WO1994016741A1

WO1994016741A1 - Immunoreactive reagents employing heterodimers

Info

Publication number: WO1994016741A1
Application number: PCT/US1994/000965
Authority: WO
Inventors: Robert Allen Snow; Christopher Douglas Valiant Black; Clyde William Shearman
Original assignee: The Wellcome Foundation Limited
Priority date: 1993-01-28
Filing date: 1994-01-26
Publication date: 1994-08-04
Also published as: EP0681484A1; CA2154896A1; AU6128694A; JPH08509955A

Abstract

In one aspect, this invention describes a non-radioactive targeting immunoreagent comprised of the residue of a proteinaceous half of a heterodimeric molecule (H1), a linking group, and the residue of an immunoreactive material together with a radioactive delivery agent comprised of a proteinaceous second half of a heterodimeric molecule (H2), a linking group, and a radioactive agent. In another aspect, this invention describes a non-radioactive targeting immunoreagent comprised of the residue of a proteinaceous half of a heterodimeric molecule (H2), a linking group, and the residue of an immunoreactive material together with a radioactive delivery agent comprised of the residue of a proteinaceous second half of a heterodimeric molecule (H1), a linking group, and a radioactive agent. These compositions comprise useful systems for the production of an amplification of delivery of the radioactive agent to tumor sites in the therapy and diagnostic imaging of cancer.

Description

IMMUNOREACTIVE REAGENTS EMPLOYING; HETERODIMERS

Field of the Invention

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 .

Background of the Invention

The various, currently available, radiolabeled immunoreactive proteins which are employed in diagnostic imaging and targeted therapeutic applications suffer from certain of the following disadvantages :

1) 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;

2) inefficient covalent bonding of the radioactive component with protein in conjugate preparation;

3) long plasma half-lives of currently available radionuclide-containing immunoreactive proteins result in prolonged exposure of normal tissue to damaging effects of radiation and can produce unacceptable toxic effects in otherwise normal and disease free tissues in the body, especially in those tissues and cells most sensitive to radiation damage, e.g., the stem cells of the bone marrow and gastrointestinal tract;

4) slow clearance of radionuclide from the body;

5) decrease in the immunoreactivity of the binding proteins with an increase in the numbers of chelating agents conjugated; 6) 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;

7) the number of chelating agents that can be attached to an immunoreactive protein is limited by the potential immunogenicity of the thus modified protein which, being highly derivatized, could be recognized by the immune, system as haptenated; and

8) the number of ionic radionuclides that can be associated with an immunoreactive protein is restricted by the number of sites of chelation available . It is an object of the present invention to overcome some of the aforementioned disadvantages of the currently available radiolabeled immunoreactive proteins .

Summary of the Invention

The present invention is directed to systems which are useful in the therapeutic treatment and diagnostic imaging of tissue, particularly of cancerous tissue. For a disease such as cancer, such systems comprise a tumor targeted sequential delivery system comprised of a primary non-radioactive targeting immunoreagent and a secondary radioactive delivery agent.

In one embodiment, the present invention is directed to a non-radioactive targeting immunoreagent

(sometimes hereinafter referred to as NRTIR) comprised of the residue of a receptor moiety, a linking group, and the residue of an immunoreactive material, which NRTIR is administered to a tissue of interest and will bind to sites on the surfaces of cells thereof.

In this embodiment, 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. This RDA is administered to the environs of the tissue which contains said NRTIR bound thereto. In particular, 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. Thus, 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 .

In another embodiment, 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.

In particular, in one aspect (sometimes hereinafter referred to as System A) , 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. Preferably, in System A, 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. 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. This limit to the degree of modification of the immunoreactive protein also applies to the NRTIR of System A, and the value of (n) will be approximately the same as the value of (c) . Thus, in this aspect, 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 . In another embodiment, 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.

In this embodiment, 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. In particular, 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. Thus, 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.

In particular, 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. Preferably, in System B, 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. 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. In these conjugates the value of (c) is limited to the number of radioactive agents that can be linked or conjugated to the immunoreactive protein while retaining the immunoreactivity for said antigen. This limit to the degree of modification of the immunoreactive protein also applies to the NRTIR of System B, and the value of (n) will be approximately the same as the value of (c) . Thus, in this aspect, 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. During the time between administrations of NRTIR and RDA said NRTIR binds to sites on cells of the target tissue and unbound NRTIR is removed from the environs of 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. During said effective period of time, said NRTIR will bind to sites on cells of said tissue of interest and unbound NRTIR will be removed from the environs of the tissue.

Subsequently, after an effective time, an image of all or part of said tissue of interest is obtained.

The present invention provides advantages compared to currently available targeting immune reagents. For example: the total amount of a therapeutically effective amount and of a diagnostic imaging effective amount of radioactive agent delivered to a tissue site can be achieved with specificity and in amplification over that which can be otherwise achieved with currently available targeting immune reagents; sequential delivery to target tissue of the NRTIR and the RDA of this invention can reduce the exposure of non-targeted tissues to damage from radiation 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;

RDA that does not bind to cell associated NRTIR is rapidly cleared from the patient; with respect to the same degree of modification of a targeting immunoreagent directly conjugated by a radionuclide or by a chelate containing a radionuclide in currently available radioimmunoconjugates, an amplification of the number of radionuclides per site of modification per targeting immune reagent can be obtained using the materials and methods of this invention; 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; and a wide variety of compositions of matter with a wide variety of sizes and molecular weights and having a specificity for accumulation in tumors can be prepared in accordance with this invention.

Other advantageous features of this invention will become readily apparent upon reference to the following description of the preferred embodiments .

Description of Preferred Embodiments In preferred embodiments, the above-described non- radioactive targeting immunoreagent (NRTIR) and radioactive delivery agent (RDA) are comprised of moieties represented in System A (4 systems) and System B (4 systems) below:

SYSTEM A

SYSTEM B

wherein:

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.

Preferred embodiments of these materials are further described below.

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. Of preferred use are 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.

As non-limiting examples, the following materials are useful sources of heterodimers :

MRP14 and MRP8 (also known as pl4 and pδ, 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; delta and gamma chains of the T cell receptor; 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. Thus, for example, in one aspect in System A and in System B, 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.

In both System A and System B 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. In addition, an immunoreactive group can be any substance which when presented to an immunocompetent host will result in the production of a specific antibody capable of binding with that substance, or the antibody so produced, which participates in an antigen-antibody reaction.

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. In addition to antibodies produced by the techniques outlined above, other antibodies and proteins produced by the techniques of molecular biology are specifically included. Preferably, the immunoreactive group does not bind to

HI so as to inhibit the binding of HI to H2 in System A, and the immunoreactive group does not bind to H2 so as to inhibit the binding of H2 to HI in System B. As used herein, 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. The term "affinity for binding" to an antigen, as used herein, 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

.3 for the antibody-antigen interaction. The term "affinity", as used herein, 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:

(a) cleavage of one or more chemical bonds of which an antibody is comprised, said 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:

*H (i) a catalysed chemical reaction comprising the action of a biochemical catalyst such as an enzyme such as papain or pepsin which to those skilled in the art are known to produce antibody fragments commonly referred to as Fab and Fab'2, respectively;

(ii) 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; (iii) 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;

(iv) 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;

(v) a chemical reaction comprising a reduction reaction such as the reduction of a disulfide bond; and

(vi) 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

(b) formation of one or more chemical bonds between one or more reactants, such as formation of one or more covalent bonds selected from, for example, carbon-nitrogen bonds (such as, for example, amide bonds, amine bonds, hydrazone bonds, and thiourea bonds) , sulfur-sulfur bonds such as disulfide bonds, carbon-carbon bonds, carbon-sulfur bonds, and carbon- oxygen bonds, and employing as reactants in said chemical bond formation one or more reagents comprised of amino acids, peptides, carbohydrates, linking groups as defined herein, spacing groups as defined herein, protein reactive groups as defined herein, lb^" and antibody fragments such as are produced as described in (a) , above; or

(c) 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

(d) an antibody fragment can be produced as a result of the methods of molecular biology or by genetic engineering of antibody genes, for example, in the genetic engineering of a single chain immunoreactive group or a Fv fragment.

An antibody fragment can be produced as a result a combination of one or more of the above methods. In certain embodiments, 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 . If desired, 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 . An especially preferred antibody is ING- 1.

π SCHEME 1A

Ab-M-S- MRP14-protein

SCHEME 1 B

Ab-M-S- MRP8-protein

As used herein, 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. With respect to use with the term "receptor" or with the term "active site in a receptor", the term "ligand" as used herein 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. Examples of 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.

In system A, 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. In system B, 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. et al. , J. Biol. Chem. 266:7706-7713 (1991); Teigelkamp, S. et al . , J. Biol. Chem. 266:13462-13467 (1991) ; Dianoux, A-C. et al . ,

Biochemistry 31:5898-5905 (1992); Odink, K. et al. , Nature 330:80-82 (1987); Lagasse, F. et al., Mol. Cell. Biol. 8:2402-2410 (1988) . 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. 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.

In one aspect in System A and in System B, the MRP14 is comprised of a human protein.

Preferably, 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.

10 Yet more preferably, 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.

In another aspect, the Z-L-X of System A is comprised of a fusion protein. As used herein, the term "fusion protein" refers to a genetically engineered material comprised of a protein whose coding region is comprised of the coding region of a residue of a first protein fused, in frame, to the coding region of a residue of a second protein. Preferably, 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. Thus, preferably, said fusion protein is comprised of a residue of an immunoreactive reagent fused to one or more residues of MRP14. In a preferred embodiment, 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. In another preferred embodiment, 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 mςjre MRP14. In yet another preferred embodiment, 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. Preferably, said coding regions are independently human and bacterial or modified by genetic engineering techniques as above. More preferably, 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. Yet more preferably, 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.

An example of a ligand that has an affinity for binding to the active site in said MRP14 is 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, 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.

In one aspect in System A and in System B, the MRP8 subunit is comprised of a human protein. Preferably, said MRP8 subunit is comprised of a recombinant human protein. More preferably, 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

30- of specific amino acids in a peptide sequence of said protein. Yet more preferably, 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. In another aspect, the Z-(Lι-D)_n of System B is comprised of a fusion protein. Thus, preferably, said fusion protein is comprised of a residue of an immunoreactive reagent fused to one or more residues of a MRP8. In a preferred embodiment, 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. In another preferred embodiment, 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. In still another preferred embodiment, 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-(Lι~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. Preferably, said coding regions are independently human and bacterial or modified by genetic engineering techniques as above. More preferably, 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. Yet more preferably, 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 .

The association of a ligand with a receptor can comprise a non-covalent interaction, or it can comprise the formation of a covalent bond. Preferably the association is non-covalent.

In System A, 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-(Lι~Rec)_n] of the System.

Preferably n is i, 2, 3, 4, 5 or 6. Most preferably n is 1 or 2.

Again in System A, in one embodiment, an MRP8 subunit is a component of a radioactive delivery agent [i.e., an RDA, Rec- (L₂-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. Preferably the chelating group is TMT (described hereinbelow) , the linking group is as described below, the radionuclide is an isotope of yttrium, and m is 2 to about 10.

In another embodiment, 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. Preferably, said covalently attached radionuclide is a

^4 radioisotope of iodine attached to an aromatic ring- containing moiety.

In yet another embodiment, 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 N₃S and N₂S₂ 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. Preferably, said covalently attached radionuclide is selected from a radioisotope of technicium and rhenium attached to a group comprised of a sulfur atom.

In System B, 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-(Lχ-Rec)_n] of the System. Preferably, n is 1, 2, 3, 4, 5 or 6. More preferably, n is 1 or 2. Again in System B, in one embodiment, a MRP14 subunit is a component of a radioactive delivery agent [i.e., an RDA, Rec- (L₂-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. Preferably the chelating group is TMT

(described hereinbelow) , the linking group is as described below, the radionuclide is an isotope of yttrium, and m is 2 to about 10.

In another embodiment, 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. Preferably,

2ff said covalently attached radionuclide is a radioisotope of iodine attached to an aromatic ring- containing moiety. In another embodiment, 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. Preferably, said covalently attached radionuclide is selected from a radioisotope of technicium and rhenium attached to a group comprised of a sulfur atom.

In the NRTIR of System A and B, chemical conjugation is achieved,by use of a linking group (Li) which, for example, is introduced through modification of, a site on an immunoreactive group. The introduction of activated groups such as activated ethylene groups (e.g., maleimide groups) on to amine groups such as lysine epsilon-amines of a protein is represented in Scheme 1. Other techniques include the use of heterobifunctional linking moieties and chemical modifications such as the examples described in U. S. Patent No. 4,719,182. Additionally, those chemicals such as SMCC which are commonly commercially available, for example, from Pierce Chemical Company are included as non-limiting examples.

In 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. In System A, reaction of the thus reduced MRP14 protein moiety with the above described maleimide modified antibody (Ab-M) results in an antibody/receptor conjugate (Ab-M-S-MRP14 protein:

Scheme IA) linked together by one or more thioether bonds. Additionally, those chemicals which are commonly commercially available, for example, from

3. C Pierce Chemical Company and the like which are useful in the covalent attachment of two proteins are included as non-limiting examples in the coupling of MRP14 to antibody in System A. In System B, reaction of the above reduced MRP14 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 MRP14/chelating agent conjugate wherein the reduced MRP14 is covalently attached to one or more chelating agents each by a thioether bond. Similarly, 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 .

In System B in another aspect, chemical conjugation is otherwise achieved by using a linking group, L₂, which is introduced through mild reduction of the MRP8 (or of the MRP8 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 MRP8 protein moiety. In System B, reaction of the thus reduced MRP8 protein moiety with the above described maleimide modified antibody (Ab-M) results in an antibody/receptor conjugate (Ab-M-S-MRP8 protein: Scheme IB) linked together by one or more thioether bonds. Additionally, those chemicals which are commonly commercially available, for example, from Pierce Chemical Company and the like which are useful in the covalent attachment of two proteins are included as non-limiting examples in the coupling of MRP8 to antibody in System B.

In System A, 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. Similarly, 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 .

In System A and B, other groups are useful in the coupling of the immunoreactive material to either the receptor moiety, (HI) , or the ligand moiety, (H2) , particularly if the above reagents are utilized. 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.

In System A, the ratio of MRP14 to immunoreactive group such as an antibody can vary widely from about 0.5 to 10 or more. In bulk, 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. In System B, the ratio of MRP8 to immunoreactive group such as an antibody can vary widely from about 0.5 to 10 or more. In bulk, 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.

In System A, in preferred embodiments, 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. In bulk, 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.

In System A and B, following the linking of the immunoreactive group, preferably of an antibody or an antibody fragment, to MRP14 (system A) or MRP8 (system B) , the conjugate is purified by passage of the material through a gel permeation column such as Superose 6 using an appropriate elution buffer or by elution from a HPLC column such as a Shodex WS-803F size exclusion column. Both these methods separate the applied materials by molecular size resulting in the elution of the antibody/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.

In System A and 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 L₂ in System A and System B are each independently a chemical bond or the residue of a linking group. In one aspect, the phrase "residue of a linking group" as used herein refers to a moiety that remains, results, or is derived from the reaction of a protein reactive group with a reactive site on a protein. The phrase "protein reactive group" as used herein refers to any group which can react with functional groups typically found on proteins. However, it is specifically contemplated that such protein reactive groups can also react with functional groups typically found on relevant nonprotein molecules. Thus, in one aspect the linking groups Li and L₂ 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. In one aspect, preferred linking groups thus formed include the linking group, Li, between the immunoreactive group, "Z", and the HI receptor- containing species "Rec", (e.g. MRP14), in the NRTIR System A; the linking group, Li, between the immunoreactive group, "Z", and H2 ligand species (e.g., MRP8) in the NRTIR in System B; the linking group, L₂, between the HI receptor- containing species in the "Rec", (e.g. MRP14), and the residue of the chelating group, "Q", in the RDA in System B; and between the H2 ligand-containing species (e.g.,MRP8) and the residue of the chelating agent, "Q", in the RDA in System A.

Preferred linking groups are derived from protein reactive groups selected from but not limited to:

(1) a group that will react directly with amine, alcohol, or sulfhydryl groups on the immunoreactive protein or biological molecule containing the reactive group, for example, active halogen containing groups including, for example, chloromethylphenyl groups and chloroacetyl [C1CH₂C (=0) -] groups, activated 2- (leaving group substituted) -ethylsulfonyl and ethylcarbonyl groups such as 2-chloroethylsulfonyl and 2- chloroethylcarbonyl; vinylsulfonyl; vinylcarbonyl; epoxy; isocyanato; isothiocyanato; aldehyde; aziridine; succinimidoxycarbonyl; activated acyl groups such as carboxylic acid halides; mixed anhydrides and the like; and other groups known to be useful in conventional photographic gelatin hardening agents; (2) a group that can react readily with modified proteins or biological molecules containing the immunoreactive group, i.e., proteins or biological molecules containing the immunoreactive group modified to contain reactive groups such as those mentioned in (1) above, for example, by oxidation of the protein to an aldehyde or a carboxylic acid, in which case the "linking group" can be derived from protein reactive groups selected from amino, alkylamino, arylamino, hydrazino, alkylhydrazino, arylhydrazino, carbazido, semicarbazido, thiocarbazido, thiosemicarbazido, sulfhydryl, sulfhydrylalkyl, sulfhydrylaryl, hydroxy, carboxy, carboxyalkyl and carboxyaryl. The alkyl portions of said linking groups can contain from 1 to about 20 carbon atoms. The aryl portions of said linking groups can contain from about 6 to about 24 carbon atoms; and

(3) a group that can be linked to the protein or biological molecule containing the immunoreactive group, or to the modified protein as noted in (1) and (2) above by use of a crosslinking agent. The residues of certain useful crosslinking agents, such as, for example, homobifunctional and heterobifunctional gelatin hardeners, bisepoxides, and bisisocyanates can become a part of, i.e., a linking group in, for example, the protein- (MRP14-containing species) conjugate in System A during the crosslinking reaction. Other useful crosslinking agents, however, can facilitate the crosslinking, for example, as consumable catalysts, and are not present in the final conjugate. Examples of such crosslinking agents are 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. With these crosslinking agents, 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. In amide bond formation, 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

3λ Immunotechnology Catalog - Protein Modification

Section,

(1991 and 1992) . Useful non-limiting examples of such reagents include:

Sulfo-SMCC

Sulfosuccinimidyl 4-(N- maleimidomethyl) cyclohexane- 1-carboxylate. Sulfo-SIAB

Sulfosuccinimidyl (4-iodoacetyl) aminobenzoate.

Sulfo-SMPB

Sulfosuccinimidyl 4- (p-maleimidophenyl)butyrate . 2-IT

2-Iminothiolane . SATA

N-Succinimidyl S-acetylthioacetate .

In addition to the foregoing description, the 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. (Palo Alto California) and include Uni-Link AminoModifier (Catalog # 5190), Biotin-ON phosphoramidite (Catalog # 5191), N-MNT-C6- AminoModifier (Catalog # 5202), AminoModifier II (Catalog # 5203), DMT-C6-3 'Amine-ON (Catalog # 5222), C6-ThiolModifier (Catalog # 5211), and the like. In one aspect, 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.- In the NRTIR of System A, one sequence of a pair of two complementary oligonucleotide sequences is attached to one component and the complementary sequence is attached to the other components of the conjugate. For example, one sequence is attached to the immune reactive agent and the complementary oligonucleotide sequence is attached to the MRP14-containing moiety. On mixing the two components, the hybrid formed between the two complementary oligonucleotide sequences then comprises the linking group between the immune reactive agent and the MRP14-containing moiety. In the RDA of System B, 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. On mixing the two compounds, 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. In System B, 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. The

3* 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. Likewise, in the NRTIR of System B, the residue of one or more MRP8-containing moieties which associate with MRP14 can be attached to the immunoreactive group using complementary olάgonucleotide hybrids as described above. In the NRTIR of System A, multiple MRP14-containing moieties can be attached to the immunoreactive protein analogously.

In the RDA of System A, 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. As is well known, 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 as triethanolamine and N-(2- hydroxyethyl)ethylenediamine; aromatic heterocyclic bases, such as 2,2 '-dipyridyl, 2,2 '-diimidazole, dipicoline amine and 1, 10-phenanthroline; phenols, such as salicylaldehyde, disulfopyrocatechol, and chromotropic acid; aminophenols, such as 8- hydroxyquinoline and oximesulfonic acid; oximes, such as dimethylglyoxime and salicylaldoxime; peptides containing proximal chelating functionality such as polycysteine, polyhistidine, polyaspartic acid, polyglutamic acid, or combinations of such amino acids;

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, (CH₃) 6-[14]-4,ll-diene-N₄, and (2.2.2-cryptate) ; and phosphonic acids, such as nitrilotrimethylene- phosphonic acid, ethylenediaminetetra (methylenephosphonic acid) , and hydroxyethylidenediphosphonic acid, or combinations of two or more of the above agents.

Preferred residues of chelating agents contain polycarboxylic acid groups and include: ethylenediamine-N, N, N¹ ,N'-tetraacetic acid (EDTA) ;

N,N,N' ,N",N"-diethylene-triaminepentaacetic acid (DTPA) ; 1, 4,7, 10-tetraazacyclododecane-N,N' ,N",N" '- tetraacetic acid (DOTA) ; 1,4,7,10- tetraazacyclododecane-N,N' ,N"-triacetic acid (D03A) ; l-oxa-4, 7, 10-triazacyclododecane-N,N' ,N"-triacetic acid (OTTA) ; and trans (1,2)-

?> C cyclohexanodiethylenetriamine pentaacetic acid (CDTPA) .

Preferred residues of chelating agents contain polycarboxylic acid groups and include: B4A, P4A, TMT, DCDTPA, PheMT, macroPheMT, and macroTMT;

TMT

DCDTPA

MacroPheMT MacroTMT

In one aspect, other 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 .

In another aspect, suitable residues of chelating agents are derived from N3S and N₂S₂ containing compounds, as for example, those disclosed

2>7 in U.S. Patent Nos. 4,444,690; 4,670,545; 4,673,562; 4,897,255; 4,965,392; 4,980,147; 4,988,496; 5,021,556 and 5,075,099.

Other suitable residues of chelating agents are described in PCT/US91/08253, the disclosure of which is hereby incorporated by reference. If Q is comprised of the residue of multiple chelating agents, such agents can be linked together by one or more linking groups such as described above. 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; phosphonate; urelene; thiourelene; urethane, i.e., iminocarbonyloxy,and oxycarbonylimino; an amino acid linkage, i.e., a

group wherein k=l and Xi, X , X₃ 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

2>S to 18, preferably 6 to 10 carbon atoms such as phenyl, hydroxyiodophenyl, hydroxyphenyl, fluorophenyl and naphthyl, aralkyl, preferably containing from 7 to 12 carbon atoms, such as benzyl, heterocyclyl, preferably containing from 5 to 7 nuclear carbon and one or more heteroatoms such as S, N, P or 0, examples of preferred heterocyclyl groups being pyridyl, quinolyl, imidazolyl and thienyl; heterocyclylalkyl, the heterocyclyl and alkyl portions of which preferably are described above; or a peptide linkage, i.e., a

group wherein k>l and each X independently is represented by a group as described for X]_, X₂, X₃ above . Two or more 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

3°ι introduced after the coupling reaction. For example, the linking group can be substituted with substituents such as halogen, such as F, Cl, Br or I; an ester group; an amide group; alkyl, preferably containing from 1 to about 18, more preferably, 1 to 4 carbon atoms such as methyl, ethyl, propyl, i-propyl, butyl, and the like; substituted or unsubstituted aryl, preferably containing from 6 to about 20, more preferably 6 to 10 carbon atoms such as phenyl, naphthyl, hydroxyphenyl, iodophenyl, hydroxyiodophenyl, fluorophenyl and methoxyphenyl; substituted or unsubstituted aralkyl, preferably containing from 7 to about 12 carbon atoms, such as benzyl and phenylethyl; alkoxy, the alkyl portion of which preferably contains from 1 to 18 carbon atoms as described for alkyl above; alkoxyaralkyl, such as ethoxybenzyl; substituted or unsubstituted heterocyclyl, preferably containing from 5 to 7 nuclear carbon and heteroatoms such as S, N, P or 0, examples of preferred heterocyclyl groups being pyridyl, quinolyl, imidazolyl and thienyl; a carboxyl group; a carboxyalkyl group, the alkyl portion of which preferably contains from 1 to 8 carbon atoms; or the residue of a chelating group. In one embodiment, multiple chelating groups, Q, can be attached to the MRP8 in system A (and to MRP14 in system B) using reagents of the form represented in structure 1.

M

Structure 1

wherein:

-t o 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, but not limited to, TMT groups or DTPA groups, macrocyclic chelating groups, chelate groups that contain sulfur atoms, chelate groups that contain nitrogen atoms, chelate groups that contain pyridine rings, and chelate groups that contain carboxylate or phosphate groups; W is selected from H, alkyl, aralkyl, alkylene, carboxylic acid groups, amino groups, amido groups, aryl groups, hydroxyaryl groups, a therapeutically effective and diagnostically effective radioisotope of an atom (such as iodine in the form of an iodo group, and the like) that can be covalently attached to a component of structure (1) [as distinct from a therapeutically effective or diagnostically effective radioisotope of an ion that can be bound to a component of structure (1) via a chelate group] , a chelate group that may contain a therapeutically effective or diagnostically effective radioisotope; M, is a metal ion such as yttrium, indium, rhenium, copper, scandium, bismuth, lead, leuticum and the like; a is zero or an integer from one to about 100; b, q, and f are independently integers from one to about 100. In the RDA of System A, preferred non-limiting examples of linking groups include the residue of a sulfur protected linear peptide H₂N- [D- (S-t-Butyl) - Cys]=Ala-Ala-Ala-Ala-Lys-Lys-OH as well as the residue of a sulfur protected linear-peptide H₂N- [L- (S-t-Butyl)-Cys]=Ala-Ala-Ala-Ala-Lys-Lys-OH, both of which may be independently synthesized via solid-phase methodology on an ABI 430A automated peptide synthesizer using the instructions provided by the manufacturer.

The linear peptide H₂N- [D- (S-t-Butyl) -Cys]=Ala-

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 FastMoc™ 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. 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_Cι₃ 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.

Additional chelating agents and radionuclides bound to chelating agents are incorporated by preparing, for example, analogous peptides comprised of additional Lys-TMT and Lys-TMT-radionuclide groups. Preferably, the number of such Lys-TMT and Lys-TMT- radionuclide residues is from 1 to about 6, and more preferably from 2 to about 6. In the System B, the NRTIR is comprised of one or more ligands such as, for example, MRP8, that each have an affinity for binding to a MRP14. Each such 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.

In one embodiment, both in System A and System B, it is desirable that 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. In the process of formation of the chelate with the metal ion, 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. Preferably, said buffer salts are selected so as not to interfere with the subsequent binding of the metal ion to the chelating agent . In therapeutic applications, the RDA of this invention preferably contains a ratio of metal radionuclide ion to chelating agent that is effective in such therapeutic applications. In preferred 3 embodiments, the mole ratio of metal ion per chelating agent is from about 1:100 to about 1:1.

In diagnostic imaging, applications, the RDA of this invention preferably contains a ratio of metal radionuclide ion to chelating agent that is effective in such diagnostic imaging applications. In preferred embodiments, the mole ratio of metal ion per chelating agent is from about -1 : 1, 000 to about 1:1.

In another embodiment, 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) .

In another embodiment, 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 ⁴⁴Sc, ⁶⁴Cu, ⁶ Cu, ^11:ι-In, ²¹²Pb, 68Ga, 8⁷Y, 9°Y, 153_Sm, 212_Bi, 99m_Tc, 177_Lu 186_{Re an}d ⁱSSRe. Of these, especially preferred is ⁹⁰Y. These radioisotopes can be atomic or preferably ionic. In yet another embodiment, the RDA of this invention comprises a diagnostically effective amount of a radionuclide and a therapeutically effective amount of a second radionuclide. Preferably, the diagnostically effective radionuclide is an radioisotope of iodine such as ¹³¹I, and the therapeutically effective isotope is a radioisotope of

Y, Sc, Cu, Pb, Ga, Bi and Re. In a further embodiment, 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.

In a still further embodiment, 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.

In Systems A and B, which describe subunit portions of heterodimeric proteins (HI and H2) , the identity of the receptor subunit and the ligand subunit are interchangeable. The preferred ligand subunit is MRP8, and the preferred receptor subunit is MRP14, but other receptor/ligand pairs are contemplated herein and, as non-limiting examples, they are described below:

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; delta and gamma chains of the T cell receptor; 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.

An example of a structure of an RDA that has utility in this invention is represented by structure 2.

Structure 2

Wherein:

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 R¹ 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 π.3 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, NH₂, a residue of a group, preferably chelating agent such as TMT chelated to a radioactive metal ion such as ⁹⁰γ⁺³, an 0-alkyl group wherein the alkyl group contains 1 to 10 carbon atoms, such as an O-methyl group, and NR_aR_b wherein each of R_a and Rj--, is independently selected from an alkyl group of 1 to 10 carbon atoms such as a methyl group, H, a hydroxyl ethyl group and a poly(alkylene oxidyl) group such as -PEG-OH and -PEG-O-alkyl (e.g., -PEG-O-CH₃) wherein the alkyl is as described above and the PEG is a poly (ethylene oxidyl) with an average molecular weight in the range between 72 and 5,000 daltons.

Another example of a structure of an RDA that has utility in this invention is structure 3 below:

Structure 3 Wherein:

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 R¹ 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 m₄, π.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_aR_b wherein each of R_a and R_b is independently selected from a alkyl groups as

4£ described above, H, a polyalkylene oxide moiety such as PEG-OH and PEG-O-alkyl (e.g., PEG-O-CH₃) wherein the PEG is described above, and a m7 an integer from 1 to about 12; In a preferred embodiment, 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 RDA is achieved.

In a preferred embodiment, 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. Subsequently, at an effective time, 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. In a preferred embodiment, 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 . Subsequently, at a therapeutically effective time of from one to about fourteen days, 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. In another embodiment of this invention, 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. For example, the use of a therapeutically effective dose of an RDA comprising a radionuclide such as ⁹⁰γ+3 together with a diagnostic imaging effective dose of an RDA comprising radionuclide such as ⁸⁷γ⁺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.

In another embodiment of this invention, the use of radioisotopes of iodine is specifically contemplated. For example, if 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. The 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. It is specifically contemplated that 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. It is also contemplated that the NRTIR can be administered by a route different from that of the RDA. 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. Examples of 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 .

S I These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose and acacia, (c) humectants, as for example, glylcerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like. Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and others well known in the art .

They may contain opacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract In a delayed manner. Examples of embedding compositions which can be used are polymeric substances and waxes.

The active compounds can also be in micro- encapsulated form, if appropriate, with one or more of the above-mentioned excipients. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, 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. Besides such inert diluents, 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. 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.

In another embodiment, 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. In this method, 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 . Subsequently, 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. The image pattern can then be visualized. Alternatively, 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

5S interest and during which time unbound NRTIR and unbound portion of reacted NRTIR will be removed from the environs of said tissue, and then obtaining an image as a function of time of all or part of said tissue of interest. When the image of all or part of said tissue of interest is optimal, a diagnostic imaging or a therapeutically effective amount of RDA containing the same or a different radionuclide as that employed on the above reacted NRTIR is administered to said tissue of interest of said patient .

In addition to human patients, the test subjects can include mammalian species such as rabbits, dogs, cats, monkeys, sheep, pigs, horses, bovine animals and the like.

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.

The following examples further illustrate the invention and are not to be construed as limiting of the specification and claims in any way. Specific embodiments of the invention are illustrated in the following examples .

EXAMPLES

The following examples for the construction of conjugates between an antibody and MRP14. In these examples ING-1 (a chimeric IgGi antibody) is chosen for the methodologies; other antibodies such as those described herein are useful. The MRP14 referred to below is of human neutrophil origin, purified as outlined below. MRP8 can be conjugated to antibodies by similar procedures .

5 Q Example 1

Purification of MRP14 and MRP8 from Human Neutrophils

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.

Rapid purification of milligram quantities of MRP14 and MRP8 from neutrophil cytoplasm is achieved by following published methods, Edgeworth, J. et al . J. Biol. Chem. 266: 7706-7713 (1991) . In brief, 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) . After centrifugation at 17,000 g for 20 minutes, the uppermost layer of cytoplasmic proteins is subjected to anion exchange protein chromatography on a MonoQ column and cation exchange chromatography on a MonoS column. The final 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. Purity of the subunits is checked by passage through one of two CNBr-activated Sepharose immunoaffinity columns containing immobilized monoclonal antibodies against either MRP14 or MRP8 (Teigelkamp, S., et al. J. Biol. Chem. 266:

S7 13462-13467 (1991)) . The purified subunits are eluted with 0.1 M glycine and immediately desalted on PD-10 columns before storage at -80°C.

ST Example 2

(2a) Preparation of Antibody-Malemide with Sulfo-SMCC (ING-1-Maleimide)

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.

(2b) Preparation of mercaptoalkyl-Antibody

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.

(2c) Preparation of mercapto-antibody using SATA

A solution containing 6 nmoles of ING-1 in PBS is vortexed while 60 nmoles of SATA (in DMSO) are added. After mixing and standing at room temperature for 60 minutes, the reaction mixture is diluted with

PBS, and eluted from a PD-10 column with PBS to afford ING-l-NH-CO-CH₂-S-COCH3. The acetylthioacetylated antibody is deprotected by the addition of 30 μL of a pH 7.5 solution containing 100 mM sodium phosphate, 25

5 mM EDTA, 50 mM NH₂OH. The reaction proceeds for two hours at room temperature after which the material is again passed down a PD-10 column by elution with PBS. The final product, ING-1 (N) -CO-CH₂-SH, is used immediately.

(2d) Radiolabeling of ING-1 with ¹²⁵I or ¹³¹I

An aliquot of ING-1 (500 μg) is labeled with either ¹²⁵j onochloride or ¹³¹I monochloride (at about 5 mCi/mg) in the presence of Iodogen (Sodium N- chlorobenzenesulfonamide: Pierce Chemical Co) beads in a volume of 500 μL lOOmM phosphate buffer (pH 7.2) at room temperature . After 15 minutes 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.

Example 3

(3a) Preparation of mercapto-MRP14 using SATA

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-CH₂-S-CO-CH3. The acetylthioacetylated MRP14 is deprotected by the addition of 25 μL of a pH 7.5 solution containing 100 mM sodium phosphate, 25 mM

EDTA, 100 mM NH OH. The reaction proceeds for two hours at room temperature after which the material is again passed down a PD-10 column by elution with PBS. The final product, MRP14 (N) -CO-CH₂-SH, is used immediately.

(3b) Preparation of mercaptoalkyl-MRP14 >0 A sample of MRP14 (50 nmoles) is dissolved in 0.1 M carbonate buffer (pH 9) and 4 μmoles of an aqueous solution of 2-iminothiolane are added. The reactants are vortex mixed and kept at room temperature for 120 minutes. The reaction mixture is quenched by the addition of 4 μmoles of ethanolamine, diluted with phosphate buffed saline. The reaction mixture is added to a prewashed PD-10 column, and eluted with PBS to afford MRP14(NH)- C (=NH₂ ⁺)CH₂CH₂CH₂SH. For use in conjugation to maleimide-derivatized ING-1, the product is eluted off the column directly into the maleimide-derivatized ING-1 solution.

(3c) Preparation of reduced MRP14 using dithiothreitol

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. For use in conjugation to maleimide- derivatized antibody, the product is eluted off the column directly into the maleimide-derivatized antibody solution. Otherwise the final product is used immediately after preparation.

(3d) Preparation of MRP14-Maleimide with Sulfo-SMCC

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 .

(3e) Radiolabeling of proteins and protein subunits with

125 _or 131

An aliquot of MRP14 (500 μg) is labeled with 125j monochloride or ^I3lι monochloride (at about 5 mCi/mg) in the presence of lodogen (Sodium N- chlorobenzenesulfonamide) beads in a volume of 500 μl 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 MRP14 is eluted with PBS and stored at 4°C until use.

An aliquot of MRP8 (500 μg) is labeled with ¹²-3ι monochloride or ^I3lι monochloride (at about 5 mCi/mg) in the presence of lodogen (Sodium N- chlorobenzenesulfonamide) beads in a volume of 500 μl 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 MRP8 is eluted with PBS and stored at 4°C until use.

An aliquot of the antibody, ING-1, (500 μg) is labeled with 25j monochloride or ^I3^lj monochloride (at about 5 mCi/mg) in the presence of lodogen (Sodium

N-chlorobenzenesulfonamide) beads in a volume of 500 μl 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.

Example 4 Conjugation of MRPs to TMT-NCS

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 sample of that MRP8 solution, containing approximately 250 μg of protein, is then pipetted into an acid washed, conical, glass reaction vial . 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 μL of the TMT-NCS solution to the MRP8 to give a 4-fold

(mole:mole) molar excess of TMT-NCS over MRP8. The solution is stirred briefly at room temperature to mix the reactants and then left in the dark at room temperature for 4 hours and then at 4°C overnight . After 16 hours, the MRP8/TMT conjugate is separated from unconjugated TMT by applying the reaction mixture to a PD-10 chromatography column which had been pre¬ washed and equilibrated with 50 mM sodium acetate in 150 mM sodium chloride buffer, pH 5.6. The pure conjugate is eluted off the column with 2.5 mL of that same buffer.

The protein concentration of MRP8 in the conjugate solutions is determined by the BCA protein assay (BioRad) using bovine immunoglobulin as the protein standard.

In order to calculate the number of TMT molecules per MRP8 molecule, 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. Thus, an aliquot of the MRP8/TMT in 2.5 ml of 0.05 M Tris HCl buffer at pH 7.5 is pipetted into a 2 ml quartz cuvette. A 20 μM

Europium chloride (Europium chloride hexahydrate: Aldrich) solution in 0.05 M Tris HCl buffer at pH 7.5 is prepared. An aliquot (50 μL) 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. As each chelating site on the MRP8/TMT conjugate binds one Europium ion, and as an Europium ion has to be in a chelate site for phosphorescence to occur, this method allows the number of functional chelation sites to be quantitated. Using this method, the ratio of TMT molecules per molecule of MRP8 in bulk solution is in the range of 1:1 to 2:1.

Example 5

Conjugation of MRPs to Antibody The following procedure is applicable to the conjugation of materials from Example 2a to the materials from Example 3a.

Similar methodologies for conjugation can be applied to the protein components of this invention irrespective of whether the maleimide group is on the antibody, on the MRP14 or on the MRP8 and irrespective of the method chosen to introduce the sulfhydryl group into said proteins. The molar ratio of MRP to antibody during the conjugation is maintained at close to unity in order to avoid over-reaction of either protein with the other.

A sample (50 nmoles) of MRP14 (N) -CO-CH₂-SH of

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.

After concentrating the protein down to a volume of approximately 500 μL by centrifugation, 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 μ filter and stored at 4°C until use.

Addition of trace amounts of either ^l25I-labeled MRP14 (Example 3e) or ^l25I-labeled ING-1 (example 3e) or both ¹²⁵I-labeled MRP14 and ^13lι-labeled ING-1 (Example 3e) to the reaction mixtures, allows the ratio of one protein to the other after conjugation to be calculated.

Example 6

Preparation of Radionuclide labeled (⁹⁰Y)MRP8/TMT

A volume of radioactive Yttrium chloride (⁹⁰Y 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 μL 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 ⁹⁰Y and controlled by a Compaq 386/20e computer. In this system, free (unchelated) ⁹⁰Y migrates at the solvent front while ⁹⁰Y-labeled MRP8/TMT remains at the origin. In excess of 97% of the added ⁹⁰Y is taken up by the MRP8/TMT to form the desired ⁹⁰Y-labeled product .

Example 7

Assays on the ING-1-Maleimide-S-MRP conjugates

(7a) Protein Concentration

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. By inclusion of trace amounts of ^l25I-labeled or ^I3lι-labeled MRP14, MRP8, or ING-1 (all prepared according to example 3e) in the reaction mixtures, and by knowing the specific activity of the preparations, the ratio of one protein to the other after conjugation is calculated.

As an alternative to radiolabeling, the MRP14 or MRP8 can be conjugated to other materials (e.g., TMT

(Example 4 : for use with ⁹⁰Y 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.

(7b) Immunoreactivity assay by Flow Cytometry

Antibody/MRP14 conjugates (e.g.ING-1/MRP14 prepared according to Example 5) 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) ) are grown to confluence in tissue culture flasks using McCoy's media supplemented with 10% fetal calf serum. The cells are harvested by scraping the flask walls with a cell scraper. Cells from many separate flasks are pooled, centrifuged to a pellet, resuspended at 5xl0⁵/mL in a solution of ice-cold 50mM sodium phosphate with 150 mM sodium chloride buffer pH 7.4 (PBS) supplemented with 0.1% bovine serum albumin (Sigma) and 0.02% sodium azide (Flow buffer) . The cells are washed in this same buffer and then counted. An antibody standard curve is constructed by diluting a stock solution of ING-1 with an irrelevant (non-binding) , isotype- matched control antibody (human IgGl) to give a number of samples ranging in ING-1 content from 10% to 100%. The standard curve is made in flow buffer so that each sample contains 1.0 μg protein per mL. Samples from the standard curve and ING-1/MRP14 unknowns are then incubated with 5xl0⁵ 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.

This procedure is repeated a further 3 times after which the cell pellet is resupended in 100 μL of flow buffer and incubated at 4°C for 1 hour with goat-anti- human antibody labelled with fluorescein isothiocyanate (FITC) . After further washing in flow buffer the cell pellet is resupended in 100 μL of flow buffer to which a drop of propidium iodide (Coulter) has been added. The samples are then analyzed by flow cytometry on a Coulter EPICS 753 flow cytometer. Fluorescein isothiocyanate and propidium iodide (PI) are excited using the 488 nm emission line of an argon laser. The output is set at 500 mw in light regulation mode. Single cells are identified by 90 degree and forward angle light scatter. Analysis

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.

(7c) Immunoreactivity assay by ELISA

The antigen to which the antibody, ING-1, binds is prepared from LS174T or HT29 cells (available from

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

25 mL ice-cold 50 mM sodium phosphate buffer, pH 7.4 supplemented with 150 mM sodium chloride (PBS) , pelleted under the same conditions and transferred in

10 mL PBS to an ice-cold glass mortar. The cells are homogenized at 4°C using a motor-driven pestle and then centrifuged at 3000 x g for 5 minutes. The antigen-rich supernatant is removed from the other

1 <Λ cell debris and subjected to further centrifugation at 100,000 x g for one hour at 4°C. The pellet (antigen fraction) from this final step is suspended in 100 μL of PBS for every million cells harvested. Following an estimate of the protein concentration (BCA protein assay (BioRad) using bovine immunoglobulin as the protein standard) the antigen is stored at -20°C until use .

Each well of a 96-well Costar microtiter plates is coated with antigen by adding 100 μL/well of cell lysate (10 μg/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 μL/well of a

1% BSA (bovine serum albumin, Sigma A-7906) solution in PBS and incubated for 1 hour at room temperature. The plates are washed five times with 0.05% Tween-20. Samples (50 μL/well in duplicate) of ING/MRP14 conjugates and standard ING-1 antibody solutions are prepared at a range of concentrations in 1% BSA in PBS and added to the wells of the plate. Biotinylated ING-1 (1.0 μg/mL in 0.1% BSA) is added to each well (50μL/well) and the plates are then incubated for 2 hours at room temperature. Following five washes with 0.05% Tween-20, the plates are blotted dry and incubated at room temperature for one hour with dilute (1:2000 in 0.1% BSA) streptavidin-alkaline phosphatase (Tago; #6567) . After a further five washes, color is developed in each well upon the addition of 100 μL per well of phosphatase substrate reagent (two Sigma 104 phosphatase tablets dissolved in 10 mL distilled water and 20 μL Sigma 221 alkaline buffer) . After one hour at room temperature, the color is read using a 405 nm filter in a Titertek Multiscan microplate reader.

(7d) SDS PAGE gel electrophoresis n o Samples of these conjugates are subjected to electrophoresis on Novex 4%-20% reduced and native polyacrylamide gels using SDS buffers to estimate their apparent molecular weight and the degree of heterogeneity of the preparation. Using standards of known molecular weight run on the same gel, a standard curve is constructed of the distance travelled (Rf) versus the log of the molecular weight . From this standard curve the relative molecular weights of the bands associated with each conjugate preparation are determined.

(7e) Determination of aggregate formation by size- exclusion HPLC.

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 (25μL) 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. Following the recovery of the final standard from the column, it is washed with a further 10 volumes of 10 mM sodium phosphate buffer pH 6.0 supplemented with 150 mM sodium chloride. Samples (50μL) of either native ING-1 antibody, ING-1/TMT, ING-1/MRP14 at 200 μg/mL are individually injected on to the column and their respective retention times are recorded. From the areas of the retained peaks and the retention time, the amounts of aggregated material in the ING-1/TMT and ING-1/MRP14 sampled are calculated.

(7f) Determination of binding of MRP8/TMT to ING- 1/MRP14 Four of the above methodologies (7b, 7c, 7d, 7e) are used, with slight modifications to demonstrate that the two stages of the delivery system recognize and stably bind each other.

In 7b, 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.

In 7c, samples (50 μL/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)) .

Following further washing with in 0.1% BSA a further one hour incubation is carried out with dilute (1:2000 in 0.1% BSA) streptavidin-alkaline phosphatase (Tago; #6567) . After five washes, color is developed in each well upon the addition of 100 μL per well of phosphatase substrate reagent (two Sigma 104 phosphatase tablets dissolved in 10 mL distilled water and 20 @L Sigma 221 alkaline buffer) . After one hour at room temperature, the color is read using a 405 nm

7d filter in a Titertek Multiscan microplate reader. The results demonstrate that MRP8/TMT is associated with the antigen in the wells of the plate. Control samples demonstrate that the association is dependent on the presence of ING-1/MRP14.

Sodium dodecylsulfate polyacrylamide gels, such as those from Example 7d, 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. At time intervals after the start of incubation, 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. Alternatively, MRP8/TMT is labeled with ⁹⁰Y (Example 6) and incubated with antibody/MRP14 in PBS or in human serum at different temperatures as above. Again, after autoradiography, binding of ⁹⁰Y-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-

1/MRP14. Samples (50μL at 200 μg/mL) of either ING-1 antibody alone, ING-1/MRP14, MRP8 or mixtures of ING- 1/MRP14 with MRP8 are injected individually on to a 30

13 cm x 7.5 mm TSK-G3000SW size-exclusion HPLC column (Supelco) as described in Example 7e. The retention times of each sample is recorded. From the areas of the retained peaks and the retention time, the amount of association between MRP8 and ING-1/MRP14 is calculated.

1

Claims

We claim :

1. A targeting immune reagent that comprises moieties represented by the structure:

Z-(Lι-X)_n wherein:

Z comprises the residue of an immunoreactive protein; Li is a chemical bond or a linking group that may contain a spacing group;

X is the residue of a proteinaceous subunit of a heterodimeric molecule; and n is an integer greater than zero.

2. A radioactive targeting reagent comprised of moieties represented by the structure

D-(L₂-Q-M)_m wherein: D is the residue of a proteinaceous subunit of a heterodimeric molecule that associates with X of claim l;

L₂ is a chemical bond or a linking group that may contain a spacing group; Q is the residue of a chelating group;

M is a radionuclide; and m is an integer greater than zero.

3. The reagent of claim 1 wherein Z is an antibody or antibody fragment.

4. The reagent of claim 3 wherein the antibody is selected from ING-1; B72.3; 9.2.27; D612; UJ13A; NRLU-10; NRCO-02; 7E11C5; CC49; TNT; PR1A3; B174; C174; B43; and anti-HLB antibodies.

5. The reagent of claim 3 wherein the antibody is ING-1.

6. The reagent of claim 1 wherein X is selected from the group consisting of the heterodimeric pairs MRP14 and MRP8; alpha and beta chains of the T cell receptor; delta and gamma chains of the T cell receptor; Subunit proteins of the cytokine, IL-2; Subunit proteins of signal recognition particle; Subunit proteins of ligandin; Subunit proteins of hepatopoietins A and B;Subunit proteins of human platelet-derived growth factor; Subunit proteins of glutathione S-transferases;

Subunit proteins of luciferase; and Subunit proteins of gamma-glutamyl transpeptidase.

7. The reagent of claim 1 wherein X is selected from the group consisting of MRP14 and MRP8.

8. The reagent of claim 2 wherein D is selected from the group consisting of the heterodimeric pairs MRP14 and MRP8; alpha and beta chains of the T cell receptor; delta and gamma chains of the T cell receptor; Subunit proteins of the cytokine, IL-2; Subunit proteins of signal recognition particle; Subunit proteins of ligandin; Subunit proteins of hepatopoietins A and B;Subunit proteins of human platelet-derived growth factor; Subunit proteins of glutathione S-transferases;

9. The reagent of claim 2 wherein D is selected from the group consisting of MRP14 and MRP8.

10. The reagent of claim 7 wherein the residue of X is derived from human neutrophils .

11. The reagent of claim 9 wherein the residue of D is derived from human neutrophils .

12. The reagent of claim 1 wherein Li is the residue of a heterobifunctional cross-linking reagent .

13. The reagent of claim 12 wherein the heterobifunctional cross-linking reagent is selected from the group consisting of sulfosuccinimidyl 4- (N- maleimidomethyl) cyclohexane-1-carboxylate, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, sulfosuccinimidyl 4- (p-maleimidophenyl)butyrate, 2- Immothiolane, and N-succinimidyl S-acetylthioacetate.

14. The reagent of claim 1 wherein Li is the residue of a modified receptor moiety containing a reactive functional group.

15. The reagent of claim 14 wherein the reactive functional group is selected from the group consisting of amino groups and sulfhydryl groups.

16. The reagent of claim 2 wherein L is the residue of a heterobifunctional cross-linking reagent .

17. The reagent of claim 16 wherein the heterobifunctional cross-linking reagent is selected from the group consisting of sulfosuccinimidyl 4- (N- maleimidomethyl) cyclohexane-1-carboxylate, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, sulfosuccinimidyl_ 4- (p-maleimidophenyl)butyrate, 2- Iminothiolane, and N-succinimidyl S-acetylthioacetate.

18. The reagent of claim 2 wherein L₂ is the residue of a modified ligand moiety containing a reactive functional group.

19. The reagent of claim 18 wherein the reactive functional group is selected from the group consisting of amino groups and sulfhydryl groups.

20. The reagent of claim 2 wherein Q contains a polycarboxylic acid group.

21. The reagent of claim 2 wherein Q is i selected from the group consisting of B4A, P4A, TMT,

DCDTPA, PheMT, macroPheMT, and macroTMT.

22. The reagent of claim 2 wherein M is a radioactive isotope of a metal.

23. The reagent of claim 22 wherein the radioactive isotope is selected from ⁴Sc, ⁶⁴Cu, ⁶ Cu, min, ^2l2Pb, ⁶⁸Ga, ⁸⁷Y, ⁰Y, ¹⁵ Sm, ^{2 2}Bi, ""-Tc, ⁷⁷LU ¹⁸⁶Re and ⁱ⁸⁸Re.

24. A method of making a compound of the structure:

Z-(Lι-X)_n wherein: Z comprises the residue of an immunoreactive protein;

Li is a chemical bond or a linking group that may contain a spacing group;

X is the residue of a proteinaceous subunit of a heterodimeric molecule; and n is an integer greater than zero; comprising: (i) derivatizing a precursor of Li with X under conditions and for a time period sufficient to form a covalent complex Lι~X; and

(ii) derivatizing Z with Lχ-X under conditions and for a time period sufficient to form a covalent complex Z- (Lι-X)„.

25. A method of making a compound of the structure : D-(L₂-Q-M)m wherein :

-l D is the residue of a proteinaceous subunit of a heterodimeric molecule that associates with X of claim

1;

L₂ is a chemical bond or a linking group that may contain a spacing group;

Q is the residue of a chelating group; M is a radionuclide; and m is an integer greater than zero, comprising: (i) derivatizing D with L₂ under conditions and for a time period sufficient to form a covalent complex D- L₂; (ii) derivatizing D-L₂ with Q under conditions and for a time period sufficient to form a covalent complex D- L -Q; (iii) derivatizing D-L₂-Q with M under conditions and for a time period sufficient to form a covalent complex D-(L₂-Q-M)m.

26. The method of claim 24 wherein Z is an antibody or antibody fragment.

27. The antibody of claim 26 wherein the antibody is selected from ING-1; B72.3; 9.2.27; D612; UJ13A; NRLU-10; NRCO-02; 7E11C5; CC49; TNT; PR1A3; B174; C174; B43; and anti-HLB antibodies.

28. The method of claim 24 wherein X is selected from the group consisting of the heterodimeric pairs MRP14 and MRP8; alpha and beta chains of the T cell receptor; delta and gamma chains of the T cell receptor; Subunit proteins of the cytokine, IL-2; Subunit proteins of signal recognition particle; Subunit proteins of ligandin; Subunit proteins of hepatopoietins A and B;Subunit proteins of human platelet-derived growth factor; Subunit proteins of glutathione S-transferases;

Subunit proteins of luciferase; and Subunit proteins of gamma-glutamyl transpeptidase. n ¹)

29. The method of claim 24 wherein X is selected from the group consisting of MRP14 and MRP8.

30. The method of claim 24 wherein Li is the residue of a heterobifunctional cross-linking reagent .

31. The method of claim 30 wherein the heterobifunctional cross-linking reagent is selected from the group consisting of sulfosuccinimidyl 4-(N- maleimidomethyl) cyclohexane-1-carboxylate, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, sulfosuccinimidyl 4- (p-maleimidophenyl)butyrate, 2- Iminothiolane, and N-succinimidyl S-acetylthioacetate.

32. The method of claim 24 wherein Li is the residue of a modified nucleotide moiety containing a reactive functional group.

33. The method of claim 24 wherein said reactive functional group is selected from the group consisting of amine groups and sulfhydryl groups.

34. The method of claim 28 wherein said MRP14 is derived from human neutrophils

35. The method of claim 28 wherein said MRP8 is derived from human neutrophils

36. The method of claim 25 wherein D is selected from the group consisting of the heterodimeric pairs MRP14 and MRP8; alpha and beta chains of the T cell receptor;delta and gamma chains of the T cell receptor; Subunit proteins of the cytokine, IL-2; Subunit proteins of signal recognition particle; Subunit proteins of ligandin; Subunit proteins of hepatopoietins A and B;Subunit proteins of human platelet-derived growth factor; Subunit proteins of glutathione S-transferases; ft) Subunit proteins of luciferase; Subunit proteins of gamma-glutamyl transpeptidase.

37. The method of claim 25 wherein D is selected from the group consisting of MRP14 and MRP8.

38. The method of claim 36 wherein said MRP14 is derived from human neutrophils

39. The method of claim 36 wherein said MRP8 is derived from human neutrophils

40. The method of claim 25 wherein L₂ is the residue of a heterobifunctional cross-linking reagent .

41. The method of claim 40 wherein the heterobifunctional cross-linking reagent is selected from the group consisting of sulfosuccinimidyl 4-(N- maleimidomethyl) cyclohexane-1-carboxylate, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, sulfosuccinimidyl 4- (p-maleimidophenyl)butyrate, 2- Iminothiolane, and N-succinimidyl S-acetylthioacetate.

42. The method of claim 25 wherein L is a modified ligand moiety containing the residue of a reactive functional group.

43. The method of claim 25 wherein the reactive functional group is selected from the group consisting of amine groups and sulfhydryl groups .

44. The method of claim 25 wherein Q contains a polycarboxylic acid group.

45. The method of claim 25 wherein Q is selected from the group consisting of B4A, P4A, TMT, DCDTPA, PheMT, macroPheMT, and macroTMT.

46. The method of claim 25 wherein M is a radioactive isotope of a metal.

47. The method of claim 46 wherein the radioactive isotope is selected from ⁴⁴Sc, ⁶⁴Cu, ⁶⁷Cu, ^U ln, ^2l Pb, 6⁸Ga, ⁸⁷Y, ⁹°Y, ¹⁵3_Sm, ^2l2Bi, ⁹⁹™Tc, ⁷⁷Lu ⁱ⁸⁶Re and ⁱ⁸⁸Re.

48. A pharmaceutical composition comprising a compound of claim 1 dissolved or dispersed in a pharmaceutically acceptable carrier.

49. A pharmaceutical composition comprising a compound of claim 2 dissolved or dispersed in a pharmaceutically acceptable medium.

50. A method of treating a tumor in a mammal comprising administering to said mammal an effective dose of a non-radioactive targeting immunoreagent of claim 1 in a pharmaceutically acceptable medium, waiting for a time period sufficient for said non- radioactive targeting immunoreagent to accumulate at the tumor site in said mammal, and subsequently, administering an effective dose of a radioactive targeting reagent of claim 2 in a pharmaceutically acceptable medium to said mammal, and waiting for a time period sufficient for said radioactive targeting reagent to accumulate at the target site, said target site being the said non-radioactive targeting immunoreagent accumulated at said tumor site in said mammal.

51. A method of diagnostic imaging in a mammal comprising administering to said mammal an imaging effective dose of a non-radioactive targeting immunoreagent of claim 1 in a pharmaceutically acceptable medium, waiting for a time period sufficient for said non-radioactive targeting immunoreagent to accumulate at the imaging site in said mammal, and subsequently, administering an effective dose of a radioactive targeting reagent of claim 2 in a pharmaceutically acceptable medium to said mammal, waiting for a time period sufficient for said radioactive targeting reagent to accumulate at the target site, said target site being the said non- radioactive targeting immunoreagent accumulated at said imaging site in said mammal.

52. The reagent of claim 1 wherein X is a residue of a receptor moiety and Z and X comprise a fusion protein.

53. The reagent of claim 52 wherein the receptor moiety is MRP14.

54. The reagent of claim 24 wherein X is a residue of a receptor moiety and Z and X comprise a fusion protein.

55. The reagent of claim 54 wherein the receptor moiety is MRP8.

S3