# Patent

## Title
Multivalent Clostridial toxin derivatives and methods of their use

## Abstract

The present invention is directed to multivalent Clostridial neurotoxin derivatives having more than one binding domain directed to a cell surface feature of a target cell. Such modified neurotoxins are useful as therapeutic compositions to prevent exocytosis and secretion by the target cell. Conditions in which such compositions man be useful include, without limitation, disorders of the sensory or motor nervous system, acute or chronic pain, cancer, pancreatitis, hyperhydrosis, glandular disorders, viral infections, cystic fibrosis and the like. The invention is also directed to methods of using and administering such a composition, and methods of treating a given condition using such a composition.

## Background
This application is a non-provisional application claiming priority pursuant to 35 U.S.C. 119(e) to provisional patent application Ser. No. 60/662,151, filed on Mar. 15, 2005 and to provisional patent application Ser. No. 60/661,953 filed on Mar. 15, 2005. Each of these provisional patent applications is hereby expressly incorporated by reference herein in its entirety. Each and every patent, patent application, and other publication cited herein is expressly incorporated by reference herein in its entirety.


The present invention is drawn to multivalent Clostridial neurotoxin derivatives having more than one binding domain directed to a cell surface feature of a target cell.
The ability of Clostridial toxins, such as, e.g., Botulinum neurotoxins (BoNTs), BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F and BoNT/G, and Tetanus neurotoxin (TeNT), to inhibit neuronal transmission are being exploited in a wide variety of therapeutic and cosmetic applications, see e.g., William J. Lipham, COSMETIC AND CLINICAL APPLICATIONS OF BOTULINUM TOXIN (Slack, Inc., 2004). As an example, BOTOX® is currently approved in one or more countries for the following indications: achalasia, adult spasticity, anal fissure, back pain, blepharospasm, bruxism, cervical dystonia, essential tremor, glabellar lines or hyperkinetic facial lines, headache, hemifacial spasm, hyperactivity of bladder, hyperhidrosis, juvenile cerebral palsy, multiple sclerosis, myoclonic disorders, nasal labial lines, spasmodic dysphonia, strabismus and VII nerve disorder. In addition, Clostridial toxin therapies are proposed for treating neuromuscular disorders, see e.g., Kei Roger Aoki et al., Method for Treating Neuromuscular Disorders and Conditions with Botulinum Toxin Types A and B, U.S. Pat. No. 6,872,397 (Mar. 29, 2005); Rhett M. Schiffman, Methods for Treating Uterine Disorders, U.S. Patent Publication No. 2004/0175399 (Sep. 9, 2004); Richard L. Barron, Methods for Treating Ulcers and Gastroesophageal Reflux Disease, U.S. Patent Publication No. 2004/0086531 (May. 7, 2004); and Kei Roger Aoki, et al., Method for Treating Dystonia with Botulinum Toxin C to G, U.S. Pat. No. 6,319,505 (Nov. 20, 2001); eye disorders, see e.g., Eric R. First, Methods and Compositions for Treating Eye Disorders, U.S. Patent Publication No. 2004/0234532 (Nov. 25, 2004); Kei Roger Aoki et al., Botulinum Toxin Treatment for Blepharospasm, U.S. Patent Publication No. 2004/0151740 (Aug. 5, 2004); and Kei Roger Aoki et al., Botulinum Toxin Treatment for Strabismus, U.S. Patent Publication No. 2004/0126396 (Jul. 1, 2004); pain, see e.g., Kei Roger Aoki et al., Pain Treatment by Peripheral Administration of a Neurotoxin, U.S. Pat. No. 6,869,610 (Mar. 22, 2005); Stephen Donovan, Clostridial Toxin Derivatives and Methods to Treat Pain, U.S. Pat. No. 6,641,820 (Nov. 4, 2003); Kei Roger Aoki, et al., Method for Treating Pain by Peripheral Administration of a Neurotoxin, U.S. Pat. No. 6,464,986 (Oct. 15, 2002); Kei Roger Aoki and Minglei Cui, Methods for Treating Pain, U.S. Pat. No. 6,113,915 (Sep. 5, 2000); Martin A. Voet, Methods for Treating Fibromyalgia, U.S. Pat. No. 6,623,742 (Sep. 23, 2003); Martin A. Voet, Botulinum Toxin Therapy for Fibromyalgia, U.S. Patent Publication No. 2004/0062776 (Apr. 1, 2004); and Kei Roger Aoki et al., Botulinum Toxin Therapy for Lower Back Pain, U.S. Patent Publication No. 2004/0037852 (Feb. 26, 2004); muscle injuries, see e.g., Gregory F. Brooks, Methods for Treating Muscle Injuries, U.S. Pat. No. 6,423,319 (Jul. 23, 2002); headache, see e.g., Martin Voet, Methods for Treating Sinus Headache, U.S. Pat. No. 6,838,434 (Jan. 4, 2005); Kei Roger Aoki et al., Methods for Treating Tension Headache, U.S. Pat. No. 6,776,992 (Aug. 17, 2004); and Kei Roger Aoki et al., Method for Treating Headache, U.S. Pat. No. 6,458,365 (Oct. 1, 2002); William J. Binder, Method for Reduction of Migraine Headache Pain, U.S. Pat. No. 5,714,469 (Feb. 3, 1998); cardiovascular diseases, see e.g., Gregory F. Brooks and Stephen Donovan, Methods for Treating Cardiovascular Diseases with Botulinum Toxin, U.S. Pat. No. 6,767,544 (Jul. 27, 2004); neurological disorders, see e.g., Stephen Donovan, Parkinson's Disease Treatment, U.S. Pat. No. 6,620,415 (Sep. 16, 2003); and Stephen Donovan, Method for Treating Parkinson's Disease with a Botulinum Toxin, U.S. Pat. No. 6,306,403 (Oct. 23, 2001); neuropsychiatric disorders, see e.g., Stephen Donovan, Botulinum Toxin Therapy for Neuropsychiatric Disorders, U.S. Patent Publication No. 2004/0180061 (Sep. 16, 2004); and Steven Donovan, Therapeutic Treatments for Neuropsychiatric Disorders, U.S. Patent Publication No. 2003/0211121 (Nov. 13, 2003); endocrine disorders, see e.g., Stephen Donovan, Method for Treating Endocrine Disorders, U.S. Pat. No. 6,827,931 (Dec. 7, 2004); Stephen Donovan, Method for Treating Thyroid Disorders with a Botulinum Toxin, U.S. Pat. No. 6,740,321 (May. 25, 2004); Kei Roger Aoki et al., Method for Treating a Cholinergic Influenced Sweat Gland, U.S. Pat. No. 6,683,049 (Jan. 27, 2004); Stephen Donovan, Neurotoxin Therapy for Diabetes, U.S. Pat. No. 6,416,765 (Jul. 9, 2002); Stephen Donovan, Methods for Treating Diabetes, U.S. Pat. No. 6,337,075 (Jan. 8, 2002); Stephen Donovan, Method for Treating a Pancreatic Disorder with a Neurotoxin, U.S. Pat. No. 6,261,572 (Jul. 17, 2001); Stephen Donovan, Methods for Treating Pancreatic Disorders, U.S. Pat. No. 6,143,306 (Nov. 7, 2000); cancers, see e.g., Stephen Donovan, Methods for Treating Bone Tumors, U.S. Pat. No. 6,565,870 (May 20, 2003); Stephen Donovan, Method for Treating Cancer with a Neurotoxin to Improve Patient Function, U.S. Pat. No. 6,368,605 (Apr. 9, 2002); Stephen Donovan, Method for Treating Cancer with a Neurotoxin, U.S. Pat. No. 6,139,845 (Oct. 31, 2000); and Mitchell F. Brin and Stephen Donovan, Methods for Treating Diverse Cancers, U.S. Patent Publication No. 2005/0031648 (Feb. 10, 2005); otic disorders, see e.g., Stephen Donovan, Neurotoxin Therapy for Inner Ear Disorders, U.S. Pat. No. 6,358,926 (Mar. 19, 2002); and Stephen Donovan, Method for Treating Otic Disorders, U.S. Pat. No. 6,265,379 (Jul. 24, 2001); autonomic disorders, see, e.g., Pankai J. Pasricha and Anthony N. Kalloo, Method for Treating Gastrointestinal Muscle Disorders and Other Smooth Muscle Dysfunction, U.S. Pat. No. 5,437,291 (Aug. 1, 1995); as well as other disorders, see e.g., William J. Binder, Method for Treatment of Skin Lesions Associated with Cutaneous Cell-proliferative Disorders, U.S. Pat. No. 5,670,484 (Sep. 23, 1997); Eric R. First, Application of Botulinum Toxin to the Management of Neurogenic Inflammatory Disorders, U.S. Pat. No. 6,063,768 (May 16, 2000); Marvin Schwartz and Brian J. Freund, Method to Reduce Hair Loss and Stimulate Hair Growth, U.S. Pat. No. 6,299,893 (Oct. 9, 2001); Jean D. A. Carruthers and Alastair Carruthers, Cosmetic Use of Botulinum Toxin for Treatment of Downturned Mouth, U.S. Pat. No. 6,358,917 (Mar. 19, 2002); Stephen Donovan, Use of a Clostridial Toxin to Reduce Appetite, U.S. Patent Publication No. 2004/40253274 (Dec. 16, 2004); and Howard I. Katz and Andrew M. Blumenfeld, Botulinum Toxin Dental Therapies and Procedures, U.S. Patent Publication No. 2004/0115139 (Jun. 17, 2004); Kei Roger Aoki, et al., Treatment of Neuromuscular Disorders and Conditions with Different Botulinum, U.S. Patent Publication No. 2002/0010138 (Jan. 24, 2002); and Kei Roger Aoki, et al., Use of Botulinum Toxins for Treating Various Disorders and Conditions and Associated Pain, U.S. Patent Publication No. 2004/0013692 (Jan. 22, 2004). In addition, the expected use of Clostridial toxins, such as, e.g., BoNTs and TeNT, in therapeutic and cosmetic treatments of humans and other mammals is anticipated to expand to an ever widening range of diseases and ailments that can benefit from the properties of these toxins.
Table 1, below, provides the amino acid sequences of the various currently known clostridial toxins. These toxins possess approximately 35% amino acid identity with each other and share the same functional domain organization and overall structural architecture. As stated above, clostridial toxins are each translated as a single chain polypeptide of approximately 150 kDa that is subsequently cleaved by proteolytic scission within a disulfide loop by a naturally-occurring protease, such as, e.g., an endogenous clostridial toxin protease or a naturally-occurring protease produced in the environment. This posttranslational processing yields a di-chain molecule comprising an approximately 50 kDa light chain (LC) and an approximately 100 kDa heavy chain (HC) held together by a single disulfide bond and noncovalent interactions.
Each mature di-chain molecule comprises three functionally distinct domains: 1) an enzymatic domain located in the LC that includes a metalloprotease region containing a zinc-dependent endopeptidase activity which specifically targets core components of the neurotransmitter release apparatus; 2) a translocation domain contained within the amino-terminal half of the H chain (HN) that facilitates release of at least the L chain of the toxin from an endosome into the cytoplasm of the target cell; and 3) a binding domain found within the carboxyl-terminal half of the H chain (HC) that determines the binding activity and binding specificity of the toxin to the receptor complex located at the surface of the target cell.
It will be understood that there exist certain strains of each of these toxins that may vary in their amino acid sequence in non-critical regions without a substantial change in the identity or activity of the indicated toxin.







TABLE 1







Clostridial Toxin Reference Sequences and Regions


(identified from amino to carboxy direction;


amino acid number to amino acid number)












SEQ ID





Toxin
NO:
LC
HN
HC





BoNT/A
1
M1-K448
A449-K871
N872-L1296


BoNT/B
2
M1-K441
A442-S858
E859-E1291


BoNT/C1
3
M1-K449
T450-N866
N867-E1291


BoNT/D
4
M1-R445
D446-N862
S863-E1276


BoNT/E
5
M1-R422
K423-K845
R846-K1252


BoNT/F
6
M1-K439
A440-K864
K865-E1274


BoNT/G
7
M1-K446
S447-S863
N864-E1297


TeNT
8
M1-A457
S458-V879
I880-D1315









Using this information it is possible to construct an expressible open nucleic acid reading frame for insertion into an expression vector and subsequent expression within a chosen host cell. Indeed, International Patent publication WO 01/14570 discloses methods of making single-chain, cleavable recombinant modified or unmodified Clostridial neurotoxin derivatives and chimeric and hybrid forms thereof using such methods. Additional publications disclosing methods of making expressible recombinant neurotoxins and derivatives thereof include U.S. Pat. Nos. 5,989,545; 6,203,794; 6,395,513; U.S. Publication Numbers U.S. 2003/0166238; U.S. 2002/169942; U.S. 2004/176299; U.S. 2004/126397; U.S. 2005/035730; U.S. 2005/068494; U.S. 2006/011966; International Patent Applications WO95/32738; WO 99/55359; WO96/33273; WO98/07864; WO99/17806; WO98/07864; WO02/44199; WO02/40506. All these publications are incorporated by reference herein in their entirety.
The use of recombinant DNA techniques permits the construction of modified clostridial neurotoxins having different functional properties from the naturally occurring toxin subtypes and strains thereof. For example, altering the naturally occurring amino acid sequence of the native neurotoxin light chain and/or adding a different therapeutic moiety permits the construction of transport proteins designed to carry a therapeutic agent within a neuron. See U.S. Pat. No. 6,203,794 (incorporated by reference herein). Altering the targeting (binding) domain permits the toxin to be transported within pancreatic cells, such as acinar cells, thereby preventing secretion of activated digestive enzymes by such cells, See U.S. Pat. No. 6,843,998 (hereby incorporated by reference herein), or sensory afferent neurons, thereby preventing neurotransmitter release and thus providing relief from pain; see U.S. Pat. No. 6,395,513 (hereby incorporated by reference herein.)
In addition, the creation of chimeric neurotoxin derivatives comprising, for example, the binding domain and the translocation domain (or modified versions thereof) of one neurotoxin subtype for example, BoNT/A, and the light chain region of another neurotoxin subtype, for example, BoNT/E. It will be seen that given the general structural homology between the neurotoxin subtypes, any combination of the three basic clostridial neurotoxin domains, may be made in a single amino acid chain (or cleaved di-chain molecule). Thus, a binding region from any of neurotoxin subtypes A, B, C1, D, E, F, G, or TeTX may be independently combined with a translocation domain from neurotoxin subtypes A, B, C1, D, E, F, G, or TeTX, and further independently combined with a endopeptidase domain from any of neurotoxin subtypes A, B, C1, D, E, F, G or TeTX.
When discussing the three general neurotoxin domains of each clostridial neurotoxin subtype (binding, translocation and endopeptidase) it will be understood that clostridial neurotoxin research is a well-developed field, and the correlation of the amino acid sequences comprising each of these domains with their functions is well known. Additionally, the nucleotide and amino acid sequences of each of these domains are known and have been disclosed in this specification. It will therefore be understood that an endopeptidase domain, translocation domain, or the binding domain(s) of a given neurotoxin subtype may respectively and independently have 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95% identity to any of the amino acid sequence regions indicated in Table 1 and disclosed in SEQ ID NO: 1-8. Further, it will be understood that the amino acid sequences set forth in Table 1 and SEQ ID NO: 1-8 provide a full disclosure of any and all nucleotide sequences encoding these amino acid sequences and indicated regions thereof. A nucleotide sequence encoding an endopeptidase domain, translocation domain, or binding domain of a given neurotoxin subtype may respectively have 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95% identity to any of such reference nucleotide sequence regions listed in Table 1 and/or SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A depicts an embodiment of the multivalent Clostridial neurotoxin derivatives of the invention in which the domains “Binding 1”, Translocation” and “Endopeptidase” correspond to the domains of the naturally occurring Clostridial neurotoxin. In this embodiment, a second binding domain, “Binding 2”, is inserted at the amino terminus of the single chain toxin prior to cleavage. The protease sensitive region separating the Translocation and Endopeptidase domains may be the naturally occurring loop region, or may contain an engineered protease sensitive amino acid sequence. Two cysteine residues create a disulfide linkage between the Translocation and Endopeptidase domains.
FIG. 1B shows the multivalent Clostridial neurotoxin derivative of FIG. 1A after protease cleavage. The Binding 1 and Binding 2 sites are part of the Translocation and Endopeptidase domains, respectively.
FIG. 2A depicts an embodiment of the multivalent Clostridial neurotoxin derivatives of the invention in which the domains “Binding 1”, Translocation” and “Endopeptidase” correspond to the domains of the naturally occurring Clostridial neurotoxin. In this embodiment, two additional binding sites, (“Binding 2” and “Binding 3”) are inserted at the amino terminus of the single chain toxin prior to cleavage. The protease sensitive regions separating the Translocation and Endopeptidase domain and the Binding 2 and Binding 3 domains may independently comprise naturally occurring loop region amino acid sequences, or may contain an engineered protease sensitive amino acid sequence. Two cysteine residues create a disulfide linkage between the Translocation and Endopeptidase domains and the Binding 1 and Binding 2 domains.
FIG. 2B shows the multivalent Clostridial neurotoxin derivative of FIG. 2A after protease cleavage. The Binding 1 and Binding 2 sites are part of the Translocation and Endopeptidase domains, respectively, while the Binding 3 site comprises a separate disulfide-linked chain.



DETAILED DESCRIPTION
The binding and/or selectivity and thus, the therapeutic utility of a naturally occurring or modified clostridial neurotoxin may be enhanced or altered by inclusion of at least 2 target cell-binding domains. All such enhanced or altered Clostridial neurotoxins are referred to herein as “multivalent Clostridial neurotoxin derivatives” or MCNDs. By adding, for example, multiple binding domains the binding constant (Kd) (which equals [toxin] [target cell]/[toxin:target cell complex]) of the MCND will be increased over that of a monovalent toxin or toxin derivative.
Each binding domain of a MCND may comprise any ligand capable of binding under physiological conditions to a cell surface receptor capable of selectively binding to the ligand; however each such binding domain is capable of binding to, or facilitating the entry of the MCND to, a single target cell type. In addition, at least one of said binding domains will bind selectively to the target cell and at least one of the binding domains will bind to a cell surface receptor capable of mediating the internalization of the MCND within the target cell.
By “cell surface receptor” is meant both a traditional receptor capable of binding a ligand and thereby causing a ligand-selective response (at e.g., the cell surface, the cytoplasm, or both), and a cell surface feature such as caveolae, membrane “patches”, or lipid rafts, which appear to serve at least in part to gather together and locally concentrate at the cell surface specific receptors and bound ligand types for later processing such as endocytosis, often mediated by a cell surface receptor. By “selective binding” between ligand and such a cell surface receptor is meant that the dissociation constant (Kd) of ligand for target is at least four fold, or preferably at least 10 fold, or preferably at least 50 fold, or preferably at least 100 fold, or preferably at least 1000, or preferably at least 100,000 fold the value of Kd of the ligand for other cell surface receptors.
In one embodiment therefore, the present invention is drawn to a composition comprising a multivalent Clostridial neurotoxin derivative having an endopeptidase domain which selectively cleaves a vSNARE or tSNARE protein; a translocation domain which assists in the migration of the endopeptidase through a vesicular membrane, and at least two binding domains, each independently capable of binding a cell surface receptor of the desired target cell. The binding domains may be identical or different; however both domains bind target cell surface receptors on the surface of the target cell.
In one broad embodiment the target cell may be a natural target of a Clostridial neurotoxin. For example, the target cell may be a neuron located at the neuromuscular junction. In such a case, one or more of the binding domains of the multivalent Clostridial neurotoxin derivative of the present invention may be the naturally-occurring HC domain of the Clostridium botulinum subtypes or of TeTX. However, one or more of the binding domains may comprise a ligand that does not comprise a naturally occurring neurotoxin HC domain. For example, one or more binding domain may comprise a domain of a hemagglutinin (HA) or non-toxin non-hemagglutinin (NTNH) protein constituent of a clostridial toxin complex.
Any of a variety of sequence alignment methods can be used to determine percent identity of a multivalent Clostridial neurotoxin derivative, or of an endopeptidase, translocation, or binding domain thereof, relative to a given naturally-occurring Clostridial neurotoxin, or of the respective domains it comprises. Such methods may include, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art and from the teaching herein.
Approaches well known to one skilled in the art on how to modify a Clostridial neurotoxin in order to increase its binding activity for an endogenous Clostridial toxin receptor system present on a naturally-occurring Clostridial toxin target cell. As described above, one approach involves identifying amino acids using computational protein design algorithms; changing specifically-identified amino acids using, without limitation, site-directed mutagenesis, oligonucleotide-directed mutagenesis and site-specific mutagenesis; and testing the relevant activity (selective endopeptidase activity, translocation activity, or binding activity) of multivalent Clostridial neurotoxin derivatives using relevant assays, e.g., heterogeneous assays, homogeneous assays and non-separating homogeneous assays. It is further envisioned that the binding activity of a multivalent Clostridial neurotoxin derivative having altered or enhanced binding domains, as disclosed in the present specification, can be determined by, for example, affinity chromatography using immobilized receptors and interfacial optical assays. In another approach described above, a binding activity of a multivalent Clostridial neurotoxin derivative for a cell surface receptor present on a target cell can be achieved using directed-evolution methods. Similar methods can be used to alter the endopeptidase domain's substrate selectivity.
A multivalent Clostridial neurotoxin derivative includes, without limitation, naturally occurring Clostridial toxin domains, such as, e.g., naturally occurring Clostridial endopeptidase, translocation and at least 2 naturally occurring Clostridial toxin binding domains, each such domain from the same or different Clostridial toxin types, isoforms; non-naturally occurring Clostridial domain variants, such as, e.g., conservative Clostridial domain variants, non-conservative Clostridial domain variants, Clostridial domain chimerics, active Clostridial domain fragments thereof, or any combination thereof.
As used herein, the term “Clostridial domain variant”, whether naturally-occurring or non-naturally-occurring, means a Clostridial endopeptidase, translocation, or binding domains that has at least one amino acid change from the corresponding region of the disclosed reference sequences (see Table 1) and can be described in terms of percent identity to the corresponding region of that reference sequence. Unless expressly indicated, all Clostridial domain variants disclosed in the present specification are capable of executing the endopeptidase, translocation, and/or target cell binding step of the intoxication process.
It is recognized by those of skill in the art that within each Clostridial bacterium there can be naturally occurring Clostridial toxin variants having Clostridial domain variants that differ somewhat in their amino acid sequence, and also in the nucleic acids encoding these proteins, from the sequences provided in Table 1 and SEQ ID NO: 1-8 above.
As used herein, the term “naturally occurring Clostridial domain variant” means any Clostridial domain (endopeptidase, translocation, and/or binding domains) produced by a naturally-occurring process, including, without limitation, Clostridial domain isoforms produced from alternatively-spliced transcripts, Clostridial domain isoforms produced by spontaneous mutation and Clostridial domain subtypes. A naturally occurring Clostridial domain variant can function in substantially the same manner as the reference Clostridial domain on which the naturally occurring Clostridial domain variant is based, and can be substituted for the reference Clostridial domain in any aspect of the present invention. A naturally occurring Clostridial domain variant may substitute one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, ten or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, 50 or more amino acids or 100 or more amino acids from the reference Clostridial domain on which the naturally occurring Clostridial domain variant is based. A naturally occurring Clostridial domain variant can also substitute at least 10 contiguous amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino acids, or at least 25 contiguous amino acids from the reference Clostridial domain on which the naturally occurring Clostridial domain variant is based, that possess at least 50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino acid identity to the reference Clostridial domain on which the naturally occurring Clostridial domain variant is based.
Non-toxin Associated Proteins (NAPs)
HA and NTNH regions, including, without limitation, regions of non-toxin associated proteins of Clostridial toxins, such as, e.g., non-toxic non-hemagglutinin (NTNH), hemagglutinin-17 (HA-17), hemagglutinin-33 (HA-33) and hemagglutinin-70 (HA-70) may provide an enhanced targeting domain that increases binding activity for an endogenous Clostridial toxin receptor system present on a naturally-occurring Clostridial toxin target cell. As indicated previously, Clostridial bacteria produce a toxin complex that comprises the approximately 150-kDa di-chain Clostridial toxin along with other proteins. These other proteins are collectively called nontoxin associated proteins (NAPs). Identified NAPs include proteins possessing hemaglutination activity, such, e.g., a hemagglutinin of approximately 17-kDa (HA-17), a hemagglutinin of approximately 33-kDa (HA-33) and a hemagglutinin of approximately 70-kDa (HA-70); as well as a non-toxic non-hemagglutinin (NTNH), a protein of approximately 130-kDa, see, e.g., Eric A. Johnson and Marite Bradshaw, Clostridial botulinum and its Neurotoxins: A Metabolic and Cellular Perspective, 39 TOXICON 1703-1722 (2001); and Stephanie Raffestin et al., Organization and Regulation of the Neurotoxin Genes in Clostridium botulinum and Clostridium tetani, 10 ANAEROBE 93-100 (2004). The toxin complex is believed to be important for the intoxication process at least in part because it appears to provide protection to the toxin molecule from adverse environmental conditions and resistance to protease digestion. Importantly, certain domains of the HA and NTNH proteins appear to coordinate with toxin binding and bind to locations on the cell surface (and may bind to the natural Clostridial neurotoxin cell surface receptor at sites other than or additional to the toxin binding site), thus facilitating binding, internalization, and activation of the toxin.
Recent crystallography experiments have revealed that HA-17, HA-33 and NTNH from various Clostridial bacteria contain a region comprising β-trefoil domains very similar to the single β-trefoil domain present in the binding domain of Clostridial toxins, see, e.g., Kaoru Inoue et al., Structural Analysis by X-Ray Crystallography and Calorimetry of a Haemagglutinin Component (HA1) of the Progenitor Toxin from Clostridium botulinum, 149 MICROBIOL. 3361-3370 (2003); and Joseph W. Arndt et al., The Structure of the Neurotoxin-Associated Protein HA33/A from Clostridial botulinum Suggests a Reoccurring β-trefoil Fold in the Progenitor Toxin Complex, 346 J. MOL. BIOL. 1083-1093 2005). For example, HA-33 from Clostridum botulinum serotype A has two β-trefoil domains, each of which consists of three potential carbohydrate binding moieties or β-trefoil folds, designated 1α (amino acids 10-55 of SEQ ID NO: 9), 1β (amino acids 56-102 of SEQ ID NO: 9) and 1γ (amino acids 103-144 of SEQ ID NO: 9) for the first β-trefoil domain and 2α (amino acids 151-197 of SEQ ID NO: 9), 2β (amino acids 198-245 of SEQ ID NO: 9) and 2γ (amino acids 246-293 of SEQ ID NO: 9) for the second β-trefoil domain. Mutations in conserved amino acids of the carbohydrate binding moiety result in a loss of carbohydrate binding, see, e.g., Kaoru Inoue et al., Structural Analysis by X-Ray Crystallography and Calorimetry of a Haemagglutinin Component (HA1) of the Progenitor Toxin from Clostridium botulinum, 149 MICROBIOL. 3361-3370 (2003).
These β-trefoil domains are also found in the HA-33 proteins produced by Clostridium botulinum serotype B, serotype C1 and serotype D and sequence alignments revealed that amino acids essential for overall carbohydrate binding are conserved. The amino acids predicted to be important or essential for carbohydrate binding are as follows Asp 263, Tyr 265, Gln 268, Gln 276, Phe 278 and Gln 286 of HA-33/A1 of SEQ ID NO: 9, HA-33/A2 of SEQ ID NO: 10, HA-33/A3 of SEQ ID NO: 11, HA-33/A5 of SEQ ID NO: 12 and HA-33/B2 of SEQ ID NO: 15; Asp 264, Tyr 266, Gln 269, Gln 277, Phe 279 and Gln 287 of HA-33/A4 of SEQ ID NO: 12; Asp 262, Tyr 264, Gln 267, Gln 275, Phe 277 and Gln 285 of HA-33/B1 of SEQ ID NO: 14; Asp 255, Val 257, Gly 260, Gln 268, Typ 270 and Gln 278 of HA-33/C1 of SEQ ID NO: 16; and Asp 256, Tyr 258, Gln 261, Ise 269, Asp 271 and Gln 279 of HA-33/C2 of SEQ ID NO: 17 and HA-33/D of SEQ ID NO: 18. Immunoaffinity column chromatography and pull-down assays have shown that HA-33 an bind synaptotagmin II, a putative Clostridial toxin receptor, see, e.g., Yu Zhou et al., Haemagglutinin-33 of Type Q Botulinum Neurotoxin Complex Binds with Synaptotagmin II, 272 FEBS LETT. 2717-2726 (2005). The amino acid sequences comprising the β-trefoil domains found in various Clostridial HA-33 proteins are shown in Tables 2 and 3.







TABLE 2







β-trefoil Domains of Clostridial HA-33 Proteins









Amino Acid Sequence Region of Carbohydrate



Binding Moieties
















1β4/β5

1β8/β9




SEQ

β-hairpin

β-hairpin


Protein
ID NO:
1α-fold
turn
1β-fold
turn
1γ-fold
















HA-33/
9
10-54
55-59
60-100
101-104
105-144


A1


HA-33/
10
10-54
55-59
60-100
101-104
105-144


A2


HA-33/
11
10-54
55-59
60-100
101-104
105-144


A3


HA-33/
12
10-56
57-61
62-102
103-106
107-146


A4


HA-33/
13
10-54
55-59
60-100
101-104
105-144


A5


HA-33/
14
10-54
55-59
60-100
101-104
105-144


B1


HA-33/
15
10-56
57-61
62-102
103-106
107-146


B2


HA-33/
16
10-54
55-59
60-98 
 99-102
103-141


C1-1


HA-33/
17
10-54
55-59
60-98 
 99-102
103-141


C1-2


HA-33/
18
10-54
55-59
60-98 
 99-102
103-141


D1
















TABLE 3







β-trefoil Domains of Clostridial HA-33 Proteins









Amino Acid Sequence Region of Carbohydrate



Binding Moieties
















2β4/β5

2β8/β9



Protein
SEQ ID NO:
2α-fold
β-hairpin turn
2β-fold
β-hairpin turn
2γ-fold
















HA-33/A1
9
151-195
196-199
200-242
243-248
249-293


HA-33/A2
10
151-195
196-199
200-242
243-248
249-293


HA-33/A3
11
151-195
196-199
200-242
243-248
249-293


HA-33/A4
12
153-197
198-201
202-243
244-249
250-294


HA-33/A5
13
151-195
196-199
200-242
243-248
249-279


HA-33/B1
14
151-195
196-199
200-241
242-247
248-292


HA-33/B2
15
153-197
198-201
200-242
243-248
249-291


HA-33/C1-1
16
148-190
191-194
195-234
235-240
241-285


HA-33/C1-2
17
148-190
191-194
195-235
236-241
242-286


HA-33/D1
18
148-190
191-194
195-235
236-241
242-286









Further analysis of the β-trefoil domain sequence of HA-33 also identified β-trefoil domains in HA-17 and NTNH, see, e.g., Joseph W. Arndt et al., The Structure of the Neurotoxin-Associated Protein HA33/A from Clostridial botulinum Suggests a Reoccurring β-trefoil Fold in the Progenitor Toxin Complex, 346 J. MOL. BIOL. 1083-1093 (2005). The HA-17 comprises a single β-trefoil domain containing three carbohydrate binding moieties or β-trefoil folds. The carbohydrate binding moieties of HA-17 exhibits the greatest sequence similarity with the 2γ carbohydrate-binding moiety of HA-33. These β-trefoil domains are also found in the HA-17 proteins produced by Clostridium botulinum serotype B, serotype C1 and serotype D and sequence alignments revealed that amino acids essential for overall carbohydrate binding are conserved. The amino acids predicted to be important or essential for carbohydrate binding are as follows Tyr 110, Typ 112, Tyr 115, Pro 130, Phe 132 and Asn 138 of HA-17/A of SEQ ID NO: 19, HA-17/B of SEQ ID NO: 20, HA-17/C1 of SEQ ID NO: 21 and HA-17/D of SEQ ID NO: 22. The amino acid sequences comprising the β-trefoil domains found in various Clostridial HA-17 proteins are shown in Table 4.







TABLE 4







β-trefoil Domains of Clostridial HA-17 Proteins









Amino Acid Sequence Region of Carbohydrate



Binding Moieties
















β4/β5

β8/β9




SEQ

β-hairpin

β-hairpin


Protein
ID NO:
α-fold
turn
β-fold
turn
γ-fold





HA-17/
19
9-50
51-54
55-91
92-94
95-146


A


HA-17/
20
9-50
51-54
55-91
92-94
95-146


B


HA-17/
21
9-50
51-54
55-91
92-94
95-146


C1


HA-17/
22
9-50
51-54
55-91
92-94
95-146


D









NTNH proteins from various Clostridial bacteria show significant sequence similarity to the β-trefoil domains present in the cell-binding domain of BoNT/A and TeNT. The high degree of structural similarity is interesting in light of the low primary sequence similarity between NTNH and the Clostridial toxins themselves. Furthermore, since NTNH of the various serotypes have greater sequence similarity than the Clostridial toxins, it is likely that the NTNH produced by other Clostridial strains will also have β-trefoil domains exhibiting high structural similarity with the binding domains of Clostridial toxins. The β-trefoil domains of various Clostridial NTNHs are as follows: amino acids 1050-1193 of NTNH/A1 of SEQ ID NO: 23; amino acids 1050-1198 of NTNH/A2 of SEQ ID NO: 24; amino acids 1050-1193 of NTNH/A3 of SEQ ID NO: 25; amino acids 1049-1197 of NTNH/B of SEQ ID NO: 26; amino acids 1049-1196 of NTNH/C1 of SEQ ID NO: 27; amino acids 1049-1196 of NTNH/D of SEQ ID NO: 28; amino acids 1014-1162 of NTNH/E of SEQ ID NO: 29; amino acids 1016-1159 of NTNH/F1 of SEQ ID NO: 30; amino acids 1017-1165 of NTNH/F2 of SEQ ID NO: 31; and amino acids 1050-1198 of NTNH/G of SEQ ID NO: 32. The amino acid sequences comprising the β-trefoil domains found in various Clostridial NTNH proteins are shown in Table 5.
The β-trefoil domains present in the Clostridial toxin, HA-33, HA-17 and NTNH collectively form nearly half the mass of a Clostridial toxin complex and underlies the apparent importance of carbohydrate binding in the cell-binding step of the intoxication process. This observation is further enhanced by the fact that a Clostridial toxin alone is not as effective in intoxicating a cell as the entire toxin complex. One potential explanation for this enhanced binding activity is the presence, both in type and in quantity, of the β-trefoil domains present in HA-33, HA-17 and NTNH. Therefore, the high prediction of structural similarity of the β-trefoil domains present in HA-33, HA-17 and NTNH relative to the β-trefoil domain found in Clostridial toxins provides a potential source of binding domains useful for developing multivalent Clostridial neurotoxin derivatives with enhanced binding activity. As a non-limiting example, such a multivalent Clostridial neurotoxin derivative may comprise one or more carbohydrate binding moiety or a β-trefoil fold from HA-33, HA-17 or NTNH. Such a NAP binding motif may be substituted for, or added to, the naturally occurring binding domain (or a variant thereof) present in a Clostridial toxin. As yet another non-limiting example, 2 or more carbohydrate binding moieties or β-trefoil folds from HA-33, HA-17 or NTNH can be substituted in a multivalent Clostridial neurotoxin derivative for the naturally occurring carbohydrate binding moiety (or a variant thereof) present in a Clostridial toxin. As still another non-limiting example, two or more carbohydrate binding moieties or β-trefoil folds from HA-33, HA-17 or NTNH can be added in addition to the naturally occurring carbohydrate binding moiety present in a Clostridial toxin. As another non-limiting example, multiple carbohydrate binding moieties or a β-trefoil folds from a Clostridial toxin binding domain can be added in addition to the naturally occurring carbohydrate binding moiety present in a Clostridial toxin.







TABLE 5







β-trefoil Domains of Clostridial NTNH Proteins









Amino Acid Sequence Region of Carbohydrate



Binding Moieties
















β4/β5

β8/β9



Protein
SEQ ID NO:
α-fold
β-hairpin turn
β-fold
β-hairpin turn
γ-fold





NTNH/A1
23
1050-1097
1098-1110
1111-1138
1139-1148
1149-1194


NTNH/A2
24
1050-1097
1098-1110
1111-1139
1140-1148
1149-1199


NTNH/A3
25
1050-1097
1098-1110
1111-1138
1139-1148
1149-1194


NTNH/B
26
1049-1096
1097-1109
1110-1138
1139-1147
1148-1198


NTNH/C1
27
1049-1096
1097-1109
1110-1138
1139-1147
1148-1197


NTNH/D
28
1049-1096
1097-1109
1110-1138
1139-1147
1148-1197


NTNH/E
29
1014-1061
1062-1074
1075-1103
1104-1113
1114-1163


NTNH/F1
30
1016-1063
1064-1076
1077-1104
1105-1114
1115-1160


NTNH/F2
31
1017-1064
1065-1077
1078-1106
1107-1116
1117-1166


NTNH/G
32
1050-1097
1098-1110
1111-1139
1140-1149
1150-1199









As used herein, the term “Non-toxin Associated Protein” is synonymous with “NAP” and means a Clostridial NAP with selective binding activity, such as, e.g., a binding affinity or a binding specificity, for an endogenous Clostridial toxin receptor system. It is envisioned that both naturally occurring NAPs as well as NAPs with enhanced binding activity can be used to practice aspects of the present invention. As used herein, the term “NAP with enhanced binding activity” means a Clostridial NAP with enhanced binding activity for an endogenous Clostridial toxin receptor system, such as, e.g., a binding affinity or a binding specificity, to a statistically significantly degree relative to an unmodified naturally occurring Clostridial toxin binding domain from a Clostridial toxin. By definition, a NAP with enhanced binding activity has at least one amino acid change from the corresponding region of the disclosed reference sequences (see Tables 2-5) and can be described in percent identity to the corresponding region of that reference sequence.
Any of a variety of sequence alignment methods can be used to determine percent identity of a modified Clostridial NAP relative to a naturally-occurring Clostridial NAP, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art and from the teaching herein.
Approaches well known to one skilled in the art on how to modify a Clostridial NAP in order to increase its binding activity for an endogenous Clostridial toxin receptor system present on a naturally-occurring Clostridial toxin target cell. As described above, one approach involves identifying amino acids using computational protein design algorithims; changing specifically-identified amino acids using, without limitation, site-directed mutagenesis, oligonucleotide-directed mutagenesis and site-specific mutagenesis; and testing the binding activity of multivalent Clostridial neurotoxin derivatives comprising a modified Clostridial NAP with enhanced binding activity using, e.g., heterogeneous assays, homogeneous assays and non-separating homogeneous assays. It is further envisioned that the binding activity of a multivalent Clostridial neurotoxin derivative disclosed in the present specification can be determined by affinity chromatography using immobilized receptors and interfacial optical assays. In another approach described above, a binding activity of a modified Clostridial NAP for a naturally occurring Clostridial toxin receptor system present on a naturally occurring Clostridial toxin target cell can be achieved using directed-evolution methods.
A Clostridial NAP includes, without limitation, naturally Occurring Clostridial NAP variants, such as, e.g., Clostridial NAP isoforms and Clostridial NAP subtypes; non-naturally occurring Clostridial NAP variants, such as, e.g., conservative Clostridial NAP variants, non-conservative Clostridial NAP variants, Clostridial NAP chimerics, active Clostridial NAP fragments thereof, or any combination thereof.
As used herein, the term “Clostridial NAP variant,” whether naturally-occurring or non-naturally-occurring, means a Clostridial NAP that has at least one amino acid change from the corresponding region of the disclosed reference sequences (see Tables 2-5) and can be described in terms of percent identity to the corresponding region of that reference sequence. Unless expressly indicated, all Clostridial NAP variants disclosed in the present specification are capable of executing the cell-binding step of the intoxication process.
It is recognized by those of skill in the art that within each Clostridial bacterium there can be naturally occurring Clostridial NAP variants that differ somewhat in their amino acid sequence, and also in the nucleic acids encoding these proteins. For example, there are presently known five Clostridial botulinum serotype A HA-33 variants, HA-33/A1, HA-33/A2, HA-33/A3, HA-33/A4 and HA-33/A5 (Tables 2 and 3), with specific HA-33 variants showing various degrees of amino acid divergence when compared to another HA-33 variant. As another example, there are presently three Clostridial botulinum serotype A NTNH-33 variants, NTNH/A1, NTNH/A2 and NTNH/A3 (Table 5), with specific NTNH variant showing various degrees of amino acid divergence when compared to another NTNH variant. As used herein, the term “naturally occurring Clostridial NAP variant” means any Clostridial NAP produced by a naturally-occurring process, including, without limitation, Clostridial NAP isoforms produced from alternatively-spliced transcripts, Clostridial NAP isoforms produced by spontaneous mutation and Clostridial NAP subtypes. A naturally occurring Clostridial NAP variant can function in substantially the same manner as the reference Clostridial NAP on which the naturally occurring Clostridial NAP variant is based, and can be substituted for the reference Clostridial NAP in any aspect of the present invention. A naturally occurring Clostridial NAP variant may substitute one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, ten or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, 50 or more amino acids or 100 or more amino acids from the reference Clostridial NAP on which the naturally occurring Clostridial NAP variant is based. A naturally occurring Clostridial NAP variant can also substitute at least 10 contiguous amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino acids, or at least 25 contiguous amino acids from the reference Clostridial NAP on which the naturally occurring Clostridial NAP variant is based, that possess at least 50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino acid identity to the reference Clostridial NAP on which the naturally occurring Clostridial NAP variant is based.
Non-limiting examples of naturally occurring Clostridial NAP variant are each of the following Clostridial NAP isoform such as, e.g., a Clostridial botulinum serotype A HA-33 isoform, a Clostridial botulinum serotype B HA-33 isoform, a Clostridial botulinum serotype C1 HA-33 isoform, a Clostridial botulinum serotype D HA-33 isoform, a Clostridial botulinum serotype A HA-17 isoform, a Clostridial botulinum serotype B HA-17 isoform, a Clostridial botulinum serotype C1 HA-17 isoform, a Clostridial botulinum serotype D HA-17 isoform, a Clostridial botulinum serotype A NTNH isoform, a Clostridial botulinum serotype B NTNH isoform, a Clostridial botulinum serotype C1 NTNH isoform, a Clostridial botulinum serotype D NTNH isoform, a Clostridial botulinum serotype E NTNH isoform, a Clostridial botulinum serotype F NTNH isoform and a Clostridial botulinum serotype G NTNH isoform. A Clostridial NAP isoform can function in substantially the same manner as the reference Clostridial NAP on which the Clostridial NAP isoform is based, and can be substituted for the reference Clostridial NAP in any aspect of the present invention.
Cell Surface Receptors
Each of the two or more binding domains in a multivalent Clostridial neurotoxin derivative may be, for example, a targeting moiety or moiety that enhances stability, solubility and/or cell penetration (see below). At least one such binding domain is selective for a cell surface receptor of a target cell. In addition, at least one cell surface receptor to which a multivalent Clostridial neurotoxin derivative binds is capable of being internalized by the target cell after ligand binding. Preferably, at least one cell surface receptors to which a multivalent Clostridial neurotoxin derivative selectively binds is capable of being internalized by the target cell after ligand binding.
Each binding domain of a multivalent Clostridial neurotoxin derivative may independently be a ligand. Non-limiting examples of ligands or binding partners include native, recombinant and modified cell surface receptor binding domains (e.g. HC from serotypes A-G and TeTx) or one or more of the BoNT complex proteins (e.g. HA, NTNH). Such ligands have been described above. In addition, peptide or protein ligands may target specific cellular receptors that do not normally bind BONT or TeTx.
Examples of the latter category of ligands include, but are not restricted to, the transforming growth factor beta (TGF-β) family of receptors and ligands (such as, without limitation, bone morphogenetic protein (BMP)); growth factor receptor ligands (e.g. (BDNF bone-derived neurotropic factor) at TrkB, insulin-like growth factor at IGF1,2); and visceral (gut) peptides (e.g. gastrin, VIP, bombesin, etc). As described herein, “ligand” is not limited to entities selectively binding to receptors, but may include any moiety that enhances binding stable binding of the multivalent Clostridial neurotoxin derivative at the cell surface.
Other ligands that may be employed in accordance with the present invention are disclosed in U.S. Pat. No. 6,843,998, No. 6,641,820, No. 6,831,059, and No. 5,989,545. The disclosures of which are incorporated in their entirety herein by reference.
In some embodiments, the ligand is CPN/A (centrally presented nociceptin-A), CPNvar/A, opioid, galanin and/or somatostatin. In some embodiments, a CPN/A, CPNvar/A, opioid, galanin and/or somatostatin is/are attached to the toxin at the nicking loop.
In some embodiments, the ligand is capable of selective binding to presynaptic receptors such as those selectively binding members of the growth factor families; for example, the fibroblast growth factor (FGF) family, the epidermal growth factor (EGF) family, the insulin-like growth factor (IGF) family; neurotrophins such as nerve growth factor (NGF), bone derived neurotrophic factor (BDNF), NT-3, NT-4/5); members of the transforming growth factor TGF-beta family, such as Gbb, BMP; Neurexin (neuroligin); Frizzled (WNTs); Neuroleukin/AMF/MF receptor; Axon guidance signaling (netrin, semaphoring, ephrin type interactions; neuroregulatory cytokines (e.g., CNTF, GPA, LIF, interleukins, oncostatin M, CT-1, CLC.
In some embodiments, the ligand is capable of selective binding to cell surface receptors pf the post-synaptic membrane; for example: sequestration proteins at neuromuscular junction such as Ng-CAM (L1), NCAM, and N-cadherin.
In some embodiments, a ligand of the multivalent Clostridial neurotoxin derivative of the present invention is Substance P or a derivative or modified polypeptide based thereupon. Substance P is an 11 amino acid peptide that has a number of different natural and synthetic precursor forms. A representative listing of certain substance P-related compositions is provided by Table 6 below; all of these amino acid sequences are based on those set forth (left to right correlating from amino to carboxyl terminus) as SEQ ID NO: 33 (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met), SEQ ID NO: 34 (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly) and SEQ ID NO: 35 (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly-Lys) and SEQ ID NO: 36 (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly-Lys-Arg). The following are representative and not exhaustive examples of Substance P variants. Naturally occurring Substance P possesses the amino acid sequence SEQ ID NO: 33, with an amidated carboxy terminus. Longer variants are also known.









Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-
(SEQ ID NO: 34)



Gly-Leu-Met-Gly


 


Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-
(SEQ ID NO: 35)


Gly-Leu-Met-Gly-Lys


 


Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-
(SEQ ID NO: 36)


Gly-Leu-Met-Gly-Lys-Arg






Each of these molecules may also possess modifications such as methyl or ethyl esters at the carboxy terminus. In addition, fragment of Substance P may function to binding receptors at cell surface; such fragments may include peptide fragments comprising the N-terminal 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 33.
In certain embodiments of the present invention, the multivalent Clostridial neurotoxin derivative of the present invention may contain two or more identical or different binding domains comprising substance P or a substance P derivative or analog; such a multivalent Clostridial neurotoxin derivative may be administered to neural cells in order to treat pain. In other embodiments the Substance P derived binding domain of the multivalent Clostridial neurotoxin derivative may be combined with different additional binding domains (such as binding domains that directly or indirectly bind to clatherin, or a caveolin-1 binding region of a glycosyl phosphatylinositol (GPI)-linked membrane protein.) engineered to provide cooperative and therefore stronger binding than would otherwise be provided using a monovalent neurotoxin.
One major route of receptor-mediated endocytosis is by means of endocytotic coated pits. These specialized sites on the plasma membrane of eukaryotic cells are responsible for internalization of many cell surface receptors, usually after ligand binding. The coated pits serve in part to concentrate the receptors for internalization within far fewer endosomes than would be the case if each receptor were individually internalized.
Coated pits consist largely of two major proteins: clatherin and the adaptor protein (adaptin) Adaptor Protein-2 (AP-2). Assembly of the coated pit is thought to be initiated by the binding of AP-2 molecules to a receptor or ligand-docking site in the membrane. The receptor:AP-2 complex then recruits the structural protein clatherin.
Sorting signals present in many membrane proteins, such as cell surface receptors, provide for recognition and binding of AP-2 to these proteins as the initial step of coated pit formation. One common sorting signal is the amino acid sequence YXXΦ (wherein Φ is an amino acid with a bulky hydrophobic side chain, such as leucine, phenylalanine, methionine and valine), which provides a signal for rapid internalization. This signal is recognized by and binds to the μ2 subunit of AP-2, and mediates rapid internalization by mammalian cells. Preferably, the signal is present ion such a way as to be displayed on the cytosolic side of the membrane. See e.g., Bonifacino et al., J. Cell Biol. 145: 923 (May 31, 1999).
At least one binding domain of a multivalent Clostridial neurotoxin derivative of the present invention may comprise at least one sorting signal for an adaptin, preferably AP-2. Preferably, the binding site comprising this sorting signal is present in addition to another distinct binding domain which will directly bind a cell surface receptor.
In certain embodiments, a multivalent Clostridial neurotoxin derivative of the present invention comprising two or more pancreatic acinar cell targeting moieties may be administered to treat pancreatitis; the use of CCK forms and derivatives capable of selective binding to CCK receptors of pancreatic acinar cells is disclosed in U.S. Pat. No. 6,843,998. In one preferred embodiment of this aspect of the present invention, two or more binding moieties of the present invention are independently a CCK or CCK derivative.
In another embodiment of the invention for treatment of, e.g., acute or chronic pancreatitis, at least one binding moiety of the multivalent Clostridial neurotoxin derivative is a CCK or CCK derivative, and at least one additional binding region comprises a region of a glycosyl phosphatylinositol (GPI)-linked membrane protein that directly or indirectly binds caveolin-1 binding. Alternatively, the additional binding region may comprise a ligand capable of binding a GPI-linked membrane protein, particularly a membrane associated cell surface receptor, as the CCK receptor, like the TNF and other known receptors, is known to be concentrated and recruited for internalization by caveolae.
Caveolae are discrete membrane domains present in many cell types; they are not thought to be present in neural cells. Caveolae are comprised largely of detergent-insoluble lipids and lipid associated proteins, including a major protein, caveolin-1. Caveolae are also known to recruit and concentrate GPI-linked membrane proteins, a family of membrane proteins that includes cell surface receptors. Further, caveolae appear to participate in a non clatherin-associated form of endocytosis, and experiments have been reported in which gene transfer vectors have been directed to GPI-linked receptor proteins to facilitate gene transfer.
Thus, for example, the urokinase plasminogen activator receptor (uPAR) is a GPI-linked membrane protein that appears to facilitate endocytosis upon binding of its cognate ligand urokinase plasminigen activator (uPA). See e.g., Drapkin et al., J. CLIN. INVEST. 105: 589-596 (Mar. 1, 2000), incorporated by reference as part of this specification. uPA is a 55 KDa protein secreted by alveolar epithelial cells that cleaves plasminogen to plasmin and is known to degrade the extracellular matrix in alveoli. Targeting such cells, which also display the uPAR, with the multivalent Clostridial neurotoxin derivative of the present invention comprising, for example, two or more binding domains comprising a uPAR-binding region derived from, for example, uPA or an anti-uPAR antibody may be of therapeutic use is the treatment of respiratory and lung diseases such as e.g., cystic fibrosis, since the ligand-bound receptor is subject to endocytosis in a ligand dependent manner. Thus for example, a patient may be administered a mist or dispersion comprising such multivalent Clostridial neurotoxin derivative as a delivery mechanism.
In certain embodiments the target cell may comprise a cell comprising a membrane protein that displays an antibody variable region at the cell surface. In such a case the multivalent Clostridial neurotoxin derivative comprises two or more binding domains, with at least one binding domain comprising an antigen able to selectively bind the antibody variable region. In a preferred embodiment, the multivalent Clostridial neurotoxin derivative may comprise more than one binding region comprising an antigen able to selectively bind to said antibody variable region.
Alternatively, the multivalent Clostridial neurotoxin derivative of the present invention (comprising more than one binding domain), may have at least one binding domain comprising an antigen-binding portion of an antibody H or L chain, wherein the antigen comprises a cell surface marker for a target cell. For example, and without limitation, the multivalent Clostridial neurotoxin derivative of the present invention may comprise at least one binding domain comprising an antibody variable region that selectively binds to an eosinophil cell surface marker (for example, the cell surface markers CD 44 and CD 69), thereby aiding in alleviating the symptoms of allergy. The nomenclature “CD” is derived from the use of monoclonal antibodies directed towards a given cell type, and stands for “Cluster of Differentiation”.
In another example, the multivalent Clostridial toxin derivative may be used to inhibit or decrease the rate of infection of T helper cells by the human immunodeficiency virus (HIV). T helper cells are commonly identified by virtue of their display of the cell surface marker CD4. The HIV virus uses the CD4 marker to gain entry into and thereby infect T helper cells, and appears to employ viral fusion proteins sharing remarkable similarity to the SNARE system to invade cells through a membrane fusion mechanism. See e.g., Duman & Forte, AM. J. PHYSIOL. CELL PHYSIOL. 285: C237-C249 (2003). A multivalent Clostridial neurotoxin derivative of the present invention comprising at least one, and preferably two or more, anti-CD4 antibody domain or similar CD4 “addressable” binding domain would permit the neurotoxin protease domain to deny the virus use of the SNARE proteins for entry and/or exit of the cell. Moreover, alteration of the toxin proteolytic domain to selectively recognize and cleave one or more of the HIV viral fusion proteins, using S such techniques as directed evolution, site directed mutagenesis or other well known molecular biology methods, could provide a method of reducing the extent of, or eliminating, HIV infection.
In some embodiments, one or more binding domain of the multivalent Clostridial neurotoxin derivative may comprise a translocation domain. As used herein, a “translocator” of the present invention comprises a protein, peptide (or a peptidomimetic) that facilitates the transport of the neurotoxin across a cell membrane. In some embodiments, the translocator may function independently of cell surface transporters or specific receptors, and therefore non-specifically. In this embodiment the multivalent Clostridial toxin derivative will also comprise at least one target cell selective ligand.
In other embodiments, a translocator may comprise a ligand selective for a cell surface transporter, feature, or receptor; in such an embodiment the multivalent Clostridial toxin derivative may comprise two or more identical binding domains, or may comprise at least one target cell selective domain and at least one target cell non-selective binding domain, so long as the number of binding domains totals at least two.
Without wishing to limit the invention to any theory or mechanism of operation, it is believed that the translocator comprises a protein translocation domain (PTD). Further, it is believed that the PTD is primarily responsible for the translocation of the neurotoxin across a cell membrane. PTDs are amino acid sequence domains that have been shown to cross biological membranes efficiently and independently of transporters or specific receptors. See e.g., Morris M. C. et al., NATURE BIOTECHNOLOGY, 19:1173-1176 (December 2001), the disclosure of which is incorporated in its entirety by reference herein. A number of PTDs have now been characterized and are well-known in the art. For example, the SV-40 virus large T-related PTD is:











CGGGPKKKRKVGG
(SEQ ID NO: 37)







an HIV TAT-related PTD is:












CGGRKKRRQRRRAP
(SEQ ID NO: 38)







an adenovirus related PTD is:












CGGFSTSLRARKA
(SEQ ID NO: 39)







a synthetic integrin binding domain-related PTD is:












CKKKKKKGGRGDMFG
(SEQ ID NO: 40)







In some embodiments, the multivalent Clostridial toxin derivative comprises at least two binding domains, wherein at least one of these domains comprises a ligand selected from the cell surface receptor-binding domains of ciliary neurotrophic factor (CNTF), caveolin, interleukin 1-β (Il-1β), thioredoxin, fibroblast growth factor-1 (FGF-1), fibroblast growth factor-2 (FGF-2), Human integrins such the β1 and β3 subunits, lactoferrin, Engrailed, Hoxa-5, Hoxb-4, or Hoxc-8. Engrailed-1, Engrailed-2, Hoxa-5, Hoxb-4 and Hoxc-8 are homeoproteins. Homeoproteins are helix-turn-helix proteins that contain a 60 amino acid DNA-binding domain, the homeodomain (HD) or homeobox. The PTD is believed to lie within the HD. When Engrailed-1 and Engrailed-2 are expressed in COS7 cells, they are secreted by the host cell and then reinternalized by other cells. Similar observations have been made for Hoxa-5, Hoxc-8 and Hoxb-4.
In some embodiments, the translocator is a PTD domain of a herpes simplex virus type 1 (HSV-1) VP22 protein, which is a transcription factor that concentrates in the nucleus and binds chromatin. It has been shown that VP22 traffics across the membrane via non-classical endocytosis and can enter cells regardless of GAP junctions and physical contacts. If VP22 is expressed in a small population of cells in culture, it will reach 100% of the cells in that culture. Fusion proteins with VP22 and for example p53, GFP, thymidine kinase, β-galactosidase and others have been generated. It has been demonstrated that the fusion proteins are taken up by several kinds of cells including terminally differentiated cells suggesting that mitosis is not a requirement for efficient entry. In addition, VP22-GFP fusion showed that the protein can shuttle in and out of the cells and enter cells that were not exposed to VP22.
The HIV-1 trans-activator gene product (TAT) was one of the earliest cell-permeating proteins described. A receptor-mediated event appears not to be required for TAT to pass into a neighboring cell; thus for selective cell targeting, a PTD is preferably used in conjunction with a more target cell-selective binding domain in the multivalent Clostridial toxin derivative of the present invention. HIV-1, as well as other lentiviruses, encodes a potent TAT protein. Embury et al., DIABETES 50:1706-1713, 2001 discuss the use the TAT PTD to transduce anti-apoptotic proteins into pancreatic islet cells. The PTD of TAT is a small peptide comprising amino acids 47-57 of the TAT protein; the PTD sequence comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO 41) or at least the nonapeptide comprising TAT amino acids 49-57. Fusion proteins comprising this peptide can transit the plasma membrane in vitro and in vivo. E.g., Schwartz, J. J. et al., Peptide-mediated cellular delivery, Curr. Opin. Mol. Therapeutics 2000, 2:162-7. The disclosures of these references are incorporated in their entirety by reference herein.
Furthermore, PTD-transported proteins and peptides, such as fusion peptides, have been shown to generally retain their biological properties and functions once inside the cells. Further, the TAT-PTD is able to carry a variety of cargo molecules including nucleic acids (DNA and RNA), and therapeutic drugs. The capability of this sequence to promote internalization of the peptide (and any cargo peptide) appears to be related to the strongly positive charge pf the PTD sequence, and not to be inhibited at 4° C. or in the presence of endocytosis inhibitors. Thus, the PTD appears not to be dependent upon active transport or recentor mediated endocytosis for this activity.
The PTD sequence is able to mediate the transduction of its cargo in a concentration dependent and receptor-, transporter-, and endocytosis-independent manner to 100% of the target cells in a given tissue or culture. Of special interest are the studies demonstrating that the PTD of TAT is able to deliver proteins in vivo to several different tissues when injected into animals.
In some embodiments, the multivalent Clostridial toxin derivative comprises at least one binding domain comprising a at least two PTDs, or at least three PTDs. Additional, non-limiting examples of PTDs are shown below.








Kaposi fibroblast growth factor membrane-



translocating sequence (kFGF MTS)









AAVALLPAVLLALLAP
(SEQ ID NO. 42)



 








Nuclear Localization Signal (NLS)










TPPKKKRKVEDP
(SEQ ID NO. 43)



 








Transportan










GWTLNSAGYLLGKINLKALAALAKKIL
(SEQ ID NO. 44)



 








Herpes Simplex Virus Type 1 Protein 22 (VP22)










DAATATRGRSAASRPTERPRAPARSASRPRRPVE
(SEQ ID NO. 45)







In some embodiments, PTDs contained in one or more binding site(s) of this invention are peptides derived from a homeoprotein. Homeoproteins are helix turn helix proteins that contain a 60 amino acid DNA-binding domain, the homeodomain (HD). PTDs may be derived from the HD. In some embodiments, PTDs are derived from the family of Drosophila homeoproteins. Drosophila homeoproteins are involved in developmental processes and are able to translocate across neuronal membranes. The third helix of the homeodomain of just 16 amino acids, known as penetratin, is able to translocate molecules into live cells. When added to several cell types in culture, 100% of the cells were able to uptake the peptide. Internalization occurs both at 37° C. and 4° C., and thus appears to be neither receptor-mediated nor energy-dependent. Several penetrating peptides, including those of the penetratin family, have been developed and used to internalize cargo molecules into the cytoplasm and nucleus of several cell types in vivo and in vitro. The results suggest that the entry of penetratin peptides is mediated by tryptophan, phenylalanine, and/or glutamine residues. In addition, the retroinverse and all D-amino acid forms are also translocated efficiently using the penetratins, and non-α-helical structures are also internalized. See Prochiantz, A., Messenger proteins:homeoproteins, TAT and others, Curr Opin Cell Biol 2000, 12:400-6; and Schwartz, J J et al., Peptide-mediated cellular delivery, Curr Opin Mol Therapeutics 2000, 2:162-7. The disclosures of these references are incorporated in their entirety by reference herein.
In some embodiments, the translocator comprises the PDT of at least one penetratin peptide. Non-limiting examples of penetratin peptides are shown below:











RQIKIWFQNRRMKWKK
(SEQ ID NO: 46)




 



KKWKMRRNQFWIKIQR
(SEQ ID NO: 47)



 



RQIKIWFQNRRMKWKK
(SEQ ID NO: 48)



 



RQIKIWFPNRRMKWKK
(SEQ ID NO: 49)



 



RQPKIWFPNRRMPWKK
(SEQ ID NO: 50)



 



RQIKIWFQNMRRKWKK
(SEQ ID NO: 51)



 



RQIRIWFQNRRMRWRR
(SEQ ID NO: 52)



 



RRWRRWWRRWWRRWRR
(SEQ ID NO: 53)



 



RQIKIFFQNRRMKFKK
(SEQ ID NO: 54)



 



TERQIKIWFQNRRMK
(SEQ ID NO: 55)



 



KIWFQNRRMKWKKEN
(SEQ ID NO: 56)






In some embodiments, a translocator comprises a synthetic protein transduction domain. Other synthetic PTD sequences that may be employed in accordance with the present invention may be found in WO 99/29721 and Ho, A. et al., Synthetic PTDs: enhanced transduction potential in vitro and in vivo, CANCER RES 2001, 61, 474-7. In addition, it has been demonstrated that a 9-mer of L-Arginine is 20 fold more efficient than the TAT-PTD at cellular uptake, and when a D-arginine oligomer was used the rate enhancement was >100 fold. See Wender, P A et al., The Design, Synthesis, And Evaluation Of Molecules That Enable Or Enhance Cellular Uptake: Peptoid Molecular Transporters, PROC. NATL. ACAD. SCI. USA 2000, 97:13003-13008. These data suggested that the guanidinium groups of TAT-PTD play a greater role than charge or backbone structure in mediating cellular uptake. Thus, a peptoid analogue containing a six-methylene spacer between the guanidine head group and backbone was synthesized. This peptoid exhibited enhanced cellular uptake when compared to TAT-PTD and even to the D-Arg peptide.
In addition to the proteins and peptides discussed above, other peptide-mediated delivery systems have been described and are well-known to those of skill in the art. These PTD-containing proteins and peptides include: MPG, SCWKn, (LARL)n, HA2, RGD, AlkCWK18, DiCWK18, DipaLytic, K16RGD, Plae and Kplae. See Schwartz, J J et al., Peptide-mediated cellular delivery, Curr Opin Mol Therapeutics 2000, 2:162-7. The disclosure of which is incorporated in its entirety by reference herein. In some embodiments, these proteins and peptides may be used as translocators in accordance with the present invention.
EXAMPLES
Example 1
Construction of a Multivalent Clostridial Neurotoxin Derivative Comprising Two Clostridial Toxin Binding Domains
This example illustrates how to make a multivalent Clostridial neurotoxin derivative comprising two modified Clostridial toxin binding domains with enhanced binding activity using site-directed mutagenesis.
A polynucleotide molecule encoding BoNT/A (SEQ ID NO: 1) and further comprising a nucleotide sequence comprising a repeat of the binding domain of BoNT/A at the amino terminus (Table 1) is synthesized using standard procedures (BlueHeron® Biotechnology, Bothell, Wash.). The basic strategy is set forth in FIG. 1A and FIG. 1B. Oligonucleotides of 20 to 50 bases in length are synthesized using standard phosphoramidite synthesis. These oligonucleotides are hybridized into double stranded duplexes that are ligated together to assemble the full-length polynucleotide molecule. This polynucleotide molecule is cloned using standard molecular biology methods into a pUCBHB1 vector at the SmaI site to generate pUCBHB1/BoNT/MCND(AA)1. The synthesized polynucleotide molecule is verified by sequencing using Big Dye TerminatorTM Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).
If desired, an expression-optimized polynucleotide molecule encoding BoNT/A (SEQ ID NO: 1) can be synthesized in order to improve expression in an Escherichia coli strain. The polynucleotide molecule encoding the BoNT/A can be modified to 1) contain synonymous codons typically present in native polynucleotide molecules of an Escherichia coli strain; 2) contain a G+C content that more closely matches the average G+C content of native polynucleotide molecules found in an Escherichia coli strain; 3) reduce polymononucleotide regions found within the polynucleotide molecule; and/or 4) eliminate internal regulatory or structural sites found within the polynucleotide molecule, see, e.g., Lance E. Steward et al. Optimizing Expression of Active Botulinum Toxin Type E, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005); Lance E. Steward et al. Optimizing Expression of Active Botulinum Toxin Type A, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005). Once sequence optimization is complete, oligonucleotides of 20 to 50 bases in length are synthesized using standard phosphoramidite synthesis. These oligonucleotides are hybridized into double stranded duplexes that are ligated together to assemble the full-length polynucleotide molecule. This polynucleotide molecule is cloned using standard molecular biology methods into a pUCBHB1 vector at the SmaI site to generate pUCBHB1/BoNT/MCND1/(AA)1:ECopt. The synthesized polynucleotide molecule is verified by sequencing using Big Dye TerminatorTM Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.). Is so desired, optimization to a different organism, such as, e.g., a yeast strain, an insect cell-line or a mammalian cell line, can be done, see, e.g., Steward, supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005); and Steward, supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005).
A similar cloning strategy is used to make pUCBHB1 cloning constructs comprising a polynucleotide molecule encoding BoNT/B of SEQ ID NO: 2; a polynucleotide molecule encoding BoNT/C1 of SEQ ID NO: 3; a polynucleotide molecule encoding BoNT/D of SEQ ID NO: 4; a polynucleotide molecule encoding BoNT/E of SEQ ID NO: 5; a polynucleotide molecule encoding BoNT/F of SEQ ID NO: 6; a polynucleotide molecule encoding BoNT/G of SEQ ID NO: 7; and a polynucleotide molecule encoding TeNT of SEQ ID NO: 8; each with an additional binding domain of any of the BONRT subtypes added to the amino terminus of the BoNT toxin sequence. In addition, one skilled in the art can modify Clostridial toxins, such as, e.g., to include an exogenous protease cleavage site within the di-chain loop region, or flexible spacer regions.
To construct pET29/MCND/A(AA)1, a pUCBHB1/MCND(AA)1 construct is digested with restriction endonucleases that 1) excise the insert comprising the open reading frame of SEQ ID NO: 1 (with the added nucleotide sequence encoding the N terminal Binding 1 site) encoding the MCND(AA)1; and 2) enable this insert to be operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert is subcloned using a T4 DNA ligase procedure into a pET29 vector that is digested with appropriate restriction endonucleases to yield pET29/MCND/A(AA)1. The ligation mixture is transformed into chemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of Kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs are identified as kanamycin resistant colonies. Candidate constructs are isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping to determine the presence and orientation of the insert. This cloning strategy yields a pET29 expression construct comprising the polynucleotide molecule encoding the BoNT/A of SEQ ID NO: 1 operably-linked to an additional N-terminal binding site comprising the native binding region of BoNT/A.
A similar cloning strategy is used to make pET29 expression constructs comprising polynucleotide molecule encoding MCNDs comprising BoNT/B of SEQ ID NO: 2; a polynucleotide molecule encoding BoNT/C1 of SEQ ID NO: 3; a polynucleotide molecule encoding BoNT/D of SEQ ID NO: 4; a polynucleotide molecule encoding BoNT/E of SEQ ID NO: 5; a polynucleotide molecule encoding BoNT/F of SEQ ID NO: 6; a polynucleotide molecule encoding BoNT/G of SEQ ID NO: 7; and a polynucleotide molecule encoding TeNT of SEQ ID NO: 8, each with an additional N-terminal binding site of the same native BONT or TeNT subtype to which the binding site is linked. It will be understood that added binding sites of Clostridial neurotoxin subtypes other than that of the subtype to which the additional binding domain is linked may easily be substituted at the N-terminus of the MCND.
To construct a MCND comprising one or more modified binding domain with enhanced binding activity, specific amino acids influencing binding activity will be changed. For example, it is already known that amino acids Trp 1101, Gly 1102, Leu 1105, Tyr 1111, Tyr 1112, Gly 1158, Ile 1163, Asp 1179, Glu 1203, Phe 1252, Ser 1264, Trp 1266, Tyr 1267, Gln 1270, Gly 1279 and Trp 1282 of SEQ ID NO: 1 (within the binding site) are important for function. To determine which amino acid substitutions could enhance the binding activity of a BoNT/A binding domain, computational protein design algorithms will generate novel binding domains with optimized properties. The crystal structure of a BoNT/A binding domain will be used as the starting template for computational calculations. Potential amino acid candidates will be identified using a combined output from Protein Design Automation® (PDA®) and Sequence Prediction AlgorithmTM (SPATM) calculations. For PDA calculations, the conformations of amino acids at variable positions will be represented as a set of backbone-independent side chain rotamers derived from the rotamer library. The energies of all possible combinations of the considered amino acids at the chosen variable positions will be calculated using a force field containing terms describing van der Waals, salvation, electrostatic, and hydrogen bond interactions. The optimal (ground state) sequence will be determined using a Dead End Elimination (DEE) algorithm, and a Monte Carlo (MC) algorithm will be used to evaluate the energies of similar sequences around the predicted ground state. SPA calculations utilize a genetic algorithm to screen for low energy sequences, with energies being calculated during each round of “evolution” for those sequences being sampled. The conformations of amino acids will be represented as a set of side chain rotamers derived from a backbone-independent rotamer library using a flexible rotamer model. SPA calculations will generate sequences which will be subsequently clustered computationally into groups of similar sequences using a nearest neighbor single linkage hierarchical clustering algorithm. Critical contact amino acids will be fixed in both sequence and conformation and calculations will be carried out to evaluate single and combinatorial substitutions at variable amino acids. All amino acids in contact with these residues will be floated, that is the amino acid conformation but not the amino acid identity will be allowed to vary to allow for conformational adjustments. Final experimental substitutions will be chosen based on their predicted energies relative to the naturally occurring BoNT/A binding domain and their occupancy, that is the number times the substitution occurred in the set of 1000 MC or genetic algorithm sequences. Two sets of design calculations will be carried out using Rosetta to identify substitutions predicted to stabilize the BoNT/A binding domain. In the first round, only single amino acid substitutions will be modeled. In a second round, interface amino acids will be allowed to change to all 20 naturally occurring amino acids including the native amino acid type, but excluding cysteine, simultaneously. In each case, amino acid side chains contacting the substituted amino acid side chains will be repacked (allowing all rotamers of the native amino acid type). Sequences and conformations with low energies will be selected using a Monte-Carlo simulated annealing procedure. All resulting protein complex models will be rescored by computing a predicted binding energy. Final sequences are selected for the lowest binding energy.
To use this information to generate one or more modified Clostridial neurotoxin binding domain, candidate amino acids identified as described above will be changed using site-directed in vitro mutagenesis. A 50 μL reaction will be assembled using pET29/MCND/A(AA)1 as a template, sense and antisense oligonucleotides encoding the desired amino acid change identified above, and reagents included with the QuickChange® II XL Site-Directed Mutagenesis kit (Stratagene, La Jolla, Calif.). The polymerase chain reaction (PCR) mix will contain 5 μL of 10× Buffer, 1 μL of deoxyribonucleotides (dNTPs), 1 μL of PfuUltraTM High Fidelity DNA polymerase (2.5 units/μL), 125 ng of each primer, 100 ng of template DNA, and nuclease-free water to a final volume of 50 μL. The thermocycler conditions will be: one cycle of 95° C. for 60 seconds; 16 cycles of 95° C. for 30 seconds, 55° C. for 60 seconds, and 72° C. for 10 minutes; one cycle of 72° C. for 5 minutes; and 4° C. to hold. Following thermocycling, 1 μL of DpnI restriction enzyme (Stratagene, La Jolla, Calif.) will be added to the reaction and will be incubated for 1 hour at 37° C. to digest the template DNA. The reaction will be purified by QIAquick kit (QIAGEN, Inc., Valencia, Calif.) and will be analysis by agarose gel electrophoresis to determine that the reaction produced full-length plasmid. The mutagenesis products will be transformed into chemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 100 μg/mL of Ampicillin, and will be placed in a 37° C. incubator for overnight growth. Candidate mutagenesis constructs will be isolated as Ampicillin resistant colonies and will be analyzed using an alkaline lysis plasmid mini-preparation procedure to isolate the expression construct and restriction endonuclease digests to determine the presence of the insert. The incorporation of the point mutation will be determined by sequence analysis of candidate plasmid constructs.
To test the binding activity of multivalent Clostridial neurotoxin derivatives comprising binding domains derived from BoNT/A, the soluble portion of FGFR3 will be expressed recombinantly for use in surface plasmon resonance (SPR) binding assays, e.g., Biacore® (Biacore Inc., Piscataway, N.J.) The soluble portion of FGFR3 will be expressed as a fusion to streptavidin and the receptor will then be immobilized on an appropriate sensor chip. Utilizing a Biacore® instrument, changes in local refractive index as a result of receptor binding will be measured as a change in the SPR angle. The rates of change in the SPR angle will then be analyzed to determine association rate (Kon), dissociation rate (Koff) and the dissociation equilibrium constant (KD=Koff/Kon). Multivalent Clostridial neurotoxin derivatives comprising binding domains derived from BoNT/A exhibit either an increased association rate, a decreased dissociation rate, both an increased association rate and a decreased dissociation rate, or a decreased dissociation equilibrium constant relative to the measurements obtained from the naturally occurring BoNT/A from which the MCND is derived.
The same methods and rationale may be used to make and test the affinity of any multivalent Clostridial neurotoxin derivatives comprising binding domains derived from a Clostridial neurotoxin. Generally, but not exclusively, the MCND is tested relative to the Clostridial neurotoxin with which it shares the greatest homology, particularly in the binding domains.
Example 2
Construction of a Multivalent Clostridial Neurotoxin Derivative Comprising a Non-Toxin Associated Protein
A polynucleotide molecule encoding BoNT/A-Nterm33/A is synthesized using standard procedures (BlueHeron® Biotechnology, Bothell, Wash.), as described in Example 1. BoNT/A-Nterm33/A is a BoNT/A modified to replace amino acids in the second binding domain (the N-terminal binding region of the MCND described in Example 1) corresponding to amino acids 1111-1296 of SEQ ID NO: 1, a BoNT/A β-trefoil domain, with amino acids 151 to 293 of SEQ ID NO: 9, a HA-33 β-trefoil domain from a Clostridial botulinum serotype A strain. If desired, an expression optimized polynucleotide molecule encoding BoNT/A-Nterm33/A can be synthesized in order to improve expression in to a different organism, such as, e.g., an Escherichia coli strain, a yeast strain, an insect cell-line or a mammalian cell line, can be done, see, e.g., Steward, supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005); and Steward, supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005). The synthesized polynucleotide molecule is verified by sequencing using Big Dye TerminatorTM Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).
A similar cloning strategy is used to make pUCBHB1 cloning constructs for MCND BoNT/B-Nterm33/A, a MCND based on BoNT/B where amino acids 1098-1291 of SEQ ID NO: 2 are replaced with amino acids 151 to 293 of SEQ ID NO: 9; MCND BoNT/C1-Nterm33/A, a MCND based on BoNT/C1 where amino acids 1112-1291 of SEQ ID NO: 3 are replaced with amino acids 151 to 293 of SEQ ID NO: 9; MCND BoNT/D-Nterm33/A, a MCND based on BoNT/D where amino acids 1099-1276 of SEQ ID NO: 4 are replaced with amino acids 151 to 293 of SEQ ID NO: 9; MCND BoNT/E-Nterm33/A, a MCND based on BoNT/E where amino acids 1086-1252 of SEQ ID NO: 5 are replaced with amino acids 151 to 293 of SEQ ID NO: 9; MCND BoNT/F-Nterm33/A, a MCND based on BoNT/F where amino acids 1106-1274 of SEQ ID NO: 6 are replaced with amino acids 151 to 293 of SEQ ID NO: 9; MCND BoNT/G-Nterm33/A, a MCND based on BoNT/G where amino acids 1106-1297 of SEQ ID NO: 7 are replaced with amino acids 151 to 293 of SEQ ID NO: 9; and MCND TeNT-Nterm33/A, a modified TeNT where amino acids 1128-1315 of SEQ ID NO: 8 are replaced with amino acids 151 to 293 of SEQ ID NO: 9.
Similarly, the β-trefoil domain from a Clostridial toxin indicated above can be replaced with a non-toxin associated protein β-trefoil domain comprising amino acids 10-144 of SEQ ID NO: 9; amino acids 10-144 of SEQ ID NO: 10; amino acids 10-144 of SEQ ID NO: 11; amino acids 10-146 of SEQ ID NO: 12; amino acids 10-144 of SEQ ID NO: 13; amino acids 10-144 of SEQ ID NO: 14; amino acids 10-146 of SEQ ID NO: 15; amino acids 10-141 of SEQ ID NO: 16; amino acids 10-141 of SEQ ID NO: 17; amino acids 10-141 of SEQ ID NO: 18; 151-293 of SEQ ID NO: 10; 151-293 of SEQ ID NO: 11; 153-294 of SEQ ID NO: 12; 151-279 of SEQ ID NO: 13; 151-292 of SEQ ID NO: 14; 153-291 of SEQ ID NO: 15; 148-285 of SEQ ID NO: 16; 148-286 of SEQ ID NO: 17; 148-286 of SEQ ID NO: 18; 9-146 of SEQ ID NO: 19; 9-146 of SEQ ID NO: 20; 9-146 of SEQ ID NO: 21; 9-146 of SEQ ID NO: 22; 1050-1194 of SEQ ID NO: 23; 1050-1199 of SEQ ID NO: 24; 1050-1194 of SEQ ID NO: 25; 1049-1198 of SEQ ID NO: 26; 1049-1197 of SEQ ID NO: 27; 1049-1197 of SEQ ID NO: 28; 1014-1163 of SEQ ID NO: 29; 1016-1160 of SEQ ID NO: 30; 1017-1166 of SEQ ID NO: 31; and 1050-1199 of SEQ ID NO: 32.
To construct pET29/MCND/BoNT/A-Nterm33/A, a pUCBHB1/BoNT/A-33/A construct is digested with restriction endonucleases that 1) excise the insert comprising the open reading frame encoding MCND/BoNT/A-Nterm33/A; and 2) enable this insert to be operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert is subcloned using a T4 DNA ligase procedure into a pET29 vector that is digested with appropriate restriction endonucleases to yield pET29/MCND/BoNT/A-Nterm33/A. The ligation mixture is transformed into chemically competent E. coli DHSA cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs are identified as kanamycin resistant colonies. Candidate constructs are isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping to determine the presence and orientation of the insert. This cloning strategy yielded a pET29 expression construct comprising the polynucleotide molecule encoding MCND/BoNT/A-Nterm33/A operably-linked to a carboxyl terminal polyhistidine affinity binding peptide.
A similar cloning strategy is used to make pET29 expression constructs comprising a polynucleotide molecule encoding for BoNT/B-33/A, BoNT/C1-33/A, BoNT/D-33/A, BoNT/E-33/A, BoNT/F-33/A, BoNT/G-33/A, TeNT-33/A, as well as the modified Clostridial toxin indicated above comprising amino acids 10-144 of SEQ ID NO: 9; amino acids 10-144 of SEQ ID NO: 10; amino acids 10-144 of SEQ ID NO: 11; amino acids 10-146 of SEQ ID NO: 12; amino acids 10-144 of SEQ ID NO: 13; amino acids 10-144 of SEQ ID NO: 14; amino acids 10-146 of SEQ ID NO: 15; amino acids 10-141 of SEQ ID NO: 16; amino acids 10-141 of SEQ ID NO: 17; amino acids 10-141 of SEQ ID NO: 18; 151-293 of SEQ ID NO: 10; 151-293 of SEQ ID NO: 11; 153-294 of SEQ ID NO: 12; 151-279 of SEQ ID NO: 13; 151-292 of SEQ ID NO: 14; 153-291 of SEQ ID NO: 15; 148-285 of SEQ ID NO: 16; 148-286 of SEQ ID NO: 17; 148-286 of SEQ ID NO: 18; 9-146 of SEQ ID NO: 19; 9-146 of SEQ ID NO: 20; 9-146 of SEQ ID NO: 21; 9-146 of SEQ ID NO: 22; 1050-1194 of SEQ ID NO: 23; 1050-1199 of SEQ ID NO: 24; 1050-1194 of SEQ ID NO: 25; 1049-1198 of SEQ ID NO: 26; 1049-1197 of SEQ ID NO: 27; 1049-1197 of SEQ ID NO: 28; 1014-1163 of SEQ ID NO: 29; 1016-1160 of SEQ ID NO: 30; 1017-1166 of SEQ ID NO: 31; and 1050-1199 of SEQ ID NO: 32.
Example 3
Construction of a Multivalent Clostridial Neurotoxin Derivative Comprising Three CCK-A Binding Sites
The following example illustrates how to make a retargeted multivalent Clostridial neurotoxin derivative comprising three CCK-A binding sites.
A polynucleotide molecule is constructed as described in Example 1 above, encoding BoNT/A with the native binding site replaced with 10 repeats of the CCK 58 amino acid sequence (see U.S. Pat. No. 6,843,998, hereby incorporated by reference herein in its entirety) and with the same CCK 58 sequence repeated twice at the N-terminus of the single chain. The two N-terminal repeats are constructed to be separated by a short amino acid region comprising the loop region of BoNT/A, and two cysteine residues spanning the protease sensitive site. The MCND is termed MCND BoNT/A/C(CCK)N(CCKx2). The basic architecture of the amino acid construct is given in FIG. 2A.
If desired, an expression-optimized polynucleotide molecule encoding MCND BoNT/A/C(CCK)N(CCKx2) can be synthesized in order to improve expression in to a different organism, such as, e.g., an Escherichia coli strain, a yeast strain, an insect cell-line or a mammalian cell line, can be done, see, e.g., Steward, supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005); and Steward, supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005). The synthesized polynucleotide molecule is verified by sequencing using Big Dye TerminatorsTM Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).
A similar cloning strategy is used to make pUCBHB1 cloning constructs for C(CCK)N(CCKx2) MCND's in which the translocation and endopeptidase domains are independently selected from BoNT/B, C1, D, E, F, G, or TeNT.
To construct pET29/MCND BoNT/A/C(CCK)N(CCKx2), a pUCBHB1/MCND BoNT/A/C(CCK)N(CCKx2) construct is digested with restriction endonucleases that 1) excise the insert comprising the open reading frame encoding MCND BoNT/A/C(CCK)N(CCKx2); and 2) enable this insert to be operably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert is subcloned using a T4 DNA ligase procedure into a pET29 vector that is digested with appropriate restriction endonucleases to yield pET29/MCND BoNT/A/C(CCK)N(CCKx2). The ligation mixture is transformed into chemically competent E. coli DH5a cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of Kanamycin, and placed in a 37° C. incubator for overnight growth. Bacteria containing expression constructs are identified as Kanamycin resistant colonies. Candidate constructs are isolated using an alkaline lysis plasmid mini-preparation procedure and analyzed by restriction endonuclease digest mapping to determine the presence and orientation of the insert. This cloning strategy yielded a pET29 expression construct comprising the polynucleotide molecule encoding MCND BoNT/A/C(CCK)N(CCKx2), operably-linked to a carboxyl terminal polyhistidine affinity binding peptide.
A similar cloning strategy is used to make pET29 expression constructs comprising a polynucleotide molecule encoding any combination of translocation and endopeptidase domains derived from BoNT/A, B, C1, D, E, F, G or TeNT with CCK 58 domains similar positioned as in MCND BoNT/A/C(CCK)N(CCKx2).
Example 4
Purification and Quantification of Modified Clostridial Toxins
The following example illustrates methods useful for purification and quantification of any modified Clostridial toxins disclosed in the present specification.
For immobilized metal affinity chromatography (IMAC) protein purification, E. coli BL21 (DE3) cell pellets used to express a modified Clostridial toxin, as described in Example 7, are resuspended in Column Binding Buffer (25 mM N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH 7.8; 500 mM sodium chloride; 10 mM imidazole; 2× Protease Inhibitor Cocktail Set III (EMD Biosciences-Calbiochem, San Diego Calif.); 5 units/mL of Benzonase (EMD Biosciences-Novagen, Madison, Wis.); 0.1% (v/v) Triton-X® 100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol), and then are transferred to a cold Oakridge centrifuge tube. The cell suspension is sonicated on ice (10-12 pulses of 10 seconds at 40% amplitude with 60 seconds cooling intervals on a Branson Digital Sonifier) in order to lyse the cells and then is centrifuged (16,000 rpm at 4° C. for 20 minutes) to clarify the lysate. An immobilized metal affinity chromatography column is prepared using a 20 mL Econo-Pac column support (Bio-Rad Laboratories, Hercules, Calif.) packed with 2.5-5.0 mL of TALON® SuperFlow Co2+ affinity resin (BD Biosciences-Clontech, Palo Alto, Calif.), which is then equilibrated by rinsing with 5 column volumes of deionized, distilled water, followed by 5 column volumes of Column Binding Buffer. The clarified lysate is applied slowly to the equilibrated column by gravity flow (approximately 0.25-0.3 mL/minute). The column is then washed with 5 column volumes of Column Wash Buffer (N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH 7.8; 500 mM sodium chloride; 10 mM imidazole; 0.1% (v/v) Triton-X® 100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol). The modified Clostridial toxin is eluted with 20-30 mL of Column Elution Buffer (25 mM N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH 7.8; 500 mM sodium chloride; 500 mM imidazole; 0.1% (v/v) Triton-X® 100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol) and is collected in approximately twelve 1 mL fractions. The amount of modified Clostridial toxin contained in each elution fraction is determined by a Bradford dye assay. In this procedure, 20 μL aliquots of each 1.0 mL fraction is combined with 200 μL of Bio-Rad Protein Reagent (Bio-Rad Laboratories, Hercules, Calif.), diluted 1 to 4 with deionized, distilled water, and then the intensity of the calorimetric signal is measured using a spectrophotometer. The five fractions with the strongest signal are considered the elution peak and are combined together. Total protein yield is determined by estimating the total protein concentration of the pooled peak elution fractions using bovine gamma globulin as a standard (Bio-Rad Laboratories, Hercules, Calif.).
For purification of a modified Clostridial toxin using a FPLC desalting column, a HiPrepTM 26/10 size exclusion column (Amersham Biosciences, Piscataway, N.J.) is pre-equilibrated with 80 mL of 4° C. Column Buffer (50 mM sodium phosphate, pH 6.5). After the column is equilibrated, a modified Clostridial toxin sample is applied to the size exclusion column with an isocratic mobile phase of 4° C. Column Buffer and at a flow rate of 10 mL/minute using a BioLogic DuoFlow chromatography system (Bio-Rad Laboratories, Hercules, Calif.). The desalted modified Clostridial toxin sample is collected as a single fraction of approximately 7-12 mL.
For purification of a modified Clostridial toxin using a FPLC ion exchange column, a modified Clostridial toxin sample that has been desalted following elution from an IMAC column is applied to a 1 mL Q1TM anion exchange column (Bio-Rad Laboratories, Hercules, Calif.) using a BioLogic DuoFlow chromatography system (Bio-Rad Laboratories, Hercules, Calif.). The sample is applied to the column in 4° C. Column Buffer (50 mM sodium phosphate, pH 6.5) and is eluted by linear gradient with 4° C. Elution Buffer (50 mM sodium phosphate, 1 M sodium chloride, pH 6.5) as follows: step 1, 5.0 mL of 5% Elution Buffer at a flow rate of 1 mL/minute; step 2, 20.0 mL of 5-30% Elution Buffer at a flow rate of 1 mL/minute; step 3, 2.0 mL of 50% Elution Buffer at a flow rate of 1.0 mL/minute; step 4, 4.0 mL of 100% Elution Buffer at a flow rate of 1.0 mL/minute; and step 5, 5.0 mL of 0% Elution Buffer at a flow rate of 1.0 mL/minute. Elution of modified Clostridial toxin from the column is monitored at 280, 260, and 214 nm, and peaks absorbing above a minimum threshold (0.01 au) at 280 nm are collected. Most of the modified Clostridial toxin will elute at a sodium chloride concentration of approximately 100 to 200 mM. Average total yields of modified Clostridial toxin will be determined by a Bradford assay.
Expression of a modified Clostridial toxin is analyzed by polyacrylamide gel electrophoresis. Samples purified using the procedure described above are added to 2×LDS Sample Buffer (Invitrogen, Inc, Carlsbad, Calif.) and are separated by MOPS polyacrylamide gel electrophoresis using NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels (Invitrogen, Inc, Carlsbad, Calif.) under denaturing, reducing conditions. Gels are stained with SYPRO® Ruby (Bio-Rad Laboratories, Hercules, Calif.) and the separated polypeptides are imaged using a Fluor-S MAX MultiImager (Bio-Rad Laboratories, Hercules, Calif.) for quantification of modified Clostridial toxin expression levels. The size and amount of modified Clostridial toxin is determined by comparison to MagicMarkTM protein molecular weight standards (Invitrogen, Inc, Carlsbad, Calif.).
Expression of modified Clostridial toxin is also analyzed by Western blot analysis. Protein samples purified using the procedure described above are added to 2×LDS Sample Buffer (Invitrogen, Inc, Carlsbad, Calif.) and are separated by MOPS polyacrylamide gel electrophoresis using NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels (Invitrogen, Inc, Carlsbad, Calif.) under denaturing, reducing conditions. Separated polypeptides are transferred from the gel onto polyvinylidene fluoride (PVDF) membranes (Invitrogen, Inc, Carlsbad, Calif.) by Western blotting using a Trans-Blot® SD semi-dry electrophoretic transfer cell apparatus (Bio-Rad Laboratories, Hercules, Calif.). PVDF membranes are blocked by incubating at room temperature for 2 hours in a solution containing 25 mM Tris-Buffered Saline (25 mM 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid (Tris-HCl)(pH 7.4), 137 mM sodium chloride, 2.7 mM potassium chloride), 0.1% TWEEN-20®, polyoxyethylene (20) sorbitan monolaureate, 2% bovine serum albumin, 5% nonfat dry milk. Blocked membranes are incubated at 4° C. for overnight in Tris-Buffered Saline TWEEN-20® (25 mM Tris-Buffered Saline, 0.1% TWEEN-20®, polyoxyethylene (20) sorbitan monolaureate) containing appropriate primary antibodies as a probe. Primary antibody probed blots are washed three times for 15 minutes each time in Tris-Buffered Saline TWEEN-20®. Washed membranes are incubated at room temperature for 2 hours in Tris-Buffered Saline TWEEN-20® containing an appropriate immunoglobulin G antibody conjugated to horseradish peroxidase as a secondary antibody. Secondary antibody-probed blots are washed three times for 15 minutes each time in Tris-Buffered Saline TWEEN-20®. Signal detection of the labeled modified Clostridial toxin are visualized using the ECL PlusTM Western Blot Detection System (Amersham Biosciences, Piscataway, N.J.) and are imaged with a Typhoon 9410 Variable Mode Imager (Amersham Biosciences, Piscataway, N.J.) for quantification of modified Clostridial toxin expression levels.
Example 5
Treatment of Hyperhidrosis Using a Multivalent Clostridial Neurotoxin Derivative
A 32-year-old woman presents complaining with chronic and excessive perspiring under the arms and in the palm of the hands. Clinical examination reveals a high degree of sweating under the arms, and stains on clothing in the same area.
The patient is injected in the eccrine glands under one arm with an approximately minimum effective dose (15 drops) of BOTOX®. The same patient is injected in the eccrine glands under the other arm with 7 drops of the Multivalent Clostridial Neurotoxin Derivative of Example 1.
The patient is observed one week later. Examination reveals that excessive sweating under the arms has been deceased by 85-95% in both cases, despite the fact that the MCND was administed at less than 50% of the BOTOX® dosage.
Example 6
Treatment of Acute Pancreatitis with a Multivalent Clostridial Neurotoxin Derivative
A 55 year-old man with a history of alcoholism presents with nausea, loss of appetite and severe abdominal pain radiating to the back. Examination reveals that the patient suffers from acute pancreatitis with a Balthazar Score of Grade D (with fluid collection in a single pancreatic location). The acute and advancing pancreatic necrosis threatens the patient's life.
The patient is administered the multivalent Clostridial neurotoxin derivative of Example 3 in an effective dose by injection directly into the pancreatic acini. Within 48 hours, there is a halt in the progression of the patient's deterioration. Within two weeks the acute pain has been relieved and the patient is able to take oral nourishment.
Although aspects of the present invention have been described with reference to the disclosed embodiments, one skilled in the art will readily appreciate that the specific examples disclosed are only illustrative of these aspects and in no way limit the present invention. Various modifications can be made without departing from the spirit of the present invention.
Any and all patents, publications, patent applications, and nucleotide and/or amino acid sequences referred to by accession numbers cited in this specification are hereby incorporated by reference as part of this specification.
Each and every feature described herein, and each and every combination of two or more of such features, is included within the scope of the present invention provided that the features included in such a combination are not mutually inconsistent.
These and other aspects of the present invention are set forth in the following claims.

## Claims

1. A polypeptide comprising:
a) an endopeptidase domain;
b) a translocation domain effective to facilitate the movement of said endopeptidase domain across an endosomal membrane; and
c) at least two binding domains, wherein:
i) a first binding domain comprises a first ligand binding, under physiological conditions, to a first cell surface receptor displayed by the target cell; and
ii) a second binding domain comprises a second ligand binding, under physiological conditions, to a second cll surface receptor displayed by the target cell;

wherein said target cell internalizes said polypeptide upon binding of said polypeptide to said target cell.

2. The polypeptide of claim 1 wherein said second cell surface receptor comprises a caveolin-binding domain.

3. The polypeptide of claim 1 wherein said second cell surface receptor comprises TNFR-1.

4. The polypeptide of claim 3 wherein said second ligand comprises TNF-α.

5. The polypeptide of claim 2 wherein said second ligand binds a glycosyl phosphatylinositol (GPI)-linked membrane protein.

6. The polypeptide of claim 1 wherein said second cell surface receptor comprises an antibody variable (V) domain region.

7. The polypeptide of claim 6 wherein said second ligand comprises an antigen that selectively binds the antibody variable domain region of said second cell surface receptor.

8. The polypeptide of claim 1 wherein said second cell surface receptor binds clatherin.

9. The polypeptide of claim 8 wherein the second cell surface receptor has the amino acid sequence tyrosine-X-arginine-phenylalanine near the carboxy terminus.

10. The polypeptide of claim 8 wherein the second cell surface receptor binds an adaptin.

11. The polypeptide of claim 10 wherein the second cell surface receptor binds Adaptor Protein-2.

12. The polypeptide of claim 1 wherein the first ligand selectively binds a Clostridial neurotoxin-binding cell surface receptor.

13. The polypeptide of claim 12 wherein the first ligand comprises a Clostridial neurotoxin binding domain.

14. The polypeptide of claim 13 wherein the first ligand comprises a Clostridial neurotoxin binding domain selected from the group consisting of
a) a TeNT receptor binding domain;
b) a BoNT-A receptor binding domain;
c) a BoNT-B receptor binding domain;
d) a BoNT-C receptor binding domain;
e) a BoNT-C1 receptor binding domain;
f) a BoNT-D receptor binding domain;
g) a BoNT-E receptor binding domain;
h) a BoNT-F receptor binding domain;
i) a BoNT-G receptor binding domain HA or NTNH β trefoil domain of a neurotoxin associated protein (NAP) associated with any Clostridial neurotoxin, and
j) variants and isoforms of any of the above.

15. The polypeptide of claim 1 wherein first or second ligand comprises a receptor-binding domain selected from the group consisting of: a nerve growth factor (NGF) receptor binding domain; a leukemia inhibitory factor (LIF) receptor binding domain; a basic fibroblast growth factor (bFGF) receptor binding domain; the brain-derived neurotrophic factor (BDNF) receptor binding domain; a neurotrophin-3 (NT-3) receptor binding domain; a hydra head activator peptide (HHAP) receptor binding domain; a transforming growth factor 1 (TGF-1) receptor binding domain; a transforming growth factor 2 (TGF-2) receptor binding domain; a transforming growth factor 3(TGF-3) receptor binding domain; an epidermal growth factor (EGF) receptor binding domain; a ciliary neurotrophic factor (CNTF) receptor binding domain; a tumor necrosis factor (TNF-) receptor binding domain; an interleukin-1 (IL-1) receptor binding domain; an interleukin-1 (IL-1) receptor binding domain; an interleukin-8 (IL-8) receptor binding domain; a bradykinin receptor binding domain; a dynorphin receptor binding domain; a β-endorphin receptor binding domain; an etorphine receptor binding domain; an endomorphin-1 receptor binding domain; an endomorphin-2 receptor binding domain; a leu-enkephalin receptor binding domain; a met-enkephalin receptor binding domain; a galanin receptor binding domain; a lofentanil receptor binding domain; a nociceptin receptor binding domain; an antigen-binding variable region of an antibody against the lactoseries carbohydrate epitopes found on the surface of dorsal root ganglion neurons; an antigen-binding variable region of an antibody binding any of the receptors for the binding domains given above and an antibody against the surface expressed antigen Thyl.

16. The polypeptide of claim 15 wherein said first and second ligands are different.

17. The polypeptide of claim 14 wherein said second ligand comprises a receptor-binding domain selected from the group consisting of: a nerve growth factor (NGF) receptor binding domain; a leukemia inhibitory factor (LIF) receptor binding domain; a basic fibroblast growth factor (bFGF) receptor binding domain; the brain-derived neurotrophic factor (BDNF) receptor binding domain; a neurotrophin-3 (NT-3) receptor binding domain; a hydra head activator peptide (HHAP) receptor binding domain; a transforming growth factor 1 (TGF-1) receptor binding domain; a transforming growth factor 2 (TGF-2) receptor binding domain; a transforming growth factor 3(TGF-3) receptor binding domain; an epidermal growth factor (EGF) receptor binding domain; a ciliary neurotrophic factor (CNTF) receptor binding domain; a tumor necrosis factor (TNF-) receptor binding domain; an interleukin-1 (IL-1) receptor binding domain; an interleukin-1 (IL-1) receptor binding domain; an interleukin-8 (IL-8) receptor binding domain; a bradykinin receptor binding domain; a dynorphin receptor binding domain; a β-endorphin receptor binding domain; an etorphine receptor binding domain; an endomorphin-1 receptor binding domain; an endomorphin-2 receptor binding domain; a leu-enkephalin receptor binding domain; a met-enkephalin receptor binding domain; a galanin receptor binding domain; a lofentanil receptor binding domain; a nociceptin receptor binding domain; an antigen-binding variable region of an antibody against the lactoseries carbohydrate epitopes found on the surface of dorsal root ganglion neurons; an antigen-binding variable region of an antibody binding any of the receptors for the binding domains given above and an antibody against the surface expressed antigen Thyl.

18. The polypeptide of claim 15 wherein said second cell surface receptor comprises a caveolin-binding domain.

19. The polypeptide of claim 15 wherein said second cell surface receptor comprises TNFR-1.

20. The polypeptide of claim 19 wherein said second ligand comprises TNF-α.

21. The polypeptide of claim 18 wherein said second ligand binds a glycosyl phosphatylinositol (GPI)-linked membrane protein.

22. The polypeptide of claim 15 wherein said second cell surface receptor comprises an antibody variable (V) domain region.

23. The polypeptide of claim 22 wherein said second ligand comprises an antigen that selectively binds the antibody variable domain region of said second cell surface receptor.

24. The polypeptide of claim 15 wherein said second cell surface receptor binds clatherin.

25. The polypeptide of claim 24 wherein the second cell surface receptor has the amino acid sequence tyrosine-X-arginine-phenylalanine near the carboxy terminus.

26. The polypeptide of claim 24 wherein the second cell surface receptor binds an adaptin.

27. The polypeptide of claim 26 wherein the second cell surface receptor binds Adaptor Protein-2.