WO2003097105A1 - Drug pre-targeting by means of bi-specific antibodies and hapten constructs comprising a carrier peptide and the active agent (s) - Google Patents

Drug pre-targeting by means of bi-specific antibodies and hapten constructs comprising a carrier peptide and the active agent (s) Download PDF

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Publication number
WO2003097105A1
WO2003097105A1 PCT/GB2003/002110 GB0302110W WO03097105A1 WO 2003097105 A1 WO2003097105 A1 WO 2003097105A1 GB 0302110 W GB0302110 W GB 0302110W WO 03097105 A1 WO03097105 A1 WO 03097105A1
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WO
WIPO (PCT)
Prior art keywords
targetable construct
antibody
lys
acid
arm
Prior art date
Application number
PCT/GB2003/002110
Other languages
French (fr)
Inventor
David M. Goldenberg
Hans Hansen
Shui-On Leung
William J. Mcbride
Zhengxing Qu
Original Assignee
Immunomedics, Inc.
Mccall, John, Douglas
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Immunomedics, Inc., Mccall, John, Douglas filed Critical Immunomedics, Inc.
Priority to US10/514,632 priority Critical patent/US20060140858A1/en
Priority to EP03725404A priority patent/EP1506018B1/en
Priority to KR1020047018606A priority patent/KR101087923B1/en
Priority to CN038168987A priority patent/CN1668335B/en
Priority to CA2486307A priority patent/CA2486307C/en
Priority to BR0310088-0A priority patent/BR0310088A/en
Priority to AU2003227939A priority patent/AU2003227939B2/en
Priority to MXPA04011422A priority patent/MXPA04011422A/en
Priority to JP2004505100A priority patent/JP4455322B2/en
Publication of WO2003097105A1 publication Critical patent/WO2003097105A1/en

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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
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    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A61K51/1048—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell determinant being a carcino embryonic antigen
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    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
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Definitions

  • the invention relates to immunological reagents for therapeutic use, for example, in radioimmunotherapy (RAIT), and diagnostic use, for example, in radioimmunodetection (RAID) and magnetic resonance imaging (MRI).
  • RAIT radioimmunotherapy
  • RAID radioimmunodetection
  • MRI magnetic resonance imaging
  • 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.
  • 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 arm 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.
  • 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.
  • a bsAb is administered and allowed to localize to target, and to clear normal tissue.
  • a radiolabeled low molecular weight hapten is given, which being recognized by the second specificity of the bsAb, also localizes to the original target.
  • the detection and/or therapy agent must remain associated with the low MW hapten throughout its application within the bsAb protocol employed.
  • 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 ⁇ l, U.S. Patent No. 4,863,713), indium-I l l ( ⁇ axhet et ⁇ i, U.S. Patent No. 5,256,395 and U.S. Patent No. 5,274,076, Goodwin et ⁇ l., J. Nucl.
  • 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, tifie 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).
  • an effector molecule e.g., a radionuclide or a drug linked to a small carrier
  • the body 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.
  • the radionuclide 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 vascu/afure and binds to the primary targeting agent. Its small size may also encourage a more uniform distribution in the tumor.
  • Pretargeting methods have used a number of different strategies, but most often involve an avidin/streptavidin-biotin recognition system 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 biotin-conjugated antibody, followed by a bridging with streptavidin/avidin, and then the biotin-conjugated effector is given.
  • These systems can be easily converted for use with a variety of effector substances so long as the effector and the targeting agent can be coupled with biotin or streptavidin/avidin depending on the configuration used.
  • This type of pretargeting has considerable advantages over other proposed systems.
  • avidin and streptavidin are foreign proteins and therefore would be immunogenic, which would limit the number of times they could be given in a clinical application.
  • bsAbs have the advantage of being able to be engineered as a relatively non-immunogenic humanized protein.
  • the binding affinity of a bsAb typically 10 ⁇ 9 to 10" ⁇ ⁇ M
  • both pretargeting systems are dependent on the binding affinity of the primary targeting agent, and therefore the higher affinity of the streptavidin/avidin-biotin systems may not offer a substantial advantage over a bsAb pretargeting system.
  • 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 ⁇ l.
  • HSG histamine-succinyl-glycl
  • Other objects of the invention are to provide pre-targeting methods of diagnosis and therapy using the combination of multi-specific antibody and targetable construct, methods of making the multi-specifics, and kits for use in such methods.
  • the present inventors have discovered that it is advantageous to raise multi-specific Abs against a targetable construct that is capable of carrying one or more diagnostic or therapeutic agents.
  • the characteristics of the chelator, metal chelate complex, therapeutic agent or diagnostic agent can be varied to accommodate differing applications, without raising new multi-specific Abs for each new application.
  • two or more distinct chelators, metal chelate complexes, diagnostic agents or therapeutic agents can be used with the inventive multi-specific Ab.
  • 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 other arm that specifically binds a targetable construct.
  • a compound of the formula X-Phe-Lys(HSG)-D-Tyr-Lys[HSG)-Lys(Y)-W_2 (SEQ ID NO: 1), where the 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 soil acid cation chelator may include a fhiol group, and may also include such chelators as Tscg-Cys and Tsca-Cys.
  • a preferred embodiment of this compound is DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)- NH2 (SEQ ID NO; 1) also known as IMP 245.
  • Other 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(DOTA)-NH 2 (SEQ ID NO: 1).
  • the compound may also include cations bound to the different chelating moeities.
  • hard acid cations may include Group Ha and Group Ilia metal cations, which commonly bind to hard acid chelators.
  • Soft acid cations that may bind to the soft acid chelators can include the transition metals, lanthanides, actinides and or Bi, Non exhaustive examples of such soft acid cations include Tc, Re, and
  • a targetable construct including X-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Y)-NH- R (SEQ ID NO: 1 ).
  • a hard acid cation chelator is positioned at either X or Y
  • 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 therapeutic or diagnostic agent or enzyme "R".
  • the linker may have at least one amino acid for conjugating the R group to the compound.
  • therapeutic agents include a drug, prodrug (e.g, epirubicin glucuronide, CPT-11.
  • etoposide glucuronide etoposide glucuronide, daunomicin glucuronide and doxorubicin glucuronide
  • toxin e.g., ricin, abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pse domonas exotoxin, ⁇ Pseudomonas endotoxi ⁇ .
  • therapeutic agents include doxorubicin, SN-38, etoposide, methotrexate, 6- mercaptopurine and/or etoposide phosphate.
  • Diagnostic agents may include nuclides, one or more agents for photodynamic therapy (e.g, a photosensitizer such as benzoporphyrin monoacid ring A (BPD-MA), tin etiopurpurin (SnET2), sulfonated aluminum phthalocyanine (AlSPc) and lutetium texaphyrin (Lutex)), contrast agents and image enhancing agents for use in magnetic resonance imaging (MRI) and computed tomography (CT).
  • Enzymes may also serve as the R group which may be capable of converting a prodrug to a drug at the target site; or capable of reconverting a detoxified drug intermediate to a toxic form to increase toxicity of said drug at a target site.
  • the invention provides a method of treating, diagnosing and/or identifying diseased tissues in a patient, comprising:
  • the invention provides a kit useful for treating or identifying diseased tissues in a patient comprising:
  • 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;
  • (C) optionally, a clearing composition usefuf for clearing non-localized antibodies and antibody fragments
  • 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.
  • 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.
  • 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.
  • Another embodiment of the invention provides a method of preparing the antibodies or antibody fragments by recombinant technology.
  • a method of preparing 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:
  • 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- specif ⁇ c fusion protein is under the control of the regulatory regions; (B) co-introducing into the host cell a recombinant DNA construct 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
  • 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 in (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 5' 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 termination regulatory region functional in the second host cell, wherein the Fd fragment is under the control of the regulatory regions;
  • 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.
  • the method utilizes a mammalian zygote, and the introduction of the recombinant DNA construct produces a transgenic animal capable of producing a bi-specific antibody or antibody fragment.
  • the present invention seeks to provide inter ⁇ li ⁇ 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.
  • a further embodiment of the invention involves using the inventive antibody or antibody fragment in photodynamic therapy.
  • a further embodiment of the invention involves using the inventive antibody or antibody fragment in radioimmunoi-naging for positron-emission tomography (PET).
  • PET positron-emission tomography
  • a further embodiment of the invention involves using the inventive antibody or antibody fragment in radioimmu ⁇ oimaging for single-photon emission.
  • a further embodiment of the invention involves using the inventive antibody or antibody fragment in magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • CT computed tomography
  • ultrasound imaging J0028 J A further embodiment of the invention involves using the inventive antibody or antibody fragment in X-ray, computed tomography (CT) or ultrasound imaging.
  • a further embodiment of the invention involves using the inventive antibody or antibody fragment for intraoperative, endoscopic, or intravascular detection and/or therapy.
  • a further embodiment of the invention involves using the inventive antibody or antibody fragment in boron neutron capture therapy (BNCT).
  • BNCT boron neutron capture therapy
  • a further embodiment of the invention involves using the inventive antibody or antibody fragment for diagnosing or treating diseased tissues (e.g., cancers, infections, inflammations, clots, atherosclerois, infarcts), normal tissues (e.g., spleen, parathyroid, thymus, bone marrow), ectopic tissues (e.g., endomet ⁇ osis), and pathogens.
  • diseased tissues e.g., cancers, infections, inflammations, clots, atherosclerois, infarcts
  • normal tissues e.g., spleen, parathyroid, thymus, bone marrow
  • ectopic tissues e.g., endomet ⁇ osis
  • the invention provides pre-targeting methods of diagnosis and therapy using the combination of bi-specific antibody and the following targetable constructs:
  • 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.
  • the characteristics of the chelator, metal chelate complex, therapeutic agent or diagnostic agent can be varied to accommodate differing applications, witiiout raising new bsAbs for each new application.
  • two or more distinct chelators, metal chelate complexes or therapeutic agents can be used with the inventive bsAb.
  • the invention relates to a method of treating or identifying diseased tissues in a subject, comprising:
  • a targetable construct which comprises a carrier 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;
  • 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.
  • the invention further relates to a method for detecting 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 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 (a) DOTA-Phe- ys(HSG)-D-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 3); (b) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH 2 (SEQ ID NO: 2); (c) Ac-Lys(HSG)-D-Tyr-Lys(HSG)- ys(Tscg-C
  • 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 arm that specifically binds a targeted tissue and at least one other arm 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 construct selected from the group consisting of:
  • 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 other arm that specifically binds a targetable construct, wherein said construct is selected from the group consisting of
  • 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;
  • (C) optionally, a clearing composition useful for clearing non-localized antibodies and antibody fragments
  • 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.
  • the invention further relates to a targetable construct selected from the group consisting of:
  • 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 ceils, tissues or pathogen or on a molecule produced by or associated therewith; and optionally incubating said mixture; and analyzing said mixture.
  • 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
  • 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 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
  • the invention further relates to a method for the endoscopic 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
  • 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
  • Figure 1 schematically illustrates various Abs and bsAbs.
  • Figure 2 provides SDS-PAGE analysis of purified hMN-14Fab-734scFv. 3 ⁇ g 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.
  • Figure 3 schematically illustrates two bi-specific fusion proteins.
  • Figure 4 illustrates the production of a DNA construct useful for producing a hMN-14Fab- 734scFv bi-specific fusion protein.
  • Figure 5 illustrates the production of a DNA construct useful for producing a hMN-14Fab- 734scFv bi-specific fusion protein.
  • Figure 6 shows the binding properties of hMN-14 x m679 bsMAb with 11 ⁇ -Iabeled IMP-241 divalent HSG-DOTA peptide.
  • Panel A ⁇ n In-IMP-241 alone on SE-HPLC;
  • Panel B U lln-IMP-241 mixed with hMN-14 x 679 bsMAb;
  • Panel C 1 1 1 In-IMP-241 added to a mixture containing hMN-14 x m679 bsMAb with an excess of CEA.
  • Chromatograms show the association of the J l 1 In-IMP-241 with the bsMAb (B) and bsMAb/CEA complex (C).
  • "a" or "an” means "one or more”.
  • the present invention provides a bi-specific antibody (bsAb) or antibody fragment (bsFab) having at least one arm that is reactive against a targeted tissue and at least one other arm that is reactive against a targetable construct.
  • the targetable construct includes a peptide having at least 2 units of a recognizable hapten. Examples of recognizable haptens include, but are not limited to, histamine succinyl glycine (HSG) and fluorescein isothiocyanate.
  • HSG histamine succinyl glycine
  • fluorescein isothiocyanate examples of recognizable haptens.
  • the targetable construct may be conjugated to a variety of agents useful for treating or identifying diseased tissue.
  • conjugated agents include, but are not limited to, chelators, metal chelate complexes, drugs, toxins (e.g., ricin, abrin, ribonuclease (e.g., RNase), DNase I, St ⁇ phylococc ⁇ l enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomon ⁇ s exotoxi ⁇ , Pseudomon ⁇ s endotoxin) and other effector molecules. Additionally, enzymes useful for activating a prodrug or increasing the target-specific toxicity of a drug can be conjugated to the targetable construct.
  • toxins e.g., ricin, abrin, ribonuclease (e.g., RNase), DNase I, St ⁇ phylococc ⁇ l enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomon ⁇ s exotoxi ⁇ , Ps
  • Bi-specific antibody (bsAb) pretargeting represents a potentially non-immunogenic, highly selective alternative for diagnostic and therapeutic applications.
  • the bsAb pretargeting system described herein represents an additional significant advantage over other pretargeting systems in that it potentially can be developed for use with a variety of different imaging or therapeutic agents.
  • the flexibility of this system is based on use of an antibody directed against histamine-succinyl-glycl (HSG) and the development of peptides containing the HSG residue.
  • HSG histamine-succinyl-glycl
  • HSG-containing peptides were synthesized with either DOTA for the chelation of 111 In, 90 ⁇ , or 177l, u or a technetium/rhenium chelate.
  • these peptides were used in combination with bi-specific antibodies using the anti-HSG Fab' chemically stabilized with the Fab' of either an anti-carcinoembryonic antigen (CEA) or an anfi-co/on-specific antigen-p (CSAp) antibody to provide tumor targeting capability for tumors expressing these antigens.
  • CEA anti-carcinoembryonic antigen
  • CSAp an anfi-co/on-specific antigen-p
  • other antigen targets may include diverse tumor-associated antigens known in the art, such as against CD19, CD20, CD21, CD22, CD23, CD30, CD74, CD SO, HLA-DR, la, MUC 1, MUC 2, MUC 3,
  • EGP-1 e.g., RS7
  • EGP-2 e.g., 17- 1A and other Ep-CAM targets
  • Le(y) e.g., B3
  • necrosis antigens folate receptors
  • angiogenests markers e.g., VEGF
  • tenascin PSMA
  • PSA tumor-associated cytokines
  • MAGE fragments thereof.
  • Tissue-specific antibodies e.g., against bone marrow cells, such as CD34, CD74, etc., parathyroglobulin antibodies, etc.
  • 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.
  • 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.
  • tumor uptake of the radiolabeled peptides increased from 28 to 175- fold with tumor/nontumor 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 99m ⁇ c _ ant i_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 combination with the 11 ⁇ In-labeled peptide, radiation dose estimates for therapeutic radionuclides, such as 90 ⁇ and l ⁇ Lu, 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.
  • 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.
  • 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 ami that specifically binds a targetable construct.
  • 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 virus, hepatitis B virus, Sendai virus, 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 virus, rubella virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus,
  • viruses e.g.
  • hyorhinis M. orale, M. arginini, Acholeplasma laidlawii, M. salivarum, and M. pneumoniae
  • protozoans e.g., Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japanicum, Babesia bovis, Elmeria tenella, 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.
  • the antibody fragments are antigen binding portions of an antibody, such as F(ab')2 .
  • the antibody fragments bind to the same antigen that is recognized by the intact antibody.
  • an anti-CD22 monoclonal antibody fragment binds to an epitope of CD22.
  • antibody fragment also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.
  • antibody fragments include isolated fragments, "Fv” fragments, consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker ("sFv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the "hypervariable region.”
  • Fv proteins recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker
  • minimal recognition units consisting of the amino acid residues that mimic the "hypervariable region.”
  • CDR complementarity-determining regions
  • 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 the CDRs accounts for the diversity of specificity of antibodies.
  • the term "subject” refers to any animal (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, turkeys, 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 any animal (i.e.,
  • the targetable construct can be of diverse structure, but is selected not only to diminish the eJicitati ⁇ fi of immune responses, but also for rapid in vivo clearance when used within the bsAb targeting method.
  • Hydrophobie agents are best at eliciting strong immune responses, whereas hydrophilic agents are preferred for rapid in vivo clearance, thus, a balance between hydrophobie and hydrophilic needs to be established. This is accomplished, in part, by relying on the use of hydrophilic chelati ⁇ g agents to offset the inherent hydrophobicity of many organic moieties.
  • sub-units of the targetable construct may be chosen which have opposite solution properties, for example, peptides, which contain amino acids, some of which are hydrophobie and some of which are hydroprji.ic. Aside from peptides, carbohydrates may be used.
  • the targetable construct may include a peptide backbone having as few as two amino-acid residues, with preferably two to ten amino acid residues, and may be coupled to other moieties such as chelating agents.
  • the targetable construct should be a low molecular weight construct, preferably having a molecular weight of less than 50,000 daltons, and advantageously less than about 20,000 daltons, 10,000 daltons or 5,000 daltons, including any metal ions that may be bound to the chelating agents.
  • the known peptide DTPA-Tyr-Lys(DTPA)-OH (wherein DTPA is diethylenetriaminepentaacetic acid) has been used to generate antibodies against the indiu -DTPA portion of the molecule.
  • new Abs against the tyrosyl-lysine dipeptide can be made.
  • the antigenic peptide of the targetable construct will have four or more residues, such as the peptide DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)- ⁇ H2 (SEQ ID NO: 2), wherein DOTA is 1 ,4,7,10-tetraazacyclododecanetetraacetic acid and HSG is the histamine succinyl glycyl group of the formula:
  • the non-metal-containing peptide may be used as an immunogen, with resultant Abs screened for reactivity against the Phe-Lys-Tyr-Lys (SEQ ID NO: 2) backbone.
  • the haptens of the targetable construct also provide an immunogenic recognition moiety, for example, a chemical hapten.
  • a chemical hapten preferably the HSG hapten
  • high specificity of the construct for the antibody is exhibited. This occurs because antibodies raised to the HSG hapten are known and can be easily incorporated into the appropriate bsAb.
  • binding of the haptens to the peptide backbone would result in a targetable construct that is specific for the bsAb or bsFab.
  • the invention also contemplates the incorporation of unnatural amino acids, e.g., D-amino acids, into the peptide backbone structure to ensure that, when used with the final bsAb/construct system, the arm of the bsAb which recognizes the targetable construct is completely specific.
  • the invention further contemplates other backbone structures such as those constructed from non-natural amino acids and peptoids.
  • 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
  • hydrophilic chelate moieties on the targetable construct helps to ensure rapid in vivo clearance.
  • 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 issue.
  • Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with 47s c , 52p e> 55 C ⁇ j 67 Ga> 68 Gaj l llj nj 8 ⁇ 90 Y) 161 ⁇ », 177 Lu> 212 ⁇ , 213 ⁇ i, and 225 Ac for radio-imaging and RAIT.
  • the same chelators when complexed with non- radioactive metals such as Mn, Fe and Gd for use with MRI, when used along with the bsAbs of the invention.
  • Macrocyclic chelators such as NOTA (l,4,7-triaza-cyclon ⁇ na ⁇ e-N,N',N"-triacetic acid), 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.
  • DTPA and DOTA-rype 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 Ila and Group Ilia 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 p ⁇ lyethers are of interest for stably binding nuclides such as 223 R a f or RAIT.
  • Porphyrin chelators may be used with numerous radiometals, and are also useful as certain cold metal complexes for bsAb-directed immuno-phototherapy.
  • 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.
  • Particularly useful diagnostic radionuclides that can be bound to the chelating agents of the targetable construct include, but are not limited to, ] ] °I ⁇ , ] * J In, ] 77 Lu, 1 $F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67Ga, 68 G a, 86 ⁇ , 90 ⁇ , 89 ⁇ 94m Tc , 94 Tc , 99m Tc , 1 0 123 ⁇ , 124 It 125 ⁇ , 131 I; 154-158 G d, 32p, 11 C , 13N, 15O, 186 Re> 188R e> 51 Mn> 52m Mll ⁇ 55c 0 , 72 As> 75 Br> 76 ⁇ r , 82mRt>, °3Sr, or other gamma-, beta-, or positron-emitters.
  • the diagnostic radionuclides include a decay energy in the range of 25 to 10,000 keV, more preferably in the range of 25 to 4,000 keV, and even more preferably in the range of 20 to 1,000 keV, and still more preferably in the range of 70 to 700 keV.
  • Total decay energies of useful positron-emitting radionuclides are preferably ⁇ 2,000 keV, more preferably under 1 ,000 keV, and most preferably ⁇ 700 keV.
  • Radionuclides useful as diagnostic agents utilizing gamma-ray detection include, but are not limited to: Cr-51, Co-57, Co-58, Fe-59, Cu-67, Ga-67, Se-75, Ru-97, Tc-99m, In-111, In- 1 24m, 1-223, 1-125, 1-131, Yb-169, Hg-297, and T ⁇ -201. Decay energies of useful gamma-ray emitting radionuclides are preferably 20-2000 keV, more preferably 60-600 keV, and most preferably 100-300 keV.
  • Particularly useful therapeutic radionuclides that can be bound to the chelating agents of the targetable construct include, but are not limited to 1 Hn, l 77 Lu, 212 Bi, 13Bi, 211 A , 62c u , 64 Cu, 67 Cu, 90 Yj 125 ⁇ , 131 ⁇ , 32 P , 33 P , 47 SCj l l l Ag , 67 Ga; 142 Pr , ]53 Sm , 161 ⁇ b, 166 Dy) l66 H ⁇ j 186 Re , 188 Re , !S9 Re , 212 Pb) 223 Ra> 225 Ac , 59 e , 75 Se> 77 ASj 89 Sr , 99 Mo , 105 ⁇ 109 Pd , 143ft., 149 P ⁇ i) 169 E ⁇ ⁇ -.4 ⁇ r?
  • the therapeutic radionuclide preferably has a decay energy in the range of 25 to 10,000 keV. Decay energies of useful beta-particle-emitting nuclides are preferably 25-5,000 ke V, more preferably 200-4,000 keV, and most preferably 500-2,500 keV. Also preferred are radionuclides that substantially decay with Auger-emitting particles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-1 19, 1-125, Ho-161, Os-189m and Ir-192.
  • Decay energies of useful beta-particle-emitting nuclides are preferably ⁇ 1 ,000 ke V, more preferably ⁇ 100 keV, and most preferably ⁇ 70 keV.
  • radionuclides that substantially decay with generation of alpha- particles.
  • Such radionuclides include, but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-21 1, Ac-225, Fr-221, At-217, Bi-213 and Fm-255.
  • Decay energies of useful alpha-particle- emitting radionuclides are preferably 2,000-9,000 keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.
  • J Chelators such as those disclosed in U.S. Patent 5,753,206, especially thiosemi- carbazonylglyoxylcysteine(Tscg-Cys) and thiosemicarbazinyl-acetylcysteine (Tsca-Cys) chelators are advantageously used to bind soft acid cations of Tc, Re, Bi and other transition metals, lanthanides and acti ⁇ ides that are tightly bound to soft base ligands, especfatfy suhur- or phosphorus-containing ligands.
  • chelator fi can be useful to link more than one type of chelator to a peptide, e.g., a hard acid chelator like DTPA for In(III) cations, and a soft acid chelator (e.g, thiol-containing chelator such as Tscg-Cys) for Tc cations.
  • a hard acid chelator like DTPA for In(III) cations and a soft acid chelator (e.g, thiol-containing chelator such as Tscg-Cys) for Tc cations.
  • thiol-containing chelator such as Tscg-Cys
  • a peptide is Ac-Lys(DTPA)-Tyr-Lys(DTPA)-Lys(Tscg-Cys)-NH 2 (SEQ ID NO: 7).
  • This peptide can be preloaded with In(III) and then labeled with 99-m-Tc cations, the In(III) ions being preferentially chelated by the DTPA and the Tc cations binding preferentially to the thiol-containing Tscg- Cys.
  • Other hard acid chelators such as NOTA, DOTA, TETA and the like can be substituted for the DTPA groups, and Mabs specific to them can be produced using analogous techniques to those used to generate the anti-di-DTPA Mab.
  • J0072J Jt will be appreciated that two different hard acid or soft acid chelators can be inco ⁇ orated into 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.
  • 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:
  • 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(HSO)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ JD 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 “F oc” used herein refer to the groups allyloxycarbonyl and fluorenylmethyloxy 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 Aloe group was then removed.
  • the peptide synthesis was then continued on the synthesizer to make the following peptide: Lys(Aloc)-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.
  • 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.
  • a hard acid cation is introduced as a solution of a convenient salt, and is taken up 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 ma be chelated therein.
  • soft acid cations such as 1 8 6 R e, 188 R ⁇ 13gj and divalent or trivalent cations of Mn, Co, Ni, Pb, Cu, Cd, Au, Fe, Ag (monovalent), Zn and Hg, especially 64 Cu and 6 GU ⁇ and the like, some of which are useful for radioimmunodetection or radioimmunotherapy.
  • Re cations also can be generated in situ from perrhenate and stannous ions or a prereduced rhenium glucoheptonate or other transchelator can be used.
  • inventive bi-specific antibodies and targetable constructs in the context of treating diseased tissue.
  • inventive bi-specif ⁇ c antibodies and targetable constructs in treating and/or imaging normal tissue and organs using the methods described in U.S. Patent Nos. 6,126,916; 6,077,499; 6,010,680; 5,776,095; 5,776,094; 5,776,093; 5,772,981; 5,753,206; 5,746,996; 5,697,902; 5,328,679; 5,128,119; 5,101,827; and 4,735,210, which are incorporated herein by reference.
  • tissue refers to tissues, including but not limited to, tissues from the ovary, thymus, parathyroid, bone marrow or spleen.
  • An important use when targeting normal tissues is to identify and treat them when they are ectopic (i.e., displaced from their normal location), such as in endometriosis.
  • 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 timing 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 form or by administering one after the other.
  • diagnostic and therapeutic reagents can be advantageously conjugated to the targetable construct.
  • 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.
  • gadolinium metal is used for magnetic resonance imaging and fiuorochromes can be conjugated for photodynamic therapy.
  • 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.
  • 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.
  • Addiffona- diagnostic agents can include fluorescent labeling compounds such as fluorescein isothiocyanate, rhodamine, phycoerytherin, 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.
  • Radionuclides can also be used as diagnostic and/or therapeutic agents, including for example, 90 ⁇ , 1 Hln, 131l, 99m ⁇ c . ISdRe, l88 Re> 277 Lu , 67 Cu> 232 B f, 2.3 B i, and 21 -At.
  • Therapeutic agents also include, for example, chemotherapeutic drugs such as vinca alkaloids, anthracyclines, epidophyllotoxinw, taxanes, antimetabolites, alkylating agents, antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic and apoptotoic agents, particularly doxorubicin, methotrexate, taxol, CPT-11, camptothecans, and others from these and other classes of anticancer agents.
  • chemotherapeutic drugs such as vinca alkaloids, anthracyclines, epidophyllotoxinw, taxanes, antimetabolites, alkylating agents, antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic and apoptotoic agents, particularly doxorubicin, methotrexate, taxol, CPT-11, camptothecans, and others from these and other classes of anticancer agents.
  • Suitable therapeutic agents for the preparation of immunoconjugates and antibody fusion proteins include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2 inhibitors, pyrimidine analogs, purine analogs, platinum coordination complexes, hormones, and the like. Suitable therapeutic agents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as well as revised editions of these publications. Other suitable therapeutic agents, such as experimental drugs, are known to those of skill in the art.
  • Therapeutic agents may also include, without limitation, others drugs, prodrugs and/or toxins.
  • drug drug
  • prodrug and/or toxins
  • diagnostic agent or “diagnosis” include, but are not limited to, detection agent, detection, or localization.
  • 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 the 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.
  • 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.
  • 511 keV gamma photons produced during positron annihilation decay are detected.
  • the targetable construct may include radioactive diagnostic agents that emit 25-10,000 keV gamma-, beta-, alpha- and auger- particles and/or positrons.
  • Such agents include, but are not limited to 18p, 52p e , 2c u , 64c u , 67 Cu , 67Q a , 68Ga, 86Y, 89zr, 94m ⁇ c .94 ⁇ C) 99m Tc , 111 In, 123 ⁇ 124 ⁇ , 125 131 154-158 G d and 175 Lu ,
  • the present bsAbs or bsFabs can be used in a method of photodynamic therapy (PDT) 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.
  • PDT photodynamic therapy
  • a photosensitizer e.g., a hematoporphyrin derivative such as dihematoporphyrin ether
  • Anti-tumor activity is initiated by the use of light, e.g., 630 nm.
  • photosensitizers can be utilized, including those useful at longer wavelengths, where skin is less photosensitized by the sun.
  • photosensitizers include, but are not limited to, benzopoiphyrin monoacid ring A (BPD- MA), tin etiopurpurin (SnET2), sulfonated aluminum phthalocyanine (AlSPc) and lutetium texaphyrin (Lutex).
  • a diagnostic agent may be injected, for example, systemically, 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 ⁇ l. Chest 76:32 (1979).
  • the antibodies and antibody fragments can be used in single photon emission.
  • a Tc- 99m-labeled diagnostic agent can be administered to a subject following administration of the inventive antibodies or antibody fragments.
  • 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 photoradiatio ⁇ , phototherapy, or photodynamic therapy (Jori et al. (eds.), Photodynamic Therapy of Tumors and Other Diseases (Libreria Progetto 1985); van den Bergh, Chem. Britain 22:430 (1986)).
  • Radiopaque and contrast materials are used for enhancing X-rays and computed tomography, and include iodine compounds, barium compounds, gallium compounds, thallium compounds, etc.
  • Specific compounds 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, and thallous chloride.
  • Ultrasound contrast material may also by used including dextran and liposomes, particularly gas-filled liposomes.
  • 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 art.
  • immunomodulator includes cytokines, stem cell growth factors, lymphotoxins, such as tumor necrosis factor (TNF), and hematopoietic factors, such as interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12 and IL-18), colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)), interferons (e.g., interferons- ⁇ , - ⁇ and - ⁇ ), the stem cell growth factor designated "SI factor,” erythropoietin and thrombopoietin.
  • suitable immunomodulator moieties include IL-2, IL-6, IL-10, IL-12, IL-18, interferon- ⁇ , TNF- ⁇ , and the like.
  • the targetable construct may also be conjugated to an enzyme capable of activating a drug/prodrug at the target site or improving the efficacy of a normal therapeutic by controlling the body's detoxification pathways.
  • an enzyme conjugated to the targetable construct having a low MW hapten is administered.
  • a cytotoxic drug is injected that is known to act at the target site.
  • the drug may be one which is detoxified by the mammaVs 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.
  • 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.
  • 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.
  • cytotoxic drugs that are useful for anticancer therapy are relatively insoluble in serum. Some are also quite toxic in an u ⁇ conjugated 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.
  • the prodrug CPT- 11 (irinotecan) is converted in vivo by carboxylesterase to the active metabolite SN-38.
  • One application of the invention is to use a bsAb targeted against a tumor and a hapten (e.g. di-DTPA) followed by injection of a di-DTPA-carboxylesterase conjugate. Once a suitable tumor-to- background localization ratio has been achieved, the CPT-11 is given and the tumor-localized carboxylesterase serves to convert CPT-11 to SN-38 at the tumor.
  • Etoposide is a widely used cancer drug that is detoxified to a major extent by formation of its glucuronide and is within the scope of the invention. See, e.g., Hande et ⁇ l CancerRes.
  • Glucuronide conjugates can be prepared from cytotoxic drugs and can be injected as therapeutics for tumors pre-targeted with mAb-glucuronidase conjugates. See, e.g., Wang et al. CancerRes. 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 giucuronidases. See, e.g., Bakina et al. J. Med Chem. ' 40:4013-4018 (1997).
  • 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- 11 and carboxypepti ase; 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.
  • 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 alternatively be conjugated to the hapten.
  • the enzyme-hapten conjugate is administered to the subject following administration of the pretargeting 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.
  • 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
  • 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.
  • 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.
  • BNCT Boron Neutron Capture Therapy
  • BNCT is a binary system designed to deliver ionizing radiation to tumor cells by neutron irradiation of tumor-localized 1 "B atoms.
  • BNCT is based on the nuclear reaction which occurs when a stable isotope, isotopically enriched 10 ⁇ (present in 19.8% natural abundance), is irradiated with thermal neutrons to produce an alpha particle and a ?Li nucleus. These particles have a path length of about one cell diameter, resulting in high linear energy transfer.
  • the peptide backbone of the targetable 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.
  • a targetable construct comprising a prodrug, for example poly-glutamic acid (SN-38-ester) ⁇ o> is given, thereby localizing the prodrug specifically at the tumor target.
  • SN-38-ester poly-glutamic acid
  • carboxylesterase activates the prodrug poly-glutamic acid (SN-38-ester)iQ by cleaving the ester bond of the poly-glutamic acid (SN-38-ester) ⁇ o releasing large concentrations of free SN-38 at the tumor.
  • the appropriate enzyme also can be targeted to the tumor site.
  • the drug After cleavage from the targetable construct, the drug is internalized by the tumor cells. Alternatively, the drug can be internalized as part of an intact complex by virtue of cross-linking at the target.
  • the targetable construct can induce intemalization of tumor-bound bsAb and thereby improve the efficacy of the treatment by causing higher levels of the drag to be internalized.
  • A-variety of peptide carriers-are-well-suited for conjugation to prodrugs including polyamino acids, such as poly lysine, polyglutamic (E) and aspartic acids (D), including D-amino acid analogs of the same, co-polymers, such as poly(Lys-Glu) [polyfKE] ⁇ , advantageously from 1:10 to 10:1.
  • Copolymers based on amino acid mixtures such as poly(Lys-Ala-Glu-Tyr (SEQ ED 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.
  • 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 hydroxyl groups, to generate species such as SN-38-O-CO(CH2)2CO-O-PEG-0-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 of PEG derivatives and the limited loading capacity due to their dimeric functionality led to the preparation of PEG co-polymers having greater hapten-bearing capacity such as those described by Poiani et ⁇ l.
  • 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.
  • HMPA N-(2- hydroxypropyl)methacrylamide
  • SMA poly(styrene-co-maleic acid anhydride
  • DIVEMA poly(divinylether aleic anhydride)
  • PVP polyolyfN-vinylpyrrolidone
  • 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-ethyl-3-(3-dimethyIaminopropyl)carbodiimide hydrochloride (EDC). and coupled to a recognition moiety bearing a free amino group.
  • EDC water-soluble peptide coupling agents
  • EDC e.g. l-ethyl-3-(3-dimethyIaminopropyl)carbodiimide hydrochloride
  • An example of the latter is histamine, to which antibodies have been raised in the past.
  • 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-I1 (irinotecan).
  • SN- 38 has an aromatic hydroxyl group that was used in the above descriptions to produce aryl esters susceptible to esterase-type enzymes.
  • 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.
  • Doxorubicin also contains aromatic hydroxyl groups that can be coupled to carboxylate- containing polymeric carriers using acid-catalyzed reactions similar to those described for the camptothecin family.
  • doxorubicin analogs like daunomycin, epirubicin and idarubicin can be coupled in the same manner.
  • Polymers bearing tree carboxylate groups can be activated in 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 bis-[2-(succinimido- oxycarbonyloxy)ethyl]sulfone (BSOCOES, Molecular Biosciences, Huntsville, AL), to cross-link the two amines as two amides after reaction with the bis(succinimidy_) ester groups. This is advantageous as these groups remain susceptible to enzymatic cleavage.
  • ethylene glycobis(succinimidylsuccinate) EHS, Pierce Chemical Co., Rockford, IL
  • BSOCOES bis-[2-(succinimido- oxycarbonyloxy)ethyl]sulfone
  • (doxorubicin-EGS) n -poly-lysine remains susceptible to enzymatic cleavage of the diester groups in the EGS linking chain by enzymes such as esterases.
  • the therapeutic conjugate comprises doxorubicin coupled to a carrier compnsmg amine residues and a chelating agent, such as DTPA, to form a DTPA-peptide- doxorubicin conjugate, wherein the DTPA forms the recognition moiety for a pretargeted bsAb.
  • the earner comprises a tyrosyl-lysme dipeptide, e.g., Tyr-Lys(DTPA)-NH2, and more preferably still it comp ⁇ ses Lys(DTPA)-Tyr-Lys(DTPA)-NH2.
  • Doxorubicin phenyl hydrazone conjugates to bis-DPTA containing peptides are particularly desirable in a therapeutic context.
  • Methotrexate also has an available ammo group for coupling to acti vated carboxylate-contaming polymers, in a similar manner to that described for doxorubicin. It also has two glutamyl carboxyl groups (alpha and gamma) that can be activated for coupling to amino-group containing polymers.
  • the free carboxylate groups of methotrexate can be activated in situ (EDC) and the activated drug mixed with an amino-contai ng polymer to directly attach the drug to the side-chains of the polymer via amide bonds. Excess unreacted or cross-reacted drug is separated readily from the polymer-drug conjugate using size- exclusion or ion-exchange chromatography.
  • Maytansinoids and calicheamicins (such as esperamycm) contain mixed di- and t ⁇ -sulfide bonds that can be cleaved to generate species with a single thiol useful for chemical manipulation.
  • the thiomaytensmoid or tluoespera-mycin is first reacted w th a cross-linking agent such as a maleimido- peptide that is susceptible to cleavage by peptidases.
  • the C-terminus of the peptide is then activated and coupled to an amino-containing polymer such as polylysine.
  • the bi-specific antibody-directed delivery of therapeutics or prodrug polymers to m vivo targets can be combined with bi-specific antibody delivery of radionuclides, such that combination chemotherapy and radioimmunotherapy is achieved.
  • Each therapy can be conjugated to the targetable construct and administered simultaneously, or the nuclide can be given as part of a first targetable construct and the drug given in a later step as part of a second targetable construct.
  • a peptide containing a single prodrug and a single nuclide is constructed.
  • the t ⁇ pepbde Ac-Glu-Gly-Lys-NHj can be used as a carrier portion of a targetable construct, whereby SN-38 is attached to the gamma glutamyl carboxyl group as an aryl ester, while the chelate DOTA is attached to the epsilon amino group as an amide, to produce the complex Ac-Glu(SN-38)-Gly- Lys(DOTA)-NH2-
  • the DOTA chelate can then be radiolabeled with various metals for imaging and therapy purposes including In-111, Y-90, Sm-153, Lu-177 and Zr-89.
  • the metal-DOTA complex may represent the recognizable hapten on the targetable construct
  • the only requirement for the metal used as part of the DOTA complex is that the secondary recognition antibody also used recognizes that particular metal-DOTA complex at a sufficiently high affinity.
  • this affinity is between 6-11.
  • Polyme ⁇ c peptides such as ⁇ oly[Glu(SN-38) ⁇ ⁇ -Lys(Y-90-DOTA)2- can be given as readily as the more chemically defined lower MW reagent above, and are indeed preferred.
  • triply substituted polymers can be used, such as poly[Glu(Sn-38) ⁇ o-Lys(Y-90-DOTA) n (histamine-succinate) m , where n and m are integers, such that the recognition agent is independent of the radioimmunotherapy agent.
  • the prodrug is activated by carboxylesterases present at the tumor site or by carboxylesterases targeted to the site using a second targetable construct.
  • a combination therapy can be achieved by administering the chemotherapy and radioimmunotherapy agents in separate steps.
  • a patient expressing CEA-tumors is first administered bsAb with at least one arm which specifically binds CEA and at least one other arm which specifically binds the targetable construct whose hapten is a conjugate of yttrium-DOTA. Later the patient is treated with a targetable construct comprising a conjugate o ytt ⁇ um-DOTA-beta-glucuronidase.
  • a second targetable construct comprising Ac-GIu(SN-38)-Gly-Lys(Y-90-DOTA)-NH2
  • the second targetable construct localizes to the tumor by virtue of bsAb at the tumor that are not already bound to a first targetable construct.
  • First targetable constructs which are localized to the target site act on the Ac-Glu(SN-38)-Gly-Lys(Y-90- DOTA)-NH2 to liberate the free SN-38 drug.
  • Localization of both the prodrug and its respective enzyme to the target site enhances the production of active drug by ensuring that the enzyme is not substrate limited. This embodiment constitutes a marked improvement of current prodrug methodologies currently practiced in the art.
  • Another advantage of administering the prodrug-polymer in a later step, after the nuclide has been delivered as part of a previously given targetable construct, is that the synergistic effects of radiation and drug therapy can be manipulated and, therefore, maximized. It is hypothesized that tumors become more eaky' after RAIT due to radiation damage. This can allow a polymer-prod ug to enter a tumor more completely and deeply. This results in improved chemotherapy.
  • the RAIT therapy agent can be attached to bsAb rather than to the targetable construct.
  • an anti-CEA x anti-DTPA bsAb conjugated to Y-90-DOTA is administered first to a patient with CEA-expressing tumors.
  • advantage is taken of the selectivity of certain anti-chelate mabs in that an anti-indium-DTPA antibody do not bind to a yttrium-DOTA chelate.
  • a conjugate of indium-DTPA-glucuronidase is injected and localized specifically to the CEA tumor sites.
  • the patient is then injected with a polymer-prodrug such as poly(Glu)(SN-38) ⁇ o. The latter is cleaved selectively at the tumor to active monomeric SN-38, successfully combining chemotherapy with the previously administered RAIT.
  • a bi-specific antibody or antibody fragment can be used in the present method, with at least one binding site specific to an antigen at a target site and at least one other binding site specific to the enzyme component of the antibody-enzyme conjugate.
  • Such an antibody can bind the enzyme prior to injection, thereby obviating the need to covalently conjugate the enzyme to the antibody, 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.
  • Multivalent target binding proteins which have at least three different target binding sites as described in Patent Appl. Serial No. 60/220,782.
  • Multivalent target binding proteins have been made by cross-linking several Fab-like fragments via chemical linkers. See U.S. Patent Nos. 5,262,524; 5,091,542 and Landsdorp et al, Euro. J. Immunol 16: 679-83 (1986).
  • Multivalent target binding proteins also have been made by covalently linking several single chain Fv molecules (scFv) to form a single polypeptide. See U.S. PatentN ' o. 5,892,020.
  • a multivalent target binding protein which is basically an aggregate of scFv molecules has been disclosed in U.S. Patent Nos. 6,025,165 and 5,837,242, A bivalent target binding protein comprising three scFv molecules has been described in Krott et ⁇ l, Protein Engineering 10(4): 423-433 (1997).
  • a clearing agent may be used which is given between doses of the bsAb and the targetable construct.
  • the present inventors have discovered that a clearing agent of novel mechanistic action may be used with the invention, namely a glycosylated anti-idiotypic Fab' fragment targeted against the disease targeting arm(s) of the bsAb.
  • Anti-CEA (MN-14 Ab) x anti-peptide bsAb is given and allowed to accrete in disease targets to its maximum extent.
  • an anti-idiotypic Ab to MN-14 termed WI2
  • WI2 an anti-idiotypic Ab to MN-14, termed WI2
  • the clearing agent binds to the bsAb in a monovalent manner, while its appended glycosyl residues direct the entire complex to the liver, where rapid metabolism takes place. Then the therapeutic or diagnostic agent which is associated with the targetable construct is given to the subject.
  • the WI2 Ab to the MN-14 arm of the bsAb has a high affinity and the clearance mechanism differs from other disclosed mechanisms (see Goodwin et ⁇ l, ibid), as it does not involve cross-linking, because the WI2-Fab' is a monovalent moiety.
  • the present invention provides a kit suitable for treating or identifying diseased tissues in a patient, comprising 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, a first targetable construct which comprises a carder 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, optionally, a clearing composition useful for clearing non-localized antibodies and antibody fragments.
  • the kit may optionally contain a prodrug when the first targetable construct comprises an enzyme capable of converting the prodrug to a drug at the target site, an enzyme that is capable of reconverting a detoxified intermediate of a drug to a toxic form, and, therefore, of increasing the toxicity of the drug at the target site, or an enzyme capable of reconverting a prodrug which is activated in the patient through natural processes and is subject to detoxification by conversion to an intermediate of lower toxicity from the detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of the drug 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 identifying or treating diseased tissue also can be included in the kit. Examples include, but are not limited 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.
  • the targetable construct may be administered intravenously, intraarterially, 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.
  • the targetable construct may include agents for other methods of detecting and treating diseased tissue including, without limitation, conjugating dextra ⁇ 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.
  • 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 supernatants of the resulting clones analyzed for anti-peptide 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., In(III)-DTPA chelates. Monoclonal mouse antibodies to an In(III)-di- DTPA are known (Barbet '395 supr ).
  • 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 markers are those disclosed by Herberman, "Immunodiagnosis of Cancer", in Fleisher ed., "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.
  • Tumor-associated markers have been categorized by Herberman, supra, in a number of categories including oncofetal antigens, place ⁇ tal antigens, oncogenic or tumor virus associated antigens, tissue associated antigens, organ associated antigens, ectopic hormones and normal antigens or variants thereof.
  • 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.
  • HCG human chorionic gonadotropin
  • CEA carcino embryonic antigen
  • VEGF tumor vasculature
  • Epstein patents of tumor necrosis
  • membrane receptors e.g., folate receptor, EGFR
  • transmembrane antigens e.g., PSMA
  • 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, CD21, 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.
  • B-cell complex antigens e.g., CD19, CD20, CD21, CD22, CD23, and HLA-DR on B-cell malignancies
  • cytokines expressed by certain tumor cells e.g., IL-2 receptor in T-cell malignancies
  • Other well-known tumor associated antigens that can be targeted 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-4, EGP-1. EGP-2, BrE3, PAM-4, KC-4,
  • TACI transmembrane activator and CAML-interactor
  • B-cell malignancies e.g., lymphoma
  • B-cell maturation antigen BCMA
  • APRIL proliferation-inducing ligand
  • 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.
  • BC A-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.
  • APRIL-TALL-I and BCMA-TACI form a two ligand-two receptor pathway involved in stimulation of B and T cell function.
  • the antibodies can be sequenced and subsequently prepared by recombinant techniques.
  • Humanization and chimerizatioii of murine antibodies and antibody fragments are well known to those skilled in the art.
  • humanized monoclonal antibodies are produced by transferring 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 counte ⁇ arts.
  • the use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions.
  • human antibodies can be obtained from transgenic non-human animals. See, e.g., Mendez et al, Nature Genetics, 15: 146-156 (1997); U.S. Patent No. 5,633,425.
  • human antibodies can be recovered from transgenic mice possessing human immunoglobulin loci.
  • the mouse humoral immune system is humanized by inactivating the endogenous immunoglobulin genes and introducing human immunoglobulin loci.
  • the human immunoglobulin loci are exceedingly complex and comprise a large number of discrete segments which together occupy almost 0.2% of the human genome. To ensure that transgenic mice are capable of producing adequate repertoires of antibodies, large portions of human heavy- and light-chain loci must be introduced into the mouse genome.
  • yeast artificial chromosomes 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 light-chain loci, are introduced separately into mice via fusion of YAC-containing yeast spheroblasts with mouse embryonic stem cells. Embryonic stem cell clones are then microinjected into mouse blastocysts.
  • Resulting chimeric males are screened for their ability to transmit the YAC through their germline and are bred with mice deficient in murine antibody production. Breeding the two transgenic strains, one containing the human heavy-chain loci and the other containing the human light-chain loci, creates progeny which produce human antibodies in response to immunization.
  • Unrearranged human immunoglobulin genes also can be introduced into mouse embryonic stem cells via microcell-mediated chromosome transfer (MMCT). See, e.g., Tomizuka et ⁇ l., Nature Genetics, 16: 133 (1997).
  • MMCT microcell-mediated chromosome transfer
  • microcells containing human chromosomes are fused with mouse embiyonic stem cells. Transferred chromosomes are stably retained, and adult chimeras exhibit proper tissue-specific expression.
  • an antibody or antibody fragment of the present invention may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, e.g., Barbas et ⁇ l, METHODS: A Companion to Methods in Enzymology 2: 119 (1991), and Winter et al, Ann. Rev. Immunol 12: 433 (1994), which are inco ⁇ orated by reference. Many of the difficulties associated with generating monoclonal antibodies by B-cell immortalization can be overcome by engineering and expressing antibody fragments in E. coli, using phage display. To ensure the recovery of high affinity, monoclonal antibodies a combinatorial immunoglobulin library must contain a large repertoire size.
  • a typical strategy utilizes mRNA obtained from lymphocytes or spleen cells of immunized mice to synthesize cDNA using reverse transcriptase.
  • the heavy- and light-chain genes are amplified separately by PCR and ligated into phage cloning vectors.
  • Two different libraries are produced, one containing the heavy-chain genes and one containing the light-chain genes.
  • Phage DNA is isolated from each library, and the heavy- and light-chain sequences are ligated together and packaged to form a combinatorial library.
  • Each phage contains a random pair of heavy- and light-chain cD As and upon infection ofE. coli directs the expression of the antibody chains in infected cells.
  • the phage library is plated, and the antibody molecules present in the plaques are transferred to filters.
  • the filters are incubated with radioactively labeled antigen and then washed to remove excess unbound ligand.
  • a radioactive spot on the autoradiogram identifies a plaque that contains an antibody that binds the antigen.
  • Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA).
  • the scFv linker (Gly-j., Ser)3, is then ligated into the phagemid upstream of the VL fragment.
  • the Vjj and linker- V ⁇ _ fragments are amplified and assembled on the JJJ region.
  • the resulting V ⁇ -linker-VL fragments are ligated into a phagemid vector.
  • the phagemid library can be panned using filters, as described above, or using immunotubes (Nunc; Maxiso ⁇ ). Similar results can be achieved by constructing a combinatorial immunoglobulin library from lymphocytes or spleen cells of immunized rabbits and by expressing the scFv constructs in P. p ⁇ storis.
  • Another form of an antibody fragment is a peptide coding for a single CDR.
  • CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells.
  • the bsAbs can be prepared by techniques known in the art, for example, an anti-CEA tumor Ab and an anti-peptide Ab are both separately digested with pepsin to their respective F(ab')2S.
  • the anti- CEA-Ab-F(ab')2 is reduced with cysteine to generate Fab' monomeric units which are further reacted with the cross-linker bis(maleimido) hexane to produce Fab'- aleimide moieties.
  • the anti-peptide Ab-F(ab')2 is reduced with cysteine and the purified, recovered anti-peptide Fab'-SH reacted with the anti-CEA-Fab'- maleimide to generate the Fab' x Fab ' bi-specific Ab.
  • the anti-peptide Fab'-SH fragment may be coupled with the anti-CEA F(ab')2 t0 generate a F(ab')2 x F ⁇ construct, or with anti-CEA IgG to generate an IgG x F ⁇ b ' bi-specific construct.
  • the IgG x F ⁇ b ' construct can be prepared in a site-specific manner by attaching the antipeptide F ⁇ b ' thiol group to anti-CEA IgG heavy- chain carbohydrate which has been periodate-oxidized, and subsequently activated by reaction with a commercially available hydrazide-maleimide cross-linker.
  • the component Abs used can be chimerized or humanized by known techniques.
  • a chimeric antibody is a recombinant protein that contains the variable domains and complementary determining regions derived from a rodent antibody, while the remainder of the antibody molecule is derived from a human antibody.
  • Humanized antibodies are recombinant proteins in which murine complementarity determining regions of a monoclonal antibody have been transferred from heavy and light variable chains of the murine immunoglobulin into a human variable domain.
  • a variety of recombinant methods can be used to produce bi-specific antibodies and antibody fragments.
  • bi-specific antibodies and antibody fragments can be produced in the milk of transgenic livestock. See, e.g., Col an, A., Biochem. Soc. Sytnp., 63; 141-147, 1998; U.S. Patent No. 5,827,690.
  • Two DNA constructs are prepared which contain, respectively, DNA segments encoding paired immunoglobulin heavy and light chains.
  • the fragments are cloned into expression vectors which contain a promoter sequence that is preferentially expressed in mammary epithelial cells.
  • a promoter sequence that is preferentially expressed in mammary epithelial cells. Examples include, but are not limited to, promoters from rabbit, cow and sheep casein genes, the cow ⁇ - lactoglobulin gene, the sheep ⁇ -lactoglobulin gene and the mouse whey acid protein gene.
  • the inserted fragment is flanked on its 3 ' side by cognate genomic sequences from a mammary-specific gene. This provides a polyadenylation site and transcript-stabilizing sequences.
  • the expression cassettes are coinjected into the pronuclei of fertilized, mammalian eggs, which are then implanted into the uterus of a recipient female and allowed to gestate.
  • 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 mouse sequences, however, and may still be immunogenic.
  • a humanized Ab contains only those mouse amino acids necessary to recognize the antigen. This product is constructed by building into a human antibody framework the amino acids from mouse complementarity determining regions.
  • Other recent methods for producing bsAbs include engineered recombinant Abs which have additional cysteine residues so that they crosslink more strongly than the more common immunoglobulin isotypes. See, e.g., FitzGerald et ⁇ l., Protein Eng. 10(10):1221-1225, 1991. Another approach is to engineer recombinant fusion proteins linking two or more different single-chain antibody or antibody fragment segments with the needed dual specificities.
  • bi-specific fusion proteins can be produced using molecular engineering.
  • the bi-specific fusion protein is monovalent, consisting of, for example, a scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen.
  • the bi-specific fusion protein is divalent, consisting of, for example, an IgG with two binding sites for one antigen and two scFv with two binding sites for a second antigen.
  • bscAb bi-specific single-chain antibodies
  • diabodies can be produced in mammalian cells using recombinant methods. See, e.g., Mack et ⁇ l, Proc. N ⁇ tl Ac d. Sci., 92: 7021- 7025, 1995.
  • bscAb are produced by joining two single-chain Fv fragments via a glycine- serine linker using recombinant methods.
  • the V light-chain (Vj_) and V heavy-chain (V jj ) domains of two antibodies of interest are isolated using standard PCR methods.
  • Vr ⁇ and Vf ⁇ cDNA's obtained from each hybridoma are then joined to form a single-chain fragment in a two-step fusion PCR.
  • the first PCR step introduces the (Gly4-Seq)3 linker (SEQ ID NO: 9), and the second step joins the VL and H a plicons.
  • Each single chain molecule is then cloned into a bacterial expression vector.
  • one of the single-chain molecules is excised and sub-cloned into the other vector, containing the second single-chain molecule of interest.
  • the resulting bscAb fragment is subcloned into an eukaiyofic expression vector.
  • Functional protein expression can be obtained by transfecting the vector into Chinese hamster ovary cells.
  • Bi-specific fusion proteins are prepared in a similar manner. Bi-specific single-chain antibodies and bi-specific fusion proteins are included within the scope of the present invention.
  • fusion proteins 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.
  • 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 the scFv are introduced into the VL and VK domains through PCR reactions.
  • the DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CHI domain.
  • the resulting scFv-Cjjl construct is excised and ligated into a vector containing a DNA sequence encoding the VJJ region of an anti-CEA antibody,
  • the resulting vector can be used to transfect mammalian cells for the expression of the bi- specific fusion protein.
  • bscAb and fusion proteins can be produced using Escherichi ⁇ coli expression systems. See, e.g., Zhenping et ⁇ l, Biotechnology, 14: 192-196, 1996.
  • a -functional bscAb can be produced by the coexpression in E. coli of two "cross-over" scFv fragments in which the VL an VJJ domains for the two fragments are present on different polypeptide chains.
  • the V light-chain (Vj_) and V heavy-chain (VH) domains of two antibodies of interest are isolated using standard PCR methods.
  • the cDNA's are then ligated into a bacterial expression vector such that C-terminus of the VL domain of the first antibody of interest is ligated via a linker to the N-terminus of the VJJ domain of the second antibody.
  • the C-teimi ⁇ us of the V domain of the second antibody of interest is Hgated via a linker to the N-tenninus of the VJJ domain of the first antibody.
  • the resulting dicistronic operon is placed under transcriptional control of a strong promoter, e.g., the E. coli alkaline phosphatase promoter which is inducible by phosphate starvation.
  • a strong promoter e.g., the E. coli alkaline phosphatase promoter which is inducible by phosphate starvation.
  • single-chain fusion constructs have successfully been expressed in E.
  • coli using the l ⁇ c promoter and a medium consisting of 2% glycine and 1% Triton X-100. See, e.g., Yang et ⁇ l., Appl. Environ. Microbiol, 64: 2869-2874, 1998.
  • An E. coli, heat-stable, enterotoxin II signal sequence is used to direct the peptides to the periplasmic space. After secretion, the two peptide chains associate to form a non-covalent heterodimer which possesses both antigen binding specificities.
  • the bscAb is purified using standard procedures known in the art, e.g., Staphylococcal protein A chromatography.
  • Functional bscAb and fusion proteins also can be produced in the milk of transgenic livestock. See, e.g., Colman, A., Biochem. Soc. Symp., 63: 141-147, 1998; U.S. Patent No. 5,827,690.
  • the bscAb fragment, obtained as described above, is cloned into an expression vector containing a promoter sequence that is preferentially expressed in mammary epithelial cells. Examples include, but are not limited to, promoters from rabbit, cow and sheep casein genes, the cow ⁇ -lactoglobulin gene, the sheep ⁇ - Iactoglobulin gene and the mouse whey acid protein gene.
  • the inserted bscAb is flanked on its 3' side by cognate g ⁇ nomic sequences from a mammary-specific gene. This provides a polyadenylation site and transcript-stabilizing sequences.
  • the expression cassette is then injected into the pronuclei of fertilized, mammalian eggs, which 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 using standard methods known in the art. Transgenic production of bscAb in milk provides an efficient method for obtaining large quantities of bscAb.
  • bscAb and fusion proteins also can be produced in transgenic plants. See, e.g., Fiedler et l, Biotech., 13: 1090-1093, 1995; Fiedler et al, Immunotechnology, 3: 205-216, 1997. Such production offers several advantages including low cost, large scale output and stable, long term storage.
  • the bscAb fragment, obtained as described above, is cloned into an expression vector containing a promoter sequence and encoding a signal peptide sequence, to direct the protein to the endoplasmic recticulum.
  • a variety of promoters can be utilized, allowing the practitioner to direct the expression product to particular locations within the plant.
  • ubiquitous expression in tobacco plants can be achieved by using the strong cauliflower mosaic virus 35S promoter, while organ specific expression is achieved via the seed specific Iegumin B4 promoter.
  • the expression cassette is transformed according to standard methods known in the art. Transformation is verified by Southern analysis. Transgenic plants are analyzed for the presence and functionality of the bscAb using standard immunological methods known in the art.
  • the bscAb can be purified from the plant tissues using standard methods known in the art.
  • transgenic plants facilitate long term storage of bscAb and fusion proteins. Functionally active scFv proteins have been extracted from tobacco leaves after a week of storage at room temperature. Similarly, transgenic tobacco seeds stored for 1 year at room temperature show no loss of scFv protein or its antigen binding activity.
  • Functional bscAb and fusion proteins also can be produced in insect cells. See, e.g., Mahiouz ef ⁇ l., J. Immunol. Methods, 212: 149- 160 (1998). Insect-based expression systems provide a means of producing large quantities of homogenous and properly folded bscAb.
  • the baculovirus is a widely used expression vector for insect cells and has been successfully applied to recombinant antibody molecules. See, e.g., Miller, EX.., Ann. Rev. Microbiol, 42: 177 (19S8); Bei et ⁇ l., J. Immunol Methods, 186: 245 (1995).
  • an inducible expression system can be utilized by generating a stable insect cell line containing the bscAb construct under the transcriptional control of an inducible promoter. See, e.g., Mahiouz et ⁇ l., J. Immunol. Methods, 212: 149-160 (1998).
  • the bscAb fragment, obtained as described above, is cloned into an expression vector containing the Drosphil ⁇ metallothionein promoter and the human HLA-A2 leader sequence.
  • the construct is then transfected into D. mel ⁇ nog ⁇ ster SC-2 cells. Expression is induced by exposing the cells to elevated amounts of copper, zinc or cadmium.
  • the presence and functionality of the bscAb is determined using standard immunological methods known in the art. Purified bscAb is obtained using standard methods known in the art.
  • bi-specific antibodies of the instant invention are those which incorporate the Fv of MAb Mu-9 and the Fv of MAb 679 or the Fv of MAb MN-14 and the Fv of MAb 679, and their human, chimerized or humanized counte ⁇ arts.
  • the MN-14, as well as its chimerized and humanized counterparts, are disclosed in U.S. Patent No. 5,874,540.
  • bi-specific antibodies which inco ⁇ orate one or more of the CDRs of Mu-9 or 679.
  • the antibody can also be a fusion protein or a bi- specific antibody that inco ⁇ orates a Class-Ill anti-CEA antibody and the Fv of 679.
  • Class-Ill antibodies, including Class-Ill anti-CEA are discussed in detail in U.S. Patent No. 4,818,709. VII.
  • the present invention encompasses the use of the bsAb and a therapeutic or diagnostic agent associated with the targetable construct discussed above in intraoperative, intravascular, and endoscopic tumor and lesion detection, biopsy and therapy as described in U.S. Patent No. 6,096,289.
  • the antibodies and antibody fragments of the present invention can be employed not only for therapeutic or imaging purposes, but also as aids in performing research in vitro.
  • the bsAbs of the present invention can be used in vitro to ascertain if a targetable construct can form a stable complex with one or more bsAbs. Such an assay would aid the skilled artisan in identifying targetable constructs which form stable complexes with bsAbs.
  • the assay is advantageously performed by combining the targetable construct in question with at least two molar equivalents of a bsAb. Following incubation, the mixture is analyzed by size-exclusion HPLC to determine whether or not the construct has bound to the bsAb. Alternatively, the assay is performed using standard combinatorial methods wherein solutions of various bsAbs are deposited in a standard 96-weZ_ plate. To each well, is added solutions of targetable constructs). Following incubation and analysis, one can readily determine which constructs) bind(s) best to which bsAb(s).
  • the order of addition of the bsAb to the targetable construct is not crucial; that is, the bsAb may be added to the construct and vice versa.
  • neither the bsAb nor the construct needs to be in solution; that is, they may be added either in solution or neat, whichever is most convenient.
  • the method of analysis for binding is not crucial as long as binding is established.
  • the peptide was synthesized as described by Karacay et. ⁇ l. Bioconjug ⁇ te Chem. H. "842-854 (2000) except D-tyrosine was used in place of the L-tyrosine and the N-trityl-HSG-OH was used in place of the DTPA.
  • the final coupling of the N-trityl-HSG-OH was carried out using a ten fold excess of N- trityl-HSG-OH relative to the peptide on the resin.
  • N-trityl-HSG-OH (0.28 M inNMP) was activated using one equivalent (relative to HSG) of N-hydroxybenzot ⁇ azole, one equivalent of benzotrazole-1-yl- oxy-tns-(d ⁇ methylammo)phos ⁇ homum hexafluorophosphate (BOP) and two equivalents of dusopropylethylamine
  • BOP benzotrazole-1-yl- oxy-tns-(d ⁇ methylammo)phos ⁇ homum hexafluorophosphate
  • a formulation buffer was prepared which contained 22.093 g hydroxypropyl- ⁇ -cyclodext ⁇ n, 0.45 g 2,4-d ⁇ hydroxybenzo ⁇ c acid, 0 257 g acetic acid sodium salt, and 10.889 g ⁇ -D-glucoheptonic 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 02 L of SnCl2 (200 mg/mL) with 3.8 mL of the formulation buffer.
  • the peptide Ac-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (0.0026g), was dissolved in 78 mL of the buffer solution and mixed with 0 52 L of the stannous buffer. The peptide solution was then filtered through a 0.22 ⁇ m Millex GV filter in 1 5 mL ahquots into 3 mL lyophihzation vials. The filled vials were frozen immediately, lyophilized and c ⁇ mp sealed under vacuum.
  • Pertechnetate solution (27 mCi) in 1.5 mL of salme was added to the kit.
  • the kit was incubated at room temperature for 10 min and heated in a boiling water bath for 25 min The kit was cooled to room temperature before use.
  • Example 3 Peptides for Carrying Therapeutic/Imaging Radioisotopes to Tumors via Bi-specific Antibody Tumor Pretargeting
  • DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH 2 (SEQ ID NO: 2) (IMP 237) was synthesized to deliver therapeutic radioisotopes such as 90 ⁇ or 177 U to tumors via bi-specific antibody tumor pretargeting
  • the bi-specific antibody is composed of one portion which binds to an antigen on the tumor and another portion which binds to the HSG peptide
  • the antibody which bmds the HSG peptide is 679.
  • This system can also be used to deliver imaging isotopes such as 11 lfr ⁇ -111
  • IMP 231 was synthesized on Sieber Amide resm (Nova-Biochem) using standard Fmoc based solid phase peptide synthesis to assemble the peptide backbone with the following protected ammo acids, in order: Fmoc-Lys(Aloc)-OH, Fmoc-Tyr(But)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Phe-OH, (Reagents from Advanced Chemtech) t ⁇ -t-butyl DOTA (Macrocyclics).
  • the peptide was cleaved from the resm and the protecting groups were removed by treatment with TFA The peptide was pu ⁇ fied by HPLC to afford 0 6079 g of peptide from 1 823 g of Fmoc-Lys(Aloc)-Tyr(But)-Lys(Aloc)-NH-S ⁇ eber amide resin.
  • N-trityl-HSG-t-butyl ester (20.620 g, 3.64 x 10" 2 mol) was dissolved in a solution of 30 L chloroform and 35 mL glacial acetic acid. The reaction was cooled in an ice bath and 15 mL of BF3*Et2 ⁇ was slowly added to the reaction solution. The reaction was allowed to warm slowly to room temperature and mix for 5 hr. The reaction was quenched by pouring into 200 mL 1M NaOH and the product was extracted with 200 mL chloroform. The organic layer was dried over Na2S ⁇ 4 and concentrated to afford a crude gum which was triturated with 100 L Et2 ⁇ to form a precipitate.
  • the crude precipitate was poured into 400 mL 0.5 M pH 7.5 phosphate buffer and extracted with 2 x 200 mL EtOAc.
  • the aqueous layer was acidified to pH 3.5 with 1 M HCI and extracted with 2 x 200 mL chloroform.
  • the precipitate was the desired product by HPLC comparison to a previous sample (ESMS MH+ 511).
  • the 9®Y ( ⁇ 400 ⁇ Ci/kit) was diluted to I L in deionized water and added to the lyophilized kits.
  • the kits were heated in a boiling water bath for 15 min, the vials were cooled to room temperature and the labeled peptides were evaluated by reverse phase HPLC (HPLC conditions: Waters Nova-Pak C-18, 8x100 mm RCM column eluted at 3 mL/min with a linear gradient from 100 % (0.1 % TFA in H2O) to 100 % (90 % CH3CN, 0.1% TFA, 10 % H2O)).
  • the HPLC analysis revealed that the minimum concentration of peptide needed for complete labeling, with this formulation, was 35 ⁇ g/mL.
  • the reverse phase HPLC trace showed a sha ⁇ 90 ⁇ labeled peptide peak.
  • the labeled peptide was completely bound when mixed with excess 679 IgG by size exclusion HPLC.
  • the labeled peptide vials were cooled to room temperature and evaluated by reverse phase HPLC (HPLC conditions: Waters Nova-Pak C-18, 8x100 mm RCM column eluted at 3 mL/mi ⁇ with a linear gradient from 100 % (0.1 % TFA in H 2 0) to 100 % (90 % CH3CN, 0.1 % TFA, 10 % H2O)).
  • HPLC analysis revealed that the minimum concentration of peptide needed for labeling (4.7 % loose ⁇ fin), with this formulation, was 35 ⁇ g/mL.
  • the reverse phase HPLC trace showed a sha ⁇ IT lfo labeled peptide peak.
  • the labeled peptide was completely bound when mixed with excess 679 IgG by size exclusion HPLC.
  • the peptides, IMP 237 and IMP 241 were labeled according to the procedure described by Karacay et. al. Bioconjugate Chem. 77:842-854 (2000).
  • the peptide, IMP 241 (0.0019 g), was dissolved in 587 ⁇ l 0.5 M NH4CI, pH 5.5.
  • a 1.7 ⁇ L aliquot of the peptide solution was diluted with 165 ⁇ l 0.5 M NH4CI, pH 5.5.
  • the ' ⁇ In (1.8 mCi) in 10 ⁇ L was added to the peptide solution and the mixture was heated in a boiling water bath for 30 min.
  • the labeled peptide was analyzed by HPLC using a Waters 8x100 mm radial-pak, nova-pak C-28 RCM cartridge column.
  • the column was eluted at 3 mL/min with a linear gradient which started with 100 % of 0.1 % TFA in water and went to 100 % of 0.1 %TFA in 90% acetonitrile and 10 % water over 10 min. There was about 6% loose H ⁇ In in this labeling which came out at the void volume of the column (1.6 min). There were also some H lln labeled peaks at 5 min and 6.6 to 8 min. The Hlln labeled peptide was eluted at 8.8 min as a single peak.
  • the HPLC profile of ⁇ lln IMP 237 was nearly identical to 1 "in IMP 241.
  • the 11 lm IMP 237 was about 70 % converted to the shorter retention time peak after incubation for 22 hr at 37° C.
  • 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(Al c)-OH.
  • the alpha-Fmoc protecting group is selectively removed and the Fmoc-Tyr(OBut), alpha-Fmoc-Lys(AIoc)-OH, and Fmoc-Phe-OH added with alternate cycles of coupling and alpha-amino group deprotection.
  • 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 ofAc-Phe-Lvs(Ac)-Tyr-Lvs(Ac)-OH (SEQ ID NO: 2) to KLH [0171]
  • Example 6 Generation of an Anti-Peptide Ah
  • Immunocompetent mice are injected with a mixture of the peptide antigen in complete Freund's adjuvant. Two booster shots of the peptide mixed with incomplete Freund's adjuvant are administered over the next several weeks.
  • Spleen cells are harvested from the animals and fused with Sp2/0-Agl4 myeloma cells. Culture supematants of the resulting clones are analyzed for anti-peptide reactivity by ELISA, using plates coated with the original peptide immunogen.
  • Enzyme-deficient hybridomas are isolated to enable selection of fused cell lines, and selected clones grown in culture media to produce the anti-peptide Abs.
  • Anti-peptide Ab is purified chromatographically using a protein A column to isolate the IgG fraction, followed by ion-exchange columns to clean the desired product.
  • the Ab of interest is finally purified by using an affinity column comprised of the peptide of interest bound to a solid support, prepared by chemically coupling said peptide to activated beads or resin.
  • the anti-peptide Ab is incubated with 200 ⁇ g/ ⁇ L of pepsin at pH 4 for one hour and purified by a tandem column of protein A, to remove undigested IgG, followed by G-50-Sephadex, to remove low molecular weight contaminants.
  • the anti-peptide-F(ab 'fa is reduced to a Fab' fragment by reaction with a freshly prepared cysteine solution in 0.1M PBS, containing lOmM 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 ⁇ 5 containing lOmM EDTA.
  • Anti-CEA Ab IgG is oxidized by reaction with 1 OmM sodium periodate for 90 minutes at 4°C, in the dark.
  • the oxidized Ab is purified by spin-column chromatography and mixed with an excess of the cross-linker 4-(4-maleimidophenyl) butyric acid hydrazide (MPBH).
  • MPBH 4-(4-maleimidophenyl) butyric acid hydrazide
  • the reaction is allowed to proceed for 2 h and the IgG-hydrazone-meleimide purified by spin-column chromatography.
  • the hydrazone bond is reduced by reaction with lOmM sodium cyanoborohydride and repurified.
  • Example 11 Preparation of anti-CEA-IeGx anti-Peptide-Fab' Bi-specific Ab
  • the IgG-hydrazide-maleimide from Example 10 is treated with an equimolar amount of anti- peptide Fab'-SH, prepared in Example 6, at pH 6.0, for 30 minutes at room temperature. Remaining free thiol groups are blocked by a 30-minute reaction with iodoacetamide.
  • the bi-specific Ab anti-CEA-IgG x anti-peptide-Fab' is purified by size-exclusion chromatography to remove unreacted Fab', followed by affinity chromatography using solid-phase-bound peptide to separate IgG x F ⁇ b ' from unreacted IgG.
  • the alpha-Fmoc protecting group is selectively removed and the 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- side-chain is removed by reaction with palladium (0) catalyst.
  • Boc-group protecting groups may be used which may be removed by reaction with TFA and the free amino groups reacted with excess of the ITC-Bz-DTPA.
  • the alpha-amino group is capped by reaction with acetic anhydride, and the entire peptide removed from the resin with TFA (with concomitant deprotection of the tyrosyl residue) to give Ac-Phe-Lys(Bz-DTPA)-Tyr-Lys(Bz-DTPA)-NH2.
  • Carboxylesterase (5 mg) in 0.2 M phosphate buffer, pH 8.0, is treated with a five-fold molar excess of the cross-linking agent sulfo-succinimidyl-[4-maleimidomethyl]-cyclohexane-l-carboxylate (sulfo-SMCC). After stirring two hours at room temperature, the activated enzyme is separated from low molecular weight contaminants using a spin-column of G-25 Sephadex and equilibrated in 0.1 M phosphate buffer, pH 7, containing 1 mM EDTA.
  • sulfo-SMCC cross-linking agent sulfo-succinimidyl-[4-maleimidomethyl]-cyclohexane-l-carboxylate
  • the tetrapeptide N-acetyl-Cys-Lys(DTPA)-Tyr- Lys(DTPA)-NH2 (ten-fold molar excess) is added to the activated enzyme and dissolved in the same buffer as used in the spin-column. After stirring for one hour at room temperature, the Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH2 (SEQ ID NO: 11) peptide carboxylesterase conjugate is purified from unreacted peptide by spin-column chromatography on G-25 Sephadex in 0.25 M acetate buffer, pH 6.0. Successful conjugation is demonstrated by indium-Ill labeling of an aliquot of the conjugate, and analysis by size-exclusion HPLC.
  • Example 15 Use of anti-CEA-IgG x anti-Peptide-Fab' Bi-specific Ab for RAIT
  • a patient with a CEA-expressing tumor burden is given anti-CEA-IgG x anti-peptide-Fab' bi- specific Ab. Seven days later, the patient is given Y-90-di-Bz-DTPA-peptide (from Example 13). The Y- 90-labeled peptide clears rapidly from non-target tissue but localizes avidly to sites pre-targeted with the anti-CEA-IgG x anti-peptide-Fab' bi-specific Ab, effecting destruction of tumors.
  • the anti-idiotypic Ab to MN-14 termed WI2 is digested to a F(ab')2 fragment using pepsin, as outlined in Example 8.
  • the F(ab')2 is reduced to a Fab' fragment using a low molecular weight thiol, as outlined in Example 9.
  • the Fab'-SH is purified by spin-column chromatography and reacted with excess iodoacetamide to block hinge-region thiol groups and prevent reassociation.
  • the Fab' is reacted with a 400-fold molar excess of the galactosylation agent, the thio-i idate of cyanomethyl-2,3,4,6-tetra-0-acetyl-l-thio-beta-D- galactopyranoside (see Karacay et al.).
  • the galactosylated protein is purified by two spin-columns and the galactose;Fab' radio determined by MALDI-MS,
  • Example 17 Use of anti-CEA-IgG x anti-Peptide Fab' Bi-specific Ab for RAIT, with a bsAb Clearing Step.
  • a patient with a CEA-expressing tumor burden is given anti-CEA-IgG (MN-14) x anti-peptide- Fab' bi-specific Ab.
  • the patient is given a clearing dose of galactose- I2-Fab'.
  • Twenty- four hours after the clearing dose of a galactose-WI2-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.
  • Aloc-Lys(Fmoc)-OH was attached to 0.2 1 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 form 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 CH2CI2, 0.75 mL glacial acetic acid and 2.5 ml diisopropylethyl amine.
  • the resin mixture was then treated with 0.8 ml tributyltin 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 resin (SEQ ID NO: 7),
  • the N-terminus was acetylated by vortex mixing the resin for 60 mm with 8 mL of a solution containing 10 mL DMF, 3 mL acetic anhydride, and 6 mL diisopropylethylamine.
  • the side chain Aloe protecting groups were then removed as described above and the resin treated with piperidine using the standard Fmoc deprotection protocol to remove any acetic acid which may have remained on the resin.
  • 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 with 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.
  • DTPA Tetra-t-butyl ester could be used with conventional coupling agents such as DIC and HBTU.
  • conventional coupling agents such as DIC and HBTU.
  • the peptide was formulated into lyophilized kits which contained 78 ⁇ g of the peptide, 0.92 mg non-radioactive InCFj, 100 ⁇ g stannous chloride, 3 mg gentisic acid, and HPCD (10 % on reconstitution).
  • An IMP 192 kit was labeled by reconstituting the contents of the vial with 1.5 mL of saline which contained 25 Ci Na99m ⁇ c ⁇ 4. The kit was incubated at room temperature for 10 mm and then heated in a boiling water bath for 15 mm. The labeled peptide solution was then cooled to room temperature. 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, the 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 min. The shift may be due to ion pairing of some serum component with the peptide.
  • This bsAb was prepared by crosslinking the hMN-14 Fab'gH ( a humanized monoclonal anti-CEA antibody) and 734 Fab' ma ⁇ (a murine anti-diDTPA) fragments, analogously to Example 8.
  • the Fab'gH fragments of hMN-14 and 734 were prepared by reduction of the F(ab')2 fragments with 10 mM 2- mercaptoethylamine in the presence of 10 mM EDTA at pH 7.3 for 60 min at 37°C Fab'sH was collected after spin column (Penefsky) purification (Sephadex G-50-80, 50 mM NaOAc, 0.5 mM EDTA, pH 5.3) Maleimide grou ⁇ (s) were introduced onto 734 Fab'sjj fragment using 4 mM N,N'-o- phenylenedimaleimide at RT for 60 min. Spin column purification was used to isolate the Fab' ma ⁇ .
  • the bsAb was radiodinated using chloramine T (Greenwood and Hunter). Binding of the radioiodinated bsAbs to CEA, WI2 (rat anti-MN-34 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 10-20x molar excess of CEA. The bsAb complexed with radiolabeled indium-DTPA chelates (IMP-156 or IMP-192).
  • Radioiodinated bsAb was tested for stability in fresh human serum at 37°C under a humidified 5 % CO2 atmosphere. Aliquots were examined on SE-HPLC. In order to detect radioiodine associated with serum proteins, the aliquots were mixed with WI2 to shift the bsAb peak to earlier retention times. The bsAbs showed 3-5 % loss of binding capacity to WI2 after 48 h incubation in serum. Slight aggregate formation (4-7 %) was observed upon incubation of the bsAbs in serum for 72 h.
  • 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 hMN-14 heavy chain Fd to 734scFv were introduced into the VL and VJJ; domains of 734 by PCR reactions using specific primer sets.
  • PC -amplification of 734Vr was performed using the primer set 734V ⁇ j scFv5'(Cys) and 734VLSCFV3 ' (polypeptide and polynucleutide sequences for such primers are shown and described in U.S. Patent Application Serial No.
  • the primer 73 VLScFv5'(Cys) represents the sense- strand sequence encoding the first four residues (PKSC) (SEQ ID NO: 12) of the human IgGl hinge, linked in-frame to the first six residues (QLVVTQ) of 734 V (SEQ ID NO: 13), via a short flexible linker, GGGS (SEQ ID NO: 10).
  • PKSC first four residues
  • QLVVTQ residues of 734 V
  • GGGS SEQ ID NO: 10
  • One cysteine of the human hinge was included because it is required for the interchain disulfide linkage between the hMN-14 heavy chain Fd-734scFv fusion and the hMN-14 light chain.
  • a Pstl site was inco ⁇ orated to facilitate ligation at the intronic sequence connecting the Cfil domain and the hinge.
  • the primer 734Vj ⁇ scFv3 ' represents the anti-sense sequence encoding the last six residues (TKLKIL) of the 734 V domain (SEQ ID NO: 14) and a portion of the flexible linker sequence (GGGGSGGGG) (SEQ ID NO: 15), which is fused in-frame downstream of the VL domain. ]0195] Following PCR amplification, the amplified product (-400 bp) first was treated with T4 DNA polymerase to remove the extra "A" residue added to the termini during PCR-arnplification and subsequently was digested with Pstl .
  • Primer 734VHSCFV5' represents the sense-strand sequence encoding the remaining part of the flexible linker sequence (SGGGGS) (SEQ ID NO: 16) connecting the V ⁇ _ and Vj-r sequences, and the first six residues (EVKLQE) of the 734 VH domain (SEQ ID NO: 17).
  • Primer 734V H scFv3'(Sacl) represents the sense-strand sequence encoding the remaining part of the flexible linker sequence (SGGGGS) (SEQ ID NO: 16) connecting the V ⁇ _ and Vj-r sequences, and the first six residues (EVKLQE) of the 734 VH domain (SEQ ID NO: 17).
  • Vfj represents the anti-sense sequence encoding the last six residues (TvTVSS) of 734 Vfj (SEQ ID NO: 18). Also included is a translation stop co ⁇ on. The restriction sites Eagl and Sacl were inco ⁇ orated downstream of the stop codon to facilitate subcloning. Similarly, the PCR-amplified Vjj product o -400 bp was first treated with T4 DNA polymerase to remove the extra "A" residues at the PCR product termini, and then digested with Sacl, resulting in a Vff DNA fragment with a blunt end-sticky end configuration.
  • a pBlueScript (Stratagene, La Jolla)-based staging vector (HClkbpSK) containing a SacII fragment of the human IgGl genomic sequence was constructed.
  • the genomic SacII fragment contains a partial 5' intron, the human IgGl Cftf domain, the intronic sequence connecting the Cyl- to the hinge, the hinge sequence, the intronic sequence connecting the hinge to the Cjj2 domain, and part of the Cjj2 domain.
  • the segment containing the hinge and part of the C] ⁇ 2 domain in HClkbpSK was removed by Pstl/Sacl digestion, and the cloning site generated was used to co-Iigate the VL (Pstl/blunt) and Vjj (blunt/Sac 1) PCR products prepared above.
  • the Cf j l domain in the resultant construct (Cjjl-734pSK) is connected to the 734scFv gene sequence via an intron ( Figure 4). Since the genomic SacII fragment for IgGl only included part of the 5' intron sequence flanking the C l domain, the full intronic sequence was restored by inserting the remaining intronic sequence as a BamHl/SacII segment, into the corresponding sites of the Cfjl-734pSK.
  • the BamHl/Eagl fragment containing the full 5' intron, Cfjl domain, connecting intron, 5 hinge- residues, short GGGS linker (SEQ ID NO: 1 ), and a 734scFv sequences was then isolated, and used to replace the Hindlll/Eagl segment containing the human genomic IgGl constant sequence in the hMN- 14pdHL2 vector.
  • a HNB linker (see Patent Serial No. 09/337,756) with a BamHl overhang on one end and a Hindlll overhang on the other was used to facilitate the BamHl Eagl fragment ligation into the Hindlll/Eag 1 site in the hMN-14pdHL2 vector.
  • the resultant vector was designated hMN-14-734pdHL2 and can be used to transfect mammalian cells for the expression of the bi-specific protein.
  • the hMN-14pdHL2 vector was derived from the vector, pdHL2, which has previously been described. See Losman et at., Cancer Supplement, 80:2660, 1997. Construction of hMN-14pdHL2 was performed by replacing the Vft and Vj domains of hLL2pdHL2 with that of hMN-14 using standard molecular biology techniques ( Figure 5). The hMN-14-734pdHL2 vector was transfected into SP2/0 cells by electroporation and the cell clones secreting bsAb were identified.
  • the bsAb purified from cell culture supernatant (clone 341.1G6) on a protein L column (Pierce, Rockford, IL) is a 75 kD protein (based on amino acid sequence calculation) that co-migrated with the 66 kD marker in non-reducing SDS-PAGE probably due to secondary structure ( Figure 2, lane 2).
  • Figure 2, lane 2 Under reducing conditions, bands corresponding to a heavy (50 kD) and a light (25 kD) chain were observed ( Figure 2, lane 4).
  • Kappa chain monomers (25 kD) and dimers (50 kD) secreted by the transfectoma also were co-pu ⁇ fied (Figure 2, lane 2) since protein L binds to kappa light chains of human, mouse and rat.
  • hMN-14Fab-734scFv shows specific binding to both CEA and In-DTPA-BSA in a dose dependent manner.
  • 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. Afterbirth, 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.
  • a bscAb fragment is cloned into an expression vector containing a shortened legumin B4 promoter plus 54 base pairs of LeB4 untranslated RNA leader from Vici ⁇ f ⁇ b ⁇ and encoding a LeB4 signal peptide, to direct the protein to the endoplasmic recticulum.
  • the expression cassette is transformed into tobacco leaf discs according to the methods described by Zambryski et ⁇ l., using Agrob ⁇ cterium-me ⁇ iated gene transfer. Transformation is verified by Southern analysis. Transgenic plants are analyzed for the presence and functionality of the bscAb using standard immunological methods known in the art.
  • the bscAb can be purified from the plant tissues using standard methods known in the art.
  • mice Female nude mice (Taconic NCRNU, 3-4 weeks old) with GW 39 tumor xenografts were used for the pretargeting experiments. Tumors were 0.3-0.8 g.
  • Control group showing the clearance of 11 l-In-indium-IMP-156 at 3 h after injection.
  • Nude mice bearing GW 39 tumor xenografts were administered 125-I-labeled bsAb (5 ⁇ Ci, 15 ⁇ g, 1.5 x 10- ⁇ 0 mol).
  • hMN-14 x 734 was allowed 24 h for localization and clearance before administering 99m-Tc- IMP-192 (10 ⁇ Ci, 1.6 x 10- ⁇ mol of peptide).
  • Biodistribution studies were performed at 30 min, I, 3 and 24 h post 99m-Tc-IMP-192 injection, five animals per time point.
  • Nude mice bearing GW 39 tumor xenografts were administered 125-I-labeled bsAb (5 ⁇ Ci, 15 ⁇ g, 1.5 x 10-10 mo i), hMN-14 x 734 was allowed 24 h for localization and clearance before administering 99m-Tc- IMP-192 (10 ⁇ Ci, 1.6 x 10" 1 * mol of peptide). Biodistribution studies were performed at 30 rain, 1, 3 and 24 h post 99m-Tc-IMP-192 injection, five animals per time point. BsAb:peptide, 1: 0.1.
  • Control group of nude mice bearing GW-39 tumors received 99m-Tc-IMP-192 (10 ⁇ Ci, 1.6 x 10"H mol of peptide) and were sacrificed 3 h later.
  • the percentage of the available DTPA binding sites on the tumor bound bsAb filled with 99m-Tc- IMP-392 was calculated from the above data assuming one peptide bound to one bsAb molecule. However, it is possible that one peptide molecule can crosslink two molecules of bsAb.
  • 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.
  • 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-Bz-DTPA-peptide, containing 50 mCi of Y-90.
  • a CT 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.
  • the eluate is purified on a TSK 3000G Supelco column using 0.2 M sodium phosphate pH 6.8 at 4 ml/min.
  • the fraction containing conjugate is concentrated on a Centricon-l ⁇ TM concentrator, and buffer-exchanged with 0.1 M sodium acetate pH 6.5. Recovery: 0.9 ml, 4.11 mg/ml (3.7 mg).
  • 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 M 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-specific 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.
  • the fractions containing product were combined and lyophilized to afford 0.0453 g of the desired product, which was confirmed by ESMS MH + 1847.
  • the peptide of Example 31 was formulated into kits for In- 111 labeling.
  • a solution was prepared which contained 5.014 g 2-hydroxypropyl- ⁇ -cyclodextrin, and 0.598 g citric acid in 85 mL.
  • the solution was adjusted to pH 4.20 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 0.22 ⁇ jm Millex GV filter into 2 mL lyophilization vials which were immediately frozen and lyophilized.
  • the In-111 was dissolved in 0.5 mL water and injected into the lyophilized kit.
  • the kit solution was incubated at room temperature for 10 min 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.
  • Kits were reconstituted with 400 ⁇ Ci 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
  • the labeled peptide was analyzed by ITLC in saturated NaCl.
  • the loose In-111 was at the top 20 % of the ITLC strip.
  • Each mouse was injected with 100 ⁇ L (20 ⁇ Ci) of the In-111 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.
  • Kits were reconstituted with 4 mCi In-Ill in 0.5 L water.
  • the In-111 kit was incubated at room temperature for 10 min and then diluted with 0.5 mL of the cold indium containing 0.5 M pH 7.2 acetate buffer.
  • the labeled peptide was analyzed by ITLC in saturated NaCl..
  • the loose In-111 was at the top 20 % of the ITLC strip.
  • Each mouse was injected with 100 ⁇ L (400 ⁇ Ci) of the In-111 labeled peptide.
  • the animals were anesthetized and sacrificed at 30 min and 1 hr using two animals per time point.
  • the serum 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
  • the reverse phase HPLC analysis showed that the radiolabeled peptide was excreted intact in the urine.
  • the amount of activity remaining in the serum was too low to be analyzed by reverse phase HPLC due to the poor sensitivity of the detector.
  • Doxorubicin has ⁇ 95 % hepatobiliary clearance.
  • 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.
  • a lyophilized kit of IMP 224 containing 10 micrograms of peptide was used.
  • the kit was lyophilized in 2 L vials and reconstituted with 1 mL sterile water. A 0.5 mL aliquot was removed and mixed with 1.0 mCi In-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.
  • each mouse was injected with 100 ⁇ L (lO ⁇ Ci) 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 tail were collected and counted.
  • 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-operative, endoscopic, intravascular or other similar procedures, using the pretargeting methods 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.
  • 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-LvstHSG)-D-Tyr-Lys( ⁇ SG)-LvsfTscg-Cvs -NH-. (SEQ ID NO: I ) (IMP 245) [0219]
  • 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 peptide with a single benzotriazole-l-yl-oxy-tris-(dimethylamino)-phosphonium hexafiuorophosphate (BOP) coupling using 5 eq of protected DOTA for 16 hr.
  • BOP benzotriazole-l-yl-oxy-tris-(dimethylamino)-phosphonium hexafiuorophosphate
  • a formulation buffer was prepared which contained 22.093 g hydroxypropyl- ⁇ -cyclodextrin, 0.45 g 2,4-dihydroxybenzofc acid, 0.257 g acetic acid sodium salt, and 10.889 g ⁇ -D-gfucoheptonic 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 SnCfj (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 InCkj in 0.1 M HC1.
  • the peptide solution was mixed with 2 mL 0.5 M NH4OAC and allowed to incubate at room temperature for 15 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 ⁇ m 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.
  • 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.
  • the peptide was analyzed by reverse phase HPLC and size exclusion HPLC (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., McBride, W.J., Karacay, H., Chang, K., Griffiths, G.L., Hansen, H.J., and Goldenberg, D.M., the entire contents of which are incoiporafed 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.
  • Rhenium oxo complex was made by mixing 0.0504 g of IMP 245 with 0.0045 g of ReOBr4
  • the peptide, IMP 245 (0.0029 g, 1.58 x 10-6 mo i) was dissolved in 2.0 mL of 0.5 M NH4OAC pH 5.5 buffer, which contained 0.0020 g of InCl ⁇ .
  • the peptide solution was heated at 50°C for 17 min.
  • a formulation buffer was prepared from 22.093 g hydroxypropyl- ⁇ -cyclodextrin (HPCD), 0.450 g 2,4- dihydroxybenzoic acid (gentisic acid), 0.257 g Acetic acid sodium salt, 10.889 g ⁇ -D-glucoheptonic acid and dissolved in 170 mL nitrogen purged DI water.
  • the solution was adjusted to pH 5.30 with a few drops of IM NaOH and diluted to a final volume of 220 mL with DI water.
  • the formulation buffer was then sterile filtered through a 0.22 ⁇ :m filter.
  • a stannous buffer was prepared by diluting 0.2 mL (200 mg mL SnCl2 m 6 M HC1) with 3.8 mL of the formulation buffer in an argon purged sterile vial.
  • the peptide solution was then mixed with 76 mL of the formulation buffer and 0.56 mL of the stannous buffer.
  • the solution was then dispensed in 1.5 mL aliquots through a Millex GV 0.22 mm filter into 3 mL lyophilization vials.
  • kits were immediately frozen on dry ice and lyophilized.
  • the kits were sealed under vacuum at the end of the lyophilization cycle.
  • Each kit contained 55 Tig of the peptide and was formulated for a 1.5 mL reconstitution volume of 99m Tc ⁇ 4" in saline.
  • Tc-99m kits formulated with ascorbic acid were prepared in the same manner as the gentisic acid kits except 0.222 g ofL-ascorbic acid was used instead of gentisic acid.
  • Example 45 Tc-99m Kit Labeling
  • the kit was reconstituted with 1.5 mL of 99m c ⁇ 4- in saline (0.5 to 70 mCi) and incubated at room temperature for 10 min, The kit was then heated in a boiling water bath for 15 min and allowed to cool to room temperature before use.
  • the peptide was dissolved in 0.5 M NH4OAC, pH 3.08 at 2.2 x 10- ⁇ M (peptide). An aliquot, 3.5 ⁇ L of the peptide solution was then mixed with 165 ⁇ L of 0.5 M NH4OAC pH 3.93 and 6 ⁇ L of the Y-90 solution. The mixture was then heated for 20 min at 85-95°C. Reverse phase HPLC showed that the peptide labeled well.
  • the bi-specific F(ab')2 antibody composed of Fab' fragments of humanized MN-14 anti-CEA or murine Mu-9 anti-CSAp and murine 679 were prepared using PDM as the crosslinker.
  • the F(ab')2 of each parental antibody was first prepared.
  • the F(ab')2 was reduced with 1 mM DTT to Fab'-SH, which was diafiltered into a pH 5.3 acetate buffer containing 0.5 mM EDTA (acetate/EDTA buffer) to remove DTT, concentrated to 5-10 mg/mL, and stored at 2-8 °C until needed.
  • the F(ab') 2 was reduced with 1 M DTT to Fab'-SH , which was then diluted with 5 volumes of 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 free PDM is minimum, and concentrated to 5-10 mg/mL. A solution of hMN-14 Fab'-SH or Mu-9 Fab'-SH was then mixed with a solution of 679 Fab'-PDM at a 1:1 ratio based on the amount of Fab'.
  • hMN-14 x 679 for HSG was determined by BIAcore analysis using a CM-5 chip derived with a peptide containing a single HSG substituent and a thiol by the method recommended by the manufacturer (Biacore, Inc., Piscataway, NJ 08854).
  • the hMN-14 x m679 F(ab')2 was radioiodinated with (Perkin Elmer Life Science, Inc. Boston, MA) by the chloramine-T method (20), and purified using centrifuged size-exclusion columns. Quality assurance testing found ⁇ 5% unbound radioiodine by ITLC, > 90% of the product migrating as a single peak by SE-HPLC (Bio-Sil SE 250, Bio Rad, Hercules, CA), and > 90% of the radiolabeled product shifting to a higher molecular weight with the addition of an excess of CEA (Scripps Laboratories, San Diego, CA).
  • 125j_ m M U - , ⁇ x m 679 bsMAb was tested in a similar manner, using a partially purified extract from GW-39 human colon xenografts as a source of CSAp, which shifted the elution profile of the mMu-9-x679 bsMAb to the void fraction of the SE-HPLC column.
  • Humanized MN-14 (hMN-14) Fab'-SH was prepared in a similar manner as described previously. 99m ⁇ c- ⁇ ertechnetate (30 mCi) was added directly to the lyophilized hMN-14- Fab'-SH (1.0 mg) and injected in animals within 30 minutes. This product had 3.0% unbound 99 ⁇ p c DV ULC and an immunoreactive fraction of 92%.
  • the divalent HSG-peptide, IMP 241 used for 90 Y-, 17?Lu- and 11 ⁇ n-radiolabeling contains a DOTA ligand to facilitate the binding of these radiometals.
  • IMP 241 was dissolved in 0.5 M ammonium acetate (pH 4.0) to a concentration of 2.2 x 10-3 jyf.
  • 90 YQ ⁇ was obtained from Perkin Elmer Life Sciences, Inc. (Boston, MA), fl ⁇ Cl3 from IsoTex Diagnostics (Friendswood. TX), and l ⁇ T u from the Research Reactor Facility, University of Missouri-Columbia, (Columbia, MO).
  • H I In-IMP 241 was prepared by mixing 3 mCi of I ⁇ InCl3 in a plastic conical vial with 0.5 M ammonium acetate, pH 4.0 (3x volume of 1 1 3 InCl3) and 2.3 ⁇ L of IMP 241 (2.2 xl0"3 jyf in 0.5 M ammonium acetate, pH 4.0), After centrifugation, the mixture was heated in a boiling water bath for 30 min and cooled. The mixture was centrifuged and DTPA was added to a final concentration of 3 mM. After 15 min at room temperature, the final volume was raised to 1.0 mL with 0.1 M sodium acetate, pH 6.5.
  • the amount of unbound isotope was determined by reverse phase HPLC and ITLC developed in saturated sodium chloride solution.
  • Reverse phase HPLC analyses were performed on a Waters 8 x 100 mm radial Pak cartridge filled with a C-18 Nova-Pak 4 ⁇ m stationary phase. The column was eluted at 1.5 mL/min with a linear gradient of 100 % A (0.075 % TFA in water) to 55 % A and 45 % B where B was 0.075 % of TFA in 75 % acetonitrile and 25 % water over 15 min. At 15 min, solvent was switched to 100%B and maintained there for 5 min before re-equilibration to initial conditions. Reverse HPLC analyses showed a single peak at 11.8 min. Analysis of 111 In-IMP 241 mixed with excess m679 IgG on a Bio-Sil SE 250 HPLC gel filtration column showed a peak at the retention time of the antibody indicating binding to the antibody.
  • IMP-241 was radiolabeled with 0 Y by adding to 15 mCi of 9 0YC13, 3-times the volume of 0.5 M ammonium acetate, pH 4.0 and 83.2 ⁇ L of IMP 241 (1.1 lQ- ⁇ M in 0.5 M ammonium acetate, pH 4.0), and ascorbic acid to a final concentration of 6.75 mg/mL. The mixture was heated in a boiling water bath for 30 min, and after cooling to room temperature, DTPA was added to a final concentration of 5 mM. Fifteen minutes later, the final volume was increased to 1.0 mL with 0.1 M sodium acetate, pH 6.5.
  • ITLC strips developed in saturated sodium chloride solution showed ⁇ 0.2 % unbound isotope.
  • Analysis of 9 ⁇ Y- IMP 241 mixed with an excess of m679 IgG by SE-HPLC showed a peak at the retention time of the antibody indicating binding to the antibody.
  • GW-39 a CEA-producing human colon cancer cell line (See, Goldenberg, D.M. and Hansen, H.J, Carcinoembryonic antigen present in human coionic neoplasms serially propagated in hamsters, Science, 175:1117-18 (1972)) was serially propagated in nude mice by mincing 1-2 grams of tumor in sterile saline, passing the minced mixture through a 50-mesh wire screen, and adjusting the saline volume to a final ration of 10 ml saline per gram tumor.
  • mice Female NCr nude mice (Charles River Laboratories, Inc., Fredrick MD or Taconic, Germantown, NY) approximately 6 weeks of age were implanted subcutaneously with 0.2 ml of this suspension. Two to three weeks after implantation of tumors, animals were injected with the radiolabeled peptide alone, or for pretargeting, with the bsMAb followed 1 to 2 days later with the radiolabeled peptide.
  • 1.5 x 10" l ⁇ moles (15 ⁇ g; 6 ⁇ Ci 125j) ⁇ f the bsMAb was injected intravenously (0.1 to 0.2 mL) followed with an intravenous injection (0.1 to 0.2 mL) of n ⁇ In-IMP-241 (1.5 x lO-H moles, 8-10 ⁇ Ci), 177L U -IMP-241 (1.5 x 10"H moles, 5 ⁇ Ci), or 99m Tc . ⁇ MP-243 (1.5 x 10- 11 , 25-30 ⁇ Ci).
  • 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.
  • IMP-241 's DOTA chelation group can be used with 1 ! fin, 90 ⁇ ; and omer radiometals, such as 17 u.
  • ⁇ h e peptide was radiolabeled with each of these radionuclides to specific activities of about 600, 1650, and 300 Ci mmol, respectively.
  • the lower specific activity for ⁇ 7L U was attributed to both the age of the product at the time it was used and the isotope production run that was not performed in a manner to optimize the specific activity of l ⁇ Lu.
  • the specific activity of the yy Tc- ⁇ eptides was between 1500 and 1600 Ci/mmol.
  • Figure 6 shows the binding of the hMN-14 x m679 bsMAb to 1 1 1 In-IMP-241 by SE-HPLC. Essentially all the radiolabeled peptide is shifted to the bsMAb elution time, and when CEA is first added to the bsMAb followed by the addition of the radiolabeled peptide, the entire amount of radioactivity shifts to the void fraction. Similar results were found with the mMu-9 x m679 bsMAb when using the CSAp preparation (not shown).
  • IMP-241 was radiolabeled with the gamma-emitting radionuclides, ⁇ ⁇ In or 177L U) t 0 facilitate the peptide's detection in tissues, while IMP-243 and IMP- 245 were radiolabeled with yym Tc.
  • the 1 7 ⁇ __ u - and * ⁇ lln-IMP-241 had 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.
  • an average kidney weight of 0.15 g there was only about 0.6% of the total injected activity in the kidney at 0.5 to 1.0 h after injection.
  • An additional group of animals given the ⁇ Lu-IMP-241 was necropsied at 48 h, but since there was only enough radioactivity in the kidneys for accurate reporting, the data are not presented in the table.
  • the T77 j --u-IMP-241 i n the kidneys had decreased to a level of 0.94 ⁇ 0.2 %ID/g, which represented about a 45% decrease compared to the level seen at 24 h.
  • the hMN-14 x m679 F(ab')2 bsMAb was used to test the pretargeting capabilities of the 99m ⁇ c . Mp_243 and 9 m ⁇ c -iMp_245.
  • the bsMAb was radiolabeled with 125 I so that its distribution could be co-registered with either the 99nv c .. ⁇ ] y rp.243 or IMP-245.
  • the bsMAb was given to animals i.v., and after 24 h, the radiolabeled peptide was given and animals were necropsied 3 and 24 hours later.
  • tumor uptake of the yym Tc-IMP-243 was nearly 28 and 70 times higher than that seen with peptide alone at 3 and 24 h after its injection (Table 23).
  • Tumor uptake was 12.25 ⁇ 3.32 % ID/g at 3 h, reducing to 7.36 ⁇ 3.19 by 24 h.
  • the reduction of 99n i Tc-IMP-243 in the tumor over this time was not as high as the reduction of the bsMAb in the tumor, which dropped from 4.78 ⁇ 1.11 %ID/g to 2.24 + 0.53 %ID/g over this same period.
  • IMP-243 were all greater than 2.0:1 within 3 hours, except for the large intestine where the peptide had not yet cleared, but this improved nearly 20-fold by 24 h.
  • Tumor/blood ratios were 2.4 ⁇ 0.6 at 3 h after peptide injection.
  • Tumor uptake for yym Tc-IMP-245 was similar to that seen with yym Tc-IMP- 243 (Table 6), but tumor/nontumor ratios favored the yym Tc-IMP-245, primarily because the bsMAb had cleared to a lower level in these animals than in the animals that had received the yym Tc-IMP-243.
  • tumor/kidney ratio using the yym Tc-labeled peptides was substantially higher than an antibody fragment directly radiolabeled with "mTc-hMN-14 Fab' 3 h after its injection (Table 25).
  • each bsMAb was prepared by chemically coupling its Fab' to the Fab' of the murine 679 MAb. For biodistribution studies, each bsMAb was radiolabeled with 125 ⁇ so j na t j ts distribution could be assessed together with the IMP-241, which was radiolabeled with ⁇ *In.
  • the amount of bsMAb and peptide injected in tumor-bearing nude mice was the same in each pretargeting system, but because the Mu-9 bsMAb took longer to clear from the blood than the hMN-14 bsMAb, the radiolabeled peptide was given at 48 h after the Mu-9 bsMAb compared to 24 h after the hMN-14 bsMAb.
  • the blood levels of each bsMAb were similar, 0.79 ⁇ 0.24 %ID/g and 0.55 ⁇ 0.10 %ID/g, respectively.
  • y ⁇ Y because of its higher beta-radiation energy (2,27 MeVr ⁇ a ), delivers a higher radiation dose to the tumor than f 7 7j_ u (495 keV max ) on a per mCi basis.
  • the radiation doses were normalized to reflect an identical radiation to a dose-limiting organ. In this case, 1500 cGy to the kidneys was selected as a dosage that should be tolerated, but could result in similar toxicities.
  • the absorbed doses to the tissues were normalized, the data suggest that 177LU-IMP-241 would potentially deliver the same dose to the tumor as y ⁇ Y-IMP-241. If the kidneys were able to tolerate 1500 cGy, then the tumor would receive nearly 12,000 cGy, a radiation dose that should be lethal to most solid tumors.
  • Additional references of interest include the following: Ara ⁇ Y, Uezono T, Akizawa H, Ono M, Wakisaka K, Nakayama M, Sakahara H, Konishi J,
  • Penefsky, H.S. A centrifuged column procedure for the measurement of ligand binding by beef heart FI . Part G. Methods En ⁇ ymo 1979; 56:527-530.

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.

Description

DRUG PRE-TARGETING BY MEANS OF BI-SPECIFIC ANTIBODIES AND HAPTEN CONSTRUCTS COMPRISING A CARRIER PEPTIDE AND THE ACTIVE AGENT ( S)
[0001 J 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 incoφorated 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 radioimmunodetection (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 arm 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
[0003] 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 rapid-y 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 αl, U.S. Patent No. 4,863,713), indium-I l l (βaxhet etαi, U.S. Patent No. 5,256,395 and U.S. Patent No. 5,274,076, Goodwin et αl., J. Nucl. Med., 33:1366-1372 (1992), and xanenborg et αl., 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 enantiomers o 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 yttrium-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 iodiπe-131, a non-metal, has been adopted for RAIT purposes by using an 1-131 -labeled indium-metal-chelate complex in the second targeting step. A second disadvantage to this methodology requires that antibodies be raised against every agent desired for diagnostic or therapeutic use.
[0006] 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, tifie 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 vascu/afure and binds to the primary targeting agent. Its small size may also encourage a more uniform distribution in the tumor.
[0007] Pretargeting methods have used a number of different strategies, but most often involve an avidin/streptavidin-biotin recognition system 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 biotin-conjugated antibody, followed by a bridging with streptavidin/avidin, and then the biotin-conjugated effector is given. These systems can be easily converted for use with a variety of effector substances so long as the effector and the targeting agent can be coupled with biotin or streptavidin/avidin depending on the configuration used. With its versatility for use in many targeting situations and high binding affinity between avidin streptavidin and biotin, this type of pretargeting has considerable advantages over other proposed systems. However, avidin and streptavidin are foreign proteins and therefore would be immunogenic, which would limit the number of times they could be given in a clinical application. In this respect, bsAbs have the advantage of being able to be engineered as a relatively non-immunogenic humanized protein. Although the binding affinity of a bsAb (typically 10~9 to 10"^υ M) cannot compete with the extremely high affinity of the streptavidin/avidin-biotin affinity (~10~^5 M), both pretargeting systems are dependent on the binding affinity of the primary targeting agent, and therefore the higher affinity of the streptavidin/avidin-biotin systems may not offer a substantial advantage over a bsAb pretargeting system. However, most bsAbs have only one arm available for binding the primary target, whereas the streptavidin/avidin-biotin pretargeting systems have typically used a whole IgG with two arms for binding the target, which strengthens target binding. By using a divalent peptide, an affinity enhancement is achieved, which greatly improves the binding of the peptide to the target site compared to a monovalent peptide. Thus, both systems are likely to provide excellent targeting ratios with reasonable retention.
[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 αl. that used an antibody directed against a histamine derivative, histamine-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 90γ, 111-Ln, and ^Hhu, as well as an alternative 99mTc-binding peptide.
[0009] Thus, there is a continuing need for imrnunological 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 that specifically binds a targetable construct that can be modified for use in a wide variety of diagnostic and therapeutic applications.
[0011] Other objects of the invention are to provide pre-targeting methods of diagnosis and therapy using the combination of multi-specific antibody and targetable construct, methods of making the multi- specifics, and kits for use in such methods.
[0012] In accomplishing the foregoing object, the present inventors have discovered that 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, the characteristics of the chelator, metal chelate complex, therapeutic agent or diagnostic agent can be varied to accommodate differing applications, without raising new multi-specific Abs for each new application. Further, by using this approach, two or more distinct chelators, metal chelate complexes, diagnostic agents or therapeutic agents can be used with the inventive multi-specific Ab. Summary of the Invention
10013] 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 other arm that specifically binds a targetable construct.
10014 ] Provided is a compound of the formula X-Phe-Lys(HSG)-D-Tyr-Lys[HSG)-Lys(Y)-W_2 (SEQ ID NO: 1), where the 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 soil acid cation chelator may include a fhiol group, and may also include such chelators as Tscg-Cys and Tsca-Cys. A preferred embodiment of this compound is DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)- NH2 (SEQ ID NO; 1) also known as IMP 245. Other 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(DOTA)-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 Ha and Group Ilia metal cations, which commonly bind to hard acid chelators. Soft acid cations that may bind to the soft acid chelators can include the transition metals, lanthanides, actinides and or Bi, Non exhaustive examples of such soft acid cations include Tc, Re, and
Bi.
[0016] 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 therapeutic or diagnostic agent or enzyme "R". The linker may have at least one amino acid for conjugating the R group to the compound. Examples of therapeutic agents include a drug, prodrug (e.g, epirubicin glucuronide, CPT-11. etoposide glucuronide, daunomicin glucuronide and doxorubicin glucuronide) or toxin (e.g., ricin, abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pse domonas exotoxin, άPseudomonas endotoxiη). [0017] Other examples of therapeutic agents include doxorubicin, SN-38, etoposide, methotrexate, 6- mercaptopurine and/or etoposide phosphate. Diagnostic agents may include nuclides, one or more agents for photodynamic therapy (e.g, a photosensitizer such as benzoporphyrin monoacid ring A (BPD-MA), tin etiopurpurin (SnET2), sulfonated aluminum phthalocyanine (AlSPc) and lutetium texaphyrin (Lutex)), contrast agents and image enhancing agents for use in magnetic resonance imaging (MRI) and computed tomography (CT). Enzymes may also serve as the R group which may be capable of converting a prodrug to a drug at the target site; or capable of reconverting a detoxified drug intermediate to a toxic form to increase toxicity of said drug at a target site.
[0018] In one embodiment, the invention provides a method of treating, diagnosing and/or identifying diseased tissues in a patient, comprising:
(A) administering to the patient 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) optionally, administering to the patient a clearing composition, and allowing the composition to clear non-focalized 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 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. [0019] 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 usefuf 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 ] Another 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 arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable 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.
/0022J 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- specifϊc fusion protein is under the control of the regulatory regions; (B) co-introducing into the host cell a recombinant DNA construct 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-speeiftc 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 Jinked to a light-chain antibody fragment, and a transcriptional and translational termination regulatory region functional in the first 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 in (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 5' 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 termination 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 the 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 zygote, 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 αliα 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 radioimmunoi-naging for positron-emission tomography (PET).
[0027] A further embodiment of the invention involves using the inventive antibody or antibody fragment in radioimmuπoimaging for single-photon emission.
[0028 J A further embodiment of the invention involves using the inventive antibody or antibody fragment in magnetic resonance imaging (MRI). J0029] 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 treating diseased tissues (e.g., cancers, infections, inflammations, clots, atherosclerois, infarcts), normal tissues (e.g., spleen, parathyroid, thymus, bone marrow), ectopic tissues (e.g., endometήosis), and pathogens.
[0033] Further, the invention provides pre-targeting methods of diagnosis and therapy using the combination of bi-specific antibody and the following targetable constructs:
(a) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 3);
(b) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 2);
(c) Ac-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 4);
(d) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 1);
(e) (Tscg-Cys)-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(DOTA)-NH (SEQ ID NO: 1);
Figure imgf000010_0001
(SEQ ID NO: 5)
Figure imgf000010_0002
(SEQ ID NO: 6)
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, witiiout raising new bsAbs 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 carrier 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, further administering to said subject
1) a prodrug, when said enzyme is capabie of converting sard 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 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 pathogen or on a molecule produced by or associated therewith; and administering a targetable construct selected from the group consisting of (a) DOTA-Phe- ys(HSG)-D-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 3); (b) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 2); (c) Ac-Lys(HSG)-D-Tyr-Lys(HSG)- ys(Tscg-Cys)-NH2 (SEQ ID NO: 4); (d) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 1); (e) (Tscg-Cys)-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(DOTA)-NH2 (SEQ ID NO: 1);
Figure imgf000012_0001
(SEQ ID NO: 5)
D-Ala-Lys(HSG)-D-Tyr-Lys(HSG)-NH2
Figure imgf000012_0002
(SEQ ID NO: 6)
[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 arm that specifically binds a targeted tissue and at least one other arm 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 construct selected from the group consisting of:
(a) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 3);
(b) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 2);
(c) Ac-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 4);
(d) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 1);
(e) (Tscg-Cys)-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(DOTA)-NH2 (SEQ ID NO: 1);
Figure imgf000013_0001
(SEQ ID NO: 5)
Figure imgf000013_0002
D-AIa-Lys(HSG)-D-Tyr-Lys(HSG)-NH2 H H
(SEQ ID NO: 6)
(003%] 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 other arm that specifically binds a targetable construct, wherein said construct is selected from the group consisting of
(a) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 3);
(b) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 2);
(c) Ac-Lys(HSG)-D-Tyr-Lys(HSG)- ys(Tscg-Cys)-NH2 (SEQ ID NO: 4);
(d) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 1);
(e) (Tscg-Cys)-Phe-Lys HSG)-D-Tyr-Lys(HSG)-Lys(DOTA)-NH2 (SEQ ID NO: i);
Figure imgf000013_0003
(SEQ ID NO: 5)
Figure imgf000014_0001
(SEQ ID NO: 6)
(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 capable 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.
[0039] The invention further relates to a targetable construct selected from the group consisting of:
(a) DOTA-Phe-Lys(HSG)-D-Tyr-Lys( SG)-NH2 (SEQ ID NO: 3);
(b) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 2);
(c) Ac-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 4);
(d) DOTA-Phe-Lys(HSG)-D-Tyr- ys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: I);
(e) (Tscg-Cys)-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(DOTA)-NH2 (SEQ ID NO: 1);
Figure imgf000015_0001
(SEQ ID NO: 5)
Figure imgf000015_0002
(SEQ ID NO: 6)
[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 ceils, 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
(a) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 3);
(b) DOTA-Phe~Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 2);
(c) Ac-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 4);
(d) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 1); (e) (Tscg-Cys)-Phe-Lys(HSG)-D-Tyr-Lys(nSG)-Lys(DOTA)-NH2 (SEQ ID NO: 1);
Figure imgf000016_0001
(SEQ ID NO: 5)
Figure imgf000016_0002
(SEQ ID NO: 6)
[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 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
(a) DOTA-Phe-Lys(HSG)-D-Tyr- ys HSG)-NH2 (SEQ ID NO: 3);
(b) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 2);
(c) Ac-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 4);
(d) DOTA-Phe-Lys(HSG)-D-Tyr- ys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 1);
(e) (Tsc -Cys)-Phe-Lys(HSG)-D-Tyr-Lys(HSG)- ys OTA)-NH2 (SEQ ID NO: 1);
Figure imgf000017_0001
(SEQ ID NO: 5)
Figure imgf000017_0002
(SEQ ID NO: 6)
(0043] The invention further relates to a method for the endoscopic 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
(a) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 3);
(b) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 2);
(c) Ac-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 4);
(d) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 1);
(e) (Tscg-Cys)-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(DOTA)-NH2 (SEQ ID NO: 1);
Figure imgf000018_0001
(SEQ ID NO: 5)
Figure imgf000018_0002
(SEQ ID NO: 6)
[0044] 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
(a) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 3);
(b) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 2);
(c) Ac-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 4);
(d) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 1);
(e) (Tscg-Cys)-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(DOTA)-NH2 (SEQ ID NO: 1);
Figure imgf000019_0001
(SEQ ID NO: 5)
D-AJa-Lys(HSG)-D-Tyr-Lys(HSG)-NH2
Figure imgf000019_0002
(SEQ ID NO: 6)
[0045] 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 1 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.
10050] Figure 5 illustrates the production of a DNA construct useful for producing a hMN-14Fab- 734scFv bi-specific fusion protein.
[0051] Figure 6 shows the binding properties of hMN-14 x m679 bsMAb with 11 ^α-Iabeled IMP-241 divalent HSG-DOTA peptide. Panel A: ι nIn-IMP-241 alone on SE-HPLC; Panel B: U lln-IMP-241 mixed with hMN-14 x 679 bsMAb; Panel C: 11 1In-IMP-241 added to a mixture containing hMN-14 x m679 bsMAb with an excess of CEA. Chromatograms show the association of the J l 1In-IMP-241 with the bsMAb (B) and bsMAb/CEA complex (C). [0052] Figure 7 shows clearance of ^Si-mMu-α x m679 F(ab')2 bsMAb and ll l-n_IMp-24-. in GW-39 tumor-bearing nude mice. Mice were injected i.v. with the radiolabeled bsMAb and 48 h later the radiolabeled peptide was given i.v. Values represent the mean and standard deviations of the percent injected dose per gram (n =5 for each time interval). Detailed Description of Preferred Embodiments [0053] Unless otherwise specified, "a" or "an" means "one or more". I. Overview
[0054] The present invention provides a bi-specific antibody (bsAb) or antibody fragment (bsFab) having at least one arm that is reactive against a targeted tissue and at least one other arm that is reactive against a targetable construct. Desirably, the targetable construct includes a peptide having at least 2 units of a recognizable hapten. Examples of recognizable haptens include, but are not limited to, histamine succinyl glycine (HSG) and fluorescein isothiocyanate. The targetable construct may be conjugated to a variety of agents useful for treating or identifying diseased tissue. Examples of conjugated agents include, but are not limited to, chelators, metal chelate complexes, drugs, toxins (e.g., ricin, abrin, ribonuclease (e.g., RNase), DNase I, Stαphylococcαl enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonαs exotoxiπ, Pseudomonαs endotoxin) and other effector molecules. Additionally, enzymes useful for activating a prodrug or increasing the target-specific toxicity of a drug can be conjugated to the targetable construct. Thus, the use of bsAb which are reactive to a targetable construct allows a variety of therapeutic and diagnostic applications to be performed without raising new bsAb for each application. [0055] Bi-specific antibody (bsAb) pretargeting represents a potentially non-immunogenic, highly selective alternative for diagnostic and therapeutic applications. The bsAb pretargeting system described herein represents an additional significant advantage over other pretargeting systems in that it potentially can be developed for use with a variety of different imaging or therapeutic agents. The flexibility of this system is based on use of an antibody directed against histamine-succinyl-glycl (HSG) and the development of peptides containing the HSG residue. HSG-containing peptides were synthesized with either DOTA for the chelation of 111 In, 90γ, or 177l,u or a technetium/rhenium chelate. For pretargeting, these peptides were used in combination with bi-specific antibodies using the anti-HSG Fab' chemically stabilized with the Fab' of either an anti-carcinoembryonic antigen (CEA) or an anfi-co/on-specific antigen-p (CSAp) antibody to provide tumor targeting capability for tumors expressing these antigens. However, other antigen targets may include diverse tumor-associated antigens known in the art, such as against CD19, CD20, CD21, CD22, CD23, CD30, CD74, CD SO, HLA-DR, la, MUC 1, MUC 2, MUC 3,
MUC 4, EGFR. HER 2/neu, PAM-4, BrE3, TAG-72 (B72.3, CC49), EGP-1 (e.g., RS7), EGP-2 (e.g., 17- 1A and other Ep-CAM targets), Le(y) (e.g., B3), A3, KS-1, Si 00, IL-2, T101, necrosis antigens, folate receptors, angiogenests markers (e.g., VEGF), tenascin, PSMA, PSA, tumor-associated cytokines, MAGE and/or fragments thereof. Tissue-specific antibodies (e.g., against bone marrow 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/nontumor 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 99mτc_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 combination with the 11 ^In-labeled peptide, radiation dose estimates for therapeutic radionuclides, such as 90γ and l^Lu, 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.
[0057/ 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 ami that specifically 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 virus, hepatitis B virus, Sendai virus, 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 virus, rubella virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus and blue tongue virus), parasites, bacteria (e.g., Anthrax bacillus, Streptococcus agaiacti'ae, Legioneila pneumophifia, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum, Ly e disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis and Tetanus toxin), mycoplasma (e.g., Mycoplas a arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarum, and M. pneumoniae) and protozoans (e.g., Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japanicum, Babesia bovis, Elmeria tenella, 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.
[0058] 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)2, 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.
[0059] The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments, "Fv" fragments, consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker ("sFv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the "hypervariable region." Three of these so-called "hypervariable" regions or "complementarity-determining 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 the CDRs accounts for the diversity of specificity of antibodies.
[0060] As used herein, the term "subject" refers to any animal (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, turkeys, 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. II. Constructs Targetable to Antibodies
[0061] The targetable construct can be of diverse structure, but is selected not only to diminish the eJicitatiøfi of immune responses, but also for rapid in vivo clearance when used within the bsAb targeting method. Hydrophobie agents are best at eliciting strong immune responses, whereas hydrophilic agents are preferred for rapid in vivo clearance, thus, a balance between hydrophobie and hydrophilic needs to be established. This is accomplished, in part, by relying on the use of hydrophilic chelatiπg agents to offset the inherent hydrophobicity of many organic moieties. Also, sub-units of the targetable construct may be chosen which have opposite solution properties, for example, peptides, which contain amino acids, some of which are hydrophobie and some of which are hydroprji.ic. Aside from peptides, carbohydrates may be used. [0062] The targetable construct may include a peptide backbone having as few as two amino-acid residues, with preferably two to ten amino acid residues, and may be coupled to other moieties such as chelating agents. The targetable construct should be a low molecular weight construct, preferably having a molecular weight of less than 50,000 daltons, and advantageously less than about 20,000 daltons, 10,000 daltons or 5,000 daltons, including any metal ions that may be bound to the chelating agents. For instance, the known peptide DTPA-Tyr-Lys(DTPA)-OH (wherein DTPA is diethylenetriaminepentaacetic acid) has been used to generate antibodies against the indiu -DTPA portion of the molecule. However, by use of the noπ-indium-containing molecule, and appropriate screening steps, new Abs against the tyrosyl-lysine dipeptide can be made. More usually, the antigenic peptide of the targetable construct will have four or more residues, such as the peptide DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-ΗH2 (SEQ ID NO: 2), wherein DOTA is 1 ,4,7,10-tetraazacyclododecanetetraacetic acid and HSG is the histamine succinyl glycyl group of the formula:
Figure imgf000023_0001
The non-metal-containing peptide may be used as an immunogen, with resultant Abs screened for reactivity against the Phe-Lys-Tyr-Lys (SEQ ID NO: 2) backbone.
[00631 The haptens of the targetable construct also provide an immunogenic recognition moiety, for example, a chemical hapten. Using a chemical hapten, preferably the HSG hapten, high specificity of the construct for the antibody is exhibited. This occurs because antibodies raised to the HSG hapten are known and can be easily incorporated into the appropriate bsAb. Thus, binding of the haptens to the peptide backbone would result in a targetable construct that is specific for the bsAb or bsFab.
[0064J The invention also contemplates the incorporation of unnatural amino acids, e.g., D-amino acids, into the peptide backbone structure to ensure that, when used with the final bsAb/construct system, the arm of the bsAb which recognizes the targetable construct is completely specific. The invention further contemplates other backbone structures such as those constructed from non-natural amino acids and peptoids.
|0065| 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. III. 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 issue.
[0067] Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with 47sc, 52pe> 55Cθj 67Ga> 68Gaj l lljnj 8 ^ 90Y) 161τ», 177Lu> 212^, 213βi, and 225 Ac for radio-imaging and RAIT. The same chelators, when complexed with non- radioactive metals such as Mn, Fe and Gd for use with MRI, when used along with the bsAbs of the invention. Macrocyclic chelators such as NOTA (l,4,7-triaza-cyclonσnaπe-N,N',N"-triacetic acid), 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-rype 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 Ila and Group Ilia 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 pβlyethers are of interest for stably binding nuclides such as 223 Ra 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.
[0069] Particularly useful diagnostic radionuclides that can be bound to the chelating agents of the targetable construct include, but are not limited to, ] ] °Iπ, ] * J In, ] 77Lu, 1 $F, 52Fe, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 86γ, 90γ, 89^ 94mTc, 94Tc, 99mTc, 1 0 123τ, 124It 125τ, 131I; 154-158Gd, 32p, 11C, 13N, 15O, 186Re> 188Re> 51Mn> 52mMllι 55c0, 72As> 75Br> 76βr, 82mRt>, °3Sr, or other gamma-, beta-, or positron-emitters. Preferably, the diagnostic radionuclides include a decay energy in the range of 25 to 10,000 keV, more preferably in the range of 25 to 4,000 keV, and even more preferably in the range of 20 to 1,000 keV, and still more preferably in the range of 70 to 700 keV. Total decay energies of useful positron-emitting radionuclides are preferably < 2,000 keV, more preferably under 1 ,000 keV, and most preferably < 700 keV. Radionuclides useful as diagnostic agents utilizing gamma-ray detection include, but are not limited to: Cr-51, Co-57, Co-58, Fe-59, Cu-67, Ga-67, Se-75, Ru-97, Tc-99m, In-111, In- 1 24m, 1-223, 1-125, 1-131, Yb-169, Hg-297, and Tϊ-201. Decay energies of useful gamma-ray emitting radionuclides are preferably 20-2000 keV, more preferably 60-600 keV, and most preferably 100-300 keV. [0070] Particularly useful therapeutic radionuclides that can be bound to the chelating agents of the targetable construct include, but are not limited to 1 Hn, l77Lu, 212Bi, 13Bi, 211 A , 62cu, 64Cu, 67Cu, 90Yj 125ι, 131ι, 32P, 33P, 47SCj l l lAg, 67Ga; 142Pr, ]53Sm, 161τb, 166Dy) l66Hθj 186Re, 188Re, !S9Re, 212Pb) 223Ra> 225Ac, 59 e, 75Se> 77ASj 89Sr, 99Mo, 105^ 109Pd, 143ft., 149Pπi) 169E^ ι-.4ιr? 198 U, 199 u, and 1 lpb. The therapeutic radionuclide preferably has a decay energy in the range of 25 to 10,000 keV. Decay energies of useful beta-particle-emitting nuclides are preferably 25-5,000 ke V, more preferably 200-4,000 keV, and most preferably 500-2,500 keV. Also preferred are radionuclides that substantially decay with Auger-emitting particles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-1 19, 1-125, Ho-161, Os-189m and Ir-192. Decay energies of useful beta-particle-emitting nuclides are preferably < 1 ,000 ke V, more preferably < 100 keV, and most preferably < 70 keV. Also preferred are radionuclides that substantially decay with generation of alpha- particles. Such radionuclides include, but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-21 1, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies of useful alpha-particle- emitting radionuclides are preferably 2,000-9,000 keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.
[0071 J Chelators such as those disclosed in U.S. Patent 5,753,206, especially thiosemi- carbazonylglyoxylcysteine(Tscg-Cys) and thiosemicarbazinyl-acetylcysteine (Tsca-Cys) chelators are advantageously used to bind soft acid cations of Tc, Re, Bi and other transition metals, lanthanides and actiπides that are tightly bound to soft base ligands, especfatfy suhur- or phosphorus-containing ligands. fi can be useful to link more than one type of chelator to a peptide, e.g., a hard acid chelator like DTPA for In(III) cations, and a soft acid chelator (e.g, thiol-containing chelator such as Tscg-Cys) for Tc cations. Because antibodies to a di-DTPA hapten are known (Barbet '395, supra) and are readily coupled to a targeting antibody to form a bsAb, it is possible to use a peptide hapten with cold di-DTPA chelator and another chelator for binding a radioisotope, in a pretargeting protocol, for targeting the radioisotope. One example of such a peptide is Ac-Lys(DTPA)-Tyr-Lys(DTPA)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 7). This peptide can be preloaded with In(III) and then labeled with 99-m-Tc cations, the In(III) ions being preferentially chelated by the DTPA and the Tc cations binding preferentially to the thiol-containing Tscg- Cys. Other hard acid chelators such as NOTA, DOTA, TETA and the like can be substituted for the DTPA groups, and Mabs specific to them can be produced using analogous techniques to those used to generate the anti-di-DTPA Mab. J0072J Jt will be appreciated that two different hard acid or soft acid chelators can be incoφorated into 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:
(a) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 3);
(b) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 2);
(c) Ac-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 4);
(d) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 1);
(e) (Tscg-Cys)-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(DOTA)-NH (SEQ ID NO: 1);
Figure imgf000026_0001
(SEQ ID NO: 5)
Figure imgf000026_0002
(SEQ ID NO: 6)
[0074] The chelator-peptide conjugates (f) and (g), above, has been shown to bind °°Qa md js thus useful in positron emission tomography (PET) applications.
[0075] 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(HSO)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ JD 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 "F oc" used herein refer to the groups allyloxycarbonyl and fluorenylmethyloxy 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 Aloe group was then removed. The peptide synthesis was then continued on the synthesizer to make the following peptide: Lys(Aloc)-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 peptide was then treated with activated N-trityl-HSG-OH until the resin gave a negative test for amines using the Kaiser test. See Karacay et al. Bioconjugate Chem. 77:842-854 (2000). The synthesis of Ac- Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 4), as well as the syntheses of DOTA- Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 3); DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 2); DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 1), are described in greater detail below.
IV. General Methods for Preparation of Metal Chelates
[0076] 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 up 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 ma be chelated therein. For example, even in the presence of an excess of cold * 1 llnC-3, labeling with 99m-Tc(V) glucoheptonate or with Tc cations generated in situ with stannous chloride and Na99m-Tcθ4 proceeds quantitatively on the soft acid chelator.
[0078] Other soft acid cations such as 186Re, 188Rβι 13gj and divalent or trivalent cations of Mn, Co, Ni, Pb, Cu, Cd, Au, Fe, Ag (monovalent), Zn and Hg, especially 64Cu and 6 GUι and the like, some of which are useful for radioimmunodetection or radioimmunotherapy. can be loaded onto the linker peptide by analogous methods. Re cations also can be generated in situ from perrhenate and stannous ions or a prereduced rhenium glucoheptonate or other transchelator can be used. Because reduction of perrhenate requires more stannous ion (typically above 200 μg/mL final concentration) than is needed for the reduction of Tc, extra care needs to be taken to ensure that the higher levels of stannous ion do not reduce sensitive disulfide bonds such as those present in disulfide-cyclized peptides. During radiolabeling with rhenium, similar procedures are used as are used with the Tc-99m. One method for the preparation of ReO metal complexes of the Tscg-Cys- ligands is by reacting the peptide with eOCl3(P(Ph3)2 but it is also possible to use other reduced species such as ReO(ethylenediamine)2. V. Methods of Administration
[0079] It should be noted that much of the discussion presented hereinbelow focuses on the use of the inventive bi-specific antibodies and targetable constructs in the context of treating diseased tissue. The invention contemplates, however, the use of the inventive bi-specifϊc antibodies and targetable constructs in treating and/or imaging normal tissue and organs using the methods described in U.S. Patent Nos. 6,126,916; 6,077,499; 6,010,680; 5,776,095; 5,776,094; 5,776,093; 5,772,981; 5,753,206; 5,746,996; 5,697,902; 5,328,679; 5,128,119; 5,101,827; and 4,735,210, which are incorporated herein by reference. As used herein, the term "tissue" refers to tissues, including but not limited to, tissues from the ovary, thymus, parathyroid, bone marrow or spleen. An important use when targeting normal tissues is to identify and treat them when they are ectopic (i.e., displaced from 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 timing 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 form or by administering one after the other.
J0081] 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 fiuorochromes 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. Addiffona- diagnostic agents can include fluorescent labeling compounds such as fluorescein isothiocyanate, rhodamine, phycoerytherin, 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. Radionuclides can also be used as diagnostic and/or therapeutic agents, including for example, 90γ, 1 Hln, 131l, 99mτc. ISdRe, l88Re> 277Lu, 67Cu> 232Bf, 2.3Bi, and 21 -At.
[0082] Therapeutic agents also include, for example, chemotherapeutic drugs such as vinca alkaloids, anthracyclines, epidophyllotoxinw, taxanes, antimetabolites, alkylating agents, antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic and apoptotoic agents, particularly doxorubicin, methotrexate, taxol, CPT-11, camptothecans, and others from these and other classes of anticancer agents. Other useful therapeutic agents for the preparation of immunoconjugates and antibody fusion proteins include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2 inhibitors, pyrimidine analogs, purine analogs, platinum coordination complexes, hormones, and the like. Suitable therapeutic agents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as well as revised editions of these publications. Other suitable therapeutic agents, such as experimental drugs, are known to those of skill in 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 the 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 keV gamma photons produced during positron annihilation decay are detected. X-ray, computed tomography (CT), MRI and gamma imaging (e.g., Single Photon Emission Computed Tomography (SPECT)) may also be utilized through use of a diagnostic agent that functions with these modalities. (0084] As discussed earlier, the targetable construct may include radioactive diagnostic agents that emit 25-10,000 keV gamma-, beta-, alpha- and auger- particles and/or positrons. Examples of such agents include, but are not limited to 18p, 52pe, 2cu, 64cu, 67Cu, 67Qa, 68Ga, 86Y, 89zr, 94mτc.94χC) 99mTc, 111 In, 123^ 124τ, 125 131 154-158Gd and 175Lu,
[0085] The present bsAbs or bsFabs can be used in a method of photodynamic therapy (PDT) 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 dihematoporphyrin 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 are not limited to, benzopoiphyrin 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, systemically, 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 αl. Chest 76:32 (1979). In another example, the antibodies and antibody fragments can be used in single photon emission. For example, a Tc- 99m-labeled diagnostic agent can be administered to a subject following administration of the inventive antibodies or antibody fragments. The subject is then scanned with a gamma camera which produces single- photon emission computed tomographic images and defines the lesion or tumor site. |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 photoradiatioπ, phototherapy, or photodynamic therapy (Jori et al. (eds.), Photodynamic Therapy of Tumors and Other Diseases (Libreria Progetto 1985); van den Bergh, Chem. Britain 22:430 (1986)). Moreover, monoclonal antibodies have been coupled with photoactivated dyes for achieving phototherapy. Mew et al., J. Immunol. 130:1473 (1983); idem., Cancer Res. 45:4380 (1985); Oseroff e. al, Proc. Natl Acad. Sci. USA 83:8744 (1986); idem., Photochem. Photobiol 46:83 (1987); Hasan et al, Prog. Clin. Biol. Res. 288:471 (1989); Tatsuta et al, Lasers Surg. Med. 9:422 (1989); Pelegrin et al, Cancer 67:2529 (1991). However, these earlier studies did not include use of endoscopic therapy applications, especially with the use of antibody fragments or subfragments. Thus, the present invention contemplates the therapeutic use of immunoconjugates comprising photoactive agents or dyes. [0088] Radiopaque and contrast materials are used for enhancing X-rays and computed tomography, and include iodine compounds, barium compounds, gallium compounds, thallium compounds, etc. Specific compounds 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, and thallous chloride. Ultrasound contrast material may also by used including dextran and liposomes, 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 art. As used herein, the term "immunomodulator" includes cytokines, stem cell growth factors, lymphotoxins, such as tumor necrosis factor (TNF), and hematopoietic factors, such as interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12 and IL-18), colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)), interferons (e.g., interferons-α, -β and -γ), the stem cell growth factor designated "SI factor," erythropoietin and thrombopoietin. Examples of suitable immunomodulator moieties include IL-2, IL-6, IL-10, IL-12, IL-18, interferon-γ, TNF-α, and the like.
[0089] The targetable construct may also be conjugated to an enzyme capable of activating a drug/prodrug at the target site or improving the efficacy of a normal therapeutic by controlling the body's detoxification pathways. Following administration of the bsAb, an enzyme conjugated to the targetable 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 drug may be one which is detoxified by the mammaVs 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. [0090] 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 uπconjugated 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.
|0092] Conversion of certain toxic substances such as aromatic or alicyclic alcohols, thiols, phenols and amines to glucuronides in the liver is the body's method of detoxifying them and making them more easily excreted in the urine. One type of antitumor drug that can be converted to such a substrate is epirubicin, a 4-epimer of doxorubicin (Adriamycin), which is an anthracycline glycoside and has been shown to be a substrate for human beta-D-glucuronidase See, e.g., Arcamone Cancer Res. 45:5995 (1985). Other analogues with fewer polar groups are expected to be more lipophilic and show greater promise for such an approach. Other drugs or toxins with aromatic or alicyclic alcohol, thiol or amine groups are candidates for such conjugate formation. These drugs, or other prodrug forms thereof, are suitable candidates for the site-specific enhancement methods of the present invention.
[0093] The prodrug CPT- 11 (irinotecan) is converted in vivo by carboxylesterase to the active metabolite SN-38. One application of the invention, therefore, is to use a bsAb targeted against a tumor and a hapten (e.g. di-DTPA) followed by injection of a di-DTPA-carboxylesterase conjugate. Once a suitable tumor-to- background localization ratio has been achieved, the CPT-11 is given and the tumor-localized carboxylesterase serves to convert CPT-11 to SN-38 at the tumor. Due to its poor solubility, the active SN-38 will remain in the vicinity of the tumor and, consequently, will exert an effect on adjacent tumor cells that are negative for the antigen being targeted. This is a further advantage of the method. Modified forms of carboxylesterases have been described and are within the scope of the invention. See, e.g., Potter et αl, Cancer Res. 58:2646-2651 (1998) and Potter et al, CancerRes. 55:3627-3632 (1998). }0094] Etoposide is a widely used cancer drug that is detoxified to a major extent by formation of its glucuronide and is within the scope of the invention. See, e.g., Hande etαl CancerRes. 45:1829-1834 (1988). Glucuronide conjugates can be prepared from cytotoxic drugs and can be injected as therapeutics for tumors pre-targeted with mAb-glucuronidase conjugates. See, e.g., Wang et al. CancerRes. 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 giucuronidases. See, e.g., Bakina 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- 11 and carboxypepti ase; 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. f0095[ 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 alternatively be conjugated to the hapten. The enzyme-hapten conjugate is administered to the subject following administration of the pretargeting 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 recycling the detoxified drug. This approach can be adopted for use with any enzyme-drug pair. [0096J 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 1 "B atoms. BNCT is based on the nuclear reaction which occurs when a stable isotope, isotopically enriched 10β (present in 19.8% natural abundance), is irradiated with thermal neutrons to produce an alpha particle and a ?Li 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 ^B 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 purposes of the present invention.
[0098] In another embodiment of the present invention, the peptide backbone of the targetable 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 targetable construct comprising a prodrug, for example poly-glutamic acid (SN-38-ester)ι o> is given, thereby localizing the prodrug specifically at the tumor target. It 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)iQ by cleaving the ester bond of the poly-glutamic acid (SN-38-ester)ι o releasing large concentrations of free SN-38 at the tumor. Alternatively, the appropriate enzyme also can be targeted to the tumor site. [0099] After cleavage from the targetable construct, the drug is internalized by the tumor cells. Alternatively, the drug can be internalized as part of an intact complex by virtue of cross-linking at the target. The targetable construct can induce intemalization of tumor-bound bsAb and thereby improve the efficacy of the treatment by causing higher levels of the drag to be internalized. |0100] A-variety of peptide carriers-are-well-suited for conjugation to prodrugs, including polyamino acids, such as poly lysine, polyglutamic (E) and aspartic acids (D), including D-amino acid analogs of the same, co-polymers, such as poly(Lys-Glu) [polyfKE]}, advantageously from 1:10 to 10:1. Copolymers based on amino acid mixtures such as poly(Lys-Ala-Glu-Tyr (SEQ ED 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 hydroxyl groups, to generate species such as SN-38-O-CO(CH2)2CO-O-PEG-0-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 of PEG derivatives and the limited loading capacity due to their dimeric functionality led to the preparation of PEG co-polymers having greater hapten-bearing capacity such as those described by Poiani et αl. See, e.g., Poiani et αl. Bioconjugαte 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 carry recognition haptens and prodrugs include N-(2- hydroxypropyl)methacrylamide (HMPA) copolymers, poly(styrene-co-maleic acid anhydride (SMA), poly(divinylether aleic anhydride) (DIVEMA), polyethyleneimine, ethoxylated polyethylene-imine, starburst dendriniers and olyfN-vinylpyrrolidone) (PVP), 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-ethyl-3-(3-dimethyIaminopropyl)carbodiimide hydrochloride (EDC). 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-I1 (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 carboxylate- 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 tree carboxylate groups can be activated in 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 bis-[2-(succinimido- oxycarbonyloxy)ethyl]sulfone (BSOCOES, Molecular Biosciences, Huntsville, AL), to cross-link the two amines as two amides after reaction with the bis(succinimidy_) 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 esterases. Doxorubicin also can be conjugated to a variety of peptides, for example, HyBnK(DTPA)YK(DTPA)-NH2, using established procedures (HyBn= P-H2NNHC6Η CO2H). See Kaneko etal, J. Bwconjugate Chem., 2: 133-141, 1991.
[0105] In one preferred embodiment, the therapeutic conjugate comprises doxorubicin coupled to a carrier compnsmg amine residues and a chelating agent, such as DTPA, to form a DTPA-peptide- doxorubicin conjugate, wherein the DTPA forms the recognition moiety for a pretargeted bsAb. Preferably, the earner comprises a tyrosyl-lysme dipeptide, e.g., Tyr-Lys(DTPA)-NH2, and more preferably still it compπses Lys(DTPA)-Tyr-Lys(DTPA)-NH2. Doxorubicin phenyl hydrazone conjugates to bis-DPTA containing peptides are particularly desirable in a therapeutic context. (0106] Methotrexate also has an available ammo group for coupling to acti vated carboxylate-contaming polymers, in a similar manner to that described for doxorubicin. It also has two glutamyl carboxyl groups (alpha and gamma) that can be activated for coupling to amino-group containing polymers. The free carboxylate groups of methotrexate can be activated in situ (EDC) and the activated drug mixed with an amino-contai ng polymer to directly attach the drug to the side-chains of the polymer via amide bonds. Excess unreacted or cross-reacted drug is separated readily from the polymer-drug conjugate using size- exclusion or ion-exchange chromatography.
[0107] Maytansinoids and calicheamicins (such as esperamycm) contain mixed di- and tπ-sulfide bonds that can be cleaved to generate species with a single thiol useful for chemical manipulation. The thiomaytensmoid or tluoespera-mycin is first reacted w th a cross-linking agent such as a maleimido- peptide that is susceptible to cleavage by peptidases. The C-terminus of the peptide is then activated and coupled to an amino-containing polymer such as polylysine.
[0108] In still other embodiments, the bi-specific antibody-directed delivery of therapeutics or prodrug polymers to m vivo targets can be combined with bi-specific antibody delivery of radionuclides, such that combination chemotherapy and radioimmunotherapy is achieved. Each therapy can be conjugated to the targetable construct and administered simultaneously, or the nuclide can be given as part of a first targetable construct and the drug given in a later step as part of a second targetable construct. In one simple embodiment, a peptide containing a single prodrug and a single nuclide is constructed. For example, the tπpepbde Ac-Glu-Gly-Lys-NHj can be used as a carrier portion of a targetable construct, whereby SN-38 is attached to the gamma glutamyl carboxyl group as an aryl ester, while the chelate DOTA is attached to the epsilon amino group as an amide, to produce the complex Ac-Glu(SN-38)-Gly- Lys(DOTA)-NH2- The DOTA chelate can then be radiolabeled with various metals for imaging and therapy purposes including In-111, Y-90, Sm-153, Lu-177 and Zr-89. As the metal-DOTA complex may represent the recognizable hapten on the targetable construct, the only requirement for the metal used as part of the DOTA complex is that the secondary recognition antibody also used recognizes that particular metal-DOTA complex at a sufficiently high affinity. Generally, this affinity (log Kg) is between 6-11. Polymeπc peptides such as ρoly[Glu(SN-38)ι ø-Lys(Y-90-DOTA)2- can be given as readily as the more chemically defined lower MW reagent above, and are indeed preferred. Also, triply substituted polymers can be used, such as poly[Glu(Sn-38)ιo-Lys(Y-90-DOTA)n(histamine-succinate)m, where n and m are integers, such that the recognition agent is independent of the radioimmunotherapy agent. The prodrug is activated by carboxylesterases present at the tumor site or by carboxylesterases targeted to the site using a second targetable construct.
[0109] Alternatively, a combination therapy can be achieved by administering the chemotherapy and radioimmunotherapy agents in separate steps. For example, a patient expressing CEA-tumors is first administered bsAb with at least one arm which specifically binds CEA and at least one other arm which specifically binds the targetable construct whose hapten is a conjugate of yttrium-DOTA. Later the patient is treated with a targetable construct comprising a conjugate o yttήum-DOTA-beta-glucuronidase. After sufficient time for bsAb and enzyme localization and clearance, a second targetable construct, comprising Ac-GIu(SN-38)-Gly-Lys(Y-90-DOTA)-NH2, is given. The second targetable construct localizes to the tumor by virtue of bsAb at the tumor that are not already bound to a first targetable construct. First targetable constructs which are localized to the target site act on the Ac-Glu(SN-38)-Gly-Lys(Y-90- DOTA)-NH2 to liberate the free SN-38 drug. Localization of both the prodrug and its respective enzyme to the target site enhances the production of active drug by ensuring that the enzyme is not substrate limited. This embodiment constitutes a marked improvement of current prodrug methodologies currently practiced in the art.
[0110] Another advantage of administering the prodrug-polymer in a later step, after the nuclide has been delivered as part of a previously given targetable construct, is that the synergistic effects of radiation and drug therapy can be manipulated and, therefore, maximized. It is hypothesized that tumors become more eaky' after RAIT due to radiation damage. This can allow a polymer-prod ug to enter a tumor more completely and deeply. This results in improved chemotherapy.
[0111] Alternatively, the RAIT therapy agent can be attached to bsAb rather than to the targetable construct. For example, an anti-CEA x anti-DTPA bsAb conjugated to Y-90-DOTA is administered first to a patient with CEA-expressing tumors. In this instance, advantage is taken of the selectivity of certain anti-chelate mabs in that an anti-indium-DTPA antibody do not bind to a yttrium-DOTA chelate. After the Y-90-DOTA-anti-CEA x anti-indium-DTPA has maximized at the tumor and substantially cleared non- target tissue, a conjugate of indium-DTPA-glucuronidase is injected and localized specifically to the CEA tumor sites. The patient is then injected with a polymer-prodrug such as poly(Glu)(SN-38)ιo. The latter is cleaved selectively at the tumor to active monomeric SN-38, successfully combining chemotherapy with the previously administered RAIT.
[0112] It should also be noted that a bi-specific antibody or antibody fragment can be used in the present method, with at least one binding site specific to an antigen at a target site and at least one other binding site specific to the enzyme component of the antibody-enzyme conjugate. Such an antibody can bind the enzyme prior to injection, thereby obviating the need to covalently conjugate the enzyme to the antibody, 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.
[0113] It should also be noted that the invention also contemplates the use of multivalent target binding proteins which have at least three different target binding sites as described in Patent Appl. Serial No. 60/220,782. Multivalent target binding proteins have been made by cross-linking several Fab-like fragments via chemical linkers. See U.S. Patent Nos. 5,262,524; 5,091,542 and Landsdorp et al, Euro. J. Immunol 16: 679-83 (1986). Multivalent target binding proteins also have been made by covalently linking several single chain Fv molecules (scFv) to form a single polypeptide. See U.S. PatentN ' o. 5,892,020. A multivalent target binding protein which is basically an aggregate of scFv molecules has been disclosed in U.S. Patent Nos. 6,025,165 and 5,837,242, A bivalent target binding protein comprising three scFv molecules has been described in Krott et αl, Protein Engineering 10(4): 423-433 (1997). [0114] A clearing agent may be used which is given between doses of the bsAb and the targetable construct. The present inventors have discovered that a clearing agent of novel mechanistic action may be used with the invention, namely a glycosylated anti-idiotypic Fab' fragment targeted against the disease targeting arm(s) of the bsAb. Anti-CEA (MN-14 Ab) x anti-peptide bsAb is given and allowed to accrete in disease targets to its maximum extent. To clear residual bsAb, an anti-idiotypic Ab to MN-14, termed WI2, is given, preferably as a glycosylated Fab'' fragment. The clearing agent binds to the bsAb in a monovalent manner, while its appended glycosyl residues direct the entire complex to the liver, where rapid metabolism takes place. Then the therapeutic or diagnostic agent which is associated with the targetable construct is given to the subject. The WI2 Ab to the MN-14 arm of the bsAb has a high affinity and the clearance mechanism differs from other disclosed mechanisms (see Goodwin et αl, ibid), as it does not involve cross-linking, because the WI2-Fab' is a monovalent moiety.
[0115] In accordance with yet another aspect of the present invention, the present invention provides a kit suitable for treating or identifying diseased tissues in a patient, comprising 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, a first targetable construct which comprises a carder 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, optionally, a clearing composition useful for clearing non-localized antibodies and antibody fragments. The kit may optionally contain a prodrug when the first targetable construct comprises an enzyme capable of converting the prodrug to a drug at the target site, an enzyme that is capable of reconverting a detoxified intermediate of a drug to a toxic form, and, therefore, of increasing the toxicity of the drug at the target site, or an enzyme capable of reconverting a prodrug which is activated in the patient through natural processes and is subject to detoxification by conversion to an intermediate of lower toxicity from the detoxified intermediate to a toxic form, and, therefore, of increasing the toxicity of the drug 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 identifying or treating diseased tissue also can be included in the kit. Examples include, but are not limited 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, intraarterially, 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 dextraπ 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
[0117] 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^- LH, wherein KLH is keyhole limpet hemocyanin, and n=l-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 supernatants of the resulting clones analyzed for anti-peptide 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., In(III)-DTPA chelates. Monoclonal mouse antibodies to an In(III)-di- DTPA are known (Barbet '395 supr ).
[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 markers are those disclosed by Herberman, "Immunodiagnosis of Cancer", in Fleisher ed., "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 incoφorated herein by reference. [011 | Tumor-associated markers have been categorized by Herberman, supra, in a number of categories including oncofetal antigens, placeπtal antigens, oncogenic or tumor virus associated antigens, tissue associated antigens, organ associated antigens, ectopic hormones and normal antigens 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 receptors (e.g., folate receptor, EGFR), of transmembrane antigens (e.g., PSMA), 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, CD21, 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 targeted 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-4, EGP-1. EGP-2, BrE3, PAM-4, KC-4, A3, KS-1, PSMA, PSA, tenascin, T101, S100, MAGE, HLA-DR, CD 19, CD20, CD22, CD30, and CD74.
[0120] Another marker of interest is transmembrane activator and CAML-interactor (TACI). See Yu et al Nat. Immunol. 1 :252-256 (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. BC A-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 form a two ligand-two receptor pathway 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 chimerizatioii of murine antibodies and antibody fragments are well known to those skilled in the art. For example, humanized monoclonal antibodies are produced by transferring 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 counteφarts. 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 Orlandi etal, Proc. Nat'lAcad. Sci. USA 86: 3833 (1989), which is incoφorated by reference in its entirety. Techniques for producing humanized Mabs are described, for example, by Jones et al. , Nature 321: 522 (1986), Riechmann et al. Nature 332: 323 (1988), Verhoeyen et al, Science 239: 1534 (1988), Carter et al, Proc. Nat'lAcad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12: 437 (1992), and Singer et al, J. Immun. 150: 2844 (1993), each of which is hereby incoφorated by reference.
[0122] Alternatively, fully human antibodies can be obtained from transgenic non-human animals. See, e.g., Mendez et al, Nature Genetics, 15: 146-156 (1997); U.S. Patent No. 5,633,425. For example, human antibodies can be recovered from transgenic mice possessing human immunoglobulin loci. The mouse humoral immune system is humanized by inactivating the endogenous immunoglobulin genes and introducing human immunoglobulin loci. The human immunoglobulin loci are exceedingly complex and comprise a large number of discrete segments which together occupy almost 0.2% of the human genome. To ensure that transgenic mice are capable of producing adequate repertoires of antibodies, large portions of human heavy- and light-chain loci must be introduced into the mouse genome. This is accomplished in a 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 light-chain loci, are introduced separately into mice via fusion of YAC-containing yeast spheroblasts with mouse embryonic stem cells. Embryonic stem cell clones are then microinjected into mouse blastocysts. Resulting chimeric males are screened for their ability to transmit the YAC through their germline and are bred with mice deficient in murine antibody production. Breeding the two transgenic strains, one containing the human heavy-chain loci and the other containing the human light-chain loci, creates progeny which produce human antibodies in response to immunization.
[0123] Unrearranged human immunoglobulin genes also can be introduced into mouse embryonic stem cells via microcell-mediated chromosome transfer (MMCT). See, e.g., Tomizuka et αl., Nature Genetics, 16: 133 (1997). In this methodology microcells containing human chromosomes are fused with mouse embiyonic stem cells. Transferred chromosomes are stably retained, and adult chimeras exhibit proper tissue-specific expression.
[01 4] As an alternative, an antibody or antibody fragment of the present invention may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, e.g., Barbas et αl, METHODS: A Companion to Methods in Enzymology 2: 119 (1991), and Winter et al, Ann. Rev. Immunol 12: 433 (1994), which are incoφorated by reference. Many of the difficulties associated with generating monoclonal antibodies by B-cell immortalization can be overcome by engineering and expressing antibody fragments in E. coli, using phage display. To ensure the recovery of high affinity, monoclonal antibodies a combinatorial immunoglobulin library must contain a large repertoire size. A typical strategy utilizes mRNA obtained from lymphocytes or spleen cells of immunized mice to synthesize cDNA using reverse transcriptase. The heavy- and light-chain genes are amplified separately by PCR and ligated into phage cloning vectors. Two different libraries are produced, one containing the heavy-chain genes and one containing the light-chain genes. Phage DNA is isolated from each library, and the heavy- and light-chain sequences are ligated together and packaged to form a combinatorial library. Each phage contains a random pair of heavy- and light-chain cD As and upon infection ofE. coli directs the expression of the antibody chains in infected cells. To identify an antibody that recognizes the antigen of interest, the phage library is plated, and the antibody molecules present in the plaques are transferred to filters. The filters are incubated with radioactively labeled antigen and then washed to remove excess unbound ligand. A radioactive spot on the autoradiogram identifies a plaque that contains an antibody that binds the antigen. Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA).
[0125[ A similar strategy can be employed to obtain high-affinity scFv. See, e.g., Vaughn et αl., Nat. Biotechnol, 14: 309-314 (1996). An scFv libraiy with a large repertoire can be constructed by isolating V- genes from non-immunized hu an donors using PCR primers coπresponήing to all known V , Vκ and V^ gene families. Following amplification, the Vκ and Vχ pools are combined to form one pool. These fragments are ligated into a phagemid vector. The scFv linker, (Gly-j., Ser)3, is then ligated into the phagemid upstream of the VL fragment. The Vjj and linker- Vτ_ fragments are amplified and assembled on the JJJ region. The resulting Vπ-linker-VL fragments are ligated into a phagemid vector. The phagemid library can be panned using filters, as described above, or using immunotubes (Nunc; Maxisoφ). Similar results can be achieved by constructing a combinatorial immunoglobulin library from lymphocytes or spleen cells of immunized rabbits and by expressing the scFv constructs in P. pαstoris. See, e.g., Riddεr et αl, Biotechnology, 13: 255-260 (1995). Additionally, following isolation of an appropriate scFv, antibody fragments with higher binding affinities and slower dissociation rates can be obtained through affinity maturation processes such as CDR3 mutagenesis and chain shuffling. See, e.g., Jackson et αl, Br. J. Cancer, 78: 181-188 (1998); Osbourn et al, Immunotechnology, 2: 181-196 (1996).
[0126] Another form of an antibody fragment is a peptide coding for a single CDR. CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick etαl., Methods: A Companion to Methods in Enzγmology 2:106 (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND 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: PRINCIPLES AND APPLICATIONS, Birch et al, (eds.), pages 137-185 (Wiley-Liss, Inc. 1995). [0127] The bsAbs can be prepared by techniques known in the art, for example, an anti-CEA tumor Ab and an anti-peptide Ab are both separately digested with pepsin to their respective F(ab')2S. The anti- CEA-Ab-F(ab')2 is reduced with cysteine to generate Fab' monomeric units which are further reacted with the cross-linker bis(maleimido) hexane to produce Fab'- aleimide moieties. The anti-peptide Ab-F(ab')2 is reduced with cysteine and the purified, recovered anti-peptide Fab'-SH reacted with the anti-CEA-Fab'- maleimide to generate the Fab' x Fab ' bi-specific Ab. Alternatively, the anti-peptide Fab'-SH fragment may be coupled with the anti-CEA F(ab')2 t0 generate a F(ab')2 x Fα construct, or with anti-CEA IgG to generate an IgG x Fαb ' bi-specific construct. In one embodiment, the IgG x Fαb ' construct can be prepared in a site-specific manner by attaching the antipeptide Fαb ' thiol group to anti-CEA IgG heavy- chain carbohydrate which has been periodate-oxidized, and subsequently activated by reaction with a commercially available hydrazide-maleimide cross-linker. The component Abs used can be chimerized or humanized by known techniques. A chimeric antibody is a recombinant protein that contains the variable domains and complementary determining regions derived from a rodent antibody, while the remainder of the antibody molecule is derived from a human antibody. Humanized antibodies are recombinant proteins in which murine complementarity determining regions of a monoclonal antibody have been transferred from heavy and light variable chains of the murine immunoglobulin into a human variable domain. [0128] A variety of recombinant methods can be used to produce bi-specific antibodies and antibody fragments. For example, bi-specific antibodies and antibody fragments can be produced in the milk of transgenic livestock. See, e.g., Col an, A., Biochem. Soc. Sytnp., 63; 141-147, 1998; U.S. Patent No. 5,827,690. Two DNA constructs are prepared which contain, respectively, DNA segments encoding paired immunoglobulin heavy and light chains. The fragments are cloned into expression vectors which contain a promoter sequence that is preferentially expressed in mammary epithelial cells. Examples include, but are not limited to, promoters from rabbit, cow and sheep casein genes, the cow α- lactoglobulin gene, the sheep β-lactoglobulin gene and the mouse whey acid protein gene. Preferably, the inserted fragment is flanked on its 3 ' side by cognate genomic sequences from a mammary-specific gene. This provides a polyadenylation site and transcript-stabilizing sequences. The expression cassettes are coinjected into the pronuclei of fertilized, mammalian eggs, which are then implanted into the uterus of a 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 standard 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 mouse sequences, however, and may still be immunogenic. A humanized Ab contains only those mouse amino acids necessary to recognize the antigen. This product is constructed by building into a human antibody framework the amino acids from mouse complementarity determining regions. [0130] Other recent methods for producing bsAbs include engineered recombinant Abs which have additional cysteine residues so that they crosslink more strongly than the more common immunoglobulin isotypes. See, e.g., FitzGerald etαl., Protein Eng. 10(10):1221-1225, 1991. Another approach is to engineer recombinant fusion proteins linking two or more different single-chain antibody or antibody fragment segments with the needed dual specificities. See, e.g., Coloma et αl., Nature Biotech. 15:159-163, 1997. A variety of bi-specific fusion proteins can be produced using molecular engineering. In one form, the bi- specific fusion protein is monovalent, consisting of, for example, a scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen. In another form, the bi-specific fusion protein is divalent, consisting of, for example, an IgG with two binding sites for one antigen and two scFv with two binding sites for a second antigen.
[0131] Functional bi-specific single-chain antibodies (bscAb), also called diabodies, can be produced in mammalian cells using recombinant methods. See, e.g., Mack etαl, Proc. Nαtl Ac d. Sci., 92: 7021- 7025, 1995. For example, bscAb are produced by joining two single-chain Fv fragments via a glycine- serine linker using recombinant methods. The V light-chain (Vj_) and V heavy-chain (Vjj) domains of two antibodies of interest are isolated using standard PCR methods. The Vr^ and Vf{ cDNA's obtained from each hybridoma are then joined to form a single-chain fragment in a two-step fusion PCR. The first PCR step introduces the (Gly4-Seq)3 linker (SEQ ID NO: 9), and the second step joins the VL and H a plicons. Each single chain molecule is then cloned into a bacterial expression vector. Following amplification, one of the single-chain molecules is excised and sub-cloned into the other vector, containing the second single-chain molecule of interest. The resulting bscAb fragment is subcloned into an eukaiyofic expression vector. Functional protein expression can be obtained by transfecting the vector into Chinese hamster ovary cells. Bi-specific fusion proteins are prepared in a similar manner. Bi-specific single-chain antibodies and bi-specific fusion proteins are included within the scope of the present invention.
[0132] Bi-specific fusion proteins linking two or more different single-chain antibodies or antibody fragments are produced in similar manner.
(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 the scFv are introduced into the VL and VK domains through PCR reactions. The DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CHI domain. The resulting scFv-Cjjl construct is excised and ligated into a vector containing a DNA sequence encoding the VJJ region of an anti-CEA antibody, The resulting vector can be used to transfect mammalian cells for the expression of the bi- specific fusion protein.
[0134] Large quantities of bscAb and fusion proteins can be produced using Escherichiα coli expression systems. See, e.g., Zhenping et αl, Biotechnology, 14: 192-196, 1996. A -functional bscAb can be produced by the coexpression in E. coli of two "cross-over" scFv fragments in which the VL an VJJ domains for the two fragments are present on different polypeptide chains. The V light-chain (Vj_) and V heavy-chain (VH) domains of two antibodies of interest are isolated using standard PCR methods. The cDNA's are then ligated into a bacterial expression vector such that C-terminus of the VL domain of the first antibody of interest is ligated via a linker to the N-terminus of the VJJ domain of the second antibody. Similarly, the C-teimiπus of the V domain of the second antibody of interest is Hgated via a linker to the N-tenninus of the VJJ domain of the first antibody. The resulting dicistronic operon is placed under transcriptional control of a strong promoter, e.g., the E. coli alkaline phosphatase promoter which is inducible by phosphate starvation. Alternatively, single-chain fusion constructs have successfully been expressed in E. coli using the lαc promoter and a medium consisting of 2% glycine and 1% Triton X-100. See, e.g., Yang et αl., Appl. Environ. Microbiol, 64: 2869-2874, 1998. An E. coli, heat-stable, enterotoxin II signal sequence is used to direct the peptides to the periplasmic space. After secretion, the two peptide chains associate to form a non-covalent heterodimer which possesses both antigen binding specificities. The bscAb is purified using standard procedures known in the art, e.g., Staphylococcal protein A chromatography.
[0135] Functional bscAb and fusion proteins also can be produced in the milk of transgenic livestock. See, e.g., Colman, A., Biochem. Soc. Symp., 63: 141-147, 1998; U.S. Patent No. 5,827,690. The bscAb fragment, obtained as described above, is cloned into an expression vector containing a promoter sequence that is preferentially expressed in mammary epithelial cells. Examples include, but are not limited to, promoters from rabbit, cow and sheep casein genes, the cow α-lactoglobulin gene, the sheep β- Iactoglobulin gene and the mouse whey acid protein gene. Preferably, the inserted bscAb is flanked on its 3' side by cognate gεnomic sequences from a mammary-specific gene. This provides a polyadenylation site and transcript-stabilizing sequences. The expression cassette is then injected into the pronuclei of fertilized, mammalian eggs, which 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 using standard methods known in the art. Transgenic production of bscAb in milk provides an efficient method for obtaining large quantities of bscAb.
[0136] Functional bscAb and fusion proteins also can be produced in transgenic plants. See, e.g., Fiedler et l, Biotech., 13: 1090-1093, 1995; Fiedler et al, Immunotechnology, 3: 205-216, 1997. Such production offers several advantages including low cost, large scale output and stable, long term storage. The bscAb fragment, obtained as described above, is cloned into an expression vector containing a promoter sequence and encoding a signal peptide sequence, to direct the protein to the endoplasmic recticulum. A variety of promoters can be utilized, allowing the practitioner to direct the expression product to particular locations within the plant. For example, ubiquitous expression in tobacco plants can be achieved by using the strong cauliflower mosaic virus 35S promoter, while organ specific expression is achieved via the seed specific Iegumin B4 promoter. The expression cassette is transformed according to standard methods known in the art. Transformation is verified by Southern analysis. Transgenic plants are analyzed for the presence and functionality of the bscAb using standard immunological methods known in the art. The bscAb can be purified from the plant tissues using standard methods known in the art.
[0137] Additionally, transgenic plants facilitate long term storage of bscAb and fusion proteins. Functionally active scFv proteins have been extracted from tobacco leaves after a week of storage at room temperature. Similarly, transgenic tobacco seeds stored for 1 year at room temperature show no loss of scFv protein or its antigen binding activity.
[0138] Functional bscAb and fusion proteins also can be produced in insect cells. See, e.g., Mahiouz ef αl., J. Immunol. Methods, 212: 149- 160 (1998). Insect-based expression systems provide a means of producing large quantities of homogenous and properly folded bscAb. The baculovirus is a widely used expression vector for insect cells and has been successfully applied to recombinant antibody molecules. See, e.g., Miller, EX.., Ann. Rev. Microbiol, 42: 177 (19S8); Bei et αl., J. Immunol Methods, 186: 245 (1995). Alternatively, an inducible expression system can be utilized by generating a stable insect cell line containing the bscAb construct under the transcriptional control of an inducible promoter. See, e.g., Mahiouz etαl., J. Immunol. Methods, 212: 149-160 (1998). The bscAb fragment, obtained as described above, is cloned into an expression vector containing the Drosphilα metallothionein promoter and the human HLA-A2 leader sequence. The construct is then transfected into D. melαnogαster SC-2 cells. Expression is induced by exposing the cells to elevated amounts of copper, zinc or cadmium. The presence and functionality of the bscAb is determined using standard immunological methods known in the art. Purified bscAb is obtained using standard methods known in the art.
[0139J Preferred bi-specific antibodies of the instant invention are those which incorporate the Fv of MAb Mu-9 and the Fv of MAb 679 or the Fv of MAb MN-14 and the Fv of MAb 679, and their human, chimerized or humanized counteφarts. The MN-14, as well as its chimerized and humanized counterparts, are disclosed in U.S. Patent No. 5,874,540. Also preferred are bi-specific antibodies which incoφorate one or more of the CDRs of Mu-9 or 679. The antibody can also be a fusion protein or a bi- specific antibody that incoφorates a Class-Ill anti-CEA antibody and the Fv of 679. Class-Ill antibodies, including Class-Ill anti-CEA are discussed in detail in U.S. Patent No. 4,818,709. VII. Other Applications
[0140] The present invention encompasses the use of the bsAb and a therapeutic or diagnostic agent associated with the targetable construct discussed above in intraoperative, intravascular, and endoscopic tumor and lesion detection, biopsy and therapy as described in U.S. Patent No. 6,096,289. [0141] The antibodies and antibody fragments of the present invention can be employed not only for therapeutic or imaging purposes, but also as aids in performing research in vitro. For example, the bsAbs of the present invention can be used in vitro to ascertain if a targetable construct can form a stable complex with one or more bsAbs. Such an assay would aid the skilled artisan in identifying targetable constructs which form stable complexes with bsAbs. This would, in turn, allow the skilled artisan to identify targetable constructs which are likely to be superior as therapeutic and/or imaging agents. [0142] The assay is advantageously performed by combining the targetable construct in question with at least two molar equivalents of a bsAb. Following incubation, the mixture is analyzed by size-exclusion HPLC to determine whether or not the construct has bound to the bsAb. Alternatively, the assay is performed using standard combinatorial methods wherein solutions of various bsAbs are deposited in a standard 96-weZ_ plate. To each well, is added solutions of targetable constructs). Following incubation and analysis, one can readily determine which constructs) bind(s) best to which bsAb(s). [0143] It should be understood that the order of addition of the bsAb to the targetable construct is not crucial; that is, the bsAb may be added to the construct and vice versa. Likewise, neither the bsAb nor the construct needs to be in solution; that is, they may be added either in solution or neat, whichever is most convenient. Lastly, the method of analysis for binding is not crucial as long as binding is established. Thus, one may analyze for binding using standard analytical methods including, but not limited to, FABMS, high-field NMR or other appropriate method in conjunction with, or in place of, size-exclusion HPLC.
[0144[ The present invention is further illustrated by, though in no way limited to, the following examples. Examples
Example 1) Synthesis of Ac-LvstHSGVD-Tyr-LvsfHSGVLvsfTscg-Cvs-VNHTjIMP 243)
[0145] The peptide was synthesized as described by Karacay et. αl. Bioconjugαte Chem. H. "842-854 (2000) except D-tyrosine was used in place of the L-tyrosine and the N-trityl-HSG-OH was used in place of the DTPA. The final coupling of the N-trityl-HSG-OH was carried out using a ten fold excess of N- trityl-HSG-OH relative to the peptide on the resin. The N-trityl-HSG-OH (0.28 M inNMP) was activated using one equivalent (relative to HSG) of N-hydroxybenzotπazole, one equivalent of benzotrazole-1-yl- oxy-tns-(dιmethylammo)phosρhomum hexafluorophosphate (BOP) and two equivalents of dusopropylethylamine The activated substrate was mixed with the resm for 15 hr at room temperature.
Example 2) Tc-99m Kit Formulation Comprising IMP 243
[0146] A formulation buffer was prepared which contained 22.093 g hydroxypropyl-β-cyclodextπn, 0.45 g 2,4-dιhydroxybenzoιc acid, 0 257 g acetic acid sodium salt, and 10.889 g α-D-glucoheptonic 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 02 L of SnCl2 (200 mg/mL) with 3.8 mL of the formulation buffer. The peptide Ac-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2 (SEQ ID NO: 4) (0.0026g), was dissolved in 78 mL of the buffer solution and mixed with 0 52 L of the stannous buffer. The peptide solution was then filtered through a 0.22 μm Millex GV filter in 1 5 mL ahquots into 3 mL lyophihzation vials. The filled vials were frozen immediately, lyophilized and cπmp sealed under vacuum.
[0147] Pertechnetate solution (27 mCi) in 1.5 mL of salme was added to the kit. The kit was incubated at room temperature for 10 min and heated in a boiling water bath for 25 min The kit was cooled to room temperature before use.
Example 3) Peptides for Carrying Therapeutic/Imaging Radioisotopes to Tumors via Bi-specific Antibody Tumor Pretargeting
10148] DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 2) (IMP 237) was synthesized to deliver therapeutic radioisotopes such as 90γ or 177 U to tumors via bi-specific antibody tumor pretargeting The bi-specific antibody is composed of one portion which binds to an antigen on the tumor and another portion which binds to the HSG peptide The antibody which bmds the HSG peptide is 679. This system can also be used to deliver imaging isotopes such as 11 lfrι-111
Synthesis of IMP 237
[0149] IMP 231 was synthesized on Sieber Amide resm (Nova-Biochem) using standard Fmoc based solid phase peptide synthesis to assemble the peptide backbone with the following protected ammo acids, in order: Fmoc-Lys(Aloc)-OH, Fmoc-Tyr(But)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Phe-OH, (Reagents from Advanced Chemtech) tπ-t-butyl DOTA (Macrocyclics). The side lysine side chains were then deprotected with Pd[PfPh)3J4 by the method of Dangles et αl J Org Chem 52:4984-4993 (1987). The HSG ligands were then added as Tπtyl HSG (synthesis described below) using the BOP/HBTU double coupling procedure used to attach the ammo acids. The peptide was cleaved from the resm and the protecting groups were removed by treatment with TFA The peptide was puπfied by HPLC to afford 0 6079 g of peptide from 1 823 g of Fmoc-Lys(Aloc)-Tyr(But)-Lys(Aloc)-NH-Sιeber amide resin. Synthesis ofN-Trit l-HSG-OH
]0150] Glycine t-butyl ester hydrochloride (15.263 g, 9.1 xl 0-2 mol) and 19.760 g 2C03 were mixed, then suspended in 50 mL H2O and cooled in an ice bath. Succinic anhydride (9.142 g, 9,14 x 10_2 mol) was then added to the reaction solution which was allowed to warm slowly to room temperature and stir for 18 hr. Citric acid (39.911 g) was dissolved in 50 mL H2O and slowly added to the reaction solution and then extracted with 2 x 150 mL EtOAc. The organic extracts were dried over Na2Sθ4, filtered and concentrated to afford 25.709 g of a white solid.
[0151] The crude product (25.709 g) was dissolved in 125 mL dioxane, cooled in a room temperature water bath and mixed with 11.244 g of -hydroxysuccinimide. Diisopropylcarbodiimide 15.0 mL was added to the reaction solution which was allowed to stir for one hour. Histamine dihydrochloride (18.402 g, 1.00 x 10"1 mol) was then dissolved in 100 mL DMF and 35 mL diisopropylethylamine. The histamine mixture was added to the reaction solution which was stirred at room temperature for 21 hr. The reaction was quenched with 100 mL water and filtered to remove a precipitate. The solvents were removed under hi-vacuum on the rotary evaporator. The crude product was dissolved in 300 mL dichloromethane and extracted with 100 mL saturated NaHCOβ. The organic layer was dried over N 2Sθ4 and concentrated to afford 34.19 g of crude product as a yellow oil.
[0152] The crude product (34.19 g) was dissolved in 50 mL chloroform and mixed with 31 mL diisopropylethylamine. Triphenylmethyl chloride (25.415 ) was dissolved in 50 ml chloroform and added dropwise to the stirred reaction solution which was cooled in an ice bath. The reaction was stirred for 45 min and then quenched with 100 L H2O. The layers were separated and the organic solution was dried over Na2Sθ4 and concentrated to form a green gum. The gum was triturated with 100 mL Et2θ to form a yellow precipitate which was washed with 3 x 50 mL portions of E 2θ. The solid was vacuum dried to afford 30.641 g (59.5 % overall yield) of N-trityl-HSG-t-butyl ester.
[0153] N-trityl-HSG-t-butyl ester (20.620 g, 3.64 x 10"2 mol) was dissolved in a solution of 30 L chloroform and 35 mL glacial acetic acid. The reaction was cooled in an ice bath and 15 mL of BF3*Et2θ was slowly added to the reaction solution. The reaction was allowed to warm slowly to room temperature and mix for 5 hr. The reaction was quenched by pouring into 200 mL 1M NaOH and the product was extracted with 200 mL chloroform. The organic layer was dried over Na2Sθ4 and concentrated to afford a crude gum which was triturated with 100 L Et2θ to form a precipitate. The crude precipitate was poured into 400 mL 0.5 M pH 7.5 phosphate buffer and extracted with 2 x 200 mL EtOAc. The aqueous layer was acidified to pH 3.5 with 1 M HCI and extracted with 2 x 200 mL chloroform. A precipitate formed and was collected by filtration (8.58 g). The precipitate was the desired product by HPLC comparison to a previous sample (ESMS MH+ 511).
Radiolabeling
9QY Kit Preparation [0154] DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 2) was dissolved in 0.25 M NH4OAC/ 10 % HPCD buffer at concentrations of 9, 18, 35, 70 and 140 μg/mL. The solutions were sterile filtered through a 0.22 μm Millex GV filter in one mL aliquots into acid washed lyophilization vials. The filled vials were frozen immediately on filling and lyophilized. When the lyophilization cycle was complete the vials were sealed under vacuum and crimp sealed upon removal from the lyophilizer. ]0155] The 9®Y (~400 μCi/kit) was diluted to I L in deionized water and added to the lyophilized kits. The kits were heated in a boiling water bath for 15 min, the vials were cooled to room temperature and the labeled peptides were evaluated by reverse phase HPLC (HPLC conditions: Waters Nova-Pak C-18, 8x100 mm RCM column eluted at 3 mL/min with a linear gradient from 100 % (0.1 % TFA in H2O) to 100 % (90 % CH3CN, 0.1% TFA, 10 % H2O)). The HPLC analysis revealed that the minimum concentration of peptide needed for complete labeling, with this formulation, was 35 μg/mL. The reverse phase HPLC trace showed a shaφ 90γ labeled peptide peak. The labeled peptide was completely bound when mixed with excess 679 IgG by size exclusion HPLC.
Labeling with 11 lln
[0156] The * 1 n (~300 μCi/ it) was diluted to 0.5 L in deionized water and added to the lyophilized kits. The kits were heated in a boiling water bath for 15 min, the vials were cooled and 0.5 mL of 2.56 x 10"5 M In in 0.5 M acetate buffer was added and the kits were again heated in the boiling water bath for 15 min. The labeled peptide vials were cooled to room temperature and evaluated by reverse phase HPLC (HPLC conditions: Waters Nova-Pak C-18, 8x100 mm RCM column eluted at 3 mL/miπ with a linear gradient from 100 % (0.1 % TFA in H20) to 100 % (90 % CH3CN, 0.1 % TFA, 10 % H2O)). The HPLC analysis revealed that the minimum concentration of peptide needed for labeling (4.7 % loose ϊ fin), with this formulation, was 35 μg/mL. The reverse phase HPLC trace showed a shaφ IT lfo labeled peptide peak. The labeled peptide was completely bound when mixed with excess 679 IgG by size exclusion HPLC.
In-Vivo Studies
[0157] Nude mice bearing GW-39 human colonic xenograft tumors (100-500 mg) were injected with the bi-specific antibody hMN-14 x m679 (1.5 x 10-10 mol). The antibody was allowed to clear for 24 hr before the 11 ^In labeled peptide (8.8 μCi, 1.5 x 10"' * mol) was injected. The animals were sacrificed at 3, 24, 48 hr post injection.
[0158] The results of the biodistribution studies of the peptide in the mice pretargeted with hMN-1 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
Pretargeting With U In Labeled Peptide 24 hr After Injection of hMN-14 x m679 % Injected/g Tissue
Figure imgf000050_0001
Table 2
Pretargeting With f 1 n Labeled Peptides 24 hr After Injection of hMN-14 x m679 Tumor/Non-Tumor Tissue Ratios
Figure imgf000050_0002
Serum Stability ofDOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 2) (IMP 237) andDOTA-Phe- Lys(HSG)-D-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 3) (IMP 241)
Peptide Labeling and HPLC Analysis
[0159] The peptides, IMP 237 and IMP 241 , were labeled according to the procedure described by Karacay et. al. Bioconjugate Chem. 77:842-854 (2000). The peptide, IMP 241 (0.0019 g), was dissolved in 587 μl 0.5 M NH4CI, pH 5.5. A 1.7 μL aliquot of the peptide solution was diluted with 165 μl 0.5 M NH4CI, pH 5.5. The ' ^In (1.8 mCi) in 10 μL was added to the peptide solution and the mixture was heated in a boiling water bath for 30 min.
[0160] The labeled peptide was analyzed by HPLC using a Waters 8x100 mm radial-pak, nova-pak C-28 RCM cartridge column. The column was eluted at 3 mL/min with a linear gradient which started with 100 % of 0.1 % TFA in water and went to 100 % of 0.1 %TFA in 90% acetonitrile and 10 % water over 10 min. There was about 6% loose H ^In in this labeling which came out at the void volume of the column (1.6 min). There were also some H lln labeled peaks at 5 min and 6.6 to 8 min. The Hlln labeled peptide was eluted at 8.8 min as a single peak. The HPLC profile of ^ lln IMP 237 was nearly identical to 1 "in IMP 241.
Serum Stability
[0161] An aliquot (30 μL) of m In IMP 241 was placed in 300 μL of fresh mouse serum and placed in a
37° C incubator. The peptide was monitored as described above by HPLC.
[0162] An aliquot (24 μL) of 1 ^In IMP 237 was placed in 230 μL of fresh mouse serum and placed in a
37° C incubator. The peptide was monitored as described above by HPLC.
[0163] The analysis showed that the TI lfo IMP 241 may have decomposed slightly (~ 5%) after heating
22 hr in mouse serum at 37° C. The 11 lm IMP 237 was about 70 % converted to the shorter retention time peak after incubation for 22 hr at 37° C.
Conclusion
[0164] The D-tyrosine in the IMP 241 peptide slows the decomposition of the peptide in mouse serum compared to IMP 237. In Vivo Stability of IMP 237 and IMP 241 Compared
[0165] The in vivo stabilities of 1 ^In IMP 237 and ^ ljn IMP 241 were compared by examining (by HPLC) urine samples from mice at 30 and 60 min. The peptides, IMP 241 and IMP 237, were 1 Hln-l 11 labeled as described above.
[0166] The labeled peptides were injected into Balb/c mice which were sacrificed at 30 min and 60 min post injection of the peptides using one mouse per time point. The attached HPLC traces indicate that H lfri IMP 241 was excreted intact while ' 1 Ifa IMP 237 was almost completely metabolized to a new 11 lfa labeled peptide.
Conclusion
[0167] The replacement of Tyr with D-Tyr in the peptide backbone minimized metabolism of the peptide in-vivo.
Additional In Vivo Studies
[0168] Nude mice bearing GW-39 human coionic xenograft tumors (100-500 mg) were injected with the bi-specific antibody mMu-9 x m679 (1.5 x 10-10 mol). The antibody was allowed to clear for 48 hr before the 11 fin labeled peptides (8.8 μCi, 1.5 x 10-11 moj) were injected. The animals were sacrificed at 3, 24, 48 hr post injection.
[0169] The results of the biodistribution studies of the peptides in the mice pretargeted with mMU-9 x m679 are shown in Table 3. The tumor to non-tumor ratios of the peptides in the pretargeting study are show in Table 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
Pretargeting With 11 n Labeled Peptides 48 hr After Injection of mMU-9 x m679 % Injected/g Tissue
Figure imgf000053_0001
Table 4
Pretargeting With 1 ! lln Labeled Peptides 48 hr After Injection of mMU-9 x m679 Tumor/Non-Tumor Tissue Ratios
Figure imgf000054_0001
Table 5
Biodistribution of " *In Labeled Peptides Alone
Figure imgf000055_0001
Example 4) Synthesis of a Peptide Antisen
[0170J 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(Al c)-OH. The alpha-Fmoc protecting group is selectively removed and the Fmoc-Tyr(OBut), alpha-Fmoc-Lys(AIoc)-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 ofAc-Phe-Lvs(Ac)-Tyr-Lvs(Ac)-OH (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 HC1, 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 (BCLH) 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 4°C. 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 [0372] Immunocompetent mice are injected with a mixture of the peptide antigen in complete Freund's adjuvant. Two booster shots of the peptide mixed with incomplete Freund's adjuvant are administered over the next several weeks. Spleen cells are harvested from the animals and fused with Sp2/0-Agl4 myeloma cells. Culture supematants of the resulting clones are analyzed for anti-peptide reactivity by ELISA, using plates coated with the original peptide immunogen. Enzyme-deficient hybridomas are isolated to enable selection of fused cell lines, and selected clones grown in culture media to produce the anti-peptide Abs.
Example 7) Purification of Anti-Peptide Ab
[0173] Anti-peptide Ab is purified chromatographically using a protein A column to isolate the IgG fraction, followed by ion-exchange columns to clean the desired product. The Ab of interest is finally purified by using an affinity column comprised of the peptide of interest bound to a solid support, prepared by chemically coupling said peptide to activated beads or resin.
Example 8) Digestion of Anti-Peptide Ab to Ffab'b
[0174] The anti-peptide Ab is incubated with 200 μg/μL of pepsin at pH 4 for one hour and purified by a tandem column of protein A, to remove undigested IgG, followed by G-50-Sephadex, to remove low molecular weight contaminants.
Example 9) Reduction of Anti-Peptide-Ab to Fab'-SH
[0175] The anti-peptide-F(ab 'fa is reduced to a Fab' fragment by reaction with a freshly prepared cysteine solution in 0.1M PBS, containing lOmM 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 < 5 containing lOmM EDTA.
Example 10) Oxidative Coupling of Anti-CEA-IsG to a Maleimide Moiety
[01 61 Anti-CEA Ab IgG is oxidized by reaction with 1 OmM sodium periodate for 90 minutes at 4°C, in the dark. The oxidized Ab is purified by spin-column chromatography and mixed with an excess of the cross-linker 4-(4-maleimidophenyl) butyric acid hydrazide (MPBH). The reaction is allowed to proceed for 2 h and the IgG-hydrazone-meleimide purified by spin-column chromatography. The hydrazone bond is reduced by reaction with lOmM sodium cyanoborohydride and repurified.
Example 11) Preparation of anti-CEA-IeGx anti-Peptide-Fab' Bi-specific Ab [0177] The IgG-hydrazide-maleimide from Example 10 is treated with an equimolar amount of anti- peptide Fab'-SH, prepared in Example 6, at pH 6.0, for 30 minutes at room temperature. Remaining free thiol groups are blocked by a 30-minute reaction with iodoacetamide. The bi-specific Ab anti-CEA-IgG x anti-peptide-Fab' is purified by size-exclusion chromatography to remove unreacted Fab', followed by affinity chromatography using solid-phase-bound peptide to separate IgG x Fαb ' from unreacted IgG.
Example 12) Synthesis of Ac-Phe-Lvs(Bz-DTPA)-Tyr-Lys(Bz-DTPAVNH? TSEO ID NO: 2) [0178] The peptide, Ac-Phe-Lys(Bz-DTPA)-Tyr-Lys(Bz-DTPA)-NH2 (SEQ ID NO: 2), is assembled using a resin for solid-phase synthesis and attaching the first residue (lysine to said resin as the differentially protected derivative alpha-Fmoc-Lys(Aloc)-OH. The alpha-Fmoc protecting group is selectively removed and the 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- side-chain is removed by reaction with palladium (0) catalyst. Alternatively, Boc-group protecting groups may be used which may be removed by reaction with TFA and the free amino groups reacted with excess of the ITC-Bz-DTPA. After removing excess Bz-DTPA, the alpha-amino group is capped by reaction with acetic anhydride, and the entire peptide removed from the resin with TFA (with concomitant deprotection of the tyrosyl residue) to give Ac-Phe-Lys(Bz-DTPA)-Tyr-Lys(Bz-DTPA)-NH2.
Example 13) Radiolabeling of Ac-Phe-LvsfBz-DTPA)-Tyr-Lvs(Bz-DTPA)-NH-> CSEQ ID NO: 2)with
Y-90
[0179] The title peptide in 100-fold molar excess is mixed with yttriurn-90 radionuclide in acetate buffer at pH 5.5. The radiolabeling is complete and quantitative after 30 minutes.
Example 14) Conjugation of a Carboxylesterase to di-DTPA-Peptide
[0180] Carboxylesterase (5 mg) in 0.2 M phosphate buffer, pH 8.0, is treated with a five-fold molar excess of the cross-linking agent sulfo-succinimidyl-[4-maleimidomethyl]-cyclohexane-l-carboxylate (sulfo-SMCC). After stirring two hours at room temperature, the activated enzyme is separated from low molecular weight contaminants using a spin-column of G-25 Sephadex and equilibrated in 0.1 M phosphate buffer, pH 7, containing 1 mM EDTA. The tetrapeptide N-acetyl-Cys-Lys(DTPA)-Tyr- Lys(DTPA)-NH2 (SEQ ID NO: 11) (ten-fold molar excess) is added to the activated enzyme and dissolved in the same buffer as used in the spin-column. After stirring for one hour at room temperature, the Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH2 (SEQ ID NO: 11) peptide carboxylesterase conjugate is purified from unreacted peptide by spin-column chromatography on G-25 Sephadex in 0.25 M acetate buffer, pH 6.0. Successful conjugation is demonstrated by indium-Ill labeling of an aliquot of the conjugate, and analysis by size-exclusion HPLC.
Example 15) Use of anti-CEA-IgG x anti-Peptide-Fab' Bi-specific Ab for RAIT [0181] A patient with a CEA-expressing tumor burden is given anti-CEA-IgG x anti-peptide-Fab' bi- specific Ab. Seven days later, the patient is given Y-90-di-Bz-DTPA-peptide (from Example 13). The Y- 90-labeled peptide clears rapidly from non-target tissue but localizes avidly to sites pre-targeted with the anti-CEA-IgG x anti-peptide-Fab' bi-specific Ab, effecting destruction of tumors.
Example 16) Preparation of a Galactose-WI2-Fab' Clearing Agent
[0182] The anti-idiotypic Ab to MN-14, termed WI2 is digested to a F(ab')2 fragment using pepsin, as outlined in Example 8. The F(ab')2 is reduced to a Fab' fragment using a low molecular weight thiol, as outlined in Example 9. At the end of the reduction, the Fab'-SH is purified by spin-column chromatography and reacted with excess iodoacetamide to block hinge-region thiol groups and prevent reassociation. After repurifϊcation from excess iodoacetamide the Fab' is reacted with a 400-fold molar excess of the galactosylation agent, the thio-i idate of cyanomethyl-2,3,4,6-tetra-0-acetyl-l-thio-beta-D- galactopyranoside (see Karacay et al.). The galactosylated protein is purified by two spin-columns and the galactose;Fab' radio determined by MALDI-MS,
Example 17) Use of anti-CEA-IgG x anti-Peptide Fab' Bi-specific Ab for RAIT, with a bsAb Clearing Step.
[0183J 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- I2-Fab'. Twenty- four hours after the clearing dose of a galactose-WI2-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-Lys(DTPA)-Tyr-Lvs('DTPA)-LvsfTscg-Cvs)-NH2 ("SEQ ID NO: 7) (IMP
192)
[0184] The first amino acid, Aloc-Lys(Fmoc)-OH was attached to 0.2 1 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 form 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 CH2CI2, 0.75 mL glacial acetic acid and 2.5 ml diisopropylethyl amine. The resin mixture was then treated with 0.8 ml tributyltin 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 resin (SEQ ID NO: 7), The N-terminus was acetylated by vortex mixing the resin for 60 mm with 8 mL of a solution containing 10 mL DMF, 3 mL acetic anhydride, and 6 mL diisopropylethylamine. The side chain Aloe protecting groups were then removed as described above and the resin treated with piperidine using the standard Fmoc deprotection protocol to remove any acetic acid which may have remained on the resin.
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 with 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, Konishi J, Yokoyama A., "Reassessment of die hylenetriaminepentaacetic acid (DTPA) as a chelating agent for indium- 111 labeling of polypeptides using a newly synthesized monoreactive DTPA derivative," J Med Chem. 1996 Aug 30;39(18):3451-60).
Cleavage and Purification
[0I86[ The peptide was then cleaved from the resin by treatment with 8 ml of a solution made from 30 ml TFA, 1 ml triisopropylsilane, and 1 ml ethanedithiol for 60 mm. The crude cleaved peptide was precipitated by pouring into 30 ml ether and was collected by centrifugation. The peptide was then purified by reverse phase HPLC using a 4 x 30 cm Waters preparative C-18 Delta-Pak column (15 μm, 100 A). The HPLC fractions were collected and lyophilized to obtain a fraction which contained the desired product by ESMS (MH±1590).
Kit Formulation
[0187] The peptide was formulated into lyophilized kits which contained 78 μg of the peptide, 0.92 mg non-radioactive InCFj, 100 μg stannous chloride, 3 mg gentisic acid, and HPCD (10 % on reconstitution).
Example 19) Tc-99m Labeling and Stability
[0188] An IMP 192 kit was labeled by reconstituting the contents of the vial with 1.5 mL of saline which contained 25 Ci Na99mχcθ4. The kit was incubated at room temperature for 10 mm and then heated in a boiling water bath for 15 mm. The labeled peptide solution was then cooled to room temperature. 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, the 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 min. The shift may be due to ion pairing of some serum component with the peptide.
Table 6
Figure imgf000060_0001
Example 20) Preparation of hMN-14 x 734 (Fab x Fab)
[0189] This bsAb was prepared by crosslinking the hMN-14 Fab'gH (a humanized monoclonal anti-CEA antibody) and 734 Fab'maι (a murine anti-diDTPA) fragments, analogously to Example 8. The Fab'gH fragments of hMN-14 and 734 were prepared by reduction of the F(ab')2 fragments with 10 mM 2- mercaptoethylamine in the presence of 10 mM EDTA at pH 7.3 for 60 min at 37°C Fab'sH was collected after spin column (Penefsky) purification (Sephadex G-50-80, 50 mM NaOAc, 0.5 mM EDTA, pH 5.3) Maleimide grouρ(s) were introduced onto 734 Fab'sjj fragment using 4 mM N,N'-o- phenylenedimaleimide at RT for 60 min. Spin column purification was used to isolate the Fab'maι. Crosslinking of 734 Fab'maι and hMN-14 Fab'sH was allowed to proceed 16 h at 4°C at 1:1 molar ratio. To break the disulfide bonds which might have formed during this time, the reaction mixture was treated with 10 mM 2-mercaptoethylamine for Ih at pH 5.3 at 23°C. The SH groups were blocked with N- ethyimaleimide at pH 6.4. The reaction mixture was applied to a spin column to remove excess small molecular weight compounds. The bsAb was then isolated after purification on an analytical size exclusion HPLC column, Bio-Sil SEC-250. The HPLC retention time of the purified bsAb was 10.23 min.
Example 21 ) HPLC binding studies
[0190] The bsAb was radiodinated using chloramine T (Greenwood and Hunter). Binding of the radioiodinated bsAbs to CEA, WI2 (rat anti-MN-34 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 10-20x molar excess of CEA. The bsAb complexed with radiolabeled indium-DTPA chelates (IMP-156 or IMP-192).
IMP 156 Ac-Phe-Lys(DTPA)-Tyr-Lys(DTPA)-NH2 (SEQ ID NO: 2)
Example 22) Serum stability
[01 1] Radioiodinated bsAb was tested for stability in fresh human serum at 37°C under a humidified 5 % CO2 atmosphere. Aliquots were examined on SE-HPLC. In order to detect radioiodine associated with serum proteins, the aliquots were mixed with WI2 to shift the bsAb peak to earlier retention times. The bsAbs showed 3-5 % loss of binding capacity to WI2 after 48 h incubation in serum. Slight aggregate formation (4-7 %) was observed upon incubation of the bsAbs in serum for 72 h.
Example 23) 99m-Tc-IMP-192
[0192] In vitro stability of the Tc-99m complex of this peptidyl chelate was established by incubations in saline, fresh human serum and 10 mM cysteine for up to 20 h. In vivo stability was examined by analysis of urine collected from a mouse injected with 99m-Tc-IMP- 192 in a pretargeting experiment. The activity excreted in the urine appears to be the intact peptide because the activity still binds to the antibody as shown by SE-HPLC. Biodistribution studies of 99m-Tc-IMP-192 in normal BALB/c mice showed rapid blood clearance, Table 7. The in vitro and in vivo studies clearly demonstrate stability of 99m-Tc-IMP- 192.
Table 7
Clearance of 99m-Tc-IMP-192 in BALB/c mice.
Tissue %ID/g
Ih 2 4Jι 24 h
Liver 0.27 ± 0.18 0.22 ± 0.16 0.09 ± 0.02 0,04 ± 0.0
Spleen 0.08 ± 0.01 0.09 ± 0.3 0.05 ± 0.02 0.03 ± 0.01
Kidney 4.16 ± 0.75 4.05 ± 0.60 3.21 ± 0.99 1.21 ± 0.08
Lungs 0.50 ± 0.23 0.29 ± 0.08 0.19 ± 0.04 0.05 ± 0.00
Blood 0.30 ± 0.09 0.21 ± 0.03 0.14 ± 0.04 0.05 ± 0.01
Stomach 0.39 ± 0.18 0.42 ± 0.18 0.27 ± 0.33 0.02 ± 0.01
Small int 1.37 ± 0.75 0.60 ± 0.06 0.21 ± 0.09 0.03 ± 0.01
Lg.Int. 0.41 ± 0.54 1.53 ± 0,45 1.58 ± 0.70 0.15 ± 0.14
Muscle 0.10 ± 0.06 0.05 ± 0.00 0.03 ± 0.01 0.00 ± 0.0
Urine 169 ± 95 57 ± 15 6.30 ± 4.53 0.20 ± 0.02 Example 24) Construction and expression of hMN-14Fab-734scFv
[0193] Recombinant methods were used to produce a monovalent bi-specific fusion protein comprising a Fab fragment derived from a humanized monoclonal anti-CEA antibody and a scFv derived from a murine anti-dtDTPA. See Figure 3. The structure of single chain 734 (734scFv) was designed as GGGS (SEQ ID NO: 10)-V -(GGGGS)3 (SEQ ID NO: 9)-VH, in which the proximal GGGS (SEQ ID NO: 10) provides a flexible linkage for the scFv to be connected to the constant region of the heavy chain of hMN-14 (Figure 1). 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 hMN-14 heavy chain Fd to 734scFv were introduced into the VL and VJJ; domains of 734 by PCR reactions using specific primer sets. [0194) PC -amplification of 734Vr, was performed using the primer set 734VτjscFv5'(Cys) and 734VLSCFV3 ' (polypeptide and polynucleutide sequences for such primers are shown and described in U.S. Patent Application Serial No. 09/337,756, filed on June 22, 1999, the contents of which are incoφorated herein by reference in their entirety). The primer 73 VLScFv5'(Cys) represents the sense- strand sequence encoding the first four residues (PKSC) (SEQ ID NO: 12) of the human IgGl hinge, linked in-frame to the first six residues (QLVVTQ) of 734 V (SEQ ID NO: 13), via a short flexible linker, GGGS (SEQ ID NO: 10). One cysteine of the human hinge was included because it is required for the interchain disulfide linkage between the hMN-14 heavy chain Fd-734scFv fusion and the hMN-14 light chain. A Pstl site was incoφorated to facilitate ligation at the intronic sequence connecting the Cfil domain and the hinge. The primer 734Vj^scFv3 ' represents the anti-sense sequence encoding the last six residues (TKLKIL) of the 734 V domain (SEQ ID NO: 14) and a portion of the flexible linker sequence (GGGGSGGGG) (SEQ ID NO: 15), which is fused in-frame downstream of the VL domain. ]0195] Following PCR amplification, the amplified product (-400 bp) first was treated with T4 DNA polymerase to remove the extra "A" residue added to the termini during PCR-arnplification and subsequently was digested with Pstl . The resultant product was a double-stranded DNA fragment with a Pstl overhang and a blunt end. PCR amplification of 73 Vpr was performed using the primer set 734VHSCFV5' and 73 VHscFV3'(Sacl). Primer 734VHSCFV5' (see Patent Serial No. 09/337,756) represents the sense-strand sequence encoding the remaining part of the flexible linker sequence (SGGGGS) (SEQ ID NO: 16) connecting the Vτ_ and Vj-r sequences, and the first six residues (EVKLQE) of the 734 VH domain (SEQ ID NO: 17). Primer 734VHscFv3'(Sacl) (see Patent Serial No. 09/337,756) represents the anti-sense sequence encoding the last six residues (TvTVSS) of 734 Vfj (SEQ ID NO: 18). Also included is a translation stop coόon. The restriction sites Eagl and Sacl were incoφorated downstream of the stop codon to facilitate subcloning. Similarly, the PCR-amplified Vjj product o -400 bp was first treated with T4 DNA polymerase to remove the extra "A" residues at the PCR product termini, and then digested with Sacl, resulting in a Vff DNA fragment with a blunt end-sticky end configuration. (0196] A pBlueScript (Stratagene, La Jolla)-based staging vector (HClkbpSK) containing a SacII fragment of the human IgGl genomic sequence was constructed. The genomic SacII fragment contains a partial 5' intron, the human IgGl Cftf domain, the intronic sequence connecting the Cyl- to the hinge, the hinge sequence, the intronic sequence connecting the hinge to the Cjj2 domain, and part of the Cjj2 domain. The segment containing the hinge and part of the C]\2 domain in HClkbpSK was removed by Pstl/Sacl digestion, and the cloning site generated was used to co-Iigate the VL (Pstl/blunt) and Vjj (blunt/Sac 1) PCR products prepared above.
[0197] The Cfjl domain in the resultant construct (Cjjl-734pSK) is connected to the 734scFv gene sequence via an intron (Figure 4). Since the genomic SacII fragment for IgGl only included part of the 5' intron sequence flanking the C l domain, the full intronic sequence was restored by inserting the remaining intronic sequence as a BamHl/SacII segment, into the corresponding sites of the Cfjl-734pSK. The BamHl/Eagl fragment containing the full 5' intron, Cfjl domain, connecting intron, 5 hinge- residues, short GGGS linker (SEQ ID NO: 1 ), and a 734scFv sequences was then isolated, and used to replace the Hindlll/Eagl segment containing the human genomic IgGl constant sequence in the hMN- 14pdHL2 vector. A HNB linker (see Patent Serial No. 09/337,756) with a BamHl overhang on one end and a Hindlll overhang on the other was used to facilitate the BamHl Eagl fragment ligation into the Hindlll/Eag 1 site in the hMN-14pdHL2 vector. The resultant vector was designated hMN-14-734pdHL2 and can be used to transfect mammalian cells for the expression of the bi-specific protein. [0198J The hMN-14pdHL2 vector was derived from the vector, pdHL2, which has previously been described. See Losman et at., Cancer Supplement, 80:2660, 1997. Construction of hMN-14pdHL2 was performed by replacing the Vft and Vj domains of hLL2pdHL2 with that of hMN-14 using standard molecular biology techniques (Figure 5). The hMN-14-734pdHL2 vector was transfected into SP2/0 cells by electroporation and the cell clones secreting bsAb were identified. The bsAb purified from cell culture supernatant (clone 341.1G6) on a protein L column (Pierce, Rockford, IL) is a 75 kD protein (based on amino acid sequence calculation) that co-migrated with the 66 kD marker in non-reducing SDS-PAGE probably due to secondary structure (Figure 2, lane 2). Under reducing conditions, bands corresponding to a heavy (50 kD) and a light (25 kD) chain were observed (Figure 2, lane 4). Kappa chain monomers (25 kD) and dimers (50 kD) secreted by the transfectoma also were co-puήfied (Figure 2, lane 2) since protein L binds to kappa light chains of human, mouse and rat. Further separation of bsAb from kappa mono- and dimers is accomplished with ion-exchange chromatography. Purified hMN-14Fab-734scFv shows specific binding to both CEA and In-DTPA-BSA in a dose dependent manner.
Example 25) Transgenic production of bscAb in milk
[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. Afterbirth, 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.
Example 26) Transgenic production of bscAb in plants
[0200] A bscAb fragment is cloned into an expression vector containing a shortened legumin B4 promoter plus 54 base pairs of LeB4 untranslated RNA leader from Viciαfαbα and encoding a LeB4 signal peptide, to direct the protein to the endoplasmic recticulum. The expression cassette is transformed into tobacco leaf discs according to the methods described by Zambryski etαl., using Agrobαcterium-meάiated gene transfer. Transformation is verified by Southern analysis. Transgenic plants are analyzed for the presence and functionality of the bscAb using standard immunological methods known in the art. The bscAb can be purified from the plant tissues using standard methods known in the art.
Example 27) Pretargeting Experiments
[0201] Female nude mice (Taconic NCRNU, 3-4 weeks old) with GW 39 tumor xenografts were used for the pretargeting experiments. Tumors were 0.3-0.8 g.
Table 8
Biodistribution of 125-I-hMN-14 x 734 bsAb and 11 l-In-indium-IMP-156 peptide in nude mice bearing GW-39 tumor xenografts: hMN-14 x 734 was allowed 48 h for localization prior to 11 l-In-indium-IMP- 156 injection. Biodistiibution was performed 3 h post 11 l-In-indium-IMP-156. bsAb:peptide ratio administered, 1: 0.03. Five animals per time point.
125-I-hMN-14 x 734 11 l-In-indium-IMP-156
Tissue % ID/g T/NT % ID/g T/NT
tumor 2.9 ±1.1 1 5.2 ±1.9 1
Liver 0.1 ±0.06 19±6 0.5 ± 0.09 10.6 ±3.5
Spleen 0.5 ± 0.03 6.3 ±1.2 0.5 ±0.1 12 ±6
Kidney 0.3 ± 0.08 9.3 ±1.8 1.9 ±0.5 2.6 ±0.5
Lungs 0.3 ±0.1 12 ±3 0.4 ±0.1 12 ±2
Blood 0.3 ±0.1 11 ±2 0.7 ± 0.2 7.6 ±1.5
Table 9
Control group showing the clearance of 11 l-In-indium-IMP-156 at 3 h after injection.
% ID/g T/NT
Tumor 0.14 ± 0.02 I
Liver 0.42 ± 0.1 0.3 ±0.1
Spleen 0.28 ± 0.09 0.5 ± 0.1
Kidney 0.93 ±0.13 0.2 ± 0.03
Lungs 0.04 ±0.01 3.5 ±0.7
Blood 0.05 ± 0.01 3.1 ± 0.7
Table 10
Nude mice bearing GW 39 tumor xenografts were administered 125-I-labeled bsAb (5 μCi, 15 μg, 1.5 x 10-^0 mol). hMN-14 x 734 was allowed 24 h for localization and clearance before administering 99m-Tc- IMP-192 (10 μCi, 1.6 x 10-^ mol of peptide). Biodistribution studies were performed at 30 min, I, 3 and 24 h post 99m-Tc-IMP-192 injection, five animals per time point. BsAb:peptide, 1: 0,1.
125-I-hMN-l 4 x 734 % ID/g
Tissue 30 min 1 h 3 h 24 h
Tumor 4.9 ±1.1 6.0 ±2.3 5.5 ±1.1 3.3 ±0.7
Liver 0.6 ±0.1 0.5 ± 0.2 0.5 ±0.1 0.1 ±0.02
Spleen 0.8 ± 0.3 0.7 ±0.3 0.7 ±0.2 0.2 ± 0.03
Kidney 0.5 ±0.1 0.5 ±0.1 0.5 ±0.1 0.1 ±0.02
Lungs 0.9 ±0.3 0.8 ± 0.2 0.8 ± 0.3 0.3 ±0.1
Blood 0.9 ±0,3 1.2 ±0.4 1.1 ±0.3 0.2 ± 0.07
99m-Tc-IMP-192 %ID/g
Tissue 30 min lh 3h 24 h
Tumor 11.4 ±4.8 14.3 ± 3.6 12.6 ±5.2 8.7 ±3.3
Liver 1.4 ±0.3 0.9 ±0.2 0.6 ±0.1 0.4 ± 0.08
Spleen 1.2 ±0.4 0.8 ± 0.2 0.5 ±0.1 0.4 ± 0.2
Kidney 9.9 ±6.1 4.6 ±0.7 2.4 ±0.5 1.2 ±0.3
Lungs 4.2 ± 3.4 3.6 ±1.9 1.0 ±0.3 0.3 ±0.1
Blood 4.3 ±1.2 3.5 ± 0.9 1.7 ±0.4 0.6 ± 0.2
Table 11
Nude mice bearing GW 39 tumor xenografts were administered 125-I-labeled bsAb (5 μCi, 15 μg, 1.5 x 10-10 moi), hMN-14 x 734 was allowed 24 h for localization and clearance before administering 99m-Tc- IMP-192 (10 μCi, 1.6 x 10"1 * mol of peptide). Biodistribution studies were performed at 30 rain, 1, 3 and 24 h post 99m-Tc-IMP-192 injection, five animals per time point. BsAb:peptide, 1: 0.1.
125-I-hMN-14 Tumor / non-tumor ratio
Tissue 30 min lh 3h 24 h
Liver 8.8 ±1.5 12.1 ±5.5 10.3 ±2.5 23.8 ± 3.5
Spleen 6.4 ±1.6 9.3 ±4.0 7.9 ±1.7 18.2 ±4.0
Kidney 10.0 ±2.6 12.5 ±4.5 11.1 ±3.0 27.3 ±4.6
Lungs 6.2 ± 2.3 8.4 ± 4,6 7,2 ± 2.3 12.4 ±6.6
Blood 5.7±2.1 4.9 ±1.2 5.1 ±1.3 14.5 ± 3,6
99m-Tc-IMP-192 Tumor / non-tumor ratio
Tissue 30 min lh 3h 24 h
Liver 7.9 ±1.7 15.7 ±5.4 20.7 ±7.6 22.3 ± 7.4
Spleen 9.4 ±1.0 19.5 ± 8.6 22.9 ± 7.5 23.8 ±3.5
Kidney 1.2 ±0.2 3.1 ±0.6 5.2 ±1.5 7.3 ±1.9
Lungs 3.7 ±1.7 5.5 ± 3.6 13.5 ±7.1 30.8 ±14.4
Blood 2.7 ±0.7 4.2 ±1.3 7.3 ±2.3 16.1 ±6.4
Table 12
Control group of nude mice bearing GW-39 tumors received 99m-Tc-IMP-192 (10 μCi, 1.6 x 10"H 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- IMP-392 was calculated from the above data assuming one peptide bound to one bsAb molecule. However, it is possible that one peptide molecule can crosslink two molecules of bsAb.
Table 13
Percentage of the available DTPA binding sites on the tumor bound bsAb filled with 99m-Tc-IMP-192
% saturation on time hMN-14 x 734
30 min 25.4
1 h 25.8
3 h 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-14 x 734 (Fab x Fab) effectively targets a tumor; the dual functional peptidyl Tc-99m chelator was stable; 99m-Tc-IMP-192 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 bsAb Clearing Step
[0203] 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-Bz-DTPA-peptide, containing 50 mCi of Y-90. A CT 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
[0204] Two vials of rabbit liver carboxylesterase (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/ml) of the latter in DMSO. The final concentration of DMSO in the conjugation mixture is 3 % (v/v). After I hour of incubation, the mixture is pre-purifϊed 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 Supelco column using 0.2 M sodium phosphate pH 6.8 at 4 ml/min. The fraction containing conjugate is concentrated on a Centricon-lθTM concentrator, and buffer-exchanged with 0.1 M sodium acetate pH 6.5. 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-5 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 M 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-specific 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 mm segments, 6 μm, 6θA ) prep column eluting with a gradient of 80:20 to 60:40 Buffer A:B over 40 min (Buffer A= 0.3 % NH OAc, 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- 111 labeling. A solution was prepared which contained 5.014 g 2-hydroxypropyl-β-cyclodextrin, and 0.598 g citric acid in 85 mL. The solution was adjusted to pH 4.20 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 0.22 μjm 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 temperature for 10 min 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
[0208] 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 mL 0.5 M citrate buffer pH 4.0, 0.9 mL 0.5 M citrate 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 reverse phase HPLC. HPLC Conditions: Waters Radial-Pak C-18 Nova-Pak 8x100 mm, Flow Rate 3 mL/min, Gradient: 100 % A= 0.3 % NH OAc to 100 % B= 90 % CH3CN, 0.3 % H4θ c over 10 min. Table 14
In-Vitro Stability of In In- 111 IMP 224
Figure imgf000072_0001
Some peptide decomposed but was not included in the calculation of the areas of the peaks
Example 34) In-vivo biodistribution of IMP 221 in BALB/c mice
J0209] Kits were reconstituted with 400 μCi 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
7.2, 0.5 M acetate buffer. The labeled peptide was analyzed by ITLC in saturated NaCl. The loose In-111 was at the top 20 % of the ITLC strip.
[0210] Each mouse was injected with 100 μL (20 μCi) of the In-111 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 15
Biodistribution in BALB/c mice %ID/g of IMP 224 (Dox=N-NH-C6H4-CO-Lys(DTPA)-Tyr- Lys(DTPA)-NH2 MH+ 1847 radiolabeled with In-111 and saturated with cold In
Figure imgf000073_0001
Example 35) In-vivo stability and clearance of IMP 224
[0211] Kits were reconstituted with 4 mCi In-Ill in 0.5 L water. The In-111 kit was incubated at room temperature for 10 min and then diluted with 0.5 mL of the cold indium containing 0.5 M pH 7.2 acetate buffer. The labeled peptide was analyzed by ITLC in saturated NaCl.. The loose In-111 was at the top 20 % of the ITLC strip.
[0212] Each mouse was injected with 100 μL (400 μCi) of the In-111 labeled peptide. The animals were anesthetized and sacrificed at 30 min and 1 hr using two animals per time point. The serum 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 serum was too low to be analyzed by reverse phase HPLC due to the poor sensitivity of the detector. Doxorubicin has ~95 % hepatobiliary clearance. Thus, by attaching the bis DTPA peptide in a hydrolyzeab-e 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
Figure imgf000074_0001
Example 36) Pretargeting experiments with IMP 224 and IMP 225
[02131 A lyophilized kit of IMP 224 containing 10 micrograms of peptide was used. The kit was lyophilized in 2 L vials and reconstituted with 1 mL sterile water. A 0.5 mL aliquot was removed and mixed with 1.0 mCi In-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.
[0214[ Female nude mice (Taconic NCRNU, 3-4 weeks old) with GW 39 tumor xenografts were used for the pretargeting experiments. Tumors were 0.3-0.8 g. Each animal was injected with 100 microliters (5 μCi, 15 μg, 1.5 x 10 ~10 mol) of the 1-125 labeled antibody F6 x 734-F(ab')2 .
[0215| Seventy two hours later, each mouse was injected with 100 μL (lOμCi) 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 tail were collected and counted.
[0216J 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-11 l-IMP-224 in nude mice bearing GW-39 tumor xenografts, previously given F6 x 734-F(ab')2 ?2 earlier. Data in % ID/g tissue. n=5.
Figure imgf000075_0001
Table 18
Biodistribution of In-11 l-IMP-224 in nude mice bearing GW-39 tamor xenografts, previously given F6 x 734-F(ab')2 72 h earlier. Data in tumor-to-normal organ ratios. n=5.
Figure imgf000076_0001
Table 39
Biodistribution of In-11 l-IMP-225 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.
Figure imgf000077_0001
[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 Ϊ88-Re, 213-Bi, 67-Cu and the like, ft 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-operative, endoscopic, intravascular or other similar procedures, using the pretargeting methods 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-LvstHSG)-D-Tyr-Lys(ΗSG)-LvsfTscg-Cvs -NH-. (SEQ ID NO: I ) (IMP 245) [0219] 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 peptide with a single benzotriazole-l-yl-oxy-tris-(dimethylamino)-phosphonium hexafiuorophosphate (BOP) coupling using 5 eq of protected DOTA for 16 hr. The resin was then capped with acetic anhydride. The Aloe 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 Formulation
[0220] A formulation buffer was prepared which contained 22.093 g hydroxypropyl-β-cyclodextrin, 0.45 g 2,4-dihydroxybenzofc acid, 0.257 g acetic acid sodium salt, and 10.889 g α-D-gfucoheptonic 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 SnCfj (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 InCkj in 0.1 M HC1. The peptide solution was mixed with 2 mL 0.5 M NH4OAC and allowed to incubate at room temperature for 15 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 (37°C)
[0222] 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 37°C 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 tHPLC) of IMP 245
[0223] The peptide was analyzed by reverse phase HPLC and size exclusion HPLC (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., McBride, W.J., Karacay, H., Chang, K., Griffiths, G.L., Hansen, H.J., and Goldenberg, D.M., the entire contents of which are incoiporafed 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:
[02241 Tc-99m IMP 245 Alone 54%,
Tc-99m IMP 245 + hMN-14 x m679 66 %, Tc-99m IMP 245+ mMU-9 x m679 66 %.
Example 41) Serum Stability of IMP 245
[0225] An aliquot of the Tc-99m IMP 245, 50 μL, was diluted with 470 μL of fresh mouse serum and incubated at 37°C. Aliquots were removed and analyzed by reverse phase HPLC at 2.5 hr and 19 hr. The peptide appeared to be relatively stable.
Example 42) Synthesis of Cold Rhenium Oxo Complex of IMP 245
[0226] The Rhenium oxo complex was made by mixing 0.0504 g of IMP 245 with 0.0045 g of ReOBr4
N(bu)4 (synthesized by the method of Cotton et. al.) and 50 μL DIEA in 1 mL DMF for five days at room temperature. The entire reaction mixture was purified by HPLC to afford 0.0118 g of the desired product.
ESMS MH+ 2031
Example 43) Tc-99m Kit Formulation (Gentisic Acid Version)
[0227] The peptide, IMP 245 (0.0029 g, 1.58 x 10-6 moi) was dissolved in 2.0 mL of 0.5 M NH4OAC pH 5.5 buffer, which contained 0.0020 g of InClβ. The peptide solution was heated at 50°C for 17 min. A formulation buffer was prepared from 22.093 g hydroxypropyl-β-cyclodextrin (HPCD), 0.450 g 2,4- dihydroxybenzoic acid (gentisic acid), 0.257 g Acetic acid sodium salt, 10.889 g α-D-glucoheptonic acid and dissolved in 170 mL nitrogen purged DI water. The solution was adjusted to pH 5.30 with a few drops of IM NaOH and diluted to a final volume of 220 mL with DI water. The formulation buffer was then sterile filtered through a 0.22 ~:m filter. A stannous buffer was prepared by diluting 0.2 mL (200 mg mL SnCl2 m 6 M HC1) with 3.8 mL of the formulation buffer in an argon purged sterile vial. The peptide solution was then mixed with 76 mL of the formulation buffer and 0.56 mL of the stannous buffer. The solution was then dispensed in 1.5 mL aliquots through a Millex GV 0.22 mm filter into 3 mL lyophilization vials. The filled vials were immediately frozen on dry ice and lyophilized. The kits were sealed under vacuum at the end of the lyophilization cycle. Each kit contained 55 Tig of the peptide and was formulated for a 1.5 mL reconstitution volume of 99mTcθ4" in saline.
Example 44) Tc-99m Kit Formulation (Ascorbic Acid Formulation)
[0228] The Tc-99m kits formulated with ascorbic acid were prepared in the same manner as the gentisic acid kits except 0.222 g ofL-ascorbic acid was used instead of gentisic acid.
Example 45) Tc-99m Kit Labeling [0229] The kit was reconstituted with 1.5 mL of 99m cθ4- in saline (0.5 to 70 mCi) and incubated at room temperature for 10 min, The kit was then heated in a boiling water bath for 15 min and allowed to cool to room temperature before use.
Example 46) Labeling & Stability of Tc-99m/In IMP 245
[0230] Early labeling attempts demonstrated that it was preferable to fill the DOTA with cold indium to afford a high yield of Tc-99m/In IMP 245 from the kits. The gentisic acid formulation gave a cleaner initial labeling of the peptide when labeled at 30 mCi Tc-99m in 1.5 mL but the ascorbic acid formulation afforded a kit with much greater stability when the peptide was stored overnight at room temperature. Early stability studies at 37CC in fresh mouse serum showed that the Tc-99m labeled peptide was as stable in serum as in the kit, HPLC analysis on an expanded gradient revealed that the labeled peptide had two peaks. The two peaks were probably due to the formation of syn and anti Tc oxo species. The ratio of the peaks can change depending on the peptide sequence, formulation and labeling conditions.
Example 47) Y-90 and In-111 Labeling of IMP 245
[02311 The peptide was dissolved in 0.5 M NH4OAC, pH 3.08 at 2.2 x 10-^M (peptide). An aliquot, 3.5 μL of the peptide solution was then mixed with 165 μL of 0.5 M NH4OAC pH 3.93 and 6 μL of the Y-90 solution. The mixture was then heated for 20 min at 85-95°C. Reverse phase HPLC showed that the peptide labeled well.
[0232] An analogous labeling process was attempted using In-111 under a number of conditions none of which led to a clean, labeled product. Subsequent HPLC analysis of the cold peptide showed that it had formed several new peaks. The peptide was probably forming disulfides on storage. The Tc Re ligand was then pre-filled with cold rhenium to stabilize the peptide for Y-90, Lu-177, and In-111 labeling.
Example 48) In-111 Labeling of ReO IMP 245
[0233] The peptide, 0.0025 g ReO IMP 245 (MH+ 2031) was dissolved in 560 μL 0.5 M NH4OAC pH 3.98 buffer (2.2 x 1 "3 M peptide). An aliquot, 2.7 μL of the ReO IMP 245 was mixed with 2 μL of In- 1 11 (573 μCi) and 150 μL of the 0.5 M NH4OAC pH 3.98 buffer. The solution was then heated in a boiling water bath for 20 min. HPLC analysis showed a clean, labeled peak with a comparable retention time to Tc-99mΔn IMP 245.
Example 49) Generation of Peptides Suitable for Radiolabeling with 0Y. * Hn. and ^Lα.
Preparation ofbsMAbs
[0234] The bi-specific F(ab')2 antibody composed of Fab' fragments of humanized MN-14 anti-CEA or murine Mu-9 anti-CSAp and murine 679 were prepared using PDM as the crosslinker. The F(ab')2 of each parental antibody was first prepared. For hMN-14 or Mu-9, the F(ab')2 was reduced with 1 mM DTT to Fab'-SH, which was diafiltered into a pH 5.3 acetate buffer containing 0.5 mM EDTA (acetate/EDTA buffer) to remove DTT, concentrated to 5-10 mg/mL, and stored at 2-8 °C until needed. For 679, the F(ab')2 was reduced with 1 M DTT to Fab'-SH , which was then diluted with 5 volumes of 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 free PDM is minimum, and concentrated to 5-10 mg/mL. A solution of hMN-14 Fab'-SH or Mu-9 Fab'-SH was then mixed with a solution of 679 Fab'-PDM at a 1:1 ratio based on the amount of Fab'. Adding cysteine to a final concentration of 2 mM quenched the conjugation reaction and the desirable bi-specific conjugate (-100 kDa) was obtained following purification on a Superdex 200-packed column (Amersbam, Pharmacia Bio, Piscataway, NJ). The bi-specific conjugates were analyzed by SE-HPLC, SDS-PAGE, and IEF. For hMN-14 x m679 F(ab')2, the bi-specificity was demonstrated by BIAcore as well as by SE-HPLC. In addition, the affinity of hMN-14 x 679 for HSG was determined by BIAcore analysis using a CM-5 chip derived with a peptide containing a single HSG substituent and a thiol by the method recommended by the manufacturer (Biacore, Inc., Piscataway, NJ 08854).
|0235] For biodistribution studies, the hMN-14 x m679 F(ab')2 was radioiodinated with
Figure imgf000081_0001
(Perkin Elmer Life Science, Inc. Boston, MA) by the chloramine-T method (20), and purified using centrifuged size-exclusion columns. Quality assurance testing found < 5% unbound radioiodine by ITLC, > 90% of the product migrating as a single peak by SE-HPLC (Bio-Sil SE 250, Bio Rad, Hercules, CA), and > 90% of the radiolabeled product shifting to a higher molecular weight with the addition of an excess of CEA (Scripps Laboratories, San Diego, CA). 125j_mMU-,α x m679 bsMAb was tested in a similar manner, using a partially purified extract from GW-39 human colon xenografts as a source of CSAp, which shifted the elution profile of the mMu-9-x679 bsMAb to the void fraction of the SE-HPLC column. [0236] Humanized MN-14 (hMN-14) Fab'-SH was prepared in a similar manner as described previously. 99mτc-ρertechnetate (30 mCi) was added directly to the lyophilized hMN-14- Fab'-SH (1.0 mg) and injected in animals within 30 minutes. This product had 3.0% unbound 99πτpc DV ULC and an immunoreactive fraction of 92%.
Radiolabeling of Peptides
[0237] The divalent HSG-peptide, IMP 241 used for 90Y-, 17?Lu- and 11 ^n-radiolabeling contains a DOTA ligand to facilitate the binding of these radiometals. IMP 241 was dissolved in 0.5 M ammonium acetate (pH 4.0) to a concentration of 2.2 x 10-3 jyf. 90 YQ^ was obtained from Perkin Elmer Life Sciences, Inc. (Boston, MA), flπCl3 from IsoTex Diagnostics (Friendswood. TX), and l^T u from the Research Reactor Facility, University of Missouri-Columbia, (Columbia, MO).
[0238] H I In-IMP 241 was prepared by mixing 3 mCi of I ^ InCl3 in a plastic conical vial with 0.5 M ammonium acetate, pH 4.0 (3x volume of 1 1 3InCl3) and 2.3 μL of IMP 241 (2.2 xl0"3 jyf in 0.5 M ammonium acetate, pH 4.0), After centrifugation, the mixture was heated in a boiling water bath for 30 min and cooled. The mixture was centrifuged and DTPA was added to a final concentration of 3 mM. After 15 min at room temperature, the final volume was raised to 1.0 mL with 0.1 M sodium acetate, pH 6.5. The amount of unbound isotope was determined by reverse phase HPLC and ITLC developed in saturated sodium chloride solution. Reverse phase HPLC analyses were performed on a Waters 8 x 100 mm radial Pak cartridge filled with a C-18 Nova-Pak 4 μm stationary phase. The column was eluted at 1.5 mL/min with a linear gradient of 100 % A (0.075 % TFA in water) to 55 % A and 45 % B where B was 0.075 % of TFA in 75 % acetonitrile and 25 % water over 15 min. At 15 min, solvent was switched to 100%B and maintained there for 5 min before re-equilibration to initial conditions. Reverse HPLC analyses showed a single peak at 11.8 min. Analysis of 111 In-IMP 241 mixed with excess m679 IgG on a Bio-Sil SE 250 HPLC gel filtration column showed a peak at the retention time of the antibody indicating binding to the antibody.
[0239] IMP-241 was radiolabeled with 0Y by adding to 15 mCi of 90YC13, 3-times the volume of 0.5 M ammonium acetate, pH 4.0 and 83.2 μL of IMP 241 (1.1 lQ-^ M in 0.5 M ammonium acetate, pH 4.0), and ascorbic acid to a final concentration of 6.75 mg/mL. The mixture was heated in a boiling water bath for 30 min, and after cooling to room temperature, DTPA was added to a final concentration of 5 mM. Fifteen minutes later, the final volume was increased to 1.0 mL with 0.1 M sodium acetate, pH 6.5. ITLC strips developed in saturated sodium chloride solution showed < 0.2 % unbound isotope. Analysis of 9^Y- IMP 241 mixed with an excess of m679 IgG by SE-HPLC showed a peak at the retention time of the antibody indicating binding to the antibody.
[0240] The stability of the radiolabeled peptides was tested in mouse serum by diluting each of the radiolabeled peptides 10-fold in mouse serum and incubating the solution at 37°C. Samples were removed at I, 3, and 24 h and analyzed by reverse-phase HPLC.
In Vivo Pretargeting Studies
[0241] GW-39, a CEA-producing human colon cancer cell line (See, Goldenberg, D.M. and Hansen, H.J, Carcinoembryonic antigen present in human coionic neoplasms serially propagated in hamsters, Science, 175:1117-18 (1972)) was serially propagated in nude mice by mincing 1-2 grams of tumor in sterile saline, passing the minced mixture through a 50-mesh wire screen, and adjusting the saline volume to a final ration of 10 ml saline per gram tumor. Female NCr nude mice (Charles River Laboratories, Inc., Fredrick MD or Taconic, Germantown, NY) approximately 6 weeks of age were implanted subcutaneously with 0.2 ml of this suspension. Two to three weeks after implantation of tumors, animals were injected with the radiolabeled peptide alone, or for pretargeting, with the bsMAb followed 1 to 2 days later with the radiolabeled peptide. For pretargeting, 1.5 x 10"lυ moles (15μg; 6 μCi 125j) αf the bsMAb was injected intravenously (0.1 to 0.2 mL) followed with an intravenous injection (0.1 to 0.2 mL) of n ιIn-IMP-241 (1.5 x lO-H moles, 8-10 μCi), 177LU-IMP-241 (1.5 x 10"H moles, 5 μCi), or 99mTc.τMP-243 (1.5 x 10- 11 , 25-30 μCi). 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.
Results
Table 20
Figure imgf000083_0001
a Radioactivity concentration below threshold of detection.
Table 21
Figure imgf000083_0002
a Ra ioact vity concentration below threshol of etection.
Figure imgf000084_0001
Table 23
00
Figure imgf000085_0001
Table 24
Figure imgf000086_0001
Table 25
Tumor/nontumor ratios for yymTc-hMN-14 Fab' in GW-39 tumor-bearing nude mice 3 h after injection, (n = 5)
Tissue y9mτc-hMN-14 Fab'
Liver 0.2 ± 0.02 Spleen 0.9 ± 0.4 Kidney 0.02 ± 0.001 Lungs 0.7 ± 0.1 Blood 1.0 ± 0.01
Table 26
Figure imgf000087_0001
Figure imgf000088_0001
Table 27
Figure imgf000088_0002
Figure imgf000089_0001
Table 28
Figure imgf000089_0002
Table 29
Figure imgf000090_0001
a Radiation absorbed doses are normalized to 1500 cGy to the kidneys
Radiolabeling of Peptides and Testing of BsMAbs
[0242] IMP-241 's DOTA chelation group can be used with 1 ! fin, 90γ; and omer radiometals, such as 17 u. τhe peptide was radiolabeled with each of these radionuclides to specific activities of about 600, 1650, and 300 Ci mmol, respectively. The lower specific activity for ^7LU was attributed to both the age of the product at the time it was used and the isotope production run that was not performed in a manner to optimize the specific activity of l^Lu. The specific activity of the yy Tc-ρeptides was between 1500 and 1600 Ci/mmol. In each instance radiolabeling conditions were developed to ensure > 98% incorporation of the radioactivity in the peptide so that no purification was required. Reverse phase HPLC indicated that when mixed with fresh mouse serum at 37°C, all of the peptides were stable over 24 h, retaining the original elution profile as seen after their preparation. HPLC analysis of the IMP-243 and 245 on an expanded gradient revealed that the labeled peptide had two peaks, likely due to the formation of syn and anti technetium oxo species.
[0243] Figure 6 shows the binding of the hMN-14 x m679 bsMAb to 111In-IMP-241 by SE-HPLC. Essentially all the radiolabeled peptide is shifted to the bsMAb elution time, and when CEA is first added to the bsMAb followed by the addition of the radiolabeled peptide, the entire amount of radioactivity shifts to the void fraction. Similar results were found with the mMu-9 x m679 bsMAb when using the CSAp preparation (not shown). The kinetic binding of hMN-14 x m679 F(ab')2 bsMAb to the mono HSG peptide on the chip was evaluated by BIAcore and found to be KD=1.5 x 10"y M. Peptide Biodistribution
[0244] For biodistribution purposes, IMP-241 was radiolabeled with the gamma-emitting radionuclides, ^ ^In or 177LU) t0 facilitate the peptide's detection in tissues, while IMP-243 and IMP- 245 were radiolabeled with yymTc. In tumor-bearing nude mice, the 1 7τ__u- and *■ lln-IMP-241 had 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 kidney weight of 0.15 g, there was only about 0.6% of the total injected activity in the kidney at 0.5 to 1.0 h after injection. An additional group of animals given the ^^Lu-IMP-241 was necropsied at 48 h, but since there was only enough radioactivity in the kidneys for accurate reporting, the data are not presented in the table. However, the T77j--u-IMP-241 in the kidneys had decreased to a level of 0.94 ± 0.2 %ID/g, which represented about a 45% decrease compared to the level seen at 24 h. The vast majority of the radioactivity was excreted in the urine, but there was also a very small fraction of the radioactivity that cleared through the GI tract. From 1.0 to 3.0 hours, about 0.6 to 0.7% of the total injected activity can be accounted for in all the GI tissues (i.e., stomach, small and large intestine). By 24 h, only 0.07% of the radioactivity was accounted for in all the GI tissues.
[0245] The tissue distribution of y9r«Tc-IMP-243 was considerably different than the IMP-241 (Table 22). There was a slower clearance from the blood, a higher uptake in the liver, and a substantial fraction in the GI tract. For example, 1 hour after injection, the small intestine contained 24.3 + 4.75 %ID/g of the 99mTc_iMP-243, and by 3 h, the activity had shifted to the large intestine. By 24 h, the activity was fully cleared from the body. Thus, the radioactivity was not associated with the GI tissues per se, but was in the GI contents, as seen with the progression of the radioactivity through the small and large intestines. Another peptide, IMP-245, had a much smaller fraction of the radioactivity in the GI tissues. Liver and renal retention were also appreciable lower than that seen with yymTc-IMP-243. .
Pretargeting Studies
[0246] The hMN-14 x m679 F(ab')2 bsMAb was used to test the pretargeting capabilities of the 99mχc. Mp_243 and 9 mτc-iMp_245. The bsMAb was radiolabeled with 125I so that its distribution could be co-registered with either the 99nv c..τ]yrp.243 or IMP-245. The bsMAb was given to animals i.v., and after 24 h, the radiolabeled peptide was given and animals were necropsied 3 and 24 hours later. In the pretargeting setting, tumor uptake of the yymTc-IMP-243 was nearly 28 and 70 times higher than that seen with peptide alone at 3 and 24 h after its injection (Table 23). Tumor uptake was 12.25 ± 3.32 % ID/g at 3 h, reducing to 7.36 ± 3.19 by 24 h. The reduction of 99niTc-IMP-243 in the tumor over this time was not as high as the reduction of the bsMAb in the tumor, which dropped from 4.78 ± 1.11 %ID/g to 2.24 + 0.53 %ID/g over this same period. Tumor/nontumor ratios for y9mχc. IMP-243 were all greater than 2.0:1 within 3 hours, except for the large intestine where the peptide had not yet cleared, but this improved nearly 20-fold by 24 h. Tumor/blood ratios were 2.4 ± 0.6 at 3 h after peptide injection. Tumor uptake for yymTc-IMP-245 was similar to that seen with yymTc-IMP- 243 (Table 6), but tumor/nontumor ratios favored the yymTc-IMP-245, primarily because the bsMAb had cleared to a lower level in these animals than in the animals that had received the yymTc-IMP-243. However, ^^mTc-IMP-245 pretargeting also had lower GI uptake, even at 1 hour, and therefore this peptide has a distinct advantage over 99mTc-IMP-243, Tumor kidney ratios for 99mTc-IMP-245 were higher than those obtained with 99nτrc-iMP-243. These biodistribution data suggest that pretargeting with yymTc-IMP-245 should provide better image contrast at an earlier time than that found with a directly radiolabeled Fab' fragment. It should also be emphasized that the tumor/kidney ratio using the yymTc-labeled peptides was substantially higher than an antibody fragment directly radiolabeled with "mTc-hMN-14 Fab' 3 h after its injection (Table 25).
[0247] Two different targeting systems were used in the evaluation of pretargeting the IMP-241 peptide, one system used a humanized anti-CEA antibody (hMN-14) while the other used a murine antibody to CSAp (mMu-9). Each bsMAb was prepared by chemically coupling its Fab' to the Fab' of the murine 679 MAb. For biodistribution studies, each bsMAb was radiolabeled with 125τ so jnat jts distribution could be assessed together with the IMP-241, which was radiolabeled with ^ *In. The amount of bsMAb and peptide injected in tumor-bearing nude mice was the same in each pretargeting system, but because the Mu-9 bsMAb took longer to clear from the blood than the hMN-14 bsMAb, the radiolabeled peptide was given at 48 h after the Mu-9 bsMAb compared to 24 h after the hMN-14 bsMAb. By using a 24-h delay for the hMN-14 x m679 construct and a 48-h delay for the mMu-9 x m679 construct, the blood levels of each bsMAb were similar, 0.79 ± 0.24 %ID/g and 0.55 ± 0.10 %ID/g, respectively. It was not unexpected to find a higher amount of the Mu-9 bsMAb in the tumor (13.1 ± 4.36 %ID/g) than the MN-14 bsMAb (2.92 ± 0.41), since earlier studies comparing the targeting of the Mu-9 and anti-CEA antibodies had found Mu-9 to have a higher uptake and a longer retention in the GW-39 xenograft model than that seen with anti-CEA antibodies. With a higher amount of Mu-9 bsMAb in the tumor, a higher concentration of the peptide was achieved, reaching a level of 17.8 ± 1.4 %ID/g in just 3 hour after the peptide injection compared to I I .3 ± 2.2 %ID/g for the peptide in animals pretargeted with the hMN-14 bsMAb. Interestingly, the hMN-14 bsMAb was more efficient at binding the peptide, since the ratio of the %ID/g of the peptide compared to the bsMAb in the tumor was 3.9 for the hMN-14 bsMAb at 3 h compared to 1.4 for the Mu-9 bsMAb at this same time. However, 11 lln-IMP-241 was retained by the tumor in Mu-9 pretargeting system for a longer period of time, which corresponded to the extended time that the bsMAb was bound to the tumor. In each system, the peptiderbsMAb ratio observed at 3 h was maintained over the 48-h observation period, suggesting that the peptide was bound specifically by the bsMAb. Pretargeting increased tumor accretion of the 1 * ! In-IMP-241 nearly 100-fold compared to the peptide alone (refer Table 20). With pretargeting, tumor/nontumor ratios were also significantly improved for all tissues as compared to that seen with the 1 lln-241 peptide alone, regardless of which bsMAb pretargeting system was used. Overall, tumor/nontumor ratios for the 1 * 1 In-IMP-241 were significantly higher in the Mu-9 bsMAb system, especially over time. [0248] Regardless of whether IMP-241 was radiolabeled with 177τ_u or 11 ^In, the pretargeting results were the same. As seen in Table 27, using the hMN-14 bsMAb pretargeting system, the %ID/g of the 177Lu-IMP-241 was identical to that seen with H lln-IMP-241. Because ^In-IMP^l's distribution mimicked 177Lu-IMP-241 , and since * * In has also been used as a surrogate for predicting y^Y- distribution, an extended biodistribution study was performed using the 1 * 1 In-IMP-241 and the mMu-9 x m679 F(ab')2 bsMAb. As shown in Figure 7, as a consequence of extended retention of the Mu-9 antibody in the tumor, there was also an excellent retention of the radiolabeled peptide in the tumor. Using these data, radiation dose estimates were modeled for y^Y and ^77Lu. yυY, because of its higher beta-radiation energy (2,27 MeVrøa ), delivers a higher radiation dose to the tumor than f77j_u (495 keVmax) on a per mCi basis. However, in order to make a better comparison, the radiation doses were normalized to reflect an identical radiation to a dose-limiting organ. In this case, 1500 cGy to the kidneys was selected as a dosage that should be tolerated, but could result in similar toxicities. When the absorbed doses to the tissues were normalized, the data suggest that 177LU-IMP-241 would potentially deliver the same dose to the tumor as y^Y-IMP-241. If the kidneys were able to tolerate 1500 cGy, then the tumor would receive nearly 12,000 cGy, a radiation dose that should be lethal to most solid tumors.
[0249] It will be apparent to those skilled in the art that various modifications and variations can be made to the compositions and processes of this invention. Thus, it is intended that the present invention cover such modifications and variations, provided they come within the scope of the appended claims and their equivalents.
[0250} The disclosure of all publications cited above are expressly incorporated herein by reference in their entireties to the same extent as if each were incorporated by reference individually.
Additional references of interest include the following: Araπσ Y, Uezono T, Akizawa H, Ono M, Wakisaka K, Nakayama M, Sakahara H, Konishi J,
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Kranenborg, M.h., Boer an, O.C., Oosterwijk-Wakka, j., weijert, M., Corstens, F., Oosterwijk, 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 I.S., Wang J., Hansen H.J., Goldenberg D.M., Leung S.O. Clin. Cancer Res. 1999; 5(10 Suppl.);3101s-3105s.
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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 Chem 1997; 8:595-604.
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All references cited herein are hereby incoφorated herein by reference in their entireties.

Claims

WHAT IS CLAIMED IS:
1. A compound of the formula: X-Phe-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.
7. The compound of claim 6, wherein the radionuclide is selected from the group consisting of 225 Ac, 11 g, 72As, 77As, 21 lAt, ^8All! 199AUj 212Bi, 2l3Bi, 75Br, 76Br, 1 lC, 55Co, 62cu, 64Cu, 67Cu> I66Dyj 169Elj 18F, 52pC} 59FCy 67QΆ> 68Ga> 254 3d, 155Gd, 156Gd, 15 Gd, 158Gd, 166Ho, 120T, 123τ; 124L 12S 131If 110τnj 111^ 194fr) I77Lu> 51Mn, 52mMπ, 99Mo, 13N> 150) 32P> 33P, 211Pb, 212Pb> 109Pd) 149Pm> 142Pr> 143Pr> 223Ra> 82mRb; 186Re> 188Re> 189Rej 105^, 47Sc> 153Sm, 75Se, 83Sr, 89Sr, 161Tb, 94mτc, 94χC) 99mTCj 86γ> 90γ, 90Yj ^d 89zr; and, when the targetable construct includes more than one radionuclide, the radionuclides may be different radionuclides.
8. The compound of claim 1 comprising: DOTA-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 Ilia 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 contract 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.
18. The compound of claim 17, wherein the radionuclide is selected from the group consisting of 25 Ac, 111 Ag, 72 As, 77As, 211 At, 198Au, 199Au, 12Bi5213Bj} 75Br> 76Br, l lC, 55Co, 62Cu, 64Cu> 67Clij 166Dyj 169Er, 18F, 52Fe, 59Fe> 67Ga> 68Ga) 154Gd, 155Gdj 156Gd, 157Gd) 158Gd, 166Ho> 120^ I23 124τ, 125 131 110m, lllin, 194^
177Lu, 5lMn, 52 Mn, 99Mo, 13N> 150. 32P) 33Pj 21 lPb) 212Pb, 109Pdj l49Pm> 142Prj 143Pr> 223Ra> 82mRb> 186R^ 188Rs> 189Re, 105^ 47SCj 153Sm, 75Se> 83Sr, 89Sr, 161Tb> 94mχC) 94τc, 99mTCj 86Y> 90γ, 90γ> and 89zr; and, when the targetable construct includes more than one radionuclide, the radionuclides may be different radionuclides.
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- A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
22. The targetable construct of claim 13, wherein said therapeutic agent comprises 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 photosensitizer.
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 (MRI).
27. The targetable construct of claim 26, wherein said enhancing agents include Mn, 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, iopa idol, iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, -otetric acid, iothalarm'c acid, iotroxic 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-filled.
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 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) 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 the 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 specifically 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.
37. The method of claim 34, wherein said therapeutic cation is selected from the group consisting of 11 I In, 177Lu, 212Bl, 213Bl, 21 lAt> 62C , 64Cn, 67Cu> 90γ, US , 131L 32P, 33P, 47SC) HlAg, 67Ga, 142Pr, 153Sm> 161 b, 166Dy, 166Ho, 186Re, 188Re, 189Re, 212pb, 223^, 225AC; 59Fe> 75Se, 77As, 89Srj 99Mo, 105Rh, 109Pd, 143Pr> 149Pm> 169Er, 194ιr) 198AuJ 199Au and 11Pb.
38. The method of claim of claim 34, wherein the diagnostic cation emits particles and/or positrons having 25-10,000 keV.
39. The method of claim 34, wherein said diagnostic cation is selected from the group consisting of 1 lOfa, 11 lln, 1 7LU, 18F, 52Fe, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 86γ, 90γ> 89Zr, 94mTc> 94Tc. 99mTC) 1201, 123τ, 124Tj 125τ, 131^ 154-158Gd, 32P; 11C, 13N, 150, 186Re> 188Re> 5 lMn, 5 mMn> 55Co, 72As, 75Br> 76Br, 82mRb and 83Sr.
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 more 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 more 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, metiizoate, 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 chemiluminescent compound, and a bioluminescent compound.
50. The method of claim 49, wherein said fluorescent compound is selected from the group consisting of fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamiπe.
51. The method of claim 49, wherein said chemiluminescent compound is selected from the group consisting of Iuminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.
52. The method of claim 49, wherein said bioluminescent compound is selected from the group consisting of luciferin, 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 2/neu, PAM-4, TAG-72, EGP-1, EGP-2, A3, KS- 1, Le(y), SI 00, 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 arm 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 arm 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.
60. The method of claim 59, wherein said therapeutic nuclide is selected from the group consisting of H lln, 177Lu, 212Bi 213Bi, 21 -Al, 62 u. WQ,, 67CU, 90Y> 1251, 131I; 32Pj 33P, 47Sc, 111 Ag, 67Ga. I42Pr, 153Sm, 161τb, 166Dy5 166Ho> 186Re> 188Re, 189Re) 212Pbj 223Ra, 225Ac, 59Fe, 75Se, 77As, 89Sr, 99Mo, 105RH, 109Pd> 143pf) 149ft,,, 169Erj 194Ir) 198Au, 199Au an 211Pb.
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 MN-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-specifw 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-Ill 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 l^B 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 antibody or antibody fragment comprising at least 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 schenc ii, Cyrptococcus neoformans, 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), heφes virus, cytomegalovirus, rabies virus, influenza virus, hepatitis B virus, Sendai virus, feline leukemia virus, Reo virus, polio virus, human serum parvo-Iike virus, simian virus 40, respiratory syncytial virus, mouse mammary tumor virus, Varicella-Zoster virus, Dengue virus, rubella virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus 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 gonoιτhoeae, 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, Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japanicum, Babesia bovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis, Onchocerca volvulus, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus and Mesocestoides corti.
85. The method of claim 79, wherein said mycoplasma is selected from the group consisting of Mycoplasma arthritidis, Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma arginini, Acholeplasma laidlawii, Mycoplasma salivarum, and Mycoplasma pneumoniae.
86. The method of claim 78, wherein said targetable construct further comprises at least one radionuclide, therapeutic agent, diagnostic agent or enzyme.
87. The method of claim 86, wherein the radionuclide is selected from the group consisting of 225Ac, H 'Ag, 7 AS, 77AS. 211AJJ 198Au> I99Au, 212Bi, 213Bi, 75Br, 76Br, 1 lC, 55Co, 62Cu, 64Cu, 67Cu> I66Dy> 169Er> 18Fj 52Fe, 59Fej 67Ga, 68Ga> 154Gd) 155Gd, 156Gd, 157Gd> 158Gd, 166Ho, 1 0 I23τ, 12 ι, 12SIf 131L 110Iπ> lllιn, 194fr, l77Lu, 5lMn, 52mMn> 99Mθι 13N> 150) 32P, 33P, 2UPθι 212Pb) 109pό< 149Prn> 142pr> 143Pr 223Ra, 82mRb> 186Re> 188Re, 189Re, 105^ 47Sc, 153Sπij 75Se, 83Sr, 89Sr, 161τ , 94mτc, 94TC; 99mTc, 86Yj 90γ, 90Y) ^ 89Zr; and, when the targetable construct includes more than one radionuclide, the radionuclides may be dffierenf radionuclides.
88. The method of claim 86, wherein the diagnostic agent includes an imaging agent.
89. 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 arm that specifically binds a targeted tissue and at least one other arm 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 construct comprising the compound of claim 1.
90. The method of claim 89, wherein said subject is mammalian.
91. 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.
93. The method of claim 92, wherein the radionuclide is selected from the group consisting of 225Ac, 11 g, 72As, 7AS, 21 IAt, 198Au, 199Au, 2I2Bi, 2I3Bi, 5Brj 76Br> He, 55Co, 62Cu, 64Cu, 67Cu, 166Dy, l69Er) 18F) 52F6j 59FC) 67Ga, 68Ga, 154Gd, 155Gd> 156Gd, 157Gd, 158Gd, 166Ho, 1 0L 123τ, 124L 125J., 131ι, HOfo, l lljn, 194IΓJ I 77 u> Sl n, 52mMnj 99Mθι 13N> 150> 32P> 33Pj 211Pb> 212Pbj 109Pdj 149Pm, 142Pr> 143Pr> 223Ra, 82mRb, 186Re, 188Re, 189Re, 105Rh, 47SCj 153Sm, 75Se, 83Sr, 89Sr, 161τb, 94mTc, 94Tc> 99mτc, 86Yj 90Y> 90γ> ^d 89Zr; and, when the targetable construct includes more than one radionuclide, the radionuclides may be different radionuclides.
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.
97. The kit of claim 96, wherein said diagnostic agent selected from the group consisting of l lOUi. l l lj . 177Lu> 18F) 52Fe, 62Cu, 64Cu, 67Cu, 67Ga, 6°Ga, 86γ, 90γ, 89Zr, 94mTc, 9 Υc> 99mTC) I20If 123 124I( I25I; 131^ 154-158Gd) 32Pj llCj 13N; 150, 186Re> 188Re, 51Mn> 52 n. 55c0, 72As, 75βr, 76βr, 82mR, md 83Sn
98. The kit of claim 96, wherein said therapeutic agent is selected from the group consisting of 11 lln, 177 u, 212Bi, 213Bi, 21 lAt, 62Cu> 64Cu, 67Cu, 90γ5 125T; 131 32P, 33P, 7Sc, iπAg, 67Ga, 142Pr, 153Sm> 161τb, l66Dy, 166Ho> 186Re, 188Re> 189Re> 212Pb? 223Ra, 225Ac, 59Fe> 75Se> 77As, 89Sr, 99Mo, 105Rh; 109Pd> 143Pr> 149Pm, 169Er, I94Irj 198Au, ∑99Au and 212Pb.
99. The method 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-specffic 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.
104. The method of claim 103, wherein the radionuclide is selected from the group consisting of 225Ac. 11 g, 72As, 77As, 211 At, 198Au, 199All) 212Bi, 213Bi> 75Br, 76Br, 1 lC, 55Co, 62Cu, 64CU) 67Cu, 166Dyj 169Er, 18F, 52Fej 59Fe> 67Ga, 68Ga, 154G , 155Gd, 15όGd. l57Gd, ^°Gd, ^Ho, 120^ 12 ^ 124τ> 125T) I31Is HOfo, I I IT^ I94τr> 177Lu, 51Mn, 52mMn, 99Mo, 13N> 15o, 32P; 33P> 211Pb> 212Pb> 109Pd, l49Pm, 142Prj 143pr> 223R3, 82mRb) 186RCj 188Rej 189Re> 105^, 47Sc> 153Sπl) 75Se, 83Sr, 89Sr>
Iδljb, 94m jc5 94Tc> 99mχCj 86γ> 90γ, 90γ> and 8 zr; and, when the targetable construct includes more than one radionuclide, the radionuclides may be different radionuclides.
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 imaging 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 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.
111. The method of claim 110, wherein said targetable construct further comprises at least one radionuclide, therapeutic agent, diagnostic agent or enzyme.
1 12. The method of claim 111, wherein the radionuclide is selected from the group consisting of 225AC, l ^A , 72 S, 7AS, 211At> 198 U, 1 9AU, 12BJ, 213Bi. 75Br, 76βr, 1 lC, 55Co> 62cu, 64 u, 67Cu, 166Dyj 169Erj 18F> 2Fej 59Fe, 67Ga, 68Ga, 154Qd, 155Gd) 156Gd, 157Gd, 158Gd, 166Ho> 120^ 123It 124τ> \25i_ lr, - -0_h, -Ufo, J9 fr, 177Lu, 51Mn, 52mMn, 99Mo> 13Nj 150, 32Pj 33P) 211Pb; 212Pb) 109P > 149Pm, 142?,-, 143Pr) 223Ra, 82mRb5 186Rβj 188Re, 189Re, 105^, 47Sc, 153Sm, 75Se, 83Sr, 89Sr, 161χ 5 94mχC; 94TCj 99mTc> 86γ> 90γ5 90YJ mά 89Zr; and, when the targetable construct includes more than one radionuclide, the radionuclides may be different radionuclides.
213. The method of claim 111, wherein the diagnostic agent includes a contrast agent.
114. The method of claim 111, 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 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 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.
118. The method of claim 117, wherein the radionuclide is selected from the group consisting of 225Ac, 111 Ag, 72AS, 77AS, 21 lAt, 198 u> 199Au> 212B}, 213BJ, 75Br, 76Br> I lC, 55Co, <5 Cu, < Cu, &Cu, 166Dy, 169Er> 18F) 52Fe> 59Fe> 67Ga, 68Ga, 154Gd, 155Gdj 156Gd, 157Gd, 158Gd, 166Hθ) 120L 123 124 12S 131Z, 110In> 111^ 194Ir> 177Lu, 51Mn, S2mMrl) 99Mθ) 13N> 150, 32P> 33P) 211Pb> 212Pb) 109Pd) 149Pπlj 142Prj 1 3pr) 223RΑj S2mRb> I86Re, I8SRe; I89Re, 105^ 47Sc, lS3Sm, S_, 83Sr, 89Sr, 161-τb, 94mTc> 94TCj 99mχCj 86Yj 90 Y) 90γj and 89zr; and; when the targetable construct includes more than one radionuclide, the radionuclides may be different radionuclides.
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 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 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.
124. The method of claim 123, wherein the radionuclide is selected from the group consisting of 225Ac, 11 lAg, 2ASj 77As> 211 At, 198Au, 199Au, 212Bi, 213Bi, 75Br, 76Br, I lC, S5Co, 6~ Cu, «Cu, < Cu, Iββpy, 169^ I8F, 52Fe, 59Fe, 67Ga, 68Ga, 154Gd, 155Gd( 156Gdj 157Gd, 158Gd, 166Ho, 120τ, 123ι, 124Tj 125], 13 IT, 110^ l llj^, 194fr> 177Lu, 51Mn, 52mMn> 99Mo> 13N> 150, 32P> 33Pι 211Pb) 212Pb> 109Pd> 149Pmj 142Pr> 143Pr> 223Ra, 82mRb, 186Re, 188Re> 189Re> 105^ 47Sc, 153Sm, 75Se, 83Sr, 89sr, 161τb, 94mTc? 94Tc, 99mTc> 86Y/ 90Yj 90γ m^ 89Zr; and, when the targetable construct includes more than one radionuclide, the radionuclides may be different radionuclides.
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, hormones, or growth factors.
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