EP0681484A1 - Immunreaktive reagenzien unter verwendung von heterodimeren - Google Patents

Immunreaktive reagenzien unter verwendung von heterodimeren

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Publication number
EP0681484A1
EP0681484A1 EP94907897A EP94907897A EP0681484A1 EP 0681484 A1 EP0681484 A1 EP 0681484A1 EP 94907897 A EP94907897 A EP 94907897A EP 94907897 A EP94907897 A EP 94907897A EP 0681484 A1 EP0681484 A1 EP 0681484A1
Authority
EP
European Patent Office
Prior art keywords
group
reagent
residue
mrp14
mrp8
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Withdrawn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
EP94907897A
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English (en)
French (fr)
Inventor
Robert Allen Snow
Christopher Douglas Valiant Black
Clyde William Shearman
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Wellcome Foundation Ltd
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Wellcome Foundation Ltd
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Publication date
Application filed by Wellcome Foundation Ltd filed Critical Wellcome Foundation Ltd
Publication of EP0681484A1 publication Critical patent/EP0681484A1/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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 .
  • 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 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 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
  • NRTIR (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 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 is directed to a NRTIR comprised of the residue of a proteinaceous subunit portion of a heterodimeric molecule (sometimes hereinafter referred to as an HI) , 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.
  • This embodiment is also directed to a RDA comprised of a second subunit portion of a heterodimeric molecule (sometimes hereinafter referred to as an H2) which associates with said subunit portion HI of the heterodimeric molecule receptor moiety, a linking group, and a radioactive agent.
  • This RDA is administered to the environs of the tissue which contains said NRTIR bound thereto.
  • the heterodimeric subunit portion H2 of said RDA will bind to the heterodimeric subunit portion HI of said NRTIR to provide an effective amount of radioactivity to said tissue. Unbound RDA is removed rapidly from the environs of said tissue.
  • the present invention comprises an NRTIR comprised of the residue of a receptor moiety which receptor moiety is comprised of the residue of the proteinaceous subunit (HI) of the myeloid differentiation protein (sometimes hereinafter referred to as MRP14), a linking group, and the residue of an immunoreactive material.
  • System A also comprises a RDA of another proteinaceous subunit (H2) of the myeloid differentiation protein (sometimes hereinafter referred to as MRP8) , a linking group, and a radioactive agent.
  • the present invention is directed to an NRTIR comprised of the residue of the proteinaceous subunit (HI) of the myeloid differentiation protein (MRP14) , a linking group, and the residue of an immunoreactive material such as a tumor targeting antibody together with an RDA comprised of another proteinaceous subunit (H2) of the myeloid differentiation protein (MRP8) , a linking group, and a radioactive agent comprised of a chelating agent and a radionuclide.
  • an NRTIR comprised of the residue of the proteinaceous subunit (HI) of the myeloid differentiation protein (MRP14) , a linking group, and the residue of an immunoreactive material such as a tumor targeting antibody together with an RDA comprised of another proteinaceous subunit (H2) of the myeloid differentiation protein (MRP8) , a linking group, and a radioactive agent comprised of a chelating agent and a radionuclide.
  • the NRTIR of system A is comprised of (n) MRP14 moieties, each of which can associate with RDA comprised of the residue of a MRP8 with an affinity for association with a MRP14 and of (m) radioactive agents where each of n and m are independently integers 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 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.
  • the present invention is directed to an NRTIR comprised of the residue of a ligand which exhibits an affinity for 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 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 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.
  • this aspect (sometimes hereinafter referred to as System B) , of the present invention comprises a NRTIR comprised of the residue of a proteinaceous subunit (H2) of the myeloid differentiation protein (MRP8) , a linking group, and the residue of an immunoreactive material and an RDA comprised of the residue of the proteinaceous subunit (HI) of the myeloid differentiation protein (MRP14) , a linking group, and a radioactive agent.
  • NRTIR comprised of the residue of a proteinaceous subunit (H2) of the myeloid differentiation protein (MRP8) , a linking group, and the residue of an immunoreactive material and an RDA comprised of the residue of the proteinaceous subunit (HI) of the myeloid differentiation protein (MRP14) , a linking group, and a radioactive agent.
  • the present invention is directed to a NRTIR comprised of the residue of a proteinaceous subunit (H2) of the myeloid differentiation protein (MRP8) , 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 the proteinaceous subunit (HI) of the myeloid differentiation protein (MRP14) , a linking group, and a radioactive agent comprised of a chelating agent and a radionuclide.
  • a NRTIR comprised of the residue of a proteinaceous subunit (H2) of the myeloid differentiation protein (MRP8) , 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 the proteinaceous subunit (HI) of the myeloid differentiation protein (MRP14) , a linking group, and a radioactive agent comprised of a chelating agent and a radionuclide.
  • H2 proteinaceous subunit
  • the NRTIR of System B is comprised of (n) residues of MRP8 that have an affinity for binding to a MRP14, each of which can bind an RDA comprised of a MRP14 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.
  • the association 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 also directed to pharmaceutical and diagnostic compositions comprising an NRTIR and a pharmaceutically acceptable carrier (excipient) , 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 vi tro or in vi vo, 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 vi tro or in vi vo, 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 NRTIR will bind to sites on cells of said tissue of interest and unbound NRTIR will be removed from the environs of the tissue.
  • 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 thus reducing the toxicity; the 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 NRTIR can comprise a wide variety of immunoreactive groups, linking groups, and HI residues in System A, and a wide variety of immunoreactive groups, linking groups, and H2 residues which associate with HI residues in System B
  • the RDA can comprise a wide variety of spacing, linking and chelating groups, radionuclides, and H2 residues which have an affinity to associate with HI residues in System A, and a wide variety of spacing, linking and chelating groups, radionuclides, and HI residues which have an affinity to associate with H2 residues in System B;
  • 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 MRP14;
  • D is the residue of a ligand, preferably a MRP8, that has an affinity for binding to a receptor, preferably to a MRP14;
  • Hi is the residue of one of two subunits of a heterodimer which comprises HI and H2, preferably HI is MRP14;
  • H2 is the residue of one of two subunits of a heterodimer which comprises HI and H2, preferably H2 is MRP8 a subunit that has an affinity association with HI, i and I_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.
  • Heterodimers are proteins composed of two nonidentical subunits, HI and H2; each subunit may serve as either a receptor subunit or a ligand subunit .
  • Any heterodimeric subunit receptor ligand pair is useful in this invention.
  • those subunit receptor ligand pairs which: i) non-covalently associate (i.e. H1/H2 or H2/H1) without inter-subunit covalent bond formation (for example, without disulfide bond formation) between the receptor and subunit; ii) have high inter subunit pair association constants, such that, once the subunits have bound to each other to form a heterodimer, the two non- covalently bonded subunits remain stably associated for prolonged periods even in the presence of other
  • I O proteins such as immunoglobulins, albumin, and other plasma proteins; iii) do not substantially self-associate to form homodimers (i.e. HI/HI or H2/H2) from identical subunits; iv) are soluble in blood; v) are free of contaminating materials such as, for example, the phospholipids, lipids, sterols, and carbohydrates of membranes; vi) are available in recombinant form; vii) have no affinity for binding to sites currently found within a mammalian blood circulatory system; viii) have no enzymatic activity when re-associated; ix) are not products of oncogenes; and x) have no cell regulatory function.
  • MRP14 and MRP8 also known as pl4 and p ⁇ , also known as the cystic fibrosis antigen, also known as L heavy chain and L*L light chain
  • alpha and beta chains of the T cell receptor also known as the cystic fibrosis antigen, also known as L heavy chain and L*L light chain
  • proteins of the cytokine IL-2 natural killer cell stimulatory factor
  • cytochrome b558 signal recognition particle
  • ligandin chaperone proteins
  • punta toro virus glycoproteins hepatopoietins A and B
  • human platelet-derived growth factor lipocortin II
  • the heterodimeric proteins of the following enzymes glutathione S-transferases; reverse transcriptase; luciferase; creatine kinase; phosphoglycerate mutase; alcohol dehydrogenase; and gamma-glutamyl transpeptidase.
  • the term "residue” is used herein in context with a chemical entity.
  • Said chemical entity comprises, for example, a ligand, or a H2, or the proteinaceous subunit of the myeloid differentiation protein MRP8, or a receptor moiety, or an HI, or the proteinaceous subunit of the myeloid differentiation protein MRP14, 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, are 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, polysaccharides, 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
  • 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.
  • the immunoreactive group does not bind to
  • the term "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 for binding refers to the thermodynamic expression of the strength of interaction or binding between an antibody combining site and an antigenic determinant and, thus, of the stereochemical compatibility between them; as such, it is the expression of the equilibrium or association constant
  • affinity also refers to the thermodynamic expression of the strength of interaction or binding between a ligand and a receptor and, thus, of the stereochemical compatibility between them; as such, it is the expression of the equilibrium or association constant for the 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 and resulting from the 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 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;
  • a chemical reaction comprising 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 comprising 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; or
  • 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.
  • 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, aspartate, 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 O-A-89/02931 and O-A-89/2932, which are directed to modification of oligonucleotides, and U.S. Patent No. 4,719,182.
  • l C Two highly preferred uses for the 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. 4,522,918 and 4,612,282) which recognize colorectal tumors; 9.2.27 and related anti-melanoma antibodies; D612 and related antibodies which recognize colorectal tumors; UJ13A and related antibodies which recognize small cell lung carcinomas; NRLU-10, NRCO-02 and related antibodies which recognize small cell lung carcinomas and colorectal tumors (Pan-carcinoma) ; 7E11C5 and related antibodies which recognize prostate tumors; CC49 and related antibodies which recognize colorectal tumors; TNT and related antibodies which recognize necrotic tissue; PR1A3 and related antibodies which recognize colon carcinoma; ING-1 and related antibodies, which are described in International Patent Publication O- A-90/02569; B174, C174 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
  • 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 or has a specific affinity for binding to a receptor, especially to an active site in a receptor.
  • receptors include one of two subunits of a heterodimeric protein, such as HI; which has an affinity for binding to the other subunit of said heterodimeric protein as in System A, one of two subunits of a heterodimeric protein, such as H2 which has an affinity for binding to the other subunit of said heterodimeric protein as in System B; cell surface receptors which bind hormones; and cell surface receptors which bind drugs.
  • the sites of specific association of one subunit, HI, of a heterodimeric protein with the other subunit H2; of specific association of one subunit, H2, of a heterodimeric protein with said Hl ; of specific binding of hormones to said cell surface receptors; and of specific binding of drugs to all surface receptors are examples of active sites of said receptors, and the heterodimers subunit HI and subunit H2, hormones, and drugs are examples of ligands for the respective receptors .
  • Preferred receptors (Rec) in System A, HI, and in System B, H2 are comprised of the residue of an active site of a subunit of a heterodimeric protein, HI and H2.
  • Preferred ligands in System A, H2, and System B, HI are comprised of the residue of the other subunit of the heterodimeric protein, H2 and HI.
  • an especially preferred receptor is comprised of the residue of the subunit MRP14 of a calcium-binding protein belonging to the S-100 protein family, of molecular weight of approximately 14,000 daltons .
  • Said MRP14 subunit, in whole or in part, can be isolated from any source and used in this invention without further modification, as long as it maintains MRP8 binding activity.
  • an especially preferred receptor is comprised of the residue of the subunit MRP8 of a calcium-binding protein belonging to the S-100 protein family, of molecular weight of approximately 8,000 daltons.
  • Said MRP8 subunit, in whole or in part can be isolated from any source and used in this invention without further modification, as long as it maintains MRP14 binding activity. See Edgeworth, J.
  • the subunits, MRP14 and MRP8 are isolated from the cytosol of human neutrophils, or the subunits are produced in a suitable organism (e.g., bacteria, yeast, insect or mammalian cells) as a recombinant human protein.
  • a suitable organism e.g., bacteria, yeast, insect or mammalian cells
  • Said subunits are chemically modified before or after isolation for use in this invention, or they can be modified by well known techniques of molecular biology and isolated for use in this invention, or said molecular biology modified subunits can be chemically modified before or ⁇ after isolation for use in this invention as long as the active site of each subunit is maintained in such use.
  • the MRP14 is comprised of a human protein.
  • said MRP14 is comprised of a recombinant human protein. More preferably, said MRP14 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 MRP14 subunit comprised of a thus modified recombinant human protein is comprised of an active site which has an affinity for binding to a MRP8 subunit.
  • a thus modified recombinant MRP14 subunit has an affinity for a MRP8 subunit which is greater than the affinity of the natural, unmodified, MRP14 subunit for a MRP8 subunit.
  • the Z-L-X of System A is comprised of a fusion protein.
  • 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 MRP14.
  • said fusion protein is comprised of a residue of an immunoreactive reagent fused to one or more residues of MRP14.
  • said fusion protein is comprised of residues of MRP14 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 MRP14.
  • said fusion protein can be comprised of one or more MRP14 fused to an immunoglobulin heavy chain in the CH2 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 m ⁇ jre MRP14.
  • said fusion protein can be comprised of one or more MRP14 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 MRP14.
  • the above genetically engineered fusion protein comprising Z-(L**_-Rec) n of System A can be comprised a
  • ⁇ ⁇ l 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 human MRP14 or a genetically engineered modified human MRP14.
  • the fusion protein is comprised of a thus modified recombinant MRP14 comprised of an active site which has an affinity for binding to a MRP8 subunit.
  • a thus modified recombinant MRP14 subunit of a fusion protein has an affinity for a MRP8 subunit which is greater than the affinity of the natural, unmodified, MRP14 subunit for a MRP8 subunit.
  • MRP8 of a calcium-binding protein belonging to the S-100 protein family, of molecular weight of approximately 8,000 daltons
  • Said MRP8 subunit in whole or in part, can be isolated from any source, as long as it maintains MRP14 binding activity (See references above) .
  • MRP8 is isolated from the cytosol of human neutrophils, or the MRP8 is produced in a suitable organism (e.g., bacteria, yeast, insect or mammalian cells) as a recombinant human protein.
  • the MRP8 subunit is comprised of a human protein.
  • said MRP8 subunit is comprised of a recombinant human protein.
  • said MRP8 subunit is comprised of a recombinant human protein which is modified by genetic engineering techniques, which modifications comprise the independent incorporation, substitution, insertion, and deletion
  • the MRP8 subunit comprised of a thus modified recombinant human protein is comprised of an active site which has an affinity for binding to a MRP14 subunit.
  • a thus modified recombinant MRP8 subunit has an affinity for a MRP14 subunit which is greater than the affinity of the natural, unmodified, MRP8 subunit for a MRP14 subunit.
  • the Z-(L ⁇ -D) n of System B is comprised of a fusion protein.
  • said fusion protein is comprised of a residue of an immunoreactive reagent fused to one or more residues of a MRP8.
  • said fusion protein is comprised of residues of MRP8 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 MRP8.
  • said fusion protein can be comprised of one or more MRP8 fused to an immunoglobulin heavy chain in the CH2 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 MRP8.
  • said fusion protein can be comprised of one or more MRP8 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 MRP8.
  • the above genetically engineered fusion protein comprising Z-(L ⁇ D) n of System B can be comprised of 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 human MRP8 or a genetically engineered modified human MRP8.
  • the fusion protein is comprised of a thus modified recombinant
  • MRP8 comprised of an active site which has an affinity for binding to a MRP14 subunit.
  • a thus modified recombinant MRP8 subunit of a fusion protein has an affinity for a MRP14 subunit which is greater than the affinity of the natural, unmodified, MRP8 subunit for a MRP14 subunit .
  • association of a ligand with a receptor can comprise a non-covalent interaction, or it can comprise the formation of a covalent bond.
  • association is non-covalent.
  • n MRP14 subunits are covalently linked, i.e., conjugated, by a linking group 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.
  • n is i, 2, 3, 4, 5 or 6. Most preferably n is 1 or 2.
  • an MRP8 subunit is a component of a radioactive delivery agent [i.e., an RDA, Rec- (L 2 -Q-M) m ] is attached to m chelating groups, each by means of a linking group, and the chelating group is associated with a radionuclide.
  • a radioactive delivery agent i.e., an RDA, Rec- (L 2 -Q-M) m
  • the chelating group is TMT (described hereinbelow)
  • the linking group is as described below
  • the radionuclide is an isotope of yttrium
  • m is 2 to about 10.
  • the RDA in System A is comprised of a MRP8 that contains one or more radionuclides that are covalently attached, either directly to one or more components of the MRP8 or to one or more components that are attached by a linking group as described below to the MRP8.
  • said covalently attached radionuclide is a
  • the RDA in System A is comprised of a MRP8 that contains one or more radionuclides that are covalently attached, either directly to one or more components of the MRP8 (such as described in U.S. Patent No. 5,078,985, the disclosure of which is hereby incorporated by reference) or to one or more components that are attached to the MRP8 by a linking group such as are derived from N 3 S and N 2 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.
  • said covalently attached radionuclide is selected from a radioisotope of technicium and rhenium attached to a group comprised of a sulfur atom.
  • n MRP8 subunits are covalently linked, i.e., conjugated, by a linking group 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.
  • a linking group 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.
  • n is 1, 2, 3, 4, 5 or 6. More preferably, n is 1 or 2.
  • a MRP14 subunit is a component of a radioactive delivery agent [i.e., an RDA, Rec- (L 2 -Q-M) m ] , and is attached to m chelating groups, each by means of a linking group, and the chelating group is associated with a radionuclide.
  • a radioactive delivery agent i.e., an RDA, Rec- (L 2 -Q-M) m
  • the chelating group is associated with a radionuclide.
  • the chelating group is TMT
  • the linking group is as described below, the radionuclide is an isotope of yttrium, and m is 2 to about 10.
  • the RDA in System B is comprised of a MRP14 that contains one or more radionuclides that are covalently attached, either directly to one or more components of the MRP14 or to one or more components that are attached by a linking group as described below to the MRP14.
  • a MRP14 that contains one or more radionuclides that are covalently attached, either directly to one or more components of the MRP14 or to one or more components that are attached by a linking group as described below to the MRP14.
  • 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 MRP14 that contains one or more radionuclides that are covalently attached, either directly to one or more components of the MRP14 or to one or more components that are attached by a linking group as described below to the MRP14.
  • said covalently attached radionuclide is selected from a radioisotope of technicium and rhenium attached to a group comprised of a sulfur atom.
  • linking group Li
  • activated groups such as activated ethylene groups (e.g., maleimide groups)
  • amine groups such as lysine epsilon-amines of a protein
  • 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 Pierce Chemical Company are included as non-limiting examples.
  • System A in one aspect, chemical conjugation is otherwise achieved by using a linking group, Li which is introduced through mild reduction of the MRP14 (or of the MRP14 chemically modified by covalent attachment of reagents which contain disulfide bonds) with a reducing reagent such as dithiothreitol to produce sulfhydryl (SH) sites in the reduced MRP14 protein moiety.
  • a linking group Li which is introduced through mild reduction of the MRP14 (or of the MRP14 chemically modified by covalent attachment of reagents which contain disulfide bonds) with a reducing reagent such as dithiothreitol to produce sulfhydryl (SH) sites in the reduced MRP14 protein moiety.
  • a linking group Li which is introduced through mild reduction of the MRP14 (or of the MRP14 chemically modified by covalent attachment of reagents which contain disulfide bonds) with a reducing reagent such as dithiothreitol to produce sulf
  • reaction of the above reduced immunoreactive protein moiety with the residue of a ligand which contains a precursor of one or more linking groups, each of which residue comprises an activated ethylene group such as a maleimide group results in the formation of an immunoreactive protein moiety/ligand conjugate linked together by one or more thioether bonds .
  • reaction of the above reduced MRP8 protein moiety with a chelating agent which contains a precursor of a linking group comprised of an activated ethylene group such as a maleimide group results in a MRP8/chelating agent conjugate wherein the reduced MRP8 is covalently attached to one or more chelating agents by a thioether bond.
  • reaction of the thus reduced immunoreactive protein moiety to the residue of a ligand which contains a precursor of one or more linking groups, each of which residue comprises an activated ethylene group such as a maleimide group results in the formation of an immunoreactive protein moiety/ligand conjugate linked together by one or more thioether bonds .
  • 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 glutamic 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 MRP14 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 MRP14 are also suitable. Such mixtures can have a bulk ratio of MRP14 to immunoreactive group of from about 0.1 to about 10.
  • the ratio of MRP8 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 MRP8 are iff also suitable.
  • Such mixtures can have a bulk ratio of MRP8 to immunoreactive group of from about 0.1 to about 10.
  • the mole ratio of MRP14 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 MRP14, especially human MRP14, a fusion protein can be made between the antibody and the MRP14, or portions thereof, through the use of genetic engineering techniques . It is specifically contemplated that in all of these compositions of MRP14 bound to antibody, the MRP14 retains a capacity to bind to the ligand subunit of the heterodimer described in the invention. In System B, in preferred embodiments, the mole ratio of MRP8 to immunoreactive group is from about 1:1 to about 10:1.
  • mixtures comprised of immunoreactive groups which are unmodified and immunoreactive groups which are modified with MRP8 are also suitable. It is specifically contemplated that with knowledge of the DNA sequence that encodes MRP8, especially human MRP8, a fusion protein can be made between the antibody and the MRP8, or portions thereof, through the use of genetic engineering techniques. It is specifically contemplated that in all of these compositions of MRP8 bound to antibody, the MRP8 retains a capacity to bind to the receptors described in the invention.
  • 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.
  • 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/MRP14 conjugate in a different fraction from any residual non-conjugated MRP14 in System A, and in the elution of the antibody/MRP8 conjugate in a different fraction from any residual non-conjugated MRP8 in System B.
  • the concentrations of the antibody in the conjugate solutions are determined by the BCA (BioRad Catalog # 500-0001) method using bovine immunoglobulin as the protein standard.
  • the ability of the antibody to bind to its target antigen following conjugation to either MRP14 (system A) or MRP8 (system B) 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.
  • Li and L 2 in System A and System B are each 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.
  • protein reactive group 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 HI receptor- containing species "Rec", (e.g.
  • 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 MRP14-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 MRP14-containing species with themselves is avoided, whereas the reaction of, for example, homobifunctional 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
  • 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, one or more of which has been incorporated into an oligonucleotide sequence, through synthesis using one of the above Clontech reagents, with, for example, one or more of the previously described protein reactive groups such as the above described heterobifunctional protein reactive groups, one or more of which has been incorporated into, for example, an immune reactive agent or a MRP14 moiety as described in system A of this invention or a MRP8 moiety as described in system B of this invention.
  • a reactive functional group such as an amine or sulfhydryl group
  • one sequence is attached to the immune reactive agent and the complementary oligonucleotide sequence is attached to the MRP14-containing moiety.
  • the hybrid formed between the two complementary oligonucleotide sequences then comprises the linking group between the immune reactive agent and the MRP14-containing moiety.
  • the complementary oligonucleotide sequences are separately 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 MRP14-containing moiety.
  • the hybrid formed between the two complementary oligonucleotide sequences then comprises the linking group between the MRP14-containing moiety and the component comprised of one or more chelating agents.
  • one or more oligonucleotides each comprising two or more oligonucleotide units each containing a copy of the same oligonucleotide sequence linked, for example, in tandem, and optionally with spacing groups and linking groups, can be covalently attached to one MRP14-containing moiety.
  • An oligonucleotide sequence containing a sub-sequence that is complementary to the above copied sequence and is comprised of one or more chelating agents can then be added to the above MRP14-containing moiety.
  • multiple hybrids formed between the parts of complementary oligonucleotide sub-sequences then comprise the linking group between the MRP14- containing moiety and the multiple chelating agents.
  • the residue of one or more MRP8-containing moieties which associate with MRP14 can be attached to the immunoreactive group using complementary ol ⁇ gonucleotide hybrids as described above.
  • multiple MRP14-containing moieties can be attached to the immunoreactive protein analogously.
  • an MRP8-containing moiety 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- X sulfosalicylic acid; polyamines, such as ethylenediamine, diethylenetriamine, triethylenetetramine, and triaminotriethylamine; aminoalcohols, such
  • Schiff bases such as disalicylaldehyde 1,2- propylenediimine
  • tetrapyrroles such as tetraphenylporphin and phthalocyanine
  • sulfur compounds such as toluenedithiol, meso-2,3- dimercaptosuccinic acid, dimercaptopropanol, thioglycolic acid, potassium ethyl xanthate, sodium diethyldithiocarbamate, dithizone, diethyl dithiophosphoric acid, and thiourea
  • synthetic macrocylic compounds such as dibenzo[18] crown- 6, (CH 3 ) 6-[14]-4,ll-diene-N 4 , and (2.2.2-cryptate)
  • phosphonic acids such as nitrilotrimethylene- phosphonic acid, ethylenediaminetetra (methylenephosphonic acid) , and hydroxyethylidenediphosphonic acid, or combinations of two or more of the
  • Preferred residues of chelating agents contain polycarboxylic acid groups and include: ethylenediamine-N, N, N 1 ,N'-tetraacetic acid (EDTA) ;
  • N,N,N' ,N",N"-diethylene-triaminepentaacetic acid DTPA
  • DTPA 1, 4,7, 10-tetraazacyclododecane-N,N' ,N",N" '- tetraacetic acid
  • D03A 1,4,7,10- tetraazacyclododecane-N,N' ,N"-triacetic acid
  • OTTA 10-triazacyclododecane-N,N' ,N"-triacetic acid
  • CDTPA C cyclohexanodiethylenetriamine pentaacetic acid
  • Preferred residues of chelating agents contain polycarboxylic acid groups and include: B4A, P4A, TMT, DCDTPA, PheMT, macroPheMT, and macroTMT;
  • 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 2 S 2 containing compounds, as for example, those disclosed
  • 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. The residues of the chelating agent Q are independently linked to the other components of this invention through a chemical bond or a linking group such as L as described above.
  • Preferred 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, i.e., iminothiocarbonyl and thiocarbonylimino; thio; dithio; phosphate;
  • k l and Xi, X , X 3 independently are H, alkyl, containing from 1 to 18, preferably 1 to 6 carbon atoms, such as methyl, ethyl and propyl, such alkyl optionally being interrupted by 1 or more heteroatoms such as oxygen, nitrogen and sulfur, substituted or unsubstituted aryl, containing from 6
  • 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 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
  • 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
  • L is the residue of a protein reactive group as defined above, wherein preferably L is a linking group comprised of 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, 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 radionuclides bound thereto by chelating groups, Q, as defined above, such as, for example,
  • Ala-Ala-Ala-Lys-Lys-OH can be synthesized via solid- phase methodology, on an ABI 430A Automated Peptide Synthesizer.
  • a solid support useful in the synthesis is a 4-Alkoxybenzyl alcohol polystyrene resin (Wang resin) .
  • the N-alpha-Fmoc protecting group can be used throughout the synthesis, with S-trityl side chain protection on D-Cys, and t-BOC protection on the side chain of Lys .
  • the peptide chain can be 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.
  • the epsilon amines of the lysine groups of these peptides are reacted with a protein reactive group on a chelating agent, for example with TMT-NCS to form a thiourea linking group to each lysine.
  • a protein reactive group on a chelating agent for example with TMT-NCS
  • the 4-Butyl protecting group on sulfur is removed with acid, and the thus produced peptide containing the SH group is then conjugated using the sulhydryl group to MRP8 and the previously described maleimide heterobifunctional chemistry.
  • the thus prepared conjugate is then exposed to a solution of a radionuclide such as 90y+3 C ⁇ 3 j_ n acetate buffer to form the RDA.
  • the delivery agent (RDA) in System B is comprised of a MRP14 moiety conjugated to one or more chelating agents via a linking group in a like manner.
  • the NRTIR is comprised of one or more ligands such as, for example, MRP8, that each have an affinity for binding to a MRP14.
  • MRP8 is conjugated by a linking group (Li) to an immunoreactive group (Z) as defined above.
  • the NRTIR preferably contains 1 to about 10 of such ligands, more preferably 2 to about 4.
  • 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 to form the RDA.
  • 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 of the RDA.
  • the chelating agent-containing moiety is preferably in an aqueous solution at a pH of between about 4 and about 9, more preferably between pH about 5 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, 6 Cu, 11: ⁇ -In, 212 Pb, 68Ga, 8 7 Y, 9°Y, 153 S m, 212 Bi , 99m Tc , 177 Lu 186 Re an d i SSRe. Of these, especially preferred is 90 Y.
  • the RDA of this invention comprises a diagnostically effective amount of a radionuclide and a therapeutically effective amount of a second radionuclide.
  • the diagnostically effective radionuclide is an radioisotope of iodine such as 131 I
  • the therapeutically effective isotope is a radioisotope of
  • 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 L . 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, Tm, Yb and Lu. Mn, Gd, and Dy are especially preferred.
  • MRP14 and MRP8 also known as pl4 and p8, also known as the cystic fibrosis antigen, also known as Li heavy chain and L ⁇ light chain
  • alpha and beta chains of the T cell receptor also known as pl4 and p8, also known as the cystic fibrosis antigen, also known as Li heavy chain and L ⁇ light chain
  • proteins of the cytokine IL-2 natural killer cell stimulatory factor
  • cytochrome b558 signal recognition particle
  • ligandin chaperone proteins
  • punta toro virus glycoproteins hepatopoietins A and B
  • human platelet-derived growth factor lipocortin II
  • the heterodimeric proteins of the following enzymes glutathione S-transferases; reverse transcriptase; luciferase; creatine kinase; phosphoglycerate mutase; alcohol dehydrogenase; and gamma-glutamyl transpeptidase.
  • structure 2 An example of a structure of an RDA that has utility in this invention is represented by structure 2.
  • MRP comprises one of the residue of a ligand, MRP8, in system A or the residue of a receptor, MRP14, in system B; each of R 1 and R" is independently selected from a component of an amino acid that comprise a natural amino acid 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, an unnatural racemate of a natural amino acids, and from H, a polyalkylene oxidyl group with a molecular weight in the range of 72 to 5000 daltons, and the alkyl units therein comprised of from 2 to 10 carbon atoms, a branched peptide group comprised of from 2 to 20 of the above amino acids and which may contain from 1 to 10 additional radioactive groups; each of mi, m , and ⁇ .3
  • MRP comprises one of a residue of a ligand (MRP8) in system A or a residue of a receptor (MRP14) in system B; each of R 1 and R" is independently selected from a component of an amino acid that comprise, for example, a natural amino acid such as glycine, alanine, leucine, serine, lysine, isoleucine, glutamine, aspartic acid, glutamic acid, proline, threonine, valine, phen'ylalanine, tyrosine, and the like, as well as an unnatural amino acid or a racemate of natural amino acid, and from H, a poly (alkylene oxidyl) group as described above, a branched peptide group which may contain from one to about 10 additional radioactive groups as described above; each of m 4 , ⁇ . 5 , and mg is independently selected from zero and an integer between 1 and 10 with the proviso that m $ is at least 1, and preferably 2 to about 5; and
  • W is selected from OH, NH , the residue of a radioactive group as described above, an O-alkyl group as described above, NR a R b wherein each of R a and R b is independently selected from a alkyl groups as
  • 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 comprising a residue of an MRP8, a linking group, and a residue of a chelating agent in System A, and a residue of a MRP14, a linking group, and a residue of a chelating agent in System B)- to a source of radioactive metal ion wherein the molar amount of said radionuclide metal ion is less than the molar amount of the chelating group comprising the RDA and wherein the duration of such exposure lasts an effective time so that uptake of said metal ion into said R
  • 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 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 or to the tissue from said patient, and is allowed to accumulate at the target site, said target site being the NRTIR accumulated at said tumor site in said patient.
  • a mixture of an RDA comprising a diagnostically effective radioactive isotope in combination with an RDA comprising a therapeutically effective radioactive isotope in a pharmaceutically acceptable formulation is specifically contemplated.
  • a therapeutically effective dose of an RDA comprising a radionuclide such as 90 ⁇ +3
  • a diagnostic imaging effective dose of an RDA comprising radionuclide such as 87 ⁇ +3 wherein the ratio of the molar concentration of the therapeutically effective radionuclide ion to the molar concentration of the diagnostically effective radionuclide ion is between 1 and 10,000, preferably between 1 and 1,000, permits the simultaneous diagnostic 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 .
  • 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 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 .
  • 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, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate
  • solution retarders as for example paraffin
  • absorption accelerators as for example, quaternary ammonium compounds
  • wetting agents as for example
  • 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 .
  • opacifying agents 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.
  • 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.
  • the composition can also include 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,
  • S3 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-multiples 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 tis.sue 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
  • a portion of an NRTIR may be reacted with a diagnostic imaging effective amount of a reagent comprised of a radionuclide prior to administration of the entire amount of said NRTIR 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 the immunoreactive group in both the NRTIR and the portion of the NRTIR reacted with the diagnostically effective reagent will bind to sites on cells of said tissue of
  • test subjects can include mammalian species such as rabbits, dogs, cats, monkeys, sheep, pigs, horses, bovine animals and the like.
  • the subject mammal After administration of the compositions of the present invention, 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.
  • ING-1 a chimeric IgGi antibody
  • MRP14 is of human neutrophil origin, purified as outlined below.
  • MRP8 can be conjugated to antibodies by similar procedures .
  • the two proteins MRP14 and MRP8 have been isolated and molecularly cloned, Odink, K. et al. , Nature 330: 80-82 (1987); Lagasse, F. et al. Mol. Cell. Biol. 8: 2402-2410 (1988) .
  • the preferred source for these materials is the recombinant form of the proteins which require no separation from their natural heterodimer complex before use.
  • a purified neutrophil preparation is obtained by centrifuging citrated human blood through dextran followed by percoll density gradient separation of the pelleted cells .
  • Neutrophil cytoplasm is obtained by disrupting the cells by nitrogen cavitation and then layering the non-sedimentable fraction onto a discontinuous Percoll gradient (1.12 about 1.05 g/L) .
  • MRP14/MRP8 complex is separated into component heterodimers by treatment with 9 M urea containing 0.5% 2-mercaptoethanol (Odink et al, supra) followed by application of the sample to a Roto for preparative IEF cell with ampholites in the pH range 5 - 7.5.
  • MRP14 is eluted with a pl of 5.5 and MRP8 is eluted with a pl of 6.7
  • the purified subunits are immediately desalted on PD-10 columns, concentrated and stored at -80°C.
  • a sulfo-SMCC solution (36 nmoles) in PBS is added to a sample of a chimeric antibody (ING-1; 6 nmoles) solution 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 ING-1-malemide. This material is stored on ice until use.
  • a sample 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.
  • ING-1 500 ⁇ g
  • 125 j onochloride or 131 I monochloride at about 5 mCi/mg
  • Iodogen Sodium N- chlorobenzenesulfonamide: Pierce Chemical Co
  • the reaction is terminated by passage of the labeled antibody down a prewashed NAP-5 column.
  • the iodinated protein is eluted with PBS and stored at 4°C until use.
  • a solution containing 50 nmoles of MRP14 in PBS is vortexed while 500 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 MRP14 (N)-CO-CH 2 -S-CO-CH3.
  • the acetylthioacetylated MRP14 is deprotected by the addition of 25 ⁇ L of a pH 7.5 solution containing 100 mM sodium phosphate, 25 mM
  • a solution containing 40 nmoles of MRP14 in PBS is vortexed and an equal volume of 500 mM dithiothreitol in PBS is added. After mixing and standing on ice for 60 minutes, the reaction mixture is eluted from a prewashed PD-10 column with PBS to afford MRP14-SH.
  • the product is eluted off the column directly into the maleimide-derivatized antibody solution. Otherwise the final product is used immediately after preparation.
  • a sulfo-SMCC solution (300 nmoles) in PBS is added to a sample of MRP14 (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 by the addition of 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 frl MRP14-maleimide . This material is stored on ice until use .
  • MRP14 500 ⁇ g
  • 125j monochloride or I3l ⁇ monochloride at about 5 mCi/mg
  • lodogen Sodium N- chlorobenzenesulfonamide
  • the reaction is terminated by passage of the labeled protein down a prewashed NAP-5 column.
  • the iodinated MRP14 is eluted with PBS and stored at 4°C until use.
  • MRP8 500 ⁇ g
  • 12 -3 ⁇ monochloride or I3l ⁇ monochloride at about 5 mCi/mg
  • lodogen Sodium N- chlorobenzenesulfonamide
  • the reaction is terminated by passage of the labeled protein down a prewashed NAP-5 column.
  • the iodinated MRP8 is eluted with PBS and stored at 4°C until use.
  • N-chlorobenzenesulfonamide beads in a volume of 500 ⁇ l of 100 mM phosphate buffer (pH 7.2) at room temperature. After 15 minutes the reaction is terminated by passage of the labeled protein down a prewashed NAP-5 column. The iodinated ING-1 is eluted with PBS and stored at 4°C until use.
  • TMT-NCS or another suitable derivative thereof can be conjugated to either MRP14 or MRP8 subunits of the MRP14/MRP8 heterodimer.
  • Each TMT-conjugated subunit exhibits an affinity for binding to the respective complementary heterodimer subunit .
  • MRP8 (at approximately 5.0 mg/mL) as produced in Example 1 is dialyzed into phosphate buffered saline at pH 7.2.
  • the conjugation of MRP8 to TMT-NCS is achieved by first adding 1.0 M carbonate, 150 mM sodium chloride buffer, pH 9.3 to MRP8 until the pH of the MRP8 solution reaches 9.0.
  • a solution of TMT-NCS is prepared by dissolving 10 mg in 10 mL of 1.0 M carbonate, 150 mM sodium chloride buffer, pH 9.0 at 4°C. The conjugation reaction is started by the addition of 100 ⁇ L of the TMT-NCS solution to the MRP8 to give a 4-fold
  • the protein concentration of MRP8 in the conjugate solutions is determined by the BCA protein assay (BioRad) using bovine immunoglobulin as the protein standard.
  • MRP8/TMT is reacted with fc3 a solution of Europium chloride until saturation of the metal-binding capacity of the TMT, as determined by flourescence emission, occurs.
  • fc3 a solution of Europium chloride until saturation of the metal-binding capacity of the TMT, as determined by flourescence emission, occurs.
  • Europium chloride (Europium chloride hexahydrate: Aldrich) solution in 0.05 M Tris HCl buffer at pH 7.5 is prepared. An aliquot (50 ⁇ L) of this Europium chloride solution is added to the cuvette containing MRP8/TMT and the resulting solution is slowly stirred on a magnetic stirrer at room temperature for 10 minutes using a small magnetic stir bar placed in the cuvette. The phosphorescence of the metal-MRP8/TMT complex is determined in a Perkin Elmer LS 50 spectrofluorometer using an excitation wavelength of 340 nm (10 nm slit width) .
  • the phosphorescent emission is monitored at 618 nm using a 10 nm slit width, a 430 nm cutoff filter and 400 msec time delay. The above procedure is repeated and phosphorescence readings are made after each addition. Aliquots of europium chloride are added until the increase in phosphorescence intensity is less than 5% of the preceding reading. A dilution correction is applied to the phosphorescence intensity measured at each mole ratio, to compensate for the change in volume of the test solution.
  • the ratio of TMT molecules per molecule of MRP8 in bulk solution is in the range of 1:1 to 2:1.
  • Example 3a is eluted off a PD-10 column directly into a solution of maleimide-derivatized ING-1 (5 nmoles) prepared according to Example 2a. 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/MRP14 conjugate thus formed is transferred to a fresh Centricon-30® ultrafiltration concentrator and diluted with PBS.
  • the retentate is again diluted with PBS to 3.0 mL and concentrated by centrifugation.
  • This procedure which separates unconjugated MRP14 and other low molecular weight materials into the filtrate and retains antibody/MRP1 conjugate and unconjugated antibody in the retentate, 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 retentate is then concentrated to approximately 1.0 mg of ING-1/MRP14 per milliliter solution and applied to a 2.6 x 60 cm Sephacryl 5-200 size- exclusion column equilibrated and eluted with a buffer containing 50mM sodium phosphate and 150mM sodium chloride at pH 7.2. This column separates unconjugated antibody from antibody/MRP14 conjugate. Fractions of the eluate containing the conjugate as determined by size exclusion HPLC are pooled, and then centrifuged in a Centricon- 30® device to a concentration of approximately 1.0 mg ING-1/MRP14 per milliliter solution. The solution of the conjugate is sterile filtered through a 0.22 ⁇ filter and stored at 4°C until use.
  • a volume of radioactive Yttrium chloride ( 90 Y in 0.04M hydrochloric acid at a specific activity of >500 Ci/mg: Amersham-Mediphysics) is buffered by the addition of two volumes of 0.5 M sodium acetate at pH 6.0 and added to a solution of MRP8/TMT (prepared according to Example 4) in 0.5 M sodium acetate, pH 6.0, at room temperature.
  • the labeling reaction is allowed to proceed for one hour. Then labeling efficiency is determined by ' removing 1.0 ⁇ L of the sample and spotting it at the origin of a Gel an ITLC- SG strip.
  • the strip is developed in a glass beaker containing 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 strip.
  • the strip is then inserted into a System 200 Imaging Scanner (Bioscan) which has been optimized for 90 Y and controlled by a Compaq 386/20e computer.
  • System 200 Imaging Scanner Bioscan
  • free (unchelated) 90 Y migrates at the solvent front while 90 Y-labeled MRP8/TMT remains at the origin. In excess of 97% of the added 90 Y is taken up by the MRP8/TMT to form the desired 90 Y-labeled product .
  • the concentrations of ING-1, MRP-14, and MRP8 for use in the conjugate reactions are determined by the BCA protein assay (BioRad) using bovine immunoglobulin as the protein standard.
  • BioRad BCA protein assay
  • bovine immunoglobulin bovine immunoglobulin as the protein standard.
  • the MRP14 or MRP8 can be conjugated to other materials (e.g., TMT
  • Example 4 for use with 90 Y or europium fluorescence, or to biotin (Pierce) , or to fluorescein isothiocyanate (FITC) : Pierce) to detect and quantify the amount of MRP14 or MRP8 present in a solution or conjugated to another protein.
  • Antibody/MRP14 conjugates 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 fo- 7 of the antibody before being subjected to modification and conjugation to MRP14.
  • Target HT29 cells a human adenocarcinoma cell line obtained from the American Type Tissue Collection (ATCC)
  • ATCC American Type Tissue Collection
  • the standard curve is made in flow buffer so that each sample contains 1.0 ⁇ g protein per mL. Samples from the standard curve and ING-1/MRP14 unknowns are then incubated with 5xl0 5 HT29 cells at 4°C for 1 hour. Unbound antibody is removed by first centrifuging the cells to a pellet (1000 x g for 5 minutes at 4°C) and then resuspending the cells in 2.0 mL of flow buffer.
  • 6& 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. 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 from a histogram displayed in the analysis window. FITC calibration beads are analyzed 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 ING-1/MRP14 sample with values from the standard curve.
  • the antigen to which the antibody, ING-1, binds is prepared from LS174T or HT29 cells (available from
  • ATCC ATCC 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. Following centrifugation of the cells at 1500 rpm for 10 minutes at 4°C , the cells are washed once in
  • Each well of a 96-well Costar microtiter plates is coated with antigen by adding 100 ⁇ L/well of cell lysate (10 ⁇ g/ml) prepared as above.
  • the microtiter plates are allowed to dry overnight in a 37°C incubator. After washing the plate five times with 0.05% Tween-20 (Sigma) they are blotted dry.
  • the wells of each plate are blocked by adding 125 ⁇ L/well of a
  • 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 PS1.
  • a sample (25 ⁇ L) 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.
  • samples of ING-1/MRP14 are incubated with 5x105 HT29 cells at 4°C for 1 hour. After extensive washing to remove unbound antibody, the cells are incubated at 4°C for 1 hour with a mouse anti-TMT antibody labeled with FITC (prepared according to standard procedures (Pierce Chemical Co. catalog)) . After further washing in flow buffer the samples are analyzed by flow cytometry as before. The average fluorescence intensity for each sample is expressed as the average FITC equivalents per cell to demonstrate that MRP8/TMT is associated with the cells.
  • samples (50 ⁇ L/well in duplicate) of ING/MRP14 conjugates are prepared at a range of concentrations in 1% BSA in PBS and added to the wells of a microtitre plate, prepared as in Example 7c and containing the HT-29 cell antigen in its wells.
  • the plates are then incubated for 1 hour at room temperature. Following three washes with 0.05% Tween- 20, the plates are blotted dry and incubated a further one hour with MRP8/TMT at room temperature. After extensive washing to remove unbound MRP8/TMT, the cells are incubated for 1 hour with a biotinylated mouse anti-TMT antibody (prepared according to standard procedures (Pierce Chemical Co. catalog)) .
  • Sodium dodecylsulfate polyacrylamide gels are also used to demonstrate the association of antibody/MRP14 with MRP8 and the association of antibody/MRP8 with MRP14.
  • the 12 -*>I- labeled MRP8, without conjugated TMT is incubated with the antibody/MRP14 in PBS or in human serum at room temperature, 37°C, and 4°C.
  • samples are withdrawn from the mixtures and subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE) .
  • the gel is examined by autoradiography for the presence of radiolabel associated with the higher molecular weight antibody/MRP14 complex.
  • MRP8/TMT is labeled with 90 Y (Example 6) and incubated with antibody/MRP14 in PBS or in human serum at different temperatures as above. Again, after autoradiography, binding of 90 Y-labeled MRP8/TMT to the higher molecular weight antibody/MRP1 complex shows the ability of the two stages of the delivery system to self assemble even in the presence of human serum at 37°C.
  • SDS PAGE is also used to assay the degree to which the process of conjugation and number of TMTs conjugated influence the ability of the subunits to recognize and associate with each other.
  • Size-exclusion column chromatography is used to quantitate the association between MRP8 and ING-

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JPH08509955A (ja) 1996-10-22
AU6128694A (en) 1994-08-15
WO1994016741A1 (en) 1994-08-04
CA2154896A1 (en) 1994-08-04

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