EP0743956A1 - Procede et composes permettant un ciblage prealable - Google Patents

Procede et composes permettant un ciblage prealable

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Publication number
EP0743956A1
EP0743956A1 EP95904859A EP95904859A EP0743956A1 EP 0743956 A1 EP0743956 A1 EP 0743956A1 EP 95904859 A EP95904859 A EP 95904859A EP 95904859 A EP95904859 A EP 95904859A EP 0743956 A1 EP0743956 A1 EP 0743956A1
Authority
EP
European Patent Office
Prior art keywords
ligand
biotin
conjugate
clearing agent
antibody
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP95904859A
Other languages
German (de)
English (en)
Other versions
EP0743956A4 (fr
Inventor
Louis J. Theodore
Donald B. Axworthy
John M. Reno
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Poniard Pharmaceuticals Inc
Original Assignee
Poniard Pharmaceuticals Inc
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 Poniard Pharmaceuticals Inc filed Critical Poniard Pharmaceuticals Inc
Publication of EP0743956A1 publication Critical patent/EP0743956A1/fr
Publication of EP0743956A4 publication Critical patent/EP0743956A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • 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
    • A61K47/54—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 organic compound
    • A61K47/555—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 organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
    • A61K47/557—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 organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells the modifying agent being biotin
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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

Definitions

  • the present invention relates to methods, compounds, compositions and kits useful for delivering to a target site a targeting moiety that is conjugated to one member of a ligand/anti-ligand pair. After localization and clearance of the targeting moiety conjugate, direct or indirect binding of a diagnostic or therapeutic agent conjugate at the target site occurs. Methods for radiometal labeling of biotin or other small molecules, as well as the related compounds, are also disclosed. Clearing agents and clearance mechanisms are discussed, which agents or mechanisms facilitate a decrease in the serum half- life of targeting moiety-ligand or targeting moiety- anti-ligand conjugates.
  • ratio of administered dose localizing to tumor versus administered dose circulating in blood or ratio of administered dose localizing to tumor versus administered dose migrating to bone marrow is low. Also, the absolute dose of radiation or therapeutic agent delivered to the tumor is insufficient in many cases to elicit a significant tumor response. Improvement in targeting ratio or absolute dose to tumor is sought.
  • 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 clearing agents that incorporate ligand derivatives or anti-ligand derivatives, wherein such derivatives exhibit a lower affinity for the complementary ligand/anti-ligand pair member than the native form of the compound.
  • preferred clearing agents incorporate either a biotin derivative exhibiting a lower affinity for avidin or streptavidin than biotin or an avidin or a streptavidin derivative exhibiting a lower affinity for biotin than avidin or streptavidin.
  • Preferred biotin derivatives for use in the practice of the present invention are 2' -thiobiotin, desthiobiotin, 1- oxy-biotin, l-oxy-2' -thiobiotin, 1-sulfoxide-biotin, l-sulfoxide-2' -thiobiotin, 1-sulfone-biotin, 1- sulfone-2' -thiobiotin, lipoic acid imminobiotin and the like.
  • the present invention further provides .methods of increasing active agent localization at a target cell site of a mammalian recipient, which methods include: administering to the recipient a first conjugate comprising a targeting moiety and a member of a ligand-anti-ligand binding pair; thereafter administering to the recipient a clearing agent capable of directing the clearance of circulating first conjugate via hepatocyte receptors of the recipient, wherein the clearing agent does not incorporate a member of the ligand-anti-ligand binding pair or a lower binding affinity derivative thereof; or thereafter administering to the recipient a clearing agent capable of directing the clearance of circulating first conjugate via hepatocyte receptors of the recipient, wherein the clearing agent incorporates a lower binding affinity derivative of a ligand/anti-ligand binding pair member, wherein the second conjugate binding pair member is complementary to that of the first conjugate; and subsequently administering to the recipient a second conjugate comprising an active agent and a ligand/anti-ligand binding pair member, wherein the second conjugate
  • the present invention provides methods of increasing active agent localization at a target cell site of a mammalian recipient, which methods include: administering to the recipient a receptor blocking agent in an amount sufficient to substantially block a subpopulation of hepatocyte receptors; administering to the recipient a first conjugate comprising a targeting moiety, a hepatocyte receptor recognizing agent, and a member of a ligand-anti- ligand binding pair; and subsequently administering to the recipient a second conjugate comprising an active agent and a ligand/anti-ligand binding pair member, wherein the second conjugate binding pair member is complementary to that of the first conjugate.
  • preferred receptor blocking agents include galactose- IgG conjugate, asialorosomucoid galactosylated biotins and other small molecule clearing agents and the like.
  • the receptor blocking agents are preferably administered in multiple doses over time to facilitate substantially continuous blockage of a substantial portion of the relevant hepatocyte receptors.
  • the receptor becomes deblocked through receptor-based clearance of the blocking agent and cessation of administration of such blocking agent.
  • the cessation/clearance events occur after a time sufficient to permit localization of the targeting moiety to target sites.
  • the second conjugate is preferably administered after a time sufficient to permit receptor-based clearance of circulating first conjugate.
  • Figure 1 illustrates blood clearance of biotinylated antibody following intravenous administration of avidin.
  • 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 comparison to a control profile of native NR- U-10 whole antibody.
  • 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 6 depicts the 5 minute limited biodistribution 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 (D, 0) 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 (V, ⁇ , D) 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 blood clearance of PIP-125 LU-10/SA with and without 50:1 biotin-sc-galactose in BALB/c mice.
  • Figure 13 depicts the blood clearance of PIP-LU- 10/SA with and without galactose-biotin analogs in BALB/c mice.
  • Figure 14 depicts the blood clearance of PIP-125 LU-IO/SA pre-complexed with galactose-biotin analogs.
  • Figure 15 also depicts the blood clearance of PIP- 125 LU-IO/SA pre-complexed with galactose-biotin analogs.
  • Figure 16 depicts blood clearance in BALB/c mice of 1-125 LU-IO/SA following administration of 100 ⁇ g of biotin-galactose analogs.
  • Figure 17 depicts blood clearance in BALB/c mice of 1-125 LU-IO/SA following administration of 100 ⁇ g of biotin-galactose analogs.
  • Figure 18 depicts the structure of a preferred biotin-galactose analog, (gal) 16 -Bt.
  • Figure 19 depicts the blood clearance of PIP-125 LU-10/SA in BALB/c mice alone or precomplexed with (gal) lg -biotin.
  • Figure 20 depicts the blood clearance of PIP-125 following intravenous injection of (gal) lg -biotin at ratios of 100, 50 or 10:1 to circulating conjugate.
  • Figure 21 depicts blood clearance of PIP-125 following intravenous injection of (gal) 16 -biotin at ratios of 100, 50 or 10:1 to circulating conjugate.
  • Figure 22a depicts blood clearance of PIP-125 LU- IO/SA with and without gal-HSA-Bt or (gal) 16 -Bt clearing agents.
  • Figure 22b contains the blood clearance data corresponding to the results depicted in Figure 22a.
  • Figure 23 depicts the biodistribution after two hours of Y-90-DOTA-biotin following LU-10/SA and either 220 ⁇ g gal-HSA-Bt or 46 ⁇ g of (gal) 16 -Bt in SW- 1222 tumored mice and SHT-1 tumored mice.
  • Figure 24 depicts biodistribution of Y-90-DOTA- biotin two hours after administration with 3 hour interval for (gal) 16 -Bt or gal-HSA-Bt clearing agent.
  • Figure 25 depicts molar ratio of DOTA-biotin to
  • 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 describing the present invention.
  • Ligand/anti-ligand pair A complementary/anti- complementary set of molecules that demonstrate specific binding, generally of relatively high affinity.
  • 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, multivalent binding of the complementary ligand.
  • the anti-ligand is large enough to avoid rapid renal clearance, and contains sufficient multivalency to accomplish crosslinking and aggregation of targeting moiety-ligand conjugates.
  • Univalent anti-ligands are also contemplated by the present invention.
  • Anti-ligands of the present invention may exhibit or be derivitized to exhibit structural features that direct the uptake thereof, e.g., galactose residues that direct liver uptake.
  • Avidin and streptavidin are used herein as prototypical anti-ligands.
  • 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 administered intravenously in an animal or human. Biotin is used as the prototypical ligand. Lower Affinity Ligand or Lower Affinity Anti-
  • Ligand A ligand or anti-ligand that binds to its complementary ligand-anti-ligand pair member with an affinity that is less than the affinity with which native ligand or anti-ligand binds the complementary member.
  • lower affinity ligands and anti- ligands exhibit between from about 10 "6 to 10 "10 M binding affinity for the native form of the complementary anti-ligand or ligand.
  • Lower affinity ligands and anti-ligands may be employed in clearing agents or in active agent-containing conjugates of the present invention.
  • 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 -..-S. y. Chelates As defined herein, the term "N -- ⁇ ,-S,y. chelates" includes bifunctional 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 ⁇ S y chelates have N 2 S 2 and N 3 S cores. Exemplary N ⁇ S y chelates are described in Fritzberg et al., Proc. Natl. Acad. Sci. USA 85:4024-29. 1988; in Weber et al. , Biocon . 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 sufficient for optimal target-to-non-target accumulation of this targeting moiety conjugate, active agent conjugated to the opposite member of the ligand/anti-ligand pair is administered and is bound (directly or indirectly) to the targeting moiety conjugate at the target site (two-step pretargeting) .
  • Three-step and other related methods described herein are also encompassed.
  • Clearing Agent An agent capable of binding, complexing or otherwise associating with an administered moiety (e.g., targeting moiety-ligand, targeting moiety-anti-ligand or anti-ligand alone) present in the recipient's circulation, thereby facilitating circulating moiety clearance from the recipient's body, removal from blood circulation, or inactivation thereof in circulation.
  • the clearing agent is preferably characterized by physical properties, such as size, charge, configuration or a combination thereof, that limit clearing agent access to the population of target cells recognized by a targeting moiety used in the same treatment protocol as the clearing agent.
  • a conjugate encompasses chemical conjugates (covalently or non-covalently bound) , fusion proteins and the like.
  • 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.
  • 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 maximize exposure of target tissue) , while remaining below the threshold of unacceptable normal organ toxicity (due to non-target tissue exposure) . Unlike radioisotopes, however, therapeutic drugs need to be taken into a target cell to exert a cytotoxic effect. In the case of 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 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.
  • pretargeting the targeting moiety at a target site
  • a rapidly clearing diagnostic or therapeutic agent conjugate that is capable of binding to the "pretargeted” targeting moiety at the target site.
  • Anti-ligand D Ugand ⁇ Ugand-active agent / Binding site i- ⁇ ., receptor, antigenic determinant
  • this three-step pretargeting protocol features administration of an antibody-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 crosslinking and aggregation of circulating antibody- ligand conjugates. The clearing by anti-ligand is probably attributable to anti-ligand crosslinking and/or aggregation of antibody-ligand conjugates that are circulating in the blood, which leads to complex/aggregate clearance by the recipient's RES (reticuloendothelial system) .
  • RES reticuloendothelial system
  • 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.
  • 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.
  • Alternate 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 administration, followed by administration of active agent conjugated to the opposite member of the ligand- anti-ligand pair.
  • a clearing agent preferably other than ligand or anti-ligand alone is administered to facilitate the clearance of circulating targeting moiety-containing conjugate.
  • the 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.
  • An example of two-step pretargeting involves the use of biotinylated human transferrin as a clearing agent for avidin-targeting moiety conjugate, wherein the size of the clearing agent results in liver clearance of transferrin-biotin- circulating avidin-targeting moiety complexes and substantially precludes association with the avidin- targeting moiety conjugates bound at target cell sites. (See, Goodwin, D.A. , Antibod. Immunoconi . Radiopharm. , 4_: 427-34, 1991) .
  • 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
  • One explanation for the observed phenomenon is that the multivalent anti-ligand crosslinks targeting moiety- ligand conjugates on the cell surface, thereby initiating or facilitating internalization of the resultant complex.
  • 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) .
  • 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.
  • the present invention recognizes that this phenomenon (apparent loss of the targeting moiety- ligand from the target cell) may be used to advantage with regard to in vivo delivery of therapeutic agents generally, or to drug delivery in particular.
  • 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.
  • a 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 moiety- anti-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 and related methods are disclosed.
  • radioiodinated biotin derivatives were of high molecular weight and were difficult to characterize.
  • the 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) .
  • biotin derivatives were radiolabeled with indium-111 for use in pretargeted immunoscintigraphy (for instance, Virzi et al., Nucl. Med. Biol. 18:719-26, 1991; Kalofonos et al., J. Nucl. Med. 31: 1791-96, 1990; Paganelli et al., Cane. Res. 51:5960-66, 1991).
  • 99m Tc is a particularly preferred radionuclide for immunoscintigraphy due to (i) low cost, (ii) convenient supply and (iii) favorable nuclear properties.
  • 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 or a thrombus site.
  • 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.
  • 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 targeting moieties that retain the capacity to bind to a defined target cell population may also be used within the claimed invention. In addition, 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 functional equivalent is a short polypeptide designated as a "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. & Phar . , 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 conventional techniques, such as hybridoma synthesis, recombinant DNA techniques and protein synthesis.
  • 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. Immunol.. 6_ : 511-19, 1976.
  • 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 determining regions thereof with the nucleotide sequence encoding a human constant domain region and an Fc region, e.g., in a manner similar to that disclosed in European Patent Application
  • 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 reduction in the possibility of adverse immune reactions.
  • single chain antibodies, FVs and dimers thereof are useful targeting moieties.
  • Still further bispecific antibodies are suitable targeting moieties.
  • 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 conjugate.
  • Preferred toxins in this regard include holotoxins, such as abrin, ricin, modeccin, Pseudomonas exotoxin A, Diphtheria toxin, pertussis toxin and Shiga toxin; and 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.
  • holotoxins such as abrin, ricin, modeccin, Pseudomonas exotoxin A, Diphtheria toxin, pertussis toxin and Shiga toxin
  • Ribosomal inactivating proteins RIPs
  • Naturally occurring protein synthesis inhibitors that lack translocating and cell-binding ability, 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.
  • 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 as described in Cancer: Principles and Practice of Oncology. 2d ed. , V.T. DeVita, Jr., S. Hellman, S.A. Rosenberg, J.B. Lippincott Co.,
  • 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. Biol. .8:41-110, 1983; Jarvis & Mazzola, Ace. Chem. Res. 1,5:338-395, 1982) . They appear to be the most toxic molecules that contain only carbon, hydrogen and oxygen (Tamm, C. Fortschr. Chem. Org.
  • Group A simple trichothecenes include: Scirpene, Roridin C, dihydrotrichothecene, Scirpen-4, 8-diol, Verrucarol, Scirpentriol, T-2 tetraol, pentahydroxyscirpene, 4-deacetylneosolaniol, trichodermin, deacetylcalonectrin, calonectrin, diacetylverrucarol, 4-monoacetoxyscirpenol, 4,15-diacetoxyscirpenol, 7-hydroxydiacetoxyscirpenol, 8-hydroxydiacetoxy-scirpenol (Neosolaniol) , 7,8-dihydroxydiacetoxyscirpenol,
  • Group B simple trichothecenes include: Trichothecolone, Trichothecin, deoxynivalenol, 3-acetyldeoxynivalenol, 5-acetyldeoxynivalenol, 3,15-diacetyldeoxynivalenol, Nivalenol, 4-acetylnivalenol (Fusarenon-X) , 4,15-idacetylnivalenol, 4,7,15-triacetylnivalenol, and tetra-acetylnivalenol.
  • Group C simple trichothecenes include: Crotocol and Crotocin.
  • Representative macrocyclic trichothecenes include Verrucarin A, Verrucarin B, Verrucarin J (Satratoxin C) , Roridin A, Roridin D, Roridin E (Satratoxin D) , Roridin H, Satratoxin F, Satratoxin G, Satratoxin H, Vertisporin, Mytoxin A, Mytoxin C, Mytoxin B, Myrotoxin A, Myrotoxin B, Myrotoxin C, Myrotoxin D, Roritoxin A, Roritoxin B, and Roritoxin D.
  • trichothecene sesquiterpenoid ring structure
  • baccharins isolated from the higher plant Baccharis megapotamica, and these are described in the literature, for instance as disclosed by Jarvis et al. (Chemistry of Alleopathy, ACS Symposium Series No. 268: ed. A.C. Thompson, 1984, pp. 149-159) .
  • Experimental drugs such as mercaptopurine, N- methylformamide, 2-amino-l,3,4-thiadiazole, melphalan, hexamethylmelamine, gallium nitrate, 3% thymidine, dichloromethotrexate, mitoguazone, suramin, bromodeoxyuridine, iododeoxyuridine, semustine, 1- (2- chloroethyl) -3- (2,6-dioxo-3-piperidyl) -1-nitrosourea, N,N' -hexamethylene-bis-acetamide, azacitidine, dibromodulcitol, Erwinia asparaginase, ifosfamide, 2- mercaptoethane sulfonate, teniposide, taxol, 3- deazauridine, soluble Baker's antifol, homoharringtonine, cyclocytidine, acivicin, ICRF-187, spirom
  • 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 f 67 Ga, 68 Ga, 64 Cu, 67 Cu, 75 Br, 76 Br, 77 Br, 99m Tc, ⁇ - ⁇ ⁇ C, 13 N, 15 0, 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 ⁇ r/ 211 At#
  • anti-tumor agents e.g., agents active against proliferating cells
  • agents active against proliferating cells are administrable in accordance with the present invention.
  • 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, S-peptide, head activator peptide (HA-peptide) , 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.
  • DOTA may also be conjugated to other ligands or to anti-ligands in the practice of the present invention. Because DOTA strongly binds Y-90 and other radionuclides, it has been proposed for use in radioimmunotherapy. For therapy, it is very important that the radionuclide be stably bound within the DOTA chelate and that the DOTA chelate be stably attached to a ligand or anti-ligand. For illustrative purposes, DOTA-biotin conjugates are described. Only radiolabeled DOTA-biotin conjugates exhibiting those two characteristics are useful to deliver radionuclides to the targets.
  • Biotinidase is a hydrolytic enzyme that catalyzes the cleavage of biotin from biotinyl peptides. See, for example, Evangelatos, et al. , "Biotinidase Radioassay Using an I-125-Biotin Derivative, Avidin, and Polyethylene
  • Drug-biotin conjugates which structurally resemble biotinyl peptides are potential substrates for cleavage by plasma biotinidase. Poor in vivo stability therefore limits the use of drug-biotin conjugates in therapeutic applications.
  • the use of peptide surrogates to overcome poor stability of peptide therapeutic agents has been an area of intense research effort. See, for example, Spatola, Peptide Backbone Modification: A Structure-Activity Analysis of Peptide Containing Amide Bond Surrogates, "Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, " vol. 7, Weinstein, ed. , Marcel Dekker, New York, 1983; and Kim et al. , "A New Peptide Bond Surrogate: 2-Isoxazoline in Pseudodipeptide Chemistry," Tetrahedron Letters, 45 : 6811-14, 1991.
  • DOTA-SC-biotin where the SC indicates the "s_hort chain” linker between the DOTA and biotin conjugate components
  • DOTA-SC-biotin exhibits significantly improved serum stability in comparison to DOTA-LC-biotin. This result does not appear to be explainable on the basis of biotinidase activity alone. The experimentation leading to this conclusion is summarized in the Table set forth below.
  • biotinidase activity measuring method uses N- (d-biotinyl) -4-aminobenzoate (BPABA) as a substrate, with the hydrolysis of BPABA resulting in the liberation of biotin and 4-aminobenzoate (PABA) .
  • BPABA N- (d-biotinyl) -4-aminobenzoate
  • PABA 4-aminobenzoate
  • DOTA-SC-biotin exhibits serum stability
  • biotinidase activity alone does not adequately explain why some conjugates are serum stable while others are not.
  • a series of DOTA-biotin conjugates was therefore synthesized by the present inventors to determine which structural features conferred serum stability to the conjugates.
  • linkage between DOTA and biotin may also have a significant impact on biodistribution
  • the DOTA-biotin conjugates of the present invention reflect the implementation of one or more of the following strategies :
  • DOTA-biotin conjugates in accordance with the present invention may be generally characterized as follows: conjugates that retain the biotin carboxy group in the structure thereof and those that do not (i.e., the terminal carboxy group of biotin has been reduced or otherwise chemically modified. Structures of such conjugates represented by the following general formula have been devised:
  • L may alternatively be substituted in one of the following ways on one of the -CH 2 -COOH branches of the DOTA structure: -CH(L)-COOH or -CH 2 COOL or -CH 2 COL) .
  • the portion of the linker bearing the functional group for binding with the DOTA conjugate component is selected for the capability to interact with either the carbon or the carboxy in the branch portions of the DOTA structure, with the serum stability conferring portion of the linker structure being selected as described below.
  • L is selected according to the following principles, with the portion of the linker designed to bind to the DOTA conjugate component selected for the capability to bind to an amine.
  • One embodiment of the present invention includes linkers incorporating a D-amino acid spacer between a DOTA aniline amine and the biotin carboxy group shown above. Substituted amino acids are preferred for these embodiments of the present invention, because alpha-substitution also confers enzymatic cleavage resistance.
  • Exemplary L moieties of this embodiment of the present invention may be represented as follows:
  • R 1 is selected from lower alkyl, lower alkyl substituted with hydrophilic groups (preferably, OH (C ⁇ ) n -OH, (CH2) n -OSO3, (CH2) n -S0 3> (CH ⁇ n- POH,
  • n 1 or 2) , glucuronide-substituted amino acids or other glucuronide derivatives; and R 2 is selected from hydrogen, lower alkyl, substituted lower alkyl (e.g., hydroxy, sulfate, phosphonate or a hydrophilic moiety (preferably OH) .
  • lower alkyl indicates an alkyl group with from one to five carbon atoms.
  • substituted includes one or several substituent groups, with a single substituent group preferred.
  • Preferred moieties incorporating the glucuronide of D-lysine and the glucuronide of amino pimelate are shown below as I and II, respectively.
  • a particularly preferred linker of this embodiment of the present invention is the D-alanine derivative set forth above.
  • Linkers incorporating alkyl substitution on one or more amide nitrogen atoms are also encompassed by the present invention, with some embodiments of such linkers preparable from L-amino acids. Amide bonds having a substituted amine moiety are less susceptible to enzymatic cleavage. Such linkers exhibit the following general formula:
  • R 4 is selected from hydrogen, lower alkyl, lower alkyl substituted with hydroxy, sulfate, phosphonate or the like and - (CH 2 ) n+1 -CO-NH-@>-CH 2 -DOTA;
  • R 3 is selected from hydrogen; an amine; lower alkyl; an amino- or a hydroxy-, sulfate- or phosphonate-substituted lower alkyl; a glucuronide or a glucuronide-derivatized amino groups; and n ranges from 0-4.
  • n 0 (bis-DOTA-SC-biotin) , synthesizable from iminodiacetic acid.
  • the synthesis of a conjugate including a linker wherein R 3 is H and R 4 is -CH 2 CH 2 OH and n is 0 is also described in Example XV. Schematically, the synthesis of a conjugate of this embodiment of the present invention wherein n is 0, R 3 is H and R 4 is
  • Bis-DOTA-LC-biotin offers the following advantages:
  • Another linker embodiment incorporates a thiourea moiety therein.
  • exemplary thiourea adducts of the present invention exhibit the following general formula:
  • R 5 is selected from hydrogen or lower alkyl
  • R 6 is selected from H and a hydrophilic moiety; and n ranges from 0-4.
  • the second preferred linker recited above can be prepared using either L-lysine or D-lysine.
  • the third preferred linker can be prepared using either N-methyl-D-lysine or N-methyl-L-lysine.
  • Another thiourea adduct of minimized lipophilicity is
  • Z is -(CH 2 ) 2 -, conveniently synthesized form glutamic acid
  • Another embodiment involves disulfide- containing linkers, which provide a metabolically cleavable moiety (-S-S-) to reduce non-target retention of the biotin-DOTA conjugate.
  • exemplary linkers of this type exhibit the following formula:
  • n and n' preferably range between 0 and 5.
  • conditionally cleavable linkers include enzymatically cleavable linkers, linkers that are cleaved under acidic conditions, linkers that are cleaved under basic conditions and the like. More specifically, use of linkers that are cleaved by enzymes, which are present in non-target tissues but reduced in amount or absent in target tissue, can increase target cell retention of active agent relative to non-target cell retention.
  • conditionally cleavable linkers are useful, for example, in delivering therapeutic radionuclides to target cells, because such active agents do not require internalization for efficacy, provided that the linker is stable at the target cell surface or protected from target cell degradation.
  • Cleavable linkers are also useful to effect target site selective release of active agent at target sites.
  • Active agents that are preferred for cleavable linker embodiments of the present invention are those that are substantially non-cytotoxic when conjugated to ligand or anti-ligand. Such active agents therefore require release from the ligand- or anti- ligand-containing conjugate to gain full potency.
  • such active agents while conjugated, may be unable to bind to a cell surface receptor; unable to internalize either actively or passively; or unable to serve as a binding substrate for a soluble (intra- or inter-cellular) binding protein or enzyme.
  • exemplary of an active agent-containing conjugate of this type is chemotherapeutic drug-cis-aconityl-biotin.
  • the cis-aconityl linker is acid sensitive.
  • Other acid sensitive linkers useful in cleavable linker embodiments of the present invention include esters, thioesters and the like.
  • conjugates wherein an active agent and a ligand or an anti-ligand are joined by a cleavable linker will result in the selective release of the active agent at tumor cell target sites, for example, because the inter-cellular milieu of tumor tissue is generally of a lower pH (more highly acidic) than the inter-cellular milieu of normal tissue.
  • Ether, thioether, ester and thioester linkers are also useful in the practice of the present invention.
  • Ether and thioether linkers are stable to acid and basic conditions and are therefore useful to deliver active agents that are potent in conjugated form, such as radionuclides and the like.
  • Ester and thioesters are hydrolytically cleaved under acidic or basic conditions or are cleavable by enzymes including esterases, and therefore facilitate improved target:non-target retention.
  • Exemplary linkers of this type have the following general formula:
  • Q is a bond, a methylene group, a -CO- group or -CO- (CH 2 ) n -NH-; and n ranges from 1-5.
  • Other such linkers have the general formula:
  • Another amino-containing linker of the present invention is structured as follows:
  • R 7 is lower alkyl
  • Polymeric linkers are also contemplated by the present invention. Dextran and cyclodextran are preferred polymers useful in this embodiment of the present invention as a result of the hydrophilicity of the polymer, which leads to favorable excretion of conjugates containing the same.
  • dextran polymers are substantially non-toxic and non-immunogenic, that they are commercially available in a variety of sizes and that they are easy to conjugate to other relevant molecules.
  • dextran-linked conjugates exhibit advantages when non-target sites are accessible to dextranase, an enzyme capable of cleaving dextran polymers into smaller units while non-target sites are not so accessible.
  • Other linkers of the present invention are produced prior to conjugation to DOTA and following the reduction of the biotin carboxy moiety. These linkers of the present invention have the following general formula:
  • An ether linkage as shown below may be formed in a DOTA-biotin conjugate in accordance with the procedure indicated below.
  • n ranges from 1 to 5, with 1 preferred.
  • This linker has only one amide moiety which is bound directly to the DOTA aniline (as in the structure of DOTA-SC-biotin) .
  • the ether linkage imparts hydrophilicity, an important factor in facilitating renal excretion.
  • This linker contains no amide moieties and the unalkylated amine may impart favorable biodistribution properties since unalkylated DOTA-aniline displays excellent renal clearance.
  • R 8 where R 8 is H; -(CH 2 ) 2 -OH or a sulfate or phosphonate
  • R 9 is a bond or - (CH 2 ) n -CO-NH-, where n ranges from 0-5 and is preferably 1 and where q is 0 or 1.
  • Conjugates containing this thiourea linker have the following advantages: no cleavable amide and a short, fairly polar linker which favors renal excretion.
  • a bis-DOTA derivative of the following formula can also be formed from amino-biotin.
  • n ranges from 1 to 5, with 1 and 5 preferred.
  • This molecule offers the advantages of the previously discussed bis-DOTA derivatives with the added advantage of no cleavable amides.
  • L -(CH 2 ) 4 -NH-, wherein the amine group is attached to the methylene group corresponding to the reduced biotin carboxy moiety and the methylene chain is attached to a core carbon in the DOTA ring.
  • Such a linker is conveniently synthesizable from lysine.
  • L - (CH 2 ) -CO-NH-, wherein q is 1 or 2, and wherein the amine group is attached to the methylene group corresponding to the reduced biotin carboxy moiety and the methylene group (s) are attached to a core carbon in the DOTA ring. This moiety is synthesizable from amino-biotin.
  • linkers set forth above are useful to produce conjugates having one or more of the following advantages : - bind avidin or streptavidin with the same or substantially similar affinity as free biotin;
  • - are stable to endogenous enzymatic or chemical degradation (e.g., bodily fluid amidases, peptidases or the like) ;
  • step eight proceeds in good yield, but the process involves copious volumes of the coreactants.
  • step six of the prior art synthesis procedure in which a tetra amine alcohol is converted to a tetra-toluenesulfonamide toluenesulfonate as shown below, is the likely result of premature formation of the O-toluenesulfonate functionality (before all of the amine groups have been converted to their corresponding sulfonamides.
  • Trifluoroacetates being much poorer leaving groups than toluenesulfonates, are not vulnerable to analogous side reactions.
  • the easy hydrolysis of trifluoroacetate groups as reported in Greene and Wuts, "Protecting Groups in Organic Synthesis," John Wiley and Sons, Inc., New York, p. 94, 1991, suggests that addition of methanol to the reaction mixture following consumption of all amines should afford the tetra-fluoroacetamide alcohol as a substantially exclusive product.
  • This alternative procedure involves the cyclization of p-nitrophenylalanyltriglycine using a coupling agent, such as diethylycyanophosphate, to give the cyclic tetraamide. Subsequent borane reduction provides 2- (p-nitrobenzyl) -1,4,7,10-tetraazacyclododecane, a common precursor used in published routes to DOTA including the Renn and Meares article referenced above.
  • This alternative procedure of the present invention offers a synthetic pathway that is considerably shorter than the prior art Renn and Meares route, requiring two rather than four steps between p-nitrophenylalanyltriglycine to the tetraamine.
  • the procedure of the present invention also avoids the use of tosyl amino protecting groups, which were prepared in low yield and required stringent conditions for removal. Also, the procedure of the present invention poses advantages over the route published by Gansow et al., U.S. Patent No. 4,923,985, because the crucial cyclization step is intramolecular rather than intermolecular. Intramolecular reactions typically proceed in higher yield and do not require high dilution techniques necessary for successful intermolecular reactions.
  • the present invention also provides an article of manufacture which includes packaging material and a clearing agent, such as a galactose-HSA-biotin, contained within the packaging material, wherein the clearing agent, upon administration to a mammalian recipient (which recipient has previously been administered a conjugate or moiety to be cleared) , is capable of decreasing circulating conjugate or moiety concentration, and wherein the packaging material includes a label that identifies the clearing agent and the component parts thereof, if any, and indicates an appropriate use of the clearing agent in human recipients.
  • a clearing agent such as a galactose-HSA-biotin
  • the packaging material indicates whether the clearing agent is limited to investigational use or identifies an indication for which the clearing agent has been approved by the U.S. Food and Drug Administration or other similar regulatory body for use in humans.
  • the packaging material may also include additional information including the amount of clearing agent, the medium or environment in which the clearing agent is dispersed, if any, lot number or other identifier, storage instructions, usage instructions, a warning with respect to any restriction upon use of the clearing agent, the name and address of the company preparing and/or packaging the clearing agent, and other information concerning the clearing agent.
  • the clearing agent is preferably contained within a vial which allows the clearing agent to be transported prior to use.
  • Such clearing agent is preferably vialed in a sterile, pyrogen-free environment.
  • the clearing agent may be lyophilized prior to packaging. In this circumstance, instructions for preparing the lyophilized clearing agent for administration to a recipient may be included on the label.
  • 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.
  • 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.
  • 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 unavailable for biotin binding due to the configuration of the streptavidin-antibody conjugate.
  • 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. Sheldon et al., Appl. Radiat. Isot.
  • Streptavidin-proteinaceous targeting moiety conjugates are preferably prepared as described in
  • Example XI below, with the preparation involving the steps of: preparation of SMCC-derivitized 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 characterized by one or more of the following techniques: HPLC size exclusion, SDS-PAGE, immunoreactivity, biotin binding capacity and in vivo studies.
  • 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-derivitized form of the other protein.
  • lysine epsilon amino moieties react with protein maleimides, prepared, for instance, by treatment of the protein with SMCC, to generate stable amine covalent conjugates.
  • 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, by oxidation of protein sugar residues or by reaction with aldehyde-containing heterobifunctional cross linkers.
  • Another method of forming streptavidin-targeting moiety conjugates involves immobilized iminobiotin that binds SMCC-derivitized streptavidin.
  • iminobiotin immobilized to streptavidin is exploited to readily separate conjugate from the unreacted targeting moiety.
  • 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.
  • 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 localization of either targeting moiety-ligand or targeting moiety-anti-ligand at target tissue. Often, peak uptake to such target tissue is achieved before the circulating level of targeting moiety-containing conjugate in the blood is sufficiently low to permit the attainment of an optimal target-to-non-target conjugate ratio. To obviate this problem, two approaches are useful.
  • the first approach allows the targeting moiety-containing conjugate to clear from the blood by "natural" or endogenous clearance mechanisms. This method is complicated by variations in systemic clearance of proteins and by endogenous ligand or anti-ligand. For example, endogenous biotin may interfere with the preservation of biotin binding sites on a streptavidin-targeting moiety conjugate.
  • the second approach for improving targeting moiety-ligand or targeting moiety-anti-ligand 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.
  • both "chase" methodologies involve the prolonged presence of aggregated moieties of intermediate, rather than large, size (which are not cleared as quickly as large size particles by RES uptake) , thereby resulting in serum retention of subsequently administered ligand- active agent or anti-ligand active agent. Such serum retention unfavorably impacts the target cell-to-blood targeting ratio.
  • 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- acetylglucosamine, pentamannosylphosphate, and the like.
  • 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.
  • the rapid clearance from the blood of asialoorosomucoid has been documented by Galli, et al. , J. of Nucl. Med. Allied Sci. 32 (2) : 110-16, 1988.
  • Treatment of orosomucoid with neuraminidase removes sialic acid residues, thereby exposing galactose residues.
  • derivatized clearing agents include, for example, galactosylated albumin, galactosylated-IgM, galactosylated-IgG, asialohaptoglobin, asialofetuin, asialoceruloplasmin and the like.
  • the present invention therefore provides clearing agents that do not incorporate ligand or anti-ligand molecules or derivatives thereof.
  • the present invention provides IgM molecules that are amenable to receptor-based clearance such as hexose residue-bearing IgM molecules.
  • Preferred hexose residue-bearing clearing agents also incorporate a moiety that is recognized by a hepatocyte receptor, such as galactose, mannose, mannose 6-phosphate, N-acetylglucosamine, glucose, N- galactosamine, N-acetylgalactosamine, thioglycosides of galactose and, generally, D-galactosides and glucosides or the like.
  • the methods of derivatization of IgM with galactose or the like is analogous to those for derivatizing HSA therewith.
  • desialyation analogous to the procedure discussed herein with respect to orosomucoid, may be employed in appropriate circumstances.
  • the present invention further provides methods of increasing active agent localization at a target cell site of a mammalian recipient, which methods include: administering to the recipient a first conjugate comprising a targeting moiety and a member of a ligand-anti-ligand binding pair; thereafter administering to the recipient a clearing agent capable of directing the clearance of circulating first conjugate via hepatocyte receptors of the recipient, wherein the clearing agent does not incorporate a member of the ligand-anti-ligand binding pair or a lower binding affinity derivative thereof; and subsequently administering to the recipient a second conjugate comprising an active agent and a ligand/anti-ligand binding pair member, wherein the second conjugate binding pair member is complementary to that of the first conjugate.
  • Human serum albumin HSA
  • HSA Human Serum Albumin
  • Ligand n
  • n is an integer from 1 to about 10
  • m is an integer from 1 to about 25
  • the hexose is recognized by Ashwell receptors.
  • Other mammalian forms of human serum albumin which differ from human serum albumin only by a few amino acid residues, may also be used in the practice of the present invention. Examples of such mammalian forms of serum albumin are bovine serum albumin, porcine serum albumin, and the like.
  • the ligand is biotin and the hexose is galactose.
  • HSA is derivatized with from 10-20 galactose residues and 1-5 biotin residues.
  • 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 sufficient to produce individual clearing agent molecules with a range of biotinylation levels that averages a recited whole number, such as 1, biotin. Derivatization with 3 biotins, for example, produces a product mixture made up of individual clearing agent molecules, substantially all of which having at least one biotin residue. Derivatization with 1 biotin produces a clearing agent product mixture, wherein a significant portion of the individual molecules are not biotin derivatized.
  • the whole numbers used in this description refer to the average biotinylation of the clearing agents under discussion.
  • 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, IgA and IgM class, and the like, are less immunogenic upon administration into the serum of a human recipient.
  • human orosomucoid is commercially available from, for example, Sigma Chemical Co, St. Louis, Missouri.
  • Human HSA (Cutter Biological) and human IgG, IgA and IgM (Sigma Chemical Co.), for example, are also commercially available.
  • Another embodiment of the clearing agent of the present invention is a small molecule clearing agent.
  • a small molecule clearing agent incorporates a hepatic clearance directing moiety; a liver retention moiety; and a member of a ligand/anti-ligand pair or a lower affinity form thereof to facilitate binding to targeting moiety-ligand/anti-ligand conjugate.
  • small molecule clearing agents of the present invention range in molecular weight from between about 1,000 and about 20,000 daltons, more preferably from about 2,000 to 16,000 daltons.
  • the clearance directing moiety component of the small molecule clearing agent of the present invention is a molecule that is recognized by a hepatocyte receptor. Exemplary molecules of this type have been discussed elsewhere herein.
  • liver retention moiety of the small molecule clearing agent of the present invention promotes retention by the liver of the clearing agent which is directed to liver clearance by the clearance directing moiety component thereof.
  • Exemplary liver retention moieties useful in the practice of the present invention include cyanuric chloride, cellobiose, polylysine, polyarginine and the like.
  • Exemplary ligand/anti-ligand pair members and lower affinity derivatives thereof have been discussed elsewhere herein.
  • 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 effectiveness of "protein-type" or "polymer-type” galactose-based clearing agents.
  • Galactose-based clearing agents include galactosylated, biotinylated proteins (to remove circulating streptavidin-targeting moiety conjugates, for example) of intermediate molecular weight (ranging from about 40,000 to about 200,000 Dal) , such as 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 1,000,000 Dal) characterized by poor target access, including galactosyl-biotinyl-IgM or -IgG (approximately 150,000 Dal) molecules, as well as galactose- and biotin-derivatized transferrin conjugates of human serum albumin, IgG and IgM molecules and the like, can also be used as clearing agents of the claimed invention.
  • a potential disadvantage associated with biotinylated galactosylated human serum albumin clearing agents is that metabolism thereof may result in the release of biotin. This is undesirable because it may result in poisoning of the targeted conjugate by biotin. Such biotin release may occur after uptake by the Ashwell receptor and catabolism of the protein.
  • One means of alleviating this potential problem is to produce conjugates which are metabolically stable and therefore do not release any catabolized biotin.
  • a non- cleavable linker comprised, e.g., of amino acid sequences, D-amino acids, teritary amines, sugars or highly charged or polar groups between the biotin linker and the HSA protein. Incorporation of such linkers should prevent biotin release, and the escape of biotin molecules from the hepatocytes and being released into the circulation.
  • linker sequence will eliminate the release of free biotin altogether, thereby eliminating the possibility of free biotin being released into the circulation and potentially adversely affecting the binding of active agent to tumor bound conjugates.
  • 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 6,6'- [(3,3' -dimethyl [1,1' -biphenyl] -4,4' -diyDbis (azo) bis [4-amino-5-hydroxy-1,3-naphthalene disulfonic acid] tetrasodium salt, mono- or poly-biotinyl-galactose- derivatized polysulfated dextran-biotin, mono- or poly-biotinyl-galactose-derivatized dextran-biotin and the like.
  • 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 conjugate, compromising target localization.
  • aggregation-mediated clearance mechanisms operating through the RES system are accomplished using a large stoichiometric excess of clearing agent.
  • the rapid blood clearance of galactose- based clearing agents used in the present invention prevents equilibration, and the high affinity ligand- anti-ligand binding allows the use of low stoichiometric amounts of such galactose-based clearing agents. This feature further diminishes the potential for galactose-based clearing agents to compromise target-associated conjugate, because the absolute amount of such clearing agent administered is decreased.
  • a preferred embodiment of the present invention is the preparation of novel small molecule clearing agents and the use thereof in pretargeting protocols. More specifically, the present invention provides novel bispecific small molecule clearing agents which have utility for the clearance of streptavidin- targeting moiety or avidin-targeting moiety conjugates from non-targeted sites, e.g., the circulation, extravascular space, etc.
  • This aspect of the invention was developed while attempting to produce further improved galactosylated HSA-biotin clearing agents. While such clearing agents are effective as described supra, they have one potential adverse side effect. Specifically, the administration of such clearing agents may potentially result in biotin poisoning of the targeted conjugate as a result of endocytosis and degradation of the clearing agent. This may occur by endocytosis and degradation, the end result of which is the production and diffusion of the small molecule metabolite biotin into the circulation.
  • lower molecular weight clearing agents could be designed to contain a sufficient number of hexose residues (e.g., galactoses) to facilitate directed clearance and which only transiently bind to conjugate (i.e., for an interval sufficient to clear non-targeted conjugate) .
  • Residual clearing agent initially bound to target associated conjugate could be expected to dissociate over time, thus allowing access for the active agent biotinylated ligand.
  • X is H, methyl, lower alkyl or lower alkyl with heteroatoms.
  • the above structures bear 4, 8, and 16 galactose respectively. Further iteration in the branching allows expansion to include 32, 64, etc., galactose residues.
  • this embodiment of the invention involves the preparation and use of bispecific small molecule agents for use in clearance of streptavidin-targeting agent (antibody) or avidin-targeting agent (antibody) from non-targeted sites, i.e., the circulation, and possibly extravascular space, etc.
  • These bispecific small molecule clearing agents will preferably consist of a "low affinity" biotin analog arm, which can bind to avidin or streptavidin in a metastable fashion, to which has been attached one or more hexose residues which provide for targeted clearance, e.g., through hepatocyte receptors.
  • Exemplary low affinity biotin molecules useful in this embodiment of the invention are identified elsewhere in this application.
  • hepatocyte receptors which provide for effective clearance include in particular Ashwell receptors, mannose/N- acetylgalactosamine receptors associated with endothelial cells and/or Kupffer cells of the liver, the mannose 6-phosphate receptor, and the like.
  • Hexoses which may be attached to such low affinity biotin analogs have been identified above and include by way of example galactose, mannose, mannose 6- phosphate, N-acetylgalactosamine, pentamannosylphosphate, and the like.
  • Hexoses recognized by Ashwell receptors include glucose, galactose, N-galactosamine, N-acetylgalactosamine, pentamannosyl phosphate, thioglycosides of galactose, D-galactosides, galactosamine, N-acetylgalactosamine, mannosyl-6-phosphate and glucosides.
  • a sufficient number of hexose residues will be attached to the selected biotin analog to provide for effective clearance, e.g., via the Ashwell receptors comprised on the surface of hepatocytes.
  • the clearance agents should be of a low enough molecular weight to provide for efficient diffusion into the extravascular space, thus providing for binding to both circulating and non-circulating conjugate. This molecular weight will preferably range from about
  • the low affinity biotin analog will be bound to at least 3 hexose residues, e.g., galactose residues or N-acetylgalactosamine residues.
  • the invention is not limited thereby and embraces the attachment of any number of hexose residues or mixture thereof which results in an efficacious bispecific small molecular weight clearance agent.
  • hexose portion of the small molecule e.g., galactose or N-actylgalactosamine also depends upon a number of factors including:
  • the small molecule clearing agent should possess at least three galactose residues and preferably more, to provide for "galactose clusters.”
  • the small molecule clearing agent will contain from about 3 to about 50 galactose residues, preferably from about 3 to 32, and most preferably 16 galactose residues.
  • each galactose receptor is separated by a distance of 15, 22 and 25 A.
  • the galactose residues within each small molecule should preferably be separated by a flexible linker which provides for a separation distance of at least 25 A, to enable the sugars to be separated by at least said distance. It is expected that this minimum spacing will be more significant as the number of sugar residues, e.g., galactoses, are decreased. This is because larger numbers of galactoses will likely contain an appropriate spacing between sugars that are not immediately adjacent to one another, thus providing for the desired receptor interaction.
  • the sugar residues should ideally be separated by a spacer of not less than about 10 bond lengths, with at least 25 bond lengths being more preferred.
  • the galactoses may be attached in a branched arrangement as follows, which is based on bis-homotris:
  • each arm is extended, and terminates in a carboxylic acid terminus as follows:
  • the linker should be long enough to alleviate adverse steric effects which may result in diminished binding of the small molecule to the conjugate and/or diminished binding of the complex to the galactose receptor.
  • ligands for biotin, ideally those having small molecular weight.
  • Such ligands may also be modified to include suitable functional groups to allow for the attachment of other molecules of interest, e.g., peptides, proteins, nucleotides, and other small molecules.
  • the clearing agent may be attached to a desired functional group via the end which is opposite to the sugar residues.
  • suitable functional groups include, e.g., maleimides, activated esters, isocyanates, alkyl halides (e.g., iodoacetate) , hydrazides, thiols, imidates and aldehydes.
  • the subject small molecule clearing agents may also be conjugated to active small molecules, e.g., radionuclides, peptides, small proteins and nucleotides, to provide for an active agent which is delivered to an active site which has been pretargeted with a first agent containing a targeting moiety attached to a ligand or anti-ligand which binds the ligand or anti-ligand contained in the small molecule clearing agent.
  • active small molecules e.g., radionuclides, peptides, small proteins and nucleotides
  • this embodiment will provide for active agents which are delivered to active sites, and are rapidly eliminated from the circulation by virtue of the clearing directing moieties, e.g., galactose residues.
  • This embodiment is particularly useful if the active agent is cytotoxic, e.g., a radionuclide.
  • Preferred galactose clusters contained in the subject small molecule clearing agents will be of the formula:
  • x is H, methyl, lower alkyl or lower alkyl withi hetero atoms.
  • the above stuctures bear 4, 8 and 16 galactose, respectively. Further iteration in the branching allows expansion to include 32, 64, etc. galactose residues.
  • branching structures may also be employed in the design of galactose clusters in accordance with the present invention.
  • the extender from the galactose to branching linker may be variable in length.
  • the ligand e.g., biotin
  • galactose cluster and optionally an active agent
  • suitable bifunctional or trifunctional linkers selection of suitable trifunctional and bifunctional linkers amenable to binding with functional groups on the ligand, galactose cluster, and optionally the active moiety, e.g., a chelate, is well within the level of skill in the art.
  • suitable bifunctional linkers include bis-N,N- (6- (1-hydroxycarbonylhexyl) amine.
  • Suitable trifunctional linkers include lysine.
  • extender moieties may be utilized in the construction of the subject small molecule clearing agents. Suitable extenders include difunctional moieties capable of binding either the ligand component and the linker or the galactose cluster component and the linker. Suitable extender moieties include an aminocaproate moiety, 4 aminobutane thiol and the like. One of skill in the art can readily select appropriate extender molecules which promote bioavailability of the galactose cluster. Alternatively, the extender function may be served by an appropriately constructed linker.
  • Clearing agent evaluation experimentation involving galactose- and biotin-derivatized clearing agents of the present invention is detailed in Examples XIII and XVI.
  • Specific clearing agents of the present invention that were examined during the Example XVI experimentation are (1) asialoorosomucoid- biotin, (2) human serum albumin derivatized with galactose and biotin, and (3) a 70,000 dalton molecular weight dextran derivatized with both biotin and galactose.
  • the experimentation showed that proteins and polymers are derivatizable to contain both galactose and biotin and that the resultant derivatized molecule is effective in removing circulating streptavidin-protein conjugate from the serum of the recipient.
  • Biotin loading was varied to determine the effects on both clearing the blood pool of circulating avidin-containing conjugate and the ability to deliver a subsequently administered biotinylated isotope to a target site recognized by the streptavidin-containing conjugate.
  • the effect of relative doses of the administered components with respect to clearing agent efficacy was also examined.
  • Examples XIX and XX relate to small molecule clearing agents comprising biotin and galactose residues.
  • 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 aggregation-mediated RES mechanism.
  • 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.
  • the present invention provides clearing agents that incorporate ligand derivatives or anti-ligand derivatives, wherein such derivatives exhibit a lower affinity for the complementary ligand/anti-ligand pair member than the native form of the compound (i.e., lower affinity ligands or anti-ligands) .
  • preferred clearing agents incorporate either lower affinity biotin (which exhibits a lower affinity for avidin or streptavidin than native biotin) or lower affinity avidin or a streptavidin (which exhibits a lower affinity for biotin than native avidin or streptavidin) .
  • Clearing agents that employ a ligand or anti- ligand moiety that is complementary to the ligand/anti-ligand pair member (previously administered in conjunction with the targeting moiety) are useful in the practice of the present invention.
  • clearing agents localize to hepatocytes, they are generally rapidly degraded. This degradation liberates a quantity of free ligand or free anti- ligand into the circulation. This bolus release of ligand or anti-ligand may compete for binding sites of targeting moiety-ligand or targeting moiety-anti- ligand with subsequently administered active agent- ligand or active agent-anti-ligand conjugate.
  • This competition can be addressed by using a clearing agent incorporating a lower affinity ligand or anti-ligand.
  • the ligand or anti- ligand employed in the structure of the clearing agent more weakly binds to the complementary ligand/anti- ligand pair member than native ligand or anti-ligand. Consequently, lower affinity ligand or anti-ligand derivatives that bind to target-localized targeting moiety-anti-ligand or targeting moiety-ligand conjugate may be displaced by the subsequently administered, active agent-native (or higher binding affinity ligand) or active agent-native (or higher binding affinity) anti-ligand conjugate.
  • lower affinity biotin, lower affinity avidin or lower affinity streptavidin may be employed.
  • Exemplary lower affinity biotin molecules exhibit the following properties: bind to avidin or streptavidin with an affinity less than that of native biotin (10 ⁇ 15 ) ; retain specificity for binding to avidin or streptavidin; are non-toxic to mammalian recipients; and the like.
  • Exemplary lower affinity avidin or streptavidin molecules exhibit the following properties: bind to biotin with an affinity less than native avidin or streptavidin; retain specificity for binding to biotin; are non-toxic to mammalian recipients; and the like.
  • Exemplary lower affinity biotin molecules include 2' -thiobiotin; 2' -iminobiotin; 1' -N-methoxycarbonyl- biotin; 3' -N-methoxycarbonylbiotin; 1-oxy-biotin; 1- oxy-2' -thiobiotin; l-oxy-2'-iminobiotin; 1-sulfoxide- biotin; l-sulfoxide-2' -thiobiotin; l-sulfoxide-2' - iminobiotin; 1-sulfone-biotin; l-sulfone-2' -thio- biotin; l-sulfone-2' -iminobiotin; imidazolidone derivatives such as desthiobiotin (d and dl optical isomers) , dl-desthiobiotin methyl ester, dl- desthiobiotinol, D-4-n-hexyl-
  • Preferred lower affinity biotin molecules for use in the practice of the present invention are 2'- thiobiotin, desthiobiotin, 1-oxy-biotin, l-oxy-2'- thiobiotin, 1-sulfoxide-biotin, l-sulfoxide-2' - thiobiotin, 1-sulfone-biotin, l-sulfone-2' -thiobiotin, lipoic acid and the like.
  • These exemplary lower affinity biotin molecules may be produced substantially in accordance with known procedures therefor. Conjugation of the exemplary lower affinity biotin molecules to HSA or other amino acid-based or polymeric moieties proceeds substantially in accordance with known procedures therefor and with procedures described herein with regard to biotin conjugation.
  • the present invention further provides methods of increasing active agent localization at a target cell site of a mammalian recipient, which methods include: administering to the recipient a first conjugate comprising a targeting moiety and a member of a ligand-anti-ligand binding pair; thereafter administering to the recipient a clearing agent capable of directing the clearance of circulating first conjugate via hepatocyte receptors of the recipient, wherein the clearing agent incorporates lower affinity complementary member of the ligand-anti-ligand binding pair; and subsequently administering to the recipient a second conjugate comprising an active agent and a ligand/anti-ligand binding pair member, wherein the second conjugate binding pair member is complementary to that of the first conjugate and, preferably, constitutes a native or high affinity form of the member.
  • Certain active agents e.g., certain cytokines, exert therapeutic activity in association with a receptor therefor on the target cell surface or on the surface of other cells in the vicinity of target cells.
  • the "sandwich" at the target cell surface including, for example, targeting moiety-anti-ligand- ligand-active agent may not provide optimal delivery of the active agent to the relevant receptor.
  • the sandwich is preferably structured to be conditionally cleavable.
  • One way to provide for conditional cleavage of the active agent is to employ lower affinity ligand or anti-ligand in the sandwich. After the sandwich is formed (e.g., from about 2 to about 8 hours following administration of ligand-active agent conjugate) , a bolus dose of native or higher affinity ligand or anti-ligand is given. This native or higher affinity (from 3-6 orders of magnitude) ligand or anti-ligand will serve to displace its lower affinity counterpart in the sandwich, thereby releasing the active agent from the sandwich.
  • the present invention therefore provides methods of increasing active agent localization at a target cell site of a mammalian recipient, which methods include: administering to the recipient a first conjugate comprising a targeting moiety and a member of a ligand-anti-ligand binding pair; thereafter administering to the recipient a clearing agent capable of directing the clearance of circulating first conjugate via hepatocyte receptors of the recipient; administering to the recipient a second conjugate comprising an active agent and a lower affinity ligand/anti-ligand binding pair member, wherein the second conjugate lower affinity binding pair member is complementary to that of the first conjugate; and administering to the recipient native or higher affinity ligand or anti-ligand corresponding to the lower affinity binding pair member of the second conjugate.
  • the present invention provides methods of increasing active agent localization at a target cell site of a mammalian recipient, which methods include: administering to the recipient a receptor blocking agent in an amount sufficient to substantially block a subpopulation of hepatocyte receptors; administering to the recipient a first conjugate comprising a targeting moiety, a hepatocyte receptor recognizing agent, and a member of a ligand-anti- ligand binding pair; and subsequently administering to the recipient a second conjugate comprising an active agent and a ligand/anti-ligand binding pair member, wherein the second conjugate binding pair member is complementary to that of the first conjugate.
  • hepatocyte receptors with respect to which this block/deblock protocol may be employed include Ashwell receptors; other receptors such as the mannose/N-acetylglucosamine receptor which are associated with endothelial cells and/or Kupffer cells of the liver; the mannose 6-phosphate receptor; or the like.
  • Exemplary receptor blocking agents of the present invention exhibit one or more of the following structural or functional characteristics: low immunogenicity; low toxicity; are recognized by a hepatocyte receptor and are processed thereby; and the like.
  • preferred receptor blocking agents include IgG- galactose; human IgG-galactose; asialoorosomucoid, galactose-HSA, with human or other mammalian HSA.
  • the receptor blocking agent is administered via intravenous, intraarterial or like routes of administration, with intravenous administration preferred.
  • Such administration is preferably conducted in continuous or via multiple administrations for a time sufficient to substantially block the relevant hepatocyte receptors and to permit localization of the targeting moiety to target sites, e.g., generally ranging from about 18 to about 72 hours.
  • the blocking agent is occupying the relevant hepatocyte receptor population to permit localization of the first conjugate.
  • the hepatocyte receptor population processes the remaining blocking agent and the hepatocyte receptor recognizing agent- bearing first conjugate.
  • the second conjugate is preferably administered after a time sufficient to permit receptor-based clearance of receptor blocking agent to deblock the receptors and receptor-based clearance of circulating first conjugate, e.g., generally ranging from about 2 to about 8 hours post-cessation of administration or post-final administration of receptor blocking agent and from about 24 to about 72 hours post- administration of first conjugate.
  • Another class of clearing agents includes agents that do not remove circulating ligand or anti- ligand/targeting moiety conjugates, but instead "inactivate” the circulating conjugates by blocking the relevant anti-ligand or ligand binding sites thereon.
  • These "cap-type” clearing agents are preferably small (500 to 10,000 Dal) highly charged molecules, which exhibit physical characteristics that dictate a volume of distribution equal to that of the plasma compartment (i.e., do not extravasate into the extravascular fluid volume) .
  • Exemplary cap-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 intravenously for a time period sufficient to clear targeting moiety-streptavidin in a continuous manner.
  • 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.
  • One embodiment of the present invention in which rapid acting clearing agents are useful is in the delivery of Auger emitters, such as 1-125, 1-123, Er- 165, Sb-119, Hg-197, Ru-97, Tl-201 and 1-125 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
  • Such internalizing receptors include EGF receptors, transferrin receptors, HER2 receptors, IL-2 receptors, other interleukins and cluster differentiation receptors, somatostatin receptors, other peptide binding receptors and the like.
  • a rapidly acting clearing agent is administered.
  • an active agent-containing ligand or anti-ligand conjugate such as a biotin-Auger emitter or a biotin- nucleus acting drug
  • an active agent-containing ligand or anti-ligand conjugate such as a biotin-Auger emitter or a biotin- nucleus acting drug
  • the clearing agent 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. 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 1-123, 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.
  • Preferable agents and protocols within the present invention are therefore characterized by prolonged residence of radionuclide at the target cell site to which the radionuclide has localized and improved radiation absorbed dose deposition at that target cell site, with decreased targeted radioactivity loss resulting from metabolism.
  • 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 radionuclides can be administered in pretargeting protocols intravenously, intraarterially or the like or injected directly into lesions.
  • BBPs include peptides containing the motif represented by CXWXPPF (K or R) XXC; peptides containing the previously identified motif without one or both terminal cysteine residues; biotin operon repressor; biotin holoenzyme synthetase; and biotin carboxylase.
  • Biotin binding peptides are well known in the art.
  • Enzymes are chiral molecules having strict selectivity for substrates with the correct stereochemical configuration. Natural enzymes are made up of L amino acids and may recognize substrates of either L or D configuration. See, for example,
  • Natural enzymes of L configuration prepared synthetically using D-amino acids exhibit specificity for substrates with the opposite stereochemistry compared to that of the natural substrate. Also, these "mirror image" enzymes convert the opposite stereochemical substrate with substantially the same efficiency or turnover rate as the naturally occurring enzyme acts on the natural substrate. See, for example, Milton et al., Science. 256: 1445-1447, 1992, for a discussion of preparation of D-enzymes having reciprocal chiral substrate specificity.
  • Natural streptavidin and natural avidin recognizes D-biotin. Consequently, streptavidin or avidin formed with D-amino acids in the manner described by Milton et al. therefore interact with L-biotin rather than D- biotin (endogenous biotin) . Consequently, any inefficiencies in in vivo operation of pretargeting protocols caused by endogenous biotin can be obviated or substantially reduced by forming streptavidin or avidin of D-amino acids.
  • the D-amino acid forms of streptavidin or avidin will show binding specificity for L-biotin rather than naturally occurring, endogenous D-biotin. Moreover, the high affinity avidin- and streptavidin-binding will be preserved in the mirror image format.
  • Biotin binding peptides may also be converted to mirror image configuration in this manner to bind L-biotin rather than D-biotin. Preparation of mirror image biotin binding peptides may be conducted by solid phase peptide synthesis in accordance with known techniques therefor.
  • mirror image streptavidin, avidin or BBPs Binding of mirror image streptavidin, avidin or BBPs to targeting moieties can be accomplished via the same techniques described herein and known in the art for natural protein-targeting moiety binding.
  • mirror image BBPs can be incorporated into fusion proteins substantially as described herein for L stereochemistry BBPs.
  • targeting moiety-avidin, -streptavidin, -BBP conjugates or fusion proteins in pretargeting protocols is accomplished as described herein for natural protein-containing forms of such conjugates and fusion proteins.
  • Monovalent antibody fragment-streptavidin 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.
  • Monovalent antibody 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 penetration.
  • 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 radionuclides 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 molecule and, therefore, enhances target retention, under appropriate conditions of antigen density at the target cell.
  • monovalent antibody 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.
  • the pretargeting 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 intraarterially using an artery supplying tissue that contains the target.
  • 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) .
  • particle emitting therapeutic radionuclides 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 invention, if they are able to bind in amounts delivering a therapeutically effective amount of radiation to the target.
  • 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 such as IL-2 and tumor necrosis factor, drug/lymphokine-carrier-biotin molecules, biotinylated drugs/lymphokines, and drug/lymphokine/ toxin-loaded, biotin-derivitized 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.
  • 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 protecting agents, such as WR 2721. As a result, even higher doses of radiation can be given, absent dose limiting bone marrow toxicity.
  • a complementary binding pair selected from the group consisting of S-peptide/S-protein, head activator peptide (which binds to itself) , cystatin C/cathepsin B, and antibody/hapen pairs
  • Conjugates containing said peptides have utility in all aspects of pretargeting methods, i.e., they may be administered in the initial pretargeting step, they may be used as novel clearing agents, and they may be administered in order to direct an active agent, e.g., a therapeutic or diagnostic agent, to a targeted site, e.g. , a tumor.
  • an active agent e.g., a therapeutic or diagnostic agent
  • S-peptide/S-protein complementary binding pair members have particular applicability in pretargeting methods given the fact that both of these moieties are well characterized, e.g., the complete amino acid sequences of both S-peptide and S-protein have been reported in the literature. Moreover, both the S-peptide and the S-protein are commercially available from Sigma Chemical (St. Louis, Missouri).
  • S-peptide and S-protein are enzymatically inactive products obtained by limited digestion of ribonuclease A with subtilisin. These moieties bind to one another with an affinity of about 10 "9 M to produce a ribonuclease S complex which catalyzes the hydrolytic cleavage of RNA similar to ribonuclease A.
  • ribonuclease S complex which catalyzes the hydrolytic cleavage of RNA similar to ribonuclease A.
  • the present invention embraces the use of conjugates containing S-peptide and/or S-protein in pretargeting methods, as well as derivatives and analogs thereof.
  • S-peptide or S-protein derivatives and analogs retain their ability to bind either S-peptide or S-protein with sufficient affinity to be useful in pretargeting methods.
  • An especially preferred S-peptide is a truncated form known in the art as S15 which consists of the following peptide sequence:
  • S-peptides and S-protein conjugates and derivatives thereof are also known in the literature.
  • Thomson et al., Biochem. 33(28), 8587-8593, (1994) describes methylene derivatized S-peptides and truncated forms which effectively complex with S-protein; Kim et al. , Protein Science. 2(3), 348-356, (1993) describes functional S-peptide derivatives where the aspartic acid at position 14 is changed to an asparagine
  • Varadarjan et al., Biochem.. 31(49) 12315-12327, (1992) describes variants of S-peptide modified at position 13, and Pease et al., Proc. Natl. Acad. Sci..
  • the conjugates will contain S-peptide or S15 and/or S-protein because all of these moieties have been extensively characterized and are commercially available. Moreover, since the amino acid sequence of each of these moieties is known, and all of these moieties are relatively small, i.e., S-peptide is 20 amino acid residues, S15 is 15 amino acid residues, and S-protein is only 104 amino acid residues, all of these moieties can readily be made synthetically, e.g., by solid-state synthesis or by recombinant methods.
  • the S-peptide and S-protein may be obtained by limited digestion of ribonuclease A with subtilisin to generate a peptide fragment containing the first 20 amino acid residues of ribonuclease A (S-peptide) and a protein fragment containing residues 21 to 124 (S-protein) .
  • S-peptide, S15 peptide and/or S-protein or derivatives and analogs thereof may be used in lieu of other ligand/anti-ligand in conjugates which are used in pretargeting strategies or in combination therewith.
  • S-peptide/S-protein may be used in lieu of biotin/avidin or biotin/streptavidin or in combination therewith.
  • the use of S-peptide and S-protein and derivatives and analogs as the ligand/anti-ligand pair in pretargeting methods should afford numerous advantages given their ready availability, relatively, low cost; high degree of characterization; the fact that they bind to one another with relatively high affinity
  • bovine pancreatic ribonuclease A a mammalian protein
  • bovine pancreatic ribonuclease A a mammalian protein
  • immunogenicity should not be as significantly reduced compared to bacterial proteins such as streptavidin.
  • bovine ribonuclease is about 70% homologous to human (Beintena et al. , Anal. Biochem.. 136, 48-64, (1984)).
  • biotin/avidin or biotin/streptavidin system there also should not be the problem of endogenously circulating ligand or anti-ligand (S-peptide or S-protein) .
  • S-peptide and S-protein have particular applicability in the preparation of novel clearing agents.
  • S-peptide or S-protein may be conjugated to or derivatized with clearance directing moieties to produce compounds which provide for enhanced clearance of a previously, concurrently or subsequently administered conjugate.
  • S-peptide or S-protein may be attached to any of the afore-described clearance directing agents.
  • a clearing agent is any agent capable of binding, complexing or otherwise associating with an administered moiety, e.g., targeting moiety-ligand, targeting moiety-anti-ligand or anti-ligand alone, present in the recipient's circulation, thereby facilitating circulating moiety clearance from the recipient's body, removal from blood circulation, or inactivation thereof in the circulation.
  • the clearing agent will comprise hepatocyte receptor binding moiety or moieties.
  • S-peptide, S-15 peptide or S-protein may be conjugated or derivatized with hexose-based or non-hexose based moieties such as are described supra .
  • Hexose-based clearing agents are molecules that have been derivatized to contain one or more hexoses (six carbon moieties) , which are preferably recognized by receptor, i.e., Ashwell receptors or other receptors such as the mannose/N-acetylgalactosamine receptor which are associated with endothelial cells and/or the mannose 6-phosphate receptor.
  • S-peptide or S-protein may be directly or indirectly attached to one or more hexoses selected from galactose, mannose, mannose 6- phosphate, N-acetylgalactosamine, pentamannosyl phosphate, thioglycosides of galactose, and more generally, D-galactosides and glucosides or the like, as well as combinations thereof.
  • galactose is the prototypical hexose clearing agent.
  • One or more such hexoses or several different hexoses may be directly or indirectly attached to an S-peptide or S-protein to provide for an effective clearance agent.
  • galactose it appears that at least three galactose residues are necessary, with about 3 to 32 being preferred.
  • Methods of attachment of hexose residues to proteins and peptides are well known in the art. For example, if galactose residues are to be attached, this may be accomplished, e.g., by galactose thioglycoside conjugation such as is described supra .
  • the efficacy of the resultant clearing agent depends upon the ability of the resultant agent, e.g., galactose derivatized S-peptide or S-protein to effectively bind its binding partner, i.e., S-protein or S-peptide.
  • the resultant agent e.g., galactose derivatized S-peptide or S-protein to effectively bind its binding partner, i.e., S-protein or S-peptide.
  • S-protein it is expected that galactose or other hexose-derivatization should not adversely affect the ability of the resultant galactose-derivatized S-protein to bind S-peptide and conjugates containing S-peptide.
  • the S-peptide or S-protein may instead be indirectly attached to galactose or other hexoses by attachment to a moiety or moieties which contain one or more exposed hexoses, e.g., galactose residues.
  • the galactose will be arranged in clusters as described elsewhere in this application.
  • moieties include proteinaceous hexose-based clearing agents which endogenously contain or have been derivatized to contain one or more exposed hexose residues. Exposed hexose residues, e.g., galactose residues, direct rapid clearance by endocytosis into the liver through specific receptors (Ashwell receptors) .
  • S-peptide or S-protein may be attached to the asialoorsomucoid derivative of human alpha-1 acid glycoprotein (orosomucoid) , galactosylated albumins such as galactosylated HSA, galactosylated-IgM, galactosylated-IgG, asialohaptoglobin, asialofetuin, asialoceruloplasmin and the like.
  • galactosylated albumins such as galactosylated HSA, galactosylated-IgM, galactosylated-IgG, asialohaptoglobin, asialofetuin, asialoceruloplasmin and the like.
  • S-peptide or S-protein will be attached to a hexose residue bearing proteinaceous clearing agent which effectively binds to hepatocyte receptors such as galactose, mannose 6-phosphate, N- acetylglucosamine, glucose, N-galactosamine, N- acetylgalactosamine, thioglycosides of galactose, and more generally D-galactosides and glucosides or the like.
  • hepatocyte receptors such as galactose, mannose 6-phosphate, N- acetylglucosamine, glucose, N-galactosamine, N- acetylgalactosamine, thioglycosides of galactose, and more generally D-galactosides and glucosides or the like.
  • S-peptide may be C-or N-terminally fused to proteinaceous moieties without loss of S-protein binding function.
  • attachment of hexose containing proteinaceous moieties should result in conjugates which effectively bind to S-protein or S-peptide and to conjugates which contain S-peptide or S-protein.
  • Attachment of either S-peptide or S-protein to hexose derivatized proteins, e.g., galactosylated human serum albumin may be effected using conventional heterobifunctional cross-linking agents.
  • conjugates will be screened to assess their ability to effectively bind conjugates containing the complementary binding partner with a sufficient binding affinity to provide for effective clearance.
  • conjugates will further preferably be designed so as to contain a number of hexose residues, e.g., galactose, which provides for optimal clearance, e.g., by the Ashwell receptor mechanism. This can be determined by variation of the number of attached hexose residues on the S-peptide or S-protein derivative and comparing clearance rates as a function of the number of attached hexose residues, e.g., galactose, after in vivo administration.
  • S-protein conjugate When S-protein conjugate is utilized as the clearing agent, exemplary embodiments are described schematically below:
  • a pretargeting step comprising the administration of a conjugate or fusion protein comprising an S-peptide, e.g., S15 peptide N-or C- terminally attached to a targeting moiety, e.g., an antibody or antibody fragment, which is optionally attached to another ligand or anti-ligand, e.g., streptavidin, avidin or biotin; and
  • a particular application for the S-peptide is for imaging of clots by modification of an annexin with S- peptide. More specifically, it should be useful in enhancing target and background ratio for improved clot/thrombus imaging with Tc-99m annexin. Technetium-99m labeled annexin has been shown to effectively localize in clots that have been induced in the pig animal model.
  • subtilisin cleaves RNase A into S- protein and S-peptide. These are of 103 and 20 residues respectively. The S-protein and S-peptide bind with high affinity.
  • S-protein would be modified with a liver targeting moiety such as galactose. This would allow a procedure as follows: (i) Tc-99m annexin-S-peptide is administered, allowed to target clots and determination of sufficient information for diagnosis made by scintigraphic imaging. (ii) If higher clot to blood background ratio is needed for diagnosis, S-protein-liver targeting moiety is injected. Tc-99m annexin in circulation would be bound by the S-protein portion while the liver targeting moiety would cause liver uptake of the bound complex.
  • a liver targeting moiety such as galactose.
  • the S-peptide and S-protein and derivatives thereof may be used alone or in combination with or as a substitute for streptavidin, avidin or biotin in pretargeting methods.
  • the S-peptide or S-protein may be mutagenized, e.g., by site-specific mutagenesis to produce analogs which bind either S-peptide or S-protein with higher affinity than the unmodified S-peptide or S-protein or complex to produce non-enzymatically active derivaties.
  • the S-peptide may be derivatized to effectively "lock in” the peptide by modification of an appropriate amino acid side chain. This may be accomplished, e.g., by reaction with an active halide such as chloromethylketone.
  • S-peptide derivatives should result in better retention of the active agent at the target site, attributable to non- reversible binding of the modified S-peptide and S-protein containing conjugate at the target site.
  • the S-peptide is so small in size, it should also be possible to attach several of these moieties to the targeting moiety, e.g., antibody or antibody fragment, thereby increasing receptor target at the target site, e.g., a tumor.
  • the S-peptide may be modified by linking two or more together to provide for multivalent binding.
  • the linking peptide may contain an additional linkage to the effector moiety.
  • Such conjugates should enhance binding affinity by providing for the crosslinking of the S-protein or S-peptide which is attached to the antibody or other targeting moiety, or by increasing the probability that the conjugate rebinds its complementary binding partner.
  • S-peptide is used in clearing agents it may particularly exhibit some liver toxicity. If toxic, ribonuclease inhibitors can be used to obviate cytotoxicity. (See, e.g., White et al. , Principles
  • the S-protein may also be oligomerized to produce conjugates having increased binding stoichiometry and affinity. Incorporation of such oligomers in conjugates for use in pretargeting should provide for both better delivery and retention of the active agent at the targeted site.
  • one disadvantage is that this may result in enhanced immunogenicity to the S-protein.
  • Yet another advantage of the S-peptide/S-protein system is that these moieties complex to produce an enzymatically active ribonuclease S complex. This enzyme activity may be exploited to provide for targeted cytotox ' ic activity.
  • an internalizing antibody e.g., slowly internalizing antibody
  • S-peptide is used as a clearing agent it may potentially by cytotoxized. However, this may potentially be obviated by administration of ribonuclease inhibitors, inhibit A??? or RNase.
  • iodoacetate reaction with ribonuclease inactivates the enzyme by alkylation of histidine 119. (White et al. , Principles Biochem. , McGraw Hill, Sin ed., p. 258) .
  • HA head activator
  • the HA peptide is derived from the freshwater coelenterate, Hydra . This peptide acts as a morphogen which controls the coelenterate' s head-specific growth and differentiation process.
  • the entire HA peptide consists of the following amino acid sequence: pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe.
  • carboxyl fragments of the HA peptide in particular a fragment containing the last six carboxyl amino acids of the HA peptide, dimerize equal to or even more efficiently than the intact HA peptide (Id. ) .
  • the HA peptide, as well as fragments and derivatives thereof, most particularly a hexameric peptide consisting of the following amino acid sequence: Ser-Lys-Val-Ile-Leu-Phe are particularly well suited as both the ligand and anti-ligand binding partners in pretargeting methods.
  • the HA peptide affords numerous advantages to other known ligands and anti-ligands in pretargeting methods. For example, given its small size, the HA peptide and fragments thereof should not be very immunogenic. Thus, the HA peptide is especially well suited for therapeutic pretargeting methods wherein immunogenicity may be a potential concern. Also, the HA peptide binds to itself with very high affinity.
  • the HA peptide or fragments thereof may be fused to a targeting moiety, e.g., an antibody or antibody fragment and then administered in the initial pretargeting step.
  • a targeting moiety e.g., an antibody or antibody fragment
  • Another advantage of the HA peptide and fragments thereof is that its small size should enable it to be inserted into targeting moiety sequences, e.g., antibody sequences and fragments thereof. Such insertion may be effected by recombinant methods or by solid state synthesis. However, recombinant methods are preferred.
  • Recombinant methods of expressing antibodies and binding fragments thereof are well known in the art.
  • methods are known in the art for the recombinant expression of antibodies, fragments and derivatives, e.g., Fab fragments, Fv's, humanized antibodies, chimeric antibodies, single chain antibodies and bispecific antibodies (See, e.g., U.S. Patent No. 4,816,567 to Cabibly et al.; U.S. Patent No. 5,132,405 to Huston et al; U.S. Patent No. 4,704,692 to Ladner, U.S. Patent No. 4,946,778 to
  • an oligonucleotide encoding the subject HA peptide, or the above-described hexameric peptide can be inserted into a DNA sequence encoding a desired antibody sequence or antibody fragment and expressed to produce a recombinant antibody or antibody fragment capable of dimerizing with another conjugate containing the HA peptide.
  • a sequence may be created by site specific mutagenesis of a recombinant antibody or antibody fragment DNA sequence.
  • sequences will preferably be inserted or created in portions of the antibody molecule which are non- essential for antigen binding.
  • Functional antigen-binding sequences can be selected by inserting the HA peptide encoding sequences into different regions of a particular antibody DNA sequence and then screening the resultant expression products in binding assays to identify those particular recombinant sequences which bind antigen with sufficient affinity.
  • HA peptide and truncated and derivative forms thereof are for "cementing" the light and variable domains of a recombinant Fv molecule. This may be accomplished by expression of fusion peptides respectively comprising the heavy variable region fused to at least one HA peptide and a light variable region fused to at least one HA peptide. These fusion peptides may be separately or co-expressed, with co-expression being preferred since this may result in formation of Fv's in the host cell.
  • the presence of the HA peptide on each of the light and variable region should facilitate the formation of a highly stable Fv molecules, given the high autoaffinity of HA peptides, e.g., the HA hexameric peptide sequence identified supra .
  • the resultant monovalent Fv sequence can additionally be dimerized by fusing several HA peptide sequences onto either or both of the variable heavy and light sequence fusion proteins. This will provide for the formation of divalent or higher valency Fv's.
  • Still another application of the HA peptide, and derivatives thereof is for increasing the avidity of single chain antibody molecules to antigen molecules.
  • single chain antibodies have not been widely used given their typically low antigen avidity relative to native antibodies and to Fab fragments. While their small size and single chain form affords some intrinsic advantages, e.g., the ability to be internalized by tumor cells, e.g., extravascular tumors and rapid renal clearance, their low avidity to antigen renders their therapeutic and diagnostic use disadvantageous.
  • HA peptide sequences into a single chain antibody molecule will result in dimerization of the single chain antibody molecule, or even multimerization if more than one HA peptide sequence is incorporated into or fused to the single chain antibody molecule. This will result in single chain antibodies containing more than one antigen binding site. Therefore, this should result in single chain antibodies having higher avidity to antigen.
  • HA peptide sequences are for the preparation of bispecific antibodies. Bispecific antibodies comprise the antigenic binding sequences of antibodies having two different antigen specificities. Therefore, such antibodies have the ability to bind to two different antigens.
  • the present invention provides a novel method for the formation of bispecific antibodies by the attachment of one or more HA peptide sequences to Fv sequences, single chain antibody sequences, or Fab sequences, wherein the fused antigen binding sequences possess different antigenic specificity.
  • fusion proteins may be made by recombinant methods or by synthetic means with recombinant methods being preferred.
  • Said HA containing sequences may be separately or co-expressed in recombinant cells. Co- expression is preferred since this may enable the fusion proteins to dimerize in the recombinant host cell to produce bispecific antibody molecules comprised of Fv's, single chain antibodies or Fab's of two different specificities.
  • the resistant HA containing antigen binding sequences may alternatively be dimerized by mixing in solution, or alternatively by contacting a solid phase to which one of the antigen binding fusion proteins has been immobilized with the other HA peptide containing antigen binding sequence having different antigenic specificity.
  • HA may be fused to another member of a complementary binding pair, e.g., biotin.
  • This HA-biotin fusion protein may be used to produce a highly stable linkage with an HA-antibody fusion protein, e.g., which has been pretargeted to a target site, e.g., a tumor cell.
  • a target site e.g., a tumor cell.
  • the presence of the biotin in the fusion protein will in addition provide for the stable attachment of avidin or streptavidin.
  • the use of two ligands in combination will also enable several different moieties to be directed to a targeted site, e.g., tumor cells.
  • the HA peptide may be incorporated in conjugates which are used in all steps of pre-targeting methods.
  • the HA peptide or a fragment thereof may be attached to or inserted in a targeting moiety, e.g., an antibody, antibody fragment or receptor binding moiety as described previously, and used in the initial pretargeting step.
  • a targeting moiety e.g., an antibody, antibody fragment or receptor binding moiety as described previously, and used in the initial pretargeting step.
  • an active agent e.g., the diagnostic or therapeutic agent, can also be attached to an HA peptide.
  • the HA peptide-active agent will bind the pretargeted HA-targeting moiety because of the affinity of the HA peptide to the HA peptide contained in the pretargeted conjugate. Also, because of its small size, several HA peptides may be attached to an active agent, or the HA peptide may be attached to different active agents. This should enable more or several different active agents to be delivered to a targeted site. This is advantageous because some therapies may require delivery of several active agents, (because of synergistic cytotoxic effects) or high dosages of the particular cytotoxin agent to be effective.
  • the HA peptide may also be utilized for the preparation of novel clearing agents.
  • the HA peptide will be directly or indirectly attached to one of the clearance directing moieties described supra, e . g . , a galactosylated protein such as galactosylated human serum albumin.
  • a galactosylated protein such as galactosylated human serum albumin.
  • proline residues may be engineered onto the particular HA peptide fusion protein given their known efficacy in enhancing the flexibility of proteins and in particular antibody fusion proteins.
  • 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 mammalian origin, chimeric, humanized or human.
  • NR-LU-10 is a 150 kilodalton molecular weight IgG2b monoclonal antibody that recognizes an approximately 40 kilodalton glycoprotein antigen expressed on most carcinomas. In vivo studies in mice using an antibody specific for the NR-LU-10 antigen revealed that such antibody was not rapidly internalized, which would have prevented localization of the subsequently administered active-agent- containing conjugate to the target site.
  • 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 x 63/Ag8UI murine myeloma cells. After establishing a seed lot, the hybridoma was grown via in vi tro cell culture methods, purified and verified for purity and sterility.
  • Radioimmunoassays, immunoprecipitation and Fluorescence-Activated Cell Sorter (FACS) analysis were used to obtain reactivity profiles of NR-LU-10.
  • the 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
  • 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," Cancer Research, 44: 681-687, 1984, and Okabe et al., "Monoclonal Antibodies to Surface Antigens of Small Cell Carcinoma of the Lung," Cancer Research. 44 : 5273-5278, 1984.
  • 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.
  • Such antibodies may be prepared by the following procedure: absorbing a first monoclonal antibody directed against a first epitope of a polyvalent antigen onto an inert, insoluble matrix capable of binding immunoglobulin, thereby forming an immunosorbent; combining the immunosorbent with an extract containing polyvalent NR-LU-10 antigen, forming an insolubilized immune complex wherein the first epitope is masked by the first monoclonal antibody; immunizing an animal with the insolubilized immune complex; fusing spleen cells from the immunized animal to myeloma cells to form a hybridoma capable of producing a second monoclonal antibody directed against a second epitope of the polyvalent antigen; culturing the hybridoma to produce the second monoclonal antibody; and collecting the second monoclonal antibody as
  • 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.
  • 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 / S--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 on the following page.
  • 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 complex. 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.
  • 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 protein) ; this percentage was unaffected by the addition of exogenous biotin. When the samples were analyzed using size exclusion H-12 FPLC with 0.2 M phosphate as mobile phase, no association of radioactivity with serum macromolecules was observed. Each radiolabeled biotin conjugate was further examined using a competitive biotin binding assay.
  • the 186 Re-chelate-biotin conjugate of Example I was studied in an animal model of a three-step antibody pretargeting protocol. Generally, this protocol involved: (i) prelocalization of biotinylated monoclonal antibody; (ii) administration of avidin for formation of a "sandwich" at the target site and for clearance of residual circulating biotinylated antibody; and (iii) administration of the 186Re-biotin conjugate for target site localization and rapid blood clearance.
  • Biotinylated NR-LU-10 was prepared according to either of the following procedures. The first procedure involved derivitization of antibody via lysine e-amino groups. NR-LU-10 was radioiodinated at tyrosines using chloramine T and either 125 ⁇ or 131 I sodium iodide. The radioiodinated antibody (5-10 mg/ml) was then biotinylated using biotinamido caproate NHS ester in carbonate buffer, pH 8.5, containing 5% DMSO, according to the scheme below. 7 * prot ⁇ in-NH "j"
  • NR-LU-10 was biotinylated using thiol groups generated by reduction of cystines. Derivitization of thiol groups was hypothesized to be less compromising to antibody immunoreactivity.
  • 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
  • biotinylated antibody species 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.
  • 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.
  • saline administration blood clearances for both biotinylated antibody species were biphasic and similar to the clearance of native NR-LU-10 antibody.
  • biotinylated antibody lysine
  • avidin administration (10:1 or 25:1) reduced the circulating antibody level to about 35% of injected dose after two hours.
  • Residual radiolabeled antibody activity in the circulation after avidin administration was examined in vi tro using immobilized biotin. This analysis revealed that 85% of the biotinylated antibody was complexed with avidin.
  • 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) .
  • Avidin (MW - 66 kD) was radiolabeled with 131 I/PIP-NHS (as described for radioiodination of NR-LU-10 in
  • Group 1 Time 0, inject 100 ⁇ g 125 I-labeled, biotinylated NR-LU-10
  • 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-step pretargeting protocol (described for Group 1, above) was then examined. More specifically, tumor uptake of the 186 Re-chelate-biotin conjugate in the presence or absence of biotinylated antibody and avidin was determined. In the absence of biotinylated antibody and avidin, 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. In contrast, at 2 h post- injection in the presence of biotinylated antibody and avidin (specific) , 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. Further, the specifically bound
  • 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.
  • Rhenium tumor uptake via the three-step pretargeting protocol was compared to tumor uptake of the same antibody radiolabeled through chelate covalently attached to the antibody (conventional procedure) .
  • the results of this comparison are depicted in Figure 2.
  • Blood clearance and tumor uptake were compared for the chelate directly labeled rhenium antibody conjugate and for the three-step pretargeted sandwich. Areas under the curves (AUC) and the ratio of AUC tumor /AUC jDloocj were determined.
  • 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.
  • a clear increase in the targeting ratio tumor exposure to radioactivity:blood exposure to radioactivity-- AUC tumor :AUC j:) - ] . ood
  • was observed for three-step pretargeting approximately 7:1) in comparison to the direct labeled antibody approach (approximately 2.4:1) .
  • Neutral MAMA Chelate/Conjugate 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 gastrointestinal routes. For instance, neutral compounds are generally cleared by the kidneys; 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 derivitized 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 interaction.
  • Polylysine derivitization is accomplished by standard methodologies. Briefly, 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) . Alternative methodology involves anhydrous conditions using nitrophenyl esters in DMSO and triethyl amine. The resultant conjugates are characterized by UV and NMR spectra.
  • 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.
  • the radiometal-chelate-biotin 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 ⁇ -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.
  • R - a sugar such as ribos ⁇ or glucose or SCfe
  • This compound is synthesized according to the standard reaction procedures. Briefly, 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. Subsequent cleavage of the N-t- BOC group with TFA and condensation with ligand NHS ester in DMF with triethylamine provides ligand- amidoserine (O-sulfonate or O-glucose)biocytinamide.
  • Radioiodinated biotin derivatives prepared by exposure of poly-L-lysine to excess NHS-LC-biotin and then to Bolton-Hunter N-hydroxysuccinimide esters in DMSO has been reported. After purification, this product was radiolabeled by the iodogen method (see, for instance, Del Rosario et al . , J. Nucl . Med. , 32 :5, 1991, 993 (abstr.)) . Because of the high molecular weight of the resultant radioiodinated biotin derivative, only limited characterization of product (i.e., radio-HPLC and binding to immobilized streptavidin) was possible.
  • radioiodinated biotin is a low molecular weight compound that is amenable to complete chemical characterization.
  • the disclosed methods for preparation involve a single step and eliminate the need for a purification step.
  • "X" may be any radiohalogen, including 125 I, 131 I, 123 I, 211 At and the like.
  • Preparation of 1 was generally according to Wilbur et al., J. Nucl. Med.. 3_0: 16- 6, 1989, using a 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. When the reaction of 1.
  • the reaction was extremely clean and complete when a suspension of _____ 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.
  • Both iodobiotin derivatives 2. exhibited ⁇ 95% binding to immobilized avidin. Incubation of the products 2 with mouse serum resulted in no loss of the ability of 2 . to bind to immobilized avidin. Biodistribution studies of 2. in male BALB/c mice showed rapid clearance from the blood (similar to 186 Re-chelate-biotin conjugates described above) .
  • the radioiodobiotin 2 had decreased hepatobiliary excretion as compared to the 186 Re-chelate-biotin conjugate; urinary excretion was increased as compared to the 186 Re-chelate-biotin conjugate.
  • Analysis of urinary metabolites of 2 indicated deiodination and cleavage of the biotin amide bond; the metabolites showed no binding to immobilized avidin.
  • metabolites of the 186 Re-chelate-biotin conjugate appear to be excreted in urine as intact biotin conjugates. Intestinal uptake of 2. is ⁇ 50% that of the 186 Re-chelate-biotin conjugate.
  • 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 biotinylation of the polylysine.
  • Certain antibodies have available for reaction endogenous sulfhydryl groups. If the antibody to be biotinylated contains endogenous sulfhydryl groups, such antibody is reacted with N-iodoacetyl-n' -biotinyl hexylene diamine (as described in Example IV.A. , above) .
  • N-iodoacetyl-n' -biotinyl hexylene diamine as described in Example IV.A. , above.
  • DTT reducing agent
  • 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. As with endogenous sulfhydryl groups (discussed above) , the detrimental effects of reducing agents on antibody are thereby avoided.
  • a NR-LU-13-avidin conjugate is prepared as follows. Initially, avidin is derivitized with N- succinimidyl 4- (N-maleimidomethyl) cyclohexane-1- carboxylate (SMCC) . 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 characterized component as the minor product.
  • SMCC N- succinimidyl 4- (N-maleimidomethyl) cyclohexane-1- carboxylate
  • 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 ⁇ c- 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.
  • 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: administering a ligand- or anti-ligand-targeting moiety conjugate to a recipient; after a time sufficient for localization of the administered conjugate to the target site, withdrawing blood from the recipient by, for example, plasmapheresis; separating cellular element from said blood to produce a serum fraction and returning the cellular elements to the recipient; and reducing the titer of the administered conjugate in the serum fraction to produce purified serum; infusing the purified serum back into the recipient.
  • 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 preferably constitutes a biodegradable polymeric carrier having a plurality of biotin molecules bound thereto.
  • 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 characteristics: - 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;
  • 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, nonenzymatic, hydrolytic scissioning. Polymers derived from the condensation of alpha hydroxycarboxylic acids and related lactones are more preferred for use in the present invention. A particularly preferred moiety is formed of a mixture of thermoplastic polyesters (e.g., polylactide or polyglycolide) or a copolymer of lactide and glycolide components, such as poly(lactide-co-glycolide) .
  • An exemplary structure, a random poly(DL-lactide-co- glycolide) 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, 18.:293, 1980) and polyorthocarbonates (U.S. Patent No. 4,093,709) and the like.
  • Preferred lactic acid/glycolic acid polymer containing matrix particulates of the present invention are prepared by emulsion-based processes, that constitute modified solvent extraction processes such as those described by Cowsar et al. , "Poly(Lactide-Co-Glycolide) Microcapsules for Controlled Release of Steroids," Methods Enzvmology.
  • 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.
  • particulate clearing agents 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 conditions.
  • 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. Advantages garnered through the use of particulate clearing agents of the type described above are as follows:
  • 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
  • - 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. Clearance of NR-LU-13-avidin may also be accelerated by an arterially inserted proteinaceous or polymeric multiloop device.
  • a catheter-like device consisting of thin loops of synthetic polymer or protein fibers derivitized 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 si tu 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.
  • An alternative procedure for clearing NR-LU-13- avidin from the circulation without induction of internalization involves administration of 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 complexes are readily cleared from the circulation via the RES.
  • biotin molecules or chelate-biotin conjugates
  • avidin crosslinking induces internalization of crosslinked complexes at the target cell surface.
  • Biotinylated NR-CO-04 (lysine) is prepared according to the methods described in Example IV.A. , above. 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. At such time, 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. Thus, 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) .
  • avidin for blood clearance and to form the middle layer of the sandwich at the target cell-bound biotinylated antibody
  • a methotrexate-biotin conjugate is administered.
  • 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) .
  • avidin for blood clearance and to form the middle layer of the sandwich at the target cell-bound biotinylated antibody
  • LU-05 is further covalently linked to methotrexate. Subsequent administration of avidin induces internalization of the complex and enhances intracellular delivery of drug to the tumor target cell.
  • 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.
  • 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) . C. Conjugation of SMCC-streptavidin to DTT- reduced NR-LU-10.
  • DTT-reduced NR-LU-10 (63 mg, 29 ml, 0.42 ⁇ mol) was diluted with 44.5 ml PBS.
  • the solution of SMCC-streptavidin (28 mg, 17 ml, 0.42 ⁇ mol) was added rapidly to the stirring solution of NR-LU-10.
  • Total protein concentration in the reaction mixture was 1.0 mg/ml.
  • the progress of the reaction was monitored by HPLC (Zorbax ® GF-250, available from
  • monosubstituted or disubstituted adduct is isolatable using DEAE ion exchange chromatography.
  • free streptavidin was removed therefrom by eluting the column with 2.5% xylitol in sodium borate buffer, pH 8.6.
  • the bound unreacted antibody and desired conjugate were then sequentially eluted from the column using an increasing salt gradient in 20 mM diethanolamine adjusted to pH 8.6 with sodium hydroxide.
  • Conjugates to be evaluated were not boiled in sample buffer containing SDS to avoid dissociation of streptavidin into its 15 kD subunits. Two product bands were observed on the gel, which correspond to the mono- and di- substituted conjugates.
  • Immunoreactivity was assessed, for example, by competitive binding ELISA as compared to free antibody. Values obtained were within 10% of those for the free antibody.
  • Biotin binding capacity was assessed, for example, by titrating a known quantity of conjugate with p- [1-125] iodobenzoylbiocytin. Saturation of the biotin binding sites was observed upon addition of 4 equivalences of the labeled biocytin.
  • 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.
  • 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:
  • Time 0 inject i.v. 200 ⁇ g NR-LU-10-StrAv conjugate
  • Time 24-48 h inject i.v. 60-70 fold molar excess of radiolabeled biotinyl molecule
  • perform biodistributions at 2, 6, 24, 72, 120 hours after injection of radiolabeled biotinyl molecule %
  • NR-LU-10-streptavidin has shown very consistent patterns of blood clearance and tumor uptake in the LS-180 animal model.
  • a representative profile is shown in Figure 4.
  • PIP-BT or Re-BT is administered after allowing the LU-10-StrAv conjugate to localize to target cell sites for at least 24 hours, the tumor uptake of therapeutic radionuclide is high in both absolute amount and rapidity.
  • 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.
  • Uptake values of about 20% ID/G were achieved at no- carrier added (high specific activity) doses of radiolabeled biotinyl molecules. At less than saturating doses, circulating LU-10-StrAv was observed to bind significant amounts of administered radiolabeled biotinyl molecule in the blood compartment.
  • 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.
  • A. Derivitization of orosomucoid 10 mg human orosomucoid (Sigma N-9885) was dissolved in 3.5 ml of pH 5.5 0.1 M sodium acetate buffer containing 160 mM NaCl. 70 ⁇ l of a 2% (w/v) CaCl solution in deionized (D.I.) water was added and 11 ⁇ l of neuraminidase (Sigma N-7885) , 4.6 U/ml, was added. The mixture was incubated at 37°C for 2 hours, and the entire sample was exchanged over a Centricon-10 ® ultrafiltration device (available from Amicon, Danvers, Massachusetts) with 2 volumes of PBS. The asialoorosomucoid and orosomucoid starting material were radiolabeled with 1-125 using PIP technology, as described in Example IV above.
  • 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.
  • two animals receiving each compound were sacrificed at 5 minutes post-administration and limited biodistributions were performed. These results are shown in Figure 6.
  • Clearing agent 200 ⁇ l PBS - group 1; 400 ⁇ g non-biotinylated asialoorosomucoid - group 2; 400 ⁇ g biotinylated asialoorosomucoid - group 3; and 200 ⁇ g biotinylated asialoorosomucoid- group 4) was administered at 25 hours following conjugate administration.
  • 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 purification on a G-25 PD-10 column.
  • mice from each group were serially bled, as described above, at 0.17, 1, 4 and 25 hours (pre- injection 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 complexation with biotinylated asialoorosomucoid.
  • asialoorosomucoid-biotin was highly effective at reducing blood levels of circulating streptavidin-containing conjugate by an in vivo complexation that was dependent upon biotin-avidin interaction.
  • Example XIV Tumor Uptake of PIP-Biocytin PIP-Biocytin, as prepared and described in Example VII above, was tested to determine the fate thereof in vivo.
  • the following data are based on experimentation with tumored nude mice (100 mg LS-180 tumor xenografts implanted subcutaneously 7 days prior to study) that received, at time 0, 200 ⁇ g of 1-125 labeled NR-LU-10- Streptavidin conjugate (950 pmol) , as discussed in Example XI above. At 24 hours, the mice received an i.v.
  • 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 is indicative of streptavidin saturation as well. In contrast, the two lowest doses produced lower absolute tumor localization of PIP- biocytin, despite equivalent localization of NR-LU-10- Streptavidin conjugate (tumors in all groups averaged about 40% ID/G for the conjugate) .
  • the lowest dose group (0.5 ⁇ g) exhibited high efficiency tumor delivery of PIP-I-131-biocytin, which efficiency increased over time.
  • 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.
  • the decrease in uptake on a % ID/G basis at the 168 hour time point resulted from significant growth of the tumors between the 120 and 168 hour time points.
  • 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 appeared insufficient to account for this phenomenon. The ratio of PIP-Biocytin to LU-10-StrAv in the tumor increased continually during the experiment, while the ratio in the blood decreased continually.
  • 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) . Consequently, an increase in radiation dose to tumor was achieved.
  • the critical step is the intermolecular cyclization between the bis-NHS ester and the diamine to give the cyclized dodecane.
  • McMurry et al. conducted the cyclization step on a 140 mmol scale, dissolving each of the reagents in 100 ml DMF and adding via a syringe pump over 48 hours to a reaction pot containing 4 liters dioxane.
  • a 5x scale-up of the McMurry et al. procedure was not practical in terms of reaction volume, addition rate and reaction time. Process chemistry studies revealed that the reaction addition rate could be substantially increased and that the solvent volume could be greatly reduced, while still obtaining a similar yield of the desired cyclization product.
  • each of the reagents was dissolved in 500 ml DMF and added via addition funnel over 27 hours to a reaction pot containing 3 liters dioxane.
  • the addition rate of the method employed involved a 5.18 mmol/hour addition rate and a 0.047 M reaction concentration.
  • the D-alanine-linked conjugate was prepared by first coupling D-alanine (Sigma Chemical Co.) to biotin-NHS ester. The resultant biotinyl-D-alanine was then activated with 1- (3-dimethylaminopropyl) -3- ethyl-carbodiimide hydrochloride (EDCI) and N- hydroxysuccinimide (NHS) .
  • This NHS ester was reacted in si tu with DOTA-aniline to give the desired product which was purified by preparative HPLC.
  • Biotinyl-D- alanine was obtained as a white solid (130 mg, 0.41 mmol) in 47% yield.
  • NHS (10 mg, 0.08 mmol) and EDCI (15 mg, 0.07 mmol) were added to a solution of biotinyl-D-alanine (27 mg, 0.08 mmol) in DMF (1 ml) .
  • the solution was stirred at 23 * C for 60 hours, at which time TLC analysis indicated conversion of the carboxyl group to the N- hydroxy succinimidyl ester.
  • Pyridine 0.8 ml was added followed by DOTA-aniline (20 mg, 0.04 mmol) .
  • the mixture was heated momentarily at approximately 100 * C, then cooled to 23 * C and evaporated.
  • the product, DOTA-aniline-D-alanyl-biotinamide was purified by preparative HPLC.
  • the residue was diluted with 150 ml of ethyl acetate and washed first with 5% aqueous sodium sulfite (2 x 100 ml) and then with 100 ml of 1 N aqueous hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated to afford a yellow oily residue.
  • the residue was diluted with 250 ml of methanol and then hydrogen chloride (gas) was rapidly bubbled into the mixture for 2-3 minutes. The resultant mixture was refluxed for 18 hours, cooled and concentrated.
  • the residue was diluted with 150 ml of methanol and washed with hexane (3 x 150 ml) to remove mineral oil previously introduced with NaH. The methanol phase was concentrated to afford 4.91 g of the product as a yellow oil (86%) :
  • NMR assay of an aliquot indicated the reaction to be incomplete. Consequently, an additional 1.00 g (4.8 mmol) of methyl 6-bromocaproate was added and the mixture stirred at reflux for 26 hours. NMR assay of an aliquot indicated the reaction to be incomplete. An additional 1.0 g of methyl 6-bromocaproate was added and the mixture stirred at reflux for 24 hours. NMR assay of an aliquot indicated the reaction to be near complete. The mixture was cooled and then directly filtered through celite. The solids were rinsed with 100 ml of tetrahydrofuran. The filtrates were combined and concentrated.
  • the residue was diluted with 100 ml of methanol and washed with hexane (3x 100 ml) to remove the mineral oil introduced with the sodium hydride.
  • the methanol phase was treated with 6 ml of 10 N aqueous sodium hydroxide and stirred at 15- 25 * C for 3 hours.
  • the mixture was concentrated.
  • the residue was diluted with 100 ml of deionized water and acidified to pH 2 with concentrated HCI.
  • the mixture was washed with ether (3 x 100 ml) .
  • the aqueous phase was concentrated, diluted with 200 ml of dry methanol and then hydrogen chloride gas was bubbled through the mixture for 2-3 minutes. The mixture was stirred at 15-25"C for 3 hours and then concentrated.
  • N-hydroxysuccinimidyl biotin in 15 ml of dry dimethyl- formamide was added 600 mg (1.94 mmol) of N,N-bis- (6-methoxy carbonylhexyl) amine hydrochloride followed by 1.0 ml of triethylamine.
  • the mixture was stirred at 80-85 * C for 3 hours and then cooled and concentrated.
  • the residue was chromatographed on silica gel, eluting with 20% methanol/ethyl acetate, to afford 620 mg of the product as a near colorless oil (85%) : H-NMR (CDC1 3 ) 5.71 (IH, s) , 5.22 (IH, s) , 4.52
  • N-methyl glycine-linked DOTA-biotin conjugate was prepared by an analogous method to that used to prepare D-alanine-linked DOTA-biotin conjugates.
  • N- methyl-glycine trivial name sarcosine, available from Sigma Chemical Co.
  • N-methyl-glycyl biotin was then activated with EDCI and NHS.
  • the resultant NHS ester was not isolated and was condensed in si tu with DOTA- aniline and excess pyridine.
  • the reaction solution was heated at 60 * C for 10 minutes and then evaporated.
  • the biotin carboxyl group is reduced with diborane in THF to give a primary alcohol.
  • Tosylation of the alcohol with tosyl chloride in pyridine affords the primary tosylate.
  • Aminobenzyl DOTA is acylated with trifluoroacetic anhydride in pyridine to give (N- trifluoroacetyl)aminobenzyl-DOTA.
  • Deprotonation with 5.0 equivalents of sodium hydride followed by displacement of the biotin tosylate provides the (N- trifluoracetamido-N-descarboxylbiotinyl)aminobenzyl- DOTA.
  • Acidic cleavage of the N-trifluoroacetamide group with HCI (g) in methanol provides the amine- linked DOTA-biotin conjugate.
  • mice Female BALB/c mice (20-25 g) .
  • 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 1-131 or 1-125 using procedures described herein.
  • PIP-biocytin tumor localization was inhibited at higher doses of AO-Bt. This effect is most likely due to reprocessing and distribution to tumor of biotin used to derivatize AO-Bt.
  • Optimal tumor to blood ratios (% injected dose of radiolabeled ligand/gram weight of tumor divided by % injected dose of radioligand/gram weight of blood were achieved at the 50 microgram dose of AO-Bt.
  • Biodistributions conducted following completion of the protocols employing a 50 microgram AO-Bt dose revealed low retention of radiolabel in all non-target tissues (1.2 pmol/g in blood; 3.5 pmol/gram in tail; 1.0 pmol/g in lung; 2.2 pmol/g in liver; 1.0 pmol/g is spleen; 7.0 pmol/g in stomach; 2.7 pmol/g in kidney; and 7.7 pmol/g in intestine) .
  • these results indicate effective decoupling of the PIP-biocytin biodistribution from that of the MAb- StrAv at all sites except tumor.
  • HSA Human Serum Albumin
  • Unreacted biotin reagent was removed from the biotin-derivatized HSA using G-25 size exclusion chromatography.
  • the crude material may be directly galactosylated.
  • the same chemistry is applicable for biotinylating non-previously biotinylated dextran.
  • 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-l-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.
  • the galactosylation of biotinylated HSA began by initial evaporation of the anhydrous methanol from a 300 fold molar excess of reactive thioimidate.
  • 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 procedure therefor described above in the discussion of HSA-based clearing agents, at a level of about 9 galactoses/molecule. The molar equivalence offering ratio of galactose to HSA was about 300:1, with about one-third of the galactose being converted to active form.
  • GAL- DEX-BT galactose-dextran-biotin
  • biotin G-HSA-B clearing agent is both effective at clearing MAb-StrAv over a broader range of doses (potentially eliminating the need for patient-to-patient titration of optimal dose) and appears to release less competing biotin into the systemic circulation than the same agent having a higher biotin loading level.
  • 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 characteristics set forth above.
  • 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.
  • the heterocyclic ring becomes protonated.
  • the ring protonation prevents the catabolite from exiting the lysosome.
  • 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.
  • 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.
  • a patient presents with ovarian cancer.
  • the MAb-streptavidin conjugate is administered to the patient in an amount sufficient to substantially saturate the available antigenic sites at the target (which amount is at least sufficient to allow the capture of a therapeutically effective radiation dose at the target and which amount may be in excess of the maximum tolerated dose of conjugate administrable in a targeted, chelate-labeled molecule protocol, such as administration of monoclonal antibody-chelate- radionuclide conjugate) .
  • the MAb-streptavidin so administered is permitted to localize to target cancer cells for 24-48 hours.
  • an amount of a clearing agent consisting of human serum albumin, exposed galactose residues and 2' -thiobiotin molecules is administered in an amount sufficient to clear non- targeted MAb-streptavidin conjugate.
  • a biotin-radionuclide chelate conjugate of the type discussed in Example XV(F) above is radiolabeled with Y-90 as set forth below.
  • Carrier free 90 YC1 3 (available from NEN-DuPont, Wilmington, Delaware) at 20-200 ⁇ l in 0.05 N HCI was diluted with ammonium acetate buffer (0.5M, pH 5) to a total volume of 0.4 ml.
  • 50 ⁇ l (500 mg/ml) of ascorbic acid and 50-100 ⁇ l (10 mg/ml) of DOTA-biotin were added to the buffered ⁇ U YC1 3 solution.
  • the mixture was incubated for one hour at 80°C. Upon completion of the incubation, 55 ⁇ l of 100 mM DTPA was added to the mixture to chelate any unbound 90 Y.
  • the final preparation was diluted to 10 ml with 0.9% NaCl.
  • the radiolabeled DOTA-biotin conjugate is administered to the patient in a therapeutically effective dose at a time point 1-4 hours post-clearing agent administration.
  • the biotin-radionuclide chelate conjugate localizes to targeted MAb-streptavidin or is substantially removed from the patient via the renal pathway.
  • An amount of asialoorosomucoid sufficient to substantially saturate galactose receptors of the patient's hepatocytes is administered in a single or multiple doses. Additional administrations of asialoorosomucoid are conducted from time to time during the protocol to maintain substantial saturation of the galactose receptors for a time sufficient to permit localization of a subsequently administered monoclonal antibody-streptavidin conjugate to target cell sites, e.g., from 18-72 hours.
  • the MAb-streptavidin conjugate is administered to the patient in an amount sufficient to substantially saturate the available antigenic sites at the target (which amount is at least sufficient to allow the capture of a therapeutically effective radiation dose at the target and which amount may be in excess of the maximum tolerated dose of conjugate administrable in a targeted, chelate-labeled molecule protocol, such as administration of monoclonal antibody-chelate-radionuclide conjugate) .
  • the MAb- streptavidin so administered is permitted to localize to target cancer cells for 20-48 hours. At this time, the MAb-streptavidin conjugate is cleared via the galactose receptors of hepatocytes, because such receptors have processed the asialoorosomucoid blocking agent.
  • biotin-radionuclide chelate conjugate of the type discussed in Example XV(F) above is radiolabeled with Y-90 as set forth in Example XVII above.
  • the radiolabeled DOTA-biotin conjugate is administered to the patient in a therapeutically effective dose.
  • the biotin-radionuclide chelate conjugate localizes to targeted MAb-streptavidin or is substantially removed from the patient via the renal pathway.
  • conjugates were synthesized using biotin rather than a "low affinity" biotin analog and galactose residues. These conjugates were synthesized using different numbers of attached galactose residues. In addition, these conjugates contained either a long chain linker (LC) or the short chain linker (SC) as depicted below: The conjugates which were synthesized are depicted below:
  • the (galactosyl) g -LC-biotin conjugate was also compared to galactosylated-HSA-biotin in a Balb/C mouse model for its ability to clear a 1-125 LU-10- streptavidin conjugate from the circulation as a function of time. These results are shown in Figures 16 and 17. These results indicate that the (galactosyl)g-LC-biotin conjugate is comparable to galactosylated-HSA-biotin in its ability to clear the streptavidin containing conjugate from the circulation. Subsequent experiments have further shown that conjugates containing 16 galactose residues provide for even better clearance than those containing 8 galactose residues.
  • This example describes a stepwise procedure by which an exemplary small molecular weight clearing agent, hexadeca-galactosyl biotin may be prepared.
  • This example is exemplary of small molecule clearing agents which may be prepared according to the present invention. Additionally, this synthetic scheme is depicted schematically below:
  • NCOCF j toluene sulfonyl chloride > ⁇ NC ⁇ f ⁇ n ⁇ ⁇ r t trriiaetthhuyliaammiinnae, m maetthhuyllaennae chloride
  • the mixture was cooled, diluted with 150 mL of 1 N aq HCI and then extracted with ethyl acetate (3 x 100 mL) .
  • the organic extracts were combined, dried over magnesium sulfate, filtered and concentrated.
  • the residue was diluted with 200 mL of methanol and then treated with 30 mL of 10 N aqueous sodium hydroxide.
  • the mixture was stirred at room temperature for 18 h and then concentrated.
  • the solution was washed with diethyl ether (3 x 100 mL) .
  • the aqueous phase was concentrated.
  • the product was initially free based by addition of 10 N aq sodium hydroxide, to a pH of 9-9.5, and then by addition of 70 g of AG 1 X-8 anion exchange resin (hydroxide form) and allowing the solution to stir for 2 h.
  • the resin was filtered off and washed with 150 mL of deionized water.
  • the aqueous filtrates were combined and concentrated.
  • the residue was diluted with 200 mL of 2-propanol and filtered.
  • the collected solids were rinsed with 100 mL of 2-propanol.
  • the organic filtrates were combined and concentrated.
  • the residue was chromatographed on C-18 reverse phase silica gel, eluting first with 20:80:0.1 acetonitrile/water/trifluoroacetic acid and then with 50:50:0.1 acetonitrile/water/trifluoroacetic acid. The fractions containing product (2 ) were combined and concentrated. The residue was diluted with 40 mL of water and 20 mL of acetonitrile.
  • the residue was chromatographed on C-18 reverse phase silica gel, eluting first with 60:40 methanol/water and then with 75:25 methanol/water.
  • the fractions containing the product (1J9) were combined, concentrated and rechromatographed on C-18 reverse phase silica gel, eluting first with 40:60:0.1 acetonitrile/water/trifluoroacetic acid and then with 50:50:0.1 acetonitrile/water/trifluoroacetic acid.
  • the fractions containing the product (1£) were again combined and concentrated.
  • the residue was dissolved in 20 mL of water.
  • This example demonstrates the in vivo efficacy of the subject small molecule clearing agents containing galactose residues and biotin and specifically the (gal) 16 -BT clearing agent for providing for clearance of conjugates during therapeutic pretargeting methods.
  • the protocol of these experiments comprised the evaluation of biodistribution of 1:L1 In-DOTA-biotin at 1 ⁇ g dose from 2 - 120 hr in SW-1222 tumored nude mice.
  • Four hundred ⁇ g* of LU-10/SA was administered I.V., and 46 ⁇ g of (GAD- ⁇ g-BT was administered 24 hr later.
  • At 27 hr post-MAb/SA 11:L In-DOTA-biotin was administered.
  • the second protocol conducted at the saturating 15 ⁇ g biotin dose further demonstrated that the (GAL) 16 - BT clearing agent does not accrete heavily in tumor at the 2 hr post-DOTA-BT time point.
  • a mole ratio of 2.65:1 DOTA-BT:MAb/SA was attained indicating that although not the ideal 4:1 biotins/streptavidin attained with BT-HSA-gal, the (GAL) 16 -BT did not compromise tumor appreciably by localizing to pretargeted conjugate.
  • the (GALj- j ⁇ g-BT clearing agent used does not have a stabilized biotin linkage. Therefore it may release BT quickly post- hepatic processing potentially blocking some of the prelocalized SA by 3 hr when the 111 In-DOTA-biotin is administered.
  • the (GAL) lg -BT is apparently being effectively cleared by dual processes of renal excretion and hepatic uptake via the Ashwell receptor. It is additionally hypothesized that the (GAL) 16 -BT clustered galactose sugars may bind more strongly to the Ashwell receptor than the gal-HSA-BT clearing agent because the array of galactose on (GAL) 16 -BT may be better matched to the structure of the Ashwell receptor.
  • Example XXII Experiments were designed and executed to evaluate a particular small molecule biotin-galactose construct: (gal) 16 -biotin (structure 1 shown in Fig. 18) .
  • mice Separate groups of mice were injected with either 120 or 12 ⁇ g of radiolabeled which had been precomplexed with (gal) 16 -biotin by mixing the biotin analog at a 20-fold molar excess with the antibody conjugate, and purifying the excess small molecule from the protein by size-exclusion chromatography. As shown in Fig. 19, both doses of precomplexed conjugate showed extremely rapid clearance from the blood, relative to the antibody conjugate control.
  • mice received 400 ⁇ g of 1-125 LU-10/streptavidin (LU-IO/SA) i.v., and approximately 22 hours later, received (gal) 16 -biotin i.v. at doses of 100, 50 or 10 : 1 molar excess to circulating LU-10/SA.
  • Fig. 20 shows the blood clearance of conjugate in each group. While it is apparent that each dose was effective at clearing conjugate, the most effective dose (both kinetic and absolute) was the 10 : 1 (45 ⁇ g) dose.
  • mice bearing either SW-1222 (colon) tumor xenografts or SHT-1 (SCLC) tumor xenografts were pretargeted with LU-IO/SA and, 22 hours later, received 46 ⁇ g of (gal)--_g-biotin. After 2 hours 90 Y- DOTA-biotin was administered and its uptake and retention in tumor and non-target tissues was evaluated by sacrifice and tissue counting for radioactivity 2 hours after administration.
  • liver levels were lower and equivalent to those seen with the HSA clearing agent (-1% ID/g) .
  • kits containing one or more of the components described above are also contemplated.
  • radiohalogenated biotin may be provided in a sterile container for use in pretargeting procedures.
  • a chelate-biotin conjugate provided in a sterile container is suitable for radiometallation by the consumer; such kits would be particularly amenable for use in pretargeting protocols.
  • radiohalogenated biotin and a chelate-biotin conjugate may be vialed in a non-sterile condition for use as a research reagent.

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Abstract

L'invention concerne des procédés, composés, compositions et trousses permettant l'apport préalablement ciblé d'agents diagnostiques et thérapeutiques. Cette invention se rapporte en particulier à des procédés permettant un marquage radiométallique de la biotine, ainsi qu'à des composés associés. Sont également décrits des agents et des mécanismes de clairance.
EP95904859A 1993-12-07 1994-12-07 Procede et composes permettant un ciblage prealable Withdrawn EP0743956A4 (fr)

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US6908903B1 (en) 1994-12-07 2005-06-21 Aletheon Pharmaceuticals, Inc. Cluster clearing agents
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US6432699B1 (en) 1997-03-28 2002-08-13 New York University Viral vectors having chimeric envelope proteins containing the IgG-binding domain of protein A
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RU2013133813A (ru) 2010-12-21 2015-01-27 Конинклейке Филипс Электроникс Н.В. Средства для выведения биомолекул из кровотока

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CA2178477A1 (fr) 1995-06-15
WO1995015978A1 (fr) 1995-06-15
JPH09506106A (ja) 1997-06-17
EP0743956A4 (fr) 1999-03-24

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