EP0906015A1 - Agents de suppression de la retention hepatique - Google Patents

Agents de suppression de la retention hepatique

Info

Publication number
EP0906015A1
EP0906015A1 EP97926844A EP97926844A EP0906015A1 EP 0906015 A1 EP0906015 A1 EP 0906015A1 EP 97926844 A EP97926844 A EP 97926844A EP 97926844 A EP97926844 A EP 97926844A EP 0906015 A1 EP0906015 A1 EP 0906015A1
Authority
EP
European Patent Office
Prior art keywords
component
liver retention
liver
binding
clearing agent
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Withdrawn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
EP97926844A
Other languages
German (de)
English (en)
Other versions
EP0906015A4 (fr
Inventor
Louis J. Theodore
Donald B. Axworthy
John M. Reno
Eric K. Yau
Linda M. Gustavson
Alan R. Fritzberg
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 EP0906015A1 publication Critical patent/EP0906015A1/fr
Publication of EP0906015A4 publication Critical patent/EP0906015A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6893—Pre-targeting systems involving an antibody for targeting specific cells clearing therapy or enhanced clearance, i.e. using an antibody clearing agents in addition to T-A and D-M

Definitions

  • the present invention relates to liver retention clearing agents (LRCAs), reagents for the preparation thereof and associated methods and compositions.
  • LRCAs impact the elimination and biodistribution of constructs that directly or indirectly become associated with, e.g., incorporate, such agents in a manner resulting in increased elimination via a hepatic route without release of certain metabolites from the liver.
  • the LRCA-associated constructs also generally exhibit a decreased serum half-life in comparison to counterpart compounds which do not incorporate or become associated with LRCAs.
  • a targeting moiety is formed of a targeting agent and a receptor.
  • the active agent is associated with a ligand for the receptor.
  • the targeting moiety is administered to a recipient, and permitted to localize to the target site with binding at that site mediated by the targeting agent.
  • the active agent-ligand is administered The ligand component ofthe construct binds to the pretargeted receptor, thereby delivering the active agent to the target
  • Pretargeting is made more efficient by administration of a clearing agent to facilitate elimination of circulating targeting moiety
  • Various clearing agents have been disclosed
  • Galactose-human serum albumin (HSA)-b ⁇ ot ⁇ n clearing agents have been used in pretargeting protocols employing a monoclonal antibody-streptavidin targeting moiety and a biotm-active agent construct
  • Such clearing agents are discussed in PCT US93/05406 Denvatization by galactose facilitates elimination of complexes of monoclonal antibody-streptavidin-biotin-HSA-galactose via Ashwell receptors in the liver
  • These clearing agents rapidly decrease circulating monoclonal antibody-streptavidin levels in patients Since pretargeting methods are enhanced using clearing agents, improvements in such clearing agents are sought
  • the present invention is directed to liver retention clearing agents (LRCAs) which are designed to reduce the level of serum-associated targeting moiety-anti- gand complex
  • LRCAs ofthe present invention are also designed to prevent release of certain metabolites, ⁇ _e_, metabolites bearing a ligand or an anti- ligand binding component respectively capable of binding pretargeted anti-hgand or ligand receptor
  • LRCAs of the present invention are preferably capable of achieving circulating targeting moiety clearance without compromising the binding potential of the pretargeted targeting moiety, either directly by binding ofthe LRCA thereto or indirectly by binding of LRCA metabolites thereto
  • Preferred LRCAs of the present invention also preferably exhibit one or more of the following characteristics
  • LRCAs of the present invention are operability over a wide LRCA dose range to avoid extensive dose optimization.
  • Preferred LRCAs of present invention incorporate (1) a hepatic clearance directing component; (2) a binding component; (3) a liver retention component associated with the binding component to promote liver retention of metabolites of LRCA constructs containing ligand or anti-ligand; and (4) a structural component.
  • the structural component serves as a scaffold for binding of the hepatic clearance directing component, the liver retention component and/or the binding component.
  • the binding component is attached to the structural component through the liver retention component. This construction facilitates the formation of LRCA metabolites containing both the binding component and the liver retention component.
  • Hepatic clearance directing components of LRCAs ofthe present invention promote clearance of moieties to which they are attached to the liver.
  • Preferred hepatic clearance directing components include sugar residues recognized by hepatocyte receptors.
  • Preferred LRCAs ofthe present invention incorporate from about 15 to about 60 sugar residues, with from about 25 to about 50 residues preferred.
  • preferred sugars include galactose and N-acetylgalactosamine.
  • the structural component is derivatized with an appropriate number of sugar residues.
  • Liver retention components ofthe present invention are designed to prevent release of binding component (such as ligand or anti-ligand)-containing LRCA metabolites to the serum compartment in a manner allowing those metabolites to accrete to pretargeted receptors.
  • binding component such as ligand or anti-ligand
  • liver retention components of the present invention are characterized by at least one ofthe following attributes: 1) Resistance to agents that cleave peptide bonds or otherwise promote catabolism of LRCA-containing moieties; 2) Retention in the cytoplasmic or a subcellular compartment following internalization into hepatic cells; or 3) Excretion upon metabolism without re-entry, or with delayed or retarded re ⁇ entry, into the serum compartment ( jj., biliary excretion without reabsorption in the intestine).
  • Protease-resistant liver retention components of the present invention are useful,
  • the ligand or anti-ligand binding component ofthe LRCA is generally retained in i ⁇ hepatocytes for a time sufficient to allow active agent-ligand or active agent-anti- ligand construct to reach and bind to the pretargeted receptor therefor.
  • a protease-resistant liver retention agent is a poly-amino acid of unnatural (D) orientation.
  • Preferred liver retention components of this type are linear chains of from about 2 to about 12 amino acids and the like.
  • 15 resistant liver retention component incorporates at least one tertiary amide (-CO-
  • N(R)-) bond wherein R is preferably lower alkyl.
  • bonds are more highly resistant to hydrolytic enzymatic activity (e.g., biotinidase activity) than secondary amide (peptide, -CO-NH-)bonds.
  • hydrophobic cellular or subcellular membranes also afford enhanced retention of binding component-containing LRCA metabolites in hepatocytes. Consequently, metabolites having the potential to bind to pretargeted receptor are retained in the liver for a time sufficient to permit later administration and accretion to targeted receptor of active agent-containing conjugate. Diffusion-restricted liver retention
  • liver retention components are moieties characterized by positive charge, negative charge or neutral charge combined with hydrophilicity.
  • Preferred liver retention components of this type are saccharides (neutral charge/hydrophilic),
  • Liver retention component-binding component metabolites that are characterized by relatively rapid excretion, without passage into the serum compartment, are also useful in the practice ofthe present invention. Metabolites excreted by a hepatobiliary route without reabsorption by the intestines are preferred for use in this aspect ofthe present invention. Examples of rapid excretion-liver retention components are pepstatin, 1,4,7, 10-tetraazacyclododecane-N,N',N",N'"-tetra acetic acid (DOT A) and the like. Pepstatin is rapidly excreted into the bile.
  • DOT A 10-tetraazacyclododecane-N,N',N",N'"-tetra acetic acid
  • liver retention components When internalized into hepatocytes, DOTA is generally either trapped therein or excreted via a hepatobiliary route.
  • Another group of moieties useful as liver retention components in the practice ofthe present invention are moieties employed in the prior art to retain radioactivity at tumor target sites. These molecules also afford resistance to enzymatic degradation. Examples of such liver retention components are cellobiose, dilactitol, lysyl-epsilon-amido 5-iodo-3-pyridinecarboxylate, other non-mammalian sugars, other radiolabel residualizing moieties and the like.
  • Liver retention components useful in the present invention may combine more than one ofthe desirable properties set forth above.
  • Preferred LRCAs ofthe present invention are characterized by the following liver retention components:
  • alpha-phosphonomethyl amino acid compounds such as the following:
  • Each of these preferred liver retention components incorporate from about 2 to about
  • liver retention components are polymers of natural amino acids ofthe (L) configuration employed in combination with a tertiary amide of a biotin binding moiety.
  • Binding components of the LRCAs ofthe present invention are moieties which recognize epitopes or components of molecules to be cleared by the LRCAs.
  • Preferred binding components of LRCAs ofthe present invention are members of ligand/anti-ligand pairs or lower affinity forms thereof that facilitate binding to targeting moiety-anti-ligand/ligand conjugate.
  • a preferred ligand/anti-ligand pair for use in the practice of the present invention is biotin-avidin.
  • Preferred LRCAs ofthe present invention incorporate from about 1 to about 10 ligand or anti-ligand molecules, with from about 1 to about 4 more preferred and with from about 1 to about 2 still more preferred.
  • Preferred structural components include proteinaceous and non-proteinaceous materials having sufficient reactive groups for derivatization with hepatic clearance directing components and binding components and/or liver retention components, such as proteins and polymers. More preferred proteinaceous structural components include those expected to elicit little response from a recipient's immune system.
  • human proteins such as human serum albumin, IgG, IgM and the like constitute structural components useful in the practice ofthe present invention.
  • Preferred polymeric structural components ofthe present invention include dextran, hydroxypropylmethylacrylamide (HPMA), hydroxypropylacrylamide, hydroxypropylethylacrylamide, poly-D-lysine, poly-D-glutamate, poly-D-aspartate, dendrimers (spherical constructs having functional units on the exterior thereof) and the like.
  • FIGS. 2a and 2b schematically illustrate the preparation of a liver retention component-binding component construct, N-methyl-N- ⁇ 5'-[methylester tris-(-(D, L)- phosphonoalanyl)-(D)-cystyl]-5-carbamylpentyl ⁇ biotinamide.
  • FIGS 3a and 3b schematically illustrate the preparation of a liver retention component-binding component construct, N-methyl-N-[5-(t ⁇ glutamylcyste ⁇ e)-5- carbamylpentylj-biotinamide
  • 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, gand/receptor, S-protein S-peptide, head activator protein (which binds to itself), cystatin-C/cathepsin B, and biotin/avidin Biotin/avidin is used throughout the specification as a prototypical example of a ligand/anti-ligand pair Anti-ligand
  • 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 is multivalent to bind a larger number of ligands
  • Uruvalent anti- gands are also contemplated by the present invention
  • Anti-ligands ofthe present invention may exhibit or be derivatized to
  • Avidin As defined herein, “avidin” includes avidin, streptavidin and de ⁇ vatives and analogs thereof that are capable of high affinity, multivalent or univalent binding ofbiotm
  • Ligand As defined herein, a "ligand” is a relatively small, soluble molecule that binds with high affinity by anti-ligand and preferably exhibits rapid serum, blood and/or whole body clearance when administered intravenously in an animal or human. Biotin constructs are used as prototypical ligands.
  • 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
  • Radionuclide therapeutic agents are used as prototypical active agent. Attachment of such radionuclide active agents to other moieties, either directly or via chelation technology, may be accomplished as described herein or as known in the art.
  • 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 ofthe 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..).
  • the clearing agent is preferably characterized by physical properties, such as size, charge, reduced affinity, 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.
  • LRCA Liver Retention Clearing Agent
  • Preferred LRCAs of the present invention are characterized by a structural component, a hepatic clearance directing component, a liver retention component and a binding component.
  • Hepatic Clearance Directing Component A plurality of sugar residues recognized by a liver receptor.
  • Hepatic clearance directing components preferably contain from 15 about 60 sugar residues, with from 25 to about 50 sugar residues preferred.
  • the structural component is preferably derivatized with an appropriate number of sugar residues.
  • Liver Retention Component A moiety designed to prevent release of ligand- or anti-ligand-containing metabolites of LRCA constructs to the serum compartment in a manner allowing those metabolites to accrete to pretargeted receptors.
  • Preferable liver retention components ofthe present invention are characterized by at least one of the following attributes :
  • Binding Component A ligand, anti-ligand or other moiety capable of in vivo association with a previously administered molecule (bearing the complementary ligand or anti-ligand, for example) or with another toxic or potentially toxic molecule present in the recipient's circulation or extravascular fluid space via recognition by the binding component of an epitope associated with the previously administered moiety or with the toxic or potentially toxic molecule.
  • Structural Component A moiety which serves as a scaffold for binding of the hepatic clearance directing component, the liver retention component and, optionally, the binding component.
  • Preferred structural components include proteinaceous and non-proteinaceous materials having sufficient reactive groups for such denvatization, such as human proteins and polymers.
  • the LRCAs ofthe present invention are preferably employed in pretargeting protocols.
  • "Two-step" pretargeting procedures feature targeting moiety-ligand or targeting moiety-anti-ligand (targeting moiety-receptor) administration, followed by administration of active agent conjugated to the opposite member of the ligand-anti- ligand pair.
  • a LRCA is administered to facilitate the clearance of circulating targeting moiety-receptor 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.
  • LRCAs ofthe present invention contain a hepatic clearance directing component, a liver retention component, a binding component and a structural component.
  • LRCAs of the present invention are bispecific in that the hepatic clearance directing component mediates interaction with heptocyte receptors and the binding component mediates binding with the moiety to be cleared. These bispecific LRCAs are capable of/// vivo binding or association with molecules to be cleared and interaction with hepatic receptors to effect clearance of LRCA-containing constructs by that route. Preferred LRCAs ofthe present invention are suitable for use as a clearing agent in pretargeting protocols, including two step protocols.
  • HSA human serum albumin
  • HSA Human Serum Albumin
  • ligands as follows: (Hexose) m ⁇ Human Serum Albumin (HSA) ⁇ (Ligand) n , wherein n is an integer from 1 to about 10 and m is an integer from 1 to about 45 and wherein the hexose is recognized by liver receptors, e.g.. Ashwell receptors.
  • the exposed hexose residues direct the clearing agent to rapid clearance by endocytosis into the liver through specific receptors therefor. These receptors bind the clearing agent or clearing agent-containing complexes, and induce endocytosis into the hepatocyte, leading to fusion with a lysosome and recycling of the receptor back to the cell surface.
  • This clearance mechanism is characterized by high efficiency, high capacity and rapid kinetics.
  • the rapid kinetics of hexose-mediated liver uptake, coupled with a relatively high affinity interaction between the binding moiety, such as a ligand, and the compound to be cleared, provide for rapid and efficient clearance.
  • LRCAs of the present invention are designed to meet the four criteria set forth above as well. Two additional performance criteria were instituted for LRCAs:
  • LRCAs o the present invention also incorporate a structural component of proteinaceous or non ⁇ protemaceous composition. Such preferred LRCAs exhibit physical properties facilitating use for in vivo complexation and blood clearance of anti-ligand/ligand- targeting moiety conjugates.
  • Other embodiments of the present invention involve the preparation and use of
  • Previously administered molecules may include active agent-containing conjugates (e.g.. radionuclide-chelate-antibody which can be cleared by a LRCA containing an anti-chelate or anti-antibody binding moiety; or radionuclide-chelate-antibody-biotin binding protein which can be cleared by a biotin-containing LRCA); targeting moiety-receptor conjugates; or the like.
  • active agent-containing conjugates e.g. radionuclide-chelate-antibody which can be cleared by a LRCA containing an anti-chelate or anti-antibody binding moiety; or radionuclide-chelate-antibody-biotin binding protein which can be cleared by a biotin-containing LRCA
  • Preferred LRCAs ofthe present invention are administered and permeate the circulation. Consequently, previously administered compounds or toxic or potentially toxic moieties that are present in the circulation are accessible to the LRCAs ofthe present invention. Circulating compounds are removed from the serum via association with the LRCA and processing by liver receptors. Previously administered compounds or toxic or potentially toxic moieties, present in extravascular fluid space but not associated with a target cell or epitope, are removed by the LRCAs ofthe present invention via liver receptors as such compounds diffuse back into the circulation and become associated with LRCAs. Toxic or potentially toxic molecules that may be removed from a recipient's circulation or extravascular fluid space include: chemotherapeutics e.g.. alkylators, heavy metals and the like.
  • Binding components useful in the practice ofthe present invention are capable of associating with the molecule to be cleared. Suitable binding components therefore include those moieties that are capable of associating with toxic or potentially toxic molecules present in the recipient's circulation, which include antibodies or fragments thereof directed to epitopes that are characteristic of such toxin or potential toxin Other useful binding components include oligonucleotides, ligands or anti-ligands Ligands and anti-ligands are preferred binding components of the present invention A particularly preferred binding component for use in the practice of the present invention is biotin or a derivative or analog thereof.
  • the binding between the binding component of the LRCAs ofthe present invention and the molecule to be cleared from the circulation need only be transient, e ⁇ . , exists for a sufficient amount of time to clear the molecule to the liver and for hepatocyte internalization
  • the binding constant ofthe binding component is determined with regard to the LRCA as a whole That is, a biotin- containing LRCA is expected to bind to avidin or streptavidin with a binding constant less than that of biotin itself. Experimentation has revealed that the LRCAs of the present invention are capable of clearance.
  • the binding constant must be sufficiently high to capture the molecule to be bound and traffic that molecule to the liver for internalization into hepatocytes. Consequently, LRCA binding components having a binding constant in excess of about 10 8 are preferred.
  • the number of binding components ranges from about 1 to about 10, and preferably from about 1 to about 4 and more preferably from about 1 to about 2
  • Binding components of the present invention include ligands, anti-ligands, and other target epitope-recognizing moieties.
  • One skilled in the art can substitute acceptable moieties for the binding components discussed specifically herein
  • binding components are characterized by a molecular weight of a Fab fragment of a monoclonal antibody or lower. Such binding components may also be modified to include suitable functional groups to allow for attachment of other molecules of interest, e g.. peptides, proteins, nucleotides, and other small molecules.
  • LRCAs ofthe present invention are designed to interact with hepatic receptors to facilitate clearance of LRCA-containing constructs via that route. The hepatic clearance directing component of the LRCA is included for this purpose.
  • Hepatocyte receptors which provide for effective clearance include in particular Ashwell receptors, mannose receptors associated with endothelial ceils and/or Kuptfer ceils of the liver, the mannose 6-phosphate receptor, and the like.
  • Hexoses which may be employed in the LRCA structure include by way of example galactose. mannose, mannose 6-phosphate, N-acetyigalactosamine, pentamannosyl-phosphate, and the like.
  • Hexoses recognized by Ashwell receptors include glucose, galactose, galactosamine, N-acetylgalactosamine, pentamannosyl phosphate, mannose 6- phosphate and thioglycosides of galactose, galactosides, galactosamine, and N- acetylgalactosamine. A sufficient number of hexose residues are incorporated into the
  • LRCA to provide for effective clearance, e.g., via the Ashwell receptors located on the surface of hepatocytes.
  • hepatic clearance directing components of this embodiment of the LRCAs of the present invention constitute between about 15 and about 60 hexose residues, e.g., galactose residues or N-acetylgalactosamine residues. More preferably hepatic clearance directing components of the present invention constitute between about 25 and 50 hexose residues.
  • the LRCA structural component is derivatized by an appropriate number of sugar 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 LRCA.
  • liver retention components of the present invention are designed to prevent release of binding component-containing metabolites of LRCA constructs to the serum compartment in a manner allowing those metabolites to accrete to pretargeted receptors.
  • liver retention components of the present invention are characterized by at least one ofthe following attributes:
  • Protease-resistant liver retention components of the present invention are useful, because formation of binding component-containing metabolites of LRCA constructs capable of accessing target-associated ligand or anti-ligand is not favored. That is, any such metabolites are unlikely to accrete from hepatocytes in a manner permitting access to pretargeted receptors.
  • the ligand or anti-ligand binding component of the LRCA is generally retained in hepatocytes for a time sufficient to allow active agent-ligand or active agent-anti- ligand construct accretion to target.
  • liver retention agent is a poly-amino acid of unnatural (D) orientation.
  • the (D) amino acid sequence provides resistance to catabolic processing, because lysosomal exopeptidases and endopeptidases recognize peptides of the natural (L) orientation.
  • the (D) amino acids constitute a poor substrate for the peptidases.
  • Preferred liver retention components of this type are linear chains of from about 2 to about 12 (D) amino acids and the like, with from about 3 to about 10 (D) amino acids preferred. It should be noted, however, that more than 12 (D) amino acids could be employed as well.
  • Preferred poly (D) amino acids are charged (D) amino acid polymers, such as poly (D) lysine, poly (D) glutamic acid, poly (D) aspartate, poly (D) ornithine and the like.
  • Other protease-resistant liver retention agents useful in the practice of the present invention include the following: alpha-aminoisobutyric acid (ALB) and N-alkyl-substituted amino acids which form tertiary amide bonds rather than secondary amide (peptide) bonds.
  • the alkyl moiety is lower alkyl, preferably methyl, ethyl, propyl or butyl, with methyl most preferred.
  • the tertiary amide can be formed using an N-phosphono substitution.
  • Liver retention moieties that are characterized by a limited capacity to traverse hydrophobic cellular or subcellular membranes also afford enhanced retention of binding component-containing metabolites of LRCA-containing constructs in hepatocytes. Consequently, metabolites having the potential to bind to pretargeted receptor are retained in the liver for a time sufficient to permit accretion to targeted receptor of active agent-containing conjugate.
  • liver retention moieties of the present invention are sufficiently polar or sufficiently charged to render passage through non-polar lipid bilayer membranes difficult under hepatocyte cellular conditions, pH 6-7
  • liver retention components are moieties characterized by positive charge, negative charge or neutral charge combined with hydrophi citv
  • Preferred liver retention components of this type are saccharides (neutral charge/hydrophi c), phosphates or phosphonates (negative charge), polylysines (positive charge), poiyglutamic acids (negative charge) and the like
  • liver retention components are sufficiently polar or sufficiently charged to render passage through the non-polar lipid bilayer membranes difficult under subcellular (e_g_, lysosomal) conditions
  • lysosomes are characterized by a pH of about 5
  • moieties expected to be highly charged at acidic pH are desirable for use as liver lysosomal retention components
  • basic proteins are generally catabolized more slowly than acidic proteins
  • liver retention components made up of basic amino acids, such as lysine, histidine, arginine, ornithine and the like are useful lysosomal membrane, diffusion-resistant liver retention components of the present invention
  • Positively charged, diffusion-resistant liver retention agents useful in the practice of the present invention include the following polylysines, polyhistidines, polyarginines and the like
  • Negatively charged, diffusion-resistant liver retention agents useful in the practice of the present invention include the following, poiyglutamic acids, poly- phosphates, polyphosphonates, such as poly-alpha-phosphonomethyl amino acids, polyaspartates and the like
  • Neutral charge/hydrophilic, diffusion-resistant liver retention agents useful in the practice of the present invention include the following disaccharides such as cellobiose and lactose, deoxysorbitol, dilactitol, amino-naphthaltyrimide-deoxysorbitol (ANTDS), unnatural polysaccharides, D-poly amino acid saccharide derivatives and the like See, for example, Ali et al., "Synthesis and Radioiodination of Tyramine Cellobiose for Labeling Monoclonal Antibodies," Nucl Med Biol . L5£5 ⁇ .
  • liver retention components involving poly-amino acids the number of such amino acids is selected to prevent passive diffusion of the binding component-liver retention component metabolite ofthe LRCA from hepatocyte iysosomes.
  • Literature indicates that certain dipeptides diffuse freely, while tripeptides generally do not. Consequently, liver retention components having three or more amino acids are generally preferred. Also, lengthy poly-amino acids present some synthetic challenges. Thus, liver retention components having 12 or fewer amino acids are generally preferred.
  • Liver retention component-binding component metabolites that are characterized by relatively rapid excretion, without passage into the serum compartment are also useful in the practice of the present invention. Metabolites excreted by a hepatobiliary route without reabsorption by the intestines are preferred for use in this embodiment ofthe present invention. Examples of excretion-liver retention components are pepstatin, l,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetra acetic acid (DOTA) and the like. Pepstatin is rapidly excreted into the bile. DOTA is excreted via a hepatobiliary route following internalization by hepatocytes.
  • DOTA is excreted via a hepatobiliary route following internalization by hepatocytes.
  • DTP A diethylene triamine penta-acetic acid
  • EDTA ethylene diamine tetra-acetic acid
  • EGTA ethylene glycol bis-(beta-aminoethylether)-N,N,N',N'- tetraacetic acid
  • liver retention components Another group of moieties useful as liver retention components in the practice ofthe present invention are moieties employed in the prior art to retain radioactivity at tumor target sites.
  • the present inventors believe that retention following internalization by hepatocytes will operate similarly to retention upon internalization by tumor cells. In tumor and hepatocyte cells, retention can be generated by a combination of lysosomal/intracellular retention and decreased susceptibility to metabolic degradation.
  • liver retention components are cellobiose, dilactitol, lysyl-epsilon-amido 5-iodo-3-pyridinecarboxylate, other non-mammalian sugars, other radiolabel residualizing moieties and the like.
  • Liver retention components useful in the present invention may combine more than one of the desirable properties set forth above.
  • Preferred LRCAs o the present invention are characterized by the following liver retention components: - poly-(D) orientation lysine residues (metabolic stability and positive charge);
  • Amino acids of natural (L) configuration may be employed in the preferred liver retention components; provided that at least one bond in the liver retention component-binding moiety construct is a tertiary amide bond. Such tertiary amide bonds are resistant to enzymatic degradation.
  • the stabilized bond should be incorporated in the construct, such that the biding component remains associated with a sufficient portion ofthe liver retention component to prevent access of the binding moiety to pretargeted receptor.
  • polyamino acids of natural (L) orientation may be employed, provided that the amide bonds between the biotin and the polyamino acid and between the individual amino acids ofthe polyamino acid incorporate a tertiary amine.
  • the polyamino acids serve to prevent egress of biotin-containing metabolites to pretargeted avidin or streptavidin.
  • the biotinidase-resistant tertiary amide ensures that biotin will remain associated with the polyamino acid.
  • Each of these preferred liver retention components incorporate from about 2 to about 12 monomers, with from about 3 to about 6 monomers, more preferred.
  • the binding component is attached to the structural component through the liver retention component. This construction facilitates the formation of LRCA metabolites containing both the binding component and the liver retention component. Examples of specific preferred embodiments of biotin binding component/liver retention component combinations for use in LRCAs o the present invention are shown below
  • n is 1 to 50, preferably about 3 to about 12, and more preferably from about 3 to about 6; R is a lower alkyl moiety of from 1 to about 6 carbon atoms; and R * is hydrogen or lower alkyl from 1 to about 6 carbon atoms.
  • Biotinidase generally cleaves a secondary amide bond adjacent to the biotin.
  • substitution of the amide nitrogen with, for example, an alkyl moiety resulted in stability with respect to biotinidase cleavage. Consequently, preferred liver retention component-bi tin constructs employ a tertiary amide nitrogen bearing an alkyl substituent, preferably of from I to about 6 carbon atoms and more preferably of from 1 to about 4 carbon atoms.
  • LRCAs of the present invention also contain a structural component of proteinaceous or non-pi oteinaceous composition.
  • Preferred structural components are characterized by or ire derivatized to contain sufficient reactive groups for binding with hepatic clearance directing components and binding components and/or liver retention components. Consequently, such structural components must incorporate from about 16 to about 70 functional groups, and more preferably from about 26 to about 52 functional groups. These ranges are derived as follows: Preferred hepatic clearance directing component derivatization is from about 15 to about 60 hexoses (more preferred, from about 25 to about 50); and preferred binding component/liver retention component derivatization is from about 1 to about 10 (most preferred, from about 1 to about 2).
  • Derivatization with both hexose and binding component are conducted in a manner sufficient to produce individual clearing agent molecules with a range of derivatization levels that averages a recited whole number.
  • biotinylation levels of a LRCA average a recited whole number, such as 1, biotin.
  • Derivatization of a structural component with 3 equivalents of biotin for example, produces a product mixture made up of individual LRCAs, substantially all of which having at least one biotin residue.
  • Derivatization with 1 biotin equivalent produces a LRCA 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 derivatization of the LRCAs under discussion.
  • LRCA ofthe present invention incorporates a proteinaceous structural component of intermediate molecular weight (ranging from about 40,000 to about 200,000 Dal), such as asialoorosomucoid, human serum albumin or other soluble natural protein, preferably those having low immunogenicity when administered to humans.
  • LRCAs may include polyglutamate, polylysine, polyarginine, polyaspartate and like structural components.
  • High molecular weight structural components(ranging from about 200,000 to about 1,000,000 Dal) characterized by poor target access, including IgM or IgG (approximately 150,000 Dal) molecules, ferritin (approximately 445 kD) may also be employed.
  • Chemically modified polymers of intermediate or high molecular weight (ranging from about 40,000 to about 1,000,000 Dal), such as dextran, hydroxypropylmethacryiamide polymers, polyvinylpyrrolidone-polystyrene copolymers, divinyl ether-maleic acid copolymers, pyran copolymers, or polyethylene glycol (PEG), also have utility as structural components of LRCAs of the present invention.
  • liposomes high molecular weight moieties with poor target access
  • LRCAs having a human protein as the structural component thereof are preferred for use in the practice ofthe present invention.
  • 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. Treatment of orosomucoid with neuraminidase removes sialic acid residues, thereby exposing galactose residues, forming asialoorosomucoid.
  • Human HSA Cosmetic Biological
  • human IgG, IgA and IgM (Sigma Chemical Co.), for example, are also commercially available.
  • Other proteinaceous structural components include albumin, IgM, IgG, asialohaptoglobin, asialofetuin, asialoceruloplasmin and the like.
  • Human serum albumin is a preferred proteinaceous structural component for LRCAs ofthe present invention.
  • 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 ofthe present invention.
  • Examples of such mammalian forms of serum albumin are bovine serum albumin, porcine serum albumin, and the like.
  • LRCAs ofthe present invention are prepared in the following manner:
  • the hepatic clearance directing component is conjugated to the structural component
  • the hepatic clearance directing component-structural component construct is derivatized, preferably with from about 1 to about 10, more preferably from about 1 to about 4, and still more preferably from about 1 to about 2 reactive groups;
  • Binding component/liver retention component constructs are synthesized and are either characterized or are derivatized to contain a complementary reactive group to those generated in step (2);
  • Binding component/liver retention component is conjugated to hepatic clearance directing component-structural component to form an LRCA.
  • reactive groups set forth herein are such groups as are generally employed in organic synthesis.
  • Complementary reactive groups are well known to those skilled in the art and include, for example, maleimide-sulfhydryl, active ester-amine, isothiocyanate-amine and the like. Selection ofuseful reactive groups is within the ordinary skill in the art.
  • n is 1 to 50, preferably about 3 to about 12, and more preferably from about 3 to about 6;
  • R is a lower alkyl moiety of from 1 to about 6 carbon atoms; and
  • R * is hydrogen or lower alkyl from 1 to about 6 carbon atoms.
  • the preferred LRCAs ofthe present invention were evaluated using two criteria:
  • LRCAs having physical properties facilitating use for in vivo complexation and blood clearance of anti- ligand/ligand (e *., avidin/biotin)-targeting moiety (e.g.. antibody) conjugates. These LRCAs are useful in improving the target:blood ratio of targeting moiety-containing conjugate.
  • target:blood ratio improvement is sought is in solid tumor imaging and therapy.
  • Other applications of these LRCAs include lesional imaging or therapy involving blood clots and the like, employing antibody or other targeting vehicle-active agent delivery modalities.
  • an efficacious anti-clotting agent provides rapid target localization and high target: non-target 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.
  • 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 first conjugate comprising a targeting moiety and a member of a ligand-anti-ligand binding pair; thereafter administering to the recipient a LRCA incorporating a hepatic clearance directing component capable of directing the clearance of circulating first conjugate via hepatocyte receptors of the recipient, a liver retention component, a structural component and a binding component; 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.
  • Example III Clearing agent evaluation experimentation involving galactose- and biotin- derivatized clearing agents is detailed in Example III.
  • the specific clearing agents examined during the Example III experimentation are human serum albumin derivatized with galactose and biotin and 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 ofthe 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.
  • Preparation of LRCAs ofthe present invention is discussed in Example IV below. Experimentation relating to the LRCAs of the present invention is set forth in
  • the present invention provides LRCAs that incorporate ligand derivatives or anti-ligand derivatives, wherein such derivatives exhibit a lower affinity than the native form of the compound, employed in the same construct, for the complementary ligand/anti-ligand pair member (i.e.. lower affinity ligands or anti-ligands).
  • preferred LRCAs 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).
  • 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 ); 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; l'-N-methoxycarbonyl-biotin; 3'-N-methoxycarbonylbiotin; 1-oxy-biotin; l-oxy-2'- thiobiotin; l-oxy-2'-iminobiotin; 1-sulfoxide-biotin; l-sulfoxide-2'-thiobiotin; 1- sulfoxide-2'-iminobiotin; 1-sulfone-biotin; 1 -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-imidazolidone, L-4-
  • Preferred lower affinity biotin molecules for use in the practice of the present invention are 2'-thiobiotin, desthiobiotin, 1-oxy- biotm, l-oxy-2'-thiobiotin, 1-sulfoxide-biotin, l-sulfoxide-2'-thiobiot ⁇ n, 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 Incorporation of the exemplary lower affinity biotin molecules into LRCAs proceeds substantially in accordance with procedures described herein in regard to biotin incorporation
  • 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 LRCA incorporating a hepatic clearance directing component capable of directing the clearance of circulating first conjugate via hepatocyte receptors of the recipient, a liver retention component and a binding component including a lower affinity complementary member of the ligand- anti-ligand binding pair employed in 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 binding pair member is complementary to that ofthe first conjugate and, preferably, constitutes a native or high affinity form thereof.
  • targeting moiety binds to a defined target cell population, such as tumor cells.
  • Preferred targeting moieties useful in this regard include antibody and antibody fragments, peptides, and hormones. Proteins corresponding to known cell surface receptors (including low density lipoproteins, transferrin and insulin), fibrinolytic enzymes, anti-HER2, platelet binding proteins such as annexins, and biological response modifiers (including interleukin, interferon, erythropoietin and colony-stimulating factor) are also preferred targeting moieties.
  • 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 ofthe present invention are antibodies (polyclonal or monoclonal), peptides, oligonucleotides or the like.
  • Polyclonal antibodies useful in the practice of the present invention are polyclonal (Vial and Callahan, Univ. Mich. Med. Bull.. 20: 284-6, 1956), affinity-purified polyclonal or fragments thereof (Chao et al., Res. Comm. in Chem. Path. & Pharm.. 9: 749-61, 1974).
  • Monoclonal antibodies useful in the practice ofthe 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:
  • Human 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 (ML, 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 No. 0,41 1,893 A2. Some murine residues may also be retained within the human variable region framework domains to ensure proper target site binding characteristics.
  • 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.
  • 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 o 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 ofthe toxin A chain-targeting protein conjugate, as compared to the corresponding hoiotoxin-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 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 imp ⁇ rfecli or isolated from Bacchants megapotamica (Bamburg, J.R. Proc. Molec. Subcell. Biol. 8:41-1 10. 1983: Jarvis & Mazzola. Ace. Chem. Res. 15:338-395. 1982V They appear to be the most toxic molecules that contain only carbon, hydrogen and oxygen (Tamm, C. Fortschr. Chem. Org. Naturst. 31 :61-1 17. 1974). They are all reported to act at the level ofthe ribosome as inhibitors of protein synthesis at the initiation, elongation, or termination phases.
  • trichothecenes There are two broad classes of trichothecenes: those that have only a central sesquiterpenoid structure and those that have an additional macrocyclic ring (simple and macrocyclic trichothecenes, respectively).
  • the simple trichothecenes may be subdivided into three groups (i.e.. Group A, B, and C) as described in U.S. Patent Nos. 4,744,981 and 4,906,452 (incorporated herein by reference).
  • Group A simple trichothecenes include: Scirpene, Roridin C, dihydrotrichothecene, Scirpen-4, 8-dioI, 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, 7-hydroxy-8-acetyldiacetoxyscirpenol,
  • Group B simple trichothecenes include: Trichothecolone, Trichothecin, deoxynivalenol, 3-acetyldeoxynivalenol, 5-acetyldeoxynivalenol, 3, 15-diacetyldeoxynivalenol, Nivalenol, 4-acetylnivalenoI (Fusarenon-X), 4, 15-idacetylnivalenol, 4,7, 15-triacetylnivalenol. and tetra- acetylnivalenol.
  • Representative examples of 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. AC. Thompson, 1984, pp. 149-159).
  • Experimental drugs such as mercaptopurine, N-methylformamide, 2-amino- 1,3,4-thiadiazole, melphalan, hexamethylmelamine, gallium nitrate, 3% thymidine, dichloromethotrexate, mitoguazone, suramin, bromodeoxyuridine, iododeoxyuridine, semustine, l-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-l-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, spiromus
  • 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-
  • Preferred therapeutic radionuclides include Re,
  • anti-tumor agents ej*., agents active against proliferating cells
  • exemplary anti-tumor agents include cytokines, such as IL-2, tumor necrosis factor or the like, lectin inflammatory response promoters (selectins), such as L-selectin, E-selectin, P-selectin or the like, and like molecules.
  • Ligands suitable for use within the present invention include biotin, haptens, lectins, epitopes, dsDNA fragments, enzyme inhibitors and analogs and derivatives thereof.
  • Useful complementary anti-ligands include avidin (for biotin), carbohydrates (for lectins) and antibody, fragments or analogs thereof, including mimetics (for haptens and epitopes) and zinc finger proteins (for dsDNA fragments) and enzymes (for enzyme inhibitors).
  • Preferred ligands and anti-ligands bind to each other with an
  • ligand/anti-ligand systems include S- protein/S-peptide, head activator protein (which binds to itself), cystatin-C/cathepsin
  • One preferred chelate system for use in the practice of the present invention is based upon a 1,4,7, 10-tetraazacyclododecane-N,N',N",N'"-tetra acetic acid (DOTA) construct.
  • DOTA 10-tetraazacyclododecane-N,N',N",N'"-tetra acetic acid
  • DOTA-biotin conjugates that are preferably employed in the practice of the present invention reflect the implementation of one or more of the following strategies:
  • DOTA-biotin conjugates in accordance with the present invention are described in published PCT Patent Application No. PCT/US93/05406.
  • a method of preparing a preferred DOTA-biotin embodiment is described in Example II hereof.
  • the preferred linkers are useful to produce DOTA-biotin or other DOTA-small molecule conjugates having one or more of the following advantages:
  • - are stable to endogenous enzymatic or chemical degradation (e.g.. bodily fluid amidases, peptidases or the like);
  • One component to be administered in a preferred two-step pretargeting protocol is a targeting moiety-anti-ligand or a targeting moiety-ligand conjugate.
  • Streptavidin-proteinaceous targeting moiety conjugates are preferably prepared as described in Example I below, with the preparation involving the steps of: preparation of SMCC-derivatized streptavidin; preparation of DTT-reduced proteinaceous targeting moiety; conjugation ofthe 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, im unoreactivity, biotin binding capacity and in vivo studies
  • LRCAs of the present invention may be administered in single or multiple doses or via continuous infusion
  • a single dose of a biotm-containing LRCA 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 LRCA doses may then be employed to provide supplemental clearance of targeting moiety-streptavidin
  • LRCA may be infused intravenously for a time period sufficient to clear targeting moiety-streptavidin in a continuous manner
  • the dose of LRCAs ofthe present invention will depend upon numerous patient-specific and clinical factors, which clinicians are uniquely qualified to assess In general, the dose of the LRCA to be administered will depend on the dose of the targeting conjugate or other previously administered component to be cleared that is either measured or expected to remain in the serum compartment at the time the LRCA is administered Alternatively, the
  • One embodiment ofthe present invention in which rapid acting LRCAs are useful is in the delivery of Auger emitters, such as 1-125, 1-123, Er-165, Sb-1 19, Hg-
  • targeting moieties that localize to internalizing receptors on target cell surfaces are employed to deliver a targeting moiety-containing conjugate (i e . a targeting moiety-anti-ligand conjugate in the preferred two-step protocol) to the target cell population
  • a targeting moiety-containing conjugate i e . a targeting moiety-anti-ligand conjugate in the preferred two-step protocol
  • internalizing receptors include EGF receptors, transferrin receptors, HER2 receptors, IL-2 receptors, other interleukins and cluster differentiation receptors, somatostatin receptors, other peptide binding receptors and the like.
  • an active agent-containing ligand or anti-ligand conjugate such as a biotin-Auger emitter or a biottn-nucleus acting drug, is administered as soon as the LRCA has been given an opportunity to complex with circulating targeting moiety-containing conjugate, with the time lag between LRCA 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.
  • 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 MacMod) After approximately 45 minutes, the reaction was quenched by adding solid sodium tetrathionate to a final concentration of 5 mM
  • 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.
  • 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-[I-125]iodobenzoylbiocytin. Saturation ofthe biotin binding sites was observed upon addition of 4 equivalences ofthe labeled biocytin.
  • Figure 1 depicts the tumor uptake profile ofthe NR-LU- 10- streptavidin conjugate (Ab/SA, referred to in this example as LU-10-StrAv) in comparison to a control profile of native NR-LU- 10 whole antibody and a control profile of streptavidin.
  • 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.
  • 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 cyciization 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.
  • N-methyl-glvcine linked conjugate The 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.) was condensed with biotin-NHS ester in DMF and triethylamine to obtain N-methyl glycyl-biotin. N-methyl-glycyl biotin was then activated with EDCI and NHS. The resultant NHS ester was not isolated and was condensed in situ with DOTA-aniline and excess pyridine. The reaction solution was heated at 60° C for 10 minutes and then evaporated. The residue was purified by preparative HPLC to give [(N-methyl- N-biotinyl)-N-glycyl]-aminobenzyl-DOTA.
  • HSA Galactose- and Biotin-Derivatization of Human Serum Albumin (HSAY HSA was evaluated because it exhibits the advantages of being both inexpensive and non-immunogenic. HSA was derivatized with varying levels of biotin ( 1 -about 9 biotins/molecule) via analogous chemistry to that previously described with respect to AO. More specifically, to a solution of HSA available from Sigma Chemical Co. (5- 10 mg/ml in PBS) was added 10% v/v 0.5 M sodium borate buffer, pH 8.5, followed by dropwise addition of a DMSO solution of NHS-LC-biotin (Sigma Chemical Co.) to the stirred solution at the desired molar offering (relative molar equivalents of reactants).
  • the final percent DMSO in the reaction mixture should not exceed 5%. After stirring for 1 hour at room temperature, the reaction was complete. A 90% incorporation efficiency for biotin on HSA was generally observed. As a result, if 3 molar equivalences ofthe NHS ester of LC-biotin was introduced, about 2 7 biotins per HSA molecule were obtained. Unreacted biotin reagent was removed from the biotin-derivatized HSA using G-25 size exclusion chromatography. Alternatively, the crude material may be directly galactosylated. The same chemistry is applicable for biotinylating non-previously biotinylated dextran.
  • HSA-biotin was then derivatized with from 12 to 45 galactoses/molecule.
  • Galactose derivatization of the biotinylated HSA was performed according to the procedure of Lee, et al., Biochemistry. 5. 3956, 1976.
  • 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.
  • 40 Micrograms of galactose-dextran-biotin (GAL-DEX-BT) was then injected i.v. into one group of mice which had received 200 micrograms MAb-StrAv conjugate intravenously 24 hours earlier, while 80 micrograms of GAL-DEX-BT was injected into other such mice.
  • GAL-DEX-BT was rapid and efficient at clearing StrAv-MAb conjugate, removing over 66% of circulating conjugate in less than 4 hours after clearing agent administration. An equivalent effect was seen at both clearing agent doses, which correspond to 1.6 (40 micrograms) and 3.2 (80 micrograms) times the stoichiometric amount of circulating StrAv conjugate present.
  • 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 ofthe present invention are short strings of D-aniino 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.
  • Time 0 administer 400 micrograms MAb-StrAv conjugate; Time 24 hours: administer 240 micrograms of G-HSA-B with one biotin and 12-45 galactoses and
  • Lu-177 is complexed with the DOTA chelate using known techniques therefor.
  • the resultant mixture was purified on 175 ml of G-25 size exclusion matrix, eluting with PBS, to afford 35 ml of 2.85 mg/ml of galactosylated HSA with, on average, 2.0 maleimides/HSA.
  • the product was concentrated via Amicon filtration (30,000 MW cutoff), commercially available from Amicon, Beverly, Mass., to a concentration of 6.4 mg/ml (14 ml total).
  • the solution was sterile filtered (using 0.2 micrometer filters), dispensed into cryogenic vials and stored at -70°C.
  • liver retention component-binding component construct N-methyl-N- ⁇ 5'-[methylester tris-(-(D, L)-phosphonoalanyl)-(D)-cystyl]-5- carbamylpentyljbiotinamide (1 1), may be prepared as described below.
  • NHS-carbonyl-pentyl)biotinamide is prepared from biotin (Sigma Chemical Company, St. Louis, Missouri) by the following process: First is BOP-mediated coupling of biotin and N-methyl-epsilon-aminocaproate methylester in DMF and DIEA at room temperature for 3 hours; followed by base hydrolysis (2 eq NaOH in MeOH, room temperature, 14 hours) of the methylester group of the corresponding intermediate, and finally treatment of the obtained N-methyl-N-(5-hydroxycarbonylpentyl)- biotinamide with N-hydroxysuccinimide in the presence of DCC in DMF at room temperature overnight.
  • N-Methyl-N- 5-rmethylester tris-(-(D. L)-phosphono-alanyl)-(D)-cvstyl " ]-5- carbamylpentyl) biotinamide (1 1).
  • a solution of 100 mg of (10) in 2.38 ml TFA, 0.1 ml anisole and 25 ⁇ l ethanedithiol was stirred at room temperature under argon for 2 hours.
  • the reaction mixture was then precipitated into 40 ml ice-cold tert-butyl methylether (deoxygenated with He at 0°C for 10 minutes) in a 50 ml glass centrifuge tube. This tube was capped and centrifuged at 2,000 g for 10 minutes.
  • liver retention component-binding component construct N-methyl-N-[5-(triglutamylcysteine)-5-carbamylpentyl]-biotinam ⁇ de, may be prepared as described below
  • the reaction mixture was precipitated into 30 ml ice-cold deoxygenated tert-butyl methylether in a 50 ml glass centrifuge tube. This tube was capped and centrifuged at 2,000 g for 10 minutes. The supernatant was removed by gentle aspiration.
  • the reaction mixture was gently shaken at room temperature for 90 minutes.
  • the mixture was transferred into a centricon tube (30K molecular weight cutoff) and centrifuged at 5000 RPM for 10-12 minutes to reduce the mixture volume to 1.2 ml.
  • the mixture plus 100 microliters rinsing was purified by PD-10 Column (Pharmacia Biotech, Inc., Piscataway, New Jersey) eluting with 6 ml PBS.
  • the protein containing fractions were pooled, and the total volume was reduced to 1.12 ml by means of centrifugation in a centricon tube.
  • the mixture was transferred into a centricon tube (3 OK molecular weight cutoff) and centrifuged at 5000 RPM for 10-12 minutes to reduce the mixture volume to 1.2 ml.
  • the mixture plus 100 microliters rinsing was purified by PD-10 Column (Pharmacia Biotech, Inc., Piscataway, New Jersey) eluting with 6 ml PBS.
  • the protein containing fractions were pooled, and the total volume was reduced to 1.1 ml by means of centrifugation
  • the animal model employed for this evaluation was female Balb/c mice (20-25 g) injected i.v with 400 ⁇ g I-125-NR-LU-10-streptavidin conjugate prepared substantially as described above. Each group of experimental animals consisted of 3 mice. Blood was sampled serially from the retro-orbital sinus
  • biotin of the LRCAs was not radiolabeled. Biotin release would be expected to compromise or fill biotin-binding sites of residual serum compartment NR-LU- 10-streptavidin conjugate. Thus, assessment of biotin release from the LRCA was indirectly determined by measuring the amount of In- 11 1 -DOTA-biotin, prepared substantially in accordance with Example III hereof, that bound to the residual serum conjugate. Such residual serum conjugate, exposed to LRCAs releasing less biotin-containing metabolites, should 5 exhibit minimal loss of capacity for binding radiolabeled biotin.
  • Criteria 2 was evaluated using the animal model and experimental procedure described for Criteria 1 evaluation, except that 4 hours after LRCA administration, the animals received 1.0 or 15.0 ⁇ g of In- 111 -DOT A-biotin, prepared substantially in accordance with procedures described herein. A blood sample 2 hours after administration of In-1 1 1 l o was used to assess residual biotin-binding capacity of circulating I- 125-NR-LU- 10- streptavidin conjugate.
  • HSA(d-lys) 3 BT HSA(d-lys-gal) 3 BT were compared to the control compound made in accordance with the procedure set forth in Example IV above, with regard to criteria 1 at clearing agent doses of 220 micrograms and 1 100
  • a dose of 220 micrograms has been found to be an optimal dose of the comparison clearing agent in a tumored mouse model to clear 400 micrograms of targeting conjugate.
  • the 1 100 microgram dose is 5x the optimal dose.
  • All three compounds cleared conjugate to 4.8-5.6% ED 6 hours after clearing agent administration.
  • Construct 3 HSA(d-glu) 3 BT was analogously evaluated. With respect to criteria 1 , serum conjugate concentrations were calculated as %ID/g, rather than %ID as set forth above for the evaluation of constructs 1 and 2. At the 220 microgram clearing agent dose, construct 3 and the reference compound cleared conjugate to 4.96 and 3.41 %ED/g, respectively, 6 hours after clearing agent administration. At the 1 100 microgram dose, construct 3 and the reference compound cleared conjugate to 1.19 and 2.02 %ED/g, respectively, 6 hours after clearing agent administration. Construct 3 and the reference compound cleared monoclonal antibody-streptavidin conjugate comparably.
  • HSA(phos) 3 BT was evaluated in a tumored mouse model with regard to criteria 2. Criteria 1 was evaluated also, but only at a single time point. At that time point, blood clearance of NR-LU- 10-streptavidin was roughly equivalent when construct 4 was compared to the control compound.
  • mice were injected with 400 micrograms of conjugate, followed 24 hours later by 200 micrograms (low dose) or 1100 micrograms (high dose) of clearing agent (LRCA or reference compound), followed 4 hours later by 15 micrograms of In-11 1 -DOTA-biotin. Animals were sacrificed 2 hours later. Tumor uptake of In-1 1 1 -DOTA-biotin was superior at both low and high clearing agent doses (8.89 and 5.01 %ED/g LRCA verses 8.04 and 0.68
  • Kits containing one or more ofthe components described above are also contemplated.
  • LRCAs may be provided in a sterile container for use in pretargeting procedures.
  • such a LRCA may be vialed in a non-sterile condition for use as a research reagent.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nanotechnology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Medical Informatics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des agents de suppression de la rétention hépatique et leur utilisation. Ces agents sont composés d'un composant assurant la clairance hépatique qui dirige la biodistribution d'un produit de recombinaison contenant des agents de suppression de la rétention hépatique de manière à assurer la clairance hépatique. Ces agents comprennent également un composant de liaison qui assure la liaison de l'agent à un composé pour lequel une clairance hépatique rapide doit être assurée, et un composant de rétention hépatique qui diminue l'accès aux sites cibles des métabolites contenant un composant de liaison, et enfin un composant structurel constituant un squelette pour les autres composants.
EP97926844A 1996-06-06 1997-06-06 Agents de suppression de la retention hepatique Withdrawn EP0906015A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US660603 1991-02-25
US66060396A 1996-06-06 1996-06-06
PCT/US1997/009400 WO1997046099A1 (fr) 1996-06-06 1997-06-06 Agents de suppression de la retention hepatique

Publications (2)

Publication Number Publication Date
EP0906015A1 true EP0906015A1 (fr) 1999-04-07
EP0906015A4 EP0906015A4 (fr) 2004-05-12

Family

ID=24650198

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97926844A Withdrawn EP0906015A4 (fr) 1996-06-06 1997-06-06 Agents de suppression de la retention hepatique

Country Status (3)

Country Link
EP (1) EP0906015A4 (fr)
CA (1) CA2257353A1 (fr)
WO (1) WO1997046099A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6172045B1 (en) * 1994-12-07 2001-01-09 Neorx Corporation Cluster clearing agents
WO2000023113A1 (fr) * 1998-10-08 2000-04-27 Stichting Voor De Technische Wetenschappen Vecteurs peptidiques pour cellules etoilees
AU6275800A (en) * 1999-07-16 2001-02-05 Mallinckrodt, Inc. Inhibition of renal uptake of molecules that are potentially damaging for the kidney
EP1375513A4 (fr) * 2001-03-09 2006-04-12 Meiji Seika Kaisha Immunostimulants
US8623324B2 (en) * 2010-07-21 2014-01-07 Aat Bioquest Inc. Luminescent dyes with a water-soluble intramolecular bridge and their biological conjugates

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993025240A2 (fr) * 1992-06-09 1993-12-23 Neorx Corporation Procedes et composes de preciblage
WO1995015770A1 (fr) * 1993-12-09 1995-06-15 Neorx Corporation Procedes et composes de preciblage
WO1995015978A1 (fr) * 1993-12-07 1995-06-15 Neorx Corporation Procede et composes permettant un ciblage prealable
WO1996017613A1 (fr) * 1994-12-07 1996-06-13 Neorx Corporation Composes a ciblage hepatique et reactifs pour leur preparation
WO1997046098A1 (fr) * 1996-06-06 1997-12-11 Neorx Corporation Agents de suppression d'amas

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993025240A2 (fr) * 1992-06-09 1993-12-23 Neorx Corporation Procedes et composes de preciblage
WO1995015978A1 (fr) * 1993-12-07 1995-06-15 Neorx Corporation Procede et composes permettant un ciblage prealable
WO1995015770A1 (fr) * 1993-12-09 1995-06-15 Neorx Corporation Procedes et composes de preciblage
WO1996017613A1 (fr) * 1994-12-07 1996-06-13 Neorx Corporation Composes a ciblage hepatique et reactifs pour leur preparation
WO1997046098A1 (fr) * 1996-06-06 1997-12-11 Neorx Corporation Agents de suppression d'amas

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HIRABAYASHI H ET AL: 'Development and pharmacokinetics of galactosylated poly-L-glutamic acid as a biodegradable carrier for liver-specific drug delivery.' PHARMACEUTICAL RESEARCH. JUN 1996 vol. 13, no. 6, June 1996, pages 880 - 884, XP008028760 ISSN: 0724-8741 *
See also references of WO9746099A1 *

Also Published As

Publication number Publication date
WO1997046099A1 (fr) 1997-12-11
CA2257353A1 (fr) 1997-12-11
EP0906015A4 (fr) 2004-05-12

Similar Documents

Publication Publication Date Title
US6172045B1 (en) Cluster clearing agents
US5630996A (en) Two-step pretargeting methods using improved biotin-active agent conjugates
US6908903B1 (en) Cluster clearing agents
US6015897A (en) Biotinamido-n-methylglycyl-seryl-o-succinamido-benzyl dota
US5847121A (en) Production of nitro-benzyl-dota via direct peptide cyclization
US6217869B1 (en) Pretargeting methods and compounds
US5976535A (en) Pretargeting protocols for the enhanced localization of cytotoxins to target sites and cytotoxic combinations useful therefore
US6022966A (en) Pretargeting methods and compounds
WO1997046098A9 (fr) Agents de suppression d'amas
US5985826A (en) Methods of using hepatic-directed compounds in pretargeting strategies
EP1346730A1 (fr) Procedes de preciblage et nouveaux conjugues pour le preciblage
US5955605A (en) Biotinidase resistant biotin-DOTA conjugates
US6287536B1 (en) Two-step pretargeting methods using improved bidtin-active agent conjugates
US5624896A (en) Clearing agents useful in pretargeting methods
WO1995015770A1 (fr) Procedes et composes de preciblage
US6075010A (en) Small molecular weight ligand-hexose containing clearing agents
US5616690A (en) Hexose derivatized human serum albumin clearing agents
US6416738B1 (en) Pretargeting methods and compounds
US20030129191A1 (en) Pretargeting methods and compounds
EP0743956A1 (fr) Procede et composes permettant un ciblage prealable
WO1997046099A1 (fr) Agents de suppression de la retention hepatique
US5914312A (en) Pretargeting methods and compounds

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19981127

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

A4 Supplementary search report drawn up and despatched

Effective date: 20040331

RIC1 Information provided on ipc code assigned before grant

Ipc: 7A 61K 51/04 B

Ipc: 7A 61K 47/48 B

Ipc: 7A 61K 31/70 B

Ipc: 7A 01N 43/04 A

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20040610

RIC1 Information provided on ipc code assigned before grant

Ipc: A61K 51/04 20060101ALI20110914BHEP

Ipc: A61K 47/48 20060101ALI20110914BHEP

Ipc: A61K 31/70 20060101ALI20110914BHEP

Ipc: A01N 43/04 20060101AFI20110914BHEP