EP1615995A2

EP1615995A2 - Morpholino imaging and therapy via amplification targeting

Info

Publication number: EP1615995A2
Application number: EP04750119A
Authority: EP
Inventors: Donald J. Hnatowich; Jiang He; Guozheng Liu; Suresh Gupta; Yumin Zhang; Mary Rusckowski
Original assignee: Immunomedics Inc
Current assignee: University of Massachusetts Boston
Priority date: 2003-04-15
Filing date: 2004-04-15
Publication date: 2006-01-18
Also published as: WO2004091525A3; EP1615995A4; WO2004091525A2; JP2006523706A

Abstract

The present invention provides a kit and a method for targeting of a diagnostic or therapeutic agent to a target site in a mammal having a pathological condition. The kit comprises, in separate containers, (A) a first conjugate comprising a targeting moiety and a Morpholino oligomer, wherein said targeting moiety selectively binds to a primary, target-specific binding site of the target site or to a substance produced by or associated with the target site; (B) optionally, a clearing agent; (C) a second conjugate comprising multiple copies of complementary Morpholino oligomer and a diagnostic agent or therapeutic agent; wherein said complementary Morpholino oligomer is bound to a polymer; and (D) a third conjugate comprising a Morpholino oligomer and a radiolabel. The method comprises administering (A), optionally (B), (C) and (D) to a mammal.

Description

MORPHOLINO IMAGING AND THERAPY VIA AMPLIFICATION TARGETING

FIELD OF THE INVENTION The present invention is directed to a kit for targeting of a diagnostic or therapeutic agent to a target site in a mammal, as well as to a method for diagnosing or treating a pathological condition using multiple copies of complementary pair of single- stranded Morpholino oligomers conjugated to a polymer.

BACKGROUND OF THE INVENTION The objective of drug targeting research is to improve the effectiveness of therapeutic drugs by delivering them directly to the targeted tumor sites and allowing a more effective dosing at these sites, thereby reducing non-tumor-related side effects. Another objective is to achieve an absolute accretion of the therapeutic agent at the target site thereby increasing the target/non-target ratio. Different targeting vectors comprising diagnostic or therapeutic agents conjugated to a targeting moiety for selective localization have long been known. Examples of targeting vectors include diagnostic agent or therapeutic agent conjugates of targeting moieties such as antibodies or antibody fragments, cell- or tissue-specific peptides, hormones and other receptor binding molecules. For examples, antibodies against different determinants associated with pathological and normal cells, as well as associated with pathogenic microorganisms, have been used for the detection and treatment of a wide variety of pathological conditions or lesions. In these methods, the targeting antibody is directly conjugated to an appropriate detecting or therapeutic agent as described, for example in, Hansen et al., U.S. Pat. No. 3,927,193 and Goldenberg, U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,460,459, 4,460,561, 4,624,846 and 4,818,709, the disclosures of all of which are incorporated herein by reference.

One of the problems encountered in direct targeting methods is that a relatively small fraction of the conjugate actually binds to the target site, while the majority of the conjugate remains in circulation and compromises in one way or another the function of the targeted conjugate. Other problems include high background and low resolution when a diagnostic agent is administered and marrow toxicity or systemic side effects when a therapeutic agent is attached to a long circulating targeting moiety.

Pretargeting methods have been developed to increase the targetbackground ratios of the detection or therapeutic agents. Examples of pretargeting and biotin/avidin approaches are described, for example, in Goodwin et al., U.S. Pat. No.4,863,713; Goodwin et al, J. Nucl Med. 29:226 (1988); Hnatowich et al, J. Nucl. Med. 28: 1294 (1987); Oehr et al, J. Nucl. Med. 29:728 (1988); Klibanov et al, J. Nucl. Med. 29:1951 (1988); Sinitsyn et al, J. Nucl Med. 30:66 (1989); Kalofonos et al, J. Nuc Med. 31:1791 (1990); Schechter et al, Int. J. Cancer. 48:167 (1991); Paganelli et al, Cancer Res. 51:5960 (1991); Paganelli et al, Nucl. Med. Commun. 12:211 (1991); Stickney et al, Cancer Res. 51:6650 (1991); and Yuan et al, Cancer Res. 51:3119 (1991); all of which are incorporated by reference herein in their entireties.

In pretargeting methods, a primary targeting species (which is not bound to a diagnostic agent or therapeutic agent) comprising a first targeting moiety which binds to the targeting site and a binding site that is available for binding by a subsequently administered second targeting species is targeted to an in vivo target site. Once sufficient accretion of the primary targeting species is accomplished, a second targeting species comprising a diagnostic or therapeutic agent and a second targeting moiety, which recognizes the available binding site of the primary targeting species, is administered. An illustrative example of pretargeting methodology is the use of a biotin-

(strept)avidin system to administer a cytotoxic radioantibody to a tumor. In the first step, a monoclonal antibody targeted against a tumor-associated antigen is conjugated to avidin (or biotin) and administered to a patient who has a tumor recognized by the antibody. In the second step, the therapeutic agent, via its attached biotin (or avidin), is taken up by the antibody-avidin (or -biotin) conjugate pretargeted to the tumor.

However, difficulties have arisen in the applications of biotin-avidin or (strept)avidin system during pretargeting. First of all, unless properly constructed, radiolabeled biotins may be subject to plasma biotinidase degradation. Furthermore, when conjugated to antibodies, strept/avidin and avidin can generate anti-strept/avidin antibodies in a patient. Finally, the potential effects of endogenous biotin during in vivo pretargeting can lead to the disappearance of biotin binding expression because of saturation by biotin. This happened, for example, when one strept/avidin-conjugated antibody localized in a nude mouse xenograft became saturated with biotin. Rusckowski et al, Cancer 80:2699-705 (1997). A three-step strategy involving administration of biotinylated monoclonal antibody, avidin, followed by radiolabeled biotin alleviates some of the drawbacks; however, this procedure is considered complex for imaging and does not address immunogenicity.

Another recognized example of pretargeting method involves the use of the bispecifϊc antibody-hapten recognition system which uses a radiolabeled hapten and a bispecifϊc antibody in place of (strept)avidin and biotin. Barbet, J. et al. Cancer Biother. Radiopharm. 14:153-166 (1999); Karacay, H. et al, Bioconj. Chem. 11: 842-854 (2000); Gautherot, E. et al, J. Nucl. Med. 41:480-487 (2000); Lubic, S.P. et al, J. Nucl. Med. 42:670-678 (2001); Gestin, J.F. et al, J. Nucl. Med. 42:146-153 (2001). The hapten is often a coordination complex, for example, indium-DTPA. The bispecifϊc antibody is the product of linking two antibodies or antibody fragments against separate determinants, the hapten and a tumor marker such as carcinoembryonic antigen. In addition to the need to prepare bispecifϊc antibodies, this approach may suffer from lower affinities. The affinity of an antibody for its hapten, particularly for a monovalent one, is orders of magnitude lower than that of (strepfjavidin for biotin. Mathematical modeling has shown that a high affinity between an antibody and its hapten is an important determinant of successful pretargeting. Zhu, H. et al, J. Nucl. Med. 39:65-76 (1998).

As an alternative to the biotin-avidin and bispecifϊc antibody-hapten systems for pretargeting, single-stranded oligomers, such as peptide nucleic acid (PNA), have been used. Single-stranded oligomers bind specifically to their complementary single-stranded oligomers by in vivo hybridization. A single-stranded PNA bound to a targeting moiety is first administered to a patient, followed by the single-stranded complementary PNA radiolabeled with a diagnostic agent. An example of this methodology is described in Rusckowski et al, Cancer 80:2699-705 (1997). An optional intermediate step can be added to the two-step method by administration of a clearing agent. The purpose of the clearing agent is to remove circulating primary conjugate which is not bound at the target site. This is disclosed by Griffiths et al, in U.S. Pat. No. 5,958,408, which is incorporated herein by reference.

Chemical modifications to the backbone of these single-stranded oligomers for attachment to radionucleotides are usually required to improve nuclease stability and decrease protein binding affinities. The influence of three distinct chemical modifications to one 18 mer phosphorothioate DNA to permit labeling with ^99mTc have been compared in vitro and in vivo in mice. Zhang, Y. M. et αl, Eur. J. Nucl. Med. 27:1700-1707 (2000). While the association rate constant for hybridization was found to be independent of labeling method, both cellular accumulations in culture and the pharmacokinetic behavior of the radiolabel in normal mice was strongly influenced by the labeling method.

These in vivo properties of oligomers may possibly be influenced by changes in their chain length and/or base sequences. Conceivably, the pharmacokinetics of an oligomer may thereby be modified in a useful manner if the influences of chain length and base sequence were to be understood. Despite this possibility (and as in the case of the chemical modifications), these additional influences have almost entirely gone uninvestigated thus far. In part, this may be attributed to constraints placed on these parameters by the application. For example, antisense chemotherapy is thought to achieve efficacy usually by the hybridization of a short, single-chain oligomer with a base sequence complementary to that of its mRNA target. Hnatowich, D.J., J. Nuc Med. 40:693-703 (1999). The base sequence, and to an extent the chain length as well, are thus restricted to those providing the desired hybridization. Nevertheless, there are combinations of bases that have received attention. One example is the presence of a G- quartet (i.e. four guanine bases in a row) in either phosphodiester or phosphorothioate

DNAs. Shafter, R.H. et al, Biopoly (Nucleic Acid Sci.) 56:209-227 (2001). In the case of these chemical forms of DNAs at least, the stacking of the guanine bases provides the oligonucleotides with a particular three dimensional quadruplex structure. This structure is apparently responsible for a variety of sequence-specific effects with significance to various biological processes, id Another example is the CpG motif, a cytosine base followed immediately by a guanine, that has been shown to be immunostimulatory. Zhao, Q. et al, Antisense Nucleic Acid Drug Dev. 7:495-502 (1997). The influences of these sequences, if any, on pharmacokinetics has yet to be established.

A variety of other published reports have appeared concerning the in vitro influences of oligomer chain length and sequence. Cytotoxicity in one cell line of phosphodiester DNAs composed entirely of guanine and thymidine bases was found to require at least a chain length of 20 bases and the cytotoxicity disappeared with the introduction of adenines or cytosines at either end. Morassutti, C. et al, Nucleosides & Nucleotides 18:1711-1716 (1999). The efficiency with which PNAs initiated transcription and gene expression in cells was found to be optimum at chain lengths of 16 to 18 bases. Wang, G. et al, J. Mol. Biol. 313:933-940 (2001). Rat liver homogenates have been used ex vivo to investigate the metabolism of a series of phosphorothioate DNAs differing in chain length and base sequence. Crooke, R.M. et al, J. Pharm. Exp. Therapeutics 292:140-149 (2000). All oligomers were degraded primarily by 3'exonucleases with the rate of metabolism increasing with increasing chain length. The rate and extent of nuclease metabolism was also related to base sequence in that pyrimidine-rich oligonucleotides were more labile. This particular investigation was unusual in that the influence of stereoisomerism was also studied. The metabolism rate was found to be more rapid for one of the diastereoisomers than the other with mixtures being digested at rates in between. Finally, a recent report described the influence of base sequence on reactivity of the phosphodiester bond in RNAs. Kaukinen, U. et al, Nucl. Acids Res. 30:468-474 (2002).

The inventors have previously disclosed the use of oligomers such as phosphorodiamidate morpholinos (MORFs) tumor imaging and therapy. Such use has been disclosed in a pending continuation-in-part application of a pending U.S. patent application serial No. 10/112,094, filed April 01, 2002, which, in turn, claims priority to U.S. provisional application serial Nos. 60/279,809, filed March 30, 2001, and 60/341,794, filed December 21, 2001. The entire contents of these applications, including their specifications, claims and drawings, are incorporated herein by reference in their entirety.

The native phosphodiester DNA differs from the phosphorothioate by the substitution of a nonbonding oxygen with a sulfur atom. In the case of PNA, the phosphate backbone of DNA has been replaced with a (2-aminoethyl) glycine polypeptide linkages to which the nitrogenous bases are attached via methylenecarbonyl groups while the phosphodiester backbone in MORFs has been substituted with a phosphorodiamidate group and the ribose sugar has been replaced with a morpholino ring. Like the DNAs, MORFs and PNAs are commercially available but, unlike DNAs, they are both uncharged and (unlike phosphodiester DNAs) stable to nucleases and (unlike phosphorothioate DNAs) nonchiral.

Amplification is a multistep pretargeting process with the potential to greatly improve targeting through the intermediate use of polymers conjugated with multiple copies of oligomers. Accordingly, there is a need for an improved kit or method that greatly increase the accumulation of radioactivity in tumor and, at the same time, improve upon the tumor/normal tissue ratios. This would first require the preparation of a MORF polymer that would ultimately be conjugated to the antibody. Only in this way is there any hope of avoiding significant denaturation of the antibody.

One alternative to amplification targeting is pretargeting using either (strept)avidin/biotin, bispecifϊc antibodies or oligomer pairs. Recently, encouraging results have been reported especially using bispecific antibodies (Karacay, H. et al, Bioconjug. Chem. 13: 1054-1070, 2002) and oligomers (Liu, G. et al, J. Nucl. Med. 43: 384-391, 2002). However, since pretargeting does not involve a multivalent polymer, amplification is not possible (other than a factor of up to four in the case of certain streptavidin/biotin protocols (Kassis, A.I. et al, J. Nucl. Med. 37: 343-352, 1996).

There have been numerous reports of antitumor antibodies conjugated directly with multiple copies of low molecular weight species such as boron for boron neutron capture (Novick, S. et al,. Nucl Med. Biol. 29:159-67, 2002) or metal chelating agents for a variety of applications (Torchilin, V.P. et al, Hybridoma 6: 229-40, 1987). The obvious disadvantage is the risk of denaturing the antibody as a result of the conjugation, especially when the aim is to attach multiple copies of a relatively high molecular weight MORF. For example, to carry 50 MORFs per molecule, the molecular weight of an IgG antibody must be raised from about 150 KDa to least 600 KDa. Under these conditions, it may not be possible to preserve immunoreactivity. Furthermore, amplification targeting may eventually be useful with tissue specific agents other than antibodies such as antitumor and antitissue peptides which, because of their low molecular weight, could not tolerate conjugation with multiple MORFs.

Neither the use of phosphodiester nor the phosphoromonothioate would be ideal for the amplification targeting and other radiopharmaceutical applications. The former being too unstable to nucleases in vivo and the latter showing too high an affinity for serum and tissue proteins (Hnatowich, D.J. et al, J. Pharmacol. Exp. Tlier. 276:326-334, 1996). We subsequently showed that PNAs are stable and with minimal protein binding affinities (Mardirossian, G. etal, J. Nucl. Med. 38: 907-913, 1997). Accordingly, our first amplification targeting study used PNAs (Wang, Y. et al, Bioconjug. Chem. 12: 807-816, 2001). However, difficulties were encountered due to a low aqueous solubility of PNAs with the selected base sequence. Because of difficulties in conjugation, the PNA study did not use an antitumor antibody (Wang, Y. et al, 2001, supra). Possibly because of higher aqueous soluble, the amine-derivitized MORFs were successfully conjugated both to the antitumor antibody MN14 and to the chelator MAG₃ to permit radiolabeling with ^99mTc. The radiolabel and the MORFs were found to be "stable" in vitro and in vivo, to show little affinity for proteins and, like the labeled PNAs, are capable of rapid hybridization (Mang'era, K. et al, Eur. J. Nucl Med. 28:1682-1689, 2001).

Amplification targeting share some similarities with pretargeting (as described herein) in the use of both a MORF-antibody and a radiolabeled MORF (cMORF in the case of pretargeting) but differs in the intermediate use of the polymer. Amplification targeting is obviously more complicated than pretargeting but with potential for signal amplification.

One important aspect of amplification targeting is in situ accessibility. To achieve high tumor radioactivity levels, the MORFs on antibody in tumor must be accessible to the polymeric cMORFs and, in turn, the cMORF on the polymer in tumor must be accessible to the radiolabeled MORF. Another important aspect results from accumulation of the polymeric cMORF in liver, spleen, kidneys and other normal organs. To lower background radiation levels in these normal organs, the polymeric cMORF expression should rapidly become inaccessible to the radiolabeled MORF.

Despite the several advantages over the strept/avidin-biotin and bispecific antibody-hapten systems, a few limitations exist in the use of these oligomers in pretargeting. These limitations include poor specificity, possible insolubility in aqueous solutions, and high costs. A need continues to exist for an improved kit and method for in vivo targeting to deliver a therapeutic or diagnostic agent to a target site in a mammal, that is more specific, affordable and inexpensive and provides higher target uptake and lower uptake in normal tissues. There is also a need to provide an improved pretargeting kit and method, wherein excess isotopically labeled oligomers, which do not bind to the cancer cells, are rapidly cleared from the body, particularly from the kidneys.

SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is to provide a kit and a method useful for amplification targeting of a diagnostic or therapeutic agent in a mammal which can be prepared from relatively inexpensive starting materials but yet provides reduced renal uptake and retention, lesser toxicity, better specificity, stability, predictable targeting and/or more desirable antigen-antibody effects than conventional and other known kits and methods. Another object of the present invention is to conjugate the multiple copies of MORF directly to the antibody, thus avoiding the second administration to the mammal. It is another object to provide an alternative method useful for tumor localization/imaging by amplification targeting using a multivalent Morpholino oligomers (MORFs, as defined below) instead of strep/avidin-biotin, peptide nucleic acids and other oligomers, wherein a radiolabeled targeting moiety is highly accreted to the primary target-specific binding site within the target thereby greatly increasing the accumulation of radioactivity in tumor and, at the same time, improve tumor/normal tissue ratios.

These and other objects are achieved, in accordance with one embodiment of the present invention by the provision of a kit for targeting of a diagnostic or therapeutic agent in a mammal comprising: (A) a first conjugate comprising a targeting moiety and a Morpholino oligomer, wherein said targeting moiety selectively binds to a primary, target-specific binding site of the target site or to a substance produced by or associated with the target site; (B) optionally, a clearing agent; (C) a second conjugate comprising multiple copies of complementary Morpholino oligomer and a diagnostic agent or therapeutic agent, wherein said complementary Morpholino oligomer is bound to a polymer; and (D) a third conjugate comprising a Morpholino oligomer and a radiolabel.

The targeting moiety of step (a) preferably comprises an antibody, especially a humanized antibody or an antigen-binding fragment of a humanized antibody. One such humanized antibody is an anti-carcinoembryonic antigen (CEA) antibody. The targeting moiety is selected from the group consisting of proteins, small peptides, polypeptides, enzymes, hormones, steroids, cytokines, neurotransmitters, oligomers, vitamins and receptor binding molecules.

The polymer of step (c) includes, but is not limited to, poly-lysine (PL), polyethyvinylether maleic acid (PA) dextran, dendrimers and N-(2- hydroxypropyl)methacrylamide (HPM A) .

In accordance with another aspect of the present invention, a kit is provided, as described above, wherein the length of the Morpholino oligomer and its complementary Morpholino oligomer is at least about 6 bases to about 100 bases. In addition, the Morpholino and its complementary Morpholino oligomer can be a 15-mer, an 18-mer or a 25-mer. The target moiety is bound to a 15-mer, an 18-mer or a 25-mer Morpholino oligomer.

In a preferred embodiment, the clearing agent is an anti-idiotypic antibody or antigen-binding antibody fragment. In another preferred embodiment, the therapeutic agent is selected from the group consisting of antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, chelators, boron compounds, photoactive agents or dyes and radionuclides. In yet another preferred embodiment, the diagnostic agent is selected from the group consisting of radionuclides, dyes, contrast agents, fluorescent compounds or molecules and enhancing agents useful for magnetic resonance imaging (MRI).

The present invention contemplates an targeting method for delivering a diagnostic or therapeutic agent to a target site in a mammal, comprising: (a) administering to said mammal a first conjugate comprising a targeting moiety and a Morpholino oligomer, wherein said targeting moiety selectively binds to a primary, target-specific binding site of the target site or to a substance produced by or associated with the target site; (b) optionally, administering to said mammal a clearing agent, and allowing said clearing agent to clear non-localized first conjugate from circulation; and (c) administering to said mammal a second conjugate comprising a polymer bound to multiple copies of complementary Morpholino oligomers and a diagnostic agent or therapeutic agent, wherein said complementary Morpholino oligomer-polymer conjugate binds its Morpholino oligomer complement on the first conjugate thereby targeting the diagnostic or therapeutic agent to the target site; and (d) administering to said mammal a third conjugate comprising a Morpholino oligomer and a radiolabel.

Other objects, features and advantages of the present invention will become apparent from the following detailed description and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Whole body images, obtained simultaneously, of LS 174T tumored mice

3 h post administration of ^99mTc-MORF and 43 h post administration of PA30KDa- cMORF to animals receiving MORF-MN14 73 h earlier. Study animal at left, animal receiving the polymer but not the antibody, on the right.

Figure 2. Whole body images, obtained simultaneously, of LS174T tumored mice 3 h post administration of ^99mTc-MORF and 21 h post administration of PA30KDa- cMORF to animals receiving MORF-MN14 51 h earlier. Study animal at right, animal receiving the polymer but not the antibody in the middle, and animal receiving neither polymer nor antibody on the left. DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise specified, "a" or "an" means "one or more".

The present invention provides a kit and a method useful for in vivo targeting of a diagnostic or therapeutic agent in a mammal (preferably human) comprising: (A) a first conjugate comprising a targeting moiety and a Morpholino oligomer, wherein said targeting moiety selectively binds to a primary, target-specific binding site of the target site or to a substance produced by or associated with the target site; (B) optionally, a clearing agent; (C) a second conjugate comprising multiple copies of complementary Morpholino oligomer and a diagnostic agent or therapeutic agent; wherein said complementary Morpholino oligomer is bound to a polymer; and (D) a third conjugate comprising a Morpholino oligomer and a radiolabel.

The targeting moiety may be, for example, an antibody or an antigen binding antibody fragment. Preferred are the monoclonal antibodies (Mabs) due to their high specificities. They are readily prepared by what are now considered conventional procedures of immunization of mammals with immunogenic antigen preparation, fusion of immune lymph or spleen cells with an immortal myeloma cell line, and isolation of specific hybridoma clones. More unconventional methods of preparing monoclonal antibodies are also contemplated, such as interspecies fusions and genetic engineering manipulations of hypervariable regions, since it is primarily the antigen specificity of the antibodies that affects their utility in the present invention. It will be appreciated that newer techniques for production of monoclonals can also be used, e.g., human monoclonals, interspecies monoclonals, chimeric (e.g., human/mouse) monoclonals, genetically engineered antibodies and the like.

Antibody fragments useful in the invention include F(ab')₂, F(ab)₂, Fab', Fab, Fv and the like including hybrid fragments. Preferred fragments are Fab', F(ab')₂, Fab, and F(ab)₂. Also useful are any subfragments retaining the hypervariable, antigen-binding region of an immunoglobulin and having a size similar to or smaller than a Fab' fragment. This will include genetically-engineered or recombinant antibodies and proteins, whether single-chain or multiple-chain, which incorporate an antigen-binding site and otherwise function in vivo as targeting vehicles in substantially the same way as natural immunoglobulin fragments. Such single-chain binding molecules are disclosed in U.S. Pat. No. 4,946,778, which is incorporated herein by reference. Fab' fragments may be conveniently made by reductive cleavage of F(ab')₂ fragments, which themselves may be made by pepsin digestion of intact immunoglobulin, under reducing conditions, or by cleavage of F(ab')₂ fragments which result from careful papain digestion of whole immunoglobulin. The fragments may also be produced by genetic engineering.

Also preferred are antibodies having a specific immunoreactivity to a marker substance produced by or associated with the cancer cells of at least 60% and a cross- reactivity to other antigens or non-targeted substances of less than 35%. A monoclonal antibody that specifically targets tumor sites by binding to antigens produced by or associated with the tumors is particularly preferred.

Antibodies against tumor antigens are known. For example, antibodies and antibody fragments which specifically bind markers produced by or associated with tumors have been disclosed, inter alia, in Hansen et al, U. S. Pat. No. 3,927,193, and Goldenberg's U. S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,818,709 and 4,624,846, the contents of all of which are incorporated herein by reference in their entirety. In particular, antibodies against an antigen, e.g., a gastrointestinal, lung, breast, prostate, ovarian, testicular, brain or lymphatic tumor, a sarcoma or a melanoma, are advantageously used.

Other targets of the targeting moiety of the present invention include, but are not limited to B-cell antigens, T-cell antigens, plasma cell antigens, HLA-DR lineage antigens, CEA, NCA, MUC1, MUC2, MUC3, and MUC4 antigens, EGP-1 antigens, EGP-2 antigens, placental alkaline phosphatase antigen, IL-6, VEGF, tenascin, CD33, CD74, PSMA, PSA, PAP, antigens associated with autoimmune diseases, infection/inflammation, and infectious diseases. The target may be a target antigen associated with a B- or T-cell lymphoma, or B- or T-cells associated with autoimmune diseases. The target may be an antigen selected from the group consisting of CD 19, CD22, CD40, CD74, CEA, NCA, MUC1, MUC2, MUC3, MUC4, HLA-DR, EGP-1, EGP-2, IL-15 and HLA-DR expressed by malignant diseases. The target may be, for example EGP-2, EGP-1, CD22, CEA, or MUC1, for certain malignant diseases. The target may be expressed by bacteria, viruses, fungi, parasites, or other microorganisms. The target may also be expressed by the host cells accumulating at the sites of infection, such as activated granulocytes (e.g., CD15, CD33, , CD66a, CD66b, CD66c (NCA), and CD66e, etc.).

The antibodies and antigen-binding antibody fragments useful in the methods of the present invention may be conjugated to the member of the binding pair by a variety of methods of chemical conjugation known in the art. Many of these methods are disclosed in the above-referenced U.S. patents and patent applications. See also Childs et al, J. Nuc. Med. 26:293 (1985), the contents of all of which are incorporated herein by reference in their entirety. One monoclonal antibody useful in the present invention is MN-14, a second generation CEA-antibody that has ten times more affinity for CEA than the first generation version, NP-4. Hansen et al, Cancer 71:3478-85, (1993). MN-14 internalizes slowly, making it suitable for targeting approach, and has been chimerized and humanized. Leung et al., U.S. Pat. No. 5,874,540. Other antibodies or antibody fragments suitable for use in the present invention may be, or may be derived from, for example, from RSI 1, 17-1A, RS7, LL1, LL2, MN-3, MN-14 or PAM4 or humanized versions thereof, when targeting malignant diseases. A suitable granulocyte antibody is MN3, used in LeukoScan®.

Other targeting moieties useful in the present invention can also be non-antibody species selecting from the group consisting of proteins, small peptides, polypeptides, enzymes, hormones, steroids, cytokines, neurotransmitters, oligomers, vitamins, and receptor binding molecules, which preferentially bind marker substances that are produced by or associated with the target site.

Morpholino oligomers (herein "Morpholinos" or "MORFs") bind and inactivate selected RNA sequences. These oligomers are assembled from four different Morpholino subunits, each of which contains one of the four genetic bases (A, G, C, T or U), linked to a six-membered morpholine ring. These subunits, as 15 - 25 mers, are joined together in a specific order by non-ionic phosphorodiamidate intersubunit linkages to produce a Moφholino oligomer. They may offer better antisense properties than do DNA, RNA, and their analogs having five-membered ribose or deoxyribose backbone moieties joined by ionic linkages. Summerton's work on Moφholinos is disclosed in U.S. Pat. Nos. 5,142,047 and 5,185,444, the contents of which are herein incoφorated by reference. Moφholinos are commercially available from Gene Tools, LLC, Corvallis, Oregon. Because they are readily delivered to the target, Moφholinos are effective tools for genetic studies and drug target validation programs. They are completely resistant to nucleases. A more rigid MORF backbone may offer better access during duplex formation when compared with a peptide backbone or with the more common sugar backbone. When compared to PNAs, Moφholinos are less expensive and more soluble in aqueous solutions, and provide better predictable targeting and higher efficacy in RNA binding affinities.

In the present invention, a Moφholino oligomer (herein MORF) bound to a targeting antibody is in vivo hybridized to the complementary MORF (herein cMORF) bound to a diagnostic or therapeutic agent. In a preferred embodiment, the length of the MORF and its complementary Moφholino (cMORF) is from 6 bases to about 100 bases, for example, MORF15 and cMORF15 (15-mer), MORF18 and cMORFlδ (18-mer) or MORF25 and cMORF25 (25-mer).

The MORFs used in the present invention include a 15-mer (5' equivalent TGT- ACG-TCA-CAA-CTA-linker-amine (herein MORF15), and TAG-TTG-TGA-CGT- ACA-linker-amine (herein complementary MORF 15 or cMOR 15)), an 18-mer (5' equivalent CGG-TGT-ACG-TCA-CAA-CTA-linker-amine (herein MORF18) and TAG- TTG-TGA-CGT-ACA-CCC-linker-amine (herein complementary MORF 18 or CMORF18)), and a 25-mer (5' equivalent T-GGT-GGT-GGG-TGT-ACG-TCA-CAA- CTA-linker-amine (herein MORF25), and TAG-TTG-TGA-CGT-ACA-CCC-ACC-ACC- A-linker-amine (herein complementary MORF25 or cMORF25)).

The Moφholino oligo structure (Summerton and Weller, Antisense Nucl. Acid DrugDev. 7:187-95, 1997) used in the present invention, is shown below as:

B = adenine, cytosine, guanine. thymine/uracil

Clearing agents known in the art may be used in accordance with the present invention. For example, if the first conjugate comprises avidin or streptavidin, biotin may be used as a clearing agent. Alternatively, if the first conjugate comprises biotin, avidin or streptavidin may be used as a clearing agent.

In a preferred embodiment, the clearing agent is an antibody which binds the binding site of the targeting moiety, wherein the targeting moiety can be an antibody, an antigen-binding antibody fragment or a non-antibody targeting moiety. In a more preferred method, the clearing agent is a monoclonal antibody that is an anti-idiotypic to the monoclonal antibody of the conjugate used in the first step, as described in U.S. application Ser. No. 08/486,166. In another preferred embodiment, the clearing agent is substituted with multiple residues of carbohydrate, such as galactose, which allow the clearing agent to be cleared quickly from circulation by asialoglycoprotein receptors in the liver.

A physiological solution of the targeting species is advantageously metered into sterile vials, e.g., at a unit dosage of about 1.0-500 mg targeting species/vial, and the vials are either stoppered, sealed and stored at low temperature or lyophilized, stoppered, sealed and stored.

Variations and modifications of these formulations will be readily apparent to the ordinary skilled artisan, as a function of the individual needs of the mammal or treatment regiment, as well as of variations in the form in which radioisotopes may be provided or may become available. Routes of administration include intravenous, intraarterial, intrapleural, intraperitoneal, intrathecal, subcutaneous or by perfusion.

Methods useful for internal detection or treatment of tumors or other lesions, such as cardiovascular lesions (clots, emboli, infarcts, etc.), infectious diseases, inflammatory diseases, and autoimmune diseases are disclosed in U.S. Pat. Nos. 4,782,8404,932,412 and 5,716,595, the disclosures of which are incoφorated herein by reference. The methods of the present invention can be used to enhance the methods disclosed in these references. The present invention also may be practiced in conjunction with intraoperative probes, endoscopic and laparoscopic uses, and in methods for imaging normal organs. The methods of the present invention can be used in other methods that will be apparent to those skilled in the art. Examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, chelators, boron compounds, photoactive agents or dyes and radionuclides.

In a further preferred embodiment, the cMORF is conjugated to a bifunctional chelator which in turn, is radiolabeled with an isotope. A chelator is radiolabeled first prior to conjugation (preconjugation labeling) to a protein, a polypeptide or an oligonucleotide which cannot withstand harsh conditions. Examples of chelators may include hydrazino nicotinamide (HYNIC), diethylenetriaminepentaacetic acid (DTP A), 1, 4, 1, 10-tetraaza-cyclododecane N, N', N", N'"-tetraacetic acid (DOT A), and mercaptoacetylglycylgly-cylglycine (MAG₃). A preferred bifunctional chelator used in the present invention is N-hydroxysuccinimidyl derivative of acetyl-S-protected mercaptoacetyltriglycine (NHS-MAG₃). NHS-MAG₃ is synthesized according to the method of Winnard, P. et al, Nucl. Med. Biol. 24:425-32 (1997). The conjugation of single-stranded moφholino oligomers with NHS-MAG₃ was accomplished as described in Mardirossian, G. et al, J. Nucl. Med. 38:907-13 (1997).

The polymer, according to the present invention, should have the following properties: (1) nontoxic; (2) commercially available with the proper molecular weight; (3) conjugation with cMORF should be achievable; (4) the required number of cMORFs should be accessible on the conjugated polymer; (5) the conjugated polymer should be sufficiently water soluble and its pharmacokinetics should be favorable with reasonably persistent blood levels; (6) hybridization of the polymer to the antibody should not encourage internalization in tumor; and (7) the polymer should metabolize in normal tissue such that the cMORF expression disappears in these tissues and the polymer should diffuse effectively in tumor. Preferably, the polymers are poly-lysines (PL) and polyethyvinylether maleic acid (PA) having all of the above-mentioned properties. Other preferred polymers include but are not limited to dextran, dendrimers and N-(2- hydroxypropyl)methacrylamide (HPMA). These polymers were selected because they are each commercially available in the variety of useful molecular weights shown below, each are water soluble and each should be readily conjugated with cMORF. In addition, both PL and PA have been used successfully. Dendrimers offer an opportunity to evaluate a nonlinear polymer. Dextrans have been in clinical use for more than 50 years for plasma volume expansion, peripheral flow promotion, and as antithrombolytic agents (Thoren, 1981; Mehvar, 2000). Evidence also exists for the safety of PLs and dendrimers especially at the low dosage to be administered in connection with amplification (Malik, 2000).

Table 7: Select commercially available polymers, their suppliers and ranges of

MWs

The polymer used by the inventors in the earlier PNA study was a polymethylvinylether maleic acid (PA) with an initial molecular weight of 80 KDa and approximately 900 carboxyl groups per molecule (Wang, Y. et al, Bioconjug. Chem. 12: 807-816, 2001). Each molecule was modified with an average of 80 PNAs (each with a 19-member polyetheφolyamide linker). Because of the limited aqueous solubility of PNA, the PA polymer showed unfavorable pharmacokinetics (i.e. high liver and low blood levels) unless the polymer was also conjugated with an average of 200 polyethylene glycol (PEG) groups. As a consequence, the molecular weight of the final polymer was raised to 1.4 MDa with more than 70% due to PEG. Increasing the molecular weight to this extent is expected to limit diffusion and penetration into tumor. Nevertheless, the most encouraging aspect of the PNA study was accessibility. While 75% of the PNAs on PA were accessible to the radiolabeled cPNA in solution and even when immobilized (on beads), between 35 and 58% of the PNAs on PA were still accessible (Wang et al, 2001, supra). The use of MORFs over PNAs simplified the synthesis of the polymers and provided a larger variety of polymer choices. In a parallel investigation, three polylysines (PLs) completely succinylated to provide the required carboxyl groups and of different initial molecular weights were synthesized and tested (unpublished observations). In addition, one PA similar to that used previously with PNAs was also studied but without the PEG such that the final molecular weight of the PA was now about 0.4 MDa compared to 1.4 MDa for only slightly fewer accessible oligomers per molecule. The biodistribution in normal mice showed the advantage of MORF over PNA since PA shows about half the liver and four times the blood levels when conjugated with MORFs compared to PNAs under nearly identical conditions. The results also suggested that a good choice for in vivo study is the 30 KDa PL which shows higher blood levels and lower liver levels than the other three polymers tested. It was for this reason that the 30 KDa PL polymer was used in the in vivo studies described herein. However, this polymer had only 12-15 cMORFs per molecule. By contrast, the larger 100 KDa PL polymer selected for the in vitro studies had 35 - 40 cMORFs per molecule.

Tumor cell accumulation studies in tissue culture are considerably simpler to perform compared to animal studies and do not suffer from decreasing concentrations due to clearance. Such studies may therefore serve as a useful preliminary test of amplification strategies. Nevertheless, as shown in Table 1, tissue culture studies in common with tumored animal studies are complicated by nonspecific accumulations, necessitating the use of controls. By correcting for nonspecific cell accumulations in tissue culture, an increase in specific accumulation of a factor of 6 compared to pretargeting was achieved.

Animal studies obviously provide a more realistic and more stringent test of amplification targeting compared to tissue culture studies. The first two animal studies were designed to quantitate antibody and polymer in tumor. By using dual radioactivity labels, it was possible to calculate that under the conditions of this investigation, about 25% of the antibody MORFs in tumor were targeted with polymeric cMORF in about 2 days and that 12% of these polymeric cMORF in tumor could be targeted with radiolabeled MORF after 3 h. Because the previous study from this laboratory with

PNAs did not use the antitumor antibody, no comparison with the 25% value is possible. However, the 12% accessibility of polymeric oligomer in tumor is lower than the 35-60% accessibility found earlier (Wang, Y. et al, 2001, supra). There are many possible explanations for this difference, including the use of a different polymer backbone (i.e. PA vs. PL), different size polymers (initial M_r of 80 KDa vs. 30 KDa), different oligomer (i.e. PNA vs. MORF), different linkers (i.e. 19-member polyetheφolyamide vs. 9- member succinylated piperidine), different tumor models (i.e. ACHN vs. LS174T) and different times between administration of the polymer and the radiolabel (i.e. 3 h vs. 18 h). Perhaps the most likely explanation is the use of different mechanism of localization. Whereas the PNA polymer localized in tumor by nonspecific diffusion and therefore was presumably free in the interstitial fluid whereas the MORF polymer was tethered to its antibody target presumably on the tumor cell surface. Some support for this possibility comes from closer agreement in accessibility of both polymers in solution of 70% for the cPNA polymer (Wang, Y. et al, 2001, supra) compared to 50% for the cMORF polymer. Over all animal studies, the only tissue consistently showing statistically significant higher values between study and control animals was tumor. Blood was often significantly higher for the study animals but this is expected as antibody and polymer remaining in circulation (and in tissues) at the time of the MORF-⁹⁹ Tc administration will hybridize and retain radiolabeled MORF. Use of a clearing agent would likely be helpful (Lubic, S.P. et al, J. Nucl. Med. 42: 670-678, 2001).

In conclusion, proof of concept of amplification targeting has been demonstrated in that it was possible to target efficiently both the MORF on MN14 in tumor with the cMORF polymer and the cMORF on polymer in tumor with the radiolabeled MORF despite the barriers to accumulation that exist in vivo and the competition between clearance and targeting. Radioactivity accumulation in tumor was more than doubled (i.e. amplification factor of 2.1) over pretargeting. Furthermore, MORF expression (on antibody) and cMORF expression (on polymer) was rapidly lost in normal organs such as liver, spleen and kidneys but not in tumor, thus improving the target nontarget ratios. These results were achieved despite an antibody conjugated with an average of only 0.2 MORFs and with what is probably a less than ideal polymer with no more than 15 cMORFs per molecule. Furthermore, no clearing agent was used.

Radionuclides useful as therapeutic agents, which substantially decay by beta- particle emission include, include but are not limited to P-32, P-33, Sc-47, Fe-59, Cu-64, Cu-67, Se-75, As-77, Sr-89, Y-90, Mo-99, Rh-105, Pd-109, Ag-111, 1-125, 1-131, Pr-142, Pr-143, Pm-149, Sm-153, Tb-161, Ho-166, Er-169, Lu-177, Re-186, Re-188, Re-189, Ir- 194, Au-198, Au-199, Pb-211, Pb-212, and Bi-213. Maximum decay energies of useful beta-particle-emitting nuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, and most preferably 500-2,500 keV. Radionuclides useful as therapeutic agents, which substantially decay with Auger- emitting particles include, but are not limited to Co-58, Ga-67, Br-80m, Tc-99m, Rh- 103m, Pt-109, In-Ill, Sb-119, 1-125, Ho-161, Os-189m and Ir-192. Maximum decay energy of these radionuclides is preferably less than 1,000 keV, more preferably less than 100 keV, and most preferably less than 70 keV.

Radionuclides useful as therapeutic agents, which substantially decay with generation of alpha-particles include, but are not limited to Dy-152, At-211, Bi-212, Ra- 223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies of useful alpha-particle-emitting radionuclides are preferably 2,000-9,000 keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.

Metals useful, as complexes, as part of a photodynamic therapy procedure include, but are not limited to zinc, aluminum, gallium, lutetium and palladium.

Radionuclides useful in therapies based on neutron capture procedures include, but are not limited to B-10, Gd-157 and U-235. Useful diagnostic agents include, but are not limited to radionuclides, dyes (such as with the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules and enhancing agents (e.g. paramagnetic ions) for magnetic resonance imaging (MRI). U.S. Patent No. 6,331,175 describes MRI technique and the preparation of antibodies conjugated to a MRI enhancing agent and is incoφorated in its entirety by reference. Preferably, the diagnostic agents are selected from the group consisting of radionuclides, enhancing agents for use in magnetic resonance imaging, and fluorescent compounds.

Radionuclides useful as diagnostic agents that are used in positron emission tomography include, but are not limited to F-18, Mn-51, Mn-52m, Fe-52, Co-55, Cu-62, Cu-64, Ga-68, As-72, Br-75, Br-76, Rb-82m, Sr-83, Y-86, Zr-89, Tc-94m, In-110, 1-120, and 1-124. Total decay energies of useful positron-emitting radionuclides are preferably less than 2,000 keV, more preferably under 1,000 keV, and most preferably less than 700 keV.

Metals useful in diagnostic agents utilizing magnetic resonance imaging techniques include, but are not limited to gadolinium, manganese, iron, chromium, copper, cobalt, nickel, dysprosium, rhenium, europium, terbium, holmium and neodymium. Radionuclides useful as diagnostic agents utilizing gamma-ray detection include, but are not limited to Cr-51, Co-57, Co-58, Fe-59, Cu-67, Ga-67, Se-75, Ru-97, Tc-99m, In-Ill, In-114m, 1-123, 1-125, 1-131, Yb-169, Hg-197, and Tl-201. Decay energies of useful gamma-ray emitting radionuclides are preferably 20-2000 keV, more preferably 60-600 keV, and most preferably 100-300 keV.

The embodiments of the invention may be further illustrated through examples which show aspects of the invention in detail. These examples illustrate specific elements of the invention and are not to be construed as limiting the scope thereof.

EXAMPLES Materials and Methods

The 25-mer MORF and cMORF were purchased (Gene Tools, Corvallis, OR) with a 3'-amine via a 9 member succinylated piperidine linker and were identical to that used by us previously (Liu, G. et al, Quart. J. Nucl. Med. 46: 233-43, 2002). The high affinity murine anti-CEA antibody (MN14, IgGi subtype, M_r 160 KDa) was obtained from Immunomedics (Morris Plains, NJ). Poly-lysines, uniformly succinylated with an average M_r ca. 30 KDa and 100 KDa, were purchased from Sigma- Aldrich, St Louis, MO. One-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were from Pierce Company, Rockford, IL.

The bifunctional chelator, N-hydroxysuccinimidyl derivative of acetyl-S-protected mercaptoacetyl-triglycine (NHS-MAG ), was synthesized according to the method of Winnard, P. et al, Nucl. Med. Biol. 24:425-32 (1997). The structure was confirmed by elemental analysis, proton NMR, and mass spectroscopy. To 0.97 ml of a 0.225 M sodium hydroxide was added 50 mg of triglycine (264 μmol) and 10 μl of a freshly- prepared 50 mM disodium ethylenetriaminetetracetic acid (EDTA). This solution was passed through a 0.2 um filter to remove amine-containing particulates. A solution of 90 mg (390 μmol) of S-acetylthioglycolic acid N-hydrosuccinimide ester (SAT A) in 340 μl of dimethy formamide (DMF; dried over molecular sieve) was prepared and was added dropwise to the stirred triglycine solution. After 15 min of stirring at room temperature, the non-aqueous solution was adjusted from an apparent pH of 8.9 to an apparent pH of approximately 2.7 (measured with a glass electrode-pH meter) by the addition of 37.6 μl of 6 M hydrochloric acid. An initial pH of about 8.9 was selected to deprotonate the amine on triglycine (pK 7.9; Fasman, G.D. [ed.] 1976, CRC Handbook of Biochemistry and Molecular Biology, Third Edition, Vol. I, p 321, CRC Press, Boca Raton, FL) but without reaching extreme basic pH values in which the acetyl group on SATA may hydrolyze. The pH was lowered as soon as possible to minimize hydrolysis of the acetyl group. A solution of 60 mg (290 μmol) of dicyclohexylcarbodiimide (DCC) in 3.6 ml of dry DMF was added rapidly to the stirred triglycine/S TA solution (apparent pH of about 5.0). The solution became cloudy within 2 min. as dicyclohexylurea began to precipitate. The reaction was stirred at room temperature in the dark for 2-4 hrs and was then cooled to -20°C for an additional hour to encourage complete precipitation. After centrifugation at 4°C, 2500 g for 15 min., the clear supernatant was removed.

Due to the presence of water in the DMF solution, the NHS-MAG preparation in this form was always used within 24 hours of preparation. For long-term storage, the NHS-MAG₃ water DMF solution was evaporated to near-dryness in 15-30 min. on a rotary flash evaporator (Rotavapur-R, Buchi, Switzerland) and was then lyophilized to dryness within 1 hr on a lyophilizer (Virtis, Gardenier, NY). After drying in this fashion, the NHS-MAG₃ can be stored indefinitely at room temperatures in a desiccator. When using the dry, powdered NHS-MAG₃ for conjugation, an arbitrary value of 50% by weight was assumed for its purity.

Size exclusion (SE) HPLC analysis was performed on a Superdex Peptide column (optimal separation range 1 X 10²-7 X 10³ Da, Amersham Pharmacia Biotech,

Piscataway, NJ) with 0.10 mol/L phosphate buffer, pH 7.0, as eluant at a flow rate of 0.6 mL/min. In-line UV absorbance at 260 nm and radioactivity detectors were used to identify and quantitate peak fractions. Recovery of radioactivity was determined routinely. Reagent grade DTPA cyclic anhydride and carbonyldiimidazole were from

Sigma-Aldrich (St Louis, MO) and were used directly. The P4 resin (Bio-Gel P4 Gel, medium) was purchased from Bio-Rad Laboratories, Hercules, CA. Sephadex GlOO resin was obtained from Pharmacia Biotech, Uppsala, Sweden. The ^mTc-pertechnetate was eluted from a ⁹⁹Mo-^99mTc generator (Bristol-Myers Squibb Medical Imaging , Billerica, MA). The ' ⁿIn was purchased as the chloride (PerkinElmer Life Science Inc., Boston, MA). All other chemicals were reagent grade and were used without further purification. Preparation of MN14-MORF, MN14-DTPA, MORF-MAG3, MORF-DTPA and Radiolabeling

The conjugation of MN14 with MORF was accomplished by reacting amine- derivitized MORF with the native antibody using EDC followed by purification on Sephadex G-100 with 0.05M pH 7.2 phosphate buffer as previously described by Liu et al. (J. Nucl. Med. 43: 384-391, 2002). The antibody conjugated with MORFs were characterized by HPLC for concentration and for the average number of MORFs per antibody molecule (groups per molecule) using a differential UV method at 265 and 280 nm (Liu et al, Quart. J. Nucl. Med. 46: 233-43, 2002). MN14-DTPA and MORF-DTPA were prepared using DTPA cyclic anhydride as described (Liu et al, Nucl. Med. Comm., in press, 2003). The average number of DTPA groups per MN14 was determined by labeling the mixture with ^ιnIn before purification assuming the identical accessibility of ^nιIn to both conjugated and free DTPA. MORF-MAG₃ ^"99mTc was prepared and analyzed as described previously (Liu et al, Quart. J. Nucl. Med. 46: 233-43, 2002). Radiolabeling was achieved by first adding ^99mTc pertechnetate generator eluate to a solution of 5-10 μl of either MORF-MAG₃ or cMORF-MAG₃ (concentrations greater than 0.1 μg/μl), 25 μL 0.25 M ammonium acetate buffer pH 5.2, 10 μl pH 9.2 tartrate solution (50 μg sodium tartrate dihydrate/μl), and 4 μl stannous chloride solution (lμg stannous chloride dihydrate and 1 μg sodium ascorbate/μl in 10 mM HCl), followed by heating in boiling water for 20 min. The product was purified on a P4 column with 0.05 M phosphate buffer pH 7.2 as eluant. MORF-¹¹ 'in was prepared by incubating MORF- DTPA with ^nιIn for 1 h at room temperature and followed by purification as described above for MORF-^99mTc. Both labeled (c)MORF were routinely analyzed by size exclusion HPLC and found to provide essentially identical chromatograms both with UV and radioactivity detection.

Preparation of PL-cMORF and Radiolabelings

Ten milligrams of uniformly succinylated polylysine polymer (PL) with initial M_r 30 KDa (animal studies) or 100 KDa (tissue culture studies) was dissolved in 1.0 ml of the aprotic solvent N-methyl pyrrolidinone (NMP) and to this was added 20.4 mg of 1 ,1'- carbonyldiimidazole and 3.0 μl of diisopropylethylamine (DIEA). The mixture was incubated at room temperature for 2 h. To 500 μl of a 2.0 mg/ml solution of cMORF in NMP, a designed amount of the activated PL mixture and equivalent mole of DIEA were added to reach a 100:1 molar ratio of cMORF to PL. The solution was incubated overnight at room temperature.

An aliquot of the solution prior to purification was analyzed by size exclusion HPLC with UV detection at 265 nm to estimate the average number of cMORF groups bonded to each PL molecule. Since PL does not absorb appreciably at 265 nm, the peak areas of free, non-conjugated cMORF and PL-coupled cMORF were compared. As an alternative method of estimating the average number of groups per molecule, radiolabeled MORF at tracer concentrations was also added to another aliquot of the PL-cMORF solution and the radioactivity of MORF-^99mTc hybridized to free, non-conjugated cMORF was compared to that hybridized to PL-coupled cMORF. The PL-cMORF conjugates were then purified by open column gel filtration chromatography on a 1 cm x 30-cm Sephadex GlOO column using water as eluant. The concentration of PL-cMORFs with respect to cMORFs in the recovered fraction(s) was estimated by UV absorbency using the molar absorbency value of cMORFs provided by the manufacture. PL-cMORFs were radiolabeled by incubation with trace amount of MORF-^99mTc or MORF-^{ι n}In for 30 min at room temperature such that on average only about one cMORF on each polymer was hybridized with MORF. Quality assurance was routinely performed based on size-exclusion HPLC chromatography in which radioactivity recovery was routinely monitored. Using the UV HPLC chromatograms and the slopes of the standard curves (8), the average MORFs per MN14 molecule for the two MN14-MORF preparations used in this investigation was calculated as 0.09 (tissue culture studies) and 0.20 (animal studies). The HPLC radiochromatograms of the MN14-MORFs showed one prominent peak when freshly prepared and purified but upon storage showed evidence of a free MORF peak. Only freshly prepared conjugated antibodies were used in this investigation. The HPLC radiochromatograph of ^nlIn labeled to MN14-DTPA before purification was used to calculate that an average of 0.7 DTPA groups was conjugated to each MN14 antibody. The ^99mTc labeling procedure employed in this investigation always provided a labeled MORF with greater than 90% radiochemical purity after purification as demonstrated by routine HPLC analysis with greater than 90% recovery in all analysis. The radiolabeled cMORFs were routinely shown to be capable of hybridizing to MORF conjugated on antibodies or polymers or immobilized on magnetic beads (Liu, G. et al, J. Nucl. Med. 43: 384-391, 2002; Mang'era, K. et al, Eur. J. Nucl. Med., 28:1682-1689, 2001). By size exclusion HPLC analysis with UV absorbance and by size exclusion HPLC analysis of the conjugated but unpurified PL-cMORF polymers before and after the addition of trace MORF-^99mTc, the 30 KDa PLpolymer was estimated to have been conjugated with an average of 12-15 cMORF groups per molecule while the 100 KDa PL polymer was conjugated with an average of 35-40 groups per molecule. The two methods of measuring groups per molecule showed 80-90% agreement. The final molecular weights were estimated as 130-160 KDa and 400-440 KDa respectively. HPLC analysis of both purified polymers showed a single peak with greater than 90% recovery.

Tissue Culture Studies The LS174T cells were grown in Minimum Essential Medium (MEM, Gibco BRL

Products, Gaithersburg, MD) with 2 mM L-glutamine, 1.5 mg/L sodium bicarbonate, 0.1 mM non-essential amino acids and 1.0 mM sodium pyruvate supplemented with 10% fetal bovine serum (FBS) and 100 mg/ml of penicillin-streptomycin (Gibco BRL Products, Gaithersburg, MD). Cells were maintained as monolayers in a humidified 5% carbon dioxide atmosphere, normally in T75 flasks (Falcon, Becton Dickinson, Lincoln Park, NJ). For uptake studies, the cells were trypsinated in the T75 flasks at 80-90% confluence using 0.05% trypsin/0.02% EDTA and were then suspended in MEM with 10% FBS to the desired density, normally 1 - 2 x 10⁶ cells in 0.1 ml. Cells exposed to antibody were incubated with 20 μg of MN14-cMORF in 0.2 ml Dulbecco's phosphate buffered saline (PBS, Gibco BRL Products, Gaithersburg, MD) and, after 1 h at 4 °C, the cell suspensions were centrifuged at 2000 φm for 4 min and the cells washed three times each with 0.5 ml of PBS (incubations were performed at 4 °C to minimize the possibility of antibody internalization). Cells receiving the polymer were incubated with 10 μg of 100 KDa PL-cMORF for 1 h at 4 °C and centrifuged and washed three times as above. All cells received either 0.60 μg of MORF-^99mTc or 0.27 μg of cMORF-^99mTc. After 30 min incubation at room temperature, the cells were centrifuged and washed three times as above and the cell pellet counted in a Nal(Tl) well counter against a standard of the radiolabeled incubation solution.

Table 1 lists the five groups of the tissue culture study. The cells within the amplification group received the antibody, the 100 KDa PL polymer and radiolabeled MORF. The remaining four groups were the controls; cells within the pretargeting control group received only the antibody and radiolabeled cMORF; cells within the polymer only control group received only the polymer and radiolabeled MORF while cells with the ^99mTc only control groups I and II received only labeled cMORF and

MORF respectively. The last two rows in the table lists the average percentage of added radioactivity and moles of (c)MORF accumulated in the cells.

Table 1 Amplification⁸ Pretargeting" Polymer" ^99mTe (I)^{d 99m}Te (II)^e

(c)MORF accumulation (%)" 2.39 0.31 1.78 0.10 0.08

(c)MORF accumulation (pmoles)^* 1.64 0.10 1.23 0.03 0.05

Average, N = 5 ^a Treated with antibody, polymer and MORF-^99mTc ^bTreated with antibody and ⁹⁹ⁿTc-cMORF ^c Treated with polymer and ^99raTc-MORF ^d' ^e Treated with ^99mTc-cMORF.

Since the results of ^99mTc only control groups I and II show that labeled (c)MORFs accumulate nonspecifically in cells, 0.030 pmoles was subtracted from that of the pretargeting group and 0.052 pmoles from both the polymer-only group and the amplification group. In addition, since the results of the polymer-only group show that the polymer also accumulates nonspecifically in cells, the accumulation in the amplification group was further reduced by 1.18 pmoles. This value of 0.41 pmoles was then compared to the 0.067 pmoles of pretargeting to obtain an amplification factor of 6.

Animal Studies

All animal studies were performed with the approval of the University of Massachusetts Medical School (UMMS) Institutional Animal Care and Use Committee. Nude mice (NIH Swiss, Taconic Farms, Germantown, NY, 30-40 g) were each injected subcutaneously in the left thigh with a 0.1 ml suspension containing 10⁶ LS174T colon tumor cells. Animals were used after 14 days when the tumors were no more than 1.5 cm in any dimension.

To estimate the optimum dosage of the MN14-MORF antibody and 30 KDa PL polymer, tumored animals were administered 60 μg (as an initial guess) of MN14 conjugated with an average of 0.2 MORFs per molecule and 3 μg of MN14-DTPA radiolabeled with about 2.0 μCi of ^{ι n}In 2 days prior to the administration of ""relabeled polymer at three dosages from 24 to 76 μg/animal. On the basis of these results, a 25 μg dosage of MN14-MORF and a 15 μg dosage of polymer were selected for subsequent animal studies. Prior to animal studies of amplification, the influence of antibody and polymer dosage on tumor accumulation was established. Tumored animals were administered 60 μg (as an initial guess) of ^πlIn-labeled MORF antibody 2 days prior to the administration of ^99mTc-labeled 30 KDa PL polymer at three dosages from 24 to 76 μg/animal. Each control animals received 24 μg of polymer but not the antibody. The ^99mTc biodistribution results are presented in Table 2 and show an increasing tumor accumulation of polymer with decreasing dosage. The ^mIn results (not presented) show an accumulation in tumor of antibody at 60 μg that is statistically identical to that shown below for 24 μg of MN14-MORF (Table 2). Therefore, 15μg polymer along with 25μg

MN14-MORF were used in all animals studies of amplification. In this and subsequent tables, statistical significance was established by the Student's T-test based on Microsoft Excel with two-tailed distribution and paired. Statistically significant values (i.e. p <

0.05) are underlined.

Table 2 Biodistribution in tumored mice 18 h post administration of ^99mTc-labeled cMORF- PL30K polymer and 66 h post administration of 60 μg of MORF-antibody

O Orrggaann Study Animals"

76 μg Polymer 52 μg Polymer 24 μg Polymer Polymer Control¹⁵ P values

[1] [2] [l]vs[2]

Liver 34.70(0.95) 34.20(0.18) 36.70(2.87) 32.80(1.78) 0.092

Heart 0.60(0.30) 1.01(0.09) 0.92(0.24) 0.71(0.11) 0.059

Kidney 4.85(2.40) 5.19(0.40) 7.99(1.62) 5.44(1.99) 0.153

Lung 0.38(0.12) 0.43(0.07) 0.56(0.09) 0.39(0.08) 0.099

Spleen 11.90(7.96) 15.20(1.62) 15.90(1.58) 14.90(1.16) 0.416

Muscle 0.16(0.09) 0.19(0.02) 0.17(0.01) 0.18(0.04) 0.834

Tumor 1.19(0.10) 1.31(0.22) 1.92(0.16) 0.75(0.03) 0.001

Blood 0.29(0.08) 0.41(0.04) 0.58(0.07) 0.21(0.03) 0.002 ^a Study animals received 76, 52 or 24 μg of ^99mTc-labeled cMORF-PL30K polymer and 66 h post administration of 60 μg of MORF-antibody. ^b Control animals did not receive the antibody. Average %ID/g, N =4; S.D. in parentheses.

In the first study, nude mice received simultaneously 25 μg of MN14-MORF mixed with 3 μg (2.0 μCi) of MN14-^{ι π}In. About 30 h later, the animals received 15 μg (250 μCi) of the 30 KDa PL-cMORF polymer labeled by hybridization with MORF- ^99mTc occupying an average of only about one of the 12-15 cMORF on the polymer. Control animals did not receive the antibody and/or the polymer. Animals were sacrificed by heart puncture under anesthesia at 18 h post administration of the polymer. Organs and blood were harvested for simultaneously counting of ' ⁿIn and ^99mTc in an automatic gamma counter (Cobra II, Packard Instrument Company, Downers Grove, IL). All counts were corrected for physical decay and for the small contribution of ¹¹ 'in activity in the ^99mTc window. Results are presented as percentage of injected dosage per gram.

The first animal study related to amplification was designed to evaluate the degree to which antibody MORF in tumor can be targeted by polymeric cMORF. Thus nude mice implanted with LS174T tamors received radiolabeled antibody (i.e. MN14 -MORF along with MN14-^ιnIn). Two days later, the animals received the PL-cMORF polymer labeled by hybridization with MORF-^99mTc. Animals were sacrificed at 18 h post administration of the polymer. Control animals did not receive the antibody and received only the labeled polymer 18 h earlier. Table 3 Biodistribution in Tumored Mice 18 h Post-Injection of ^99mTc-Labeled Polymer

Organ Study Animals³ Control Animals^b P value ^{π 99m}Tc [1] ^99mTc [2] [1] vs. [2]

Liver 9.20(0.74) 29.1(1.69) 29.4(1.04) 0.745

Heart 1.36(0.33) 0.66(0.04) 0.77(0.10) 0.091

Kidney 12.2(0.62) 5.28(0.84) 5.36(0.79) 0.888

Lung 2.12(0.10) 0.43(0.09) 0.40(0.06) 0.646

Spleen 3.96(0.63) 12.7(1.97) 11.8(0.98) 0.419

Muscle 0.68(0.02) 0.15(0.04) 0.16(0.04) 0.566

Tumor 11.0(0.57) 1.17(0.17) 0.77(0.10) 0.002

Blood 3.46(0.40) 0.32(0.03) 0.21(0.04) 0.004 ^a Study animals received In labeled antibody 48h earlier before receiving "Tc-polymer. ^b Control animals received only ^99mTc-polymer 18 h earlier. (Average %ID/g, N = 8, (s.d in parentheses)). From the biodistributions results in Table 3, the number of antibody MORFs per gram of tumor may be calculated using the ^ιnIn values while the ^99mTc values in tumor for the study minus the control may be used to calculate the number of polymeric cMORFs per gram of tumor localized specifically due to the antibody. These calculations show that under the conditions of this study, 25% of the antibody MORFs in tumor were targeted with polymeric cMORFs. This calculation assumes that one polymer molecule can bind to no more than one antibody molecule. If this assumption is incorrect and a polymer can cross link antibodies on the tumor, the above value would then be greater than 25%. While the polymer may be capable of cross-linking multiple antibody molecules in tumor, in a separate study, hybridization of the antibody MORFs in tumor by free (i.e. not conjugated to a polymer and therefore monovalent) labeled cMORF was found to be only 50% (10). Since it is unlikely that the polymeric cMORFs target antibody MORFs more effectively than free cMORF, cross-linking may be judged as unlikely and, if it occurs at all, probably results in crosslinking no more than two antibody molecules.

In connection with a previous pretargeting investigation, this laboratory had reported on similar ^99mTc and ^ιπIn dual label studies in LS17 T tumored animals to estimate accessibility as a function of time of MN14-MORF in tumor, liver and spleen to radiolabeled cMORF (10). The results showed that access to MORF in liver and spleen by radiolabeled cMORF was limited to about 6% or less after 24 h post administration of the antibody. Apparently, the MORF on MN14 quickly becomes largely "invisible" once the antibody is localized in these organs. Fortunately this was not the case in tamor where more than 50% was accessible throughout.

In a second study, targeting were performed in a similar manner with 25 μg of the MN14-MORF (now unlabeled) administered to LS174T tumored mice 30 h before the administration of 15 μg of the PL-cMORF polymer (now radiolabeled with trace [3μCi] MORF-^nιIn). Animals received 1.5 μg (about 300 μCi) of MORF-^99mTc at 41 hpost administration of the polymer and were sacrificed 3 h later. The labeled MORF dosage was selected to be reasonably low yet capable of carrying sufficient radioactivity. For controls, the antibody was eliminated in one set of animals while both the antibody and polymer were eliminated in another set.

The second animal study related to amplification was designed to evaluate the degree to which polymeric cMORF in tumor can be targeted by radiolabeled MORF. Thus nude mice implanted with LS174T tumors first received 25 μg of unlabeled antibody and, 30 h later, received 15 μg of the 30 KDa PL-cMORF polymer labeled by hybridization with trace ^ιnIn-MORF. Forty-one hours later, animals received 1.5 μg of MORF-^99mTc and were sacrificed 3 h later (i.e. 74 h post antibody administration, 44 h post administration of ¹¹ 'in-polymer). The labeled MORF dosage was selected to be reasonably low yet capable of carrying sufficient radioactivity for imaging. For controls, the antibody was eliminated in one set of animals (control I) while both the antibody and polymer were eliminated in another set (control II). Animals were imaged before sacrifice. Biodistribution results are presented in Table 4. Table 4 Biodistribution in Tumored Mice 3-h Post-Injection of MORF- 99 *m^m _πTc

Organ Study Group" Control I^b Control IF P values values

'In ⁿTc[l] 99m T. c[2] 99m T- c[3] [l]vs[2]

[l]vs[3]

Liver 38.30(3 .21) 0.77(0.40) 0.32(0.03) 0 .26(0.01) 052 0.048

Heart 0.51(0 •02) 0.07(0.00) 0.07(0.01) 0.06(0.01) 183 0.215

Kidney 2.41(0.67) 13.20(6.32) 15.70(3.91) 12.20(5.47) 675 0.480

Lung 0.27(0.00) 0.16(0.00) 0.27(0.01) 0.14(0.02) 012 0.117

Spleen 8.30(1.61) 0.35(0.04) 0.62(0.19) 0.13(0.01) 110 0.001

Muscle 0.10(0.02) 0.05(0.01) 0.14(0.06) 0.13(0.08) 048 0.059

Tumor 0.52(0.02) 0.50(0.10) 0.26(0.05) 0.14(0.02) 0 043 0.001

Blood 0.05(0.01) 0.12(0.01) 0.12(0.00) 0.08(0.00) 0 510 0.002 ^a Study animals (amplification) received unlabeled antibody 74 h earlier and In labeled polymer 44 h earlier. ^b Control I animals did not receive the antibody and received only the polymer. ^c Control II animals received neither the antibody nor the polymer. Average % ID/g (S.D.), N = 4.

That the ^99mTc values in tamor are significantly higher with compared to without antibody administration (group [1] vs. group [2]) illustrates the specific nature of amplification targeting. The number of polymeric cMORFs per gram of tamor may be calculated from the ^nιIn values while the ^99mTc values in tumor for the study animals receiving the antibody (group [1]) minus that of animals not receiving the antibody (group [2]) may be used to calculate that 12% of the polymeric cMORFs in tamor were targeted with MORFs-^99mTc.

The ^ιπIn and ^99mTc results may also be used to calculate that after 44 h post administration of the PL-cMORF polymer less than 1% of the polymeric cMORFs in liver, spleen and kidneys were targeted by the radiolabeled MORF. Just as MORF on antibody carried into liver and spleen was shown to become "invisible" rapidly to the radiolabeled cMORF (10), these latest results show that polymeric cMORF also rapidly become invisible to radiolabeled MORF. Fortunately, once again this phenomenon of disappearing expression is much less evident in tamor. Even after more than 40 h, 12% of polymeric cMORFs were targeted in tamor. The above calculation may also be used on the blood values to show that about 30% of the polymeric cMORFs were targeted in circulation with the labeled MORF.

That the polymer in normal tissues is rapidly sequestered may also be seen by comparison in the table of MORF-⁹⁹ Tc values with and without the polymer (group [2] vs. group [3]). With the exception of spleen and blood, the differences are not significant. Thus once again, the evidence is that cMORF expression carried by the polymer into these tissues is sequestered within the 41h and becomes effectively invisible to the radiolabeled MORF. In the case of MORF expression on antibody and cMORF expression on polymer, the result is a lowering in normal tissue of the radioactivity levels. Fortunately, in both cases, accessibility in tamor was largely maintained over this period. A third animal study was designed specifically to evaluate amplification. Nude mice first received 25 μg of the unlabeled antibody 30 h prior to administration of 15 μg of the unlabeled 30 KDa PL-cMORF polymer. Animals were sacrificed 3 h post administration of 1.5 μg of MORF-^99raTc at 21 h or 43 h post administration of the polymer. Control animals either did not receive the antibody or received neither the antibody nor the polymer. As an additional control (pretargeting), animals did not received the polymer but received the MN14-MORF antibody followed by cMORF-^99mTc 51 h later. After carefully voiding the bladder with a syringe, the animals were imaged on an Elscint APEX 409 M large view gamma camera (Hackensack, NJ). After imaging, animals were sacrifice and dissected as above. Statistical significance was established by the Stadent's T-test based on Microsoft Excel with two-tailed distribution and paired.

The third animal study was designed specifically to evaluate amplification. Thus nude mice implanted with LS174T tumors first received 25 μg of the unlabeled antibody and, 30 h later, received 15 μg of the unlabeled 30 KDa PL-cMORF polymer. Animals then received 1.5 μg of MORF-^99mTc and were sacrificed 3 h later at either at 21 h (Table 5) or 43 h (Table 6) post administration of the polymer. Control animals either did not receive the antibody (group [2]) or received neither the antibody nor the polymer (group [3]). As an additional control (pretargeting), animals did not received the polymer but received the MN14-MORF antibody followed by cMORF-^99mTc 51 h later (Table 5). Animals were imaged before sacrifice.

Table 5 Biodistribution in Tumored Mice 3 H Post-Administration of (C)Morf-^99mtc

Organ Study Group" Control I^b Control If Control III^d P values P values Amplification Pretargeting Polymer Only Radiolabel

[ϊ] PΪ [3] [l]vs[2] [l]vs[3]

Liver 0.64(0.04) 0.60(0.18) 0.41(0.07) 0.44(0.15) 0.040 0.069

Heart 0.22(0.02) 0.40(0.12) 0.16(0.00) 0.14(0.03) 0.046 0.008

Kidney 13.80(4.30) 22.60(0.66) 15.40(2.50) 11.80(0.90) 0.365 0.878

Lung 0.50(0.07) 0.66(0.15) 0.38(0.01) 0.33(0.10) 0.027 0.078

Spleen 0.76(0.21) 0.45(0.16) 0.69(0.06) 0.23(0.11) 0.224 0.020

Muscle 0.18(0.05) 0.19(0.05) 0.17(0.18) 0.05(0.01) 0.815 0.022

Tumor 0.65(0.13) 2.03(0.23) 0.24(0.04) 0.18(0.07) 0.001 0.005

Blood 0.67(0.06) 1.48(0.42) 0.31(0.01) 0.22(0.05) 0.006 0.004 ^a Study animals (amplification) received unlabeled antibody 51h earlier and the unlabeled polymer 21 h earlier. ^b Control I (pretargeting)animals. received the antibody but not the polymer. ^c Control II animals did not receive the antibody. ^d Control III animals received neither the antibody nor the polymer. Average % ID/g (S.D.), N = 4. Table 6 Biodistribution in Tumored Mice 3-hr Post-Administration of MORF-

^99raTc.

Organ Amplification Polymer Only P values

[1] [2] [1] vs

[2]

Liver 0.55(0.04) 0.38(0.11) 0.064

Heart 0.19(0.01) 0.13(0.02) 0.016

Kidney 15.20(0.38) 10.50(2.49) 0.255

Lung 0.38(0.02) 0.31(0.02) 0.479

SSpplleeeenn 00..8899((00..1166)) 0.88(0.19) 0.723

Muscle 0.08(0.00) 0.14(0.04) 0.038

Tumor 0.60(0.08) 0.18(0.03) 0.003

Blood 0.44(0.03) 0.22(0.03) 0.001 ^a Study animals (amplification) received unlabeled antibody 73 h earlier and the unlabeled polymer 43 h earlier. Control animals did not receive the antibody. Average % ID/g (s.d.), N = 4

An in vivo amplification factor relative to pretargeting in the above study may be estimated by assuming that the in vivo behavior of MORF-^99mTc and cMORF-^99mTc are sufficiently similar. Thus, the absolute accumulation in tamor of MORF-^99mTc in the amplification study group (0.65%-0.24%) x 1.5 μg is 6.15 ng compared to the accumulation of cMORF-^99mTc by pretargeting (2.03%-0.18%) x 0.15 μg or 2.78 ng. The ratio provides an amplification factor over pretargeting of 2.1.

In summary, these results provide evidence that in vivo amplification targeting is feasible and has been achieved. Under the conditions of these studies, about 25% of the MORFs localized in tamor via the MN14 antibody are accessible and can be targeted with the PL cMORF polymer. Furthermore, about 12% of the cMORFs localized in tamor in this way can be successfully targeted with radiolabeled MORF. Proof-of-concept for amplification is apparent in that the tamor accumulation in each study is consistently statistically higher than the controls. Equally important with the absolute accumulation in the target are the target/normal tissue ratios. The calculation of these ratios for each tissue for each of the three amplification studies shows that amplification targeting provided higher tamor/nontarget ratios in 33 of the 35 evaluations compared to the polymer-only and radiolabeled cMORF-only controls.

Animal Imaging.

Figure 1 presents whole body images obtained simultaneously of two nude mice each bearing LS 174T tumors in the right thigh. Both animals received MORF-^99mTc (3 h before imaging) and both received the cMORF-polymer (43 h before imaging). Only the study (amplification) animal on the left received the MN14-MORF antibody (73 h before imaging).

Figure 2 presents whole body images obtained simultaneously of three nude mice each bearing LS 174T tumors in the right thigh under identical conditions as that of figure 1. Thus the animal on the left received only the MORF-^99mTc (3 h before imaging), the animal in the middle received the MORF-^99mTc and the cMORF-polymer (21h before imaging), while the study animal (amplification) on the right received the MORF-⁹⁹ Tc, cMORF-polymer and the MN14-MORF (51 h before imaging). The images show tamor only in the study animals receiving both the antibody and the polymer.

It will be apparent to those skilled in the art that various modifications and variations can be made to the products, compositions, methods, and processes of this invention. Thus, it is intended that the present invention cover such modifications and variations, provided they come within the scope of the appended claims and their equivalents. The disclosure of all publications cited above are expressly incorporated herein by reference in their entireties to the same extent as if each were incorporated by reference individually.

Claims

CLAIMSWhat is claimed is

1. A method of delivering a diagnostic or therapeutic agent to a target site in a subject, comprising:

(a) administering to said subject a first conjugate comprising a targeting moiety and a first moφholino oligomer, wherein said targeting moiety selectively binds to a primary, target-specific binding site of the target site or to a substance produced by or associated with the target site;

(b) administering to said subject a second conjugate comprising a polymer bound to a plurality of copies of a second moφholino oligomer, wherein said second moφholino oligomer is complementary to said first moφholino oligomer; and

(c) administering to said mammal a third conjugate comprising a third moφholino oligomer and a diagnostic or therapeutic agent, wherein said third moφholino oligomer is complementary to said second moφholino oligomer.

2. The method of claim 1, wherein said first and said third moφholino oligomer are the same.

3. The method according to claim 1 , further comprising at a point after step (a) administering to said mammal a clearing agent, and allowing said clearing agent to clear non-localized first conjugate from circulation.

4. The method according to claim 1 wherein said targeting moiety is an antibody or antibody fragment.

5. The method according to claim 4 wherein said antibody or antibody fragment is a human or humanized antibody or antibody fragment.

6. The method according to claim 1 wherein said targeting moiety is selected from the group consisting of proteins, small peptides, polypeptides, enzymes, hormones, steroids, cytokines, neurotransmitters, oligomers, vitamins and receptor binding molecules.

7. The method according to claim 5 wherein said antibody or antibody fragment binds to a target selected from the group consisting of CEA, B-cell antigens, T- cell antigens, plasma cell antigens, HLA-DR lineage antigens, NCA, MUCl, MUC2, MUC3, and MUC4 antigens, EGP-1 antigens, EGP-2 antigens, placental alkaline phosphatase antigen, IL-6, VEGF, tenascin, CD33, CD74, PSMA, PSA, PAP, antigens associated with autoimmune diseases, infection inflammation, and infectious diseases, antigens associated with a B- or T-cell lymphoma, or with B- or T-cells associated with autoimmune diseases, CD19, CD22, CD40, CD74, HLA-DR, IL-15, and HLA-DR expressed by malignant diseases, CD15, CD33, , CD66a, CD66b, and CD66e.

8. The method according to claim 1 wherein said polymer is selected from the group consisting of poly-lysine (PL), polyethyvinylether maleic acid (PA) dextran, dendrimers and N-(2-hydroxypropyl)methacrylamide (HPMA).

9. The method according to claim 1 wherein each of said moφholino oligomers has a length of at least about 6 bases to about 100 bases.

10. The method of claim 9 wherein said first oligomer has a length of between about 15 and about 25 bases.

11. The method of claim 9 wherein said second oligomer has a length of between about 15 and about 25 bases.

12. The method of claim 9 wherein said third oligomer has a length of between about 15 and about 25 bases.

13. The method according to claim 3 wherein said clearing agent comprises an anti-idiotypic antibody or antigen-binding antibody fragment.

14. The method according to claim 1 wherein said third conjugate comprises a therapeutic agent selected from the group consisting of antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, chelators, boron compounds, photoactive agents or dyes and radionuclides.

15. The method according to claim 1 wherein said third conjugate comprises a diagnostic agent selected from the group consisting of radionuclides, dyes, contrast agents, fluorescent compounds or molecules and enhancing agents useful for magnetic resonance imaging (MRI).

16. The method according to claim 1 wherein said second conjugate comprises at least 5, at least 10, at least 25 or at least 50 copies of said second moφholino oligomer.

17. The method according to claim 1 wherein said second conjugate comprises between about 25 and 50 copies of said second moφholino oligomer.

18. A kit for targeting of a diagnostic or therapeutic agent in a subject comprising:

(a) a first conjugate comprising a targeting moiety and a first moφholino oligomer, wherein said targeting moiety selectively binds to a primary, target-specific binding site of the target site or to a substance produced by or associated with the target site;

(b) a second conjugate comprising a polymer bound to a plurality of copies of a second moφholino oligomer wherein said second moφholino oligomer is complementary to said first moφholino oligomer; and (c) a third conjugate comprising a third moφholino oligomer and a radiolabel, wherein said third moφholino oligomer is complementary to said second moφholino oligomer.

19. The kit according to claim 18, wherein said first and said third moφholino oligomer are the same.

20. The kit according to claim 18, further comprising a clearing agent.

21. The kit according to claim 18 wherein said targeting moiety is an antibody or antibody fragment.

22. The kit according to claim 21 wherein said antibody or antibody fragment is a human or humanized antibody or antibody fragment.

23. The kit according to claim 18 wherein said targeting moiety is selected from the group consisting of proteins, small peptides, polypeptides, enzymes, hormones, steroids, cytokines, neurotransmitters, oligomers, vitamins and receptor binding molecules.

24. The kit according to claim 22 wherein said antibody or antibody fragment binds to human carcinoembryonic antigen.

25. The kit according to claim 18 wherein said polymer is selected from the group consisting of poly-lysine (PL), polyethyvinylether maleic acid (PA) dextran, dendrimers and N-(2-hydroxypropyl)methacrylamide (HPMA).

26. The kit according to claim 18 wherein each of said moφholino oligomers has a length of at least about 6 bases to about 100 bases.

27. The kit according to claim 26 wherein said first oligomer has a length of between about 15 and about 25 bases.

28. The kit according to claim 26 wherein said second oligomer has a length of between about 15 and about 25 bases.

29. The kit according to claim 26 wherein said third oligomer has a length of between about 15 and about 25 bases.

30. The kit according to claim 20 wherein said clearing agent comprises an anti-idiotypic antibody or antigen-binding antibody fragment.

31. The kit according to claim 18 wherein said third conjugate comprises a therapeutic agent selected from the group consisting of antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, chelators, boron compounds, photoactive agents or dyes and radionuclides.

32. The kit according to claim 18 wherein said third conjugate comprises a diagnostic agent selected from the group consisting of radionuclides, dyes, contrast agents, fluorescent compounds or molecules and enhancing agents useful for magnetic resonance imaging (MRI).

33. The kit according to claim 18 wherein said second conjugate comprises at least 5, at least 10, at least 25 or at least 50 copies of said second moφholino oligomer.

34. The kit according to claim 18 wherein said second conjugate comprises between about 25 and 50 copies of said second moφholino oligomer.