EP0680340A1 - Orientation amplifiee de groupes effecteurs vers des cellules cibles chez un animal - Google Patents

Orientation amplifiee de groupes effecteurs vers des cellules cibles chez un animal

Info

Publication number
EP0680340A1
EP0680340A1 EP94907865A EP94907865A EP0680340A1 EP 0680340 A1 EP0680340 A1 EP 0680340A1 EP 94907865 A EP94907865 A EP 94907865A EP 94907865 A EP94907865 A EP 94907865A EP 0680340 A1 EP0680340 A1 EP 0680340A1
Authority
EP
European Patent Office
Prior art keywords
reagent
functional group
effector
group
specific binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94907865A
Other languages
German (de)
English (en)
Inventor
Amin I. Kassis
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Harvard College
Original Assignee
Harvard College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Harvard College filed Critical Harvard College
Publication of EP0680340A1 publication Critical patent/EP0680340A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • A61K47/66—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 the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A61K47/665—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 the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells the pre-targeting system, clearing therapy or rescue therapy involving biotin-(strept) avidin systems
    • 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
    • A61K47/68—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6897—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
    • A61K47/6898—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies using avidin- or biotin-conjugated antibodies
    • 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
    • A61K47/68—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6899—Antibody-Directed Enzyme Prodrug Therapy [ADEPT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo

Definitions

  • the invention relates to the optimization of diagnostic and therapeutic uses of specific binding molecules, such as monoclonal antibodies. More particularly, the invention relates to use of such molecules to direct effector groups in an amplified fashion to specific target cells in an animal, including a human.
  • MAbs Monoclonal antibodies
  • the invention relates to targeting certain effector molecules to specific living cells, such as cancer cells, or their constituents in an animal, including a human.
  • sequential formation of specific binding pairs targets effector molecules in an amplified fashion to a particular target cell. Initially, a specific binding pair if formed between a target molecule on the target cell and a first reagent. This specific binding pair formation results from specific binding between the target molecule and a first functional group on the first regent. Sequential specific binding pairs are then formed between the first reagent and a second reagent, the second reagent and a third reagent, and so on.
  • each reagent has a second functional group that specifically binds to a first functional group present in the next reagent in the sequence.
  • Amplification occurs because the first and second functional groups on each reagent are multivalent.
  • the last reagent in the sequence has a first functional group, but has an effector group in place of the second functional group.
  • the effector group may be either a diagnostic marker or a therapeutic molecule.
  • Figure 1 shows a bound complex formed by the method according to the invention carried out through the third reagent step, using a diagnostic effector group.
  • T is a target cell
  • C is a chemical constituent thereon
  • FI and F2 are first and second functional groups respectively
  • N is a nuclide
  • the encircled numbers 1, 2 and 3 are first, second and third reagents, respectively. Dots indicate specific binding while straight lines extending from reagents indicate covalent conjugation.
  • Figure 2 shows a bound complex formed by the method according to the invention carried out through the third reagent step, using a therapeutic effector group. Symbols are as in Figure 1, except that R is a radionuclide.
  • Figure 3 shows a bound complex formed by an embodiment of the method according to the invention in which a therapeutic drug is an effector group on a third reagent, and an enzyme capable of releasing that drug from the third reagent is administered after formation of the bound complex.
  • the left panel shows the bound complex prior to enzyme addition, the right panel shows the bound complex and vicinity thereafter. Symbols are as in Figure 1 , except that D is a therapeutic drug and encircled E is an enzyme capable of releasing the drug from the third reagent.
  • Figure 4 shows a bound complex formed by an embodiment of the method according to the invention in which an enzyme that catalyzes conversion of a specific inactive prodrug to an active drug is conjugated to a third reagent, and the specific prodrug is added after formation of the bound complex.
  • Symbols are as in Figure 1 , except that encircled P is an inactive prodrug and D is an active drug.
  • the invention relates to a method for optimizing diagnostic and therapeutic uses of monoclonal antibodies (MAbs) and other specific binding ligands in animals afflicted with various diseases. More particularly, the invention relates to a method of targeting certain effector molecules to living cells, such as cancer cells, or their constituents in an animal, including a human.
  • the method according to the invention provides certain advantages over currently available methods for targeting effector molecules to specific cells. First, the method does not depend so heavily on preserving the immunointegrity of each and every antibody molecule, since it allows wasting of a significant proportion of the first reagents administered. These first reagents that have been damaged during chemical manipulation will simply fair to bind the target and will be swept from the animal's circulation.
  • the method of the invention uses the sequential formation of specific binding pairs to target effector molecules in amplified fashion to particular target cells or cell constituents in an animal, including a human.
  • a first specific binding pair is formed between a target molecule on a cell and a first reagent having a first functional group that specifically binds to that target molecule.
  • this first reagent will be an antibody, most preferably a monoclonal antibody, that specifically binds the target molecule on the cell and the first functional group will be an antigen binding site.
  • target molecules such as ligands or receptors
  • the first reagent is an antibody, receptor or ligand, it will be conjugated, preferably covalently, to a second functional group that allows the first reagent to form a specific binding pair with a second reagent.
  • preferred second functional groups include double stranded oligonucleotide (DSO), biotinylated DSO (BN-DSO), avidin-conjugated DSO (AV-DSO), single strand oligonucleotide-conjugated DSO (SSO-DSO), biotinylated SSO (BN-SSO), DNA intercalating agents (INT), biotinylated INT (SSO-INT), biotin that is conjugated to a peptide, protein or carbohydrate or otherwise rendered multimeric (BN), avidin (AV), single stranded oligonucleotide (SSO), and avidin-conjugated SSO (AV-SSO).
  • DSO double stranded oligonucleotide
  • BN-DSO biotinylated DSO
  • AV-DSO avidin-conjugated DSO
  • SSO-DSO single strand oligonucleotide-conjugated DSO
  • the reagents according to the invention may have first and second functional or effector groups directly linked together, or they may be connected via another molecule, such as human serum albumin, or any other non-antigenic molecule.
  • the first reagent is administered to an animal, preferably parenterally, and allowed to form a specific binding pair with a target molecule on a cell, to which the first functional group of the first reagent specifically binds. Any first reagent that has neither formed a specific binding pair with the target molecule nor yet been excreted from the body will be in the circulation of the animal.
  • a second reagent is then administered to the animal. Administration of the second reagent preferably involves the use of an amount of second reagent that exceeds (in molar terms) the amount of the first reagent remaining in the circulation at the time that the second reagent is administered.
  • the second reagent forms a specific binding pair with the first reagent, which is bound to the target molecule on a cell.
  • the ability of the first and second reagents to form a specific binding pair arises in the following manner.
  • the second functional group of the first reagent is selected, for example, from the functional groups shown in the left hand column of Table I.
  • the first functional group of the second reagent is selected, for example, from the functional groups shown in the right hand column of Table I, and is a functional group to which the second functional group of the first reagent specifically binds, as shown in Table I.
  • the second functional group of the first reagent specifically binds to the first functional group of the second reagent, resulting in the formation of a specific binding pair between the first and second reagents.
  • the second reagent does not have a second functional group, but rather has an effector group.
  • the effector group can have either a diagnostic function or a therapeutic function.
  • diagnostic effector groups useful in the method according to the invention include nuclides and radionuclides.
  • Therapeutic effector groups useful in the method according to the invention include therapeutic enzymes, drugs, toxins, radionuclides, nuclides, enzymes that catalyze the conversion of a prodrug into an active drug, and antisense oligonucleotides.
  • a third reagent may optimally be administered to the animal after allowing the second reagent to form a specific binding pair with the first reagent, such that the amount of third reagent exceeds the amount of second reagent remaining in the circulation of the animal at the time that the third reagent is administered.
  • this third reagent will comprise an enzyme that is capable of cleaving the chemical bond between the effector group and the second reagent.
  • the effector group of the second reagent is an enzyme capable of catalyzing conversion of a prodrug to an active drug, then the third reagent is the prodrug that is a substrate for the enzyme.
  • the second reagent has a second functional group that allows it to form a specific binding pair with a third reagent.
  • the second functional group of the second reagent is selected, for example, from the functional groups shown in the left hand column of Table I.
  • the method is carried out as described above, using the alternative embodiment in which the second reagent has a second functional group that allows it to form a specific binding pair with a third reagent.
  • a third reagent is administered, preferably parenterally, to the animal, such that the total amount of the third reagent exceeds the total amount of the second reagent that is in the circulation of the animal at the time that the third reagent is administered.
  • This third reagent is capable of forming a specific binding pair with the second reagent, because the third reagent has a first functional group to which the second functional group of the second reagent specifically binds.
  • This first functional group of the third reagent may be selected, for example, from the functional groups shown in the right hand column of Table I, and will be chosen to specifically bind to the second functional group of the second reagent, as shown in Table I.
  • the third reagent does not have a second functional group, but rather has an effector group.
  • the effector group can have either a diagnostic function or a therapeutic function.
  • diagnostic effector groups useful in the method according to the invention include nuclides and radionuclides.
  • Therapeutic effector groups useful in the method according to the invention include therapeutic enzymes, drugs, toxins, radionuclides, nuclides, enzymes that catalyze the conversion of a prodrug into an active drug, and antisense oligonucleotides.
  • a fourth reagent may optionally be administered to the animal after allowing the second reagent to form a specific binding pair with the first reagent, such that the amount of fourth reagent exceeds the amount of third reagent remaining in the circulation of the animal at the time that the fourth reagent is administered.
  • the effector group of the third reagent is a drug, toxin, radionuclide with short range emissions, therapeutic enzyme or antisense oligonucleotide
  • this fourth reagent will comprise an enzyme that is capable of cleaving the chemical bond between the effector group and the third reagent, thereby releasing the effector group.
  • the effector group of the third reagent is an enzyme capable of catalyzing the conversion of a prodrug to an active drug, then the fourth reagent is the prodrug that is a substrate for the enzyme.
  • the third reagent has a second functional group that allows it to form a specific binding pair with a fourth reagent.
  • the second functional group of the third reagent is selected, for example, from the functional groups shown in the left hand column of Table I.
  • the fourth reagent is administered to the animal, preferably parenterally, such that the total amount of the fourth reagent exceeds the total amount of the third reagent that is in the circulation of the animal at the time that the fourth reagent is administered.
  • This fourth reagent is capable of forming a specific binding pair with the third reagent, because the fourth reagent has a first functional group to which the second functional group of the third reagent specifically binds.
  • This first functional group of the fourth reagent may be selected, for example, from the functional groups shown in the right hand column of Table I, and will be chosen to specifically bind to the second functional group of the third reagent, as shown in Table I.
  • the fourth reagent does not have a second functional group, but rather has an effector group.
  • a fifth reagent may optionally be administered to the animal after allowing the fourth reagent to form a specific binding pair with the third reagent, such that the amount of fifth reagent exceeds the amount of fourth reagent present in the circulation of the animal at the time that the fifth reagent is administered.
  • this fifth reagent will comprise an enzyme that is capable of cleaving the chemical bond between the effector group and the fourth reagent, thereby releasing the effector group.
  • the effector group of the fourth reagent is an enzyme capable of catalyzing the conversion of a prodrug to an active drug, then the fifth reagent is the prodrug that is a substrate for the enzyme.
  • the fourth reagent has a second functional group that allows it to form a specific binding pair with a fifth reagent.
  • the second functional group of the fourth reagent is selected, for example, from the functional groups shown in the left hand column of Table I.
  • the fifth reagent is administered to the animal, preferably parenterally, such that the total amount of the fifth reagent exceeds the total amount of the fourth reagent that is in the circulation of the animal.
  • This fifth reagent is capable of forming a specific binding pair with the fourth reagent, because the fifth reagent has a first functional group to which the second functional group of the fourth reagent specifically binds.
  • This first functional group of the fifth reagent may be selected, for example, from the functional groups shown in the right hand column of Table I, and will be chosen to specifically bind to the second functional group of the fourth reagent, as shown in Table I.
  • the fifth reagent does not have a second functional group, but rather has an effector group.
  • the fifth reagent has a second functional group that allows it to form a specific binding pair with an additional reagent.
  • his amplification system can be extended beyond a fifth reagent to include a larger number of reagents, using the types of functional and effector groups described herein.
  • the point of the method is to target diagnostic or therapeutic effector molecules, to the vicinity of the target cell.
  • the method according to the invention both creates and amplifies a signal in the vicinity of the target cell.
  • the signal is created by specifically binding to the target cell a chemical constituent that can be detected or that can be specifically bound by yet another chemical constituent, including an effector molecule.
  • the signal is amplified by having multiple copies of this chemical constituent on the reagents used in the method of the invention.
  • FIG. 1 An example of such signal creation and amplification using a diagnostic effector group is shown in Figure 1.
  • a target cell, T has a chemical constituent, C, that is specifically bound by a first functional group, FI, of a first reagent, 1.
  • a second functional group, F2 on the first reagent are specifically bound by a first functional group, FI , on a second reagent, 2.
  • multiple copies of the second functional group, F2, on the second reagent are bound by a first functional group, FI , on a third reagent, 3, which in turn has multiple detectable nuclides attached.
  • a signal is created, since the signal C has been converted to a detectable signal N.
  • the signal has been amplified, since a single copy, in this case, of signal C has been replaced by many copies of signal N.
  • this system works equally well for use with a therapeutic effector molecule, in this case a therapeutic radionuclide, R.
  • many therapeutic effector groups are brought into the vicinity of the target cells. If, for example, the target cell is a cancer cell, such a high concentration of effector groups, in this case radionuclides, would greatly improve the chances of killing the target cancer cell.
  • Figure 3 illustrates a situation in which the effector group is a chemotherapeutic drug, D, that must be released from reagent 3 to be taken up by the target cell, T, in this case a cancer cell.
  • D chemotherapeutic drug
  • localization of high concentrations of a drug in the vicinity of a target cell can be achieved by conjugating to a final reagent an enzyme that is capable of catalyzing conversion of a prodrug to an active drug, as shown in Figure 4.
  • the enzyme, E. is conjugated to a third reagent, 3, and the inactive prodrug, P, is converted to an active drug, D, only in the vicinity of the target cell, T.
  • the terms set forth below are intended to have the following meanings, for purposes of defining the invention.
  • a specific binding pair is a pair of chemical constituents having a binding affinity for each other of at least 10 6 /_mole, or greater, including affinities beyond about 10 15 //mole. Examples illustrating this range are the specific binding pairs DSO-INT, having a binding affinity of about 10 * 7_mole, and AV-BIO, having a binding affinity of at least 10 l ⁇ /mole. A number of specific binding pairs within this range are set forth in Table I, above.
  • Specific binding for purposes of the invention, is binding between two chemical constituents with an affinity of at least about 10 6 /mole, including binding with affinities beyond about 10 10 /mole.
  • a first reagent for purposes of the invention, is a molecule that specifically binds to a chemical constituent on a target cell.
  • first reagents are antibodies that specifically bind antigens on the cell surface, ligands that specifically bind receptors on the cell surface, and receptors that specifically bind ligands on the cell surface.
  • antibody is intended to include monoclonal, chimeric, humanized and human antibodies, or fragments thereof, including Fab, F(ab) 2 ' and F(v) fragments.
  • Such first reagents have a first functional group that specifically binds to the chemical constituent on the target cells, and a second functional group that specifically binds to a second reagent.
  • a functional group is intended to mean a chemical constituent that mediates specific binding between a chemical constituent on a target cell and a first reagent, or between a first reagent and a second reagent, or between a second reagent and either a third reagent or an effector cell, and so on.
  • the second functional group of the first reagent specifically binds to the first functional group of the second reagent
  • the second functional group of the second reagent if present, specifically binds to the first functional group of a third reagent or to an effector cell
  • the second functional group of the third reagent if present, specifically binds to the first functional group of a fourth reagent or to an effector cell, and so on.
  • all reagents will be multivalent, i.e.. will have multiple copies of first and second functional groups, except that certain preferred first reagent may have as few as one or two first functional groups.
  • Preferred functional groups are set forth in the left hand column of Table I.
  • Some second, third, fourth, fifth, etc., reagents may have only a first functional group, with the second functional group being replaced by an effector group.
  • the effector group is a chemical constituent that confers upon a reagent a therapeutic or diagnostic function at or near the site of the target cell. Effector groups conferring diagnostic function include nuclides such as gadolinium and fluorine, which are detectable by MRI, and radionuclides, such as position emitters detectable by PET (e.g..).
  • Effector groups conferring therapeutic function include drugs, toxins, radionuclides, therapeutic enzymes and proteins, antisense oligonucleotides and enzymes capable of catalyzing conversion of prodrugs to active drugs.
  • Drugs include, but are not limited to common chemotherapeutic agents and antibiotics.
  • Therapeutic enzymes and proteins include, but are not limited to, tissue plasminogen activator, streptokinase and human DNase.
  • Antisense oligonucleotides include oligonucleotides having nuclease-resistant internucleotide linkages, such as phosphorothioate, phosphorodithioate, alkylphosphonate, phosphoramidate, and phosphotriester linkages, among other linkages and modifications well known in the art.
  • Enzymes capable of catalyzing conversion of a prodrug to an active drug include carboxypeptidase-19, which cleaves phenylalanine-linked methotrexate to yield active methotrexate, carboxypeptidase G2, which can cleave glutamate-linked bis-chlorobenzoic acid mustard to yield the active mustard, an alkylating agent, B-lactamase, which cleaves desacetyl vinblastine hydrazide-linked cephalosporine 20, and alkaline phosphatase, which dephosphorylates (and thus activates) phosphorylated anti-tumor drugs, such as mitomycin and etoposide.
  • carboxypeptidase-19 which cleaves phenylalanine-linked methotrexate to yield active methotrexate
  • carboxypeptidase G2 which can cleave glutamate-linked bis-chlorobenzoic acid mustard to yield the active mustard
  • an alkylating agent B-lac
  • Anti-human mammary cancer MAb B72.3 an IgG ⁇ antibody that reacts avidly with glycoprotein TAG72 is obtained by culturing in vitro the hybridoma cell line producing it (ATCC MB8108) in a Mini Flo-PathTM bioreactor (Amicon, Danvers, MA). The MAb is dissolved in 0.1 M NaHCO s 0.2 M NaCl (pH 8.5) at a concentration of 2 mg/ml. Biotinyl-N-hydroxysuccinimide ester (Sigma, St.
  • the MAb binds about 1 to 20 biotin groups per MAb molecule.
  • the carbohydrate moieties on the IgG sample ( 1-10 mg/ml, 30 mM acetate buffer pH 5.0) are oxidized by the addition of sodium periodate (100-200 ⁇ g in 100-200 ⁇ g water) in a 1:15 Ab:periodate molar ratio.
  • the mixture is kept on ice for 90 minutes and the oxidized IgG run on a Sephadex G-25 column.
  • ethidium bromide 400 ⁇ l of a 1 mg/ml water is added and the mixture kept in the dark at room temperature for 4 days.
  • the data indicate that when ethidium is added to IgG (100: 1 molar ratio), 30 moles of ethidium are conjugated per mole of IgG.
  • the fluorescent signal of the ethidium-IgG conjugate is enhanced 3-4 times in the presence of calf thymus DNA indicating the ability of the conjugated ethidium to intercalate with DNA.
  • HSA Human serum albumin
  • CNBr fragments Human serum albumin (HSA) (Sigma, St. Louis, MO) is digested to yield 7 CNBr fragments (HSA-f) as follows. First 100 mg HSA is denatured in 10 ml 0.5 M triethanolamine acetate buffer (pH 8.1 ) containing 6 M guanidine-HCl and 0.1% EDTA for 30 minutes at 50° C, then reduced by adding 200 mg dithiothreitol (DTT) and incubating at 50° C for 4 hours. Iodoacetamide is then added to 260 mM final concentration to alkylate the protein and prevent mixed disulf ide formation. The reaction is then allowed to proceed in the dark for 20 minutes at room temperature, and is stopped by the addition of excess DTT. The reaction mixture is then dialyzed against distilled water and incubated at 4° C overnight under nitrogen-saturated 70% formic acid with 100 mg cyanogen bromide (CNBr) to produce HSA-f (s
  • Two complementary 40-mer oligonucleotide phosphorothioate strands are prepared on a Model 8700 automated synthesizer (Milligen-Biosearch, Burlington, MA) using H-phosphonate chemistry on controlled pore glass (CPG), followed by oxidation with 0.2 M sulfur in carbon disulfide/pyridine/triethylamine (9:9:1 , v/v). Synthesis is carried out on a 5x 10 micromolar scale. Oligonucleoside phosphorothioates are purified by low pressure ion exchange chromatography (DEAE-cellulose, DE-50 Whatman), followed by reverse phase chromatography (C 18 ) and dialysis.
  • the maleimide-conjugated butyramide oligos are then added to HSA-f, following mild reduction in 2-mercaptoethanol.
  • the mixture is incubated overnight at 0 to 4° C, then separated by HPLC, as described for HSA- f, above.
  • DTPA diethylenetriaminepentaacetic acid, Aldrich; 7.1 mgs/ml
  • NHS-biotin N-hydroxysuccinimidobiotin ester, Sigma; 6.8 mgs/ml
  • DMF N,N-dimethylformamide, anhydrous, Aldrich
  • HSA human serum albumin, Sigma; 1.32 mgs/ml in 0.1 M NaHC0 3 , 0.2 M NaCl, pH 8.5
  • the reactions were carried out in glass tubes which had been washed in 6N HNO s and aqueous buffers were stored over chelating resin (Iminodiacetic acid, Sigma).
  • biotinylation To determine the extent of biotinylation, tritiated biotin (d-[8,9- 3 H] biotin, succinimidyl ester, Amersham; 100,000 cpm) was added to each reaction tube. After one hour at room temperature, an aliquot of each solution was counted, and the solutions were dialyzed into 0.15 M NaCl to remove unbound biotin. Aliquots of the dialysate were counted and the final biotin/HSA ratios determined by multiplying the fraction of counts recovered in the dialysate by the initial biotin:HSA ratio.
  • Biotin- or DSO-conjugated HSA-f is prepared as described in Examples 3 and 4.
  • Diethylenetriamine pentaacidic dianhydride (DTPA) (Aldrich,
  • m In acetate is prepared by addition of 1M sodium acetate to an equal volume of m In radionuclide solution for a final acetate concentration 0.5 M, and pH is adjusted to 5.5-6.0 using 2 M NaOH.
  • the u:* In acetate is mixed 1:1 with the conjugated HSA-f in 0.01 M acetate buffer (pH 6.0) and left at room temperature for 30 minutes.
  • Formed third reagent is separated from free lu In by HPLC under conditions described in Example 3.
  • Biotin- or DSO-conjugated HSA-f is prepared according to Examples 3 and 4.
  • One ml of a 1 mg/ml conjugated HSA-f solution is added to 0.67 ml of a solution containing 5 mM SnCl 2 , 40 mM potassium phhalate and 13.4 m sodium potassium tartrate (pH 5.6).
  • the mixture is incubated at room temperature for 24 hours, then I mCi pertechnetate is added and incubation at room temperature is continued for 1 hour.
  • Radioactive iodine (0.1 -1.0 mCi Na 123 I or Na 125 I) is mixed in a 5:1 ratio with DSO-conjugated HSA-f ( 1 to 10 mg) in PBS and transferred to a glass tube coated with 1 mg lodogen. After 5 minutes at room temperature, the formed reagent is separated from free iodine by HPLC, under conditions described in Example 3.
  • Biotin- or DSO-conjugated HSA-f is prepared according to Examples 3 and 4.
  • the radioastato group is then added using either N-succinimidyl-p- p[ 211 At]-Astatobenzoate, as described by Khawli and Kassis, Nucl. Med. Biol.
  • the LS 174T human adenocarcinoma (colon) cell line which expresses TAG72, is grown as a solid subcutaneous tumor in nude mice by injecting 10 7 cells subcutaneously into the flanks of 5 to 8 week old male Swiss-nu/nu nude mice. The tumors are allowed to grow to 3 to 5 mm in size. The mice are then injected intravenously with a first reagent having a first functional group that is a TAG72-binding site and a second functional group that is biotin. This reagent is prepared as described in Example 1 and is administered at a concentration of 10-100 mg/kg body weight. After 1 -2 days, the mice are injected with 10-100 ⁇ g avidin (Sigma,
  • mice which is a second reagent having the biotin binding sites of avidin as first and second functional groups.
  • the mice are injected with 10-100 mg/kg of a third reagent having a first functional group that is biotin and an effector group that is 99m Tc.
  • This third reagent is prepared as described in Example 6. The localization of the 99m Tc tumor is confirmed by tomography, within 24 hours.
  • mice bearing a 3 to 5 mm LS 174T tumor are prepared as described in Example 9. The mice are then injected with 10-100 mg/kg of a first reagent having a first functional group that is a TAG72 binding site and a second functional group that is avidin. After 24-48 hours, the mice are injected with
  • a second reagent having a first functional group that is biotin and a second effector group that is DTPA This reagent is prepared according to Example 3. After 1 -6 additional hours, the mice are injected with ln InCl 3 . After 1 -48 additional hours, localization of the lu In effector group to the tumor site is confirmed using tomography.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Nanotechnology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • General Engineering & Computer Science (AREA)
  • Medical Informatics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne le ciblage de certaines molécules effectrices sur des cellules vivantes spécifiques telles que des cellules cancéreuses ou leurs constituants chez l'animal y compris l'homme. Dans le procédé selon l'invention, la formation séquentielle de paires de liaison spécifiques cible des molécules effectrices de manière amplifiée sur une cellule cible particulière. Initialement une paire de liaison spécifique est formée entre une molécule cible sur la cellule cible et un premier réactif. Cette formation de paire de liaison spécifique résulte de la liaison spécifique entre la molécule cible et un premier groupe fonctionnel sur le premier réactif. Des paires de liaison séquentielles spécifiques sont ensuite formées entre le premier réactif et un deuxième réactif, le deuxième réactif ainsi qu'un troisième réactif et ainsi de suite. Ces paires de liaison spécifiques se forment du fait que chaque réactif comprend un deuxième groupe fonctionnel qui se lie spécifiquement à un premier groupe fonctionnel présent dans le réactif suivant dans la séquence. L'amplification se produit car le premier et le deuxième groupes fonctionnels sont multivalents. Généralement, le dernier réactif de la séquence comprend un premier groupe fonctionnel ainsi qu'un groupe effecteur à la place du deuxième groupe fonctionnel. Le groupe effecteur peut être soit un marqueur diagnostique soit une molécule thérapeutique.
EP94907865A 1993-01-21 1994-01-21 Orientation amplifiee de groupes effecteurs vers des cellules cibles chez un animal Withdrawn EP0680340A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US663693A 1993-01-21 1993-01-21
US6636 1993-01-21
PCT/US1994/000813 WO1994016734A1 (fr) 1993-01-21 1994-01-21 Orientation amplifiee de groupes effecteurs vers des cellules cibles chez un animal

Publications (1)

Publication Number Publication Date
EP0680340A1 true EP0680340A1 (fr) 1995-11-08

Family

ID=21721861

Family Applications (1)

Application Number Title Priority Date Filing Date
EP94907865A Withdrawn EP0680340A1 (fr) 1993-01-21 1994-01-21 Orientation amplifiee de groupes effecteurs vers des cellules cibles chez un animal

Country Status (6)

Country Link
EP (1) EP0680340A1 (fr)
JP (1) JPH08508713A (fr)
AU (1) AU6126794A (fr)
CA (1) CA2154471A1 (fr)
IL (1) IL108388A0 (fr)
WO (1) WO1994016734A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0670728A4 (fr) * 1992-11-12 1996-04-17 Molecular Dynamics Inc Porteurs lipophiles a base de peptides destines a l'administration ciblee de medicaments selon un concept rationnel de fixation des medicaments.
WO1997005266A1 (fr) * 1995-07-25 1997-02-13 Introgene B.V. Procedes et moyens d'apport cible de genes
IL133053A0 (en) * 1998-03-23 2001-03-19 Conjuchem Inc Local delivery of long lasting therapeutic agents
JP2008064475A (ja) * 2006-09-04 2008-03-21 Osaka Univ 標的物質の高感度検出方法、検出用キットおよび検出装置

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4863713A (en) * 1986-06-23 1989-09-05 The Board Of Trustees Of Leland Stanford Jr. Univ. Method and system for administering therapeutic and diagnostic agents
GB8809616D0 (en) * 1988-04-22 1988-05-25 Cancer Res Campaign Tech Further improvements relating to drug delivery systems
IT1245748B (it) * 1990-12-21 1994-10-14 Mini Ricerca Scient Tecnolog Preparazione includente anticorpi monoclonali biotinilati, avidina e biotina, per la diagnosi di affezioni tumorali e suo impiego
ATE210464T1 (de) * 1992-06-09 2001-12-15 Neorx Corp BIOTIN-DOTA KONJUGATE UND DEREN VERWENDUNG IN ßPRETARGETINGß VERFAHREN

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9416734A1 *

Also Published As

Publication number Publication date
AU6126794A (en) 1994-08-15
JPH08508713A (ja) 1996-09-17
CA2154471A1 (fr) 1994-08-04
WO1994016734A1 (fr) 1994-08-04
IL108388A0 (en) 1994-04-12

Similar Documents

Publication Publication Date Title
CA2163107C (fr) Detection et therapie ameliorees pour les lesions, a l'aide de conjugues d'avidine ou de biotine d'un metal chelate et de proteines
CA2223261C (fr) Apport ameliore d'agents diagnostiques ou therapeutiques a un site cible
EP0954340B1 (fr) Immunotherapie combinee ciblee contre le cancer
US5630996A (en) Two-step pretargeting methods using improved biotin-active agent conjugates
EP0505357B1 (fr) Procede de ciblage par anticorps d'agents diagnostiques ou therapeutiques
WO1989007269A1 (fr) Procede de preparation de conjugues de fragments d'anticorps
EP0920623B1 (fr) Therapie par capture de neutrons de bore dans laquelle des procedes de pre-ciblage sont utilises
US20020114808A1 (en) Methods and compositions for increasing the target-specific toxicity of a chemotherapy drug
US5578289A (en) In vivo binding pair pretargeting
US20020034511A1 (en) Pretargeting methods and compounds
US20070010014A1 (en) Compositions and methods for enhanced delivery to target sites
EP1507562B1 (fr) Compositions destinees a la radio-immunotherapie des tumeurs du cerveau
EP0832111A1 (fr) Avidine et streptavidine modifiees et leurs procedes d'utilisation
EP0680340A1 (fr) Orientation amplifiee de groupes effecteurs vers des cellules cibles chez un animal
WO1991011201A2 (fr) Composes
JP2942776B2 (ja) 蛋白質の放射標識に関する改良
JP4616991B2 (ja) 化学療法剤の標的特異的な毒性を増加させるための方法および組成物
US7713528B1 (en) Method for in vivo delivery of active compounds using reagent conjugate
JPH06505480A (ja) 安定化抗体断片
US20070009435A1 (en) Compositions and methods for enhanced delivery to target sites
CA2005162A1 (fr) Methode de ciblage par anticorps des agents de diagnostic ou des agents therapeutiques

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19950821

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL PT SE

17Q First examination report despatched

Effective date: 19960705

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19971229