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WO1995015770A1 - Procedes et composes de preciblage - Google Patents

Procedes et composes de preciblage Download PDF

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Publication number
WO1995015770A1
WO1995015770A1 PCT/US1994/014223 US9414223W WO9515770A1 WO 1995015770 A1 WO1995015770 A1 WO 1995015770A1 US 9414223 W US9414223 W US 9414223W WO 9515770 A1 WO9515770 A1 WO 9515770A1
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WO
WIPO (PCT)
Prior art keywords
conjugate
biotin
ligand
antibody
streptavidin
Prior art date
Application number
PCT/US1994/014223
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English (en)
Inventor
Scott S. Graves
Michael J. Bjorn
John M. Reno
Donald B. Axworthy
Alan R. Fritzberg
Louis J. Theodore
Original Assignee
Neorx Corporation
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 Neorx Corporation filed Critical Neorx Corporation
Publication of WO1995015770A1 publication Critical patent/WO1995015770A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • A61K47/51—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
    • A61K47/68—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6893—Pre-targeting systems involving an antibody for targeting specific cells clearing therapy or enhanced clearance, i.e. using an antibody clearing agents in addition to T-A and D-M
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • A61K47/51—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
    • A61K47/68—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6897—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • A61K47/51—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
    • A61K47/68—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6897—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
    • A61K47/6898—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies using avidin- or biotin-conjugated antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • 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
    • A61K51/10—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
    • A61K51/1093—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 conjugates with carriers being antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo

Definitions

  • the present invention relates to methods
  • the absolute dose of radiation or therapeutic agent delivered to the tumor is insufficient in many cases to elicit a significant tumor response.
  • the present invention is directed to diagnostic and therapeutic pretargeting methods, moieties useful therein and methods of making those moieties.
  • Such pretargeting methods are characterized by an improved targeting ratio or increased absolute dose to the target cell sites in comparison to conventional cancer therapy.
  • the present invention provides targeting moiety-anti-ligand, such as avidin or streptavidin, compounds useful in diagnostic and therapeutic pretargeting methods. Preparation and purification of such anti-ligand-targeting moiety compounds are also discussed. Reduction of the immunogenicity of this targeting moiety-anti-ligand component is also provided in accordance with the present invention (two-step pretargeting). Reducing the immunogenicity of targeting moiety-ligand conjugate is at issue for three-step pretargeting.
  • targeting moiety-anti-ligand such as avidin or streptavidin
  • Immunogenicity reduction may be accomplished for conjugates having an antibody component by employing antibodies or fragments thereof exhibiting human character (e.g., chimeric, human or humanized
  • human or humanized antibodies exhibit lower immunogenicity when administered to humans than antibodies produced in or from other mammalian species.
  • targeting moieties of lower molecular weight are generally less immunogenic than their higher molecular weight counterparts.
  • antibody fragments are generally less immunogenic than whole antibodies as are small molecule targeting moieties.
  • the targeting moiety-anti-ligand conjugate may be chemically altered to decrease the immunogenicity thereof. Either or both the targeting moiety or the anti-ligand portion of the conjugate may be so altered; provided that the targeting moiety retains its targeting ability and the anti-ligand retains its capability to bind ligand with high affinity.
  • the conjugate or any component thereof may be chemically modified via
  • PEG polyethyleneglycol
  • Chemical modification in accordance with the present invention includes charge modification, such as modification via succinyl derivatization (i.e, succinylation).
  • Immunogenicity-reducing modification of the tertiary structure of a proteinaceous immunogen may also be employed in the practice of the present invention. Such tertiary structure modification is preferably conducted by recombinant techniques to reduce the number or immunogenicity of immunogenic sites on the proteinaceous immunogen.
  • immunosuppressive agents may be administered prior to, concurrently with, or following administration of the targeting moiety-anti-ligand conjugate.
  • Preferred immunosuppressive agents include cyclosporin A, verapamil, mycophenolic acid,
  • transforming growth factor-beta deoxyspergualin
  • FK506, rapamycin azathioprine cyclophosphamide
  • immunophilins such as FK binding protein 12, peptide derivatives of non-erythroid spectin, and combinations thereof.
  • Liposome encapsulated or particulate immunosuppressive agents may also be employed.
  • immunosuppressive agents in the practice of the methods of the present invention.
  • Extracorporeal, particulate and other clearing agents and mechanisms are also contemplated for use within the present invention.
  • immunogenic therapeutic or diagnostic agents may be employed to reduce the immunogenicity of such components.
  • Non-specific pretargeting techniques are also contemplated by the present invention. Such as
  • pretargeting techniques contemplate administration of a non-specific targeting moiety-ligand or a non-specific targeting moiety-anti-ligand conjugate.
  • the non-specific targeting moiety becomes trapped
  • the non-specific targeting moiety-containing conjugate is administered, equilibrates between the
  • extravascular compartment and the vascular compartment and the conjugate in the vascular space is cleared using a clearing agent or a clearing mechanism.
  • Active agent-anti-ligand or active agent-ligand conjugate is then administered and such active agent-containing conjugate accretes to the extravascular compartment localized non-specific targeting moiety-containing conjugate.
  • Non-specific pretargeting allows the use of large, non-immunogenic proteins (e.g., IgG and IgM) for radioimmunotherapy, for example.
  • Irrelevant antibodies and other non-specific targeting moieties may also be used in accordance with this aspect of the present invention.
  • Short half-life active agents such as alpha-emitters, exert a
  • the present invention also provides an article of manufacture which includes packaging material and a first conjugate contained within the packaging
  • the first conjugate incorporates a targeting moiety and streptavidin, and wherein the first conjugate is capable of localizing at a target site upon administration to a mammalian recipient, and streptavidin retains the ability to bind to biotin, and further wherein the packaging material includes a label, which label identifies the targeting moiety component of the first conjugate, identifies the streptavidin component of the first conjugate and indicates an appropriate use of the first conjugate in human recipients.
  • Figure 2 depicts radiorhenium tumor uptake in a three-step pretargeting protocol, as compared to administration of radiolabeled antibody (conventional means involving antibody that is covalently linked to chelated radiorhenium).
  • Figure 3 depicts the tumor uptake profile of NR-LU-10-streptavidin conjugate (LU-10-StrAv) in
  • Figure 4 depicts the tumor uptake and blood clearance profiles of NR-LU-10-streptavidin conjugate.
  • Figure 5 depicts the rapid clearance from the blood of asialoorosomucoid in comparison with
  • orosomucoid in terms of percent injected dose of I-125-labeled protein.
  • Figure 7 depicts NR-LU-10-streptavidin conjugate blood clearance upon administration of three controls (O, ⁇ , ⁇ ) and two doses of a clearing agent ( ⁇ , ⁇ ) at 25 hours post-conjugate administration.
  • Figure 8 shows limited biodistribution data for LU-10-StrAv conjugate upon administration of three controls (Groups 1, 2 and 5) and two doses of clearing agent (Groups 3 and 4) at two hours post-clearing agent administration.
  • Figure 9 depicts NR-LU-10-streptavidin conjugate serum biotin binding capability at 2 hours post-clearing agent administration.
  • Figure 10 depicts NR-LU-10-streptavidin conjugate blood clearance over time upon administration of a control (O) and three doses of a clearing agent ( ⁇ , ⁇ , ⁇ ) at 24 hours post-conjugate administration.
  • Figure 11A depicts the blood clearance of LU-10-StrAv conjugate upon administration of a control (PBS) and three doses (50, 20 and 10 ⁇ g) of clearing agent at two hours post-clearing agent administration.
  • Figure 11B depicts LU-10-StrAv conjugate serum biotin binding capability upon administration of a control (PBS) and three doses (50, 20 and 10 ⁇ g) of clearing agent at two hours post-clearing agent administration.
  • Figure 12 depicts the prolonged tumor retention of NR-LU-10-streptavidin conjugate ( ⁇ ) relative to NR-LU-10 whole antibody ( ⁇ ) over time.
  • Figure 13 depicts the prolonged liver retention of a pre-formed complex of NR-LU-10-biotin ( O ;
  • Figure 14 depicts the prolonged liver retention of Biotin-PIP-I-131 label relative to the streptavidin-NR-LU-10-(PIP-I-125) label.
  • Figure 15A depicts tumor uptake for increasing doses of PIP-Biocytin in terms of %ID/G.
  • Figure 15B depicts tumor uptake for increasing doses of PIP-Biocytin over time in terms of pMOL/G.
  • Figure 16A depicts tumor versus blood localization of a 0.5 ⁇ g dose of PIP-Biocytin over time in terms of %ID/G.
  • Figure 16B depicts tumor versus blood localization of a 0.5 ⁇ g dose of PIP-Biocytin in terms of %ID.
  • Figure 17A depicts tumor uptake of LU-10-StrAv and PIP-Biocytin over time in terms of %ID/G.
  • Figure 17B depicts blood clearance of LU-10-StrAv and PIP-Biocytin over time in terms of %ID/G.
  • Figure 18 depicts PIP-Biocytin:LU-10-StrAv ratio in tumor and blood over time.
  • Targeting moiety A molecule that binds to a defined population of cells.
  • the targeting moiety may bind a receptor, an oligonucleotide, an enzymatic substrate, an antigenic determinant, or other binding site present on or in the target cell population.
  • Antibody is used throughout the specification as a prototypical example of a targeting moiety.
  • Tumor is used as a prototypical example of a target in
  • Non-specific targeting moiety A molecule that binds to a variety of cells including target cells of diagnostic or therapeutic protocols as well as non-target cells.
  • the target cells are located within a separate physiological compartment than non-target cells (e.g., the target cells are located in the extravascular space while non-target cells are not).
  • Non-specific targeting moieties may be antibody-based, peptide-based, polymer-based, microparticulate-based, aggregate-based (e.g., aggregated streptavidin, avidin or the like) or the like.
  • Ligand/anti-ligand pair A complementary/anti-complementary set of molecules that demonstrate specific binding, generally of relatively high
  • Exemplary ligand/anti-ligand pairs include zinc finger protein/dsDNA fragment, enzyme/inhibitor, hapten/antibody, lectin/carbohydrate, ligand/receptor, and biotin/avidin. Biotin/avidin is used throughout the specification as a prototypical example of a ligand/anti-ligand pair.
  • Anti-ligand As defined herein, an "anti-ligand” demonstrates high affinity, and preferably,
  • the anti-ligand is large enough to avoid rapid renal clearance, and contains sufficient
  • Anti-ligands of the present invention may exhibit or be derivatized to exhibit structural features that direct the uptake thereof, e.g., galactose residues that direct liver uptake.
  • Avidin and streptavidin are used herein as
  • Avidin As defined herein, “avidin” includes avidin, streptavidin and derivatives and analogs thereof that are capable of high affinity, multivalent or univalent binding of biotin.
  • Ligand As defined herein, a "ligand” is a relatively small, soluble molecule that exhibits rapid serum, blood and/or whole body clearance when
  • Biotin is used as the prototypical ligand.
  • a diagnostic or therapeutic agent A diagnostic or therapeutic agent
  • the payload including radionuclides, drugs, anti-tumor agents, toxins and the like. Radionuclide therapeutic agents are used as prototypical active agents.
  • N x S y Chelates As defined herein, the term "N x S y chelates" includes buoy chelators that are capable of (i) coordinately binding a metal or radiometal and (ii) covalently attaching to a targeting moiety, ligand or anti-ligand. Particularly preferred N x S y chelates have N 2 S 2 and N 3 S cores. Exemplary N X S chelates are described in Fritzberg et al., Proc. Natl. Acad. Sci. USA 85:4024-29, 1988; in Weber et al., Bioconj. Chem. 1:431-37, 1990; and in the references cited therein, for instance.
  • Pretargeting involves target site localization of a targeting moiety that is conjugated with one member of a ligand/anti-ligand pair; after a time period
  • Clearing Agent An agent capable of binding, complexing or otherwise associating with an
  • the clearing agent is preferably characterized by physical
  • Target Cell Retention The amount of time that a radionuclide or other therapeutic agent remains at the target cell surface or within the target cell.
  • Catabolism of conjugates or molecules containing such therapeutic agents appears to be primarily responsible for the loss of target cell retention.
  • a conjugate encompasses chemical conjugates (covalently or non-covalently bound), fusion proteins and the like.
  • Immunogenicity A measure of the ability of a targeting protein or therapeutic moiety to elicit an immune response (humoral or cellular) when
  • the present invention is concerned with the immunogenicity of the conjugates and their component parts.
  • a recognized disadvantage associated with in vivo administration of targeting moiety-radioisotopic conjugates for imaging or therapy is localization of the attached radioactive agent at both non-target and target sites.
  • the administered radiolabeled conjugate clears from the circulation, normal organs and tissues are transitorily exposed to the attached radioactive agent.
  • radiolabeled whole antibodies that are administered in vivo exhibit relatively slow blood clearance; maximum target site localization generally occurs 1-3 days post-administration.
  • the longer the clearance time of the conjugate from the circulation the greater the radioexposure of non-target organs.
  • radiosensitive is the dose-limiting organ of non-specific toxicity.
  • potential targeting moieties In order to decrease radioisotope exposure of non-target tissue, potential targeting moieties generally have been screened to identify those that display minimal non-target reactivity, while retaining target specificity and reactivity.
  • increased doses of a radiotherapeutic conjugate may be administered; moreover, decreased non-target accumulation of a radiodiagnostic conjugate leads to improved contrast between background and target.
  • Therapeutic drugs administered alone or as targeted conjugates, are accompanied by similar disadvantages. Again, the goal is administration of the highest possible concentration of drug (to
  • targeting moiety-therapeutic drug conjugates it would be advantageous to combine the relative target specificity of a targeting moiety with a means for enhanced target cell internalization of the targeting moiety-drug conjugate.
  • diagnostic agent-targeting moiety conjugates In contrast, enhanced target cell internalization is disadvantageous if one administers diagnostic agent-targeting moiety conjugates. Internalization of diagnostic conjugates results in cellular catabolism and degradation of the conjugate. Upon degradation, small adducts of the diagnostic agent or the
  • diagnostic agent per se may be released from the cell, thus eliminating the ability to detect the conjugate in a target-specific manner.
  • One method for reducing non-target tissue exposure to a diagnostic or therapeutic agent involves
  • pretargeting the targeting moiety at a target site, and then subsequently administering a rapidly clearing diagnostic or therapeutic agent conjugate that is capable of binding to the "pretargeted" targeting moiety at the target site.
  • a description of some embodiments of the pretargeting technique may be found in US Patent No. 4,863,713 (Goodwin et al.).
  • this three-step pretargeting protocol features administration of an antibody-ligand
  • anti-ligand conjugate which is allowed to localize at a target site and to dilute in the circulation. Subsequently administered anti-ligand binds to the antibody-ligand conjugate and clears unbound antibody-ligand conjugate from the blood.
  • Preferred anti-ligands are large and contain sufficient multivalency to accomplish
  • Anti-ligand clearance of this type is preferably accomplished with a multivalent molecule; however, a univalent molecule of sufficient size to be cleared by the RES on its own could also be employed.
  • receptor-based clearance mechanisms e.g., Ashwell receptor or hexose residue, such as galactose or mannose residue, recognition mechanisms, may be responsible for anti-ligand clearance via the liver.
  • Such clearance mechanisms are less dependent upon the valency of the anti-ligand with respect to the ligand than the RES complex/aggregate clearance mechanisms. It is preferred that the ligand-anti-ligand pair displays relatively high affinity binding.
  • a diagnostic or therapeutic agent-ligand conjugate that exhibits rapid whole body clearance is then administered.
  • anti-ligand binds the circulating active agent-ligand conjugate and produces an antibody-ligand : anti-ligand : ligand-active agent "sandwich" at the target site.
  • the diagnostic or therapeutic agent is attached to a rapidly clearing ligand (rather than antibody, antibody fragment or other slowly clearing targeting moiety), this technique promises decreased non-target exposure to the active agent.
  • pretargeting methods eliminate the step of parenterally administering an anti-ligand clearing agent.
  • These "two-step” procedures feature targeting moiety-ligand or targeting moiety-anti-ligand
  • a clearing agent preferably other than ligand or anti-ligand alone is administered to facilitate the clearance of circulating targeting moiety-containing conjugate.
  • clearing agent preferably does not become bound to the target cell population, either directly or through the previously administered and target cell bound
  • targeting moiety-anti-ligand or targeting moiety-ligand conjugate is an example of two-step pretargeting.
  • two-step pretargeting involves the use of biotinylated human transferrin as a clearing agent for avidin-targeting moiety
  • the two-step pretargeting approach overcomes certain disadvantages associated with the use of a clearing agent in a three-step pretargeted protocol. More specifically, data obtained in animal models demonstrate that in vivo anti-ligand binding to a pretargeted targeting moiety-ligand conjugate (i.e., the cell-bound conjugate) removes the targeting moiety-ligand conjugate from the target cell.
  • a pretargeted targeting moiety-ligand conjugate i.e., the cell-bound conjugate
  • the apparent loss of targeting moiety-ligand from the cell might result from internal degradation of the conjugate and/or release of active agent from the conjugate (either at the cell surface or intracellularly).
  • An alternative explanation for the observed phenomenon is that permeability changes in the target cell's membrane allow increased passive diffusion of any molecule into the target cell.
  • some loss of targeting moiety-ligand may result from alteration in the affinity by subsequent binding of another moiety to the targeting moiety-ligand, e.g., anti-idiotype monoclonal antibody binding causes removal of tumor bound monoclonal antibody.
  • a targeting moiety may be covalently linked to both ligand and therapeutic agent and administered to a recipient. Subsequent administration of anti-ligand crosslinks targeting moiety-ligand-therapeutic agent tripartite conjugates bound at the surface, inducing internalization of the tripartite conjugate
  • targeting moiety-ligand may be delivered to the target cell surface, followed by administration of anti-ligand-therapeutic agent.
  • targeting moiety-anti-ligand conjugate is administered in vivo; upon target localization of the targeting moiety-anti-ligand conjugate (i.e., and clearance of this conjugate from the circulation), an active agent-ligand conjugate is parenterally administered.
  • This method enhances retention of the targeting moietyanti-ligand : ligand-active agent complex at the target cell (as compared with targeting moiety-ligand : anti-ligand : ligand-active agent complexes and targeting moiety-ligand : anti-ligand-active agent complexes).
  • ligand/anti-ligand pairs may be suitable for use within the claimed invention, a preferred ligand/anti-ligand pair is biotin/avidin.
  • radioiodinated biotin described herein is a low molecular weight compound that has been easily and well characterized.
  • a targeting moiety-ligand conjugate is administered in vivo; upon target localization of the targeting moiety-ligand conjugate (i.e., and clearance of this conjugate from the circulation), a drug-anti-ligand conjugate is parenterally administered.
  • This two-step method not only provides pretargeting of the targeting moiety conjugate, but also induces internalization of the subsequent targeting moiety-ligand-anti-ligand-drug complex within the target cell.
  • another embodiment provides a three-step protocol that produces a targeting moiety-ligand : anti-ligand : ligand-drug complex at the surface, wherein the ligand-drug conjugate is administered simultaneously or within a short period of time after administration of anti-ligand (i.e., before the targeting moiety-ligand-anti-ligand complex has been removed from the target cell surface).
  • methods for radiolabeling biotin with technetium-99m, rhenium-186 and rhenium-188 are disclosed.
  • biotin derivatives were radiolabeled with indium-111 for use in pretargeted immunoscintigraphy (for instance, Virzi et al., Nucl. Med.
  • Rhenium-186 displays chelating chemistry very similar to 99m Tc, and is considered to be an excellent therapeutic radionuclide (i.e., a 3.7 day half-life and 1.07 MeV maximum particle that is similar to 131 I). Therefore, the claimed methods for technetium and rhenium radiolabeling of biotin provide numerous advantages.
  • targeting moiety binds to a defined target cell population, such as tumor cells.
  • Preferred targeting moieties useful in this regard include antibody and antibody fragments, peptides, and hormones. Proteins corresponding to known cell surface receptors (including low density lipoproteins, transferrin and insulin), fibrinolytic enzymes, anti-HER2, platelet binding proteins such as annexins, and biological response modifiers (including interleukin, interferon, erythropoietin and colony-stimulating factor) are also preferred targeting moieties. Also, anti-EGF receptor antibodies, which internalize following binding to the receptor and traffic to the nucleus to an extent, are preferred targeting moieties for use in the present invention to facilitate delivery of Auger emitters and nucleus binding drugs to target cell nuclei.
  • Oligonucleotides e.g., antisense oligonucleotides that are complementary to portions of target cell nucleic acids (DNA or RNA), are also useful as targeting moieties in the practice of the present invention. Oligonucleotides binding to cell surfaces are also useful. Analogs of the above-listed target cell nucleic acids (DNA or RNA).
  • targeting moieties that retain the capacity to bind to a defined target cell population may also be used within the claimed invention.
  • synthetic targeting moieties may be designed.
  • targeting moieties of the present invention are also useful as targeting moieties of the present invention.
  • One targeting moiety functional equivalent is a "mimetic" compound, an organic chemical construct designed to mimic the proper configuration and/or orientation for targeting moiety-target cell binding.
  • Another targeting moiety is a "mimetic" compound, an organic chemical construct designed to mimic the proper configuration and/or orientation for targeting moiety-target cell binding.
  • minimal polypeptide constructed using computer-assisted molecular modeling and mutants having altered binding affinity, which minimal polypeptides exhibit the binding affinity of the targeting moiety.
  • Preferred targeting moieties of the present invention are antibodies (polyclonal or monoclonal), peptides, oligonucleotides or the like.
  • Polyclonal antibodies useful in the practice of the present invention are polyclonal (Vial and Callahan, Univ. Mich. Med. Bull., 20: 284-6, 1956), affinity-purified polyclonal or fragments thereof (Chao et al., Res. Comm. in Chem. Path. & Pharm., 9: 749-61, 1974).
  • Monoclonal antibodies useful in the practice of the present invention include whole antibody and fragments thereof. Such monoclonal antibodies and fragments are producible in accordance with
  • Useful monoclonal antibodies and fragments may be derived from any species (including humans) or may be formed as chimeric proteins which employ sequences from more than one species. See, generally, Kohler and Milstein, Nature, 256: 495-97, 1975; Eur. J.
  • Murine monoclonal antibodies or "humanized” murine antibody are also useful as targeting moieties in accordance with the present invention.
  • murine monoclonal antibody may be "humanized” by genetically recombining the nucleotide sequence encoding the murine Fv region (i.e., containing the antigen binding sites) or the complementarity
  • Humanized targeting moieties are recognized to decrease the immunoreactivity of the antibody or polypeptide in the host recipient, permitting an increase in the half-life and a
  • An additional aspect of the present invention is directed to the use of targeting moieties that are monoclonal antibodies or fragments thereof that localize to an antigen that is recognized by the antibody NR-LU-10.
  • Such monoclonal antibodies or fragments may be murine or of other non-human
  • NR-LU-10 is a 150 kilodalton molecular weight IgG2b monoclonal antibody that recognizes an
  • NR-LU-10 is a well characterized pancarcinoma antibody that has been safely administered to over 565 patients in human clinical trials.
  • the hybridoma secreting NR-LU-10 was developed by fusing mouse splenocytes immunized with intact cells of a human small cell lung carcinoma with P3 ⁇ 63/Ag8UI murine myeloma cells. After establishing a seed lot, the hybridoma was grown via in vitro cell culture methods, purified and verified for purity and sterility.
  • NR-LU-10 target antigen was present on either fixed cultured cells or in detergent extracts of various types of cancer cells.
  • the NR-LU-10 antigen is found in small cell lung, non-small cell lung, colon, breast, renal, ovarian, pancreatic, and other carcinoma tissues.
  • Tumor reactivity of the NR-LU-10 antibody is set forth in Table A, while NR-LU-10 reactivity with normal tissues is set forth in Table B. The values in Table B are obtained as described below.
  • Positive NR-LU-10 tissue reactivity indicates NR-LU-10 antigen expression by such tissues.
  • the NR-LU-10 antigen has been further described by Varki et al., "Antigens Associated with a Human Lung Adenocarcinoma Defined by Monoclonal Antibodies,"
  • the tissue specimens were scored in accordance with three reactivity parameters: (1) the intensity of the reaction; (2) the uniformity of the reaction within the cell type; and (3) the percentage of cells reactive with the antibody. These three values are combined into a single weighted comparative value between 0 and 500, with 500 being the most intense reactivity. This comparative value facilitates comparison of different tissues.
  • Table B includes a summary reactivity value, the number of tissue samples examined and the number of samples that reacted positively with NR-LU-10.
  • Additional antibodies reactive with the NR-LU-10 antigen may also be prepared by standard hybridoma production and screening techniques. Any hybridoma clones so produced and identified may be further screened as described above to verify antigen and tissue reactivity.
  • Types of active agents (diagnostic or therapeutic) useful herein include toxins, anti-tumor agents, drugs and radionuclides.
  • toxins include toxins, anti-tumor agents, drugs and radionuclides.
  • Several of the potent toxins useful within the present invention consist of an A and a B chain.
  • the A chain is the cytotoxic portion and the B chain is the receptor-binding portion of the intact toxin molecule (holotoxin).
  • toxin B chain may mediate non-target cell binding, it is often advantageous to conjugate only the toxin A chain to a targeting protein.
  • elimination of the toxin B chain decreases non-specific cytotoxicity, it also generally leads to decreased potency of the toxin A chain-targeting protein conjugate, as .compared to the corresponding holotoxin-targeting protein
  • Preferred toxins in this regard include
  • holotoxins such as abrin, ricin, modeccin,
  • Pseudomonas exotoxin A Diphtheria toxin, pertussis toxin and Shiga toxin
  • a chain or "A chain-like" molecules such as ricin A chain, abrin A chain, modeccin A chain, the enzymatic portion of Pseudomonas exotoxin A, Diphtheria toxin A chain, the enzymatic portion of pertussis toxin, the enzymatic portion of Shiga toxin, gelonin, pokeweed antiviral protein, saporin, tritin, barley toxin and snake venom peptides.
  • Ribosomal inactivating proteins are also suitable for use herein.
  • Extremely highly toxic toxins such as palytoxin and the like, are also contemplated for use in the practice of the present invention.
  • toxin moieties that are charge-modified in accordance with the description of protein charge modification set forth below, e.g., succinic anhydride modification, also find utility in the practice of the present invention.
  • Preferred drugs suitable for use herein include conventional chemotherapeutics, such as vinblastine, doxorubicin, bleomycin, methotrexate, 5-fluorouracil, 6-thioguanine, cytarabine, cyclophosphamide and cis-platinum, as well as other conventional chemotherapeutics, such as vinblastine, doxorubicin, bleomycin, methotrexate, 5-fluorouracil, 6-thioguanine, cytarabine, cyclophosphamide and cis-platinum, as well as other conventional chemotherapeutics, such as vinblastine, doxorubicin, bleomycin, methotrexate, 5-fluorouracil, 6-thioguanine, cytarabine, cyclophosphamide and cis-platinum, as well as other conventional chemotherapeutics, such as vinblastine, doxorubicin, ble
  • a particularly preferred drug within the present invention is a trichothecene.
  • Trichothecenes are drugs produced by soil fungi of the class Fungi imperfecti or isolated from Baccharus megapotamica (Bamburg, J.R. Proc. Molec. Subcell.
  • trichothecenes may be subdivided into three groups (i.e., Group A, B, and C) as described in U.S. Patent Nos. 4,744,981 and 4,906,452 (incorporated herein by reference).
  • Group A simple trichothecenes include: Scirpene, Roridin C,
  • Group B simple trichothecenes include: Trichothecolone,
  • Trichothecin deoxynivalenol, 3-acetyldeoxynivalenol, 5-acetyldeoxynivalenol, 3,15-diacetyldeoxynivalenol, Nivalenol, 4-acetylnivalenol (Fusarenon-X),
  • Group C simple trichothecenes include: Crotocol and Crotocin.
  • Representative macrocyclic trichothecenes include Verrucarin A, Verrucarin B, Verrucarin J
  • Experimental drugs such as mercaptopurine, N-methylformamide, 2-amino-1,3,4-thiadiazole, melphalan, hexamethylmelamine, gallium nitrate, 3% thymidine, dichloromethotrexate, mitoguazone, suramin,
  • pentostatin PALA, carboplatin, amsacrine, caracemide, iproplatin, misonidazole, dihydro-5-azacytidine, 4'-deoxy-doxorubicin, menogaril, triciribine phosphate, camphor, tiazofurin, teroxirone, ethiofos, N-(2-hydroxyethyl)-2-nitro-1H-imidazole-1-acetamide, mitoxantrone, acodazole, amonafide, fludarabine phosphate, pibenzimol, didemnin B, merbarone,
  • Radionuclides useful within the present invention include gamma-emitters, positron-emitters, Auger electron-emitters, X-ray emitters and fluorescence-emitters, with beta- or alpha-emitters preferred for therapeutic use.
  • Radionuclides are well-known in the art and include 123 I, 125 I, 130 I, 131 I, 133 I, 135 I, 47 Sc, 72 As, 72 Se, 90 Y, 88 Y, 97 Ru, 100 Pd, 101m Rh, 119 Sb, 128 Ba, 197 Hg, 211 At, 212 Bi, 153 Sm, 169 Eu, 212 Pb, 109 Pd, 111 In, 67 Ga, 68 Ga, 64 Cu, 67 Cu, 75 Br, 76 Br, 77 Br, 99m Tc, 11 C, 13 N, 15 O, 166 Ho and 18 F.
  • Preferred therapeutic radionuclides include 188 Re, 186 Re, 203 Pb, 212 Pb, 212 Bi , 109 Pd, 64 Cu, 67 Cu, 90 Y , 125 I , 131 I , 77 Br , 211 At , 97 Ru, 105 Rh , 198 Au , 166 Ho and 199 Ag or 177 Lu .
  • anti-tumor agents e.g., agents active against proliferating cells
  • exemplary anti-tumor agents include cytokines, such as IL-2, tumor necrosis factor or the like, lectin inflammatory response promoters (selectins), such as L-selectin, E-selectin, P-selectin or the like, and like molecules.
  • Ligands suitable for use within the present invention include biotin, haptens, lectins, epitopes, dsDNA fragments, enzyme inhibitors and analogs and derivatives thereof.
  • Useful complementary anti-ligands include avidin (for biotin), carbohydrates (for lectins) and antibody, fragments or analogs thereof, including mimetics (for haptens and epitopes) and zinc finger proteins (for dsDNA fragments) and enzymes (for enzyme inhibitors).
  • Preferred ligands and anti-ligands bind to each other with an affinity of at least about k D ⁇ 10 9 M.
  • One component to be administered in a preferred two-step pretargeting protocol is a targeting moiety-anti-ligand or a targeting moiety-ligand conjugate.
  • a preferred component for administration is a targeting moiety-ligand conjugate.
  • the present invention provides an article of manufacture which includes packaging material and a targeting moiety-anti-ligand (e.g., streptavidin) conjugate or a targeting moiety-ligand (e.g., biotin) conjugate contained within the packaging material, wherein the conjugate is capable of localizing at a target site upon administration to a mammalian
  • the ligand or anti-ligand retains the capability to bind to the complementary ligand/anti-ligand binding pair member, and wherein the packaging material includes a label that identifies the packaging material
  • targeting moiety component of the conjugate identifies the ligand or anti-ligand component of the conjugate and indicates an appropriate use of the conjugate in human recipients.
  • the packaging material indicates whether the conjugate is limited to investigational use or
  • the packaging material may also include additional information including the amount of conjugate, the medium or environment in which the conjugate is dispersed, if any, lot number or other identifier, storage instructions, usage instructions, a warning with respect to any restriction upon use of the conjugate, the name and address of the company
  • the targeting moiety-ligand conjugate or targeting moiety-anti-ligand conjugate is preferably contained within a vial which allows the first conjugate to be transported prior to use.
  • Such conjugate is
  • the conjugate may be lyophilized prior to packaging.
  • instructions for preparing the lyophilized conjugate for administration to a recipient may be included on the label.
  • the targeting moiety of the targeting moiety-ligand or targeting moiety-anti-ligand conjugate is a monoclonal antibody or antibody fragment of murine origin
  • a host immune response will be generated against the antibody component or the component conjugated to the antibody component upon conjugate administration to a mammal of differing species.
  • B-cells recognize antigen by a mechanism whereby early B-cells bind the circulating foreign protein. A signal is transduced via recognition of IgM, and the B-cell is stimulated to multiply and form memory cells. Upon subsequent binding of antigen, higher affinity B-cells are selected and produced in higher quantity. B-cell antigen recognition is therefore independent of MHC cell presentation.
  • Nonspecific uptake into normal tissues results in catabolism of the conjugate to peptides by macrophages or other antigen presenting cells with presentation of the peptides to immunocompetent T-cells. These peptides could then be recognized as non-self with respect to the recipient by T-cell dependent or independent mechanisms and augment the antibody response generated by B-cells. Biodistribution, serum clearance, normal organ accumulation and excretion impact the degree of conjugate immunogenicity and, therefore, the ability to administer conjugate multiple times to a recipient.
  • moieties e.g., Fab or Fab' fragments
  • chimeric, humanized or human antibodies or fragments are used instead of their murine or other mammalian
  • Antibodies or fragments thereof exhibiting more human character are less likely to be recognized by the human immune system as non-self entities. Such antibodies or fragments are therefore generally less immunogenic than antibodies or
  • murine variable regions are prepared in accordance with known procedures described in U.S. Patent No. 4,816,397, issued to Boss et al. and U.S. Patent No. 4,816,567, issued to Cabilly et al.
  • Antibody humanization techniques i.e., methods for the preparation of antibodies characterized by human constant regions and variable framework regions and, generally, murine complementarity determining regions with some retained murine framework residues are discussed in United Kingdom Patent No. 2,188,638 issued to Winter; PCT Patent Application No.
  • Monoclonal antibody fragments e.g., Fv, Fab,
  • F(ab') 2 or other antigen or receptor targeting vehicles (e.g., molecular recognition units (generally less than 5 kD in molecular weight), lymphokines or factors such as epidermal growth factor), rather than whole monoclonal antibodies, may be employed as the targeting moieties of the present invention.
  • antigen or receptor targeting vehicles e.g., molecular recognition units (generally less than 5 kD in molecular weight), lymphokines or factors such as epidermal growth factor), rather than whole monoclonal antibodies, may be employed as the targeting moieties of the present invention.
  • Antibody fragments are lower in molecular weight (generally ranging from about 25-30 kD (e.g., single chain Fv as described, for example, in Biochem., 31(6): 1579, 1991) to about 45-50 kD (e.g., Fab) to about 100-105 kD (F(ab') 2 )) than their whole antibody counterparts (generally ranging from about 150-160 kD (e.g., IgG) to about 900 kD (e.g., IgM)). Also, antibody
  • fragments generally exhibit shorter serum half-lives in comparison to their whole antibody counterparts. A shorter serum half-life is believed to be related to reduced immunogenicity.
  • the antibody fragment presents a smaller and more rapidly excreted immunogenic target and, therefore, generally exhibits less immunogenicity than the larger and slower
  • an antibody generally exhibit more immunogenicity than the Fab portion thereof.
  • Antibody fragments offer the additional advantage faster, albeit less efficient, accretion to target sites, thereby enabling the time between pretargeting
  • protocol agent administrations to be reduced For example, a pretargeting protocol which employs Fab-streptavidin conjugate; clearing agent and active agent-biotin conjugate may be accomplished in mice by administering clearing agent at from about 2 to about 6 hours post-Fab-streptavidin conjugate
  • %ID/g On a percent initial dose/gram (%ID/g) basis, whole antibody generally exhibits a higher %ID/g than antibody fragment.
  • the molecular weight of Fab fragments for example is about one-third that of whole antibody, however. Consequently, the moles of the lower molecular weight fragment or fragment-ligand or fragment-anti-ligand molecules accreting to a target site is generally greater than the number of moles of whole antibody or whole antibody-ligand or whole antibody-anti-ligand molecules so accreting, thereby affording a higher concentration of ligand or anti-ligand molecules associated with the target site.
  • Antibody fragmentation techniques are known in the art and routinely practiced.
  • somatostatin and derivatives thereof such as octreotide and the like, annexins, cytokines such as IL-6, and the like are useful as targeting moieties in the practice of the present invention.
  • immunogenicity is impacted by biodistribution, serum clearance, normal organ accumulation and excretion, which parameters are somewhat interrelated. Generally, decreases in serum clearance and excretion will result in increased immunogenicity, because the recipient remains exposed to the immunogen for a longer period of time. Normal organ accumulation (non-specific uptake) results in antigen presentation of macrophage-metabolized
  • conjugate to T-cells.
  • Conjugation of the targeting moiety to molecules that enhance RES uptake of the conjugate also generally increases immunogenicity.
  • Whole conjugate or individual conjugate components of targeting moiety-anti-ligand or targeting moiety-ligand conjugates may be chemically modified to reduce the immunogenicity thereof. Chemical modification may be employed so long as the targeting moiety retains the capability to localize to target sites and the ligand or anti-ligand retains the ability to bind to the complementary member of the ligand/anti-ligand pair with high affinity.
  • PEGylation Enzymes covalently attached to methoxy-polyethylene glycol (5000 molecular weight), PEGylated enzymes, have been shown generally to be non-immunogenic. See, for example, Abuchowski et al., "Effect of Covalent Attachment of Polyethylene Glycol on Immunogenicity and Circulating Life of Bovine Liver Catalase," J. Biol. Chem., 252: 3581 (1977). Such PEGylated enzymes retained enzyme activity and
  • proteinaceous moieties e.g., proteinaceous ligands or anti-ligands, such as the anti-ligand streptavidin, antibodies, antibody fragments and other proteinaceous targeting moieties
  • proteinaceous ligands or anti-ligands such as the anti-ligand streptavidin, antibodies, antibody fragments and other proteinaceous targeting moieties
  • PEGylated by substantially the same procedures by which enzymes are PEGylated may be PEGylated by substantially the same procedures by which enzymes are PEGylated.
  • One useful PEGylation procedure employs previously derivatized cyanuric chloride-PEG
  • Protein or protein-bearing conjugate is buffer exchanged into 0.1M borate, pH 9.2;
  • the PEGylated product is purified by anion exchange chromatography, wherein the product passed through while underivatized protein or protein-bearing conjugate bound to the matrix.
  • non-proteinaceous moieties having appropriate functional groups may also be PEGylated.
  • a hydroxyl group of poly(ethyleneglycol) monomethylether is activated to form an active ester for reaction with an amine or other appropriate functional group on a proteinaceous or non-proteinaceous moiety.
  • poly(ethyleneglycol) monomethylether commercially available from Sigma Chemical Company, St. Louis, Missouri, is reacted to succinic anhydride to form poly (ethyleneglycol) monomethylether mono succinic acid.
  • the acid is reacted with DCC and N-hydroxy succinimide to form the product ester.
  • activating groups may be employed to produce a poly(ethyleneglycol) molecule amenable to conjugation with proteins, peptides or other moieties having suitable functional groups.
  • activating groups are cyanuric chloride (described in Anal. Biochem., 165: 114 (1987) and J. Biol. Chem., 252: 3578 (1977)); carbonate active esters such as para-nitrophenylcarbonate, succinimidyl carbonate or the like (see, generally, Appl. Biochem. Biotech., 11: 141 (1985)); other activated carbonates such as imidazolyl carbonate (Anal. Biochem., 131; 25 (1983)).
  • the offering ratio of PEG to protein impacts the results, with increased PEG rendering the protein both non-antigenic (i.e., unable to react with antibodies formed against the native protein) as well as non-immunogenic.
  • offering ratios of PEG:streptavidin and PEG: antibody will range between from about 5:1 to about 10:1.
  • PEGylated ligands and PEGylated anti-ligands to bind to the complementary member of the ligand/anti-ligand pair is tested in accordance with know procedures for testing ligand/anti-ligand binding affinity. See Example XI (E)(4), for example. Also, immune response profiles against the ligand or anti-ligand may be developed. Likewise, the ability of PEGylated proteinaceous targeting moiety to bind to target may be tested in accordance with known
  • fragments are basic compared to whole antibody, with the general exception that antibodies of the gamma-2b subclass undergo an acidic shift upon fragmentation. Fragments exhibiting a basic shift are characterized by greater kidney localization and shorter serum half life. Such fragments, being faster clearing and less immunogenic than their whole
  • antibody counterparts are particularly useful in diagnostic pretargeting protocols with or without clearing agent administration.
  • Such fragments can also be used in therapeutic pretargeting protocols of the present invention.
  • Preferred charge modification in accordance with the present invention involves treatment of a
  • charge-modified conjugate exhibiting an acidic shift in isoelectric point.
  • the shift in isoelectric point is one-tenth of a pH unit or greater.
  • charge-modified proteins exhibit a serum half-life that is at least 10% greater than the half-life of native proteins. A 50% or greater increase in half-life is not uncommon following charge modification to a protein.
  • Charge modification introducing more negative charge on a proteinaceous targeting moiety or on both components of a proteinaceous ligand- or anti-ligand-targeting moiety conjugate is a reduction in non-specific binding. Such a reduction directly reduces the ability of macrophages to metabolize and present peptides to immunocompetent cells, thereby resulting in reduced immunogenicity as well as slower metabolism and elimination of charge-modified conjugate.
  • peptides with a more homogenous charge distribution are characterized by an exposed surface of increased uniformity and are therefore less
  • Anion-forming agents useful in the practice of the present invention are structured to react with
  • anion-forming agents useful in the practice of the present invention are structured to react with primary amines on lysine residues of the protein to be charge modified.
  • anion-forming agents include active esters (carboxylic acid and imidate), maleimides and anhydrides.
  • active esters include N-hydroxysuccinimidyl, thiophenyl, 2,3,5,6-tetrafluorophenyl, and 2,3,5,6,-tetrafluorothiophenyl esters.
  • Derivatization of other protein residues may also be employed in the practice of the present invention (e.g., derivatization of arginine residues with glyoxal, phenyl glyoxal or cyclohexanedione).
  • Negatively charged groups which may be used to impart an acidic shift to proteinaceous targeting moieties, ligands or anti-ligands include phosphates, phosphonates, sulfates, nitrates, borates, silicates, carbonates, and carboxyl groups such as native carboxyl groups or carboxyl groups generated from an anhydride during the reaction of the anion-forming agent with the protein.
  • Useful anion-forming agents include compounds incorporating an anhydride and/or at least one COOH group, such as succinic anhydride, other cyclic acid anhydrides, phthalic anhydride, maleic anhydride, N-ethyl maleimide substituted with carboxyl groups, aliphatic anhydrides (e.g., acetic anhydride), aromatic anhydrides, pH-reversible anhydrides (e.g., citraconic anhydride and dimethyl maleic anhydride), alpha halo acids such as bromoacetate and iodoacetate, and diacids or triacids substituted with a functional group that reacts with an amino acid on a protein to be charge-modified.
  • succinic anhydride other cyclic acid anhydrides
  • phthalic anhydride maleic anhydride
  • N-ethyl maleimide substituted with carboxyl groups aliphatic anhydrides (e.g., acetic anhydride), aromatic anhydr
  • succinic anhydride is dissolved in DMSO or another dry organic solvent at a concentration of 40 mg per 200 microliters.
  • This succinic anhydride solution (or a dilution thereof up to 2.5 ml in anhydrous DMSO, 1.73 ⁇ 10 -2 M) is added to a protein (e.g., antibody, antibody fragment, ligand, anti-ligand or conjugate containing one or more of these components) solution (e.g., 3-5 mg/ml in carbonate/bicarbonate buffer, pH 8.5-9.0) at molar ratios of protein to succinic anhydride of 1:5, 1:10 and 1:25 (with higher molar ratios preferred).
  • the reaction is carried out at room temperature for 15-30 minutes.
  • succinic acid is removed by ultrafiltration or by gel filtration.
  • the degree of isoelectric shift is determined by isoelectric
  • polymer derivatization Another chemical modification that may be employed in the practice of the present invention is polymer derivatization.
  • Polymers exhibit a repetitive structure and have, in some instances, been found to be poorly immunogenic or non-immunogenic. For example, highly polymerized molecules of bacterial origin are T-cell independent, and a low affinity response can be generated against such molecules.
  • Such polymers may be conjugated to either or both of the components of the targeting moiety-ligand or targeting moiety-anti-ligand conjugates of the present invention, rendering them less immunogenic.
  • polymers having a small repetitive unit, a long serum half-life and relatively low molecular weight are useful in the practice of the present invention.
  • Exemplary polymers useful in the practice of the present invention are alpha-amino acid polymers, vinyl polymers and dextrans.
  • the immunogenicity of poly-alpha-amino acid polymers depends upon the structure of the polymer and upon the immunized species. In general, the more structurally homogeneous a molecule is, the less immunogenic it is. While antigens having one
  • determinant may elicit a delayed type
  • homopolymers of alpha-amino acids are therefore generally non-immunogenic.
  • Exemplary polymers of this type include poly-D-glutamic acid, poly-D-alanine, poly-glycine, poly-D-aspartic acid, poly-D-lysine, poly-D-proline, and poly-D-hydroxyproline. Copolymers of two amino acids are more immunogenic than
  • homopolymers and polymers formed of greater numbers of amino acids exhibit even greater immunogenicity.
  • molecular weight molecules may be rendered immunogenic by conjugation to an immunogen. Consequently, the alpha-amino acid homopolymers useful in the practice of the present invention range in molecular weight from about 5 kD to about 200 kD, with from about 10 kD to about 50 kD preferred.
  • homopolymers exhibiting a high net electric charge are generally characterized by low immunogenicity.
  • the high charge interferes with the presentation of the homopolymer-derivatized conjugates to immunocompetent cells.
  • D-amino acids are less immunogenic than L-amino acids. D-amino acids may exhibit some
  • D-amino acids are poorly metabolized and, therefore, are characterized by a long serum half-life. Homopolymers incorporating D-amino acids are preferred for use in the practice of the present invention.
  • Quaternary structure of alpha-amino acid polymers is of most importance with regard to immunogenicity, while antigenicity is highly influenced by secondary structure. Consequently, the most immunogenic portion of the polymer are antigenic determinants expressed on the surface of the quaternary structure and therefore exposed to the environment of the polymer. If such exposed polymer portions correspond to exposed
  • the conjugate is likely to be less immunogenic than an non-polymer-containing conjugate.
  • Targeting moieties such as antibodies or antibody fragments, ligands or anti-ligands may be derivatized with homopolymers of alpha-amino acids by known procedures. The ability of alpha amino acid
  • homopolymer-derivatized ligands and polymer-derivatized anti-ligands to bind to the complementary member of the ligand/anti-ligand pair is tested in accordance with known procedures for testing
  • ligand/anti-ligand binding affinity This procedure is based on ELISA techniques in which the derivatized molecule is tested for binding in a competitive assay against a non-derivatized molecule. Likewise, the ability of homopolymer-derivatized targeting moiety to bind to target may be tested in accordance with similar procedures for testing targeting moiety/target cell or target antigen binding.
  • Vinyl polymers are nondegradable under
  • polyvinylpyrrolidone similar to peptide polymers with regard to substituted amino groups and heterocyclic rings
  • poly-methacrylic acid-2-dimethylaminoethyl methacrylate a polyampholyte with protein-like molecular charge distribution and glutamic acid-lysine copolymer-like structure
  • polymethacrylic acid and polyvinylamine fully charged vinyl analogs of homopolymers of glutamic acid and lysine
  • Targeting moieties such as antibodies or antibody fragments, ligands or anti-ligands may be derivatized with vinyl polymers by known procedures.
  • Vinyl polymers generally exhibit alcohol and carboxylic acid functionalities.
  • the vinyl polymer derivatization procedure is as follows: vinyl polymer carboxylic acid functional group is converted to active ester form through the use of reactive alcohols or phenols in the presence of water soluble
  • vinyl polymer-derivatized targeting moiety to bind to target may be tested in accordance with known procedures for testing targeting moiety/target cell or target antigen binding.
  • Dextrans are naturally occurring homopolymers which are formed as exopolysaccharides by some Gram-negative bacteria.
  • Dextrans include alpha-D-glucose residues linked by glycosidic linkages selected from ⁇ (1->6), ⁇ (1->2), ⁇ (1->3) and ⁇ (1->4), with the majority of such linkages being ⁇ (1->6).
  • the degree of branching of the alpha-D-glucose chains in the dextran structure is the basis of classification of dextrans as class I (characterized by a linear
  • Dextrans are hydrophilic, water soluble molecules and are not easily degraded in biological systems. Dextrans with a molecular weight above 90,000 are generally immunogenic in man. Lower molecular weight dextrans are not immunogenically active (i.e., generally are unable to induce either an immune response or tolerance). Low molecular weight dextrans ( ⁇ 10,000 daltons) exhibit immunogenicity when
  • molecular weights ranging from about 5 kD to about 200 kD, with from about 10 kD to about 50 kD preferred.
  • Dextrans of varying molecular weights are commercially available from Sigma Chemical Co., St. Louis, Missouri, for example.
  • Targeting moieties such as antibodies or antibody fragments, ligands or anti-ligands may be derivatized with dextrans by known procedures.
  • the dextran derivatization procedure is as follows:
  • lysine-derivatized dextran polymers (commercially available from Sigma Chemical Co.) are activated by reacting a lysine residue thereof with the
  • bifunctional reagent SMCC (commercially available from Pierce Chemical Co.), preferably at room temperature for 0.5 to 1.0 hours in a 0.1M sodium borate buffer, pH 8.0-8.5; the activated dextran polymer contains a maleimide group available for conjugation with
  • endogenous sulfhydryl groups of targeting moieties generated by reaction of the targeting moiety with DTT for example.
  • Alternative functional groups may be introduced for conjugation with non-sulfhydryl-bearing targeting moieties, ligands or anti-ligands in accordance with procedures that are known in the art.
  • dextran-derivatized ligands and dextran-derivatized anti-ligands to bind to the complementary member of the ligand/anti-ligand pair is tested in accordance with know procedures for testing ligand/anti-ligand binding affinity.
  • dextran-derivatized targeting moiety to bind to target may be tested in accordance with known procedures for testing targeting moiety/target cell or target antigen binding.
  • conjugate component could be rendered less immunogenic using one chemical modification procedure, while another conjugate component is rendered less immunogenic using another.
  • the targeting moiety could be rendered less immunogenic via charge modification with succinic anhydride, while a ligand or anti-ligand is rendered less immunogenic via class I dextran derivatization.
  • both conjugate components could be rendered less immunogenic using the same chemical modification procedure, which modification preferably is conducted following conjugation.
  • antibodies with increased human character may be employed along with chemical modification of the ligand or anti-ligand.
  • the streptavidin portion may be PEGylated while the monoclonal antibody portion may be humanized.
  • the chemical modification is preferably conducted prior to
  • the chemically-modified conjugates useful in the present invention generally exhibit longer serum half-lives than their non-chemically-modified counterparts.
  • the drawback of an extended serum half-life is increased non-target site exposure to the active agent.
  • the present invention addresses this drawback in that the kinetics of the active agent is decoupled from that of the targeting moiety.
  • the optional clearing agent of the present invention provides the basis for removing targeting moiety-ligand conjugate or targeting moiety-anti-ligand conjugate from the recipient regardless of the extended half-life of the conjugate.
  • immunogenicity-reducing modification of the tertiary structure of a proteinaceous immunogen may be employed in the
  • Such tertiary structure modification is preferably conducted by recombinant techniques to reduce the number or
  • a preferred modification involves altering the charge of surface amino acids from a net negative charge to a neutral charge.
  • One method to accomplish this preferred modification is surface amino acid substitution conducted in a manner to substantially preserve the tertiary structure of the protein.
  • Another method to decrease the immunogenicity of administered conjugates is to administer an
  • immunosuppressive agents of the present invention are administered before, during and after conjugate administration.
  • Immunosuppressive agents such as cyclosporin A, which is commercially available from Sandoz Pharmaceuticals Corporation in various
  • formulations are typically non-targeted, systemically acting agents that generally reduce the recipient's immune response to non-self antigens.
  • Cyclosporin A is a neutral, hydrophobic cyclic peptide of 11 amino acids exhibiting a highly
  • Cyclosporin A can also restore the sensitivity of cell lines and experimental tumors that are resistant to several cancer therapeutics.
  • Cyclosporin A has been tested in humans and approved by the U.S. Food and Drug Administration for the prophylaxis of organ rejection in kidney, liver and heart allogenic transplants and in the treatment of chronic rejection in patients previously treated with other immunosuppressive agents. Cyclosporin A has also been tested in humans for the ability to decrease the immunogenicity of murine antibodies.
  • the results of some experiments involving the impact of cyclosporin A on human-anti-mouse antibody (HAMA) are set forth in Ledermann et al., "Repeated Antitumor Antibody Therapy in Man with Suppression of the Host Response by Cyclosporin A" and in Example XX. As the article and the material presented in Example XX indicate, cyclosporin A has utility in managing HAMA.
  • cyclosporin A is a candidate for immunosuppression of human anti-streptavidin antibody (HASA) as well as for immunosuppression of conjugates incorporating an immunogenic targeting moiety.
  • HASA human anti-streptavidin antibody
  • Cyclosporin A is commercially available in 25 mg soft gelatin capsules, as an oral solution of 100mg/mL, and in 5 ml sterile ampules containing 50 mg/ml for intravenous administration.
  • a dosing regimen for cyclosporin A to suppress the recipient's immune response to non-self moieties involves one or more pre-immunogen administration doses ranging from about 5 to about 15 mg/kg/day taken orally and ranging from about 1.5 to about 5.5 mg/kg/day f or intravenous and daily post-immunogen administrations thereof for approximately 7 days weeks.
  • pre-immunogen administration doses ranging from about 5 to about 15 mg/kg/day taken orally and ranging from about 1.5 to about 5.5 mg/kg/day f or intravenous and daily post-immunogen administrations thereof for approximately 7 days weeks.
  • the exact dosing regimen and the doses employed therein will be selected by the attending physician on a patient-by-patient basis depending on a variety of factors known to practicing attending physicians.
  • immunosuppressive agents have shown utility in suppressing a recipient's response to non-self antigens.
  • immunosuppressive agents useful in the practice of the present invention exhibit one or more of the following characteristics: low
  • azathioprine is known to directly inhibit B cell function and is approved for human use.
  • Cyclophosphamide is also approved for human use.
  • immunosuppressive agents include:
  • verapamil mycophenolic acid, transforming growth factor-beta, deoxyspergualin, FK506, rapamycin, immunophilins such as FK binding protein 12, peptide derivatives of non-erythroid spectin, fluorinated cyclosporin analogs, such as those discussed in U.S. Patent No. 5,227,467, and combinations thereof.
  • Clearing agents are also useful as immunosuppressive agents in the practice of the present invention.
  • immunosuppressive agent is administered both prior to and following administration of the immunogen.
  • the exact immunosuppressive agent dosing regimen and the doses employed therein for suppressing the immune response of a recipient to administered targeting moiety-ligand or targeting moiety-anti-ligand conjugate will be selected by the attending physician on a patient-by- patient basis depending on a variety of factors known to practicing attending physicians.
  • Immunosuppressive agents characterized by one or more of the following: short serum half-life; low therapeutic index; high cost; and significant dose-related toxicity, are preferably employed in liposomal or particulate form for use in the practice of the present invention. Sustained release dosage forms of this type will extend the serum half-lives of the immunosuppressive agents encapsulated therein. The extended serum half-life will increase the
  • Such dosage forms should also permit fewer administrations of immunosuppressive agent and/or a lower total immunosuppressive agent dose to constitute effective immunosuppressive
  • sustained release dosage forms facilitate release of immunosuppressive agents over time, thereby decreasing the maximum exposure of the recipient to that agent. That is, the immunosuppressive agent recipient is not exposed to a large initial dose, rather the recipient is exposed to a gradually
  • liposomal and particulate immunosuppressive agent dosage forms can be prepared using known techniques therefor. Liposome encapsulation of compounds is known in the art and discussed, for example, in U.S. Patent Nos. 4,948,590, 5,047,245 and 4,885,172. The preparation of particulate dosage forms is discussed with respect to particulate clearing agent dosage forms in Example IX.
  • Cyclosporin A for example, has a blood clearance half-life of about 6 hours.
  • the major toxicities of cyclosporin A are renal, and nephrotoxicity occurs in 25-75% of patients treated with the drug. This toxicity frequently mandates cessation or modification of cyclosporin A treatment.
  • a treatment regimen of cyclosporin A is also costly.
  • a liposomal formulation of cyclosporin A is expected to show an increased serum residence time, increased bioavailability, and decreased toxicity in comparison to the free drug. Such liposomal dosage forms would therefore exhibit an increase in therapeutic index over that attainable through administration of free immunosuppressive drug compositions. Increased bioavailability should result in a decrease in the amount of drug and/or drug administrations necessary for efficacious treatment, thereby decreasing overall treatment cost.
  • Non-specific pretargeting techniques are also contemplated by the present invention. Such as
  • pretargeting techniques contemplate administration of a non-specific targeting moiety-ligand or a non-specific targeting moiety-anti-ligand conjugate.
  • the non-specific targeting moiety equilibrates between the extravascular and vascular physiological compartment.
  • Non-specific targeting moiety-containing conjugate becomes trapped in the extravascular compartment and is cleared from the vascular compartment by the recipient's excretory mechanisms or via the use of a clearing agent or clearing mechanism in accordance with the present invention.
  • the non-specific targeting moiety-containing conjugate is administered, equilibrates between the extravascular compartment and the vascular compartment, and the conjugate in the vascular space is cleared using a clearing agent or a clearing mechanism.
  • Active agent-anti-ligand or active agent-ligand conjugate is then administered and such active agent-containing conjugate accretes to the
  • the active agent is maintained in the extravascular space for a time sufficient to exert a therapeutic benefit at the target site.
  • Non-specific pretargeting allows the use of large, non-immunogenic proteins (e.g., IgG and IgM) for radioimmunotherapy, for example.
  • IgG and IgM immunoimmunogenic proteins
  • conjugates may be administered in large doses, greater than the dose administered using specific murine antibody-containing conjugates.
  • a clearing agent or clearing mechanism is preferably administered or conducted between from about 18 to about 48 hours following non-specific targeting moiety containing conjugate administration.
  • Active agent-anti-ligand or active agent-ligand is administered between from about 2 to about 6 hours after the clearing agent administration (or between about 18 to 120 hours following the non-specific targeting moiety-containing conjugate if a clearing agent or mechanism is not used).
  • Short half-life active agents such as alpha-emitters, rapidly degraded active agents and the like, exert a therapeutic effect within a short time frame.
  • Such active agents are therefore particularly amenable to use in non-specific pretargeting protocols, because the conjugate need not reside in the extravascular space for an extended amount of time to deliver a therapeutically effective dose to target sites.
  • Example XXII describes experimentation involving a non-specific antibody-streptavidin conjugate; clearing agent; Y-90 chelate-labeled biotin pretargeting protocol that produced anti-tumor effects in mice, apparently through non-specific localization into the tumor interstitial fluid located in the extravascular compartment.
  • Such protocols are useful for other diagnostic or therapeutic purposes for target sites that are located in a physiological compartment other than the vasculature. Inflammation imaging and the like are amenable to non-specific pretargeting diagnostic or therapeutic protocols.
  • one conjugate component could be rendered less immunogenic using one chemical modification procedure, and an immunosuppressive agent could be employed to render the chemically modified conjugate less immunogenic.
  • an immunosuppressive agent could be employed to render the chemically modified conjugate less immunogenic.
  • targeting moiety could be rendered less immunogenic via charge modification with succinic anhydride, and cyclosporin A could be administered both before and after administration of the succinylated conjugate.
  • Other combinations of immunogenicity-reducing measures are also contemplated by the present invention.
  • a preferred targeting moiety useful in these embodiments of the present invention is a monoclonal antibody. Protein-protein conjugations are generally problematic due to the formation of undesirable byproducts, including high molecular weight and cross-linked species, however. A non-covalent synthesis technique involving reaction of biotinylated antibody with streptavidin has been reported to result in substantial byproduct formation. Also, at least one of the four biotin binding sites on the streptavidin is used to link the antibody and streptavidin, while another such binding site may be sterically
  • covalent streptavidin-antibody conjugation is preferred, but high molecular weight byproducts are often obtained.
  • the degree of crosslinking and aggregate formation is dependent upon several factors, including the level of protein derivitization using heterobifunctional crosslinking reagents.
  • Streptavidin-proteinaceous targeting moiety conjugates are preferably prepared as described in Example XI below, with the preparation involving the steps of: preparation of SMCC-derivatized
  • streptavidin preparation of DTT-reduced proteinaceous targeting moiety; conjugation of the two prepared moieties; and purification of the monosubstituted or disubstituted (with respect to streptavidin) conjugate from crosslinked (antibody-streptavidin-antibody) and aggregate species and unreacted starting materials.
  • the purified fraction is preferably further
  • thioether conjugates useful in the practice of the present invention may be formed using other thiolating agents, such as SPDP, iminothiolane, SATA or the like, or other thio-reactive
  • heterobifunctional cross linkers such as m-maleimidobenzoyl-N-hydroxysuccinimide ester, N-succinimidyl(4-iodoacetyl)aminobenzoate or the like.
  • Streptavidin-proteinaceous targeting moiety conjugates of the present invention can also be formed by conjugation of a lysine epsilon amino group of one protein with a maleimide-derivatized form of the other protein. For example, at pH 8-10, lysine epsilon amino moieties react with protein maleimides,
  • conjugates can be prepared by reaction of lysine epsilon amino moieties of one protein with aldehyde functionalities of the other protein.
  • the resultant imine bond is reducible to generate the corresponding stable amine bond.
  • Aldehyde functionalities may be generated, for example
  • streptavidin-targeting moiety conjugates Another method of forming streptavidin-targeting moiety conjugates involves immobilized iminobiotin that binds SMCC-derivatized streptavidin. In this conjugation/purification method, the reversible binding character of iminobiotin (immobilized) to streptavidin is exploited to readily separate
  • Iminobiotin binding can be reversed under conditions of lower pH and elevated ionic strength, e.g., NH 2 OAc, pH 4 (50 mM) with 0.5 M NaCl.
  • ionic strength e.g., NH 2 OAc, pH 4 (50 mM) with 0.5 M NaCl.
  • DTT-reduced antibody preferably free of reductant
  • a molar excess (with respect to streptavidin) of DTT-reduced antibody is added to the nitrogen-purged, phosphate-buffered iminobiotin column wherein the SMCC-streptavidin is bound (DTT-reduced antibody will saturate the bound SMCC-streptavidin, and unbound reduced antibody passing through the column can be reused);
  • targeting moiety-mediated ligand-anti-ligand pretargeting involves the
  • the first approach allows the targeting moiety-containing conjugate to clear from the blood by "natural” or endogenous clearance
  • conjugate target-to-blood ratio "chases” the conjugate from the circulation through in vivo complexation of conjugate with a molecule constituting or containing the complementary anti-ligand or ligand.
  • biotinylated antibodies are used as a ligand-targeting moiety conjugate, for example, avidin forms relatively large aggregated species upon complexation with the circulating biotinylated antibody, which aggregated species are rapidly cleared from the blood by the RES uptake. See, for example, U.S. Patent No. 4,863,713.
  • One problem with this method is the potential for cross-linking and internalizing tumor-bound biotinylated antibody by avidin.
  • poly-biotinylated transferrin When avidin-targeting moiety conjugates are employed, poly-biotinylated transferrin has been used to form relatively large aggregated species that are cleared by RES uptake. See, for example, Goodwin, J. Nucl. Med. 33(10) : 1816-18. 1992). Poly-biotinylated transferrin also has the potential for cross-linking and internalizing tumor-bound avidinylated-targeting moiety, however. In addition, both "chase"
  • the present invention provides clearing agents of protein and non-protein composition having physical properties facilitating use for in vivo complexation and blood clearance of anti-ligand/ligand (e.g., avidin/biotin)-targeting moiety (e.g., antibody) conjugates.
  • anti-ligand/ligand e.g., avidin/biotin
  • targeting moiety e.g., antibody
  • These clearing agents are useful in improving the target:blood ratio of targeting moiety conjugate.
  • Other applications of these clearing agents include lesional imaging or therapy involving blood clots and the like, employing antibody-active agent delivery modalities.
  • efficacious anti-clotting agent provides rapid target localization and high target: non-target targeting ratio.
  • Active agents administered in pretargeting protocols of the present invention using efficient clearing agents are targeted in the desirable manner and are, therefore, useful in the imaging/therapy of conditions such as pulmonary embolism and deep vein thrombosis.
  • Clearing agents useful in the practice of the present invention preferably exhibit one or more of the following characteristics:
  • Hexose-based clearing agents include hexose-based and non-hexose based moieties.
  • Hexose-based clearing agents are molecules that have been derivatized to incorporate one or more hexoses (six carbon sugar moieties) recognized by Ashwell receptors or other receptors such as the mannose/N-acetylglucosamine receptor which are associated with endothelial cells and/or Kupffer cells of the liver or the mannose 6-phosphate receptor.
  • hexoses are galactose, mannose, mannose 6-phosphate, N-acetylglusosamine and the like.
  • Ashwell receptors include glucose, N-galactosamine, N-acetylgalactosamine, thioglycosides of galactose and, generally, D-galactosides and glucosides or the like may also be used in the
  • Galactose is the prototypical clearing agent hexose derivative for the purposes of this description.
  • Galactose thioglycoside conjugation to a protein is preferably accomplished in accordance with the teachings of Lee et al., "2-Imino-2-methoxyethyl 1-Thioglycosides: New Reagents for Attaching Sugars to Proteins," Biochemistry, 15(18): 3956, 1976.
  • Another useful galactose thioglycoside conjugation method is set forth in Drantz et al, "Attachment of Thioglycosides to Proteins: Enhancement of Liver Membrane Binding," Biochemistry, 15(18) : 3963, 1976.
  • galactose-based and non-galactose based molecules are discussed below.
  • Protein-type galactose-based clearing agents include proteins having endogenous exposed galactose residues or which have been derivatized to expose or incorporate such galactose residues. Exposed
  • galactose residues direct the clearing agent to rapid clearance by endocytosis into the liver through specific receptors therefor (Ashwell receptors).
  • This clearance mechanism is characterized by high efficiency, high capacity and rapid kinetics.
  • An exemplary clearing agent of the protein-based/galactose-bearing variety is the
  • orosomucoid with neuraminidase removes sialic acid residues, thereby exposing galactose residues.
  • derivatized clearing agents include, for example, galactosylated albumin, galactosylated-IgM, galactosylated-IgG,
  • HSA Human serum albumin
  • HSA Human Serum Albumin
  • n is an integer from 1 to about 10 and m is an integer from 1 to about 25 and wherein the hexose is recognized by Ashwell receptors.
  • the ligand is biotin and the hexose is galactose. More preferably, HSA is derivatized with from 10-20
  • HSA clearing agents of the present invention are derivatized with from about 12 to about 15 galactoses and 3 biotins. Derivatization with both galactose and biotin are conducted in a manner
  • clearing agents based upon human proteins especially human serum proteins, such as, for example, orosomucoid and human serum albumin, human IgG, human-anti-antibodies of IgG and IgM class and the like, are less immunogenic upon administration into the serum of a human recipient.
  • human serum proteins such as, for example, orosomucoid and human serum albumin, human IgG, human-anti-antibodies of IgG and IgM class and the like
  • One way to prevent clearing agent compromise of target-bound conjugate through direct complexation is through use of a clearing agent of a size sufficient to render the clearing agent less capable of diffusion into the extravascular space and binding to target- associated conjugate.
  • This strategy is useful alone or in combination with the aforementioned recognition that exposed galactose residues direct rapid liver uptake.
  • This size-exclusion strategy enhances the effectiveness of non-galactose-based clearing agents of the present invention.
  • the combination (exposed galactose and size) strategy improves the
  • galactosylated, biotinylated proteins to remove circulating streptavidin-targeting moiety conjugates, for example
  • intermediate molecular weight ranging from about 40,000 to about 200,000 Dal
  • biotinylated asialoorosomucoid galactosyl-biotinyl-human serum albumin or other galactosylated and biotinylated derivatives of non-immunogenic soluble natural proteins, as well as biotin- and galactose-derivatized polyglutamate, polylysine, polyarginine, polyaspartate and the like.
  • High molecular weight moieties ranging from about 200,000 to about
  • conjugates of human serum albumin, IgG and IgM molecules and the like can also be used as clearing agents of the claimed invention.
  • Chemically modified polymers of intermediate or high molecular weight ranging from about 40,000 to about 1,000,000 Dal
  • galactose- and biotin-derivatized dextran hydroxypropylmethacrylamide polymers
  • polyvinylpyrrolidone-polystyrene copolymers divinyl ether-maleic acid copolymers, pyran copolymers, or PEG, also have utility as clearing agents in the practice of the present invention.
  • rapidly clearing biotinylated liposomes can be derivatized with galactose and biotin to produce clearing agents for use in the practice of the present invention.
  • a further class of clearing agents useful in the present invention involve small molecules (ranging from about 500 to about 10,000 Dal) derivatized with galactose and biotin that are sufficiently polar to be confined to the vascular space as an in vivo volume of distribution. More specifically, these agents exhibit a highly charged structure and, as a result, are not readily distributed into the extravascular volume, because they do not readily diffuse across the lipid membranes lining the vasculature.
  • Exemplary of such clearing agents are mono- or poly-biotin-derivatized
  • the galactose-exposed or -derivatized clearing agents are preferably capable of (1) rapidly and efficiently complexing with the relevant ligand- or anti-ligand-containing conjugates via ligand-anti-ligand affinity; and (2) clearing such complexes from the blood via the galactose receptor, a liver specific degradation system, as opposed to aggregating into complexes that are taken up by the generalized RES system, including the lung and spleen. Additionally, the rapid kinetics of galactose-mediated liver uptake, coupled with the affinity of the ligand-anti-ligand interaction, allow the use of intermediate or even low molecular weight carriers.
  • Non-galactose residue-bearing moieties of low or intermediate molecular weight (ranging from about 40,000 to about 200,000 Dal) localized in the blood may equilibrate with the extravascular space and, therefore, bind directly to target-associated
  • Protein-type and polymer-type non-galactose-based clearing agents include the agents described above, absent galactose exposure or derivitization and the like. These clearing agents act through an
  • the clearing agent used will be selected on the basis of the target organ to which access of the clearing agent is to be excluded. For example, high molecular weight (ranging from about 200,000 to about 1,000,000 Dal) clearing agents will be used when tumor targets or clot targets are involved.
  • Another class of clearing agents includes agents that do not remove circulating ligand or anti-ligand/targeting moiety conjugates, but instead
  • eap-type clearing agents are poly-biotin-derivatized 6,6'-[(3,3'-dimethyl[1,1'-biphenyl]-4,4'-diyl)bis(azo) bis[4-amino-5-hydroxy-1,3-naphthalene disulfonic acid] tetrasodium salt, poly-biotinyl-derivatized
  • polysulfated dextran-biotin mono- or poly-biotinyl-derivatized dextran-biotin and the like.
  • Cap-type clearing agents are derivatized with the relevant anti-ligand or ligand, and then administered to a recipient of previously administered ligand/ or anti-ligand/targeting moiety conjugate. Clearing agent-conjugate binding therefore diminishes the ability of circulating conjugate to bind any subsequently administered active agent-ligand or active agent-anti-ligand conjugate.
  • the ablation of active agent binding capacity of the circulating conjugate increases the efficiency of active agent delivery to the target, and increases the ratio of target-bound active agent to circulating active agent by preventing the coupling of long-circulating serum protein kinetics with the active agent. Also, confinement of the clearing agent to the plasma compartment prevents compromise of target-associated ligand or anti-ligand.
  • Clearing agents of the present invention may be administered in single or multiple doses.
  • a single dose of biotinylated clearing agent for example, produces a rapid decrease in the level of circulating targeting moiety-streptavidin, followed by a small increase in that level, presumably caused, at least in part, by re-equilibration of targeting moiety-streptavidin within the recipient's physiological compartments.
  • a second or additional clearing agent doses may then be employed to provide supplemental clearance of targeting moiety-streptavidin.
  • clearing agent may be infused
  • clearing agents and clearance systems are also useful in the practice of the present invention to remove circulating targeting moiety-ligand or -anti-ligand conjugate from the recipient's circulation.
  • Particulate-based clearing agents for example, are discussed in Example IX.
  • extracorporeal clearance systems are discussed in Example IX.
  • In vivo clearance protocols employing arterially inserted proteinaceous or polymeric multiloop devices are also described in Example IX.
  • the targeting moiety portion of the conjugate may be humanized or may constitute a fully human antibody to address the immunogenicity issue.
  • Such techniques cannot be employed with streptavidin, a bacterial protein.
  • streptavidin immunogenicity One such possibility is that the streptavidin-containing conjugate is simply cleared from the recipient too quickly for anti-streptavidin antibody formation to occur. In this case, any method of rapid serum clearance, such as particulate clearing agents or extracorporeal
  • the clearance mechanism utilized by the clearing agent e.g., an Ashwell receptor mechanism, delivers the streptavidin-containing conjugate to a physiological compartment that is less readily exposed to the immune system. In these latter cases, any clearing agent that operates using such mechanisms would be expected to attenuate streptavidin-containing conjugate immunogenicity.
  • One embodiment of the present invention in which rapid acting clearing agents are useful is in the delivery of Auger emitters, such as I-125, I-123, Er-165, Sb-119, Hg-197, Ru-97, Tl-201 and I-125 and Br-77, or nucleus-binding drugs to target cell nuclei.
  • Auger emitters such as I-125, I-123, Er-165, Sb-119, Hg-197, Ru-97, Tl-201 and I-125 and Br-77, or nucleus-binding drugs to target cell nuclei.
  • targeting moieties that localize to internalizing receptors on target cell surfaces are employed to deliver a targeting moiety-containing conjugate (i.e., a targeting moiety-anti-ligand conjugate in the preferred two-step protocol) to the target cell population.
  • a targeting moiety-containing conjugate i.e., a targeting moiety-anti-ligand conjugate in the preferred two-step protocol
  • internalizing receptors include EGF receptors, transferrin receptors, HER2 receptors, IL-2 receptors, other interleukins and cluster
  • an active agent-containing ligand or anti-ligand is administered.
  • conjugate such as a biotin-Auger emitter or a biotin-nucleus acting drug
  • a biotin-Auger emitter or a biotin-nucleus acting drug is administered as soon as the clearing agent has been given an opportunity to complex with circulating targeting moiety-containing conjugate, with the time lag between clearing agent and active agent administration being less than about 24 hours.
  • active agent is readily internalized through target cell receptor-mediated internalization.
  • Auger emitters While circulating Auger emitters are thought to be non-toxic, the rapid, specific targeting afforded by the pretargeting protocols of the present invention increases the potential of shorter half-life Auger emitters, such as I-123, which is available and capable of stable binding.
  • the radionuclide is preferably retained at the tumor cell surface. Loss of targeted radiation occurs as a consequence of metabolic degradation mediated by metabolically active target cell types, such as tumor or liver cells.
  • Radionuclides that are particularly amenable to the practice of this aspect of the present invention are rhenium, iodine and like "non +3 charged" radiometals which exist in chemical forms that easily cross cell membranes and are not, therefore, inherently retained by cells.
  • radionuclides having a +3 charge such as In-111, Y-90, Lu-177 and Ga-67, exhibit natural target cell retention as a result of their containment in high charge density chelates.
  • streptavidin-associated radionuclide can be administered in pretargeting protocols or injected directly into lesions.
  • streptavidin-associated radionuclide e.g., streptavidin-radionuclide and streptavidin-biotin-radionuclide
  • streptavidin-associated radionuclide may be administered as such (in pretargeting protocols) or as conjugates incorporating targeting moieties (intralesional injection and pretargeting protocols) specific for stable target cell surface antigens (such as NR-LU-10 antibody, L6, anti-CEA antibodies or the like) or target cell internalizing antigens (such as anti-HER2 neu ; anti-epidermal growth factor; anti-Lewis Y, including B-1, B-3, BR-64, BR-96 and the like; or the like) to target the streptavidin to the appropriate target cell population.
  • target cell surface antigens such as NR-LU-10 antibody, L6, anti-CEA antibodies or the like
  • target cell internalizing antigens such as anti-HER2 neu ; anti-epidermal growth factor; anti-Lewis Y, including
  • high molecular weight carriers such as biodegradable particles, dextran, albumin or the like, may be employed (e.g., conjugated to streptavidin) to limit leakage of the administered streptavidin from the injection site.
  • such carriers are biotinylated, thereby constituting suitable targets or carriers for radionuclide-streptavidin molecules.
  • streptavidin-associated radionuclide in intralesional injection protocols provides the following
  • radionuclide is used to better advantage, because the therapeutic efficacy of the administered radionuclide is improved as a result of retention at the target cell site; - microdiffusion from the injection site results in expansion of the field of radiation deposition;
  • - target sites are imageable post-injection to allow dosimetry determinations to be made
  • streptavidin-associated radionuclide in pretargeting protocols provides the following
  • radionuclide less radionuclide is used to better advantage, because the therapeutic efficacy of the administered radionuclide is improved as a result of retention at the target cell site;
  • - target sites are imageable post-injection to allow dosimetry determinations to be made
  • target cell retention-enhancing aspect of the present invention is applicable to a hybrid pretargeting/intralesional injection protocol.
  • targeting moiety-biotin conjugate is administered and an intralesional injection of
  • streptavidin follows after a time sufficient to permit localization of the targeting moiety-biotin conjugate to target cell sites of reasonably determinable location.
  • a radionuclide-biotin molecule is administered, wherein this administration is conducted by intralesional, intravenous or other convenient route.
  • conjugate may be used to pretarget streptavidin, preferably in additional embodiments of the two-step aspect of the present invention.
  • exemplary monovalent antibody fragments useful in these embodiments are Fv, Fab, Fab' and the like.
  • fragments typically exhibiting a molecular weight ranging from about 25 kD (Fv) to about 50 kD (Fab,
  • Fab' are smaller than whole antibody and, therefore, are generally capable of greater target site
  • monovalent binding can result in less binding carrier restriction at the target surface (occurring during use of bivalent antibodies, which bind strongly and adhere to target cell sites thereby creating a barrier to further egress into sublayers of target tissue), thereby improving the homogeneity of targeting.
  • a multivalent, with respect to ligand, moiety is preferably then administered.
  • This moiety also has one or more radionuclide associated therewith.
  • the multivalent moiety serves as both a clearing agent for circulating anti-ligand-containing conjugate (through cross-linking or aggregation of conjugate) and as a therapeutic agent when associated with target bound conjugate.
  • cross-linking at the tumor cell surface stabilizes the monovalent fragment-anti-ligand
  • fragments generally do not internalize as do bivalent or whole antibodies.
  • the difficulty in internalizing monovalent antibodies permits cross-linking by a monovalent moiety serves to stabilize the bound monovalent antibody through multipoint binding.
  • This two-step protocol of the present invention has greater flexibility with respect to dosing, because the decreased fragment immunogenicity allows more
  • streptavidin-containing conjugate for example, to be administered, and the simultaneous clearance and therapeutic delivery removes the necessity of a separate controlled clearing step.
  • methodologies of the present invention involves the route of administration of the ligand- or anti-ligand-active agents.
  • the active agent-ligand e.g., radiolabeled biotin
  • -anti-ligand is administered
  • the high extraction efficiency provided by avidin-biotin interaction facilitates delivery of very high radioactivity levels to the target cells, provided the radioactivity specific activity levels are high.
  • the limit to the amount of radioactivity delivered therefore becomes the biotin binding capacity at the target (i.e., the amount of antibody at the target and the avidin equivalent attached thereto).
  • radionuclide resulting from transmutation processes are preferred.
  • exemplary radionuclides include Y-90, Re-188, At-211, Bi-212 and the like.
  • Other reactor-produced radionuclides are useful in the practice of these embodiments of the present
  • a therapeutically effective amount of radiation ranges from about 1500 to about 10,000 cGy depending upon several factors known to nuclear medicine practitioners.
  • Intraarterial administration pretargeting can be applied to targets present in organs or tissues for which supply arteries are accessible.
  • Exemplary applications for intraarterial delivery aspects of the pretargeting methods of the present invention include treatment of liver tumors through hepatic artery administration, brain primary tumors and metastases through carotid artery administration, lung carcinomas through bronchial artery administration and kidney carcinomas through renal artery administration.
  • Intraarterial administration pretargeting can be conducted using chemotherapeutic drug, toxin and anti-tumor active agents as discussed below.
  • High potency drugs, lymphokines, such as IL-2 and tumor necrosis factor, drug/lymphokine-carrier-biotin molecules, biotinylated drugs/lymphokines, and drug/lymphokine/toxin-loaded, biotin-derivatized liposomes are exemplary of active agents and/or dosage forms useful for the delivery thereof in the practice of this embodiment of the present invention.
  • intraarterial administration discussed above and direct intralesional administration, of conjugate generally renders that conjugate less immunogenic.
  • Other examples include intraperitoneal administration for ovarian cancer, intralymphatic administration for lymph node metastases secondary to cervical cancer, intrapleural administration for intractable pleural effusion, and intrapericardially for pericardial effusion and impending cardiac tamponade.
  • the rapid clearance of nontargeted therapeutic agent decreases the exposure of non-target organs, such as bone marrow, to the therapeutic agent. Consequently, higher doses of radiation can be administered absent dose limiting bone marrow toxicity.
  • pretargeting methods of the present invention optionally include administration of short duration bone marrow
  • a protocol such as administration of
  • streptavidin-targeting moiety conjugate followed by administration of biotinylated cytokine, is also contemplated by the present invention.
  • pretargeting of anti-ligand serves to improve the performance of cytokine therapeutics by increasing the amount of cytokine localized to target cells.
  • Streptavidin-antibody conjugates generally exhibit pharmacokinetics similar to the native antibody and localize well to target cells, depending upon their construction.
  • Biotinylated cytokines retain a short in vivo half-life; however, cytokine may be localized to the target as a result of the affinity of biotin for avidin.
  • biotin-avidin experience a pH-dependent dissociation which occurs at a slow rate, thereby permitting a relatively constant, sustained release of cytokine at the target site over time.
  • cytokines complexed to target cells through biotin-avidin association are available for extraction and internalization by cells involved in cellular-mediated cytotoxicity.
  • a pre-formed antibody-streptavidin-biotin-cytokine preparation may also be employed in the practice of these methods of the present invention.
  • a three-step protocol of the present invention may also be employed to deliver a cytokine, such as IL-2, to a target site.
  • selectins include L-selectin, P-selectin and E-selectin.
  • cytokines stimulates cells, such as endothelial cells, to express selectins on the surfaces thereof.
  • Selectins bind to white blood cells and aid in delivering white blood cells where they are needed. Consequently, a protocol, such as administration of streptavidin- or avidin-targeting moiety conjugate followed by
  • biotinylated selectins administration of biotinylated selectins, is also contemplated by the present invention.
  • Such pretargeting of anti-ligand serves to improve the performance of selectin therapeutics by increasing the amount of selectin localized to target cells. In this manner, the necessity of cytokine induction of
  • selectin expression is obviated by the localization and retention of selectin at a target cell population.
  • Chemotherapeutic drugs also generally exhibit short in vivo half-lives at a therapeutically
  • a three-step protocol of the present invention may also be employed to deliver a chemotherapeutic drug, such as methotrexate,
  • trichothecenes such as esperamycins and calicheamycins, cytoxan, vinca alkaloids,
  • actinamycin D, taxol, taxotere or the like to a target site.
  • a chelating compound that contains an N 3 S chelating core was attached via an amide linkage to biotin.
  • Radiometal labeling of an exemplary chelate-biotin conjugate is illustrated below.
  • the spacer group "X" permits the biotin portion of the conjugate to be sterically available for avidin binding.
  • R 1 " is a carboxylic acid substituent (for instance, CH 2 COOH)
  • the conjugate exhibits improved water solubility, and further directs in vivo excretion of the radiolabeled biotin conjugate toward renal rather than hepatobiliary clearance.
  • N- ⁇ -Cbz-N- ⁇ -t-BOC protected lysine was converted to the succinimidyl ester with NHS and DCC, and then condensed with aspartic acid ⁇ - - t-butyl ester.
  • the resultant dipeptide was activated with NHS and DCC, and then condensed with glycine t-butyl ester.
  • the Cbz group was removed by hydrogenolysis, and the amine was acylated using tetrahydropyranyl mercaptoacetic acid succinimidyl ester, yielding S-(tetrahydropyranyl)-mercaptoacetyl-lysine.
  • Trifluoroacetic acid cleavage of the N-t-BOC group and t-butyl esters, followed by condensation with LC- biotin-NHS ester provided ( ⁇ -caproylamide biotin)-aspartyl glycine. This synthetic method is illustrated below.
  • the chelate-biotin conjugate of Example I was radiolabeled with either 99m Tc pertechnetate or 186 Re perrhenate. Briefly, 99m Tc pertechnetate was reduced with stannous chloride in the presence of sodium gluconate to form an intermediate Tc-gluconate
  • Example II The chelate-biotin conjugate of Example I was added and heated to 100°C for 10 min at a pH of about 1.8 to about 3.3. The solution was neutralized to a pH of about 6 to about 8, and yielded an N 3 S-coordinated 99m Tc-chelate-biotin conjugate.
  • C-18 HPLC gradient elution using 5-60% acetonitrile in 1% acetic acid demonstrated two anomers at 97% or greater radiochemical yield using ⁇ (gamma ray) detection.
  • 186 Re perrhenate was spiked with cold ammonium perrhenate, reduced with stannous chloride, and complexed with citrate.
  • the chelate-biotin conjugate of Example I was added and heated to 90°C for 30 min at a pH of about 2 to 3.
  • the solution was neutralized to a pH of about 6 to about 8, and yielded an N 3 S-coordinated 186 Re-chelate-biotin conjugate.
  • C-18 HPLC gradient elution using 5-60% acetonitrile in 1% acetic acid resulted in radiochemical yields of 85-90%. Subsequent purification over a C-18 reverse phase hydrophobic column yielded material of 99% purity.
  • a 99m Tc-biotin conjugate was subjected to various chemical challenge conditions. Briefly, 99m Tc-chelate-biotin conjugates were combined with avidin and passed over a 5 cm size exclusion gel filtration column. The radiolabeled biotin-avidin complexes were subjected to various chemical challenges (see Table 1), and the incubation mixtures were centrifuged through a size exclusion filter. The percent of radioactivity retained (indicating avidin-biotin-associated
  • Radiolabel is presented in Table 1.
  • Table 1 Thus, upon chemical challenge, the radiometal remained associated with the macromolecular complex.
  • each radiolabeled biotin conjugate was incubated at about 50 ⁇ g/ml with serum; upon completion of the incubation, the samples were subjected to instant thin layer chromatography (ITLC) in 80% methanol. Only 2-4% of the radioactivity remained at the origin (i.e., associated with
  • Each radiolabeled biotin conjugate was further examined using a competitive biotin binding assay. Briefly, solutions containing varying ratios of D-biotin to radiolabeled biotin conjugate were combined with limiting avidin at a constant total biotin: avidin ratio. Avidin binding of each radiolabeled biotin conjugate was determined by ITLC, and was compared to the theoretical maximum stoichiometric binding (as determined by the HABA spectrophotometric assay of Green, Biochem. J. 94:23c-24c, 1965). No significant difference in avidin binding was observed between each radiolabeled biotin conjugate and D-biotin.
  • Biotinylated NR-LU-10 was prepared according to either of the following procedures. The first procedure involved derivitization of antibody via lysine ⁇ -amino groups. NR-LU-10 was radioiodinated at tyrosines using chloramine T and either 125 I or 131 I sodium iodide. The radioiodinated antibody (5-10 mg/ml) was then biotinylated using biotinamido
  • biotinylation The impact of lysine biotinylation on antibody immunoreactivity was examined. As the molar offering of biotin:antibody increased from 5:1 to 40:1, biotin incorporation increased as expected (measured using the HABA assay and pronase-digested product) (Table 2, below). Percent of biotinylated antibody
  • NR-LU-10 was biotinylated using thiol groups generated by reduction of cystines.
  • NR-LU-10 was radioiodinated using p-aryltin phenylate NHS ester (PIP-NHS) and either 125 I or 131 I sodium iodide. Radioiodinated NR-LU-10 was incubated with 25 mM dithiothreitol and purified using size exclusion chromatography. The reduced antibody (containing free thiol groups) was then reacted with a 10- to 100-fold molar excess of N-iodoacetyl-n'-biotinyl hexylene diamine in phosphate-buffered saline (PBS), pH 7.5, containing 5% DMSO (v/v).
  • PBS phosphate-buffered saline
  • biotin antibody molar offering, only 6 biotins per antibody were incorporated. No significant impact on immunoreactivity was observed.
  • Biotinylated antibody (lysine) had an apparent molecular weight of 160 kD, while
  • biotinylated antibody species was performed using non-reducing SDS-PAGE, using a 4% stacking gel and a 5% resolving gel.
  • Biotinylated samples were either radiolabeled or unlabeled and were combined with either radiolabeled or unlabeled avidin or streptavidin. Samples were not boiled prior to SDS-PAGE analysis. The native antibody and biotinylated antibody (lysine) showed similar migrations; the biotinylated antibody (thiol) produced two species in the 50-75 kD range. These species may represent two thiol-capped species. Under these SDS-PAGE conditions, radiolabeled streptavidin migrates as a 60 kD tetramer. When 400 ⁇ g/ml
  • radiolabeled streptavidin was combined with 50 ⁇ g/ml biotinylated antibody (analogous to "sandwiching" conditions in vivo), both antibody species formed large molecular weight complexes.
  • biotinylated antibody (thiol)-streptavidin complex moved from the stacking gel into the resolving gel, indicating a decreased molecular weight as compared to the biotinylated antibody (lysine)-streptavidin complex.
  • Radioiodinated biotinylated NR-LU-10 (lysine or thiol) was intravenously administered to non-tumored nude mice at a dose of 100 ⁇ g.
  • mice were intravenously injected with either saline or 400 ⁇ g of avidin. With saline
  • blood clearances for both biotinylated antibody species were biphasic and similar to the clearance of native NR-LU-10 antibody.
  • biotinylated antibody lysine 2 h post-avidin or post-saline administration were performed.
  • Avidin administration significantly reduced the level of biotinylated antibody in the blood (see Figure 1), and increased the level of biotinylated antibody in the liver and spleen. Kidney levels of biotinylated antibody were similar.
  • NR-LU-10 antibody (MW ⁇ 150 kD) was radiolabeled with 125 I/Chloramine T and biotinylated via lysine residues (as described in Example IV.A, above).
  • Group 1 Time 0, inject 100 ⁇ g 125 I-labeled
  • Group 2 Time 0, inject 400 ⁇ g 131 I-labeled avidin (control) Time 2 h, inject 60 ⁇ g 186 Re-chelate- biotin conjugate
  • Group 3 Time 0, inject 60 ⁇ g 186 Re-chelate- (control) biotin conjugate
  • the three radiolabels employed in this protocol are capable of detection in the presence of each other. It is also noteworthy that the sizes of the three elements involved are logarithmically different ⁇ antibody ⁇ 150,000; avidin ⁇ 66,000; and biotin ⁇
  • Fig. 1 At 25 h, about 350 pmol/g biotinylated antibody was present at the tumor; at 32 h the level was about 300 pmol/g; at 48 h, about 200 pmol/g; and at 120 h, about 100 pmol/g. Avidin uptake at the same time points was about 250, 150, 50 and 0 pmol/g, respectively. From the same experiment, tumor to blood ratios were determined for biotinylated antibody and for avidin. From 32 h to 120 h, the ratios of tumor to blood were very similar.
  • Fig. 1 a 10-fold reduction in blood pool antibody concentration was noted (Fig. 1), resulting in a sharp increase in tumor to blood ratios.
  • Avidin is cleared rapidly, with greater than 90% of the injected dose cleared from the blood within 1 hour after administration.
  • the Re-186-biotin chelate is also very rapidly cleared, with greater than 99% of the injected dose cleared from the blood by l hour after administration.
  • the three-step pretargeting protocol (described for Group 1, above) was then examined. More
  • tumor uptake of the 186 Re-chelate-biotin conjugate in the presence or absence of biotinylated antibody and avidin was determined.
  • the 186 Re-chelate-biotin conjugate displayed a slight peak 2 h post-injection, which was substantially cleared from the tumor by about 5 h.
  • the 186 Re-chelate-biotin conjugate reached a peak in tumor approximately 7 times greater than that observed in the absence of biotinylated antibody and avidin.
  • the specifically bound 186 Re-chelate-biotin conjugate was retained at the tumor at significant levels for more than 50 h. Tumor to blood ratios determined in the same experiment
  • T:B ⁇ 8 at 30 h; ⁇ 15 at 100 h; ⁇ 35 at 140 h).
  • Tumor uptake of the 186 Re-chelate-biotin conjugate has further been shown to be dependent on the dose of biotinylated antibody administered.
  • biotinylated antibody At 0 ⁇ g of biotinylated antibody, about 200 pmol/g of 186 Re-chelate-biotin conjugate was present at the tumor at 2 h after administration; at 50 ⁇ g antibody, about 500 pmol/g of 186 Re-chelate-biotin conjugate; and at 100 ⁇ g antibody, about 1,300 pmol/g of 186 Re-chelate-biotin conjugate.
  • Tumor uptake results are best taken in context with radioactivity exposure to the blood compartment, which directly correlates with bone marrow exposure.
  • the very rapid clearance of the small molecule (Re-186-biotin) from the blood minimizes the exposure to Re-186 given in this manner.
  • direct labeled (conventional procedure) NR-LU-10 whole antibody yielded greater exposure to rhenium than did the 100-fold higher dose given in the three-step protocol.
  • radioactivity blood exposure to radioactivity
  • AUC tumor :AUC blood was observed for three-step pretargeting (approximately 7:1) in comparison to the direct labeled antibody approach (approximately 2.4:1).
  • a neutral MAMA chelate-biotin conjugate is prepared according to the following scheme.
  • the resultant chelate-biotin conjugate shows superior kidney excretion. Although the net overall charge of the conjugate is neutral, the polycarboxylate nature of the molecule generates regions of hydrophilicity and hydrophobicity. By altering the number and nature of the carboxylate groups within the conjugate, excretion may be shifted from kidney to
  • anionic compounds are generally cleared through the GI system.
  • Conjugates containing polylysine may also exhibit beneficial biodistribution properties. With whole antibodies, derivitization with polylysine may skew the biodistribution of conjugate toward liver uptake. In contrast, derivitization of Fab fragments with polylysine results in lower levels of both liver and kidney uptake; blood clearance of these conjugates is similar to that of Fab covalently linked to chelate.
  • An exemplary polylysine derivatized chelate-biotin conjugate is illustrated below.
  • polylysine derivatives are preferably succinylated following biotinylation.
  • Polylysine derivatives offer the further advantages of: (1) increasing the specific activity of the radiometal-chelate-biotin conjugate; (2) permitting control of rate and route of blood clearance by varying the molecular weight of the polylysine polymer; and (3) increasing the circulation half-life of the conjugate for optimal tumor
  • poly-L-lysine is acylated according to standard amino group acylation procedures (aqueous bicarbonate buffer, pH 8, added biotin-NHS ester, followed by chelate NHS ester).
  • the number of biotins attached to polylysine is determined by the HABA assay. Spectrophotometric titration is used to assess the extent of amino group derivitization.
  • conjugate is characterized by size exclusion.
  • linkers that are cleaved by enzymes present in normal tissue but deficient or absent in tumor tissue can increase tumor retention.
  • the kidney has high levels of ⁇ -glutamyl transferase; other normal tissues exhibit in vivo cleavage of 7-glutamyl prodrugs.
  • tumors are generally deficient in enzyme peptidases.
  • the glutamyl-linked biotin conjugate depicted below is cleaved in normal tissue and retained in the tumor.
  • biocytin is condensed with N-t-BOC-(O-sulfonate or O-glucose) serine NHS ester to give N-t-BOC-(O-sulfonate or O-glucose) serine biocytinamide.
  • N-t-BOC-(O-sulfonate or O-glucose) serine NHS ester to give N-t-BOC-(O-sulfonate or O-glucose) serine biocytinamide.
  • ligand-amidoserine O-sulfonate or O-glucose
  • Radioiodinated biotin derivatives prepared by exposure of poly-L-lysine to excess NHS-LC-biotin and then to Bolton-Hunter N-hydroxysuccinimide esters in
  • radioiodinated biotin Preparation of radioiodinated biotin according to the present invention provides certain advantages.
  • the radioiodobiotin derivative is a low
  • the disclosed methods for preparation involve a single step and eliminate the need for a purification step.
  • "X" may be any radiohalogen, including
  • tributyltin intermediate Water soluble carbodiimide was used in the above-depicted reaction, since the NHS ester 1 formed intractable mixtures with DCU.
  • the NHS ester was not compatible with chromatography; it was insoluble in organic and aqueous solvents and did not react with biocytin in DMF or in buffered aqueous acetonitrile.
  • the reaction between 1 and biocytin or 5-(biotinamido) pentylamine was sensitive to base.
  • the reaction of 1 and biocytin or the pentylamine was performed in the presence of triethylamine in hot DMSO, formation of more than one biotinylated product resulted.
  • the reaction was extremely clean and complete when a suspension of 1 and biocytin (4 mg/ml) or the pentylamine (4 mg/ml) was heated in DMSO at 117°C for about 5 to about 10 min.
  • the resultant 125 I-biotin derivatives were obtained in 94% radiochemical yield.
  • the radioiodinated products may be purified using C-18 HPLC and a reverse phase hydrophobic column.
  • mice conjugate labeled with 125 I using PIP-NHS (see Example IV.A.).
  • the mice received 42 ⁇ g of 131 I-PIP-biocytin.
  • the data showed immediate, specific tumor localization, corresponding to ⁇ 1.5 131 I-PIP-biocytin molecules per avidin molecule.
  • radiohalogenated biotin compounds are amenable to the same types of modifications described in Example VI above for 186 Re-chelate-biotin conjugates.
  • the following PIP-polylysine-biotin molecule is made by trace labeling polylysine with 125 I-PIP, followed by extensive
  • one or more sulfhydryl groups are attached to a targeting moiety through the use of chemical compounds or linkers that contain a terminal sulfhydryl group.
  • An exemplary compound for this purpose is iminothiolane.
  • a NR-LU-13-avidin conjugate is prepared as
  • SMCC-derived avidin is then incubated with NR-LU-13 in a 1:1 molar ratio at pH 8.5 for 16 h. Unreacted NR-LU-13 and SMCC-derived avidin are removed from the mixture using preparative size exclusion HPLC. Two conjugates are obtained as products ⁇ the desired 1:1 NR-LU-13-avidin conjugate as the major product; and an incompletely
  • a 99m Tc-chelate-biotin conjugate is prepared as in Example II, above.
  • the NR-LU-13-avidin conjugate is administered to a recipient and allowed to clear from the circulation.
  • One of ordinary skill in the art of radioimmunoscintigraphy is readily able to determine the optimal time for NR-LU-13-avidin conjugate tumor localization and clearance from the circulation.
  • the 99m Tc-chelate-biotin conjugate is administered to the recipient. Because the 99m Tc- chelate-biotin conjugate has a molecular weight of ⁇ 1,000, crosslinking of NR-LU-13-avidin molecules on the surface of the tumor cells is dramatically reduced or eliminated. As a result, the 99m Tc diagnostic agent is retained at the tumor cell surface for an extended period of time. Accordingly, detection of the
  • diagnostic agent by imaging techniques is optimized; further, a lower dose of radioisotope provides an image comparable to that resulting from the typical three-step pretargeting protocol.
  • NR-LU-13-avidin clearance of NR-LU-13-avidin from the circulation may be accelerated by plasmapheresis in combination with a biotin affinity column.
  • a biotin affinity column Through use of such column, circulating NR-LU-13-avidin will be retained extracorporeally, and the recipient's immune system exposure to a large, proteinaceous immunogen (i.e., avidin) is minimized.
  • an example of an extracorporeal clearance methodology may include the following steps:
  • withdrawing blood from the recipient by, for example,
  • NR-LU-13-avidin Clearance of NR-LU-13-avidin is also facilitated by administration of a particulate-type clearing agent (e.g., a polymeric particle having a plurality of biotin molecules bound thereto).
  • a particulate clearing agent e.g., a polymeric particle having a plurality of biotin molecules bound thereto.
  • Such a particulate clearing agent preferably constitutes a biodegradable polymeric carrier having a plurality of biotin
  • Particulate clearing agents of the present invention exhibit the capability of binding to circulating administered conjugate and removing that conjugate from the recipient.
  • Particulate clearing agents of this aspect of the present invention may be of any configuration suitable for this purpose.
  • Preferred particulate clearing agents exhibit one or more of the following
  • microparticulate e.g., from about 0.5
  • micrometers to about 100 micrometers in diameter, with from about 0.5 to about 2 micrometers more preferred), free flowing powder structure;
  • biodegradable structure designed to biodegrade over a period of time between from about 3 to about 180 days, with from about 10 to about 21 days more preferred, or non-biodegradable structure;
  • binding moieties preferably, the complementary member of the ligand/anti-ligand pair.
  • the total molar binding capacity of the particulate clearing agents depends upon the particle size selected and the ligand or anti-ligand substitution ratio.
  • the binding moieties are capable of coupling to the surface structure of the particulate dosage form through covalent or non-covalent modalities as set forth herein to provide accessible ligand or anti-ligand for binding to its previously administered circulating binding pair member.
  • Preferable particulate clearing agents of the present invention are biodegradable or non-biodegradable microparticulates. More preferably, the particulate clearing agents are formed of a polymer containing matrix that biodegrades by random,
  • Polymers derived from the condensation of alpha hydroxycarboxylic acids and related lactones are more preferred for use in the present invention.
  • thermoplastic polyesters e.g., polylactide or polyglycolide
  • a copolymer of lactide and glycolide components such as poly(lactide-co-glycolide).
  • An exemplary structure, a random poly(DL-lactide-coglycolide), is shown below, with the values of x and y being manipulable by a practitioner in the art to achieve desirable microparticulate properties.
  • agents suitable for forming particulate clearing agents of the present invention include polyorthoesters and polyacetals (Polymer Letters,
  • the procedure for forming particulate clearing agents of the present invention involves dissolving the polymer in a halogenated hydrocarbon solvent and adding an additional agent that acts as a solvent for the halogenated hydrocarbon solvent but not for the polymer.
  • the polymer precipitates out from the polymer-halogenated hydrocarbon solution.
  • they are washed and hardened with an organic solvent. Water washing and aqueous non-ionic surfactant washing steps follow, prior to drying at room temperature under vacuum.
  • particulate For biocompatibility purposes, particulate
  • the particulates are sterilized prior to packaging, storage or administration. Sterilization may be conducted in any convenient manner therefor.
  • the particulates can be irradiated with gamma radiation, provided that exposure to such radiation does not adversely impact the structure or function of the binding moiety attached thereto. If the binding moiety is so adversely impacted, the particulate clearing agents can be produced under sterile
  • the preferred lactide/glycolide structure is biocompatible with the mammalian physiological environment. Also, these preferred sustained release dosage forms have the advantage that biodegradation thereof forms lactic acid and glycolic acid, both normal metabolic products of mammals.
  • Functional groups required for binding moiety -particulate bonding are optionally included in the particulate structure , along with the non-degradable or biodegradable polymeric units .
  • Functional groups that are exploitable for this purpose include those that are reactive with ligands or anti-ligands , such as carboxyl groups , amine groups , sulfhydryl groups and the like .
  • Preferred binding enhancement moieties include the terminal carboxyl groups of the preferred (lactide-glycolide) polymer containing matrix or the like .
  • a practitioner in the art is capable of selecting appropriate functional groups and monitoring conjugation reactions involving those functional groups.
  • - particles in the "micron" size range localize in the RES and liver, with galactose derivatization or charge modification enhancement methods for this capability available, and, preferably, are designed to remain in circulation for a time sufficient to perform the clearance function; - the size of the particulates facilitates central vascular compartment retention thereof, substantially precluding equilibration into the peripheral or extravascular compartment;
  • ligand- or anti-ligand-particulate linkages having desired properties (e.g., serum biotinidase resistance thereby reducing the release of biotin metabolite from a particle-biotin clearing agent) and
  • - multiple ligands or anti-ligands can be bound to the particles to achieve optimal cross-linking of circulating targeting agent-ligand or -anti-ligand conjugate and efficient clearance of cross-linked species. This advantage is best achieved when care is taken to prevent particulate aggregation both in storage and upon in vivo administration.
  • a catheter-like device consisting of thin loops of synthetic polymer or protein fibers derivatized with biotin, is inserted into a major artery (e.g., femoral artery) to capture NR-LU-13-avidin. Since the total blood volume passes through a major artery every 70 seconds, the in situ clearing device is effective to reduce circulating NR-LU-13-avidin within a short period of time.
  • This device offers the advantages that NR-LU-13-avidin is not processed through the RES; removal of NR-LU-13-avidin is controllable and measurable; and fresh devices with undiminished binding capacity are insertable as necessary. This methodology is also useful with intraarterial administration embodiments of the present invention.
  • biotinylated, high molecular weight molecules such as liposomes, IgM and other molecules that are size excluded from ready permeability to tumor sites.
  • biotinylated, high molecular weight molecules aggregate with NR-LU-13-avidin, the aggregated
  • Example X
  • conjugates is advantageously used to improve delivery of therapeutic agents. More specifically, avidin crosslinking induces internalization of crosslinked complexes at the target cell surface.
  • Biotinylated NR-CO-04 (lysine) is prepared
  • Doxorubicin-avidin conjugates are prepared by standard conjugation chemistry.
  • the biotinylated NR-CO-04 is administered to a recipient and allowed to clear from the circulation.
  • One of ordinary skill in the art of radioimmunotherapy is readily able to determine the optimal time for biotinylated NR-CO-04 tumor localization and clearance from the circulation.
  • the doxorubicin-avidin conjugate is administered to the recipient.
  • the avidin portion of the doxorubicin-avidin conjugate crosslinks the biotinylated NR-CO-04 on the cell surface, inducing internalization of the complex.
  • doxorubicin is more efficiently delivered to the target cell.
  • a standard three-step pretargeting methodology is used to enhance intracellular delivery of a drug to a tumor target cell.
  • biotinylated NR-LU-05 is administered, followed by avidin (for blood clearance and to form the middle layer of the sandwich at the target cell-bound
  • biotinylated antibody Shortly thereafter, and prior to internalization of the biotinylated NR-LU-05-avidin complex, a methotrexate-biotin conjugate is
  • biotinylated NR-LU-05 is further covalently linked to methotrexate. Subsequent administration of avidin induces
  • NR-CO-04-avidin is administered to a recipient and allowed to clear from the circulation and localize at the target site. Thereafter, a polybiotinylated species (such as biotinylated poly-L-lysine, as in Example IV.B., above) is administered.
  • a polybiotinylated species such as biotinylated poly-L-lysine, as in Example IV.B., above
  • the drug to be delivered may be covalently attached to either the antibody-avidin component or to the polybiotinylated species.
  • the polybiotinylated species induces internalization of the (drug)-antibody-avidin-polybiotin-(drug) complex.
  • Example XI Example XI
  • the pH of the solution was adjusted to 8.5 by addition of 0.9 ml of 0.5 M berate buffer, pH 8.5.
  • a DMSO solution of SMCC (3.5 mg/ml) was prepared, and 477 ⁇ l (4.8 ⁇ mol) of this solution was added dropwise to the vortexing protein solution. After 30 minutes of stirring, the solution was purified by G-25 (PD-10, Pharmacia, Piscataway, New Jersey) column chromatography to remove unreacted or hydrolyzed SMCC.
  • the purified SMCC-derivatized streptavidin was
  • DTT-reduced NR-LU-10 Preparation of DTT-reduced NR-LU-10. To 77 mg NR-LU-10 (0.42 ⁇ mol) in 15.0 ml PBS was added 1.5 ml of 0.5 M borate buffer, pH 8.5. A DTT solution, at 400 mg/ml (165 ⁇ l) was added to the protein solution. After stirring at room temperature for 30 minutes, the reduced antibody was purified by G-25 size exclusion chromatography. Purified DTT-reduced NR-LU-10 was obtained (74 mg, 2.17 mg/ml).
  • conjugate were then sequentially eluted from the column using an increasing salt gradient in 20 mM diethanolamine adjusted to pH 8.6 with sodium
  • Biotin binding capacity was assessed, for example, by titrating a known quantity of conjugate with p-[I-125]iodobenzoylbiocytin. Saturation of the biotin binding sites was observed upon addition of 4 equivalences of the labeled biocytin.
  • streptavidin-NR-LU-10 whole antibody conjugate that exhibits blood clearance properties similar to native NR-LU-10 whole antibody, and tumor uptake and
  • Figure 3 depicts the tumor uptake profile of the NR-LU-10-streptavidin conjugate (LU-10-StrAv) in comparison to a control profile of native NR-LU-10 whole antibody.
  • LU-10-StrAv was radiolabeled on the streptavidin component only, giving a clear indication that LU-10-StrAv localizes to target cells as efficiently as NR-LU-10 whole antibody itself.
  • Re-BT Re-chelate-biotin conjugate
  • PIP-BT has the ability to bind well to avidin and is rapidly cleared from the blood, with a serum half-life of about 5 minutes. Equivalent results were observed for both molecules in the two-step pretargeting experiments described herein.
  • NR-LU-10 antibody (MW ⁇ 150 kD) was conjugated to streptavidin (MW ⁇ 66 kD) (as described in Example XI above) and radiolabeled with 125 I/PIP-NHS (as described for radioiodination of NR-LU-10 in Example IV.A., above).
  • streptavidin MW ⁇ 66 kD
  • radiolabeled with 125 I/PIP-NHS as described for radioiodination of NR-LU-10 in Example IV.A., above.
  • the experimental protocol was as follows:
  • tumor uptake was above 500 pMOL/G at the 40 hour time point and peaked at about 700 pMOL/G at 45 hours post-LU-10-StrAv administration.
  • radionuclide at its highest specific activity. Also, the rapid clearance of radionuclide that is not bound to LU-10-StrAv conjugate permits an increased
  • clearing agents were sought that are capable of clearing the blood pool of targeting moiety-anti-ligand conjugate (e.g., LU-10-StrAv), without compromising the ligand binding capacity thereof at the target sites.
  • targeting moiety-anti-ligand conjugate e.g., LU-10-StrAv
  • biotinylated asialoorosomucoid which employs the avidin-biotin interaction to conjugate to circulating LU-10-StrAv, was tested.
  • the two radiolabeled preparations were injected i.v. into female BALB/c mice (20-25 g), and blood clearance was assessed by serial retro-orbital eye bleeding of each group of three mice at 5, 10, 15 and 30 minutes, as well as at 1, 2 and 4 hours post-administration.
  • the results of this experiment are shown in Figure 5, with asialoorosomucoid clearing more rapidly than its orosomucoid counterpart.
  • asialoorosomucoid and the specificity of uptake of asialoorosomucoid in the liver (86%), as opposed to other tissues.
  • a fifth group received PIP-I-131-LU-10-StrAv conjugate which had been saturated prior to injection with biotin - group 5.
  • the 400 ⁇ g dose constituted a 10:1 molar excess of clearing agent over the initial dose of LU-10-StrAv conjugate, while the 200 ⁇ g dose constituted a 5:1 molar excess.
  • the saturated PIP-I-131-LU-10-StrAv conjugate was produced by addition of a 10-fold molar excess of D-biotin to 2 mg of LU-10-StrAv followed by size exclusion
  • mice from each group were serially bled, as described above, at 0.17, 1, 4 and 25 hours (preinjection of clearing agent), as well as at 27, 28, 47, 70 and 90 hours. Two additional animals from each group were sacrificed at 2 hours post-clearing agent administration and limited biodistributions were performed.
  • Biodistribution data are shown in tabular form in Figure 8.
  • the biodistribution data show reduced levels of conjugate for groups 3 and 4 in all tissues except the liver, kidney and intestine, which is consistent with the processing and excretion of radiolabel associated with the conjugate after
  • biotinylated asialoorosomucoid resulted in a 50-fold reduction in serum biotin-binding capacity and, in preliminary studies in tumored animals, has not exhibited cross-linking and removal of prelocalized LU-10-StrAv conjugate from the tumor.
  • asialoorosomucoid-biotin was highly effective at reducing blood levels of circulating streptavidin-containing conjugate by an in vivo
  • mice subcutaneously with LS-180 human colon carcinoma xenografts as described above, were randomized into groups of 4 animals/timepoint. The mice were
  • mice paraiodophenyl (PIP) I-125, as described in Example IV above.
  • Groups of mice were sacrificed at 26, 30, 48, 96 and 144 hours post-conjugate injection. Tissues were isolated, weighed and counted with respect to iodine radionuclide content using conventional
  • mice bearing LS-180 xenografts were also randomized into groups of 4 animals/timepoint. These mice were intravenously injected with 50 ⁇ g of NR-LU-10 monoclonal antibody radiolabeled with paraiodophenyl (PIP) 1-131 (MAB), as described in Example IV above. Mice were sacrificed at 4, 24, 48, 128 and 168 hours post-radiolabeled monoclonal antibody injection. Tissues were isolated, weighed and counted with respect to iodine
  • NR-LU-10-streptavidin-PIP-I-125 and NR-LU-10-PIP-I-131 in LS-180 tumors over time.
  • the NR-LU-10-streptavidin conjugate exhibits higher tumor uptake and a longer retention time as compared to NR-LU-10 alone.
  • mice Female nude mice xenografted with LS-180 tumor cells, as discussed above, were randomized into groups of 4 animals/timepoint. Mice were intravenously injected with 50 ⁇ g of biotinylated NR-LU-10
  • the antibody portion of the complex was radiolabeled with 1-125 using chloramine-T, and the streptavidin portion was labeled with paraiodophenyl (PIP) 1-131, both of the labeling procedures having been described above.
  • PIP paraiodophenyl
  • Mice were sacrificed at 4, 24, 48, 96 and 144 hours post-conjugate injection. Tissues were isolated, weighed and counted with respect to the content of each iodine radionuclide using conventional procedures therefor.
  • FIG. 13 shows the percent injected dose per gram of streptavidin-PIP-I-131 (STREPT) and NR-LU-10-biotin-Chloramine-T-I-125 (MAB-BT) in liver over time.
  • STREPT streptavidin-PIP-I-131
  • MAB-BT NR-LU-10-biotin-Chloramine-T-I-125
  • xenografted with xenografted with LS-180 tumor cells as discussed above, and were intravenously injected with 200 ⁇ g of 1:1 mol/mol NR-LU-10 monoclonal antibody covalently coupled to streptavidin, prepared as described in Example XI above.
  • the antibody portion of the conjugate was radiolabeled with
  • mice received an injection of 0.5 ⁇ g of PIP-I-125.
  • PIP I-131 paraiodophenyl
  • FIG. 14 shows the percent injected dose per gram of streptavidin-monoclonal antibody-PIP-I-125 (STREP-MAB-I-125) and biocytin-PIP-I-131 (BT-I-131) in liver over time.
  • Figures 15A (%ID/G v. Dose) and 15B (pMOL/G v. Dose).
  • the three highest doses produced PIP-biocytin tumor localizations of about 600 pmol/g. Histology conducted on tissues receiving the two highest doses indicated that saturation of tumor-bound streptavidin was achieved. Equivalent tumor localization observed at the 5.7 ⁇ g dose ( Figure 15B) is indicative of streptavidin saturation as well. In contrast, the two lowest doses produced lower absolute tumor
  • the lowest dose group (0.5 ⁇ g) exhibited high efficiency tumor delivery of PIP-I-131-biocytin, which efficiency increased over time, as shown in Figure 16A.
  • a peak uptake of 85.0 % ID/G was observed at the 120 hour time point (96 hours after administration of PIP-biocytin).
  • the absolute amount of PIP-biocytin, in terms of % ID showed a continual increase in the tumor over all of the sampled time points ( Figure 16B).
  • the decrease in uptake on a % ID/G basis ( Figure 16A) at the 168 hour time point resulted from significant growth of the tumors between the 120 and 168 hour time points.
  • Figure 17A shows the co-localization of NR-LU-10-Streptavidin conjugate (LU-10-StrAv) and the subsequently administered PIP-Biocytin at the same tumors over time.
  • the localization of radioactivity at tumors by PIP-biocytin exhibited a pattern of uptake and retention that differed from that of the antibody-streptavidin conjugate (LU-10-StrAv).
  • LU-10-StrAv exhibited a characteristic tumor uptake pattern that is equivalent to historical studies of native NR-LU-10 antibody, reaching a peak value of 40% ID/G between 24 and 48 hours after administration.
  • the PIP-Biocytin exhibited an initial rapid accretion in the tumor, reaching levels greater than those of LU-10-StrAv by 24 hours after PIP-Biocytin administration. Moreover, the localization of PIP-Biocytin continued to increase out to 96 hours, when the concentration of radioactivity associated with the conjugate has begun to decrease. The slightly greater amounts of circulating PIP-Biocytin compared to LU-10-StrAv at these time points (shown in Figure 17B) appeared insufficient to account for this phenomenon.
  • the AUC tumor /AUC blood for PIP-Biocytin is over twice that of the conjugate (4.27 compared to 1.95, where AUC means "area under the curve"). Further, the absolute AUC tumor for PIP-Biocytin is nearly twice that of the conjugate (9220 compared to 4629).
  • mice were conducted using female BALB/c mice (20-25 g).
  • mice Female nude mice were injected subcutaneously with LS-180 tumor cells, and, after 7 d, the mice displayed 50-100 mg tumor xenografts.
  • the monoclonal antibody used in these experiments was NR-LU-10.
  • radiolabeled the NR-LU-10-streptavidin conjugate was radiolabeled with 1-125 using procedures described herein.
  • PIP-biocytin was labeled with I-131 or I-125 using procedures described herein.
  • LS-180 colon tumor xenografts were injected with 200 micrograms NR-LU-10 antibody-streptavidin (MAb-StrAv) conjugate at time 0, which was allowed to prelocalize to tumor for 22 hours. At that time, 20 micrograms of AO-Bt was administered to one group of animals. Two hours later, 90 micrograms of a radioisotope-bearing, ligand-containing small molecule (PIP-biotin-dextran prepared as discussed in part B hereof) was
  • the clearing agent was radiolabeled in a separate group of animals and found to bind directly to tumor at very low levels (1.7 pmol/g at a dose of 488 total pmoles (0.35%ID/g), indicating that it does not significantly compromise the ability of tumor-bound MAb-StrAv to bind subsequently administered radiolabeled ligand.
  • HSA Human Serum Albumin
  • the final percent DMSO in the reaction mixture should not exceed 5%. After stirring for 1 hour at room temperature, the reaction was complete. A 90% incorporation efficiency for biotin on HSA was generally observed. As a result, if 3 molar equivalences of the NHS ester of LC-biotin was introduced, about 2.7 biotins per HSA molecule were obtained. Unreacted biotin reagent was removed from the biotin-derivatized HSA using G-25 size exclusion chromatography. Alternatively, the crude material may be directly galactosylated. The same chemistry is applicable for biotinylating non-previously
  • HSA-biotin was then derivatized with from 12 to 15 galactoses/molecule.
  • Galactose derivatization of the biotinylated HSA was performed according to the procedure of Lee, et al., Biochemistry. 15: 3956, 1976. More specifically, a 0.1 M methanolic solution of cyanomethyl-2,3,4,6-tetra-O-acetyl-1-thio-D-galactopyranoside was prepared and reacted with a 10% v/v 0.1 M NaOMe in methanol for 12 hours to generate the reactive galactosyl thioimidate.
  • HSA in PBS buffered with 10% v/v 0.5 M sodium borate, was added to the oily residue. After stirring at room temperature for 2 hours, the mixture was stored at 4oC for 12 hours.
  • the galactosylated HSA-biotin was then purified by G-25 size exclusion chromatography or by buffer exchange to yield the desired product. The same chemistry is exploitable to galactosylating dextran. The incorporation efficiency of galactose on HSA is approximately 10%.
  • G-HSA-B Galactose-HSA-Biotin
  • Non-Protein Clearing Agent A commercially available form of dextran, molecular weight of 70,000 daltons, pre-derivatized with approximately 18 biotins/molecule and having an equivalent number of free primary amines was studied. The primary amine moieties were derivatized with a galactosylating reagent, substantially in accordance with the
  • asialoorosomucoid clearing agent regarding plots of tumor/blood ratio were found with respect to G-HSA-B, in that an optimal balance between blood clearance and tumor retention occurred around the 40 microgram dose.
  • a retention linker has a chemical structure that is resistant to agents that cleave peptide bonds and, optionally, becomes protonated when localized to a catabolizing space, such as a lysosome.
  • Preferred retention linkers of the present invention are short strings of D-amino acids or small molecules having both of the
  • An exemplary retention linker of the present invention is cyanuric chloride, which may be interposed between an epsilon amino group of a lysine of a proteinaceous primary clearing agent component and an amine moiety of a reduced and chemically altered biotin carboxy moiety (which has been discussed above) to form a compound of the structure set forth below.
  • biotin catabolites containing the heterocyclic ring are restricted to the site(s) of catabolism and, therefore, do not compete with active-agent-biotin conjugate for prelocalized targeting moiety-streptavidin target sites.
  • Another key result of the dose ranging experimentation is that G-HSA-B with an average of only 1 biotin per molecule is presumably only clearing the MAb-StrAv conjugate via the Ashwell receptor mechanism only, because too few biotins are present to cause cross-linking and aggregation of MAb-StrAv conjugates and clearing agents with such aggregates being cleared by the reticuloendothelial system.
  • Time 0 administer 400 micrograms MAb-StrAv conjugate
  • Time 24 hours administer 240 micrograms of G-HSA-B with one biotin and 12-15 galactoses and Time 26 hours: administer 6 micrograms of
  • Lu-177 is complexed with the DOTA chelate using known techniques therefor, and the DOTA chelate is prepared in accordance with the following procedure.
  • N-methylglycine trivial name sarcosine, available from Sigma Chemical Co.
  • biotin-NHS ester in DMF and triethylamine to obtain N-methyl glycylbiotin.
  • N-methyl-glycyl biotin was then activated with EDCI and NHS.
  • the resultant NHS ester was not isolated and was condensed in situ with DOTA-aniline preparable using known techniques (e.g., McMurry et al., Bioconjugate Chem., 3 : 108-117, 1992) and excess pyridine.
  • the reaction solution was heated at 60oC for 10 minutes and then evaporated.
  • the residue was purified by preparative HPLC to give [(N-methyl-N- biotinyl)-N-glycyl]-aminobenzy
  • the AUC tumor/AUC blood obtained for this non-optimized clearing agent dose was 300% greater than that achievable by comparable direct MAb-radiolabel administration. Subsequent experimentation has resulted in AUC tumor/AUC blood over 1000% greater than that achievable by comparable conventional MAb-radiolabel administration.
  • the HSA-based clearing agent is expected to exhibit a low degree of immunogenicity in humans.
  • mice Three test groups (Groups I, II and III) of female Balb/C mice between 16 and 20 grams were used in the study, with each group containing 15 mice and split into 3 equal subgroups containing 5 mice each. Each of the mice in the three test groups received murine NR-LU-10-streptavidin conjugate, which conjugate approximates humanized or human NR-LU-10-streptavidin conjugate administration in a human.
  • Group I received 120 ⁇ g NR-LU-10-streptavidin conjugate prepared substantially as described in
  • Example XI above by intravenous injection via the tail vein in 4 weekly injections (at weeks 0, 1, 2 and 3).
  • Group II also received 120 ⁇ g NR-LU-10-streptavidin conjugate by intravenous injection via the tail vein in 4 weekly injections.
  • Group II mice also received 60 ⁇ g of a biotinylated and galactosylated murine serum albumin administered intravenously 24 hours after each of the 4 NR-LU-10-streptavidin conjugate administrations.
  • Group III mice received 120 ⁇ g of NR-LU-10-streptavidin conjugate intramuscularly.
  • Serum samples were collected on weeks 2, 4 and 6 from Groups I-III and control mice. Anti-streptavidin levels were detected by ELISA using streptavidin-coated plates and serial dilutions of serum followed by goat anti-mouse horseradish peroxidase (HRPO) detection.
  • HRPO horseradish peroxidase
  • the clearing agent is able to remove non-tumor associated antibody-streptavidin conjugate from circulation to the hepatobiliary system prior to the formation of anti-streptavidin antibodies by the mouse immune system.
  • the immunosuppressive effect of the clearing agent should be enhanced, because the levels of circulating biotin are 2 to 3 orders of magnitude lower in humans than in mice.
  • more circulating streptavidin will be able to bind to biotin-containing clearing agent and be rapidly cleared from
  • Immunohistochemical analysis showed tumor localization of Re-186, antibody NR-LU-10 and streptavidin.
  • a peroxidase-conjugated anti-murine probe was used to detect NR-LU-10 in the tumor section and a chromogen/substrate was then administered, which colorant yielded a brown stain at sites of NR-LU-10 localization.
  • a negative result appeared as sections devoid of brown staining.
  • a known positive tumor control (ovarian carcinoma) was also run concurrently with the clinical specimens. Degree of reactivity of clinical specimens was compared to the positive control.
  • Streptavidin was shown to localize in the same pattern as NR-LU-10 by rabbit-anti-streptavidin antibody which was detected by a goat anti-rabbit horse radish peroxidase conjugate (HRPO).
  • HRPO horse radish peroxidase conjugate
  • NR-CO-02 is a murine IgG1 monoclonal antibody that recognizes a CEA-like antigen. NR-CO-02 was pepsin cleaved to the Fab') 2 fragment which was labeled with Tc-99m for imaging and Re-196 for therapy.
  • NR-LU-10 is a murine IgG2b monoclonal antibody that recognizes a glycoprotein antigen expressed by several epithelial tumors. The Fab fragment of NR-LU-10 was labeled with Tc-99m and used for diagnostic imaging. The intact murine antibody was labeled with Re-186 and
  • Cyclosporin was given orally at an initial dose of 8.6-15 mg/kg/day. The daily dose was divided into two doses taken 12 hours apart and begun 48 hours prior to administration of the Tc-99m-labeled murine antibody fragment. Cyclosporin was continued for 14 days after the second (Re-186-labeled) murine antibody
  • HAMA results in patients receiving cyclosporin were compared with those previously reported in 28 patients who received two doses of NR-CO-02 F(ab') 2 or 15 patients who received a single dose of NR-LU-10 Fab followed by a single dose of intact NR-LU-10 antibody (Breitz et al. referenced above).
  • the geometric mean titer of HAMA formed by patients who were given cyclosporin was substantially less than that of patients given NR-LU-10 without cyclosporin.
  • Cyclosporin given from two days before until two weeks after administration can slow down or suppress HAMA formation.
  • a patient presents with colon cancer.
  • An oral dose of cyclosporin ranging between 10 and 15
  • a monoclonal antibody (MAb) directed to a colon cancer cell antigen such as NR-LU-10 is conjugated to streptavidin to form a NR-LU-10-streptavidin conjugate.
  • MAb monoclonal antibody directed to a colon cancer cell antigen
  • streptavidin to form a NR-LU-10-streptavidin conjugate.
  • the NR-LU-10-streptavidin conjugate is administered to the patient in an amount in excess of the maximum
  • chelate labeled molecule protocol e.g., administration of monoclonal antibody-chelate-radionuclide conjugate
  • Galactose-human serum albumin-biotin as described in Example XVIII above is then administered as a clearing agent to remove circulating NR-LU-10-streptavidin conjugate.
  • Y-90-DOTA-N-methyl-glycine-biotin conjugate of the type discussed in Example XXII below is dispersed in a pharmaceutically acceptable diluent and administered to the patient in a therapeutically effective dose.
  • biotin-radionuclide chelate conjugate localizes to the targeted NR-LU-10-streptavidin moiety or is removed from the patient via the renal pathway.
  • Example XXII is administered, preferably in two daily administrations as described above, to reduce HAMA response to NR-LU-10 if murine or chimeric antibody is employed and/or to reduce HASA to streptavidin.
  • Example XXII is administered, preferably in two daily administrations as described above, to reduce HAMA response to NR-LU-10 if murine or chimeric antibody is employed and/or to reduce HASA to streptavidin.
  • mice bearing SHT-1 tumors were employed in this study.
  • the average initial tumor volume for the mice employed in this study was in excess of 250 mm 3 , constituting a large tumor burden.
  • Three groups of test mice were studied as follows:
  • Group I 400 ⁇ Ci of Y-90-DOTA-N-methyl-glycine-biotin, preparable in accordance with the synthetic procedure described below, was administered.

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Abstract

L'invention concerne des procédés des composés, des compositions et des kits permettant d'effectuer l'administration préciblée d'agents diagnostiques et thérapeutiques. Elle concerne en particulier des procédés et des agents servant à limiter l'immunogénicité de conjugués de ciblage fraction-anti-ligand ou d'autres constituants utilisés dans des protocoles de préciblage diagnostiques et thérapeutiques.
PCT/US1994/014223 1993-12-09 1994-12-09 Procedes et composes de preciblage WO1995015770A1 (fr)

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Cited By (22)

* Cited by examiner, † Cited by third party
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EP0743956A1 (fr) * 1993-12-07 1996-11-27 NeoRx Procede et composes permettant un ciblage prealable
WO1996037516A1 (fr) * 1995-05-26 1996-11-28 Meir Strahilevitz Procedes de traitement, de diagnostic et de visualisation, notamment de tumeurs cancereuses
EP0813425A1 (fr) * 1993-07-12 1997-12-29 Neorx Corporation Proteines marquees avec des radionucleides metalliques destinees a une utilisation diagnostique et therapeutique
EP0906015A1 (fr) * 1996-06-06 1999-04-07 Neorx Corporation Agents de suppression de la retention hepatique
EP0914042A1 (fr) * 1996-06-06 1999-05-12 Neorx Corporation Agents de suppression d'amas
WO2000075333A1 (fr) * 1999-06-07 2000-12-14 Neorx Corporation Fusions de genes exprimees par la streptavidine et methodes pour les utiliser
US6908903B1 (en) 1994-12-07 2005-06-21 Aletheon Pharmaceuticals, Inc. Cluster clearing agents
US7144991B2 (en) 1999-06-07 2006-12-05 Aletheon Pharmaceuticals, Inc. Streptavidin expressed gene fusions and methods of use thereof
WO2017214339A1 (fr) 2016-06-08 2017-12-14 Abbvie Inc. Anticorps anti-b7-h3 et conjugués anticorps-médicaments
WO2017214322A1 (fr) 2016-06-08 2017-12-14 Abbvie Inc. Anticorps anti-b7-h3 et conjugués anticorps-médicaments
WO2017214456A1 (fr) 2016-06-08 2017-12-14 Abbvie Inc. Anticorps anti-cd98 et conjugués anticorps-médicament
WO2018195302A1 (fr) 2017-04-19 2018-10-25 Bluefin Biomedicine, Inc. Anticorps anti-vtcn1 et conjugués anticorps-médicament
US10640563B2 (en) 2016-06-08 2020-05-05 Abbvie Inc. Anti-B7-H3 antibodies and antibody drug conjugates
US11160821B2 (en) 2017-05-19 2021-11-02 Lunella Biotech, Inc. Antimitoscins: targeted inhibitors of mitochondrial biogenesis for eradicating cancer stem cells
US11197872B2 (en) 2017-04-21 2021-12-14 Lunella Biotech, Inc. Vitamin C and doxycycline: a synthetic lethal combination therapy for eradicating cancer stem cells (CSCs)
US11229657B2 (en) 2017-04-21 2022-01-25 Lunella Biotech, Inc. Targeting hypoxic cancer stem cells (CSCs) with doxycycline: implications for improving anti-angiogenic therapy
US11260027B2 (en) 2015-07-29 2022-03-01 Musc Foundation For Research Development Donor organ pre-treatment formulation
US11667639B2 (en) 2017-06-26 2023-06-06 Lunella Biotech, Inc. Mitoketoscins: mitochondrial-based therapeutics targeting ketone metabolism in cancer cells
EP4218929A1 (fr) 2014-03-21 2023-08-02 AbbVie Inc. Anticorps anti-egfr et conjugués anticorps-médicament
US11759527B2 (en) 2021-01-20 2023-09-19 Abbvie Inc. Anti-EGFR antibody-drug conjugates
US12006553B2 (en) 2017-05-19 2024-06-11 Lunella Biotech, Inc. Companion diagnostics for mitochondrial inhibitors
WO2025027529A1 (fr) 2023-07-31 2025-02-06 Advesya Conjugués médicament-anticorps anti-il-1rap et leurs procédés d'utilisation

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Cited By (31)

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Publication number Priority date Publication date Assignee Title
EP0813425A1 (fr) * 1993-07-12 1997-12-29 Neorx Corporation Proteines marquees avec des radionucleides metalliques destinees a une utilisation diagnostique et therapeutique
EP0813425A4 (fr) * 1993-07-12 1998-09-09 Neorx Corp Proteines marquees avec des radionucleides metalliques destinees a une utilisation diagnostique et therapeutique
EP0743956A4 (fr) * 1993-12-07 1999-03-24 Neorx Corp Procede et composes permettant un ciblage prealable
EP0743956A1 (fr) * 1993-12-07 1996-11-27 NeoRx Procede et composes permettant un ciblage prealable
US6172045B1 (en) 1994-12-07 2001-01-09 Neorx Corporation Cluster clearing agents
US6908903B1 (en) 1994-12-07 2005-06-21 Aletheon Pharmaceuticals, Inc. Cluster clearing agents
US7166295B1 (en) 1995-05-26 2007-01-23 Meir Strahilevitz Methods of treatment and diagnostic visualization, particularly in cancer
WO1996037516A1 (fr) * 1995-05-26 1996-11-28 Meir Strahilevitz Procedes de traitement, de diagnostic et de visualisation, notamment de tumeurs cancereuses
EP0906015A1 (fr) * 1996-06-06 1999-04-07 Neorx Corporation Agents de suppression de la retention hepatique
EP0914042A1 (fr) * 1996-06-06 1999-05-12 Neorx Corporation Agents de suppression d'amas
EP0914042A4 (fr) * 1996-06-06 2000-10-25 Neorx Corp Agents de suppression d'amas
EP0906015A4 (fr) * 1996-06-06 2004-05-12 Neorx Corp Agents de suppression de la retention hepatique
WO2000075333A1 (fr) * 1999-06-07 2000-12-14 Neorx Corporation Fusions de genes exprimees par la streptavidine et methodes pour les utiliser
US7144991B2 (en) 1999-06-07 2006-12-05 Aletheon Pharmaceuticals, Inc. Streptavidin expressed gene fusions and methods of use thereof
EP4218929A1 (fr) 2014-03-21 2023-08-02 AbbVie Inc. Anticorps anti-egfr et conjugués anticorps-médicament
US11260027B2 (en) 2015-07-29 2022-03-01 Musc Foundation For Research Development Donor organ pre-treatment formulation
WO2017214339A1 (fr) 2016-06-08 2017-12-14 Abbvie Inc. Anticorps anti-b7-h3 et conjugués anticorps-médicaments
US10640563B2 (en) 2016-06-08 2020-05-05 Abbvie Inc. Anti-B7-H3 antibodies and antibody drug conjugates
WO2017214456A1 (fr) 2016-06-08 2017-12-14 Abbvie Inc. Anticorps anti-cd98 et conjugués anticorps-médicament
WO2017214322A1 (fr) 2016-06-08 2017-12-14 Abbvie Inc. Anticorps anti-b7-h3 et conjugués anticorps-médicaments
WO2018195302A1 (fr) 2017-04-19 2018-10-25 Bluefin Biomedicine, Inc. Anticorps anti-vtcn1 et conjugués anticorps-médicament
US11865124B2 (en) 2017-04-21 2024-01-09 Lunella Biotech, Inc. Vitamin c and doxycycline: a synthetic lethal combination therapy for eradicating cancer stem cells (CSCS)
US11197872B2 (en) 2017-04-21 2021-12-14 Lunella Biotech, Inc. Vitamin C and doxycycline: a synthetic lethal combination therapy for eradicating cancer stem cells (CSCs)
US11229657B2 (en) 2017-04-21 2022-01-25 Lunella Biotech, Inc. Targeting hypoxic cancer stem cells (CSCs) with doxycycline: implications for improving anti-angiogenic therapy
US11160821B2 (en) 2017-05-19 2021-11-02 Lunella Biotech, Inc. Antimitoscins: targeted inhibitors of mitochondrial biogenesis for eradicating cancer stem cells
US12006553B2 (en) 2017-05-19 2024-06-11 Lunella Biotech, Inc. Companion diagnostics for mitochondrial inhibitors
US11865130B2 (en) 2017-05-19 2024-01-09 Lunella Biotech, Inc. Antimitoscins: targeted inhibitors of mitochondrial biogenesis for eradicating cancer stem cells
US11667639B2 (en) 2017-06-26 2023-06-06 Lunella Biotech, Inc. Mitoketoscins: mitochondrial-based therapeutics targeting ketone metabolism in cancer cells
US11667640B2 (en) 2017-06-26 2023-06-06 Lunella Biotech, Inc. Mitoketoscins: mitochondrial-based therapeutics targeting ketone metabolism in cancer cells
US11759527B2 (en) 2021-01-20 2023-09-19 Abbvie Inc. Anti-EGFR antibody-drug conjugates
WO2025027529A1 (fr) 2023-07-31 2025-02-06 Advesya Conjugués médicament-anticorps anti-il-1rap et leurs procédés d'utilisation

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