JP2006523706A - Morpholino imaging and treatment with amplification targeting - Google Patents

Abstract

The present invention provides kits and methods for targeting a diagnostic or therapeutic agent to a target site in a mammal in a pathological state. The kit comprises (A) a primary target-specific binding site of a target site, or a targeting moiety that selectively binds to a substance generated by the target site or a substance related to the target site, contained in a separate container; A first complex comprising a morpholino oligomer, (B) optionally comprising a clearing agent, (C) a plurality of copies of a complementary morpholino oligomer bound to a polymer and a diagnostic or therapeutic agent. A second complex, and (D) a third complex comprising a morpholino oligomer and a radioisotope. The method comprises administering (A), optionally (B), (C) and (D) to the mammal.

Description

Field of Invention

  The present invention relates to a kit for targeting a diagnostic or therapeutic agent to a target site in a mammal and to diagnose or treat a pathological condition using multiple copies of a single-stranded morpholino oligomer complementary pair conjugated to a polymer. Concerning the method.

Background of the Invention

  The goal of drug target research is to deliver therapeutic agents directly to the targeted tumor sites, allowing more effective dosing at these sites, thereby reducing non-tumor related side effects and It is to improve effectiveness. Another objective is to achieve complete accumulation of therapeutic agent at the target site, thereby increasing the target / non-target ratio.

  Different targeting vectors comprising a diagnostic or therapeutic agent linked to a targeting moiety for selective localization have been known for some time. Examples of targeting vectors include conjugates of targeting moieties such as antibodies or antibody fragments, cell-specific or tissue-specific peptides, hormones and other receptor binding molecules with diagnostic or therapeutic agents. For example, antibodies against different determinants associated with diseased and normal cells and also with pathogenic microorganisms have been used to detect and treat disease states or lesions. In these methods, for example, Hansen et al., U.S. Pat. No. 3,927,193 and Goldenberg, U.S. Pat. No. 4, which are hereby incorporated by reference in their entirety. 331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,460,459, As described in US Pat. No. 4,460,561, US Pat. No. 4,624,846 and US Pat. No. 4,818,709, the targeting antibody is directly conjugated with a suitable detection or therapeutic agent.

  One of the problems encountered in the direct targeting method is that only a small portion of the complex actually binds to the target site, with the majority of the complex remaining in circulation, and the function of the targeted complex. Is somehow suppressed. Other problems include high background and low resolution when administering a diagnostic agent, and myelotoxicity or systemic side effects when the therapeutic agent is combined with a long-circulating targeting moiety.

  The prelettering method was developed with the aim of increasing the target: background ratio of detection or therapeutic agents. Examples of pre-getting and biotin / avidin approaches are, for example, Goodwin et al., US Pat. No. 4,863,713, all of which are incorporated herein by reference in their entirety. Goodwin et al., J. Nucl. Med. 29: 226 (1988); Hnatowich et al., J. Nucl. Med. 28: 1294 (1987); 0ehr 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. Nucl. 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).

  In the pretargeting method, a first targeted species comprising a first targeting moiety that binds to a target site and a binding site available for binding of a second targeted species that is subsequently administered ( (Not bound to a diagnostic or therapeutic agent) is directed to the target site in vivo. Once the first targeted species has fully accumulated, a second targeting comprising a diagnostic or therapeutic agent and a second targeting moiety that recognizes an available binding site for the first targeted species Administer seeds.

  An example of a pretargeting methodology is the use of the biotin- (strept) avidin system to administer cytotoxic radioactive antibodies to a tumor. In the first step, a monoclonal antibody directed against a tumor-associated antigen is conjugated with avidin (or biotin) and administered to a patient having a tumor recognized by the antibody. In the second step, the therapeutic agent is incorporated into the antibody-avidin (or -biotin) complex pretargeted to the tumor via the bound biotin (or avidin).

  However, problems have arisen in the application of biotin-avidin or (strept) avidin systems during pretargeting. First of all, unless properly constructed, radiolabeled biotin is subjected to plasma biotinidase degradation. Further, when bound to antibodies, strept / avidin and avidin can produce anti-strept / avidin antibodies in patients. Finally, the potential effect of endogenous biotin during in vivo pretargeting can cause loss of biotin binding expression due to biotin saturation. This occurs, for example, when a certain strept / avidin-conjugated antibody localized in a nude mouse xenograft becomes saturated with biotin. Rusckowski et al., Cancer 80: 2699-705 (1997). The three-step strategy of administering a biotinylated monoclonal antibody, avidin, followed by radiolabeled biotin alleviates these deficiencies to some extent, but this approach is thought to be laborious as an imaging method. This technique does not solve the problem of immunogenicity.

  Another example of a recognized pretargeting method is the use of a bispecific antibody-hapten recognition system that uses a radiolabeled hapten and a bispecific antibody instead 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, SP et al., J. Nucl. Med. 42: 670-678 (2001); Gestin, JF et al., J. Nucl. Med. 42 : 146-153 (2001). Haptens are often coordination complexes, for example indium-DTPA. Bispecific antibodies are products in which two antibodies or antibody fragments bind to tumor markers such as independent determinants, haptens and carcinoembryonic antigens. This approach is hampered by the low affinity in addition to the need to generate bispecific antibodies. The affinity of an antibody for its hapten, especially the monovalent one, is orders of magnitude lower than the affinity of (strept) avidin for biotin. Mathematical modeling has shown that the 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 biotin-avidin and bispecific antibody-hapten systems for lettergetting, single-stranded oligomers such as peptide nucleic acids (PNA) have been used. Single-stranded oligomers bind specifically to their complementary single-stranded oligomers by in vivo hybridization. A single-stranded PNA associated with a targeting moiety is first administered to the patient, followed by a 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 may be added to the two-step method by administration of the clearing agent. The purpose of the clearing agent is to remove the circulating first complex that is not bound at the target site. This is disclosed by Griffiths et al. In US Pat. No. 5,958,408, which is incorporated herein by reference.

To improve nuclease stability and reduce protein binding affinity, chemical modifications to the backbone of these single-stranded oligomers that normally bind to radioactive nucleotides are required. ^The effects on 18-mer phosphorothioate DNA with three different chemical modifications that allow labeling with ^99m Tc have been compared in vitro and in vivo in mice. Zhang, YM et al., Eur. J. Nucl. Med. 27: 1700-1707 (2000). Although it has been found that the binding rate constant in hybridization is independent of the labeling method, both intracellular accumulation in culture and the pharmacokinetic behavior of radiolabel in normal mice are stronger by the labeling method. Affected.

  These in vivo properties of oligomers may be affected by changes in their chain length and / or base sequence. It is conceivable that understanding the effects of chain length and base sequence can effectively modify the pharmacokinetics of oligomers. Despite this possibility (as in the case of chemical modification), these further effects have so far been largely unexplored. This may be due in part to the constraints imposed on these parameters by application. For example, antisense chemotherapy usually appears to be efficacious by hybridizing a short single-stranded oligomer with a base sequence complementary to that of its mRNA target. Hnatowich, D. J., J. Nucl. Med. 40: 693-703 (1999). Thus, the base sequence and, to some extent, the chain length are also limited to those that provide the desired hybridization. Nevertheless, there are base combinations that have attracted attention. An example is the G quartet present in phosphodiester or phosphorothioate DNA (ie, four guanine bases in a row). Shafter, R.H. et al., Biopoly (Nucleic Acid Sci.) 56: 209-227 (2001). At least in the case of these chemical forms of DNA, guanine base stacking provides oligonucleotides with a specific three-dimensional quadruple structure. It is clear that this structure is involved in various sequence-specific effects that are important for various in vivo processes. Ibid. Another example is the CpG motif, which is immediately followed by a guanine immediately following a cytosine base, which has been shown to have immunostimulatory activity. Zhao, Q. et al., Antisense Nucleic Acid Drug Dev. 7: 495-502 (1997). If these sequences have any effect on pharmacokinetics, the effects are not yet established.

  Various other published reports summarize the in vitro effects of oligomer chain length and sequence. It was found that the cytotoxic effect in one cell line of phosphodiester DNA completely composed of guanine base and thymidine base requires a chain length of at least 20 bases. In addition, its cytotoxic effect disappeared by introducing adenine or cytosine into either end. Morassutti, C. et al., Nucleosides & Nucleotides 18: 1711-1716 (1999). The efficiency with which PNA initiates transcription and gene expression in cells has been found to be optimal for chain lengths of 16-18 bases. Wang, G. et al., J. Mol. Biol. 313: 933-940 (2001). Rat liver homogenates were used ex vivo to investigate the metabolism of a series of phosphorothioate DNAs with different chain lengths and base sequences. Crooke, R. M. et al., J. Pharm. Exp. Therapeutics 292: 140-149 (2000). Initially, degradation of all oligomers with 3 'exonuclease increased the chain length and increased metabolic rate. Since oligonucleotides rich in pyrimidines are more unstable, the rate and extent of nuclease metabolism has also been related to the base sequence. This particular survey was unusual in that it also studied the effects of stereoisomerism. It has been found that the metabolic rate of some diastereoisomers is faster than others, and that mixtures are digested at rates in between. Finally, recent reports describe the effect of base sequence on the reactivity of phosphodiester bonds in RNA. Kaukinen, U. et al., Nucl. Acids Res. 30: 468-474 (2002).

  We have previously disclosed the use of oligomers such as phosphorodiamidate morpholino (MORF) tumor imaging and therapy. Such use, in turn, claims the priority of US Provisional Application No. 60 / 279,809, filed March 30, 2001, and 60 / 341,794, filed December 21, 2001, April 4, 2002. Disclosed in pending continuation-in-part of pending US patent application Ser. No. 10 / 112,094. The entire disclosure, including the specifications, claims and drawings of these applications, is hereby incorporated by reference.

  Natural phosphodiester DNA differs from phosphorothioates in that non-binding oxygen is replaced with a sulfur atom. In PNA, the phosphate backbone of the DNA is replaced by a (2-aminoethyl) glycine polypeptide bond to which a nitrogenous base is attached via a methylenecarbonyl group, while the phosphodiester backbone of the MORF is phosphodiester. Substituted with a rosamidate group, the ribose sugar is replaced by a morpholino ring. Like DNA, MORF and PNA are generally commercially available, but unlike DNA, they are both uncharged and stable to nucleases (unlike phosphodiester DNA) ( It is achiral (unlike phosphodithioate).

  Amplification is a multi-stage pre-targeting process that has the potential to greatly improve targeting through the intermediate use of polymers coupled with multiple copies of the oligomer. Therefore, there is a need for improved kits or methods that significantly increase the accumulation of radioactivity in tumors while at the same time increasing the tumor / normal tissue ratio. To do this, it is first necessary to prepare a MORF polymer that is finally attached to the antibody. In such a method, we only want to avoid significant denaturation of the antibody.

  One alternative to amplification targeting is pre-targeting using either (strept) avidin / biotin, bispecific antibodies or oligomer pairs. Recently, in particular, 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), promising results have been reported. However, since multivalent polymers are not involved in pretargeting, no amplification can occur (other than up to 4 times for specific streptavidin / biotin protocols) (Kassis, AI et al., J. Nucl. Med. 37 : 343-352, 1996).

  Boron-like low molecular weight species (Novick, S. et al., Nucl. Met. Biol. 29: 159-67, 2002) or metal chelators (Torchilin, VP) for various applications et al, Hybridoma 6: 229-40, 1987), many reports have been made on anti-tumor antibodies bound directly to multiple copies. As an obvious disadvantage, there is a risk that the antibody will denature as a result of the binding, especially if it is intended to bind multiple copies of a relatively high molecular weight MORF. For example, to retain 50 MORFs per molecule, the molecular weight of an IgG antibody should increase from about 150 KDa to a minimum of 600 KDa. It may not be possible to retain immune activity under these conditions. In addition, amplification targeting is effective even with tissue specific agents other than antibodies such as anti-tumor and anti-tissue peptides that cannot accept binding to multiple MORFs due to their low molecular weight.

Neither the use of phosphodiester nor the use of phosphoromonothioate is ideal for amplification targeting and other radiopharmaceutical applications. The former is too unstable for nucleases in vivo, and the latter shows too high affinity for serum and tissue proteins (Hnatowich, DJ et al., J. Pharmacol. Exp. Ther. 276 : 326-334, 1996). We have subsequently shown that PNA is stable and has the lowest protein binding affinity (Mardirossian, G. et al., J. Nucl. Med. 38: 907-913, 1997). Accordingly, PNA was used in our first amplification targeting study (Wang, Y. et al., Bioconjug. Chem. 12: 807-816, 2001). However, problems were encountered due to the low water solubility of PNAs with selected base sequences. Due to problems with binding, anti-tumor antibodies were not used in PNA studies (Wang, Y. et al., 2001, supra). In some cases, due to the higher water solubility, amine derivatized MORF was coupled with both anti-tumor antibody MN14 and chelator MAG ₃ and was successfully radiolabeled with ^99m Tc. Radiolabels and MORF have shown little affinity for proteins and have been found to be “stable” in vitro and in vivo, which, like labeled PNA, is capable of rapid hybridization. (Mang'era, K. et al., Eur. J. Nucl. Med. 28: 1682-1689, 2001).

  Amplification targeting shares some similarities with pretargeting (described herein) using both MORF-antibodies and radiolabeled MORF (cMORF in the case of pretargeting), but intermediates the polymer It is different in use. Amplification targeting is clearly more complex than pretargeting, but there is the potential for signal amplification.

  One important aspect of amplification targeting is in situ accessibility. In order to achieve high radioactivity levels in the tumor, it is necessary to make the antibody MORF in the tumor accessible to the multimeric cMORF and to make the polymer cMORF in the tumor accessible to the radiolabeled MORF. Another important aspect arises from the accumulation of multimeric cMORF in the liver, spleen, kidney and other normal organs. In order to reduce background radiation levels in these normal organs, the expression of multimeric cMORF should be rapidly inaccessible to radiolabeled MORF.

  Despite several advantages over the strept / avidin-biotin and bispecific antibody-hapten systems, there are some limitations when using these oligomers in pretargeting. These limitations include poor specificity, possible insolubility in aqueous solutions, and high cost.

  Used for in vivo targeting to deliver therapeutic or diagnostic agents to target sites in mammals, providing more specific, affordable and cheap uptake to targets and low uptake into normal tissues, There remains a need for improved kits and methods. There is also a need to provide improved pretargeting kits and methods in which excess isotope-labeled oligomers that are not bound to cancer cells are rapidly cleared from the body, particularly from the kidney.

Summary of the Invention

  Accordingly, one object of the present invention is kits and methods useful for the amplification targeting of diagnostic or therapeutic agents in mammals, which can be prepared from relatively inexpensive starting materials, Lesser uptake and retention in the kidney, lower toxicity, better specificity, stability, predictable targeting and / or desired antigen-antibody than conventional kits and methods and other known kits and methods It is to provide a highly effective kit and method. Another object of the present invention is to directly bind multiple copies of MORF to the antibody, thereby avoiding a second administration to the mammal.

  Another objective is a method useful for tumor localization / imaging by amplification targeting using multivalent morpholino oligomers (MORF, defined below) instead of strept / avidin-biotin, peptide nucleic acids and other oligomers. So that the radiolabeled targeting moiety is highly accumulated at the main target-specific binding sites within the target, thereby greatly increasing the accumulation of radioactivity in the tumor, while at the same time increasing the tumor / normal tissue ratio. It is to provide an alternative way to enhance.

These and other objects are, according to one embodiment of the invention, a kit for targeting a diagnostic or therapeutic agent in a mammal, comprising:
(A) a primary target-specific binding site of a target site, or a targeting moiety that selectively binds to a substance generated by the target site or a substance related to the target site, and a morpholino oligomer Complex,
(B) A clearing agent, if desired,
(C) a second complex comprising a plurality of copies of a complementary morpholino oligomer linked to a polymer and a diagnostic or therapeutic agent; and (D) a third complex comprising a morpholino oligomer and a radiolabel. Realized by providing a kit comprising the body.

  The targeting moiety of step (a) preferably comprises an antibody, in particular 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), polyethyl vinyl ether maleic acid (PA) dextran, dendrimer and N- (2-hydroxypropyl) methacrylamide (HPMA). Can be mentioned.

  According to another aspect of the present invention, there is provided a kit 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 oligomer and its complementary morpholino oligomer may be 15-mer, 18-mer or 25-mer. The targeting moiety binds to a 15mer, 18mer or 25mer morpholino oligomer.

  In a preferred embodiment, the clearing agent is an anti-idiotype 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, chelating agents, boron compounds, photoactive substances or dyes and radionuclides. The

  In yet another preferred embodiment, the diagnostic agent is selected from the group consisting of radionuclides, dyes, contrast agents, fluorescent compounds or molecules and enhancers useful for magnetic resonance imaging (MRI).

According to the present invention, a targeting method for delivering a diagnostic or therapeutic agent to a target site in a mammal, comprising:
(A) the mammal includes a targeting moiety that selectively binds to a main target-specific binding site of the target site, or a substance generated by the target site or a substance related to the target site, and a morpholino oligomer; Administering a first complex comprising:
(B) optionally administering a clearing agent to the mammal and eliminating from the circulation a first complex not localized by the clearing agent; and (c) complementary to the mammal Administering a second complex comprising a polymer coupled to multiple copies of a morpholino oligomer and a diagnostic or therapeutic agent, wherein the complementary morpholino oligomer-polymer complex is the first complex. Targeting a diagnostic or therapeutic agent to the target site by binding to its morpholino oligomer complement of), and
(D) There is provided a method comprising administering to said mammal a third complex 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 the appended claims.

Detailed description of the invention

  Unless otherwise noted, terms in the singular refer to one or more.

The present invention provides kits and methods useful for targeting a diagnostic or therapeutic agent in vivo in a mammal (preferably a human) comprising:
(A) a primary target-specific binding site of a target site, or a targeting moiety that selectively binds to a substance generated by the target site or a substance related to the target site, and a morpholino oligomer Complex,
(B) A clearing agent, if desired,
(C) a second complex comprising a plurality of copies of a complementary morpholino oligomer linked to a polymer and a diagnostic or therapeutic agent; and (D) a third complex comprising a morpholino oligomer and a radioactive label. Kits and methods comprising the complexes are provided.

  The targeting moiety can be, for example, an antibody or an antigen-binding antibody fragment. A monoclonal antibody (Mab) is preferred because of its high specificity. These are considered conventional procedures consisting of immunization of mammals with immunogenic antigen preparations, fusion of immunolymph or spleen cells with immortal myeloma cell lines and isolation of specific hybridoma clones Easily prepared. More novel monoclonal antibody preparation methods such as interspecies fusion and manipulation of hypervariable regions by genetic engineering are also contemplated, primarily because the antigen specificity of antibodies affects their utility in the present invention. Of course, state-of-the-art techniques for producing monoclonals, such as human monoclonals, interspecies monoclonals, chimeric (eg, human / mouse) monoclonals, recombinant antibodies, etc. can also be used.

Antibody fragments useful in the present 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 . Similarly, any subfragment of an immunoglobulin that possesses a hypervariable region that binds to an antigen and has a size that is smaller than or equal to the Fab ′ fragment is useful. This includes a genetically engineered antibody that incorporates an antigen binding site, whether single-chained or multi-chained, or otherwise functions as a targeting vehicle in vivo substantially similar to a natural immunoglobulin fragment. Protein or recombinant antibodies and proteins are included. Such single chain binding molecules are disclosed in US Pat. No. 4,946,778, which is hereby incorporated by reference.

Fab ′ fragments can conveniently be made by F (ab ′) ₂ fragment cleavage by pepsin digestion of intact immunoglobulins under reducing conditions or by careful papain digestion of complete immunoglobulins. ') It may be produced by reductive cleavage of _two fragments. Further, the fragment may be prepared by genetic engineering.

  In addition, antibodies having specific immunoreactivity for marker substances produced by or associated with at least 60% of cancer cells and cross-reactivity for less than 35% of other antigens or non-target substances. Also preferred is an antibody having it. Particularly preferred are monoclonal antibodies that are specifically targeted to the tumor site by binding to an antigen produced by or associated with the tumor.

  Antibodies against tumor antigens are known. For example, antibodies and antibody fragments that are produced by a tumor or that specifically bind to a marker associated with a tumor, among others, are incorporated herein by reference in their entirety. Hansen et al., US Pat. No. 3,927,193 and Goldenberg US Pat. Nos. 4,331,647, 4,348,376, 4,361,544 and 4,468. No. 4,457, No. 4,444,744, No. 4,818,709, and No. 4,624,846. In particular, it is advantageous to use antibodies against antigens such as gastrointestinal tumors, lung tumors, breast tumors, prostate tumors, ovarian tumors, testicular tumors, brain or lymph glandular tumors, sarcomas or melanomas.

  Other targets of the targeting moiety of the present invention include, but are not limited to, B cell antigen, T cell antigen, plasma cell antigen, HLA-DR lineage antigen, CEA, NCA, MUC1, MUC2, MUC3 and MUC4 antigen , EGP-1 antigen, EGP-2 antigen, placental alkaline phosphatase antigen, IL-6, VEGF, tenascin, CD33, CD74, PSMA, PSA, PAP, autoimmune diseases, infection / inflammation and antigens associated with infection It is done. In some cases, the target is associated with a B cell or T cell lymphoma, or a target antigen associated with a B cell or T cell associated with an autoimmune disease. Group consisting of CD19, CD22, CD40, CD74, CEA, NCA, MUC1, MUC2, MUC3, MUC4, HLA-DR, EGP-1, EGP-2, IL-15 and HLA-DR expressed by malignant diseases Or an antigen selected from: Targets are, for example, EGP-2, EGP-1, CD22, CEA or MUC1 for specific malignancies.

  The target may be expressed by bacteria, viruses, fungi, parasites or other microorganisms.

  The target may also be expressed by host cells such as activated granulocytes that increase at the site of infection (eg, CD15, CD33, CD66a, CD66b, CD66c (NCA) and CD66e etc.). .

  Antibodies and antigen-binding antibody fragments useful in the methods of the invention may be conjugated to members of a binding pair by a variety of chemical coupling methods known in the art. Many of these methods are disclosed in the above US patents and patent applications, the entire disclosures of which are incorporated herein by reference. See also Childs et al., J. Nuc. Med. 26: 293 (1985).

  One monoclonal antibody useful in the present invention is MN-14, a second generation CEA-antibody that has a 10-fold higher affinity for CEA than the first generation NP-4. Hansen et al., Cancer 71: 3478-85, (1993). MN-14 is taken up slowly, making it suitable for targeting approaches. This is chimerized and humanized. Leung et al., US Pat. No. 5,874,540.

  Other antibodies or antibody fragments suitable in the present invention are, for example, RS11, 17-1A, RS7, LL1, LL2, MN-3, MN-14 or PAM4 or humanized forms thereof when targeting a malignant disease Or it may be derived from RS11, 17-1A, RS7, LL1, LL2, MN-3, MN-14 or PAM4 or a humanized form thereof. A preferred granulocyte antibody is MN3 used in LeukoScan®.

  Other targeting moieties useful in the present invention are proteins, small peptides, polypeptides, enzymes, hormones, steroids, cytokines, that are preferentially bound to a marker substance that is generated by or associated with the target site. It may be a non-antibody species selected from the group consisting of neurotransmitters, oligomers, vitamins and receptor binding molecules.

  Morpholino oligomers (herein “morpholino” or “MORF”) bind to and inactivate a selected RNA sequence. These oligomers are composed of four different morpholino subunits, each containing one of four gene bases (A, G, C, T or U) linked to a six-membered morpholine ring. Has been. In the case of 15-25 mer, the nonionic phosphorodiamidate intersubunit linkages connect these subunits in a specific order, resulting in morpholino oligomers. They can provide superior antisense properties over DNA, RNA, and their analogs with five-membered ribose or deoxyribose backbone moieties joined by ionic bonds. Summerton's work on morpholinos is disclosed in US Pat. Nos. 5,142,047 and 5,185,444, the disclosures of which are hereby incorporated by reference. . Morpholino is commercially available from Gene Tools, LLC, Corvallis, Oregon.

  Because they are easily delivered to the target, morpholinos are an effective tool for genetic research and drug target demonstration programs. They are completely resistant to nucleases. A more rigid MORF backbone can provide better accessibility when forming a duplex compared to a peptide backbone or a common sugar backbone. Compared to PNA, morpholinos are less expensive, have higher aqueous solubility, and therefore better predictable targeting and higher RNA binding affinity effects.

  In the present invention, a morpholino oligomer (herein MORF) conjugated with a targeting antibody is hybridized in vivo with a complementary MORF (herein cMORF) conjugated with a diagnostic or therapeutic agent. In a preferred embodiment, the length of MORF and its complementary morpholino (cMORF) is from 6 bases to about 100 bases, eg, MORF15 and cMORF15 (15 mer), MORF18 and cMORF18 (18 mer) or MORF25 and cMORF25 (25 Ma).

  MORF used in the present invention includes 15-mer (5 ′ equivalent TGT-ACG-TCA-CAA-CTA-linker-amine (herein, MORF15) and TAG-TTG-TGA-CGT-ACA-linker-amine ( Complementary MORF15 or cMORF15)), 18mer (5 'equivalent CGG-TGT-ACG-TCA-CAA-CTA-CTA-linker-amine (herein MORF18) and TAG-TTG-TGA-CGT-ACA -CCC-linker-amine (here complementary MORF18 or cMORF18)) and 25mer (5 'equivalent T-GGT-GGT-GGG-TGT-ACG-TCA-CAA-CTA-linker-amine (herein) MORF25) and TAG-TTG-TGA-CGT-ACA-CC -ACC-ACC-A- linker - amine (complementary MORF25 or cMORF25 herein)) may be mentioned.

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

  In accordance with the present invention, clearing agents known in the art may be used. For example, biotin can be used as a clearing agent when the first complex comprises avidin or streptavidin. When the first complex contains biotin, avidin or streptavidin can be used as a clearing agent.

  In a preferred embodiment, the clearing agent is an antibody that binds to the binding site of a targeting moiety that is an antibody, antigen-binding antibody fragment or non-antibody targeting moiety. In a more preferred method, the clearing agent is a monoclonal antibody that is an anti-idiotype to the complex monoclonal antibody used in the first step, as described in US application Ser. No. 08 / 486,166. In another preferred embodiment, the clearing agent is replaced with multiple residues of carbohydrates such as galactose, so that the clearing agent is rapidly cleared from the circulation by the asialoglycoprotein receptor in the liver. I try to do it.

  The physiological solution of the targeted species is advantageously placed in a sterile vial at a metered amount, eg, a unit dose of about 1.0-500 mg targeted species / vial. The vial is capped, sealed, and stored cold, or lyophilized, capped, sealed, and stored.

  Variations and modifications of these formulations will be readily apparent to those skilled in the art depending on the requirements of each mammal or treatment plan, as well as variations in the form supplying the radioisotope or the available form of the radioisotope. .

  Administration routes include intravenous, intraarterial, intrapleural cavity, intraperitoneal, intrathecal, subcutaneous or perfusion administration.

  The disclosures should be cited for methods useful for in-vivo detection or treatment of tumors or cardiovascular lesions (clots, emboli, infarcts, etc.), infections, inflammatory diseases and autoimmune diseases. U.S. Pat. Nos. 4,782,840, 4,932,412 and 5,716,595, which are hereby incorporated by reference. The method of the present invention may be used to augment the methods disclosed in these references. In addition, the present invention may be implemented using an intraoperative probe, an endoscope, and a laparoscope in combination, or may be performed by a method for imaging a normal organ. The method of the present invention may be used in other ways that will be apparent to those skilled in the art.

  Examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, chelating agents, boron compounds, photoactive substances or dyes and radionuclides.

In a further preferred embodiment, cMORF is coupled to a bifunctional chelator, after which the chelator is radiolabeled with an isotope. For proteins, polypeptides, or oligonucleotides that cannot withstand harsh conditions, the chelator is first radiolabeled (pre-binding labeling) prior to conjugation with them. Examples of chelating agents include hydrazinonicotinamide (HYNIC), diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraaza-cyclododecane N, N ′, N ″, N ′ ″-4 Examples include acetic acid (DOTA) and mercaptoacetylglycylglycylsylglycine (MAG ₃ ). A preferred bifunctional chelating agent for use in the present invention is the N-hydroxysuccinimidyl derivative of acetyl-S-protected mercaptoacetyltriglycine (NHS-MAG ₃ ). NHS-MAG ₃ is synthesized according to the method of Winnard, P. et al., Nucl. Med. Biol. 24: 425-32 (1997). The conjugation of single chain morpholino oligomers to NHS-MAG ₃ was performed as described in Mardirossian, G. et al., J. Nucl. Med. 38: 907-13 (1997).

  One important aspect of amplification targeting is in situ accessibility. In order to achieve high radioactivity levels in the tumor, it is necessary to make the antibody MORF in the tumor accessible to the multimeric cMORF and to make the polymer cMORF in the tumor accessible to the radiolabeled MORF. Another important aspect arises from the accumulation of multimeric cMORF in the liver, spleen, kidney and other normal organs. In order to reduce background radiation levels in these normal organs, the expression of multimeric cMORF should be rapidly inaccessible to radiolabeled MORF.

  The polymers of the present invention should have the following properties: (1) non-toxic; (2) have the appropriate molecular weight and are commercially available; (3) binding to cMORF is feasible. (4) be accessible through the polymer to which the required number of cMORF is attached; (5) the pharmacokinetics of the attached polymer having sufficient water solubility and moderately sustained blood concentration. (6) hybridization of the polymer with the antibody does not promote tumor uptake; and (7) the polymer is metabolized in these normal tissues so that cMORF expression is lost in normal tissues. The polymer diffuses effectively in the tumor. Preferably, the polymers are poly-lysine (PL) and polyethyl vinyl ether maleic acid (PA) having all the above properties. Other preferred polymers include, but are not limited to, dextran, dendrimers and N- (2-hydroxypropyl) methacrylamide (HPMA). Each of these polymers is commercially available as a class having the useful molecular weights shown below, each selected to be water soluble and easily conjugated to each cMORF. In addition, both PL and PA were successfully used. Dendrimers provide an opportunity to evaluate nonlinear polymers. Dextran has been used in clinical practice for more than 50 years to increase plasma volume, promote peripheral blood flow, and as an antithrombotic drug in practice (Thoren, 1981; Mehvar, 2000). In particular, the safety of PL and dendrimers at low doses administered in connection with amplification has been proven (Malik, 2000).

  The polymer we used in previous PNA studies was polymethylvinylethermaleic acid (PA) with an initial molecular weight of 80 KDa and about 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 polyether polyamide linker. PA polymers have unfavorable pharmacokinetics (ie high liver concentration and low blood concentration) unless the polymer is also coupled with an average of 200 polyethylene glycol (PEG) groups due to the limited water solubility of PNA )showed that. As a result, PEG increased the final polymer molecular weight to 1.4 MDa for greater than 70%. Increasing molecular weight to this extent is expected to limit tumor diffusion and penetration. Nevertheless, accessibility was the most promising aspect of PNA research. Radiolabeled cPNA in solution was accessible to 75% of PNA in PA, and even when immobilized (to beads) 35-58% of PNA in PA was still accessible (Wang et al., 2001, supra).

  For PNA, the use of MORF simplified the synthesis of the polymer and provided a wider selection of polymer types. In parallel studies, we synthesized and tested three polylysine (PL) with different initial molecular weights that were fully succinylated to provide the required carboxyl groups (unpublished findings). Furthermore, the final molecular weight of PA, similar to that used with PNA so far, is about 0.4 MDa, compared to 1.4 MDa when the number of accessible oligomers per molecule is only slightly lower. PA that did not contain was also examined. In the normal mouse biodistribution, when PANA is combined with MORF under almost the same conditions, the liver concentration of PA is about half and the blood concentration is 4 times, so MORF superior to PNA The advantage of was shown. These results further suggested that 30 KDa PL with higher blood levels and lower liver concentrations than the other three polymers tested was suitable for in vivo studies. This is why the 30 KDa PL polymer was used in the in vivo studies described herein. However, this polymer had only 12-15 cMORF per molecule. In contrast, the larger 100 KDa PL polymer selected for in vitro studies had 35-40 cMORF per molecule.

  Tumor cell accumulation studies with tissue culture are much simpler than performing animal studies and do not suffer from concentration reduction due to clearance. As such, such a test can be a useful preliminary test for an amplification strategy. Nonetheless, as the tissue culture test is accompanied by non-specific accumulation, as shown in Table 1, as in the tumor animal test, it is necessary to use a control. By eliminating non-specific cell accumulation in tissue culture, a 6-fold increase in specific accumulation was achieved compared to pretargeting.

Animal testing clearly provides a more realistic and rigorous amplification targeting test compared to tissue culture tests. The first two animal studies were designed to quantify antibodies and polymers within the tumor. By using dual radiolabeling, under this research condition, about 25% of the antibody MORF in the tumor is targeted by multimeric cMORF in about 2 days, and after 3 hours these multimeric cMORF in the tumor. Could be predicted to be targeted by radiolabeled MORF. Previous work on PNA by this lab did not use anti-tumor antibodies and is therefore not comparable to the 25% value. However, the 12% accessibility of multimeric oligomers in tumors is lower than the 35-60% accessibility found so far (Wang, Y. et al., 2001, supra). There are many possible reasons for this difference, including the use of different polymer backbones (ie PA vs. PL), the use of polymers of different sizes (first M _r 80 KDa vs. 30 KDa), different oligomers Use (ie, PNA vs. MORF), use of different linkers (ie, 19-member polyether polyamide vs. 9-membered succinylated piperidine), use of different tumor models (ie, ACHN vs. LS174T) and polymer administration and radiolabeling A change in the time taken between doses (ie, 3 hours versus 18 hours). The most likely reason is that it uses a different localization mechanism. The PNA polymer was localized to the tumor by non-specific diffusion, so it was probably free in the interstitial fluid, but the MORF polymer was probably immobilized on its antibody target on the tumor cell surface Let's go. This is because the accessibility of both polymers in solution is approximately 50% for cMORF polymer and 70% for cPNA polymer (Wang, Y. et al., 2001, supra). The possibility is supported.

In all animal studies, the only tissue that consistently showed statistically significantly higher values between the test and control animals was the tumor. The blood is often significantly higher in the test animals, which is that the antibody and polymer remaining in the circulation (and in the tissue) upon MORF- ^99m Tc administration hybridize and retain the radiolabeled MORF it is conceivable that. Use of a clearing agent would be useful (Lubic, SP et al., J. Nucl. Med. 42: 670-678, 2001).

  Finally, amplification targeting proof-of-concept shows that MORF in MN14 in tumors is cMORF polymer in tumors and cMORF in polymers in tumors, despite obstacles to accumulation present in vivo and competition between clearance and targeting. Evidence is that it can be effectively targeted by radiolabeled MORF. Radioactivity accumulation in the tumor was more than double that of pretargeting (ie, amplification factor 2.1). Furthermore, MORF expression (in the antibody) and cMORF expression (in the polymer) disappeared rapidly in normal organs such as the liver, spleen and kidney, but did not disappear in the tumor, increasing the target / non-target ratio. Despite the binding of the antibody to what is probably a less than ideal polymer with an average of only 0.2 MORF and 15 cMORF per molecule, these results Obtained. In addition, no clearing agent was used.

  Examples of radionuclides that are useful as therapeutic agents and that substantially decay by β-ray radiation 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, I-125, I-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. The maximum decay energy of useful β-ray radionuclides is preferably 20 to 5,000 keV, more preferably 100 to 4,000 keV, and most preferably 500 to 2,500 keV.

  Radionuclides that are substantially disrupted by Auger radiation particles that are useful as therapeutic agents include, but are not limited to, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt- 109, In-111, Sb-119, I-125, Ho-161, Os-189m and Ir-192. The 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 that are useful as therapeutic agents and that substantially decay by generating alpha particles include, but are not limited to, Dy-152, At-211, Bi-212, Ra-223, Rn-219, Examples include Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, and Fm-255. The decay energy of useful α-ray radionuclides is preferably 2,000 to 9,000 keV, more preferably 3,000 to 8,000 keV, and most preferably 4,000 to 7,000 keV. is there.

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

  Radionuclides useful for therapy based on neutron capture methods include, but are not limited to, B-10, Gd-1557, and U-235.

  Useful diagnostic agents include, but are not limited to, radionuclides, dyes (eg, use of biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules and enhancements used for magnetic resonance imaging (MRI). Agents (for example, paramagnetic ions). US Pat. No. 6,331,175 describes MRI methods and the preparation of antibodies conjugated with MRI enhancers, the entire disclosure of which is hereby incorporated by reference. Preferably, the diagnostic agent is selected from the group consisting of radionuclides, enhancers used in magnetic resonance imaging and fluorescent compounds.

  Radionuclides useful as diagnostic agents for use 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, I-120 and I- 124. The total decay energy of useful positron emitting nuclides is preferably less than 2,000 keV, more preferably less than 1,000 keV, and most preferably less than 700 keV.

  Metals useful as diagnostics when utilizing magnetic resonance imaging include, but are not limited to, cadolinium, manganese, iron, chromium, copper, cobalt, nickel, dysprosium, rhenium, europium, terbium, holmium and Neodymium.

  Radionuclides useful as diagnostic agents when 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-111, In-114m, I-123, I-125, I-131, Yb-169, Hg-197 and TI-201. The decay energy of useful γ-ray radionuclides is preferably 20 to 2000 keV, more preferably 60 to 600 keV, and most preferably 100 to 300 keV.

  In the following, embodiments of the present invention will be described by way of examples showing in detail the embodiments of the present invention. These examples are illustrative of specific elements of the invention and are not intended to limit the scope thereof.

Materials and Methods 25-mer MORF and cMORF with 3′-amine were purchased via a 9-membered succinylated piperidine linker (Gene Tools, Corvallis, OR). These were the same as those used so far by the present inventors (Liu, G. et al., Quart. J. Nucl. Med. 46: 233-43, 2002). High-affinity murine anti-CEA antibody (MN14, IgG _{1 subtype,} M r 160 kDa) were obtained from Immunomedics (Morris Plains, NJ). Was purchased lysine Sigma-Aldrich, St Louis, from MO - having an average _{M r} of about 30KDa and 100 KDa, uniformly been succinylated poly. 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) hydrochloride was from Pierce Company, Rockford, IL.

N of bifunctional chelating agent, acetyl-S-protected mercaptoacetyl-triglycine (NHS-MAG ₃ ) according to the method of Winnard, P. et al., Nucl. Med. Biol. 24: 425-32 (1997). -A hydroxysuccinimidyl derivative was synthesized. This structure was confirmed by elemental analysis, proton NMR and mass spectrometry. To 0.97 ml of 0.225 M sodium hydroxide was added 50 mg (264 μmol) of triglycine and 10 μl of freshly prepared 50 mM ethylenetriaminetetraacetic acid (EDTA) disodium. This solution was passed through a 0.2 μm filter to remove amine-containing particles. A solution of 90 mg (390 μmol) of S-acetylthioglycolic acid N-hydrosuccinimide ester (SATA) in 340 μl of dimethylformamide (DMF; dried with molecular sieves) was prepared, and the solution was added dropwise to the triglycine stirring solution. After stirring for 15 minutes at room temperature, 37.6 μl of 6M hydrochloric acid was added to adjust the apparent pH of the non-aqueous solution from 8.9 to about 2.7 (measured with a glass electrode-pH meter). The initial pH of about 8.9 was selected to deprotonate the amine of triglycine but not reach a strongly basic pH value where the acetyl group of SATA is hydrolyzed (pK7.9; Fasman, GD [ed 1976, CRC Handbook of Biochemistry and Molecular Biology, Third Edition, Vol. I, p 321, CRC Press, Boca Raton, FL). The pH was lowered as soon as possible to minimize acetyl group hydrolysis.

  A solution of 60 mg (290 μmol) of dicyclohexylcarbodiimide (DCC) in 3.6 ml of dry DMF was quickly added to the triglycine / SATA stirred solution (apparent pH about 5.0). Since dicyclohexylurea began to precipitate, the solution became turbid in 2 minutes. The reaction was stirred in a dark room at room temperature for 2-4 hours and then cooled to -20 ° C for an additional hour to facilitate complete settling. After centrifuging at 2500 g at 4 ° C. for 15 minutes, the clear supernatant was removed.

Due to the presence of water in the DMF solution, this form of NHS-MAG ₃ preparation was used within 24 hours of preparation. For long-term storage, the NHS-MAG ₃ water / DMF solution is almost evaporated to dryness in a rotary flash evaporator (Rotavapur-R, Buchi, Switzerland) in 15 to 30 minutes, and then freeze-dried (Virtis, Gardenier, NY) and lyophilized within 1 hour. NHS-MAG ₃ can be stored indefinitely at room temperature indefinitely after being dried in this way. When NHS-MAG ₃ made into a dry powder for complex formation was used, an arbitrary value of 50% by weight was considered as its purity.

Size exclusion (SE) HPLC analysis was performed on a Superdex peptide column (optimal separation range 1 × 10 ^{2 to} 7 × 10 ³ Da, Amersham Pharmacia Biotech, Piscataway, NJ) as 0.10 mol / L phosphate buffer, pH 7 0.0 was performed at a flow rate of 0.6 mL / min. Peak fractions were identified and quantified using in-line UV absorbance at 260 nm and a radioactivity detector. Radioactivity recovery was measured as usual.

Reagent DTPA cyclic anhydride and carbonyldiimidazole were from Sigma-Aldrich (St Louis, MO) and were used as they were. P4 resin (Biogel P4 gel, medium) was purchased from Bio-Rad Laboratories, Hercules, CA. Sephadex G100 resin was obtained from Pharmacia Biotech Uppsala, Sweden. ^99m Tc-pertechnetate was eluted from a ⁹⁹ Mo- ^99m Tc generator (Bristol-Myers Squibb Medical Imaging, Billerica, Mass.). ¹¹¹ In was purchased as chloride (PerkinElmer Life Science Inc., Boston, MA). All other chemicals were for reagents and were used without further purification.

Preparation of MN14-MORF, MN14-DTPA, MORF-MAG ₃ , MORF-DTPA and complexation of radiolabeled MN14 with MORF has been previously described by Liu et al. (J. Nucl. Med 43: 384-391, 2002), the amine-derivatized MORF was reacted with the natural antibody using EDC, and then purified with Sephadex G-100 using 0.05M phosphate buffer, pH 7.2. It carried out by doing. MORF-conjugated antibodies were characterized by HPLC using differential UV methods at 265 nm and 280 nm for concentration and average number of MORFs per antibody molecule (groups per molecule) (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). For both complexed DTPA and free DTPA, ¹¹¹ In accessibility was the same, and the average number of DTPA groups per MN14 was determined by labeling the mixture with ¹¹¹ In prior to purification. _MORF-MAG ^{3 - 99m} Tc has been described previously (.... Liu et al , Quart J. Nucl Med 46: 233-43, 2002) As described above, were prepared and analyzed. Radiolabeling was performed by first using ^99m Tc pertechnetate generator eluate, MORF-MAG ₃ or cMORF-MAG ₃ (concentration above 0.1 μg / μl) (5-10 μl), 0.25 M ammonium acetate buffer, pH 5.2 (25 μL), pH 9.2 tartrate solution (50 μg sodium tartrate dihydrate / μl) (10 μl), and tin chloride solution (1 μg tin chloride dihydrate in 1 μl 10 mM HCl and 1 μg sodium ascorbate) ) (4 μl) was added to the solution followed by heating the solution in boiling water for about 20 minutes. The product was purified on a P4 column using 0.05M phosphate buffer, pH 7.2 as eluent. MORF- ¹¹¹ In was prepared by incubating MORF-DTPA with ¹¹¹ In for 1 hour at room temperature, followed by purification as described above for MORF- ^99m Tc. Both labeled MORF and labeled cMORF were routinely analyzed by size exclusion HPLC, and it was found that both UV and radioactivity detection yielded essentially the same chromatograph.

Preparation and Radiolabeling of PL-cMORF Ten milligrams of uniformly succinylated polylysine polymer (PL) with initial M _r 30 KDa (animal test) or 100 KDa (tissue culture test) as aprotic solvent N-methylpyrrolidinone Dissolved in 1.0 ml of (NMP), 20.4 mg of 1,1′-carbonyldiimidazole and 3.0 μl of diisopropylethylamine (DIEA) were added thereto. This mixture was incubated at room temperature for 2 hours. When a planned amount of activated PL mixture and equimolar DIEA were added to 500 μl of a 2.0 mg / ml cMORF solution in NMP, a molar ratio of cMORF to PL of 100: 1 was reached. This solution was incubated overnight at room temperature.

Prior to purification, one aliquot of the solution was analyzed by size exclusion HPLC using UV detection at 265 nm to estimate the average number of cMORF groups bound to each PL molecule. Since PL does not absorb appreciably at 265 nm, the peak areas of unbound cMORF in the free state and cMORF bound to PL were compared. As an alternative to estimating the average number of groups per molecule, a tracer concentration of radiolabeled MORF is also added to another aliquot of PL-cMORF solution, and MORF- hybridized with unbound cMORF in the free state. The radioactivity of ^99m Tc was compared to that of hybridized with cMORF coupled to PL. The PL-cMORF complex was then purified by open column gel filtration chromatography on a 1 cm × 30 cm Sephadex G100 column using water as the eluent. The concentration of PL-cMORF for cMORF in the collected fractions was estimated by UV absorbance using the molar absorbance of cMORF provided by the manufacturer.

PL-cMORF is radiolabeled by incubating with trace amounts of MORF- ^99m Tc or MORF- ¹¹¹ In for 30 minutes at room temperature so that on average only about 1 cMORF in each polymer is hybridized with MORF. Turned into. Quality assurance was performed as usual based on size exclusion HPLC chromatography where radioactivity recovery was monitored as usual.

Using the UV HPLC chromatogram and the slope of the standard curve (8), the average MORF per MN14 molecule for the two MN14-MORF preparations used in this study were 0.09 (tissue culture test) and 0.20. Calculated as (animal test). The HPLC radiochromatogram of MN14-MORF showed one prominent peak when freshly prepared and purified, but showed evidence of free MORF peak when stored. Only newly prepared synthetic antibodies were used in this study. Using a ¹¹¹ In-labeled HPLC radiochromatograph attached to MN14-DTPA before purification, it was calculated that an average of 0.7 DTPA groups were bound to each MN14 antibody.

The ^99m Tc labeling technique used in this study was always labeled with a radiochemical purity greater than 90% after purification, as indicated by the normal HPLC analysis with a recovery of greater than 90% in all analyses. It was a technique for providing MORF. It has been shown routinely that radiolabeled cMORF is hybridizable to MORF bound to 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).

30 KDa PL polymer averaged 12-15 per molecule by size exclusion HPLC analysis utilizing UV absorbance and by size exclusion HPLC analysis of bound but unpurified PL-cMORF polymer before and after addition of trace MORF- ^99m Tc. It was estimated that 100 KDa PL polymer was bound to an average of 35-40 groups per molecule, while bound to cMORF groups. Two methods of measuring groups per molecule showed 80-90% agreement. The final molecular weight was estimated as 130-160 KDa and 400-440 KDa, respectively. HPLC analysis of both purified polymers showed a single peak, with a recovery greater than 90%.

Tissue culture test LS174T cells were supplemented with 10% fetal bovine serum (FBS) and 100 mg / ml penicillin-streptomycin (Gibco BRL Products, Gaithersburg, MD), 2 mM L-glutamine, 1.5 mg / L sodium bicarbonate, Grow in minimal essential medium (MEM, Gibco BRL Products, Gaithersburg, MD) containing 0.1 mM non-essential amino acids and 1.0 mM sodium pyruvate. Cells were usually maintained as a monolayer in a humidified 5% carbon dioxide atmosphere in a T75 flask (Falcon, Becton Dickinson, Lincoln Park, NJ). In the uptake test, cells were trypsinized with 0.05% trypsin / 0.02% EDTA in a T75 flask at 80-90% confluence and then the desired density with MEM containing 10% FBS. Usually suspended in 0.1 ml up to 1-2 × 10 ⁶ cells. Cells exposed to antibody were cultured with 20 μg of MN14-cMORF in 0.2 ml Dulbecco's phosphate buffered saline (PBS, Gibco BRL Products, Gaithersburg, MD) and after 1 hour at 4 ° C., the cell suspension was Centrifugation was performed at 2000 rpm for 4 minutes, and the cells were washed 3 times with 0.5 ml of PBS each time (incubation was performed at 4 ° C. to minimize the possibility of antibody internalization). Cells fed with polymer were incubated with 10 μg of 100 KDa PL-cMORF for 1 hour at 4 ° C., centrifuged as above and washed 3 times. All cells received 0.60 μg MORF- ^99m Tc or 0.27 μg cMORF- ^99m Tc. After incubation at room temperature for 30 minutes, cells were centrifuged as described above, washed three times, and cell pellets were counted against a radiolabeled culture standard with a NaI (Tl) well counter.

Table 1 lists five groups for tissue culture tests. Cells belonging to the amplification group were given antibody, 100 KDa PL polymer and radiolabeled MORF. The remaining 4 groups served as controls. Cells belonging to the pre-targeting control group receive only antibody and radiolabeled cMORF, cells belonging to the polymer alone control group receive only polymer and radiolabeled MORF, and cells belonging to ^99m Tc alone control group I and II Each gave only labeled cMORF and MORF. The last two columns in the table show the average added radioactivity and moles of (c) MORF accumulated in the cells.

The results of ^99m Tc alone control groups I and II show that labeled (c) MORF accumulates non-specifically in the cells, so subtract 0.030 pmol from the pretargeting group value and 0.052 pmol was subtracted from the values of the single group and the amplification group. Furthermore, the results for the polymer alone group showed that the polymer also accumulated non-specifically in the cells, so the accumulation in the amplified group was even lower by 1.18 pmol. As a result, this value of 0.41 pmol was compared with 0.067 pmol of pretargeting to obtain an amplification factor of 6.

Animal testing All animal studies were conducted with the approval of the University of Massachusetts Medical School (UMMS) Institutional Animal Care and Use Committee. Each nude mouse (NIH Swiss, Taconic Farms, Germantown, NY, 30-40 g) was injected subcutaneously into the left thigh with 0.1 ml of a suspension containing 10 ⁶ LS174T colon tumor cells. Animals were used 14 days after the tumor size was 1.5 cm or less.

To estimate the optimal dose of MN14-MORF antibody and 30 KDa PL polymer, tumor animals were radioactive with 60 μg of MN14 (as an initial estimate) and approximately 2.0 μCi of ¹¹¹ In coupled with an average of 0.2 MORF per molecule. 3 μg of labeled MN14-DTPA was administered, and 2 days later, ^99m Tc-labeled polymer was administered at 3 doses of 24-76 μg / animal. Based on these results, a 25 μg dose of MN14-MORF and a 15 μg dose of polymer were selected for subsequent animal studies.

Prior to animal studies for amplification, the effects of antibody and polymer doses on tumor accumulation were established. Tumor animals were dosed with 60 μg of ¹¹¹ In-labeled MORF antibody (as an initial guess) and two days later, ^99m Tc-labeled 30 KDa PL polymer was administered at 3 doses of 24-76 μg / animal. Each control animal received 24 μg polymer but no antibody. ^{The 99m} Tc biodistribution results are shown in Table 2, which shows that the polymer tumor accumulation increases with decreasing dose. ^{From the 111} In results (not shown) it can be seen that the intratumor accumulation of 60 μg of antibody is statistically the same as that shown below (Table 2) for 24 μg of MN14-MORF. Thus, in all animal studies for amplification, 15 μg of polymer was used with 25 μg of MN14-MORF. Statistical significance in this and subsequent tables was determined by two-sided distribution with Microsoft Excel and Student's t-test when paired. Statistically significant values (ie, p <0.05) are underlined.

In the first study, it was mixed with the ^{MN14-MORF 25μg MN14- 111 In 3μg} (2.0μCi), gave them simultaneously into nude mice. After about 30 hours, the animals receive 15 μg (250 μCi) of 30 KDa PL-cMORF polymer labeled by hybridization with MORF- ^99m Tc, which occupies only about 1 out of 12-15 cMORF of the polymer on average. It was. Control animals received no antibody and / or polymer. Animals were sacrificed by cardiac puncture under anesthesia at 18 hours after polymer administration. Organs and blood were collected for simultaneous counting of ¹¹¹ In and ^99m Tc by an automatic gamma counter (Cobra II, Packard Instrument Company, Downers Grove, IL). All counts were corrected for physical decay and a small contribution of ¹¹¹ In activity in the ^99m Tc window. The results are shown as a percentage of the amount injected per gram.

The first animal test for amplification is designed to assess the extent to which antibody MORF in the tumor is targeted by multimeric cMORF. Therefore, nude mice transplanted with LS174T tumors received a radiolabeled antibody (ie, together with MN14-MORF, MN14-111In). Two days later, the animals received PL-cMORF polymer labeled by hybridization with MORF- ^99m Tc. Animals were sacrificed 18 hours after polymer administration. Control animals received no antibody but only labeled polymer 18 hours before.

From the results of biodistribution in Table 3, the number of antibody MORFs per gram of tumor was calculated using ¹¹¹ In value, while the ^99m Tc value of the tumor of the test animal minus the value of the control animal was used to be more specific to the antibody. The number of multimeric cMORF per gram of tumor localized in can be calculated. These calculations show that under the conditions of this study, multimeric cMORF targeted 25% of the antibody MORF in the tumor. This calculation is based on the assumption that one polymer molecule binds to no more than one antibody molecule. If this assumption is wrong and the polymer is cross-linked with an antibody in the tumor, the above value will be greater than 25%. While the polymer is capable of cross-linking with multiple antibody molecules of the tumor, another study has shown that the tumor antibody MORF with labeled cMORF in a free state (ie, monovalent because it is not conjugated to the polymer) Was found to be only 50% (10). Multimeric cMORF is unlikely to target antibody MORF more effectively than cMORF in the free state, so crosslinking is less likely, but if it happens, it will probably crosslink with no more than two antibody molecules It will be judged.

In connection with previous pretargeting studies, the lab has reported similar ^99m Tc and ¹¹¹ In dual labeling studies in LS174T tumor animals, and the accessibility to radiolabeled cMORF (10) Estimated as a function of time MN14-MORF was present in the tumor, liver and spleen. These results indicated that access to the liver and intrasplenic MORF by radiolabeled cMORF was limited to less than about 6% 24 hours after antibody administration. Clearly, once antibodies are localized in these organs, the MN14 MORF rapidly “mostly disappears”. Fortunately, this was not the case for tumors with accessibility far exceeding 50%.

In the second study, targeting was performed in the same manner, 25 μg of MN14-MORF (here unlabeled) was administered to LS174T tumor mice, and 30 hours later, PL-cMORF polymer (wherein trace [3 μCi] MORF 15 μg (radiolabeled with ¹¹¹ In) was administered. Animals were given MORF- ^99m Tc 1.5 μg (approximately 300 μCi) at 41 hours after polymer administration and the animals were sacrificed 3 hours later. The labeled MORF dose was selected to be reasonably low but have sufficient radioactivity. For controls, antibodies were excluded in one animal set and both antibodies and polymers were excluded in another animal set.

A second animal study for amplification was designed to assess the extent to which multimeric cMORF within the tumor is targeted by radiolabeled MORF. Therefore, nude mice transplanted with LS174T tumor were first given 25 μg of unlabeled antibody, and 30 hours later, 15 μg of 30 KDa PL-cMORF polymer labeled by hybridization with a minute amount of ¹¹¹ In-MORF was given. Forty-one hours later, animals received 1.5 μg of MORF- ^99m Tc and sacrificed 3 hours later (ie, 74 hours after antibody administration and 44 hours after ¹¹¹ In-polymer administration). The labeled MORF dose was selected to be reasonably low but sufficient for imaging. For the control, the antibody was excluded in one animal set (Control I) and in another animal set (Control II) both antibody and polymer were excluded. Images were taken before the animals were sacrificed. The biodistribution results are shown in Table 4.

Tumor ^99m Tc values are significantly higher for antibody-free administration (group [1] vs. group [2]), indicating the specificity of amplification targeting. ^The number of multimeric cMORF per gram of tumor was calculated from the ¹¹¹ In value, while the tumor of the test animal (group [1]) given the antibody minus the value of the animal not given the antibody (group [2]) ^{Using 99m} Tc values, it can be estimated that MORF- ^99m Tc targeted 12% of the multimeric cMORF in the tumor.

^{Using 111} In and ^99m Tc results, it can also be estimated that less than 1% of multimeric cMORF in the liver, spleen and kidney targeted by radiolabeled MORF 44 hours after PL-cMORF polymer administration . At the same time that the MORF of the antibody delivered to the liver and spleen was rapidly “disappeared” from the radiolabeled cMORF (10), these latest studies also showed that the multimeric cMORF was also rapidly Is turned off. Fortunately, this loss of expression is not so obvious in tumors. In the tumor, 12% of the multimeric cMORF was targeted beyond 40 hours. The above calculations can also be used for blood values, indicating that about 30% of circulating multimeric cMORF was targeted by labeled MORF.

The rapid loss of normal tissue polymer can also be seen by comparison in the MORF- ^99m Tc value table with and without polymer (group [2] vs. group [3]). Except for the spleen and blood, the difference is not significant. Thus, again, the evidence is that cMORF expression carried to these tissues by the polymer disappears within 41 hours and effectively disappears from the radiolabeled MORF. For MORF expression with antibodies and cMORF expression with polymers, the resulting radioactivity levels in normal tissues are reduced. Fortunately, in all cases, accessibility in the tumor was significantly maintained during this period.

A third animal study was specially designed to assess amplification. Nude mice were first given 25 μg of unlabeled antibody, and 30 hours later, 15 μg of unlabeled 30 KDa PL-cMORF polymer was administered to the mice. The animals were sacrificed 3 hours after administration of 1.5 μg of MORF- ^99m Tc at 21 or 43 hours after administration of the polymer. Control animals received no antibody or received neither antibody nor polymer. As an additional control (pretargeting), animals received no polymer but received MN14-MORF antibody followed by cMORF- ^99m Tc after 51 hours. After carefully aspirating urine from the bladder with a syringe, the animals were imaged with an Elscint APEX 409 M large-view gamma camera (Hackensack, NJ). After imaging, the animals were sacrificed and dissected as described above. Statistical significance was determined by two-sided distribution with Microsoft Excel and Student's t-test when paired.

The third animal study is specifically designed to assess amplification. Therefore, nude mice transplanted with LS174T tumor were first given 25 μg of unlabeled antibody, and 30 hours later, 15 μg of unlabeled 30 KDa PL-cMORF polymer. The animals were then given 15 μg of MORF- ^99m Tc at 21 hours (Table 5) or 43 hours (Table 6) after polymer administration, and the animals were sacrificed 3 hours later. Control animals received no antibody (group [2]) or received neither antibody nor polymer (group [3]). As an additional control (pretargeting), animals received no polymer but received MN14-MORF antibody followed by cMORF- ^99m Tc after 51 hours (Table 5). Images were taken before the animals were sacrificed.

If the in vivo behavior of MORF- ^99m Tc and cMORF- ^99m Tc are sufficiently similar, the in vivo amplification factor for pretargeting in the above study can be inferred. Therefore, the absolute tumor accumulation (0.65% to 0.24%) × 1.5 μg of MORF- ^99m Tc in the amplification study group is 6.15 ng, whereas the accumulation of cMORF- ^99m Tc by pretargeting. (2.03% to 0.18%) × 0.15 μg is 2.78 ng. This ratio provides an amplification factor of 2.1 for pretargeting.

  In short, these results prove that in vivo amplification targeting is feasible and has been achieved. Under the conditions of these studies, approximately 25% of the MORF localized to the tumor via the MN14 antibody is accessible and is targeted by the PL cMORF polymer. Furthermore, about 12% of cMORFs thus localized to tumors are successfully targeted by radiolabeled MORF. The proof of concept for amplification is clear in that the tumor accumulation in each study is consistently statistically higher than the controls. Also important for the absolute accumulation at the target is the target / normal tissue ratio. By calculating these ratios for each tissue for each of the three amplification studies, amplification targeting was higher in 33 out of 35 evaluations than the polymer alone control or the radiolabeled cMORF alone control. It is shown to provide a tumor / non-target ratio.

Animal Imaging FIG. 1 is a whole body image obtained simultaneously of two nude mice each having an LS174T tumor in the right thigh. Both animals received MORF- ^99m Tc (3 hours before imaging), and both animals received cMORF-polymer (43 hours before imaging). Only the left test (amplified) animal received MN14-MORF antibody (73 hours before imaging).

FIG. 2 is a whole body image of three nude mice, each having an LS174T tumor in the right thigh, obtained simultaneously under the same conditions as FIG. Therefore, the left animal received only MORF- ^99m Tc (3 hours before imaging), the central animal received MORF- ^99m Tc and cMORF-polymer (21 hours before imaging), and the right test animal ( Amplification) was given MORF- ^99m Tc, cMORF-polymer and MN14-MORF (51 hours before imaging). Only test animals given both antibody and polymer show tumors in the image.

  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 the present invention. Thus, it is intended that the present invention covers the modifications and variations that come within the scope of the appended claims and their equivalents. The disclosures of all publications cited above are to the same extent as if each was individually incorporated by reference, and all of their disclosures are hereby expressly incorporated by reference. Part of the book.

Whole body images of these animals were obtained simultaneously 3 hours after ^99m Tc-MORF administration and 43 hours after PA30KDa-cMORF administration to LS174T tumor mice given MORF-MN14 73 hours ago. The left is the test animal, the right is the animal that received the polymer but not the antibody. Whole body images of these animals were obtained simultaneously 3 hours after ^99m Tc-MORF administration and 21 hours after PA30KDa-cMORF administration to LS174T tumor mice given MORF-MN14 51 hours ago. The right is the test animal, the middle is the animal that received the polymer but did not receive the antibody, and the left is the animal that received no polymer or antibody.

Claims (34)

A method of delivering a diagnostic or therapeutic agent to a target site in a subject comprising:
(A) a target moiety that selectively binds to a main target-specific binding site of a target site, or a substance produced by the target site, or a substance related to the target site; and a first morpholino Administering a first complex comprising an oligomer,
(B) administering to said subject a second complex comprising a polymer coupled to a plurality of copies of a second morpholino oligomer complementary to said first morpholino oligomer; and (c) said Administering to the mammal a third complex comprising a third morpholino oligomer complementary to the second morpholino oligomer and a diagnostic or therapeutic agent.   The method of claim 1, wherein the first morpholino oligomer and the third morpholino oligomer are the same.   Further comprising administering a clearing agent to the mammal at a time point after step (a), and removing the non-localized first complex from the circulation by the clearing agent. Item 2. The method according to Item 1.   The method of claim 1, wherein the targeting moiety is an antibody or antibody fragment.   5. The method of claim 4, wherein the antibody or antibody fragment is a human antibody or antibody fragment or a humanized antibody or antibody fragment.   2. The method of claim 1, wherein 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 antibody or antibody fragment is CEA, B cell antigen, T cell antigen, plasma cell antigen, HLA-DR lineage antigen, NCA, MUC1, MUC2, MUC3 and MUC4 antigen, EGP-1 antigen, EGP-2 antigen, placental alkaline phosphatase Antigen, IL-6, VEGF, tenascin, CD33, CD74, PSMA, PSA, PAP, autoimmune disease, antigen associated with infection / inflammation and infection, B cell or T cell lymphoma, or autoimmune disease Antigens associated with B cells or T cells, CD19, CD22, CD40, CD74, HLA-DR, IL-15 and HLA-DR expressed by malignant diseases, CD15, CD33, CD66a, CD66b and CD66e Binds to a target selected from the group The method of claim 5.   The method of claim 1, wherein the polymer is selected from the group consisting of poly-lysine (PL), polyethyl vinyl ether maleic acid (PA) dextran, dendrimer and N- (2-hydroxypropyl) methacrylamide (HPMA). .   2. The method of claim 1, wherein each of the morpholino oligomers has a length of at least about 6 bases to about 100 bases.   The method of claim 9, wherein the first oligomer has a length of about 15 to about 25 bases.   The method of claim 9, wherein the second oligomer has a length of about 15 to about 25 bases.   10. The method of claim 9, wherein the third oligomer has a length of about 15 to about 25 bases.   4. The method of claim 3, wherein the clearing agent comprises an anti-idiotype antibody or an antibody fragment that binds an antigen.   The third complex comprises a therapeutic agent selected from the group consisting of antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, chelating agents, boron compounds, photoactive substances or dyes and radionuclides. The method of claim 1, comprising:   2. The diagnostic agent selected from the group consisting of a radionuclide, a dye, a contrast agent, a fluorescent compound or molecule and an enhancer useful for magnetic resonance imaging (MRI), wherein the third complex comprises The method described.   The method of claim 1, wherein the second complex comprises at least 5 copies, at least 10 copies, at least 25 copies, or at least 50 copies of the second morpholino oligomer.   2. The method of claim 1, wherein the second complex comprises about 25-50 copies of the second morpholino oligomer. A kit for targeting a diagnostic or therapeutic agent in a subject comprising:
(A) a primary target-specific binding site of the target site, or a targeting moiety that selectively binds to a substance generated by the target site or a substance related to the target site, and a first morpholino oligomer The first complex,
(B) a second complex comprising a polymer coupled to a plurality of copies of a second morpholino oligomer complementary to the first morpholino oligomer, and (c) complementary to the second morpholino oligomer A kit comprising a third complex comprising a third morpholino oligomer and a radioactive label.   The kit according to claim 18, wherein the first morpholino oligomer and the third morpholino oligomer are the same.   19. A kit according to claim 18 further comprising a clearing agent.   The kit according to claim 18, wherein the targeting moiety is an antibody or an antibody fragment.   The kit according to claim 21, wherein the antibody or antibody fragment is a human antibody or antibody fragment or a humanized antibody or antibody fragment.   19. A kit according to claim 18, wherein 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.   24. The kit of claim 22, wherein the antibody or antibody fragment binds to a human carcinoembryonic antigen.   19. The kit of claim 18, wherein the polymer is selected from the group consisting of poly-lysine (PL), polyethyl vinyl ether maleic acid (PA) dextran, dendrimer and N- (2-hydroxypropyl) methacrylamide (HPMA). .   19. The kit of claim 18, wherein each of the morpholino oligomers has a length of at least about 6 bases to about 100 bases.   27. The kit of claim 26, wherein the first oligomer has a length of about 15 to about 25 bases.   27. The kit of claim 26, wherein the second oligomer has a length of about 15 to about 25 bases.   27. The kit of claim 26, wherein the third oligomer has a length of about 15 to about 25 bases.   21. The kit of claim 20, wherein the clearing agent comprises an anti-idiotype antibody or an antibody fragment that binds an antigen.   The third complex comprises a therapeutic agent selected from the group consisting of antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, chelating agents, boron compounds, photoactive substances or dyes and radionuclides. The kit according to claim 18, comprising:   19. The diagnostic agent selected from the group consisting of a radionuclide, a dye, a contrast agent, a fluorescent compound or molecule and an enhancer useful for magnetic resonance imaging (MRI), wherein the third complex comprises The described kit.   19. The kit of claim 18, wherein the second complex comprises at least 5 copies, at least 10 copies, at least 25 copies or at least 50 copies of the second morpholino oligomer.   19. The kit of claim 18, wherein the second complex comprises about 25-50 copies of the second morpholino oligomer.

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