WO1999045020A1 - Isolement de peptides ligands specifiques et leur utilisation pour diriger certains produits pharmaceutiques sur des organes - Google Patents

Isolement de peptides ligands specifiques et leur utilisation pour diriger certains produits pharmaceutiques sur des organes Download PDF

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Publication number
WO1999045020A1
WO1999045020A1 PCT/US1999/004691 US9904691W WO9945020A1 WO 1999045020 A1 WO1999045020 A1 WO 1999045020A1 US 9904691 W US9904691 W US 9904691W WO 9945020 A1 WO9945020 A1 WO 9945020A1
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WIPO (PCT)
Prior art keywords
peptide
agent
artery
vein
organ
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PCT/US1999/004691
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English (en)
Inventor
Tikva Vogel
Amos Panet
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Bio-Technology General Corp.
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Priority claimed from PCT/US1998/004188 external-priority patent/WO1998039469A1/fr
Application filed by Bio-Technology General Corp. filed Critical Bio-Technology General Corp.
Priority to AU28012/99A priority Critical patent/AU2801299A/en
Publication of WO1999045020A1 publication Critical patent/WO1999045020A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Advantages of local drug delivery are the lowering of the amount of drug needed to achieve therapeutic efficacy and the minimizing of undesired side effects.
  • tissue specific targeting is either to infuse the drug through a catheter or a balloon (PTCA) to a site of the vasculature, or through linking of a drug to a protein ligand with affinity for a predetermined target .
  • PTCA catheter or a balloon
  • Phage libraries have been used to select random peptides that bind to isolated pre-determined target proteins such as antibodies, hormone receptors, and the erythropoietin receptor.
  • the process of phage selection from a random peptide library does not require prior knowledge of the target cell or its receptors.
  • This approach also has the advantage that molecular recognition and ligand selection are not dependent on the immunogenicity of the candidate target protein, as required in the monoclonal antibody approach.
  • Pasqualini and Ruoslahti (Nature 380: 364-366, 1996) injected phage libraries intravenously into mice and subsequently rescued the phage from individual organs.
  • Peptides capable of mediating selective localization of phage to brain and kidney blood vessels were identified and were shown to exhibit up to 13 -fold higher sensitivity for these mouse organs.
  • One of the peptides displayed by the brain-localized phage was chemically synthesized and shown to specifically inhibit the localization of the homologous phage into the brain. When coated onto glutaraldehyde fixed red blood cells, the peptide caused selective localization of intravenously injected red blood cells into the brain of the mouse.
  • the subject invention discloses the use of phage display peptide (epitope) libraries to identify peptides useful as ligands for targeting drugs, cells or genes to specific human tissue and various human organs.
  • the novelty of the subject approach is inter alia the application of the peptide library methodology to isolated perfused human tissues.
  • a phage library is included in the organ perfusion fluid, and after ex vivo organ perfusion, phages are extracted from the human tissue, amplified and the displayed peptide - 3 - sequence is determined.
  • This ex vivo approach is applied to human organs such as placenta, umbilical cord artery and vein as well as blood vessels removed during surgery. This approach is further applied to diseased tissue removed during surgery and to organs such as kidney, heart and liver available following transplantation procedures.
  • Endothelium which lines the inner surface of blood vessels expresses multiple surface proteins and receptors for diverse types of ligands.
  • Endothelial cells derived from different tissues or even from veins and arteries of the same tissue, have been shown to be phenotypically and functionally distinct.
  • the unique distinctive, characteristic surface proteins and receptors expressed by endothelial cells of the various tissues are exploited to discover novel, defined peptide ligands which are subsequently linked to drugs or radioactive isotopes for targeting to the desired tissue.
  • peptide sequences of the subject invention specific for different human organs and tissue cells are linked to various pharmaceutical agents to form drug-peptide conjugates and to radioactive isotopes for diagnostic and therapeutic purposes.
  • the subject invention provides novel peptides comprising the sequence Glu Gly Arg and the use of these peptides in the treatment of various diseases and conditions.
  • the novel peptides specifically bind to endothelial cells and various tumor cells and tissues.
  • compositions comprising novel peptides and a pharmaceutical agent linked to the peptide, wherein the pharmaceutical agent is a polypeptide and is linked to the peptide by a peptide linkage.
  • the pharmaceutical agent may also be a toxin, an anti-cancer agent, an anti-angiogenic compound, a cardiovascular agent, an agent used in a neurological disorder, a liver disease agent, a kidney disease agent or a radioisotope .
  • the subject invention provides for a method for the identification of a peptide comprising incubating a phage display peptide library with an isolated organ; washing the isolated organ to remove unbound phages; eluting bound phage from the isolated organ; amplifying the resulting bound phage; and determining the displayed peptide sequence of the bound phage so as to identify the peptide.
  • the organ may be an artery, a vein, placenta, tumor tissue, kidney, heart, liver, or central nervous system.
  • the organ may also be a perfused organ.
  • the phage display library may be a 15-mer library or a 6-mer library.
  • the elution medium may be a compound selected from acid, urea, octyl , trypsin or tween.
  • the subject invention also provides for a method of producing the novel peptides comprising identifying the peptide as described above, and synthesizing the peptide by joining the amino acids of the peptide in the proper order.
  • the subject invention additionally provides an imaging agent which comprises a peptide of the subject invention with an imageable marker.
  • an imaging agent may be used for diagnostic purposes.
  • the subject invention further provides a composition comprising an effective imaging amount of an imaging agent of the invention and a physiologically acceptable carrier.
  • the subject invention also encompasses a composition comprising an effective imaging amount of an imaging agent of the invention, a pharmaceutical agent linked thereto and a physiologically acceptable carrier.
  • the marker may be a radioactive isotope, an element which is opaque to X-rays or a paramagnetic ion.
  • the radioactive isotope may be indium-Ill, technetium-99 , iodine-123, iodine-125, iodine-131, krypton-81m, xenon-33 or gallium-67.
  • the subject invention also provides for a method for imaging an organ comprising contacting the organ to be imaged with an imaging agent under conditions such that the imaging agent binds to the organ, imaging bound imaging agent; and thereby imaging the organ.
  • the subject invention also provides for a method of treating an organ in vivo comprising contacting the organ to be treated with a composition of the invention under conditions such that the composition binds to the organ, and thereby treating the organ.
  • FIG. 1 Organ distribution of selected phages Clone KSC#3 (KSCR3#3) is a phage clone that was enriched on Kaposi Sarcoma cells after three rounds of biopanning in culture.
  • Clone R4B * #1 (TUN-R4B*#1) is a phage clone that was enriched on umbilical vein and artery.
  • Clone #P13 (R6P#13) is a sporadic non-enriched phage clone as a negative control . The in vivo binding of these three clones to tumor tissue and brain tissue was compared.
  • Figure 2 Binding of TUV-R4B-#1 (the EGR-phage) to mouse melanoma cells and to brain cells.
  • Figure 3 Ratio of binding of TUV-R4B-#1 to melanoma tumor cells and to brain cells.
  • Figure 4 Binding of TUV-R4B-#1 and of 14-4 phage to mouse mammary tumor cells.
  • Figure 5 Binding of TUN-R4B-#1 to mouse mammary tumor cells and to brain cells.
  • Figure 6 Comparison of binding of TUN-R4B-#1 to mouse mammary tumor cells and to brain cells.
  • the subject invention provides novel peptides comprising the sequence Glu Gly Arg and the use of these peptides in the treatment of various diseases and conditions.
  • the novel peptides specifically bind to endothelial cells and various tumor cells and tissues.
  • Phage display is a technique in which a peptide, antibody or protein is expressed on the surface of a bacteriophage, while the D ⁇ A encoding the displayed protein resides within the phage virion.
  • a phage display peptide library (also termed phage peptide library or phage display library or phage library or peptide library) is constructed wherein the virions display a wide range of protein residues of specific lengths. This technology, known to one skilled in the art, is more specifically described in the following publications: Smith (1985) Science 228: 1315, Scott et al . (1990), Science 249: 386-390, Cwirla et al . (1990), P. ⁇ .A.S. 87: 6378-6382; Devlin et al . (1990), Science 249: 404-406, U.S. Patent ⁇ os 5,427,908, 5,432,018, 5,223,409 and 5,403,484.
  • Biopanning is a procedure comprising many steps, one of which is selection; biopanning is carried out by incubating phages displaying protein ligand variants (a phage display library) with a target, washing away unbound phage and specifically eluting the bound phage.
  • the eluted phage is amplified and taken through additional cycles of binding and amplification which enrich the pool of eluted specific sequences in favor of the best binding peptide bearing phages.
  • individual phages are characterized, and the sequence - 7 - of the peptides displayed is determined by sequencing of the corresponding DNA of the phage virion.
  • a peptide obtained in this manner may be called a "lead-compound”.
  • One way of obtaining a peptide with a higher affinity relative to a lead-compound is to construct an extension phage display peptide library based on a core amino acid sequence of the lead-compound.
  • an extension library random amino acids are added to each side of the core sequence.
  • oligonucleotides are synthesized so that each amino acid of the core sequence is independently substituted by any other amino acid.
  • Cancer tissue as used herein may be obtained from any form of cancer such as carcinoma, sarcoma, leukemia, adenoma, ly ⁇ nphoma, myeloma, blastoma, seminoma or melanoma.
  • Diseased tissue as used herein may be obtained from any diseased organ such as liver, kidney, lung, heart, ovary, colon and so forth.
  • the organ may be diseased as a result of an autoimmune disorder.
  • the organ may be diseased as a result of any other disease, such as cardiovascular disease or cancer .
  • a neurologic disorder as used herein encompasses any neurologic disorder as defined and described in "The Merck
  • muscular dystrophy myasthenia gravis, multiple sclerosis, Alzheimer's disease, neuropathy, Parkinson's disease and amyotrophic lateral sclerosis (Lou Gehrig ' s disease) are neurologic disorders.
  • a vein as used herein may originate from any tissue.
  • An example of a vein is safenal vein or femoral vein.
  • An artery as used herein may originate from any tissue, e.g. radial artery, coronary artery, mammary artery and so forth.
  • a peptide of the subject invention may be administered to a patient, alone, radiolabeled, linked to a pharmaceutical agent (drug), or in the form of a peptidomimetic .
  • the mode of administration of a peptide of the subject invention is intravenous, intramuscular, subcutaneous, topical, intratracheal , intrathecal , intraperitoneal , rectal, vaginal or intrapleural .
  • the pharmaceutical agent may inter alia be a radioactive label (radio-isotope) .
  • the peptide or the peptide-drug combination is administered orally, it is administered in the form of a tablet, a pill or a capsule.
  • compositions comprising the peptides produced in accordance with the invention may comprise conventional pharmaceutically acceptable diluents or carriers.
  • Tablets, pills and capsules may include conventional excipients such as lactose, starch and magnesium stearate .
  • Suppositories may include excipients such as waxes and glycerol .
  • injectable solutions comprise sterile pyrogen-free media such as saline and may include buffering agents, stabilizing agents or preservatives. Conventional enteric coatings may also be used.
  • compositions for topical administration may be in the form of creams, ointments, lotions, solutions or gels.
  • the mode of administration of the peptide or drug-peptide linkage is a solid dosage form, a liquid dosage form, or a - 9 - sustained-release formulation.
  • the subject invention provides a polypeptide which comprises a peptide of the subject invention which corresponds to a peptide displayed on a phage virion and wherein both the polypeptide and the peptide have the same biological activity.
  • a Fv fragment of a human antibody of about 100 amino acids is displayed on the N-terminus of piII of M13 bacteriophage and contains the Glu Gly Arg sequence.
  • the subject invention provides peptides comprising Glu Gly Arg having the following sequences:
  • R x and R 2 are amino acid sequences ranging from 1-50 amino acids, preferably 1-15 amino acids, most preferably 1-5 amino acids in length;
  • amino acid at the extreme left represents an amino acid on the amino terminal side of the peptide and the amino acid at the extreme right represents an amino acid on the carboxy terminal side of the peptide.
  • the invention further provides a composition comprising a peptide of the subject invention and a pharmaceutical agent - 10 - linked thereto.
  • the composition optionally also comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical agent is a polypeptide and is linked to the peptide by a covalent linkage, preferably a peptide linkage.
  • the pharmaceutical agent is a toxin, an anti-cancer agent, an anti-angiogenic compound, a cardiovascular agent, an agent used in a neurological disorder, a liver disease agent or a kidney disease agent or a radio isotope.
  • the pharmaceutical agent is a recombinant protein.
  • the subject invention further provides a composition comprising a peptide of the subject invention and a pharmaceutically acceptable carrier.
  • the subject invention also provides a chimeric polypeptide comprising a first peptide and a second peptide wherein the first peptide is a peptide of the subject invention.
  • the second peptide is a toxin, an anti-cancer agent, an anti-angiogenic compound, a cardiovascular agent, an agent used in a neurological disorder, a liver disease agent or a kidney disease agent.
  • the second peptide is a recombinant protein.
  • the subject invention also encompasses a method of synthesizing a peptide of the subject invention which comprises joining the amino acids of the peptide in the proper order . - 11 -
  • the subject invention further provides a method of producing a peptide which comprises identifying the peptide by the above described method and synthesizing the peptide by joining the amino acids of the peptide in the proper order.
  • the isolated organ is a perfused organ .
  • the isolated organ is an artery, a vein, placenta, tumor tissue, kidney, heart, liver or central nervous system.
  • the artery is umbilical cord artery, a radial artery, a coronary artery, a mammary artery or a damaged artery.
  • the damaged artery is a damaged coronary artery.
  • the vein is umbilical cord vein, safenal vein or femoral vein.
  • the phage display peptide library is a 15-mer library, a 6-mer library or a synthetic human antibody library.
  • Preferred elution medium is a compound selected from acid, urea, octyl, trypsin or Tween.
  • the subject invention additionally provides an imaging agent which comprises a peptide of the subject invention with an imageable marker.
  • an imaging agent may be used for diagnostic purposes.
  • the subject invention further provides a composition comprising an effective imaging amount of an imaging agent of the invention and a physiologically acceptable carrier. - 12 -
  • the subject invention also encompasses a composition comprising an effective imaging amount of an imaging agent of the invention, a pharmaceutical agent linked thereto and a physiologically acceptable carrier.
  • the marker is a radioactive isotope, an element which is opaque to X-rays or a paramagnetic ion.
  • the radioactive isotope is indium-Ill, technetium-99, iodine-123, iodine-125, iodine- 131, krypton-81m, xenon-33 or gallium-67.
  • the subject invention also provides a method for imaging an organ which comprises contacting the organ to be imaged with an imaging agent of the invention under conditions such that the imaging agent binds to the organ, imaging the bound imaging agent and thereby imaging the organ.
  • the organ is an artery, a vein, placenta, tumor tissue, kidney, heart or liver.
  • the artery is umbilical cord artery, a radial artery, a coronary artery, a mammary artery or a damaged artery.
  • the damaged artery is a damaged coronary artery.
  • the vein is umbilical cord vein, safenal vein or femoral vein.
  • the pharmaceutical agent is a polypeptide and is linked to the imaging agent by a peptide linkage .
  • the pharmaceutical agent is a - 13 - toxin, an anti-cancer agent, an anti-angiogenic compound, a cardiovascular agent, an agent used in a neurological disorder, a liver disease agent or a kidney disease agent.
  • the subject invention further provides a composition wherein the pharmaceutical agent is a recombinant protein.
  • the subject invention further provides a method of treating an organ in vi vo which comprises contacting the organ to be treated with a composition of the invention under conditions such that the composition binds to the organ and thereby treating the organ.
  • the organ is an artery, a vein, placenta, tumor tissue, kidney, heart, liver, or central nervous system.
  • the artery is umbilical cord artery, a radial artery, a coronary artery or a mammary artery or a damaged artery.
  • the damaged artery is a damaged coronary artery.
  • the vein is umbilical cord vein, safenal vein or femoral vein.
  • novel peptides of the subject invention or their corresponding peptidomimetics are also used in the manufacture of compositions to treat various diseases and conditions.
  • the subject invention also provides a method for the identification of peptides or antibodies by biopanning which comprises incubating a phage display library with lymphocytes derived from blood, washing to remove unbound phages, eluting the bound phages from the lymphocytes, amplifying the resulting bound phage and determining the displayed peptide -14- sequence of the bound phage so as to identify the peptide.
  • E.XAMPLE 1 Isolation and Selection of Tissue-Specific Epitopes which Specifically Bind to Undetermined Targets in Umbilical Cord Vein and Artery
  • Freshly obtained full term umbilical cord was cut into fragments of 4-5 cm in length, the blood was removed and the umbilical cord was cannulated and connected either through the vein (V) or the artery (A) to a small circulating pump for perfusion at a rate of lml/min (Pharmacia peristaltic pump) .
  • Perfusion temperature was either 4°C or 23°C and the perfusion buffer composition is indicated below.
  • phage display peptide libraries (6-mer and 15-mer) were kindly provided by G. Smith (Virology 167: 156-165, 1988) . The libraries were amplified to form phage library working stock.
  • the libraries were originally constructed by splicing the 2.8 kilo-base pair tetracycline resistance determinant of TnlO into the minus-strand origin of replication of the wild type fd phage.
  • the resulting defect in minus strand synthesis reduced the intracellular replicative form (RF) copy number to 1.
  • RF replicative form
  • the filamentous phage including fd-tet do not kill their host.
  • the infected cell which becomes resistant to tetracycline, continues to grow and secret tetracycline resistant progeny particles (about 10 tetracycline transducing units (TTU) per bacteria) .
  • TTU tetracycline transducing units
  • the calculated theoretical number of - 16 - primary clones for the 6 and 15-mers libraries are 4xl0 7 and lxlO 19 , respectively. However, the number of primary clones obtained from each library was about 2xl0 8 .
  • the phage titer is determined by infecting starved K91/kan bacterial culture and selecting tetracycline resistant clones on kan/tetracycline agar plates. The biopanning yield is calculated as the % of the output phage from the total input .
  • Bacterial strain K91/Kan The preparation of starved bacterial culture for phage infection was essentially as described by G. Smith (1993), Methods of Enzymology 217:228.
  • Phages selection and amplification Phages that expressed epitopes of specific interest were selected from the libraries by biopanning in an essentially 4 -step procedure:
  • Sequencing selected phage The DNA sequence of the insert was determined by the dideoxy DNA sequencing method (Sanger et al . (1977), P.N.A.S. 74: 5463-5467) using Sequenase Version 2.0 (DNA Sequencing Kit, Amersham) and a primer of 18 nucleotides (5': TGAATTTTCTGTATGAGG) (Seq. ID. No. 1).
  • Protocol Tl (Acid/Urea/Octyl elution) 1.5.2. Protocol Rl (Trypsin-EDTA/Acid-Tween elution)
  • Protocol Nl (Acid-Tween/Tissue elution)
  • Protocol R2 (Trypsin-EDTA/Acid-Tween/Tissue elution)
  • Protocol EC-1 (Acid/Tissue/Urea elution)
  • Protocol Tl (Acid/Urea/Octyl elution)
  • Prewashin ⁇ of the umbilical cord was carried out with 20 ml cold PBS 1 followed by 20ml cold DMEM-5% BSA.
  • PBS is 136 mM NaCl , 2.7 mM KCl , 100 mM Na 2 HP0 4 . 2H 2 0, 1.4 mM KH 2 P0 4 TTU is phage infection units, conferring tetracycline resistance in the E. coli host.
  • DMEM is Dulbecco's Modified Eagle Medium with D-glucose
  • BSA 4 -Protease inhibitors mixture (Pi 5 ) . (50 ⁇ l of the selection solution is used for titration of the input phage) .
  • Wash 1+ Wash 2 (W1,W2) was carried out in 2 x 25ml DMEM-5% BSA-Pi. When indicated, 0.2% Tween-20 was included.
  • Wash 3 was carried out in 3 ml Wl+2 containing TBS ⁇ - Octyl 7 (0.05%) .
  • Steps 2-4 were carried out at 4°C or at 23 C as indicated below.
  • Acid elution was carried out in 3 ml 0.2M glycine-HCl, pH 2.2.
  • BSA bovine serum albumin fraction V (Sigma)
  • Pi Phenyl Methyl Sulfonyl Fluoride (PMSF) ImM, Aprotinin (20 ⁇ g/ml) , Leupeptin (1 ⁇ g/ml)
  • PMSF Phenyl Methyl Sulfonyl Fluoride
  • TBS is 50 mM Tris-HCl, pH 7.5 and 150 mM NaCl
  • Octyl is octyl - ⁇ -D-glucopyranoside (Sigma) - 19 - out in liquid medium by mixing the eluate with equal volume of 10 10 starved bacteria. After 10 minutes, 100ml 8 NZY solution containing 0.2 ⁇ g/ml tetracycline was added and bacterial suspension was mixed vigorously at 37°c for 30 minutes. Diluted samples were plated immediately on agar/kanamycin/tetracycline plates for titration of the output phage. To the rest of the bacterial cell suspension lOO ⁇ l of 20mg/ml tetracycline were added and incubation/amplification continued over-night.
  • Protocol Rl (Trypsin-EDTA/Acid-T een elution)
  • the acidic fraction was neutralized with 2M Trisma base (about 270 ⁇ l) .
  • 2M Trisma base about 270 ⁇ l
  • NZY is a medium consisting of 10 g NZ amine A, 5 g yeast extract and 5 g NaCl . -20 -
  • Protocol Nl (Acid-T een/Tissue elution)
  • Prewashing was carried out with 30ml DMEM-heparin (5u/ml) containing 10 8 M13 phage followed by 30ml DMEM-5% human serum.
  • Elution 2 tissue elution (i.e. bacteria elution), was carried out by clamping one side of the vein and adding 0.5ml NAP 9 buffer containing 2.5 x 10 10 starved bacteria. After clamping the other end of the vein, the blood vessel was immersed into DMEM- 1% BSA solution and shaken at 37°C for 45 minutes. The bacteria were removed to a 50ml tube, the blood vessel was washed twice with l-2ml NAP buffer, and the two washes were combined. 10ml NZY was added, mixed gently and left at room temperature for 10 minutes.
  • tissue elution i.e. bacteria elution
  • Steps 1-4 were carried out on ice and step 5 at room temperature (using a lamp from above) .
  • NAP consists of 80mM NaCl and 50 mM NH 4 H 2 P0 4 , pH 7.0 - 21 - added .
  • Protocol R2 (Trypsin-EDTA/Acid-Tween/Tissue elution) Protocol R2 is essentially identical to protocol Rl apart from the addition of tissue elution with bacteria following the trypsin and acid-tween elutions.
  • Protocol EC-1 Primary human endothelial cells seeded in 35cm tissue culture bottles (3rd passage of full term umbilical cord) were used.
  • Prewashing was carried out with 5ml serum-free medium followed by 10 ml blocking solution (DMEM-BSA (1%) -Pi) for 90 minutes at 37°C.
  • Elutions 5a. Elution 1 was carried out with 2ml acid-glycine pH 2.2 containing 0.2% Tween for 10 minutes at room temperature. 170 ⁇ l of 2M Trizma base was added to the bottle for neutralization, and the acid elution obtained was transferred to another tube. The bottle was washed with 2ml DMEM-BSA (1%) . 0.2ml of the eluate was mixed with NAP buffer containing lxlO 10 starved bacteria, and after 10 minutes absorption at room temperature, 2ml NZY-0.2% tetracycline was added and the - 22 - suspension was shaken at 37°c. Aliquots were plated on agar- kanamycin-tetracycline plates for phage titration.
  • Elution 2 was carried out with 2ml NaP0 4 buffer containing 5xl0 10 starved bacteria. Incubation was carried out at room temperature for 45 minutes on a rocker. Cell eluate was transferred to another tube. The bottle was washed with 2ml medium. 0.4ml suspension was mixed with 2ml NZY 0.2%- tetracycline and incubated and titrated as described for elution 1 (6a) .
  • Elution 3 was carried out with 2ml urea (6M, pH 3) . Incubation at room temperature for 10 minutes while rocking. 170 ⁇ l of 2M Trizma base was added directly to the bottle and urea eluate was transferred to another tube containing 36ml medium. The bottle was washed with 2ml medium. 0.2ml was mixed with 0.2ml NAP buffer containing lxlO 10 starved bacteria and then continued as described for elution l(6a) .
  • the working stock of the 15-mer library was the product of several cycles of amplifications.
  • Several clones of the working stock i.e. input of round 1
  • were isolated, amplified and their single stranded DNA was purified and sequenced. None of the phage clones was identical to specifically selected clones described below.
  • Table 1 Comparison of phage binding to umbilical cord vein and non-specific phage adsorption to peristaltic pump plastic tubes following biopanning with 15-mer library (yield after acid and urea elution, protocol Tl, room temperature (23°C) ) .
  • Plastic tubes 1 yield % Vein 2 (TTU) yield % input 2x10 9 3xl0 10
  • Table 2 Effect of temperature and Tween-20 on the binding of the 6-mer and 15-mer phage libraries to umbilical artery and vein (acid/urea/Octyl elution, protocol Tl) .
  • vein 2 i.e. biopanning at 23°C and washing with a buffer containing 0.2% Tween followed by washing with a buffer containing 0.05% Octyl
  • Tl based experiments i.e. biopanning at 23°C and washing with a buffer containing 0.2% Tween followed by washing with a buffer containing 0.05% Octyl
  • Table 3 Biopanning of 15-mer library with human umbilical vein (Vein 1 and Vein 2) and 6-mer library with human umbilical artery (Artery 1) - yield after urea elution, protocol Tl (experiment B) .
  • amino acids are aromatic (bolded) forming two hydrophobic clusters (amino acids 4 , 5 , 6 , ...13 , 14 , 15) , accompanied by a linker peptide (underlined) region containing the charged sequence Glu Gly Arg (9,10,11) followed by Ser Phe. Peptides based on this charged core sequence, Glu Gly Arg, are discussed in Example 2. In addition, the amino end of the 15- mer peptide is positively charged having the sequence Arg, Gin, His (position 2, 3, 4, double underlining) .
  • Clones #1, #2, and #4 were identical at nucleotide positions 1-27 (amino acids 1-9) .
  • Clones #1 and #2 also have an identical amino acid at position 13 (proline) and clones #1 and #4 have an identical amino acid at position 14 (leucine) .
  • TUV-R4D-#1, TUV-R4D-#2 and TUV-R4D-#4 are the amino acids which the displayed peptide sequences have in common.
  • 3/6 clones were unique (#3, #5, and #6) clones.
  • TUV-R4D-#3 was also identical to six of the clones selected at R5D, to two of the clones selected at R5E and to seven clones selected at R5H.
  • 1/10 clone was identical to clone TUV-R4B*-#3 and 1/10 clone was a unique clone.
  • Phage library was first biopanned on the umbilical vein (8- 10cm length) , and only the unbound (vein-excluded) phage was then biopanned with umbilical artery (4-5 cm length) . This sequence was repeated for all rounds. Tissue elution with starved bacteria was conducted according to protocol Nl .
  • Table 11 Binding of clone TUV-R4B*-#1 and clone TUV-R4B*-#3 (see section 3 below) to endothelial cell culture (Protocol EC-1) ; results of acid, tissue and urea elutions.
  • a phage mixture (4xl0 10 ) of a selected clone, either clone TUV-R4B*-#1 or clone TUV-R4B*-#3 or both clones together, with the 15-mer library (in a ratio of 1:10- 1:100) was injected through the tail vein of a rat (in 0.5ml DMEM-1% BSA) . After 4 minutes, the animal was sacrificed, the chest was opened, and extensive washing of the blood vessels was carried out by flushing isotonic salt solution through the left ventricle.
  • tissue-bacterial suspension was diluted in 10ml NZY containing 0.2% tetracycline and after 45 minutes of vigorous mixing at 37°C, aliquots were plated on agar plates containing 40 ⁇ g/ml tetracycline- 20 ⁇ g/ml kanamycin and incubated at 37°c for 16 hours. After monitoring tet R colonies on each plate, the colonies were transferred to a millipore sheet for colony hybridization as described in "Molecular Cloning: A Laboratory Manual", J, Sambrook, E.S. Fritsch, and T. Maniatis, Cold Spring Harbor Laboratory Press, 2nd edition, 1989.
  • Table 12 Tissue distribution of TUV-R4B*-#1 in a rat model (the ratio of clone TUV-R4B*-#1 to 15-mer library in the input phage was approximately 1:10).
  • Tables 13A and 13B Tissue distribution of clone TUN-R4B* -#1 and TUV-R4B*-#3 in rat model
  • the ratio of TUV-R4B*-#1 and TUV-R4B*-#3 to the 15-mer library was approximately 1:1:98.
  • Table 13A Tissue distribution of TUN-R4B* -#1
  • Preferred peptides of the subject invention contain the core sequence Glu Gly Arg (EGR) which binds specifically to endothelial cells and to tumor tissue.
  • EGR Glu Gly Arg
  • a peptide is a cyclic peptide Cys Glu Gly Arg Cys where the Cys residues form a -S-S- bond.
  • Another example of such a peptide is the 15-mer peptide derived from clone TUV-R4B-#1 described in Example 1:
  • An additional example is a peptide having the sequence Glu Gly Arg Ser Phe (Seq. ID. No. 11) .
  • peptides with higher affinity for binding to cells/tissue may be constructed based on the core sequence Glu Gly Arg. These peptides may be identified, e.g. by preparing an extension phage display library or by a phagemid display mutagenesis library. They may also be identified by varying the above 15-mer peptide by additions, deletions or mutations, while maintaining the Glu Gly Arg core sequence.
  • Peptides of the invention have the amino acid sequence R- L -GIU Gly Arg-R 2 , where R ⁇ and R 2 comprise amino acid sequences from 0-50 amino acids, preferably 1-15 amino acids, most preferably 1-5 amino acids. All peptides of the invention bind to cells/tissue by means of the Glu Gly Arg core sequence.
  • Ri and ⁇ are amino acid sequences which do not negatively affect the specific binding of the EGR sequence to tissue/cells.
  • -42- EXAMPLE 3 Preparation of a peptide
  • a peptide of the subject invention which contains the Glu Gly Arg (EGR) core sequence, is prepared by the following methods:
  • the chemically synthesized peptide is linked to a pharmaceutical agent (drug) inter alia by a covalent or by a non-covalent bond forming a drug-peptide conjugate.
  • the peptide may also be incorporated into a liposome.
  • the covalent bond may be inter alia a peptide, an amide, an ester, a disulfide or an anhydride covalent bond.
  • the non covalent bond is inter alia an ionic bond or a hydrophobic complex.
  • the pharmaceutical agent may be inter alia a radio isotope label . -43 -
  • the choice of the peptide linkage is determined based on the functional groups of the individual drug.
  • the peptide may also be cyclic (see e.g. Example 2) .
  • Cyclic peptides having a disulfide bond may be prepared as described in U.S. Patent No. 4,903,773 (Partoliano and Ladner) . Cyclic peptides may also be prepared as described in Schiller et al . , Int. J. Peptide-Protein Re. 25:171 (1985) and in U.S. Patent No. 5,648,330 (Pierschbacher et al . ) .
  • peptides are produced by methods known in the art.
  • the DNA encoding the peptide is prepared by synthetic oligonucleotides based on the amino acid sequence of the peptide and their known nucleotide codons (see e.g. U.S. Patent No. 5,221,619).
  • the peptide is then produced by expression of the nucleotide sequence encoding the polypeptide.
  • the recombinantly produced peptide is linked to a pharmaceutical agent.
  • a hybrid fusion polypeptide is constructed comprising the peptide linked by a peptide bond to the drug as follows: a DNA molecule is prepared comprising DNA encoding the drug and DNA encoding the peptide.
  • the DNA encoding the peptide is prepared by synthetic oligonucleotides based on the amino acid sequence of the peptide and their known nucleotide codons.
  • the synthetic oligonucleotide encoding the peptide is ligated either to the 5 ' end of the DNA strand encoding the drug or to the 3' end.
  • an ATG nucleotide sequence is added to the synthetic oligonucleotide encoding the peptide.
  • a DNA termination codon (TAA or TGA) is added at the 3 ' end of the synthetic oligonucleotide encoding the peptide and the termination codon at the 3 ' end of the DNA encoding the drug is removed using conventional recombinant DNA technology methods by use of Mung bean nuclease or cleavage -44- with an appropriate restriction enzyme.
  • hybrid polypeptide encoded by the above described recombinant DNA molecule is expressed and produced by recombinant technology by methods known in the art, e.g. in bacteria, yeast, insect, plant or mammalian cells in culture or in a genetically engineered transgenic animal or plant.
  • a recombinant protein Upon injection, a recombinant protein is targeted to tissue specific endothelial cells and is converted from a soluble protein to a cell surface bound protein (ectoenzyme) .
  • ectoenzyme a cell surface bound protein
  • arteriosclerosis and thrombogenicity of the vessel wall in certain clinical indications is reduced.
  • Conversion of these soluble proteins into ectoenzymes also reduces blood clearance time and hence a lower amount of recombinant protein is injected to achieve efficacy.
  • peptidomimetics or peptide analogs that display more favorable pharmacological properties than their prototype native peptides.
  • the native peptide itself the pharmacological properties of which have been optimized, generally serves as a lead for the development of these peptidomimetics.
  • a lead structure for development of a peptidomimetic can be optimized, for example, by molecular modeling programs.
  • a mimotope i.e. a peptide mimetic of a peptide of the subject invention, the chemical structure of which is different from the peptide, but the biological activity of which remains similar to that of the peptide.
  • U.S. Patent Nos . 4,879,313, 4,992,463 and 5,0191,396 describe examples of such peptide mimetic compounds.
  • amino acid residues may be added, -45 - deleted or substituted using established well known procedures, thereby producing extended peptides.
  • DNA encoding a peptide of the subject invention may be mutated by methods known to those skilled in the art, thereby produced mutated peptides.
  • a peptide of the subject invention may be administered to a patient, alone, radiolabeled, or linked to a pharmaceutical agent (drug) .
  • the following pharmaceutical agents are examples of drugs which are linked to a peptide of the subject invention corresponding to a peptide displayed by a phage virion to form drug-peptide conjugates.
  • Other pharmaceutical agents not mentioned below may also be used.
  • Peptides of the subject invention are linked to a toxin, an anti -cancer drug or an anti-angiogenic compound in order to target and destroy tumor tissue. This specific use of such drug-peptide conjugates represents an alternative approach to current attempts to apply immunotoxins against cancer.
  • Solid tumor growth in vivo is associated with recruitment of new blood vessels. Targeting the tumor-vasculature is an attractive possibility for anti -cancer therapy for the following reasons: First, most anti-cancer drugs are given by the systemic intravenous route and drug concentration at the tumor tissue is the crucial factor for effective therapy.
  • anti-cancer drugs that are linked to the peptides of the subject invention are adriamycin, cis-Platinum, taxol , bleomycin and so forth.
  • anti-angiogenic compounds that are linked to peptides of the subject invention are cortisone, heparin and so forth.
  • toxins that are linked to peptides of the subject invention are Pseudomonas exotoxin A, ricin and so forth.
  • thrombolytic enzymes such as tissue plasmmogen activator (tPA) , streptokinase (SK) , and anti-thrombotic agents such as heparin, ticlopidine or antiplatelet monoclonal antibodies.
  • tPA tissue plasmmogen activator
  • SK streptokinase
  • anti-thrombotic agents such as heparin, ticlopidine or antiplatelet monoclonal antibodies.
  • Peptides of the subject invention are further linked to recombinant proteins such as CuZnSOD, MnSOD, Factor Xa Inhibitors, erythropoietin, von Willebrand Factor or fragments thereof, ecto-enzymes such as Apyrase and so forth.
  • recombinant proteins such as CuZnSOD, MnSOD, Factor Xa Inhibitors, erythropoietin, von Willebrand Factor or fragments thereof, ecto-enzymes such as Apyrase and so forth.
  • recombinant proteins are described inter alia in U.S. Patent Nos. 5,126,252 and 5,360,729 (CuZnSOD) , U.S. Patent Nos . 5,270,195 and 5,246,847 (MnSOD), WO 91/01416 (von Willebrand factor fragment), U.S. Patent No. 5,783,421 (Factor Xa Inhibitor), and U
  • BBB blood-brain barrier
  • CNS drugs that are linked to the peptides of the subject invention are L-Dopa, Cortisone, tPA or phenobarbital .
  • Both the liver and the kidney are suitable for targeted drug delivery via the vascular system since they are well perfused tissues, involved in the metabolism of many endogenous and exogenous compounds. Targeting of liver and kidney is important particularly for cancer therapy and hepatitis B and C virus infection of the liver.
  • drugs for treatment of liver and kidney disease that are linked to peptides of the subject invention are antiviral drugs such as Interferon, Iododeoxyuridine, and Adenine arabinoside.
  • Adriamycin can be used for the treatment of renal cancer and Glucocerebrosidase 6-thioguanine for the treatment of Gaucher ' s disease.
  • EXAMPLE 4 Isolation of peptides which specif cally bind to undetermined targets on the blood vessels of Kaposi sarcoma (KS) tumor-bearing nude mice in vivo .
  • KS Kaposi sarcoma
  • KS cell line which was isolated from a non-HIV-infected patient, and which was recently shown to carry several markers of HIV- infected KS cells (Herndier et al . (1994), Aids 8(5): 575-581).
  • the KS cells (5xl0 6 ) as a mixture m Matri-gel (1:1 v/v) were injected into nude mice. After approximately 12-14 days, when well defined localized tumors were visualized (about 1.2 cm m diameter) , a mixture of the 15-mer library (described Example 1) together with M13 bacteriophage (10 12 :10 13 , respectively) was injected into the tail-vein.
  • the animal vascular system was perfused with PBS for an additional 4 minutes and then sacrificed. Tumors were excised, weighed, and kept on ice for all subsequent manipulations. Following homogenization and extensive washing, phage elution was carried out with starved bacteria. The eluated phage was titrated, amplified, and then subjected to additional rounds of biopanning m KS tumor bearing mice.
  • the KS-tumor eluted phages from two independent experiments (designated MKS1 and MKS2) , following three rounds of biopanning -in vivo, were analyzed by DNA sequencing.
  • TUV-R4B*#1 which comprises an EGR-containing peptide, binds specifically to KS-tumor.
  • EXAMPLE 5 Binding of EGR-containing peptides to melanoma and mammary tumor cells
  • Fig. 2 The results are demonstrated in Fig. 2.
  • the dose response curve of the binding of clone TUV-R4B-#1 demonstrated a different binding profile for the two organs tested.
  • the binding to the tumor was much higher than to the brain (Fig 2) , with a tumor/brain ratio of approximately 7.5 (Fig. 3) .
  • Each point is based on an average of 3 animals.
  • DA-3-p Mouse mammary carcinoma cells, DA-3-p (Fu X.-Y. Et al , Cancer Res., 1990, 50:227) were injected subcutaneously into the hind leg of Balb/C female mice, 6 weeks old, at 1 million cells/animal. After 10-15 days when tumor size was approximately 0.3-0.6g, phage mixture was injected into the tail vein in triplicates. The yield (%) of the binding [eluted phage (output) over input phage x 100] of the EGR- phage (clone TUV-R4B-#1) and of the unselected control phage, clone 14-4, are shown in Fig. 4.
  • the yield (%) of binding of the EGR-phage is several fold higher than that of the unselected control phage 14-4, indicating a selective enrichment of the EGR- phage on the mammary tumor vascular bed.
  • the comparison of binding of the EGR-phage to the mammary tumor and to the brain is demonstrated in Fig. 5.
  • the yield (%) of the binding to the mammary tumor is many fold higher than to the brain in all three animals tested.
  • the enrichment factor of the EGR-phage in tumor and brain i.e., the yield (%) of the EGR- phage over the yield (%) of the 14-4 phage in the tumor and in the brain
  • Fig. 6 The enrichment factor of the EGR-phage in the tumor was approximately 4 fold higher than enrichment factor in the brain, indicating a preferential homing of the EGR-phage to the mammary tumor vascular bed.
  • mice were separately injected into the foot pad of 8- 10 weeks old female Balb/C nude mice at 0.1-0.5 million cells per animal. After 8-14, weeks when tumor size was approximately 0.3-0.8g, phage mixture was injected into the tail vein. Phage elution and titration was monitored as described in Example 4.
  • Example 4 show that the EGR- containing phage binds specifically to various tumor tissues.lt is likely that the binding is to endothelial cells in the vascular bed. Such specific binding is a characteristic of the EGR-containing peptides of the subject invention.

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Abstract

L'invention porte sur de nouveaux peptides contenant de l'EGR, et leur utilisation pour le traitement de différents troubles et états. Lesdits peptides se fixent spécifiquement aux cellules épithéliales et à différentes cellules et différents tissus tumoraux.
PCT/US1999/004691 1998-03-04 1999-03-04 Isolement de peptides ligands specifiques et leur utilisation pour diriger certains produits pharmaceutiques sur des organes WO1999045020A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001057069A2 (fr) * 2000-02-02 2001-08-09 Transgene S.A. Peptides capables de ciblage
EP1315517A1 (fr) * 2000-09-04 2003-06-04 Hyo-Joon Kim Peptides mimetiques pour l'epitope de l'apolipoproteine b-100, concatemeres et ses peptides modifies, et composition de vaccin les comprenant
WO2010012850A1 (fr) * 2008-07-29 2010-02-04 Universitat Pompeu Fabra Peptides à capacité de pénétration cellulaire et utilisations de ceux-ci
AU2007200834B2 (en) * 2000-09-04 2010-10-14 Hyo-Joon Kim Mimetic peptides for epitope of apolipoprotein B-100, concatemer and modified peptides thereof, and the vaccine composition comprising the same

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5047333A (en) * 1987-12-22 1991-09-10 Eniricerche S.P.A. Method for the preparation of natural human growth hormone in pure form

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5047333A (en) * 1987-12-22 1991-09-10 Eniricerche S.P.A. Method for the preparation of natural human growth hormone in pure form

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001057069A2 (fr) * 2000-02-02 2001-08-09 Transgene S.A. Peptides capables de ciblage
WO2001057069A3 (fr) * 2000-02-02 2002-02-21 Transgene Sa Peptides capables de ciblage
EP1315517A1 (fr) * 2000-09-04 2003-06-04 Hyo-Joon Kim Peptides mimetiques pour l'epitope de l'apolipoproteine b-100, concatemeres et ses peptides modifies, et composition de vaccin les comprenant
EP1315517A4 (fr) * 2000-09-04 2005-02-09 Kim Hyo Joon Peptides mimetiques pour l'epitope de l'apolipoproteine b-100, concatemeres et ses peptides modifies, et composition de vaccin les comprenant
AU2007200834B2 (en) * 2000-09-04 2010-10-14 Hyo-Joon Kim Mimetic peptides for epitope of apolipoprotein B-100, concatemer and modified peptides thereof, and the vaccine composition comprising the same
WO2010012850A1 (fr) * 2008-07-29 2010-02-04 Universitat Pompeu Fabra Peptides à capacité de pénétration cellulaire et utilisations de ceux-ci
ES2334315A1 (es) * 2008-07-29 2010-03-08 Universitat Pompeu Fabra Peptidos con capacidad de penetracion celular y sus usos.

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