid sequence, to reduce any unwanted immunogenic reaction from human host of administration of the antibody fragment based antifungal conjugate for treatment.

[0062] Hence, another aspect of the present invention is to provide a novel antibody fragment based antifungal conjugate targeting Candida spp. which has the ability to bind to the surface of individual microbes and their biofilms and assert antifungal properties. Accordingly, the invention provides an antibody fragment based antifungal conjugate, comprising of at least at least one antibody specifically recognizing biofilm abundant antigens, more specifically, enolase. Enolase is secreted by Candida spp. as a virulent protease and is present in extracellular matrix of biofilms. It has also been shown that C. albicans enolase is an immunodominant antigen. Hence, directing the antifungal conjugate by anti-enolase VHH enables the conjugate to bind to the surface of and to the biofilms of Candida spp. successfully, thereby enabling it to assert the antifungal properties of the antimicrobial peptide. Moreover, it is essential to ensure target specific activity of the conjugate which is non-toxic to host. The antifungal conjugate is activated only after it encounters the target pathogen. To remove the toxic effect at undesired place, the antimicrobial peptide is fused to a larger antibody fragment that is wrapped around it, reducing its toxic effects both during the bacterial drug production stage and the therapeutic application stage. The antibody is specific to the pathogen which targets the antimicrobial peptide and the antibody conjugate to the surface of the pathogen, thereby reducing non-target action of the antimicrobial peptide. Additionally, the VHH targeting Candida spp. may be humanized by mutating amino acid sequence to reduce chance of eliciting an immune response in human host against the antifungal molecule.

[0063] Additionally, the invention provides an antibody fragment based antifungal conjugate, which acts as a prodrug and is activated only upon encountering the pathogen to reduce non-toxicity to host and non-target activation. Therefore, the invention provides an antibody fragment based antifungal conjugate, comprising of at least one antimicrobial peptide at one end of the conjugate either on the C or the N terminal end, at least one antibody fragment (VHH) at the other end of the fragment, at least one protease cleavage sequence, and at least one flexible polypeptide linker, wherein, the protease cleavage specific sequence is cleaved upon interaction with membrane, cell wall associated, or secreted protease of Candida spp., or host neutrophil specific proteases, more preferably, SAP1 of Candida spp. The protease cleavage sequence and the flexible polypeptide linker sequence are placed in tandem in between the antimicrobial peptide and antibody fragment. The conjugate is a non-toxic prodrug and gets activated only upon interaction of the VHH of the conjugate with Candida spp., thereby initiating a cascade of reactions leading to cleavage of the protease cleavage site of the conjugate releasing the antimicrobial peptide from the conjugate to act against the Candida spp. The antimicrobial peptide is now released from the prodrug and is capable to assert antimicrobial properties against the pathogen. Optionally, the protease cleavage sequence may be specific to host neutrophil proteases, to clear neutrophil ingested pathogen. Optionally, a combination of antibody fragment based antifungal conjugate comprising of conjugates having pathogen specific protease cleavage sequence, and conjugates having host neutrophil specific proteases cleavage sequence can be used to defend against both free pathogens and neutrophil ingested pathogens.

[0064] The three most significant extracellular hydrolytic enzymes produced by C. albicans are the secreted aspartyl proteinases (SAP), phospholipase B enzymes, and lipases. Of these, the SAP proteins, encoded by a family of 10 SAP genes, have been the most comprehensively studied as key virulence determinants of C. albicans. C. albicans is not the only Candida species known to produce extracellular proteinases. Many of the pathogenic Candida species have been shown to possess SAP genes, hence SAP is common to many pathogenic Candida spp.

Example 1

Antibody Fragment Based Antifungal Conjugate Design and its Mode of Action against pathogen

[0065] As depicted in FIG. 1a the antibody fragment based antifungal conjugate (100) comprises of antimicrobial peptide (101) followed by a linker (103) sequence and a pathogen specific antibody fragment (102). The linker (103) additional comprises of a small protease cleavage sequence (103) susceptible to cleavage by pathogen specific proteases (105) such as membrane, cell wall associated, or secreted proteases, or are host neutrophil specific proteases (105). The antibody fragment (102) preferably a camelid VHH fragment targeting the pathogen surface or extracellular matrix antigen. The conjugate (100) is a prodrug which on encountering the pathogen initiates a cascade of reactions leading to cleavage of the protease cleavage site (103) and releases the antimicrobial peptide (101) from the antibody fragment (102).

[0066] The antibody fragment based antifungal conjugate (100) may act against the pathogen in three different modes based on the kind of protease cleavage sequence (103).

[0067] Mode 1 is depicted in FIG. 1b, where the protease cleavage sequence (103) of antibody fragment based antifungal conjugate (100) is specific to membrane or cell wall associated proteases (105). The antibody fragment based antifungal conjugate (100) targets the pathogen in the host organism, because of the affinity of the antibody fragment (102) to the antigen on the pathogen, which upon coming in contact with the pathogen membrane or cell wall (104) is susceptible to cleavage by membrane or cell wall associated proteases (105), thereby releasing the antimicrobial peptide (101).

[0068] Mode 2 is depicted in FIG. 1c, where the protease cleavage sequence (103) of antibody fragment based antifungal conjugate (100) is specific to proteases secreted by pathogens (105). The antibody fragment based antifungal conjugate (100) targets the pathogen in the host organism which upon coming in vicinity of the pathogen is susceptible to cleavage by pathogen secreted proteases, thereby releasing the antimicrobial peptide (101).

[0069] Mode 3 is depicted in FIG. 1d, where the protease cleavage sequence (103) of antibody fragment based antifungal conjugate (100) is specific to host neutrophil specific proteases (105). The antibody fragment based antifungal conjugate (100) is internalized by the host neutrophils (106) and inside the neutrophil the antibody fragment based antifungal conjugate (100) targets neutrophil ingested pathogen which upon being internalized by host neutrophil is susceptible to cleavage by host neutrophils proteases (105), thereby releasing the antimicrobial peptide (101). In all the above three modes, once the antimicrobial peptide is released from the antibody fragment based antifungal conjugate, the peptide can assert its antimicrobial properties against the pathogen.

[0070] The antibody fragment based antifungal conjugate specific against Candida albicans comprises of amino acids represented by Seq. ID 19 and Seq. ID 20,

TABLE-US-00002 Seq.ID19: MetGlyAlaLysArgHisHisGlyTyrLysArgLys PheHisGluLeuTyrLeuValGlyGlyGlyGlySer MetAspValGlnLeuGlnGluSerGlyGlyGlySer ValGlnAlaGlyGlySerLeuArgLeuSerCysThr IleSerGlyTyrSerGlyTyrSerPheArgThr LeuCysValTyrTrpPheArgGlnAlaProGlyLys GlyLeuGluGlyValAlaArgIleAsnIleGly SerThrSerThrTyrTyrGlyAspSerValLysGly ArgPheThrValSerSerAspAsnAspLysAsnThr ValTyrLeuGluMetAsnSerLeuGluProGlu AspThrValThrTyrTyrCysAlaAlaGlnProAsn TyrCysTyrLeuPheSerTyrSerAsnLysLeu ThrTyrTrpGlyGlnGlyThrGlnValThrValSer SerLeuGlu; and Seq.ID20: MetGlyGlyCysLeuAlaHisGlnLysProPheIle ArgLysSerTyrLysCysLeuHisLysArgCys ArgLysArgGluAlaGlyGlyGlyGlySerGlyGly GlyGlySerMetAspValGlnLeuGlnGluSer GlyGlyGlySerValGlnAlaGlyGlySerLeuArg LeuSerCysValAlaSerGlyValThrTyrSer ProTyrTyrCysMetGlyTrpPheArgGlyGlnGlu ArgGluAlaValAlaSerIleThrIleGlyArg IleGlyGlyGlyGlyThrPheValAlaAspSerVal LysGlyArgPheThrIleSerGlnAspAlaAla LysAsnSerAlaTyrLeuGlnLeuAsnSerLeu ArgProGluAspSerAlaIleTyrTyrCysAla AlaGlyValGlyTyrCysTyrThrArgArgLeu AspTyrAspHisTrpGlyTyrGlyThrGlnVal ThrValSerSerLeuGlu

[0071] As depicted in FIG. 2a, the Seq. ID 19 comprises of 155 amino acids of which amino acids 1-14 correspond to the antimicrobial peptide, human Histatin 5 active region, amino acids 15-19 (ELYLV) is the SAP1 specific cleavage sequence, amino acid sequence 20-24 correspond to the linker with Glycine and Serine residues (Gly).sub.4Ser in tandem, and amino acid sequence 25-155 is Seq. ID 9 which is humanized Camelid heavy chain antibody variable region fragment (VHH) specific to Candida albicans antigen enolase.

[0072] As depicted in FIG. 2b, the Seq. ID 20 comprises of 165 amino acids of which amino acids 1-24 correspond to the antimicrobial peptide, Mucin 7, amino acids 25-28 (KREA) is the fungal signal peptidase 3 specific cleavage sequence, amino acid sequence 29-38 correspond to the linker with Glycine and Serine residues((Gly).sub.4Ser).sub.2 in tandem, and amino acid sequence 38-165 is Seq. ID 8 which is Camelid heavy chain antibody variable region fragment (VHH) specific to Candida albicans surface antigen to which it shows a strong binding.

[0073] The human Histatin 5 active region amino sequence is AKRHHGYKRKFH to with two amino acids at the N terminal and two amino acids at the C terminal end may be optionally added to give stability to the peptide after it has been released from the conjugate and also to facilitate fusion to the antibody during the cloning steps.

Example 2

Anti-Candida Camelid Heavy Chain Antibody Variable Region Fragment (VHH)

[0074] Heavy chain antibody based anti-Candida molecules was developed with the ability to kill Candida spp. that possibly disrupt biofilms as well. For this purpose, camels were immunized with the extracts of Candida albicans isolated from clinical samples. The antibody library was prepared in a phage display vector in E. coli and hits were isolated after panning against microbial cell wall components and strong binders assayed for their Candida neutralizing ability.

[0075] Camelid monoclonal antibody fragments are derived from single heavy chain antibody molecules derived from camels, with low immune signature in humans, extremely small (15 kDa), with excellent stability and tissue penetrability properties. These antibodies do not need cold chain for transportation and remain stable for years at room temperature, a property, that can be exploited to develop and formulate stable antimicrobials. Furthermore, being small, they can be engineered to add value, have the ability of deep tissue penetration and disruption of biofilms. Four antibodies were isolated and sequenced with the Seq. ID 6, Seq. ID 7, Seq. ID 8, and Seq. ID 9. These antimicrobial antibodies can be used to control topical as well as invasive Candida infections.

[0076] The target for antimicrobial antibodies with amino acid sequence represented by Seq. ID 6 was identified to be extracellular matrix enolase present on the surface of the pathogen as a virulent protease also present in biofilms of Candida spp. and is an important immune antigen for Candida biofilms. This antibody was used as a backbone to produce the antimicrobial peptide antibody conjugate.

[0077] Camelid antibodies have a low immune signature but have been shown to elicit an anti-camel immune response when applied in high doses. To overcome this problem, the antibody may be optionally humanized by replacing amino acids of antibodies by mutation.

[0078] Accordingly, anti-Candida antibody with amino acid sequence represented by Seq. ID 6 was humanized by mutation to generate anti-Candida antibody with amino acid sequence represented by Seq. ID 9, wherein, the Glutamic acid (E) at position 49 of was replaced by Glycine (G), and the Arginine (R) at position 50 was replaced with Leucine (L). Hence, VHH with Seq. ID 9 is a humanized form VHH Seq. ID 6. This VHH with Seq. ID 9 is expected to have a low immune signature and can be used for large dose parenteral applications as well.

Example 3

Expression, Purification, and Specificity Test of Antibody Fragment Based Antifungal Conjugates

[0079] Conjugate with Seq. ID 19 was expressed in pET28c+ vector in the E. coli BL21(DE3) system as inclusion bodies, solubilized and purified using metal affinity and ion exchange chromatography as depicted in FIG. 3a and the Purified final product as depicted by SDS PAGE in FIG. 3b and used for further analysis.

[0080] The antibody fragment based antifungal conjugates comprise of VHH fragments against Candida specific enolase. Hence, to confirm the specificity of the VHH fragments towards enolase the following experiment was conducted. VHH fragment represented by Seq. ID 9 was expressed in E. coli strains, E2B (Rosetta Gami2 DE3), C41 (DE3), and BL21(DE3), using a suitable His-vector and isolated. The Candida enolase gene (NCBI No GenBank: M93712.1) 44 kDa, was expressed in a pET expression vector in the above mentioned three strains of E coli, the cell lysates were subjected to SDS-PAGE followed by western blotting using the VHH fragment represented by Seq. ID 9 as the primary antibody and anti-His Mouse as secondary antibody and anti-mouse HRP as tertiary antibodies. As depicted in FIG. 3c, strong signal in all the three clones in lanes 3, 6, and 9 of the western blots, confirmed the target to be the Candida specific Enolase, the glycolytic enzyme and the immunodominant antigen.

Example 4

The Conjugate is a Prodrug

[0081] The antibody fragment based antifungal conjugate which is initially a prodrug and inactive because the antimicrobial peptide is partially or wholly enclosed by the antibody component.

[0082] As depicted in FIG. 4, the purified antibody fragment based antifungal conjugate with Seq. ID 19 was compared to antibody fragment based antifungal conjugate with Seq. ID 19 exposed to pathogen Candida albicans and subjected to SDS PAGE analysis using camelid antibody of Seq. ID 9. The purified antibody fragment based antifungal conjugate with Seq. ID 19 (Lane 1) showed larger sized molecule (22 kDa) compared to antibody fragment (17 kDa) based antifungal conjugate with Seq. ID 19 exposed to Candida albicans (Lanes 2 and 3). The difference in size was comparable to that of the antimicrobial peptide, Histatin 5 active region of amino acid sequence represented by Seq. ID2. This confirmed that the conjugate was cleaved when it encountered the proteases released by the pathogen thereby releasing Histatin 5 active region.

Example 5

A. Efficiency Test of the VHH Antibody with Seq. ID 9

[0083] Purified VHH fragment against Candida enolase represented by Seq. ID 9 showed ?99% inhibition of growth of against C. albicans, C. tropicalis, C. krusei, C. parasilopsis, and C. glabrata with the MIC dose of 125 ug/ml.

TABLE-US-00003 TABLE 2 Efficacy of Seq. ID 9 against different spp. of Candida % Growth inhibition* with Seq. ID 9 (125 ug/mL) Candida albicans (Lab strain) 99.81% Candida albicans (MTCC 227) 99.54% Candida tropicalis 99.56% Candida krusei 98.16% Candida glabrata 98.85% Candida parasilopsis 99.50% *Individual controls maintained

B. Efficiency Test of the Antibody Fragment Based Antifungal Conjugate with Seq. ID20

[0084] Microbiology assays were done with the purified VHH with Seq. ID9 and the antibody fragment based antifungal conjugate of Seq. ID 19 to see their antifungal activity. As depicted in FIG. 5, the MIC-99 of the Seq. ID9VHH fragment alone was found to be 6.5 ?M (in anaerobic conditions) but the value was reduced by 20-fold for the conjugate of Seq. ID 19 with value of 0.25 ?M.

[0085] While certain exemplary embodiments have been described and shown in the accompanying drawings, it is to be understood that such embodiments are merely illustrative of, and not restrictive on, the broad invention, and that this invention not be limited to the specific constructions and arrangements shown and described, since various other changes, combinations, omissions, modifications and substitutions, in addition to those set forth in the above paragraphs, are possible. Those skilled in the art will appreciate that various adaptations and modifications of the just described embodiments can be configured without departing from the scope and spirit of the invention. Therefore, it is to be understood that, within the scope of the appended claims, the invention may be practiced other than as specifically described herein.