EA045463B1 - NON-NEURAL SNARE CLEAVING BOTULINUM NEUROTOXINS - Google Patents

Description

Introduction

The present invention relates to a modified botulinum neurotoxin protease having the ability to cleave a non-neuronal SNARE protein, and its use for inhibiting unwanted secretion from a mammalian cell by cleaving said non-neuronal SNARE protein in said mammalian cell.

Toxins are generally classified into one of two classes, namely cytotoxic toxins (eg, a plant toxin such as ricin), which kill their natural target cells, and noncytotoxic toxins (eg, botulinum neurotoxins), which do not kill their natural target cells. . Noncytotoxic toxins exert their effects on the target cell by inhibiting a cellular process other than protein synthesis.

Botulinum neurotoxin proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (eg, SNAP-25, VAMP, or syntaxin). The abbreviation SNARE comes from the term soluble attachment protein receptor NSF, where NSF stands for N-ethylmaleimide sensing factor. SNARE proteins are a large super family of proteins. An important function of SNARE proteins is to mediate the exocytosis of neurotransmitter molecules to the postsynaptic junction. Therefore, SNARE proteins are integral to the secretion of molecules via vesicular transport out of the cell.

Clostridium botulinum produces seven (A to G) different neurotoxins (BoNTs), which are differentiated serologically by the absence of antiserum serotype cross-neutralization. BoNTs cause neuron-specific atonic paralysis by targeting neurons and cleaving neuron-specific SNARE proteins.

BoNTs have a 150 kDa polypeptide chain containing a 100 kDa heavy chain and a 50 kDa light chain linked by a disulfide bond. BoNTs are organized into three functional domains: the N-terminal proteolytic light chain (L chain); and a C-terminal heavy chain (H-chain), the latter consisting of a translocation domain ( _HN ) and a C-terminal neuron-binding domain ( _HC ). The toxic effect of BoNT (nerve intoxication) occurs through a three-stage mechanism of action. First, a portion of the _HC binds to the cholinergic nerve cell and is internalized by receptor-mediated endocytosis. Then, part of the H _N translocates the L chain through the endosomal membrane into the cytosol of the nerve cell. Finally, the L chain binds to and cleaves the neuronal SNARE protein in the cytosol, thereby inhibiting the release of the neurotransmitter from the nerve cell and leading to toxicity of the nerve cell.

Seven BoNT serotypes cleave specific residues in one of three SNARE proteins:

serotypes B, D, F and G cleave VAMP-2;

serotypes A and E cleave SNAP-25; and serotype C cleaves SNAP-25 and syntaxin Ia.

While native BoNTs are able to target and cleave neuronal SNARE isoforms such as VAMP-2, SNAP-25, and syntaxin-1a, these proteases have little or no cleaving effect on most non-neuronal SNARE proteins. This substrate specificity for neuronal SNAREs is consistent with and is understood to reflect the natural neuronal cell binding specificity exhibited by BoNT. For example, BoNT/A cleaves human SNAP-25 but not its human non-neuronal isoforms.

Back in 1989, the US Food and Drug Administration (FDA) approved BoNT/A for the treatment of strabismus, blepharospasm and hemifacial spasm, and later for the treatment of cervical dystonia, cosmetic use, for the treatment of facial glabellar lines and axillary hyperhidrosis. The effectiveness of BoNT/A for dystonia and other disorders associated with involuntary skeletal muscle activity, coupled with a satisfactory safety profile, has prompted its empirical/off-label use for a variety of discharges, as well as for pain and cosmetic disorders.

Clinical applications of BoNT are focused on disorders associated with neuromuscular activity. More recently, pioneering research by Syntaxin Ltd has enabled the development of redirected BoNTs that bind to a unique group of neurons (e.g. nociceptive afferents - see International Patent Application WO 96/33273, the contents of which are incorporated herein in its entirety) and/or cells that are not neurons (for example, respiratory epithelial cells - see International Patent Application WO 00/10598, the contents of which are fully incorporated into the present invention). This technology, known as targeted secretion inhibition (TSI) technology, involves replacing the native BoNT binding domain with another targeting moiety (such as a growth factor or other signaling molecule), and has opened the door to new BoNT-based treatments and therapies. .

However, the selective cleavage of neuron-specific SNARE proteins by BoNT has limited the development of new therapies in these non-neuronal systems. Neuronal and non-neuronal SNARE proteins are thought to be equally important for the process of intracellular vesicle fusion and, consequently, for the secretion of molecules through transport of vesicles out of the cell. Accordingly, the use of traditional BoNT-based therapeutics to inactivate neuronal SNARE protein-driven secretion will not target any corresponding cellular secretion driven by non-neuronal SNAREs.

Accordingly, there is a need for an engineered BoNT L chain protease that efficiently degrades non-neuronal SNARE protein.

The present invention solves one or more of the above problems by providing an engineered BoNT/A L chain protease that cleaves an isoform of the SNARE protein that is primarily expressed in non-neuronal cells, namely human SNAP-23 (hSNAP-23). The present invention thus relates to a new class of noncytotoxic therapeutic agents.

Detailed Description of the Invention

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have the meanings commonly understood by those skilled in the art. In addition, unless the context indicates otherwise, the cell and tissue culture nomenclatures and techniques used herein are well known and commonly used in the art.

However, with regard to the use of various terms herein, the following definitions are more specifically applicable.

Terms used in the singular include the plural meaning, such as one or more or at least one, unless the context otherwise requires.

As used herein, the term protease means an enzyme that is capable of hydrolytically degrading proteins and/or peptides. In the context of the present invention, said protease is in particular a botulinum neurotoxin light chain (L chain) protease (BoNT), that is, a protease (also called proteolytic domain) derived from a botulinum neurotoxin, in particular from botulinum neurotoxin A (BoNT/ A). As is well known to one skilled in the art, the light chain of botulinum neurotoxin has a protease function (also known as a noncytotoxic protease function) and typically has a molecular weight of approximately 50 kDa. Such noncytotoxic proteases typically act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g. SNAP-25, VAMP or syntaxin) - see Gerald K (2002) Cell and Molecular Biology (4th edition) John Wiley & Sons, Inc. The naturally occurring (i.e., wild-type) BoNT/A L chain is particularly capable of efficiently degrading SNAP-25, but is only marginally capable of degrading hSNAP-23, as further explained below. In contrast, the BoNT/A L chain protease of the present invention, as described in more detail below, differs from the naturally occurring BoNT/A L chain in that it has an improved ability to cleave hSNAP-23 and is referred to herein as a modified L chain BoNT/A, which cleaves hSNAP-23.

The ability to cleave hSNAP23 can be confirmed using a conventional assay, such as the assay described in Example 2 below. As used herein, the term hSNAP-23 cleavage more specifically means that the modified BoNT/A L chain of the present invention exhibits improved hSNAP-23 cleavage compared to the wild type BoNT/A L chain (SEQ ID NO: 1). Any simple comparative assay can be used for this purpose, such as the hSNAP-23 assay described in Example 2. The wild-type BoNT/A L chain is only able to cleave hSNAP-23 to a small extent (ie, at background level).

Thus, the modified BoNT/A L chain of the present invention has one or more (preferably two) of the following properties:

a) more than 0.2% (e.g., more than 0.5%, preferably more than 1%, more preferably more than 5%) hSNAP-23 cleavage in a cell-free assay (1 μmol modified BoNT/A L-chain; 20 μmol hSNAP -23; preferably incubated at a temperature of about 37°C for about 1 hour) - see example 2, Fig. 1; and/or

b) Km less than 225 (e.g. less than 200, preferably less than 150) µmol in a cell-free assay (1 µmol modified BoNT/A L-chain; 20 µmol hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see example 2, fig. 1; and/or

c) Kcat (1/min) less than 0.2 (e.g. less than 0.18, preferably less than 0.1) in a cell-free assay (1 µmol modified BoNT/A L-chain; 20 µmol hSNAP-23; preferably incubated at approximately 37°C for approximately 1 hour) - see example 2, fig. 1.

The modified BoNT/A L chain protease of the present invention can optionally cleave not only hSNAP-23 but also SNAP-25. Cleavage of SNAP-25 can be confirmed using a conventional assay, such as the assay described in Example 3 below. In accordance with this optional embodiment of the invention, the term SNAP-25 cleavage as used herein means that the modified BoNT/A L chain of the present invention preferably exhibits at least 0.5%, at least 1%, at least 2%, preferably at least

- 2045463 at least 3%, even more preferably at least 10% cleavage of SNAP-25 compared to wild type BoNT/A L chain (SEQ ID NO: 1). Any simple comparative assay, such as the SNAP-25 assay described in Example 3, can be used for this purpose.

In a preferred embodiment, the modified BoNT/A L chain protease of the present invention cleaves hSNAP-23 but does not cleave SNAP-25, such as hSNAP25.

As used herein, the term SNAP-23 (synaptosome associated protein 23) refers to a SNARE protein that is capable of binding to various other SNARE proteins and forming a complex with these proteins with high affinity in a cell, preferably a nerve cell, thereby regulating intracellular membrane fusion in the cell. cage. The term hSNAP-23 more specifically refers to human SNAP-23 and preferably to the protein having the sequence SEQ ID NO: 2.

As used herein, the term SNAP-25 (synaptosome associated protein 25) refers to a SNARE protein that is capable of binding to various other SNARE proteins and forming a complex with these proteins with high affinity in a cell, preferably a nerve cell, thereby regulating intracellular membrane fusion in said cell. cage. The term hSNAP-25 more specifically refers to human SNAP-25 and preferably to the protein having the sequence SEQ ID NO: 3.

The terms modification, substitution or mutation may be used interchangeably herein and refer to a change in amino acid sequence compared to a reference protein sequence, i.e., in the present invention, compared to the wild type BoNT/A L chain sequence (SEQ ID NO: 1). The amino acid sequence shown here as SEQ ID NO: 1 is 438 amino acid residues long and ends at K438. It is clear that K438 is the first lysine amino acid residue in the activation loop and most likely represents the C-terminal end of the L chain after proteolytic cleavage of the activation loop. Thus, SEQ ID NO: 1 represents the (naturally) activated wild-type form of the BoNT/A L chain. Therefore, prior to proteolytic activation, the wild-type BoNT/A L chain is typically ~448 amino acid residues long and includes a short C-terminal extension of the activation loop amino acid residues.

As further explained below, the present invention discloses the identification of critical amino acid positions in the wild-type BoNT/A L chain that require rational substitution with another amino acid residue to render the BoNT/A L chain capable of cleavage by hSNAP-23. In this regard, introducing an amino acid substitution (ie mutation) can be accomplished by insertion, deletion or substitution of an amino acid, and preferably by substitution of an amino acid. Methods for introducing such a mutation are known to one skilled in the art. For example, it is possible to introduce a mutation by random or site-directed mutagenesis, using PCR using degenerate primers, for example, in the nucleotide sequence encoding a reference protein. These methods are, in particular, described in Sambrook et al. Molecular Cloning: A laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press, (2012, and updated 2014), and in Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons (2012).

The amino acid substitution occurs in one or more binding pockets of the L chain relative to the wild type BoNT/A L chain (SEQ ID NO: 1). As used herein, the term binding pocket means a region of the BoNT/A L chain that contains one or more amino acids that are points of contact (e.g., via a hydrogen bond, a salt bridge, and/or a hydrophobic contact) for binding to the corresponding hSNAP-23 binding site. and/or which provide space to accommodate other amino acid residues of the substrate (eg, by modification such as substitution) capable of binding hSNAP-23. As used herein, the term binding means suitable for binding and forms part of the applicant's scientific rationale for the present invention - said rationale is not an essential technical feature of the present invention. For example, the BoNT/A L chain protease binding pocket defined by amino acid residues E148, T307, A308 and Y312 of SEQ ID NO: 1 refers to the BoNT/A L chain protease region containing amino acids E148, T307, A308 and/or Y312 , and/or mutants thereof, as described in the present invention, which the applicant believes are involved in binding to a predicted binding site on hSNAP-23 (eg, the P182/D178 binding site of hSNAP-23).

As used herein, the term binding site refers to a region of hSNAP-23 that contains one or more amino acids that can be bound by the corresponding binding pocket of the BoNT/A L chain. For example, the P182/D178 hSNAP-23 binding site contains amino acids P182 and/or D178 of hSNAP-23. As used herein, the term binding site simply means a hypothetical binding site (as theoretically predicted by Applicant) and is part of Applicant's scientific rationale for the present invention - said rationale is not an essential technical feature of the present invention.

Sequence identity between amino acid or nucleotide sequences can be determined by comparing positions in each of the sequences, which can be aligned for comparison purposes. When a position in the sequences being compared is occupied by the same nucleotide or amino acid, then the sequences at that position are identical. The degree of identity between amino acid sequences is a function of the number of identical amino acid sequences that are common to those sequences. The degree of sequence identity between nucleic acids is a function of the number of identical nucleotides at positions common to those sequences.

To determine the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison. For example, spaces may be introduced into the sequence of the first amino acid sequence or the first nucleotide sequence for optimal alignment with the second amino acid sequence or the second nucleotide sequence. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules at that position are considered identical.

The percentage (%) identity between two sequences is a function of the number of identical positions shared by the sequences. Therefore, percent identity can be calculated by multiplying the number of identical positions by 100 and dividing by the length of the aligned region (overlapping positions), including gaps (only internal gaps, not gaps at the ends of the sequence).

In this comparison, the sequences may be the same length or may be of different lengths. The identity score only considers perfect matches and does not take into account the degree to which amino acids are similar to each other.

In accordance with the present invention, optimal sequence alignment may preferably be performed using a global homology alignment algorithm if the alignment is performed using sequences of the same or similar length, for example, the algorithm described in Needleman and Wunsch (Journal of Molecular Biology; 1970, 48 (3) : 44353), by computer implementations of this algorithm (for example, using DNASTAR® Lasergene software) or by visual inspection. Alternatively, if the alignment is performed using sequences of varying lengths (eg, the light chain amino acid sequence of the present invention is compared to the complete amino acid sequence of a naturally occurring botulinum neurotoxin), then optimal sequence alignment may preferably be performed using a local homology alignment algorithm such as algorithm described in Smith and Waterson (Journal of Molecular Biology; 1981, 147: 195-197), by computer implementations of that algorithm (eg, using DNASTAR® Lasergene software) or by visual inspection. The best alignment (i.e., resulting in the highest percentage of identity between the sequences being compared) generated by the various methods is selected. Examples of global and local homology-based alignment algorithms are well known to one skilled in the art and include, but are not limited to, ClustalV (global alignment), ClustalW (local alignment), and BLAST (local alignment).

One skilled in the art will also readily appreciate that the present invention includes modified BoNT/A L chains that are substantially homologous and that retain the ability to cleave hSNAP-23, that is, functional variants or homologs. These functional variants or homologues can be characterized as having one or more amino acid mutations (such as deletion, addition and/or substitution of amino acids) other than those disclosed herein in connection with hSNAP-23 cleavage and which do not significantly affect folding or protease activity. in particular the cleavage of hSNAP-23. For example, such mutations include, but are not limited to, conservative substitutions, small deletions (typically 1 to 30 amino acids), small amino- or carboxy-terminal extensions (such as an amino-terminal methionine residue), addition of a small linker peptide up to about 20-25 residues or affine tag.

Functional variants or homologs according to the present invention preferably contain mutations of a minor nature, such as conservative amino acid substitutions. Conservative amino acid substitutions are well known to one skilled in the art and include, without limitation:

Main: arginine, lysine, histidine;

Acidic: glutamic acid, aspartic acid;

Polar: glutamine, asparagine;

Hydrophobic: leucine, isoleucine, valine, methionine;

- 4 045463

Aromatic: phenylalanine, tryptophan, tyrosine;

Minor: glycine, alanine, serine, threonine.

In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline and α-methylserine) can replace amino acid residues of the polypeptides of the present invention. A limited number of non-conserved amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids can be replaced by amino acid residues of the clostridial polypeptide. The polypeptides of the present invention may also contain unnatural amino acid residues.

Non-naturally occurring amino acids include, but are not limited to, trans-3methylproline, 2,4-methanolpoline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allothreonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4azaphenylalanine and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins.

An amino acid substitution may involve replacing an amino acid having a particular physicochemical property (eg, hydrophobicity) with an amino acid having a similar or alternative property. Examples of such substitutions are given below:

An acidic amino acid replaced by a neutral polar amino acid;

A polar amino acid replaced by a non-polar amino acid;

A non-polar amino acid substituted with a non-polar amino acid;

A non-polar amino acid replaced by a polar amino acid;

A polar amino acid substituted for a basic amino acid;

A non-polar amino acid substituted by an acidic amino acid;

A non-polar amino acid replaced by a polar amino acid.

Accordingly, the present invention includes the L chain of all BoNT/A subtypes, such as any of the BoNT/A1-BoNT/A8 L chains that contain one or more mutations as described herein to cleave hSNAP-23. Said BoNT/A L chain may further contain additional mutations to create a non-native cleavage activation site, such as the cleavage site of enterokinase (SEQ ID NO: 10), PreScission protease, factor Xa, thrombin, TEV protease, etc.

Additional definitions are provided in the description.

The present invention can be described as follows.

In a first aspect, the present invention relates to a modified botulinum neurotoxin A L chain protease (BoNT/A) that cleaves human SNAP-23 (hSNAP-23) having a modified amino acid sequence compared to the wild type BoNT/A L chain (SEQ ID NO: 1), which contains:

a) at least one substitution of an amino acid residue located in the first binding pocket of the BoNT/A L chain protease for binding to the P182/D178 binding site of hSNAP-23;

b) wherein said first binding pocket of the BoNT/A L chain protease is defined by amino acid residues E148, T307, A308 and Y312 of the wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said at least one amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of asparagine and tyrosine, at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue E148 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or ii) an amino acid residue selected from the group consisting of phenylalanine, isoleucine and leucine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue T307 of the wild type BoNT/A L chain (SEQ ID NO: 1 ); and/or iii) an amino acid residue selected from the group consisting of proline, asparagine, threonine and isoleucine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue A308 of the wild type BoNT/A L chain (SEQ ID NO : 1); and/or iv) an amino acid residue selected from the group consisting of lysine, valine, methionine and leucine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue Y312 of the wild type BoNT/A L chain (SEQ ID NO : 1).

Without wishing to be bound by any theory, the Applicant believes that the BoNT/A L chain binding pocket defined above influences an important enzyme-substrate relationship with the hSNAP-23 recognition sequence.

Thus, the present invention is based on the unexpected discovery (e.g., unexpected technical effect) that targeted amino acid substitutions are claimed to produce BoNT/A L chain(s) with increased hSNAP-23 cleavage efficiency (compared to wild-type BoNT L chain). type). The inventors of the present invention have not only successfully identified suitable amino acid positions of the BoNT/A L chain that can be changed (eg, substituted) to increase the efficiency of hSNAP-23 cleavage, but have also identified the exact amino acid positions

- 5 045463 substitutions that provide this effect.

In a first aspect, the present invention relates to a modified botulinum neurotoxin A L chain protease (BoNT/A) that cleaves human SNAP-23 (hSNAP-23) having a modified amino acid sequence compared to the wild type BoNT/A L chain (SEQ ID NO: 1), which contains:

a) at least one substitution of an amino acid residue located in the first binding pocket of the BoNT/A L chain protease to bind to the P182/D178 binding site of hSNAP-23;

b) wherein said first BoNT/A L chain protease binding pocket is defined by amino acid residue E148 of wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said at least one amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of asparagine and tyrosine, at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue E148 of the wild type BoNT/A L chain (SEQ ID NO: 1).

In one aspect, the present invention provides a modified botulinum neurotoxin A L chain protease (BoNT/A) that cleaves human SNAP-23 (hSNAP-23) having a modified amino acid sequence compared to the wild type BoNT/A L chain. (SEQ ID NO: 1) which contains:

a) at least one substitution of an amino acid residue located in the first binding pocket of the BoNT/A L chain protease to bind to the P182/D178 binding site of hSNAP-23;

b) wherein said first binding pocket of the BoNT/A L chain protease is defined by amino acid residues E148, T307, A308 and Y312 of the wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said at least one amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of phenylalanine, isoleucine and leucine at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue T307 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or ii) an amino acid residue selected from the group consisting of proline, asparagine, threonine and isoleucine at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue A308 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or iii) an amino acid residue selected from the group consisting of lysine, valine, methionine and leucine at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue Y312 of the wild type BoNT/A L chain (SEQ ID NO: 1).

The modified BoNT/A L chain may contain one amino acid substitution (compared to wild type SEQ ID NO: 1). The modified BoNT/A L chain may contain two amino acid substitutions (compared to wild type SEQ ID NO: 1). The modified BoNT/A L chain may contain three amino acid substitutions (compared to wild type SEQ ID NO: 1). The modified BoNT/A L chain may contain four amino acid substitutions (compared to wild type SEQ ID NO: 1).

The modified BoNT/A L chains of the present invention containing a binding pocket mutation for the P182/D178 binding site of hSNAP-23 typically exhibit at least 1.15-fold increased hSNAP-23 cleavage efficiency (compared to BoNT L chain /A wild type) - see 1st data column in Fig. 1A. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A containing substitution E148Y or E148N;

modified L-chain BoNT/A containing substitution T307I, A308P and Y312V;

modified L-chain BoNT/A containing substitution T307F, A308N and Y312L;

modified L-chain BoNT/A containing substitution E148N, T307I and A308P, Y312V;

modified L-chain BoNT/A containing substitution E148Y, T307F, A308N and Y312L;

modified L-chain BoNT/A containing substitution E148Y, T307I, A308P and Y312V;

modified L-chain BoNT/A containing substitution E148Y, T307L, A308T and Y312M;

modified L-chain BoNT/A containing substitution E148Y, T307L, A308I and Y312M.

In one embodiment, the modified BoNT/A L chains of the present invention containing a binding pocket mutation for the hSNAP-23 P182/D178 binding site typically exhibit at least 1.35-fold increased hSNAP-23 cleavage efficiency (compared to wild type BoNT/A L chain) - see 1st data column in FIG. 1A. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A containing substitution E148Y or E148N;

modified L-chain BoNT/A containing substitution T307I, A308P and Y312V;

modified L-chain BoNT/A containing substitution T307F, A308N and Y312L;

modified L-chain BoNT/A containing substitution E148N, T307I, A308P and Y312V;

- 6 045463 modified L-chain BoNT/A containing replacement E148Y, T307F, A308N and Y312L;

modified L-chain BoNT/A containing substitution E148Y, T307I, A308P and Y312V; modified L-chain BoNT/A containing substitution E148Y, T307L, A308T and Y312M; modified L-chain BoNT/A containing substitution E148Y, T307L, A308I and Y312M.

In another embodiment, modified BoNT/A L chains of the present invention containing a binding pocket mutation for the hSNAP-23 P182/D178 binding site typically exhibit at least 1.7-fold increased hSNAP-23 cleavage efficiency (compared to wild type BoNT/A L chain) - see 1st data column in FIG. 1A. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A containing the E148Y substitution;

modified L-chain BoNT/A containing T307F substitution;

modified L-chain BoNT/A containing T307I substitution;

modified L-chain BoNT/A containing the Y312K substitution;

modified L-chain BoNT/A containing the Y312K, E148Y substitution;

modified L-chain BoNT/A containing substitution T307I, A308P and Y312V;

modified L-chain BoNT/A containing substitution E148N, T307I, A308P and Y312V;

modified L-chain BoNT/A containing substitution E148Y, T307F, A308N and Y312L;

modified L-chain BoNT/A containing substitution E148Y, T307L, A308T and Y312M.

In an additional embodiment, modified BoNT/A L chains of the present invention containing a binding pocket mutation for the hSNAP-23 P182/D178 binding site can exhibit at least a 2.0-fold increase in hSNAP-23 cleavage efficiency (compared to wild type BoNT/A L chain) - see 1st data column in FIG. 1A. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A containing the E148Y substitution;

modified L-chain BoNT/A containing substitution E148N, T307I, A308P and Y312V;

modified L-chain BoNT/A containing substitution E148Y, T307F, A308N and Y312L;

modified L-chain BoNT/A containing substitution E148Y, T307L, A308T and Y312M.

In an additional embodiment, modified BoNT/A L chains of the present invention containing a binding pocket mutation for the hSNAP-23 P182/D178 binding site can exhibit at least a 4.0-fold increase in hSNAP-23 cleavage efficiency (compared to wild type BoNT/A L chain) - see 1st data column in FIG. 1A. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A containing the E148Y substitution;

modified L-chain BoNT/A containing substitution E148N, T307I, A308P and Y312V;

modified L-chain BoNT/A containing substitution E148Y, T307F, A308N and Y312L;

modified L-chain BoNT/A containing substitution E148Y, T307L, A308T and Y312M.

In another embodiment, the modified BoNT/A L chains of the present invention containing a binding pocket mutation for the hSNAP-23 P182/D178 binding site exhibit at least 6.0, preferably at least 7.0, more preferably at least 8-fold increase in hSNAP-23 cleavage efficiency (compared to wild-type BoNT/A L chain) - see 1st data column in FIG. 1A. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include: modified BoNT/A L chain containing the E148Y substitution .

The modified BoNT/A L chains of the present invention containing a binding pocket mutation for the P182/D178 binding site of hSNAP-23 typically exhibit greater than 1.5% hSNAP-23 cleavage (% at 1 μM modified BoNT/A L chain; 20 μM hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2nd data column in FIG. 1A. For reference, wild-type BoNT/A L chain (e.g., SEQ ID NO: 1) exhibits less than 0.5% hSNAP-23 cleavage (% at 1 μM modified BoNT/A L chain; 20 μM hSNAP-23; preferably incubated at about 37°C for about 1 hour). Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A containing the E148Y substitution;

modified L-chain BoNT/A containing T307F substitution;

modified L-chain BoNT/A containing T307I substitution;

modified L-chain BoNT/A containing the Y312K substitution;

modified L-chain BoNT/A containing the Y312K and E148Y substitution.

In one embodiment, the modified BoNT/A L chains of the present invention, with

- 7 045463 harboring a binding pocket mutation for the P182/D178 binding site of hSNAP-23 typically exhibit greater than 2% hSNAP-23 cleavage (% at 1 µM modified BoNT/A L-chain; 20 µM hSNAP-23; preferably incubated at temperature approximately 37°C for approximately 1 hour) - see 2nd data column in FIG. 1A. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A containing the E148Y substitution;

modified L-chain BoNT/A containing T307I substitution;

modified L-chain BoNT/A containing the Y312K substitution.

In yet another embodiment, the modified BoNT/A L chains of the present invention containing a binding pocket mutation for the P182/D178 binding site of hSNAP-23 exhibit at least 9% increased hSNAP-23 cleavage (% at 1 μmol of modified BoNT L chain /A; 20 µmol hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2nd data column in FIG. 1A. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include: a modified BoNT/A L chain containing the E148Y substitution.

Additional examples of modified BoNT/A L chain mutants having a binding pocket mutation for the hSNAP-23 P182/D178 binding site (designated by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1 ) include:

modified L-chain BoNT/A containing the A308L substitution;

a modified BoNT/A L-chain containing an A308V substitution;

modified L-chain BoNT/A containing the A308I substitution;

modified L-chain BoNT/A containing the A308P substitution;

modified L-chain BoNT/A containing the A308N substitution;

modified L-chain BoNT/A containing the A308T substitution;

modified L-chain BoNT/A containing Y312V substitution;

modified L-chain BoNT/A containing the Y312M substitution;

modified L-chain BoNT/A containing the Y312L substitution.

The modified BoNT/A L chain of the present invention having a binding pocket mutation for the hSNAP-23 P182/D178 binding site may contain one or more amino acid residue substitutions relative to the wild type BoNT/A L chain as previously defined herein. By way of illustration, the modified BoNT/A L chain of the present invention may have a single amino acid residue mutation (within the binding pocket for the hSNAP23 P182/D178 binding site as defined above), for example, a mutation corresponding to amino acid residue E148 of the wild BoNT/A L chain type (SEQ ID NO: 1). Likewise, the modified BoNT/A L chain of the present invention may contain more than one amino acid residue mutation (within the binding pocket for the hSNAP-23 P182/D178 binding site as defined above), for example mutations corresponding to amino acid residues T307, A308 and Y312 L -wild type BoNT/A chain (SEQ ID NO: 1).

In a preferred embodiment, the modified BoNT/A L chain of the present invention having one or more binding pocket mutations for the hSNAP-23 P182/D178 binding site further comprises one or more mutations in one or more different BoNT/A L chain binding pockets for hSNAP-23, as further described below.

Said one or more different BoNT/A L chain binding pockets for hSNAP-23 include a second BoNT/A L chain protease binding pocket for binding to the D189/D192 binding site of hSNAP-23; a third binding pocket of the L chain protease BoNT/A for binding to the I198 binding site of hSNAP-23; a fourth binding pocket of the L chain protease BoNT/A for binding to the K185 binding site of hSNAP-23; a fifth binding pocket of the L chain protease BoNT/A for binding to the R186 binding site of hSNAP-23; a sixth binding pocket of the L chain protease BoNT/A for binding to the K206 binding site of hSNAP-23; a seventh binding pocket of BoNT/A L chain protease for binding to the D210 binding site of hSNAP-23; the eighth binding pocket of the L chain protease BoNT/A for binding to the D168 binding site of hSNAP-23.

Alternatively, according to various technical features of the present invention, the modified BoNT/A L chain may contain one or more mutations within one or more BoNT/A L chain binding pockets other than the binding pocket for the hSNAP23 P182/D178 binding site as defined above .

The indicated BoNT/A L chain binding pockets for hSNAP-23 and corresponding mutations of interest are further described below.

Accordingly, as an additional technical feature or as an alternative technical feature, the present invention includes BoNT/A L chain mutants containing one or more mutations in the BoNT/A L chain pocket as defined herein. As an example, the modified BoNT/A L chain of the present invention, which cleaves human SNAP-23 (hSNAP23), has a modified amino acid sequence compared to the wild type BoNT/A L chain (SEQ ID NO: 1) which includes:

a) replacing an amino acid residue located in the fifth binding pocket of the BoNT/A L chain protease to bind to the R186 binding site of hSNAP-23;

b) wherein said fifth binding pocket of the BoNT/A L chain protease is defined by amino acid residue S143 of the wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of glutamine, glutamate and aspartate at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue S143 of the wild type BoNT/A L chain (SEQ ID NO: 1).

Without being bound by any theory, Applicant believes that the BoNT/A L chain binding pocket defined above provides a stabilizing salt bridge between residues R186 on hSNAP-23 and S143 on BoNT/A.

The modified BoNT/A L chains of the present invention containing a binding pocket mutation for the hSNAP-23 R186 binding site typically have a KM for hSNAP-23 of less than 100 μM, e.g., less than 95 μM - 3rd data column in FIG. . 1A. For reference, wild type BoNT/A L chain (eg, SEQ ID NO: 1) has a K _M for hSNAP-23 greater than 150 μM, eg, greater than 200 μM. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

a modified BoNT/A L-chain containing a V304D substitution;

modified L-chain BoNT/A containing the V304E substitution;

modified L-chain BoNT/A containing G305D substitution;

modified L-chain BoNT/A containing G305E substitution;

modified L-chain BoNT/A containing the S143D substitution;

modified L-chain BoNT/A containing the S143E substitution;

modified L-chain BoNT/A containing the S143Q substitution;

modified L-chain BoNT/A containing the K166F substitution.

In a preferred embodiment, the modified BoNT/A L chain of the present invention having a binding pocket mutation for the hSNAP-23 R186 binding site may further comprise one or more mutations within one or more different BoNT/A L chain binding pockets as described here (eg, in the binding pocket for the P182/D178 binding site of hSNAP-23, as defined above). Such mutants typically exhibit at least a 0.5-fold reduction in hSNAP-23 cleavage efficiency (compared to the E148Y-modified BoNT/A L chain) or, in other words, at least a 4.0-fold increase in hSNAP-23 cleavage. 23 (compared to wild type BoNT L chain) - see 1st data column for multipocket mutants in FIG. 1B; or at least 5% hSNAP-23 cleavage (% at 1 µmol modified BoNT/A L-chain; 20 µmol hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2- th data column in Fig. 1B. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A containing substitution E148Y and S143E;

modified L-chain BoNT/A containing substitution E148Y and S143D;

modified L-chain BoNT/A containing substitution E148Y and S143Q.

Thus, in one embodiment, the present invention provides a modified BoNT/A L chain protease that cleaves human SNAP-23 (hSNAP-23) and has a modified amino acid sequence compared to the wild type BoNT/A L chain (SEQ ID NO : 1), which contains:

a) replacing an amino acid residue located in the first binding pocket of the BoNT/A L chain protease to bind to the P182/D178 binding site of hSNAP-23; wherein said first BoNT/A L chain protease binding pocket is defined by amino acid residue E148 of wild type BoNT/A L chain (SEQ ID NO: 1); And

b) replacing an amino acid residue located in the fifth binding pocket of the BoNT/A L chain protease to bind to the R186 binding site of hSNAP-23; wherein said fifth BoNT/A L chain protease binding pocket is defined by amino acid residue S143 of wild type BoNT/A L chain (SEQ ID NO: 1); And

c) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of tyrosine at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue E148 of the wild type BoNT/A L chain (SEQ ID NO: 1); and ii) an amino acid residue selected from the group consisting of glutamate, at a position in the mod-

- 9 045463 fixed amino acid sequence of the L chain protease, which corresponds to amino acid residue S143 of the wild type BoNT/A L chain (SEQ ID NO: 1).

In another embodiment, the present invention provides a modified BoNT/A L chain protease that cleaves human SNAP-23 (hSNAP-23) and has a modified amino acid sequence compared to the wild type BoNT/A L chain (SEQ ID NO: 1) , which contains:

a) replacing an amino acid residue located in the first binding pocket of the BoNT/A L chain protease to bind to the P182/D178 binding site of hSNAP-23; wherein said first binding pocket of the BoNT/A L chain protease is defined by amino acid residue E148 of the wild type BoNT/A L chain (SEQ ID NO: 1); And

b) replacing an amino acid residue located in the fifth binding pocket of the BoNT/A L chain protease to bind to the R186 binding site of hSNAP-23; wherein said fifth BoNT/A L chain protease binding pocket is defined by amino acid residue S143 of wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of tyrosine at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue E148 of the wild type BoNT/A L chain (SEQ ID NO: 1); and ii) an amino acid residue selected from the group consisting of aspartate at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue S143 of the wild type BoNT/A L chain (SEQ ID NO: 1).

In another embodiment, the modified BoNT L chain of the present invention having a binding pocket mutation for the hSNAP-23 R186 binding site further comprises one or more mutations in one or more different BoNT/A L chain binding pockets as described herein (e.g. within the binding pocket for the hSNAP-23 P182/D178 binding site as defined above). Such mutants typically exhibit at least a 2.0-fold increase in hSNAP-23 cleavage efficiency (compared to the E148Y modified BoNT/A L chain) see 1st data column for multipocket mutants in FIG. 1B. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A having E148Y and S143E substitution;

modified L-chain BoNT/A, having replacement E148Y and S143D.

In another embodiment, the modified BoNT L chain of the present invention having a binding pocket mutation for the hSNAP-23 R186 binding site further comprises one or more mutations in one or more different BoNT/A L chain binding pockets as described herein (e.g. within the binding pocket for the hSNAP-23 P182/D178 binding site as defined above). Such mutants typically exhibit at least a 3.0-fold increase in hSNAP-23 cleavage efficiency (as a percentage compared to the E148Y modified BoNT/A L chain) - see 1st data column for multipocket mutants in FIG. 1B. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include: modified BoNT/A L chain containing the E148Y substitution , S143D.

As an additional technical feature or alternative technical feature, the present invention includes BoNT/A L chain mutants containing one or more mutations in the BoNT/A L chain pocket as defined herein. For example, the modified BoNT/A L chain of the present invention, which cleaves human SNAP-23 (hSNAP-23), has a modified amino acid sequence compared to the wild type BoNT/A L chain (SEQ ID NO: 1), which contains:

a) at least one substitution of an amino acid residue located in the fourth binding pocket of the L-chain protease BoNT/A for binding to the K185 binding site of hSNAP-23;

b) wherein said fourth BoNT/A L chain protease binding pocket is defined by amino acid residues V304 and G305 of wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said at least one amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of glutamate and aspartate, at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue V304 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or ii) an amino acid residue selected from the group consisting of glutamate and aspartate, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue G305 of the wild type BoNT/A L chain (SEQ ID NO: 1).

Without being bound by any theory, the Applicant believes that the BoNT/A L chain binding pocket defined above provides a stabilizing salt bridge between the K185 residue on hSNAP-23 and BoNT/A.

- 10 045463

The modified BoNT/A L chains of the present invention containing a binding pocket mutation for the K185 binding site of hSNAP-23 typically have a KM for hSNAP-23 of less than 100 μM see 3rd data column in FIG. 1A. For reference, wild type BoNT/A L chain (eg, SEQ ID NO: 1) has a KM for hSNAP-23 greater than 150 μM, eg, greater than 200 μM. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

a modified BoNT/A L-chain containing a V304D substitution;

modified L-chain BoNT/A containing the V304E substitution;

modified L-chain BoNT/A containing G305D substitution;

modified L-chain BoNT/A containing the G305E substitution.

The modified BoNT/A L chain of the present invention having a binding pocket mutation for the K185 binding site of hSNAP-23 contains one or more amino acid residue substitutions relative to the wild type BoNT/A L chain as previously defined herein. By way of illustration, the modified BoNT/A L chain of the present invention may have a single amino acid residue mutation (within the binding pocket for the K185 hSNAP-23 binding site as defined above), for example, a mutant corresponding to amino acid residue G305 of the BoNT/A L chain. A wild type (SEQ ID NO: 1). Likewise, the modified BoNT/A L chain of the present invention may contain more than one mutation (within the binding pocket for the hSNAP-23 K185 binding site as defined above), for example corresponding to amino acid residues V304 and G305 of the wild type BoNT/A L chain (SEQ ID NO: 1).

In a preferred embodiment, the modified BoNT/A L chain of the present invention having a binding pocket mutation for the K185 binding site of hSNAP-23 may further comprise one or more mutations within one or more different BoNT/A L chain binding pockets for hSNAP-23. 23 as described herein (eg, within the binding pocket for the hSNAP-23 P182/D178 binding site as defined above). Such mutants typically exhibit at least a 2.0-fold, preferably at least a 2.5-fold increase in hSNAP-23 cleavage efficiency (compared to E148Y-modified BoNT/A L chain) - see 1 The th data column for multipocket mutants in Fig. 1B. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A, having replacement E148Y and G305D.

Thus, in another embodiment, the present invention provides a modified BoNT/A L chain protease that cleaves human SNAP-23 (hSNAP-23) and has a modified amino acid sequence compared to wild type BoNT/A L chain (SEQ ID NO: 1), which includes:

a) replacing an amino acid residue located in the first binding pocket of the BoNT/A L chain protease to bind to the P182/D178 binding site of hSNAP-23; wherein said first binding pocket of the BoNT/A L chain protease is defined by amino acid residue E148 of the wild type BoNT/A L chain (SEQ ID NO: 1); And

b) replacing an amino acid residue located in the fourth binding pocket of the BoNT/A L chain protease to bind to the K185 binding site of hSNAP-23; wherein said fourth BoNT/A L chain protease binding pocket is defined by amino acid residue G305 of wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of tyrosine at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue E148 of the wild type BoNT/A L chain (SEQ ID NO: 1); and ii) an amino acid residue selected from the group consisting of aspartate at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue G305 of the wild type BoNT/A L chain (SEQ ID NO: 1).

Also, as an additional technical feature or as an alternative technical feature, the present invention includes BoNT/A L chain mutants containing one or more mutations in the BoNT/A L chain pocket as defined herein. As an example, the modified BoNT/A L chain of the present invention, which cleaves human SNAP-23 (hSNAP-23), has a modified amino acid sequence compared to the wild type BoNT/A L chain (SEQ ID NO: 1), which includes:

a) replacing an amino acid residue located in the second binding pocket of the BoNT/A L chain protease to bind to the D189/D192 binding site of hSNAP-23;

b) wherein said second BoNT/A L chain protease binding pocket is defined by amino acid residue Q29 of wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said amino acid residue substitution includes:

i) an amino acid residue of alanine at a position in the modified amino acid sequence

- 11 045463 L chain protease activity, which corresponds to amino acid residue Q29 of the wild type BoNT/A L chain (SEQ ID NO: 1).

Without wishing to be bound by any theory, the Applicant believes that the BoNT/A L chain binding pocket defined above provides a stabilizing interaction between D189/D192 on hSNAP-23 and amino acid 29 of BoNT/A or neighboring amino acids.

Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified BoNT/A L-chain with Q29A substitution.

In a preferred embodiment, the modified BoNT L chain of the present invention having a binding pocket mutation for the hSNAP-23 D189/D192 binding site further comprises one or more mutations in one or more different BoNT/A L chain binding pockets as described herein (eg , within the binding pocket for the hSNAP-23 P182/D178 binding site as defined above). Such mutants typically exhibit at least a 1.50-fold increase in hSNAP-23 cleavage efficiency (compared to E148Y-modified BoNT/A Lchain) - see 1st column of data presented for multipocket mutants in FIG. 1B. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A, having the replacement E148Y, Q29A.

Thus, in another embodiment, the present invention provides a modified BoNT/A L chain protease that cleaves human SNAP-23 (hSNAP-23) and has a modified amino acid sequence compared to the wild type BoNT/A L chain (SEQ ID NO : 1), which includes:

a) replacing an amino acid residue located in the first binding pocket of the BoNT/A L chain protease to bind to the P182/D178 binding site of hSNAP-23; wherein said first BoNT/A L chain protease binding pocket is defined by amino acid residue E148 of wild type BoNT/A L chain (SEQ ID NO: 1); And

b) replacing an amino acid residue located in the second binding pocket of the BoNT/A L chain protease to bind to the D189/D192 binding site of hSNAP-23; wherein said second BoNT/A L chain protease binding pocket is defined by amino acid residue Q29 of wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of tyrosine at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue E148 of the wild type BoNT/A L chain (SEQ ID NO: 1); and ii) an amino acid residue selected from the group consisting of alanine at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue Q29 of the wild type BoNT/A L chain (SEQ ID NO: 1).

In another embodiment, the modified BoNT L chain of the present invention having a binding pocket mutation for the hSNAP-23 D189/D192 binding site may further contain one or more mutations in at least two different BoNT/A L chain binding pockets, as described herein (eg, within the binding pocket for the P182/D178 binding site of hSNAP-23 and within the binding pocket for the R186 binding site of hSNAP-23, as defined above). Such mutants typically exhibit at least a 2.5-fold increase in hSNAP-23 cleavage efficiency (compared to the E148Y-modified BoNT/A L-chain - see 1st data column presented in Fig. 1B. Examples of such modified L-chain BoNT/A chains (identified by amino acid substitution(s) relative to the corresponding wild-type BoNT/A L chain SEQ ID NO: 1) include: a modified BoNT/A L chain containing the E148Y, Q29A and S143D substitution.

Thus, in another embodiment, the present invention provides a modified BoNT/A L chain protease that cleaves human SNAP-23 (hSNAP-23) and has a modified amino acid sequence compared to wild type BoNT/A L chain (SEQ ID NO: 1), which includes:

a) replacing an amino acid residue located in the first binding pocket of the BoNT/A L chain protease to bind to the P182/D178 binding site of hSNAP-23; wherein said first BoNT/A L chain protease binding pocket is defined by amino acid residue E148 of wild type BoNT/A L chain (SEQ ID NO: 1); And

b) replacing an amino acid residue located in the second binding pocket of the BoNT/A L chain protease to bind to the D189/D192 binding site of hSNAP-23; wherein said second binding pocket of the BoNT/A L chain protease is defined by amino acid residue Q29 of the wild type BoNT/A L chain (SEQ ID NO: 1); And

c) replacing an amino acid residue located in the fifth binding pocket of the BoNT/A L chain protease to bind to the R186 binding site of hSNAP-23; wherein said fifth BoNT/A L chain protease binding pocket is defined by amino acid residue S143 of the wild BoNT/A L chain

- 12 045463 type (SEQ ID NO: 1);

d) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of tyrosine at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue E148 of the wild type BoNT/A L chain (SEQ ID NO: 1); and ii) an amino acid residue selected from the group consisting of alanine at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue Q29 of the wild type BoNT/A L chain (SEQ ID NO: 1); and iii) an amino acid residue selected from the group consisting of aspartate at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue S143 of the wild type BoNT/A L chain (SEQ ID NO: 1).

In another embodiment, a modified BoNT L chain of the present invention having a binding pocket mutation for the D189/D192 binding site on hSNAP-23 may further contain one or more mutations in at least two different BoNT/A L chain binding pockets (e.g. , within the binding pocket for the hSNAP-23 P182/D178 binding site and within the binding pocket for the hSNAP-23 K185 binding site, as defined above). Such mutants typically exhibit at least a 3.0-fold, preferably at least a 3.4-fold increase in hSNAP-23 cleavage efficiency (compared to E148Y-modified BoNT/A L chain) - see 1st data column shown for multipocket mutants in Fig. 1B. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include: modified BoNT/A L chain containing E148Y substitutions , Q29A and G305D.

Thus, in another embodiment, the present invention provides a modified BoNT/A L chain protease that cleaves human SNAP-23 (hSNAP-23) and has a modified amino acid sequence compared to wild type BoNT/A L chain (SEQ ID NO: 1), which includes:

a) replacing an amino acid residue located in the first binding pocket of the BoNT/A L chain protease to bind to the P182/D178 binding site of hSNAP-23; wherein said first BoNT/A L chain protease binding pocket is defined by amino acid residue E148 of wild type BoNT/A L chain (SEQ ID NO: 1); And

b) replacing an amino acid residue located in the second binding pocket of the BoNT/A L chain protease to bind to the D189/D192 binding site of hSNAP-23; wherein said second BoNT/A L chain protease binding pocket is defined by amino acid residue Q29 of wild type BoNT/A L chain (SEQ ID NO: 1); And

c) replacing an amino acid residue located in the fourth binding pocket of the BoNT/A L chain protease to bind to the K185 binding site of hSNAP-23; wherein said fourth BoNT/A L chain protease binding pocket is defined by amino acid residue G305 of wild type BoNT/A L chain (SEQ ID NO: 1);

d) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of tyrosine at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue E148 of the wild type BoNT/A L chain (SEQ ID NO: 1); and ii) an amino acid residue selected from the group consisting of alanine at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue Q29 of the wild type BoNT/A L chain (SEQ ID NO: 1); and iii) an amino acid residue selected from the group consisting of aspartate at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue G305 of the wild type BoNT/A L chain (SEQ ID NO: 1).

As an additional technical feature or alternative technical feature, the present invention includes BoNT/A L chain mutants containing one or more mutations in the BoNT/A L chain pocket as defined herein. As an example, the modified BoNT/A L chain of the present invention, which cleaves human SNAP-23 (hSNAP-23), has a modified amino acid sequence compared to the wild type BoNT/A L chain (SEQ ID NO: 1), which includes:

a) at least one substitution of an amino acid residue located in the sixth binding pocket of the BoNT/A L chain protease to bind to the K206 binding site of hSNAP-23;

b) wherein said sixth binding pocket of the BoNT/A L chain protease is defined by amino acid residues Y251, L256, V258, L367 and F369 of the wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said at least one amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of glutamate and aspartate, at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue Y251 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or

- 13 045463 ii) an amino acid residue selected from the group consisting of glutamate, aspartate, glutamine, glycine, alanine and arginine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue L256 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or iii) an amino acid residue selected from the group consisting of serine, alanine, proline, leucine and glutamate, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue V258 of the wild type BoNT/A L chain (SEQ ID NO : 1); and/or iv) an amino acid residue selected from the group consisting of alanine and glycine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue L367 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or

v) an amino acid residue selected from the group consisting of glycine, serine and leucine, at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue F369 of the wild type BoNT/A L chain (SEQ ID NO: 1).

As an additional technical feature or alternative technical feature, the present invention includes BoNT/A L chain mutants containing one or more mutations in the BoNT L chain A pocket as defined herein. As an example, the modified BoNT/A L chain of the present invention , which cleaves human SNAP-23 (hSNAP-23), has a modified amino acid sequence compared to the wild-type BoNT/A L chain (SEQ ID NO: 1), which includes:

a) at least one substitution of an amino acid residue located in the sixth binding pocket of the BoNT/A L chain protease to bind to the K206 binding site of hSNAP-23;

b) wherein said sixth binding pocket of the BoNT/A L chain protease is defined by amino acid residues Y251, L256, V258, L367 and F369 of the wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said at least one amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of glutamate and aspartate, at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue Y251 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or ii) an amino acid residue selected from the group consisting of glutamine and arginine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue L256 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or iii) an amino acid residue selected from the group consisting of serine, leucine and glutamate, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue V258 of the wild type BoNT/A L chain (SEQ ID NO: 1 ); and/or iv) an amino acid residue selected from the group consisting of alanine and glycine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue L367 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or

v) an amino acid residue selected from the group consisting of glycine, serine and leucine at a position in the amino acid sequence of the modified L chain protease that corresponds to amino acid residue F369 of the wild type BoNT/A L chain (SEQ ID NO: 1).

Without wishing to be bound by any theory, the Applicant believes that the BoNT/A L-chain binding pocket defined above influences an important enzyme-substrate relationship to the position (S3') of the hSNAP-23 recognition sequence.

As an additional technical feature or alternative technical feature, the present invention includes BoNT/A L chain mutants containing one or more mutations in the BoNT/A L chain pocket as defined herein. As an example, the modified BoNT/A L chain of the present invention, which cleaves human SNAP-23 (hSNAP-23), has a modified amino acid sequence compared to the wild type BoNT/A L chain (SEQ ID NO: 1), which includes:

a) at least one substitution of an amino acid residue located in the sixth binding pocket of the BoNT/A L chain protease to bind to the K206 binding site of hSNAP-23;

b) wherein said sixth binding pocket of the BoNT/A L chain protease is defined by amino acid residues Y251, L256, V258, L367 and F369 of the wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said at least one amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of glutamate and aspartate, at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue Y251 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or ii) an amino acid residue selected from the group consisting of glutamine and arginine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue L256 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or iii) an amino acid residue selected from the group consisting of serine, leucine and glutamate, at a position in the modified L chain protease amino acid sequence that corresponds to

- 14 045463 corresponds to amino acid residue V258 of the wild-type BoNT/A L chain (SEQ ID NO: 1); and/or iv) an amino acid residue selected from the group consisting of alanine and glycine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue L367 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or

v) an amino acid residue selected from the group consisting of glycine, serine and leucine, at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue F369 of the wild type BoNT/A L chain (SEQ ID NO: 1).

The modified BoNT/A L chain of the present invention having a binding pocket mutation for the K206 binding site of hSNAP-23 may contain one or more amino acid residue substitutions compared to the wild type BoNT/A L chains previously described herein. By way of illustration, the modified BoNT/A L chain of the present invention may have a single amino acid residue mutation (within the binding pocket for the K206 binding site of hSNAP-23), for example, a mutant corresponding to amino acid residue Y251 of the wild type BoNT/A L chain ( SEQ ID NO: 1). Likewise, the modified BoNT/A L chain of the present invention may contain more than one mutation (within the binding pocket for the K206 binding site of hSNAP-23), for example, mutants corresponding to amino acid residues Y251 and L256 of the wild type BoNT/A L chain (SEQ ID NO: 1).

The modified BoNT/A L-chain of the present invention, containing a binding pocket mutation for the K206 hSNAP-23 binding site, typically exhibits greater than 1.5% hSNAP-23 cleavage (% at 1 μM modified BoNT/A L-chain; 20 μM hSNAP- 23; preferably incubated at about 37°C for about 1 hour) - see 2nd data column in FIG. 1A. For reference, wild-type BoNT/A L chain (e.g., SEQ ID NO: 1) exhibits less than 0.5% hSNAP-23 cleavage (% at 1 μM modified BoNT/A L chain; 20 μM hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2nd column of data presented in FIG. 1A. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A containing the Y251D substitution;

modified L-chain BoNT/A containing the Y251E substitution;

modified L-chain BoNT/A containing the L256D substitution.

In one embodiment, the modified BoNT/A L chains of the present invention exhibit at least 3% hSNAP-23 cleavage (% at 1 μM modified BoNT/A L chain; 20 μM hSNAP-23; preferably incubated at about 37°C for approximately 1 hour) - see 2nd column of data presented in FIG. 1A. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include: modified BoNT/A L chain containing the Y251E substitution .

Additional examples of modified BoNT/A L chain mutants having a mutation in the hSNAP-23 K206 binding pocket (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

a modified BoNT/A L-chain containing a V245D substitution;

modified L-chain BoNT/A containing the L256E substitution;

modified L-chain BoNT/A containing the L256G substitution;

modified L-chain BoNT/A containing the L256Q substitution;

modified L-chain BoNT/A containing the L256A substitution;

modified L-chain BoNT/A containing the V258A substitution;

modified L-chain BoNT/A containing the V258P substitution;

modified L-chain BoNT/A containing the V258L substitution;

modified L-chain BoNT/A containing substitution L256E and V258P;

modified L-chain BoNT/A containing substitution L256Q and V258P;

modified L-chain BoNT/A containing substitution L256A and V258L;

modified L-chain BoNT/A containing substitution L256G and V258L.

In a preferred embodiment, the modified BoNT L chain of the present invention having a binding pocket mutation for the hSNAP-23 K206 binding site may further contain one or more mutations in one or more different BoNT/A L chain binding pockets as described herein (eg , within the binding pocket for the hSNAP-23 P182/D178 binding site as defined above). Such mutants typically show at least a 0.5-fold reduction in hSNAP-23 cleavage efficiency (compared to the E148Y-modified BoNT/A L chain) or, in other words, at least a 4.0-fold increase cleavage efficiency of hSNAP23 (compared to wild type BoNT/A L chain) - see 1st column of data presented for multipocket mutants in FIG. 1B, or at least 5% hSNAP-23 cleavage (% at 1 µM modified BoNT/A L-chain; 20 µM hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2nd column of data in Fig. 1B. Preferred examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A containing substitutions E148Y and Y251D;

modified L-chain BoNT/A containing substitutions E148Y and L256D;

modified L-chain BoNT/A containing substitutions E148Y and Y251E.

Thus, in another embodiment, the present invention provides a modified BoNT/A L chain protease that cleaves human SNAP-23 (hSNAP-23) and has a modified amino acid sequence compared to wild type BoNT/A L chain (SEQ ID NO: 1), which includes:

a) replacing an amino acid residue located in the first binding pocket of the BoNT/A L chain protease to bind to the P182/D178 binding site of hSNAP-23; wherein said first BoNT/A L chain protease binding pocket is defined by amino acid residue E148 of wild type BoNT/A L chain (SEQ ID NO: 1); And

b) replacing an amino acid residue located in the fifth binding pocket of the BoNT/A L chain protease to bind to the K206 binding site of hSNAP-23; wherein said fifth BoNT/A L chain protease binding pocket is defined by wild type BoNT/A L chain amino acid residue Y251 (SEQ ID NO: 1);

c) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of tyrosine at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue E148 of the wild type BoNT/A L chain (SEQ ID NO: 1); and ii) an amino acid residue selected from the group consisting of aspartate at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue Y251 of the wild type BoNT/A L chain (SEQ ID NO: 1).

In another embodiment, the modified BoNT L chain of the present invention having a binding pocket mutation for the hSNAP-23 K206 binding site may further contain one or more mutations in at least three different BoNT/A L chain binding pockets as described here (eg, in the binding pocket for the P182/D178 binding site of hSNAP23, in the binding pocket for the R186 binding site of hSNAP-23, and in the binding pocket for the D189/D192 binding site, as defined above). Such mutants typically exhibit at least a 1.3-fold increase in hSNAP-23 cleavage efficiency (compared to E148Y-modified BoNT/A L-chain)—see 1st column of data presented for multipocket mutants in FIG. 1B. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include: modified BoNT/A L chain containing E148Y substitutions , S143D, Q29A and Y251E.

Thus, in another embodiment, the present invention provides a modified BoNT/A L chain protease that cleaves human SNAP-23 (hSNAP-23) and has a modified amino acid sequence compared to wild type BoNT/A L chain (SEQ ID NO: 1), which includes:

a) replacing an amino acid residue located in the first binding pocket of the BoNT/A L chain protease to bind to the P182/D178 binding site of hSNAP-23; wherein said first binding pocket of the BoNT/A L chain protease is defined by amino acid residue E148 of the wild type BoNT/A L chain (SEQ ID NO: 1); And

b) replacing an amino acid residue located in the fifth binding pocket of the BoNT/A L chain protease to bind to the R186 binding site of hSNAP-23; wherein said fifth BoNT/A L chain protease binding pocket is defined by amino acid residue S143 of wild type BoNT/A L chain (SEQ ID NO: 1); And

c) replacing an amino acid residue located in the second binding pocket of the BoNT/A L chain protease to bind to the D189/D192 binding site of hSNAP-23; wherein said second binding pocket of the BoNT/A L chain protease is defined by amino acid residue Q29A of the wild type BoNT/A L chain (SEQ ID NO: 1); And

d) replacing an amino acid residue located in the sixth binding pocket of the BoNT/A L chain protease to bind to the K206 binding site of hSNAP-23; wherein said sixth BoNT/A L chain protease binding pocket is defined by wild type BoNT/A L chain amino acid residue Y251 (SEQ ID NO: 1);

e) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of tyrosine at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue E148 of the wild type BoNT/A L chain (SEQ ID NO: 1); and ii) an amino acid residue selected from the group consisting of aspartate, at a position in the modified amino acid sequence of the L chain protease that corresponds to the amino acid

- 16 045463 to the S143 residue of the wild-type BoNT/A L chain (SEQ ID NO: 1); and iii) an amino acid residue selected from the group consisting of alanine at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue Q29 of the wild type BoNT/A L chain (SEQ ID NO: 1); and iv) an amino acid residue selected from the group consisting of glutamate at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue Y251 of the wild type BoNT/A L chain (SEQ ID NO: 1).

As an additional technical feature or alternative technical feature, the present invention includes BoNT/A L chain mutants containing one or more mutations in the BoNT/A L chain pocket as defined herein. As an example, the modified BoNT/A L chain of the present invention, which cleaves human SNAP-23 (hSNAP-23), has a modified amino acid sequence compared to the wild type BoNT/A L chain (SEQ ID NO: 1), which includes:

a) replacing an amino acid residue located in the third binding pocket of the BoNT/A L chain protease to bind to the I198 binding site of hSNAP-23;

b) wherein said third BoNT/A L chain protease binding pocket is defined by amino acid residue K166 of wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of valine, phenylalanine, leucine and isoleucine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue K166 of the wild type BoNT/A L chain (SEQ ID NO: 1) .

Without being bound by any theory, the Applicant believes that the BoNT/A L chain binding pocket defined above may provide a stabilizing hydrophobic interaction between I198 in hSNAP-23 and amino acid 166 of BoNT/A.

The modified BoNT/A L chains of the present invention containing a binding pocket mutation for the I198 binding site of hSNAP-23 typically exhibit greater than 9% hSNAP-23 cleavage (% at 1 μM modified BoNT/A L chain; 20 μM hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2nd column of data presented in FIG. 1A. For reference, wild-type BoNT/A L chain (e.g., SEQ ID NO: 1) exhibits less than 0.5% hSNAP-23 cleavage (% at 1 μM modified BoNT/A L chain; 20 μM hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2nd column of data presented in FIG. 1A. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

a modified BoNT/A L-chain containing a K166V substitution;

modified L-chain BoNT/A containing the K166F substitution;

modified L-chain BoNT/A containing the K166L substitution;

modified L-chain BoNT/A containing the K166I substitution.

In one embodiment, the modified BoNT/A L chains of the present invention containing a binding pocket mutation for the hSNAP-23 I198 binding site typically exhibit greater than 40% hSNAP-23 cleavage (% at 1 μM modified BoNT/A L chain; 20 μM hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2nd column of data presented in FIG. 1A. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A containing the K166F substitution;

modified L-chain BoNT/A containing the K166L substitution.

In a further embodiment, the modified BoNT/A L chains of the present invention containing a binding pocket mutation for the hSNAP-23 I198 binding site typically exhibit greater than 60% hSNAP-23 cleavage (% at 1 μM modified BoNT/A L chain; 20 μM hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2nd column of data presented in FIG. 1A. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include: modified BoNT/A L chain containing the K166F substitution .

In a preferred embodiment, the modified BoNT L chain of the present invention having a binding pocket mutation for the hSNAP-23 I198 binding site may further contain one or more mutations in one or more different BoNT/A L chain binding pockets as described herein (eg , in at least two different binding pockets of the BoNT/A L chain, such as a binding pocket for the P182/D178 binding site of hSNAP-23 and a binding pocket for the K185 or R186 binding site of hSNAP-23). Such mutants typically exhibit at least 40% hSNAP-23 cleavage (% at 1 μM modified BoNT/A L chain; 20

- 17 045463 µmol hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2nd column of data presented in FIG. 1B. Examples of such modified

BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A containing substitutions E148Y, K166F and G305D;

modified L-chain BoNT/A containing substitutions E148Y, K166V and G305D;

modified L-chain BoNT/A containing substitutions E148Y, S143D and K166F.

Thus, in another embodiment, the present invention provides a modified BoNT/A L chain protease that cleaves human SNAP-23 (hSNAP-23) and has a modified amino acid sequence compared to wild type BoNT/A L chain (SEQ ID NO: 1), which includes:

a) replacing an amino acid residue located in the first binding pocket of the BoNT/A L chain protease to bind to the P182/D178 binding site of hSNAP-23; wherein said first BoNT/A L chain protease binding pocket is defined by amino acid residue E148 of wild type BoNT/A L chain (SEQ ID NO: 1); And

b) replacing an amino acid residue located in the third binding pocket of the BoNT/A L chain protease to bind to the I198 binding site of hSNAP-23; wherein said third BoNT/A L chain protease binding pocket is defined by amino acid residue K166 of wild type BoNT/A L chain (SEQ ID NO: 1); And

c) replacing an amino acid residue located in the fourth binding pocket of the BoNT/A L chain protease to bind to the K185 binding site of hSNAP-23; wherein said fourth BoNT/A L chain protease binding pocket is defined by amino acid residue G305 of wild type BoNT/A L chain (SEQ ID NO: 1);

d) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of tyrosine at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue E148 of the wild type BoNT/A L chain (SEQ ID NO: 1); and ii) an amino acid residue selected from the group consisting of valine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue K166 of the wild type BoNT/A L chain (SEQ ID NO: 1); and iii) an amino acid residue selected from the group consisting of aspartate at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue G305 of the wild type BoNT/A L chain (SEQ ID NO: 1).

In another embodiment, a modified BoNT L chain of the present invention having a binding pocket mutation for the hSNAP-23 I198 binding site may further comprise one or more mutations in at least two different BoNT/A L chain binding pockets (e.g. within the binding pocket for the P182/D178 binding site of hSNAP-23 and within the binding pocket for the K185 or R186 binding site of hSNAP-23). Such mutants typically exhibit at least 15% hSNAP-23 cleavage (% at 10 nmol modified BoNT/A L-chain; 20 µmol hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2nd column of data** presented in Fig. 1B. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A containing substitution E148Y, K166F and G305D;

modified L-chain BoNT/A containing substitution E148Y, S143D and K166F.

In one embodiment, a modified BoNT L chain of the present invention having a binding pocket mutation for the hSNAP-23 I198 binding site may further contain one or more mutations in at least three different BoNT/A L chain binding pockets (e.g. in the binding pocket for the P182/D178 binding site of hSNAP-23, in the binding pocket for the D189/D192 binding site of hSNAP23, and in the binding pocket for the K185 binding site of hSNAP-23, as defined above). Such mutants typically exhibit at least 60% hSNAP-23 cleavage (% at 1 µM modified BoNT/A L-chain; 20 µM hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2 The th data column shown in FIG. 1B. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A containing substitutions E148Y, Q29A, K166V and G305D;

modified L-chain BoNT/A containing substitutions E148Y, Q29A, K166F and G305D.

Thus, in another embodiment, the present invention provides a modified BoNT/A L chain protease that cleaves human SNAP-23 (hSNAP-23) and has a modified amino acid sequence compared to wild type BoNT/A L chain (SEQ ID NO: 1), which includes:

- 18 045463

a) replacing an amino acid residue located in the first binding pocket of the BoNT/A L chain protease to bind to the P182/D178 binding site of hSNAP-23; wherein said first BoNT/A L chain protease binding pocket is defined by amino acid residue E148 of wild type BoNT/A L chain (SEQ ID NO: 1); And

b) replacing an amino acid residue located in the third binding pocket of the BoNT/A L chain protease to bind to the I198 binding site of hSNAP-23; wherein said third BoNT/A L chain protease binding pocket is defined by amino acid residue K166 of wild type BoNT/A L chain (SEQ ID NO: 1); And

c) replacing an amino acid residue located in the second binding pocket of the BoNT/A L chain protease to bind to the D189/D192 binding site of hSNAP-23; wherein said second BoNT/A L chain protease binding pocket is defined by wild type BoNT/A L chain amino acid residue Q29A (SEQ ID NO: 1); And

d) replacing an amino acid residue located in the fourth binding pocket of the BoNT/A L chain protease to bind to the K185 binding site of hSNAP-23; wherein said fourth BoNT/A L chain protease binding pocket is defined by amino acid residue G305 of wild type BoNT/A L chain (SEQ ID NO: 1);

e) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of tyrosine at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue E148 of the wild type BoNT/A L chain (SEQ ID NO: 1); and ii) an amino acid residue selected from the group consisting of valine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue K166 of the wild type BoNT/A L chain (SEQ ID NO: 1); and iii) an amino acid residue selected from the group consisting of alanine at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue Q29 of the wild type BoNT/A L chain (SEQ ID NO: 1); and iv) an amino acid residue selected from the group consisting of aspartate at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue G305 of the wild type BoNT/A L chain (SEQ ID NO: 1).

In another embodiment, the modified BoNT L chain of the present invention having a binding pocket mutation for the I198 binding site on hSNAP-23 may further contain one or more mutations in at least three different BoNT/A L chain binding pockets (eg , within the binding pocket for the P182/D178 binding site of hSNAP-23, within the binding pocket for the D189/D192 binding site of hSNAP23, and within the binding pocket for the K185 binding site of hSNAP-23). Such mutants typically exhibit at least 10% hSNAP-23 cleavage (% at 10 nmol modified BoNT/A L-chain; 20 µmol hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2nd column of data** presented in Fig. 1B. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include: modified BoNT/A L chain containing the E148Y substitution , Q29A, K166F and G305D.

Additional examples of modified BoNT/A L chain mutants having a mutation in the hSNAP-23 I198 binding pocket (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include : modified Lchain BoNT/A containing replacement E148Y, Q29A, K166F, Y251E and G305D.

In one embodiment, a modified BoNT L chain of the present invention having a binding pocket mutation for the hSNAP-23 I198 binding site may further comprise one or more mutations in one or more different binding pockets of the BoNT/A L chain (e.g., within the binding pocket for the P182/D178 binding site of hSNAP-23 or within the binding pocket for the K185 binding site of hSNAP-23 as defined above). Such mutants typically exhibit at least 3% hSNAP-23 cleavage (% at 10 nmol modified BoNT/A Lchain; 20 µmol hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2 The th data column** shown in FIG. 1B. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A containing substitution E148Y and K166F;

modified BoNT/A L-chain containing G305D and K166F substitution.

In another embodiment, a modified BoNT L chain of the present invention having a binding pocket mutation for the hSNAP-23 I198 binding site may further comprise one or more mutations in one or more different binding pockets of the BoNT/A L chain (e.g., within the binding pocket for the P182/D178 binding site of hSNAP-23 or within the binding pocket for the K185 binding site of hSNAP-23). Such mutants usually show

- 19 045463 at least 5% hSNAP-23 cleavage (% at 10 nmol modified BoNT/A L-chain; 20 µmol hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2- th data column** presented in FIG. 1B. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A containing the E148Y and K166F substitution.

As an additional technical feature or alternative technical feature, the present invention includes BoNT/A L chain mutants containing one or more mutations in the BoNT/A L chain pocket as defined herein. As an example, the modified BoNT/A L chain of the present invention, which cleaves human SNAP-23 (hSNAP-23), has a modified amino acid sequence compared to the wild type BoNT/A L chain (SEQ ID NO: 1), which includes:

a) replacing an amino acid residue located in the seventh binding pocket of the BoNT/A L chain protease to bind to the D210 binding site of hSNAP-23;

b) wherein said seventh binding pocket of the BoNT/A L chain protease is defined by amino acid residue S254 of the wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said amino acid residue substitution includes:

i. for example, an alanine amino acid residue at a position in the modified L chain protease amino acid sequence that corresponds to wild type BoNT/A L chain amino acid residue S254 (SEQ ID NO: 1).

Without being bound by any theory, the Applicant believes that the BoNT/A L chain binding pocket defined above may provide a hydrogen bond to the D210 binding site of hSNAP23. In addition, the applicant believes that modification of the specified binding pocket (as defined herein) eliminates the formation of a hydrogen bond between D210 of hSNAP-23 and amino acid 254 of BoNT/A. It is believed that this, in turn, generates the C-terminal cleavage product of hSNAP-23 in the form of a better leaving group and thus increases the rate of hSNAP-23 cleavage.

Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include: modified BoNT/A L chain containing the S254A substitution .

In one embodiment, a modified BoNT L chain of the present invention having a binding pocket mutation for the hSNAP-23 D210 binding site may further contain one or more mutations in one or more different BoNT/A L chain binding pockets as described herein (e.g. , within at least two or three different binding pockets of the BoNT/A L chain, such as a binding pocket for the hSNAP-23 P182/D178 binding site and a binding pocket for the hSNAP-23 I198 binding site and, optionally, a binding pocket for the K185 binding site of hSNAP23). Such mutants typically exhibit at least 10% hSNAP-23 cleavage (% at 10 nmol modified BoNT/A L-chain; 20 µmol hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2nd column of data** presented in Fig. 1B. Examples of such modified BoNT/A L-chains include:

modified L-chain BoNT/A containing substitution E148Y, K166F and S254A;

modified L-chain BoNT/A containing substitution E148Y, K166F, S254A and G305D.

In a preferred embodiment, the modified BoNT L chain of the present invention having a binding pocket mutation for the hSNAP-23 D210 binding site may further contain one or more mutations in one or more different BoNT/A L chain binding pockets as described herein (eg , within at least three different binding pockets of the BoNT/A L chain, such as the binding pocket for the P182/D178 binding site of hSNAP-23, the binding pocket for the I198 binding site of hSNAP-23, and the binding pocket for the K185 binding site of hSNAP23 ). Such mutants typically exhibit at least 25% hSNAP-23 cleavage (% at 10 nmol modified BoNT/A L-chain; 20 µmol hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2nd column of data** presented in Fig. 1B. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include: modified BoNT/A L chain containing the E148Y substitution , K166F, S254A and G305D.

As an additional technical feature or alternative technical feature, the present invention includes BoNT/A L chain mutants containing one or more mutations in the BoNT/A L chain pocket as defined herein. As an example, the modified BoNT/A L chain of the present invention, which cleaves human SNAP-23 (hSNAP-23), has a modified amino acid sequence compared to the wild type BoNT/A L chain (SEQ ID NO: 1), which includes:

a) replacing an amino acid residue located in the eighth binding pocket of the BoNT/A L chain protease to bind to the D168 binding site of hSNAP-23;

- 20 045463

b) wherein said eighth BoNT/A L chain protease binding pocket is defined by amino acid residue K340 of wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said amino acid residue substitution includes:

i) for example, a histidine amino acid residue at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue K340 of the wild type BoNT/A L chain (SEQ ID NO: 1).

Without being bound by any theory, the Applicant believes that the BoNT/A L chain binding pocket defined above may provide a salt bridge between D168 of hSNAP-23 and amino acid 340 of BoNT/A.

Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include:

modified L-chain BoNT/A containing the K340H substitution.

In one embodiment, a modified BoNT L chain of the present invention having a binding pocket mutation for the hSNAP-23 P182/D178 binding site may further contain one or more mutations in the BoNT/A L chain binding pocket of the hSNAP-23 D168 binding site. Such mutants typically exhibit at least 3% hSNAP-23 cleavage (% at 1 µM modified BoNT/A L-chain; 20 µM hSNAP-23; preferably incubated at about 37°C for about 1 hour) - see 2nd column of data presented in Fig. 1B. Examples of such modified BoNT/A L chains (identified by amino acid substitution(s) relative to the corresponding wild type BoNT/A L chain SEQ ID NO: 1) include: modified BoNT/A L chain containing the E148Y substitution and K340H.

The modified BoNT/A L chain protease, as described above, may comprise an amino acid sequence having at least 70%, such as at least 80%, or at least 85%, or at least , 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99%, sequence identity with wild-type BoNT/A L chain (SEQ ID NO: 1). The modified amino acid sequence of the BoNT/A L chain has less than 100% sequence identity with the wild type BoNT/A L chain (eg, SEQ ID NO: 1). As previously stated, reference in this specification to the modified L chain protease BoNT/A includes its functional fragments, that is, fragments of said protease that cleave hSNAP-23. For example, the modified BoNT/A L chain protease of the present invention contains at least 300 (eg, at least 350, or at least 400, or at least 410) amino acids. For example, the N-terminal eight amino acids and/or the carboxyl terminus (eg, the last 32 amino acids) of botulinum neurotoxin L chain protease are not essential for proteolytic activity.

The modified BoNT/A L chain of the present invention may be pegylated to increase stability, eg, duration of action of the protease moiety. PEGylation preferably involves adding PEG to the N-terminus of the L chain. As an example, the N-terminus of the L chain may be extended by one or more amino acid (eg, cysteine) residues. One or more of these amino acid residues may have its own PEG molecule attached (eg, covalently linked) to it. An example of this technology is described in International Patent Application WO 2007/104567, the contents of which are incorporated by reference in their entirety.

The modified BoNT/A L-chain of the present invention may include the addition (or removal) of secondary modification sites - see patent documents WO 2002/040506, US7223577 and WO 2005/068494, each of which is incorporated herein by reference in its entirety. The additional presence or absence (compared to the wild-type BoNT L chain) of such sites alters the biological persistence (eg, biological half-life) of the modified L chain of the present invention.

In a second aspect, the present invention provides a nucleic acid construct comprising or consisting of a nucleic acid sequence that encodes a modified BoNT/A L chain as described herein. Said nucleic acid sequence may preferably encode a TSI delivery vehicle, as further described below. The nucleic acid construct of the present invention may include conventional regulatory elements such as a promoter and/or terminator.

In one embodiment, the present invention relates to a nucleic acid construct in the form of a bacterial plasmid or viral vector. Said nucleic acid design may optionally be codon optimized to optimize expression (eg, recombinant expression) in the desired host cell (eg, E. coli).

In one embodiment, a nucleic acid construct encoding a modified BoNT/A L chain as described herein can be used for administration to a target cell of interest, for example, for therapeutic or cosmetic purposes. To this end, said nucleic acid design can generally be optimized using conventional

- 21 045463 methodology for delivery into (and subsequent expression within) a target cell, preferably a human cell. The target cell is preferably a cell that is not a neuron.

In a third aspect, the present invention provides a delivery vehicle for a modified BoNT/A L chain, thereby facilitating passage of the modified BoNT/A L chain into a target cell of interest. In accordance with this aspect, the present invention particularly relates to a delivery device comprising:

a) a modified BoNT/A L chain protease of the present invention or a nucleic acid construct comprising or consisting of a nucleic acid sequence encoding a modified BoNT/A L chain of the present invention; And

b) a means for delivering said modified BoNT/A L-chain protease or said nucleic acid construct to a target cell.

Preferred vehicles for such delivery include any conventional delivery vehicle known in the art, such as liposomes, biolistic particles, cell penetrating peptides, gene transfer vectors, etc.

One particularly preferred delivery method that is most suitable for use with the present invention is Applicant's targeted secretion inhibitor (TSI) technology. The main methods used to create TSI are well documented and are now considered traditional (see, for example, International Patent Applications WO 98/07864, WO 2006/059113, WO 2009/150469, WO 2010/020811, WO 2009/150470, WO 2010/094905, WO 2012/156743, each of which is incorporated by reference in its entirety). TSI technology is based on a delivery mechanism that mimics the same basic steps that a clostridial neurotoxin uses when it intoxicates a host cell (i.e., binding to the target cell, endosome formation, translocation of the L chain into the cytosol, proteolytic cleavage of the SNARE protein using an L-chain). TSI delivery vehicles are based on a simple clostridial neurotoxin structure having three main components:

1) L-chain of clostridial neurotoxin;

2) a targeting fragment (TF) to direct the delivery vehicle to the selected target cell. Typically, the native binding domain of a clostridial neurotoxin ( _HCC ) may be replaced with a ligand to provide selective binding of the delivery vehicle to a desired target cell other than the native target cell of said _HCC . In a preferred embodiment, more than one NP (optionally including the clostridial neurotoxin binding domain) may be used;

3) a translocation peptide (eg, clostridial neurotoxin H _N translocation domain) to ensure delivery of the clostridial neurotoxin L chain to the target cell, where it can then exert its proteolytic effects (ie, cleavage of the SNARE protein).

Thus, in a preferred embodiment of the invention, means b) of the delivery means of the present invention may comprise:

i) a targeting moiety (TF) that binds the delivery vehicle to the target cell. Said targeting moiety may be either a native clostridial neurotoxin ( _HCC ) binding domain or, more preferably, a ligand that provides binding to a target cell other than the native target cell of said _HCC ; and ii) a translocation peptide that translocates the modified BoNT/A L chain protease of the present invention into a target cell, preferably into the cytosol of said cell.

The delivery vehicle of the present invention typically includes one or more targeting moieties (TFs). Reference to NF includes any structure (usually a peptide) that operably interacts with a site (e.g., receptor or acceptor) causing physical association between the modified BoNT/A L chain protease of the present invention and the surface of a target mammalian cell (e.g., human cell ). The site is preferably a site capable of internalization (eg, endosome formation), also called receptor-mediated endocytosis. NF may have the function of endosomal membrane translocation, in which case there is no need to use separate NF components and the translocation domain.

The NP of the present invention binds (eg, specifically binds) to a selected target cell. The term specifically binds preferably means that the NP binds to the target cell with a binding affinity (Ka) of 106 M ^-1 or more, for example, 10 ⁷ M ^-1 or more, 10 ⁸ M ^-1 or more, or 109 M ^-1 or more.

That NF binds to the selected target cell is confirmed in a standard manner. For example, a simple radioactive displacement experiment may be used in which tissue or cells representing the target cell of interest are exposed to labeled (eg, tritiated) NF in the presence of an excess of unlabeled NF. In such an experiment, the relative proportions of nonspecific and specific binding can be assessed, thereby confirming that NF is binding to the target cell. Optionally, the analysis may include one

- 22 045463 or more binding antagonists, and the analysis may further include monitoring for loss of NF binding. Examples of experiments of this type can be found in Hulme, E.C. (1990), Receptor-binding studies, a brief outline, pp. 303-311, In Receptor biochemistry, A Practical Approach, Ed. E.C. Hulme, Oxford

University Press.

The NP of the present invention preferably binds to a non-neuronal target cell (for example, a mast cell and/or an epithelial cell - see, for example, International Patent Applications WO 00/10598 and WO 01/21213, each of which is incorporated herein by reference in its entirety) . In this case, said NF is capable of directing the delivery vehicle to a selected non-neuronal target cell that expresses the undesirable hSNAP-23 phenotype (and, optionally, the undesirable SNAP-25 phenotype). In parallel, the NP of the present invention can separately bind (for example, through the same NP or through a second NP) to a second selected target cell, for example, to a second non-neuronal target cell or to a target neuronal cell expressing an undesirable hSNAP-23 and/or SNAP phenotype -25.

Suitable NPs include: ligands to mammalian cell receptor binding sites such as cytokines, growth factors, neuropeptides, lectins and antibodies - this term includes monoclonal antibodies, single chain antibodies and antibody fragments such as Fab, F(ab)' 2, Fv, ScFv, etc.

As a further example, NFs include leptin peptide, ghrelin receptor, somatostatin peptide, insulin growth factor peptide, ErbB peptide (e.g., EGF), VIPglucagon-GRF-secretin peptide (e.g., PACAP peptide), interleukin peptide (e.g., IL-1, IL-2, IL-6 or IL-10), NGF peptide, VEGF peptide, bombesin peptide, urotensin peptide, melanin-concentrating hormone peptide, prolactoliberin peptide, KiSS-1 peptide, CRF peptide, GHRH peptide, substance P peptide , beta-2-adrenergic receptor peptide, gastrin-releasing peptide, calcitonin gene-related peptide, platelet-derived growth factor peptide, keratinocyte growth factor peptide, hepatocyte growth factor peptide, TNF-alpha peptide, TNF-beta peptide, atrial natriuretic peptide and peptide integrin.

The delivery vehicle of the present invention generally does not have a (functional) clostridial neurotoxin binding domain (as a NF).

Alternatively, the delivery vehicle of the present invention may include a (functional) clostridial neurotoxin binding domain (as NF). Reference to the clostridial neurotoxin binding domain includes the _HC (more precisely, H _CC ) portion of the clostridial neurotoxin, as well as mutants thereof that retain the binding ability of the _HC domain (for example, the ability to bind rat synaptosomal membranes in conventional binding assays such as those described in Shone et al (1985) Eur J Biochem 151, 75-82.

The HC binding domain/peptide of native clostridial neurotoxin contains approximately 400-440 amino acid residues and consists of two functionally distinct domains of approximately 25 kDa each, namely the N-terminal region (usually called the peptide or H _CN domain) and the C-terminal region (usually called the peptide or domain H _CC ) - It is the C-terminal region (H _CC ), which is formed by C-terminal amino acid residues 160-200, that is responsible for the binding of clostridial neurotoxin to nerve endings at the neuromuscular junction. Typical H _CC peptides include:

botulinum neurotoxin type A - amino acid residues (Y1111-L1296);

botulinum neurotoxin type B - amino acid residues (Y1098-E1291);

botulinum neurotoxin type C - amino acid residues (Y1112-E1291);

botulinum neurotoxin type D - amino acid residues (Y1099-E1276);

botulinum neurotoxin type E - amino acid residues (Y1086-K1252);

botulinum neurotoxin type F - amino acid residues (Y1106-E1274);

botulinum neurotoxin type G - amino acid residues (Y1106-E1297);

tetanus neurotoxin - amino acid residues (Y1128-D1315).

The above reference sequences should be considered as approximate data as slight variations may occur depending on the subserotype.

The delivery vehicle of the present invention typically includes a translocation peptide that enables translocation of the modified L chain into the target cytosol. Whether a peptide has the required translocation function according to the present invention can be determined by any of several conventional assays. For example, Shone C. (1987) describes an in vitro assay using liposomes that are loaded with the test molecule. The presence of the required translocation function is confirmed by the release of K+ and/or labeled NAD from liposomes, which can be easily controlled [see Shone C. (1987) Eur. J Biochem; vol. 167(1): pp. 175-180]. Another example can be found in Blaustein R. (1987), which describes a simple in vitro assay using planar phospholipid bilayer membranes. The membranes are loaded with the test molecule and the required translocation function is confirmed by an increase in conductance across the said membranes [see Blaustein (1987) FEBS Letts; vol. 226, no. 1: pp. 115-120]. Additional techniques to assess membrane fusion and thereby identify translocation domains suitable for use in the present invention are described in Methods in Enzymology Vol 220 and 221.

Membrane Fusion Techniques, Parts A and B, Academic Press 1993.

The translocation domain may be of clostridial origin, such as the H _N domain/portion of a neurotoxin. The term H _N domain refers to a fragment of the clostridial neurotoxin H chain approximately equivalent to the amino terminal half of the H chain. The H _N domain of clostridial neurotoxin lacks the natural binding function of the H _C H chain component. Thus, the H _N domain is unable to bind to the binding site on the target cell to which the native clostridial neurotoxin (ie holotoxin) binds.

Examples of suitable (reference) translocation domains include:

botulinum neurotoxin type A - amino acid residues (449-871);

botulinum neurotoxin type B - amino acid residues (441-858);

botulinum neurotoxin type C - amino acid residues (442-866);

botulinum neurotoxin type D - amino acid residues (446-862);

botulinum neurotoxin type E - amino acid residues (423-845);

botulinum neurotoxin type F - amino acid residues (440-864);

botulinum neurotoxin type G - amino acid residues (442-863);

tetanus neurotoxin - amino acid residues (458-879).

Studies have shown that the full length of the H _N portion of the clostridial neurotoxin heavy chain is not necessary for translocation activity. Thus, aspects of this embodiment may include clostridial toxin HN regions comprising a translocation domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, and at least at least 425 amino acids. For more information on the genetic basis of toxin production in Clostridium botulinum and C. tetani, see Henderson et al (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic press.

The term H _N includes naturally occurring portions of the H _N neurotoxin, as well as H _N variants having amino acid sequences that do not occur in nature, provided that these H _N variants retain the above-mentioned translocation function. For example, the H _N portion of a clostridial neurotoxin includes various amino acid sequences having at least 70% (e.g., at least 80% or at least 85% or at least 90% or at least 95% or at least 97% or at least 98% or at least 99%) sequence identity with the HN portion of the wild-type clostridial neurotoxin, although provided that they retain translocation function .

Alternatively, the translocation peptide may be of non-clostridial origin, for example it may be the translocation domain of diphtheria toxin [O. Keefe et al., Proc. Natl. Acad. Sci. USA (1992) 89, 6202-6206; Silverman et al., J. Biol. Chem. (1993) 269, 22524-22532; and London, E. (1992) Biochem. Biophys. Acta., 1112, pp.25-51], translocation domain of exotoxin type A of Pseudomonas aeruginosa [Prior et al. Biochemistry (1992) 31, 3555-3559], translocation domains of anthrax toxin [Blanke et al. Proc. Natl. Acad. Sci. USA (1996) 93, 8437-8442], a variety of fusogenic or hydrophobic peptides with translocation function [Plank et al. J Biol. Chem. (1994) 269, 12918-12924; and Wagner et al (1992) PNAS, 89, pp.7934-7938] and amphiphilic peptides [Murata et al (1992) Biochem., 31, pp.1986-1992].

Reference to non-clostridial neurotoxin translocation peptides includes fragments and variants of amino acid sequences having at least 70% (e.g., at least 80% or at least 85% or at least 90% or, according to at least 95% or at least 97% or at least 98% or at least 99%) sequence identity with the corresponding wild-type non-clostridial translocation peptide sequence, provided that the variant retains translocation function.

- 24 045463

Source translocation domain Amino acid residues Links Diphtheria toxin 194-380 Silverman et al., 1994, J. Biol. Chern. 269, 2252422532 London E., 1992, Biochem. Biophys. Acta., 1113, 25-51 Domain II of Pseudomonas aeruginosa exotoxin 405-613 Prior et al., 1992, Biochemistry 31, 3555-3559 Kihara & Pastan, 1994, Bioconj Chem. 5, 532-538 Influenza virus hemagglutinin GLFGAIAGFIENGWEGMIDG WYG and its variants Plank et al., 1994, J. Biol. Chem. 269, 12918-12924 Wagner et al., 1992, PNAS, 89, 7934-7938 Murata et al., 1992 Fusogenic protein of Semliki forest virus Glycoprotein G of vesicular stomatitis virus Protein F of SER virus Translocation domain 118-139 Translocation domain Biochemistry 31, 1986-1992 Kielian et al., 1996, J Cell Biol. 134(4), 863-872 Yao et al., 2003, Virology 310(2),319-332 Seth et al., 2003, J Virol 77(11)6520-6527 Foamy virus envelope glycoprotein Translocation domain Picard-Maureau et al., 2003, J Virol. 77(8), 47224730

The polypeptides of the present invention may further comprise a translocation facilitating domain. This domain facilitates delivery of the noncytotoxic protease into the cytosol of the target cell and is described, for example, in International Patent Applications WO08/008803 and WO08/008805, each of which is incorporated herein by reference.

For example, suitable translocation facilitating domains include a viral envelope fusogenic peptide domain, e.g., suitable fusogenic peptide domains include an influenza virus fusogenic peptide domain (e.g., the 23 amino acid influenza A virus fusogenic peptide domain), an alphavirus fusogenic peptide domain (e.g., Semliki Forest Virus 26 amino acid fusogenic peptide domain), vesiculovirus fusogenic peptide domain (e.g. vesicular stomatitis virus 21 amino acid fusogenic peptide domain), respiratory virus fusogenic peptide domain (e.g. Sendai virus 25 amino acid fusogenic peptide domain), fusogenic peptide domain morbilivirus (e.g. canine distemper virus 25 amino acid fusogenic peptide domain), avulavirus fusogenic peptide domain (e.g. Newcastle disease virus 25 amino acid fusogenic peptide domain), henipavirus fusogenic peptide domain (e.g. Hendra virus 25 amino acid fusogenic peptide domain), a metapneumovirus fusogenic peptide domain (eg, the 25 amino acid human metapneumovirus fusogenic peptide domain) or a spumavirus fusogenic peptide domain, such as a simian foamy virus fusogenic peptide domain; or fragments or variations thereof.

As a further example, the translocation facilitating domain may include the H _CN domain of a clostridial neurotoxin or a fragment or variant thereof (having at least 70% sequence identity with the corresponding wild-type sequence), although provided that it retains enhanced translocation function. In more detail, the translocation facilitating domain H _CN of a clostridial toxin may have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, at least 275 amino acids. In this regard, the translocation-facilitating domain H _CN of clostridial toxin,

- 25045463 preferably has a length of no more than 200 amino acids, no more than 225 amino acids, no more than 250 amino acids or no more than 275 amino acids. Specific (reference) examples include:

botulinum neurotoxin type A - amino acid residues (872-1110);

botulinum neurotoxin type B - amino acid residues (859-1097);

botulinum neurotoxin type C - amino acid residues (867-1111);

botulinum neurotoxin type D - amino acid residues (863-1098);

botulinum neurotoxin type E - amino acid residues (846-1085);

botulinum neurotoxin type F - amino acid residues (865-1105);

botulinum neurotoxin type G - amino acid residues (864-1105);

tetanus neurotoxin - amino acid residues (880-1127).

In a fourth aspect, the present invention relates to a method for cleaving hSNAP-23, wherein the method comprises contacting hSNAP-23 with a modified BoNT/A L chain protease or nucleic acid construct or delivery vehicle as described herein, thereby allowing the modified BoNT/A L chain to bind to said hSNAP-23, followed by proteolytic cleavage of hSNAP-23 by the modified BoNT/A L chain protease. In one embodiment of the invention, the method is performed in vitro.

In one embodiment of the invention, said hSNAP-23 cleavage method includes the preliminary steps of:

1) binding of the delivery vehicle to the target cell using its targeting fragment (NF); And

2) translocation of the modified BoNT/A L chain into a target cell, preferably into the cytosol of said target cell, using a delivery vehicle translocation peptide.

Preferably, NP binds to a site on a target cell (eg, a protein, sugar and/or lipid molecule), which site is capable of receptor-mediated endocytosis, and the delivery vehicle is then internalized by the target cell through endosome formation. The delivery vehicle translocation peptide can then translocate the modified BoNT/A L chain across the endosomal membrane and into the cytosol of the target cell.

In another aspect, the present invention provides a modified L chain protease (BoNT/A) or nucleic acid construct or delivery vehicle as described herein for use in the hSNAP23 cleavage method as described above.

In another aspect, the present invention relates to a modified L-chain protease (BoNT/A) described herein for use in a method of treatment, preferably a method of treating a secretory disorder.

Thus, in one aspect, the present invention relates to a BoNT/A L chain protease described herein, or a nucleic acid construct described herein, or a delivery vehicle described herein for use as a drug.

In such aspects, said modified BoNT/A L chain protease is preferably contained in a BoNT further including a heavy chain, that is, a full-length BoNT. Such full-length BoNTs typically have a 150-kDa polypeptide chain containing a 100-kDa heavy chain and a 50-kDa light chain linked by a disulfide bond, and are organized into three functional domains: an N-terminal proteolytic light chain (L-chain); and a C-terminal heavy chain (H-chain), the latter consisting of a translocation domain ( _HN ) and a C-terminal neuron-binding domain ( _HC ).

Preferred secretory disorders include muscle spasticity/overactive muscle movement (including spasticity after stroke, spasticity after spinal cord injury, spasms of the head and neck, eyelid, vagina, limbs, jaws and vocal cords), strabismus, hyperhidrosis and severe primary axillary hyperhidrosis.

The present invention will be better understood in light of the following detailed examples. However, one skilled in the art will appreciate that this detailed description is not limiting and that various modifications, substitutions, departures and changes may be made without departing from the scope of the invention.

Description of drawings

Fig. 1 - hSNAP-23 and hSNAP25 cleavage data for the modified BoNT/A L chain proteases of the present invention. (A) data for BoNT/A L chain protease having mutations in one binding pocket, (B) data for BoNT/A L chain protease having mutations in multiple binding pockets.

Fig. 2 - shaded 3D image of the interaction of the L-chain protease BoNT/A (contour) with the binding sites of SNAP-25/23 (visualization line).

- 26 045463

Amino acid sequences

SEQ ID NO: 1 - Wild type BoNT/A light chain (amino acid residues 1-438 Uniprot A5HZZ9)

MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDFTFTNPE EGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGI PFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTR NGYGSTQYIRFSPDFTF GFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAIN PNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTL NKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFV KFFKVLNRKTYLNFDKAVFKINIVPKVNYTI YDGFNLRNTNLAANFNGQNTEINNMNFT KLKNFTGLFEFYKLLCVRGIITSK

SEQ GO NO: 2 - human SNAP23

MDNLSSEEIQQRAHQITDESLESTRRILGLAIESQDAGIKTITMLDEQKEQLNRIEE GLDQINKDMRETEKTLTELNKCCGLCVCPCNRTKNFESGKAYKTTWGDGGENSPCNVV SKQPGPVTNGQLQQPTTGAASGGYIKRITNDAREDEMEENLTQVGSILGNLKDMALNIG NEIDAQNPQIKRITDKADTNR DRIDIANARAKKLIDS

SEQ GO NO: 3 - SNAP25 human and rodent

MAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVMLDEQ GEQLERIEEGMDQINKDMKEAEKNLTDLGKFCGLCVCPCNKLKSSDAYKKAWGNNQD GVVASQPARVVDEREQMATSGGFIRRVTNDARENEMDENLEQVSGTTGNLRHMALDMG NEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGS G

SEQ GO NO: 4 - IgA protease site His6 tag (artificial)

PPTPGHHHHHH

SEQ GO NO: 5 - Twin Strep Tag (artificial)

MASWSHPQFEKGGGSGGGSGGGSWSHPQFEKGAGS

SEQ ID NO: 6 - His6 tag (artificial)

GHHHHHH

SEQ ID NO: 7 - V-IgA protease site His6 tag (artificial)

VPPTPGHHHHHH

SEQ ID NO: 8 - Wild Type BoNT/Al

MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDFTFTNPE EGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGI PFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTR NGYGSTQYIRFSPDFTF GFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAIN PNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTL NKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFV KFFKVLNRKTYLNFDKAVFKINIVPKVNYTI YDGFNLRNTNLAANFNGQNTEINNMNFT KLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTND LNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFP NGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTF FSSDYVKKVNK ATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALI FSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWL AKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLN ESIN KAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRL KDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEY TIINC MENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNR LNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEI KDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPR GSVMTTNI YLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQ AGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNI AKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

SEQ ID NO: 9 - LHn linker (artificial)

- 27 045463

VRGIITSKTKSLDKGYNKALNDL

SEQ ID NO: 10 - enterokinase activation site

DDDDK

SEQ ID NO: 11 - BoNT/ΑΙ activation loop

VDGIITSKTKSDDDDKNKALNLQ

Examples

Example 1 - Preparation of modified BoNT/A L-chains (LC BoNT/A) of the present invention.

Plasmid pBN3, encoding the wild-type BoNT/A L chain (amino acids 1-448, SEQ ID NO: 1), was prepared by PCR and appropriate oligonucleotide primers using bacterial DNA of strain 62A as a template. DNA encoding the amino acid sequence PPTPGHHHHHH (SEQ ID NO: 4) was inserted after the codon for the amino acid Ala-449. E. coli strain M15pREP4 (Qiagen, Hilden, Germany) was transfected with plasmid pBN3 containing wild-type LC BoNT/A or its mutants, that is, protease mutants SEQ ID NO: 1, as described herein. For each transfected E. coli strain, one bacterial colony grown overnight in 5 ml of 2YT medium was used to inoculate 500 ml of 2YT medium.

After the culture reached an OD of 0.7 at 600 nm, BoNT/A L chains were produced through a 15 h induction using 0.2 mM IPTG at 21°C. Bacteria were collected by centrifugation and frozen at −20°C overnight. Bacteria were resuspended in lysis buffer (300 mM NaCl, 50 mM phosphate, pH 8.0) with the addition of benzamidine, pepstatin A and PMSF at final concentrations of 5 mM, 1 μg/ml and 0.5 mM, respectively, lysed by ultrasound, and the lysate was purified by centrifugation for 30 minutes at 29,000 g, and the BoNT/A L chain was coupled to M2+-nitrilotriacetic acid and agarose beads. The beads were washed with 20 bed volumes of lysis buffer containing 10 mM imidazole, and the BoNT/A L chain was eluted with lysis buffer containing 100 mM imidazole. Fractions containing the desired protein were dialyzed against toxin assay buffer (150 mM MSG, 10 mM Hepes-KOH, pH 7.2) and finally the purified L chain, frozen in liquid nitrogen and stored at -70°C. C.

Example 2 - Cell-free hSNAP-23 cleavage assay.

Plasmid hSNAP-23 (SEQ ID NO: 2) was prepared for expression in E. coli and in vitro transcription/translation, pS3-hSNAP-23His6.

It encodes an N-terminal Twin Strep Tag fusion (MASWSHPQFEKGGGSGGGSGGGSWSHPQFEKGAGS, SEQ ID NO: 5) and a C-terminal His6 fusion tag (GHHHHHH, SEQ ID NO: 6) after the carboxy-terminal serine 211 codon.

To obtain and purify the protein, E. coli strain BL21-DE3 (Stratagene Europe, Ebsdorfergrund, Germany) was transfected with the plasmid pS3-hSNAP-23His6 according to the same protocol that is described in detail for the L-chain protease BoNT/A in example 1. However, the protein eluted from M2+nitrilotriacetic acid-agarose beads, was further purified on strep-tactin-agarose beads (IBM Lifesciences, Göttingen, Germany) by washing with 20 bed volumes of 0.1 M Tris, pH 8.0 and eluting with 10 mM desthiobiotin at 0. 1 M Tris, pH 8.0. In addition, 10 mM P -mercaptoethanol was added to all buffers used to purify hSNAP-23.

Radiolabeled hSNAP-23 was then produced by in vitro transcription/translation using pS3-hSNAP-23His6, T7-coupled TNT reticulocyte lysate system (Promega) and [35S]methionine (370 kBq/μl, >37 TBq/mmol; Hartmann Analytic, Braunschweig, Germany) according to the manufacturer's instructions.

The cell-free hSNAP-23 cleavage assay included recombinant hSNAP-23 at a final concentration of 20 μM plus 1 μl of a transcription/translation mixture consisting of [35S]methionine-labeled hSNAP-23 and each modified or wild-type BoNT/A L chain. at final concentrations of 1 μmol or 10 nmol, which were incubated for 60 min at 37°C in a total volume of 10 μl of toxin assay buffer. Reactions were stopped by adding an equal volume of 2× sample buffer [120 mM Tris-HCl (pH 6.75), 10% (v/v) β-mercaptoethanol, 4% (v/v) SDS, 20% (wt) ./vol.) glycerin and 0.014% (wt./vol.) bromophenol blue]. After incubation for 30 minutes at 37°C, each sample was analyzed by SDS-PAGE using 15% Tris-glycine gels (acrylamide/bis-acrylamide ratio: 73.5:1).

The gels were dried and the radiolabeled protein was visualized using a FLA-9000 phosphoimager (Fuji Photo Film, Co., Ltd., Tokyo, Japan). Quantification of radiolabeled protein and its degradation products was performed using Multigauge 3.2 software (Fuji Photo Film). To determine the kinetic parameters of the wild-type BoNT/A L-chain enzyme and its mutants, substrate concentrations were varied from 5 to 100 μM using hSNAP-23 produced in E. coli. To each of the different substrate concentrations, 1 μl of radiolabeled hSNAP-23 produced by in vitro transcription/translation was added. Incubation was performed in a final volume of 25 μl of toxin assay buffer. After 2 and 4 min of incubation at 37°C, 10 μl aliquots were taken and the enzymatic reaction was stopped by mixing with 10

- 28 045463 µl of pre-cooled 2× SDS-PAGE sample buffer. The percentage of degradation was determined from the conversion of the radiolabeled substrate as detailed above and was used to calculate the initial rate of substrate hydrolysis. The values of Km, Kcat and Vmax were calculated by nonlinear regression using the GraphPad Prism 4.03 program (GraphPad Software

Inc., San Diego, USA).

The obtained data is shown in Fig. 1.

Example 3 - Cell-free hSNAP-25 cleavage assay.

Plasmid pBNIO for expression of hSNAP-25 (SEQ ID NO: 3) in E. coli was described in Binz et al. (J Biol Chem. 1994;269:1617-20). Following the codon for carboxy-terminal glycine-206 is DNA encoding the amino acid sequence VPPTPGHHHHHH (SEQ ID NO: 7). The in vitro transcription/translation plasmid, pSNAP-25His6, was subsequently generated by subcloning the EcoRI-SalI fragment of pBNIO into the appropriately digested plasmid pSP73 (Promega, Mannheim, Germany).

To obtain the protein and purify SNAP-25, E. coli strain M15pREP4 (Qiagen, Hilden, Germany) was transfected with the pBNIO plasmid according to the same protocol that is described in detail for the BoNT/A L chain protease in example 1.

Radiolabeled SNAP-25 was produced by in vitro transcription/translation using pSNAP-25His6, TNT reticulocyte lysate system coupled to SP6 (Promega) and [35S]methionine (370 kBq/μL, >37 TBq/mmol; Hartmann Analytic, Braunschweig , Germany) according to the manufacturer's instructions.

The hSNAP25 cleavage assay was performed exactly as described for hSNAP-23 in Example 2.

The obtained data is shown in Fig. 1.

Example 4 - Preparation of LH _N domains containing the modified light chain A (BoNT/A LC) of the present invention.

This example describes the construction of LH _N translocation domains containing a modified light chain A (BoNT/A LC) exhibiting hSNAP23 cleavage activity in accordance with the present invention. Such LH _N domains can be used to create families of TSI delivery vehicles by adding appropriate targeting moieties.

Briefly, for each BoNT/A LC mutant of the present invention, cloning vectors were first constructed by chemically synthesizing DNA (GeneArt, ThermoFisher) that encodes said BoNT/A LC mutant and that is optimized for expression in E. coli, which DNA was subcloned into the vector pCR4 (Invitrogen). In parallel, a cloning vector for the H _N BoNT/A domain was constructed in a similar manner by chemically synthesizing codon-optimized DNA encoding the H _N /A domain (corresponding to amino acid residues 449-872 SEQ ID NO: 8, UniprotKB accession number A5HZZ9), and subcloned into a standard vector such as pCR4 vector (Invitrogen). A cloning vector for the LHN linker was also constructed by chemically synthesizing codon-optimized DNA encoding the linker and subcloning into a standard vector such as the pCR4 vector (Invitrogen). Specifically, the LH _N VRGIITSKTKSLDKGYNKALNDL linker (SEQ ID NO: 9) suitable for the BoNT/ serotype (this is an interdomain polypeptide region that exists between the cysteines of the disulfide bridge between LC and the H _N domain of BoNT/A) was used to construct the LH _N linker vector. Alternative LH _N linker constructs can also be created: in fact, as is well known to one skilled in the art, to obtain a cleavage site for a particular protease, one can either exploit the native susceptibility to proteolysis by the LysC protease or insert an enterokinase activation site (e.g. DDDDK, SEQ ID NO: 10) into the activation loop to produce a sequence such as VDGIITSKTKSDDDDKNKALNLQ (SEQ ID NO: 11), or a protease site for any other protease well known in the art, such as PreScission factor, can be inserted into this activation loop Xa, thrombin, TEV protease, etc.

The LHN domains were then assembled by cloning into a modified pET expression vector (Novagen) in 2 main steps. DNA encoding each of the modified LC BoNT/A of the present invention was inserted before the DNA encoding the LH _N linker, which linker was further before the DNA encoding the H _N /A domain.

Example 5 - Preparation of TSI delivery vehicles that bind to a non-neuronal cell in accordance with the present invention.

This example describes the construction of TSI delivery vehicles by adding a suitable targeting moiety (in this case, human GHRP) to each C-terminal end of the LHN domains containing the modified light chain A of the present invention, as described above in Example 2. To do this, between the targeting group and a flexible linker was introduced with the LHN domain.

Briefly, linker-hGHRP cloning vectors were constructed by chemically synthesizing codon-optimized DNA encoding a flexible linker fused in frame to an hGHRP targeting moiety and subcloning into the pCR4 vector (Invitrogen).

The TSI constructs were then assembled by cloning the DNA encoding the linker-hGHRP into each of the pET expression vectors containing the LH _N domains described in Example 2, such that the linker-hGHRP would be fused in frame to the C-terminal end of each LHN domain.

For protein expression of each TSI carrier, 100 ml of modified Terrific Broth (TB) medium containing 0.2% glucosamine and 30 μg/ml kanamycin was incubated in a 250 ml flask with one bacterial colony (E. coli BL21 (DE3)) transfected with TSI Each culture was grown at 37°C and 225 rpm for 16 hours, followed by 10 ml overnight culture in 1 liter of modified TB medium containing 0.2% glucosamine and 30 µg/ml kanamycin in a 2 liter flask. The resulting culture was then grown at 37°C until an approximate OD value of 0.5 was reached at 600 nm, after which the temperature was reduced to 16°C. After 1 hour, each culture was induced with 1 mM IPTG and further grown at 16°C for another 16 hours. Bacteria were collected by centrifugation and frozen at -20°C overnight.

Subsequent purification of each expressed TSI was performed as follows.

The bacteria were thawed and the cell pellet was treated with ultrasound to lyse the cells. After centrifugation, the supernatant was loaded onto a 0.1 M NiSO ₄ chelate column equilibrated with 50 mM HEPES, pH 7.2, 200 mM NaCl. The column was washed with a buffer containing 40-100 mM imidazole (step gradient) to elute unbound protein, and a buffer containing 200 mM imidazole to elute the TSI protein. Fractions containing the desired protein (TSI) were then dialyzed against a buffer containing 50 mM HEPES, pH 7.2, 200 mM NaCl. Protease (in this case LysC) was then added to 1 mg of purified TSI in the appropriate amount to activate it (i.e., so that TSI forms a di-chain capable of binding to GHRP, translocating the light chain into the cytoplasm, and catalytically degrading hSNAP23). The resulting mixture was then further purified by loading it onto a 0.1 M NiSO4 chelate column equilibrated with 50 mM HEPES, pH 7.2, 200 mM NaCl. The column was first washed with 50 mM HEPES, pH 7.2, 200 mM NaCl, then with a buffer containing 40-100 mM imidazole to elute nonspecifically bound protein, and a buffer containing 200 mM imidazole to elute activated TSI. Fractions containing the desired activated protein (TSI) were subsequently dialyzed against a buffer containing 50 mM HEPES, pH 7.2, 150 mM NaCl. The dialyzed protein was then concentrated to approximately 2 mg/ml, aliquoted and finally frozen at -80°C.

Clause.

1. Modified botulinum neurotoxin A L chain protease (BoNT/A) that cleaves human SNAP-23 (hSNAP-23) having a modified amino acid sequence compared to wild type BoNT/A L chain (SEQ ID NO: 1) , which contains:

a) at least one substitution of an amino acid residue located in the first binding pocket of the BoNT/A L chain protease for binding to the P182/D178 binding site of hSNAP-23;

b) wherein said first binding pocket of the BoNT/A L chain protease is defined by amino acid residues E148, T307, A308 and Y312 of the wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said at least one amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of asparagine and tyrosine, at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue E148 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or ii) an amino acid residue selected from the group consisting of phenylalanine, isoleucine and leucine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue T307 of the wild type BoNT/A L chain (SEQ ID NO: 1 ); and/or iii) an amino acid residue selected from the group consisting of proline, asparagine, threonine and isoleucine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue A308 of the wild type BoNT/A L chain (SEQ ID NO : 1); and/or iv) an amino acid residue selected from the group consisting of lysine, valine, methionine and leucine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue Y312 of the wild type BoNT/A L chain (SEQ ID NO : 1).

2. Modified L-chain protease BoNT/A according to claim 1, additionally containing:

a) replacing an amino acid residue located in the second binding pocket of the BoNT/A L chain protease to bind to the R186 binding site of hSNAP-23;

b) wherein said second BoNT/A L chain protease binding pocket is defined by amino acid residue S143 of wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of glutamine, glutamate and aspartate, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue S143 of the wild type BoNT/A L chain (SEQ ID NO: 1).

3. Modified L-chain protease BoNT/A according to claim 1 or claim 2, additionally containing:

a) at least one substitution of an amino acid residue located in the third binding pocket of the L-chain protease BoNT/A for binding to the K185 binding site of hSNAP-23;

b) wherein said third binding pocket of the BoNT/A L chain protease is defined by an amino acid

- 30 045463 residues V304 and G305 of the wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said at least one amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of glutamate and aspartate, at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue V304 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or ii) an amino acid residue selected from the group consisting of glutamate and aspartate, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue G305 of the wild type BoNT/A L chain (SEQ ID NO: 1).

4. Modified L-chain protease BoNT/A according to any of the preceding paragraphs, additionally containing:

a) replacing an amino acid residue located in the fourth binding pocket of the BoNT/A L chain protease to bind to the D189/D192 binding site of hSNAP-23;

b) wherein said fourth BoNT/A L chain protease binding pocket is defined by amino acid residue Q29 of wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of alanine at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue Q29 of the wild type BoNT/A L chain (SEQ ID NO: 1).

5. Modified L-chain protease BoNT/A according to any of the preceding paragraphs, additionally containing:

a) at least one substitution of an amino acid residue located in the fifth binding pocket of the BoNT/A L chain protease to bind to the K206 binding site of hSNAP-23;

b) wherein said fifth binding pocket of the BoNT/A L chain protease is defined by amino acid residues Y251, L256, V258, L367 and F369 of the wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said at least one amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of glutamate and aspartate, at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue Y251 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or ii) an amino acid residue selected from the group consisting of glutamate, aspartate, glutamine, glycine, alanine and arginine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue L256 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or iii) an amino acid residue selected from the group consisting of serine, alanine, proline, leucine and glutamate, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue V258 of the wild type BoNT/A L chain (SEQ ID NO : 1); and/or iv) an amino acid residue selected from the group consisting of alanine and glycine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue L367 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or

v) an amino acid residue selected from the group consisting of glycine, serine and leucine, at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue F369 of the wild type BoNT/A L chain (SEQ ID NO: 1).

6. Modified L-chain protease BoNT/A according to any of the preceding paragraphs, additionally containing:

a) replacing an amino acid residue located in the sixth binding pocket of the BoNT/A L chain protease to bind to the I198 binding site of hSNAP-23;

b) wherein said sixth BoNT/A L chain protease binding pocket is defined by amino acid residue K166 of wild type BoNT/A L chain (SEQ ID NO:

1);

c) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of valine, phenylalanine, leucine and isoleucine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue K166 of the wild type BoNT/A L chain (SEQ ID NO: 1) .

7. Modified L-chain protease BoNT/A according to any of the preceding paragraphs, additionally containing:

a) replacing an amino acid residue located in the seventh binding pocket of the BoNT/A L chain protease to bind to the D210 binding site of hSNAP-23;

b) wherein said seventh binding pocket of the BoNT/A L chain protease is defined by amino acid residue S254 of the wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of alanine, at a position in the modified amino acid sequence of the L chain protease that corresponds to the amino acid

- 31045463 residue S254 of the wild-type BoNT/A L chain (SEQ ID NO: 1).

8. Modified L-chain protease BoNT/A according to any of the preceding paragraphs, additionally containing:

a) replacing an amino acid residue located in the eighth binding pocket of the BoNT/A L chain protease to bind to the D168 binding site of hSNAP-23;

b) wherein said eighth BoNT/A L chain protease binding pocket is defined by amino acid residue K340 of wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of histidine at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue K340 of the wild type BoNT/A L chain (SEQ ID NO: 1).

9. Modified botulinum neurotoxin A L chain protease (BoNT/A) that cleaves human SNAP-23 (hSNAP-23) having a modified amino acid sequence compared to wild type BoNT/A L chain (SEQ ID NO: 1) , which contains:

a) replacing an amino acid residue located in the fourth binding pocket of the BoNT/A L chain protease to bind to the D189/D192 binding site of hSNAP-23;

b) wherein said fourth BoNT/A L chain protease binding pocket is defined by amino acid residue Q29 of wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of alanine at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue Q29 of the wild type BoNT/A L chain (SEQ ID NO: 1).

10. Modified botulinum neurotoxin A L chain protease (BoNT/A) that cleaves human SNAP-23 (hSNAP-23) having a modified amino acid sequence compared to wild type BoNT/A L chain (SEQ ID NO: 1) , which contains:

a) replacing an amino acid residue located in the sixth binding pocket of the BoNT/A L chain protease to bind to the I198 binding site of hSNAP-23;

b) wherein said sixth BoNT/A L chain protease binding pocket is defined by amino acid residue K166 of wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of valine, phenylalanine, leucine and isoleucine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue K166 of the wild type BoNT/A L chain (SEQ ID NO: 1) .

11. Modified botulinum neurotoxin A L chain protease (BoNT/A) that cleaves human SNAP-23 (hSNAP-23) having a modified amino acid sequence compared to wild type BoNT/A L chain (SEQ ID NO: 1) , which contains:

a) at least one substitution of an amino acid residue located in the fifth binding pocket of the BoNT/A L chain protease to bind to the K206 binding site of hSNAP-23;

b) wherein said fifth binding pocket of the BoNT/A L chain protease is defined by amino acid residues Y251, L256, V258, L367 and F369 of the wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said at least one amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of glutamate and aspartate, at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue Y251 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or ii) an amino acid residue selected from the group consisting of aspartate, glutamine, glycine, alanine and arginine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue L256 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or iii) an amino acid residue selected from the group consisting of serine, alanine, proline, leucine and glutamate, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue V258 of the wild type BoNT/A L chain (SEQ ID NO : 1); and/or iv) an amino acid residue selected from the group consisting of alanine and glycine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue L367 of the wild type BoNT/A L chain (SEQ ID NO: 1); and/or

v) an amino acid residue selected from the group consisting of glycine, serine and leucine, at a position in the amino acid sequence of the modified L chain protease that corresponds to amino acid residue F369 of the wild type BoNT/A L chain (SEQ ID NO: 1).

12. Modified botulinum neurotoxin A L chain protease (BoNT/A) that cleaves human SNAP-23 (hSNAP-23) having a modified amino acid sequence compared to wild type BoNT/A L chain (SEQ ID NO: 1) , which contains:

a) replacement of an amino acid residue located in the seventh binding pocket of protease L-

- 32 045463 BoNT/A chain, for binding to the D210 binding site of hSNAP-23;

b) wherein said seventh binding pocket of the BoNT/A L chain protease is defined by amino acid residue S254 of the wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of alanine at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue S254 of the wild type BoNT/A L chain (SEQ ID NO: 1).

13. Modified botulinum neurotoxin A L chain protease (BoNT/A) that cleaves human SNAP-23 (hSNAP-23) having a modified amino acid sequence compared to wild type BoNT/A L chain (SEQ ID NO: 1) , which contains:

a) replacing an amino acid residue located in the eighth binding pocket of the BoNT/A L chain protease to bind to the D168 binding site of hSNAP-23;

b) wherein said eighth BoNT/A L chain protease binding pocket is defined by amino acid residue K340 of wild type BoNT/A L chain (SEQ ID NO: 1);

c) and wherein said amino acid residue substitution includes:

i) an amino acid residue selected from the group consisting of histidine at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue K340 of the wild type BoNT/A L chain (SEQ ID NO: 1).

14. Modified L-chain protease of botulinum neurotoxin A (BoNT/A) according to any one of claims 9-13, additionally containing at least one replacement of an amino acid residue located in another binding pocket of the L-chain protease BoNT/A wherein said amino acid residue substitution and said other BoNT/A L-chain protease binding pocket are defined by the technical features listed in any one of claims 1 to 13.

15. A nucleic acid construct comprising or consisting of a nucleic acid sequence encoding a modified BoNT/A L chain protease according to any one of the preceding claims.

16. Delivery vehicle containing:

a) a modified L-chain protease BoNT/A according to any one of claims 1 to 14, or a nucleic acid construct according to claim 15; And

b) means for delivering said modified BoNT/A L chain protease or said nucleic acid construct to a target cell, preferably a non-neuronal target cell.

17. The delivery device according to claim 16, where the means b) for delivering said modified L-chain protease BoNT/A to the target cell contains:

i) a targeting moiety that binds the delivery vehicle to the target cell; and ii) a translocation peptide that translocates the modified BoNT/A L chain protease or nucleic acid construct into a target cell, preferably a non-neuronal target cell.

18. A method for cleaving hSNAP-23, comprising contacting hSNAP-23 with an L-chain protease (BoNT/A) according to any one of claims 1 to 14, or with a nucleic acid construct according to claim 15, or with a delivery vehicle according to paragraph 16 or 17.

19. L-chain protease (BoNT/A) according to any one of claims 1 to 14, or a nucleic acid construct according to claim 15, or a delivery vehicle according to claim 16 or 17, for use in the method of claim 18.

Sequence List <110> IPSEN BIOPHARM LIMITED <12 0> CLEAVING NON-NEURAL SNARE BOTULINUM NEUROTOXINS <130> P48382WO <160>11 <170> Patent version 3.5 <210>1 <211>438 <212> Protein <213> Clostr idium botulinum < 400 >1

Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly

- 33 045463

Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn

25

Val Lys Ala Phe Lys Ile His Asn Lys Ile 35 40

Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp

55

Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp 65 70

Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly

90

Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met

100 105

Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr

115 120

Val Ile Asp Thr Asn Cys Ile Asn Val Ile

130 135

Arg Ser Glu Glu Leu Asn Leu Val Ile Ile 145 150

Ile Gln Phe Glu Cys Lys Ser Phe Gly His

165 170

Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile

180 185

Thr Phe Gly Phe Glu Glu Ser Leu Glu Val

195 200

Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala

210 215

Leu Ile His Ala Gly His Arg Leu Tyr Gly

225 230

Arg Val Phe Lys Val Asn Thr Asn Ala Tyr

245 250

Glu Val Ser Phe Glu Glu Leu Arg Thr Phe

260 265

Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe

275 280

Gly Gln Met Gln Pro 30

Val Ile Pro Glu Arg 45

Asn Pro Pro Pro Glu 60

Thr Tyr Leu Ser Thr 80

Thr Lys Leu Phe Glu 95

Leu Thr Ser Ile Val

110

Asp Thr Glu Leu Lys 125

Pro Asp Gly Ser Tyr 140

Pro Ser Ala Asp Ile

160

Val Leu Asn Leu Thr

175

Phe Ser Pro Asp Phe 190

Thr Asn Pro Leu Leu

205

Thr Leu Ala His Glu 220

Ala Ile Asn Pro Asn

240

Glu Met Ser Gly Leu

255

Gly His Asp Ala Lys

270

Leu Tyr Tyr Tyr Asn

285

- 34 045463

Lys Phe Lys Asp Ile Ala Ser Thr Leu

290 295

Asn Lys Ala Lys Ser Ile

300

Gly Thr Thr Ala Ser Leu Gln

305 310

Tyr Met Lys Asn Val Phe Lys Glu

315

Tyr Leu Leu Ser Glu Asp Thr 325

Ser Gly Lys Phe Ser Val Asp Lys

330 335

Lys Phe Asp Lys

340

Asn Phe Val Lys

355

Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu 345 350

Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu 360 365

Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn

370 375 380

Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala

385 390 395

Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys

405 410 415

Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val

420 425 430

Val

Lys 320

Leu

Asp

Asn

Tyr

Asn 400

Leu

Arg

Gly Ile Ile Thr Ser Lys 435 <210>2 <211>211 <212> Protein <213> Homo sapiens <400>2

Met Asp Asn Leu Ser Ser Glu 15

Glu Ile Gln Gln Arg Ala His Gln

15

Thr Asp Glu Ser Leu Glu Ser 20

Thr Arg Arg Ile Leu Gly Leu Ala

thirty

Glu Ser Gln Asp Ala Gly Ile 35

Lys Thr Ile Thr Met Leu Asp Glu 40 45

Lys Glu Gln Leu Asn Arg Ile

55

Glu Glu Gly Leu Asp Gln Ile Asn 60

Asp Met Arg Glu Thr Glu Lys

70

Gly Leu Cys Val Cys Pro Cys 85

Thr Leu Thr Glu Leu Asn Lys Cys 75

Asn Arg Thr Lys Asn Phe Glu Ser 90 95

Ile

Ile

Gln

Lys

Cys 80

Gly

- 35 045463

Lys Ala Tyr Lys Thr Thr Trp Gly Asp Gly

100 105

Asn Val Val Ser Lys Gln Pro Gly Pro Val

115 120

Gln Pro Thr Thr Gly Ala Ala Ser Gly Gly

130 135

Asn Asp Ala Arg Glu Asp Glu Met Glu Glu 145 150

Ser Ile Leu Gly Asn Leu Lys Asp Met Ala

165 170

Ile Asp Ala Gln Asn Pro Gln Ile Lys Arg

180 185

Thr Asn Arg Asp Arg Ile Asp Ile Ala Asn

195 200

Ile Asp Ser

210 <210>3 <211>206 <212> Protein <213> Homo sapiens <400>3

Met Ala Glu Asp Ala Asp Met Arg Asn Glu

510

Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu

25

Leu Gln Leu Val Glu Glu Ser Lys Asp Ala

40

Met Leu Asp Glu Gln Gly Glu Gln Leu Glu 50 55

Asp Gln Ile Asn Lys Asp Met Lys Glu Ala 65 70

Leu Gly Lys Phe Cys Gly Leu Cys Val Cys

90

Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly

100 105

Val Ala Ser Gln Pro Ala Arg Val Val Asp

115 120

Glu Asn Ser Pro Cys 110

Asn Gly Gln Leu Gln 125

Ile Lys Arg Ile Thr 140

Leu Thr Gln Val Gly

160

Asn Ile Gly Asn Glu

175

Thr Asp Lys Ala Asp 190

Arg Ala Lys Lys Leu 205

Glu Glu Met Gln Arg

Ser Thr Arg Arg Met 30

Ile Arg Thr Leu Val 45

Ile Glu Glu Gly Met 60

Lys Asn Leu Thr Asp 80

Cys Asn Lys Leu Lys

Asn Gln Asp Gly Val 110

Arg Glu Gln Met Ala 125

- 36 045463

Ile Ser Gly Gly Phe Ile Arg Arg Val Thr

130 135

Glu Met Asp Glu Asn Leu Glu Gln Val Ser

145 150

Arg His Met Ala Leu Asp Met Gly Asn Glu

165 170

Gln Ile Asp Arg Ile Met Glu Lys Ala Asp

180 185

Asp Glu Ala Asn Gln Arg Ala Thr Lys Met

195 200 <210> 4 <211> 11 <212> Protein <213> Artificial sequence <220>

<223> IgA protease site His6 tag <400> 4

Pro Pro Thr Pro Gly His His His His His 1 5 10 <210> 5 <211> 35 <212> Protein <213> Artificial sequence <220>

<223> Twin Strep Tag <400> 5

Met Ala Ser Trp Ser His Pro Gln Phe Glu

5 10

Gly Gly Ser Gly Gly Gly Ser Trp Ser His

25

Asp Ala Arg Glu Asn 140

Ile Ile Gly Asn Leu

160

Asp Thr Gln Asn Arg

175

Asn Lys Thr Arg Ile 190

Gly Ser Gly

205

Gly Gly Gly Ser Gly

Gln Phe Glu Lys Gly 30

Ala Gly Ser <210> 6 <211> 7 <212> Protein <213> Artificial sequence <220>

<223> His6 tag <400> 6

Gly His His His His His

5

- 37 045463 <210> 7 <211> 12 <212> Protein <213> Artificial sequence <220>

<223> V-IgA protease site His6 tag <400>7

Val Pro Pro Thr Pro Gly His His His His His

510 <210>8 <211>1296 <212> Protein <213> Clostridium botulinum <400> 8

Met Pro Phe

Val Asp Ile

Val Asn Lys 5

Gln Phe Asn

Tyr Lys Asp 10

Pro Val Asn

Val Lys Ala

Asp Thr Phe 50

Ala Lys Gln 65

Asp Asn Glu

Arg Ile Tyr

Arg Gly Ile

115

Val Ile Asp

130

Arg Ser Glu 145

Ile Gln Phe

Arg Asn Gly

Ala Tyr Ile 20

Lys Ile Pro 25

Asn Ala Gly

Gln Met Gln

Phe Lys Ile

His Asn Lys 40

Ile Trp Val

Ile Pro Glu 45

Thr Asn Pro

Glu Glu Gly 55

Asp Leu Asn

Pro Pro Pro

Val Pro Val

Lys Asp Asn 85

Ser Thr Asp 100

Pro Phe Trp

Thr Asn Cys

Glu Leu Asn

150

Glu Cys Lys 165

Tyr Gly Ser 180

Ser Tyr Tyr

Tyr Leu Lys

Leu Gly Arg

105

Gly Gly Ser

120

Ile Asn Val 135

Leu Val Ile

Ser Phe Gly

Thr Gln Tyr

185

Asp Ser Thr 75

Gly Val Thr 90

Met Leu Leu

Thr Ile Asp

Ile Gln Pro

140

Ile Gly Pro

155

His Glu Val 170

Ile Arg Phe

Tyr Leu Ser

Lys Leu Phe 95

Thr Ser Ile

110

Thr Glu Leu 125

Asp Gly Ser

Ser Ala Asp

Leu Asn Leu

175

Ser Pro Asp

190

Gly

Pro

Arg

Glu

Thr 80

Glu

Val

Lys

Tyr

Ile 160

Thr

Phe

- 38 045463

Thr Phe Gly Phe Glu Glu Ser Leu Glu Val

195 200

Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala

210 215

Leu Ile His Ala Gly His Arg Leu Tyr Gly

225 230

Arg Val Phe Lys Val Asn Thr Asn Ala Tyr

245 250

Glu Val Ser Phe Glu Glu Leu Arg Thr Phe

260 265

Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe

275 280

Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn

290 295

Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys

305 310

Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys

325 330

Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr

340 345

Asn Phe Val Lys Phe Phe Lys Val Leu Asn

355 360

Phe Asp Lys Ala Val Phe Lys Ile Asn Ile

370 375

Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn

385 390

Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn

405 410

Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr

420 425

Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu

435 440

Ala Leu Asn Asp Leu Cys Ile Lys Val Asn

450 455

Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp

Thr Asn Pro Leu Leu

205

Thr Leu Ala His Glu 220

Ala Ile Asn Pro Asn

240

Glu Met Ser Gly Leu

255

Gly His Asp Ala Lys

270

Leu Tyr Tyr Tyr Asn

285

Ala Lys Ser Ile Val 300

Val Phe Lys Glu Lys

320

Ser Val Asp Lys Leu

335

Ile Tyr Thr Glu Asp

350

Lys Thr Tyr Leu Asn

365

Pro Lys Val Asn Tyr 380

Asn Leu Ala Ala Asn

400

Asn Phe Thr Lys Leu

415

Leu Leu Cys Val Arg 430

Lys Gly Tyr Asn Lys

445

Trp Asp Leu Phe Phe 460

Asn Lys Gly Glu Glu

- 39 045463

465

Ile

Asp

Glu

Glu

Leu 545

His

Leu

Lys

Gln

Asp 625

Leu

Ile

Ile

Val

Lys 705

Val

Glu

470

475

480

Thr Ser Asp Thr Asn

485

Leu Ile Gln Gln Tyr 500

Asn Ile Ser Ile Glu

515

Leu Met Pro Asn Ile 530

Asp Lys Tyr Thr Met

550

Gly Lys Ser Arg Ile

565

Asn Pro Ser Arg Val 580

Val Asn Lys Ala Thr

595

Leu Val Tyr Asp Phe 610

Lys Ile Ala Asp Ile

630

Asn Ile Gly Asn Met

645

Phe Ser Gly Ala Val 660

Pro Val Leu Gly Thr

675

Leu Thr Val Gln Thr 690

Trp Asp Glu Val Tyr

710

Asn Thr Gln Ile Asp

725

Asn Gln Ala Glu Ala

740

Ile Glu Ala Ala Glu

490

Tyr Leu Thr Phe Asn

505

Asn Leu Ser Ser Asp 520

Glu Arg Phe Pro Asn 535

Phe His Tyr Leu Arg

555

Ala Leu Thr Asn Ser

570

Tyr Thr Phe Phe Ser

585

Glu Ala Ala Met Phe

600

Thr Asp Glu Thr Ser 615

Thr Ile Ile Ile Pro

635

Leu Tyr Lys Asp Asp

650

Ile Leu Leu Glu Phe

665

Phe Ala Leu Val Ser

680

Ile Asp Asn Ala Leu 695

Lys Tyr Ile Val Thr

715

Leu Ile Arg Lys Lys

730

Thr Lys Ala Ile Ile 745

Glu Asn Ile Ser Leu

495

Phe Asp Asn Glu Pro 510

Ile Ile Gly Gln Leu

525

Gly Lys Lys Tyr Glu 540

Ala Gln Glu Phe Glu

560

Val Asn Glu Ala Leu

575

Ser Asp Tyr Val Lys 590

Leu Gly Trp Val Glu 605

Glu Val Ser Thr Thr 620

Tyr Ile Gly Pro Ala

640

Phe Val Gly Ala Leu

655

Ile Pro Glu Ile Ala

670

Tyr Ile Ala Asn Lys 685

Ser Lys Arg Asn Glu 700

Asn Trp Leu Ala Lys

720

Met Lys Glu Ala Leu

735

Asn Tyr Gln Tyr Asn 750

- 40 045463

Gln

Leu

Asn 785

Ile

Asp

Gln

Ile

Thr 865

Leu

Lys

Gln

Lys

Phe 945

Glu

Ser

Ile

Asp

Leu

Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp

755 760 765

Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile

770 775 780

Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met

790 795 800

Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys

805 810 815

Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly

820 825 830

Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp

835 840 845

Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser

850 855 860

Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn

870 875 880

Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser

885 890 895

Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn

900 905 910

Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu

915 920 925

Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser

930 935 940

Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn

950 955 960

Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val

965 970 975

Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu

980 985 990

Lys Gln Arg Val Val

995

Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser

1000 1005

Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg

1010 1015 1020

Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln

- 41 045463

1025

1030

1035

Lys Pro 1040 Ile Ser Asn Leu Gly 1045 Asn Ile His Ala Ser 1050 Asn Asn Ile Met Phe 1055 Lys Leu Asp Gly Cys 1060 Arg Asp Thr His Arg 1065 Tyr Ile Trp Ile Lys 1070 Tyr Phe Asn Leu Phe 1075 Asp Lys Glu Leu Asn 1080 Glu Lys Glu Ile Lys 1085 Asp Leu Tyr Asp Asn 1090 Gln Ser Asn Ser Gly 1095 Ile Leu Lys Asp Phe 1100 Trp Gly Asp Tyr Leu 1105 Gln Tyr Asp Lys Pro 1110 Tyr Tyr Met Leu Asn 1115 Leu Tyr Asp Pro Asn 1120 Lys Tyr Val Asp Val 1125 Asn Asn Val Gly Ile 1130 Arg Gly Tyr Met Tyr 1135 Leu Lys Gly Pro Arg 1140 Gly Ser Val Met Thr 1145 Thr Asn Ile Tyr Leu 1150 Asn Ser Ser Leu Tyr 1155 Arg Gly Thr Lys Phe 1160 Ile Ile Lys Lys Tyr 1165 Ala Ser Gly Asn Lys 1170 Asp Asn Ile Val Arg 1175 Asn Asn Asp Arg Val 1180 Tyr Ile Asn Val Val 1185 Val Lys Asn Lys Glu 1190 Tyr Arg Leu Ala Thr 1195 Asn Ala Ser Gln Ala 1200 Gly Val Glu Lys Ile 1205 Leu Ser Ala Leu Glu 1210 Ile Pro Asp Val Gly 1215 Asn Leu Ser Gln Val 1220 Val Val Met Lys Ser 1225 Lys Asn Asp Gln Gly 1230 Ile Thr Asn Lys Cys 1235 Lys Met Asn Leu Gln 1240 Asp Asn Asn Gly Asn 1245 Asp Ile Gly Phe Ile 1250 Gly Phe His Gln Phe 1255 Asn Asn Ile Ala Lys 1260 Leu Val Ala Ser Asn 1265 Trp Tyr Asn Arg Gln 1270 Ile Glu Arg Ser Ser 1275 Arg Thr Leu Gly Cys 1280 Ser Trp Glu Phe Ile 1285 Pro Val Asp Asp Gly 1290 Trp Gly Glu

-

Claims (9)

Arg Pro Leu 1295 <210> 9 <211> 23 <212> Protein <213> Artificial sequence <220> <223> linker LHN <400>9 Val Arg Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr 1 5 1015 Asn Lys Ala Leu Asn Asp Leu <210>10 <211>5 <212> Protein <213> Homo sapiens <400>10 Asp Asp Asp Asp Lys 15 <210>11 <211>23 <212> Protein <213> Artificial sequence <220> <223> Modified activation loop BoNT/A1 <400> 11 Val Asp Gly Ile 1 Ile Thr Ser Lys Thr Lys Ser Asp Asp Asp Asp Lys Asn Lys Ala Leu Asn Leu Gln CLAIM 1. A modified botulinum neurotoxin A (BoNT/A) L chain protease that exhibits cleavage of human SNAP-23 (hSNAP-23) with increased efficiency compared to the wild type BoNT L chain (SEQ ID NO: 1) having a modified amino acid sequence compared to the wild type BoNT/A L chain (SEQ ID NO: 1), which contains: a) at least one substitution of an amino acid residue located in the first binding pocket of the BoNT/A L chain protease for binding to the P182/D178 binding site of hSNAP-23; b) wherein said first BoNT/A L chain protease binding pocket is defined by amino acid residue E148 of wild type BoNT/A L chain (SEQ ID NO: 1); c) and wherein said at least one amino acid residue substitution involves the amino acid tyrosine at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue E148 of the wild type BoNT/A L chain (SEQ ID NO: 1). 2. Modified L-chain protease BoNT/A according to claim 1, additionally containing: a) replacing an amino acid residue located in the second binding pocket of the BoNT/A L chain protease to bind to the D189/D192 binding site of hSNAP-23; b) wherein said second binding pocket of the BoNT/A L chain protease is defined by an amino acid - 43 045463 lot residue Q29 of the L chain of wild type BoNT/A (SEQ ID NO: 1); c) and wherein said amino acid residue substitution includes: i) an amino acid residue selected from the group consisting of alanine at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue Q29 of the wild type BoNT A L chain (SEQ ID NO: 1). 3. Modified L-chain protease BoNT/A according to claim 1 or 2, additionally containing: a) replacing an amino acid residue located in the third binding pocket of the BoNT/A L chain protease to bind to the I198 binding site of hSNAP-23; b) wherein said third BoNT/A L chain protease binding pocket is defined by amino acid residue K166 of wild type BoNT/A L chain (SEQ ID NO: 1); c) and wherein said amino acid residue substitution includes: i) an amino acid residue selected from the group consisting of phenylalanine and valine, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue K166 of the wild type BoNT/A L chain (SEQ ID NO: 1). 4. Modified L-chain protease BoNT/A according to any of the preceding paragraphs, additionally containing: a) at least one substitution of an amino acid residue located in the fourth binding pocket of the BoNT/A L chain protease to bind to the K185 binding site of hSNAP-23; b) wherein said fourth BoNT/A L chain protease binding pocket is defined by amino acid residue G305 of wild type BoNT/A L chain (SEQ ID NO: 1); c) and wherein said at least one amino acid residue substitution includes: i) an amino acid residue selected from the group consisting of glutamate and aspartate, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue G305 of the wild type BoNT/A L chain (SEQ ID NO: 1). 5. Modified L-chain protease BoNT/A according to any of the preceding paragraphs, additionally containing: a) replacing an amino acid residue located in the fifth binding pocket of the BoNT/A L chain protease to bind to the R186 binding site of hSNAP-23; b) wherein said fifth BoNT/A L chain protease binding pocket is defined by amino acid residue S143 of wild type BoNT/A L chain (SEQ ID NO: 1); c) and wherein said amino acid residue substitution includes: i) an amino acid residue selected from the group consisting of glutamine, glutamate and aspartate, at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue S143 of the wild type BoNT/A L chain (SEQ ID NO: 1). 6. Modified L-chain protease BoNT/A according to any of the preceding paragraphs, additionally containing: a) at least one substitution of an amino acid residue located in the sixth binding pocket of the BoNT/A L chain protease to bind to the K206 binding site of hSNAP-23; b) wherein said sixth BoNT/A L chain protease binding pocket is defined by amino acid residue Y251 of wild type BoNT/A L chain (SEQ ID NO: 1); c) and wherein said at least one amino acid residue substitution includes: i) an amino acid residue selected from the group consisting of glutamate and aspartate, at a position in the modified amino acid sequence of the L chain protease that corresponds to amino acid residue Y251 of the wild type BoNT/A L chain (SEQ ID NO: 1). 7. Modified L-chain protease BoNT/A according to any of the preceding paragraphs, additionally containing: a) replacing an amino acid residue located in the seventh binding pocket of the BoNT/A L chain protease to bind to the D210 binding site of hSNAP-23; b) wherein said seventh binding pocket of the BoNT/A L chain protease is defined by amino acid residue S254 of the wild type BoNT/A L chain (SEQ ID NO: 1); c) and wherein said amino acid residue substitution includes: i) an amino acid residue selected from the group consisting of alanine at a position in the modified L chain protease amino acid sequence that corresponds to amino acid residue S254 of the wild type BoNT/A L chain (SEQ ID NO: 1). 8. A vector comprising a nucleic acid sequence encoding a modified L-chain protease BoNT/A according to any of the preceding paragraphs. 9. Delivery vehicle containing: a) a modified L-chain protease BoNT/A according to any one of claims 1-7 or a vector according to claim 8; And b) means for delivering said modified BoNT/A L-chain protease or said vector into a target cell, where means b) for delivering said modified BoNT/A L-chain protease into a target cell comprises: i) a targeting moiety that binds the delivery vehicle to the target cell; And -