Title of Invention	A COMPOUND OR A TARGETABLE CONSTRUCT FOR THERAPEUTIC USE
Abstract	The present invention relates to targetable constructs which may be bound by a bi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds the targetable construct. The targetable construct comprises a carrier portion which comprises or bears at least one epitope recognizable by at least one arm of said bi-specific antibody or antibody fragment. The targetable construct further comprises one or more therapeutic or diagnostic agents or enzymes. The invention provides constructs and methods for producing the targetable constructs and bi-specific antibodies or antibody fragments, as well as methods for using them.

Title of Invention

A COMPOUND OR A TARGETABLE CONSTRUCT FOR THERAPEUTIC USE

Abstract

The present invention relates to targetable constructs which may be bound by a bi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds the targetable construct. The targetable construct comprises a carrier portion which comprises or bears at least one epitope recognizable by at least one arm of said bi-specific antibody or antibody fragment. The targetable construct further comprises one or more therapeutic or diagnostic agents or enzymes. The invention provides constructs and methods for producing the targetable constructs and bi-specific antibodies or antibody fragments, as well as methods for using them.

Full Text	DRUG PRE-TARGETING BY MEANS OF BI-SPECIFIC ANTIBODIES AND HAPTEN CONSTRUCTS COMPRISING A CARRIER PEPTIDE AND THE ACTIVE AGENT (S) [0001] This application is a continuation-in-part of United States Serial No. 09/382,186, filed August 23, 1999 and a continuation-in-part of United States Serial No. 09/823,746, filed April 3,2001, both of which are continuations-in-part of United States Serial No. 09/337.756, filed June 22,1999, the contents of which are incorporated herein by reference in their entirety. Background of the Invention [0002] Field of the Invention. The invention relates to immunological reagents for therapeutic use, for example, in radioimmunotherapy (RAIT), and diagnostic use, for example, in radioimmunodctection (RAID) and magnetic resonance imaging (MRI). In particular, the invention relates to bi-specific antibodies (bsAb) and bi-specific antibody fragments (bsFab) which have at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct Further, the invention relates to monoclonal antibodies that have been raised against specific immunogens, humanized and chimeric monoclonal bi-specific antibodies and antibody fragments having at least one aim that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct, DNAs that encode such antibodies and antibody fragments, and vectors for expressing the DNAs, Earlier provisional patent applications, U.S.S.N. 60/090,142 and U.S.S.N. 60/104,156 disclose a part of what is now included in this invention and are incorporated herein by reference in their entireties. Related Art 10003] An approach to cancer therapy and diagnosis involves directing antibodies or antibody fragments to disease tissues, wherein the antibody or antibody fragment can target a diagnostic agent or therapeutic agent to the disease site. One approach to this methodology which has been under investigation, involves the use of bsAbs having at least one arm that specifically binds a targeted diseased tissue and at least one other arm that specifically binds a low molecular weight hapten. In this methodology, a bsAb is administered and allowed to localize to target, and to clear normal tissue. Some time later, a radiolabeled low molecular weight hapten is given, which being recognized by the second specificity of the bsAb, also localizes to the original target. \|0004\| Although low MW haptens used in combination with bsAbs possess a large number of specific imaging and therapy uses, it is impractical to prepare individual bsAbs for each possible application-Further, the application of a bsAb/low MW hapten system has to contend with several other issues. First, the arm of the bsAb that binds to the low MW hapten must bind with high affinity, since a low MW hapten is designed to clear the living system rapidly, when not bound by bsAb. Second, the non-bsAb-bound low MW hapten actually needs to clear the living system rappidly to avoid non-target tissue uptake and retention. Third, the detection and/'or therapy agent must remain associated with the low MW hapten throughout its application within the bsAb protocol employed. (0005) Of interest with this approach are bsAbs that direct chelators and metal chelate complexes to cancers using Abs of appropriate dual specificity. The chelators and metal chelate complexes used are often radioactive, using radionuclides such as cobalt-57 (Goodwin et al, U.S. Patent No. 4,863,713), indium-111 (Barbet et aL, U.S. Patent No. 5,256,395 and US. Patent No. 5,274,076, Goodwin et al.J. Nucl Med., 33:1366-1372 (1992), and Kranenborg et ai, Cancer Res (suppL), 55;5864s-5867s (1995) and Cancer (suppl.) 80:2390-2397 (1997)) and gallium-68 (Boden et al., Bioconjugate Chem., 6:373-379, (1995) and Schuhmacher et al., Cancer Res., 55:115'123 (1995)) for radioimmuno-imaging. Because the Abs were raised against the chelators and metal chelate complexes, they have remarkable specificity for the complex against which they were originally raised. Indeed, the bsAbs of Boden et aL have specificity for single cnantiomere of enantiomeric mixtures of chelators and metal-chelate complexes. This great specificity has proven to be a disadvantage in one respect, in that other nuclides such as yrrrium-90 and bismuth-213 useful for radioimmunotherapy (RAIT), and gadolinium useful for MRI, cannot be readily substituted into available reagents for alternative uses. As a result iodine-231, a non-metal, has been adopted for RAIT purposes by using an 1-13 l-labeled indium-metal-chelate complex in die second targeting step. A second disadvantage to this methodology requires that antibodies be raised against every agent desired for diagnostic or therapeutic use, [00061 Pretargeting methodologies have received considerable attention for cancer imaging and therapy. Unlike direct targeting systems where an effector molecule (e.g., a radionuclide or a drug linked to a small carrier) is directly linked to the targeting agent, in pretargeting systems, the effector molecule is given some time after the targeting agent This allows time for the targeting agent to localize in tumor lesions and, more importantly, clear from the body. Since most targeting agents have been antibody proteins, they tend to clear much more slowly from the body (usually days) than the smaller effector molecules (usually in minutes). In direct targeting systems involving therapeutic radionuclides, the body, and in particular the highly vulnerable red marrow, is exposed to the radiation all the while the targeting agent is slowly reaching its peak levels in the tumor and clearing from the body. In a pretargeting system, the radionuclide is usually bound to a small "effector" molecule, such as a chelate or peptide, which clears very quickly from the body, and thus exposure of normal tissues is minimized. Maximum tumor uptake of the radionuclide is also very rapid because the small molecule efficiently transverses the tumor vasculature and binds to the primary targeting agent. Its small size may also encourage a more uniform distribution in the tumor. (0007 j Pretargeting methods have used a number of different strategies, but most often involve an avidin/slreptavidin-biotin recognition systern or bi-specific antibodies that co-recognize a tumor antigen and the effector molecule. The avidin/streptavidin system is highly versatile and has been used in several configurations. Antibodies can be coupled with streptavidin or biotin, which is used as the primary targeting agent. This is followed sometime later by the effector molecule, which conjugated with biotin or with avidin/streptavidin, respectively. Another configuration relies on a 3-step approach first targeting a [0008] Pretargeting with a bsAb also requires one arm of the antibody to recognize an effector molecule. Most radionuclide targeting systems reported to date have relied on an antibody to a chelate-metal complex, such as antibodies directed indium-loaded DTPA or antibodies to other chelates. Since the antibody is generally highly selective for this particular chelate-metal complex, new bsAbs would need to be constructed with the particular effector antibody. This could be avoided if the antibody was not specific to the effector, but instead reacted with another substance. In this way, a variety of effectors could be made so long as they also contained the antibody recognition substance. We have continued to develop the pretargeting system originally described by Janevik-Ivanovska et ah that used an antibody directed against a histamine derivative, histaminc-succinyl-glycl (HSG) as the recognition system on which a variety of effector substances could be prepared. Excellent pretargeting results have been reported using a radioiodinated and a rhenium-labeled divalent HSG-containing peptide. In this work, we have expanded this system to include peptides suitable for radiolabeling 90Y, 11 111In, and 177Lu as well as an alternative 99mTc.binding peptide. [0009] Thus, there is a continuing need for immunological agents which can be directed to diseased tissue and can specifically bind to a subsequently administered targetable diagnostic or therapeutic conjugate, and a flexible system that accommodates different diagnostic and therapeutic agents without alteration to the bi-specific or multi-specific antibodies. Objects of the Invention \|0010] It is one object of the present invention to provide a multi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one other arm feat specifically binds a targetable construct feat can be modified for use in a wide variety of diagnostic and feerapeutic applications. (0011] Other objects of fee invention are to provide pre-targeting methods of diagnosis and therapy using fee combination of multi-specific antibody and targetable construct, mefeods of making fee multi-specifics, and kits for use in such methods. [0012] In accomplishing fee foregoing object, fee present inventors have discovered feat it is advantageous to raise multi-specific Abs against a targetable construct that is capable of carrying one or more diagnostic or therapeutic agents. By utilizing this technique, fee characteristics of fee chelator, metal chelate complex, feerapeutic agent or diagnostic agent can be varied to accommodate differing applications, wifeout raising new multi-specific Abs for each new application. Further, by using feis approach, two or more distinct chelators, metal chelate complexes, diagnostic agents or feerapeutic agents can be used wife the inventive multi-specific Ab. Summary of the Invention \|D0I3] The present invention relates to a multi-specific or bi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one ofeer ann feat specifically binds a targetable construct. [0014] Provided is a compound of the formula X-phe-Ly5(HSG)-D-T-Lys(HSG)-Lys(y)-NH2 (SEQ ID NO: 1), where fee compound includes a hard acid cation chelator positioned at X or Y and a soft acid cation chelator positioned at remaining X or Y, The hard acid cation chelator may include a carboxylate or amine group, and may include such chelators as NOTA, DOTA, DTPA, and TETA. The soft acid cation chelator may include a thiol group, and may also include such chelators as Tscg-Cys and Tsca-Cys. A preferred embodiment of feis compound is DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 3) also known as IMP 245. Ofeer embodiments may have a hard acid cation chelator and a soft acid cation chelator in switched positions as provided in (Tscg-Cys)-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(D0TA)-NH2 (SEQ ID NO: 1). [0015] The compound may also include cations bound to the different chelating moeities. For example, hard acid cations may include Group IIa and Group IIIa metal cations, which commonly bind to hard acid chelators. Soft acid cations that may bind to fee soft acid chelators can include the transition metais, lanthanides, actinides and/or Bi, Non exhaustive examples of such soft acid cations include Tc, Re, and Bi. 10016] Also provided is a targetable construct including X-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Y)-NH-R (SEQ ID NO:1). Again, a hard acid cation chelator is positioned at either X or Y, and a soft acid cation chelator is positioned at remaining X or Y. The targetable construct also includes a linker to conjugate the compound to a feerapeutic or diagnostic agent or enzyme "R". The linker may have at least one amino I 1 (B) optionally, administering to the patient a clearing composition, and allowing the composition to clear non-localized antibodies or antibody fragments from circulation; (C) administering to the patient a first targetable construct which comprises a carrier portion which comprises or bears at least one epitope recognizable by the at least one other arm of the bi-specific antibody or antibody fragment, and one or more conjugated therapeutic or diagnostic agents, or enzymes; and (D) when the targetable construct comprises an enzyme, further administering to the patient 1) a prodrugs when the enzyme is capable of converting the prodmg to a drug at the target site; or 2) a drug which is capable of being detoxified in the patient to form an intermediate of lower toxicity , when the enzyme is capable of reconverting the detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of the drug at the target site, or 3) a prodrug which is activated in the patient through natural processes and is subject to detoxification by conversion to an intermediate of lower toxicity, when the enzyme is capable of reconverting the detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of the drrier portion which comprises or bears at least one epitoprug at the target site, or 4) a second targetable construct which comprises a recognizable by the at least one other arm of the bi-specific antibody or antibody fragment, and a prodrug, when the enzyme is capable of converting the prodrug to a drug at the target site. [00191 In another embodiment, the invention provides a kit useful for treating or identifying diseased tissues in a patient comprising: (A) a bi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct; (B) a first targetable construct which comprises a carrier portion which comprises or bears at least one epitope recognizable by the at least one other arm of the bi-specific antibody or antibody fragment, and one or more conjugated therapeutic or diagnostic agents, or enzymes; and (C) optionally, a clearing composition useful for clearing non-localized antibodies and antibody fragments; and (D) optionally, when the first targetable construct comprises an enzyme, 1) a prodrug, when the enzyme is capable of converting the prodrug to a drug at the target site; or 2) a drug which is capable of being detoxified in the patient to form an intermediate of lower toxicity, when the enzyme is capable of reconverting the detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of the drug at the target site, or 3) a prodrug which is activated in the patient through natural processes and is subject to detoxification by conversion to an intermediate of lower toxicity, when the enzyme is capable of reconverting the detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of the drug at the target site, or 4) a second targetable construct which comprises a carrier portion which comprises or bears at least one epitope recognizable by the at least one other arm of the bi-specific antibody or antibody fragment, and a prodrug, when the enzyme is capable of converting the prodrug to a drug at the target site. [0020] Another embodiment of the invention is to provide DNA constructs which encode such antibodies or antibody fragments. Specifically, DNA constructs which produce the variable regions which provide the advantageous properties of reactivity to a targetable construct and reactivity to a disease tissue. In accordance with this aspect of the present invention, there is provided a recombinant DNA construct comprising an expression cassette capable of producing in a host cell a bi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct, wherein the construct comprises, in the 5' to 3' direction of transcription, a transcriptional initiation regulatory region functional in the host cell, a translational initiation regulatory region functional in the host cell, a DNA sequence encoding the bi-specific antibody or antibody fragment, and a transcriptional and translational termination regulatory region functional in the host cell, wherein the bi-specific antibody or antibody fragment is under the control of the regulatory regions. [0021] Anotlier embodiment of the invention provides a method of preparing the antibodies or antibody fragments by recombinant technology. In accordance with this aspect of the present invention, there is provided a method of preparing a bi-specific antibody or antibody fragment having at least one ann that specifically binds a targeted tissue and at least one other arm that specifically binds a targecable construct, comprising: (A) introducing the recombinant DNA construct described above into a host cell; (B) growing the cell and isolating the antibody or antibody fragment [0022] In another embodiment of the present invention there is provided a method of preparing a bi- specific fusion protein having at least one arm that specifically binds to a targeted tissue and at least one other arm that is specifically binds to a targetable construct, comprising: (1) (A) introducing into a host cell a recombinant DNA construct comprising an expression cassette capable of producing in the host cell a fragment of the bi-specific fusion protein, wherein the construct comprises, in the 5' to 3". direction of transcription, a transcriptional initiation regulatory region functional in the host cell, a translational initiation regulatory region functional in the host cell, a DNA sequence encoding a scFv linked to a light-chain antibody fragment, and a transcriptional and translational termination regulatory region functional in the host cell, wherein the fragment of the bi-specific fusion protein is under the control of the regulatory regions; (B) co-introducing into the host cell a recombinant DNA constmct comprising an expression cassette capable of producing in the host cell a Fd fragment which is complementary to the light-chain antibody fragment in (A) and which when associated with the light-chain antibody fragment forms a Fab fragment whose binding site is specific for the targeted tissue, wherein the construct comprises, in the 5' to 3' direction of transcription, a transcriptional initiation regulatory region functional in the host cell, a translational initiation regulatory region functional in the host cell, a DNA sequence encoding a Fd fragment, and a transcriptional and translational termination regulatory region functional in the host cell, wherein the Fd fragment is under the control of the regulatory regions; (C) growing the cell and isolating the bi-specific fusion protein, or (2) (A) introducing into a first host cell a recombinant DNA construct comprising an expression cassette capable of producing in the first host cell a fragment of the bi-specific fusion protein, wherein the construct comprises, in the 5' to 3' direction of transcription, a transcriptional initiation regulatory region functional in the first host cell, a translational initiation regulatory region functional in the first host cell, a DNA sequence encoding a scFv linked to a light-chain antibody fragment, and a transcriptional and translational termination regulatory region functional in the fust host cell, wherein the fragment of the bi-specific fusion protein is under the control of the regulatory regions; (B) introducing into a second host cell a recombinant DNA construct comprising an expression cassette capable of producing in the second host cell a Fd fragment which is complementary to the light-chain antibody fragment ia (2)(A) and which when associated with the light-chain antibody fragment forms a Fab fragment whose binding site is specific for the targeted tissue, wherein the construct comprises, in the S' to 3' direction of transcription, a transcriptional initiation regulatory region functional in the second host cell, a translational initiation regulatory region functional in the second host cell, a DNA sequence encoding a Fd fragment, and a transcriptional and translational tennination regulatory region functional in the second host cell, wherein the Fd fragment is under the control of the regulatory regions; (C) growing the first and second host cells; (D) optionally isolating Che bi-specific fusion protein fragment and the Fd fragment; and (E) combining the fragments to produce a bi-specific fusion protein and isolating the bi'Specific fusion protein. [0023] A variety of host cells can be used to prepare bi-specific antibodies or antibody fragments, including, but not limited to, mammalian cells, insect cells, plant cells and bacterial cells. In one embodiment, the method utilizes a mammalian Tygote, and the introduction of the recombinant DNA construct produces a transgenic animal capable of producing a bi-specific antibody or antibody fragment. 10024] The present invention seeks to provide inter alia a bi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct that can be modified for use in a wide variety of diagnostic and therapeutic applications. [0025] A further embodiment of the invention involves using the inventive antibody or antibody fragment in photodynamic therapy. [0026] A further embodiment of the invention involves using the inventive antibody or antibody fragment in radioimmunoimaging for positron-emission tomography (PET). [00271 A further embodiment of the invention involves using the inventive antibody or antibody fragment in radioimmunoimaging for single-photon emission. \|002SI A further embodiment of the invention involves using the inventive antibody or antibody fragment in magnetic resonance imaging (MRI). [ 0029] A further embodiment of the invention involves using the inventive antibody or antibody fragment in X-ray, computed tomography (CT) or ultrasound imaging. [0030] A further embodiment of the invention involves using the inventive antibody or antibody fragment for intraoperative, endoscopic, or intravascular detection and/or therapy. [0031] A further embodiment of the invention involves using the inventive antibody or antibody fragment in boron neutron capture therapy (BNCT), [0032] A further embodiment of the invention involves using the inventive antibody or antibody fragment for diagnosing or creating diseased tissues (e.g., cancers, infections, inflammations, clots, atherosclerois, infarcts), normal tissues (e.g., spleen, parathyroid, thymus, bone marrow), ectopic tissues (e.g., endometriosis), and pathogens, as well as methods of making the bi-specifics, and kits for use in such methods. [0034] The present inventors have discovered that it is advantageous to raise bsAbs against a targetable construct that is capable of carrying one or more diagnostic or therapeutic agents. By utilizing this technique, the characteristics of the chelator, metal chelate complex, therapeutic agent or diagnostic agent can be varied to accommodate differing applications, without raising new bsAhs for each new application. Further, by using this approach, two or more distinct chelators, metal chelate complexes or therapeutic agents can be used with the inventive bsAb. [0035] The invention relates to a method of treating or identifying diseased tissues in a subject, comprising: (A) administering to said subject a bi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct comprising at least two HSG haptens; (B) optionally, administering to said subject a clearing composition, and allowing said composition to clear non-localized antibodies or antibody fragments from circulation; (C) administering to said subject a targetable construct which comprises a earner portion which comprises or bears at least two HSG haptens and at least one chelator, and may comprise at least one diagnostic and/or therapeutic cation, and/or one or more chelated or chemically bound therapeutic or diagnostic agents, or enzymes; and (D) when said targetable construct comprises an enzyme, fizrther administering to said subject 1) a prodrug, when said enzyme is capa We of converting said prodrug to a drug at the target site; or 2) a drug which is capable of being detoxified in said subject to form an intermediate of lower toxicity, when said enzyme is capable of reconverting said detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of said drug at the target site, or 3) a prodrug which is activated in said subject through natural processes and is subject to detoxification by conversion to an intermediate of lower toxicity, when said enzyme is capable of reconverting said detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of said drug at the target site. \|0036] The invention further relates to a method for detecting or treating target cells, tissues or pathogens in a manunal, comprising; administering an effective amount of a bi-specific antibody or antibody fragment comprising at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct; wherein said at least one arm is capable of binding to a complementary binding moiety on the target cells, tissues or pathogen or on a molecule produced by or associated therewith; and administering a target [0037] The invention further relates to a method of treating or identifying diseased tissues in a subject, comprising: administering to said subject a bi-specific antibody or antibody fragment having at least one ami that specifically binds a targeted tissue and at least one other ann that specifically binds a targetable construct; optionally, administering to said subject a clearing composition, and allowing said composition to clear non-localized antibodies or antibody fragments from circulation; and administering to said subject a targetable constmct selected from the group consisting of: [0038] The invention further relates to a kit useful for treating or identifying diseased tissues in a subject comprising: (A) a bi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one ot\her arm that specifically binds a targetable construct, wherein said construct is selected from the group consisting of (B) a targetable construct which comprises a carrier portion which comprises or bears at least one epitope recognizable by said at least one other arm of said bi-specific antibody or antibody fragment, and one or more conjugated therapeutic or diagnostic agents, or enzymes; and (C) optionally, a clearing composition useful for clearing non-localized antibodies and antibody fragments; and (D) optionally, when said first targetable construct comprises an enzyme 1) a prodrug, when said enzyme is enable of converting said prodrug to a drug at the target site; or 2) a drag which is capable of being detoxified in said subject to foim an intermediate of lower toxicity, when said enzyme is capable of reconverting said detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of said drug at the target site, or 3) a prodrug which is activated in said subject through natural processes and is subject to detoxification by conversion to an intermediate of lower toxicity, when said enzyme is capable of reconverimg said detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of said drug at the target site. (0039) The invention further relates to a targetable construct selected from the group consisting of: [0040] The invention further relates to a method of screening for a targetable construct comprising: contacting said targetable construct with a bi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds said targetable construct to give a mixture; wherein said at least one arm is capable of binding to a complementary binding moiety on the target cells, tissues or pathogen or on a molecule produced by or associated therewith; and optionally incubating said mixture; and analyzing said mixture. (0041] The invention further relates to a method for imaging normal tissue in a mammal, comprising: administering an effective amount of a bi-specific antibody or antibody fragment comprising at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct; wherein said at least one arm is capable of binding to a complementary binding moiety on the target cells, tissues or on a molecule produced by or associated therewith; and administering a targetable construct selected from the group consisting of [0042] The invention further relates to a method of intraoperatively identifying or treating diseased tissues, in a subject, comprising: administering an effective amount of a bi-specific antibody or antibody fragment comprising at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct; wherein said at least one arm is capable of binding to a complementary binding moiety on die target cells, tissues or pathogen or on a molecule produced by or associated therewith; and administering a targetable construct selected from the group consisting of i \|0043] The invention further relates to a method for the endoscopic identification or treatment of diseased tissues, in a subject, comprising: administenng an effective amount of a bi-specific antibody or antibody fragment comprising at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct; wherein said at least one arm is capable of binding to a compiementary binding moiety on the target cells, tissues or pathogen or on a molecule produced by or associated therewith; and administering a targetable construct selected from the group consisting of I0044J The invention further relates to a method for the intravascular identification or treatment of diseased tissues, in a subject, comprising: administering an effective amount of a bi-specific antibody or antibody fragment comprising at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct; wherein said at least one arm is capable of binding to a complementary binding moiety on the target cells, tissues or pathogen or on a molecule produced by or associated therewith; and administering a targetable construct selected from the group consisting of [00451 Additional aspects, features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The embodiments and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the appended claims. Brief Description of the Drawings [0046] Figure 2 schematically illustrates various Abs and bsAbs. [0047] Figure 2 provides SDS-PAGE analysis of purified hMN-14Fab-734scFv. 3 µg of hMN-14 IgG (lanes 1 and 3) or bsAb (lanes 2 and 4) was applied in each lane of a 4-20% polyacrylamide gel under non- reducing (lanes 1 and 2) and reducing (lanes 3 and 4) conditions. [0048] Figure 3 schematically illustrates two bi-specific fusion proteins. [0049] Figure 4 illustrates the production of a DNA construct useful for producing a hMN-14Fab- 734scFv bi-specific fusion protein, \|0050] Figure 5 illustrates the production of a DNA construct useful for producing a hMN-14Fab- 734scFv bi-specific fusion protein, (00511 Figure 6 shows the binding properties of hMN-14 x m679 bsMAb with 111In-labeled IMP-24I divalent HSG-DOTA peptide. Panel A: min.lMP-241 alone on SE-HPLC; Panel B: 111IN-IMP-241 mixed with hMN-14 x 679 bsMAb; Panel C: 111n-IMP-241 added to a mixture containing hMN-14 x m679 bsMAb with an excess of CEA. Chromatograms show the association of the 111n-IMP-241 with the bsMAb (B) and bsMAb/CEA complex (C), lA and other Ep-CAM targets), Le(y) (e.g., B3), A3, KS-1, S100, IL-2, T101, necrosis antigens, folate receptors, angiogenesis markers (e.g., VEGF), tenascin, PSMA, PSA, tumor-associated cytokines, MAGE and/or fragments thereof. Tissue-specific antibodies (e.g., against bone man-ow cells, such as CD34, CD74, etc., parathyroglobulin antibodies, etc.) as well as antibodies against non-malignant diseased tissues, such as fibrin of clots, macrophage antigens of atherosclerotic plaques (e.g., CD74 antibodies), and also specific pathogen antibodies (e.g., against bacteria, viruses, and parasites) are well known in the art. [0056] The peptides can be radiolabeled to a high specific activity in a facile manner that avoids the need for purification. In vivo studies in tumor bearing nude mice showed the radiolabeled peptides cleared rapidly from the body with minimal retention in tumor or normal tissues. When administered 1 to 2 days after a pretargeting dose of the bsAbs, tumor uptake of the radiolabeled peptides increased from 28 to 175-fold with tumor/nontunior ratios exceeded 2:1 to 8:1 within just 3 hour of the peptide injection, which represented a marked improvement over that seen with a 99mTc-anti-CEA Fab' at this same time. The anti-CSAp x anti-HSG F(ab')2 bsAb had the highest and longest retention in the tumor, and when used in combinarioh with the IIIIn-labeled peptide, radiation dose estimates for therapeutic radionuclides, such as 90 and 1500cGy, suggested that as much 12,000 cGy could be delivered to tumors with the kidneys receiving 1500 cGy, but all other tissues receiving 500 cGy, Thus, this pretargeting system is highly flexible, being capable of using a wide array of compounds of diagnostic imaging and therapeutic interest, and by achieving excellent tumor uptake and targeting ratios, is highly promising for use in these applications. [0057J Additionally, encompassed is a method for detecting and/or treating target cells, tissues or pathogens in a mammal, comprising administering an effective amount of a bi-specific antibody or antibody fragment comprising at least one arm that specifically binds a targeted tissue and at least one other arm that spec/fically binds a targetable construct. As used herein, the term pathogen" includes, but is not limited to fungi (e.g., Microsporum, Trichophyton, Epidermophyton, Sporothrix schenckii, Cryptococcus neoformans, Coccidioides immitis, Histoplasma Capsulatum, Blastomyces dermatitidis, Candida albicans), viruses (e.g., human immunodeficiency virus (HIV), herpes virus, cytomegalovirus, rabies vims, influenza vims, hepatitis B vims, Sendai vims, feline leukemia virus, Reo virus, polio virus, human serum parvo-like vims, simian virus 40, respiratory syncytial virus, mouse mammary tumor virus, Varicella-Zoster virus, Dengue virus, rubella virus, measles virus, adenovims, human T-cell leukemia viruses, Epslein-Barr vims, murine leukemia vims, mumps vims, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis vims, wart vims and blue tongue virus), parasites, bacteria (e.g.. Anthrax bacillus, Streptococcus agalactiae, Legionella pneumophilia. Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae. Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum. Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae. Brucella abortus, Mycobacterium tuberculosis and Tetanus toxin), mycoplasma (e.g.. Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivamm, and M. pneumoniae) and protozoans (e.g., Plasmodium falciparum, Plasmodium vivax. Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma bmcei. Schistosoma mansom'. Schistosoma japanicum, Babesia bovis, Elmena lenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis, Onchocerca volvulus, Theileria parva, Taenia hydatigena. Taenia ovis. Taenia saginata, Echinococcus granulosus and Mesocestoides corti). See U.S. Patent No. 5,332,567. 10058] Also provided herein are antibodies and antibody fragments. The antibody fragments are antigen binding portions of an antibody, such as F(ab')2, F(ab)2i Fab', Fab, and the like. The antibody fragments bind to the same antigen that is recognized by the intact antibody. For example, an anti-CD22 monoclonal antibody fragment binds to an epitope of CD22, 10059] The term "antibody fragment" also includes any s}'nthetic or genetically engineered protein feat acts like an antibody by binding to a specific antigen to form a conq)lex. For example, andbody fragments include isolated fragments, "Fv" fragments, consisting of the variable regions of tiie heavy and light chains, recombinant single chain polypeptide molecules in which Jight and heavy diain variable rsgions ars connected by a peptide linker ("sFv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the "hypervariable regioa" Three of these so-called "hypervariable" regions or "complementarity-detennining regions" (CDR) are found in each variable region of the light or heavy chain. Each CDR is flanked by relatively conserved framework regions (FR). The FR are thought to maintain the structural integrity of the variable region. The CDRs of a light chain and the CDRs of a corresponding heavy chain form the antigen-binding site. The "hypervariability" of fee CDRs accounts for the diversity of specificity of antibodies. [0060] As used herein, the term "subject" refers to any ammal (i.e., vertebrates and invertebrates) including, but not limited to humans and other primates, rodents (e-g-, mice, rats, and guinea pigs), lagamorphs (e.g., rabbits), bovines (e.g, cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., swine), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), domestic fowl (e.g., chickens, tuikcys, ducks, geese, other gallinaceous birds, etc.), as well as feral or wild animals, including, but not limited to, such animals as ungulates (e.g., deer), bear, fish, lagamorphs, rodents, birds, etc. It is not intended that the term be limited to a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are encompassed by the term IL Constructs Targetable to Antibodies [0061] The targetable construct can be of diverse structure, but is selected not only to diminish fee elicitation of immune responses, but also for rapid in vivo clearance when used within the bsAb targeting method. Hydrophobic agents are best at eliciting strong immune responses, whereas hydrophilic agents are preferred for rapid in vivo clearance, thus, a balance between hydrophobic and hydrophilic needs to be established. This is accomplished, in part, by relying on fee use of hydrophilic chelating agents to offset the inherent hydrophobicity of many organic moieties. Also, sub-units of fee targetable construct may be chosen which have opposite solution properties, for example, peptides, which contain amino acids, some of which are hydrophobic and some of which are hydrophih'c. Aside from peptides, carbohydrates may be used. j0065\| The peptides to be used as immunogens are synthesized conveniently on an automated peptide synthesizer using a solid-phase support and standard techniques of repetitive orthogonal deprotection and coupling. Free amino groups in the peptide, that are to be used later for chelate conjugation, are advantageously blocked with standard protecting groups such as an acetyl group. Such protecting groups wilL be known to the skilLed artisan. See Greene and Wuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons, N.Y.). When the peptides are prepared for later use within the bsAb system, they are advantageously cleaved from the resins to generate the corresponding C-terminal amides, in order to inhibit in vivo carboxypeptidase activity. in. Chelate Moieties (0066] The presence of hydrophilic chelate moieties on the targetable construct helps to ensure rapid in vivo clearance. In addition to hydrophilicity, chelators are chosen for their metal-binding properties, and may be changed at will since, at least for those targetable constructs whose bsAb epitope is part of the peptide or is a non-chelate chemical hapten, recognition of the metal-chelate complex is no longer an "- " ~"—" J"— — .^_,,,. __, - ,, ,_ _ ^, DOT A, and TETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) are of use with a variety of metals and radiometals, most particularly with radionuclides of Ga, Y and Cu, respectively. [0068] DTPA and DOTA-type chelators, where the ligand includes hard base chelating functions such as carboxylate or amine groups, are most effective for chelating hard acid cations, especially Group IIa and Group IIIa metal cations. Such metal-chelate complexes can be made very stable by tailoring the ring size to the metal of interest. Other ring-type chelators such as macrocyclic polyethers are of interest for stably binding nuclides such as 223Ra for RAIT. Porphyrin chelators may be used with numerous radiometals, and are also useful as certain cold metal complexes for bsAb-directed immuno-phototherapy. Also, more than one type of chelator may be conjugated to the targetable construct to bind multiple metal ions, e.g., cold ions, diagnostic radionuclides and/or therapeutic radionuclides. the linker, e.g., with different chelate ring sizes, to bind preferentially to two different hard acid or soft acid cations, due to the differing sizes of the cations, the geometries of the chelate rings and the preferred complex ion structures of the cations. This will permit two different metals, one or both of which may be radioactive or useful for MRI enhancement, to be incorporated into a linker for eventual capture by a pretargeted bsAb. [0073] Preferred chelators include NOT A, DOTA and Tscg and combinations thereof. These chelators have been incorporated into a chelator-peptide conjugate motif as exemplified in the following constructs: (0074) The chelator-peptide conjugates (f) and (g), above, has been shown to bind 68Ga and is thus useful in positron emission tomography (PET) applications. 10075] Chelators are coupled to the peptides of the targetable construct using standard chemistries, some of which are discussed more fully in the working examples below. Briefly, the synthesis of the peptide Ac-Lys(HSG)-D-Tyr-Lys(HSO)-Lys(Tscg-Cys)-NH2 (SEQ ID NO; 4)was accomplished by first attaching Aloc-Lys(Fmoc)-OH to a Rink amide resin on the peptide synthesizer. The protecting group abbreviations "Aloe" and "Fnioc" used herein refer to die groups aliyloxycarbonyl and fluorenyimethyloxy carbonyl. The FmoC"Cys(Trt)-OH and TscG were then added to the side chain of the lysine using standard Fmoc automated synthesis protocols to form the following peptide: Aloc-Lys(Tscg-Cys(Trt))-rink resin. The Aloc group was then removed. The peptide synthesis was then continued on the synthesizer to make the ibJIowing peptide: Lys(A]oc)-D-Tyr-Lys(Aloc).Lys(Tscg"Cys(Trt)-rink resin (SEQ ID NO: 4). Following N-terminus acylation, and removal of the side chain Aloe protecting groups. The resulting 10076] Chelator-peptide conjugates may be stored for long periods as solids. They may be metered into unit doses for metal-binding reactions, and stored as unit doses either as solids, aqueous or semi-aqueous solutions, frozen solutions or lyophilized preparations. They may be labeled by well-known procedures, [0077] Typically, a hard acid cation is introduced as a solution of a convenient salt, and is taken vp by the hard acid chelator and possibly by the soft acid chelator. However, later addition of soft acid cations leads to binding thereof by the soft acid chelator, displacing any hard acid cations which may be chelated therein. For example, even in the presence of an excess of cold IIIInCl3, labeling with 99m-Tc(V) glucoheptonate or with Tc cations generated in situ with stannous chloride and Na99m-Tc04 proceeds quantitatively on the soft acid chelator. identify and treat them when they are ectopic (i.e,, displaced torn their normal location), such as in endometriosis. [0080] The administration of a bsAb and the targetable construct discussed above may be conducted by administering the bsAb at some time prior to administration of the therapeutic agent which is associated with the linker moiety. The doses and liming of the reagents can be readily devised by a skilled artisan and are dependent on the specific nature of the reagents employed. If a bsAb-F(ab')2 derivative is given first, then a waiting time of 1-6 days before administration of the targetable construct may be appropriate. If an IgG-Fab' bsAb conjugate is the primary targeting vector, then a longer waiting period before administration of the linker moiety may be indicated, in the range of 3-15 days. Alternatively, the bsAb and the targetable construct may be administered substantially at the same time in either a cocktail fonn or by administering one after the other. \|0081] A wide variety of diagnostic and therapeutic reagents can be advantageously conjugated to the targetable construct. Generally, diagnostic and therapeutic agents can include isotopes, drugs, toxins, cytokines, conjugates with cytokines, hormones, growth factors, conjugates, radionuclides, contrast agents, metals, cytotoxic drugs, and immune modulators. For example, gadolinium metal is used for magnetic resonance imaging and fluorochromes can be conjugated for photodynamic therapy. Moreover, contrast agents can be MRI contrast agents, such as gadolinium ions, lanthanum ions, manganese ions, iron, chromium, copper, cobalt, nickel, dysprosium, rhenium, europium, terbium, holmium, neodymium or other comparable label, CT contrast agents, and ultrasound contrast agents. Addtional diagnostic agents can include fluorescent labeling compounds such as fluorescein isothiocyanate, rhodamine, phycocrytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine, chemiluminescent compounds including luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester, and bioluminescent compounds including luciferin, luciferase and aequorin. Radicmuclides the art. Therapeutic agents may also include, without limitation, others drugs, prodrugs and/or toxins. The terms "drug," 'prodrug," and "toxin" are defined throughout the specification. The terms "diagnostic agent" or "diagnosis" include, but are not limited to, detection agent, detection, or localization. \|0083] When the targetable construct includes a diagnostic agent, the bsAb is preferably administered prior to administration of the targetable construct with the diagnostic agent After sufficient time has passed for the bsAb to target to the diseased tissue, the diagnostic agent is administered, by means of (he targetable construct, so that imaging can be performed. Tumors can be detected in body cavities by means of directly or indirectly viewing various structures to which light of the appropriate wavelength is delivered and then collected, or even by special detectors, such as radiation probes or fluorescent detectors, and the like. Lesions at any body site can be viewed so long as nonionizing radiation can be delivered and recaptured from these structures. For example, PET which is a high resolution, non-invasive, imaging technique can be used with the inventive antibodies and targetable constructs for the visualization of human disease. In PET, 511 ke V gamma photons produced during positron annihilation decay are detected. X-ray, computed tomography (CT), MRl and gamma imaging (e.g.. Single Photon Emission Computed Tomogr^hy (SPECT)) may also be utilized through use of a diagnostic agent that functions with these modalities. 10084] As discussed eariier, the targetable construct may include radioactive diagnostic agents that emit [0085] The present bsAbs or bsFabs can be used in a method of photodynamic therapy (PUT) as discussed in U.S. Patent Nos, 6,096,289; 4,331.647; 4,818,709; 4,348,376; 4,361,544; 4,444,744; 5,851,527. In PDT, a photosensitizer, e.g., a hematoporphyrin derivative such as dihematopoiphyiin ether, is administered to a subject. Anti-tumor activity is initiated by the use of light, e.g., 630 nm. Alternate photosensitizers can be utilized, including those useful at longer wavelengths, where skin is less photosensitized by the sun. Examples of such photosensitizers include, but arc not limited to, benzoporphyrin monoacid ring A (BPD-MA), tin etiopurpurin (SnET2), sulfonated aluminum phthalocyanine (AlSPc) and lutetium texaphyrin (Lutex). [0086] Additionally, in PDT, a diagnostic agent may be injected, for example, systematically and laser-induced fluorescence can be used by endoscopes including wireless capsule-sized endoscopes or cameras to detect sites of cancer which have accreted the light-activated agent. For example, this has been applied to fluorescence bronchoscopic disclosure of early lung tumors. Doiron et al Chest 76:32 (1979). In another example, the antibodies and antibody fragments can be used in single photon emission. For example, a Tc- 00871] Therapeutically useful immunoconjugates can be obtained by conjugating photoactive agents or dyes to an antibody composite. Fluorescent and other chromogens, or dyes, such as porphyrins sensitive to visible light, have been used to detect and to treat lesions by directing the suitable light to the lesion. In therapy, this has been termed photoradiation, phototherapy, or photodynamic therapy (Jori et al (eds.), include iodine compounds, barium compounds, gallium compounds, thallium compounds, etc. Specific compounds include barium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid, iocetamic acid, iodamide, iodipamlde, iodoxamic acid, iogulamide, iohexol, iopamidol, iopanoic acjd, ioprocemic acid, josefamic acid, ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, totetric acid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallous chloride. Ultrasound contrast materia] may also by used including dcxtrsn and Uposomes, particularly gas-filled liposomes. In one embodiment, an immunomodulator, such as a cytokine, may also be conjugated to the targetable construct by a linker or through other methods known by those skilled in the construct having a low MW hapten is administered. After the enzyme is pretargeted to the target site by bsAb;targetable construct binding, a cytotoxic drug is injected that is known to act at the target site. The dnig may be one which is detoxified by the mammal's ordinary detoxification processes to form an intermediate of lower toxicity. For example, the drug may be converted into the potentially less toxic glucuronide in the liver. The detoxified intermediate can then be reconverted to its more toxic form by the pretargeted enzyme at the target site, and this enhances cytotoxicity at the target site. (00901 Alternatively, an administered prodrug can be converted to an active drug by the pretargeted enzyme. The pretargeted enzyme improves the efficacy of the treatment by recycling the detoxified drug. This approach can be adopted for use with any enzyme-drug pair. Alternatively, the targetable construct with enzyme can be mixed with the targeting bsAb prior to administration to the patient. After a sufficient time has passed for the bsAb:targetable construct- conjugate to localize to the target site and for unbound targetable construct to clear from circulation, a prodrug is administered. As discussed above, the prodrug is then converted to the drug in situ by the pre-targeted enzyme. [0091] Certain cytotoxic drugs that are useful for anticancer therapy are relatively insoluble in serum Some are also quite toxic in an unconjugated form, and their toxicity is considerably reduced by conversion to prodrugs. Conversion of a poorly soluble drug to a more soluble conjugate, e.g:, a glucuronide, an ester of a hydrophilic acid or an amide of a hydrophilic amine, will improve its solubility in the aqueous phase of serum and its ability to pass through venous, arterial or capillary cell walls and to reach the interstitial fluid bathing the tumor. Cleavage of the prodrug deposits the less soluble drug at the target site. Many examples of such prodrug-to-drug conversions are disclosed in U,S, Patent No. 5,851,527, to Hansen. [0094] Eloposide is a widely used cancer drug that is detoxified to a major extent by fonnation of its glucuronide and is within the scope of the invention. See, e.g., Hande et aL Cancer Res. 48:1829-1834 (1988). Glucuronide conjugates can be prepared from cytotoxic dmgs and can be injected as therapeutics for tumors pre-targeted with mAb-glucuronidase conjugates. See, e.g., Wang et aL Cancer Res, 52:4484 4491 (1992). Accordingly, such conjugates also can be used with the pre-targeting approach described here. Similarly, designed prodrugs based on derivatives of daunomycin and doxorubicin have been described for use with carboxylesterases and glucuronidases. See, c.g., Baikina et aL J. Med Chem. ' 40:4013-4018 (1997), Other examples of prodrug/enzyme pairs that can be used within the present invention include, but are not limited to, glucuronide prodrugs of hydroxy derivatives of phenol mustards and beta-g/ucuronidase; phenol mustards or CPT-l I and carboxypeptidase; methotrexate-substituted alpha-amino acids and carboxypeptidase A; penicillin or cephalosporin conjugates of drugs such as 6-mercaptopurine and doxorubicin and beta-lactamase; etoposide phosphate and alkaline phosphatase.[ [0095] The enzyme capable of activating a prodrug at the target site or improving the efficacy of a normal therapeutic by controlling the body's detoxification pathways may altematively be conjugated to the hapten. The enzyme-hap en conjugate is administered to the subject following administration of the pre-targeting bsAb and is directed to the target site. After the enzyme is localized at the target site, a cytotoxic drug is injected, which is known to act at the target site, or a prodrug form thereof which is converted to the drug in situ by the pretargeted enzyme. As discussed above, the drug is one which is detoxified to form an intermediate of lower toxicity, most commonly a glucuronide, using the mammal's ordinary detoxification processes. The detoxified intermediate, e.g., the glucuronide, is reconverted to its more toxic form by the pretargeted enzyme and thus has enhanced cytotoxicity at the target site. This results in a recycling of the drug. Similarly, an administered prodrug can be converted to an active drug through normal biological processes. The pretargeted enzyme improves the efficacy of the treatment by lecycling the detoxified drug. This approach can be adopted for use with any enzyme-drug pair. [0096] In an alternative embodiment, the enzyme-hapten conjugate can be mixed with the targeting bsAb prior to administration to the patient. After a sufficient time has passed for the enzyme-hapten-bsAb conjugate to localize to the target site and for unbound conjugate to clear from circulation, a prodrug is administered. As discussed above, the prodrug is then converted to the drug in situ by the pre-targeted enzyme, [0097] The invention further contemplates the use of the inventive bsAb and the diagnostic agent(s) in the context of Boron Neutron Capture Therapy (BNCT) protocols, BNCT is a binary system designed to deliver ionizing radiation to tumor cells by neutron irradiation of tumor-localized 10B atoms. BNCT is based on the nuclear reaction which occurs when a stable isotope, isotopically eiuiched 10B (present in 19.8% natural abundance), is irradiated with thermal neutrons to produce an alpha particle and a 7Li nucleus. These particles have a path length of about one cell diameter, resulting in high linear energy transfer. Just a few of the short-range 1.7 MeV alpha particles produced in this nuclear reaction are sufficient to target the cell nucleus and destroy it. Success with BNCT of cancer requires methods for localizing a high concentration of 10B at tumor sites, while leaving non-target organs essentially boron-free. Compositions and methods for treating tumors in subjects using pre-targeting bsAb for BNCT are described in U.S. Patent No. 6,228,362 and can easily be modified for the pujposes of the present invention. (00981 In another embodiment of the present invention, the peptide backbone of the targetabie construct is conjugated to a prodrug. The pre-targeting bsAb is administered to the patient and allowed to localize to the target and substantially clear circulation. At an appropriate later time, a targetabie construct comprising a prodrug, for example poly-glutamic acid (SN-38-ester)10 is given, thereby localizing the prodrug specifically at the tumor target. Jt is known that tumors have increased amounts of enzymes released from intracellular sources due to the high rate of lysis of cells within and around tumors. A practitioner can capitalize on this fact by appropriately selecting prodrugs capable of being activated by these enzymes. For example, carboxylesterase activates the prodrug poly-glutamic acid (SN-38-ester)10 by cleaving the ester bond of the poly-glutamic acid (SN-38-ester)10 releasing large concentrations of free SN-38 at the tumor. Alternatively, the appropriate enzyme also can be targeted to the tumor site. /0099J After cleavage from the targetabie construct, the drug is intemalized by the tumor cells. Alternatively, the drug can be intemalized as part of an intact complex by virtue of cross-linking at the target. The targetabie construct can induce internalization of tumor-bound bsAb and thereby improve the efficacy of the treatment by causing higher levels of the drug to be internalized, [0100] A-vaiiety of peptide caniers-are-well-suited for conjugation to prodrugs,, including.polyaniino_ acids, such as polylysine, polyglutamic (E) and aspartic acids (D), including D-amino acid analogs of the same, co-polymers, such as poly(Lys-G3u} {poly[KE]}, advantageously from 1:10 to 10:1. Copolymers based on amino acid mixtures such as poly(Lys-Ala-Glu-Tyr (SEQ ID NO: 8) (KAEY; 5:6:2:1) can also be employed. Smaller polymeric carriers of defined molecular weight can be produced by solid-phase peptide synthesis techniques, readily producing polypeptides of from 2-50 residues in chain length. A second advantage of this type of reagent, other than precise structural definition, is the ability to place single or any desired number of chemical handles at certain points in the chain. These can be used later for attachment of recognition and therapeutic haptens at chosen levels of each moiety. [0101] Poly(ethylene) glycol [PEG] has desirable in vivo properties for a bi-specific antibody prodrug approach. Ester linkages between the hydroxyl group of SN-38 and both ends of a standard di-hydroxyl PEG can be introduced by insertion of diacids such as succinic acid between the SN-38 and PEG hydroxy] groups, to generate species such as SN-38-O-CO(CH2)2CO-O-PEG-O-CO(CH2)2CO-OSN-38. The di-SN-38-PEG produced can be considered as the shortest member of the class of SN-38-polymer prodrugs. The desirable in vivo properties ofPEG derivatives and the limited loading capacity due to their dimeric functionality led to the preparation of PEG co-polymers having greater haptcn-bearing capacity such as those described by Poiani et al See, e.g., Poiani et al Bioconjugate Chem,, 5:621-630,1994. PEG derivatives are activated at both ends as their bis(succinimidyl)carbonate derivatives and co-polymerized with multi-functional diamines such as lysine. The product of such co-polymerization, containing (-Lys(COOH)-PEG-Lys(COOH)-PEG-)n repeat units wherein the lysyl carboxyl group is not involved in the polymerization process, can be used for attachment of SN-38 residues. The SN-38 residues are reacted with the free carboxyl groups to produce SN-38 esters of the (-Lys-(COOH)-PEG-Lys(COOH)-PEG-)n chain. [0102] Other synthetic polymers that can be used to cany recognition haptens and prodrugs include N-(2-hydroxypropyl)methacrylamide (HMPA) copolymers, poly(styrene-co-maleic acid/anhydride (SMA), poly(divinylether maleic anhydride) (DIVEMA), polyethyleneimine, ethoxylated polyethylene-imine, starburst dendrimers and poly(N-vinylpyrrolidone) fPVP). As an example, DIVEMA polymer comprised of multiple anhydride units is reacted with a limited amount of SN-38 to produce a desired substitution ratio of drug on the polymer backbone. Remaining anhydride groups are opened under aqueous conditions to produce free carboxylate groups. A limited number of the free carboxylate groups are activated using standard water-soluble peptide coupling agents, e.g. l-ethyI-3-(3-dimethyIaminopropyl)carbodiimide hydrochloride (EDO),-and coupled to a recognition moiety bearing a free amino group. An example of the latter is histamine, to which antibodies have been raised in the past [0103] A variety of prodrugs can be conjugated to the targetable construct The above exemplifications of polymer use are concerned with SN-38, the active metabolite of the prodrug CPT-l 1 (irinotecan). SN-38 has an aromatic hydroxyl group that was used in the above descriptions to produce aryl esters susceptible to esterase-type enzymes. Similarly the camptothecin analog topotecan, widely used in chemotherapy, has an available aromatic hydroxyl residue that can be used in a similar manner as described for SN-38, producing esterase-susceptible polymer-prodrugs. [0104] Doxorubicin also contains aromatic hydroxyl groups that can be coupled to caiboxylate-containing polymeric carriers using acid-catalyzed reactions similar to those described for the camptothecin family. Similarly, doxorubicin analogs like daunomycin, epirubicin and idarubicin can be coupled in the same manner. Doxorubicin and other drugs with amino 'chemical handles, active enough for chemical coupling to polymeric carriers can be effectively coupled to carrier molecules via these free amino groups in a number of ways. Polymers bearing free carboxylate groups can be activated en situ (EDC) and the activated polymers mixed with doxorubicin to directly attach the drug to the side-chains of the polymer via amide bonds. Amino-containing drugs can also be coupled to amino-pendant polymers by mixing commercially available and cleavable cross-linking agents, such as ethylene glycobis{succinimidylsuccinate) (EGS, Pierce Chemical Co., Rockford, IL) or bi5-[2-(5uccinimido-oxycarbonyloxy)ethyl]sulfone {BSOCOES, Molecular Biosciences, Huntsville, AL), to cross-link the two amines as two amides after reaction with the his(succinimidyl) ester groups. This is advantageous as these groups remain susceptible to enzymatic cleavage. For example, (doxorubicin-EGS)n-poly-lysine remains susceptible to enzymatic cleavage of the diester groups in the EGS linking chain by enzymes such as or It can be injected and localized at the target site and, after non-targeted antibody has substantially cleared from the circulatory system of the mammal, enzyme can be injected in an amount and by a route which enables a sufficient amount of the enzyme to reach a localized antibody or antibody fragment and bind to it to form the antibody-enzyme conjugate in situ. toxicity from the detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of the dnig at the target site. A second targetable construct may also be used which comprises a carrier portion which comprises or bears at least one epitope recognizable by the at least one other arm of the bi-specific antibody or antibody fragment, and a prodrug, when the enzyme is capable of converting the prodrug to a drug at the target site. Instruments which facilitate identiiying or treating diseased tissue also can be included in the kit. Examples include, but are not linuted to application devices, such as syringes. Solutions required for utilizing the disclosed invention for identifying or treating diseased tissue also can be included in the kit. (01161 The targetable construct may be administered intravenously, intraaterially, intraoperatively, endoscopically, intraperitoneally, intramuscularly, subcutaneously, intrapleurally, intrathecally, by perfusion through a regional catheter, or by direct intralesional injection, and can be by continuous infusion or by single or multiple boluses, or through other methods known to those skilled in the art for diagnosing (detecting) and treating diseased tissue. Further, the targetable construct may include agents for other methods of detecting and treating diseased tissue including, without limitation, conjugating dextran or liposome formulations to the targetable construct for use with ultrasound, or other contrast agents for use with other imaging modalities, such as X-ray, CT, PET, SPECT and ultrasound, as previously described VI. Methods for Raising Antibodies j01l7) Abs to peptide backbones and/or haptens are generated by well-known methods for Ab production. For example, injection of an immunogen, such as (peptide)n-KLH, wherein KLH is keyhole limpet hemocyanin, and n=I-30, in complete Freund's adjuvant, followed by two subsequent injections of the same immunogen suspended in incomplete Freund's adjuvant into immunocompetent animals, is followed three days after an i.v. boost of antigen, by spleen cell harvesting. Harvested spleen cells are then fused with Sp2/0-Agl4 myeloma cells and culture supematants of the resulting clones aruilyzed for anti-pepdde reactivity using a direct-binding ELISA. Fine specificity of generated Abs can be analyzed for by using peptide fragments of the original immunogen. These fragments can be prepared readily using an automated peptide synthesizer. For Ab production, enzyme-deficient hybridomas are isolated to enable selection of fused cell lines. This technique also can be used to raise antibodies to one or more of the chelates comprising the linker, e.g., ln(ni)-DTPA chelates. Monoclonal mouse antibodies to an In(in)-di-DTPA are known (Barbel 395 supra). [0118] The antibodies used in the present invention are specific to a variety of cell surface or intracellular tumor-associated antigens as marker substances. These markers may be substances produced by the tumor or may be substances which accumulate at a tumor site, on tumor cell surfaces or within tumor cells, whether in the cytoplasm, the nucleus or in various organelles or sub-cellular structures. Among such tumor-associated maricers are those disclosed by Herberznan, "Immunodiagnosis of Cancer", in Fleishercd., 'The Clinical Biochemistry of Cancer", page 347 (American Association of Clinical Chemists, 1979) and in U.S. Patent Nos. 4.150,149; 4,361.544; and 4,444,744. See also U,S. Patent No. 5,965,132, to Thorpe et aL, U.S. Patent 6,004,554, to Thorpe et al., U.S. Patent No. 6,071,491, to Epstein et al., U.S. Patent No. 6,017,514, to Epstein et al., U.S. Patent No. 5,882,626, to Epstein et al., U.S. Patent No. 5,019,368, to Epstein et al., and U.S. Patent No. 6,342,221, to Thorpe et al, all of which are incorporated herein by reference. [0119] Tumor-associated markers have been categorized by Herberman, supra, in a number of categories including oncofetal antigens, placental antigens, oncogenic or tumor vims associated antigens, tissue associated andgens, organ associated antigens, ectopic hormones and normal andgens or variants thereof. Occasionally, a sub-unit of a tumor-associated marker is advantageously used to raise antibodies having higher tumor-specificity, e.g., the beta-subunit of human chorionic gonadotropin (HCG) or the gamma region of carcino embryonic antigen (CEA), which stimulate the production of antibodies having a greatly reduced cross-reactivity to non-tumor substances as disclosed in U.S. Patent Nos, 4,361,644 and 4,444,744. Markers of tumor vasculature (e.g,, VEGF), of tumor necrosis (Epstein patents), of membrane receptor (e.g., folate receptor, EGFR), of transmembrane antigens (e.g., PSNfA), and of oncogene products can also serve as suitable tumor-associated targets for antibodies or antibody fragments. Markers of normal cell constituents which are expressed copiously on tumor cells, such as B-cell complex antigens (e.g., CD19, CD20, C021, CD22, CD23, and HLA-DR on B-cell malignancies), as well as cytokines expressed by certain tumor cells (e.g., IL-2 receptor in T-cell malignancies) are also suitable targets for the antibodies and antibody fragments of this invention. Other well-known tumor associated antigens that can be taigeted by the antibodies and antibody fragments of this invention include, but are not limited to, CEA, CSAp, TAG-72, MUC-1, MUC-2, MUC-3, MUC-A, EGP-I, EGP-2, BrE3, PAM-4, KC-4, A3. KS-1, PSMA, PSA, tenascin, TlOl, SlOO, MAGE, HLA-DR, CD19, CD20, CD22, CD30, and CD74. [0120] Another marker of interest is transmembrane activator and CAML-interactor (TAd). See Yu et al Nat Immunol 7252-256 (2000). Briefly, TACI is a marker for B-cell malignancies (e.g,, lymphoma). Further it is known that TACI and B-cell maturation antigen (BCMA) are bound by the tumor necrosis factor homolog a proliferation-inducing ligand (APRIL). APRIL stimulates in vitro proliferation of primary B and T cells and increases spleen weight due to accumulation of B cells in vivo. APRIL also competes with TALL-I (also called BLyS or BAFF) for receptor binding. Soluble BCMA and TACI specifically prevent binding of APRIL and block APRIL-stimulated proliferation of primary B cells, BCMA-Fc also inhibits production of antibodies against keyhole limpet hemocyanin and Pneumovax in mice, indicating that APRIL and/or TALL-I signaling via BCMA and/or TACI are required for generation of humoral immunity. Thus, APRIL-TALL-I and BCMA-TACI foim a two ligand-two receptor patiiway involved in stimulation of B and T cell function. \|0121] After the initial raising of antibodies to the immunogen, the antibodies can be sequenced and subsequently prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art. For example, humanized monoclonal antibodies are produced by transfenring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then, substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by the publication of Oriandi et al, Proc, Nat'lAcad. Set USA 86:3833 (1989), which is stepwise process beginning with the formation of yeast artificial chromosomes (YACs) containing either human heavy- or light-chain immunoglobulin loci in germline configuration. Since each insert is approximately 1 Mb in size, YAC construction requires homologous recombination of overlapping fragments of the immunoglobulin loci. The two YACs, one containing the heavy-chain loci and one containing the CLINICAL APPLICATION, Ritter et al (eds.), pages 166-179 (Cambridge University Press 1995); and Ward et aL "Genetic Manipulation and Expression of Antibodies,** in MONOCLONAL ANTIBODIES: recipient female and allowed to gestate. After birth, the progeny are screened for the presence of both transgenes by Southern analysis. In order for the antibody to be present, both heavy and light chain -genes must be expressed concurrently in the same cell. Milk from transgenic females is analyzed for the presence and functionality of the antibody or antibody fragment using standanrd immunological methods known in the art The antibody can be purified from the milk using standard methods known in the art [0129] A chimeric Ab is constructed by ligating the cDNA fragment encoding the mouse light variable and heavy variable domains to fragment encoding the C domains from a human antibody. Because the C domains do not contribute to antigen binding, the chimeric antibody will retain the same antigen specificity as the original mouse Ab but will be closer to human antibodies in sequence. Chimeric Abs still contain some [0133] Recombinant methods can be used to produce a variety of fusion proteins. For example a fusion protein comprising a Fab fragment derived from a humanized monoclonal anti-CEA antibody and a scFv derived from a murine anti-diDTPA can be produced A flexible linker, such as GGGS (SEQ ID NO: 10) connects the scFv to the constant region of the heavy chain of the anti-CEA antibody. Alternatively, the scFv can be connected to the constant region of the light chain of hMN-14. Appropriate linker sequences necessary for the in-frame connection of the heavy chain Fd to Ae scFv are introduced into the VL and gestate. After birth, the progeny are screened for the presence of the introduced DNA by Southern analysis. Milk from transgenic females is analyzed for the presence and functionality of the bscAb using standard immunological methods known in the art The bscAb can be purified from the milk using standard methods known in the art. Transgenic production of bscAb in milk provides an efficient method for obtaining large quantities of bscAb, counterparts, are disclosed in U.S. Patent No, 5,874,540. Also preferred are bi-specific antibodies which incorporate one or more of the CDRs of Mu-9 or 679, The antibody can also be a fusion protein or abi-specific antibody that incorporates a Class-Ill anti-CEA antibody and the Fv of 679. Class-Ill antibodies, including Class-Ill anti-CEA are discussed in detail in US, Patent No. 4,818,709, using one equivalent (relative to HSG) of N-hydroxybenzotriazole, one equivalent of benzotrazole-1-yl-oxy-tris-(dimethylamino)phosphoniuni hexafluorophosphate (BOP) and two equivalents of diisopropylethylamine. The activated substrate was mixed with the resin for 15 hr at room temperature. [0158] The results of the biodistribution studies of the peptide in the mice pretargeted with hMN-14 x m679 are shown in Table 1, The tumor to non-tumor ratios of the peptides in the pretargeting study are show in Table 2. Table 1 Prelargeting With III Labeled Peptide 24 hr After Injection of hMN-l4 x m679 % Injected/g Tissue show in 1 able 4, The data in Table 5 shows the biodistribution of the peptides in mice that were not pretreated with the bi-specific antibody. Table 3 Table 4 Table S Biodistribution of 111 In Labeled Peptides Alone Example 4) Synthesis of a Peptide Anticen (01701 The peptide, Ac-Phe-Lys(Ac)-Tyr-Lys(Ac)-OH (SEQ ID NO: 2), is assembled using a resin for solid-phase synthesis and attaching the first residue (lysine) to the resin as the differentially protected derivative alpha-Fmoc-Lys(Aloc)-OH. The alpha-Fmoc protecting group is selectively removed and die Fmoc-Tyr(OBut), alpha-Fmoc-Lys(Aloc)-OH, and Fmoc-Phe-OH added with alternate cycles of coupling and alpha-amino group deprotection. The Aloe - and OBut- side-chain protecting groups are then removed by reaction with TFA and the free alpha- and epsilon-amino groups are capped by reaction with acetic anhydride to give Ac-Phe-Lys(Ac)-Tyr.Lys(Ac)-OH (SEQ ID NO: 2). Example 5) Coupling of Ac-Phe-Lys)-Ac)-Tvr-Lys(AcM)H(SEQ ID NO: 2) to KLH [0171] The peptide, Ac-Phe-Lys(Ac)-Tyr-Lys(Ac)-OH (SEQ ID NO: 2), dissolved in water and pH-adjusted to 4.0 with IN HCl, is treated with a molar equivalent of l-ethyl-3(3-dimethylaminopropyl) carbodiimide and allowed to react for 1 h at 4°C. Keyhold limpet hemocyanin(KLH) buffered at pH 8.5 is treated with a 100-fold molar excess of the activated peptide and the conjugation reaction is allowed to proceed for 1 h at 4oC. The peptide-KLH conjugate is purified from unreacted peptide by size-exclusion chromatography and used for antibody production. Example 6) Generation of an Anti-Peptide Ah Example 9) Reduction of Anti-Peptide-Ab to Fab'-SH DTi [0175] The anti-peptide-F(ab ') is reduced to a Fab' fragment by reaction with a freshly prepared cysteine solution in 0,1M PBS, containing 10mM EDTA. The progress of the reaction is followed by HPLC, and when complete, in about 1 h, the Fab*-SH is purified by spin-column chromatography and stored in deoxygenated buffer at pH chromatography and reacted with excess iodoacetamide to block hinge-region thiol groups and prevent reassociation. After repurification from excess iodoacetamide the Fab' is reacted with a 400-fold molar excess of the galactosylation agent, the ihio-imidate of cyanomethyl-23,4,6-tetra-0-acctyl-l-thio-beta-D-galactopyranoside (see Karacay et aJ.). The galactosylated protein is purified by two spin-columns and the galactose:Fab' radio detennined by MALDI-MS. Example 17) Use of anti-CEA-IgG x anti-Peptide Fab' Bi-specific Ab for RAIT, with absAb Clearing Step [01831 A patient with a CEA-expressing tumor burden is given anti-CEA-IgG (MN-14) x anti-peptide-Fab' bi-specific Ab. Three days later, the patient is given a clearing dose of galactose-WI2-Fab', Twenty-four hours after the clearing dose of a galactose-W12-Fab', the patient is given Y-90-di-Bz-DTPA-peptide. The y-90-labeled peptide clears rapidly from non-target tissue but localizes avidly to sites pretargeted with the anti-CEA-IgG x anti-peptide-Fab' bi-specific Ab, effecting destruction of tumors. Example 18) Synthesis of Ac-Lvs(DTPA)Tyr-Lys(DTPA)-Ly(Tscg-Cys)-NH2 (SEQ ID NO: 7)- (IMP 192) \|0184] The first amino acid, Aloc-Lys(Fmoc)-OH was attached to 0.21 mmol Rink amide resin on the peptide synthesizer followed by the addition of the Tc-99m ligand binding residues Fmoc-Cys(Trt)-OH and TscG to the side chain of the lysine using standard Fmoc automated synthesis protocols to fbnn the following peptide: AloC-Lys(TscG-Cys(Trt)-rink resin. The Aloe group was then removed by treatment of the resin with 8 mL of a solution containing 100 mg Pd[P(Ph)3]4 dissolved in 10 mL CH2Cl2, 0.75 mL glacial acetic acid and 2.5 ml diisopropylethyl amine. The resin mixture was then treated with 0.8 ml tributyhin hydride and vortex mixed for 60 min. The peptide synthesis was then continued on the synthesizer to make the following peptide: Lys(Aloc)-Tyr-Lys(Aloc)-Lys(Tscg-Cys)-rink resm Activated DTPA and DTPA Addition [0185] The DTPA, 5 g was dissolved in 40 mL 1.0 M tetrabutylammonium hydroxide in methanol. The methanol was removed under hi-vacuum to obtain a viscous oil. The oil was dissolved in 50 mL DMF and the volatile solvents were removed under hi-vacuum on the rotary evaporator. The DMF treatment was repeated two more times. The viscous oil was then dissolved in 50 ml DMF and mixed wife 5 g HBTU. An 8 ml aliquot of the activated DTPA solution was then added to the resin which was vortex mixed for 14 hr. The DTPA treatment was repeated until the resin gave a negative test for amines using the Kaiser test. Alternatively, DTPA Tetra-t-butyl ester could be used with conventional coupling agents such as DIC and HBTU. {See Arano Y, Uezono T. Akizawa H, Ono M, Wakisaka K, Nakayama M, Sakahara H, Aliquots were removed for stability studies. The aliquots were diluted 1:10 in saline, 1 mM cysteine in 0.05M phosphate pH 7.5, and fresh human serum. The original kit solution, die saline dilution, and the cysteine challenge were incubated at room temperature while the serum sample was incubated at 37°C. The samples were monitored by HPLC and ITLC. The labeled peptide was stable in the in vitro tests. The retention time of the labeled peptide in serum was shifted from 6.3 mm to 7.3 nun. The shift may be due to ion pairing of some serum component with the peptide. Table 6 radioiodinated bsAbs to CEA, WI2 (xat anti-MN-14 idiotypic antibody) and radiolabeled peptidyl DTPA chelate was examined on analytical size exclusion HPLC. Approximately 90 % of the radioiodinated bsAb bound to CEA upon treatment with l0-20x molar excess of CEA, The bsAb complexed with radiolabeled indium-DTPA chelates (IMP-156 or IMP-192). IMP 156 Ac-Phe-Lys(DTPA>Tyr-Ly5(DTPA)NH2 (SEQ ID NO: 2) Example 24) Construction and expression of hMN-]4Fab-734scFv [0193] Recombinant methods were used to produce a monovalent bi-specific fusion protein comprising a i 1 4 ( I t c [0199] A bscAb fragment is cloned into an expression vector containing a 5' casein promoter sequence and 3' untranslated genomic sequences that flank the insertion site. The expression cassette is then injected into the pronuclei of fertilized, mouse eggs, using procedures standard in the art The eggs are then implanted into the uterus of a recipient female and allowed to gestate. After birth, the progeny are screened for the presence of the introduced DNA by Southern analysis. Milk from transgenic females is analyzed for the presence and functionality of the bscAb using standard immunological methods known in the art. The bscAb can be purified from the milk by complementary binding to an immobilized antigen, column chromotography or other methods known in the art Table 8 Biodistribution of 125-I-hMN-14 x 734 bsAb and 11 l-In-indiuin-lMP-156 peptide in nude mice bearing GW-39 tumor xenografts: hMN-14 x 734 was allowed 48 h for localization prior to 11 l-ln-indium-IMP-156 injection. Biodistribution was performed 3 h post 11 l-In-indium-IMP-156. bsAbipeptide ratio administered, 1:0.03. Five animals per time point. l25-I-bMN-14x 734 111-In-indium-IMP-ISe Table 9 Control group showing the clearance of 11 l-In-indium-IMP-156 at 3 h after injectioa Table 10 Kude mice bearing GW 39 tumor xenografts were administered 125-Wabeled bsAb (5 µCi, 15 µg, 1.5 x 10-10 mol). hMN-14 x 734 was allowed 24 h for localization and clearance before administering 99m-Tc-IMP-192 (10 µCi, 1.6 A 10" 11 mol of peptide). Biodistribution studies were performed at 30 min, 1,3 and 24 h post 99m-Tc-lMP-192 injection, five animals per time point BsAbrpeptide, 1:0.1. Table 11 Nude mice bearing GW 39 tumor xenografts were administered 12S-I-labeled bsAb (5µCi, 15 µg, 1.5 x 10-10 mol), hMN-14 x 734 was allowed 24 h for localization and clearance before administering 99m-Tc. IMP-192 (10µCi, 1.6 X 10-11 mol of peptide). Biodistribution studies were performed at 30 min, 1,3 and 24 h post 99m-Tc-rMP-192 injection, five animals per time point BsAb:peptide, 1:0.1. Table 12 Control group of nude mice bearing GW-39 tumors received 99in-Tc-IMP-192 (10µCi, 1.6 x 10-11 mol of peptide) and were sacrificed 3 h later. 99m-Tc-IMP-192 Tissue % ID/g Tumor 0,2 ±0.05 Liver 0,3 ± 0.07 Spleen 0.1 ± 0.05 Kidney 2.6 ±0.9 Lungs 0.2 ± 0.07 Blood 0.2 ±0.09 The percentage of the available DTPA binding sites on the tumor bound bsAb filled with 99m-Tc-JMP-192 was calculated from the above data assuming one peptide bound to one hsAb molecule. However, it is possible that one peptide molecule can crosslink two molecules of bsAb. Table 13 Percentage of the available DTPA bmding sites on the tumor bound bsAb filled with 99m-Tc-IMP-192 % saturation on time hMN.14x734 30 min 25.4 1 h 25.8 3h 25 24 h 28 [0202] The foregoing experimental data show that: the humanized x murine bsAb retained its binding capability to CEA and indium-DTPA; the hMN-] 4 x 734 (Fab x Fab) effectively targets a tumor; the dual functional peptidyl Tc-99m chelator was stable; 99m-Tc-IMP-l92 complexed to tumor-localized hMN-14 X 734 and was retained for at least 24 h; and imaging of tumors is possible at early time points (l-3h) post 99m-Tc-IMP-192 injection. Example 28) Use of anti-CEA Fab x anti-peptide scFv fusion protein for RAIT, with a bsAfa Clearing Step [02031] A 69-year-old man with colon cancer that had undergone resection for cure, after a year is found to have a CEA blood serum level of 50 ng/mL. The patient undergoes a CT scan, and 5 tumor lesions ranging from 1 cm to 3 cm are present in the left lobe of the liver. The patient is given 100 mg of hMN14-Fab/734-scFv fusion protein. Three days later, the patient is given a clearing dose of galactose-WI2-Fab'. Twenty-four hours after the clearing dose of agalactose-WI2-Fab', the fusion protein in the blood is reduced 20-fold the concentration of the protein just prior to injection of the clearing agent The patient is then infused with the IMP 245 Y-90-di-B2-DTPA-peptide, containing 50 mCi of Y-90. ACT scan performed three months later demonstrates three of the lesions have disappeared, and the remaining two have not increased in size. The CEA blood serum level is decreased to 10 ng/mL at this time. No increase is seen in the CEA blood serum level for the following 6 months, and CT scans demonstrate no growth of the two tumor lesions. The therapy is repeated a year after the first therapy, when an increase in CEA is observed, and the two tumor lesions are observed to decrease in size at 3 months and six months after the second therapy. The blood serum CEA level after six months is less than 5 ng/mL. Example 29) Preparation of a carboxylesterase-DTPA conjugate 10204] Two vials of rabbit liver caiboxylesterase (SIGMA; protein content -17 mg) are reconstituted in 2.2 ml of 0.1 M sodium phosphate buffer, pH 7.7 and mixed with a 25-fold molar excess of CA-DTPA using a freshly prepared stock solution (- 25 mg/mJ) of the latter in OMSO. The final concentration of DMSO in the conjugation mixture is 3 % (v/v). After 1 hour of incubation, the mixture is pre-purified on two 5-mL spin-columns (Sephadex G50/80 in 0.1 M sodium phosphate pH 7.3) to remove excess reagent and DMSO. The eluate is purified on a TSK 3000G Supeico column using 0.2 M sodium phosphate pH 6.8 at 4 ml/min. The fraction containing conjugate is concentrated on a Centricon-10TM concentrator, and buffer-exchanged with 0.1 M sodium acetate pH 6.S. Recovery: 0.9 ml, 4.11 mg/ml (3.7 mg)'. Analytical HPLC analysis using standard conditions, with in-line UV detection, revealed a major peak with a retention time of 9.3 min and a minor peak at 10.8 min in 95-to-S ratio. Enzymatic analysis showed 115 enzyme units/mg protein, comparable to unmodified carboxylesterase. Mass spectral analyses (MALDI mode) of both unmodified and DTPA-modified CE shows an average DTPA substitution ratio near 1.5. A metal-binding assay using a known excess of indium spiked with radioactive indium confirmed the DTPA-.enzyme ratio to be 1.24 and 1.41 in duplicate experiments. Carboxylesterase-DTPA is labeled with In-111 acetate at a specific activity of 12.0 mCi/mg, then treated with excess of non-radioactive indium acetate, and finally treated with 10 mM EDTA to scavenge off excess non-radioactive indium. Incorporation by HPLC and ITLC analyses is 97.7%. A HPLC sample is completely complexed with a 20-fold molar excess of bi-speciiic antibody hMN-14 Fab' x 734 Fab', and the resultant product further complexes with WI2 (anti-ID to hMN-14), with the latter in 80-fold molar excess with respect to bi-specific antibody. Example 30) Synthesis of IMP 224 [0205] An amount of 0.0596 g of the phenyl hydrazine containing peptide IMP 221 (H2N-NH-C6H4-CO-Lys(DTPA).Tyr-Lys(DTPA)-NH2 MH+ 1322. made by Fmoc SPPS) was mixed with 0.0245 g of Doxorubicin hydrochloride in 3 mL of DMF. The reaction solution was allowed to react at room temperature in the dark. After 4 hours an additional 0.0263 g of IMP 221 was added and the reaction continued overnight. The entire reaction mixture was then purified by HPLC on a Waters Nova-Pak (3-40X100 nun segments, 6 µm, 60A ) prep column eJuting with a gradient of 80:20 to 60:40 Buffer A:B over 40 min (Buffer A= 0.3 % NH4OAC, Buffer B= 0.3 % NH4OAC in 90 % CH3CN). The fractions containing product were combined and lyophilized to afford 0.0453 g of the desired product, which was confirmed by ESMS MH+ 1847. Example 31) IMP 224 Kit Formulation [0206] The peptide of Example 31 was formulated into kits for In-l 11 labeling. A solution was prepared which contained 5.014 g 2-hydroxypropyl-P-cyclodextrin, and 0.598 g citric acid in 85 mL. The solution was adjusted to pH 4.0 by the addition of 1 M NaOH and diluted with water to 100 mL. An amount of 0.0010 g of the peptide IMP 224 was dissolved in 100 mL of the buffer, and 1 mL aliquots were sterile filtered through a 0.22µm Millex GV filter into 2 mL lyophilization vials which were immediately frozen and lyophilized. Example 32) In-111 Labeling of IMP 224 Kits [0207] The In-111 was dissolved in 0.5 mL water and injected into the lyophilized kit The kit solution was incubated at room temprature for 10 mm then 0.5 mL of a pH 7.2 buffer which contained 0,5 M NaOAc and 2.56 x 10-5 M cold indium was added. Example 33) In-Vitro Stability of IMP 224 Kits I0208I An IMP 224 kit was labeled as described with 2.52 mCi of In-111. Aliquots (0.15 mL, 370 µCi) were withdrawn and mixed with 0.9 xnL 0.5 M citrate buffer pH 4.0, 0.9 mL 0.5 M dtrste buffer pH 5.0, and 0-9 mL 0.5 M phosphate buffer pH 7.5. The stability of the labeled peptide was followed by inverse phase HPLC. HPLC Conditions: Waters Radial-Pak C-18 Nova-Pak 8x100 mm. Flow Rate 3 mL/min, Gradient: 100 % A= 0.3 % NH4OAC to 100 % B= 90 % CH3CN, 0.3 % NH4OAC over 10 min. Table 14 In-Vitro Stability of In/In-111 IMP 224 Example 34) In-vivo biodistribution of IMP 221 in BALB/c mice 102091 Kits were reconstituted with 400 pCi In-111 in 0.5 mL water- The In-111 kit solution was incubated at room temperature for 10 min and then diluted with 1.5 mL of the cold indium containing pH 12,0.5 M acetate buffer. The labeled peptide was analyzed by ITLC in saturated NaCl. The loose In-Ill was at tbe top 20 % of the ITLC strip, [0210] Each mouse was injected with 100 µL (20 µCi) of the In-III labeled peptide. The animals were anesthetized and sacrificed at 30 minutes, 1 hours, 2 hours, 4 hours, and 24 hours using three mice per time point. Blood, muscle, liver, lungs, kidneys, spleen, large intestine, small intestine, stomach, urine, and tail were collected and counted. The results of the biodistribution study are shown in the following table. Table IS Biodistribution in B ALB/c mice %ID/g of IMP 224 (Dox=N-NH-C6H4-CO-Lys(DTPA)-Tyr--LyspTPA)-NH2 MH+ 1847 radiolabeled with In-111 and saturated with cold In Example 35) In-vivo stability and clearance of IMP 224 \|0211) Kits were reconstituted with 4 mCi InIII in 0.5 mL water. The In-III kit was incubated at room temperature for 10 min and then diluted with 0.5 mL of the cold indium containing 0.S M pH 7.2 acetate buffer. The labeled peptide was analyzed by ITLC in saturated NaCL. The loose bi-III was at the top 20 % of fuel ITLC strip. (02121 Bach mouse was injected with 100 µL (40µCi) of the In-111 labeled peptide. The animals were anesthetized and sacrificed at 30 min and I hr using two animals per time point. The srum and urine samples were collected, stored on ice, and sent on ice as soon as possible for HPLC analysis. The HPLC (by size exclusion chromatography) of the urine samples showed that the In-111 labeled peptide could still bind to the antibody. The reverse phase HPLC analysis showed that the radiolabeled peptide was excreted intact in the urine. The amount of activity remaining in the scrum was too low to be analyzed by reverse pliase HPLC due to the poor sensitivity of the detector. Doxorubicin has -95 % hepatobiliary clearance. Thus, by attaching the his DTPA peptide in a hydrolyzeable manner, the biodistribution of the drug is altered to give - 100 % renal excretion. This renders the drug far less toxic because all of the nontargeted drug is rapidly excreted intact Table 16 Activity Recovered in The Urine and Serum. Example 36) Pretargeting experiments with IMP 224 and IMP 225 [0213] A lyophilized kit of IMP 224 containing 10 micrograms of peptide was ued. The kit was lyophilized in 2 mL vials and reconstituted with 1 mL sterile water, A 0.5 mL aliquot was removed and mixed with 1.0 mCi ln-111. The In-111 kit solution was incubated at room temperature for 10 minutes then 0.1 mL was removed and diluted with 1.9 mL of the cold indium containing acetate buffer BM 8-12 in a sterile vial. The labeled peptide was analyzed by ITLC in saturated NaCl. The loose In-111 was at the top 20% of the ITLC strip. (02141 Female nude mice (Taconic NCRNU, 3-4 weeks old) with GW 39 tumor xenografis were used for the pretargeting experiments. Tumors were 0.3-0.8 g. Each animal was injected with 100 microliters (5 µCi, 15µg. 1.5 X 10 -10 mol) of the 1-125 labeled antibody F6 x 734-F(ab')2. (02151 Seventy two hours later, each mouse was injected with 100 µL (10µCi) of the In-111 labeled peptide. The animals were anesthetized and sacrificed at 1 hour, 4 hours and 24 hours using five mice per time point. Tumor, blood, muscle, liver, lungs, kidneys, spleen, large intestine, small intestine, stomach, urine and tall were collected and counted. (02161 The experiment was repeated with a lyophilized kit of IMP 225 Ac-Cys(Dox-COCH2)- Lys(DTPA)-Tyr-Lys(DTPA)-NH2 (SEQ ID NO: 11) MNa+ 1938), containing 11 micrograms of peptide. Table 17 Biodistribution of In-111 "IMP-224 in nude mice bearing GW-39 tumor xenografts, previously given F6 x 734-F(ab')2 72 h earlier. Data in % ID/g tissue. n=5. Table 18 Biodistribution of In-11 l-IMP-224 in nude mice bearing GW-39 tumor xenografts, previously g^ven F6 x 734-F(ab'2 72hrlier. Data in tiunor-to-nonnal organ ratios. n=5. Table 39 Biodistribution of In-111-IMP-22S in nude mice bearing GW-39 tumor xenografts, previously given F6 x 734-F(ab')2 72 h earlier. Data in % ID/g tissue. n=. [0217] Combinations of the bi-specific constructs described in the present invention or others of similar specificities are suitable for pretargeted RAIT, where IMP-192 peptide and its analogues are labeled with therapeutic radioisotopes such as IS8-Re, 2I3-Bi, 67-Cu and the like. It will be recognized that therapeutic chelates can be conjugated to peptides that have other than chelate epitopes for recognition by bsAbs, as described above. (0218] It will be appreciated as well that detectable radiolabels can be directed to a site of interest, e.g. a tumor, which is to be excised or otherwise detected and /or treated in intra-operati, endoscopi intravascular or other similar procedures, using the pretargeting meths of the present invention, in combination with various linkers. The pretargeting is effected with non-radioactive bsAbs and the eventual administration and localization of the low molecular weight radiolabeled linker, and clearance of unbound linker, are both comparatively rapid, compatible with surgical procedures that should avoid needless delay and which can use radioisotopes with short half-lives. Additionally, the disclosed therapies can be used for post-surgical radioimmunotherapy protocols to ensure the eradication of residual tumor cells. Example 37) Synthesis of DOTA-Phe-Lvs(GVD-Tvr-Ly(SGVLvsnrseg-Cys)NHo/SEQ ID N: I) (IMP245) [219] The peptide was synthesized by the usual double coupling procedure as described for the synthesis of IMP 192. The tri-t-butyl DOTA was added to the C-terminus of the pepde with a single benzotria2ole-l"yl-oxy"triS"(dimethylamino)-phosphonium hcxafluorophosphate (BOP) coupling using 5 eq of protected DOTA for 16 hr. The resin was then capped with acetic anhydride. The Aloc groups on the side chains were removed using the palladium catalyst and the N-trityl-HSG groups were added as described for the synthesis of IMP 243. The product was cleaved from the resin and purified by HPLC to afford 0.2385 g of product, from four fractions, after lyophilization.. ESMS MH+ 1832 Example 38) Tc-99m Kit Fonnulation (0220] A formulation buffer was prepared which contained 22.093 g hydroxypropyl-p-cyclodextrin, 0.45 g 2,4-d]hydroxybenzoic acid, 0.257 g acetic acid sodium salt, and 10.889g aplpha-g(ucoheptonic acid sodium salt dissolved in 170 mL nitrogen degassed water. The solution was adjusted to pH 5.3 with a few drops of 1 M NaOH then further diluted to a total volume of 220 mL. A stannous buffer solution was prepared by diluting 0.2 mL of SnCl2 (200 mg/mL) with 3.8 mL of the formulation buffer. The peptide. IMP 245 (0.0029g), was dissolved in 1 mL 1.6 x 10-3 M InCl3 in 0.1 M HCl. The peptide solution was mixed with 2 mL 0.5 M NH4OAC and allowed to incubate at room temperature for IS min. The formulation buffer, 75 mL, and 0.52 mL of the stannous buffer were then added to the peptide solution. The peptide solution was then filtered through a 0.22 µm Millex GV filter in 1.5 mL aliquots into 3 mL lyophilization vials. The filled vials were frozen immediately, lyophilized and crimp sealed under vacuum. Example 39) Tc-99m Labeling of IMP 245 High Temperature (Boiling Water Bath) [0221] The pertechnetate solution (29 mCi) in 1.5 mL of saline was added to the kit The kit was incubated at room temperature for 10 min and heated in a boiling water bath for 15 min. The kit was cooled to room temperature before use. Low Temperature (3 loC) 10222] The pertechnetate solution (25 mCi) in 1.5 mL of saline was added to the kit The kit was incubated at room temperature for 14 min and heated in a 37oC water bath for 18 min. The kit was cooled to room temperature before use. The HPLC retention time for this label is slightly different because a different injector was used. Example 40) Peptide Analysis (HPLC) of IMP 245 10223] The peptide was analyzed by reverse phase HPLC and size exclusion Iff LC (shown below). The size exclusion HPLC traces indicated that the peptide binds to two mMU-9 x m679 and two hMN-14 x m679 bi-specific antibodies {see "A Universal Pre-Targeting System for Cancer Detection and Therapy Using Bi-specific Antibody," Sharkey, R.M.. IVfcBride, W.J., Karacay, H., Chang, K., Griffidis, Gi., Hansen, H.J., and Goldenberg, D.M., the entire contents of which are incojporated by reference herein). The reverse phase HPLC analysis shows several small peaks before the main peak and heat did not seem to significantly alter the ratio of the small peaks to the large peak. Recovery from SEC: [0224] Tc-99m IMP 245 Alone 54%, TC-99m IMP 24S + hA4N-14 x m679 66 %, Tc-99m IMP 245+ mMU-9 x m679 66 %. the acetate/EDTA buffer, followed by a rapid addition of 20 mM PDM (prepared in 90% DMF) to a final concentration of 4 mM. After stirring at room temperature for 30 minutes, the resulting solution (containing 679 Fab'-PDM) was diafiltered into the acetate/EDTA buffer until tree PDM is minimum, and concentrated to 5-10 mg/mL. A solution of hMN-14 Fab'-SH or Mu-9 Fab'-SH was the mixed with a 3 i 25-20µ/ci). At the designated times after the peptide injection, animals were anesthetized, bled by cardiac puncture, and then euthanized prior to necropsy. Tissues were removed, weighed and counted by gamma scintillation using appropriate windows for each radionuclide along with standards prepared from the injected materials. When dual isotope counting was used, appropriate backscatter correction was made. GI tissues (stomach, small intestine and large intestine were weighed and counted with their contents. Data are expressed as the percent injected dose per gram tissue (%ID/g) and the ratio of the percentages in the tumor to the normal tissues (T/NT). All values presented in the tables and figures represent the mean and standard deviation of the calculated values with the number of animals used for each study provided therein. Table 22 Table 25 Tumor/nontumor ratios for 99m7c-hMN-14 Fab' in GW-39 tumor-bearing nude mice 3 h after injection, (n = 5) Table 28 similar distribution and clearance properties (Tables 20 and 21). In both instances, the peptide was cleared so rapidly from blood that within 3 hour after its injection, there was insufficient radioactivity in the blood to quantify accurately, but there was sufficient radioactivity in the major organs to permit quantitation. The radioactivity was eliminated from the body through renal excretion, with a small percentage of the injected activity lingering in the kidneys over the monitoring period. At an average Bos, ES., Kuijpers, WHA., Meesters-Winters, M., Pham, DT., deHaan, AS., van DoonnaIen,Am., Kasperson, F..M.,vanBoeckel, CAA and Gouegeon-Bertrand, F. In vitro evaluation of DNA-DNA hybridization as a two-step approach in radioimmunotherapy of cancer. Cancer Res, 1994; 54:3479-3486. Carr et al..WO00/34317. Gautherot, E., Bouhou, J., LeDoussai, J-M., Manetti, C, Martin, M-, Rouvier, E., Barbet, J. Therapy for colon carcinoma xenografts with bi-specific antibody-targeted, iodine-131-IabeIed bivalent hapten. Cancer suppl 1997;80:2618-2623. Gautherot, E., Bouhou, J., Loucif, E., Manetti, C, Martin, M., LeDoussal, J Jvl,, Rouvier, E,, Baibet, J. Radioimmunotherapy of LS174T colon carcinoma in nude mice using an iodine-131-abeIed bivalent hapten combined with an anti-CEA x anti-indium-DTPA bi-specific antibody. J,Nucl, Med. Suppl. 1997; 38: 7p. Goodwin, D.A., Meares, CF„ McCall, MJ., McTigue,M., Cbaovapong, W. Pre-targetd immunoscintigraphy of murine tumors with indium-l 11-labeled bifunctional haptens. J Nucl. Med, 1988; 29:226-234. Greenwood, F.C. and Hunter, W.M. The preparation of 1-131 labeled human growth hormone of high specific radioactivity. Biochem. 1963; 89:114-123. Hawkins, G.A., McCabe, R.P., Kim, C.-H., Subramanian,R., Bredehorst, R., McCuUers, GA., Voge],C.-W., Hanna, M.G. Jr., and Pomata, N. Delivery of radionuclides to pretargeted monodona] antibodies using dihydrofolate reductase and methotrexate in an affinity system. Cancer Res. 1993; 53: 2368-2373. Kranenborg, M.h-, Boerman, O.C, Oosterwijk-Waklca, j., weijert, M., Corstens, F., Gosterwijk, E. Development and characterization of anti-renal cell carcinoma x antichelate bi-specific monoclonal antibodies for two-phase targeting of renal cell carcinoma. Cancer Res.(suppl) 1995; 55:5864s-5867s Losman M.J., Qu Z., Krishnan LS., Wang J., Hansen HJ., Goldenbeig D.M., Leung S.O. Clin. Cancer Res. 1999; 5(10 Suppl):3101s.3105s. Penefsky, H.S. A centrifuged column procedure for the measurement of ligand binding by beef heart Fl. Part G. Methods Enzymol. 1979; 56:527-530. Schuhmacher, J., Klivenyi,G., Matys JL, Stadler, M., Regiert, T., Hauser,H., Doll, J-, Maier-Borst, W., Zoller, M. Multistep tumor targeting in nude mice using bi-specific antibodies and a gallium chelate suitable for immunocintigraphy with positron emission tomography. Cancer Res. 1995; 55,115-123. Sharkey, RM., Karacay, Griffiths, GL., Behr, TM., Blumenthal, RD., Mattes,MJ.,Hansen, HJ., Goldenberg. Development of a streptavidin-anti-carcinoembryonic antigen antibody, radiolabeled biotin pretargeting method for radioimmunotherapy of colorectal cancer. Studies in a human colon cancer xenograft model. Bioconjugate Ghent 1997; 8:595-604, Stickney, DR., Anderson, LD., Slater, JB., Ahlem, CN.,Kirk, GA,, Schweighardt, SA and Frincke, JM. Bifunctional antibody: a binary radiopharmaceutical delivery system for imaging colorectal carcinoma. Cancer Res. 1991;51:6650-6655. All references cited herein are hereby incorporated herein by reference in their entireties. WHAT IS CLAIMED IS: 1. A compound of the formula: X-Phc-Lys(HSG)-D-Tyr.Lys(HSG)-Lys(Y)-NH2; wherein the compound includes a hard acid cation chelator at X or Y, and a soft acid cation chelator at remaining X or Y. 2. The compound of claim 1, wherein the hard acid cation chelator includes a carboxylate or amine group. 3. The compound of claim 1, wherein the hard acid cation chelator is selected from the group consisting of NOTA, DOTA, DTPA, and TETA. 4. The compound of claim 1, wherein the soft acid cation chelator includes a thiol group. 5. The compound of claim 1, wherein the soft acid cation chelator is selected from the group consisting of Tscg-Cys and Tsca-Cys. 6. The compound of claim 1, further comprising at least one radionuclide, therapeutic agent or diagnostic agent. 8. The compound of claim 1 comprising: D0TA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2. 9. The compound of claim 1 comprising: Tscg-Cys-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(DOTA)-NH2. 10. The compound of claim 1, wherein the hard acid cation chelator includes a cation selected from the group consisting of Group Ha and Group IIIa metal cations. 11. The compound of claim 1, wherein the soft acid cation chelator includes a cation selected from the group consisting of transition metals, Bi, lanthanides and actinides. 12. The compound of claim 1, wherein the soft acid cation chelator includes a cation selected from the group consisting of Tc, Re, and Bi, 13. A targetable construct comprising: X-Phe-Lys(HSG)-D.Tyr.Lys(HSG)-Lys(Y)-NH-.R; wherein the targetable contruct includes a hard acid cation chelator- at X or Y; a soft acid cation chelator at remaining X or Y; and a therapeutic agent, diagnostic agent or enzyme at R. 14. The targetable construct of claim 13, wherein R is covalently linked to the targetable construct. 15. The targetable cosntruct of claim 13, wherein R is linked to the targetable construct by a linker moiety. 16. The targetable construct of claim 15, wherein the linker moiety includes at least one amino acid. 17. The compound of claim 13, further comprising at least one radionuclide bound to at least one of the hard acid chelator and soft acid chelator. 19. The targetable construct of claim 13, wherein said therapeutic agent includes a radionuclide, drug, prodrug or toxin. 20. The targetable construct of claim 19, wherein said prodrug is selected from the group consisting of epirubicin glucuronide, CPT-11, etoposide glucuronide, daunomicin glucuronide and doxorubicin glucuronide. 21. The targetable construct of claim 19, wherein said toxin is selected from the group consisting of ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-!, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. 22. The targetable construct of claim 13, wherein said therapeutic agent compsrises doxorubicin, SN-38, etoposide, methotrexate, 6-mercaptopurine or etoposide phosphate. 23. The targetable construct of claim 13, wherein the diagnostic agent includes one or more agents for photodynamic therapy. 24. The targetable construct of claim 23, wherein said agent for photodynamic therapy is a photo sensitizer. 25. The targetable construct of claim 24, wherein said photosensitizer is selected from the group consisting of benzoporphyrin monoacid ring A (BPD-MA), tin etiopurpurin (SnET2), sulfonated aluminum phthalocyanine (AlSPc) and lutetium texaphyrin (Lutex). 26. The targetable construct of claim 13, wherein said diagnostic agent comprises one or more image enhancing agents for use in magnetic resonance imaging (MRl). 27. The targetable construct of claim 26, wherein said enhancing agents include Nfn, Fe, La and Gd. 28. The targetable construct of claim 13, wherein said diagnostic agent comprises one or more radiopaque or contrast agents for X-ray or computed tomography. 29. The targetable construct of claim 28, wherein said radiopaque or contrast agents include barium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid, iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide, iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetric acid, iothalamic acid, fotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine, metrizamide, metrizoate, propyliodone, or thallous chloride. 30. The targetable construct of claim 13, wherein said diagnostic agent comprises one or more ultrasound contrast agents. 31. The targetable construct of claim 30, wherein said ultrasound contrast agent includes a liposome or dextran. 32. The targetable construct of claim 31, wherein the liposome is gas-fiUed. 33. The targetable construct of claim 13, wherein said enzyme includes an enzyme capable of converting drug intermediate to a toxic form to increase toxicity of said drug at a target site. 34. A method of treating or diagnosing or treating and diagnosing a disease or a condition that may lead to a disease comprising: (A) administering to said subject a bi-specific antibody or antibody feagment having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct. (B) optionally, administering to said subject a clearing composition, and allowing said composition to clear non-localized antibodies or antibody fragments from circulation; and (C) administering to said subject a targetable construct comprising rge compound of claim 1 which further comprises at least one diagnostic or therapeutic cation, and/or one or more chelated or chemically bound therapeutic agent, diagnostic agent, or enzyme. 35. The method of claim 34, wherein the bi-specific antibody or antibody fragment having at least one arm that speciiically binds a targeted tissue and at least one other arm that specifically binds a targetable construct and the targetable construct are administered at substantially the same time. 36. The method of claim 34, wherein the therapeutic cation emits particles and/or positrons having 20 to 10,000 keV. 38. The method of claim of claim 34, wherein the diagnostic cation emits particles and/or positrons having 25-10,000 keV. 40. The method of claim 34, wherein said diagnostic agent is used to perform positron- emission tomography (PET). 41. The method of claim 34, wherein said diagnostic agent is used to perform SPECT imaging. 42. The method of claim 34, wherein said diagnostic cation or agent includes one or nnore image enhancing agents for use in magnetic resonance imaging (MRI). 43. The method of claim 42, wherein said enhancing agent is selected from the group consisting of Mn, Fe, La and Gd. 44. The method of claim 34, wherein said diagnostic agent comprises one or niore radiopaque or contrast agents for X-ray or computed tomography. 45. The method of claim 44, wherein said radiopaque or contrast agents include barium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid, iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide, iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetric acid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine, metrizamide, metrizoate, propyliodone, or thallous chloride. 46. The method of claim 34, wherein said diagnostic agent comprises one or more ultrasound contrast agents. 47. The method of claim 46, wherein said ultrasound contrast agent includes a liposom or dextran. 48. The method of claim 47, wherein said liposome is gas-filled. 49. The method of claim 34, wherein said diagnostic agents are selected from the group consisting of a fluorescent compound, a chemilmninescent compound, and a bioiuminescent compound. 50. The method of claim 49, wherein said fluorescent compound is selected from the group consisting of fluorescein isothiocyanate, rhodamine, phycoetytherin, phycocyanin, alJophycocyanin,o-phthaldehyde and fluorescamine. 5 2. The method of claim 49, wherein said chemily\uminescent compound is selected from the group consisting of luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester. 52. The method of claim 49, wherein said bioiuminescent con:q)ound is selected from the group consisting of lucifeiin, luciferase and aequorin. 53. The method of claim 34, wherein said targeted tissue is a tumor. 54, The method of claim 53, wherein said tumor produces or is associated with antigens selected from the group consisting of colon-specific antigen-p{CSAp), carcinoembryonic antigen (CEA). CD19, CD20, CD21, CD22, CD23, CD30, CD74, CD80, HLA-DR, la, MUC 1. MUC 2, MUC 3, MUC 4, EGFR, HER 2Meu, PAM-4, TAG-72, EGP-1, EGP-2, A3, KS- 1, Le(y), S100, PSMA, PSA, tenascin, folate receptor, VEGFR, necrosis antigens, IL-2, T101, MAGE, 55. The method of claim 34, wherein said at least one arm that specifically binds a targeted tissue is a monoclonal antibody or a fragment of a monoclonal antibody. 56. The method of claim 34, wherein said at least one other ann that specifically binds a targetable construct is a monoclonal antibody or a fragment of a monoclonal antibody. 57. The method of claim 34, wherein said at least one ann that specifically binds a targeted tissue is a human, chimeric or humanized antibody or a fragment of a human, chimeric or humanized antibody. 58. The method of claim 34, wherein said at least one other arm that specifically binds a targetable construct is a human, chimeric or humanized antibody or a fragment of a human, chimeric or humanized antibody. 59. The method of claim 34, wherein said bi-specific antibody or antibody fragment further comprises a therapeutic nuclide. 61. The method of claim 34, wherein the bi-specific antibody comprises the Fv of MAb Mu-9 and the Fv of MAb 679. 62. The method of claim 61, wherein Mu-9 and/or 679 are chimerized or humanized. 63. The method of claim 61, wherein Mu-9 and/or 679 are human Mu-9 and 679. 64. The method of claim 61, wherein the bi-specific antibody comprises one or more of the CDRs of Mu-9. 65. The method of claim 61, wherein the bi-specific antibody comprises one or more of the CDRs of 679. 66. The method of claim 61, wherein the bi-specific antibody is a fusion protein. 67. The method of claim 34, wherein the bi-specific antibody comprises the Fv of MAb MN-14 and the Fv of MAb 679. 68. The method of claim 67, wherein MN-14, and/or 679 are chimerized or humanized. 69. The method of claim 67, wherein MN-14, and/or 679 are human MN4-14 and 679. 70. The method of claim 67, wherein the bi-specific antibody comprises one or more of the CDRs of MN-14. 71. The method of claim 67, wherein the bi-specific antibody comprises one or more of the CDRs of 679. 72. The method of claim 67, wherein the bi-specifi c antibody is a fusion protein. 73. The method of claim 34, wherein the fusion protein is trivalent, and incorporates the Fv of an antibody reactive with CSAp. 74. The method of claim 34, wherein the bi-specific antibody incorporates a Class-III anti-CEA antibody and the Fv of 679, 75. The method of claim 34, wherein said targetable construct comprises one or more radioactive isotopes useful for killing diseased tissue. 76. The method of claim 34, wherein said targetable construct comprises 10B atoms, and said method further comprises the step of irradiating said boron atoms localized at said diseased tissue, thereby effecting BNCT of said diseased tissue. 77. The method of claim 34, when said targetable construct comprises an enzyme, further administering to said subject a drug which said enzyme is capable of converting to a toxic form, and, therefore, increasing the toxicity of said drug at the target site. 78. A method for detecting or treating target cells, tissues or pathogens in a mammal, comprising: administering an effective amount of a bi-specific aiitibody or antibody fragment comprising at hast one arm that specifically binds a target and at least one other arm that specifically binds a targetable construct; and administering a targetable construct comprising the compound of claim 1; wherein said target includes a target cell, tissue, pathogen or a molecule produced by or associated therewith and at least one arm that specifically binds said target is capable of binding to a complementary binding moiety on the target 79. The method of claim 78, wherein said pathogen is a fungus, virus, parasite, bacterium, protozoan, or mycoplasm. 80. The method of claim 79, wherein said fungus is selected from the group consisting of Microsporum, Trichophyton, Epidermophyton, Ssporothrix scbenckii, Cyrptococcus neofomians, Coccidioides immitis, Histoplasma capsulatum, Blastomyces dermatitidis, Candida albicans. 81. The method of claim 79, wherein said virus is selected from the group consisting of human immunodeficiency virus (HIV), heipes virus, cytomegalovirus, rabies virus, influenza virus, hepatitis B virus, Sendai vims, feline leukemia virus, Reo virus, polio virus, human serum parvo-like virus, simian virus 40, respiratory syncytial virus, mouse mammary tumor virus, Varicella-Zoster virus. Dengue vims, rubella virus, measles virus, adenovirus, human T-cell leukemia vimses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart vims and blue tongue virus. 82. The method of claim 79, wherein said bacterium is selected from the group consisting of Anthrax bacillus. Streptococcus agalactiae, Legionella pneumophilia. Streptococcus pyogenes, Escherichia coli. Neisseria gonorrhoeae. Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae. Brucella abortus, Mycobacterium tuberculosis and Tetanus toxin. 83. The method of claim 79, wherein said parasite is a helminth or a malarial parasite. 84. The method of claim 79, wherein said protozoan is selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Tiypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma 86. The method of claim 78, wherein said targetable construct further comprises at least one radionuclide, therapeutic agent, diagnostic agent or enzyme 90. The method of claim 89, wherein said subject is mammalian. 9\. The method of claim 90, wherein said mammalian subject is selected from the group consisting of humans, primates, equines, canines and felines. 92. The method of claim 89, wherein said targetable construct further comprises at least one radionuclide, therapeutic agent, diagnostic agent or enzyme. 94. The method of claim 92, wherein the diagnostic agent includes an imaging agent 95. The method of claim 92, wherein the therapeutic agent includes drugs, toxins, cytokines, hormones, or growth factors. 96. A kit useful for treating or identifying diseased tissues in a subject comprising: (A) a targetable construct comprising the compound of claim 1; (B) a bi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds the targetable construct; wherein the targetable construct includes a carrier portion which comprises or bears at least one epitope recognizable by said at least one other arm of said bi-specific antibody or antibody fragment, and one or more conjugated therapeutic or diagnostic agents, or enzymes; and (C) optionally, a clearing composition useful for clearing non-localized antibodies and antibody fragments. 99. The kit of claim 96, when said targetable construct comprises an enzyme, optionally, the kit further comprising a drug which enzyme is capable of converting to a toxic form to increase the toxicity of said drug at the target site. 100. A targetable construct comprising the compound of claim 1. 101. A method for imaging normal tissue in a mammal comprising: administering an effective amount of a bi-specific antibody or antibody fragment; and administering a targetable construct comprising the compound of claim 1; wherein the bi-specific antibody or antibody fragment comprises at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds the targetable construct; and wherein said at least one arm is capable of binding to a complementary binding moiety on the normal tissue or target cells produced by or associated therewith. 102. The method of claim 101, wherein said normal tissue is tissue from the ovary, thymus, parathyroid, endometrium, bone marrow, or spleen. 103. The method of claim 101, wherein said targetable construct further comprises at least one radionuclide, therapeutic agent, diagnostic agent or enzyme. 105. The method of claim 103, wherein the diagnostic agent includes a contrast agent, 106. The method of claim 103, wherein the diagnostic agent includes an imaging agent. 107. The method of claim 106, wherein said imagine agent is an agent used for PET. 108. The method of claim 106, wherein the imaging agent is an agent used for SPECT. 109. The method of claim 103, wherein the therapeutic agent includes drugs, toxins, cytokines, hormones, or growth factors. 110. A method of intraoperatively identifying diseased tissues, in a subject, comprising administering an effective amount of a bi-specific antibody or antibody fragment; and administering a targetable construct comprising the compound of claim 1; wherein the bi-specific antibody or antibody fragment comprises at least one atm that specifically binds a targeted tissue and at least one other arm that specifically binds the targetable construct; and wherein said at least one arm is capable of binding to a complementary binding moiety on the target cells, tissues or pathogen or on a molecule produced by or associated therewith. 111. The method of claim 110, wherein said targetable construct further comprises at least one radionuclide, therapeutic agent, diagnostic agent or enzyme. 213. The method of claim 111. wherein the diagnostic agent includes a contrast agent. 114. The method of claim HI, wherein the diagnostic agent includes an imaging agent 115. The method of claim 111, wherein the therapeutic agent includes drugs, toxins, cytokines, hormones, or growth factors. 116. A method for the endoscopic identification of diseased tissues, in a subject, comprising: administering an effective amount of a bi-specific antibody or antibody fragment; and administering a targetable construct comprising the compound of claim I; wherein the bi-specific antibody or antibody fragment comprises at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds the targetable construct; and wherein said at least one arm is capable of binding to a complementary binding moiety on the target cells, tissues or pathogen or on a molecule produced by or associated therewith. 117. The method of claim 116, wherein said targetable construct further comprises; at least one radionuclide, therapeutic agent, diagnostic agent or enzyme. 119. The method of claim 117, wherein the diagnostic agent includes a contrast agent 120. The method of claim 117, wherein the diagnostic agent includes an imaging agent 121. The method of claim 117, wherein the therapeutic agent includes drugs, toxins, cytokines, hormones, or growth factors. 122. A method for the intravascular identification of diseased tissues, in a subject, comprising: administering an effective amount of a bi-specific antibody or antibody fragment comprising at least one arm that specifically binds a targeted tissue and at least one other aim that specifically binds a targetable construct; wherein said at least one ami is capable of binding to a complementary binding moiety on the target cells, tissues or pathogen or on a molecule produced by or associated therewith; and administering a targetable construct comprising the compound of claim 1. 123, The method of claim 122, wherein said targetable construct further comprises at least one radionuclide, therapeutic agent, diagnostic agent or enzyme. 125. The method of claim 123, wherein the diagnostic agent includes a contrast agent 126. The method of claim 123, wherein the diagnostic agent includes an imaging agent 127. The method of claim 123, wherein the therapeutic agent includes drugs, toxins, cytokines, honnones, or growth factors. 128. A compound of the formula substantially as herein described with reference to the accompanying drawings. 129. A method of treating or diagnosing or treating an diagnosing a disease or a condition that may lead to a disease substantially as herein described with reference to the accompanying drawings. Dated this 13 day of December 2004

Full Text

DRUG PRE-TARGETING BY MEANS OF BI-SPECIFIC ANTIBODIES AND HAPTEN CONSTRUCTS COMPRISING A CARRIER PEPTIDE AND THE ACTIVE AGENT (S)
[0001] This application is a continuation-in-part of United States Serial No. 09/382,186, filed August 23, 1999 and a continuation-in-part of United States Serial No. 09/823,746, filed April 3,2001, both of which are continuations-in-part of United States Serial No. 09/337.756, filed June 22,1999, the contents of which are incorporated herein by reference in their entirety. Background of the Invention
[0002] Field of the Invention. The invention relates to immunological reagents for therapeutic use, for example, in radioimmunotherapy (RAIT), and diagnostic use, for example, in radioimmunodctection (RAID) and magnetic resonance imaging (MRI). In particular, the invention relates to bi-specific antibodies (bsAb) and bi-specific antibody fragments (bsFab) which have at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct Further, the invention relates to monoclonal antibodies that have been raised against specific immunogens, humanized and chimeric monoclonal bi-specific antibodies and antibody fragments having at least one aim that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct, DNAs that encode such antibodies and antibody fragments, and vectors for expressing the DNAs, Earlier provisional patent applications, U.S.S.N. 60/090,142 and U.S.S.N. 60/104,156 disclose a part of what is now included in this invention and are incorporated herein by reference in their entireties. Related Art
10003] An approach to cancer therapy and diagnosis involves directing antibodies or antibody fragments to disease tissues, wherein the antibody or antibody fragment can target a diagnostic agent or therapeutic agent to the disease site. One approach to this methodology which has been under investigation, involves the use of bsAbs having at least one arm that specifically binds a targeted diseased tissue and at least one other arm that specifically binds a low molecular weight hapten. In this methodology, a bsAb is administered and allowed to localize to target, and to clear normal tissue. Some time later, a radiolabeled low molecular weight hapten is given, which being recognized by the second specificity of the bsAb, also localizes to the original target.
|0004| Although low MW haptens used in combination with bsAbs possess a large number of specific imaging and therapy uses, it is impractical to prepare individual bsAbs for each possible application-Further, the application of a bsAb/low MW hapten system has to contend with several other issues. First,
the arm of the bsAb that binds to the low MW hapten must bind with high affinity, since a low MW hapten is designed to clear the living system rapidly, when not bound by bsAb. Second, the non-bsAb-bound low MW hapten actually needs to clear the living system rappidly to avoid non-target tissue uptake and retention. Third, the detection and/'or therapy agent must remain associated with the low MW hapten throughout its application within the bsAb protocol employed.

(0005) Of interest with this approach are bsAbs that direct chelators and metal chelate complexes to cancers using Abs of appropriate dual specificity. The chelators and metal chelate complexes used are often radioactive, using radionuclides such as cobalt-57 (Goodwin et al, U.S. Patent No. 4,863,713), indium-111 (Barbet et aL, U.S. Patent No. 5,256,395 and US. Patent No. 5,274,076, Goodwin et al.J. Nucl Med., 33:1366-1372 (1992), and Kranenborg et ai, Cancer Res (suppL), 55;5864s-5867s (1995) and Cancer (suppl.) 80:2390-2397 (1997)) and gallium-68 (Boden et al., Bioconjugate Chem., 6:373-379, (1995) and Schuhmacher et al., Cancer Res., 55:115'123 (1995)) for radioimmuno-imaging. Because the Abs were raised against the chelators and metal chelate complexes, they have remarkable specificity for the complex against which they were originally raised. Indeed, the bsAbs of Boden et aL have specificity for single cnantiomere of enantiomeric mixtures of chelators and metal-chelate complexes. This great specificity has proven to be a disadvantage in one respect, in that other nuclides such as yrrrium-90 and bismuth-213 useful for radioimmunotherapy (RAIT), and gadolinium useful for MRI, cannot be readily substituted into available reagents for alternative uses. As a result iodine-231, a non-metal, has been adopted for RAIT purposes by using an 1-13 l-labeled indium-metal-chelate complex in die second targeting step. A second disadvantage to this methodology requires that antibodies be raised against every agent desired for diagnostic or therapeutic use,
[00061 Pretargeting methodologies have received considerable attention for cancer imaging and therapy. Unlike direct targeting systems where an effector molecule (e.g., a radionuclide or a drug linked to a small carrier) is directly linked to the targeting agent, in pretargeting systems, the effector molecule is given some time after the targeting agent This allows time for the targeting agent to localize in tumor lesions and, more importantly, clear from the body. Since most targeting agents have been antibody proteins, they tend to clear much more slowly from the body (usually days) than the smaller effector molecules (usually in minutes). In direct targeting systems involving therapeutic radionuclides, the body, and in particular the highly vulnerable red marrow, is exposed to the radiation all the while the targeting agent is slowly reaching its peak levels in the tumor and clearing from the body. In a pretargeting system, the radionuclide is usually bound to a small "effector" molecule, such as a chelate or peptide, which clears very quickly from the body, and thus exposure of normal tissues is minimized. Maximum tumor uptake of the radionuclide is also very rapid because the small molecule efficiently transverses the tumor vasculature and binds to the primary targeting agent. Its small size may also encourage a more uniform distribution in the tumor. (0007 j Pretargeting methods have used a number of different strategies, but most often involve an
avidin/slreptavidin-biotin recognition systern or bi-specific antibodies that co-recognize a tumor antigen and the effector molecule. The avidin/streptavidin system is highly versatile and has been used in several configurations. Antibodies can be coupled with streptavidin or biotin, which is used as the primary targeting agent. This is followed sometime later by the effector molecule, which conjugated with biotin or with avidin/streptavidin, respectively. Another configuration relies on a 3-step approach first targeting a

[0008] Pretargeting with a bsAb also requires one arm of the antibody to recognize an effector molecule. Most radionuclide targeting systems reported to date have relied on an antibody to a chelate-metal complex, such as antibodies directed indium-loaded DTPA or antibodies to other chelates. Since the antibody is generally highly selective for this particular chelate-metal complex, new bsAbs would need to be constructed with the particular effector antibody. This could be avoided if the antibody was not specific to the effector, but instead reacted with another substance. In this way, a variety of effectors could be made so long as they also contained the antibody recognition substance. We have continued to develop the pretargeting system originally described by Janevik-Ivanovska et ah that used an antibody directed against a histamine derivative, histaminc-succinyl-glycl (HSG) as the recognition system on which a variety of effector substances could be prepared. Excellent pretargeting results have been reported using a radioiodinated and a rhenium-labeled divalent HSG-containing peptide. In this work, we have expanded this system to include peptides suitable for radiolabeling 90Y, 11 111In, and 177Lu as well as an alternative 99mTc.binding peptide.
[0009] Thus, there is a continuing need for immunological agents which can be directed to diseased tissue and can specifically bind to a subsequently administered targetable diagnostic or therapeutic conjugate, and a flexible system that accommodates different diagnostic and therapeutic agents without alteration to the bi-specific or multi-specific antibodies. Objects of the Invention

|0010] It is one object of the present invention to provide a multi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one other arm feat specifically binds a targetable construct feat can be modified for use in a wide variety of diagnostic and feerapeutic applications.
(0011] Other objects of fee invention are to provide pre-targeting methods of diagnosis and therapy using fee combination of multi-specific antibody and targetable construct, mefeods of making fee multi-specifics, and kits for use in such methods.
[0012] In accomplishing fee foregoing object, fee present inventors have discovered feat it is advantageous to raise multi-specific Abs against a targetable construct that is capable of carrying one or more diagnostic or therapeutic agents. By utilizing this technique, fee characteristics of fee chelator, metal chelate complex, feerapeutic agent or diagnostic agent can be varied to accommodate differing applications, wifeout raising new multi-specific Abs for each new application. Further, by using feis approach, two or more distinct chelators, metal chelate complexes, diagnostic agents or feerapeutic agents can be used wife the inventive multi-specific Ab. Summary of the Invention
|D0I3] The present invention relates to a multi-specific or bi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one ofeer ann feat specifically binds a targetable construct.
[0014] Provided is a compound of the formula X-phe-Ly5(HSG)-D-T-Lys(HSG)-Lys(y)-NH2 (SEQ ID NO: 1), where fee compound includes a hard acid cation chelator positioned at X or Y and a soft acid cation chelator positioned at remaining X or Y, The hard acid cation chelator may include a carboxylate or amine group, and may include such chelators as NOTA, DOTA, DTPA, and TETA. The soft acid cation chelator may include a thiol group, and may also include such chelators as Tscg-Cys and Tsca-Cys. A preferred embodiment of feis compound is DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 3) also known as IMP 245. Ofeer embodiments may have a hard acid cation chelator and a soft acid cation chelator in switched positions as provided in (Tscg-Cys)-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(D0TA)-NH2 (SEQ ID NO: 1).
[0015] The compound may also include cations bound to the different chelating moeities. For example, hard acid cations may include Group IIa and Group IIIa metal cations, which commonly bind to hard acid chelators. Soft acid cations that may bind to fee soft acid chelators can include the transition metais, lanthanides, actinides and/or Bi, Non exhaustive examples of such soft acid cations include Tc, Re, and
Bi.
10016] Also provided is a targetable construct including X-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Y)-NH-R (SEQ ID NO:1). Again, a hard acid cation chelator is positioned at either X or Y, and a soft acid cation chelator is positioned at remaining X or Y. The targetable construct also includes a linker to conjugate the compound to a feerapeutic or diagnostic agent or enzyme "R". The linker may have at least one amino

I
1
(B) optionally, administering to the patient a clearing composition, and allowing the
composition to clear non-localized antibodies or antibody fragments from circulation;
(C) administering to the patient a first targetable construct which comprises a carrier portion
which comprises or bears at least one epitope recognizable by the at least one other arm of the bi-specific
antibody or antibody fragment, and one or more conjugated therapeutic or diagnostic agents, or enzymes;
and
(D) when the targetable construct comprises an enzyme, further administering to the patient
1) a prodrugs when the enzyme is capable of converting the
prodmg to a drug at the target site; or
2) a drug which is capable of being detoxified in the patient
to form an intermediate of lower toxicity , when the enzyme is capable of reconverting the detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of the drug at the target site, or
3) a prodrug which is activated in the patient through
natural processes and is subject to detoxification by conversion to an intermediate of lower toxicity, when the enzyme is capable of reconverting the detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of the drrier portion which comprises or bears at least one epitoprug at the target site, or
4) a second targetable construct which comprises a
recognizable by the at least one other arm of the bi-specific

antibody or antibody fragment, and a prodrug, when the enzyme is capable of converting
the prodrug to a drug at the target site. [00191 In another embodiment, the invention provides a kit useful for treating or identifying diseased tissues in a patient comprising:
(A) a bi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct;
(B) a first targetable construct which comprises a carrier portion which comprises or bears at least one epitope recognizable by the at least one other arm of the bi-specific antibody or antibody fragment, and one or more conjugated therapeutic or diagnostic agents, or enzymes; and
(C) optionally, a clearing composition useful for clearing non-localized antibodies and antibody fragments; and
(D) optionally, when the first targetable construct comprises an enzyme,

1) a prodrug, when the enzyme is capable of converting the prodrug to a drug at the target site; or
2) a drug which is capable of being detoxified in the patient
to form an intermediate of lower toxicity, when the enzyme is capable of reconverting the detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of the drug at the target site, or
3) a prodrug which is activated in the patient through
natural processes and is subject to detoxification by conversion to an intermediate of lower toxicity, when the enzyme is capable of reconverting the detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of the drug at the target site, or
4) a second targetable construct which comprises a carrier portion which comprises
or bears at least one epitope recognizable by the at least one other arm of the bi-specific
antibody or antibody fragment, and a prodrug, when the enzyme is capable of converting
the prodrug to a drug at the target site.
[0020] Another embodiment of the invention is to provide DNA constructs which encode such antibodies or antibody fragments. Specifically, DNA constructs which produce the variable regions which provide the advantageous properties of reactivity to a targetable construct and reactivity to a disease tissue. In accordance with this aspect of the present invention, there is provided a recombinant DNA construct comprising an expression cassette capable of producing in a host cell a bi-specific antibody or antibody
fragment having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct, wherein the construct comprises, in the 5' to 3' direction of transcription, a transcriptional initiation regulatory region functional in the host cell, a translational initiation regulatory region functional in the host cell, a DNA sequence encoding the bi-specific antibody or antibody fragment, and a transcriptional and translational termination regulatory region functional in the

host cell, wherein the bi-specific antibody or antibody fragment is under the control of the regulatory
regions.
[0021] Anotlier embodiment of the invention provides a method of preparing the antibodies or antibody
fragments by recombinant technology. In accordance with this aspect of the present invention, there is
provided a method of preparing a bi-specific antibody or antibody fragment having at least one ann that
specifically binds a targeted tissue and at least one other arm that specifically binds a targecable construct,
comprising:
(A) introducing the recombinant DNA construct described above into a host cell;
(B) growing the cell and isolating the antibody or antibody fragment
[0022] In another embodiment of the present invention there is provided a method of preparing a bi-
specific fusion protein having at least one arm that specifically binds to a targeted tissue and at least one other arm that is specifically binds to a targetable construct, comprising:
(1) (A) introducing into a host cell a recombinant DNA construct comprising an
expression cassette capable of producing in the host cell a fragment of the bi-specific fusion protein, wherein the construct comprises, in the 5' to 3". direction of transcription, a transcriptional initiation regulatory region functional in the host cell, a translational initiation regulatory region functional in the host cell, a DNA sequence encoding a scFv linked to a light-chain antibody fragment, and a transcriptional and translational termination regulatory region functional in the host cell, wherein the fragment of the bi-specific fusion protein is under the control of the regulatory regions;
(B) co-introducing into the host cell a recombinant DNA constmct comprising an
expression cassette capable of producing in the host cell a Fd fragment which is
complementary to the light-chain antibody fragment in (A) and which when associated
with the light-chain antibody fragment forms a Fab fragment whose binding site is
specific for the targeted tissue, wherein the construct comprises, in the 5' to 3' direction
of transcription, a transcriptional initiation regulatory region functional in the host cell, a
translational initiation regulatory region functional in the host cell, a DNA sequence
encoding a Fd fragment, and a transcriptional and translational termination regulatory
region functional in the host cell, wherein the Fd fragment is under the control of the
regulatory regions;
(C) growing the cell and isolating the bi-specific fusion protein, or
(2) (A) introducing into a first host cell a recombinant DNA
construct comprising an expression cassette capable of producing in the first host cell a fragment of the bi-specific fusion protein, wherein the construct comprises, in the 5' to 3' direction of transcription, a transcriptional initiation regulatory region functional in the first host cell, a translational initiation regulatory region functional in the first host cell, a

DNA sequence encoding a scFv linked to a light-chain antibody fragment, and a transcriptional and translational termination regulatory region functional in the fust host cell, wherein the fragment of the bi-specific fusion protein is under the control of the regulatory regions;
(B) introducing into a second host cell a recombinant DNA construct comprising an expression cassette capable of producing in the second host cell a Fd fragment which is complementary to the light-chain antibody fragment ia (2)(A) and which when associated with the light-chain antibody fragment forms a Fab fragment whose binding site is specific for the targeted tissue, wherein the construct comprises, in the S' to 3' direction of transcription, a transcriptional initiation regulatory region functional in the second host cell, a translational initiation regulatory region functional in the second host cell, a DNA sequence encoding a Fd fragment, and a transcriptional and translational tennination regulatory region functional in the second host cell, wherein the Fd fragment is under the control of the regulatory regions;
(C) growing the first and second host cells;
(D) optionally isolating Che bi-specific fusion protein fragment and the Fd fragment; and
(E) combining the fragments to produce a bi-specific fusion protein and isolating the
bi'Specific fusion protein.
[0023] A variety of host cells can be used to prepare bi-specific antibodies or antibody fragments,
including, but not limited to, mammalian cells, insect cells, plant cells and bacterial cells. In one
embodiment, the method utilizes a mammalian Tygote, and the introduction of the recombinant DNA
construct produces a transgenic animal capable of producing a bi-specific antibody or antibody fragment.
10024] The present invention seeks to provide inter alia a bi-specific antibody or antibody fragment
having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically
binds a targetable construct that can be modified for use in a wide variety of diagnostic and therapeutic
applications.
[0025] A further embodiment of the invention involves using the inventive antibody or antibody fragment
in photodynamic therapy.
[0026] A further embodiment of the invention involves using the inventive antibody or antibody fragment
in radioimmunoimaging for positron-emission tomography (PET).
[00271 A further embodiment of the invention involves using the inventive antibody or antibody fragment
in radioimmunoimaging for single-photon emission.
|002SI A further embodiment of the invention involves using the inventive antibody or antibody fragment
in magnetic resonance imaging (MRI).

[ 0029] A further embodiment of the invention involves using the inventive antibody or antibody fragment
in X-ray, computed tomography (CT) or ultrasound imaging.
[0030] A further embodiment of the invention involves using the inventive antibody or antibody fragment
for intraoperative, endoscopic, or intravascular detection and/or therapy.
[0031] A further embodiment of the invention involves using the inventive antibody or antibody fragment
in boron neutron capture therapy (BNCT),
[0032] A further embodiment of the invention involves using the inventive antibody or antibody fragment
for diagnosing or creating diseased tissues (e.g., cancers, infections, inflammations, clots, atherosclerois,
infarcts), normal tissues (e.g., spleen, parathyroid, thymus, bone marrow), ectopic tissues (e.g.,
endometriosis), and pathogens,

as well as methods of making the bi-specifics, and kits for use in such methods.

[0034] The present inventors have discovered that it is advantageous to raise bsAbs against a targetable construct that is capable of carrying one or more diagnostic or therapeutic agents. By utilizing this technique, the characteristics of the chelator, metal chelate complex, therapeutic agent or diagnostic agent can be varied to accommodate differing applications, without raising new bsAhs for each new application. Further, by using this approach, two or more distinct chelators, metal chelate complexes or therapeutic agents can be used with the inventive bsAb.
[0035] The invention relates to a method of treating or identifying diseased tissues in a subject, comprising:
(A) administering to said subject a bi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct comprising at least two HSG haptens;
(B) optionally, administering to said subject a clearing composition, and allowing said composition to clear non-localized antibodies or antibody fragments from circulation;
(C) administering to said subject a targetable construct which comprises a earner portion which comprises or bears at least two HSG haptens and at least one chelator, and may comprise at least one diagnostic and/or therapeutic cation, and/or one or more chelated or chemically bound therapeutic or diagnostic agents, or enzymes; and
(D) when said targetable construct comprises an enzyme, fizrther administering to said subject

1) a prodrug, when said enzyme is capa We of converting said prodrug to a drug at the target site; or
2) a drug which is capable of being detoxified in said subject
to form an intermediate of lower toxicity, when said enzyme is capable of reconverting said detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of said drug at the target site, or
3) a prodrug which is activated in said subject through
natural processes and is subject to detoxification by conversion to an intermediate of lower
toxicity, when said enzyme is capable of reconverting said detoxified intermediate to a toxic form,
and, therefore, of increasing the toxicity of said drug at the target site. |0036] The invention further relates to a method for detecting or treating target cells, tissues or pathogens in a manunal, comprising;
administering an effective amount of a bi-specific antibody or antibody fragment comprising at
least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct;
wherein said at least one arm is capable of binding to a complementary binding moiety on the target cells, tissues or pathogen or on a molecule produced by or associated therewith; and
administering a target

[0037] The invention further relates to a method of treating or identifying diseased tissues in a subject, comprising:
administering to said subject a bi-specific antibody or antibody fragment having at least one ami that specifically binds a targeted tissue and at least one other ann that specifically binds a targetable construct;
optionally, administering to said subject a clearing composition, and allowing said composition to clear non-localized antibodies or antibody fragments from circulation; and
administering to said subject a targetable constmct selected from the group consisting of:

[0038] The invention further relates to a kit useful for treating or identifying diseased tissues in a subject
comprising:
(A) a bi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one ot\her arm that specifically binds a targetable construct, wherein said construct is selected from the group consisting of

(B) a targetable construct which comprises a carrier portion which comprises or bears at least
one epitope recognizable by said at least one other arm of said bi-specific antibody or antibody fragment,
and one or more conjugated therapeutic or diagnostic agents, or enzymes; and
(C) optionally, a clearing composition useful for clearing non-localized antibodies and
antibody fragments; and
(D) optionally, when said first targetable construct comprises an enzyme
1) a prodrug, when said enzyme is enable of converting said
prodrug to a drug at the target site; or
2) a drag which is capable of being detoxified in said subject to foim an
intermediate of lower toxicity, when said enzyme is capable of reconverting said
detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of said
drug at the target site, or
3) a prodrug which is activated in said subject through natural processes and is
subject to detoxification by conversion to an intermediate of lower toxicity, when said
enzyme is capable of reconverimg said detoxified intermediate to a toxic form, and,
therefore, of increasing the toxicity of said drug at the target site.
(0039) The invention further relates to a targetable construct selected from the group consisting of:

[0040] The invention further relates to a method of screening for a targetable construct comprising:
contacting said targetable construct with a bi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds said targetable construct to give a mixture;
wherein said at least one arm is capable of binding to a complementary binding moiety on the target cells, tissues or pathogen or on a molecule produced by or associated therewith; and
optionally incubating said mixture; and
analyzing said mixture. (0041] The invention further relates to a method for imaging normal tissue in a mammal, comprising:
administering an effective amount of a bi-specific antibody or antibody fragment comprising at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct;
wherein said at least one arm is capable of binding to a complementary binding moiety on the
target cells, tissues or on a molecule produced by or associated therewith; and
administering a targetable construct selected from the group consisting of

[0042] The invention further relates to a method of intraoperatively identifying or treating diseased tissues, in a subject, comprising:
administering an effective amount of a bi-specific antibody or antibody fragment comprising at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct;
wherein said at least one arm is capable of binding to a complementary binding moiety on die target cells, tissues or pathogen or on a molecule produced by or associated therewith; and
administering a targetable construct selected from the group consisting of

i
|0043] The invention further relates to a method for the endoscopic identification or treatment of diseased tissues, in a subject, comprising:
administenng an effective amount of a bi-specific antibody or antibody fragment comprising at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct;
wherein said at least one arm is capable of binding to a compiementary binding moiety on the target cells, tissues or pathogen or on a molecule produced by or associated therewith; and
administering a targetable construct selected from the group consisting of

I0044J The invention further relates to a method for the intravascular identification or treatment of diseased tissues, in a subject, comprising:
administering an effective amount of a bi-specific antibody or antibody fragment comprising at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct;
wherein said at least one arm is capable of binding to a complementary binding moiety on the target cells, tissues or pathogen or on a molecule produced by or associated therewith; and
administering a targetable construct selected from the group consisting of

[00451 Additional aspects, features and advantages of the invention will be set forth in the description which
follows, and in part will be obvious from the description, or may be learned by practice of the invention. The
embodiments and advantages of the invention may be realized and obtained by means of the instrumentalities
and combinations particularly pointed out in the appended claims.
Brief Description of the Drawings
[0046] Figure 2 schematically illustrates various Abs and bsAbs.
[0047] Figure 2 provides SDS-PAGE analysis of purified hMN-14Fab-734scFv. 3 µg of hMN-14 IgG
(lanes 1 and 3) or bsAb (lanes 2 and 4) was applied in each lane of a 4-20% polyacrylamide gel under non-
reducing (lanes 1 and 2) and reducing (lanes 3 and 4) conditions.
[0048] Figure 3 schematically illustrates two bi-specific fusion proteins.
[0049] Figure 4 illustrates the production of a DNA construct useful for producing a hMN-14Fab-
734scFv bi-specific fusion protein,
|0050] Figure 5 illustrates the production of a DNA construct useful for producing a hMN-14Fab-
734scFv bi-specific fusion protein,
(00511 Figure 6 shows the binding properties of hMN-14 x m679 bsMAb with 111In-labeled IMP-24I
divalent HSG-DOTA peptide. Panel A: min.lMP-241 alone on SE-HPLC; Panel B: 111IN-IMP-241
mixed with hMN-14 x 679 bsMAb; Panel C: 111n-IMP-241 added to a mixture containing hMN-14 x
m679 bsMAb with an excess of CEA. Chromatograms show the association of the 111n-IMP-241 with
the bsMAb (B) and bsMAb/CEA complex (C),

lA and other Ep-CAM targets), Le(y) (e.g., B3), A3, KS-1, S100, IL-2, T101, necrosis antigens, folate receptors, angiogenesis markers (e.g., VEGF), tenascin, PSMA, PSA, tumor-associated cytokines, MAGE and/or fragments thereof. Tissue-specific antibodies (e.g., against bone man-ow cells, such as CD34, CD74, etc., parathyroglobulin antibodies, etc.) as well as antibodies against non-malignant diseased

tissues, such as fibrin of clots, macrophage antigens of atherosclerotic plaques (e.g., CD74 antibodies), and also specific pathogen antibodies (e.g., against bacteria, viruses, and parasites) are well known in the art. [0056] The peptides can be radiolabeled to a high specific activity in a facile manner that avoids the need for purification. In vivo studies in tumor bearing nude mice showed the radiolabeled peptides cleared rapidly from the body with minimal retention in tumor or normal tissues. When administered 1 to 2 days after a pretargeting dose of the bsAbs, tumor uptake of the radiolabeled peptides increased from 28 to 175-fold with tumor/nontunior ratios exceeded 2:1 to 8:1 within just 3 hour of the peptide injection, which represented a marked improvement over that seen with a 99mTc-anti-CEA Fab' at this same time. The anti-CSAp x anti-HSG F(ab')2 bsAb had the highest and longest retention in the tumor, and when used in combinarioh with the IIIIn-labeled peptide, radiation dose estimates for therapeutic radionuclides, such as 90 and 1500cGy, suggested that as much 12,000 cGy could be delivered to tumors with the kidneys receiving 1500 cGy, but all other tissues receiving 500 cGy, Thus, this pretargeting system is highly flexible, being capable of using a wide array of compounds of diagnostic imaging and therapeutic interest, and by achieving excellent tumor uptake and targeting ratios, is highly promising for use in these applications.
[0057J Additionally, encompassed is a method for detecting and/or treating target cells, tissues or pathogens in a mammal, comprising administering an effective amount of a bi-specific antibody or antibody fragment comprising at least one arm that specifically binds a targeted tissue and at least one other arm that spec/fically binds a targetable construct. As used herein, the term pathogen" includes, but is not limited to fungi (e.g., Microsporum, Trichophyton, Epidermophyton, Sporothrix schenckii, Cryptococcus neoformans, Coccidioides immitis, Histoplasma Capsulatum, Blastomyces dermatitidis, Candida albicans), viruses (e.g., human immunodeficiency virus (HIV), herpes virus, cytomegalovirus, rabies vims, influenza vims, hepatitis B vims, Sendai vims, feline leukemia virus, Reo virus, polio virus, human serum parvo-like vims, simian virus 40, respiratory syncytial virus, mouse mammary tumor virus, Varicella-Zoster virus, Dengue virus, rubella virus, measles virus, adenovims, human T-cell leukemia viruses, Epslein-Barr vims, murine leukemia vims, mumps vims, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis vims, wart vims and blue tongue virus), parasites, bacteria (e.g.. Anthrax bacillus, Streptococcus agalactiae, Legionella pneumophilia. Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae. Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum. Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae. Brucella abortus, Mycobacterium tuberculosis and Tetanus toxin), mycoplasma (e.g.. Mycoplasma arthritidis, M. hyorhinis,
M. orale, M. arginini, Acholeplasma laidlawii, M. salivamm, and M. pneumoniae) and protozoans (e.g., Plasmodium falciparum, Plasmodium vivax. Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma bmcei. Schistosoma mansom'. Schistosoma japanicum, Babesia bovis, Elmena lenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis, Onchocerca

volvulus, Theileria parva, Taenia hydatigena. Taenia ovis. Taenia saginata, Echinococcus granulosus and Mesocestoides corti). See U.S. Patent No. 5,332,567.
10058] Also provided herein are antibodies and antibody fragments. The antibody fragments are antigen binding portions of an antibody, such as F(ab')2, F(ab)2i Fab', Fab, and the like. The antibody fragments bind to the same antigen that is recognized by the intact antibody. For example, an anti-CD22 monoclonal antibody fragment binds to an epitope of CD22,
10059] The term "antibody fragment" also includes any s}'nthetic or genetically engineered protein feat acts like an antibody by binding to a specific antigen to form a conq)lex. For example, andbody fragments include isolated fragments, "Fv" fragments, consisting of the variable regions of tiie heavy and light chains, recombinant single chain polypeptide molecules in which Jight and heavy diain variable rsgions ars connected by a peptide linker ("sFv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the "hypervariable regioa" Three of these so-called "hypervariable" regions or "complementarity-detennining regions" (CDR) are found in each variable region of the light or heavy chain. Each CDR is flanked by relatively conserved framework regions (FR). The FR are thought to maintain the structural integrity of the variable region. The CDRs of a light chain and the CDRs of a corresponding heavy chain form the antigen-binding site. The "hypervariability" of fee CDRs accounts for the diversity of specificity of antibodies.
[0060] As used herein, the term "subject" refers to any ammal (i.e., vertebrates and invertebrates) including, but not limited to humans and other primates, rodents (e-g-, mice, rats, and guinea pigs), lagamorphs (e.g., rabbits), bovines (e.g, cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., swine), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), domestic fowl (e.g., chickens, tuikcys, ducks, geese, other gallinaceous birds, etc.), as well as feral or wild animals, including, but not limited to, such animals as ungulates (e.g., deer), bear, fish, lagamorphs, rodents, birds, etc. It is not intended that the term be limited to a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are encompassed by the term IL Constructs Targetable to Antibodies
[0061] The targetable construct can be of diverse structure, but is selected not only to diminish fee elicitation of immune responses, but also for rapid in vivo clearance when used within the bsAb targeting method. Hydrophobic agents are best at eliciting strong immune responses, whereas hydrophilic agents are preferred for rapid in vivo clearance, thus, a balance between hydrophobic and hydrophilic needs to be established. This is accomplished, in part, by relying on fee use of hydrophilic chelating agents to offset the inherent hydrophobicity of many organic moieties. Also, sub-units of fee targetable construct may be chosen which have opposite solution properties, for example, peptides, which contain amino acids, some of which are hydrophobic and some of which are hydrophih'c. Aside from peptides, carbohydrates may be used.

j0065| The peptides to be used as immunogens are synthesized conveniently on an automated peptide synthesizer using a solid-phase support and standard techniques of repetitive orthogonal deprotection and coupling. Free amino groups in the peptide, that are to be used later for chelate conjugation, are advantageously blocked with standard protecting groups such as an acetyl group. Such protecting groups wilL be known to the skilLed artisan. See Greene and Wuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons, N.Y.). When the peptides are prepared for later use within the bsAb system, they

are advantageously cleaved from the resins to generate the corresponding C-terminal amides, in order to
inhibit in vivo carboxypeptidase activity.
in. Chelate Moieties
(0066] The presence of hydrophilic chelate moieties on the targetable construct helps to ensure rapid in
vivo clearance. In addition to hydrophilicity, chelators are chosen for their metal-binding properties, and
may be changed at will since, at least for those targetable constructs whose bsAb epitope is part of the
peptide or is a non-chelate chemical hapten, recognition of the metal-chelate complex is no longer an
"- " ~"—" J"— — .^_,,,. __, - ,, ,_ _ ^,
DOT A, and TETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) are of use with a variety of metals and radiometals, most particularly with radionuclides of Ga, Y and Cu, respectively. [0068] DTPA and DOTA-type chelators, where the ligand includes hard base chelating functions such as carboxylate or amine groups, are most effective for chelating hard acid cations, especially Group IIa and Group IIIa metal cations. Such metal-chelate complexes can be made very stable by tailoring the ring size to the metal of interest. Other ring-type chelators such as macrocyclic polyethers are of interest for stably binding nuclides such as 223Ra for RAIT. Porphyrin chelators may be used with numerous radiometals, and are also useful as certain cold metal complexes for bsAb-directed immuno-phototherapy. Also, more than one type of chelator may be conjugated to the targetable construct to bind multiple metal ions, e.g., cold ions, diagnostic radionuclides and/or therapeutic radionuclides.

the linker, e.g., with different chelate ring sizes, to bind preferentially to two different hard acid or soft acid cations, due to the differing sizes of the cations, the geometries of the chelate rings and the preferred complex ion structures of the cations. This will permit two different metals, one or both of which may be radioactive or useful for MRI enhancement, to be incorporated into a linker for eventual capture by a pretargeted bsAb.

[0073] Preferred chelators include NOT A, DOTA and Tscg and combinations thereof. These chelators have been incorporated into a chelator-peptide conjugate motif as exemplified in the following constructs:
(0074) The chelator-peptide conjugates (f) and (g), above, has been shown to bind 68Ga and is thus useful in positron emission tomography (PET) applications.
10075] Chelators are coupled to the peptides of the targetable construct using standard chemistries, some of which are discussed more fully in the working examples below. Briefly, the synthesis of the peptide Ac-Lys(HSG)-D-Tyr-Lys(HSO)-Lys(Tscg-Cys)-NH2 (SEQ ID NO; 4)was accomplished by first attaching Aloc-Lys(Fmoc)-OH to a Rink amide resin on the peptide synthesizer. The protecting group abbreviations
"Aloe" and "Fnioc" used herein refer to die groups aliyloxycarbonyl and fluorenyimethyloxy carbonyl. The FmoC"Cys(Trt)-OH and TscG were then added to the side chain of the lysine using standard Fmoc automated synthesis protocols to form the following peptide: Aloc-Lys(Tscg-Cys(Trt))-rink resin. The Aloc group was then removed. The peptide synthesis was then continued on the synthesizer to make the ibJIowing peptide: Lys(A]oc)-D-Tyr-Lys(Aloc).Lys(Tscg"Cys(Trt)-rink resin (SEQ ID NO: 4). Following N-terminus acylation, and removal of the side chain Aloe protecting groups. The resulting

10076] Chelator-peptide conjugates may be stored for long periods as solids. They may be metered into unit doses for metal-binding reactions, and stored as unit doses either as solids, aqueous or semi-aqueous solutions, frozen solutions or lyophilized preparations. They may be labeled by well-known procedures, [0077] Typically, a hard acid cation is introduced as a solution of a convenient salt, and is taken vp by the hard acid chelator and possibly by the soft acid chelator. However, later addition of soft acid cations leads to binding thereof by the soft acid chelator, displacing any hard acid cations which may be chelated therein. For example, even in the presence of an excess of cold IIIInCl3, labeling with 99m-Tc(V) glucoheptonate or with Tc cations generated in situ with stannous chloride and Na99m-Tc04 proceeds quantitatively on the soft acid chelator.

identify and treat them when they are ectopic (i.e,, displaced torn their normal location), such as in endometriosis.
[0080] The administration of a bsAb and the targetable construct discussed above may be conducted by administering the bsAb at some time prior to administration of the therapeutic agent which is associated with the linker moiety. The doses and liming of the reagents can be readily devised by a skilled artisan and are dependent on the specific nature of the reagents employed. If a bsAb-F(ab')2 derivative is given first, then a waiting time of 1-6 days before administration of the targetable construct may be appropriate. If an IgG-Fab' bsAb conjugate is the primary targeting vector, then a longer waiting period before administration of the linker moiety may be indicated, in the range of 3-15 days. Alternatively, the bsAb and the targetable construct may be administered substantially at the same time in either a cocktail fonn or by administering one after the other.
|0081] A wide variety of diagnostic and therapeutic reagents can be advantageously conjugated to the targetable construct. Generally, diagnostic and therapeutic agents can include isotopes, drugs, toxins, cytokines, conjugates with cytokines, hormones, growth factors, conjugates, radionuclides, contrast agents, metals, cytotoxic drugs, and immune modulators. For example, gadolinium metal is used for magnetic resonance imaging and fluorochromes can be conjugated for photodynamic therapy. Moreover, contrast agents can be MRI contrast agents, such as gadolinium ions, lanthanum ions, manganese ions, iron, chromium, copper, cobalt, nickel, dysprosium, rhenium, europium, terbium, holmium, neodymium or other comparable label, CT contrast agents, and ultrasound contrast agents. Addtional diagnostic agents can include fluorescent labeling compounds such as fluorescein isothiocyanate, rhodamine, phycocrytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine, chemiluminescent compounds including luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester, and bioluminescent compounds including luciferin, luciferase and aequorin. Radicmuclides

the art. Therapeutic agents may also include, without limitation, others drugs, prodrugs and/or toxins. The terms "drug," 'prodrug," and "toxin" are defined throughout the specification. The terms "diagnostic agent" or "diagnosis" include, but are not limited to, detection agent, detection, or localization. |0083] When the targetable construct includes a diagnostic agent, the bsAb is preferably administered prior to administration of the targetable construct with the diagnostic agent After sufficient time has passed for the bsAb to target to the diseased tissue, the diagnostic agent is administered, by means of (he targetable construct, so that imaging can be performed. Tumors can be detected in body cavities by means of directly or indirectly viewing various structures to which light of the appropriate wavelength is delivered and then collected, or even by special detectors, such as radiation probes or fluorescent detectors, and the like. Lesions at any body site can be viewed so long as nonionizing radiation can be delivered and recaptured from these structures. For example, PET which is a high resolution, non-invasive, imaging technique can be used with the inventive antibodies and targetable constructs for the visualization of human disease. In PET, 511 ke V gamma photons produced during positron annihilation decay are detected. X-ray, computed tomography (CT), MRl and gamma imaging (e.g.. Single Photon Emission Computed Tomogr^hy (SPECT)) may also be utilized through use of a diagnostic agent that functions with these modalities. 10084] As discussed eariier, the targetable construct may include radioactive diagnostic agents that emit

[0085] The present bsAbs or bsFabs can be used in a method of photodynamic therapy (PUT) as discussed in U.S. Patent Nos, 6,096,289; 4,331.647; 4,818,709; 4,348,376; 4,361,544; 4,444,744; 5,851,527. In PDT, a photosensitizer, e.g., a hematoporphyrin derivative such as dihematopoiphyiin ether, is administered to a subject. Anti-tumor activity is initiated by the use of light, e.g., 630 nm. Alternate photosensitizers can be utilized, including those useful at longer wavelengths, where skin is less photosensitized by the sun. Examples of such photosensitizers include, but arc not limited to, benzoporphyrin monoacid ring A (BPD-MA), tin etiopurpurin (SnET2), sulfonated aluminum phthalocyanine (AlSPc) and lutetium texaphyrin (Lutex).
[0086] Additionally, in PDT, a diagnostic agent may be injected, for example, systematically and laser-induced fluorescence can be used by endoscopes including wireless capsule-sized endoscopes or cameras to detect sites of cancer which have accreted the light-activated agent. For example, this has been applied to fluorescence bronchoscopic disclosure of early lung tumors. Doiron et al Chest 76:32 (1979). In another
example, the antibodies and antibody fragments can be used in single photon emission. For example, a Tc-

00871] Therapeutically useful immunoconjugates can be obtained by conjugating photoactive agents or dyes to an antibody composite. Fluorescent and other chromogens, or dyes, such as porphyrins sensitive to visible light, have been used to detect and to treat lesions by directing the suitable light to the lesion. In therapy, this has been termed photoradiation, phototherapy, or photodynamic therapy (Jori et al (eds.),

include iodine compounds, barium compounds, gallium compounds, thallium compounds, etc. Specific compounds include barium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid, iocetamic acid, iodamide, iodipamlde, iodoxamic acid, iogulamide, iohexol, iopamidol, iopanoic acjd, ioprocemic acid, josefamic acid, ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, totetric acid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallous chloride. Ultrasound contrast materia] may also by used including dcxtrsn and Uposomes, particularly gas-filled liposomes. In one embodiment, an immunomodulator, such as a cytokine, may also be conjugated to the targetable construct by a linker or through other methods known by those skilled in the

construct having a low MW hapten is administered. After the enzyme is pretargeted to the target site by bsAb;targetable construct binding, a cytotoxic drug is injected that is known to act at the target site. The dnig may be one which is detoxified by the mammal's ordinary detoxification processes to form an
intermediate of lower toxicity. For example, the drug may be converted into the potentially less toxic

glucuronide in the liver. The detoxified intermediate can then be reconverted to its more toxic form by the pretargeted enzyme at the target site, and this enhances cytotoxicity at the target site. (00901 Alternatively, an administered prodrug can be converted to an active drug by the pretargeted enzyme. The pretargeted enzyme improves the efficacy of the treatment by recycling the detoxified drug. This approach can be adopted for use with any enzyme-drug pair. Alternatively, the targetable construct with enzyme can be mixed with the targeting bsAb prior to administration to the patient. After a sufficient time has passed for the bsAb:targetable construct- conjugate to localize to the target site and for unbound targetable construct to clear from circulation, a prodrug is administered. As discussed above, the prodrug is then converted to the drug in situ by the pre-targeted enzyme.
[0091] Certain cytotoxic drugs that are useful for anticancer therapy are relatively insoluble in serum Some are also quite toxic in an unconjugated form, and their toxicity is considerably reduced by conversion to prodrugs. Conversion of a poorly soluble drug to a more soluble conjugate, e.g:, a glucuronide, an ester of a hydrophilic acid or an amide of a hydrophilic amine, will improve its solubility in the aqueous phase of serum and its ability to pass through venous, arterial or capillary cell walls and to reach the interstitial fluid bathing the tumor. Cleavage of the prodrug deposits the less soluble drug at the target site. Many examples of such prodrug-to-drug conversions are disclosed in U,S, Patent No. 5,851,527, to Hansen.

[0094] Eloposide is a widely used cancer drug that is detoxified to a major extent by fonnation of its glucuronide and is within the scope of the invention. See, e.g., Hande et aL Cancer Res. 48:1829-1834 (1988). Glucuronide conjugates can be prepared from cytotoxic dmgs and can be injected as therapeutics for tumors pre-targeted with mAb-glucuronidase conjugates. See, e.g., Wang et aL Cancer Res, 52:4484 4491 (1992). Accordingly, such conjugates also can be used with the pre-targeting approach described here. Similarly, designed prodrugs based on derivatives of daunomycin and doxorubicin have been described for use with carboxylesterases and glucuronidases. See, c.g., Baikina et aL J. Med Chem. ' 40:4013-4018 (1997), Other examples of prodrug/enzyme pairs that can be used within the present invention include, but are not limited to, glucuronide prodrugs of hydroxy derivatives of phenol mustards and beta-g/ucuronidase; phenol mustards or CPT-l I and carboxypeptidase; methotrexate-substituted alpha-amino acids and carboxypeptidase A; penicillin or cephalosporin conjugates of drugs such as 6-mercaptopurine and doxorubicin and beta-lactamase; etoposide phosphate and alkaline phosphatase.[
[0095] The enzyme capable of activating a prodrug at the target site or improving the efficacy of a normal therapeutic by controlling the body's detoxification pathways may altematively be conjugated to the hapten. The enzyme-hap en conjugate is administered to the subject following administration of the pre-targeting bsAb and is directed to the target site. After the enzyme is localized at the target site, a cytotoxic drug is injected, which is known to act at the target site, or a prodrug form thereof which is converted to the drug in situ by the pretargeted enzyme. As discussed above, the drug is one which is detoxified to form an intermediate of lower toxicity, most commonly a glucuronide, using the mammal's ordinary detoxification processes. The detoxified intermediate, e.g., the glucuronide, is reconverted to its more toxic form by the pretargeted enzyme and thus has enhanced cytotoxicity at the target site. This results in a recycling of the drug. Similarly, an administered prodrug can be converted to an active drug through normal biological processes. The pretargeted enzyme improves the efficacy of the treatment by lecycling the detoxified drug. This approach can be adopted for use with any enzyme-drug pair. [0096] In an alternative embodiment, the enzyme-hapten conjugate can be mixed with the targeting bsAb prior to administration to the patient. After a sufficient time has passed for the enzyme-hapten-bsAb conjugate to localize to the target site and for unbound conjugate to clear from circulation, a prodrug is administered. As discussed above, the prodrug is then converted to the drug in situ by the pre-targeted enzyme,
[0097] The invention further contemplates the use of the inventive bsAb and the diagnostic agent(s) in the context of Boron Neutron Capture Therapy (BNCT) protocols, BNCT is a binary system designed to
deliver ionizing radiation to tumor cells by neutron irradiation of tumor-localized 10B atoms. BNCT is based on the nuclear reaction which occurs when a stable isotope, isotopically eiuiched 10B (present in 19.8% natural abundance), is irradiated with thermal neutrons to produce an alpha particle and a 7Li nucleus. These particles have a path length of about one cell diameter, resulting in high linear energy transfer. Just a few of the short-range 1.7 MeV alpha particles produced in this nuclear reaction are

sufficient to target the cell nucleus and destroy it. Success with BNCT of cancer requires methods for localizing a high concentration of 10B at tumor sites, while leaving non-target organs essentially boron-free. Compositions and methods for treating tumors in subjects using pre-targeting bsAb for BNCT are described in U.S. Patent No. 6,228,362 and can easily be modified for the pujposes of the present invention.
(00981 In another embodiment of the present invention, the peptide backbone of the targetabie construct is conjugated to a prodrug. The pre-targeting bsAb is administered to the patient and allowed to localize to the target and substantially clear circulation. At an appropriate later time, a targetabie construct comprising a prodrug, for example poly-glutamic acid (SN-38-ester)10 is given, thereby localizing the prodrug specifically at the tumor target. Jt is known that tumors have increased amounts of enzymes released from intracellular sources due to the high rate of lysis of cells within and around tumors. A practitioner can capitalize on this fact by appropriately selecting prodrugs capable of being activated by these enzymes. For example, carboxylesterase activates the prodrug poly-glutamic acid (SN-38-ester)10 by cleaving the ester bond of the poly-glutamic acid (SN-38-ester)10 releasing large concentrations of free SN-38 at the tumor. Alternatively, the appropriate enzyme also can be targeted to the tumor site. /0099J After cleavage from the targetabie construct, the drug is intemalized by the tumor cells. Alternatively, the drug can be intemalized as part of an intact complex by virtue of cross-linking at the target. The targetabie construct can induce internalization of tumor-bound bsAb and thereby improve the efficacy of the treatment by causing higher levels of the drug to be internalized, [0100] A-vaiiety of peptide caniers-are-well-suited for conjugation to prodrugs,, including.polyaniino_ acids, such as polylysine, polyglutamic (E) and aspartic acids (D), including D-amino acid analogs of the same, co-polymers, such as poly(Lys-G3u} {poly[KE]}, advantageously from 1:10 to 10:1. Copolymers based on amino acid mixtures such as poly(Lys-Ala-Glu-Tyr (SEQ ID NO: 8) (KAEY; 5:6:2:1) can also be employed. Smaller polymeric carriers of defined molecular weight can be produced by solid-phase peptide synthesis techniques, readily producing polypeptides of from 2-50 residues in chain length. A second advantage of this type of reagent, other than precise structural definition, is the ability to place single or any desired number of chemical handles at certain points in the chain. These can be used later for attachment of recognition and therapeutic haptens at chosen levels of each moiety. [0101] Poly(ethylene) glycol [PEG] has desirable in vivo properties for a bi-specific antibody prodrug approach. Ester linkages between the hydroxyl group of SN-38 and both ends of a standard di-hydroxyl PEG can be introduced by insertion of diacids such as succinic acid between the SN-38 and PEG hydroxy] groups, to generate species such as SN-38-O-CO(CH2)2CO-O-PEG-O-CO(CH2)2CO-OSN-38. The di-SN-38-PEG produced can be considered as the shortest member of the class of SN-38-polymer prodrugs. The desirable in vivo properties ofPEG derivatives and the limited loading capacity due to their dimeric functionality led to the preparation of PEG co-polymers having greater haptcn-bearing capacity such as those described by Poiani et al See, e.g., Poiani et al Bioconjugate Chem,, 5:621-630,1994. PEG

derivatives are activated at both ends as their bis(succinimidyl)carbonate derivatives and co-polymerized with multi-functional diamines such as lysine. The product of such co-polymerization, containing (-Lys(COOH)-PEG-Lys(COOH)-PEG-)n repeat units wherein the lysyl carboxyl group is not involved in the polymerization process, can be used for attachment of SN-38 residues. The SN-38 residues are reacted with the free carboxyl groups to produce SN-38 esters of the (-Lys-(COOH)-PEG-Lys(COOH)-PEG-)n chain.
[0102] Other synthetic polymers that can be used to cany recognition haptens and prodrugs include N-(2-hydroxypropyl)methacrylamide (HMPA) copolymers, poly(styrene-co-maleic acid/anhydride (SMA), poly(divinylether maleic anhydride) (DIVEMA), polyethyleneimine, ethoxylated polyethylene-imine, starburst dendrimers and poly(N-vinylpyrrolidone) fPVP). As an example, DIVEMA polymer comprised of multiple anhydride units is reacted with a limited amount of SN-38 to produce a desired substitution ratio of drug on the polymer backbone. Remaining anhydride groups are opened under aqueous conditions to produce free carboxylate groups. A limited number of the free carboxylate groups are activated using standard water-soluble peptide coupling agents, e.g. l-ethyI-3-(3-dimethyIaminopropyl)carbodiimide hydrochloride (EDO),-and coupled to a recognition moiety bearing a free amino group. An example of the latter is histamine, to which antibodies have been raised in the past
[0103] A variety of prodrugs can be conjugated to the targetable construct The above exemplifications of polymer use are concerned with SN-38, the active metabolite of the prodrug CPT-l 1 (irinotecan). SN-38 has an aromatic hydroxyl group that was used in the above descriptions to produce aryl esters susceptible to esterase-type enzymes. Similarly the camptothecin analog topotecan, widely used in chemotherapy, has an available aromatic hydroxyl residue that can be used in a similar manner as described for SN-38, producing esterase-susceptible polymer-prodrugs. [0104] Doxorubicin also contains aromatic hydroxyl groups that can be coupled to caiboxylate-containing polymeric carriers using acid-catalyzed reactions similar to those described for the camptothecin family. Similarly, doxorubicin analogs like daunomycin, epirubicin and idarubicin can be coupled in the same manner. Doxorubicin and other drugs with amino 'chemical handles, active enough for chemical coupling to polymeric carriers can be effectively coupled to carrier molecules via these free amino groups in a number of ways. Polymers bearing free carboxylate groups can be activated en situ (EDC) and the activated polymers mixed with doxorubicin to directly attach the drug to the side-chains of the polymer via amide bonds. Amino-containing drugs can also be coupled to amino-pendant polymers by mixing commercially available and cleavable cross-linking agents, such as ethylene
glycobis{succinimidylsuccinate) (EGS, Pierce Chemical Co., Rockford, IL) or bi5-[2-(5uccinimido-oxycarbonyloxy)ethyl]sulfone {BSOCOES, Molecular Biosciences, Huntsville, AL), to cross-link the two amines as two amides after reaction with the his(succinimidyl) ester groups. This is advantageous as these groups remain susceptible to enzymatic cleavage. For example, (doxorubicin-EGS)n-poly-lysine remains susceptible to enzymatic cleavage of the diester groups in the EGS linking chain by enzymes such as

or It can be injected and localized at the target site and, after non-targeted antibody has substantially cleared from the circulatory system of the mammal, enzyme can be injected in an amount and by a route which enables a sufficient amount of the enzyme to reach a localized antibody or antibody fragment and bind to it to form the antibody-enzyme conjugate in situ.

toxicity from the detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of the dnig at the target site. A second targetable construct may also be used which comprises a carrier portion which comprises or bears at least one epitope recognizable by the at least one other arm of the bi-specific antibody or antibody fragment, and a prodrug, when the enzyme is capable of converting the prodrug to a drug at the target site. Instruments which facilitate identiiying or treating diseased tissue also can be included in the kit. Examples include, but are not linuted to application devices, such as syringes. Solutions required for utilizing the disclosed invention for identifying or treating diseased tissue also can be included in the kit.
(01161 The targetable construct may be administered intravenously, intraaterially, intraoperatively, endoscopically, intraperitoneally, intramuscularly, subcutaneously, intrapleurally, intrathecally, by perfusion through a regional catheter, or by direct intralesional injection, and can be by continuous infusion or by single or multiple boluses, or through other methods known to those skilled in the art for diagnosing (detecting) and treating diseased tissue. Further, the targetable construct may include agents for other methods of detecting and treating diseased tissue including, without limitation, conjugating dextran or liposome formulations to the targetable construct for use with ultrasound, or other contrast agents for use with other imaging modalities, such as X-ray, CT, PET, SPECT and ultrasound, as previously described VI. Methods for Raising Antibodies
j01l7) Abs to peptide backbones and/or haptens are generated by well-known methods for Ab production. For example, injection of an immunogen, such as (peptide)n-KLH, wherein KLH is keyhole limpet hemocyanin, and n=I-30, in complete Freund's adjuvant, followed by two subsequent injections of the same immunogen suspended in incomplete Freund's adjuvant into immunocompetent animals, is followed three days after an i.v. boost of antigen, by spleen cell harvesting. Harvested spleen cells are then fused with Sp2/0-Agl4 myeloma cells and culture supematants of the resulting clones aruilyzed for anti-pepdde reactivity using a direct-binding ELISA. Fine specificity of generated Abs can be analyzed for by using peptide fragments of the original immunogen. These fragments can be prepared readily using an automated peptide synthesizer. For Ab production, enzyme-deficient hybridomas are isolated to enable selection of fused cell lines. This technique also can be used to raise antibodies to one or more of the chelates comprising the linker, e.g., ln(ni)-DTPA chelates. Monoclonal mouse antibodies to an In(in)-di-DTPA are known (Barbel 395 supra).
[0118] The antibodies used in the present invention are specific to a variety of cell surface or intracellular tumor-associated antigens as marker substances. These markers may be substances produced by the tumor or may be substances which accumulate at a tumor site, on tumor cell surfaces or within tumor cells, whether in the cytoplasm, the nucleus or in various organelles or sub-cellular structures. Among such tumor-associated maricers are those disclosed by Herberznan, "Immunodiagnosis of Cancer", in Fleishercd., 'The Clinical Biochemistry of Cancer", page 347 (American Association of Clinical Chemists, 1979) and in U.S. Patent

Nos. 4.150,149; 4,361.544; and 4,444,744. See also U,S. Patent No. 5,965,132, to Thorpe et aL, U.S. Patent 6,004,554, to Thorpe et al., U.S. Patent No. 6,071,491, to Epstein et al., U.S. Patent No. 6,017,514, to Epstein et al., U.S. Patent No. 5,882,626, to Epstein et al., U.S. Patent No. 5,019,368, to Epstein et al., and U.S. Patent No. 6,342,221, to Thorpe et al, all of which are incorporated herein by reference. [0119] Tumor-associated markers have been categorized by Herberman, supra, in a number of categories including oncofetal antigens, placental antigens, oncogenic or tumor vims associated antigens, tissue associated andgens, organ associated antigens, ectopic hormones and normal andgens or variants thereof. Occasionally, a sub-unit of a tumor-associated marker is advantageously used to raise antibodies having higher tumor-specificity, e.g., the beta-subunit of human chorionic gonadotropin (HCG) or the gamma region of carcino embryonic antigen (CEA), which stimulate the production of antibodies having a greatly reduced cross-reactivity to non-tumor substances as disclosed in U.S. Patent Nos, 4,361,644 and 4,444,744. Markers of tumor vasculature (e.g,, VEGF), of tumor necrosis (Epstein patents), of membrane receptor (e.g., folate receptor, EGFR), of transmembrane antigens (e.g., PSNfA), and of oncogene products can also serve as suitable tumor-associated targets for antibodies or antibody fragments. Markers of normal cell constituents which are expressed copiously on tumor cells, such as B-cell complex antigens (e.g., CD19, CD20, C021, CD22, CD23, and HLA-DR on B-cell malignancies), as well as cytokines expressed by certain tumor cells (e.g., IL-2 receptor in T-cell malignancies) are also suitable targets for the antibodies and antibody fragments of this invention. Other well-known tumor associated antigens that can be taigeted by the antibodies and antibody fragments of this invention include, but are not limited to, CEA, CSAp, TAG-72, MUC-1, MUC-2, MUC-3, MUC-A, EGP-I, EGP-2, BrE3, PAM-4, KC-4, A3. KS-1, PSMA, PSA, tenascin, TlOl, SlOO, MAGE, HLA-DR, CD19, CD20, CD22, CD30, and CD74.
[0120] Another marker of interest is transmembrane activator and CAML-interactor (TAd). See Yu et al Nat Immunol 7252-256 (2000). Briefly, TACI is a marker for B-cell malignancies (e.g,, lymphoma). Further it is known that TACI and B-cell maturation antigen (BCMA) are bound by the tumor necrosis factor homolog a proliferation-inducing ligand (APRIL). APRIL stimulates in vitro proliferation of primary B and T cells and increases spleen weight due to accumulation of B cells in vivo. APRIL also competes with TALL-I (also called BLyS or BAFF) for receptor binding. Soluble BCMA and TACI specifically prevent binding of APRIL and block APRIL-stimulated proliferation of primary B cells, BCMA-Fc also inhibits production of antibodies against keyhole limpet hemocyanin and Pneumovax in mice, indicating that APRIL and/or TALL-I signaling via BCMA and/or TACI are required for generation of humoral immunity. Thus, APRIL-TALL-I and BCMA-TACI foim a two ligand-two receptor patiiway involved in stimulation of B and T cell function.
|0121] After the initial raising of antibodies to the immunogen, the antibodies can be sequenced and subsequently prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art. For example, humanized monoclonal antibodies are produced by transfenring mouse complementary determining regions from heavy and light

variable chains of the mouse immunoglobulin into a human variable domain, and then, substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by the publication of Oriandi et al, Proc, Nat'lAcad. Set USA 86:3833 (1989), which is

stepwise process beginning with the formation of yeast artificial chromosomes (YACs) containing either human heavy- or light-chain immunoglobulin loci in germline configuration. Since each insert is approximately 1 Mb in size, YAC construction requires homologous recombination of overlapping fragments of the immunoglobulin loci. The two YACs, one containing the heavy-chain loci and one containing the

CLINICAL APPLICATION, Ritter et al (eds.), pages 166-179 (Cambridge University Press 1995); and Ward et aL "Genetic Manipulation and Expression of Antibodies,** in MONOCLONAL ANTIBODIES:
recipient female and allowed to gestate. After birth, the progeny are screened for the presence of both
transgenes by Southern analysis. In order for the antibody to be present, both heavy and light chain -genes must be expressed concurrently in the same cell. Milk from transgenic females is analyzed for the presence and functionality of the antibody or antibody fragment using standanrd immunological methods known in the art The antibody can be purified from the milk using standard methods known in the art

[0129] A chimeric Ab is constructed by ligating the cDNA fragment encoding the mouse light variable and heavy variable domains to fragment encoding the C domains from a human antibody. Because the C domains do not contribute to antigen binding, the chimeric antibody will retain the same antigen specificity as the original mouse Ab but will be closer to human antibodies in sequence. Chimeric Abs still contain some

[0133] Recombinant methods can be used to produce a variety of fusion proteins. For example a fusion protein comprising a Fab fragment derived from a humanized monoclonal anti-CEA antibody and a scFv derived from a murine anti-diDTPA can be produced A flexible linker, such as GGGS (SEQ ID NO: 10)

connects the scFv to the constant region of the heavy chain of the anti-CEA antibody. Alternatively, the scFv can be connected to the constant region of the light chain of hMN-14. Appropriate linker sequences necessary for the in-frame connection of the heavy chain Fd to Ae scFv are introduced into the VL and
gestate. After birth, the progeny are screened for the presence of the introduced DNA by Southern analysis. Milk from transgenic females is analyzed for the presence and functionality of the bscAb using

standard immunological methods known in the art The bscAb can be purified from the milk using standard methods known in the art. Transgenic production of bscAb in milk provides an efficient method for obtaining large quantities of bscAb,

counterparts, are disclosed in U.S. Patent No, 5,874,540. Also preferred are bi-specific antibodies which incorporate one or more of the CDRs of Mu-9 or 679, The antibody can also be a fusion protein or abi-specific antibody that incorporates a Class-Ill anti-CEA antibody and the Fv of 679. Class-Ill antibodies, including Class-Ill anti-CEA are discussed in detail in US, Patent No. 4,818,709,

using one equivalent (relative to HSG) of N-hydroxybenzotriazole, one equivalent of benzotrazole-1-yl-oxy-tris-(dimethylamino)phosphoniuni hexafluorophosphate (BOP) and two equivalents of diisopropylethylamine. The activated substrate was mixed with the resin for 15 hr at room temperature.

[0158] The results of the biodistribution studies of the peptide in the mice pretargeted with hMN-14 x m679 are shown in Table 1, The tumor to non-tumor ratios of the peptides in the pretargeting study are show in Table 2.

Table 1
Prelargeting With III Labeled Peptide 24 hr After Injection of hMN-l4 x m679 % Injected/g Tissue

show in 1 able 4, The data in Table 5 shows the biodistribution of the peptides in mice that were not
pretreated with the bi-specific antibody.

Table 3

Table 4

Table S Biodistribution of 111 In Labeled Peptides Alone

Example 4) Synthesis of a Peptide Anticen
(01701 The peptide, Ac-Phe-Lys(Ac)-Tyr-Lys(Ac)-OH (SEQ ID NO: 2), is assembled using a resin for solid-phase synthesis and attaching the first residue (lysine) to the resin as the differentially protected derivative alpha-Fmoc-Lys(Aloc)-OH. The alpha-Fmoc protecting group is selectively removed and die Fmoc-Tyr(OBut), alpha-Fmoc-Lys(Aloc)-OH, and Fmoc-Phe-OH added with alternate cycles of coupling and alpha-amino group deprotection. The Aloe - and OBut- side-chain protecting groups are then removed by reaction with TFA and the free alpha- and epsilon-amino groups are capped by reaction with acetic anhydride to give Ac-Phe-Lys(Ac)-Tyr.Lys(Ac)-OH (SEQ ID NO: 2).
Example 5) Coupling of Ac-Phe-Lys)-Ac)-Tvr-Lys(AcM)H(SEQ ID NO: 2) to KLH [0171] The peptide, Ac-Phe-Lys(Ac)-Tyr-Lys(Ac)-OH (SEQ ID NO: 2), dissolved in water and pH-adjusted to 4.0 with IN HCl, is treated with a molar equivalent of l-ethyl-3(3-dimethylaminopropyl) carbodiimide and allowed to react for 1 h at 4°C. Keyhold limpet hemocyanin(KLH) buffered at pH 8.5 is treated with a 100-fold molar excess of the activated peptide and the conjugation reaction is allowed to proceed for 1 h at 4oC. The peptide-KLH conjugate is purified from unreacted peptide by size-exclusion chromatography and used for antibody production.
Example 6) Generation of an Anti-Peptide Ah

Example 9) Reduction of Anti-Peptide-Ab to Fab'-SH
DTi
[0175] The anti-peptide-F(ab ') is reduced to a Fab' fragment by reaction with a freshly prepared cysteine solution in 0,1M PBS, containing 10mM EDTA. The progress of the reaction is followed by HPLC, and when complete, in about 1 h, the Fab*-SH is purified by spin-column chromatography and stored in deoxygenated buffer at pH

chromatography and reacted with excess iodoacetamide to block hinge-region thiol groups and prevent reassociation. After repurification from excess iodoacetamide the Fab' is reacted with a 400-fold molar excess of the galactosylation agent, the ihio-imidate of cyanomethyl-23,4,6-tetra-0-acctyl-l-thio-beta-D-galactopyranoside (see Karacay et aJ.). The galactosylated protein is purified by two spin-columns and the galactose:Fab' radio detennined by MALDI-MS.
Example 17) Use of anti-CEA-IgG x anti-Peptide Fab' Bi-specific Ab for RAIT, with absAb Clearing Step
[01831 A patient with a CEA-expressing tumor burden is given anti-CEA-IgG (MN-14) x anti-peptide-Fab' bi-specific Ab. Three days later, the patient is given a clearing dose of galactose-WI2-Fab', Twenty-four hours after the clearing dose of a galactose-W12-Fab', the patient is given Y-90-di-Bz-DTPA-peptide. The y-90-labeled peptide clears rapidly from non-target tissue but localizes avidly to sites pretargeted with the anti-CEA-IgG x anti-peptide-Fab' bi-specific Ab, effecting destruction of tumors.
Example 18) Synthesis of Ac-Lvs(DTPA)Tyr-Lys(DTPA)-Ly(Tscg-Cys)-NH2 (SEQ ID NO: 7)- (IMP 192)
|0184] The first amino acid, Aloc-Lys(Fmoc)-OH was attached to 0.21 mmol Rink amide resin on the peptide synthesizer followed by the addition of the Tc-99m ligand binding residues Fmoc-Cys(Trt)-OH and TscG to the side chain of the lysine using standard Fmoc automated synthesis protocols to fbnn the following peptide: AloC-Lys(TscG-Cys(Trt)-rink resin. The Aloe group was then removed by treatment of the resin with 8 mL of a solution containing 100 mg Pd[P(Ph)3]4 dissolved in 10 mL CH2Cl2, 0.75 mL glacial acetic acid and 2.5 ml diisopropylethyl amine. The resin mixture was then treated with 0.8 ml tributyhin hydride and vortex mixed for 60 min. The peptide synthesis was then continued on the synthesizer to make the following peptide: Lys(Aloc)-Tyr-Lys(Aloc)-Lys(Tscg-Cys)-rink resm Activated DTPA and DTPA Addition
[0185] The DTPA, 5 g was dissolved in 40 mL 1.0 M tetrabutylammonium hydroxide in methanol. The methanol was removed under hi-vacuum to obtain a viscous oil. The oil was dissolved in 50 mL DMF and the volatile solvents were removed under hi-vacuum on the rotary evaporator. The DMF treatment was repeated two more times. The viscous oil was then dissolved in 50 ml DMF and mixed wife 5 g HBTU. An 8 ml aliquot of the activated DTPA solution was then added to the resin which was vortex mixed for 14 hr. The DTPA treatment was repeated until the resin gave a negative test for amines using the Kaiser test. Alternatively, DTPA Tetra-t-butyl ester could be used with conventional coupling agents such as DIC and HBTU. {See Arano Y, Uezono T. Akizawa H, Ono M, Wakisaka K, Nakayama M, Sakahara H,

Aliquots were removed for stability studies. The aliquots were diluted 1:10 in saline, 1 mM cysteine in 0.05M phosphate pH 7.5, and fresh human serum. The original kit solution, die saline dilution, and the cysteine challenge were incubated at room temperature while the serum sample was incubated at 37°C. The samples were monitored by HPLC and ITLC. The labeled peptide was stable in the in vitro tests. The retention time of the labeled peptide in serum was shifted from 6.3 mm to 7.3 nun. The shift may be due to ion pairing of some serum component with the peptide.

Table 6

radioiodinated bsAbs to CEA, WI2 (xat anti-MN-14 idiotypic antibody) and radiolabeled peptidyl DTPA

chelate was examined on analytical size exclusion HPLC. Approximately 90 % of the radioiodinated bsAb bound to CEA upon treatment with l0-20x molar excess of CEA, The bsAb complexed with radiolabeled indium-DTPA chelates (IMP-156 or IMP-192).
IMP 156 Ac-Phe-Lys(DTPA>Tyr-Ly5(DTPA)NH2 (SEQ ID NO: 2)

Example 24) Construction and expression of hMN-]4Fab-734scFv
[0193] Recombinant methods were used to produce a monovalent bi-specific fusion protein comprising a
i
1
4 ( I t
c

[0199] A bscAb fragment is cloned into an expression vector containing a 5' casein promoter sequence and 3' untranslated genomic sequences that flank the insertion site. The expression cassette is then injected into the pronuclei of fertilized, mouse eggs, using procedures standard in the art The eggs are then implanted into the uterus of a recipient female and allowed to gestate. After birth, the progeny are

screened for the presence of the introduced DNA by Southern analysis. Milk from transgenic females is analyzed for the presence and functionality of the bscAb using standard immunological methods known in the art. The bscAb can be purified from the milk by complementary binding to an immobilized antigen, column chromotography or other methods known in the art

Table 8
Biodistribution of 125-I-hMN-14 x 734 bsAb and 11 l-In-indiuin-lMP-156 peptide in nude mice bearing GW-39 tumor xenografts: hMN-14 x 734 was allowed 48 h for localization prior to 11 l-ln-indium-IMP-156 injection. Biodistribution was performed 3 h post 11 l-In-indium-IMP-156. bsAbipeptide ratio administered, 1:0.03. Five animals per time point.
l25-I-bMN-14x 734 111-In-indium-IMP-ISe

Table 9
Control group showing the clearance of 11 l-In-indium-IMP-156 at 3 h after injectioa

Table 10
Kude mice bearing GW 39 tumor xenografts were administered 125-Wabeled bsAb (5 µCi, 15 µg, 1.5 x 10-10 mol). hMN-14 x 734 was allowed 24 h for localization and clearance before administering 99m-Tc-IMP-192 (10 µCi, 1.6 A 10" 11 mol of peptide). Biodistribution studies were performed at 30 min, 1,3 and 24 h post 99m-Tc-lMP-192 injection, five animals per time point BsAbrpeptide, 1:0.1.

Table 11
Nude mice bearing GW 39 tumor xenografts were administered 12S-I-labeled bsAb (5µCi, 15 µg, 1.5 x 10-10 mol), hMN-14 x 734 was allowed 24 h for localization and clearance before administering 99m-Tc. IMP-192 (10µCi, 1.6 X 10-11 mol of peptide). Biodistribution studies were performed at 30 min, 1,3 and 24 h post 99m-Tc-rMP-192 injection, five animals per time point BsAb:peptide, 1:0.1.

Table 12
Control group of nude mice bearing GW-39 tumors received 99in-Tc-IMP-192 (10µCi, 1.6 x 10-11 mol of peptide) and were sacrificed 3 h later.
99m-Tc-IMP-192
Tissue % ID/g
Tumor 0,2 ±0.05
Liver 0,3 ± 0.07
Spleen 0.1 ± 0.05
Kidney 2.6 ±0.9
Lungs 0.2 ± 0.07
Blood 0.2 ±0.09
The percentage of the available DTPA binding sites on the tumor bound bsAb filled with 99m-Tc-JMP-192 was calculated from the above data assuming one peptide bound to one hsAb molecule. However, it is possible that one peptide molecule can crosslink two molecules of bsAb.
Table 13
Percentage of the available DTPA bmding sites on the tumor bound bsAb filled with 99m-Tc-IMP-192
% saturation on
time hMN.14x734
30 min 25.4
1 h 25.8
3h 25
24 h 28
[0202] The foregoing experimental data show that: the humanized x murine bsAb retained its binding capability to CEA and indium-DTPA; the hMN-] 4 x 734 (Fab x Fab) effectively targets a tumor; the dual functional peptidyl Tc-99m chelator was stable; 99m-Tc-IMP-l92 complexed to tumor-localized hMN-14

X 734 and was retained for at least 24 h; and imaging of tumors is possible at early time points (l-3h) post 99m-Tc-IMP-192 injection.
Example 28) Use of anti-CEA Fab x anti-peptide scFv fusion protein for RAIT, with a bsAfa Clearing
Step
[02031] A 69-year-old man with colon cancer that had undergone resection for cure, after a year is found to have a CEA blood serum level of 50 ng/mL. The patient undergoes a CT scan, and 5 tumor lesions ranging from 1 cm to 3 cm are present in the left lobe of the liver. The patient is given 100 mg of hMN14-Fab/734-scFv fusion protein. Three days later, the patient is given a clearing dose of galactose-WI2-Fab'. Twenty-four hours after the clearing dose of agalactose-WI2-Fab', the fusion protein in the blood is reduced 20-fold the concentration of the protein just prior to injection of the clearing agent The patient is then infused with the IMP 245 Y-90-di-B2-DTPA-peptide, containing 50 mCi of Y-90. ACT scan performed three months later demonstrates three of the lesions have disappeared, and the remaining two have not increased in size. The CEA blood serum level is decreased to 10 ng/mL at this time. No increase is seen in the CEA blood serum level for the following 6 months, and CT scans demonstrate no growth of the two tumor lesions. The therapy is repeated a year after the first therapy, when an increase in CEA is observed, and the two tumor lesions are observed to decrease in size at 3 months and six months after the second therapy. The blood serum CEA level after six months is less than 5 ng/mL.
Example 29) Preparation of a carboxylesterase-DTPA conjugate
10204] Two vials of rabbit liver caiboxylesterase (SIGMA; protein content -17 mg) are reconstituted in 2.2 ml of 0.1 M sodium phosphate buffer, pH 7.7 and mixed with a 25-fold molar excess of CA-DTPA using a freshly prepared stock solution (- 25 mg/mJ) of the latter in OMSO. The final concentration of DMSO in the conjugation mixture is 3 % (v/v). After 1 hour of incubation, the mixture is pre-purified on two 5-mL spin-columns (Sephadex G50/80 in 0.1 M sodium phosphate pH 7.3) to remove excess reagent and DMSO. The eluate is purified on a TSK 3000G Supeico column using 0.2 M sodium phosphate pH 6.8 at 4 ml/min. The fraction containing conjugate is concentrated on a Centricon-10TM concentrator, and buffer-exchanged with 0.1 M sodium acetate pH 6.S. Recovery: 0.9 ml, 4.11 mg/ml (3.7 mg)'. Analytical HPLC analysis using standard conditions, with in-line UV detection, revealed a major peak with a retention time of 9.3 min and a minor peak at 10.8 min in 95-to-S ratio. Enzymatic analysis showed 115 enzyme units/mg protein, comparable to unmodified carboxylesterase. Mass spectral analyses (MALDI mode) of both unmodified and DTPA-modified CE shows an average DTPA substitution ratio near 1.5. A metal-binding assay using a known excess of indium spiked with radioactive indium confirmed the DTPA-.enzyme ratio to be 1.24 and 1.41 in duplicate experiments. Carboxylesterase-DTPA is labeled with In-111 acetate at a specific activity of 12.0 mCi/mg, then treated with excess of non-radioactive indium acetate, and finally treated with 10 mM EDTA to scavenge off excess non-radioactive indium. Incorporation by HPLC and ITLC analyses is 97.7%. A HPLC sample is completely complexed with a 20-fold molar excess of bi-speciiic antibody hMN-14 Fab' x 734 Fab', and the resultant product further

complexes with WI2 (anti-ID to hMN-14), with the latter in 80-fold molar excess with respect to bi-specific antibody.
Example 30) Synthesis of IMP 224
[0205] An amount of 0.0596 g of the phenyl hydrazine containing peptide IMP 221 (H2N-NH-C6H4-CO-Lys(DTPA).Tyr-Lys(DTPA)-NH2 MH+ 1322. made by Fmoc SPPS) was mixed with 0.0245 g of Doxorubicin hydrochloride in 3 mL of DMF. The reaction solution was allowed to react at room temperature in the dark. After 4 hours an additional 0.0263 g of IMP 221 was added and the reaction continued overnight. The entire reaction mixture was then purified by HPLC on a Waters Nova-Pak (3-40X100 nun segments, 6 µm, 60A ) prep column eJuting with a gradient of 80:20 to 60:40 Buffer A:B over 40 min (Buffer A= 0.3 % NH4OAC, Buffer B= 0.3 % NH4OAC in 90 % CH3CN). The fractions containing product were combined and lyophilized to afford 0.0453 g of the desired product, which was confirmed by ESMS MH+ 1847.
Example 31) IMP 224 Kit Formulation
[0206] The peptide of Example 31 was formulated into kits for In-l 11 labeling. A solution was prepared which contained 5.014 g 2-hydroxypropyl-P-cyclodextrin, and 0.598 g citric acid in 85 mL. The solution was adjusted to pH 4.0 by the addition of 1 M NaOH and diluted with water to 100 mL. An amount of 0.0010 g of the peptide IMP 224 was dissolved in 100 mL of the buffer, and 1 mL aliquots were sterile filtered through a 0.22µm Millex GV filter into 2 mL lyophilization vials which were immediately frozen and lyophilized.
Example 32) In-111 Labeling of IMP 224 Kits
[0207] The In-111 was dissolved in 0.5 mL water and injected into the lyophilized kit The kit solution was incubated at room temprature for 10 mm then 0.5 mL of a pH 7.2 buffer which contained 0,5 M NaOAc and 2.56 x 10-5 M cold indium was added.
Example 33) In-Vitro Stability of IMP 224 Kits
I0208I An IMP 224 kit was labeled as described with 2.52 mCi of In-111. Aliquots (0.15 mL, 370 µCi) were withdrawn and mixed with 0.9 xnL 0.5 M citrate buffer pH 4.0, 0.9 mL 0.5 M dtrste buffer pH 5.0, and 0-9 mL 0.5 M phosphate buffer pH 7.5. The stability of the labeled peptide was followed by inverse phase HPLC. HPLC Conditions: Waters Radial-Pak C-18 Nova-Pak 8x100 mm. Flow Rate 3 mL/min, Gradient: 100 % A= 0.3 % NH4OAC to 100 % B= 90 % CH3CN, 0.3 % NH4OAC over 10 min.

Table 14
In-Vitro Stability of In/In-111 IMP 224

Example 34) In-vivo biodistribution of IMP 221 in BALB/c mice
102091 Kits were reconstituted with 400 pCi In-111 in 0.5 mL water- The In-111 kit solution was
incubated at room temperature for 10 min and then diluted with 1.5 mL of the cold indium containing pH
12,0.5 M acetate buffer. The labeled peptide was analyzed by ITLC in saturated NaCl. The loose In-Ill
was at tbe top 20 % of the ITLC strip,
[0210] Each mouse was injected with 100 µL (20 µCi) of the In-III labeled peptide. The animals were
anesthetized and sacrificed at 30 minutes, 1 hours, 2 hours, 4 hours, and 24 hours using three mice per
time point. Blood, muscle, liver, lungs, kidneys, spleen, large intestine, small intestine, stomach, urine,
and tail were collected and counted. The results of the biodistribution study are shown in the following
table.

Table IS
Biodistribution in B ALB/c mice %ID/g of IMP 224 (Dox=N-NH-C6H4-CO-Lys(DTPA)-Tyr--LyspTPA)-NH2 MH+ 1847 radiolabeled with In-111 and saturated with cold In

Example 35) In-vivo stability and clearance of IMP 224
|0211) Kits were reconstituted with 4 mCi InIII in 0.5 mL water. The In-III kit was incubated at room
temperature for 10 min and then diluted with 0.5 mL of the cold indium containing 0.S M pH 7.2 acetate
buffer. The labeled peptide was analyzed by ITLC in saturated NaCL. The loose bi-III was at the top 20
% of fuel ITLC strip.
(02121 Bach mouse was injected with 100 µL (40µCi) of the In-111 labeled peptide. The animals were
anesthetized and sacrificed at 30 min and I hr using two animals per time point. The srum and urine
samples were collected, stored on ice, and sent on ice as soon as possible for HPLC analysis. The HPLC
(by size exclusion chromatography) of the urine samples showed that the In-111 labeled peptide could still
bind to the antibody. The reverse phase HPLC analysis showed that the radiolabeled peptide was excreted
intact in the urine. The amount of activity remaining in the scrum was too low to be analyzed by reverse pliase HPLC due to the poor sensitivity of the detector. Doxorubicin has -95 % hepatobiliary clearance. Thus, by attaching the his DTPA peptide in a hydrolyzeable manner, the biodistribution of the drug is altered to give - 100 % renal excretion. This renders the drug far less toxic because all of the nontargeted drug is rapidly excreted intact

Table 16
Activity Recovered in The Urine and Serum.

Example 36) Pretargeting experiments with IMP 224 and IMP 225
[0213] A lyophilized kit of IMP 224 containing 10 micrograms of peptide was ued. The kit was
lyophilized in 2 mL vials and reconstituted with 1 mL sterile water, A 0.5 mL aliquot was removed and
mixed with 1.0 mCi ln-111. The In-111 kit solution was incubated at room temperature for 10 minutes
then 0.1 mL was removed and diluted with 1.9 mL of the cold indium containing acetate buffer BM 8-12
in a sterile vial. The labeled peptide was analyzed by ITLC in saturated NaCl. The loose In-111 was at
the top 20% of the ITLC strip.
(02141 Female nude mice (Taconic NCRNU, 3-4 weeks old) with GW 39 tumor xenografis were used for
the pretargeting experiments. Tumors were 0.3-0.8 g. Each animal was injected with 100 microliters (5
µCi, 15µg. 1.5 X 10 -10 mol) of the 1-125 labeled antibody F6 x 734-F(ab')2.
(02151 Seventy two hours later, each mouse was injected with 100 µL (10µCi) of the In-111 labeled
peptide. The animals were anesthetized and sacrificed at 1 hour, 4 hours and 24 hours using five mice per
time point. Tumor, blood, muscle, liver, lungs, kidneys, spleen, large intestine, small intestine, stomach,
urine and tall were collected and counted.
(02161 The experiment was repeated with a lyophilized kit of IMP 225 Ac-Cys(Dox-COCH2)-
Lys(DTPA)-Tyr-Lys(DTPA)-NH2 (SEQ ID NO: 11) MNa+ 1938), containing 11 micrograms of peptide.

Table 17
Biodistribution of In-111 "IMP-224 in nude mice bearing GW-39 tumor xenografts, previously given F6 x 734-F(ab')2 72 h earlier. Data in % ID/g tissue. n=5.

Table 18
Biodistribution of In-11 l-IMP-224 in nude mice bearing GW-39 tumor xenografts, previously g^ven F6 x 734-F(ab'2 72hrlier. Data in tiunor-to-nonnal organ ratios. n=5.

Table 39
Biodistribution of In-111-IMP-22S in nude mice bearing GW-39 tumor xenografts, previously given F6 x 734-F(ab')2 72 h earlier. Data in % ID/g tissue. n=.

[0217] Combinations of the bi-specific constructs described in the present invention or others of similar specificities are suitable for pretargeted RAIT, where IMP-192 peptide and its analogues are labeled with therapeutic radioisotopes such as IS8-Re, 2I3-Bi, 67-Cu and the like. It will be recognized that therapeutic chelates can be conjugated to peptides that have other than chelate epitopes for recognition by bsAbs, as described above.
(0218] It will be appreciated as well that detectable radiolabels can be directed to a site of interest, e.g. a tumor, which is to be excised or otherwise detected and /or treated in intra-operati, endoscopi intravascular or other similar procedures, using the pretargeting meths of the present invention, in combination with various linkers. The pretargeting is effected with non-radioactive bsAbs and the eventual administration and localization of the low molecular weight radiolabeled linker, and clearance of unbound linker, are both comparatively rapid, compatible with surgical procedures that should avoid needless delay and which can use radioisotopes with short half-lives. Additionally, the disclosed therapies can be used for post-surgical radioimmunotherapy protocols to ensure the eradication of residual tumor cells.
Example 37) Synthesis of DOTA-Phe-Lvs(GVD-Tvr-Ly(SGVLvsnrseg-Cys)NHo/SEQ ID N: I) (IMP245)

[219] The peptide was synthesized by the usual double coupling procedure as described for the synthesis of IMP 192. The tri-t-butyl DOTA was added to the C-terminus of the pepde with a single benzotria2ole-l"yl-oxy"triS"(dimethylamino)-phosphonium hcxafluorophosphate (BOP) coupling using 5 eq of protected DOTA for 16 hr. The resin was then capped with acetic anhydride. The Aloc groups on the side chains were removed using the palladium catalyst and the N-trityl-HSG groups were added as described for the synthesis of IMP 243. The product was cleaved from the resin and purified by HPLC to afford 0.2385 g of product, from four fractions, after lyophilization.. ESMS MH+ 1832
Example 38) Tc-99m Kit Fonnulation
(0220] A formulation buffer was prepared which contained 22.093 g hydroxypropyl-p-cyclodextrin, 0.45 g 2,4-d]hydroxybenzoic acid, 0.257 g acetic acid sodium salt, and 10.889g aplpha-g(ucoheptonic acid sodium salt dissolved in 170 mL nitrogen degassed water. The solution was adjusted to pH 5.3 with a few drops of 1 M NaOH then further diluted to a total volume of 220 mL. A stannous buffer solution was prepared by diluting 0.2 mL of SnCl2 (200 mg/mL) with 3.8 mL of the formulation buffer. The peptide. IMP 245 (0.0029g), was dissolved in 1 mL 1.6 x 10-3 M InCl3 in 0.1 M HCl. The peptide solution was mixed with 2 mL 0.5 M NH4OAC and allowed to incubate at room temperature for IS min. The formulation buffer, 75 mL, and 0.52 mL of the stannous buffer were then added to the peptide solution. The peptide solution was then filtered through a 0.22 µm Millex GV filter in 1.5 mL aliquots into 3 mL lyophilization vials. The filled vials were frozen immediately, lyophilized and crimp sealed under vacuum.
Example 39) Tc-99m Labeling of IMP 245 High Temperature (Boiling Water Bath)
[0221] The pertechnetate solution (29 mCi) in 1.5 mL of saline was added to the kit The kit was incubated at room temperature for 10 min and heated in a boiling water bath for 15 min. The kit was cooled to room temperature before use.
Low Temperature (3 loC)
10222] The pertechnetate solution (25 mCi) in 1.5 mL of saline was added to the kit The kit was incubated at room temperature for 14 min and heated in a 37oC water bath for 18 min. The kit was cooled to room temperature before use. The HPLC retention time for this label is slightly different because a different injector was used.
Example 40) Peptide Analysis (HPLC) of IMP 245
10223] The peptide was analyzed by reverse phase HPLC and size exclusion Iff LC (shown below). The size exclusion HPLC traces indicated that the peptide binds to two mMU-9 x m679 and two hMN-14 x m679 bi-specific antibodies {see "A Universal Pre-Targeting System for Cancer Detection and Therapy Using Bi-specific Antibody," Sharkey, R.M.. IVfcBride, W.J., Karacay, H., Chang, K., Griffidis, Gi., Hansen, H.J., and Goldenberg, D.M., the entire contents of which are incojporated by reference herein).

The reverse phase HPLC analysis shows several small peaks before the main peak and heat did not seem to significantly alter the ratio of the small peaks to the large peak.
Recovery from SEC:
[0224] Tc-99m IMP 245 Alone 54%,
TC-99m IMP 24S + hA4N-14 x m679 66 %, Tc-99m IMP 245+ mMU-9 x m679 66 %.

the acetate/EDTA buffer, followed by a rapid addition of 20 mM PDM (prepared in 90% DMF) to a final concentration of 4 mM. After stirring at room temperature for 30 minutes, the resulting solution (containing 679 Fab'-PDM) was diafiltered into the acetate/EDTA buffer until tree PDM is minimum, and concentrated to 5-10 mg/mL. A solution of hMN-14 Fab'-SH or Mu-9 Fab'-SH was the mixed with a
3 i

25-20µ/ci). At the designated times after the peptide injection, animals were anesthetized, bled by cardiac puncture, and then euthanized prior to necropsy. Tissues were removed, weighed and counted by gamma scintillation using appropriate windows for each radionuclide along with standards prepared from the injected materials. When dual isotope counting was used, appropriate backscatter correction was made. GI tissues (stomach, small intestine and large intestine were weighed and counted with their

contents. Data are expressed as the percent injected dose per gram tissue (%ID/g) and the ratio of the percentages in the tumor to the normal tissues (T/NT). All values presented in the tables and figures represent the mean and standard deviation of the calculated values with the number of animals used for each study provided therein.

Table 22

Table 25
Tumor/nontumor ratios for 99m7c-hMN-14 Fab' in GW-39 tumor-bearing nude mice 3 h after injection, (n = 5)

Table 28

similar distribution and clearance properties (Tables 20 and 21). In both instances, the peptide was cleared so rapidly from blood that within 3 hour after its injection, there was insufficient radioactivity in the blood to quantify accurately, but there was sufficient radioactivity in the major organs to permit quantitation. The radioactivity was eliminated from the body through renal excretion, with a small percentage of the injected activity lingering in the kidneys over the monitoring period. At an average

Bos, ES., Kuijpers, WHA., Meesters-Winters, M., Pham, DT., deHaan, AS., van DoonnaIen,Am., Kasperson, F..M.,vanBoeckel, CAA and Gouegeon-Bertrand, F. In vitro evaluation of DNA-DNA hybridization as a two-step approach in radioimmunotherapy of cancer. Cancer Res, 1994; 54:3479-3486.

Carr et al..WO00/34317.
Gautherot, E., Bouhou, J., LeDoussai, J-M., Manetti, C, Martin, M-, Rouvier, E., Barbet, J. Therapy
for colon carcinoma xenografts with bi-specific antibody-targeted, iodine-131-IabeIed bivalent hapten.
Cancer suppl 1997;80:2618-2623. Gautherot, E., Bouhou, J., Loucif, E., Manetti, C, Martin, M., LeDoussal, J Jvl,, Rouvier, E,, Baibet, J.
Radioimmunotherapy of LS174T colon carcinoma in nude mice using an iodine-131-abeIed bivalent
hapten combined with an anti-CEA x anti-indium-DTPA bi-specific antibody. J,Nucl, Med. Suppl.
1997; 38: 7p.
Goodwin, D.A., Meares, CF„ McCall, MJ., McTigue,M., Cbaovapong, W. Pre-targetd
immunoscintigraphy of murine tumors with indium-l 11-labeled bifunctional haptens. J Nucl. Med,
1988; 29:226-234.
Greenwood, F.C. and Hunter, W.M. The preparation of 1-131 labeled human growth hormone of high specific radioactivity. Biochem. 1963; 89:114-123.
Hawkins, G.A., McCabe, R.P., Kim, C.-H., Subramanian,R., Bredehorst, R., McCuUers, GA., Voge],C.-W., Hanna, M.G. Jr., and Pomata, N. Delivery of radionuclides to pretargeted monodona] antibodies using dihydrofolate reductase and methotrexate in an affinity system. Cancer Res. 1993; 53: 2368-2373.
Kranenborg, M.h-, Boerman, O.C, Oosterwijk-Waklca, j., weijert, M., Corstens, F., Gosterwijk, E. Development and characterization of anti-renal cell carcinoma x antichelate bi-specific monoclonal antibodies for two-phase targeting of renal cell carcinoma. Cancer Res.(suppl) 1995; 55:5864s-5867s
Losman M.J., Qu Z., Krishnan LS., Wang J., Hansen HJ., Goldenbeig D.M., Leung S.O. Clin. Cancer Res. 1999; 5(10 Suppl):3101s.3105s.
Penefsky, H.S. A centrifuged column procedure for the measurement of ligand binding by beef heart Fl. Part G. Methods Enzymol. 1979; 56:527-530.
Schuhmacher, J., Klivenyi,G., Matys JL, Stadler, M., Regiert, T., Hauser,H., Doll, J-, Maier-Borst, W., Zoller, M. Multistep tumor targeting in nude mice using bi-specific antibodies and a gallium chelate suitable for immunocintigraphy with positron emission tomography. Cancer Res. 1995; 55,115-123.
Sharkey, RM., Karacay, Griffiths, GL., Behr, TM., Blumenthal, RD., Mattes,MJ.,Hansen, HJ., Goldenberg. Development of a streptavidin-anti-carcinoembryonic antigen antibody, radiolabeled biotin pretargeting method for radioimmunotherapy of colorectal cancer. Studies in a human colon cancer xenograft model. Bioconjugate Ghent 1997; 8:595-604,
Stickney, DR., Anderson, LD., Slater, JB., Ahlem, CN.,Kirk, GA,, Schweighardt, SA and Frincke, JM. Bifunctional antibody: a binary radiopharmaceutical delivery system for imaging colorectal carcinoma. Cancer Res. 1991;51:6650-6655.
All references cited herein are hereby incorporated herein by reference in their entireties.

WHAT IS CLAIMED IS:
1. A compound of the formula:
X-Phc-Lys(HSG)-D-Tyr.Lys(HSG)-Lys(Y)-NH2;
wherein the compound includes a hard acid cation chelator at X or Y, and a soft acid cation chelator at remaining X or Y.
2. The compound of claim 1, wherein the hard acid cation chelator includes a
carboxylate or amine group.
3. The compound of claim 1, wherein the hard acid cation chelator is selected from the
group consisting of NOTA, DOTA, DTPA, and TETA.
4. The compound of claim 1, wherein the soft acid cation chelator includes a thiol
group.
5. The compound of claim 1, wherein the soft acid cation chelator is selected from the
group consisting of Tscg-Cys and Tsca-Cys.
6. The compound of claim 1, further comprising at least one radionuclide, therapeutic
agent or diagnostic agent.

8. The compound of claim 1 comprising:
D0TA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2.
9. The compound of claim 1 comprising:
Tscg-Cys-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(DOTA)-NH2.

10. The compound of claim 1, wherein the hard acid cation chelator includes a cation selected from the group consisting of Group Ha and Group IIIa metal cations.
11. The compound of claim 1, wherein the soft acid cation chelator includes a cation selected from the group consisting of transition metals, Bi, lanthanides and actinides.
12. The compound of claim 1, wherein the soft acid cation chelator includes a cation
selected from the group consisting of Tc, Re, and Bi,
13. A targetable construct comprising:
X-Phe-Lys(HSG)-D.Tyr.Lys(HSG)-Lys(Y)-NH-.R;
wherein the targetable contruct includes a hard acid cation chelator- at X or Y; a soft acid cation chelator at remaining X or Y; and a therapeutic agent, diagnostic agent or enzyme at R.
14. The targetable construct of claim 13, wherein R is covalently linked to the targetable
construct.
15. The targetable cosntruct of claim 13, wherein R is linked to the targetable construct
by a linker moiety.
16. The targetable construct of claim 15, wherein the linker moiety includes at least one
amino acid.
17. The compound of claim 13, further comprising at least one radionuclide bound to at
least one of the hard acid chelator and soft acid chelator.

19. The targetable construct of claim 13, wherein said therapeutic agent includes a
radionuclide, drug, prodrug or toxin.
20. The targetable construct of claim 19, wherein said prodrug is selected from the group
consisting of epirubicin glucuronide, CPT-11, etoposide glucuronide, daunomicin
glucuronide and doxorubicin glucuronide.
21. The targetable construct of claim 19, wherein said toxin is selected from the group
consisting of ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-!,
pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas
endotoxin.
22. The targetable construct of claim 13, wherein said therapeutic agent compsrises doxorubicin, SN-38, etoposide, methotrexate, 6-mercaptopurine or etoposide phosphate.
23. The targetable construct of claim 13, wherein the diagnostic agent includes one or more agents for photodynamic therapy.
24. The targetable construct of claim 23, wherein said agent for photodynamic therapy is a photo sensitizer.
25. The targetable construct of claim 24, wherein said photosensitizer is selected from the group consisting of benzoporphyrin monoacid ring A (BPD-MA), tin etiopurpurin (SnET2), sulfonated aluminum phthalocyanine (AlSPc) and lutetium texaphyrin (Lutex).
26. The targetable construct of claim 13, wherein said diagnostic agent comprises one or more image enhancing agents for use in magnetic resonance imaging (MRl).
27. The targetable construct of claim 26, wherein said enhancing agents include Nfn, Fe, La and Gd.
28. The targetable construct of claim 13, wherein said diagnostic agent comprises one or more radiopaque or contrast agents for X-ray or computed tomography.
29. The targetable construct of claim 28, wherein said radiopaque or contrast agents include barium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid, iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide, iohexol, iopamidol, iopanoic acid,

ioprocemic acid, iosefamic acid, ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetric acid, iothalamic acid, fotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine, metrizamide, metrizoate, propyliodone, or thallous chloride.
30. The targetable construct of claim 13, wherein said diagnostic agent comprises one or more ultrasound contrast agents.
31. The targetable construct of claim 30, wherein said ultrasound contrast agent includes a liposome or dextran.
32. The targetable construct of claim 31, wherein the liposome is gas-fiUed.
33. The targetable construct of claim 13, wherein said enzyme includes an enzyme capable of converting drug intermediate to a toxic form to increase toxicity of said drug at a target site.
34. A method of treating or diagnosing or treating and diagnosing a disease or a condition that may lead to a disease comprising:

(A) administering to said subject a bi-specific antibody or antibody feagment having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable construct.
(B) optionally, administering to said subject a clearing composition, and allowing said composition to clear non-localized antibodies or antibody fragments from circulation; and
(C) administering to said subject a targetable construct comprising rge compound of claim 1 which further comprises at least one diagnostic or therapeutic cation, and/or one or more chelated or chemically bound therapeutic agent, diagnostic agent, or enzyme.
35. The method of claim 34, wherein the bi-specific antibody or antibody fragment
having at least one arm that speciiically binds a targeted tissue and at least one other arm that
specifically binds a targetable construct and the targetable construct are administered at substantially the same time.
36. The method of claim 34, wherein the therapeutic cation emits particles and/or positrons
having 20 to 10,000 keV.

38. The method of claim of claim 34, wherein the diagnostic cation emits particles and/or positrons having 25-10,000 keV.

40. The method of claim 34, wherein said diagnostic agent is used to perform positron-
emission tomography (PET).
41. The method of claim 34, wherein said diagnostic agent is used to perform SPECT
imaging.
42. The method of claim 34, wherein said diagnostic cation or agent includes one or nnore image enhancing agents for use in magnetic resonance imaging (MRI).
43. The method of claim 42, wherein said enhancing agent is selected from the group consisting of Mn, Fe, La and Gd.
44. The method of claim 34, wherein said diagnostic agent comprises one or niore
radiopaque or contrast agents for X-ray or computed tomography.
45. The method of claim 44, wherein said radiopaque or contrast agents include barium,
diatrizoate, ethiodized oil, gallium citrate, iocarmic acid, iocetamic acid, iodamide,
iodipamide, iodoxamic acid, iogulamide, iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetric acid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine, metrizamide, metrizoate, propyliodone, or thallous chloride.

46. The method of claim 34, wherein said diagnostic agent comprises one or more ultrasound contrast agents.
47. The method of claim 46, wherein said ultrasound contrast agent includes a liposom or dextran.
48. The method of claim 47, wherein said liposome is gas-filled.
49. The method of claim 34, wherein said diagnostic agents are selected from the group consisting of a fluorescent compound, a chemilmninescent compound, and a bioiuminescent compound.
50. The method of claim 49, wherein said fluorescent compound is selected from the group consisting of fluorescein isothiocyanate, rhodamine, phycoetytherin, phycocyanin, alJophycocyanin,o-phthaldehyde and fluorescamine.
5 2. The method of claim 49, wherein said chemily\uminescent compound is selected from the group consisting of luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.
52. The method of claim 49, wherein said bioiuminescent con:q)ound is selected from the group consisting of lucifeiin, luciferase and aequorin.
53. The method of claim 34, wherein said targeted tissue is a tumor.
54, The method of claim 53, wherein said tumor produces or is associated with antigens
selected from the group consisting of colon-specific antigen-p{CSAp), carcinoembryonic
antigen (CEA). CD19, CD20, CD21, CD22, CD23, CD30, CD74, CD80, HLA-DR, la, MUC
1. MUC 2, MUC 3, MUC 4, EGFR, HER 2Meu, PAM-4, TAG-72, EGP-1, EGP-2, A3, KS-
1, Le(y), S100, PSMA, PSA, tenascin, folate receptor, VEGFR, necrosis antigens, IL-2,
T101, MAGE,

55. The method of claim 34, wherein said at least one arm that specifically binds a
targeted tissue is a monoclonal antibody or a fragment of a monoclonal antibody.
56. The method of claim 34, wherein said at least one other ann that specifically binds a
targetable construct is a monoclonal antibody or a fragment of a monoclonal antibody.
57. The method of claim 34, wherein said at least one ann that specifically binds a
targeted tissue is a human, chimeric or humanized antibody or a fragment of a human,
chimeric or humanized antibody.
58. The method of claim 34, wherein said at least one other arm that specifically binds a
targetable construct is a human, chimeric or humanized antibody or a fragment of a human,
chimeric or humanized antibody.
59. The method of claim 34, wherein said bi-specific antibody or antibody fragment
further comprises a therapeutic nuclide.

61. The method of claim 34, wherein the bi-specific antibody comprises the Fv of MAb Mu-9 and the Fv of MAb 679.
62. The method of claim 61, wherein Mu-9 and/or 679 are chimerized or humanized.
63. The method of claim 61, wherein Mu-9 and/or 679 are human Mu-9 and 679.
64. The method of claim 61, wherein the bi-specific antibody comprises one or more of
the CDRs of Mu-9.
65. The method of claim 61, wherein the bi-specific antibody comprises one or more of the CDRs of 679.
66. The method of claim 61, wherein the bi-specific antibody is a fusion protein.

67. The method of claim 34, wherein the bi-specific antibody comprises the Fv of MAb
MN-14 and the Fv of MAb 679.
68. The method of claim 67, wherein MN-14, and/or 679 are chimerized or humanized.
69. The method of claim 67, wherein MN-14, and/or 679 are human MN4-14 and 679.
70. The method of claim 67, wherein the bi-specific antibody comprises one or more of
the CDRs of MN-14.
71. The method of claim 67, wherein the bi-specific antibody comprises one or more of the CDRs of 679.
72. The method of claim 67, wherein the bi-specifi c antibody is a fusion protein.

73. The method of claim 34, wherein the fusion protein is trivalent, and incorporates the Fv of an antibody reactive with CSAp.
74. The method of claim 34, wherein the bi-specific antibody incorporates a Class-III anti-CEA antibody and the Fv of 679,
75. The method of claim 34, wherein said targetable construct comprises one or more radioactive isotopes useful for killing diseased tissue.
76. The method of claim 34, wherein said targetable construct comprises 10B atoms, and said method further comprises the step of irradiating said boron atoms localized at said diseased tissue, thereby effecting BNCT of said diseased tissue.
77. The method of claim 34, when said targetable construct comprises an enzyme, further administering to said subject a drug which said enzyme is capable of converting to a
toxic form, and, therefore, increasing the toxicity of said drug at the target site.
78. A method for detecting or treating target cells, tissues or pathogens in a mammal,
comprising:

administering an effective amount of a bi-specific aiitibody or antibody fragment comprising at hast one arm that specifically binds a target and at least one other arm that specifically binds a targetable construct; and
administering a targetable construct comprising the compound of claim 1;
wherein said target includes a target cell, tissue, pathogen or a molecule produced by or associated therewith and at least one arm that specifically binds said target is capable of binding to a complementary binding moiety on the target
79. The method of claim 78, wherein said pathogen is a fungus, virus, parasite, bacterium, protozoan, or mycoplasm.
80. The method of claim 79, wherein said fungus is selected from the group consisting of Microsporum, Trichophyton, Epidermophyton, Ssporothrix scbenckii, Cyrptococcus neofomians, Coccidioides immitis, Histoplasma capsulatum, Blastomyces dermatitidis, Candida albicans.
81. The method of claim 79, wherein said virus is selected from the group consisting of human immunodeficiency virus (HIV), heipes virus, cytomegalovirus, rabies virus, influenza virus, hepatitis B virus, Sendai vims, feline leukemia virus, Reo virus, polio virus, human serum parvo-like virus, simian virus 40, respiratory syncytial virus, mouse mammary tumor virus, Varicella-Zoster virus. Dengue vims, rubella virus, measles virus, adenovirus, human T-cell leukemia vimses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart vims and blue tongue virus.
82. The method of claim 79, wherein said bacterium is selected from the group consisting of Anthrax bacillus. Streptococcus agalactiae, Legionella pneumophilia. Streptococcus pyogenes, Escherichia coli. Neisseria gonorrhoeae. Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae. Brucella abortus, Mycobacterium tuberculosis and Tetanus toxin.
83. The method of claim 79, wherein said parasite is a helminth or a malarial parasite.
84. The method of claim 79, wherein said protozoan is selected from the group consisting of Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Tiypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma

86. The method of claim 78, wherein said targetable construct further comprises at least one radionuclide, therapeutic agent, diagnostic agent or enzyme

90. The method of claim 89, wherein said subject is mammalian.
9\. The method of claim 90, wherein said mammalian subject is selected from the group consisting of humans, primates, equines, canines and felines.

92. The method of claim 89, wherein said targetable construct further comprises at least one radionuclide, therapeutic agent, diagnostic agent or enzyme.

94. The method of claim 92, wherein the diagnostic agent includes an imaging agent
95. The method of claim 92, wherein the therapeutic agent includes drugs, toxins,
cytokines, hormones, or growth factors.
96. A kit useful for treating or identifying diseased tissues in a subject comprising:
(A) a targetable construct comprising the compound of claim 1;
(B) a bi-specific antibody or antibody fragment having at least one arm that
specifically binds a targeted tissue and at least one other arm that specifically binds the
targetable construct;
wherein the targetable construct includes a carrier portion which comprises or bears at least one epitope recognizable by said at least one other arm of said bi-specific antibody or antibody fragment, and one or more conjugated therapeutic or diagnostic agents, or enzymes; and
(C) optionally, a clearing composition useful for clearing non-localized
antibodies and antibody fragments.

99. The kit of claim 96, when said targetable construct comprises an enzyme,
optionally, the kit further comprising a drug which enzyme is capable of converting to a toxic form to increase the toxicity of said drug at the target site.
100. A targetable construct comprising the compound of claim 1.
101. A method for imaging normal tissue in a mammal comprising:
administering an effective amount of a bi-specific antibody or antibody fragment;
and
administering a targetable construct comprising the compound of claim 1;
wherein the bi-specific antibody or antibody fragment comprises at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds the targetable construct; and wherein said at least one arm is capable of binding to a complementary binding moiety on the normal tissue or target cells produced by or associated therewith.
102. The method of claim 101, wherein said normal tissue is tissue from the ovary, thymus, parathyroid, endometrium, bone marrow, or spleen.
103. The method of claim 101, wherein said targetable construct further comprises at least one radionuclide, therapeutic agent, diagnostic agent or enzyme.

105. The method of claim 103, wherein the diagnostic agent includes a contrast agent,
106. The method of claim 103, wherein the diagnostic agent includes an imaging agent.

107. The method of claim 106, wherein said imagine agent is an agent used for PET.
108. The method of claim 106, wherein the imaging agent is an agent used for SPECT.
109. The method of claim 103, wherein the therapeutic agent includes drugs, toxins,
cytokines, hormones, or growth factors.
110. A method of intraoperatively identifying diseased tissues, in a subject, comprising
administering an effective amount of a bi-specific antibody or antibody fragment;
and
administering a targetable construct comprising the compound of claim 1;
wherein the bi-specific antibody or antibody fragment comprises at least one atm that specifically binds a targeted tissue and at least one other arm that specifically binds the targetable construct; and wherein said at least one arm is capable of binding to a complementary binding moiety on the target cells, tissues or pathogen or on a molecule produced by or associated therewith.
111. The method of claim 110, wherein said targetable construct further comprises at least one radionuclide, therapeutic agent, diagnostic agent or enzyme.

213. The method of claim 111. wherein the diagnostic agent includes a contrast agent.
114. The method of claim HI, wherein the diagnostic agent includes an imaging agent
115. The method of claim 111, wherein the therapeutic agent includes drugs, toxins,
cytokines, hormones, or growth factors.

116. A method for the endoscopic identification of diseased tissues, in a subject, comprising:
administering an effective amount of a bi-specific antibody or antibody fragment; and
administering a targetable construct comprising the compound of claim I;
wherein the bi-specific antibody or antibody fragment comprises at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds the targetable construct; and wherein said at least one arm is capable of binding to a complementary binding moiety on the target cells, tissues or pathogen or on a molecule produced by or associated therewith.
117. The method of claim 116, wherein said targetable construct further comprises; at least one radionuclide, therapeutic agent, diagnostic agent or enzyme.

119. The method of claim 117, wherein the diagnostic agent includes a contrast agent
120. The method of claim 117, wherein the diagnostic agent includes an imaging agent
121. The method of claim 117, wherein the therapeutic agent includes drugs, toxins,
cytokines, hormones, or growth factors.
122. A method for the intravascular identification of diseased tissues, in a subject,
comprising:
administering an effective amount of a bi-specific antibody or antibody fragment comprising at least one arm that specifically binds a targeted tissue and at least one other aim that specifically binds a targetable construct;

wherein said at least one ami is capable of binding to a complementary binding moiety on the target cells, tissues or pathogen or on a molecule produced by or associated therewith; and
administering a targetable construct comprising the compound of claim 1.
123, The method of claim 122, wherein said targetable construct further comprises at least one radionuclide, therapeutic agent, diagnostic agent or enzyme.

125. The method of claim 123, wherein the diagnostic agent includes a contrast agent
126. The method of claim 123, wherein the diagnostic agent includes an imaging agent
127. The method of claim 123, wherein the therapeutic agent includes drugs, toxins,
cytokines, honnones, or growth factors.

128. A compound of the formula substantially as herein described with reference to the accompanying drawings.
129. A method of treating or diagnosing or treating an diagnosing a disease or a condition that may lead to a disease substantially as herein described with reference to the accompanying drawings.
Dated this 13 day of December 2004

Documents:

2815-chenp-2004-abstract.pdf

2815-chenp-2004-assignement.pdf

2815-chenp-2004-claims filed.pdf

2815-chenp-2004-claims granted.pdf

2815-chenp-2004-correspondnece-others.pdf

2815-chenp-2004-correspondnece-po.pdf

2815-chenp-2004-description(complete) filed.pdf

2815-chenp-2004-description(complete) granted.pdf

2815-chenp-2004-drawings.pdf

2815-chenp-2004-form 1.pdf

2815-chenp-2004-form 18.pdf

2815-chenp-2004-form 26.pdf

2815-chenp-2004-form 3.pdf

2815-chenp-2004-form 5.pdf

2815-chenp-2004-pct.pdf

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Patent Number

209394

Indian Patent Application Number

2815/CHENP/2004

PG Journal Number

38/2007

Publication Date

21-Sep-2007

Grant Date

28-Aug-2007

Date of Filing

13-Dec-2004

Name of Patentee

M/S. IMMUNOMEDICS, INC

Applicant Address

300 American Road, Morris Plains, NJ 07950

Inventors:

#	Inventor's Name	Inventor's Address
1	LEUNG Shui-on	10C, University Residence No. 16- The Chinese University of Hong Kong, Shatin, N.T. 07059,
2	QU Zhengxing	15 Sycamore Way, Warren, NJ 07059
3	GOLDENBERG David M	1 Charolais Farm Road, Mendham, NJ 07945
4	HANSEN Hans	6014 Angler Drive, Picayune, MS 39466
5	MCBRIDE William J	116 Glover Street, Boonton, NJ 07005

PCT International Classification Number

A61K 47/48

PCT International Application Number

PCT/GB2003/002110

PCT International Filing date

2003-05-16

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	10/150,654	2002-05-17	U.S.A.