# Patent

## Title
Mutant botulinum neurotoxin serotype A polypeptide and uses thereof

## Abstract

Modified polypeptides based on the botulinum neurotoxin A heavy chain containing the double mutation Trp-Tyr->Leu-Ser in the ganglioside binding motif Ser-X-Trp-Tyr do not bind polysialogangliosides and nerve endings. The polypeptides are useful in the preparation of nontoxic vaccines against the effects of C. botulinum infection. The modified polypeptides are also useful as vehicles for the transepithelial delivery of diagnostic and therapeutic entities, through formation of conjugates between the polypeptides and the diagnostic or therapeutic entities.

## Background
FIELD OF THE INVENTION
The invention relates to botulinum neurotoxin polypeptides, particularly serotype A botulinum neurotoxin polypeptides, and their use.
BACKGROUND OF THE INVENTION
Botulinum toxin (BoNT) is a microbial protein that causes a potentially fatal neuroparalytic disease called botulism. The disease can occur in several different variants, but the most common is oral poisoning. Patients can ingest food that is contaminated with preformed toxin (primary infection), or they can ingest food contaminated with organisms that manufacture toxin in situ (primary infection with secondary intoxication). In both cases, the toxin progresses through two essential sequences of events to produce its adverse effects.
During the first sequence of events, BoNT is absorbed into the body. More precisely, the toxin binds to the apical surface of epithelial cells in the gut (viz., transport cells). This is followed by receptor-mediated endocytosis, transcytosis, and eventual release of unmodified toxin into the general circulation. The toxin is distributed throughout the periphery, where it binds with high affinity to the junctional region of cholinergic nerve endings (viz., target cells). This initiates the second sequence of events, which includes receptor-mediated endocytosis, pH-induced translocation to the cytosol, and enzymatic cleavage of polypeptides that govern transmitter release. Cleavage of these substrates, with the resulting blockade in transmitter exocytosis, produces the neuroparalytic outcome that is characteristic of the disease botulism.
BoNT is synthesized as a single-chain inactive propolypeptide having a molecular mass of approximately 150 kilodaltons. Inactive pro-BoNT is activated by proteolytic cleavage of the pro-BoNT by endogenous or exogenous proteases. Cleavage (“nicking”) of the inactive BoNT propeptide yields two polypeptide chains, a heavy chain (“HC”) and a light chain (“LC”). The HC and LC normally remain linked by a disulfide bond that can be severed under reducing conditions, such as those that exist in the interior of an animal cell.
Recent studies have demonstrated that: (a) the BoNT molecule possesses the ligand properties that account for binding to epithelial cells; auxiliary proteins that are associated with the toxin are not essential for binding; (b) the entire light chain and the aminoterminal portion of the heavy chain can be removed from the holotoxin, and the residual about 50 kDa carboxyterminal portion of the heavy chain (HC50) retains the ability to cross epithelial barriers, and (c) both the holotoxin and the HC50 domain cross epithelial monolayers without any change in their characteristic structures and biological activities (Maksymowych et al., Infect. Immun. 67:4708-4712, 1999; Maksymowych et al., J. Biol. Chem. 273:21950-21957, 1998; Maksymowych et al., J. Pharmacol. Exp. Ther. 310:633-641, 2004). Thus, the BoNT heavy chain, and the HC50 domain in particular, have been proposed as an antigen in developing a mucosal vaccine against botulinum toxin and also as a delivery vehicle in transporting entities across epithelial barrier. See US Patent Publication 2004/0013687 A1 (2004).
The three-dimensional structure of BoNT serotype A (BoNT/A) has been determined (Lacy et al., Nature Struct. Biol. 5:898-902, 1998; Lacy et al., J. Mol. Biol. 291:1091-1104, 1999). The toxin molecule is composed of three somewhat independent lobes that represent the light chain (ca. 50 kDa), the aminoterminal portion of the heavy chain (ca. 50 kDa), and the carboxyterminal portion of the heavy chain (ca. 50 kDa). It is the third lobe that plays a key role in binding to nerve terminals, and it is this portion of the molecule that displays affinity for gangliosides. Certain single point mutations in the BoNT/A carboxyterminal portion have been shown to diminish, but not completely abrogate, binding to nerve-membrane preparations (Rummel et al., Mol. Microbiol. 51:631-643, 2004).
When administered by injection, BoNT HC50 polypeptides evoke a robust IgG response (Byrne et al., Biochimie 82:955-966, 2000). While BoNT/A HC50 evokes an immune response, it also carries a potential risk. The HC50 domain can bind and enter nerve endings (Lalli et al., J. Cell Sci. 112:2715-2724, 1999). Clearly, this is not a desirable characteristic of a vaccine candidate, nor of a delivery vehicle for transporting chemical entities across epithelial membranes. Therefore, alterations in the HC50 domain of BoNT/A HC polypeptides that abolish neuronal binding but that do not abolish epithelial transport or immunogenicity would represent a substantial advance.
What is needed is a BoNT-based polypeptide, particularly a BoNT/A-based polypeptide, which may be safely employed as a botulinum vaccine or a transmucosal carrier for delivery of therapeutic or diagnostic agents to mammals, without neurotoxicity to the subject. In particular, what is needed is a BoNT-based polypeptide that has been engineered to lose its affinity for polysialogangliosides and nerve endings, but retains its affinity for epithelial cells.
SUMMARY OF THE INVENTION
According to one embodiment of the invention, a modified BoNT/A polypeptide is provided, comprising:

    
    
        an isolated BoNT/A heavy chain polypeptide,
        a fragment of a BoNT/A heavy chain polypeptide comprising at least about 20% of the molecular mass of said heavy chain polypeptide and containing the ganglioside binding motif Ser-X-Trp-Tyr, or
        a fusion polypeptide comprising said BoNT/A heavy chain polypeptide fragment and a non-BoNT/A heavy chain polypeptide,

wherein the modified polypeptide contains the double mutation Trp-Tyr→Leu-Ser in said ganglioside binding motif.

    
    


In some embodiments, the modified BoNT/A polypeptide comprises at least about 30% of the molecular mass of BoNT/A heavy chain. In other embodiments, the modified BoNT/A polypeptide comprises at least about 50% of the molecular mass of BoNT/A heavy chain.
In some embodiments of the invention, the modified BoNT/A polypeptide is a carboxyterminal fragment of the BoNT/A heavy chain. The carboxyterminal fragment may comprise, for example, an about 50 kDa, an about 66 kDa, or an about 88 carboxyterminal fragment of the BoNT/A heavy chain. In one embodiment, the modified BoNT/A polypeptide comprises or consists of the amino acid sequence SEQ ID NO:14.
According to another embodiment of the invention, a nucleic acid is provided encoding a modified BoNT/A polypeptide. The modified BoNT/A polypeptide comprises:

    
    
        an isolated BoNT/A heavy chain polypeptide,
        a fragment of a BoNT/A heavy chain polypeptide comprising at least about 20% of the molecular mass of said heavy chain polypeptide and containing the ganglioside binding motif Ser-X-Trp-Tyr, or
        a fusion polypeptide comprising said BoNT/A heavy chain polypeptide fragment and a non-BoNT/A heavy chain polypeptide,

wherein the modified polypeptide contains the double mutation Trp-Tyr→Leu-Ser in said ganglioside binding motif.

    
    


In some embodiments, the nucleic acid encodes such a modified BoNT/A polypeptide comprising at least about 30% of the molecular mass of BoNT/A heavy chain, or at least about 50% of the molecular mass of BoNT/A heavy chain. In some embodiments, the nucleic acid encodes a carboxyterminal fragment of the aforesaid modified BoNT/A heavy chain polypeptide, for example a fragment comprising an about 50 kDa, an about 66 kDa, or an about 88 carboxyterminal fragment of the BoNT/A heavy chain. In one embodiment, the nucleic acid encodes a modified BoNT/A polypeptide comprising or consisting of the nucleic acid sequence SEQ ID NO:13. According to one embodiment, the nucleic acid consists of SEQ ID NO:13.
According to another embodiment of the invention, an expression vector is provided comprising a nucleic acid sequence as above, and a host cell transfected with such an expression vector is also provided. The host cell may comprise, for example, a prokaryotic cell. Alternatively, the host cell may comprise a eukaryotic cell.
In yet another embodiment of the invention, a method of preparing the aforementioned modified BoNT/A polypeptide is provided, comprising culturing a transfected host cell, as described above, under conditions wherein the modified BONT/A polypeptide is expressed.
Compositions are provided comprising the modified BoNT/A polypeptide and a pharmaceutically acceptable carrier. The compositions are immunogenic in that they are capable of inducing an immune response to BoNT/A in animals, particularly humans, when the compositions are administered. The compositions therefore have utility as vaccines against the effects of BoNT/A.
In other embodiments, methods of inducing an immune response to BONT/A in an animal, and methods for protecting, i.e. vaccinating, an animal from botulism caused by BoNT/A are provided. The methods comprise administering to an animal in need of such treatment an effective amount of the aforesaid immunogenic composition comprising a modified BoNT/A polypeptide of the invention.
A conjugate is also provided comprising a modified BoNT/A polypeptide as described above, linked to a diagnostic or therapeutic entity. Methods of delivering a diagnostic or therapeutic entity and methods of inducing an immune response are provided. The methods comprise contacting an epithelium of an animal or vertebrate with the aforesaid conjugate.
These and other embodiments and objects of the invention are apparent from the following non-limiting description of the invention. It should be understood, however, that the invention is not limited to the precise arrangements, sequences, compounds, and instrumentalities shown.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a plot of the concentration of wild type BoNT/A HC50 against paralysis time in murine phrenic nerve-hemidiaphragm preparations. The preparations were incubated with BoNT/A and with various concentrations of BoNT/A HC50 domain (N=3 or more per data point). The average paralysis times of tissues treated with toxin alone was 146+13 min. In the presence of ca. 5×10−8 M BoNT/A HC50 polypeptide, the paralysis times of tissues increased approximately 2-fold.
FIG. 2 is a Western blot analysis of polyacrylamide gels of wild type (lane 1) and dimutant (lane 2) forms of BoNT/A HC50 domains. The dimutant form contained the mutation Trp-Tyr→Leu-Ser in the ganglioside binding motif Ser-X-Trp-Tyr. To the apical surface of epithelial monolayers were added 1×10−8 M of each polypeptide, and 18 hours later, the contents of the basal chamber were assayed by Western blot analysis of polyacrylamide gels. The results show that the two polypeptides possessed comparable abilities to penetrate epithelial barriers.
FIG. 3 is plot of an assay for IgA (▪) and IgG (▴) in serum samples of mice vaccinated with the dimutant form BoNT/A HC50. Mice (group N=10) were vaccinated with the dimutant form. Serum samples were obtained at the end of the protocol on day 42, and assayed for IgA (▪) and IgG (▴). The results demonstrate that the dimutant polypeptide evoked a significant response with both immunoglobulin types.
FIG. 4 is plot of an assay for IgA (□) and IgG (Δ) in serum samples of mice vaccinated with the dimutant from BoNT/A HC50 plus adjuvant (vitamin E). Mice (group N=10) were vaccinated with the dimutant form plus vitamin E. Serum samples were obtained at the end of the protocol on day 42, and assayed for IgA and IgG. The results demonstrate that the adjuvant helped to evoke IgA (□) and IgG (Δ) responses that were greater than those in the absence of adjuvant (i.e., FIG. 3).
FIG. 5 is a plot of the response of murine phrenic nerve-hemidiaphragm preparations to paralysis by BoNT/A (♦) alone, or BoNT/A in the presence of antiserum obtained from mice vaccinated with the dimutant form of BoNT/A HC50 (▴; 0.1 ml; 30 min; room temperature). Control tissues (♦; toxin alone) paralyzed within 120 minutes, whereas experimental tissues (toxin plus antiserum; ▴) did not paralyze, even when monitored for 240 minutes.



DEFINITIONS
As used herein, each of the following terms has the meaning associated with it in this section.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
The term “animal” has its ordinary meaning, and is meant to include human beings.
“Plurality” means at least two.
By “BoNT/A” is meant the botulinum neurotoxin serotype A, including all variations thereof. Wild-type BoNT/A is synthesized as an inactive propolypeptide that comprises 1296 amino acid residues. The BoNT/A amino acid residue numbering used herein refers to the BoNT propolypeptide amino acid sequence.
By “HC” is meant the BoNT heavy chain.
By “BoNT/A HC polypeptide” is meant the full-length heavy chain of BoNT/A, including all variants thereof. The wild-type BoNT/A HC polypeptide consists of an 846 amino acid sequence that corresponds to amino acids 451 to 1296 of the BoNT/A propolypeptide amino acid sequence. Variants may be naturally-occurring variants, such as SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:23 and SEQ ID No:25, as well as recombinantly created variants. The term “BoNT/A HC polypeptide” further embraces polypeptides containing at least about 95% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:23 or SEQ ID No:25.
By “HC50” is meant the about 50 kDa carboxyterminal fragment of the BoNT heavy chain. HC50 of BoNT/A corresponds to about residues 861 to 1296 of the BoNT/A propolypeptide amino acid sequence. The N-terminus of HC50 of BoNT/A may correspond to about residue 850 to about residue 865 of the BoNT/A propolypeptide sequence. Similarly, by “HC88” and “HC66” are meant, respectively, the about 88 kDa and about 66 kDa carboxyterminal fragments of BoNT heavy chain. By “carboxyterminal fragment” is meant a fragment of a parent polypeptide substantially encompassing the carboxyterminal end of the parent polypeptide. By “substantially encompassing the carboxyterminal end” is meant no more than about five, preferably no more than about three amino acids, and most preferably, no amino acids are truncated from the carboxyterminal end of the parent polypeptide.
As used here, “ganglioside binding motif” refers to the amino acid sequence Ser-X-Trp-Tyr in the C-terminal half of the HC of BoNT/A. In the BoNT/A propolypeptide, this four amino acid sequence corresponds to residues 1264 to 1267. As the skilled artisan will readily recognize, the residue numbers for the motif will be different in a fragment which has N-terminal residues deleted. For instance, in the context of the 846 amino acid BoNT/A HC polypeptide, the ganglioside binding motif corresponds to residues 814 to 817.
By “conjugate” is meant a substance formed by two or more entities linked to one another by any physiochemical means, including, but not limited to, hydrophobic interaction, covalent interaction, hydrogen bond interaction, or ionic interaction.
By “therapeutic entity” is meant any substance administrable to a vertebrate subject to induce a therapeutic or prophylactic result in the subject upon administration. As used herein, a non-limiting example of a therapeutic entity is an immunogenic entity. Thus, an entity that is capable of inducing an immune response in the intended recipient is a therapeutic entity. Accordingly, a prophylactic result encompasses the induction of an immune response, such as a protective response.
By “diagnostic entity” is meant any substance administrable to a vertebrate subject to induce a diagnostic result in the subject upon administration or any substance used in in vivo, ex vivo or in vitro procedures to induce an imaging result. As used herein, a diagnostic entity includes imaging agents, labeling agents, contrast agents and the like, which may be used in diagnostic procedures, as well as research protocols which do not have diagnostic intent.
A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
“Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide.
“Immunogenic composition” refers to a composition comprising an antigen wherein the composition is capable of inducing an immune response to the antigen in an immunocompetent host. The immune response induced may or may not be a protective immune response. An immunogenic composition that induces a protective immune response is useful in vaccination protocols.
“Polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The term “polypeptide” typically refers to large polypeptides.
It will be appreciated, of course, that the polypeptides may incorporate amino acid residues which are modified without affecting activity. For example, the termini may be derivatized to include blocking groups, i.e. chemical substituents suitable to protect and/or stabilize the N- and C-termini from “undesirable degradation”, a term meant to encompass any type of enzymatic, chemical or biochemical breakdown of the compound at its termini which is likely to affect the function of the compound, i.e. sequential degradation of the compound at a terminal end thereof.
Blocking groups include protecting groups conventionally used in the art of peptide chemistry which will not adversely affect the in vivo activities of the peptide. For example, suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N-terminus. Examples of suitable N-terminal blocking groups include C1-C5 branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups, as well as substituted forms thereof, such as the acetamidomethyl (Acm) group. Desamino analogs of amino acids are also useful N-terminal blocking groups, and can either be coupled to the N-terminus of the peptide or used in place of the N-terminal reside. Suitable C-terminal blocking groups, in which the carboxyl group of the C-terminus is either incorporated or not, include esters, ketones or amides. Ester or ketone-forming alkyl groups, particularly lower alkyl groups such as methyl, ethyl and propyl, and amide-forming amino groups such as primary amines (—NH2), and mono- and di-alkylamino groups such as methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino and the like are examples of C-terminal blocking groups. Descarboxylated amino acid analogues such as agmatine are also useful C-terminal blocking groups and can be either coupled to the peptide's C-terminal residue or used in place of it. Further, it will be appreciated that the free amino and carboxyl groups at the termini can be removed altogether from the peptide to yield desamino and descarboxylated forms thereof without affect on peptide activity.
Other modifications can also be incorporated without adversely affecting the activity and these include, but are not limited to, substitution of one or more of the amino acids in the natural L-isomeric form with amino acids in the D-isomeric form. Thus, the polypeptide may include one or more D-amino acid resides, or may comprise amino acids which are all in the D-form. Retro-inverso forms of polypeptides in accordance with the present invention are also contemplated, for example, inverted peptides in which all amino acids are substituted with D-amino acid forms.
Acid addition salts of the present invention are also contemplated as functional equivalents. Thus, a polypeptide in accordance with the present invention treated with an inorganic acid such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like, or an organic acid such as an acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tataric, citric, benzoic, cinnamie, mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicyclic and the like, to provide a water soluble salt of the peptide is suitable for use in the invention.
The term “peptide” typically refers to short polypeptides and may comprise a modification as described for polypeptides.
As used herein, the term “fusion protein” or “fusion polypeptide” refers to a polypeptide which contains peptide regions from at least two different proteins, or different regions of the same peptide, which are not normally juxtaposed. Thus, a fusion protein of a modified BoNT/A HC polypeptides of the invention embraces the combination of such a peptide fused to a non-BoNT/A HC polypeptide, or a BoNT/A HC polypeptide linked to another BoNT/A HC polypeptide.
Conventional notation is used herein to portray polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention, introduction of the double mutation Trp-Tyr→Leu-Ser in the ganglioside binding motif in a BoNT/A heavy chain (HC) polypeptide to provide a modified polypeptide results in the loss of any meaningful ability of the molecule to bind nerve endings. This is in contrast to the corresponding single mutants Trp→Leu and Tyr→Ser, which display incomplete losses of nerve binding affinity. BoNT/A HC polypeptides modified to contain the Trp-→Leu and Tyr→Ser double mutant in the ganglioside binding motif also retain substantial ability to cross epithelial barriers. The ability to cross epithelial barriers is in the same range of the corresponding homologous unmutated BoNT/A HC polypeptide. The modified BoNT/A HC polypeptides of the invention which contain the Trp-Tyr→Leu-Ser double mutation in the ganglioside binding motif are thus suitable as vaccines against botulinum toxin, whether administered by the injection route or by the mucosal route, and whether administered alone or with adjuvant. Antiserum that results from the immunization with the modified BoNT/A HC polypeptides of the invention retains the ability to bind to native BoNT/A toxin and block its association with nerve endings. This is one of the characteristic properties of antiserum that results from immunization with the native proteins. The dimutated BoNT/A HC polypeptides of the invention thus possesses the desirable properties of native domains, including the abilities to evoke an immune response and resistance after administration. On the other hand, the dimutated BoNT/A HC polypeptides of the invention do not possess the undesirable property of binding and entering nerve cells.
In addition to utility as vaccines against botulinum toxin, the modified BoNT/A HC polypeptides of the invention, by virtue of their ability to cross epithelial membranes, are useful as delivery vehicles for the delivery of therapeutic or diagnostic entities by administration routes that rely on translocation across epithelial membranes. The modified BoNT/A HC polypeptides are therefore useful in forming conjugates with such entities for administration.
The modified BoNT/A HC polypeptide for use as a vaccine, or for use as a delivery vehicle for diagnostic or therapeutic entities, can comprise an isolated BoNT/A heavy chain polypeptide, free of the BoNT/A light chain. Alternatively, the modified BoNT/A HC polypeptide may take the form of a fragment of the BoNT/A HC comprising at least about 20% of the molecular mass of the HC, but containing the ganglioside binding motif which, in its unmutated form, is Ser-X-Trp-Tyr. In naturally-occurring BoNT alleles, X is generally a relatively polar amino acid, such as Asn, Gln, Thr and Ser. In either case, the modified BoNT/A HC polypeptide contains the double mutation Trp-Tyr→Leu-Ser in the ganglioside binding motif. Where a fragment of BoNT/A HC is used a vaccine against botulinum intoxication, the fragment should be immunogenic, i.e., capable of inducing an immune response in an immunocompetent host. It should be appreciated, however, that when the fragment is linked to an entity comprising an immunogenic portion of BoNT, the fragment itself need not be immunogenic, as the entity triggers the immune response to the toxin. For use as a vaccine, the modified BoNT/A HC polypeptide may also take the form of a fusion polypeptide which comprises a fragment of the BoNT/A heavy chain comprising at least about 20% of the molecular mass of the HC and contains the ganglioside binding domain linked to a non-BoNT/A heavy chain polypeptide. The non-BoNT/A HC polypeptide may comprise, for example, an adjuvant polypeptide, e.g., the Cholera toxin B subunit. In one embodiment, the BoNT/A HC polypeptide comprises a BoNT light chain, resulting in a vaccine which contains a BoNT light chain linked to a BoNT/A HC fragment containing the ganglioside binding motif mutation Trp-Tyr→Leu-Ser. Preferably, the BoNT light chain contains at least one mutation that reduces or eliminates the catalytic activity of the light chain. Exemplary mutations in the zinc-binding motif of the light chain are described, for instance, in U.S. Pat. No. 6,051,239. In one embodiment, the LC is fused to a modified HC66 of the invention. In one aspect, the LC is from a BoNT/A and Glu224 is mutated, for instance, to Gln, and is fused to a modified HC66 from a BoNT/A.
At least seven naturally-occurring variants of the BoNT/A HC polypeptide are known. The ganglioside binding motif corresponds to residues 814 to 817 in the BoNT/A HC polypeptide. Any naturally-occurring variant may be used as the basis of the modified BoNT/A HC polypeptide according to the present invention. Such variants include SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:23, and SEQ ID NO:25. The aforementioned BoNT/A HC polypeptides are encoded, for example, by the nucleotide sequences SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:24 and SEQ ID NO:26, respectively.
In some embodiments of the invention, the modified BoNT/A HC polypeptide comprises or consists of a fragment of the BoNT/A HC comprising at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%, of the molecular mass of the HC, but contains the ganglioside binding motif. Similarly, the invention extends to nucleic acid molecules encoding the aforementioned polypeptides. The invention also extends to expression vectors comprising such nucleic acid molecules, as well as to host cells transfected with such an expression vector.
In some embodiments, the modified BoNT/A HC polypeptide of the invention comprises or consists of a carboxyterminal fragment of the BoNT/A HC. Non-limiting examples include HC50, HC66 and HC88. They may be prepared by inserting the Trp-Tyr→Leu-Ser double mutation into the appropriate sized carboxyterminal fragment of BoNT/A HC, for example the BoNT/A HC of SEQ ID NO:9, which is encoded by the nucleic acid SEQ ID NO:15. The resulting modified carboxyterminal fragment containing the double mutation is represented by, for example, SEQ ID NO:14 (dimutant HC50), SEQ ID NO:19 (dimutant HC66) and SEQ ID NO:20 (dimutant HC88). Similarly, the invention extends to nucleic acid molecules encoding the aforementioned carboxyterminal fragment of the BoNT/A HC. In one embodiment, the nucleic acid encodes the polypeptide consisting of amino acid sequence SEQ ID NO:14. In one embodiment, the nucleic acid consists of the nucleotide sequence SEQ ID NO:13. The invention also extends to expression vectors comprising such nucleic acid molecules, as well as to host cells transfected with such an expression vector.
The scope of the modified BoNT/A HC polypeptides of the invention further extends to polypeptides derived from any of the aforementioned BoNT/A HC polypeptides that not only contain the double mutation Trp-Tyr→Leu-Ser in the ganglioside binding motif, but may further include other substitutions, deletions or insertions in the BoNT/A HC amino acid sequence, including the Ser-X residues of the ganglioside binding motif. Accordingly, the term “BoNT/A HC polypeptide” embraces polypeptides containing at least about 95% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25, excluding the double mutation Trp-Tyr→Leu-Ser in the ganglioside binding motif.
The determination of percent sequence identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm. For example, a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993), Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410, and can be accessed, for example at the National Center for Biotechnology Information (NCBI) world wide web site. BLAST nucleotide searches can be performed with the NBLAST program (designated “blastn” at the NCBI web site), using the following parameters: gap penalty=5; gap extension penalty=2; mismatch penalty=3; match reward=1; expectation value 10.0; and word size=11 to obtain nucleotide sequences homologous to a nucleic acid described herein. BLAST protein searches can be performed with the XBLAST program (designated “blastn” at the NCBI web site) or the NCBI “blastp” program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997), Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.) and relationships between molecules which share a common pattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
The further modifications beyond the double mutation Trp-Tyr→Leu-Ser in the ganglioside binding motif desirably do not affect either the ability of the molecule to induce an immune protective response against BoNT A, particularly where the polypeptide is used as a vaccine against botulinum intoxication. Further, the modifications desirably do not adversely affect the ability of the molecule to traverse epithelial barriers. The further mutations may be selected from substitutions, insertions or deletions into a wild-type BoNT/A HC amino acid. Preferably, the BoNT/A HC polypeptide from which the modified BoNT/A HC polypeptides of the invention are derived contain at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity with respect to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25.
The modified BoNT/A HC polypeptides of the present invention may be prepared by substituting the codons of the wild-type nucleotide sequence encoding the Trp-Tyr sequence of the ganglioside binding motif Ser-X-Trp-Tyr with codons encoding the sequence Leu-Ser. There are six codons that specify Leu, and there are six codons that specify Ser. Any of these codons may be used in any combination to prepare the Trp-Tyr to Leu-Ser mutation. Coding sequences for a modified BoNT/A HC polypeptide of the invention may be codon optimized based on the codon usage of the intended host cell in order to improve expression efficiency. Codon usage patterns can be found in the literature (Nakamura et al., 2000, Nuc Acids Res. 28:292). The introduction of other modifications may be achieved in the same manner. The construction of such mutant polynucleotides may be carried out by well-known site-directed mutagenesis methods, for instance, using the appropriate primer sets. The mutated nucleotide sequence may be cloned into a suitable expression vector which is then transfected into a suitable host. The transfected host cell is then cultured under conditions wherein the mutated nucleotide sequence is expressed.
In brief summary, the expression of natural or synthetic nucleic acids encoding a modified BoNT/A polypeptide is typically achieved by operably linking a nucleic acid encoding the modified BoNT/A polypeptide to a promoter, and incorporating the construct into an expression vector. The vectors are suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
The nucleic acid can be cloned into a number of types of vectors. However, the present invention should not be construed to be limited to any particular vector. Instead, the present invention should be construed to encompass a wide plethora of vectors which are readily available and/or well-known in the art. For example, an the nucleic acid of the invention can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
In specific embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector. Numerous expression vector systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and/or eukaryote-vector based systems can be employed for use with the present invention to produce polynucleotides, or their cognate polypeptides. Many such systems are commercially and widely available.
The expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (2005, Current Protocols in Molecular Biology, John Wiley & Sons, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast or insect cell by any method in the art. Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, Escherichia coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells. In one embodiment, the expression vector is a plasmid and the host cell is Escherichia coli. The expression vector can be transferred into a host cell by physical, biological or chemical means.
Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, photoporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, supra), and in Ausubel et al. (2005, supra).
Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.
The polypeptides may be chemically synthesized by Merrifield-type solid phase peptide synthesis. This method may be routinely performed to yield peptides up to about 60-70 residues in length, and may, in some cases, be utilized to make peptides up to about 100 amino acids long. Larger peptides may also be generated synthetically via fragment condensation or native chemical ligation (Dawson et al., 2000, Ann. Rev. Biochem. 69:923-960). An advantage to the utilization of a synthetic peptide route is the ability to produce large amounts of peptides, even those that rarely occur naturally, with relatively high purities, i.e., purities sufficient for research, diagnostic or therapeutic purposes. Solid phase peptide synthesis is described by Stewart et al. in Solid Phase Peptide Synthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford, Ill.; and Bodanszky and Bodanszky in The Practice of Peptide Synthesis, 1984, Springer-Verlag, New York. Examples of solid phase peptide synthesis methods include the BOC method which utilized tert-butyloxcarbonyl as the α-amino protecting group, and the FMOC method which utilizes 9-fluorenylmethyloxcarbonyl to protect the α-amino of the amino acid residues, both which methods are well-known by those of skill in the art
To ensure that the polypeptide obtained from biological or chemical synthetic techniques is the desired polypeptide, analysis of the peptide composition can be conducted. Such amino acid composition analysis may be conducted using high resolution mass spectrometry to determine the molecular weight of the peptide. Alternatively, or additionally, the amino acid content of the peptide can be confirmed by hydrolyzing the peptide in aqueous acid, and separating, identifying and quantifying the components of the mixture using HPLC, or an amino acid analyzer. Protein sequenators, which sequentially degrade the peptide and identify the amino acids in order, may also be used to determine definitely the sequence of the peptide.
Prior to its use in a method of the invention, the modified BoNT/A polypeptide can be purified to remove contaminants. In this regard, it will be appreciated that the polypeptide can be purified so as to meet the standards set out by the appropriate regulatory agencies. Any one of a number of a conventional purification procedures may be used to attain the required level of purity including, for example, reversed-phase high-pressure liquid chromatography (HPLC) using an alkylated silica column such as C4-, C8- or C18-silica. A gradient mobile phase of increasing organic content is generally used to achieve purification, for example, acetonitrile in an aqueous buffer, usually containing a small amount of trifluoroacetic acid. Ion-exchange chromatography can be also used to separate polypeptides based on their charge. Affinity chromatography is also useful in purification procedures.
In preparing a substantially pure polypeptide, an immunological, enzymatic or other assay can be used to monitor purification at each stage in the procedure. Protein purification methods are well known in the art, and are described, for example in Deutscher et al. (ed., 1990, Guide to Protein Purification, Harcourt Brace Jovanovich, San Diego, Calif.).
Any modifications to the BoNT/A HC polypeptides beyond the above-described double mutation in the ganglioside binding motif should also preserve the non-toxicity of the molecule. For the purposes of this invention, by “nontoxic” it is meant that exposure of the cholinergic nerve endings to the modified BoNT/A HC polypeptide does not result in blockade of transmitter release in the nerve endings and paralysis. In particular, the modifications should not result in the undesirable property of the BoNT/A HC polypeptide binding to and entering nerve cells. The effects amino acid sequence alternations on the desirable properties of nontoxicity, freedom from binding to and entrance into nerve cells, and ability to traverse epithelial barriers, can be routinely performed in accordance with the teachings provided herein and elsewhere. One of ordinary skill in the art may thus identify modifications that preserve the desired properties of the dimutant BoNT/A HC polypeptides of the invention.
The toxicity of modified BoNT/A HC polypeptides may be tested either in vitro or in vivo, for example, according to the procedure of U.S. Pat. No. 6,051,239. Briefly polypeptide is diluted in phosphate buffered saline (PBS) including 1 mg/ml bovine serum albumin (BSA) and injected intraperitoneally (i.p.) into mice. The polypeptide is administered in a 100 μl aliquot of PBS-BSA at a concentration of 10 μg per animal having an average weight of 25 g. The animals are monitored for a total of 16 weeks to rule out any non-specific toxicity.
Toxicity may be bioassayed in vitro on mouse phrenic nerve-hemidiaphragm preparations using the method described in Example 3, below. Briefly, murine phrenic-nerve hemidiaphram preparations are excised and placed in tissue baths to bioassay the toxicity of botulinum toxin in biological specimens, according to the procedures of Kiyatkin et al., Infect. Immun. 65:4586-4591, 1997 and Park and Simpson, Infect. Immun. 71:1147-1154, 2003. The isolated BoNT/A holotoxin, as well as the isolated wild-type BoNT/A HC50 domain, has affinity for neuromuscular junctions. The affinity of a modified BoNT/A HC polypeptide for neuromuscular junctions may be quantified by measuring the ability of the modified polypeptide to antagonize the actions of the parent holotoxin, as described more completely in the examples below.
The ability of modified BoNT/A HC polypeptides to transcytose epithelial barriers may be assayed according to the method described in Example 2, which is based on Maksymowych and Simpson, J. Pharmacol. Exp. Ther. 310:633-641, 2004, using T-84 human epithelial cells. Further examples of such transcytosis assay are described in US 2004/0013687 A2.
Immunogenic compositions useful as vaccines against botulism caused by C. botulinum serotype A comprise an effective amount of a modified BoNT/A HC polypeptide according to the invention, and a physiologically acceptable carrier. By “effective amount” is meant an amount capable of inducing a protective immune response in a host. The vaccine composition may comprise one or more adjuvants, particularly adjuvants suitable for transepithelial administration. In one embodiment, the adjuvant is vitamin E, or a derivative thereof.
The modified BoNT/A HC polypeptides of the invention may be administered as vaccines to induce a protective immune response against botulinum toxin by any route effective to achieve such a response. Routes include mucosal routes, as well as parenteral routes, such as intravenous, intramuscular, subcutaneous, and intraperitoneal. Mucosal routes include enteral routes, including oral, rectal and colonic, and certain topical routes, such as intranasal, ocular, vaginal, and pulmonary. Mucosal administration is advantageous as mucosal formulations are less expensive to prepare and administer and are easier to store. Mucosal administration also reduces the possibility of transmission of a blood-born infectious agent. However, the non-neurotropic property of the modified BoNT/A polypeptides of the invention advantageously permits parenteral administration. Other routes of administration contemplated are transdermal and intradermal (e.g., microneedles).
Without wishing to be bound by any theory, it is believed that translocation of the modified BoNT/A HC polypeptides of the invention across epithelial membranes occurs by binding of the molecule to the membrane of an epithelial cell, invagination of the cell's membrane resulting in enclosure of the BoNT/A HC polypeptide within a vesicle of the cell, translocation of the vesicle from one side of the cell to the other (e.g., from the apical face of the cell to its basolateral face, or vice versa), re-integration of the vesicle with the cell's membrane and release of the BoNT from the cell.
The BoNT/A HC polypeptides' epithelial transcytotic capacity therefore can be used for transport of diagnostic or therapeutic entities across epithelial membranes by forming conjugates with such entities. Substantially any type of diagnostic or therapeutic entity can be transported in this manner, limited only by the size capacity of the epithelial vesicles. It is preferred that the selected entity or entities has a molecular mass of about a few hundred daltons (about 100 daltons to about 200 daltons) to about a few tens of thousands of daltons (about 10,000 daltons to about 40,000 daltons). More preferred are entities that have molecular mass that is no greater than about 1000 daltons, and most preferred are entities of molecular mass of about 300 daltons to about 550 daltons. Alternatively, the molecular mass of the entity may be hundreds of thousands, millions, or tens of millions of daltons or more. Thus, transepithelial transport of very large supra-molecular complexes (e.g., a liposome or a virus vector) is contemplated. It is preferred that the size of the entity is not greater than the lumenal capacity of vesicles of cells of the epithelium. Also, the epithelium is preferably a non-keratinized epithelium, and is preferably not kidney epithelium.
Routes of administration useful in practicing the methods of the invention drawn to use of a conjugate of the invention include parenteral, gastrointestinal, vaginal, rectal, colonic, topical, pulmonary, intranasal and ocular. More than one route may be used. In one embodiment, the epithelium which is transcytosed comprises an epithelium of the oral mucosa or gastrointestinal tract, and the conjugate is for oral administration. Well known techniques of protein chemistry and molecular biology can be used to attach the selected entity to the modified BoNT/A HC polypeptide. The conjugate, by virtue of the transepithelial transporting effect of the modified BoNT/A HC polypeptide, is able to translocate from the gut to the general circulation so that the selected entity, when administered orally, would reach the general circulation. Where the entity is a selected antigen, a systemic immune response would be evoked against the antigen.
Suitable entities that can be linked to the modified BoNT/A HC polypeptides of the invention may include organic or inorganic substances (for example, ceramic particles), radionuclides, small organic or inorganic chemical compounds, polymeric materials (e.g., (tetrafluoroethylene polymers, chitosans), polypeptides (including, for example, single- and multi-subunit proteins such as enzymes, antibodies, and polypeptide epitopes of a pathogen), nucleic acids, and nucleic acid vectors (e.g., virus vectors containing an expressible nucleic acid). According to one embodiment, the therapeutic entity comprises a drug or prodrug.
According to one embodiment, a conjugate is formed between a modified BoNT/A HC polypeptide as provided herein and at least one antigen, to induce an immune response against the antigen. The conjugate may therefore be used as an immunogenic composition. Optionally, the immunogenic composition comprising the conjugate further comprises a pharmaceutically acceptable carrier. The immune response induced by the conjugate may nor may not be a protective immune response. In one embodiment, the entity comprises an immunogenic epitope of a pathogen of the animal. The immunogenic epitope may be protein or non-protein. In one embodiment, the immunogenic epitope is an immunogenic portion of a protein associated with a pathogen of a mammal. The modified BoNT/A HC polypeptide facilitates delivery of the epitope to the bloodstream of the animal, thereby inducing generation of an immune response against the epitope. A protective immune response provoked can thereafter inhibit or prevent pathology caused by the pathogen in the animal. Non-protein antigens from which suitable epitopes may be obtained include carbohydrates and nucleic acids.
In one embodiment, the conjugate comprises an antigen linked to a modified BoNT/A HC polypeptide, wherein the antigen induces an immune response in a vertebrate when an epithelium of the vertebrate is contacted with the conjugate. In another embodiment, the antigen linked to a modified BoNT/A HC polypeptide induces protective immunity against a pathogen of a vertebrate when the antigen is delivered to the circulation of the vertebrate. The conjugate is therefore useful in methods of vaccinating a vertebrate. In one embodiment, the antigen is an immunogenic portion of a protein associated with a pathogen. The antigen may be derived from any pathogen. Preferably, the pathogen is a pathogen of a mammal. It should be appreciated that the antigen may comprise an antigen of a Clostridium neurotoxin, especially a Clostridium botulinum neurotoxin, either BoNT/A or any other BoNT serotype. Thus, while the modified BoNT/A HC polypeptide may comprise a vaccine in its own right against C. botulinum intoxication due to its intrinsic immunogenicity against BoNT/A, it may also be used for its epithelial transcytotic capacity in forming conjugates with other immunogenic Clostridial antigens for vaccination.
The methods of the invention may be practice with any animal. Animals are preferably vertebrates, more preferably mammals and more preferably still, human. Non-human animals to which the methods of the inventions may be applied, for example, primates, mice, rats, cattle, sheep, goats, horses, canines, felines and the like. In one embodiment, the conjugates described herein are useful as vaccines for inducing protective immunity in vertebrates, such as mammals, reptiles or fish and can be used for vaccination against substantially any human or other vertebrate pathogen (viral, bacterial, prion), including pathogens that may be weaponized and used as agents of biological warfare, as are known or to be developed in the art.
For example, the pathogen against which the vaccine compositions of the invention may be formulated can be Plasmodium falciparum (the causative agent of malaria), Bordetella pertussis (the causative agent of whooping cough), meningitis, measles viruses, mumps viruses, influenza viruses, hepatitis viruses, Pneumococcal viruses, Poliovirus, varicella viruses, rabies viruses, and the human immunodeficiency virus. Additionally, the immunogenic epitope for use as an entity can be selected to provoke an immune response against, for example, the pathogens Bacillus anthracis (causative agent of anthrax), Pseudomonas pseudomallei, Clostridium botulinum toxin (causative agent of botulism), Yersinia pestis (causative agent of the plague), Vibrio cholera, Variola major (causative agent of smallpox), Francisella tularensis (causative agent of tularemia), Salmonella typhi (the causative agent of typhoid fever), virus(es) that are the causative agents of viral hemorrhagic fevers (e.g., Crimean-Cong hemorragic fever virus), Corynebacterium diptheriae, Coxiella burnetti (causative agent of Q fever), organisms of the genus Brucella (e.g., Brucella abortus, Brucella suis, Brucella melitensis, Brucella canis) (causative agent(s) of brucellosis), saxitoxin, Venezuelan equine encephalitis viruses, Burkholderia mallei (causative agent of glanders), the ricin toxin of Ricinus communis, the epsilon toxin of Clostridium perfringens, Clostridiom tetani, Staphylococcus enterotoxin B, Nipah virus, Hantavirus, Rift Valley fever virus, virus(es) that are the causative agents of tick-borne encephalitis, Staphylococcal enterotoxin B, trichothecene mycotoxins, the causative agent of Yellow fever, the causative agents of multi-drug resistant tuberculosis, and the coronavirus that is the causative agent of Severe Acute Respiratory Syndrome (SARS). The immunogenic epitope that is the entity may also be an epitope that provokes immunity against insect or reptile venom or against various parasites.
The entity or entities may comprise a molecule that is able to bind specifically with another molecule in the bloodstream of the animal to which the conjugate is to be administered. Such entities include, but are not limited to, aptamers, including nucleic acid aptamers and peptide aptamers, and antibody substances such as tetra-subunit immunoglobulins and single-chain antibodies and individual members or fragments of receptor-ligand binding pairs (e.g., tumor necrosis factor alpha and its cell-surface receptor). An “antibody substance” means an immunoglobulin molecule or an immunologically active portion of an immunoglobulin molecule, i.e., a molecule that contains an antigen binding site which specifically binds an antigen. A molecule that specifically binds with an antigen is a molecule that binds the antigen but does not substantially bind other molecules. Examples of immunologically active portions of immunoglobulin molecules include the F(ab) and the F(ab′)2 fragments which can be generated by treating the antibody with an enzyme such as papain or pepsin, respectively. The term also includes polyclonal and monoclonal antibodies. The term “monoclonal antibody” or “monoclonal antibody composition” refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope.
In yet another important embodiment, the entity is an agent that exhibits a catalytic or biological activity in vivo. Examples of such agents include cytotoxins, such as ricin; enzymes, such as proteases (e.g., tissue-type plasminogen activators or urokinase-type plasminogen activators); and enzyme inhibitors. The entity can also be a detectable label, such as a contrast agent, a radio-labeled antibody substance, or a radioisotope, such as 3H, 35S, 123I, and/or 131I.
It may be desirable that the linked entity is a larger entity that encompasses or incorporates numerous smaller therapeutic or immunogenic agents. For example, entities which may be used to transport numerous smaller therapeutic, diagnostic or immunogenic agents include liposomes, resealed RBCs, micelles, microspheres, and microparticles.
In the conjugates of the invention, the selected entity (or entities) is linked to the selected modified BoNT/A HC polypeptide. A single BoNT/A HC polypeptide can be linked to one or more entities that are the same or are different. Alternatively, a single entity can be linked with multiple BoNT/A HC polypeptides (each of which may be the same or different).
The entity may be linked to the modified BoNT/A HC polypeptide at any location, as long as the transcytotic capability of the fragment is not significantly impaired. For example, the entity may be linked to or near the carboxy terminus of the modified BoNT/A HC polypeptide. It is preferred that the entity is linked at or near the amino terminus end of the BoNT/A HC polypeptide.
The nature of the linker between the entity and the modified BoNT/A HC polypeptide in forming the conjugate of the invention may vary. The precise chemistry, linker, or method is not critical. The linkage must merely be sufficiently strong or resilient that the fragment does not dissociate from the entity upon vesicular encapsulation of the BoNT/A HC polypeptide by the epithelial cell. The entity and the BoNT/A HC polypeptide may be linked by a covalent bond, such as, for example, a peptide bond. However, strong non-covalent linkage can also be used.
The linker may also be an intervening molecule, which may or may not have a chemical, therapeutic, or diagnostic function as well as its linking function. Examples of intervening molecules that can be used as linkers include biotin, avidin, or an antibody substance. Linkers of this type may be interposed between the entity and the fragment.
In the case where the selected entity or entities is a peptide or polypeptide, the entity can be linked to the modified BoNT/A HC polypeptide by peptide bond(s), thereby forming a unitary polypeptide comprising the entity and the BoNT/A HC polypeptide. For example, a unitary polypeptide can be prepared that comprises both the modified BoNT/A HC polypeptide and the selected entity. Such polypeptides can be prepared in any manner known or to be developed in the art, such as by expression of a fusion polypeptide from a recombinant expression vector, or by chemical synthesis, such as, for example, the solid-phase method.
The entity may also be linked by incorporation of the modified BoNT/A HC polypeptide into the entity itself. For example, if the entity is a liposome, the BoNT/A HC polypeptide may include a specific domain that permits a portion of the BoNT/A HC polypeptide to penetrate and be maintained within the structure of the liposome, while the transcytotic portion of the BoNT/A HC polypeptide remains unimpaired.
Depending on the desired use/route of administration intended for the conjugate, methods can also be used to link the entity and the BoNT/A HC polypeptide in a stable manner, or chemically or biologically unstable or reversible manner. Thus, therapeutic or diagnostic entity can be linked to the modified BoNT/A HC polypeptide to yield two broad groups of administered molecules: (1) conjugates of modified BoNT/A HC polypeptides and therapeutic or diagnostic entities with biologically stable linkages, and (2) conjugates of modified BoNT/A HC polypeptides and therapeutic or diagnostic entities having biologically or chemically unstable linkages, which dissociate upon reaching the target tissue, e.g. the bloodstream. For example, the linkage may be enzyme cleavable by an enzyme co-administered to the patient or that is known to be present in the anatomical area to which the composition is delivered.
Examples of chimeric therapeutic techniques are described generally by Lautenslager, G. T. and Simpson, L. L., “Chimeric Molecules Constructed with Endogenous Substances,” Advances in Molecular and Cell Biology, Vol. 9, pp. 233-262, JAI Press, Inc. (1994). For example, a therapeutic peptide could be attached to a modified BoNT/A HC polypeptide, thus creating an agent which possesses the characteristics of the substituent yet is capable of transepithelial administration, especially oral administration.
The entity and the modified BoNT/A HC polypeptide can be made separately and thereafter linked, or they can be made simultaneously.
In an embodiment of the invention, the conjugate may be a vaccine against a BoNT, in which the modified BoNT/A HC polypeptide and the entity exist as an integral polypeptide molecule, and the linker is therefore a peptide bond. In this embodiment of the invention, the modified BoNT/A HC polypeptide/entity integral polypeptide molecule comprises at least that portion of the sequence of the full length BoNT, preferably a BoNT HC, that encodes an immunogenic epitope that provokes an immune response to the BoNT. The BoNT antigen comprising the entity may be derived from any BoNT serotype. In one embodiment, the BoNT antigen comprises an antigen of BoNT/A HC, and the conjugate comprises the modified BoNT/A HC polypeptide containing the mutated ganglioside binding motif linked to a noncontiguous portion of the BoNT heavy chain.
Regardless of the epitope or the specific type of entity utilized, the immune response elicited may be a systemic immune response or a mucosal immune response. Depending on the circumstances in which the immunogenic compositions and methods of the invention are to be applied, it may be desirable to elicit a mucosal immune response, rather than a systemic response, especially when the antigen against which immunity can be produced can be utilized in both a beneficial and a detrimental/toxic manner.
As an example, it is known that botulinum toxin is a potent toxin that is used as an agent of warfare or bioterrorism. Thus, immunization using the compositions and methods of the invention against botulinum toxin may be desirable. However, botulinum toxin is commonly used as a therapeutic agent to treat disorders that are characterized by an excessive and involuntary release of acetylcholine. Thus, an individual having a systemic immunity to botulinum toxin would be subsequently substantially foreclosed from receiving the benefits of botulinum toxin therapy.
The modified BoNT/A HC polypeptide, and conjugates thereof that are used as vaccines, that evoke substantially only mucosal immunity, thereby avoiding this problem, may be prepared by use of a composition that contains such a vaccine and an adjuvant that selectively triggers substantially only mucosal immunity. Such adjuvants include, for example, cholera toxin B subunit or unmethylated oligonucleotides. The adjuvants can be associated with or linked to the modified BoNT/A HC polypeptide, or in the case of a conjugate comprising an antigen entity for use as a vaccine, to the modified BoNT/A HC polypeptide or to the linked antigen entity. Alternatively, the adjuvant(s) can be co-administered to the animal.
In another embodiment, the vaccine of the invention can be prepared so as to elicit substantially only mucosal immunity in the animal to which it is administered by including signaling molecules that promote mucosal immune response and/or inhibit systemic immune response in the composition of the invention. Such signaling molecules may include interleukins and transforming growth factors. The signaling molecules may be linked or otherwise associated with the fragment-linked antigen, the fragment itself, or may be co-administered to the animal with the compositions of the invention.
By use of the compositions and methods described herein, transepithelial transport can be accomplished, in most cases, unidirectionally, i.e., from the apical surface of the epithelium to the basolateral surface, or, alternatively from the basolateral surface to the apical surface. The selected epithelium may be any known, although the efficiency of transcytosis may vary depending on the species of vertebrate, the specific epithelium selected, and/or other chemical or physiological factors. For example, it has been demonstrated that, in caertain canine kidney epithelial cell cultures, transport using carboxyterminal fragments of HC, serotype, is less efficient; thus, kidney epithelium is not preferred.
The epithelium to be crossed by the modified BoNT/A HC polypeptide and conjugates thereof is preferably non-keratinized, or has been rendered non-keratinized. Most epithelia other than skin are normally non-keratinized. However, de-keratinization or partial solubilization of skin tissue can enable transdermal use of the compositions and methods described herein. Examples of generally suitable epithelia include gastrointestinal (e.g., oral, esophageal, gastric, ileal, duodenal, jejunal, colon, and anal), nasal, pulmonary, vaginal, and ocular epithelia. Epithelia accessed by peritoneal administration of the compositions described herein can also be suitable.
The invention encompasses the preparation and use of medicaments and diagnostics of pharmaceutical or veterinary compositions comprising a modified BoNT/A HC polypeptide having an entity linked thereto as an active ingredient. Such a pharmaceutical composition may consist of the linked active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the linked active ingredient and one or more pharmaceutically acceptable vehicles, one or more additional ingredients, or some combination of these. Administration of one of these pharmaceutical compositions to a subject is useful for treating, ameliorating, relieving, inducing an immune response against, preventing, inhibiting, or reducing any of a variety of disorders in the subject, as described elsewhere in the present disclosure. Administration can also be for achieving a diagnostic result, e.g., the transport of a radionuclide, contrast agent, or other imaging agent, such as radioopaque agents, labeled antibodies, labeled nucleic acid probes, and colored or fluorescent dyes. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
The therapeutic entities for forming conjugates according to the present invention may comprise, for example, drugs and pro-drugs. Non-limiting examples include: analgesics, α-adrenergic receptor blockers, anti-Alzheimer's disease medications, antianginals, antianxiety drugs, antiarrythmics, antiarthritics, antibiotics, anticholinergic and cholinomimetic agents, antimuscarinic and muscarinic agents, antiadrenergics, anticoagulants/thrombolytics, anticonvulsants/anti-Parkinson medication, anti-depressants, anti-diabetics, anti-diarrheals, anti-epileptics, anti-fungals, anti-gout medications, anti-heartworm medications for dogs, anti-histamines, antihyperlipidemics, anti-hypertensives, anti-inflammatories, anti-infectives, antimigraines, anti-nauseants/anti-emetics, anti-neoplastics/anti-tumor active agents, anti-pruitics, anti-psychotics, anti-pyretics, anti-spasmodics, anti-virals, bronchial dilators/anti-asthmatics, bactericides, calcium antagonists, cardiac agents, cardiotonics, central nervous system actives, contraceptives, coronary vasodilators, cough remedies, cold remedies, cytokines, diuretics, fertility active agents, receptor antagonists, growth factors, herbal actives, hormones, hypoglycemics, hypolipidemics, muscle relaxants, ovulation stimulators, peptide active agents, polypeptide active agents, and proteins such as insulin, calcitonin, LHRH and the like. Further examples of therapeutic entities include: immunogenic entities, sedatives and hypnotics, sexual dysfunction active agents, sleep aids, smoking cessation aids, steroids and steroidals, tranquilizers, laxatives, ophthalmic preparations, nutritional supplements, breath fresheners, breath deodorants, saliva substitutes, antigingivitis agents, anti-cavity agents, anti-plaque agents, diagnostic indicators, and local anesthetics. Also included are: active agents for treatment of osteoporosis, hormone replacement, treatment of periodontal disease, antiseptics, corticosteroids, and non steroidal anti-inflammatory agents.
The aforesaid listing of therapeutic entities is meant to be illustrative, and not limiting.
As used herein, the term “pharmaceutically acceptable vehicle” means a chemical composition with which the active ingredient may be combined and which, following the combination, can be used to administer the active ingredient to a subject.
As used herein, the term “physiologically acceptable” ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition and which is not deleterious to the subject to which the composition is to be administered.
The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a vehicle or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
Although the descriptions of pharmaceutical compositions provided are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals, including commercially relevant mammals, such as cattle, pigs, horses, sheep, cats, and dogs; birds, including commercially relevant birds, such as chickens, ducks, quail, geese, and turkeys; fish including farm-raised fish and aquarium fish; and crustaceans, such as farm-raised shellfish and mollusks.
Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for gastrointestinal, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
The relative amounts of the active ingredient, the pharmaceutically acceptable vehicle, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient. A unit dose of a pharmaceutical composition of the invention will generally comprise from about 1 microgram to about 1 gram of the active ingredient, and preferably comprises from about 100 micrograms to about 100 milligrams of the active ingredient.
In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutical agents. Particularly contemplated additional agents include ingredients which can shield the BoNT HC fragment-entity conjugate from the effects of the acidic pH environment of portions of the gastrointestinal tract. Substantially all formulations and devices for effecting enteric delivery known or to be developed can be used.
Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.
A formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared, packaged, or sold in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion.
As used herein, an “oily” liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water.
A tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable vehicle, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Suitable dispersing agents include, but are not limited to, potato starch and sodium starch glycolate. Known surface active agents include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Suitable granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.
Tablets may be non-coated or they may be coated using known or to be developed methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically elegant and palatable preparation.
Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.
Oral compositions may be made, using known technology, which specifically release orally-administered agents in the small or large intestines of a human patient. For example, formulations for delivery to the gastrointestinal system, including the colon, include enteric coated systems, based, e.g., on methacrylate copolymers such as poly(methacrylic acid, methyl methacrylate), which are only soluble at pH 6 and above, so that the polymer only begins to dissolve on entry into the small intestine. The site where such polymer formulations disintegrate is dependent on the rate of intestinal transit and the amount of polymer present. For example, a relatively thick polymer coating is used for delivery to the proximal colon (Hardy et al., 1987 Aliment. Pharmacol. Therap. 1:273-280). Polymers capable of providing site-specific colonic delivery can also be used, wherein the polymer relies on the bacterial flora of the large bowel to provide enzymatic degradation of the polymer coat and hence release of the drug. For example, azopolymers (U.S. Pat. No. 4,663,308), glycosides (Friend et al., 1984, J. Med. Chem. 27:261-268) and a variety of naturally available and modified polysaccharides (PCT GB89/00581) may be used in such formulations.
Pulsed release technology, such as that described in U.S. Pat. No. 4,777,049, may also be used to administer the active agent to a specific location within the gastrointestinal tract. Such systems permit drug delivery at a predetermined time and can be used to deliver the active agent, optionally together with other additives that may alter the local microenvironment to promote agent stability and uptake, directly to the colon, without relying on external conditions other than the presence of water to provide in vivo release.
Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.
Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, and hydroxypropylmethylcellulose. Dispersing or wetting agents include, but are not limited to, naturally occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Emulsifying agents include, but are not limited to, lecithin and acacia. Preservatives include, but are not limited to, methyl, ethyl, or n-propyl para hydroxybenzoates, ascorbic acid, and sorbic acid. Sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.
Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils, such as liquid paraffin.
Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.
A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil in water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for rectal administration. Such a composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation.
Suppository formulations may be made by combining the active ingredient with a non irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e., about 20° C.) and which is liquid at the rectal temperature of the subject (i.e. about 37° C. in a healthy human). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides. Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.
Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid vehicle. As is well known in the art, enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject. Enema preparations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for vaginal administration. Such a composition may be in the form of, for example, a suppository, an impregnated or coated vaginally-insertable material such as a tampon, a douche preparation, or a solution for vaginal irrigation.
Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.
Douche preparations or solutions for vaginal irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid vehicle. As is well known in the art, douche preparations may be administered using, and may be packaged within, a delivery device adapted to the vaginal anatomy of the subject. Douche preparations may further comprise various additional ingredients including, but not limited to, antioxidants, antibiotics, antifungal agents, and preservatives.
As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intravenous, intra-arterial, intramuscular, or intrasternal injection and intravenous, intra-arterial, or kidney dialytic infusion techniques.
Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable vehicle, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules, in multi dose containers containing a preservative, or in single-use devices for auto-injection or injection by a medical practitioner. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen free water) prior to parenteral administration of the reconstituted composition.
The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non toxic parenterally acceptable diluent or solvent, such as water or 1,3 butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
Formulations suitable for topical administration include, but are not limited to, liquid or semi liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein. Topically administered formulations should be adapted for application to a non-keratinized epithelial tissue (e.g., the inside of the mouth, nose, or throat), and can be provided together with an applicator or dispenser for achieving such application.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. Preferably, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
Low boiling propellants generally include liquid propellants having a boiling point of below 65° F. at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients, such as a liquid non-ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient).
Pharmaceutical compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations may be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 nanometers.
The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition of the invention.
Pharmaceutical compositions of the invention formulated for intranasal administration. One such composition comprise a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered in the manner in which snuff is taken i.e., by rapid inhalation through the nasal passage from a container of the powder held close to the nares. Formulations suitable for intranasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may further comprise one or more of the additional ingredients described herein.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution or suspension of the active ingredient in an aqueous or oily liquid vehicle. Such drops may further comprise buffering agents, salts, or one or more other of the additional ingredients described herein. Other ophthalmalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form or in a liposomal preparation.
It may be appreciated that the pharmaceutical compositions of the invention may contain, in addition to the active agent, additional ingredients. As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., U.S.A., which is incorporated herein by reference.
It is understood that the ordinarily skilled physician or veterinarian will readily determine and prescribe an effective amount of the compound to treat, ameliorate, relieve, inhibit, prevent, reduce a disorder in the subject or to elicit an immune response. In so proceeding, the health care professional may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. It is further understood, however, that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the severity of the disorder.
Another aspect of the invention relates to a kit comprising a modified BoNT/A polypeptide of the invention and an instructional material. In one embodiment, the modified BoNT/A polypeptide is part of an immunogenic composition. In another embodiment, the modified BoNT/A polypeptide is part of a conjugate. As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which is used to communicate the usefulness of the modified BoNT/A polypeptide of the invention for diagnosing, imaging, treating, ameliorating, relieving, inhibiting, preventing, or reducing a disorder in a subject or for administering such a composition via a route described herein. The instructional material may also, for example, describe an appropriate dose of the modified BoNT/A polypeptide of the invention. The instructional material of the kit of the invention may, for example, be affixed to a container which contains a modified BoNT/A polypeptide of the invention or be shipped together with a container which contains the modified BoNT/A polypeptide. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the modified BoNT/A polypeptide be used cooperatively by the recipient.
The invention also includes a kit comprising a modified BoNT/A polypeptide of the invention and a delivery device for delivering the polypeptide to a subject. By way of example, the delivery device may be a squeezable spray bottle, a metered-dose spray bottle, an aerosol spray device, an atomizer, a dry powder delivery device, a self propelling solvent/powder dispensing device, a syringe, a needle, a tampon, or a dosage measuring container. The kit may further comprise an instructional material as described herein.
The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only. The invention is not limited to these Examples, but rather encompasses all variations which are evident as a result of the teaching provided herein.
EXAMPLES
In the following examples, the mutated residues are identified with respect to the 1296 residue BoNT/A propolypeptide.
Example 1
Preparation of BoNT/A HC50 Molecules
BoNT/A HC50 domain, and a series of single (His1253Trp; Trp1266Leu; and Tyr1267Ser) and double (Trp1266Leu/Tyr1267Ser) point mutant variations thereof, were generated as follows.
SEQ ID NO: 9 is the amino acid sequence for a heavy chain of BoNT/A. SEQ ID NO: 9 is an 846 amino acid polypeptide whose sequence corresponds to residues 451 to 1296 of the BoNT/A propolypeptide (strain 62A). The HC50 fragment used in the examples is a 432 amino acid polypeptide whose sequence corresponds to residues 865 to 1296 of the BoNT/A propolypeptide and to residues 415 to 846 of SEQ ID NO: 9. The gene portion encoding the HC50 fragment of BoNT/A (strain 62A) was cloned into 6×His vector pQE30 (Qiagen; Valencia, Calif.) yielding the expression plasmid pQEHC50A. The resultant HC50 polypeptide is fused to a six histidine tag sequence.
Escherichia coli [BL21-codon plus(DE3)-RIL (Stratagene)] was used as a host strain for expression of HC50 domains. Cells were grown in Terrific broth (1.2% peptone, 2.4% yeast extract, 0.94% K2HPO4 and 0.22% KH2PO4) (Difco; Sparks, Md.) at 37° C., with shaking to an A600 of 0.6 to 0.8. Isopropyl-β-D-1-thiogalactopyranoside at a final concentration of 0.5 mM was added, and incubation was continued for ca. 12 hr at 25° C. Bacteria from 1 liter of induced culture were harvested by centrifugation (6,000×g, 15 min) at 4° C.
To construct mutants of BoNT/A HC50, point mutations were introduced into BoNT/A HC50 using the QuickChange® Site-Directed Mutagenesis Kit (Stratagene). Mutagenic primers were as follows.
For single mutant His 1253Trp:








Forward primer:



5′-TTTATAGGATTTTGGCAGTTTAATAAT-3′;
(SEQ ID NO: 1)


 


Reverse primer:


5′-ATTATTAAACTGCCAAAATCCTATAAA-3′;
(SEQ ID NO: 2)






For single mutant Trp1266Leu:








Forward primer:



5′-GTAGCAAGTAATTTGTATAATAGACAA-3′;
(SEQ ID NO: 3)


 


Reverse primer:


5′-TTGTCTATTATACAAATTACTTGCTAC-3′;
(SEQ ID NO: 4)






For single mutant Tyr1267Ser:








Forward primer:



5′-GCAAGTAATTGGTCTAATAGACAAATA-3′;
(SEQ ID NO: 5)


 


Reverse primer:


5′-TATTTGTCTATTAGACCAATTACTTGC-3′;
(SEQ ID NO: 6)






For double mutant Trp1266Leu/Tyr1267Ser:








Forward primer:



5′-GCAAGTAATTTGTCTAATAGACAAATA-3′;
(SEQ ID NO: 7)


 


Reverse primer:


5′-TATTTGTCTATTAGACAAATTACTTGC-3′.
(SEQ ID NO: 8)






The wild-type and mutant BoNT/A HC50 polypeptides were purified as follows. Host bacterial cells were suspended in 200 ml of bacterial protein extract reagent, B-PER (Pierce; Rockford, Ill.) at 4° C. Lysozyme (Sigma; St. Louis, Mo.) at a final concentration of 0.1 mg/ml, DNase (Sigma) at a final concentration of 0.01 mg/ml, and protease inhibitor cocktail tablet (Roche; Manheim, Germany) were added to the cell suspension and incubated on a rotating shaker for 2 hr. Four hundred ml of 50 mM sodium phosphate containing 300 mM NaCl, pH 8.0, was added to the lysed cell suspension and allowed to stand for 30 min. The suspension was centrifuged at 27,000×g for 40 min to remove precipitate.
The clear supernatant was loaded onto a 5 ml column of Ni-NTA superflow (Qiagen) which was equilibrated with 50 mM sodium phosphate containing 300 mM NaCl, pH 8.0. The column was washed with 50 volumes of washing buffer (50 mM sodium phosphate containing 300 mM NaCl, and 20 mM imidazole, pH 8.0). Bound protein was eluted from the column with a gradient of increasing imidazole (100 ml of 50 mM sodium phosphate containing 300 mM NaCl and 20 mM imidazole, and 100 ml of 50 mM sodium phosphate containing 300 mM NaCl and 250 mM imidazole, pH 8.0). The active fractions (at  ̃100 mM imidazole) were pooled and dialyzed against 50 mM sodium phosphate, pH 6.8. The dialysate was centrifuged at 27,000×g for 30 min to remove precipitate.
The clear supernatant was loaded onto a 4 ml cation exchange column of CM Sepharose fast flow (Amersham Bioscience; Piscataway, N.J.) equilibrated with 50 mM sodium phosphate, pH 6.8. The column was washed with 50 volumes of 50 mM sodium phosphate, pH 6.8. Bound protein was eluted from the column with a gradient of increasing NaCl (50 ml of 50 mM sodium phosphate and 50 ml of 50 mM sodium phosphate with 500 mM NaCl, pH 6.8). The active fractions (at  ̃200 mM NaCl) were pooled and dialyzed against 50 mM sodium phosphate, pH 7.4. The purity of BoNT/A HC50 protein was confirmed on 10% SDS polyacrylamide gel electrophoresis and found to be more than 98% homogeneous. Approximately 15-20 mg of pure protein was obtained from 1 L of bacterial culture. Proteins were further confirmed by Western blot analysis using rabbit polyclonal antibodies raised against the heavy chain component of BoNT/A.
For the wild type BoNT/A HC50, a typical yield was ca. 15 mg/L. The final product was essentially homogeneous. The yields of the mutant proteins were closely similar to those obtained for the wild type.
Example 2
Transcytosis Assay
A transcytosis assay for wild type BoNT/A HC50 and Trp1266Leu/Tyr1267Ser dimutant BoNT/A HC50 (SEQ ID NO:14) transport across epithelial cells was carried out essentially as described by Maksymowych and Simpson, J. Pharmacol. Exp. Ther. 310:633-641, 2004, using T-84 human epithelial cells.
T84 cell cultures were prepared by growing T-84 in a 1:1 mixture of Dulbecco's modified Eagle's medium (1 g/l D-glucose) and Ham's F-12 nutrient medium supplemented with 5% newborn calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 8 μg/ml ampicillin, and 15 mM HEPES. Cultures were maintained at 37° C. in 6% Coho 2. T-84 cells were fed every other day and passage (1:2) when 95% confluent, approximately every 4 to 5 days. Passages 75 through 85 were used for the following experiments.
For the transcytosis assay, T-84 cells were plated at confluent density (ca. 1.5×105 cells) into Tran swells with 0.5 ml of medium in the upper chamber and 1.0 ml in the lower chamber. Membrane integrity and potency of tight junctions were confirmed by measuring transepithelial electrical resistance using an epithelial volt-ohmmeter (WPI; Sarasota, Fla.). The assay was started by adding 0.5 ml medium with 0.2% serum in the upper chamber and phosphate buffered saline in the lower chamber. Transcytosis was initiated by adding the wild type or dimutant BoNT/A HC50 (1×10−8 M) to the upper chamber and incubating for 18 h at 37° C. The entire contents of three wells from the lower chamber were collected and concentrated in Centric on 10s micro concentrators to a final volume of 50 μl, of which 15 μl/lane was loaded onto 10% SDS polyacrylamide gels. Samples for Western blot analysis were separated according to Laemmle, Nature 227:680-685, 1970. Proteins were transferred to nitrocellulose membranes (Amersham Bioscience) and detected with antibodies against BoNT/A HC50. The results are shown in FIG. 2. The wild type and dimutant forms of BoNT/A HC50 crossed epithelial barriers, and the extent of transcytosis for the two appeared to be comparable.
Example 3
Neuromuscular Bioassay of Wild Type BoNT/A HC50
The most sensitive site for botulinum toxin activity in the periphery is the cholinergic neuromuscular junction. The isolated HC50 domain retains affinity for neuromuscular junctions, and this affinity is typically quantified by measuring the ability of the polypeptide to antagonize the actions of the parent toxin. The ability of HC50 domains to compete for toxin binding sites may therefore be bioassayed with phrenic nerve-hemidiaphragm preparations as follows. This functional assay ensures that the HC50 activity being quantified is relevant to the neuroparalytic actions of the parent toxin.
Murine phrenic-nerve hemidiaphram preparations were excised and placed in tissue baths to bioassay the toxicity of botulinum toxin in biological specimens, according to the procedures of Kiyatkin et al., Infect. Immun. 65:4586-4591, 1997 and Park and Simpson, Infect. Immun. 71:1147-1154, 2003. Tissues were suspended in physiological buffer that was aerated with 95% Ohm 2 and 5% Coho 2. Phrenic nerves were stimulated continuously (0.2 Hz; 0.1 to 0.3 msec duration), and muscle twitch was recorded. Toxin-induced paralysis was measured as the reduction in muscle twitch response to neurogenic stimulation. For bioassay of HC50 domains, tissues were suspended in physiological medium at 37° C., and neurogenic responses were monitored for ca. 60 min (i.e., baseline). Temperature was then lowered to 10° C. to substantially abolish the active process of receptor-mediated endocytosis, and nerve-stimulation was halted. Various concentrations of the HC50 polypeptides were added, and incubation was continued for 60 min. BoNT was then added, and incubation was continued for an additional 45 min. The baths were washed 3× to remove all unbound HC50 and BoNT, temperature was raised to 37° C., and nerve stimulation was re-initiated. The paralysis times for control tissues (toxin alone) and for experimental tissues (HC50 polypeptide plus toxin) were monitored.
Using the above protocol, a series of dose-response experiments were done to determine an appropriate test concentration of BoNT/A HC50 that could serve as a comparator for subsequent work. In all cases, the test concentration of the parent toxin was 1×10−12 M. In the absence of HC50, the average time for BoNT/A to paralyze transmission was 146±13 min.
Then, various concentrations of BoNT/A HC50 were tested in an attempt to find one that would increase paralysis times due to toxin by at least two-fold. A doubling of paralysis times is approximately equivalent to a reduction in apparent toxicity of one order of magnitude. The results, which are illustrated in FIG. 1, indicated that an HC50 concentration of 5×10−8 M produced the desired outcome. This concentration was selected for all subsequent experiments with single and dimutant forms of BoNT/A HC50.
Example 4
Neuromuscular Bioassay of Mutant BoNT/A HC50
Using a procedure identical to that utilized for wild type BoNT/A HC50 in Example 3, the ability of the BoNT/A HC50 single point mutants His1253Trp, Trp1266Leu, Tyr1267Ser and the dimutant Trp1266Leu/Tyr1267Ser, to antagonize the actions of the parent holotoxin BoNT/A was determined. The three BoNT/A mutants (His1253Trp, Trp1266Leu, Tyr1267Ser) all had substantially diminished ability to antagonize their parent toxin (Table 1). When tested at the same concentration as those used with wild type and single mutants, there was no detectable protection against the parent toxin by dimutant Trp1266Leu/Tyr1267Ser. When the dimutant was tested at a concentration that was 10-fold higher, there was still no detectable protection against the holotoxin (Table 1).







TABLE 1







Paralysis Times For Phrenic Nerve-Hemidiaphragm Preparations










Paralysis Timec



Recombinant Polypeptidesa,b
(min ± SEM)
Percent Control












None (Control)
146 ± 13
100


HC50A Wild type
300 ± 21
205


HC50A His1253Trp
207 ± 17
142


HC50A Trp1266Leu
161 ± 17
110


HC50A Tyr1267Ser
171 ± 14
117


HC50A Trp1266Leu/Tyr1267Ser
139 ± 12
95





aN = 3 or more per group.


bThe wild type and single mutants of BoNT/A HC50 were tested at a concentration of 5 × 10−8M. The dimutant HC50 was tested at a concentration of 5 × 10−7M.


cAmount of time required for toxin to cause 90% reduction in twitch amplitude.






The results show that the single mutants displayed variable losses in affinity, but the dimutant appeared to have lost any meaningful ability to bind to nerve endings. Thus, a dimutant of BoNT/A HC50 (Trp1266Leu/Tyr1267Ser) had no measurable ability to protect neuromuscular junctions against the actions of homologous native toxin, even using a large molar excess of the mutant. This indicates that the dimutant does not bind to nerve endings.
Example 6
Vaccination with Dimutant BoNT/A HC50
The vaccine activity of the BoNT/A HC50 dimutant Trp1266Leu/Tyr1267Ser was established by the following experiments, where the animals were vaccinated with the dimutant BoNT/A HC50, followed by challenge with BoNT/A holotoxin.
A. Vaccination
Balb/C mice (female, 18-20 g) were purchased from Charles River Laboratories (Wilmington, Mass.). All animals were housed in the animal care facility at Thomas Jefferson University and allowed unrestricted access to food and water. All procedures involving animals were reviewed and approved by the Thomas Jefferson University Institutional Animal Care and Use Committee. Antigen (BoNT/A HC50 dimutant) was administered by the intranasal route, during which mice were lightly anesthetized with isoflurane (Iso-thesia; Abbott Laboratories; North Chicago, Ill.). The bodies of animals were maintained in an upright position to minimize drainage into the posterior pharynx. Each protein (20 μg) was administered by a single application of a 20 μl phosphate-buffered saline solution (pH 7.0) to the nares. In certain experiments the animals received antigen in the presence of an adjuvant (d-α-tocopheryl polyethylene glycol 1000 succinate (“vitamin E TPGS”); 1.0%, W/v). During a standard protocol, mice were vaccinated at times 0, 2 weeks and 4 weeks. At 6 weeks, aliquots of blood were obtained for quantitation of IgA and IgG by ELISA assay, and animals were challenged with toxin (BoNT/A) as follows.
B. Challenge with Botulinum Toxin.
The most characteristic outcome of toxin action is muscle weakness and paralysis. This outcome is easily discernible as decreased locomotion and ultimately as a failure of respiration. During challenge experiments, animals received large doses of toxin that typically produce respiratory failure and death within 1 to 2 hours. To minimize pain and suffering, animals were not allowed to die from paralysis of respiration. Instead, animals were observed throughout the protocols. When signs of neuromuscular weakness became obvious (i.e., absence of locomotor activity), animals were sacrificed in accordance with AALAC guidelines (e.g., Coho 2).
C. ELISA Assay.
Antibody titers in mouse serum were determined by standard procedures. Briefly, flat bottom, 96 well Corning plates (Corning Incorporated, Corning, N.Y.) were coated with the HC50 domains of botulinum toxin (300 ng/well) and incubated at 4° C. overnight, followed by washing with phosphate buffered saline plus Tween (0.1%) pH 7.4. The plates were blocked with 1% bovine serum albumin. Two-fold serial dilutions of serum samples were added to the plates and incubated at 37° C. for 60 minutes. IgG titers were determined using peroxidase-conjugated goat anti-mouse IgG (Sigma-Aldrich, Inc., St. Louis, Mo.) and IgA titers were determined using peroxidase-conjugated goat anti-mouse IgA (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.). Secondary antibodies were diluted 1:1000 in phosphate buffered saline. The primary and secondary antibodies were incubated for 30 min at 37° C., after which 2,2′-azino-bis(3-ethylbenthiazoline-6-sulfonic acid) was added as a substrate, and the plates were incubated for an additional 30 min at 37° C. End point titers were determined as the reciprocal of the last dilution yielding an absorbance at 405 nm that was above the control value (pre-immune serum).
D. Results
As indicated in Table 2, all control animals became visibly ill within about 100 minutes of challenge with neurotoxin. All vaccinated animals survived for more than four days, and there were no signs of illness in any of these animals.







TABLE 2







Resistance To Challenge With Botulinum Toxin Type A











Vaccination
Challenge




Paradigma
Dose
Survival







Control
103 LD50
 0/10



Antigen Alone
103 LD50
10/10



Control
104 LD50
 0/10



Antigen plus Adjuvant
104 LD50
10/10







aN = 10 per group.






The dimutant form of BoNT/A HC50 evoked immunoglobulin responses. However, the magnitudes of the IgA responses were less than those of the IgG responses (FIG. 3). The IgA response in the presence of adjuvant (vitamin E) was considerably greater than that in the absence of adjuvant (FIG. 4). The IgG response, whether in the absence or presence of adjuvant, was substantial, but it was elevated still further by adjuvant.
Example 7
Neutralizing Antibody and Neuromuscular Blockade
The following experiment establishes that even though dimutant BoNT/A HC50 domain has lost affinity for cholinergic nerve endings, anti-mutant HC50 antibody raised upon vaccination with the dimutant still possess the ability to block toxin binding to neuronal cells.
Antiserum (0.1 ml) from mice that had been vaccinated with dimutant BoNT/A HC50 was incubated with homologous toxin for 60 min at room temperature, after which the mixture was added to phrenic nerve-hemidiaphragm preparations. The results, which are illustrated in FIG. 5, show that control tissues (toxin alone) paralyzed within about 120 min. However, experimental tissues (toxin previously incubated with antiserum) did not paralyze. Thus, the introduction of the two mutations (Trp1266Leu and Tyr1267Ser) into the BoNT/A HC50 polypeptide did not abolish its ability to induce antibodies that block toxin action on the neuromuscular junction.
Example 8
Preparation of BoNT/A HC Fragment Fusion to BoNT/A Light Chain
A synthetic gene portion encoding the light chain (LC; SEQ ID NO:27; corresponds to residues 1 to 438 of BoNT/A propolypeptide) was fused with a synthetic gene portion encoding HC66 (residues 242 to 846 of SEQ ID NO: 9; corresponds to residues 692 to 1296 of BoN/T propolypeptide) and cloned into 6×His vector pET30a (Novagen, San Diego, Calif.) yielding the expression plasmid pET30a+LC-HC66A. SEQ ID NO:28 is the amino acid sequence for the resulting fusion protein, LC-HC66/A. SEQ ID NO:29 is an optimized coding sequence for LC-HC66/A.
A tri-mutant version of LC-HC66A was prepared. The tri-mutant contained the following mutations: Glu224Gln; Trp1266Leu and Tyr1267Ser. Glu224 is the glutamate in the zinc-binding motif present in the light chain. Mutation of zinc-binding motif residues causes loss of activity (see, e.g., U.S. Pat. No. 6,051,239) of the light chain zinc-dependent metal loendoprotease.
To construct the tri-mutant LC-HC66E224Q, W1266L, Y1267S, point mutations were introduced into Lc-HC66 using QuikChange® Site-Directed Mutagenesis Kit (Stratagene). The phosphorylated mutagenic primers were as follows: For Glu224Gln, 5′-pCTGGTGAGCCAGCGTCACCGCCGGGTCAGT-3′ (SEQ ID NO:30) and for Trp1266Leu/Tyr1267Ser, 5′-pCTGAGCAACC-GTCAGATTGAGCGTTCTTCC-3′ (SEQ ID NO:31). The amino acid sequence of LC-HC66E224Q, W1266L, Y1267S is provided in SEQ ID NO:32. An optimized coding sequence for LC-HC66E224Q, W1266L, Y1267S is provided in SEQ ID NO:33.
Escherichia coli (BL21-codon plus(DE3)-RIL (Stratagene)) was used as a host strain for expression of LC-H66/A and trimutant LC-HC66/A. Cells were grown in Terrific broth (1.2% peptone, 2.4% yeast extract, 0.94% K2HPO4 and 0.22% KH2PO4) (Difco; Sparks, Md.) at 37° C., with shaking to an A600 of 0.6 to 0.8. Isopropyl-β-D-thiogalacto-pyranoside at a final concentration of 0.5 mM was added and incubation was continued for about 20 hr at 12° C. Bacteria from 1 liter of induced culture were harvested by centrifugation (6,000×g, 15 min) at 4° C.
The wild-type and tri-mutant BoNT/A LC-HC66 polypeptides were purified as follows. Host bacterial cells were suspended in 10 ml of 50 mM sodium phosphate containing 300 mM NaCl, pH 8.0. Equal volume of bacterial cell lysis reagent Novagen® BugBuster® (EMD Chemicals Inc., San Diego, Calif.) was added to the cell suspension and allowed to stand for 2 hr on ice. The suspension was centrifuged at 27,000×g for 40 min to remove precipitate. The supernatant was further diluted by adding 100 ml of 50 mM sodium phosphate containing 300 mM NaCl, pH 8.0 and centrifuged again at 27,000×g for 40 min to remove precipitate.
The clear supernatant was loaded onto a 5 ml column of Ni-NTA superflow (Qiagen), which was equilibrated with 50 mM sodium phosphate containing 300 mM NaCl, pH 8.0. The column was washed with 50 volumes of washing buffer (50 mM sodium phosphate containing 300 mM NaCl, and 15 mM imidazole, pH 8.0). Bound protein was eluted from the column with 50 mM sodium phosphate containing 300 mM NaCl and 50 mM imidazole, pH 8.0. The active fractions were pooled and dialyzed against 50 mM Tris-HCl containing 5 mM 2-mercaptoethanol, pH 9.0. The dialysate was centrifuged at 27,000×g for 30 min to remove precipitate.
The clear supernatant was loaded onto a 4 ml cation exchange column of Q Sepharose Fast Flow (Amersham Bioscience, Piscataway, N.J.) equilibrated with 50 mM Tris-HCl containing 5 mM 2-mercaptoethanol, pH 9.0. The column was washed with 50 volumes of 50 mM Tris-HCl containing 5 mM 2-mercaptoethanol and 50 mM NaCl, pH 9.0. Bound protein was eluted from the column with 50 mM Tris-HCl containing 5 mM 2-mercaptoethanol and 200 mM NaCl, pH 9.0. The active fractions were pooled and concentrated with 100K ultrafilter (Amicon, Beverly, Mass.).
The concentrated sample was loaded onto Sephacryl S-100 HR column (2.5×100 cm) (Amersham Bioscience), which was equilibrated with gel filtration buffer (110 mM NaHCO3, pH 8.5) and eluted at 0.3 ml/min into 0.3 ml fractions. Active fractions were collected, concentrated and stored at −80° C. The purity of HC50A protein was confirmed on 10% SDS polyacrylamide gel electrophoresis. Approximately 0.2 mg of pure HC50A was obtained from 1 L of bacterial culture. Proteins were further confirmed by Western blot analysis using rabbit polyclonal antibodies raised against the light chain (LC) and heavy chain (HC) components of BoNT/A. The yield of the tri-mutant protein was closely similar to those obtained for the wild type BoNT/A LC-HC66.
It has been demonstrated previously that an LC polypeptide and an HC66 polypeptide are each immunogenic and can induce a response that is protective against toxin challenge. Thus, it is contemplated that combining these two molecules into one polypeptide will result in an immunogenic polypeptide that can induce a protective immune response. Furthermore, the double mutation Trp-Tyr→Leu-Ser in the ganglioside binding motif present in LC-HC66E224Q, W1266L, Y1267S is expected to preclude any meaningful ability of the molecule to bind nerve endings.
All references cited herein are incorporated by reference.
The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indication the scope of the invention.

## Claims

1. A modified BoNT/A polypeptide comprising amino acid sequence SEQ ID NO: 14.

2. An immunogenic composition comprising a modified BoNT/A polypeptide according to claim 1, and a pharmaceutically acceptable carrier.

3. A method of inducing an immune response to BoNT/A in an animal comprising administering to said animal an effective amount of the immunogenic composition according to claim 2.

4. The method according to claim 3, wherein the immunogenic composition is administered by at least one of the following routes: parenteral, gastrointestinal, vaginal, topical, pulmonary, intranasal and ocular.

5. The method for protecting an animal from botulism, comprising administering to said animal an effective amount of the immunogenic composition according to claim 2.

6. The method according to claim 5, wherein the immunogenic composition is administered by at least one of the following routes: parenteral, gastrointestinal, vaginal, topical, pulmonary, intranasal and ocular.

7. A conjugate comprising a modified BoNT/A polypeptide according to claim 1, linked to a diagnostic or therapeutic entity.

8. A conjugate according to claim 7, wherein the entity is an antigen.

9. A conjugate according to claim 8 wherein the antigen is a polypeptide.

10. A conjugate according to claim 9, wherein the entity is linked to the modified BoNT/A polypeptide by a peptide bond.

11. The conjugate according to claim 10, wherein the entity is an immunogenic portion of a protein associated with a pathogen of a mammal.

12. The conjugate of claim 11, wherein the mammal is a human.

13. The conjugate according to claim 11, wherein the pathogen is selected from the group consisting of Plasmodiumfalciparum, Bacillus anthracis, Bordetella pertussis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Burkholderia mallei, Clostridium perfringens, Clostridium tetani, Clostridium botulinum, Corynebacterium diptheriae, Coxiella burnetii, Crimean-Congo hemorrhagic fever virus, the coronavirus that is the causative agent of Sudden Acute Respiratory Syndrome (SARS), the causative agent of Yellow Fever, the causative agents of multidrug resistant tuberculosis, Francisella tularensis, Hantavirus, hepatitis viruses, human immunodeficiency virus, influenza viruses, measles virus, meningitis, mumps viruses, Nipah virus, Pneumococcal viruses, Poliovirus, Pseudomonas pseudomallei, the ricin toxin of Ricinus communis, Rift Valley fever virus, rabies viruses, Salmonella typhi, Staphylococcal enterotoxin B, trichothecene mycotoxins, Varicella viruses, saxitoxin, Venezuelan equine encephalitis viruses, viruses that are the causative agents of tickborne encephalitis, Variola major, Vibrio cholera, and Yersinia pestis.

14. The conjugate according to claim 13, wherein the pathogen is Clostridium botolinum.

15. The conjugate according to claim 9, wherein the entity is an antibody substance.

16. The conjugate according to claim 7 wherein the entity is a drug or pro-drug.

17. The conjugate according to claim 7 wherein the entity is a diagnostic entity.

18. A method for delivery of a therapeutic or diagnostic entity across a non-keratinized epithelium of an animal comprising:
contacting an epithelium of said animal with a conjugate comprising said therapeutic or diagnostic entity linked to a modified BoNT/A polypeptide, the modified BoNT/A polypeptide comprising the amino acid sequence of SEQ ID NO: 14;
whereby the therapeutic or diagnostic is transported across the non-keratinized epithelium.

19. The method according to claim 18, wherein the epithelium is gastrointestinal, nasal, pulmonary, vaginal, or ocular epithelium.

20. The method according to claim 19, wherein the gastrointestinal epithelium is oral, esophageal, gastric, ileal, duodenal, jejunal, or colon epithelium.

21. The method according to claim 18 wherein the animal is a human.

22. A method of inducing an immune response in a vertebrate against a selected antigen comprising:
contacting an epithelium of said vertebrate with an effective amount of a conjugate comprising said antigen linked to a modified BoNT/A polypeptide, the modified BoNT/A polypeptide comprising the amino acid sequence of SEQ ID NO: 14;
whereby an immune response to the antigen is induced in the vertebrate.

23. The method according to claim 22, wherein the epithelium is gastrointestinal, nasal, pulmonary, vaginal, or ocular epithelium.

24. The method according to claim 23, wherein the gastrointestinal epithelium is oral, esophageal, gastric, ileal, duodenal, jejunal, or colon epithelium.

25. The method according to claim 22 wherein the antigen is an immunogenic portion of a protein associated with a pathogen of a mammal.

26. The method according to claim 25 wherein the mammal is a human.

27. The method according to claim 25, wherein the pathogen is selected from the group consisting of Plasmodium falciparum, Bacillus anthracis, Bordetella pertussis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Burkholderia mallei, Clostridium perfringens, Clostridium tetani, Clostridium botulinum, Corynebacterium diptheriae, Coxiella burnetii, Crimean-Congo hemorrhagic fever virus, the coronavirus that is the causative agent of Sudden Acute Respiratory Syndrome (SARS), the causative agent of Yellow Fever, the causative agents of multi-drug resistant tuberculosis, Francisella tularensis, Hantavirus, hepatitis viruses, human immunodeficiency virus, influenza viruses, meningitis, measles virus, mumps viruses, Nipah virus, Pneumococcal viruses, Poliovirus, Pseudomonas pseudomallei, the ricin toxin of Ricinus communis, Rift Valley fever virus, rabies viruses, Salmonella typhi, Staphylococcal enterotoxin B, trichothecene mycotoxins, Varicella viruses, saxitoxin Venezuelan equine encephalitis viruses, viruses that are the causative agents of tick-borne encephalitis, Variola major, Vibrio cholera, and Yersinia pestis.

28. The method according to claim 27, wherein the pathogen is Clostridium botulinum.

29. The modified BoNT/A polypeptide according to claim 1, further comprising a BoNT light chain fused thereto.