WO2000045850A2 - Drug delivery vehicle - Google Patents

Drug delivery vehicle Download PDF

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Publication number
WO2000045850A2
WO2000045850A2 PCT/US2000/003045 US0003045W WO0045850A2 WO 2000045850 A2 WO2000045850 A2 WO 2000045850A2 US 0003045 W US0003045 W US 0003045W WO 0045850 A2 WO0045850 A2 WO 0045850A2
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WO
WIPO (PCT)
Prior art keywords
protein
drug
vehicle
release
uptake
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PCT/US2000/003045
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French (fr)
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WO2000045850A3 (en
WO2000045850A9 (en
Inventor
Gary Calton
Michael Kulka
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Aurx Inc.
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Priority to AU29831/00A priority Critical patent/AU2983100A/en
Publication of WO2000045850A2 publication Critical patent/WO2000045850A2/en
Publication of WO2000045850A3 publication Critical patent/WO2000045850A3/en
Publication of WO2000045850A9 publication Critical patent/WO2000045850A9/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell

Definitions

  • the present invention relates to a drug delivery vehicle comprising a protein sequence for influenza virus hemagglutinin which is covalently attached to a ligand which will bind to a receptor which is further covalently or ionically attached to a non- protein drug.
  • the vehicle can also contain a lysine containing peptide so to provide additional drug attachment sites and/or an intranuclear localized signal peptide so as to enhance intranuclear localization of the nucleic acid.
  • Drugs are used to treat diseases but often fail to reach or enter the cell or cell type in which the diseased state is present. This is especially true in the treatment of viral infections and cancer.
  • Such delivery systems have often been attempted with antibodies, however, the immune response and the lack of penetration of the drug or toxin has been low.
  • Liposomes have often been used but lack specificity of delivery. Therefore, there has been a desire in the art for the development of effective delivery systems which allow for intracellular bio-availability of drug molecules.
  • endosomolytic peptides for gene delivery has been the subject of a number of patents including US Patent 5,547,932, for transfecting eucaryotic cells; WO 94/04696 for introducing proteins or nucleotide sequences into the nucleus; and WO95/28494 for gene transduction in target cells.
  • An object of the present invention is to provide a novel drug delivery vehicle.
  • Another object of the present invention is to provide a DNA construct encoding a drug delivery vehicle.
  • Still another object of the invention is to provide a method of intracellular delivery of drug molecules using the drug delivery vehicle.
  • DDVE drug delivery vehicle
  • the endosomolytic protein is the hemagglutinin of influenza virus or a mutant thereof containing at least a 90% sequence homology and retaining the endosomolytic activity (HA).
  • the DDVE also comprises: (iii) a covalently attached peptide containing one to 100 lysines for the attachment site of the drug.
  • the peptide is a lysyl-leucyl peptide.
  • the lysyl-leucyl peptide has 1 to 100 repeating units.
  • the DDVE also comprises: (iv) an intranuclear localized signal peptide.
  • the above-described objects of the present invention have been met by a DNA construct encoding a DDVE, thus providing an amide linkage for the method of covalent attachment.
  • the above-described objects of the present invention have been met by a method of delivery of drugs comprising contacting cells with a DDVE.
  • the present invention relates to a DDVE comprising:
  • an endosomolytic protein covalently attached to (ii) a ligand which will bind to a receptor, and (iii) a drug bound ionically or covalently to (i) or (ii).
  • the ligand is for targeting the cell surface, while the HA causes endosomolysis and increases membrane permeability for the drug.
  • Any endosomolytic protein could be used for insertion of the vehicle including those of influenza virus hemagglutinin, G protein of visicular stomatitus virus, rhinovirus, (P28 of targeted genetics), exotoxin A of Pseudomonas aeruginosa, streptolysin O, bacterial cytolysins, the entry protein of Mycobacterium tuberculosis, the internalin protein of Listeria monocytogenes, the invasin protein of Yersinia enterocolitica, gp36 from mouse mammary tumor virus, gp37 from Rous sarcoma virus, pl5E from Moloney murine leukemia virus, gp20 from Mason Pfizer monkey virus, gp41 from human immunodeficiency virus, gp21 from human T-Cell leukemia virus, retrovirus transmembrane proteins, and lentivirus transmembrane proteins or synthetic analogs thereof.
  • the DDVE also comprises:
  • a lysine containing peptide (L) m where L is lysine and m is 1 to 100.
  • the (L) m peptide provides additional sites for attachment of the drug molecule via an appropriate linker.
  • the value of m is not critical to the present invention, but generally represents from 1 to 100 lysine residues and preferably from 1 to 20 lysine residues.
  • a preferred peptide is a lysyl-leucyl (KL) repeating unit with 1 to 100 KL units and preferably 1 to 20 KL units (with the number of repeats being designated by the subscript m).
  • the DDVE also comprises:
  • NLS intranuclear localized signal
  • the orientation of the elements in the DDVE is not critical to the present invention.
  • the DDVE can contain the protein covalently linked via peptide bonds in any sequence.
  • the mechanism of action of the DDVE involves increased intracellular delivery of the drug molecules, and increased release of the drugs from endocytic-like vesicles in which they are entrapped during intracellular delivery.
  • the particular drug is not critical to the present invention.
  • examples of such drugs include daunomycin, doxarubicin, VP16, teniposide, paclitaxel, docetaxel, vincristine, vinblastine, and tretinoin.
  • Other drugs which would benefit from site directed delivery due to solubility, toxicity, or insufficient cell selectivity would be advantageously delivered by the DDVE.
  • DDVE design and versatility enables variation of its construction in order to address these important issues. This includes: (i) increasing the flexibility of the molecule by insertion of a variable number of Gly residues adjacent to the HA region of the DDVE,
  • ligands which can bind specifically to cell surface receptors may be covalently or ionically cross-linked to DDVE so as to alter/increase the binding specificity and uptake of the DDVE with the target cell/tissue.
  • Cross -linking can be effected by any one of a variety of commercially available cross-linking/derivatizing agents and procedures, such as those shown in the Pierce catalog (Pierce Chemical Co., Rockford, IL).
  • the particular protein ligand employed is not critical to the present invention.
  • the ligand sequence for a receptor may be any such protein sequence such as, epidermal growth factor (EGF) which will bind to EGF receptor which receptors are often present in high abundance on certain tumor cells.
  • EGF epidermal growth factor
  • Other examples of such protein ligands include transferrin, cholera toxin B subunit, Adenoviral penton base protein.
  • Still other protein ligands which react, for instance, with platelet derived growth factor, transforming growth factor- ⁇ , transforming growth factor- ⁇ , basic fibroblast growth factor, c-erbB- 1 , c-erb-2, hepatocyte growth factor, epidermal growth factor receptor, p21-ras-related proteins, ras, p62/64 protein of c-myc, myc, mutant p53 or a ligand reacting with the gene abl, erbB l, erbB-2, gip, gsp, myc, L-myc, N-myc, H-ras, N-ras, ret, ros, K-sam, sis, src, and trk or any other cell marker may also be used.
  • Drug molecules may be bound to the uptake and release protein ionically or covalently.
  • the drug is covalently bound at a site located on the protein. Suitable binding sites include, but are not limited to, amino, carboxy and thio sites.
  • the drug is covalently bound to an epsilon amino group of a lysine contained with the protein.
  • the drug is bound covalently to a thiol group of a cystine contained within the drug uptake and release protein.
  • the drug may be attached in the DDVE by means of an appropriate linker, e.g. an alcohol linkage, an amine linkage, a carboxylic acid linkage or a thiol linkage.
  • an appropriate linker e.g. an alcohol linkage, an amine linkage, a carboxylic acid linkage or a thiol linkage.
  • the present invention also provides a method of intracellular delivery of therapeutic drug molecules comprising contacting cells with a DDVE.
  • the DDVE can be administered topically either proximal and/or distal to the site of disease, to skin, mucous membranes and/or eye in the absence or presence of creams/ointments/lipid carriers, e.g., polyethylene glycol or liposomes, designed to facilitate complex uptake and/or stability at the site of topical application and/or disease.
  • creams/ointments/lipid carriers e.g., polyethylene glycol or liposomes
  • the DDVE can also be administered intradermally either proximal and/or distal to the site of disease in the presence of buffered physiologic saline and/or other solutions containing (or not) lipid carriers designed to facilitate complex uptake and/or stability at the site of injection and/or disease.
  • the DDVE can also be administered subcutaneously either proximal and/or distal to the site of disease in the presence of buffered physiologic saline and/or other solutions containing (or not) lipid carriers designed to facilitate complex uptake and/or stability at the site of injection and/or disease.
  • the DDVE can also be administered intramuscularly either proximal and/or distal to the site of disease in the presence of buffered physiologic saline and/or other solutions containing (or not) lipid carriers designed to facilitate complex uptake and/or stability at the site of injection and/or disease.
  • the DDVE can also be administered intravenously by injection and/or infused intravenously either proximal and/or distal to the site of disease in the presence of buffered physiologic saline and/or other solutions containing (or not) lipid carriers designed to facilitate complex uptake and/or stability at the site of injection and/or disease.
  • the DDVE can also be administered nasally or orally by inhalation and/or ingestion either proximal or distal to the site of disease either contained within (or not) biodegradable capsules in the presence of buffered physiologic saline and/or other solutions containing (or not) lipid carriers designed to facilitate complex uptake and/or stability at the tissue/organ of administration and/or disease.
  • the amount of DDVE to be administered will vary depending upon the age, weight, sex, and species of the subject (cells), as well as the disease to be treated and the drug molecule to be used. However, typically, the DDVE will be administered in an amount of from about 0.1 nmole to 10,000 ⁇ moles, preferably from about 0.1 nmole to 1 ,000 ⁇ mole.
  • DDVE Construction of PBP containing DDVE
  • a DDVE was constructed by assembly of DNA sequences encoding: (i) the adenovirus type 2 penton base protein (PBP), which binds the cell surface receptors-integrin and (ii) an endosomolytic peptide derived from influenza virus HA. More specifically, a dsDNA encoding the HA endosomolytic peptide (20 amino acids) plus 5' and 3' flanking sequences (Gly) were generated by PCR amplification of a primary single-stranded 70 nucleotide (nt) DNA sequence.
  • PBP adenovirus type 2 penton base protein
  • nt flanking sequence
  • the Gly residues permit formation of alpha-helices flanking the HA peptide (Gly 2 -HA-Gly 2 ), thereby imparting flexibility to the secondary structure at the HA-penton base junction.
  • the sequence for Gly 2 -HA-Gly 2 was first cloned into pGEMT (Promega, Madison, WI), a PCR cloning vector.
  • the resulting plasmid, PETllaHA/PB codes for a chimeric protein that begins with Gly 2 -HA-Gly 2 (initiator Met is encoded by vector), followed by expression vector and penton base protein coding sequences. Positive clones were identified by sequencing.
  • pETl laHA/PB was subjected to collapse ligation at the Ndel located upstream of the penton base protein initiator Met.
  • the resulting plasmid pETl la ⁇ HA/PB codes for a chimeric protein that begins with an initiator Met, Ala, Ser and Thr all encoded by vector followed by the penton base protein coding sequence. Positive clones were identified by sequencing.More specific details as to the construction of the above DDVE are set forth below.
  • the Ad2 penton base protein reading frame begins pETl laPB, which also encodes a four-amino acid extension (Met-Ala-Ser-Thr) at the N-terminal of the Ad2 penton base protein, and continues with the first amino acid (Met) of the Ad2 penton base protein sequence. All of the signals for transcriptional and translational regulation of the Ad2 penton base protein gene are present within pETllaPB.
  • This plasmid also contains an ampicillin resistance gene for positive (bacterial transformation) selection, as well as sequences important for its growth and replication in E. coli.
  • the HA Peptide A DNA molecule encoding the influenza HA peptide was synthesized and then cloned into pETllaPB.
  • dsDNA which encodes the 20 amino acid HA plus, 5' and 3' flanking sequences that provide the nucleotides suitable for cloning into either pETl la or pETllaPB, as well as encoding for HA flanking glycine residues (Gly 2 -HA-Gly 2 ).
  • the dsDNA was generated by PCR amplification of the following primary single-stranded 70 bp DNA sequence for HA: 5-GAGGTGGACTCTTCGAAGCAATTGCAGGTTTAATCGAAAACGGCTGGGA AGGCATGATCGACGGTGGTGG-3' (SEQ ID NO: 1 ), using the following sense and anti-sense primers, respectively:
  • primers each contain seven 5' terminal nucleotides designed to encode for 2 glycines upon ligation into the Ndel site of pETl laPB.
  • the dsDNA was ligated into pGEMT according to manufacturer's (Promega, Madison, WI) recommended procedures and transformed into E. coli DH5 ⁇ (Life Technologies) followed by growth selected on culture media containing ampicillin and 5-bromo-4-chloro-3-indoyl- ⁇ -D- galactose (X-GAL). Growth selection of pGEMT transformed DH5 ⁇ on this culture media yields blue colonies. Disruption of lacZ expression in pGEMT via insertion/ligation of a PCR product can result in the formation of white colonies on this media.
  • Plasmid DNA was extracted from white colonies (putative positive recombinants) and subjected to secondary screening using Ndel and Mfel restriction enzyme analysis. Positive clones (pGEMT HA) were subjected to ds DNA sequencing using universal primer which targets vector sequences upstream of the insert.
  • the Gly -HA-Gly encoding ds DNA sequence was removed from pGEMT by Ndel restriction digestion and cloned into Ndel digested pETl laPB to give plasmid pETl laHA/PB.
  • the Ndel cleavage site in pETl laPB overlaps the vector encoded initiation (Met) codon.
  • pETl laHA/PB encodes for a fusion protein that begins with a start Met followed by I) Gly 2 -HA-Gly 2 , ii) His, Met, Ala, Ser and Thr residues encoded by the expression vector and iii) the Ad2 penton base protein coding sequence.
  • E. coli DH5 ⁇ was transformed with PET 1 1 aHA/PB, and growth selected on Amp containing culture media. Plasmid DNA was extracted from isolated colonies, and subjected to initial screening (for positive recombinants) using Ndel restriction enzyme analysis. Positive recombinants were subjected to a secondary screening using Mfel and Nhel restriction analysis in order to confirm the correct orientation of Gly 2 -HA-Gly sequence within pETl laHA/PB. The Mfel restriction site is present in the HA encoding sequence and the Nhel site is located 5' adjacent to the AD2 PBP sequence.
  • DDVE multiple cloning site
  • Ndel fragment encoding Gly 2 -HA-Gly 2 from pGEMT/HA was cloned into the Ndel site of pETl la to give plasmid pETl laHA.
  • This plasmid was further modified through restriction digestion with BamHI and Nhel, which cut downstream of the Gly 2 -HA-Gly 2 encoding sequence, both sites made blunt-ended followed by a collapse ligation of these sites.
  • the unique HindDI site in this plasmid was deleted through a subsequent modification involving digestion with Hindm followed by mung bean nuclease treatment and blunt-end ligation of this site to generate plasmid pETl laHA+.
  • This cloning strategy creates a single site (BamHI) downstream of Gly 2 -HA-Gly 2 for ligation/insertion of the MCS.
  • a ds DNA molecule was prepared which contains endonuclease cleavage sites for several enzymes including Bgi ⁇ , Xhol, Eel 136m, Pmll, Ascl, Ncol, Sail, Bsrgl and BamHI as well as encoding for translational stop codons in all three reading frames.
  • the ds DNA was generated by PCR amplification of the following primary single-stranded 54 bp DNA sequence 5' - CTCGAGCTCACGTGGCGCGCCATGGTCGACTGTACAGGATCCTAACTAGGT AAG-3' (SEQ ID 4) using the following sense and antisense primers, respectively 5'- AGATCTCTCGAGCTCACGTGGCGCGC 3' (SEQ ID 5) and 5'- AGATCTCTTACCTAGTTAGGATCCTG-3' (SEQ ID 6).
  • the dsDNA was ligated into pGEMT according to manufacturer's recommended procedures and protocols and transformed into DH5 ⁇ and growth selected on culture media containing ampicillin and 5-Bromo-4-chloro-3-indoyl- ⁇ -D-galactose (X-GAL). Growth selection of pGEMT transformed DH5 ⁇ on this culture media yields blue colonies. Disruption of lacZ expression in pGEMT via insertion/ligation of a PCR product can result in the formation of white colonies on this media. Plasmid DNA was extracted from white colonies (putative positive recombinants) and subjected to secondary screening using BamHI restriction enzyme analysis which cuts only once (in the MCS) in a positive recombinant.
  • pGEMT/MCS Positive clones
  • pGEMT/MCS Positive clones
  • the MCS was removed from pGEMT/MCS and cloned into the unique BamHI site of pETl laHA+ to create plasmid pETl laHAmcs.
  • flanking glycine residues may be varied according to the length of the primary nucleotide sequences which flank the HA encoding region.
  • the Gly 2 -HA-Gly sequence may be extended at the 5' and 3' ends by PCR amplification using sense and anti-sense primers that contain terminal sequences which encode for additional glycines.
  • the strategy used to clone Gly contest-HA-Glyking sequences (where n > 2) into pGEMT, pETllaPB and pETl laHAmcs and confirm the success of the cloning is similar to that used for Gly 2 -HA-Gly 2 .
  • Another DDVE was constructed to include 1 (KL) ⁇ o unit for which details are set forth below.
  • a polylysylleucyl (KL) ⁇ o was constructed that was suitable for cloning in-frame with the Ad2 penton base protein coding sequence of PETl 1 aHA/PB.
  • DNA molecule encoding (KL) ⁇ o was prepared using the following sense and anti-sense primers, respectively:
  • the KLio encoding fragment was removed from pGEMT/KL by Sall/Bgl ⁇ digestion and cloned into the Sall/BamHI sites in the MCS of pETl laHAmcs to give plasmid pETl laHA/KL. Transformation into DH5 and growth selection on Amp containing media followed by HindlH restriction analysis was used to identify positive clones. Final confirmation through ds DNA sequencing provided nucleotide sequence confirmation. Cloning the Sall Bgi ⁇ fragment into pETl laHAmcs affects a BsrGI to Acc65I change downstream of the Sail site in the MCS.
  • the KLio encoding fragment may be cloned into pETl laHAmcs as a BamHI/Bgl ⁇ fragment thereby retaining the Bsrgl site within the MCS.
  • the utility of this alternate cloning strategy is to provide the potential for generating translational frame-dependent expression of a Cys codon (present in a BfrGI but not Acc65I recognition sequence) upstream of the translational stop codons.
  • a Cys amino acid can provide an alternative to Lys as a site for derivatization/complexation of drug moieties. 13
  • pETl laHA/PBP/KL was created to contain DNA sequences encoding in frame for Gly 2 -HA-Gly , PBP and KL i0 .
  • This DNA sequence was constructed by cloning the PBP sequence from pETl laPBP in pETl laHA/KL using a two-step strategy.
  • the 1.6kb Mfel/Ascl fragment of pETl laPB which encodes the carboxyl region of Gly 2 -HA-Gly and the coding sequence of PBP from amino acids 1 through 507, was cloned directionally into Mfel/Ascl digested pETl laHA KL to create plasmid pETl laHA/PBpart/KL.
  • Recombinants were growth selected on Amp containing media and positive recombinants identified by restriction analysis with BamHI wherein positives recombinant plasmids are cut twice with this enzyme.
  • a ds DNA PBP sequence encoding amino acids 495 to 571 was amplified by PCR using the following sense and antisense primers 5'- CGTGTTCAATCGCTTTCCCGAGAA-3' (SEQ ID 10) and 5'- GTCGACAAAAGTGCGGCTCGATAGGACG-3' ( ID 1 1), respectively.
  • the antisense primer contains six 5' terminal nucleotides designed to create a Sail restriction site 3' adjacent to the codon for PBP amino acid 571 with concomitant elimination of the translational stop codon.
  • the dsDNA was ligated into pGEMT according to manufacturer's recommended procedures and protocols and transformed into DH5 ⁇ and growth selected on culture media containing ampicillin and 5-Bromo-4-chloro-3-indoyl- ⁇ -D-galactose (X-GAL). Growth selection of pGEMT transformed DH5 ⁇ on this culture media yields blue colonies. Disruption of lacZ expression in pGEMT via insertion/ligation of a PCR product can result in the formation of white colonies on this media. Plasmid DNA was extracted from white colonies (putative positive recombinants) and subjected to secondary screening using Ascl restriction enzyme analysis. Positive clones (pGEMT/PBterm) were subjected to ds DNA sequencing using universal primer which targets vector sequences upstream of the insert.
  • PGEMT/PBterm was digested with Ascl and Sail and the 204bp fragment encoding PBP amino acids 507 to 571 cloned into the Ascl/Sall sites of pETl laHA/PBpart/KL to generate pETl laHA/PB/KL.
  • E. Coli DH5 was transformed with this recombinant and growth selected on Amp containing culture media. Plasmid DNA is extracted from isolated colonies, and subjected to screening (for positive recombinants) using BamHI restriction enzyme analysis. Positive clones are subjected to ds DNA sequencing using the universal forward primer to confirm nucleotide sequence.
  • pETl laHA/PB/KL encodes for a fusion protein that begins with a Met followed by i)Gly 2 -HA-Gly 2 , ii) His, Met, Ala, Ser, and Thr residues encoded by vector sequences, iii) the Ad2 penton base protein, iv) Val, Asp, Gly, Thr, Gly and Ser encoded by the MCS, v) KLio and vi) end terminal amino acids Arg and Ser. C2.
  • the EGF Protein A DNA molecule encoding the mature EGF protein plus an additional amino acid, Ala, was first produced as two individual fragments and then joined to one complete molecule.
  • the complete molecule was then cloned into pETl laHAmcs and pETl laHA/KL. While encoding for the mature EGF sequence, the complete DNA sequence was designed, based on codon degeneracy, to have a primary sequence different from that of the native sequence.
  • a dsDNA molecule was prepared which encodes for Ala followed by the first 25 amino acids of EGF plus 5' flanking sequences that provide the nucleotides suitable for cloning into the appropriate site in vectors such as pETl laHAmcs or pETHA/KL and 3' flanking sequences that provide for cloning (ligation) adjacent with the second EGF fragment (below).
  • the dsDNA was generated by PCR amplification of the following primary single-stranded 73 bp DNA sequence: 5'-GCCAACTCAGATTCAGAATGTCCACTGTCACACGATGGCTACTGCCTCCA TGACGGAGTGTGCATGTATATCG-3' (SEQ ID NO: 12), using the following sense and anti-sense primers, respectively: 5'-CTCGAGGCCAACTCAGATTCAGAATG-3' (SEQ ID NO:13); and 5'-AGGCCTCGATATACATGCACACTCCG-3' (SEQ ID NO:14).
  • the dsDNA was ligated into pGEMT according to manufacturer's recommended procedures and protocols and transformed into DH5 ⁇ and growth selected on culture media containing ampicillin and 5-Bromo-4-chloro-3-indoyl- ⁇ -D- galactose (X-GAL). Growth selection of pGEMT transformed DH5 ⁇ on this culture media yields blue colonies. Disruption of lacZ expression in pGEMT via insertion/ligation of a PCR product can result in the formation of white colonies on this media. Plasmid DNA was extracted from white colonies (putative positive recombinants) and subjected to secondary screening using Xhol and Pstl restriction enzyme analysis.
  • pGEMT/EGFl Positive clones
  • pGEMT/EGFl Positive clones
  • a dsDNA molecule was prepared which encodes for amino acids 25 to 53 of EGF plus 5' flanking sequences that provide the nucleotides suitable for cloning
  • the dsDNA was generated by PCR amplification of the following primary single-stranded 83 bp DNA sequence: 5'-TGGACAAATACGCATGCAACTGTGTTGTTGGATACATCGGCGAACGATGT CAATACCGCGATCTGAAATGGTGGGAACTGCGA-3' (SEQ ID NO: 15), using the following sense and annti-sense primers, respectively: 5'-AGGCCTTGGACAAATACGCATGCAAC-3' (SEQ ID NO: 16); and 5'-GTCGACTCGCAGTTCCCACCATTTCA-3' (SEQ ID NO: 17).
  • the dsDNA was ligated into pGEMT according to manufacturer's recommended procedures and protocols and transformed into DH5 and growth selected on culture media containing ampicillin and 5-Bromo-4-chloro-3-indoyl- ⁇ -D- galactose (X-GAL). Growth selection of pGEMT transformed DH5 on this culture media yields blue colonies. Disruption of lacZ expression in pGEMT via insertion/ligation of a PCR product can result in the formation of white colonies on this media. Plasmid DNA was extracted from white colonies (putative positive recombinants) and subjected to secondary screening using Sail restriction enzyme analysis. Positive clones (pGEMT/EGF2) were subjected to ds DNA sequencing using universal primer which targetsvector sequences upstream of the insert.
  • the ds DNA sequence the second EGF fragment was removed from pGEMT/EGF2 by Stul/Sall restriction digestion and cloned into the Stul Sall sites of pGEMT/EGFl .
  • the resulting recombinant plasmid pGEMT/EGF contains a novel DNA sequence which encodes for a native EGF protein plus a 5' Ala amino acid.
  • E. coli DH5 ⁇ was transformed with pGEMT/EGF, and growth selected on Amp containing culture media. Plasmid DNA was extracted from isolated colonies, and subjected to initial screening (for positive recombinants) using Xhol/Ndel restriction enzyme analysis. Positive recombinants were subjected to a secondary screening using Xhol Stul and Xhol/Sall restricition analysis in order to screen for the presence of one complete copy of EGF encoding DNA sequence within the vector. Positive clones were subjected to double-stranded sequencing using the universal forward primers described-above to confirm the nucleotide sequence of the EGF encoding DNA.
  • pET 1 1 aHA/EGF was created to contain a DNA sequence which encodes inframe for Gly 2 -HA-Gly 2 and EGF.
  • the Ala-EGF encoding dsDNA sequence was removed from pGEMT/EGF by Xhol/Sall restriction digestion and ligated into the Xhol/Sall sites of pETl laHAmcs to give pETl 1 aHA EGF.
  • E. coli DH1 was transformed with putative recombinant, pETl 1 aHA EGF, and growth selected on Amp containing culture media. Plasmid DNA was extracted from isolated colonies, and subjected to initial screening (for positive recombinants) using Xhol/Sall restriction enzyme analysis.
  • pETl 1 aHA/EGF encodes for a fusion protein that begins with Met followed by i) Gly 2 -HA-Gly 2 , (ii) His, Met, Gly, Ser, Leu and Glu encoded by vector and MCS sequences, iii) Ala followed the EGF protein and iv) Val, Asp, Cys, Thr, Gly and Ser encoded by MCS.
  • pETl laHA/EGF/KL was created to contain DNA sequences encoding for Gly 2 -HA-Gly 2 inframe with EGF followed by Lio.
  • 0 encoding fragment was removed from pGEMT/KL by Sall/Bgi ⁇ digestion and cloned into the Sall/BamHI sites in the MCS of pETl 1 aHA/EGF to give pETl laHA EGF/KL.
  • the Ala-EGF containing Xhol/Sall fragment from pGEMT/EGF could be cloned into Xhol/Sall sites of pETl 1 aHA/KL to generate the same recombinant plasmid.
  • pETl laHA/EGF/KL encodes for a fusion protein that begins with Met followed by i) Gly 2 -HA-Gly 2 ii) His, Met, Gly, Ser, Leu and Glu encoded by vector and MCS sequences, iii) Ala followed by EGF protein, iv) Val, Asp, Gly, Thr, Gly and Ser, v) KLio and vi) end terminal amino acids Arg and Ser.
  • the DDVEs were transformed in E. coli strain BL21(DE3 and the DDVE protein produced, isolated and purified as described by Bai et al, J. Virol, 67:5198-5205 (1993).
  • the insoluble fraction containing DDVE was collected by centrifugation and resuspended in buffer comprising 20 mM Tris-HCl (pH 7.5), 1.0 mM EDTA and 0.1 % (w/v) Nonidet P-40. After three additional cycles of washing, the final pellet was resuspended in a small volume of 6.0 M urea, diluted with 9 volumes of buffer comprising 50 mM K 2 PO 4 (pH 10.7), 50 mM NaCl, 1.0 mM EDTA, and dialyzed against phosphate buffered saline (PBS) and then 50 mM phosphate buffer (pH 7.5).
  • buffer comprising 20 mM Tris-HCl (pH 7.5), 1.0 mM EDTA and 0.1 % (w/v) Nonidet P-40. After three additional cycles of washing, the final pellet was resuspended in a small volume of 6.0 M urea, diluted with 9 volumes of buffer compris
  • the resulting dialyzate was sterilized by filtration and stored at 4°C or frozen at -70°C.
  • the purity of the DDVE protein was confirmed by SDS-PAGE and Coomassie staining. Only one protein band was observed, representing the purified DDVE. A similar band was seen for DDVE- ⁇ HA.
  • Example 1 was linked to doxarubicin via disulfosuccimidyl tartrate (Pierce Chemical, Rockford, IL).
  • DDVE Intracellular Delivery of DDVE Delivered Drug Molecules
  • the DDVE lacking the (KL) m and a drug, but having HA and PBP (Pro- DDVEl) was tested for intracellular uptake and localization.
  • Pro-DDVEl is internalized by HeLa and Vero cells which differ in the levels of cell surface receptors (A549 > HeLa > Vero). Internal ization is dose dependent as evidenced by staining with anti-PBP antibody (100% staining cells at 60 min and 15 min of exposure to 80 and 800 nM of Pro-DDVEl protein respectively). Internalized Pro-DDVEl persists for 24 hrs in A549 cells (stained after Pro-DDVEl removal) but only for 10 and 2 hrs in HeLa and Vero cells respectively. Staining is cytoplasmic. It is primarily diffuse, indicative of protein release from endocytic vesicles.
  • Pro-DDVEl lacking HA is similarly internalized and processed except that its intracellular distribution is primarily granular, indicative of sequestration within cytoplasmic granules consistent with endocytic vesicles.
  • a weak diffuse component is consistent with a low level of endosomolysis . This shows that the Pro-DDVEl functions endosomolytically.
  • A431 cells which express the EGF receptor, were grown overnight at 37° C in DMEM-10% FBS supplemented with ImM glutamine to approximately 60% confluency. The growth media was removed and the monolayers washed once with CMF-PBS. The monolayers were incubated for 5, 10 or 30 minutes at 37° C with EGF-free media containing either 0.3, 1.5 or 3ug/ml of partially purified HA-EGF. Control monolayers were incubated in EGF-free media in the absence of HA-EGF. The incubation media was removed, the monolayers washed three times with CMF-PBS and fixed in acetone.
  • MOLECULE TYPE synthetic
  • HYPOTHETICAL NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 1 : GAGGTGGACT CTTCGAAGCA ATTGCAGGTT TAATCGAAAA CGGCTGGGAA GGCATGATCG ACGGTGGTGG 70

Abstract

A drug delivery vehicle comprising a protein sequence for a receptor and an influenza virus hemagglutinin peptide is disclosed, which optionally may also comprise a polylysylleucyl peptide, as well as an intranuclear localized signal. The vehicle can be utilized to enhance the delivery of drug molecules.

Description

DRUG DELIVERY VEHICLE
CROSS-REFERENCE TO RELATED APPLICATION
None
FIELD OF THE INVENTION The present invention relates to a drug delivery vehicle comprising a protein sequence for influenza virus hemagglutinin which is covalently attached to a ligand which will bind to a receptor which is further covalently or ionically attached to a non- protein drug. In a preferred embodiment, the vehicle can also contain a lysine containing peptide so to provide additional drug attachment sites and/or an intranuclear localized signal peptide so as to enhance intranuclear localization of the nucleic acid.
BACKGROUND OF THE INVENTION Drugs are used to treat diseases but often fail to reach or enter the cell or cell type in which the diseased state is present. This is especially true in the treatment of viral infections and cancer. Such delivery systems have often been attempted with antibodies, however, the immune response and the lack of penetration of the drug or toxin has been low. Liposomes have often been used but lack specificity of delivery. Therefore, there has been a desire in the art for the development of effective delivery systems which allow for intracellular bio-availability of drug molecules. The use of endosomolytic peptides for gene delivery has been the subject of a number of patents including US Patent 5,547,932, for transfecting eucaryotic cells; WO 94/04696 for introducing proteins or nucleotide sequences into the nucleus; and WO95/28494 for gene transduction in target cells.
The use of targeting proteins has also been the subject of a number of patents including US Patent 5,736,687, for targeting a retroviral vector for gene delivery. SUMMARY OF THE INVENTION An object of the present invention is to provide a novel drug delivery vehicle. Another object of the present invention is to provide a DNA construct encoding a drug delivery vehicle.
Still another object of the invention is to provide a method of intracellular delivery of drug molecules using the drug delivery vehicle.
These and other objects of the present invention, which will be apparent from the detailed description of the invention provided hereinafter, have been met in one embodiment, by a drug delivery vehicle (hereinafter "DDVE") comprising: (i) an endosomolytic protein covalently attached to (ii) a protein sequence comprising a ligand for a receptor to provide an uptake and release protein for a drug, and (iii) a drug bound ionically or covalently to the uptake and release protein.
In a more preferred embodiment, the endosomolytic protein is the hemagglutinin of influenza virus or a mutant thereof containing at least a 90% sequence homology and retaining the endosomolytic activity (HA).
In a preferred embodiment, the DDVE also comprises: (iii) a covalently attached peptide containing one to 100 lysines for the attachment site of the drug.
In a more preferred embodiment, the peptide is a lysyl-leucyl peptide. In a still more preferred embodiment the lysyl-leucyl peptide has 1 to 100 repeating units. In yet another preferred embodiment, the DDVE also comprises: (iv) an intranuclear localized signal peptide.
In another embodiment, the above-described objects of the present invention have been met by a DNA construct encoding a DDVE, thus providing an amide linkage for the method of covalent attachment. In still another embodiment, the above-described objects of the present invention have been met by a method of delivery of drugs comprising contacting cells with a DDVE.
DETAILED DESCRIPTION OF THE INVENTION
As discussed above, in one embodiment, the present invention relates to a DDVE comprising:
(i) an endosomolytic protein covalently attached to (ii) a ligand which will bind to a receptor, and (iii) a drug bound ionically or covalently to (i) or (ii).
The ligand is for targeting the cell surface, while the HA causes endosomolysis and increases membrane permeability for the drug.
Any endosomolytic protein could be used for insertion of the vehicle including those of influenza virus hemagglutinin, G protein of visicular stomatitus virus, rhinovirus, (P28 of targeted genetics), exotoxin A of Pseudomonas aeruginosa, streptolysin O, bacterial cytolysins, the entry protein of Mycobacterium tuberculosis, the internalin protein of Listeria monocytogenes, the invasin protein of Yersinia enterocolitica, gp36 from mouse mammary tumor virus, gp37 from Rous sarcoma virus, pl5E from Moloney murine leukemia virus, gp20 from Mason Pfizer monkey virus, gp41 from human immunodeficiency virus, gp21 from human T-Cell leukemia virus, retrovirus transmembrane proteins, and lentivirus transmembrane proteins or synthetic analogs thereof.
We have found that HA is an exceptionally useful endosomolytic protein for intracellular release of the DDVE after its receptor mediated endocytosis. As discussed above, in a preferred embodiment, the DDVE also comprises:
(iv) a lysine containing peptide (L)m where L is lysine and m is 1 to 100. The (L)m peptide provides additional sites for attachment of the drug molecule via an appropriate linker. In the (L)m peptide, the value of m is not critical to the present invention, but generally represents from 1 to 100 lysine residues and preferably from 1 to 20 lysine residues. A preferred peptide is a lysyl-leucyl (KL) repeating unit with 1 to 100 KL units and preferably 1 to 20 KL units (with the number of repeats being designated by the subscript m).. In still a more preferred embodiment, the DDVE also comprises:
(v) an intranuclear localized signal peptide.
The particular intranuclear localized signal (NLS) peptide employed is not critical to the present invention. Examples of such NLS peptides include the NLS peptide of the SV40 large T antigen (Van Dermonne et al, 77RS, 21:59-64 (1996)), the NLS peptides listed in said references are incorporated by reference herein in their entirety).
The orientation of the elements in the DDVE is not critical to the present invention. For example, the DDVE can contain the protein covalently linked via peptide bonds in any sequence. The mechanism of action of the DDVE involves increased intracellular delivery of the drug molecules, and increased release of the drugs from endocytic-like vesicles in which they are entrapped during intracellular delivery.
The particular drug is not critical to the present invention. Examples of such drugs include daunomycin, doxarubicin, VP16, teniposide, paclitaxel, docetaxel, vincristine, vinblastine, and tretinoin. Other drugs which would benefit from site directed delivery due to solubility, toxicity, or insufficient cell selectivity would be advantageously delivered by the DDVE.
DDVE design and versatility enables variation of its construction in order to address these important issues. This includes: (i) increasing the flexibility of the molecule by insertion of a variable number of Gly residues adjacent to the HA region of the DDVE,
(ii) using various methods of drug linkage to DDVE in order to achieve optimal intracellular release of the drug molecule from the DDVE. Thus, in another embodiment, ligands which can bind specifically to cell surface receptors may be covalently or ionically cross-linked to DDVE so as to alter/increase the binding specificity and uptake of the DDVE with the target cell/tissue. Cross -linking can be effected by any one of a variety of commercially available cross-linking/derivatizing agents and procedures, such as those shown in the Pierce catalog (Pierce Chemical Co., Rockford, IL).
The particular protein ligand employed is not critical to the present invention. The ligand sequence for a receptor may be any such protein sequence such as, epidermal growth factor (EGF) which will bind to EGF receptor which receptors are often present in high abundance on certain tumor cells. Other examples of such protein ligands include transferrin, cholera toxin B subunit, Adenoviral penton base protein. Still other protein ligands which react, for instance, with platelet derived growth factor, transforming growth factor-α, transforming growth factor-β, basic fibroblast growth factor, c-erbB- 1 , c-erb-2, hepatocyte growth factor, epidermal growth factor receptor, p21-ras-related proteins, ras, p62/64 protein of c-myc, myc, mutant p53 or a ligand reacting with the gene abl, erbB l, erbB-2, gip, gsp, myc, L-myc, N-myc, H-ras, N-ras, ret, ros, K-sam, sis, src, and trk or any other cell marker may also be used.
Linkage of drug molecules in the DDVE is accomplished so that:
(i) levels of intracellular DDVE and drug molecules are optimized/maximized,
(ii) release of the drug molecules after intracellular penetration is optimized, and
(iii) the effect on the targeted cell is maximized.
Drug molecules may be bound to the uptake and release protein ionically or covalently. In a preferred embodiment, the drug is covalently bound at a site located on the protein. Suitable binding sites include, but are not limited to, amino, carboxy and thio sites. In one embodiment, the drug is covalently bound to an epsilon amino group of a lysine contained with the protein. In another embodiment, the drug is bound covalently to a thiol group of a cystine contained within the drug uptake and release protein.
In addition, the drug may be attached in the DDVE by means of an appropriate linker, e.g. an alcohol linkage, an amine linkage, a carboxylic acid linkage or a thiol linkage.
The present invention also provides a method of intracellular delivery of therapeutic drug molecules comprising contacting cells with a DDVE.
The particular means of administration of the DDVE to effect contacting of the cells is not critical to the present invention. For example, the DDVE can be administered topically either proximal and/or distal to the site of disease, to skin, mucous membranes and/or eye in the absence or presence of creams/ointments/lipid carriers, e.g., polyethylene glycol or liposomes, designed to facilitate complex uptake and/or stability at the site of topical application and/or disease.
The DDVE can also be administered intradermally either proximal and/or distal to the site of disease in the presence of buffered physiologic saline and/or other solutions containing (or not) lipid carriers designed to facilitate complex uptake and/or stability at the site of injection and/or disease.
The DDVE can also be administered subcutaneously either proximal and/or distal to the site of disease in the presence of buffered physiologic saline and/or other solutions containing (or not) lipid carriers designed to facilitate complex uptake and/or stability at the site of injection and/or disease.
The DDVE can also be administered intramuscularly either proximal and/or distal to the site of disease in the presence of buffered physiologic saline and/or other solutions containing (or not) lipid carriers designed to facilitate complex uptake and/or stability at the site of injection and/or disease.
The DDVE can also be administered intravenously by injection and/or infused intravenously either proximal and/or distal to the site of disease in the presence of buffered physiologic saline and/or other solutions containing (or not) lipid carriers designed to facilitate complex uptake and/or stability at the site of injection and/or disease.
The DDVE can also be administered nasally or orally by inhalation and/or ingestion either proximal or distal to the site of disease either contained within (or not) biodegradable capsules in the presence of buffered physiologic saline and/or other solutions containing (or not) lipid carriers designed to facilitate complex uptake and/or stability at the tissue/organ of administration and/or disease.
The amount of DDVE to be administered will vary depending upon the age, weight, sex, and species of the subject (cells), as well as the disease to be treated and the drug molecule to be used. However, typically, the DDVE will be administered in an amount of from about 0.1 nmole to 10,000 μmoles, preferably from about 0.1 nmole to 1 ,000 μmole.
The following examples are provided for illustrative purposes only and are in no way intended to limit the scope of the present invention.
EXAMPLE 1 Construction of PBP containing DDVE A DDVE was constructed by assembly of DNA sequences encoding: (i) the adenovirus type 2 penton base protein (PBP), which binds the cell surface receptors-integrin and (ii) an endosomolytic peptide derived from influenza virus HA. More specifically, a dsDNA encoding the HA endosomolytic peptide (20 amino acids) plus 5' and 3' flanking sequences (Gly) were generated by PCR amplification of a primary single-stranded 70 nucleotide (nt) DNA sequence. The Gly residues permit formation of alpha-helices flanking the HA peptide (Gly2-HA-Gly2), thereby imparting flexibility to the secondary structure at the HA-penton base junction. The sequence for Gly2-HA-Gly2 was first cloned into pGEMT (Promega, Madison, WI), a PCR cloning vector. The sequence for Gly2-HA-Gly was removed from the pGEMT vector and cloned into pETl laPB (Bai M, Harfe B, Freimuth P, Mutations that alter an Arg-Gly-Asp (RGD) sequence in the adenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flat cells. J. Virology , 67(9):5198-205(1993 ), a bacterial expression vector which contains the entire Ad2 penton base protein under the control of a T7 phage promoter, by ligation in the Ndel site located upstream of the penton base protein initiator Met. The resulting plasmid, PETllaHA/PB, codes for a chimeric protein that begins with Gly2-HA-Gly2 (initiator Met is encoded by vector), followed by expression vector and penton base protein coding sequences. Positive clones were identified by sequencing.
To generate DDVEΔHA, pETl laHA/PB was subjected to collapse ligation at the Ndel located upstream of the penton base protein initiator Met. The resulting plasmid pETl laΔHA/PB codes for a chimeric protein that begins with an initiator Met, Ala, Ser and Thr all encoded by vector followed by the penton base protein coding sequence. Positive clones were identified by sequencing.More specific details as to the construction of the above DDVE are set forth below.
A. The Ad2 Penton Base Protein
The Ad2 penton base protein reading frame begins pETl laPB, which also encodes a four-amino acid extension (Met-Ala-Ser-Thr) at the N-terminal of the Ad2 penton base protein, and continues with the first amino acid (Met) of the Ad2 penton base protein sequence. All of the signals for transcriptional and translational regulation of the Ad2 penton base protein gene are present within pETllaPB. This plasmid also contains an ampicillin resistance gene for positive (bacterial transformation) selection, as well as sequences important for its growth and replication in E. coli.
B. The HA Peptide A DNA molecule encoding the influenza HA peptide was synthesized and then cloned into pETllaPB.
This was accomplished by preparing a dsDNA molecule which encodes the 20 amino acid HA plus, 5' and 3' flanking sequences that provide the nucleotides suitable for cloning into either pETl la or pETllaPB, as well as encoding for HA flanking glycine residues (Gly2-HA-Gly2). The dsDNA was generated by PCR amplification of the following primary single-stranded 70 bp DNA sequence for HA: 5-GAGGTGGACTCTTCGAAGCAATTGCAGGTTTAATCGAAAACGGCTGGGA AGGCATGATCGACGGTGGTGG-3' (SEQ ID NO: 1 ), using the following sense and anti-sense primers, respectively:
5'-CATATGGGAGGTGGACTCTTCGAAGCA-3' (SEQ ID NO:2); and 5'-CATATGGCCACCACCGTCGATCATGCC-3' (SEQ ID NO: 3)
These primers each contain seven 5' terminal nucleotides designed to encode for 2 glycines upon ligation into the Ndel site of pETl laPB. The dsDNA was ligated into pGEMT according to manufacturer's (Promega, Madison, WI) recommended procedures and transformed into E. coli DH5α (Life Technologies) followed by growth selected on culture media containing ampicillin and 5-bromo-4-chloro-3-indoyl-β-D- galactose (X-GAL). Growth selection of pGEMT transformed DH5α on this culture media yields blue colonies. Disruption of lacZ expression in pGEMT via insertion/ligation of a PCR product can result in the formation of white colonies on this media. Plasmid DNA was extracted from white colonies (putative positive recombinants) and subjected to secondary screening using Ndel and Mfel restriction enzyme analysis. Positive clones (pGEMT HA) were subjected to ds DNA sequencing using universal primer which targets vector sequences upstream of the insert.
The Gly -HA-Gly encoding ds DNA sequence was removed from pGEMT by Ndel restriction digestion and cloned into Ndel digested pETl laPB to give plasmid pETl laHA/PB. The Ndel cleavage site in pETl laPB overlaps the vector encoded initiation (Met) codon. pETl laHA/PB encodes for a fusion protein that begins with a start Met followed by I) Gly2-HA-Gly2, ii) His, Met, Ala, Ser and Thr residues encoded by the expression vector and iii) the Ad2 penton base protein coding sequence.
E. coli DH5α was transformed with PET 1 1 aHA/PB, and growth selected on Amp containing culture media. Plasmid DNA was extracted from isolated colonies, and subjected to initial screening (for positive recombinants) using Ndel restriction enzyme analysis. Positive recombinants were subjected to a secondary screening using Mfel and Nhel restriction analysis in order to confirm the correct orientation of Gly2-HA-Gly sequence within pETl laHA/PB. The Mfel restriction site is present in the HA encoding sequence and the Nhel site is located 5' adjacent to the AD2 PBP sequence. Positive clones were subjected to double-stranded sequencing using the universal forward primers described-above to confirm in-frame ligation of Gly -HA-Gly2 sequence to the Ad2 penton base protein sequence in pETllaHA/PB. Another DDVE was constructed to contain a DNA molecule encoding Gly2-HA-Gly2 followed by a multiple cloning site (MCS) cloned into pETl la. The
Ndel fragment encoding Gly2-HA-Gly2 from pGEMT/HA was cloned into the Ndel site of pETl la to give plasmid pETl laHA. This plasmid was further modified through restriction digestion with BamHI and Nhel, which cut downstream of the Gly2-HA-Gly2 encoding sequence, both sites made blunt-ended followed by a collapse ligation of these sites. The unique HindDI site in this plasmid was deleted through a subsequent modification involving digestion with Hindm followed by mung bean nuclease treatment and blunt-end ligation of this site to generate plasmid pETl laHA+. This cloning strategy creates a single site (BamHI) downstream of Gly2-HA-Gly2 for ligation/insertion of the MCS. To generate the MCS, a ds DNA molecule was prepared which contains endonuclease cleavage sites for several enzymes including Bgiπ, Xhol, Eel 136m, Pmll, Ascl, Ncol, Sail, Bsrgl and BamHI as well as encoding for translational stop codons in all three reading frames. The ds DNA was generated by PCR amplification of the following primary single-stranded 54 bp DNA sequence 5' - CTCGAGCTCACGTGGCGCGCCATGGTCGACTGTACAGGATCCTAACTAGGT AAG-3' (SEQ ID 4) using the following sense and antisense primers, respectively 5'- AGATCTCTCGAGCTCACGTGGCGCGC 3' (SEQ ID 5) and 5'- AGATCTCTTACCTAGTTAGGATCCTG-3' (SEQ ID 6). The dsDNA was ligated into pGEMT according to manufacturer's recommended procedures and protocols and transformed into DH5α and growth selected on culture media containing ampicillin and 5-Bromo-4-chloro-3-indoyl-β-D-galactose (X-GAL). Growth selection of pGEMT transformed DH5α on this culture media yields blue colonies. Disruption of lacZ expression in pGEMT via insertion/ligation of a PCR product can result in the formation of white colonies on this media. Plasmid DNA was extracted from white colonies (putative positive recombinants) and subjected to secondary screening using BamHI restriction enzyme analysis which cuts only once (in the MCS) in a positive recombinant. Positive clones (pGEMT/MCS) were subjected to ds DNA sequencing using universal primer which targets vector sequences upstream of the insert. The MCS was removed from pGEMT/MCS and cloned into the unique BamHI site of pETl laHA+ to create plasmid pETl laHAmcs.
As discussed above, the number of flanking glycine residues may be varied according to the length of the primary nucleotide sequences which flank the HA encoding region. Alternatively the Gly2-HA-Gly sequence may be extended at the 5' and 3' ends by PCR amplification using sense and anti-sense primers that contain terminal sequences which encode for additional glycines. The strategy used to clone Gly„-HA-Gly„ sequences (where n > 2) into pGEMT, pETllaPB and pETl laHAmcs and confirm the success of the cloning is similar to that used for Gly2-HA-Gly2. Another DDVE was constructed to include 1 (KL)ιo unit for which details are set forth below.
C. The Polylsylleucyl Peptide
In order to generate additional attachment sites for drug molecules, a polylysylleucyl (KL)ιo was constructed that was suitable for cloning in-frame with the Ad2 penton base protein coding sequence of PETl 1 aHA/PB.
More specifically, a DNA molecule encoding (KL)ιo was prepared using the following sense and anti-sense primers, respectively:
5'-GTCGACGGTACCGGATCCAAG-3' (SEQ ID NO:7); and 5'-GAATTCAGATCTGAGCTTAAG-3' (SEQ ID NO:8) so as to PCR amplify a single-stranded 90 base DNA molecule:
5'-GTCGACGGTACCGGATCCAAGCTCAAGCTTAAACTCAAGCTTAAG CTCAAACTGAAGCTTAAGCTCAAGCTTAAGCTCAGATCTGAATTC-3' (SEQ ID NO:9), which encodes for 10 polylysylleucyl repeat sequences (identified in brackets) flanked by sequences for restriction endonuclease cleavage by Sail (bold), BamHI (italics), Bgiπ (underlined) and Hindiπ (underlined within brackets). After PCR amplification, the dsDNA was ligated into pGEMT, transformed and grown as above. Growth selection of pGEMT (no insert) transformed DH5α on this culture media yields blue colonies. Disruption of lacZ expression in pGEMT via insertion/ligation of a PCR product can result in the formation of white colonies on this media. Plasmid DNA was extracted from white colonies and subjected to secondary screening using HindDI restriction enzyme analysis. Positive clones (pGEMT/KL) were subjected to ds DNA sequencing using universal primer which targets vector sequences upstream of the insert.
The KLio encoding fragment was removed from pGEMT/KL by Sall/Bglϋ digestion and cloned into the Sall/BamHI sites in the MCS of pETl laHAmcs to give plasmid pETl laHA/KL. Transformation into DH5 and growth selection on Amp containing media followed by HindlH restriction analysis was used to identify positive clones. Final confirmation through ds DNA sequencing provided nucleotide sequence confirmation. Cloning the Sall Bgiπ fragment into pETl laHAmcs affects a BsrGI to Acc65I change downstream of the Sail site in the MCS. Alternatively, the KLio encoding fragment may be cloned into pETl laHAmcs as a BamHI/Bglϋ fragment thereby retaining the Bsrgl site within the MCS. The utility of this alternate cloning strategy is to provide the potential for generating translational frame-dependent expression of a Cys codon (present in a BfrGI but not Acc65I recognition sequence) upstream of the translational stop codons. A Cys amino acid can provide an alternative to Lys as a site for derivatization/complexation of drug moieties. 13
Another DDVE, pETl laHA/PBP/KL was created to contain DNA sequences encoding in frame for Gly2-HA-Gly , PBP and KLi0. This DNA sequence was constructed by cloning the PBP sequence from pETl laPBP in pETl laHA/KL using a two-step strategy. In the first step the 1.6kb Mfel/Ascl fragment of pETl laPB, which encodes the carboxyl region of Gly2-HA-Gly and the coding sequence of PBP from amino acids 1 through 507, was cloned directionally into Mfel/Ascl digested pETl laHA KL to create plasmid pETl laHA/PBpart/KL. Recombinants were growth selected on Amp containing media and positive recombinants identified by restriction analysis with BamHI wherein positives recombinant plasmids are cut twice with this enzyme. In the second step, a ds DNA PBP sequence encoding amino acids 495 to 571 was amplified by PCR using the following sense and antisense primers 5'- CGTGTTCAATCGCTTTCCCGAGAA-3' (SEQ ID 10) and 5'- GTCGACAAAAGTGCGGCTCGATAGGACG-3' ( ID 1 1), respectively. The antisense primer contains six 5' terminal nucleotides designed to create a Sail restriction site 3' adjacent to the codon for PBP amino acid 571 with concomitant elimination of the translational stop codon. The dsDNA was ligated into pGEMT according to manufacturer's recommended procedures and protocols and transformed into DH5α and growth selected on culture media containing ampicillin and 5-Bromo-4-chloro-3-indoyl- β-D-galactose (X-GAL). Growth selection of pGEMT transformed DH5α on this culture media yields blue colonies. Disruption of lacZ expression in pGEMT via insertion/ligation of a PCR product can result in the formation of white colonies on this media. Plasmid DNA was extracted from white colonies (putative positive recombinants) and subjected to secondary screening using Ascl restriction enzyme analysis. Positive clones (pGEMT/PBterm) were subjected to ds DNA sequencing using universal primer which targets vector sequences upstream of the insert.
PGEMT/PBterm was digested with Ascl and Sail and the 204bp fragment encoding PBP amino acids 507 to 571 cloned into the Ascl/Sall sites of pETl laHA/PBpart/KL to generate pETl laHA/PB/KL. E. Coli DH5 was transformed with this recombinant and growth selected on Amp containing culture media. Plasmid DNA is extracted from isolated colonies, and subjected to screening (for positive recombinants) using BamHI restriction enzyme analysis. Positive clones are subjected to ds DNA sequencing using the universal forward primer to confirm nucleotide sequence. pETl laHA/PB/KL encodes for a fusion protein that begins with a Met followed by i)Gly2-HA-Gly2, ii) His, Met, Ala, Ser, and Thr residues encoded by vector sequences, iii) the Ad2 penton base protein, iv) Val, Asp, Gly, Thr, Gly and Ser encoded by the MCS, v) KLio and vi) end terminal amino acids Arg and Ser. C2. The EGF Protein A DNA molecule encoding the mature EGF protein plus an additional amino acid, Ala, was first produced as two individual fragments and then joined to one complete molecule. The complete molecule was then cloned into pETl laHAmcs and pETl laHA/KL. While encoding for the mature EGF sequence, the complete DNA sequence was designed, based on codon degeneracy, to have a primary sequence different from that of the native sequence.
More specifically, a dsDNA molecule was prepared which encodes for Ala followed by the first 25 amino acids of EGF plus 5' flanking sequences that provide the nucleotides suitable for cloning into the appropriate site in vectors such as pETl laHAmcs or pETHA/KL and 3' flanking sequences that provide for cloning (ligation) adjacent with the second EGF fragment (below). The dsDNA was generated by PCR amplification of the following primary single-stranded 73 bp DNA sequence: 5'-GCCAACTCAGATTCAGAATGTCCACTGTCACACGATGGCTACTGCCTCCA TGACGGAGTGTGCATGTATATCG-3' (SEQ ID NO: 12), using the following sense and anti-sense primers, respectively: 5'-CTCGAGGCCAACTCAGATTCAGAATG-3' (SEQ ID NO:13); and 5'-AGGCCTCGATATACATGCACACTCCG-3' (SEQ ID NO:14).
The dsDNA was ligated into pGEMT according to manufacturer's recommended procedures and protocols and transformed into DH5α and growth selected on culture media containing ampicillin and 5-Bromo-4-chloro-3-indoyl-β-D- galactose (X-GAL). Growth selection of pGEMT transformed DH5α on this culture media yields blue colonies. Disruption of lacZ expression in pGEMT via insertion/ligation of a PCR product can result in the formation of white colonies on this media. Plasmid DNA was extracted from white colonies (putative positive recombinants) and subjected to secondary screening using Xhol and Pstl restriction enzyme analysis. Positive clones (pGEMT/EGFl) were subjected to ds DNA sequencing using universal primer which targetsvector sequences upstream of the insert. A dsDNA molecule was prepared which encodes for amino acids 25 to 53 of EGF plus 5' flanking sequences that provide the nucleotides suitable for cloning
(ligation) adjacent to the first EGF fragment (above) and 3' flanking sequences that provide for cloning inot the appropriate site in vectors such as pETl l aHAmcs or pETl laHA/KL. The dsDNA was generated by PCR amplification of the following primary single-stranded 83 bp DNA sequence: 5'-TGGACAAATACGCATGCAACTGTGTTGTTGGATACATCGGCGAACGATGT CAATACCGCGATCTGAAATGGTGGGAACTGCGA-3' (SEQ ID NO: 15), using the following sense and annti-sense primers, respectively: 5'-AGGCCTTGGACAAATACGCATGCAAC-3' (SEQ ID NO: 16); and 5'-GTCGACTCGCAGTTCCCACCATTTCA-3' (SEQ ID NO: 17). The dsDNA was ligated into pGEMT according to manufacturer's recommended procedures and protocols and transformed into DH5 and growth selected on culture media containing ampicillin and 5-Bromo-4-chloro-3-indoyl-β-D- galactose (X-GAL). Growth selection of pGEMT transformed DH5 on this culture media yields blue colonies. Disruption of lacZ expression in pGEMT via insertion/ligation of a PCR product can result in the formation of white colonies on this media. Plasmid DNA was extracted from white colonies (putative positive recombinants) and subjected to secondary screening using Sail restriction enzyme analysis. Positive clones (pGEMT/EGF2) were subjected to ds DNA sequencing using universal primer which targetsvector sequences upstream of the insert.
The ds DNA sequence the second EGF fragment was removed from pGEMT/EGF2 by Stul/Sall restriction digestion and cloned into the Stul Sall sites of pGEMT/EGFl . The resulting recombinant plasmid pGEMT/EGF contains a novel DNA sequence which encodes for a native EGF protein plus a 5' Ala amino acid.
E. coli DH5α was transformed with pGEMT/EGF, and growth selected on Amp containing culture media. Plasmid DNA was extracted from isolated colonies, and subjected to initial screening (for positive recombinants) using Xhol/Ndel restriction enzyme analysis. Positive recombinants were subjected to a secondary screening using Xhol Stul and Xhol/Sall restricition analysis in order to screen for the presence of one complete copy of EGF encoding DNA sequence within the vector. Positive clones were subjected to double-stranded sequencing using the universal forward primers described-above to confirm the nucleotide sequence of the EGF encoding DNA. Another protype DDVE, pET 1 1 aHA/EGF was created to contain a DNA sequence which encodes inframe for Gly2-HA-Gly2 and EGF. The Ala-EGF encoding dsDNA sequence was removed from pGEMT/EGF by Xhol/Sall restriction digestion and ligated into the Xhol/Sall sites of pETl laHAmcs to give pETl 1 aHA EGF. E. coli DH1 was transformed with putative recombinant, pETl 1 aHA EGF, and growth selected on Amp containing culture media. Plasmid DNA was extracted from isolated colonies, and subjected to initial screening (for positive recombinants) using Xhol/Sall restriction enzyme analysis. Positive clones were subjected to double-stranded sequencing using the universal forward primers described-above to confirm nucleotide sequence. pETl 1 aHA/EGF encodes for a fusion protein that begins with Met followed by i) Gly2-HA-Gly2, (ii) His, Met, Gly, Ser, Leu and Glu encoded by vector and MCS sequences, iii) Ala followed the EGF protein and iv) Val, Asp, Cys, Thr, Gly and Ser encoded by MCS.
Another protype DDVE, pETl laHA/EGF/KL was created to contain DNA sequences encoding for Gly2-HA-Gly2 inframe with EGF followed by Lio. The L|0 encoding fragment was removed from pGEMT/KL by Sall/Bgiπ digestion and cloned into the Sall/BamHI sites in the MCS of pETl 1 aHA/EGF to give pETl laHA EGF/KL. Alternatively, the Ala-EGF containing Xhol/Sall fragment from pGEMT/EGF could be cloned into Xhol/Sall sites of pETl 1 aHA/KL to generate the same recombinant plasmid. E. coli DH5 was transformed with putative recombinant pETl laHA/EGF/KL and growth selected on Amp containing culture media. Plasmid DNA was extracted from isolated colonies ans subjected to screening (for positive recombinants) using HindHI restricition analysis. Positive clones were subjected to ds DNA sequenceing using universal forward primer to confirm nucleotide sequence. pETl laHA/EGF/KL encodes for a fusion protein that begins with Met followed by i) Gly2-HA-Gly2 ii) His, Met, Gly, Ser, Leu and Glu encoded by vector and MCS sequences, iii) Ala followed by EGF protein, iv) Val, Asp, Gly, Thr, Gly and Ser, v) KLio and vi) end terminal amino acids Arg and Ser. D. Isolation and Purification of DDVE
The DDVEs were transformed in E. coli strain BL21(DE3 and the DDVE protein produced, isolated and purified as described by Bai et al, J. Virol, 67:5198-5205 (1993).
More specifically, expression was induced in late log phase cultures (the optical density at 600 nm was 0.8) in LB broth at 37°C by adjusting the medium to 0.4 mM isopropylthiogalactopyranoside (IPTG). After 3 hrs shaking, the cells were collected by centrifugation, washed in STE buffer comprising 10 mM Tri-HCl (pH 8.0), 1.0 mM EDTA and 100 mM NaCl, lysed by freezing and thawing in the presence of 0.1 mg/ml lysozyme and sonicated (2 x 30 sec each) at 40% maximum output in a Branson Sonifier. The insoluble fraction containing DDVE was collected by centrifugation and resuspended in buffer comprising 20 mM Tris-HCl (pH 7.5), 1.0 mM EDTA and 0.1 % (w/v) Nonidet P-40. After three additional cycles of washing, the final pellet was resuspended in a small volume of 6.0 M urea, diluted with 9 volumes of buffer comprising 50 mM K2PO4 (pH 10.7), 50 mM NaCl, 1.0 mM EDTA, and dialyzed against phosphate buffered saline (PBS) and then 50 mM phosphate buffer (pH 7.5). The resulting dialyzate was sterilized by filtration and stored at 4°C or frozen at -70°C. The purity of the DDVE protein was confirmed by SDS-PAGE and Coomassie staining. Only one protein band was observed, representing the purified DDVE. A similar band was seen for DDVE-ΔHA.
EXAMPLE 2 Interaction of DDVE and Drug Molecules The DDVE pro-drug, lacking the (KL)m. but having HA and PBP, obtained in
Example 1 , was linked to doxarubicin via disulfosuccimidyl tartrate (Pierce Chemical, Rockford, IL).
EXAMPLE 3
Intracellular Delivery of DDVE Delivered Drug Molecules The DDVE lacking the (KL)m and a drug, but having HA and PBP (Pro- DDVEl) was tested for intracellular uptake and localization.
Pro-DDVEl is internalized by HeLa and Vero cells which differ in the levels of cell surface receptors (A549 > HeLa > Vero). Internal ization is dose dependent as evidenced by staining with anti-PBP antibody (100% staining cells at 60 min and 15 min of exposure to 80 and 800 nM of Pro-DDVEl protein respectively). Internalized Pro-DDVEl persists for 24 hrs in A549 cells (stained after Pro-DDVEl removal) but only for 10 and 2 hrs in HeLa and Vero cells respectively. Staining is cytoplasmic. It is primarily diffuse, indicative of protein release from endocytic vesicles. Pro-DDVEl lacking HA is similarly internalized and processed except that its intracellular distribution is primarily granular, indicative of sequestration within cytoplasmic granules consistent with endocytic vesicles. A weak diffuse component is consistent with a low level of endosomolysis . This shows that the Pro-DDVEl functions endosomolytically.
EXAMPLE 4 Intracellular Delivery of DDVE Delivery The DDVE lacking the (KL)m and a drug, but having HA and EGF (Pro-
DDVE2) was tested for intracellular uptake and localization
A431 cells, which express the EGF receptor, were grown overnight at 37° C in DMEM-10% FBS supplemented with ImM glutamine to approximately 60% confluency. The growth media was removed and the monolayers washed once with CMF-PBS. The monolayers were incubated for 5, 10 or 30 minutes at 37° C with EGF- free media containing either 0.3, 1.5 or 3ug/ml of partially purified HA-EGF. Control monolayers were incubated in EGF-free media in the absence of HA-EGF. The incubation media was removed, the monolayers washed three times with CMF-PBS and fixed in acetone. Cells were stained for the presence of HA-EGF by indirect immunofluorescence using rabbit anti-human EGF antibody (Chemicon International, Temecula, CA) and goat FITC-labeled anti-rabbit IgG antibody (Accurate Chemical and Scientific Corp, Westbury, NY).
Dose-dependent intracellular fluorescent staining was observed in A431 cells with a maximum of approximately 70% positive stainng cells observed after treatment for 10 minutes with 3ug/ml of partially purified Pro-DDVE2. Treatment with 0.3ug/ml for 10 minutes resulted in approximately 5% positive staining cells. These results indicate that Pro-DDVE2 targets the EGF receptor and is taken into the cytoplasm of cells bearing these receptors.
While the invention has been described in detail, and with reference to specific embodiments thereof, it will be apparent to one with ordinary skill in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. SEQUENCE LISTING (1) GENERAL INFORMATION:
(i) APPLICANT: Calton, Gary
Kulka, Micheal (ii)TITLE OF INVENTION: DRUG DELIVERY VEHICLE
(iii) NUMBER OF SEQUENCES: 17 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Beverly J. Artale, Esq.
(B) STREET: 3826 Sunflower Circle (C) CITY: Mitchel lle
(D) STATE: Maryland
(E) COUNTRY: United States of America
(F) ZIP: 20721
(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: MS-WD-97 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER:
(B) FILING DATE: 6-FEB- 1999
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Artale, Beverly J.
(B) REGISTRATION NUMBER: 32,366
(C) REFERENCE/DOCKET NUMBER: AuRx-10 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (301 ) 352-8577
(B) TELEFAX: (301 ) 352-7539
(2) INFORMATION POR SEQ ID NO: 1 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 base pairs
(B) TYPE: drug
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 : GAGGTGGACT CTTCGAAGCA ATTGCAGGTT TAATCGAAAA CGGCTGGGAA GGCATGATCG ACGGTGGTGG 70
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: drug
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CATATGGGAG GTGGACTCTT CGAAGCA 27 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: drug
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CATATGGCCA CCACCGTCGA TCATGCC 27
(2 ) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs
(B) TYPE: drug (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CTCGAGCTCA CGTGGCGCGC CATGGTCGAC TGTACAGGAT CCTAACTAGG TAAG 54
(2) INFORMATION FOR SEQ ID NO:5
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs
(B) TYPE: drug
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: AGATCTCTCG AGCTCACGTG GCGCGC 26
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: drug (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: AGATCTCTTA CCTAGTTAGG ATCCTG 26
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: drug
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GTCGACGGTA CCGGATCCAA G 21
(2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: drug
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GAATTCAGAT CTGAGCTTAA G
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS: A) LENGTH: 90 base pairs (B) TYPE: drug (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GTCGACGGTA CCGGATCCAA GCTCAAGCTT AAACTCAAGC TTAAGCTCAA ACTGAAGCTT AAGCTCAAGC TTAAGCTCAG ATCTGAATTC 90
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs
(B) TYPE: drug
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: CGTGTTCAAT CGCTTTCCCG AGAA 24
(2) INFORMATION FOR SEQ ID NO: 11 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: drug (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 GTCGACAAAA GTGCGGCTCG ATAGGACG 28
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 73 base pairs (B) TYPE: drug
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12
GCCAACTCAG ATTCAGAATG TCCACTGTCA CACGATGGCT ACTGCCTCCA TGACGGAGTG TGCATGTATA TCG 73 (2) INFORMATION FOR SEQ ID NO: 13 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: drug
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13
CTCGAGGCCA ACTCAGATTC AGAATG 26
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs
(B) TYPE: drug
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: AGGCCTCGAT ATACATGCAC ACTCCG 26
(2) INFORMATION POR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 83 base pairs
(B) TYPE: drug
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: TGGACAAATA CGCATGCAAC TGTGTTGTTG GATACATCGG CGAACGATGT CAATACCGCG ATCTGAAATG GTGGGAACTG CGA 83
(2) INFORMATION FOR SEQ ID NO: 16
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs
(B) TYPE: drug
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: AGGCCTTGGA CAAATACGCA TGCAAC 26
(2) INFORMATION FOR SEQ ID NO: 17 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: drug
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic
(iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: GTCGACTCGC AGTTCCCACC ATTTCA 26

Claims

WE CLAIM:
1. A drug delivery vehicle comprising:
(i) a drug uptake and release protein comprising an endosomolytic protein covalently attached to a protein comprising a ligand capable of binding to a receptor, bound to
(ii) a drug selected from the group consisting of proteins and non- oligonucleotide drugs.
2. The vehicle of Claim 1 wherein said endosomolytic protein is selected from the group of endosomolytic proteins consisting of influenza virus hemagglutinin, G protein of visicular stomatitus virus, rhinovirus, (P28 of targeted genetics), exotoxin A of Pseudomonas aeruginosa, streptolysin O, bacterial cytolysins, the entry protein of Mycobacterium tuberculosis, the internalin protein of Listeria monocytogenes, the invasin protein of Yersinia enterocolitica, gp36 from mouse mammary tumor virus, gp37 from Rous sarcoma virus, pl5E from Moloney murine leukemia virus, gp20 from Mason Pfizer monkey virus, gp41 from human immunodeficiency virus, gp21 from human T-Cell leukemia virus, retrovirus transmembrane proteins, and lentivirus transmembrane proteins or synthetic analogs therof.
3. The vehicle of Claim 1 wherein said endosomolytic protein is influenza virus hemagglutinin.
4. The vehicle of Claim 3 wherein said influenza virus hemagglutinin has been changed less than 50% in its protein sequence but retains its endosomolytic activity.
5. The vehicle of Claim 1 wherein said endosomolytic protein is attached to the protein ligand by an amide bond.
6. The vehicle of Claim 1 wherein said drug is bound ionically to said drug uptake and release protein.
7. The vehicle of Claim 1 wherein said drug is bound covalently to an amino site located on said drug uptake and release protein.
8. The vehicle of Claim 1 wherein said drug is bound covalently to an carboxy site located on said drug uptake and release protein.
9. The vehicle of Claim 7 wherein said drug is bound covalently to an epsilon amino group of a lysine contained within said drug uptake and release protein.
10. The vehicle of Claim 1 wherein said drug is bound covalently to a thiol group of a cystine contained within said drug uptake and release protein.
1 1. The vehicle of Claim 1, wherein said drug uptake and release protein additionally comprises a peptide containing one to 100 lysine molecules covalently attached thereto.
12. The vehicle of Claim 1 1 wherein said peptide is covalently attached to said drug uptake and release protein by an amide bond.
13. The vehicle of Claim 1 1 wherein said peptide is a lysyl-leucyl peptide containing 1 to 100 lysyl-leucyl units.
14. The vehicle of Claim 1 wherein said uptake and release protein additionally comprises an intranuclear localized signal peptide.
15. The vehicle of Claim 14, wherein said intranuclear localized signal peptide is derived from SV40 large T antigen.
16. The vehicle of Claim 1 , wherein said protein ligand is selected from the group consisting of ligands for the receptors for epidermal growth factor, c-kit, adenovirus penton base protein, platelet derived growth factor, transforming growth factor-α, transforming growth factor-β, basic fibroblast growth factor, c-erbB-1 , c-erb- 2, hepatocyte growth factor, epidermal growth factor receptor, p21 -ras-related proteins, or a ligand reacting with ras, p62/64 protein of c-myc, myc, mutant p53, mutant rb, abl, erbB 1 , erbB-2, gip, gsp, myc, L-myc, N-myc, H-ras, N-ras, ret, ros, K-sam, sis, src, and trk.
17. The vehicle of Claim 1, wherein said protein ligand is epidermal growth factor.
18. The vehicle of Claim 1 , wherein said protein ligand is adenovirus penton base protein.
19. The vehicle of Claim 1, wherein said drug is a protein.
20. The vehicle of Claim 1, wherein said drug is a non-oligonucleotide other than a protein.
21. The vehicle of Claim 1, wherein said drug is selected from the group consisting of daunomycin, doxarubicin, VP16, teniposide, paclitaxel, docetaxel, vincristine, vinblastine, and tretinoin or a combinations thereof.
22. The vehicle of Claim 1 , wherein said drug is covalently attached to the drug uptake and release protein by means of an amine linkage.
23. The vehicle of Claim 1, wherein said drug is covalently attached to the drug uptake and release protein by means of an alcohol linkage.
24. The vehicle of Claim 1 , wherein said drug is covalently attached to the drug uptake and release protein by means of an carboxylic acid linkage.
25. The vehicle of Claim 1, wherein said drug is covalently attached to the the drug uptake and release protein by means of a thiol linkage.
26. A method of delivery of a non-oligonucleotide drug molecules comprising contacting a cell with a drug delivery vehicle wherein said vehicle comprises: (i) a drug uptake and release protein comprising an endosomolytic protein covalently attached to a protein comprising a ligand capable of binding to a receptor, bound to
(ii) a non-oligonucleotide drug.
27. The method of Claim 26, wherein said drug uptake and release protein additionally comprises a peptide containing one to 100 lysine molecules covalently thereto.
28. The method of Claim 26, wherein said protein ligand is epidermal growth factor.
29. The method of Claim 26, wherein said protein ligand is an adenovirus penton base.
30. The method of Claim 26, wherein said drug uptake and release protein additionally comprises an intranuclear localized signal peptide.
PCT/US2000/003045 1999-02-06 2000-02-04 Drug delivery vehicle WO2000045850A2 (en)

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