EP0675737A1 - Reactifs a reactivite immunologique utilisant la dihydrofolate reductase - Google Patents

Reactifs a reactivite immunologique utilisant la dihydrofolate reductase

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Publication number
EP0675737A1
EP0675737A1 EP94905922A EP94905922A EP0675737A1 EP 0675737 A1 EP0675737 A1 EP 0675737A1 EP 94905922 A EP94905922 A EP 94905922A EP 94905922 A EP94905922 A EP 94905922A EP 0675737 A1 EP0675737 A1 EP 0675737A1
Authority
EP
European Patent Office
Prior art keywords
residue
group
reagent
dhfr
comprised
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Withdrawn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
EP94905922A
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German (de)
English (en)
Inventor
Robert A. Snow
Lawrence I. Kruse
Christopher D.V. Black
Clyde W. Shearman
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Wellcome Foundation Ltd
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Wellcome Foundation Ltd
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Publication of EP0675737A1 publication Critical patent/EP0675737A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being 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
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6881—Cluster-antibody conjugates, i.e. the modifying agent consists of a plurality of antibodies covalently linked to each other or of different antigen-binding fragments covalently linked to each other
    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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

  • This invention relates to the therapeutic treatment and diagnostic imaging of cancer by means of a tumor targeted sequential delivery system comprised of a primary non-radioactive targeting immunoreagent and a secondary radioactive delivery agent.
  • the number of chelating agents that can be attached to an immunoreactive protein is limited by the need to restrict chemical conjugations to sites removed from the immunoreactive recognition or binding sites of the protein;
  • the number of chelating agents that can be attached to an immunoreactive protein is limited by the number of available groups such as, for example, amino groups suitable for use in attachment of the chelating agents;
  • the number of chelating agents that can be attached to an immunoreactive protein is limited by the potential immunogenicity of the thus modified protein which, being highly derivatized, could be recognized by the immune system as haptenated;
  • radioimmunotherapy and diagnostic imaging with the various currently available radionuclide containing immunoreactive proteins can be less than optimal because these radiopharmaceuticals may bind to non-target normal tissue, which binding can result in undesirable toxicity to normal tissue during therapeutic applications as well as in high background signals during diagnostic imaging applications.
  • the present invention is directed to systems which are useful in the therapeutic treatment and diagnostic imaging of tissue, particularly of cancerous tissue.
  • tissue particularly of cancerous tissue.
  • systems comprise a tumor targeted sequential delivery system comprised of a primary non-radioactive targeting immunoreagent and a secondary radioactive delivery agent.
  • the present invention is directed to a non-radioactive targeting immunoreagent (sometimes hereinafter referred to as NRTIR) comprised of the residue of a receptor moiety, a linking group, and the residue of an immunoreactive material, which
  • NRTIR non-radioactive targeting immunoreagent
  • NRTIR is administered to a tissue of interest and will bind to sites on the surfaces of cells thereof.
  • the present invention is also directed to a radioactive delivery agent (sometimes hereinafter referred to as RDA) comprised of the residue of a ligand which has an affinity for non-covalent binding to a receptor moiety, a linking group, and the residue of a radioactive agent.
  • RDA radioactive delivery agent
  • This RDA is administered to the environs of the tissue which contains said NRTIR bound thereto.
  • the ligand residue of this RDA will non-covalently bind to the receptor of said NRTIR which is bound to the cells of said tissue of interest.
  • an effective amount of radioactivity is provided to said tissue.
  • RDA which is unbound to NRTIR can be removed rapidly from the environs of the tissue.
  • the present invention comprises an NRTIR comprised of the residue of a receptor moiety which is comprised of the residue of a proteinaceous active site of a dihydrofolate reductase enzyme (sometimes hereinafter referred to as a DHFR) , a linking group, and the residue of an immunoreactive material.
  • the present invention also comprises an RDA comprised of a ligand which has an affinity for non-covalent binding to a DHFR receptor moiety, a linking group, and the residue of a radioactive agent.
  • the present invention is directed to an NRTIR comprised of a residue of the proteinaceous active site of a dihydrofolate reductase enzyme, a linking group, and the residue of an immunoreactive material such as a tumor targeting antibody together with an RDA comprised of the residue of a ligand which has an affinity for non-covalent binding to said DHFR receptor moiety, a linking group, and the residue of a radioactive agent comprised of the residue of a chelating agent and a radionuclide.
  • the NRTIR of system A is comprised of (n) DHFR moieties, each of which can non-covalently bind an RDA comprised of the residue of a ligand with an affinity for non-covalent binding to a DHFR and of (m) radioactive agents where each of n and m is independently an integer greater than zero.
  • the total number of radioactive agents capable of being bound per antigen is then the product of (n) multiplied by (m) .
  • radioimmunoconjugates comprised of an immunoreactive protein conjugated to (c) radioactive agents wherein the value of (c) is an integer greater than zero and is limited to the number of conjugations that can be performed on said immunoreactive protein while retaining the immunoreactivity for said antigen.
  • This limit to the degree of modification of the immunoreactive protein also applies to the NRTIR of System A, and the value of (n) will be approximately the same as the value of (c) .
  • the non-covalent binding of the RDA to the antigen-bound NRTIR of the present invention will amplify the maximum number of radioactive agents bound per antigen by a factor of approximately (m) over the maximum value (c) available in previously available radioimmunoconjugates.
  • the present invention is directed to an NRTIR comprised of the residue of a ligand which exhibits an affinity for non-covalent binding to a receptor moiety, a linking group, and the residue of an immunoreactive material, which NRTIR is administered to a tissue of interest and will bind to sites on the surfaces of cells thereof.
  • the present invention is also directed to an RDA comprised of the residue of a receptor moiety for which a ligand has an affinity for non-covalent binding, a linking group, and the residue of a radioactive agent, which RDA is administered to the environs of the tissue which contains the NRTIR of this embodiment bound thereto.
  • the ligand of the RDA of this embodiment will non-covalently bind to the receptor of the NRTIR which is bound to the surface of the cells of said tissue of interest.
  • an effective amount of radioactivity is provided to said tissue.
  • RDA which is unbound to NRTIR can be removed rapidly from the environs of the tissue.
  • the present invention comprises a NRTIR comprised of the residue of a ligand which has an affinity for non- covalent binding to a DHFR receptor moiety, a linking group, and the residue of an immunoreactive material.
  • the present invention also comprises an RDA comprised of the residue of a DHFR receptor moiety, a linking group, and the residue of a radioactive agent.
  • the present invention is directed to a NRTIR comprised of the residue of a ligand which has an affinity for non-covalent binding to a DHFR receptor moiety, a linking group, and the residue of an immunoreactive material such as a tumor targeting antibody.
  • the present invention also comprises an RDA comprised of the residue of a DHFR receptor moiety, a linking group, and the residue of a radioactive agent comprised of the residue of a chelating agent and a radionuclide.
  • the NRTIR of System B is comprised of (n) residues of ligands that have an affinity for non-covalent binding to a DHFR, each of which can non-covalently bind an RDA comprised of a DHFR and of (m) radioactive agents where each of n and m is independently an integer greater than zero.
  • the total number of radioactive agents capable of being bound per antigen is then the product of (n) multiplied by (m) . This is in contrast to the binding to cell surface antigen of previously available radioimmunoconjugates comprised of an immunoreactive protein conjugated to (c) radioactive agents.
  • the value of (c) is limited to the number of radioactive agents that can be linked or conjugated to the immunoreactive protein while retaining the immunoreactivity for said antigen.
  • This limit to the degree of modification of the immunoreactive protein also applies to the NRTIR of System B, and the value of (n) will be approximately the same as the value of (c) .
  • NRTIR of the present invention will amplify the maximum number of radioactive agents bound per antigen by a factor of approximately (m) over the maximum value (c) available in previously available radioimmunoconjugates.
  • the present invention is also directed to pharmaceutical and diagnostic compositions comprising an NRTIR and a pharmaceutically acceptable carrier, and to pharmaceutical and diagnostic compositions comprising an RDA and a pharmaceutically acceptable carrier.
  • the present invention is further directed to therapeutic methods comprising the administration, in vitro or in vivo, of a therapeutically effective amount of NRTIR to the environs of a tissue of interest of a patient undergoing such therapy, followed after the lapse of an effective period of time by the subsequent administration of a therapeutically effective amount of RDA to said tissue.
  • a therapeutically effective amount of NRTIR to the environs of a tissue of interest of a patient undergoing such therapy, followed after the lapse of an effective period of time by the subsequent administration of a therapeutically effective amount of RDA to said tissue.
  • the present invention is further directed to diagnostic imaging methods comprising the sequential administration, in vitro or in vivo, of a diagnostic imaging effective amount of an NRTIR to the environs of a tissue of interest of a patient undergoing such diagnostic imaging, followed after a lapse of an effective period of time by the subsequent administration of a diagnostic imaging effective amount of RDA to said tissue.
  • diagnostic imaging methods comprising the sequential administration, in vitro or in vivo, of a diagnostic imaging effective amount of an NRTIR to the environs of a tissue of interest of a patient undergoing such diagnostic imaging, followed after a lapse of an effective period of time by the subsequent administration of a diagnostic imaging effective amount of RDA to said tissue.
  • said effective period of time said NRTIR will bind to sites on cells of said tissue of interest and unbound NRTIR will be removed from the environs of the tissue.
  • an image of all or part of said tissue of interest is obtained.
  • the present invention provides advantages compared to currently available targeting immune reagents. For example: the total amount of a therapeutically effective amount and of a diagnostic imaging effective amount of radioactive agent delivered to a tissue site can be achieved with specificity and in amplification over that which can be otherwise achieved with currently available targeting immune reagents; sequential delivery to target tissue of the NRTIR and the RDA of this invention can reduce the exposure of non-targeted tissues to damage from radiation; the non-covalent binding of the ligand to the receptor occurs with high affinity and is selective; the NRTIR and the RDA can be used in both therapeutic and diagnostic imaging applications; the above-described NRTIR can accumulate at a tumor tissue site in vivo while it is not substantially accumulated at normal tissue sites; the in vivo residence half life of the above- described NRTIR is long enough to permit its accumulation at a tumor site; the in vivo residence half life of the above- described RDA is shorter than the residence half life of the above-described NRTIR;
  • the NRTIR can comprise a wide variety of immunoreactive groups, linking groups, and DHFR active site residues in System A, and a wide variety of immunoreactive groups, linking groups, and ligand residues which have an affinity for non-covalent binding to DHFR active site residues in System B
  • the RDA can comprise a wide variety of spacing, linking and chelating groups, radionuclides, and ligand residues which ligands have an affinity for non-covalent binding to DHFR in System A, and a wide variety of spacing, linking and chelating groups, radionuclides, and ligand residues which ligands have an affinity for non-covalent binding to DHFR in System A, and a wide variety of spacing, linking and chelating groups, radionucli
  • NRTIR non- radioactive targeting immunoreagent
  • RDA radioactive delivery agent
  • Z is the residue of an immunoreactive group
  • Rec is the residue of a receptor, preferably a DHFR;
  • D is the residue of a ligand that has an affinity for non-covalent binding to a receptor, preferably to a DHFR receptor;
  • DHFR ligand is the residue of a ligand that has an affinity for non-covalent binding to a DHFR active site
  • TMP is the residue of a ligand comprised of a trimethoprim analog
  • MTX is the residue of a ligand comprised of a methotrexate analog
  • Li and L 2 are each independently the residue of a linking group that may independently contain a spacing group
  • Q is the residue of a chelating group
  • M is a radionuclide; and n and m are each independently an integer greater than zero. Preferred embodiments of these materials are further described below.
  • residue is used herein in context with a chemical entity.
  • Said chemical entity is comprised of, for example, a ligand, or a trimethoprim analog, or a methotrexate analog, or a receptor moiety, or a proteinaceous active site of a dihydrofolate reductase enzyme, or a DHFR, or a chelating group, or a radioactive agent, or a linking group, or a protein reactive group, or an immunoreactive group, or an immunoreactive material, or an immunoreactive protein, or an antibody, or an antibody fragment, or a cross ⁇ linking agent such as a heterobifunctional cross-linking agent, or a spacing group.
  • the term "residue” is defined as that portion of said chemical entity which exclusively remains when one or more chemical bonds of which said chemical entity is otherwise comprised when considered as an independent chemical entity, is altered, modified, or replaced to comprise one or more covalent bonds to one or more other chemical entities.
  • the residue of a chelating group is comprised of a chelating group which is at least monovalently modified through attachment to the residue of another chemical entity such as, for example, to the residue of a linking group.
  • the immunoreactive group, Z can be selected from a wide variety of naturally occurring or synthetically prepared materials, including, but not limited to enzymes, amino acids, peptides, polypeptides, proteins, lipoproteins, glycoproteins, lipids, phospholipids, hormones, growth factors, steroids, vitamins, polysaccarides, viruses, protozoa, fungi, parasites, rickettsia, molds, and components thereof, blood components, tissue and organ components, pharmaceuticals, haptens, lectins, toxins, nucleic acids (including oligonucleotides) , antibodies (monoclonal and polyclonal) , anti-antibodies, antibody fragments, antigenic materials (including proteins and carbohydrates), avidin and derivatives thereof, biotin and derivatives thereof, and others known to one skilled in the art.
  • an immunoreactive group can be any substance which when presented to an immunocompetent host will result in the production of a specific antibody capable of binding with
  • Preferred immunoreactive groups are antibodies and various immunoreactive fragments thereof, as long as they contain at least one reactive site for reaction with the reactive groups on the residue of the receptor moiety in System A or ligand species in System B or with linking groups (L) as described herein. That site can be inherent to the immunoreactive species or it can be introduced through appropriate chemical modification of the immunoreactive species.
  • L linking groups
  • antibody fragment refers to an immunoreactive material which comprises a residue of an antibody, which antibody characteristically exhibits an affinity for binding to an antigen.
  • affinity refers to the thermodynamic expression of the strength of interaction or binding between an antibody combining site (or other ligand) and an antigenic determinant (or receptor)and, thus, of the sterochemical compatibility between them; as such it is the expression of the equilibrium or association constant for the antibody-antigen (or ligand-receptor) interaction.
  • Antibody fragments exhibit at least a percentage of said affinity for binding to said antigen, said percentage being in the range of 0.001 per cent to 1,000 per cent, preferably 0.01 per cent to 1,000 per cent, more preferably 0.1 per cent to 1,000 per cent, and most preferably 1.0 per cent to 1,000 per cent, of the relative affinity of said antibody for binding to said antigen.
  • An antibody fragment can be produced from an antibody by a chemical reaction comprising one or more chemical bond cleaving reactions; by a chemical reaction comprising one or more chemical bond forming reactions employing as reactants one or more chemical components selected from a group comprised of amino acids, peptides, carbohydrates, linking groups as defined herein, spacing groups as defined herein, protein reactive groups as defined herein, and antibody fragments such as are produced as described herein; and by a molecular biological process, a bacterial process, or by a process comprised of a genetic engineering of antibody genes.
  • An antibody fragment can be derived from an antibody by a chemical reaction comprised of one or more of the following reactions:
  • bonds being selected from, for example, carbon-nitrogen bonds, sulfur-sulfur bonds, carbon-carbon bonds, carbon-sulfur bonds, and carbon-oxygen bonds, and wherein the method of said cleavage is selected from:
  • a catalysed chemical reaction comprising the action of a biochemical catalyst such as an enzyme such as papain or pepsin which to those skilled in the art is known to produce antibody fragments commonly referred to as Fab and Fab'2, respectively;
  • a biochemical catalyst such as an enzyme such as papain or pepsin which to those skilled in the art is known to produce antibody fragments commonly referred to as Fab and Fab'2, respectively;
  • a catalysed chemical reaction comprising the action of an electrophilic chemical catalyst such as a hydronium ion which, for example, favorably occurs at a pH equal to or greater than 7;
  • a catalysed chemical reaction comprising the action of a nucleophilic catalyst such as a hydroxide ion which, for example, favorably occurs at a pH equal to or greater than 7; and
  • a chemical reaction comprised of a substitution reaction employing a reagent which is consumed in a stoichiometric manner such as a substitution reaction at a sulfur atom of a disulfide bond by a reagent comprised of a sulfhydryl group;
  • a chemical reaction comprised of a reduction reaction such as the reduction of a disulfide bond
  • a chemical reaction comprised of an oxidation reaction such as the oxidation of a carbon-oxygen bond of a hydroxyl group or the oxidation of a carbon-carbon bond of a vicinal diol group such as occurs in a carbohydrate moiety
  • an antibody fragment can be derived by formation of one or more non-covalent bonds between one or more reactants.
  • Such non-covalent bonds are comprised of hydrophobic interactions such as occur in an aqueous medium between chemical species that are independently comprised of mutually accessible regions of low polarity such as regions comprised of aliphatic and carbocyclic groups, and of hydrogen bond interactions such as occur in the binding of an oligonucletide with a complementary oligonucletide; or
  • an antibody fragment can be produced as a result of the methods of molecular biology or by genetic engineering of antibody genes, for example, in the genetic engineering of a single chain immunoreactive group or a Fv fragment.
  • An antibody fragment can be produced as a result a combination of one or more of the above methods.
  • the immunoreactive group can be an enzyme which has a reactive group for attachment to the receptor moiety in System A or ligand species in System B or to a linking group as described below.
  • Representative enzymes include, but are not limited to, aspartane, aminotransaminase, alanine aminotransaminase, lactate dehydrogenase, creatine phosphokinase, gamma glutamyl transferase, alkaline acid phosphatase, prostatic acid phosphatase, horseradish peroxidase and various esterases.
  • the immunoreactive group can be modified or chemically altered to provide reactive groups for attaching to the residues of the receptor moiety in System A or ligand species in System B or to a linking group as described below by techniques known to those skilled in the art.
  • Such techniques include the use of linking moieties and chemical modification such as described in WO-A-89/02931 and WO-A-89/2932, which are directed to modification of oligonucleotides, and U.S. Patent No. 4,719,182.
  • compositions of this invention are for the diagnostic imaging of tumors and the radiological treatment of tumors .
  • Preferred immunological groups therefore include antibodies (sometimes hereinafter referred to as Ab) to tumor- associated antigens.
  • Specific non-limiting examples include B72.3 and related antibodies (described in U.S. Patent Nos.
  • ING-1 which are described in International Patent Publication O-A-90/02569, B174 and related antibodies which recognize squamous cell carcinomas, B43 and related antibodies which are reactive with certain lymphomas and leukemias, and anti-HLB and related monoclonal antibodies.
  • An especially preferred antibody is ING-1.
  • the term "receptor” refers to a chemical group in a molecule which comprises an active site in said molecule, or to an array of chemical groups in a molecule which comprise one or more active sites in said molecule, or to a molecule comprised of one or more chemical groups or one or more arrays of chemical groups, which group or groups or array of groups comprise one or more active sites in said molecule.
  • An "active site” of a receptor has a specific capacity to bind to or has an affinity for binding to a ligand.
  • ligand refers to a molecule comprised of a specific chemical group or a specific array of chemical groups which molecule, group, or array of groups is complementary to and has a specific affinity for binding to a receptor, especially to an active site in a receptor.
  • receptors include enzymes which catalyze chemical reactions, and cell surface receptors which bind hormones and drugs.
  • the sites of specific binding of substrates for said enzymes and of hormones and drugs of said cell surface receptors are examples of active sites of said receptors, and the respective substrates, hormones, and drugs are examples of ligands for said receptors.
  • Preferred receptors (Rec) in System A and System B are comprised of the residue of an active site of an enzyme, and preferred ligands in System A and System B are comprised of the residue of a substrate for said active site of said enzyme.
  • An especially preferred receptor is comprised of the active site of any protein having dihydrofolate reductase (DHFR) activity.
  • Said DHFR can be isolated, in whole or in part, from any source and used in this invention without further modification, or it can be chemically modified before or after isolation for use in this invention, or it can be modified by well known techniques of molecular biology and isolated for use in this invention, or said molecular biology modified DHFR can be chemically modified before or after isolation for use in this invention as long as the DHFR active site is maintained in such use.
  • DHFR activity is defined in terms of units; one said unit of DHFR enzyme activity is defined as the amount of material needed to convert 1.0 icromole of 7,8-dihydrofolate and NADPH to 5,6,7,8- tetrahydrofolate and NADP per minute at pH 6.5 and 25°C.
  • a chemical species comprised of a group or array of groups that catalyze the conversion of
  • 7,8-dihydrofolate and NADPH to 5, 6,7,8-tetrahydrofolate and NADP has DHFR activity and such group or array of groups comprises a dihydrofolate reductase active site, i.e., a DHFR.
  • a DHFR dihydrofolate reductase active site
  • the modification of a DHFR by known techniques of molecular biology or by chemical modification may reduce the protein's ability to catalyze the conversion of 7, 8-dihydrofolate and NADPH to 5, 6,7,8-tetrahydrofolate and NADP yet retain the protein's capacity to bind a ligand.
  • ligands that have an affinity for binding to the active site in said DHFR include 7,8- dihydrofolate, derivatives and analogs of 7,8- dihydrofolate, and residues of said derivatives and analogs which may be agonists or antagonists of DHFR activity with respect to 7,8-dihydrofolate.
  • Preferred examples of ligands which exhibit an affinity for binding to a DHFR are comprised of antifolate drugs as described in "Principals of Drug Action, The Basis of Pharmacology", Third Edition, Churchill Livingstone, New York, 1990, Pratt W. B. and Taylor P., Editors, page 623 which are antagonists of DHFR activity with respect to
  • the DHFR is comprised of a non-human protein, preferably comprised of a bacterial or protozoal protein, and more preferably of a bacterial protein.
  • said DHFR is comprised of protein derived from S. aureus or from E. Coli. More preferably, said DHFR is comprised of protein derived from E. Coli (strain CV634) infected with the plasmid pCV29 which harbors the E. coli DHFR gene.
  • the DHFR is comprised of a human protein.
  • said DHFR is comprised of a recombinant human protein.
  • said DHFR is comprised of a recombinant human protein which is modified by genetic engineering techniques, which modifications comprise the independent incorporation, substitution, insertion, and deletion of specific amino acids in a peptide sequence of said protein.
  • the DHFR comprised of a thus modified recombinant human protein is comprised of an active site which has an affinity for binding to substrates, which affinity is greater than the affinity of natural human protein for said substrates.
  • the DHFR comprised of a thus modified recombinant human protein is comprised of an active site which has an affinity for binding to residues of the antifolate drugs, methotrexate and trimethoprim, which affinity is greater than the affinity of natural human protein for binding to the residues of said antifolate drugs.
  • the DHFR comprised of a thus modified recombinant human protein is comprised of an active site which has an affinity for binding to residues of the antifolate drug, trimethoprim, which affinity is greater than the affinity of natural human protein for binding to the residues of said antifolate drug.
  • the Z-L-X of System A is comprised of a fusion protein.
  • a fusion protein As used herein, the term
  • fusion protein refers to a genetically engineered material comprised of a protein whose coding region is comprised of the coding region of a residue of a first protein fused, in frame, to the coding region of a residue of a second protein.
  • said fusion protein is comprised of a protein whose coding region is comprised of the coding region of a residue of an immunoreactive reagent fused, in frame, to the coding region of one or more residues of a DHFR.
  • said fusion protein is comprised of a residue of an immunoreactive reagent fused to one or more residues of a DHFR.
  • said fusion protein is comprised of residues of DHFR fused to an immunoglobulin heavy chain in the CHi region, such that when combined with an appropriate light chain the said fusion protein comprises an Fab fragment linked to one or more DHFR.
  • said fusion protein can be comprised of one or more DHFR fused to an immunoglobulin heavy chain in the CH 2 or in the CH3 region.
  • said fusion protein when comprised of an immunoglobulin light chain, can be comprised of a Fab'2 fragment linked to one or more DHFR.
  • said fusion protein can be comprised of one or more DHFR fused to the C-terminal end of an immunoglobulin single-chain construct and thus be comprised of an Fv fragment linked to one or more DHFR.
  • the above genetically engineered fusion protein comprising Z-L-X of System A can be comprised a protein whose coding region is independently comprised of the coding region of a residue of a human or of a non-human first protein fused, in frame, to the coding region of a residue of a human or non-human second protein.
  • said coding regions are independently human and bacterial or modified by genetic engineering techniques as above.
  • the fusion protein is comprised of a protein whose coding region is comprised of the coding region of a residue of a human immunoreactive reagent fused, in frame, to the coding region of one or more residues of a bacterial or human DHFR or a genetically engineered modified bacterial or human DHFR.
  • the fusion protein is comprised of a thus modified recombinant human DHFR comprised of an active site which has an affinity for binding to a ligand, which affinity is greater than the affinity of natural human protein for said ligand.
  • the fusion protein is comprised of a thus modified recombinant human DHFR comprised of an active site which has an affinity for binding to a ligand comprised of a residue of an antifolate drug such as methotrexate or trimethoprim, which affinity is greater than the affinity of natural human protein for binding to the residue of said ligand.
  • the fusion protein is comprised of a thus modified recombinant human DHFR comprised of an active site which has an affinity for binding to a ligand comprised of a residue of trimethoprim, which affinity is greater than the affinity of natural human protein for binding to the residue of said ligand.
  • the binding of a ligand to a receptor can comprise the formation of a covalent bond or it can comprise a non-covalent interaction.
  • the binding of a ligand to a receptor comprises a non- covalent interaction, sometimes herein referred to as non-covalent binding.
  • the DHFR is covalently linked, i.e., conjugated, to an immunoreactive group, preferably to an antibody or to an antibody fragment, most preferably to ING-1, to form the NRTIR [i.e., Z-(L ⁇ -Rec) n ] of the System.
  • the DHFR as a component of a radioactive delivery agent i.e., an RDA, Rec-(L2 ⁇ Q-M) m
  • a radioactive delivery agent i.e., an RDA, Rec-(L2 ⁇ Q-M) m
  • the chelating group is associated with a radionuclide.
  • the chelating group is TMT (described hereinbelow)
  • the linking group is as described below
  • the radionuclide is 90 ⁇
  • the RDA in System B is comprised of a DHFR that contains one or more radionuclides that are covalently attached, either directly to one or more components of the DHFR or to one or more components that are attached by a linking group as described below to the DHFR.
  • said covalently attached radionuclide is a radioisotope of iodine attached to an aromatic ring-containing moiety.
  • the RDA in System B is comprised of a DHFR that contains one or more radionuclides that are covalently attached, either directly to one or more components of the DHFR or to one or more components that are attached by a linking group as described below to the DHFR.
  • said covalently attached radionuclide is selected from a radioisotope of technicium and rhenium attached to a group comprised of a sulfur atom.
  • chemical conjugation can be achieved, for example, by a technique comprising the use of a linking group (Li) which is introduced through modification of, for example, a site on an immunoreactive group.
  • a linking group Li
  • activated groups such as activated ethylene groups (e.g., maleimide groups) on to amine groups such as lysine epsilon-amines of a protein is represented in Scheme 1.
  • Other techniques include the use of heterobifunctional linking moieties and chemical modifications such as the examples described in U. S. Patent No. 4,719,182.
  • those chemicals such as SMCC which are commonly commercially available, for example, from
  • Suitable reactive sites on the immunoreactive material and on the receptor moiety include: amine sites of lysine; terminal peptide amines; carboxylic acid sites, such as are available in aspartic acid and gluta ic acid; sulfhydryl sites; carbohydrate sites; activated carbon-hydrogen and carbon-carbon bonds which can react through insertion via free radical reaction or nitrene or carbene reaction of a so activated residue; sites of oxidation; sites of reduction; aromatic sites such as tyrosine; and hydroxyl sites.
  • the ratio of DHFR to immunoreactive group such as an antibody can vary widely from about 0.5 to 10 or more.
  • mixtures comprised of immunoreactive groups which are unmodified and immunoreactive groups which are modified with DHFR are also suitable.
  • Such mixtures can have a bulk ratio of DHFR to immunoreactive group of from about 0.1 to about 10.
  • the mole ratio of DHFR to immunoreacative group is from about 1:1 to about 6:1. It is specifically contemplated that with knowledge of the DNA sequence that encodes DHFR, especially bacterial and human DHFR, a fusion protein can be made between the antibody and the DHFR, or portions thereof, through the use of genetic engineering techniques. It is specifically contemplated that in all of these compositions of DHFR bound to antibody, DHFR retains a capacity to bind to the ligands described in the invention.
  • the ratio of ligand to immunoreactive group such as an antibody can vary widely from about 0.5 to 10 or more.
  • mixtures comprised of immunoreactive groups which are unmodified and immunoreactive groups which are modified with ligand are also suitable.
  • Such mixtures can have a bulk ratio of ligand to immunoreactive group of from about 0.1 to about 10.
  • the mole ratio of ligand to immunoreactive group is from about 1:1 to about 6:1.
  • the conjugate is purified by passage of the material through a gel permeation column such as Superose 6 using an appropriate elution buffer or by elution from a HPLC column such as a Shodex WS- 803F size exclusion column. Both these methods separate the applied materials by molecular size resulting in the elution of the antibody/DHFR conjugate in a different fraction from any residual non-conjugated DHFR.
  • the concentrations of the antibody in the conjugate solutions are determined by the Bradford (BioRad Catalog # 500-0001) method using bovine immunoglobulin as the protein standard.
  • the conjugate is purified by passage of the material through a gel permeation column such as Superose 6 using an appropriate elution buffer or by elution from a HPLC column such as a Shodex WS-803F size exclusion column. Both these methods separate the applied materials by molecular size resulting in the elution of the antibody/ligand conjugate in a different fraction from any residual non-conjugated ligand.
  • the concentrations of the antibody in the conjugate solutions are determined by the BioRad protein assay using bovine immunoglobulin as the protein standard.
  • the ability of the antibody to bind to its target antigen following conjugation to DHFR can be assayed by ELISA or flow cytometry.
  • a 30 cm x 7.5 mm TSK-G3000SW size-exclusion HPLC column (Supelco) fitted with a guard column of the same material can be used to determine the amount of aggregation in the final conjugate.
  • the ability of the antibody to bind to . its target antigen following conjugation to the residue of a ligand can be assayed by ELISA or flow cytometry.
  • a 30 cm x 7.5 mm TSK-G3000SW size-exclusion HPLC column (Supelco) fitted with a guard column of the same material can be used to determine the amount of aggregation in the final conjugate.
  • the dihydrofolate reductase enzymic activity of the antibody-associated DHFR can be assayed by following the oxidation of pyridine nucleotides during the reduction of folate to tetrahydrofolate by DHFR as described by Mathews and Huennekens ( J. Biol. Chem. 238, 3436-3442 (1963)).
  • This method can also be used to assay the DHFR inhibitory effects of the novel DHFR binding ligands which are modified to include chelating agents as described in this invention.
  • the dihydrofolate reductase enzymic activity of the chelating agent-associated DHFR can be assayed by following the oxidation of pyridine nucleotides during the reduction of folate to tetrahydrofolate by DHFR as described by Mathews and Huennekens ( J. Biol. Chem. 238, 3436-3442 (1963)).
  • This method can also be used to assay the DHFR inhibitory effects of the novel ligands which have an affinity for binding to DHFR and which are linked to immunoreactive groups as described in this invention.
  • Li and L 2 in System A and System B are independently a chemical bond or the residue of a linking group.
  • the phrase "residue of a linking group” as used herein refers to a moiety that remains, results, or is derived from the reaction of a protein reactive group with a reactive site on a protein.
  • the phrase "protein reactive group” as used herein refers to any group which can react with functional groups typically found on proteins. However, it is specifically contemplated that such protein reactive groups can also react with functional groups typically found on relevant nonprotein molecules.
  • the linking groups Li and L 2 useful in the practice of this invention derive from those groups which can react with any relevant molecule "Z" or "Rec” as described above containing a reactive group, whether or not such relevant molecule is a protein, to form a linking group.
  • preferred linking groups thus formed include the linking group, Li, between the immunoreactive group, "Z", and the DHFR active site containing species, "Rec", in the NRTIR System A; the linking group, Li, between the immunoreactive group, "Z", and DHFR ligand species (e.g., "TMP” or “MTX") in the NRTIR in System B; and the linking group, L 2 , between the DHFR active site containing species, "Rec", and the chelating agent, "Q", in the RDA in System Br ⁇ and between the DHFR ligand species (e.g., "TMP” or “MTX”) and the chelating agent, "Q", in the RDA in System A.
  • Preferred linking groups are derived from protein reactive groups selected from but not limited to:
  • crosslinking agents examples include carbodiimide and carbamoylonium crosslinking agents as disclosed in U.S. Patent No. 4,421,847 and the ethers of U.S. Patent No. 4,877,724.
  • one of the reactants such as the immunoreactive group must have a carboxyl group and the other such as the oligonucleotide containing species must have a reactive amine, alcohol, or sulfhydryl group.
  • the crosslinking agent first reacts selectively with the carboxyl group, then is split out during reaction of the thus "activated" carboxyl group with an amine to form an amide linkage between, for example, the protein and DHFR active site containing species, thus covalently bonding the two moieties.
  • An advantage of this approach is that crosslinking of like molecules, e.g., proteins with proteins or DHFR active site containing species with themselves is avoided, whereas the reaction of, for example, homo-bifunctional crosslinking agents is nonselective and unwanted crosslinked molecules are obtained.
  • Preferred useful linking groups are derived from various heterobifunctional cross-linking reagents such as those listed in the Pierce Chemical Company Immunotechnology Catalog - Protein Modification Section, (1991 and 1992) .
  • Useful non-limiting examples of such reagents include:
  • Sulfo-SMCC Sulfosuccini idyl 4-(N- maleimidomethyl)cyclohexane-1- carboxylate.
  • Sulfo-SIAB Sulfosuccinimidyl (4- iodoacetyl)aminobenzoate.
  • Sulfo-SMPB Sulfosuccinimidyl 4-(p- maleimidophenyl)butyrate.
  • linking groups in whole or in part, can also be comprised of and derived from complementary sequences of nucleotides and residues of nucleotides, both naturally occurring and modified, preferably non-self-associating oligonucleotide sequences.
  • Particularly useful, non- limiting reagents for incorporation of modified nucleotide moieties containing reactive functional groups, such as amine and sulfhydryl groups, into an oligonucleotide sequence are commercially available from, for example, Clontech Laboratories Inc.
  • linking groups of this invention are derived from the reaction of a reactive functional group such as an amine or sulfhydryl group as are available in the above Clontech reagents, one or more of which has been incorporated into an oligonucleotide sequence, with, for example, one or more of the previously described protein reactive groups such as heterobifunctional protein reactive groups, one or more of which has been incorporated into, for example, an immune reactive agent or DHFR active site containing moiety of this invention.
  • a reactive functional group such as an amine or sulfhydryl group as are available in the above Clontech reagents, one or more of which has been incorporated into an oligonucleotide sequence
  • protein reactive groups such as heterobifunctional protein reactive groups
  • complementary oligonucleotide sequences are attached to two components of the conjugate, respectively, one sequence to the immune reactive agent and the complementary oligonucleotide sequence to the DHFR active site containing moiety.
  • the hybrid formed between the two complementary oligonucleotide sequences then comprises the linking group between the immune reactive agent and the DHFR active site containing moiety.
  • the complementary oligonucleotide sequences are attached to two components of the conjugate, one sequence to the residue comprised of one or more chelating agents and the complementary oligonucleotide sequence to the DHFR active site containing moiety.
  • the hybrid formed between the two complementary oligonucleotide sequences then comprises the linking group between the DHFR active site containing moiety and the component comprised of one or more chelating agents.
  • System B two or more copies of the same oligonucleotide sequence can be linked, for example, in tandem to one DHFR active site containing moiety and a complementary oligonucleotide sequence comprised of multiple chelating agents can be added.
  • the multiple hybrids formed between the two complementary oligonucleotide sequences then comprises the linking group between the DHFR active site containing moiety and multiple chelating agents .
  • the residue of one or more ligands which have an affinity for non-covalent binding to DHFR can be attached to the immunoreactive group using complementary oligonucleotide hybrids as described above.
  • multiple DHFR sequences can be attached to the immunoreactive protein.
  • one or more ligands which have an affinity for non-covalent binding to DHFR can be attached to multiple chelating agents using complementary oligonucleotide hybrids as described above.
  • Q in System A and in System B represents the residue of a chelating group.
  • the chelating group of this invention can comprise the residue of one or more of a wide variety of chelating agents that can have a radionuclide associated therewith.
  • a chelating agent is a compound containing donor atoms that can combine by coordinate bonding with a metal atom to form a cyclic structure called a chelation complex or chelate. This class of compounds is described in the Kirk-Othmer Encyclopedia of Chemical Technology, Vol. 5, 339-368.
  • the residues of suitable chelating agents can be independently selected from polyphosphates, such as sodium tripolyphosphate and hexametaphosphoric acid; aminocarboxylic acids, such as ethylenediaminetetraacetic acid, N-(2- hydroxyethyl)ethylene-diaminetriacetic acid, nitrilotriacetic acid, N,N-di(2-hydroxyethyl)glycine, ethylenebis (hydroxyphenylglycine) and diethylenetriamine pentacetic acid; 1,3-diketones, such as acetylacetone, trifluoroacetylacetone, and thenoyltrifluoroacetone; hydroxycarboxylic acids, such as tartaric acid, citric acid, gluconic acid, and 5-sulfosalicylic acid; polyamines, such as ethylenediamine, diethylenetriamine, triethylenetetramine, and triaminotriethyla ine; aminoalcohols, such as triethanolamine
  • Preferred residues of chelating agents contain polycarboxylic acid groups and include: ethylenediamine-N,N N' ,N'-tetraacetic acid (EDTA) ; N,N,N' ,N",N"-diethylene-triaminepentaacetic acid (DTPA); 1,4, 7, 10-tetraazacyclododecane-N,N' ,N",N"'-tetraacetic acid (DOTA) ; 1,4,7,10-tetraazacyclododecane-N,N' ,N"- triacetic acid (D03A) ; l-oxa-4,7,10-triazacyclododecane- N,N',N"-triacetic acid (OTTA) ; and trans(1,2)- cyclohexanodiethylenetriamine pentaacetic acid (CDTPA) .
  • Preferred residues of chelating agents contain polycarboxylic acid groups and include: B4A, P4
  • Suitable residues of chelating agents are comprised of proteins modified for the chelation of metals such as technetium and rhenium as described in U.S. Patent No. 5,078,985, the disclosure of which is hereby incorporated by reference.
  • suitable residues of chelating agents are derived from N3S and N S 2 containing compounds, as for example, those disclosed in U.S. Patent Nos. 4,444,690; 4,670,545; 4,673,562; 4,897,255; 4,965,392; 4,980,147; 4,988,496; 5,021,556 and 5,075,099.
  • Other suitable residues of chelating agents are described in PCT/US91/08253, the disclosure of which is hereby incorporated by reference. If Q is comprised of the residue of multiple chelating agents, such agents can be linked together by one or more linking groups such as described above.
  • linking groups also include nitrogen atoms in groups such as amino, imido, nitrilo and imino groups; alkylene, preferably containing from 1 to 18 carbon atoms such as methylene, ethylene, propylene, butylene and hexylene, such alkylene optionally being interrupted by 1 or more heteroatoms such as oxygen, nitrogen and sulfur or heteroatom-containing groups; carbonyl; sulfonyl; sulfinyl; ether; thioether; ester, i.e., carbonyloxy and oxycarbonyl; thioester, i.e., carbonylthio, thiocarbonyl, thiocarbonyloxy, and oxythiocarboxy; amide, i.e., iminocarbonyl and carbonylimino; thioamide,
  • linking groups can be used, such as, for example, alkyleneimino and iminoalkylene. It is contemplated that other linking groups may be suitable for use herein, such as linking groups commonly used in protein heterobifunctional and homobifunctional conjugation and crosslinking chemistry as described for Li or L 2 above.
  • Especially preferred linking groups include amino groups which when linked to the residue of a chelating agent via an isothiocyanate group on the chelating agent form thiourea groups.
  • the linking groups can contain various substituents which do not interfere with the coupling reaction between the chelating agent Q and the other components of this invention.
  • the linking groups can also contain substituents which can otherwise interfere with such reaction, but which during the coupling reaction, are prevented from so doing with suitable protecting groups commonly known in the art and which substituents are regenerated after the coupling reaction by suitable deprotection.
  • the linking groups can also contain substituents that are introduced after the coupling reaction.
  • the linking group can be substituted with substituents such as halogen, such as F, Cl, Br or I; an ester group; an amide group; alkyl, preferably containing from 1 to about 18, more preferably, 1 to 4 carbon atoms such as methyl, ethyl, propyl, i-propyl, butyl, and the like; substituted or unsubstituted aryl, preferably containing from 6 to about 20, more preferably 6 to 10 carbon atoms such as phenyl, naphthyl, hydroxyphenyl, iodophenyl, hydroxyiodophenyl, fluorophenyl and methoxyphenyl; substituted or unsubstituted aralkyl, preferably containing from 7 to about 12 carbon atoms, such as benzyl and phenylethyl; alkoxy, the alkyl portion of which preferably contains from 1 to 18 carbon atoms as described for alkyl above; alkoxy
  • ligands that have an affinity for non-covalent binding to a DHFR active site useful in this invention include residues of trimethoprim analogs and methotrexate analogs listed in Rahman et al, Methotrexate and Its Analogs; Medicinal Research Reviews, Vol 8, No. 1, 95-157, and Kuyper et al, Carboxysubstituted Trimethoprim Analogs; J. Med. Chem. Vol 28, 303, (1985) .
  • Preferred analogs include those defined in structure (i) and in structure (ii) , each comprising a reactive group to permit or facilitate binding of one portion of the ligand to the immunoreactive species, Z, in System B and to the chelating agent, Q, in System A.
  • Ligands comprised of trimethoprim analogs that are useful in the practice of this invention include derivatives defined in structure (i) above where: Ri, R2, and R3 are independently selected from H, alkyl (such as methyl as a preferred group for one or two of Ri R2 and R3) , aralkyl, aryl (including substituted aryl), an alkylene carboxylic acid or amide derivative thereof, an alkylene group that contains one or more heteroatoms (such as, but not limited to, for example, oxygen [as an ether or a hydroxyl group] or sulfur [as a thioether group] ) , an amino acid group and a peptide group, and at least one of -Ri, -R2, and -R3 is of the form represented in structure 13:
  • X is an alkylene group from 1 to 12 carbon atoms which may contain one or more heteroatoms such as oxygen,, sulfur or nitrogen;
  • L is a linking group as defined above, preferably the residue of an amide group, a chemical bond, an amino acid residue, or an arylene group which may be substituted by one or more hydroxyl groups;
  • A is an alkylene group, a polyalkylene oxidyl group, an amino acid residue, a peptide residue, X, or a group containing pendant substituents which contain heteroatoms (such as, for example, oxygen in the form of one or more hydroxyl groups, carboxylic acid groups or salts thereof, amido groups, ether groups, sulfur in the form of thioether, sulfone, sulfoxide or sulfonate, nitrogen in the form of amino groups, amido groups or a diazo linkage, or phosphorous in the form of phosphate) ;
  • B is selected from A but modified to contain one or more radion
  • a preferred non-limiting example of a linking group includes the residue of a protected (D)-Glu- (Ala) 4 -Lys-Lys (4) as well as the residue of a protected (L)-Glu-(Ala)4 ⁇ (Lys)2-
  • Both 4'- carboxymethyloxy-trimethoprim (structure 5) and methotrexate analog (structure iia) undergo coupling reactions (see SCHEMES 2, 3 and 4) followed by deprotection to afford trimethoprim-(D)-Glu-(Ala) -Lys- Lys (structure iii) and methotrexate (structure iv) and the analogous L-Glu containing species, respectively.
  • the synthetic schemes (2,3,4) describe the synthetic methods for trimethoprim-heptapeptide (structure iii) and methotrexate-heptapeptide (structure iv) .
  • the t-butoxycarbonyl (tBoc) blocked Lys-Lys (structure 2) is prepared using a dehydrative coupling method, for example, using DCC (dicyclohexylcarbodiimide) and two protected lysine groups (SCHEME 2) .
  • the FMOC (9- Fluorenylmethoxycarbonyl) group of Lys-Lys is then removed via base treatment, and 4 units of alanine are introduced as shown in SCHEME 2 to afford the blocked Ala-Ala-Ala-Ala-Lys-Lys (structure 3) .
  • the resulting heptapeptide (structure 4) is coupled with 4*-carboxymethyloxy- trimethoprim (structure 5) to yield trimethoprim- heptapeptide (structure 6) .
  • trimethoprim-D-Glu-(Ala) -Lys-Lys affords the desired trimethoprim-D-Glu-(Ala) -Lys-Lys (structure 7) .
  • trimethoprim-D-Glu-(Ala) -Lys-Lys upon treatment with TMT-NCS (structure 8) (SCHEME 3) yields a desirable ligand-to-chelating agent conjugate, trimethoprim-D-Glu- (Ala) 4 -[Lys-(TMT) ]-[Lys-(TMT) ] ,
  • a coupling reaction (Scheme 4) of the heptapeptide (structure 4) and the benzoic acid derivative, 4*-carboxy-methotrexate (structure iia) yields the desired blocked methotrexate-D-Glu-(Ala) 4 -Lys-Lys (structure 10).
  • methotrexate-heptapeptide (structure 11) is obtained from acid deblocking of (structure 10) , and reacts with TMT-NCS to afford the desirable methotrexate-D-Glu- (Ala) 4 -[Lys-(TMT) ]-[Lys- (TMT) ] (structure 12) .
  • a solution of methotrexate-D-Glu-(Ala) 4 -[Lys-(TMT) ]-[Lys- (TMT) ] (structure 12) in deionized water buffered with 0.5 M sodium acetate at a pH of about 6.0 at room temperature is treated with an aqueous HC1 solution of 90 YCl3 to afford a radionuclide labeled ( 90 Y) methotrexate-D-Glu- (Ala) 4 -[Lys-(TMT) ]-[Lys-(TMT) ] .
  • Racemic mixtures of D and L enantiomers of the above described trimethoprim and methotrexate derivatives are also useful in this invention.
  • Additional chelating agents and radionuclides bound to chelating agents are incorporated by preparing, for example, analogous peptides comprised of additional Lys- TMT and Lys-TMT-radionuclide groups.
  • the number of such Lys-TMT and Lys-TMT-radionuclide residues is from 1 to about 6, and more preferably from 2 to about 6.
  • the NRTIR is comprised of one or more ligands that have an affinity for non-covalent binding to a DHFR active site each with a suitably substituted linking group (Li) conjugated to the immunoreactive group (Z) .
  • said ligand that has an affinity for non-covalent binding to a DHFR active site is comprised of a residue of (structure i) or (structure ii) linked to Z by a linking group Li as defined above.
  • the NRTIR preferably contains 2 to about 10 of such groups, more preferably 2 to about 4.
  • the delivery agent in the System B is comprised of a DHFR active site moiety conjugated to one or more chelating agents via a linking group as described above.
  • the radionuclide be a metal ion and that said metal ion be easily complexed to the chelating agent, for example, by merely exposing or mixing an aqueous solution of the chelating agent-containing moiety with a metal salt in an aqueous solution preferably having a pH in the range of about 4 to about 11.
  • the salt can be any salt, but preferably the salt is a water soluble salt of the metal such as a halogen salt, and more preferably such salts are selected so as not to interfere with the binding of the metal ion with the chelating agent.
  • the chelating agent-containing moiety is preferrably in aqueous solution at a pH of between about 5 and about 9, more preferably between pH about 6 to about 8.
  • the chelating agent-containing moiety can be mixed with buffer salts such as citrate, acetate, phosphate and borate to produce the optimum pH.
  • said buffer salts are selected so as not to interfere with the subsequent binding of the metal ion to the chelating agent.
  • the RDA of this invention preferably contains a ratio of metal radionuclide ion to chelating agent that is effective in such therapeutic applications .
  • the mole ratio of metal ion per chelating agent is from about 1:100 to about 1:1.
  • the RDA of this invention preferably contains a ratio of metal radionuclide ion to chelating agent that is effective in such diagnostic imaging applications.
  • the mole ratio of metal ion per chelating agent is from about 1:1,000 to about 1:1.
  • the RDA of this invention can comprise a non-radioisotope of a metal ion.
  • the metal ions can be selected from, but are not limited to, elements of groups IIA through VIA. Preferred metals include those of atomic number 12, 13, 20, the transition elements 21 - 33, 38 - 52, 56, 72 - 84 and 88 and those of the lanthanide series (atomic number 57 - 71) .
  • the RDA of this invention can comprise a radionuclide.
  • the radionuclide can be selected, for example, from radioisotopes of Sc, Fe, Pb, Ga, Y, Bi, Mn, Cu, Cr, Zn, Ge, Mo, Tc, Ru, In, Sn, Sr, Sm, Lu, Sb, W, Re, Po, Ta and Tl.
  • Preferred radionuclides include 44 Sc, 64 Cu, 67 Cu, U1 ln, 212 Pb, fi 8Ga, 90 Y, "Ssm, 212 Bi/ 99m TCf 186 Re and 188 Re . 0f these, especially preferred is 90 Y.
  • These radioisotopes can be atomic or preferably ionic.
  • the RDA of this invention can comprise a fluorescent metal ion.
  • the fluorescent metal ion can be selected from, but is not limited to, metals of atomic number 57 to 71. Ions of the following metals are preferred: La, Ce, Pr, Nd, Pm, Sm, Eu, Gd,
  • Tb Dy, Ho, Er, Tm, Yb, and Lu. Eu is especially preferred.
  • the RDA of this invention can comprise one or more paramagnetic elements which are suitable for the use in MRI applications.
  • the paramagnetic element can be selected from elements of atomic number 21 to 29, 43, 44 and 57 to 71. The following elements are preferred: Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, T , Yb and Lu. Mn, Gd, and Dy are especially preferred.
  • R l , R 2 and R3 are as defined above and preferably wherein one of Ri, R2, and R3 is represented by structure 14.
  • R' and R" are selected from components of amino acids that comprise, for example, natural amino acids such as glycine, alanine, leucine, serine, lysine, isoleucine, glutamine, aspartic acid, glutamic acid, proline, threonine, valine, phenylalanine, tyrosine, and the like, as well as unnatural amino acids and racemates of natural amino acids, and R' and R n can independently be selected from H, a polyalkylene oxidyl group, and branched peptide groups which may contain additional chelate groups such as TMT; mi is an integer between 1 and 10, m 2 , m3, and ⁇ t 4 are independently selected from zero and an integer between 1 and 10 with the proviso that m2 is at least 1, and preferably 2 to about 5; and W is selected from OH, NH 2 , a residue of TMT, an O-alkyl group such as an O-methyl group, and NR a R f
  • R ⁇ , R2, and R3 are as defined above and preferably wherein one of Ri, R2, or R3 is represented by structure 15.
  • R* and R" are selected from components of amino acids that comprise, for example, natural amino acids such as glycine, alanine, leucine, serine, lysine, isoleucine, glutamine, aspartic acid, glutamic acid, proline, threonine, valine, phenylalanine, tyrosine, and the like, as well as unnatural amino acids or racemates of natural amino acids, and R' and R" can be independently selected from H, a polyalkylene oxidyl group, a branched peptide group which may contain one to about 10 additional chelate groups such as TMT; ins is an integer between 1 and 10, mg, m , and m ⁇ are independently selected from zero and an integer between 1 and 10 with the proviso that m 7 is at least 1, and preferably 2 to about 5; is selected from OH, NH 2 , a TMT moiety, an O-alkyl group such as an O-methyl group, NR a R b where
  • R' * * is selected from H and the residue of a TMT moiety with or without a radionuclide, said TMT moiety being linked via a thiourea group.
  • an RDA comprised of at least two metal ions in combination with one another in the same formulation is specifically contemplated.
  • a therapeutically effective dose of a radionuclide such as 90 ⁇ +3
  • a diagnostic imaging effective dose of a paramagnetic ion such as Gd +3
  • the ratio of the molar concentration of the diagnostic imaging effective ion to the molar concentration of the radionuclide ion being typically greater than one
  • a pharmaceutically effective formulation of said RDA permits the simultaneous magnetic resonance imaging of at least a portion of the tissue of a host patient during therapeutic treatment of said patient.
  • radioisotopes of iodine is specifically contemplated.
  • the RDA of System A or of System B is comprised of substituents that can be chemically substituted by iodine in a covalent bond forming reaction, such as, for example, substituents containing hydroxyphenyl functionality, such substituents can be labeled by methods well known in the art with a radioisotope of iodine.
  • the thus covalently linked iodine species can be used in the aforementioned fashion in therapeutic and diagnostic imaging applications.
  • an effective dose of an RDA of System A or of System B as described above in a pharmaceutically acceptable medium is prepared by exposing a composition of a precursor of an RDA (said precursor being comprised of a residue of a ligand that has an affinity for non-covalent binding to a DHFR active site, a linking group, and a residue of a chelating agent in System A and of a residue of a DHFR active site, a linking group, and a residue of a chelating agent in System B) to a composition containing a radioactive metal ion such that the molar amount of said radionuclide metal ion is less than the molar amount of the chelating groups comprising the RDA in said composition, said duration of exposure lasting an effective time to permit uptake of said metal ion into said RDA.
  • an effective dose of a NRTIR of System A or System B as described above in a pharmaceutically acceptable medium is administered to a patient and said NRTIR is allowed to accumulate at the target site such as at a tumor site in said patient.
  • an effective dose of a RDA as described above in a pharmaceutically acceptable medium is administered to said patient, and said RDA is allowed to accumulate at the target site, said target site being the said NRTIR accumulated at said tumor site in said patient.
  • a therapeutically effective dose of a NRTIR of System A or System B as described above in a pharmaceutically acceptable medium is administered to a patient or to a tissue from a patient and said NRTIR is allowed to accumulate at the target site such as at a tumor site in said patient.
  • a therapeutically effective dose of a RDA as described above in a pharmaceutically acceptable medium is administered to said patient, and said RDA is allowed to accumulate at the target site, said target site being the said NRTIR accumulated at said tumor site in said patient.
  • the present invention also comprises one or more NRTIR as described above formulated into compositions together with one or more non-toxic physiologically acceptable carriers, adjuvants or vehicles which are collectively referred to herein as carriers, for parenteral injection for oral administration in solid or liquid form, for rectal or topical administration, or the like.
  • the present invention also comprises one or more RDA as described above formulated into compositions together with one or more non-toxic physiologically acceptable carriers, adjuvants or vehicles which are collectively referred to herein as carriers, for parenteral injection, for oral administration in solid or liquid form, for rectal, or topical administration, or the like.
  • compositions can be administered to humans and animals either orally, rectally, parenterally (intravenous, by intramuscularly or subcutaneously) , intracisternally, intravaginally, intraperitoneally, intravesically, locally (powders, ointments or drops) , or as a buccal or nasal spray.
  • the NRTIR and the RDA can be administered by the same route such as orally, rectally, parenterally (intravenous, by intramuscularly or subcutaneously) , intracisternally, intravaginally, intraperitoneally, intravesically, locally (powders, ointments or drops), or as a buccal or nasal spray.
  • compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like) , suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or
  • fillers or extenders as for example, starches, lactose, sucrose, glucose, mannitol and silicic acid,
  • binders as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose and acacia
  • humectants as for example, glylcerol
  • disintegrating agents as for example, agar-agar.
  • the dosage forms may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like.
  • Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain opacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions which can be used are polymeric substances and waxes.
  • the active compounds can also be in micro- encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.
  • inert diluents commonly used in the art, such as water or other solvents, solubilizing agents
  • composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • suspending agents as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • compositions for rectal administrations are preferably suppositories which can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component.
  • Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays and inhalants.
  • the active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers or propellants as may be required.
  • Opthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
  • Actual dosage levels of active ingredients in the compositions of the present invention may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration. The selected dosage level therefore depends upon the desired therapeutic effect, on the route of administration, on the desired duration of treatment and other factors.
  • the total daily dose of the compounds of this invention administered to a host in single of divided dose may be in amounts, for example, of from about 1 nanomol to about 5 micromols per kilogram of body weight.
  • Dosage unit compositions may contain such amounts of such sub ultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.
  • the present invention is directed to a method of diagnosis comprising the administration of a diagnostic imaging effective amount of the compositions of the present invention to a mammal or to a tissue from said mammal in need of such diagnosis.
  • a method for diagnostic imaging for use in medical procedures in accordance with this invention comprises administering to the body of a test subject in need of a diagnostic image an effective diagnostic image producing amount of the above-described compositions.
  • an effective diagnostic image producing amount of a non-radioactive targeting immunoreagent (NRTIR) as described above in a pharmaceutically acceptable medium is administered to a patient and said non-radioactive targeting immunoreagent is allowed to accumulate at the target site such as at a tumor site in said patient.
  • NRTIR non-radioactive targeting immunoreagent
  • a diagnostic imaging effective dose of a radioactive delivery reagent (RDA) as described above in a pharmaceutically acceptable medium is administered to said patient, and said radioactive targeting reagent is allowed to accumulate at the target site, said target site being the said non-radioactive targeting immunoreagent accumulated at said tumor site in said patient.
  • RDA radioactive delivery reagent
  • the NRTIR may be reacted with a diagnostic imaging effective amount of a reagent comprised of a radionuclide prior to administration to the environs of a tissue of interest of a patient undergoing such diagnostic imaging, waiting for an effective period of time during which time said NRTIR will bind to sites on cells of said tissue of interest and during which time unbound NRTIR will be removed from the environs of said tissue and then obtaining an image as a function of time of all or part of said tissue of interest.
  • a diagnostic imaging or a therapeutically effective amount of RDA containing the same or a different radionuclide as that employed on the NRTIR is administered to said tissue of interest of said patient.
  • test subjects can include mammalian species such as rabbits, dogs, cats, monkeys, sheep, pigs, horses, bovine animals and the like.
  • the subject mammal is maintained for a time period sufficient for the administered compositions to be distributed throughout the subject and enter the tissues of the mammal.
  • a sufficient time period is generally from about 1 hour to about 2 weeks or more and, preferably from about 2 hours to about 1 week.
  • the solid support used in the synthesis was a 4-Alkoxybenzyl alcohol polystyrene resin (Wang resin) .
  • the N-alpha- Fmoc protecting group was used throughout the synthesis, with t-butyl side chain protection on D-Glu, and t-BOC protection on the side chain of Lys.
  • the peptide chain was assembled using the ABI FastMocTM software protocols (0.25 mmole scale, HBTU activated couplings, 4 fold excess of amino acid, 1 hour) for Fmoc-chemistry.
  • trimethoprim-4'-O-acetic acid of Example 3 was carried out manually by adding in order: 335 mg trimethoprim (1 mmole) in 25 ml of DMSO, 525 uL of diisopropylethylamine (3 mmole) followed by 380 mg of HBTU (1 mmole) .
  • the mixture was allowed to react for 2 hours at room temperature, at which time the mixture was filtered, and the peptide resin washed 3 x 50 ml DMF and then by 3 x 50 ml MeOH.
  • Removal and deprotection of the peptide was accomplished by adding 15 ml of a 95:5 TFA/H20 solution to the peptide-resin, in a.sealed vessel and shaken at room temperature for 2 hours. At the end of 2 hours, the mixture was filtered by pouring into a scintered glass funnel. The filtrate volume was then reduced to an oil (@ 3 ml) by rotoevaporation. The peptide was then precipitated by dropping the oil into a centrifuge tube containing 50 ml of Et2 ⁇ . The peptide was spun down and the ether decanted, and the peptide was allowed to air dry.
  • Prppara inn of radJnmifilide labeled ( Y - ri ⁇ nP hop ⁇ -i ⁇ n- n-fi1 ⁇ -fA1 « ⁇ -1 -Lvs-(TMTl-Lvs- ⁇ 'MT) (Scheme 1 & 2 .
  • trimethoprim-4 *-O-acetic acid heptapeptide (7) is stirred 2 days, and the solvent and the urea are removed to afford the blocked trimethoprim-4 '-O-acetic acid heptapeptide (6), which upon mild hydrolysis with trifluoroacetic acid, yields trimethoprim-4 *-O-acetic acid heptapeptide (7) .
  • trimethoprim-4 '-O-acetic acid heptapeptide (7) (20 mM) in 50 ml of a saturated aqueous sodium bicarbonate at about pH 9 is allowed to react with TMT-isothiocyanate (8; 20mM) at room temperature for 12 hours to afford trimethoprim-4 '-O-acetic acid-D- Glu-(ALA) 4 -[Lys- (TMT) ]-[Lys- (TMT) ] (9) .
  • 0.04M hydrochloric acid at a specific activity of >500 Ci/mg: Amersham-Mediphysics is neutralized using two volumes of 0.5 M sodium acetate pH 6.0 and added to a solution of the above 4 '-carboxymethotrexate-D-Glu- (ALA) -[Lys-(TMT) ]-[Lys-(TMT)] (12) in deionized water buffered with 0.5 M sodium acetate at pH 6.0 at room temperature. The labeling is allowed to proceed for one hour and then the labeling efficiency is determined by thin layer chromatography on a Gelman ITAL-SG strip developed in 0.1 M sodium citrate, pH 6.0.
  • DHFR dihydrofolate reductase
  • ING-1 a chimeric IgGi antibody
  • the DHFR referred to below is of bacterial origin, produced as a recombinant product from the cloned DHFR gene which is overexpresed in Escherchia coli (E. coli), or of human origin, available as a recombinant protein. Examples of System A
  • Z is the residue of an immunoreactive group
  • Rec is the residue of a receptor, preferably DHFR;
  • D is the residue of a ligand that has an affinity for non-covalent binding to the receptor, preferably to a DHFR receptor;
  • DHFR ligand is the residue of a ligand that has an affinity for non-covalent binding to a DHFR active site;
  • TMP is the residue of a trimethoprim analog;
  • MTX is the residue of a methotrexate analog;
  • Li and L2 are each independently the residue of a linking group that may independently contain spacing group,
  • Q is the residue of a chelating group
  • M is a radionuclide
  • n and m are each independently an integer greater than zero.
  • a sulfo-SMCC solution (36 nmoles) in PBS was added to a sample of a chimeric antibody (ING-1; 6 nmoles) solution in phosphate buffer (pH7) .
  • the resulting mixture was allowed to stand for 30 minutes with occasional mixing at room temperature.
  • the reaction mixture was diluted with phosphate buffered saline, added to a prewashed PD-10 column (Pharmacia) , and eluted with PBS to afford ING-1-maleimide. This material was stored on ice until use.
  • a sample of of a chimeric antibody (ING-1; 6 nmoles) solution in 0.1 M carbonate buffer (pH 8.8) is mixed with 200 nmoles of an aqueous solution of 2- iminothiolane.
  • the resulting mixture is allowed to stand for 30 minutes with occasional mixing at room temperature.
  • the reaction mixture is diluted with phosphate buffed saline, added to a prewashed PD-10 column, and eluted with PBS to afford mercaptoalkyl-ING- 1. This material is stored on ice until use.
  • a solution containing 6 nmoles of ING-1 in PBS is vortexed while 60 nmoles of SATA (in DMSO) are added. After mixing and standing at room temperature for 60 minutes, the reaction mixture is diluted with PBS, and eluted from a PD-10 column with PBS to afford ING-l-CO- CH2-S-CO-CH3.
  • the acetylthioacetylated antibody is deprotected by the addition of 30 uL of a pH 7.5 solution contaning 100 mM sodium phosphate, 25 mM EDTA, 50 mM NH20H. The reaction proceeds for two hours at room temperature after which the material is again passed down a PD-10 column by elution with PBS. The final product (ING-1-CO-CH2-SH) is used immediately.
  • ING-1 500 ug
  • 125 I monochloride at about 5 mCi/mg
  • iodogen Sodium N-chloro-benzenesulfonamide
  • DHFR DHFR
  • 0.1 M carbonate buffer pH 9
  • 2-iminothiolane 2 mmoles of an aqueous solution of 2-iminothiolane are added.
  • the reactants are vortex mixed and kept at room temperature for 120 minutes.
  • the reaction mixture is quenched by the addition of 2 mmoles of ethanolamine, diluted with phosphate buffed saline.
  • the reaction mixture is added to a prewashed PD-10 column, and eluted with PBS to afford DHFR-HC(NH 2 + )CH2CH2CH2SH.
  • the product is eluted off the column directly into the antibody solution.
  • a sulfo-SMCC solution (300 nmoles) in PBS is added to a sample of DHFR (50 nmoles) in phosphate buffer (pH7) .
  • the resulting mixture is allowed to stand for 30 minutes with occasional mixing at room temperature.
  • the reaction is stopped with 60 nmoles basic tris buffer.
  • the reaction mixture is diluted with phosphate buffed saline, added to a prewashed PD-10 column, and eluted with PBS to afford DHFR-maleimide. This material is stored on ice until use.
  • DHFR DHFR
  • 125 I monochloride at about 5 mCi/mg
  • iodogen Sodium N-chloro-benzenesulfonamide
  • the active site of the enzyme is blocked to prevent the entry of the reagents.
  • the final centrifuged pellet is mixed with DHFR (1 mmole/5 mL) in 5.0 mL of buffer containing (50 mM KPO 4 , 1.0 mM K 2 EDTA and 0.5 mM dithioerythritol at pH 6.0) and left for one hour.
  • the resin with the now attached DHFR is centrifuged to a pellet and washed x3 with washing buffer (50 mM KPO 4 , 1.0 mM K ⁇ EDTA at pH 6.0).
  • the resin pellet is suspended in a sulfo-SMCC solution (6 mmoles) in PBS (pH 7) .
  • the resulting mixture is stirred very slowly for 60 minutes at room temperature.
  • the reaction is stopped by dilution with 10 mL of washing buffer and the resin is again centrifuged to a pellet and washed 2x with the washing buffer.
  • the resin is poured into a narrow glass Pasteur pipette whose exit is obstructed with a plug of glass wool.
  • DHFR-maleimide derivatized DHFR is removed from the resin by elution with 30 mL of eluting buffer (100 mM folic acid, 200 mM KBO 3 , 1.0 M KC1, 1.0 mM K 2 EDTA at pH 9.0) to afford DHFR-maleimide.
  • eluting buffer 100 mM folic acid, 200 mM KBO 3 , 1.0 M KC1, 1.0 mM K 2 EDTA at pH 9.0
  • This material is pooled, dialysed against dialysis buffer (20 mM Tris, 1.0 mM K ⁇ EDTA; pH 7.2) at 4°C overnight and then concentrated in a Centricon-10 ®
  • the folate is removed from the active site of the enzyme by passage of the maleimide DHFR (Example 7a) or antibody-DHFR conjugate (see Example 8 below) through a DEAE-SEPHACEL column (Pharmacia) .
  • the column (-50 mL resin) is prewashed with DEAE-washing buffer (10 mM Tris, 1.0 mM K 2 EDTA, 0.2 mM dithioerythritol; pH 7.2) and the protein loaded on in the same buffer.
  • the protein is removed from the column with a non-linear gradient of lOOOmL of DEAE-washing buffer (10 mM Tris, 1.0 mM K ⁇ EDTA, 0.2 mM dithioerythritol; pH 7.2) to 1000 mL of 10 mM Tris, 0.5 M KC1, 1.0 mM K 2 EDTA, 0.2 mM dithioerythritol; pH 7.2. Fractions eluting from the colum are collected and monitored at 280 nm for protein content. Fractions containing protein are pooled, concentrated as before and dialysed overnight against PBS to produce folate-free material.
  • DEAE-washing buffer 10 mM Tris, 1.0 mM K ⁇ EDTA, 0.2 mM dithioerythritol; pH 7.2
  • the methodologies for conjugation are essentially the same, irrespective of whether the maleimide group is on the antibody or on the DHFR, and irrespective of the method chosen to introduce the sulfhydryl group into the protein.
  • the final molar ratio during the conjugation is maintained at close to equimolar antibody:DHFR in order to control over-conjugation of the proteins which could result in inactivation of one or other or both.
  • the following procedure is applicable to the conjugation of materials in Example 6 to the materials in Example 7.
  • a sample (50 nmoles) of DHFR(N)-CO-CH2-SH is eluted off a PD-10 column directly into a solution of maleimide- deriviatitzed ING-1 (5 nmoles) prepared according to Example 6a. After a brief mixing the solution is rapidly concentrated by centrifugation in a Centricon-10® device to a concentration of approximately 3.0 mg/mL protein. The reaction then is allowed to proceed for 4 hours at room temperature. The antibody-DHFR conjugate is transfered to a Centricon-30® concentrator and diluted with PBS.
  • the material After concentrating the protein down to a volume of approximately 500 uL by centrifugation, the material is again diluted with PBS to 3.0 mL and recentrifuged. This procedure, which separates unconjugated DHFR and other low molecular weight products from the retained antibody-DHFR and unconjugated antibody products, is repeated 4 times or until spectrophotometric monitoring of the filtrate at 280 nm shows that no further protein is being filtered.
  • the material in the Centricon-30® is concentrated to approximately 1.0 mg, ING-1/DHFR, per milliliter solution and applied to a 2.6 x 60 cm Sephacryl S-200 size-exclusion column equilibrated and eluted with 50 mM sodium phosphate buffer at pH 7.2 supplemented with 150 mM sodium chloride. This column separates unconjugated antibody from antibody-DHFR conjugate. Fractions of the eluate containing the conjugate are pooled and then centrifuged in a Centricon-30 device to a concentration of approximately 1.0 mg, ING-1/DHFR, per milliliter solution. The conjugate is sterile filtered through a 0.22 ⁇ filter and stored at 4°C until use.
  • Example 7c was eluted off a PD-10 column directly into a solution of maleimide-deriviatitzed ING-1 (5 nmoles) prepared according to Example 6a. After a brief mixing, the reaction was allowed to proceed for 4 hours at room temperature.
  • the antibody-DHFR conjugate was transfered to a Centricon-30® concentrator, diluted with PBS, and concentrated down to a volume of approximately 500 ⁇ L by centrifugation. The concentrated protein was then diluted with PBS to a volume of 3.0 mL and recentrifuged to a volume of 500 uL.
  • Z is the residue of an immunoreactive group
  • Rec is the residue of a receptor, preferably a DHFR receptor;
  • D is the residue of a ligand that has an affinity for non-covalent binding to the receptor, preferably to a
  • DHFR ligand is the residue of a ligand that has an affinity for non-covalent binding to a DHFR active site
  • TMP is the residue of a trimethoprim analog
  • MTX is the residue of a methotrexate analog
  • Li and L2 are each independently the residue of a linking group that may independently contain spacing group
  • Q is the residue of a chelating group
  • M is a radionuclide; and n and m are each independently an integer greater than zero.
  • Trimethoprim-4'-O-acetic acid cysteine (50 nmoles) prepared according to Example 9b is added directly into a solution of maleimide-derivatized ING-1 (5 nmoles) prepared according to Example 9a. After a brief mixing the solution is then allowed to proceed for 4 hours at room temperature with intermittent stirring. The antibody-trimethoprim conjugate is transferred to a Centricom-30® concentrator and diluted with PBS. After concentrating the protein down to a volume of approximately 500 ⁇ L by centrifugation, the material is again diluted with PBS to 3.0 mL and recentrifuged.
  • Fractions of the eluate containing the conjugate are pooled and then centrifuged in a Centricon-30® device to a concentration of approximately 1.0 mg ING-1- trimethoprim per milliliter solution.
  • the conjugate is sterile filtered through a 0.22 filter and stored at 4°C until use.
  • Terpyridine methylenetetraacetic acid (TMT) or a suitable derivative thereof can be conjugated to a protein molecule (DHFR) to yield a protein-TMT conjugate.
  • DHFR protein molecule
  • the DHFR referred to below is of either bacterial origin, produced as a recombinant product from the cloned DHFR gene which is overexpressed in E.coli, or of human origin available as a recombinant protein.
  • DHFR (50 nmoles) is allowed to react with TMT- isothiocyanate (250 nmoles in 1.0 M carbonate, 150 mM sodium chloride buffer, pH 9.3 in an acid washed, conical, glass reaction vial. The solution is stirred briefly to mix the reactants and then left in the dark at room temperature. After 16 hours, the DHFR/TMT conjugate is separated from unconjugated TMT, by applying the reaction mixture to a PD-10 chromatography column, which has been pre-washed and equilibrated with 50 mM sodium acetate buffer containing 150 mM sodium chloride at pH 5.6. The pure conjugate is eluted off the column with 2.5 mL of that same buffer, and concentrated on a Centricon-10 ® concentration device.
  • a volume of radioactive Yttrium chloride ( 90 Y in 0.04 M hydrochloric acid at a specific activity of >500 Ci/g: Amersham-Mediphysics) is neutralized using two volumes of 0.5 M sodium acetate pH 6.0.
  • the neutralized 90 Y (1.0 mCi) is added to 1.0 mL of DHFR/TMT (1 mg/mL) in 50 mM sodium acetate buffer containing 150 mM sodium chloride at pH 5.6.
  • the labeling is allowed to proceed for one hour and then the reaction mixture is loaded on to a PD-10 chromatography column which has been pre ⁇ washed and equilibrated in a buffer contaiing SO mM sodium phosphate with 150 mM sodiumchloride pH 7.4 (PBS) .
  • the sample is eluted from the column with 1.5 mL of PBS.
  • Fractions of radiolabeled DHFR/TMT (0.5 mL) are collected, assayed for radioactivity, and pooled.
  • the labeling efficiency is determined by removing 1.0 uL of the sample and spotting it on to a Gelman ITLC-SG strip.
  • the strip is developed in a glass beaker contining 0.1 M sodium citrate, pH 6.0 for a few minutes until the solvent front has reached three-quarters of the way to the top of the paper.
  • the strip is inserted into a System 200 Imaging Scanner (Bioscan) which has been optimized for 90 Y and is controlled by a Compaq 386/20e computer.
  • System 200 Imaging Scanner Bioscan
  • free 90 Y migrates at the solvent front while the DHFR/TMT ( 90 Y) remains at the origin.
  • DHFR/TMT 90 Y
  • Using this system more than 98% of the total 90 Y radioactivity is found associated with DHFR/TMT at the origin.
  • the concentrations of ING-1 and DHFR for use in the conjugate reactions are determined by the BioRad protein assay (BioRad Catalog # 500-0001) using bovine immunoglobulin as the protein standard. By inclusion of trace amounts of 125 I-labeled DHFR or ING-1 in the reaction mixtures, and by knowing the specific activity of the preparations, the ratio of one protein to the other after conjugation is calculated.
  • the DHFR can be tagged with other materials (e.g., TMT (for use with 50 ⁇ or Europium fluorescence), biotin, fluorescene isothiocyanate (FITC) etc. to detect and quantify the amount of DHFR present in a solution or conjugated to another protein.
  • TMT for use with 50 ⁇ or Europium fluorescence
  • biotin for use with 50 ⁇ or Europium fluorescence
  • Conjugates of antibody-DHFR (e.g.. Example 8) or antibody-trimethoprim (e.g.. Example 10) are examined for their ability to bind to antigens on the surface of a human tumor cell line to which the antibody had been raised.
  • the immunoreactivity of the conjugates is compared by flow cytometry with a standard preparation of the antibody before being subjected to modification.
  • Target HT-29 cells (a human adenocarcinoma cell line obtained from the American Type Tissue Collection: ATTC) are grown to confluency in tissue culture flasks using McCoy's media supplemented with 10% fetal calf serum. The cells are harvested by scraping the flask walls with a cell scraper.
  • the standard curve is made in flow buffer so that each sample contains 1.0 mg protein per mL.
  • Samples from the standard curve and ING-1-DHFR unknowns, or ING-1- trimethoprim unknowns are then incubated with 5xl0 5 HT29 cells at 4°C for 1 hour. After extensive washing to remove unbound antibody, the cells are resuspended in 100 uL flow buffer and incubated at 4°C for 1 hour with goat-anti-human antibody labelled with fluorescene isothiocyanate. After further washing in flow buffer the samples are analyzed by flow cytometry on a Coulter EPICS 753 flow cyto eter.
  • Fluorescein isothiocyanate (FITC) and propidium iodide (PI) are excited using the 488 nm emission line of an argon laser. The output is set at 500 mw in light regulation mode. Single cells are identified by 90 degree and forward angle light scatter. Analysis windows are applied to these parameters to separate single cells from aggregates and cell debris. Fluorescence from FITC and propidium are separated with a 550 nm long pass dichroic filter and collected through a 530 nm band pass filter (for FITC), and a 635 nm band pass filter (for PI). Light scatter parameters are collected as integrated pulses and fluorescence is collected as log integrated pulses.
  • FITC Fluorescein isothiocyanate
  • PI propidium iodide
  • Dead cells are excluded from the assay by placing an analysis window on cells negative for PI uptake.
  • the mean fluorescence per sample (weighted average from 2500 cells) is calculated for each histogram.
  • FITC calibration beads are analysed in each experiment to establish a fluorescence standard curve.
  • the average fluorescence intensity for each sample is then expressed as the average FITC equivalents per cell.
  • Immunoreactivity is calculated by comparing the average fluorescence intensity of the ING1-DHFR or ING-1-trimethoprim sample with values from the standard curve.
  • the antigen to which the antibody, ING-1, binds is prepared from LS174T or HT-29 cells (available from ATTC) by scraping confluent monolayers of cells from the walls of culture flasks with a cell scraper. The cells from many flasks are combined and a sample is taken and counted to estimate the total number of cells harvested. At all times the cells are kept on ice.
  • the cells are washed once in 25 mL ice-cold 50 mM sodium phosphate buffer, pH 7.4 supplemented with 150 mM sodium chloride (PBS), pelleted under the same conditions and transfered in 10 mL PBS to an ice-cold glass mortar.
  • the cells are homogenized at 4°C using a motor-driven pestle and then centrifuged at 3000 x g for 5 minutes. The antigen-rich supernatant is removed from the other cell debris and subjected to further centrifugation at 100,000 x g for one hour at 4°C.
  • the pellet (antigen fraction) from this final step is suspended in 100 ⁇ L of PBS for every million cells harvested. Following an estimate of the protein concentration (BioRad BCA protein assay using bovine immunoglobulin as the protein standard) the antigen is stored at -20°C until use.
  • Each well of a 96-well Costar microtiter plates is coated with antigen by adding 100 ⁇ L/well of cell lysate
  • Biotinylated ING-1 (1.0 mg/mL in 0.1% BSA) is added to each well (50 ⁇ L/well) and the plates are then incubated for 2 hours at room temperature. Following five washes with 0.05% Tween-20, the plates are blotted dry and incubated at room temperature for one hour with dilute (1:2000 in 0.1% BSA) streptavidin-alkaline phosphatase (Tago) . After a further five washes, color is developed in each well upon the addition of 100 ⁇ L per well of phosphatase substrate reagent (Sigma) . After one hour at room temperature, the color wis read using a 405 nm filter in a Titertek Multiscan microplate reader.
  • a 30 cm x 7.5 mm TSK-G3000SW size-exclusion HPLC column (Supelco) fitted with a guard column of the same material is equilibrated with 12 column volumes of 10 mM sodium phosphate buffer pH 6.0 supplemented with 150 mM sodium chloride using a Waters 600E HPLC system with a flow rate of 1.0 mL per minute at 400-600 PSI.
  • a sample (25 uL) of BioRad gel filtration protein standards is injected on to the column. The retention time of each standard is monitored by a Waters 490 UV detector set at 280 nm.
  • the column is washed with a further 10 volumes of 10 mM sodium phosphate buffer pH 6.0 supplemented with 150 mM sodium chloride.
  • Samples (50 ⁇ L) of either native ING- 1 antibody or ING-1-DHFR at 200 ⁇ g/mL are injected on to the column and their retention times recorded. From the areas of the retained peaks and the retention time, the amount of aggregated material in the ING-1-DHFR or ING- 1-trimethoprim conjugates sampled are calculated.
  • the enzymatic activity of DHFR is used to monitor: i) Preservation of enzyme activity in a manner analogous to measuring the binding of the antibody to it's antigen, the activity of the enzyme before and after conjugation is assayed to ensure that the act of conjugation does not inhibit the enzyme; ii) inhibitory effect of drugs (e.g., trimethoprim and the trimethoprim analogs described above in Examples
  • DHFR activity is defined in terms of units; one said unit of DHFR enzyme activity is defined as the amount of material needed to convert 1.0 micromole of 7,8-dihyrofolic acid and the reduced form of ⁇ -nicotinamide adenine dinucleotide phosphate (NADPH) to 5,6,7,8-tetrahydrofolate and the oxidized form of ⁇ - nicotinamide adenine dinucleotide phosphate (NADP) per minute at pH 6.5 and 25°C)
  • the activity of the enzyme was measured spectrophotometrically at 340 nm by following the the rate of oxidation of NADPH to NADP during the reduction of dihydrofolate to tetrahydrofolate.
  • An aliquot (362 ⁇ L) of a freshly thawed solution of the enzyme substrate (7,8, dihyrofolic acid (5.44 mM) ) was treated with 38 ⁇ L ⁇ -mercaptoethanol and 600 ⁇ L of lOOmM Imidazole buffer pH 7.0.
  • the reaction mixture contained 20 ⁇ l (3.2 mg/mL) of NADPH, 20 ⁇ L of the above 7,8, dihyrofolic acid mixture, and 955 ⁇ L lOOmM Imidazole buffer pH 7.0.
  • a sample (5 ⁇ L; 120 ng of protein) of DHFR was added to the prewarmed (25°C) reaction mixture, mixed rapidly, and the change in optical density at 340 nm was monitored every 5 seconds for a total of 120 seconds in a Shimadzu 160U ultraviolet spectrophotometer. The activity (units/mL) was calculated from the slope of the optical density vs time plot.
  • Example 8b A sample (1.2 ⁇ g of protein) of ING-1/DHFR conjugate (from Example 8b) was used in place of DHFR in the assay for enzyme activity (Example llf) .
  • the activity of the conjugate (units/mL) was calculated from the slope of the optical density vs time plot as before. From the activity of the conjugate prepared in Example 8b and the activity of a known standard amount of unconjugated DHFR, the average number of molecules of DHFR conjugated to each antibody molecule was calculated and found to be 0.34 moles of DHFR per mole of antibody.

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Abstract

L'invention se rapporte à un immunoréactif de ciblage non radioactif, qui est constitué par le reste d'un site actif protéiné d'une enzyme appelée dihydrofolate réductase (DHFR), par un groupe de liaison et par le reste d'une substance immunoréactive en association avec un agent d'apport radioactif constitué par le reste d'un ligand ayant une affinité pour la fixation non covalente à une fraction réceptrice de cette enzyme DHFR, par un groupe de liaison et par le reste d'un agent radioactif. Cette invention décrit également un immunoréactif de ciblage non radioactif constitué par le reste d'un ligand ayant une affinité pour la fixation non covalente à une fraction réceptrice du site actif protéiné de l'enzyme DHFR, par un groupe de liaison et par le reste d'une substance immunoréactive en association avec un agent d'apport radioactif constitué par le reste d'une fraction réceptrice du site actif protéiné de l'enzyme DHFR, par un groupe de liaison et par un agent radioactif. Ces compositions comprennent des systèmes utiles pour la production d'une amplification de l'apport de l'agent radioactif sur des sites de tumeurs dans le domaine de la thérapie et de l'imagerie diagnostique du cancer.
EP94905922A 1992-12-15 1993-12-06 Reactifs a reactivite immunologique utilisant la dihydrofolate reductase Withdrawn EP0675737A1 (fr)

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US99042392A 1992-12-15 1992-12-15
US990423 1992-12-15
PCT/US1993/011842 WO1994013327A1 (fr) 1992-12-15 1993-12-06 Reactifs a reactivite immunologique utilisant la dihydrofolate reductase

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US5688488A (en) * 1989-04-03 1997-11-18 Purdue Research Foundation Composition and method for tumor imaging
DE19910102B4 (de) * 1999-03-08 2006-06-01 Fischer, Markus, Dr.rer.nat. Proteinkonjugate, Verfahren, Vektoren, Proteine und DNA zu deren Herstellung, deren Verwendung, sowie Arzneimittel und Impfstoffe mit einem Gehalt derselben
DE60230046D1 (de) * 2001-08-10 2009-01-08 Novartis Ag Peptide, die atherosklerotische schädigungen binden
CA2559188C (fr) * 2004-03-05 2013-01-08 Denki Kagaku Kogyo Kabushiki Kaisha Conjugue acide hyaluronique-methotrexate
JP5001645B2 (ja) * 2004-04-02 2012-08-15 電気化学工業株式会社 ヒアルロン酸−メトトレキサート結合体
EP1991577A2 (fr) 2006-01-31 2008-11-19 Parkinson, John F. Modulation de l'activite mdl-1 pour le traitement de maladies inflammatoires
EP2005970A1 (fr) 2007-06-22 2008-12-24 Berlin Science Partners GmbH i.V. Imagerie Diagnostique par combinaison de moyens de contraste
EP2072060A1 (fr) 2007-12-18 2009-06-24 Institut Curie Procédés et compositions pour la préparation et l'utilisation de conjugués de toxines
CN112649600A (zh) * 2021-01-13 2021-04-13 青岛农业大学 基于dhfr的磺胺增效剂类药物多残留荧光偏振免疫分析方法

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