AU2005333666A1 - Delivery of active proteins to the central nervous system using phage vectors - Google Patents

Description

WO 2007/001302 PCT/US2005/022955 DELIVERY OF ACTIVE PROTEINS TO THE CENTRAL NERVOUS SYSTEM USING PHAGE VECTORS by Kim J. Janda BACKGROUND OF THE INVENTION [0001] This invention is directed to methods and compositions for the delivery of active proteins to the central nervous system using vectors derived from bacteriophage, particularly filamentous bacteriophage. [0002] The delivery of active proteins to the central nervous system is desired in many diagnostic or therapeutic applications. Among the proteins that it would be desirable to deliver to the central nervous system are enzymes, antibodies, receptor proteins, ligands for receptor proteins, reporter proteins, proteins regulating gene expression or other metabolic processes, and membrane proteins. The delivery of such proteins to the central nervous system can be used, for example, to treat diseases and conditions associated with the absence of a normally-functioning protein or with the undesired presence of a substance that could inactivated by binding or degradation if a protein with the proper specificity were introduced. [0003] However, the delivery of proteins to the central nervous system is greatly complicated by the existence of the blood-brain barrier (BBB). The capillaries that supply blood to the tissues of the brain constitute the blood-brain barrier (Goldstein et al., Scientific American 255:74-83 (1986); W.M. Pardridge, Endocrin. Rev. 7:314-330 (1986)). The endothelial cells which form the brain capillaries are different from those found in other tissues in the body. Brain capillary endothelial cells are joined together by tight intercellular junctions which form a continuous wall against the passive movement of substances from the blood to the brain. These cells 1 WO 2007/001302 PCT/US2005/022955 are also different in that they have few pinocytic vesicles which in other tissues allow somewhat unselective transport across the capillary wall. Also lacking are continuous gaps or channels running through the cells which would allow unrestricted passage. [0004] The blood-brain barrier functions to ensure that the environment of the brain is constantly controlled. The levels of various substances in the blood, such as hormones, amino acids and ions, undergo frequent small fluctuations which can be brought about by activities such as eating and exercise (Goldstein et al., supra). If the brain were not protected by the blood brain barrier from these variations in serum composition, the result could be uncontrolled neural activity. [0005] The isolation of the brain from the bloodstream is not complete. If this were the case, the brain would be unable to function properly due to a lack of nutrients and because of the need to exchange chemicals with the rest of the body. The presence of specific transport systems within the capillary endothelial cells assures that the brain receives, in a controlled manner, all of the compounds required for normal growth and function. In many instances, these transport systems consist of membrane-associated receptors which, upon binding of their respective ligand, are internalized by the cell (W.M. Pardridge, W. M., supra). Vesicles containing the receptor-ligand complex then migrate to the abluminal surface of the endothelial cell where the ligand is released. [0006] The problem posed by the blood-brain barrier is that, in the process of protecting the brain, it excludes many potentially useful therapeutic agents. Presently, only substances which are sufficiently lipophilic can penetrate the blood brain barrier (Goldstein et al., supra; W.M. Pardridge, W. M., supra). Some drugs can be modified to make them more lipophilic and thereby increase their ability to cross the blood brain barrier. However, each modification has to be tested individually on each drug and the modification can alter the activity of the drug. The modification can also have a very general effect in that it will increase the ability of 2 WO 2007/001302 PCT/US2005/022955 the compound to cross all cellular membranes, not only those of brain capillary endothelial cells. However, this is not readily feasible for most biologically active proteins. Such proteins typically have structures with a large number of charged or polar residues on the outside of the protein structure and thus are transported as water-soluble, polar molecules. These proteins could not readily be modified to make them more lipophilic without seriously disrupting their secondary, tertiary, and quaternary structures and thus greatly reducing, if not eliminating, their biological activity. [0007] Among the contexts in which it would be desirable to introduce proteins into the central nervous system is the treatment of drug addiction, particularly cocaine addiction. Cocaine is highly addictive and may be the most reinforcing of all drugs of abuse (1-3). Despite intensive efforts, effective therapies for cocaine craving and addiction remain elusive. Unlike the historically successful methadone treatment for heroin addiction, there is no proven pharmacotherapy for cocaine abuse (4). A number of medications acting as agonists, antagonists, or anti depressants have been evaluated in both animal models and humans, with only limited success (5-11). In the absence of a single highly effective drug, available pharmacological agents must be part of a comprehensive approach toward treatment. [0008] Unquestionably, an improved pharmacotherapy would increase the effectiveness of such programs and alternative strategies for treating cocaine addiction are needed if progress is to be made. One such strategy is to use protein based therapeutics, whereby proteins are designed to bind cocaine, thereby blocking its effects, and/or degrade cocaine via hydrolysis of the benzoyl ester, thus rendering it less psychoactive (12). Over the last decade, several groups have reported the successful blocking of the psychostimulatory effects of cocaine by anti-cocaine antibodies with both active and passive immunization in rodent models. These results demonstrate that anti-cocaine antibodies bind to cocaine in circulation, retarding its ability to enter the brain (13-17). Both strategies reduce cocaine 3 WO 2007/001302 PCT/US2005/022955 induced locomotor activity and self-administration in rats. A different antibody-based approach to cocaine addiction treatment uses catalytic antibodies specific for cocaine and the cleavage of its benzoyl ester (18-23). The efficacy of catalytic antibodies has been demonstrated in rodent models of cocaine overdose and reinforcement, but kinetic constants for all reported antibody catalysts are marginal and thus improved rates will be required before clinical development is warranted (24). Finally, groups using butyrylcholinesterase (BChE), the major cocaine metabolizing enzyme present in the plasma of humans and other mammals (25,26) have reported that intravenous pretreatment with either wild-type or genetically engineered BChE can mitigate the behavioral and physiological effects of cocaine and accelerate its metabolism (27-29). One drawback common to all of these protein-based approaches is that none can act directly within the CNS; thus, their success depends solely on peripheral contact between the enzyme or antibody with ingested cocaine. This greatly limits the ability of these enzymes or antibodies to treat cocaine addiction and the consequences of cocaine abuse. [0009] Accordingly, there is a need for improved compositions and methods that can deliver active proteins to the central nervous system and allow them to pass through the blood-brain barrier without denaturing the proteins or otherwise disrupting or destroying their biological activity. SUMMARY OF THE INVENTION [0010] One aspect of the invention is a method of delivering a protein to the central nervous system in active form comprising the steps of: (1) preparing a single-stranded filamentous phage vector comprising a nucleic acid construct in which a protein to be delivered to the central nervous system is encoded as a fusion protein with a coat protein of a filamentous phage; (2) preparing phage particles incorporating the nucleic acid construct as the phage genome and in which the fusion protein is expressed as a coat protein; and 4 WO 2007/001302 PCT/US2005/022955 (3) delivering the phage particles to a mammal by a route such that the phage particles reach the central nervous system so that the protein is delivered to the central nervous system in active form. [0011] Typically, the single-stranded filamentous bacteriophage vector is derived from a bacteriophage selected from the group of M13, fd, and fl 1. Preferably, the bacteriophage is M13. [0012] Typically, the filamentous phage vector is a phagemid. [0013] Typically, the coat protein that is incorporated into the fusion protein is selected from the group consisting of pill, pVII, pVll, and plX. More typically, the coat protein is pVIIl. [0014] Typically, the protein is selected from the group consisting of an antibody, an enzyme, a reporter protein, a receptor protein, a ligand for a receptor protein, a regulatory protein, and a membrane protein. [0015] Typically, the protein is delivered by a route selected from the group consisting of intranasal delivery, intravenous delivery, intraperitoneal delivery, and intramuscular delivery. Preferably, the protein is delivered by intranasal delivery. [0016] Another aspect of the invention is a nucleic acid construct comprising: (1) an origin of replication of a filamentous bacteriophage; (2) a nucleic acid framework allowing replication of the construct into circular single-stranded DNA molecules operably linked to the origin of replication; and (3) at least one nucleic acid sequence encoding a fusion protein such that the fusion protein can be expressed during replication of the construct and assembled into chimeric bacteriophage particles. 5 WO 2007/001302 PCT/US2005/022955 [0017] Yet another aspect of the invention is a bacteriophage particle displaying a fusion protein comprising: (1) a single-stranded DNA molecule; and (2) at least one fusion protein including: (a) a coat protein of a single-stranded filamentous bacteriophage; and (b) a protein to be delivered. [0018] Yet another aspect of the invention is a pharmaceutical composition comprising: (1) the bacteriophage particle displaying a fusion protein as described above; and (2) a pharmaceutically acceptable carrier. [0019] Still another aspect of the invention is a fusion protein comprising: (1) a first domain that is pVllII protein of a filamentous bacteriophage; and (2) a second domain that is a protein that is deliverable to the central nervous system of a mammal. BRIEF DESCRIPTION OF THE DRAWINGS [0020] The following invention will become better understood with reference to the specification, appended claims, and accompanying drawings, where: [0021] Figure 1 is a diagram of filamentous bacteriophage fd architecture (this applies equally well to M13 and fl 1, which are closely related). [0022] Figure 2 is a graph showing the affinity of a fusion protein incorporating an anti-cocaine antibody, GNC 92H2, with pVIII (GNC 92H2-pVIII) for cocaine as determined by equilibrium dialysis. 6 WO 2007/001302 PCT/US2005/022955 [0023] Figure 3 is a graph showing locomotor activity (crossovers; Upper) and stereotyped behavior (sniffing and rearing; Lower) after i.p. injection of cocaine after nasal immunization with GNC 92H2-pVIIlI (0) or RCA 60 28-pVllI (0), at 10 mg/kg, 15 mg/kg, and 30 mg/kg of cocaine. [0024] Figure 4 is a graph showing Ambulatory behavior (crossovers) elicited by increasing doses of systemic cocaine (i.p.): 10 (a), 15 (b), and 30 (c) mg/kg in a between-subject design and the effect of phage infusion with phage displaying anti cocaine antibody as part of the fusion protein. [0025] Figure 5 is a scheme showing the action of a catalytic antibody that hydrolyzes the benzoyl ester of cocaine into benzoate and methylecgonine. DETAILED DESCRIPTION [0026] The present invention is directed to methods and compositions that can deliver proteins to the central nervous system in active form. [0027] One aspect of the present invention is a method of delivering a protein to the central nervous system in active form. In general, this method comprises the steps of: (1) preparing a single-stranded filamentous bacteriophage vector comprising a nucleic acid construct in which a protein to be delivered to the central nervous system is encoded as a fusion protein with a coat protein of a filamentous phage; (2) preparing phage particles incorporating the nucleic acid construct as the phage genome and in which the fusion protein is expressed as a coat protein; and 7 WO 2007/001302 PCT/US2005/022955 (3) delivering the phage particles to a mammal by a route such that the phage particles reach the central nervous system so that the protein is delivered to the central nervous system in active form. [0028] The single-stranded filamentous bacteriophage vector is preferably derived from a bacteriophage selected from the group of M13, fd, and fl 1. A particularly preferred single-stranded filamentous phage is M13. However, M13, fd, and fl are extremely closely related. The genomes of these three phages are more than 98% identical; most of the differences occur at the third position of codons and do not alter the sequence of the protein encoded. [0029] Preferably, the filamentous phage vector is a phagemid. These vectors carry origins of replication derived from a single-stranded filamentous bacteriophage. Such vectors have the advantage of two modes of replication: as a conventional double-stranded DNA plasmid and as a template to produce single stranded copies of one of the phagemid strands. A phagemid can be used to produce filamentous phage particles that contain single-stranded copies of cloned segments of DNA, such as DNA encoding the fusion proteins described above. A particularly suitable phagemid is pCGMT or a derivative of pCGMT. Phagemids are described in J. Sambrook & D.W. Russell, "Molecular Cloning: A Laboratory Manual"

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3 rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001), v. 1, pp. 3.42-3.49, incorporated herein by this reference. Segments of foreign DNA can be cloned in plasmids and propagated as plasmids. However, when a suitable male strain of Escherichia coli carrying a phagemid is infected with a suitable filamentous bacteriophage, typically known as a helper virus, the mode of replication of the phagemid changes as a result of the expression of gene products by the incoming bacteriophage and the activity of these gene products in initiating DNA replication. The gene II protein encoded by the helper virus introduces a nick at a specific site in the intergenic region of the phagmid and thus initiates rolling circle DNA replication. This generates copies of one strand of the phagemid DNA. These single-stranded copies of the phagemid DNA are packaged into progeny 8 WO 2007/001302 PCT/US2005/022955 bacteriophage particles, which are then extruded into the medium. These particles can be recovered by precipitation with polyethylene glycol and the single-stranded DNA purified by standard techniques, such as phenol extraction. [0030] Therefore, the step of preparing phage particles incorporating the nucleic acid construct as the phage genome and in which the fusion protein is expressed as a coat protein typically comprises: (a) transforming a bacterial host cell with a phagemid incorporating the nucleic acid construct; and (b) producing phage particles by infection with a helper virus. [0031] When the filamentous bacteriophage is M13, the coat protein that is incorporated into the fusion protein is typically one of pill, pVll, pVlll, and plX. However, it is generally preferred to use pVlll as the coat protein, as this is the major coat protein and offers the potential of expressing up to 2800 copies/phage particle. However, in some cases, steric constraints can lead to a preference for use of another coat protein, such as pll or plX. [0032] The expression of cloned proteins, such as fusion proteins, by filamentous bacteriophages or phagemids is known as phage display, and such techniques are well known in the art. Phage display is described, for example, in J. Sambrook & D.W. Russell, "Molecular Cloning: A Laboratory Manual" ( 3 rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001), v. 3 pp. 18.115-18.122, incorporated herein by this reference. [0033] The protein to be expressed can be any protein whose size renders it amenable to incorporation into a fusion protein with a coat protein of a filamentous phage and to subsequent phage display. Typically, the protein is monomeric, homodimeric, or homomultimeric; however, as discussed below, it is possible to express heterodimeric or heteromultimeric proteins, such as native antibodies, by the use of several populations of phagemids, each engineered to express one chain of the heterodimer or heteromultimer. For example, the protein can be a chain of an 9 WO 2007/001302 PCT/US2005/022955 antibody molecule, such as a heavy chain or a light chain, which can then reassemble to form an intact native antibody molecule. However, it is generally preferred that the protein is monomeric. [0034] The protein can be, but is not limited to, an antibody, an enzyme, a reporter protein, a receptor protein, a ligand for a receptor protein, a regulatory protein, or a membrane protein. If the protein is an antibody, it is typically in the form of a scFv or Fab' fragment. The term "antibody" is used herein to refer to all protein molecules having affinity and cross-reactivity substantially equivalent to native antibodies having a four-chained L 2

H

2 structure, whether monomeric or multimeric, and thus includes scFv or Fab' fragments unless such fragments are specifically excluded. The term "antibody" as used herein further encompasses catalytic antibodies. [0035] Enzymes suitable for delivery into the central nervous system include, but are not limited to: enzymes having a therapeutic effect, such as asparaginase, which has been used in cancer treatments; enzymes that degrade molecules that act as toxins or drugs of addiction, such as cocaine esterase or butyrylcholinesterase, which hydrolyze cocaine; and enzymes that replace cellular enzymatic activity that is missing or diminished because of a mutation or cellular damage, such as Tay-Sachs disease, in which the oa subunit of hexosaminidase is lacking, or Gaucher's disease, in which the enzyme glucocerebroside j3-glucosidase is lacking. Other enzymes can also be delivered. [0036] The protein to be delivered can be a wild-type protein or can be a protein modified by mutagenesis, such as site-specific mutagenesis; i.e., it can be a mutein. These techniques are well known in the art. [0037] Alternatively, the protein to be delivered can be itself a fusion protein that has been prepared by techniques known in the art and described further below. 10 WO 2007/001302 PCT/US2005/022955 For example, the protein can be a fusion protein between an antibody and a protein toxin, which can be administered for therapeutic purposes [0038] A preferred route of delivery is intranasal delivery. However, other delivery routes, such as intravenous, intraperitoneal, or intramuscular delivery, can be used. [0039] The mammal can be a human, or a socially or economically important non-human mammal selected from the group consisting of a dog, a cat, a horse, a cow, a sheep, a goat, a rat, a mouse, and a rabbit. Methods according to the present invention are not limited to use on humans. [0040] Methods according to the present invention, therefore, can be used for diagnostic or therapeutic purposes as described above. If the protein to be delivered is a reporter protein, the method is typically used for diagnostic purposes, or to monitor the effect of another therapeutic method or process. If the protein to be delivered is other than a reporter protein, such as an antibody, an enzyme, a receptor protein, a ligand for a receptor protein, a regulatory protein, or a membrane protein, the method is typically used to treat a disease or condition that is affected by the protein to be delivered. As used herein, the term "treatment" does not require a complete cure or remission of the disease or condition, but only requires that at least one measurable physical or psychological parameter associated with the disease or condition be improved by the treatment. [0041] As used herein, "isolated," when referring to a molecule or composition, such as, e.g., a nucleic acid or polypeptide of the invention, means that the molecule or composition is separated from at least one other compound, such as a protein, DNA, RNA, or other contaminants with which it is associated in vivo or in its naturally occurring state. Thus, a nucleic acid sequence is considered isolated when it has been isolated from any other component with which it is naturally associated. An isolated composition can, however, also be substantially pure. An 11 WO 2007/001302 PCT/US2005/022955 isolated composition can be in a homogeneous state. It can be in a dry or an aqueous solution. Purity and homogeneity can be determined, e.g., using analytical chemistry techniques such as, e.g., polyacrylamide gel electrophoresis (SDS-PAGE) or high performance liquid chromatography (HPLC). [0042] The term "nucleic acid" or "nucleic acid sequence" refers to a deoxy ribonucleotide or ribonucleotide oligonucleotide or polynucleotide, including single- or double-stranded forms, and coding or non-coding (e.g., "antisense") forms. The term encompasses nucleic acids containing known analogues of natural nucleotides. The term also encompasses nucleic-acid-like structures with synthetic backbones. DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research and Applications (1993, CRC Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described, e.g., by U.S. Pat. Nos. 6,031,092; 6,001,982; 5,684,148; see also, WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197. Other synthetic backbones encompassed by the term include methylphosphonate linkages or alternating methylphosphonate and phosphodiester linkages (see, e.g., U.S. Pat. No. 5,962,674; Strauss-Soukup (1997) Biochemistry 36:8692-8698), and benzylphosphonate linkages (see, e.g., U.S. Pat. No. 5,532,226; Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156). [0043] As used herein the term "protein" includes "conservative variants" with structures and activity that substantially correspond to the protein to be delivered. Such a conservative variant has a modified amino acid sequence, such that the change(s) do not substantially alter the protein's (the conservative variant's) 12 WO 2007/001302 PCT/US2005/022955 structure and/or activity, e.g., antibody activity, enzymatic activity, or receptor activity. These include conservatively modified variations of an amino acid sequence, i.e., amino acid substitutions, additions or deletions of those residues that are not critical for protein activity, or substitution of amino acids with residues having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non polar, etc.) such that the substitutions of even critical amino acids does not substantially alter structure and/or activity. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, one exemplary guideline to select conservative substitutions includes (original residue followed by exemplary substitution): Ala/Gly or Ser; Arg/Lys; Asn/Gln or His; Asp/Glu; Cys/Ser; Gln/Asn; Gly/Asp; Gly/Ala or Pro; His/Asn or Gin; Ile/Leu or Val; Leu/lle or Val; Lys/Arg or Gin or Glu; Met/Leu or Tyr or Ile; Phe/Met or Leu or Tyr; Ser/Thr; Thr/Ser; Trp/Tyr; Tyr/Trp or Phe; Val/Ile or Leu. An alternative exemplary guideline uses the following six groups, each containing amino acids that are conservative substitutions for one another: (1) alanine (A or Ala), serine (S or Ser), threonine (T or Thr); (2) aspartic acid (D or Asp), glutamic acid (E or Glu); (3) asparagine (N or Asn), glutamine (Q or Gin); (4) arginine (R or Arg), lysine (K or Lys); (5) isoleucine (I or lie), leucine (L or Leu), methionine (M or Met), valine (V or Val); and (6) phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W or Trp); (see also, e.g., Creighton (1984) Proteins, W. H. Freeman and Company; Schulz and Schimer (1979) Principles of Protein Structure, Springer-Verlag). One of skill in the art will appreciate that the above-identified substitutions are not the only possible conservative substitutions. For example, for some purposes, one may regard all charged amino acids as conservative substitutions for each other whether they are positive or negative. In addition, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence can also be considered "conservatively modified variations" when the three-dimensional structure and the function of the protein to be delivered are conserved by such a variation. 13 WO 2007/001302 PCT/US2005/022955 [0044] Techniques for the isolation of nucleic acids encoding the protein of interest and generation of appropriate fusions with a coat protein of a filamentous phage are well known in the art. The nucleic acid sequences, whether RNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof, may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed recombinantly. Any recombinant expression system can be used, including, in addition to insect and bacterial cells, e.g., mammalian, yeast or plant cell expression systems. Alternatively, these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066. [0045] Techniques for the manipulation of nucleic acids, such as, e.g., generating mutations in sequences, subcloning, labeling probes, sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., J. Sambrook & D.W. Russell, "Molecular Cloning: A Laboratory Manual"

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3 rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001);"Current Protocols in Molecular Biology" (F.W. Ausubel, ed. John Wiley & Sons, Inc., New York 1997); "Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, (Tijssen, ed. Elsevier, N.Y. 1993)). [0046] Accordingly, another aspect of the invention is a nucleic acid construct comprising: (1) an origin of replication of a filamentous bacteriophage; (2) a nucleic acid framework allowing replication of the construct into circular single-stranded DNA molecules operably linked to the origin of replication; and 14 WO 2007/001302 PCT/US2005/022955 (3) at least one nucleic acid sequence encoding a fusion protein such that the fusion protein can be expressed during replication of the construct and assembled into chimeric bacteriophage particles. [0047] The fusion protein typically includes at least one domain having an activity selected from the group consisting of antibody activity, enzymatic activity, reporter protein activity, receptor protein activity, ligand activity for a receptor protein, regulatory protein activity, and membrane protein activity. [0048] The nucleic acid construct can also be incorporated into a vector and used to transfect or transform suitable host cells, as is well known in the art. This is the basis of phagemid vectors, as described above. Host cells that are transformed or transfected with the vector are also within the scope of the invention. Typically, the host cell is a bacterial host cell that is capable of producing filamentous bacterial phage particles. Methods of transforming or transfecting host cells, including bacterial host cells, are well known in the art and are described, for example, in B.R. Glick & J.J. Pasternak, "Molecular Biotechnology: Principles and Applications of Recombinant DNA" (2d ed., ASM Press, Washington, 1998), pp. 74-75, incorporated herein by this reference. [0049] The fusion protein encoded by the nucleic acid sequence is as described above. Typically, the filamentous bacteriophage is M13. [0050] Still another aspect of the invention is a bacteriophage particle displaying a fusion protein. In general, the bacteriophage particle comprises: (1) a single-stranded DNA molecule; and (2) at least one fusion protein including: (a) a coat protein of a single stranded filamentous bacteriophage; and (b) a protein to be delivered. 15 WO 2007/001302 PCT/US2005/022955 [0051] In this bacteriophage particle displaying a fusion protein, the fusion protein is as described above. If the bacteriophage is M13, the coat protein is preferably the major coat protein, pVIIl. [0052] Yet another aspect of the present invention is a pharmaceutical composition comprising: (1) the bacteriophage particle displaying a fusion protein as described above; and (2) a pharmaceutically acceptable carrier. [0053] A pharmaceutically acceptable carrier can be chosen from those generally known in the art, including, but not limited to, human serum albumin, ion exchangers, alumina, lecithin, or buffer substances such as phosphates, glycine, sorbic acid, or potassium sorbate. Other carriers can be used and are known in the art. [0054] The pharmaceutical composition can be formulated for intranasal delivery, intravenous delivery, intraperitoneal delivery, or intramuscular delivery as described above. Typically, intranasal delivery is preferred. [0055] Still another aspect of the present invention is a fusion protein comprising: (1) a first domain that is pVIII protein of a filamentous bacteriophage; and (2) a second domain that is a protein that is deliverable to the central nervous system of a mammal. [0056] In one alternative, the first domain and the second domain are linked so that they are expressed in one polypeptide without a linker. In another alternative, the fusion protein further comprises a linker between the first domain and the second domain. Suitable linkers for fusion proteins are well known in the art and 16 WO 2007/001302 PCT/US2005/022955 need not be described further here. Such linkers typically comprise short oligopeptide regions that typically assume a random coil conformation. The linker typically consists of less than about 15 amino acid residues, more typically about 4 to 10 amino acid residues. [0057] The invention is illustrated by the following Examples. These Examples are included for illustrative purposes only, and are not intended to limit the invention. Example 1 Delivery of Anti-Cocaine Antibodies to the Central Nervous System [0058] Cocaine is highly addictive and may be the most reinforcing of all drugs of abuse (1-3). Despite intensive efforts, effective therapies for cocaine craving and addiction remain elusive. Unlike the historically successful methadone treatment for heroin addiction, there is no proven pharmacotherapy for cocaine abuse (4). A number of medications acting as agonists, antagonists, or anti depressants have been evaluated in both animal models and humans, with only limited success (5-11). In the absence of a single highly effective drug, available pharmacological agents must be part of a comprehensive approach toward treatment. [0059] Unquestionably, an improved pharmacotherapy would increase the effectiveness of such programs and alternative strategies for treating cocaine addiction are needed if progress is to be made. One such strategy is to use protein based therapeutics, whereby proteins are designed to bind cocaine, thereby blocking its effects, and/or degrade cocaine via hydrolysis of the benzoyl ester, thus rendering it less psychoactive (12). Over the last decade, several groups have reported the successful blocking of the psychostimulatory effects of cocaine by anti-cocaine antibodies with both active and passive immunization in rodent models. These 17 WO 2007/001302 PCT/US2005/022955 results demonstrate that anti-cocaine antibodies bind to cocaine in circulation, retarding its ability to enter the brain (13-17). Both strategies reduce cocaine induced locomotor activity and self-administration in rats. A different antibody-based approach to cocaine addiction treatment uses catalytic antibodies specific for cocaine and the cleavage of its benzoyl ester (18-23). The efficacy of catalytic antibodies has been demonstrated in rodent models of cocaine overdose and reinforcement, but kinetic constants for all reported antibody catalysts are marginal and thus improved rates will be required before clinical development is warranted (24). Finally, groups using butyrylcholinesterase (BChE), the major cocaine metabolizing enzyme present in the plasma of humans and other mammals (25, 26) have reported that intravenous pretreatment with either wild-type or genetically engineered BChE can mitigate the behavioral and physiological effects of cocaine and accelerate its metabolism (27-29). One drawback common to all of these protein-based approaches is that none can act directly within the CNS; thus, their success depends solely on peripheral contact between the enzyme or antibody with ingested cocaine. [0060] Bacteriophage are viruses that infect bacteria, and are distinct from animal and plant viruses in that they lack intrinsic tropism for eukaryotic cells (30). Filamentous bacteriophage fd can be produced at high titer in bacterial culture, making production simple and economical. Furthermore, phage are extremely stable to a variety of harsh conditions, such as extremes in pH and treatment with nucleases or proteolytic enzymes (30). But, perhaps the most significant importance is the genetic flexibility of filamentous phage. In 1985, Smith reported a method that physically linked genotype and phenotype in a protein display system, and this technology has become known as phage display (31); it allows a wide variety of proteins, antibodies, and peptides to be displayed on the phage coat (Fig. 1). [0061] Advances in filamentous phage display for in vitro application have been described wherein phage displaying a random peptide library were intravenously injected into mice and subsequently rescued from the internal organs, 18 WO 2007/001302 PCT/US2005/022955 showing that the integrity of the phage was not compromised (32, 33); and a report in which filamentous phage were shown to penetrate the CNS (34). In this later study, Solomon and co-workers were able to deliver phage-displayed anti-3-amyloid antibodies via intranasal administration into the brain of mice. This publication is significant as it provides the following findings: (1) Filamentous phage can access the CNS. (2) Phage can display foreign proteins on its surface and still penetrate the CNS. (3) Bacteriophage can be injected multiple times into the same animals without visible toxic effects. [0062] Previously, it has been shown that sequestering of cocaine by anti cocaine antibodies can suppress the psychomotor and reinforcing actions of the drug (13-15). A murine monoclonal antibody termed GNC 92H2 emerged from these studies that has exquisite affinity and specificity to cocaine (Kd = 40 nM and benzoyl ecgonine Kd = 1.4 IM), (35). It has also been demonstrated that antibody libraries can be displayed on the filamentous phage coat proteins pill, pVII, and plX for the selection of high affinity antibodies to a wide variety of antigens (36, 37). Herein we detail the therapeutic potential of a phage-displayed protein (GNC 92H2-pVIII) that is designed to be not only highly specific for binding of cocaine but that can also access and act directly within the CNS as an additional mode of drug abuse therapy. Materials and Methods [0063] Preparation of Phage display vectors. The phage display vector pCGMT-p8 that was used in this study was derived from the phagemid pCGMT (38). The DNA sequence encoding the C-terminus of the coat protein III (pill) gene in pCGMT was replaced with the major coat protein VIII (pVIII) gene. The vector also contains the cloning site for the single chain (scFv) genes with two Sfil restriction sites. The genes for the scFv antibodies GNC 92H2 and RCA 60 28 were amplified using PCR methodology (36,37). The PCR reaction products were agarose gel purified, recovered (Qiagen), digested with the restriction enzyme Sfil (New England 19 WO 2007/001302 PCT/US2005/022955 Biolabs), and ligated into pCGMT-p8. DNA sequencing was used to confirm the new construct. [0064] Preparation and Purification of Phage Particles Displaying scFv GNC 92H2. E. coliTG1 cells (Stratagene) were transformed with the phagemid encoding the appropriate scFv antibody. E. coliTG1 cultures were grown in 2 x 0.5 L of 2YT broth in the presence of the antibiotic carbenicillin (100 gg/mL). Upon an optical density at a wavelength of 600 nm (OD 600 ) of 0.8, the cells were infected with 0.5 mL of VCSM13 helper phage (Stratagene) (1012 pfu/mL). After 30 min incubation at room temperature, the culture was grown for 2 h at 30'C. Kanamycin/IPTG were then added to a final concentration of 70 gg/mL, and the culture was grown overnight at 300C. Following growth overnight, the bacterial cells were removed by centrifugation and phage particles were harvested from the supernatant by precipitation with NaCI (3% w/v) and polyethylene glycol (PEG) 8000 (4% w/v). The phage pellet was re-suspended in sterile, endotoxin-free PBS (Invitrogen) and re precipitated again. Upon re-suspension of the pellet in 4 mL PBS, the phage solution was filtered through a pyrogen-free 0.45 pm cellulose-acetate filter to remove any remaining bacterial cells. The phage preparation was titered, i.e., the number of colony forming units (cfu) was determined according to standard protocols (39). [0065] Affinity Measurements of Phage Displayed Proteins. Equilibrium dialysis was performed using [ 3 H]-cocaine as the ligand and phage scFv GNC 92H2 pVIII; helper phage VCS M13 and RCA 60 28-pVIII were also measured and used as controls. Phage samples were serially diluted on a 96-well microtiter plate (80 t/well). Wells (12 per sample) were then filled with another 80 LL of [ 3 H]-cocaine in PBS, (2 nM per well). A second plate was prepared with 2 times 12 wells containing just PBS (160 g.L/well). The two plates were very tightly connected with filled wells facing each other and separated with a dialysis membrane (cutoff 6000-8000 Da). The plates were attached vertically to a shaker and were shaken at high frequency for 24 h at room temperature, after which they were carefully separated. The 20 WO 2007/001302 PCT/US2005/022955 membrane was discarded and from each well 100 RL was transferred to a scintillation vial. 5 mL scintillation fluid was added to each vial and radiation was counted for each sample for 5 min. The experiment was repeated twice for each serum sample. The average in differences in DPM (dosage per minute) between opposite wells was determined for each dilution of phage particles. The number of phage particles was determined spectrophotometrically (A 266 nm - A 320 nm) using a molar extinction coefficient of 1.006 x 104 M-1cm 1 , and a genome size of 3722 bases for the modified phage. [0066] Animals. Male Wistar rats (n = 16; Charles River Breeding Laboratories), weighing 200-225 g on arrival, were housed in groups of 2 in a humidity- and temperature-controlled (220C) vivarium on a 12 h light/dark cycle (lights on at 10 p.m.) with free access to food and water. All behavioral procedures were performed during the light cycle. Before behavioral testing, each rat was handled by the experimenter for 10 min. All procedures were conducted in strict adherence to the National Institutes of Health Guide of the Care and Use of Laboratory Animals. [0067] Intranasal Phage Administration Protocols. Animals were anesthetized with a bolus i.p. injection of Pentobarbital (sodium salt Sigma) 60 mg/kg diluted in physiological saline. Rats were placed dorsally with heads positioned to maximize residency of exogenous substances on the olfactory epithelium. Intranasal (i.n.) administration of GNC 92H2-pVII (wt 1.0 x 1014 pfu/ml) diluted in PBS (50 pl/naris) was administered over 15-30 min using a Hamilton micro-syringe (100 p1) with scilastic tubing. During i.n. administration, the opposite naris was closed to induce natural aspiration of the injected substance. Control animals received 50 pl of phage/sc RCA 60 28-pVIII, a single chain antibody that binds to RCA 60 , ricin. Injections were delivered twice per day for three consecutive days. [0068] Detection and Identification of Filamentous Phage in Rat Brain. Brain samples were obtained in a time continuum throughout the phage-infusion 21 WO 2007/001302 PCT/US2005/022955 regimen, testing days, and post-challenge days as to ascertain the time frame of introduction, residence and clearance of phage in the neural tissue. To this end, brains were collected on days 2, 3, 4, 8, 10, 13, 15 and 17 after onset of phage infusion. Animals were deeply anesthetized with Halothane vapor, and rapidly decapitated. Brains were harvested from cerebellum to olfactory tubercule and immediately ground to a fine, homogenous consistency, washed with, and incubated in 3ml of PBS (pH 7.4) for 1 h at room temperature. After the incubation time, the tubes were spun down at 1000 rpm in a benchtop GS-6R centrifuge (Beckman). E. coliTG1 cells were infected with serial dilutions of the supernatant for lh at room temperature and plated onto Luria Bertani (LB) broth LB) agar plates containing carbenicillin (100 .g/ml). Colonies were counted the following day and the titer was calculated based on serial dilution. Phagemids isolated from phage particles on days 4 and 7 were analyzed using DNA sequencing at the Protein and Nucleic Acid Core facility of The Scripps Research Institute in order to positively identify the phage particles as well to confirm the presence of the antibody gene. [0069] Detection of Anti-Filamentous Phage Antibodies in Rat Serum. Blood was drawn from the jugular vein for serum IgG measurements following phage injections at day 28. Filamentous phage particles displaying scFv GNC 92H2 or

RCA

60 28 on pVIII were coated on 96-well microtiter plates (NuncMaxiSorp) overnight at 4°C. Wells were washed five times, blocked with 5%(w/v) BLOT-QuickBlocker (Oncogene) in PBS (pH 7.4), at ambient temperature for 1 h. After subsequent washing, rat serum (serially diluted) was added into the wells and incubated for 1 h at room temperature. Repeated washing of the plate was followed by the addition of goat-anti-rat IgG-HRP (Pierce) and goat-anti-rat IgM-HRP (Pierce) which was diluted 1:5000 in blocking solution and was added followed by incubation for 1 h at ambient temperature. The plates were copiously washed and HRP substrate (3,3',5,5' tetramethyl benzidine (TMB) and hydrogen peroxide) was added according to the manufacturer's instructions (Pierce). Color reactions were read at a wavelength of 450 nm using a Thermomax ELISA plate reader (Molecular Devices). 22 WO 2007/001302 PCT/US2005/022955 [0070] Behavioral Procedures. Locomotor activity was measured in a bank of 16 wire cages, each cage 20 cm high x 25 cm wide x 36 cm long, with two horizontal infrared beams across the long axis 2 cm above the floor. Total photocell beam interruptions and crossovers, the number of times breaking on the photocell beam followed directly by breaking the other photocell beam, were recorded by a computer every 10 minutes; background noise was provided by a white noise generator. [0071] Before the phage-treatment regime, each rat was habituated to the photocell cages overnight, and prior to drug injection the rats were habituated again to the photocell cages for 90 min. To determine pre-immunization drug-response (baseline), animals received an i.p. injection of 15 mg/kg cocaine HCI mixed in saline solution (bolus 1 mL/kg) and their locomotor responses were measured during a 90 min session. Based on locomotor activity scores, animals were assigned to the experimental or control group in ranking order. [0072] Stereotypic behavior (sniffing and rearing) was rated for 10 seconds every 10 minutes as previously described (40). Data were arranged in contingency tables in the following way: (1) For each response category and for each 10 min interval, the number of rats showing a particular category was tabulated. (2) The degree of heterogeneity in each contingency table was then calculated by a likelihood ratio method (see Statistical Analysis). [0073] On challenge days, animals received an i.p. injection of isotonic saline (bolus 1 mg/kg) and were habituated for 90 min prior to the drug injection. Locomotor activity was measured during habituation and the testing session as described above. The experimental design consisted of a 2 X 3 between-subjects design, where two different phage infusions and three cocaine doses were administered. Animals received either RCA 60 28-pVIll or GNC 92H2-pVlll, the anti-cocaine mAb displaying phage. Cocaine doses ranged from 10, 15 and 30 mg/kg i.p. In all experiments, animals were challenged with their corresponding dose of cocaine at 23 WO 2007/001302 PCT/US2005/022955 the fourth day from the onset of phage infusions, that is, the next day after the last phage treatment. Animals were subjected to cocaine challenges for three consecutive days. [0074] Statistical Analysis. Locomotor activity data were analyzed by subjecting 10-minute total means for locomotor activity to a two-factor analysis of variance (ANOVA) (group X time) with repeated measures on the within-group factor, time. Individual means comparisons for the main treatment effects were analyzed using a Newman-Keuls a posterioritest. Stereotyped behavior data were analyzed by a likelihood ratio method, the "information statistic" (41, 42). Results [0075] Validation of GNC 92H2-pVIII Affinity for Cocaine. The affinity of GNC 92H2-pVlll for cocaine was determined using a radioimmunoassay based on equilibrium dialysis with tritium labeled cocaine and serial dilutions of phage. A comparison was made between the cocaine binding ability of GNC 92H2-pVII with VCS M13 helper phage and with RCA 60 28-pVlII; the latter two phage constructs in theory are not expected to bind cocaine. As shown in Figure 2, phage GNC 92H2 pVll clearly binds cocaine, whereas control phage do not. Based on this binding curve, we estimate the Kd-avg of phage GNC 92H2-pVIII to be between 50 nM and 5 pM, depending on the number of scFv 92H2 antibodies displayed on each phage particle. [0076] In Figure 2, The affinity of phage displaying GNC 92H2-pVll for cocaine, as determined using equilibrium dialysis with [ 3 H]-cocaine and serial dilutions of phage, is shown (GNC 92H2-pVlll (0), RCA 60 28-pVIII (0) and VCS M13 (A)). [0077] Psychomotor Response to Systemic Cocaine. The average weight of the animals upon completion of the studies was 365 ± 42 g (n = 16). The 24 WO 2007/001302 PCT/US2005/022955 photocell cage habituation procedure resulted in consistent patterns of activity after saline injection: transient arousal (less than 20 min) followed by typically low levels of ambulation. Cocaine injection pre-treatment baseline values were (RCA 60 28-pVlll), 523 ± 98.6; (GNC 92H2-pVlIIl) 594 ± 121.5. Intranasal administration of phage required an average of 20 min of infusion time per naris. Spillage due to sneezing of abrupt movement was monitored by degree of wetness on tissue rostral drape. Substantial wetness merited dosing de novo. Figure 3 shows the psychomotor response to cocaine after the intranasal phage administration regime. At the low dose of 10 mg/kg, cocaine elicited a significant motor differential between groups [Fig.3a Upper RCA 60 28-pVlll, 513 ± 94.29; GNC 92H2-pVIII, 317.25 ± 78.95; F (1, 14) = 5.3, P< .05). Significance in treatment x time interaction was not observed. However, simple effects analysis revealed a significant difference in behavior between the RCA 60 28-pVIII and GNC 92H2-pVIII groups 20 min into the session [F(1,8) = 6.826, P < 0.05). This time-dependent effect was reflected in the stereotypy levels displayed by animals at this dose [Fig.3a Lower 21= 76.2, df = 1, 9). In a different group, 15 mg/kg of cocaine resulted in a highly pronounced difference in both psychomotor measures [locomotor: Fig. 3b Upper RCA 60 28-pVIll, 1064.375 ± 213.52; GNC 92H2-pVll, 550.125 ± 135.89; F (1, 14) = 6.875, P< .05; with significant main effects of treatment x time interaction F (1, 8) = 4.268, P < 0.001; stereotypy: Fig. 3b Lower, 2! = 82.2, df = 1, 9; P < 0.05]. According to simple main effects analysis, differences between groups were greater from the 10 to the 40 min time points of the session [time 10-20: F (1, 8) = 7.27, P < 0.017; time 20-30: F(1, 8) = 9.03, P< 0.009; time 30-40: F(1, 8) = 4.18, P< 0.05]. The group receiving the higher dose of cocaine (30 mg/kg) displayed a contrasting pattern of behavior in relation to the former groups in the locomotor category (Fig. 3c Upper). Although the overall ANOVA test statistic did not reach significance, there were treatment x time interaction main effects [F(1, 8) = 4.81; P < 0.001], with simple main effects during the first 10 min of the session [time 0-10 F(1, 8) = 4.12, P< 0.05; and a marginal effect at time 10-20 F(1, 8) = 3.996, P < 0.06). Lastly, a dramatic difference was observed in the stereotypy measure at this group as shown in Fig. 3c Lower (2 = 91.7, df = 1,9; P < 0.01). 25 WO 2007/001302 PCT/US2005/022955 [0078] In Figure 3, locomotor activity (crossovers; Upper) and stereotyped behavior (sniffing and rearing; Lower) after i.p. injection of cocaine after nasal immunization with GNC 92H2-pVlll (0) or RCA 60 28-pVlll (0) are shown. The figure shows the response to post-nasal immunization cocaine challenge at 10 (a), 15 (b) and 30 (c) mg/kg. Uppervalues represent means +/- SEM of 16 animals (n = 8). *, P < 0.05 ANOVA, significant difference between groups. Lower data represent the percentage of incidence of the observed behavior. *, P < 0.05. [0079] Subsequent challenges with cocaine resulted in sustained suppressive effects in some animals, albeit non-significant as a group. Figure 4 depicts the pattern of mean activity as a total, (90 min session), in a 2-within x 2 between subject's design where time (90 min), cocaine challenge day (1 or 4) in the within factor, and cocaine dose (10, 15 or 30 mg/kg) and treatment (RCA 60 28-pVlll or GNC 92H2-pVIII) are the between factors. The significance achieved in this study was time-dependent with regards to days post-phage infusion (cocaine challenge day 1 = day 4 post-infusion; cocaine challenge day 4 = day 7 post-infusion). As evidenced in Figure 4, and complementarily with Figure 3, psychomotor effects were significantly blocked by phage treatment only on the cocaine challenge day 1 for doses 10 and 15 mg/kg of the drug (left-most columns on a and b) but not 30 mg/kg (right-most columns): [Day 1/10 mg/kg, RCA 60 28-pVllI: 513.75 ± 94.29; GNC 92H2 pVIII: 317.25 ± 78.95; Day 1/15 mg/kg, RCA 60 28-pVIII: 1064.38 ± 213.52; GNC 92H2-pVlll: 550.13 + 213.52; Day 1/30 mg/kg, RCA 60 28-pVlll: 675.1 ± 222.74, GNC 92H2-pVll: 778.25 ± 225.71; cocaine challenge day 4 [Day 4/10 mg/kg, RCA 60 28 pVllI: 592.64 ± 82.51; GNC 92H2-pVlll: 622.41 ± 105.43; Day 4/15 mg/kg, RCA 60 28 pVIII: 862.25 ± 235.28; GNC 92H2-pVlll: 1391.38 ± 255.25; Day 4/30 mg/kg,

RCA

60 28-pVIII: 592.25 ± 299.5, GNC 92H2-pVll: 839.75 ± 235.77]. [0080] In Figure 4, ambulatory behavior (crossovers) elicited by increasing doses of systemic cocaine (i.p.): 10 (a), 15 (b), and 30 (c) mg/kg in a between subject design is shown. Data is represented as total mean activity ± SEM of 48 26 WO 2007/001302 PCT/US2005/022955 animals (n = 8) from cocaine challenges on days 1 and 4 after phage infusion (days 4 and 7 post-initial infusion). *, P < 0.05, ANOVA, significant difference between groups. [0081] Analysis of Filamentous Phage Found in the Brain. To investigate the capability of filamentous phage to enter the CNS, the amount of phage that accumulates, and its duration of stay in the CNS, a phage titer experiment was conducted. Thus, on days 1 through 3, 1 x 1015 phage were administered intranasally twice daily to each rat. Whole brains were removed, washed, serially diluted, and allowed to infect bacteria. Phage were counted, and all numbers reported in Table 1 are based on a total of number of four brains used per day and an average number was calculated from this total. The threshold of phage detection was 105 cfu; phage were not detected until day 3 while the highest level of phage was found on day 4. Phage-titer dropped off rapidly on day 7 but was persistent until day 13. Phagemids isolated from phage particles on days 4 and 7 were analyzed using DNA sequencing and the presence of the scFv antibody genes was confirmed, while no phage was detected on day 17, or under the same experimental conditions with the brains of rats unimmunized. Table 1. Phage titer detected in the rat brains after intranasal treatment of GNC92H2-pVll. Day* Phage titert 2 None detected 3 2.8 x 10 9 4 2.5 x 10 1 3 7 1.3 x 1010 8 4.6x 10 9 10 2.6 x 10 11 13 2.1 x 10 11 15 6.6 x 10 7 17 None detected * On days 1-3 an average of 1 x 1015 phage were administered intranasally into each rat. 27 WO 2007/001302 PCT/US2005/022955 t A total of four rat brains were used for each day examined and the titer was estimated based on the total counts divided by four. Discussion: [0082] To assess the efficacy of immunization with phage display antibodies within the CNS the psychostimulant effects of cocaine were measured in the rat. This psychostimulant effect is a dose-dependant increase in locomotor activity and stereotyped behavior as a result from cocaine's actions on dopaminergic neurons in the brain. Male Wistar rats were tested in photocell cages after treatment with i.p. cocaine (15 mg/kg) to determine pre-immunization drug response. Three different doses of cocaine were chosen: 10, 15 and 30 mg/kg. These doses of cocaine represent a broad range of both locomotor and behavioral responses. Thus, the lowest dose produces little locomotor and virtually no stereotyped behavior, where the medium dose produces a significant locomotor activation and modest stereotyped behavior and the highest dose produces less locomotor activity but more robust stereotyped behavior. [0083] Animals were administered intranasally twice per day for three consecutive days with phage displaying single chain antibodies on their pVIII surfaces and include GNC 92H2-pVlII and RCA 60 28-pVlIl (Figs. 1 & 2). The pVlll gene contains 2,800 copies as such was anticipated to provide an overall higher concentration of protein on the phage surface versus using the more common display gene pill which can only exhibit up to five copies on its surface (39). Protein surface concentration was considered to be a key element in success of our approach as the antibodies displayed were not catalytic; hence, sequestering was the only means of inhibiting cocaine from reaching its target. Monoclonal antibody GNC 92H2 has previously been shown to have excellent avidity and specificity to cocaine and has yielded outstanding results in previous passive immunization behavioral studies (13-15). RCA 60 28 is a single chain antibody that has excellent affinity (400 nM) and selectivity to RCA 60 (Ricinus communis Agglutinin, "Ricin") and thus was considered a control (36). 28 WO 2007/001302 PCT/US2005/022955 [0084] Animals received 4 consecutive daily cocaine challenges of one of three doses of the drug, 4 days after the onset of the phage-infusion regime. Intranasal administration of phage GNC 92H2-pVIII versus RCA 60 28-pVII resulted in significant psychomotor differences between groups in response to cocaine (Fig. 3). At the 10 mg/kg dose, a 30% reduction in ambulatory behavior (crossovers) compared to baseline values was observed in the GNC 92H2-pVIIl group but not in controls [Fig. 3(a) Upper] and this difference was reflected in the stereotypy measurement during the first 10 min of the session [Fig. 3(b) Lower]. This modest difference in ambulation is not surprising given the tenuous hypermotility elicited at this dose. Furthermore, the paucity of the observed stereotypy is consistent with the reported negligible presence of this behavior at the low dose of cocaine (43). In contrast, a marked 47% decrease in locomotor activity was measured in GNC 92H2 pVlll-treated animals versus baseline values, while controls increased their overall responses by 11% [Fig. 3(b) Upper]. This quantitative trend was also observed in the percent stereotypy displayed by this 15 mg/kg-treated group, where the behavior was rated in controls up to 70 min in to the session, as opposed to 50 min in the GNC 92H2-pVlll group [Fig. 3 (b) Lower]. These results bear a striking similarity to those previously reported by our group using both active immunization with two different cocaine conjugates (13-15) and passive immunization with the mAb GNC 92H2 (15). This similarity probably is contingent upon two main experimental factors. First, the same cocaine dose was used, therefore, the patterns of hyperactivity are congruent. Second, the cocaine-blocking mechanism, albeit central versus peripheral, still obeys an immune-mediated dynamic, which is subject to the same elements of affinity and titer surmountability. This so-called element of surmountability is most evident in the data depicted by Figure 3 (c). At the 30 mg/kg cocaine dose, a reversal of behavioral profile was obtained, whereby control animals showed diminishing levels of locomotion compared to both their own baseline values and GNC 92H2-pVIllI-treated rats during the first 30 min of the session [Fig. 3 (c) Upper]. Interestingly, stereotyped behavior was sustained by controls significantly longer and at higher percentages than by the GNC 92H2-pVIl group [Fig. 3 (c) 29 WO 2007/001302 PCT/US2005/022955 Lower] reflecting the typical emergence of increased levels of this measure at the higher doses of cocaine (43). Therefore, the apparent absence of a blunting effect in locomotor activity in the GNC 92H2-pVll-treated animals versus controls may instead be interpreted as an absence of group differences by virtue of a decrease in ambulation by control animals as their repetitive (stereotypic) behavior increased and endured. [0085] To confirm the basis of the behavioral suppression we determined the presence of phage particles in the brain before, during and after the time span of animal behavioral studies (Table 1). The earliest time-point at which phage were observed in the brain was at day 2, while the highest titer of phage observed in the brain was at day 4. We note that high phage titers dropped precipitously from day 5 to day 7 (103), but was relatively constant at this number until day 15 and was not detected by day 17. Thus, upon subsequent challenges ie day 4, there were no significant differences in either motor measure between groups. Figure 4 provides comparative analysis between RCA 60 28- and GNC 92H2-pVII-treated animals at day 4-post infusion-regime completion. Although no statistical significances were reached, the large error bars at the 2 higher doses denote sustained hypermotor suppression in 3 out of the 8 GNC 92H2-pVIII-treated rats [Fig. 4 (b, c)]. It would thus appear that a threshold of phage displaying protein must be present in the brain for a full-blunted behavioral response to be observed. We anticipate that this amount may be reduced if catalytic proteins for the drug of abuse are displayed on the appropriate gene's surface; this will be a basis of future research from our laboratories. [0086] In understanding the role of our nasal vaccine, we felt it was important to investigate potential limitations. The CNS is considered an immune privileged site, however, the possibility of phage entering the periphery cannot be ruled-out. Filamentous phage in itself, and with displayed proteins on its surface, comprises a foreign entity to the immune system. Additionally, there is a growing body of research wherein nasal vaccination has become increasingly popular (44, 45). 30 WO 2007/001302 PCT/US2005/022955 Gratifyingly, ELISA analysis of rat serum from vaccinated animals showed no appreciable titer to phage and thus provides further evidence that potential toxic side effects are not being manifested in animals that were administered filamentous phage (32, 34). [0087] We have shown a promising new strategy in the continuing effort to find effective treatments for cocaine addiction. While previous protein-based treatments have relied on peripheral drug-protein interactions, our new approach delivers the therapeutic protein agent directly into the CNS, the site of drug action. Thus, convergence of phage display and immunopharmacotherapy has enabled us to investigate for the first time how a protein-based therapeutic acting within the CNS can influence the effects of cocaine in animal models. Future investigations will include the combination of this phage-based approach with either passive or active immunization protocols to determine whether any synergistic benefits can be obtained. Other tantalizing scenarios might comprise the display of two different proteins of interest on the phage using one protein to target the phage to a specific area of the brain, while the other protein provides the actual therapeutic function, effectively increasing the concentration of the therapeutic protein in specific regions in the CNS. The application of this new protein-based treatment for cocaine abuse may also serve as a therapeutic for other drug abuse syndromes as well as any xenobiotic intoxication in which areas of the CNS are targeted. However, this technique is neither limited to antibodies nor to the treatment of cocaine addiction. It is of general significance for the delivery of proteins to the central nervous system. 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K., Lukas, S. E., Kamien, J. B., Mendelson, J. H., Drieze, J. & Cone, E. J. (1992) J. Pharmaco. Exper. Therapeutics 260, 1185-1193. 11. Arndt, I. O., Dorozynsky, L., Woody, G. E., McLellan, A. T. & O'Brien, C. P. (1992) Arch. Gen. Psychiatry 49, 888-893. 12. Cashman, J. R. (1997) NIDA Research Monograph 173, 225-58. 13. Carrera, M. R. A., Ashley, J. A., Parsons, L. H., Wirsching, P., Koob, G. F. & Janda, K. D. (1995) Nature 378, 727-730. 14. Carrera, M. R. A., Ashley, J. A., Zhou, B., Wirsching, P., Koob, G. F. & Janda, K. D. (2000) Proc. Natl. Acad. Sci. USA 97, 6202-6206. 15. Carrera, M. R. A., Ashley, J. A., Wirsching, P., Koob, G. F. & Janda, K. D. (2001). Proc. Natl. Acad. Sci. USA 98, 1988-1992. 16. Fox, B. S., Kantak, K. M., Edwards, M. A., Black, K. M., Bollinger, B. K., Botka, A. J., French, T. L. Thompson, T. L., Schad, V. C. Greenstein, J. L., et al. (1996) Nature Med. 2, 1129-1132. 32 WO 2007/001302 PCT/US2005/022955 17. Kantak, K. M., Collins, S. L., Lipman, E. G., Bond, J., Giovanoni, K. & Fox, B. S. (2000) Psychopharm. 148, 251-262. 18. Landry, D. W., Zhao, K., Yang, G. X.-P., Glickman, M. & Georgiadis, T. M. (1993) Science 259, 1899-1901. 19. Cashman, J. R., Berkman, C. E. & Underiner, G. E. (2000) J. Pharm. And Experimental Ther. 293, 952-961. 20. Yang, G., Chun, J., Arakawa,-Uramoto, H., Wang, X., Gawinowicz, M. A., Zhao, K. & Landry, D.W. (1996) J. Am. Chem. Soc. 118, 5880-5890. 21. Baird, T. J., Deng, S-X, Landry, D. W., Winger, G., & Woods, J. H. (2000) J. Pharmacol. Exp. Ther. 295,1127-1134. 22. Matsushita, M., Hoffman, T. Z., Ashley, J. A., Zhou, B., Wirsching, P., & Janda, K. D. (2001) Bioorg. Med. Chem. Lett. 11, 87-90. 23. Isomura, S., Hoffman, T. Z., Wirsching, P., & Janda, K. D. (2002) J. Amer. Chem. Soc. 124, 3661-3668. 24. Meijler, M. M., Matsushita, M., Wirsching, P. & Janda, K. D. Curr. Drug Discov. Tech. (2004) 1, 77-89. 25. Gorelick, D. A. (1997) Drug Alcohol Dependence 48, 159-165. 26. Mattes, C. E., Belendiuk, G. W., Lynch, T. J., Brady, R. O. & Dretchen, K. L. (1998) Addiction Biology 3, 171-188. 27. Nachon, F., Nicolet, Y., Viquie, N., Masson, P., Fontecilla-Camps, J. D. & Lockridge, O. (2002) Eur. J. Biochem. 269, 630-637. 28. Mattes, C. E., Lynch, T. J., Singh, A., Bradley, R. M., Kellaris, P. A., Brady, R. O. & Dretchen, K. L. (1997) Tox. Applied Pharm. 145, 372-380. 29. Sun, H., Pang, Y.-P., Lockridge, O. & Brimijoin, S. (2002) Mol. Pharmacol. 62, 220-224. 30. Larocca, D., Burg, M. A., Jensen-Pergakes, K., Ravey, E. P., Gonzales, A. M. & Baird, A. (2002) Curr. Pharm. Biotech 3, 45-57. 31. Smith, G. P. (1985) Science 228, 1315-1317. 32. Pasqualini, R. & Ruoslahti, E. (1996) Nature, 380, 364-366. 33. Essler, M. & Ruoslahti, E. (2002) Proc. Natl. Acad. Sci. USA 99, 2252 2257. 33 WO 2007/001302 PCT/US2005/022955 34. Frenkel, D. & Solomon, B. (2002) Proc. Natl. Acad. Sci. USA 99, 5675 5679. 35. Moss, J. A., Coyle, A. R., Ahn, J.-M., Meijier, M. M., Offer, J. & Janda, K. D. (2003) J. Immun. Meth. 281, 143-148. 36. Gao, C., Mao, S., Lo, C.-H. L., Wirsching, P., Lerner, R. A. & Janda, K. D. (1999) Proc. Natl. Acad. Sci. USA 96, 6025-6030. 37. Gao, C., Mao, S., Kaufmann, G. F., Wirsching, P., Lerner, R. A. & Janda, K. D. (2002) Proc. Natl. Acad. Sci. USA 99,12612-12616. 38. Gao, C., Lin, C.-H., Lo, C.-H. L, Mao, S., Wirsching P., Lerner, R. A. & Janda, K. D. (1997) Proc. Natl. Acad. Sci. USA 94, 11777-11782. 39. Barbas, C. F., Burton, D. R., Scott, J. K. & Silverman G. J. (2001) Phage display: A laboratory manual. Cold Spring Harbor Lab. Press; Plainview, NY. 40. Fray, P. J., Sahakian, B. J., Robbins, T. W., Koob, G. F. & Iversen, S. D. (1980) Psychopharmacology 69,153-539. 41. Robbins, T. W. (1977) in Handbook of Psychopharmacology, eds. Iversen, L., Iversen, S. D. & Snyder, S. (Plenum, New York), pp. 37-82. 42. Kullback, S. (1968) Information Therory and Statistics (Dover, New York). 43. Lyon, M. & Robbins, T. W. (1975) in Current Developments in Psychopharmacology Vol. 2, eds Essman, W. & Valzelli, L. (Spectrum, New York), pp. 89-163. 44. Illum, L. & Davis, S. S. (2001) Adv. Drug Deliv. Rev. 51, 1-3. 45. Jones, N. (2001) Adv. Drug Deliv. Rev. 51, 5-19. Example 2 Delivery of Active Cocaine Esterase Enzyme to the Central Nervous System [0089] Cocaine is a powerful stimulant and may be the most reinforcing of all drugs. Consequently, the abuse of cocaine continues to be a major societal and health problem. A myriad of medical problems, including death, often accompany cocaine use and the association of the drug with the spread of AIDS is of concern 34 WO 2007/001302 PCT/US2005/022955 (1). Cocaine acts as an indirect dopamine agonist by blocking the dopamine transporter in the pleasure/reward center of the brain (2). This obstruction leads to an excess of dopamine in the synapses, amplifying pleasure sensation. Despite intensive effort, there is yet no generally available and effective pharmacology for cocaine abuse (3). The inherent difficulties in antagonizing a blocker have led to the development of protein-based therapeutics designed to treat cocaine abuse. In one approach (4, 5), anti-cocaine antibodies have been shown to sequester cocaine, retarding its ability to enter the CNS, in an approach termed immonopharmacotherapy. A parallel strategy utilizes catalytic antibodies that are specific for the hydrolysis of the benzoyl ester of cocaine to give the non psychoactive products benzoate and methyl ecgonine (Figure 5) (6). While the potential of this method has been demonstrated in rodent models of cocaine overdose and reinforcement, the kinetic constants of these antibodies must be improved to be a viable clinical treatment (6a, 7). Alternatively, potential enzymatic therapeutics have been explored and include butyrylcholinesterase (BChe), the major cocaine-metabolizing enzyme present in the plasma of humans and other mammals (8), and the bacterial enzyme cocaine esterase (cocE) (9). The efficacy of any protein-based cocaine treatment is limited by the inability of these proteins to access the CNS. Thus, their success depends on peripheral contact between the protein and ingested cocaine. [0090] An improved treatment would contact cocaine both in circulation as well as within the CNS. Filamentous bacteriophages with foreign proteins displayed on their surfaces are able to penetrate the CNS of mice after various routes of administration (e.g., intravenous, intraperitoneal, intranasal, intramuscular) and can be administered multiple times without visible toxic effects (10). Furthermore, bacteriophage can also diffuse into a wide variety of peripheral organs including the lung, kidney, spleen, liver, and intestine (11). The genetic flexibility of filamentous bacteriophage allows for a wide variety of protein, including antibodies, as well as peptides, to be displayed on the protein phage coat in a methodology known as phage display (12). Filamentous bacteriophage fd (Figure 1), as well as its close 35 WO 2007/001302 PCT/US2005/022955 relatives M13 and fl, can be produced in high titer in bacterial culture, making production simple and economical. The therapeutic potential of a phage-displayed cocaine-binding antibody has been shown (13). However, due to the requisite 1:1 stoichiometry of any traditional antibody pharmacology, it is difficult to obtain a meaningful concentration of the therapeutic agent in vivo. However, another approach is to use a phage-displayed catalytic protein, i.e., an enzyme, as a cocaine therapeutic. Accordingly, this Example describes the preparation and kinetics of the first catalytic phage-displayed therapeutic with suitable rates of activity to treat cocaine addiction. [0091] Cocaine esterase is a globular, 574-amino-acid bacterial enzyme with a molecular weight of ~65 kDa and is the most efficient protein catalyst for the hydrolysis of cocaine characterized to date (9). The specificity rate constant of this enzyme (kcat/Km) is 10 3 -fold higher than BChE, and 10 5 -fold and 10 6 -fold faster than catalytic antibodies 15A10 (14) and GNL3A6 (6a), respectively. The size and catalytic efficiency of cocE make it an ideal candidate for an improved cocaine therapy. However, an exogenous bacterial enzyme would be rapidly cleared by proteolysis and immune surveillance. Also, available protein would not be able to enter the CNS, limiting its efficacy. Bacteriophage, on the other hand, readily enter the bloodstream and cross the blood-brain barrier (11) and are stable to a variety of harsh conditions, such as extremes of pH and treatment with nucleases and proteolytic enzymes. Furthermore, the immune response against filamentous bacteriophage is generally slow (11, 13). Thus, displaying cocE on the phage surface may overcome the inherent disadvantages of the natural enzyme and endow it with more favorable immuno/proteodynamics. [0092] Expression of cocE was performed using protein III (pill) and protein IX (plX) of the phage coat. These ~42 kDa and ~3.7 kDa proteins, respectively, are expressed in three to five copies on opposite ends of the phage (Figure 1). These proteins were chosen because they could best accommodate a protein of the size of cocE, in contrast to major coat protein pVlll. CocE was expressed on phage by 36 WO 2007/001302 PCT/US2005/022955 ligating the vector pCocE between two flanking Sfil restriction sites on phagemid pCGMT for cocE-pill (16) or pCGMT9 for cocE-plX (17, 18). Escherichia colicells were transformed with either phagemid and then infected with VCSM13 helper phage. After incubation and centrifugation, the pellet was resuspended in bacterial media and the culture grown at 280C. Since both phage and cocE expression are temperature-sensitive, 280C was chosen as a compromise between optimal phage growth (370C) and cocE expression (240C). Under these conditions, both cocE-pIll and cocE-plX were reproducibly grown in high titers (~1011-1 012 cfu/mL) with consistent cocaine hydrolysis activity. [0093] The rate of hydrolysis for cocE-plIlI and cocE-plX was measured by monitoring the increase in benzoic acid concentration over time by reversed-phase HPLC. zBoth cocE-plII and cocE-PIX displayed classic Michaelis-Menten steady state kinetics (Table 2). Estimated values of kcat and kcat/Km are reported as ranges, assuming an average of between 0.1-5 copies of cocE per phage particle. While five copies of cocE per phage is the theoretical maximum, the lower limit of the range is a more reasonable estimate based on previous reports (19). It is encouraging to note that based on the activity of the wild-type enzyme, no less than 10% of the phage displayed an average of one copy of cocE. Furthermore, the enzymatic activity of the phage does not depend on the coat protein on which cocE is expressed. Therefore, there is no interference of the enzyme due to the local conditions of the phage, such as antagonistic effects from the tethering protein or nearby pVI or pVII coat proteins. In both cases, however, cocE-pIII and cocE-plX are less active than the natural enzyme, primarily due to a 10 3 -fold reduction in apparent Km. While we can exclude local phenomena on the phage surface, the reduced activity may be caused by phage itself. It is more likely that the reduction in kinetic parameters is due to misfolded enzyme, as phage expression requires higher temperatures than that for cocE. Indeed, expression of native cocE at higher temperature (370C) gave good yield of protein, but with little activity (data not shown). Identical to the native enzyme, cocE-phage is also able to hydrolyze cocaethylene (9). However, due to 37 WO 2007/001302 PCT/US2005/022955 the extremely poor solubility of this substrate, it was impossible to determine the kinetic parameters for this reaction. Table 2 Kinetic Parameters for cocE Enzymes Catalyst K. (p4 M) kafonintx) ,/ (MV ) cocE-plEX 586 ± 63 415-8.3 11.8 x 10'.2 x 10' cccE-pmI 412 ± 43 181-3.6 7.3 x 10-0.1 x 10: cccE 0.64 ± 0.02 468 ± 6 12 ± 0.04 x 10 7 ' 'See Supporting Information for procedures of kinetic experiments. "Apparent K . Estimated range of km or kdJI based on the possibility of 0.1-5 copies of cocE displayed per phage particle. ralues taken from ref. 9. [0094] Assuming the frequency of cocE incorporation relative to native phage is low (i.e., the lower estimate is accurate), the koat of cocE-phage approaches that of the natural enzyme. In this case, cocE-plX achieves a therapeutically relevant kcat/Km (~104 M-' s1) (6a); importantly, this value is greater than that of any known catalytic anti-cocaine antibodies and only recently obtained by a designed mutant BChE (20). [0095] While the relevance of phage-displayed cocE in vivo has not been examined in this Example, unlike Example 1, these results demonstrate a potential method for catalytic cocaine degradation in both the CNS and the periphery possessing both suitable kinetic parameters and pharmacological profile for mammalian administration. 38 WO 2007/001302 PCT/US2005/022955 References [0096] The following references are used for Example 2 only. (1) (a) Brody, S. L.; Slovis, C. M.; Wrenn, K. D., Am. J. Med. 1990, 88, 325 331. (b) Des Jarlais, D. C.; Frieland, S. R., Science 1989, 245, 578. (2) (a) Ritz, M. C.; Lamb, R. C.; Goldberg, S. R.; Kuhar, M. J., Science 1987, 237, 1219-1223. (b) Withers, N. W.; Pulvirenti, L.; Koob, G. F.; Gillin, J. C., J. Clin. Psychopharmacol. 1995, 15, 63-78. (3) Mendelson, J. H.; Mello, N. K., New Eng/. J. Med. 1996, 334, 965-972. (4) (a) Carrera, M. R. A.; Ashley, J. A.; Parsons, L. H.; Wirching, P.; Koob, G. F.; Janda, K. D., Nature 1995, 378, 727-730. (b) Carrera, M. R. A.; Ashley, J. A.; Zhou, B. Wirsching, P.; Koob, G. F.; Janda, K. D., Proc. Natl. Acad. Sci. USA 2000, 97, 6202-6206. (c) Carrera, M. R. A.; Ashley, J. A.; Wirsching, P.; Koob, G. F.; Janda, K. D., Proc. Natl. Acad. Sci USA 2001, 98, 1988-1992. (d) Carrera, M. R. A.; Trigo, J. M.; Roberts, A. J.; Janda, K. D., Pharmacol. Biochem, Behav. 2005, in press. (5) (a) Fox, B. S.; Kantak, K. M.; Edwards, M. A.; Black, K. M.; Bollinger, B. K.; Botka, A. J.; French, T. L.; Thompson, T. L.; Schad, V. C.; Greenstein, J. L.; Gefter, M. L.; Exley, M. A.; Swain, P. A.; Bringer, T. J., Nat. Med. 1996, 2,1129-1132. (b) Kantak, K. M.; Collins, S. L.; Lipman, E. G.; Bond, J. Giovanoni, K.; Fox, B. S., Psychopharmacology2000, 148, 251-262. (6) (a) Matsushita, M.; Hoffman, T. Z.; Ashley, J. A.; Zhou, B.; Wirsching, P.; Janda, K. D., Bioorg. Med. Chem. Lett. 2001, 11, 87-90. (b) Landry, D. W.; Zhao, K.; Yang, G. X.Q.; Glickman, M.; Georgiadis, T. M.; Science 1993, 259, 1899-1901. (c) Cashman, J. R.; Berkman, C. E.; Underiner, G. 39 WO 2007/001302 PCT/US2005/022955 E., J. Pharm. Exp. Ther. 2000, 293, 952-961. (d) Baird, T. J.; Deng, S.-X; Landry, D. W.; Winger, G.; Woods, J. H., J. Pharmacol. Exp. Ther. 2000, 295, 1127-1134. (7) Meijler, M. M.; Matsushita, M.; Wirsching, P.; Janda, K. D., Curr. Drug Discovery TechnoL. 2004, 1, 77-89. (8) (a) Nachon, F.; Nicolet, Y.; Viquie, N.; Masson, P.; Fontecilla-Camps, J. C.; Lockridge, O., Eur. J. Biochem. 2002, 269, 630-637. (b) Mattes, C. E.; Lynch, T. J.; Singh, A.; Bradely, R. M.; Kellaris, P. A.; Brady, R. O.; Dretchen, K. L., Tox. Appl. Pharmacol. 1997, 145, 372-380. (c) Sun, H.; Pang, Y.-P; Lockridge, O.; Brimijoin, S. MoL. Pharmacol. 2002, 62, 220 224. (9) (a) Bresler, M. M.; Rosser, S. J.; Basran, A.; Bruce, N. C., AppL. Environ. MicrobioL. 2000, 66, 904-908. (b) Larsen, N. A.; Turner, J. M.; Stevens, J.; Rosser, S. J.; Basran, A.; Lerner, R. A.; Bruce, N. C.; Wilson, I. A., Nat. Struct. BioL. 2002, 9, 17-21. (c) Turner, J. M.; Larsen, N. A.; Basran, A.; Barbas, C. F., III; Bruce, N. C.; Wilson, I. A.; Lerner, R. A., Biochemistry, 2002, 41, 12297-12307. (10) Frenkel, D.; Solomon, B., Proc. Natl. Acad. Sci. USA 2002, 99, 5675-5679. (11) Dabrowska, K.; Switala-Jelen, K.; Opolski, A.; Weber-Dabrowska, B.; Gorski, A., J. App. Microbiol. 2005, 98, 7-13. (12) Smith, G. P. Science 1985, 228,1315-1317. (13) Carrera, M. R. A.; Kaufmann, G. F.; Mee, J. M.; Meijler, M. M.; Koob, G. F.; Janda, K. D., Proc. Natl. Acad. Sci. USA 2004, 101, 10416-10421. 40 WO 2007/001302 PCT/US2005/022955 (14) Deng, S. X.; De Prada, P.; Winger, G.; Landry, D.W., J. Immunol. Methods 2002, 269, 299-310. (15) Larocca, D.; Burg, M. A.; Jensen-Pergakes, K.; Ravey, E. P.; Gonzales, A. M.; Baird, A., Curr. Pharm, Biotechnol. 2002, 3, 45-57. (16) Gao, C.; Mao, S.; Lo, C.-H. L.; Wirsching, P.; Lerner, R. A.; Janda, K. D., Proc. Nat/. Acad. ScL. USA 1997, 94, 11777-11782. (17) Gao, C.; Mao, S.; Kaufmann, G. F.; Wirsching, P.; Lerner, R. A.; Janda, K. D., Proc. Natl. Acad. Sci. USA 1999, 96, 6025-6030. (18) Gao, C.; Mao, S.; Lo, C.-H. L.; Wirsching, P.; Lerner, R. A.; Janda, K. D., Proc. Natl. Acad. Sci. USA 1997, 94, 11777-11782. (19) Baek, H.; Suk, K.-H.; Kim, Y.-H; Cha, S., Nucleic Acids Res. 2002, 30, el18. (20) Sun. H.; Pang, Y.-P.; Lockridge, O.; Brimijoin, S., Mol. Pharmacol. 2002, 62, 220-224. INDUSTRIAL APPLICABILITY [0097] Methods and compositions according to the present invention possess industrial applicability, as such methods and compositions are useful for the delivery of proteins to the central nervous system. The compositions in and of themselves have industrial applicability because of the activity of the proteins contained in the compositions, or because of the ability of nucleic acid constructs to be expressed to produce proteins. 41 WO 2007/001302 PCT/US2005/022955 ADVANTAGES OF THE INVENTION [0098] Methods and compositions according to the present invention provide a greatly improved way to deliver active proteins to the central nervous system. Such methods and compositions enable the delivery of a wide range of proteins to the central nervous system, including antibodies, enzymes, receptor proteins, ligands to receptor proteins, regulatory proteins, and membrane proteins. The methods and compositions enable delivery of such proteins despite the blood-brain barrier and also enable delivery of such proteins without generating significant immune responses or other side effects. The ability to prepare high titers of filamentous bacteriophage allows the rapid preparation of large quantities of bacteriophage carrying the desired protein for administration. [0099] Methods and compositions according to the present invention allow the delivery of proteins to the central nervous system in active form without degradation or denaturation. [0100] Methods and compositions according to the present invention also allow the delivery of proteins to the central nervous system without creating an immune response or toxicity. Bacteriophage administration is well tolerated. [0101] The inventions illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the future shown and described or any portion thereof, and it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, 42 WO 2007/001302 PCT/US2005/022955 modification and variation of the inventions herein disclosed can be resorted by those skilled in the art, and that such modifications and variations are considered to be within the scope of the inventions disclosed herein. The inventions have been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the scope of the generic disclosure also form part of these inventions. This includes the generic description of each invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised materials specifically resided therein. [0102] In addition, where features or aspects of an invention are described in terms of the Markush group, those schooled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. It is also to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of in the art upon reviewing the above description. The scope of the invention should therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. The disclosures of all articles and references, including patent publications, are incorporated herein by reference. REMAINDER OF PAGE LEFT INTENTIONALLY BLANK 43

Claims (45)

1. A method of delivering a protein to the central nervous system in active form comprising the steps of: (a) preparing a single-stranded filamentous phage vector comprising a nucleic acid construct in which a protein to be delivered to the central nervous system is encoded as a fusion protein with a coat protein of a filamentous phage; (b) preparing phage particles incorporating the nucleic acid construct as the phage genome and in which the fusion protein is expressed as a coat protein; and (c) delivering the phage particles to a mammal by a route such that the phage particles reach the central nervous system so that the protein is delivered to the central nervous system in active form.

2. The method of claim 1 wherein the single-stranded filamentous bacteriophage vector is derived from a bacteriophage selected from the group of M13, fd, and fl.

3. The method of claim 2 wherein the bacteriophage is M13.

4. The method of claim 1 wherein the filamentous phage vector is a phagemid.

5. The method of claim 4 wherein the phagemid is pCGMT or a derivative of pCGMT.

6. The method of claim 1 wherein the step of preparing phage particles incorporating the nucleic acid construct as the phage genome and in which the fusion protein is expressed as a coat protein comprises: (i) transforming a bacterial host cell with a phagemid incorporating the nucleic acid construct; and 44 WO 2007/001302 PCT/US2005/022955 (ii) producing phage particles by infection with a helper virus.

7. The method of claim 3 wherein the coat protein that is incorporated into the fusion protein is selected from the group consisting of pill, pVll, pVIII, and plX.

8. The method of claim 7 wherein the coat protein is pill.

9. The method of claim 7 wherein the coat protein is pVlll.

10. The method of claim 7 wherein the coat protein is plX.

11. The method of claim 1 wherein the protein is monomeric, homidimeric, or homomultimeric.

12. The method of claim 11 wherein the protein is monomeric.

13. The method of claim 1 wherein the protein is selected from the group consisting of an antibody, an enzyme, a reporter protein, a receptor protein, a ligand for a receptor protein, a regulatory protein, and a membrane protein.

14. The method of claim 13 wherein the protein is an enzyme.

15. The method of claim 14 wherein the enzyme is an enzyme hydrolyzing cocaine.

16. The method of claim 15 wherein the enzyme is selected from the group consisting of bacterial cocaine esterase and butyrylcholineresterase. 45 WO 2007/001302 PCT/US2005/022955

17. The method of claim 14 wherein the enzyme is selected from the group consisting of an enzyme having a therapeutic effect and an enzyme that replaces missing or diminished cellular enzymatic activity.

18. The method of claim 13 wherein the protein is an antibody.

19. The method of claim 18 wherein the antibody is a single-chain antibody selected from the group consisting of scFv or Fab' antibodies.

20. The method of claim 19 wherein the antibody is a scFv antibody.

21. The method of claim 1 wherein the protein is a mutein.

22. The method of claim 1 wherein the protein is a fusion protein.

23. The method of claim 1 wherein the protein is delivered by a route selected from the group consisting of intranasal delivery, intravenous delivery, intraperitoneal delivery, and intramuscular delivery.

24. The method of claim 23 wherein the protein is delivered by intranasal delivery.

25. The method of claim 1 wherein the mammal is a human.

26. The method of claim 1 wherein-the mammal is a socially or economically important non-human mammal selected from the group consisting of a dog, a cat, a horse, a cow, a sheep, a goat, a rat, a mouse, and a rabbit.

27. The method of claim 1 wherein the delivery of the protein treats a disease or condition that is affected by the protein to be delivered. 46 WO 2007/001302 PCT/US2005/022955

28. A nucleic acid construct comprising: (a) an origin of replication of a filamentous bacteriophage; (b) a nucleic acid framework allowing replication of the construct into circular single-stranded DNA molecules operably linked to the origin of replication; and (c) at least one nucleic acid sequence encoding a fusion protein such that the fusion protein can be expressed during replication of the construct and assembled into chimeric bacteriophage particles.

29. The nucleic acid construct of claim 28 wherein the origin of replication of the filamentous bacteriophage is selected from the group consisting of the origins of replication of M13, fd, and fl.

30. The nucleic acid construct of claim 29 wherein the origin of replication is the origin of replication of M13.

31. The nucleic acid construct of claim 28 wherein the fusion protein includes at least one domain having an activity selected from the group consisting of antibody activity, enzymatic activity, reporter protein activity, receptor protein activity, ligand activity for a receptor protein, regulatory protein activity, and membrane protein activity.

32. A vector comprising the nucleic acid construct of claim 28.

33. Host cells transformed or transfected with the vector of claim 32.

34. A bacteriophage particle displaying a fusion protein comprising: (a) a single-stranded DNA molecule; and (b) at least one fusion protein including: (i) a coat protein of a single-stranded filamentous bacteriophage; and 47 WO 2007/001302 PCT/US2005/022955 (ii) a protein to be delivered.

35. The bacteriophage particle displaying a fusion protein of claim 34 wherein the single-stranded filamentous bacteriophage is M13.

36. The bacteriophage particle displaying a fusion protein of claim 35 wherein the coat protein is pVIll.

37. The bacteriophage particle displaying a fusion protein of claim 34 wherein the protein to be delivered is selected from the group consisting of protein is selected from the group consisting of an antibody, an enzyme, a reporter protein, a receptor protein, a ligand for a receptor protein, a regulatory protein, and a membrane protein.

38. A pharmaceutical composition comprising: (a) the bacteriophage particle displaying a fusion protein of claim 34; and (b) a pharmaceutically acceptable carrier.

39. The pharmaceutical composition of claim 38 wherein the pharmaceutical composition is formulated for intranasal delivery, intravenous delivery, intraperitoneal delivery, or intramuscular delivery.

40. The pharmaceutical composition of claim 39 wherein the pharmaceutical composition is formulated for intranasal delivery.

41. A fusion protein comprising: (a) a first domain that is pVll protein of a filamentous bacteriophage; and (b) a second domain that is a protein that is deliverable to the central nervous system of a mammal. 48 WO 2007/001302 PCT/US2005/022955

42. The fusion protein of claim 41 wherein the first domain and the second domain are linked so that they are expressed in one polypeptide without a linker.

43. The fusion protein of claim 41 wherein the fusion protein further comprises a linker between the first domain and the second domain.

44. The fusion protein of claim 41 wherein the protein that is deliverable to the central nervous system of a mammal is selected from the group consisting of an antibody, an enzyme, a reporter protein, a receptor protein, a ligand for a receptor protein, a regulatory protein, and a membrane protein.

45. The fusion protein of claim 41 wherein the filamentous bacteriophage is M13. 49