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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
  • C12Y301/01008—Cholinesterase (3.1.1.8), i.e. butyrylcholine-esterase
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
  • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2795/00—Bacteriophages
  • C12N2795/00011—Details
  • C12N2795/14011—Details ssDNA Bacteriophages
  • C12N2795/14111—Inoviridae
  • C12N2795/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2795/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• 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.
• the delivery of active proteins to the central nervous system is desired in many diagnostic or therapeutic applications.
• 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.
• BBB blood-brain barrier
• 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 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.
• 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.
• the problem posed by the blood-brain barrier is that, in the process of protecting the brain, it excludes many potentially useful therapeutic agents.
• 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.
• 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 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.
• 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.
• One aspect of the invention is a method of delivering a protein to the central nervous system in active form comprising the steps of:
• the single-stranded filamentous bacteriophage vector is derived from a bacteriophage selected from the group of M 13, fd, and f1.
• the bacteriophage is M13.
• the filamentous phage vector is a phagemid.
• the coat protein that is incorporated into the fusion protein is selected from the group consisting of pill, pVII, pVIII, and plX. More typically, the coat protein is pVIII.
• 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.
• the protein is delivered by a route selected from the group consisting of intranasal delivery, intravenous delivery, intraperitoneal delivery, and intramuscular delivery.
• the protein is delivered by intranasal delivery.
• nucleic acid construct comprising:
• a bacteriophage particle displaying a fusion protein comprising:
• composition comprising:
• Still another aspect of the invention is a fusion protein comprising:
• Figure 1 is a diagram of filamentous bacteriophage fd architecture (this applies equally well to M 13 and f1 , which are closely related).
• 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.
• Figure 3 is a graph showing locomotor activity (crossovers; Uppe ⁇ and stereotyped behavior (sniffing and rearing; Lower) after i.p. injection of cocaine after nasal immunization with GNC 92H2-pVIII (•) or RCA 60 28-pVIII (D), at 10 mg/kg, 15 mg/kg, and 30 mg/kg of cocaine.
• Figure 4 is a graph showing Ambulatory behavior (crossovers) elicited by increasing doses of systemic cocaine (i.p.): 10 (a), 15 (h), 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.
• Figure 5 is a scheme showing the action of a catalytic antibody that hydrolyzes the benzoyl ester of cocaine into benzoate and methylecgonine.
• the present invention is directed to methods and compositions that can deliver proteins to the central nervous system in active form.
• One aspect of the present invention is a method of delivering a protein to the central nervous system in active form.
• this method comprises the steps of:
• 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;
• the single-stranded filamentous bacteriophage vector is preferably derived from a bacteriophage selected from the group of M13, fd, and f1.
• a particularly preferred single-stranded filamentous phage is M13.
• M13, fd, and f1 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.
• 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.
• the gene Il 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 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.
• 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.
• the coat protein that is incorporated into the fusion protein is typically one of pill, pVII, pVIII, and plX.
• pVIII is the major coat protein and offers the potential of expressing up to 2800 copies/phage particle.
• steric constraints can lead to a preference for use of another coat protein, such as pill or plX.
• phage display 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.
• 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.
• 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.
• the protein can be a chain of an 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.
• 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.
• 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.
• 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 a subunit of hexosaminidase is lacking, or Gaucher's disease, in which the enzyme glucocerebroside ⁇ -glucosidase is lacking. Other enzymes can also be delivered.
• 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
• 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.
• 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.
• the protein can be a fusion protein between an antibody and a protein toxin, which can be administered for therapeutic purposes
• a preferred route of delivery is intranasal delivery.
• other delivery routes such as intravenous, intraperitoneal, or intramuscular delivery, can be used.
• 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.
• Methods according to the present invention can be used for diagnostic or therapeutic purposes as described above.
• the method is typically used for diagnostic purposes, or to monitor the effect of another therapeutic method or process.
• 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.
• 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.
• 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.
• 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 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).
• 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.
• 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.
• protein includes “conservative variants” with structures and activity that substantially correspond to the protein to be delivered.
• 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) structure and/or activity, e.g., antibody activity, enzymatic activity, or receptor activity.
• 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.
• amino acids having similar properties e.g., acidic, basic, positively or negatively charged, polar or non- polar, etc.
• Conservative substitution tables providing functionally similar amino acids are well known in the art.
• 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; lie/Leu or VaI; Leu/lle or VaI; Lys/Arg or GIn or GIu; Met/Leu or Tyr or He; Phe/Met or Leu or Tyr; Ser/Thr; Thr/Ser; Trp/Tyr; Tyr/Trp or Phe; Val/lle 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 GIu); (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 VaI); and (6) phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W or Trp); (see also, e.g., Creighton (1984) Proteins, W.
• 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.
• 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.
• nucleic acid sequences 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.
• 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.
• 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” (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)).
• nucleic acid construct comprising:
• 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.
• 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.
• 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 & JJ. Pasternak, "Molecular Biotechnology: Principles and Applications of Recombinant DNA" (2d ed., ASM Press, Washington, 1998), pp. 74-75, incorporated herein by this reference.
• the fusion protein encoded by the nucleic acid sequence is as described above.
• the filamentous bacteriophage is M13.
• Still another aspect of the invention is a bacteriophage particle displaying a fusion protein.
• the bacteriophage particle comprises:
• the fusion protein including: (a) a coat protein of a single- stranded filamentous bacteriophage; and (b) a protein to be delivered.
• the fusion protein is as described above. If the bacteriophage is M13, the coat protein is preferably the major coat protein, pVIII.
• Yet another aspect of the present invention is a pharmaceutical composition
• a pharmaceutical composition comprising:
• 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.
• the pharmaceutical composition can be formulated for intranasal delivery, intravenous delivery, intraperitoneal delivery, or intramuscular delivery as described above. Typically, intranasal delivery is preferred.
• Still another aspect of the present invention is a fusion protein comprising:
• the first domain and the second domain are linked so that they are expressed in one polypeptide without a linker.
• 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 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.
• 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 antidepressants 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.
• 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.
• phage display 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 ).
• GNC 92H2-pVIII phage-displayed protein
• 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 6 o28 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 Biolabs), and ligated into pCGMT-p8. DNA sequencing was used to confirm the new construct.
• E. coli TG 1 cells (Stratagene) were transformed with the phagemid encoding the appropriate scFv antibody.
• E. coli TG 1 cultures were grown in 2 x 0.5 L of 2YT broth in the presence of the antibiotic carbenicillin (100 ⁇ g/mL).
• the cells were infected with 0.5 ml_ of VCSM13 helper phage (Stratagene) (10 12 pfu/mL). After 30 min incubation at room temperature, the culture was grown for 2 h at 30 0 C.
• Kanamycin/IPTG were then added to a final concentration of 70 ⁇ g/mL, and the culture was grown overnight at 30 0 C. 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 ⁇ m 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).
• 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 membrane was discarded and from each well 100 ⁇ l_ 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 26 6 nm - A32 0 nm) using a molar extinction coefficient of 1.006 x 10 4 M "1 cm '1 , and a genome size of 3722 bases for the modified phage.
• PBS pH 7.4
• 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.
• 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.
• 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.
• mice 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 6 o28-pVIII or GNC 92H2-pVIII, the anti-cocaine mAb- displaying phage.
• Cocaine doses ranged from 10, 15 and 30 mg/kg i.p.
• animals were challenged with their corresponding dose of cocaine at 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.
• 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 posteriori test. Stereotyped behavior data were analyzed by a likelihood ratio method, the "information statistic" (41 , 42).
• ANOVA analysis of variance
• GNC 92H2-pVIII Affinity for Cocaine.
• the affinity of GNC 92H2-pVIII 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-pVIII with VCS M 13 helper phage and with RCA 60 28-pVIII; the latter two phage constructs in theory are not expected to bind cocaine.
• phage GNC 92H2- pVIII clearly binds cocaine, whereas control phage do not.
• Figure 3 shows the psychomotor response to cocaine after the intranasal phage administration regime.
• 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 6 o28-pVIII or GNC 92H2-pVIII) are the between factors.
• FIG 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.
• the threshold of phage detection was 10 5 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.
• 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 6 o28 is a single chain antibody that has excellent affinity (400 nM) and selectivity to RCA 6 O ⁇ Ricinus communis Agglutinin, "Ricin") and thus was considered a control (36).
• 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 6 o28-pVIII resulted in significant psychomotor differences between groups in response to cocaine (Fig. 3).
• 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.
• 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.
• 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 (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.
• 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).
• 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 relatives M13 and f1 , 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).
• a phage-displayed catalytic protein i.e., an enzyme
• this Example describes the preparation and kinetics of the first catalytic phage-displayed therapeutic with suitable rates of activity to treat cocaine addiction.
• 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 (k cat /K m ) 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.
• 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.
• CocE was expressed 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 pVIII.
• CocE was expressed on phage by ligating the vector pCocE between two flanking Sfi ⁇ restriction sites on phagemid pCGMT for cocE-plll (16) or pCGMT9 for cocE-plX (17, 18).
• Escherichia coli cells 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 28 0 C. Since both phage and cocE expression are temperature-sensitive, 28 0 C was chosen as a compromise between optimal phage growth (37°C) and cocE expression (24°C). Under these conditions, both cocE-plll and cocE-plX were reproducibly grown in high titers ( ⁇ 10 11 -10 12 cfu/mL) with consistent cocaine hydrolysis activity.
• cocE-plX achieves a therapeutically relevant kca t /K m (-104 M "1 s "1 ) (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).
• 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.
• 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.
• 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.
• 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.

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